**3. CRISPR**/**Cas9 System**

Recently, a fascinating GE tool CRISPR/Cas9 was identified for targeted genome manipulations and to express desired genes in numerous organisms [6,65]. The CRISPR/Cas9 system has emerged as the most powerful tool for GE in many species including plants [26]. The latest ground-breaking technology of CRISPR/Cas9 is basically present as an adaptive immune system of type II prokaryotes and protects them against invading organisms during phage infection by spacer acquisition, biogenesis, and target degradation [9]. The toolbox of CRISPR/Cas9 was adapted from bacteria as well as Archaea and in included in the toolbox of engineered nucleases [23,66]. There are two main components of the CRISPR/Cas9 system: a single guide RNA (sgRNA) that identifies a specific DNA sequence and the Cas9 protein which produces DSBs at a targeted site [9]. Therefore, when changing the design of sgRNA, numerous desired sites can be targeted, which makes it simpler to handle than TALENs and ZFNs [10].

## *3.1. Discovery of CRISPR*/*Cas9 Wonder*

The discovery of the CRISPR/Cas9 system dates back to 1987 when Ishino and his colleagues first identified CRISPR while studying the *iap* gene in the genome of *E. coli*. During the cloning of the *iap* gene, they unexpectedly cloned a specific portion of CRISPR, and at the conclusion of their experiment, revealed that the bacterial genome consisted of a successive array of repeats [67]. After this discovery, an Archaea (*Haloferax mediteranii*) was also found to contain the CRISPR sequences [68]. Mojica et al. (2000) reported a similar type of regularly spaced repeats in *Haloferax mediterranei* and *Haloferax volcanii*, having interrelated functions [69]. Only prokaryotes were considered to have such repetitive sequences, which were named as CRISPR but were not present in eukaryotes and viruses [70]. These short repeats have an average length of 32 bp but are of different sizes from 21 to 47 bp in different organisms. Every repeat has a unique sequence of nucleotides that are extremely conserved in specific species [71]. It was unveiled that the short regular repeats are transcribed into small RNAs [72].

Four *Cas* genes (*Cas1*–*4*) were discovered in prokaryotes having CRISPR DNA sequences during that period [70]. From then on, many CRISPR/Cas sequences and multiple Cas proteins were identified [72]. In 2005, CRISPR spacers were discovered in plasmids and phages by three independent research groups by applying computational and sequencing technologies [73–75]. The function of CRISPR/Cas was still ambiguous before Barrangou et al. (2007) successfully demonstrated for the first time that CRISPR protected *Streptococcus thermophilus* from viral attack [76]. It was revealed that the CRISPR defense mechanism prevents the horizontal gene flow in *Staphylococci* [77]. In another study, it was observed that CRISPR RNAs regulate the CRISPR interference [78]. The presence of the CRISPR/Cas system in the bacterial genome was identified to cut specific sites in plasmid DNA and bacteriophages [79]. In 2011, the CRISPR/Cas machinery of *S. thermophilus* was exploited to confer immunity in *E. coli* [80]. Some of these important events are highlighted in Figure 4.

**Figure 4.** Historical chart illuminating key developments in the CRISPR/Cas9 system.
