rel="nofollow" href="#ulink_9147fee9-259b-57ea-83e3-2ef8ee4873ab">1.4 CRISPR‐Cas System
1.1 Introduction
Agricultural practices, combined with advanced plant breeding and modern technologies, provided food security to millions of people. However, increasing global population demands significant increase in world food production (Parry and Hawkesford 2012). Nevertheless, climate change, depletion of natural resources, increased pollution, and political instabilities are a threat to the food and nutritional security for future generations in the twenty‐first century. Unfortunately, the amount of remaining arable land is limited, necessitating an increase in food production on currently‐used land. Compounding these challenges are the predicted crop losses due to extreme temperatures, pest attacks, and pathogen outbreaks. A powerful approach that may help overcome these challenges is to modify DNA sequences within plant chromosomes for trait improvement (Sedeek et al. 2019). Further, plants can be engineered to have increased tolerance to environmental stresses and pathogens (Han and Kim 2019; Ji et al. 2015; Makarova et al. 2011). In addition to improving the genetic makeup of the crops to meet increasing food demands and control crop loss, genome engineering can also be used to produce valuable plants or products for non‐agricultural purposes (Chen et al. 2019). For example, there is great potential for plants to be used as bioreactors for pharmaceutical proteins. Genetic engineering for increasing the secondary metabolite production in plants would be another use of this technology which would help the perfumery, cosmetic and medical industries, as the secondary metabolites produced from plants have a number of uses (El‐Mounadi et al. 2020). However, to realize the potential benefits of these applications, we must generate effective tools and approaches for editing plant DNA (Miroshnichenko et al. 2019; Tang and Tang 2017).
Introduction of programmed sequence‐specific nucleases (SSNs) and their applications in precise genome editing unfurled a new dimension in genome engineering (Kim and Kim 2014; Voytas 2013). Over the last few decades, researchers reported a few important SSNs, which could be easily engineered and reprogrammed to create double‐stranded breaks (DSBs) at the desired location inside the chromosome. There are three major genome engineering methods, ZFNs, TALENs, and CRISPR‐Cas system (Figure 1.1A) (Jang and Joung 2019; Mahfouz et al. 2014), that have been utilized so far for a variety of purposes, and these have been discussed in detail in the coming sections. Further, we have also described the recently added CRISPR‐Cpf1 system of genome engineering.
1.2 ZFNs
Zinc‐finger nucleases are chimeric fusion proteins consisting of a DNA‐binding domain and a DNA‐cleavage domain. The DNA‐binding domain is composed of a set of Cys2His2 zinc fingers (usually three to six). Each zinc finger primarily contacts 3 bp of DNA and a set of three to six fingers recognize 9–18 bp, respectively. The DNA‐cleavage domain is derived from the cleavage domain of the FokI restriction enzyme. FokI activity requires dimerization; therefore, to site‐specifically cleave DNA, two zinc‐finger nucleases are designed in a tail‐to‐tail orientation (Kim et al. 1996).
Zinc‐finger nucleases can be remodified to recognize different DNA sequences. However, one limitation with redirecting targeting is that it depends on the context of the host. For example, a zinc finger that recognizes GGG may not recognize this sequence when fused to other zinc fingers. As a result, the modular assembly of zinc fingers has had limited success (Ramirez et al. 2008). One of the more successful methods for redirecting targeting involves generating a library of three zinc‐finger variants from a pre‐selected pool of zinc‐finger monomers (Maeder et al. 2008). The resulting library of zinc‐finger arrays can then be interrogated using a bacterial two‐ hybrid screen, where binding of the zinc‐finger array to a pre‐determined sequence results in the expression of a selectable marker gene. This method has generated highly‐active zinc‐finger nuclease (ZFN) pairs for sites within animal and plant genomes. Since the development of ZFN technology, several studies have been done to engineer specific zinc‐finger modules for each of the 64 codon triplets (Bae et al. 2003; Dreier et al. 2001; Pabo et al. 2001). Until now, several ZFNs have been designed and used in numerous species. The developments for more specific and efficient technologies also gave rise to fewer off‐target effects. There are three most commonly available tools for engineering the ZF domains: context‐dependent Assembly (CoDA), Oligomerized Pool Engineering (OPEN), and Modular Assembly (MA). Several softwares are available for designing engineered ZFs (ZiFiT), containing the database of ZFs (ZiFDB) and identification of potential targets for ZFNs in several model organisms (ZFNGenome) (Kim et al. 2009; Mandell and Barbas 2006; Sander et al. 2007).
Figure 1.1 (A) Diagrammatic representation of (a) Zinc‐finger nucleases (ZFNs), (b) Transcription activator‐like effector nucleases (TALENs) and (c) Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 mediates DSBs formation. (B) dCas9‐based targeted genome regulation by (a) activation of gene expression, (b) repression of gene expression and (c) DNA methylation.
Source: Adapted from Mahfouz et al. (2014) © 2014. Reproduced with the permission of John Wiley & Sons.
Zinc‐finger nucleases have been widely used for plant genome engineering. Plant species that have been modified using zinc‐finger nucleases include, Arabidopsis, maize, soybean, tobacco, etc. (Ainley et al. 2013; Cai et al. 2009; Curtin et al. 2011; Lloyd et al. 2005; Marton et al. 2010; Osakabe et al. 2010; Shukla et al. 2009; Townsend et al. 2009; Wright et al. 2005; Zhang et al. 2010). With their relatively small size (~300 amino acids per zinc‐finger nuclease monomer), and the further advancements in methods for redirecting targeting (Sander et al. 2011a), zinc‐finger nucleases should continue to be an effective technology for editing plant.
1.3 TALENs
Transcription activator‐like effectors nucleases (TALENs) are fusion proteins, consisting of a DNA‐binding domain and a DNA‐cleavage domain. Whereas the DNA‐cleavage domain is the same between zinc‐finger nucleases and TALENs (the catalytic portion of FokI), the DNA binding domains are different. The TALEN DNA‐binding domain is derived from TALE proteins found in the plant pathogen Xanthomonas. These proteins are composed of direct repeats of 33–35 amino acids, and nearly all arrays found in Xanthomonas contain a final, half repeat, consisting of the first 20 amino acids from the normal repeat. Two amino acids within these repeats (positions 12 and 13) are responsible for recognizing a single nucleotide base (these amino acids are referred to as repeat‐variable diresidues; RVDs). When the TALE effector code was broken (i.e. the relationship between the RVD and corresponding target base) (Boch et al. 2009; Moscou and Bogdanove 2009), the ability to redirect targeting, and their use as a genome engineering tool was realized (Christian et al. 2010; Li et al. 2011; Mahfouz et al. 2011). To make TALENs useful in gene targeting, the basic requirement is the modular assembly of repeat sequences containing the appropriate RVD corresponding to the nucleotide target. The most widely used RVDs and their nucleotide targets are HD, cytosine; NG, thymine; NI, adenine; NN, guanine, and adenine; NS, adenine, cytosine, and guanine; N*, all four nucleotides. This one‐to‐one correspondence of a single RVD to a single DNA base has eliminated construction challenges due
