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Biology and Biotechnology of Environmental Stress Tolerance in Plants, Volume 3

V targets DNA; type VI targets RNA and type III targets both the DNA and

RNA (Samai et al., 2015; Koonin et al., 2017). In several organisms, Cas loci

do not fit into current classification due to their complex modular structure

and variability, e.g., target of Cas13d is not DNA, but RNA. Class 2 includes

Cas9, Cas12, and Cas13 endonucleases which are widely used as genome

editing tools (Wu et al., 2018).

11.3 MECHANISM OF ACTION OF CRISPR/Cas SYSTEMS

CRISPR/Cas system has a basis from the natural phenomena of bacterial

and archaeal adaptive immune system for invasion against external plasmids

or sequences of viruses (Marraffini & Sontheimer, 2010). CRISPR/Cas

editing system has two components which comprise Cas endonuclease and

single guide RNA (sgRNA). Cas-sgRNA complex binds to the target DNA

sequences. The sgRNA is made of CRISPR RNA (crRNA) with program­

mable 20-nt RNA target sequences and trans-activating crRNA (tracrRNA).

The sgRNA recognizes and directs Cas protein to specific genomic target

(Hsu et al., 2014). Cas induces double-strand break (DSB) at the loci of

interest in the genome. The specificity of the system is due to 20 nt elon­

gated replaceable guide sequence within sgRNA, complementary to the

target sequence (Li et al., 2013). The most generally used Cas protein is

Cas9 endonuclease derived from the gram-positive Streptococcus pyogenes

bacterium. Cas9 endonuclease cleavage is based on Protospacer Adjacent

Motif (PAM, 5’-NGG-3’) present directly downstream of 20-nt target DNA

sequence (Zhang et al., 2018) and PAM requirement is a bottleneck in it.

PAM sequence varies on the basis of type of CRISPR/Cas, e.g., in Cas9 vari­

ants and Cas9 orthologs (Leenay & Beisel, 2017). The Cas9 endonuclease

cleaves at 3-nt upstream of PAM within DNA target site, generating blunt

ended double-strand breaks (DSBs) (Jiang & Doudna, 2017). CRISPR from

Prevotella and Francisella (Cpf1) known as Cas12a endonuclease of class 2

type V lacks HNH domain, needs T rich PAM and generates staggered cuts

for the plant genetic manipulations (Zetsche et al., 2015; Stella et al., 2017).

DSBs activated intrinsic DNA repair mechanism of the cell through NHEJ

or homology-directed repair (HDR) to generate gene knockout or gene

knock-in. HDR requires repair donor DNA template and leads to knock-in.

NHEJ is most dominant and active error prone DNA repair pathway and

generates insertions and deletions (indels) or substitutions resulting in the

gene knockout or gene silencing (Rong & Golic, 2000; Hasley et al., 2021).