Over the last few decades, various techniques have been developed to alter the properties of plants and animals. While the targeted transfer of recombinant DNA into crop plants remains a valuable tool to achieve a desirable breeding outcome, integration of transgenes into the host genome has been random, which in part, leads to reduced acceptance of GMOs by the general population in some parts of the world. Likewise, methods of induced mutagenesis, such as TILLING, have the disadvantage that many mutations are induced per plant, which has to be removed again by expensive backcrossing. Advances in genome sequencing have provided more and more information on differences between susceptible and resistant varieties, which can now be directly targeted and modified using CRISPR/Cas9 technology. By selecting specific gRNAs occurrence of off-target modifications are comparatively low. ZFNs and TALENs- based approaches required re-engineering a new set of assembled polypeptides for every new target site for each experiment. The difficulty in cloning and protein engineering prevented these tools from being broadly adopted by the scientific community. Compared to these technologies, designing the CRISPR toolbox is much simpler and more flexible. CRISPR/Cas9 is versatile, less expensive and highly efficient. It has become the most widely used technology for genome editing in many organisms.
Since its inception as a powerful genome-editing tool in late 2012, this breakthrough technology has completely changed how science is performed. The first few chapters in this book introduce the basic concept, design and implementation of CRISPR/Cas9 for different plant systems. They are followed by in-depth discussions on the legal and bio-safety issues accompanying commercialization and patenting of this emerging technology. Lastly, this book covers emerging areas of new tools and potential applications. We believe readers, novice and expert alike, will benefit from this all-in-one resource on genome editing for crop improvement.
Chapter 17 is available open access under a Creative Commons Attribution 4.0 International License via link.springer.com.
Author(s): Shabir Hussain Wani, Goetz Hensel
Publisher: Springer
Year: 2022
Language: English
Pages: 336
City: Cham
Foreword
Preface
Contents
Part I: Current Status and Challenges of Plant Genome Editing Using CRISPR/Cas Technology
Genome Engineering as a Tool for Enhancing Crop Traits: Lessons from CRISPR/Cas9
1 Introduction
2 Zinc-Finger Nucleases
3 Transcription Activator-Like Effector Nucleases
4 CRISPR/Cas9 System
5 Mechanism of CRISPR/Cas9 System
6 Targeted Improvement of Crop Traits Using CRISPR/Cas9-Based Genome Editing in Cereals
6.1 Resistance Against Bacterial Disease
6.2 Resistance Against Fungal Disease
6.3 Resistance Against Viruses
6.4 Resistance and Tolerance Against Herbicides
6.5 Improved Quality and Yield
7 CRISPR/Cas9 Mediated Genome Editing in Horticultural Crops
7.1 Resistance Against Bacterial Disease
7.2 Resistance Against Fungal Disease
7.3 Resistance Against Viruses
7.4 Resistance and Tolerance Against Herbicide
7.5 Improved Quality and Yield
8 Conclusion
References
Vegetable Crop Improvement Through CRISPR Technology for Food Security
1 Introduction
2 Applications of Genome Editing in Improvement of Vegetable Crops
2.1 Qualitative Traits
2.1.1 Starch Content
2.1.2 Pigmentation
2.1.3 Saturated Fatty Acid Content
2.1.4 Improvement of Shelf-Life and Quality
2.1.5 Other Qualitative Traits
2.2 Abiotic Stress Tolerance
2.2.1 Drought and Extreme Temperature
2.2.2 Salinity and Mineral Deficiency
2.3 Pest and Disease Resistance
2.4 Biosafety and Legal Regulations
2.5 Conclusion
References
CRISPR/Cas9-Mediated Targeted Mutagenesis in Medicinal Plants
1 Introduction
2 CRISPR/Cas9 Mechanism
2.1 Cleavage Activity of Cas9
3 CRISPR/Cas9 Vector System for Plants
3.1 sgRNA Expression Cassettes
3.2 Cas9 Expression Cassettes
4 CRISPR/dCas9 and Epigenome Editing in Plants
4.1 Nuclease-Dead Cas9
4.2 sgRNA
4.3 Transcriptional Effectors
5 Analysis and Efficiency of Targeted Mutations
5.1 Reporter Genes
5.2 Single-Strand Conformation Polymorphism (SSCP)
5.3 High-Resolution Melting (HRM)
5.4 High-Throughput Sequencing (HTS or Deep Sequencing)
5.5 Sanger Sequencing
6 Medicinal Plants Modified Using CRISPR/Cas9
6.1 Salvia militorrhiza
6.2 Dendrobium officinale
6.3 Dioscorea zingiberensis
7 Applications of Genome Editing in Medicinal Plants
8 Conclusion
References
Genome Editing: A Review of the Challenges and Approaches
1 Introduction
2 Mechanisms of Repairing Double-Stranded Breaks
2.1 Non-Homologous End Joining (NHEJ)
2.2 Genome editing and Homologous Recombination
2.2.1 History of Genome Editing
2.2.2 Homologous Recombination in E. coli
2.2.3 HR in Saccharomyces cerevisiae
2.2.4 HR in Higher Organisms
3 Different Genome Editing Techniques
3.1 Meganucleases (MNs)
3.2 Zinc Finger Nucleases (ZFNs)
3.3 Transcription Activator-Like Effector Nucleases (TALENs)
3.4 Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR Associated Protein
4 Challenges in the CRISPR/cas9 System
4.1 Complex Designing
4.2 Inefficient Delivery
4.3 Selection of Target Site and gRNA Design
4.4 Off-Target Effect
4.5 Weak Repair Efficiency of HDR in Eukaryotes
4.6 Cas9 Endonuclease Activity and Cytotoxicity
5 Approaches
5.1 Base Editing
5.2 Prime Editing
6 Conclusion and Future Prospects
References
Part II: Developments in the Field of CRISPR/Cas Technology
Recent Advances and Application of CRISPR Base Editors for Improvement of Various Traits in Crops
1 Introduction
2 The Discovery of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Based Genome-Editing System, Its Components, and Mode of Action
3 Classification of CRISPR/Cas Systems
4 An Overview of CRISPR-Cas9 in Crop Improvement
5 Base Editors: Structure and Mechanisms
6 The Development and Evolution of Base Editors
6.1 Cytosine Base Editors (CBE)
6.2 Adenine Base Editor (ABE)
7 RNA Base Editors
8 Application of Base Editors in Crop Improvement
8.1 CBE-Mediated Base Editing in Plants
8.2 ABE-Mediated Base Editing in Plants
8.3 Best of Both Worlds: Combining CBEs and ABEs for More Efficient Crop Gene Editing
9 RNA Editing in Plants
10 Limitations and Prospects
10.1 Off-Target Activity of Base Editors
10.2 Generation of Indels
10.3 Need for a More Relaxed PAM Requirement
10.4 Limited Application of RNA Base-Editing Technology in Plants
10.5 Future Perspectives of the Emerging Technology
11 Conclusion
References
New Cas Endonuclease Variants Broadening the Scope of the CRISPR Toolbox
1 Introduction
2 The Natural Origin of Cas Proteins
2.1 Cas Proteins Have Different PAM Requirements
2.2 Protein Engineering of Cas Proteins
2.3 New Cas Functionality Through Fusion with Exogenous Protein Domains
2.4 Base Editors and Prime Editing
References
Multiplexed Genome Editing in Plants Using CRISPR/Cas-Based Endonuclease Systems
1 Introduction
2 Strategies for the Expression of Multiple gRNAs
2.1 Two-Component Transcriptional Unit Multiplex System
2.1.1 Cas9 and Multiple gRNA-TU Arrays
2.1.2 Cas9 and Multiple gRNAs Using Single Pol III Promoter
2.1.3 Cas9 and gRNA Using Bidirectional Promoters
2.2 Single Transcriptional Unit
2.2.1 Ribozymes-gRNA Ribozyme Array
2.2.2 Csy4 gRNA Array
3 Parameters Influencing Successful Multiplex Targeting
4 Conclusion
References
Transgene-Free Genome Editing in Plants
1 Introduction
2 Targeted Nucleases
3 From Natural DNA Modifiers to Programmable Genome Editors
4 Base Editors
5 Mito TALEs (Plastid Editing)
6 Delivery of Genome Editing Reagents into the Cells
7 Viral Vector Delivery
8 RNP or Protein Delivery into Plants
9 Protoplast Transformation
10 Biolistic Delivery
11 Additional Methods
12 Current Regulatory Views on DNA-Free Genome Editing
References
Genome Editing by Ribonucleoprotein Based Delivery of the Cas9 System in Plants
1 Introduction
2 Delivery Methods
2.1 Protoplast Transformation Methods
2.2 Biolistics
3 Validation of gRNAs and Target Edits
4 Tissue Types to Edit
4.1 Protoplasts
4.2 Immature Embryos
4.3 Shoot Apical Meristem
5 Benefits of RNP Delivery
6 Future Prospects
References
Virus-Mediated Delivery of CRISPR/CAS9 System in Plants
1 Introduction
2 Plant Viruses as Vectors
3 Virus as Vectors for Their Use in Genome Editing
4 Conclusions and Future Prospects
References
Characterization of Gene Edited Crops via Metabolomics
1 Introduction
2 Metabolomics
3 Analytical Tools for Metabolomic Studies
4 The Advent and Adoption of Genome Editing in Plants
5 Metabolomics for Improvement of Fruits
6 Metabolomics for Improvement of Legume Crops
7 Metabolomics for Improvement of Cereal Crops
8 Effect of Biotic and Abiotic Stress on Plant
9 Metabolomics: An Integral Part of Knowledge-Based Plant Breeding
10 Concluding Remarks and Future Perspectives
References
Part III: Applications of CRISPR/Cas Technology for Biotic and Abiotic Stress Tolerance
Genome Editing in Plants for Resistance Against Bacterial Pathogens
1 Introduction
2 CRISPR/Cas9 Genome Editing
3 Plant-Pathogen Interaction
4 CRISPR/Cas9-Mediated Editing of S Genes
5 Adoption of Wider Approaches to Improve Resistance in Plants
6 RNA Silencing
7 Phytohormones
8 Conclusions and Future Perspectives
References
Improvement of Resistance in Plants Against Insect-Pests Using Genome Editing Tools
1 Introduction
2 Genome Editing for the Management of Insect-Pests
2.1 Genome Editing to Diminish The Insect-Pests
2.2 Gene Drive Based on CRISPR in Insect for Crop Protection
2.3 Genome Editing in Plant for Insect-Pest Management
3 Future Studies
4 Conclusion
References
Applications of Gene Drive for Weeds and Pest Management Using CRISPR/Cas9 System in Plants
1 Introduction
2 Weed Management Through the Use of Gene Drives
3 Natural Gene Drives
3.1 Gene Drives That Occur at the Pre-gametic Phase
3.2 Gene Drives Occurring at the Post-gametic Phase
4 Weed Control Using Indirect Genetic Engineering Programs
5 Controlling Agricultural Pests Using Direct Genetic Engineering Programs
6 Weed Control Achieved Through the CRISPR/Cas9 Gene Drives
7 Weed Gene Drives
8 Genetic Modification of Polyploids
9 Risks of Weed Gene Drives
10 The Potential of Gene Drives to Reduce Vector-Borne Diseases (VBD)
11 Using Gene Drive to Combat Lyme Disease
12 Other Examples of Gene Drives for Pest Management
13 Conclusions and Future Prospects
References
Recent Trends in Targeting Genome Editing of Tomato for Abiotic and Biotic Stress Tolerance
1 Introduction
2 Productivity and Total Acreage
3 Diversification of Tomato
4 Tomato Nutritional Value and Applications
5 Factors Limiting Tomato Production
6 Impact of Abiotic and Biotic Stress Factors
7 Plant Genome Editing
8 Genome Editing Tools for Biotic and Abiotic Stress in Tomato
9 Conclusion
References
Part IV: Legal Framework for CRISPR/Cas Technology
Biosafety Issue Related to Genome Editing in Plants Using CRISPR-Cas9
1 Introduction
2 Genome Editing and CRISPR-Cas9
3 CRISPR Technology for Crop Improvement
4 Biosafety Concerns to Humans
4.1 Delivery of CRISPR-Cas Technology and Biosafety Concerns
4.1.1 Off-Targets and Their Effects
4.1.2 Players to Reduce the Off-Target Activity
5 Role of Specificity of Genome Editing on Limiting the Off-Target Activity
5.1 Preventing Off-Target Mutations
6 Identification of Potential Off-Target Activity
7 Less Risky Vector Applications
8 Develop a Reversal Mechanism to Revert in Cases of Unintended Effects
9 Develop Detection Mechanism to Ensure Safety Before Field Application
10 Monitoring Mechanism for Post-release Stability
11 Safe Alternatives from This Technology
11.1 Regulation Country-Wise
11.1.1 EU Regulatory Framework
11.2 Community Awareness
12 Conclusions
References
Regulatory Constraints and Differences of Genome-Edited Crops Around the Globe
1 Introduction
2 Paving the Way
3 The Need for Clarity
4 The Regulatory Climate
4.1 South America
4.1.1 Argentina
4.1.2 Uruguay
4.1.3 Paraguay
4.1.4 Chile
4.1.5 Brazil
4.1.6 Ecuador
4.1.7 Colombia
4.2 Central America
4.2.1 Honduras, Guatemala, and El Salvador
4.3 North America
4.3.1 Mexico
4.3.2 USA
4.3.3 Canada
4.4 Europe
4.4.1 The European Union (EU)
4.4.2 The UK
4.4.3 Norway
4.5 Israel
4.6 Africa
4.7 Russia
4.8 Asia
4.8.1 China
4.8.2 Japan
4.8.3 India
4.9 Southern Hemisphere
4.9.1 New Zealand
4.9.2 Australia
5 Conclusion
References
Index
