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- Herausgeber: John Wiley & Sons
- Kategorie: Wissenschaft und neue Technologien
- Sprache: Englisch
Applications of Genome Engineering in Plants Understand the keys to creating the food of the future Genome engineering in plants is a field that has made enormous strides in recent years. In particular, the CRISPR-Cas system has been used in a number of crop species to make significant leaps forward in nutritional improvement, stress tolerance, crop yield, and more. As scientists work to meet global food needs and foster sustainable agriculture in a changing world, genome engineering promises only to become more important. Applications of Genome Engineering in Plants details the history of, and recent developments in, this essential area of biotechnology. It describes advances enabling nutritional improvement, nutraceuticals improvement, flavonoid enrichment, and many more crop enhancements, as well as subjects such as biosafety and regulatory mechanisms. The result is a thorough and essential overview for researchers and biotech professionals. Applications of Genome Engineering in Plants readers will also find: * Chapters on trans-gene free editing or non-transgenic approaches to plant genomes * Detailed discussion of topics including nanotechnology-facilitated genome editing, engineering for virus resistance in plants, and more * Applications of genome editing in oil seed crops, vegetables, ornamental plants, and many others Applications of Genome Engineering in Plants is ideal for academics, scientists, and industry professionals working in biotechnology, agriculture, food science, and related subjects.
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Veröffentlichungsjahr: 2023
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Table of Contents
Cover
Table of Contents
Title Page
Copyright Page
List of Contributors
Preface
About the Editor
1 CRISPR/Cas‐Mediated Genome Editing in Plants: A Historical Perspective
1.1 Introduction
1.2 Historical Background
1.3 Mechanism of CRISPR/Cas System
1.4 Breakthrough Studies in CRISPR/Cas System
1.5 CRISPR Types
1.6 Type of Cas Proteins
1.7 CRISPR/Cas Modification
1.8 CRISPR/Cas as a Genome Editing Tool and Its Application
1.9 Conclusion
References
2 CRISPR/Cas‐Mediated Multiplex Genome Editing in Plants and Applications
2.1 Introduction
2.2 Construct Design for Multiplex CRISPR/Cas Genome Editing
2.3 Strategies for Processing Multiple‐Guide RNAs
2.4 Delivery of CRISPR/Cas Construct into Plant Cells
2.5 Broader Implications of CRISPR/Cas Multiplex Gene Editing
2.6 Application of CRISPR/Cas Multiplex Gene Editing in Generating Disease Resistant Plants
2.7 Application of CRISPR/Cas Multiplex Gene Editing in Abiotic Stress‐Tolerant Crop Production
2.8 Application of CRISPR/Cas Multiplex Gene Editing in Enhancing Crop Yield, Nutrition, and Related Traits
2.9 Conclusion
Acknowledgments
References
3 Cas Variants Increased the Dimension of the CRISPR Tool Kit
3.1 Introduction
3.2 General Architecture and Mechanism of CRISPR‐Cas System
3.3 Classification of CRISPR‐Cas System
3.4 Different Application‐Based CRISPR‐Cas System
3.5 Advancement and Reengineering of CRISPR‐Cas System
3.6 Conclusions
Acknowledgments
References
4 Advancement in Delivery Systems and Vector Selection for CRISPR/Cas‐Mediated Genome Editing in Plants
4.1 Introduction
4.2 Advancement in Delivery Systems and Vector Selection for CRISPR/Cas‐Mediated Genome Editing in Plants
4.3 Emerging Advanced CRISPR/Cas Systems and the Increased Demand for Quick Transformation Protocols
4.4 Advancements in
Agrobacterium
‐Meditated Stable Transformation of Plants
4.5 Improvement of Agrobacterium‐Mediated Transformation System by Developmental Regulators and Modular
Agrobacterium
Strains
4.6 Non‐
Agrobacterium
Systems for Plant Transformation
4.7 Viral Vectors for Delivery of CRISPR Reagents and Increasing Donor Titer
4.8
De novo
Meristem Induction
4.9 Biolistics and Protoplast Systems for CRISPR‐Based Genome Editing
4.10 Generation of Transgene‐Free CRISPR‐Edited Lines
References
5 Role of Nanotechnology in the Advancement in Genome Editing in Plants
5.1 An Overview of Plant Genome Editing
5.2 Nanoparticles used as Genome Editing Tools in Plants
5.3 Point of View: The Nanotechnology and Plant Genome Editing
5.4 The Approach to Transferring Biomolecules to Plants and Its Limitations
5.5 Role of Nanotechnology in Agriculture
5.6 Conclusion
References
6 Genome Editing for Crop Biofortification
6.1 Introduction
6.2 Current Global Status of Micronutrient Malnutrition
6.3 Importance of Biofortification in Ensuring Food Security
6.4 Strategies for Biofortification
6.5 Biofortification Through Agronomic Practices
6.6 Genome Editing Is a Powerful Tool
6.7 Examples of Biofortification Using Genome Editing Technologies
6.8 Regulation of Genome Editing
6.9 Conclusions and Future Prospects
References
7 Genome Editing for Nutritional Improvement of Crops
7.1 Introduction
7.2 Evolution of Techniques for Improvement of Crops’ Genomes
7.3 Genome Editing for Nutritional Improvement
7.4 Regulation of Genome Edited Crops: Current Status
7.5 Future Perspectives and Conclusion
Author Contribution
Acknowledgment
References
8 Genome‐Editing Tools for Engineering of MicroRNAs and Their Encoded Peptides, miPEPs, in Plants
8.1 Introduction
8.2 CRISPR–Cas9‐Mediated DNA Interference in Bacterial Adaptive Immunity
8.3 CRISPR/Cas9 Effector Complex Assembly
8.4 The Mechanism of CRISPR/Cas9‐Mediated Genome Engineering
8.5 Role of Genome‐Editing in miRNA Expression
8.6 Applications of the CRISPR/Cas9 System in miRNA Editing
8.7 miPEPs Act as the Master Regulator in Plant Growth and Development
8.8 Conclusions and Future Prospect
Acknowledgments
References
9 Genome Editing for Trait Improvement in Ornamental Plants
9.1 Introduction
9.2 Application of Gene Editing Technology in Color Regulation of Ornamental Plants
9.3 Application of Gene Editing Technology in Ornamental Plants Preservation
9.4 Application of Gene Editing Technology in Shape and Organ Regulation of Ornamental Plants
9.5 Application of Gene Editing Technology in Other Traits of Ornamental Plants
9.6 Conclusions and Perspectives
Acknowledgments
References
10 Abiotic Stress Tolerance in Plants by Genome Editing Applications
10.1 Introduction
10.2 Drought Tolerance
10.3 Salinity Tolerance
10.4 Temperature Stress Tolerance
10.5 Conclusions
References
11 Genome Editing for Improvement of Nutrition and Quality in Vegetable Crops
11.1 Vegetables and Human Nutrition
11.2 Important Quality Parameters of Vegetables
11.3 Approaches for Improving Nutrition Content in Vegetables
11.4 Applications of Genome Editing for Improvement of Vegetable Nutrition and Quality
11.5 Challenges and Future Prospects
11.6 Conclusion
References
12 Insight into the Flavonoids Enrichment in Plants by Genome Engineering
12.1 The Importance of Flavonoids
12.2 Flavonoid Biosynthesis Pathway
12.3 In Planta Flavonoid Enrichment via Genome Editing
12.4 Biotechnological Production of Flavonoids
12.5 Conclusions
References
13 Genome Engineering in Medicinal Plants for Improved Therapeutics: Current Scenario and Future Perspective
13.1 Introduction
13.2 Genome Engineering in Plants
13.3 Genome Editing in Plants
13.4 Medicinal Plants: Comparison of Traditional and Scientific Use
13.5 Chemical Components of Medicinal Plants
13.6 Using Biotechnological Techniques in Medicinal Plant Production
13.7
In Vitro
Culture Techniques in Herbal Medicine
13.8 Pharmaceutical Products from Medicinal Plants: Current Situation
13.9 Future Perspective and Conclusion
References
14 Nutraceuticals Enrichment by Genome Editing in Plants
14.1 Introduction
14.2 Functional and Biofortified Foods: Phytochemicals, Nutraceuticals, and Micronutrients
14.3 Metabolic Engineering to Enhance the Production of Phenolic Compounds
14.4 Metabolic Engineering to Enhance the Production of Terpenes
14.5 Metabolic Engineering to Enhance the Production of Alkaloids
14.6 Metabolic Engineering to Enhance the Production of Vitamins and Minerals
14.7 Metabolic Engineering to Enhance the Production of Polyunsaturated Fatty Acids
14.8 Metabolic Engineering to Enhance the Production of Bioactive Peptides
14.9 Conclusions
References
15 Exploration of Genome Editing Tools for microRNA Engineering in Plants
15.1 Introduction
15.2 The Biogenesis of the miRNA and RNA Silencing in Plant
15.3 MIRs as a Family in Plant
15.4 The miRNA Engineering Methods in Plant
15.5 The PAM of CRISPR/Cas and Strategy in Construct Design for miRNA Knock‐Out
15.6 Evolving CRISPR/Cas Tools, Strategies, and Their Potential Uses in MIR Regulation
15.7 Conclusion and Future Perspectives
References
16 Application of Genome Editing in Pulses
16.1 Introduction
16.2 Genome Editing for Crop Improvement in Pulses
16.3 Conclusion and Future Prospects
References
17 Genome Editing for Microbial Pathogens Resistance in Crops
17.1 Introduction
17.2 Effects of Climate Change on Crop Productivity
17.3 CRISPR/Cas‐Mediated Genome Editing in Plants
17.4 CRISPR‐Based Engineering of Crop Plants
17.5 CRISPR/Cas in Imparting Tolerance to Biotic Factors
17.6 CRISPR/Cas in Abiotic Stress Tolerance in Crops
17.7 Conclusion
Author Contributions
Funding
Acknowledgements
Conflicts of Interest
References
18 Genome Editing for Raising Crops for Arid Lands: A Perspective of Increasing Stress Tolerance
Abbreviations
18.1 Introduction
18.2 Genome Editing Toolbox
18.3 Plants’ Responses to Drought and Heat
18.4 Increasing Drought Tolerance in Plants Through Genome Editing
18.5 Increasing Heat Tolerance in Plants Through Genome Editing
18.6 Conclusion and Future Perspective
Author Contributions
Conflicts of Interest
Acknowledgment
References
19 Genome Engineering for the Development of Climate‐Resilient Crop Plants
19.1 Introduction
19.2 Effect of Climate Change on Crop Plants
19.3 Genome Engineering in Crop Improvement
19.4 Traditional and Modern Molecular Breeding for Crop Improvement
19.5 Genome Engineering in Development of Climate Resilient Crops
19.6 Status of Improved Crops with Genetic Engineering
19.7 Problems Associated with Genetic Engineering
19.8 Future Aspects
19.9 Conclusion
References
Index
End User License Agreement
List of Tables
Chapter 3
Table 3.1 Recent classification of CRISPR‐Cas systems.
Table 3.2 Different modes of nucleotide modification using CRISPR system.
Chapter 5
Table 5.1 Nanomaterials (NMs) are thought to be advantageous to some of agr...
Chapter 6
Table 6.1 Biofortification of tomato using genome editing and RNAi technolo...
Chapter 7
Table 7.1 List of genes edited for nutritional improvement of crops.
Chapter 8
Table 8.1 Summary of the mechanisms governing the genome‐editing processes ...
Chapter 10
Table 10.1 Examples of plants improved using the CRISPR‐Cas system.
Chapter 11
Table 11.1 Status of genome edited vegetable crops for nutrition and qualit...
Chapter 12
Table 12.1 Effects of the editing of flavonoid biosynthesis genes.
Chapter 13
Table 13.1 Comparison of genome editing techniques.
Table 13.2 Medicinal plants and edited genes with CRISPR‐Cas9 delivery syst...
Table 13.3 Active secondary metabolites isolated from medicinal plants via
Table 13.4 Some of commercial molecules produced by plant tissue cultures u...
Chapter 14
Table 14.1 Improved plants and crops for the accumulation and production of...
Table 14.2 Plants and crops bred for terpene accumulation and production.
Table 14.3 Metabolic engineering to enhance de production of alkaloids.
Table 14.4 Genetic engineering strategies to enhance the production of vita...
Chapter 16
Table 16.1 Examples of genome editing in pulses.
Chapter 17
Table 17.1 CRISPR/Cas‐ based crop improvement with resistance to bacterial,...
Chapter 18
Table 18.1 Improvement of drought tolerance in plants through genome editin...
Table 18.2 Increasing heat tolerance in plants through genome editing.
Chapter 19
Table 19.1 Examples of crop plants showing climate resilient traits develop...
List of Illustrations
Chapter 2
Figure 2.1 A graphical representation of different multiplex gRNA expression...
Figure 2.2 Overview of applications of multiplex CRISPR/Cas gene editing in ...
Figure 2.3 Strategy for multiplex CRISPR/Cas genome editing. (a) Flow chart ...
Chapter 4
Figure 4.1 Schematic representation of the genome editing process in plants ...
Figure 4.2 Schematic representation of the different kinds of plant transfor...
Figure 4.3 An overview of
Agrobacterium‐
mediated transformation for th...
Chapter 5
Figure 5.1 Nanoparticles of different shapes are used for genome editing. To...
Chapter 7
Figure 7.1 Representation of different genome editing tools and their applic...
Chapter 8
Figure 8.1 Mechanism of zinc finger nuclease (ZFN). ZFNs recognize the targe...
Figure 8.2 Mechanism of transcription activator‐like effector nucleases (TAL...
Figure 8.3 Clustered regularly interspaced short palindromic repeats‐Cas9 sy...
Figure 8.4 miRNA biogenesis and regulation in plants. miR gene transcribed t...
Figure 8.5 miPEP biogenesis and their transcriptional regulation. Schematic ...
Chapter 10
Figure 10.1 The effects of different abiotic stresses on plants.
Chapter 12
Figure 12.1 Basic skeleton structure of flavonoids.
Figure 12.2 Flavonoid biosynthesis pathway.
Chapter 13
Figure 13.1 NGS and different biotechnological applications.
Figure 13.2 Bioactive molecule production via
in vitro
culture techniques....
Chapter 14
Figure 14.1 Main phenylpropanoid compounds. (a) Resveratrol. (b) Esculetin....
Figure 14.2 Some monoterpenes and aromatic terpenes molecules are used in th...
Figure 14.3 Chemical structures of pharmacologically potent alkaloids. (a) Q...
Figure 14.4 Fruits and vegetables rich in vitamins and minerals. (a) Vitamin...
Figure 14.5 Seeds and grains rich in PUFAs and their biological activities....
Figure 14.6 Biosynthesis of polyunsaturated fatty acids.
Chapter 15
Figure 15.1 The biogenesis of miRNA and RISC complex‐mediated RNAi. The MIR ...
Figure 15.2 The miRNA engineer tool kits in plant. The miRNA abundance can b...
Chapter 16
Figure 16.1 Approaches for enhancing genetic gains in pulses by genome editi...
Chapter 19
Figure 19.1 Schematic representation of effect of the different abiotic stre...
Figure 19.2 Different tools and techniques to develop genome engineered crop...
Guide
Cover Page
Table of Contents
Title Page
Copyright Page
List of Contributors
Preface
About the Editor
Begin Reading
Index
WILEY END USER LICENSE AGREEMENT
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Applications of Genome Engineering in Plants
Edited by
Santosh Kumar Upadhyay
Department of Botany
Panjab University
Chandigarh, India
This edition first published 2024© 2024 John Wiley & Sons Ltd
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List of Contributors
Mehtap AYDINGenetics and Bioengineering DepartmentYeditepe UniversityIstanbul, Turkey
Uzma BadarDepartment of GeneticsUniversity of KarachiKarachi, Pakistan
Purva BhalothiaBirla Institute of Scientific Research (BISR)Jaipur, Rajasthan, India
Mudasir Ahmad BhatSchool of Biosciences and BiotechnologyBaba GhulamShah Badshah University, RajouriJammu and Kashmir, India
Dipul Kumar BiswasDepartment of BiologyFaculty of Arts and Science, Bolu Abantİzzet Baysal ÜniversitesiGölköy, Turkey
Amit ChauhanInstitute of Biochemistry and BiologyUniversity of Potsdam, Potsdam‐GolmGermany
Max Planck Institute of MolecularPlant PhysiologyPotsdam‐Golm, Germany
Prasanta K. DashICAR‐National Institute for PlantBiotechnology, New DelhiIndia
Tapasya DattaCSIR‐Central Institute of Medicinal andAromatic Plants (CSIR‐CIMAP), LucknowUttar Pradesh, India
Rupesh DeshmukhDepartment of BiotechnologyCentral University of HaryanaHaryana, India
Sameer DixitNational Institute of PlantGenome ResearchNew Delhi, India
Jesus Christian Grimaldi‐OlivasCiencia y Tecnología de Alimentos(Food Science and Technology)Alimentos Funcionales y Nutracéuticos(Nutraceuticals and Functional Foods)Centro de Investigación en Alimentación yDesarrollo, AC (CIAD), (Research Centerfor Food and Development), CuliacánSinaloa, México
Buket Çakmak GünerDepartment of Medical BiologySchool of MedicineAnkara UniversityAnkara, Turkey
Bhavuk GuptaNational Centre for Cell Science, PuneMaharashtra, India
Payal GuptaICAR‐National Institute for PlantBiotechnology, New Delhi India
Kathleen HefferonDepartment of Microbiology, CornellUniversity, IthacaNY, USA
J. Basilio HerediaCiencia y Tecnología de Alimentos(Food Science and Technology)Alimentos Funcionales y Nutracéuticos(Nutraceuticals and Functional Foods)Centro de Investigación en Alimentación yDesarrollo, AC (CIAD), (Research Centerfor Food and Development), CuliacánSinaloa, México
Arif Tasleem JanSchool of Biosciences and BiotechnologyBaba Ghulam Shah BadshahUniversity, RajouriJammu and Kashmir, India
Saima JanSchool of Biosciences and BiotechnologyBaba Ghulam Shah BadshahUniversity, RajouriJammu and Kashmir, India
Pooja JangirDepartment of BotanyUniversity of Rajasthan, JaipurRajasthan, India
Luis Alfonso Jiménez‐OrtegaCiencia y Tecnología de Alimentos(Food Science and Technology)Alimentos Funcionales y Nutracéuticos(Nutraceuticals and Functional Foods)Centro de Investigación en Alimentación yDesarrollo, AC (CIAD), (Research Centerfor Food and Development), CuliacánSinaloa, México
Suhas G. KarkuteDivision of Vegetable ImprovementICAR‐Indian Institute of VegetableResearch, VaranasiUttar Pradesh, India
Sukhmandeep KaurNational Agri‐Food BiotechnologyInstitute (NABI), MohaliPunjab, India
Department of biotechnologyPanjab University, ChandigarhIndia
Ayush KhandelwalSouth Asian UniversityNew Delhi, India
Purva KhandelwalDepartment of BotanyUniversity of Rajasthan, JaipurRajasthan, India
Anil KumarNational Institute of Plant GenomicResearch (NIPGR), Aruna Asaf Ali MargNew Delhi, India
Brijesh KumarPatanjali Herbal Research DivisionPatanjali Research Institute, HaridwarUttarakhand, India
Ravi Shankar KumarCSIR‐Central Institute of Medicinal andAromatic Plants (CSIR‐CIMAP), LucknowUttar Pradesh, India
Yuxin LiSchool of HorticultureHainan University/Key Laboratoryfor Quality Regulation of TropicalHorticultural Crops of Hainan ProvinceHaikou, China
Wen LiuSchool of HorticultureHainan University/Key Laboratoryfor Quality Regulation of TropicalHorticultural Crops of Hainan ProvinceHaikou, China
Nikhil MalhotraICAR‐National Bureau of Plant GeneticResources Regional Station, ShimlaHimachal Pradesh, India
Elena V. MikhaylovaInstitute of Biochemistry and GeneticsUfa Federal Research Center RASUfa, Russia
Brandon Estefano Morales‐MeridaCiencia y Tecnología de Alimentos(Food Science and Technology)Alimentos Funcionales y Nutracéuticos(Nutraceuticals and Functional Foods)Centro de Investigación enAlimentación y Desarrollo, AC(CIAD), (Research Center for Foodand Development), CuliacánSinaloa, México
Mahendra PawarNational Institute of PlantGenome ResearchNew Delhi, India
R. PrajapatiDepartment of Plant PathologyKansas State UniversityManhattan, KS, USA
Hasthi RamNational Institute of Plant GenomeResearch, New DelhiDelhi, India
Sumreen Amin ShahSchool of Biosciences and BiotechnologyBaba Ghulam Shah BadshahUniversity, RajouriJammu and Kashmir, India
Ashish SharmaCSIR‐Central Institute of Medicinal andAromatic Plants (CSIR‐CIMAP), LucknowUttar Pradesh, India
Pooja Kanwar ShekhawatDepartment of BotanyUniversity of Rajasthan, JaipurRajasthan, India
Erum ShoebDepartment of GeneticsUniversity of KarachiKarachi, Pakistan
Akanchha ShuklaAlthea DRF LifesciencesRouse Avenue Institutional AreaNew Delhi, India
ShumaylaDepartment of BotanyKashyap BlockPanjab University, ChandigarhIndia
Achuit K. SinghDivision of Vegetable ImprovementICAR‐Indian Institute of VegetableResearch, VaranasiUttar Pradesh, India
Hiteshwari SinhaCSIR‐National Botanical ResearchInstitute, Council of Scientific andIndustrial Research (CSIR‐NBRI)Rana Pratap Marg, LucknowUttar Pradesh, India
Academy of Scientific and InnovativeResearch (AcSIR), GhaziabadUttar Pradesh, India
Praveen SoniDepartment of BotanyUniversity of Rajasthan, JaipurRajasthan, India
Prabodh Kumar TrivediCSIR‐Central Institute of Medicinal andAromatic Plants (CSIR‐CIMAP), LucknowUttar Pradesh, India
CSIR‐National Botanical ResearchInstitute, Council of Scientific andIndustrial Research (CSIR‐NBRI)Rana Pratap Marg, LucknowUttar Pradesh, India
Academy of Scientific and InnovativeResearch (AcSIR), GhaziabadUttar Pradesh, India
K. TyagiDepartment of Plant PathologyUniversity of Minnesota, St PaulMinneapolis, MN, USA
Santosh Kumar UpadhyayDepartment of Botany, Kashyap BlockPanjab University, ChandigarhIndia
Elif Karlik UrhanInstitution of ScienceDepartment of BiotechnologyIstanbul UniversityIstanbul, Turkey
Jyothilakshmi VadasseryNational Institute of PlantGenome ResearchNew Delhi, India
Sanskriti VatsNational Agri‐Food BiotechnologyInstitute (NABI), MohaliPunjab, India
Regional Centre forBiotechnology, FaridabadHaryana (NCR Delhi), India
Srividhya VenkataramanDepartment of Cell andSystems BiologyUniversity of TorontoToronto, Canada
Hengyi XuDepartments of Molecular Biosciencesand Oncology, Institute of Cellular andMolecular BiologyUniversity of Texas at AustinAustin, TX, USA
Yang ZhouSchool of HorticultureHainan University/Key Laboratoryfor Quality Regulation of TropicalHorticultural Crops of Hainan ProvinceHaikou, China
Preface
Recent advances in the genome editing methods, especially the CRISPR‐Cas system for the precise engineering of the target plant genes/genomes, enabled numerous applications of this technology for crop improvement programs and functional genomics studies. In the last few years, several advances have been done in the CRISPR‐Cas tool kit including the identification of new Cas proteins having a diverse range of PAM specificity, base editing technologies and prime editing without any stand break, trans‐gene free editing, nano‐particle‐mediated delivery of the CRISPR tool kit, development of various ready to use vectors for numerous purposes, etc. These advancements make this system more efficient and effective for genome engineering, both in vivo and in vitro.
The modified tool kit has been utilized in several agricultural and horticultural crop species including rice, tomato, banana, etc., either for nutritional improvement or for stress tolerance. Further, it has been demonstrated that the CRISPR tool kit can also be utilized in the non‐transgenic mode for trans‐gene‐free editing, which has been recently deregulated by various regulatory bodies in several countries including India and USA. These announcements of deregulation have opened a new avenue and strongly boosted the research and developments by utilizing the genome engineering tools for numerous purposes in recent years.
Nutritional improvement and stress tolerance have been of prime importance in the last few decades due to increasing nutritional deficiency in the various regions and changes in climatic conditions. The use of genome engineering methods has been efficiently demonstrated for nutritional improvement in various agricultural and horticultural crops including rice, tomato, potato, banana, etc. Development of carotenoid‐ and flavonoid‐rich cereals, tomato and fruit crops like banana, iron‐enriched rice, maize, wheat, etc., are few examples of successful utilization of technology. Further, it is being utilized for abiotic and biotic stress tolerance, disease resistance, virus resistance, and ultimately for the improvement of yield in numerous important crop species.
In this book, the updated information related to the various technological advancements and applications for the nutritional improvements, disease resistance, abiotic stress tolerance, and yield improvement in various agricultural and horticultural crops by utilizing the genome engineering tool kits have been discussed.
Santosh Kumar Upadhyay,
Department of Botany, Panjab University,Chandigarh, India.
About the Editor
Dr. Santosh Kumar Upadhyay is currently working as an Assistant Professor in the Department of Botany, Panjab University, Chandigarh, India. Prior to this, Dr. Upadhyay was DST‐INSPIRE faculty at the National Agri‐Food Biotechnology Institute, Mohali, Punjab, India. He did his doctoral work at the CSIR‐National Botanical Research Institute, Lucknow, and received his PhD in Biotechnology from UP Technical University, Lucknow, India. He has been working in the field of Plant Biotechnology for more than 14 years. His present research focuses in the area of functional genomics. He is involved in the bio‐prospecting and characterization of various insect toxic proteins from plant biodiversity and defence and stress signaling genes in bread wheat. His research group at PU has characterized numerous important gene families and long non‐coding RNAs related to the abiotic and biotic stress tolerance and signaling in bread wheat. He has also established the method for genome editing in bread wheat using CRISPR‐Cas system and developed a tool SSinder for CRISPR target site prediction. His research contribution led to the publication of more than 80 research papers in leading journals of international repute. Further, there are more than 5 national and international patents, 40 book chapters and eleven books in his credit. In recognition of his substantial research record, he has been awarded INSA Associate Fellow (2023), NAAS Young scientist award (2017–2018) and NAAS‐Associate (2018) from the National Academy of Agricultural Sciences, India, INSA Medal for Young Scientist (2013) from the Indian National Science Academy, India, NASI‐ Young Scientist Platinum Jubilee Award (2012) from the National Academy of Sciences, India, and Altech Young Scientist Award (2011). He has also been the recipient of the prestigious DST‐INSPIRE Faculty Fellowship (2012), and SERB‐Early Career Research Award (2016) from the Ministry of Science and Technology, Government of India. Dr. Upadhyay also serves as a member of the editorial board and reviewer of several peer‐reviewed international journals.
1CRISPR/Cas‐Mediated Genome Editing in Plants: A Historical Perspective
Anil Kumar 1#, Shumayla 2#, and Santosh Kumar Upadhyay 2
1 National Institute of Plant Genomic Research (NIPGR), Aruna Asaf Ali Marg, New Delhi, India
2 Department of Botany, Kashyap Block, Panjab University, Chandigarh, India
1.1 Introduction
The production of food on a global scale is currently faced with enormous obstacles. By the year 2050, the population of the world is expected to reach 9.6 billion, with a corresponding rise in staple food crop demand by 60% (Tilman et al. 2011). There is a decline in the rate of increase in yield brought about by the “Green Revolution” and adverse climatic changes that are limiting plant production. Therefore, it is a prerequisite to generate cultivars with enhanced stress tolerance, improved quality, and increased yields. Moreover, the conventional methods of crop breeding are time‐consuming, complicated, and laborious. There is a need for cost‐effective and time‐saving methods (Chen et al. 2019).
Nowadays, crop plant improvement and their sustainable production have been achieved by a newly emerged targeted genome engineering approach, an alternative to breeding and transgenic methods. Genome editing tool utilizes sequence‐specific nucleases (SSNs) to introduce double‐strand breaks (DSBs) at the desired loci within the genome (Wei et al. 2022). The introduced DSBs are immediately repaired either by a nonhomologous end joining (NHEJ) pathway or a homologous recombination (HR) mechanism. NHEJ is an error‐prone pathway ideally recruited by most organisms, including higher plants that typically involves the ligation of broken ends that ultimately generates insertions and deletions (indels). NHEJ can also introduce double‐stranded templates in the DSBs‐generated overhangs (Li et al. 2019; Park and Beal 2019; Permyakova et al. 2022). Since the HR pathway uses homologous sequence as a template to repair the DSBs, this pathway can be utilized for the precise integration of the desired genes into the genome (Park and Beal 2019). Until recently, techniques such as zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENS) were utilized by researchers. But due to their complex protein engineering processes, these are not widely adopted by the plant research community. However, due to advancements in sequencing technology and the availability of genomic information for a large number of plant species, genome editing with precision for crop improvement has become feasible.
CRISPR (clustered regularly interspaced palindromic repeats)/Cas (CRISPR‐associated protein) has become the most widely used genome editing system due to its low cost, easy adaptability, and high specificity. It is an adaptive immune system from archaea and bacteria (Mali et al. 2013b; Yin et al. 2017). This technology mainly requires two major components: Cas9 enzyme and a single guide RNA (sgRNA). CRISPR/Cas is an RNA‐guided machinery that exists naturally as a part of a prokaryotic defense system that cleaves foreign DNA in a sequence‐specific manner. CRISPR repeats were first discovered as an unusual structure in the Escherichia coli IAP gene (Ishino et al. 1987), and later investigations revealed that the spacer sequences in CRISPR loci had similarities with phage and plasmid sequences (Bolotin et al. 2005; Mojica et al. 2005; Pourcel et al. 2005). The spacers are short fragments of the sequences integrated between the two adjacent repeats at the proximal end of a CRISPR locus. The CRISPR arrays, including the spacers, are transcribed and processed into small interfering CRISPR RNAs (crRNAs). These crRNAs further combine with the transactivating RNA (tracrRNA) to activate and guide a Cas9 nuclease to cleave complementary genomic target sequences, also known as protospacers (Barrangou 2007). However, the only requirement for the cleavage is the presence of a conserved protospacer‐adjacent motif (PAM) downstream of the target DNA usually 5′‐NGG‐3′ (Gasiunas et al. 2012; Jinek et al. 2012) and less frequently 5′‐NAG‐3′ (Hsu et al. 2013). The CRISPR/Cas system of archaea and bacteria is classified into three major types (I–III) (Makarova et al. 2015), of which the simplest is the type II system from Streptococcus pyogenes. The type II system only requires individual expression of crRNA, tracrRNA, and Cas9. This is followed by the hybridization of crRNA with trans‐activating crRNA (tracrRNA) and requires a host factor RNaseIII, which is important for crRNA maturation and recruitment of Cas9 to the target (Deltcheva et al. 2011; Jinek et al. 2012; Sushmita et al. 2021). However, the complexity of CRISPR/Cas has been made facile for genome editing by the fusion of crRNA and tracrRNA into a single‐guide RNA (sgRNA/gRNA) (Jinek et al. 2012; Makarova and Koonin 2015).
Soon after its demonstration in bacteria, yeast, mammals, and model plants, CRISPR/Cas is being widely applied as a genome editing tool (Tripathi et al. 2021; Lu et al. 2022; Pant et al. 2022; Vicencio et al. 2022). The application appears to offer a significant promise for modifying crops to improve their nutritional content, resistance to pathogens, or metabolic pathways leading to important end products. This chapter of the book provides an outline of the evolution, discovery, classification, and application of CRISPR/Cas as well as its mechanism of action.
1.2 Historical Background
In 1985, a novel assembly of repetitive sequences in the 3′‐end flanking regions of the IAP gene in E. coli, which plays a role in the isozyme conversion of alkaline phosphatase, was discovered for the first time. This was the beginning of the CRISPR framework. The structure consisted of direct repeats of five highly similar sequences, each of which contained 29 nucleotides, with 32 nucleotides serving as spacers between the repeats (Ishino et al. 1987). The sequencing of the bacterial genome in the following decade led to the discovery of many CRISPR loci. However, the biological function of the CRISPR array was not appreciated until 2005, when it was found that spacers contained sequences that were identical to those of viruses and plasmids (Bolotin et al. 2005; Mojica et al. 2005; Pourcel et al. 2005). In the latter part of the year 2007, it was discovered that the CRISPR array may be utilized by a strain of Streptococcus thermophiles to confer resistance against an invading bacteriophage by making use of the Cas genes (Barrangou 2007). It was confirmed that the spacer region in the CRISPR loci is a form of RNA‐mediated immune system that is used by bacteria to defend themselves against nucleic acids that have invaded their territory. Similar observations were reported for other organisms. This technique assists bacteria in the process of acquiring short sequences into the CRISPR locus, which may then be utilized by transcription later on. CRISPR loci in Streptococcus pyogenes have been studied, and the results have shown that numerous Cas genes are involved in the recognition and cleavage of invading DNA (Heler et al. 2015). However, the role that Cas proteins play in CRISPR‐based resistance is not entirely known as of yet.
Further research into the structure of the Cas protein uncovered similarities with endonuclease domains, which hints at a possible function in viral resistance via DSBs. Upon activation, spacers are translated into short crRNA, and Cas proteins then form an association with crRNA to create DSBs in the invading sequences (Garneau et al. 2010). Later on, CRISPR was split up into three major categories (I, II, and III) based on the gene conservation and locus arrangement of the different Cas genes that were discovered in bacteria and archaea. In 2005, the classification of the Cas genes was originally suggested by Haft et al. and then in 2011, Makarova et al. did a good job of expanding on that classification (Haft et al. 2005; Makarova et al. 2011). The structural functioning of Cas9 as an RNA‐guided endonuclease was demonstrated by Jinek et al. (2012). Cas9 is comprised of two primary nuclease domains that primarily execute cleavage functions on both strands of DNA. This research was bolstered even further by the direct confirmation of the connection between the Cas9‐guide RNA and target DNA complexes that was made possible by the imaging of their crystal structures (Nishimasu et al. 2014). Multiple authors demonstrated the use of CRISPR/Cas immune system based on RNA‐mediated DNA targeting soon after understanding its biological role in the year 2007 (Marraffini and Sontheimer 2008; Garneau et al. 2010; Deltcheva et al. 2011).
The transition of the CRISPR/Cas from the immune system to the genome editing technique was not by fate, but it was designed and confirmed that editing could be performed by changing only 20 nt in the crRNA and further specificity of targeting by crRNA could be combined with tracrRNA into a chimeric single guide RNA (gRNA), thus reducing the system from three to two components (Jinek et al. 2012). Shortly thereafter, this two‐component system was functionally demonstrated in eukaryotes such as humans, mice, and zebrafish, suggesting the other functions of the CRISPR locus gene were supported by eukaryotic endogenous enzymes (Cho et al. 2013; Cong et al. 2013; Hwang et al. 2013; Jinek et al. 2013; Mali et al. 2013b). Interestingly, it was shown that multiple gRNA could be used to achieve high‐efficiency multiplex genome engineering simultaneously at different loci (Cong et al. 2013; Mali et al. 2013a). The first applications of CRISPR/Cas were reported in August 2013 by five research groups separately in the model plants Arabidopsis, tobacco, and rice using transient and stable transformation strategies (Feng et al. 2013; Li et al. 2013; Nekrasov et al. 2013; Shan et al. 2013; Xie and Yang 2013). Immediately, research on higher crop species including sorghum (Jiang et al. 2013), wheat (Upadhyay et al. 2013), and maize (Liang et al. 2014) were published. All these milestones in genome editing substantiate that CRISPR/Cas is a simple, versatile, and inexpensive tool for targeted genome editing in different organisms including plants.
1.3 Mechanism of CRISPR/Cas System
The CRISPR/Cas system recognizes and cleaves foreign DNA or RNA in a sequence‐specific manner. The defense mechanism can be explained easily by first understanding the components of the CRISPR/Cas unit.
The CRISPR/Cas system can be broken down into two classes, Class I and Class II, according to the structure and functions of the Cas proteins. Class I includes types I, III, and IV, while Class II includes types II, V, and VI. The Class I systems are made up of Cas‐protein complexes that contain multiple subunits, whereas the Class II systems only make use of a single Cas‐protein. Because of type II CRISPR/Cas‐9 simplicity, it has been the subject of extensive research and has seen considerable application in genetic engineering (Liu et al. 2020). Guide RNA, also known as gRNA, and CRISPR‐associated proteins, also known as Cas‐9 proteins, are the two parts that are necessary for the CRISPR/Cas‐9 system. Cas9 is a multidomain endonuclease isolated from Streptococcus pyogenes (SpCas‐9). It is known as a genetic scissor (Mei et al. 2016). It is responsible for cutting the target DNA to generate a double‐stranded break in the DNA. There are three steps in the mechanism by which the type II CRISPR/Cas system (van der Oost et al. 2009; Garneau et al. 2010; Horvath and Barrangou 2010; Marraffini and Sontheimer 2010) recognizes and targets foreign genetic material.
1.3.1 Acquisition of Spacers
It is the first stage in CRISPR/Cas‐mediated defense. At this point, the CRISPR/Cas system is modified to include the foreign genetic material, allowing the host to recognize and respond effectively to future invasions by the same phage strain. This stage is mediated by the proteins Cas1 and Cas2, which are found in all the CRISPR/Cas systems (Makarova et al. 2006). Foreign DNA is recognized and integrated into the CRISPR locus as a spacer during the acquisition process. Typically, the spacer is recognized by a short (2–5 bp) sequence of conserved nucleotides (PAMs) in the protospacer. The selection of the protospacers is determined by the presence of protospacer‐adjacent short motifs, also known as PAMs, in proximity to the target sequence. PAMs are short sequences of two to five nucleotides that are unique to each CRISPR/Cas subtype and bacteria (Mojica et al. 2009). A single copy of 30 bp spacer is inserted at the leader side of the CRISPR array followed by its duplication (Garneau et al. 2010). The immunity against pathogen attack via CRISPR/Cas system can be suppressed by mutations in the PAM region of the viral genome (Garneau et al. 2010).
1.3.2 Biogenesis
During the expression phase of the CRISPR/Cas system functioning, the lengthy pre‐crRNA is transcribed from the CRISPR locus and processed into crRNAs with the aid of Cas proteins (Cas1, Cas2, Cas9, and Cas4/Casn2). Karvelis et al. (2013) recently discovered that the tracrRNA also has a role in the synthesis of pre‐crRNA in S. pyogenes (Karvelis et al. 2013). The pre‐crRNA into crRNA processing is aided by the tracrRNA, which forms base‐complementary pairings with the crRNA repeat region (Deltcheva et al. 2011). For defense against any foreign invader, the processed crRNA integrates into the CRISPR‐associated complex and recognizes and base pairs with an exact target region of the foreign DNA (Deltcheva et al. 2011).
1.3.3 Interference with the Target
In the final stage, crRNA combines with Cas protein to form an effector complex that base pairs with the complementary strand in the invading DNA and induces DSB of the specific target region (Garneau et al. 2010). Hence, the proliferation and propagation of foreign pathogens are prevented by these effector ribonucleoprotein (RNP) complexes. The type II effector complexes function as RNA‐guided endonucleases that recognize the target sequence by crRNA and guide the Cas protein for cleavage of DNA within the target proto‐spacer (Garneau et al. 2010; Marraffini and Sontheimer 2010). Repairing this DSB is necessary for maintaining the host system. Now, if the break is repaired via NHEJ, the gene may become defective due to insertion or deletion mutations (INDELs) (Symington and Gautier 2011). Homologous direct repair (HDR) allows DNA insertion at the target gene, permitting gene insertion or substitution, if the repair mechanism is aided by another template DNA that has homologous sequence flanking to the target site. However, the implementation of a bacterial CRISPR/Cas system in any organism requires the assembly of a functional crRNA‐tracrRNA‐Cas9 unit.
1.4 Breakthrough Studies in CRISPR/Cas System
S. no
Breakthrough study
Approach
Species
References
1
Base editing
Cytosine base editor
Oryza sativa, maize
Li et al. (
2020
)
Adenine base editor
Triticum aestivum, O. sativa, rapeseed, and Arabidopsis thaliana
Li et al. (
2018
), Kang et al. (
2018
), Hua et al. (
2020
)
Cytosine‐dependent DNA manipulation
2
Prime editing
Nick in the unmodified DNA strand
O. sativa and T. aestivum
Lin et al. (
2020b
), Xu et al. (
2020
)
3
Multiplexing
Multiple gRNA delivery within single CRISPR/Cas complex
T. aestivum, Oryza sativa, A. thaliana, and Bombyx mori
Ma et al. (
2014
), Debbarma et al. (
2019
), Zhou et al. (
2019
)
4
Chromosome engineering
18 kb stretch rearrangement via CRISPR/Cas; reciprocal chromosomal translocations
A. thaliana
Schmidt et al. (
2019
), Beying et al. (
2020
)
5
Homologous recombination
Gene replacement
Nicotiana tobaccum
Barakate et al. (
2020
)
High‐efficiency gene targeting with stable inheritance
A. thaliana
Hahn et al. (
2018
)
6
Epigenetic engineering
Histone modification
A. thaliana
Kuppu et al. (
2020
)
DNA methylation editing
Cucumis melo
L.
Giordano et al. (
2022
)
Transposon silencing
Hordeum vulgare
Galli et al. (
2022
)
7
Conditional CRISPR/Cas systems
Efficient mutagenesis in specific cell types, tissues, or organs
A. thaliana
Decaestecker et al. (
2019
)
8
Crop improvement
Abiotic and biotic stress tolerant crops, biofortified crops, high yield crops, etc.
O. sativa, Musa acuminata, T. aestivum
, etc.
Zhang et al. (
2019
), Lacchini et al. (
2020
), Kaur et al. (
2020
)
1.5 CRISPR Types
CRISPR/Cas system is categorized into two classes including Class I and Class II. The categorization is based on the number of subunits and the complexity of the system. Class‐I CRISPR/Cas system has a multi‐subunit effector protein and thus is more complex; however, Class‐II system has a single subunit effector protein. Besides this classification, based on the type of effector Cas proteins and/or modules, both classes are further divided into their types and subtypes. Types I, III, and IV are part of the Class‐I CRISPR system that has Cas3, Cas1, and CsfI as effector proteins, respectively. Types II, V, and VI are the types of Class‐II with Cas9, Cas12, and Cas13 proteins as their effector modules, respectively. Moreover, based on their functional mechanisms, these types are further divided into their subtypes. The three stages of bacterial adaptation for the viral attack include the adaptation of spacer DNA, expression of CRISPR array, and then the action of effector complex called interference. The multi‐subunit effector complex of Type‐I and subtypes III‐A and III‐B CRISPR/Cas systems is called CRISPR‐associated complex for antiviral defense (Cascade) complex including Cas3, Casm, and Cmr, respectively. These cascade complexes perform expression and maturation followed by interference. On the other hand, in Types II, III, and V, single multidomain proteins including Cas9, Cas10, and Cpf1, respectively, perform the expression, maturation, and interference stages.
1.6 Type of Cas Proteins
1.6.1 Cas1
Cas1 is a metal‐dependent DNase and is one of the most conserved proteins that constitute the CRISPR‐based bacterial immune system. Cas1 is the universal part of all types of CRISPR systems, and it has the capacity to bind to RNA in a non‐sequence‐specific manner. It is one of the important proteins that is requisite for the acquisition of spacer DNA (Makarova et al. 2011; Nuñez et al. 2014).
1.6.2 Cas2
Cas1 is a metal‐dependent endoribonuclease and is essential for acquisition of spacer DNA. Cas2 is a universal protein of all the CRISPR/Cas system of bacteria. Cas1 and Cas2 function by forming a complex and recognize the CRISPR array. The symmetrical dimerized Cas2 binds to asymmetrical dimer of Cas1 and forms a complex that is involved in the identification of viral DNA invasion (Makarova et al. 2011; Nuñez et al. 2014).
1.6.3 Cas3
Cas3 is a translocase/helicase, and it is a part of type I CRISPR/Cas system and an important component of CRISPR interference for precise priming as well as deleting DNA into smaller fragments (Nuñez et al. 2014; He et al. 2020; Yoshimi et al. 2022). Cas3 has distinguished helicase and nuclease activities due to the presence of helicase domain known as Cas’ and HD nuclease domain named as Cas’ domain, respectively. It is involved in unidirectional and long‐range deletion of genomic DNA upstream to the PAM region (Makarova et al. 2011; Morisaka et al. 2019).
1.6.4 Cas4
Cas4 is one of the important proteins that form the core of CRISPR/Cas system. It is a DNA exonuclease and can show 5′–3′ and 3′–5′ exonuclease activity (Zhang et al. 2012; Lemak et al. 2013). It has two domains including RecB nuclease domain responsible for nuclease activity and Fe‐S cluster binding domain (Dixit et al. 2021). It is involved in the identification of 5′PAM sequence and shows a direct association with Cas1–Cas2 complex (Lee and Dhingra 2019).
1.6.5 Cas5
Cas5 is recognized as an endoribonuclease that is involved in the processing of pre‐crRNA and the production of individual crRNA by binding to its 5′ handle. It is one of the components of CRISPR/Cas core complex that shows an interaction with the larger subunit of the cascade and Cas7 (Brendel et al. 2014).
1.6.6 Cas6
Cas6 is recognized as a metal‐independent endoribonuclease. It is the part of CRISPR/Cas systems 1 and 3. It is recognized as an endoribonuclease and is found to be involved in the biogenesis of crRNA against the DNA of the invading viruses. It helps in stabilizing the crRNA and the processing of individual crRNA units (Carte et al. 2008; Makarova and Koonin 2015).
1.6.7 Cas7
These are the multicopy proteins that are the integral part of the Cascade complex. Cas7 is involved in the transcription process of pre‐crRNA and also helps in the production of discrete crRNA (Makarova and Koonin 2015). In a recent study, Cas7–11 that comprised of Cas7.1–4, Cas11, INS, and CTE domains was reported to be used for programmable targeted RNA cleavage (Katoh et al. 2002; Kato et al. 2022).
1.6.8 Cas8
Cas8 is an integral member of CRISPR/Cas interference system that is required for the identification of PAM sequence in the invading bacteriophage DNA. Cas8 shows DNA binding by interacting with Cas5‐Cas‐cRNA complex. Cas8 is functionally analogous to the CasA module in type I CRISPR/Cas system (Cass et al. 2015; Kalwani et al. 2020).
1.6.9 Cas9
Cas9 is one of the extensively studied and utilized protein in precise genome editing. This is the signature protein of type II CRISPR/Cas system. Cas9 has the nuclease activities driven by RuvC and HNH domains for double‐stranded break (Jinek et al. 2012). The most studied Cas9 protein was isolated from Streptococcus pyogenes that requires a PAM sequence of ‐NGG at the 3′ end of the target site. Several modifications have been done in Cas9 protein to design new variants with different functions and utilities. Cas9 and its variants have been used extensively for CRISPR/Cas9‐mediated targeted genome editing (Liu et al. 2020).
1.6.10 Cas10
Cas10 is the signature effector protein of Type‐III CRISPR/Cas system that is required for the crRNA processing and interference. Cas10 can create a non‐PAM‐based single‐stranded break both in RNA and DNA. Three different domains have been identified in Cas10 including a nuclease domain, a palm domain responsible for the production of secondary messengers, and a Zn ribbon that shows homology with the cascade module (Lin et al. 2020a).
1.6.11 Cas11
Cas 11 is the integral member of type III and two type I systems including 1A and 1E CRISPT/Cas systems. Previous studies suggest that certain type I systems that do not have Cas11 subunits as effector proteins produce Cas11d small subunit variant from the Cas10d ORFs (Schunder et al. 2013; McBride et al. 2020).
1.6.12 Cas12
Cas12 is also known as Cpf1 or Cas12a (class 2 type V‐A system). This is one of the most important and studied Cas proteins after Cas9. Cas12 does not require tracrRNA and can effectively cleave the target DNA. Cas12 has RNase activity, and hence it is involved in the processing of self‐pre‐crRNA processing. Due to its smaller size, it has a significant importance as genome editing protein (Zetsche et al. 2015). Cas12b (C2c1) is the recently introduced genome editing effector protein that has a T‐rich preferred PAM. This protein creates staggered ends at double‐stranded DNA breaks. Among Cas9, Cas12a, and Cas12b, which are major genome editing effector proteins, Cas12b has a smaller size (Ming et al. 2020).
1.6.13 Cas13
Cas13 is an RNA‐guided RNA endonuclease that cleaves single‐stranded RNA at the precise target site. Cas13 has two domains including higher eukaryotic‐prokaryotic nucleotide (HEPN) binding domain that is responsible for its RNase activity. It has gRNA maturation activity that is potentially provided by HEPN2 and Halical‐1 domains. Cas13 has four small subtypes including Cas13a (C2c2) (Type VI‐A), Cas13b (C2c4) (Type VI‐B), Cas13c (C2c7) (Type VI‐C), Cas13d, Cas13X (Type VI‐X), and Cas13Y (Type VI‐Y) (Abudayyeh et al. 2017; O’Connell 2019).
1.6.14 Cas14
Cas14 proteins size ranged from 40–70 kDa that act as effector proteins and cleave only single‐stranded DNA without the requirement PAM sequence. Cas14 is highly specific to the middle nucleotides of its target sites, and hence this property can be utilized to mark a specific locus at genomic level by fusing any fluorophore to the protein. Moreover, this can be used to detect SNPs (Savage 2019).
1.7 CRISPR/Cas Modification
After the discovery of CRISPR/Cas9 system as a genome editing tool, several researchers engineered Cas proteins for their versatile utilization.
1.7.1 Nickase
Cas9 nickases are the modified versions of Cas9 that create a nick in either of the strands of double‐stranded DNA. For instance, two variants of Cas9 nickases are generated by creating a D10A mutation in RuvC domain that cleaves target strand and a H840A mutation in HNH domain that cleaves nontarget strands (Jinek et al. 2012). These variants can be used in pairs to minimize the off‐target efficiency of CRISPR/Cas9 system. Moreover, these can further be modified by making fusion proteins and generating new variants such as base editors.
1.7.2 Dead Cas9 (dCas9)
This variant of Cas9 was created by the inactivation of both the nuclease domains of Cas9. dCas9 does not have nuclease activity; however, this could make a complex with sgRNA that further binds to the DNA at target site (Jinek et al. 2012). These variants are further modified by making a fusion with other proteins such as reporter proteins.
1.7.3 Base Editors
Taking the advantages of dCas9 and nickases, base editors were generated by fusing these proteins with base editing proteins. There are primarily two variants of base editors including cytosine base editors (CBEs) and adenine base editors (ABEs). In CBEs, cytidine deaminase is fused to dCas9 to create cytidine (C) to thymidine (T) conversion; however, in ABEs, the TadA (tRNA‐specific adenosine deaminase) from E. coli is fused to dCas9 that creates the conversion of adenosine (A) to inosine (I), which is translated as guanosine (G) resulting in A to G conversion. The primary reason to use TadA domain is that adenine deaminase base editors arenatively absent in nature (Wolf et al. 2002). Studies have shown base editing in plants such as wheat and rice (Li et al. 2018).
1.7.4 Prime Editors
The prime editors are the fusion proteins of Cas9 nickase (H840A) and a modified reverse transcriptase. The system also requires a guide RNA that is extended by addition modified sequences serving as template for reverse transcription that would lead to a mismatch at the target site. This mismatch can either be retained by the host system or repaired to its original sequence that would serve as another chance for editing (Scholefield and Harrison 2021).
1.8 CRISPR/Cas as a Genome Editing Tool and Its Application
After the discovery of this system, several researchers have used it for various molecular biology research (Upadhyay 2021). The system was initially used for precise genome editing for gene knockout; however, the further advancements paved new ways for its utilization in gene knockin, localization, fine tuning of genes, and RNA editing. Some of the primary applications are discussed further.
1.8.1 Gene Knockout
As described in the section above, CRISPR/Cas9 system of Streptococcus pyogenes is the extensively studied system. Researchers further modified this system to be used as a precise genome engineering tool. The target site is first fused with a gRNA scaffold; the fused product is known as single‐guide RNA (sgRNA). This cassette is cloned into a binary vector system that also harbors the Cas9 open reading frame (ORF) driven by an appropriate constitutive and/or inducible promoter depending upon the nature of the work. Because Cas9 is originally a bacterial protein, the coding sequence of Cas9 is first codon optimized according to the host such as plants and animals. The expression constructs are transformed into the host system. For instance, in plants, the expression constructs are transferred extensively by agrobacterium‐mediated plant transformation and particle bombardment methods. However, the transformation is not limited to these methods. In successful transformants, Cas9 will bind to the target sites based on the homology to sgRNA to the target site; however, the cleavage of DNA will be done based on the PAM sequence present adjacent to the target site. Cas9 cleaves the double‐stranded DNA at the precise position guided by sgRNA. These breaks are repaired by either of the host DNA repair mechanisms including non‐homologous end joining and homologous direct repair that are often error prone, leading the indel mutation at the cleavage site that in turn disrupt the gene function either by frame shift mutation or stop codon gain, etc. Cas9, Cas9 nickase, Cas12a, and Cas12b can be utilized for gene knockout (Liang et al. 2017; Ming et al. 2020).
1.8.2 DNA Insertion
Taking advantage of homologous direct repair, it is possible to insert DNA precisely at the targeted site on the host genome. To perform the gene insertion, a donor DNA template is provided to the cell and this switches the repair mechanism to HDR instead of NHEJ (Jasin and Haber 2016; Gallagher and Haber 2018; Nambiar et al. 2019). PCR‐amplified DNA template, single‐stranded oligonucleotides, and plasmids are being used for donor templates. The donor template is designed in such a way that it harbors the homologous sequences known as the homologous arms from the upstream and downstream regions of the target site, and in between these arms, the desired DNA with mutation is present. To avoid the cleavage of the donor template, the PAM sequence can be altered in the donor template with missense mutation. The double‐stranded breaks are created by Cas9 at the target site, and the break is repaired by the insertion of donor DNA template. Although HDR mechanism can intentionally be switched, however, it has a very low efficiency, and therefore double‐stranded DNA can also be repaired by NHEJ. The desired cells with mutation can further be screened. CRISPR/Cas‐based DNA insertion has been reported in different studies (Dahan‐Meir et al. 2018; Lu et al. 2020).
1.8.3 Base Editing
It is possible to utilize the CRISPR/Cas system for genome editing without creating a double‐stranded break and create a precise mutation at a single nucleotide instead (Zhang et al. 2019). The binary vectors that harbor a fusion protein of dCas9 and base editors’ as effector proteins are used in such studies. The complex of sgRNA and base editors is guided to its target loci at the genome and the effector proteins (CBE/ABE) make the targeted single base editing. The complementary to the converted base is repaired by the host systems that lead to permanent base conversion at the target loci. Several modifications such as incorporation of DNA glycosylase inhibitor (UGI) or bacteriophage Mu‐GAM protein have been done in the fusion protein to enhance the expression and hence the efficiency of base editing (Komor et al. 2017). Moreover, base editing of RNA is the further advancement of the tool. The fusion protein of dCas13b and adenosine deaminase is an effector protein for RNA base editing that converts A–I. Because of the functional equivalency of I–G, the final modification is A–G in RNA.
1.8.4 Gene Activation and/or Repression
The ability of dCas9 to bind to the DNA at the targeted site and not showing any nuclease activity created several utilizations of the protein such as the activation and/or repression of genes. dCas9 can be fused to different activators and repressors, and the promoter region of the genes can be targeted for gene regulation. The fusion of dCas9 with one or more activators will result in the activation of the downstream genes known as CRISPRa and fusion with one or more repressors will repress the expression of downstream genes known as CRISPRi (KREB‐dCAS9) (Alerasool et al. 2020
