List of Contributors xv
Preface xix
About the Editor xx
1 CRISPR/Cas-Mediated Genome Editing in Plants: A Historical Perspective 1
Anil Kumar, Shumayla, and Santosh Kumar Upadhyay
1.1 Introduction 1
1.2 Historical Background 2
1.3 Mechanism of CRISPR/Cas System 4
1.3.1 Acquisition of Spacers 4
1.3.2 Biogenesis 5
1.3.3 Interference with the Target 5
1.4 Breakthrough Studies in CRISPR/Cas System 5
1.5 CRISPR Types 6
1.6 Type of Cas Proteins 7
1.6.1 Cas 1 7
1.6.2 Cas 2 7
1.6.3 Cas 3 7
1.6.4 Cas 4 7
1.6.5 Cas 5 7
1.6.6 Cas 6 8
1.6.7 Cas 7 8
1.6.8 Cas 8 8
1.6.9 Cas 9 8
1.6.10 Cas 10 8
1.6.11 Cas 11 8
1.6.12 Cas 12 9
1.6.13 Cas 13 9
1.6.14 Cas 14 9
1.7 CRISPR/Cas Modification 9
1.7.1 Nickase 9
1.7.2 Dead Cas9 (dCas9) 10
1.7.3 Base Editors 10
1.7.4 Prime Editors 10
1.8 CRISPR/Cas as a Genome Editing Tool and Its Application 10
1.8.1 Gene Knockout 10
1.8.2 DNA Insertion 11
1.8.3 Base Editing 11
1.8.4 Gene Activation and/or Repression 12
1.8.5 Epigenetic Modifications 12
1.8.6 Localization 12
1.8.7 RNA Editing 13
1.9 Conclusion 13
References 13
2 CRISPR/Cas-Mediated Multiplex Genome Editing in Plants and Applications 20
R. Prajapati and K. Tyagi
2.1 Introduction 20
2.2 Construct Design for Multiplex CRISPR/Cas Genome Editing 22
2.3 Strategies for Processing Multiple-Guide RNAs 23
2.4 Delivery of CRISPR/Cas Construct into Plant Cells 24
2.4.1 Agrobacterium-Mediated Delivery 24
2.4.2 Virus-Mediated Delivery 24
2.4.3 Particle Bombardment-Based Delivery 25
2.5 Broader Implications of CRISPR/Cas Multiplex Gene Editing 25
2.5.1 Simultaneous Knockout of Multiple Genes 25
2.5.2 Targeted Chromosomal Deletions 26
2.5.3 Transcriptional Activation or Repression of Genes 26
2.5.4 Base Editing 26
2.6 Application of CRISPR/Cas Multiplex Gene Editing in Generating Disease Resistant Plants 27
2.6.1 Disease Resistance Against Viruses 27
2.6.2 Disease Resistance Against Fungi 28
2.6.3 Disease Resistance Against Bacteria 29
2.7 Application of CRISPR/Cas Multiplex Gene Editing in Abiotic Stress-Tolerant Crop Production 29
2.7.1 Drought Tolerance 30
2.7.2 Salinity Tolerance 30
2.7.3 Herbicide Resistance 31
2.8 Application of CRISPR/Cas Multiplex Gene Editing in Enhancing Crop Yield, Nutrition, and Related Traits 31
2.9 Conclusion 32
Acknowledgments 32
References 34
3 Cas Variants Increased the Dimension of the CRISPR Tool Kit 40
Sameer Dixit, Akanchha Shukla, Mahendra Pawar, and Jyothilakshmi Vadassery
3.1 Introduction 40
3.2 General Architecture and Mechanism of CRISPR-Cas System 41
3.3 Classification of CRISPR-Cas System 42
3.3.1 Class 1 CRISPR-Cas System 44
3.3.2 Class 2 CRISPR-Cas System 45
3.4 Different Application-Based CRISPR-Cas System 45
3.4.1 Cas 9 46
3.4.2 Cas 12 46
3.4.3 Cas 14 46
3.4.4 Cas 13 47
3.4.5 Cas 3 47
3.5 Advancement and Reengineering of CRISPR-Cas System 47
3.6 Conclusions 48
Acknowledgments 49
References 49
4 Advancement in Delivery Systems and Vector Selection for CRISPR/ Cas-Mediated Genome Editing in Plants 52
Sanskriti Vats, Sukhmandeep Kaur, Amit Chauhan, Dipul Kumar Biswas, and Rupesh Deshmukh
4.1 Introduction 52
4.2 Advancement in Delivery Systems and Vector Selection for CRISPR/ Cas-Mediated Genome Editing in Plants 53
4.2.1 Vector Selection Based on Application and Availability in Plants 53
4.2.2 Plant Transformation Methodologies 56
4.3 Emerging Advanced CRISPR/Cas Systems and the Increased Demand for Quick Transformation Protocols 57
4.4 Advancements in Agrobacterium-Meditated Stable Transformation of Plants 59
4.5 Improvement of Agrobacterium-Mediated Transformation System by Developmental Regulators and Modular Agrobacterium Strains 61
4.6 Non-Agrobacterium Systems for Plant Transformation 62
4.7 Viral Vectors for Delivery of CRISPR Reagents and Increasing Donor Titer 63
4.8 De novo Meristem Induction 65
4.9 Biolistics and Protoplast Systems for CRISPR-Based Genome Editing 66
4.9.1 Biolistic Approach 66
4.9.2 Protoplast Approach 67
4.10 Generation of Transgene-Free CRISPR-Edited Lines 68
4.10.1 Mendelian Segregation Analysis 68
4.10.2 Programmed Self-Elimination Method 68
4.10.3 Transient Expression of CRISPR/Cas9 Cassette 68
References 69
5 Role of Nanotechnology in the Advancement in Genome Editing in Plants 78
Mehtap AYDIN
5.1 An Overview of Plant Genome Editing 78
5.1.1 Meganuclease 79
5.1.2 Zinc Finger Nucleases 79
5.1.3 Transcription Activator-Like Effectors Nucleases 80
5.1.4 CRISPR/Cas9 Based Genome Editing 80
5.2 Nanoparticles used as Genome Editing Tools in Plants 80
5.2.1 Mesoporous Silica Nanoparticles 82
5.2.2 Carbon Nanotubes Carbon 82
5.2.3 Lipid-Based Nanoparticles 83
5.2.4 Polymer-Based Nanoparticles 83
5.3 Point of View: The Nanotechnology and Plant Genome Editing 83
5.4 The Approach to Transferring Biomolecules to Plants and Its Limitations 84
5.5 Role of Nanotechnology in Agriculture 84
5.6 Conclusion 86
References 86
6 Genome Editing for Crop Biofortification 91
Erum Shoeb, Srividhya Venkataraman, Uzma Badar, and Kathleen Hefferon
6.1 Introduction 91
6.2 Current Global Status of Micronutrient Malnutrition 92
6.3 Importance of Biofortification in Ensuring Food Security 92
6.4 Strategies for Biofortification 93
6.4.1 Chloroplast Metabolic Engineering for Developing Nutrient-Dense Food Crops 94
6.5 Biofortification Through Agronomic Practices 96
6.6 Genome Editing Is a Powerful Tool 98
6.6.1 Meganucleases (MegNs) 99
6.6.2 Zinc Finger Nucleases 100
6.6.3 TALENs 100
6.6.4 CRISPR/Cas- 9 101
6.7 Examples of Biofortification Using Genome Editing Technologies 102
6.7.1 Amino Acid Biofortification 102
6.7.2 GABA Biofortification 102
6.7.3 Improvement of Oil Content and Quality 105
6.7.4 Improvement of Resistant Starch Content 105
6.7.5 Improvement of Micronutrient Bioavailability 105
6.7.6 Crops Enriched in Iron 105
6.7.7 Zn-enriched Crops 106
6.7.8 Crops Enriched in Vitamin A 106
6.7.9 Crops Enriched in Vitamin E 107
6.7.10 Engineering Crops Adapted to Growing in Toxic Environments 107
6.7.11 CRISPR-Cas9-enabled Decrease in Anti-nutrients 107
6.7.12 Benefits of Genome Editing over Other Technologies for Biofortification 108
6.8 Regulation of Genome Editing 108
6.9 Conclusions and Future Prospects 109
References 109
7 Genome Editing for Nutritional Improvement of Crops 122
Pooja Kanwar Shekhawat, Hasthi Ram, and Praveen Soni
Abbreviations 122
7.1 Introduction 124
7.2 Evolution of Techniques for Improvement of Crops’ Genomes 124
7.3 Genome Editing for Nutritional Improvement 125
7.3.1 Improvement in Cereal Crops 126
7.3.2 Improvement in Oilseed Crops 138
7.3.3 Improvements in Horticulture Crops 139
7.4 Regulation of Genome Edited Crops: Current Status 141
7.5 Future Perspectives and Conclusion 142
Author Contribution 142
Acknowledgment 142
References 143
8 Genome-Editing Tools for Engineering of MicroRNAs and Their Encoded
Peptides, miPEPs, in Plants 153
Ravi Shankar Kumar, Hiteshwari Sinha, Tapasya Datta, Ashish Sharma, and Prabodh Kumar Trivedi
8.1 Introduction 153
8.1.1 ZINC Finger Nucleases 154
8.1.2 TALE Nucleases 155
8.1.3 CRISPR/Cas 9 156
8.2 CRISPR–Cas9-Mediated DNA Interference in Bacterial Adaptive Immunity 157
8.2.1 Types of CRISPR Systems 158
8.2.2 The Cas9 Enzyme 158
8.3 CRISPR/Cas9 Effector Complex Assembly 159
8.4 The Mechanism of CRISPR/Cas9-Mediated Genome Engineering 159
8.4.1 Comparison with Other Technologies for Genome Editing 160
8.4.2 Limitations of the Cas9 System 160
8.4.3 miRNAs 162
8.4.4 Biogenesis of miRNA 162
8.4.5 miRNA and Gene Regulations 163
8.5 Role of Genome-Editing in miRNA Expression 164
8.6 Applications of the CRISPR/Cas9 System in miRNA Editing 165
8.6.1 microRNA-Encoded Peptide 166
8.6.2 Biogenesis of miPEPs 166
8.6.3 Role of miPEP 167
8.7 miPEPs Act as the Master Regulator in Plant Growth and Development 167
8.8 Conclusions and Future Prospect 168
Acknowledgments 169
References 169
9 Genome Editing for Trait Improvement in Ornamental Plants 177
Yang Zhou, Yuxin Li, and Wen Liu
9.1 Introduction 177
9.2 Application of Gene Editing Technology in Color Regulation of Ornamental Plants 178
9.3 Application of Gene Editing Technology in Ornamental Plants Preservation 179
9.4 Application of Gene Editing Technology in Shape and Organ Regulation of Ornamental Plants 180
9.5 Application of Gene Editing Technology in Other Traits of Ornamental Plants 180
9.6 Conclusions and Perspectives 181
Acknowledgments 181
References 181
10 Abiotic Stress Tolerance in Plants by Genome Editing Applications 185
Elif Karlik Urhan
10.1 Introduction 185
10.2 Drought Tolerance 187
10.3 Salinity Tolerance 191
10.4 Temperature Stress Tolerance 196
10.4.1 Heat Stress Tolerance 196
10.4.2 Cold Stress Tolerance 199
10.5 Conclusions 202
References 203
11 Genome Editing for Improvement of Nutrition and Quality in Vegetable Crops 222
Payal Gupta, Suhas G. Karkute, Prasanta K. Dash, and Achuit K. Singh
11.1 Vegetables and Human Nutrition 222
11.2 Important Quality Parameters of Vegetables 223
11.3 Approaches for Improving Nutrition Content in Vegetables 223
11.3.1 Breeding for Improving Nutrition in Vegetable Crops 224
11.3.2 Genome Editing Technologies 225
11.3.2.1 CRISPR/Cas9 and Advances in Genome Editing 225
11.3.2.2 Mechanism of CRISPR/Cas-Mediated Genome Editing in Plants 226
11.4 Applications of Genome Editing for Improvement of Vegetable Nutrition and Quality 227
11.4.1 Improvement in the Appearance in Terms of Shape and Size 229
11.4.2 Improvement of the Shelf-Life 229
11.4.3 Improvement of the Ripening Time 230
11.4.4 Improvement in Colour of the Fruit/Vegetable 230
11.4.5 Biofortification of Vegetable Crops Through Genome Editing 231
11.4.5.1 Metabolic Engineering of Carotenoid Biosynthesis Pathway 231
11.4.5.2 Increasing γ-Amino Butyric Acid and Vitamin D Content 232
11.4.6 Improvement of Starch Content 232
11.4.7 Elimination of Anti-Nutritional Factors 232
11.5 Challenges and Future Prospects 233
11.6 Conclusion 234
References 234
12 Insight into the Flavonoids Enrichment in Plants by Genome Engineering 242
Elena V. Mikhaylova
12.1 The Importance of Flavonoids 242
12.2 Flavonoid Biosynthesis Pathway 244
12.3 In Planta Flavonoid Enrichment via Genome Editing 247
12.4 Biotechnological Production of Flavonoids 252
12.5 Conclusions 253
References 253
13 Genome Engineering in Medicinal Plants for Improved Therapeutics: Current Scenario and Future Perspective 260
Buket Çakmak Guner
13.1 Introduction 260
13.2 Genome Engineering in Plants 261
13.2.1 Agrobacterium-Mediated Transformation 261
13.2.2 Biolistic or Particle Bombardment-Mediated Transformation 262
13.2.3 Electroporation-Mediated Transformation 262
13.2.4 Chemical-Mediated Transformation 262
13.3 Genome Editing in Plants 263
13.3.1 Applications in Medicinal Plants 264
13.4 Medicinal Plants: Comparison of Traditional and Scientific Use 266
13.5 Chemical Components of Medicinal Plants 266
13.6 Using Biotechnological Techniques in Medicinal Plant Production 267
13.7 In Vitro Culture Techniques in Herbal Medicine 268
13.7.1 Plant Tissue Culture in Herbal Medicine 268
13.7.2 Hairy Root Cultures in Herbal Medicine 269
13.7.3 Callus and Cell Suspension Culture in Herbal Medicine 270
13.7.4 Micropropagation in Herbal Medicine 270
13.7.5 Elicitation 270
13.7.6 Bioreactors for Large Scale Up 270
13.8 Pharmaceutical Products from Medicinal Plants: Current Situation 271
13.8.1 Antimicrobial Molecules 271
13.8.2 Antioxidant Molecules 271
13.8.3 Anticancer Molecules 273
13.8.4 Cardiovascular Molecules 273
13.9 Future Perspective and Conclusion 274
References 275
14 Nutraceuticals Enrichment by Genome Editing in Plants 282
Luis Alfonso Jimenez-Ortega, Jesus Christian Grimaldi-Olivas, Brandon Estefano Morales-Merida, and J. Basilio Heredia
14.1 Introduction 282
14.2 Functional and Biofortified Foods: Phytochemicals, Nutraceuticals, and Micronutrients 283
14.3 Metabolic Engineering to Enhance the Production of Phenolic Compounds 283
14.3.1 Biosynthetic Pathway of Phenolic Compounds 283
14.3.1.1 Phenolic Acids 283
14.3.1.2 Flavonoids 284
14.3.2 Tools to Increase the Production of Phenolic Compounds in Plants and Crops 285
14.4 Metabolic Engineering to Enhance the Production of Terpenes 286
14.4.1 Biosynthetic Pathway of Terpenes 287
14.4.2 Tools to Increase the Production of Terpenes in Plants and Crops 287
14.5 Metabolic Engineering to Enhance the Production of Alkaloids 289
14.5.1 Biosynthetic Pathway of Alkaloids 289
14.5.2 Tools to Increase the Production of Alkaloids in Plants and Crops 291
14.6 Metabolic Engineering to Enhance the Production of Vitamins and Minerals 292
14.6.1 Tools to Increase the Production of Vitamins in Plants and Crops 292
14.6.2 Tools to Increase the Production of Minerals in Plants and Crops 295
14.7 Metabolic Engineering to Enhance the Production of Polyunsaturated Fatty Acids 296
14.7.1 Biosynthetic Pathway of Polyunsaturated Fatty Acids 296
14.7.2 Tools to Increase the Production of Polyunsaturated Fatty Acids in Plants and Crops 297
14.8 Metabolic Engineering to Enhance the Production of Bioactive Peptides 298
14.8.1 Tools to Increase the Production of Bioactive Peptides in Plants and Crops 298
14.9 Conclusions 299
References 299
15 Exploration of Genome Editing Tools for microRNA Engineering in Plants 310
Hengyi Xu
15.1 Introduction 310
15.2 The Biogenesis of the miRNA and RNA Silencing in Plant 311
15.3 MIRs as a Family in Plant 313
15.4 The miRNA Engineering Methods in Plant 315
15.5 The PAM of CRISPR/Cas and Strategy in Construct Design for miRNA Knock-Out 316
15.6 Evolving CRISPR/Cas Tools, Strategies, and Their Potential Uses in MIR Regulation 317
15.7 Conclusion and Future Perspectives 319
References 320
16 Application of Genome Editing in Pulses 326
Nikhil Malhotra
16.1 Introduction 326
16.2 Genome Editing for Crop Improvement in Pulses 327
16.2.1 Chickpea (Cicer arietinum) 327
16.2.2 Cowpea (Vigna unguiculata) 328
16.2.3 Soybean (Glycine max) 328
16.2.4 Non-Edited Grain Legumes 329
16.2.4.1 Common Bean (Phaseolus vulgaris) 329
16.2.4.2 Dry Pea (Pisum sativum) 330
16.2.4.3 Faba Bean (Vicia faba) 330
16.2.4.4 Mung Bean (Vigna radiata) 331
16.2.4.5 Lentil (Lens culinaris) 332
16.3 Conclusion and Future Prospects 332
References 333
17 Genome Editing for Microbial Pathogens Resistance in Crops 339
Mudasir Ahmad Bhat, Saima Jan, Sumreen Amin Shah, and Arif Tasleem Jan
17.1 Introduction 339
17.2 Effects of Climate Change on Crop Productivity 340
17.3 CRISPR/Cas-Mediated Genome Editing in Plants 341
17.3.1 CRISPR/cpf 1 342
17.3.2 CRISPRi 342
17.4 CRISPR-Based Engineering of Crop Plants 343
17.4.1 Gene Disruption via Indel in Coding Sequences 343
17.4.2 Gene Disruption via Indel in Promoter Regions 343
17.4.3 Gene Deletion via Multiplex sgRNAs 344
17.4.4 Gene Insertion via Homology-Directed Repair 344
17.5 CRISPR/Cas in Imparting Tolerance to Biotic Factors 344
17.5.1 CRISPR in Developing Resistance to Viruses 345
17.5.2 CRISPR in Developing Resistance to Fungal Pathogens 345
17.5.3 CRISPR in Developing Resistance to Different Bacteria 349
17.6 CRISPR/Cas in Abiotic Stress Tolerance in Crops 350
17.6.1 CRISPR/Cas in Temperature Stress Tolerance 350
17.6.2 Drought Stress Responses 352
17.6.3 Salinity Stress Responses 353
17.6.4 Metal Stress Tolerance 354
17.7 Conclusion 355
Author Contributions 356
Funding 356
Acknowledgements 356
Conflicts of Interest 356
References 356
18 Genome Editing for Raising Crops for Arid Lands: A Perspective of Increasing
Stress Tolerance 369
Pooja Jangir, Purva Khandelwal, and Praveen Soni
Abbreviations 369
18.1 Introduction 370
18.2 Genome Editing Toolbox 371
18.3 Plants’ Responses to Drought and Heat 373
18.4 Increasing Drought Tolerance in Plants Through Genome Editing 375
18.4.1 Transcription Factors 375
18.4.2 Phytohormone Signaling 381
18.4.3 Morphology and Drought Avoidance 382
18.4.4 MicroRNAs 382
18.4.5 Nutrient and Yield Traits 383
18.5 Increasing Heat Tolerance in Plants Through Genome Editing 383
18.6 Conclusion and Future Perspective 385
Author Contributions 386
Conflicts of Interest 386
Acknowledgment 386
References 386
19 Genome Engineering for the Development of Climate-Resilient Crop Plants 394
Bhavuk Gupta, Ayush Khandelwal, Brijesh Kumar, and Purva Bhalothia
19.1 Introduction 394
19.2 Effect of Climate Change on Crop Plants 395
19.2.1 Effect on Photosynthesis and CO 2 Fixation 397
19.2.2 Effect of Temperature 397
19.2.3 Effect of Change in Precipitation 398
19.2.4 Effect of Salinity 398
19.3 Genome Engineering in Crop Improvement 398
19.4 Traditional and Modern Molecular Breeding for Crop Improvement 400
19.4.1 Classical Plant Breeding 400
19.4.2 Genetic Engineering 401
19.4.3 RNA Interference 401
19.4.4 Phenomics and Genomics 401
19.4.5 Role of miRNAs 402
19.4.6 Zinc Finger Nucleases 402
19.4.7 TALENs 403
19.4.8 CRISPR/Cas 9 403
19.5 Genome Engineering in Development of Climate Resilient Crops 404
19.6 Status of Improved Crops with Genetic Engineering 405
19.7 Problems Associated with Genetic Engineering 406
19.8 Future Aspects 407
19.9 Conclusion 407
References 408
Index 412
