Genome Editing Technologies for Crop Improvement 9811905991, 9789811905995

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Kaijun Zhao Rukmini Mishra Raj Kumar Joshi   Editors

Genome Editing Technologies for Crop Improvement

Genome Editing Technologies for Crop Improvement

Kaijun Zhao • Rukmini Mishra • Raj Kumar Joshi Editors

Genome Editing Technologies for Crop Improvement

Editors Kaijun Zhao Institute of Crop Science Chinese Academy of Agricultural Sciences Beijing, Beijing, China Shandong Shunfeng Biotechnology Co. Ltd Jinan, China

Rukmini Mishra Department of Botany, School of Applied Sciences Centurion University of Technology and Management Bhubaneswar, Odisha, India

Raj Kumar Joshi Department of Biotechnology Rama Devi Women’s University Bhubaneswar, Odisha, India

ISBN 978-981-19-0599-5 ISBN 978-981-19-0600-8 https://doi.org/10.1007/978-981-19-0600-8

(eBook)

© Springer Nature Singapore Pte Ltd. 2022 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Foreword

From the later part of the twentieth century, increasingly accumulated knowledge of plant genes/genomes and their relationship with phenotypes has guided crop breeding programs to efficiently create new elite cultivars. Creation of genetic variations through chemical mutagens and physical irradiation, exploration of genetic recombination by intentional crossing/hybridization, transfer of beneficial genes via genetic engineering, and coupled marker-aided selection are instrumental to the development of elite crop varieties. However, modern crop breeding methods still rely heavily on limited genetic diversity in nature or randomly induced rare mutations with breeding value, which makes the breeding programs highly laborious, time-consuming, and unable to satisfy the ever-increasing demand for food by the ever-growing world population. Today’s agriculture is facing a tremendous challenge to improve the global food security, which requires 50% more food by 2050. This challenge becomes even greater because of climate change that leads to extremely high or low temperatures and shrinking water resources. Fortunately, crop breeding has reached a technological inflection point. The recently emerged genome editing technologies have been advancing in an extremely fast track. The genome editing is not only revolutionizing the fields of basic research in life science and medicine but also holds enormous potential to initiate a new era of crop improvement because this technology can precisely generate genetic changes at virtually any site in a genome and can generate desired plant phenotypes without foreign DNA remained in the genome. Particularly, CRISPR/Cas-based genome editing tools, including multiplex genome editing (MGE) reagents, base editors, and prime editors, have advanced at a breath-taking pace in the past decade. The applications of various genome editing tools have not only broadened basic research, but also spawned new opportunities in developing novel germplasms and crop cultivars with improved productivity, product quality, and resistance/tolerance to bio- and abio-stresses. Many case studies on genome editing of agriculturally important crops have been reported. In this regard, the editors of this book attempt to highlight the recent advances and huge potentials in genome editing-based crop breeding. This book offers a balanced set of chapters. v

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Foreword

After an opening chapter that provides an overview of advances in genome editingbased breeding of crops, eight chapters (Chaps. 2–9) describe various editing tools and approaches used in crop genome editing-based crop improvement. The off-target effect in crop genome editing and strategies to minimize it are discussed in Chap. 10. The last nine chapters are devoted to the recent progresses in applications of various genome editing tools to improvement of various crops, including cereal crops (rice, wheat, maize, and sorghum), oil crops (soybean, oil seeds), and vegetable crops (potato and tomato), involving improvement of a wide range of traits associated with crop yield, quality, and stress tolerance. I would like to congratulate the editors of this book on bringing together a valuable collection of chapters regarding the most important aspects of genome editing-based crop breeding. This book will be of particular interest and value to plant biologists working in the field of genome editing, crop breeders, students, policy makers, and other researchers in both academia and industry. Division of Plant Sciences, Bond Life Sciences Center, University of Missouri, Columbia, MO, USA Donald Danforth Plant Science Center, St. Louis, MO, USA

Bing Yang, PhD

Preface

Over the centuries, crop plants have been the primary source of fundamental foods for human life; especially in modern agriculture, most of animal-sourced foods are even derived from crop products. Crop cultivars have been dramatically transformed for higher yield and better quality in the past century to meet the ever-increasing demand for food. However, the human population is still growing and is projected to reach 9.7 billion by 2050. The drastic change in climatic conditions possesses additional threats to agricultural productivity worldwide. Thus, sustainable agricultural production is the need of the hour. Scientific innovations and technological breakthroughs in crop production are needed to ensure food security of the growing planet. Crop improvement is very essential to meet the increasing global food demands and food nutrition. Classical breeding methods, molecular marker-based breeding approaches, and the use of genetically modified crops have played a crucial role in strengthening the food security worldwide. However, their usages in crop improvement have been highly limited due to multiple caveats. The advent of genome editing, however, marks a turning point. Genome editing technology helps scientists to precisely modify genome sequences, facilitating novel insights into the functional genomics of plants and trait improvement of crops. Genome editing encompasses a wide variety of tools such as the zinc-finger nucleases (ZFNs), transcription factorlike effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems, which have been developed and advanced at a breathless pace. Although the most common usage of the editing platforms has been the generation of gene knockout or null alleles, advanced versions of CRISPR/Cas tools have myriads of innovative applications, including targeted gene replacement, multiplex editing, base editing, and prime editing which offers new opportunities to develop improved crop varieties with addition of valuable traits or removal of undesirable traits with high precision. Such versatility of the CRISPR/Cas systems has made genome editing a method of choice for plant genome modification by research institutions and biotechnologybased companies across the world. vii

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This book is a compilation of chapters from eminent scientists across the globe who have established expertise working in genome editing technologies towards efficient crop improvement. It has been divided into three sections. The introduction section gives an overview about the genome editing platforms and their broad applications in crop improvement (Chap. 1). The second section consists of chapters that describe various editing tools and approaches involved in crop genome editing. Some of the recently developed innovative CRISPR/Cas platforms such as base editors and prime editors are described in detail (Chaps. 7 and 8). Also, Chap. 10 is specifically devoted to recent strategies to minimize off-target effects in crop genome editing. The final section of the book is dedicated to the establishment of genome editing systems in a wide range of crop species including rice, wheat, maize, sorghum, soybean, oil seeds, potato, and tomato with specific focus on the improvement of crop yield, quality, and stress tolerance. We thank all the experts from different countries with experiences in various aspects of genome editing-mediated improvement of different crop plants for enriching this book with their valuable contributions. This book will serve as a valuable source of information for graduate and post-graduate students in the field of molecular biology and biotechnology, academicians, researchers, and scientists from biotechnology industries associated with plant breeding and crop improvement, and policy makers involved in the development of biosafety regulations and intellectual properties. Beijing, China Bhubaneswar, India Bhubaneswar, India

Kaijun Zhao Rukmini Mishra Raj Kumar Joshi

Contents

Part I 1

Introduction to Genome Editing and Crop Improvement

Genome Editing Is Revolutionizing Crop Improvement . . . . . . . . . Rukmini Mishra, Raj Kumar Joshi, and Kaijun Zhao

Part II

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Genome Editing Tools and Approaches

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Genome Editing Tools for Food Security . . . . . . . . . . . . . . . . . . . . . Kashaf Zafar, Muhammad Zuhaib Khan, Imran Amin, and Shahid Mansoor

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CRISPR-Cas9/Cpf1-Based Multigene Editing in Crops . . . . . . . . . . Sanjeev Kumar, Yogita N. Sarki, Johni Debbarma, and Channakeshavaiah Chikkaputtaiah

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CRISPR/Cas9 Tools for Multiplex Genome Editing in Crops . . . . . Naoki Wada, Tomoko Miyaji, Chihiro Abe-Hara, Keishi Osakabe, and Yuriko Osakabe

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Plant Genome Editing Mediated by CRISPR/Cas12a System . . . . . 109 Rongfang Xu, Juan Li, Ruiying Qin, and Pengcheng Wei

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Genome Editing in Crops Via Homology-Directed Repair Using a Geminivirus-Based CRISPR/Cas9 System . . . . . . . . . . . . . 119 Amir Hameed, Bareera Faazal, Muhammad Awais, and Ahad Naveed

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Targeted Gene Replacement in Plants Using CRISPR-Cas Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 Zheng Wei, Rukmini Mishra, Raj Kumar Joshi, and Kaijun Zhao

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Expanding the Scope of Base Editing in Crops Using Cas9 Variants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 Rukmini Mishra, Muntazir Mushtaq, and Raj Kumar Joshi

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Contents

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Plant Precise Genome Editing by Prime Editing . . . . . . . . . . . . . . . 177 Ruiying Qin and Pengcheng Wei

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Off-Target Effects of Crop Genome Editing and Its Minimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 Ali Movahedi, Bahram Barati, Shuang Wang, Hui Wei, Honghua Ruan, and Qiang Zhuge

Part III

Genome Editing Towards Crop Improvement

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Genome Editing Toward Rice Improvement . . . . . . . . . . . . . . . . . . 211 Kaijun Zhao, Rukmini Mishra, Raj Kumar Joshi, and Yao-Guang Liu

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Genome Editing Toward Wheat Improvement . . . . . . . . . . . . . . . . 241 Xingguo Ye, Ke Wang, Huiyun Liu, Huali Tang, Yuliang Qiu, and Qiang Gong

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The Use of CRISPR Technologies for Crop Improvement in Maize . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 Joshua Young, Pierluigi Barone, Stephen Gasior, Spencer Jones, Vesna Djukanovic, and Marissa Simon

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Genome Editing Towards Sorghum Improvement . . . . . . . . . . . . . 295 Albert Chern Sun Wong, Yasmine Lam, Jessica Hintzsche, Jemma Restall, and Ian D. Godwin

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Accelerating Cereal Breeding for Disease Resistance Through Genome Editing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 M. Raghurami Reddy, Joan Taaca Acaso, Amos E. Alakonya, Satendra Kumar Mangrauthia, Raman Meenakshi Sundaram, Sena M. Balachandran, and Akshaya Kumar Biswal

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Genome Editing Technologies Contribute for Precision Breeding in Soybean . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349 Yupeng Cai, Li Chen, and Wensheng Hou

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Genome Editing for the Improvement of Oilseed Crops . . . . . . . . . 367 Ananya Sarkar, Raj Kumar Joshi, Urmila Basu, Habibur Rahman, and Nat N. V. Kav

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Genome Editing Tools for Potato Improvement . . . . . . . . . . . . . . . 393 Karl Ravet, François Sevestre, Laura Chauvin, Jean-Eric Chauvin, Gisèle Lairy-Joly, Andrew Katz, Pierre Devaux, Nicolas Szydlowski, Jean-Luc Gallois, Stephen Pearce, and Florian Veillet

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Genome Editing for Tomato Improvement . . . . . . . . . . . . . . . . . . . 429 Priya Gambhir, Sanskriti Ravi, and Adwaita Prasad Parida

Editors and Contributors

About the Editors Kaijun Zhao obtained his Ph.D. from the Graduate School of Chinese Academy of Agricultural Sciences (CAAS) majoring in plant genetics and breeding. He conducted postdoctoral researches in the Department of Botany at the University of Hong Kong, where he experienced intensive researches on plant molecular biology. Since 2000 he led his own research group at the Institute of Crop Science, CAAS, in China. Kaijun’s laboratory is mainly interested in the molecular mechanisms of rice-pathogen interaction and consequently developing resources for rice breeding. He and colleagues cloned the prestigious rice bacterial blight resistance gene Xa23 which has been widely used in rice breeding programmes and brought billions of benefits to farmers in China and beyond. Since 2010, Kaijun’s laboratory has been focused on application of genome editing technologies for rice improvement. Based on investigations on the transcription activator-like effector AvrXa23, the lab has developed a simplified method for assembly of TALENs. Meanwhile, the lab has also established the CRISPR-Cas9 system for plant genome editing, which has been successfully adopted for improvement of rice resistance to diseases, rice grain yield and quality. Kaijun is the recipient of several prestigious awards such as Outstanding Scientific and Technological Innovation Award of CAAS, First Prize of China Agricultural Science & Technology Award and First Prize of DBN Science & Technology Award. Prof. Zhao has published more than 130 research papers and serves on the editorial board of several plant biotechnology journals. He was the liaison Scientist of International Rice Research Institute (IRRI) and currently serves as a Member of PROGRAMME ADVISORY COMMITTEE (PAC), MPOB, Malaysia. Rukmini Mishra currently serves as Associate Professor and Head in the Department of Botany, School of Applied Sciences, Centurion University of Technology and Management, Odisha, India, where she teaches graduate level courses in Plant Genomics and Biotechnology. She is also the coordinator of the Centre for Genetics xi

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and Genomics and in this capacity supervises the overall research related to plant genomics, next generation sequencing and data analysis. Dr. Mishra has a Ph.D. in Agricultural Biotechnology from National Rice Research Institute (NRRI-ICAR), India. She is the recipient of the prestigious SERB Young Scientist project Award and SERB Power grant from the Department of Science & Technology, Government of India, and the Talented Young Scientist Fellowship under the TYSP programme of the Ministry of Science and Technology, Government of China. Her research primarily focuses on molecular marker development and CRISPR/Cas9-based genome editing towards development of resistance against fungal phytopathogens in rice, pepper and other crop species. She has published more than 30 research articles in journals of international repute. Raj Kumar Joshi currently serves as Associate Professor and Head of the Department of Biotechnology, Rama Devi Women’s University, India, where he teaches graduate level courses on Plant Genomics and Genetic Engineering. He also serves as the Group Leader of the Plant Functional Genomics Group, and in that capacity supervises the overall research activities on the functional aspects of molecular plant-microbe interactions. He has been awarded with the prestigious SERB early career grant and SERB extramural grant from the Department of Science and Technology, Government of India, and CREST award from Department of Biotechnology, Government of India. Dr. Joshi is currently running a successful programme on the delineation of molecular genetic networks in the interaction between plants and fungal phytopathogens. The crop of interest includes Capsicum annuum and Allium cepa. His recent forays into genome editing and precise base editing towards improvement of these crops have been highly productive. Dr. Joshi has published more than 60 research papers and serves on the editorial board of many prestigious plant biotechnology journals.

Contributors Chihiro Abe-Hara Faculty of Bioscience and Bioindustry, Tokushima University, Tokushima, Japan Joan Taaca Acaso Department of Biological Sciences and Environmental Studies, University of the Philippines Mindanao, Davao City, Philippines Amos E. Alakonya International Maize and Wheat Improvement Center (CIMMYT) Carretera México-Veracruz, Texcoco, México Imran Amin Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan Muhammad Awais Akhuwat-Faisalabad Institute of Research Science and Technology, Faisalabad, Pakistan

Editors and Contributors

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Sena Munuswamy Balachandran Indian Institute of Rice Research, Hyderabad, India Pierluigi Barone Corteva Agriscience, Johnston, IA, USA Urmila Basu Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada Behram Berati School of Energy and Power Engineering, Jiangsu University, Jiangsu, China Akshaya Kumar Biswal International Maize and Wheat Improvement Center (CIMMYT) Carretera México-Veracruz, Texcoco, México Yupeng Cai National Center for Transgenic Research in Plants, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China Jean-Eric Chauvin IGEPP, INRAE, Institut Agro, Univ Rennes, Ploudaniel, France Laura Chauvin IGEPP, INRAE, Institut Agro, Univ Rennes, Ploudaniel, France Li Chen National Center for Transgenic Research in Plants, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China Channakeshavaiah Chikkaputtaiah Biological Sciences and Technology Division, CSIR-North East Institute of Science and Technology, Jorhat, Assam, India Johni Debbarma Biological Sciences and Technology Division, CSIR-North East Institute of Science and Technology, Jorhat, Assam, India Pierre Devaux Germicopa Breeding, Kerguivarch, Chateauneuf Du Faou, France Vesna Djukavic Corteva Agriscience, Johnston, IA, USA Bareera Faazal Akhuwat-Faisalabad Institute of Research Science and Technology, Faisalabad, Pakistan Jean-Luc Gallois INRAE, GAFL, Montfavet, France Priya Gambhir Department of Plant Molecular Biology, University of Delhi, New Delhi, India Stephen Gasior Corteva Agriscience, Johnston, IA, USA Ian D. Godwin Centre for Crop Science, Queensland Alliance for Agriculture and Food Innovation, University of Queensland, Brisbane, QLD, Australia Qiang Gong Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing, China Amir Hameed Akhuwat-Faisalabad Institute of Research Science and Technology, Faisalabad, Pakistan

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Jessica Hintzsche Centre for Crop Science, Queensland Alliance for Agriculture and Food Innovation, University of Queensland, Brisbane, QLD, Australia Wensheng Hou National Center for Transgenic Research in Plants, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China Spencer Jones Corteva Agriscience, Johnston, IA, USA Raj Kumar Joshi Department of Biotechnology, Rama Devi Women’s University, Bhubaneswar, Odisha, India Andrew Katz Colorado State University, Fort Collins, CO, USA Nat Kav Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada Muhammad Zuhaib Khan Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan Sanjeev Kumar Department of Biosciences and Bioengineering, Indian Institute of Technology, Guwahati, India Gisele Lairy-Joly Germicopa Breeding, Kerguivarch, Chateauneuf Du Faou, France Yasmine Lam Centre for Crop Science, Queensland Alliance for Agriculture and Food Innovation, University of Queensland, Brisbane, QLD, Australia Juan Li Key Laboratory of Rice Genetic Breeding, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei, China Huiyun Liu Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing, China Yao-Guang Liu State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Sciences, South China Agricultural University, Guangzhou, China Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, China Satendra Mangrauthia Indian Institute of Rice Research, Hyderabad, India Shahid Mansoor Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan Rukmini Mishra Department of Botany, School of Applied Sciences, Centurion University of Technology and Management, Bhubaneswar, Odisha, India Tomoko Miyaji Faculty of Bioscience and Bioindustry, Tokushima University, Tokushima, Japan Ali Movahedi Key Laboratory of Forest Genetics and Biotechnology, Nanjing Forestry University, Nanjing, China

Editors and Contributors

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Muntazir Mustaq ICAR-National Bureau of Plant Genetic Resources, Division of Germplasm Evaluation, Pusa Campus, New Delhi, India Ahad Naveed Akhuwat-Faisalabad Institute of Research Science and Technology, Faisalabad, Pakistan Keishi Osakabe Faculty of Bioscience and Bioindustry, Tokushima University, Tokushima, Japan Yuriko Osakabe School of Life Science and Technology, Tokyo Institute of Technology, Kanagawa, Japan Adwaita Prasad Parida Department of Plant Molecular Biology, University of Delhi, New Delhi, India Stephen Pearce Colorado State University, Fort Collins, CO, USA Ruiying Qin Key Laboratory of Rice Genetic Breeding, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei, China Karl Ravet Colorado State University, Fort Collins, CO, USA Sanskriti Ravi Department of Plant Molecular Biology, University of Delhi, New Delhi, India Raghuramy Reddy Indian Institute of Rice Research, Hyderabad, India Jemma Restall Centre for Crop Science, Queensland Alliance for Agriculture and Food Innovation, University of Queensland, Brisbane, QLD, Australia Honghua Ruan Key Laboratory of Forest Genetics and Biotechnology, Nanjing Forestry University, Nanjing, China Ananya Sarkar Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada Yogita Sarki Biological Sciences and Technology Division, CSIR-North East Institute of Science and Technology, Jorhat, Assam, India François Sevestre Unité de Glycobiologie Structurale et Fonctionnelle, Univ. Lille, CNRS, UMR8576, UGSF, Lille, France Marissa Simon Corteva Agriscience, Johnston, IA, USA Raman Minakshi Sundaram Indian Institute of Rice Research, Hyderabad, India Nicolas Szydlowski Unité de Glycobiologie Structurale et Fonctionnelle, Univ. Lille, CNRS, UMR8576, UGSF, Lille, France Huali Tang Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing, China Florian Veillet Germicopa Breeding, Kerguivarch, Chateauneuf Du Faou, France

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Naoki Wada Faculty of Bioscience and Bioindustry, Tokushima University, Tokushima, Japan Ke Wang Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing, China Shuang Wang School of Energy and Power Engineering, Jiangsu University, Jiangsu, China Hui Wei Key Laboratory of Forest Genetics and Biotechnology, Nanjing Forestry University, Nanjing, China Pengcheng Wei Key Laboratory of Rice Genetic Breeding, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei, China Zheng Wei National Key Facility for Crop Gene Resources and Genetic Improvement (NFCRI), Institute of Crop Science, Chinese Academy of Agriculture Sciences (CAAS), Beijing, China Albert Chern Sun Wong Centre for Crop Science, Queensland Alliance for Agriculture and Food Innovation, University of Queensland, Brisbane, QLD, Australia Rongfang Xu Key Laboratory of Rice Genetic Breeding, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei, China Xingguo Ye Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing, China Joshua K. Young Corteva Agriscience, Johnston, IA, USA Kashaf Zafar Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan Kaijun Zhao National Key Facility for Crop Gene Resources and Genetic Improvement (NFCRI), Institute of Crop Science, Chinese Academy of Agriculture Sciences (CAAS), Beijing, China Shandong Shunfeng Biotechnology Co. Ltd, Jinan, China Qiang Zhuge Key Laboratory of Forest Genetics and Biotechnology, Nanjing Forestry University, Nanjing, China

Part I

Introduction to Genome Editing and Crop Improvement

Chapter 1

Genome Editing Is Revolutionizing Crop Improvement Rukmini Mishra, Raj Kumar Joshi, and Kaijun Zhao

Abstract The ever-increasing human population together with emerging environmental cues from climate change demand for novel innovations in plant breeding and agriculture. Targeted genome editing technologies especially the CRISPR/Cas systems have revolutionized basic research and crop breeding by enabling precise targeted modification of an organism’s genome. Lately, genome editing has been widely utilized in myriads of plant species to evaluate gene functions and improve valued agronomic traits including pathogen resistance, abiotic tolerance, yield and quality. In this chapter, we provide a brief overview of genome editing technologies with a special focus on CRISPR/Cas systems. In addition, we discuss about novel innovations in CRISPR-based technologies and their subsequent usage in the development and commercialization of improved genome-edited crops. Further, we have also pointed out major challenges faced by plant genome editing and have predicted possible solutions for speeding of plant breeding and improving crop productivity. Keywords Genome editing · CRISPR/Cas · Targeted mutagenesis · Plant breeding · Crop improvement · Base editing · Prime editing · Agronomic traits

Rukmini Mishra and Raj Kumar Joshi contributed equally with all other contributors. R. Mishra Department of Botany, School of Applied Sciences, Centurion University of Technology and Management, Bhubaneswar, Odisha, India e-mail: [email protected] R. K. Joshi Department of Biotechnology, Rama Devi Women’s University, Bhubaneswar, Odisha, India e-mail: [email protected] K. Zhao (*) National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agriculture Sciences (CAAS), Beijing, China Shandong Shunfeng Biotechnology Co. Ltd, Jinan, China e-mail: [email protected]; [email protected] © Springer Nature Singapore Pte Ltd. 2022 K. Zhao et al. (eds.), Genome Editing Technologies for Crop Improvement, https://doi.org/10.1007/978-981-19-0600-8_1

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1.1

R. Mishra et al.

Introduction

Current agricultural approaches are facing tremendous challenges towards improvement of the yield, quality, and productivity of food crops to feed 9.7 billion people by 2050 (Hickey et al. 2019). The presently most widely used breeding approaches including screening genetic variations through chemical mutagens, physical irradiation, spontaneous mutations, cross hybridization and recombination are highly laborious, time consuming and cannot satisfy the ever-increasing demand for food (Bradshaw 2017). Although transfer of beneficial genes through genetic engineering has been instrumental in the development of elite crop varieties, the widespread adoption of genetically modified (GM) crops is largely restricted by regulatory frameworks based on mostly unsubstantiated health and environmental biosafety concerns (Goldsbrough 2017). Therefore, it is imperative to continuously develop and adopt multiple innovations in crop breeding for sustainable food production. The advances in genome editing technologies entail precise and efficient genetic manipulation of crops at the targeted genomic loci and have the capability to facilitate precision breeding for crop improvement (Mishra et al. 2020). While genome editing approaches like zinc-finger nucleases (ZFNs) (Urnov et al. 2010) and transcription activator-like effector nucleases (TALENs) (Christian et al. 2010) are in use for a long time, but complex in engineering, the recently developed CRISPR/Cas (cluster regularly interspaced short palindromic repeats/Cas) technologies provide greater simplicity, no complex structural chemistry and easiness in targeted gene modification (Fig. 1.1a). All these technologies make use of sequencespecific nucleases (SSNs) to identify specific endogenous DNA sequences and introduce double-stranded breaks (DSBs). The DSBs are subsequently repaired by the endogenous natural DNA repair mechanism either by non-homologous end joining (NHEJ) or by homologous recombination (HR) (Symington and Gautier 2011). While NHEJ is an error-prone pathway which creates insertion and deletion leading to gene knockouts, the HR pathway is a precise approach which makes use of donor template to facilitate gene knockin or gene replacements (Fig. 1.1b). Unlike the GM crops, majority of the genome editing experiments involves the modification of only a few nucleotides that do not significantly alter the natural population of a species (Voytas and Gao 2014). Besides, genome editing follows a natural Mendelian segregation pattern, making them in offspring plants indistinguishable from the naturally occurring mutations. In recent times, these technologies have been used in a wide variety of plants for the improvement of multiple traits including grain yield, stress tolerance and crop domestication (Zhang et al. 2020a, b, c; Li et al. 2021a, b). In other words, the usage of genome editing tools in current plant breeding programmes holds great promise in accelerating crop improvement.

1 Genome Editing Is Revolutionizing Crop Improvement

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Fig. 1.1 Sequence-specific nucleases used as genome editing tools. (a) Zinc-finger nucleases (ZFNs) and transcriptional activator-like effector nucleases (TALENs) create double-stranded breaks (DSBs) at the target site when two ZFNs or TALENs bring two FokI monomers into close vicinity. CRISPR/Cas9 recognizes the target site using a 100 nt single guide (sgRNA) comprising of crisprRNA (crRNA) and transacting crispr RNA (tracrRNA) and create DSB using Cas9 endonuclease adjacent to a NGG protospacer motif (PAM). CRISPR/Cas12a recognizes the target site using a 40–45 nt long crRNA and creates DSB using Cas12a endonuclease. (b) DSBs are subsequently repaired either by non-homologous end joining (NHEJ) or homologous recombination (HR) resulting in multiple outcomes including Indel mutations, gene deletion, NHEJ-mediated gene insertion/replacement, dsDNA/ssODN-mediated gene correction and dsDNA-mediated gene insertion/replacement. crRNA CRISPR RNA, DSB double-stranded break, dsDNA double-stranded DNA, HDR homology-directed repair, NHEJ non-homologous end joining, PAM protospaceradjacent motif, sgRNA single-guide RNA, ssODN single-stranded oligodeoxynucleotide

1.2

Brief Overview of Genome Editing Technologies

Genome editing systems make use of advanced molecular platforms to facilitate precise and targeted editing of specific DNA sequences in a genome. While meganucleases identified from yeast were the first genome editing tool (Pâques and Duchateau 2007), they were the least efficient and could not be used in crop improvement programmes owing to unsubstantial communication and poor

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recognition between meganuclease protein complex and the corresponding target genomic loci (Hsu et al. 2014). Apart from it, ZFNs, TALENs, CRISPR/Cas system, and its associated platforms have been widely used for precise crop improvement.

1.2.1

Zinc-Finger Nucleases (ZFNs)

ZFNs consist of an engineered array of zinc-finger DNA-binding motif fused with the DNA-cleavage domain of the FokI restriction enzyme (Fig. 1.1a). An individual zinc-finger (ZF) domain identifies and binds to a nucleotide triplet, and fingers are assembled into array of 3-6 ZF domains to recognize specific DNA sequences (Urnov et al. 2010). FokI functions only in dimeric; a pair of ZFNs are assembled to bring the two FokI monomers within the specific target DNA to introduce DSBs. So far, ZFNs have facilitated genome modification in rice, maize, soybean, rapeseed and apple (reviewed in Martínez-Fortún et al. 2017). For instance, ZFN-mediated transgene technology has been used for stacking multiple herbicide resistance genes in maize (Ainley et al. 2013). Nonetheless, ZFNs-based genome editing often has low efficacy, mostly because of a complex process of SSN development, high off-target activity and complicated interaction between the protein motifs and the target DNA sequences. Moreover, the high expense involved in the development of commercial ZFNs has significantly limited their usage in crop improvement.

1.2.2

Transcriptional Activator-Like Effector Nucleases (TALENs)

As like ZFNs, a TALEN also consists of FokI restriction enzyme-based cleavage domain fused with the transcriptional activator-like effector (TALE) repeats (Fig. 1.1a). However, unlike ZFNs, a TALE repeat usually binds to a single nucleotide which allows greater flexibility in designing the target regions thereby providing more potential target genomic loci (Bogdanove and Voytas 2011). Furthermore, the construction of TALENs is more simplified by Golden Gate cloning and other available proficient DNA assembly techniques. TALENs-based genome editing has been carried out in a variety of crop plants including potato, barley, tomato, rice, maize, wheat, soybean and rapeseed (reviewed in Zhang et al. 2018a, b, c, d). TALENs-based editing was first used for rice improvement by disruption of the rice OsSWEET14 gene which resulted in mutant rice with enhanced resistance to the bacterial blight pathogen, Xanthomonas oryzae pv. oryzae (Li et al. 2012). Likewise, TALENs were used to knock out three homeoalleles of the resistance specific TaMLO gene in wheat that conferred broad-spectrum resistance to powdery mildew (Wang et al. 2014a, b). In sugarcane, TALENs-mediated multiallelic mutagenesis of caffeic acid O-methyltransferase (COMT) genes resulted in

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improved saccharification efficiency (Kannan et al. 2018). In potato, TALENs were used to knock out potato vacuolar invertase (StVInv) gene which improved the postharvest self-life of cold storage potato by depleting the reducing sugars (Clasen et al. 2016). In tomato, TALENs-based geminivirus replicons were used for HR-based repair of the tomato anthocyanin mutant 1 (Slant1) leading to the development of purple tomatoes with high anthocyanin accumulation (Čermák et al. 2015). TALEN technology has also been used to target and disrupt the rice betaine aldehyde dehydrogenase (OsBADH) gene resulting in improved fragrance due to enhanced 2-acetyl-1-pyrroline (2AP) accumulation (Shan et al. 2015). All these studies establish the significance of TALENs in crop improvement. Yet, TALENs-based genome editing remains a challenging approach with variable gene targeting efficiency. TALENs are designed using repetitive elements with the need of a thymidine residue at the first position which can trigger in vivo HR (Doyle et al. 2012). Further, each TALEN pair must be experimentally authenticated as some of them fails to introduce the expected mutations (Hwang et al. 2013). With the recent surge of the CRISPR/ Cas technology in terms of accessibility, simplicity and versatility, the TALEN technology has taken a backseat.

1.2.3

CRISPR/Cas System: A Genome Editing Marvel

The CRISPR/Cas technology is based on RNA-guided DNA targeting demonstrated by the adaptive immune system found in bacterial and archaeal genomes (Marraffini and Sontheimer 2010). It is a two-component genome engineering tool having a Cas9 DNA endonuclease and a single-guide RNA (sgRNA) molecule to enable target specificity (Jinek et al. 2012) (Fig. 1.1a). The presence of a protospacer adjacent motif (PAM) sequence downstream of the target site facilitates the recognition and binding of the Cas9-sgRNA complex to the site of interest and subsequent cleavage at the genomic locus. Therefore, multiple sgRNAs with specificity for different spacer sequences could be utilized to achieve efficient modification of different loci simultaneously, making it a simple, rapid, versatile and multiplex genome engineering platform (Cong et al. 2013). Since its establishment as a genome engineering tool in eukaryotic system, a CRISPR/Cas craze has resulted in overwhelming research to solve a variety of problems in plant biology and crop improvement (reviewed in Zhang et al. 2018a, b, c, d, 2020a, b, c; Mishra et al. 2020). The most common use has been in the development of gene knockouts or null alleles through insertion and deletion leading to frame shift mutations in many crop plants including soybean, barley, rice, wheat, maize, lettuce, sorghum, cucumber, potato, tomato, grapes, oranges and watermelon (reviewed in Li et al. 2021a, b). While the efficiency of SpCas9-based CRISPR system is limited by their specificity to recognize a 50 -NGG-30 PAM, multiple Cas9 orthologues have been characterized with greater flexibility in the recognition of the target sites and widen the scope of editing remote genomic loci (Wrighton 2018). For instance, NmCas9 (from Neisseria meningitidis) specifies for 50 -N4GATT-30 PAM (Hou et al. 2013), StCas9

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(from Streptococcus thermophilus) prefers 50 -NGGNG-30 PAM (Müller et al. 2016), CjCas9 (from Campylobacter jejuni) recognizes 50 -N4ACAC-30 PAM (Kim et al. 2017), and ScCas9 (derived from Streptococcus canis) requires 50 -NNG-30 PAM (Chatterjee et al. 2018). Additionally, numerous SpCas9 variants including Cas9VRER, Cas9-VQR, Cas9-EQR, Cas9-RVR, xCas9 and the SaCas9-KKH (from Staphylococcus aureus) have also been engineered to introduce novel PAM recognition spots (Kleinstiver et al. 2015a, b; Zhong et al. 2019). Most recently, new SpCas9 variants such as the SpG and SpRY that recognize 50 -NGN-30 and 50 -NRN-30 /50 -NYN-30 PAMs as well as the SpCas9-NRRH, SpCas9-NRTH and SpCas9-NRCH that collectively specifies for 50 -NRNH-3 PAM have further eliminated the limitation of PAM sequences in CRISPR/Cas9 system (Walton et al. 2020; Miller et al. 2020). The emergence of the type II, group V CRISPR/Cas12a system has significantly substantiated the constraints with SpCas9 and has proven to be highly effective and competent in plant genome editing (Endo et al. 2016). It is characterized by a Cas12a enzyme derived from Francisella tularensis subsp. novicida (FnCas12a), and its orthologue from a Lachnospiraceae bacterium (LbCas12a) prefers a T-rich PAM sequence (50 -TTTN-30 ) and cleaves the target DNA using only a 20 nt crRNA sequence resulting in a 4-5 nt staggered DSB, which enables more accurate gene modification through NHEJ repair pathway (Zetsche et al. 2015) (Fig. 1.1a). Like Cas9, many Cas12a variants such as the AsCas12a-RR and AsCas12a-RVR (both derived from Acidominococcus sp. BV3L6), LbCas12a-RR and LbCas12a-RVR, have also greatly overcome the target restriction to allow broad-spectrum multiplex genome editing in plants (reviewed in Li et al. 2021a, b). Also, compared to Cas9, Cas12a can stimulate HR more efficiently when a dsDNA repair template is available in conjunction with a plasmid expressing Cas12a or orthologous enzymes (Begemann et al. 2017). What more, the CRISPR/Cas12a system has confirmed efficient editing of important agronomic traits in multiple crops including rice, soybean, wheat and tobacco (Tang et al. 2017a, b; Zhang et al. 2018a, b, c, d). In a recent study, six orthologues of Cas12a, namely, ErCas12a, Lb5Cas12a, BsCas12a, Mb2Cas12a, TsCas12a and MbCas12a, were reported to have highly editing efficiency in rice. Further, the Mb2Cas12-RVRR variant demonstrated more relaxed PAM requirement and higher genome coverage as compared to SpCas9. In another study, three engineered variants of ErCas12a (also known as MAD7 nuclease), namely, MAD7-RR and MAD7-RVR, revealed comparable or higher editing efficiency and increased target range to LbCas12a in both rice and wheat (Lin et al. 2021a, b). A MAD7-APOBEC fusion-induced deletion (M-AFID) system also reported predictable deletions from 50 deaminated cytosines to the cleavage site (Lin et al. 2021a, b). Taken together, CRISPR/Cas system and the associated Cas protein variants have the potential to accelerate plant breeding by allowing desirable modification of multiple traits directly in an elite background.

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9

Application of CRISPR/Cas for Crop Improvements

By 2013, at least five groups had demonstrated that CRISPR/Cas9 technology has immense scope in plant biology by targeting various genes and by exploiting both NHEJ and HR to generate targeted insertions, deletions and multiplex modifications (Bortesi and Fischer 2015). Over the last several years, the CRISPR/Cas9 technology has been established in a wide range of crop species including maize, soybean, tomato, potato, sugarcane and sorghum with specific focus in the improvement of crop yield, quality and stress resistance through simple knockout of one or more genes (reviewed in Li et al. 2021a, b).

1.3.1

Resistance to Biotic and Abiotic Stresses

Biotic agents like bacteria, fungi, viruses and insects, and abiotic conditions including drought, temperature stress, salinity and submergence are primary factors causing agricultural losses and have been identified as chief threats to global food security (Pandey et al. 2017). Therefore, the improvement of agriculturally important traits for stress tolerance has been an uninterrupted activity for many decades. While conventional breeding together with marker-based selection technology and genetic engineering has been quite effective in improving the crops against the stresses, there are still many agricultural challenges which demand for novel technologies that can facilitate the development of climate-resilient plants. CRISPR/Cas technology is revolutionizing the field of agriculture through precise breeding of important traits resulting in tolerance to biotic and abiotic stresses. The dawn of next-generation sequencing together with functional genomics studies had resulted in the identification of both coding and non-coding genes that respond to different biotic stresses. The overexpression of numerous genes and transcription factors associated with molecular mechanisms governing the plantpathogen interactions confers enhanced pathogen resistance in plants (Mukhtar 2013). Instead, a wide number of susceptibility (S) genes are involved in negative regulation of plant defense leading to successful pathogen infection (Zaidi et al. 2018). While the resistance (R) genes exhibit a dominant disease resistance, pathogen defense in plants can also be realized by manipulation of S-genes by altering the compatibility of the plant and inhibiting the pathogen for recognition, penetration and proliferation in the host tissues. For instance, the Mildew resistance locus O (MlO) genes are prominent plant susceptibility trait responsible for severe powdery mildew infection across the monocots and dicots (Acevedo-Garcia et al. 2014). Wang et al. (2014a, b) used the CRISPR/Cas9 technology for simultaneous modifications of three homeoalleles of MlO genes in wheat that resulted in resistance to powdery mildew in the mutant lines. Powdery mildew resistance in wheat has also been realized by CRISPR/Cas9-based modification of three homologs of the Triticum aestivum-enhanced disease resistance (TaEDR1) gene (Zhang et al.

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2017). Subsequently, a similar toolkit was used to develop Tomelo, a non-transgenic tomato variety by editing the SlMlO locus resulting in broad resistance to the powdery mildew pathogen (Nekrasov et al. 2017). More recently, CRISPR/Cas9targeted mutagenesis of the tomato powdery mildew resistance 4 (PMR4) gene resulted in reduced loss of susceptibility to the powdery mildew pathogen Oidium neolycopersici (Santillán Martínez et al. 2020). Fusarium wilt caused by Fusarium oxysporum f.sp niveum (Fon) is a major fungal disease responsible for 3080% of yield losses in cucurbits, especially watermelon (Egel and Martyn 2007). A CRSIPR/Cas9 toolkit was successfully employed to precisely edit the phytosulfokine (PSK) precursor gene, Clpsk1 in watermelon, and the developed knockout mutants of watermelon exhibited significantly enhanced resistance to Fon (Zhang et al. 2020a, b, c). In another study, CRISPR/Cas9-based simultaneous modification of two copies of Gh14-3-3d genes enhanced tolerance to Verticillium dahliae in cotton as evidenced by significant decrease in fungal sporulation, fungal biomass together with upregulated expression of defense genes (Zhang et al. 2018a, b, c, d). In rice, bacterial blight (BB) and rice blast are the two major diseases causing sever yield losses across major rice-growing regions of the world. CRISPR/ Cas9-based mutagenesis of OsSWEET13 and OsERF922 has resulted in enhanced rice blight and blast resistance, respectively (Zhou et al. 2015; Wang et al. 2016). In a recent study, a CRISPR/Cas9-based multiplex vector system generated broadspectrum resistance to both bacterial blight and rice blast through simultaneous modification of a BB S-gene, Xa13 and blast S-gene Pi21 (Li et al. 2019a, b). Potyvirus is the largest genus of the plant viruses causing significant yield losses in a wide range of crop species. The eukaryotic translation initiation factor 4E (elf4E) proteins are central molecules in the infection of major viruses from the family Potyviridae. Disruption of elf4Es by the CRISPR/Cas9 system resulted in ipomovirus as well as potyvirus resistance in cucumber (Chandrasekaran et al. 2016). In another study, the CRISPR/Cas9-based in-frame mutation of the elf4G gene generated new sources of resistance against rice tungro spherical virus (RTSV) together with improved grain yield (Macovei et al. 2018), while CRISPR/Cas9 system normally targets dsDNA and is unable to directly cleave the viral RNA genome in most plant viruses. To overcome this, the CRISPR/Cas13 system has been recently used to target the viral RNA genome, resulting in reduced virus accumulation and enhanced resistance against the turnip mosaic virus (TuMV) in tobacco (Aman et al. 2018). Likewise, the CRISPR/Cas-based knockout of several other genes responsible for viral, bacterial and fungal diseases has resulted in improved stress resistance in different crop species (reviewed in Li et al. 2021a, b). The genes that have been modified in various crop plants for resistance to virus, bacterial and fungal diseases are listed in Table 1.1. The ever-changing climatic conditions including extreme temperature conditions, dwindling water resources, drought and high salinity of the agricultural fields are the greatest threats to global food security as they directly affect the plant growth, development, yield and quality. However, unlike the biotic stresses, wide networks of regulatory genes and transcription factors in different plants species contribute to the alleviation of these stresses. As such, only a handful of genes have been modified

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Table 1.1 Application of CRISPR/Cas technology towards development of resistance to bacterial, viral and fungal diseases Crop species Oryza sativa

Triticum aestivum

Solanum lycopersicon

Targeted gene(s) OsERF922

Pathogen type Fungi

OsMPK5

Fungi

OsSWEET13

Bacteria

Xa13

Bacteria

Os8N3

Bacteria

OsSWEET13 promoter, OsSWEETT11 and OsSWEEt14 genes OsSWEET11, OsSWEET13 and OsSWEET14 OsSWEET11, OsSWEET14

Bacteria

Bacteria

Bacteria

Eif4g

Virus

Virus RNA genome

Virus

TaMlo-A1, TaMloB1 and TaMlo-D1 TaMlo

Fungi

TaEdr1

Fungi

SlPmr4

Fungi

SlMAPK3

Fungi

Solyc08g075770

Fungi

Fungi

Improved trait Resistance to Magnaporthe oryzae Resistance to Magnaporthe oryzae Resistance to Xanthomonas oryzae pv. oryzae (Xoo) Resistance to Xanthomonas oryzae pv. oryzae (Xoo) Resistance to Xanthomonas oryzae pv. oryzae (Xoo) Resistance to Xanthomonas oryzae pv. oryzae (Xoo)

References Wang et al. (2016) Xie and Yang (2013) Zhou et al. (2015)

Li et al. (2020a, b) Kim et al. (2019)

Xu et al. (2019)

Resistance to Xanthomonas oryzae pv. oryzae (Xoo) Resistance to Xanthomonas oryzae pv. oryzae (Xoo) Resistance to rice tungro spherical virus Reduction in virus level for Southern rice black streaked dwarf virus, tobacco mosaic virus Resistance to powdery mildew

Oliva et al. (2019)

Resistance to powdery mildew Resistance to powdery mildew Resistance to powdery mildew caused by Oidium neolycopersici Resistance to Botrytis cinerea Resistance to Fusarium wilt

Shan et al. (2013)

Jiang et al. (2013)

Macovei et al. (2018) Zhang et al. (2019a, b, c, d, e)

Wang et al. (2014a, b)

Zhang et al. (2017) Santillán Martínez et al. (2020) Zhang et al. (2018a, b, c, d) Prihatna et al. (2018) (continued)

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Table 1.1 (continued) Targeted gene(s) SlMlo1

Pathogen type Fungi

Jaz2

Bacteria

Dmr6

Bacteria

Virus DNA Rep, IR and Cp RGA2, Ced9

Virus

Virus sequences in the host plantain genome nCBP-1, nCBP-2

Virus

eIF4E

Virus

Vitis vinifera

VvWRKY52

Fungi

Citrus paradisi Wanjincheng orange Malus domestica Theobroma cacao Carica papaya

CsLOB1

Bacteria

CsLOB1

Bacteria

DIPM-1, DIPM2, and DIPM-4 TcNPR3

Bacteria

CpalEPIC8

Fungi

Crop species

Musa paradisica

Manihot esculenta Cucumis sativus

Fungi

Virus

Fungi

Improved trait Resistance to powdery mildew Resistance to bacterial speck disease caused by Pseudomonas syringae pv. tomato DC 3000 Resistance to Pseudomonas syringae, Phytophthora capsici, and Xanthomonas spp. Resistance to tomato yellow leaf curl virus Resistance to Fusarium oxysporum f. sp. cubense tropical race 4 (TR4) Tolerance to endogenous banana streak virus Suppression of cassava brown streak disease Cumulative resistance to cucumber vein yellowing virus, zucchini yellow mosaic virus, and papaya ring spot mosaic virus-W Increased resistance to B. cinerea Increased resistance to citrus canker Increased resistance to citrus canker Mild tolerance to Erwinia amylovora Resistance to Phytophthora tropicalis Resistance to Phytophthora palmivora

References Nekrasov et al. (2017) Ortigosa et al. (2019)

deToledo Thomazella et al. (2016) Tashkandi et al. (2018) Dale et al. (2017)

Tripathi et al. (2019) Gomez et al. (2019) Chandrasekaran et al. (2016)

Wang et al. (2018) Jia et al. (2017) Peng et al. (2017) Malnoy et al. (2016) Fister et al. (2018) Gumtow et al. (2018)

by using the CRISPR/Cas technology for improving plant tolerance to abiotic stresses (Zafar et al. 2020) (Table 1.2). As a proof of concept, CRISPR/Cas9 system was used to generate new alleles of the OPEN STOMATA 2 (OST2) gene, a prominent plasma membrane H+ATPase involved in stomatal response in Arabidopsis (Osakabe et al. 2016). Under ABA induction, the ost2 mutants facilitated drought tolerance through high degree of stomatal closure. ABA being a primary factor of drought response, alteration in the expression pattern of the

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Table 1.2 Application of CRISPR/Cas technology towards development of abiotic stress tolerance in crops Crop species Oryza sativa

Target gene(s) OsNramp5

Improved trait Tolerance to cadmium toxicity

OsSAPK2 OsSRL1, OsSRL2 OsRR22

Drought tolerance Drought tolerance Salinity tolerance

OsDST

Tolerance to salinity and osmotic stress Tolerance to salt, ABA and dehydration Tolerance to drought, salinity and low temperature Tolerance to low temperature

Zeng et al. (2020)

Drought tolerance Tolerance to high temperature

Shi et al. (2017) Klap et al. (2017)

Tolerance to salinity and osmotic stress

Bouzroud et al. (2020)

OsmiR535 GS3 and DEP1

Zea mays Solanum lycopersicon

OsPIN5b, GS3 and OsMYB30 ARGOS8 SlAGL6 ARF4

References Tang et al. (2017a, b) Lou et al. (2017) Liao et al. (2019) Zhang et al. (2019a, b, c, d, e) Kumar et al. (2020) Yue et al. (2020) Cui et al. (2020)

ABA-responsive element-binding protein/ABRE-binding factors (AREBs/ABFs) is significant to controlling stomatal closure (Osakabe et al. 2014). Roca Paixão et al. (2019) used a CRISPR/Cas9HAT-based histone modification system for targeting the AREB1 gene in Arabidopsis. The binding of the catalytic domain exposed the AREB1 promoter to the transcriptional machinery through acetylation of core histone in the region. This in turn resulted in drought tolerance through positive regulation of drought-responsive genes. Ethylene is a major phytohormone involved in the physiological network for regulating responses to high temperature and drought conditions in plants (Kawakami et al. 2010). Of late, the CRISPR/Cas9-based HDR pathway was used to insert the maize native GOS2 promoter into the 50 untranslated region of ARGOS8, encoding a prominent negative regulator ethylene response (Shi et al. 2017). The ARGOS8 variants of maize conferred beneficial traits, including drought tolerance and enhanced grain yield. CRISPR/Cas-mediated knockout of SlAGAMOUS-Like 6 (SlAGL6) gene in tomato has resulted in better fruit and improved tolerance to high temperature (Klap et al. 2017). Simultaneous knockdown and knockout of ARF4 transcription factor in tomato using a CRISPR/Cas vector have demonstrated enhanced tolerance to salinity and osmotic stress through improved water usage efficiency (Bouzroud et al. 2020). Rice being the major staple food for two-third of the world population, a majority of experiments related to genome editing to assist abiotic stress tolerance have been carried in it. CRISPR/ Cas9-mediated knockout of the OsNramp5, a prominent cadmium (Cd) transporter, generated rice lines with significantly low Cd accumulation in both hydroponic and field conditions (Tang et al. 2017a, b). Zhang et al. (2019a, b, c, d, e) employed the

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CRISPR/Cas9 technology to target the OsRR22 gene, a prominent negative regulator of salt stress in the japonica rice cultivar WPB106. The generated homozygous mutants displayed enhanced tolerance to salinity. Likewise, the osdst rice mutant lines generated through CRISPR/Cas9-mediated mutagenesis in the indica rice cultivar MTU1010 showed reduced stomatal density, enhanced water retention in the leaves and higher level of tolerance to osmotic and salinity stress as compared to the wild-type plants (Kumar et al. 2020). In another study, the gene encoding the OsmiR535 was knocked out by CRISPR/Cas9, and the edited lines revealed enhanced tolerance to salt, ABA and dehydration stresses (Yue et al. 2020). Drought adaptability is also associated with leaf-rolling phenotypes which decreases water loss from the plants. Recently, CRISPR/Cas9 was used to generate rolled leaf mutants in rice with reduced stomatal number, stomatal conductance and transpiration rate through precise editing of the semi-rolled leaf 1 (SRL1) and SRL2 genes (Liao et al. 2019). Likewise, two heterotrimeric G protein genes GS3 and DEP1 in the japonica rice cultivar Sasanishiki were simultaneously edited using the CRISPR/ Cas9 (Cui et al. 2020). The generated double mutants demonstrated improved rice tolerance to multiple abiotic stresses, including drought, chilling and salinity stresses. Also, triple mutant rice plants generated through CRISPR/Cas9-mediated mutagenesis of OsPIN5b, GS3 and OsMYB30 genes reported enhanced cold tolerance along with increased panicle length and grain size (Zeng et al. 2020).

1.3.2

Tolerance to Herbicides

The competitive and allopathic interaction between the weeds and crop species makes it a significant problem in agricultural system, and the management of weeds is essential to circumvent yield losses and to ensure food security (Bajwa et al. 2015). Traditional practices for weed management including chemical treatment, seed predation and the use of bioherbicides haven’t met with tangible success. Genetically engineered herbicide-tolerant plants are expensive and complex and face regulatory issues and inherent concerns for horizontal or vertical gene flow (Schütte et al. 2017). In contrast, CRSIPR/Cas9-mediated editing could be suitable for creating transgene-free herbicide-tolerant crops. A large number of studies in this regard have focused on the precise modification of the acetolactate synthase (ALS), a vital protein involved in the biosynthesis of branched chain amino acids (Table 1.3). Endo et al. (2016) developed highly efficient herbicide-tolerant rice mutants by using a CRISPR/Cas9 complex to disrupt the DNA ligase 4 gene followed by homology-directed repair-mediated gene targeting of the OsALS gene. Sun et al. (2016) used two sgRNAs to introduce multiple distinct mutations in the OsALS gene using the CRISPR/Cas9-mediated HR pathway. CRISPR/Cas9 was also used to facilitate the insertion of a donor DNA template into the rice 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, through an intronmediated site-specific gene replacement approach (Li et al. 2016a, b). Rice plants with desired substitution in the EPSPS gene were found resistant to glyphosate, a

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Table 1.3 Application of CRISPR/Cas technology towards development of herbicide tolerance in crops Crop species Oryza sativa

Target gene ALS1

Targeted herbicide Bispyribac sodium

ALS1 EPSPS ALS1

Bispyribac sodium Glyphosate Bispyribac sodium

ALS1 OsACCase

Bispyribac sodium Gallant

OsACCase

Halocyfop-Rmethyl Imazamox

ALS ACCase

Triticum aestivum

Zea mays

OsALS1 ALS1 ALS1

Halocyfop-Rmethyl Bispyribac sodium Imazethapyr Nicosulfuron

TaALS, ACCase ALS2

Sulfonylurea, imidazolinone Chlorsulfuron

ALS1 StALS1

Chlorsulfuron Imidizolinone

LyALS

Chlorsulfuron

LuEPSPS

Glyphosate

Glycine max Solanum tuberosum Lycopersicon esculentum Linum usitatissimum Manihot esculenta Citrullus lanatus

MeEPSPS

Glyphosate

ClALS

Trebenuron

Brassica napus

BnaALS1

Trebenuronmethyl

CRISPR editing method CRISPR/Cas9HDR CRISPR/Cas9 CRISPR/Cas9 cgRNA-mediated HDR Prime editing Base editing (CBE) Base editing (ABE) Target-AID STEMEs BEMGE CRISPR/Cas9 Base editing (CBE) dCas9/PBE CRISPR/Cas9 CRISPR/Cas9 GV-mediated editing Base editing (CBE) ssODN and CRISPR/Cas9 Cas9-gRNA Base editing (CBE) Base editing (CBE)

Reference Endo et al. (2016) Sun et al. (2016) Li et al. (2016a, b) Butt et al. (2017) Butt et al. (2020) Liu et al. (2020a, b) Li et al. (2018a, b, c, d, e) Shimatani et al. (2018) Li et al. (2020a, b) Kuang et al. (2020) Wang et al. (2021) Zhang et al. (2019a, b, c, d, e) Zong et al. (2018a, b) Svitashev et al. (2015) Li et al. (2015) Butler et al. (2016) Veillet et al. (2019) Sauer et al. (2016) Hummel et al. (2018) Tian et al. (2018) Wu et al. (2020a, b)

popular broad-spectrum herbicide. In a separate study, a chimeric sgRNA carrying both the sequences for repair template and target specificity was used in a CRISPR/ Cas9 HDR approach and generated successful substitutions in the OsALS locus conferring herbicide tolerance of rice plants (Butt et al. 2017). Recently, a combination of nCas9-based adenine and cytosine base editors (ABEs and CBEs) and sgRNAs known as base editing-mediated gene evolution (BEMGE) was used to

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develop robust point mutations at the OsALS1 gene (Kuang et al. 2020). The resultant mutants showed varying levels of resistance to bispyribac-sodium. Most recently, CRISPR/Cas9 technology was used for modifying the OsALS gene to develop a novel G628W herbicide-tolerant allele in rice that conferred significant tolerance to herbicide imazethapyr (Wang et al. 2021). Acetyl-CoA carboxylase (ACC), a catalytic enzyme in the fatty acid biosynthetic pathway, is often used as a target by major group of herbicides as its mutation leads to serious developmental arrest in plants (Baud et al. 2004). A so-called STEMEs (engineered saturated targeted endogenous mutagenesis editors) approach was used to produce nearsaturated mutagenesis for a 56-amino acid portion of the OsACC gene (Li et al. 2020a, b). The directed evolution of OsACC gene resulted in herbicide resistance mutations and demonstrated significant tolerance to haloxyfop-R-methyl. Targeted base editing of OsACC gene using both ABEs and CBEs has also been used to generate rice mutants with resistance to multiple aryloxyphenoxypropionate (APP) group ACC-inhibited chemicals (Li et al. 2018a, b, c, d, e; Liu et al. 2020a, b). Apart from rice, herbicide-tolerant maize and soybean were produced through co-transformation of a CRISPR/Cas9 vector and donor template through particle bombardment method (Li et al. 2015; Svitashev et al. 2015). Butler et al. (2016) used a geminivirus-based CRISPR/Cas9 replicon structure to develop herbicide-resistant potato by introducing herbicide-inhibiting point mutations in the ALS1 gene. Recently, the ALS1 gene was targeted in potato and tomato using a dCAs9-CBE system leading to chlorosulfuron-resistant plants (Veillet et al. 2019). Similar CBEs were used for target-specific substitution in the ALS1 gene of watermelon and rapeseed resulting in trebenuron-resistant lines (Tian et al. 2018; Wu et al. 2020a, b). Furthermore, a combination of single-stranded oligonucleotides (ssODNs) and CRISPR/Cas9 was used to develop herbicide tolerance in flax by precise editing of EPSPS genes (Sauer et al. 2016). Targeted base editing in the ALS and ACC genes has also resulted in herbicide-tolerant wheat lines (Zong et al. 2018a, b; Zhang et al. 2019a, b, c, d, e). Also, a combination of promoter swap and substitution of dual amino acids at the endogenous EPSPS locus using the CRISPR/Cas9-based HR method has generated robust glyphosate tolerance in cassava (Hummel et al. 2018).

1.3.3

Improvement of Crop Yield and Quality

The commercial viability of a crop variety is directly associated with its yield and quality. Being a complex trait with the involvement of multiple genes, molecular breeding is yet to make significant progress to improve the crop yield. However, the recent surge in genome sequencing and whole genome analysis has led to the identification of specific genes acting as negative regulators of crop yield. As could be expected, CRISPR/Cas technology offers a perfect instrument for knockout of these negative regulators to improve the yield in crop species (Table 1.4). Three prominent negative regulatory genes, Gn1a (GRAIN NUMBER 1a), DEP1 (DENSE

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Table 1.4 Application of CRISPR/Cas technology towards improvement of crop yield Crop species Oryza sativa

Target gene(s) Gn1a, DEP1, GS3 GW2, GW5, TGW6 QTL for GS3 & Gn1a L QTL for GS3, GW2 & Gn1a OsPYL Cytochrome p450 OsEHD1

Triticum aestivum

Lycopersicon esculentum Brassica napus Glycine max

Panicum virgatum

Improved trait Grain number, larger grain size and erect panicles Grain weight, grain size Grain number, larger grain size Grain number, larger grain size, grain weight Grain productivity Grain productivity

OsSPL16

Vegetative growth, grain productivity Grain productivity

OsPAO5 TaGASR7

Grain weight, grain number Grain weight

TaGW2

Grain weight

CLV-WUS BnaMAX1 GmLHY

Fruit size, inflorescence architecture Plant architecture and yield Plant height and internode length

AP1 GmNMHC5 Pvtb1

Flowering time and plant height Flowering and maturity Tiller number, biomass

References Li et al. (2016a, b) Xu et al. (2016) Shen et al. (2018) Zhou et al. (2019) Miao et al. (2018) Usman et al. (2020) Wu et al. (2020a, b) Usman et al. (2021) Lv et al. (2021) Zhang et al. (2016) Zhang et al. (2018a, b, c, d) Rodríguez-Leal et al. (2017) Zheng et al. (2020) Cheng et al. (2019) Chen et al. (2020) Wang et al. (2020) Liu et al. (2020a, b)

AND ERECT PANICLE 1) and GS3 (GRAIN SIZE 3) were simultaneously knocked out using CRISPR/Cas9 that resulted in enhanced grain number, larger grain size and erect panicles in rice (Li et al. 2016a, b). Additionally, simultaneous knockout of three negative grain weight regulation genes, GW2 (grain weight 2), GW5 and TGW6 (thousand grain weight 6) reported enhanced grain weight and size in the edited rice lines (Xu et al. 2016). Recently, CRISPR/Cas9-mediated editing of the grain yield-associated QTL consisting of GS3 and Gn1a genes demonstrated diverse effect on grain yield in different japonica rice cultivars (Shen et al. 2018). Contrary to this, precise mutagenesis of grain yield QTL consisting of GS3, GW2 and Gn1a showed significant rise in grain yield in several other japonica cultivars (Zhou et al. 2019). In another study, CRISPR/Cas9 platform was used to edit the PYL gene, involved in ABA biosynthetic pathway, and the edited pyl rice mutants demonstrated improved grain productivity (Miao et al. 2018). CRISPR/Cas9-targeted mutagenesis of three homologs of the cytochrome P450 gene family has generated high yield indica rice lines (Usman et al. 2020). Deletion mutants of four rice cultivars

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generated through CRISPR/Cas editing of the early heading date 1 (OsEHD1) gene displayed longer vegetative growth period and improved yield potential in low latitude regions (Wu et al. 2020). Most recently, CRISPR/Cas9-guided mutation in the OsSPL16 gene reported significant boost in grain yield of rice through modulation of the pyruvate enzyme activity (Usman et al. 2021). Likewise, CRISPRinduced ospao5 rice mutant lines conferred enhanced grain weight and grain number (Lv et al. 2021). Among other crops, improved grain weight was realized in wheat through CRISPR/Cas9-mediated modification of three homologues of TaGASR7 gene (Zhang et al. 2016). CRISPR/Cas9-mediated editing of the cis-regulatory element of the CLAVATA-WUSCHEL (CLV-WUS) stem cell circuit resulted in multiple novel variants with improved fruit size, inflorescence architecture and better growth habits in tomato (Rodríguez-Leal et al. 2017). Three independent researches made use of CRISPR/Cas9 technology to produce soybean mutants with reduced flowering time, better plant height and internode length leading to better productivity (Cheng et al. 2019; Chen et al. 2020; Wang et al. 2020). In switchgrass (Panicum virgatum L.), CRISPR/Cas9-mediated nonchimeric mutation at the teosinte branched 1(tb1) gene produced more tillers and higher biomass as compared to wild-type plants (Liu et al. 2020). Also, the precise modification of two homologs of Brassica napus more axillary growth 1 (BnaMAX1) gene generated semi-dwarf, multi-branched phenotypes with higher silique-bearing rapeseed mutants which contributed to increased yield compared to the wild-type genotype (Zheng et al. 2020). CRISPR/Cas technology has been widely employed to regulate the concentration of several proteins, carbohydrates, oil and secondary metabolites which are crucial for maintaining crop quality (Table 1.5). Resistant starch (RS) and high amylose content (AC) are helpful for patients with diabetes, hypothyroidism and other dietrelated non-infectious chronic diseases. CRISPR/Cas was employed for targeted knockout of the rice starch branching genes, OsBEI and OsSBEIIb, encoding the key enzyme for determining the fine structure of starch (Sun et al. 2017). While the sbeI mutants did not show any observable difference, sbeIIb mutants showed significantly increased RS and AC content. Similarly, CRISPR/Cas-induced mutation of the sweet potato IpSBEII gene reported high amylose percentage with no significant change in the total starch contents (Wang et al. 2019). In a separate study, CRISPR/ Cas was used to edit the regulatory elements in the promoter of the O. sativa waxy gene (OsWaxy) in japonica rice cultivars (Zhang et al. 2018a, b, c, d). Multiple oswaxy mutant lines resulted in glutenous rice with varied levels of AC and better eating and cooking quality. Also, CRISPR/Cas-mediated editing of all four alleles of the granule bound starch synthase (StGBSS) gene in tetraploid potato and the mutant lines displayed altered starch phenotypes and other agronomic properties (Andersson et al. 2017). Multiplex editing of seven homologues of lysophosphatidic acid acyltransferase 2 (BnLPAT2) and four homologues of BnLPAT5 generated double mutant lines with increased accumulation of starch in the mature seed of Brassica napus (Zhang et al. 2019a, b, c, d, e). Gluten protein in wheat flour used for making breads and noodles often causes allergenic and hypersensitivity reactions. Therefore, gluten-free cereals are in greater

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Table 1.5 Application of CRISPR/Cas technology towards improvement of qualitative traits in crops Crop species Oryza sativa

Target gene(s) OsBEI and OsSBEIIb

OsWaxy OsCYP97A4, OsDSM2, OsCCD4a, OsCCD4b, and OsCCD7 fad2 OsBADH2

Triticum aestivum

Brassica napus

TaSBEIIa

α-gliadin α-gliadin and γ-gliadin BnLPAT2 fad2 BnTT8 BnSFAR4 and BnSFAR5 BnITPK

Lycopersicon esculentum

SlGAD2 and GlGAD3 GABA-TP1, GABA-TP2, GABA-TP3, CAT9 SGR1, LCY-E, Blc,LCYB2 and LCY-B1 SlDDB1, SlDET1 andSlCYC-B CycB, FW2.2 GGP1, GLV3

Solanum tuberosum Ipomoea batatas Banana

StGBSS IpSBEII

LCYε

Improved trait Resistant starch and amylose content Amylose content Carotenoid accumulation High oleic acid content Improved fragrance Resistant starch and amylose content Low gluten Low gluten Starch and oil content High oleic acid content Oil and protein Seed oil content Phytic acid content GABA content

References Sun et al. (2017)

Zhang et al. (2018a, b, c, d) Dong et al. (2020)

Abe et al. (2018) Usman et al. (2020); Ashokkumar et al. (2020); Tang et al. (2020) Li et al. (2020a, b)

Sánchez-León et al. (2018) Jouanin et al. (2019) Zhang et al. (2019a, b, c, d, e) Huang et al. (2020) Zhai et al. (2019) Karunarathna et al. (2020) Sashidhar et al. (2020) Nonaka et al. (2019)

GABA content

Li et al. (2018a, b, c, d, e)

Lycopene content Accumulation of carotenoid Lycopene content Vitamin C content Amylose content Resistant starch and amylose content β-carotene

Li et al. (2018b) Hunziker et al. (2020) Zsögön et al. (2018) Li et al. (2018a, b, c, d, e) Andersson et al. (2017) Wang et al. (2019)

Kaur et al. (2020) (continued)

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Table 1.5 (continued) Crop species Camelina sativa Arachis hypogea Glycine max

Target gene(s) fad2

Cotton

fad2

fad2 fad2

Improved trait High oleic acid content High oleic acid content High oleic acid content High oleic acid content

References Jiang et al. (2017) Yuan et al. (2019) Al Amin et al. (2020) Chen et al. (2020)

demand in the current diet requirements. Sanchez-Leon and colleagues employed the CRISPR/Cas technology to successfully knock out the α-gliadin genes and generated genome-edited wheat lines with significantly reduced gluten content (Sánchez-León et al. 2018). Consequently, a similar approach was used for targeted modification of both the α- and β-gliadins genes leading to the development of low-gluten transgenic-free bread wheat lines (Jouanin et al. 2019). Similarly, ɣ-aminobutyric acid (GABA) is a non-proteinogenic amino acid with beneficial effect in the treatment of hypertension. CRISPR/Cas9 has been employed for targeted modification of GABA biosynthetic genes GAD2 and GAD3, and the edited tomato plants displayed significant accumulation of GABA in fruits (Nonaka et al. 2019). Up to 19-fold higher GABA accumulation has been realized in tomato mutant lines obtained through precise modification of multiple GABA metabolism genes including GABA-TP1, GABA-TP2, GABA-TP3 and CAT9 (Li et al. 2018a, b, c, d, e). Likewise, lycopene is a carotenoid with antioxidant activities and often recommended for the treatment of cardiovascular disorders. Therefore, increasing lycopene content in fruits and vegetables will have significant health benefits. CRISPR/Cas9-mediated multiplex editing of lycopene biosynthetic genes, like SGR1, lycopene α-cyclase, lycopene β-cyclase and LYC-B2 genes, resulted in more than fivefold increase in tomato lycopene content (Li et al. 2018a, b, c, d, e). This was achieved through simultaneous synthesis of lycopene and inhibiting the formation of α- and β-carotene. Using a similar process, genome-edited bananas were developed with sixfold higher β-carotene, the primary precursor for provitamin A (Kaur et al. 2020). Recently, CRISPR/Cas9 has been utilized for insertion of a carotenoid biosynthesis cassette into the rice genome, and the edited lines showed high accumulation of carotenoid in rice grain without any compromise in the phenotype or yield (Dong et al. 2020). Oleic oil with high amount of mono- and polyunsaturated fatty acids have greater health benefits. Hence, genome editing can be used for the targeted medication of fatty acid desaturase 2 (fad2) genes, whose encoded proteins are involved in the natural switch of linoleic acid to oleic acid in many oil yielding crops. Jiang et al. (2017) obtained fad2 knocked out lines of Camelina sativa with more than 50% increase in oleic acid in the total fatty acid composition. Since then, CRISPR/Cas9-induced fad2 knockout plants with high oiled acid content has been reported in rice, rapeseed, soybean, peanut and cotton (reviewed in Li et al. 2021a, b). All these studies suggest that CRISPR/Cas genome

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editing is an indispensable approach to facilitate precise plant breeding for the improvement of crop yield and quality.

1.3.4

Crop Domestication

Domestication of wild plants into new crop varieties could be significant in the expansion of agricultural practices in degraded and unfavourable site and regions with climatic extremes (Zhang et al. 2018a, b, c, d). De novo domestication can also facilitate genetic and species diversification of agrarian systems and greater fit between crops and specific ecological niches (Fernie and Yan 2019). However, the traditional process of domestication is a time-consuming and labour-intensive process that involves only a limited number of progenitor species and selection of specifically improved traits such as the yield. As the core traits of domestication syndrome from food crop plants include higher harvestable roots, tubers, seeds and fruits, such traits could be used as suitable targets for rapid de novo domestication through genetic modification (Van Tassel et al. 2020). The availability of CRISPR/ Cas genome editing technology has usher in a new era for rapid de novo domestication of novel crops. The early works in this regard have been carried out to accelerate the domestication of the wild tomato, Solanum pimpinellifolium (Li et al. 2018a, b, c, d, e; Zsögön et al. 2018). In one study, CRISPR/Cas9 genome engineering was used for precise alteration of six loci related to yield and productivity in S. pimpinellifolium (Zsögön et al. 2018). The engineered lines demonstrated significant threefold increase in fruit number, tenfold increase in fruit size and up to 500% increase in fruit lycopene accumulation as compared to the cultivated S. lycopersicon. Likewise, Li et al. (2018a, b, c, d, e) performed multiplex CRISPR/Cas9 editing of the coding sequences, cis-regulatory regions and upstream open reading frames (uORFs) of domestication-associated traits in four stresstolerant wild tomato accessions. In addition to retaining the parental properties of disease resistance and salt tolerance, the edited progenies exhibited domesticationrelated phenotypes including increased flower and fruit production, higher day-length sensitivity, reduced shoot architecture and ascorbic acid synthesis (Li et al. 2018a, b, c, d, e). Other than the wild species, many orphan crops although rich in nutritional properties are highly underexploited for wider cultivation and improvement due to undesirable characteristics such as fruit shattering, high vegetative growth and low yield. In a recent study, CRISPR technology was used to mutate multiple orthologues of tomato domestication and improvement genes in the solanaceous orphan crop ‘groundcherry’ (Physalis pruinosa) (Lemmon et al. 2018). The modified lines showed improved domestication traits including increased flower production and fruit size and plant architecture and paved the way for rapid development of targeted allelic diversity in distantly related orphan crops. Trait improvement in potato is hindered by tetrasomic inheritance and vegetative propagation. Ye et al. (2018) knocked out a self-incompatibility gene S-RNase using the CRISPR/ Cas9 system and created self-compatible diploid potatoes that can be propagated by

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seeds. These diploid potatoes can be subsequently used to produce inbred lines that could facilitate genetic gains through molecular breeding. While seed shattering is an advantageous behaviour in wild rice, the trait is responsible for major yield losses in elite domesticated rice varieties. To overcome this, new indica rice lines with lower rates of seed shattering have been developed using the targeted CRISPR/Cas9mediated mutagenesis of the qSH1 gene (Sheng et al. 2020). The qsh1 homozygous mutant lines showed significantly reduced seed shattering without any change in other agronomic traits. In another study, CRISPR/Cas9-based vectors were used for simultaneous multiple gene knockouts in the cultivated African landrace Kabre to address the shortcomings of the unpredictable yield and grain quality albeit with higher tolerance to biotic and abiotic stresses (Lacchini et al. 2020). Disruption of HTD1 homologue resulted in reduced plant stature and diminished lodging, while targeted modification of ‘domestication loci’ GS3, GW2 and GN1A reported improved seed yield. Most recently, genome editing has been utilized for generating new crops with multiple sets of chromosomes to increase the adaptability and directed evolution (Yu et al. 2021). CRISPR/Cas9 technology was used for editing a suite of domestication-related traits in a polyploidy rice strain, PPR1 selected from the tetraploid rice Oryza alta. Homozygous edited progenies reported significant loss of shattering, reduced awn length, increased grain length and greater yield, decreased height and thickened stem and modified flowering times (Yu et al. 2021). This broadly established the role of genome editing in fast tracking the process of evolution and domestication which naturally occurs over thousands of years. Overall, with the availability of reference genome and information about the domestication loci together with efficient transformation system, CRISPR/Cas technology can significantly engineer required traits into any crops to increase the global food security in the face of changing climates.

1.4

Novel CRISPR/Cas-Based Breakthrough for Crop Improvement

While CRISPR/Cas technology has proven itself with great potentials in crop improvement, many desirable crop traits can only be realized through precise DNA insertion and replacement through HR. Therefore, it is imperative to develop robust and novel CRISPR/Cas-based gene editing systems to address the limitations of the existing genome editing toolkits. For instance, many recent researches are concentrated on modifying the cleavage domain of the Cas9 enzyme to exploit its binding activity and simultaneously facilitate cleavage and/or nucleotide modification by a supplementary enzyme. Such modified Cas proteins such as Cas9 Nickase (nCas9, with single cleavage domain) and dead Cas9 (dCas9, without functional cleavage domain) fused with other functional proteins have repurposed the genome engineering approaches by facilitating the development of novel genome editors.

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Base-Editing Technology

A large number of elite agronomic traits in crop plants are associated with single base variations (Henikoff and Comai 2003). Therefore, editing technique that could facilitate precise single base variations in crops is the absolute necessity of the time. The base editors provide a new genome editing approach that facilitates the conversion of one base to another, under the supervision of a sgRNA and without the requirement of a DNA repair template (Komor et al. 2016). A base editor primarily consists of an inactive CRISPR/Cas9 domain (nCas9 or dCas9) for recognizing the target sequence and a deaminase enzyme for base substitution activity (Fig. 1.2a). Among the available base editing techniques, the first recognized approach is the cytosine base editors (CBEs) that catalyzes the conversion of cytosine (C) to uracil (U) which in turn mediates the substitution from C/G pair to T/A pair after subsequent DNA repair and replication by the usage of a cytidine deaminase and uracil glycosylase inhibitor (UBI) (Komor et al. 2016; Zong et al. 2018a, b) (Fig. 1.2a). Additionally, at least four generations of CBEs have been developed with improved sequence structure and cytidine deaminases for greater ability of substitution (Komor et al. 2016; Endo et al. 2019). CBE-mediated editing has been demonstrated in many crops including rice, maize, tomato, wheat and watermelon (reviewed in Mishra et al. 2020). Alternatively, adenine base editors (ABEs) use adenine deaminase to substitute adenine (A) to inosine (I) ultimately leading to A/T to G/C base pair conversion (Gaudelli et al. 2017) (Fig. 1.2a). While no adenine deaminase that can facilitate DNA deamination naturally are available, Prof. David Liu and his group were able to develop an evolved transfer RNA adenosine deaminase (TadA) for the purpose (Gaudelli et al. 2017). ABE 7.10, ABE 8 and ABE 8e have demonstrated significant catalytic activity in the conversion of A/T to G/C base pairs in plants (Ren et al. 2018; Li et al. 2020a, b; Richter et al. 2020). ABEs have been used for multiplex base editing in rice and alteration of desired phenotypes in rapeseed (Kang et al. 2018; Hua et al. 2020a, b). ABE has also been used for developing point mutation within the acetyl-coenzyme A carboxylase (ACC) gene for herbicide-resistance in rice and wheat (Li et al. 2018a, b, c, d, e). In another study, an rBE5 (hAID*¨-XTEN-Cas9n-UGI-NLS) base editor was adopted to introduce point-based genetic variations in the Pi-d2 locus that modulated rice resistance to blast fungus Magnaporthe oryzae (Ren et al. 2018). Most recently, the C to G base editors (CGBE1) equipped with nCas9, Escherichia coli-derived uracil DNA glycosylase (eUNG) and rAPOBEC1 variant (R33A) have reported induced C to G base transversions with reduced indel mutations (Kurt et al. 2021). Likewise, the glycosylase base editors (GBEs) with a specific uracil DNA glycosylase (Ung) for efficient production of base transversions (both C to A and C to G) in mammalian cells (Zhao et al. 2021). However, both of these transversion base editors are yet to be applied in targeting G/C-based mutations in plant species. Overall, base editing platforms have provided a new facet to plant genome editing and associated crop improvement through precise base modification at the targeted genomic loci.

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Fig 1.2 Novel CRISPR technologies used for plant genome editing. (a) CRISPR/Cas9-mediated base editing. Cytidine base editors (CBEs) have nCas9 (D10A) fused with cytidine deaminase (rAPOBAC1/hAID/PmCDA1/hA3A) and an uracil glycosylase inhibitor (UGI) to convert cytosine (C) to uracil (U) causing a C-G to T-A substitution. Adenine base editors (ABEs) have nCas9 (D10A) fused with E. coli-derived adenine deaminase (ecTadA(WT)-ecTadA* heterodimer) to convert adenine (A) to inosine (I) causing a T-A to C-G substitution. (b) CRISPR/Cas9-mediated prime editing. Prime editors (PEs) consist of nCas9 (H840A) fused with a reverse transcriptase (RT) prime editing RNA (contains an sgRNA and an RNA template that will replace sgRNA). The nCas9-RT complex recognizes the target site and replaces the target DNA sequence with new genetic information

1.4.2

Prime-Editing Technology

Prime editors (PEs) are typically based on a search and replace approach by directly placing a new DNA sequence at the target genomic site without the requirement of a DNA template (Anzalone et al. 2019). The prime editors comprise of a catalytically inactive Cas9 enzyme (nCas9 H840A) fused with a reverse transcriptase (RT) domain and a programmable prime editing guide RNA (pegRNA) that exhibit

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the dual role of a template DNA for reverse transcription as well a sgRNA for defining the target site for the RT-mediated modification (Anzalone et al. 2019) (Fig. 1.2b). The creation of a single-stranded break (SSB) on the non-complementary strand of the target site is subsequently followed by the transfer of desired edits from the pegRNA to the target DNA. Multiple strategies of primer editing including the PE1 (with M-MuLV RT fused with nCas9), PE2 (with a pentamutant version of MM-MuLV-RT fused with nCas9) and the PE3 (with added gRNA for concurrent cleavage of the non-edited strand) have significantly increased the efficiency of the primer editor as per the current genome editing requirements (Anzalone et al. 2019). Unlike the base editors which are constrained by their capability for insertion, deletion and development of other transversion mutations, prime editors have the ability for target-specific DNA deletions, insertions as well as 12 different types of base modification without the need of a donor template or creation of DSBs (Anzalone et al. 2019). Prime editing holds great potential for future plant genome editing as it has been demonstrated in wheat and rice (Lin et al. 2020; Li et al. 2020a, b; Tang et al. 2020; Xu et al. 2020; Hua et al. 2020a, b; Butt et al. 2020). Yet, the editing efficiency of the current prime editors is very much lower in plants as compared to animals (reviewed in Li et al. 2021a, b). A recent study has shown that the usage of two pegRNAs encoding the same edits in trans condition together with prime binding sites with a melting temperature of 30  C resulted in 17-fold increase in the PE editing efficiency in rice (Lin et al. 2021a, b). Future research should be focused on more similar study for optimizing the editing efficiency as well as by targeting multiple genes in different crop species to realize the full potential of prime editing technology in crop improvement.

1.5

Perspectives and Future Challenges

Over the last decade, genome editing technologies predominantly the CRISPR/Cas systems have become the most important biotechnological tools, and their applications have facilitated rapid progress in myriads of fields including crop improvement. More than 45 genera of plants across 24 families have been improved or demonstrated a scope for improvement by using the CRISPR/Cas9 and associated genome editing platforms (Shan et al. 2020). Yet, plant genome editing faces major challenges in understanding the molecular mechanism of genome editing for continuous plant improvement, crop evolution and domestications, technical limitations for editing experiments and uncertainty of the regulatory landscape regarding the gene-edited crops. Unlike animals, genome editing in plants makes use of species- and genotypedependent strenuous transformation and regeneration protocols which limit its wider application in agriculture and crop improvement. The tissue culture features of a plant genotype significantly contribute to the successful transformation process. For instance, a large number of elite indica rice cultivars are recalcitrant to the tissue culture process making them inaccessible for improvement through genome editing.

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In a recent study, plant developmental regulators-WUSCHEL (WUS) and SHOOT MERISTEMLESS (STM) were combined with gene editing reagents to develop gene-edited shoots through de novo meristem induction (Maher et al. 2020). Further, the gene-edited shoots were also induced in the soil-grown plants and produced flowers and seeds excluding the need for an aseptic tissue culture process. In another study, a sgRNA fused to an endogenous mobile RNA sequence of the FLOWERING LOCUS T (FT) gene cloned to an RNA virus was used to generate in planta geneedited tobacco lines (Ellison et al. 2020). Also, a sonchus yellow net rhabdovirus (SYNV) was used for DNA-free in planta delivery of the entire CRISPR/Cas9 cassette and achieved 90% of targeted mutations in the regenerated tobacco geneedited plants (Ma et al. 2020). These studies could open ways for extending in planta transformation to a large range of species including the tissue culture recalcitrant types for rapid production of gene-edited germplasm. However, it would be interesting as well as challenging to visualize the adaption of this technology to a wider group of crop plants. Epigenetic modifications including DNA methylation and histone modifications are important controlling factors for multiple biological pathways (Hauser et al. 2011). Recently, CRISPR/Cas toolkit consisting of dCas9 fused with many epigenetic modifier has been used to introduce epigenetic modifications and alteration of targeted gene expression in the parental plants as well as their progenies (Fig. 1.3a). For instance, an epigenome editor comprising the SunTag system, dCas9 fused with a GCN peptide repeats and DNA demethylase TET1cd fused with a single-chain antibody recognizing GCN peptide resulted in targeted demethylation leading to upregulated expression of the FLOWERING WAGENINGEN (FWA) gene in Arabidopsis thaliana (Gallego-Bartolomé et al. 2018). The resultant genome-edited plants showed late-flowering phenotypes which were stably transferred into the progenies without any transgenic effects. Likewise, precise mutation of the Cas9 endonuclase domains, HNH-H841A and RuVC-D10A, has repurposed the CRISPR/ Cas9 system from a site-specific genome editing tool to a targeted genome regulation tool (Qi et al. 2013). This has led to the emergence of new CRISPR/dCas9 tool kit, namely, CRISPR activator (CRISPRa), for inducing the gene expression and CRISPR inhibitors (CRISPRi) for inhibiting the target gene expression (reviewed in Li et al. 2021a, b; Karlson et al. 2021) (Fig. 1.3b). Recently, a CRISPRa toolkit consisting of dCas9-VP64 and dCas9-TV was used for transcriptional activation of Thioredoxin H (TrxH), leading to increased transcription of the target locus and conferred enhanced resistance to sugarcane mosaic virus (SMV) in maize (Gentzel et al. 2020). Additionally, a CRISPRi toolkit consisting of dCas9-SRDX was also used for inactivation of the magnesium chelatase gene (ChlH) resulting in yellow seedling phenotype (Gentzel et al. 2020). Thus, the epigenome and CRISPRa/I technologies have open up new avenues to regulate gene function of multiple beneficial traits in agriculturally important plant species. However, both of these bioengineering strategies depend on the constitutive expression of the introduced transgenes, and the edited plants are technically genetically modified. Therefore, it would be interesting to see how they could be adapted for wide application in crop breeding programmes.

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Fig. 1.3 Overview of deactivated Cas9 (dCAs9)- and RNA- based editing. (a) CRISPRa/CRISPRi editing. dCas9 fused with transcriptional activators (VPR1/VP64) or repressors (KRAB/SRDX) can be used for gene regulation through targeted gene activation or repression. (b) Epigenome editing. dCas9 fused with epigenetic modifying factors such as p300/TET1 and DNMT3/EZH2 can be used for epigenetic regulation through targeted DNA demethylation and histone modification. (c) DNA imaging. dCas9 fused with fluorescent protein can be used for fluorescent imaging and labelling of specific chromosomal regions, (d) RNA-editing. Catalytically inactive Cas 13 (dCas13b) fused with a naturally occurring adenosine deaminase acting on RNA (ADARdd) facilitate adenosine (A) to Inosine (I) substitution in RNA molecule

While CRISPR/Cas9 has precisely edited myriads of genes in different crop species, it is also limited by the requirement of a PAM which restricts its access to many important genetic loci. To overcome this limitation, multiple studies have led to the development of novel Cas variants to have relaxed PAM preferences (Kleinstiver et al. 2015a, b). However, these sites are still limited albeit it has expanded the DNA target sites for CRISPR/Cas platforms. Therefore, development of a PAM-independent CRISPR/Cas system could boost the application of genome editing even at the most inaccessible target sites. A recent research by Walton et al.

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(2020) used a serial structure guided engineering approach to develop a modified SpCas9 variant, SpG with relaxed third nucleotide position (50 -NGN-30 ) for PAM that exhibited robust targeting and editing activities. They further optimized the SpCas9 variant to relax the secondary nucleotide and developed the SpRY variant (50 -NRN/NYN-30 ) that could recognize most of the PAM sequences in the genome. Such near-PAMless variants could be significant in editing all most all DNA sequences with negligible side effects. However, this can also lead to more off-target editing. Off-targeting of genes could potentially affect the precision breeding of agriculturally important traits especially those associated with yield and productivity. Hence it is imperative to reduce if not eliminate off-target effects as much as possible for efficient genome editing results. Currently, genome editing through ribonucleoprotein (RNP) transformation significantly reduce off-target effect mainly due to limited lifespan of the CRISPR/Cas in the targeted cells. Further modification of the RNA-based delivery system with the addition of special enzymes like DNA polymerase having proofreading function could increase the delivery efficiency without the off-target effects. Also, combining the PAM relaxed Cas9 variants with high-fidelity SpCas9 variants could also eliminate off-target effect without disturbing the gene-editing activity (Kleinstiver et al. 2015a, b). The availability of desired genes for specific traits is crucial for fine tuning of complex biological mechanisms including crop yield and quality (Kwon et al. 2019). Unfortunately, the current available sets of target genes for genome editing are insufficient due to lack of information about genes, pathways and their interaction with environmental factors in many plant species. Therefore, development of largescale mutant libraries at the whole genome level could open up new possibilities for crop improvement. At least two groups have used CRISPR/Cas genome editing technologies to construct genome-wide mutant libraries in rice. Lu et al. (2017) used a pooled set of sgRNAs targeting 39045 genes and generated 91004 targeted loss-offunction mutants. Likewise, Meng et al. (2017) selected 12802 rice genes and targeted them with 25604 corresponding sgRNAs to develop a mutant library consisting of 14000 loss-of-function mutants. Similar studies could be undertaken in other crop species to identify targets with useful agronomic traits as well as compatible for precise genome editing. Also, a majority of superior traits in plants are governed by single-base variations (SNPs). While genome editing-induced targeted insertion and deletion of genes is a reality, base substitution is still out of reach for a majority of crop plants owing to its low efficiency. The recently developed ‘base editing’ and ‘prime editing’ holds greater promise in this regard (Rees and Liu 2018; Anzalone et al. 2019). Base editing with specificity for C to T and A to G substitution is already established in different crop species (Rees and Liu 2018). Compared to this, prime editing has the advantage of facilitating 12 different base substitutions within a small genomic region without DSBs (Anzalone et al. 2019). Establishment of these technologies in varied crop species will broaden the scope of genome editing in crop improvement. Lastly, the actual potential of the genome editing technology depends upon its ultimate transfer from the lab to field. However, the ambiguity in the regulatory frameworks for genome-edited plants across different countries is the biggest

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bottleneck in global acceptability of the gene-edited crops. Transgene-free geneedited plants could be practically indistinguishable from those obtained through natural mutagenesis. Therefore, they are not currently subjected to the regulatory framework of genetically modified organisms (GMOs) in some countries including in the United States of America (Wolt et al. 2016). However, the European Union Court of Justice has categorized the CRISPR-induced plant mutants as GMOs (Callaway 2018). Therefore, a science-based regulatory framework for gene-edited crops may be proposed for continued innovation in crop breeding and better public and regulatory acceptance for genome-edited plants. A similar step has already been taken by a group of scientists from leading biotech nations (Huang et al. 2016). More efforts in this regard will certainly strengthen public trust and sway the regulatory policies which in turn will influence the application of genome editing in agriculture and crop improvement. Acknowledgement This research was supported by grants from the Key R & D Project of Science and Technology of Sichuan Province (2021YFN0003), National Natural Science Foundation of China (U20A2035) (to K. Zhao) and the Talented Young Scientist Program of China (to R. Mishra). Conflict of Interest Statement The authors declare that they have no conflict of interest. Author Contributions The manuscript was written by RM and RKJ. The manuscript was critically revised by KZ. All the authors have read and approved the final manuscript.

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Yue E, Cao H, Liu B (2020) OsmiR535, a potential genetic editing target for drought and salinity stress tolerance in Oryza sativa. Plants (Basel) 9(10):1337. https://doi.org/10.3390/ plants9101337 Zafar SA, Zaidi SS, Gaba Y, Singla-Pareek SL, Dhankher OP, Li X, Mansoor S, Pareek A (2020) Engineering abiotic stress tolerance via CRISPR/Cas-mediated genome editing. J Exp Bot 71(2):470–479. https://doi.org/10.1093/jxb/erz476 Zaidi SS, Mukhtar MS, Mansoor S (2018) Genome editing: targeting susceptibility genes for plant disease resistance. Trends Biotechnol. 36(9):898–906. https://doi.org/10.1016/j.tibtech.2018. 04.005 Zeng Y, Wen J, Zhao W, Wang Q, Huang W (2020) Rational improvement of rice yield and cold tolerance by editing the three genes OsPIN5b, GS3, and OsMYB30 with the CRISPR-Cas9 system. Front Plant Sci 10:1663. https://doi.org/10.3389/fpls.2019.01663 Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin EV, Zhang F (2015) Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163(3):759–771. https://doi.org/10.1016/j. cell.2015.09.038 Zhai Y, Yu K, Cai S, Hu L, Amoo O, Xu L, Yang Y, Ma B, Jiao Y, Zhang C, Khan MHU, Khan SU, Fan C, Zhou Y (2019) Targeted mutagenesis of BnTT8 homologs controls yellow seed coat development for effective oil production in Brassica napus L. Plant Biotechnol J 18(5): 1153–1168. https://doi.org/10.1111/pbi.13281 Zhang Y, Liang Z, Zong Y, Wang Y, Liu J, Chen K, Qiu JL, Gao C (2016) Efficient and transgenefree genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA. Nat Commun 7:12617. https://doi.org/10.1038/ncomms12617 Zhang Y, Bai Y, Wu G, Zou S, Chen Y, Gao C, Tang D (2017) Simultaneous modification of three homoeologs of TaEDR1 by genome editing enhances powdery mildew resistance in wheat. Plant J 91(4):714–724. https://doi.org/10.1111/tpj.13599 Zhang H, Li Y, Zhu JK (2018a) Developing naturally stress-resistant crops for a sustainable agriculture. Nat Plants 4:989–996. https://doi.org/10.1038/s41477-018-0309-4 Zhang J, Zhang H, Botella JR, Zhu JK (2018b) Generation of new glutinous rice by CRISPR/Cas9targeted mutagenesis of the Waxy gene in elite rice varieties. J Integr Plant Biol 60(5):369–375 Zhang S, Wang L, Zhao R, Yu W, Li R, Li Y, Sheng J, Shen L (2018c) Knockout of SlMAPK3 reduced disease resistance to Botrytis cinerea in tomato plants. J Agric Food Chem 66(34): 8949–8956. https://doi.org/10.1021/acs.jafc.8b02191 Zhang Z, Ge X, Luo X, Wang P, Fan Q, Hu G, Xiao J, Li F, Wu J (2018d) Simultaneous editing of two copies of Gh14-3-3d confers enhanced transgene-cean plant defense against Verticillium dahliae in allotetraploid upland cotton. Front Plant Sci 9:842. https://doi.org/10.3389/fpls.2018. 00842 Zhang A, Liu Y, Wang F, Li T, Chen Z, Kong D, Bi J, Zhang F, Luo X, Wang J, Tang J, Yu X, Liu G, Luo L (2019a) Enhanced rice salinity tolerance via CRISPR/Cas9-targeted mutagenesis of the OsRR22 gene. Mol Breed 39:47. https://doi.org/10.1007/s11032-019-0954-y Zhang K, Nie L, Cheng Q, Yin Y, Chen K, Qi F, Zou D, Liu H, Zhao W, Wang B, Li M (2019b) Effective editing for lysophosphatidic acid acyltransferase 2/5 in allotetraploid rapeseed (Brassica napus L.) using CRISPR-Cas9 system. Biotechnol Biofuels 12:225. https://doi.org/10. 1186/s13068-019-1567-8 Zhang R, Liu J, Chai Z, Chen S, Bai Y, Zong Y, Chen K, Li J, Jiang L, Gao C (2019c) Generation of herbicide tolerance traits and a new selectable marker in wheat using base editing. Nat Plants 5(5):480–485. https://doi.org/10.1038/s41477-019-0405-0 Zhang T, Zhao Y, Ye J, Cao X, Xu C, Chen B, An H, Jiao Y, Zhang F, Yang X, Zhou G (2019d) Establishing CRISPR/Cas13a immune system conferring RNA virus resistance in both dicot and monocot plants. Plant Biotechnol J 17(7):1185–1187. https://doi.org/10.1111/pbi.13095 Zhang Y, Massel K, Godwin ID, Gao C (2019e) Applications and potential of genome editing in crop improvement. Genome Biol 19(1):210. https://doi.org/10.1186/s13059-018-1586-y

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Zhang D, Zhang Z, Unver T, Zhang B (2020a) CRISPR/Cas: A powerful tool for gene function study and crop improvement. J Adv Res 29:207–221. https://doi.org/10.1016/j.jare.2020.10.003 Zhang M, Liu Q, Yang X, Xu J, Liu G, Yao X, Ren R, Xu J, Lou L (2020b) CRISPR/Cas9-mediated mutagenesis of Clpsk1 in watermelon to confer resistance to Fusarium oxysporum f.sp. niveum. Plant Cell Rep 39(5):589–595. https://doi.org/10.1007/s00299-020-02516-0 Zhang Y, Pribil M, Palmgren M, Gao C (2020c) A CRISPR way for accelerating improvement of food crops. Nat Food 1:200–205. https://doi.org/10.1038/s43016-020-0051-8 Zhao D, Li J, Li S, Xin X, Hu M, Price MA, Rosser SJ, Bi C, Zhang X (2021) Glycosylase base editors enable C-to-A and C-to-G base changes. Nat Biotechnol 39(1):35–40. https://doi.org/10. 1038/s41587-020-0592-2 Zheng M, Zhang L, Tang M, Liu J, Liu H, Yang H, Fan S, Terzaghi W, Wang H, Hua W (2020) Knockout of two BnaMAX1 homologs by CRISPR/Cas9-targeted mutagenesis improves plant architecture and increases yield in rapeseed (Brassica napus L.). Plant Biotechnol J 18(3): 644–654. https://doi.org/10.1111/pbi.13228 Zhong Z, Sretenovic S, Ren Q, Yang L, Bao Y, Qi C, Yuan M, He Y, Liu S, Liu X, Wang J, Huang L, Wang Y, Baby D, Wang D, Zhang T, Qi Y, Zhang Y (2019) Improving plant genome editing with high-fidelity xCas9 and non-canonical PAM-targeting Cas9-NG. Mol Plant 12(7): 1027–1036. https://doi.org/10.1016/j.molp.2019.03.011 Zhou J, Peng Z, Long J, Sosso D, Liu B, Eom JS, Huang S, Liu S, Vera Cruz C, Frommer WB, White FF, Yang B (2015) Gene targeting by the TAL effector PthXo2 reveals cryptic resistance gene for bacterial blight of rice. Plant J 82(4):632–643. https://doi.org/10.1111/tpj.12838 Zhou J, Xin X, He Y, Chen H, Li Q, Tang X, Zhong Z, Deng K, Zheng X, Akher SA, Cai G, Qi Y, Zhang Y (2019) Multiplex QTL editing of grain-related genes improves yield in elite rice varieties. Plant Cell Rep 38(4):475–485. https://doi.org/10.1007/s00299-018-2340-3 Zong Y, Song Q, Li C, Jin S, Zhang D, Wang Y (2018a) Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A. Nat Biotechnol 36:950–953. https://doi.org/ 10.1038/nbt.4261 Zong Y, Song Q, Li C, Jin S, Zhang D, Wang Y, Qiu JL, Gao C (2018b) Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A. Nat Biotechnol 36:950–953. https://doi.org/10.1038/nbt.4261 Zsögön A, Čermák T, Naves ER, Notini MM, Edel KH, Weinl S, Freschi L, Voytas DF, Kudla J, Peres LEP (2018) De novo domestication of wild tomato using genome editing. Nat Biotechnol 36:1211–1216. https://doi.org/10.1038/nbt.4272

Part II

Genome Editing Tools and Approaches

Chapter 2

Genome Editing Tools for Food Security Kashaf Zafar, Muhammad Zuhaib Khan, Imran Amin, and Shahid Mansoor

Abstract Food security is a very important issue to meet the demands of continuously increasing population. To fulfil the global demand, current improvement rate is not enough. There is a dire need of improving food crops to enhance production and deal with changing climate, reduction in existing water resources and less arable land. Traditional approaches are also being used for improvement in agricultural productivity, but the dawn of genome editing technologies has transformed the plant research. In this chapter, we have discussed different genome editing tools including some latest tools like prime editor which can be used for food security. Various examples from different food crops have been discussed where crop traits like yield, quality, nutrition, biotic and abiotic stress resistance were improved using genome editing tools. The prospects of these technologies have also been addressed including factors affecting release of genome-edited crops in the field. Keywords Genome editing · CRISPR-Cas9 · Food security · Agriculture

2.1

Introduction

Food security is estimated to become one of the greatest problems that mankind will face in the future. The environmental change and continuously growing population have raised further concerns regarding global food security. The world population is increasing unceasingly and expected to cross 9.6 billion till 2050 (Tilman et al. K. Zafar Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Constituent College of Pakistan Institute of Engineering and Applied Sciences, Faisalabad, Pakistan Department of Biotechnology, Balochistan University of Information Technology, Engineering and Management Sciences (BUITEMS), Quetta, Pakistan M. Z. Khan · I. Amin · S. Mansoor (*) Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Constituent College of Pakistan Institute of Engineering and Applied Sciences, Faisalabad, Pakistan © Springer Nature Singapore Pte Ltd. 2022 K. Zhao et al. (eds.), Genome Editing Technologies for Crop Improvement, https://doi.org/10.1007/978-981-19-0600-8_2

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2011). To feed this rapidly increasing population, food security is an important concern (Khoury et al. 2014; Tilman et al. 2011). The future of human life is dependent on crops because they not only provide food, but fuel and many other consumable resources are also dependent on crops. Thus, they have enormous potential to serve society. But the current improvement rate is not enough to meet the future demands (Ray et al. 2013). So, there is a dire need of improving food crops to enhance production and deal with changing climate, reduction in existing water resources and less arable land. Innovations in crop breeding technology are needed to resolve current challenges. The main strategies which are being used for this purpose include traditional cross breeding, mutation breeding and genetic engineering. Although conventional plant breeding approaches have played a key role in agricultural productivity improvement and all of these methods have their own importance, creating desirable traits in crops through traditional breeding can take years (Ansari et al. 2017; Scheben et al. 2017). In this chapter, we will briefly discuss how we are trending from traditional methods of crop improvement towards the use of genome editing tools to deal with food security.

2.2

Traditional Approaches for Crop Improvement

There are various approaches which have been used for improvement in agricultural productivity. Mutation breeding is one of them, which uses a chemical or radiation source as a mutagen to create desired traits in the crops, but this method is also laborious and time consuming due to screening of large populations to find the one with desired traits (Pacher and Puchta 2017). Other traditional methods including marker-assisted breeding cannot be speed up to meet the growing demands in improvement of food crops (Scheben et al. 2017). Genetic engineering has been established in different crops for various improvements (Hashmi et al. 2011). To improve traits, different genes related to different traits can be engineered into elite varieties, but the limitation of this method is expensive regulatory evaluation. Many food crops do not reach the end users due to public concern and problems in commercialization of genetically modified crops (Prado et al. 2014), although improved crop yields, lesser use of pesticides, reduced poverty and enhanced nutrition were observed by smallholder farmers after acceptance of the genetically modified (GM) crops (Qaim 2016). Furthermore, years of research has shown the similar risk factor associated with GM and conventional crops. But still the incorporation of GM crops has been done by only a few emerging technologies like Pakistan, India, China, South Africa and Bangladesh (Nicolia et al. 2014). Many Asian and African countries are resilient to endorse the use of GM crops mainly because of incorrectly perceived hazards and uncertainties of losing European export markets.

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Trending Towards Genome Editing

The idea of developing tools for targeted modification in plants came from the outcomes of gene targeting in Nicotiana tabacum protoplast as well as from the identification of double-stranded breaks (DSBs) (Prado et al. 2014; Puchta et al. 1993). With these discoveries, the researchers came to know that sequence-specific nucleases (SSNs) are reprogrammable having the ability to create double-stranded breaks (DSBs) at target site. The development of such tools enables researchers to modify endogenous genes to alter genetic functions of various organisms (Barrangou and Horvath 2017; Doudna and Charpentier 2014; Gaj et al. 2013; Hsu et al. 2014; Joung and Sander 2013; Kim and Kim 2014; Sander and Joung 2014; Voytas 2013). There are four major classes of DNA-binding proteins which have been used so far to accomplish genome editing. These four classes comprise of meganucleases (Voytas 2013; Puchta and Fauser 2014), zinc-finger nucleases (Urnov et al. 2010), TALENs (Boch and Bonas 2010; Boch et al. 2009; Moscou and Bogdanove 2009; Schornack et al. 2013) and CRISPR tools (Li et al. 2013; Nekrasov et al. 2013; Shan et al. 2013; Shimatani et al. 2017; Zong et al. 2017). These tools are presented in Fig. 2.1, and details are presented in other chapters of this book.

2.4

Combining Genome Editing with Speed Breeding

To deal with food security, genome editing platforms can be combined with speed breeding. Speed breeding is a method which was developed in 2018 and has the potential to reduce generation time. In this way, it can fast-track the research and breeding programmes. With speed breeding, it is possible to get six generations per year of wheat, chickpea, barley and pea and four generations per year for canola. Under normal glasshouse conditions, only two to three generations are achieved for these crops. Speed breeding can greatly accelerate plant growth for research purpose including phenotyping, mutant screening and transformation. This strategy has the potential to be used for large-scale crop improvement programmes using costeffective light-emitting diode (LED) supplemental lighting. So, speed breeding can also be combined with genome editing and genomic selection for accelerating the rate of crop improvement (Watson et al. 2018). This can be a very important step for future food security.

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Fig. 2.1 Genome editing tools. (a) Meganucleases, (b) zinc-finger nucleases (ZFNs), (c) transcription activator-like effector nucleases (TALEN), (d) CRISPR-Cas9 and (e) CRISPR-Cpf1. The components of these tools are also presented here. These tools recognize a target site and create a double-stranded break at the target site (represented by red triangles). The type of double-stranded break can be blunt or staggered depending upon the tool used like CRISPR-Cas9 resulted in blunt whereas CRISPR-Cpf1 cause staggered cuts at the target side. These DSBs are then repaired either by non-homologous end joining or homology-directed repair pathway. As a result of this repair, the target site is modified. The modifications can be of any type including insertions, deletions, point mutations and replacements

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Improving Crop Traits for Food Security

Genome editing has been used in last years for developing crop varieties with improved traits. These traits include enhancement in yield, increased disease resistance, abiotic stress tolerance and improved nutritional quality (Fig. 2.2).

2.5.1

Yield Improvement

Yield is primarily determined by gain size, weight and gain number. Many genes have been characterized so far which are involved in affecting crop yield (Xing and Zhang 2010; Bai et al. 2012). The genes which are known to negatively affect yield can be knocked down or knocked out to improve crop yield. The examples of such genes in rice crop are Gn1a, GW2, GS5, DEP1, GS3 and TGW6. Rice genes Gn1a, DEP1, GS3 and IPA1 were targeted for improvement in yield using CRISPR-Cas9, and predicted phenotype was observed in edited plants (Li et al. 2016a). Quantitative trait loci (QTL) editing was also done for this purpose in different genetic backgrounds showing diverse effects on yield (Shen et al. 2018; Zhou et al. 2019). Superior alleles were created using CRISPR-Cas9-based genome editing to deliver higher yield than the non-edited alleles (Huang et al. 2018). Phytohormones, i.e. abscisic acid (ABA), have the ability to control crop yield. ABA encoding

Fig. 2.2 Genome editing applications for food security. There are many examples of genome editing in food crops. Different types of modifications were done to improve yield, quality and nutritional value by targeting various genes related to these traits. Many biotic stress-tolerant food crops were developed having resistance against different bacterial, viral and fungal pathogens. Resistance against abiotic stresses was also developed in food crops using genome editing toolkit. With all these efforts, a normal variety can be transformed into improved variety

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genes were edited to create a high-yielding rice variety with up to 31% more grains (Miao et al. 2018). Hybrid rice breeding is also very important for increase in rice yield. A very important rice line which can be used in hybrid breeding system is thermo-sensitive genetic male sterility (TGMS) line (Zhou et al. 2012). Using CRISPR-Cas9 technology, 11 TGMS lines were created in 1 year, which showed the potential of this system to quicken hybrid rice breeding (Zhou et al. 2016). Another rice gene CCD7 involved in strigolactone biosynthesis was edited resulting in dwarf habit and high-tillering plants (Butt et al. 2018). The phenotypes of developed elite high-yielding cultivars are usually lost during genetic segregation. CRISPR-Cas9 was also used to preserve these phenotypes (Khanday et al. 2019; Wang et al. 2019a). Three alleles of GASR7 gene were targeted in wheat using genome editing (CRISPR-Cas9) showing increase in thousands of grain weight. GASR7 was chosen because it is a negative regulator of kernel weight and width (Zhang et al. 2016). Grain weight and size are very important yield-related traits. Recently, CRISPRCas9-mediated gene editing of TaGW7 was done in wheat. The mutations in B and D genomes showed an increase in grain weight and width (Wang et al. 2019b). These examples revealed the potential of CRISPR-Cas9 for editing wheat, which is very important food crop. In maize, erect leaves can capture more light than conventional leaf structure. Thus, they have the ability to grow at high density. The homozygous recessive genotype of LG1 can result in erect leaf structure (Johnston et al. 2014). So, this gene is important in this regard and can be a potential candidate of genome editing. For proof-of-concept study, this gene was targeted using RNA-guided endonuclease (RGEN) system to begin a desired target mutator (DTM) in maize. These were then shifted through hybridization into receiver lines. The successful editing was done in recipient lines, resulting in maize plant growth with significantly increased density and hence better yield potential (Li et al. 2017). This study also unrevealed an alternative yet valuable approach to perform genome editing. ARGOS 8 is negative regulator of ethylene responses in maize. Overexpression of ARGOS 8 can result in reduced ethylene sensitivity and yield improvement. ARGOS 8 variants were created using CRISPR-Cas9, which showed improved gain yield in drought stress. This indicated that drought-tolerant maize crop with high yield can be developed using CRISPR-Cas9 (Shi et al. 2017). The regulation of flowering is a very important yield factor. In several crops, flowering genes and their interactions can be modified for regulation of flowering time (Subudhi et al. 2018). CRISPR-Cas9 system has been used to knockout flowering-related genes-FT and SP5G to later the flowering time and induce early yield in soybean (Cai et al. 2018) and tomato (Soyk et al. 2017). While CRISPRCas9 accounts for considerable success in yield improvement, a few other phenotypic modifications with potential role in breeding and development of new varieties has been created in Brassica oleraceae, Brassica napus and barley (Lawrenson et al. 2015; Braatz et al. 2017), although considerable success has been made in using CRISPR-Cas9 for yield improvement. But an important point to note is that increased thousand grain weight or enhanced grain yield per plant does not

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essentially result in upgraded crop yield. So, it is necessary to do large-scale field trials to demonstrate the possible agronomic enhancements.

2.5.2

Biotic Stress Tolerance

The biotic stresses are yield-limiting factor because they result in reduced production of food crops, creating a threat for food security. Various pathogens including virus, bacteria and fungus cause different diseases in crops ultimately resulting in crop loss. This disease pressure can be controlled in field via various strategies (Heinrichs and Muniappan 2017). The genome editing had played a very important role in developing disease-resistant crops in the last years. Here we will enlist few examples of genome editing to develop biotic stress tolerance in different food crops. Rice is a very important food crop, but it faces various diseases in the field. One of them is rice bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo). This disease can cause 50% to complete yield loss in promising infection environments (i.e. high humidity) (Mew et al. 1993). Xoo have effector binding elements (EBE) in the promoter region of OsSWEET gene family (OsSWEET11, OsSWEET13 and OsSWEET14). This region can be targeted using genome editing tools to develop resistance against bacterial blight (Makino et al. 2006; Boch and Bonas 2010; Doyle et al. 2013; Chen et al. 2012). TALENs technology was used to do such type of editing to deal with this disease (Li et al. 2012). Among SWEET genes, OsSWEET14 is a major target of Xoo because it has 4 EBEs to interact with various Xoo strains. So different attempts were made to edit the EBEs present in the promoter of this gene using TALENs and CRISPR-Cas9 (Blanvillain-Baufumé et al. 2017; Zafar et al. 2020a). The coding region of OsSWEET13 gene was targeted using CRISPR-Cas9, and the generated mutants showed increased resistance without any phenotypic defects (Zhou et al. 2015b). In another study, OsSWEET11 and OsSWEET14 were targeted for the same purpose causing reduced symptom in edited plants (Jiang et al. 2013). The same genome editing tool was then used to create wide spectrum resistance towards diverse Xoo strains causing bacterial blight (Xu et al. 2019; Oliva et al. 2019). The genome editing was also done in rice to create resistance against viral disease “rice tungro disease” (Macovei et al. 2018). An effort was made to develop resistance against “rice blast” a very damaging fungal disease of rice caused by Magnaporthe oryzae (Liu et al. 2014) using CRISPR-Cas9. The OsERF922 gene was chosen for editing to create resistant lines showing considerably reduced symptoms (Wang et al. 2016). These examples highlighted the importance of genome editing to improve rice crop (Zafar et al. 2020b). Bread wheat is a very important food crop which faces 15–50% decrease in yield due to biotic stresses (leaf rust), depending upon infection occurrence stage (HuertaEspino et al. 2011; Roelfs and Bushnell 1985). CRISPR-Cas9 has been used effectively to fight various pathogens by targeting susceptibility genes. Mildew resistance locus o (MLO) gene is a susceptibility gene which makes the plant susceptible to fungal powdery mildew caused by Blumeria graminis f. sp. The

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resistance was generated by downregulation of this gene through genome editing (Wang et al. 2014). Recently multiplexed genome editing was done in wheat using CRISPR-Cas9 to knockout TaGW2, TaLpx-1 and TaMLO genes, simultaneously (Wang et al. 2018a). Among these, the silencing of TaLpx-1 and TaMLO gene showed resistance against Fusarium graminearum and powdery mildew, respectively (Wang et al. 2018a). A very important and second most consumable vegetable is tomato (Schwartz et al. 2015), which is threatened by different plant pathogens. A potential susceptibility gene SlDMR6-1 was edited using CRIPSR-Cas9 in tomato to develop resistance against Phytophthora spp., Pseudomonas syringae and Xanthomonas spp. (de Toledo Thomazella et al. 2016). In another attempt, resistance was developed against Pseudomonas syringae (PTO) strain DC3000 using CRISPRCas9 (Ortigosa et al. 2019). The powdery mildew-resistant tomato was developed using genome editing by targeting the susceptible allele of powdery mildew “SlMlo1” (Nekrasov et al. 2017). There are various other examples where genome editing was done in grapes (Malnoy et al. 2016), citrus cultivars (Jia et al. 2016; Jia et al. 2017; Peng et al. 2017), banana (Tripathi et al. 2019), cucumber (Chandrasekaran et al. 2016), cassava (Gomez et al. 2019) and barley (Kis et al. 2019; Ji et al. 2015; Zhang et al. 2018) to develop disease resistance.

2.5.3

Abiotic Stress Tolerance

Abiotic stresses are of different types which a crop can face in the field. To deal with these stresses, different approaches including genome editing were used in the past years. Here we will discuss few examples of genome editing to develop abiotic stress tolerance in food crops. Herbicides are important to control various types of grass and dicot weeds. Bispyribac-sodium (BS) and chlorosulfuron are important herbicides developed to deal with various weeds, which mainly target acetolactate synthase (OsALS) gene (Zhou et al. 2007). This gene is involved in the synthesis of branched chain amino acids (valine, leucine and isoleucine), and loss of its activity can result in blockage of branched chain amino acid synthesis. Point mutations were introduced in OsALS gene of rice using TALEN-based genome editing with up to 6.3% efficiency (Li et al. 2016b). CRISPR-Cas9 was also used to target similar gene to develop herbicide resistance (Sun et al. 2016). The phenotypic screening showed that edited plants were resistant against BS, whereas wildtype plants died after 36 days of spraying. There are also many recent examples which showed the potential of different genome editing tools to create herbicide tolerance in rice, wheat and maize (Zhang et al. 2019b; Butt et al. 2020; Li et al. 2020c). Developing cold-tolerant varieties is also very important to improve yield and crop quality. Rice crop is very sensitive to cold at seedling stage. So, different efforts were made in the past using genome editing tools to open new avenues in creating cold-tolerant rice (Huang et al. 2017; Shen et al. 2017). Genome editing was also

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used to create drought resistance in different crops. The mitogen-activated protein kinases3 (SlMAPK3) gene was mutated in tomato using CRIPSR-Cas9. The analysis of edited lines showed the involvement of SLMAPK3 in drought resistance (Wang et al. 2017). In rice, the function of SAPK2 gene was studied in detail by means of CRISPR-Cas9. The SAPK2 mutants were unresponsive to ABA, more sensitive to drought and oxidative stress. These findings suggested that SAPK2 can be an important candidate gene in future crop improvement (Lou et al. 2017). The knowledge of gene function is very important to develop stress-tolerant crops. CRISPR-Cas9 was used in maize to modify ARGOS8 to create drought tolerance. This gene has negative effect on ethylene responses and has low expression in maize plants. The native promoter of this gene was changed into GOS2 promoter using genome editing and homology-directed repair method. Under drought conditions, the ARGOS8 variants showed higher yield than the control maize plants (Shi et al. 2017). Salinity is also an important abiotic factor which is one of the main problems affecting the world crop production. Developing salt-tolerant varieties is the best approach to deal with this problem. CRISPR-Cas9 was used to edit OsRR22 gene of rice, and the seedlings of edited lines were tolerant to salinity as compared to wildtype plants (Farhat et al. 2019a; Zhang et al. 2019a). These examples showed that genome editing tools have great potential to target various genes to confer abiotic stress tolerance in different crops (Abdelrahman et al. 2018).

2.5.4

Quality and Nutritional Improvement

Quality and nutrition of crops are very important for health of people consuming them as food. The valuable component of rice bran oil is oleic acid, which has the potential of suppressing diseases and improving health. For high oleic and low linoleic acid production, OsFAD2-1 knockout rice lines were developed using CRISPR-Cas9. In this way, the fatty acid composition of rice was improved (Abe et al. 2018). Rice storability was upgraded by targeting LOX3 using CRISPR-Cas9, and the deficit of this gene enhanced seed storability (Ma et al. 2015). The rice fragrance is very important trait for quality and good market value. The main gene responsible for fragrance is betaine aldehyde dehydrogenase (Badh2). Suppression of this gene increases fragrance in rice. So TALEN- and CRIPSR-based genome editing was used to disrupt Badh2 and increasing rice aroma (Shan et al. 2015; Shao et al. 2017). Using same technology Osor was directed for alteration in rice, which results in increasing β-carotene in rice calli (Endo et al. 2019). There is also an example of genome editing where the quality of rice was also improved by increasing amylose content in rice by targeting SBEIIb gene (Sun et al. 2017). The quality of a crop can also be improved by eliminating heavy metal accumulation in grains of food crops. Excessive amount of cadmium (Cd) is present in rice grains, so people intake Cd while consuming rice which is hazardous for health (Clemens et al. 2013). CRISPR-Cas9 was used to develop low Cd lines by targeting OsNramp5. The field

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trials of edited lines showed relatively less Cd concentration than the control plants. Tang et al. (2017) reported that that this type of editing did not affect plant yield, whereas plant height was affected in another report by editing similar gene causing lower yield. So Yang et al. (2019) concluded that OsNramp5 should be handled carefully for getting low Cd and high-yielding lines (Tang et al. 2017; Yang et al. 2019). Genome editing was used to create red rice which has high levels of health-promoting nutrients (anthocyanins and proanthocyanidins). The white grains of cultivated rice are due to frameshift deletion in Rc gene. The deletion was revered back using CRISPR-Cas9 in edited lines. These lines exhibited higher anthocyanins and proanthocyanidins accumulation without compromising agronomic traits (Zhu et al. 2019). This type of restoring approach can also be used in other food crops to improve quality. CRISPR-Cas9 was used to target granule-bound starch synthase (GBSS) gene in potato. The disruption of this gene revealed reduced amylose content and higher amylopectin/amylose ratio (Andersson et al. 2017). The same tool was used to develop potato varieties with reduced enzymatic browning by editing StPPO gene (González et al. 2020). The transgene-free genome editing was established in tomato and potato using base editors (Veillet et al. 2019). All of these examples disclosed the ability of genome editing to be used as a convenient and vigorous tool for the improvement of polyploid crops. A health encouraging compound c-aminobutyric acid (GABA) is produced by GABA shunt metabolic pathway (Takayama and Ezura 2015). The genes of this pathway were modified in tomato (Nonaka et al. 2017) and potato (Li et al. 2018a) using CRIPR-Cas9 to improve GABA accumulation. The edited tomatoes can be used as parental lines for hybrid tomato breeding (Lee et al. 2018). The above approaches are examples of using genome editing as an effective tool in turning poor quality crops into improved ones.

2.6

Regulatory Concerns and Status of Genome-Edited Crops

With the development of first genetically modified organism (GMO) in 1995, guidelines were imposed globally to regulate genetically modified crops. In most European countries, marketable production and release of GM crops in field and consumer markets are forbidden. Genetically modified crops are inciting widespread public misunderstanding regardless of their cultivation on 189.8 million hectares (2017) and financial profit of US $18.2 billion in 2016. To differentiate between genetically engineered and transgenic crops, strict laws may require swift action. Modern breeding technology, especially genome editing, is highly capable of providing an alternative to GM crops for reducing regulatory hurdles. New and constant discoveries of GE technology and advances in systems to deliver genome editing components that do not have to introduce a foreign DNA into host crop can seriously undermine legislation on GM crops (Wolter and Puchta 2017). Alien genes

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are transmitted to elite varieties to attain desired characteristics via transgenic cultivation. In comparison to traditional agriculture, transgenic technology has removed all the barriers of crossing and also possesses better genetic variability. But it can also affect human health and the environment. Therefore, GMOs are under close supervision, and their commercialization is limited due to long-term and costly regulatory evaluation procedures (Hartung and Schiemann 2014). Advanced genome editing technology helps create accurate and targeted mutations without DNA sequence integration into the plant’s genome. These technologies enable the production of non-transgenic crops with higher yield, better quality and tolerant to various stresses (Xu et al. 2015). It also reduces the time (4–6 years) to get desired traits into the plant in comparison to other breeding methods which takes 8–12 years (Scheben et al. 2017). There is an initial requirement to evaluate the existing rules and regulations on GMOs. Genome manipulation performed using GE tools is very different from the transgenic approaches. The CIRSPR-Cas9 produces small indels as compared to the insertion of large gene sequences (Feng et al. 2014). These small indels are also commonly found in plants in normal growth environments or can also be created via conventional mutagenic substances. In addition, unlike GMOs, there is no need for stable incorporation of foreign DNA into the genome using CRISPRCas9-mediated genome editing. With regard to regulatory issues, there is not any international regulatory framework present for regulation of genome-edited plants. The United States (US) and European Union (EU), the main stakeholders, have conflicting rules to regulate the release of genome-edited plants. The GE crops have been exempted from firm rules and regulations by the United States Department of Agriculture (USDA) (Waltz 2016), whereas the EU policies have same regulatory procedure for GE plants and GMOs (Callaway 2018). Such strict procedures may prevent investment in GE techniques and restrict its usage in European countries. In addition to the United States, various countries, such as Argentina, Chile and Brazil, have introduced innovative regulatory rules for GE crops. The new discoveries in biotechnology have been permitted effortlessly in Canada due to Canadian traitbased Scheme. A robust and genuine regulatory policy is needed to discriminate between GE plants and GMOs. But many countries do not have such regulatory policies for GE plants. The widespread use of GE strategies poses many challenges for regulators as this requires widespread technical knowledge and reliable assessment of procedures for regulation of GE plants. Manifestly, scientific guidelines to assess GE plants in a similar way as plants produced through conventional breeding are needed to increase GE applications to improve crops. So many countries have modified their regulatory rulebooks and relieved GE crops from firm regulatory rules of GMOs depending on some stringent requirements like having no alien gene, type of modified trait and having no pest characteristics (Ishii and Araki 2017). The USDA has recognized GE as a much quicker form of traditional crop improvement. They also allowed more than a dozen case studies on genome editing for cultivation without regulatory authorizations (Waltz 2016). In recent years, investments in bio-companies have improved five times (Brinegar et al. 2017). In conclusion, comprehensive regulatory frameworks may be developed to guide the use of GE

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tools without any restriction which is essential for overall food supply and security (Shao et al. 2018).

2.7

Conclusion and Prospects

Genome editing tools hold a great potential of improving crops for future food security. Using these tools, the target genes can be directly modified without the need of mating process. With many other benefits, the genome editing tools are becoming an excellent choice due to ease and efficacy of using them to improve food crops. Beside the usage of CRISPR-Cas9 in crop improvement through gene replacement and disruption, regulation of gene expression is also critical for growth and development of plants. For instance, the induced expression of gene(s) in maize exhibit significant resistance against ear mould and leaf spot disease (Chintamanani et al. 2008). So, CRISPR-based gene regulation (CRISPRi or CRISPRa) can also be used to enhance or reduce gene expression for trait improvement. The information of genes, understanding of function and their involvement in different pathways are also very important. With incredible progression in next-generation sequencing (NGS), the total number of sequences from different food crops is continuously increasing in databases (Buermans and Den Dunnen 2014). Using sequencing information, the more accurate experiments of genome editing can be designed with minimum off-target effects. There are many bioinformatic tools which provide an ease in designing CRISPR-related experiments. These tools help in designing gRNA libraries, identification of off-targets and predicting mutations at the target site (Cancellieri et al. 2020; Li et al. 2020b; Allen et al. 2019). All of these advancements will be helpful for improving food crops using genome editing tools. Besides having all the designing and genome editing tools, an efficient delivery system is also required to transfer genome editing components efficiently and accurately. Although many delivery systems have been used in the past, still the research is going on to explore more systems. The genome editors are in continuous struggle to improve tools for efficient, transgene-free genome editing with no need of tissue culturing (Li et al. 2020a; Ji et al. 2020). The transgene-free genome editing is very important, because there are many biosafety and regulatory concerns especially for food crops. Although transgene-free genome editing has been achieved successfully in many crops (Li et al. 2020a; He et al. 2018; Zhang et al. 2016; Liang et al. 2017; Veillet et al. 2019), still there is a dire need to educate general public and address different societal and biosafety concerns to effectively use genome editing for food crops. There is also a need to reconsider regulatory issues on genome-edited crops for their successful release in the field. Besides all these reservations, genome editing tools can provide a promising way to develop new crop varieties with improved traits to deal with future food security (Table 2.1).

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Table 2.1 Examples of using new technologies to deal food security Sr. no. 1

Food crop/ plant Tomato

2

Tomato, potato

3

Wheat

4

Bread wheat, maize Spring wheat

5 6

Rice

Method ZFN CRISPR-Cas9 CRISPR-Cas9

Effect Cholesterol reduction Leaf development Resistance against powdery mildew Transgene-free edited plants development

CRISPRCas9base editing TALEN CRISPR-Cas9

Heritable modification Low-gluten variety

CRISPR-Cas9

Herbicide resistance

Speed breeding Speed breeding CRISPR-Cas9 CRISPR-Cas9 CRISPR-Cas9 CRISPR-Cpf1 CRISPR-Cpf1

Resistance to stem rust Four to six generations per year Enhanced fragrance Increased grain weight Resistance against salt stress Stomatal density regulation in leaf Stable mRNA equal Herbicide resistance Early flowering Herbicide resistance Mutation frequencies doubled Four to six generations/year Lignin content Controlling pod shattering resistance in oilseed rape Resistance to leaf rust Four to six generations/year Two to three generations/year Four to six generations/year Decreased carotenoid content

7

Soybean

8

Maize

CRISPR-Cas9 CRISPR-Cas9 CRISPR-Cas9 CRISPR-Cpf1 Speed breeding

9 10

Poplar Rapeseed

CRISPR-Cas9 CRISPR-Cas9

11

Barley

12 13 14 15 16

Peanut Canola Sweet orange Apple Watermelon

Speed breeding Speed breeding Speed breeding Speed breeding CRISPR-Cas9

17

Banana

CRISPR-Cas9

18

Kiwifruit

CRISPR-Cas9

19

Wild strawberry

CRISPR-Cas9

CRISPR-Cas9 CRISPR-Cas9

Resistance to fire blight disease Albino phenotype and decrease of carotenoid content Albino phenotype and decrease of carotenoid content Albino phenotype and decrease of carotenoid content Auxin biosynthesis and signalling

References Sawai et al. (2014) Brooks et al. (2014) Nekrasov et al. (2017) Veillet et al. (2019)

Luo et al. (2019) Sánchez-León et al. (2018) Svitashev et al. (2015)

Riaz (2018) Watson et al. (2018) Zhou et al. (2015a) Xu et al. (2016) Farhat et al. (2019b) Yin et al. (2019) Li et al. (2018b), Malzahn et al. (2019) Li et al. (2015) (Han et al. (2019) Svitashev et al. (2015) Malzahn et al. (2019) O’Connor et al. (2013)) Zhou et al. (2015c) Zaman et al. (2019) Watson et al. (2018) Watson et al. (2018) O’Connor et al. (2013) Watson et al. (2018) Zhang et al. (2017) Malnoy et al. (2016) Tian et al. (2017) Kaur et al. (2018) Wang et al. (2018b) Zhou et al. (2018)

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Chapter 3

CRISPR-Cas9/Cpf1-Based Multigene Editing in Crops Sanjeev Kumar, Yogita N. Sarki, Johni Debbarma, and Channakeshavaiah Chikkaputtaiah

Abstract Genome editing with CRISPR/Cas9 has now become an effective tool for gene function analysis and crop improvement. Recent advances in this technology enable efficient targeted modification for various trait improvements in crop plants. CRISPR/Cas-based multigene editing in plants is known to edit the targets in various ways that include introduction of alien DNA inserts, generation of knockout mutations and altering of nitrogenous bases via deamination. The most crucial steps of CRISPR/Cas-based genome editing are the selection of appropriate target region, designing suitable guide RNAs, generation of genetically engineered constructs and finally transformation of the plant system by CRISPR/Cas components. In this chapter, we have focused on recent developments in CRISPR/Cas9 and its variants, their applications in plant genome editing, various base-editing tools that enable targeted nucleotide substitutions, delivery of CRISPR/Cas9 components into living cells, their editing specificity and homology-directed repair in crop improvement. We have also summarized the applications of multigene editing for various trait improvements and techniques involved for fine-tuning gene regulation in crop plants, current limitation of CRISPR/Cas technology and future prospects of genome editing for its bright future in agriculture. Keywords CRISPR/Cas · Cpf1 · Cas13 · Multiplex · Multigene · Target specificity · Crop improvement

S. Kumar (*) Department of Biosciences and Bioengineering, Indian Institute of Technology, Guwahati, India e-mail: [email protected] Y. N. Sarki · J. Debbarma · C. Chikkaputtaiah (*) Biological Sciences and Technology Division, CSIR-North East Institute of Science and Technology, Jorhat, Assam, India Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 K. Zhao et al. (eds.), Genome Editing Technologies for Crop Improvement, https://doi.org/10.1007/978-981-19-0600-8_3

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Abbreviations CRELs DSB GMO HDR MSBSP-PCR NHEJ PAM

3.1

CRISPR-edited lines Double-stranded breaks Genetically engineered organism Homology-directed repair Mutation sites-based specific primers polymerase chain reaction Non-homologous end joining Photospacer active motif

Introduction

Over the last decade, the CRISPR/Cas9-based genome editing has seen a lot of use to target the genome precisely for creating mutations at particular locations. Genome editing, as it is widely known, has become prominent as a common and feasible technology that is carried out in labs worldwide on numerous organisms such as crop plants. The mechanism of CRISPR/Cas9 involves using synthetic nucleases that have the ability to generate double-stranded breaks (DSBs) in DNA which leads to mutations when the endogenous DNA repair mechanism repairs the breakage. Targeted gene editing technologies, in contrast to traditional random mutagenesis and screening procedures, can greatly speed up the process of developing target gene mutants. Previous research has revealed that DSBs in nuclear DNA can activate two distinct endogenous DNA repair pathways: homologous recombination (HR) and non-homologous end joining (NHEJ), which are both responsible for small or major chromosomal alterations (Sonoda et al. 2006). Initially, technologies like homing-endonucleases or meganucleases (Antunes et al. 2012; Gao et al. 2010), zinc-finger nucleases (ZFN) (Osakabe et al. 2010; Townsend et al. 2009) and transcription activator-like effector nucleases (TALENs) (Bogdanove and Voytas 2011; Li et al. 2011) were adopted for genome-targeted editing. ZFNs and TALENs have been employed to successfully target genes in both plants and animals (Li et al. 2012; Wang et al. 2013). The construction of the binding domain of ZFNs, on the contrary, is technically difficult. The clustered regularly interspaced short palindromic repeats system (CRISPR), a revolutionary technology extracted and derived from the prokaryotic immune system, has had a significant impact on expanding the possibility of precision genome editing in recent years (Feng et al. 2013). The most appealing feature of the CRISPR system is its flexible nature that allows to edit the targets at specific locations within the genome, and hence it is widely adopted (Belhaj et al. 2015; Bortesi and Fischer 2015). The CRISPR technique has the added benefit of being able to make transformed plants that bypass the regulatory categories that are frequently used in conjunction with transgenic plants in some countries (Voytas and Gao 2014). Only two components are required for CRISPR/

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Cas to function: single-guide RNA (sgRNA) and CRISPR-associated protein or effector. Genome editing has been accomplished on plants with considerable success, keeping in mind the current agricultural manifesto as well as the constant need to produce crops with stronger and improved characteristics such as improved yield and enhanced stress tolerance, either for biotic or abiotic stress. In the last few years, the CRISPR/Cas system has been widely employed with plants, with studies involving plants from a diverse group of plant families (Cong et al. 2013; Feng et al. 2013; Jiang et al. 2013). In 2013, the first reports of CRISPR/Cas9 editing in plants surfaced, describing successful applications to Arabidopsis (Arabidopsis thaliana) (Li et al. 2013). This was followed by. tobacco (Nicotiana benthamiana) (Jiang et al. 2013), sorghum (Sorghum bicolor), wheat (Triticum aestivum) (Jiang et al. 2013), rice (Jiang et al. 2013; Shan et al. 2013), tomato (Solanum lycopersicum L.) (Brooks et al. 2014), soybean (Glycine max) (Jacobs et al. 2015) and various other crops. Newer and better variations can be obtained by introducing targeted mutations and new features through a CRIPSR-Cas-based advanced technique; therefore, consistent research on food crops will considerably help globally. In this chapter, we described a quick glance on mechanism of CRISPR-Cas9/Cpf1 genome editing, overview of multiplex-multigene editing, applications in crop plants, limitations of this technology, off-target effects, bio-safety regulations-related issues and future prospects of this technology.

3.2

The Mechanism of CRISPR/Cas9 System

The CRISPR/Cas system consists of Cas proteins that perform different activities, such as those of helicases or nucleases (Makarova et al. 2002). CRISPR/Cas9 cleaves foreign DNA via two components, Cas9 and sgRNA. Cas9 is a DNA endonuclease that has originated from different bacterias. The sgRNA is a synthetic RNA of approximately 100 nt length. Its 50 -end has a 20-nt sequence that acts as a guide sequence to identify the specific target sequence accompanied by a protospacer adjacent motif (PAM) sequence, which is often the consensus NGG (N, any nucleotide; G, guanine) and extremely important for selection of targets. In type II-A, the signature protein, Cas9 nuclease, is given the charge of identification of PAM in potential targets (Heler et al. 2015).

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CRISPR/Cas Produce Homozygous (Biallelic) Mutants at T0 and Multiple Knockouts

The majority of modifications caused by the CRISPR/Cas9 system are insertions or deletions that occur 3 bp upstream of the PAM region and are usually close to the DSB site (Cong et al. 2013). The targeted gene region is amplified by polymerase chain reaction (PCR) and sequenced to investigate the nature of mutations induced by CRISPR. The efficiency of CRISPR-mediated mutagenesis varies depending on the targeted sequences and plant species; hence, a variety of approaches for mutant screening have been developed, including the T7 endonuclease I (T7EI) assay, surveyor nuclease assay, PCR/restriction enzyme (RE) assay, the fluorescent PCR-capillary gel electrophoresis methods, high-resolution melting (HRM) analysis-based assays, PAGE-based methods and annealing at critical temperature PCR (ACT-PCR) assays (Cong et al. 2013; Montgomery et al. 2007; Shan et al. 2014; Thomas et al. 2014; Zhu et al. 2014). Individuals having mutations in the T0 or following generations would be identified using these methods. All of these methods have been successfully implemented, but they all have drawbacks, such as being time-consuming, having low detection specificity, labour intensive, requiring expensive equipment or being unable to identify individuals with homozygous mutations in PAGE-based methods. The usefulness of the mutation sites-based-specific primers polymerase chain reaction (MSBSP-PCR) method was verified by targeting NtCRTISO gene by CRISPR/Cas9 construct cloned in a binary vector (Wang et al. 2015b). A total of 39 putative T0 transgenic tobacco lines were produced by the process of Agrobacterium-mediated transformation. The amplification of the fragment comprising the targeted region in NtCRTISO was carried out from pure genomic DNA using the CRTISO-F/CRTISO-R primers to validate the NtCRTISO in the T0 putative events. A fragment comprising the targeted location in NtCRTISO was amplified from genomic DNA using the CRTISO-F/CRTISO-R primers for the validation of NtCRTISO in the T0 putative events. The genomic DNA was used as template for the first round of PCR using primers CRTISO-F/CRTISO-R, and the same amounts of amplification products were used as templates in another set of PCR using CRTISO-T/CRTISO-R set of primers for the direct screening of the T0 transgenic lines. The presence of mutations in both the alleles were determined by the absence of amplification in PCR reactions, although this method is unable to distinguish between homozygous and biallelic mutations. The second PCR analysis was carried out to further analyze the data by sequencing the primary amplification products (primers CRTISO-F/CRTISO-R). Sequence analysis identified 3 plants previously identified by MSBSP-PCR to be homozygous mutants, while the rest 13 plants contained heterozygous mutations (Guo et al. 2018).

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Rise of CRISPR 2.0: An Improved CRISPR/Cas9 Tool for Genome Editing in Plants

Recently, CRISPR system has emerged as a versatile molecular tool for genome editing in many organisms. Therefore, the selection of targeting sites is the critical step in CRISPR/Cas9 technology. Till date, a large number of bioinformatic tools have been created to optimize sgRNA design for various organisms (Ding et al. 2016), including CRISPR-P, which was previously developed for plant sgRNA design (Lei et al. 2014) with minimal off-target potentials which are similar as the previous version but include many new features for guide sequence analysis such as: (1) It supports sgRNA design for 49 plant genomes, covering almost all available plant species that have well-assembled genomes so far. (2) CRISPR-P 2.0 makes use of a modified scoring system for the calculation of the on-targeting efficiency and the off-targeting potential of sgRNAs for Streptococcus pyogenes Cas9, which is the most commonly used Cas9 protein till date. The scoring system in CRISPR-P 2.0 is founded on the latest studies on SpCas9 specificity and efficiency in genome editing. (3) It supports the design of guide sequences for various CRISPR/Cas systems, including various Cas9 endonucleases and Cpf1 (Zetsche et al. 2015a). (4) It also supports the comprehensive analysis of the guide sequence in CRISPR-P 2.0, which includes the restriction endonuclease site, GC content in the sequences, microhomology sequences flanking the targeting site and the secondary structure of sgRNA. (5) It is also possible to identify sgRNA from specific sequences. It also allows users to submit custom sequences if their genome/sequence is not included in the list of selectable genomes, as well as identify sgRNAs. With these unique features, CRISPR-P 2.0 provides a more efficient bioinformatic tool to help in the designing of CRISPR/Cas9 genome editing in plants.

3.5

Cas9 and Cpf1: The Lead Players in the Game of Genome Editing

The CRISPR/Cas system has been widely utilized for genome editing efficiently in the past couple of years. The ribonucleoproteins Cas9 (Jinek et al. 2012) and Cpf1 (Zetsche et al. 2015a) have been ground-breaking discoveries that are propelling the genome-editing tool CRISPR into greater applications day by day. The CRISPR effector molecules such as Cas9/Cpf1 do not require any processing. It can function with just the presence of a mature crRNA, so it is possible to target varied regions of the genome by designing a synthetic guide RNA, which can be an assembly of both the crRNA:tracrRNA (Cas9) or only crRNA (Cpf1). It possesses a 20 nt sequence complementary to the target region with a protospacer adjacent motif site (PAM site). PAM identifies the target and plays a significant role in target binding. Cas9 requires a GC-rich PAM sequence being characterized by the sequence of NGG.

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Recently, different Cas9 varieties have been reported and designed. Also, Cas9 from other species are widely used. A few mentions of the different variants of Cas9 are Cas9 nickase and Cas9n (Fauser et al. 2014). In this particular variant, there is inactivation of one of the endonuclease domains of Cas9 (RuvC) which leads to the generation of singlestrand cuts instead of double cuts. The NmCas9 or Cas9 isolated from N. meningitides has an alternative PAM site and decreased off-target potential (Hou et al. 2013). A catalytically dead Cas9 (dCas9) can be used for transcriptionbased studies (Qi et al. 2013). With the invention of new methods and technologies, the CRISPR/Cas variants have been developed, and Cas12a (formerly named Cpf1) is added to the list, and, more recently, the RNase Cas13 (formerly named C2C2) have the potential to be used in the field of plant research and breeding. The Class 2 type V CRISPR effector from Prevotella and Francisella 1, or Cpf1, is slowly gaining popularity for its ability to create double-stranded DNA breaks. In spite of the fact that it belongs to the same class of effectors as Cas9, there is a remarkable difference between the two Cas. Cpf1 does not require an additional tracrRNA for mature crRNA function; second, Cpf1 is linked with a A/T-rich PAM site, with the sequence being TTN or TTTN. The location of seed region for Cpf1 at the 50 end of the protospacer is advantageous because Cpf1 cleavage will occur away from the seed region at the target. Hence, this will make the target site available for other rounds of Cpf1-based targeting and cleavage as the indels embedded will be away from the target. The preceding CRISPR effectors and their other variants are useful in broadening the application of CRISPR in genome editing. They have the ability to target multiple parts of the genome, including both G/C- and A/T-rich regions; they will be able to generate more accurate genome editing at numerous sites. Furthermore, because complimentary overhangs could significantly change the repair from NHEJ to HR, the CRISPR/Cas12a system could be a useful tool for plants to increase the induction of genomic modifications via HR. Targeted insertions via HR utilizing FnCas12a and LbCas12a were achieved in rice, and at least for FnCas12a, with greater rates than SpCas9-based experiments (Begemann et al. 2017). The recent additions to the CRISPR toolbox are Cas13, formerly known as C2c2 and C2c6 in case of Cas13a and Cas13b, respectively. Due to its unique characteristics, Cas13 was assigned to class II type VI (Shmakov et al. 2015). However, this becomes an important discovery in the field of genome engineering. This promiscuous RNA cleavage after activation could not be detected in eukaryotic cells, at least with the Cas13 orthologues that showed high activity in these cells (Cox et al. 2017). Moreover, Cas13 proteins, like Cas12a, have the ability to process pre-crRNA without the use of a tracrRNA, which is mediated by a different domain called the Helical1 domain. This crRNA maturation activity could be used to address several targets simultaneously in a straightforward and efficient manner (Cox et al. 2017). A wide range of targeted RNA alterations are now possible due to the availability of an efficient and specific RNA targeting CRISPR system.

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Assembly and Delivery of CRISPR/Cas9 Components into Living Cells Physical and Chemical Delivery of CRISPR/Cas Components

The simplicity of designing CRISPR/Cas9 for the editing or altering specific genomic loci proposes a new way to learn about the functions of gene on genome-wide scale and repairing genetic defects. The CRISPR/Cas9 technology has demonstrated a lot of promise in gene editing, and this gene editing machinery can be delivered into cells in three different ways: (1) plasmid DNA that encodes both the Cas9 protein and the guide RNA, (2) messenger RNA (mRNA) for Cas9 translation and a separated single-guide RNA, and (3) ribonucleoprotein complex, composed of the guide RNA and Cas9 protein (Carboni et al. 2019). The most straightforward delivery approach is represented by the ribonucleoprotein complex since it bypasses additional transcription and/or translation passages and provides the most transient functionality of genome editing with reduced off-target effects. Moreover, the delivery and uptake of Cas9 protein are constrained by a number of physiological barriers due to the large size of the Cas9 protein, the positive charge of Cas9 protein and the strong negative charge of guide RNA (Carboni et al. 2019). CRISPR/Cas delivery platforms are divided into two groups based on the nature of delivery vectors: (a) viral and (b) non-viral vectors. As the viral vectors are highly immunogenic, they are more efficient in delivery. Non-viral vectors are less immunogenic, and they can be described as physical or chemical, based on the the nature of the process of the carriers involved. The physical methods serve as an option for CRISPR/Cas9 complex delivery as they enable cell transfection by the creation of transient defects in the phospholipid bilayer of cell membrane or by direct injection. Microinjection is another easy physical technique which involves the direct injection of CRISPR/Cas9 at the cellular level by using micro-scale needles. It is a very efficient transfection technique which allows precise delivery into cytoplasm or nucleus by directly overcoming all the extracellular barriers that usually hinder efficient delivery. However, the process requires manual injection on each targeted cell, and this is translated in high degree of sophistication and time-consuming for the preparation of samples which also make it impractical for experiments in which a large number of cells are involved (Carboni et al. 2019). The other most common method of delivery (both in vitro and in vivo) is electroporation, which involves the application of electrical field onto cell membrane, due to which there is an increase in the cell membrane permeability towards biomolecules. This is very efficient and safe, but it also has several disadvantages, such as irreversible transformations in cell membrane physiology, loss of cell mobility and cell death. There are several other approaches for delivery of CRISPR/Cas such as mechanical deformation (Ran et al. 2013), sonoporation (Fan et al. 2012), laser irradiation

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(Tirlapur and König 2002) and osmocytosis (D’Astolfo et al. 2015), but these methods can result in the damage of cells. On the contrary, chemical methods represent a viable strategy for in vivo delivery of CRISPR/Cas9 complex, due to their flexibility and versatility. The process involves the chemical modification of deliverable cargos (Staahl et al. 2017) or self-assembly of Cas9-sgRNA complex with various organic or hybrid materials, allowing access to different delivery nanoplatforms which can be achieved by encapsulation into lipid vesicles or other hybrid and polymeric/organic nanosized systems or by coordination onto the surface of metallic nanoparticles, then coated with polymeric shells. In all cases, the cargo is protected from external degradation and recognition by the host immune system due to the presence of an external protective layer, thus ensuring prolonged circulation times. The majority of the delivery performance is determined by the physical properties of the delivery entity, and this can be customized by proper molecular design of components, offering the advantage of enabling the platforms to release their cargos upon application of external triggers or in response to the cytosolic environment of targeted cells (Carboni et al. 2019). Recently, non-viral vectors in the form of nanoparticles have received a great attention. The simplest vector used for CRISPR/Cas9 delivery is based on Ca3 (PO4)2 (Kingston et al. 2003). The ease of tailoring nanoparticles’ design and properties according to CRISPR/Cas9 conformation is the main advantage of using nanoparticles in the process of CRISPR/Cas9 delivery.

3.6.2

Agrobacterium tumefaciens-Mediated Delivery of CRISPR/Cas Components

The Agrobacterium tumefaciens-mediated binary vector system is by far the most successful system to introduce foreign gene in both monocotyledons and dicotyledons (Kuluev et al. 2019). What more, the stable or transient expression of T-DNA vector carrying the CRISPR/Cas components has become widely used method for plant genome editing. A. tumefaciens-mediated stable delivery of CRISPR/Cas components has been realized in Arabidopsis thaliana, Nicotiana tabacum, Oryza sativa and Sorghum bicolor (Jiang et al. 2013; Miao et al. 2013; Feng et al. 2013, 2014; Fauser et al. 2014). At the same time, agroinfiltration (Schob et al. 1997)based transient expression of Cas9/guide RNA has been achieved in N. benthamiana (Nekrasov et al. 2013). According to theory, due to relatively short transgene expression, transient expression has the advantage of fewer edited off-target sites. In some citrus species, agroinfiltration was employed for genome editing under conditions of transient expression (Jia and Wang 2014). In other studies, Agrobacterium-based vectors have facilitated multiplex genome editing in multiple crop species including rice, tomato, maize, grapes and cassava (Ma et al. 2015; Pan et al. 2016; Char et al. 2017; Nakajima et al. 2017; Odipio et al. 2017).

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Delivery of CRISPR/Cas Components into Plant Cells Using Agrobacterium rhizogenes

Another approach allows the use of bacterium Agrobacterium rhizogenes, as a platform for delivery of CRISPR/Cas components. The A. rhizogenes can induce hairy root formation that may indirectly confirm successful genome editing and aid in selecting genetically modified forms (Ron et al. 2014). For example, the successful editing of a reporter gene (variants of green fluorescent protein GFP) in tomato using a plant binary vector carrying CRISPR/Cas components was reported (Ron et al. 2014). This delivery method was used earlier to deliver components of genome editing based on a “zinc finger” nuclease (Curtin et al. 2011). In comparison with A. tumefaciens, the use of A. rhizogenes better indicates a successful editing event. While the rol- genes within wild A. rhizogenes cause root growth, the T-DNA carrying Cas9 gene and guide RNA cassette facilitate the actual targeted mutation at the same time (Jacobs and Martin 2016; Kiryushkin et al. 2022). Thus, the appearance of hairy roots is an indication that foreign DNA has been introduced into a plant cell.

3.6.4

Plant Virus-Mediated Delivery of CRISPR/Cas Components

Another common approach was the use of plant viruses as delivery agents; in this case, viruses were primarily used to produce an increased amount of guide RNAs, which significantly improved the efficiency of genome editing. Genetically modified plant geminiviruses allow greater transformation efficiency without requiring a stable insertion into the plant genome (Baltes et al. 2014). Geminiviruses belong to a large plant virus family Geminiviridae, which infect a wide range of monocotyledon and dicotyledon plants. This viral species’ genome is represented as singlestranded DNA. When a plant cell is introduced, it becomes double-stranded and actively replicates itself using a rolling-circle mechanism and host enzymes. However, owing to their small genome sizes (2.5–2.8 kb), they are frequently unable to carry extended foreign DNA fragments (such as genes encoding Cas nucleases), even if their modified genome contains only viral DNA replication regions. As a result, it was proposed that geminiviruses be used primarily to produce more guide RNA. Virus-induced gene silencing (VIGS) refers to the conventional practice of inducing gene silencing. (Kumagai et al. 1995; Ruiz et al. 1998), and VIGE (virusbased gRNA delivery system for CRISPR/Cas9-mediated plant genome editing) was the name given to one of the variants of genome editing using plant viruses (Yin et al. 2015). The modified genome of the cabbage leaf curl virus (CaLCuV), which belongs to the genus Begomovirus, contained a gene encoding guide RNA; the genome was inserted into T-DNA within a binary Agrobacterium-based vector and used for

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transformation on tobacco (N. benthamiana) (Yin et al. 2015). Similarly, the use of other geminivirus strain, such as bean yellow dwarf virus (BeYDV), wheat dwarf virus (WDV), were used in other studies for the genome editing of rice (Oryza sativa) (Wang et al. 2017b), tomato (S. lycopersicum) (Čermák et al. 2015), potato (Solanum tuberosum L.) (Butler et al. 2016) and wheat (Triticum aestivum L.) (Gil-Humanes et al. 2017).

3.6.5

Delivery of CRISPR/Cas Components by Biolistics

A common method employed to obtain transgenic plants and for transformation in monocotyledons is the biolistic delivery of CRISPR/Cas components into explants. This approach is also being used to modify plant genomes. For instance, Shan et al. (2013) used particle bombardment to insert a CRISPR/Cas9 vector targeting the Phytoene desaturase gene. The resultant rice plant showed the expected white phenotype (Shan et al. 2013). Other studies have shown that editing genes in all three sub-genomes of soft hexaploid wheat (Tricticum aestivum) are responsible for attracting the infectious causative agent of powdery mildew (Wang et al. 2014; Zhang et al. 2017). Similarly, physical delivery of a CRISPR/Cas9 vector targeting the Acatolactate synthase in rice demonstrated effective tolerance to herbicide (Sun et al. 2016). Subsequently, this was popularized as the transiently expressing CRISPR/Cas9 DNA system (TECCDNA) (Zhang et al. 2016).

3.6.6

Delivery of CRISPR/Cas Components Via Protoplast Transformation

Several methods for delivering CRISPR/Cas components into plants include biolistic gene delivery and protoplast PEG-mediated transformation. This approach is used to create transgenic plants by altering the genomes with few modifications. Previous studies reported the successful genome editing of rice (Oryza sativa) protoplasts by inserting the constructs containing the requisite CRISPR/Cas components (Feng et al. 2013). Along with using Agrobacterium-mediated transformation to edit the genomes of rice and Arabidopsis using stably produced Cas9 nuclease and guide RNA, Arabidopsis protoplast transformation was also used to give transitory production of the comparable editing components (Feng et al. 2013). The protoplast transfection approach using ribonucleoprotein (RNP) particles consisting of guide RNA/Cas9 nuclease complex was applied for a directed genome editing of grape (V. vinifera), apple tree (Malus domestica Borkh.) and petunia (Petunia Juss) (Malnoy et al. 2016; Subburaj et al. 2016).

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CRISPR/Cas9 and Cpf1 for Single-, Dual-Gene Multigene Editing in Crop Plants

The CRISPR-associated Cas9 system has been widely used to perform genetic studies and crop improvement. Over the last few years, significant progress has been made by enhancing the CRISPR/Cas9 systems in plants (Puchta 2017; Mao et al. 2019). A variety of genome editing approaches including the single-, dual- and multigene editing CRISPR/CAs9 system has been carried out in many crops (Lloyd and Meinke, 2012; Chikkaputtaiah et al. 2017; Debbarma et al. 2019; Saikia et al. 2020).

3.7.1

Dual-Gene Editing

Since the introduction of the CRISPR/Cas system, various reports have surfaced about the simultaneous targeting of two genes. Do et al. (2019) reported the editing of homologous genes GmFAD2–1A and GmFAD2–1B present in soybean that regulate the content of monounsaturated fats in soybean seeds via CRISPR/Cas9 gene editing. The genes encode fatty acid desaturase 2, which converts monosaturated oleic acid (C18:1) to polyunsaturated linoleic acid (C18:2). To edit the homologues simultaneously, two gRNAs were designed targeting the second exons of GmFAD2–1A and GmFAD2–1B. The two guides could cleave two sites with a spacing of 1Kb apart. The editing was performed both in the transgenic and stable soybean plants. In the stable lines, ten randomly selected T0 events were characterized. All the selected, edited lines observed various mutations via genotyping, including insertions, small and large deletions and inversions in the GmFAD2 genes. While 77.8% of the T1 progenies displayed the GmFAD2 mutation, 40% of T1 progenies exhibited null alleles for both GmFAD2 genes (Do et al. 2019). Further, the CRISPR-mediated modification of both GmFAD2 genes resulted in 80% increase in oleic acid content. In another study, CRISPR editing of GABA-TP1 and GABA-TP3 reported significant increase in ɤ-amino butyric acid in Solanum lycopersicum (Li et al. 2018a). Likewise, the CRISPR/Cas9 system facilitated effective development of full knockout of all four alleles of GBSS gene in tetraploid potato (Andersson et al. 2017). Also, the full-length 10 kb deletion was observed while targeting in-between DENSE AND ERECT PANICLE1 (DEP1) and Os09g0442100 gene downstream of DEP1 in rice (Wang et al. 2017a). Similarly, the TaDREB and TaERF3 genes targeted by CRISPR/Cas9 in wheat showed gradual increase in response to dehydration condition (Kim et al. 2018).

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Multigene Editing

Multiplex approaches that target many genes/loci simultaneously can help researchers figure out what role individual genes play in intracellular signalling networks and contribute to the development of complex, multigenic agronomic features in crop plants. CRISPR/Cpf1 is a very recent addition to genome editing tools. Its application has been widely reported in plant and animal system, thus, making it feasible and the easiest way for multiplexing. It targets multiple genes than Cas9 system that requires large constructs for expression of multiple sgRNA cassettes, which are more strenuous to construct and unstable resulting in reduction of transformation efficiency (Zetsche et al. 2015a; Fonfara et al. 2016; Wang et al. 2017b). A detailed scheme of workflow of multiplex-multigene CRISPR/Cas genome editing system for the development of multi-stress tolerance is given in Fig. 3.1. In recent past, some studies are carried using multigene editing approach in crop plants (summarized in Table 3.1). Wang et al. (2017b) demonstrated the feasibility of multiplex gene editing in rice crop model. They used FnCpf1 targeting four members of receptor-like kinases (OsRLKs): OsRLK-798 (LOC_Os02g04430), OsRLK-799 (LOC_Os02g07960), OsRLK-802 (LOC_Os01g39600) and OsRLK803 (LOC_Os06g04370) and LbCpf1 to edit four OsBEL genes of the CYP81A family: OsBEL-230 (LOC_Os03g55230), OsBEL-240 (LOC_Os03g55240), OsBEL-250 (LOC_Os03g55250) and OsBEL-260 (LOC_Os03g55260). The construct of the FnCpf1/LbCpf1 multiplex gene editing system consisted of FnCpf1/ LbCpf1 expression cassette and a multi-crRNA expression cassette that included each of 23–24 bp guide sequence separated by 20 bp (FnCpf1) or 21 bp (LbCpf1) direct repeats (DR) that were ligated in tandem and driven by one OsU6 promoter. The T0 CRISPR-edited plants showed robust multiplex gene editing events. The four OsRLK genes targeted by FnCpf1 showed the mutation frequency of 43.8–75% and 40–60% for the four OsBEL genes targeted by LbCpf1. Hu et al. (2019) exhibited multi-site editing and multiple gene editing to produce various types of mutations in tomato. They developed a Cas9 system consisting of two binary vectors pHNCas9HT and pHNCas9. pHNCas9HT was utilized to construct sgRNA expression cassettes without PCR and for direct transformation of the Agrobacterium tumefaciens. The ligation of several sgRNA expression cassettes into the pHNCas9 in one pot reaction was possible by Golden Gate mechanism. SlACS2 and SlACS4 genes are involved in the process of ethylene synthesis during the ripening of tomato fruit. Two sgRNAs were designed to target the genes driven by AtU3b and AtU3d promoters. The sgRNA expression cassettes were amplified by one-step PCR. Construction of pHNCas9:SlACS2 and 4 was performed via the Golden Gate cloning followed by transformation of tomato. A total of nine T0 lines were obtained for these constructs. The SlACS2 gene was mutated in five lines (L1, L2, L4, L6, L7 and L9), while the SlACS4 gene was altered in four lines (L1, L2, L4, L6, L7 and L9), according to direct PCR sequencing data (L2, L4, L6 and L9). However, only

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Fig. 3.1 A schematic workflow of development of multi-stress tolerance in plants through multiplex-multigene CRISPR-Cas genome editing system

Oryza sativa

Oryza sativa

Oryza sativa

Solanum lycopersicum L. Solanum lycopersicum L.

2

3

4

5

6

Crops Oryza sativa

Sl. no. 1

SlGRAS8, SlACS2 and 4

1. Gn1a 2. GS3 3. IPA1 4. LCD1 5. LCT1 6. NrAMP5 7. NrAMP1 Solyc08g075770

Target genes 1. OsSBEIIb 2. OsPDS 1. OsRLK-798 2. OsRLK-799 3. OsRLK-802 4. OsRLK-803 5. OsBEL-230 6. OsBEL-240 7. OsBEL1. OsEPSPS 2. OsBEL 3. OsPDS

1. Function as transmembrane ion transport or as a cell surface receptor 2. Susceptible to Fusarium wilt 1. Plant development and defense

1. 5-Enolpyruvylshikimate 3-phosphate synthase 2. Bentazon-sensitive lethal 3. Phytoene desaturase (4)–(6) cadmium accumulation-related genes

Annotation 1. Encodes a starch branching enzyme IIb 2. Encodes a phytoenedesaturase (1)–(4) Receptor-like kinases (5)–(8) Bentazon sensitive lethal

5–16 12–62 1–73

0–100

42.9–100

1. 2. 3.

(1)– (4) 43.8–75 (5)– (8) 40–60

Editing efficiency (%) 9–17

Cas9

Cas9

Cpf1

Cpf1

Cpf1

Cas9/ Cpf1 Cpf1

Multigene

Single

Multigene

Single

Single gene

Single/ dual/ multigene Dual gene

Table 3.1 Summary of single-, dual- and multigene editing in plants using CRISPR-Cas9/Cpf1 genome editing system

Multiplex

Single

Multiplex

Single

Multiplex

Single/ multiplex Single

Hu et al. (2019)

Prihatna et al. (2018)

Xu et al. (2019)

Wang et al. (2017b)

References Li et al. (2018b) Wang et al. (2017b)

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Zea mays

Hordeum vulgare

Brassica oleracea Gossypium hirsutum

11

13

14

15

17

16

12

Glycine max

Nicotiana tabacum Nicotiana attenuata Zea Mays

10

9

8

Solanum lycopersicum L. Triticum aestivum Triticum aestivum

7

Cloroplastos alterados (GhCLA) FAD2-1A FAD2-1B

BolC.GA4.a

HvPM19

1. Maize glossy2 (gl2)

Allen oxide cyclase ZmIPK

NtPDS

TaGW2-A1, B1 and D1 1. GW2T2, 2. LPX1T2 3. MLOT1

SlEIN2 and SlERFE1

Conversion of oleic acid to linoleic acid ( fatty acid desaturase)

Chloroplast synthesis

HvPM19 encodes an ABA-inducible plasma membrane protein which acts as a positive regulator of grain dormancy Dwarf stature and affect the pod valve margin

1. Mutated inositol phosphate kinase (IPK) enzyme reduced phosphorous production in maize seeds 2. Help in digestion to mono gastric animal Epicuticular wax formation in juvenile leaves

1. Increase in grain size and weight 2. TaLPX1-1 gene encodes 9-lipoxygenase, rendered wheat resistant to Fusarium graminearum 3. Powdery mildew disease resistance Phytoene desaturase (NtPDS)-induced albino phenotype Key enzyme for jasmonic acid biosynthesis

Negative regulator of grain size and weight

1. Fruit development 2. Ethylene signalling

0–11.7

1–94.12

10

10–23

1. 90–100 2. 0–60

2–19

0.1–1

Cpf1

Cpf1

Cas9

Single

1. Cas9 2. Cpf1 Cas9

Dual

Single

Single

Single

Single

Single

Single

Multigene

Multigene

Multigene

Cas9

Cpf1

Cas9

Cas9

>30

15

Cas9

Cas9

5.5

0–100

Single

Single

Multiplex

Multiplex

Single

Multiplex

Single

Single

Single

Single

Multiplex

Kim et al. (2017)

Lawrenson et al. (2015) Li et al. (2019)

Lawrenson et al. (2015)

Lee et al. (2019)

Lin et al. (2018) Kim et al. (2017) Liang et al. (2014), Lin et al. (2018)

Wang et al. (2018b) Wang et al. (2018a)

Hu et al. (2019)

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about 66 percent of SlACS2 and 4 have mutated at target 2. SlACS2 and 4 at target 1 caused only a few base modifications.

3.8

Application of CRISPR/Cas Technology in Crop Plants

Agricultural plants were the next to benefit following the successful use of the CRISPR method for genome editing and validation of its potential in model plants such as Arabidopsis and tobacco. Also, due to the precise nature of the CRISPR system, it is possible to generate new traits efficiently. This CRISPR technique has been applied to many crop plants across a wide spectrum by targeting single or multiple genes. Plants such as Sorghum bicolor (Jiang et al. 2013), Glycine max or soybean (Jacobs et al. 2015; Li et al. 2015), Solanum lycopersicum (Brooks et al. 2014), Solanum tuberosum (Wang et al. 2015a), and even plants such as grapes (Vitis vinifera L.) have been targeted and modified (Ren et al. 2016). The CRISPR system has not been limited to vegetable crops, as previously stated, but has also been used to essential food crops such as wheat and rice. Both wheat and rice have been considered important both economically and agriculturally as they are responsible for feeding most of the world’s population. Accelerated research in Triticum aestivum (Upadhyay et al. 2013) and Oryza sativa (Mikami et al. 2015) is being conducted to generate better varieties of these crop plants to satisfy the growing need for them. With CRISPR/Cas9, around 25 plant species and over 100 genes have been successfully edited, and numerous desired features in important crops have been developed.

3.9

Construction of Multigene Editing Vectors

The versatility of CRISPR/Cas system has been successfully established for targeted mutagenesis in rice (Li et al. 2018b; Xu et al. 2019), Arabidopsis (Schindele and Puchta 2019), tobacco (Endo et al. 2016), maize (Lee et al. 2019), soybean (Kim et al. 2017), cotton (Li et al. 2019), etc. Consecutive rounds of conventional cloning can insert a few sgRNA expression cassettes that contain multiple targets into a single binary vector (Zhou et al. 2014; Yan et al. 2015). Alternately, multiple restriction enzymes that form sequential compatible palindromic sticky ends can be used to clone a few (usually up to three) sgRNA expression cassettes into a vector (Zhang et al. 2015; Wang et al. 2015b). Thus, the traditional cloning method can be a disadvantage in multiplexing as it requires multiple rounds of cloning, which is timeconsuming. Only a few guide RNA expression cassettes can be cloned into the CRISPR binary vectors. According to Engler et al. (2008), another cloning strategy known as the Golden Gate cloning method can concurrently and proficiently ligate various DNA fragments in a given order. This cloning method employs distinguished type II

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restriction enzymes that cleave outside of their recognition sequence, such as BsaI, leading to generation of sequential compatible, non-palindromic four base flanking overhangs among multiple DNA fragments which can prevent self-ligation and non-compatible end ligation. Thus, this method is feasible for the linking of several DNA fragments in a designed order in a single reaction. On the basis of the Golden Gate method, Ma et al. (2015) developed two sets of CRISPR/Cas9 vector systems to prepare CRISPR/Cas9 binary constructs with multiple PCR-prepared sgRNA expression cassettes in a single cloning reaction. In addition, the Gibson Assembly based cloning approach involving the assembling of multiple DNA fragments with overlapping ends has also been used. The co-ordinated activity of T5 exonuclease, Phusion DNA polymerase and Taq DNA ligase can efficiently join multiple DNA fragments with homologous termini (Gibson et al. 2009). Utilizing the above explained two cloning techniques, 20 pYLCRISPR/Cas9Pubi-H-based constructs were developed to target genes in rice. Each construct carried up to eight sgRNA expression cassettes driven by the OsU6 and OsU3 promoters. Additionally, four pYLCRISPR/Cas9P35S-H-based constructs carrying one to three sgRNA expression cassettes driven by the AtU3 and/or AtU6 promoters were also designed to target genes in Arabidopsis. An alternate strategy to develop CRISPR/Cas9 vector for sgRNA cassette construction and assembly was designed. The multiple single sgRNA cassettes were cloned in intermediate vectors followed by digestion, and then using Golden Gate ligation, the recovered sgRNA cassettes were cloned into a binary vector (Lowder et al. 2015). Multigene targeting will lead to highly efficient editing, resulting in a high chance of desirable phenotypic characters in less time. Precise key genes selection can target more than one pathway resulting in multi-stress tolerance in crops, increased crop production, etc. Yu et al. (2018) has reported that the use of single sgRNA seed in multiplex CRISPR/Cas systems can be advantageous to reduce off-target gene editing in Arabidopsis. Multiplexing will result in a high frequency of large deletions of the target gene. Despite the tremendous capacity and widespread application of CRISPR technology multigene editing, it has certain limitations. Molecular screening to select the transgene-free CRISPR-edited lines (CRELs) will be challenging due to multiplex-multigene editing.

3.10

Current Limitation of CRISPR/Cas Technology

When examining the likelihood of off-target edits during crop plant genome editing, it’s critical to evaluate the source and magnitude of genome modifications that are found in plants under natural conditions. Plants are continually exposed to various kinds of environmental stresses, including desiccation and rehydration, UV-B radiation, ozone, soil and air pollution, which can cause a range of DNA damage products, including single-stranded breaks (SSBs) and double-stranded DNA breaks (DSBs) as a result of stress response (Galhardo et al. 2007; Rosenberg et al. 2012).

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DSBs are one of the most important types of DNA damage because if they are not repaired exactly, they can cause cell death, genetic information loss or genotoxic effects (Roy et al. 2013). In higher plants, the DSBs are mainly repaired by NHEJ mechanisms (Waterworth et al. 2011; Singh et al. 2011). The presence of accurate DNA repair mechanisms in the exonic region is essential to ensure genomic stability, fertility and genetic diversity, and error-prone repair acts as a resource of natural mutational variety which is crucial for plant evolution and valuable for crop improvement (Christensen 2013). Plant breeders have typically used chemical or physical genotoxins to create plant mutations. In this instance, DSBs occur at random in the genome, and error-prone repair produces just a small percentage of favourable phenotypes (Pacher and Puchta 2017). Radiation mutagenesis has developed over 3000 crop cultivars that are utilized worldwide without regulatory risk evaluation. CRISPR/Cas9 or other genome-editing reagents are far more sitespecific than analogous alterations sought by traditional plant breeding procedures. DSBs occurring in off-target regions are likely comparable to spontaneously generated mutations (Pacher and Puchta 2017).

3.10.1 Off-Target Effects Off-target mutation is undoubtedly a major concern in the evolution of the CRISPR/ Cas9 system in plants. In many plants, it has been observed that the frequency of undesired mutations using the CRISPR/Cas system is minimal (Peng et al. 2017; Wolt et al. 2016). Whole genome sequencing (WGS) and deep sequencing have been used to investigate the CRISPR/Cas9 specificity in Arabidopsis thaliana (Feng et al. 2014; Peterson et al. 2016). The results showed that low Cas9 protein expression levels are responsible for the high specificity of CRISPR/Cas9 in plants, which resulted in undetectable quantities of off-target mutation (Peterson et al. 2016; Feng et al. 2014). Most CRISPR/Cas9-based studies in plants show a low frequency of off-target mutation, which could be related to its prevalence in noncoding areas and, as a result, the difficulty to detect off-target consequences in the phenotype (Wang et al. 2014). In one study, despite the use of high-specificity sgRNA, high frequencies of undesirable mutations induced by CRISPR/Cas9 were reported in A. thaliana (Zhang et al. 2018). In higher plants, it is rare to find undesirable mutations resulting from the CRISPR/Cas9; if they persists, then it can be detected by whole genome sequencing (WGS). Thus, it is critical that while developing a gRNA for a specific target, consideration be given to the possibility of off-targeting. Software and programmes accessible on the web that can predict potential off-targets of a particular gRNA sequence have been developed. The Cas-OFFinder is one of them (Bae et al. 2014). Cas-OFFinder can be used to generate off-target sites by adjusting the parameters of stringency, which helps in evaluation of base pair mismatches. Ten targets were examined to identify off-target loci with the highest probability against the IR64 genome (Yin et al. 2017). The amplicons were generated in both the transgenic and wild type using primers designed for the flanking

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areas of the target site. The transgenic and wild-type sequencing chromatograms were identical at all ten loci, showing no off-target effect against IR64 genome (Yin et al. 2017). To develop highly precise sgRNAs with low off-target activity, several bioinformatic techniques have been introduced. CRISPR-PLANT v2 is an important prediction tool for detecting off-target mutations in plants (Minkenberg et al. 2019). This online software works in very precise ways, which work by combining the results of both the global and local alignment with NGG and NAG spacer sequences for the accession of the unwanted mutation probability. Among all off-target prediction tools, this software has the highest sensitivity and can be used in seven plant genomes, including Oryza sativa, Medicago truncatula, Glycine max, Solanum lycopersicum, Brachypodium distachyon, Arabidopsis thaliana and Sorghum bicolor (Minkenberg et al. 2019). Several other approaches for detecting off-targets, including deep sequencing and online prediction software, have been introduced in eukaryotes (Cho et al. 2013; Heigwer et al. 2014; Singh et al. 2015). However, in silico and in vitro methods for detecting potential off-target sites have been developed, but it is difficult to predict the exact unwanted mutations in vivo (Cho et al. 2014). Further studies are extremely necessary to create a precision off-target prediction system.

3.11

Future Prospects of Genome Editing in Plants

The CRISPR-based genome editing approach is widely recognized because it can produce transgene-free and homozygous plant kinds in fewer generations, allowing it to escape the present regulatory environment for genetically modified species. This technology has the capacity to target a specific DNA site in any genome, allowing for faster crop variety development by enhancing the simplicity with which new traits can be added by knockout of a trait of choice, which helps speed up plant breeding. Crops with improved insect resistance, increased nutritional content in crop plants and the ability to live in changing climates are among the prospective future crops for sustainable productive agriculture using CRISPR/Cas-based genome editing. Crop development by genome editing via targeted mutagenesis and induced manipulation for generation of biotic and abiotic stress resistance in plants is the future of crop improvement (Kissoudis et al. 2014). Genome editing will be vital for the formation of new bio-energy crops that can deliver maximum yield on wastelands while also adapting to changing climates (Bosch and Hazen 2013). The CRISPR/Cas technology could offer any possible novel genome-editing concept for plants in order to aid in improvement of crops for enhanced food security and nutrition values. There are some possible concepts where CRISPR/Cas-based multigene editing system can play vital role for the crop improvement. The use of nanoparticles for direct delivery of Cas9 and gRNA using Agrobacterium and viral replicons should be implemented (Hiei et al. 2014; Khatodia et al. 2014; Nonaka and

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Ezura 2014). The chemically inducible Cas9 system for improved transcription modulation using split-Cas9 and light-activated Cas9 effector (LACE) could be utilized as tools for crop improvement in the future (Zetsche et al. 2015b). In the future, model plants based on the CRISPRi system like RNAi may be able to generate a whole-genome targeted sgRNA library for high-throughput loss-of-function screening applications (Heintze et al. 2013). Production of improved root architecture employing CRISPR/Cas9 genome editing in cereal crops that can capture unevenly distributed water and nutrient resources in climate instability and resource scarcity is an important aspect of crop improvement. Targeted mutagenesis using the CRISPR-Cas9 system can help to drive the progress of legume functional genomic research, particularly by creating target mutants of genes involved in the development of roots and nodules (Sun et al. 2015). To assess whether T-DNAs confer a beneficial phenotype in naturally transgenic sweet potatoes, CRISPR/Cas9 genome editing approaches can be used to delete T-DNAs for “non-transgenic” sweet potato production (Jones 2015). By modifying the histone proteins found inside centromeric nucleosomes, the CRISPR/Cas9 genome editing method can also be used to create haploid plants (Kumar and Jain 2015). Targeting several locations with the same CRISPR effector is now possible to achieve due to the multiplexing platform, allowing researchers to build vectors with multiple gRNAs to target multiple loci simultaneously and reducing the effort (Cong et al. 2013). Plants typically use the NHEJ repair mechanism to repair their DNA, but utilizing a homology arm delivered right after the CRISPR system cleavage can result in the induction of HDR. The new homology arm serves as a template for the repair mechanism in this case. Allele swapping can be accomplished by changing the homology arm’s nucleotide sequence and employing the CRISPR system’s precise targeting (Endo et al. 2016). A different approach would be the use of singlestranded oligonucleotides in conjunction with the CRISPR effector to induce HDR and consequently impart new traits (Sauer et al. 2016), despite the fact that vectorbased plant transformation is the most generally utilized method of delivering the CRISPR system of foreign genetic components in the plant genome. On the contrary, scientists are also using a procedure in which the CRISPR ribonucleoprotein (purified CRISPR effector protein) is combined with the sgRNA in RNA form, which results in the formation of ribonucleo-protein and RNA-guided endonuclease (RNP:RGEN) complex that is then delivered into the plant system. This combination can then be transfected into a protoplast, and host endonucleases almost certainly eliminate it to restrict the possibility of off-targeting. The applications of CRISPR system are tremendously increasing due to discovery of CRISPR effectors from diverse species and the derivation of existing CRISPR effectors to develop effectors that can perform specialized roles. Cas9 nickase, or Cas9n, is created by the inactivation of the RuvC-like endonuclease domain, which forms an effector that can only cleave a single strand (Fauser et al. 2014). Modifications and innovations of this nature will be instrumental in helping us to perform gene discovery and functional characterization in an easy manner. These modifications and further research will be very of great help to the researchers, who perform site-specific

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integration of the desired traits and conduct gene expression regulation research and, more importantly, create transgene-free edited plants in the future.

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Chapter 4

CRISPR/Cas9 Tools for Multiplex Genome Editing in Crops Naoki Wada, Tomoko Miyaji, Chihiro Abe-Hara, Keishi Osakabe, and Yuriko Osakabe

Abstract Multiplex gene editing—the genetic modification of multiple sites simultaneously using the CRISPR/Cas9 system—is very useful not only in crop breeding to improve multiple traits but also for functional analysis of gene families. To solve the problem of delivery of many T-DNAs with separated binary vectors into plant cells at the same time, several approaches have been developed to express multiple gRNA cassettes from a single CRISPR/Cas9 vector. Generally, two approaches are used for CRISPR/Cas9 multiplex genome editing in plants: (1) expression of individual gRNA expression cassettes and (2) expression of the multiple gRNAs from one promoter on a single transcript, followed by processing into individual gRNAs. For the latter, several different systems, including endogenous tRNA processing, hammerhead self-cleaving ribozyme, Cys4 endoribonuclease, and intron processing-based systems, have been developed to yield the mature gRNA. Optimization of the Cas9 expression promoter is another important approach to further improve multiplex genome editing, and the tissue-specific expression of Cas9 leads to the induction of precise mutations with low levels of mosaicism. The development of multiplex gene editing provides increased opportunity to produce useful and important crops to enhance productivity and resistance to climate change in the future. Keywords Multiple genome editing · CRISPR/Cas9 · gRNA cassettes · Csy4 ribonuclease

N. Wada · T. Miyaji · C. Abe-Hara · K. Osakabe Faculty of Bioscience and Bioindustry, Tokushima University, Tokushima, Japan Y. Osakabe (*) School of Life Science and Technology, Tokyo Institute of Technology, Kanagawa, Japan e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 K. Zhao et al. (eds.), Genome Editing Technologies for Crop Improvement, https://doi.org/10.1007/978-981-19-0600-8_4

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Introduction

Genome editing is a very powerful technology that can induce mutations into a target gene specifically without introducing undesirable changes in the genome (Osakabe and Osakabe 2015; Wang et al. 2016; Jaganathan et al. 2018; Wada et al. 2020). The CRISPR/Cas9 [clustered regularly interspaced short palindromic repeat/CRISPRassociated protein 9 nuclease] that utilizes the bacterial immune defense system against phage infection has become the most popular genome editing tool in recent years and can be applied as a programmable genome editing tool (Jinek et al. 2012; Cong et al. 2013; Mali et al. 2013). The Cas9 protein is a RNA-dependent DNA nuclease that has both gRNA-dependent DNA-binding activity and nuclease activity. Recognition of the target sequence is mediated by simple Watson-Crick base pairing between the target DNA and gRNA. This latter point distinguishes CRISPR/ Cas9 from other genome editing tools, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), which recognize the target DNA sequence through DNA–protein interactions. This difference makes it easier to design the constructs necessary for genome editing and speeds up genome editing experiments. Any 20-bp DNA sequence upstream of the protospacer adjacent motif (PAM) sequence, for example, 50 -NGG-30 in the case of Streptococcus pyogenes Cas9 (spCas9), can be selected as a target sequence. The Cas9/gRNA complex that binds to target DNA cleaves the double-stranded DNA (dsDNA) and induces the mutations at target sites through the errors that occur during endogenous DNA repair processes. Its ease, simplicity, and high efficiency have accelerated application of the CRISPR/Cas9 system to genome editing in various organisms. The applicability and flexibility of the CRISPR/Cas9 system have also enabled the simultaneous editing of multiple genes, a process termed “multiplex genome editing.” In this chapter, we first briefly introduce recent studies on plant genome editing using the CRISPR/Cas9 system, before introducing the development of CRISPR/Cas9 tools for multiplex genome engineering in plants.

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CRISPR/Cas9-Mediated Plant Genome Editing

Successful genome editing in plants has been reported in various plant species to date, such as Arabidopsis, barley, rice, tobacco, sorghum, wheat, maize, soybean, tomato, potato, poplar, apple, etc. (see reviews, Osakabe and Osakabe 2015; Jaganathan et al. 2018). However, optimization of vector systems has been required to obtain efficient mutagenesis. For example, the constitutive Cauliflower mosaic virus (CaMV) 35S promoter is most general promoter used for the expression of transgenes in plants. However, some studies have reported that transformation of the CaMV35S pro::Cas9 gene by the floral-dip method in Arabidopsis resulted in high mosaicism in the T1 generation (Feng et al. 2013; Jiang et al. 2013; Li et al. 2013). High mosaicism makes it difficult and time-consuming to establish knockout plants

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using the CRISPR-Cas9 system. Several studies evaluating the effects of promoter activity have found that selection of a meristematic and germ-tissue-specific promoter resulted in high mutation efficiency with low mosaicism in Arabidopsis (Osakabe et al. 2016; Tsutsui and Higashiyama 2017; Mao et al. 2016; Castel et al. 2019). Several types of terminators have also been tested in Arabidopsis (Castel et al. 2019), of which the rbcS E9 terminator gave higher mutagenesis efficiency than other terminators tested. These results indicated that optimization of the vector system is a useful strategy to achieve high genome editing efficiency in plants. We optimized the vector system for genome editing in one of the most important vegetable crops: tomato (Solanum lycopersicum L. cv. Micro-Tom) (Ueta et al. 2017). We constructed two types of vector: 2x35S CaMV pro with Ω enhancer:: Cas9_3xNLS::2A::GFP::Ter and Parsley ubiquitin 4-2 (Pubi4) pro::Cas9_3xNLS:: 2A:: GFP::Ter. These vectors were then introduced into tomato leaf discs by Agrobacterium-mediated infection. The SlIAA9 gene, which plays an important role in controlling parthenocarpy, was selected as a target gene. Both vectors gave high somatic mutation efficiency, up to 100%, in calli and shoots. The regenerated plants indicated biallelic and homozygous mutation with parthenocarpic phenotypes in the T0 generation, suggesting that our system is efficient for mutagenesis in tomato plants. Recently, we expanded the application of our vector system to other commercial tomato varieties, such as Alisa Craig (AC) (Ueta et al. 2017), Money Maker (MM), and Rio Grande (RG). Like our previous results in MicroTom, mutations in the SlIAA9 gene were induced efficiently in these cultivars in the T0 generation. Phenotypic analysis indicated changes in leaf morphology (a simple leaf in mutants in contrast to a compound leaf in wild-type plants) (Fig. 4.1a) and the production of seedless fruits (Fig. 4.1b). The absence of transgenes in genome-edited plants can also be checked by PCR and Southern blot analyses. These studies demonstrated that our CRISPR/Cas9 system can produce SlIAA9 knockout plants efficiently in a wide variety of cultivars and also suggest that CRISPR/Cas9 technology will be a useful tool for efficient crop genome engineering.

4.3

Multiplex Genome Editing Systems in Plants

Multiplex genome editing is a powerful tool in plant genome editing. Since the first report of multiple genome editing in mammalian cells in 2013 (Li et al. 2013), the application of multiplex genome editing has expanded widely, including into plant genome editing. Plants generally have a complex genome structure, including multiple gene families, gene duplication, and polyploidy. Therefore, multiplex genome editing is expected to be useful to investigate gene functions by allowing the construction of knockout lines in plants (Najera et al. 2019). Kannan et al. (2018) induced a mutation into 107 of 109 members of the caffeic acid O-methyltransferase (COMT) gene family in sugarcane using the TALEN system, indicating the power of

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Fig. 4.1 Genome editing of commercial tomato cultivars. (a) Leaf morphology in young sliaa9crispr T1 plants (3 weeks after sowing). Bars ¼ 1 cm. (b) Phenotypes of sliaa9-crispr T1 fruits. Bar ¼ 10 mm. AC Alisa Craig, MM Money Maker, RG Rio Grande, WT wild-type plant

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Fig. 4.2 Polycistronic gRNA expression system used for plant genome editing. Representative systems are shown; (a) individual expression cassette-based system (b) endogenous tRNA processing-based system (c) ribozyme-based system (d) Csy4 endoribonuclease-based system. For a detailed explanation of each system, please refer to the main text. The multiple gRNAs expressed from these systems produce individual gRNAs either without processing (a) or with processing via the pathway specific for each system (b–d). The gRNAs make a complex with Cas9 protein and cleave the target DNA, resulting in mutagenesis at the target sites

multiplex genome editing. Considering the ease and flexibility of the CRISPR/Cas9 system compared with the TALEN system, the CRISPR/Cas9 system appears more suitable for multiplex genome editing and has the potentials to edit more multiple genes easily and simultaneously. Multiplex genome editing by CRISPR/Cas9 in plants was first reported in Arabidopsis and Nicotiana benthamiana in 2013. Li et al. (2013) expressed individual gRNAs in different expression cassettes and succeeded in the simultaneous targeting of two sites (Fig. 4.2a). This strategy has since been applied to multiplex genome editing in many studies (Zhang et al. 2016; Hanania et al. 2017; Li et al. 2018a; Li et al. 2017b, 2018b; Rodriguez-Leal et al. 2017; Yang et al. 2018; Wang et al. 2018a, b, c; Ma et al. 2015; Li et al. 2017a; Xu et al. 2016; Lawrenson et al. 2015; Xing et al. 2014; Lowder et al. 2015). However, the drawback of this approach is that many gRNA expression cassettes are needed, depending on the number of gRNAs. In particular, genetic transformation in plants has been performed mainly by the Agrobacterium-mediated method, in which it is difficult to deliver large DNAs and also to deliver many T-DNAs simultaneously. Therefore, several polycistronic expression systems that can express many gRNAs from only one promoter from a single transcript have been developed for multiplex genome engineering (McCarty et al. 2020; Najera et al. 2019). Such polycistronic expression systems include the endogenous tRNA processing-based system (Fig. 4.2b, Xie et al. 2015), a ribozymebased system (Fig. 4.2c, Gao and Zhao 2014) and the Cys4 endoribonucleasemediated system (Fig. 4.2d, Čermák et al. 2017; Tsai et al. 2014). Polycistronic expression systems using tRNA are based on endogenous tRNA processing systems (Fig. 4.2b). It has long been known that the tRNA structure is

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processed at specific sites by RNase P and RNase Z to remove extra 50 and 30 sequences in eukaryotic cells (Phizicky and Hopper 2010). Utilizing this endogenous tRNA processing system, expression of multiple gRNAs from a single transcript has been achieved in mammalian cells (McCarty et al. 2020). In this system, each gRNA is flanked by tRNA sequences, and multiple tRNA–gRNA sequences have been expressed as a single transcript in cells. After transcription, the tRNA structure is processed by endogenous RNase P and RNase Z. The processing results in site-specific cleavage of the transcript and the release of individual functional gRNAs. Released gRNAs can make a complex with Cas9 proteins and induce mutagenesis at target sequences. This strategy has been applied successfully to multiplex gene editing in plants (Mercx et al. 2017; Jansing et al. 2019; Wang et al. 2018a, b, c; Minkenberg et al. 2017; Hashimoto et al. 2018; Xie et al. 2015; Čermák et al. 2017). Expression of the tRNA–gRNA was generally performed under RNA polymerase III (Pol III) promoters, such as U6 promoters, because endogenous tRNA genes are transcribed under Pol III promoters in cells. However, the use of Pol III promoters limits the promoter selection in the endogenous tRNA processingbased system because the number of Pol III promoters whose characteristics have been identified is limited. In addition, Pol III promoter requires a specific nucleotide at the first position of the transcript. Furthermore, Pol III promoters are expressed ubiquitously in plants, thus precluding spatiotemporal control of gRNA expression under this promoter. Recently, these limitations have been overcome by applying a Pol II promoter for tRNA–gRNA expression. Čermák et al. (2017) tested the Pol II promoter from Centrum yellow leaf curling virus (CYLCV) for expression of a tRNA–gRNA and showed that this system could induce mutations at target genes in tomato. Ribozyme-gRNA-ribozyme (RGR) is a polycistronic expression system that uses ribozymes (Fig. 4.2c, Gao and Zhao 2014). In this system, a single transcript containing gRNAs flanked by a ribozyme sequence is processed by ribozymes, e.g., the Hammerhead (HH) self-cleaving enzyme (Scott et al. 1996) and a hepatitis delta virus (HDV) ribozyme (Nakano et al. 2000). One of the advantages of this system is that a Pol II promoter can be used to express the gRNAs. In fact, Gao et al. (2015) successfully induced mutations in target genes in Arabidopsis by expressing the HH ribozyme-gRNA-HDV ribozyme from the CaMV 35S promoter. The ribozyme sequences also can be modified as long as the secondary structure is maintained (He et al. 2017). Tang et al. (2016) expressed the Cas9 gene with gRNAs and HH ribozymes as a single transcript under a single Pol II promoter by flanking each gene with a ribozyme cleavage site, resulting in efficient mutagenesis at multiple target genes in rice, Arabidopsis, and tobacco. They also demonstrated that the estrogen-inducible (XVE) promoter can be used to drive expression of the Cas9-sgRNAs-HH ribozyme cassette with subsequent mutagenesis at target sites. As mentioned later, the design of simple coordinated Cas9–gRNA transcription unit is an attractive approach for efficient mutagenesis in plants. This strategy would be useful for the coordinated expressions of Cas9 gene and multiple gRNAs under Pol II promoters.

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Fig. 4.3 Coordinated expression system for gRNA and Cas9 gene expression from a Pol II promoter as a single transcript. (a) Single transcript unit (STU)-Cas9 system. Cas9 gene and multiple gRNAs are expressed under one Pol II promoter. Each gene is connected by a cleavage site specific for each polycistronic expression system; (Top) ribozyme-based system (middle) Csy4 endonuclease-based system (Bottom) tRNA processing-based system. (b) Intron-based system. Introns are designed in Cas9 gene, and polycistronic expression systems are designed in the intron. Splicing and polycistronic processing produce the functional Cas9 genes and gRNAs. (c) In planta processing system. Cas9 gene and gRNAs are fused with or without linker sequence and expressed under one Pol II promoter. An unknown in planta processing system produces functional Cas9 genes and gRNAs

The type III CRISPR/Cas-associated Csy4 endoribonuclease also can be applied to multiplex genome editing (Fig. 4.2d, Tsai et al. 2014; Čermák et al. 2017; Tang et al. 2019). In the Type III CRISPR/Cas system (e.g., Csy4 from Pseudomonas aeruginosa, Haurwitz et al. 2010), the endoribonuclease Csy4 cleaves pre-crRNA to generate mature crRNAs. Csy4 recognizes 20 bp Csy4 hairpin sequences and cleaves them at a specific site. Utilizing this function, multiple gRNAs can be expressed and processed by constructing arrays of gRNAs and 20 bp Csy4 hairpin sequences. Co-expression of Csy4 protein with such arrays results in cleavage of the Csy4 hairpin sequences and release of the individual gRNAs. Using this system, multiple genome editing has been achieved in plants (Čermák et al. 2017; Tang et al. 2019). The drawback of this system is the requirement for additional expression of the Csy4 protein. Comparing three polycistronic expression systems in plants, Čermák et al. (2017) indicated that Csy4 and tRNA expression systems were twice as effective as individual gRNAs expressed from Pol III promoters in tomato. The ribozyme-based expression system showed lowest mutation frequency in their experiment. Recently, simple expression systems that can express both multiple gRNAs and a Cas9 gene as a single transcript under one Pol II promoter have been reported (Fig. 4.3, Tang et al. 2016, 2019; Mikami et al. 2017; Ding et al. 2018; Zhong et al. 2020; Wang et al. 2018a, b, c). As mentioned above, Tang et al. (2016) reported a single transcript unit (STU)-Cas9 system using HH ribozymes. In addition, Tang’s group has reported two versions of the STU-Cas9 system: a Csy4 ribonucleasebased system and endogenous tRNA processing system in rice (Fig. 4.3a) (Tang et al. 2019). In these systems, the Cas9 gene and multiple gRNAs can be transcribed

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as a single transcript, keeping the total size of the construct small and allowing coordinated expression of the Cas9 gene and gRNAs. Ding et al. (2018) and Zhong et al. (2020) inserted the multiplex gRNAs expression system into a specially designed intron in the Cas9 gene to express them as a single transcript under one Pol II promoter (Fig. 4.3b). Furthermore, even simpler systems have been reported by Mikami et al. (2017) and Wang et al. (2018a, b, c) (Fig. 4.3c). They connected the Cas9 gene and gRNAs either without additional sequences (Mikami et al. 2017) or with only a 6-bp linker sequence (Wang et al. 2018a, b, c). Surprisingly, their system successfully induced mutations at the target sites in rice. It was speculated that the Cas9 RNA and multiple gRNAs were processed by unknown ribonucleases in rice. Thus, multiple expression systems have been developed towards the establishment of simple and efficient expression systems for multiple gRNAs and Cas9 gene. These efforts will make it easier to perform multiple genome editing in plants and contribute to speeding up and expanding new possibilities in plant breeding.

4.4

Precise Deletions Induced by Multiplex Genome Editing

Multiplex genome editing cannot only induce mutations into multiple genes simultaneously but can also generate predictable deletions between target sites (Li et al. 2013; Hashimoto et al. 2018; Čermák et al. 2017). We performed the multiplex

Fig. 4.4 Effect on mutation patterns induced by multiple genome editing of promoter activity. (a) CaMV35S promoter expresses Cas9 gene ubiquitously in various tissues, resulting in various mutations patterns at target sites (high mosaicism). (b) SlEF1 promoter expresses Cas9 gene specifically in meristems, resulting in precise deletion between target sites (low mosaicism)

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genome editing using a tRNA-gRNA expression system to induce mutations in the SlNADK2A gene in tomato (Hashimoto et al. 2018). Evaluation of several promoters [2xCaMV35S, parsley ubiqutin4-2 (Pcubi4), Slp16, SlELONGATION FACTOR-1α (SlEF1α)] for genome editing activity in tomato indicated that mutation patterns differed depending on the promoters used (Fig. 4.4). Expression of the Cas9 gene from the 2xCaMV35S, Pcubi4 or Slp16 promoters resulted in various mutation patterns with high mosaicism (Fig. 4.4a). On the other hand, expression of Cas9 gene under the SlEF1α promoter resulted in deletions between the target sites with low mosaicism (Fig. 4.4b). The activity of these promoters was tested using two kinds of expression vector: CaMV35S pro::GFP and SlEF1α pro::GFP, to investigate the localization of GFP proteins expressed under each promoter. CaMV35S pro::GFP gene expression resulted in the ubiquitous accumulation of GFP protein in calli (Fig. 4.4a). On the other hand, SlEF1α promoter::GFP gene expression yielded specific accumulation of GFP protein in developing shoot buds (Fig. 4.4b). The meristem-specific promoter activity of the SlEF1α promoter has also been reported in other studies (Pokalsky et al. 1989). These data indicate that tissue specificity of promoter activity influences the mutation patterns induced by multiplex genome editing. Tissue-specific expression of the Cas9 gene in the early stage of shoot buds could effectively induce predictable deletions at target sites. These results indicate that optimization of the Cas9 gene expression system will be useful in the control of mutation patterns. Induction of precise deletions will certainly be useful to knock out specific target genes.

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Conclusion

CRISPR/Cas9 tools for multiplex genome editing have been developed for the improvement of crop plants, which often have complex genomes. Expression of individual gRNAs in separate expression cassettes is one option for the expression of multiple gRNAs, but it is labor intensive and requires the construction and delivery of large vectors. On the other hand, polycistronic expression systems have been developed towards efficient and simple multiplex genome editing. In particular, strategies to express multiple gRNAs and the Cas9 gene under a Pol II promoter as a single transcript have been reported recently. Such strategies would enable the coordinated and controlled expression of gRNAs and the Cas9 gene, contributing to the development of simple and efficient multiplex genome editing systems. Multiplex genome editing cannot only induce mutations into multiple genes simultaneously and target a single gene with multiple gRNAs but also induce precise deletions of expected size at the target site. These would be advantageous in the construction of knockout plants. An interesting application of multiple genome editing is de novo domestication of wild plants. By targeting multiple key genes for domestication of plants, natural evolution can be mimicked in a short time frame (Zsögön et al. 2018). This strategy would accelerate plant breeding by enabling us to

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add characteristics suitable for the cultivation to the wild plants with useful traits such as abiotic/biotic stress resistance. Recently, a new genome editing strategy, prime editing, has also been reported to function in mammalian and plants cells (Anzalone et al. 2019; Lin et al. 2020). Prime editing is a CRISPR/Cas9-based technology with an engineered gRNA (prime editing gRNA, pegRNA) that can induce insertion, deletion, and substitution precisely at the target site. Prime editing also can be performed with multiple pegRNAs, suggesting that multiplex genome editing systems could further expand the possibilities of prime editing. Novel CRISPR/Cas9-based multiplex genome editing tools continue to be developed, facilitating studies on plant gene function as well as promoting crop improvements. Acknowledgments This work was supported by Program on Open Innovation Platform with Enterprises, Research Institute and Academia (OPERA, funding agency: Japan Science and Technology Agency; to K.O.).

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Chapter 5

Plant Genome Editing Mediated by CRISPR/Cas12a System Rongfang Xu, Juan Li, Ruiying Qin, and Pengcheng Wei

Abstract The clustered regularly interspaced short palindromic repeats (CRISPR)Cas system has emerged as an efficient genome editing tool. CRISPR/Cas12a system can induce the cohesive end with a single crRNA and T-rich PAM. Due to the characteristic, the scientists expanded the edit scope and developed the multiplex genes editing system and base editing system by CRISPR-Cpf1. In addition, they increased gene editing efficiency by the single transcription unit domain with different linkers in plants. So far, the CRISPR-Cas12a system is with higher efficiency, specificity, and easier for gene insertion and large fragment deletion. In this chapter, we summarize the current knowledge of CRISPR-Cas12a system, including found, mechanism, development, application in plants, and prospects. Keywords CRISPR/Cas12a · Plants · Genome editing · Efficiency

5.1

Introduction

Ever since the discovery that double-stranded breaks (DSBs) can induce gene targeting (Puchta et al. (1993)), scientists have continually worked on developing target-editing tools for use in the genome. In 2005, zinc-finger nucleases (ZFNs) were adapted for tobacco (Wright et al. 2005) and then used for trait improvement in several other plants species. In 2010, transcription activator-like effector nucleases (TALENs) were added to the plant genome-editing toolbox (Christian et al. 2010). However, these two nucleases have several disadvantages, so they have not been widely used in plants. Until 2013, many published articles showed that the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) system can induce DSBs in specific targets of organism genomes (Cong et al. 2013; Jinek et al. 2014; Li et al. 2013). The CRISPR/Cas9 system has several advantages: time-saving in vector construction, efficient in editing, and easily applicable for use

R. Xu · J. Li · R. Qin · P. Wei (*) Key Laboratory of Rice Genetic Breeding, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei, China © Springer Nature Singapore Pte Ltd. 2022 K. Zhao et al. (eds.), Genome Editing Technologies for Crop Improvement, https://doi.org/10.1007/978-981-19-0600-8_5

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in different animal and plant species (Esvelt et al. 2014; Jinek et al. 2012; Li et al. 2013; Mali et al. 2013; Nekrasov et al. 2013; Shan et al. 2013). In 2015, scientists identified another Cas protein—called Cas12a (Zetsche et al. 2015). In this section, we review the mechanism of the CRISPR-Cas12a system and its use in crop species.

5.2

Mechanism of CRISPR/Cas12a

In 2015, Cas12a was reported that can be harnessed to facilitate genome editing in human cells (Zetsche et al. 2015). Unlike the CRISPR/Cas9 system, the CRISPR/ Cas12a system needs short mature crRNAs of 42
44 nt in length, with 19 nt of the direct repeat followed by 23
25 nt of the spacer sequence, and T-rich protospacer adjacent motifs (PAMs) are used to recognize the target. The structure of the Cas12a endonuclease differs from that of the Cas9 endonuclease, as Cas12a has a RuvC-like nuclease domain but does not have any of the HNH nuclease domains that are present in Cas9. While Cas9 generates blunt-ended DSBs, Cas12a generates DSBs with staggered ends, which may be useful for gene insertion and replacement via homology-directed repair (HDR). The Cas12a-mediated cleavage results in a 5 nt overhang, and the cleavage site is distant from the PAM, which differs from the blunt cleavage products and relatively close proximity to the PAM by Cas9. According to different sources, Cas12a proteins can divide of Acidaminococcus sp. Cas12a (AsCas12a), Lachnospiraceae bacterium Cas12a (LbCas12a), and Francisella novicida Cas12a (FnCas12a). Until that, scientists had been devoted to cracking the structure and elucidating the underlying mechanism. First, two groups analyzed the recognition and cutting mechanisms of AsCas12a, LbCas12a, and FnCas12a in 2016 and 2017. AsCas12a contains the RuvC domain and a putative novel nuclease domain, which are responsible for cutting nontarget and target strands, respectively. AsCas12a is recognized the TTTN PAM by a base-and-shape readout mechanism (Yamano et al. 2016), and LbCas12a has a triangle-shaped architecture with a large positively charged channel at the center. Recognized by the oligonucleotide-binding domain of LbCas12a, the crRNA adopts a highly distorted conformation stabilized by extensive intramolecular interactions and (Mg(H2O)6)2+ ions. The oligonucleotide-binding domain also harbors an outward-looping helical domain that is important for LbCas12a substrate binding. Binding of crRNA or crRNA lacking the guide sequence induces marked conformational changes but no oligomerization of LbCas12a (Dong et al. 2016). In FnCas12a, the PAM is recognized by a PAM-interacting domain. The loop-lysine helix-loop motif in this domain contains three conserved lysine residues that are inserted in a dentate manner into the doublestranded DNA. Unzipping of the double-stranded DNA occurs within a cleft arranged by acidic and hydrophobic residues facilitating crRNA-DNA hybrid formation. The PAM single-stranded DNA is funneled toward the nuclease site through a mixed hydrophobic and basic cavity. In these catalytic conformations, the PAM-interacting domain and the helix-loop-helix motif in the REC1 domain adopt a “rail” shape and a “flap-on” conformation, respectively, channeling the

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PAM strand into the cavity. A steric barrier between the RuvC-II and REC1 domains forms the “septum,” separating the displaced PAM strand and the crRNA-DNA hybrid, avoiding DNA reannealing (Stella et al. 2017).

5.3

Development of CRISPR/Cas12a

Although CRISPR/Cas12a can complement CRISPR/Cas9, it still has some limitations, such as a limited number of target sites and low editing efficiency. To expand the number of editing targets in the genome, two AsCas12a variants that recognize PAM sequences from TTTN to TYCV and TATV with two or three amino acids mutations are used: AsCas12a-RR (S542R, K607R) and AsCas12a-RVR (S542R, K548V, N552R) (Gao et al. 2017). This technique greatly increases the number of editable sites in cells. The crystal structures of RR/RVR variants bound to the crRNA and target DNA with the TYCV/TATV PAM at 2.0. Å resolution adopts a bilobed architecture consisting of a recognition (REC) lobe and a nuclease (NUC) lobe, in which the crRNA-target DNA heteroduplex is bound to the central channel between the two lobes. In the two structures, the target DNA strand and the PAM-containing nontarget DNA strand form a PAM duplex, which is bound to the narrow channel formed by the WED, REC1, and PI domains (Nishimasu et al. 2017). To expand the number of Cas12a proteins available for use, 16 Cas12a orthologs were identified and tested for activity in cells. Four of these new enzymes have activity in targets, one (Moraxella bovoculi AAX11-00205 [Mb3Cas12a)] of which exhibits robust indel formation with a short TTN PAM from 16 Cas12a orthologs (Zetsche et al. 2017b). The other four Cas12a orthologs were identified in 2019; these enzymes use the 4n96 crRNA scaffold-carrying nucleotide substitution in the loop region for increased genome editing efficiency (Teng et al. 2019b). Other research groups have found that the combination of cr30 5F (containing five 20 -fluororiboses at the 30 terminus) and ψ-modified AsCas12a or LbCas12a mRNA augmented gene-cutting efficiency by more than 300% compared with that of the control (Li et al. 2017). Moon et al. reported an engineered crRNA for highly efficient genome editing by Cas12a, including a 20 nt target complementary sequence and a uridinylate-rich (U4AU4) 30 -overhang (Moon et al. 2018). Cas12b from the other subunit family is from Alicyclobacillus acidophilus (AaCas12b); this enzyme maintains optimal nuclease activity across a wide temperature range (31–59  C). AaCas12b can be repurposed to engineer mammalian genomes for various applications, including single and multiplex genome editing, gene activation, and generation of mutant mouse models (Teng et al. 2018). The ability of Cas12a to process its own crRNA could be exploited for multiplex gene editing in mammalian cells in conjunction with a crRNA array (Zetsche et al. 2017a). Based on the CRISPR/Cas12a system different from CRISPR/Cas9, it may be used in the following:

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Establishment and Utilization of CRISPR/Cas12a System in Plants

To date, CRISPR/Cas12a has been used for gene targeting in many plant species, as shown in Table 5.1. FnCas12a can induce NtPDS and NtSTF1 gene targeting in tobacco at a success rate of 4.3–65.2% (Endo et al. 2016) and four genes in rice at a success rate of 4%–80% (Begemann et al. 2017; Wang et al. 2017, 2018). In 2017, many research groups applied genome editing to different crop species via LbCas12a or AsCas12a. Six teams successfully targeted the rice genome (Hu et al. 2017; Tang et al. 2017; Xu et al. 2017; Yin et al. 2017), five of the six by nonhomologous end joining (NHEJ) repair and one by HDR. Not only was gene editing achieved but also gene loss-of-function mutations were obtained, such as OsPDS and albino leaf genes. Similarly, dAsCas12a-SRDX or dLbCas12a-SRDX can also induce gene transcriptional repression in Arabidopsis. Although Cas12a can efficiently be used to edit genes plants, it results in the formation of T-DNA insertions in T0 plants. To achieve transgene-free plants, scientists induced FAD2 gene mutations with LbCas12a and AsCas12a mediated by RNP (Kim et al. 2017). Cas12a has also been used to target genes and induce loss-of-function gene mutations in non-model plant species, such as tomato and cotton (Bernabé-Orts et al. 2019; Li et al. 2019b). To expand the targeting scope in plants, Li et al. and Xu et al. used a LbCas12aRR/RVR variant-induced gene targeting that recognized the TYCV/TATV PAM sequence. These authors analyzed the whole rice genome and verified that compared with wild-type (WT) Cas12a, the Cas12a variants can increase the number of editable sites by more than twofold (Li et al. 2018c; Xu et al. 2019). Scientists have made many attempts to optimize the efficiency of Cas12a. Using a single transcription unit, one group fused a crRNA domain behind a Cas12a domain driven by the Ubi promoter in protoplasts (Tang et al. 2019). Two other groups induced highly multiplex gene mutations in rice via expression of a single crRNA (Wang et al. 2018). Another group improved targeting efficiency in rice by the use of particle bombardment, which increased the concentration of the vector (Li et al. 2020b). Schindele et al. improved targeting efficiency by Cas12a variants via temperature tolerance in Arabidopsis mediated by the floral-dip method (Schindele and Puchta 2020). Another group reported the application of Cas12a temperature sensitivity for improved genome editing in rice, maize, and Arabidopsis (Malzahn et al. 2019). In plants, HDR efficiency by CRISPR/Cas9 system is very low, perhaps because of the donor concentration. Xia lab obtained precise gene replacement in rice via RNA transcript-templated homologous recombination by particle bombardment together with CRISPR/Cas12a and ultimately obtained herbicide-resistant mutant plants (Li et al. 2019a). Similarly, Wolter et al. also used the CRISPR/ LbCas12a system to mediate gene substitution by HDR in Arabidopsis (Wolter and Puchta 2019). Although crop improvement by CRISPR/Cas12a has not been reported until now, the researchers still have done a lot of groundwork. They obtained the ALS mutation plants, which determine herbicide resistance in rice and Agrobacterium, by

FnCas12a LbCas12a LbCas12aRR/RVR

FnCas12a LbCas12a AsCas12a LbCas12a LbCas12a RR

LbCas12a LbCas12a LbCas12a

FnCas12a LbCas12a

Agrobacterium

Agrobacterium

Rice

Rice

TTCA TTCC TTC TTTC TYCV TATV

PEG-mediated RNP

Agrobacterium

Soybean

TTTA

Agrobacterium

Agrobacterium Agrobacterium Agrobacterium

Agrobacterium

Agrobacterium

Agrobacterium

Delivery Agrobacterium

Rice

Rice

Rice Rice Rice

Rice

Rice

Rice

Crop Tobacco

TTTC

TTTC TTTG TTTA TTTC TTTG TTTA

TTA TTC TTTG TTTC

FnCas12a

LbCas12a AsCas12a

PAM TTN

Orthologs FnCas12a

Table 5.1 The genome editing in plants by CRISPR/Cas12a

OsPDS OsSBEIIb LEA family OsPDS OsDL

FAD2

C1
C5 OsEPFL9 OsPDS OsBEL OsCAO1

Gene NtPDS NtSTF1 OsDL OsALS OsPDS OsROC5 OsDEP1 OsRLK OsBEL

Deletions

Deletions

Deletions

Deletions

Insertions

Deletions Deletions Deletions

Deletions

Deletions

Indels

Mutation types Deletions

Stomatal density Albino seedlings

Photo-bleaching; curly leaf

Phenotypes

NHEJ

NHEJ

NHEJ

NHEJ

NHEJ

NHEJ NHEJ NHEJ

NHEJ

NHEJ

NHEJ

Repairing pathway NHEJ

(continued)

12.5–70.8% 4.2–70.8% 13.9%–69.4%

5–12.1% 10% 21.4% 41.2% 8% 3% 0.6–1.6% 9.1–11.7% 16.5–31.1%

43.8–75% 40–60%

85.7% 90.0% 100.0%

Editing efficiency 4.3–65.2%

5 Plant Genome Editing Mediated by CRISPR/Cas12a System 113

TTTG

TTTA

LbCas12a

LbCas12a variants AsCas12a

LbCas12a AsCas12a LbCas12a LbCas12a

TTTG

LbCas12a

Arabidopsis Tomato

Cotton

TTTG

Arabidopsis Maize Rice

Rice

Rice

Crop Rice

TTTC TTTC

TTTC

PAM N.A

Orthologs LbCas12a

Table 5.1 (continued)

Agrobacterium

Agrobacterium Agrobacterium

Agrobacterium

Delivery Protoplasts transformation Particle bombardment Particle bombardment Floral dip

GhCLA1

AtGL2 ZmGL2 OsDEP1 OsROC5 AtALS MYB2

OsALS

Gene OsDEP1 OsROC5 OsALS

Deletions

Substitutions Deletions

Deletions

Indels Substitutions Replacements Deletion Deletions

Mutation types Deletions

Albino seedlings

Herbicide resistance

Phenotypes

NHEJ

HDR NHEJ

NHEJ

NHEJ HDR HDR NHEJ NHEJ

Repairing pathway NHEJ

87%

77.8% 92.8% 1.47% 8.8–82.7%

69.0% 1.8% 4.6% N.A 4.1–90.3%

Editing efficiency 29.2–82.4%

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Cas12a-mediated HDR or NHEJ. In soybean, the scientists got the FAD2 mutation lines by Cas12a with DNA-free. FAD2 proteins convert oleic acid, a monounsaturated fatty acid, to linoleic acid, a polyunsaturated fatty acid, in seeds. In addition, some gene mutations related to photosynthesis and leaf phenotype were obtained by CRISPR/Cas12. The CRISPR/Cas12a not only provides a new method for the study of crop gene function but also provides a new technical means for crop variety improvement.

5.5

Other Applications and Future Prospect by CRISPR/Cas12a

Due to Cas12a’s ability to cleave nontargeted ssDNAs upon the formation of the Cas12a-crRNA-target DNA complex (Li et al. 2018a), Li et al. used a quenched fluorescent ssDNA reporter as a probe and developed the 1-HOur Low-cost Multipurpose highly Efficient System (HOLMES), which can be used for the rapid detection of target DNA as well as target RNA (Li et al. 2018b). Two groups subsequently developed the CDetection and Cas12aVDet systems for rapid and visual nucleic acid detection (Teng et al. 2019a; Wang et al. 2019). CRISPR/Cas12a can realize homologous repairs, but the efficient was low. According to improving the efficiency about HR by CRISPR/Cas12a, we may be aimed at developing shear capacity by Cas12a with combining ability. The donor content is also significant for HR efficient. Due to CRISPR/Cas12a with different PAM from Cas9, it also has widely application prospects in base editing and primer editing. In base editing, we can try to using differently deaminase conversion C/G to T/A reciprocally. In addition, C to G or A substitutions can come true by CRISPR/ Cas9 (Kurt et al. 2021), but no reports by CRISPR/Cas12a. The primer editing system can induce substitutions, insertions, and deletions at the specific sites and was not limited by the base numbers (Anzalone et al. 2019). Although this tool has great application value, it still has certain limitations, which are mainly manifested in limited application scope and low efficiency (Hua et al. 2020; Li et al. 2020a; Lin et al. 2020; Lu et al. 2021; Tang et al. 2020; Xu et al. 2020). The core elements in primer editing system were Cas9-sgRNA protein, reverse transcriptase MMLV, and pegRNA. We can use the Cas12a-crRNA replacing the Cas9-sgRNA. It can expand the editable range and maybe can increase the efficient.

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Chapter 6

Genome Editing in Crops Via Homology-Directed Repair Using a Geminivirus-Based CRISPR/Cas9 System Amir Hameed, Bareera Faazal, Muhammad Awais, and Ahad Naveed

Abstract In recent years, the CRISPR/Cas technology has proven a great potential for targeted genome editing (GE) in plants. From simple random mutagenesis through gene knockout (KO) breeding, the next-shifts are focused on the precise gene knock-in (KI) through homology-directed repair (HDR), a DNA repair pathway in which the CRISPR/Cas-induced DNA cleavage is repaired in the presence of homologous repair templates. To facilitate the delivery of these repair templates to the targeted cells, viral DNA-based replicons have been engineered as efficient vectors for CRISPR/Cas-mediated HDR. Here, we summarize the available delivery vehicles for CRISPR/Cas9 reagents and discuss the applications of geminivirus replicons (GVRs) for HDR-mediated GE in crop plants. Moreover, we provide a general layout for designing the experiment using CRISPR-GVRs and the prospects and applications of this technology. Using the full potential of the CRISPR toolbox will have more impact on enhancing food crops and ensuring food security in eco-friendly agriculture. Keywords CRISPR/Cas9 · Crop plants · Genome editing · Geminiviral replicons · Homology-directed repair

6.1

Introduction

The prokaryote-derived clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has emerged as a powerful tool for genome editing (GE) in numerous organisms. Since its first demonstration outside the prokaryotic cell (Jinek et al. 2012), several orthologs of CRISPR/Cas systems have been discovered and practically used in molecular research (Pickar-Oliver and Gersbach 2019). Among these, CRISPR/Cas9, a class II, type II nuclease system from Streptococcus pyogenes, has been extensively used for plant GE (Ma et al.

A. Hameed (*) · B. Faazal · M. Awais · A. Naveed The Plant Breeding and Acclimatization Institute (IHAR)–National Research Institute, Radzikow, Blonie, Poland © Springer Nature Singapore Pte Ltd. 2022 K. Zhao et al. (eds.), Genome Editing Technologies for Crop Improvement, https://doi.org/10.1007/978-981-19-0600-8_6

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2016; Puchta 2016). The precision, robustness, and ease of use of CRISPR technology have extended the genetic manipulations of diverse plant species from basic to translational science. The CRISPR/Cas system is an adaptive immune system of prokaryotes that relies on the homology-specific cleavage of the invading nucleic acids (Barrangou et al. 2007). In an engineered CRISPR/Cas9 system, Cas9 proteins interact with the backbone of the experimentally designed single-guide RNA (sgRNA, a lab variant of naturally occurring CRISPR RNA–crRNA). Upon nucleic acids invasion, the spacer part of the sgRNA hybridizes with a complementary target sequence positioned adjacent to a 50 -NGG protospacer adjacent motif (PAM, specific to each Cas protein). Next, the Cas9 protein induces the double-stranded breaks (DSBs) in the hybridized nucleic acids (crRNA/sgRNA-target sequence) by employing two of its Cas9 nuclease domains, RuvC and HNH (Lander 2016). These DSBs are endogenously repaired by either cell’s native nonhomologous end joining (NHEJ) pathway creating knockout (KO) mutations or by precise homologous recombination (HR) to edit/knock-in (KI) genes, or through homology-directed repair (HDR, a DNA repair pathway in which the induced DSBs are repaired in the presence of homologous repair templates (Symington and Gautier 2011)). To further optimize the target-specific cleavage, and delivery of Cas9, various customized versions of Cas9 were also employed in diverse applications (Kleinstiver et al. 2016; Kim et al. 2017). Several review papers describe the evolution of CRISPR systems (Koonin et al. 2017; Galizi and Jaramillo 2019; Pickar-Oliver and Gersbach 2019; Adli 2018; Yan et al. 2019) and provide a comprehensive overview of some of the recently characterized CRISPR-associated endonucleases such as Cpf1 (Zetsche et al. 2015), Cas13a (Abudayyeh et al. 2016), Cas13b (Smargon et al. 2017), Cas14 (Harrington et al. 2018), etc. The current chapter describes the delivery of CRISPR reagents into the plant cell. Here, we focus on the HDR-mediated gene replacement in crop plants facilitated through geminiviral replicons (GVRs). The use of viral replicons has shown potential for efficient gene replacement where an exogenous template is supplied through viral particles resulting in enhanced gene editing frequency (Baltes et al. 2014). Also, we describe a brief methodology to design the GVRs-mediated CRISPR experiment and the potential applications and prospects of this technique for crop improvement.

6.2

Expression and Delivery of CRISPR Reagents

Most CRISPR/Cas9 systems use Cas9 and gRNA to express separately; promoters such as 35S from cauliform mosaic virus derive strong expression of Cas9 by RNA polymerase II, and U6/U3 promoters (small nuclear RNA promoters) derive the expression of gRNA by RNA polymerase III. However, this system requires elements for promoter and terminator when multiple gRNAs are to be expressed (Paul 3rd and Qi 2016). The possible way is to harness the ribozyme sequences adjacent to the gRNA sequence in transcribed RNA and to precisely cleave out a functional gRNA. Two ribozymes, one from hepatitis delta virus (HDV) and the other

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ubiquitous hammerhead type (HH), are cloned, upstream and downstream of the gRNA, respectively, to accomplish this (Paul 3rd and Qi 2016). This system is independent of the promoter that’s why either constitutive or inducible promoter can be used for gRNA expression. Another approach is to use tRNA leader and trailer sequences in gRNA sequence. These sequences are cleaved by RNase P and Z, respectively (Paul 3rd and Qi 2016). The delivery of CRISPR/Cas9 reagents is categorized into cargo and delivery vehicle systems. There are three approaches for cargo: (1) DNA plasmid for both Cas9 and gRNA; (2) mRNA for translation into Cas9, and a separate gRNA; (3) ribonucleoprotein complex of Cas9 protein and gRNA (where 2 and 3 are DNA-free methods (Liu et al. 2017; Yin et al. 2017)). However, further optimization is required to stabilize RNA by adding some protectants (Yin et al. 2017). The vehicle system usually specifies the cargo that either it could be packed or to what extent it could be delivered for the in vitro/in vivo study. Vehicles for the CRISPR-reagent cargoes could be classified into physical delivery, viral vector, and nonviral vector systems (Lino et al. 2018).

6.2.1

Microinjection

In physical delivery, microinjection is considered best approaching 95–100% efficiency in some cases (Yang et al. 2013; Gu et al. 2018; Horii et al. 2014). Any cargo system can be directly injected into a cell using a 0.5–5.0 μm diameter needle and a microscope (Lino et al. 2018). The system is good for the targeted delivery of reagents to the specified site and bypasses the extracellular matrix, plasma membrane, and cellular organelles generating the transfer constraints. The other significant advantage of using microinjection is the liberty of taking any molecular weight of the cargo which is a big problem with some other methods. The microinjection method is best suited for in vitro and/or ex vivo work due to its specificity to the targeted cell sites only, and nucleic acid cargo is most common for it. The DNA transfer to the nucleic acid is mostly used approached through microinjection. However, while injecting RNA, it is ideal to inject gRNA into the nucleus and Cas9 coding RNA into the cytoplasm of the cell (Crispo et al. 2015; Raveux et al. 2017). However, two microinjections can make this laborious and even may result in nonviable cells (Lino et al. 2018; Ma et al. 2014).

6.2.2

Electroporation

Electroporation is one of the most routinely used physical methods for delivering the gene-editing components into a population of cells. In electroporation delivery, cells suspended in the buffer are treated with a pulsed high-voltage electric field to open the nanosized pores within the plasma membranes which allow the molecular components to move into the cell. Electroporation-based transfection is relatively

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simple, less cumbersome, and more efficient for in vitro studies. However, cell types, buffer composition, and the applied electric pulse (pulse voltage, duration, width, and number) are the critical factors determining the electroporation efficiency. For example, bacterial cells are more tolerant to the electric voltage and can be easily managed during electroporation as compared to plant cells and mammalian cells which are more sensitive (Lino et al. 2018). Voltage optimization is a very important step in electroporation protocol. Common cargo for this technique is DNA plasmid and mRNA (encoding gRNA and cas9) (Matano et al. 2015). Neon-transfection system is an electroporation device engineered for plant cells that have been used to deliver plasmid containing Cas9 into wheat microspores (Bhowmik et al. 2018), and CRISPR/Cas9 ribonucleoproteins (RNPs) into cabbage protoplasts (Lee et al. 2020).

6.2.3

Agrobacterium-Mediated Transformation

Agrobacterium-mediated transformation is the most employed method for delivering the CRISPR/Cas9 cassettes into the plant cells (Paul 3rd and Qi 2016; Liu et al. 2017). Agrobacterium directly integrates the transfer DNA (T-DNA) templates into the plant genome through activation of several virion genes located on the tumorinduced (Ti) plasmid (Gelvin 2003). The gene cassette harboring the Ti plasmid sequences, Cas9-sgRNA, and selectable marker sequences is transfected into plant cell through Agrobacterium suspension cultures (Liu et al. 2017). The most commonly used strains for Agrobacterium-mediated transformation are A. tumefaciens, EHA105, GV3101/pMP90, and GV2260, respectively. Some Agrobacterium-vector including binary vector, super binary vector, the dual binary vector has been engineered for the efficient delivery of gene-editing components into plant cells, comprehensively reviewed by (Zhang et al. 2020).

6.2.4

Other Nonviral Vehicles

The nonviral approaches enlist a number of different techniques including lipoplexes/polyplexes (Miller et al. 2017), cell-penetrating peptides (Axford et al. 2017), DNA nanoclew (Sun et al. 2014), and gold nanoparticles (AuNPs) (Lee et al. 2017). It has been demonstrated that Cas9/gRNA-RNPs associated with AuNPs create a complex that can be efficiently delivered into cells and produce the desired mutation at the rate of 30% (Lee et al. 2017). Direct delivery of Cas9/gRNA using nanoparticles is very useful for simplifying genome editing and reducing GMO-related issues (Lino et al. 2018).

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Viral-Based Approaches

Several viral-based approaches have been developed to combat the challenges of transgene delivery in live plants especially when HDR-mediated GE becomes difficult (Ali et al. 2015; Huang and Puchta 2019; Yin et al. 2015). However, viral-based delivery has a major limitation of carrying only small-sized templates due to the viral genome size. For example, the genome size of geminivirus is ∼2.5–3.0 kb, whereas the Cas9 (from S. pyogenes) alone has a nucleotide sequence of ∼4.2 kb. Thus, the cloned-sgRNAs are more efficient to be delivered using the viral particles in those plants which have already cloned Cas9 particles (Paul 3rd and Qi 2016). Still, the viral-based delivery is more desirable providing a surplus amount of template sequences and remained valuable in plant genetic engineering (Paul 3rd and Qi 2016; Begemann et al. 2017).

6.3

Geminivirus as Delivery Vehicles

Geminiviruses are plants infecting DNA viruses containing a genome of 2.8 kb encoding limited overlapping genes. After entering the host nucleus, the geminiviral single-stranded genome is replicated into a double-stranded form by using the host DNA polymerase enzymes (Hanley-Bowdoin et al. 1999; Lozano-Duran 2016; Baltes et al. 2014). This double-stranded template triggers the rolling-circle replication (RCR) to form the new viral genome and starts the transcription of viral genes (Hanley-Bowdoin et al. 1999; Johne et al. 2009). Only Rep/RepA (replicationassociated protein; mastrevirus Rep, RepA) of geminivirus is necessary to initiate the viral replication process (Baltes et al. 2014). Rep/RepA recognize the intergenic region (IR) sequence on the geminiviral genome and produce a site-specific cleavage to mark the start site for replication (Hanley-Bowdoin et al. 1999). For a complete overview of geminiviral association with the host factors/protein, we recommend reading this comprehensive review entitled “Geminiviruses: masters at redirecting and reprogramming plant processes” (Hanley-Bowdoin et al. 2013).

6.4

Geminivirus Replicons (GVRs) for Crop Improvements

A schematic representation of the CRISPR/Cas9-GVRs-based GE has been illustrated in Fig. 6.1. The delivery of sufficient CRISPR reagents to the target sites is one of the most important factors determining the gene-targeting efficiency. CRISPR systems facilitated with GVRs have demonstrated a higher potential of GE frequency as compared to the conventional T-DNA transformations (Baltes et al. 2014). The removal of geminiviral infectious genes with any exogenous sequence, such as nuclease-expression cassette or repair templates, would generate

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Fig. 6.1 Schematic representation of CRISPR/Cas9-geminiviral replicons (GVRs) for homologydirected repair (HDR)-mediated genome editing in a plant cell. Geminivirus genome containing Rep (replication-associated protein), LIR (long intergenic region), and SIR (short intergenic region) sequences along with SSN (sequence-specific nucleases), and repair template is cloned in T-DNA (transfer DNA) and transformed in Agrobacterium tumefaciens. Upon plant transformation, the engineered GVRs are released into the plant-cell nucleus and start replication (RCR; rolling circle replication) to make double-stranded DNA. The RCR in a circular way amplifies thousands of copies of GVRs containing the repair templates. DSBs at the target loci by CRISPR/Cas9 system are repaired by HDR, and the donor template is integrated into the plant genome

noninfectious viral replicons. Movement protein (MP) replacement restricts its cellular and insect-mediated transmission from one plant to others. Coat protein (CP) elimination also increases replicon intermediates. The absence of CP and MP in these replicons will facilitate their enhanced replications inside the host cells without having any counter interactions of CP/Rep, viral transcripts packaging, or cell-to-cell movement (Zaidi and Mansoor 2017). Once inside the host, any sequence (expression cassette/repair templates) flanked by GVRs-Rep/RepA-binding sites and IR sequences will start the RCR via homologous recombination-dependent replication (HDR; Fig. 6.1) (Richter et al. 2016). This will revert the host cells to the S phase making it favorable for HR in the presence of abundantly produced homologous repair templates, thus leading to higher targeting efficiency (Baltes et al. 2014). GVRs-mediated GE has been achieved in several crops due to certain advantages. The broad-spectrum infectivity of geminiviruses makes the GVRs the best choice for

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GE for multiple hosts such as rice, tomato, potato, cotton, wheat, etc. (Table 6.1). The first demonstration of GVRs-based GE was achieved in tobacco plants with engineered bean yellow dwarf virus (BeYDV) replicons delivering the ZFNs and repair templates efficiently at a host reporter site (Baltes et al. 2014). The BeYDV replicons were able to deliver a sufficient sized cassette to the target cells for HR (Steinert et al. 2016). The same year, the CRISPR/Cas9-mediated HDR was approached for a kanamycin resistance cassette into the ADH1 gene locus in Arabidopsis thaliana (Schiml et al. 2014). This opened the new avenue for CRISPR-GVRs associations and was used for promoter replacement in tomato adjacent to anthocyanin synthesis gene (Cermak et al. 2015). The BeYDV replicon delivered the repair template (strong promoter sequence) at a 12-fold higher frequency as compared to the conventional Agrobacterium T-DNA transfer (Cermak et al. 2015). Gil-Humanes et al. (2017) deconstructed the wheat dwarf virus (WDV) for delivering the CRISPR reagents in cereal crops. The WDV replicons produced a 110-fold increase in targeted gene expression as compared to the non-replicating controls. The WDR replicon-mediated delivery of CRISPR/Cas9 and repair templates produced a 12-fold higher GE in the targeted ubiquitin locus in hexaploid wheat as compared to other nonviral delivery methods (Gil-Humanes et al. 2017). The HDR-mediated GE was achieved in all homoeoalleles (A, B, and D) of polyploid wheat by using the WDR replicons (Gil-Humanes et al. 2017). In rice, Wang et al. (2017) used WDV replicons for HDR-mediated targeting of two genomic loci (Actin-1, and glutathione S-transferase) and reported the targeting efficiency of up to 19.4%. Similarly, Dahan-Meir et al. (2018) used BeYDV replicons for tomato GE with 90% efficiency in gene KI. The carotenoid isomerase (CRTISO) and phytoene synthase 1 (PSY1) genes of tomato were targeted for HR due to their phenotypic response. The BeYDV replicons resulted in 25% higher amplification of donor templates in transformed tomato lines (Dahan-Meir et al. 2018), elucidating the potential of viral replicons for GE. Butler et al. (2016) utilized GVRs for HDR-mediated targeting of Acetolactate synthase1 (ALS1) gene in potato with improved transformation. The GVRs-CRISPR system incorporated herbicideinhibiting point mutations in the targeted loci with enhanced efficacy as compared to the conventional T-DNAs transfer and proved a novel technique for vegetatively propagated plants (Butler et al. 2016). In another study, the cabbage leaf curl virus (CaLCuV) vector was engineered for “virus-based gRNA delivery system for CRISPR/Cas9 mediated plant genome editing (VIGE)” in N. benthamiana plants (Yin et al. 2015). The VIGE system was developed to deliver the sgRNA targeting the host NbPDS3 and NbIspH genes producing the KO mutations. This proved to be a successful alternative to the virus-induced gene silencing (VIGS) (Yin et al. 2015). In addition, plant RNA viruses such as tobacco rattle virus (TRV) has been extensively used for plant genome editing coupled with RNAi (Brigneti et al. 2004; Sha et al. 2014), ZFNs (Marton et al. 2010), and CRISPR/Cas (Ali et al. 2015). However, TRV replicon has limitations of carrying limited capacity of SSNs and/or sgRNAs.

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Table 6.1 GVR vector used for plant research Plant species Nicotiana tabacum, Nicotiana benthamiana

GVRvector AYVV

Delivery Whiteflies, electroporation, biolistic

Targeted gene NPTII, GUS

Nicotiana benthamiana

BeYDVm

Agrobacterium tumefaciens

GFP, HPV-16 CP L1, HIV-1 Cp24

Nicotiana benthamiana

BeYDV

Agrobacterium tumefaciens

mAb 6D8

Lactuca sativa

BeYDV

Agrobacterium tumefaciens

NVCP, mAB hE16, and mAB hE6D8

Nicotiana tabacum

BeYDV

Agrobacterium tumefaciens

P-GUS: NPTII

Applications Development and evaluation of a plant Escherichia coli shuttle vector using the replicon of the monopartite begomovirus AYVV and a bacterial replicon derived from pUC19 to ensure a high copy number in E. coli Production of subunit vaccines of HIV-1 cp24 and HPV-16 cp L1. Also provides a platform for the development of new vaccines and pharmaceutical products Production of mAb 6D8 against Ebola (0.5 mg per g of leaf mass). Also, support the idea of multi replicon vectors for the production of VLP with two or more subunits BeYDV-based GVR, high expression of NVCP from Norwalk virus coat protein, and therapeutic monoclonal antibody production against Ebola and West Nile viruses For transient expression of SSNs and DNA repair templates for enhanced HDR efficiency (one to two orders higher as compared to conventional T-DNA transfer)

References Tamilselvi et al. (2004)

Regnard et al. (2010)

Chen et al. (2011)

Lai et al. (2012)

Baltes et al. (2014)

(continued)

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Table 6.1 (continued) Plant species Solanum lycopersicum

GVRvector BeYDV

Delivery Agrobacterium tumefaciens

Targeted gene ANT1

Nicotiana benthamiana

CaLCuV

Agrobacterium tumefaciens

NbPDS3, NbIspH

Solanum tuberosum

BeYDV

Agrobacterium tumefaciens

StALS1

Nicotiana benthamiana

BeYDV

Agrobacterium tumefaciens

mAb rituximab, NVCP

Nicotiana benthamiana

BeYDV

Agrobacterium tumefaciens

P19 p, HBc, NVCP

Applications Higher frequency of precise modification in tomato using GVR to remove efficiency constraints in GE To demonstrate the stable expression and efficiency of CaLCuV vector in plants through developing photo-bleached phenotype to overcome the deficiencies of VIGE To explore GVR-based CRISPR against conventional T-DNA in diploid and tetraploid potato by targeting stAL1 5’UTR and 3’UTR enhance recombinant production and improve NVCP production by ~fourfold and mAb rituximab by 1 mg per gram leaf. 50 untranslated region (UTR) reduced Rep/RepA expression, reduced cell death, and enhanced the production of monoclonal antibodies Enhanced production of virus-like particles (VLP) hepatitis B core antigen and Norwalk virus capsid protein with co-expression of P19 protein of tomato bush stunt virus as VIGS

References Čermák et al. (2015)

Yin et al. (2015)

Butler et al. (2016)

Diamos et al. (2016)

Wang et al. (2017)

(continued)

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Table 6.1 (continued) Plant species Triticum aestivum

GVRvector WDV, ToLCV

Oryza sativa

WDV

Agrobacterium tumefaciens

GFP, GUS

Manihot esculenta

BeYDV

Agrobacterium tumefaciens

EPSPS

Solanum lycopersicum

BeYDV

Agrobacterium tumefaciens

CRTISO, PSY1

Nicotiana benthamiana

SPLCV

Agrobacterium tumefaciens

GFP

6.5

Delivery Biolistic

Targeted gene Ubi, MLO, GFP

Applications Transient expression of WDV and ToLCV replicons for cereal genome engineering (promote multiplex GT and achieved a >tenfold increase in expression To demonstrate high frequency, precise KI of template DNA at DSB through GVR (nearly 19.4% targeted frequency achieved) Induction of glyphosate resistance in cassava Precisely targeted modification with 90% efficiency in the absence of selection markers or reporter genes Development of SPLCV repliconbased vector to increase the efficiency of SVs by 35% than TVs

References GilHumanes et al. (2017)

Wang et al. (2017)

Hummel et al. (2018) DahanMeir et al. (2018)

Yu et al. (2020)

Layout for Engineering CRISPR-GVRs Cassette

Certain characteristics of geminiviruses make them remarkable for genetic engineering. The broad host range (Nawaz-ul-Rehman and Fauquet 2009), ease of engineering with minor viral components (Baltes et al. 2014), and the ability to perform RCR for multiple copy number inside host (Hanley-Bowdoin et al. 2013) make it perfect for multiple genetic engineering works. The detailed methodology for GVRs engineering for CRISPR-Cas is written for potato crop in a book chapter entitled “Genome Editing in Potato with CRISPR/Cas9” (Nadakuduti et al. 2019). Here, we simply describe the general layout for designing a CRISPR-GVRs cassette that could be modified according to the crop or cloning vector’s availability.

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Fig. 6.2 (a) Native viral genome having movement protein (MP) and coat protein (CP) responsible for infections. (b) MP and CP are replaced by SSNs sequences for CRISPR reagents translations and template sequences for HDR repairing mechanism

6.5.1

Deconstruction of Geminivirus for Designing the DNA Replicon

For GVRs replicon, coding sequences of MP and CP are removed in the viral genome to prevent the systematic spread including cell-to-cell and plant-to-plant movements (Fig. 6.2). Geminiviruses such as BeYDV, WDV, and tomato leaf curl virus (ToLCV) have been deconstructed for DNA replicons in many studies (Baltes et al. 2014; Gil-Humanes et al. 2017) (Dahan-Meir et al. 2018; Butler et al. 2016). The removal of CP and MP not only restricts the viral movement but also facilitates the increase in copy number of the DNA replicon (Padidam et al. 1999), probably due to loss of CP interactions with the synthesized DNA replicons (to be packed) and CP/Rep associations repressing the viral replications (Malik et al. 2005). After sequence removal, a reporter gene such as GFP could be cloned in viral genome adjacent to the viral promoter to create the reporter plasmid such as pWDV2-GFP and ToLCV-GFP (Gil-Humanes et al. 2017). The reporter replicon will be used as appositive control carrying a GFP gene and will be replicated and expressed in the transformed cell lines.

6.5.2

CRISPR/Cas Vector Construction

For cassette development, the GVRs elements (LIR, SIR, and Rep/RepA) are cloned into a plant binary vector. For this purpose, PCR amplification could be achieved for

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GVRs elements from some commercially available vector such as pWI11 (Ugaki et al. 1991). After this, the PCR-amplified product will be cloned into the multicloning site of a binary vector in LIR-SIR-Rep-LIR orientation (Fig. 6.2). The binary vectors with Gateway cloning sites (attR1 and attR2) remained more suitable for GVRs sequences due to the ease of Gibson assembly (see step-by-step vector construction in the detailed protocol (Nadakuduti et al. 2019)). For CRISPR-Cas9 cassette, different reagents, such as plant codon-optimized Cas9 sequence (pCas9) with N-terminal 3x Flag and N- and C-terminal nuclear localization signals, sgRNAs (20-nt) adjacent to a U6 RNA polymerase III promoter, reporter gene, could be cloned into a vector (pCLEAN-G185) (Gil-Humanes et al. 2017). The designing of an expression cassette depends upon several factors including the targeted plant, transformation methods, and the availability of reagents. Based on these facts, the CRISPR-cassette could be easily modified. For HDR-mediated genome editing, an exogenous template is cloned in the CRISPR-GVRs cassette to replace the targeted endogenous gene. The donor template could range from single nucleotide insertion to several bases (~500–1000 bp) and must have left to right homology with the targeted sequences to be replaced. Care must be taken while designing sgRNAs to not hybridize to the donor template provided with the expression cassette. The donor template with the desired modifications could be cloned from another organism through PCR amplification or could be commercially synthesized from companies such as Integrated DNA Technologies® or GenScript®. Once we got the donor template, it could be assembled with the targeted cassette through Gibson assembly.

6.5.3

Transformation in Ex-Plants

There are several ways to introduce the cassette to the targeted explant. Biolistic transformation is one of the approaches used for cereal crops (Sanford 1990). For example, in the case of wheat, immature wheat scutella (0.5–1.5 mm) were used for biolistic transformation through gene gun (Gil-Humanes et al. 2017). Gold nanoparticles (0.6 μm) coated with CRISPR expression cassette could be used for stable transformation to produce heritable mutations (Shan et al. 2013). Moreover, in vitro synthesized gRNAs could be directly transformed to the plants expressing the Cas9 proteins in the targeted cells (Svitashev et al. 2015). The most common method of plant transformation in multiple systems (RNAi/ZFNs/CRISPR-Cas) is Agrobacterium-mediated transformation. The T-DNA vectors carrying the sgRNAs, pCas9, and GVRs sequences could be integrated into the plant genome by using this technique. For this purpose, any T-DNA vector could be introduced into A. tumefaciens strains (LB4404 or GV3101) through electroporation (Nadakuduti et al. 2019). The internodal stem cuttings of explants (Nadakuduti et al. 2019) or leave disks (Yin et al. 2015) could be soaked in Agrobacterium culture (0.6 OD) for plant transformation. Other methods to introduce the CRISPR-GVRs reagents in crop plants include vacuum infiltration (Jiang et al. 2014) and protoplast transformation (Woo et al. 2015).

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Prospects and Conclusion

Targeted GE has certainly emerged as an alternative to classical plant breeding which promises the precise molecular editing of plant DNA to facilitate gene function studies, induction of novel traits, abiotic, and biotic stress resistance (Belhaj et al. 2013; Baltes et al. 2014). CRISPR/Cas9 is an efficient molecular suite to precisely modify the genome in a defined manner (Li et al. 2020). CRISPR/Cas9 system has been functionally applied in model plants and crops including Nicotiana benthamiana (Li et al. 2013; Belhaj et al. 2013), Nicotiana tabacum, Arabidopsis (Belhaj et al. 2013), wheat, maize, rice, sorghum, tomato, potato, and sweet orange (Jia and Wang 2014). GVRs-based CRISPR/Cas9 gene KI/KO and base modification have been diversely exploited for crop improvement and augmentation of several traits, as well as promoter regulation for altered gene expression. Future developments will likely include multigene functional studies and the emergence of non-transgenic GE researches through improvements in HDR efficiency (Wang et al. 2018). CRISPR applications are limited concerning challenges to HDR (Zaidi and Mansoor 2017; Cermak et al. 2015) due to lack of efficient cargo delivery system (Lino et al. 2018) to deliver sufficient repair template (Wang et al. 2017; Baltes et al. 2014) and the large size of cassette which impose a problem for viral-vector engineering (Schaeffer and Nakata 2015). Several vector systems have been developed for delivering CRISPR/Cas9 cassette in cells utilizing the NHEJ DNA repair mechanism for GE (Zaidi and Mansoor 2017; Lino et al. 2018). HDR is highly desirable in plants as this integrates template DNA at DSBs precisely as compared to NHEJ which is “error-prone” (Jiang et al. 2013; Puchta 2005). HDR pathways are often described as “error-free and exploit homologous DNA templates to fix the targeted DNA sequence precisely”, however, with low competency as compared to natural NHEJ (Puchta et al. 1993; Puchta 2005). Research is focused to improve the efficiency of HDR concerning the limited template sequences in plants (Zaidi and Mansoor 2017). The efficient use of SSN via geminivirus vectors might prove to be a potential solution for this limitation (Zaidi and Mansoor 2017). GVRs contribute to higher frequencies of HDR in several ways. Perhaps the most significant is by greatly increasing availability of the donor molecule (Ellison 2021*: 38). With several successful examples, GVR-based CRISPR/Cas9 GE through HDR has become the hotspot of research, yet there are some issues with using geminivirus as cargo delivery vehicles. The cargo capacity of these viruses is quite restricted, limited to only infiltrated leaves, and cell-to-cell movement is restricted as per removal of cell-to-cell movement proteins to remove the cargo size constraints. A plausible solution could be to engineer betasatellites for delivery of GE reagents or separately expressing the MP/s under another vector or using bipartite begomovirus vectors that have movement-related proteins on a separate genomic component. An alternative approach is to employ single-stranded donor oligonucleotide (ssODN) by covalently bonded Cas9/gRNAribonucleoprotein complex. Spatial and temporal co-localization of T-DNA and Cas9 via SNAP-tag could increase the efficiency of HDR through GVR-based

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CRISPR/Cas9. No doubt, currently available GVRs can target several plants, but BeYDV- and WDV-derived replicons may not function in all target crops which is a probable sign of host-specificity requirement. Advancements in CRISPR/Cas9 gears themselves and delivery vectors/vehicles can efficiently remove constraints in HDR through geminivirus-based vectors. There is a need to address host-specificity limitation while deconstructing GVR for broadening the spectrum of target hosts. This challenge can be addressed by leveraging expanding viral sequence resources made available through metagenomics. Multiplex genome editing which enables quick integration of multiple genes in elite varieties dramatically improves agronomic traits (Yin et al. 2017) has been applied through CRISPR/Cas9 (Zhang et al. 2016). But the GE system requires large constructs to express multiple sgRNA cassettes, which are more laborious to construct and may cause instability and reduce transformation efficiency (Paul 3rd and Qi 2016). Recent studies showed that the Lachnospiraceae bacterium Cas12a (LbCas12a or LbCpf1) presents an advantage in HDR-based GE, over conventional CRISPR/Cas9-based GVR approach (Vu et al. 2020). CRISPR/Cpf1 complexes have been successfully applied for GE in rice (Li et al. 2018) and many other commercially important crops. These studies demonstrate the feasibility of highefficiency multiplex gene editing in plants using engineered CRISPR-Cpf1. This can be a possible alternative for conventional CRISPR/Cas9 T-DNA cassette in GVRs. In conclusion, the CRISPR system is evolving at an extraordinary pace (Lino et al. 2018). GVRs for CRISPR/Cas9 have the potential for HDR-based GE and present a user-friendly technique with much-improved efficacy. With time, the novel improvements in GVRs can help targeted HDR by expressing the Cas9/sgRNA and template strand with ample amounts (Zaidi and Mansoor 2017). GVR-based GE might be able to alleviate the limitations/constraints of RNA viruses, e.g., TVR with a limited cargo capacity and delivery of DNA sequences. The geminivirusassociated satellites, like betasatellites, might have the potential to act as efficient co-vectors, however, yet unproven for GE.

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Chapter 7

Targeted Gene Replacement in Plants Using CRISPR-Cas Technology Zheng Wei, Rukmini Mishra, Raj Kumar Joshi, and Kaijun Zhao

Abstract The CRISPR-Cas based genome editing systems have been widely used in plant functional genomics research and crop improvement due to its simplicity, high efficiency and specificity. For example, the CRISPR-Cas9 system has been extensively used to generate gene knock-out and knock-in mutants of various plant species, because this system can efficiently recognize the targeted site and cause double strand breaks (DSBs), which can be repaired by the error-prone non-homologous end joining (NHEJ) pathway or the precisely homologous recombination (HR) pathway. Compared to the broadly applied error-prone small insertion or deletion (InDel) mutations of targeted site by NHEJ pathway, precisely targeted gene replacement by HR is lagged by its low efficiency. However, precise modification by HR is favored by scientists because of its precision and flexibility. In this chapter, we reviewed the mechanism and the recent technical achievements of the CRISPR-Cas meditated HR repair, its applications and future prospect in plant research and crop improvement. Keywords CRISPR-Cas · Crop improvement · Genome editing · Gene targeting · Homologous recombination Z. Wei National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing, China R. Mishra Department of Botany, School of Applied Sciences, Centurion University of Technology and Management, Bhubaneswar, Odisha, India e-mail: [email protected] R. K. Joshi Department of Biotechnology, Rama Devi Women’s University, Bhubaneswar, Odisha, India e-mail: [email protected] K. Zhao (*) National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing, China Shandong Shunfeng Biotechnology Co. Ltd, Jinan, China e-mail: [email protected]; [email protected] © Springer Nature Singapore Pte Ltd. 2022 K. Zhao et al. (eds.), Genome Editing Technologies for Crop Improvement, https://doi.org/10.1007/978-981-19-0600-8_7

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Introduction

The identification of gene function and the breeding of new crop varieties are inseparable from the acquisition of mutants. Previously, the acquisition of mutants mainly depended on natural mutation, physical or chemical mutation, and random insertion of T-DNA. These methods have some defects, such as low mutation efficiency, random mutation sites, time-, and labor-consuming. Therefore, the introduction of nucleotide variation at specific sites in a genome to achieve site-specific gene editing can efficiently generate the desired mutants, thus speeding up the process of basic research and crop breeding. Genome editing technology mainly uses sequence-specific nucleases (SSNs) to produce double-stranded breaks (DSBs) at specific sites in a genome; the DSBs can be repaired by the nonhomologous end joining (NHEJ) to introduce error-prone small insertions or deletions (InDels) or be repaired by the homologous recombination (HR) pathway to introduce precisely targeted gene replacement after the insertion of corresponding repair template. Among all editing tools, CRISPR-Cas systems have been widely used to generate gene knockout/ knock-in mutants of various plant species, due to its simple chemistry and high editing efficiency. In a broad sense, the HR in vivo can generally be classified into two different pathways, the RNA-mediated transcript template homologous-directed repair (TT-HDR) and the DNA-mediated HR, which can be further categorized into the single-strand annealing (SSA), the microhomology-mediated end joining (MMEJ), break-induced replication (BIR), the synthesized dependent strand annealing (SDSA), and the canonical HR which is featured by the formation of the double Holliday junctions (dHJs) (Anabelle 2013; Ranjha et al. 2018). The main drawback of HR method is the low efficiency. To increase the efficiency, researchers from all over the world have shown no-less enthusiasm on tattering the new technology to overcome the barriers. The recent attempts to increase the editing efficiency are to create precisely repair-prone CRISPR editing tools, supply the sufficient repair templates, provide the precisely repair-prone donor, and express Cas9 under germline-specific promoters. The application of CRISPR-Cas system-mediated HR, which is also referred to as gene targeting (GT), arouse great interest among researchers to create the precisely edited plants with the ideal features (Paszkowski et al. 1988; Kumar and Fladung 2002; Dong and Ronald 2021). Despite the low efficiency of this homologous recombination-based repair, successful applications have been reported in various species of plants. Scientists have successfully applied this method in creation of herbicide tolerance, abiotic resistance, quality improvement, and disease resistance. The recent advancements in the field of medicine to increase the GT efficiency could be a guide to future technological breakthroughs in plant science.

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7.2

141

The Homologous Recombination Pathways

The RNA-mediated HR is mostly referred to as TT-HDR. RNA can be used as an intermediate product of DNA recombination after DNA damage (Derr et al. 1991). In yeast and human, RAD52 proteins effectively catalyze RNA annealing to DSBs like DNA ends in vitro and revealed a new mechanism of homologous recombination and DNA repair, in which transcriptional RNA is used as a template for DSBs repair. Considering that RNA:DNA hybridization is more stable than DNA:DNA double strand and the abundance of RNA transcripts in cells, RNAs may have a significant effect on the stability and plasticity of genome (Keskin et al. 2014). The DNA-mediated HR in vivo is currently categorized into five different types: the SSA, the MMEJ, the BIR, the SDSA, and the canonical HR (Ranjha et al. 2018). Unlike the NHEJ, the HR mostly was initiated by the DNA resection at the break site to produce long exposure 30 single-strand DNA (ssDNA) overhang, which will anneal each other between the homologous sequence (SSA and MMEJ), or invade the double-stranded DNA (dsDNA) of the repair template to form the displacement loop (D-loop) structure at the following step (BIR, SDSA, and canonical HR) (Fig. 7.1). SSA and MMEJ are the repair mechanisms that directly anneal the same direction homologous sequences on both sides of the DSBs site. When a double-stranded break occurs between two homologous repeats, these ends are resected, creating ssDNA. When the excision proceeds to the repeated sequence, such as the homologous 30 single-strand tail, these single strands can be complementary annealing each other and finally leaving only one copy of the repeat sequence. In this mechanism, when the length of the homologous sequences is 4–20 bp, it is usually called MMEJ, and when the homologous sequence length is longer than 20 bp, it is considered SSA mechanism (Mcvey and Lee 2008; Truong et al. 2013). BIR is a unique HR pathway for the repair of a single end of the DSBs. After the DSBs formed, the newborn ssDNA with the 30 hydroxyl created by the 50 –30 exonuclease will invade the double-stranded DNA of the repair template and start to synthesize new DNA stands. This synthesis will last to the end of the repair template or even the whole chromosome arm, which makes it as a unique feature of the BIR. The BIR can also maintain the length of the telomere without the assistance of telomerase (Sakofsky and Malkova 2017). The SDSA pathway shares the same procedures: resection at the DSBs, ssDNA invasion, and D-loop formation. But when the synthesis of invasion strand proceeds to the homologous sequence on the repair template, it will be apart from the repair template. Then the homologous sequence on the newly synthesized DNA strand anneals to the originally damaged DNA sequence. The lesion on the original DNA strands is then repaired by DNA polymerase to produce non-crossover product (Knoll et al. 2014). The canonical HR is the most intensively studied mechanism. It is classified into two categories of dHJs processing: the resolution of dHJs and the dissolution of the dHJs. The resolution of dHJs usually happens in the G2/S stage of leptotene. The invading DNA strands synthesize and extend new DNA strands using the DNA

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Fig. 7.1 The overview of homologous recombination pathways in vivo. Homologous recombination repairs the DSBs in the DNA by (a) transcript-templated HDR in which transcript RNA is used as a template and mediated by RaD52 protein. (b) Single-strand annealing pathway is facilitated to repair DSBs between two repeat sequences. As the DNA around the DSBs is resected, the single 30 overhangs are coated with replication protein A (RPA). Rad52 protein aligns the two complementary repeat sequences, while the leftover nonhomologous flaps are cut away by a set of Rad1/Rad10 nucleases. In the absence of a repeat sequence in the 30 overhangs, the ssDNA invades the repair template of homologous DNA and induces the break-induced replication (BIR) pathway (c) or the synthesis-dependent strand annealing (SDSA) pathway (d). The strand invasion followed with DNA extension results in two cross-shaped structures called as the double Holliday junctions (dHJs). Resolution of dHJs, one in horizontal and the other along the vertical axis, results in both crossover and non-crossover products (e), while the dissolution of both dHJs along the horizontal axis produces non-crossover product (f)

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strands of homologous chromosomes as templates to form double Holliday junctions. The resolution of the double Holliday junctions produced two different types of repair results, namely, crossover product and non-crossover product (Wright et al. 2018). Unlike the resolution of the dHJs, the dissolution of dHJs produces non-crossover product by the assistant of helicase/topoisomerase. The dissolution of dHJs or the SDSA may be the main repair mechanism in plant’s gene targeting by the CRISPR technology (Andersen and Sekelsky 2010; Mitchel et al. 2013; Mao et al. 2019).

7.3

The Approaches of Increasing GT Efficiency in Plants

Scientists have made many attempts to increase the GT efficiency in plants. The efforts to increase the GT efficiency can be categorized in four aspects: creating precisely repair-prone CRISPR editing tools (like the CRISPR/Cas12a and the Cas9VirD2 variant), supplying the sufficient repair templates (like the geminivirus replicon-based donor), providing the precisely repair-prone donors (like the ssDNA donor, the RNA donor, and the dsODN donor) and expressing Cas9 under germline-specific promoters.

7.3.1

The Application of CRISPR/Cas12a

Different from the CRISPR/Cas9 system that produces blunt ends after cutting, the CRISPR/Cas12a (CRISPR/Cpf1) system usually results in a sticky end of 4–5 nucleotides at the DSBs (Zetsche et al. 2015). The production of sticky ends is very conducive to single-stranded invasion and recognition during homologous recombination repair. In 2017, the rice chlorophyllide-a oxygenase gene of rice (CAO1) was selected as the target, and GT with hygromycin phosphotransferase gene (hpt) as a repair template has been successfully achieved (Begemann et al. 2017). In addition, GT in the acetolactate synthase gene (OsALS) locus in rice has also been obtained with high efficiency through the biolistic method (Li et al. 2020). The combined use of CRISPR/Cpf1 and geminivirus replicon in tomato enhanced the GT efficiency at ANT1 gene, a key transcription factor controlling the anthocyanin pathway (Vu et al. 2020).

7.3.2

The Application of Cas9-VirD2 Variant

To facilitate the recognition of donor fragment in nucleus during the DSBs repair, Cas9 is combined with VirD2 relaxase to produce a chimeric Cas9-VirD2 protein. This chimeric protein contains the VirD2 relaxase domain, which is derived from the

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VIR protein produced by Agrobacterium tumefaciens and can recognize the right border (RB) region of T-DNA. The nuclear localization signal (NLS) in VirD2 also helps to import the repair template into the nucleus and brings the repair template near to DSBs to enhance HR. The Cas9-VirD2 system has been used to create precise modification at the acetolactate synthase (OsALS) locus, carotene lyase dioxygenase-7 (OsCCD7) locus and the histone deacetylase (OsHDT) locus. The Cas9-VirD2 system expands the ability to improve important agricultural traits of crops and opens up new possibilities for precise genome engineering across different eukaryotic species (Ali et al. 2020) (Table 7.1).

7.3.3

Supply of Sufficient Repair Templates Via Geminivirus Replicon

Geminivirus replicon-based donor molecules are applied to provide enough repair templates for GT in plants. After integrating the viral genome elements such as long intergenic region (LIR), short intergenic region (SIR), and replication initiator proteins (Rep) into Ti-plasmid of Agrobacterium, the gene of interest can be selfsynthesized like viral genome replication to increase the gene copies (Grimsley et al. 1986). In the genome editing field, scientists build the geminivirus repliconbased donor which contains two repeated LIRs, Rep gene, SIR, donor fragment, and necessary CRISPR components between the left and right borders (LB and RB) of the Ti plasmid. In this way, after infecting the plant with the geminivirus repliconbased donor vector by Agrobacterium, the repair template is copied in large quantities (Baltes et al. 2014). For dicots, soybean yellow dwarf virus (BeYDV) is generally an accepted tool for GT. The first attempt was done to replace the promoter of the transcription factor ANT1 with a 35S promoter in tomato, which resulted in the accumulation of anthocyanins and the formation of purple callus and seeds (Cermák et al. 2015). The efficiency of GT was further improved by the application of geminivirus replicon-based multi-replicon system (Vu et al. 2020). Similarly, this technology also achieved the expected GT at the StALS1 gene locus in potato (Butler et al. 2016). In addition, the technology has successfully restored the functions of the GUS and NPTII genes previously integrated into the tobacco genome (Cermák et al. 2017). For monocots, wheat dwarf virus (WDV) was used to supply enough repair donors for GT. It was reported that GT was achieved for TaMLO, TaUBI, and TaEPSPS gene loci in wheat protoplasts and embryogenic calli (Gil-Humanes et al. 2017). Geminivirus has also been successfully used in rice calli to achieve GT with high efficiency (Wang et al. 2017). However, although the use of geminivirus can improve the efficiency of GT, there is less report on whether a heritable mutation can be formed after the GT event of T0 generation (Huang and Puchta 2019) (Fig. 7.2).

GL1

ALS

ROS1

Arabidopsis

Arabidopsis

Arabidopsis

ANT1

ctisto

ANT1

SIHK1;2 ALS

Tomato

Tomato

Tomato

Potato

DD20 DD43

NbPDS gus:npt II

Tobacco Tobacco

Soybean

AtTFL1

Arabidopsis

DME

Targeted gene ADH1

Plant species Arabidopsis

T-DNA + BeYDV replicons Biolistic

PEG protoplast T-DNA + BeYDV replicons T-DNA + BeYDV replicons T-DNA + BeYDV replicons T-DNA + BeYDV replicons

T-DNA

T-DNA

T-DNA + BeYDV replicons T-DNA

Delivery method T-DNA

Table 7.1 The GT in plant by CRISPR-Cas system

dsDNA: LHA (~1 kb) + RT (~2.6 kb) + RHA (~1 kb) dsDNA: LHA (~1 kb) + RT (~2.6 kb) + RHA (~1 kb)

dsDNA: 1851 bp homology sequence with 7 substitutions dsDNAe

dsDNA: LHA (989 bp) + RT (2424 bp) + RHA (740 bp)

dsDNA: LHA (1778 bp) + RT (281 bp) + RHA (1737 bp)

dsDNA: LHA (987 bp) + RT (1949 bp) + RHA (719 bp)

dsDNA: LHA (533 bp) + RT (6 bp) + RHA (114 bp) dsDNAe

dsDNA: 1542 bp homology sequence with (10 + 2) bp substitutions dsDNA: LHA (801 bp) + RT (720 bp) + RHA (325 bp) dsDNA: LHA (801 bp) + RT (1653 bp) + RHA (325 bp) dsDNA: LHA (812 bp) + RT (720 bp) + RHA (742 bp) dsDNA: LHA (609 bp) + RT (720 bp) + RHA (751 bp) dsDNA: LHA (801 bp) + RT (1915 bp) + RHA (853 bp)

dsDNA: LHA (813 bp) + RT (10 bp) + RHA (830 bp)

Donor structure dsDNA: LHA (674 bp) + RT (~1.8 kb) + RHA (673 bp)

Up to 12.79  0.37%a 0.66%a 12.5% 32.2%d 4.6% 3.8%

25.0%

3.65–11.66%

e

9.0% Successful GTb,

0.8%

5.3–9.1%

6.3–8.3%

~1.0%

0.12%

GT efficiency Successful GTe

(continued)

Butler et al. (2016) Li et al. (2015)

Zhao et al. (2016) Li et al. (2013) Cermák et al. (2017) Cermák et al. (2015) Dahan-Meir et al. (2018) Vu et al. (2020)

References Schiml et al. (2014) Hahn et al. (2018) Wolter et al. (2018) Miki et al. (2018)

7 Targeted Gene Replacement in Plants Using CRISPR-Cas Technology 145

OsPDS

OsALS

OsALS

OsGTS

Rice

Rice

Rice

Rice

Rice

Rice

OsALS OsHDT701 OsALS OsCAO

OsACT1

CTL1

Maize

Biolistic + WDV replicons Biolistic + WDV replicons PEG protoplast PEG protoplast T-DNA Biolistic

Biolistic T-DNA T-DNA

Biolistic

Biolistic

T-DNA

Biolistic Biolistic

LIG1 ARGOS8

Maize

Maize

Biolistic

Delivery method

Targeted gene ALS1 ALS2

Plant species

Table 7.1 (continued)

RNA: 200 nt homology sequence with 2 substitutions RNA: LHA (75 nt) + RT (87 nt) + RHA (75 nt) RNA: 200 nt homology sequence with 2 substitutions dsDNA: LHA (1 kb) + RT (3.3 kb) + RHA (1 kb)

dsDNA: LHA (~500 bp) + RT (~2.6 kb) + RHA (~500 bp)

dsDNA: LHA (~500 bp) + RT (~2.6 kb) + RHA (~500 bp)

ssDNA1 and ssDNA2: 80 nt homology sequence with 2 substitutions dsDNA: LHA (100 bp) + RT (330 bp) + RHA (46 bp) dsDNA: LHA (100 bp) + RT (330 bp) + RHA (46 bp) dsDNA: 1935 bp homology sequence with 4 substitutions

ssDNA: 72 nt homology sequence with 12 nt mutations

dsDNA: LHA (411 bp) + RT (~3.8 kb) + RHA (422 bp)

Donor structure dsDNA: 1084 bp homology sequence with 5 substitutions dsDNA: 794 bp homology sequence with 4 substitutions ssDNA: 127 nt homology sequence with 4 or 7 substitutions dsDNA: LHA (1 kb) + RT (3.2 kb) + RHA (1 kb) dsDNA: LHA (~400 bp) + RT (~600 bp) + RHA (~400 bp)

Up to 16.88% 2.13–4.69% 2.14% 3.0–8.0%a

Up to 19.4%

Up to 7.7%

Successful GTe Successful GTe 0.147–1%

0.2% (1/480)

6.8% (2/29)

GT efficiency Successful GTe 0.2% (2/1000) 0.4% (4/1000) 2.5–4.1% ~1.0%(2/194,3/ 334) Up to 4.7%

Begemann et al. (2017)

Butt et al. (2017)

Endo et al. (2016) Wang et al. (2017)

Barone et al. (2020) Shan et al. (2013) Sun et al. (2016)

Shi et al. (2017)

Svitashev et al. (2015)

References

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xa23

TaMLO

Rice

Rice

Wheat

EPSPS and ubiquitin

Ubiquitin and TaMLO

Ubiquitin

Biolistic Biolistic Biolistic Biolistic Biolistic

OsNRT OsUBQ6 OsALS OsHDT OsCCD7

PEG protoplast + WDV replicons PEG protoplast + WDV replicons Biolistic + WDV replicons PEG protoplast + WDV replicons Biolistic + WDV replicons

Biolistic

Biolistic Biolistic Biolistic Biolistic

OsALS OsALS OsSLR1 OsTT1

Rice Rice Rice

Biolistic

OsNRT1.1B

Rice

dsDNA: LHA (210 bp) + RT (981 bp) + RHA (646 bp) & dsDNA: LHA (747 bp) + RT (1058 bp) + RHA (773 bp)

0.4%

1.1%

5.74%

dsDNA: LHA (747 bp) + RT (1058 bp) + RHA (773 bp) dsDNA: LHA (747 bp) + RT (1058 bp) + RHA (773 bp) & dsDNA: LHA (674 bp) + RT (1027 bp) + RHA (647 bp)

3.8%

6.4%

1.8%

1.56–9.87%c Enhancedc,e 1.5–8.8%c

4.6%a 1.8%a Average frequency: 6.1%

6.7%

dsDNA: LHA (747 bp) + RT (1058 bp) + RHA (773 bp)

dsDNA: LHA (674 bp) + RT (1027 bp) + RHA (647 bp)

RNA: LHA (97 nt) + RT (381 nt) + RHA (121 nt) dsDNA: LHA (196 bp) + RT (~484 bp) + RHA (74 bp) dsODN: 96 bp with 87 bp homology sequence dsODN: 99 bp with 93 bp homology sequence, dsODN: 130 bp with ~40 bp homology sequence dsODN: 98 bp with 80 bp homology sequence dsODN: 128 bp with ~40 bp homology sequence ssDNA: 94 nt homology sequence with 4 substitutions ssDNA: LHA (25 nt) + RT (43 nt) + RHA (27 nt) ssDNA: 95 nt homology sequence With 4 substitutions dsDNA: LHA (176 bp) + RT (34 bp) + RHA (349 bp)

dsDNA: LHA (100 bp) + RT (251 bp) + RHA (100 bp)

(continued)

Wei et al. (2021) Gil-Humanes et al. (2017)

Ali et al. (2020)

Li et al. (2018a, b) Li et al. (2019) Li et al. (2020) Lu et al. (2020)

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Targeted gene Ubiquitin

Delivery method PEG + WDV replicons

Donor structure dsDNAe e

GT efficiency Successful GTb,

References Cermák et al. (2017)

LHA left homologous arm, RT repair template, RHA right homologous arm, GT gene targeting, dsDNA double-stranded DNA, ssDNA single-stranded DNA, T-DNA the Agrobacterium-mediated transformation, PEG protoplast the protoplast transformation, BeYDV soybean yellow dwarf virus, WDV wheat dwarf virus a By the application of CRISPR-Cas12a system b By the application of CRISPR-Cas9 nickase c By the application of CRISPR-Cas9-VirD system d Enhanced by multiple rounds of regeneration e Unspecific clarified in the original manuscript

Plant species Wheat

Table 7.1 (continued)

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Fig. 7.2 Geminivirus replicon provides enough repair templates

7.3.4

The ssDNA Donor

Single-stranded DNA templates were shown to be efficient substrates for extrachromosomal recombination because they could directly facilitate the initial annealing step between the donor and targeted DNAs (Vu et al. 2019). Successful achievement of GT was reported in rice by transient expression in 2013 (Shan et al. 2013). A similar strategy was also used to co-transform with the Cas9 editing system to obtain replacement plants for the target gene site OsALS (Sun et al. 2016). In addition to the above application in rice, GT was also successfully applied in maize by using ssDNA as repair template (Svitashev et al. 2015) (Table 7.1).

7.3.5

The RNA Donor

To increase the repair efficiency, scientists have developed the template-annealingprone donor, which can facilitate the homologous sequence recognition between the targeted genomic loci and the repair donor. The RNA-DNA hybrid is more stable than the DNA-DNA structure (Chien and Davidson 1978). In vivo, transcription template HDR (TT-HDR) can be used for homologous directed DNA repair (HDR) of DSBs (Keskin et al. 2014). But primary transcripts are often processed and transported to the cytoplasm, making them impossible for HDR. The donor fragment of TT-HDR was combined with the ribozyme fragment by using the characteristics of self-cleavage of ribozyme. RNA repair templates that can be self-processed and released can be produced in the nucleus. After cutting the target gene OsALS by CRISPR-Cpf1 system, TT-HDR was applied to obtain the desired editing type (Li et al. 2019) (Table 7.1). The RNA-mediated GT was reported by using chimeric

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guide RNA (cgRNA) repair method. The chimeric single-guide RNA (cgRNA) contained both sequences for target site specificity (to generate the double-stranded breaks) and repair template sequences (to direct HDR), flanked by regions of homology to the target. Researchers have successful applied this cgRNA repair method to generate herbicide resistance in rice (Butt et al. 2017).

7.3.6

The dsODN Donor

Robust targeted integration of oligonucleotide has also been achieved through the use of blunt, 50 -phosphorylated, double-stranded oligonucleotide (dsODN) beading phosphorothioate linkages (Tsai et al. 2015). The 50 phosphorylation facilitates NHEJ repairing of DSBs. Considering that NHEJ act as the predominant repair method in plant cells, attempts have been made to introduce chemically modified dsODNs to improve the efficiency of targeted insertion in plant system (Lu et al. 2020). A 60-bp translational enhancer from the 50 untranslated region (UTR) of rice ADH1 (alcohol dehydrogenase 1) was flanked by two additional nucleotides with phosphorothioate-linkage and 50 -phosphorylation modifications and used as donor DNA for insertion of three nucleotide polymorphisms in rice (Lu et al. 2020). The successful insertion of the chemically modified translational enhancer of rice ADH1 (ADHE) by NHEJ indicated feasibility of using chemically modified dsODNs as a donor. Subsequently, a tandem-repeat HDR strategy (TR-HDR) was devised using dsODN having homology to the target sequence. The insertion of dsODN in the target site results in a tandem repeat structure with the flanking genomic sequence. Then, the sgRNA targeting the genomic DNA creates DSBs which trigger HDR between the tandem repeats, leading to replacement of the target sequence with the homologous sequence (Lu et al. 2020). Collectively, the use of chemically modified donor DNA together with TR-HDR can achieve precise sequence insertion and replacement in plants (Table 7.1).

7.3.7

The Application of Germline-Specific Promoters to Drive Cas9

Beside the innovation in modifying the Cas9 and donors, manipulating the HR in the reproductive cell by expressing Cas9 under the egg cell and early embryo-specific promoter can also rise the replacement efficiency. Stable genetic GT was successfully achieved at ALS, ROS1, and DME target gene loci in Arabidopsis thaliana. Using egg cell and early embryo-specific promoters to drive the expression of Cas9 or other site-specific nuclease, combined with the strategy of effective delivery of donor DNA, not only makes GT in Arabidopsis simple and efficient but also can be

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further applied in other plants, including crops (Miki et al. 2018; Wolter et al. 2018) (Table 7.1).

7.4

Applications of CRISPR-Cas System-Mediated GT in Crop Improvement

With the ability to create targeted mutations, the CRISPR-Cas system-mediated GT is well accepted by the breeders of the world. Till date, the CRISPR-Cas systemmediated GT has been used in tobacco, rice, soybean, corn, and other crops to achieve precise insertion or replacement of gene fragments.

7.4.1

Herbicide Resistance

With the widespread use of herbicides in crop production, the breeding of new crop varieties resistant to herbicides has become an important breeding target. Unlike the conventional transgenic methods which generally introduce foreign herbicideresistant genes into crops, CRISPR–Cas editing targets plant endogenous genes to confer herbicide resistance with higher speed and flexibility and resulted in transgene-free plants (Zhu et al. 2020). For example, specific amino acid mutations in acetolactate synthase (ALS) can increase the resistance of plants to acetolactate synthase herbicides. In maize, the chlorosulfuron-resistant mutations were achieved by precise modification at the ALS gene (P165S) (Svitashev et al. 2015). A similar study was conducted where herbicide-tolerant rice was created using a gene replacement strategy in the ALS coding region (Sun et al. 2016; Endo et al. 2016). CRISPR/ Cas9-mediated gene editing technology has been effectively used to generate herbicide-resistant plants by targeting multiple genes such as cellulose synthase A catalytic subunit 3 (CESA3), 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and splicing factor 3B subunit 1 (SF3B1) that are endogenous in nature (Han and Kim 2019). These valuable gene loci can be chosen as target site for future GT applications.

7.4.2

Drought Tolerance

Due to the growing scarcity of global water resources, researchers are trying to breed the crop with drought tolerance. Unveiling the molecular mechanism of drought response in plants using CRISPR/Cas genome-editing system has led to improvement in tolerance to drought stress. Maize ARGOS8 gene is a negative regulator of ethylene response, contributing to the drought tolerance of the crop. The maize

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GOS2 gene promoter, which confers a moderate level of constitutive expression, was inserted into the 50 -untranslated region of the ARGOS8 gene, resulting in a significant increase in ARGOS8 gene expression. The mutants with GOS2 promoter fragment showed a significant increase in final yield compared to the wild-type maize in arid environments (Shi et al. 2017). In rice, the dsODN was used to precisely repair the TT1 gene in rice in order to obtain high-temperature-resistant rice mutants (Lu et al. 2020).

7.4.3

Improvement of Crop Quality

To fulfill the aim of creating carotenoid-enriched rice, scientists analyzed the whole genome sequencing data of rice mutant library and then selected genomic safe harbors as targeted locus to insert a 5.2 kb carotenoid biosynthesis cassette and obtain marker-free rice plants with high carotenoid content in seeds (Dong et al. 2020). In addition, by using dsODN as the donor, the researchers accurately repair the SLR1 gene in rice, producing expected dwarf-phenotype variant (Lu et al. 2020). At the nitrate-transporter gene, NRT1.1B locus, which is related to nitrogen absorption in rice roots, precise repair was achieved through gene gun method (Li et al. 2018a, b). GT results have also been obtained by using dsODN for this locus (Lu et al. 2020).

7.4.4

Disease Resistance

Rice bacterial blight caused by Xanthomonas oryzae pv. oryzae is a devastating bacterial disease worldwide. Rice bacterial blight resistance gene Xa23 is usually not expressed due to the lack of EBEavrxa23, which can detect the appearance of the pathogen effector protein AvrXa23, in the core element of the Xa23 gene promoter. After the pathogen infection, the AvrXa23 will bind to the EBEavrxa23 and immediately initiate the Xa23 gene expression and trigger the hypersensitive reaction. Even if the universal existence of Xa23 ORF sequence in most rice cultivars, the EBEavrxa23 is just preserved in a few rice cultivars. To generate broad-spectrum resistance to bacterial blight disease, we have successfully converted rice varieties from susceptible into resistant by the replacement of EBEAvrXa23 via CRISPR/Cas9mediated HDR (Wei et al. 2021).

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Challenges and Future Implications

CRISPR/Cas system has been widely applied to various species in plants, which enables us to manipulate the genome as we desired. However, the low efficiency of CRISPR/Cas system-mediated GT limited its further application. Drawing on the experience of the medical research, there may be some potential breakthroughs to increase GT efficiency in plants.

7.5.1

Inhibition of NHEJ

DSBs caused by CRISPR-Cas system is mainly repaired by NHEJ in plants. Therefore, inhibition of key enzymes of NHEJ (such as DNA ligase IV) seems to be a reasonable way to improve GT efficiency. A ligase IV inhibitor, SCR7, which targets the DNA-binding domain of ligase IV and hinders its function in NHEJ repair, has been used to inhibit the NHEJ (Srivastava et al. 2012). It has been demonstrated that SCR7 could effectively promote GT in human epithelial melanoma cell line (A549) and mouse bone marrow-derived dendritic cell line (DC2.4) (Maruyama et al. 2015). In addition, E1B55K and E4orf6 proteins from adenovirus type 4 can also be used to inhibit NHEJ. These two proteins can mediate ubiquitination and proteasome degradation of DNA ligase IV (Forrester et al. 2011). When e1b55k and E4orf6 were co-expressed with the CRISPR-Cas system, the efficiency of HDR was increased eight times, and NHEJ activity in human and mouse cell lines was basically eliminated. This finding provides a useful tool for increasing the frequency of precise gene modification in mammalian cells (Chu et al. 2015). Similar attempt to inhibit the activity of DNA ligase IV was also reported in rice, where the GT efficiency can also be promoted once the DNA ligase IV was knocked out (Nishizawa-Yokoi et al. 2016). NHEJ can also be inhibited by using small interfering RNA (siRNA). In porcine fetal fibroblasts, the GT efficiency could be increased by 1.6–3 times by expressing the siRNA which can inhibit the expression of Ku70 or Ku80 (Li et al. 2018a). Short hairpin RNA (shRNA) could also be applied to inhibit the key NHEJ pathway proteins such as Ku70, Ku80, and DNA ligase IV in HEK293 cells. The results showed that when transfected with single shRNA targeting Ku70, Ku80, or DNA ligase IV, HDR efficiency increased from 5% to 8–14% (Chu et al. 2015).

7.5.2

Promotion of HR

CTIP is a key protein in the early stage of DSBs resection, which is crucial for HR initiation. To take the advantage of CTIP in HR repair pathway, the N-terminal fragment of CTIP was combined to dCas9 or Cas9 protein to form dCas9-CTIP or

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Cas9-CTIP fusion protein. After transfected by this new fusion protein, the GT efficiency in human fibroblast cells, iPS cells, and mouse fertilized eggs was increased by two times (Charpentier et al. 2018). Besides CTIP, many other proteins were involved in HR procedure, such as Rad51, Rad50, and BRCA1. Overexpression of Rad51 or Rad50 with CRISPR-Cas9 system can be used to increase the GT efficiency (Yu et al. 2011). Further, the researchers fused the D-loop formation supporter hRad51 with nickase Cas9 to create hRad51-Cas9 (D10A) fusion protein, improving GT without double-stranded breaks (Rees et al. 2019). The GT efficiency was also increased two- to threefold by overexpression of BRCA1 (Pinder et al. 2015). Furthermore, it has been found that the overexpressed yeast chromatin remodeling gene RAD54 can significantly promote the GT efficiency in the Arabidopsis (Shaked et al. 2005). RS-1 is a chemical compound which can stimulate Rad51 bind to ssDNA during DSBs repair (Song et al. 2016). Application of RS-1 can enhance HDR by three to six times in HEK293 and U2OS cell lines (Pinder et al. 2015). Moreover, there are two small molecules (L755507 and brefeldin A) that can facilitate HR repair pathway after DSBs. L755507, a β3-adrenergic receptor agonist, can be used to triple the GT efficiency in embryonic stem cell by the concentration of 5 μM. Meanwhile, brefeldin A, an inhibitor of intracellular protein transport from endoplasmic reticulum to Golgi apparatus, can increase the GT efficiency in embryonic stem cell by twofold at a concentration of 0.1 μM (Yu et al. 2015).

7.5.3

Control of Cell Cycle to Facilitate GT

As HR repair usually happened in S/G2 phase of cell cycle, the chemical methods can be used to make the cells stay in S/G2 phase to improve GT efficiency. In the field of medical research, by blocking cells in S/G2 phase of cell cycle with chemical inhibitors, the GT efficiency could be increased to 38% in HEK293T cells (Lin et al. 2014). Similarly, the GT efficiency was enhanced three- to sixfold by using microtubule polymerization inhibitor nocodazole or ABT-751 to control the cell cycle (Yang et al. 2016).

7.5.4

Optimizing Length of Homologous Arms

The length of homologous arms also has important effect on GT efficiency. Researchers found that when the length of the homologous arm of the repair template was 350 bp, the repair efficiency of homologous recombination was significantly lower than that of the vector with the length of homologous arm of 1450 bp (Rong et al. 2014). Moreover, it was reported that the homologous arm with the length no less than 1 kb was an effective donor of HR (Chu et al. 2015) (Table 7.2).

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Table 7.2 The elements used for increasing the GT efficiency Improvement of GT efficiency Inhibition of NHEJ

Targeted factors in DNA repair progress Ku70, Ku80

DNA ligase IV

Applications siRNA-mediated suppression of NHEJ siRNA-mediated suppression of NHEJ NHEJ suppression by SCR7 NHEJ suppression by E1B55K and E4orf6 protein Knockout

Promotion of HR

7.6

Rad51, Rad50

Overexpression hRad-Cas9 nickase fusions Enhanced by RS-1

Rad54

Overexpression

CTIP

Fused with Cas9

BRCA1

Overexpression

References Li et al. (2018a, b) Chu et al. (2015) Srivastava et al. (2012) Chu et al. (2015) NishizawaYokoi et al. (2016) Yu et al. (2011) Rees et al. (2019) Song et al. (2016) Pinder et al. (2015) Shaked et al. (2005) Charpentier et al. (2018) Pinder et al. (2015)

Conclusions

The gene editing technology via homology-directed repair method can accurately and efficiently change the traits of crops. At the same time, it has some limitations, mainly the low GT efficiency. In order to overcome the shortcomings of low GT efficiency, scientists have made many innovations, such as creating precisely repair-prone CRISPR editing tools (like the CRISPR/Cas12a and the Cas9-VirD2 variants), supplying the sufficient repair templates (like the geminivirus replicon-based donor), providing the precisely repair-prone donors (like the ssDNA donor, the RNA donor, and the dsODN donors), and expressing Cas9 under germline-specific promoters. In recent years, the research on improving the GT efficiency in medical field has provided new insights for further improving GT efficiency in plants. In contrast to the reported achievements in CRISPR/Cas-mediated gene knockout applications, the applications of GT in crops were still limited, mostly focusing on the selection marker to detect the GT efficiency. But the various applications of CRISPR technology in conventional knockout editing also provide the target sites of valuable gene locus, which can be used for reference in GT in crops to create variants

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for herbicide tolerance, abiotic resistance, quality improvement, and disease resistance.

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Chapter 8

Expanding the Scope of Base Editing in Crops Using Cas9 Variants Rukmini Mishra, Muntazir Mushtaq, and Raj Kumar Joshi

Abstract Genome editing technologies hold tremendous possibilities towards crop improvement and food security for the future. Among others, base editors have developed as novel editing platforms that facilitates specific base modification minus the introduction of double stranded break or homologous recombination. Base editors typically consisted of non-functional CRISPR–Cas9 motif (Cas9 variants) fused with cytosine (CBEs) or adenosine deaminase (ABEs) protein. While the combinations of cytosine and adenine base editors can produce the four possible base transitions, the necessity of a specific protospacer adjacent motif (PAM) sequence restrict the number of genomic locations that could be altered by CBEs and ABEs. The recent surge in the development of new ABEs and CBEs with multiple Cas9 variants has meaningfully improved the effectiveness of inducing specific and targeted point mutation linked to important agronomic traits in many crops. In this chapter, we have presented a concise idea on the base editing platforms and focused on the application of new ABEs and CBEs with Cas9 variants in crop improvement. Keywords Base editors · CRISPR · Cas12a · Cas12b · PAM · SpCas9

R. Mishra Department of Botany, School of Applied Sciences, Centurion University of Technology and Management, Bhubaneswar, Odisha, India e-mail: [email protected] M. Mushtaq ICAR-National Bureau of Plant Genetic Resources, Pusa Campus, New Delhi, India R. K. Joshi (*) Department of Biotechnology, Rama Devi Women’s University, Bhubaneswar, Odisha, India e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2022 K. Zhao et al. (eds.), Genome Editing Technologies for Crop Improvement, https://doi.org/10.1007/978-981-19-0600-8_8

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Introduction

By 2050, the global population is projected to touch 9.6 billion, and the requirement for staple food crops will be increased by 60% (Zhu et al. 2020). Therefore, crop varieties with better resilience to detrimental environments and with better yield improved quality are the need of the hour. However, the conventional crop breeding approaches used so far are arduous, time-consuming and complex (Mushtaq et al. 2019), and more efficient and time-saving breeding tools are obligatory. The rapid development in genome sequencing methods has made available powerful genomic information for various plant species, and genome editing technologies put forward the opportunity to edit genes of interest with precision, thus providing the potential for crop improvement. Genome editing includes the use of sequence-specific nucleases (SSNs) to create DNA double-strand breaks (DSBs) which are subsequently repaired by endogenous repair mechanisms comprising the homology-directed repair (HDR) or the non-homologous end joining (NHEJ). Although the initial SSNs like the meganucleases, zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) (reviewed in Mishra et al. 2018) were found to be effective genome edit tools in plants, their usage was limited due to the involvement of complex protein engineering in their development. CRISPR/Cas9 system provides an exceedingly accurate and robust genome editing strategy that has been efficaciously employed for precise editing in crops and model plants (Zhang et al. 2018). Nonetheless, undesirable modifications at the non-targeted loci and specificity of the PAM sequences are major caveats for universal application of this technology. Targeted editing of genes in plants is mostly carried out through HDR, which facilitates precise genome engineering via base insertions, substitution and sequence replacements (Atkins and Voytas 2020). CRISPR/Cas9-mediated genetic substitution via HDR has been reported to induce precise point mutations in several crop plants (Zhang et al. 2018). However, irrational HDR together with poor competency in the delivery of template DNA and inability to convert one base to another has been major challenges to achieve trait-specific point mutations in plants (Ran et al. 2017). Base editing is a novel alternate gene editing technology that facilitates precise base substitution with no significant gene disruption or necessity of template DNA sequence (Komor et al. 2016). After the development of base editors for animal cells, base editing platforms with precise DNA deletion properties were established in plants (Zhu et al. 2020). A fusion of inactive nuclease alternates with several deaminases has created different base editing structures that intend to deal with the prime precincts such as PAM compatibility, accuracy, length of editing site and sequence inclination (Molla and Yang 2019; Mishra et al. 2020). Since the latest base editors considerably relegate inadvertent editing in the plant genomes, they exhibit a great promise for creating better agricultural crop plants. In this chapter, we summarize the different kinds of base editors that are precisely employed to edit plant genomes and the Cas9 variants that have augmented the possibility of singlebase modification.

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Base Editors: Overview DNA Base Editors

Base editors are synthetically modified proteins adept for creating single nucleotide modifications (SNMs) in a precise manner, made up of Cas9, a DNA targeting element and a deaminase protein which deaminates adenine or cytosine base in a targeted region (Gaudelli et al. 2017). Based on the design of base editing system, Cas9 is modified either to an enzymatically inactive or dead Cas9 (dCas9) or a Cas9 nickase (nCas9). The dCas9 is developed by Asp10Ala and His840Ala modification that leads to inactivation of its endonuclease activity but maintains the capacity to bind to DNA. A single-guide RNA (sgRNA)-guided attachment of dCas9 to the specific region in DNA forms an ‘R-loop’ by unzipping a fragment of DNA. This 5–8 bp ssDNA fragment acts as an editing site for dCas9-bound deaminase enzyme to substitute the cytosine bases. The base editors have the ability to introduce singlebase modification minus a DSB, thus restricting the formation of indels. DNA base editors are broadly classified as either CBEs or ABEs based on the targeted base substitution induced by them.

8.2.2

Cytosine Base Editors

These are first-generation base editors (BE1) which alter cytosine for a thymine by removing the amino group from a targeted cytosine to create uracil. To deaminate the amine of cytosine base, David Liu employed an apolipoprotein B mRNA editing enzyme catalytic subunit 1 (APOBEC1) cytidine deaminase, which acts on single stranded DNA (ssDNA). A 16 amino acid-long XTEN linker was used to fuse APOBEC1 with dCas9 generating the first cytidine base editor (Komor et al. 2016). The main problem in employing base editors is bypassing DNA repair mechanism that resists base pair alteration. It is observed that editing efficiency of BE1 dropped from 25–40% in vitro to 0.8–7.7% in vivo. This occurs due to cellular base excision repair of U-G. Base excision repair (BER) mechanism involves uracil N glycosylase (UNG), which recognizes the U-G mispairing and breaks the glycoside bond DNA backbone and uracil. Thus, the BER reverts the BE1-generated U-G intermediate to C-G. To inhibit the UNG activity, the C terminus of the dCas9 was attached with a uracil DNA glycosylase inhibitor (UGI), thereby forming the BE2 platform APOBEC-XTEN-dcas9-UGI) (Komor et al. 2016). UGI is an 83 amino acid residue protein from Bacillus subtilis bacteriophage PBS1 which inhibits UNG in both human and bacterial. Thus, UGI-mediated prevention of BER improved the base editing efficacy by threefold. BE2 base editor is restricted to edit cytosine present on target strand of the DNA. To substitute G (guanine) present on the non-target strand of the DNA with A, the BE3 base editor was generated that particularly nicks the non-targeted strand. This

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nicking feature of BE3 inclines BER of U-G mismatch to promote U-A product, improving editing efficiency up to sixfold compared to BE2. To nick the non-edited strand, BE2 was modified by replacing dCas9 with nCas9 (Cas9 nickase). Thus, BE3 was made from the fusion of UGI to the C terminus and rAPOBEC1 to the N terminus of a nickase Cas9 D10A (Komor et al. 2016; Fig. 8.1a). The nCas9 also displayed a rise in editing efficiency by 1.1% likened to BE2. Thus, CBE changes C-G to T-A in a specific fashion (Fig. 8.1a). Nevertheless, the presence of multiple cytosines (Cs) inside the target site can lead to off-target editing and non-target C to U modification. To prevent the off-target conversion of C to U, the BE3 has been further developed through the usage of different Cas9 variants. The SpCas9 mutants such as EQR-Cas9, VQR-Cas9, VRER-Cas9 and SaKKH-Cas9 were used to modify BE3. David Liu and co-workers substituted these four Cas9 variants for SpCas9 in BE3 to generate EQR-BE3, VQR-BE3, SaKKH-BE3 and VRER-BE3 which target NGAG, NGAN, NNNRRT and NGCG PAMs, respectively. These modified four BE3 variants showed a 2.5-fold increase in the editing efficiency (Kim et al. 2017). It is reported that mutation in cytosine deaminase influences the specificity and off-target editing. Various APOBEC1 cytidine deaminase mutants including YEE-BE2 and YEE-BE3 with altered range of editing region have been developed to improve the preciseness of the target and minimization of the off-target effects. The APOBEC1 triple mutant W90Y + R126E + R132E (YEE-BE3) showed the highest editing competence with an editing region of approximately two bases (Kim et al. 2017) (Fig. 8.1b). Base editors have been further improvised with the development of Target-AID (activation-induced cytidine deaminase) containing an nCas9 and pmCDA1 cytidine deaminase protein (Nishida et al. 2016) (Fig. 8.1c). Target-AID facilitated the modification of immunoglobulin (Ig) locus and developed varied mutations which are chosen using antigen binding. Thus, the Target-AID system was employed to develop base editors and enhanced efficacy in mouse and human cells. Successively, the S. pyogenes Cas9-derived fourth-generation base editor SpBE4 and S. aureus Cas9-derived base editor SaBE4 were generated by fusing rAPOBEC1 to nCas9 using a linker of 32-aa. Also, BE4 had two UGI protein molecules combined specifically to both C and N terminal of nCas9 (Komor et al. 2017). Additionally, to improve precision in editing by minimizing indel, bacteriophage Mu-derived Gam protein that binds to the free ends of DSB was combined to N terminus of nCas9. SpBE4-Gam and SaBE4-Gam (Fig. 8.1d) showed a decrease in non-T outcome and rise in cytosine to thiamine editing in comparison to SpBE4 and SaBE4. Thus, C to T editing with minimum indel and maximum purity outcome was achieved by employing fourth-generation base editors. Furthermore, deaminases are also employed to produce an assorted library of single-base mutations restricted to a specific region of the genome. For instance, the Targeted AID-mediated mutagenesis (TAM) and CRISPR-X were used to localize diversity in genome (Hess et al. 2016; Ma et al. 2016) (Fig. 8.1e, f). The TAM base editing platform was developed by fusing dCas9 to human AID used for systematic DNA modification in mammals (Ma et al. 2016). It is a standard base editing system to study protein functions,

Fig. 8.1 Base editing toolboxes. (a) The base editor BE3 constituted a Cas9 nickase (nCas9D10A) fused with a cytidine deaminase rAPOBAC1 and a uracil DNA glycosylase inhibitor (UGI). (b) Third-generation cytidine BE (YEE-BE3) with YEE-rAPOBAC1. (c) Target-AID BE employs PmCDA1. (d) SaBE4Gam consists of a SaCas9D10A fused with two UGI and a Gam protein. (e) and (f) The TAM and CRISPR-X use dCas9 to engage multiple variants of deaminase AID (AIXx or MS2-AID*D). (g) Adenine base editors (ABEs) are composed of heterodimeric structure (ectadA(WT)-ectadA*) attached to nCas9 that are developed through complex protein chemistry. (h) ADAR2 are RNA base editors made up of dead Cas13 (dCas13) attached with natural adenosine deaminase acting on RNA 2 (ADAR2) protein

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identify drug targets and elucidate novel drug resistance mechanisms. In the same year, David Liu and group also developed the CRISPR-X system to induce localized, diverse base mutation by dCas9-medicated targeting of hyperactive AID variant (Hess et al. 2016). CRISPR-X platform consisted of dual MS2 RNA hairpins which individually inscribe two MS2 proteins directed to AID. The AID displayed a 50-bp wider editing window from the PAM sequence and reported a sixfold increase in the mutation frequency. CRISPR-X has reported effective mutagenesis of the genetic locus targeted by the chemotherapeutic drug bortezomib (PSMB5) (Hess et al. 2016). CRISPR-X also uses dCas9 for DNA targeting. However, the need of G/ C-rich PAM was a major constraint in this system. Later on, Li and co-workers developed the first cytidine deaminase base editor using the Cpf1 endonuclease to further increase the range of PAM sequence (Li et al. 2018a, b). The dLbCpf1-BE0 had an editing window of 8–13 bp prior to PAM and displayed a 20–22% editing efficiency. Subsequently, a series of Cpf1-type base editors including dCpf1-eBE, dCpf1-BE-YE and dCpf1-eBE-YEb were developed that provided multiple PAM choices and broadened the possibility of base editing (Li et al. 2018a, b).

8.2.3

Adenine Base Editors

In 2017, several generations of ABEs were developed that could deaminate adenine nucleotides (Gaudelli et al. 2017). These ABEs were made using E. coli transfer RNA adenosine deaminase (tadA) through robust strategies involving significant protein modification and directed evolution. The tRNA adenine deaminase derived from E. coli shares significant homology with the APOBEC enzyme and modifies adenine to inosine (A to I) in the anticodon loop of arginine tRNA (Fig. 8.1g). tadA was fused with an inactivated CRISPR/Cas9 mutant to develop the first-generation ABEs (Gaudelli et al. 2017). Among others, the seventh-generation ABEs, namely, ABE7.7, ABE7.8 and ABE7.9, are recognized as the most effective ABEs with wider sequence compatibility. ABE7.10 modifies A.T to G.C in a large number of targets with higher specificity and purity. What more, ABEs have significantly extended the possibility of base editing by facilitating all the transition mutation (C to T, A to G, T to C and G to A) in a suitable genome.

8.2.4

RNA Base Editors

RNA base editors were developed by fusing a dead Cas13 (dCas13) with an adenosine deaminase acting on RNA (ADAR) to convert adenosine to inosine (Cox et al. 2017) (Fig. 8.1h). Cas13 is a type VI CRISPR-RNA-guided RNase. Cas13b, an orthologue of Cas13 obtained from Prevotella sp. (PspCas13b), was reported as the most efficient editors in RNA knockdown. The ADAR enzymes facilitate precise modification of RNA sequences through the removal of amino

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group leading to the conversion of adenosine to inosine (Nishikura 2010; Fig. 8.1h). RNA Editing for Programmable A to I Replacement (REPAIR) is the system that is used to edit RNA transcripts. REPAIR platform has been efficiently used to simulate specific alleles that protects against autoimmune ailments (Ferreira et al. 2013). Also, the REPAIRv2 generated via dCas13b-ADAR2DD was found to be highly specific as compared to other RNA editing platforms (Cox et al. 2017).

8.3

Cas9 Variants

Cas9, a characteristic type II CRISPR-Cas system-based protein, is a single DNA-specific nuclease often guided to a specific targeted DNA sequence near the PAM sequence by two noncoding RNAs: CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA) (Jinek et al. 2012). In August 2012, Prof. Jennifer Doudna and her group demonstrated that a synthetic RNA fusion (single-guide RNA or sgRNA) created by combination of crRNA and tracrRNA functions to the same level as the crRNA and tracrRNA complex (Jinek et al. 2012). The system works on the basis of Cas9 enzyme and a contrived single-guide RNA (sgRNA) targeting a specific DNA sequence. As a single RNA is needed to develop specificity of the target, the CRISPR/Cas platforms are highly relevant to genome editing experiments as compared to TALENs and ZFNs (Mishra et al. 2018). However, the availability of only a limited specificity of PAM site together with high off-target effects of Cas9 protein (Wada et al. 2020) has limited the scope of the original CRISPR/Cas9 system. The SpCas9-gRNA normally identifies with the 20 nucleotide sequence upstream of the 50 -NGG-30 PAM sequence and as such limits the availability of wider target sites. Consequently, various strategies have been used to expand the PAM recognition and specificity to overcome the problems. These strategies include engineering of SpCas9 protein, identification of novel Cas9 orthologues and associated CRISPR/Cas system from other organisms. The SpCas9 protein has been comprehensively modified in the recent times to increase the specificity and compatibility of PAM sites as well as dipping the rate of off-target modifications (Wada et al. 2020). The crystal structure modification of SpCas9 with gRNA together with retention of binding to target DNA has led to the development of multiple CRISPR/Cas platform with diverse PAM inclinations. Kleinstiver et al. (2016a, b) demonstrated the development of SpCas9-EQR, SpCas9-VRER and SpCas9-VQR with NGAG-PAM, NGCG-PAM and NGAPAM, respectively. These modified Cas9 variants have also been used in Arabidopsis and rice although their activity was low as compared to wild-type SpCas9 (Hu et al. 2016; Hu et al. 2018). Nishimasu et al. (2018) developed the SpCas9-NG, a SpCas9 with a wider compatibility, recognizing NG PAM. A large number of studies have already demonstrated the application of SpCas9-NG for targeted mutagenesis in rice and Arabidopsis (Endo et al. 2019; Zhong et al. 2019; Ge et al. 2019; Hua et al. 2019; Niu et al. 2020).

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Additionally, other SpCas9 proteins such as SpCas9-HF1 (Kleinstiver et al. 2016a, b), eSpCas9 (Slaymaker et al. 2016) and HypaCas9 (Chen et al. 2017) have also been established. eSpCas9 and SpCas9-HF1 have reported reduced off-target effects in rice, signifying higher specificity of these Cas9 variants in plant cells (Zhang et al. 2017). Directed evolution methods have resulted in the production of modified SpCas9 such as xCas9 (Hu et al. 2018), evoCas9 (Casini et al. 2018) and Sniper-Cas9 (Lee et al. 2018) that exhibited high specificity. xCas9 has wider PAM preferences (NG, GAA and GAT-PAM) that has been demonstrated in Arabidopsis and rice (Zhong et al. 2019; Ge et al. 2019; Hua et al. 2019; Niu et al. 2020; Wang et al. 2019). Both xCas9 and Cas9-NG can create mutations at non-canonical PAMs in plants albeit with different efficiency and specificity in plant cells. Although, Hua et al. (2019) showed that xCas9 functions efficiently in rice, other reports (Zhong et al. 2019; Niu et al. 2020; Wang et al. 2019) have confirmed that xCas9 has significantly lower efficiency in rice calli as compared to mammalian cells (Wang et al. 2019) and is incapable of recognizing NG PAM in tomato (Niu et al. 2020). Zhong et al. (2019) also demonstrated that xCas9 displayed similar activity to Cas9-WT at NGG PAM and higher specificity as compared to Cas9-WT; however activity of xCas9 showed lower activity at NGH (A, T, C) PAM in rice. Alternatively, Cas9-NG has been found to show higher activity as compared to xCas9 in almost all NG PAM sites in rice (Zhong et al. 2019). Hua et al. (2019) also reported robust editing activity of Cas9-NG at multiple NG PAM (CGG, AGC, TGA, CGT) sites. These studies suggested that Cas9-NG could be applicable for genome engineering at the NG PAM site in plants. Base editing with Cas9 variants has also been reported. SpCas9-VQR and SpCas9-NG have been efficiently implicated in the base editing of plants (Hua et al. 2019; Wu et al. 2019; Endo et al. 2019; Zhong et al. 2019; Hua et al. 2019; Negishi et al. 2019). Cas9 variants with diverse PAM compatibility have been discovered in other bacteria (Wang et al. 2019) such as NmCas9 from Neisseria meningitidis (Hou et al. 2013), SaCas9 from Staphylococcus aureus (Ran et al. 2015), FnCas9 from Francisella novicida (Hirano et al. 2016), StCas9 from Streptococcus thermophilus (Müller et al. 2016a, b) and CjCas9 from Campylobacter jejuni (Kim et al. 2017). Novel Cas9 variants including StCas9, SaCas9 and FnCas9 have been employed for genome editing in Arabidopsis and tobacco (Steinert et al. 2015; Zhang et al. 2018; Murovec et al. 2017). Interestingly, in a study carried out by Steinert et al. (2015), it has been demonstrated that SaCas9 and SpCas9 do not intervene with each other in Arabidopsis. In another study, SaCas9-derived base editors have been effectively applied for rice base editing (Hua et al. 2019). Overall, targeting sites with different PAM sequences concurrently with Cas9 orthologues result in multiplex gene editing, thereby widening the use of the CRISPR/Cas system in different plants.

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Increasing the Scope of Base Editing Towards Crop Improvement

CRISPR/Cas9 base editing has developed into a novel strategy for installing point mutations at the specific locations in the DNA or RNA with high precision without incorporating a foreign DNA template or making double-strand breaks (DSBs) (Mishra et al. 2020). Base altering could be more proficient by focusing more on base conversion rather than HDR-interceded quality supplanting with low paces of indel development. Moreover, the creation of a HDR plasmid is more cumbersome as compared to the construction of a base altering vector. Ever since the development of the first base editing platform, several base editing tools have been implicated in the insertion of point mutations in different organisms belonging to dissimilar kingdoms. Efficient and rapid advancements in base editing technology have been proved to reduce the undesirable editing. Existing CRISPR/Cas BEs can establish all four base transitions G ! A, A ! G, C ! T and T ! C in the genome. While CBEs has facilitated C–G to T–A conversion in rice, wheat and Arabidopsis, ABEs have enabled A–T to G–C conversion. Additionally, RBEs could be sued for converting A to inosine (I) (Mishra et al. 2020). CRISPR/Cas-mediated base editing has realized improvement of various crops as has been represented in Table 8.1. CRISPR/Cas-based base conversion in the coding regions has contributed to herbicide tolerance in plants. Plant adenine base editor (Target-AID) has been used for precise base modification in the acetyl-coenzyme A carboxylase ACCase gene resulting in haloxyfop-R-methyl tolerance in rice (Hunziker et al. 2020). In another study, homozygous base substitution using CBE has resulted in the development of marker-free tomato (Shimatani et al. 2017). Likewise, Li et al. (2017) successfully introduced precise point mutations at three target sites in rice. Sitespecific C to T conversion can be performed in different plant species through codon-optimized base editors. The human APOBEC3A-derived CBEs have been employed in the conversion of C to T base independent of the sequence framework of wheat, rice and potato (Li et al. 2018a, b; Zong et al. 2018). Moreover, deamination window for base editors is reported to be wider in case of plants suggesting that the BEs could be a suitable alternative to HDR for inducing single-base modification in plants. Cytidine deaminase not only facilitates the conversion of desired C but also alters other Cs inside the deamination window (Yin et al. 2017). Additionally, a number of engineered and natural Cas9 variants with diverse PAM requirements were used to widen the targeting array of base editors. Until now, all the current base editors mediate CG to TA conversion. Seventh-generation adenine base editors (ABEs) have been developed that perform targeted conversion of A to G (Gaudelli et al. 2017). The evolution of tRNA adenosine deaminase (ectadA) in E. coli and the introduction of mutations gave rise to tadA* which is capable of deaminating adenine in DNA with efficiency rate of 53%. Among the four classes of ABEs, ABE7.10 is the most effective and favours to target A at the protospacer positions 4–7, while the other three Bes insert efficient point mutation when A is at position

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Table 8.1 CRISPR/Cas-mediated base editing applications for crop improvement Plant species Rice

Target gene PDS, OsSBEIIb

Base editor BE3

Editing frequency 0.1–20%

Indel frequency 9.61%

NRT1.1B, SLR1

BE3 (-UGI)

0–13.3%

10%

CDC48

BE3

43.5%

–

ALS, FTIP1e

Target-AID

6–89%

10–62%

CERK1, SERK1, SERK2, ipa1, Pita, BRI-1 ACC, ALS, CDC48, DEP1, NRT1.1B MPK6, MPK13, SERK2, WRKY45, Tms9-1 SPL14, SLR1, SPL16, SPL18, SPL14, SPL17, SPL16, SPL18 SPL13, SPL14, SPL16, SPL17, SPL18, GRF4, TOE1, IDS1, MTN1, SNB, PMS1, PMS3, SNB

BE3

0–38.9%

Nil

ABE7.10

3.2– 59.1% 0– 62.26% 12.5– 26% 0–74.3%

Nil

44–83%

Nil

CDC48, NRT1.1B EPSPS, ALS, DL

ABE7.10 ABE7.8 ABE7.10 ABE-Sa ABE-Sa, ABE-VQR, ABEVRER, ABESaKKH, BE3,VQRBE3, SaKKHBE3 hA3A-BE

Nil Nil Nil

5–95.5%

0–68%

LOX2

TargetAID-NG BE3

1.25%

Nil

DEP1, GW2

ABE7.10

0.4–1.1%

Nil

ALS, MTL

hA3A-BE

Nil

Maize

CENH3

BE3

16.7– 22.5% 10%

Tomato

DELLA, ETR1

Target-AID

41–92%

16–69%

Potato

GBSS

hA3A-BE

6.5%

Nil

Canola

ALS, PDS

ABE7.10, ABE6.3, ABE7.8, ABE7.9

8.8%