Genome Editing in Plants

Principles and Applications

Om Prakash Gupta, Suhas Gorakh Karkute, Om Prakash Gupta, Suhas Gorakh Karkute

• Publisher: CRC Press
• Year: 2021
• ISBN:
  9781000410198
• Edition: 1
• Topic: Biological Sciences
• Subtopic: Genetics & Genomics
• Index: Biological Sciences

Genome Editing in Plants: Principles and Applications addresses the information of genome editing starting from principles and historical aspects to the latest advancements in the field. As genome-editing technology has emerged as promising and cutting edge, researchers around the world have started producing original research outputs, which have significantly improved our current understanding and potential of this technology. The initial chapters of this book describe different genome-editing tools as well as their principles and applications. Other chapters are dedicated to the present status and future applications of genome-editing techniques in various crop improvement programmes. Some of the advanced applications of CRISPR/Cas tools, such as base editing and RNA detection, along with regulatory aspects of genome-edited crops are described in detail.

This book serves as a valuable resource to researchers in the field of crop improvement; graduate and postgraduate students engaged in plant molecular biology and biotechnology; academicians; and policy makers.

Key Features:

• Addresses topics associated with historical development and principles of genome-editing technology

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• Addresses basic mechanisms operating under each genome-editing technology

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• Addresses its application in plants to design crops as per the current and future demands

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• Addresses the regulatory mechanisms of genome-edited crops

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1 Historical Developments of Genome Editing in Plants

Ravindra Ramrao Kale

ICAR-Indian Institute of Rice Research

Prashant Raghunath Shingote

Vasantrao Naik College of Agricultural Biotechnology
Dr. Panjabrao Deshmukh Krishi Vidyapeeth

Dhiraj Lalji Wasule

Vasantrao Naik College of Agricultural Biotechnology

Shriram Jagannath Mirajkar

Dr. D. Y. Patil Arts, Commerce and Science College

Darasing Ramsing Rathod and Mangesh Pradeep Moharil

Dr. Panjabrao Deshmukh Krishi Vidyapeeth

Contents

1.1 Genesis

1.2 Techniques for Genome Editing

1.2.1 Cre-Lox System

1.2.2 Meganucleases

1.2.3 Zinc Finger Nucleases

1.2.4 Transcription Activator-Like Effector Nucleases (TALENs)

1.2.5 Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs)

1.3 Applications of Genome Editing

1.3.1 Applications for Developing Biotic Stress Tolerance

1.3.2 Applications for Developing Abiotic Stress Tolerance and Nutritional Quality

1.4 Conclusion

References

1.1 Genesis

An increase in population and the limitation of available resources for food production have caused food security problems globally. Crop improvement through conventional and molecular breeding approaches can help to tackle the problem of food security through increased food production. Conventional breeding is a very widely used approach for crop improvement and found to be promising for the establishment and increased crop production. Major constraints with conventional breeding are time required for crop improvement; laborious; huge population size; less accurate and reliable; phenotypic selection (PS)-dependent, which is affected by environment; less successful for low-heritable multigenic quantitative traits and the problem with distant crosses, etc. (Tester and Langridge, 2010). Thus, to fasten the crop improvement programme, various molecular breeding approaches such as transgenic, RNAi technology, marker-assisted breeding, gene editing and so on were proven to be effective in terms of the current scenario (Limbalkar et al., 2019). The transgenic approach was found to be effective as it helps to transfer genes from distant species or genera and reduces the time for improvement of crops for a particular trait, but due to societal distrust, biosafety and regulatory issues, the use of transgenic is limited to few countries (Limbalkar et al., 2019). Genome editing has the potential to alter an organism’s genetic makeup through precise changes in the DNA sequence. Genome editing is done using enzymes, especially nucleases to make cuts into the DNA strands, allowing the removal or insertion of the target DNA. It is one of the leading approaches for crop improvement that allows for the modification of a specific region of the genome and allows for the removal, alteration and insertion of DNA sequences at a particular location in the genome with very high precision. Genome editing is based on natural enzymes, including site-directed recombinase (SDR) and site-specific nuclease (SSN) repair enzymes. Genome editing has many benefits, such as site-driven mutagenesis rather than random mutation, site-driven insertion of transgenes with small copy numbers, analysis of functional genomics research, virus or pathogen gene disruption and lesser biosafety issues as compared to transgenics (Pohare et al., 2019).

In the 1960s, scientists started to search for ways to alter genomes. Working in test tubes, researchers in the University of California, San Francisco and Stanford, bombarded DNA with complex variations of molecular gadgets, all borrowed from bacteria. For the first time in 1972, it had shown that genes could be mixed and matched to establish hybrid sequences called recombinant DNA, which had never existed before (Jackson et al., 1972). The initial experiments in the direction of genome editing were carried out by Scherer and Devis (1979), where they demonstrated in vitro replacement of target DNA, which involved the double-stranded break (DSB) repair and recombination mechanisms. There are four main subtypes of SSNs developed to make cuts in genomic sequences, viz. meganucleases, zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat/CRISPR-associated proteins (CRISPR/Cas) nuclease system. The timeline of major historical discoveries in genome editing is summarized in Table 1.1.

TABLE 1.1
Timeline of Major Scientific Breakthroughs in Genome Editing

Year	Major Discovery
1978	Ishino and colleagues for the first time observed an unusual repetitive sequence activity in bacteria
1979	In vitro genome editing with the replacement of chromosome segment with other DNA sequence
1985	Identification of zinc-binding domain in protein transcription factor
1987	CRISPRs, first described as a bacterial immune system in Escherichia coli
1989	Studied the double-stranded break repair and recombination mechanism in yeast
1993	Francisco Mojica was the first to characterize what is now called a CRISPR locus
1996	Use of hybrid restriction enzyme including zinc finger and FokI cleavage domain
2001	Spanish researchers at University of Alicante proposed the name CRISPR
2005	Discovered the involvement of CRISPR as a form of adaptive immunity in bacteria and the discovery of Cas9 and PAM sequences
2007	Experimental demonstration of adaptive immunity of CRISP-Cas9 in bacteria
2008	Spacer sequences are transcribed into guide RNAs (crRNAs) and CRISP target DNA
2009	Identified action of TAL-type III effecters in Xanthomonas
2010	Role of type II, CRISPR-Cas identified
2011	Discovery of tracrRNA for Cas9 system
2012	Duodna and Charpentier use the CRISPR-Cas9 system as a tool for genome editing, by employing genetically engineered CRISPR-Cas system
2013	First-time demonstration of use of the CRISPR/Cas system for genome editing in plants, humans and other eukaryotic
2015	DNA-free genome editing technique in plants

1.2 Techniques for Genome Editing

1.2.1 Cre-Lox System

Cre-lox system has many applications involving controlled removals of DNA fragments and the targeted insertion of DNA into specific genome sites, whereas recent advances, including controlled creative expression and innovative use of wild-type and modified lox sites, have enhanced its applicability. Cre-lox is a member of a broad family of recombinases of tyrosine that includes many other common recombinases, including Flp and Int that are derived from the bacteriophage P1. Cyclization recombination (Cre) gene encodes a 38-kDa protein, which identifies 34-base pair (bp) sites known as lox (locus of crossing over), which includes 13-bp inverted repeats at both sites of 8-bp non-palindromic core region and catalyses recombination between two lox sites (Abremski et al., 1983). The role of such a system involves a precise recombination, removal of a selectable gene (transgene) and site-specific gene transfer for crop improvement (Abremski et al., 1983; Gilbertson, 2003). It is observed that prokaryotic recombinase can be used in the eukaryotes where it enters into the nucleus and Cre recombinase is capable of performing successful recombination events on the chromosomes (Sauer, 1987). A Cre-recombinase protein from microbes may be useful to study the genome modification in other higher organisms (Sauer and Henderson, 1988; Orban et al., 1992).

1.2.2 Meganucleases

Meganucleases are a class of endonucleases characterized by a large recognition site and act like ‘molecular DNA scissors’ that can be used to remove, delete or alter sequences in a highly targeted way. Meganucleases possess recognition sites of 12–40 bp, consequently occur only once in any given genome and are known to be the most common naturally occurring restriction enzymes (Figure 1.1a). By altering their recognition sequence through protein engineering, the enzymes can be specified to the specific desired sequence. Meganucleases can be expressed in different compartments of the cell such as mitochondria or chloroplasts of archaebacteria, bacteria, phages, fungi, yeast, algae and some plants, and are mainly represented by the two main enzyme families, namely intron endonucleases and intein endonucleases. Meganucleases are most commonly found in Saccharomyces cerevisiae (I-SceI), Chlamydomonas reinhardtii and Desulfurococcus mobilis, named as I-CreI and Dmo, respectively (Choulika et al., 1995; Roberts et al., 2003; Marcaida et al., 2008). Chimeric meganucleases, derived from homing endonucleases that can induce recombination and cleave a selected new DNA target site, have been used in genome modification (Epinat et al., 2003). Sylvain et al. (2007) used an engineered homing endonuclease approach for the first time to modify the I-CreI meganuclease DNA-binding interface to target sequences of a chromosomal locus. In another study, Grizot et al. (2009) customized the chimeric meganuclease, DmoCre, by successfully assembling mutants with locally altered specificities, and these newly engineered variants of DmoCre were found selective with low toxicity level, which help to induce successful homologous recombination events. Further, meganuclease engineering was used to make a fram...