Agnès Ricroch · Dennis Eriksson Dragana Miladinović · Jeremy Sweet Katrijn Van Laere · Ewa Woźniak-Gientka *Editors*

# A Roadmap for Plant Genome Editing

A Roadmap for Plant Genome Editing

Agnès Ricroch • Dennis Eriksson Dragana Miladinović • Jeremy Sweet Katrijn Van Laere • Ewa Woźniak-Gientka Editors

# A Roadmap for Plant Genome Editing

*Editors* Agnès Ricroch The Jean-Monnet Faculty. Idest Laboratory Paris-Saclay University Sceaux, France

Dragana Miladinović Institute of Field and Vegetable Crops National Institute of Republic of Serbia Novi Sad, Serbia

Katrijn Van Laere Flanders Research Institute for Agriculture, Fisheries and Food (ILVO) Melle, Belgium

Dennis Eriksson Department of Plant Breeding Swedish University of Agricultural Sciences Alnarp, Sweden

Jeremy Sweet Sweet Environmental Consultants Cambridge, UK

Ewa Woźniak-Gientka Institute of Bioorganic Chemistry Polish Academy of Sciences Poznań, Poland

ISBN 978-3-031-46149-1 ISBN 978-3-031-46150-7 (eBook) https://doi.org/10.1007/978-3-031-46150-7

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# **Editorial**

Genome editing is a technology that has been embraced swiftly and enthusiastically by plant researchers, innovators and breeders across the world in the past decade. Its impact can hardly be overestimated. A quick search for "plant genome editing" in the Web of Science shows over 8300 scientifc publications in 2013–2023, with a steady and considerable growth in numbers per year. The COST Action PlantEd (CA18111), funded by the European Cooperation in Science and Technology programme (www.cost.eu), represents a European-based network of more than 600 experts on plant genome editing that has monitored, and led the progress in this feld since 2019. When it started, highly controlled CRISPR/Cas-based advancements such as base editing and prime editing were barely more than conceptual ideas in plants whereas these are now being used for various applications (Betül Kaya). Novel delivery methods have been developed (Doyle Prestwich et al.), guide RNA design and validation is steadily improving (Dorso) and the screening as well as functional prediction of resulting mutations are increasingly effcient (Vereecke et al.). This technology now also allows for the editing of entire gene families (Avci and Sipahi) as well as avoiding linkage drag and other undesired side effects (Wind), which are considerable improvements to conventional plant breeding. All these novel methods and improvements of genome editing offer to plant scientists and breeders multiple strategies for enhancing crop production, crop protection, food quality and climate change adaptation (citing Part V, Future Outlook).

To date, genome editing has been applied in a wide range of crops, including wheat (Nigro et al.; Smulders et al.), barley (Jakobson et al.; Pouramini and Hensel), maize (Varotto), legumes (Das and Acharjee; Dervishi et al.), soybean (Oztolan Erol), oilseed rape (Boniecka), tomato (Scarano and Santino; García-Caparrós), chicory (Cankar et al.), various horticultural plants (Mahna and Nayeri), fruit trees (Claessen et al.), forest trees (Bruegmann et al.) and also algae (Opsahl-Sorteberg and Evju; Evju and Opsahl-Sorteberg).

The plant breeding sector stresses the importance of innovations in breeding with genome editing for contributing to solving many important societal challenges (Jorasch and Vangheluwe). At the same time, the technology is surrounded by controversy, particularly in Europe (Kuntz), and the number of concerned EU citizens

seems to be increasing (Ichim). In addition, acceptability of collaborative licensing platforms to overcome the diffculties created by the complex patent landscape of CRISPR technology is lacking (Ricroch). A survey conducted in Poland identifed knowledge gaps and low awareness on genome editing (Molodziejko et al.); however, a study in Germany suggests that strong moral convictions may hinder effective science communication (Waldhof). One result of this societal controversy is that plant genome editing remains regulated in the EU to the extent that innovations are hindered (Zimny; Sprink and Wilhelm). It is therefore important to discuss what types of reforms are necessary to allow society to reap the benefts that this technology can bring. In this context, it is important to compare the EU views with the approaches to genome editing and other novel gene technologies being taken in other parts of the world (Sprink and Wilhelm; Rosado).

This book represents a snapshot of the latest advancements in the feld of plant gene editing, together with perspectives from many other disciplines such as social sciences and law. It is our hope that this book will contribute to illuminate the road ahead for this very important technology and that it will serve as inspiration and guidance for a wide range of stakeholders.


# **Contents**


**7 Genome Editing of a Macroalgae with Possible Global Impacts** . . . . 121 Hilde-Gunn Opsahl-Sorteberg and Espen Evju



Contents

ix


Jeremy Sweet, Katrijn Van Laere, and Ewa Woźniak-Gientka

# **Part I Plant Breeding and Technological Advances**

# **Chapter 1 Genome Editing of Gene Families for Crop Improvement**

**Utku Avci and Hülya Sipahi**

**Abstract** Crop improvement has been a long-standing focus of agricultural research, aiming to enhance nutritional richness, aroma, visual appeal, and yield to meet the growing global food demand. Recent advances in molecular biology and genetic engineering, particularly genome editing, offer precise and targeted tools for modifying crop genomes. Traditional plant breeding methods, while successful in the past, are time-consuming, and techniques like mutagenesis and transgenesis have limitations. Genome editing techniques provide unprecedented precision and enable scientists to make desired modications to a plant's DNA. This chapter explores the role of genome editing, specically in gene families, for crop improvement, highlighting its potential benets and challenges.

Gene families are crucial for important crop traits like yield, disease resistance, and environmental adaptation. However, conventional breeding methods often struggle to effectively manipulate gene families due to their complex nature. Genome editing offers a promising solution by allowing targeted modications to specic gene family members. The precision of genome editing tools can help unravel the functions of gene family members in diverse plant species.

With the challenges posed by climate change, global conicts, and population growth, the conventional food system falls short of meeting future demands sustainably. Genome-edited crops hold promise in obtaining elite genotypes with desirable traits, contributing to a resilient and sustainable agriculture and food system. Moreover, genome editing facilitates the study of genetic diversity that governs desirable crop characteristics, beneting both genome-edited and conventionally bred crops.

U. Avci (\*) · H. Sipahi

Faculty of Agriculture, Agricultural Biotechnology Department, Eskisehir Osmangazi University, Eskisehir, Türkiye

# **1 Introduction**

Crop improvement has been the main target of agricultural research for centuries in comprehensive areas such as increasing the nutritional richness of foods, obtaining aromatic foods with pleasant smell and taste, producing decorative products that appeal to visual pleasures, while increasing the yield, especially to meet the increasing food demand of the global population. In recent years, advances in molecular biology and genetic engineering have provided new tools for crop improvement. Genome editing, in particular, has emerged as a promising technique for modifying the genome of crops in a precise and targeted manner [1, 2].

The process of conventional plant breeding is time-consuming despite its signifcant contribution to the frst green revolution in the 1960s. The introduction of molecular marker-assisted selection has helped to speed up the process of plant breeding. Techniques such as mutagenesis and transgenesis have helped to reduce the time required to develop new varieties. However, mutagenesis is a random process that can have undesirable side effects or can result in incomplete and off-target disruption of genes. Genetically modifed transgenic plants can also face regulatory challenges. Unlike traditional genetic engineering methods, which involve the insertion of foreign DNA into an organism's genome, genome editing allows scientists to make precise modifcations to the existing DNA sequence. Genome editing can be accomplished through different techniques, including CRISPR-Cas9, TALENs, and Zinc Finger Nucleases (ZFNs) [2]. These tools offer an unprecedented level of precision, enabling scientists to make precise, targeted changes to a plant's DNA, resulting in desired traits [3].

Gene families are groups of genes that share a similar DNA sequence and function. They often play critical roles in crop traits such as yield, disease resistance, and environmental adaptation. Although members of a gene family have different specialized functions, they share a common purpose. Therefore, it is important to understand and defne the functions of gene family members involved in important plant development or stress resistance in different plant species. Traditional breeding methods are often unable to effectively target and manipulate gene families, as they may contain many members that have overlapping functions or redundancies [1].

The food system faces challenges such as sustainability and food supply stemming from climate change, global conficts and increasing population [4]. Conventional breeding and agricultural practices come short in terms of meeting the current and future food demand. Therefore, it is necessary to have different approaches to ensure a more resilient and sustainable agrifood system. In this sense, genome-edited crops have the potential to play a role in obtaining elite genotypes with desirable traits in plant breeding. Besides, genome editing has another advantage, as it enables the study of genetic diversity that governs desirable crop characteristics. The knowledge that comes out of such studies could be used in the development of a genome-edited crop as well as in conventional breeding.

In this chapter, we explore the role of genome editing of gene families in crop improvement, its potential benefts, and its challenges.

# **2 Benefts of Genome Editing in Crop Improvement**

Genome editing has several potential benefts for crop improvement. One of the most signifcant advantages is its precision. Unlike traditional genetic engineering techniques, which often involve the insertion of foreign DNA into an organism's genome, genome editing allows scientists to make precise modifcations to the existing DNA sequence. This precision means that genome editing can be used to introduce desirable traits into crops without introducing unwanted traits or disrupting existing genes [1, 3].

Another advantage of genome editing is its effciency. Traditional breeding methods can take many years to develop new crop varieties with desirable traits. In contrast, genome editing can produce changes in the DNA sequence in a matter of weeks or months. This speed and effciency mean that genome editing can be used to rapidly develop new crop varieties that are better adapted to changing environmental conditions, such as drought or disease resistance.

According to a survey of experts on the added potential benefts of genomeedited crops compared to those developed through genetic modifcation and conventional breeding, there is a consensus among experts on the enhanced agronomic performance and product quality of genome-edited crops over alternatives [5]. The majority of experts indicated that genome editing enables faster trait development, lower R&D costs, and trait innovation [6]. High-oleic soybean in the US and tomato with increased φ-aminobutyric acid (GABA) levels in Japan are two current genome edited crops in the market. New ones are expected to follow in the coming years.

In recent studies, the functions of members of gene families have been investigated by many different types of mutation, including knock-out, base editing and allele exchange with multiplex sgRNAs for targeted genes with CRISPR/Cas9 technology. Their role in crop improvement has been summarized in Table 1.1 and discussed below in terms of improved agronomic traits, disease resistance, quality, and nutritional content.

# *2.1 Improved Agronomic Traits*

Genome editing can be used to enhance the yield of crops by improving their resistance to environmental stressors, such as drought, heat, and cold. By modifying specifc genes, scientists can increase the plant's ability to cope with adverse conditions, leading to improved yield.

Allelic variants of the *ARGOS8* gene belonging to the ARGOS gene family were obtained by CRISPR gene editing in maize. These allelic variants were the negative regulator of ethylene responses [7]. The ARGOS8 variants had high levels of *ARGOS8* transcripts relative to the native allele and increased grain yield under fowering stress conditions and had no yield loss in arid conditions.


6

**Table 1.1** Summary of gene families targeted by genome editing for crop improvement in terms of improved agronomic traits, disease resistance, quality, and


#### 1 Genome Editing of Gene Families for Crop Improvement

In order to understand the function of *BnaPLDα1* genes of Phospholipase D Gene Family in *Brassica napus*, *BnaPLDα1* gene was over-expressed and knocked out by CRISPR. Four lines were edited at one sgRNA site and two lines were edited at two sgRNA sites [8]. Compared to the wild type, the PLDα1 protein was found to be more expressed in the over-expressed edited lines while less or not expressed in the knockout lines. When the plants were planted in the feld, the plant height was not different between overexpression lines and wild type, but the plant heights of the knocked-out lines were shorter, indicating that the appropriate dose of PLDα1 was required to maintain the normal growth of the plant, indicating that further increase of PLDα1 level had limited effect on it. Many agronomic traits such as plant height, effective branches, silique number in inforescence, silique seed number were adversely affected in lines with reduced PLDα1 levels [8]. These fndings proved that *BnaPLDα1* regulation changes the agronomic traits of *B. napus*.

The loss of function lines of *OsOTS1* gene enhanced sensitivity to salt with reduced root and shoot biomass, suggesting it plays a role in salinity stress. Mutagenesis of *OsOTS1* gene of SUMO proteases gene family to reveal its role against salinity stress was targeted in rice [9]. To investigate the usability of transcription factor (basic helix-loop-helix (bHLH)) sub-U family genes in breeding rice varieties under salinity stress conditions in rice (*Oryza sativa* L.), loss of function lines of *OsbHLH024* and *OsbHLH044* genes was generated by the CRISPR/Cas9 system [10, 11]. The gene editing mutant lines revealed altered morphological and physiological phenotypes. *OsbHLH024* mutant lines had increased shoot weight, the total chlorophyll content, and the chlorophyll fuorescence. They had less reactive oxygen species and stabilized levels of MDA, fewer Na+ but more K+, and a balanced level of Ca2+, Zn2+, and Mg2+ in the shoot and root. Therefore, it was concluded that the *OsbHLH024* gene improves salt stress resistance by playing a role as a negative regulator [10]. Considering *OsbHLH044* mutant lines, they showed reduced morphological and physiological parameters, lower antioxidant activities and higher lipid peroxidation and hydrogen peroxide (H2O2) accumulation. The loss-of-function of this gene also altered the expression of ion homeostasis-related genes (*OsHKTs*, *OsHAK*, *OsSOSs*, and *OsNHX*) and ABA-responsive gene (*OsLEA3*). The mutant synthesized less stored starch and proteins because of decreased expression of genes coding for starch (*OsAGPL1*, *OsSSIIa*, *OsWx*, and *OsFLO2*) and seed storage proteins (*GluA1* and *Globulin 1*). It was concluded that the *OsbHLH044* gene plays a role as a positive regulator of salt stress and grain quality [11].

In rice, three genes (*Os03g0603100*, *Os03g0568400*, *GL3.2*) of cytochrome P450 family and *OsBADH2 were* multiplex edited with CRISPR/Cas9 and their novel alleles were generated [12]. The high performance of mutant plants homozygous for grain width, grain length, and grain fragrance was recorded. Also, the mutant plants showed high level 2-acetyl-1-pyrroline (2AP). It has been concluded that the edited plant in this study could be used as valuable genetic material for crop breeding to improve rice yield and quality.

Indel mutations in the *TaSPL13* gene of SQUAMOSA promoter-binding proteinlike (SPL) transcription factors gene family were created to increase grain yield in bread wheat (*Triticum aestivum*) [13]. The microRNA 156 recognition element indel mutations in 3′ UTR of *TaSPL13* brought about the abundance of TaSPL13 transcripts and a decrease in fowering time, tiller number, and plant height and the consequent grain size and number increased in the mutant plants.

Eleven members of the Cytokinin oxidase/dehydrogenase gene family (*OsCKX1- OsCKX11*) were knocked out using CRISPR/Cas9 and CRISPR-Cas12a to reveal their functions in rice [14]. CRISPR-Cas12a performed better than CRISPR-Cas9 for generating multigene mutants. The *OsCKX* members showed functional redundancy and affected plant development, seed quality, and starch composition by regulating endogenous cytokinins. *OsCKX1/2* and *OsCKX3/8/10* genes had roles in the control of panicle architecture and grain number. *OsCKX4/5/9* genes regulated the development of roots and plant height and tillers.

Knock-out and over-expression of *SlLBD40* (*Solyc02g085910*) gene of The Lateral Organ Boundaries Domain (Lbd) Protein Family were created to understand its role against drought stress in tomato [15]. The results showed that *SlLBD40* knock-out lines had improved water-holding ability and relieved the physical harm of the photosynthetic system of tomato seedlings, while *SlLBD40* overexpressing plants showed vigorous wilting symptoms under drought stress, suggesting that *SlLBD40* is a negative regulator of drought tolerance.

In order to increase photosynthetic effciency in tetraploid tobacco (*Nicotiana tabacum*), it was aimed to obtain novel alleles of genes (*rbcS*) coding the small subunit of CO2-fxing enzyme Rubisco family so that the effciency of carbon assimilation through better Rubisco enzymes could be increased [16]. The phenotyping analysis of mutant plants showed reduced Rubisco content, photosynthetic rates, and biomass accumulation. In this study, it has been shown that it is possible to produce superior non-native Rubisco enzymes by multiplex gene editing with CRISPR/Cas9 in polyploid species.

The function of the *OsPMEI12* gene, which belongs to the pectin methylesterase inhibitors (*OsPMEI*) gene family for growth, cell wall development, and response to phytohormone and heavy metal stress, was elucidated by mutating it in rice (*Oryza sativa* L.) [17]. It has been revealed that the *OsPMEI12* gene plays a role in the regulation of methyl esterifcation during growth, thus pectin status is controlled by hormones and cadmium stress.

Discovery of genetic male sterility (GMS) genes will allow to unravel the molecular mechanism of anther development and to develop male sterility systems for crop breeding and hybrid seed production. Therefore, the researchers focused on elucidating the roles of the transcription factors genes (*bHLH*, *bZIP*, *MYB*, *PHD* and *LBD* gene families) responsible for the GMS agronomic trait in highly heterosis maize [18]. Mutation of transcription factor gene families with CRISPR yielded the following results; that *ZmbHLH51* and *ZmbHLH122* are necessary for male fertility, *ZmTGA10* has affected anther dehiscence. *ZmMYB84* is essential for maize pollen development. *ZmPHD11* is responsible for the formation of anther cuticle and Ubisch bodies. Two MYB33 paralogs are important for GMS. *ZmLBD27* has an impact on the reduced ratio of normal pollen grains. ZmPHD11/27 infuenced viable pollen formation.

Two homologous genes (*Bra018924* and *Bra014296*) of PP2A, a 55 kDa B regulatory subunit (PR55/B), were edited through the CRISPR/Cas9 system to develop self-compatible lines controlled by type 2A serine/threonine protein phosphatases (PP2As) in Chinese cabbage [19]. The *PR55/B* gene knock-out line did not show self-incompatibility and produced seeds, suggesting that the *PR55/B* gene was responsible for the self-incompatibility.

# *2.2 Improved Disease Resistance*

Plant diseases are a signifcant threat to crop productivity, with losses estimated to cost billions of dollars annually. Genome editing can be used to enhance the resistance of crops to diseases by editing genes that encode for disease resistance.

MicroRNAs act as regulators of the plant immune system by silencing the genes involved in pathogen virulence or by regulating the expression of target genes. MicroRNAs belonging to the miRNA482/2118 superfamily targeted R-genes of the class NBS-LRR (nucleotide-binding site-leucine rich repeat). In tomato, knockout mutants for *SlymiR482e-3p* gene, a member of the miR482/2118 family, were generated to detect its role against tomato wilt disease resistance [*Fusarium oxysporum* f. sp. *lycopersici* (race 2) (Fol)] [20]. The results showed that *SlymiR482e-3p* gene negatively regulated the resistance to wilt disease. In another study with miR482 gene family in tomato, mutations in the two genes (*miR482b* and *miR482c*) were generated [21]. The expression levels of their target NBS-LRR genes were increased, followed by reduced late blight, caused by *Phytophthora infestans,* disease symptoms in mutant plants. Interestingly, it was concluded that knocking out these two genes could lead to expression perturbation of other miRNAs, suggesting cross-regulation between miRNAs. In cotton (*Gossypium hirsutum* L.), the MIR482 mutant collection was obtained by knocking out *MIR482* genes in miR482 gene family with dozens of members using CRISPR/Cas9. This mutant collection allow us to examine the role of individual *MIR482* genes against pathogen response and to identify miR482-NLR module(s) that respond to, particularly fungal pathogen *Verticillium dahliae* agent for Verticillium wilt and other pathogens. It has also been reported that this collection may be a useful genetic resource for the development of new disease-resistant cotton varieties [22].

# *2.3 Improved Quality*

Quantitative trait loci (QTLs) are genomic regions that are associated with complex traits such as crop quality. QTLs can be used to improve crop quality by identifying genomic regions associated with desirable traits. Many genes in QTLs have been targeted with genome editing.

Starch branching enzymes (SBEs) are involved in the biosynthesis of starch in plants and there have been several studies on the use of genome editing *SBE* genes in crops to modify starch content and properties. For example, one study used CRISPR-Cas9 RNP-method to induce mutations in *SBE* genes to develop a unique potato starch lacking branching [23]. Another study used CRISPR/Cas9 to generate high-amylose rice by targeting *SBEI* and *SBEIIb* genes [24].

In *Brassica napus*, an oilseed plant that is the raw material for both cooking oil and biodiesel production, loss-of function of the *BnLPAT2* and *BnLPAT5* genes belonging to lysophosphatidic acid acyltransferase (LPAT) family allowed to lay out their precise roles in oil biosynthesis. That is, the oil content decreased, the size of oil body increased in mutant lines [25]. The results of this study illustrated that these genes could be used for oil production improvement.

In order to prevent enzymatic browning, negatively affecting tubers during harvest and post-harvest procedures, researchers targeted the Polyphenol Oxidases (PPOs) gene family and mutated the *StPPO2* gene in *Solanum tuberosum* L. [26]. A lower PPO causing oxidation of polyphenols (natural antioxidants) in tuber was observed in mutant lines and enzymatic browning was reduced. Another study with the polyphenol oxidases (PPOs) gene family was carried out in eggplant (*Solanum melongena* L.) [27]. In this study, the researchers stated that it would be possible to develop eggplant varieties that retain a high polyphenol content benefcial to human health in the berries during harvest and post-harvest processes by knocking out the three genes (*SmelPPO4*, *SmelPPO5*, *SmelPPO6*).

# *2.4 Improved Nutritional Content*

Genome editing can also be used to enhance the nutritional content of crops by increasing the levels of essential nutrients, such as oil, phenolic acid, vitamins and minerals [4]. This is particularly important in developing countries, where nutrient defciencies are prevalent and access to a diverse diet is limited.

The oleic acid content of soybean seeds was modulated by knocking out GmFAD2-1A and GmFAD2 genes belonging to the fatty acid desaturase (FAD2) family [28]. The loss of function of these genes has paved the way for cultivating soybean genotypes with high oleic acid content.

In *Salvia miltiorrhiza*, the *SmLAC*s genes of the laccase family, which has 29 family members with high homology, were multiplex silenced by targeting their conserved sequences to reveal their role in the production of medicinal phenolic acid compounds [29]. It has been concluded that *SmLAC*s play a role in lignin formation and phenolic acid biosynthesis in the roots due to decreased expression of target genes and delayed hairy root development, larger and looser xylem cells, low RA and SAB accumulation, and very low lignin content in edited lines compared to wild types.

By knocking out six genes (*BBLa*, *BBLc*, *BBLd.2*, *BBLb*, *BBLd.1*, *BBLe*) belonging to the berberine bridge enzyme-like (BBL) family using a single sgRNA, a nicotine-free tobacco variety was developed and the amount of nicotine was reduced [30]. In this way, the use of nicotine-free tobacco by smokers may protect their health.

The genes in α- and γ-gliadin gene families were silenced using six sgRNAs, and a less allergenic bread wheat variety containing gluten with fewer immunogenic epitopes, which would be benefcial in the diet of coeliac patients, was obtained [31]. A less allergenic durum wheat variety was produced [32]. The multiplex editing of *CM3* and *CM16* genes belonging to α-amylase/trypsin inhibitors (ATI) family subunit U form was carried out using seven gRNAs targeting the two genes of interest. The editing of these genes decreased the amount of ATI transcripts. It was reported that this might have happened due to nonsense-mediated mRNA decay. Interestingly, it was noted that ATI 0.28 pseudogene were activated after knocking out *CM3* and *CM6* genes.

# **3 Challenges of Genome Editing in Crop Improvement**

Despite its potential benefts, genome editing in crops also faces several challenges. One of the biggest challenges is the regulatory landscape. Many countries have regulations that govern the use of genetically modifed organisms (GMOs), and it is not yet clear how gene-edited crops will be regulated. In some cases, gene-edited crops may be subject to the same regulations as GMOs, even though the changes made to the DNA sequence are much smaller and more precise.

Another challenge is the potential for unintended consequences. Although genome editing is a precise technique, there is still a risk of off-target changes in the host genome [4]. These unintended changes could have unforeseen consequences for the crop's performance or safety. To mitigate this risk, scientists must employ rigorous testing and validation processes to ensure that the desired changes are made without causing any unintended effects. Therefore, it is important to carefully assess the safety and effcacy of gene-edited crops before they are released into the environment or consumed by humans.

# **4 Summary**

Genome editing is a promising technique for crop improvement that has the potential to produce crops that are better adapted to changing environmental conditions and have improved nutritional content. However, there are also challenges that need to be addressed, including regulatory issues and potential unintended consequences. With careful consideration and regulation, genome editing has the potential to revolutionize crop improvement.

The use of genome editing in crop improvement also raises ethical considerations. For instance, there are concerns around the ownership and control of the technology, as well as the potential for gene-edited crops to create new monopolies in the seed industry. Inadequate stewardship, enhanced inequity between rich and poor, lack of transparency, an unclear intellectual property landscape, and inadequate public sector institutional infrastructures to support the use of genome-editing technologies are also challenges that need to be addressed [33].

Regulatory policy is another challenge. The global regulatory policy for genomeedited crops is still emerging and will shape the path of genome editing innovation. Genome editing is a relatively new technology, and its regulatory framework is still evolving. The regulation of gene-edited crops varies between countries, with some countries adopting a more stringent approach than others. As genome editing techniques become more widespread, there is a need for a coordinated global approach to regulating their use in agriculture to ensure that the technology is used responsibly and safely.

# **References**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **Chapter 2 Base Editing and Prime Editing**

**Hilal Betul Kaya**

**Abstract** The development of new adaptations of CRISPR-based genome editing platforms, such as base editing and prime editing, made it possible to broaden the scope and applications of genome editing in plants. First base editing and, more recently, prime editing evade the creation of double-stranded breaks in deoxyribonucleic acid (DNA) and the requirement of donor template of DNA for repair while enhancing editing efciency and product purity over CRISPR/Cas9. As base-pair changes in genomic DNA determine many signicant agronomic traits, crop varieties can be developed by precisely converting specic single bases in plant genomes. While base editing can introduce specic nucleotide changes, such as transition and transversion mutations in the targeted region, prime editing can create precise insertions, deletions, and all 12 types of point mutations using the "search-andreplace" method.

This chapter provides the basic principles of base editing and prime editing technologies and their practical applications in plants. The chapter also summarizes the recent breakthroughs in applying base and prime editors in diverse plant species, including their use in improving disease resistance, herbicide resistance, nutritional quality, crop yield, and quality. Finally, this chapter aims to clearly understand base editing and prime editing in plants by outlining potential developments.

# **1 Base Editing**

Base editing is a novel genome editing method that creates transition and transversion mutations at the single-base level without double-stranded DNA breaks, donor templates, or undesirable effects of NHEJ and HDR mechanisms [1, 2] (Fig. 2.1a). Since base editors (BEs) considerably minimize unintended modications, they show great potential in plant genome editing applications [3]. Base editor combines a catalytically impaired Cas protein with a nucleotide deaminase to convert one

H. B. Kaya (\*)

Department of Bioengineering, Manisa Celal Bayar University, Manisa, Turkey e-mail: hilalbetul.kaya@cbu.edu.tr

**Fig. 2.1** Schematic diagrams of base editors. (**a**) Graphical overview of the transition and transversion base-pair substitutions in base editing, (**b**) Cytosine base editors (CBE) mediate C-to-T conversion, (**c**) Adenine base editors (ABE) mediate A-to-G conversion, (**d**) Glycosylase base editors (CGBE) mediate transversion mutations

base to another at a target locus in DNA or RNA [4, 5]. First, the Cas protein-gRNA complex binds to its target locus in DNA, and then the Cas protein denatures the double-stranded DNA resulting in an R-loop that exposes a small segment of singlestranded DNA [6]. Next, the deaminase enzyme catalyzes the specifc base conversion in this single-stranded DNA. Finally, the permanent introduction of single-base substitutions resulted in the target region through DNA repair and replication [5].

The frst developed base editors, cytosine base editors (CBEs), convert a cytosine (C) to thymine (T) and guanine (G) to adenine (A) in the opposite strand) in the target region [5] (Fig. 2.1b). In 2016, David Liu group created the frst-generation base-editor (CBE1) by fusing a rat cytidine deaminase (rAPOBEC1) to the N terminus of dCas9 (dead Cas9) via a 16 amino acid XTEN linker [5, 7]. Although CBE1 successfully converts C:G to T:A *in vitro*, the base excision repair mechanism (BER) recognizes any G:U base pair as a mismatch and removes the uracils with the help of uracil N-glycosylases (UNGs) in vivo. To address this limitation and improve its effciency, CBE2 was developed by adding a uracil DNA glycosylase inhibitor (UGI) to the C-terminus of dCas9 (dead Cas9) via a 4-amino acid linker [5, 8]. With this new version, editing effciency was increased three times compared to CBE1 due to the inhibition of uracil DNA glycosylase (UDG) in the organism by UGI. To increase editing effciency, CBE3 was developed by replacing the dCas9 with a nCas9 (H840, HNH catalytic domain) nickase variant. In this new version, nCas9 would induce a nick in the G-containing DNA strand, which activated the cellular mismatch repair (MMR) mechanism [5, 7]. This MMR mechanism replaces the G on the nicked strand with an A, forming a U:A pair with the target strand. The U:A pair was later corrected, leading to the desired T:A substitution. While inhibition of BER by using UGI in CBE2 enhanced editing effciencies approximately threefold, the nicking strategy of CBE3 increased effciencies by up to sixfold compared to CBE2 in human cells [5]. CBE in plants was frst implemented in rice [9–11] and then adapted to various plants species such as wheat [11, 12], *Arabidopsis* [13, 14], maize [11], potato [15, 16], tomato [15, 16], cotton [17], watermelon [18], soybean [19], apple [20], pear [20], strawberry [21], rapeseed [22], *P. patens* [23] and poplar [24] quickly CBE-mediated genome editing used for different purposes, such as obtaining disease and herbicide resistance, accelerating crop domestication, and increasing yield and nutrient use effciency in various plants, has been summarized in Table 2.1 (also reviewed in [25, 26]).

# *1.1 Improving the Base Editing Effciency*

With a better understanding of the molecular functions of deaminases, adenine base editors (ABEs) are developed for inducing A•T to G•C conversions with high effciency [27] (Fig. 2.1c). ABEs use engineered transfer RNA adenosine deaminases (TadA) derived from *E. coli*, which bind to ssDNA and deaminates A into inosine I. The use of ABEs overcomes the limitation of CBEs, which can only edit C or G bases, and provides a broader range of base transformation options. Unlike CBEs, ABEs do not need to suppress the activity of alkyl adenine DNA glycosylase (AAG) [28, 29]. Over time, various optimization strategies were implemented, including TadA mutations and using varying lengths of the linker between TadA and nCas9 (D10A) to enhance the editing effciencies of ABEs [25, 27, 30]. Various variants (ABE 6.3, ABE 7.8, ABE 7.9, ABEmax, etc.) of ABEs have been developed and implemented in mammalian cells [8, 27] and then rapidly adapted to plant cells. Li et al. [31] used ABE-mediated base editing to edit rice's acetyl-coenzyme A carboxylase (*ACC*) gene to confer herbicide resistance. To create A·T to G·C conversion in *OsMPK6*, *OsSERK2*, and *OsWRKY45* in rice, a forescence-tracking ABE was developed and successfully implemented by obtaining up to 62.3% editing effciency [32]. In a proof-of-concept study, Kang et al. [33] edited the *PDS* gene in *Arabidopsis thaliana* and *Brassica napus* by creating a single amino acid substitution using ABE. Hua et al. [34] compared two different ABE versions (ABE-P1S and ABE-P1) to increase effciency in rice. Like CBEs, ABEs were also implemented in various plants, as shown in Table 2.1.


**Table 2.1** Selected applications of base editors in plants


Type of base


**Table 2.1** (continued)


Type of base


24

**Table 2.1**

(continued)


#### 2 Base Editing and Prime Editing

Since CBEs and ABEs mainly generate base transitions, C-to-G base editors (CGBEs) were developed by modifying CBEs to generate a new tool suitable for C·G to-G·C transversion (Fig. 2.1d). Instead of the UGI inhibitor used in CBEs, CGBEs include Uracil-N-glycosylase (UNG), which locates U in the DNA and eliminates it [35], promoting the BER pathway and improving uracil glycosylation. CGBEs were frst developed in human cells, including a UNG fused to nCas9 nickase (D10A) and a cytidine deaminase rAPOBEC1 or its engineered form rAPO-BEC1 (R33A). Because of the promising editing results of CBEs and ABEs in plants, Sretenovic et al. [24] modifed the CGBE method for plants which is proven to work in human cells [36–38]. They tested three versions of CGBEs in rice, tomato, and poplar and obtained different editing effciencies from CGBEs in different plants. Tian et al. [39] developed CGBEs for rice by optimizing the codon of UNG and by using three highly active deaminases, hAID, hA3A, and Anc689. They tested optimized CGBEs in fve different rice genes and obtained successful C-to-G conversions with an average frequency of 21.3% [39]. Another CGBE-mediated genome editing in rice was reported by Zeng et al. using the highly active cytidine deaminase evoFENRY and the PAM-relaxed Cas9-nickase variant Cas9n-NG with rice and human UNG [40]. Although their CGBEs achieved C-to-G conversions up to 27.3% in rice, they did not achieve signifcant C-to-G performance, contrary to previous studies on mammalian cells [40]. Recently, monocot plant-compatible CGBEs were developed in rice protoplasts, and low editing effciency was obtained [41]. Similar to previous CGBE studies in plants, this study also emphasizes that further improvements are necessary to enhance the editing effciency of plant CGBEs for versatile applications [41].

As a result of several attempts to increase base editing effciency, dual base editing technology which combines both ABEs and CBEs into a single base editor, was developed. Dual base editors convert C-G to T-A and A-T to G-C mutations simultaneously in the target site using a single gRNA [42]. Various dual-base editing platforms have been developed for mammalian cells (SPACE, A&C-BEmax, Target-ACEmax, and ACBE) [42–45] and plants (STEMEs) [46]. STEMEs (saturated targeted endogenous mutagenesis editors), a fusion of nCas9 with both deaminases, APOBEC3A/ecTadA, was frst tested in the OsACC gene to obtain herbicideresistant rice mutants [46].

Applications of base editors in plants are presented in Table 2.1 by highlighting the plant species, target genes, type of BE, purpose of the targeted mutation, and delivery technique of the reagents.

# **2 Prime Editing**

In 2019, David Liu's group introduced prime editing, a 'search and replace' tool that can perform any intended changes, including all 12 possible base-to-base conversions, insertions, and deletions without requiring DSBs or donor DNA templates [78] (Fig. 2.2a). Prime editor is a versatile, precise genome editing tool that is

**Fig. 2.2** Schematic diagrams of prime editors, (**a**) Graphical overview of the edits including insertions, deletions, transition, and transversion base-pair substitutions in prime editing, (**b**) Prime editors consist of a Cas9 nickase domain fused to a reverse transcriptase domain

composed of a Cas9 nickase (nCas9; H840A mutation) fused to an engineered reverse transcriptase (RT) and a prime editing guide RNA (pegRNA) containing a primer binding site (PBS) and an RT template (Fig. 2.2b). When pegRNA is delivered into a cell, Cas9 nickase (nCas9; H840A mutation) recognizes and breaks the non-complementary strand of the DNA three bases upstream of the PAM site. The PBS hybridizes with the bases upstream of the nCas9 (H840A)-generated nick and RT template encodes desired edits and directs reverse transcription. Then the new DNA containing the desired edit is integrated, and the unedited strand is repaired to match the edited strand by a cellular DNA repair system [78].

Anzalone et al. [78] presented three versions of prime editing system in their frst article on prime editing. The frst prime editor (PE1) incorporates wild-type reverse transcriptase from commercial Moloney murine leukemia virus (M-MLV) with Cas9 (H840A) nickase and a pegRNA. Various RT mutations have been investigated to increase the effciency of prime editing by altering thermostability, processivity, DNA–RNA substrate affnity, and RNaseH activity. PE1 effciency was increased by harboring engineered M-MLV-RT pentamutant (M-MLV RT (D200N/ L603W/T330P/T306K/ W313F)) after which it is called "prime editor 2" (PE2). PE2 enhanced the editing effciency by 1.6- to 5.1-fold to harbor point mutations on average. In addition, it showed higher editing effciency in indels and reported that it was compatible with shorter PBS sequences. To further increase the effciency, an optimized prime editor called PE3 used an additional sgRNA to direct Cas9 (H840A) nickase to produce a nick in the non-edited DNA strand and increased approximately three times the editing effciency of PE2. Since a high level of indels could be formed depending on the location of the additional sgRNA, the authors resolved this issue by designing the additional sgRNA to target the edited strand but not the original one. This variant of the PE3 system is called PE3b, as it achieved a 13-fold reduction in the average number of indels in human cell lines compared to PE3 while maintaining editing effciency [78].

# *2.1 Improving the PE Effciency*

Since it was frst published, the possibilities of the use of PE have advanced and broadened. Prime editing mechanism is a complex process infuenced by multiple factors, including prime editor structure, pegRNA design, and cellular determinants [79]. Different groups have been developing new strategies to increase the editing effciency of prime editing in animal and human cells [80]. For example, it is known that the 3′ extension of a pegRNA is critical for priming reverse transcription and templating the desired edit. Nelson et al. [81] discovered that exonucleases could hinder prime editing effciency by degrading the 3′ extension of pegRNA [81]. pegRNA optimization by incorporating structured RNA motifs to the 3′ terminus of pegRNAs enhanced stability by preventing degradation of the 3′ extension. This strategy is called engineered pegRNAs (epegRNAs) that improved prime editing effciency three to fourfold in human cells without increasing off-target editing activity [81].

Another strategy was manipulating the DNA repair pathway to increase PE effciency and reduce indels [82]. The temporary inhibition of DNA mismatch repair (MMR) by MLH1dn signifcantly improves the effectiveness of PE and reduces the occurrence of indels in various cell types [82]. Transient co-expression of MLH1dn (a dominant-negative variant of the MMR protein MLH1) with PE2 and PE3 yielded PE4 (PE2 + MLH1dn) and PE5 (PE3 + MLH1dn), respectively [82]. PE4 and PE5 versions enhanced the editing effciency by sevenfold over PE2 and twofold over PE3, respectively [79, 82]. With further efforts to enhance prime editing effciency, an improved prime editor architecture, "PEmax," was obtained by optimizing RT codon usage, Cas9 mutations, linker length /composition, and nuclear localization signals (NLS) tags based on the PE2 protein. The combination of PEmax with PE4/ PE5 systems (known as PE4max and PE5max, respectively) along with epegRNAs signifcantly improved editing effciency [80, 82, 83]. Additional optimization strategies were used in various cell types, including prime editing protein engineering, pegRNA structure, stability improvements, repair mechanism suppression, and twopegRNA implementation [80].

Most pioneering studies in prime editing have been implemented in animal and human cells, and applications of prime editing in plants are mostly proof of concept and optimization studies (Table 2.2). The frst report on prime editing in plants was published by Lin et al. [84]. They obtained a variety of edits, including insertions up to 15 bp in wheat and rice protoplast, by optimizing codon, promoter, and editing conditions. This study was followed by studies demonstrating the applicability of prime editors in various plant species, including tomato [85], potato [86], maize [87], *Arabidopsis* [88], *N. benthamiana* [88], and rice [89, 90]. However, these studies showed that the application of prime editing is limited by the low effciency and optimization studies required to reach its full potential. Therefore, various approaches have been rapidly developed and applied in plants to overcome the limitations of prime editors [91, 92].


**Table 2.2** Selected applications of prime editors in plants (continued)


**Table 2.2** (continued)


Type of

prime

Delivery

Dual-pegRNA strategy employs two pegRNAs in trans to simultaneously encode the same edits, increasing PE effciency by expanding the size and type of genomic mutations in rice [93]. Lin et al. [93] also optimized the melting temperature (Tm) of the PBS combined with a dual-pegRNA strategy and increased the editing effciency from 2.9-fold to 17.4-fold in rice protoplasts [93]. Xu et al. [94] reported that changing the C-terminal reverse transcriptase Cas9 nickase fusion with N- terminal fusion improved the editing effciency at some target sites in rice and maize [94]. In addition to this modifcation, codon optimization of M-MLV RT by introducing multiple-nucleotide substitutions enhanced editing frequency up to 24.3% and 6.2% in rice and maize, respectively [94]. Zong et al. [95] indicated that engineering the M-MLV reverse transcriptase by deletion of the RT RNase H domain and the addition of a virus-derived protein which is called engineered plant prime editor (ePPE) improved prime editing effciency by ~1.8–3.4-fold in rice and wheat [95]. Zou et al. [96] optimized prime editing by combining PE3 system and epegRNAs, which include a structured RNA motif (evopreQ1 or mpknot) with an 8 bp linker at the 3′ terminus of the pegRNA [96]. Their PPE3-evopreQ1 and PPE3-mpknot systems improved the prime editing effciencies in rice protoplast, and PPE3-evopreQ1system showed a more signifcant increase compared to PPE3-mpknot system [96]. This study also increased PE effciency by at least 2.8 times by applying an appropriate high-temperature treatment. Although each modifcation enhances PE effciency, combining these approaches could result in even more signifcant effciency improvements [81].

Different prime editing systems in plants, targeted genes, the purpose of the study, and plant delivery technique are summarized in Table 2.2 and also reviewed in [97, 98].

# **3 Future Prospects and Limitations**

# *3.1 Base Editors*

The precise and effcient conversion of single bases at targeted genomic sites is made possible by the CRISPR/Cas base editing technology, which has found wide applications across various plants, as shown in Table 2.1. Although this technology holds great promise for plant trait development, it needs to be improved in order to overcome several limitations, including off-target activity, editing window length, PAM site compatibility, bystander effect, sequence preferences, and the limited capability in editing only four types of base changes [4, 7, 25]. In recent years, substantial efforts have been dedicated to reducing these limitations and enhancing the specifcity of base editors in mammalian cells and plants [2, 7].

Comprehensive whole-genome sequencing studies have revealed that base editors can induce gRNA-dependent and gRNA-independent off-target mutations throughout the entire genome [98, 107]. Several effective strategies have been reported to reduce gRNA-dependent off-target effects, such as employing alternative Cas9 variants, enlarging the gRNA sequence, and delivering base editors through RNP (ribonucleoprotein) complexes [98, 108, 109]. Moreover, gRNAindependent off-target mutations were observed in mice and rice using cytosine base editors (CBEs) but not adenine base editors (ABEs). This occurrence is likely attributed to the excessive expression of the deaminase, resulting in random mutations throughout the genome, particularly in gene-enriched regions. Effective strategies to mitigate gRNA-independent off-target effects involve employing alternative deaminases instead of rAPOBEC1 or modifying the deaminase domain through engineering [110].

Jin et al. [111] reported unexpected and unpredictable genome-wide off-target mutations induced by CBEs BE3 and high-fdelity BE3 (HF1-BE3) in rice [111]. The study emphasized the need to optimize the cytidine deaminase domain and/or UGI components to minimize the occurrence of off-target mutations. Additionally, improved variants of CBEs, such as YEE-BE3, were suggested as a potential approach for reducing off-target edits in plants [111]. In another study, upon analyzing off-targets of ABE, considering the predicted top off-target sites with 1- or 2-nt mismatches, it was found that the TadA\* (modifed version of TadA) deaminases displayed negligible off-target activity (0–4.65% frequency). Furthermore, they suggested that TadA variants exhibit minimal off-target effects dependent on sgRNA [71]. Target selection can be restricted in base editing applications because of the limitations of PAM site compatibility and editing window length [98]. In order to surpass these limitations, various Cas orthologs and engineered variants with altered PAM specifcities have been employed to expand the scope of base editors [62, 112]. However, although these variants expand the scope of base editors, they can decrease editing effciency and enhance the target dependence [98]. In addition to these limitations, large genomes of plants with duplicated regions and genes could pose additional obstacles in selecting target genes and plant transformation steps in base editing [98].

# *3.2 Prime Editors*

While prime editing represents a signifcant advancement in plant genome editing, the technology is still in its early stages, necessitating further research and studies to unlock its capabilities and potential. One signifcant challenge with prime editing is its relatively low effciency [84, 97]. The editing effciency frequencies observed in plants were considerably lower than those reported in mammalian cells, and numerous sites exhibited a lack of editing, particularly in dicot species [113]. It is also reported that editing effciencies for insertions were lower than for deletions and substitutions [114]. Although it is possible to obtain targeted mutation in stable transgenic lines by prime editing, as shown in rice and tomato, the occurrence of homozygous and biallelic edits is infrequent, highlighting the ineffciency of prime editing in plants [84, 90, 102, 113]. Researchers have devised various strategies to overcome these limitations, including using engineered prime-editing proteins, enhancements in prime-editing guide RNA design, manipulation of the mismatch repair pathway, and optimization of delivery strategies [92, 97]. These approaches aim to enhance the effectiveness and effciency of prime editing for more precise and robust genome modifcations. Ensuring high effciency in prime editing relies heavily on the careful design of the pegRNA [78, 93]. Selecting an appropriate combination of the primer binding site (PBS) and reverse transcriptase (RT) template is crucial for optimizing prime editing effciency. Typically, effcient PBSs range from 8 to 15 nucleotides, while RT templates are between 10 and 20 nucleotides long [78, 93]. Although the specifc matrix of optimal PBS and RT template combinations is determined through empirical observations, several factors contribute to selecting the ideal pegRNA design, including GC content, primary sequence motifs, and secondary structures within the pegRNA 3′ extensions [84]. The design of pegRNA is considerably more complex than sgRNA design for other CRISPRbased editing techniques, as it requires adherence to multiple fundamental rules and the various combinations of PBS and RT templates [92, 93]. As a result, its manual design is time-consuming, error-prone, and challenging in high-throughput applications [92]. Several design tools have been developed to overcome this limitation [93, 115, 116].

In conclusion, the development of base editing and prime editing technologies has revolutionized the feld of plant genome editing, providing effcient and precise tools for targeted genetic modifcations. In addition, the ability to introduce single nucleotide changes without the need for double-stranded DNA breaks has opened new possibilities for crop improvement, disease resistance, and trait engineering. However, despite the signifcant progress made in this feld, challenges still need to be addressed, such as improving editing effciency and specifcity, optimizing delivery methods, and addressing off-target effects. Nevertheless, with continued research and development, base editing, and prime editing hold great promise for advancing the feld of plant biotechnology and crop improvement.

# **References**


#### 2 Base Editing and Prime Editing


#### 2 Base Editing and Prime Editing


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# **Chapter 3 Novel Delivery Methods for CRISPR-Based Plant Genome Editing**

**Barbara Doyle Prestwich, Teodoro Cardi, Allah Bakhsh, Alessandro Nicolia, and Kaushal Kumar Bhati**

**Abstract** Using modern genome editing tools, scientists are increasingly able to engineer animals and plants for better traits and improved downstream outcomes that benet humans. As part of the CRISPR-Cas system, guide RNA (gRNA) is used to identify the target sequence, while Cas is an endonuclease that performs the nucleotide cleavage. It is imperative that these two components are delivered to the nucleus of the cell in order to ensure an optimal editing process. As a consequence of differences in the cellular structure and biomolecular composition of the outer membrane, plants are not capable of being cloned genetically in the same manner as animal cells. A more optimized method and pipeline must be developed to improve the efciency of transformations and genome editing for plants. In this book chapter, we highlight traditional and novel delivery methods used for optimal delivery of plant genome editing components. We discuss the potential and limitations of these methods in the light of recent literature and available experimental validations.

T. Cardi CNR-IBBR, Institute of Biosciences and Bioresources, Portici (NA), Italy

A. Bakhsh Centre of Excellence in Molecular Biology, University of Punjab, Lahore, Pakistan

A. Nicolia

K. K. Bhati (\*) Louvain Institute of Biomolecular Science, University of Louvain, Louvain la Neuve, Belgium

B. D. Prestwich

School of Biological Earth and Environmental Science, University College Cork, Cork, Ireland

Council for Agricultural Research and Economics, Research centre for Vegetable and Ornamental Crops, Pontecagnano, Italy

# **1 Introduction**

CRISPR/Cas systems are a vital component of genome engineering tools for animals and plants due to their ability to analyse gene function and accelerate trait development [1]. Effcient editing, convenient design, cost-effectiveness, and simplicity provide an edge to CRISPR-Cas over other editing tools. The system consists of guide RNA (gRNA), for target sequence recognition, and Cas, a CRISPRassociated protein endonuclease, which exhibits different versions and is sometimes modifed and/or linked to other molecules to achieve specifc targets. All relevant components must be delivered into the cell nucleus to achieve genome editing or other modifcations.

Compared to animal systems, plants have distinctive characteristics which may limit the application of those methods developed for animal cells, thus requiring the development of specifc plant-based methods. In this chapter, a number of traditional and novel delivery methods are discussed with reference to their potential and their limitations, with the ultimate aim of increasing effciency and accuracy, and broadening the application of genotype-independent delivery systems in plants.

# **2 Biological Delivery Methods**

# *2.1 Bacteria Based Methods*

Successful editing outcomes in crops are reliant on the availability of effcient genotype-independent delivery systems which facilitate the introduction of individual genome editing components. Bacterial-mediated delivery of CRISPR/Cas components into plant cells is probably the most popular method in use, and one that incorporates a foreign gene into the plant genome using a binary vector system. The natural ability of *Agrobacterium tumefaciens* to engineer plant cells in the wild, and whose mechanism has been exploited by plant biotechnologists in the lab, means that this bacterial-based delivery system has been used effciently for transforming both monocot and eudicot plants, notwithstanding the recalcitrant nature of some plant species to *Agrobacterium* infection. To address this recalcitrance with some plant species, Raman et al. [2] developed a strategy whereby a type III secretion system from *Pseudomonas* was expressed in *Agrobacterium* to allow for the effcient delivery of several effectors to suppress the plant response system and aid transformation. They found an increase of up to 400% in transformation effciency using this approach. With a unique mode of action, which is reliant on an interplay of host and bacterial functions to effect transformation [3], *Agrobacterium* retains the title of most popular delivery system in use today. There are many excellent published reviews on what is known to date on the mechanisms of *Agrobacterium-mediated* delivery to plant cells [4–7]. Here we will examine the role *Agrobacterium* plays as an effcient delivery system in the introduction of CRISPR/Cas components.

Cas9 was frst discovered in 2005 and the frst genome edited plants using CRISPR followed less than a decade later [8]. Since then, plants drawn from over 24 families (including those containing the major food crops) have been edited using CRISPR/Cas9 components [9] in *Agrobacterium* (both *tumefaciens* and *rhizogenes*) where it continues to hold a prominent position as an effcient delivery system including in transient assays where leaf cell agroinfltration has been used [9]. *Agrobacterium*-mediated transformation (AMT) relies on the plant being inoculated with the bacteria containing the requisite reagents for gene editing. This means a binary plasmid containing a gene-editing cassette inserted between the border sequences on the T-DNA integrates into the plant genome leading to stable transformation as referenced in earlier reviews [6]. In general, AMT relies on tissue culture for the regeneration of a stable transformant. With this comes the possibility of producing unwelcome somaclonal variants. One way to circumvent this is to use AMT with a Floral Dip procedure. Here, transformed seeds can be obtained outside of the tissue culture process. However, this method has had limited success beyond *Arabidopsis thaliana* [10]. Another report of successful transformation outside of a tissue culture system is referenced in the same paper by Laforest and Nadakuduti [10]. Here an improved method of gene-editing effciency is described whereby developmental regulators are co-delivered with CRISPR components and overexpressed leading to improved regeneration and transformation of soil-grown plants using *Agrobacterium* which had been injected into plants creating de novo meristems. An effcient transformation system using AMT was developed in wheat by Zhang et al. [11]. Earlier transformation studies in wheat were focused on the gene gun which has drawbacks in terms of copy number insertions and gene silencing effects. Generally, with AMT events, single copy insertions are the norm with low levels of gene silencing detected. In the study by Zhang et al. [11], the authors report effcient editing events in wheat. Generally, if DNA-free genome editing is required, alternative methods such as PEG must be employed. One of the drawbacks of Agrobacterium-mediated transformation is the fact that T-DNA is integrated into the genome. And this can be problematic for regulators. A report by Dalla Costa et al. [12] examined two strategies for producing T-DNA free CRISPRed fruit trees using *Agrobacterium*. The frst strategy was based on the site specifc recombinase Flp/FRT system. This system was reliant on the recognition of a 34-bp long sequence which was excised along with any undesired sequences from an optimised T-DNA vector system. The second strategy focused on the use of synthetic cleavage target sites (CTS) which were engineered adjacent to the left and right border sequences of the vector. These CTS were recognised by the Cas9 cleavage system and removed. Molina-Risco et al. [13] describe an improved method for the AMT and gene editing of tropical japonica rice where *Oryza sativa* is seen as a model monocot species.

# *2.2 Virus Vector-Based Methods*

Plant viruses-based vectors are known for being reliable, effcient tools for transient protein expression and VIGS (Virus induced gene silencing) [14, 15]. More recently, plant viruses are being used as a delivery vehicle for genome editing tools, such as CRISPR/Cas9, to introduce specifc mutations in the plant genome. These approaches are collectively known as VIGE (*Viruses induced genome editing*) [16–22]. VIGE allows DNA integration free methods to study gene function, modify plant traits, and develop novel crop features [23, 24]. The transiently introduced recombinant viral clones replicate within the plant, which then spread and infect the cells, delivering the genome editing machinery to the desired target site. This method has the advantage of being able to reach cells in different tissues and organs of the plant, leading to high effciency genome editing. The overall choice of virus vector depends on the targeted host plant species and the size of the cargo insert that infectious viral replicon could sustainably carry while replicating in the plant cell. The replicon size threshold also limits the recombinant cargo size (nuclease and sgRNA cassette). The past few years have witnessed development of plants-based VIGE approaches [25, 26].

Several known plant viral vectors have been tested for their functional effciency in delivering the CRISPR/Cas9 cassette for precise genome editing. These recent studies have optimised critical factors for success in the genome editing process such as choice of host species, infection method and replication of recombinant clones and copy number of sgRNAs. The viruses have better replication and transfection effciency in their specifc hosts, and this limits the utilisation of VIGE vectors in a broad host range. However, if used with the right host the VIGE approach expands beyond gene knockout and recent work proved its application in the precise gene replacement using geminivirus replicon of wheat dwarf virus (WDV) [27]. The WDV replicon could carry Cas9, sgRNA and a donor DNA and the editing effciency was ~12 fold higher than non-viral methods. Up to that point, viral vectors from two RNA viruses viz. Tobacco etch virus (TEV) and potato virus X (PVX) had been used in tandem to express Cas12a and sgRNA respectively [19, 20]. All reported viral vectors have their own limitations and benefts [26]. Broadly, the use of DNA viruses-based vectors could lead to integration of a viral genome in plants, although the beneft is that they provide more cargo space due to the bigger replicon size. On the other hand, RNA viruses ensure integration-free genome editing but have smaller genomes hence the cargo size is limited. In the following sections, we have discussed some of the most successful VIGE viral vectors across model and crop plants.

#### **2.2.1 Tobacco Rattle Virus (TRV)**

Tobacco Rattle Virus (TRV) is a positive single-strand plant virus (Bipartite, RNA1 and RNA2) that infects the roots of tobacco plants and has a broad range of host plants. TRV could systematically infect and replicate in different plant cells and tissues. These attributes make TRV an ideal RNA virus candidate to deliver CRISPR/

Cas9 modules. Several recent studies have engineered this virus to express the guide RNA and Cas9 nuclease, which then target specifc genes to demonstrate heritable editing. TRV has been successfully tested in *Arabidopsis* (targets: *AtAGL1, AtTT4;* [28]) and Tobacco (*NbPDS, NbAG* and *NbPCNA*; [17]). These methods provide a rapid and effcient way to introduce genetic modifcations in plants and have been applied in various crops, including tobacco, to produce plants with improved traits. TRV has been exclusively used for the delivery of sgRNAs to plants expressing Cas9. The TRV vector system is successfully used in model hosts like *N. benthamiana* and Arabidopsis. The targeting and editing effciency were positively impacted by 3′ end sgRNAs tagging with *Flowering Locus T (FT)* transcript [17].

#### **2.2.2 Barley Stripe Mosaic Virus (BSMV)**

Barley Stripe Mosaic Virus (BSMV) vector is a plant virus that has been used as a delivery system for genetic engineering in plants. BSMV is a positive-sense RNA virus with a tripartite genome (alpha, beta and gamma RNA) that infects barley and other cereal crops, causing a mosaic pattern on the leaves. Researchers have utilised the natural replication and movement properties of the virus to deliver transgenes into plant cells, making it an effective tool for plant genetic engineering. BSMV has been used to transiently express sgRNAs and the editing process has been tested successfully in *N. benthamiana*, wheat and maize plants [21, 29, 30]. For wheat, multiple genes (*TaGW2/7, TaUPL3 and TaQ*) were targeted by expressing sgRNA scaffold from the gamma chain of BSMV replicon [29]. These engineered viral particles were then used for infection of Cas9 expressing wheat plants. The authors evaluated that sgRNA fusion with mobile RNAs like *tRNA*, *AtFT*, and *Vern3* did not result in improved editing effciency. On the contrary, another study on different wheat varieties confrmed the enhanced editing effciency for BSMV sgRNA fusion with TaFT mRNA [21]. The authors argued that mobile mRNA fusion effciency could be infuenced by choice of wheat genotype and expression level of Cas9 nuclease.

#### **2.2.3 Bean Yellow Dwarf Virus (BeYDV)**

Bean yellow dwarf virus (BeYDV) belongs to the plant geminivirus family. It infects legume crops such as beans and peanuts. The virus can replicate and move within the plant along with its satellite replicons, making it an effective tool for delivering bigger transgenes cargos into plant cells. Bigger cargo carrying ability has prompted the use of BeYDV to deliver Cas9 together with sgRNA scaffold. By engineering the virus to contain a specifc transgene, the virus can be used to infect the plant, delivering the transgene into the plant genome. These methods have been improvised now to introduce precise genome modifcations. So far, BeYDV based CRISPR/Cas9 delivery has been validated in tobacco, potato, and tomato [31–33].

# **3 Physical and Chemical Methods**

# *3.1 Physical Methods*

Effcient delivery of the CRISPR/Cas9 complex into the nucleus of the targeted cell is an essential aspect to make it functional. The various forms of delivery approaches are adopted for instance messenger RNA, ribonucleoprotein (RNP-complex of sgRNA and Cas proteins preassembled *in vitro*), and plasmid DNA (pDNA) [34]. RNPs are usually constituted by a complex of protein (*e.g.*, Cas9, Cas12a) and an RNA, such as the sgRNA-scaffold RNA single strand. Delivery of the complex via RNP avoids drawbacks related to mRNA and pDNA. RNP delivery avoids intracellular transcription and translation and speeds up genome editing. Apart from this, it not only edits effciently but also decreases the immune response, off-target effects, and insertional mutagenesis, making it a promising method of genome editing [35, 36]. Amongst the options to deliver CRISPR systems into plant cells, biolistic is one which is widely adopted [37, 38].

#### **3.1.1 Gene Gun/Biolistic-Based Delivery**

Gene gun based delivery or biolistics is a direct physical delivery method for microprojectiles carrying foreign DNA into plant cells or tissues at high velocity. DNA is coated onto gold or tungsten microprojectiles before gearing up for cell wall penetration of the target plant. Upon entry into the cell, dissociation from the particles takes place to either integrate stably or express transiently in the host genome [39].

To substantially breach the cell wall barrier, the gene gun method is widely adopted in order to deliver foreign DNA into the plant cell. Previously, RNA-guided Cas9 endonuclease was effectively used to modify the genome of various plants. Regardless of the success, particle bombardment of the plasmids containing the Cas9, gRNA, and marker genes often incorporated in the genome resulting in offsite cutting, gene disruption, and plant mosaicism [40]. Moreover, DNA molecules can also integrate at the double-strand break site hence decreasing the effcacy of gene insertion and gene editing. To address these undesirable effects, one suggested solution was to pre-integrate the Cas9 nuclease in plants to deliver the gRNA in the form of RNA molecules. Though successful the process was laborious and resourcedemanding to develop and characterise the pre-integrated lines.

There are several documented reports which demonstrate the effective delivery of CRISPR reagents for genome editing using biolistics. They comprise the *in planta* genome editing of wheat via SAM and the production of novel variants of the maize ARGOS8 gene. Shi et al. [41] used gene editing approach to develop new allelic variants of ARGOS8 in maize. ARGOS8 encodes a negative regulator of ethylene responses, that is expressed at low levels in most inbred maize lines. The group increased ARGOS8 expression by substituting or knocking in GOS promoter replacing native promoter via HDR to drive ARGOS8 expression. The feld evaluation of hybrids exhibited increased yield under stress regimes [41]. In wheat, a group of researchers bombarded 210 plants and found 11 transgenic lines with the mutant TaGASR7 allele, and the mutation was transferred to the next generation of three transgenic lines with no observation of the presence of Cas9 and guide RNA [38, 42]. Recent research in wheat showed some promising results regarding transgene-free genome editing by means of transient expression [43].

RNP complexes were delivered by biolistics into maize embryo cells [44]. Cas9-gRNA RNPs were employed to target four different genes: MS26, MS45 (male fertility genes), acetolactate synthase (ALS2), and liguleless 1 (LIG). The results were comparable to DNA plasmids and ranged from 0.21% to 0.69% in all four immature embryo cells of maize [44]. Similar results have been reported for wheat using the same method where 0.18% was obtained for TaGW2-B1 and 0.21% for TaGW2-D1 [45]. The RNP complex is of similar or greater effcacy as compared to the plasmid-mediated editing method and gives transgene-free plants with reduced off-target frequency along with the ability to directly target the genomic region of interest with the concomitant degradation of the RNP complex within hours [38].

In order to decrease uneven bombardments, a double-barrelled gene gun along with software that counts the cells was applied as a technical improvement [10].

In case of RNP transfections, a single nucleotide mismatch between the sgRNA and the target site greatly reduces the off-targeting of homolog sequences. Besides, RNPs were also found to accommodate the hefty heritable inversion of 75.7 Mb in maize chromosome 2, when constructed with gRNAs fanking the junctions of the required inversion [38, 46]. This highly specifc engineering of chromosomes is invaluable to crop breeding.

Apart from all the above-mentioned success stories of RNP transport using biolistic, delivery systems for CRISPR/Cas9 continue to be a considerable hindrance concerning its effcient use. For now, an all-purpose delivery system is still lacking. Each method possesses both merits and demerits. Moreover, options are there for the transport of small cargos as compared to massive protein-nucleic acid complexes. Although the gene gun method is capable of transforming a myriad of cell types and tissues in the absence of a binary vector, some obvious limitations include the laborious work of explant preparation and random incorporation of cargo at various sites in the genome. In the case of RNPs, they offer DNA-free gene editing that instantly alters the target site by sidestepping the transcription and translation machinery of the cell and degrades rapidly. Although there is a possibility that the bombarded explant and resultant phenotypes experience uncertain downstream effects [10]. Additionally, particle bombardment has a high consumable and equipment cost along with complicated integration patterns and gives relatively low throughput [39]. Moreover, the costlier method to deliver Cas9/gRNA RNP lacks the control of bombardment sites like the nucleus, plastid and mitochondria [47].

#### **3.1.2 Electroporation**

In electroporation, electrical pulses are used to generate transient pores in the plasma membrane in order to allow nucleic acids to enter the cell [48]. These microscopic pores allow not only the micro-, but also the macromolecules to move either inside or outside the cell. An electroporator device performs the electroporation activity that comprises three basic portions: a power supply for the pulse, electroporation cuvettes, and electrodes. According to the simple mechanism of electroporation, water molecules are the frst to pierce the lipid bilayer and make unstable hydrophilic pores. Then reorientation of adjacent lipids with their polar heads towards these water molecules takes place due to the increase in transmembrane voltage that in turn lowers the energy required to form an aqueous aperture leading to the formation of metastable hydrophilic pores [48, 49]. Fortunately, these electropores can be recovered and resealed in the case of optimised electric pulses. It has been observed that the duration of the electric feld and its intensity are of immense importance concerning the healing of the cells as unsuitable electric currents may lead to cell death [50].

Ensuring uniform electroporation can be diffcult to achieve in plant tissue due to the presence of variable cell types and different three-dimensional organisations due to gap junctions. So, this non-homogeneous distribution makes some cells experience a greater degree of electroporation than others. To address this issue, optimization needs to be performed with respect to electrode position, size, shape, and different cells [51].

Some examples of application of electroporation include tobacco protoplasts with cucumber mosaic viral RNA using exponential and square wave electroporation pulses [52]. Similarly, electro-pulsed colt cherry protoplasts demonstrated effcient regeneration capability and a greater number of shoots per callus along with a prolifc root system in comparison to non-electro-pulsed ones. Besides, protoplastderived tissues of *Solanum dulcamara L.,* which is a woody medicinal plant, showed improved morphogenesis when compared to the untreated protoplasts. Apart from this, roots of regenerated shoots were established effciently with prolifc root systems [50]. Recently, electroporation has been used for gene editing with CRISPR technology [53].

#### **3.1.3 Sonication and Pulsed-Laser**

Sonication can involve acoustic and ultrasound and has been observed to enhance the growth processes in plants [54]. Moreover, acoustic microstreaming and cavitation are caused by ultrasound and can modify enzyme stability, cell growth, and ultrastructure. It has the potential to discharge DNA from the nucleus, modify the permeability of the cell membrane, cleave the extracellular polymers, enhance cell surface charges, and reduce the stability of the cell. Furthermore, the duration of sound irradiation, frequency, and intensity of sonication are some of the factors to

be considered [54]. During the process, the cell wall interacts frst with the sound waves and probably experiences the variation followed by the cell membrane [55]. Ultrasound irradiates the bubbles and then leads to the crumpling of those cavities resulting in the release of a large amount of energy. This activity provokes various physical and chemical modifcations like microstreaming in the plant cells and cell suspensions enhancing mass transport [54]. However, high-intensity irradiation can spoil the cell structure and inhibit plant growth [56]. The properties of sound waves that infuence sonication include duration, intensity, pressure level, and frequency. Apart from these, the distance between the source and the target plant, sensitivity of the cells, genotype, and species are other factors affecting organogenesis and plant growth [57]. Moreover, ribonucleoproteins comprising the sgRNA and Cas9 protein were inserted into the extracellular vesicles by sonication for gene therapy [58]. Plant species modifed by ultrasound *in vitro* include rice, aloe, carrot, commercial squash, gerbera, hazelnut, and red microalga [55, 56, 59]. To further strengthen our understanding of the technique, advanced knowledge about various processes sparked by sonication and more insight into sounds would generate greater yield and better growth [60].

Recently, RNPs have been delivered into tobacco cells via cavitation bubbles generated using a pulsed laser [61]. The resulting shockwave achieves the effcient transfection of walled cells in tissue explants by creating transient membrane pores. Genome-edited plants were produced with an effciency of 35.2 and 16.5% for phytoene desaturase (PDS) and actin depolymerizing factor (ADF) genes, respectively.

#### **3.1.4 Silicon Carbide Whiskers**

Different types of silicon nanoparticles are used for different purposes. Some of them are relatively more diffcult to synthesise and process than others and hence their applications are limited. Those that are relatively inexpensive and uncomplicated fnd more usage and have wider applications. Silicon Carbide (SiC) whiskers/fbres are among those nanoparticles belonging to a wide variety of silicon-based nanoparticles that ft this description. They are needle-like structures attached to a base. They appeared as a tool for the physical delivery of DNA in the 1990s where they were used to create pores in tobacco and maize cells through abrasion allowing the penetration of the exogenous DNA when mixed with the cell suspension [62]. The SiC fbres do not carry the genes themselves, but rather help the foreign DNA slide into the target cells. To date many crops have been transformed using this method. Crops, like wheat and rice that are recalcitrant to the Agrobacterium infection/transfection, have not only shown higher effcacy but more stable transgenic expression with SiC whiskers when compared to the particle bombardment method (cDNA coated microprojectiles) [63]. The transformation has been conducted on cell suspensions, embryos, embryogenic cells, and calli of different plant species such as cotton, maize, tobacco, onion, and rice [64–67].

Several types of SiC whiskers have been used for research on different plant species. These types differ based on their diameter and length. To achieve optimum results through this method some precautions like the pretreatment of the explant with osmotic agents and proper mixing of cells with SiC whiskers etc., have been adopted [68].

Some of the characteristics of SiC whiskers that make them desirable for DNA transfer into plant cells include compatibility with almost all plant species, the ability to help transform a wide range of cells, enhanced regeneration rate of transformants, ease of setup, and quick and inexpensive. The indirect involvement of SiC whiskers in DNA delivery can also be attributed to their positive/desired character because the amount of DNA available for transformation can be controlled. On the contrary, there are some limitations to this method that include their moderate transformation effciency, the need for tissue culture and regeneration, and the requirement of a sophisticated protocol for successful transformation.

So far, most of the effort has been directed towards showcasing the effectiveness of this approach through the utilisation of reporter genes like GUS and GFP. Although, SiC-mediated plant transformation has been frequently reported, no studies have been conducted to deliver CRISPR/Cas for genome editing experiments in plants using this technology [69]. However, SiC whiskers have been used to edit the genome of other organisms such as algae and viruses through CRISPR/Cas [70, 71].

# *3.2 Chemical Methods*

#### **3.2.1 PEG-Mediated Delivery**

Polyethylene glycol (PEG) is a polyether composed of repeated ethylene glycol units [-(CH2CH2O)n]. PEG is available with different structures (*e.g.*, branched, star) having different molecular weights (MW) and showing different aggregation states: PEGs below MW 700 are liquid, PEGs comprised between MW 1000 and MW 2000 are soft solids, PEG above MW 2000 are hard crystalline solids. Polyethylene oxide (PEO) is a synonym for PEG, however macromolecules with MW below 20,000 are usually referred as PEG while those with MW above 20,000 are called PEO. PEG is biocompatible, highly soluble in water as well as in organic/inorganic solvents therefore being extremely important in solubilization and permeation [72]. In plant science, PEG properties were initially exploited in cell fusion experiments to obtain somatic hybrids (reviewed in [73]), but PEG is also capable of precipitating DNA molecules and stimulating their effcient cellular uptake by endocytosis [74]. Suitable PEG (usually MW 6000) and divalent cations (Ca2+) concentrations were initially identifed to achieve genetic transformation [75, 76] with further improvements in effciencies by Shillito and coauthors [77]. Besides genetic transformation of plant protoplasts, with stable integration in genomic DNA, it became evident that PEG-mediated transfection allowed episomal transient expression of the introduced DNA [78]. These fundamental experiments paved the way for thousands of studies that made large use of PEG-mediated transformation/transfection of isolated plant cells by different types of reagents (i.e., DNA, RNA, protein), with the establishment of consolidated platforms such as the Transient Expression in Arabidopsis Mesophyll Protoplast (TEAMP) system [79]. Typically, the concentration of PEG ranges from 12.5% to 20% (fnal concentration). The interest in using PEG for transfecting plant protoplasts, revamped in recent years because its application in the site directed mutagenesis protocols of various model species and crops (reviewed in [73, 80]).

Indeed, there are several examples of successful PEG-mediated delivery of RNPs in protoplasts of important crops such as potato, tomato, rice, and others (reviewed in [80]). The use of plant protoplasts requires an established regeneration protocol to obtain mutated adult plants, that additionally may show genetic instability and undesired somaclonal variation [36]. To bypass these issues, in pioneering work, Toda et al. [81] reported the direct delivery of RNPs (Cas9 based) in rice zygotes by PEG+Ca2+ mediated transfection thus achieving somatic mutagenesis with high frequencies (up to 64%).

#### **3.2.2 Lipofection**

The delivery of RNPs in plant cells need further development and lipofection could represent a promising delivery method. Briefy, lipofection (*i.e.*, lipid transfection, liposome-based transfection) takes advantage of tiny lipid vesicular structure called liposomes that can be multilamellar or unilamellar and neutral, positively, or negatively charged [74]. Liposomes are easily produced, and they can form lipoplexes by encapsulating DNA, RNA, or proteins; lipoplexes will further release their content into the cells upon endocytosis or fusion with membranes. Lipofection has been extensively used in mammalian cell transfection, while, in plants, following the initial demonstration of exogenous DNA transfer in isolated plant protoplasts by liposomes [82], lipofection by cationic lipids (positively charged liposomes) has been used exclusively for DNA or RNA transfer through the negatively charged plant cell membranes (protoplasts) with limited examples [83]. Recently, sweet orange genome edited plants have been obtained by delivering lipid-based nanostructures, produced with the cationic lipid-based transfection agent Lipofectamine™, in protoplasts. In detail, lipid particles encapsulated the CRISPR/ Cas9 DNA construct, protecting it from endosomal and enzymatic degradation [84]. While in a frst attempt, Liu et al. [83] tested the delivery of RNPs (Cas9 based) in tobacco protoplasts isolated from BY-2 cells, by using two different reagents (Lipofectamine™ 3000, Lipofectamine™ RNAiMAX) with a transfection effciency of 66% and a mutation frequency of 6%, but without regenerating plants. RNP delivery in plant cells by lipofection is still in its infancy, but it is considered an easy and inexpensive method for cell transfection that will probably largely beneft in the future by the technological improvements achieved in other research felds.

# **4 Emerging Technologies**

Apart from a few applications, current approaches to precisely modify higher plant genomes rely on *de novo* regeneration from tissues or cell-derived calli. The lack of genotype-independent protocols and/or the induction of somaclonal variation during the *in vitro* growth phase call for the development of genotype-independent, simple, and economic *in planta* procedures. The latter are particularly necessary in view of routinely implementing editing approaches in breeding programs. Some emerging technologies aiming to overcome present limitations of available procedures have recently emerged.

# *4.1* **In planta de novo** *Induction of Meristems*

Two methods to edit higher plant genomes have been recently developed in *Nicotiana benthamiana* and validated in tomato, potato, and grape [85]. Both rely on the co-delivery of plasmids with the gene of interest and genes encoding Developmental Regulators (*Wus2*, *STM* and others) to somatic tissues of germinating seedlings or mature plants grown *in vivo*. When specifc sgRNAs were tested in transgenic Cas9+ plants, edited shoots, able to transmit the induced mutations to the progeny, were regenerated from *de novo* formed meristems. Nevertheless, to obtain shoot formation, while reducing negative pleiotropic effects on their developments, the method requires a careful combination of different Developmental Regulators in genotypes tested. Further, the co-transfer of the nuclease remains to be demonstrated.

# *4.2 Editing During Haploid Induction*

In several crops, either maternal or paternal haploids by chromosome elimination of one parent after fertilisation can be induced by interspecifc crosses, knock-out of specifc genes (e.g.*, MATL* in maize or rice) or the manipulation of the gene encoding the centromere-specifc histone CENH3 protein [86]. Capitalising on this information, site-directed mutagenesis in maize, *Arabidopsis*, and wheat has been achieved through the expression of editing reagents in the zygote, prior to elimination of chromosomes derived from the haploid-inducer parent and ploidy doubling in derived plants [86–88]. This method is attractive because it allows the production of DNA-free edited plants in just two generations but requires the availability of genotypes able to stably express editing reagents in the gametes and to induce chromosome elimination in the zygotes.

# *4.3 Editing Through Grafting*

A breakthrough approach has been demonstrated recently by Yang and colleagues [89]. The authors produced transgenic *Arabidopsis* plants expressing a modifed version of Cas9 and sgRNAs to which a tRNA sequence had been added. The latter allowed *in planta* long-distance movement of guide RNAs and nuclease transcripts from rootstock to scion, where translation occurred, producing edits visible both in the mother plant and derived progenies. Interestingly, the same was observed in interspecifc graft combinations involving *Arabidopsis* and *Brassica rapa*, enlarging the potential scope of the technology to all species where graft compatibility occurs. It is, however, necessary to produce transgenic rootstocks for each editing target, although alternative ways to deliver and express mobile editing reagents in the rootstock could be attempted.

# *4.4 Nanotechnologies*

Innovative nanoparticle-based methods *for in vivo* delivery of CRISPR/Cas have been recently developed in animal cells [90, 91]. Due to the presence of the cell wall, which shows a Size Exclusion Limit (SEL) of 5–20 nm and other differences with animals (f.i., the absence of a true circulatory system), research for similar alternative methods in plant cells lags behind. Nevertheless, the feld is quite dynamic and nanomaterials (e.g., metallic/magnetic, silicon-based, carbon-based, lipid-based, polymeric, DNA nanostructures, peptide-based), showing at least one dimension below 100 nm and adjustable physico-chemical properties, could be adapted to deliver various cargoes precisely and in a controlled way to different plant tissues and without genotype-dependency, as reviewed elsewhere [92–96]. The possibility to directly transfer proteins or multiple biomolecules would allow the exploitation of RNPs without using laborious protoplast or biolistic-based approaches. The nanoparticle-mediated delivery of proteins in plants, however, presents diffculties not strictly related to their size [97].

Magnetic nanoparticles (MNP) have been tested, with results not always reproducible, to deliver plasmid DNA in pollen of cotton and other species using a magnetic feld (magnetofection) [98–100]. In maize, the pre-treatment of pollen grains to open aperture was critical to obtain positive results. Single-walled carbon nanotubes (SWCNT) show high aspect and surface area-to-volume ratios. Functionalized with polyethyleneimine (PEI), chitosan or imidazolium, they have been assessed to deliver GFP/YFP plasmid DNA in leaves, chloroplasts, and pollen of various species, respectively [101–103], obtaining protein expression without gene integration. Intriguingly, in case of chloroplast expression, the delivery system was designed to release the loading DNA based on the stroma pH. Carbon dots (CD) with a 5–10 nm spherical shape showed internalisation and transient plasmid DNA expression in roots, leaves, and embryogenic callus cells [104], while the delivery of Cas9 and gRNA plasmid DNA, leading to somatic editing of *SPO11*, has been reported by [105] after spraying plasmid coated CDs on wheat leaves*.* Rosette nanotubes (RNTs) derive from the self-assembly of complementary guanine and cytosine motifs, in solution self-organised in rosettes which eventually form biocompatible hollow nanotubes with an internal and external diameter of 1.1 and 3.4 nm, respectively. They could represent a less toxic alternative to SWCNTs and have been used to express mCherry plasmid DNA in wheat microspores [106].

RNA molecules (siRNA, dsRNA) have also been delivered to leaves and pollen by using SWCNTs, gold nanoparticles of different shape and size, DNA nanostructures with different characteristics, LDH clay nanosheets*,* inducing gene silencing and virus protection, when a viral gene was targeted [107–111]. Interestingly, with DNA nanostructures as carriers, RNA interference was induced at the transcript or protein level depending on the DNA nanostructure shape and the siRNA attachment locus [108], while, among gold particles, only nanorods entered the cells, but silencing was obtained only with the non-internalised spheres colocalized with the cell wall [109].

The co-delivery of a protein and plasmid DNA has been demonstrated in onion, tobacco and teosinte cells using gold functionalized Mesoporous Silica Nanoparticles (MSN) and the biolistic method [112]. A similar approach allowed the delivery of a functional CRE recombinase in maize embryos, determining the excision of DNA sequences fanked with *loxP* [113]. Recently, preliminary results of RNP delivery in wheat pollen have been reported by using nanoassemblies (5–10 nm in diameter) with polycation linear homopolymer PDMAEMA [114].

Cell penetrating peptides (CPP) are natural (protein derived) or artifcially designed molecules generally between 5 and 30 amino acids, with the ability to be translocated through the cell membrane either directly or *via* endocytosis. Based on physicochemical properties they are classifed as cationic, hydrophobic or amphipathic. They can bind to various kinds of molecular cargoes, either covalently or noncovalently, allowing their transfection in various recipient cells to transiently modify gene expression and metabolic pathways [115, 116]. In relation to genome editing, the feasibility of delivering proteins of varying sizes, including ADH (150 kDa) or GUS (272 kDa), has been demonstrated in triticale microspores as well as in intact *Arabidopsis* leaves [117, 118]. The transfer of the *GUS* linear plasmid DNA or of multiple biomolecules at the same time has been also shown [117, 119]. Proof of genome editing of the *IPK1* gene, albeit at low frequency, has been obtained in wheat microspores and derived haploid embryos by delivering ZFNs complexed with two different CPPs. Edited plants, however, could not be regenerated [115, 120]. Polyion complex vesicles synthesised by mixing two oligolysine peptides and displaying a CPP (CPP-PICsome) have been recently shown to be able to encapsulate Cas9 RNP complexes [121]. When introduced into *Arabidopsis* calli by vacuum and compression, editing of the target Phytoene Desaturase gene *PDS3* was obtained, although mutation rate was around 0.007%.

With reference to the use of nanotechnologies for genetic engineering and genome editing, quite a lot of research developed in the last years trying to adapt concepts and technologies derived from investigation in the human feld to plants, although in most cases only at the proof-of-concept level. Nucleic acids or proteins could be successfully delivered to plant cells, constituting the basis to develop methods for delivering editing reagents. Nevertheless, moving from one system to another, e.g., from small plasmids encoding reporter genes to large constructs encoding Cas encoding genes, is not straightforward, while, concerning DNA binding conditions on Nanoparticless, the trade-off between cell wall traffcking and accessibility of the transcription machinery can be a challenge [122]. The size and other characteristics of Cas molecules generally used are an obstacle for the fast development of plant-based nanotechnological approaches, but the continuous discovery of new nucleases, some of them hypercompact, open new perspectives [123]. The recent results with CPP [121] are also promising. Nevertheless, the possibility to transiently edit *in planta* the germline, without relying on undifferentiated growth *in vitro* and *de novo* regeneration, and passing only the induced mutations to the progeny, remains a desirable objective not yet achieved.

# **5 Conclusions and Perspectives**

Plant genome editing has been successfully demonstrated and applied to add or delete gene(s) in crop plants for functional genomic studies. The technology has gained momentum recently because of simplicity, effciency, low cost and ability to target multiple genes. The variation in mutation effciency in plants is associated with a number of different factors. Most importantly, the mode of delivery of the CRISPR/Cas9 components is of crucial importance for genome modifcation in plants. Plants have a complex genome structure due to common occurrence of polyploidy and other genomic re-arrangements. Thus, the delivery of CRISPR/Cas components to plant cells is still a challenge for researchers to attain high editing effciency. There are many CRISPR delivery methods currently in use and development (Fig. 3.1). The various delivery methods such as biological (*Agrobacterium*mediated, virus vector based), physical and chemical methods (PEG or biolistic based) are being applied to obtain effcient genome editing effciency. Each delivery method has advantages and disadvantages. These methods deliver CRISPR systems to cells with the aid of chemicals or devices that make cells more amenable to delivery. Besides that, scientists are developing new ways and means for the delivery of CRISPR components to plant cells with the aim of establishing highly effcient and genotype-independent delivery systems for genome editing (summarised in Table 3.1). We assume the novel delivery methods those provide the opportunity for generating transgene-free genome edited plants will be most preferred in the future. These transgene free methods will boost acceptance of CRISPR/Cas technologies in food and agriculture.

**Fig. 3.1** Different delivery methods for components and T-DNA constructs to achieve genome editing in plant tissues. The left part of the scheme represents the delivery methods for in-vitro genome editing. While the right part of the infographic shows the in-planta methods for the delivery of genome editing tools, those potentially could give edits in early generations without integration of T-DNA. Additionally, the cargos for delivery of the different genome editing components are presented at the bottom



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# **References**


mediated ribonucleoprotein delivery increases osmotic stress tolerance. Physiol. Plant. **173**, 993–1007 (2021)


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# **Chapter 4 Balancing Trait Improvement with Tradeoff Side-Effects Using Genome Editing Technology**

**Julia Johanna Wind**

**Abstract** Improving traits by breeding involves compromising between desired traits and possible undesired side effects. Often encountered examples include pathogen resistance versus yield, shelf life time versus fruit quality, and seed number versus seed weight. Genome editing can be used to reduce the effect of some of these tradeoffs. Different genetic reasons underlying a tradeoff require different approaches: important to note is whether a detrimental effect is caused by a unique gene, or several analogs/ homologs, because the strategy needs to be adopted accordingly. Unique genes, for example, can be substituted by analogs, and homologs have the advantage that, amongst the available options, the gene causing the fewest pleiotropic effects can be altered in its activity. When the detrimental effect of a tradeoff is caused by two genetically linked genes, this can lead to linkage drag. To break this type of tradeoff genome editing can be used to force a crossover event. Overcoming a tradeoff can generate a new one, but can nevertheless result in an improved crop variety.

# **1 Different Genetic Reasons Underlying a Tradeoff**

The rst plant breeding technique was the selection and propagation of plants with improved heritable traits, which were based on the cumulative net positive outcome of genetic changes. In more modern breeding, Quantitative Trait Loci (QTL) studies have led to the discovery of alleles responsible for the improvement of traits. Molecular breeding was then developed to cross such alleles into the crop variety of interest, thereby increasing speed and accuracy of the breeding process. Studying the effects that these alleles had on a trait made apparent how common pleiotropic effects are due to the introduction of an allele from a wild donor to a receiving elite line: often the improvement of one trait goes hand in hand with negative effects on other traits. The association between breeding values of linked traits can be positive

© The Author(s) 2024 69

J. J. Wind (\*)

Keygene N.V., Wageningen, Gelderland, Netherlands e-mail: julia.wind@keygene.com

A. Ricroch et al. (eds.), *A Roadmap for Plant Genome Editing*, https://doi.org/10.1007/978-3-031-46150-7\_4

or negative so that if one trait changes, other traits will change as well. A positive value indicates a win-win situation. For example, shorter plants have been shown to be easier for the mechanical harvest and also lead to a yield gain because plants put more energy into seed production. A negative value indicates a tradeoff: for example, an average increase in seed number often leads to a decrease in average seed weight. Genome wide association study (GWAS) experiments are very effective in indicating which genes or alleles are important for a given trait. Trait correlations are often caused by a set of genes that form the connection between two traits. However, some alleles have been discovered which cause breaks in trait associations. They can be the key to overcoming tradeoffs. However, such fndings are rare and can often not be translated to crop plants. Transgenic approaches can be used to introduce these alleles. Alternative approaches to infuence trait associations include selection of novel alleles from mutant populations or targeted mutagenesis with genome editing. Mutant populations have been created by random mutagenesis with a mutagen such as ethyl methanesulfonate (EMS), and such populations are very expensive to make because they require thousands of chemically treated individuals that are grown for multiple generations. Therefore, targeted mutagenesis is attractive as it can make targeted mutations in the gene or promoter of interest, so that only a few plants need to be edited instead of a large population. A few types of editing are discussed here: editing to create genetic variation including knockouts, editing to alter expression of target genes and cutting DNA to induce crossovers in recombination cold spots. These techniques are discussed in the context of a few well-known tradeoffs.

# **2 Unique Genes**

During plant evolution, two important whole genome duplications have occurred. First, in the ancestral seed plant whole genome duplication has led to the divergence of the seed plants. Second, whole genome duplication in the ancestral angiosperm plant has led to the angiosperm radiation. These whole genome duplications lead to gene duplication. Changes accumulate in the gene copies over time, because the alleles are under different selective pressure. This can result in divergence of gene function. It was estimated that 65% of genes have at least one duplicated copy. Genes are often part of gene families, but this does not mean that they can always be substituted by a homolog or an ortholog: this is clear when a knockout or an allele of the gene results in a phenotype. Such genes can be important regulators of plant growth and development, and often have pleiotropic effects when they are modifed. Therefore, reducing the negative effects of a tradeoff is most diffcult for these types of genes. Most of the time, fnding another gene that affects the trait in a similar way, but with fewer pleiotropic effects would be preferable. Three examples of tradeoffs caused by unique genes are discussed in the next sections.

# *2.1 Flowering Time vs. Yield*

FLOWERING LOCUS T (FT) is a well-known and often studied fowering time regulator that is unique for its central role in the induction of fowering in most plant species [1]. Knockouts of FT exhibit a late fowering phenotype, and overexpression reduces time before fowering. FT also plays a role in control of growth, heterosis, tuberization and the regulation of stomatal opening in a variety of plants [1]. Therefore, FT can have pleiotropic effects on plant growth and development. Natural variation for fowering time in canola is frequently associated with FT alleles and comes with a biomass tradeoff [2]. Swinnen and colleagues have summarized examples from literature of genetic variation in cis-regulatory elements in genes that underly initial domestication of various crops [3]. A large part of these alleles are in the FT promoter, suggesting that altered expression pattern of this gene is of major importance in the domestication of crops, very likely due to its effect on fowering time and fowering synchrony. Pleiotropic effects are also evident in other fowering time regulators, such as TERMINAL FLOWER 1 (TFL1). *Brassica napus* knockouts for BnaC03.TFL1 does not only have reduced fowering time but also reduced plant height, as well as reduced branch number, number of siliques, and seeds per silique [4]. Variation in the FT promoter through genome editing technologies could result in more genetic variation aimed at reducing pleiotropy. Changing expression of FT is also likely to improve synchronous fowering as a positively correlated trait, but it might be hard to limit the negatively correlated traits that result from early maturity, because of the link between early fowering and early maturity. If earlier fowering time is needed, it could be preferred to fnd genes that can speed the overall growth so that the plant matures faster. A knockout in cpn60β4 in *Arabidopsis thaliana* was shown to accelerate plant development [5]. Using CRISPR, cpn60β4 orthologs could be knocked out in the species for which accelerated fowering is needed, as this gene is conserved in angiosperms [5]. This might lead to a similar effect on reducing fowering time without early maturity.

# *2.2 Fruit Shelf Life vs. Lycopene Content*

The Food and Agriculture Organization from the United Nations has calculated that tomato has become the third most grown vegetable worldwide in the last decade and is therefore considered a very valuable crop [6]. Two properties are indispensable for the success of tomato: frmness and high lycopene content. Delayed ripening improves frmness and reduces the damage during the shipping of tomatoes as well as storability, while high lycopene improves the attractiveness for consumers, the value for the processing industry, as well as its nutritional value. Transcription factors that are important in improving shelf life are NON-RIPENING (NAC-NOR) and RIPENING-INHIBITOR (MADS-RIN). Both proteins are not knockouts but

alleles causing a phenotype: NOR is a partially mistranslated but functional protein and RIN is a fusion between two proteins. They both cause physiological, transcriptional and hormonal changes in ripening tomato fruits. Both of these regulators delay fruit ripening in similar but not identical ways. Crossing the RIN mutation to eight different wild type tomato lines clearly demonstrated that the improvement of shelf life negatively correlates with reddening of the tomato in the F1 progeny, and it was shown that the reduced reddening is due to a twofold reduction of lycopene. Conclusively, RIN and NOR mutations can improve shelf life at the cost of lycopene production. However, transgenic lines aiming to improve astaxanthin content surprisingly accumulated high levels of lycopene and also displayed an improvement in shelf life [7]. The authors explain the phenotype as the result of the extended duration of lycopene synthesis, so lycopene can accumulate, whereas the lack of β-carotene or its metabolic products possibly reduced the feedback inhibition. This phenotype demonstrates that frmness and lycopene content are not necessarily always correlated. The genes that were overexpressed originated from marine bacteria from the *Brevundimonas* genus and these genes are not present in tomato. However, the enzyme that forms β-carotene or its metabolic products are present in the tomato genome and hence could be knocked out. Alternatively, reducing the expression with an RNAi construct of a fruit-specifc expressed pectate lyase (Solyc03g111690) reduced the softening of the tomato fruit, showing that it is possible to bypass the overall climacteric ripening program [8]. This could be easily reproduced with a genome edit aiming to knockout or knockdown this gene. A third example that frmness and carotenoid levels are not always correlated is the phenotype of the *hp1* and *hp1-w* mutants. These plants produce tomatoes with delayed ripening, higher levels of carotenoid and other phytonutrients due to altered light transduction [9]. However, the authors point out that this results in undesirable whole plant phenotypic changes and therefore they suggest that the knockdown of the gene responsible for the phenotype, DNA damage-binding protein 1 (*DDB1*), should be occurring only during fruit ripening. This demonstrates that the tradeoff of high lycopene content vs. delayed ripening can be resolved by taking one of several possible approaches that could bypass the tradeoffs: either by rerouting the lycopene pathway, directly targeting the enzymes involved in softening, or by altering the light signaling pathway specifcally in the tomato.

# *2.3 Seed Number vs. Seed Weight*

For many crops, an increase in seed number leads to a reduction in seed weight. Grain Weight 2 (TaGW2-A1), is a well-characterized gene in wheat that has a positive effect on seed weight but reduces the number of seeds. The introduction of the wild emmer allele *GNI-A1* into wheat was demonstrated to break this correlation because this allele could improve seed weight without affecting seed number, as reviewed by [10]. It was demonstrated that a single amino acid substitution is responsible for this (G182R). Alternatively, overexpressing an α-expansin gene in wheat led to transgenic lines with a higher seed weight, no signifcant change in seed number, and hence an increase in grain yield of 12.3% [10]. These two examples show that an increase in seed weight does not always lead to a reduced seed number. The opposite was also demonstrated. In rice, elevated *NOG1* expression was shown to increase the number of grains per plant without affecting the grain weight, and this was due to a natural variation of a 12 bp insertion in the promoter [10]. Crossing an allele that improves seed weight to another allele that improves seed number would be a good strategy to improve total yield. The above examples are in wheat and rice, and thus analogs/homologs should be found in other grain crops of interest. A list of candidates that could have a comparable function could be made, and these genes can be edited, either by SNP introduction (to GNI-A1 homologs) or through promoter editing (for α-expansin or NOG1 expression). This could be done simultaneously with genome editing technologies aimed at multiplexing of editing tools. Together these examples show that the genetic link of two traits causing a tradeoff can be broken through introducing/creating/knocking out alleles that do not affect both traits. Different tools can be used to achieve this effect. The detailed molecular studies of tradeoffs allow for the use of targeted mutagenesis. Hence, genome editing can be very helpful when multiple alleles need to be modifed or when natural variants are not available in the available germplasm.

# **3 Making Use of Expression Diversity in Orthologs/ Homologs**

As mentioned before, many genes are part of gene families. This essentially means that a mutation in such a gene, if is not dominant or dominant-negative, does not lead to a phenotype because the function is compensated by a homolog with a similar function. For breeders this means that for many genes, single knock-outs are not effective for improving traits. However, if a gene that is important for a trait, is part of a gene family, and causes additional negative effects on other traits, there is an opportunity to substitute such a gene with a homolog.

# *3.1 Fruit Size vs. Inforescence Branching*

Using genome editing technology to modify promoters can be aimed at introducing single nucleotide polymorphisms (SNPs), small and larger deletions and even rearranging the promoter randomly though multiplex editing. Generating a population of plants with variations in the promoter sequence of a single gene can be used for fne-tuning the desired effect. A series of deletions in the promoter of CLAVATA3 in tomato (SlCLV3) was linked to altered expression and it was shown that the altered expression coincided with an altered level of locule number and thus fruit size, though not predictably [11]. Reducing SlCLV3 activity also promoted inforescence branching [12], which can lead to excessive number of fruits, dampening the effect that reduced SlCLV3 expression can have on fruits size due to disturbed source to sink transport. It was shown that a null mutant for SlCLV3 in tomato is compensated by elevated expression of the ortholog SlCLE9 [12]. The authors also show that SlCLE9 has a similar function as SlCLV3, though the SlCLE9 knockout has a weak phenotype with only a subtle effect on locule number. This means that an edited promoter population for either SlCLV3 or SlCLE9 could be a valid strategy to obtain a tomato plant with a high number of locules and regular inforescence branching.

# *3.2 Shorter Plants vs. Plant Morphology*

Mutants in the gibberellic acid biosynthesis and catabolism pathway often result in a dwarf or elongated phenotype respectively, due to modifed gibberellic acid levels. However, this often comes with pleiotropic effects such as increased tillering in rice, increased culm bending in sorghum, and in one case can result in complete inhibition of fowering in rice. Tomato *internode elongated-1* (*EI-1*) is a splice-site mutation in the SlGA2ox7 gene. SlGA2ox7 is a gibberellin 2-oxidase that catalyzes the breakdown of certain bioactive gibberellins and *EI-1* results in a dwarf phenotype. *EI-1* leads to an increase in bioactive gibberellins in stems and petioles [13]. However, since SlGA2ox7 is more highly expressed in hypocotyls and internodes than in petioles, the elongated internode mutation phenotype is stronger here. Due to this organ specifc expression, *EI-1* exhibits a reduced elongation specifc effect only, while its ortholog Solyc10g005360 has a different expression pattern and is therefore expected to have more pleiotropic effects, including in the leaves. Consequently, the former gene would be a preferred candidate for genome editing as higher expression could lead to shorter plants without affecting the morphology of the leaves. Similarly, in pea the mutant *Le-1* has a mutation in a gibberellin 3-oxidase, which results in a shoot specifc phenotype, while the roots are unaffected. In summary, to shorten the height of a crop species, unwanted pleiotropic phenotypes could be avoided by learning about the expression patterns in all genes that are affecting this phenotype, and choosing a gene that has a tissue specifc expression.

# *3.3 Vitamin C vs. Growth*

Ascorbate peroxidases (APX) catalyze the H2O2-dependent oxidation of ascorbic acid (vitamin C) in plants, and can therefore reduce ascorbic acid levels. In Arabidopsis, a knocked out major cytosolic isoform of APX led to severe growth retardation. In contrast, in tomato there are nine homologs that encode for APX enzymes that catalyze the breakdown of ascorbate, with one being highest expressed in red ripe tomato fruits. Specifc mutation of *SlAPX4* by genome editing led to an increase of ascorbic acid in fruit with no detected growth impairment [14]. Hence, this approach might be easily copied to other crops for the biofortifcation of fruits, in case their genomes contain a family of *APX* homologs.

# *3.4 Blast Resistance vs. Yield*

Rice blast is a disease caused by the fungal pathogen *Magnaporthe oryzae* and is a huge problem in rice cultivation. Resistance to this disease has a tradeoff with yield. *Pigm* is a locus that has been used in breeding for durable and broad-spectrum resistance to rice blast. The locus consists of 13 homologs coding for NLR receptors and two of these, *PigmR* and *PigmS* are separated by just two genes. While *PigmR* confers blast resistance but at a yield cost, *PigmS* attenuates the blast resistance, and therefore counteracts the yield cost by promoting seed set [15]. Interestingly, increased expression by transgenic overexpression of both loci, overcomes this tradeoff. Hence, the *Pigm* locus effect on yield could be improved by editing their promoters for improved expression of both.

# **4 Overcoming Linkage Drag**

Sometimes a tradeoff is caused by two genes that are in close proximity to each other on a chromosome. Such genes might genetically be linked if recombination between the genes is low or absent. This would make separation of these genes diffcult or even impossible. If one of the two genes has a positive effect on a trait, but the other one has a negative effect on the same or another important trait, this presents a tradeoff known as linkage drag. Linkage drag examples include virus resistance vs. yield in tobacco, viral resistance vs. bacterial resistance in tomato, abiotic stress resistance vs. yield and quality traits in sunfower, and heading date vs. root biomass in wheat. In a series of near isogenic lines, the precise site was determined where recombination could break the tradeoff between viral resistance and bacterial resistance in tomato [16]. Recombination of the *I-3* gene out of the donor *Solanum pennelli* introgression at the end of the chromosome would remove the unknown gene from *S. pennelli* that causes bacterial spot susceptibility. The technology to induce precise crossover events is still in development, but some progress has been made. Controlling the recombination event has been shown in yeast. In plants linkage drag could be broken by swopping chromosome arms between linked loci [17]. The authors also demonstrated how two chromosome arms were exchanged in the *ALS2* locus for the *Solanum pennellii* and *Solanum lycopersicum cv. M82.* This shows that performing controlled recombination to remove a gene with a deleterious effect while maintaining the novel introgressed allele in plants is a promising new breeding tool.

# **5 Concluding Remarks and Future Perspectives**

Applying genome editing technology to reduce tradeoff side-effects has potential as a tool in plant breeding. Especially knockout editing or promoter editing has been proposed due to their broad applicability. More precise edits can result in altered protein function, and to obtain this, different editing technologies are being developed [18]. Due to the nature of DNA editing enzymes and the properties of chromosomes in alive cells, not every nucleotide can be edited in such a controlled manner. Also, off-target effects are a problem for genome editing success. Solving this remains a challenge for the future. It should be considered that not all crops can be edited yet. For example, incomplete sequenced genomes, diffculty in editing itself, resistance to regeneration or obligatory outcrossing are hurdles to overcome. Some tradeoffs were broken through an allele that was discovered with a QTL experiment. Other tradeoffs can be broken through editing (promoters) of genes, especially if tradeoff-breaking alleles have not been discovered in the crop of interest. Both approaches can be useful additions to the breeders' toolbox. Breaking tradeoffs is not easy. In fact, breaking one tradeoff could create a novel one. An example is the GNP1TQ allele in rice which has broken the seeds number vs. seed weight tradeoff. When this allele was introduced in the Lemont background, this did not result in a higher yield because the variety could not meet the increased sink capacity needed for the flling of the grains [10]. So next, the grain number vs. sink capacity tradeoff needs to be addressed. In the end, by balancing tradeoff side-effects with trait improvement, new crop varieties can be bred that meet novel breeding demands.

**Acknowledgements** Thanks to Dr. Nathalie Dollé-van Orsouw, Dr. Matthias Berens, Dr. Paul Bundock and Dr. Thea van der Wijk for the critical reading of the manuscript.

# **References**


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# **Chapter 5 CRISPR/Cas Mutation Screening: From Mutant Allele Detection to Prediction of Protein Coding Potential**

**Elke Vereecke, Katrijn Van Laere , and Tom Ruttink**

**Abstract** CRISPR/Cas is a gene-editing technique that allows for the precise and specic introduction of a mutation into a DNA sequence. The outcome of a mutation on encoded protein depends on the type of mutation (deletion, insertion and/or substitution) and the position of the mutation in the DNA sequence. It can be predicted by using screening methods that are able to identify a mutation at nucleotide level. Here, several screening methods are discussed with a difference in complexity, resolution and scalability and the results are interpretated by taken into account the central dogma of the molecular biology. Two modules of the SMAP package, SMAP *haplotype-window* and SMAP *effect-prediction,* are proposed and implemented in a high-throughput screening workow that allows for the automated and streamlined screening of CRISPR experiments.

# **1 Precision Gene Editing: Design Guided by Gene Structural Features**

Gene editing, *e.g.,* by CRISPR/Cas, is widely used for plant functional genomics research and has huge potential for targeted improvement of desired heritable traits in crops [1]. The precision and specicity of CRISPR/Cas allows for dedicated

E. Vereecke (\*)

Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), Melle, Belgium

VIB Center for Plant Systems Biology, Ghent, Belgium e-mail: elke.vereecke@ilvo.vlaanderen.be

K. Van Laere

T. Ruttink

Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), Melle, Belgium

Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium e-mail: tom.ruttink@ilvo.vlaanderen.be

Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), Melle, Belgium e-mail: katrijn.vanlaere@ilvo.vlaanderen.be

**Fig. 5.1 The central dogma of molecular biology posits that the genetic information encoded in a genic DNA sequence is transcribed into messenger RNA (mRNA) and then translated into protein.** Transcription factors bind to regulatory elements in the promoter of a gene and the fanking genomic DNA sequence is transcribed into pre-messenger RNA (pre-mRNA) by an RNA polymerase. This pre-mRNA molecule matures through the removal of introns resulting in the juxtaposition of exons to form mature mRNA. The mature mRNA is translated into a protein by ribosomes that start from the translation initiation (START) codon and follow the open reading frame (ORF) until the frst translation termination (STOP) codon. Transcription and translation are tightly interrelated dynamic processes that are regulated at different levels and rely on various structural features in a gene [2]. Therefore, mutations in a DNA sequence may affect any of these processes and gRNAs and CRISPR/Cas DNA modifers may be designed to create specifc changes in the expression, structure, stability, activity, and/or function of a protein

screening for induced mutant alleles at target sites. Combinations of mutant alleles detected by molecular screens on the one hand, with the central dogma of molecular biology that explains the relationship between the primary DNA sequence, gene structural features, and expression of the encoded protein (Fig. 5.1) on the other, allows prediction of the effect of the mutation on protein functionality. Thus, a comprehensive gene editing screening workfow to characterize the generated mutants ideally spans the entire path between "gene structure based" CRISPR/Cas and gRNA design, molecular detection of the mutant alleles, and prediction of the effect of the mutation on the encoded protein sequence, hence capturing the actual outcome of gene editing. Here, we review the different types of mutations that can be introduced by variants of CRISPR/Cas gene editing, highlight several molecular screening and detection techniques and place them in this overarching perspective.

# **2 Types of Mutations Introduced by CRISPR/Cas Mediated Gene Editing**

In its basic form, CRISPR/Cas mediated gene editing introduces a double-stranded DNA break at a specifc genomic position defned by the gRNA. Subsequent nonperfect repair via non-homologous end-joining (NHEJ) or via homology-directed repair (HDR) results in the introduction of mutations in the target DNA sequence [3]. Mutations induced by CRISPR/Cas occur within a short range fanking the protospacer adjacent motif (PAM) site (*e.g.*, 3–4 bp upstream of the PAM site for Cas9). NHEJ creates allelic series of mutations, typically in the range of short deletions and insertions (one up to tens of nucleotides), or few substitutions. Potential target sites, cleavage effciencies, and induced scarring patterns may be predicted based on the gRNA sequence using machine learning, but accurate models for plants require further training on large-scale data sets [4, 5]. CRISPR/Cas is widely used to create targeted gene knockouts in several plant species. For example, Wang et al. [6] used CRISPR/Cas to knock out the susceptibility genes of the *mildewresistance locus* (*MLO*) in wheat, generating wheat that is resistant to powdery mildew [6]. Mutations in a gene that result in a non-functional protein encoded by that gene, as described for the mutations in the *MLO* genes, are called loss-of-function (LOF) mutations and can occur when the ORF downstream of the mutation is disrupted (out-of-frame indel or frameshift) [7].

The CRISPR/gRNA complex may also be used as location-specifc vehicle to deliver DNA sequence modifers to a given location and modify the primary sequence (like base-editing or prime-editing) or epigenetic state [8, 9]. In baseediting, a cytidine deaminase (C:G-to-T:A) or adenosine deaminase (A:T-to-G:C) is linked to the CRISPR/gRNA complex and is used for the conversion of a single nucleotide at a specifc position [8]. Base-editing can be used to create a specifc point mutation, which may result in a single amino acid change in the protein sequence, a premature STOP codon or alter a splicing acceptor or donor site [10]. For example, in tomato and potato, base-editing was successfully used to convert a cytidine into a thymine in the *acetolactate synthase* (*ALS*) gene, conferring resistance to herbicides [11]. Mutations in a gene that result in an enhanced activity or functionality of the protein encoded by that gene, as described for the mutation in the *ALS* gene, are called gain-of-function (GOF) mutations.

While base-editing can only be used for two types of nucleotide substitution, prime-editing can introduce all kinds of predefned mutations, including the deletion, insertion, and/or substitution of specifc nucleotides [12]. A prime-editing system consists of a Cas enzyme with nickase activity, reverse transcriptase, and prime-editing guide RNA (pegRNA) with a primer binding site for the specifcation of the genomic target site and an RNA template that encodes the desired edit [13]. It was already successfully used in rice for the insertion of a fragment up to 15 bp in the OsCDC48-T1 gene [12] and for the triple amino acid substitution in the *EPSPS* gene in rice to confer a higher level of glyphosate resistance [14].

CRISPR/Cas and its variants are also able to target DNA sequences at gene regulatory sites, *e.g.,* transcription factor binding sites, splicing sites, and translation initiation and/or termination codons, thus changing gene structural features or coding potential. This will affect the different processes driving transcription, mRNA maturation, and translation (Fig. 5.1). In addition, the epigenetic state can be modulated by fusing the CRISPR protein with an epigenetic modifer that can affect the methylation state at DNA level or affect the methylation and/or acetylation state at nucleosome level (histone modifcation) [15, 16]. For example, Gallego-Bartolomé et al. [17] were able to reactivate the transcription of the *FWA* gene by demethylation of the *FWA* promotor using a dead Cas9 fused to the human demethylase TET2cd [17].

In short, CRISPR/Cas and its variants can be used to introduce a range of mutations into a DNA sequence that have different effects on the encoded protein. LOF or GOF mutations can be generated to study the role of certain proteins in biological processes, to confer resistance to pathogens or herbicides, to divert the metabolic fux of biosynthesis pathways towards valuable compounds, etc.

# **3 Screening Methods: Complexity, Resolution, and Scalability**

Different screening methods are available to detect the outcome of CRISPR/Cas gene editing, to identify which plant material contains a desired gene edited sequence, and to evaluate the mutation effciency. Screening methods may apply different detection methods (physical properties of an amplifed allele vs sequencingbased), comprise targeted or untargeted screening (local sequencing of the predicted edited site (*e.g.,* amplifcation or capture of the gRNA binding site and fanking regions), or global sequencing (*e.g.,* WGS, RNA-Seq)), and with different levels of throughput and automation (via locus and/or sample multiplexing).

Simply put, any standard molecular detection technique that can discriminate DNA sequence variants (alleles) can also be used to detect CRISPR-induced mutations (Fig. 5.2). PCR-amplifcation of the target region, coupled to a detection method such as high-resolution melting (HRM) [18], fuorescent probe binding (qPCR or ddPCR [19]), or amplicon length polymorphism (agarose gel-electrophoresis, capillary fragment analysis, or mismatch detection assay [20] (a variant of Cleaved Amplifed Polymorphic Sequences (CAPS) markers), or IDAA [20]) can be used to identify mutated alleles (Fig. 5.2). In addition, Kompetitive Allele-Specifc PCR (KASP [21]) or primer–extension assays [22] may be used to screen for expected SNPs. These techniques are cheap, easy to implement, and allow for quick routine screening of gene edited mutant collections [23]. However, they only indirectly show the presence of a mutation, and not the actual, exact mutant DNA sequence at the nucleotide level, a prerequisite to interpret the effect of the mutation on the encoded protein.

Amplifcation and sequencing of target loci of mutants provides information on the specifc nucleotides that are deleted, inserted and/or substituted (Fig. 5.2). Sanger (dideoxy-) sequencing generates electropherograms allowing for the determination of the DNA sequence and the identifcation of mutations [23, 24]. The interpretation of the electropherogram can be challenging, as multiple nucleotides can be called at the same position due to heterozygous insertions, deletions and/or substitutions. Therefore, several computational tools have been developed to deconvolute the electropherograms, such as Tracking of Insertions and Deletions (TIDE) [25], CRISP-ID [26], Deconvolution of Complex DNA Repair (DECODR) [27],

**Fig. 5.2 A targeted mutation screening workfow.** Mutations can be introduced into a DNA sequence using CRISPR/Cas and the CRISPR/Cas-based variants base-editing and prime-editing. Screening methods are needed to evaluate if a mutation has occurred. In this screening workfow, the different steps needed to: (1) identify if a mutation occurred (differential detection based on physical attributes of amplicons); (2) identify which mutation (deletion, insertion, substitution) occurred at nucleotide level (sequencing-based methods); and (3) evaluate the effect of the mutation on encoded protein sequence (Fig. 5.1) are illustrated together with the expected outcome of the different steps

and Inference of CRISPR Edits (ICE) [28]. These tools utilize distinct algorithms to analyze electropherograms from a wild-type and a gene edited sample, generating a list with predicted mutated sequences [24]. The sensitivity of Sanger sequencing for alternative alleles in a heterozygous or otherwise mixed sample is about 15% [29]. Consequently, low-effciency editing is likely to be overlooked. Furthermore, these methods are typically performed with a separate amplifcation and detection reaction for each sample and each locus (simplex), limiting the scalability for mutation screens to large collections at multiple target loci.

Next Generation Sequencing NGS allows for massive parallel sequencing and analysis of heterogeneous samples and substantially lowers the per-sample and per-locus costs in high-throughput mutation screens [3]. Because of its deep read coverage, NGS sensitivity for alternative alleles is 0.1–1% and thus enables screening of bulk samples (*e.g.,* protoplasts after transfection), and effcient 1D, 2D or 3D

pooling schemes [28]. In addition, multiplex amplicon sequencing combined with incorporation of sample-specifc barcodes during library preparation facilitates parallel sequencing at hundreds of loci, in hundreds of samples per sequencing run. NGS yields targeted resequencing data that can be analyzed via bioinformatics tools such as CRISPResso2 [30] and SMAP *haplotype-window* [31] (Fig. 5.3). In SMAP *haplotype-window*, sequencing reads are mapped to a reference and the entire read sequence spanning the region between borders (typically the amplicon primer binding sites) is considered as an allele [31]. All the unique alleles are sorted and counted for the calculation of relative allele frequency per locus per sample. A region of interest (ROI) can be defned to focus the analysis on mutations introduced by the gene editing technique in a narrow nucleotide window and ignore additional sequence variants at distance from the edit site. Every allele is compared to the reference in its entirety, allowing for the detection of any combination of insertion, deletion, and/or substitution. SMAP *haplotype-window* will generate an integrated genotype call table with all the observed alleles per locus per sample. Since it is agnostic to the length of the deletion, insertion, or substitution, it can detect any mutation resulting from an edit in the primary DNA sequence in a given window, as long as the amplicon or read length spans the mutated allele. SMAP *haplotypewindow* can also process probe-capture enriched, WGS, and RNA-Seq read data from global resequencing screens, for a given list of target loci. PacBio sequencing [32] and nanopore MinION sequencing [33] can be used to detect long-range insertions and deletions, as well as epigenetic DNA modifcations introduced by CRISPR/Cas.

# **4 Mutation Screening in a Broader Perspective: From Nucleotide to Protein**

The current repertoire of CRISPR/Cas DNA modifers combined with gRNA specifcity, generates a huge array of design possibilities, especially when based on the principles that predict how protein sequences may be altered by editing the genomic nucleotide sequence. A mutation screening workfow that draws on clever CRISPR design, in turn, should be able to consider detected mutated alleles in their respective gene context and classify the mutated alleles based on predefned desired alleles (*e.g.* a unique base-edit) or on percentage protein sequence similarity to the original wild-type allele [7].

The SMAP *effect-prediction* module from the SMAP package estimates the novel encoded protein sequence and is the fnal step in the mutation screening workfow (Fig. 5.3) [31]. SMAP *haplotype-window* generates a list of all observed haplotypes per locus per sample, which is directly used as input for SMAP *effectprediction*, together with all positional information on gene structural features. SMAP *effect-prediction* replaces a segment of the original reference gene sequence by the observed mutated sequence and evaluates all the splicing sites, the translation initiation codon, open reading frame, and translation termination codon [31]. After translation of the most likely ORF in the mutated allele, the amino acid

**Fig. 5.3 SMAP** *haplotype-window* **and SMAP** *effect-prediction* **can be used to analyze highly multiplex amplicon sequencing data and to estimate the novel encoded protein sequence.** SMAP *haplotype-window* is a module of the SMAP package that is used to analyze the sequencing reads obtained from NGS. It maps the sequencing reads (in this fgure illustrated for a bulk sample) to the reference genome, groups all the alleles with the same mutations, determines a ROI and calculates the mutation frequency. SMAP *effect-prediction* is used to provide biological interpretation of the different mutations that were introduced by substituting the wild-type allele with the mutant allele(s) in the reference genome and translating the novel genic sequence. Mutation types include a frameshift mutation, in-frame indel, missense mutation, deletion, silent mutation and nonsense mutation

sequence is aligned to the original reference protein and the percentage protein sequence similarity is estimated as quantitative score for the remaining protein functionality. Proteins may no longer resemble the original reference protein (frameshift mutation or nonsense mutation), proteins can be identical to the original reference protein (silent mutation), etc. (Fig. 5.3). A threshold value can be set for the percentage sequence similarity to the original reference protein still needed for a protein to perform its function. DNA mutations that result in a protein with a lower percentage of similarity as a given threshold value can be defned as a loss-offunction mutation [34].

# **5 Conclusions**

CRISPR/Cas and CRISPR/Cas variants are widely used to introduce mutations into a DNA sequence. However, mutations can have different effects on the function of a gene and its encoded protein. Here, we describe a molecular screening workfow that focuses on the path from CRISPR/Cas and gRNA design, through screening for mutant alleles, and prediction of the effect of the DNA sequence mutation on the encoded protein, all implemented in modules of the SMAP package. By using SMAP *haplotype-window* and SMAP *effect-prediction,* the detected mutated alleles are placed in their respective gene context, and the mutated alleles can be classifed based on percentage of protein sequence similarity to the original wild-type allele. This high-throughput screening workfow allows for the automated and streamlined screening of multiplex CRISPR experiments, in large mutant collections (locus and/ or sample multiplexing) and enables fast and easy interpretation of the effect of the mutant alleles on the protein sequence, and automated routine identifcation of carriers of desired alleles.

# **References**


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# **Chapter 6 Methods and Techniques to Select Efcient Guides for CRISPR-Mediated Genome Editing in Plants**

**Fabio D'Orso, Valentina Forte, Simona Baima, Marco Possenti, Daniela Palma, and Giorgio Morelli**

**Abstract** CRISPR technology is revolutionizing genomic engineering by enabling scientists to precisely modify plant DNA, thus representing a powerful tool for plant breeding.

This chapter provides a summary of the approaches and constraints of CRISPRmediated genome editing in plants, with a focus on the critical prerequisite of ef cient CRISPR reagents for successful gene editing in plants.

While computational tools have tremendously improved our ability to design specic guides, their limitations make guide effectiveness prediction unreliable, especially for plants. Therefore, it is strongly recommended to validate CRISPR reagents before investing time and resources in the challenging process of plant transformation.

A number of *in vitro* and *in planta* assays coupled with analytical methods have been proposed to assess the editing performances. Each approach has its own strengths and weaknesses, so the choice of the most suitable system depends on the specic plant species and the type and depth of the genotypic data required.

In many cases, the hairy root assay can provide a good compromise between rapidity, reliability and cost-effectiveness for assessing editing performance in numerous plant species.

**Keywords** CRISPR · Genome editing · Guide design · Guide efciency · Computational tools · Agroinltration · Protoplasts · Hairy roots · Genotyping

F. D'Orso (\*) · V. Forte · S. Baima · M. Possenti · D. Palma · G. Morelli CREA – Research Centre for Genomics and Bioinformatics, Rome, Italy e-mail: fabio.dorso@crea.gov.it

# **1 Introduction**

With the introduction of precision genome editing using Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) technology, we have entered a new era of genetic engineering. CRISPR technology has allowed straightforward, cost-effective and effcient gene editing compared with technologies as zinc fnger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). CRISPR represents a new perspective for plant breeding and a powerful alternative for genetic engineering to speed up the introduction of improved traits by precise and predictable modifcations (deletions, substitutions, insertions) directly in an elite background [38]. Major challenge for the application of genome editing in crop breeding is generating plants without introducing a transgene, and this has led to new challenges for the regulation and social acceptance of genetically modifed organisms (GMO). Furthermore, CRISPR technology can produce novel plants transgene-free indistinguishable from natural variants or generated by conventional breeding techniques [42]. Nowadays it has emerged as a powerful tool for applications in medicine, agriculture, and basic studies of gene function. In plants, since the frst demonstration of CRISPR in DNA editing in 2013 there has been much progress in basic plant science and crop improvement with applications for biotic and abiotic tolerance, yield performance improvement, biofortifcation and enhancement of plant quality [118] ranging from model plant, like *Arabidopsis thaliana*, to food crops [38].

In addition, CRISPR technology can accelerate crop domestication, a labourintensive process involving alteration of a plant from its wild state to a new form that can serve human needs. Recently, CRISPR was used to domesticate wild tomato, *Solanum pimpinellifolium*, which is remarkably stress tolerant but is defective in terms of fruit production [77]. In one study, six loci that are important for yield and productivity were targeted, and the engineered lines displayed increased fruit size, fruit number and fruit lycopene accumulation [157].

In prokaryotes, CRISPR/Cas are a family of DNA sequences found in bacteria and archaea genomes and as adaptive immune system that naturally protects cells from DNA virus infections [38]. As biotechnological tool, the CRISPR/Cas9 system is comprised of the Cas9 endonuclease and a synthetic single guide RNA (sgRNA), which combines functions of CRISPR RNA (cRNA) and trans-activating CRISPR RNA (tracrRNA) to direct the Cas9 protein to the DNA target sequence preceding the protospacer-associated motif (PAM) [153].

Various strategies and different types of CRISPR/Cas systems, as genetic manipulation tools, have been attempted to generate and study the impact of functional mutations in crop improvement [92].

Furthermore, the requirement for a specifc PAM sequence is a major factor restricting the selection of target sequences. For this reason, different engineered SpCas9 or orthologous Cas9 nucleases derived from different organisms, able to recognize different PAM sequences, have been used for genome editing [28].

The desired genetic modifcation is initiated by inducing double stranded breaks (DSBs) into a target sequence by using nucleases, and it is subsequently attained by DNA repair through Non-Homologous End Joining (NHEJ) or homology-directed repair (HDR) [36].

The events of genome editing in plant according to the NHEJ mechanism described in the literature, are mainly represented by gene knock-out [117]. The rate of knock-in is much lower [41]. In fact, NHEJ plays a major role in the repair of DSBs through insertions and deletions (indels) at the junction, which change the nucleotide sequence information surrounding the repair region [140]. These indels may cause frameshifts and lead to knock-out of the corresponding gene [122].

HDR-mediated genome editing, also known as gene targeting (GT), is the approach to introduce precise insertion (knock-in) using information from an exogenously supplied DNA donor template to repair the break. The information is copied from the donor template to the chromosome, achieving the desired DNA sequence modifcation [22]. GT in higher plants was extremely diffcult for decades. One of the obstacles in achieving HDR has been the ability to deliver suffcient donor templates to the plant cell to repair the DSB [51]. Development of approaches to improve GT effciencies in plants are in progress; however, there is still no universal and effcient method for increasing the knock-in frequency. Currently the best approach for precise modifcation of plant genomes is geminivirus-mediated Gene targeting, but also other non HDR-mediated approaches like Base Editing (BE) and Prime Editing (PE) are promising [44].

Irrespective to the nuclease or editing system, a major concern of CRISPR/Cas technology is the guide effciency and specifcity for a given gene [74]. Moreover, despite the huge repertoire of CRISPR-based molecular tools and their great potential for improving food and nutrition security, genome editing application in agriculture is still slowed down by some limitations. One of the main bottlenecks is the ability to regenerate transformed plants. This process is very time consuming and labour intensive; in many cases leading to very low transformation frequency especially for those species and genotypes which are recalcitrant to plant regeneration or suffer poor transformability. Due to this reason, it becomes essential to rely on highly effcient editing systems and, consequently, execute a fawless experimental design. This should include a careful choice of the most suitable editing system and the validation of the CRISPR reagents before undertaking the stable transformation. In particular, the evaluation of the guide effciency is of utmost importance to ensure a successful CRISPR experiment.

# **2 Approaches and Constraints of Genome Editing Using CRISPR Technology**

# *2.1 CRISPR-Based Editors*

Many CRISPR-based genome editing tools have been developed to facilitate effcient plant genome engineering. Thanks to its fexibility, effciency and low cost, CRISPR/Cas technology has been widely used in plants for fundamental and applied research.

CRISPR/Cas systems are divided into two distinct classes according to their structures and functions: class 1 systems (including types I, III, and IV) that use multiprotein complexes to destroy foreign nucleic acids, and class 2 systems (including types II, V, and VI) that use single proteins [93].

The CRISPR/Cas9 system (type II) is the most frequently used. It is composed of a Cas9 nuclease and an engineered single guide RNA (sgRNA). The sgRNA comprises a scaffold sequence necessary for Cas binding and a specifc DNA sequence designed to be complementary to the target DNA site, followed by a short DNA sequence acting as a binding signal for Cas9 (Protospacer Adjacent Motif – PAM). Every Cas9 requires a specifc PAM to recognize and cleave the target DNA: for example the widely used Cas9 from *Streptococcus pyogenes* (SpCas9) recognizes an 5′-NGG-3′ PAM sequence [134]. Cas9 contains two nuclease domains, HNH and RuvC, which break the double-stranded DNA (dsDNA) site primarily near the PAM in the target DNA. The resulting DSB is then usually repaired by the NHEJ repair pathway that is the most active repair mechanism; this usually leads to nucleotide insertions or deletions (indels) at the cleavage position. This system enables targeted mutations to be introduced into genomes with high effciency, but the resulting mutations can vary and are not easy to predict.

Although SpCas9 is very effcient, its specifc PAM requirement, target specifcity and the large protein size limit its applications. To overcome this limitation, several Cas9 orthologs and variants have been studied for their suitability in genome editing, exhibiting diverse preferences for PAM sequences, and varying in their molecular weights. These include *Staphylococcus aureus* (SaCas9), *Neisseria meningitides* (NmeCas9), *Streptococcus thermophilus* (StCas9), *Campylobacter jejuni* (CjeCas9) and others [88]. The PAM sequences recognized by these Cas9s are relatively complex, which limits the widespread use of these nucleases in genome editing. However, some engineered Cas9 variants with an increased PAM compatibility have been reported, such as VQR-Cas9 (NGA PAM), VRER- Cas9 (NGCG PAM), EQR-Cas9 (NGAG PAM), xCas9 (NG, GAA and GTA PAM), SpCas9-NG (NG PAM), SpG, and SpRY, a near-PAMless SpCas9 variant (NRN>NYN PAMs) [88]. These variants allow the targeting of simple, non-canonical PAM sites, thus expanding the range of targetable sequences.

In addition to the Cas9 proteins, class 2 type V CRISPR-Cas systems involving Cas12a (or Cpf1) and Cas12b (or C2c1) have been adopted for modifying genomes at AT-rich PAM sequences [10], and presently AsCpf1, LbCpf1, and FnCpf1 are the most common types of Cpf1 used in genome editing. Another interesting alternative Cas nuclease recently identifed is the type I CRISPR/Cas10 that causes long-range deletions [143]. An applicative example can be found in Osakabe et al. [111] where a 7.2 kb deletion has been obtained in tomato.

During CRISPR experiment design, a major concern is the guide effciency and specifcity. In fact, although this system can be programmed to virtually cleave any sequence preceding the PAM site sequence, it does not always succeed to target all the predicted sites [91]. Multiple guides designed with different target sequences determine the rate at which simultaneous modifcations can be introduced in the genome and therefore the ability to perform comprehensive genome engineering at corresponding specifc sites [30]. This feature is especially important to edit multiple loci simultaneously in the same individual.

CRISPR system has also been used to achieve a precise change in the plant genome by different approaches like homology-directed DNA repair (HDR) mediated GT [44], BE [50, 82] and PE [6, 44, 82].

GT is an HDR-mediated targeted gene replacement that requires the presence of a DNA donor template containing the desired sequence delivered with the guide and Cas9. GT enables specifc nucleotide changes ranging from a single nucleotide change to large insertions or deletions. However, GT is ineffcient because HDR occurs with extremely low frequency in plants, limiting the widespread use of this process for gene modifcation.

Different strategies have been applied to improve GT effciencies, such as increasing the copy number of the repair template using geminivirus replicons or releasing the template from a T-DNA or manipulating the DNA repair pathway to improve HDR frequency [44], but despite these attempts, effciencies are still low.

Unlike HDR, base editors (BE) do not require the formation of DSBs and a repair template. In general, a base editor is composed of an impaired nuclease, nickase (nCas9) or dead (dCas9) fused with a deaminase that can convert a nucleotide into a different one. According to the action types of deaminases, the BE systems are classifed as the cytosine BE (CBEs convert C to T), the adenine BE (ABEs convert A to G) and dual-base editors. In these years several base editors have been developed using Cas protein variants with different PAM requirements (SpCas9, SaCas9, SaKKH-Cas9, VQR-Cas9, SpRY, SpCas9-NG), testing several deaminases to improve editing effciency (APOBEC, BE3, AID, CDA1, A3A, ABE7.10, ABEmax, ABE8e, ABE9) and engineering their connection to Cas9 to alter position and width of the editing window [50, 102]. Currently, effcient editing from C to T and A to G have been achieved with the BE, but not all BE systems work equally in plants and the technology has still some limits. BE is limited by the targeting scope of Cas, it can only work in a narrow activity window and it has a low accuracy when multiple target nucleotides of the deaminase are present within the activity window. In addition, the purity of the cytosine base editing (CBE) product depends on the uracil N-glycosylase inhibitor (UGI).

Prime editing (PE) is a precise genome editing technology capable of introducing a predefned change in a genome without the need for a DSB. PE can achieve a variety of edits, including all 12 types of base substitution, small indels, and replacements. The PE system consists of a Cas9 nickase (nCas9) fused with a reverse transcriptase (RT) enzyme Moloney Murine Leukemia Virus RT (M-MLV RT), and a PE gRNA (pegRNA), an engineered standard sgRNA targeting the specifc genome sites that specifes the target site and encodes the desired edit [6]. The nCas9/ pegRNA complex binds to the desired target region and nicks one strand of the targeted DNA, providing a primer to initiate the production of edited DNA, while the pegRNA act as a template for reverse transcription.

Different versions of PEs have been developed in plants, such as PE1, PE2, PE3 e PE3b [102]. Unfortunately, the editing effciency of PE is low. Different strategies have been used to increase PE effciency, such as the use of alternative promoters for

the expression of Cas9 and RT, as well as the codon optimization of their coding sequences and the fusion with nuclear localization signals [50]. Furthermore, a recent study in rice has shown that designing primer binding site with a melting temperature of 30 °C and the use of two pegRNAs in trans encoding the same edits enhanced the editing effciency [86]. However, the editing effciency of PE is still low and further improvement is needed to broaden its application in plants, by optimizing key parameters such as RT enzyme type, experimental condition, and pegRNA design [50].

# *2.2 Plant Transformation and Regeneration Bottlenecks*

To initiate a CRISPR-mediated genome editing experiment, CRISPR reagents are delivered into plant cells through various methods such as agrobacterium-based delivery [2], particle bombardment [112], or protoplast-based delivery [152]. Regardless of the delivery method, plant cells must undergo tissue culture procedures after transformation or transfection to obtain fully edited plants. The process of organogenesis involves three phases: cell dedifferentiation, cell reprogramming, and the development of new apical meristems (root apical meristem or shoot apical meristem) [11, 73]. These steps are challenging, time-consuming, and labor-intensive.

The success of the regeneration process depends on the ability of explant cells to overcome their programmed cell type. Once reprogramming and regeneration are activated, cells acquire a new fate, leading to the generation of new meristems and organs. However, the process is not linear and can encounter obstacles in each phase, affecting organ or plant development.

Factors such as the ratio of auxins and cytokinins [128], carbon sources, salts, vitamins ([25, 151], hormones [40], and the type of explant used [33] can infuence the success of regeneration. Epigenetic factors [90] and other intrinsic factors like hormones, hormone receptors, transcription factors, and hormone signal transducers [11, 73] also play a role in guiding cell fate during the regeneration process.

Certain plant species, like *Nicotiana* and tomato [37, 48], can be regenerated *in vitro* with relatively high effciency, while others like pepper [97] or fruit trees [95, 100, 137] exhibit strong recalcitrance to regeneration. One strategy to enhance regenerative capacity is the expression of key transcription factors involved in meristem organization and development, such as BABY BOOM [14, 131], WUSCHEL [13, 18, 58], and SHOOT MERISTEMLESS [18, 156].

The regenerative capacity of each species can be a limiting factor when using genome editing technologies. These processes can act as bottlenecks in the gene editing pipeline, particularly for plants that are diffcult to culture and regenerate *in vitro*. Therefore, it is crucial to test and validate the effciency of the CRISPR guides used for mutagenesis to increase the likelihood of successful mutagenesis events and reduce the number of plants needed for analysis after transformation and regeneration.

# **3** *In Silico* **Designing of a Successful CRISPR Experiment**

# *3.1 Features Affecting CRISPR-Mediated Editing*

When designing a CRISPR experiment, the main issue to be taken into account for successful gene editing is the optimal trade-off between effciency and specifcity of CRISPR machinery. The ultimate goal is to maximize the on-target mutation rate and to avoid off-target mutations, which can occur when unintended genomic sites are cleaved due to sequence homology with the target site(s).

The specifcity issue is less problematic in plant breeding than in clinical research because unwanted mutations can be segregated away from the on-target mutation(s) by crossing mutants with wild-type plants. However, the crossing procedure can be laborious, time-consuming, or even impossible for perennial plants and asexually propagated crops. Consequently, as general rule, it is important to choose the RNA guides with highest specifcity scores (minimum off-target risk) [35, 103]. Being tightly dependent on the sequence homology, the off-target risk is normally predictable by *in silico* analysis and nowadays many bioinformatic tools are freely available. While SpCas9 tolerates single-base mismatches in the PAM-distal region, the proximal region is much more sensitive, even single mismatches can inhibit the cleavage [47, 62]. Then only guides whose off-sites have at least 1–2 mismatches within the PAM-proximal region should be considered highly specifc.

On the contrary, mutagenesis effciency is another bottleneck for genome editing in plants. In fact, low mutation frequency coupled with poor regeneration performance may jeopardize the success of the experiment leading to no edited plants.

Several aspects can affect a CRISPR-mediated editing experiment and concern intrinsic features of the nucleases and, most importantly, those of the RNA guides, which are different depending on the DNA sequence to be mutagenized.

When designing a CRISPR experiment, the frst step is deciding which type of nuclease to use. In plants, the codon-optimized versions of SpCas9 from *Streptococcus pyogenes* are the most used nucleases. Normally its PAM (NGG) is well distributed in the genome [12], therefore, unless specifc requirements are needed, NGG-PAM is usually suitable for generating loss-of-function mutants. While for T-rich regions, Cpf1 has found a wide application in plant genome editing [10]. However, in some cases it is necessary to edit specifc genomic regions for which SpCas9 or Cpf1 cannot be used, e.g. in Gene-Targeting, Base Editing or Prime Editing experiments. Having a wide repertoire of gene editing tools at one's disposal guarantees a better chance of obtaining the desired mutation. To this end, many efforts have been made to engineer SpCas9 to expand its ability to recognize different [70] or more fexible PAMs [144]. Furthermore, Cas9 orthologues with different PAM preferences have been identifed and used for GE [23, 46, 104, 120, 133].

Considering the huge genetic variability in microorganisms, it is expected to see an increasing number of Cas nucleases available for genome editing in the near future. To this end, Ciciani et al. [27] developed a computational pipeline to identify and isolate sequence-tailored Cas9 nucleases expanding the genome editing toolbox to respond to possibly any PAM requirement.

# *3.2 Selection of the Gene Region for Mutagenesis*

The availability/distribution of PAMs within the gene of interest identifes potential target sites for mutagenesis. However, not all positions within the gene are equivalent. The choice of the sequence target depends on the specifc aim of the editing. In certain cases, such as the removal or disruption of a specifc regulatory element on the promoter or intron, or when modifying a particular sequence by Base Editing, Prime Editing or Gene-Targeting, there may be limited fexibility for selecting the target sequence. To knock-out gene function, it is generally recommended to target the coding sequence relatively close to the N-terminus to generate a premature termination codon (PTC). However, targeting of regions too close to the initial ATG might be impractical due to a lack of PAMs. Even if it were possible, there is a risk that other ATG codons downstream of the mutation could act as a translation reinitiation site, leading to N-terminally truncated proteins with partial activity [129]. If the functional or structural domain(s) of the protein are already characterized, the guide can be designed to target those specifc domains, rendering the protein nonfunctional [125]. However, designing guides on a single exon might not guarantee a successful knockout if alternative splicing eliminates the mutated exon and produces a partially functional protein [129]. Other strategies include designing guides that bind on the exon-intron junctions to disrupt the splicing site and generate misprocessed mRNA, or attempting to delete the entire gene by designing target sequences upstream and downstream of the coding sequence. However, the latter approach may occur at a low frequency due to the length of the gene. Point mutations on the promoter and untranslated regions (UTRs) may have just a little effect on the expression and stability of the mRNA, respectively. For instance, it has been observed in mammalian cells that sgRNAs targeting the 5′ and 3′ UTRs were highly ineffective [34]. To ensure gene disruption, the best strategy is to employ multiple guides targeting different positions along the gene, leading to multiple mutations or deletions of varying size.

In addition to the selection of the nuclease and the position on target gene, another critical aspect in a CRISPR experiment is the guide's ability to form the complex with the nuclease and to trigger the cut on the target site. Several papers have investigated the characteristic of an ideal guide for optimal mutation induction, primarily based on SpCas9 system. It has been observed that nucleotide composition, GC content and secondary structure play a pivotal role in determining guide effciency.

Wang et al. [145] found that the nucleotide composition at the PAM proximal region was an important factor determining cutting effciency, in particular the purines (G/A) are preferred in the last 4 bases of the guide, while the pyrimidines are disfavored. These data have been substantially confrmed in other papers; in particular, there is a strong evidence about the preference of a G in the frst position before the PAM [34, 103, 148] and the disfavoring of the T at the last 4 bases of the spacer [34, 148]. In plants, Liang et al. [83] did not fnd a specifc relationship between spacer nucleotide composition and guide effciency. This fnding suggests

that there might be a distinction between animals and plants in this regard. However, it is worth noting that further analysis is required as the authors used fewer guides in their study.

CG content has a great impact on sgRNA effcacy [34, 91]. Doench et al. [34] found that sgRNAs with low (35%) or high (75%) CG content were less active in mammalian cells. Similarly, in plants Liang et al. [83] showed that for the 97% of the effcient guides, the CG content spanned between 30% and 80%; then this is the range to be taken into consideration when designing a guide.

In addition to base composition, secondary structures of sgRNA can affect the ability to form the ribonucleoproteic complex and/or its activity. Alterations in the canonical secondary structure of the sgRNA, can impede the interaction between the sgRNA scaffold and the Cas9 or the binding of the sgRNA seed sequence with the target DNA. Hairpin formation in the spacer region of the guide can prevent the recognition of the target DNA and so its cleavage [91]. Liang et al. [83] evaluated the secondary structure of a population of effective sgRNAs and found some common features which allowed to determine some criteria to design the guides. Based on this analysis, it was suggested to check the secondary structure and select guides with an overall intact tetra loop structure (especially for the loops 2, 3 and RAR), with no more than 12 spacer bases pairing with other bases of the sgRNA and no more than 7 consecutive base pairs. Moreover, the spacer sequence should have a low level of self-pairing with no more than 6 base pairs.

# *3.3 Computational Tools for Guide Activity Prediction*

Conventionally, computational methods for effcient and specifc guide designing can be classifed into three groups: (i) alignment-based methods; suitable guides are designed based only on the alignment, and retrieving on the genome by locating the PAM sequence; (ii) hypothesis-driven; guide activity is predicted by using empirically rules (GC content or nucleotide composition at position 20); (iii) machine and deep learning-based; guides are scored by using training models which consider several features [71, 98, 149].

The most reliable predictions come from the hypothesis-driven and learningbased methods [146, 150, 154] because they are driven by previously described features [149]. However, learning-based models are considered the current cutting edge for *in silico* guide effcacy prediction. The development of reliable models requires large datasets of guides and their respective cut performance determined experimentally. Many algorithms have been developed to design guide suitable for SpCas9 like Azimuth 2.0, CRISPRpred, TUSCAN, CRISPRscan, sgDesigner and many others reviewed in Konstantakos et al. [71]. Furthermore, some computational algorithms emerged also for Cpf1, deepCpf1 [66] and SaCas9 [106]. To date, many web-based bioinformatic platforms are available for guide design and estimation of on- and off-target activity that rely on one or more above-mentioned computational methods [43].

Considering the wide availability of software tools for guide design, choosing the most appropriate is challenging. There are several features which should be taken into consideration such as the kind of input that the program allows, type of nucleases they support, algorithms used to predict on- and off-target activity, availability of genome of interest. The latter is a crucial point as a large number of software tools do not operate with guide design for plants. The most used and recommended tools for plants are: CRISPOR, CRISPR-P, RGEN Cas designer, or CHOPCHOP [43]. Among them, CRISPOR is one of the most complete and reliable. It supports hundreds of species and tens of different nucleases, giving a wide coverage in terms of organisms and molecular tools. Also, it integrates multiple scoring models for sgRNA effciency prediction for SpCas9, in particular it uses CRISPRscan algorithm [103] and Azimuth 2.0 algorithm [35], considered a stateof-the-art model [71], but also deepCpf1 and Namj's models for Cpf1 and SaCas9, respectively. Furthermore, CRISPOR predicts CRISPR/Cas outcomes in terms of probability to obtain out-of-frame deletions based on the microhomology around the target site [7] or to obtain frameshift due to any type of insertion or deletions [24]. About the specifcity, CRISPOR includes scores, such as MIT [47] and CFD [35], for off-targeting prediction and gives support for off-sites identifcation in the genome and for primer designing.

Albeit machine learning-based guide design algorithms represent exceptionally useful tool, they have some limitations. Most of computational models have been built by using datasets regarding guide performances of SpCas9. With the exception of SaCas9 and Cpf1, other cas nucleases remain completely deprived of reliable models for an optimal guide design. Furthermore, it is important to emphasize that the effciency prediction reliability may depend on the cell type or on species [80], and that many algorithms have been developed with human or animal datasets. Therefore, it is not obvious that they work equally well for plants. Indeed, Naim et al. [105] examined the prediction performances of several on-line tools and they did not fnd a statistical correlation between software rankings and *in vivo* effectiveness measured in several plants. This suggests that current algorithms based on rules designed for guides in animals do not perform well for plants.

Consequently, further efforts are required to improve *in silico* guide design for plant genome editing. With this in mind, after a preliminary *in silico* evaluation of guide effciency by using the most suitable tools, it is advisable to experimentally test their performance before starting with a plant stable transformation.

About prime editing, in addition to usual features of sgRNAs which mediate the binding on the target sequence, other aspects must be evaluated during pegRNA design. In fact, it has two more components affecting the editing effciency, the RT template which guides the DNA repair and a PBS which anneals to the nicked target DNA strand [6]. It has been recommended that the length range should be between 9 and 15 nt for PBS and 10–15 for RT templates [68]. Moreover, Lin et al. [86] found that, in rice, the optimal melting temperature of PBS should be 30 °C.

Many computational tools have been developed for pegRNA design, Easy-Prime [78], PrimeDesign [48], pegFinder [26], PnB Designer [127], PINE-CONE [132], PE-Analyzer [53], peg-IT [4], PlantPegDesigner [61, 86]. Unfortunately, very few are suitable for plants. The most advanced one is PlantPegDesign [61, 86] which was developed on the basis of rice experimental data and takes into consideration a series of known parameters such as optimal Tm, the exclusion of the frst C in the 3′ extension of the RT template, length of RT, GC content of the PBS and the PE window. This tool is promising, but at the moment it has been used to predict effcient pegRNAs only in rice. More study would be necessary to investigate if PlantPegDesign algorithm parameters are suitable for other plants or if they need to be reevaluated and adjusted accordingly.

At present, the application of prime editing in plants presents signifcant challenges. Therefore, it would be appropriate to experimentally validate pegRNAs before starting stable transformation in order to save time and resources.

# **4 Experimental Approaches for CRISPR Reagent Validation**

Stable transformation and plant regeneration are tedious, time-consuming and cost intensive. Moreover, for certain species, especially crops, they still pose a challenge [1]. Having effcient CRISPR reagents, particularly guides, is a crucial aspect to realistically obtain edited plants. Since reliable computational software, based on plant specifc training datasets is lacking, the evaluation and selection of the most effcient guides should be carried out experimentally. Over time, a number of procedures have been proposed to validate the effectiveness of CRISPR reagents in plants (Fig. 6.1).

# *4.1 Endonuclease Cleavage* **In Vitro** *Assay*

An easy way to test guide effciency is through the endonuclease cleavage *in vitro* assay. It has been demonstrated that CRISPR reagents can successfully work *in vitro*, and protocols have been developed to produce and purify recombinant cas nucleases by heterologous expression. Additionally, guides can be prepared by *in vitro* transcription followed by ribonucleoprotein complex assembly and cleavage activity assays [3, 101]. This approach has been used to assess the effectiveness of the CRISPR system with the specifc guide [60, 65, 67, 94]. Such a system is very simple and rapid, and the availability of commercial purifed nucleases and *in vitro* transcription kits make even easier and attractive its application for guide evaluation. However, the reliability of this system is undermined by several aspects. The effciency of T7-mediated *in vitro* transcription can be affected by the nucleotide composition of the initially transcribed region [31]. Consequently, the altered amount of guides transcript can affect the effciency of the cleavage leading to potentially misleading results. Moreover, *in vitro* cleavage assay cannot simulate the *in vivo* expression level of nuclease and guide, which is one of the well-known factors affecting the mutagenesis rate. It has been shown that low concentrations of

**Fig. 6.1 Outline of CRISPR-mediated genome editing design experiments** (**a**) Schematic representation of the frst step in the CRISPR-mediated genome editing experiments, involving the use of bioinformatic tools to design specifc and effcient guides suitable for plant genomes. The selected guides meeting the desired specifcity and effciency criteria undergo experimental validation as shown in (**b**). (**b**) Experimental validation methods for the selected guides, including *in vitro* cleavage assay as a pre-screening system to reduce the number of guides for *in planta* validation. Depending on the species and desired genotyping depth, different assays such as agroinfltration, protoplasts, or hairy roots can be employed. (**c**) Analytical methods used for genotyping in genome editing experiments. Various techniques can be selected based on the specifc requirements of the experiment. (Image sources: Some images have been obtained from the freely available collection on the pixabay.com website)

SpCas9 reduce on-target cleavage activity [47]. In addition, the *in vitro* assay does not replicate the accessibility to the genomic target sequence and the *in vivo* biochemical conditions for the folding of guide secondary structures. Recently, Sagarbarria and Caraan [123] found a discrepancy between *in vitro* and *in vivo* evaluation of sgRNA effciency, as guides that appeared to be functional *in vitro* did not lead to successful mutagenesis in stable transformed plants of *S. melongena*.

Lastly, the *in vitro* approach, while can provide insights into cut effciency, has limitations in predicting the types of mutations induced by the repair systems after the DSB. It also does not offer information regarding the ability of Base or Prime editors to introduce the desired edits, nor does it provide any information on the risk of off-targets in the genome.

For these reasons, the *in vitro* approach cannot be considered truly predictive of mutagenesis effciency and should be taken into account only as pre-screening method. To better simulate physiological conditions and obtain reliable estimates of the mutagenesis rates, *in vivo* systems should be preferred.

# *4.2 Agroinfltration Assay*

Transient expression systems constitute a good compromise between ease and reliability for testing the effcacy of CRISPR reagents before proceeding with stable transformation. Acting directly on the genome, these systems provide more detailed and realistic information about the *in vivo* cleavage and mutagenesis effciency. Additionally, they offer the advantage of being rapid to execute, as they do not involve time-consuming tissue culture.

Among the *in vivo* approaches used with plants, agroinfltration is a rapid method that involves the use of special strains of *Agrobacterium tumefaciens*. These modifed bacteria, that have inserted the CRISPR machinery genes into the T-DNA, are infltrated into the intercellular space of plant tissues by syringe or vacuum infltration. This process leads to the transient expression of nuclease and guide. Agroinfltration can be performed in various parts of the plant, such as fruit [110], petal [52], whole seedling [75] and pollen [32]. However, the most commonly used organ for agroinfltration is the leaf, for which numerous of protocols have been optimized (reviewed in [63]) and high level of expression and high transformation effciency have been reached [130]. The main advantage of this technique is its rapidity, with high expression levels of transgenes typically achieved within a few days [63]. For this reason, transient agroinfltration has been widely used as a preliminary experiment to test the effectiveness of constructs expressing guides and cas nucleases for *in vivo* targeting of specifc genes through CRISPR-mediated genome editing.

In some cases, this system has been used as proof-of-concept study for assessing whether the CRISPR machinery was active in different species of interest [16, 59, 75, 107, 136], or for testing new cloning approaches to assemble CRISPR constructs [64, 141]. Moreover, several studies have reported the use of agroinfltration as a technique to experimentally validate constructs before proceeding with stable transformation. Baltes et al. [8] tested the activity of Cas9 and sgRNAs in *Nicotiana benthamiana* to target BeYDV replicons. Zhang et al. [155] used the agroinfltration to verify the mutagenesis effciency of hundreds of guides in tomato and *N. benthamiana* to edit 63 immunity associated genes.

Overall, the agroinfltration method has several limitations. The transformation effciency may depend on many factors, including the biological compatibility and tissue accessibility of the plant species (and genotypes) with *A. tumefaciens* [147]. For example, comparing some CRISPR constructs harboring guides which recognize the identical target sites in both species, Zhang et al. [155] found a much lower mutagenesis in tomato than *N. benthamiana,* concluding that agroinfltration system in tomato leaves can give misleading results underestimating the guides effciency.

To overcome the incompatibility, the recalcitrance or the inaccessibility which make agroinfltration problematic for some plant species, the guide effciency could be determined by co-transforming the CRISPR system (Cas9 and guide) with its target DNA in *N. benthamiana*. This method was developed by Khan et al. [64] as a proof of concept that an exogenous gene (YFP) transiently co-transformed in

*N. benthamiana* together with CRISPR apparatus can be successfully mutated. This system could allow to assess virtually any guide for its target DNA from any species.

Another limitation of agroinfltration is the inability to accurately estimate the mutagenesis effciency due to the presence of genomic DNA from non-transformed cells. This can introduce variability in the results, as there is no selection of cells that have integrated the T-DNA. In particular, Li et al. [75] showed that the mutagenesis effciency appeared to be higher when using protoplast transfection compared to foliar agroinfltration in both *A. thaliana* and *N. benthamiana*. They proposed that this difference might be attributed to a higher gene transfer effciency in protoplasts which leads to a different dilution ratio between transformed and untransformed genomic DNA.

# *4.3 Protoplast Assay*

Protoplasts, plant cells deprived of cell wall, are very useful biological and biotechnological tools for both basic and applied plant science [152]. They are mainly, but not exclusively, obtained from leaf through mannitol-mediated plasmolysis and exposure of mesophyll cells to cell-wall-digesting enzymes (macerozyme and cellulase). Due to the high transfection effciency and rapidity, protoplasts have become an excellent system to evaluate the effectiveness of CRISPR vectors in plants before attempting to transform an entire organism [152]. PEG-mediated protoplast transfection has been employed in a multitude of plant species to check the effcacy of designed CRISPR tools, not only in model plants like *Arabidopsis* [60], *N. benthamiana* [60] or *N. tabacum* [85], but also in several crops, including both dicotyledonous [88, 103] and monocotyledonous plants [15, 85].

Once the protocols for protoplast isolation and transfection are established, the protoplast platform to validate CRISPR constructs is simple, reliable, and not expensive. The yield of viable protoplasts is normally high, and this allows several tests to be carried out from the same preparation, e.g. to evaluate many CRISPR constructs or to examine different experimental conditions [9]. Moreover, protoplast transfection is suitable for both RNPs and plasmids, making it possible to assess not only the effectiveness of the cas-guide complex activity, but also different plasmid architectures, and to make a comparison between RNPs and plasmids. Jiang et al. [60] showed that RNPs are more effcient than plasmids, suggesting that, despite production and purifcation of cas nuclease and guides may be more challenging (or costly, if purchased) than extracting plasmid from bacteria, RNPs may offer better mutagenesis performance, providing a greater guarantee of successful editing of target genes. Another important feature of protoplast platform is its versatility. Many studies have focused on the evaluation of mutagenesis rate caused mainly by SpCas9 (and its variants or orthologs) and Cpf1 to give random mutations on target sites. Furthermore, it has been proven that protoplasts can be useful to detect also precise mutagenesis events, like those determined by HDR-mediated Gene-Targeting, Prime Editing [60] and Base Editing [39].

Although the plant protoplast platform is robust and widely applicable in multiple plant species, it does have certain limitations. Protoplasts, despite their high transfection effciency, require specifc expertise for their careful handling, from isolation to transfection. The transfection of protoplast generates thousands of independent events, which can be genotyped using various systems. For precise and detailed information about the mutations generated by the CRISPR machinery, amplicon sequencing by NGS is the preferred technology, but it requires specifc expertise that may not always be readily available, unless an external service is used. Furthermore, even with NGS technology, there is still some uncertainty regarding the exact mutation rate and composition. This is because it is not possible to differentiate between transfected and non-transfected protoplasts from the total DNA extracted, unless a transfection marker, such as a GFP reporter, is employed.

# *4.4 Hairy Root Assay*

A possible way to overcome the weaknesses of agroinfltration and protoplast transfection can be given by *Agrobacterium rhizogenes* mediated hairy roots generation.

The hairy root system is a rapid and convenient approach for obtaining stably transformed roots. It is based on the natural ability of *Agrobacterium rhizogenes* (*Rhizobium rhizogenes*) to infect injured parts of the plant, triggering root organogenesis from the wounded sites and giving origin to the well-known "hairy root disease". The combination of hairy root approach and CRISPR/Cas techniques represents an excellent platform for an easy, rapid, accurate and cost-effective evaluation of CRISPR reagents, with the added value of a possible functional analysis of genes of interest in roots.

Currently, in the literature there are several articles reporting the use of hairy root transformation to deliver CRISPR vectors in a multitude of plant species such as tomato [56, 81, 121], potato [19], cucumber [108], soybean ([20];), *Brassica napus* [57], peanut [126], papaya [45], *Populus* [139], rubber dandelion [54], *Brassica carinata* [69], *Salvia miltiorrhiza* [76], *Medicago truncatula* [99].

The main advantage of the hairy root assay counts on the fact that transformed roots can serve as a simulation of stable whole plant transformation. Each root can be considered an independent transformation event, and the transformation effciency is very high, as escapes can be avoided by antibiotic-mediated selection. These characteristics facilitate straightforward and rapid genotyping, enabling the calculation of the mutagenesis rate as the proportion of roots harboring at least one edited allele among the total number of transformed roots.

The drawbacks of this system include the need for some tissue culture procedures and a waiting time between the infection of the explant and formation of the hairy roots, which can range from 10 days to several weeks depending on the plant species. However, these drawbacks are offset by the simplicity and speed of genotyping using PCR followed by Sanger sequencing. The availability of commercial PCR kits with engineered DNA polymerase that are resistant to PCR inhibitors

present in plant extracts allows for direct genotyping of root fragments without the need for genomic DNA purifcation, using minimal amounts of plant material. This enables the amplifcation of numerous samples in a short period of time.

Many plant species, mostly dicotyledonous, are *A. rhizogenes* susceptible, consequently from these plants it is easy to obtain hairy roots transformed with T-DNA harboring CRISPR machinery. This characteristic makes the hairy root system a promising method of choice for evaluating guide effciency or new CRISPR systems in a rapid and reliable way. Furthermore, it has been shown that tomato hairy roots exhibit morphological similarity to normal adventitious roots, making them a valuable tool for studying the function of genes in this organ [121]. In cases where target genes are completely inactivated, hairy roots can provide insights into the potential phenotypic changes that may arise in roots after stable transformation with CRISPR constructs.

A typical hairy root protocol workfow, established in our lab to evaluate CRISPR approaches in tomato, is described in the Fig. 6.2. With some adaptations, this protocol can serve as a roadmap for designing and validating genome editing experiment in other plant species.

**Fig. 6.2 Workfow of CRISPR construct assembly and hairy root generation from tomato explants**. The workfow illustrates the steps involved in the generation of CRISPR constructs and the induction of hairy roots in tomato explants. First, computational tools are used to design the guides required for the CRISPR experiment. The guide expression cassette is then produced and assembled with the nuclease and antibiotic resistance expression cassettes. These components are combined to form the fnal constructs. The CRISPR constructs are cloned into *Agrobacterium rhizogenes* cells, which are used to infect cotyledon and/or leaf explants from 10–15 day old tomato plantlets. The infected explants undergo a co-cultivation process with *Agrobacterium rhizogenes* for 3 days. Subsequently, the explants are transferred to a selective medium and incubated. After approximately 2 weeks, the frst hairy roots start to develop and can be collected. The collected hairy roots are then subjected to direct genotyping using PCR followed by Sanger sequencing to analyze the desired genomic modifcations. (Image sources: Some images have been obtained from the freely available collection on the pixabay.com website)

# **5 Analytical Techniques to Estimate the Editing Effciency**

Genotyping is a pivotal step to assess the editing effciency of CRISPR constructs or RNPs. Effective screening methods are necessary before undertaking the timeconsuming transformation and regeneration procedures to validate the effcacy of genome-editing reagents (guide and nuclease) in order to increase the effciency of genome editing. For this purpose, many experimental methods and bioinformatic tools have been developed to detect and analyze indels generated after CRISPRmediated targeting.

The choice of analytical technique depends mainly on the type of information required in terms of mutagenesis occurrence, precise identifcation of indels and the accurate frequency of each type of mutation. Some editing approaches, such as Base Editing, Prime Editing and HDR-mediated knock-in, are not very effcient and the desired mutations may occur at very low frequencies. In these cases, the detection and quantifcation of induced mutations are very challenging and require specifc methods for large-scale mutation detection. Conversely, when gene inactivation with typical out-of-frame mutations is desired, frequencies are normally higher, partly because a wide range of indels can result in a knockout effect. In these cases, techniques that can provide qualitative (or semi-quantitative) information about mutagenesis occurrence are preferred. Such techniques should be simple and costeffective, should not require special equipment, and should enable the detection of a mutated allele in a background of wild-type alleles even when mutations are obtained at a very low frequency or in a complex polyploid plant genome.

Although sequencing will ultimately be required to confrm and identify the exact sequence of the mutant DNA, the availability of powerful high-throughput strategies during the screening stage can signifcantly reduce the cost involved in generating and identifying mutants.

A commonly used method for detecting modifed genes relies on the enzymatic cleavage of heteroduplexes formed, after melting and re-annealing, by hybridization of wild-type and mutated DNA strands or two differently mutated DNA strands. A bacteriophage resolvase, such as T7E1, or single-strand-specifc plant endonucleases, such as those of the CEL family (commercialized under the brand Surveyor) are used to recognize and digest unpaired heteroduplex DNA independently of the sequence. Therefore, they are suitable for screening of any target sequence [142]. Enzymatic digestion of heteroduplexes has been utilized, for example, by Cai et al. [20] to test target site editing effciency in soybean hairy roots, by Khan et al. [64] for sgRNA effciency testing in infltrated leaves of *N. benthamiana*, and by Brandt et al. [15] to optimize wheat protoplasts transformation with CRISPR-Cas ribonucleoprotein complexes. A side-by-side comparison has shown that T7E1 identifes preferentially insertions and deletions, whereas Surveyor has better sensitivity to recognize substitutions [142]. The T7E1/Surveyor assays are reproducible, inexpensive, and easy to use, but are unable to differentiate between identically mutated (homozygous mutants) and wild-type alleles, or between biallelic mutants and heterozygous monoallelic mutants. Therefore, additional testing is necessary to detect biallelic mutations, and homozygous mutant clones may be discarded as falsely reported wild type. Furthermore, the T7EI and Surveyor nuclease assays do not provide information about the type of induced mutation or the number of indels, making it impossible to exclude indels of (multiples of) three nucleotides. In addition, in polyploid species, false positive signals can arise from formation of heteroduplex between non-identical paralogs. In some cases, a heteroduplex mobility assay (HMA) using PAGE has been used to analyze heteroduplex formation instead of the enzymatic digestion, as reported by Hoang et al. [45] and Nguyen et al. [108] to test the effciency of genome editing in hairy roots of papaya and cucumber, respectively.

Another classic method used for mutation detection is polymerase chain reaction (PCR)/restriction enzyme (RE) assay. If a restriction site is present in the target locus, the region around it can be amplifed by PCR and run on an agarose gel after digestion with the RE to display the digestion pattern. When a mutation disrupts the restriction site, the amplifed fragment remains uncut and appears as a single undigested band. Although it is straightforward and accessible to most laboratories, the PCR/RE method is heavily dependent on the availability of a restriction enzyme site near the target sequence, which is already constrained of a PAM for the nuclease at the cleavage site. Furthermore, each target sequence requires a specifc set-up that hinders the general optimization of the protocol. Nevertheless, this approach is widely used for rapid and inexpensive screening of mutagenesis events, especially when optimizing protocols in less explored species such as in wild diploid potato relative [19] and Taraxacum [54] hairy roots, in tobacco and maize protoplasts [85], soybean protoplasts and hairy roots [135] or in agroinfltrated *N. benthamiana* leaves [141].

Recently, a method based on PCR followed by digestion with purifed ribonucleoprotein complexes of SpCas9 or FnCpf1 (known as PCR/RNP method) was reported to detect nuclease induced mutations in both polyploid and diploid plants [84]. This method is more applicable than PCR/RE as the CRISPR nucleases RNP will digest PCR products identical to the guide (wild type) but fail to digest PCR products with mutated sequences (mutants) without the need for the presence of the additional restriction site. According to the authors, the PCR/RNP method is less effective in detecting SNPs than indels, but the latter can still be distinguished from the wild-type sequence. In addition, the PCR/RNP method appears to be superior to the T7EI assay in terms of accuracy, and to Sanger sequencing in terms of sensitivity [84]. The main drawback of this screening strategies is that it requires purifed *in vitro* transcribed guides and purifed nucleases to preassemble CRISPR ribonucleoprotein (RNP) complexes.

To overcome the limitations of the PCR/RE method, several other PCR-based protocols have been developed, such as annealing at critical temperature PCR (ACT-PCR) [49], double-strand break site-targeted PCR (DST-PCR) [55], and bindel-PCR [124]. In addition, real-time PCR [114] and droplet digital PCR [115] are interesting alternatives to conventional PCR methods because they do not require post-PCR product manipulation. They are fast, high-throughput, and reduce the risk of laboratory contamination. Both protocols require the design of two differently labelled probes recognizing different parts of the same amplicons. The probe designed outside the expected mutation position will bind to all alleles, allowing the assessment of the total amount of amplicons present in the sample, while the probe designed on the gene-editing target site will only bind to the wild-type sequence, thus revealing the presence of a new mutation. The ratio of the relative amplifcation values of the two probes in qPCR or the ratio of mutant droplets (positive for one fuorophore) to wild-type droplets (positive for a double fuorophore) in dPCR can then be used to distinguish wild-type, homozygous, and heterozygous mutations and quantify the mutation frequency of gene editing. qPCR and dPCR can detect single nucleotide indels or single nucleotide mutations with high sensitivity, especially qPCR. The cost of the labelled probes, the inability to detect large deletions in homozygous samples, and the need for direct sequencing to determine the exact mutated sequence are the major drawbacks of these two protocols.

Fluorescent PCR-capillary gel electrophoresis/DNA fragment analysis [119] and high-resolution melting curve analysis (HRM) [138] are also PCR based approaches that are successfully employed for genotyping genome editing events. Fluorescent PCR-capillary gel electrophoresis/DNA fragment analysis, of which several variants are available, employs fuorophore-labelled primers to amplify the genomic region containing the expected edited site. The labelled amplicons are then separated by capillary electrophoresis, and sized by comparison to an internal standard mixed with the sample before proceeding with electrophoresis. Data analysis guided by software identifes mutants based on the shifts in fragment size compared to the wild-type fragment. Recently, Carlsen et al. [21] successfully employed a specifc fragment analysis strategy called Indel Detection by Amplicon Analysis (IDAA) to evaluate editing effciency in tetraploid potato protoplasts in a study aimed at improving guide effciency design [21]. Additionally, High Resolution Fragment Analysis (HRFA) described by Andersson et al. [5] has been utilized to test mutation effciency during genome editing in tomato protoplasts [89], and the editing and regeneration protocol in rapeseed protoplasts [79]. Fragment analysis effciently and accurately detects the number of nucleotides inserted or deleted at the target site, but is not accurate in detecting indels larger than 30 base pairs, and does not detect base substitutions or single nucleotide polymorphisms (SNPs). By providing information about the size and the relative abundance of different amplicons, fragment analysis allows detection of different alleles, homo- and heterozygosity, chimerism, sample mixtures, and genome editing effciency. In addition, it offers a high sensitivity and resolution, being able to discriminate fragments that differ by a single base pair, and is a fast, high-throughput, automatable and multiplexable. On the other hand, it requires specialized equipment and software that are not commonly available and accessible to all laboratories. This also applies for another easy and fast techniques called High Resolution Melting (HRM), that combines PCR and heteroduplex formation. By increasing the temperature after PCR of small amplicons (about 100 bp) containing the target region, a specifc and different dissociation profle is observed for homoduplex and heteroduplex of double stranded DNA fuorescently labelled. Subsequently, the Tm and the characteristic different signatures of PCR products are used by dedicated software to identify the presence of

mutant alleles without the need of additional manipulations after PCR. The main advantage of HRM protocol is that it can distinguish individual mutant alleles within a complex background, allowing detection of even rare mutation and chimerism as low as 5%. On the other hand, in addition to the cost of the equipment, a limitation of HRM is that it cannot detect large deletions due to the small size of the amplicons.

Certainly, Sanger sequencing of PCR products is the most direct and defnitive approach to obtain detailed information about the type(s) of mutation in a sample, and is used in almost all studies, both to directly analyze amplifed target regions and to accurately determine the mutated sequence after screening with one of the aforementioned methods. Although highly informative, sequencing methods are costly for high-throughput genotyping and require bioinformatic skills to analyze the data. In fact, chromatograms of PCR amplicons derived from complex samples (heterozygotes, biallelic mutants, chimera, polyploid species) can contain multiple traces, and several online bioinformatics tools, such as TIDE [17], DSDecode [87], and ICE [29], have been developed to decode the underlying mutation types.

Finally, thanks to its high sensitivity (0.01%), deep sequencing of the amplicons using NGS-based methods represents the gold standard for mutation detection, especially when targeted mutagenesis frequencies are low and rare editing events must be detected among a high background of unmodifed alleles. The genomic region around the target site is amplifed with a proofreading DNA polymerase, and the PCR products are barcoded (to distinguish the reads from independent amplifcations of the target site) and indexed (to enable library multiplexing in the fow cell) to allow high-throughput sequencing. The raw data are then analyzed to calculate mutation effciency as the percentage of reads containing indels in a defned window around the cleavage site, with the help of specifc bioinformatic pipelines such as Cas-Analyzer [113], CRISPResso [116] or CRISPAltRations [72]. Several examples of deep sequencing of amplicons for assessing editing effciency can be found in the literature, specifcally in studies involving protoplast assay [9, 39, 60] or agroinfltration assay [8, 96].

Unfortunately, due to the high costs and the need for specialized skills and bioinformatic tools, NGS methods are not widely applicable for the initial screening of edited lines.

# **6 Summary and Conclusions**

CRISPR/Cas technology is considered the cutting-edge tool for genome editing for both fundamental and applied research in plants. It relies on a wide repertoire of molecular tools, including nucleases with the ability to target a large spectrum of PAM sequences, as well as precise editing approaches such as Base Editing and Prime Editing. This versatility makes CRISPR/Cas an invaluable technology. However, the challenge lies in obtaining transformed plants that can effectively express the editing machinery, as this step remains challenging for many plant species. Without verifying the actual effcacy of the selected CRISPR reagents, undertaking a stable plant transformation can be demanding and risky using current transformation and regeneration protocols. Therefore, knowing the actual editing frequencies is crucial in selecting the most effcient guides and planning the editing experiment accordingly.

The development of computational tools enormously facilitates the selection of putative editing sites on target genes by predicting the most specifc and effcient guides. The development of these computational models requires the use of large datasets regarding the performance of the experimentally determined guide. To date, computational models are not available for all editing approaches, especially for new nucleases, and it will be diffcult to keep up with the continuous emergence of new molecular tools. Furthermore, bioinformatic tools built by using plantspecifc datasets are almost completely lacking. For this reason, it is essential to have rapid systems capable of evaluating the performance of CRISPR reagents *in planta*. This allows for screening different guides and choosing the most effective one, as well as testing new editing systems quickly.

The *in vitro* test is extremely simple, but has many limitations, so it can only be considered as a pre-screening system, while the *in vivo* tests, such as agroinfltration, protoplasts and hairy roots are to be preferred. The choice of the *in vivo* system depends mostly on the species and the desired type of genotypic data, which in turn relies on the analytical method used to determine the editing rate. The disadvantage of agroinfltration and protoplasts is that they require NGS for accurate genotyping.

Among the *in vivo* platforms, the hairy roots represent a promising tool to test the effcacy of CRISPR reagents in *A. rhizogenes*-susceptible plants. This system combines the advantage of stable transformation with the rapid analysis of transient systems, allowing for accurate and rapid genotyping using PCR-based methods, and potentially Sanger sequencing, without the need for regenerating entire plants.

**Acknowledgements** The writing of this manuscript was supported by BIOTECH-Cisget project from the Italian Ministry of Agriculture (MiPAAF D.M. 15924) and SMART-BREED project (A0375E0166, POR FESR LAZIO 2014 – 2020), it was carried out within the Agritech National Research Center and received funding from the European Union Next-GenerationEU (PIANO NAZIONALE DI RIPRESA E RESILIENZA (PNRR) – MISSIONE 4 COMPONENTE 2, INVESTIMENTO 1.4 – D.D. 1032 17/06/2022, CN00000022). This manuscript refects only the authors' views and opinions, neither the European Union nor the European Commission can be considered responsible for them.

# **References**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

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# **Part II Applications of Genome Editing**

# **Chapter 7 Genome Editing of a Macroalgae with Possible Global Impacts**

**Hilde-Gunn Opsahl-Sorteberg and Espen Evju**

**Abstract** Kelp forests are major marine ecosystems and key sources of biodiversity comparable to tropical forests, as pointed out by Darwin on the Beagle in 1834: *"Yet if in any country a forest was destroyed, I do not believe nearly so many species of animals would perish as would here, from the destruction of the kelp"*. Despite the key roles supporting marine life, our understanding of their biology lags far behind that of land plants. Kelp mitigates the effects of climate change, sequesters CO2, reduces eutrophication while providing biomass for food, feed, and materials. Genome editing together with functional genomics can map genetic diversity potentials for temperature tolerance, important since they already face the upper tolerance limits in some regions. This chapter considers the major genome editing prerequisites; the transformation methods for introducing DNA/RNA and annotated genomes for predicting results. Risk assessments are discussed. These uses of genome editing show how widely applicable the techniques can be used from basic science to securing the global environment for our existence.

# **1 Genome Editing Prerequisites**

Disappearance of species due to climate change is causing catastrophic changes to **biodiversity** within our ecosystems, affecting environmental and human health including threatening our survival. Policy makers recognize the need for actions to halt these losses and prevent extreme weather catastrophes, hopefully acting, leading to increased **science**-based activities **to secure our globe's future**. Species loss threatens human survival and quality of life directly by losing space, food/feed, and water resources and indirectly by reducing our health. Actions to halt or reverse planetary warming must involve policy leaders from across the globe and across the globe's environments. In the marine environment, **kelp forests** are highly relevant environments because they represent essential ecosystems supporting high levels of

H.-G. Opsahl-Sorteberg (\*) · E. Evju

Faculty of Biosciences, Norwegian University of Life Sciences (NMBU), Ås, Norway e-mail: hilde-gunn.sorteberg@nmbu.no

biodiversity that cover extensive regions of our coastlines. Kelp species have many uses from **harvested wild** habitats and **aquaculture** production globally, where sugar kelp is **facing extinction** from the warmer ranges of its habitat and arctic populations are challenged by melting ice caps and reduced sea salt content [1, 2]. Losses of kelp forests affects **whole ecosystems** because these environments are habitats for numerous species ranging from microorganisms through photosynthetic plants to a range of marine animals including mammals. Kelp forest disappearance would also be disastrous for the global ecosystem because kelp is highly effcient at sequestering CO2. **Newly available technologies** provide tools to evaluate genetic diversity and resilience. Uses of kelp have a long history, however fundamental and applied research are underfnanced, and progress lags behind other felds of plant and animal sciences. This situation is improving with the advent of increasing numbers of annotated genome sequences for kelp and the development of associated genomics-enabled tools including **genome editing**. The **aquaculture potential** of kelps in the blue economy should ideally be kept in balance with and isolated from wild populations [3, 4]. In addition, cultivation of local strains as material for rewilding needs to be considered. However, Europe is still missing clear guidelines for macroalgae management ranging from harvesting wild populations, cultivation systems, and breeding strategies and methods including genome editing. The Phycomorph COST action produced a report [5] providing a background for the development of guidelines for good practice in macroalgae exploitation. This chapter presents an updated overview of the **potential of applying genome editing** to secure future benefts from **macroalgae for global health** in the face of climate change.

The CRISPR/Cas9 system, discovered from the adaptive immune system of bacteria, has revolutionized the feld of genome editing by providing a simple, effcient, and precise method for targeted gene modifcation [6].

Genome editing for research and applied purposes still depends on (1) adapted **gene transfer methods** and (2) **annotated genomes** for effcient and safe predictable uses. To secure reliable results and predictable outcomes, independent of positional effects and additional genome variation caused by somaclonal variation due to time in vitro casing mutations, robust gene transfer methods with minimal time in vitro are paramount [7]. This gene transfer is still species dependent in plants. The gene transfer methods are mostly limited to two; particle bombardment and agrobacterial T-DNA transfer. The number, accuracy and annotation quality of plant genome sequences are rapidly increasing since the Arabidopsis genome was released in 2000 [8]. Since the techniques for sequencing whole genomes have improved massively this prerequisite is mostly met both regarding directing the location of gene transfer when using CRISPR/Cas9 and the means to do predictable high quality risk assessment of well characterized modifed land-plants. However, some species are lagging behind such as macroalgae where the molecular characterization is just emerging with promising insights to basic biological understanding and correlated applied opportunities [9].

# *1.1 Genetic Transformation Methods*

Gene transfer was frst achieved by a range of techniques in the 1970s and 1980s [10]. The frst GM bacteria were produced in 1973 [11], human insulin production from GM bacteria was achieved in 1978 and FDA approved 1982 and in 1994 the frst GM crop plant was approved (see e.g. ISAAA statistics: https://www.isaaa.org/ resources/publications/briefs/05/download/isaaa-brief-05-1997.pdf). Since then, transgenics and resulting plants have been established for most species including many economically important crops, generating over 30 GM crop plants which are approved in over 40 countries including the USA, Canada, Argentina, EU, Norway, UK and Switzerland covering over 190 million hectares.

The two preferred gene transfer routes are still agrobacterium transfection if it is adapted to the plant species and particle bombardment mostly when agrobacterium cannot be applied. Agrobacterium is preferred since it is a "cleaner" method transferring less transgene copies, and into more appropriate genome locations causing less unintended effects. This could be because of natural selection pressure favoring successful integration for Agrobacterium [12]. *Agrobacterium tumefaciens* is a plant pathogenic bacterium commonly found in soil, which has a long history of transferring a portion of its Ti-plasmid, the T-DNA segment into host genomes for infection and survival. In nature the T-DNA allow the introduction of exogenous genes and subsequent expression of the corresponding proteins [13]. This has been adapted for research and gene transfer purposes, by replacing the agrobacterium tumor causing T-DNA and introducing any DNA of interest. The cloned DNA in the T-DNA region is placed into a binary vector before being introduced to the *Agrobacterium* cells. As the plant tissue becomes infected, the T-DNA integrates itself into the host's genome, facilitating the stable expression of new genes [14].

Tobacco and tomato easily incorporate added DNA, while crop plants like cereals took decades of research before predictable transformation frequencies and reproducible results were achieved. Frequencies of regenerated transgenic plants have recently improved considerably for such recalcitrant species. This was partly due to an improved understanding of the cell cycles achieved by the introduction of related meristem developmental regulatory genes. Adding a growth regulating factor and a cofactor, greatly increased regeneration frequencies in wheat, triticale, rice and citrus [15]. CRISPR/Cas9 modifcations of plant stem cells and the foral meristems have shown how the cell cycle and stem cells are related to growth and biomass production as well as yield from fowers, fruits and/or seeds [16–19]. As also seen in medical science, stem cell research is central to both basic and applied science, and plant breeding to meet sustainability goals for food production [20]. Further improvement by directing integration or integrating transgene constructs by homologous recombination by CRISPR/Cas9 has greatly improved predictability of targeted genome knock-out or transgene integration, while simultaneously reducing off-target effects by avoiding multiple inserted copies that are common when using particle bombardment for transgenesis.

Risk assessments and approval for commercialization of transgenic crops require characterization of the transgene integration, to secure single copy integration and detect possible unintended positional effects. This minimizes the risk of silencing the transgene as well as other parts of the genome. Somaclonal variation due to mutant effects of the transformation or time in tissue culture, has led to technological improvements that minimize time in culture, reducing unintended effects. Stable transfer depends on integration of transgenes into chromosomes. Some methods such as those to produce maximal amounts of transgene products, might have transgenes transiently active without nuclear integration or integration in organelles such as plastids.

# *1.2 Annotated Genomes*

Genome sequences and analyses keep revolutionizing our understanding of basic biology such as stem cell functions and cell cycle implications, phylogeny, and evolution, providing a robust basis for deeper understanding of basic biological relationships and applications [21, 22]. This is because sequencing techniques have become increasingly accurate, effcient, and accessible. Also, shot-gun sequencing combined with long sequence reads provides reading through long repetitive sequences, such as centromeres, making it possible to identify unique regions for CRISPR/Cas9 specifc alterations within any targeted genome part. This allows mapping the unknown parts of genomes, changing them from what some still refer as "junk-DNA" to informed molecular understanding [23]. Updated knowledge shows most of the genome is being transcribed to variable sized RNA molecules involved in gene regulation by RNAi like mechanisms [24, 25].

Specifcally, such genomic methods are promising to fll the gaps in our understanding of microtubule directed cell cycle control, division planes, meristem functions and growth. The information generated is of great importance to medicine, food production and global health through allowing informed decisions for optimal sustainable uses of resources. Additionally, genome editing depends on well annotated genomes for risk assessments and applications. Thus, genome sequencing currently developed for new species allows adapting the CRISPR/Cas9 system in macroalgae. The other main limiting factor to applying genome editing to macroalgae are the gene transfer methods. Currently gene transfer is still only established for the green algae *Ulva* and the model brown algae *Ectocarpus*. This is expected to be extended to more species shortly like *S. latissima* from current efforts in Europe and Japan [9, 26].

# *1.3 Macroalgae Cultivation and Breeding*

Macroalgae vegetate coastal and inshore areas and have been exploited by humans since prehistoric times. They are also important for providing a range of ecosystem services. Research is lagging behind land plants possibly partly because they are positioned between land-based and fshery interests that are mainly further out at sea. They additionally compete with fsh aquaculture e.g., the salmon industry, which often has higher fnancial signifcance in sea water areas. To secure native macroalgae populations from non-sustainable exploitation, some technology-based aquaculture production is developed for the dominant species such as the brown algae *S japonica* in the Eastern coastal regions of China, Japan, and Australia while *S latissima* occurs in the Atlantic cooler sea regions. Challenges for the exploitation of GM and GE algae are that sea regions interconnect and so cannot be easily contained as land plants. This means that regulations need to be established and agreed internationally.

Genome sequences are important to map genetic potential to survive strong selection pressures. Genome editing would be the best method to do both functional studies linking genes to temperature tolerance, and to enable breeding to meet requirements for sustainable algal strains that meet human needs.

The European Commission is responsible for EU's joint marine resources, but no regulatory frameworks and laws for macroalgae exploitation have been established at neither national nor European level [27]. The Phycomorph EU COST action FA1406 (2015–2019) drafted an extensive 200 page guideline "Pegasus" on sustainable aquaculture of seaweeds [5]. The impact of this on the EU Commission is unknown. An issue raised in several European countries is whether breeding can be applied to macroalgae being cultivated, where current cultivation of seaweed is largely carried out by local populations. Breeding would result in pre-cultivated gametophytes intercrossed in laboratory facilities to generate sporophyte "seedlings" for sea-based aquaculture cultivation of novel strains. So far only minimal breeding has been exploited, by crossing selected parental strains in *S. japonica*, and hardly any for *S. latissima* yet. Breeding might be needed to adapt sugar kelp for survival in warming sea locations; since temperature rise appears to be the main reason that sugar kelp populations have decreased.

# **2 Genome Editing – Important to Global Health for Mapping and Increasing Biodiversity to Survive Increasing Temperatures**

Brown algae (*Phaeophyceae*) are ubiquitous, covering app. 25% of the world's coastlines and about half of them are in the Laminariales and Fucales. Kelps are effcient CO2 sequesters and very effcient energy producers since they do not require terrestrial space, fresh water or added fertilizer. They therefore represent an important resource in the effort to mitigate the effects of global warming. In addition, kelps are effective purifying systems, removing organic pollutants from marine waters and they can also serve as monitors of the impact of climate change, particularly increased temperatures. Kelp growth is controlled by carbon allocation, primarily infuenced by light, temperature, nutrient availability, and their genomic competence. The growth of the endemic Brazilian deep kelp *L. abyssalis*, for example, is limited to the Austral summers while they tend to decrease in the Austral winter [1]. Consequently, little is known regarding the temporal variations in density and biomass of standing stock (individuals per m2 ) for harvesting, commercial applications, or conservation of these species and, consequently, protecting the environment they inhabit. This is addressed in an ongoing Biodiversa+ project with 11 European and 2 Brazilian partners with some additional French and Norwegian associated partners in RESTORESEAS (https://www.restoreseas.net).

Generally, organisms can respond to changes in the environment by acclimation (phenotypic plasticity), or evolution (local adaptation). Local adaptation requires heritable genetic variation for traits that increase tolerance to the new environment, therefore, mapping the genetic variation for temperature tolerance is important to predict effects of rising seawater temperature and to possibly take actions to meet expected future sea water temperatures. Temperature tolerance in kelps has been shown to vary and is linked to genetic variation in the northern hemisphere for *Saccharina latissima* [28, 29] and *Laminaria digitata* [30]. For the cold-water kelps and the Laminaria pockets by the Brazilian and Moroccan coasts, the populations are declining and expected to disappear if they cannot adapt to warming sea regions. Comparative crosses within and between an arctic (Spitsbergen) and a temperate North Sea (Helgoland) population have shown heterosis effects positively affecting increased temperature tolerance in both populations, probably caused by increased heterozygosity. This even occurs if the introduced new alleles are from the northern population with less high temperature tolerance [30]. Previously genome sequencing in *Saccharina japonica* showed that the genetic variation within cultivated types were low compared to the genetic variation in the wild, since all cultivated populations descended from a few collected individuals from the wild [31]. This demonstrates that it is important to generate whole genome mapping/understanding to get accurate understanding of the genetic potential, also to avoid possible inbreeding depression in cultivated macroalgae due to accumulated reduced levels of genetic variation.

Pan genomes help anchoring newly sequenced individuals or closely related species as deeply sequenced and well annotated new accessions are generated, in addition to new genotypes from which to utilize the application of new tools including genome editing. Genome editing can facilitate fundamental functional studies to unravel seaweed biology from single genes to genomes and systems biology, including the kelps' coexisting biota. Annotated genomes will give us better mapping of genetic variation for important traits for survival of wild populations, as well as the productivity of cultivated populations. Mapping genetic variation for elevated temperature tolerance and variation in iodine content would build a solid foundation for selective breeding. The importance of understanding biodiversity was demonstrated when whole genomes of wild and cultivated *S. japonica* were re-sequenced in China, since that revealed existing genetic variation and the potentials from the populations [31].

A high quality annotated brown algae genome sequence is available for *Ectocarpus* (https://bioinformatics.psb.ugent.be/orcae/overview/EctsiV2). A chromosome-scale assembly of the *S. latissima* genome generated by the French Genomique large-scale sequencing project Phaeoexplorer (https://phaeoexplorer.sb-roscoff.fr/home/) is expected to be released in early 2024 while the *L. ochroleuca* genome is currently being sequenced by the Restoresea project. This will add to the two available genomes: *S. japonica* and *Undaria pinnatifda* [32, 33], for interspecifc comparisons. Genes that potentially play key roles in determining resilience to climate change, particularly climate warming, can be identifed by combining results of the transcriptomic analyses with geographically correlated polymorphism information from the genomic analysis of population structure. Genes differentially regulated under temperature stress compared with control conditions and that show geographically correlated patterns of polymorphism, can be functionally assessed by CRISPR/Cas9 to select genes that are correlated with temperature tolerance. This approach may allow functionally annotate genes linked to resilience to temperature stress, with applied applications for breeding and survival in increasingly warmer seas [30].

# **3 Future Perspectives on Macroalgae Socioeconomics: From Ecology to Ecosystem Services**

Genome editing is a powerful tool to meet important challenges in plant improvement that we face. GE allows **modelling** to predict the effects of temperature tolerance in wide crosses of kelp populations, to assess the effects of macroalgal growth on ecosystem dynamics [34, 35] and determine the potential for carbon sequestration under different climate scenarios [36]. The impact of climate change can be assessed based on no intervention on current kelp populations, a 2 °C temperature rise and the effects of above 2 °C increase in sea temperature [34, 37].

Growth as an integrative parameter of all physiological processes is controlled by carbon allocation that is primarily infuenced by light, temperature, and nutrient availability, and their seasonal variations and interaction. Generally, most Northern Atlantic kelp species exhibit rapid growth from mid-winter to spring or early summer [38]. The pressure from anthropogenic activities on marine ecosystems have been driving severe changes in kelp distribution and abundance globally [39]. The decline in kelps forests can additionally affect a wide range of ecosystem services which are vital to human well-being (e.g., recreational and commercial fsheries activities). Examples of indirect infuence on human well-being are: habitat provision for marine species, primary production, climate control, carbon storage, nutrient fltering and coastline protection [40].

Marine macroalgae (seaweed) net primary productivity (NPP) is of major ecological importance in the global carbon balance. Seaweeds form the largest and most productive underwater vegetated habitat on Earth, comparable to the terrestrial Amazon rain forest [36]. Global NPP datasets for 246 seaweed taxa from 429 individual sites distributed on all continents, from the intertidal to 55 m depth, underpin our increasing understanding of the importance of our ocean forests ecosystem services. Their ecological contribution to annual aerial carbon production as well as to carbon production volumes are estimated to global averages of 656 and 1711 gC m-2 year-1 in the subtidal and intertidal regions [41]. More than half of the macro algae species are brown algae; mainly Laminariales and Fucales. Brown algae (Phaeophyceae) are ubiquitous, dominating app. 25% of the world's coastlines and representing the major foundation of temperate coastal ecosystems, conservatively estimated to amount to an ecosystem value of \$500,000–\$1,000,000 per year per km of coastline [42]. The brown algae provide ecosystem services indirectly by increasing coastal production and habitat provision, and directly as fuel, feed, food and specialized products [43]. Well annotated genomes, functional genomics linking gene sequences with functions and application of this in breeding might be crucial for future survival of many macroalgae species and populations. GE is our best tool to answer fundamental questions about their biology, predict possibilities and generate solutions for their many services to the globe's ecology, climate, and more direct human interests.

# **References**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

### **Espen Evju and Hilde-Gunn Opsahl-Sorteberg**

**Abstract** Plant-based antigen manufacturing procedures have transformed vaccine research and industry by offering a cost-effective, scalable, and safe alternative to traditional protein production systems. This chapter discusses genome editing applications for plant-based protein production systems, antigen, and antibody manufacturing, as well as their future and current developments. The chapter briey summarizes the several advantages of plant-based protein manufacturing platforms, including lower production costs, faster response to developing risks, and the absence of animal-derived components, which contributes to a lower risk of contamination and allergic responses. The chapter provides a basic overview of recent advances in plant-based antigen production, with a focus on vaccine antigens generated from CRISPR/Cas9 genome edited *Nicotiana benthamiana* to improve immunogenicity by altering plant glycosylation patterns to be more compatible with human glycosylation. Solving this could revolutionize existing vaccine production from plants to meet sustainable production objectives while also beneting human health. These applications of genome editing demonstrate how versatile the approaches may be, from basic science to improving human health.

E. Evju (\*) · H.-G. Opsahl-Sorteberg

Faculty of Biosciences, Norwegian University of Life Sciences (NMBU), Ås, Norway e-mail: espen.evju@nmbu.no

# **1 Production of Plant-Based Therapeutic Antigens and Antibodies**

The growing demand for recombinant proteins, particularly for therapeutic uses, drives the development of alternative effcient and cost-effective production systems. Current widely used platforms for recombinant protein production include bacterial, yeast, insect, and mammalian cells. These traditional systems are limited by high production costs, human pathogen contamination, improper protein folding, and glycosylation patterns [1].

# *1.1 Background to Plant-Based Protein Production Systems*

Plant-based expression systems exhibit increasing interest due to their innate ability to generate complex proteins with precise folding patterns and post-translational modifcations, and their potential for scalability and reduced risk of pathogenic contamination [2]. In 2012, the United States Food and Drug Administration (FDA) approved taliglucerase alfa, a therapeutic protein produced in plants, a groundbreaking milestone for plant-based protein production [3]. Numerous other biopharmaceuticals have been generated since and been through rigorous clinical evaluations, exhibiting the practicality and viability of plant-based protein manufacturing platforms [4]. Plant-based production can be based on transient, chloroplast, or stable nuclear expression [5]. **Transient** expression systems involve the temporary activity from transferred genes of interest into plant cells' cytoplasm, typically via Agrobacterium-mediated infltration or viral vectors [6]. This approach enables rapid protein production and has been used to produce a wide range of proteins, including monoclonal antibodies, vaccines, enzymes, and therapeutic proteins, such as human serum albumin and human somatotropin, in crops like rice and tobacco [7]. **Stable** transformation systems involve the integration of the target gene into the plant's nuclear or chloroplast genomes, generating transgenic plants that express the protein of interest through their life span and following generations if transferred to offspring by classical cross-breeding [8].

# *1.2 The Use of* **Nicotiana benthamiana** *in Transient Expression Systems*

*Nicotiana benthamiana*, a close relative of tobacco (*Nicotiana tabacum*), is a diploid herbaceous plant native to Australia, which can grow through 6–8 weeks from seed to harvest of vegetative parts. The small genome size, approximately 3 Gb, facilitates genetic modifcation, while its high rate of inbreeding promotes genetic uniformity [9]. *Nicotiana benthamiana* is there for one preferred model plant for

**Fig. 8.1** An overview of the steps to produce proteins in a transient plant-based expression system

plant-based protein production due to its high transformation frequencies, rapid growth, high biomass yield, and "accepting" immune system, making it susceptible to many common biotechnology tools [10].

*N. benthamiana* has been widely employed for the expression of a diverse range of recombinant proteins, including antibodies, enzymes, and vaccine antigens. The transient expression system further allows for rapid production and evaluation of protein candidates, which is particularly valuable to respond to emerging infectious diseases, such as the production of ZMapp, a monoclonal antibody mix against Ebola virus [11], and generation of vaccine antigens in response to infuenza viruses [12] (Fig. 8.1).

# **2 Antigen/Antibody Production for Sustainable Health Solutions**

The *N. benthamiana* was decoded and compiled in 2012, offering an essential tool for exploring the functional genomics of *N. benthamiana* [13]. The primary assembly, Nb-1.0, spanned 3.1 Gb and comprised 46,220 anticipated protein-encoding genes. In 2023, a de novo whole-genome assembly was carried out in *N.*  *benthamiana* using Hif reads, resulting in 1668 contigs with a combined length of 3.1 Gb [14]. The 21 lengthiest scaffolds, considered pseudomolecules, held a 2.8- Gb sequence, covering 95.6% of the assembled genome. A sum of 57,583 gene sequences with high confdence was anticipated.

The functional annotation of the *N. benthamiana* genome is a process that predicts gene functions by comparing them to known genes from other species and assessing experimental data. This method is vital for determining gene functionality and control, as well as aiding in pinpointing potential genome editing targets. Various tools have been established for the functional annotation of the *N. benthamiana* genome, such as the Sol Genomics Network (SGN) [15]. These repositories grant researchers access to genomic data, gene annotations, and functional projections, thus laying the groundwork for experimental inquiries guided by hypotheses and applications in biotechnology.

# *2.1 The Role of Glycosylation in Protein Function and Stability*

Glycosylation is a post-translational modifcation of macromolecules, such as proteins and lipids, that involves the addition of carbohydrates to the fnal molecule. This modifcation affects the stability, activity, and immunogenicity of the molecule, and is particularly important in the production of recombinant proteins [16].

Despite the many benefts of plant-based protein manufacturing systems, the glycosylation patterns of plant and human proteins differ, which is a considerable challenge for plant-based production systems [17]. Protein folding, stability, and function depend on glycosylation, while such glycosylation patterns can affect plant-produced protein effcacy and immunogenicity in humans [18]. As such, the comprehension and modifcation of plant glycosylation patterns to emulate those of humans is essential and must be solved for effcient development and application of plant-derived biopharmaceuticals.

# *2.2 Key Differences Between Human and Plant Glycosylation Patterns*

N-linked glycosylation, the covalent attachment of oligosaccharides to asparagine residues within the consensus sequence Asn-X-Ser/Thr, is a conserved modifcation among eukaryotes, including humans and plants [18]. However, despite conserving the core glycan structure, signifcant differences exist in the processing and maturation of N-glycans between humans and plants [19].

In humans, the intricate N-glycans exhibit prominent adornment with sialic acid residues and β1,4-galactose, which are nonexistent in plants. Additionally, the

**Fig. 8.2** The key differences in post-translational glycosylation patterns between humans and plants

human N-glycans display core α1,6-fucosylation, while devoid of plant-specifc β1,2-xylose and core α1,3-fucose residues (Fig. 8.2). These variances in N-glycan confgurations can signifcantly impact the effectiveness and immunogenicity of recombinant proteins manufactured by plant-based expression systems, thus creating a signifcant obstacle that must be solved to develop plant-based biopharmaceuticals [20].

# *2.3 CRISPR/Cas9-Mediated Engineering of Glycosylation Patterns in* **N. benthamiana**

In *Nicotiana benthamiana*, CRISPR/Cas9-mediated knockouts of the XylT and FucT genes have effectively eliminated plant-specifc glycan structures, resulting in recombinant proteins with glycosylation profles that more closely resemble human glycoproteins [21].

The study utilized CRISPR-Cas9 to create knockout lines of *N. benthamiana* plants to produce biopharmaceutical glycoproteins. The target genes XylT1, XylT2, FucT1, FucT2, FucT3, FucT4, and FucT5 were identifed, and gRNAs were designed to target all genes in each group [21]. The gRNAs were tested by transient expression and then inserted into the binary pPAM vector carrying a plant-codon optimized cas9 cassette with intron and a gRNA expression cassette under the control of the *A. thaliana* U6 promoter. Three variants of the knockout construct were prepared, one targeting XylT1 and 2, one targeting FucT1-4, and one with all seven gRNAs combined. The CRISPR constructs were then transformed into *N. benthamiana* plants using agroinfltration. High-resolution melt analysis and Western blotting were used to confrm successful knockout of the targeted genes [21].

The CRISPR-Cas9 system works by introducing a double-strand break at a specifc location in the genome, which is then repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR). In the case of this study, the goal was to disrupt the function of specifc genes involved in glycosylation, which can affect the effcacy of biopharmaceuticals. By creating knockout lines of *N. benthamiana* plants using CRISPR-Cas9, the researchers were able to produce glycoproteins with reduced or eliminated plant-specifc glycans, which have comparable affnity to gold standard biopharmaceuticals produced in by using cells from Chinese hamster ovaries. (CHO).

A very recent paper utilizing this newly double knockout line of *N. benthamiana* investigated the impact of plant N-glycosylation on the immunogenic properties of a chimeric Hepatitis B Virus (HBV) S/L vaccine candidate produced in wild-type and XylT and FucT knockout lines of *N. benthamiana* [22]. The study found that prevention of b-1,2-xylose, and a-1,3-fucose attachment to the HBV antigen signifcantly increased the immune response in mice compared to the wild-type plantproduced counterpart. Notably, the antibodies triggered by the knockout-made antigen neutralized both wild-type HBV and a clinically relevant vaccine escape mutant more effciently. The study validates the glycoengineered *N. benthamiana* as a substantially improved host for plant production of glycoprotein vaccines.

This work provides evidence that glyco-engineering of plants can signifcantly enhance the immunogenicity of plant-produced vaccines. Further research is ongoing to explore the potential of the CRISPR/Cas9 system in enhancing the immunogenicity of plant-produced antigens for human use. Using CRISPR/Cas9 in plant-based vaccine production provides an innovative avenue for developing novel cost-effective vaccines.

# **3 Socioeconomics of Plant-Based Protein Production Including Regulatory Issues**

CRISPR/Cas9-mediated engineering of plant glycosylation patterns can produce human-like therapeutic proteins at lower cost, higher scalability, and with less contamination risk than traditional protein production platforms [23]. However, to ensure the safe and responsible development and use of plant-produced recombinant proteins, this technology's introduction into the biopharmaceutical industry raises several ethical, regulatory, and commercial concerns.

CRISPR/Cas9 enables quick and easy alteration of glycosylation patterns in plants. Consequently, apprehensions regarding health and environmental hazards associated with genetically modifed organisms (GMOs) emerge [24]. While not exclusive to plant-based protein production, considering long-term repercussions of introducing genetically altered fora into ecosystems and devising strategies to mitigate potential risks is vital. CRISPR/Cas9-mediated plant engineering also falls under GMO regulations, which exhibit considerable variation across nations. Such regulations impact the development, manufacture, and distribution of plant-made recombinant proteins. Businesses must adapt their operations to ensure compliance within this intricate framework. Streamlining international regulations and enhancing the transparency of the approval process could facilitate wider utilization of this technology.

Regulatory approval and patient safety are contingent upon the safety and quality of plant-produced recombinant proteins. Regulatory bodies, including the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA), mandate comprehensive safety and effcacy data for the authorization of therapeutic proteins—even those generated in plants. Due to the lower costs associated with plant-based protein production, life-saving medications could become accessible in low-income regions where cutting-edge medical treatments often remain elusive. From an ethical perspective, it is crucial to ensure that technological advancements' benefts are distributed equitably, preventing exacerbation of existing global health disparities.

The recent research into the intricacies of plant-specifc glycosyltransferases, such as β1,2-xylosyltransferase (XylT) and core α1,3-fucosyltransferase (FucT), has laid the foundation for gene-editing methods to further engineer plant glycosylation patterns. By employing CRISPR/Cas9 technology, the modifcation of plant glycosylation patterns becomes more streamlined, enabling alterations to specifc genes with exceptional accuracy and effectiveness, ultimately obtaining engineered plants that produce glycoproteins resembling their human equivalents. The ongoing fne-tuning of CRISPR/Cas9, which include the creation of new Cas9 variations, guide RNA designs, and the adoption of high-throughput screening methods, has the capacity to further advance gene-editing procedures within plants, making them even more precise and effcient.

The modifcation of glycosylation patterns in plants holds the promise to transform the biopharmaceutical industry. Plant-based expression systems can contribute to the discovery of innovative therapeutic proteins that boost enhanced pharmacological properties by offering an affordable, scalable, and secure alternatives to mammalian cell culture. Continued efforts in this area could potentially give rise to new treatments for a broad spectrum of illnesses.

# **References**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **Chapter 9 Precise Gene Editing of Cereals Using CRISPR/Cas Technology**

**Pouneh Pouramini and Goetz Hensel**

**Abstract** Targeted mutagenesis using CRISPR/Cas technology has become routine in elucidating biological processes or their application in breeding and agriculture. This means that the change to be achieved can be accurately predicted. However, knockout of a gene function is not always desirable, as reducing activity or affecting a protein domain can inuence its properties and, thus, the phenotype. This chapter will therefore focus on precise genome modi cation in temperate cereals. The methods used, including some representative examples, are summarised here.

Time is an essential factor to consider in developing new agricultural varieties. Since domestication, plant breeders have steadily expanded their toolbox, but establishing a new cereal variety takes an average of 8–10 years [1]. Developments in genome sequencing (barley [2], wheat [3], rye [4]), oat [5], and molecular biology methods for genome-assisted breeding (marker-assisted breeding [6]) have provided tools and techniques for the breeding process that positively inuence the process and workload.

In this chapter, temperate cereals of the botanical tribe Triticeae (barley, wheat, rye, and oats) grown in Europe will be considered. Due to limitations in the availability of genomic sequences (rye and oat only in the last two years) and the lack of ef cient transformation protocols, there are currently only reports from barley and wheat. These are also among the more essential cereals in Europe in terms of cultivated area (FAO Stat). While diploid barley is mainly used for animal feed, beer, and whisky, tetra- and hexaploid wheat are essential for pasta and bakery products. Oat and rye are the main components of breakfast cereals.

Targeted mutagenesis induced by endonucleases such as TALEN [7] and CRISPR/Cas [8] has enabled an incredible number of applications in a wide range of species since their rst biotechnological application in 2012 [9]. Thus, the results have helped many new insights into basic research and show promise for

P. Pouramini · G. Hensel (\*)

Heinrich-Heine-University, Düsseldorf, Germany e-mail: goetz.hensel@hhu.de

applications. Targeting an organism's genomic sequence has never been so easy. CRISPR/Cas technology is a two-component system in which a target-specifc guide RNA guides a double-strand-inducing Cas enzyme to the desired location in the target genome [8]. The cell's repair mechanisms then repair the induced doublestrand breaks. A more detailed description of the technology and repair mechanisms is described in Chap. 1.

Applications of precise genome editing in plants have been summarised several times (for review, see [10–12]). These reach into all areas of modern plant research. Starting with pure basic research, i.e., the identifcation and characterization of genes and their phenotypic expressions, the methods are also used to master the current challenges of agriculture. Here, agronomic parameters play a similarly important role in improving product characteristics. Plants with enhanced resistance to fungal or viral pathogens are being developed and tested. Plants should be better adapted to changing climatic conditions and thus have better water and nutrient utilization. Plants should become heat or drought-tolerant but also be able to grow with an increased salt concentration in the soil. There are examples of all these experiments in barley and/or wheat (Fig. 9.1). The only publication on applying CRISPR/Cas technology in triticale, a cross between wheat and rye, shows its functionality exclusively in protoplasts [13].

Even if the targeted induction of a double-strand break can be carried out precisely, the result is random. According to previous reports, the most common

**Fig. 9.1** Summary of barley and wheat genes with functions in agronomic features [14–47], gene discovery [48–59], product quality [21, 34, 60–74], and abiotic and biotic stress [46, 75–92]

outcome for CRISPR/Cas9 is a deletion of a few (≤10) nucleotides or the insertion of one nucleotide (InDel) [93]. However, it is impossible to predict precisely whether loss or insertion will occur, and it is also somewhat random which base is inserted. Although there are reports (for review, see [94]) that a certain percentage can be predicted using microhomology-dependent repair mechanisms, the outcome remains undetermined in most applications.

To achieve a prediction of the mutation result and thus precise genome editing, one can consider several possibilities. When using two gRNAs, one gets an exact deletion between the two induced double-strand breaks in a part of the mutated cells. In this case, the choice of gRNA binding sites can, for example, infuence the function of a protein domain [49]. By not inducing a knockout, a reduced or altered functionality remains, similar to the RNAi effect but is genetically fxed in contrast.

Another possibility is to transfer a repair template with the desired sequence simultaneously with the double-strand-inducing reagent. However, the challenge here is to bring a suffcient number of repair templates to this site at the time of the double-strand break repair. One possibility is the biolistic transfer of the repaired DNA [95]. However, this method has all the previously described disadvantages that have led to the preferential use of *Agrobacteria*-mediated transformation [96].

Initial results at the cellular level in barley showed targeted allelic exchange of the fuorescence protein GFP. GFP and YFP differ in only one amino acid; thus, exchanging two nucleotides causes a change in the emission spectrum [97]. It was shown that 3% of the mutant epidermal cells had integrated the non-functional YFP fragment in the genome, thus exhibiting a shift in the lambda scan. These results were even surpassed when pre-assembled RNP complexes were biolistically transferred with Cas9 instead of plasmid DNA [95]. Here, it was shown that up to 8% of GFP-mutated epidermal cells exhibited such an allelic exchange. A typical application for allelic exchange is the creation of herbicide resistance [98]. Since this allows the selection of the correctly modifed cells, such a method is easier to apply. However, the effciency is expected to be lower if the modifcation has no selection advantage during creation.

An improvement here is the prime editing method [99]. In contrast to the Cas9 technology, the Moloney Murine Leukemia Virus reverse transcriptase (MLV-RT) domain was added to the Cas endonuclease. At the same time, the gRNA was extended by the part of the repair template. However, there have been few reports of plant applications so far, suggesting that the technology still needs improvement.

To precisely incorporate large DNA fragments in plants, a PrimeRoot-named method was recently described [100]. Third-generation PrimeRoot editors use optimized prime editing guide RNA designs, an improved plant prime editor, and superior recombinases to enable precise large DNA insertions of up to 11.1 kilobases into plant genomes. The authors describe using PrimeRoot to introduce gene regulatory elements into the rice. Applications in temperate cereals have not yet been described.

Base editing (BE) is another technology for the precise modifcation of genomes (DNA) or transcriptomes (RNA) of living cells at single-base resolution (for review, see [101]). BEs comprise a catalytically impaired cas nuclease fused with a

nucleotide deaminase and sometimes DNA repair proteins. BEs can introduce single nucleotide variants at desired sites into the DNA (nuclear or organellar) or RNA of both dividing and non-dividing cells. There are two types of BEs – DNA BEs, which directly induce targeted point mutations in DNA, and RNA BEs, which convert one ribonucleotide to another in RNA. The currently available DNA BEs can be further divided into cytosine BEs (CBEs), adenine BEs (ABEs), C-to-G BEs (CGBEs), dual-base editors, and organellar BEs. These categories are discussed below [101]. After protoplast testing, C to T substitutions was successfully detected in two heterozygous wheat plants [102]. To increase the effciency, further improvements such as the NLS, crRNA, LbCas12a nuclease, adenine deaminase, and linker were undertaken, achieving up to 55% effciency in stable mutants (*TaLOX* and *TaMLO*) [103]. In other plants, further improvements, such as placing a N-terminal reverse transcriptase–Cas9 nickase fusion performed better in rice than the commonly applied C-terminal fusion [104]. In addition, introducing multiple-nucleotide substitutions in the reverse transcriptase template stimulated prime editing with enhanced effciency. Additionally, it was shown that using two pegRNAs that encode the same edits but target complementary DNA strands highly promotes the desired outcome [105].

However, all the previously mentioned methods are still subject to particular challenges. These concern all parts of the process, such as selecting, using, and transferring appropriate gRNAs and Cas and proteins, general tissue culture, genotype dependence, detection of induced mutations, and identifcation of transgenefree, etc. homozygous progeny [106]. Further sequencing of genomes and enzyme evolution will undoubtedly lead to other plant improvements. It is crucial that the plants produced in this way also fnd use in European agriculture and that outdated regulations do not prevent their use.

# **References**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **Chapter 10 Implementing Genome Editing in Barley Breeding**

**Liina Jakobson, Signem Oney Birol, and Ljudmilla Timofejeva**

**Abstract** This chapter summarizes the status of the genome editing efforts in *Hordeum vulgare* L. and provides an overview of the technical advances and obstacles of applying genome editing in barley. It also highlights the potential of genome editing in barley breeding with the focus on breeding for high yielding, disease resistant and stable varieties. The CRISPR/Cas technology is a breakthrough in genome editing due to its robustness and easy to use programming, especially for generating targeted mutations to switch off genes that have a negative impact on food quality, increase susceptibility to pathogens, or divert metabolic ux away from useful end products. Genome editing studies are expected to advance barley breeding by accelerating the breeding process and enabling easier multiplexing of traits. The chapter offers an outlook on the future of barley genome editing techniques based on CRISPR/Cas system.

# **1 Barley Breeding**

Barley (*Hordeum vulgare* L.) is one of the rst crops to be domesticated and on the other hand one of the most genetically diverse cereal species [1]. The cultivation history of the barley was started with the rst seeding about 10,000 years ago by the farmers in Near East [1, 2]. In Europe, barley is nowadays the second most important cereal crop. It is mainly used for animal feed and beverage production. Although human diet is not the primary use, barley offers several health benets and is still an important source of calories in Northern and Eastern Europe as well as in other parts of the world such as North Africa, Middle East and Asia.

L. Jakobson (\*) · L. Timofejeva

Plant Biotechnology Department, Centre of Estonian Rural Research and Knowledge, Jõgeva, Estonia e-mail: liina.jakobson@metk.agri.ee

S. Oney Birol

Department of Molecular Biology and Genetics, Faculty of Science, Burdur Mehmet Akif Ersoy University, Burdur, Turkiye

Over decades, breeders have utilized many strategies to introduce novel heritable mutations into plant genomes in order to develop new improved varieties. The use of numerous physical, chemical and biological mutagens such as gamma rays [3], X-rays [4], ethyl methanesulfonate [5], sodium azide [6], *Agrobacterium tumefaciens* [7] and transposon-based molecular markers [8] has facilitated the rapid extension of genetic diversity throughout the last century. However, these approaches have signifcant limitations, including the non-specifc character of the produced mutations, the huge number of unnecessary mutations and the occasional undesired deletions, duplications or rearrangements of large genomic fragments [9]. Methods of haploid production are also an important tool in barley breeding, being timesaving and providing genetically fxed breeding lines.

Current breeding goals for barley depend on local conditions and vary a lot. In order to meet the increasing demand for livestock feed, starch, and a range of alcoholic (such as beer, whisky, and ethanol) and non-alcoholic (such as barley tea, barley coffee, and malt drink) beverages, barley breeding must focus on developing high-yielding and stress-tolerant varieties that can thrive even in challenging climatic conditions. Global changes such as the predicted increase in human population and diet, set some common goals both for breeding and agricultural crop production [10]. Thus, breeding for disease resistance, high yielding and stability are crucial components worldwide to ensure food security and satisfy the increasing demand. Together with the accelerating global changes, barley breeding has been challenged to speed up the process and multiplex a variety of traits in new varieties.

# **2 Genome Editing Advancements in Barley**

Among gene editing technologies there are three major classes of synthetic endonucleases applied in plants: (a) **zinc fnger nucleases (ZFNs)** are endonucleases linked to a multi-zinc-fnger DNA-binding domain [11]; (b) **transcription activator-like effector nucleases (TALENs)** are composed of multiple transcriptionfactor-like DNA-binding domains linked to endonuclease domain [12]; (c) the widely used **CRISPR/Cas** (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system is composed of single guide RNAs (sgRNAs) enabling targeting multiple genes simultaneously and the Cas nuclease [13, 14]. First, TALEN-mediated genetic modifcations in barley were induced in embryogenic pollen and leaf epidermis of winter barley variety 'Igri' [15, 16]. At the same time, Lawrenson and colleagues demonstrated for the frst time the use of CRISPR/ Cas9 technology in immature embryos of barley variety 'Golden Promise' to generate stable and inheritable mutations [17]. Biolistic transformation method for inducing CRISPR/Cas9-mediated InDels in 'Golden Promise' was presented only couple of years later [18].

Barley genome assembly was completed on the North American barley variety 'Morex' in 2017 [19]. Soon thereafter several protocols optimizing CRISPR/Cas protocols for barley were published [20–23]. The frst Cas nuclease utilised in sitedirected mutagenesis of barley was *Sp*Cas9 originated from *Streptococcus pyogenes*. Recently, the use of two versions of high effciency endonuclease *Lb*Cas12a from *Lachnospiraceae bacterium* coupled with CRISPR was reported in barley [24].

Most CRISPR/Cas-mediated approaches focus on "negative effects" and generation of null alleles or loss-of-function alleles by targeting coding regions, while many agronomically important traits are associated with gain-of-function alleles. New strategies employed in other plants **target non-coding promoter regions**. Li and co-workers [25] engineered allelic variation by editing tomato KLUH promoter around a single-nucleotide polymorphism (SNP) located in a conserved putative cis-regulatory element [25]. Among twenty-one mutant alleles with various insertions and deletions, fve mutant alleles showed a consistent increase in fruit weight. Moreover, promoter editing has proven useful in altering plant architecture in tomato [26], in developing resistance against *Xanthomonas* in rice [27, 28] and citrus [29], and in engineering drought tolerance in maize [30, 31]. Editing of noncoding cis-regulatory elements (CRE) offers considerable potential for crop improvement via fne-tuning of gene expression that cannot be achieved by simple knockout mutations. However, its widespread application is still hampered by the lack of precise knowledge about functional motifs in CRE [32]. Recent advancement CRISPR-Combo enables genome editing (targeted mutagenesis or base editing) and gene activation in plants simultaneously [33].

The above-mentioned fndings have opened the great potential of rapid characterisation of gene function in barley, followed by advancement in precision and increased speed in breeding. **New breeding techniques (NBTs)** now enable switching on/off target genes in barley or convert allelic variants into more advantageous alleles without genetic linkage drag. These approaches could support traditional breeding by overcoming the limits of random mutagenesis and at the same time without developing transgenic plants. However, there are several bottlenecks in the successful utilisation of CRISPR/Cas system in barley such as transformation effciency and the risk for off-targets. Transformation effciency is one of the obstacles hindering the use of CRISPR/Cas in the production of new barley varieties. The risk of off-target mutations can be minimized with the help of large number of bioinformatic and computational tools developed to date, which facilitate gRNA site selection and evaluation of the probability for off-target events [34].

Thus, genome editing with the CRISPR/Cas system has been presented to be applicable in barley and a suitable technology for precision breeding. So far, the examples of using genome editing in barley have mainly focused on the validation of mutagenesis protocols and gene function, however, many genes and traits applicable in breeding are yet to be tested and verifed.

# **3 Using Genome Editing to Target Disease Resistance, Yield and Stability**

The discovery of the CRISPR/Cas system about ten years ago has brought an extensive precision to the portfolio of site-specifc mutagens and a wide range of inducible modifcations, which have many putative applications in breeding. Effcient, easy to use and highly target-specifc single-guide RNAs (sgRNA) enable crop breeders to boost specifcally either yield, biomass, abiotic/biotic stress tolerance, disease, pest resistance or any other trait [35–37]. Recent experiments combining customisable endonucleases and doubled haploid technology facilitate and accelerate the induction of multiple homozygous and inheritable mutations even further [38]. There are also several recent reviews available about the potential of using CRISPR/Cas genome editing in barley [39–45]. Here we shall highlight the potential of genome editing in barley breeding with the focus on breeding for high yielding, disease resistant and stable varieties.

Overall plant immunity and genome stabilization could be one of the targets to induce disease resistant barley varieties. There are a few examples in barley, where disease resistance has been tackled with the aid of CRISPR/Cas technology. The cosmopolitan fungal pathogen *Fusarium graminearum* causes fusarium head blight (FHB), which not only reduces crop yield but also accumulates mycotoxins in barley grains. 2-oxoglutarate Fe(II)-dependent oxygenase (2OGO) has been identifed as a susceptibility factor and plant immunity suppressor in *Arabidopsis*. Barley orthologue *Hv*2OGO was shown to complement the CRISPR/Cas9-induced knock out mutation in *Arabidopsis* and may have a similar role in controlling resistance to FHB in barley [46]. In addition, seven MORC proteins in barley, paralogs of Microrchidia (MORC) protein family, were shown to be involved in plant immunity. CRISPR/Cas-induced double knockout mutants of *HvMORC1* and *HvMORC6a* showed increased disease resistance to fungal pathogens *Blumeria graminis* and *Fusarium graminearum* [47, 48]. A large number of *mildew locus o* (*mlo*) mutants have been found or generated in various barley varieties, which exhibit strong resistance to powdery mildew fungus *Blumeria graminis f. sp. hordei*. Recently, CRISPR/ Cas9-mediated reverse genetics approach was employed to elucidate the molecular function of *MLO* [49].

Wheat dwarf virus (WDV) is an economically important, insect-transmitted DNA virus, which infects also barley, causing severe yield losses. Direct antiviral utilization of the CRISPR/Ca9 system was presented in barley by establishing WDV resistance via targeting sgRNA sequences against viral genome [50]. Eukaryotic virus translation initiation factor E (eIF4E) is a plant cellular translation initiation factor and an essential target in potyvirus infection. Barley plants with modifed *HveIF4E* were generated, but viral resistance is yet to be tested [51].

Soil-borne bymoviruses *barley yellow mosaic virus* (BaYMV) and *barley mild mosaic virus* (BaMMV) infect young winter barley seedlings in autumn and can

cause yield loss up to 50%. PROTEIN DISULFIDE ISOMERASE LIKE 5–1 (PDIL5-1) from ancient landraces and wild relatives of barley confers resistance to all known strains of these viruses. Novel genome-edited *PDIL5-1* alleles were shown also to be resistant to BaMMV, without any adverse effects on growth or yield [52].

Yield stability is the genotype's ability to produce consistently high yield in diverse environments. Breeding for high yield and stability is a complex process that requires the consideration of various factors such as genetics, environment and management practices. Breeding for yield requires the selection of high-yielding genotypes with desirable agronomic traits such as plant height [53, 54], physiological maturity [55], disease and pest resistance [56], and lodging resistance [57]. Previous studies on CRISPR/Cas editing technology in barley targeted *HvPM19* multi-copy genes (*PM19-1 and PM19-3*), associated with grain dormancy [17]. Lawrenson and colleagues transformed the two *PM19* genes independently into variety 'Golden Promise'. Genome-editing of the cytokinin oxidase/dehydrogenases (*HvCKX1* and *HvCKX3*) in barley, which are regulating endogenous cytokinin metabolism, has shown their importance in regulating root length, tillering and yield [20, 58]. Galli and colleagues described transformation of *HvMORC1* and *HvMORC6a* CRISPR/SpCas9 constructs to regulate transposable elements to increase biotic stress resistance and agronomic traits in barley [48]. Cellulose synthase-like gene superfamily (*HvCslF3, HvCslF6, HvCslF9* and *HvCslH1*) genes are related to low grain (1,3; 1,4)-β-glucan content in barley, which is a preferred trait in brewing and distillation processes [59, 60]. D-hordein gene *(HvHor3)* has also been targeted to change the D-hordein composition and other grain phenotypic features [61, 62]. Gene-editing of the caffeic acid O-methyltransferase 1 (*COMT1*) for use in lignocellulosic biomass and lignin biosynthesis has also been utilized [63–65].

The terms "phenotypic stability and yield stability" are often used to refer to phenotypic variations of the genotypes. Moreover, according to Becker and Leon a stable genotype is one that performs consistently despite signifcant statistical differences in environmental variables [66]. Stability of quality traits to produce superior varieties in cereals is very important for breeders because genotype ranks affect selection effciency for genotypes that perform well under different environmental stress factors such as drought [67], salinity [68], diseases [69] to produce superior varieties in cereals. Thus, several studies have reported that the yield stability is correlated with the biomass [70], photosynthetic capacity [71], fowering time [72]. Newly developed targeting and genome editing technologies provide an opportunity to manipulate specifc genomic sequences for improved yield stability. Recently, most of the genome editing studies in barley focus on grain quality, by targeting phytase activity [73], favoenzyme activity [20], high amylose content [74], D-hordein content [62], grain size and composition [75], lignocellulosic content in secondary cell walls [63] and vitamin E biosynthesis in grains [76].

# **4 Challenges in Barley Genome Editing**

Although, the CRISPR/Cas is currently actively studied in many research centres and breeding companies, there are still a number of limitations in the application of the protocol. The limiting factors are: small number of suitable barley genotypes, low transformation effciency, possible off-targets in other parts of the genome exhibiting high sequence similarity, availability of mutable cut sites in the target sequence, and biallelic mutations due to ineffcient cutting of the genomic DNA [20, 77, 78].

Callus regeneration is the fnal step in the *Agrobacterium*-mediated transformation and CRISPR/Cas genome editing protocols used mainly for barley. However, the varietal dependency in the effciency of barley callus regeneration was observed already in 1980-ies both for winter and spring barley [79]. Barley variety 'Golden Promise' has since then been used as the standard for callus regeneration and transformation. Thus, genotypic restrictions on plant regeneration have hindered the implementation of transformation and genome editing tools on most barley varieties for over four decades.

Barley transformation protocols have been optimized, updated and improved over time. Several enhanced protocols have been published, mainly for *Agrobacterium*-mediated transformation of immature embryos, which provide average transformation effciencies of 25% in the background of 'Golden Promise' [80–83]. For instance dicamba in the callus induction and maintenance media was generally superior to 2,4-D in promoting transformation and addition of CuSO4 resulted in formation of more green plants [84]. The addition of L-cysteine as an antioxidant was reported to hinder the browning of embryos and boost the effciency of transformation [85]. Albinism, which can appear among regenerated barley, is caused by the inability of proplastids to transform into chloroplasts. Pre-treatment with mannitol may help to reduce albino barley plants [86]. Recently, anther culture-based system was shown to enable effective creation of transgenic plants not only from 'Golden Promise' but also from four other Australian commercial barley varieties [87].

In addition to changing hormone and nutrient levels, techniques that modify the innate gene expression of plants could enhance the effectiveness of transformation. There are examples from maize, rice, sorghum and sugarcane (*Saccharum offcinarum*) that overexpressing *Baby boom* (*Bbm*) and *Wuschel2* (*Wus2*) genes produced high transformation frequencies in previously nontransformable lines [88]. BBM is a transcription factor among the superfamily of the APETALA 2/ETHYLENE RESPONSE FACTOR (AP2/ERF) DNA-binding domain, subfamily AP2 [89, 90]. Two *TaBBM* genes in wheat have been identifed as orthologues for maize *Bbm* [91], however, barley counterparts are still to be uncovered. WUS is a bifunctional homeodomain transcription factor, which mainly acts as a repressor but can become also an activator [92, 93].

Another promising approach for increasing transformation effciency, could be the expression of fusion protein combining wheat GROWTH-REGULATING FACTOR 4 (GRF4) and its cofactor GRF-INTERACTING FACTOR 1 (GIF1) [94]. The concept was proven to have the desired effect in wheat, rice, triticale as well as in the dicot crop citrus hybrid Carrizo citrange [94]. Studying the effect of GRF-GIF fusion protein in reducing varietal gap in barley transformation would be a compelling avenue for exploration.

Lately, there have been several reports addressing the transformation effciency by aiming to dissect its genetic determinants in transformable genotypes. Three signifcant and seven suggestive Transformation Amenability (TFA) regions were identifed in 'Golden Promise', which likely include necessary factors for *Agrobacterium*-mediated transformation in barley [7, 95]. Additionally, the transformation effciency (*TRA1*) locus was identifed in the barley mutant M1460 on chromosome 2H incorporating 225 gene sequences [96]. Thus, there are suggestions in the literature that certain genetic components could affect the amenability to *Agrobacterium*-mediated transformation. However, further research is needed before this data could be used to overcome recalcitrance.

# **5 Social and Legislative Aspects of Using Genome Editing in Barley Breeding**

Transgenic and genome editing technologies have a number of challenges, including regulatory barriers, public acceptance and the time and cost of risk assessments needed prior to commercialization. Genome edited products do not align with the prevailing defnitions of genetically modifed organisms (GMOs) in the majority of legal frameworks [75, 97]. The United States Department of Agriculture (USDA) [98] and the Australian Government Offce of the Gene Technology Regulator [99] have determined that CRISPR-edited crops without foreign DNA are exempt from regulation as genetically modifed organisms with regard to the regulation and commercialization of such products that enables to support and usage of the genome edited crop production. Afterwards, the European Parliamentary Research Service has ruled the legislation process based on the societal acceptance to promote the safety of humans, other animals, the non-living environment, and safe agriculture based on current developments in international genome editing laws in any genome editing application organized with the CRISPR/Cas system [100]. Recent developments in England reveal a change in the law permitting the commercial use of the gene edited products [101]. Cambridge-based researchers support utilizing this technology to develop crop varieties that are resilient to climate change and anticipate that it will create new employment opportunities. By enabling the production of better-adapted crops in less time and facilitating prompt market access, this approach holds signifcant promise.

**Fig. 10.1** Implementing genome editing in barley breeding. NBTs such as ZFNs, TALENs and CRISPR/Cas system enable to accelerate breeding and multiplex different traits. Application of NBTs in breeding is yet accompanied by the challenges in legislative rules

Consequently, genome editing enables to breed in less time and with greater precision, maximize crop genetic potential, generate germplasms that are more resistant to pests, biotic and abiotic challenges, and extend shelf life of plant products to reduce food waste. However, barley genome editing protocols still have a number of limitations. Acquiring knowledge about genome editing techniques and their applications is crucial for shaping regulatory frameworks that will impact the feasibility of utilizing novel barley varieties and support food security (Fig. 10.1).

# **References**


table mutations in barley (Hordeum vulgare L.). Plant Method. **14**, 111 (2018). https://doi. org/10.1186/s13007-018-0382-8


fowering time in barley. Theor. Appl. Genet. **118**, 259–273 (2009). https://doi.org/10.1007/ s00122-008-0896-y


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **Chapter 11 Current Status and Future Prospective of Genome Editing Application in Maize**

**Serena Varotto**

**Abstract** Maize (*Zea mays* ssp. *mays*) is the most world-widely cultivated agricultural crop and over the past century, its yield per unit land area has increased consistently due to both breeding efforts and improvement in management. Cross breeding, mutation breeding, and transgenic breeding are the main methodologies adopted for maize improvement. The rst maize transgenic hybrids were commercialized more than 20 years ago, and till now more than 150 different events of transgenic maize have been approved for commercial cultivation. The sequencing of the maize genome and the development of advanced genomic tools provided the biologists with the theoretical information necessary to attempt the genome modication at the pre-intended genomic loci. The tremendous advances brought about by CRISPR/ Cas systems from rst applications to nowadays has made genome editing a powerful tool for precise maize improvement. Although many CRISPR-Cas-edited genes have been documented to improve maize traits of agronomic interest, only a few lines have been tested in eld trials; additional work for determining potential breeding values of edited maize lines must be done in terms of eld tests. The integration of CRISPR-Cas technology in the breeding of new maize varieties also depends on existing and future regulatory policies that will be adopted worldwide.

# **1 Introduction**

Maize (*Zea mays* ssp. *mays*) is the most world-widely cultivated agricultural crop and a renowned experimental model plant for molecular and genetic studies. Maize domestication started about 9000 years ago from the wild grass Z. mays subsp. Parviglumis in the Balsas region of southwest Mexico [1, 2]. Morphological observations, genetic and genomic studies have elucidated how from *Z. mays* subsp*. Parviglumis* (also called "teosinte") *Z. mays* subsp*. mays* was

S. Varotto (\*)

Department of Agronomy Animal, Food Natural Resources and Environment (DAFNAE), University of Padova – Agripolis Viale dell'Università, Legnaro (PD), Italy e-mail: serena.varotto@unipd.it

domesticated. Although a few major morphological differences distinguish these two subspecies, and genes responsible for these traits have been identifed, it has emerged that several genomic regions have contributed to maize domestication [3]. Among the grasses, maize has a medium-sized genome of approximately 2.4 Gb [4], which is characterized by an exceptional genomic structural diversity. Large insertions and deletions that includes tandem repeats cluster and transposable elements are common between maize inbred lines. This structural diversity, characterized by copy number variants (CNVs) and presence/absence variants (PAVs) is important for maize adaptation and has offered a rich pool of genetic diversity to breeders for creating improved germplasm [5]. At the gene sequence level, single nucleotide polymorphisms (SNPs) are frequent in introns and untranslated regions of maize genome. SNPs surveys and subsequent quantitative traits genome wide association studies (QTL/GWAS) were adopted for linking genetic and phenotypic variations. In parallel, the sequencing of the B73 reference genome, which quality has been greatly improved during the last few years by the development of long-read sequencing (mainly by Pacifc Biosciences' PacBio) single molecule real-time (SMRT) sequencing [6] have shed light on both the complexity of the maize genome and the contributions of different variations to phenotypic differences. In addition to the characterization of genetic variations, recent research in maize was aimed at understanding the relationships among gene expression, epigenetic modifcations, chromatin interactions, and metabolic, proteome, and phenotype variations. The development of high-quality genome assembly tools together with the precise characterization of genomic diversity and the association of genetic variants with yield-related traits has greatly improved maize genomic research. Till now, thousands of diverse and representative maize lines have been genotyped [7] and the integration of multiple annotated reference genomes has been facilitated by comprehensive databases that store, maintain, analyze, and visualize the multi-omics data, such as MaizeGDB [8] and ZEAMAP [9]. The development of genomic technologies has allowed a deeply exploration of diversity at all levels in different environments and the identifcation of genes that determine maize yield: the mechanistic understanding of gene function and the precise modifcation of genes in different genomic background can accelerate breeding for yield in the coming years.

# **2 A Glance Over Conventional Maize Breeding**

Over the past century, maize yield per unit land area increased consistently (over sevenfold) due to both breeding efforts and improvement in management [7]. Breeding of hybrid crops was pioneered in maize since the observation, in early 1900s, that hybrid cultivars provided higher yield compared to pure lines and open pollinated varieties [10]. The constitution of modern maize hybrids relays on development of elite parental inbred lines and their subsequent evaluation in single cross combination. The original methods for hybrid production introduced by Shull in 1908 (referred as "pure line method of corn breeding") [11] underwent several modifcations over years. This allowed both a more effcient production of inbred lines and identifcation of superior hybrid combinations between them [12]. In modern maize breeding, the activity of evaluation of inbred lines for hybrid performance is the most critical and expensive phase. Inbred lines are collected in heterotic groups with the aim of facilitating the identifcation of superior hybrid combinations [10]. Additionally, to increase the number of lines having good potential for hybrid performance, population improvement methods are adopted, and double haploid (DH) technology is used to generate homozygous lines [13]. To introduce desirable alleles into a desired elite inbred line background by genetic crosses, 8–10 selfng generations are required. Moreover, extensive background screening and evaluation of large-sized progenies are necessary to increase the chance of genetic recombination and reduce possible linkage drag effects, when the desirable trait is closely linked to an undesirable trait [7]. The advantage of DH technology relays on the much quicker development of homozygous lines compared to 8–10 generations of inbreeding by selfng or sib-crossing necessary to develop inbreds [14]. Nowadays in maize breeding programs, DH are routinely obtained by pollinating the plants with haploid inducer (HI) lines. Subsequently, chromosomes can be doubled spontaneously or artifcially, by treatment with mitotic inhibitors such as colchicine, for generating DH lines.

Cross breeding, mutation breeding, and transgenic breeding are the main methodologies adopted for maize improvement. As illustrated above, in maize to introduce desirable alleles by cross breeding, and use genetic recombination to produce genetic variability are long and costly procedure. Additionally, for some agronomic traits genetic variability has been greatly reduced by domestication and directed selection [15]. To overcome these limits, mutation breeding has created genetic variation by introducing random mutations in the maize genome [16]. However, the stochastic nature of the mutations produced and the need to screen large numbers of mutant genotypes makes mutation breeding a time-consuming and laborious procedure that cannot enhance selection effciency, even if markerassisted breeding approach are adopted. Transgenic breeding through the transfer of exogenous genes into commercial elite varieties can accelerate the improvement of important agronomic traits. However, along with some limitations of the methodology, such as the random insertion of the transgene and the low number of sequences that can be introduce in the genome by genetic transformation, the long and costly deregulation processes, and public concerns about transgenic crops, limit the commercialization of genetically modifed maize plants [17]. In the following chapter we will highlight how the introduction of targeted mutagenesis and the combination of these novel technologies with conventional breeding procedures can solve some of the main limiting factors for a more sustainable maize breeding.

# **3 Maize Genetic Transformation**

Technological innovation and scientifc discoveries have always had a big impact on maize research and breeding, and genetic transformation has been an indispensable biotechnology in both applied and basic maize research. In basic research transformation technologies were widely adopted to study gene regulation and function, mainly through the obtention of mutant plants in which the target sequences are over-expressed, expressed ectopically, downregulated or silenced. Additionally, transgenesis allows the study of gene promoters and other regulatory sequences regulating gene expression in the coding portion of the genome. As already mentioned above, in applied maize research, the introgression of benefcial target genes from one line (donor) to another (recipient) by conventional breeding requires many years of backcrossing after hybridization and may lead to linkage drug effect on the recipient line genome [7]. Conversely, genetic transformation introduces well characterized DNA regulatory and coding sequences into the plant genome. The goal to transform maize with a high effciency technology, providing high-quality transgenic events has been essential to improve specifc maize traits [18]. Initially, the production of genetically modifed maize varieties has encountered enormous diffculties, mainly for the genotype-associated recalcitrance to transformation. In late 80', progresses in genetic engineering and biotechnology resulted in stable transformation of maize [19]. Fromm and colleagues stably transformed maize cells for resistance to kanamycin by electroporation-mediated DNA transfer of a chimeric gene encoding neomycin phosphotransferase. In 1987 Grimsley and colleagues reported that maize plants developed symptoms of viral infection when inoculated with strains of Agrobacterium carrying copies of maize streak virus (MSV) genomes in their T-DNA, thus demonstrating that Agrobacterium could transfer DNA to maize [20]. The frst genetically transformed infertile maize plants were obtained from embryogenic cell derived protoplasts treated with plasmid DNA containing a gene coding for neomycin phosphotransferase (NPT II) driven by the 35S promoter region of caulifower mosaic virus [21]. Finally, fertile transgenic maize plants were produced from embryogenic cell suspension transformed with the bacterial gene bar, encoding for phosphinothricin acetyltransferase (PAT), using microprojectile bombardment [22]. Although several protocols for Agrobacterium- and polyethylene glycol (PEG) protoplast-mediated transformation were developed before 2000s [23, 24] transformation effciency and successful *in vitro* plantlet regeneration through tissue culture was highly dependent on genotypes. Therefore, hybrid lines showing the ability to produce highly transformable calluses were selected: High type II callus (Hi II), containing both A188 and B73 inbred genetic background, became one of the most widely used hybrids for maize transformation in both academic and plant industrial labs [25, 26]. Commonly for maize transformation, embryogenic callus started from immature embryos and cell suspension cultures of embryogenic callus were used [27–29]. Nowadays, although maize transformation is routinely performed, a few genotypes (A188, B104) have been reported to have acceptable transformation effciency comparable to Hi-II, which have remained the most popular lines for commercial transformation. For instance, the maize inbred line B73 that is the frst inbred to be sequenced and an important genetic resource is strongly recalcitrant to transformation, as well as most of the commercial elite maize inbred lines. The discovery that transgenic maize genotypes overexpressing BABY Boom (ZmBbm), WUSHEL (ZmWus2) and OVULE DEVELOPMENT PROTEIN 2 (ODP2) genes can enable high transformation frequencies in numerous recalcitrant genotypes was an important milestone for maize transformation [30]. Morphogenic Regulator-Mediated Transformation (MRMT) vectors containing these morphogenetic genes can be introduced into Agrobacterium strains and used for immature embryo transformation. Through MRMT increased plant regeneration rates, recovery of transformed plants from recalcitrant genotypes, and a shortening in time needed for transformation by avoiding the callus culture step have been obtained [31]. However, since the constitutive expression of MRs can have a negative pleiotropic effect on important developmental traits, its expression has to be restricted to the embryogenesis induction step, either by excision of the MR expression cassette through a recombination system or driving the expression of MRs using specifc promoters [31].

The frst maize transgenic hybrids were commercialized more than 20 years ago, and till now more than 150 different events of transgenic maize have been approved for commercial cultivation or food/feed use. Most of the released events concerns simple traits, such as herbicide tolerance, insect resistance, modifed product quality, pollination control system and abiotic stress tolerance [32]. It is a matter of fact that applying transgenic approaches for the improvement of complex traits is diffcult, because these traits are controlled by numerous genomic loci with a small effect and are strongly infuenced by the environment. Evidently this represents a limitation for maize improvement because the limitation concerns the integration of biotechnology and traditional breeding in the improvement of complex traits [33]. One more limitation of transgenic approaches is that foreign DNA integrates into random sites of the host genome. Random integration of transgene might affect the transgene expression, although some recent observations did not prove the assumption of this risk [34]. Moreover, from the frst transgenic hybrid commercialization new techniques were developed, new regulations were adopted, and despite their signifcant benefcial impact on modern agriculture, public perception is still controversial about transgenic crops. The high costs necessary for the deregulation of genetically modifed commercial maize plants can be afford only by the largest agricultural biotechnology companies, with a consequent increasing concentration of maize seed providers [35].

# **4 Mutagenesis in Maize**

Traditionally in maize, mutagenesis has been an impressive useful tool for both broadening genetic variation and understanding gene function. Numerous strategies were developed for creating mutations and identifying genes based on phenotypes (forward genetics). The same strategies have also been used to assignee a phenotype to target coding sequences (reverse genetics). While in early mutagenesis experiments UV and X rays [36, 37], ethyl methanesulfonate (EMS) and other chemical mutagens were used, the main strategies for comprehensive mutagenesis of maize genes comprise TILLING (Targeting Induced Local Lesions IN Genomes) [38], RNAi [39] and transposons mutagenesis. The discovery and characterization of transposon families as well as their massive presence into maize genome facilitated the development of transposable element systems, particularly useful for two maize inbreds, namely B73 and W22. In recent years these systems based on Class II "cut and paste" maize transposons were implemented for creating based genetic resources, such as Ac/Ds families and UniformMu available through MaizeGDB (https://www.maizegdb.org/) and Maize Genetics Cooperation Stock Center (http:// maizecoop.crops ci.uiuc.edu/) respectively. In the genomic era maize transposons have been the premier method for gene discovery and phenotypic-related characterization in a whole -organism context [40].

The sequencing of the maize genome and the development of advanced genomic tools provided the biologists with the theoretical information necessary to attempt the genome modifcation at the pre-intended genomic loci in a more precise way than random mutation breeding, which can be time consuming and expensive for the large screens needed. Targeted mutagenesis initial attempts were rarely successful in maize, due to the very low frequency of homologous recombination (HR) events involving either endogenous target or exogenous donor DNA [41]. However, the adaptation to maize of Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) two novel technologies successfully applied in mammalian cells and in a few model plants, such as Arabidopsis and tobacco, allowed the frst genome editing events on maize endogenous target genes. Targeted genome editing tools use nucleases to induce DNA double-strand breaks (DSBs). In plant cells, DSBs can be repaired by two main pathways, nonhomologous end-joining (NHEJ) or homology-directed repair (HDR). The NHEJ pathway usually generates in/dels at the repair sites. Differently, when a template DNA is provided HDR can be adopted for precise sequence replacement or insertion [42]. The ZFNs are chimeric proteins with two domains: the N-terminal domain is a synthetic zinc fnger-based domain that recognizes a 3-base-pair (bp) target sequence and binds to DNA; the C-terminal domain is a non-specifc DNA cleavage domain using Fok1 a type IIS class of restriction endonucleases [43]. Because FokI functions as a dimer, ZFNs are designed as two ZFN monomers bound to an 18- or 24-bp sequence with a 5–7-nucleotide spacer. This spacing is a critical part of ZFN design as it allows Fok1 monomer to dimerise and create a DSB in the target sequence. A pair of Zinc fnger arrays (ZFAs) binds to respective sequences targeted and get aligned in reverse fashion with each other. In 2009, Schukla et al. [44] reported the use of designed (ZFNs) that induced a double-stranded break to modify a target endogenous locus in maize. The simultaneous expression of ZFNs and delivery of a simple heterologous donor molecule allowed the targeted addition of an herbicide-tolerance gene, one of the phytic acid biosynthesis genes, namely inositol-1,3,4,5,6-pentakisphosphate kinase 1 (IPK1). ZFN-modifed maize plants could pass these genetic changes to their progeny: in developing maize seeds the targeted cleavage of IPK1 gives the characteristics of both herbicide tolerance and desired alteration of the inositol phosphate [44]. About fve years later, the TALEN technology was successful applied for targeted gene mutagenesis in a proof-ofconcept study in maize [45]. Similarly to ZFNs, TALENs are fusion proteins of native or artifcial TAL effector DNA-binding domains and the DNA-cleavage domain of FokI. The modular TAL effector repeats can be custom-tailored into DNA recognizing domains for virtually any sequence in a genome [46]. When expressed in plant cells, the paired TALENs recognize and bind to two adjacent, opposite subsites, enabling the FokI domains (homo- or heterodimeric) to dimerize to an approximately 50–60-bp target sequence within a 14–18-bp spacer, which is necessary for its function. In maize, TALENs were employed to generate heritable mutations at the glossy2 (gl2) locus. Hi-II transgenic lines containing mono- or diallelic mutations were produced at a frequency of about 10%. Three modifed alleles were functionally tested in progeny seedlings, demonstrating that they conferred the glossy phenotype. The authors reported that the integrated TALEN T-DNA segregated independently from the loss of function gl2 alleles in most of the events, generating mutated null-segregant progeny in T1 generation [45].

These results confrmed ZFNs and TALENs as new strategies for maize genome editing in basic science, with potential in breeding applications. However, owing to construction complexity, high off target rate of ZFNs and high costs of result screenings they had limited applications in maize till now.

# **5 The CRISPR Technology Application in Maize**

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPRassociated (Cas) system, comprising CRISPR repeat-spacer arrays and Cas proteins, is an RNA- mediated adaptive immune system in bacteria and archaea. It provides defense against invasive genetic elements by cleaving their nucleic acid genome [47]. The detailed description of the system and its classifcation in classes and types based on Cas genes are provided in other chapters of this book. Here we will focus on the CRISPR system that has been developed and improved for genome editing in maize, which is generally based on RNA-guided interference with DNA. Briefy, the CRISPR/Cas9 approach uses an RNA-guided endonuclease to generate DNA DSBs at the target sites of the plant genome. The type II CRISPR/ Cas9 system adopted in plants requires the hetero-duplex RNA of CRISPR RNA (crRNA) and auxiliary trans-activating crRNA (tracrRNA) to guide the non-specifc nuclease, Cas9, for DNA cleavage. In Cas9 the nuclease domains RuvC and HNH cleave the complementary DNA strands [48]. The crRNA and tracrRNA can be further replaced by a single chimeric guide RNA (sgRNA) [47] that can recognize any genomic locus that is followed by a 5′-NGG protospacer-adjacent motif (PAM), and a 20-nt sequence preceding the PAM directs the Cas9 and cleave the target sequence by complementary base pairing. Differently from ZFNs and TALENs that require sophisticated protein engineering to defne precise target site recognition, Cas9-sgRNA components are simple to design and clone. Additionally, the fewer limitations of potential target sites in plant genomes make the CRISPR/Cas system widely applicable. When using this technology, the NHEJ repair pathway is the preferred way to disrupt genes by producing small indels at specifc sites in target genes. Moreover, NHEJ can also be exploited to produce insertions of donor DNA sequences in a homology-independent manner [49]. An important choice to make for the application of this technology in maize is the system of delivery to cells of editing reagents, which include DNA, RNA, and ribonucleoproteins (RNPs). Protoplast transfection is commonly uses for transient expression, while Agrobacterium-mediated transfer DNA (T-DNA) transformation, or particle bombardment are the delivery methods of choice to produce edited plants.

In 2014 Xing et al. reported the development of a toolkit to facilitate transient or stable expression of the CRISPR/Cas9 system and generate mutants bearing multiple gene mutations in a variety of plant species, which comprised maize [50]. This system was composed by a CRISPR/Cas9-based binary vector set and a sgRNA module vector set and was validated using maize protoplasts and maize transgenic lines for the targeting of the same maize genomic DNA site (ZmHKT1). In the same year, a comparison between two systems of targeted mutagenesis TALENs and the CRISPR/Cas was conducted by Liang and colleagues [51]. Five TALENs targeting the genes ZmPDS, ZmIPK1A, ZmIPK, ZmMRP4 and two sgRNAs targeting the ZmIPK gene were constructed. The effciency in inducing targeted mutations was similar for the two systems: 13.1% CRISPR/Cas and 9.1% TALENs, respectively, in maize protoplasts.

The frst detailed report in a scientifc journal of Cas9-gRNA genome editing technology application in maize, with direct delivery of sgRNA in the form of RNA molecules, was published by Svitashev et al. in 2015 [52]. For targeting fve different maize genomic regions, the biolistic transformation of maize immature embryos was conducted with DNA vectors expressing a maize codon optimized Cas9 endonuclease and sgRNAs, with or without DNA repair templates. The genomic regions were located upstream of the liguleless1 (LIG1) gene, at male fertility genes (Ms26 and Ms45) and acetolactate synthase (ALS) genes (ALS1 and ALS2). Following transformations, the authors could identify mutations at all sites targeted, as well as plants containing biallelic multiplex mutations at LIG1, Ms26, and Ms45. When immature embryo cells containing pre-integrated Cas9 were directly used for biolistic delivery of sgRNAs (as RNA molecules) targeted mutations could also be detected. The same authors also reported the editing of ALS2 gene and the recovery of chlorsulfuron-resistant plants, using either single-stranded oligonucleotides or double-stranded DNA vectors as repair templates yielded. Moreover, RNA-guided Cas9 endonuclease- generated double-strand breaks at a site near LIG1 stimulated insertion of a trait gene by homology-directed repair. In all cases of genes knockouts, edits, and insertions, T1 plants genetic analysis showed that the mutations followed a Mendelian segregation in subsequent generations.

CRISPR/Cas9 has been also applied for the targeted knockout of the endogenous ZmPSY1 gene in maize T0 transgenic plants [53]. Interestingly in this work, the authors confgured and optimized the CRISPR-Cas9 system for targeted genome editing in maize and accurately tested the activity of their customized system. Appropriate vectors to express Cas9 and sgRNAs for maize were generated. Cas9 open reading frame was maize codon-optimized and driven by maize Ubiquitin2 promoter. Additionally, to the N terminus of Cas9 a monopartite SV40 nuclear localization signal (NLS) was added and, to further facilitate nuclear localization, a bipartite nucleoplasmin nuclear localization signal (BiNLS) was added to the Cas9 C terminus. Similarly, the authors accurately studied the maize promoter that was used to transcribe the short noncoding sgRNA. A reasonably high mutation rate was frstly established in maize protoplasts. Subsequently, the mutations occurred in germ cells and were transmitted to the next generation with high effciency. No offtarget effect could be detected at the computationally predicted putative off-target loci and no signifcant difference between the transcriptomes of the Cas9 expressed and non-expressed lines was reported [53].

In maize, multiplex gene editing was achieved by expressing Cas9 together with multiple gRNAs, each targeting different sites and using conventional delivery methods. In principle, single construct containing more than one guide RNA can accelerate and improve the transformation procedures. Two procedures have been developed: the frst was based on a multi-guide RNA activated by a single promoter and processed by tRNA motif-mediated self-cleavage into several sgRNAs [54] the second based on tandem repeats of different U3 and U6 promoters each controlling one guide RNA [55]. In both cases, the mutations resulting from targeted mutagenesis were mainly deletions or insertions of a few nucleotides probably due to NHEJ.

In the works described above, to produce edited maize plants, transformation mediated by Agrobacterium or particle bombardment was used for delivering CRISPR/Cas DNA and a selectable marker gene into recipient maize cells and these DNA constructs were stably integrated into the plant genome. However, this strategy of stable integration might increase off-target changes, gene disruption and plant mosaicism, as well as limit commercial applications. Transgene-free edited plants can be selected through genetic segregation by selfng and crossing, which is time consuming in maize hybrid breeding. To avoid these negative effects, transgenic maize plants with pre-integrated Cas9 nuclease have been generated and used for delivery of sgRNAs in the form of RNA molecules. However, this strategy requires the specifc development and characterization of Cas9 pre-integrated lines. Transient gene expression of DNA constructs in protoplasts could represent an alternative approach for achieving transgene-free editing in plant. Till now, maize protoplast transient transformation experiments serve mainly for the evaluation of the effciency of different Cas9 and sgRNA designs, due to the lack of an effcient protocol for the regeneration of maize plants from protoplasts. In 2016 Svitashev [56] and colleagues reported for the frst time the biolistic delivery of in vitro assembled Cas9–sgRNA ribonucleoproteins (RNPs) into maize embryo cells and regeneration of plants with both mutated and edited alleles. Purifed Cas9 protein pre-assembled with in vitro transcribed gRNAs were delivered into maize immature embryo cells. The previously studied four genomic regions, liguleless1 (LIG), acetolactate synthase (ALS2) and two male fertility genes (MS26 and MS45) [52] were targeted. Using this method of delivery, they produced DNA- and selectable markerfree maize plants with mutated alleles at high frequencies [56]. As observed in other systems, delivery of RNP complexes led to a signifcantly decreased undesired mutation frequencies in comparison to DNA vectors. While the frequency of off-site mutations was about 50% for MS45 target site for both immature embryos and mature plants when Cas9 and sgRNA were delivered on DNA vectors, off-site mutations were not detected in regenerated plants when RNPs were used. These results demonstrated that Cas9–gRNA delivered as RNP complex has a signifcant advantage over DNA vector delivery by promoting high mutation frequencies in a more precise manner also in maize, as already observed for other organisms and plant species [57, 58].

Although involving genes and loci of agronomic interest, many of the initial works using CRISPR/Cas9 were proof-of-principle studies to test genome editing different strategies and effciency for applications in precision breeding. For an effcient targeted genome editing in maize, the promoters for driving both Cas9 and sgRNA expression were proven to be an essential factor. In early works, the maize ubiquitin gene promoter was used in construct containing a maize codon optimized Cas9. In the same experiments the rice U3 or wheat U3 promoters were used for driving sgRNAs [50, 51]. Different promoter combinations, such as maize ubiquitin1 and U6 promoters, CaMV35S and maize U3, and maize ubiquitin1 gene promoter and two rice U6 promoters, were utilized for Cas9 and sgRNAs in other studies [53, 55, 59] providing additional evidence that the optimization of the promoters used for the CRISPR/Cas9 system is an essential step for effcient targeted genome editing procedures. Furthermore, the mutation effciency of CRISPR/Cas9 system appeared largely depending upon both the expressions of Cas9 and sgRNAs. Feng and colleagues observed that when the 35S promoter was used for driving the expression of a human codon optimized Cas9 a low mutation rate was obtained and most of the regenerated T0 plants were mosaic [59]. For increasing the mutation effciency and concomitantly avoiding mosaicism, in a subsequent work they used the promoter of dmc1 gene. DMC1 was thought to be expressed specifcally in meiocytes and was used for expressing the Cas9, combined with the U3 promoter for driving the sgRNA expression. The authors reasoned that utilizing these constructs gametes could be mutated, and T1 homozygous or bi-allelic mutants could have been recovered at high frequency at the three loci selected for targeting in the maize genome. However, during their transformation experiments, they realized that the dmc1 promoter could drive the Cas9 to be highly expressed also in maize callus. This observation was confrmed by expression analysis of the endogenous dmc1 gene in different maize tissues including callus. The dmc1 gene was found to have the highest expression level in tassel but was also highly expressed in callus. Using this transformation approach, the T0 plants regenerated were highly effciently edited at the target sites with homozygous or bi-allelic mutants accounting for about 66%; mutations could be stably transmitted to the T1 generation, while no off-target mutations could be detected in the predicted loci with sequence similarity to the targeted site [60].

After having established the CRISPR/Cas9 technology platform and confrmed its effciency for single and multiple GT in maize, subsequent studies focused on exploitation of targeted editing for hybrid-breeding technique improvement. In maize, male-sterile maternal lines are an essential prerequisite for generating highquality commercial hybrid varieties. ZmTMS5 gene controls a thermosensitive male-sterility trait in maize and is the orthologue of the previously characterized and edited TMS5 rice gene [61]. The targeted knockout of the ZmTMS5 gene using immature Hi-II embryos transformation by particle bombardment for delivering CRISPR/Cas9 elements, produced T1 tms5 mutant plants, male-sterile at 32 °C, but male-fertile at 24 °C. T1 plants were Cas9-free through segregation and carried only the desired tms5 mutation. They provide a useful germplasm that can potentially be used to simplify hybrid maize seed production [62].

A potential application of the genome-editing technology concerns the reduction of so-called linkage drag during back-cross breeding. Direct genome-editing technology provides the opportunity of stacking favorable genes without introgression breeding. An experimental proof-of-concept to validate this strategy was provided by the work of Li and coworkers [63]. They established an RNA-guided endonuclease (RGEN) system as an in vivo desired-target mutator (DTM) in maize, to reduce the linkage drag during breeding procedure, using the LIGULELESS1 (LG1) locus as target. The RNA-guided Cas9 system showed 51.5–91.2% mutation frequency in T0 transgenic plants. The T1 plants stably expressing DTM were crossed with six diverse recipient maize lines producing 11.79–28.71% of mutants. Furthermore, the analysis of F2 plants showed that the mutations induced by the DTM effect were heritable. The results were confrmed by the phenotypical characterization of the mutant plants in the feld [63].

A further major technical limitation of utilizing gene targeting technologies resides on the recalcitrant nature of most elite maize inbred lines for genetic transformation. To facilitate GT techniques, initials functional tests and transformations are usually done in maize lines with relatively high transformation effciencies, such as Hi-II and B104. Subsequently, the selected and desired transformation events must be introgressed into elite commercial inbred lines, through at least six backcrossing for achieving more than 99% background purity, requiring additional work and time.

Doubled haploid (DH) technology based on in vivo haploid induction (HI) is an important modern approach for maize breeding. Therefore, there is the need to identify candidate genes underlying HI in maize genome for their detailed functional characterization. Several genes involved in HI were recently validated through CRISPR–Cas9 system [64–66].

In 2018 Wang and colleagues reported the validation of a new strategy for the development of a haploid-inducer mediated genome editing system (IMGE) for accelerating maize breeding. This system uses a maize haploid inducer line carrying a CRISPR/Cas9 cassette targeting for a desired agronomic trait to pollinate an elite maize inbred line. The pollination can generate genome edited haploid maize plants in the elite background. During the process, HI genome is degraded, and no editing tools are present in edited plants [67]. Within two generations homozygous pure DH lines improved for the desired trait could be obtained, avoiding repeated crossing and backcrossing used in traditional breeding for introgressing a desirable trait into elite commercial backgrounds [67]. Similarly, Kelliher and colleagues co-opted the aberrant reproductive process of haploid induction (HI) to induce edits in nascent seeds in maize and other monocot and dicot species. Their method, named HI-Edit, made direct genomic modifcation of commercial crop varieties possible, and was tested in feld and sweet corn using a native haploid-inducer line. Also in this case, edited haploid plants lack both the haploid-inducer parental DNA and the editing machinery and could be used in trait testing and directly integrated into commercial variety development [68].

# *5.1 Novel Approaches for Maize Trait Improvement*

Based on experimental proof-of-concept reported above, the application of CRISPR/ Cas technology not only allows to modify the agronomic traits of interest through the insertion or deletion of single or few nucleotides, but it can also facilitate maize breeding by inserting new alleles in the genome without any linkage drag. Additionally, precise gene modifcations can generate novel allelic variants by knock-ins and replacements, thus having great value for crop trait improvement Moreover, knock-in can be used to alter multiple elite traits by stacking genes in a single variety. CRISPR/Cas- systems are currently being applied for enhancing yield, product quality, resistance to diseases and abiotic stress [69].

Novel allelic variants for breeding drought-tolerant plants have been generated in maize [70]. Starting from the observation that maize transgenic plants constitutively overexpressing ARGOS8, which is a negative regulator of ethylene responses, have reduced ethylene sensitivity and improved grain yield under drought stress conditions [70], new variants of ARGOS8 were generated employing CRISPR-Cas technology. Precise genomic DNA modifcation at the ARGOS8 locus was produced by inserting the native maize GOS2 promoter into the 5′-untranslated region of the native ARGOS8 gene or was used to replace the promoter of ARGOS8. The modifed ARGOS8 variants was highly expressed in all plant tissues and increased grain yield under fowering stress conditions when plants were grown in the feld [71].

CRISPR–Cas9 can be used for editing of cis-elements, such as in promoters, alternatively to the generation of weak alleles by targeting coding regions. The fnetuning of gene expression by editing of cis-regulatory elements can lead to quantitative trait variation. Weak promoter alleles of CLV3/EMBRYO-SURROUNDING REGION (ZmCLE) and ZmFON2-LIKE CLE PROTEIN1 (ZmFCP1) were engineered in maize with the aim to create quantitative variation for yield-related traits [72]. For CLE and FCP1 promoter mutagenesis, Cas9 with nine sgRNAs targeting promoter in accessible chromatin regions in developing ears were designed. Multiple maize grain-yield-related traits were successfully increased by using this strategy. In the same work, Liu and colleagues demonstrated in an elegant way that, in addition to weak allele promoter editing, exploitation of compensation among paralogs can be used for improving maize traits through genome editing [72].

The construction of whole-genome-scale mutant libraries is a modern approach for both functional genomic studies and pre-breeding improvement. Commonly in plants mutant libraries are based on random mutations induced by different mutagenesis procedures like irradiation, T-DNA insertions, (EMS), and transposons. In all these cases many generations to stabilize loss-of-function mutations are required, and additionally the process for determining the relationship between phenotype and genotype in all mutants is a long and critical process.

Liu and co-workers [72] reported the development of a CRISPR/Cas9-based editing platform adapted to high-throughput gene targeting in maize. Li and colleagues established a low-cost optimized and quality-controlled pipelines that includes the design of guide RNAs (sgRNAs) up to the identifcation of targeted genes and edited sequences. They selected knowledge-driven candidate genes and screened a large number of mutants up to T1 or follow-up generations, showing that their platform allowed functional gene cloning and validation [63].

# **6 Prospects**

In 2016, company researchers from Iowa–based DuPont Pioneer (currently Corteva) using the gene-editing tool CRISPR-Cas9, knocked out the endogenous maize waxy gene Wx1, which encodes the endosperm's granule-bound starch synthase responsible for producing amylose. Engineered maize contains starch composed exclusively of the branched polysaccharide amylopectin and not amylose. DuPont Pioneer expected the CRISPR-edited variety to have improved yields than conventional waxy maize and to commercialize the improved variety within fve years [73]. Due to the high costs associated with the deregulation of genetically modifed commercial maize hybrids only the largest agricultural biotechnology companies can afford these costs, with a consequent increasing concentration of maize seed providers [35]. It has been suggested that this scenario could be modifed by both the benefts of a wider application of gene-editing technologies and reduced regulatory oversight of CRISPR-derived varieties in comparison to transgenic GM breeding technologies [74].

Using maize as a model species and CRISPR/Cas9 technology, a very recent interesting European initiative has developed a pipeline called BREEDIT to generate a collection of multiplex gene-edited plants [75]. BREEDIT combines multiplex genome editing of whole gene families with crossing schemes to improve maize quantitative traits. The researchers were able to knock out 48 growth-related maize genes and produced a collection of over 1000 gene-edited plants which displayed 5–10% increases in leaf length and up to 20% increases in leaf width compared with the controls. BREEDIT has the potential to generate diverse collections of mutants for the identifcation of allelic variants for use in breeding programs.

The tremendous advances in both basic plant research and crop breeding brought about by CRISPR/Cas systems from frst applications to nowadays has made genome editing a powerful tool for precise maize improvement through multiple approaches, comprising point mutation, gene knock-out -in, allele replacement, fne-tuning of gene expression, and other modifcations at any potential genome locus. It is expected that in the next years novel strategies will be designed to improve the specifcity of Cas9-linked base editors, such as extending sgRNA guide sequences, and delivering base editors via RNP in many crops and in maize as well [58, 69].

Although many CRISPR-Cas-edited genes have been documented to improve maize traits of agronomic interest, only a few lines have been tested in feld trials, with only CRISPR-waxy maize hybrids having had a limited diffusion in United States [76]. Thus, much additional work for determining potential breeding values of edited maize lines must be done in terms of feld tests. For an effcient transfer of technologies from the bench to the feld there is still the need to elucidate the genetic and regulatory architecture of important traits as well as to increase the effciency of all steps of gene targeting and subsequent plant regeneration procedures. Finally, the integration of CRISPR-Cas technology in the breeding of new maize varieties also depends on existing and future regulatory policies that will be adopted worldwide.

# **References**


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# **Chapter 12 Using Gene Editing Strategies for Wheat Improvement**

**Domenica Nigro, Mark A. Smedley, Francesco Camerlengo, and Sadiye Hayta**

**Abstract** Despite wheat's global importance, it has trailed behind the other major cereals regarding genomic tools and resources as well as gene transformation. As each gene usually exists as two copies in the tetraploid durum wheat or as three in hexaploid bread wheat, it is very difcult assess gene function and improve important agronomic traits in polyploid wheat with traditional breeding methods. Recent advances allow researchers to use gene editing technologies in wheat which facilitates the opportunity to knockout or modify one, two or all three gene homoeologs simultaneously, which is important to clarify the function and contribution of gene copies in a specic phenotype or trait. CRISPR-Cas technology is now being used routinely for gene knockout. Technological advancement has been rapid within the eld, and recently more advance and precise methods have been deployed such as cytidine base editing, adenosine base editing, and prime editing in wheat. Here we summarised gene editing strategies that are presently being applied for wheat improvement.

# **1 Introduction**

Modern domesticated wheats are derivatives from ancient hybridisation events between ancestral progenitor species. The two most extensively cultivated wheats are the tetraploid durum or pasta wheat (*Triticum turgidum* ssp*. durum* L.) and hexaploid bread wheat (*Triticum aestivum* L.) Matsuoka and Nasuda [1]. Bread wheat is

D. Nigro

M. A. Smedley · S. Hayta (\*) John Innes Centre, Department of Crop Genetics, Norwich Research Park, Norwich, Norfolk, UK e-mail: sadiye.hayta@jic.ac.uk

#### F. Camerlengo Department of Agricultural and Food Sciences, Alma Mater Studiorum, University of Bologna, Bologna, Italy

Department of Soil, Plant and Food Sciences, University of Bari Aldo Moro, Bari, Italy

the major cultivated wheat species, while durum wheat accounts for about 5% of the total wheat production [2]. Both species have large genomes, durum ∼12 and hexaploidy ∼16 Gbp consisting mostly of repetitive elements. Within these polyploid species each gene usually exists as two copies in the tetraploid durum wheat or as three in hexaploid bread wheat. Homoeologous gene copies are usually highly conserved in gene structure and sequence among the subgenomes >95% [3]. Due to the polyploid nature of wheat, functional redundancy between homoeologs often occurs [4]. The traits controlled by recessive genes are particularly diffcult to observe due to their multiple homeologs. This means that it may be necessary to manipulate all homoeologs and paralogs simultaneously to measure a phenotypic effect and this is very diffcult to do it with traditional breeding methods [3]. A very low probability exists of the simultaneous mutation of genes in the A, B, and D genomes by natural processes or induced mutagenesis. Gene editing approaches gives the opportunity to knockout one, two or all three of the homoeologs of a gene, which is important to clarify the function and contribution of each homoeolog to a specifc phenotype or trait.

Unrivalled in its geographic range of cultivation, wheat accounts for ∼20% of the calorifc value and ∼25% of the daily protein intake of the world's population [5, 6]. In the 2020/2021 cropping season, over 770 million tons of wheat grain was harvested from over 220 million ha of arable land [7]. One of the three major cereal crops, along with maize (*Zea mays*) and rice (*Oryza sativa*), wheat has more infuence on global food security than any other crop [5, 8]. Notwithstanding wheat's global importance, however, it has, until recently, trailed behind the other major cereals in regard to the development of genomic tools and resources for its improvement [2, 3]. One such tool is wheat transformation, a prerequisite for many CRISPR-Cas gene editing applications, which until lately had been languishing with low transformation effciency ~5% and suffered with genotype dependence. Developments in open access robust transformation protocols and the use of morphological genes to improve regeneration, transformation effciency and reduce genotype dependence have recently made outstanding improvements [9–11].

High quality DNA reference sequences of target genes are required for CRISPR gene editing, recent advances in sequencing technologies and bioinformatic tools have expediated wheat gene editing studies. Researchers are able to target multiple homoeoalleles simultaneously by CRISPR-Cas which enables the production of targeted mutations in all copies of a gene; therefore, the system holds great promise in the characterisation of genes endowing important agronomic traits in polyploid wheat. Furthermore, gene editing has been used to modify multiple genes simultaneously controlling different agronomic traits [12].

In wheat, CRISPR-Cas is being used for yield enhancement, improvement of grain quality, biofortifcation, development of resistance against diseases, and tolerance against abiotic factors (Table 12.1). The promising outcomes of the CRISPRbased multiplexing approach circumvent the constraint of targeting merely one gene at a time. Moreover, deployment of CRISPR-Cas variant systems such as cytidine base editing, adenosine base editing, and prime editing in wheat has been used to induce precise point mutations. The combination of these novel technologies


**Table 12.1** Some agronomic traits improved by *CRISPR*/*Cas* in wheat

(continued)


**Table 12.1** (continued)

addresses some of the most important limiting factors for sustainable and climatesmart wheat that should lead to the second "Green Revolution" for global food security.

Here we summarised advanced gene-editing tools to facilitate sustainable wheat production and use of these tools for the improvement of genetic traits related to the agronomic performance.

# **2 CRISPR-Cas Gene Editing**

During the last two decades, there has been rapid development of genome-editing strategies that make it possible to directly target regions of genes in a DNA sequencespecifc manner. Site-directed nucleases (SDNs)-based gene editing technologies considerably enhance the precision of gene modifcation in plants [34]. This set of tools, comprising zinc fnger nucleases (ZFNs), transcription-activator-like effector nucleases (TALENs), and clustered regulatory interspaced short palindromic repeats associated protein (CRISPR-Cas) [35, 36], allow to repress or activate gene expression, modifcation of gene function, or create gene knockouts, mediating the targeted manipulation of DNA sequences [37]. Gene editing involves the introduction of targeted DNA double-strand breaks (DSBs) at the specifc targeted site within a gene by using an engineered nuclease, which induces cellular DNA repair mechanisms. Once a DSB is induced repair pathways, such as, the non-homologous end-joining (NHEJ) mechanism or homologous recombination (HR) pathway can repair the induced DSBs introducing simultaneously the desired modifcations at the target locus [38]. Due to its easiness, accuracy and effectiveness, along with its ability to produce transgene-free, gene edited crops, CRISPR-Cas rapidly diffused as the most used site-directed nucleases (SDNs)-based gene editing technology. The CRISPR-Cas system is usually introduced into plants as transgenes, however, in the following generation the transgene can be segregated away, leaving a transgene-free plant containing the desired mutations. Zhang et al. [13] developed a highly effcient transient expression-based gene-editing system for producing transgene-free and homozygous wheat mutants in the T0 generation. Liang et al. [40] reported an effcient gene edited method to produce transgene-free plants by CRISPR/Cas9 ribonucleoproteins (RNPs) in wheat [30, 39, 40].

In nature, CRISPR-Cas exists as a microbial adaptive immune system that uses RNA-guided nucleases to cleave foreign genetic elements such as bacterial phage. Biotechnologists have harnessed and modifed this system to enable gene editing applications in multiple species, including plants. There are over 30 different CRISPR-Cas systems naturally occurring that have been reported [41], however, the majority of gene editing applications are performed by CRISPR-Cas9 based systems, followed closely by CRISPR-Cas12a, previously known as CpfI. The Cas9 nuclease was frst thought to make a blunt DSB of DNA, three base pairs upstream of the PAM, however, evidence strongly suggests that Cas9 leaves a single nucleotide 5′ overhang [42]. Cas12a on the other hand leaves 5 bp staggered overhang at the opposite end of the PAM motif making it more favoured for applications such as gene targeting [43] or recently reported disruption of *cis*-regulatory elements within promoter regions [44].

Since the frst report of CRISPR-Cas technology being used for gene editing, technological advancement has been rapid within the feld. There are experimentally derived protocols for the selection of sgRNA targets, construct assembly, and screening analysis for genome editing in hexaploid wheat such as Smedley et al. [45]. Although, the majority of reports are for gene knockout, researchers are now able to perform targeted base changes through base editing, rewrite small length of DNA using prime editing, insertion of DNA via gene targeting, upregulation or suppression of gene expression as well as multi-gene knockouts.

# **3 Multiplex Gene Editing**

CRISPR-Cas based gene editing technologies enable the easy modifcation of two or more specifc DNA loci in a genome with high precision. These tools have greatly increased the feasibility of introducing desired changes in specifc but different genes, resulting in the development of new plant genotypes carrying multiple mutations in a single generation. There are three main strategies to produce multiple single-guide RNAs (sgRNAs), the conventional multiplex system with separate U3

or U6 promoters driving individual sgRNAs, the tRNA-processing system [46] and the ribozyme-processing system [47]. The polycistronic tRNA-sgRNA system consists of the sgRNAs linked together by tRNA sequences, the guides are transcribed in a single transcript. The tRNA sequences are then recognized and processed by endogenous RNases that excise the individual sgRNAs from the transcript [46]. The ribozyme system consists of a single transcript of multiple sgRNA, where individual sgRNAs are fanked by self-cleaving ribozyme sequences such as the hammerhead ribozyme (HH) and the hepatitis delta virus (HDV) ribozyme, [47, 48]. Transcripts are cleaved by the cis-acting ribozymes post-transcriptionally. Wang et al. [49] deployed a multiplexing gene editing approach based on the tRNA system in wheat to target *TaGW2*, *TaLpx-1,* and *TaMLO* genes, simultaneously. A similar tRNA approach was used in durum wheat cultivar Svevo by Camerlengo et al. [26] to edit the α-amylase/trypsin inhibitor subunits WTAI-CM3 and WTAI-CM16 in the grain to reduce allergen proteins. Abdallah et al. [50] created *TaSal1* mutants using this multiplex system to address drought tolerance in wheat. The three main multiplex CRISPR-Cas systems for simultaneous gene editing at 8 target sites in bread wheat were tested by Li et al. [51]. The ribozyme and tRNA systems were found to be more effective at gene editing than the conventional multiplex system with individual promoter driving individual guides [51].

# **4 Base Editing**

Base editing enables the generation of targeted point mutations without DSBs, DNA donor templates, or the reliance of the homologous repair (HR) pathway [52, 53]. Base editors consist of a DNA deaminase fused to a catalytically impaired Cas nuclease such as deactivated Cas9 (dCas9) or a Cas9 nickase (nCas9). The Casdeaminase fusion is guided to the target site by the guide RNA, where a single stranded DNA R-loop is formed allowing access for the DNA deaminase [54]. Where the deaminase is fused to SpCas9 variants, the 'activity window' for base editing spans approximately protospacer positions 4–8 (position 1 being the frst nucleotide of the protospacer, the PAM being at positions 21–23). There are two main classes of base editors which have been developed so far: cytosine base editors (CBEs), which catalyse the conversion of C/G base pairs to T/A base pairs; and adenine base editors (ABEs), which catalyse A/T-to-G/C conversions [52, 53]. Therefore, CBEs and ABEs can facilitate four possible transition mutations (C → T, A → G, T → C, G → A). Improvements in base editor effciency such as the inclusion of uracil glycosylase inhibitor proteins (UGI) typically CBEs such as BE3 [53] and optimisation of linker sequences Komor et al. [55] have substantially increased base editing yield, extensively reviewed in Anzalone et al. [54]. Zong et al. [56] used a CBE in both protoplasts and regenerated rice, wheat, and maize plants at frequencies of up to 43.48 in rice%. Li et al. [57], from same research group, described optimisation of an ABE for application in plant systems, demonstrating its high

effciency in creating targeted point mutations at multiple endogenous loci in rice and wheat. Han et al. [58] compared two ABEs, ABE7.1 and the new ABE8e containing a high-activity adenosine deaminase TadA8e, both were codon optimised for wheat. To aid nuclear localisation Bipartite-SV40-Nuclear-Localization-Signals (bpNLS) were added at the N-terminus of TadA\* and also a bpNLS followed by a nucleoplasmin NLS (npNLS) at the C-terminus of SpCas9n. Calling them wheat high-effciency ABEs (WhieABE7.1 and WhieABE8e), it was found in the study that WhieABE8e out performed WhieABE7.1 when targeting 5 wheat tubulin alleles.

# **5 Prime Editing**

Prime-editing technology enables targeted insertions, deletions and all 12 types of point mutation without requiring double-strand breaks or donor DNA templates. It expands the scope and capabilities of directly targeting and modifying genomic sequences. Prime editing is achieved by the Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) fused to the C-terminus of Cas9 H840A nickase (nCas9) together with engineered prime editing guide RNA (pegRNA). Primer Editors (PEs) directly copy the desired genetic information from the pegRNA into the target genomic locus [59]. The pegRNA contains two parts: a primer binding site (PBS) and an RT template (RTT) and guides the nickase to the target site by homology to a genomic DNA locus. nCas9 recognizes and nicks the nontarget DNA strand of the target site and releases a single-strand DNA, and the PBS then hybridises with the released DNA and serves as a primer for reverse transcription. The desired edits encoded by the RTT are then reverse transcribed and transferred to the nontarget DNA strand, generating a DNA fap that is subsequently incorporated into the target site by DNA repair [60].

Initially, PE1, PE2, PE3 and PE3b were characterized by Anzalone et al. [54] who developed the technology in mammalian cells. PE1 is a fusion of Cas9 nickase to the wild-type M-MLV RT. PE2 substitutes for the wild-type M-MLV RT an engineered pentamutant M-MLV RT. PE3 combines the PE2 fusion protein and pegRNA with an additional sgRNA that targets the non-edited strand for nicking. A variant of the PE3 system called PE3b uses a nicking sgRNA that targets only the edited sequence, resulting in decreased levels of indel products by preventing nicking of the non-edited DNA strand until the other strand has been converted to the edited sequence [54].

Lin et al. [61] compared PE2, PE3, and PE3b in wheat protoplasts and rice, they produced a wide variety of edits at genomic sites in rice and wheat. Prime-edited effciencies up to 21.8% were reported for regenerated rice plants [61]. An N-terminal RT–Cas9 nickase fusion PE and multiple synonymous nucleotide substitutions introduced into the RT template increased the average effciency of prime editing in rice to 24.3% Xu et al. [59]. An engineered plant prime editor (ePPE) was

developed by removing the ribonuclease H domain from M-MLV RT and incorporating a viral nucleocapsid protein with nucleic acid chaperone activity. This enhanced the frequency of various targeted modifcations, including base substitutions, insertions (34 bp) and deletions (90 bp) by an average of 5.8-fold in rice and wheat protoplast compared to PE2 [62]. An online design tool (PlantPegDesigner) was developed for designing effcient strategies for PE in plants, including dualpegRNA designs [60]. The pegRNA primer-binding site length, RT template length and nicking sgRNA position effects editing frequencies in plants [60, 61].

# **6 Other Strategies**

Strategies have been developed to deliver CRISPR system components into plant germline or meristematic cells that achieves genotype-independent editing. Plant RNA virus-based vector systems can deliver gene editing reagents into plant leaves. Based on this approach an engineered *Barley stripe mosaic virus*–based sgRNA delivery vector (BSMV-sg) was used by Li et al. [63] to perform heritable gene editing in Cas9 expressing transgenic wheat plants. The progeny in the next generation had editing at frequencies ranging from 12.9% to 100% in three different wheat varieties, and 53.8–100% of edited plants were virus free. The group achieved multiplex editing in the progeny using a pool of BSMV-sg vectors harbouring different sgRNAs and were able to generate Cas9-free wheat mutants by crossing BSMVinfected Cas9-transgenic wheat pollen with wild-type wheat.

Other delivery methods were developed in wheat. Nanomaterials have emerged as a promising candidate for delivery of genetic cargoes to intact plant cells. Functionalised high-aspect-ratio carbon nanotube (CNT) nanoparticles (NPs) have been successfully used in wheat leaves for effcient DNA delivery [64]. The use of nanomaterials for GE studies has yet to be reported.

Liu et al. [65] developed an *in planta* particle bombardment (iPB) method which has increased process effciency since no culture steps are required to create stably genome-edited wheat plants. The biolistic delivery of gold particles coated with plasmids expressing CRISPR-Cas9 components designed to target *TaQsd1* were bombarded into the embryos of imbibed seeds with their shoot apical meristem (SAM) exposed. A total of 2.51% of the bombarded plants (cv. "Haruyokoi," spring type) carried mutant alleles in the tissue. The method utilised transient expression of CRISPR-Cas and no detectable transgene integration was identifed. Kumagai et al. [66] reported an iPB-ribonucleoprotein (RNP) method which represents an alternative approach for creating genome-edited wheat varieties with an editing effciency comparable to the iPB-DNA method. Since no DNA is used, and therefore no transgene integration occurs, the iPB-RNP method has the potential for use in modern agricultural applications and commercialisation.


**Fig. 12.1** Pathway for the improvement of genetic traits in wheat by gene-editing and conventional plant breeding

# **7 Recent Applications of Gene Editing in Wheat**

Since the domestication of cereal crops, farmers selected plants with favorable agronomic traits that led to increased crop yield and performance. The introduction of semi-dwarf varieties and agrochemicals, starting from the second half of the last century, led to a leap forward in determining substantial increases in grain yield. In the modern agrifood context, crop yield remains one of the major traits to be improved to meet growing demands for food production and climate change effects. More recently, breeders and scientists also focused on the quality values of wheat grains to improve technological end-use quality of wheat four and the nutritional value of derived foods in addition to the aim to produce safer genotypes with reduced toxic, immunogenic and antinutritional compounds.

To date, gene-editing tools have been demonstrated to extensively contribute to the study and improvement of genetic traits related to the agronomic performance and end-use quality of wheat, decreasing the time for the generation of new genotypes harboring precise mutations that can enhance grain yield and quality (Fig. 12.1).

# **8 Gene Editing for Grain Quality Improvement**

Grain quality refers to the technological behavior of fours and doughs obtained from wheat as well as to internal quality factors such as nutrients and bioactive compounds. In recent years aspects related to nutritional and healthy values have gained more prominence since several disorders have been associated with wheat derived food consumption. In this respect, gene editing tools provide the ability to fne regulate the chemical composition of wheat kernels modulating biosynthetic pathways of major components such as protein, starch and lipids as well as offer the

opportunity to produce wheat with improved nutritional value, in which minor components play a predominant role.

Starch modifcations in wheat were widely explored in the last two decades leading to wheat genotypes with diverse amylose/amylopectin ratio that infuence not only the rheological behavior of doughs but also the nutritional value of derived foods. In general, starch contains two major glucose polymers, amylose and amylopectin, which differ in the degree of polymerization (DP) of glucan chains and in the frequency of branches. In wheat endosperm, starch consists of approximately 70–80% amylopectin and 20–30% amylose. Increasing the amount of amylose at the expense of amylopectin, the fraction of starch not digested and absorbed in the gastrointestinal trait (referred to as resistant starch) can be enhanced with a consequent benefcial effect on human health, correlated to blood glycemic index after wheat derived food consumption. Li et al. [24], using CRISPR-Cas9 in bread wheat, generated a series of transgene-free mutant lines with partial or triple-null *TaSBEIIa* alleles, an isoform of starch-branching enzymes. The triple-null lines (aabbdd) showed signifcantly increased amylose content, resulting in higher content of resistant starch, protein and soluble pentosan whereas a slight decrease of total starch was observed. On the other hand, they observed a series of pleiotropic effects related to plant growth and grain morphology traits besides negative effects on baking qualities of derived fours. Decreased plant height and tiller number, lower grain length, width and lower grain number associated with reduced thousand grain weight were observed in mutant lines compared to the control. In addition, the decrease in amylopectin content, generally recognized to be related to good end-use quality, strongly infuenced the viscosity parameters and negatively affected the rheological proprieties of doughs.

Dough stability time (ST) and SDS-sedimentation values (SV) are two of the major quality parameters used to determines the fnal quality of bread wheat fours. These two parameters are mostly infuenced by protein content and composition and are positively correlated to dough rheological properties. Sun et al. [27] reported for the frst time the effect of proteinaceous inhibitors of endo-xylanases. In particular, they found a QTL for dough stability time and SDS-sedimentation value on chromosome 6A (*QSt/Sv-6A-2851*) and identifed the *xylanase inhibitory protein* (*TaXip*) gene as the principal genetic component of the QTL producing variation in the above-mentioned parameters. They validated all the three homeoalleles (*TaXip-6A*, *TaXip-6B*, and *TaXip-6D*) producing CRISPR-Cas9 knock-outs, albeit they produced only two mutant genotypes (aaBBDD and AAbbdd). The SDSsedimentation value was signifcantly higher in both mutant lines compared to the control whereas the stability time value was signifcantly higher only for aaBBDD genotype compared to the control. Based on these results and on the observation of homoeologous gene expression in the grain at the later stage of grain development, the authors claim that *TaXip-6A* has a greater effect on quality parameters compared to *TaXip-6B* and *TaXip-6D.*

Zhang et al. [15] observed an increase in the SDS sedimentation volume of bread wheat lines in which they edited *TaGW2* homoeologous genes. Grain protein content and four protein content resulted considerably elevated in all the mutant lines, particularly in AAbbDD and AAbbdd genotypes. Glutenins and gliadins were also increased. Otherwise, they observed that the grains of double and triple mutants were morphologically wrinkled compared to the control plants and single mutants.

Grain quality defects such as late maturity amylase (LMA) and pre-harvest sprouting (PHS) are correlated with low Hagberg falling number (FN), which is considered an indicator of the amount of sprout damage caused by enzyme activity (α-amylase) producing the decrease of dough viscosity. PHS determines the germination of grains while they are still on the spike causing signifcant decrease of grain quality. Abe et al. [67] produced loss-of function mutations of *TaQsd1* gene that controls seed dormancy in wheat and barley resulting in longer seed dormancy. They generated all triple homozygous transgene-free genotypes (AABBDD, aaBBDD, AAbbDD, AABBdd, aabbDD, aaBBdd, AAbbdd, and aabbdd) but only the triple mutant (aabbdd) showed signifcantly different germination rates and reduced PHS.

The consumption of wheat derived foods is associated to the increasing incidence of wheat related pathologies such as Coeliac Disease (CD), allergies and Non-Coeliac Wheat Sensitivity (NCWS) but also food processing of wheat four can lead to the formation of antinutritional and toxic compounds.

Free asparagine is converted to acrylamide, a carcinogenic contaminant, during high-temperature processing of food made from wheat four. Raffan et al. [25] knocked out the asparagine synthase gene, *TaASN2,* in bread wheat genotypes to reduce the concentration of free asparagine in the grain. They observed an almost total reduction of free asparagine concentrations in the grain of triple-null mutants genotypes (aabbdd). In contrast, an increase in free glutamine, glutamate and aspartate was found in all the edited lines. Raffan et al. [68] used the low asparagine edited lines for the frst feld trial of genome-edited wheat lines in Europe.

The main triggering factors of CD are prolamins, glutenins and gliadins that are proteins contained in wheat grain endosperm and responsible for the gluten matrix formation. Sánchez-León et al. [14] used the CRISPR/Cas9 technology to reduce the content of α-gliadins in wheat kernels. Since α-gliadins represent a large protein family with high sequence homology among members, they produced multiplex editing of gliadins targeting conserved regions among the gene family members. Although pleiotropic effects on the other gliadins classes (ω- and γ-) were observed, the mutant lines could be used to produce low-gluten wheat derived foods.

Other endosperm protein families such as a-amylase/trypsin inhibitors (ATI), which are structural and metabolic proteins involved in plant defense mechanisms, can trigger the onset of wheat allergies and NCWS. Camerlengo et al. [26] used a multiplexing strategy to edit the ATI subunits WTAI-CM3 and WTAI-CM16 in durum wheat producing transgene-free wheat lines with a reduced amount of ATI. The mutant lines completely lacked target ATI subunits resulting in a decrease of their allergenic potential.

# **9 Gene Editing for Grain Yield**

One of the major goals of wheat breeding remains the improvement of yield. It is related to both the agronomic performance of crops as well as to environmental conditions. The constitution of semi-dwarf wheat varieties along with the introduction of chemicals and modern agronomic practice lead to a substantial increase of grain yield starting from the second half of last century.

Nowadays, a second "Green-Revolution" is required to face the global food demand due to the rapid increase of world population and to the worsening of climate change.

Grain morphology traits such as grain weight (GW), grain width (GWH) and grain length (GL) constitute a breeding target to enhance grain productivity. The knock-out of *TaGW2* homoeologous genes in two bread wheat cultivars had higher GWH and GL values and resulted in increased thousand grain weight (TGW) values of mutant lines compared to the control [15, 16]. Mutations in the homoeologous *TaGW2* genes had dosage-dependent effects on phenotypes in both bread wheat cultivar; although each homoeologous gene had different effect in the two genotypes, signifcant changes of TGW were associated with changes in gene dosage rather than with specifc combination of mutated alleles.

*TaGW7* encodes a TONNEAU1-recruiting motif (TRM) protein that affects grain morphology and weight in wheat and other cereal species. Wang et al. [17] demonstrated, by editing *TaGW7* in bread wheat, that mutations in the homoeologous genes of the B and D genomes increased the GWH and TGW but reduced the GL. They produced single (AABBdd) and double mutants (AAbbdd) with wider and shorter grains compared to the control, and *TaGW7-D1* seems to contribute at a greater extent to the phenotypic effects affecting grain size. Similar to *TaGW2*, *TaGW7* had dosage-dependent effects on phenotypes.

Another important target for improving grain yield is the *SQUAMOSA promoterbinding protein-like* (SPL) genes encoding for transcription factors that regulates a plethora of plant developmental and yield-related traits. The *SPL* family members are often negatively regulated by micro-RNA 156/157 (miR156). Gupta et al. [18] identifed the microRNA 156 recognition elements (MRE) in the 3′-untraslated region of the *TaSPL13* gene and, using CRISPR-Cas9, they generated mutations in the three homoeologous genes in bread wheat. Mutations in MRE led to a higher expression of *TaSPL13* which produced a decrease in fowering time, tiller number and plant height but increased grain size and number.

To improve grain yield farmers are used to applying nitrogen fertilizers, but this type of agriculture practice leads to aggravating environmental pollution and ecological deterioration. Furthermore, most of the modern wheat varieties show low Nitrogen Use Effciency (NUE) and absorb less than 40% of the supplied nitrogen. Zhang et al. [19] isolated and characterized the *abnormal cytokinin response1 repressor1* (*TaARE1*) gene in a Chinese winter wheat cultivar and then used CRISPR-Cas9 to generate a series of transgene-free mutant lines either with partial or triple-null *TaARE1* alleles. Loss of function mutations in this gene result in delayed senescence, enhanced NUE and increased grain yield under normal feld conditions. All the edited lines showed enhanced tolerance to N starvation with the AABBdd and aabbDD genotypes exhibiting signifcantly improved NUE without growth penalties compared to the control that results in increased TWG.

W eed competition is related to yield loss in wheat cultivation. Zhang et al. [20] generated transgene-free wheat germplasm by base editing the *acetolactate synthase* (*ALS*) and *acetylcoenzyme A carboxylase* genes. TaALS is a key enzyme in the biosynthesis of branched-chain amino acids and is known as an ideal herbicide tolerance target in wheat. Edited wheat lines, harboring different point mutations, were evaluated for the tolerance to multiple herbicides; homozygous mutants with four or six edited alleles showed enhanced tolerance to herbicides and grew normally whereas control plants died in few weeks after herbicide application. This strategy could be directly applied to produce wheat varieties tolerant to herbicides but also can be exploited as selection marker in wheat transformation and in vitro regeneration.

A number of important agronomic traits have been targeted by gene editing in wheat, although obtaining new high-yielding cultivars also resistant to biotic and abiotic stresses remains the main objective of most plant breeding programs. In the next sections, gene editing system successfully used for gene editing in wheat are reported.

# **10 Gene Editing for Biotic Stress Resistance**

Pest and disease, among biotic constraints, are estimated to determine 21.5% of current yield losses [69]. Modern agriculture relies on chemical compounds to avoid and prevent yield losses due to fungal diseases, but the extensive application of such chemicals severely affects both human health and the environment. So far, the development of fungus-resistant wheat cultivars has been a noteworthy goal in wheat breeding programs. Gene editing in wheat has targeted several genes for improvement of resistance against diseases caused by fungi, specifcally *Blumeria graminis* and *Fusarium graminearum*.

The frst successful experiment using the CRISPR-Cas system in wheat was reported by Shan et al. [70], who edited the *MLO* gene, which encodes for the MILDEW-RESISTANCE LOCUS (MLO) protein. The authors reported a mutation frequency of *TaMLO* in protoplasts of 28.5%. MLO has a negative resistance function, thereby causing susceptibility to powdery mildew in plants expressing this gene [71]. Powdery mildew diseases are caused by *Blumeria graminis* f. sp. *tritici* and result in signifcant wheat yield losses. As knockout of the *TaMLO* leads to disease resistance, this gene was an ideal target for RNA-guided Cas9 knockouts to improve powdery mildew tolerance. The approach was successfully demonstrated by Wang et al. [28], who simultaneously targeted the three homoeoalleles of *TaMLO* in hexaploid bread wheat with both CRISPR-Cas9 and TALEN technologies using particle bombardment, which resulted in powdery mildew resistant plants. The mutation frequency of regenerated *TaMLO*-edited wheat (5.6%) was similar for both editing methods.

More recently, Zhang et al. [30] demonstrated an improved powdery mildew resistance in wheat by simultaneously modifying the three homoeologous of *TaEDR1* by CRISPR-Cas9 gene editing. The *enhanced disease resistance1* (*EDR1*) gene is a negative regulator of resistance to powdery mildew in Arabidopsis Frye et al. [72]. The authors targeted a highly conserved region within the coding sequence of *TaEDR1*, obtaining homoeologous stable *Taedr1* mutations inherited in the T1 generation with a transmission rate of 97–100%. *Taedr1* plants were resistant to powdery mildew and did not show mildew-induced cell death.

Several studies have also been focused on *Fusarium gramineaurm* resistance, a fungus causing one of the most detrimental diseases in wheat. The lipoxygenase genes, *TaLpx* and *TaLox*, have been found to be good targets for gene editing resistance to Fusarium. These enzymes play a key role in the jasmonic acid-mediated defense responses in plants by catalyzing the hydrolysis of polyunsaturated fatty acids and then activating oxylipins biosynthesis. Nalam et al. [73] found that *TaLpx-1* gene silencing resulted in resistance to *Fusarium graminearum* in wheat. Shan et al. [16, 70] respectively edited *TaLpx* and *TaLox* genes in protoplasts, detecting a mutation frequency of 9% and 45%. Further studies by Zhang et al. [13] allowed to obtain wheat plants with mutated *TaLOX* with a frequency of 9.5%, of which homozygous mutants accounted for 44.7%.

Fusarium head blight (FHB) resistance was also investigated by targeting three wheat genes including an ABC transporter (*TaABCC6*), the nuclear transcription factor X box-binding like1 (*TaNFXL1*) both associated with FHB susceptibility, and a gene encoding a nonspecifc lipid transfer protein (nsLTP), *TansLTP9.4*, which correlates with FHB resistance. PCR amplicons from protoplasts transformed with editing constructs were sequenced, showing that the three genes had been successfully edited with effciencies of up to 42.2% [74]. Another target for Fusarium resistance was reported by Su et al. [32], who identifed HISTIDINE RICH CALCIUM-BINDING PROTEIN (*TaHRC*) as a quantitative trait locus (QTL) responsible for resistance to Fusarium head blight. By cloning and sequencing a candidate gene they found a single deletion on the B genome homeolog, which was suffcient to determine resistance to Fusarium head blight. This result was confrmed by CRISPR-Cas9 approach targeting the *TaHRC* homeolog from the B subgenome, which caused frameshift mutations resulting in more than 40% reduction in fusarium head blight disease in wheat. This study showed that it is not always necessarily to knocked out all three homeologs in wheat, and in some cases the inactivation of one single homeolog is enough to successfully induce disease resistance.

More recently, Brauer et al. [31] followed a different approach to generate mutants resistant to Fusarium head blight. They focused on the NUCLEAR TRANSCRIPTION FACTOR, X-BOX BINDING 1-LIKE (NFXL1), a transcription factor used by Fusarium to repress defense responses upon infection, which is present as two copies on each of the wheat subgenomes. The authors showed that NFXL1 downregulation via RNAi in barley confers partial resistance upon

infection. Wheat NFXL1 knockout mutants with edits in all six homoeologous in the T1 generation were then obtained by designing sgRNA pair targeting all six loci simultaneously (one homeolog remained in heterozygous state), thus showing an increased Fusarium head blight resistance, similarly to that obtained through RNAi.

# **11 Gene Editing for Abiotic Stress Resistance**

Using CRISPR-Cas to design resilience to abiotic stress in an era of global warming and extreme weather events is a justifable aim. Abiotic stresses such as drought, extreme temperatures and salinity affect plant growth, survival, reproducibility and production, furthermore, it was shown that abiotic stresses modulate epigenetic changes in plants [75].

Genome engineering represent an effective approach to increase plant growth in response to abiotic stress in wheat, especially considering that abiotic stressassociated genes involved in cellular and molecular responses can easily be targeted with the available gene editing tools. So far, only few studies have shown the application of gene editing to improve abiotic stress response. CRISPR-Cas9 gene editing was successfully used in transient transformation in wheat protoplast. Two important abiotic stress-responsive transcription factor genes were targeted: *TaDREB2*, (wheat dehydration responsive element binding protein 2) and *TaERF3*, a wheat ethylene responsive factor 3 [33]. The results suggested that CRISPR-Cas gene editing has huge potential for manipulation of wheat genome to improve stress tolerance and obtain better crop performances, despite the challenging ploidy level of wheat. Abdallah et al. [50] used a multiplex gene editing approach to knockout the fve active homologous gene copies of *TaSal1* in wheat variety Giza 168. In the primary transgenic plants 34% showed editing, these edits were heritable, and, in the progeny, fve lines were identifed with all fve copies of *TaSal1* edited. Young leaves of edited *TaSal1* lines showed closed stomata, increased stomata width and increase in the size of the bulliform cells. *TaSal1* edited seedlings germinated and grew better on media containing polyethylene glycol than wildtype seedlings [50].

Further research and breeding programs will still be necessary to better elucidate the genetic and physiological bases of metabolic and signaling pathways of stress tolerance mechanisms. The identifcation of gene regulatory networks involved in stress responses and their targeting by gene editing tools will allow the development of new stress tolerant and high yielding varieties.

# **12 Summary**

Recent advances in the production of high-quality genome sequences and effcient genotype- independent transgenic methods, developments in CRISPR-Cas based gene editing tools bring functional genomics and rational design-based molecular

breeding of polyploid wheat to unlock its hidden potential. Gene edited transgene free wheat has a critical role in addressing environmental issues while promoting sustainable agriculture and global food security. Gene editing is just one of the methods advancing wheat breeding programs and supporting wheat biology, which can be used alongside more conventional commercial breeding methods for delivering sustainability.

# **References**


#### 12 Using Gene Editing Strategies for Wheat Improvement


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# **Chapter 13 Gene Editing of Wheat to Reduce Coeliac Disease Epitopes in Gluten**

**Marinus J. M. Smulders, Luud J. W. J. Gilissen, Martina Juranić, Jan G. Schaart, and Clemens C. M. van de Wiel**

**Abstract** By using gene editing technologies such as CRISPR/Cas, precise modi cations can be made in the genome. CRISPR/Cas is especially valuable for targeted mutagenesis in polyploids, as it can induce mutations of multiple alleles simultaneously, to obtain regenerants that are homozygous for the desired mutation. A range of gene-edited traits have been developed in hexaploid bread wheat, including various nutrition and health-related traits, plant architecture, pest and disease resistance, tolerance to abiotic stress, and traits that enable more efcient breeding. Wheat is also known as a cause of some human diseases, particularly coeliac disease (CD), with a prevalence of 1–2% of the population. In the EU alone, at least 4.5 million people suffer from it. CD is a chronic inammation of the small intestine, induced and maintained in genetically predisposed individuals by the consumption of gluten proteins from wheat, barley and rye. As there is no cure, patients must follow a life-long gluten-free diet. The dominant epitopes in gluten proteins that trigger the disease, have been characterized, but they cannot be removed by classical breeding without affecting baking quality, as it concerns over 100 gluten genes that occur partly as blocks of genes in the genome of wheat. Using gene editing, two studies have shown that it is possible to modify the epitopes in several alpha- and gamma-gliadins simultaneously, while deleting some of the genes completely. In some lines more than 80% of the alpha-gliadin genes were modied. These proofof-principle studies show that it is feasible to use gene editing, along with other breeding approaches, to completely remove the CD epitopes from bread wheat. Gene-edited coeliac-safe wheat will have economic, social and environmental impact on food security, nutrition and public health, but the realisation will (partially) depend on new European legislation for plants produced by gene editing.

M. J. M. Smulders (\*) · L. J. W. J. Gilissen · M. Juranić · J. G. Schaart C. C. M. van de Wiel

Plant Breeding, Wageningen University & Research, Wageningen, The Netherlands e-mail: rene.smulders@wur.nl

# **1 Introduction**

Bread wheat (*Triticum aestivum*, a hexaploid wheat species with the AABBDD genome) originated as a hybridization between tetraploid *Triticum* and (the) diploid *Aegilops* species during early agriculture in the Fertile Crescent. *Triticum* and *Aegilops* are closely related: Triticum is considered the domesticated form of *Aegilops*. Interspecifc hybrids are found in nature and may also be produced artifcially. Long before the onset of agriculture, a diploid wheat (*T. urartu*) carrying an AA genome hybridized with a grass-like wheat with a BB genome into the tetraploid *T. turgidum* with the combined AABB genome constitution. Near the Caspian Sea area, in an ancient agricultural feld, a cultivated free-threshing tetraploid wheat (AABB) hybridized with a local wild diploid *Aegilops tauschii* with its DD genome, which led to the new hexaploid species *T. aestivum* (AABBDD). Spelt wheat is considered the result of one of the most recent natural hybridizations in which *T. aestivum* and a tetraploid *T. turgidum* (an emmer wheat type) were involved. Presently, synthetic hexaploid wheat lines are under production through various artifcial hybridization programs, to increase the diversity in the D genome in hexaploid germplasm [1–3].

Wheat is a self-pollinating crop. Farmers can cultivate it by sowing seed material saved from the preceding year. Initially, cultivation practice applied mixtures of tetraploid and hexaploid genotypes. Through conscious or unconscious selection of spontaneous mutants, landraces gradually adapted to local environmental conditions, but there is a limit to the improvement of end-use quality that can be achieved in that way. Since the early twentieth century, genetics has been used in professional wheat breeding, including pure line selection and targeted breeding. Breeders are always interested in new genetic variation. This can be achieved through introgression (trait transfer through hybridization followed by back-crossing) from other wheat species, but this process also introduces many undesired traits that subsequently must be selected against. Alternatively, genetic variation can be induced within a cultivar through the application of mutagenic chemicals or ionizing irradiation (mutation breeding).

Presently, next to the hexaploid bread wheat and spelt wheat also diploid einkorn and various tetraploid *T. turgidum* subspecies (including emmer and durum wheat) are still being cultivated. However, almost 95% of the world's annual volume of 700–750 million metric tons is bread wheat; durum wheat makes up only 5%, the other species have a minimal volume. Bread wheat and durum wheat are also referred to as soft wheat and hard wheat, respectively, due to the difference in hardness of the kernel and further technological qualities regarding milling and baking: bread, cakes and breakfast cereals are made from soft wheat, pasta from durum wheat.

Its three homoeologous genomes make bread wheat fexible and adaptive in cultivation and versatile in its applications, due to its high genetic and allelic redundancy. This versatility becomes apparent in the specifc qualitative and quantitative protein composition of its water-insoluble gluten, as used in various food and nonfood processing applications, and, together with the grain's starch, in dough and

derived baked foods. Remarkable is the interdependence of the gluten protein structure, the gluten quantity and the gluten applicability regarding genetic, environmental and processing factors [4–6].

In keeping with its wide cultivation and application, bread wheat is the species of choice for further improvement of agricultural and food technological traits through conventional breeding, using various breeding tools developed in the twentieth and twenty-frst century, including marker-assisted breeding and speed breeding [7].

In wheat breeding, the main focus was traditionally directed to yield and quality trait improvements. This has culminated in the 1960s in the Green Revolution through the introduction of dwarf genes. The dwarf genes (actually, dwarfng mutations) reduce energy investment in stem growth and increase grain yield. These new varieties were highly recognized worldwide. Today's breeding aims still include yield (especially starch quantity) and gluten and starch quality (for improved milling and baking quality), but adaptations to the biotic and abiotic environment, such as disease resistance genes, are receiving more attention because of the spread of major diseases and the threats of climate change, e.g., increased drought.

Impacts on export markets appeared to have played a role in the lack of commercialized GM wheat. The frst GM (herbicide-tolerant) wheat applied for approval in the USA was abandoned in 2004 [8]. HB4 wheat, a GM wheat variety with drought tolerance has been approved for cultivation in Argentina in 2020 and subsequently for import in Brazil in 2021, which was important as for commercialization of GM events Argentina takes the impact on export markets into account. Since then, import has also been approved in the USA, Australia, New Zealand, Colombia, and Nigeria. Recently, Brazil has also approved cultivation of HB4 wheat.

# **2 Gene Editing in Polyploid Crops**

To facilitate the time-consuming breeding of polyploid crops, alternative methods for introgression breeding and mutation breeding are important for polyploid crop improvement. Genome editing is such an alternative method. By using gene editing technologies such as CRISPR/Cas, precise modifcations can be made to specifc genes in the genome. CRISPR/Cas is especially valuable for targeted mutagenesis in polyploids, as it can induce mutations of multiple alleles simultaneously, so that regenerants homozygous for the desired mutation may be obtained. Moreover, the intrinsic specifcity of CRISPR/Cas allows for the precise targeting of specifc homoeologous genes. This enables allele-specifc modifcations, where only one copy of a gene is edited while leaving the others unchanged, or where all homoeologous copies are modifed simultaneously. This ultimately allows for the introduction of desirable traits or the removal of undesirable ones without the need for laborious backcrossing to eliminate unwanted genetic material. Several studies have demonstrated the effectiveness and robustness of gene editing for targeted mutagenesis in auto- and allopolyploids [9], including wheat ([10]; see below and Table 13.1).

Trait Gene(s) Reference **Health** Low asparagine to suppress acrylamide production during heating *ASN2* [20–22] High-amylopectin & low-discolouring Waxy *GBSS* & *PPO*, respectively [23] High-fber wheat pipeline ? Calyxt news releases Coeliac Disease (CD) epitopes Alpha-gliadins [24] Coeliac Disease (CD) epitopes Alpha- and gamma-gliadins [25–27] **Biofortifcation** Carotenoids *Psy1* [23] **Polyploidy/Recombination (incl. homoeologous pairing)** Higher homoeologous CO (crossover) frequency (to facilitate introgressions from wild genomes) *Tazip4-B2* [28] Testing effects on CO *SPO11–1* [29] **Haploid induction** Paternal haploid induction by hetero-allelic combinations of genome-edited TaCENH3α *TaCENH3α* [30] Haploid induction combined with targeted mutagenesis by using maize pollen (sperm cells) transgenically expressing *cas9* and selected gRNAs Tested using *BRI1* or *SD1* involved in dwarfng [31] **Male sterility** Male sterility *TaDCL4*, *TaDCL5*, *TaRDR6* [32] Male sterility *Ms1* [33] Maize (and rice) counterpart Ms45 encoding strictosidine synthase-like enzyme potentially enabling an SPT-like hybrid seed production in wheat *Ms45* [34] **Plant architecture** Alternative semi-dwarfng allele *Rht-B1b* combined with *tasd1* [35] Possibly reproducing semidwarf alleles not negatively affecting coleoptile length hampering deep sowing in dry environments, such as Rht18, e.g., recessive semi-dwarfng in rice by GA20ox2 knockout *GA20ox2* [36] TaQ alleles involved in domestication, impacting plant height, spike architecture AP2-like TFs *TaAQ* and/or *TaDQ* [37] **Yield** Grain size and number, plant architecture, fowering timing miRNA156 site in *TaSPL13* [38] Higher fower bearing spikelet number & higher grain protein miRNA165/166 site in *HB-2* (HD-ZIP III) [39]

**Table 13.1** Overview of research and trials using gene-edited traits in wheat. This overview was compiled in April 2023, and it is not exhaustive

(continued)



# **3 Improving Regeneration of Wheat in Tissue Culture**

Effcient protocols for plant transformation and regeneration in tissue culture are essential prerequisites for utilizing gene editing techniques in wheat. However, the process of regeneration in tissue culture presents diffculties, particularly when dealing with recalcitrant species and is often dependent on identifying genotypes with capacity to regenerate. Overcoming these hurdles is crucial for wheat tissue culture as well. Promising advancements have been made in this area, such as the utilization of the developmental regulators that stimulate somatic embryogenesis and de novo meristem formation. Notably, recent studies have demonstrated that overexpressing a *GRF4-GIF1* chimera construct and the *TaWOX5* gene can substantially increase the regeneration capacity of wheat tissue cultures, while reducing the dependence on specifc genotypes [11, 12]. These fndings open new avenues for improving the regeneration effciency of wheat in tissue culture and pave the way for more accessible and genotype-independent regeneration protocols. Continued research and exploration of innovative strategies, along with the advancement of gene editing techniques, can contribute to further enhancing the regeneration potential of wheat and accelerating the development of gene-edited varieties.

# **4 Current Research and Trials Using Gene Edited Traits in Wheat**

A range of gene edited traits have been developed in wheat, largely in the last 5 years (Table 13.1). These include various nutrition and health-related traits such as low asparagine content, high fbre content, reduced coeliac disease epitopes in gluten, and biofortifcation. Next to these, plant architecture, yield improvement, pest and disease resistance, tolerance to abiotic stress, and herbicide tolerance are also investigated. The studies used Cas9 and Cpf1 [13–16] nucleases or prime editing [17] for targeted mutagenesis, as well as some base editors (see Table 13.1). Luo et al. [18] worked towards homology-directed repair enabling the introduction of larger sequences such as complete genes or promoters.

A series of breeding-related traits are also subject of gene editing research, including recombination, crossing over, haploid induction, and male sterility. These are related on one hand to the need to introgress traits, such as disease resistances and quality traits, from wild relatives, on the other hand to the desire to develop hybrid wheat varieties. When compared to traditionally inbred varieties, hybrid wheat is estimated to have a potential yield increase of 10% [19]. Furthermore, hybrid wheat, as with other hybrid crops, offers enhanced resilience and provides greater yield stability, even in the presence of environmental stresses and the extreme impacts of climate change. However, the breeding and widespread adoption of hybrid wheat is lagging behind. One reason is a costly production of hybrid seeds due to wheat's inherent tendency for self-pollination and the production of only one seed per fower [19]. Overcoming these barriers and establishing effective breeding techniques for large-scale hybrid wheat breeding requires substantial research and development efforts. For example, male sterility provides the method to block selffertilization, while modifying fower architecture can facilitate pollination.

Below we will in detail describe why and how gene editing and other approaches that target wheat gluten are used to reduce or remove epitopes that cause coeliac disease in susceptible people.

# **5 Wheat Gluten**

Flour obtained by milling of whole wheat grains can be separated into four major protein fractions: albumins plus globulins, gliadins, glutenins, and a group of residual proteins. The gliadins and the glutenins, both water-insoluble, can be further subdivided into subtypes (Table 13.2). Cysteine residues, although relatively low in number, are characteristic to most gluten molecules (except to the omega-gliadins) as they form intrachain bonds in the alpha- and gamma-gliadins, stabilizing their tertiary structure, and interchain bonds among LMW and HMW glutenins. Gluten polymerization, a unique characteristic of gluten from wheat, is crucial for doughmaking and baking. The building of large gas-retaining gluten networks may even increase through opening of intrachain gliadin S-S bonds by heating, enabling their binding to glutenin polymers [5]. High hydrostatic pressure (extrusion) of isolated guten to increase further polymerization can be applied in the production of meat analogues [67].

Wheat four plus water produces a viscoelastic mass (a starchy dough) in which the gliadins are especially responsible for viscosity, whereas the glutenins are responsible for elasticity of the network that retains the gas from the yeast or sourdough fermentation and makes the dough rise. The degree of viscosity relates to the ratio of gliadins to glutenins: a high ratio (around 3.0) results in a soft and less


**Table 13.2** Classifcation of gluten proteins and their main characteristics

Adapted from Ref. [5]

a As monomer, but as (combined) oligo- and polymers in stable 3D structures with MWs from 700 to 10,000 kDa [70]

viscous dough; a low ratio (around 2.0) results in a highly viscous and strong dough [5]. Heating during baking makes the gluten rigid by the loss of its water to the starch, keeping the baked bread shape stable. The presence of glutenin in doughs is essential for baking of bread and cookies, and in batter for cakes. Gliadins may be exchanged for other proteins or even partly omitted without signifcant loss of baking quality [68]. Guzmán-López et al. [69] managed to bake bread from wheat lines in which most gliadins had been silenced by RNAi.

Removing the starch from unheated dough by repeated washings with water yields in a purifed gluten mass, called vital wheat gluten (VWG), which is applied in the food (bakery) and non-food industry.

# **6 Health-Related Wheat Components in Food**

Due to its food technological versatility, wheat and its isolated components (vital wheat gluten, wheat starch, and derivatives such as glucose syrups, maltodextrin, sorbitol) are applied in numerous food products. They appear in about 30% of the labelled and mostly highly processed food items [71]. Highly processed foods, including those with wheat-derived ingredients, are often classifed as unhealthy as they often contain a too high content of non-wheat ingredients such as sugar, salt and fat, and a too low content of fbre. However, whole grain wheat foods are recognized as healthy for their proven contribution to the reduction of risk of several 'western lifestyle'-related chronic diseases including obesity and diabetes, heart and vascular diseases, immune-related diseases, and certain forms of cancer. Therefore, consumption of whole grain (wheat) foods is stimulated by governmental food authorities in many countries [72, 73].

# **7 Wheat-Related Human Diseases**

Wheat is also known as a cause of some human diseases: wheat allergy with a prevalence of 0.25% is relatively rare; non-coeliac wheat sensitivity (NCWS) with a self-reported prevalence of 10% and a clinically estimated prevalence of 1% has mostly mild symptoms; and coeliac disease (CD), the most severe but also the bestknown and well-documented disease, has a prevalence of 1–2% [74, 75]. CD is a chronic infammation of the small intestine, induced and maintained in genetically predisposed individuals by the consumption of gluten proteins from wheat, barley and rye. As there is no cure, patients must follow a life-long gluten-free diet. In the EU alone, at least 4.5 million people suffer from CD.

The general daily intake of gluten from the consumption of bread and related foods is about 15 g [76], whereas the safe daily amount of gluten in foods for individuals with CD is estimated between 10 and 100 mg gluten [77]. To stay below that load, food products should not surmount the 20 ppm threshold as is defned

according to extensive research and established in EU regulation EC828/2014. Note the huge gap between current foods in a gluten containing and a gluten-free lifestyle, especially when considering the high number of food items in supermarkets and elsewhere processed with wheat and wheat-derived components. As a correct diagnosis of CD is diffcult because of the great variety of occurring symptoms, it is estimated that 85% of the patients remain undiagnosed, which means that they daily consume gluten, unaware that their bad chronic health status is caused by this food ingredient.

# **8 Gluten-Free Lifestyle**

During the last decades, a gradual but signifcant increase has been observed in the number of consumers that embrace a gluten-free or wheat-free diet. This gluten/ wheat avoiding population varies globally according to geographic location between 3.7% and 17.2% [78]. The main incentive for this diet choice is the desire for better health. This trend creates a decreasing bread and wheat-product market with a negative impact on the whole wheat value chain on the one hand, and an increasing and diversifying gluten-free product market on the other hand, with an annual growth rate of 10.4% between 2014 and 2019 in Europe, and an estimated compound annual growth rate (CAGR) of 8,1%. It had a market value of 8.3 Billion US dollars in 2025 (https://www.statista.com/statistics/248467/global-gluten-free-foodmarket-size/). The global gluten-free packaged food market now represents about 3% of the total global packaged food market, refecting the population share that is interested in gluten-free foods, which is 15–20 times higher than the diagnosed CD population.

The problem remains to the 85% undiagnosed CD population. This group would be really served with the development, global cultivation and processing of safegluten wheat varieties replacing current high CD-immunogenic wheat. Gluten safety of such varieties should be guaranteed quantitatively (elimination of all immunogenic gluten) or qualitatively (inactivation of CD-immunogenic fragments (epitopes) from individual gluten proteins). However, maintenance of the full food technological and health qualities combined with unimpaired agronomic quality in such varieties remains a prime prerequisite for a crop to be competitive on the (global) market.

# **9 Gluten Epitopes for Coeliac Disease**

What makes gluten intolerable to certain genetically predisposed individuals developing CD? The high abundance of glutamine and proline amino acids in gluten proteins hinders degradation by the proteolytic enzymes in the digestive tract, leaving relatively long peptides in the small intestine. Several of these peptides contain a core sequence of nine amino acids that can be recognized by antigen-presenting cells and presented to T cells. Upon recognition, these T cells become activated, resulting in infammation of the mucosa followed by degeneration of the mucosal villi with serious consequences for uptake of nutrients and leading to a variety of symptoms. Several epitopes are recognized by T cells from many patients, called 'major' epitopes. A comprehensive list of epitopes was recently updated [79].

Gluten proteins are encoded by large gene families located at different sites on the wheat genome (Table 13.2) with the alpha-gliadin loci on the short arm of the homoeologous chromosomes 6A, 6B and 6D, and the gamma-gliadin genes on the short arm of 1A, 1B and 1D. A single bread wheat variety genome (e.g., from the variety Chinese Spring for which the genome has been sequenced [80]), may contain as many as ~100 different gluten genes of which about sixty are expressed into proteins, as was shown by mass spectrometry analysis [4, 81–83]. Comparing different wheat varieties, variation exists in the number of expressed gluten genes, in the sequences of the encoded proteins, and in the amount of protein produced per gene. Additional variation in the gluten composition (quantitatively and qualitatively) is induced by environmental factors in the feld during the growing season, such as the temperature during certain stages of crop development, and nutrient availability from the soil, in particular nitrogen and sulphur [4].

Although both gliadins and glutenins harbour immunogenic epitopes that can trigger CD, the alpha-, gamma- and omega-gliadins contain by far the highest number and the most dominant (severe) epitopes. For alpha-gliadins the dominant epitopes are related to the genes from the D-genome, followed by the genes from the A-genome, whereas some genes from the B-genome are free from epitopes [84]. D-genome alpha-gliadins contain the so-called 33-mer peptide carrying up to six overlapping CD epitopes and is a strong binder and activator of human T cells; similarly, some gamma-gliadins have a highly immunogenic 26-mer peptide.

# **10 Breeding Methods for Coeliac-Safe Wheat**

Due to its genetic fexibility, several advanced breeding techniques are being used in wheat [85]. Wide hybridizations may result in irregularities (translocations as well as deletions) in the genomes of embryos and their later plant gametes. Chromosomal deletion lines, with impacts regarding reduction of CD-immunogenic gliadin loci, have been found and tested [86]. Chromosomal translocations and deletions may be induced technologically (as was done in the varieties Chinese Spring and Paragon, amongst others) but they also occur naturally. They are interesting for genetic research, but often the deletions impact the performance of the plants in the feld.

Synthetic hexaploids can be obtained from hybridizations of wheat varieties and species of different ploidy levels (e.g., a tetraploid durum female with AABB genome with a diploid *Aegilops* male with DD genome) requiring duplication of the originally allotriploid embryonic genome (ABD into AABBDD) to form a fertile hexaploid hybrid. An *Aegilops* line selected from the very wide D-genome diversity with a favourable CD profle may be hybridized with a low-immunogenic durum mother line. The resulting new hexaploids can be tested for their epitope profle at the gene and protein level, and applied in further breeding programs if desired [3]. This would be one way to eliminate the 33-mer in the D-genome alpha-gliadins.

EMS mutation breeding can generate large numbers of random mutations and could be applied to mutate gliadin genes, but it would be very resource-intensive to trace and combine mutations in multiple genes, from many plants, into one single, coeliac-safe and well-performing wheat plant [87]. Using γ-irradiation, large chromosomal deletions have been induced in two mutant lines of the wheat variety Paragon. Both mutants revealed a lack of sequence coverage in a large part of the gluten loci [25]. However, a clear need for more sophisticated approaches remains.

# **11 RNA Interference**

RNA interference (RNAi) is a system that may be used to interfere with the synthesis of gluten proteins through their RNA transcripts, while the DNA still contains the intact genes. With a single RNAi construct designed for a conserved region that is common for many gluten genes, Gil-Humanes et al. [88] achieved up to 92% reduction of the gliadins, and a 10–100 fold reduction of epitopes as detected in T-cell tests. Similarly, the expression of twenty α-gliadin genes was decreased, although the production of other storage proteins increased [89]. Some of the wheat lines with reduced immunogenicity but with the baking quality largely intact [90] were suffciently low in gliadins for successful clinical trials with patients [69]. Unfortunately, as RNAi requires stable genetic modifcation (GM) of the construct into wheat to silence the gliadins, and no transgenic wheat has been commercialised yet, it is unlikely that this transgenic line will be brought to the market.

# **12 Gene Editing of Gluten in Wheat**

Two successful proof-of-concept studies with gene editing have been carried out in wheat. Sánchez-León et al. [24] targeted conserved sites in alpha-gliadin genes of a single wheat line. In this study, 47 offspring gene-edited plants were genotyped, and they found smaller and larger deletions at the target site, and plants with varying numbers of genes edited. In one plant 35 of the 45 alpha-gliadin genes were mutated, and seeds of this plant showed 85% reduction of total gluten protein as measured with the R5 gluten quantifcation assay, which is the type I method for gluten detection in the Codex Alimentarius. Guzmán-López et al. [91] developed a bioinformatics pipeline to show that in some offspring lines alpha-gliadin genes were deleted.

In the other study, Jouanin et al. [27] simultaneously targeted multiple sites in alpha-gliadin as well as gamma-gliadin genes, and confrmed 117 gene-edited

plants, including plants with mutations in both gene families. Some gluten genes were deleted, as evidenced by droplet digital PCR (ddPCR) results for alphagliadins [27]. Evidence that in some regenerated plants blocks of genes had been deleted came from mapping of sequence data obtained with GlutEnSeq, a variant to RenSeq that targets gluten genes, to the Chinese Spring reference genome [25]. This is presumably the result of double-strand breaks in different gliadin genes that occur in tandem arrays in the wheat genome, leading to the deletion of tandem gene blocks in between the genes with double strand breaks.

As not all target sites in the gliadin genes will be mutated in a single progeny plant, gene editing will lead to populations of plants that each may contain a mosaic of unaffected, edited, and lost genes. The edits on different chromosomes will segregate in the next self-pollinated generations. Hence, it is useful to have a rigorous selection program aimed at maintaining only the few most promising plants (genotypes) for multiplication, cultivation and eventual application in coeliac-safe(r) food [92]. In this process, gene-edited loci may be combined with loci that are lost through smaller or large deletions.

# **13 Impacts of Gene-Edited Low CD-Immunogenic ('Low-Gluten') Wheat**

Producing low CD-immunogenic (hypo-immunogenic or 'low-gluten') wheat is technically feasible with gene editing using CRISPR/Cas technology. Such wheat varieties and the food products made with them have the potential to make a signifcant contribution to food security and public health for a group of individuals that suffer from coeliac disease, and for others that prefer to consume gluten-free products for health or diet related reasons. Such products could ft well in the gluten-free product market of packaged foods, which currently is growing fast.

Gene editing with CRISPR/Cas initially requires foreign DNA or proteins to be inserted into the plant genome or plant cell, as a temporary step to perform editing (read: to induce a directed and desired mutation) of target genes. In many jurisdictions such plants are not regulated as GM, provided that no foreign DNA is present in the fnal plant. In Europe, however, the process-based approach is applied, and because CRISPR/Cas includes a temporary GM step, gene-edited wheat will fall under the strict GM regulation (EC Directive 2001/18), whereas plants and derived products from chemically and irradiation induced mutagenesis are exempted from GM regulation according to EC Directive 2001/18, Annex 1B [93]. Presently, in 2023, the European Commission is preparing a proposal on changing the legislation for plants derived from cisgenesis or targeted mutagenesis. The possible impacts that gene-edited low CD-immunogenic wheat, once on the market, may have on the economy, society and the environment, were reviewed by Sánchez et al. (in press) [94].

One aspect of low-gluten wheat is that it will require the establishment of a separate supply chain under Hazard Analysis and Critical Control Points (HACCP) conditions [92]. Cross-contamination with regular wheat and other gluten-containing cereals (rye, barley) should be strictly avoided, requiring continuous control throughout the entire production chain. Further, labelling should emphasize the unique properties of the derived food products, particularly to CD-patients and NCWS patients. Importantly, the current legislations (EU 828/2014 and EU 41/2009) on gluten-free labelling requiring a threshold at 20 ppm gluten would need to be re-evaluated for low-gluten, low CD-immunogenic wheat products, because these regulations are based on quantitative measurement of the total amount of gluten in 'gluten-free' products which should be below the threshold for coeliac safety of 20 ppm. Gene-edited low CD-immunogenic wheat still will retain a gluten content far above this threshold and would require a separate 'CD-safe gluten' label.

Adoption of gene-edited coeliac-safe wheat might generate many new business opportunities for the entire wheat value chain, leading to diversifcation of production and products. In all, healthier and more safe foods for the CD patient population and reduced health care costs to society can be envisioned when gene-edited low-gluten CD-safe wheat would become mainstream on the food market ([94, 95] and refs therein).

**Acknowledgements** The writing of this chapter was partially made possible by the Well on Wheat (WoW) project (https://www.um-eatwell.nl/wow/), an international research project under the umbrella of the Health Grain Forum and ICC-Vienna and partially funded by the Dutch Topsector AgriFood (project TKI 1601P01 to LJWJG), and partly by the Netherlands' Ministry of Agriculture, Nature and Food Quality (BO-43-121-001 to MJMS and CMMvdW).

# **References**


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# **Chapter 14 Genome Editing in Horticultural Plants: Present Applications and Future Perspective**

**Nasser Mahna and Shahnoush Nayeri**

**Abstract** This chapter considers the genome editing technologies that have been utilized for breeding horticultural plants. Many examples of the successful application of genome editing technologies including ZFN, TALEN, and especially CRISPR/Cas systems in improving diverse characteristics of horticultural plants are mentioned and discussed. Based on the literature review, CRISPR/Cas technology has proved its potential in altering many genes of interest in horticultural plants including fruits, vegetables, and ornamental plants for improving agronomically important traits and attributes such as growth rate, seed size, owering time, ower color, storage time, resistance to biotic stresses, tolerance to abiotic stresses, herbicide tolerance, metabolism, fruit color, fruit ripening, and so forth. This advanced technology paves the way for more favorable and precise manipulation of plant genomes to improve crop performance.

# **1 Introduction**

Horticulture goes back to ancient times as an important sector within agriculture and has improved a lot during human civilization. Horticultural crops are typically known as vegetables, fruits, and ornamental plants which are planted and harvested for food, medical, and cosmetic purposes [1]. Nevertheless, horticultural crop cultivation continues to face a variety of challenges, such as serious environmental concerns, the spread of viruses and pests, climate change, and increasing population growth [2]. Conventional breeding, molecular markers, and genetic modication

N. Mahna (\*)

Department of Horticultural Sciences, Faculty of Agriculture, University of Tabriz, Tabriz, Iran e-mail: mahna@tabrizu.ac.ir

S. Nayeri

Department of Plant Sciences and Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran

have been used for improving traits in horticultural plants. However, faster development of better varieties requires more precise and safe techniques such as genome editing systems.

# **2 Non-CRISPR/Cas Genome Editing Systems and Their Applications in Horticultural Plants**

Before the discovery of CRISPR/Cas, other genome editing techniques were used to modify traits in plants, but not so frequently in horticultural crops. Strictly speaking, there is no report on the application of meganucleases [3], the frst developed genome editing tool, in horticultural crops. Later, after the rise of Zinc Finger Nuclease (ZFN) technology, two groups applied ZFN in the functional analysis and characterization of genes in apple and fg [4] as well as tomato [5] plants. Thereafter, TALEN-based gene editing technology was developed and effectively employed in some economically important horticultural crops such as rapeseed (*Brassica oleracea*) [6], potato (*Solanum tuberosum*) [7–12] and tomato (*Solanum lycopersicum*) [13, 14]. The frst instance of genome editing in a horticulture crop was accomplished in 2013 using TALEN in *Brassica oleracea* [6]. Later, in potato, TALEN was used for silencing the vacuolar invertase gene to improve the processing and cold storage of potato tubers [12].

# **3 CRISPR/Cas Systems in Horticultural Plants**

The CRISPR/Cas genome editing technology was developed almost two years later than the discovery of TALEN proteins [15]. The CRISPR/Cas technology is expected to open up novel opportunities for the improvement of horticultural plants. This technology has already been used widely in horticultural plant breeding projects (see Table 14.1). For example, CRISPR/Cas9 was employed in the genome editing of sweet orange and tomato hairy roots [16, 17]. Several works have endeavored to enhance foricultural characteristics in ornamental plants, such as fower size, shape, color, aroma, shelf life, stress tolerance, etc. Through knocking down the *Argonaute7*, CRISPR/Cas9 was exploited to develop the frst targeted mutation in the needle-leaf of tomato [18]. Since then, some research have been published on the potential uses of CRISPR/Cas in improving fruit quality, plant architecture, and shelf life as well as protecting plants from biotic and abiotic stresses [19]. The technique is now being exploited for a variety of fruits and vegetable crops, including watermelon, mustard, tomato, potato, and cabbage, etc. Table 14.1 presents a wide variety of genome-edited horticultural plants including vegetables, ornamentals, and fruit crops for different types of traits. Until now, just a handful number of teams have utilized CRISPR/Cas-mediated genome-editing in ornamental plants,


225

(continued)


226


(continued)


**Table 14.1** (continued)



230

**Table 14.1**

(continued)


Crop species

**Table 14.1**

(continued)

**Tomato**

Scientifc name

Plant type

Gene name

Trait modifcation

Classifcation


232

**Table 14.1**


(continued)

234


because it is challenging to edit genomes of the plants without any genome sequence information. Thus, more whole-genome sequencing data should be generated. In addition, for the improvement of polyploid plant species, such as tetraploid roses and hexaploid chrysanthemum plants, a very effective editing platform has yet to be developed. Eleven research papers concerning genetic manipulation through CRISPR/Cas in ornamental plants have been collected from previously published research articles (Table 14.1). Specialized application of CRISPR/Cas such as baseediting has been reported for tomato [20]; however, any research generating mutations in horticultural plants other than tomato have not been reported using either the base-editing or prime-editing approaches yet.

# **4 Making Horticultural Plants More Tolerant to Abiotic and Biotic Stresses**

Pathogens including bacteria, fungi, and viruses can cause a variety of diseases in plants. This hinders plant growth and development, which can result in signifcant losses and hence increase agricultural production costs. Nevertheless, plant tolerance to biotic stresses can be signifcantly improved through the utilization of the CRISPR/Cas technology.

Two separate strategies are utilized to develop virus-resistant plants: editing the virus genome and modifying the plant genes responsible for susceptibility to viruses. Viruses typically employ the transcription and translation machinery found in the host plant. Using CRISPR/Cas tools, sensitivity (S) genes may have their expression altered to protect plants from viruses, for example by knocking off translation initiation factors. For example, producing bananas with endogenous banana streak virus resistance was made possible by the CRISPR/Cas9 technique [25] through introucing mutation in the integrative viral components that inhibited the transcription and translation of viral proteins in banana trees. An example of modifying plant genes to introduce more resistance is apple for which CRISPR/Cas9 RNPs were introduced into protoplasts to target the *DIPM-1, -2,* and *-4* genes which are fre blight resistance inhibitors [26]. The beneft of this transient expression was revealed through a decrease in undesirable mutations. The *MdDIPM-4* gene was also knocked out in apple plants by the other researchers. Surprisingly, the FLP/FRT recombination system was used to eliminate the T-DNA harboring the expression cassettes for CRISPR/Cas9 from the genome of the transformed plants [27]. Also Citrus (*Citrus sinensis* orange and *C*. *paradisi* grapefruit) mutants were created by genome editing showing signifcant tolerance to *Xanthomonas* causing citrus canker [28, 29]. Citrus plants have the *CsLOB1* gene, which makes them susceptible to *Xanthomonas citri* subsp. *citri* [30]. This gene's promoter region contains elements necessary for the pathogenicity factor *PthA4* of the bacterium to interact, which promotes the emergence of disease symptoms [31]. The PthA4 factor's binding sites were edited using CRISPR/Cas9, which reduced the bacteria's capacity to infect *Citrus sinensis* [29].

The Wanjincheng orange variety's *CsLOB1* gene's promoter region was altered using some vector constructs. The rate of identifed mutations varies between 11.5% and 64.7% according to the structure. As a result, four canker-resistant citrus mutant lines were selected. Signifcant plant resistance was obtained when a complete deletion happened in the promoter region of *CsLOB1* where the *PthA4* effector binds. Both Cas9 and Cpf1 have been used in similar investigations [32]. The CRISPR/ Cas9-mediated editing of the *CsWRKY22* gene, which codes for a different transcription factor, was another strategy to improve the bacterial canker resistance in Wanjincheng orange [33]. Genome editing also helped in the development of mutant bananas containing the *DMR6* gene that are resistant to the *Xanthomonas* bacteriacaused banana wilt [34]. Many plant diseases are brought on by fungal infections. The introduction of CRISPR/Cas9 technology has brought novel promises for breeding crops resistant to a variety of fungal diseases by altering pathogensensitivity genes. It is well known that plants' sensitivity genes make it easier for pathogens to invade and infect them. For example, CRISPR/Cas9 has made possible to knock out the *MLO-7* gene, which negatively regulates resistance to powdery mildew pathogen, *Erysiphe necator,* in grapevine [26]. RNPs were used to deliver sgRNA to plants, and the mutagenesis frequency was reported quite low (0.1–6.9%). The RNP-mediated editing approach has been developed in recent studies [35]. Three *MLO* gene mutations caused grapevine plants to be 77% less sensitive to powdery mildew [36]. Moreover, by the deletion of the WRKY52 gene, a jasmonic acid pathway's negative regulator, highly resistant grapevine plants to *Botrytis cinerea* have been produced [37, 38]. A noteworthy number of sgRNAs targeting various sequences within the frst exon of the *WRKY52* gene, were generated, and it was discovered that mutations in two alleles of the gene were more protective against the pathogen than those in only one allele. It is helpful to employ genome editing to explain how certain genes contribute to bring resistance to a disease. For instance, through knocking out the *pathogenesis-related protein 4b* gene (*VvPR4b*), grapevines became less resistant to *Plasmopara viticola* [39]. The *VvPR4b* gene produces the chitinase II-like protein which is required to stop hyphae development in *P. viticola*.

The fungal pathogen *Botryosphaeria dothidea* infects apple plants, causing serious damage. Increased response to this pathogen has been observed in apple calli after *CNGC2* gene knockout [40]. Salicylic acid levels were found to be increasing at the same time as the expression of the gene encoding PR protein was still being inhibited. The gene *CNGC2* was selected for targeted mutagenesis, but this was not the right alternative because mutations in this gene can have negative infuences, like signifcantly reduced fertility. In another research, transient expression of the CRISPR/Cas9 in *Theobroma cacao*, the cacao plant, resulted in more resistant embryos and leaves against *Phythophtora tropicalis* [41]. The *TcNPR3* gene was selected as the candidate for modifcation because it is an inhibitor of the defense mechanism. These fndings support the outlook of cacao breeding for resistance to *P. tropicalis*.

It has been indicated that a *Clpsk1* gene mutation makes watermelon plants more resistant to the fungus *Fusarium oxysporum f. sp. niveum* [42]. With CRISPR/Cas9 technology, the alteration of the genes controlling the pathogen-sensitivity in plants seems to be an effcient and fast method to make resistant plants against viral, bacterial and fungal diseases.

A few studies report that horticultural plants had been improved for abiotic stress tolerance using genome editing. As an example, the watermelon acetolactate synthase (*ClALS*) gene knocked out by CRISPR/Cas9, promoted the development of watermelons with herbicide tolerance [43]. The *ALS* gene was employed as an indicator for CRISPR/Cas9-mediated base editing, resulting in the development of herbicide (chlorosulfuron) tolerant pear trees (*Pyrus communis* L.) [44]. Similar *CsALS* gene editing in orange (*Carrizo citrange*) led to the mutant varieties that were tolerant to the herbicide imazapyr [45].

Therefore, editing the genomes of horticultural plants by CRISPR/Cas9 can be advantageous for developing crops resistant to a variety of (a)biotic stresses. Nevertheless, it seems essential to obtain stable mutants and completely study how genome editing infuences traits and metabolic processes in plants.

# **5 Editing of Phenotypic Characteristics of Horticultural Plants**

There are reports that genome editing can alter a variety of plant characteristics, including plant growth and morphology, fruit maturation time, fruit color, metabolism, and shelf life. The semi-dwarf banana plant (*Musa acuminat*a "Gros Michel") was produced through CRISPR/Cas9-mediated editing of the *MaGA20ox2* genes regulating gibberellin biosynthesis [46]. The mutant plants displayed less growth than the wild-type plants, however thicker and deep-green leaves. The modifed plants' cells were structurally different compared to the wild-type plants. The fndings of these works are crucial for the screening of dwarf banana genotypes because tall trees are susceptible to strong winds, which causes signifcant harvest losses. In grapevine plants, the targeted mutagenesis of the *VvCCD8* gene resulted in more branches per shoot than in wild-type plants [47]. Plant phytohormones known as strigolactones prevent the development of axillary buds. The CRISPR/Cas9 editing system helped to functionally characterize the *VvCCD8* gene's role in the regulation of shoot branching. It is then planned to look into additional mechanisms for controlling the morphology and development of shoots in grapevines. In strawberry, it appeared feasible to produce white colored fruits using CRISPR/Cas9. The *RAP* (reduced anthocyanins in petioles) gene, which encodes a glutathione S-transferase enzyme, was targeted using CRISPR/Cas9 gene editing system [48]. This enzyme binds anthocyanins allowing them to move from the cytosol to the vacuole. It may be possible to produce strawberry types with popular white berries by editing the *RAP* gene.

CRISPR/Cas9-mediated genome editing is also considered as an effcient method for enhancing the nutritional value of fruits by editing their genomes. By mutating the lycopene epsilon-cyclase (*LCYε*) locus, it was possible to produce bananas with higher levels of β-carotene [49]. The amount of β-carotene rose six-fold in the fruit pulp of the mutant lines, whereas the α-carotene and lutein levels considerably diminished. Limited efforts have been made to apply CRISPR/Cas9 to create plants with improved phenotypes in red raspberry (*Rubus idaeus* L.). The favone 3-hydrolase (F3′H), one of the important enzymes in favonoid biosynthetic pathway, was knocked out in an investigation [50]. The gene *MYB-16*-like, functioning as a prickle formation regulator in raspberries, was another candidate for editing [51]. However, in both examples, it proved challenging for researchers to develop new seedlings from the generated raspberry calli.

Watermelon (*Citrullus lanatus*) seeds with mutations in the *ClBG1* (*β-glucosidase*) gene had smaller seeds but better emerging due to decreased abscisic acid levels [52]. This gene can infuence seed size and germination, which is a crucial characteristic of watermelon breeding. In another research, the role of the genes involved in fruit ripening such as 1-aminocyclopropane-1-carboxylate oxidase 1 (*MaACO1*) was evaluated and its sequences altered using CRISPR/Cas9 to fnd a way to extend the shelf life of bananas [53]. The obtained mutants developed smaller fruits with a slower maturity (60 days in comparison with 21 days for control bananas), which improved the storage capacity. The modifed banana fruits additionally contained a higher vitamin C content. The *CTR1-like*, *ROS1*, and *CmNAC-NOR* genes in the melon (*Cucumis melo* var. *cantalupensis*) were knocked out employing CRISPR/ Cas9, resulting in the development of melons with prolonged maturation and a long shelf life [54–56]. Thus, the reviewed studies demonstrated the sustainability of utilizing CRISPR/Cas9 to modify numerous characteristics in horticultural plants, for instance to improve fruit color and favor, alter ripening and storage times, and alter growth-related traits.

# **6 Modifcation of the Flowering Period and Lifetime**

Some studies indicate that horticultural plants can have their fowering time, foral life span, and fower shape and color altered by employing the CRISPR/Cas9. The function of numerous genes in the formation of fowers and fruits has been successfully identifed in both wild and cultivated strawberry plants using the CRISPR/ Cas9 technology. Since auxins are essential to the development of strawberries, *FveARF8* and *FveTAA1* genes were modifed resulting in homozygous *FveARF8* mutant plants with larger size and faster growth compared to the control plants [57]. Furthermore, mutations in *FaTM6* and *FveSEP3* strawberry genes resulted in parthenocarpy and an inaccurate fruit phenotype, as well as abnormal petals, anthers, and pollen grains [58, 59]. Thus, it has been demonstrated how these genes contribute to the growth of strawberry fowers and berries.

The fowering mechanisms of fruit plants can be manipulated with the use of genome editing. Growth traits have been manipulated in tomato to alter fruit development and fowering patterns. The transcriptional cofactors encoding genes such as *BLADE-ON-PETIOLE* (*BOP*) in tomato, can infuence the inforescences structure, and gene editing can be used to reduce the number of fowers per inforescence by editing *SlBOP* genes [60]. Rapid fowering and early harvest are induced by CRISPR/Cas9-mediated targeted mutagenesis in the fowering inhibitor *SELF-PRUNING 5G* [61]. Editing the coding sequences of *SlDML2* [62], *SlORRM4* [63], the *RIN* locus [64], or the cis-regulatory domain of *SlCLV3* [65] also affects the fruit development and ripening. The characteristics of the wild tomato relative (*Solanum pimpinellifolium*) were signifcantly altered by multiplex targeting of many genes crucial for tomato domestication, resulting in mutants that resembled domesticated tomatoes [66, 67]. Apple and pear mutant plants were obtained via knocking out of the *TFL1* fowering repressor gene [68]. Only a few pear mutant plants (9% of all) but many apple mutant tree lines (93%) showed early fowering. Genome editing was used to clarify the signifcance of the *AcCen4*, *AcCen*, and *SyGl* genes role in delaying fowering in kiwifruit plants (*Actinidia chinensis*) [69, 70]. So there is a chance to get horticultural plants that fower earlier, which would decrease the period it takes for them to yield fruit. Likewise, in blueberry (*Vaccinium corymbosum* L.) plants, gene editing was performed on the gene *CENTRORADIALIS* (*CEN*) [71]. Based on the evidence for *TFL1/CEN*-like genes in kiwifruit, pear, and apple, the researchers hypothesized that the knocking out of this gene would result in early fowering [68–70]. However, attempts to use *CEN* gene editing to infuence blueberry plant fowering were ineffective. Moreover, compared to the control plants, mutant plants grew far more slowly. Additional examination are yet to be done to explain the dwarf phenotype resulted in these mutant seedlings of *CEN*-edited blueberry plants [71].

Genes that control aging and corollas color in petunias, gentian, lily, ipomoea, chrysanthemum, and orchids have been studied by some researchers using genome editing [40, 72–89]. For instance, plants of the petunia cultivar "Mirage Rose" have been modifed targeting the *PhACO1* gene, which regulates the biosynthesis of the hormone ethylene [86]. As a result, petunia plants with less ethylene synthesis and fowers that lasted longer were developed. In Japanese morning glory (*Ipomoea nil*, "Violet") plants, the *EPH1* gene, a regulator of petal aging, was also knocked out to decrease fower wilting [76]. The problem of altering the color of fower corollas in ornamentals has been the center of many investigations. The genes *carotenoid cleavage dioxygenase 4* (*CCD4*) and *dihydrofavonol-4-reductase* (*DFR*) were knocked out in *Ipomoea nil* plants [84, 85], which allowed for the modifcation of fower color. One of the key enzymes responsible for the production of favonoids, *favone 3-hydrolase* (*F3′H*), was knocked out by another study [74]. As a result, *Torenia fournieri*'s fower color converted from bright blue to white. In another research, the essential enzyme of carotenoid synthesis, *PDS*, was mutated, resulting in the creation of mutant *Lilium pumilum* and *L. longiforum* with chimeric morphologies and different fower colors [87]. CRISPR/Cas9 gene editing has only

been used in a few orchid species [73, 83]. Hence, it has been demonstrated that it is possible to modify horticultural plants' fowering time, fower color, and fowering period using the CRISPR/Cas9 technology.

# **7 Conclusion**

CRISPR/Cas9 technology has so far demonstrated its effcacy to modify horticultural plant genomes. The genomes of these plants have been edited to regulate the period of fruit ripening and fowering, to increase plant resistance to biotic and tolerance to abiotic stresses, and to enhance the characteristics related to plant growth and fruit favor. Better editing and transcription-activation techniques as well as improved enzymes for more effective genome editing are now available for the development of novel types of horticultural plants.

# **References**


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# **Chapter 15 Application of CRISPR/Cas-Mediated Genome Editing Techniques in Leguminous Crops**

#### **Debajit Das and Sumita Acharjee**

**Abstract** Grain legumes are prized for their high protein content and abundance of phytochemicals, which are essential in the human diet. Scientists have made signi cant advancements in discovering novel genetic features in legumes, including, but not limited to, productivity, tolerance/resistance to various environmental stresses, and improved nutritive value. The contemporary surge in genetic resources of grain legumes has facilitated the integration of advanced molecular breeding techniques such as transgenic methodologies, genome modication, and genomic selection, to augment the crop's overall performance. This chapter discusses the application of CRISPR/Cas9-based genome editing tools for the improvement of grain legumes. Furthermore, it elaborates upon the latest developments in plant-specic genetic modication techniques, while also addressing the challenges and prospective benets that come with enhancing grain legumes with signicant agronomical attributes. Genome editing techniques have been prociently employed in diverse legumes, encompassing model legumes such as Medicago, alfalfa, and lotus, alongside other widely cultivated legumes like soybean, cowpea, and chickpea. The advent of gene-editing methodologies in legume breeding has presented exciting opportunities for enhancing important agronomic characteristics.

# **1 Introduction**

Legumes, comprising over 19,500 species and 751 genera, are the third-largest angiosperm family [1]. They encompass a diverse range of food crops that are crucial sources of plant-based proteins and essential amino acids. Grain legumes play a crucial role in agricultural sustainability by promoting soil fertility through symbiotic nitrogen xation (SNF) and the discharge of abundant organic matter into the ground. Notwithstanding their environmental benets and health advantages, poor

D. Das · S. Acharjee (\*)

Department of Agricultural Biotechnology, Assam Agricultural University, Jorhat, Assam, India e-mail: sumita.acharjee@aau.ac.in

productivity has impacts on legume agronomy. The implementation of innovative approaches like genomics-assisted selection (also known as marker-assisted selection or genomic selection) and genome editing can help address the challenging problem of boosting productivity and reliability.

Over the course of the past 10 years, signifcant advancements have been achieved in the sequencing of the genomes of leguminous crops. The draft genomes and transcriptome data of about 35 different legume species have been substantially assembled [2]. To select for intricate traits and generate better cultivars of grain legume crops, these genomic resources are proving invaluable [3]. The detailed assessment of the application of marker-assisted backcrossing (MAB), marker-assisted selection (MAS), and genomic selection (GS) techniques for improving the production of legume crops have been exhaustively investigated [3–5]. This chapter focuses on the latest developments in genome-editing technology and its effcacy as a precision breeding method for improving leguminous crops.

Genome editing involves making use of engineered nucleases and the inherent DNA repair mechanisms of cells to effectuate accurate and tailored modifcations to an organism's genetic makeup. The conception of gene-editing techniques commenced approximately thirty years ago, following the important revelation that targeted double-stranded breaks can be instigated in chromosomes using a meganuclease, namely I-SceI [6]. The effective use of meganucleases in genome editing was constrained by the limited number of target regions in the majority of genes. With the arrival of customizable zinc fnger nucleases (ZFNs) [7] and transcription activator-like effector nucleases (TALENs) [8], the development of effective genome editing technologies witnessed a major acceleration. The remarkable progress in this state-of-the-art methodology was attained by successfully incorporating RNA-guided Cas9 nuclease, which originates from the type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR) adaptive defense mechanism, to facilitate genetic modifcation in eukaryotic organisms [9, 10].

# **2 Genome Editing Technology**

The implementation of genome editing is a groundbreaking technological innovation that has the potential to signifcantly advance the feld of plant biology and agricultural breeding [11]. This method relies on site-directed nucleases (SDNs) such as meganucleases, ZFNs, TALENs, and the CRISPR/Cas system [12, 13]. The CRISPR/Cas system's growing popularity in genome modifcation tool development can be attributed to its simplistic nature and facile manipulability [14]. The fundamental CRISPR/Cas mechanism necessitates the presence of two integral constituents: a Cas endonuclease, such as Cas9 or Cpf1, and a guide RNA (gRNA) [15, 16]. The gRNA may be directed to bind to the desired DNA sequence and then use the Cas nuclease to create a double-strand break (DSB) at that specifc location. The predominant mechanism for DSB repair in plants is the non-homologous end joining (NHEJ) pathway, which is known to be error-prone and often results in the insertion or deletion of bases, leading to genetic changes at the site of repair [16]. Thus far, a multitude of base and prime editor tools have been devised leveraging CRISPR/Cas technology to achieve greater precision in the editing process [17].

Many Cas9 variants have been synthesized that exhibit great fdelity, including those with paired nickase, point mutations, chimeric dCas9-FokI, etc. These modifed Cas9 variants have cleavage activity with decreased off-target effects (refer to www.addgene.org). Thus choosing an appropriate Cas9 endonuclease variant is important yet diffcult. With the use of these editing tools, breeders can manipulate target genes in the way they want to boost crop production and quality, endurance to biotic and abiotic stress, and herbicide resistance [11]. As a result, genome editing is seen as the breeding approach of the future.

The approval of the commercialization of genome-edited crops necessitates a framework of legislation and regulation [18]. Genome editing generates minor indels, base-pair changes, and targeted short sequence modifcations by homologydirected repair (HDR), which are identical to those caused by natural mutations. As a result, in several countries, these sorts of mutants are not classifed as genetically modifed organisms (GMOs) and hence are excluded from GMO restrictions [19, 20].

# **3 Limitations in Genetic Modifcation of Legumes**

The availability of a suitable genetic transformation protocol to introduce DNA into the plant is a prerequisite for achieving a fruitful plant transformation. Most of the grain legumes are grouped as recalcitrance to *in vitro* regeneration and transformation [21]. This phenomenon is further compounded by the intricate fact that only certain tissues (immature cotyledons, mature cotyledons with embryonic axis, embryonic axis or hypocotyl) within leguminous crops exhibit the ability to transform, while others possess the capacity for regeneration. It is noteworthy that these two distinct events do not invariably manifest within the same tissue. Achieving successful in vitro rooting can be a signifcant obstacle, particularly for legumes with large seeds. The impediments to large-scale transformation in legumes include method specifcity and other factors. Traditional methods of genetic transformation are inadequate for achieving optimal results. Nevertheless, regeneration procedures for several legumes have not been successful, mostly because of poor *in vitro* roots during regeneration. It has been posited that conventional breeding methods are insuffcient in tackling these obstacles [22, 23].

The development of effcient transformation techniques is crucial for validating the role of genes in targeted crops [24]. Various methodologies, including sonicationassisted *Agrobacterium* transformation, have garnered attention as a means to augment the genetic transformation procedure in leguminous plants. Improving the pace of genetic transformation in leguminous crops can be achieved through signifcant approaches such as optimizing explant, improving the affnity of host plant interaction, and refning culture media additives [25]. Additional research is necessary to explore the barriers to legume transformation and potential remedies. Advancements in the feld of molecular science are expected to generate novel ideas and provide insights into the rapid enhancement of legume transformation rates.

The *Agrobacterium*-mediated transformation method is the primary means of genetic modifcation for the majority of grain legumes, with the biolistic method being utilized in a limited number of instances. Of the various grain legumes, genetic modifcation has been successfully achieved in soybean, and the CRISPR/ Cas9 system has also been extensively utilized [26]. Nevertheless, the utilization of CRISPR/Cas9 technology has the potential to overcome these limitations and obstacles [27]. The development of reliable and repeatable regeneration techniques results in the commercially successful production of several species of genetically engineered grain legumes.

# **4 Application of CRISPR/Cas9 in Grain Legumes**

The CRISPR-mediated gene-editing technique has been successfully applied to various legumes, such as soybean, chickpea, lentil, and *Medicago truncatula*, among others. Leguminous plants are cultivated extensively across the globe. The implementation of CRISPR/Cas9 technology has demonstrated enhancements in crop productivity, quality, and resilience against both biotic and abiotic stressors.

Legume species domestication has been extensively researched, and currently, the CRISPR/Cas9 mechanism is being employed in the process of domestication to introduce and enhance diverse characteristics. It is anticipated that the forthcoming process of crop domestication shall witness a signifcant acceleration, with the aid of CRISPR/Cas9, which shall effectively enhance numerous crop traits of commercial signifcance [28]. In recent times, there have been major advances in improving the nutritional value of legumes through the application of CRISPR/Cas9 technology.

# *4.1* **Medicago truncatula** *(Alfalfa)*

A forage crop, *Medicago truncatula* is used as a model crop due to its self-fertility, short life cycle, relatively simple transformation, diploidy, and smaller genome,. It is an excellent model for studying the molecular and physiological bases of leguminous crops. The CRISPR/Cas9 system exhibits a sub-optimal level of effcacy when applied to the polyploid alfalfa genome such as the tetraploid *M. sativa and M. falcata*. For effective gene editing in alfalfa, an improved CRISPR/Cas9 gene-editing system will be required. The *MsSGR* gene of *M. truncatula* has been effectively edited using CRISPR/Cas9. The outcomes indicated substantial differences in colour among the mutants. The presence of colour variation plays a crucial role in the attraction of insects and birds, thereby facilitating successful pollination. The experimental results indicated that mutations exhibited a greenish colour and suggested that the CRISPR/Cas9 method of knocking out alfalfa genes holds signifcant potential for future research [29]. Bottero and colleagues employed the CRISPR/Cas9 system to modify the NOD26 gene in alfalfa to enhance protein levels [30]. *Phytoene desaturase* (*PDS*) genes are a popular choice among researchers due to their easily observable phenotypic traits, which can be used to assess the effcacy of CRISPR/Cas9 gene-editing tools. Meng and Haji [31] devised a CRISPR/ Cas9 mechanism to induce specifc changes in the *MtPDS* gene of *M. truncatula*. Their fndings revealed that out of 309 T0 transgenic plants, 32 displayed the albino phenotype. Sixteen of these 32 transgenic plants were arbitrarily chosen for sequencing, and the results confrmed that all the tested albino plants had alterations in the intended region of the *MtPDS* gene. The genome-wide association studies (GWAS) employing CRISPR/Cas9 were carried out to study the function of potential genes for nodulation in *M. truncatula* [32]. Additionally, Trujillo et al. [33] and Wang et al. [34] have conducted mutational studies on fve different nodule-specifc PLAT domain (*NPD1–5*) and nitrate peptide family (*NPD*) genes.

# *4.2* **Glycine max** *(Soybean)*

Soybean (*G. max* (L.) Merr.) holds signifcant value as a crop for its oil and protein content, making it a prime candidate for genetic enhancement through the CRISPR/ Cas9 technology [35]. The soybean plant is a diploid species that has undergone an evolution from a palaeotetraploid ancestor. The soybean genome exhibits signifcant duplication, thereby presenting a signifcant barrier to conventional genetic methodologies aimed at elucidating gene functionality. One of the diffculties encountered in soybean transformation is the limited effcacy of *Agrobacterium*mediated techniques, which is infuenced by the type of tissue or cultivar used. Consequently, *Agrobacterium rhizogenes*-mediated hairy root transformation has acquired signifcant attention since it can be used to effciently assess the effectiveness of gRNAs before the whole-plant transformation. This is due to the prompt way of obtaining transgenic hairy roots, which can be obtained in a matter of weeks [36]. In 2015, there were multiple reports of the effective utilization of the CRISPR/ Cas9 technology for gene-editing purposes in soybean [37–40]. After these preliminary accomplishments, soybean researchers have attempted to improve gene-editing technology. Di et al. [41], for instance, used eleven different GmU6 promoters to determine which would be the most effective for driving gRNA expression in soybean hairy roots and found that the GmU6–8 and GmU6–10 promoters were the most active in improving editing effciency (20.3% and 20.6%, respectively) than the rest of the nine GmU6 promoters (ranging from 2.8% to 17%). Additionally, the CRISPR/Cas9 technique has been effective in targeting three *GmLox* genes (*GmLox1*, *GmLox2*, and *GmLox3*) that encode three lipoxygenases (LOX1, LOX2, and LOX3) that produce a beany favour that limits human intake [42]. They observed that the associated lipoxygenase activities had been eliminated in 60

T0-positive transgenic plants harboring various sgRNAs and mutations (two of them are triple mutants and one is a double mutant). Li et al. [36] employed the CRISPR/ Cas9 methodology to modify the conglycinin (7S) and glycinin (11S) storage protein genes in soybean. The researchers observed gene-editing effciencies of 5.8%, 3.8%, and 43.7% for *Glyma.20 g148400*, *Glyma.03 g163500*, and *Glyma.19 g164900* genes, respectively. Furthermore, the manipulation of soybean plant architecture has been observed through the utilization of the CRISPR/Cas9 system.

The study carried out by Bao et al. [35] involved the selective targeting of squamosal promoter-binding protein-like genes, namely *GmSPL9a*, *GmSPL9b*, *GmSPL9c*, and *GmSPL9*. The results of the study indicated that the T2 double homozygous mutant spl9a/spl9b exhibited a reduced plastochron length. Also, it has been observed that T4 mutant plants exhibit an increment in the number of nodes on the main stem as well as an increase in branch numbers. Zheng et al. [43] described simple binary vector systems using Cas9 and egg cell-specifc promoters (ECp). The two genes, *GmAGO7a* and *GmAGO7b*, which encode ARGONAUTE7 (AGO7), were specifcally targeted due to their signifcant role in regulating leaf patterns in soybean. Their fndings indicate that the promoters can generate mutations and that it is possible to obtain several distinct mutations independently. Virdi et al. [44] conducted a study wherein they employed CRISPR/Cas9 mutagenesis to generate several knockout alleles including one in-frame allele for the *β-ketoacyl synthase 1* (*KASI*) gene, which is involved in the conversion of sucrose to oil. The fndings of the study revealed that the function of the genes was lost.

Because of the importance of Soybean, a substantially higher number of CRISPR studies have been conducted on it compared to other legumes. These studies primarily focus on modifying its nutritional value and plant architecture, specifcally leaf patterns and nodule numbers. Nonetheless, the establishment of stable soybean genetic transformation remains elusive due to the recalcitrant nature of this crop towards transformation. The enhancement of transformation effciency has the potential to propel CRISPR research in soybean toward subsequent genetic research, owing to its effciency, multiplex editing, and high-throughput mutagenesis capability [35].

# *4.3* **Cicer arietinum** *(Chickpea)*

Chickpea is a crop of signifcant commercial importance on a global scale. The application of genome-editing techniques presents a viable solution for addressing the challenges encountered during its cultivation. Badhan et al. [45] conducted a study with the objective of editing genes associated with drought tolerance, namely *4-coumarate ligase* (*4CL*) and *Reveille 7* (*RVE7*), using CRISPR/Cas9 technology in chickpea protoplasts. The results suggested that the knock-out of the RVE7 gene displayed a high level of editing effciency in vivo. The fndings of this study indicate that the utilization of protoplasts enables the application of the CRISPR/ Cas9 DNA-free gene editing technique for enhancing drought tolerance in chickpea genes. This study represents the initial and singular instance in which CRISPR/Cas9 gene editing has been employed in chickpea research.

# *4.4* **Arachis hypogea** *(Peanuts or Groundnut)*

Groundnut is an important leguminous crop that exhibits a notable concentration of oleic acid. One of the primary breeding goals for peanuts is to increase their oil content. The oil possesses signifcant industrial utility and benefts, such as prolonged shelf life and antioxidant properties. The CRISPR/Cas9 gene-editing system was leveraged in a study that edited the *ahFAD28* gene, which is responsible for the conversion of oleic acid to linoleic acid in fatty acids. The CRISPR/Cas9 system has effectively modifed the gene to introduce desirable traits. The targeted mutation of this gene was achieved using groundnut protoplast and culture [46]. In a separate experiment, the utilization of the CRISPR/Cas9 tool was explored to edit the allergen gene (*Ara h 2*) in peanuts. The altered version of this gene resulted in enhanced nutritional qualities of peanuts for individuals with peanut allergies. Shu et al. [47] performed a study wherein CRISPR/Cas9 was employed to investigate the functions of Nod factor receptors (NFRs) in peanut nodule formation, particularly in the initiation of a symbiotic relationship with rhizobia. The mutants that underwent editing to contain two *AhNFR5* genes exhibited a Nod-phenotype. Conversely, the mutants that were picked for containing two *AhNFR1* genes were still capable of forming nodules following inoculation.

# *4.5* **Vigna radiata** *(Mungbean)*

Given the current accessibility of complete genome sequencing and the extensive collection of 1481 mung bean entries that have undergone comprehensive evaluation for different agronomical characteristics [48], there exists a signifcant opportunity for harnessing CRISPR/Cas9 gene editing in mung bean breeding initiatives. The successful implementation of CRISPR/Cas9-mediated gene editing in cowpea (*V. unguiculata*) has been reported, wherein a symbiosis receptor-like kinase gene was targeted to disrupt symbiotic nitrogen fxation [49]. The effcacy of CRISPR/ Cas9 in a *Vigna* system implies the potential applicability of genome editing in additional species, such as mung bean. The initial objectives for gene editing in mung bean would encompass resistance to diseases and other desirable traits. The development of mung bean cultivars that exhibit resilience to fuctuating climate conditions would facilitate the global expansion of mung bean agriculture.

# *4.6* **Vigna ungiculata** *(Cowpea)*

Cowpea is a leguminous crop that is well-suited for cultivation in warm and arid regions [50]. It is considered as an orphan grain legume. Globally, the production of cowpeas increased from 9.7 MT in 2009 to 14.4 MT in 2019. Cowpeas exhibit a protein level of 25% by dry mass and exhibit a high-lysine content, thereby enabling them to serve as complementary dietary ingredients in cereal crop-based diets.

The recalcitrance of cowpea towards transformation has been observed to impede the regular application of CRISPR/Cas9 techniques. Che et al. [51] have reported enhanced transformation effciencies ranging from 4.5% to 37% across nine cowpea varieties. Che and associates employed a CRISPR/Cas9 framework that drives the expression of Cas9 under the control of soybean elongation factor (GmEF1A2) promoter and a gRNA under the promoter of VuU6 to generate a total of 35 T1 plants. Che and colleagues specifcally targeted the *VuSPO11-1* gene to facilitate the production of asexual plants that are suitable for hybridization. Juranić et al. [52] designed a transient approach to evaluate CRISPR/Cas9 constructs within a 48-h timeframe using *Agrobacterium* infltration of detached leafets, as a means of surmounting the challenge of testing such constructs in stable cowpea transformants.

# **5 Conclusion**

Legumes have been a staple in human and livestock diets since ancient times. The uniqueness of these plants lies in their possession of multiple nutritional benefts and their ability to withstand various diseases. Population growth has exerted a major supply burden for these crops on the food supply network. Researchers are introducing new variations for various crop traits [53, 54]. The application of the CRISPR/Cas9 tool has become increasingly signifcant in contemporary genome editing, with potential implications for achieving global food security. This widely used tool demonstrates crop modifcation without the use of transgenes. Legume crop genome editing using the CRISPR/Cas system has not yet reached its full potential [55] and it is anticipated that CRISPR/Cas9 in combination with other molecular approaches would signifcantly improve legumes and increase their yield.

CRISPR/Cas9 technology has undergone signifcant advancements, which have expanded the range of possibilities for accurately and effectively manipulating genes through genetic material addition or deletion. The utilization of CRISPR/ Cas9 technology presents innovative opportunities for the exploration of functional genomics and the enhancement of diverse characteristics in grain legume crops. The effcacy of genome editing in enhancing legume quality depends on the presence of profcient procedures for plant transformation and complete plant regeneration, as well as a favorable regulatory framework and substantiation of societal endorsement of gene-edited crops.

255 15 Application of CRISPR/Cas-Mediated Genome Editing Techniques in Leguminous…

# **References**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

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# **Chapter 16 Genetic Improvement in Leguminous Crops Through Genome Editing**

#### **Aida Dervishi, Muhammad Waqas Choudry, Rabia Riaz, and Allah Bakhsh**

**Abstract** Leguminous crops belong to the Fabaceae family, known for their high nutritional properties and use for human and livestock consumption and for their ability to symbiotically xing nitrogen which plays an important role in soil enrichment and sustainable agriculture. Over the years the genetic improvement of legumes has been carried out using conventional techniques of breeding based on the QTL and MAS selection mainly to increase the yield. Genome editing techniques have been successfully used in different legume crops, mainly on model crops and grain legumes such as chickpea, soybean and cowpea. The recent advancement in gene editing tools such as CRISPR-Cas technology contributed to the improvement of important agronomic traits in legume species and provide a great potential for studies of the traits such as tolerance to biotic and abiotic stress, increased yield and improvement of seed content chave recently been introduced in legumes utilizing genome editing tools. This chapter provides an overview of the main developments in genetic improvement methods applied and the achievements so far achieved in leguminous crops. Further studies aiming at the genetic improvement of minor or underutilized legumes are pivotal and a challenge in the future.

A. Dervishi

M. W. Choudry · R. Riaz · A. Bakhsh (\*)

Department of Biotechnology, Faculty of Natural Sciences, University of Tirana, Tiranë, Albania

Centre of Excellence in Molecular Biology, University of Punjab, Lahore, Pakistan e-mail: allahbakhsh@cemb.edu.pk

# **1 Introduction**

# *1.1 Nutritional and Ecological Value of Leguminous Crops*

Legume is any plant species of Fabaceae (*Leguminosae*) family which constitutes 5% of the total 400,000 plant species known so far on earth. The term 'legume' has derived from Latin word *Legūmen* that means "beans inside pods". The Fabaceae family constitute major "founder crops", the ones domesticated earliest in known human history [1]. Among the "Big Eight' founder crops' group that known to be cultivated as early as 10,000 BC, four are legumes including; Pea (*Pisum sativum*), chick pea (*Cicer arietinum*), lentils (*Lens culinaris*), and bitter vetch (*Vicia ervilia*) [2]. Legume cultivation holds key importance in terms of worldwide grain food and forage production demands. The edible seed fruits of any leguminous plant are generally termed as pulses. The commonly cultivated grain legumes among the 20,000 worldwide distributed species are; common bean, faba bean, pea, chickpea, cowpea, pigeon pea, lentils, peanut, grass pea and horse gram [3]. According to Food and Agriculture organization (FAO) 2018 stats, common bean (dry bean) was the most cultivated grain legume worldwide with 34.5 million hectares of total cultivation area followed by chickpea (17.8 million ha) and cowpea (12.5 million ha). The estimated grain legume production exceeded 92 million tons worldwide. The major portions of grain legumes production are coming from India, China, Canada, Australia, USA, Brazil, Argentina and Russia. India is the largest producer of grain legumes contributing 1/4th of the total global production [4]. In Europe, soybeans, faba beans and feld peas are presently the most cultivated legumes. Particularly, soybean production has dramatically increased in the last decade because of its high demand in livestock feed. According to European Commission (EC) reports, 943,000 ha of land was under soya cultivation in 2019 and expected to increase by 44% by 2030. However, only 43% of the total legume consumption in Europe is indigenously produced [5].

**Nutritional Impact** The United Nations celebrated 2016 as "International year of Pulses" to highlight the importance of nutritious seeds produced by legumes. Grain legumes are also sometimes referred to as 'poor man's meat' because of the high protein content they possess. The storage proteins in pulses make them the richest plant-based source of proteins which vary from 16–50% of the total dry weight. Along with this high protein content, the presence of adequate amounts of dietary fbers, vitamins, complex carbohydrates, sugars, minerals, and fatty acids making legume seeds as one of the healthiest foods sources [6]. Beans also contain some of the non-nutritional phytochemicals such as phytosterols, polyphenols, trypsin inhibiters, phytate, lectin and saponins. Some of these non-nutritional components of legume grains have been termed as anti-nutrients which means making nutrients less available in the body. This may result from reduced digestion, absorption, and bioavailability of nutrients. Enzyme inhibitors like trypsin/a-amylase inhibitors block the active site of these enzymes and hence no digestibility. Further, lectins are found to interfere with nutrients absorption in the small intestine by attaching specifc epithelial cell receptors [7]. However, in recent research, these phytochemicals are found to play a critical role in body's normal homeostasis through their antioxidant and anti-infammatory activities [8]. Also, the consumption of pulses in daily diet is found to prevent cardiovascular diseases, obesity and type 2 diabetes control, as well as reduce risk of certain cancers including prostate and breast cancer [9].

**Ecological Impact** The members of the *Fabaceae* family are well known for their agronomic and ecological role related to their ability to fxate nitrogen [1]. They possess specialized structures in their roots called 'nodules'. Root nodules are symbiotic association structures that develop in the root hairs of leguminous plants with diazotrophic rhizobia through a complex bilateral signaling pathway initiated in nitrogen defcient soils. Rhizobia are a group of gram-negative bacteria that colonize root hairs in a host specifc manner and fx gaseous atmospheric nitrogen into a usable form (i.e., ammonia) for plants. Diazotroph is any bacteria or archaea that assimilates atmospheric nitrogen into usable form. This process of harvesting gaseous nitrogen by the rhizosphere rhizobium colonies is termed biological nitrogen fxation (BNF). The symbiotic association is a two-way relation as the bacteria living in root nodules feeds upon the carbon-rich metabolites produced by the host plants. Moreover, legumes cropping has also been found to improve the soil structure (i.e., water retention), nutrients bioavailability (i.e., phosphorous mobilization) and breaking disease cycles through the activities of these rhizobial communities in root nodules. These unique features of legume crops play a critical part in maintaining soil health and adequate soil-nitrogen levels thus reducing the needs of artifcial nitrogen fertilizers and pesticides. Further, this capacity of legumes has been extensively utilized in crop rotation systems for sustainable use of agricultural land, mitigating the Greenhouse gas (GHG) emissions and enhanced carbon sequestration processes [4]. The term 'crop rotation' refers to the process of growing different crops on the same land area in a specifc sequence cycle so to maintain soil fertility, rhizosphere microbial diversity, nutrients availability and resistance to plant pests. It has been found that legumes rotation with cereal crops reduces greenhouse gas emissions by 5–7 times compared to non-legumes cultivation [5].

**Challenges in Production** Despite all these advantages global legumes cultivation has declined over the last few decades. According to FAO stats of 2015, only in Europe, the legumes cultivation area was reduced to 1.8 M ha from 5.8 M ha that was recorded in 1961 [10]. This decline in production was mainly attributed to the socio-economic factors and to the limited yield of the legumes in the feld. Inconsistent yields effect the proftability of the crop in comparison to other crops grown at the same season [11]. Legumes output value is highly variable per unit of area and relatively low as compared to major cereals like rice, wheat and corn [12]. Pea crop, for example, has observed variability in market outputs ranging between 25–78% in Europe [10]. This low proftability of legumes is highly associated with unstable production yields rather than market prices. The factors limiting the production yields include both; (i) abiotic stresses i.e., salinity, drought, low soil fertility, extreme temperatures, and (ii) biotic stresses i.e., bacterial/fungal/viral diseases, and other pests' infestation.

# **2 Improvement in Legumes**

# *2.1 Breeding of Legumes*

The economy-driven trends of extensive cereal breeding programs, after the green revolution, is one of the reasons for the lack of focus on legumes breeding and cultivation in the last few decades. Furthermore, conventional breeding of legumes is constrained by the limited genetic diversity of available germplasms, collection facilities, and reference genome databases [13]. The natural inclination of legumes towards self-pollination is the main factor of their germplasm relatively low diversity. Breeders, in general, screen and select the favorable combination of genes/ mutations in plant genotypes to provide the desired set of characteristics. This is done by the genome-wide screening of all the available wild and adapted germplasm of that species for the resistance against biotic and abiotic stresses [14]. Rapid advancement in genome sequencing and analysis technologies has helped breeders to link a variable phenotype in a population with a particular locus, termed quantitative trait loci (QTL). In the last decade, genome of around 35 legume crops have been sequenced along with complete transcriptome profle analysis [15]. The availability of this huge genomic data has built into valuable tools for marker-assisted selection (MAS) for desired phenotypes in leguminous crops by employing various genetic markers including simple sequence repeats (SSR), single nucleotide polymorphism (SNP), amplifed fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP) etc. [16].

# *2.2 Modern Genome Editing Tools*

After the discovery of restriction endonucleases and their successful use in making a recombinant DNA molecule, the term 'Genetic Engineering' came to limelight. Genetic engineering (GE) essentially means the introduction of a foreign genetic material, using restriction enzymes and ligases, in a suitable host to provide the desired trait [17]. Since its development, technology has been continuously utilized in the various felds of crop improvement, having a signifcant global impact. The transformation of CRY1 family genes from *Bacillus Thuringiensis* into crops for insect pest resistance is one of the mainstream success of GE technology so far [18]. The next development in applied genetics was the discovery of homing endonuclease (HEs) or meganuclease I-SceI encoded by mobile genetic elements (MGEs) of the DNA for recombination process [19]. These meganuclease generate double stranded breaks at specifc sites in the DNA and then breaks are repaired by natural DNA repair pathways such as Homology directed repair (HDR) or Non-homologous ends joining (NHEJ) repair depending upon the repair mechanism available in the given cell. This double stranded break repair is generally accompanied with insertions or deletions (Indels) of some base pairs leading to genome editing in a locusspecifc manner [20]. The genome editing success by using meganucleases was limited because of low frequency of its recognition sites in the DNA [21]. To overcome this limitation synthetic genomic scissors, Zinc fnger nucleases (ZFN) were developed in 2002. ZNF is a hybrid molecule containing DNA binding domains, zinc fngers (His2Cys2) and a nuclease domain of Fok1 endonuclease. These zinc fngers can be engineered to recognize specifc sites in the genome and this capacity is being utilized for genome editing by engineering ZNFs for a specifc DNA regions [22]. One of the limitations in using zinc fnger nuclease (ZFN) technology was compromised specifcity depending upon the fanking DNA sequence of the recognition sites, called context-dependent specifcity. On the similar pattern, transcription activator like effector nucleases (TALENs) were developed by combing DNA-recognizing domains of transcription activator (TAL) like effectors with nuclease domain of restriction enzyme Fok1 for site specifc mutagenesis [23]. Despite similarity in basic concept, the major difference in TALENs and ZFNs is the frequency and accuracy of cleavage sites. Zinc fnger domains recognize 3–4 bases while TALENs has specifcity to a single bases conferred by the repeat variable di residues (RVD) of TALE proteins' DNA-binding domain making it possible to join several modules without interference in recognition sequence. Moreover, the design of TALENs is relatively simple making the DNA recognition and binding process less complex compared with ZFNs [24]. Now, coming to the most advanced genome editing tool termed as Cluster regularly interspaced palindromic sequences (CRISPR) – Crispr associated system (CAS) system. CRISPR-Cas genome editing system is tailored from the natural adaptive defense mechanism in most of the bacterial and archaeal species against consecutive infections of bacteriophages. CRISPR are simply virus-specifc sequences, termed 'spacers' and are placed between regularly clustered repeats present throughout the bacterial and archaeal genomes called CRISPR loci [25]. CRISPR Loci are associated with a set of specialized proteins called CAS-proteins. These CAS proteins have highly specifc nuclease activity mediated by RNA-DNA complementarity. The spacer sequences upon infection by bacteriophage got transcribed into crispr RNA (crRNA) and act as a guide for CAS protein to cleave the viral DNA and hence infectivity stops [26]. The adoption of this technology by artifcially designing guide RNA (gRNA) and CAS protein constructs leads to an era of third generation genome editing. The RNA-DNA base pair complementarity makes CRISPR-Cas system more precise genome editing tool compared with ZFN and TALENs where protein-DNA interaction was used for targeting [24].

ZFN technology was employed in soybean to generate heritable mutations on Dicer-like 1 (DCL1) loci that lead to a defective miRNA precursor transcript processing [27], on Dicer-like 4 (DCL4a and DCL4b) loci for hairy root transformation by increasing the growth of lateral root [28]. TALEN technology have been effectively used to improve seed nutritional characteristics, increasing oleic acid content in soybean [29] and peanut [30] or reducing linoleic acid content in soybean [31] by targeting fatty acid desaturase 2-1A (FAD2-1A) 2-1B, (FAD2-1B), and FAD3A gene family. TALEN was successfully applied in soybean to produce mutants by targeting Dicer-like2 gene [32].

# **3 CRISPR-Mediated Genome Improvement in Legumes**

CRISPR-Cas based genome editing is a relatively simple process requiring only a Cas9 endonuclease activity and a guide RNA (gRNA). The gRNA further consists of i) a crisper RNA (crRNA) that binds the target sequence and, ii) a transactivating RNA (tracer RNA) that mediates target recognition and cleavage. Some variations on the basic pattern may include the use of a different Cas-protein (i.e., Cas12a, Cas13 etc.) with different Protospacer adjacent motif (PAM) requirements. PAM is 2–5 bp sequence that fanks the target sequence to facilitates the Cas-protein bindings and is often a limitation to CRISPR-Cas system's applicability [33]. The critical steps in plant genomic improvement through CRISPR-Cas includes; (i) optimization of gRNA and Cas9 constructs, (ii) successful transformation/use of suitable delivery vehicle, (iii) detection of resulting mutations and, (iv) regeneration of mutated callus [16]. CRISPR-based genetic improvements have been extensively explored in various crops to generate mutations and then selection of high-quality cultivars. However, the lack of optimized protocols for the successful transformation and regeneration of legumes plantlets from callus is a major barrier in their genome editing [34]. Browning of tissues are recalcitrant in-vitro rooting behavior are the key barriers in regeneration of many pulses [35]. Agrobacterium-mediated transformation using seed-tissues have achieved some of success in recent years, particularly in soybean [36] and model legumes *Medicago truncatula* and *Lotus japonicus* [37]. Other legume plants with successful reports of CRISPR-based editing includes cowpea, pea, chickpea, alfalfa and peanut as mentioned in Table 16.1.

Use of Cas9 variants to achieve diverse targeted sites, optimized guide RNA vector constructs, multiple gRNA targeting the same gene or different genes at the same time [52], and development of regeneration protocols are key areas of research in legume genetic improvements. Further, ongoing regulatory debates are focusing on non-specifc complementarity binding of gRNA and off-target cleavages which may result in unwanted effects in the host [50].


**Table 16.1** Genome editing of various legume crops for improved varieties

(continued)


**Table 16.1** (continued)

# **References**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **Chapter 17 Soybean Improvement and the Role of Gene Editing**

**Nihal Öztolan Erol**

**Abstract** Soybean is a major agricultural crop that is used for food, feed, and industrial products. However, soybean production is facing several challenges, including pests, diseases, and environmental factors. In recent years, there has been a growing interest in using gene editing technologies to improve soybean traits. Gene editing technologies offer a promising new approach to improving soybean production and quality.

Gene editing technologies can be used to precisely alter the soybean genome. There are a number of different gene editing technologies that can be used to improve soybeans. One of the most commonly used technologies is CRISPR/Cas9, which uses a protein called Cas9 to cut DNA at a specic location. This can be used to insert, delete, or modify genes. Other gene editing technologies include zinc nger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs). Gene editing technologies have the potential to revolutionize soybean breeding. This can be used to introduce new traits, such as resistance to pests and diseases, or to improve existing traits, such as yield and oil content.

The use of gene editing technologies in soybean improvement is still in its early stages, but the potential benets are signicant. Gene editing technologies offer a more precise and efcient way to improve soybean production than traditional breeding methods. They also offer the potential to create new varieties of soybeans that are better able to meet the challenges of a changing world.

# **1 Soybean Production and Its Economic Value**

Soybean is a very important crop that provides substantial oil and protein nutrition for the increasing human population. Soybean cultivation has been rooted back in ancient times c. 6000–9000 years ago, in East Asia [1]. Its massive production has reached its highest in the last century with the help of improving breeding

N. Ö. Erol (\*)

Sabancı University Nanotechnology Research and Application Center, İstanbul, Turkey e-mail: nihal.oztolan@sabanciuniv.edu

A. Ricroch et al. (eds.), *A Roadmap for Plant Genome Editing*, https://doi.org/10.1007/978-3-031-46150-7\_17

techniques. Soybean production has increased since 1961 from 20–30 million tons to 350 million tons per year [2].

Soybean is very rich in oil and protein compared to other members of the legume family. Therefore, it meets a considerable demand for animal feed and oil production. Over three-fourths of soybean by weight is used for feeding livestock, poultry, and aquaculture production, so some countries increasingly export soybean products while others import to meet the demand for soybean-based animal feed. The rest is consumed by humans as an industrial oil, biofuel, food ingredients (lecithin, emulsifer, and proteins), and food (soy sauce, tempeh, soy milk, and tofu). Soybean is introduced as a rich protein source for plant-based diets as it consists of 40% of the dry matter including nine essential amino acids. Therefore, it is very important for the vegetarian and vegan diet, it provides high nutrition with its protein content [3]. Soybean seeds are the most important part of the plant, so throughout the domestication process, traits improving soybean seed quality and yield have been artifcially selected for effcient utilization in the food industry and agriculture.

Soybean domestication has led to a signifcant reduction in genetic diversity due to selective sweeps, resulting in the fxation of benefcial traits. Studies have shown that nucleotide fxation during soybean domestication and improvement has resulted in a reduction of genetic diversity compared to wild soybean populations. Furthermore, the fxation of key genes involved in the regulation of traits such as seed size, pod dehiscence, and photoperiodic fowering has played a crucial role in shaping the morphology and adaptation of soybean to different environments. These genetic changes have contributed to increased yield and better adaptation to a range of environmental conditions, making soybean a globally important crop. However, the reduced genetic diversity resulting from selective sweeps also raises concerns regarding the resilience and adaptability of soybean crops in the face of new and changing environmental challenges.

# **2 Genetically Modifed Soybean**

Genetic modifcation of an organism traces back to the domestication of organisms. However, public perception misinterprets this term and people think that genetic modifcation of organisms came out with the developments in biotechnology in the late twentieth century. Breeding practices have long been used by humans and are striking evidence of genetic modifcation. With the discovery of recombination techniques in bacteria, genetic modifcation techniques have been gradually improved over the years and used frst in producing medicines and then crops.

Recombinant DNA technologies are the fundamentals of genetic modifcations in living organisms. Briefy, a target gene cassette-containing vector is transferred by a virus or a bacterium into living cells of an organism to insert a specifc genetic sequence into that organism's genome. The frst genetically modifed soybean was produced in the 1990s. Glyphosate-resistant soybean cultivation together with the glyphosate herbicide dramatically decreased labour that occurred due to tillage of

the soil, at the same time this dual application increased genetically modifed soybean production.

Despite its recalcitrant nature of regeneration under tissue culture techniques, several studies showed that soybean has been used for gene editing of fowering time, seed oil content, lateral root growth, and defence mechanism [4] (Table 17.1).

Curtin et al. (2011) frst published the research about hairy-root and whole-plant transformation mediated by *Agrobacterium rhizogenes* using the zinc-fnger nuclease (ZFN) method to target *DICER LIKE (DCL), RNA-DEPENDENT RNA POLYMERASE (RDR),* and *HUA ENHANCER1 (HEN1)* genes in the root cells [5].

**Table 17.1** Soybean traits and associated genes that are modifed by gene editing techniques CRISPR/Cas9, zinc-fnger nuclease (ZFN), transcription activator-like effector nucleases (TALEN), *Agrobacterium tumafaciens,* and bean pod mottle virus mediated transformations


They continued using the ZFN method to create double mutants of *DCL1a* and *DCL1b* for *Agrobacterium rhizogenes*-mediated whole-plant transformation [6]. ZFNs were also used to deliver multiple different DNA donors in *the FAD2-1a* locus (Glyma.10 g278000) by using a biolistic bombardment technique on immature embryo explants [7]. This research successfully regenerated fertile plants and transmitted the insert to the next generation.

*FAD2-1a* and *FAD2-1b* loci were mutated by using transcription activator-like effector nucleases (TALEN), to convert oleic acid into linoleic acid to increase the polyunsaturated fatty acid. The study used the hairy-root transformation method mediated by *Agrobacterium rhizogenes* [8]*.* The same research group also targeted *FAD2-1a, FAD2-1b,* and *FAD3* to convert linoleic acid into oleic acid by using the TALEN technique and again successfully transformed soybean immature embryo explants [9].

Du et al. (2016) conducted a study to compare two gene editing techniques, TALEN and CRISPR-Cas9 in parallel with testing the transformation effciency by using soybean-specifc *U6-10* and *Arabidopsis*-specifc *U6-26* promoters in soybean [10]. They targeted a gene encoding phytoene desaturase (PDS), a rate-limiting enzyme involved in the carotenoid biosynthesis pathway. Hairy root transformation mediated by *Agrobacterium rhizogenes* successfully resulted in mutated buds. The study suggested the usage of CRISPR/Cas9 with species-specifc promoters to acquire a highly transformation-effcient, cost-effcient, and easy-to-construct transformation technique [10].

CRISPR/Cas9 technique outperforms other precise gene editing techniques by its cost-effcient and easily applicable features. This technique accelerates soybean breeding and supports soybean production. Cytoplasmic male sterility (*GmAMS1*) [11], fowering time (*LNK2*) [12], seed oil content (*GmFAD2*) [13, 14])*,* seed lipoxygenase, raffnose, and stachyose contents (*GmLox, GmRS, GmSTS*) [15–17], plant growth and structure (*GmLHY* and *GmSPL9*) [18, 19] are some of the traits, which have a signifcant role in soybean breeding, that were studied in the last decade.

Investigation of causal alleles of certain traits has provided the most crucial information for gene editing applications. QTL mapping, using GWAS and linkage mapping analysis, along with functional genetic mutations unravel the causative nucleotide changes. With the introduction of nucleic acid-cutting enzymes and nucleases, gene editing breathes new life into plant breeding by precise editing, and soybean breeding will defnitely beneft from this transformative new breeding techniques.

# **3 Agronomically Important Soybean Traits and the Use of Gene Editing**

Soybean breeding plays a crucial role in the production of soybeans around the world as it helps to develop soybean varieties that can adapt to different environmental conditions. By breeding soybean varieties that are good quality, and tolerant to various biotic and abiotic stresses, soybean production can be increased and stabilized. Additionally, breeding efforts have resulted in soybean varieties with

desirable traits such as high yield, improved nutritional quality, and enhanced oil and protein content, which are important for meeting the increasing demand for soybean products worldwide. Overall, soybean breeding has been instrumental in improving soybean production by developing varieties that are better adapted to the diverse environmental conditions in different regions of the world. A rapid and precise gene editing might help to improve elite soybean varieties. Soybean improvement might be accelerated by introducing a non-synonymous mutation with the help of gene editing.

Although trading and migrating routes had caused the dissemination of a certain type of cultivated soybean seeds towards Eastern Asia and North America, local landraces had provided effcient genetic resources for soybean breeding in adaptation to the environment. The idea of a single origin of soybean domestication does not completely explain the existence of allelic variation among cultivated varieties. Because local genetic diversity had provided location-specifc causal alleles associated with the traits of interest. Therefore, to improve plants' yield capacity and seed quality in terms of oil and protein, there were several genes functionally identifed to be responsible for plant architectural, physiological, and morphological changes in organs by using CRISPR/Cas9 method.

# *3.1 Pod Shattering Resistance*

Pod shattering resistance, to prevent seed dispersal and yield loss, is an important agronomical trait that has come along with domestication [20]. Angiosperms develop their seeds within the fruit and disperse them when there is an abscission between pedicel and lemma. This decreases the harvest output and was taken under control by artifcially selecting pod-shattering resistant plants. Four pod-shattering resistance-associated genes were identifed in soybean: *GmSHAT1-5, Pdh1, NST1A,* and *Glyma09g06290*.

Dong et al., (2014) identifed a causal polymorphism in *the GmSHAT1-5* gene and the pod-shattering resistant domesticated soybeans, which were diversifed from wild soybeans, derived from this single haplotype [21]. *GmSHAT1-5* is responsible for the lignifcation of fber cap cells in the pod ventral suture which causes thickening in domesticated soybeans. The sample collection included both *Glycine max* and *Glycine soja* varieties gathered from the seed bank of the Chinese Academy of Agricultural Sciences (Beijing). The pod indehiscent allele from *Glycine max* showed a 13-fold higher expression than *Glycine soja*. It seems like domestication signifcantly affects pod-shattering traits; however, this research did not reveal the origin of the indehiscent allele.

Zhang and Singh (2020) identifed a locus called *NST1A,* which showed epistasis with *Pdh1. NST1A* was a NAC family gene, a paralog of *GmSHAT1-5* [22]*.* Likewise, in NAC family transcription factors in *Arabidopsis thaliana*, a premature stop codon was identifed to be responsible for gain-of-function mutation, where it provided pod shattering-resistance despite the existence of *the Pdh1* allele [22, 23]. The indehiscent *NST1A* allele was predominantly found in Southern China and Japan, this implies that local wild cultivars in those regions were selected for the indehiscent *NST1A* allele independent of low humid conditions.

A genome-wide association study genotyped 211 soybean accessions including modern and wild cultivars collected from the National Center for Soybean Improvement of China by using NJAU 355 K SoySNP array containing 282,469 SNPs. A quantitative trait locus was identifed on chromosome nine and within that locus, a candidate gene *Glyma09g06290* was found homologous to *Arabidopsis thaliana* basic helix-loop-helix, a gene responsible for silique dehiscence. Quantitative polymerase chain reaction analysis also indicated that *the Glyma09g06290* gene was highly expressed in pod indehiscent varieties [24].

Another gene regulating pod shattering in the domesticated soybean is *Pdh1. Pdh1* showed high homology to dirigent family genes, which were initially known as a stereoselective bimolecular phenoxy radical coupling of (E)-coniferyl alcohol, for producing lignan [25]. The functional *Pdh1* was found to be highly expressed in the lignifed inner-sclerenchyma cells of the seed pod [26]. The inner sclerenchyma physical properties changed when *Pdh1* expression increased, and pod shattering started. As the relation between *Pdh1* and lignin was not clear yet, the gene might be responsible for lignin deposition in the seed pod. A non-synonymous nucleotide substitution that produces a stop codon results in pod-shattering-resistant varieties. Under low humidity *pdh1* allele containing soybeans showed signifcantly lower shattering scores than those with the *Pdh1* allele. This pod shattering-resistance associated allele was seen in more than 50% of Chinese and a considerable proportion of South Asian and North American landraces. However, Japanese and Korean landraces showed a very low frequency of this allele. The origin of domestication by selecting the indehiscent *Pdh1* allele might be originated from Huang-Huai-Hai Valley [22]. This infers that low humidity conditions provided selective pressure on *the pdh1* allele to protect seeds from dispersion. Zhang et al. (2022) provided a CRISPR/Cas9 gene editing solution for pod shuttering-susceptibility in a summer adapted soybean cultivar HC6 found in Huang-Huai-Hai [27]. They performed QTL mapping by using a recombinant inbred line population of HC6 and a pod shatteringresistant variety JD12 and they found a reproducible major allele at the *Pdh1* locus, SNP A/T that causes a nonsense variant (HC6/JD12). The resistant allele T was associated with low humidity regions in China, whereas the susceptible one A with high humidity regions in China, Japan, and Korea. Having known the contrasting effect, causal allele in different haplogroups facilitated the application of CRISPR/ Cas9, the precise gene editing. This fnally provided a gene therapy for pod shattering in soybean cultivars.

# *3.2 Shoot Growth Habit*

Planting and harvesting time remarkably affect soybean yield, therefore, farmers must choose the appropriate maturity type regarding the environmental conditions. Soybean determinacy is an important agronomic trait that identifes the maturity type. Determinacy is governed by genes and environmental signals, which control

the generation of shoot apical meristem and transition to foral meristem. Soybean can be classifed into three groups of determinacies: determinate, semi-determinate, and indeterminate. Indeterminate varieties, which are late maturing, show a prolonged vegetative phase with active stem and branch apices producing new nodes with leaves. Whereas determinate varieties, which are early maturing, cease stem and branch apical growth with photo-periodical foral induction.

Phenotypic variation amongst soybean landraces provided a good genetic resource for soybean breeding. Soybean planting management aims to maximize yield capacity and quality. It was found that, when indeterminate varieties are earlyplanted, they maintain an active vegetative growth for a long time and adequately accumulate amino acids and nutrients to allocate them towards seeds to increase yield quality and capacity. On the other hand, when determinate varieties are lateplanted, yield capacity and moisture decrease. However, early planting of indeterminate soybean varieties can carry some risks. For example, late frost or extended pathogen infection might cause to decrease in yield capacity and even delay harvesting. To avoid the risks, determinate and indeterminate varieties are planted accordingly to maximize soybean production in the feld [28–30].

In the cultivated soybean varieties, two genetic loci were identifed to be associated with the determinacy trait: *Dt1* and *Dt2*. The *Dt1* allele is dominant or incompletely dominant on the *dt1* allele; the *Dt2* allele is dominant on the *dt2* allele. Soybean plants with *Dt1/Dt1* genotype are identifed as indeterminate with *dt2/dt2* and semi-determinate with *Dt2/Dt2.* However, the *dt1/dt1* genotype shows a determinate phenotype when the *Dt2* locus is either recessive or dominant homozygous or heterozygous. Therefore, the *Dt1* locus has an epistatic effect on the *Dt2* locus [31, 32]. Their antagonistic behavior regulates fowering time and plant stem growth.

*Dt1* is induced by *E3* and *E4* under long day conditions, interacts with bZIP family transcription factor FDc1, and binds to the promoter of *APETALA1* for delaying fowering. On the other way, when *APETALA1* binds to the promoter of *Dt1*, it inhibits its expression, thus promotes fowering [33]. *Dt1* locus encodes a phosphatidylethanolamine-binding protein (PEBP) family protein called GmTf1 (or GmTf1b) which is an ortholog of Arabidopsis TERMINAL FLOWER1 that controls plant height and internode length. *GmTf1b* is expressed in the shoot apical meristem until fowering initiation [34]. Four independent single nucleotide polymorphisms on *GmTf1* were identifed in *Glycine max*, which makes nonsynonymous amino acid changes, whereas these nucleotide changes were not observed in *Glycine soja.* This infers that the determinacy trait was introduced by the domestication process of soybean.

Four homologous *Tf1* genes have been found in soybean. *Tf1b* has been already known but the functions of other homologous genes are still not clear. Wang et al. (2023) identifed the function of *Tf1c* and *Tf1d* by using CRISPR/Cas9 knock out double mutation [33]. Results showed that *tf1c/tf1d* double mutant soybeans fowered earlier than the wild type. The interaction of these homologous genes with *APETALA1* was also confrmed. Likewise*, Tf1c* and *Tf1d* interacted with FDc1 and inhibited four homologous genes of *APETALA1* and thus delaying fowering.

*Dt2* encodes the MADS-domain factor that binds to the promoter of *Dt1* and inhibits the plant stem growth to start the transition from the vegetative phase to the reproductive phase in soybean. There are two homologs of *the SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1)* gene, known as the positive regulator of fowering which is activated downstream of *FLOWERING LOCUS T* (*FT*) in *Arabidopsis thaliana* [35]*,* in soybean: *GmSOC1a* and *GmSOC1b.* These homologous genes interact in parallel with the *Dt2* locus and initiate fowering and inhibit stem elongation and node generation. A CRISPR/Cas9 gene knock out application mediated by *Agrobacterium tumefaciens* produced homozygous *soc1a, soc1b,* and *soc1a/soc1b* null mutants. Due to the lack of interaction between *Dt2* and *SOC1* in these mutant soybeans, *Dt1* expression was increased that caused delayed fowering.

Gibberellic acid biosynthesis is also very important for prolonged shoot growth. Plant height regulator genes in soybean, such as the dwarf gene *GmDW1, LATE ELONGATED HYPOCOTYL* (*GmLHY*), and an R2R3 MYB transcription factor *GmGAMYB* positively regulate gibberellic acid pathway [32]. A CRISPR/Cas9 knock out mutation application mediated by *Agrobacterium tumefaciens* produced a quadruple *LHY* mutant soybean (*Gmlhy1a,1b,2a,2b*), where it showed decreased plant height and gibberellic acid (GA3) level [19].

These functionally identifed genes have provided insights into the molecular mechanisms underlying determinacy in soybean. Further studies utilizing techniques like CRISPR/Cas9 gene editing have deepened our understanding of these genes' functions and their interactions. Overall, unravelling the genetic and environmental factors controlling determinacy in soybean will contribute to the development of improved varieties with enhanced yield capacity and quality.

# *3.3 Photo-Periodicity*

Soybean is a photo-period sensitive short-day plant, so it does not fower under long-day conditions. However, its adaptation to large latitudes requires a range of genetic variations of short-day activated genes. A group of genes has been identifed to be involved in soybean latitudinal adaptation by regulating foral initiation.

Cryptochromes are generally known as blue-light receptor proteins involved in plant development and circadian clock. In *Arabidopsis thaliana* CRY1 was found to be responsible for mediation of blue-light induced de-etiolation, and CRY2 for photo-periodic fowering. *GmCRY1a* promotes blue-light-induced cell-wall elongation inhibition and, in contrast to *Arabidopsis thaliana,* regulates photo-periodic fowering by increasing the expression of *FT* mRNA [36].

The maturity loci in soybean, *E1, E2, E3,* and *E4* have a predominant effect on mediating photo-periodic fowering and maturity. The dominant *E1, E2, E3,* and *E4* allele delay fowering and the recessive allele facilitates adaptation to high latitudes by promoting early maturing, whereas *J, FT2a, FT5a, Time of fowering (Tof)16,* and *Tof18* facilitate adaptation to low latitudes [1, 37]. *J* locus, encoding a homolog of *EARLY FLOWERING 3 (ELF3)* in *Arabidopsis thaliana,* functions as a suppressor of the *E1* allele and promotes early fowering. On the other hand, *E1* suppresses *FT2a* and *FT5a,* and also, two homologs of the red-light photoreceptor phytochrome A (phyA) *E3* and *E4* interact with plant circadian clock evening complex (LUX)

and *E1* to delay fowering [37, 38]. Therefore, the phyA-LUX-E1-FT pathway regulates photo-periodic foral initiation in soybean. Cai et al. (2018) produced *GmFT2a* frameshift mutant soybeans having a 1 base-pair insertion or short deletion by applying sgRNAs CRISPR/Cas9 vectors through *Agrobacterium tumefaciens* mediated transformation [39]. They showed that the *ft2a* soybeans were fowering later than the wild types under short and long day photoperiodic conditions. Another study that targets the photoperiodicity of a short-day soybean variety showed that a frameshift mutation of *E1* caused truncated protein production. This unfunctional proteins thus disinhibited *GmFT2a/5a* and therefore, initiated fowering under long day conditions [40]. Another fowering repressor gene *GmPRR37* was identifed by Wang et al. (2020) [41]. A CRISPR/Cas9 knock-out mutant *gmprr37* showed early fowering under long day conditions. Wang et al. (2023) clarifed the function of *E2* and its homologous genes *E2-Like a* and *E2-Like b* [42]*.* They designed a single and double mutants of *E2* and its homologous genes through CRISPR/Cas9 knock out method to investigate their function in fowering and grain yield and their interaction with *E1.* It was found that *E2* is the major regulator of fowering but *E2-Like a* and *E2-Like b* were redundant. Only double mutants *e2/e2-like a* or *e2/e2-like b* initiated fowering earlier than *e2* types. Additionally, Li et al. (2022) discovered the interaction between photoperiodicity and plant protection by identifying the function of *GmCDPK38*, a calcium-dependent protein kinase encoding gene [43]. A cutworm susceptible soybean was mutated through CRISPR/Cas9 and *GmCDPK38* sgRNA vectors delivered by *Agrobacterium tumefaciens. gmcdpk38* soybeans delayed fowering and produced more defence-related metabolites under long-day conditions. This might suggest a benefcial soybean improvement strategy for growing resistant soybean at low latitudes. Recently, a new locus identifed from GWAS, named *Tof8,* encodes a homolog of *the Arabidopsis thaliana FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1)* gene. There are two orthologous *FKF1* genes were found in soybean: *FKF1a* and *FKF1b.* These orthologous genes were found to be involved in fowering time and maturity by activating *E1* transcription through CRISPR/Cas9 knock-out mutation and two haplotypes of *FKF1b* were identifed to be a part of regulating the latitudinal effect on fowering which might be a cause of natural selection during environmental adaptation [37].

Soybean fowering is dependent on a short photoperiod, so it usually fowers in spring or fall. Unravelling the mechanism underlying the latitudinal effect on fowering time will support soybean breeding across a wide range of latitudes. Moreover, it will be possible to produce long-day soybean varieties through genetic manipulation to speed up the breeding process.

# *3.4 Seed Quality*

Seed traits determine the quality of the product. The seed coat, seed oil, oligosaccharides, lipoxigenases and protein contents are the important seed traits, which were improved during the domestication process. Breeding practices has shaped the modern soybean seed traits and improved the plant for a better human digestibility, consumption, harvestability and endurance. Gene editing accelerates this process by functionally identifying the genes of interest associated with the desired seed traits.

Improved seed coat water impermeability protects embryos for sustainable seed storage and provides resistance to environmental deterioration in the feld, such as pathogens, and mechanical and imbibition damages. Seed coat impermeability correlates with seed viability and longevity in soybean breeding programs. Additionally, hard seeds contain high calcium levels and increase the nutritional value of soyfoods [44]. As an adverse effect, hard seededness is unfavourable during postharvest processing for vegetable oil and soyfood [45, 46]. Indeed, high seed water permeability facilitates water absorption and makes the seed easy to germinate. Jang et al. (2015) identifed a seed hardness locus q*HS1*, which encodes an endo-1,4 β-glucanase [45]. A single nucleotide substitution from A to G in this gene dysfunctions the substrate-enzyme cleft domain and causes permeable seed coat in soybean. Likewise, a single nucleotide substitution from C to T by using *Agrobacterium tumefaciens* mediated transformations in *the GmHs1–1* gene, which encodes a calcineurin-like metallophosphoesterase transmembrane protein, showed increased permeability in the soybean seed coat [44]. Chandra et al. (2020) made inter-specifc crosses between *Glycine max* and *Glycine soja* to understand the genetic inheritance of seed coat impermeability by using 217 recombinant inbred lines [46]. They phenotyped seed coat impermeability by looking into slow and rapid imbibition rates of the offspring. They identifed three linked markers on chromosome 2, this locus was previously identifed by Sun et al. (2015) and Jang et al. (2015) [44, 45]. Additionally, the phenotyping results revealed semi-permeable genotypes that might cause by minor alleles, and one of them was found to be associated with leafet width, phytophthora resistance, and seed tocopherol. This implies that seed coat-identifying genes diversify in nature, and they maintain seed protection and coat-related alleles. The process of soybean breeding to improve seed coat impermeability should consider the involvement of the minor alleles as a potential genetic gain.

Another seed trait that was subjected to artifcial selection during the domestication process is seed oil content. Cultivated soybean seeds contain more oil than wild seeds, which shows the effect of domestication on selecting high oil capacity in the seeds [47]. Soybean oil has a great economical value in the market, after palm oil, it is the second most-produced vegetable oil in the world between 2018 and 2023. China is the world leader in the production and consumption of soybean oil (USDA, 2023). It is used for human consumption and shows tremendous health benefts. Soybean oil consists of 15% saturated and 85% unsaturated fatty acids, which is responsible for lowered blood cholesterol levels, and decreased coronary heart disease [48]. On the other hand, soybean seeds became one of the most consumed vegan proteins for vegetarian and vegan diets. Seed oil increases with sugar mobilization towards the embryo. The underlying reason was investigated and found that sugar fux-controlling genes are upregulated and provide precursors for fatty acid biosynthesis. However, increasing seed oil content adversely affects seed protein content, which eventually infuences the consumption of soybean as a source of protein.

Sugars Will Eventually be Exported Transporters (SWEET) gene family is responsible for the sugar transportation across cellular membranes having a role in the mobilization of carbohydrates from source to sink organs to sustain healthy growth and development of a plant [49, 50]. Bi-parental QTL mappings and genome-wide association studies dissect the genetic mechanisms by revealing QTLs on chromosomes 5, 7, 8, 10, 15, and 20, which are associated with seed oil, sugar, and protein contents [15, 51]. A major QTL has been identifed on chromosome 15, *GmSWEET10a* (Glyma.15G049200), that affects seed oil, size, and protein content [15, 47]. This locus encodes a member of the SWEET gene family, a sugar transporter gene, ensuring sucrose effux and allocating sugar from the mother seed coat to the flial embryo. Wang et al. (2020) showed that the frequency of the allele, which is signifcantly associated with seed oil content increase, is higher among landraces and cultivated soybeans than wild varieties [52]. Therefore, there was a strong artifcial selection during the domestication of soybean. Zhang et al. (2020) identifed a two-base-pair CC deletion in exon 6 of the cultivated and highseed oil-containing soybean varieties [47]. They also unravelled that this gene shows a pleiotropic effect on seed oil and protein contents since the varieties having CC alleles available are signifcantly rich in protein. Additionally, Wang et al. (2020) identifed a homologous locus of *GmSWEET10a*, named *GmSWEET10b*, by conducting a knock-out mutation through CRISPR/Cas9 showing a similar effect on seed oil and protein content while changing seed size; however, they could not fnd a signifcant artifcial selection for this locus [52]. Cai et al. (2023) showed a similar antagonistic effect between seed oil and protein contents by indicating contrasting synthesis of oil and protein under changing expression of *the GmMFT* gene through CRISPR/Cas9 mediated knock-out mutants [48]. The reason underlying this antagonistic behavior might be that during the domestication process, carbohydrate transportation to seeds enhances fatty acid biosynthesis rather than protein synthesis. Wang et al. (2019) showed that the metabolic composition of soybean seeds unravelled how protein-rich seeds produced nitrogen-assimilating amino acids -free asparagine, free 3-cyanoalanine, free glutamic acid, L-malic acid, free glutamine, and free aspartic acid- and at the same time, they expressed the negative correlation to seed oil content [15]. Therefore, for a rich protein seed content synthesis of these amino acids are crucial.

Research on soybean seed oil-rich varieties always shows big seed in size. This might explain why seed size-regulating genes co-segregate with seed oil-regulating genes. However, seed size-regulating genes do not always correlate with seed oil or protein contents. Silencing of the *GmFAD3* gene, encoding omega-3 fatty acid desaturase, showed increased seed size without changing seed protein and oil content [53]. This implies that the seed size trait controlling *GmFAD3* is not linked with seed sugar effux-controlling genes. Likewise, *the soybean seed size 1* (*GmSSS1*) gene, a homolog of *the Arabidopsis thaliana SPY* gene encodes an O-GlcNAc transferase protein and controls seed and pod size by showing pleiotropy, that was identifed through CRISPR/Cas9 mediated knock-out mutants, and it might be achieved through cell expansion and division [54]. The independence of seed size-regulating genes implies the epistatic effect of the size-regulating genes on *GmSWEET10a, GmSWEET10b,* and *GmMFT*.

Seed protein content is not the sole trait correlating with seed oil content, seed shape and coat color regulating genes are also co-segregating with the oil content. Soybean seed shape associated seed thickness (*ST1*) locus, encoding a UDP-Dglucuronate 4-epimerase, shows a pleiotropic effect on seed color and oil content through CRISPR/Cas9 mediated knock-out mutants. It acts upon seed shape by turning a fat seed into a round and catalyses the biosynthesis of UDP-galacturonic acid and participates in the glycolytic pathway. The conversion of UDP-glucuronic acid to UDP-galacturonic acid, which is a pectin precursor, promotes cell wall protein production. Simultaneously, glycerol-3-phosphate esterifcation produces triacylglycerol, which is a lipid biosynthesis precursor, and seeds show high oil content. The population analysis of 1209 soybean accessions, including 122 wild accessions, 542 landraces, and 545 cultivated soybeans, one haplotype, which shows a C to T polymorphism at nucleotide 203 of the *ST1* locus, was found the most frequent within the population and was signifcantly associated with high seed oil content and round shape. Another very interesting fnding was that seed coat color determining locus *l*, a highly unstable transposon-induced locus, when reversed, cosegregates with *ST1.* This might explain the high frequency of yellow, round, and high oil involving cultivated soybean seeds [74].

The anthropogenic impact of oil-rich soybean domestication cannot be overseen. One reason could be that seeds rich in oil might be a good source of energy storage in the human body, so a simultaneous increase in seed oil and size might drive people to select oil-rich varieties. Another reason might be that carbon and nitrogen are primarily required for high seed protein content, so under limited nitrogen resources, oil accumulating large seeds might have been selected by humans. However, this hypothesis does not explain why seed oil accumulation still competes with protein accumulation under nitrogen-suffcient conditions. Soybean protein is a crucial supplement for vegetarian diet. Therefore, upregulating the production of nitrogen assimilating amino acids will enhance the soybean protein level. Gene editing will provide confrmation of the gene functions that are involved in soybean protein production.

# *3.5 Abiotic and Biotic Stress Resistance*

Changing climatic conditions and pollution of air, soil, and water create devastating effects on soybean production. Abiotic and biotic stress conditions cause vulnerable soybean plants; indeed, their negative effects can be inherited and result in yield loss. Major abiotic stress conditions around the world for soybean are drought, salinity, cold, and fooding stresses [55]. For example, soybean production has been declining in Argentina, one of the major soybean producers, due to drought stress, which decreases the production, and so does the export and crushing [56]. QTL studies identifed causal loci, which can promote marker-assisted breeding for abiotic and biotic stress resistance. However, only a few of them have been validated through gene editing; otherwise, most of them are still in the candidate status.

The improved root system, enhanced water uptake, effective stomatal conductance, and slow wilting are some of the avoidance strategies in soybean from drought stress. Soybean breeding in light of genetic mechanisms can provide droughtresistant soybean crops and save soybean production under drought conditions. Throughout the investigations, several drought resistance-conferring genes have been functionally identifed. A crosstalk between plant hormones and transcription factors regulates plant response to stress conditions. NAC (NAM, ATAF, and CUC) [57, 58], MYB [59], WRKY [59], AREB [60], DREB [61, 62], AP2/ERF [63] transcription factors were found to be involved in this collaboration. Overexpression of soybean *GmNFYA13,* a nuclear localization protein, was found to be responsible for gaining resilience to salt and drought stress in transgenic soybean plants. Abscisic acid (ABA) is one of the plant hormones which control the physiological adaptations of a plant under stress conditions. For example, stomatal closure is induced by increasing ABA to prevent water loss. When ABA is artifcially induced in soybean, *GmNFYA13* expression was increased. This infers that *the GmNFYA13* gene is involved in abscisic acid-mediated stress response in soybean plants [64].

Soybean is a salt-sensitive plant, increased Na+ ions change cellular ion balance and damage cells. *Cation Diffusion Facilitator 1, Arabidopsis K+ Transporter 1,* and also some transcription factors that are generally involved in abiotic and biotic stress conditions, such as MYB, WRKY, AP2/ERF, and NAC are associated with salt stress resistance in soybean [65]. Wang et al. (2021) identifed an ABA and salt induced transcription repressor *GmAITR* in soybean to reduce the salinity stress related phenotypes without losing its ftness [66]. A CRISPR/Cas9 knock out mutant technique, *gmaitr* inhibited the expression of ABA and showed tolerance to salt stress.

Moreover, fooding stress is another abiotic stress that affects soybean production under ill-drained soils. Soybean roots are primary organs that are affected by fooding stress, limited oxygen uptake causes hypoxia and reduced energy production. To overcome this stress, plants undergo alternative energy-producing metabolic activities. Transcriptomic and proteomic studies unravel a group of proteins that are involved in cell wall modifcation, methylglyoxal detoxifcation, hypoxia reduction, pathogen defence, reactive oxygen species scavengers and chaperons, and energy production through glycolysis induction and alcohol fermentation [67–69].

Soybean production around the world is challenged by increasing negative impacts of fungus, bacterium, phytoplasma, nematode, and virus infections. Natural and artifcial selection strategies improved soybean resistance over the years and sustain its development and reproduction despite dynamic spatial and temporal conditions. Soybean breeding for biotic stress resistance is a very active process. Due to changing climate conditions, pathogen populations shift, and new races are introduced to host plants. This activates new protection mechanisms and benefcial mutations in resistance-conferring genes provide endurance to plants. The selection pressure on benefcial mutations can occur both naturally and artifcially. Zhao et al. (2015) investigated nucleotide fxation of pathogen resistance in wild and cultivated varieties, and their study revealed that *Glyma20g08290* (homolog of *Arabidopsis thaliana RPM1* gene) is a naturally selected locus, which is associated with *Pseudomonas syringae* in soybean (Ashfeld et al., 1995) and found in wild soybean varieties [70, 71].

Marker-assisted selection provides causal QTLs for vertical and horizontal resistance. It unravels major R genes, which maintain vertical resistance in soybean to soybean cyst nematode (*Rhg*), Phytophthora root and stem rot (*Rps*), soybean rust (*Rpp*), frog eye leaf spot (*Rcs*), bacterial blight (*Rpg*), and soybean mosaic virus (*Rsv* and *Rsc*) [65, 70, 72, 73]. R genes provide full protection in a race-specifc manner. Horizontal resistance is controlled by multiple minor effect genes and confers resistance against many soybean diseases such as sudden death syndrome, Sclerotinia stems rot, root-knot nematode, and most Pythium species [73]. However, this type of protection is not pathotype specifc so it is more long-lasting than vertical resistance. In vertical resistance, environmental conditions might cause genetic changes in avirulent proteins, which are recognized by pathotype-specifc R genes, or shift in pathogen populations. On the other hand, utilization of molecular markers associated with R genes is more feasible than pursuing a soybean breeding strategy for minor allelic resistance.

# **4 Conclusion**

QTLs have been identifed for a number of traits in soybean. These QTLs can be used to develop marker-assisted breeding programs to improve soybean cultivars for resistance to these stresses. Gene editing is a newer technology that can be used to rapidly and effciently introduce and edit specifc genes. Gene editing is a complementary approach to marker-assisted breeding, and the two technologies can be used together to accelerate the development of improved soybean cultivars. The advantages of using gene editing for soybean improvement:


The challenges of using gene editing for soybean improvement:


Despite the challenges, gene editing is a promising technology that has the potential to revolutionize soybean improvement. By combining gene editing with markerassisted breeding, we can develop soybean cultivars that are more resistant to abiotic and biotic stresses, have improved yield and nutritional quality, and are better suited to the changing climate.

# **References**


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# **Chapter 18 CRISPR/Cas-Based Precision Breeding of Oilseed Rape (***Brassica napus* **L.) – Recent Improvements**

### **Justyna Boniecka**

**Abstract** The seeds of oilseed rape (*Brassica napus* L.) are used in large-scale production of one of the most health-promoting plant oils in the food industry, as well as for animal feed and biofuel production. Thus, increasing the yield of this crop is of crucial economic and ecological importance. However, conventional breeding programs are slow, laborious and time-consuming. Hence, along with the discovery of the possibility to apply CRISPR/Cas technology to edit plant genomes and to accelerate the breeding process, much effort has been put into applying this technology to study specic genes and biosynthetic pathways, especially in species with many gene copies such as *B. napus*. Here, recent improvements in generating CRISPR/Cas-induced mutations in the *B. napus* genome, delivering CRISPR/Cas reagents into oilseed rape plant cells, fast-checking the efciency of targeted mutagenesis of CRISPR/Cas reagents, and oilseed rape transformation and regeneration procedures are described. Finally, new applications of CRISPR/Cas tools in oilseed rape precision breeding are discussed, focusing mainly on applications veried in eld.

Oilseed rape (canola; *Brassica napus* L.) belongs to the genus *Brassica*. It is a domesticated allotetraploid, which originated from spontaneous hybridization between two diploid species – turnip rape (*B. rapa*) and cabbage (*B. oleraceae*) – about 7500 years ago [1]. Today, oilseed rape is one of the most important oil crops worldwide, being cultivated in Europe and Asia predominantly as winter forms and in Australia, Canada and northern Europe as spring ones, providing food, feed and biofuel. This success is attributable to intensive breeding for seed quality traits in the last century. The rst milestone in oilseed rape quality breeding was achieved by the introduction of the rst low-erucic acid (EA – has a bitter taste and in high doses

J. Boniecka (\*)

Department of Genetics, Faculty of Biological and Veterinary Sciences, Nicolaus Copernicus University in Toruń, Toruń, Poland

Centre for Modern Interdisciplinary Technologies, Nicolaus Copernicus University in Toruń, Toruń, Poland e-mail: jboniecka@umk.pl

has been implicated in cardiac health problems) varieties (0-varieties), at the beginning of the 1970s, resulting in high-quality oil with high levels of desirable unsaturated fatty acids. The second milestone was the establishment (in the mid-1980s) of cultivars with low seed glucosinolates (GSL – in monogastric animals their digestion results in the release of toxic by-products that can cause liver and kidney damage along with lymph dysfunction) content (low EA and GSL together – 00-varieties), making possible the use of oilseed rape press residues as protein-rich fodder for animals [2].

However, these breeding programs have signifcantly reduced genetic diversity in modern breeding pools [3–5]. Both of the mentioned seed quality traits – low EA and low GSL content – originated from single genetic resources. Low EA derived from the German spring cultivar (cv.) "Liho", which carried spontaneous mutations in two *B. napus FATTY ACID ELONGASE 1* (*FAE1*) homologs, while low GSL content was frst identifed in the Polish spring-type cv. "Bronowski", which was found to possess at least three recessive genes for low GSL content. Large-scale crossing programs facilitated the introgression of these traits into all the different ecogeographical forms of oilseed *B. napus*. The result was the release in 1974 of the frst 00-quality spring variety, "Tower", with zero EA and low GSL content (in 1978, the term "canola", derived from "Canadian oil", was adopted to identify these varieties). The frst 00 winter oilseed rape cultivar, "Librador", was released in Germany in 1981. Consequently, modern oilseed rape breeding material has a relatively narrow genetic diversity [2].

This narrow genetic diversity markedly restricts breeding progress, which relies on the availability of genetic variation to introduce new desirable traits into crops. Initially, breeders sought genetic variation from landraces and heirloom varieties. The emergence of mutation breeding in the 1940s allowed for the artifcial induction of new genetic variation into plant genomes. This approach is extremely crude, as it introduces thousands of random mutations, both wanted and unwanted, with the latter requiring several rounds of backcrossing to remove. Moreover, in polyploid species, where most genes are multiple-copy genes with redundant functions, it is very ineffcient to change traits through random mutations. However, all in all, this technology greatly increased the amount of genetic variation available to breeders, and, interestingly, falls within the defnition of "conventional and traditional" breeding and thus is lightly regulated [6]. The frst commercial varieties developed through mutation breeding were registered in the 1950s, and now over 3400 varieties are listed on the FAO/IAEA Mutant Variety Database, including 21 of *B. napus* obtained mainly by applying gamma rays [7].

New breeding technologies, such as genome editing, are providing plant breeders with access to a far broader range of genetic variation. Moreover, compared with the traditional random mutagenesis, genome editing not only reaches the same end point as conventional breeding but gets there with a greater: i) degree of precision – no/limited number of unexpected mutations (hence it being termed "precision breeding"), and ii) speed, which is very important as, with conventional plant breeding, it may take 10–15 years or more to get new crops to the market [6]. The current legislation in the EU and many other countries puts plants improved by means of

genome editing "in the same box" as typical Genetically Modifed Organisms (GMOs). However, very favorable legislature for the production and consumption of such plants in the USA, Japan, Australia, some countries from South and Central America, and others such as Canada [8] is stimulating changes in the legislation in the EU. It is important to understand that simple genome edited crops, i.e., where no foreign genetic sequence is introduced, are indistinguishable and equivalent to conventionally bred crops and therefore could be regulated in a similar way [6].

Genome editing technologies encompass sequence changes within an organism or incorporation of valuable sequences into a germplasm, both resulting in an alteration of genotype without extensive backcross. These technologies are based on sequence-specifc nucleases (SSNs) such as zinc fnger nucleases (ZFNs), TAL effector nucleases (TALENs), and CRISPR (Clustered, regularly interspaced, short, palindromic repeats)/Cas (CRISPR associated nuclease; with *Streptococcus pyogenes* Cas9 being the most commonly applied). The frst two are relatively timeconsuming to construct, as protein engineering of their DNA binding domains is required to achieve the requisite target specifcity. The latter is the only RNA-guided endonuclease that targets DNA sites through nucleotide base pairing and, thus, has become the preferred SSN for genome editing in plants [9, 10]. SSNs create DNA double strand breaks (DSBs) at predefned genomic loci, which are then repaired through intrinsic erroneous, non-homologous end joining (NHEJ) or a donor DNA introducing homology-directed repair (HDR) pathways [11].

The CRISPR/Cas9 application uses a ~100-nt single guide RNA (gRNA or sgRNA), which is a combination of the crRNA and trans-activating crRNA (tracrRNA) originally encoded by the CRISPR loci [12], to direct Cas9 nuclease to a specifc DNA site. The complex *Sp*Cas9-sgRNA targets with its 20 nt guide sequence, being part of the crRNA (the rest of the crRNA sequence and the tracrRNA is a scaffold), genomic sequence of 20 nt – protospacer – upstream to a 5'-NGG-3′ protospacer adjacent motif (PAM), and introduces a DSB near the PAM sequence and originates blunt ends [13] that, in plants, will be repaired preferentially by NHEJ, often introducing indel mutations.

Due to the possibility of introducing mutations at multiple sites concurrently, genome editing is an excellent option to signifcantly accelerate the breeding process of, especially, polyploid species such as *B. napus,* with many gene targets (at least one homolog from each of the A and C genomes) [1]. CRISPR/Cas technology offers great potential when working on complex traits such as crop yield and disease tolerance, which requires simultaneously targeting multiple loci, related or unrelated, within a single cell. The technology has already been successfully applied to generate mutations in multiple locations in oilseed rape genomes by co-expressing multiple guide RNAs [14–17].

Oilseed rape is a high-value commodity crop, which has already been modifed and produced as traditional GMOs, despite the costs associated with regulatory compliance for such plants [6, 18]. Hence, it is not surprising that there is interest in investing in its precision breeding with the use of genome editing technologies. Here, recent works attempting to improve various steps on the way towards *B. napus* precision breeding are reviewed, as well as propositions/solutions that could

potentially improve the system even further. Since reviews, some of them very comprehensive, on the application of CRISPR/Cas in oilseed rape for gene function research and genetic improvements have been published only recently [16, 17, 19– 21], here, only some work that included feld trials with *Brassica* CRISPR/Cas modifed species will be described.

# **1 Optimization of CRISPR/Cas Reagents and Ways of Their Expression**

Mutating homeologs from both subgenomes of *B. napus* is necessary due to their functional genetic redundancy. An optimization of CRISPR/Cas constructs/reagents is essential, as the gene editing effciency depends to a large extent on the selection of a Cas protein and/or guide RNAs. Constructs encoding *Sp*Cas9 or *Lachnospiraceae bacterium* Cas12a (Cpf1; *Lb*Cas12a) endonucleases, with one or more guide RNA expression cassettes enabling production of either a single one-guide transcript, multiple one-guide transcripts or a single multi-guide transcript are the most widely used for CRISPR/Cas-mediated genome editing in most plant species. An example of the single, multi-guide transcript production is the CRISPR/Cas9 system based on endogenous tRNA processing that was used, for example, to introduce mutations in the genome of *Brassica oleracea* – a construct with tandemly arrayed tRNAsgRNA architecture to express multiple sgRNAs [22]. Another example of the single, multi-guide transcript production is the CRISPR/Cas system, where the multi-guide transcript produced by a Pol III from only one promoter is processed by the same nuclease involved in the generation of DSBs – Cpf1 [23]. The variety of CRISPR/Cas based nucleases and their corresponding guide RNA backbones [24] can be explored applying expanded cloning toolkits that contain not only modules encoding some of the mentioned elements but a number of promoters that allow expression of CRISPR/Cas nucleases and their guide RNAs in monocots and dicots, e.g. [25].

In plants, like in other organisms, not all transformants containing Cas-guide RNA encoding sequences display high levels of mutations in the target gene, with effciencies reported to vary from a few percent to close to 100% [26, 27]. The effciency with which mutations are generated in target genes depends on multiple factors, including the choice of target sites in selected genes, as well as the nature of the coding and regulatory sequences of the Cas gene and guide RNA construct. In recent studies, several architectural parameters of Cas constructs designed for plants have been investigated, including the codon usage of the Cas gene, the number of nuclear localization signals (NLSs) in the Cas enzyme, the nature of the promoters and terminators of the Cas gene, the length and sequence of the conserved region of the guide RNA and the terminator sequence of the guide RNA, and the relative orientations of the various expression cassettes in the fnal transfer DNA (T-DNA) [24, 28].

Marked improvements in the effciency of targeted mutagenesis (even up to 80% more mutated plants) of *Brassica* species – *B. oleracea* and *B. napus* – were observed when using a modifed version of Cas9. The application of a plantoptimized Cas9 CDS with one intron coupled with a tRNA guide architecture [22, 29] and with two guides per gene targeting two unrelated *B. oleracea* genes helped to outperform the system targeting these genes with two constructs each carrying information on two guides, each under its own Pol III promoter, and the humancodon-optimized *Hs*Cas9. The potato intron IV was originally added to avoid expression in bacteria during cloning and, as a side effect, can also increase expression *in planta* [24]. The application of the improved construct resulted in 100% of plants mutated in two of the four loci [29]. The same improved system but with one guide to edit two copies of a gene was very successful in *B. napus* and outperformed the system targeting these copies with the same guide but not coupled with the tRNA guide architecture and instead using the *Hs*Cas9 [29]. It is worth noting that, in the improved system, the transcription of sgRNA and Cas9 was in head-to-head divergent orientation, which has been shown previously to often result in a highly active CRISPR/Cas system. It was proposed that a weak terminator after Cas9 enables Pol II read through that could interfere with Pol III transcription of sgRNAs in some T-DNA construct architectures, and that this limiting factor can be corrected by divergent transcription of Cas9 and sgRNAs [24]. Another version of Cas9 with 13 introns – ZCas9 + 13int – this time prepared by introducing 13 *Arabidopsis thaliana* introns into the *Zea mays* codon-optimized version [30] (again coupled with tRNA guide architecture, with a single guide targeting a *B. oleracea* gene *GA4* or two copies of the gene in *B. napus*, with divergent transcription of Cas9 and sgRNAs) improved markedly the number of edited plants in comparison to the system not coupled with the tRNA guide architecture, head-to-tail orientation, and using the *Hs*Cas9 [29].

However, no matter what kind of modifcations of the Cas9 are proposed, the CRISPR/Cas9 system still has some limitations. One of these is the strict PAM dependence, which constrains the availability of target sites, especially to coding regions, as noncoding regions are relatively poor in 5′-NGG-3′ sites. Other limitations of *Sp*Cas9 include its large size for viral delivery and the low effciency in gene targeting caused by blunt DSBs. A solution comes with the CRISPR/Cas12a (Cpf1) system with i) a single crRNA (~42 nt, which is less than half that of Cas9, making it more suitable for multiplexed genome editing and packaging into viral vectors) and ii) Cas12a, an endonuclease smaller than *Sp*Cas9, which might facilitate viral delivery. This system requires a 5′-TTTN-3′ PAM sequence and introduces 5′ staggered ends, with 4–5 nt overhangs, at sites distal from 5′ T-rich PAM, at the end of the protospacer sequence, which has been proposed to favor gene insertions [31, 32].

Recently, *Lachnospiraceae bacterium* Cas12a (*Lb*Cas12a) was shown to generate edits in the genome of *Brassica oleracea* [29], which is a good indication that this endonuclease may function in *B. napus* as well. Working with this nuclease, four different constructs were analyzed for their effciency in inducing mutations in a *B. oleracea* gene. In the case of all constructs, the nuclease encoding sequence was under the UBI10 promoter and was followed by the sequence of the *Pisum*  *sativum rbcS E9* terminator. These sequences were in a head-to-head orientation with the RNA guide expressing cassette under the *At*U6–26 promoter.

In the case of the frst construct, an *Arabidopsis*-optimized *Lb*Cas12a CDS carrying a "temperature tolerant'' D156R mutation (ttAtCas12a) was encoded. The codon-optimized *Lb*Cas12a CDS carrying the mentioned mutation was shown to have increased activity in comparison to a non-mutated variant of the protein when *Arabidopsis* plants were grown for 2 weeks at 22°C or, especially, at 28°C. It is noteworthy that even the non-mutated variant, when put at 28°C, performed better at 4 of 5 loci tested [33]. Similarly, repeated heat stress treatments of *Arabidopsis* or *Citrus* plants had a major effect on the rate of mutagenesis by CRISPR/*Sp*Cas9 – in *Arabidopsis* it increased approximately fve-fold in somatic tissues and up to 100 fold in the germline. It was also proposed that *Sp*Cas9 is more active in creating double-stranded DNA breaks at 37°C than at 22°C [34]. The frst construct encoding the temperature-tolerant enzyme had a four guide (targeting one gene) expressing cassette, serving to produce transcript to be processed by the ttAtCas12a nuclease. The second construct encoded ttAtCas12a nuclease and a pre-gRNA transcript carrying information on one guide RNA (4 separate constructs each containing one of the four guides used in the frst construct scenario). The pre-gRNA is a self-processing ribozyme-fanked guide expression cassette named RibozymegRNA-Ribozyme (RGR). The RGR molecule contains ribozymes (possess nuclease activity) – Hammerhead (HH) type ribozyme at the 5′-end, and the terminal hepatitis delta virus (HDV) ribozyme at the 3′-end of the RGR, and thus undergoes selfcatalyzed cleavage to generate the desired gRNA. However, the introduction of a self-cleavable ribozyme in the 5′ end of the transcript is unnecessary when a 5′-G is added to the Cas12a DR (direct repeat – region of the crRNA whose proper folding is important for nuclease activity), which is compatible with the Pol III promoter AtU6–26 transcription start site. The HDV ribozyme removes the poly-A tail from the transcript, leaving no additional nucleotides at the 3′ position of the crRNA with a classical construct carrying unprocessed U6 termination signal, which conserves a spurious tail of adenines at the 3′ position of the Cas12 crRNA [29, 32, 35–37]. The third construct contained the human-codon-optimized CDS, with modifed CDS by inserting the D156R mutation to give ttHsCas12a, and the singular guide RGR expression cassette (one construct to target only one of the four target sites). The fourth construct contained ttAtCas12a, with an addition of 8 *Arabidopsis* introns generating ttAtCas12a + int, and the singular guide RGR expression cassette (one construct to target only one of the four target sites). The application of the singular guide architecture markedly increased the number of mutant plants, when applying constructs with ttAtCas12a, in two target regions from 0 to 10% and from 3 to 50% of the screened plants. The other two systems were applied only to compare the effciency of the system in one target region, where the application of the second construct increased the number of mutant plants from 3 to 50%. By using the third constructs, where instead of ttAtCas12a, ttHsCas12a CDS was applied, researchers showed that both nucleases performed equally well. Next, the application of ttAtCas12a + int further increased the number of plants with edits in the target region from 50 to 68% [29].

Although it is clear that the temperature-tolerant version of the Cas nuclease works well in *B. oleracea,* it would be interesting to compare its activity with the *Arabidopsis*-codon-optimized version. Based on the results presented, it is hard to conclude whether the heat tolerant enzyme works better than the "wild type" version and it is not clear whether heat stress increases the effciency of the CRISPR/ Cas system in *B. oleracea*, and thus potentially in *B. napus*, as was shown for *Arabidopsis* [33, 34].

# **2 Delivery of CRISPR/Cas Reagents and Elements Carrying Information on CRISPR/Cas Reagents into Oilseed Rape Plant Cells**

Oilseed rape plants bearing edited alleles have been predominantly obtained using *Agrobacterium tumefaciens*-mediated transformation. In one study, several essential factors that affect the transformation effciency, such as *Agrobacterium* strains, selection marker genes and genotypes of oilseed rape were analyzed. Comparison of different *Agrobacterium* strains showed that the GV3101 had higher transformation effciency than C58C1 and EHA105. The transformation effciency was 3.7–4.8%, 2.2–22.5%, and 1.6–5.9% when the hypocotyl of Westar was infected by GV3101 and screened under hygromycin, kanamycin and basta, respectively. The transformation effciency of Westar was highest and ZS11 was lowest when fve different genotypes of oilseed rape (Westar, ZS9, ZS11, GY284 and WH3417) were infected by GV3101 [38].

The auxotrophic *A. tumefaciens* strain LBA4404 Thy-, which exhibits the inability to survive, proliferate or grow in the absence of thymidine, provides a method for the transformation and regeneration of plant cells that does not need an *Agrobacterium* counter-selective agent to cure plant tissue of *Agrobacterium* [39], and recently this strain has been used in a genotype-independent method of oilseed rape plant transformation and regeneration [40]. Another important element of this genotypeindependent method is the application of the ternary pVir system with the T-DNA binary vector (with sequences encoding, e.g. CRISPR/Cas reagents) and an improved accessory plasmid [40] that is characterized by small size, enhanced vector stability, an improved bacterial selectable marker and amended *vir* genes. Application of the pVir system resulted in more effcient T-DNA delivery and stable plant transformation in diffcult-to-transform maize elite inbreds [40, 41].

However, some drawbacks remain in these strategies, as they involve delivery of DNA-based CRISPR/Cas reagents that are frst integrated into the genome, and expressed as a transgenic construct, and then segregated away by breeding as null segregants to leave only the desired edited allele/s [16]. T-DNA removal is crucial because regulatory constraints for gene-edited crops are likely to be less for those that do not contain foreign DNA [6]. Moreover, it can be important when a loss-offunction phenotype must be confrmed by complementation of the CRISPR/

Cas-induced mutation. A CRISPR/Cas construct still present in the mutant can target the complementation transgene and interfere with the resulting phenotypes [24]. Furthermore, as long as the T-DNA encoding the reagents of the CRISPR/Cas system is present in a genome, a progressive gene-editing process can take place, both during selection on appropriate media with antibiotics (which may lead to chimeras) and during the growth of the transgenic plants, and even in next generations [16, 42]. Therefore, DNA-free gene editing has received extensive attention in recent years.

One of the commonly applied T-DNA free oilseed rape genome editing methods is protoplast transfection, which is a transient (no T-DNA integration) alternative for delivery of CRISPR/Cas vectors [43, 44]. CRISPR/Cas reagents can be delivered into plant cells as ribonucleoproteins (RNPs) composed of purifed recombinant enzyme Cas and *in vitro*-transcribed or synthesized gRNA as well. Particle bombardment can be used to deliver RNPs into explants, whereas polyethylene glycol (PEG)-mediated transfection and lipofection can be used to deliver RNPs into protoplasts. Although CRISPR/Cas RNPs has become an attractive approach for genetic engineering, its editing effciency remains modest [45]. As an example, although Murovec et al. [46] were able to obtain mutation frequencies of 0.09 to 2.25% and 1.15 to 24.51% in *B. oleracea* and *B. rapa*, respectively, no mutations were detected after PEG-mediated transfection of oilseed rape (cv. "Topaz") protoplasts [46].

Although researchers have used protoplast systems to show the potential of CRISPR/Cas reagents to edit a locus or loci of interest, lack of protoplast regeneration protocols to obtain edited plants has been a major bottleneck in this system [16, 43]. Although *B. napus* protoplast regeneration was shown to be possible before the era of genome editing [47], only recently was a protocol for regeneration of protoplasts of oilseed rape in combination with genome editing proposed. Targeted genes – *BnGTR* – controlling glucosinolate transport from the vegetative tissues to seeds were mutated with high frequency [48]. According to this protocol, relatively high concentrations of auxins are essential for protoplasts to form cell walls and maintain cell division, and thereafter auxin should be reduced for callus formation and shoot induction. For shoot regeneration, relatively high concentrations of cytokinin are required, with the best combinations resulting in up to 45% shoot regeneration [48].

# **3 Fast-Checking the Effciency of Targeted Mutagenesis of CRISPR/Cas Reagents**

The process of plant genome editing is usually time-consuming as, in most instances, there is a need for regeneration of plants from tissue culture. Therefore, to have confdence that specifc genome editing components will work, it is important to test the CRISPR/Cas system of choice and, especially, the gRNAs, before plant genome editing.

To assess the effciency of CRISPR/Cas mutagenesis, the plant protoplast system is commonly applied. This system was used to analyze the effciency of CRISPR/ Cas vectors designed and built to target oilseed rape loci encoding proteins involved in the metabolism of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) – RelA/SpoT Homeologs (RSH]), which are likely to be involved in seed development, maturation and longevity [44, 49, 50]. Importantly, before checking the effciency of editing, the transfection effciency of the protoplast system can be determined with a GFP expressing vector, with the help of a microscope. Similarly, one can actually check whether CRISPR/Cas constructs were delivered into protoplasts and with what frequency. It has been shown that the effciency of PEG-mediated transfection depends on cultivar and concentration of PEG used for the transfection. Using this approach, followed with multiplex amplicon sequencing, it was determined that the rate of particular CRISPR/Cas-mediated edits at one of the *RSH* loci was in the range of 1–3% [44]. Genome editing events can be detected in many different ways, many of which were mentioned in Shillito et al. [51].

Very recently, another interesting system was proposed to avoid the traditional lengthy explant transformation and regeneration process to study the gene-editing effciency of various CRISPR/Cas constructs in oilseed rape – hairy-root cultures [52]. In this system, plant infection with *Agrobacterium rhizogenes* strains harboring a hairy-root-inducing (Ri) plasmid causes an abnormal rooting on hosts' tissues. After an agrobacterial infection at wounded sites, a T-DNA from the Ri plasmid is transferred to the host cells, where it is stably integrated into the plant genome. Subsequently, the expression of T-DNA genes leads to the induction of hairy roots. Agrobacterial strains carrying both Ri plasmid and artifcial binary vector have been widely used for delivering foreign DNA into plant cells. Recently, using this system, researchers examined the mutation effciency of nine different CRISPR/Cas9 constructs to edit the auxin biosynthetic gene *TRYPTOPHAN AMINOTRANSFERASE* (*BnTAA1*; two paralogs). They showed that the plant-codon-optimized *Sp*Cas9 with the potato IV2 intron (pcoCas9) is more effcient in mutating the targeted loci than *Staphylococcus aureus* Cas9 (*Sa*Cas9; PAM – 5′-NNGRRT-3′) encoded by a gene that is about 1 kb shorter than *pcoCas9* – 83.05% of mutated loci versus 47.98%. Moreover, they observed a slight increase in effciency when using a longer version of 35S promoter (1.3 kb) compared to the shorter one (0.4 kb) – 67.29% mutated loci vs 58.7%, respectively. The effciency of mutagenesis was also increased in the presence of the SV40 nuclear localization signal (NLS) – by 25% (75.82% with NLS-Cas9 versus 50.53% with Cas9). With the most effcient construct – NLSpcoCas9 – 96.95% loci were mutated, with less infuence on the promoter choice. Next, among pcoCas9 constructs, NLSpcoCas9 induced homozygous mutations with the highest effciency (33% of the mutated loci) [52].

The hairy-root system is a fast and straightforward system – it makes it possible to evaluate the most effective gene-editing construct within approximately 2 months after transformation. According to the protocol of Jedličková et al. [52], hypocotyls of 18-day-old seedlings are used for *Agrobacterium* injection, and the frst calli and hairy roots are detected after 2 weeks. Another advantage of this system is the

relatively high transformation effciency (number of seedlings with emerging hairy roots over the number of injected seedlings). Three cultivars were tested (DH12075, Westar and Topas), and it was observed that the transformation effciency was 97%, 84% and 42%. It is worth noting that Topas is a cultivar recalcitrant to petiole-based transformation. Hence, this system seems to work in a genotype-independent manner. Furthermore, hairy roots with homozygous/biallelic mutations can be used to some degree for functional gene studies, e.g. when the analyzed genes encode proteins involved in the production of metabolites, as the hairy roots have been used for the production of secondary metabolites or to investigate phytoremediation processes. Next, as it has been shown in the case of *B. napus*, using the edited hairy roots, one can regenerate plants applying appropriate protocols [52].

# **4 Oilseed Rape Plant Regeneration**

One of the milestones of oilseed rape molecular breeding has been an effcient *in vitro* regeneration, though regeneration rates are genotype-dependent. Several elite *B. napus* varieties have not been easy to transform. At present, the reported varieties for *A. tumefaciens*-mediated genetic transformation include spring oilseed rape varieties (e.g., cv. "Westar", "862" and "Haydn") and semi-winter varieties (e.g., cv. "J9707", "J9712" and "ZS6") [17]. For many years, researchers aiming to improve *B. napus* plant regeneration protocols have focused on applying explant/chemical approaches, which are based on testing explants and different ratios of auxin and cytokinin to induce cells to differentiate into whole plants [53]. So far, the most widely used method for the development of genetically modifed *B. napus* plants has been the *Agrobacterium*-mediated hypocotyl transformation [e.g., 16, 17, 38, 54–57]. However, this method, even after modifcations and other methods applied so far are genotype-dependent [40, 58].

Recently, an alternative, genotype-independent *Agrobacterium*-mediated *B. napus* transformation method was developed that is rapid and amenable for highthroughput transformation and CRISPR/Cas-mediated genome editing; the method is based on epicotyl and higher stem (internodal) segments (3–4 mm), and it has been successfully implemented in multiple oilseed rape genotypes, though with varying transformation effciencies. Epicotyl segments produced signifcantly higher rates of shoot formation compared to hypocotyl segments across all genotypes tested [40].

An effcient and likely genotype-independent system to obtain edited plants is plant regeneration from hairy roots. Recently, an optimized regeneration protocol for *B. napus* cultivar DH12075 was proposed [52]. Using this system, in combination with embryo rescue (21–28 days after pollination) from seeds containing torpedo-stage embryos or older, it is possible to obtain transgene-free T1 plants with desired mutations roughly 1 year after agrobacterial transformation. Moreover, the protocol was used for regenerating plants from hairy-root cultures of Topas, a variety referred to as being in many instances recalcitrant to transformation and plant

regeneration. The system nonetheless has some disadvantages. The *rol* genes, which are crucial for hairy root formation, encoded on the T-DNA of the Ri plasmid, are integrated into the plant genome, and their presence in regenerated plants is responsible for altered growth characteristics called "Ri phenotype". Thus, to obtain transgene free and Ri phenotype free plants, one has to carry out segregation analyses to obtain plants with neither the Ri T-DNA nor the CRISPR/Cas T-DNA [52].

Recently, to improve plant regeneration, molecular genetic-based methods have been studied [53]. These methods require transfer of sequences encoding morphogenic factors/developmental regulators, including *WUSCHEL* (*WUS2*) or *BABY BOOM* (*BBM*), that signifcantly improve the effciency of plant regeneration and allow the regeneration of thus-far recalcitrant genotypes [53, 59]. However, in order to avoid genotype-specifc pleiotropic effects, including abnormal plant growth and infertility, the expression of these genes must be regulated. To solve this problem, one can use the system based on overexpressing of a fusion protein combining transcription factor GROWTH REGULATING FACTOR 4 (GRF4) and its co-factor GRF-INTERACTING FACTOR 1 (GIF1) or of *Arabidopsis GRF5* and/or its homologs, which helps to enhance plant regeneration and transformation without affecting plant growth and fertility [60, 61]. In oilseed rape (cv. "BNS3"), overexpression of *AtGRF5*, *AtGRF6*, *AtGRF9* or *BnGRF5-LIKE* was shown to signifcantly increase transgenic callus production of hypocotyl explants; however, it had no signifcant impact of shoot formation [61].

Tissue culture procedures are often technically demanding, time-consuming and laborious. Hence, no-tissue-culture-required delivery methods, which are genotypeindependent, such as nanoparticles [62] or virus delivery [63, 64], could be very helpful to further extend the application of CRISPR/Cas in oilseed rape genome editing. The foral-dip method, commonly used for *Agrobacterium*-mediated transformation of *A. thaliana* plants [65], is another such method. This approach was already applied to transform *B. napus* plant but not in combination with genome editing [66, 67]. The drawback of this method is that, to transform oilseed rape plants in this way, one must wait a relatively long time until the plants reach inforescence, which is much longer for *B. napus* than for *A. thaliana*. To overcome this problem, it is possible to generate and use rapid fowering lines. One such example is fast-fowering mini maize, amenable to transformation and editing, with a seedto-T1-seed time of 5.5 months compared to over 9 months for other genotypes [68].

# **5 New Applications of CRISPR/Cas Technology in Oilseed Rape Precision Breeding**

CRISPR/Cas9 system was frst applied in oilseed rape to target two *ALCATRAZ* (*ALC*) homologous genes for site-directed mutagenesis to avoid seed loss during mechanical harvest by increasing shatter resistance [69]. Since that time, this system has been applied for oilseed rape gene function research and genetic improvement relating to weed control, fowering, self-incompatibility, plant hormone biology, abiotic and biotic stress resistance, grain composition and pod shatter reduction. Since most of these achievements were well described in recent review reports [16, 17, 19–21], here, only the most recent applications of the CRISPR/Cas technology in *B. napus* (and closely related species) precision breeding that have been accompanied by feld experiments will be summarized.

CRISPR/Cas9 technology was used to create targeted mutations on two homologous copies of the *FAE1* gene (on the A08 and C03 chromosomes), which plays a decisive role in the synthesis of erucic acid, in three *B. napus* germplasms with high EA (>30%) and high oil (>50%). The EA content was signifcantly reduced by more than 10 percentage points in the mutant of *BnC03.FAE1*, while the double mutation of *BnA08.FAE1* and *BnC03.FAE1* resulted in nearly zero EA in three *BnFAE1* edited germplasms, and the oleic acid content was increased in different degrees. The confrmed homozygous T2 mutant lines without Cas9 were grown in an experimental farm in China, and the feld management was performed in line with standard breeding practice. The agronomic yield-related traits, including plant height, branch height, branch number, silique length, number of siliques per plant, 1000 seed weight and yield per plant, were measured. It was concluded that growth and yield of the mutant plants were not signifcantly different in comparison to wild type plants. These results provide a way for future low-EA breeding, broadening the resources of *B. napus* with low EA [57].

CRISPR/Cas9 *MYB28-*edited *Brassica oleracea* plants were the subject of the frst CRISPR/Cas feld trial in the United Kingdom approved and regulated by the UK Department for Environment, Food & Rural Affairs after the reclassifcation of gene-edited crops as genetically modifed organisms by the European Court of Justice on July 25, 2018. The *MYB28* gene encodes a transcription factor characterized as a key regulator of aliphatic glucosinolate (A-GSL) biosynthesis in *Brassica* genus. A-GSL derivatives may contribute to the putative health-promoting effects of cruciferous plant vegetables. Knocking out of *MYB28* resulted in downregulation of A-GSL biosynthesis genes and reduction in accumulation of the methionine-derived glucosinolate – glucoraphanin, the precursor for isothiocyanate sulforaphane, which is believed to have health-promoting effects – in leaves and forets of feld-grown broccoli plants [70]. These results demonstrate the potential for the gene-edited plants to express the improved traits when grown in feld conditions.

# **6 Conclusions**

CRISPR/Cas-mediated genome-editing offers great potential for both genetic improvement and biological research. Hence, this technology is being constantly developed, especially to make it more widely applicable and effcient for economically important crop species, such as oilseed rape. One of the improvements in the CRISPR/Cas technology is the application of new or improved CRISPR/Cas reagents (e.g., Cas12a, with its differing PAM requirement comparing to Cas9) and/ or ways of their expression, which have increased the scope and effciency of

targeted mutagenesis. An important step was developing and improving methods of delivering CRISPR/Cas reagents, or of sequences encoding them, which has a tremendous impact on the fnal time that it takes to obtain transgene-free CRISPR/Cas edited plants and/or the effciency of transformation and regeneration. Next, important protocols have been developed for analyses of different CRISPR/Cas variants/ gRNAs to assess their effciencies before applying to obtain edited plants. Lastly, setting up genotype-independent regeneration protocols to obtain mutations in a desired elite germplasm has been a tremendous achievement.

However, although the presented tools and methods serve to make a major contribution to more effcient and rapid gene discovery and functional characterization, some CRISPR/Cas variants and methods applied in the frame of genome editing in plants await implementation in *B. napus* precision breeding. These include the application of editing technologies such as homology-directed gene editing or prime-editing to enable the most precise and defned edits in this crop. Furthermore, increasing the overall effciency of regeneration protocols remains an active area of research.

The CRISPR/Cas system has even further scalability; one can use it to either regulate gene expression or introduce a single base change or introduce/remove epigenetic marks, by using a system where, respectively, a transcriptional activator/ repressor or a base editor or a protein introducing/removing epigenetic marks is linked to a Cas endonuclease that functions as a nickase or a "dead" Cas [71, 72]. While some of these applications have already been applied in *B. napus* precision breeding [16, 20], some still await implementation.

The next reasonable step is to introduce the gene-edited traits through introgression breeding into elite varieties or, when genotype dependency does not play a role, apply the CRISPR/Cas technique to directly modify these elite varieties.

**Acknowledgments** The research experiments conducted by JB, fnanced from the funds of the National Science Centre in Poland (2017/01/X/NZ1/01981) and the Ministry of Science and Higher Education in Poland (1189-B research grant from the Faculty of Biological and Veterinary Sciences at the Nicolaus Copernicus University in Toruń), were the inspiration to write this publication. The Author acknowledges the European COST Action CA18111 – Genome Editing in Plants for providing an excellent platform to discuss genome editing-related topics.

# **References**


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# **Chapter 19 Targeted Gene Editing in Pome Fruit Genetics and Breeding: State-of-the-Art, Application Potential and Perspectives**

**Hanne Claessen, Pollien Aert, and Nico De Storme**

**Abstract** Even though traditional breeding of perennial fruit trees such as apple and pear has resulted in high performing cultivars in the past, it is a very lengthy and costly process that is unable to keep up with the increasing demands for improved yield, resistance and fruit quality posed by the growing world population and the rapidly changing climate. In the last decade, signicant research advances have been made that can revolutionize pome fruit breeding to meet current needs, including the sequencing of apple and pear genomes, the increased understanding of associations between gene(s) and traits of interest, and the advancement in genetic engineering tools. In particular the emergence of genome-editing tools such as the CRISPR/Cas9 technology can signicantly improve the speed and accuracy of pome fruit breeding programs. This chapter reviews the progress, opportunities and challenges of genome editing tools in apple and pear, and discusses the genetic basis of several important breeding goals to nd possible targets for new gene-editing applications.

# **1 Introduction**

The term "pome" refers to an accessory fruit produced by temperate tree species belonging to the maleae tribe of the Rosaceae family. The best-known and economically most important pome fruits are apple and pear. In 2020, apples were among the ve most produced fruit crops worldwide together with watermelons, bananas,

H. Claessen · P. Aert

Laboratory for Plant Genetics and Crop Improvement (PGCI), Division of Crop Biotechnics, Department of Biosystems, University of Leuven, Leuven, Belgium

N. De Storme (\*)

Laboratory for Plant Genetics and Crop Improvement (PGCI), Division of Crop Biotechnics, Department of Biosystems, University of Leuven, Leuven, Belgium

Leuven Plant Institute (LPI), KU Leuven, Leuven, Belgium e-mail: nico.destormde@kuleuven.be

oranges and grapes with 88.3 million metric tons [1]. Both apples and pears are commercially grown in over 50 countries with China being the top producer for both fruit crops [2]. Due to their economic importance and the wide geographic distribution of their production, ongoing breeding programs are present all over the world aiming to improve various traits including higher yield, increased disease resistance, and improved fruit quality [3–5].

Pome fruit breeding is an expensive and lengthy process due to biological characteristics typical of woody tree species, namely a long juvenile period (5–7 years), self-incompatibility, high heterozygosity, a limited available gene pool for new traits, and a large genome size and chromosome number [6, 7]. In a classical breeding scheme, selected parents are intercrossed to create hybrid seedling populations, consisting of a pool of unique genotypes, followed by a strict selection of the best performing progeny clone. Due to the limited genetic variation present in advanced breeding material and established cultivars, it is often necessary to use wild or semiwild gene pools as sources for the introduction of new traits. Pre-breeding to obtain suitable parents from these (semi-)wild gene pools that can be crossed with elite germplasm takes decades due to the long life cycle and the need for repeated cycles. More specifcally, the introgression of desired alleles originating from donor varieties with a minimum of linkage drag and concomitant selection for various other traits, requires multiple generations of hybridizations followed by selection, and thus signifcantly adds to the breeding time [7]. Alternatively, non-GMO breeding techniques that are used include interspecifc hybridization, induced mutagenesis and polyploidization. Also, a wide range of genetic and molecular tools have been used to improve the effciency and selection accuracy of pome fruit breeding programs such as genetic linkage maps, molecular markers and whole genome sequencing [8–11]. Each of these techniques have their own advantages and drawbacks, but many of the limitations remain similar to traditional breeding.

Recently, the introduction of genome editing techniques such as CRISPR/CAS has provided effcient ways to introduce precise mutations in plants. These techniques are especially benefcial in clonally propagated fruit tree species since they can generate improved breeding outcomes compared to conventional techniques without the extensive backcrossing and associated linkage drag that is necessary when introgressing new traits in established cultivars [12]. These genome editing techniques have the potential to greatly accelerate and improve the breeding process of woody fruit tree species, even though important challenges and limitations still need to be overcome to allow their broad scale application.

This chapter reviews the recent progress in the application of genome editing techniques in apple and pear, as well as the specifc opportunities and challenges. We also outline and discuss important genes underlying economically interesting traits which could be used as valuable targets for gene-editing.

# **2 Genome Editing Technologies in Pome Fruit Trees**

Genome editing refers to the use of genetic transformation techniques that can be used to precisely edit the plant genome [13]. There are three main genome editing approaches that are all based on the use of engineered nucleases, namely zinc fnger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN) and clustered regularly interspaced short palindromic repeats associated nucleases (CRIPSR/cas) [3]. These nucleases can be designed to bind a specifc target DNA sequence in the genome of the plant where they induce a double-strand break (DSB) which is subsequently repaired by one of the two following processes, homology directed DNA break repair (HDR) or non-homologous end joining (NHEJ) [14, 15]. Plant transformation is generally performed using *Agrobacterium tumefaciens* followed by regeneration of transgenic tissue *in vitro*. This paragraph describes the current use, adaptations and limitations of these three genome editing techniques in pome fruit breeding.

# *2.1 Zinc Finger Nucleases (ZFNs)*

Zinc fnger nucleases were one of the frst editing technologies [16]. They are artifcial enzymes generated by fusing a zinc-fnger DNA-binding domain to a nonspecifc DNA-cleavage domain of the Fok I endonuclease enzyme [13, 17]. A pair of custom designed ZFNs bind to the DNA at the target location and together form an active dimer nuclease complex [18]. ZFNs have been successfully used for targeted mutagenesis in many species including *Arabidopsis* [19, 20], soybean [21], rice [22], and populus [23]. To our knowledge, only a single successful instance of ZFN application in pome fruit has been published [13]. The authors validated the use of ZFNs in apple and fg using a visual transgenic repair assay based on activation of a mutated uidA gene, which encodes the GUS reporter protein. The overall effciency in apple was around 10% and the authors concluded that the genome editing approach was suitable for application in fruit tree species. However, although the technique has been around for almost three decades, the adoption of ZFNs in plant breeding is limited mainly due to their low effciency, the complex construction of the zinc fnger region that interacts with DNA and severe off-target effects [24–27].

# *2.2 Transcription Activator-Like Effector Nucleases (TALENs)*

Another genome editing approach is based on the family of proteins known as transcription activator-like effectors (TALEs) which are produced in bacteria including plant pathogenic Xanthomonas species. These effector proteins have a DNA binding domain which is linked with a catalytic domain of an endonuclease like Fok I [18, 25]. TALE nucleases (TALENs) with desired specifcities can be created through modifcation of the DNA binding domain to target specifc locations in the genome. The nucleases there act like molecular scissors that produce DSBs which are repaired with the cellular repair mechanisms leading to deletions, insertions, replacements or rearrangements [18, 25].

TALEN-mediated genome editing has been successfully applied in rice [28, 29], wheat [30], maize [31], and sugarcane [32, 33] among others. However, to our knowledge, no application of TALEN-based transformation has been applied in apple or pear or even any other woody fruit tree species such as citrus or prunus. Although TALENs have some advantages over ZFNs, such as lower toxicity and somewhat simplifed construction, the CRISPR/Cas approach has quickly surpassed both ZFNs and TALENs as method of choice in plant genome editing [26, 27].

# *2.3 CRISPR/Cas Systems*

Currently, clustered regularly interspaced short palindromic repeats (CRISPR) based genome editing has become the most reliable and cost-effective approach in plant research. Unlike ZFN and TALEN, the DNA-recognition, based on RNA-DNA interactions, is faster, cheaper and generally more effcient [17, 34]. In plants, CRISPR/Cas 9 is the most used system and consists of a Cas 9 nuclease and a single guide RNA which replaces the original CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracr RNA). The gRNA contains a unique sequence of 20 bp which preceeds a protospacer adjacent motif (PAM). This gRNA binds the Cas 9 nuclease and directs it to a complementary target sequence on the genomic DNA. The two nuclease domains of the Cas 9 protein (RuvC and HNH) will then cleave the target sequence at three nucleotides upstream of the PAM site, leaving predominantly blunt ends [26, 35].

The CRISPR/Cas9 method has been widely adopted in plant research and has been used in many crops including pome fruit species. It was frst used in apple to modify an apple phytoene desaturase (*PDS*) gene which encodes an essential plant carotenoid biosynthetic enzyme required for chlorophyll biosynthesis. Knock-out of this gene leads to an albino phenotype making it an easy visual marker [34, 36]. CRISPR/Cas9 was also used to reduce susceptibility to fre blight in apple [37], and induce early fowering in apple and pear [38]. It has also been applied in wild apple to target the *MsPDS* gene [39]. To date, nearly all applications in pome fruit use the CRISPR/Cas9 system, however alternative CRISPR systems can be applied as well [40]. One example is the CRISPR/Cas12 system, previously known as Cpf1, which also belongs to the class 2 CRISPR systems, but lacks the HNH domain and generates a staggered cut with a 5 nt 5′ overhang [41]. CRISPR/Cas12 has been successfully applied in other woody, perennial tree species such as poplar [42] and citrus [43]. Alternative CRISPR/Cas systems may be more suitable in specifc situations, for example the CRISPR/Cas12 can be used to target T-rich regions of the genome which is diffcult with CRISPR/Cas9 [44].

Base-editors are another type of genome-editing tools. They are derived from the CRISPR/Cas9 approach and allow precise nucleotide substitutions without double stranded breaks [45]. In plants, DSBs are generally repaired using the NHEJ mechanism which creates small insertions or deletions in the target sequence and usually results in gene knock-out. The alternative HDR mechanism can precisely introduce point mutations using template DNA, however it is very ineffcient, especially in plants [45]. Base editors don't use DSBs, but create precise base substitutions of A-to-G or C-to-T using a nickase Cas9 (nCas) coupled to either an adenine deaminase (ABE) or a cytidine deaminase (CBE), respectively [45, 46]. The CBE-nCa9s fusion converts cytosine to uracil without cutting the DNA and this uracil is later converted to thymine through DNA replication or repair. Similarly, the ABE-nCa9s fusion converts adenine to inosine which is later converted to guanine. A uracil DNA glycosylase inhibitor protein (UGI) can be added to the construct to prevent uracil excision which lowers the effciency of the transformation [46]. Malabarba et al. [45] applied a CBE base editing system including a UGI sequence and the nCas9-PmCDA1 fusion for the frst time in apple and pear on two targets, *acetolactate synthase* (*ALS*) and *phytoene desaturase* (*PDS*). The authors induced a stopcodon in *PDS* and an amino-acid substitution in *ALS* in apple and pear resulting in chlorsulfuron herbicide resistant, dwarfed, albino plants. The study proved the feasibility of targeting multiple genes with base editing in apple and pear but also revealed important challenges that still need to be addressed.

# *2.4 Limitations of Genome Editing*

Despite the advantages and many potential applications of these new genomeediting tools, important challenges still remain. While most studies currently use CRISPR-based methods for genome editing, many of the limitations listed here also apply to ZFN and TALEN methods.

A frst major drawback of these genome-editing tools, including CRISPR-based methods, is insuffcient target specifcity and accuracy. Off-target cleavage can occur when the frst 17–20 nucleotides of the sgRNA match with other regions in the genome instead of solely with the target and also in apple, these off-target mutations have been reported [45].

Another problem is the production of chimeras. Several of the studies on the genome editing of pear and apple discussed in this chapter report the production of chimeras during regeneration of transformed apple or pear plants [45]. Chimeras consist of genotypically distinct cells or tissues which may refer to transformed vs non-transformed cells, but also to transformed cells with different mutant alleles where the genome editing machinery has introduced distinct mutations in the same gene of different cells [45]. For example, Charrier et al. [38] reported a high rate of phenotypic chimeras (64% of regenerated transgenic plants) and editing chimeras (88% of pure albino phenotype transgenic plants) after CRISPR/Cas9 KO of the *PDS* gene in apple. The number of editing chimeras also seemed to increase during

the regeneration period, possibly due to the continued expression of the CRISPR/ Cas9 cassette. Elimination of chimeras is necessary to establish stable mutants that can reliably pass on the desired trait to progeny. However, exclusion of chimerism through sexual reproduction of the transgenic plants is diffcult in the highly heterozygous and self-incompatible woody fruit tree species. Alternatively, adventitious shoot regeneration may be used, but this process takes several rounds of regeneration [45]. Another possible method is the direct delivery of CRISPR/Cas9 ribonucleoprotein (RNPs) into protoplasts which could decrease the number of produced chimera [47]. Direct transfection of apple and grapevine protoplasts has already been performed by delivery of cas9 and gRNA using PEG solution [48, 49].

Transformation effciency in fruit trees is generally still very low compared to herbaceous plants even using CRISPR/Cas9. One major obstacle contributing to this low effciency is the highly recalcitrant nature of these species to both genetic transformation and *in vitro* regeneration [50]. In addition, this transformation and regeneration effciency seems to vary signifcantly based on the target, transformed cell type, delivery method, regeneration method, species and even genotype. For example, pear transformation seems to happen at lower effciency compared to apple: the use of the CRISPR-Cas9 system to knock out Terminal Flower 1 (TFL1) genes in apple and pear to obtain the early-fowering genotype, resulted in 93% of apple transgenic lines compared to 9% of pear transgenic lines [38]. A recent study attempted to increase transformation effciency in apple through ectopic expression of *MdBBM1* which promotes plant regeneration [50]. However, it seems that extensive protocol optimization remains necessary for transformation and regeneration of different species and even different cultivars. This also makes obtaining a transformed plant, albeit less laborious than conventional transgenesis, still very cumbersome, especially since regenerated plants still require screening and quality control.

Finally, there is considerable public concern regarding transgenic plants. After the custom-designed nucleases are transformed into transgenic plants and have induced the desired DSB and mutation, they have to be removed to obtain transgenefree plants. This can happen via genetic segregation and back-crossing, but this method is less suitable for fruit trees [13]. Better approaches include transient expression of CRISPR/Cas9 or the use of preassembled CRISPR/Cas9 ribonucleoproteins (RNPs) [37, 51].

# **3 Application Potential of Genome-Editing for the Advancement of Important Pome Fruit Breeding Goals**

# *3.1 Yield Improvement*

Higher yield is one of the most important traits to achieve in plant breeding, albeit also one of the most diffcult since it is a quantitative trait that is determined by many underlying small-effect genes and is highly infuenced by environmental conditions and management practices [52]. The complex background of yield makes this trait in itself very diffcult to use as a specifc breeding target, especially for targeted gene-editing. Instead, it can be considered as a combination of two traits, fruit number (crop load) and fruit size, which need to be carefully balanced and which are both still the result of a complex, multigenic regulation with signifcant environmental impact.

No known pome fruit breeding programs have increased crop load as a breeding target. Instead, a lot of research is focused on crop load management to minimize biennial bearing and low fruit size due to overcropping. Good management practices to optimize fruit load include cultivar-adapted pruning to optimize the light penetration into the canopy and the distribution of spurs versus extension shoots, thinning of fower buds, fowers and fruitlets, and well-considered root-stock choices to control vegetative growth [53, 54]. High and stable crop load in apple and pear are therefore mainly indirectly targeted in breeding programs through more specifc objectives such as root stock breeding, optimized tree architecture and decreased biennial bearing tendency. Tree architecture and biennial bearing are discussed more elaborately later in this chapter.

Similarly, fruit size is also largely determined by environmental factors including orchard management, fower fertilization success, seed number and current crop load [55]. However, more information is available on the genetic basis of fruit size potential compared to crop load since studies often use single fruit weight as an indicative measurement. Fruit size is a complex quantitative trait that is controlled by multiple genes [56]. It depends on the number of cells, the size of the cells and the size of the intracellular spaces [57]. Many QTLs and several major-effect genes have been identifed in different studies [56]. Some interesting major-effect genes in apple were homologous to *Arabidopsis* cell expansion gene *AtSAUR19* and tomato fruit size/shape determining genes *SlOVATE* and *SUN* [58]. Also, several miRNAs have been associated with fruit weight. For example, overexpression of miR172p in transgenic "Royal Gala" apple signifcantly reduced fruit size [56]. These miRNAs are part of a large superfamily of transcription factors that play important roles in growth, development and stress response in higher plants [59]. However, the exact functions of most of these genes and miRNAs remain elusive and more research is necessary before they can be reliably used to improve fruit size using genome-editing.

Still, yield as such is often not a primary breeding objective since many other traits are considered at least as important as high yield for the acceptance of a cultivar for commercial production. These traits include many practical aspects of fruit production such as effcient orchard management (trees must be easy to harvest and cheap to maintain), disease resistance, production stability, storability, and transportability. Also many fruit quality characteristics such as favor, texture, frmness, fruit size and novelty are important for marketability. These traits are breeding objectives in their own right and the most important ones are further discussed in this chapter.

# *3.2 Fruit Quality Attributes*

#### **3.2.1 Sensorial Fruit Quality**

The sensory or organoleptic quality of apple and other fresh fruits is determined by three main parameters, namely taste, texture and aroma. These three major sensory fruit quality attributes each independently impact the sensorial evaluation of the fruit, but also exhibit an intricate interplay that largely determines the overall taste and appreciation of fresh fruit. Some organoleptic quality attributes, like soluble sugar content, titratable acid (TA) and fruit frmness can be quantifed using biochemical assays. However, these singular quality attributes only provide partial insights of the fruit quality and the evaluation of organoleptic fruit quality is therefore still mainly performed via sensory panels. Research on existing cultivars suggests that, in general, consumers prefer apples with frm, crisp texture, that are moderately juicy and that have a balance of sweet to acid taste. These sensory fruit quality attributes, together with a specifc aroma profle, present one of the main targets for consumer-focused plant breeding in apples.

Genetic mapping and association studies in apple and other fruit species have revealed that many of these sensory fruit quality attributes including juiciness, crispness, mealiness, skin color, russet frequency, titratable acidity and soluble solids content have a complex regulation, and are quantitatively determined by a broad range of genomic regions with signifcant environmental impact [60–64]. This complex regulation with involvement of many genes as well as signifcant impact from the environment makes it more diffcult to improve these fruit quality attributes through targeted gene editing, since (i) key regulatory genes have to be known and sequence-identifed, (ii) desired alleles have to be characterized (additivity) together as well as the most optimal combination of alleles at different loci (dominant and epistatic effects), and (iii) all desired alleles have to be genetically engineered within the same generation.

Despite this complex regulation of various aspects of sensorial fruit quality, some major causative genes have been found to regulate specifc aspects of fruit taste, texture or aroma and often the most desired allele, i.e. conferring an optimal level of a specifc fruit quality parameter, is already known (as well as the genetic source). However, for most of these genes, a specifc allelic DNA sequence variant conferring fne-tuned activity of the encoded protein, is desired, rather than a functional knock-out or null mutant, implying that desired traits can only be genetically introduced by advanced editing methods that either introduce nucleotide base pair switches (base editing) or enable allele replacement (HDR – homology directed recombination). These advanced gene editing techniques are only recently available for apple and other perennial fruit trees, and their use and applicability to modulate organoleptic fruit quality has, up till now, not yet been demonstrated. However, studies in other fruit species have shown that targeted editing of specifc genes can be used to improve the sensorial quality of fresh fruit. In the following sections, key genes that determine the expression of different fruit quality attributes are outlined together with their desired genetic confguration (allelic variants), with a predominant focus on genes that can be functionally depleted to obtain the desired phenotype.

Fruit taste refers to the basic sensory evaluation of the fruit commodity (sweet, sour, bitter, etc.) and for apple and other pome fruit species mainly depends on the content and composition of soluble sugars and organic acids [65]. Although the overall content of both these types of macromolecules is important, it is mainly the relative sugar/acidity ratio (TSS/acidity balance) of the fruit that determines the fruit's characteristic taste, implying that both aspects are linked and always need to be improved in parallel. The sweetness of the apple fruit is predominantly determined by sorbitol and the total content of soluble sugars (SSC), with no direct effect of single sugars (like sucrose, glucose, fructose, xylose), though with signifcant contribution from several volatile compounds, like esters and farnesene [66]. In line with the complex metabolic pathways of each of these sweetness-contributing chemicals, genetic assays have revealed that the sweetness of pome fruit is determined by multiple genetic loci, with each loci only having minor effects [56, 67– 70]. For the fraction of fructose and sucrose in the total sugar pool, however, a major locus was identifed on linkage group 1 that respectively explains 47% and 27% of the total variance [67]. This locus harbors the VIN1 vacuolar invertase (MDP0000149570), which enzymatically confers hydrolysis of sucrose into glucose and fructose to regulate the entry of sugars into different metabolic pathways [71]. Similarly, a pedigree-based QTL mapping approach in a "Honeycrisp"-derived germplasm identifed and validated three large effect QTLs, i.e. on LG1, 13 and 16, that were consistent across multiple years for the total SSC content in apple fruit [70]. However, the causative genes have not yet been identifed. Targeted gene expression studies throughout apple fruit development have shown that the accumulation of fructose in later stages coincides with an enhanced expression of the tonoplast monosaccharide transporters (TMTs) *MdTMT1* and *MdTMT2* which convert the excess amount of imported sugars into starch. At fnal fruit maturation, the accumulation of sucrose overlaps with an elevated expression and enhanced activity of the sucrose-phosphate synthases (SPS) *MdSPS5* and *MdSPS6*, which catalyze the transfer of a hexosyl group from UDP-glucose to D-fructose 6-phosphate to form UDP and D-sucrose-6-phosphate [72]. A combined QTL and transcriptomics study in Asian pear (*Pyrus pyrifolia*) identifed two sucrose transport genes (*PpSUT*, LOC103964096, and LOC103940043) that are negatively correlated with the sugar content in ripening fruit [73]. In addition, two sorbitol dehydrogenase genes (*PpSDH genes*, LOC103960512 and LOC103960513), were also found to be negatively coexpressed with total sugar content in the fruit, indicating that these act as antagonists of fruit sweetness [73]. Plants harbor different types of sugar transporters, and the SWEET-class of sugar transporters (i.e., Sugar Will Eventually Be Exported Transporters) have been found to play a major role in sugar accumulation in the fruit of apple as well as various other fruit tree crops. Marker-based association studies thereby revealed that in particular three SWEET genes, i.e. namely *MdSWEET2e, MdSWEET9b, MdSWEET15a*, are signifcantly associated with total sugar content in the fruit, with MdSWEET15a and MdSWEET9b accounting for a relatively large

portion of the phenotypic variation (and located on a region harboring a QTL for sugar content) [74, 75].

Fruit acidity is a major determinant of the overall apple favor and strongly infuences the perception of other favor traits such as sweetness and aroma. The pH of fresh apples generally ranges from 3.3 and 4.0, making them mildly acidic, and this is almost exclusively attributed to the accumulation of organic acids, with malic acid forming the major fraction (±90% of all organic acids) followed by quinic acid (±5%), citric acid (±1,5%) and small amounts of ascorbic, shikimic, maleic and tartaric acid [76, 77]. While in wild apple varieties fruit acidity is determined by the content of both malic and citric acid, in cultivated apple varieties citric acid is almost completely absent and fruit acidity is almost exclusively determined by malic acid [76, 78]. The concentration of malic acid can be measured sensorially through panel tasting, or more quantitatively by pH measurement, analytical methods (HPLC) or via titration of fruit juice, with the latter actually indicating the total content of organic acids as expressed in malic acid equivalents per fruit mass (TA: titratable acidity) [79]. Although acidity of freshly harvested apples is a complex trait, genetic studies have revealed that it is predominantly determined by two large effect QTLs together with various minor QTLs that mainly determine the variance in high acidity apple varieties (e.g. Ma4, Ma6, M2, and M3) [80–83]. More specifcally, apple fruit acidity is genetically determined by one QTL on LG16, referred to as the *Ma* locus [84], and one on LG8, called the *Ma3* locus [85], with both QTLs jointly explaining 66% ± 5% of the phenotypic variation through an additive allele dosage model with incomplete dominance [86–88]. For the Ma3 locus on LG8, up till now, no candidate genes have been identifed that control apple fruit acidity. In contrast, high resolution mapping and expression assays provided strong evidence that the aluminum-activated malate transporter gene (ALMT1 or Ma1) is the fruit acidity determining-gene in the Ma locus [89]. ALMT1 encodes a membrane-associated protein that is targeted to the tonoplast and actively transports malic acid molecules from the cytosol to the vacuole, which serves as major subcellular repository for organic acids, hence contributing to its cellular accumulation [90]. The low acidity *ma1* allele still localizes to the tonoplast but exhibits reduced malate transport functionality as compared to the pseudo-dominant high acidity *Ma1* allele due to a 84-AA truncation in the conserved C-terminal end domain [90]. Diversity studies in apple also showed that expression of Ma1 is signifcantly correlated with the fruit titratable acidity at harvest [89], making it a highly suitable candidate for direct modulation of fruit acidity through gene editing approaches. Recent studies provided more insights into the regulation of these tonoplast transporters in apple fruit and identifed several MYB transcription factors, including MdMYB1 and MdMYB73, as important regulators of vacuolar accumulation of malate. MdMYB1 promotes the expression of two genes encoding B subunits of vacuolar H+-ATPase (VHA), MdVHA-B1 and MdVHA-B2, and thereby transcriptionally activates its H+ pumping activity and enhances the transport of malate into the vacuoles [91]. Similarly, MdMYB73 transcriptionally activates MdALMT9, MdVHA-A and MdVHP1 (vacuolar pyrophosphatase 1) to enhance their activity, leading to increased concentrations of malate and vacuolar pH [92]. The activity of MdMYB73 towards its downstream targets was thereby found to be positively infuenced by the interaction with MdCIbHLH1. This transcriptional cascade (that promotes malate accumulation in the vacuole) is antagonized by the BTB-BACK-TAZ domain protein MdBT2 which targets MdCIbHLH1 and MdMYB73 for ubiquitination and subsequent degradation by the 26S proteasome pathway, hence negatively regulating accumulation of malate and vacuolar acidifcation [93]. Recently, another R2R3 − MYB transcription factor, namely *MdMYB44,* was found to control the fruit malate content and acidity in apple, though in a negative manner. MdMYB44 represses the promoter activity of the malate-associated genes *Ma1* (*Aluminum-Activated Malate Transporter 9*), *Ma10* (*P-type ATPase 10*), *MdVHA-A3* (*V-type ATPase A3*), and *MdVHA-D2* (*V-type ATPase D2*), and thus suppresses vacuolar import and accumulation of malate. Importantly, specifc SNPs in the promotor region of MdMYB44 thereby showed strong association with fruit malate content, i.e. either through their effect on basal activity or by altering affnity towards the basic-helix–loop–helix TF MdbHLH49 [94]. Parallel to MdMYB44, the protein phosphatase MdPP2CH also negatively regulates accumulation of malate in the fruit by post-transcriptionally suppressing the activity of the vacuolar H+-ATPases MdVHA-A3, MdVHA-B2 and MdVHA-D2 as well as the malate transporter MdALMTII through dephosphorylation [95]. As MdSAUR37 was thereby found to promote malate accumulation in the apple fruit by negatively regulating the MdPP2HC phosphatase activity, the MdSAUR37/MdPP2CH/MdALMTII chain was found to precisely determine apple fruit malate contents through hierarchical epistatic genetic effects [95]. Overall, multiple genetic factors that contribute to metabolism and vacuolar accumulation of organic acids, and particularly malic acid, in apple fruit have been retrieved with identifcation of both positive and negative regulators and associated characterization of allelic effects. Despite the absence of concrete examples, these genetic insights provide a strong basis for precisely modulating the titratable acidity as well as the overall organoleptic appreciation of the apple fruit through targeted gene editing approaches such as CRISPR and TALENs.

#### **3.2.2 Nutritional Quality and Food Functionality**

During the last decade there has been a paradigm shift regarding consumer acceptance towards fruits. Due to the general awareness of the impact of food consumption on personal health and overall increased welfare, consumers now also take into account the nutritional, functional, and physio-chemical factors of fruits, in particular for fresh produce. Also for pome fruit, such as apple and pear, there is an increasing preference for varieties that have high levels of health-promoting compounds, such as essential vitamins, minerals, dietary fbers, antioxidants and other key phytochemicals. The visual characteristics (e.g. skin characteristics), eating quality (e.g. texture and favour), and storability are among the main fruit quality traits being targeted in apple breeding programs, but the enhancement of phytochemicals is now gaining traction to select "bio-fortifed" apple cultivars. However, despite

their relevance for public health, almost no directed selection for specifc fruit biochemicals or overall augmented nutritional value has yet been performed. As a result, current apple varieties generally do not have high food functionality, and instead, for most nutritional parameters, have reduced contents or values as compared to their wild counterparts. For example, several studies reported that modern apple varieties have drastically reduced polyphenol content (particularly stilbenes, hydroxycinnamic acids, and dihydrochalcones) compared with the ancestral heritage, wild progenitors (*Malus sieversii*) and germline cultivars [96, 97]. Similar observations were made for organic acids, including malic and ascorbic acid, indicating signifcant counter-selection during domestication and breeding, most likely as indirect effect of selection against bitterness [96]. In contrast, some relevant favonoids (favonols and favan-3-ols) and triterpenoids (ursolic, oleanolic, and betulinic acids) did not show this selection-induced reduction in modern apple varieties [96]. Moreover, the few incentives that projected to increase the nutritional fruit quality in apple via conventional ways failed or only had limited success, mainly due to the complexity of the underlying biochemical pathways and adverse side effects on other agronomic or consumer-related attributes.

Now, gene editing approaches allow specifc enhancement of the health-related nutritional composition and food functionality of commercial apple cultivars, without affecting their typical favor and fruit quality attributes, and therefore form an easy and straightforward method to improve general public health. Germplasm characterization studies have revealed dramatic variation in the content of various nutritional compounds in apple, including polyphenols, vitamin C, etc., indicating that the biochemical composition of the fruit is predominantly genetically determined. However, genetic studies have shown that both the content and composition of these phytochemicals is generally under polygenetic control with multiple small effect genetic loci. This diluted genetic control largely impairs the genetic enhancement of these compounds through single gene editing approaches. Though, for some biochemicals, like polyphenols, the polygenetic control is mainly determined by a small number of genetic loci that have a small effect [98]. In these cases, knowledge of these genes (the relatively simple genetic architecture) may provide a basis to signifcantly optimize the content or composition of the specifc compound of interest through targeted gene editing approaches. For many fruit bio-chemicals, underlying genetic loci (QTLs) and associated candidate genes have been identifed in apple through genetic linkage mapping or association studies [63, 99, 100]. Using a combined genomics-metabolomics approach, both Khan et al. [100] and Bilbrey et al. [99] hereby detected a large number of metabolite quantitative trait loci (mQTL) spread along all chromosomes, with hot spots on the linkage groups 16 and 17 for apple phytochemicals. However, up till now, only a few regulatory genes have actually been identifed and were validated to play a functional role in the determination of the biochemical composition of apple fruit.

For polyphenols, candidate genes for the production of quercetin, epicatechin, catechin, chlorogenic acid, 4-*O*-caffeoylquinic acid and procyanidins B1, B2, and C1 have been retrieved via mapping [98], however, no further functional validation has yet been performed and actual genetic regulators have not yet been identifed. Specifcally for the dihydrochalcone phloridzin (phloretin 2′-O-glucoside), i.e. the most abundant phenolic compound in apple trees (Malus × domestica), regulatory enzymes have been identifed via genomics and *in vitro* studies with validation through transgenic approaches. In particular, six glucosyltransferases (UGTs) have been identifed which are able to selectively glucosylate phloretin, i.e. the direct precursor of phloridzin [101]. As a follow up, one recent study used genetic approaches, including RNAi and CRISPR, to analyze the function of one of these UDP-2′-O-glucosyltransferases, namely MdPGT1, in phenol metabolism, and thereby demonstrated that PGT1 stimulates the production of phloridzin in the leaves of apple with distinct morphological differences between knock-down and genome-edited mutant lines [102].

Anthocyanins form an important group of phenolic compounds, as they confer health-related benefts due to their antioxidant activity but also contribute to sensorial fruit acceptance due to their role as a pigment. Several molecular regulators of anthocyanin metabolism have been identifed, though most act in the biosynthesis and thus have a promotive effect on anthocyanin accumulation. For example, the anthocyanin biosynthesis pathway is transcriptionally regulated by the MYBbHLH-WD40 (MBW) complex, with allelic variants of the enclosed MYB10/ MdMYB1 TF [103] determining tissue-specifc expression and thus controlling apple fruit (peel and fesh) as well as foliage color. Overexpression of *MYB10* leads to a signifcant increase in foliar, fower and fruit anthocyanins, especially in the fruit peel, with no negative impact on sensorial quality and other consumer-related quality traits. However, due to their promotive effect, these regulatory genes do not form suitable candidates for CRISPR-based gene editing for enhancing anthocyanin contents. Besides these promotive proteins, two other MYBs have recently been identifed as transcriptional inhibitors of anthocyanin biosynthesis; namely MdMYB6 and MdMYB306. Xu et al. (2020) showed that MdMYB6 inhibits anthocyanin synthesis by directly inhibiting MdANS and MdGSTF12, i.e. two positive regulators of anthocyanin production, and by reducing contents of the precursors UDP-glucose and UDP-galactose by regulating the monosaccharide transporter MdTMT1 [104]. In addition, a second R2R3-MYB TF, namely MYB306-like, was found to act as an anthocyanin repressor gene. More specifcally, the MdMYB306 like protein activates the expression of an anthocyanin repressor gene, MdMYB17, and inhibits the expression of the anthocyanin structural gene MdDFR through direct promotor binding, and additionally interacts with MdbHLH33 and MdMYB17 to enhance its TF regulatory activities [105]. In line with this, transient silencing of MdMYB6, MdBY306-like and MdMYB17 leads to increased anthocyanin concentrations, indicating that these genes form interesting targets for CRISPR-based mutagenesis to obtain increased anthocyanin contents in apple fruit [105]. Besides these antagonistic regulators of anthocyanin identifed in apple, genetic studies in various other fruit crop systems, i.e. in particular tomato, have also identifed several other factors that operate in the favonoid metabolic pathways to suppress or reduce anthocyanin production. These include the *ATROVIOLACEA* (*ATV*) R3-MYB protein [106] and the nuclear protein DE-ETIOLATED1 (DET1) [107]. Genetic loss of function of these proteins is associated with a signifcant increase in anthocyanins in the mature fruit, making them excellent candidates for CRISPR based mutagenesis of their orthologous proteins in pome fruit for increasing anthocyanin contents in the peel and the pulp.

Another bio-chemical component with strong nutritional value as antioxidant is vitamin C or ascorbic acid (AsA). Besides its general role in oxidative stress mitigation, dietary L-AsA also has various other important health benefts. Increased intake of vitamin C has been associated with a decreased incidence of several important human diseases, such as cataract, cardiovascular diseases, and cancers. Vitamin C also promotes the uptake of iron and zinc, which is particularly relevant in meat-poor diets. Humans cannot synthesize Vitamin C due to the absence of the gene encoding L-guluronic acid-1,4-lactone oxidase, which catalyzes the last step in the AsA synthesis pathway, and therefore completely rely on dietary intake of AsA to meet their daily requirements [108]. Fruits are the main source of human AsA intake, though fruit AsA levels in commercial pome fruit cultivars are generally quite low as compared to other fruit species, such as lemon, orange and strawberry, with apple and pear only containing 0.05–1.0 and 5–10 mg AsA per 100 g fresh weight, respectively [109]. The ascorbic acid metabolic pathway has been extensively studied in plants, and has been found to be rather complex involving several parallel pathways that include multiple enzymatic steps [108], with total AsA accumulation being regulated by transcription factors, protein interactions, phytohormones, and environmental factors. Despite the fact that most of the regulators have already been identifed in model systems, their functional role and contribution in the AsA metabolism of pome fruit has not yet been resolved, although there are a few exceptions. For example paralogs of GDP-l-Galactose Phosphorylase (GGP) have been found to act as a major determinant of Vitamin C concentration in apple fruit, with specifc alleles leading to a signifcantly higher level of Vitamin C content in the pulp [110]. However, as these alleles do not confer a functional GGP knock-out, but instead promote AsA biosynthesis through formation of specifc protein variants, these GGP paralogs do not form suitable candidate genes to boost AsA levels in the pome fruit via CRISPR mutagenesis. Therefore, it is still unclear which genetic factors can be used for the engineering of increased fruit AsA levels in pome fruit through CRISPR editing.

# *3.3 Agronomic Traits*

#### **3.3.1 Disease and Pest Resistance**

Apple and pear orchards are routinely plagued by insect pests, including aphids, mites, and caterpillars. Also many bacterial and fungal diseases are prominent in pome fruits. By far the most destructive bacterial disease is fre blight (*Erwinia amylovora*). Major fungal diseases include apple scab (*Venturia inaequalis*), pear scab (*Venturia pirina* and *Venturia nashicola*) and powdery mildew (*Podosphaera*  *leucotricha*). Viral diseases can generally be suffciently controlled through the use of certifed virus-free plant material. Economic losses due to these and other pests and diseases include loss of produce, reduced fruit quality, loss of trees or even orchards, disruption of orchard production, management costs, and costs related to stringent quarantine and international trade regulations. In addition, preventive and curative treatments through chemical fungal sprays or antibiotic sprays raise important environmental, biosafety and health concerns [111–114]. Disease and pest resistance is therefore an important breeding objective in pome fruit species.

A frst approach to improve disease resistance is through the introgression of resistance genes into elite cultivars. There are two main types of disease resistance, namely quantitative and qualitative resistance. Quantitative resistance is conferred by many small-effect genes and results in partial resistance to multiple pathogen strains making it highly durable [111]. On the other hand, qualitative resistance is usually based on a gene-for-gene interaction between the pathogen avirulence (Avr) gene and the plant resistance (R) gene and often leads to a hypersensitive response against the pathogen. This form of single major gene disease resistance is easier to obtain for breeders, but can also be more easily broken by newly evolving virulent pathogen strains [111, 115]. It is therefore preferred to stack disease resistance genes or to combine qualitative and quantitative resistance [111, 114]. Resistance genes have been identifed for several important pear and apple diseases. For fre blight, the only functionally characterized resistance gene is FB\_MR5, however resistance conferred by this gene has regrettably already been overcome [116, 117]. Several other putative candidate resistance genes include FB\_Mfu10, NBS-LRR genes of ornamental cultivar Evereste, and several genes with disease-related domains in the FB\_Mar12 region [117–119]. For apple scab resistance, around 20 R genes are known, but not all confer equally durable resistance. Through worldwide monitoring of *Rvi* breakdown, the genes *Rvi5, Rvi11, Rvi12, Rvi14 and Rvi15* were identifed as rarely overcome, possibly because of an associated ftness cost to the pathogen [120]. In pear, identifed resistance genes for *Venturia nashicola* include *RVnk* [121], *Rvn2* [122] and *Rvn3* [123]. For *V. pirina*, *Rvp1* was identifed [124]. Powdery mildew is mainly studied in apple where several resistance genes are known: *Pl1, Pl2, Plw, Pld* [125]. However, current advanced breeding material generally lacks major resistance genes and therefore, wild *Malus* and *Pyrus* species remain the most important source of disease resistance alleles. This hampers application of conventional breeding because of problems with the long life cycle of pome fruit species and linkage drag [111], especially when pyramiding multiple resistance genes. This process can be sped up through transgenic approaches. There are several examples of genetic transformation to bring resistance genes into apple and pear [126–128]. However, this approach is more diffcult to apply using geneediting techniques which are more suited for knock-out of negative regulators of resistance.

Alternatively, disease resistance in plants may be achieved through the silencing or knock-out of susceptibility (S) genes. Several studies have successfully obtained resistance cultivars through genetic transformation with S genes as target. For example silencing of the *HIPM* gene (HrpN-interacting protein from *Malus*) through RNA interference resulted in increased fre blight resistance in apple cv. "Galaxy" [129]. Knockout of susceptibility genes is well suited for genome editing applications and has already been applied in apple cultivars "Gala" and "Golden Delicious" to signifcantly reduce bacterial fre blight symptoms through inactivation of *MdDIPM4* which interacts with pathogen effector protein DspA/E [37, 130]. Similarly, another study targeted *DIPM1, DIPM2* and *DIPM4* in "Golden Delicious" to increase resistance to fre blight [49]. Susceptibility genes don't always have to encode proteins that directly interact with plant effectors, but may also be more broadly involved in plant immunity. For example, targeted silencing of a frequently used susceptibility gene *MdMLO* increased powdery mildew resistance in "Gala" [131]. MLO is a transmembrane protein located in the plasma membrane and is presumed to be involved in plant defense and immunity responses [132].

Plant susceptibility genes may also be found for insect pests. For example, such genes may be involved in the induction of defense signaling pathways, food accessibility and food quality [133]. For example, in *A. thaliana*, the transcription factor *WRKY22* is involved in pathogen-triggered immunity and knock-out mutants were more diffcult to colonize by aphid populations [134]. However, knowledge of S-genes against insect pests is limited and to our knowledge there have been no applications in pome fruit species to date.

Finally, another alternative approach is to target pathogen or microbial genomes using CRISPR/Cas9 technologies as compared to focusing solely on the host. For example, the gene drive system, which was shown to be successful in mosquito [135] may be adapted and applied in sexually inheriting plant pathogens [136]. Original gene drive systems are based on homing endonuclease genes (HEGs) which encode proteins that cleave a recognition site of around 20–30 nucleotides on the genome. The HEG itself is inserted in the middle of its own recognition site thereby protecting itself from further cleavage. When the HEG comes in contact with the target site on the wild-type homologous chromosome it will induce a DNA DSB. The DSB is repaired using the homologous chromosome as a template and the HEG allele is copied into the broken chromosome. As a result, these HEGs can spread rapidly through populations [137]. The gene drive system can be made more accurate and fexible using CRISPR-based adaptations [138] and there are many agricultural applications, including the sensitization of a population to pesticides (local sensitizing gene drive), manipulating plant pathogen-vector relationships, and knock-out of virulence genes [139]. The latter has already been shown successful under *in vitro* conditions for the wheat pathogen *Fusarium graminearum* [140]. Similarly, the CRISPR/Cas9 gene drive approach may also be applied to enhance performance of biocontrol agents or benefcial organisms which can heighten plant immunity [136]. However, this system is subject to major concerns regarding biosafety and bioethics and should not be applied without extensive risk assessment [141].

#### **3.3.2 Abiotic Stress Tolerance**

As sessile organisms, plants continuously endure a whole range of environmental stressors including drought, waterlogging, extreme temperatures, soil salinity, wind, and hail, which can severely affect growth and production. Improved abiotic stress tolerance allows fruit trees to be grown in suboptimal environments which is especially relevant in light of climate change and the associated occurrence of more extreme weather events worldwide. Important breeding objectives include tolerance to drought, cold, and soil salinity.

Drought tolerance in apple and pear trees is often tackled through root stock breeding in an attempt to achieve enhanced uptake of water through a betterdeveloped root system. A recent study describes the knock-out of GRETCHEN HAGEN3.6 (*MdGH3.6*), an indole-3-acetic acid conjugating enzyme, in apple rootstocks through RNAi to increase tolerance to prolonged drought periods without impacting fruit quality. Knocking out this gene increased IAA content, adventitious root number and root length [142, 143]. Other potential drought tolerance related targets for CRISPR genome editing that were successful in other species include genes involved in stomatal density (VvEPFL9-1) [144], stomatal response (AtOST2) [145, 146], genes involved in ABA signaling (AtAREB1, SlMAPK3, OsSAPK2), and leaf rolling (OsSRL1 and OsSRL2) [145].

Although adult pome fruit trees are generally quite cold-hardy, severe production losses can still occur due to frost damage to fower buds, fowers, young shoots and fruits. Cold hardiness can differ between cultivars [147], but little is known about the genetic and biochemical basis. Several genes for general cold-hardiness have been identifed in apple. Putative positive regulators of cold hardiness in apple include *MdHYL1, Mdm-miR172* [148], and *CBF* genes (C-repeat binding factors) like *MdCBF1-5* [149]. Knock-out of negative regulators, such as *Mdm-miR156*, can cause increased cold-hardiness, which is more interesting for genome editing application [148]. Many of the genes and pathways involved in cold-tolerance are also involved in other (a)biotic stress responses. For example, the previously mentioned *CBF* genes are considered hub genes that are involved in drought, salinity and cold responses [149, 150]. Targeting these genes may result in simultaneously increased tolerance to other abiotic stress factors, however caution must be taken to avoid undesired side effects.

Heat stress in pome fruits can lead to sunburn of leaves and fruits, scorching of leaves and in extreme cases early fruit drop and even leaf drop [151, 152]. Damage can be especially severe in combination with drought. In high temperature conditions, damage may occur in plant cells due to protein misfolding and denaturation, damage to membranes and accumulation of ROS species [153]. Gene targets to enhance heat tolerance in pome fruits are likely to be found in plant antioxidant defense systems or osmotic adjustment pathways [154]. Heat stressed plants will also generally synthesize a variety of heat-shock proteins (HSP). Such HSPs are also found in apple and have been identifed as important regulators of temperature stress responses. Members of the *HSP20* gene family appear to be especially important and are highly upregulated in response to heat stress [151]. Another heatstress related gene is *MdPRP6* which was shown to enhance heat stress tolerance when overexpressed in transgenic tobacco plants [153]. Possible genome editing targets are negative regulators of these heat shock related genes.

Finally, soil salinity is detrimental to plants due to osmotic stress and salt toxicity [155]. It mainly occurs due to irresponsible irrigation and fertilizer application. The plant may try to overcome salt stress by inducing osmoregulating and antioxidant systems [156]. In apple, *MdINT1* has been shown to confer salinity tolerance by regulating antioxidant systems, and homeostasis of ions and osmosis [156]. Several other genes involved in response to salt stress have been identifed in other plants, but relatively little knowledge is available in apple and pear. For example, knockout of *AITR* (ABA induced transcription repressors) gene family members in *A. thaliana* increased drought and salinity resistance [157]. Also in *A. thaliana*, a nucleoporin gene *NUP85* seems to modulate the response to salt stress [158]. Improved salt tolerance was also achieved in rice by CRISPR/Cas9-targeted mutagenesis of the *OsRR22* gene involved in cytokinin transduction and metabolism [159], and in tomato through down regulation of Auxin Response factor 4 (ARF4) [160]. Homologous gene targets may be explored in pome fruit species.

#### **3.3.3 Tree Architecture**

Tree architecture is infuenced by four main factors: primary growth, branching patterns, fowering location and meristem and shoot mortality [161]. In commercial pome fruit production, tree architecture can infuence important factors such as fruit quality, yield and orchard management requirements including planting density, pesticide application effciency, harvesting effciency, and requirements for thinning, pruning, branch-bending and tying [161]. The ideal tree form would allow high density plantings and maximum automatization [162]. The main breeding goals regarding tree architecture in pear and apple are dwarfng (mainly achieved through dwarfng root stocks) and optimal branch orientation [163]. Causative genes have been identifed for the dwarfng trait, branch orientation and the columnar growth habit in apple [162].

Dwarfng root stocks limit tree size, enable high density plantings, increase fower density and allow more effcient mechanization [164]. Mainly genes associated with growth-related plant hormones are linked to the dwarfsm trait and may be identifed as putative targets of gene editing. For example, in peach, a nonsense mutation in the GA receptor *GID1c* was found to result in dwarfsm [165]. In apple, overexpression of the transcription factors *WRKY9* and *NAC1*, which are negatively involved in the brassinosteroid biosynthesis pathway, was shown to result in dwarfing [162, 164, 166].

Several gene families are associated with branch orientation, namely *WEEP*, and IGT family genes *TAC1* and *LAZY1* [167–169]. Tiller Angle Control1 (TAC1) promotes lateral shoots to grow outward and reduced or eliminated expression of this gene causes more upright growth habits [162]. In contrast, LAZY1 promotes upward shoot orientation. Plants with reduced or no *LAZY1* expression show wide shoot angles [170, 171]. A weeping growth habit was found in birch and plum with a defect or silenced *LAZY1* gene [162, 172]. In theory, a desired branching phenotype may be obtained in trees by balancing the effects of targeted editing of one or several of these genes.

One of the most ideal branching habits in apple is the columnar type. These trees grow upwards as a column with very short branches and an increased number of spurs [162]. They are ideally suited for high density plantings and automatization, although they are generally very susceptible to biennial bearing and produce lower quality fruits [173–175]. Columnar growth habits have been achieved in apple through mutation of the *MdCoL* gene, which encodes a putative 10G-FEII oxygenase [161, 176], and overexpression of *LEAFY* [177].

# *3.4 Pollination and Fertilization*

#### **3.4.1 Self-Incompatibility**

Generally, fruit set in pome fruit species is dependent on cross-pollination between two cross-compatible cultivars due to the S-RNase dependent gametophytic selfincompatibility system (GSI) which prevents self-fertilization [178–180]. This system is genetically controlled by the S-locus which carries a pistil-expressed *S-RNase* gene and multiple pollen-expressed *SFBBs* (S-locus F-box brothers) [178, 181– 183]. According to the currently accepted non-self-recognition mechanism, SFBB proteins expressed by the pollen haplotype recognize non-self *S*-RNases in the style, but not self-S-RNases, and mediate their degradation through the ubiquitin-26S proteasome [184, 185]. Problems with this innate self-incompatibility arise when cross-pollination is hampered. To ensure suffcient cross-pollination and thus economically viable yields, several conditions must apply including (1) adequate presence of a diverse pollinating insect population, (2) presence of well-distributed trees of a cross-compatible pollen donor cultivar with overlapping fowering period in the orchard, and (3) optimal weather conditions during fowering. However, these conditions cannot always be met and even then, fruit set after self-pollination is expected to be more stable compared to cross-pollination [186]. Therefore, self-compatibility (SC) has become an important breeding objective [5].

Spontaneous self-compatible (SC) mutant apple and pear varieties are rare and hardly ever suitable for commercial fruit production. One exception is "Osanijisseiki" which is a natural SC mutant of the *Pyrus pyrifolia* cultivar "Nijisseiki". "Osanijisseiki" was released as cultivar in 1979 and has been used as a parent to breed new SC cultivars using conventional breeding strategies [5]. Spontaneous SC mutants in pome fruit species are either the result of pistil-function breakdown, essentially meaning knock-out of the *S-RNase* gene [181, 187, 188] or alternatively of competitive interaction due to polyploidy or segmental duplications in the S-locus [189, 190]. The latter approach is less suited as genome-editing application,

however pistil-function breakdown of self-incompatibility has already been successfully applied in apple to create a transgenic SC Elstar mutant using a co-suppression approach resulting in *S-RNase* gene silencing [186]. Knock-out of the *S-RNase* gene could be performed relatively easily using genome-editing approaches to introduce the self-fertility trait into established commercial cultivars.

#### **3.4.2 Parthenocarpy**

Parthenocarpy refers to natural or artifcially induced fruit development without fertilization of the ovule [191]. In commercial pome fruit production, this is a very interesting trait because it can alleviate problems with pollination, selfincompatibility, biennial bearing and spring frost. In addition, parthenocarpic fruits are seedless and often have more edible pulp and less core which is often preferred by consumers and can be an advantage for industrial food processing applications [192, 193]. Parthenocarpy is mainly genetically determined and can vary between species and cultivars. In pear, natural parthenocarpy is more common in *Pyrus communis* cultivars compared to the Asian pear species, such as *Pyrus pyrifolia* [194]. *Pyrus communis* parthenocarpic cultivars include "Conference" [195] and "Bartlett" [196]. In apple, some parthenocarpic fruit can develop on "Cox's Orange Pippin", "Wellington Bloomless" and "Spencer Seedless" [197].

Natural parthenocarpic fruit set in pome fruit species is usually relatively low, but can be stimulated using plant growth regulators (PGR) during early fruit development to obtain economically viable yields. These PGRs generally impact the auxin, cytokinin or gibberellin pathways that are involved in fruit set and development [193, 198, 199]. Correspondingly, parthenocarpic mutations in a variety of species are generally found in the synthesis and metabolism pathways of these hormones [193]. Overexpression of auxin biosynthesis or receptor genes and silencing of auxin signal repressor genes or negative regulators of auxin signaling are associated with parthenocarpy in tomato (*Solanum lycopersicum*) and eggplant (*Solanum melongena*) [193, 200–203]. In the gibberellin pathway, overexpression of GA biosynthesis genes, such as gibberellin 20-oxidase genes (*GA20ox*), and suppression of GA respressor genes like *DELLA* and *GA2ox* genes have been shown to lead to parthenocarpy in *A. thaliana* and tomato [204–208].

Alternatively, foral homeotic genes are also often associated with parthenocarpy. For example, the silencing of genes responsible for stamen identity has been associated with parthenocarpy in tomato, possibly because stamens act as negative regulators to restrict ovary development before pollination and fertilization have occurred [209, 210]. These genes include, amongst others, several class B MADSbox genes such as TOMATO APETALA3 (*TAP3*), DEFICIENS (*sl*DEF), TOMATO MADS BOX GENE6 (*TM6*), and TOMATO PISTILLATA (*TPI*) [209, 211–213]. In apple, two parthenocarpic cultivars showed similar splicing variants of the *AtPI* homolog which were causally related to their parthenocarpic trait [214, 215]. In pear, several transcriptomic studies on induced parthenocarpic fruit development in

pear showed possible involvement of homologs of many of the above listed tomato genes. For example, key GA-associated genes related to parthenocarpy in pear include *GA20ox, GA3ox, GA2ox*, GA receptor *GID1*, and *DELLA* [216, 217], as well as MADS-box class B gene *DEF*. However, the genetic basis for parthenocarpic fruit set is still largely unclear in woody fruit tree species and mutations in many of the mentioned genes cause undesired pleiotropic effects. In addition, pathways leading to parthenocarpy in pome fruits may be different compared to *A. thaliana* which produces siliques or tomato which produces botanical fruits compared to accessory fruits. Therefore necessary caution must be taken when choosing possible gene targets for parthenocarpy in pome fruit trees based on studies in these model species.

# *3.5 Tree Phenology*

As perennial species, pome fruit trees require immaculate regulation of their phenology to survive and reproduce in temperate climates with seasonally changing climatic conditions. In order to anticipate these seasonal changes, temperate tree species take climatic cues to regulate important transitions in their life-cycle, including dormancy, bud burst, fowering, fruit development, and leaf-drop. Phenology characteristics greatly determine the success of a cultivar in a given location due to their impact on the tree life-cycle, including many reproductive traits that are essential for fruit production. As a result, considerable effort has been devoted to elucidate the molecular mechanisms that underly important phenological transitions. In the following paragraphs three aspects of tree phenology are briefy discussed which have major relevance to pome fruit production: juvenility, dormancy and biennial bearing. We also discuss potential applications of gene editing techniques based on current knowledge.

#### **3.5.1 Juvenility**

Juvenility in most pome fruit species can take 5–6 years which signifcantly extends the breeding cycle and delays research on reproductive biology [218]. Therefore, a shortened juvenile period is of great interest to breeders and researchers, and several attempts have been made to obtain early fowering apple and pear mutants with the goal of accelerating conventional pome fruit breeding and research. For example, apple and European pear *TFL1-1* mutants showing an early fowering phenotype were successfully obtained using CRISPR-Cas9 technology [38]. These mutants fower continuously *in vitro*, do not require cold accumulation to induce fowering and completely by-pass the juvenile period. However, the benefts of cultivars with a short juvenile period are limited in the context of fruit production, since mature tissue can be clonally propagated onto rootstocks once juvenility is broken.

#### **3.5.2 Dormancy**

To survive the harsh winter conditions, temperate fruit trees go through both endoand ecodormancy. Dormancy can be defned as the "temporary suspension of visible growth of any plant structure containing a meristem" [219]. In pome fruits, both mixed and vegetative buds are formed during the fowering period [220]. In autumn, low temperatures and short day conditions induce a state of endodormancy in these buds which can only be broken by the fulfllment of the cultivar-dependent chilling requirement [221]. Once this chilling requirement is reached, buds transition into a state of ecodormancy. Ecodormancy is broken by higher temperatures, initiating bud break. This two-factor regulatory system prevents early bud break on a warm day in late autumn or during cold days in early spring [222]. Ideally, this break of two dormancy states initiates uniform fowering, which is advantageous to both the plant and the grower. Dormancy cycles can be disturbed when pome fruit trees cannot reach the required amount of chilling after a mild winter. This can lead to abnormal bud break, resulting in extended fowering periods, delayed leaf formation and asynchronous fruit development [223]. Currently, problems associated with abnormal bud break are mainly observed in orchards grown in subtropical climates, but may also start to occur in more temperate regions as a consequence of climate change. Several chemical treatments can be used to break dormancy in pome fruits including Dormex, potassium nitrate, and mineral oil [224]. However, these products are associated with environmental and health concerns and pose signifcant costs to growers. Therefore, the development of commercial apple and pear cultivars with low chilling requirements would be benefcial.

Several key hormones and some regulatory genes involved in the maintenance and release of endodormancy have been identifed [222]. In pear, abscisic acid (ABA) levels increase during endodormancy induction and remain high during endodormancy. During cold accumulation, transcription of *PpCYP707A-3*, which encodes an ABA 8-hydrolase enzyme, sharply increases. Simultaneously, ABA levels in the buds decrease initiating transition from endodormancy to ecodormancy in pear [225]. The onset of this transition is believed to result from the release of inhibition on GA biosynthesis, regulated by *PpGAST1*. Transcription of *PpGAST1* is inhibited by ABA and decreasing ABA levels during dormancy transition allow *PpGAST1* levels to rise. Increased *PpGAST1* transcription levels were accompanied by increased transcription of *PpGA20OX2*, a GA biosynthesis gene. Additionally, high ABA levels also indirectly induce the GA catabolism gene *PpGA2OX1*, resulting in decreasing GA levels [226]. Another important gene group associated with dormancy regulation is the *dormancy-associated MADS-box* (*DAM*) gene family. Their expression is tightly regulated by ABA through several transcription factors including ABA response element (ABRE)-binding transcription factor 1 (AREB1), which represses *PpDAM1* transcription in pear. *PpDAM* genes promote ABA biosynthesis by upregulating the expression of *PpNCED3*, an ABA biosynthesis gene [227]. In transgenic pear calli, *DAM3* was found to also inhibit cell division and cell growth, supporting their role in pear bud dormancy. Interestingly, two Asian pear cultivars (*P. pyrifolia*) "Suli" and "Cuiguan" with respectively a high and a low chilling requirement, showed different expression patterns of *PpDAM3* during endodormancy [228]. Additionally, epigenetic regulation is also found to be involved in bud dormancy. In peach (*Prunus persica*) and sweet cherry (*Prunus avium*), *DAM* genes were found to be under epigenetic regulation by histone modifcations and/or DNA methylations during the dormancy process [222]. In apple and pear, bud break was associated with a decrease in DNA methylation under ideal high chill conditions [229, 230]. However, the precise mechanisms underlying epigenetic regulation of dormancy in pome fruits remain unknown.

As negative regulators of dormancy release, *DAM* genes appear to be good candidates for targeted gene editing. *DAM* gene expression may be altered through CRISPR transcriptional repression to adapt the cold requirement of various pome fruit cultivars. Alternatively, CRISPR-Cas9 may be used for targeted mutagenesis of the *DAM* genes. To our knowledge, no previous work has targeted *DAM* genes in perennial fruit trees to alter dormancy. However, there is a known, natural *DAM evergrowing* (*evg*) peach mutant which is the result of a genomic deletion of four *DAM* genes and which does not enter dormancy when exposed to low temperatures or shortening days [231]. More research is needed to determine the effects of targeted *DAM* mutagenesis on dormancy in pome fruits and its applicability in commercial fruit production. Also, alternative targets need to be identifed to more precisely fne tune chilling requirements.

It is predicted that winter warming and resulting disturbed dormancy will be an issue for temperate fruit production in increasingly larger areas of the world [232]. For example, due to rising winter temperatures in Japan, fowering disorders occur more frequently in *P. pyrifolia* "Hosui" trees which as a result show erratic fowering, asynchronous bud-break and bud loss due to inadequate chilling during the dormancy phase [223]. However, increased temperatures are not always negative for pome fruit production. For example, European pear (*P. communis*) production of the cultivar "Conference" in Belgium is predicted to be at a lower risk of frost damage-related production losses because of the decreased occurrence of frost days during the fowering period [233]. These two examples show that the consequences of climate change on pome fruit phenology trees are complex and region- and cultivar-specifc. In given examples, a decreased chilling requirement may improve uniform bud break in "Hosui" pears grown in Japan, but could expose "Conference" pears grown in Belgium to frost damage due to early fowering. So, when adapting pome fruit tree phenology by breeding or gene editing, it is recommended to obtain an accurate view of the specifc challenges present in the crop and region of interest.

#### **3.5.3 Biennial Bearing**

Biennial bearing (BB) occurs when a fruit tree has an alternating pattern of low and high fruit production over consecutive years. This is caused by the inhibition of fower induction (FI) in the meristems by growing fruits, since FI occurs simultaneously with fruit development during 4–8 weeks after full bloom [234]. In pome fruit trees, each individual spur is biannual and can only fruit every other year, commonly named 'ON' and 'OFF' years. During 'ON' years there is abundant fowering and potentially a heavy fruit set, followed by an 'OFF'-year with mostly vegetative growth and thus less potential for fruit set. Ideally, a fruit tree will have a balanced proportion of 'OFF' spurs and 'ON' spurs each year, resulting in a predictable and constant fruit production. If a certain event triggers low fruit set or causes early loss of fruitlets, all spurs on a tree will be synchronized to an 'OFF' status and, consequently, an 'ON' status next year. This starts a cycle of BB which causes variability in yield and fruit size over the years and is therefore undesired by the grower. A frequently applied measure to avoid the continuation of the BB cycle is early crop thinning in 'ON' years by removing fowers or young fruitlets. This can be done chemically by using compounds that damage fower organs and so inhibit fruit set or by mechanically removing the young fruitlets [235, 236]. During 'OFF' years gibberellic acids such as GA7 can be applied to the trees after bloom to repress excess FI [237]. In addition to crop thinning, optimal pruning of the fruit trees will maintain young spurs which are less susceptible to BB than older spurs [235]. But despite these precautions and good management, there are still differences in susceptibility of different pome fruit cultivars to BB, especially in apple. This implies that there is a genetic basis determining susceptibility or resistance to BB in certain cultivars [238].

BB was long thought to be the result of hormonal signaling from the developing fruits to nearby developing buds. In 1998, diffusible auxins were shown to be present in the seeds of developing fruits during the period of FI, and the levels of these auxins in fruits increased with an increased number of seeds [239]. A recent study compared the apple cultivars "Gala" (a regular bearer) and "Fuji" (a biennial bearer) and found that "Fuji" had a higher average seed number per fruit compared to "Gala" [240]. The "Fuji" fruits did not only have a higher number of seeds per fruit, each seed also had higher levels of cytokinins and auxins than seeds from "Gala" fruits. On the contrary, "Gala" seeds had increased levels of GA3 and GA19 compared to "Fuji". These plant hormones were also found to be exported by diffusion through the stem of the apple fruit in both cultivars. These differences in hormone production in seeds could explain the susceptibility of "Fuji" to BB, since some GAs repress FI in apple trees trough currently unknown mechanisms [241]. Several QTL's were identifed for biennial bearing in apple, some included fowering genes like *BFTa*, *SOC1-like* and *COL1*, others included hormonal factors such as genes involved in GA biosynthesis *GIBBERELLIN 2-OXIDASE* (*GA2ox*), *GIBBERELLIN 20-OXIDASE* (*GA20ox*) and *GA3ox-like-b* and auxin related genes such as *AFB6* [242].

A study in "Gala" apple trees identifed many differentially expressed genes (DEGs) between 'ON' and 'OFF' buds in trees by artifcially inducing biennial bearing through manual fower removal during bloom. The authors found many DEGs among the fowering genes, including fowering repressor genes such as *TEMPRANILLO1* (*TEM1*) and *MAF2* (*MADS AFFECTING FLOWERING 2*). Also fowering promoting transcription factors, *SQUAMOSA PROMOTER BINDING-LIKE 5* (*SPL5*) and *SPL9* and *FLORAL TRANSITION AT MERISTEM* (*FTM1*) were upregulated in 'OFF' trees, simultaneously with *APELLATA1* (*AP1*) genes. Additionally, secondary metabolism genes were downregulated in the shoot apical meristem (SAM) of 'ON' trees, possibly due to the developing fruits causing carbohydrate depletion in the SAM. This downregulation of secondary metabolism genes is an indication of reduced cell division in the apical tissues and is supported by the observed up-regulation of *KNOTTED-LIKE FROM ARABIDOPSIS THALIANA* (*KNAT1*) and *NO APICAL MERISTEM* (*NAM*) genes during 'ON' years. *KNAT1* and *NAM* are both genes which prevent meristematic tissue differentiation and thus maintain the SAM, preventing FI and differentiation of foral tissues under low-carbon conditions. Also axillary meristem (AM) regulating genes were upregulated in 'ON' buds, such as *MORE AXILLARY BRANCHES 1* (*MAX1*) and *BRANCHED1* (*BRC1*), inhibiting AM formation and axillary bud outgrowth, respectively [243, 244]. This reduction in AM formation and axillary bud outgrowth results in reduced vegetative growth during 'ON' years with heavy fruit bearing. The study also found DEGs related to auxin, abscisic acid, brassinosteroid and ethylene. The Gibberellic Acid (GA) biosynthesis genes *GA2ox* and *GA20ox* were shown to be upregulated in 'ON' years, confrming the previously mentioned studies that showed these genes as QTLs for BB [242, 244]. This is not surprising, since GA treatments during 'OFF' years are used in horticulture to inhibit FI [245].

Also epigenetic modifcations are involved in regulation of BB, as it was shown that differentially methylated regions (DMRs) are present between 'ON' and 'OFF' trees in buds of the apple cultivar "Fuji" [246]. Many fowering genes showed to be differentially methylated such as *MADS-box*, *COL*, *B-box*, *NFY* and *SPL*. Also genes involved in hormonal signaling such as gibberellin, auxin and jasmonic acid showed to be DMRs, again highlighting the importance of hormonal regulation of FI in BB in apple.

Differences in gene expression in developing buds during 'ON' and 'OFF' years are very valuable in understanding FI and the genes involved, but it does not provide evidence to identify the genetic factors responsible for susceptibility of a cultivar to BB. Since the exact molecular mechanisms of BB are not yet well characterized, it is diffcult to put forward a good candidate gene for CRISPR gene editing. Since all genes that were identifed so far in BB are involved in crucial plant functions as fowering and hormonal signaling, the generation of knock-out (KO) mutants may not be a suitable approach due to the possible occurrence of undesirable side effects in tree phenology and/or fower morphology [247]. A better understanding of the genetic control of BB is needed to apply innovative gene editing techniques such as CRISPR base editing. Since there are observations that GA and auxin production in the seeds play a role in BB, CRISPR transcriptional activation could be used to mimic expression patterns of hormone biosynthesis genes of resistant cultivars in susceptible cultivars.

# **4 Concluding Remarks**

Apple and pear are among the most cultivated temperate fruit crops, but the threats to their production remain signifcant. Many of the currently grown cultivars still suffer from high susceptibility to pests, diseases and abiotic stress, low fruit quality, biennial bearing, suboptimal yields and other problems. Effective breeding strategies are necessary to tackle these problems. However, current breeding approaches are too time-consuming and expensive to proftably develop improved cultivars and to meet the rapidly evolving breeding requirements, especially in light of impending climate change. In addition, apple and pear production are traditionally dominated by just a handful of cultivars with a high market recognition, such as "Gala", "Golden Delicious" and "Fuji" for apple, and "Conference", "Bartlett" and "Doyenné du Comice" for pear. It has proven diffcult to introduce new cultivars in the market, therefore, enhancing important traits in established cultivars is a very effective strategy, albeit extremely diffcult using conventional breeding techniques. There is therefore a great need for faster and more accurate breeding approaches.

The recent advancement of gene-editing techniques such as CRISPR/Cas related technologies can help address these issues. Although successful gene-editing applications in pome fruit are still limited compared to many model crops, several proofof-concept studies in apple, pear and some other perennial tree species have shown that gene-editing is feasible in pome fruits and that there are numerous potential breeding applications. This chapter discussed the current state-of-the-art, challenges and opportunities regarding gene-editing in pome fruit, as well as potential genetargets for new applications in light of important breeding objectives. Several technical challenges remain to be solved in order to successfully apply gene-editing in pome fruit breeding including the high recalcitrant nature of pome fruit plantlets to *in vitro* propagation and regeneration, the low transformation effciencies, and the complex removal of transgenes by crossing due to the long life cycle and high heterozygosity of pome fruit tree species. Improvements to the original gene-editing approaches that are tailored to the specifc problems in pome fruit and other perennial tree species will help improve the application potential in pome fruit breeding.

# **References**


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# **Chapter 20 Genome Editing in Forest Trees**

### **Tobias Bruegmann, Alexander Fendel, Virginia Zahn, and Matthias Fladung**

**Abstract** Since the rst CRISPR/Cas-mediated genome editing of poplar in 2015, an increasing number of tree species are being genome-edited. Although the availability of genome sequences, tissue culture and transformation systems are limiting factors, research is ongoing on advanced methods such as DNA-free genome editing and gene targeting approaches in addition to the optimisation of single gene knockouts. These can be used to address ambitious issues and perform genome editing more accurately, which has implications for the legal assessment of edited trees. Once technically established, CRISPR/Cas can be used to circumvent specic challenges related to forest tree species, e.g., longevity and extended vegetative phases, and to modify traits relevant for breeding, whether for direct application or to elucidate the genetic basis of individual traits. Not least due to climate change, adaptation to abiotic stress such as drought stress as well as biotic stresses caused by pathogens are strongly in focus. For the use as a renewable resource and as a carbon sink, wood productivity in forest trees as well as wood properties are of interest. In biosafety assessments, tree-specic aspects have to be considered, which result, among other aspects, from the long lifespan.

# **1 Prerequisites to Use Genome Editing in Trees**

After CRISPR/Cas was rst used for genome editing in plants as published in 2013 [1, 2], the rst genome editing of a tree species was published only a short time later. Fan et al. [3] described the knockout of the visual marker gene *PDS* (encoding for phytoene desaturase) in *Populus* × *tomentosa*, resulting in albino phenotypes. For this, a Cas9 expression vector including matching guide-RNAs (gRNAs) was used

T. Bruegmann (\*) · A. Fendel · V. Zahn · M. Fladung

Thünen Institute of Forest Genetics, Grosshansdorf, Germany e-mail: tobias.bruegmann@thuenen.de

Alexander Fendel and Virginia Zahn contributed equally to this publication.

for stable *Agrobacterium*-mediated transformation of *in vitro* poplars. Even though innovative methods are being developed, in practice mostly functioning tissue culture systems remain an essential basis for successful editing in trees. This involves three steps with respective hurdles: (1) establishment of an *in vitro* culture system that is able to regenerate plant shoots, (2) establishment of a protocol for the transfer of the Cas/gRNA into living cells, i.e., by classical genetic transformation, and (3) establishment of a protocol for genome editing.

Many tree species are considered *in vitro*-recalcitrant. It is diffcult to transfer them to the *in vitro* culture and, once this initial step is accomplished, to regenerate them in large quantities and within manageable time frames for biotechnological purposes. The term recalcitrance summarizes many problems, some of them are still unidentifed because the physiological basis of recalcitrance is not fully understood to date [4]. A fundamental diffculty is the need for clean (often generalised as "sterile") cultivars, i.e., without overwhelming bacterial or fungal contamination. In a few tree species, the transfer from sterilised vegetative organs such as leaves into *in vitro* culture has worked, e.g., for poplars. Sterilised embryos or somatic meristems from shoots are more suitable for many tree species [4]. For this purpose, younger starting material seems to be more suitable than older trees if plant material is to be used for organogenesis by the cultivation of meristems (unpublished data, [5]). If organogenesis cannot be induced directly, somatic embryogenesis is often used for plant regeneration. Here, the development of complete embryos with radicle, shoot, and cotyledons is induced from somatic cells, e.g., callus. These somatic embryos are similar to zygotic embryos. They can be easily separated from the mother tissue, have all the necessary structures for regeneration into a whole plant [6].

The ability to regenerate single cells into complete plants is necessary for genetic transformation and the generation of genetically uniform regenerates. For many tree species, regeneration media with tuned hormone contents can be used to induce the totipotency of somatic cells and stimulate regenerating callus for organogenesis. Cytokinins, such as 6-Benzylaminopurine (BAP), and/or auxins, such as Indole-3 butyric acid (IBA), Indole-3-acetic acid (IAA) and 2,4-Dichlorophenoxyacetic acid (2,4-D), are usually used for this purpose. Their concentrations should be optimised for each species [7]. The use of protoplasts with appropriate regeneration medium is also suitable. At this point, brief attention should be drawn to somaclonal variation that can be induced by *in vitro* techniques [8]. Even though these mutations usually have no effect on the transformability or editability of the plants, it should be considered that, in practice, some genetic variability is unavoidable.

If an *in vitro* culture system is achievable, the basis for genetic transformation is given. However, establishing the transformation method is far from easy, as existing transformation protocols can only be utilised in tree species to a limited extent. If modifcations are needed, an establishment process follows which, if at all, can lead to success over a certain amount of time. Three conventional methods of genetic transformation are commonly used, with ascending relevance for tree species: ballistic transformation by particle gun, polyethylene glycol (PEG)-mediated transformation of protoplasts, and *Agrobacterium*-mediated transformation. The use of a particle gun requires good mechanical tuning to determine the bombardment

parameters that produce many transformations and little damaged plant material [9]. In addition, special equipment is required, especially the particle gun itself. Provided protoplasts can be generated, PEG-mediated uptake of plasmid DNA works reliably. To give some examples, PEG-mediated transformation of protoplasts, partly combined with electroporation, works in *Eucalyptus* species [10, 11], a poplar hybrid (*Populus tremula* × *Populus alba* [12]) and rubber tree (*Hevea brasiliensis* [13]). The challenge of this method is the isolation of vital protoplasts and their regeneration via callus stages into whole plants. For *Agrobacterium*mediated transformation, bacteria with a natural competence to transfer genetic material are used. Although the term *Agrobacterium*-mediated transformation continues to endure in the research community, the associated bacterial species have been renamed *Rhizobium radiobacter* (formerly known as *Agrobacterium tumefaciens*) and *Rhizobium rhizogenes* (formerly known as *Agrobacterium rhizogenes*). This commonly used method works for many tree species including conifers such as *Abies koreana* [14], *Larix decidua* [15], *Picea abies* [16], *Picea glauca* and *Picea mariana* [17], *Pinus radiata* [18], *Pinus taeda* [16], *Pseudotsuga menziesii* [19], and broadleaf trees such as *Ailanthus altissima* [20], *Castanea dentata* [21], *Castanea sativa* [22], *Eucalyptus globulus* [23], *Fraxinus americana* [24], *Fraxinus excelsior* [25], *Ginkgo biloba* [26], Poplar and aspen hybrids (*Populus* spp.) [27– 29], *Quercus robur* [30], *Quercus suber* [31], *Robinia pseudoacacia* [32], and *Ulmus americana* [33].

After the establishment of the transformation method, the genome editing technique needs to be established. For forest trees, only CRISPR/Cas actually plays a signifcant role. The three other genome editing mechanisms using transcription activator-like effector nucleases (TALEN), zinc fnger nucleases (ZFN) and oligodirected mutagenesis (ODM) are of minor importance in trees. TALEN and ODM have not been used to date. In poplars, ZFN was used experimentally before CRISPR/Cas technology was made accessible [34]. However, the results indicated that further technical improvements would be needed to increase the moderate mutation frequency.

For CRISPR/Cas-mediated genome editing, suitable and effciently editable targets need to be identifed based on gene sequencing, following the selection of a suitable Cas nuclease. Sequence information of the individual to be edited is required for accurate and reliable genome editing. Even though the number of genome-sequenced tree species is steadily increasing since the frst tree genome of the poplar species *Populus trichocarpa* [35], reference genomes are still quite limited to tree species with a manageable genome size. In particular, sequencing the complex genomes of some conifers remains a hurdle [36]. The availability of a reference genome is a prerequisite for selecting an editing target and verifying the presence of the protospacer-adjacent motif (PAM), as well as for identifying loci in the genome that might be considered as off-target sites. Avoiding off-target editing is one of the determining factors for the reliability of genome editing and its safety assessment. Meanwhile, some online tools for predicting potential off-targets have emerged for plants such as Cas-OFFinder, available at http://www.rgenome.net/casoffnder/ [37]. Furthermore, it should be remembered that trees are nearly

undomesticated plants and therefore have high genetic diversity. Thus, it cannot be assumed that the sequence of a target gene is identical in all individuals of a species. Although a reference genome can assist genome editing by providing evidence for targeting, the target sequence should be verifed in the individual being targeted before the fnal design. This challenge is addressed by the concept of the pangenome, but this is still in the distant future for trees [38].

Genome editing in forest trees, despite all the challenges described, is a promising technology that can bring benefts to plant molecular genetics research in particular. CRISPR/Cas and the other editing techniques are excellent for gene characterization studies. In these classical knockout approaches, genome editing serves as a tool to knock out genes as described by Fan et al. [3], Zhou et al. [39], and Bruegmann et al. [40]. With knockout approaches, the function of genes can simply be characterised which could subsequently be good starting points for conventional forest plant breeding. With corresponding genetic markers, individuals can be selected from natural populations and used as crossing partners to introgress favourable traits. If legal conditions permit, genome-edited trees could be used directly, too. The targeted genetic modifcations can be regarded as optimised breeding. Although the breeding goal can be achieved with conventional breeding methods based on crossing and selection, genome editing can accelerate genetic adaptation because trees take up to several decades to fower and fruit, depending on the species. Accelerating the adaptation process offers the possibility of adapting trees to rapidly advancing climate change and associated environmental conditions in foreseeable time frames. Plants' natural adaptation mechanisms such as genetic adaptation by recombination, mutation, and selection, or migration are likely to be too slow for adaptation to the new environmental challenges associated with climate change [41, 42].

# **2 Genome-Edited Forest Tree Species**

Forests have signifcant ecological and economic functions, so their preservation and vitality are of great importance. Thus, by any method, breeding forest trees is an important human mission. The term "tree" is indistinctly defned. In general, it refers to perennial plants that have wood formation and secondary thickening growth and - to distinguish them from shrubby woody plants – a single main stem. The tree forms a more or less defnite crown [43]. Some defnitions add the size: The stem grows to a height of at least six meters without external disturbance [44]. As previously indicated, TALEN and ODM have not been applied in trees to date. A ZFN was used experimentally in poplar hybrids (*P. tremula* × *P. alba* [referred to as *Populus* × *canescens*] and *P. tremula* × *P. tremuloides*) to mutagenize poplar orthologs of *LEAFY* and *AGAMOUS*. The editing rate in this *Agrobacterium*-based approach was among the lowest of all experiments with plants overall [34]. Due to the technical advantages offered by CRISPR/Cas and the boost of these techniques

in plant research throughout, from which woody plant research also benefted and still benefts, research work focused on this technique.

The frst CRISPR/Cas-edited tree was a Chinese white poplar (*P.* × *tomentosa*) in which the phytoene desaturase gene (*PDS*) was knocked out by mutation [3]. In subsequent years, *PDS* continues to serve as a marker gene in different annual and perennial plant species, as *PDS*-defcient plants are albinos [45]. Table 20.1 lists the forest tree species genome-edited to date. In trees, even though alternative Cas nucleases such as Cas12a have been available in principle for several years, Cas9 is the tool of choice. To our knowledge, the frst Cas12a editing of a tree species was performed in 2020 using the *PDS* knockout in the poplar hybrid *Populus alba* × *Populus glandulosa* [46].

# **3 Advanced Editing Technologies and Current Developments**

Since the frst genome editing experiments that resulted in Cas9-mediated knockouts, CRISPR-based methods have continuously improved and evolved, particularly in annual crop plants and model species. Due to the tree-specifc bottlenecks described above, the development of novel editing techniques in trees is not progressing as rapidly as in other model plants.

# *3.1 Effecting CRISPR/Cas Editing During Transformation and Regeneration*

Despite the expanding range of tree species that can undergo genome editing, most forest tree species still exhibit low transformation effciencies [58, 59]. Much time can be spent obtaining a suffcient number of transgenic and genome-edited plants by scaling up transformation experiments or optimising the transformation method. Those optimisations include the transfer of the DNA as well as regeneration during tissue culture.

To enhance transformation effciency in recalcitrant species by boosting regeneration, morphogenic regulator genes like *WUSCHEL* or *BABY BOOM* can be coexpressed with CRISPR/Cas components [60]. In poplar, Pan et al. [61] signifcantly increased callus and root initiation as well as shoot growth by co-activation of endogenous morphogenic genes *WUSCHEL* (*PtWUS*) or *WUSCHEL-RELATED HOMEOBOX 11* (*PtWOX11*). While morphogenic regulator genes are facing the bottleneck of regeneration, using nanoparticles can enhance the direct delivery of plasmid DNA into the target tissue, making transformation more effcient or even independent of expensive and complicated laboratory equipment [62]. In *Paulownia tomentosa*, the polysaccharide nanoparticle Chitosan was used as a carrier for direct plasmid transfection of nodular segments. Since Chitosan has a positive charge, a


**Table 20.1** CRISPR/Cas-edited forest tree species. For poplar trees, only inventions are mentioned here. More editing events are described in the section on traits and breeding

*RNP* — ribonucleoprotein

complex was formed with negatively charged DNA improving the uptake through the negatively charged cell membrane [63].

Because a shoot often develops from more than one single transformed cell, regenerated transgenic plants are often chimeric resulting in possibly non-uniformly edited regenerates in CRISPR/Cas approaches. This can affect the resulting phenotype and experiments based on genetically uniform tissue. A second step of regeneration decreased those chimeras in poplar and increased the frequency of homozygous CRISPR/Cas-mediated mutations [64].

Additionally, the activity of Cas9 and Cas12a can be enhanced by including a heat treatment during regeneration after transformation. This way, the editing effciencies in poplar as well as birch have been increased [46, 47].

# *3.2 Optimising CRISPR/Cas Vectors for Forest Tree Editing*

Many aspects infuence the effciency of CRISPR/Cas editing. On the one hand, editing effciency depends on the accessibility of the genomic target sequence to the Cas-enzyme, a factor depending on chromosome structure [65]. On the other hand, it depends on the expression of the Cas-gene as well as the activity of the Casenzyme and the transcription of gRNA, which can be infuenced by proper vector design.

In reports of CRISPR/Cas experiments in non-model forest trees like birch, chestnut, or walnut, the 35S promoter is the promoter of choice to drive Cas expression [45, 47, 51]. Because of its broad host range and well-studied functionality, as in classical gene technique experiments, this promoter is often used for the establishment of methods. In CRISPR/Cas attempts with the model tree genus *Populus*, Cas expression under control of a synthetic 35S-MAS fusion promoter increased editing effciency by 11% compared to the 35S promoter, which still is one of the standard promoters in poplars to date [66]. Driving the Cas expression under heterologous ubiquitin promoters resulted in editing effciencies of up to 95% in poplar [61, 67]. Those examples show the potential of optimisation by promoter choice in non-model forest tree species.

To enhance the translation of the Cas endonuclease in forest trees, codonoptimised variants like the plant codon optimised and the *Arabidopsis thaliana* codon-optimised Cas9 are routinely used [3, 40, 51, 53]. The use of a poplar codonoptimised AsCas12a, the frst report of target organism specifc codon optimisation for forest trees, resulted in editing effciencies of up to 70% [46]. For this kind of optimisation, knowledge about the codon usage of the target tree species must be available. In the Codon Usage Database, codon usage tables for different forest trees like poplar (*Populus* spp.), beech (*Fagus* spp.), chestnut (*Castanea* spp.), pine (*Pinus* spp.) or eucalyptus (*Eucalyptus* spp.) are available [68]. These tables can be used as a query for a codon usage analysis and optimisation of Cas genes. To predict Cas expression levels based on codon usage, the Codon Adaptation Index (CAI) can be calculated [69]. A value of one implements an optimal translation rate. If values are low, the online tool Optimizer can be used to create a sequence with a maximum CAI [70]. If only the rare codons that limit translation are to be identifed, the graphical codon usage analyser can be used to predict relative adaptiveness of each codon [71].

The most used promoter to drive gRNA transcription in forest trees is the U6-26 promoter from *A. thaliana* (*At*U6-26) [40, 51, 53], which is known for expressing high levels of gRNA in various plant species [72, 73]. In pine, a U6 promoter from *Pseudotsuga menziesii,* another member of the conifer family, is used [55]. Contrastively, in rubber tree (*Hevea brasiliensis*) endogenous U6 promoters were used for gRNA transcription resulting in editing effciencies of *HbPDS* of up to 67% [49], representing higher effciency compared to reports of *PDS* editing with *A. thaliana* U6 promoters in forest trees [3, 51]. These results correspond with reports in other tree species and plants indicating that endogenous polymerase III promoters can enhance editing effciency in general [74, 75].

To obtain a knockout mutant or to edit multiple genes at once, it is often required to target multiple sites in the genome. In consequence, the ability to express multiple gRNAs at once is desired. In chestnut, poplar or eucalyptus, individual transcription units were used to drive sgRNA transcription for Cas9 under the same or different polymerase III promoters [3, 49, 76]. To avoid usage of repetitive sequences, and therefore recombination, silencing and large vector size, multiple gRNAs separated by tRNAs can be combined in one transcription unit [77]. In this way, fve crRNAs for Cas12a were transcribed by a single *At*U6-26 promoter in poplar [46]. Pan et al. [61] in turn, used a polymerase II promoter to drive gRNA transcription for Cas9 in poplar, which is recommended for more than two gRNAs and offers the opportunity of inducible gRNA transcription.

In plant species having a long regeneration time like forest trees, the proper choice of spacer sequence and, therefore, the functionality of the corresponding gRNA is essential due to time-consuming and ecological reasons. In poplar, spacer sequences and secondary structures of multiple gRNAs were associated with Cas9 editing effciencies resulting in recommendations for favourable gRNA structure and spacer sequence. These recommendations can be used to design gRNA candidates for Cas9-mediated genome editing by *in silico* prediction of secondary structure to avoid non-functional sgRNAs [40]. For Cas12a, the effect of secondary structure and spacer sequence on editing effciency has been analysed in human cell lines, *E. coli* and maize, but not yet specifcally for forest tree species [78–80]. Potential gRNAs for different Cas variants and their effciencies can also be predicted *in silico* using online tools. But it must be considered that most of them like CRISPOR or CHOPCHOP are based on editing effciency data from mammalian cells or zebrafsh [81]. Although some tools, e.g., CHOPCHOP, use reference genome data from tree species, they are not specifcally designed for forest tree species [82].

To evaluate gRNAs in the target organism, transient expression systems can be used prior to stable transformations. In rubber tree, protoplast transfection and amplicon deep sequencing of the target region were combined to check editing effciency of Cas9 before stable transformation [83]. In poplar and birch, *Agrobacterium* inoculation of whole *in vitro* plantlets and quantitative PCR of the target locus are used [47]. It is likely that these transient systems can easily be adapted to other Cas nucleases, such as Cas12a. However, especially when using protoplasts, the cell type dependence on editing effciency must be considered [65].

If not being able to test in advance, a second (or even more) gRNA targeting the same genomic region can be used as backup to increase the probability of inducing at least one mutation [40, 67, 76]. Moreover, a 1.3 kb deletion at the target site was obtained using two sgRNAs for Cas9-mediated genome editing in Monterey pine (*P. radiata*, [55]). This large deletion is more likely to completely destroy the gene function.

# *3.3 DNA-Free Editing*

In conventional CRISPR/Cas9 approaches, the Cas9 nuclease and gRNA are stably transferred into the target organism as genetic information. However, integration of CRISPR/Cas-related transgenes can be disadvantageous. Continuous cleavage activity of the Cas nuclease can increase formation of chimeric plants and off-target cleavage. In addition, transgenes are a limiting factor in terms of legal regulation.

Since the outcrossing of transgenes in tree species is not an option in practice due to long reproduction cycles, it could be promising to obtain transgene-free edited plants in the frst generation by avoiding transgene integration. Therefore, recombinant Cas-ribonucleoproteins (RNPs) can be used instead of plasmid-encoded CRISPR components. Since RNPs do not depend on gene expression and effective promoters, vector optimisation is omitted. However, the use of RNPs has its own parameters to be optimised, such as RNP concentration, protein-gRNA ratio and incubation temperatures. For proof of principle, RNPs for CRISPR/Cas editing were introduced into protoplasts of poplar (*P. alba* × *P. glandulosa*), chestnut (*C. sativa*) and rubber tree (*H. brasiliensis*) using PEG [45, 50, 56]. A biolistic approach was used to co-deliver Cas-RNPs and a plasmid-encoded selection marker to somatic embryos of Monterey pine (*P. radiata*). Editing effciencies of up to 33% were observed in selection marker resistant plantlets [55].

# *3.4 Gene Sequence Modifcation*

CRISPR/Cas-knockout mutants are primarily based on random indels obtained by error-prone non-homologous end joining (NHEJ) during the repair of DNA doublestrand breaks (DSBs) [84]. But if interested in specifc insertions, deletions, or exchanges of DNA, a more precise and accurate way of DNA modifcation is needed. Such ambitious techniques are at the very beginning in forest trees.

For the conversion of single base pairs, CRISPR base editors (BEs) have been invented. BEs combine DNA binding domains with a nucleotide base deaminase that chemically modifes certain nucleotide bases. Using cytosine (CBEs) or adenine base editors (ABEs), conversions of C•G to T•A or A•T to G•C are possible, respectively [85]. Expression of Cas9 nickase-based BE in poplar (*P. tremula* × *P. alba*) led to the precise conversion effciency of up to 100% for CBE and 95% for ABE. Thereby indicating that effciency depends on the target and can be improved by using a U3 instead of a U6 *A. thaliana* promoter for sgRNA transcription [67].

To enable insertion or replacement of sequences larger than single base pairs and up to several kilobases, gene targeting based on homology-directed repair (HDR) is applied. Here, a donor DNA containing the intended DNA modifcation as well as fanking sequences homologous to the target site can be co-delivered with the CRISPR/Cas components. With the simultaneous inhibition of NHEJ by knocking *XRCC4* out and the enhancement of HDR by overexpressing *CtlP* and *MRE11* in poplar (*P. trichocarpa*), a bleomycin resistance gene was seamlessly integrated in frame of an endogenous promoter by Cas9 with knock-in effciency of up to 48% [86].

# *3.5 CRISPR Activation*

CRISPR/Cas can be used for activation of target genes by recruiting transcription activators, independent of CRISPR/Cas-mediated changes to the DNA sequence [87]. Because a DSB is not required, CRISPR activation is achieved by nucleaseinactive deadCas9 (dCas9). For gene activation in poplar (*P. alba* × *P. tremula*), a CRISPR-Combo system based on CRISPR-Act3.0 was used to enable gene editing and activation at the same time. Therefore, Cas9 endonuclease activity was deactivated by using short protospacer sequences of 14 to 16 nucleotides. Activators were acquired by gRNA using MS2-SunTags. Editing effciencies of 100% and gene activation of up to 100-fold expression were achieved [61].

# **4 Forest Tree Relevant Traits as Breeding Objectives**

Forest trees are important sequesters of CO2 into biomass and components of terrestrial biodiversity. As sessile organisms with prolonged growth, forest trees are frequently exposed to diverse stresses derived from the abiotic and biotic environment. Climate change-related weather conditions contribute to novel and increasingly severe environmental stresses for forest trees, such as drought periods or increased soil and water salinisation in certain climate zones. Although trees are evolutionarily adapted to local environments, fast-changing fuctuations of local climate conditions strongly affect their viability. Moreover, the establishment of novel tree pathogens caused by climate fuctuations and the increased vulnerability of already stressed forest trees to domestic pathogens also put forest trees under stress [88]. Trees are increasingly required to withstand specifc stresses and to remain upright, providing, in part, irreplaceable both ecological and economic value for countries and their people. The ability of trees to assimilate CO2 in great quantities displays a natural mechanism to mitigate global warming effects. However, climate change-derived stresses may reduce carbon fxation due to reduced photosynthesis rates when water is scarce or temperatures are too high [89].

Forest tree tolerances, resistances, and the refned breeding of commercial traits display the present-day breeding objectives. Improved and especially accelerated breeding strategies and genetic research on traits need more attention if species or products are to be sustained in the near future. Even though implementing genome editing in forest trees to improve and accelerate breeding purposes is still young, some research has displayed this mechanism's fast potential, especially on functional analyses of single genes and their correlation with desired traits.

# *4.1 Abiotic Stress Tolerances*

Abiotic stress tolerance-related research deals with elucidating and improving traits for tolerance of abiotic environmental factors, especially drought or salinity, that trees are increasingly confronted with. The continuing incorporation of genome editing mechanisms (particularly CRISPR/Cas) in this research feld drastically improves the understanding of single gene functions and their impact on tolerance traits by subsequently isolated phenotype analyses. Even though, to date, the research on abiotic stress tolerance-related traits is still limited in trees, an increase is observable and will gain more attention in future. However, some research has been done regarding single or multiple stresses and their higher-ordered adaption mechanisms.

#### **4.1.1 Drought Stress**

Drought stress describes the stress caused by the absence of water supply, which can reduce biomass production and the energy-providing mechanism of photosynthesis. While the research on annual model plants already revealed important mechanisms and genes involved in drought stress tolerance, the research on forest trees (especially under the application of genome editing) is in its infancy. The research of recent years mainly applied CRISPR/Cas-mediated knockouts in the model genus *Populus* to verify observable phenotypes derived from overexpression of putative drought tolerance-related genes. Even though CRISPR/Cas-mediated knockout mutants did not improve traits of drought stress tolerance, the precise knockouts of candidate genes helped insights into gene functions and their further use for tree tolerance breeding purposes. As a trait of putative drought stress tolerance, Zhou et al. [90] analysed the mechanism of root growth under drought stress. Overexpression and CRISPR/Cas9 knockouts of the root-specifc *NUCLEAR FACTOR-Y* transcription factor (TF) *NF-YB21* were analysed in the poplar hybrid 84 K (*P. alba* × *P. glandulosa*). Comparative analyses of one-month-old *nf-yb21* mutants and WT poplars revealed a reduced drought stress tolerance of the mutants by signifcantly reduced overall root growth and biomass, as well as thinner xylem vessels with tyloses and lower lignin contents, which reduced the hydraulic conductivity, an important indicator of water transport from soils [90].

As stomata regulate the fow of gases and thereby a plants water status, Shen et al. [91] analysed the impact of the TF *Pd*GNC on stomatal aperture in *P.* × *canescens*. 60-days-old CRISPR/Cas9-mediated *gnc* mutants exhibited increased stomatal aperture and water loss with reduced drought stress tolerance under drought stressed experimental conditions of 75 days. Analyses explained the droughtsusceptible phenotype with lower nitric oxide (NO) levels and hydrogen peroxide (H2O2) production in guard cells, increasing the stomatal aperture and, thereby water loss [91]. Similarly, Bai et al. [92] studied the function of the TF gene *OSMOTIC STRESS INDUCED C2H2 1* (*OSIC1*) in *P. alba* var. *pyramidalis* that is likewise involved in the pathway of stomatal aperture. CRISPR/Cas9-mediated knockout mutants showed signifcantly reduced performance under drought stress by greater stomatal aperture and water loss. This was explained by the involvement of *OSIC1* in a newly discovered transcriptional regulatory mechanism of H2O2 production in guard cells. The results enrich the understanding of how perennial woody plants respond to drought-induced osmotic stress, which can be further used for refned breeding approaches [92].

#### **4.1.2 Salt Stress**

The salinisation of forest soils describes the excessive accumulation of watersoluble salts in upper soil horizons. It mainly derives from extreme weather conditions, with dry climates and low precipitation. If prolonged over a long time or at high concentrations, the salinity of the soil results in reduced water and increased salt uptake of trees, resulting in stress through ionic, osmotic, oxidative, and other secondary changes. Salt stress can, when exposed for a long time, end in plant dieoffs [93]. Here, damage severity depends on salt concentrations, the growth stage of trees or the tree species, with *Populus euphratica* known to tolerate specifc salt concentrations and growing in saline semi-arid areas [94].

Efforts have been made to study the impact of single gene modifcations on salt stress tolerance by using genome editing in *Populus*. CRISPR/Cas9-mediated knockout of the TF gene *WRKY77* in *P. alba* var. *pyramidalis* signifcantly improved the salt tolerance of poplars under *in vitro* salt stress conditions [95]. After growing for one month in liquid medium with 150 mM NaCl, wild-type (WT) poplars showed leaf chlorosis symptoms, while *palwrky77* mutant leaves remained green with little discolourations. Further, signifcantly higher electrolyte leakage measurements suggested a higher cell disruption in WT compared to mutant poplars. *In vivo* and *in vitro* assays revealed the differences in salt stress tolerance by the *Pal*WRKY77-induced repression of abscisic acid-related genes. Therefore, *Pal*WRKY77 was found to be a negative regulator of salt stress response in poplars, providing a potential basis for genetic modifcation to generate salt-tolerant poplars in saline habitats [95].

To further elucidate salt stress tolerance in trees, candidate genes could be selected by the orientation of promising genes in annual plants such as *A. thaliana*, *Oryza sativa,* or *Solanum lycopersicum*. Shelake et al. [96] illustrate the potential of genome editing in crop plants to increase salinity tolerance. Here, CRISPR/Casmediated knockouts of *AtAITR* genes involved in abscisic acid signalling in *A. thaliana* [97], the *OsPQT3* gene, an E3 ubiquitin ligase involved in the regulation of oxidative stress in *O. sativa* [98], or the *SlARF4* gene, an auxin response factor in *S. lycopersicum* [99], resulted in improved salinity tolerance and may be promising candidate genes for genetic modifcations in forest trees if sequence information and genetic transformability are given.

# *4.2 Biotic Stress Resistance*

Plant pathogens, including viruses, fungi, bacteria, or oomycetes, are part of every functional ecosystem. However, globalisation or changing environmental factors contribute to the spread and mutation of pathogens, leading to pathogens harmful to single species or whole plant ecosystems. Resulting emerging infectious diseases can cause landscape-level mortality and, subsequently, ecosystem-wide changes [100]. The importance of tree pathogen control can be exemplifed in the interference of the fungus *Hymenoscyphus fraxineus*, causing ash dieback with severe mortality of common ash trees (*F. excelsior*) in most parts of the ash distribution range in Europe [101]. Other prominent examples of forest tree pathogens are chestnut blight, Dutch elm disease, myrtle rust, white pine blister rust, poplar leaf rust, and sudden oak death [100].

To date, the research on forest pathogen resistance by genome editing mechanisms is in its infancy. In addition to technical limitations in working with forest trees, the lack of knowledge of tree-pathogen interactions may restrict genome editing applications, as it denotes the basis for advanced research. Wang et al. [102] analysed the involvement of the TF MYB115 on the production of proanthocyanidins (PAs), a class of defence phenolic compounds in the leaves of poplars (*Populus* spp.) in response to abiotic and biotic stresses. CRISPR/Cas9 knockouts of *MYB115* in *P.* × *tomentosa* revealed signifcantly reduced levels of PAs and decreased expression of PA biosynthesis genes, suggesting the positive contribution of MYB115 to PA biosynthesis. After infection with *Dothiorella gregaria,* leaves from the poplar mutant *myb115* showed signifcantly higher damage [102].

Widespread biotrophic rust fungi of the genus *Melampsora* can reduce the economic value of trees, such as for *Populus* in natural stands and plantations, by reducing signifcant amounts of biomass [103, 104]. To elucidate genes involved in *Melampsora* resistance, Jiang et al. [105] focused on the TFs WRKY18 and WRKY35. The WRKY group is well known for being involved in abiotic and biotic stress responses in plants. Constitutive overexpression of *WRKY18* and *WRKY35* in *P.* × *tomentosa* led to increased resistance to *Melampsora* by elevated expression levels of downstream genes and lower H2O2 accumulation. In contrast, CRISPR/ Cas9-mediated knockout mutants did not differ from WT poplars regarding the expression levels of downstream genes and H2O2 accumulation, concluding an unaltered *Melampsora* resistance [105].

# *4.3 Commercial Traits: Productivity and Wood Properties*

Trees provide biomaterials with benefts for human life. Especially wood production and wood composition are interesting traits for refned breeding purposes due to the broad range of applications, ranging from the area of building and other industries up to energy supply. Economically important tree species include *P. abies*, *Tectona grandis*, or *Cedrus deodara* for timber production or *Picea rubens*, *Abies balsamea* or *Populus tremuloides* for the production of pulp and paper [106]. Prominent representatives of trees for biofuel production are species of the genus *Populus* or *Salix* due to their fast growth, allowing the production of signifcant amounts of biomass [107, 108].

#### **4.3.1 Wood Productivity**

The productivity of a forest is defned by the standing forest volume at a specifc time and referred to as yield, expressed by the accumulation of aboveground stem wood in standing trees. This biomass formation and correlated wood production exhibit an essential trait to improve due to the increasing wood utilisation demands, especially under increasing climate change-related disturbances on wood supply [109].

Genome editing mechanisms have been used to investigate the growth and development of woody plants. Thereby, conclusions could be made of genes involved in productivity, mainly in wood formation, to meet the demands of future wood production. CRISPR/Cas9-mediated knockouts of the *A. thaliana BRANCHED* orthologs *BRANCHED1-1* (*PcBRC1-1*) and *BRANCHED2-1* (*PcBRC2-1*) genes in *P.* × *canescens* strongly enhanced bud outgrowth [110]. *Pcbrc2-1* mutants revealed a signifcantly higher number of branches, whereas mutants of *Pcbrc1-1* revealed signifcantly higher shoots. As poplar trees are used for bioenergy production on short rotation coppices (SRCs), the enhanced sylleptic branching of the mutants may be an improved trait regarding the critical plantation establishment phase in the frst year. It may increase the biomass yield through the early closure of the canopy and, subsequently, the reduction of competing weeds by shading [110]. However, longterm biomass evaluation is still needed.

Fladung [111] generated CRISPR/Cas9-mediated knockouts of the rice ortholog *TILLER ANGLE CONTROL 1* (*TAC1*) in *P.* × *canescens* to investigate the function of the gene on the pyramidal plant growth, as could be seen by reduced expression levels of *TAC1* in *Prunus* species [112]. After a growth period of 3 years in the greenhouse, mutated poplars showed an altered phenotype compared to WT trees, with leaves of a narrower angle and an upright growth of shoots [111]. Even though no increased biomass production was detectable, upright-grown poplar trees may be interesting for SRCs, as the erect leaf or shoot growth allows more trees per area and, therefore, higher yield per area.

Further experiments of CRISPR/Cas knockouts of poplars were conducted to investigate the function of genes on wood productivity and growth [113]. However, knockouts were not correlated with improved biomass production.

#### **4.3.2 Wood Composition**

Wood mainly comprises the polymers lignin and the polysaccharidic cellulose and hemicelluloses, enriched in secondary-thickened cell walls. Polysaccharides are the desired substances of industries producing paper and pulp. In contrast, even though increasingly used for aromatic building blocks in the chemical industry, lignin impairs the extraction of cellulose and hemicelluloses, therefore being declared as a factor of biomass recalcitrance [114].

To reduce the amount of lignin in the wood composition, several genome editing attempts were conducted targeting lignin biosynthesis, ranging from involved transcription factors and oxidative enzymes up to the lignin biosynthesis genes themselves. Early genome editing via the CRISPR/Cas9 system in the *P.* × *canescens* produced biallelic knockouts of the *4-COUMARATE:COA LIGASE 1* (*4CL1*) gene, which was shown to be involved in the lignin biosynthesis [39]. The poplar mutants revealed 23% less lignin in stem wood. Xu et al. [115] genetically modifed the TF gene *PtoMYB170* in *P.* × *tomentosa* by creating knockout mutants generated by three target sites for CRISPR/Cas9 endonuclease. Knockout mutants of *PtoMYB170* displayed inability for upright growth, resulting in a pendant phenotype due to signifcantly reduced lignin deposition in the stem's secondary xylem growth. Expression analyses of lignin biosynthesis genes indicated strongly reduced expression levels in the knockout mutants, demonstrating that *Pto*MYB170 is strongly infuential on the downstream genes and lignin deposition in *P.* × *tomentosa* [115].

Other strategies involved the manipulation of genes involved in the direct biosynthesis of lignin. Vries et al. [116] conducted CRISPR/Cas9 knockouts of the *CAFFEOYL SHIKIMATE ESTERASE 1* and *2* (*CSE1, CSE2*) genes in *P.* × *canescens*. After 4 months of growth in the greenhouse, double mutants *cse1cse2* showed a height reduction of 35%, with further reduced stem diameter by 14%, stem fresh weight (not debarked) by 52% and stem dry weight (debarked) by 69% as compared to WT trees [116]. However, lignin contents were decreased by 35%, which translated into a fourfold increase in cellulose-to-glucose conversion upon limited saccharifcation. That indicates that the saccharifcation effciency (hydrolysis from polysaccharides to monosaccharides), positively affects the fermentation of monosaccharides to ethanol, a favourable trait of biofuel production [117]. Jang et al. [118] conducted a comparable CRISPR/Cas9 knockout approach of *CSE1* and *CSE2* in the closely related poplar hybrid 84 K (*P. alba* × *P. glandulosa*). In contrast to Vries et al. [116], they found a reduction of lignin deposition of up to 29.1% in either *cse1* or *cse2* single mutants, along with reduced expression levels of lignin biosynthesis genes. Simultaneously, the genome-edited lines showed no growth retardation and a morphologically indistinguishable phenotype to WT trees in a long-term living modifed organism feld test covering four seasons [118]. In addition, mutant poplars showed up to 25% higher saccharifcation effciency than the WT control. The difference between both conducted studies from Vries et al. [116] and Jang et al. [118] may rely on the different species (*P.* × *canescens; P. alba* × *P. glandulosa*, respectively) or the amount of lignin reduction (35%, 29.1%, respectively) and thereby a specifc threshold, under which no phenotypic changes are observable [118].

Within several years, genome editing positively affected the understanding of lignin-related genes in the model tree genus *Populus* and successfully established poplar trees with limited amounts of lignin and no growth retardation, providing essential insights into the future breeding of lignin-reduced wood composition in trees.

Apart from the genetic modifcation of wood composition-related genes, parameters of wood anatomy, particularly xylem fbre and vessel length were analysed by genome editing of Fasciclin-like arabinogalactan proteins (FLAs) in *P. trichocarpa* [119]. Out of 50 *in vivo* characterised poplar FLAs, two genes (*PtrFLA40* and *PtrFLA45*) were selected due to their high expression in the developing xylem as well as their high similarity in amino acids of 95.2%. Selected *FLA* genes were knocked out by CRISPR/Cas9. Double mutants *ptrfa40ptrfa45* revealed signifcantly increased stem length and diameter and enlarged cell sizes of xylem fbres and vessels of 4-month-old grown greenhouse poplars compared to unmodifed WT trees [119]. These fndings may be relevant for the paper and pulp industry, as the fbre length is an important quality trait due to its positive effect on sheet strength [120].

# **5 Biosafety of Genome-Edited Trees**

Trees differ from most agricultural crop plants in many characteristics, such as long lifespan and long generation cycles, complex habitat, and low degree of domestication. As with genetically modifed (GM) trees, biosafety has to be considered before their deployment [121, 122], but well-documented knowledge on specifc biosafety aspects is rare for genome-edited trees. Thus, information on biosafety protocols for genome-edited trees is required which provide a scientifc basis for future European Union regulations on environmental risk assessment to ensure the safe development and use of genome-edited trees.

Biosafety-relevant aspects comprise four main technological issues that need to be discussed for genome-edited trees [123]: (i) Are the gene-edited and naturally emerged modifcations in fact identical? (ii) If not, are the differences potentially hazardous? (iii) Are effcient containment strategies required to avoid possible adverse outcomes from vertical and horizontal gene transfer? (iv) Are off-target effects probable, and if yes, is the selective inclusion of "omics"-technologies needed to study cellular effects following the expression of the gene-edited gene(s)?

Like other plant species, genome-edited trees have the potential for gene fow when they fower. Thus, edited gene(s) could be spread to wild relatives through gene fow (vertical gene transfer). Many tree species are wind pollinators releasing pollen into the environment, which can sometimes be transported over very long distances. In case that the edited gene(s) have a developmental/evolutionary advantage, this could pose an invasive potential of these trees. Unintended ecological consequences could occur, such as the unintended spread of invasive genes or of the whole tree (by vegetative propagation, e.g., root suckers [124]) into natural populations. Thus, at least theoretically, the necessity of establishing containment strategies has to be considered. Therefore, the establishment of confnement systems, i.e., by making trees sterile by suppressing either pollen production (in male stamens) or female ovule development, is considered to prevent the uncontrolled spread of the edited genes [125].

In addition, possible unintended effects of genome-edited trees on non-target organisms must be considered. For example, if a tree is modifed in a gene involved in the secondary metabolism and, as consequence, produces a new ingredient, this could act as a toxin to the tree-interacting organisms. Trees fulfl numerous ecosystem services, such as carbon sequestration, soil conservation, and water regulation. Again, genome-edited trees producing a new ingredient, could impact these services. For example, if a tree is modifed to grow faster, it may sequester more carbon, but it could also deplete soil nutrients faster.

If a mutation has been detected in the plant genome without knowing whether it was natural or induced, to date, there are no detection methods to distinguish between gene-edited and natural mutations. In addition, if a cultivar carrying a natural mutation has been assessed as being safe in biosafety testing, there is no reason to assume a hazard if the cultivar has an induced mutation similar to a natural one. However, the mechanisms leading to the mutations are different, thus, because of the longevity of trees, long-term effects of genome-edited trees have to be considered, at least theoretically. This includes, for example, the long-term stability of the gene-edited modifcation or the epigenetics of the whole edited DNA region. To study the stability of the edited gene, the establishment of feld trials under natural conditions are necessary. Such feld trials could (i) deliver results about phenotypic effects resulting from expression of the gene edited genes, (ii) validate observations made under greenhouse conditions, and (iii) unravel putative non-target effects when the trees are grown within the range of natural variation. However, similar to GM trees, feld trials with gene-edited trees will be the exception rather than the rule in Europe. Reasons for this are manifold. Firstly, a high level of public concern exists against genetic engineering technologies. Thus, many consumers are reluctant to accept products made from genetically modifed organisms. In addition, regulatory hurdles which are based on the precautionary principle are high in Europe, making the regulatory process for feld trials extremely long and making it diffcult to obtain approval for a feld trial with gene-edited trees. And fnally, similar to GM plants in general, many companies and researchers fear that anti-GMO activists will destroy feld trials with gene-edited trees.

Off-target effects are unintended changes in the DNA resulting from gene editing in "wrong" genes. This could occur, for example, when gene editing is aimed at duplicated genomes and the target genes are present in duplicated or even in multiple copies and are highly similar in their sequence. As a consequence, it could be that the gRNA was not specifcally designed for the one gene to be edited and that also sequence-homologous genomic regions (e.g., paralogous genes) are targeted by the gRNA. This could lead to mutations in other parts of the genome, potentially causing gene knockouts or activation or silencing of genetic regulatory elements. However, such off-target modifcations could simply be avoided through improvement in the gRNA design [40], based on reliable genome sequences of the tree species to be edited.

It is common silvicultural practice to perform a formal evaluation of the behaviour of new tree varieties under natural feld conditions. Accordingly, a number of feld trials have to be set up to assess the safety of gene-edited trees modifed for different genes, similarly as it was performed with GM trees [126]. However, in contrast to classical GM-technology, genome editing modifed genomic information is targeted and precise, thus, organisms (microbes, plants and animals) harbouring mutations created by genome editing are indistinguishable from organisms carrying an identical but naturally emerged mutation. This could lead to the question whether the biosafety of gene-edited trees needs to be tested in the feld at all.

# **References**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **Chapter 21 Genome Editing for Reduction of Bitterness and for Production of Medicinal Terpenes in** *Cichorium* **Species**

### **Katarina Cankar, Katrijn Van Laere , and Dirk Bosch**

**Abstract** *Cichorium* varieties are cultivated both as leafy vegetables as well as industrial root crop for extraction of the food bre inulin. *Cichorium* is a typical European crop and grown on a relatively small scale. However, due to its distinctive taste and health benets and its capacity to produce multiple bioactive compounds, *Cichorium* has great potential if varieties could be optimised for these properties by breeding. In recent years it has been demonstrated in several laboratories that chicory is very amenable to genome editing. Different protocols were developed and implemented to adapt bitterness as well as to accumulate medicinal terpenes, generating potential socio-economic benets over the entire value chain from farmers to consumers, as well as for the environment. In addition, scientic knowledge on chicory biology, particularly on the biosynthesis of secondary metabolites was signicantly increased. This demonstrates how genome editing can contribute to breeding of niche crops such as *Cichorium*, which have relatively little investment leverage for extensive breeding programs.

# **1** *Cichorium* **Species and Their Sesquiterpene Lactones**

The genus *Cichorium* belongs to the Asteraceae family of plants. About 10 *Cichorium* species have been described, with *C. intybus* being the best known and being cultivated for different applications (Fig. 21.1).

*C. intybus* var. *foliosum* (e.g., witloof), *C*. *intybus* var. *latifolium* (e.g., radicchio) and the closely related *C. endivia* (e.g., endive), are well-known leafy vegetables appreciated for their characteristic bitter taste and richness in nutritionally relevant

Wageningen University & Research, Wageningen Plant Research, Wageningen, The Netherlands

e-mail: katarina.cankar@wur.nl

K. Cankar (\*) · D. Bosch

K. Van Laere Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), Melle, Belgium

**Fig. 21.1** Overview of different *Cichorium* species and varieties: (**a**) *C. intybus* var. *sylvestre* (Catalogne); (**b**) *C. endivia* (endive); (**c**) *C. intybus* var. *porphyreum* (sugerloaf); (**d**, **f**) *C. intybus* var. *latifolium* (radicchio); (**e**) *C. intybus* var. *latifolium* (radicchio early red of Treviso); (**g**) *C. intybus* var. *latifolium* (radicchio late red of Treviso); (**h**) *C. intybus* var. *foliosum* (redloof); (**i**) *C. intybus* var. *foliosum* (witloof); (**j**) *C. intybus* 'variegata di Castelfranco'; (**k**) *C. intybus* var. *sativum* (industrial chicory); (**l**) Flowers of *C. intybus*. (Pictures provided by Bram Van de Poel, KU Leuven, Belgium)

compounds with benefcial effects on human health. However, the bitter taste can have a negative infuence on consumer acceptability, especially among young adults and children. Industrial chicory (*C. intybus* var. *sativum*) is grown, particularly in the south of the Netherlands, Belgium and north of France, for its large taproots from which inulin is extracted. *C*. *intybus* is grown on a relatively small area with a total global acreage of around 14,500 ha [1, 2]. Inulin is a prebiotic food fbre which stimulates gut health [3] and is used as food ingredient in products such as yogurts and granola bars. Chicory is actually an ancient crop and its use as food and for its medicinal properties is documented already since Roman times [4]. Apart from inulin extraction, industrial chicory roots also have potential as raw material to make chicory four for use in food industry as the four will contain the nutritional benefts of inulin and does not contain gluten. But the use of this four is still limited due to the high bitterness of the roots.

The bitter taste of the *Cichorium* crops is caused by the presence of sesquiterpene lactones (STLs) that accumulate both in leaves and roots. STLs represent a large and diverse group of terpenoids with more than 5000 different STLs being identifed in Asteraceae [5]. STLs are specialized isoprenoid metabolites that contain 15 carbon atoms and which have a lactone function. The presence of a γ-lactone ring containing an α-methylene group, is a signifcant characteristic of STLs. They were shown to have a defence role in plants as protection against herbivorous insects and fungi [6–8].

Based on their skeleton, STLs are classifed in six major groups namely germacranolides, eudesmanolides, eremophilanolides, guaianolides, pseudoguaianolides and hypocretenolides and many subtypes [8]. Leaves and especially roots of *Cichorium* species contain high concentrations of the bitter guaianolides lactucopicrin, lactucin, and 8-deoxylactucin. Eudesmanolides and germacranolides are present in smaller amounts [9]. 8-Deoxylactucin, lactucin, and lactucopicrin predominantly occur in the oxalated form [10], but also their derivatives such as 11,13-dihydro-analogues are present. Several health-related bioactivities of chicory STLs have been documented. Lactucin and lactucopicrin display analgesic and sedative effects [11] and have antimalarial activity [12] and natural extracts from chicory roots present antibacterial and antifungal properties [13]. Purifed fractions from chicory roots containing a mixture of 8-deoxylactucin and dihydro-8 deoxylactucin possess promising anti-infammatory properties [14]. Also dihydrolactucin, a lactucin derivative found in chicory, revealed anti-infammatory potential [15]. It is not yet clear which STLs contribute the most to the bitterness perceived by consumers [5].

The biosynthesis of STLs in chicory has been partly elucidated (Fig. 21.2). The common sesquiterpene precursor farnesyl pyrophosphate (FPP) is frst cyclized to germacrene A by the germacrene A synthase (CiGAS) [16]. The biosynthesis continues by oxygenation by presumably as many as six cytochrome P450 enzymes of which the genes encoding germacrene A oxidase (CiGAO), costunolide synthase (CiCOS), kauniolide synthase (CiKLS) and lactucin synthase (CiLCS) have been identifed [17–23], all belonging to the CYP71 clan of cytochrome P450 enzymes. The STLs are further modifed by conjugations with 4-hydroxyphenyl acetate and oxalate and by reduction of the 11,13-double bond. Enzymes involved in 4-hydroxyphenyl acetate and oxalate transfer and reduction of the double bond have not yet been identifed. The basic STL structures in different chicory subspecies are identical but between varieties their relative abundance and the extent of modifcations can differ. The downstream steps in the STL biosynthesis, from the tricyclic

**Fig. 21.2** Biosynthetic pathway of chicory STLs. Enzymes to be inactivated for reduction of bitterness are indicated in red font and enzymes to be inactivated for accumulation of medicinal compounds are indicated by a blue arrow. Dashed arrows indicate uncharacterized enzymatic conversions. FPP – farnesyl pyrophosphate, CiGAS – germacrene A synthase, CiGAO – germacrene A oxidase, CiCOS – costunolide synthase, CiKLS – kauniolide synthase, CiLCS – lactucin synthase

kauniolide onward, appear to take place in the latex itself, as the *CiKLS* and *CiLCS* genes show latex-specifc expression [17, 18].

In summary, chicory species are producing several compounds that are highly relevant for food, namely the dietary fbre inulin and the bitter tasting terpenes, with some of these terpenes displaying benefcial health properties. Generating varieties with an optimized composition of either of these compounds would therefore be highly interesting and open new markets for food industry with great economic impact. Today, for industrial chicory hot water extraction is used to extract inulin and the bitter tasting STLs are co-extracted in this process [24]. Since the presence of bitter compounds in inulin limits its use as a prebiotic dietary fbre and sweetener in food products, the STLs are removed in subsequent purifcation steps, increasing the costs and input for processing. Moreover, related to leafy *Cichorium* species as witloof and radicchio, product differentiation by creating a more diverse range of favours would maximize witloof acceptance and beneft of its health promoting compounds. However, chicory species are relatively small crops with little investment leverage to enter lengthy breeding programs of about 10 years to produce a new cultivar. Although *Cichorium* species are self-incompatible, selfng at a low rate can still occur [25, 26]. Thanks to the high number of fower heads produced during a generative period, a quite high amount of selfed seeds can thus still be formed. However, creating true hybrids remains cumbersome. New breeding techniques, e.g. CRISPR/Cas genome editing, could make a difference to create chicory varieties with consumer traits in a faster and more effcient manner.

# **2 Genome Editing in** *Cichorium* **Species**

Genome editing methods for *Cichorium* have frst been described in 2019 [27]. Since then several studies describe the use of CRISPR/Cas-based genome editing in *Cichorium* species (Table 21.1). Protocols for four different delivery methods of CRISPR/Cas reagents have been established. High effciency of genome editing using *Agrobacterium rhizogenes*-mediated stable transformation in hairy roots was frst demonstrated for the chicory phytoene desaturase gene (*CiPDS*). This optimized protocol was later used to elucidate the function of biosynthetic enzymes involved in the synthesis of tetracoumaroyl spermine in chicory [28]. Introduction of CRISPR/Cas system via *Agrobacterium tumefaciens*-mediated stable transformation was frst described targeting the *CiGAS* gene [29]. While stable transformation methods lead to high editing effciency, thanks to the continuous editing after stable genome integration of CRISPR/Cas, they have a disadvantage for chicory breeding due to chicory self-incompatibility (although incomplete) and long time needed for outcrossing the T-DNA. Genome editing of the same gene in two compatible varieties is needed followed by segregation of transgenes after crossing. In addition, detailed genotyping of the resulting plants revealed that plants obtained by stable transformation often showed chimerism and a mixture of *CiGAS* genotypes in the same plant was observed.

In parallel, transient delivery methods for chicory protoplasts combined with whole plant regeneration were established. Transient delivery of plasmids encoding CRISPR/Cas reagents was used for editing of the *CiPDS* gene and genes involved in the biosynthesis of chicory STLs [17, 18, 21, 23, 27, 30]. Although effcient generation of biallelic mutants was described even for multi-gene families of the STL pathway, unintended integration of plasmid DNA into the chicory genome in up to 30% of plants was observed in several studies [17, 18, 27, 30]. Alternatively, delivery of preassembled ribonucleoprotein complexes (RNPs) was established for chicory protoplasts [17, 29, 30]. This DNA-free delivery method is effcient while at the same time eliminating the risk of unintended DNA integration. Therefore, it can be concluded that delivery by RNPs is the most suitable delivery method for genome editing in chicory.

When comparing the three methods, stable *Agrobacterium* transformation, transient plasmid delivery and RNP transfection, for off-target editing at six potential off-target sites in the chicory genome showing similarity to the target sequence in the *CiGAS* gene, no off-target editing was observed for either of the three delivery methods [29]. In conclusion, both stable transformation and transient transfection have successfully been used in chicory with high effciency, also for editing multigene families (Table 21.1). This advance will facilitate functional analysis and genetic improvement of chicory.


**Table 21.1** Studies describing use of genome editing in *Cichorium* sp.

a nd = not determined

# **3 Genome Editing for Reduction of Bitterness in** *Cichorium* **Crops**

Although there is some natural variation in the STL content among *Cichorium* species [31, 32], variants with a considerable low STL content have not yet been identifed. Using CRISPR/Cas targeting genes involved in the STL biosynthetic pathway would facilitate the generation of less or zero bitterness in *Cichorium*.

The frst dedicated step of the pathway (Fig. 21.2) is catalysed by CiGAS enzyme which is encoded in the chicory genome in four gene copies of the terpene synthase gene family, i.e., *CiGAS*-*S1*, *CiGAS*-*S2*, *CiGAS*-*S3* and *CiGAS*-*L* [33]. Genome and transcriptome analysis identifed two additional *CiGAS* paralogs belonging to the same gene clade [23]. Effcient editing of the *CiGAS* genes was achieved by several approaches (Table 21.1). The interruption of all *CiGAS*-*S1*, *CiGAS*-*S2*, *CiGAS*-*S3* and *CiGAS*-*L* alleles has been achieved and led to elimination of the STLs in chicory leaves and taproots [30]. The edited plants showed no other directly observable morphological phenotypic changes under greenhouse conditions.

To investigate if inactivation of downstream steps in the STL biosynthetic pathway could modulate bitterness, genes from the CYP71 clan of the cytochrome P450 gene family *CiGAO*, *CiCOS* and *CiKLS* were functionally characterized in *Cichorium* [17–20, 22, 23]. In conclusion, taken all these studies together, two paralogous *CiGAO* genes, three paralogous *CiCOS* genes, and three paralogous *CiKLS* genes displayed catalytic activity in the upstream STL pathway to produce costunolide and kauniolide. De Bruyn et al. [23] used CRISPR/Cas genome editing to simultaneously target multiple CYP71 genes in protoplasts of witloof, followed by regeneration of plants. A broad spectrum of plant genotypes containing different (loss of function) mutants in multiple genes were obtained. Several genotypes containing mutations in one or more paralogous CYP71 genes (*CiGAO* mutants, *CiCOS* mutants and *CiKLS* mutants) were profled for their STL composition. The production of 14 out of 16 detectable STL metabolites [34] was eliminated in the mutant types containing a homozygous loss of function mutation in the three functional paralogous *CiKLS* genes [22, 23] as was also observed when the three *CiKLS* paralogs were edited in root chicory [17]. Other mutants only showed shifts in STL compounds concentrations, but no clear effects or decreases were detected. It is likely that the mutation effect on STL compound quantities is masked if at least one functional allele and/or paralog is still present.

Studies indicate that the perception of bitterness is not only related to the guaianolide STL metabolite quantities and composition. François et al. [35] showed a strong correlation between glucose and sucrose with crunchiness and bitterness and a correlation between fructose and sweetness. Also the balance between STL compounds and phenolic compounds is described to affect bitterness [36]. So STLs are probably not the only metabolites to consider when aiming to modify bitterness in *Cichorium*. Cellular assays and human sensory tests should be used to link bitterness to the STL concentrations in CRISPR/Cas mutants [37].

# **4 Genome Editing to Produce Medicinal Terpenes in Chicory**

The use of industrial chicory for extraction of inulin fbre is well established, with the chicory STLs being discarded during the purifcation of inulin due to their bitterness. These terpenes could however be obtained from the chicory root as a secondary product via a procedure compatible with inulin processing, yielding both inulin and terpenes which can be used for e.g., health applications. Depending on the homogeneity and quality of the terpene fraction and on the application, this fraction might need further processing. Recently, a sustainable method of terpene extraction from chicory taproots using supercritical CO2 was established [14]. The socio-economic impacts of a multi-product process compared to a reference inulin process indicate that a multi-product process leads to higher added value and increased socio-economic impact compared to the reference inulin process [24]. Therefore, it is interesting to increase not only the inulin content of the roots by breeding, but also the terpene amounts. Genome editing can be used to facilitate the generation of chicory varieties that accumulate higher amounts of medicinal terpenes.

A frst example is a chicory variety with an increased accumulation of costunolide [17], which is a natural STL from the Asteraceae family and exhibits anticancer activity through its functional moiety α-methylene-γ-butyrolactone that reacts with the cysteine sulfhydryl group of various proteins [38]. In chicory, costunolide is an intermediate in the biosynthesis of STLs and does not accumulate since it is effciently converted to downstream STLs. To accumulate costunolide the kauniolide synthase (CiKLS) that converts costunolide to kauniolide, needs to be inactivated. Genome editing of the three *CiKLS* paralogs in chicory was performed by transient transfection of both plasmids and RNPs to chicory protoplasts, both leading to small indel mutations but only the former sometimes to unintended DNA integration. As a result the biosynthesis of the chicory sesquiterpenes was interrupted and costunolide accumulation was observed [17]. Interestingly, next to free costunolide predominantly costunolide conjugates were found to be accumulating, presumably mitigating the toxicity of costunolide to chicory cells.

A second example of chicory varieties with increased medicinal compounds are chicory lines with increased amounts of anti-infammatory 8-deoxylactucin and its derivatives [18]. 8-deoxylactucin is a natural STL of chicory that accumulates in chicory taproots next to lactucin and lactucopicrin, predominantly in oxalated form [10]. To increase 8-deoxylactucin accumulation in the taproots the gene encoding the lactucin synthase (*CiLCS*) that hydroxylates 8-deoxylactucin on position 8 to generate lactucin needs to be inactivated. Genome editing was used to inactivate three candidate cytochrome P450 genes with putative LCS activity resulting in the identifcation of one of the genes as *CiLCS* and generation of chicory lines that predominantly accumulate 8-deoxylactucin and its derivatives [18].

These two examples show that genome editing can be successfully applied in chicory to inactivate single genes or gene families, elucidating enzyme function and resulting in novel terpene chemotypes. In both examples the target medicinal STLs in genome edited roots accumulated to the similar high amounts of total STLs as found in wild type roots, namely about 1.5 mg/g FW. The availability of the chicory lines with modifed terpene composition can also be used to generate more knowledge on the natural role of terpenes in chicory development and defence.

# **5 Potential of Genome Editing for Chicory Breeding**

Chicory is very amenable to genome editing. Highly effcient protocols have recently been developed in different laboratories. Using these protocols scientifc knowledge on chicory biology, particularly of the biosynthesis of secondary metabolites was increased. Implementation of these protocols resulted in generation of multiple chicory lines with altered terpene profles. These lines could have potential benefts for consumers (healthy terpenes, varieties with altered taste), food producers (lack of bitter compounds simplifes inulin extraction or alternatively, bitter terpenes currently discarded as waste can be used as secondary product), farmers (agricultural diversifcation), breeders (creation of new cultivars with added value), the environment (reduction in energy demand and greenhouse gas emissions due to avoiding bitter compound extraction), and the economy (higher added value and more jobs).

Using the now available genome editing protocols as breeding tool, also other important biological processes for *Cichorium* breeding, e.g., male sterility, haploid induction, inulin quality and yield, etc., can be investigated and used to facilitate the long breeding process and generate novel cultivars in relatively shorter time frame with commercial interesting traits related to e.g., root shape, root weight, inulin chain length, disease resistance, bolting resistance, etc. In a broader perspective, genome editing can be a powerful tool to help to stimulate agricultural biodiversity in Europe by improving niche crops, like chicory, which have relatively little investment leverage. In synergism with other breeding and farming methods, this is highly relevant for securing crop diversity and improving sustainable production while at the same time dealing with the challenges like that of climate change.

**Acknowledgement** This work has received funding from the European Union's Horizon 2020 Research and Innovation Programme under grant agreement No. 760891 [H2020-NMBP-BIOTEC-07-2017: New Plant Breeding Techniques (NPBT) in molecular farming: Multipurpose crops for industrial bioproducts]. The authors want to thank all researchers and technicians that contributed to this work.

# **References**

1. Wilson, R.G., Smith, J.A., Yonts, C.D.: Chicory root yield and carbohydrate composition is infuenced by cultivar selection, planting, and harvest date. Crop Sci. **44**(3), 748–752 (2004)


8-deoxylactucin and its derivatives in chicory (*Cichorium intybus L.*). J. Agric. Food Chem. **71**(15), 6061–6072 (2023)


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **Chapter 22 Engineering Phytonutrient Content in Tomato by Genome Editing Technologies**

**Aurelia Scarano and Angelo Santino**

**Abstract** The application of precise genome editing represents an important stepforward in plant functional genomics research and crop improvement by generating tailored modications within a target genome sequence. Among the genome editing technologies, the CRISPR/Cas9 system has been the most largely one applied in many crop species, thanks to its high customizable specicity. Tomato is one of the most cultivated and consumed horticultural crops worldwide and an ideal model plant for studying different physiological processes (e.g., plant development, response to biotic/abiotic stresses, fruit quality) by using different approaches, such as conventional breeding, classical transgenesis and genome editing technologies. In recent years, the number of studies on the genome editing application in tomato has increasingly grown, particularly for the improvement of fruit quality and nutritional value. In this chapter, we report about the main achievements provided by such technologies for engineering the content of nutritionally relevant compounds, such as polyphenols, carotenoids, vitamins, and other important phytonutrients in tomato fruit.

# **1 Introduction**

Targeted genome engineering has emerged as an alternative to classical plant breeding and transgenic (GMO) methods to apply in both model and crop plants [1]. Precise genome editing provides great advantages in plant functional genomics research and crop improvement by generating tailored modications at a target genome sequence [2]. Different genome-editing tools have been developed in the past decade, including zinc-nger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced palindromic

A. Scarano (\*) · A. Santino (\*)

Institute of Science of Food Production, C.N.R. Unit of Lecce, Lecce, Italy e-mail: aurelia.scarano@ispa.cnr.it; angelo.santino@ispa.cnr.it

A. Ricroch et al. (eds.), *A Roadmap for Plant Genome Editing*, https://doi.org/10.1007/978-3-031-46150-7\_22

repeat/CRISPR-associated protein 9 system (CRISPR/Cas9). Zinc fnger nucleases (ZFNs) and TAL effector nucleases (TALENs) can be used to mutagenize genomes at specifc loci, and such systems require two different DNA binding proteins fanking a sequence of interest, each with a C-terminal FokI nuclease module. However, in plant research community CRISPR/Cas9 system has been the most largely preferred, thanks to its ability to reprogram the endonuclease specifcity using customizable small noncoding RNAs, named single guide RNAs (sgRNAs), specifc for a given targeted nucleic acid sequence [3, 4].

ZFNs, TALENs and CRISPR/Cas9 systems are able to induce site-specifc double-strand breaks (DSBs) within the genome, generating modifcations through homologous recombination (HR) or non-homologous end-joining (NHEJ) repair mechanisms [1]. HR can accurately repair DSBs using the homologous sequence fanking a DSB or an exogenously supplied DNA 'donor template' as the template. However, NHEJ repair is error-prone and frequently causes insertions or deletions (indels) around the sites of DNA breaks. In eukaryotic cells, and particularly in plant cells, DSBs are preferentially repaired by NHEJ mechanism, thus becoming the most used pathway to knockout genes in plants (e.g., via introducing frameshift mutation or a deletion) [5]. As preferential application, the gene knockouts can be implemented by using multiple sgRNAs easily assembled into a single delivery vector (for example using Golden Gate cloning, GoldenBraid, BioBrick technology) [6–8], thus allowing the knockout of all the members within a specifc gene family. The specifcity of CRISPR/Cas9 is guaranteed by a perfect match between the last 8–12 bases of the sgRNAs sequence, referred as the 'seed sequence' and the corresponding region of the target DNA (i.e., the region proximal to the 50 end of the PAM). Such specifcity is particularly important for the cleavage effciency target of Cas9 [4, 9, 10,] and to possibly limit potential off-target activities. Furthermore, the effciency of CRISPR/Cas9 may be infuenced by the sequence, location, context of the target (e.g., epigenetic factors, such as DNA methylation or histone modifcation, that might limit the DNA binding), beside the transformation effciency. Unfortunately, not many crop plants are easily transformable, although new delivery systems (for instance, DNA-free in vitro systems consisting in the direct delivery of a ribonucleoprotein complex, formed by the Cas9 protein and the guide RNA) have been recently proposed [11, 12].

# **2 CRISPR-Cas9 System for the Nutritional Improvement in Tomato**

Tomato is one of the most important commercial horticulture crops cultivated and consumed worldwide, and an ideal model plant for studying plant stress responses, plant reproductive development, functional genomics, and quality improvement [2]. Tomato fruit is a good source of lycopene, β-carotene, phenolic compounds, and micronutrients [13]. Most of these phytonutrients play an important role in human nutrition and signifcant health benefts associated with their antioxidant activities, beside other potential ones, such as anticancer and anti-infammatory activities [14]. Therefore, different metabolic pathways and several genes encoding important enzymatic activities or regulatory factors can be designated as genome editing targets, with the fnal aim of quality and nutritional improvement of tomato fruits.

Together with the nutritional improvement, tomato is also a relevant challenge for breeders, as fruit yield or (a)biotic stress tolerance are controlled by several genes acting independently or in concert [15]. In the past decades, many different approaches have been used for improvement of tomato fruit quality and nutritional value, including conventional breeding and genetic modifcations [14]. Such approaches, together with the identifcation of genes and alleles and their rapid integration in elite cultivars, have been often laborious, time-consuming, and expensive, as the desired mutations or insertion of new allelic versions in the lines require several crossings along the generations [16]. The recent development of gene editing technologies using programmable nucleases appears therefore to be a promising and effcient approach for both applicative and basic research involving forward and reverse genetics in tomato [15]. Some examples of genome editing have been successfully applied in tomato, by using TALENs [17], CRISPR/Cas9 systems [18– 20], CRISPR/Cpf1 [21], Cas9 variants like dCas9 [22], or base editing (Target-AID technology) [15].

# **3 Genome Editing for the Improvement of Carotenoid Content in Tomato Fruits**

Several genes involved in carotenoid accumulation have been identifed in tomato during the last two decades and used as target genes for genome editing applications. Improved lycopene content has been achieved inhibiting the conversion from lycopene to β- and α-carotene, by knocking down different genes associated with the carotenoid metabolic pathway (*SGR1*, *LCY-E*, *Blc*, *LCY-B1* and *LCY-B2* genes: *stay-green 1*, *lycopene δ-cyclase*, *β-lycopene cyclase*, *lycopene β-cyclase 1* and *2*, respectively) [18]. The tomato fruits from *SlSGR1* knockout null line obtained via CRISPR/Cas9 have showed a turbid brown color with signifcantly higher chlorophyll and carotenoid levels than in wild-type fruits. The strong changes in pigment and carotenoid content resulted in the accumulation of key primary metabolites, such as sucrose and its derivatives, and tricarboxylic acid cycle intermediates, such as malate and fumarate [23].

In another work, a series of tomato genotypes with different fruit colors, including yellow, brown, pink, light-yellow, pink-brown, yellow-green, and light green, has been generated by CRISPR/Cas9-mediated multiplex gene editing of three fruit color-related genes, two of them implicated in the lycopene biosynthesis in tomato (*PSY1* and *SGR1: phytoene synthase1* and *stay-green1*, respectively) [24].

The application of the CRISPR/Cas9 system further contributed to show the contribution of *PSY1* and *CRTR-B2* genes in fruit and fower colors changes in tomato fruit due to the carotenoid pigmentation. In fact, by targeting these genes, the obtained edited plants showed a loss-of function yellow-like phenotype caused by the suppression of lycopene biosynthesis and accumulation [25]. The CRISPR/Cas9 system has been also used to test the genome-editing effciency by targeting the tomato *phytoene desaturase* gene (*slyPDS*), allowing to observe a severe albino/ photobleached phenotype in most of transgenic tomato seedlings [18].

A Target activation-induced cytidine deaminase (Target-AID) base-editing technology has also been used to target genes related to carotenoid accumulation, such as tomato *DNA Damage UV Binding protein 1* (*SlDDB1*), *deetiolated1* (*SlDET1*), and *Lycopene beta cyclase* (*SlCYC-B*) [15]. This approach resulted in the generation of several lines showing substitutions in each target gene, resulting in lycopene, β-carotene, lutein and neoxanthin increased content. Multiplexed targeted plants in *SlDDB1*, *SlDET1* and *SlCYC-B* genes resulted in generation of several lines with improved lycopene accumulation via stacking of mutations [26].

# **4 Genome Editing for the Improvement of Polyphenol Content in Tomato Fruits**

Different genome editing strategies have been applied in tomato targeting the polyphenol biosynthetic pathway at different levels and with different scopes. Some studies were aimed to better understand the regulatory genes encoding transcription factors modulating the polyphenol biosynthesis, in other cases the application of genome editing consisted in the improvement of such compounds associated with the enhancement of fruit quality and their benefts for human health. Zhi et al. [27] have investigated the anthocyanin 2 (SlAN2) transcription factor function conducing a CRISPR/Cas9- mediated targeted mutagenesis in the purple tomato cultivar "Indigo Rose". Although the fruit and the anthocyanin content were comparable with cv "Indigo Rose", the *SlAN2* tomato mutants resulted in lowered gene expression related to anthocyanins biosynthesis in the vegetative tissues. By using the genome editing approach, this study therefore provides a proof-of-concept that *SlAN2* regulates the anthocyanin biosynthesis in tomato vegetative tissues and that anthocyanin biosynthesis differs in terms of regulatory mechanisms between cotyledons and hypocotyls [27]. In another study, the CRISPR/Cas9 system was applied to generate pink-fruited tomato plants by disrupting the *SlMYB12* gene in different inbred lines [19]. Different homozygous, heterozygous, biallelic and chimeric mutants carrying the deletion in *SlMYB12* were obtained among the T0 regeneration plants and carrying different types of mutations, including an expected large deletion in the target site. T1 progeny obtained by self-pollination of three large-deletion mutants exhibited pink fruits and *y*-like colorless peels, with naringenin chalcone and transcript levels of two key biosynthetic enzymes (CHS1 and CHS2)

signifcantly reduced. Changes in the fruit color and naringenin chalcone levels have been also observed following a CRISPR/Cas9-mediated multiplex gene editing involving *SlMYB12* gene together with other genes implicated in carotenoid biosynthesis (*PSY* and *SGR1*). Among the different colored fruits obtained with this strategy, the tomato *myb12* single mutants exhibited the expected pink color, whereas the *psy myb12* double mutant displayed a light-yellow fruit color, *myb12 sgr1* double mutants a pink-brown color and the *psy myb12 sgr1* triple mutant a light green color, suggesting a key role for *SlPSY* and *SlSGR1* in the carotenoid metabolism and *SlMYB12* gene in polyphenol metabolism [24]. Different genome editing approaches have been applied to improve the polyphenol content in tomato, with a particular focus on anthocyanin biosynthesis. Gene targeting genes involving anthocyanins biosynthesis has implied a double advantage, represented by either a technical visual screen to facilitate the selection of targeting events, or the nutritional improvement of tomato fruits as anthocyanins carry benefts for human health. In their study, Čermák and co-workers [17] used TALENs and CRISPR/ Cas9 systems to target *SlANT1* gene and introduce the 35S promoter by homologous recombination (HR) for inducing gene over-expression. In order to deliver suffcient donor template, geminivirus-based DNA replicons were generated to achieve high frequency, targeted modifcation in tomato plants. The obtained mutants showed dark purple coloration in fowers, fruit and foliage resulting from the expected targeted promoter insertion and from the boosted anthocyanin biosynthesis [17]. By using a multi-replicon system equipped with CRISPR/LbCpf1, Van Vu and collaborators [21] also applied a HR-base genome editing of tomato *ANT1* gene, obtaining purple plants and fruits enriched in anthocyanins. In another study, the tomato *DFR* gene was chosen for a targeted transgene insertion using CRISPR/ Cas9 in a two-step strategy. The frst step consisted of a targeted deletion within the endogenous *DFR* gene, resulting in green plantlets (instead of the usual purple color), and the second step consisting of an HR-mediated insertion in the *dfr* deletion to restore the original gene function. Following such insertion, the regenerating plantlets recovered a purple color phenotype, indicating that *DFR* function was regained [28].

# **5 Genome Editing for the Improvement in Vitamins and Other Phytonutrients in Tomato Fruits**

Highly effcient CRISPR/Cas9 systems have been also employed for metabolic engineering of other important micronutrients in tomato. One example is represented by the *7-dehydrocholesterol reductase* (*7DR2)* gene targeting that allowed the increase the level of provitamin D3 in tomato. Leaves and fruits – especially the green ones – from the homozygous mutant lines showed increased levels of 7-dehydrocholesterol (7DHC), and reduced levels of α-tomatine. 7-DHC content was lower in ripe fruits but high enough compared to wild-type tomato fruits, and suffcient to be equivalent to that in two medium-sized eggs, if converted to vitamin

D3 by UVB treatment [20]. Genome editing can therefore support food nutritional security and help to develop novel foods enriched in vitamins to be easily consumed and directly address vitamin defciency in susceptible individuals, as in this case of tomato fruits representing a good vitamin D source.

Another example in which genome editing can implement food nutritional quality is represented by the enhancement of soluble sugar content in tomato by targeting the *invertase inhibitor* and *vacuole processing enzyme* (*SlINVINH1* and *SlVPE5*) genes. By knocking down these two genes implicated in inhibition of soluble sugar accumulation, the glucose, fructose and total soluble solid contents were improved compared to wild-type tomato fruits, with a synergistic effect when the two genes were simultaneously knock-out and providing a key strategy to improve the sweetness in tomato fruits and related byproducts [29].

Finally, another important example of genome editing for phytonutrient improvement is the CRISPR/Cas9 application to increase GABA content in tomato fruits. By targeting fve genes (*GABA-TP1, GABA-TP2, GABA-TP3, CAT9, SSADH: gamma-aminobutyrate transaminase subunit precursor isozyme 1, 2* and *3*, *cationic amino acid transporter 9*, *succinate semialdehyde dehydrogenase*, respectively), several editing events were obtained in different tomato mutants, displaying signifcant increases in GABA accumulation, especially in the leaves, and different glutamate contents signifcantly variable among the GABA mutants [30]. However, since GABA represent a signal molecule involved in various physiological processes, the GABA mutants presented vegetative growth and fower/fruit setting severely affected [30]. Similar results, including suppression of plant growth, fowering and fruit yield, were obtained by CRISPR/Cas9-mediated gene targeting of autoinhibitory domains in *SlGAS2* and *SlGAD3* genes [31]. By introducing a stop codon before such autoinhibitory domains, the GABA accumulation was dramatically increased in tomato fruits, with variable effects on the mutated plants [31].

# **6 Conclusions and Future Perspectives**

The use of genome editing technologies offers an effective strategy to ameliorate agronomic and quality fruit traits bypassing the conventional breeding programs and transgenic approaches causing some ethical problems in the public acceptance [32]. Transgene-free genome editing provide a promising alternative to rapidly innovate germplasm and possibly introduce new traits for agrobiodiversity. The above presented studies in the tomato model represent examples of how different genome editing technologies can be easily applied for the improvement of carotenoids, polyphenols, and other important phytonutrients with valuable benefts for human health. Even though many progresses have been achieved in comparison to other crops (for example those recalcitrant to plant transformation), more efforts are still required to effciently target other genes involved in tomato fruit quality and nutritional value, and to overcome the barrier of gene activation/overexpression, system deliveries, and high-throughput screening of the novel mutant lines.

# **References**


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# **Chapter 23 Breeding for Yield Quality Parameters and Abiotic Stress in Tomato Using Genome Editing**

**Pedro García-Caparrós**

**Abstract** Growing tomatoes is an important aspect of agriculture around the world because of the positive effects it has on people's health and the economy. Tomato breeders and growers have always been inspired by the market's insatiable desire for high-yielding and high-quality tomatoes. Crop production, yield, and quality are all negatively affected by abiotic stress, which includes factors like drought, salinity, heat, and cold. As climate change alters weather patterns throughout the world, farmers around the world are increasingly worried about the effects of abiotic stress on their tomato crops. The CRISPR/Cas9 gene-editing tool has attracted attention as an alternative for solving the need for high-yield and superior-quality tomatoes, as well as for managing abiotic stress in tomato plants. This method of gene editing offers new possibilities for the development of stress-tolerant tomato varieties. The present book chapter provides a comprehensive review of the current knowledge on CRISPR/Cas9 and its potential implications in tomato agriculture, with a particular emphasis on enhancing yield quality and conferring resistance to abiotic stresses. The CRISPR/Cas9 technology has the potential to enhance the taste, appearance, and nutritional value of tomatoes by accurately altering the genes responsible for avor, color, aroma, and nutrition. The previously mentioned condition could end up in the cultivation of tomatoes that exhibit heightened levels of sweetness, as well as elevated concentrations of crucial vitamins, minerals, and antioxidants. The application of CRISPR/Cas9-mediated modications has the possibility to augment the plant's capacity to endure abiotic stress conditions through the introduction of genes implicated in different pathways that contribute to enhanced resilience to such challenging surroundings. In conclusion, the use of CRISPR/Cas9 offers an intriguing chance for improving tomato farming through the enhancement of crop quality and yield, as well as the strengthening of tomato plants against adverse abiotic conditions.

P. García-Caparrós (\*)

Agronomy Department of Superior School Engineering, University of Almeria, Almeria, Spain

# **1 Introduction**

The fruit of the tomato plant (*Solanum lycopersicum* Mill.), which is a multicarpellar berry, has emerged as the model species for feshy fruits as a consequence of specifc advantages for use in agronomic research along with basic research [1].

The tomato plant is a diploid organism that comprises 12 chromosomes and has a genome approximately 950 Mb in size. Because of the outstanding nutritional value of the plant and the plethora of benefcial industrial products capable of being derived from it, it is one of the most signifcant horticultural crops grown worldwide [2]. It is forecasted that the global tomato production, which was 41.52 million tons in 2020, will expand to 51.93 million tons in 2026 based on the universal scenario of tomato production [3].

Despite the fact that tomatoes have the potential to provide signifcant nutrition and income opportunities, it is crucial to address all possibilities associated with the application of genetic engineering to assure the feeding of the worldwide population as well as tomato growers' benefts. Consequently, CRISPR/Cas9 is a robust genome editing tool that enables researchers to make targeted and precise changes to DNA sequences in a relatively short period of time when compared to conventional breeding techniques.

The CRISPR/Cas9 system is a canonical genome editing system that consists of a shortened synthetic guide RNA (gRNA) sequence of 20 nucleotides that generates a complex with the Cas9 nuclease and guides it to a specifc DNA sequence, in which it inserts a double-stranded break (DSB) [4]. Targeted genome editing (GE) technologies, particularly clustered regularly interspaced short palindromic repeats (CRISPR)/(CRISPR-associated protein 9) (Cas9), have the ability to yield elite cultivars with higher resistance to biotic and abiotic stress [5].

CRISPR/Cas9 is also exceptional due to its versatility and adaptability, which enables more precise editing of an organism's own genes, making it a more natural and effective method for achieving desired characteristics [6]. Moreover, the simplicity and low cost of this technique compared to cumbersome and less precise conventional engineering techniques based on the introduction of foreign genes into an organism's genome aiming to achieve desired traits led us to compile in this book chapter the most recent studies based on the application of this system (CRISPR-Cas9) in tomato for the enhancement of yield quality and abiotic stress resistance.

# **2 CRISPR/Cas9 Gene Editing: Unlocking the Potential to Boost Tomato Yield and Quality**

There are several external and internal factors to consider when assessing the standard quality of tomatoes. The output, quality, marketability, plant health, and timing of tomato production are all infuenced by the plant's ability to set and mature fruit. Tomatoes' yield and quality, as well as their marketing success, are directly tied to

**Fig. 23.1** Schematic overview about the research topic of the book chapter

how well these phases are handled [7]. The fruit size, color, and texture or frmness of fruit (external factors) have also an important impact in consumer preferences, as consumers prefer fruits those are visually appealing as well as having uniform color, size, and texture. Nonetheless, internal fruit quality parameters, such as taste, sweetness, nutraceutical properties, and the presence of bioactive compounds (lycopenes and carotenoids), are gaining importance among consumers [8]. A schematic overview of the different points explained appears in Fig. 23.1.

# *2.1 Fruit Set and Ripening Period*

In tomato plants, the fruit set process is essential to ensure an adequate yield. Nevertheless, this physiological stage is widely affected by various factors including genetics, environment, and plant hormones [9]. A high fruit set results in a larger number of marketable tomatoes, which increases tomato growers' production and profts. In the tomato industry, the maturing period also infuences the timing of harvesting and logistics.

For instance, Guo et al. [10] worked with CRISPR-Cas9-mediated SlCMT4 mutants aiming to improve the fruit set in tomato. The gene SlCMT4 (*Solanum lycopersicum* CAFFEIC ACID O-METHYLTRANSFERASE 4) is involved in the biosynthesis of lignin and also participate in the fruit set process. The loss of function of mutated plants resulted in defective stamens and pistils, small fruit size with reduced setting rate, demonstrating the crucial role of this gene in reproductive organs in tomato.

Tomato ripening is indeed a carefully orchestrated process of growth that aligns with seed maturation, and its modifcation through genetic engineering is of great importance because it affects fruit quality attributes such as taste and texture, nutritional value, and shelf life [11]. Using CRISPR/Cas9, Gao et al. [12] overexpressed the SlNAM1 locus in tomato plants. This gene governs the ethylene generation and fruit maturing processes in tomato plants. The overexpression of SlNAM1 did result in an earlier onset of fruit maturation, as well as quicker softening and color changes, in comparison with control plants.

SlNAC9 is a NAC transcription factor implicated in several physiological processes as well as in pigment metabolism in tomato fruit. The loss of function in SlNAC9 tomato mutants generated by genetic engineering (CRISPR/Cas9) resulted in exhibited delayed fruit ripening and reduced accumulation of carotenoids, which are important pigments that contribute to the color and nutritional quality of tomato fruit [13]. Similarly, Gao et al. [14] investigated the role of the tomato SlNAC4 (NAC transcription factor) gene in fruit maturation. Using the clustered regularly interspaced short palindromic repeats genomic targeting system (CRISPR/Cas9), overexpressed tomato mutant plants displayed differences in fruit texture, deferring the onset of softness, in contrast to the wild type or the control treatment.

The RIN gene, which encodes a MADS-box transcription factor, is activated late in tomato fruit development and boosts the transcription of genes that produce the ripening hormone ethylene [15]. Due to the potential impact of RIN gene mutations on tomato fruit ripening, Ito et al. [16] used CRISPR/Cas9 to generate RIN gene knockout mutants in tomato, and reported that the mutant fruits ripened more slowly and generated less ethylene than the control treatment. Similarly, using CRISPR/ Cas9 technology, Jung et al. [17] created mutant tomato plants with LeMADS-RIN knocked out. The mutant fruits matured later than wild-type fruits and produced less ethylene. The mutant fruits also had decreased gene expression associated with ethylene synthesis.

Both FUL1 and FUL2 are considered to be homologous evolutionarily conserved in tomatoes. Both of these proteins are members of the MADS-box transcription factor family. They perform a crucial role in the management of the ripening of the fruit as well as the development of the fowers in tomato plants. The creation of double knockout mutants for these two homologous proteins using CRISPR/Cas9 slowed down the ripening of the fruit by inhibiting ethylene synthesis as well as decreasing the accumulation of carotenoid [18].

Lin et al. [19] evaluated the role of SlMIR164A (a microRNA gene involved in regulating fruit ripening and quality by infuencing the expression of two target genes, SlNAM2 and SlNAM3) in tomatoes. Researchers discovered that by silencing this particular microRNA gene in tomato plants, they were capable of accelerating fruit ripening and enhance fruit nutritional quality by boosting the content of soluble sugars and acids.

# *2.2 Fruit Size*

Fruit size is an important quality parameter for tomatoes, as it can impact factors such as yield, appearance, and taste [20]. It is also an important trait for tomato growers since determines the proftability of their crops. The packing and presentation of tomatoes are also impacted by their size. Breeding programs in tomato are focused on this aspect and in the literature, there are many references based on the improvement of this parameter [21, 22]. The methodology of CRISPR/Cas9 has been widely used in the improvement of this parameter in tomato fruit as it showed in the different references explained. For instance, Cui et al. [23] investigated the role of the tomato CONSTANS-LIKE 1 (SlCOL1) protein in regulating fruit yield in tomato plants. CONSTANS-LIKE proteins are transcription factors involved in the regulation of the fowering time and reproductive development of plants. The loss of function or the overexpression of the gene studied showed a high positive correlation with the fruit development and size.

The GLOBE gene is a major gene responsible of the shape of the fruit in tomato plants. The GLOBE gene is located on chromosome 2 in the tomato genome and encodes a transcription factor that regulates cell division and expansion during fruit development [24]. The loss of function of Solyc12g006860 generated via CRISPR/ Cas9 showed that other fruit shape attributes were negatively affected while fruit size showed a positive answer [25].

BZR1-like transcription factor, BZR1.7 is a determinant trans-acting factor of the SUN gene promoter responsible of the tomato fruit elongation. Yu et al. [26] investigated the role of this TF in tomato plants generating overexpressing lines via CRISPR/Cas9 and they noted that in edited plants with overexpression of BZR1.7, tomato fruits were longer compared to control plants.

# *2.3 Fruit Color*

The color of tomato fruit is an important quality attribute that affects consumer preference and marketability. Tomatoes are available in a range of colors including red, orange, yellow, green, and purple, and each color has unique favor and nutritional characteristics [27]. For instance, Yang et al. [28] targeted three fruit colorrelated genes (PSY1, MYB12, and SGR1) to develop tomato genotypes via CRISPR/Cas9 with different fruit colors ranging from light green, brown and also pink-brown. The fndings reported by these researchers is of interest to the tomato industry since they provide a strategy to obtain transgene-free plants with different colored fruits in less than 1 year.

# *2.4 Fruit Firmness*

Fruit frmness is an important parameter for determining the quality and shelf life of tomato fruits. The frmness of the fruit is dependent on the chemical composition of the cell wall. In tomato fruits there are three types of polysacharide involved: cellulose, hemicellulose being the most abundant xyloglucan; and pectins [29]. Consumers prefer tomatoes with a frm and crisp texture because they perceive them to be more appealing and fresher. Tomatoes with a frm texture provide a pleasant and satisfying eating experience. Conversely, tomatoes with low frmness may be perceived as overripe, watery, or inferior.

In order to comprehend and exert some level of control over fruit tomato softening, it is imperative that the transcription factors (TFs) that regulate the process be identifed and characterized. In this sense, by using CRISPR/Cas9 to silence the NOR-like1 (NAC Transcription factor) (Solyc07g063420) gene in tomato plants, Gao et al. [30] were able to create fruit with decreased ethylene generation, increased frmness, and decreased lycopene and other ripening-related compound levels. The result was a two-week delay in ripening compared to the control treatment.

In their study, Nie et al. [31] generated two mutants with over and down expression of CR-SlNAC4 (transcription factor involved in the softening of tomato) via CRISPR/Cas9. In both mutants, there was a strong effect of tomato fruit softening compared to the control fruit. The study also showed that SlNAC4 regulates the expression of several genes involved in fruit ripening and softening, including pectinases and cell wall-modifying enzymes.

Wang et al. [32] noted that SlXTH5, a member of the xyloglucan endotransglucosylase/hydrolase (XTH) gene family, plays a critical role in tomato fruit softening since xyloglucan is the most dominant hemicellulose in tomato fruits. Knockout mutants of SlXTH5 generated via CRISPR/Cas9 reduced fruit frmness and color index compared to control fruits.

SlBES1 transcription factor plays a crucial role in regulating tomato fruit softening. In this sense, Liu et al. [33] reported that knockout mutants of SlBES1 had frmer fruits with reduced pectin degradation compared to wild-type plants. Moreover, the increase in frmness in tomato fruits also improved the shelf life in the postharvest.

# *2.5 Fruit Taste and Sweetness*

Taste is an important feature of tomato fruits and is linked to a complex interplay of factors such as sugar content, acidity, aroma, texture, and other chemical compounds. The taste of tomatoes is crucial in the consumer preference and purchase decisions, and therefore should be of high relevance in tomato breeding programs. Nevertheless, the improvement of this feature has been replaced by other factors such as fruit ripening, fruit size, etc. [34].

FLORAL4 gene is responsible for the production of several important foral aroma volatiles such as 2-phenylethanol, phenylacetaldehyde and 1-nitro-2 phenylethane in tomato fruit. The study conducted by Tikunov et al. [35] reported that generating mutants of FLORAL4 gene via CRISPR/Cas9 the foral aroma and overall favor of commercial tomato fruit was severely affected.

Brix is a measure of fruit's sweetness and in the case of tomato is dependent of several factors such as cultivar, growing conditions or the stage of ripeness at which the tomato is harvested [36]. This important trait in tomato fruit quality has been improved via CRISPR/Cas9 in different experiments. For instance, Nguyen et al. [37] investigated the effect of targeted mutations via dual-gRNAs CRISPR/Cas9 system in uORF regions of the SlbZIP1, a gene involved in the sucrose-induced repression of translation (SIRT) mechanism. Tomato plants with disrupted bZIP1 uORFs had signifcantly higher levels of sugars and amino acids in their fruits compared to control plants. Moreover, these changes in metabolic pathways were associated with changes in the expression of several other genes involved in sugar and amino acid metabolism. Regarding the enhancement of the sweetness in tomato fruit, there are other references about CRISPR/Cas9 system design such as modifcation of SlINVINH1, a cell-wall invertase inhibitor resulting in tomatoes with a high sugar content without a reduction in fruit weight or plant growth [38] or the loss of function in SlVPE5, a vacuolar processing enzyme involved in sugar accumulation, in which tomato mutant lines increased soluble sugar content in fruit [39].

The brachytic phenotype (BR) in tomato is characterized by a reduction in plant height and increased compactness, which can be benefcial for certain agricultural applications. Lee et al. [40] found that tomato plants carrying the mutant FPF1 allele (Solyc01g066980) had a signifcant reduction in plant height although they produced fruits with better quality parameters such as higher size, increase in total soluble solids (°Brix) favoring them the commerciality of the tomato fruit.

# *2.6 Bioactive Compounds in Fruits*

Lycopene is a natural pigment responsible for the characteristic red color of tomatoes and is also a power antioxidant with many benefts for human health, therefore tomato breeding programs are currently also focused on the enhancement of the synthesis of this pigment [41]. For instance, in one study, Li et al. [42] used CRISPR/ Cas9 gene editing technology to simultaneously target fve genes involved in the carotenoid metabolic pathway in tomato plants. By introducing mutations in these genes, the researchers were able to increase the levels of lycopene in tomato fruit about fvefold.

# **3 Empowering Tomato Crop Resilience: CRISPR/Cas9 for Abiotic Stress Adaptation**

Abiotic stresses are complex in their nature and controlled by networks of different factors (e.g., genetic and environmental) that impede crop plant breeding strategies, therefore the use of the most novel genes editing technologies emphasizes the importance of this task [43].

The incidence of crop losses resulting from adverse environmental conditions has steadily increased over recent decades, attributable to both climate change and human activities. The intricate nature of abiotic stresses is governed by a multitude of factors, including genetic and environmental infuences, which pose signifcant challenges to crop plant breeding strategies. As a result, the utilization of cuttingedge gene editing technologies is of the utmost signifcance. The implementation of CRISPR/Cas9 technologies is expected to facilitate plant genetic transformation, thereby improving resistance to various abiotic stresses [4].

In this section, we shall focus on the most recent publications of genome edited tomato generated via CRISPR/Cas9 with improved resistance to the main following abiotic stresses: salinity, drought, heat and chilling conditions. A schematic overview of the different points explained appears in Fig. 23.1.

# *3.1 Salt Stress*

Salinity is considered to be a signifcant abiotic stressor for plants, causing considerable harm [44]. According to a recent publication by Hassani et al. [45], the estimated area of salt-affected soils in Europe is 24 Mha, which accounts for approximately 2.05% of the total worldwide salt-affected area of 1171.8 Mha. In light of these numbers, it is clear that developing via genetic engineering salttolerant varieties of most crops, and notably tomatoes, which are only moderately robust to salt stress, is a priority. The development of these salt tolerant cultivars allows the cultivation of tomato in soils moderately saline and also reduces the damages caused by salt stress in this crop. In this sense, we will be focusing on the most recent research that were carried out in tomato using the CRISPR/Cas9 system. For instance, Ding et al. [46] identifed an uncharacterized gene named SlABIG1 from tomato, which they identifed to be essential for salt tolerance and to be a component of the HD-ZIP II subfamily. The CRISPR/Cas9-generated knockout mutant exhibited higher salt tolerance, enhanced photosynthetic effciency, reduced ROS generation, as well as improved Na+ exclusion effciency. On the other hand, the study conducted by Tran et al. [47] attempted to boost tomato plants' resistance to salt. Here, they used CRISPR/Cas9 to generate SlHyPRP1 (hydrid proline-rich protein 1 gene) knockout mutants to investigate the effect of salt tolerance. The data showed that the modifed plants were more resistant to salt, with higher rates of survival and enhanced growth throughout the face of salt stress.

# *3.2 Drought Stress*

The impact of drought stress on crop growth, development, and productivity is a noteworthy environmental factor. It occurs when there is an inadequate supply of water to meet the demands of crops, leading to soil moisture defcit and plant water stress. Comprehending the impact of drought stress in crops and employing genetic engineering to develop more resilient cultivars could aid in alleviating its effects and ensuring sustainable crop production amidst the altering climate patterns [48]. The susceptibility of tomatoes to water defcit is attributed to their high-water requirements and shallow root systems. Consequently, the development of modifed genetically cultivars with improved drought resistance is imperative at present [49]. The development of these drought resistant cultivars is positive for the growers of tomato since they must invest in supplementary irrigation systems or water resources to combat water scarcity. Numerous recent examples in the literature demonstrate the generation of cultivars with increased tolerance to drought stress through the utilization of CRISPR/Cas9 technology. For instance, the drought tolerance of tomato plants cv. Alisa Craig was evaluated by Li et al. [50] using CRISPR/Cas9-mediated mutation of the SlNPR1 gene. This gene mutation reduces plants' resistance to water stress in different aspects such as increased stomatal conductance, generation of lipid peroxides and the higher susceptibility to oxidative damage linked to the lower levels of antioxidant enzymes. Similarly, Using CRISPR/Cas9, Chen et al. [51] created SlARF4 knockout mutants to investigate the physiological responses to drought stress in tomato plants in comparison to wild-type plants. The results indicate an improvement in the growth and water status of mutant tomato plants. In addition, arf4 mutants exhibited increased photosynthetic effciency, decreased oxidative damage, increased antioxidant enzyme activity, and the upregulation of numerous stress-responsive genes.

Tomato's sensitivity to drought was examined by Liu et al. [52] who hypothesized that SlLBD40, a lateral organ boundaries domain transcription factor, was responsible for it. Using CRISPR/Cas9, they established mutant lines of tomato with a loss of function of the SlLBD40 gene, and those plants showed enhanced resistance to drought compared to the wild type. The mutants were more capable to deal with drought conditions, as seen through their delayed withering, reduced water loss rate, and higher survival rates.

Wang et al. [53] created two independent T1 mutant lines with the loss of function of the gene SlMAPK3 using CRISPR/Cas9 in order to compare their drought tolerance to that of the wild type. This study emphasizes the importance of MAPKs (mitogen-activated protein kinases) as crucial signaling components in plant responses to multiple stresses, including drought stress. The biochemical analysis revealed that the knock-out mutants displayed earlier wilting, increased H2O2 production, and decreased scavenging of reactive oxygen species, resulting in increased membrane damage.

# *3.3 Heat and Cold Conditions*

Environmental factors such as heat and cold stress can have a signifcant impact on crop growth, development, and yield [54]. The ongoing climatic changes have resulted in frequent episodes of extreme heat or cold waves which dramatically affect the yields of crops, sometimes leading to their death. As a result, genetic engineering solutions are increasingly being recognized as appropriate way of dealing with this issue. Regarding CRISPR/Cas9 in tomato to counteract these abiotic stresses it can be highlighted in the recent studies. In the case of heat stress, Wang et al. [55] generated overexpressed mutants for the gene SlGRXS1 by activating the transcription factor SlWRKY3, which confers thermotolerance to tomato. Mutant tomato plants exhibited more ROS scavenging and less oxidative damage relative to their wild type counterparts. This research has confrmed that SlGRXS1 and SlWRKY3 act together to enhance the stress response network in tomato plants when they are subjected to high temperatures.

Due to its osmoprotectant properties, the capacity to maintain the cellular water status, the reduction in the synthesis of reactive oxygen species (ROS), and the regulation of gene expression, sucrose can play a crucial role in heat-stressed plants [56]. Zhang et al. [57] opted to investigate Sucrose phosphate synthase (SPS), a key rate-limiting enzyme in the sucrose synthesis pathway in crops. The researchers reported that the gain or loss of function of a novel gene (SlSPS) conferred increased or decreased thermotolerance to tomato plants, making them more or less susceptible to heat stress.

To suppress SlMAPK3 expression in tomato plants, Yu et al. [58] adopted CRISPR/Cas9 gene editing technology. The wild-type and mutant tomato plants were then subjected to heat stress. The thermotolerance of the edited plants compared to WT was confrmed by their survival in temperatures ranging from 25 to 45°. Under heat stress circumstances, the mutant plants survived longer and developed more successfully while minimizing the synthesis of potential reactive oxygen species (ROS).

The purpose of the study by Huang et al. [59] was to identify the function of the BAG9 gene in tomato heat tolerance. Tomato mutants (bag9) were able to withstand higher temperatures after overexpressing this gene using CRISPR/Cas9. Increased antioxidant enzyme activity and heat sock protein synthesis also resulted from this shift in higher gene expression in tomato plants.

As far as chilling conditions is concerned, Li et al. [60] generated CRISPR/Cas9 mutant tomato plants for the SlCBF1 gene aiming to develop a greater awareness of the tomato's chilling tolerance. The authors emphasize the important role of the CBF (C-repeat binding factor) genes in regulating plant responses to chilling stress, focusing in particular on the SlCBF1 gene, which is a known key regulator gene of chilling tolerance in tomatoes. The loss of function resulted in diminished physiological and biochemical responses of tomato plants to chilling stress, including altered levels of stress-related hormones such as jasmonic acid and abscisic acid.

Non-expresser of pathogenesis-related genes 1 (NPR1) is known for its role in crop resistance to diseases, but its function in cold-adapted tomato plants was investigated by Shu et al. [61]. It was found that CRISPR/Cas9-obtained tomato knockout mutants exhibited more cold stress tolerance than their wild-type counterparts, as evidenced by lower oxidative damage, higher antioxidant enzyme activity, and greater development of the plants.

# **4 Conclusion and Future Perspectives**

This manuscript presents intriguing future prospects for the application of CRISPR/ Cas9 technology to enhance tomato quality and abiotic stress resistance. Further investigation of the molecular mechanisms and pathways involved in tomato quality and abiotic stress response could yield important insights. Using functional genomics and systems biology approaches, such as transcriptomics, proteomics, and metabolomics, could reveal the complex molecular networks and regulatory mechanisms governing these traits, leading to a greater understanding of how CRISPRmediated modifcations affect tomato quality and stress tolerance. Future research might explore the concurrent editing of multiple genes aiming to improve tomato traits in a more comprehensive way. Combining genetic modifcations for enhanced favor, increased nutritional value, and enhanced stress tolerance, for instance, could result in the creation of tomatoes with superior overall quality and resilience.

# **References**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **Chapter 24 Genome Editing-Based Strategies Used to Enhance Crop Resistance to Parasitic Weeds**

# **Kubilay Yıldırım, Musa Kavas, Melekşen Akın, and İlkay Sevgen Küçük**

**Abstract** In contrast to most autotrophic plants, which produce carbohydrates from carbon dioxide using photosynthesis, parasitic weed plants rely on host plants to form vascular connections through which they withdraw the required nutritive resources and water. Many important crop plants are infested by these heterotrophic plants leading to tremendous yield losses and rendering agricultural lands uncultivable. The parasitic weeds are physically attached to the host plants and therefore their control is challenging due to the lack of selective methods for killing the weeds without damaging the host crop. Fortunately, many host plants have pre-haustorium resistance, host initiation responses and post-attachment tolerance to these parasitic weeds. However, parasitic weeds have high fecundity, dispersal efciency, and persistent seed storage in the soil all of which enable them to adapt to new hosts and break down the crop resistance. Recent discoveries in genome editing and gene silencing-based technologies open new opportunities to enhance crop resistance to parasitic weeds. Some genome editing-based studies targeting the seed germination of parasitic weeds created almost complete resistance in crop species. In this chapter, we give an overview of the host-parasitic interaction and host defence responses that can be targeted by genome editing or gene silencing technologies.

**Keywords** Parasitic weeds · Host · Defence response · Genome editing · CRISPR · RNAi

K. Yıldırım (\*) · İ. S. Küçük

Department of Molecular Biology and Genetics, Ondokuz Mayıs University, Samsun, Türkiye e-mail: kubilay.yildirim@omu.edu.tr

M. Kavas Department of Agricultural Biotechnology, Ondokuz Mayıs University, Samsun, Türkiye

M. Akın Department of Horticulture, Iğdır University, Igdır, Türkiye

# **1 Introduction**

Plants are autotrophic organisms using light as energy to convert inorganic carbon into carbohydrates by photosynthesis. However, some plants have evolved specialized organs (haustorium) to attach and form vascular connections with autotrophic plants to absorb their water and nutrients. This heterotrophic lifestyle is used by parasitic plants/weeds and has a profound negative impact on many agriculturally important crops, forests and whole dynamics of ecological systems [1]. Parasitic plants could be grouped as facultative or obligatory according to their dependency on the host. Facultative parasitic plants (hemiparasitic) have their own chlorophyll and can complete their life cycle independently of a host. However, if there is an available host plant to obtain nutrients and water with less investment in the assimilation system, they become parasitic. Obligate parasitic plants (holoparasites) lack chlorophyll and they depend completely on their hosts for seed germination and survival. Parasitic plants can also be separated as root feeders or shoot feeders based on the invaded host tissue. Depending on their vascular connections with their host, they could be xylem feeders, phloem feeders, or both [2].

Parasitic plants in lower diversifed agricultural systems can cause tremendous yield losses rendering agricultural lands uncultivable [3]. Traditional control methods such as hand weeding and herbicide treatment are too expensive and labourintensive to regulate parasitic plant infestations in crops. These methods also are ineffective due to the tight physiological connection between the host and the parasitic weed and the re-emergence of parasitic plants after damaging of the host. Parasitic plants generally produce plentiful small seeds contaminating the soil or the crop seeds before parasitism is established. The seeds of parasitic plants remain viable in the dormant state for many years and germinate after receiving the host signals [4].

Reducing the impact and spread of parasitic weeds on crops and agricultural production requires an understanding of the molecular machinery behind the interactions between the parasite and the host plants. Pre and post-attachment as well as haustorium initiation resistance mechanisms in specifc cultivars, mutants, or species have been identifed and many host metabolites required for the germination of parasitic weed seeds have been identifed. The availability of whole-genome sequences and transcriptomes of several parasitic plants facilitated the investigation of genes responsible for host–parasite interactions, and the identifcation of the genes involved in resistance or susceptibility responses of crops [5–7]. All this knowledge can be used to enhance resistance in crop species to these weeds by deploying molecular breeding and advanced genome editing strategies. In this chapter, we provide a comprehensive overview of new genome editing or gene silencingbased approaches applied to crops to enhance parasitic weed resistance and their prospective applications on the molecular mechanisms involved in host-parasitic weed interaction.

# **2 Genome Editing-Based Strategies Used to Enhance Parasitic Weeds Resistance in Crops**

Many agriculturally important plants are attacked by specifc parasitic plants, which induces a host defence response to inhibit the attachment of parasitic weeds or reduce the infestation. Based on whether the resistance mechanism functions before or after parasitic plants attach to their hosts, resistance responses can be classifed as pre-haustorium resistance, haustorium initiation resistance or post-attachment resistance [8]. In addition to the classical transgenic approach, newly discovered biotechnological strategies (RNAi, VİGS and CRISPR) have been implemented to develop a high level of crop resistance to parasitic weeds in recent years [9–11]. In the review, we grouped these studies according to their target resistance mechanisms.

# *2.1 Genome Editing for Pre-HAUSTORIUM Resistance in Crops*

The discovery of some terpenoid lactones in crops such as strigolactones (SLs) and sesquiterpene lactones (STLs), [11, 12] is a milestone in understanding the interaction between parasitic weeds and their hosts. Secondary metabolites synthesized by host roots in trace amounts have several important physiological processes in host plants from shoot branching to arbuscular mycorrhizal symbiosis. Terpenoid lactones were then realized to be also the germination stimulants for several obligate root parasitic plants [13, 14]. The seeds of these parasitic plants do not germinate unless they receive terpenoid lactones as a chemical signal from their host roots. Therefore, the parasite-host interaction has evolved in a sophisticated way to detect the presence of STLs or SLs by parasitic weeds and coordinate their germination and development with the host's lifecycle [15, 16]. Receiving the signal molecule from the host for seed germination and growth towards the host organs are critical steps in the parasitic plant life cycle. Resistant host plants take a preventive preattachment strategy by making themselves invisible to parasitic plants by decreasing or completely stopping the production of germination stimulant molecules [17]. Therefore, reducing the number of stimulants exuded by host plants is considered to be a key factor for the host resistance achieved by inhibition of parasitic weed seed germination. Clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9)-mediated mutagenesis, virus-induced gene silencing (VIGS) and RNA interference (RNAi) mediated gene silencing strategies have been used to disrupt strigolactones (SLs) biosynthesis in host plants [18–23]. In this way, the germination of seeds of parasitic plants was suppressed and almost complete resistance to parasitic weeds was achieved in genome-edited host plants (Table 24.1).


**Table 24.1** Crops and parasitic plants modifed by genome editing mediated strategies to enhance host resistance to parasitic weeds

SLs were frst isolated in cotton root exudates as a germination stimulant of *Striga lutea* [24]. Subsequent research revealed that these compounds also function as endogenous hormones to inhibit shoot branching or tillering. SL biosynthesis begins with the convertion of all-trans-β-carotene to 9-cis-β-carotene with an enzyme called β-carotene isomerase (DWARF27 or D27). Then, carotenoid cleavage dioxygenase 7 (CCD7) cleaves 9-cis-β-carotene into the volatile β-ionone and 9-cis-β-apo-10′-carotenal. This former intermediate is catalyzed by CCD8 to yield carlactone which is the precursor for all SLs (Fig. 24.1). In *Arabidopsis thaliana*, carlactone is converted into carlactonoic acid by the cytochrome P450 monooxygenase (MORE AXILLARY GROWTH 1-MAX1) (Fig. 24.1a). In rice, MAX1 homologs convert carlactone into 4-deoxyorobanchol and orobanchol [25].

RNA interference (RNAi) uses an antisense siRNA strand to associate with the RNA-induced silencing complex (RISC) to target homologous RNA molecules for degradation and gene silencing in plants [26]. RNAi was previously used to silence several key genes encoding critical enzymes functional in SL biosynthesis. Gene silencing of CCD7 and CCD8 transcripts in tomatoes using antisense siRNA resulted in decreased levels of SL in the host, leading to reduced germination of the root parasitic weed [18, 19, 27].

Kohlen et al. (2012) showed that silencing of the host CCD8 gene in tomato lines by hpRNA technique reduces infestation of *P. ramosa* by 90% in the transgenic plants [19]. In another study, Aly et al. (2014) used a tobacco rattle virus–VIGS system for the transient knockdown of CCD7 or CCD8 in *P. aegyptiaca*. The result of the study demonstrated signifcant inhibition of parasite-tubercle development and the infestation of *Nicotiana benthamiana* plants [20]. A similar approach was used for the control of root parasitic weeds based on the simultaneous trans-specifc

**Fig. 24.1** Strigolactones (SLs) released from the host roots are the main stimulants for seed germination of parasitic weeds. Therefore the genes encoding the enzymes functional in SL biosynthesis (**a**) were the targets for CRISPR-mediated gene knockout studies. CRISPR-mediated disruption of the CC7, CCD8 and MAX1 genes in rice (**b**) and tomato (**c**, **d**) to reduce SL content in the root exudates. All the SL-defcient mutant plants exhibited reduced or poor germination in the seeds of parasitic plants such as *S. hermonthica, O. crenata* and *P. aegyptiaca*

gene silencing of parasite genes [29]. In this study, multiple DNA fragments (ACS, M6PR, and Prx1) of *P. aegyptiaca* genes were targeted by RNAi. The results of the experiment showed the movement of mobile exogenous siRNA from the host to the parasite, which lead to the decreased expression of parasitic genes essential for the parasite tubercles growing on the host plants.

CRISPR/Cas9 is the newest genome editing approach used to silence or modify the genes of plant species to enhance resistance to parasitic plants. This effcient and simple genome editing tool requires a small-guided RNA (sgRNA) complementary to a target gene sequence and Cas9 enzyme that recognize sgRNA for precise cutting of DNA and leading to dsDNA breaks [30]. During DNA repair by nonhomologous end joining, insertion or deletion may occur at the break sites, silencing the protein's function [31]. CRISPR/Cas9 has been recently applied to knock out the CCD7 gene in rice (*Oryza sativa*) to reduce SL content in the roots [21]. CCD7 mutants exhibited increased tillering, combined with reduced height and extremely poor levels of SL production compared to the wild-type control. Striga seed germination was almost completely inhibited by the root exudates of some CCD7 mutants compared to that of control and the standard SL analogue GR24 (Fig. 24.1b). In another study, CRISPR/Cas9-mediated mutagenesis of the CCD8 gene was used to enhance host resistance to the parasitic weed *P. aegyptiaca* [22]. In this study, Cas9/ single guide RNA constructs were targeted to the second exon of CCD8 in tomato plants. Several mutant tomato lines with heritable insertions or deletions in CCD8 gene were recorded to be SL-defcient. Compared to control tomato plants, the CCD8 mutant lines had morphological changes such as dwarfng, excessive shoot branching and adventitious root formation. In addition, some SL-defcient CCD8 mutants exhibited an almost complete reduction in seed germination of *P. aegyptiaca* and its infestation compared to non-mutated tomato plants (Fig. 24.1c) [22]. Wakabayashi et al. (2019) knocked out the cytochrome P450 (MAX1) gene, SlCYP722C, coding for an orobanchol synthase enzyme in tomato, by using a CRISPR system (Fig. 24.1d) [23]. Indels in the gene that resulted in biallelic frameshift mutations were identifed in the T1 transgenic plants and T2 progeny lines. Orobanchol production was completely inhibited in the root exudates of MAX1 mutant tomato plants. Unlike the CCD8 mutant tomato lines created by Bari et al. [22], MAX1 mutants did not show prominent phenotypes such as increased shoot branching and reduced stem length. Production of the fruits and seeds was normal in the T1 MAX1 mutant tomato lines normally, and no serious yield loss occurred in mutant T2 progeny. Most importantly, root exudates of MAX1 mutant tomato plants reduced the induction of germination of seeds of root parasitic weeds, *Striga hermonthica*, *Orobanche crenata*, and *Phelipanche aegyptiaca*, compared to WT without changing the plant architecture.

Secretion of toxic compounds inhibiting the seed germination of parasitic weeds is another strategy for host resistance against parasitic weeds. Many phytotoxins or natural amino acids were found to interfere with the early growth stages of the parasitic weeds. These metabolites have negative effects on seed germination or germ tube elongation [32]. Serghini et al. (2001) found that the resistant sunfower genotypes release defensive secondary metabolites called 7-hydroxylated coumarins from their root to create a toxic environment for *O. cernua* [33]. In another study, transgenic tobacco overexpressing an antibacterial peptide sarcotoxin IA enhanced resistance to *Phelipanche spp*. by its toxic effects on this parasitic weed [34].

#### **2.1.1 Genome Editing for HAUSTORIUM Initiation Resistance in Crops**

Once a germination signal is released from the host and detected by the parasitic plants, a haustorium contact is established between the host and the parasite. Therefore, instead of reducing the germination of parasitic seeds, inhibition of haustorium formation via several Haustorium Induction Factors (HIFs) could be also another strategy for crop resistance. HIFs are released from the parasitic plants to enable haustoria penetration into host organs following haustorium attachment [35]. A quinone molecule, 2,6-dimethoxy-1,4-benzoquinone (DMBQ), released from sorghum root extract was the frst defned HIF molecule in the parasitic plants. DMBQ was recorded to induce both obligate and facultative parasitic haustoria development within hours [36]. Interestingly, two genes (TvQR1 and TvQR2) encoding a type of quinone oxidoreductases in *Triphysaria versicolor* (facultative parasitic plants) were identifed to be responsible for the induction of DMBQ. TvQR1 was estimated to generate the frst step in the signal-transduction pathway for haustorium development while TvQR2 was thought to be responsible for the removal of the signal with a detoxifcation system [37]. RNA interference (RNAi) technology was used to silence TvQR1and TvQR2 transcripts in *Triphysaria* roots for the evaluation of their functional role in haustoria formation. In the study, RNAi vectors designed to target TvQR1 and TvQR2 were transformed into *Triphysaria* roots via *Agrobacterium rhizogenes.* The competence of transgenic *Triphysaria* roots was accomplished by *Arabidopsis* root contact test. The results of haustoria formation in response to host contact indicated a signifcant decrease in haustorium development in roots silenced for QR1 but not in roots silenced for QR2. This experiment implicates QR1 as the frst identifed gene necessary for the redox bioactivation of haustorial-inducing factors [38].

#### **2.1.2 Genome Editing to Enhance Post-attachment Resistance in Crops**

Even after the seeds of parasitic weeds germinate and attach to the host roots, hormone-mediated defence response in host plants can be triggered to cope with this parasite attack. Defence-related plant hormones, especially jasmonic acid (JA) and salicylic acid (SA), are known to contribute to crop resistance to parasitic weeds by direct inhibition of their contact with the host or enhancing the host plant vascular body. For instance, treatment of SA on red clover roots reduced the houstaria formation of *O. minor* by lignifcation in the host endodermis cell layers [39]. Induction of SA and pathogenesis-related gene transcripts were also reported to enhance the resistance response of sunfowers to *O. cumana* [40]. JA is known to be involved in cell wall damage–induced lignin biosynthesis and, therefore, it directly contributed to the host resistance by a hypersensitive-like response in plants [35]. Several studies have concentrated on the loss of function analysis of these hormones in crop species. For instance, Brading et al. (2000) created a transgenic tomato expressing salicylate hydroxylase. This enzyme converted SA immediately to

inactive catechol and created SA-defcient tomato [41]. In another study, the radiation-based mutation was created on the tomato CORONATINE-INSENSITIVE1 gene which reduced the expression of JA-responsive genes [42]. Runyon et al. (2010) used both mutant tomato genotypes to test their resistance to parasitic weeds. The results indicated that parasitic plants grown on the SA and JA mutant tomatoes were more aggressive and had more biomass than those grown on their wild-type counterparts [43].

In another study, RNAi was used to knock down (kd) the expression JA-inducible WRKY transcription factor in rice [44]. Remarkably, WRKY45-kd rice genotypes exhibited severe susceptibility to *S. hermonthica*. The size and number of the *S. hermonthica* seedlings that attached and developed in mutant rice genotypes were almost threefold higher compared with wild-type rice. Therefore, a reduction in endogenous JA levels resulted in enhanced susceptibility to *S. hermonthica.* External application of JA was found to completely recover the resistance ability of mutant rice to this parasitic plant [5, 45, 46].

Hypersensitive response (HR) is a common mechanism which leads to localized cell death and necrosis at the infectious site to defend against pathogens and prevent the spread of infection in the plant body [47]. Some studies indicated that hosts have evolved the ability to detect parasitic plant–specifc signals to initiate signal transduction cascades that lead to an HR and prevent the haustorium penetration process of parasitic plants [48]. For instance, a cowpea cultivar resistant to *S. gesnerioides* was found to trigger a downstream signalling cascade to activate the avirulence (Avr) proteins, which is a positive regulator of the HR [49]. A similar case was also reported for the interaction between sunfowers and *O. cumana*. Sunfower recognizes an avirulence protein (AVROR7) from *O. cumana* via the kinase domain of the HAOR7 protein, which then activates signalling cascades for the induction of HR [50].

# **3 Prospective Applications of Genome Editing-Based Systems for the Control of Parasitic Plants in Crops**

Genome editing-based strategies used to silence host or parasite genes may serve as an important strategy to obtain more effective and durable crop resistance to parasitic weeds. Unlike other types of natural resistance, genome editing-based strategies could be easily applied to susceptible crop cultivars. Moreover, parasite species share homology in the target gene sequence and, therefore, an established strategy could be effective against other parasitic weed species. For instance, Aly et al. (2009) revealed that M6PR gene has high sequence similarity between *P. aegyptiaca, P. ramosa* and *O. crenata* species, suggesting that a single RNAi or CRISPR-based protocol can be used to manipulate sensitivity to several species at the same time. In addition, multiple candidate parasitic genes can be cloned in the same construct and pyramided in susceptible hosts for gene editing, thus

signifcantly reducing the risk of the development of new virulent parasitic weeds [28]. A limited number of studies described in-vitro transformation and regeneration systems for *P. aegyptiaca* [51]. and *P. ramosa* [52]. However, the establishment of genome editing-based protocols needs more effort to acquire high-quality genomic data, reverse-genetics information and reliable parasite transformation systems to target key processes in the host–parasite interaction. Nevertheless, current molecular knowledge could still be a target for CRISPR-based genome editing studies. For instance, RNAi or CRISPR-based silencing of parasite genes functional in host cell-wall degradation and penetration (pectin methylesterase, polygalacturonase, rhamnogalacturonase or peroxidases) may reduce host penetrability during haustorium formation and initial parasitic stages. Another promising strategy could be the reduction of the parasite's seed productivity by silencing the genes involved in fower and seed formation pathways. A general conclusion emerging from research in the last 20 years is that the intimate physical and physiological connection of parasites with their hosts can be used as a key target point, where its greatest potential lies in developing parasite resistance utilizing molecule or macromolecule exchange. Since host-released stimulants such as hormones, seconder metabolites and signals are the most critical key factor in germination and infestation of parasitic weeds, genes involved in these stimulant biosynthesis communications, signalling, and perception should be further studied and identifed for the best targets for genome editing. A more thorough understanding of molecular interaction between host and parasite will enable manipulation of their in-vivo interactions and activity to control root parasitic weed germination without damaging the crop plant.

# **References**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **Part III Policies and Regulations**

# **Chapter 25 Genome Editing in Biotech Regulations Worldwide**

**Thorben Sprink and Ralf Wilhelm**

**Abstract** Since the introduction of genome editing techniques in breeding and the rst commercial products on the market, various governments or jurisdictions have attempted to clarify the legal classication of genome editing in relation to their genetic engineering regulations. Only a few countries, including Europe, fully apply their strict genetic engineering laws to genome-edited organisms or products derived from them. Most countries with liberal regulations base classication on the absence of foreign DNA in the nal product (including the USA and Canada, which de facto have no specic GMO laws). Countries such as Australia and Japan have introduced subcategories when sequence templates have been used in the genome editing process. Several countries, including Europe, are in the process of revising their GMO legislation. The international legislative landscape is thus dynamic. The heterogeneity of regulatory regimes poses a challenge for international trade. This chapter summarises the status as of June 2023 and provides a brief introduction to the main legal concepts.

# **1 Introduction**

The commercial use of genome editing technologies is closely linked to national legislation in the eld of genetic engineering and biotechnology, as well as to consumer acceptance. The rst genome editing techniques in plant breeding were developed before the beginning of this century, but it was not until the use of CRISPR-Cas systems that they became widely established in the range of applications. Since the technique represents an active intervention in the genome to insert a modication at a specic genomic site, the question also arose as to how the techniques should be classied under genetic engineering law. The rapid development of genome editing techniques poses a challenge to national regulations and

T. Sprink · R. Wilhelm (\*)

Julius Kühn-Institut (JKI), Federal Research Centre for Cultivated Plants, Institute for Biosafety in Plant Biotechnology, Quedlinburg, Germany e-mail: ralf.wilhelm@julius-kuehn.de


**Table 25.1** Genome-edited products/organisms released to national markets (until May 2023)

a The product pipeline and business model of Calyxt has changed. The product was marketed by Calyxt until 2021

international treaties worldwide, as most existing laws and regulations do not provide explicit reference to the techniques and their applications as such. To date few genome-edited products have entered the market in the North America (USA, Canada) and Japan (Table 25.1).

Current legislations or guidelines may distinguish between the genome editing techniques, SDN-1, SDN-2, SDN-3, respectively [1] and whether foreign DNA and/ or sequence templates have been used during generation of the genome-edited organism.

# **2 Countries/Regions with Strict Regulations**

Few countries/regions i.e. European Union (EU), New Zealand, South Africa, Venezuela, Peru and Costa Rica (Fig. 25.1) consider genome edited organisms (plant, animals, respectively; microorganisms maybe unclear) as GMO sensu stricto [7–14]. For these countries genome edited organisms bear broader challenges for law enforcement and compliance especially in relation to international trade. Though mutations can be detected with established molecular biotic methods the identifcation of the technique or the natural event that caused the mutation is bound to a priori information about the uniqueness of the event and the modifed genome sequence(s) [15, 16]. Such data are rarely readily available and hence the detection of unintended residues from (prohibited) genome-edited plants in internationally traded commodities becomes erratic. This problem of law enforcement cannot be solved by labelling regulations and it is also challenging liability and redress. Nevertheless, labelling of "GMO" and the provision of a specifc detection method prior to market release is mandatory e.g. in the EU regulatory framework. The EU as well as New Zealand are currently reviewing their respective GMO-regulations. The European Commission has published a legislative proposal in July 2023 that

**Fig. 25.1** Regulations on genome edited food around the world (updated [7]). The regulatory status of genome-edited (GEd) crops on a global schema

suggests regulatory relaxations for genome-edited and cisgenetic plants and starts the legislation process in the EU [17]. De facto or purposely interrelated, the EU initiated two further policy actions. There is the "Sustainable food system framework initiative" [18] as a framing regulation and the "Revision of the plant and forest reproductive material legislation" [19]. The latter is less acknowledged in the public discussions, but of considerable relevance for the breeding sector as well.

Several countries are developing a liberal handling of organisms derived with new genomic techniques (NGTs) based on the Cartagena Protocol [20] that defnes a living modifed organism as "any living organism that possesses a novel combination of genetic material obtained through the use of modern biotechnology", emphasizing the novelty of a (re)combined sequence. Nevertheless, different countries tend to deal with the novelty aspect considerably differently. This raises challenges for law enforcement, compliance and liability that result when the specifc genome editing method would need to be identifed from a DNA-sequence of a sample from unknown products or commodities.

# **3 Countries/Regions with Liberal Regulations for Specifc SDN Applications**

In Japan three genome-edited organisms have already been introduced into the market (Table 25.1) and crosses using these already registered organisms do not need to be evaluated again and are free from obligations. Japan [21] refers to the Cartagena protocol and exempt SDN-1, SDN-2 and Oligonucleotide-Directed Mutagenesis (ODM) as long as the absence of foreign DNA integration is proven in the latter cases. A guidance on how to proof the absence of foreign DNA with legal suffciency is pending.

Australia [22] published legal guidelines that essentially exempt organisms derived by SDN-1 mechanisms from restrict regulations of GMO (i.e. if an external sequence template was not used and if the organisms are free from foreign sequences).

# **4 Countries with Liberal Regulations (for Organisms Free of Foreign DNA)**

Several countries followed Argentina deregulating organisms derived by genome editing if they do not contain introduced foreign DNA-sequences. Argentina has updated the regulation several times and it now includes animals and microorganisms [23]. Nevertheless, a pre-assessment or notifcation may be necessary to assess compliance. This does not mean that sequence information is published as such. Since 2015 more than 35 Prior Consultation Instances has been raised, 66% of those from local developers. Similar regulations are established in Chile in 2017 [24], Brazil and Colombia in 2018 [24], Paraguay, Honduras, Guatemala and El Salvador in 2019 [24].

Likewise, Nigeria [25], Kenia [26] and Malawi [27] published guidelines based on notifcation and a case-by-case assessment which comes to a decision within few weeks. Essentially, the regulations refer to the absence of "novel combination of DNA" in the genomes whereas small InDels and substitutions are not seen as such.

There are no specifc legislations for bioengineering in Canada and USA and at least one product is currently on the market (see Table 25.1). With regards to seeds, Canada follows a product based approach to regulation as recently confrmed by the Canadian Food Inspection Agency (CFIA): "*It is the scientifc opinion of the CFIA that genome editing technologies do not present any unique or specifcally identifable environmental or human health safety concerns as compared to other technologies of plant development. For this reason, genome-edited plants are regulated using a product-based approach, like any other product of plant breeding. Namely, it is the traits that a plant exhibits and whether these traits would have a signifcant negative impact on environmental safety that are used to determine whether a plant would be subject to Part V of the Seeds Regulations*." [28] Nevertheless, following the Division 28 of Part B of the Food and Drug Regulations about novel foods Health Canada states "*Foods derived from plants that have been genetically modifed such that they contain foreign DNA in the fnal plant product require pre-market notifcation and assessment as novel foods*." [29] "Novelty" essentially relates to terms like "history of safe use" and "familiarity" with the composition of the fnal food product. Residues of the CRISPR/Cas-System in the genome would trigger additional safety assessments like any other transgenic organism.

The legislation and regulation of genome-edited crops in the USA is more complex while a specifc regulation for bioengineering does not exist but a "Coordinated Framework for regulation of Biotechnology" [30]. Based on the Plant Protection Act (PPA) the Animal and Plant Health Inspection Service of the U.S. Department of Agriculture (USDA/APHIS), based on the Food, Drug and Cosmetics Act (FDCA), the Food and Drug Agency (FDA) and based on the FDCA and the Federal Insecticide, Fungicide, and Rodenticide Act [FIFRA of the Environmental Protection Agency (EPA)] regulate products of biotechnology applications. For several years there are governmental activities to streamline the application. Since 2021 USDA APHIS implemented the Revised Biotechnology Regulations (previously SECURE rule) to provide clear and effcient regulatory pathways for applicants, when the plant products are unlikely to pose a plant pest risk. Products derived by means of genome editing under in most conditions are free from restrictions based on the PPA when changes in the plant product's genome are either deletion(s), targeted substitutions of a single base pair or solely introductions from sequences derived from the plant's natural gene pool or edits from sequences which are known to correspond in the plants natural gene pool.

The Philippines established a procedure to regulate genome-edited plants based on the key criteria are whether they possess novel combinations of genetic material not achievable by conventional breeding [31].

# **5 Other Regulatory Frameworks**

China has released guidelines for the safety evaluation of genome-edited plants for agricultural use that do not harbour exogenous DNA-sequences (SDN-1, SDN 2). It provides a tiered assessment based on the risk profle of the target trait. The frst category (low risk) refers to plants/traits that do not increase the risk to environmental and food safety, the second to increased environmental risks, the third to increased food safety risks, and the fourth to increases in both environmental and food risks. Different requirements apply for cultivation and or import aside from some general items describing the plant and trait: (1) molecular characterization, editing method applied, data on the edited sequence, presence of residual vector sequences, and off-target analysis; (2) stability of the edit and the trait over at least three generations. At present the guidelines do not specify how to classify a product according to the four categories, what may indicate a case-by-case decision procedure. These requirements are in line with the ones requested in the guidelines for safety evaluation of GMOs. However, genome-edited crops are still managed under GMO regulation, but may require much less complicated food and environmental safety evaluations compared to classical GMOs and may reduce time for regulation from six down to one to two years. However, these guidelines also differentiate between local and foreign developers as foreign frms are not allowed to invest in Chinas biotech sector [32].

A risk based concept was also implemented by India and an appropriate tiered based risk assessment is foreseen to categorizing genome editing in three categories. In a frst category, products should be addressed with single or few base pair edits or In/Dels. The assessment confrms targeted edits as well as absence of any biological relevant off target genomic changes, and, if necessary, a phenotypic equivalence to a comparator will be checked on a case by case basis. The second category addressed targeted base pair edits in which the assessment is compiled by phenotypic equivalence and trait effcacy through appropriate contained and/or confned feld trials. The third category addresses products harbouring targeted edit(s) with synthetic/foreign DNA. The assessment is the same as for traditional GMOs [33].

Thailand also drafted a risk assessment of genome editing products in which a liberal assessment of SDN1 has been foreseen. However, this draft has not passed offcial release, yet [34]**.**

# **6 Other Countries – Ongoing Consultations**

In Europe the policy of the EU is considerably important for non-EU countries – which are trading partners. Nevertheless, England, Norway and Switzerland have discussed somewhat differing regulations for genome-edited plants.

The Norwegian Biotechnology Advisory Board initiated a reconsideration of the regulatory framework for GMOs. It sketched a tiered scheme for risk assessment of GMOs including genome-edited organisms combining biological, environmental as well as social criteria [35].

The Swiss Genetic Technology Act (Art. 37a) [36] mandates the Federal Council to develop a draft decree for a risk-based approval procedure for transgene-free GMOs by mid-2024. The current genetic engineering act considers genome-edited organisms as GMO and the year-long moratorium for cultivation of GMO in Switzerland will apply. Nevertheless, feld trials are supported.

In 2020 UK left the EU and England – independent of Scotland, Wales and Northern Ireland – moved towards specifc regulations for genome-edited organisms. On 23rd March 2023 the "Genetic Technology (Precision Breeding) Act 2023" [37] came into force. It allows for genetic changes that could also have been produced naturally or through "traditional" breeding. It rules that genome-edited (precision bred) plants and animals can be released or marketed in England based on notifcation and risk assessment provisions.

It is expected that South Korea as well as Taiwan intend to publish clarifcations on the status of genome-edited organisms in 2023. Several African countries are reconsidering the regulatory status of GE plants, but it remains open when decisions will be taken.

# **7 Compliance, Law Enforcement, Detection and Identifcation**

As mentioned above, the legal framework is closely linked to the issue of enforcement and compliance as well as labelling demands, which in turn is linked to the issue of detection, i.e., identifcation of the genome editing process associated with the DNA sequence of an organism. Unlike organisms obtained by classical transgenesis, organisms edited by ODM, SDN-1, or SDN-2 do not contain sequence elements belonging to elements associated with tranformations (e.g. S35-promotor, Nos-terminator) that simplify broader screening. Moreover, a targeted mutation often does not differ from a random mutation. Hence, a precondition for identifcation is the information about the uniqueness of a sequence caused by genome editing. The unique sequence length cannot be freely set as recombination events (crossover at meiosis during seed propagation) as well as random mutations may alter the sequence "naturally" [38]. Even large modifcations e.g. introgressions, occur in conventional breeding programs [39]. Therefore, there are calls for an international sequence database [40] for unique sequences that identify a genomeedited organism to support screening and detection. The crucial dependence on this information and detection challenges are nicely depicted by the debate about the detection of Cibus herbicide tolerant canola. There is no doubt that any SNP can be sensitively detected in a sample with mixed background. Since at various time Cibus provided contradictory information about the origin of the SNP in its modifed canola variety, the sequence itself does not reveal the actual process of modifcation [41–44]. For genome-edited organisms that are not considered GMOs in various legislations there is neither a legal obligation to pass detailed sequence information to public international repositories nor an obligation to provide a detection method, as is requested for market release of GMOs in the EU. Within a territory with a uniform legal basis (e.g. in the EU), the establishment of a register may be considered possible, but considering international trade with mixed commodities, law enforcement is challenging. In jurisdictions with differing regulations for SDN-1 and SDN-2 the handling of genome-edited organisms will become even more problematic. In addition, laws that refer to equivalence of genome-edited sequences with sequences that may be generated through conventional breeding will need to specify borderlines. As large introgressions (>>1000 bp) occur naturally and deliberately in conventional breeding, the legally fxed sequence length may affect trade with conventional breeding products as well.

A positive or negative certifcation regime may be considered that is based on documentation. Such certifcation systems have been established e.g. in the organic food sector or for certifed regional products. To scrutinize detailed compliance, audits based on extended documentation at each step of the supply or value chain would be necessary. Employing blockchain-based tracking systems is recently discussed for agro-food value chains [e.g. 45]. Nevertheless, monitoring the actual physical compliance would need appropriate identifcation methods (see above).

# **8 Conclusions**

The majority of countries currently do not explicitly regulate genome edited organisms – for various reasons. The legal practice may stay unclear for some countries for the next years. Several major players in international trade (of agricultural goods) already clarifed their legal classifcation of genome-edited organisms in specifc legislations, guidance documents, decrees or else related to the handling of biotechnology and bioengineering (GMO). The countries are applying separate regulatory requirements for organisms that do not harbour foreign gene sequences while strict GMO regulations apply in a few countries and the EU. This challenges international trade and national law enforcement and compliance (detection and identifcation of genome-edited organisms) due to the international heterogeneity of regulations. Hence, there are frequent calls for international harmonisation of legislation on genome-edited organisms to end divergent national regulations.

This situation is likely continuing for at least a few more years, affecting international trade between some countries and preventing or delaying the application and use of genome editing and its products in regions with restrictive regulations. Therefore, the prospects and progress of genome editing in breeding will vary from region to region and also for international and regional players.

**Acknowledgements** The paper is a continuing update of the initial work performed in the course of the projects "CHIC" funded by the European Union's Horizon 2020 research and innovation programme, grant agreement No. 760891 and "ELSA-GEA" funded by the German Federal Ministry of Education and Research (BMBF) grant agreement No. (01GP1613B).

# **References**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

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# **Chapter 26 Interpreting Precision Breeding: Key Legal Concepts Under International Law and Current Domestic Regulatory Approaches in the Global South**

#### **André Rosado**

**Abstract** In various countries, especially in the Global South, there is legal uncertainty about which products or organisms derived from precision breeding, also called new breeding technologies, are subject to biosafety regulation. It is not clear whether which precision breeding products are classied as Living Modied Organisms (LMOs), or Genetically Modied Organisms (GMOs), and therefore be subject to regulatory oversight under biosafety laws.

This section, under Chap. 3 of Policies and Regulations, provides an overview of key denitions under international and national legislation to clarify the regulatory status of precision breeding products. This is done by assessing provisions under international biosafety law and national legislation in selected countries in the Global South. The outcome of this section is to provide a baseline for further discussion about the regulatory status of precision breeding globally.

First, the background is presented, covering the development of international and national legal frameworks governing biosafety of LMOs and GMOs. Second, the legal denition of LMO and related terms under international law is discussed. Third, the GMO denition of national law in selected jurisdictions is presented. Fourth, a snapshot of the emergence of regulations governing precision breeding in the Global South is analyzed. Finally, key future perspectives to the regulatory status of precision breeding products are suggested.

A. Rosado (\*)

Faculty of Science, KU Leuven, Leuven, Belgium

Centro de Investigaciones de Zonas Aridas, Universidad Nacional Agraria La Molina, Lima, Peru e-mail: ayrtonandre.rosadohuaynasi@student.kuleuven.be

# **1 International and National Legal Frameworks for Biosafety**

Biotechnology products, such as LMOs and GMOs, are subject to regulation. Rules to regulate the safe use of LMOs and GMOs are established under international and national biosafety law.

For instance, the Convention on Biological Diversity (CBD) requires in Art. 8(g) that Parties "*to establish or maintain means to regulate, manage or control the risks associated with the use and release of living modifed organisms resulting from biotechnology which are likely to have adverse environmental impacts that could affect the conservation and sustainable use of biological diversity, taking also into account the risks to human health*"*.*

In addition to the obligation pertaining to national biosafety systems, the Cartagena Protocol on Biosafety (CPB), a subsequent protocol to the CBD, governs the transboundary movements of LMOs [1].

According to Art. 1 CPB, the objective to the CPB is: "*to contribute to ensuring an adequate level of protection in the feld of the safe transfer, handling and use of living modifed organisms resulting from modern biotechnology that may have adverse effects on the conservation and sustainable use of biological diversity, taking also into account risks to human health, and specifcally focusing on transboundary movements*"*.*

The CPB was adopted in 2000 and entered into force in 2003 [2]. Currently, 173 countries are Parties to the CPB. This means that such countries have either ratifed, accepted, approved or accessed the CPB [3]. It is relevant to note that most Parties to the CPB are located in the Global South. In fact, both the text of the CPB and its adoption by jurisdictions in the Global South were promoted by infuential actors in the Global North such as the European Union. In fact, the CPB stipulations were contested by other Global North prominent actors in the international environmental system, such as the United States of America [4].

According to Art. 2 CPB, CPB's Parties are required to, *inter alia*, adopt national legal measures to implement their obligations under the CPB. Since the ratifcation of the CBD and the CPB, an increasing number of Parties have adopted domestic measures to govern the use of the LMO and to implement the CPB.

With the adoption of the CPB in 2000, some Parties to the CPB rapidly started to adopt national legislation to regulate the use of LMOs within their territories [5]. In the Global South, some countries have adopted a national biosafety legislation to govern LMOs. For example, in 2007, Malaysia adopted the Biosafety Act No. 678 to, *inter alia*, regulate the use of LMOs with the objectives of protecting human, plant and animal health, the environment and biological diversity. Similarly, in 2015, Dominican Republic adopted the Law No. 219-15 regarding the safety of biotechnology to ensure safe use of LMOs and prevent adverse effects that they could cause to biodiversity, human health and the environment.

Other countries in the Global South, which are also Parties to the CPB, have designed biosafety laws to regulate the safe use of GMOs. These countries regulate

GMOs instead of LMOs. For instance, in 2005, Mexico enacted the Biosafety Law of Genetically Modifed Organisms with the purpose of regulating various activities of GMOs to prevent, avoid or reduce the possible risks that these activities might entail to human health, environment and biological diversity. Similarly, in 2009, Kenya adopted the Biosafety Act No. 2 to facilitate responsible research into, and minimize the risks that may be posed by the GMOs to human health and the environment. Therefore, regardless the ratifcation of the CPB, some Parties have opted to use the term GMO, instead of LMOs, to design national measures to implement the CPB.

In the Global South, biotechnology products have been subject to regulatory oversight even before the adoption of the CPB. For instance, in 1999, Peru enacted the Law No. 27104 to prevent risks associated with the use of biotechnology and to protect the human health, the environment and biodiversity by regulating, managing and controlling the risks associated with LMOs. Similarly, in 1997, South Africa adopted the Genetically Modifed Organism Act No. 15 to promote the responsible development, production, and use of GMOs to limit possible harmful consequences to the environment.

However, not all countries in the Global South have a biosafety legal framework to govern LMOs or GMOs. In fact, in the Global South, the purpose to adopt biosafety policies, legislation and regulations is diverse. For instance, most countries have adopted biosafety legal frameworks as an end product of the ratifcation and entry into force of the CPB [5, 6]. Other countries, such as Argentina and Brazil are major exporters of agrobiotechnology commodities, and this may be a contributing element to their advanced level of trade-related biosafety frameworks [7, 8]. Some other countries have opted to design biosafety policies to restrict the use of biotechnology products or have yet to promulgate any biosafety legal instrument [5]. Clearly, the economic importance of the agricultural sector and the role of biotechnology in each country are fundamental triggers for the rapid adoption of biosafety policies and laws [7]. Additionally, political will would be a key factor in the success of adoption biosafety policies and legislation [5].

Overall, some countries are designing bills of legal instruments to regulate LMOs, or GMOs, and implement the CBD and the CPB at a national level. For example, most Small Island States in the Caribbean, and the Pacifc, are drafting biosafety legal measures [5]. Similarly, most Least Developed countries in Africa lack biosafety legislation [9].

# **2 What Is a Living Modifed Organism (LMO)?**

The term LMO has a universal legal defnition that has been described under international environmental law. The concept of LMO was initially introduced in international law by the CBD in 1992 [10]. Two articles under the CBD presented the notion of LMO.

Firstly, Art. 8(g) CBD, concerning *in situ* conservation, indicates that each contracting party shall, as far as possible and as appropriate, "*establish or maintain means to regulate, manage or control the risks associated with the use and release of Living Modifed Organisms resulting from biotechnology which are likely to have adverse environmental impacts that could affect the conservation and sustainable use of biological diversity, taking also into account the risks to human health*"*.*

Second, the concept of LMO is also presented in Art. 19(3) CBD referring to handling of biotechnology and distribution of its benefts. Art. 19(3) CBD notes that "*the Parties shall consider the need for and modalities of a protocol setting out appropriate procedures, including, in particular, advance informed agreement, in the feld of the safe transfer, handling and use of any Living Modifed Organism resulting from biotechnology that may have adverse effect on the conservation and sustainable use of biological diversity*"*.*

The CBD only mentions the notion of LMO with the aim to develop appropriate measures to control their possible risks to biodiversity and human health. However, the defnition of LMO is subsequently clarifed in the CPB in 2003. According to Art. 3(g) CPB, a LMO refers to "*any living organism that possesses a novel combination of genetic material obtained through the use of modern biotechnology*"*.*

Some Parties to the CPB have included the legal defnition of LMO into their national biosafety legislation [11, 12].

#### **Activity 26.1: Adoption of the LMO Defnition Under National Laws**


# **3 Key Features of the LMO Defnition**

Based on Art. 3(g) CPB, the LMO defnition contains three key features. A product is considered to be a LMO when it owns all these three characteristics:


Each feature of the LMO defnition includes relevant legal terms that have also been defned under international law. Overall, the LMO defnition contains four legal terms that are described by international law. These are: "living organism", "genetic material", "biotechnology" and "modern biotechnology".

# *3.1 What Is a Living Organism?*

According to the LMO defnition of the CPB, LMO are living organisms. Art. 3(h) CPB defnes the concept of living organism as "*any biological entity capable of transferring or replicating genetic material, including sterile organisms, viruses and viroids*". According to this defnition, a living organism possesses two joint characteristics.

Firstly, a living organism is any biological entity, including sterile organisms, virus and viroids. Living organisms are alive beings which are also referred to as biological entities, living beings, living systems or natural entities [13]. They occur in various forms [14]. For example, animals (including human beings), plants, bacteria, protozoa and fungi are living organisms [15].

Furthermore, entities such as sterile organisms, viruses and viroids are also included as living organisms according to Art. 3(h) CPB. It is noteworthy that some viruses and viroids are not alive entities [10]. However, the defnition of living organisms, as stipulated by Art. 3(h) CPB, is broad as it includes non-living things, such as viruses and viroids.

Secondly, a living organism must be capable of transferring or replicating genetic material. Biological entities possess various capacities, including the ability to transfer or replicate their genetic material [13]. Similarly, viruses and viroids also possess this ability [10].

# *3.2 What Is a Novel Combination of Genetic Material?*

Based on the LMO defnition of the CPB, a LMO has to possess a novel combination of genetic material. However, there is no defnition of the concept of novel combination of genetic material under international law. Rather, the term genetic material is in:

Art. 2 CBD, genetic material refers to "*any material of plant, animal, microbial or other origin containing functional units of heredity*"*.* Similarly, Art. 2 International Treaty on Plant Genetic Resources for Food and Agriculture (ITPGRFA) defnes genetic material as: "*any material of plant origin, including reproductive and vegetative propagating material, containing functional units of heredity*"*.*

Overall, the defnition of genetic material by the CBD is broad, as it encompasses any genetic material of various types of living organisms, including plants, animals or microbes. In contrast, the defnition of the ITPGRFA exclusively refers to genetic material of plant origin. However, both defnitions agree that genetic material must contain functional units of heredity, which are composed of nucleic acids, such deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) carrying genetic information or genes [10, 16].

# *3.3 What Is Modern Biotechnology?*

Before explaining the concept of modern biotechnology, it is relevant to describe what biotechnology means. Art. 2 CBD defnes biotechnology as "*any technological application that uses biological systems, living organisms, or derivates thereof, to make or modify products or processes for specifc use*"*.*

The term biotechnology was frst introduced by natural sciences in 1919 to refer the scientifc methodologies that allow products to be developed from raw materials with the support of living organisms [17]. For instance, biotechnology is used for the selective breeding of plants and animals or the use of microorganisms in the production of beer, bread and wine [18].

Modern biotechnology is one type of biotechnology. According to Art. 3(i) CPB, modern biotechnology refers to the application of "*in vitro nucleic acid techniques, including recombinant DNA (rDNA) and direct injection of nucleic acid into cells or organelles, or fusion of cells beyond the taxonomic family, that overcome natural physiological reproductive or recombination barriers and that are not techniques used in traditional breeding and selection*"*.* Both, *in vitro* nucleic acid techniques and cell fusion are primarily used for agriculture, textile, food and feed production, including the development of LMOs [19].

Additionally, the defnition of modern biotechnology makes reference that these techniques are not same as methods used in traditional breeding and selection. However, the meaning of traditional breeding and selection is not defned under international law. Conventional, traditional, or natural could refer to traditional breeding and selection techniques, such as natural selection, cross-breeding, protoplast fusion, and chemical- or radiation-induced mutation [20]. These techniques modify the genetic material within the crossable gene pool of a species, but they do not introduce genetic information from other organisms [20].

# **4 What Is a Genetically Modifed Organism (GMO)?**

There is no international legal defnition of GMO. Instead, the term GMO is introduced and defned only under domestic and regional law in certain jurisdictions. As such, some countries have designed their own defnition of GMO, which is incorporated into their national biosafety laws. For instance, in the Global South, biosafety legislations in selected countries in Africa, Asia and Latin America and the Caribbean use the term GMO instead of LMOs (Table 26.1). It is relevant to note that, regardless the ratifcation to the CPB, some Parties to the CPB have decided to use the term GMO instead of LMO.

As shown in Table 26.1, the defnition of GMO varies between jurisdictions. Therefore, it is not possible to describe main characteristics of the GMO defnition that can be universally valid. This is because there is no standard defnition of the term GMO. In fact, the GMO defnition contains different or similar features among countries with a biosafety law to regulate GMOs. Interestingly, these features of the


**Table 26.1** GMO defnition under national biosafety laws in certain jurisdictions

(continued)


**Table 26.1** (continued)

GMO defnition can be similar or different in comparison to the LMO defnition of the CPB. Overall, the GMO defnition can be equal, similar or different to the LMO defnition.

First, for some countries with a biosafety law to regulate GMOs, the defnition of GMOs is equal to the LMO defnition of the CPB. This phenomenon occurs when revising biosafety laws in The Philippines and El Salvador. In fact, under such legislation, there is a clear indication that GMO is a synonym of the term LMO under the CPB.

Second, for other countries that have adopted a biosafety law of GMOs, the term GMO is very similar to the LMO defnition of the CPB. the GMO concept under biosafety legislation of GMOs in Mexico, Nigeria and Kenya contains almost all the features of the LMO defnition. For example, according to these laws, a GMO has to possess a novel combination of genetic material and have to be developed through the use of modern biotechnology.

Third, biosafety legislation in some other countries have design a GMO defnition that is very different to the concept of LMO. This occurs in countries such as Brazil, Panama, South Africa and Vietnam. Here, there is no indication of modern biotechnology; instead, other scientifc terms are mentioned such as genetic engineering, gene transfer technology or in a way that does not occur naturally. Similarly, these legal instruments only mention that the genetic material of the GMO has to be altered or modifed. There is no reference that GMO has to possess a novel combination of genetic material.

#### **Activity 26.2: Adoption of the GMO Defnition Under National Laws**


# **5 Regulatory Approaches Governing Precision Breeding**

Under international law, there is no binding specifc provision to regulate precision breeding or new breeding techniques. Some international organizations including the Secretariat to the CBD, Organization Economic Cooperation and Development (OECD), Food and Agriculture Organization of the United Nations (FAO) have made efforts to support countries when defning what precision breeding refers to. For instance, these organizations have designed guidelines to explain some precision breeding or other related techniques such as genome editing, synthetic biology and/or gene drive systems [21–23]. However, the defnitions proposed by these organizations are not legally binding as those concepts have been developed as guidance only.

However, under national law, rules to regulate some techniques that could be placed under the umbrella term precision breeding exist. Recently, few countries, including in the Global South, with a biosafety legislation of LMOs or GMOs have adopted a new regulation to clarify the regulatory status of precision breeding [24] (Fig. 26.1). Interestingly, most of these countries are located in Latin America and the Caribbean region.

Overall, 13 countries have designed guidelines or regulations that have been adopted in certain countries in the Global South (Table 26.2). The development of these guidelines and regulations results is a consequence that some precision breeding techniques can generate products that can be classifed as LMOs or GMOs under biosafety legislation and they can result in organisms that can also occur in nature or through conventional breeding.

Regulations in most of these countries acknowledge the fact that some precision breeding techniques could develop LMOs or GMOs, while others may not. Therefore, the purpose of these regulations is to develop an administrative procedure, and an application form for applicants to query to the National Competent Authority (NCA) whether or not their products are considered as LMOs or GMOs. The NCAs in these countries are:


**Fig. 26.1** Overview of countries in the Global South with a regulation or guidelines to regulate some technologies of precision breeding. Countries in yellow have designed a similar approach to regulate precision breeding products. Whereas countries in blue opted to adopt other type of approach

• In Latin America and the Caribbean: Ministry of Agriculture, Livestock and Fisheries in Argentina, National Biosafety Technical Commission in Brazil, Service for Agriculture and Livestock in Chile, Colombian Institute of Agriculture and Livestock in Colombia, Ministry of Environment in Ecuador, Ministry of Agriculture and Livestock in Paraguay, Ministry of Agriculture, Livestock and Food in Guatemala and National Service of Agri-Food Health and Quality in Honduras.

Overall, according to [7], the applications forms that are developed by the NCAs request the following information including:


The NCAs in these countries analyze the information provided by the applicant and evaluate whether the products have a novel combination of genetic material or not. If the fnal organism has a novel combination of genetic material, it is considered as LMO or GMOs, and falls under the biosafety regulatory oversight [7, 12, 25].

Therefore, a common defnition in most of these countries is what constitutes a novel combination of genetic material. This is because for all countries in Fig. 26.1


**Table 26.2** Regulations governing precision breeding in selected countries

(continued)


**Table 26.2** (continued)

(in yellow), excluding South Africa, precision breeding products that lack a novel combination, or foreign, of genetic material are not defned as LMOs, or GMOs, according to their national biosafety legislation. The presence of novel combination, foreign or exogenous, of genetic material in the fnal product is the main consideration to classify a product as a LMO or GMO. Clearly, the characteristics of this defnition may slightly or signifcantly vary between countries as indicated in Table 26.2.

To the contrary, the NCA of South Africa, the Executive Council for Genetically Modifed Organisms in South Africa, had adopted a communication indicating that all products derived from new breeding techniques will be subject to regulation. Therefore, all products of new breeding techniques will be treated similarly to GMOs, and they will fall under subject to the Genetically Modifed Organism Act No. 15 of 1997.

Some other countries in the Global South are currently discussing the need to adopt regulations for precision breeding or are designing drafts of guidelines to regulate precision breeding products. For example, this occurs in various countries in Latin America and the Caribbean [7]. Similarly, few countries in South East Asia are discussing whether or not regulate precision breeding products [26]. Additionally, ongoing discussions are taking place in some West African countries [27].

#### **Activity 26.3: Approval of Regulations Concerning Precision Breeding**


# **6 Future Regulatory Perspectives on Precision Breeding**

Some countries in the Global South, especially countries in Latin America and the Caribbean region, have designed regulations to assess the regulatory status of some precision breeding techniques. In fact, these countries have an extensive experience governing LMOs and/or GMOs since the 90s. Additionally, these countries are major producers of agricultural and biotechnological commodities [5]. As such, it was expected that these countries would rapidly adopt such new measures on precision breeding, as they have an interest to insert such these products into national and international markets. Clearly, the rapid adoption of procedures relevant to precision breeding is strongly linked to the role of agriculture and biotechnology in the countries and with national economic, social and political perspectives [7]. As such, it is likely that other countries with a similar attitude would likely be willing to adopt similar or slightly similar regulations on precision breeding.

However, countries in the Global South have diverse positions in terms of biosafety law and the role of agrobiotechnology in their economies. Countries that rely in other types of agriculture, e.g. organic, or have developed bans to the use of biotechnology products for agriculture would continue excluding the governance of precision breeding products. This is simply because precision breeding products are one alternative that can be utilized by certain countries but not necessarily the only one that can be used across all countries in the Global South.

It is fundamental to highlight the right of sovereignty of countries over their territory. For the Global South, clearly, there is no one-size-fts-all approach to promote the use of precision breeding in all countries. Nor all countries should follow a singular and common approach to govern precision breeding products. This would occur only when such approaches, if relevant and needed, ft the conditions and national interest of the countries about precision breeding.

Specially in the Global South, countries are complex actors and their sociopolitical and economic context and priorities would play a key role when deciding to what extent the adoption of regulation of precision breeding is relevant or not. For instance, countries that currently lack a biosafety regulatory system to assess the use of LMOs or GMOs such as Small Island States or most Least Developed Countries in Africa would probably, in the future, also lack precision breeding regulations as such matter is not a key priority for these countries. Clearly, this phenomenon could obviously change over time.

# **7 Summary**

In the Global South, 13 nations have adopted regulations or guidelines to govern some precision breeding techniques. Most of these countries have designed a similar approach to assess the regulatory status of precision breeding products. The main criterion for such countries is whether or not the fnal product contains a novel combination of genetic material. To this aim, countries have designed a concept to legally defne what novel combination of genetic material refers to. Such defnition may vary or may be similar among nations.

However, overall, most jurisdictions in the Global South require legal clarity about whether products or organisms derived from precision breeding would be subject to biosafety regulation. For such countries, it is still not clear whether precision breeding products would be classifed as LMO or GMOs, and therefore be subject to regulatory oversight under biosafety laws. Further studies are required to explore the interest or current developments of such countries to design regulations or guidelines to regulate some precision breeding techniques.

This section provided an overview of key defnitions under international and national legislation to clarify the regulatory status of precision breeding products. This was done by assessing provisions under the CPB and national biosafety laws in selected countries in the Global South. This hopefully would aim to provide a baseline and a global perspective for further discussion about the regulatory status of precision breeding in other nations.

# **8 Further Reading**

For a detailed overview of the LMO defnition see [10] and revise the CPB. With regards to the GMO defnition, read the national biosafety legislation and regulations of GMOs of the country of interest. Such legal instruments are available in the national gazettes or offcial journals of each country.

**Acknowledgements** The author would like to thank (in alphabetic order) to Dennis Eriksson, Jale Tosun, Noelia Valderrama-Bhraunx and Pieter Van der Meer for their helpful comments and suggestions.

# **References**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

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# **Chapter 27 CRISPR Processes Patents in Green Biotechnology: Collaborative Licensing Models**

### **Agnès Ricroch**

**Abstract** A strong patent system is necessary to enable innovation by incentivising investments in research and development (R&D) and promoting the dissemination of knowledge, including through licensing, as this is considered vital for the development and commercialisation of new products. The new genomic techniques (NGTs) dened by the European Union (EU) in 2021 include the CRISPR system which can selectively modify DNA sequences in a genome. More than 11,000 CRISPR-related patent applications have already been led worldwide mainly in USA and China. The proliferation of patents on CRISPR for green biotechnology applications and the dispute between two of the technology's inventors (UC Berkeley and the Broad Institute of MIT, USA) could be barriers to innovation. With regard to intellectual property (IP) and the patent protection, the conditions and opportunities of alternative licensing models to overcome the difculties created by the complex patent landscape of CRISPR technology are examined. Patent pools and clearing houses are the two models attracting most interest that leads to a onestop licensing point allowing cross-licensing and facilitating freedom to operate. The conditions for success and acceptability of collaborative licensing platforms are discussed.

# **1 The Protection of New Genomic Techniques (NGTs)**

Plant biotechnology research for new traits and thus the development of new varieties is risky and costly. Plant innovation needs a return on investment to encourage private research and public-private partnerships [1]. Intellectual Property (IP) is a tool used to: (i) disseminate knowledge and innovation to speed-up innovation cycles, (ii) encourage collaboration and open innovation, (iii) build a sustainably growing knowledge and innovation pool, (iv) enable fair access and benet sharing, and (v) prevent creation misappropriation and, when owned or licensed, allows for

© The Author(s) 2024 453

A. Ricroch (\*)

The Jean-Monnet Faculty. Idest Laboratory, Paris-Saclay University, Sceaux, France e-mail: agnes.ricroch@universite-paris-saclay.fr

A. Ricroch et al. (eds.), *A Roadmap for Plant Genome Editing*, https://doi.org/10.1007/978-3-031-46150-7\_27

so-called freedom-to-operate (FTO). An FTO analysis (or clearance/infringement search) clarifes if a product or its potential commercialization infringes on other existing IP rights. It is an expensive undertaking. It begins by searching for issued or pending patents and thereafter, involves analysing the claimed scope of protection to get a legal opinion as to whether the product, process, or service potentially infringes on any patents owned by others. It establishes a list of potential patent holders, with whom the future (prospective) licensee must then contact and successfully negotiate a license for each patent. The negotiation process involves costs for both the licensee and the patent holders. On 29 April 2021, the European Commission (EC) published a study on the status of new genomic techniques (NGTs) that defned NGTs as "techniques capable of modifying the genetic material of an organism that have emerged or been developed since 2001", *i.e.* after the existing EU legislation on genetically modifed organism (GMO) was adopted in 2001 (Directive 2001/18/ EC of the European Parliament and of the Council of 12 March 2001 on the deliberate release into the environment of genetically modifed organisms and repealing Council Directive 90/220/EEC). NGTs include (i) CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), TALEN (Transcription Activator-Like Effector Nucleases), zinc-fnger nucleases, meganuclease techniques, and prime editing; (ii) mutagenesis techniques, such as oligonucleotide directed mutagenesis; and (iii) epigenome-editing techniques, such as RNA-dependent DNA methylation.

With regard to IP and the patent protection of biotechnological inventions (under Directive 98/44/EC54), this EU study acknowledged the benefts of patents and licensing in promoting innovation and the development of NGTs and their products. The licensing landscape is developing rapidly with licensing agreements, some exclusive, some non-exclusive, on a diverse range of CRISPR technologies and application felds, from agriculture to therapeutics. More than 11,000 CRISPRrelated patent applications have already been fled worldwide, with the majority being fled in USA and China [2] leading to some concerns about the complex patent landscape for NGTs with multiple players holding patents, and uncertainty surrounding the IP situation. *IPStudies*, a company based in Switzerland, has compiled a list of more than 100 variants of CRISPR enzymes beyond the best-known discovery of Cas9 in 2012, with some commercial players attempting to claim them exhaustively to ensure as much IP exclusivity as possible (dCas, dCas9, Cas12a, Cas13a…).

Prime editing, using a modifed Cas9 protein that makes a single-stranded cut substantially expands the scope and capabilities of genome editing, a versatile and precise method that directly writes new genetic information into a specifed DNA site (see Chap. 3). Prime editing shows higher or similar effciency and fewer byproducts than homology-directed repair, has complementary strengths and weaknesses compared to base editing, and induces much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. CRISPR-Cas-based techniques are still evolving and the list of NGTs is expected to expand further in the coming years (*e.g.* base editing). New CRISPR-associated enzymes have been engineered, such as base editors that are better able to make specifc edits.

However, optimized tissue culture and transformation protocols should be in place to make genome editing a routine tool for plant breeding. More than 370,000 higher plant species exist *in natura*. But scientists can only make transformation (transgenesis and genome editing) successful in a few dozens of these.

# *1.1 Patents Characteristics*

The protection conferred by a patent to biological material possessing specifc characteristics that result from an invention [1]. (i) Patents usually have a term of protection for 20 years (starting from the fling of the application). As with all IP rights (IPRs), patent protection is also territorial, meaning that it provides protection only on a given territory. The scope of a patent on an invention is determined by the socalled patent claims. (ii) The protection conferred by a patent to biological material possessing specifc characteristics that result from an invention, shall extend to any biological material which has derived from the original biological material through propagation or multiplication and possessing those same properties. Noting that in the case of a self-replicating biological material, the right of exhaustion does not apply at the frst sale. (iii) A patented biotechnological invention incorporated into a variety remains protected in this variety but by no means is the variety itself patented, as this would be contrary to EU legislation prohibiting the patenting of varieties. Thus, the genome of this variety, when it no longer contains the patented biotechnological invention, is completely free of patent rights.

Under the European Patent Convention which has been amended by the decisions of the Administrative Council since the publication of the 17th edition (November 2020), patents are granted only for inventions that are new, involve an inventive step and are industrially applicable. An invention meets these requirements if it: (i) was not known to the public in any form, (ii) is not obvious to a person skilled in the art, and (iii) can be manufactured or used industrially. They are valid in individual countries, for a specifed period. New plant varieties in the EU are completely excluded from patentability. Plant variety rights are regulated under Council Regulation (EC) No 2100/94 of 27 July 1994 on Community plant variety rights. In Europe, transgenic or edited plants carrying a patented event (transgenic or edited trait) fall within the scope of the patent because this element is not limited to a single variety and at the same time a transgenic or edited variety can also be individually protected by a Plant Variety Protection (PVP) certifcate.

# *1.2 The CRISPR Patent Dispute and Legal Uncertainty*

Biotechnology in agriculture is a lucrative industry and its growth is going to continue. Universities hold most of the key patents using the CRISPR technology needed to deploy this technology [3]. Thus, the role of the public sector in fundamental research and surrogate companies such as the University of California, Berkeley, the Broad Institute of MIT, and Harvard, USA, are very relevant in holding fundamental/key patents of CRISPR technology. Key patents have no economically viable substitutes; a party needs a license to utilize the Intellectual Property Right (IPR) [4].

There has been extensive interest in IP issues surrounding CRISPR, including a patent dispute between two of the technology's inventors, Jennifer Doudna (the University of California, Berkeley, USA) and Emmanuelle Charpentier (currently in Max-Planck Institute, Germany) on one side, and Feng Zhang (the Broad Institute of MIT and Harvard, USA) on the other over who frst invented the gene-editing system and who should beneft from key patents [2]. This CRISPR-Cas9 dispute, which began in 2016, is still ongoing.

Companies now also have the option of avoiding these key patents (UC Berkeley, the Broad Institute and Harvard) altogether by using different CRISPR systems. Such systems occur naturally in many bacteria and Achaea and can have various properties. Relatively few of these use CRISPR–Cas9; instead, they use alternative enzymes such as Cas12a, Cas13a or Cas14, the latter being remarkably small and easy to transport into human cells. Labs have also engineered new CRISPRassociated enzymes, such as base editors, that are better able to make specifc edits.

The IP around CRISPR is becoming increasingly complex. Commercial deployments are complicated by the legal uncertainty associated with the lack of patent clarity on CRISPR. Indeed, in the context of the dispute, patent offces in the USA and the EU have issued different decisions on the validity of CRISPR patents. The United States Patent and Trademark Offce (USPTO) has repeatedly ruled in favour of the Broad Institute and, on February 28, 2022, determined that it was the frst inventor. The decisions are on who is entitled to receive patent protection [5]. In the EU, since the main CRISPR-Cas9 patents in the Broad Institute's portfolio were rejected due to lack of documentation, UC Berkeley is considered frst by the European Patent Offce (EPO) ("EP2800811" (November 10, 2020)). There is a need for greater clarity in patent rights in order to make researchers feel secure in developing further technological innovations using the CRISPR system.

Despite this legal uncertainty as to the fnal determination of ownership and patentability, the licensing landscape is developing rapidly with licensing agreements, some exclusive, some non-exclusive, on a diverse range of CRISPR technologies and application felds, from agriculture to therapeutics.

# **2 Alternative Models: Licensing Platforms and IP Clearing Houses**

The current CRISPR patent landscape presents a variety of barriers to research, innovation and proft. In order to overcome the diffculties created by the overall presence of patents, academics and breeders of the private sector are debating

alternative licensing models. Patent pools and clearing houses are the two models attracting most interest.

# *2.1 Concerns*

**Patent Profusion** With a steady increase of more than 200 patent families published each month IP studies shows the emergence of several new trends concerning both the enabling technology itself, and application developments. There are so many property rights in the hands of various owners—with whom parties must reach agreements to enable them to aggregate the rights they need in order to legally perform their activities—that it will prove diffcult to negotiate licences for patented inventions successfully. High transaction costs may stand in the way of an agreement. If a high number of agreements with rights holders is required, transaction costs may lead parties to decide that the bargaining process is not worthwhile. Hence, a socially optimum level of consumption of the resource may not be achieved, resulting in "under-utilization" of the property which will have a blocking effect on further innovation. Moreover, the fact that licensees have to acquire many licences in order to avoid patent infringements, may lead to elevated royalty fees, caused by royalty stacking. Because the licensee will usually pass on the cost of these fees to the fnal consumer, the fnal development and manufacture of products may be obstructed [6]**.**

A growing cause for concern in the CRISPR sphere is the so-called "tragedy of the anticommons." There are two reasons why anti-commons could affect the CRISPR patent pool; a thicket of patents, and the current licensing model being implemented. (i) There is a potential "thicket" of patents in the CRISPR sphere with some fragmentation of IPRs because the technology tends to be patented and licensed on a gene-by-gene basis. (ii) The surrogate licensing model currently in use raises concerns about research bottlenecks [7]. A research bottleneck could occur because the exclusive licensing to surrogates has limited the availability of CRISPR technology as a global platform, and traditional protections against the overly broad surrogate licenses will not work.

**Concentration of Players** The concentration of players in the seed market has led to high seed prices, reduced seed variety choices, and great dependence on farmers. Transgenesis technology has led to an increased concentration of ownership and power in agri-food systems through patents, contracts and licence agreements. Patent rights and the way they are granted and exercised contribute to a decrease in the diversity of breeding companies and threaten innovation in plant breeding. It is argued that the position of patents, combined with technological developments, has led to substantial consolidation of breeding companies in recent decades.

Farmers and producers fear that their freedom of choice will be threatened and that no varieties specifcally meeting their needs will be developed for certain crops (orphan or underutilized crops). Genome editing holds great promise for increasing crop productivity, and there is particular interest in advancing the breeding of orphan crops, which are often burdened by undesirable characteristics resembling wild relatives. In order to diversify cropping systems, orphan or underutilized crops are better adapted to local or marginalized environments [8].

**Access for Small and Medium-Sized Enterprises (SMEs) to Patents** The high cost of patenting innovations and high patent licence fees can be a barrier to market entry for SMEs (together with high business concentration) due to; (i) the complexity of patenting and the resulting monopolization, (ii) the licensing of patented products and the respective transaction costs, and (iii) the lack of transparency and FTO analyses, *e.g.* due to the complex patent landscape of the CRISPR technology. These same aspects may limit access to NGTs as mentioned in the EC study on the status of new genomic techniques in 2021.

# *2.2 Some Solutions of Collaborative Licensing Models: The Patent Pools*

Alternative collaborative licensing models such as patent pools and IP clearing houses may play a signifcant role in facilitating access to patents and untangling a potential patent "thicket".

A patent pool for the purpose of joint package licensing is an agreement between two or more patent owners to license one or more of their patents as a package either to one another or to third parties willing to pay the associated royalties [9]. The package is managed either directly by patentees to licensees, or indirectly through a new entity specifcally established to administer the pool. The frst licensing pool was established in 1856 among members of the sewing machine industry. More recently some pools are being established in biotechnology, such as the Golden Rice pool or the SARS-1 (severe acute respiratory syndrome) pool.

This package of IPRs is then licensed on a non-exclusive basis, providing licensees with affordability and FTO, while giving licensors adequate royalty returns [10]. A patent pool can provide competitive advantages by integrating complementary technologies, reducing transaction costs, clearing blocking positions, and avoiding costly infringement litigation. By promoting the dissemination of technology, patent pools can be pro-competition.

Classically, the creation of a patent pool involves four major characteristics. (1) It is built around the voluntary inclusion of key and specifc IPR holders. (i) "Key" patents are those that have no economically viable substitute; a party needs a license to use the IPR. Key patents are those for which the application is general, such as a technique that applies to all genomes. (ii) "Specifc" patents are those for which the application is specifc (such as a modifcation of a genetic sequence for a given trait in a given species). (2) It relies on a model. A model is an attempt to encapsulate the details and patents necessary to enable uniformity of practice across a diverse range

of implementations. (3) It requires pool administrators to conduct an in-depth and continual search of the patent landscape. Thus, a patent pool must be open to all IPR holders, but each patent must be analysed individually to determine if it is needed before possible inclusion; independent high-level scientists and lawyers analyse both the patent landscape and the potential key patents. (4) It can be subject to anticompetition laws. Any issues regarding anti-competition laws and patent pools have largely been resolved following the decision of the Department of Justice to grant MPEG LA pro-competitive clearance in 1997. The starting point for an anti-trust analysis of any patent pool is an investigation of the validity of the patents and their relationship with each other.

In the absence of a patent pool, users (licensees) have to enter into negotiations with all relevant patent holders, which is a time-consuming and expensive process. In the presence of a patent pool, licensees turn to the patent pool for the rights as one package, which results in simplifcation and a signifcant reduction of transaction costs.

#### **Example: MPEG LA**

The platform MPEG LA, an independent licensing agent based in Denver (USA), aims to pool CRISPR-Cas9 patents into a one-stop licensing point (accessibility to a multi-user market will maximize CRISPR's life-enhancing potential) [11]. Its model offers fair, reasonable, and non-discriminatory access to essential IP from multiple patent holders under a single license as an alternative to separate licenses.

By assisting users with the implementation of their technology choices, MPEG LA offers licensing solutions that provide access to fundamental IP, freedom to operate (FTO), reduced risk of litigation, and predictability in the business planning process. In turn, this enables inventors, research institutions, and other owners to monetize and accelerate market adoption of their assets in the global market while also substantially reducing the cost of licensing.

The CRISPR-Cas9 Reference Model is authored by MPEG LA for MPEG LA's use in the formation of *a CRISPR-Cas9 Joint Licensing Platform* which aims to provide one-stop, worldwide licenses to CRISPR-Cas9 patent rights as a convenient alternative to negotiating separate licenses with individual patent owners, and pursues the broader purpose of fostering innovation in genome engineering and accelerating the development and deployment of CRISPR-based products, therapies, and services. The Reference Model is intended only to support the effciency of a single licensing transaction that allows access to as many specifc patents as possible for the beneft of the market and is consistent with applicable legal requirements. Inclusion requires that at least one claim is directed to the CRISPR-Cas9 System. MPEG LA's *CRISPR Cas-9 Joint Licensing Platform* will give technology owners the opportunity to share in mass-market royalties from their CRISPR technology while also enjoying, with other developers, broad access to other important CRISPR technologies. As a voluntary, market-based business solution to the problems relating to patent access, designed to balance and resolve competing market and public interests, an independently managed patent pool represents the greatest opportunity to unleash CRISPR's full potential for the beneft of humanity.

To ensure that no single party has control over the licensing of the package, the organisation works with all the included patent holders to create a single set of licensing terms and conditions, upon which all included patent holders must agree. The patent holders also hold most of the enforcement powers. While MPEG LA can enforce contractual provisions, it does not fle patent enforcement lawsuits on its own; instead, it must notify the patentees that they may want to fle an enforcement suit.

MPEG LA develops its "CRISPR-Cas9 Reference Model", which describes how to patent essentiality will be determined with respect to the CRISPR platform (the CRISPR-Cas9 System is defned). This outline of essentiality in MPEG LA's Reference Model is meant to encompass all patents key and specifc to the underlying CRISPR platform. If a patent meets the established criteria, it will be eligible for inclusion in the pool. To collect these platform patents, MPEG LA is seeking "target-agnostic" patents that do not require a specifc genome. The Reference Model discloses the criteria for pool inclusion. MPEG LA's initiative was to provide a worldwide non-exclusive license to multiple patents held by multiple entities in a single transaction.

# *2.3 Some Solutions of Collaborative Licensing Models: The Clearing House Models*

An alternative mechanism supporting licensing negotiations is the clearing house model. The term clearing house is derived from banking institutions and refers to the mechanism by which cheques and bills are exchanged among member banks in order to transfer only the net balances in cash. The platform may provide information on patented technologies, bring together potential providers (licensors) and users (licensees) of patented technologies, and may provide additional services, *e.g.* negotiating licensing conditions, and collecting and distributing royalties. An IP clearing house was analysed for agricultural biotechnology [12].

They are fve models of clearing houses: (1) The information clearing house, (2) The technology exchange clearing house, (3) The open access clearing house, (4) The standardized licenses clearing house and (5) The royalty collection clearing house (RCCH). The RCCH model could be useful in providing access to and use of patented inventions in NGTs and novel traits.

The RCCH comprises all the features of the previous models (1, 2 and 4), but also collects license fees from users on behalf of the patent holder in return for the use of certain technologies or services. For the user, RCCH organizations would simplify licensing negotiations and, therefore, facilitate access to and use of patented inventions. For the patent holder, increased visibility of their patent rights and the streamlining of royalty collection and monitoring may lead to a rise in licensing and thus, licensing revenue. At the same time, awareness and respect for IPRs may grow among researchers and their public and private institutions, leading to decreased enforcement costs through fewer infringements. Hence, a reasonable price for licensees (royalties, transaction costs) and licensors (royalties, transaction costs, and enforcement costs) may be achieved. The patent holder is reimbursed by the clearing house pursuant to a set allocation formula. In addition, a RCCH may offer other services such as the monitoring of patents transferred to the clearing house or an independent dispute resolution mechanism.

However, the RCCH may have some drawbacks. (i) Patent holders may be reluctant to voluntarily participate in it. They would have to grant a licence to the clearing house which would then issue licences to all applicants without discrimination and on a non-exclusive basis in accordance with competition law. As a consequence, patent holders would lose some control over their business licensing strategy. (ii) Unless the RCCH represents a high proportion of all relevant patented inventions, it might not be a viable and effective alternative, nor could it prevent the emergence of an anti-commons effect. (iii) RCCH might be more complicated and costly to set up in comparison with the other clearing house models. Experts (high-level scientists and lawyers) will have to be hired to evaluate the often very complex patents, to match licensees with the patented inventions, to develop standardized licence agreements, and for monitoring and dispute resolution purposes. (iv) The standardized licences might not allow for measures highly appreciated in commercial licensing practices, such as the setting of milestones, due diligence, and the maintenance of long-term business relationships. (v) The exchange of relevant technical know-how is often fundamental for the smooth application and further development of a patented invention. Know-how is generally protected as a trade secret, but the clearing house will probably not be able to guarantee the preservation of secrecy when know-how is exchanged. Thus, with respect to complex technologies, direct negotiations between the licensor and the licensee on the issue of know-how may still be required, which may diminish some of the advantages of the royalty collection clearing house. This drawback might be a reason to advocate for the establishment of an RCCH that is limited to inventions that do not require the exchange of technical know-how, such as patented DNA sequences and mutations.

In the absence of a clearing house, licensees must enter into negotiations with all patent holders. In the presence of a clearing house, licensees turn to the patent pool for the acquisition of required rights.

#### **Example: Agricultural Crop Licensing Platform (ACLP)**

The ACLP, located in Brussels (Belgium), is open to all private or public sector organizations involved in plant breeding or trait research and development, and having employees and tangible assets in the 'territory' (the geographical scope of the ACLP is 38 member states of the EPO along with Russia and Ukraine). The initiative is currently driven by 10 European plant breeding companies and trait developers representing a wide range of agricultural crops and includes small, medium-sized, and large companies. The ACLP is fnanced by membership fees. Small members enjoy free membership during the fve frst years of the existence of the ACLP. The scope is all patented traits present in commercial varieties that are sold on the open market in the 'territory' ("Trait") and all agricultural crops as defned by CPVO (The *Community Plant Variety Offce* is the EU agency responsible for protecting plant variety rights (PVR) in the EU). The ACLP provides an innovative, simple legal framework for all breeders to use and enables transparent access to patented traits including genome edited traits.

This initiative provides breeding and guaranteed marketing rights for commercial traits which in turn fosters the transfer of technology. Transparent information on all commercial varieties that contain patented traits within the territory is shared via PINTO (*Patent Information and Transparency On-line*). PINTO was established with the aim of improving transparency regarding plant varieties that might fall under the scope of patents or patent applications and is updated continuously by *Euroseeds*.

The entitlement of members to a commercial variety containing a patented trait is established as soon as it is sold by a member on the open market in the Territory. Member's rights are; (i) to obtain a non-assertion agreement from the patent holder for breeding in the Territory with the patented trait(s) including using its specifc markers, (ii) to obtain a commercial license from the patent holder for the production and sale of the varieties, bred under the non-assertion agreement and containing the patented trait(s), within in the Territory.

The key expected outcomes of the ACLP are as follows; (i) novel patented plant traits, including those produced by new genomic technologies, are available among ACLP members on fair conditions, (ii) the sustainable development of novel varieties be enabled by information and rights shared amongst members, under the framework of the ACLP, (iii) the facilitation of technology transfer will make it easier for the ACLP members to further innovate.

Principal rights and obligations applicable to members of the ACLP Initiative are; (i) members are entitled to any commercial variety containing a patented trait, as soon as is sold by a member on the open market in the Territory; (ii) for members to obtain from the patent holder, a non-assertion agreement for the breeding of plants containing patented trait(s), including using its specifc markers, within the Territory, (iii) for members to obtain from the patent holder, a commercial license for the production and sale of varieties, bred under the non-assertion agreement and containing the patented trait(s), within the Territory.

Licensing can take the form of the *Standard License Agreement* (SLA) of the ACLP in which case only royalties need to be agreed between the patented trait holder and the interested member. In circumstances where no agreement on royalties is reached within a prescribed period of time, a process known as "Baseball" Arbitration [13] begins.

The ACLP will foster bilateral agreements between members as the standard licensing agreement will not ft all specifc factual situations best. The ACLP gives never less assurance that a commercial license will be available under the *Standard License Agreement* (SLA). For breeding activities, the ACLP acts as a technology exchange clearing house: all members get a non-assert for breeding activities which is not available in all states of the territory. The standardized licenses clearing house is also a component of the ACLP.

# **3 Success and Acceptability of Licensing Platforms and IP Clearing Houses**

# *3.1 The Public Sector*

The public sector plays an important role in fundamental research and represents a substantial source of IP in agricultural biotech. There are quantitative and qualitative distinctions between the public- and private-sector IP portfolios (1/3 of CRISPR patents are held by private companies) [2]. It is important to distinguish between 'enabling' technologies representing the research tools needed to create NGT varieties, and 'trait' technologies which provide the genetic basis for new functionalities. The public sector contributes more and more to the discovery of new trait technologies. The extent to which private and public-sector inventions can be used to assemble a platform of enabling technologies and gene-trait technologies suffcient to develop (new edited varieties) have been examined [12]. A major challenge facing the management of public-sector IP is the high degree of fragmentation of technology ownership across numerous institutions, especially in light of the need for multiple technology components to provide FTO in edited crops. Based in part on some of the economic principles of an 'IP clearing-house,' a model of publicsector collaboration, including data sharing and patent pooling, has recently been suggested as a solution that could directly address this issue. It seems that the technologies patented by the public sector might indeed be capable of providing a platform of technologies that could be suffcient to enable the development of new NGT varieties and cultivars. Such a strategy may be particularly important in the future for sharing access to key enabling technologies and enabling innovators to develop and deploy the trait technology projects with the public sector. Public funding for research emphasizes the discovery of a wide spectrum of previously unknown gene functions whereas private research focuses on application-driven research in a narrower range of established product lines.

# *3.2 Ethical Licensing*

Ethical licensing is one way of thinking about the role of universities and other public institutions in the regulatory process, but there is a wealth of other initiatives promoting ethical licensing. IP pooling, clearing houses and open source initiatives are examples of private ordering mechanisms that differ from solutions aimed at changing or harmonizing the legislative framework in that they are generated by the users themselves. Thus, a voluntary pool or a clearing house model could promote commercialization and include provisions for royalty-free research use by public institutions, while addressing ethical concerns about particular CRISPR applications.

A successful patent pool could do more than solve licensing issues (fragmentation and downstream litigation, the patent thicket and licensor issues such as the surrogate licensing model). It could also mitigate ethical concerns. Point 9 in the *Nine Points* to Consider in Licensing University Technology (AUTM, Washington, DC, 2007) states: "*Through thoughtful management and licensing of intellectual property, however, drugs, therapies, and agricultural technologies developed at universities can at least help to alleviate suffering from disease or hunger in historically marginalized population groups*"**.**

License restrictions could be used, in theory, to require that R&D funds be used, in part, to ethically promote access to NGTs (access for SMEs or to research/biotech institutes in developing countries) and also to develop traits for orphan crops in developing countries.

MPEG LA and its prospective licensors could potentially solve said ethical dilemmas through ethical constraints in their licensing agreement (ethical limitations in their licensing agreements by granting licenses for only very specifc applications). This is largely the case with *Monsanto*'s license from the Broad Institute covering the use of CRISPR-Cas9 for a variety of agricultural purposes in 2016. This non-exclusive license agreement will deliver a wide array of crop improvements to global agriculture. The Broad Institute's license to *Monsanto* covering the use of CRISPR-Cas9 for a variety of agricultural purposes requires *Monsanto* to allow its farming customers to save and re-sew seed from one season to the next, in contrast to some of *Monsanto's* past practices. Requiring this of Monsanto provides greater access to the outcomes of CRISPR technology to farmers, who would otherwise be required to purchase expensive new seeds each year from *Monsanto*. The Broad Institute has used ethical limitations in their licensing agreements by granting licenses for only very specifc applications (discussing Broad's non-exclusive licensing with *Bayer* for specifc agricultural applications such as genetic modifcation of plant varieties). In October 2017 *DuPont* entered into a joint non-exclusive licensing agreement with the Broad Institute for use in commercial agricultural research and product development (except gene drive and tobacco for human use) on the CRISPR-Cas9 IP held by the Broad Institute and its collaborators.

Only genetic modifcation (mutation, allele replacement or gene insertion) by NGTs can be patented. Prohibiting patents on native sequence, crossover processes and random mutations is important in defending the principle that all the information contained in a plant genome belongs to the scientifc heritage of humanity.

# *3.3 Open Licensing Systems (with CRISPR IP Research Tools Available)*

Companies would beneft from a CRISPR patent pool that would provide a nonexclusive license to CRISPR as a research tool, and it would appear that many of the exclusive licenses already granted would permit this feld of use to be included in a pool license.

In the context of CRISPR, this non-commercial use is done through a non-proft repository and licensor of patents on CRISPR technologies for academic organizations. *AddGene*, a company (a non-proft organization) based in the USA and UK, serves academic and non-proft institutions that provide access to CRISPR constructs and plasmids through a standardized Biological Material Transfer Agreement (BMTA). *AddGene*'s BMTAs contain patent licenses for the academic use of the underlying technology. For example, the University of California, the Broad Institute, and hundreds of other institutions have agreed to make many of their CRISPR IP research tools available for free or at a reduced cost through *AddGene* [14].

# *3.4 Patent Quality*

The scope of patents should be clearly defned for two major reasons. (i) When the product (a trait in crops) is only vaguely defned and its essential character is diffcult to determine, the utility of the pool may be diminished in upstream research, as it may be too narrow to be useful for product development. (ii) Biotech patents tend to be incomplete meaning that the innovation must be completed before the fnal product is incorporated and brought to market. Incompleteness and long development cycles can make it diffcult to defne specifc patents. This can be a problem because, if a pool does not contain specifc rights, the pool may no longer be a onestop licensing point for potential licensees.

# **4 Conclusion**

Although patent pooling (licensing platforms and IP clearing houses) is an attractive solution for the licensing of IPR in fragmented felds such as NGTs, there are many challenges that could hinder the formation of such a pool. Public perception of NGTs is critical to their adoption by the market. Understanding and awareness enable consumers to make informed choices, therefore it is necessary to provide consumers with information. With respect to IP and patent protection, alternative licensing models could overcome the diffculties caused by the complex patent landscape of CRISPR technology. Patent pools and clearing houses are a promising approach. When access and use to a certain technology are hindered by the existence of multiple patents, a patent pool could be a useful model to facilitate access and reduce potential litigation risk. The package of IPRs is licensed on a nonexclusive basis, allowing licensees to beneft from affordability and freedom to operate while providing licensors with adequate royalties. Non-exclusive licensing schemes could allow many companies to enter the market, creating a commercial ecosystem that has strengthened innovation and the economy. The ACLP, a clearing house, can be mentioned as a solution for products produced by the CRISPR technology for plant breeding in the agricultural sector in Europe. A CRISPR patent pool depends on the willingness of a suffcient number of IP owners to join the pool to ease licensing burdens and costs. The CRISPR platform will take off in a big way when a major company and all key players (major research institutions) announce that they will join the patent pool. The platform should be a low-complexity platform that lawyers and breeders are familiar and comfortable with.

**Acknowledgements** The author gratefully acknowledges the help of the 4iP Council and Dr. Marcel Kuntz. The views expressed in this paper are solely those of the author.

# **References**


# *Resources*

Addgene: https://www.addgene.org/crispr/

Agricultural Crop Licensing Platform (ACLP): https://aclp.eu/

Association of University Technology Managers, AUTM: https://autm.net/about-tech-transfer/ principles-and-guidelines/nine-points-to-consider-when-licensing-university

Broad Institute: https://www.broadinstitute.org/

EC Study on the status of new genomic techniques under Union law and in light of the Court of Justice ruling in Case C-528/16: https://food.ec.europa.eu/system/les/2021-04/gmo\_modbio\_ngt\_eu-study.pdf

European Patent Ofce*:* https://register.epo.org/application?lng=fr&number=EP13793997 Euroseeds: https://euroseeds.eu

IP Studies: https://www.ipstudies.ch/media/crispr-patent-analytics/

MPEG LA: https://www.mpegla.com

**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **Part IV Public and Stakeholder Perceptions**

# **Chapter 28 The View of the European Seed Sector on Genome Editing Tools in Plant Breeding**

**Petra Jorasch and Nick Vangheluwe**

**Abstract** Reconciling sustainability with agricultural productivity relies strongly on crop improvement. The continuous development of new plant breeding methods and high investment in crop research has triggered an unprecedented acceleration of progress in crop productivity. Companies active in the seed sector are innovative to meet the challenging and ever-evolving needs of growers, farmers, consumers, and other actors of the agri-food value chain. Communication and stakeholder engagement are extremely important to identify shared values and goals in the transition to more sustainable food systems. The seed sector is characterised by a multitude of collaborations and partnerships that contribute to the development of improved, high-quality, and diverse plant varieties that address specic market needs. In this article, we describe on behalf of Euroseeds the view of the European seed sector on plant breeding innovation.

**Keywords** European seed sector · Plant breeding · Genome editing

# **1 Plant Breeding Has a Track Record to Contribute to Sustainability**

Plant breeding involves processes by which new technologies and ndings from plant sciences and other research domains are transformed into improved plant varieties. Plant breeding has strongly contributed to increased yields and production in arable farming, and subsequently to improved market and trade conditions, increased food availability, higher economic prosperity and additional farm income while avoiding additional land use, greenhouse gas (GHG) emissions, and loss of biodiversity [1].

Scientists have shown for instance for wheat, which is a major staple food around the world, that resistance breeding can contribute to achieving the United Nations

Euroseeds, Avenue des Arts, Brussels, Belgium

P. Jorasch (\*) · N. Vangheluwe

e-mail: PetraJorasch@euroseeds.eu; NickVangheluwe@euroseeds.eu

A. Ricroch et al. (eds.), *A Roadmap for Plant Genome Editing*, https://doi.org/10.1007/978-3-031-46150-7\_28

Sustainable Development Goals (SDGs) [2]. They were able to show that resistance breeding in wheat has increased crop production, and hence the economic proftability due to a reduction in the use of fungicides. Another study could verify that fve decades of wheat breeding progress in western Europe has enhanced cultivar performance not only under optimal production conditions but also in production systems with reduced agrochemical inputs. New cultivars incrementally accumulated genetic variants conferring favourable effects on key yield parameters, disease resistance, nutrient use effciency, photosynthetic effciency, and grain quality [3].

Europe's seed sector is committed to delivering on the SDGs (2, 3, 8, 9, 12, 13 and 15) by improving the sustainability of food production, by maintaining and promoting Europe's high food quality and standards, by ensuring that the European agri-food sector can remain fair and competitive, and by contributing to Europe's climate, environmental and biodiversity goals. The European Union (EU) Farm to Fork (F2F) strategy includes several policy objectives that have implications for agricultural production in the EU and beyond.

Plant breeding in the EU contributes to socio-economic and environmental sustainability and can partially compensate production losses potentially resulting from the implementation of the EU F2F and Biodiversity strategies [4]. Policy makers seem to have implicitly concluded that the additional net benefts resulting from the EU F2F and Biodiversity Strategies outweigh the losses in production surplus. But studies do not support this claim without further technological and institutional changes, such as supporting the application of modern biotechnology by reducing regulatory hurdles for plant-based innovations [5]. Modern plant breeding including biotechnological tools must be enabled to provide farmers more effciently with improved plant varieties to sustainably secure their productivity [1].

# **2 The Ever-Evolving Plant Breeders' Toolbox**

Like evolution itself plant breeding depends upon genetic variability within crops and their relatives as a basis for developing new plant varieties with improved characteristics. For thousands of years, humans have been improving crops to suit better their needs. Conventional plant breeding methods, transgenesis or newer plant breeding methods are all important components of the plant breeders' toolbox (Fig. 28.1). By building on the mechanisms created by nature, most of the latest innovations in plant breeding methods simply reduce the complexity of breeding and achieve the relevant breeding goals in less time and with greater precision.

**Fig. 28.1** Integration of new plant breeding tools into the breeding cycle. Plant breeding depends upon genetic variability within crops and their relatives as a basis for developing new plant varieties with improved characteristics. Plant breeders are continuously integrating the latest methods in plant biology and genetics into their breeding toolbox to effciently use existing diversity but also to induce new genetic variation. Over the past years ever more precise and effcient plant breeding methods have been developed. This plant breeding innovation leap is based on an in-depth understanding of plant genomes and refnement of breeding methods, enabling more effcient, more precise, and faster progress in achieving the desired breeding goals

# **3 The Role of New Breeding Techniques According to the European Seed Sector**

The targeted development of improved plant varieties is important to mitigate climate change effects like new plant pests or diseases. These can be devastating to crops and lead to huge pre-harvest losses. Other new plant varieties provide quality improvements, such as better taste (*e.g.*, in fruits and vegetables), processing advantages or nutritional enhancements, such as desirable proteins or lower saturated fats. In addition, New Genomic Techniques (NGTs) -such as genome editing- are technologies that could also facilitate the improvement of so-called orphan species that have beneftted little from innovations in genetic selection as well as the domestication of related wild species thus increasing the genetic diversity within crop species [6].

The results of a survey among 62 private plant breeding companies conducted by Euroseeds highlights the enormous interest of companies in using NGTs for a wide range of crop species and traits and the negative impact of the current regulatory situation in the EU on companies' decisions for investments in NGT-related research and development (R&D) activities for the EU market and beyond [7].

The Commission study on NGTs [8] confrmed that plants resulting from NGTs have the potential to contribute to a more sustainable food system as part of the

objectives of the European Green Deal and the F2F Strategy. At the same time, the study found that the current genetically modifed organism (GMO) legislation, adopted in 2001, is not ft for purpose for these innovative technologies. This was also confrmed by the results of the public consultation conducted by the Commission as part of the impact assessment to draft a policy proposal. Almost 80% of the participants who responded to the consultation acknowledged that the current regulatory framework is not adequate for plants resulting from targeted mutagenesis and cisgenesis [9].

# **4 Communication Practices and Needs for Plant Breeding Innovation**

The EU-funded Horizon 2020 project CropBooster-P assessed European stakeholders' information and communication behaviour on plant genome editing including survey results of a total of 100 respondents from the seed and plant breeding sector [10].

The most referred crop characteristics communicated about by breeders and seed and plant breeding organisations relate to yield and yield stability. This suggests that communication about economic aspects of crop production and economic sustainability of farmers (as the customers of new seeds) are of utmost importance. Breeders and seed and plant breeding organisations stressed the importance to communicate safety as well as sustainability aspects in future efforts (Fig. 28.2). This does not necessarily imply that safety communication is a consequence of a

**Fig. 28.2** Ranking of topics for future communication efforts by breeders and seed and plant breeding organisations. N = 94

**Fig. 28.3** Word cloud of examples for effective communication based upon the free text answers in the survey from breeders and seed and plant breeding organisations

risk-focused approach. For breeders, safety and quality are important because seeds are at the basis of food safety and health requirements related to crops that are grown for food, feed, and bio-based production [11].

The potential benefts, examples of applications, and comparisons of genome editing and conventional breeding methods were identifed as the most important topics to communicate about NGTs. This indicates that the ongoing policy discussions in Europe about a differentiated regulatory framework for plants resulting from targeted mutagenesis and cisgenesis resonate with breeders.

Finally, respondents could share in the survey examples of communication relating to plant research, crop improvement and breeding or crop production which, in their experience was effective. The answers were thematically analysed to identify recurring topics, which are depicted in the word cloud below (Fig. 28.3). Most shared experiences by the seed and plant breeding sector are explaining the basics of plant breeding and its benefts to consumers and primary producers as well as providing specifc examples (*e.g.*, the need to reduce pesticide use) and engaging with interested stakeholders on feld days and participatory events.

# **5 A Multitude of Collaborations in the Seed Sector**

The seed and plant breeding sector engages with many different stakeholders as R&D activities take place along the value -and process chains from the feld to the shop counter to meet the challenging and ever-evolving needs of farmers and other actors of the agri-food value chain, including consumer preferences and policy objectives. In addition, cooperation between companies, public research institutes and other actors is important to address research needs and gaps in crop improvement strategies. A multi-stakeholder perspective on the role of crop improvement in future-proofng the European food system was developed in the CropBooster-P project, which revealed that for instance examining downstream sustainability impacts will be valuable to identify concrete targets for plant breeding innovation as a food systems solution [12].

COST actions are interdisciplinary research networks that bring together researchers and innovators from academia, small and medium-sized enterprises, public institutions, and other relevant organisations or interested parties to investigate a topic of their choice for 4 years [13]. The COST Action PlantEd has been a unique platform for multi-stakeholder engagement on the topic of plant genome editing [14]. The multi-actor approach put forward in H2020 and Horizon Europe research programmes, helps to facilitate the involvement of the seed and plant breeding sector in research projects to co-develop solutions, which could be more readily applied in.

Sustainability, safety-related aspects, and transparency appear to be the most important topics for communication about plant genome editing by the surveyed stakeholders in the CropBooster-P project [10]. These stakeholders include academia, farmers, seed and breeding sector, environmental and consumer organisations, journalists, and policy makers. The topic of sustainability is primarily addressed in a supportive manner for NGTs [10]. However, environmental organisations prefer to use rejective or neutral argumentation in communication about sustainability in the context of plant genome editing applications. Interestingly, respondents from consumer organisations address the topic impartially, presumably because no genome-edited products have been released on the European market yet [10].

The survey results suggest that focussing on sustainability in future communication activities might increase the opportunity to agree on shared values among different agri-food stakeholders. This is in line with the results of another survey where a majority of those surveyed at farm-level (70%), consumer-level (66%), and plant scientist (60%) respondents to the survey chose sustainability as the most important aim for crop improvement in Europe [11]. Sustainability could thus provide a good starting point for constructive discussions about the regulatory framework in Europe. This recommendation is supported by the outcome of two citizen juries with consumer experts and societal stakeholders. They concluded that the awareness of NGTs by society might be increased when their application serves goals with a societal dimension such as environmental sustainability, resilience, and quality [15, 16].

# **6 It's Time to Act**

Europe's seed sector, technology developers and public researchers have been contributing to the development of improved plant breeding methods. The private seed sector is highly innovative and invests an average of 20% and up to 30% of its turnover in R&D of improved plant varieties [7]. Each year more than 4000 new varieties are registered for cultivation in the EU [17]. In addition, plant breeding has a

proven track record for supporting sustainable agricultural production and NGTs can provide additional sustainability options by increasing effciency and reducing complexity in plant breeding. Communicating the benefts of plant breeding, and the potential of NGTs specifcally, can be a starting point for a constructive public dialogue.

NGT applications are versatile and can be used in the development of a wide range of different plant varieties. While NGTs may for some purposes be used to introduce a transgene and consequently result in a transgenic organism (transgenes are DNA fragments outside the plant species' gene pool) [18], many other types of NGT-derived plants, e.g. those derived from targeted mutagenesis and cisgenesis, are similar to those that could occur in nature or be produced by conventional breeding methods [18], e.g. by induced random mutagenesis or backcross breeding. The European Food Safety Authority (EFSA) [19] concluded that certain plants obtained by targeted mutagenesis and cisgenesis do not pose any new hazards compared to plants developed by conventional breeding.

Plant varieties developed through the latest breeding methods should therefore not be subject to different or additional regulations if they could also be obtained through earlier breeding methods or result from spontaneous processes in nature [20].

Worldwide, there is a growing number of countries that implement differentiating and enabling policy approaches for NGTs. Their regulatory decisions consider the genetic characteristics of the genome edited organism, and whether the changes introduced in its genome can (or cannot) occur naturally [21]. One of the most recent examples of such enabling policies is the United Kingdom (UK) Precision Breeding Act. [22]. The bill excludes precision bred organisms from the GMO legislation and aims to encourage agricultural and scientifc innovation in the UK and could unlock the potential of new technologies like genome editing to promote sustainable and effcient farming and food production. EU agriculture and some other of the EU's most innovative sectors are at risk of being deprived of scientifc progress, putting them at a competitive disadvantage compared to their counterparts in other countries.

Therefore, Europe should join the increasing number of countries that pursue a differentiated and effcient regulatory approach. The future legislation must provide clarity through principal defnitions and unambiguous criteria that allow to clearly determine whether plants resulting from certain NGTs fall into the same category as conventionally bred plants (and thus should be regulated alike) or constitute GMOs according to the respective regulatory framework.

The regulatory framework for GMOs in the EU (2001/18) is unworkable, specifcally for SMEs which constitute 90% of the EU's seed and breeding sector [7]. The lengthy and costly procedures risk neutralizing any effciency gains in breeding. Currently a GMO import approval takes more than 5 years between application and approval of the GMO dossier, the last and the only cultivation approval that realized into GMO cultivation in the EU dates to the end of the 1990ies. That makes it unattractive for companies to invest in those technologies. Under such conditions, NGTs will not deliver on the goals of the F2F and Biodiversity strategies.

Consequently, Europe's leading position in innovative breeding is at stake, as are the EU's sustainability goals, jobs in agriculture, their associated value chains, and international trade fows.

# **References and Resources**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **Chapter 29 The Awareness of the Polish Society on New Genomic Techniques**

**Wiktoria Mołodziejko, Justyna Nowakowska, and Anna Linkiewicz**

**Abstract** New genomic techniques (NGTs) are challenging from an ethical perspective as well as from the legal and regulatory point of view. Acceptance of solutions by society is a factor that can determine the success of a technology. Increasingly, there is a growing gap between the solutions offered by biotechnology and the public, which with certain ambiguity evaluates biotechnological innovations. Here we investigate the level of knowledge of Polish society on NGTs and the perception of this techniques. Our survey was carried out on a representative group of Polish citizens from August 2022 till January 2023, and was based on a 3-part self-developed questionnaire. Questions concerned: (i) demographic characteristics, (ii) the knowledge of the NGTs including the legal state of the NGT after a ruling European Court of Justice (Case C-528/16) and (iii) respondent's attitudes towards the NGTs. As result, only 15% of respondents (n = 194) gave the correct denition of NGTs, 43% the respondents never encountered the term NGTs, and 35% could not dene it. The majority of respondents recognised the potential benets of using the technology in agriculture as well as in healthcare, but they were not convinced about the potential personal use of NGTs if they were allowed in Poland.

# **1 Introduction**

New genomic techniques (NGTs) represent a signicant breakthrough and longawaited methods for site-specic mutations in DNA and targeted genome editing, offering promising new tools for plant and animal breeding and nally for more

W. Mołodziejko

Faculty of Biology and Environmental Sciences, Cardinal Stefan Wyszynski University in Warsaw, Warsaw, Poland e-mail: wiktoriamolodziejko1@gmail.com

J. Nowakowska · A. Linkiewicz (\*)

Laboratory of Molecular Biology and Genetics, Institute of Biological Sciences, Cardinal Stefan Wyszynski University in Warsaw, Warsaw, Poland e-mail: j.nowakowska@uksw.edu.pl; a.linkiewicz@uksw.edu.pl

A. Ricroch et al. (eds.), *A Roadmap for Plant Genome Editing*, https://doi.org/10.1007/978-3-031-46150-7\_29

sustainable food production. The use of NGTs allows to increase the effciency and precision of mutations compared to the current breeding methods and can lead to the rapid obtaining of desired plant varieties and animal breeds [1, 2]. However, like any new technology, it has its pros and cons that can be highlighted in the public debate and infuence consumer decisions. So far, there is no international consensus on whether and how the products of certain NGTs techniques like CRISPR/Cas9 or TALENs should be regulated and whether their use would fall within the genetically modifed organisms (GMOs) regulatory framework [3]. The European Union (EU) defnes NGTs as 'techniques capable of changing the genetic material of an organism and that have emerged or have been developed since 2001, that is, after the existing EU legislation on GMOs was adopted' and the products of NGTs are regarded as GMOs after the controversial decision of European Court of Justice (Case C-528/16) [4, 5].

The authors are interested in how much EU citizens are aware of the possibilities brought by the use of the CRISPR/Cas9 and other NGTs methods, and whether they know how much EU law regulates the use of these methods. This paper presents the frst comprehensive survey of public opinion of Polish citizens on NGTs. The survey was carried out from August 2022 till January 2023 on the basis of a selfdeveloped questionnaire, inspired by the questions present in the public consultation study on NGTs launched by the European Commission (EC) in April 2022 [6]. This modelling will allow for a comparative analysis of public attitudes in Poland and other EU countries. The main purpose of the study was to obtain broad information on the reception of the latest biotechnological inventions that may affect people in Poland. Our results can also be used in assessing the attitudes of Poles towards biotechnology advances, including GMOs, a study that has been conducted since the late 1990s [7–10].

# **2 Material and Methods**

# *2.1 The Survey*

The survey was conducted in Poland between August 2022 and January 2023 using an anonymous self-designed questionnaire, planned as a 3-part study. The frst part of the survey was related to the socio-demographic characteristics of the respondents such as gender, age, education, occupation and place of residence (city size and information on the region of residence in Poland) (Table 29.1).

The second part of the survey concerned general knowledge about NGTs. The questionnaire contained closed single- or multiple-choice questions, where people frst explained the meaning of the term NGT (Table 29.2). In case of an answer other than "I do not know", respondents took part in the rest of the questionnaire, which included questions 2–6 (Table 29.2). This third part of the survey included detailed questions on the use of and attitudes towards NGTs and products. The design of the survey was coordinated with sociologists from Cardinal Stefan Wyszyński University in Warsaw. The survey was conducted in Polish.

**Table 29.1** Demographic characteristics of the Polish population involved in the study


**Table 29.2** Questions and choice of answers included in the survey


(continued)


# *2.2 Characteristics of the Respondents*

The survey was addressed to farmers, agricultural and food workers and professionals supporting agricultural production, as well as students of environmental sciences. All contacts and emails to which the questionnaires were sent were found in the recipients' websites or came from the website of the Ministry of Agriculture and Rural Development.

The survey was sent via Gmail (Google, California, USA) to agricultural associations, the dairy industry and other food producers, biology students and teachers (N = 194). Everyone was able to contribute via an online questionnaire available at the link to Google Forms (Google, California, USA).

The authors selected this group of respondents based on their potential level of knowledge in the feld of biology and biotechnology. In addition, professionals and students were selected because they represent a population group that is receptive to current trends. As consumers, they can initiate positive changes in the development of agricultural biotechnology. The authors assumed that such a group of recipients would be less likely to drop out after the frst question of the test because of their education and profession.

# *2.3 Statistical Analyses*

The data reported here have been weighted to be nationally representative, with a margin of error of 5%. Pearson's chi-square test was used to compare the frequency of responses between the different groups, with P values of <0.05 considered statistically signifcant. All statistical analyses were performed using Statistica 13.3 (StatSoft, Krakow, Poland).

# **3 Results**

# *3.1 Demographic Characteristics*

Most of the Polish respondents were male (51%), lived in rural areas (52%) in the central (36%) and eastern (33%) parts of the country and had completed tertiary education (56%). Among the professional groups surveyed, animal technicians dominated (51%). Respondents came from different parts of Poland and represented all regions (Table 29.1).

# *3.2 Knowledge About NGTs Among Polish Citizens*

A total of 194 Polish citizens participated in the second part of the questionnaire (Table 29.2, question 1), while 111 respondents were included in the third part (Table 29.2, questions 2–6; Table 29.3). The results show that 111 respondents (57%) knew the term NGT, but only 30 (15%) of the respondents gave the correct defnition of NGT. Almost half of the respondents, 83 people (43%), had never heard of NGTs (Table 29.2).

Respondents answered questions 1–4 regardless of gender, age, education, occupation and place of residence (Table 29.3). For question 2, respondents were almost evenly split between the answers: "agri-food industry, e.g. to produce food with lower levels of pesticides" (28%), "they allow more precise and faster plant breeding and obtaining new varieties that ensure yield progress" (30%), and "medicine, e.g. in the fght against certain diseases such as cancer or genetic diseases" (32%) (Table 29.2).

Regarding knowledge about the availability of NTG products on the market, 38% of the respondents indicated that they are available outside the EU, e.g. "soybeans with an improved fatty acid profle", while 40% indicated that they are "available in the EU and in Poland" (Table 29.2, question 3). Almost half of the Polish respondents (48%) were aware of EU legislation on products produced using NGTs (Table 29.3, question 4).

When asked in which sectors of the economy NGTs could possibly be used, most responses pointed to use in healthcare (32%), but a similar number of responses (30% and 28%, respectively) pointed to use in plant breeding and in the agro-food industry. In addition, 2% of respondents do not think NGTs should be used or have no opinion (8%).

The survey revealed that majority of respondents (48%) correctly recognised that NGT organisms are subject to GMO requirements under Polish and EU law. Unfortunately, a 42% of respondents had no answer to this question, while a similar number of respondents believed that NGTs are fully allowed and widely used in agricultural and food production in the EU, or that products from NGTs methods are prohibited by law and cannot be used at all. The only signifcant differences in responses regarding demographic characteristics were found in questions 5 and 6 (Table 29.3).


**Table 29.3** Effect of gender, age, education, occupation and place of residence on responses to each of six questions by 111 respondents, based on chi-squared test (signifcant P-value highlighted)

Questions after Table 29.2

After analysing the data in question 5 regarding the relationship between place of residence and respondents' opinion on whether NGT products should be used in agriculture, it was found that the majority of respondents (64%) indicated that NGT products can be used but should be subject to European Union law. Residents of urban areas, especially cities with <20,000 inhabitants, were in favour of this solution (89%). Interestingly, it was found that 39% of people from rural areas were not interested in legal regulations for NGTs products (Table 29.3, p = 0.01294).

The next statistically signifcant result was the correlation between gender and willingness to use NGT products once they appear on the market (Table 29.3, p = 0.04930). The results obtained indicate a discrepancy between the responses of women and men. In the survey, about 56% of women had no opinion, while 39% of men were willing to use NGTs products (data not shown). The relationship between education and willingness to use NGTs products once they are on the market was also observed (p = 0.01368). Those with tertiary education were more likely to use NGTs products (37%) than those with secondary education (14%).

# **4 Discussion**

Genome editing methods are already applied for a diverse range of plants and animals, and at the same time there is an active debate about the possible use of these techniques in the EU. The authors of this study wanted to know to what extent EU citizens are aware of the possibilities offered by the use of CRISPR/Cas9 or other NGT methods and whether they know to what extent EU law regulates the use of these methods. The aim of the survey was to determine the knowledge and attitudes of Polish citizens, who have come into contact with agriculture as a result of their education or profession, towards the practical applications of genome editing methods, in particular CRISPR/Cas9 and TALEN methods. Questions were asked about the knowledge of the applications of the aforementioned technologies, about the legal aspects of using the methods and about people's attitudes towards the NGTs.

The survey revealed a low level of awareness of NGTs among Polish citizens. Almost half of the respondents (43%) said they had never heard of the term NGTs, and another 35% had heard of it but could not defne what it meant. Only 15% of respondents gave the correct defnition of NGTs. When the group was asked whether NGTs products are already on the EU market, 40% of respondents said that they are now present in Poland and on EU markets. An even lower level of awareness of NGTs was reported in Japan, where only 7% of respondents who did not have genetic diseases were aware of the term "gene editing" [11]. In Costa Rica, 4% of respondents had heard or read a little (2%), some (1%) or a lot (0.6%) about CRISPR/Cas9 [12]. Education may also infuence responses. Individuals with higher education were more aware of genetically modifed products [13–15], in contrast to some data from the defcit model, which showed that scientifc knowledge was only weakly associated with attitudes towards the technology [16]. In the current study, the authors were not able to fnd this correlation, but there is a clear link between education and willingness to use NTG products.

To better understand the public's attitude towards NGT technology, we asked for their opinion on the legal aspects of using NGT products by asking the question, 'What legal aspects should be fulflled for the technology to be used in agriculture'? Here, most respondents (63%) were willing to allow the use of this technology if it was regulated by Polish and EU law. Interestingly, 9% of respondents did not want the products of these techniques to be regulated by law, but wanted them to be treated like the products of classical breeding methods. The majority of participants of the EU consultation survey [6] believed that the existing provisions of GMO legislation are not appropriate for plants obtained by targeted mutagenesis or cisgenesis. According to the study published in 2021 by EC, based on targeted consultations on the status of NGTs in EU legislation and in the light of the Court of Justice's ruling in Case C-528/16, the main concerns were related to safety aspects raised by consumers, farmers, food producers and other stakeholders [5]. Most respondents expressed different, sometimes conficting, views on the level of safety of NGT and their products, and on the need and requirements for risk assessment. Some ethical questions concerned the application of these techniques, not the techniques themselves.

In response to the question "In which economic sectors can NGTs be used?" 32% of respondents indicated medical purposes, which is in line with the results of similar work in the EU [17, 18]. However, a smaller number of respondents (2%) in our survey thought that NGTs should not be used at all or had no opinion (8%). A more personal question concerned the willingness to use existing NGT products if they were available in Poland. Most of the respondents (46%) had no opinion on this, 27% would use them, while 14% would not use NGTs products and 13% saw no use for these techniques. Women were more conservative, as only 16% would use them, while men were less sceptical (39%). The observed results are in line with the research of scientists from USA or Israel [19, 20]. They pointed out that women have a stronger aversion to innovation, a lower interest in technology, a greater concern for the environment and a higher perception of environmental risks.

In 2020, Müller et al. [20] summarised the total number of tweets (in English) ever published about CRISPR/Cas9 since the frst information about the genome editing technology came out on the social media platform Twitter. The applied analysis model BERT (Bidirectional Encoder Representations from Transformers) generated groups of positive/neutral/negative sentiments expressed in n = 1,311,544 tweets about the CRISPR/Cas9 technology. The information conveyed in the tweets generally concerned unspecifed organisms (51%) or humans (30%), and plants were represented in only 5% of the tweets. The study concluded that overall, thematic tweets were initially received very positively (52%) compared to natural (40%) and negative (7.5%) tweets, but decreased over time [20]. Since 2015, various ethical concerns have been raised, especially in the feld of human genetics, which also shed a negative light on NGTs applied in the plant sector. Nevertheless, the topics of disease and medical treatment were considered important in the discussion, and was associated with a positive attitude among people, which was also evident in this study.

The further development of the new genomic techniques is dependent on legal decisions restricting or supporting the technology, but will likely depend as well, to a certain extent, on the acceptance and the decision that a citizen makes in an election and a consumer makes in a purchase. It seems that an analysis of public acceptance of these biotechnological solutions is important for predicting the economic development and economic prospects of companies and investors operating in this feld [21].

# **5 Conclusions**

The study aimed to answer the question of whether the new genomic techniques, referred to by the acronym NGTs, are known in Polish society, and if so, whether their use is accepted, in what areas and to what extent. Opinion polls, despite their limitations in terms of sampling, reliability and accuracy of conclusions, are an important means of obtaining data for comparative analysis.

The results of our survey, which analysed a total of 194 questionnaires, offer some new insights into the current awareness and perception of NGTs by Polish citizens. In general, it was found that concepts such as genome editing, CRISPR/ Cas9 and TALEN are hardly known. The majority of respondents acknowledged the potential benefts of using the technology in agriculture or healthcare, but with appropriate regulations that would be guaranteed by the EU. Nevertheless, respondents are not convinced about the personal use of CRISPR/Cas9 if it were available in Poland. For most of the questions asked, there were no statistically signifcant differences in the answers, regardless of gender, age, place of residence, occupation or education.

In order to solve the problems of modern agriculture, healthcare or environmental protection, new biotechnology solutions should be developed and disseminated. Despite considerable interest in research on NGTs in the EU [5, 22], Poland, as part of the EU, is on the way to accepting and implementing this technology in agriculture or in the medical feld. We believe that the dissemination of information and advisory services, along with research and development, are crucial factors for the promotion and adoption of any technology, especially in agriculture. The presented study has shown a big gap in knowledge, therefore more efforts should be made, starting with education at school and university level, easy access to comprehensive and neutral information in the mother tongue, including both technical and legal issues, to inform Polish citizens about the possibilities offered by NGTs.

**Acknowledgements** The authors thank COST Action CA18111 "Genome editing in plants – a technology with transformative potential" for the possibility of getting and exchanging knowledge about NGTs. We would like to thank Dr. Justyna Marchewka-Długońska (UKSW) for the valuable comments on the statistical data, Dr. Izabela Bukalska (UKSW) and Dr. Joanna Wróblewska-Skrzek (UKSW) for the help in the formation of the questionnaire.

# **References**


22. Parisi, C., Rodriguez Cerezo, E.: Current and Future Market Applications of New Genomic Techniques, EUR 30589 EN, Publications Ofce of the European Union, Luxembourg, ISBN 978-92-76-30206-3 (2021)

**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

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# **Chapter 30 Improving Science Communication About Genome Editing – Mitigating Strong Moral Convictions Through Shared Moral Goals**

### **Gabi Waldhof**

**Abstract** A large share of natural scientists considers the application of genetic engineering (GE) to be necessary for a sustainable transformation of agriculture. However, there is a stark divergence between scientic assessment and public perception of this technology, particularly in Germany. So far, science communication has not been able to reconcile this divergence. The chapter suggests that strong moral convictions hinder effective science communication and provides scientic evidence for this claim. Furthermore, the chapter presents research suggesting that strong moral convictions can be mitigated through shared moral goals. Building on this insight, recommendations for improved science communication about GE are formulated.

# **1 Introduction**

In the coming decades, the agricultural and food industry will face major challenges such as climate change that will affect the cultivation of agricultural crops, for example through more extreme weather conditions [1, 2]. In addition, the food supply of the steadily growing world population must be secured [3]. Also, the European Commission's goal to make the European Union (EU) economy climate-neutral by 2050 [4] also contributes to the fact that people require a far-reaching transformation to enable a sustainable economy.

G. Waldhof (\*)

Faculty of Business, Economics and Social Sciences, Department of Socioeconomics, Universität Hamburg, Hamburg, Germany

# *1.1 Genetic Engineering as a Potential Tool*

Such a sustainable economy, e.g. a bioeconomy, can in part be enabled by innovative, effcient and sustainable technologies. One such technology with high potential – that is even considered necessary by many experts [5] – is genetic engineering (GE). Here, GE refers to those genomic techniques that include gene transfer and gene editing [6]. In the broader debate, its products are often referred to as "genetically modifed organisms", "GMO", "GM foods", or pejoratively "genetically manipulated organisms" [7]. Here, the term "genetically engineered" (GE) is used, because "genetically modifed" can refer to any type of genetic modifcation, including conventional breeding [8].

Scientists involved in GE argue that GE and its products can have many benefcial trades: according to numerous scientifc studies, GE can greatly increase crop yields and thus create economic opportunities for farmers in poorer countries [9]. One reason for this is its potential to make plants more resistant to environmental stress such as extreme weather conditions like drought [10, 11]. Moreover, GE can increase the soil compatibility of cultivation and protect biodiversity, e.g. by reducing the use of chemical pesticides [9]. It can also be used to develop healthier food. One of the most famous examples is Golden Rice, which is enriched with betacarotene [12].

Furthermore, an extensive study by the National Academies of Sciences, Engineering, and Medicine [8] concludes that GE crops are not riskier than conventionally produced crops, but have generally favorable social, economic and ecological effects. In addition, among natural scientists, there is almost consensus on the safety of this technology: 88% of the members of the American Association for the Advancement of Science (AAAS) consider GE foods safe to eat [13]. German science academies are also uniformly in favor of the use of GE, for example, the Academies of Sciences Leopoldina or the Berlin-Brandenburg Academies of Sciences [14, 15]. In general, scientifc fndings convey a positive picture of the effects of GE. For example, in a joint statement, the German National Academy of Sciences Leopoldina, the German Research Foundation, and the Union of the German Academies of Sciences report increased yields and income for farmers through GE, as well as reduced pesticide use [16]. Qaim also notes that the technology can contribute substantially to sustainable agriculture and food security [17].

These study results signal that GE can address some of the current challenges, such as climate change, malnutrition, and economic hardship. For example, the German Ministry for Education and Research (BMBF) even considers GE necessary to manage such topics [18].

# *1.2 Public Perception Stands in Stark Contrast to the Scientifc Assessment*

Despite the quasi-consensus among scientifc academies, there is a large discrepancy between the natural scientists and the public's opinions on GE, particularly in Germany.

Numerous studies on the societal acceptance of GE indicate people's general rejection of such biotechnologies. For example, a recent survey shows that about half of the German population is concerned about GE food [19]. In another survey, respondents were asked to name problems and risks associated with food: GE foods were amongst the most frequently named causes of worry [20]. Additionally, even when asked for spontaneous associations with food risks, GE foods were among the most frequently mentioned items (i.e. 9%) [20]. To assess the public perception of GE, the Author of this chapter conducted a series of studies with their colleagues. Among the population, rejection remains high. A survey that is representative for the German population according to age, gender, income, education and region (N = 619), showed a rejection rate of 67% [21]. In another representative survey from 2020, 75% of participants (N = 653) stated to be more likely to reject GE [22]. In another study, this rate (76%, N = 636) is replicated. Others even found a rejection rate of 83% in Germany [23, 24]. In 2022, a study by the Federal Institute for Risk Evaluation found that 64% of consumers (N = 1001) said that they were "somewhat" to "very" concerned about GE foods [25].

# *1.3 Public and Consumer Support Is Necessary for Adequate Policies and Their Adoption*

The stable and stark rejection of GE by the German public stands in contrast to the assessments of scientists involved in GE research. This is problematic because scientifc consensus on the technology does not suffce for widespread adoption. It also requires the support of the public and consumers because public rejection can lead to adverse policies, and consumer rejection additionally hinders adoption. Without public support there are serious challenges in terms of policy making, agricultural development and science, as GE products cannot be benefcially developed and implemented.

Studies show that bans on GE prevent their potential positive effects and can even have negative consequences such as high opportunity costs, vitamin defciency, hunger, economic losses, disproportionate bureaucratic efforts, etc. [9, 11, 26–29]. A prominent example is the earlier mentioned Golden Rice, which could be part of the solution for famine in poor countries since it counteracts common malnourishment by supporting the human body to produce vitamin A [12]. However, the stark public rejection has so far prevented the cultivation of Golden Rice on a large-scale. Since this technology cannot be used for food production in Europe, or rather in Germany, its advantages cannot be used either.

If the application of GE really brings all these positive effects for environment and society, urgent action is required. In order to contribute to solving this problem, the present chapter hypothesizes that a decisive driver for the stark divergence between scientifc assessment and public perception was the way the German GE debate has been conducted in the public arena. Consequently, the present chapter suggests that this divergence can be mitigated by improving the public debate about biotechnologies, as well as its science communication.

# *1.4 Advocates in the German GE Debate Are Rarely Heard*

In Germany, particularly the terms "Grüne Gentechnik" (green gene technology) and "GMO" – pejoratively understood by the public as "genetically manipulated organisms" – have become popular [30, 31]. Here, GE foods have been heavily criticized since their emergence. Probably no other foodstuff has ever been discussed as fercely in Germany. The public debate has been ongoing since the late 1980s, and a settlement is not in sight. The arguments in the debate are manifold and often surprisingly contradictory. While opponents fear risks to human health [32], advocates report on health-promoting properties of such plants, e.g. through vitamin enrichment [12]. While proponents are convinced that increasing yields through GE increases the freedom and autonomy of farmers [33], opponents see poor smallholders in developing countries threatened by patents on GE products [34]. One might think that such contradictory views could be clarifed by examining the facts and comparing the evidence. But the public discourse could not have been resolved so easily.

In the wake of this rejection, numerous demonstrations have been organized. The most prominent is the "Wir haben es satt!" [We are fed up.] event in Berlin, where approximately 30 thousand participants demonstrate against GE every year, among other issues.

A large number of Non-Governmental Organizations (NGOs) are involved in organizing this event, such as Bread for the World or the Nature and Biodiversity Conservation Union of Germany [35]. In general, NGOs position themselves homogeneously against GE in the public debate [36]. Organic associations such as "Demeter" also reject GE [37]. Organic organizations publicly represent this rejectionist stance prominently. For example, the initiative "Ich stehe auf Essen ohne Gentechnik" [I stand for food without GE] collected more than 100 thousand signatures in 2018 and handed them over to the Federal Environment Ministry [38].

This gives the impression that not only the majority of the public but also the majority of offcial organizations reject GE. This impression is created because proponents of GE are less present in the social debate. Although agricultural associations such as Deutscher Bauernverband position themselves in favor of GE [39], it is unknown if they launched any media campaigns or initiatives. The situation is similar for biotechnology companies such as Syngenta or Bayer: they are in favor of GE, but publicly accessible information is brief and rare [40]. Political supporters also largely consider the debate to be settled – and are quite obviously withdrawing their involvement [41]. Thus, on the advocating side, for the most part, only scientists and science representatives are involved in the discourse, such as the Association for Biology, Biosciences and Biomedicine [42].

# *1.5 Moral Convictions Can Hinder Policy Debates and Science Communication*

The current situation of the public debate about GE, as well as the stark public resistance against this technology, particularly in Germany, suggest that science communication about GE has not been effective. This chapter aims to expand on why this is happening and how to solve this problem.

Particularly, based on recent research, this chapter proposes that science communication has not been effective due to strong moral convictions. This chapter further proposes that these strong moral convictions can be mitigated by providing credible, fact-based, scientifc reasons for GE that address people's dearest moral concerns.

Accordingly, this chapter takes the German debate about GE as an example to learn about the debate, improve science communication about GE more generally, and about newer genomic techniques such as "CRISPR"1 [43] in particular.

For this purpose, this chapter proceeds as follows: First, the German GE debate is discussed. Based on this, as well as on research about judgement and opinion formation, it is argued that a closer look into moral convictions related to GE is sensible. Then, it is reported on research showing that GE attitudes in Germany are mainly based on such moral convictions. It is then suggested that a ban (or approval) of GE has become a moral goal in itself, rather than a tool. However, as this chapter argues, this strong moralization can be overcome if the focus can be turned towards common moral goals. It is reported on research that successfully tested this. Building on this, suggestions of how to improve science communication about GE are made.

<sup>1</sup> ""CRISPR" (pronounced "crisper") stands for Clustered Regularly Interspaced Short Palindromic Repeats, which are the hallmark of a bacterial defense system that forms the basis for CRISPR-Cas9 genome editing technology. In the feld of genome engineering, the term "CRISPR" or "CRISPR-Cas9" is often used loosely to refer to the various CRISPR-Cas9 and -CPF1, (and other) systems that can be programmed to target specifc stretches of genetic code and to edit DNA at precise locations, as well as for other purposes, such as for new diagnostic tools. With these systems, researchers can permanently modify genes in living cells and organisms [for example to make food crops more drought resistant] and, in the future, may make it possible to correct mutations at precise locations in the human genome in order to treat genetic causes of disease." [43]

# **2 GE Attitudes Are Likely Guided by Moral Intuitions**

# *2.1 Complex Topics Such as GE Are Diffcult to Assess as a Single Person with Time Constraints and Without Expert Knowledge*

GE is a collective term for several methods which can be used for a variety of applications. From a theoretical perspective, these can result in applications with normatively desirable results as well as with normatively undesirable results. This chapter therefore takes the position that GE can yield many benefcial contributions to society and nature. At the same time, this also means that abuse of this technology is conceivable, and appropriate measures should be taken that help prevent such abuse.

For a single person not involved in the topic, and with their own time constraints, it is diffcult to make an informed assessment of which applications should be used under which circumstances. Even experts involved in GE do not make any of these assessments alone. On the contrary, it usually takes different groups that can assess e.g. economic, social, or environmental aspects. Consumers, therefore, need an appropriate complexity reduction to formulate an informed opinion about GE.

Conversely, this means that potential benefts are diffcult to be effectively communicated in the public debate: on the one hand, sweeping endorsements of GE lack credibility. Since the majority of the German population already rejects GE, such diametrically opposed blanket statements would only meet with even more resistance. On the other hand, differentiated, deliberative views may not entail the kind of complexity reduction that consumers, for example, need in their everyday, non-primary decision-making behavior. They are therefore diffcult to communicate.

# *2.2 Public Debate Should Provide Guidance, But Mutual Accusations Make It Unclear Who to Trust*

A public debate about new technologies can serve as a complexity reduction that informs the public or consumers and provides fact-based arguments about the risks and benefts in relation to the adoption of a technology. At the same time, public debates sometimes do not seem to move towards a consensus. This may leave the non-expert consumer with questions as to what to make of a technology.

In this, scientists – as the experts in their respective feld or technology – have the task to make assessments about potential risks and benefts, and to make those publicly available. These assessments should provide guidance in trade-offs in public debates about new technologies. In fact, about GE, there are a number of such studies by scientifc academies. The National Academies in the United States of America (USA) have evaluated the evidence available on GE and found no evidence that GE products are less safe for humans and animals than other products [8]. The World Health Organization states that no adverse health effects of GE are known to date [44]. Moreover, the German Ministry of Education and Research, BMBF reports on more than 300 studies that assess the safety of GE [45].

In their statements, the German national academies and the European Academies Science Advisory Council note the high potential of GE, as did NASEM, in their report [8, 16, 46]. In 2019, the German Research Foundation, the Union of the German Academies of Sciences and Humanities, and the Leopoldina repeatedly pointed to the opportunities offered by GE [14].

However, in the German GE debate, scientists and their offcial statements appear to not have been successful in contributing their expert-mediator role to the debate. One potential explanation for this is the situation of the German GE debate mentioned above, in which the opposition is large, strong, present, and well-organized [35, 36], while the proponents are not very present but almost non-existent in the public debate [47]. This led to the curious situation in which scientists involved in GE research are almost the only group publicly providing arguments in favor of GE.

This situation in turn – so it is argued in this chapter – has made it diffcult to perceive scientists in their role as mediators. In the case of GE, they may often not be perceived in a neutral position as informing experts, but rather, as is argued here, as a **supporting party within the debate** [47]. This may make it diffcult for a consumer who wishes unbiased guidance on a complex topic.

This problem is fortifed by the way the debate is conducted – in part. For example, the molecular biologist Ralph Bock asks in one of his essays whether politics wanted to "bully" the researchers in Germany until they were driven to despair [48]. The geneticist Wolfgang Nellen speaks of "hysteria" and "ignorance" in one of his articles [49]. Another example can be found in an article by geneticist Reinhard Szibor: "Green genetic engineering apparently serves as a projection surface for all the fears and frustrations of this world." [50].

In 2016, this situation led 124 Nobel laureates to a rather extreme measure: they signed an offcial letter urging Greenpeace and the governments of the world to cease the campaigns against Golden Rice [51].

In this letter, the authors position themselves against opposition based not on scientifc facts but on "emotion and dogma" [51]. They also call on governments to reject such campaigns. The criticism of Greenpeace in the letter is sharp: the NGO is accused of ignoring or misinterpreting scientifc evidence, and of supporting feld destruction. Implicitly, the Nobel laureates accuse Greenpeace of being responsible for the deaths of up to 500 thousand children annually. They also speak of a "crime against humanity" in this context. To date, 160 Nobel laureates signed this letter.

These examples show that scientists do not always maintain their neutrality as expert-mediator on the issue, but also argue with strong emotions and sharp criticisms against GE opponents. Such emotional debating does not strengthen the public's trust in scientifc expertise but may make science appear biased.

As a further intensifer of the problem, it can be observed that some GE opposing organizations doubt the integrity of scientists. For example, in response to the above-mentioned statement of the German Research Foundation and the National Academies of 2019, Testbioech e.V. spoke of an obvious "interest in the application of genetic engineering and its commercial exploitation." [52]. Similarly, in response to the Nobel laureates' letter, Greenpeace does not show the desired reaction, but insinuates bias: the NGO asked the lead author to be honest, and to disclose economic interests in the technology [53]. This reinforces an impression of partisanship.

At the same time, while science communication is necessary for progress and education, and is increasingly demanded and promoted, it is diffcult for scientists to always argue in an unrestrictedly objective manner since they also want to defend their work, their community, and their identity. The current GE debate in Germany runs the risk to lead to further frustration and resignation among scientists.

On top of this, both sides are not involved in a dialogue, but address **topics that are unrelated** [36]. For example, while supporters of GE foods usually point out the technological advancements and the increased economic effciency of GE, opponents of GE foods usually refer to potential environmental risks and health risks [54, 55].

Moreover, supporters and opponents also appear to use different **concepts of naturalness** in their arguments. On the opponents' side, nature is seen as something sacred and pure that should remain untouched [56]. For advocates of naturalness, the GE of plants feels like interfering with nature and contaminating our fora [57]. Thus, the advocates of this view reject any human processing in nature [58]. On the other side, supporters affrm that GE foods do not pose any risks to nature and can even have benefcial properties for nature [45]. While GE supporters attribute some characteristics to nature that should be sustained, such as biodiversity, GE opponents see nature as something sacred that does not allow human processing [59].

With reference to the previous point, the debating parties may **emphasize distinct values in their arguments**. For example, while opponents point out aspects of fairness in relation to GE foods, supporters may rather focus on loyalty. More specifcally, opponents state that corporations involved in GE food products disregard the rules for fair competition by lobbying for their own interests [60]. Supporters on the other hand promise the commitment of scientists to the use of GE for the beneft of society [45].

# *2.3 People Likely Resort to Moral Values and Emotions as Guidance*

The current situation of the public GE debate, as well as the fact that GE assessment requires expert knowledge and years of training, suggests the following assumption: the public and consumers resort to intuitive guidance such as moral values and moral emotions. And indeed, research on factors that infuence people's opinion towards GE suggests that knowledge does not have a substantial infuence on how people perceive this technology [61]. Rather, intuitions, such as emotions, play a much bigger role in GE acceptance [62].

The importance of emotions as intuitions for people's opinion formation can be explained by adopting the distinction between two modes of thought: System 1 and System 2 [63]. System 1 operates automatically and quickly, with little or no effort and no sense of voluntary control. This refers to anything that we do intuitively or with a lot of practice, such as recognizing a friend or understanding the facial expression of anger. System 2 comprises effortful mental activities, such as complex computations. Operating System 2 entails conscious reasoning and requires agency, choice, and concentration [63]. For example, we use it when we make plans or check a logical argument for its validity. System 1 may be an evolutionary adaptation [64]: in order to survive, emotions needed to signal us fast and reliably when we are facing a threat, such as rotten food. Since emotions lead to much quicker judgments than rational thinking, they serve as a guide in matters of immediate danger.

There is a special type of emotions, moral emotions*,* that are neatly intertwined with moral values. In general, these are such emotions that motivate us to behave morally, and that respond to violations of moral values [65]. Following moral values means setting aside pure self-interest and orientating behavior and judgment according to the interest of the greater social welfare [66]. In human nature, **moral values take priority over non-moral concerns** [67]. Since we are social beings and could not survive without the protection and help of a group, moral values also fulfl an adaptive task: they make us liable for our actions and hence make it possible for us to live in groups and resist temptations which may be destructive for the survival of the entire group [68].

Since we relied on emotions and moral emotions for survival, it seems plausible that we also listen to them when we form our opinions on overly complex issues such as new technologies that may have an unprecedented impact on our lives.

And indeed, there is scientifc evidence that opinion on GE foods is guided by moral values and moral emotions. Research on public opinions on agriculture fnds that GE easily evokes emotions such as anger [69] and is usually connected with normative demands [70]. Moreover, it could be found that knowledge about GE is rather low in the public and that perceived fairness and trust in food producers enhances GE acceptance [54]. Furthermore, as mentioned earlier, a perceived violation of nature deemed sacred seems to be one of the main reasons for the rejection of GE foods [59, 61]. This can be grounded in seeing nature as an intrinsic value that needs protection, or in religious reasons such as human interference in god's will [56].

# *2.4 For Many, Trying GE Is Not Worth the Risk*

Above, it was proposed that the complexity of the issue, as well as the situation of the public GE debate, lead people to resort to moral intuitions in their opinion formation. And indeed, for example, people's individual predisposition towards risks plays a decisive role in opinion formation about GE: research has shown that risk-averse individuals are signifcantly more likely to oppose GE than risk-averse individuals [21].

Many people tend to want to avoid future uncertainties in principle [71]. The introduction of new technology represents such a future uncertainty, as one deviates from the known status quo. It seems to be the case that the majority of the German public prefers to maintain the status quo with regard to GE, rather than risk unanticipated consequences as a result of the adoption of the technology. In this chapter it is suggested that this is possible because in Germany, for the average consumer, it comes at no perceptible cost to maintain the status quo because there are enough affordable alternatives. Simply, there is the opportunity to be against GE. Under these circumstances, it does not seem "worth the risk" to try something that is perceived as unknown, compared to conventional breeding.

This importance of moral intuition implies that a debating party will be more convincing if they speak to those moral emotions and values that are most relevant to the public. Arguments that are less relevant with regard to moral emotions and values have much less persuasive power.

And at the moment, GE opponents are more successful in addressing people's worries and risk aversion, by feeding into them. For example, a meta-analysis found that studies made by NGOs without scientifc peer review appear to systematically reduce the estimated positive effects of GMOs [9]. Such measures reinforce the public perception that came to see these products as much more threatening than research would suggest.

Moreover, GE opponents address moral emotions such as disgust or anger, and thus further stimulate a rejection of GE foods in the public. For example, in some campaigns, GE foods have been associated with strong, disgust-provoking images, such as a woman eating 'Frankenfood', i.e. a genetically modifed tomato which looks like a fsh [72]. From eating this tomato, her skin is turning green. With that, the moral emotion of disgust is triggered, which relates to a violation of nature as an intrinsic value that no one should interfere with. So, moral opposition is the consequence.

Moreover, using language that is easy to grasp is another common strategy for many GE opponents [36]. Polemic terms such as "Gene-contamination" [31] or "Frankenfood" [73] produce memorable pictures and dramatize the topic. The consequence is predominantly negative associations with GE foods. Generally, some NGOs opposing GE make heavy use of emotionalized language. For example, in a book published by Earth Open Source, the authors describe GMO supporters as 'unpleasant, angry, and hostile' [74]. Another recent booklet states that GE 'failed' [31].

In contrast, arguments put forward by GE supporters seem to have less intuitive appeal [64].

Moreover, media reporting on GE likely contributes to this focus on risks associated with GE. Scientifc experiments show that consumers prefer negative news and react more strongly to it [75, 76]. Negative news are therefore easier to sell, which means that they are likely overrepresented in the media landscape [77]. This is where the media's incentive to report on potential risks of GE – rather than on potential benefts of the products – becomes apparent.

# **3 Moral Intuitions About GE Have Resulted in Strong Moral Convictions**

As proposed above, the stark rejection of GE in the public and the ferce debate have led to a moralization of the topic. In our representative surveys, the majority of participants indicated that the question of GE was a moral one for them [21]. Furthermore, in recent survey (N = 619; N = 636), up to 90% of the opponents stated that they reject GE, no matter how great the benefts [21, 23]. This is in line with previous research by Scott et al. [59], who measured GE attitudes in the USA. Of their representative sample, 64%, were opposed. Of this opposition, 71% (i.e. 46% of the entire sample) stated opposition regardless of the consequences, claiming that GE (in this study: GMOs) "should be prohibited no matter how great the benefts and minor the risks from allowing it" were [59]. Other studies achieved similar results [24]. This means that participants state to maintain their attitude regardless of possible consequences. Many in Germany have become consequenceinsensitive with regard to their GE position.

Taken literally, this means that the perceived beneft from a ban of GE, or approval, respectively, is infnite [59, 78]. Thus, there is no cost-beneft trade-off regarding the use of GE. Rather, their GE position has been elevated to a sacred value worthy of protection [79].

Interestingly, research has shown that attitudes toward GE are consequenceinsensitive for a signifcant proportion of supporters as well [22, 23]. Here, it is important to emphasize that, contrary to popular belief, GE is not only a moral issue for opponents. Rather, for a signifcant proportion of supporters, their GE position has become a moral issue as well.

Such strong moral conviction makes it hard to engage in a constructive discourse about the risks and benefts of the technology and its potential applications. Here likely lies one reason for why science communication about GE is not effective at the moment. Rozin refers to such phenomena as "moral piggybacking" [80]. Here, objects or facts that are in themselves value-free are morally charged, as can be observed, for example, among vegetarians on the topic of meat consumption [80, 81]. For many, GE opposition, but also GE support, has become a moral goal in itself.

# *3.1 In the Public Eye, GE Has Become a Moral Goal, Rather Than a Means to Moral Goals*

Among consumers and the public, the moralization of GE has grown so strong that a ban or approval of it now has an intrinsic moral value, good or evil. This means that the public does not perceive the properties of the technology, but the technology itself as the central interest in the discourse. Rather than looking at potential

consequences of the technology, GE has become the moral goal of the debate. Here, moral goals are confused with their potential means [82]. GE has become the moral goal in itself [47].

This normativity of GE in the population is readily taken up by the parties to the discourse and forms the central problem of the confict: in a generalized normative ban or command on GE are mutually exclusive, there is no room for agreement.

It can be observed that positions in the public debate are generally quite generalized. These general evaluations create two fronts of "right/good" and "wrong/evil", in which the goals of each side appear incompatible: it appears that one can either be in favor or against GE. The fact that many arguments in the debate are not weighed up in a differentiated way creates the impression that one can only decide for or against one side. This creates a moral confict of objectives for the public in which the potential positions appear mutually exclusive: It appears as if one can position oneself either for a ban on GE, or an approval of GE.

In the case of mutually exclusive demands, however, it is not possible to resolve the discourse to the beneft of both sides. Since it is then not in the interest of either side to deviate from their positions, the discourse is stuck [47].

In this situation, the debating parties perceive the debate as a tradeoff thinking with little room for a solution. The debate resembles a social dilemma: the strategies of the participants lead to a mutually worse position because this stalemated debate benefts no one and is tiresome for all participants and observers [82]. This stuck discourse puts pressure on policymakers and ultimately leads to legislation that refects the goal-means confusion. As an institutional framework for research and development, this legislation blocks innovation. The results are opportunity costs because possible solutions cannot be found and urgent problems remain unsolved [29, 83].

# *3.2 Moral Convictions Are Especially Hard to Mitigate*

These strong moral convictions provide another explanation for why science communication and arguments of scientists involved in the debate do not seem to reach the public: research in moral psychology has shown that moral judgments are much more stable and long-term than other judgments [84]. This is because, for humans, moral concerns take priority over non-moral concerns [67]. Such strong moral beliefs have real effects on daily actions: For example, people with different moral beliefs are more likely to be avoided, and they are also less likely to be believed [85, 86]. Over time, some moral beliefs can become so strongly attached to a person's core moral constitution that they become linked to that person's identity [80, 81, 85].

And indeed, in recent research, a large proportion of subjects view their position towards GE as central to their personal moral beliefs [87].

# **4 Can Strong Moral Convictions Be Mitigated?**

If science communication has diffculties reaching the majority of the German public because of strong moral convictions about GE, a solution that mitigates these strong moral convictions and allows GE to be discussed as a possible means, rather than a moral goal is necessary.

Similar suggestions can also be found in "Ethical Tensions from New Technology" [88]. It is not the technology that should be discussed, but its applications. And this with regard to questions such as: What problems do we face? For which of these problems can GE be an appropriate tool? Under what conditions? What are the risks? Are there sensible alternatives? Similarly, scientifc academies in Germany are already calling for it [14].

However, if the rejection of GE is moral, is there a way to overcome this moral rejection of this technology?

Since the beginning of modernity, many technological innovations have enriched society. For example, medical diagnostics was able to make great progress through X-ray or MRI technologies. Everyday life has also been made easier by household appliances such as the washing machine or the dishwasher. Some technologies are quickly adapted because of their obvious high utility (e.g., X-ray machine, household appliances). New technologies are not always greeted with openness; in some cases, their development is viewed skeptically. For example, Kenneth Olsen saw no reason why anyone would need a computer at home [89]. Sir Preece, the chief engineer at the British Post Offce, thought the telephone was unnecessary because there were enough letter carriers [90]. Thus, some technologies experience initial skepticism, but then prevail because of their advantages.

In the eighties and nineties, there were very heated debates in Germany about the medical application of GE [91]. The focus here was particularly on human insulin, which has been used since the 1980s to treat diabetes. Concerning this, negative reports circulated in the media. A newspaper called "TAZ" poke of a "gateway drug" [92] and reported on side effects and dubious competitive practices surrounding the drug [93]. In 1991, a report of the public television broadcasting service "ARD" spoke of frequent and serious risks to patients from human insulin [94]. These reports were contradicted by physicians and scientists. For example, in the German Medical Journal, the German Medical Association confrmed the successful and problem-free treatment of patients since 1983 [94].

By now, this great initial skepticism has dissolved into acceptance. In the meantime, Germany is one of the world's leading producers of genetically engineered pharmaceuticals**,** which are no longer an issue in the public debate [91].

Thus, it is proposed here that consumers and the public need to be provided with tangible, credible reasons to try GE foods or adopt GE technology in spite of their worries related to the technology. Specifcally, the advantages of GE have to become salient and tangible enough for people to outweigh perceived potential risks.

# *4.1 Moral Goals Can Mitigate Strong Moral Convictions*

Recently, it was tested whether there are such consequences of GE that would make the public and consumers abandon their moral convictions about the technology [87]. Specifcally, in an online experiment in Germany in 2020 (based on own research), representative of the population (N = 1900) according to age, gender, and level of education, it was tested whether respondents could identify consequences that are so important to them, that they would abandon their moral convictions towards GE. This experiment only included people who indicated a strong moral conviction towards GE. This led to the inclusion of around 1500 GE opponents who stated that they would reject GE regardless of the consequences. Additionally, this also led to inclusion of around 400 GE supporters who stated they approve of GE regardless of the consequences.

These "consequence-insensitive" participants were randomly assigned to four probes that tested their moral conviction. Here, two of those probes are presented.

In the open-ended refection task, consequence-insensitive participants were asked whether they could think of any circumstances that would make them abandon their position towards GE. Respondents could then provide their answers in free-text boxes. Table 30.1 shows examples of circumstances stated, the moral goals these address, as well as their frequencies.

In this probe, a little less than 50% did not state any consequence that would make them abandon their consequence-insensitive position towards GE – but more than 50% did!

In another, the counterexamples probe, consequence-insensitive participants were provided with a list of moral goals and asked them to select all of those that would make them abandon their consequence-insensitive position towards GE. Table 30.2 shows how frequently each moral goal has been selected.

In this probe, almost every opponent (361/369; 97.8%) and supporter (100/101; 99%) selected at least one option.

The results of these probes show that nearly everyone who previously proclaimed consequence insensitivity indicated that there are circumstances for which they would abandon their position.

After these probes, participants were asked to state their positions towards GE again. Interestingly, a signifcant proportion of those who initially stated to maintain their position regardless of the consequences abandoned this strong moral conviction after the probes. Moreover, the attitudes towards GE among those previously morally convicted also became less extreme after the probes [87].

Therefore, while moral convictions about GE are both common and hard to change, still the majority of consequence-insensitive participants state that there are at least some circumstances that would make them abandon their position. This was particularly true when common moral goals were made salient to them. Examples of such moral goals that are dearest to participants are life, health, and environmental protection.


**Table 30.1** Open-ended refection (share of consequence-insensitive respondents mentioning a topic)

Note*.* Totals do not match the number of respondents (and percentages do not sum to 100) because participants could provide up to fve responses [87]

This means that consensus in the GE debate is possible and that it is possible for science communication to reach even the most morally convicted – if people's actual moral goals would become the focus of the debate, and GE would then be discussed as a potential means of these goals, rather than the goal itself.


**Table 30.2** Counterexamples (percentage of consequence-insensitive respondents selecting each counterexample)

Note. Totals do not match the number of respondents (and percentages do not sum to 100) because participants could select multiple responses [87]

# **5 Improved Science Communication About GE in Public Debate**

# *5.1 Provide Credible and Tangible Advantages That Address Common Moral Goals*

The abovementioned study indicates that in many cases, both supporters and opponents of GE actually share moral goals, such as life, health and environmental protection. This is also one result of another content analysis [95]. Moreover, our studies indicate that strong moral convictions about GE can be mitigated if people are provided with **credible, tangible moral reasons** to try GE products in spite of their worries.

If the common moral goals are identifed, it is possible to examine whether GE is a suitable means for reaching these goals. Here, appropriate measurement tools can be used to clarify this perceived confict. These are, for example, scientifc studies, meta-analyses, risk analyses, or long-term observations of the plants under discussion.

Then, science communication about GE is likely effective if science communicators provide credible evidence that GE can be a tool to reach these moral goals.

For example, one of the common moral goals that Waldhof identifed is effective development aid [95]. However, at the moment, some public statements against GE state that the technology causes disadvantages for small farmers [31]. Evidently, many conclude from such statements that if one wants to pursue effective development aid, GE would be a hindrance. Consequently, the moral goal development aid would be a reason to ban GE.

Therefore, at the moment, the argumentation can be presented as follows [47]:


However, a meta-study on the effects of GE found that the technology especially benefts farmers in developing countries [9].

Similarly, Ahmed et al. concluded in their study in Bangladesh that farmers save their costs, obtain more yield, and likewise protect their health through reduced pesticide use by using a GE eggplant [26]. This scientifc evidence requires that the positive assumption (2) needs to be changed: GE benefts small farmers in developing countries, particularly. As a consequence, the conclusion (3) also changes: GE should be used for development aid. Now, the argumentation is as follows:


The same procedure can also be applied to the moral goal of nature conservation. GE crops are often associated with increased pesticide use [96]. Pesticides can be a burden on soils and can also put insects at excessive risk. For this reason, Brookes and Barfoot examined pesticide use associated with GE from 1996 to 2015 [97]. They found that pesticide use decreased by 8.1% due to the use of green genetic engineering. Overall, their study concludes that biotechnology has a positive impact on the environment. GE thus contributes to nature conservation.

# *5.2 Include Credible and Salient Science Communicators That Truly Care About the Moral Goals*

It is argued above that scientists involved in GE research are currently not perceived as expert mediators that can solve the discourse block, but as – a somewhat biased – party within the debate. It was argued that this may be in part because of the way the debate is conducted, and because scientists and scientifc organizations are the only ones bringing forward supporting arguments for GE. Consequently, scientists alone cannot resolve the strong moral conviction and the resulting discourse block. They need support. In the same vein, in a newsletter of the Leopoldina, Hans-Georg Dederer, demands more support from politicians [41].

It is argued here that, alongside science-based and moral arguments, the debate about GE needs additional actors that truly care about the moral goals, and not about the technology or its ban. By now, more and more such actors are joining the debate. One example is Professor Urs Niggli, who was the director of the Research Institute for Organic Agriculture for 30 years since 1990 and is also known as the "organic pope" [98, 99]. He is considered one of the world's leading scientists in organic agriculture and is known for his commitment to sustainable food and farming systems [99, 100]. According to him, agriculture is at a turning point where a stronger orientation towards technology is necessary [101, 102]. For him, this also includes the use of biotechnological processes in agriculture. Niggli believes that the use of CRISPR means that goals such as food security and biodiversity no longer have to be mutually exclusive, but can be achieved together [99].

The NGO "Progressive Agrarwende" takes a similar view. This young group of scientists, students and politicians, founded in 2019, is pursuing the goal of implementing an agricultural turnaround toward socially and ecologically sustainable agriculture according to the latest scientifc and technological standards [103]. They observe that many environmentally conscious people act against their own goals by a blanket rejection of modern agricultural techniques. For this reason, they have founded a dialogue platform to address applications, regulations and patenting of agricultural technologies in an open-ended and transparent manner. To this end, Progressive Agrarwende regularly writes articles, organizes events or reports on scientifc fndings.

More and more actors that call for a science-based evaluation of products developed from GE technology are joining the debate or gaining more attention. These are for example maiLab (youtube.com/@maiLab), Grain Club (grain-club.de/) or transGEN (transgen.de/). Coalitions with such professional science communicators help scientifc evidence gain salience in the debate. They will also help scientists to communicate their fndings in an understandable and enjoyable way that is easy to follow.

Moreover, the call for a science-based assessment of GE is receiving more media attention. For example, the TV show MaiThink X dedicated an entire episode to arguing for a science-based evaluation of GE [104]. Similarly, in December 2022, the TV show Heute Show [Today Show] argued that a general fear of GE is not science-based and should be reconsidered [105].

Moreover, recent research has shown that in many arguments, the debate deviates from discussing the risks and benefts of the technology to discussing the behavior of the actors involved [95]. For example, while GE opposition criticizes unfair behavior of corporations, GE supporters claim loyal commitment of scientists to use GE research for the common welfare. However, ensuring the trustworthiness of scientists seems unlikely to solve reservations towards corporations or the market. Understandably, scientists involved in GE research cannot speak to the governance of involved actors. Here, it would help if social scientists joined the debate to discuss market mechanisms and the behavior of companies and other actors.

Furthermore, self-binding mechanisms offer a further opportunity to increase the quality of discourse. As described above, organizations involved in trade-offs also have a long-term interest in objectifying the discourse because of the loss of reputation that can otherwise be expected. If NGOs join forces and commit themselves to collectively agreed standards and fair discourse processes, they can signal the high quality of their contributions [106]. Corresponding initiatives already exist, such as Accountable Now! [83, 107, 108]. The NGOs involved regularly submit reports that are reviewed and commented on by a panel.

Generally, it seems that the German GE debate is changing, moving away from a generalized debate towards a more differentiated debate about what GE can and cannot do. In this, science-based arguments appear to be gaining increasing salience in the debate.

# **6 Summary**

A large share of natural scientists consider the application of GE to be necessary for a sustainable transformation of agriculture. However, there is a stark divergence between scientifc assessment and public perception of this technology, particularly in Germany. So far, science communication has not been able to resolve this divergence. Research on technology acceptance shows that people have a general tendency to follow their emotions and intuitions when forming their opinions on technologies [61]. The present chapter suggests that strong moral convictions hinder effective science communication and provides scientifc evidence for this claim. This is because moral convictions are more stable than other judgements. This led to a situation, in which the approval or rejection of GE are perceived as a moral goal in itself. The analysis thus suggests that a way has to be found in which GE is perceived as a potential tool for moral goals, rather than a goal in itself. For this purpose, the chapter also reports on research which suggests that strong moral convictions can be mitigated through shared moral goals. Building on this insight, recommendations for improved science communication about GE are formulated.

Generally, GE should be perceived as a potential tool to reach moral goals such as food security or sustainability. With every tool, there are advantages and disadvantages. This is what makes thorough risk assessments, and a debate that weighs potential risks and benefts, indispensable. One result of such debates would be procedures that reduce potential risks of new technologies such as GE. At the same time, potential risks should be addressed symmetrically:

The use of [new genomic techniques] NGTs raises ethical concerns but so does missing opportunities as a result of not using them. […] Any further policy action should be aimed at reaping benefts from innovation while addressing concerns. A purely safety-based risk assessment may not be enough to promote sustainability and contribute to the objectives of the European Green Deal […] [109].

# **References**


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# **Chapter 31 The Citizens' Awareness and Concerns During the Transition from Genetically Modied to Genome Edited Plants in Europe About Their Use in Agriculture and Food Production**

### **Mihael Cristin Ichim**

**Abstract** The genome edited crops and foods are commercially cultivated and marketed already at global level, rapidly expanding towards new applications and plant species, and successfully complementing the genetically modied ones. In the European Union, the genome edited plants have to follow the two-decade-old regulatory framework for genetically modied organisms. The decrease of both, number of notications for eld trials, and hectarage and countries commercially cultivating genetically modied plants, registered in the last decade in the European Union, has been closely followed by lower levels of awareness and concerns expressed by the European Union citizens about their use in farming and food production, as recorded by a long time series of Eurobarometers surveys. In contrast, in the four recent years only, the awareness about genome editing among the European Union citizens has signicantly increased, reaching more than the half of the one about genetically modied products, along with the number of concerned European Union citizens about genome editing which also has doubled. The public opinion about genome edited crops and food products needs to be monitored further as it decisively inuences the new regulatory framework to be proposed by the European Commission and therefore the extent to which the European consumer will benet from the new biotechnologies.

M. C. Ichim (\*)

<sup>&</sup>quot;Stejarul" Research Centre for Biological Sciences, National Institute of Research and Development for Biological Sciences, Piatra Neamt, Romania e-mail: cichim@hotmail.com

# **1 From Genetically Modifed to Genome Edited Plants: The Global Context**

The global production status of genetically modifed (GM) crops has steadily grew since their commercial adoption in 1996, to reach a 112-fold increase [1] to around 190 million hectares in 2019, almost equally split between 21 developing countries and fve industrial countries [2]. In terms of the share of the main crops in which transgenic traits have been commercialized, GM traits accounted for 47% of the global plantings to soybeans, maize, cotton, and canola in 2020 [3]. The global economic benefts, over the period 1996 to 2020, have been signifcant with farm incomes for those using the GM crop technology, having increased by 261.3 billion USD [4]. All the above are based on a sharp increase in the approval of the number of plant species with GM varieties. At global level, 44 countries, plus the European Union (EU) Member States (MS), have approved a total of 46 commercial GM traits introduced, as single or stacked transformation events, in 32 GM crop species for use in commercial cultivation, food, and feed.1

Genome editing (GE) was being applied to more than 40 crops across 25 countries [5]. The GE plant varieties in advanced development pipeline span now a wide range of crops, including alfalfa, camelina, canola, citrus, fax, maize, pennycress, potato, lettuce, tomato, and watermelon, and are suggesting the fexibility of the new breeding techniques (NBTs) in crop improvement [6]. Despite the apparent potential, however, only six GE crop traits - in soybean, canola, rice, maize, mushroom and camelina - have been approved for commercialization to date [7]. The United States (US) farmers are growing from 2016 a oligonucleotide-directed mutagenesis (ODM)- based GE sulfonylurea tolerant weed control canola (Cibus)*,* a transcription activator-like effector nucleases (TALEN)- based genome edited soybean with modifed oil composition (Calyxt) [5] but also a Clustered Regularly Interspaced Short Palindromic Repeats -associated protein 9 (CRISPR–Cas9) edited waxy corn (Corteva Agriscience) [8]. The hectarage cultivated with Calyxt genome edited soybean alone has increased signifcantly, to approximately 40,000, from 17,000 in 2019 [5, 9]. The two genome editing pioneering companies, Cibus and Calyxt, have recently announced they had entered into a defnitive merger agreement [10]. Genome edited food was frst sold on the open market in 2021, when the Sicilian Rouge tomatoes, genetically edited to contain high amounts of γ-aminobutyric acid (GABA), was sold direct to consumers in Japan by Sanatech Seed [11]. Two years before that, was marketed the frst genome edited food product, the high-oleic soybean oil Calyno™, obtained from plants that have been edited to produce fewer saturated fats and zero trans fats (Calyxt) [9]. To date, the GE crops and products have still limited commercial prevalence because they are new and unfamiliar to consumers relative to other breeding techniques and the regulatory process is ill defned and shifting in many countries [12].

<sup>1</sup> ISAAA: ISAAA GM Approval Database, https://www.isaaa.org/gmapprovaldatabase/. Accessed: 14 May 2023.

To reach the market, each GM plant event must undergo a long and costly process. Two decades ago, it was estimated that 8–12 years of research and development were needed prior to commercialization of GM plants [13]. A recent agri-biotech market survey has reported that the cost of discovery, development and authorization of a new plant biotechnology derived single genetic trait has declined from 136 million USD in the 2008–2012 period to the current value of 115 million USD, but the time required to complete the process has increased from 13.1 to 16.5 years [14]. This data suggest a twice as long period needed nowadays, compared with the early 2000s, for a new GM single trait plant to reach the commercialization phase. On the other hand, early commercial applications of genome editing for crop improvement appear to confrm the inherent speed and overall cost effciency of genome editing [6]. For comparison, the cost for GE crops development to commercialization were estimated to cost ten million USD and the time needed is 5 years [15]. The genome editing is faster and cheaper approach to market than conventional GM technology [16].

Consumers response to GM products is largely infuenced by the decision of the governments to ban or approve the GM crops cultivation and between acceptance for cultivation of GM crops and market exists a wide gap across countries, a comprehensive bibliometric analysis has concluded [17]. On the other hand, a recent systematic global overview has concluded that GE foods are often more accepted than the GM foods, which is partially due to the fact that genome editing is perceived as more natural [16, 18].

# **2 The Commercial Cultivation of Genetically Modifed Plants in the EU**

In 1998, only two years after the GM crops were frst marketed worldwide [3], their cultivation in the EU started, being among the frst territories to commercially adopt the cultivation of biotech crops at global level [19] (Fig. 31.1), well in advance of countries such as Brazil, India, Paraguay, and Pakistan, each of them cultivating millions of hectares with GM crops nowadays [20].

Only two GM maize varieties, both producing Cry1Ab toxin, insecticidal protein from *Bacillus thuringiensis*, have been authorized for commercial cultivation in EU: Bt176 (1998–2005), and MON810 (since 2003). The maize hybrids derived from event Bt176 were offcially withdrawn from the European market in 2006 because this event contained an ampicillin resistance gene as selectable marker [23]. It took 15 years to reach the cultivation peak, in 2013, with 148,013 ha, on all growing YieldGard™ maize (single trait event MON-ØØ81Ø-6) which, since 2006, continues to be the only GM plant commercially cultivated in the EU [20]. Yet, this GM maize line is one of the 49 insect resistant (IR) transgenic events approved for food

**Fig. 31.1** Commercial cultivation of GM crops and GM plant feld trial notifcations in EU (1998–2022) [19, 21, 22]

and feed use in the EU Register of authorised GMOs.2 Since then, the hectarage cultivated with GM maize has gradually decreased to approx. 70,000 ha in 2022, a comparable value with the one register 15 years ago. Last year was also registered the biggest reduction of the cultivated area, with approx. 30%, the most abrupt yearly variation ever recorded in EU [21, 22].

The gradually decrease of the cultivated area with GM plants in Europe was accompanied by a reduction of the countries growing these biotech crops, as farmers from Romania (in 2015) [24], Czech Republic, and Slovakia (in 2017), have voluntarily stopped the commercial cultivation of GM maize [19]. Only two countries, Spain [22] and Portugal [21], are commercially cultivating GM crops in Europe for the last 5 years.

Worth mentioning that the provisions of the EU GMO opt-out Directive 2015/412 [25], which have allowed to a signifcant number of EU MSs to offcially restrict the cultivation of genetically modifed organisms (GMOs) in their territory, did not changed the *status quo* of the EU-28 countries, as 11 countries have offcially chosen to legally allow the commercial cultivation of GM crops [19]. Yet, in 2018 the Court of Justice of the EU (CJEU) ruled (case C-528/16) that organisms obtained by mutagenesis, as can be achieved using New Plant Breeding techniques (NPBTs), including genome editing techniques, are genetically modifed organisms (GMOs) as defned in the European Directive 18/2001/EC [26]. This decision, imposing for the genome edited plants to follow exactly the same authorization procedure as the GM plants, was expected to delay their development, testing, approval and

<sup>2</sup>European Commission: EU Register of authorised GMOs, https://webgate.ec.europa.eu/dyna2/ gm-register/. Accessed: 14 May 2023.

availability of these new plant varieties on the EU market and their commercial cultivation [19].

# **3 The Notifcations for Field Trials with Genetically Modifed and Genome Edited Plants in the EU**

The feld trial under feld conditions is a preliminary, but essential step for developing GM plants, especially if they are intended for commercial cultivation. In the EU, a new GM crop event has to be compared to its closest non-GM counterpart as a corner stone of the pre-market risk assessment. This is a critical fnal test to monitor their effects on the receiving environment (e.g. non-target organisms) and also detect possible occurrence of differences, caused by intended and unintended effects, in composition, as well as in agronomic, phenotypic and molecular characteristics [27]. The EU continues to have the broadest and most stringent regulations in the world governing these feld trials as an essential part of the risk assessment before market approval [28].

During the regulatory framework set by the Directive 90/220/EEC (1991–2001) [29] 1687 feld trial notifcations were registered and their number dropped by 76% between 1998 and 2001, mainly due to the *de facto* moratorium in place since 1999 [13]. The notifcations documented by the dedicated European Commission' GM Plants Register,3 containing the list of summary notifcations (SNIFs) submitted to the competent authority of the EU MS under the Directive 2001/18/EC (from 2002) [30], has also dramatically dropped by 91% in the last 9 years (Fig. 31.1).

The CJEU's decision from 25 July 2018, stating that plants obtained with the new genome editing techniques are GMOs from the regulatory point of view, has immediately changed the legal status of the frst feld trial of a CRISPR-Cas-9 genome edited crop, *i.e*., *Camelina sativa*, in Europe (EU-28) [31], that began on 5 June the same year at Rothamsted Research (UK) [32]. Yet, the frst notifcation of a feld trial with a genome edited plant in Europe recorded in the dedicated GMO Register was carried out in 2018 by the Flemish Institute for Biotechnology (VIB) (Ghent, BE) with maize with an impaired DNA-repair mechanism and maize with modifed growth characteristics (Fig. 31.2).3

That frst feld trial with a GE plant has immediately raised a strong concern that limiting their feasibility by expanding the complexity of the regulatory process and the associated fnancial burden of dedicated experimental sites will certainly hinder research [32].

The evolution of the feld trial notifcation with GE plants since than (2018–2022) (Fig. 31.2) has fully confrmed, to date, that assumption: their yearly total number being maximum 5 (in 2019).

<sup>3</sup>European Commission: Eurobarometer – Public opinion in the European Union, https://europa. eu/eurobarometer/screen/home. Accessed: 14 May 2023.

**Fig. 31.2** The notifcation for feld trials with GM and GE plants under Directive 2001/18/EC (2018–2022)

The feld trial notifcations with GM plants in EU proved to be a sensitive indicator of the overall research and development activities in the past [33]. Moreover, the dramatic reduction of feld trials in the EU has also coincided with increasing safety demands, decreases in funding, and changes in the European directives [34]. Under the regulatory framework set by the Directive 2001/18/EC, the last 20 years can be generally divided into two separate periods: the frst years characterized by a sharp increase of both number of feld test notifcations and hectarage of commercial cultivation followed by a decrease of both of them (Fig. 31.1). This data suggests a high degree of interdependence of their overall trend in the EU. While the notifcations for feld tests with GM plants do follow a clear descendent trend in the last 4 year, the notifcations for GE are variable but their status in 2022, compared with the total number, was bigger than of the feld trials with GM plants, for the frst time (Fig. 31.2), offering hopes for an ascendant trend to be expected in the coming years, hopefully translated into their commercial cultivation status in EU. Worth remembered that all the feld trials, including with GE plants, are carried out following the regulatory framework developed more than two decades ago [30], well before the NBTs to be proposed, developed and commercially validated, as it is the situation nowadays. The current regulatory regime for feld trials was found ill-ftted to breeding activities [35].

# **4 The Awareness and Concerns of the EU Citizens About the Use of Genetically Modifed and Genome Edited Plants in Agri-Food**

Eurobarometer is the polling instrument used by the European Commission (EC), and other EU institutions, to monitor regularly the state of public opinion in Europe on issues related to the EU as well as attitudes on subjects of political or social nature.4 It is a cross-temporal and cross-national comparative program of regularly repeated cross-sectional surveys [36]. Additionally, special Eurobarometers are used to monitor if citizens want the EU to act on a specifc policy topic or support the EC's solution to a policy problem [37]. In order to guarantee the representativeness of results, Eurobarometer surveys rely on a randomly selected sample of persons and the total sample is weighted to ensure demographic and geographical representativeness and the EU average is calculated taking into account the relative weight of each country.4

# *4.1 The Concern of the EU Citizens About the Use of GM Plants for Farming*

The attitude of EU citizens toward the release of GMOs into the environment was assessed through a special series of four Eurobarometers during a 9-year period (2002–2011). In 2002, when they were frst asked about, the "use of GMOs" was a concern ranked 18th, from 25 different environmental issues, representing 30% from the worries expressed by the EU citizens [38]. The following three surveys (2004, 2007, 2011) have specifcally asked for concerns related to the use of GMOs for farming [39–41]. The percentage of worried EU citizens about the use of GM plants in the environment, for farming purposes, has decreased, constantly, down to 19% (Fig. 31.3).

# *4.2 The Awareness and Concern of the EU Citizens About the Use of GM and GE Ingredients in Food*

A series of four Eurobaromer surveys (2005, 2010, 2019, and 2022), initiated by the European Food Safety Agency (EFSA), aimed to investigate Europeans' perceptions of and attitudes towards food safety by exploring the Europeans' interest in

<sup>4</sup>European Commission: GMO Register / Part B notifcations (experimental releases) – GM Plants, https://webgate.ec.europa.eu/fp/GMO\_Registers/GMO\_Part\_B\_Plants.php. Accessed: 14 May 2023.

**Fig. 31.3** Concerns expressed by the EU citizens toward the use of GM plants in farming (2004–2011)

food safety-related topics and factors affecting food-related decisions and awareness of and main concerns about food-safety topics [42], including "genetically modifed ingredients in food or drinks" (2005, 2010, 2019, and 2022) and, more recently, "genome editing" in food (2019, and 2022).

#### **4.2.1 The Awareness of the EU Citizens About the Use of GM and GE Ingredients in Food**

Awareness of food safety topics remains high among EU citizens [42]. The awareness among the EU consumers of the use of both GM and GE in food production was investigated in the last two Special Eurobarometers [42, 43], offering a valuable insight on how the two plant biotech technologies, GMOs, used for almost three decades for farming, and food and feed production in Europe, and GE, only recently has entered into the EU consumer's attention, but with no farming and food production applications in Europe, yet, are perceived and aware off at the consumers' level (Fig. 31.4).

As expected, in 2019, the awareness level about GM were considerably higher (three fold) than about GE-food associated issue, but only 3 years later, due to a marginal decrease (4%) of the declared awareness about GMO, and a signifcant increase (38%), of the GE among the EU citizens, the difference between the two food safety-associated topics decreased by one third from the previous levels. Notably, in 23 of the 27 EU MSs, awareness of the use of new biotechnology in food production, *i.e*., GE, has risen since 2019 [42].

527

**Fig. 31.4** Awareness expressed by the EU citizens about the use of GM and GE in food production

**Fig. 31.5** Concerns expressed by the EU citizens about the use of GM ingredients and genome editing in food and drinks

#### **4.2.2 The Concerns Expressed by EU Citizens About the Use of GM and GE Ingredients in Food**

Around one quarter (26%) of the EU citizens indicate the presence of genetically modifed ingredients in food or drinks as a concern in 2022 while only a smaller proportions (8%), the use of new biotechnology in food production, i.e. genome editing (Fig. 31.5).

Compared with 2019, for most of the concerns expressed by EU consumers, listed in the Special Eurobaromer survey, there have been increases in the

**Fig. 31.6** Trends of the awareness and concern expressed by the EU citizens about the use of GM ingredients in food

proportions and this is particularly the case for the use of GE in food production which has doubled, from 4% to 8% from the total, among the EU citizens [42].

#### **4.2.3 Trends and Evolution of the Awareness and Concerns Expressed by the EU Citizens About the Use of GM and GE in Food Production**

The time series of Special Eurobaromer surveys on Food Safety, spanning over 18 years (2005–2022), offer a consistent and clear overview on the opinions the EU citizens have on the most relevant food-safety related issues. Included right from the start among the investigated topics, the opinion about the concerns about the presence of GM ingredients in food was recorded and it reveals a clear and signifcant reduction of them, from approximately 2/3 (62–66%) of the citizens to less than 1/3 (27–26%) (Fig. 31.6).

# *4.3 The EU Citizens' Awareness and Concerns During the Transition from GM to GE Plants*

Gathered in the last three decades, scientifc evidence about the perception of GM plants and derived food products is large enough, the EU especially being one of the territories well represented in such analyses [16]. Comprehensive analyses have concluded that EU consumers have more negative perception and less purchase intention toward GM foods in contrast to the consumer perception in North America while in developing nations, the positive perception arises owing to the persistent demand for food [17].

In Europe, during the frst decade of the new millennium (1999–2010) a 12% increase was recorded, by a series of Special Eurobarometers, among the EU citizens optimistic about biotechnology [44]. Unfortunately, this share of optimism was not refected towards the use of GM plants in agri-food in EU. The awareness levels, recorded lately among the citizens regarding the use of GM ingredients in food, were at medium levels and have even decreased slightly [45]. This could be correlated with the abrupt decrease of both GM crop testing and commercial cultivation in Europe. The lower level of awareness was inevitably followed by the proportion of EU citizens concerned about the use of GM crops in farming and for food production, as shown by a long time series of Special Eurobarometers.

The available data about the perception from the public and relevant stakeholders of GE is very limited [16] and, in this context, the recent surveys of the awareness and concerns among the EU citizens regarding the use of GE in food production is all the more relevant considering the large territory covered and the trends revealed [42]. In only 4 years (2019–2022), the awareness about GE among the EU citizens has signifcantly increased, reaching more than the half of the one about GM products – a three decade old technology in Europe though. Moreover, the number of concerned EU citizens about GE has doubled in the same period, as well as the proportion compared with the level of concerns about the GM ingredients.

NBTs and their products tend to be valued more highly than their GMO counterparts [12, 16] but because large differences occurs within target groups, regions and products [16] a large, highly and multidimensional heterogeneous territory, as the EU proved to be along the years with respect to biotechnology, is scientifcally and practically relevant for all stakeholders. It is critically important the public attitude towards new breeding techniques, and their feld testing, commercial cultivation and products, to be extensively investigated as they could be misperceived and rejected even though they hold much promise to improve food supply chains' sustainability, foster better health outcomes for consumers and the environment [12] and their acceptance will be a key factor for policy support [16].

# **5 Conclusion and Future Perspectives**

Relevant time series of Eurorometers have surveyed the awareness and concerns of the EU citizens about the use of GM crops for farming and food production. The public opinion has shown a steady decrease of both indicators along with the notifcation for feld trials and commercial cultivated of GM plants in the EU. More recently, both the awareness level and concerns expressed by the EU citizens about the use of GE in food production has increased signifcantly in only 4-year period.

The public opinion is fundamental for the acceptance and adoption of any new technology, including NBTs, in Europe. The long term evolution of the public attitude and acceptance of the GE plants and foods is particularly relevant considering the substantial opposition to GMOs recorded in the past, among the European citizens. The current level of awareness and concerns about GE, and their short term

evolution, is important as a new regulatory framework is in preparation by the European Commission.

**Acknowledgments** This work was supported by a grant of the Ministry of Research, Innovation and Digitization through Program 1 - Development of the National R&D System, Subprogram 1.2 - Institutional Performance - Projects for Excellence Financing in RDI, contract no. 2PFE/2021.

# **References**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **Chapter 32 What Is the Problem with Europe in a Philosophical Point of View?**

**Marcel Kuntz**

**Abstract** It is well known that developing plant biotechnological products is far more difcult in Europe than in the United States, for example. Of course, the different regulatory rationales impact technological development in both cases. This chapter discusses the reasons for such a difference, in relation with the historical background of Europe *vs*. USA, and in the philosophical context of 'postmodernism'. The latter is inuent in both the European Union (EU) and the USA, but does not politically express itself in the same way. The central pillar of the doctrine currently dominant in the EU being to prevent repetition of the tragedies of the past, especially wars, which includes avoiding becoming a political power in the old sense. This chapter proposes that this political thought has also inuenced the way technological risks are considered (Precautionary Principle) while benets are sometimes ignored, such as those of plant biotechnology. The April 2021 EU Commission report on gene editing is discussed as an example of postmodern framing.

# **1 Introduction**

Comparing how the transgenic plant innovation has been dealt with in the European Union (EU) *vs*. in the USA is of high interest in order to understand the possible perspective of genome editing in Europe.

While the transgenic plant technologies were co-invented in the early 80's by laboratories in both Europe and the USA [1, 2], the EU was clearly the centre of the plant biotechnology backlash which started in the middle of the 90s [3]. Earlier, in 1990, the EU, through its 90/220/EEC Directive, has created a new judicial object

M. Kuntz (\*)

Laboratoire de Physiologie Cellulaire et Végétale, Université Grenoble Alpes, CNRS, CEA, INRAE, Grenoble, France

called a 'genetically-modifed organism' (GMO).1 Although this Directive was replaced by Directive 2001/18/EC,2 it retained what many consider as a scientifc meaningless defnition of a GMO [4]. A central element of the defnition of a GMO according to the EU is that it is an "*Organism in which the genetic material has been altered in a way that does not occur naturally*". Such a concept has become highly damaging for public acceptance in the wake of the 'mad cow' crisis in Europe (when what was considered as an unnatural way of feeding cattle led to bovine spongiform encephalopathy, a disease potentially transmittable to humans). The temporal coincidence of the media coverage of this disease and the arrival on the market of the frst GMO harvests provided anti-GMO activists the opportunity to spread fears about food derived from GMOs. As a political response, the abovementioned new Directive was adopted in 2001. It states: "*In accordance with the precautionary principle, the objective of this Directive is to approximate the laws, regulations and administrative provisions of the Member States and to protect human health and the environment*". In other words, in the view of the EU, GMOs are intrinsically different from traditionally bred organisms and are therefore more risky, thus justifying a precautionary approach.

In the USA, the Federal government established a formal biotechnology policy as early as 1986, known as the 'Coordinated Framework for Regulation of Biotechnology'. It has since been updated 3 but remains based on existing laws, not a law in itself, in contrast with the path taken by the EU. Although one may consider that this Coordinated Framework has limited the deployment of transgenic crops to some extent, it did not have an inhibiting effect as laws in the EU had. Activists also attempted to propagate fears in the USA, which eventually lead to Public Law 114–216 on GMO labelling in 2016. However, the latter had only minimal labelling requirements, in contrast to what occurred in the EU under the 2001 Directive.

The advent of gene editing techniques gave rise to the question whether or not such a type of mutagenesis should be subject to specifc biosafety regulatory provisions. Many papers discussed the possible 'natural' occurrence of such 'edited' mutations [5]. It should also be noted that the 2001 GMO Directive lists 'mutagenesis' in "*Techniques/methods of genetic modifcation yielding organisms* to be excluded *from the Directive, on the condition that they do not involve the use of recombinant nucleic acid molecules*…".

However, in July 2018 the judgment of the Court of Justice of the European Union (CJEU) ruled that "*Organisms obtained by mutagenesis are GMOs and are, in principle, subject to the obligations laid down by the GMO Directive*" unless they

<sup>1</sup>Council Directive 90/220/EEC of 23 April 1990, https://eur-lex.europa.eu/legal-content/en/ ALL/?uri=CELEX%3A31990L0220. Accessed 12 october 2023.

<sup>2</sup>Directive 2001/18/EC of the European Parliament and of the Council of 12 March 2001, https:// eur-lex.europa.eu/legal-content/en/TXT/?uri=CELEX%3A32001L0018. Accessed 5 June 2023.

<sup>3</sup>EPA (USA), Update to the Coordinated Framework for the Regulation of Biotechnology, 2017, https://www.epa.gov/regulation-biotechnology-under-tsca-and-ffra/update-coordinated-framework-regulation-biotechnology. Accessed 5 June 2023.

"*have a long safety record*".4 This excludes gene editing, with an explicit reference to "*the precautionary principle which that directive seeks to implement*". The CJEU reasoning was that gene editing is closer to transgenesis than to conventional mutagenesis ("*since the direct modifcation of the genetic material of an organism through mutagenesis makes it possible to obtain the same effects as the introduction of a foreign gene into the organism (transgenesis) and those new techniques make it possible to produce genetically modifed varieties at a rate out of all proportion to those resulting from the application of conventional methods of mutagenesis*").

This view again clearly differentiates the EU from the USA. In the latter country, simple mutations, including those obtained by gene editing are usually considered as not necessitating regulatory oversight [5].

The question arising from these considerations is why are there such fully opposed approaches in the EU *vs*. the USA for the same biotechnologies? To address this question, this chapter will attempt to highlight the underlying ideological views and proposes that the latter are linked to a broader historical background.

# **2 A Brief Look Back at the History of Europe During the Twentieth Century**

Alongside undeniable progress (in civil rights, social conditions, medicine, etc.), Twentieth century Europe is characterized by two World Wars, with destruction at levels never seen before, and the mass crimes of two totalitarian states, including their respective concentration camps and genocides. Drawing lessons from the past, Europe has undertaken to avoid the repetition of such tragic events. One of the political instruments of this project was the European integration, which progressed from the Treaty of Rome in 1957 up to the current EU.5

The aim of the 1957 treaty was "*To work towards integration and economic growth, through trade*" and had 'specifc goals', amongst them to "*Pool their resources to* preserve and strengthen peace *and liberty*". The Consolidated Version of the Treaty on European Union (in 2016) goes further in presenting its 'values'.6 Its Preamble states: "*Recalling the historic importance of the ending of the division of the European continent and the need to create frm bases for the construction of the future Europe*", and "*Confrming their attachment to the principles of liberty, democracy and respect for human rights and fundamental freedoms and of the rule of law*".

<sup>4</sup>Court of Justice of the European Union, press release No 111/18, 25 July 2018, https://curia. europa.eu/jcms/upload/docs/application/pdf/2018-07/cp180111en.pdf. Accessed 5 June 2023.

<sup>5</sup>Treaty of Rome (EEC), https://www.europarl.europa.eu/about-parliament/en/in-the-past/the-parliament-and-the-treaties/treaty-of-rome. Accessed 5 June 2023.

<sup>6</sup>Consolidated version of the Treaty on European Union (2016), https://eur-lex.europa.eu/resource. html?uri=cellar:9e8d52e1-2c70-11e6-b497-01aa75ed71a1.0006.01/DOC\_2&format=PDF. Accessed 5 June 2023.

In other words, these values are now the 'Big Principles' on which the EU has been founded, in order to avoid the repetition of the disasters of the past. Signifcantly, "*The main goal of the European Union is to defend these values in Europe and promote peace and the wellbeing of the citizens*".7

# **3 'Big Principles' Have Also Been Applied to Science and Technologies**

Although the contributions of science and technologies to improving the human condition were considerable during the twentieth century, accidents and disasters did occur [6], with science being used to develop weapons of mass destruction. For the German-born philosopher Günther Anders and for others, the main events of the twentieth century were 'Auschwitz' and 'Hiroshima' [7].

The ideology on which the EU is founded, namely a political dream of 'no tragedy' has also encouraged an utopia of 'no technological risk', illustrated by the 'Precautionary Principle'. Together with what can be seen as a Principle of Participation (of 'stakeholders', 'citizens'…), which will not be developed here (see below and for more details see [8]), these represent new 'Big Principles' which were designed to avoid repetition of accidents caused by technologies.

In concrete terms, this precautionary ideology inspired the drafting of the GMO Directives (the General Principles of the 2001 Directive state: "*In accordance with the precautionary principle, the following general principles should be followed*… [for risk assessment]"). Gene editing is a new biotechnology which was not anticipated in these Directives, thus requiring an *ex-post* legal interpretation. In other words, the *letter* of the Directive being unclear, the CJEU (see above) reasoned within the *spirit* of these Directives, namely applying the Precautionary Principle.

# **4 Postmodernism as a Philosophical Background**

If one wishes to analyse further what has been described above in a philosophical context, the concept of postmodernism seems relevant, despite the fact that it is a polysemic term (it is also used in art, for example). Here, postmodernism refers to a type of thought that is largely a 'deconstruction' of the general Enlightenment philosophical viewpoints and values. The latter became dominant in the Western world progressively from the eighteenth century to the mid twentieth century. Regarding postmodernism, one can cite two French philosophers; Emmanuel Lévinas who criticized the Enlightenment philosophy as a "*totalizing*" system of thought and

<sup>7</sup>European Parliament in plain language: Values, https://europarlamentti.info/en/values-and-objectives/values/. Accessed 5 June 2023.

Jean-François Lyotard who defned postmodernism as the rejection of (western) "*metanarratives*" of human biological, historical, and social development. More generally, the modern 'universalist' thoughts have been accused to impose western conformity on other perspectives, thereby oppressing them, which is supposed to justify the 'deconstruction' of the European Enlightenment (for more details see [9]). The term 'deconstruction' is actually a translation of the concept of "*Abbau*" developed by German philosopher Martin Heidegger [10].

In a more recent phase, the concepts of 'deconstruction' and postmodernism moved from what was an intellectual debate to a form of more general rejection of the Western past. Such thoughts were largely fed by the tragic events mentioned above, to which one may add for some countries; colonialism, slavery and, more generally, discriminations of any type. From the 80's onwards, postmodernism insidiously became the dominant ideology of the European 'Elites'. It is largely infuenced by what can be termed 'Western Guilt' [11], which can manifest itself in various forms, from repentance to self-hatred. Its political project is a form of new redeemed society.

Such a redeemed society would not only be a 'no tragedy' one, but can also be extended to a 'post-history' one in the postmodern dream. It is worth mentioning here the book by the American political scientist Francis Fukuyama, entitled *The End of History and the Last Man*. Interestingly, its author commented the following: "*I believe that the European Union more accurately refects what the world will look like at the end of history than the contemporary United States. The EU's attempt to transcend sovereignty and traditional power politics by establishing a transnational rule of law is much more in line with a 'post-historical' world than the Americans' continuing belief in God, national sovereignty, and their military*" [12].

This is not to say that postmodern thoughts have not reached the USA; they are simply different (the past inspiring guilt and rejection is rather linked to racial issues in that country). It has provided the intellectual background of 'identity politics'. The broad implications of this politics will not be discussed here, only its implication for science.

# **5 Postmodernism Effects on Science**

Regarding science, 'identity politics' clash with Enlightenment epistemology which values open debates and merit in science. In contrast, postmodern views justify 'affrmative action' in favour of minorities and more generally politicization of science. This has led to an outcry from some scientists [13].

The general ideas of postmodernism ('deconstruction' of the Western past) have been adopted by various sociological felds, leading to the creation of new 'studies', such as 'cultural studies', 'gender studies', 'science and technology studies' (STSs), etc. [14].

While the aim of science within modern framing is to determine what is true and what is false, within the STSs feld some deconstructionist thinkers questioned the validity of claims of scientifc truth and criticized scientifc method. In their opinion, scientifc objectivity is reduced to 'claims' that are the expression of one community (the scientists) which shares preconceptions. Thus, science is simply one 'construct' of reality amongst many others (for more details, taken from the GMO case, see [15]). In such a relativist framing, politically-constructed claims by anti-GMO activists should have the same validity as science-based risk assessment of GMOs. For example, how it permeated the European Parliament was discussed [15].

Such postmodern views encourage a 'participative' approach in science, which draws scientists into the political arena, with no observed benefts for science and risk assessment as far as the GMO dispute is concerned [16]. Regarding interactions between science and society, the move from modernism to postmodernist can be summarized by a shift from the concept that 'the public (in its own interest) should rely on the judgments of expert scientists' to a reverse one, namely that 'scientists should listen to society'. In addition, concepts of 'justice', 'fairness', 'democratic deliberation', etc., will compete with the truth seeking approach of science (for an example taken in the USA, namely a report on 'gene drive' by the National Academy of Science, see [17]).

In summary, the transgenic technology has been affected by postmodernism in different manners: the Precautionary approach and cognitive relativism, which could not be attenuated due to the simultaneous 'deconstruction' of certain modern values, such as distinguishing truth and falseness, and trust in scientifc progress.

# **6 Fundamental Differences Between USA and Europe Regarding the Use of Technology**

Although the USA was involved in both World Wars experiencing great human loss, mass devastation and the above-mentioned tragedies actually occurred in Europe, not in the USA. These facts provide a likely historical explanation for the dream of 'no tragedy' in Europe but not in the USA (the outcomes in other countries such as Japan or Russia will not be considered here). In addition, the USA became the leading power in the world. To maintain this status, their authorities will always privilege their national interest and hence their industries [18].

In parallel, the former European 'imperialist' powers disappeared and the *raison d'être* of the EU was not to recreate a new bellicose empire, but was just the opposite. The EU's ambition is limited to a soft power which attempts to export its well-thinking norms and 'values' to other regions of the world, when the large size of its market allows it to do so (for discussion on the EU soft power, see resource notes.8,9,10,11 It also export its fears, as the GMO case has shown.

Based on good intentions and decked with its moral values, the EU has given a greater importance to consumers and the environment than to 'realpolitik' (for references on consumer protection and environmental policies in the EU, see notes).12,13,14,15 These EU priorities justify its reasoning on the necessity of regulation that others judge excessive (*e.g.* on biotechnologies).

However, one can note that the EU is embracing 'realpolitik' when it can hardly do otherwise. For example, it imports more than 20 million tons of GM soybeans per year to feed part of its livestock, thus supporting the production of transgenic plants in South America, while European farmers cannot do so because of the Precautionary Principle.

Besides the infuences of postmodernism, the ban on GMO cultivation was also the product of demagogy and short-term electoral alliances of certain governments in Europe, which will not be discussed here (for Germany, see [19]; for France, see [20, 21]). However, the fact that such political manoeuvres could have the adhesion of so many people is linked to the dissemination of the postmodern ideology beyond the 'Elites'.

# **7 The Implication for 'Poor' Countries**

The rejection of GMO cultivation by some 'developing' countries, such as some African countries which could beneft from plant biotechnology, can have different causes, from a concern of their leaders to preserve export opportunities to the EU, to the importation of European health fears by these countries. This situation should not be viewed as the adoption of 'postmodernism' by these countries, since the latter type of thought is inseparable from the 'Western Guilt'. It seems rather to be the consequence of a 'modern' type of reasoning (although not science-based), such as: 'why should we, the poor, adopt a technology that is rejected by the rich'.

<sup>8</sup> https://www.eeas.europa.eu/sites/default/fles/eugs\_review\_web\_0.pdf. Accessed 5 June 2023.

<sup>9</sup> https://www.unav.edu/documents/16800098/17755721/DT-05-2018\_EU-soft-power.pdf. Accessed 5 June 2023.

<sup>10</sup> https://www.academia.edu/30914935/Is\_there\_a\_European\_Soft\_Power. Accessed 5 June 2023.

<sup>11</sup> https://cepa.org/article/europe-too-soft-not-enough-power/. Accessed 5 June 2023.

<sup>12</sup> https://european-union.europa.eu/priorities-and-actions/actions-topic/environment\_en. Accessed 5 June 2023.

<sup>13</sup> https://commission.europa.eu/live-work-travel-eu/consumer-rights-and-complaints/sustainableconsumption\_en. Accessed 5 June 2023.

<sup>14</sup> https://www.efta.int/eea/policy-areas/flanking-horizontal-policies/consumer-protection. Accessed 5 June 2023.

<sup>15</sup> https://www.beuc.eu/blog/the-eu-has-become-an-environmental-policy-champion-time-to-gothe-last-mile/. Accessed 5 June 2023.

# **8 The EU Commission Report on Gene Editing as an Example of Postmodern Framing**

On 29th April 2021, the European Commission published a report regarding the status of what is called 'New Genomic Techniques' (NGTs, *i.e*. gene editing) under Union law.16 This report includes excellent overviews of research and innovation, as well as of risk assessment in this feld.

Interestingly, it also provides an illustration of the postmodern views dominant in the EU. This report appears rather positive about the potential benefts of the gene editing technology, but is not considered an asset for some kind of European power (industrial, agricultural…). Instead, gene editing is viewed as a potential contributor to its own pre-set goals: "*Several of the plant products obtained from NGTs have the potential to contribute to the objectives of the EU's Green Deal and in particular to the 'farm to fork' and biodiversity strategies and the United Nations' sustainable development goals (SDGs) for a more resilient and sustainable agri-food system*". Similarly, when benefts for farmers are mentioned, such as "…*plants more resistant to diseases*…", it is in a context "*of reduced use of agricultural inputs (including plant protection products)*", *i.e.* more constrains imposed on farmers by the EU policy.

The following sentence also illustrates the framing of the Commission: "*The Communication on the 'farm to fork' strategy stated that new innovative techniques, including biotechnology and the development of bio-based products, may play a role in increasing sustainability,* provided they are safe for consumers and the environment *while bringing benefts for society as a whole*". One can wonder whether a technology can be proven "*safe*" and whether any technology in its initial phase has ever brought "*benefts for society as a whole"…*

The following sentence illustrates that the Precautionary Principle is not viewed as a guiding tool for risk assessment, but as an aim in itself: "*Directives 2001/18/EC and 2009/41/EC* [on the contained use of genetically modifed micro-organisms] *share very similar aims (protection of health and the environment, application of the precautionary principle)*". The report does not mention that NGTs could be important for 'European power', which as discussed above is not part of the EU ideology.

In Sect. 4.6.2, the report summarizes the view of "*Stakeholders that see beneft in NGTs*" and, in Sect. 4.6.3, those of "*Stakeholders that do not see benefts in NGTs*". It concludes that "*Stakeholders are divided on the need to maintain the current legislation and reinforce its implementation, or to adapt it to scientifc and technological progress and the level of risk of NGT products*." In a somewhat utopic view, the report states that "…*efforts should be made to reconcile opposing views in order to fnd common ground to address the issues identifed in this study*".

<sup>16</sup>EC study on new genomic techniques (2021), https://food.ec.europa.eu/plants/genetically-modifed-organisms/new-techniques-biotechnology/ec-study-new-genomic-techniques\_en. Accessed 5 June 2023.

In a relativist (postmodern) framing (but it could simply be a political tactic), the report does not attempt do separate what is true and what is false. It simply wishes that "*Finally, more effort should be made to inform and engage with the public on NGTs and assess their views*".

Recently, a document from the European Commission proposes new approaches to regulating NGT plants. This proposal states that "*the Union GMO legislation is not ft for the purpose of regulating the deliberate release of plants obtained by certain NGTs*".17 In line with the 2021 report by the Commission, gene editing is viewed as "*a possible tool to increase sustainability*", that is to say its own pre-set well-thinking goals. However, a certain recognition of the reality of the world can now be observed: "*the Covid-19 pandemic and Russia's war of aggression against Ukraine have also revealed the EU's external dependencies*". It also mentions that "*The Union risks being excluded to a signifcant extent from the technological developments and economic, social and environmental benefts that these new technologies can potentially generate*". In addition, regarding Sustainable Development Goals (SDGs), SDG9 (industry, Innovation and Infrastructure) is now specifcally mentioned. Furthermore, the general objectives of this draft include to "*enhance the competitiveness of the EU agri-food sector*", while keeping (as expected with regard to the above-mentioned domination of a postmodern framing in the EU) the reference to the Precautionary Principle, the Green Deal and the Farm to Fork and Biodiversity strategies. Whether these economic objectives will clash with sustainability objectives remains to be seen.

# **9 Conclusions and Perspectives**

As discussed previously, "*The EU has thus given the absolute priority to consumers and perceived environmental care, based on good intentions and moral values, but to do so it has indulged itself in excessive regulations for ideological reasons*" [8].

In this context, it is unlikely that the EU will take into account scientifc facts presented in a 'modern' framing of truth. This implies that scientists, in future narratives, should also put forward 'values' not merely scientifc facts, bearing in mind that in the foreseeable future modifying the EU directives will not be possible if this contradicts the spirit of the European 'Big Principles' and primarily the Precautionary Principle.

<sup>17</sup>EC (2023). Proposal for a regulation of the European Parliament and of the Council on plants obtained by certain new genomic techniques and their food and feed, and amending Directives 68/193/EEC, 1999/105/EC, 2002/53/EC, 2002/55/EC, and Regulation (EU) 2017/625. https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A52023PC0411. Accessed 12 October 2023.

# **References**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

**Tomasz Zimny**

**Abstract** This chapter presents potential consequences of the adoption of strict detection, identication or traceability requirements in the EU legislation regarding NGT products featuring single nucleotide variants. The context considered encompasses changes in the biosafety legislations not only in countries which were traditionally accepting of modern biotechnology products, but also in countries which were reluctant to use classic GMOs. Due to shifts in the approach to the regulation of NGT products not featuring stable inserts of foreign DNA, the EU risks becoming an isolated market with provisions not harmonized with those of its various trade partners or falling into a situation where regulated products ofcially not present on the market will enter due to a lack of efcient detection and identication methods and enforcement systems. Recent changes in the laws of such countries as Nigeria, Kenya or Japan are presented, as well as the recent jurisprudence of the Court of Justice of the EU.

# **1 Introduction**

Following its study on the legal status of new genomic techniques' (NGT) products in the EU [1], the European Commission (EC) embarked on a mission to reform the current legislation on GMOs in order to facilitate the development and marketing of NGT products and to introduce an act, in which the level of regulation would be proportional to the risks related to the use of a given NGT product. The study considers NGT products as GMOs including: products of directed mutagenesis techniques, products of cisgenesis and intragenesis, as well as products featuring

T. Zimny (\*)

Institute of Law Studies, Polish Academy of Sciences, Warsaw, Poland e-mail: tzimny@inp.pan.pl

interventions in the genotype without changing the nucleotide sequence (epigenetic changes). The situation occurred in the aftermath of the C-528/16 judgment [2] of the Court of Justice of the European Union (CJEU), where the court opted for a narrow interpretation of the exemption of mutagenesis products from the GMO legislation, limiting it only to products of such methods, which were widely used at the time of adoption of the 2001/18/EC Directive on the deliberate release of GMOs [3]. After the publication of the study of the EC, the CJEU was tasked with deciding yet another legal problem resulting from the sentence of the former judgment – namely whether the mutagenesis exception in the 2001/18/EC Directive applies to products of random mutagenesis *in vitro* as well as *in vivo*. The response of the CJEU states that the exception should be interpreted "as meaning that organisms obtained through the application of a technique/method of mutagenesis which is based on the same processes of modifcation, by the mutagenic agent, of the genetic material of the organism concerned as a technique/method of mutagenesis which has conventionally been used in a number of applications and has a long safety record, but which differs from that second technique/method of mutagenesis by virtue of other characteristics, shall, in principle, be excluded from the exemption laid down in that provision, provided that it is established that those characteristics are likely to lead to modifcations of the genetic material of that organism which differ, by their nature or by the rate at which they occur, from those obtained by the application of that second technique/method of mutagenesis. However, the effects inherent in in vitro cultures do not, as such, justify the exclusion from that exemption of organisms obtained by the in vitro application of a technique/method of mutagenesis which has conventionally been used in a number of in vivo applications and has a long safety record with regard to those applications" [4].

In other words, the CJEU stated that the rule of thumb is that if a mutagenesis technique deviates in terms of effciency or potential for modifcation from methods conventionally applied in 2001, then its products shall not be exempted from the GMO legislation. **However, the fact that random mutagenesis was performed** *in vitro* **does not seem to justify such a notion.**

It is not the goal of this study to perform a thorough and rigorous interpretation of the judgment. Rather it serves as another iteration of conceptual problems the EU institutions need to solve, when faced with the matter of the current GMO legislation [5]. The criteria for exemption of certain products of mutagenesis from the legislation seem to be connected with the time of the development of the method that brought about a particular mutation and the effciency of the method. Given the fuzzy nature of such criteria, it is not surprising that the EC has decided to amend the current legislation. The recently published project of a new regulation on the matter, which envisages a confrmation procedure for products featuring minor changes (NGT type 1 plants) and their exclusion from organic production, as well as the questions regarding traceability and detection of NGT products posed by the EC in a recently closed public opinion poll [6], and also other activities, such as the recently closed Horizon Europe call for the development of new detection methods on products derived from new genomic techniques for traceability, transparency and innovation in the food system [7], suggest that the institution wishes to introduce a system that would at least feature some sort of detection and identifcation mechanism, possibly also labelling, even if the provisions relating to the introduction of certain NGT product to the market were to be relaxed. While such solutions might be based on a wish to honor consumer choice, and address safety concerns, the technical diffculties connected with the performance of detection and identifcation of NGT products featuring point mutations, together with the regulatory tendencies in third countries, might lead to some undesired consequences. The success of any reform of the current legislation is also not guaranteed, since the recently leaked internal EC documents show [8] that the Commission is concerned about several issues connected with the reform, such as its infuence on organic farming, the public rejection of GMOs in general and other issues, which might become an obstacle to the adoption of any amendment of the current laws.

# **2 Problems with Detection and Identifcation of NGT Products**

If the EC fails to introduce a reform of the legislation, which would somehow exempt NGT products from authorization procedures, or if the reform will contain a relaxation of the current provisions but with the maintenance of detection, identifcation, traceability or labelling requirements, the compliance with such requirements might be technically challenging. Some researchers advocate a rather strict approach to the use of such techniques, including case-by-case risk assessment and *inter alia* whole genome sequencing for the detection of potential unintended consequences of editing [9]. Others seem much more skeptical as to the feasibility of such postulates, particularly, when it comes to detection or identifcation of products featuring single nucleotide variants, as well as the ability to prove that a given mutation (even if detected) was caused by a regulated technique rather than by an exempted one (e.g. random mutagenesis) or was spontaneous.

Detection of single nucleotide mutations in plant material, using PCR methods, depending on the method used, might require previous knowledge about the edit or might require data about the sequence surrounding the edit [10, 11]. It is also pointed out that even whole genome sequencing supported by bioinformatics and database access might be prone to errors especially for heterogenous samples, and that the effciency of such detection methods also depends on the size of the genome that is being sequenced, making detection of potential contaminations in samples of such species as wheat or maize, less feasible [12].

These problems will gain practical signifcance with a broader adoption of NGT products featuring single nucleotide variants or even lacking changes in the nucleotide sequence, worldwide. The lack of applicability of existing laboratory methods for enforcement has already been stressed by the ENGL. It needs to be noted that, should EU legislation not change, unauthorized NGT products in the EU will be treated as unauthorized GMOs and essentially banned from the market. Should some requirements regarding detection, identifcation, labelling, coexistence with conventional varieties remain, these will need to be enforced somehow, in a situation of lack of easily accessible, effcient and economic detection methods. Such outcomes, in the context of trade exchange with the currently biggest producers of GMO products imported to the EU have already been described [13]. It seems however that the problems might also apply to exchange in agricultural goods with other countries, which were hitherto reluctant to introduce classic GMOs, but have decided to relax the legislation or even exempt products featuring single nucleotide variants or not featuring stable foreign DNA inserts.

# **3 Situation in Third Countries**

Countries, which are already well known for excluding certain NGT products from their GMO legislations, such as the USA, Canada, Argentina or Brazil [14, 15] have introduced changes in their legislations that will be diffcult to harmonize with an EU solution that will require the authorization of such products and more importantly their identifcation through molecular methods. Also, several countries, which were so far reluctant to adopt products of genetic engineering, are changing their policies in such a way that they allow products featuring single nucleotide variants to be less regulated than classic GMOs.

Some African nations have been reluctant to adopt the GMO technology [16], partially due to the restrictive policies of the EU, a major trading partner in agricultural goods. However when it comes to NGT products, some African countries are adopting policies, which are more permissive for plants featuring single nucleotide variants or more generally: mutations akin to those achievable through random mutagenesis or conventional breeding. For instance Nigeria issued new guidelines regarding the procedures for administrative handling of certain NGT products in December 2020 [17], according to which the applicant shall receive a biosafety approval if the method used for obtaining a product does not involve recombinant DNA or if such DNA is not present in the fnal product [see also 18].

Kenya issued an interpretation of its existing legislation [18, 19],: "modifcations by inserting genes from sexually compatible species and where regulatory elements (promoters and terminators) are also from the same species; deletions/knock outs provided that there is no insertion of foreign genetic material in the end-product; processed products whose inserted foreign genetic material cannot be detected; – do not fall under the Biosafety Act, which would otherwise require them to undergo an authorization procedure. The applicant is expected to submit an Early Consultation Form to the competent authority, in order to determine whether their product will fall under the biosafety legislation or not."

Japan is another example of a country introducing a leeway for products of directed mutagenesis without stable inserts of foreign DNA [20]. Relatively recent amendments to the biosafety policies provide that food products derived through a gene editing technology that do not contain remnants of foreign DNA fragments (e.g. SDN-1 products) fall under a notifcation rather than authorization procedure, hence do not require to go through a safety assessment procedure. Conventional crosses with such plants do not require to be notifed anymore. This solution, based on a preemptive confrmation of status, results in a release of certain products of gene editing and their progeny, to the market, without a requirement for traceability or any sort of identity preservation. Hence their products can freely circulate on the market, once they were initially notifed to the competent authority. This step means that SDN-1 products would not fall under the legislation [21, 22]. In Japan classic GMOs can be authorized for cultivation, but their use would subsequently be thwarted through the decisions of regional governments, who had the last voice in the matter [23]. The currently adopted solution allows developers to avoid the administrative burdens to a large extent and some gene editing products were actually already accepted according to the new provisions, most notably fsh [24] and tomatoes [25], which were already placed on the market [26].

Similar provisions were also adopted in high volume GMO trading countries. In Argentina and Brazil, it is the introduction of a stable construct of foreign DNA, which determines the regulatory status of the product [13, 14]. In the USA exemptions are *inter alia*:


The consultation or notifcation of the authorities is not mandatory [27, 28].

England and Wales have also introduced relaxed provisions regarding feld trials and marketing of "precision bred organisms", which could have been obtained from traditional processes [29].

Such solutions, as the ones presented above will likely result in products derived through e.g. SDN-1 techniques to fall out of the legislation, and are not required to meet the provisions associated with classic GMOs, such as traceability or development of detection or identifcation methods.

# **4 A Global Conceptual Shift**

The situation presented above – where not only countries that were liberal towards cultivation and other use of traditional GMOs relax their legislation, but also countries, which used to oppose the use of such products for various reasons – constitutes a conceptual shift, where the need for regulation is altered signifcantly.

The EU is a trendsetter in the case of many technological standards, in that entrepreneurs from third countries tend to comply with the EU-set standards, due to the size of the common market and the purchasing power of EU citizens. Complying with such standards seems economically more viable than being effectively locked out of such a market, due to lack of compliance. A recent example of this phenomenon was observed with the introduction of the USB-C port as the common charger port for all phones and tablets [30], which resulted in a decision to install such ports in iPhones, a step hitherto resisted by the manufacturer. In the case of "classic" GMOs the EU was also a *de facto* trendsetter for many third countries, which would prohibit the cultivation and often other uses of such products in order to prevent accidental infux of such products to the EU and so as to not to endanger their exports to the Union [16]. This seems not to be the case anymore, at least when it comes to NGT products featuring single nucleotide variants or lacking stable inserts of foreign DNA. In this sense the EU instead of being a standard setter is at risk of becoming an isolated island surrounded by regions with a fundamentally different approach to NGT products, namely a permissive rather than a precautionary one. This scenario is more likely to be realized if no changes are made in the current legislation or if the changed legislation will still require detection, identifcation, labelling or coexistence measures for such products. In the latter 2 cases, two not mutually exclusive scenarios can be presented for the behavior of foreign exporters, local importers or operators of those products. Firstly, they might attempt to adhere to the restrictive provisions, which might result in increased costs as well as lowering the competitive position of their products on the market. The development of a rigorous documentation system for all the steps of the production chain, capable of creating a reliable paper trail for each imported product would probably be required to support compliance with such provisions. Secondly, some entrepreneurs might choose to ignore such requirements, counting on the lack of rapid detection methods and a general lack of effciency of the enforcement authorities in the detection of NGT products. The latter scenario might result not only in formally "unauthorized GMOs" circulating in the common market, or even authorized products not being properly labelled, but might cause damage further down production chains, if unauthorized products get eventually detected at the later stages of processing or marketing of processed products. A situation, where such plant material is used in breeding activities and is only later detected as a component of registered "conventional" or organic varieties needs also to be considered.

# **5 Conclusions**

While the fnal shape of the new EU legislation regarding the development and use of NGT products is unknown and its fate also remains uncertain, the latest CJEU jurisprudence did not bring about any signifcant change to the existing status quo and the proposed legislative changes still feature authorisation procedures and labelling of some products, such as reproductive material of NGT type 1 plants. Concurrently a conceptual shift in the approach to NGT products featuring single nucleotide variants takes place in third countries, in that such products are no longer considered to be GMOs requiring strong regulation. This phenomenon becomes characteristic not only for nations, which were traditionally accepting of modern biotechnology products, but also in countries, which were reluctant when it came to the adoption of classic GMOs. This shift might result in serious practical problems for EU entrepreneurs and enforcement authorities alike, due to practical problems with the development of effcient detection methods and also might lead to a situation of legal fction, where certain products, although offcially regulated, will circulate on the market due to the aforementioned defciencies in cost- and technically-effcient detection and identifcation methods.

This work was fnanced from a National Science Centre Grant no: UMO-2020/39/D/HS5/03144, Transformation of biosafety legislation in agriculture in the EU law and the laws of its selected trade partners, with respect to the scientifc development.

# **Literature**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **Part V Future Outlook**

# **Chapter 34 Prospects for Plant Genome Editing**

**Agnès Ricroch, Dennis Eriksson , Dragana Miladinović , Jeremy Sweet, Katrijn Van Laere , and Ewa Wo**ź**niak-Gientka**

Genome editing – the focus of this book – is a set of methods that provide opportunities to precisely and efciently improve crop traits by tailoring genes and regulatory domains. In combination with genetic modications (GM), RNA interference (RNAi), epigenetics and the range of -omics technologies, they offer multiple methods for enhancing crop production, crop protection, crop quality and climate change adaptation. Combining these technologies with traditional breeding and careful management of crop cultivation methods in integrated systems can make major contributions to improving the sustainability of agricultural production, particularly in response to climate change. These technologies could also contribute to achieving United Nations' Sustainable Development Goals and national/EU policy objectives for agriculture, food safety, food security and the environment.

A. Ricroch (\*)

D. Eriksson Department of Plant Breeding, Swedish University of Agricultural Sciences, Alnarp, Sweden

D. Miladinović Institute of Field and Vegetable Crops, National Institute of Republic of Serbia, Novi Sad, Serbia

J. Sweet Sweet Environmental Consultants, Cambridge, UK

K. Van Laere Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), Melle, Belgium

E. Woźniak-Gientka Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland

The Jean-Monnet Faculty. Idest Laboratory, Paris-Saclay University, Sceaux, France e-mail: agnes.ricroch@universite-paris-saclay.fr

# **1 Technology**

Genome editing in plants is often referred to as precision breeding due to the precise nature of the methods used compared with some other genetic modifcation and mutation breeding techniques. Genome editing technologies exceeded initial expectations as a tool in plant science and breeding and are quickly adopted in most life-science laboratories as a precise and high-throughput tool for targeted crop improvement. They became the method of choice in many research studies. The future of genome editing relies on expanding the technology toolbox with more effcient multiplexing and high-throughput strategies, applications towards chromosomal rearrangements and epigenetic changes together with more tools for effcient delivery of editing components and regeneration of edited cells.

# **2 Crop Improvement**

Genome editing has already been applied for numerous trait improvements in crops. By now, there are more than 100 applications of genome editing on at least 28 different plant species, some of which have been described in this book. In planta genome editing methods still need to be optimized in a broader range of plant species before to achieve a signifcant commercial impact. This especially stands for time and costs related to the development of specialized regeneration protocols for individual plant lines. It is therefore important to broaden the targets for crop improvement by gaining knowledge about biological processes and genes involved, as well as the interaction of biological processes with the environment.

Although being known as a very precise technology, off-target, pleiotropic and other unintended effects have occasionally been reported. The regular plant breeding procedures, however, are expected to manage this by careful screening and testing of new breeding lines through several generations prior to multiplication and commercialisation.

Technical innovations will expand the genome editing toolbox and further strengthen its technical and economic advantages in crop improvement. However, genome editing will still mostly complement rather than replace conventional plant breeding methods in crop improvement. Further applications of genome editing technologies in crop improvement depend largely on the economic and legal framework, as well as public perceptions of the technology itself.

# **3 Regulations**

Government regulation for new genomic techniques (NGTs) that comprise, among others, genome editing must be science-based, predictable, risk-proportional, and harmonized with international trading partners. Harmonization and predictability would reduce investment risk and increase spending in R&D whereas riskproportionality would balance the costs of respective innovation with the social benefts secured through risk avoidance. That is why it is important that appropriate and science-based policies and regulations are in place that allow rapid and thorough assessment of any risks associated with the products of.

A recent draft European Commission proposal in July 2023 defnes an NGT (Category 1) plant as a plant obtained by directed mutagenesis, cisgenesis, and intragenesis, that is equivalent to a conventionally bred plant and does not contain any genetic material imported from outside the breeders' gene pool and contains a very limited number of changes to the plants genome. Therefore, the Commission defnes NGT plants as GMOs that do not contain any "foreign" DNA. For NGT crops, the Commission introduces two categories that are regulated differently:


According to the draft document Category 1 NGT plants are exempt from the existing GMO regulations. From this it can be deduced that Category 1 NGT plants are not subject to any specifc risk assessments for health and the environment and will not require monitoring, labelling or traceability along supply chains. However, Category 1 NGT plants (and their products) are not unregulated. They will require notifcation to the relevant competent authority in order to allow confrmation of the categorisation before release or marketing. In addition, regulations governing activities with conventionally bred plants apply to them and -depending on the type of modifcation- other obligations such as imposed by the Novel Food Regulation (EU) 2015/22 must be met.

For Category 2 NGT plants, there are different procedures for release and placing on the market. The approval process is the usual GMO procedure based on the guidelines for genetically modifed organisms (VO (EC) 1829/2003) with associated detection methods and traceability.

This proposal would potentially bring the EU more in line with regulatory authorities from other parts of the world and facilitate the commercialisation of many genome edited crops. This is especially important for controlled release of products of new breeding systems and technologies in feld trials to assess the performance and net contribution that new varieties can make to sustainable farming systems and for environmental impact assessments. NGT varieties could also be assessed for their contribution towards managing crop production in relation to climate change and other externalities infuencing food production and supply chains. However, while this proposal fnally provides a concrete basis for an adapted regulatory framework, it will take several years before the new Regulation will be

fnalized and become effective, meanwhile stifing the dynamic research, development and market environment.

# **4 Public Perception**

Public perception is one of the critical parameters infuencing the development and commercialization of plants produced with the use of NGTs. General positive attitudes towards genome editing, both in the public and stakeholder acceptance, can support the implementation of relevant regulations in different countries. Like the legal situation and state of genome editing that is diverse worldwide, the public perception of plant gene technologies differs across regions. These differences in opinions are not grounded in science but rather in politics, psychological, social, cultural, personal and economic factors.

Engaging citizens in the development of innovations in life sciences is critical, and there is a potential advantage in communicating biotechnology and genome editing to society. The engagement of scientists and experts in public debates about the future of NGTs is crucial and may encourage scientists to make more effort in public debates regarding the benefts of genome editing products. Scientists, policymakers and entrepreneurs should create more opportunities for the public to participate in relevant meetings and activities (e.g., citizen science projects). Moreover, these interactions facilitate monitoring shifts in the acceptance of NGTs by the public.

# **5 PlantEd**

The COST Action PlantEd (CA 18111) has since 2019 brought together scientists, plant breeders and other stakeholders to discuss many aspects of plant genome editing and this book refects many to the issues considered during this Action. In a survey circulated among the 608 experts involved in PlantEd to estimate the value and impact of the network, 90% agree (completely or somewhat) that they have obtained new ideas and knowledge about plant genome editing by being part of the PlantEd network, 86% agree that they have obtained new connections and potential collaborators through the network, and 88% agree that PlantEd has contributed to the development of plant genome editing in Europe and beyond. This emphasizes the importance of the Action itself, as well as the importance of constant delivery of broad knowledge about genome editing technology to general public and stakeholders. This should facilitate the adoption of NGTs in crop improvement and agricultural production that should further contribute to food security and sustainability of agricultural production in changing climate and unstable market conditions.

**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.