TALENs is that the target sequence must have thymine at the −1 position (Boch et al. 2009). Further, the long and repetitive nature of TALENs puts a strain on delivery methods where cargo capacity or stability is a limitation.
The assembly of engineered TALE repeat arrays can be challenging from nearly similar repeat sequences; therefore, a number of platforms have been designed to facilitate this assembly. These can be classified into three categories: standard restriction enzyme and ligation‐based cloning methods (Huang et al. 2011; Sander et al. 2011); Golden Gate assembly methods (Briggs et al. 2012; Cermak et al. 2011; Engler et al. 2008) and solid‐phase assembly methods (Heigwer et al. 2013; Wang et al. 2012).
Several online tools are available for designing TALE effectors to target specific gene sequence and off‐target analysis. For example‐ E‐TALEN (Lin et al. 2014), Scoring Algorithm for Predicting TALEN Activity (SAPTA) (Neff et al. 2013), Mojo‐hand (Coordinators 2013), TAL Effector‐Nucleotide Targeter (TALE‐NT), etc. TALE‐NT is a collection of versatile web‐based tools like‐TALEN Targeter, TAL Effector Targeter, Target finder, Paired Target Finder, and TALEN Targeter Off‐Target Counter (Christian et al. 2013).
Several studies have demonstrated the usefulness of TALENs in different plant species, including Arabidopsis (Zhang et al. 2013), tobacco (Wang et al. 2012; Wendt et al. 2013), barley (Li et al. 2012), rice (Shan et al. 2013a) and Brachypodium (Reyon et al. 2011). Taken together, the modular nature of TALE repeats, along with efficient methods for assembling repetitive DNA sequences (Garneau et al. 2010; Wang et al. 2012), have enabled TALENs to become one of the premier tools for plant genome engineering.
1.4 CRISPR‐Cas System
The most recent addition to the SSN family is the CRISPR/Cas system that is normally present within bacteria and archaea, and provides an adaptive immunity against invading plasmids or viruses. CRISPR/Cas system functions to destroy invading nucleic acids by introducing targeted DNA breaks (Garneau et al. 2010).
There are three major types of CRISPR/Cas system: Types I – III (Makarova et al. 2011). The Type II system was adopted for genome engineering a few years ago (Cong et al. 2013; Zhang et al. 2011). In this system, two components enable targeted DNA cleavage: a Cas9 protein and an RNA complex consisting of a CRISPR RNA (crRNA; contains 20 nucleotides of RNA that are homologous to the target site) and a trans‐activating CRISPR RNA (tracrRNA). Cas9 protein causes double‐stranded DNA break at the sequences homologous to the crRNA sequence and upstream of a protospacer‐adjacent motif (PAM) (PAM; e.g. NGG for Streptococcus pyogenes Cas9). For genome engineering purposes, the complexity of the system was reduced by fusing the crRNA and tracrRNA to generate a single‐guide RNA (gRNA). Moreover, off‐target cleavage is a limitation of the CRISPR/Cas system (Cho et al. 2014; Fu et al. 2013).
The target site recognition in CRISPR‐Cas system is facilitated through RNA: DNA interaction (as opposed to a protein: DNA interaction used by meganucleases, zinc‐finger nucleases, and TALENs). Redirecting of Cas9 targets involves modification of 20 nucleotides within the crRNA or gRNA. These 20 nucleotides are used to direct Cas9 binding and cleavage, the system has been shown to tolerate mismatches, with a higher tolerance closer to the 5′ end of the target sequence (Fu et al. 2013). Results from recent studies suggest the first 8–12 nucleotides, in addition to the PAM sequence, are most critical for target site recognition (Sternberg et al. 2014; Wu et al. 2014). To reduce off‐targeting, several methods have been developed, including dual‐nicking of DNA (Mali et al. 2013; Ran et al. 2013), a fusion of catalytically‐dead Cas9 to FokI (Guilinger et al. 2014; Tsai et al. 2014) and shortening of gRNA sequence (Fu et al. 2014). Several softwares and programs have been developed in recent years for the identification of target sequences in the genome and the design of specific gRNA, which are listed in Table 1.1.
The Cas9 is an endonuclease consisting of two discrete nuclease domains: the HNH domain which is responsible for the cleavage of the DNA strand complementary to the guide RNA sequence (target strand) and the RuvC‐like domain that cleaves the DNA strand opposite the complementary strand (Chen et al. 2014; Gasiunas et al. 2012; Jinek et al. 2012). The double‐strand breaks (DSBs) are repaired through Non‐Homologous End Joining or Homology directed Repair in the presence of a template. Mutations in both nuclease domains (Asp10 → Ala, His840 → Ala) result in an RNA‐guided DNA‐binding protein without endonuclease activity that is called dCas9 (Jinek et al. 2012; Qi et al. 2013). This dCas9 is then supplemented with effector domains for the execution of distinct functions in the genome (Figure 1.1B). Fusion of a transcriptional activator VP64 with dCas9 exhibited targeted gene activation by altering the flowering time regulation in Arabidopsis (Xu et al. 2019). Similarly, dCas9‐VP64 regulated transcriptional activation of endogenous genes and dCas9‐SRDX‐regulated transcriptional repression in Arabidopsis and tobacco (Lowder et al. 2015, 2018). These regulatory domains can also perform multiplex gene targeting using multiple sgRNAs. As a new dimension to CRISPR/Cas technology, there are the base editing enzymes, for example, cytidine deaminase fused with the dCas9, which can replace specific bases in the targeted region of DNA and RNA.
Table 1.1 List of available softwares and programs for designing gRNA.
| Software | Features | Link | References |
|---|---|---|---|
| Cas‐OFFinder | Identifies gRNA target sequence from an input sequence and checks off‐target binding site | http://www.rgenome.net/cas‐offinder | Bae et al. (2014) |
| Cas‐Designer | Identifies gRNA target sequence from an input with low probability of off‐target effect | http://www.rgenome.net/cas‐designer/ | Park et al. (2015) |
| Cas9 Design | Designs gRNA | http://cas9.cbi.pku.edu.cn/database.jsp | Ma et al. (2013) |
| E‐CRISP | Designs gRNA | http://www.e‐crisp.org/E‐CRISP/designcrispr.html | Heigwer et al. (2014) |
| CRISPR‐P | Designs gRNA | http://cbi.hzau.edu.cn/crispr2/ | Lei et al. (2014) |
| CHOP | Identifies target site | https://chopchop.rc.fas.harvard.edu/ | Montague et al. (2014) |
| CRISPR‐PLANT | Designs gRNA | http://www.genome.arizona.edu/crispr/ | Xie et al. (2014) |
| CCTop |
