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CRISPR/Cas and Its Potentiality as an Effective Tool

target specificity on DNA strand and Cas9 endonuclease creates a double-

stranded break (DSB). DSBs can be repaired by endogenous DNA repair

mechanism of cells involving error prone non-homologous end joining

(NHEJ) and homologous recombination (HR) or homology directed repair

(HDR) (Chen & Gao, 2014). NHEJ creates nucleotide insertions or deletions

causing gene knockouts. HDR pathway generates precise base modifications

or gene replacement in the presence of donor DNA sequence (Chen & Gao,

2014). In turn novel mutants get generated (Voytas & Gao, 2014). Various

reviews and chapters describe CRISPR/Cas genome-editing platform-based

relevance in crop improvement (Zhang et al., 2018; Sedeek et al., 2019;

Pandita, 2021a–c).

11.2 CLASSIFICATION OF CRISPR/Cas SYSTEMS

CRISPR/Cas system, a defensive mechanism for the degradation of foreign

genomes was discovered in year 1987 as a set of 29 nucleotide repeats

downstream of iap gene (Ishino et al., 1987). Almost 44% of 703 archaeal

systems own one or more CRISPR/Cas modules in their genomes (Koonin

& Makarova, 2009; Deveau et al., 2010). CRISPR/Cas loci involve CRISPR

array. CRISPR array has two to several 100 direct, partially palindromic,

normally exact repeats (of 25–35 bp each). These repeats are disconnected

by inimitable spacers (of 30–40 bp) and end-to-end clusters of multiple

cas genes organized in one or more operons which encode both the adap­

tation and effector modules, often with accessory genes (Makarova et al.,

2013, 2015). CRISPR/Cas systems are broadly categorized into two major

classes: Class 1 and Class 2 on the basis of design principles of the effector

modules as well as signature cas genes/cas loci structure, organization of

cas operons and phylogeny of conserved Cas proteins. Class 1 systems

rely on heteromeric multisubunit effector complexes comprising numerous

Cas proteins and class 2 systems in which effector consists of single, large,

multi-domain effector protein (Makarova et al., 2015, 2018, 2020). Two

CRISPR-Cas classes in the next hierarchical level divide into 6 main Types

I, III, and IV for class 1 including 16 subtypes, and types II, V, and VI for

class 2 includes 3 types and 17 subtypes (Makarova et al., 2020). Each type

has distinctive architecture of effector modules including unique signature

proteins. Each of these contains a total of 33 multiple subtypes and multiple

variants distinguished by subtler differences in locus organization and often

encode subtype-specific Cas proteins (Makarova et al., 2020). Type I, II, and