Applications of Genome Engineering in Plants

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 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 Güner

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 Jiménez-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