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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.

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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	Identifies candidate gRNA target sites with reduced off‐target quality	http://crispr.cos.uni‐heidelberg.de/	Stemmer et al. (2015)
CRISPRdirect	Identifies candidate gRNA target sequences	http://crispr.dbcls.jp/	Naito et al. (2015)
COSMID	Identifies target sites	https://crispr.bme.gatech.edu	Cradick et al. (2014)
CRISPR Finder	Identifies CRISPR	http://crispr.u‐psud.fr/Server	Grissa et al. (2007)
CrisprGE	Identifies target sites	http://crdd.osdd.net/servers/crisprge	Kaur et al. (2015)
CRISPR Multitargeter	Identifies target sites	http://www.multicrispr.net	Prykhozhij et al. (2015)
CRISPRseek	Identifies target specific guide RNAs	http://www.bioconductor.org/packages/release/bioc/html/CRISPRseek.html	Zhu et al. (2014)
flyCRISPR	Identifies target sites and evaluate its specificity	http://flycrispr.molbio.wisc.edu	Gratz et al. (2014)
GT‐SCAN	Identifies target sites and ranking them with their potential off target sites	http://flycrispr.molbio.wisc.edu	O'Brien and Bailey (2014)
sgRNAcas9	Identifies target sites with their potential off target sites	www.biootools.com	Xie et al. (2014)
SSFinder	Identifies target sites	https://code.google.com/p/ssfinder	Upadhyay and Sharma (2014)
ZiFiT	Identifies target sites	http://zifit.partners.org/ZiFiT	Mandell and Barbas (2006)