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CRISPR/Cas and Its Potentiality as an Effective Tool

11.4 CRISPR/Cas-MEDIATED ADVANCEMENTS IN PLANT

PROTECTION

Plant improvement by gene knockout and replacement of CRISPR/Cas9

has been very efficient. But 5’-NGG-3’ PAM sequence specificity has set

limitations on usage of CRISPR/Cas9 to prospective targets. Multiple Cas

variants having diverse specificity of PAM sequences have been character­

ized (Wrighton, 2018). SpCas9-NAG and xCas9 find use in Oryza sativa

(Meng et al., 2018; Hu et al., 2018). CRISPR/Cas12a system has widened

the skyline of genome editing. Cas12a (Cpf1) endonuclease was derivative of

Francisella novicida (FnCpf1) and its ortholog from Lachnospiraceae bacte­

rium (LbCpf1). FnCpf1 and LbCpf1 recognize T-rich (5’-TTTN-3’) PAM

sequence and generate cohesive (4–5 nucleotide overhangs) ended DSBs.

CRISPR/Cas12a system overpowers target limits of system of the CRISPR/

Cas9 (Zetsche et al., 2015). The CRISPR/Cas12a has been efficiently used

for targeted mutagenesis in Arabidopsis thaliana, Nicotiana tabacum, Oryza

sativa L. and Glycine max L. (Endo et al., 2016; Tang et al., 2017). The

Cas12a variants facilitate multiplex gene editing and recognize TYCV PAM

sequences (Li et al., 2018). CRISPR/Cas edited plants show off-target muta­

tions and inefficient ability of regeneration. These are major bottlenecks for

CRISPR/Cas system applications in the crop improvement programs. These

roadblocks can be overcome by procedure of edited pollens and immature

embryos which outdo in-vitro tissue culturing and use of stress-inducible

CRISPR/Cas methods consequential of trivial off-target activities (Kelliher

et al., 2019; Nandy et al., 2019).

11.4.1 POTENTIALITY OF CRISPR/Cas-MEDIATED EFFECTIVE TOOL

IN UNDERSTANDING ABIOTIC STRESS RESPONSE

For the first time, CRISPR/Cas9 based genome editing was reported in model

plants for instance Arabidopsis thaliana L., Nicotiana benthamiana and

Oryza sativa L. (Li et al., 2013; Shan et al., 2013; Nekrasov et al., 2013). The

CRISPR/Cas9 based manipulation of genes enabled abiotic stress response

and generation of stress resilient crops in plant science. Improved expression

of ARGOS8 which negatively regulates ethylene responses in maize was

achieved by the precise genome editing through CRISPR/Cas9 resulting in

enhanced drought tolerance and improved yield (Shi et al., 2017). A tissue-

specific AtEF1 promoter derived truncated gRNAs (tru-gRNAs) and Cas9