Santosh Kumar Upadhyay is an Assistant Professor in the Department of Botany, Panjab University, Chandigarh, India. His research focuses on functional genomics in plants, especially the use of the CRISPR-Cas system for genetic engineering.

• List of Contributors xvPreface xixAbout the Editor xx1 CRISPR/Cas-Mediated Genome Editing in Plants: A Historical Perspective 1Anil Kumar, Shumayla, and Santosh Kumar Upadhyay1.1 Introduction 11.2 Historical Background 21.3 Mechanism of CRISPR/Cas System 41.3.1 Acquisition of Spacers 41.3.2 Biogenesis 51.3.3 Interference with the Target 51.4 Breakthrough Studies in CRISPR/Cas System 51.5 CRISPR Types 61.6 Type of Cas Proteins 71.6.1 Cas 1 71.6.2 Cas 2 71.6.3 Cas 3 71.6.4 Cas 4 71.6.5 Cas 5 71.6.6 Cas 6 81.6.7 Cas 7 81.6.8 Cas 8 81.6.9 Cas 9 81.6.10 Cas 10 81.6.11 Cas 11 81.6.12 Cas 12 91.6.13 Cas 13 91.6.14 Cas 14 91.7 CRISPR/Cas Modification 91.7.1 Nickase 91.7.2 Dead Cas9 (dCas9) 101.7.3 Base Editors 101.7.4 Prime Editors 101.8 CRISPR/Cas as a Genome Editing Tool and Its Application 101.8.1 Gene Knockout 101.8.2 DNA Insertion 111.8.3 Base Editing 111.8.4 Gene Activation and/or Repression 121.8.5 Epigenetic Modifications 121.8.6 Localization 121.8.7 RNA Editing 131.9 Conclusion 13References 132 CRISPR/Cas-Mediated Multiplex Genome Editing in Plants and Applications 20R. Prajapati and K. Tyagi2.1 Introduction 202.2 Construct Design for Multiplex CRISPR/Cas Genome Editing 222.3 Strategies for Processing Multiple-Guide RNAs 232.4 Delivery of CRISPR/Cas Construct into Plant Cells 242.4.1 Agrobacterium-Mediated Delivery 242.4.2 Virus-Mediated Delivery 242.4.3 Particle Bombardment-Based Delivery 252.5 Broader Implications of CRISPR/Cas Multiplex Gene Editing 252.5.1 Simultaneous Knockout of Multiple Genes 252.5.2 Targeted Chromosomal Deletions 262.5.3 Transcriptional Activation or Repression of Genes 262.5.4 Base Editing 262.6 Application of CRISPR/Cas Multiplex Gene Editing in Generating Disease Resistant Plants 272.6.1 Disease Resistance Against Viruses 272.6.2 Disease Resistance Against Fungi 282.6.3 Disease Resistance Against Bacteria 292.7 Application of CRISPR/Cas Multiplex Gene Editing in Abiotic Stress-Tolerant Crop Production 292.7.1 Drought Tolerance 302.7.2 Salinity Tolerance 302.7.3 Herbicide Resistance 312.8 Application of CRISPR/Cas Multiplex Gene Editing in Enhancing Crop Yield, Nutrition, and Related Traits 312.9 Conclusion 32Acknowledgments 32References 343 Cas Variants Increased the Dimension of the CRISPR Tool Kit 40Sameer Dixit, Akanchha Shukla, Mahendra Pawar, and Jyothilakshmi Vadassery3.1 Introduction 403.2 General Architecture and Mechanism of CRISPR-Cas System 413.3 Classification of CRISPR-Cas System 423.3.1 Class 1 CRISPR-Cas System 443.3.2 Class 2 CRISPR-Cas System 453.4 Different Application-Based CRISPR-Cas System 453.4.1 Cas 9 463.4.2 Cas 12 463.4.3 Cas 14 463.4.4 Cas 13 473.4.5 Cas 3 473.5 Advancement and Reengineering of CRISPR-Cas System 473.6 Conclusions 48Acknowledgments 49References 494 Advancement in Delivery Systems and Vector Selection for CRISPR/ Cas-Mediated Genome Editing in Plants 52Sanskriti Vats, Sukhmandeep Kaur, Amit Chauhan, Dipul Kumar Biswas, and Rupesh Deshmukh4.1 Introduction 524.2 Advancement in Delivery Systems and Vector Selection for CRISPR/ Cas-Mediated Genome Editing in Plants 534.2.1 Vector Selection Based on Application and Availability in Plants 534.2.2 Plant Transformation Methodologies 564.3 Emerging Advanced CRISPR/Cas Systems and the Increased Demand for Quick Transformation Protocols 574.4 Advancements in Agrobacterium-Meditated Stable Transformation of Plants 594.5 Improvement of Agrobacterium-Mediated Transformation System by Developmental Regulators and Modular Agrobacterium Strains 614.6 Non-Agrobacterium Systems for Plant Transformation 624.7 Viral Vectors for Delivery of CRISPR Reagents and Increasing Donor Titer 634.8 De novo Meristem Induction 654.9 Biolistics and Protoplast Systems for CRISPR-Based Genome Editing 664.9.1 Biolistic Approach 664.9.2 Protoplast Approach 674.10 Generation of Transgene-Free CRISPR-Edited Lines 684.10.1 Mendelian Segregation Analysis 684.10.2 Programmed Self-Elimination Method 684.10.3 Transient Expression of CRISPR/Cas9 Cassette 68References 695 Role of Nanotechnology in the Advancement in Genome Editing in Plants 78Mehtap AYDIN5.1 An Overview of Plant Genome Editing 785.1.1 Meganuclease 795.1.2 Zinc Finger Nucleases 795.1.3 Transcription Activator-Like Effectors Nucleases 805.1.4 CRISPR/Cas9 Based Genome Editing 805.2 Nanoparticles used as Genome Editing Tools in Plants 805.2.1 Mesoporous Silica Nanoparticles 825.2.2 Carbon Nanotubes Carbon 825.2.3 Lipid-Based Nanoparticles 835.2.4 Polymer-Based Nanoparticles 835.3 Point of View: The Nanotechnology and Plant Genome Editing 835.4 The Approach to Transferring Biomolecules to Plants and Its Limitations 845.5 Role of Nanotechnology in Agriculture 845.6 Conclusion 86References 866 Genome Editing for Crop Biofortification 91Erum Shoeb, Srividhya Venkataraman, Uzma Badar, and Kathleen Hefferon6.1 Introduction 916.2 Current Global Status of Micronutrient Malnutrition 926.3 Importance of Biofortification in Ensuring Food Security 926.4 Strategies for Biofortification 936.4.1 Chloroplast Metabolic Engineering for Developing Nutrient-Dense Food Crops 946.5 Biofortification Through Agronomic Practices 966.6 Genome Editing Is a Powerful Tool 986.6.1 Meganucleases (MegNs) 996.6.2 Zinc Finger Nucleases 1006.6.3 TALENs 1006.6.4 CRISPR/Cas- 9 1016.7 Examples of Biofortification Using Genome Editing Technologies 1026.7.1 Amino Acid Biofortification 1026.7.2 GABA Biofortification 1026.7.3 Improvement of Oil Content and Quality 1056.7.4 Improvement of Resistant Starch Content 1056.7.5 Improvement of Micronutrient Bioavailability 1056.7.6 Crops Enriched in Iron 1056.7.7 Zn-enriched Crops 1066.7.8 Crops Enriched in Vitamin A 1066.7.9 Crops Enriched in Vitamin E 1076.7.10 Engineering Crops Adapted to Growing in Toxic Environments 1076.7.11 CRISPR-Cas9-enabled Decrease in Anti-nutrients 1076.7.12 Benefits of Genome Editing over Other Technologies for Biofortification 1086.8 Regulation of Genome Editing 1086.9 Conclusions and Future Prospects 109References 1097 Genome Editing for Nutritional Improvement of Crops 122Pooja Kanwar Shekhawat, Hasthi Ram, and Praveen SoniAbbreviations 1227.1 Introduction 1247.2 Evolution of Techniques for Improvement of Crops’ Genomes 1247.3 Genome Editing for Nutritional Improvement 1257.3.1 Improvement in Cereal Crops 1267.3.2 Improvement in Oilseed Crops 1387.3.3 Improvements in Horticulture Crops 1397.4 Regulation of Genome Edited Crops: Current Status 1417.5 Future Perspectives and Conclusion 142Author Contribution 142Acknowledgment 142References 1438 Genome-Editing Tools for Engineering of MicroRNAs and Their EncodedPeptides, miPEPs, in Plants 153Ravi Shankar Kumar, Hiteshwari Sinha, Tapasya Datta, Ashish Sharma, and Prabodh Kumar Trivedi8.1 Introduction 1538.1.1 ZINC Finger Nucleases 1548.1.2 TALE Nucleases 1558.1.3 CRISPR/Cas 9 1568.2 CRISPR–Cas9-Mediated DNA Interference in Bacterial Adaptive Immunity 1578.2.1 Types of CRISPR Systems 1588.2.2 The Cas9 Enzyme 1588.3 CRISPR/Cas9 Effector Complex Assembly 1598.4 The Mechanism of CRISPR/Cas9-Mediated Genome Engineering 1598.4.1 Comparison with Other Technologies for Genome Editing 1608.4.2 Limitations of the Cas9 System 1608.4.3 miRNAs 1628.4.4 Biogenesis of miRNA 1628.4.5 miRNA and Gene Regulations 1638.5 Role of Genome-Editing in miRNA Expression 1648.6 Applications of the CRISPR/Cas9 System in miRNA Editing 1658.6.1 microRNA-Encoded Peptide 1668.6.2 Biogenesis of miPEPs 1668.6.3 Role of miPEP 1678.7 miPEPs Act as the Master Regulator in Plant Growth and Development 1678.8 Conclusions and Future Prospect 168Acknowledgments 169References 1699 Genome Editing for Trait Improvement in Ornamental Plants 177Yang Zhou, Yuxin Li, and Wen Liu9.1 Introduction 1779.2 Application of Gene Editing Technology in Color Regulation of Ornamental Plants 1789.3 Application of Gene Editing Technology in Ornamental Plants Preservation 1799.4 Application of Gene Editing Technology in Shape and Organ Regulation of Ornamental Plants 1809.5 Application of Gene Editing Technology in Other Traits of Ornamental Plants 1809.6 Conclusions and Perspectives 181Acknowledgments 181References 18110 Abiotic Stress Tolerance in Plants by Genome Editing Applications 185Elif Karlik Urhan10.1 Introduction 18510.2 Drought Tolerance 18710.3 Salinity Tolerance 19110.4 Temperature Stress Tolerance 19610.4.1 Heat Stress Tolerance 19610.4.2 Cold Stress Tolerance 19910.5 Conclusions 202References 20311 Genome Editing for Improvement of Nutrition and Quality in Vegetable Crops 222Payal Gupta, Suhas G. Karkute, Prasanta K. Dash, and Achuit K. Singh11.1 Vegetables and Human Nutrition 22211.2 Important Quality Parameters of Vegetables 22311.3 Approaches for Improving Nutrition Content in Vegetables 22311.3.1 Breeding for Improving Nutrition in Vegetable Crops 22411.3.2 Genome Editing Technologies 22511.3.2.1 CRISPR/Cas9 and Advances in Genome Editing 22511.3.2.2 Mechanism of CRISPR/Cas-Mediated Genome Editing in Plants 22611.4 Applications of Genome Editing for Improvement of Vegetable Nutrition and Quality 22711.4.1 Improvement in the Appearance in Terms of Shape and Size 22911.4.2 Improvement of the Shelf-Life 22911.4.3 Improvement of the Ripening Time 23011.4.4 Improvement in Colour of the Fruit/Vegetable 23011.4.5 Biofortification of Vegetable Crops Through Genome Editing 23111.4.5.1 Metabolic Engineering of Carotenoid Biosynthesis Pathway 23111.4.5.2 Increasing γ-Amino Butyric Acid and Vitamin D Content 23211.4.6 Improvement of Starch Content 23211.4.7 Elimination of Anti-Nutritional Factors 23211.5 Challenges and Future Prospects 23311.6 Conclusion 234References 23412 Insight into the Flavonoids Enrichment in Plants by Genome Engineering 242Elena V. Mikhaylova12.1 The Importance of Flavonoids 24212.2 Flavonoid Biosynthesis Pathway 24412.3 In Planta Flavonoid Enrichment via Genome Editing 24712.4 Biotechnological Production of Flavonoids 25212.5 Conclusions 253References 25313 Genome Engineering in Medicinal Plants for Improved Therapeutics: Current Scenario and Future Perspective 260Buket Çakmak Güner13.1 Introduction 26013.2 Genome Engineering in Plants 26113.2.1 Agrobacterium-Mediated Transformation 26113.2.2 Biolistic or Particle Bombardment-Mediated Transformation 26213.2.3 Electroporation-Mediated Transformation 26213.2.4 Chemical-Mediated Transformation 26213.3 Genome Editing in Plants 26313.3.1 Applications in Medicinal Plants 26413.4 Medicinal Plants: Comparison of Traditional and Scientific Use 26613.5 Chemical Components of Medicinal Plants 26613.6 Using Biotechnological Techniques in Medicinal Plant Production 26713.7 In Vitro Culture Techniques in Herbal Medicine 26813.7.1 Plant Tissue Culture in Herbal Medicine 26813.7.2 Hairy Root Cultures in Herbal Medicine 26913.7.3 Callus and Cell Suspension Culture in Herbal Medicine 27013.7.4 Micropropagation in Herbal Medicine 27013.7.5 Elicitation 27013.7.6 Bioreactors for Large Scale Up 27013.8 Pharmaceutical Products from Medicinal Plants: Current Situation 27113.8.1 Antimicrobial Molecules 27113.8.2 Antioxidant Molecules 27113.8.3 Anticancer Molecules 27313.8.4 Cardiovascular Molecules 27313.9 Future Perspective and Conclusion 274References 27514 Nutraceuticals Enrichment by Genome Editing in Plants 282Luis Alfonso Jiménez-Ortega, Jesus Christian Grimaldi-Olivas, Brandon Estefano Morales-Merida, and J. Basilio Heredia14.1 Introduction 28214.2 Functional and Biofortified Foods: Phytochemicals, Nutraceuticals, and Micronutrients 28314.3 Metabolic Engineering to Enhance the Production of Phenolic Compounds 28314.3.1 Biosynthetic Pathway of Phenolic Compounds 28314.3.1.1 Phenolic Acids 28314.3.1.2 Flavonoids 28414.3.2 Tools to Increase the Production of Phenolic Compounds in Plants and Crops 28514.4 Metabolic Engineering to Enhance the Production of Terpenes 28614.4.1 Biosynthetic Pathway of Terpenes 28714.4.2 Tools to Increase the Production of Terpenes in Plants and Crops 28714.5 Metabolic Engineering to Enhance the Production of Alkaloids 28914.5.1 Biosynthetic Pathway of Alkaloids 28914.5.2 Tools to Increase the Production of Alkaloids in Plants and Crops 29114.6 Metabolic Engineering to Enhance the Production of Vitamins and Minerals 29214.6.1 Tools to Increase the Production of Vitamins in Plants and Crops 29214.6.2 Tools to Increase the Production of Minerals in Plants and Crops 29514.7 Metabolic Engineering to Enhance the Production of Polyunsaturated Fatty Acids 29614.7.1 Biosynthetic Pathway of Polyunsaturated Fatty Acids 29614.7.2 Tools to Increase the Production of Polyunsaturated Fatty Acids in Plants and Crops 29714.8 Metabolic Engineering to Enhance the Production of Bioactive Peptides 29814.8.1 Tools to Increase the Production of Bioactive Peptides in Plants and Crops 29814.9 Conclusions 299References 29915 Exploration of Genome Editing Tools for microRNA Engineering in Plants 310Hengyi Xu15.1 Introduction 31015.2 The Biogenesis of the miRNA and RNA Silencing in Plant 31115.3 MIRs as a Family in Plant 31315.4 The miRNA Engineering Methods in Plant 31515.5 The PAM of CRISPR/Cas and Strategy in Construct Design for miRNA Knock-Out 31615.6 Evolving CRISPR/Cas Tools, Strategies, and Their Potential Uses in MIR Regulation 31715.7 Conclusion and Future Perspectives 319References 32016 Application of Genome Editing in Pulses 326Nikhil Malhotra16.1 Introduction 32616.2 Genome Editing for Crop Improvement in Pulses 32716.2.1 Chickpea (Cicer arietinum) 32716.2.2 Cowpea (Vigna unguiculata) 32816.2.3 Soybean (Glycine max) 32816.2.4 Non-Edited Grain Legumes 32916.2.4.1 Common Bean (Phaseolus vulgaris) 32916.2.4.2 Dry Pea (Pisum sativum) 33016.2.4.3 Faba Bean (Vicia faba) 33016.2.4.4 Mung Bean (Vigna radiata) 33116.2.4.5 Lentil (Lens culinaris) 33216.3 Conclusion and Future Prospects 332References 33317 Genome Editing for Microbial Pathogens Resistance in Crops 339Mudasir Ahmad Bhat, Saima Jan, Sumreen Amin Shah, and Arif Tasleem Jan17.1 Introduction 33917.2 Effects of Climate Change on Crop Productivity 34017.3 CRISPR/Cas-Mediated Genome Editing in Plants 34117.3.1 CRISPR/cpf 1 34217.3.2 CRISPRi 34217.4 CRISPR-Based Engineering of Crop Plants 34317.4.1 Gene Disruption via Indel in Coding Sequences 34317.4.2 Gene Disruption via Indel in Promoter Regions 34317.4.3 Gene Deletion via Multiplex sgRNAs 34417.4.4 Gene Insertion via Homology-Directed Repair 34417.5 CRISPR/Cas in Imparting Tolerance to Biotic Factors 34417.5.1 CRISPR in Developing Resistance to Viruses 34517.5.2 CRISPR in Developing Resistance to Fungal Pathogens 34517.5.3 CRISPR in Developing Resistance to Different Bacteria 34917.6 CRISPR/Cas in Abiotic Stress Tolerance in Crops 35017.6.1 CRISPR/Cas in Temperature Stress Tolerance 35017.6.2 Drought Stress Responses 35217.6.3 Salinity Stress Responses 35317.6.4 Metal Stress Tolerance 35417.7 Conclusion 355Author Contributions 356Funding 356Acknowledgements 356Conflicts of Interest 356References 35618 Genome Editing for Raising Crops for Arid Lands: A Perspective of IncreasingStress Tolerance 369Pooja Jangir, Purva Khandelwal, and Praveen SoniAbbreviations 36918.1 Introduction 37018.2 Genome Editing Toolbox 37118.3 Plants’ Responses to Drought and Heat 37318.4 Increasing Drought Tolerance in Plants Through Genome Editing 37518.4.1 Transcription Factors 37518.4.2 Phytohormone Signaling 38118.4.3 Morphology and Drought Avoidance 38218.4.4 MicroRNAs 38218.4.5 Nutrient and Yield Traits 38318.5 Increasing Heat Tolerance in Plants Through Genome Editing 38318.6 Conclusion and Future Perspective 385Author Contributions 386Conflicts of Interest 386Acknowledgment 386References 38619 Genome Engineering for the Development of Climate-Resilient Crop Plants 394Bhavuk Gupta, Ayush Khandelwal, Brijesh Kumar, and Purva Bhalothia19.1 Introduction 39419.2 Effect of Climate Change on Crop Plants 39519.2.1 Effect on Photosynthesis and CO 2 Fixation 39719.2.2 Effect of Temperature 39719.2.3 Effect of Change in Precipitation 39819.2.4 Effect of Salinity 39819.3 Genome Engineering in Crop Improvement 39819.4 Traditional and Modern Molecular Breeding for Crop Improvement 40019.4.1 Classical Plant Breeding 40019.4.2 Genetic Engineering 40119.4.3 RNA Interference 40119.4.4 Phenomics and Genomics 40119.4.5 Role of miRNAs 40219.4.6 Zinc Finger Nucleases 40219.4.7 TALENs 40319.4.8 CRISPR/Cas 9 40319.5 Genome Engineering in Development of Climate Resilient Crops 40419.6 Status of Improved Crops with Genetic Engineering 40519.7 Problems Associated with Genetic Engineering 40619.8 Future Aspects 40719.9 Conclusion 407References 408Index 412