Genome Engineering for Crop Improvement E-Book
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- Herausgeber: John Wiley & Sons
- Kategorie: Wissenschaft und neue Technologien
- Sprache: Englisch
In recent years, significant advancements have been made in the management of nutritional deficiency using genome engineering--enriching the nutritional properties of agricultural and horticultural crop plants such as wheat, rice, potatoes, grapes, and bananas. To meet the demands of the rapidly growing world population, researchers are developing a range of new genome engineering tools and strategies, from increasing the nutraceuticals in cereals and fruits, to decreasing the anti-nutrients in crop plants to improve the bioavailability of minerals and vitamins. Genome Engineering for Crop Improvement provides an up-to-date view of the use of genome editing for crop bio-fortification, improved bioavailability of minerals and nutrients, and enhanced hypo-allergenicity and hypo-immunogenicity. This volume examines a diversity of important topics including mineral and nutrient localization, metabolic engineering of carotenoids and flavonoids, genome engineering of zero calorie potatoes and allergen-free grains, engineering for stress resistance in crop plants, and more. Helping readers deepen their knowledge of the application of genome engineering in crop improvement, this book: * Presents genetic engineering methods for developing edible oil crops, mineral translocation in grains, increased flavonoids in tomatoes, and cereals with enriched iron bioavailability * Describes current genome engineering methods and the distribution of nutritional and mineral composition in important crop plants * Offers perspectives on emerging technologies and the future of genome engineering in agriculture Genome Engineering for Crop Improvement is an essential resource for academics, scientists, researchers, agriculturalists, and students of plant molecular biology, system biology, plant biotechnology, and functional genomics.
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Table of Contents
Cover
Title Page
Copyright Page
Dedication Page
List of Contributors
Preface
About the Editor
Acknowledgments
1 An Overview of Genome‐Engineering Methods
1.1 Introduction
1.2 ZFNs
1.3 TALENs
1.4 CRISPR‐Cas System
1.5 CRISPR‐Cpf1
1.6 Conclusions
Acknowledgements
References
2 Distribution of Nutritional and Mineral Components in Important Crop Plants
2.1 Introduction
2.2 Exploring Nutrient Distribution in Grain
2.3 Exploring the Mineral Distribution in Grain
2.4 Prospect
Acknowledgement
References
3 Application of Genome Engineering Methods for Quality Improvement in Important Crops
3.1 Introduction
3.2 Evolution and Historical Perspective of Genome Engineering
3.3 CRISPR/Cas Genome Editing Systems
3.4 Application of CRISPR/Cas System for Crops Quality Improvement
3.5 Regulatory Measures for Genome Engineering Crops
3.6 Conclusion
Acknowledgement
References
4 Genome Engineering for Enriching Fe and Zn in Rice Grain and Increasing Micronutrient Bioavailability
4.1 Introduction
4.2 Genes Related to Uptake of Fe and Zn from the Soil
4.3 Fe and Zn Biofortification using the SDN‐1 Approach
4.4 Fe and Zn Biofortification Using the SDN‐2 Approach
4.5 Fe and Zn Biofortification Using the SDN‐3 Approach
4.6 Future Thrust and Implications of SDN‐1, ‐2, and ‐3
References
5 Development of Carotenoids Rich Grains by Genome Engineering
5.1 Introduction
5.2 Nutritional Quality Improvement Through Pathway Engineering
5.3 Crop Improvement through Genetic Engineering Techniques
5.4 Improvement of Carotenoid in Grain Crops through CRISPR/Cas9
5.5 Improvement of Carotenoid in Grain Crops Through RNAi
5.6 Future Perspectives and Conclusion
References
6 CRISPR‐Cas9 System for Agriculture Crop Improvement
6.1 Introduction
6.2 Genome Engineering
6.3 Tools for Genome Engineering
6.4 CRISPR/Cas Beyond Genome Editing
6.5 CRISPR/Cas and Crop Improvement
6.6 Application of Genome Engineering Tools in Metabolic Engineering
6.7 Future Prospective
References
7 Contribution of Crop Biofortification in Mitigating Vitamin Deficiency Globally
7.1 Introduction
7.2 Effect of Vitamins on Human Health and Their Sources
7.3 Plan Biofortification to Overcome Vitamin Deficiency
7.4 Conclusion
Acknowledgments
References
8 Genome Editing Approaches for Trait Improvement in the Hairy Root Cultures of the Economically Important Plants
8.1 Introduction
8.2 Secondary Metabolites and Hairy Root Culture: An Insight
8.3 Genome Editing Process in Plants
8.4 Plant Hairy Root Culture as a Model for Genome Engineering
8.5 Conclusions
Acknowledgements
References
9 Phytic Acid Reduction in Cereal Grains by Genome Engineering
9.1 Introduction
9.2 Genes Involved in Phytic Acid Biosynthesis
9.3 Potential Targets and Strategies to Achieve Low Phytate Wheat
9.4 Evolution of Genome Engineering for Trait Development in Wheat
9.5 Future Implications
Acknowledgements
References
10 Genome Engineering for Nutritional Improvement in Pulses
10.1 Introduction
10.2 Need for Nutritional Improvement in Pulses
10.3 Nutritional Defects in Pulses Targeted for Genetic Engineering
10.4 Genome Engineering as an Alternate to Conventional Breeding
10.5 Conclusive Discussion
References
11 The Survey of Genetic Engineering Approaches for Oil/Fatty Acid Content Improvement in Oilseed Crops
11.1 Background
11.2 Soybean: Triumph Oil Crop
11.3
Camelina sativa:
Biofuel and Future Ready Crop
11.4 Conclusion
Acknowledgments
References
12 Genome‐Editing Mediated Improvement of Biotic Tolerance in Crop Plants
12.1 Introduction
12.2 Plant Defense Response
12.3 Genome Engineering Tools for Engineering Disease Resistance
References
13 Genome Engineering and Essential Mineral Enrichment of Crops
13.1 Introduction
13.2 Root Engineering of Cereals: A Promising Strategy to Improve Nutrient Efficiency, Biofortification, and Drought Tolerance
13.3 Use of Genome Edited Plants in Phytoremediation
13.4 Genetic Engineering and Crop Biofortification
13.5 Genome Engineering Technology and Its Use in Essential Mineral Enrichment of Crop Plants
13.6 Conclusion
References
14 Genome Editing to Develop Disease Resistance in Crops
14.1 Introduction
14.2 Traditional Approaches to Develop Disease Resistance in Crops
14.3 Genome Editing‐a New Way Forward
14.4 Genome Editing Examples to Develop Disease Resistance in Crops
14.5 Recent Trends in Genome Editing
14.6 Conclusion and Prospects
References
15 Biotechnological Approaches for Nutritional Improvement in Potato (
Solanum tuberosum
L.)
15.1 Introduction
15.2 Genetic Transformation of Potato
15.3 Protein Engineering of Potato for Protein Content
15.4 Genetic Engineering of Potato for Starch Modification
15.5 Lipids Biosynthesis Engineering in Potato
15.6 Vitamins Genetic Engineering in Potato
15.7 Metabolic Engineering of Potato for Enhanced Mineral Content
15.8 Pathway Engineering for the Functional Secondary Metabolites
15.9 Future Prospective and Conclusions
References
16 Genome Engineering Strategies for Quality Improvement in Tomato
16.1 Introduction
16.2 Genome Editing Systems in Plants
16.3 Current Applications of Genome Editing in Tomato Improvement
16.4 Challenges and Future of Genome Editing in Tomato
References
17 Genome Editing for Biofortification of Rice
17.1 Introduction
17.2 Genome Editing and its Tools
17.3 Genome Editing for Biofortification of Rice
17.4 Genome Editing for Improvement of Agronomic Traits in Rice
17.5 Conclusion and Future Aspects
Acknowledgment
Conflicts of Interest
References
18 Genome Editing for Improving Abiotic Stress Tolerance in Rice
18.1 Introduction
18.2 Recent Developments in Genome Editing Technology
18.3 Challenges of Different Genome‐Editing Systems
18.4 Application of Genome‐Editing Technology for the Improvement of Abiotic Stress Tolerance in Rice
18.5 Challenges of Genome Editing in Rice
18.6 Conclusion and Future Prospects
Acknowledgment
Conflicts of Interest
References
19 Role of Genome Engineering for the Development of Resistant Starch‐Rich, Allergen‐Free and Processing Quality Improved Cereal Crops
19.1 Introduction
19.2 Starch Characteristics
19.3 Starch Biosynthesis
19.4 Starch Digestibility and Resistant Starch
19.5 Genetic Modification in Relation to RS
19.6 Genetic Modification in Relation to Allergen‐Free Cereals
19.7 Genetic Modification in Relation to Improved Processing Quality Cereals
19.8 Conclusions
Acknowledgments
References
20 Engineering of Plant Metabolic Pathway for Nutritional Improvement
20.1 Introduction
20.2 Methods for Metabolic Engineering
20.3 Vitamin A
20.4 Vitamin E
20.5 Vitamin C
20.6 Vitamin B
20.7 Amino Acids and Proteins
20.8 Plant Volatiles Compound
20.9 Phytic Acid
20.10 Condensed Tannin
20.11 Conclusions
Acknowledgments
References
21 Genome Engineering for Food Security
21.1 Introduction
21.2 Plant Breeding for Food Security
21.3 Conclusion
Conflict of Interest
Acknowledgement
References
Index
End User License Agreement
List of Tables
Chapter 1
Table 1.1 List of available softwares and programs for designing gRNA.
Table 1.2 List of examples of genes edited by CRISPR Cas system in various pl...
Chapter 3
Table 3.1 Online platform for tools of CRISPR/Cas system.
Chapter 4
Table 4.1 Potential genes and strategies that can be used for editing to incr...
Chapter 5
Table 5.1 Major cereal crops, gene used for increasing Vit A content and meth...
Chapter 6
Table 6.1 Epigenetic regulation by dCas9.
Table 6.2 Plant gene edited using CRISPR/Cas9 for improved agronomically impo...
Chapter 7
Table 7.1 Recommended dietary allowance (RDA) of vitamin A (retinol equivalen...
Table 7.2 Recommended dietary allowance (RDA) for vitamin D.
Table 7.3 Recommended dietary allowance (RDA) for vitamin E.
Table 7.4 Recommended adequate intake (AI) for vitamin K.
Table 7.5 Vitamin B complex, dietary source, role, and deficiency disorders.
Table 7.6 Recommended dietary allowance (RDA) of Vitamin C (mg/day).
Table 7.7 Most common sources and solubility of different vitamins.
Table 7.8 Advantages and disadvantages of major biofortification approaches.
Chapter 8
Table 8.1 List of edited genes in different plant species by using the hairy ...
Chapter 9
Table 9.1 Enlisting the genes that are good potential target for achieving lo...
Chapter 10
Table 10.1 Major genome engineering events for nutritional improvement in dif...
Chapter 11
Table 11.1 Summary of various genetic improvement approaches employed to oil ...
Table 11.2 The complied summary of genetic engineering approaches adopted in ...
Chapter 14
Table 14.1 Engineered nucleases applications for disease‐resistance developme...
Chapter 15
Table 15.1 Various nutritional constituents of the potato.
Table 15.2 Gene(s) used to enhance nutritional value in transgenic potato (
Solan
...
Chapter 16
Table 16.1 Current status of genome editing in tomato.
Chapter 17
Table 17.1 Application of genome‐editing tools for improvement of nutritional...
Chapter 18
Table 18.1 Application of genome‐editing technology for generating abiotic st...
Chapter 20
Table 20.1 Summary of the biofortification for different Vitamins in plants t...
List of Illustrations
Chapter 1
Figure 1.1 (A) Diagrammatic representation of (a)
Zinc‐finger nucleases
...
Figure 1.2 Comparison of various features of CRISPR‐Cpf1 (a) and CRISPR‐Cas9...
Chapter 2
Figure 2.1 Spatial distribution of organic compound fitting the peak of m/z ...
Figure 2.2 Comparison of the infrared spectra of common buckwheat (pink) and...
Figure 2.3 Representative SR‐FTIR chemical mapping of the Tartary buckwheat ...
Figure 2.4 Representative SR‐FTIR chemical mapping of the wheat grain cross‐...
Figure 2.5 Quantitative mineral‐element distribution maps of a Tartary buckw...
Figure 2.6 Element (K, Ca, Fe, Mn, and Zn) localization in Khorasan wheat (
T
...
Figure 2.7 LA‐ICPMS images of the Ca and K distribution in the maize (
Zea ma
...
Chapter 3
Figure 3.1
Comparison of Gets.
Classical methods include natural mutation vi...
Figure 3.2
The basic scheme of CRISPR/Cas system
(Fiaz et al. 2019). (1a) Pr...
Chapter 4
Figure 4.1 Current most promising candidate genes to increase grain Fe and Z...
Chapter 5
Figure 5.1 General structure of a carotenoid: polyene tail with double bonds...
Figure 5.2 Diagrammatic representation about Learn/Reconstruct/Test cycle.
Chapter 6
Figure 6.1 dCas9 as a transcriptional regulator: Transcriptional activator o...
Figure 6.2 dCas9 DNMT3A system for targeted DNA methylation.
Chapter 8
Figure 8.1 Outline model of the genome engineering approaches to modify the ...
Chapter 9
Figure 9.1 Schematic representation of the target areas that are important t...
Chapter 10
Figure 10.1 Pulses (a) Pigeon pea (b) Chickpea. (d) Rajmash and legumes (c) ...
Figure 10.2
Scheme for Agrobacterium‐mediated genetic transformation in pigeo
...
Chapter 11
Figure 11.1 Schematic illustration of generalized lipid biosynthesis/storage...
Chapter 14
Figure 14.1 Schematic picture of the basal plant immunity.
Figure 14.2 CRISPR/Cas‐based genome‐editing tools (GETs) for the development...
Chapter 15
Figure 15.1 Schematic representation for the starch biosynthesis and modific...
Figure 15.2 Schematic representation of various carotenoids and tocopherol b...
Figure 15.3 Biosynthetic pathways involved in tocopherols biosynthesis at ge...
Figure 15.4 schematic representation of the ascorbic acid (Vit‐C) biosynthes...
Chapter 16
Figure 16.1 Schematic of the three nucleases used for genome engineering.
Chapter 19
Figure 19.1 Genes involved in starch metabolism in cereal seed. Abbreviation...
Chapter 20
Figure 20.1 Biosynthesis pathways of different vitamins in plants. (a) Provi...
Figure 20.2 (a) Phytic acid synthetic pathway (Zm MRP4: Transporter of phyti...
Chapter 21
Figure 21.1 A schematic representation of CRISPR/Cas9 system to edit genes c...
Guide
Cover Page
Title Page
Copyright Page
Dedication
List of Contributors
Preface
About the Editor
Acknowledgments
Table of Contents
Begin Reading
Index
WILEY END USER LICENSE AGREEMENT
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Genome Engineering for Crop Improvement
Edited by
Santosh Kumar Upadhyay
Department of Botany
Panjab University
Chandigarh, India
This edition first published 2021© 2021 John Wiley & Sons Ltd
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Library of Congress Cataloging‐in‐Publication Data
Name: Upadhyay, Santosh Kumar, editor. Title: Genome engineering for crop improvement / edited by Santosh Kumar Upadhyay, Department of Botany, Panjab University, Chandigarh, India. Description: First edition. | Hoboken, NJ : Wiley, 2021. | Includes bibliographical references and index. Identifiers: LCCN 2020029680 (print) | LCCN 2020029681 (ebook) | ISBN 9781119672364 (hardback) | ISBN 9781119672388 (adobe pdf) | ISBN 9781119672401 (epub) Subjects: LCSH: Crops–Genetic engineering. | Plant biotechnology. Classification: LCC SB123.57 .G4835 2021 (print) | LCC SB123.57 (ebook) | DDC 631.5/233–dc23 LC record available at https://lccn.loc.gov/2020029680LC ebook record available at https://lccn.loc.gov/2020029681
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List of Contributors
Sipla AggarwalDepartment of BiotechnologyNational Institute of Plant Genome ResearchJawaharlal Nehru University CampusNew Delhi, India
Sibtain AhmadUniversity of Agriculture FaisalabadSub‐Campus DepalpurOkaraPunjab, PakistanAnimal Breeding and GeneticsFaculty of Animal husbandryUniversity of Agriculture FaisalabadPunjab, Pakistan
Habib AliDepartment of Agricultural EngineeringKhawaja Fareed University of Engineering and Information TechnologyRahim Yar KhanPunjab, Pakistan
Kazim AliNational Institute for Genomics and Advanced BiotechnologyNational Agricultural Research CentrePark Road, Islamabad 45500Pakistan
Mohsin AliUniversity of Agriculture FaisalabadSub‐Campus DepalpurOkaraPunjab, Pakistan
Imran AminAgricultural Biotechnology DivisionNational Institute for Biotechnology and Genetic Engineering (NIBGE)Constituent College of Pakistan Institute of Engineering and Applied SciencesFaisalabad, Pakistan
Galal Bakr AnisRice Research and Training Center (RRTC)Rice Research DepartmentField Crops Research InstituteAgricultural Research CenterSakhaKafr El‐Sheikh, Egypt
Naveed AnjumAgricultural Biotechnology DivisionNational Institute for Biotechnology and Genetic Engineering (NIBGE)Constituent College of Pakistan Institute of Engineering and Applied SciencesFaisalabad, PakistanDepartment of BiotechnologyUniversity of SialkotSialkotPunjab, Pakistan
Bilal Ahmad AsadUniversity of Agriculture FaisalabadSub‐Campus DepalpurOkaraPunjab, Pakistan
Muhammad Qasim AslamAgricultural Biotechnology DivisionNational Institute for Biotechnology and Genetic Engineering (NIBGE)Constituent College of Pakistan Institute of Engineering and Applied SciencesFaisalabad, Pakistan
Muhammad Jawad Akbar AwanAgricultural Biotechnology DivisionNational Institute for Biotechnology and Genetic Engineering (NIBGE)Constituent College of Pakistan Institute of Engineering and Applied SciencesFaisalabad, Pakistan
Uzma BadarCell and Systems BiologyUniversity of TorontoTorontoOntario, Canada
Poorwa Kamal BadolaCSIR‐National Botanical Research InstituteCouncil of Scientific and Industrial Research (CSIR‐NBRI)LucknowUttar Pradesh, IndiaAcademy of Scientific and Innovative Research (AcSIR)Ghaziabad,Uttar Pradesh, India
Deepak Singh BagriLaboratory of Plant Molecular BiologyDepartment of BiotechnologyDr Harisingh Gour Central UniversitySagarMadhya Pradesh, IndiaDepartment of BotanyGovernment Degree CollegeAmanganj, PannaMadhya Pradesh, India
Kaushal Kumar BhatiLouvain Institute of Biomolecular ScienceCatholic University of LouvainLouvain‐la‐Neuve, Belgium
Siddhant ChaturvediNational Agri‐Food Biotechnology Institute (NABI)Department of BiotechnologyMinistry of Science and Technology (Government of India)MohaliPunjab, IndiaDepartment of BiotechnologyPanjab UniversityChandigarh, India
Roni ChaudharyNational Agri‐Food Biotechnology Institute (NABI)Department of BiotechnologyMinistry of Science and Technology (Government of India)MohaliPunjab, India
Sameer DixitDepartment of BiologyUniversity of Western OntarioLondonOntario, Canada
Conrado Dueñas, Jr.Trait and Genome Engineering ClusterStrategic Innovation PlatformInternational Rice Research InstituteManila, Philippines
Sajid FiazDepartment of Plant Breeding and GeneticsThe University of Haripur 22620HaripurKhyber Pakhtunkhwa, Pakistan
Mahmoud Mohamed GaballahRice Research and Training Center (RRTC)Rice Research DepartmentField Crops Research InstituteAgricultural Research CenterSakhaKafr El‐Sheikh, Egypt
Vikrant GautamICAR‐NBPGRNew Delhi, 110012, India
Ghyda Murad HashimCell and Systems BiologyUniversity of TorontoTorontoOntario, Canada
Kathleen HefferonCell and Systems BiologyUniversity of TorontoTorontoOntario, Canada
Boštjan JenčičJozef Stefan InstituteLjubljana, Slovenia
Ajinder KaurSchool of Agricultural BiotechnologyPunjab Agricultural UniversityLudhianaPunjab, India
Gurminder KaurInstitute of Bioscience and TechnologyShri Ramswaroop Memorial UniversityBarabankiUttar Pradesh, India
Sher Aslam KhanDepartment of Plant Breeding and GeneticsThe University of Haripur 22620HaripurKhyber Pakhtunkhwa, Pakistan
Muhammad Zuhaib KhanAgricultural Biotechnology DivisionNational Institute for Biotechnology and Genetic Engineering (NIBGE)Constituent College of Pakistan Institute of Engineering and Applied SciencesFaisalabad, Pakistan
Ivan KreftNutrition InstituteLjubljana, Slovenia
Ajay KumarMolecular Biology and BiotechnologyCouncil of Scientific and Industrial ResearchNational Botanical Research Institute (CSIR‐NBRI)LucknowUttar Pradesh, India
Anil KumarDepartment of BiotechnologyNational Agri‐Food Biotechnology InstituteMohaliPunjab, India
Pankaj KumarICAR‐CPRIShimla, 171001, India
Peter KumpJozef Stefan InstituteLjubljana, Slovenia
Nirbhay Kumar KushwahaDepartment of Plant BiologySwedish University of Agricultural SciencesUppsala, Sweden
Shahid MansoorAgricultural Biotechnology DivisionNational Institute for Biotechnology and Genetic Engineering (NIBGE)Constituent College of Pakistan Institute of Engineering and Applied SciencesFaisalabad, Pakistan
Varsha MeenaDepartment of BiotechnologyNational Agri‐Food Biotechnology InstituteMohaliPunjab, India
Gela Myan B. MotaTrait and Genome Engineering ClusterStrategic Innovation PlatformInternational Rice Research InstituteManila, Philippines
Mehmood Ali NoorInstitute of Crop SciencesChinese Academy of Agricultural SciencesKey Laboratory of Crop Physiology and EcologyMinistry of AgricultureBeijing 100081, China
Azka NoureenAgricultural Biotechnology DivisionNational Institute for Biotechnology and Genetic Engineering (NIBGE)Constituent College of Pakistan Institute of Engineering and Applied SciencesFaisalabad, PakistanUniversity Institute of Biochemistry and Biotechnology (UIBB)Pir Mehar Ali Shah Arid Agriculture UniversityRawalpindi, Pakistan
Norman OlivaTrait and Genome Engineering ClusterStrategic Innovation PlatformInternational Rice Research InstituteManila, Philippines
Ajay K. PandeyDepartment of BiotechnologyNational Agri‐Food Biotechnology InstituteMohaliPunjab, India
Ashish Kumar PathakDepartment of BotanyCharles UniversityPraha 2Czech Republic
Primož PeliconJozef Stefan InstituteLjubljana, Slovenia
Paula PongracBiotechnical FacultyUniversity of LjubljanaLjubljana, SloveniaJozef Stefan InstituteLjubljana, Slovenia
Pravin PrakashMolecular Biology and BiotechnologyCouncil of Scientific and Industrial ResearchNational Botanical Research Institute (CSIR‐NBRI)LucknowUttar Pradesh, India
Krishan Mohan RaiDepartment of Plant and Microbial BiologyUniversity of MinnesotaSaint PaulMN, USA
Hasthi RamDepartment of Agri‐BiotechnologyNational Agri‐Food Biotechnology InstituteMohaliPunjab, India
Gurwinder Singh RanaAFCNew Delhi, India
Marjana RegvarBiotechnical FacultyUniversity of LjubljanaLjubljana, Slovenia
Riyazuddin RiyazuddinDepartment of Plant Biology and Doctoral School in BiologyFaculty of Science and InformaticsUniversity of SzegedSzeged, Hungary
Joy K. RoyNational Agri‐Food Biotechnology Institute (NABI)MohaliPunjab, India
Shweta RoyDepartment of Cell and Molecular BiologyUppsala UniversityUppsala, Sweden
Suchismita RoySkaggs School of Pharmacy and Pharmaceutical SciencesUniversity of CaliforniaSan DiegoCA, USA
Ashish SharmaCSIR‐National Botanical Research InstituteCouncil of Scientific and Industrial Research (CSIR‐NBRI)LucknowUttar Pradesh, IndiaAcademy of Scientific and Innovative Research (AcSIR)GhaziabadUttar Pradesh, India
Chhaya SharmaSchool of Agricultural BiotechnologyPunjab Agricultural UniversityLudhianaPunjab, India
Erum ShoebCell and Systems BiologyUniversity of TorontoTorontoOntario, Canada
Akanchha ShuklaDepartment of BiologyUniversity of Western OntarioLondonOntario, Canada
Anuradha SinghFrench Associates Institute for Agriculture and Biotechnology of Drylands (FAAB)Ben‐Gurion University of the NegevSede Boqer CampusSede Boqer, Israel
Harpal SinghDepartment of NematologyUniversity of CaliforniaRiversideCA, USA
Sudhir P. SinghCenter of Innovative and Applied Bioprocessing (DBT‐CIAB)MohaliPunjab, India
Vinayak SinghPlant Molecular Biology and Genetic Engineering DivisionCSIR‐National Botanical Research InstituteLucknowUttar Pradesh, India
Inez H. Slamet‐LoedinTrait and Genome Engineering ClusterStrategic Innovation PlatformInternational Rice Research InstituteManila, Philippines
Praveen SoniDepartment of BotanyUniversity of RajasthanJaipurRajasthan, India
SushmitaMolecular Biology and Biotechnology Council of Scientific and Industrial ResearchNational Botanical Research Institute (CSIR‐NBRI)LucknowUttar Pradesh, IndiaAcademy of Scientific and Innovative Research (AcSIR)GhaziabadUttar Pradesh, India
Rakesh SrivastavaMolecular Biology and BiotechnologyCouncil of Scientific and Industrial ResearchNational Botanical Research Institute (CSIR‐NBRI)LucknowUttar Pradesh, India
Siddharth TiwariNational Agri‐Food Biotechnology Institute (NABI)Department of BiotechnologyMinistry of Science and Technology (Government of India)MohaliPunjab, India
Prabodh Kumar TrivediCSIR‐National Botanical Research InstituteCouncil of Scientific and Industrial Research (CSIR‐NBRI)LucknowUttar Pradesh, IndiaAcademy of Scientific and Innovative Research (AcSIR)GhaziabadUttar Pradesh, IndiaCSIR‐Central Institute of Medicinal and Aromatic Plants (CSIR‐CIMAP)LucknowUttar Pradesh, India
Johannes Tuen van ElterenNational Institute of ChemistryLjubljana, Slovenia
Santosh Kumar UpadhyayDepartment of BotanyPanjab UniversityChandigarh, India
Chandrama Prakash UpadhyayaLaboratory of Plant Molecular BiologyDepartment of BiotechnologyDr Harisingh Gour Central UniversitySagarMadhya Pradesh, India
Chirag UppalSchool of Agricultural BiotechnologyPunjab Agricultural UniversityLudhianaPunjab, India
Primož VavpetičJozef Stefan InstituteLjubljana, Slovenia
Srividhya VenkataramanCell and Systems BiologyUniversity of TorontoTorontoOntario, Canada
Praveen Chandra VermaMolecular Biology and BiotechnologyCouncil of Scientific and Industrial ResearchNational Botanical Research Institute (CSIR‐NBRI)LucknowUttar Pradesh, IndiaAcademy of Scientific and Innovative Research (AcSIR)GhaziabadUttar Pradesh, India
Katarina Vogel‐MikušBiotechnical FacultyUniversity of LjubljanaLjubljana, SloveniaJozef Stefan InstituteLjubljana, Slovenia
Tian WangKey Laboratory of Food Nutrition and Safety of Shandong Normal UniversityCollege of Life ScienceShandong Normal UniversityJinan, China
Santosh WatpadeICAR‐IARI Regional Station (CHC)Shimla, 171004, India
Amit YadavZuckerberg Institute for Water Research (ZIWR)Ben‐Gurion University of the NegevIsrael
Afifa YounasDepartment of BotanyLahore College for Women UniversityLahore, Pakistan
Kashaf ZafarAgricultural Biotechnology DivisionNational Institute for Biotechnology and Genetic Engineering (NIBGE)Constituent College of Pakistan Institute of Engineering and Applied SciencesFaisalabad, PakistanDepartment of BiotechnologyBalochistan University of Information TechnologyEngineering and Management Sciences (BUITEMS)Quetta, Pakistan
Hongyan ZhangKey Laboratory of Food Nutrition and Safety of Shandong Normal UniversityCollege of Life ScienceShandong Normal UniversityJinan, China
Hongliang ZhuCollege of Food Science and Nutritional EngineeringChina Agricultural UniversityBeijing, China
Preface
About half of the world population is suffering from nutritional deficiency, which is due to either improper diet uptake, or non‐availability and unaffordability of balanced nutritive food. Therefore, nutritional enrichment of common food items has been of great interest to fulfill the demands of nutritional deficiency. For instance, development of carotenoids‐rich banana and tomato, and iron‐rich rice and wheat has been proposed to solve major issues of vitamin A deficiency and anemia. Further, the distribution of important mineral elements in grains is also a major challenge for their bioavailability. For example, the majority of elements like iron are enriched in the aleurone layer of wheat grain and, therefore, it is not in flour for consumers. These are the major challenges which can be addressed by utilizing genome‐engineering mechanisms.
Genome engineering has been evolved as an efficient method for targeted mutation and modification in the genome of various organisms. During the last decade, genome engineering has been utilized for numerous trait improvement programs in several important crop plants. Genome‐engineering tools like ZFNs, TALENs, and CRISPR‐Cas system have been used for in‐vivo gene editing, as well as much biological pathway engineering for the nutritional improvement of various agricultural and horticultural crop plants. They have been of greater interest due to their precise editing mechanisms and very high specificity. Over the past few years, they have been utilized in numerous agricultural and horticultural crop plants including rice, wheat, legumes, tomato, potato, banana, grapes, etc. They are found to be highly specific and precise in their function. They have also been used for the engineering of numerous metabolic pathways to develop nutrient‐rich produce. They are used to decrease the anti‐nutrients in crop plants to improve bioavailability of minerals and vitamins, development of zero calorie/ sugar‐free potato, indigestible starch‐rich grains, allergenic gluten‐free wheat and various other processes. Further, they can also be used for the mobilization of minerals from unavailable locations to the bioavailable location for grains.
Researchers have been working hard to increase the nutritional value of various important crop species by increasing the nutraceuticals and minerals such as carotenoids and flavonoid‐rich cereals, tomato and fruit crops, iron‐enriched rice, maize, and wheat, etc. On the other hand, people are also working to decrease the anti‐nutrients such as phytic acids and other chelating elements in crops to increase the bioavailability of the available micronutrients. Further, development of sugar‐free potato and gluten‐free wheat, resistant starch rich rice and wheat is in great demand by people suffering from various diseases or as preventive measures.
The development of new strategies to cope up with the various challenges has always been a top priority over recent years. Agronomical practices, chemical applications, biofortifications and transgenic expression of protein‐coding genes have been explored to alleviate these problems to some extent. However, new and specific technology is always in demand. Genome engineering can be the best alternative for nutritional improvement of agricultural and horticultural crops. Further, it will be highly specific to a particular gene and plant, therefore, it has minimal bio‐safety risk. Taking this, together with progress made in crop improvement using genome engineering methods, this book has covered all the recent knowledge and development in the area related to the nutritional value, mineral and nutrient localization, and engineering for stress resistance as well as nutritional improvement of various crop plants. None of the currently available books have covered this topic at such a broad scale. Further, this area is of tremendous importance, not only in the present scenario but also for future decades.
Much effort has been made by all those involved to provide a well‐informed and knowledgeable book. I hope that this stimulates discussion and inspires future research and discussion on such an important topic.
About the Editor
Dr. Santosh Kumar Upadhyay is currently working as an Assistant Professor at 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 characterization of various insect toxic proteins from plant biodiversity, and defense 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 55 research papers in leading journals of international repute. Further, there are more than five national and international patents,17 book chapters and four books to his credit.
In recognition of his strong research record he has been awarded 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 a number of peer‐reviewed international journals.
Acknowledgments
I am thankful to the Panjab University, Chandigarh, India for providing the facility to complete this book. I am grateful to all the esteemed authors for their exceptional contributions and reviewers for their critical evaluation and suggestions to improve the quality of the text.
I would like to thank Miss Rebecca Ralf (Commissioning Editor), Miss Kerry Powell (Managing Editor) and Nora Naughton (Copy Editor) from John Wiley & Sons, Ltd for their excellent management of this project, and anonymous reviewers for their positive recommendations about the book.
I also appreciate the support of my research students whose discussion and comments were very useful in shaping this book. I thank Dr. Prabodh K. Trivedi, Dr. Praveen C. Verma, Dr. Krishan Mohan Rai, Dr. Sameer Dixit, Dr. Sudhir P. Singh and Dr. Prashant Misra for direct or indirect help with this project. I wish to express my gratitude to my parents and my beloved wife for her endless support, patience, and inspiration. I thank my daughter, who missed me during this project. I would like to warmly thank the faculties and staff of the department and university for providing a great working environment. Last, but not least, my sincere thanks to Lord Krishna for endowing me to live with joy and success in the form of this book.
1An Overview of Genome‐Engineering Methods
Sushmita1,3#, Gurminder Kaur2#, Santosh Kumar Upadhyay4, and Praveen Chandra Verma1,3
1 Molecular Biology and Biotechnology, Council of Scientific and Industrial Research, National Botanical Research Institute (CSIR‐NBRI), Lucknow, Uttar Pradesh, India
2 Institute of Bioscience and Technology, Shri Ramswaroop Memorial University, Barabanki, Uttar Pradesh, India
3 Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India
4 Department of Botany, Panjab University, Chandigarh, India
CHAPTER MENU
1.1 Introduction
1.2 ZFNs
1.3 TALENs
1.4 CRISPR‐Cas System
1.5 CRISPR‐Cpf1
1.6 Conclusions
1.1 Introduction
Agricultural practices, combined with advanced plant breeding and modern technologies, provided food security to millions of people. However, increasing global population demands significant increase in world food production (Parry and Hawkesford 2012). Nevertheless, climate change, depletion of natural resources, increased pollution, and political instabilities are a threat to the food and nutritional security for future generations in the twenty‐first century. Unfortunately, the amount of remaining arable land is limited, necessitating an increase in food production on currently‐used land. Compounding these challenges are the predicted crop losses due to extreme temperatures, pest attacks, and pathogen outbreaks. A powerful approach that may help overcome these challenges is to modify DNA sequences within plant chromosomes for trait improvement (Sedeek et al. 2019). Further, plants can be engineered to have increased tolerance to environmental stresses and pathogens (Han and Kim 2019; Ji et al. 2015; Makarova et al. 2011). In addition to improving the genetic makeup of the crops to meet increasing food demands and control crop loss, genome engineering can also be used to produce valuable plants or products for non‐agricultural purposes (Chen et al. 2019). For example, there is great potential for plants to be used as bioreactors for pharmaceutical proteins. Genetic engineering for increasing the secondary metabolite production in plants would be another use of this technology which would help the perfumery, cosmetic and medical industries, as the secondary metabolites produced from plants have a number of uses (El‐Mounadi et al. 2020). However, to realize the potential benefits of these applications, we must generate effective tools and approaches for editing plant DNA (Miroshnichenko et al. 2019; Tang and Tang 2017).
Introduction of programmed sequence‐specific nucleases (SSNs) and their applications in precise genome editing unfurled a new dimension in genome engineering (Kim and Kim 2014; Voytas 2013). Over the last few decades, researchers reported a few important SSNs, which could be easily engineered and reprogrammed to create double‐stranded breaks (DSBs) at the desired location inside the chromosome. There are three major genome engineering methods, ZFNs, TALENs, and CRISPR‐Cas system (Figure 1.1A) (Jang and Joung 2019; Mahfouz et al. 2014), that have been utilized so far for a variety of purposes, and these have been discussed in detail in the coming sections. Further, we have also described the recently added CRISPR‐Cpf1 system of genome engineering.
1.2 ZFNs
Zinc‐finger nucleases are chimeric fusion proteins consisting of a DNA‐binding domain and a DNA‐cleavage domain. The DNA‐binding domain is composed of a set of Cys2His2 zinc fingers (usually three to six). Each zinc finger primarily contacts 3 bp of DNA and a set of three to six fingers recognize 9–18 bp, respectively. The DNA‐cleavage domain is derived from the cleavage domain of the FokI restriction enzyme. FokI activity requires dimerization; therefore, to site‐specifically cleave DNA, two zinc‐finger nucleases are designed in a tail‐to‐tail orientation (Kim et al. 1996).
Zinc‐finger nucleases can be remodified to recognize different DNA sequences. However, one limitation with redirecting targeting is that it depends on the context of the host. For example, a zinc finger that recognizes GGG may not recognize this sequence when fused to other zinc fingers. As a result, the modular assembly of zinc fingers has had limited success (Ramirez et al. 2008). One of the more successful methods for redirecting targeting involves generating a library of three zinc‐finger variants from a pre‐selected pool of zinc‐finger monomers (Maeder et al. 2008). The resulting library of zinc‐finger arrays can then be interrogated using a bacterial two‐ hybrid screen, where binding of the zinc‐finger array to a pre‐determined sequence results in the expression of a selectable marker gene. This method has generated highly‐active zinc‐finger nuclease (ZFN) pairs for sites within animal and plant genomes. Since the development of ZFN technology, several studies have been done to engineer specific zinc‐finger modules for each of the 64 codon triplets (Bae et al. 2003; Dreier et al. 2001; Pabo et al. 2001). Until now, several ZFNs have been designed and used in numerous species. The developments for more specific and efficient technologies also gave rise to fewer off‐target effects. There are three most commonly available tools for engineering the ZF domains: context‐dependent Assembly (CoDA), Oligomerized Pool Engineering (OPEN), and Modular Assembly (MA). Several softwares are available for designing engineered ZFs (ZiFiT), containing the database of ZFs (ZiFDB) and identification of potential targets for ZFNs in several model organisms (ZFNGenome) (Kim et al. 2009; Mandell and Barbas 2006; Sander et al. 2007).
Figure 1.1 (A) Diagrammatic representation of (a) Zinc‐finger nucleases (ZFNs), (b) Transcription activator‐like effector nucleases (TALENs) and (c) Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 mediates DSBs formation. (B) dCas9‐based targeted genome regulation by (a) activation of gene expression, (b) repression of gene expression and (c) DNA methylation.
Source: Adapted from Mahfouz et al. (2014) © 2014. Reproduced with the permission of John Wiley & Sons.
Zinc‐finger nucleases have been widely used for plant genome engineering. Plant species that have been modified using zinc‐finger nucleases include, Arabidopsis, maize, soybean, tobacco, etc. (Ainley et al. 2013; Cai et al. 2009; Curtin et al. 2011; Lloyd et al. 2005; Marton et al. 2010; Osakabe et al. 2010; Shukla et al. 2009; Townsend et al. 2009; Wright et al. 2005; Zhang et al. 2010). With their relatively small size (~300 amino acids per zinc‐finger nuclease monomer), and the further advancements in methods for redirecting targeting (Sander et al. 2011a), zinc‐finger nucleases should continue to be an effective technology for editing plant.
1.3 TALENs
Transcription activator‐like effectors nucleases (TALENs) are fusion proteins, consisting of a DNA‐binding domain and a DNA‐cleavage domain. Whereas the DNA‐cleavage domain is the same between zinc‐finger nucleases and TALENs (the catalytic portion of FokI), the DNA binding domains are different. The TALEN DNA‐binding domain is derived from TALE proteins found in the plant pathogen Xanthomonas. These proteins are composed of direct repeats of 33–35 amino acids, and nearly all arrays found in Xanthomonas contain a final, half repeat, consisting of the first 20 amino acids from the normal repeat. Two amino acids within these repeats (positions 12 and 13) are responsible for recognizing a single nucleotide base (these amino acids are referred to as repeat‐variable diresidues; RVDs). When the TALE effector code was broken (i.e. the relationship between the RVD and corresponding target base) (Boch et al. 2009; Moscou and Bogdanove 2009), the ability to redirect targeting, and their use as a genome engineering tool was realized (Christian et al. 2010; Li et al. 2011; Mahfouz et al. 2011). To make TALENs useful in gene targeting, the basic requirement is the modular assembly of repeat sequences containing the appropriate RVD corresponding to the nucleotide target. The most widely used RVDs and their nucleotide targets are HD, cytosine; NG, thymine; NI, adenine; NN, guanine, and adenine; NS, adenine, cytosine, and guanine; N*, all four nucleotides. This one‐to‐one correspondence of a single RVD to a single DNA base has eliminated construction challenges due to context‐dependency seen with zinc‐fingers and meganucleases. However, one limitation when using TALENs is that the target sequence must have thymine at the −1 position (Boch et al. 2009). Further, the long and repetitive nature of TALENs puts a strain on delivery methods where cargo capacity or stability is a limitation.
The assembly of engineered TALE repeat arrays can be challenging from nearly similar repeat sequences; therefore, a number of platforms have been designed to facilitate this assembly. These can be classified into three categories: standard restriction enzyme and ligation‐based cloning methods (Huang et al. 2011; Sander et al. 2011); Golden Gate assembly methods (Briggs et al. 2012; Cermak et al. 2011; Engler et al. 2008) and solid‐phase assembly methods (Heigwer et al. 2013; Wang et al. 2012).
Several online tools are available for designing TALE effectors to target specific gene sequence and off‐target analysis. For example‐ E‐TALEN (Lin et al. 2014), Scoring Algorithm for Predicting TALEN Activity (SAPTA) (Neff et al. 2013), Mojo‐hand (Coordinators 2013), TAL Effector‐Nucleotide Targeter (TALE‐NT), etc. TALE‐NT is a collection of versatile web‐based tools like‐TALEN Targeter, TAL Effector Targeter, Target finder, Paired Target Finder, and TALEN Targeter Off‐Target Counter (Christian et al. 2013).
Several studies have demonstrated the usefulness of TALENs in different plant species, including Arabidopsis (Zhang et al. 2013), tobacco (Wang et al. 2012; Wendt et al. 2013), barley (Li et al. 2012), rice (Shan et al. 2013a) and Brachypodium (Reyon et al. 2011). Taken together, the modular nature of TALE repeats, along with efficient methods for assembling repetitive DNA sequences (Garneau et al. 2010; Wang et al. 2012), have enabled TALENs to become one of the premier tools for plant genome engineering.
1.4 CRISPR‐Cas System
The most recent addition to the SSN family is the CRISPR/Cas system that is normally present within bacteria and archaea, and provides an adaptive immunity against invading plasmids or viruses. CRISPR/Cas system functions to destroy invading nucleic acids by introducing targeted DNA breaks (Garneau et al. 2010).
There are three major types of CRISPR/Cas system: Types I – III (Makarova et al. 2011). The Type II system was adopted for genome engineering a few years ago (Cong et al. 2013; Zhang et al. 2011). In this system, two components enable targeted DNA cleavage: a Cas9 protein and an RNA complex consisting of a CRISPR RNA (crRNA; contains 20 nucleotides of RNA that are homologous to the target site) and a trans‐activating CRISPR RNA (tracrRNA). Cas9 protein causes double‐stranded DNA break at the sequences homologous to the crRNA sequence and upstream of a protospacer‐adjacent motif (PAM) (PAM; e.g. NGG for Streptococcus pyogenes Cas9). For genome engineering purposes, the complexity of the system was reduced by fusing the crRNA and tracrRNA to generate a single‐guide RNA (gRNA). Moreover, off‐target cleavage is a limitation of the CRISPR/Cas system (Cho et al. 2014; Fu et al. 2013).
The target site recognition in CRISPR‐Cas system is facilitated through RNA: DNA interaction (as opposed to a protein: DNA interaction used by meganucleases, zinc‐finger nucleases, and TALENs). Redirecting of Cas9 targets involves modification of 20 nucleotides within the crRNA or gRNA. These 20 nucleotides are used to direct Cas9 binding and cleavage, the system has been shown to tolerate mismatches, with a higher tolerance closer to the 5′ end of the target sequence (Fu et al. 2013). Results from recent studies suggest the first 8–12 nucleotides, in addition to the PAM sequence, are most critical for target site recognition (Sternberg et al. 2014; Wu et al. 2014). To reduce off‐targeting, several methods have been developed, including dual‐nicking of DNA (Mali et al. 2013; Ran et al. 2013), a fusion of catalytically‐dead Cas9 to FokI (Guilinger et al. 2014; Tsai et al. 2014) and shortening of gRNA sequence (Fu et al. 2014). Several softwares and programs have been developed in recent years for the identification of target sequences in the genome and the design of specific gRNA, which are listed in Table 1.1.
The Cas9 is an endonuclease consisting of two discrete nuclease domains: the HNH domain which is responsible for the cleavage of the DNA strand complementary to the guide RNA sequence (target strand) and the RuvC‐like domain that cleaves the DNA strand opposite the complementary strand (Chen et al. 2014; Gasiunas et al. 2012; Jinek et al. 2012). The double‐strand breaks (DSBs) are repaired through Non‐Homologous End Joining or Homology directed Repair in the presence of a template. Mutations in both nuclease domains (Asp10 → Ala, His840 → Ala) result in an RNA‐guided DNA‐binding protein without endonuclease activity that is called dCas9 (Jinek et al. 2012; Qi et al. 2013). This dCas9 is then supplemented with effector domains for the execution of distinct functions in the genome (Figure 1.1B). Fusion of a transcriptional activator VP64 with dCas9 exhibited targeted gene activation by altering the flowering time regulation in Arabidopsis (Xu et al. 2019). Similarly, dCas9‐VP64 regulated transcriptional activation of endogenous genes and dCas9‐SRDX‐regulated transcriptional repression in Arabidopsis and tobacco (Lowder et al. 2015, 2018). These regulatory domains can also perform multiplex gene targeting using multiple sgRNAs. As a new dimension to CRISPR/Cas technology, there are the base editing enzymes, for example, cytidine deaminase fused with the dCas9, which can replace specific bases in the targeted region of DNA and RNA.
Table 1.1 List of available softwares and programs for designing gRNA.
Software
Features
Link
References
Cas‐OFFinder
Identifies gRNA target sequence from an input sequence and checks off‐target binding site
http://www.rgenome.net/cas‐offinder
Bae et al. (
2014
)
Cas‐Designer
Identifies gRNA target sequence from an input with low probability of off‐target effect
http://www.rgenome.net/cas‐designer/
Park et al. (
2015
)
Cas9 Design
Designs gRNA
http://cas9.cbi.pku.edu.cn/database.jsp
Ma et al. (
2013
)
E‐CRISP
Designs gRNA
http://www.e‐crisp.org/E‐CRISP/designcrispr.html
Heigwer et al. (
2014
)
CRISPR‐P
Designs gRNA
http://cbi.hzau.edu.cn/crispr2/
Lei et al. (
2014
)
CHOP
Identifies target site
https://chopchop.rc.fas.harvard.edu/
Montague et al. (
2014
)
CRISPR‐PLANT
Designs gRNA
http://www.genome.arizona.edu/crispr/
Xie et al. (
2014
)
CCTop
Identifies candidate gRNA target sites with reduced off‐target quality
http://crispr.cos.uni‐heidelberg.de/
Stemmer et al. (
2015
)
CRISPRdirect
Identifies candidate gRNA target sequences
http://crispr.dbcls.jp/
Naito et al. (
2015
)
COSMID
Identifies target sites
https://crispr.bme.gatech.edu
Cradick et al. (
2014
)
CRISPR Finder
Identifies CRISPR
http://crispr.u‐psud.fr/Server
Grissa et al. (
2007
)
CrisprGE
Identifies target sites
http://crdd.osdd.net/servers/crisprge
Kaur et al. (
2015
)
CRISPR Multitargeter
Identifies target sites
http://www.multicrispr.net
Prykhozhij et al. (
2015
)
CRISPRseek
Identifies target specific guide RNAs
http://www.bioconductor.org/packages/release/bioc/html/CRISPRseek.html
Zhu et al. (
2014
)
flyCRISPR
Identifies target sites and evaluate its specificity
http://flycrispr.molbio.wisc.edu
Gratz et al. (
2014
)
GT‐SCAN
Identifies target sites and ranking them with their potential off target sites
http://flycrispr.molbio.wisc.edu
O'Brien and Bailey (
2014
)
sgRNAcas9
Identifies target sites with their potential off target sites
www.biootools.com
Xie et al. (
2014
)
SSFinder
Identifies target sites
https://code.google.com/p/ssfinder
Upadhyay and Sharma (
2014
)
ZiFiT
Identifies target sites
http://zifit.partners.org/ZiFiT
Mandell and Barbas (
2006
)
sgRNA Designer
Guide RNA design based on efficiency score
http://broadinstitute.org/rnai/public/analysis‐tools/sgrna‐design
Doench et al. (
2014
)
Several plant species have been edited using CRISPR/Cas system, including rice, wheat (Shan et al. 2013b; Upadhyay et al. 2013), sorghum (Jiang et al. 2013), tobacco (Li et al. 2013), Arabidopsis (Fauser et al. 2014; Feng et al. 2014; Li et al. 2013), Brassica napus (Kang et al. 2018), watermelon (Tian et al. 2018), etc. (Table 1.2). Moreover, dCas9 can be fused with various epigenetic regulatory factors which can modulate DNA acetylation/methylation, post‐ translational histone modification, ubiquitination and protein sumoylation and phosphorylation to carry out epigenetic modifications (Shrestha et al. 2018; Yamamuro et al. 2016). This has been more recently explored in Arabidopsis for demethylation (Gallego‐Bartolomé et al. 2018).
Table 1.2 List of examples of genes edited by CRISPR Cas system in various plant species.
Plant system
Gene
Description of Experiment
References
Arabidopsis thaliana
GSS21/2
Host adaptation against
P. xylostella
Chen et al. (
2020
)
Rice
EPFL9
a positive regulator of stomatal development
Yin et al. (
2017
)
OsDEP1 OsROC5 OsPDS
Carotenoid biosynthesis, leaf morphology
Tang and Tang (
2017
)
OsDL
and
OsALS
loss of midrib in the leaf blade
Endo et al. (
2016
)
OsPDS, OsBEL
Herbicide resistant and Nutritional improvement
Xu et al. (
2017
)
OsRLK, OsBEL
receptor‐like kinases
Wang et al. (
2017
)
OsPDS OsDL
Herbicide resistant and loss of midrib in the leaf blade
Tang et al. (
2019
)
OsERF922
Enhanced resistance to blast disease
Wang et al. (
2016
)
GW2, GW5, and TGW6
Improvement of grain weight
Xu et al. (
2016
)
ALS
Enhanced herbicide resistance
Sun et al. (
2016
)
SBEIIb and SBEI
Generation of high amylose rice
Sun et al. (
2017
)
Hd 2, Hd 4, and Hd 5
Early maturity of rice varieties
Li et al. (
2017
)
OsMATL
Induction of haploid plants
Yao et al. (
2018
)
ALS
Herbicide resistance
Butt et al. (
2017
)
EPSPS
Herbicide resistance
Li et al. (
2016
)
ALS
Herbicide resistance
Endo et al. (
2016
)
Gn1a, GS3, DEP1
Enhanced yield, dense erect panicles
Li et al. (
2016
)
LAZY1
Tiller‐spreading
Miao et al. (
2013
)
OsSWEET13
Bacterial blight resistance
Zhou et al. (
2015
)
OsDEP1 OsROC5
Herbicide resistant
Yao et al. (
2018
)
Soybean
FAD2‐1A, FAD2‐1B
Biosynthesis of lipids
Kim et al. (
2017
)
ALS
Herbicide resistance
Li et al. (
2015
)
GmPDS11&18
Carotenoid Biosynthesis
Du et al. (
2016
)
Tobacco
FAD2‐1A, FAD2‐1B
Lipid biosynthesis
Kim et al. (
2017
)
NtPDS and NtPDR6
etiolated leaves for the psd mutant and more branches for the pdr6 mutant
Gao et al. (
2015
)
Cotton
Cloroplastos alterados (GhCLA)
Photosynthesis
Li et al. (
2019
)
CABs, replication associated protein (Rep) and non‐coding intergenic regions (IR), a‐Satellite Rep and b‐Sat IR.
CLCuD associated Begomoviruses (CABs) and Helper begomoviruses a and b satellites.
Iqbal et al. (
2016
), Uniyal et al. (
2019
)
Ashbya gossypii
HIS3, ADE2, TRP1, LEU2 and URA3
auxotrophic markers
Jiménez et al. (
2020
)
Maize
Maize glossy2 gene
Cuticular wax deposition
Lee et al. (
2019
)
ARGOS8
Novel variants of ARGOS8 for drought‐tolerance
Shi et al. (
2017
)
ALS
Herbicide resistance
Svitashev et al. (
2015
)
ZmIPK
Reduction of anti‐nutritional compound phytic acid
Liang et al. (
2014
)
TMS5
Thermosensitive male‐sterile
Li et al. (
2017
)
Wheat
MLO
Resistance to powdery mildew
Wang et al. (
2014
)
GW2
Enhanced yield
Zhang et al. (
2018
)
EDR1
Powdery mildew resistance
Zhang et al. (
2017
)
Barley
HvPM19
Positive regulation of grain dormancy
Lawrenson et al. (
2015
)
HvCKX1/3
Cytokinin metabolism and root morphology
Gasparis et al. (
2019
)
Tomato
SlMlo1
Resistant to powdery mildew
Nekrasov et al. (
2017
)
SlWUS
Increased fruit size
Rodríguez‐Leal et al. (
2017
)
SlAGL6
Facultative parthenocarpy
Klap et al. (
2017
)
SP5G
Day neutrality and early flowering
Soyk et al. (
2016
)
SP, SP5G, CLV3, WUS, GGP1
Tomato domestication
Li et al. (
2018
)
SIAN2
Anthocyanin biosynthesis
Zhi et al. (
2020
)
SlJAZ2
Bacterial speck resistance
Ortigosa et al. (
2019
)
Potato
VInv
Reduction of sugar accumulation
Clasen et al. (
2016
)
ALS
Herbicide resistance
Butler et al. (
2016
)
Wx1
High amylopectin content
Andersson et al. (
2017
)
StPPO
Reduced Enzymatic Browning in Tubers
González et al. (
2020
)
Cassava
MePDS
Carotenoid Biosynthesis
Odipio et al. (
2017
)
Citrus paradise
CsLOB1
Citrus canker resistance
Jia et al. (
2017
)
Citrus sinensis
CsLOB1 promoter
Citrus canker resistance
Peng et al. (
2017
)
Citrus paradise
CsLOB1 promoter
Alleviated citrus canker
Jia et al. (
2016
)
Manihot esculenta
EPSPS
Herbicide resistance
Hummel et al. (
2018
)
Cucumas sativus
eIF4E
Virus resistance
Chandrasekaran et al. (
2016
)
Camelina sativa
FAD2
Low polyunsaturated fatty acids
Jiang et al. (
2017
)
Linumusitatissimum
EPSPS
Herbicide resistance
Sauer et al. (
2016
)
Carrot
DcPDS and DcMYB113‐like genes
depigmented carrot plants
Xu et al. (
2019
)
Strawberry
PDS
Carotenoid Biosynthesis
Wilson et al. (
2019
)
Grapes
VvPDS
Carotenoid Biosynthesis
Nakajima et al. (
2017
)
CsLOB1
Increased resistance to citrus canker
Jia et al. (
2017
)
MLO‐1
Negative regulator of resistance to Powdery mildew
Malnoy et al. (
2016
)
Pear
MdPDS and TFL1.1
Carotenoid Biosynthesis & Floral repression
Charrier et al. (
2019
)
Apple
PDS
Carotenoid Biosynthesis
Charrier et al. (
2019
, Nishitani et al. (2016)
DIMP‐1/2/3
Negative regulator of resistance to blight disease
Malnoy et al. (
2016
)
Banana
eBSV
Resistance against Banana streak virus (eBSV)
Tripathi et al. (
2019
)
Chicory
phytoene desaturase gene (CiPDS)
Fruit ripening
Jansing et al. (
2019
)
Rubber
HbFT HbTFL1
Delayed‐flowering and early‐flowering
Fan et al. (
2020
)
Brassica napus
L.)
BnLPAT2 and BnLPAT5
size of the oil bodies increased
Zhang et al. (
2019
)
Populus
phytoene desaturase gene 8
Carotenoid Biosynthesis
Fan et al. (
2015
)
Flax
EPSPS
Aromatic amino acid biosynthesis
Sauer et al. (
2016
)
1.5 CRISPR‐Cpf1
Introduction of CRISPR‐Cpf1 also known as CRISPR‐Cas12a further diversified the genome engineering methods (Figure 1.2). Cpf1 is an endonuclease which belongs to the class II CRISPR family (Alok et al. 2020; Zaidi et al. 2017). It was identified from Prevotella and Francisella1, therefore named as named as Cpf1. This system become popular as it was able to fill the gaps of previous genome editing tools and can substitute the CRISPR‐Cas9 in an efficient way (Moon et al. 2018). It is smaller in size than Cas9 and required a shorter CRISPR RNA for proper functioning (Liu et al. 2017). Unlike the CRISPR/Cas9, tracrRNA is no longer necessary to process Cpf1 associated mature CRISPR RNAs (Zetsche et al. 2015). Further, in contrast to the G rich PAM at 3′ end in CRISPR Cas9, it requires a T‐rich PAM sequence at the 5′‐end to perform cleavage efficiently, which enables the targeting of AT‐rich regions in the genome. Further, Cpf1 creates staggered cuts where insertion of a DNA fragment is easily possible via HDR (Gao et al. 2017). The off‐target binding of CRIPSR‐Cpf1 is comparatively less than CRISPR‐Cas9 which could be an additional benefit (Kim et al. 2016; Kleinstiver et al. 2016; Yan et al. 2017). A number of studies showed the application of CRISPR‐Cpf1 for targeted genome editing in various eukaryotes including plants (Kim et al. 2017; Zetsche et al. 2015). It has also been used for multiple targets in the genome (Wang et al. 2017).
Figure 1.2 Comparison of various features of CRISPR‐Cpf1 (a) and CRISPR‐Cas9.
Source: Adapted from Zaidi et al. (2017) © 2017. Reproduced with the permission of Elsevier.
1.6 Conclusions
Genome‐editing technologies enable us to make precise changes in the genome of any living organism. These changes may be diverse‐insertion, deletion or even replacement of a particular stretch of DNA from the genome. Targeting these changes in accordance with our needs has always been the ambition of the scientific community. It was a challenging job until the development of recent genome‐editing technologies. Among these, the most popular and successful ones till now have been ZFNs, TALENs, and CRISPR‐Cas9. The breakthrough in the field of genome editing came after the discovery of CRISPR/Cas9 system as it is an RNA‐ guided and easy‐to‐design system. This system is fascinatingly repurposed as a genome editing tool and is till now the most efficient, cost effective and least demanding genome editing technique. Moreover, the recent addition of CRISPR‐Cpf1 that is a variant of the CRISPR‐Cas system further diversified the application of genome engineering tools by overcoming the various shortcomings of earlier systems.
Acknowledgements
Ms. Sushmita is grateful to DST‐INSPIRE fellowship Program, DST, New Delhi, India for providing financial support. Authors are also thankful to CSIR, New Delhi for financial support in the form of “FBR Genome Editing Network Project” (MLP‐007).
Institute's Manuscript Number is 'CSIR‐NBRI_MS/2020/06/24.
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