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at the National Agri‐Food Biotechnology Institute, Mohali, Punjab, India. He did his doctoral work at the CSIR‐National Botanical Research Institute, Lucknow and received his PhD in Biotechnology from UP Technical University, Lucknow, India. He has been working in the field of Plant Biotechnology for more than 14 years. His present research focuses in the area of functional genomics. He is involved in the characterization of various insect toxic proteins from plant biodiversity, and defense and stress‐signaling genes in bread wheat. His research group at PU has characterized numerous important gene families and long non‐coding RNAs related to the abiotic and biotic stress tolerance and signaling in bread wheat. He has also established the method for genome editing in bread wheat using CRISPR‐Cas system and developed a tool, SSinder, for CRISPR target‐site prediction. His research contribution led to the publication of more than 55 research papers in leading journals of international repute. Further, there are more than five national and international patents,17 book chapters and four books to his credit.
In recognition of his strong research record he has been awarded NAAS Young scientist award (2017–2018) and NAAS‐Associate (2018) from the National Academy of Agricultural Sciences, India, INSA Medal for Young Scientist (2013) from the Indian National Science Academy, India, NASI‐ Young Scientist Platinum Jubilee Award (2012) from the National Academy of Sciences, India, and Altech Young Scientist Award (2011). He has also been the recipient of the prestigious DST‐INSPIRE Faculty Fellowship (2012), and SERB‐Early Career Research Award, (2016) from the Ministry of Science and Technology, Government of India. Dr. Upadhyay also serves as a member of the editorial board and reviewer of a number of peer‐reviewed international journals.
Acknowledgments
I am thankful to the Panjab University, Chandigarh, India for providing the facility to complete this book. I am grateful to all the esteemed authors for their exceptional contributions and reviewers for their critical evaluation and suggestions to improve the quality of the text.
I would like to thank Miss Rebecca Ralf (Commissioning Editor), Miss Kerry Powell (Managing Editor) and Nora Naughton (Copy Editor) from John Wiley & Sons, Ltd for their excellent management of this project, and anonymous reviewers for their positive recommendations about the book.
I also appreciate the support of my research students whose discussion and comments were very useful in shaping this book. I thank Dr. Prabodh K. Trivedi, Dr. Praveen C. Verma, Dr. Krishan Mohan Rai, Dr. Sameer Dixit, Dr. Sudhir P. Singh and Dr. Prashant Misra for direct or indirect help with this project. I wish to express my gratitude to my parents and my beloved wife for her endless support, patience, and inspiration. I thank my daughter, who missed me during this project. I would like to warmly thank the faculties and staff of the department and university for providing a great working environment. Last, but not least, my sincere thanks to Lord Krishna for endowing me to live with joy and success in the form of this book.
1 An Overview of Genome‐Engineering Methods
Sushmita1,3#, Gurminder Kaur2#, Santosh Kumar Upadhyay4, and Praveen Chandra Verma1,3
1 Molecular Biology and Biotechnology, Council of Scientific and Industrial Research, National Botanical Research Institute (CSIR‐NBRI), Lucknow, Uttar Pradesh, India
2 Institute of Bioscience and Technology, Shri Ramswaroop Memorial University, Barabanki, Uttar Pradesh, India
3 Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India
4 Department of Botany, Panjab University, Chandigarh, India
CHAPTER MENU
1.1 Introduction
Agricultural practices, combined with advanced plant breeding and modern technologies, provided food security to millions of people. However, increasing global population demands significant increase in world food production (Parry and Hawkesford 2012). Nevertheless, climate change, depletion of natural resources, increased pollution, and political instabilities are a threat to the food and nutritional security for future generations in the twenty‐first century. Unfortunately, the amount of remaining arable land is limited, necessitating an increase in food production on currently‐used land. Compounding these challenges are the predicted crop losses due to extreme temperatures, pest attacks, and pathogen outbreaks. A powerful approach that may help overcome these challenges is to modify DNA sequences within plant chromosomes for trait improvement (Sedeek et al. 2019). Further, plants can be engineered to have increased tolerance to environmental stresses and pathogens (Han and Kim 2019; Ji et al. 2015; Makarova et al. 2011). In addition to improving the genetic makeup of the crops to meet increasing food demands and control crop loss, genome engineering can also be used to produce valuable plants or products for non‐agricultural purposes (Chen et al. 2019). For example, there is great potential for plants to be used as bioreactors for pharmaceutical proteins. Genetic engineering for increasing the secondary metabolite production in plants would be another use of this technology which would help the perfumery, cosmetic and medical industries, as the secondary metabolites produced from plants have a number of uses (El‐Mounadi et al. 2020). However, to realize the potential benefits of these applications, we must generate effective tools and approaches for editing plant DNA (Miroshnichenko et al. 2019; Tang and Tang 2017).
Introduction of programmed sequence‐specific nucleases (SSNs) and their applications in precise genome editing unfurled a new dimension in genome engineering (Kim and Kim 2014; Voytas 2013). Over the last few decades, researchers reported a few important SSNs, which could be easily engineered and reprogrammed to create double‐stranded breaks (DSBs) at the desired location inside the chromosome. There are three major genome engineering methods, ZFNs, TALENs, and CRISPR‐Cas system (Figure 1.1A) (Jang and Joung 2019; Mahfouz et al. 2014), that have been utilized so far for a variety of purposes, and these have been discussed in detail in the coming sections. Further, we have also described the recently added CRISPR‐Cpf1 system of genome engineering.
1.2 ZFNs
Zinc‐finger nucleases are chimeric fusion proteins consisting of a DNA‐binding domain and a DNA‐cleavage domain. The DNA‐binding domain is composed of a set of Cys2His2 zinc fingers (usually three to six). Each zinc finger primarily contacts 3 bp of DNA and a set of three to six fingers recognize 9–18 bp, respectively. The DNA‐cleavage domain is derived from the cleavage domain of the FokI restriction enzyme. FokI activity requires dimerization; therefore, to site‐specifically cleave DNA, two zinc‐finger nucleases are designed in a tail‐to‐tail orientation (Kim et al. 1996).
Zinc‐finger nucleases can be remodified to recognize different DNA sequences. However, one limitation with redirecting targeting is that it depends on the context of the host. For example, a zinc finger that recognizes GGG may not recognize this sequence when fused to other