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The CRISPR/Cas9 system is made of Cas9 nuclease and single-guide RNA (sgRNA). The sgRNA is an engineered single RNA molecule containing crispr RNA and tracr RNA parts. The sgRNA recognizes the target sequence by standard Watson-Crick base pairing. It has to be followed by a DNA motif called a protospacer adjacent motif (PAM). The commonly used wild-type Streptococcus pyogenes Cas (SpCas9) protein has a specific PAM sequence, 5’-NGG-3’, where “N” can be any nucleotide base followed by two guanine (“G”) nucleobases. This sequence is located directly downstream of the target sequence in the genomic DNA, on the non-target strand. Targeting is constrained to every 14 bp (12 bp from the seed sequence and 2 bp from PAM). SpCas9 variants may increase the specificity of genome modifications at DNA targets adjacent to NGG PAM sequences when used in place of wild-type SpCas9 (Lino et al., 2018).
The CRISPR was first reported by Yushizumi Ishino (Ishino et al.,1987), but its biological application was unknown at the time (Mira et al.,2019). According to effector proteins, this system has been categorized into two main classes with six subtypes (Manghwar et al., 2019). The type2 CRISPR-Cas9 system is the most widely used item in the field of genome editing with three main components: a CRISPR RNA (cRNA), an endonuclease named Cas9, and a transactivating crRNA (tracrRNA) . This system consists of two components: (1) the Cas9 protein which can cleave the DNA and (2) the guide RNA that distinguishes the sequence of DNA to be rectified. To apply CRISPR-Cas9, sequences of the intended target genome are first identified. Then, the guide RNA is tailored to recognize a particular stretch of As, Ts, Gs, and Cs in the DNA. The guide RNA is affiliated to the DNA cutting enzyme Cas9, and then this complex is presented to the target cells. Cas9 locates the target letter and cuts the DNA at that point, allowing alteration of the existing genome by either modifying or adding to the sequence (Figure 6). As such, CRISPR-Cas9 functions as a cut and paste tool for DNA editing (Doudna et al., 2014). Using this technology, any genomic sequence identified by a short strand of guide RNA can be exactly modified . This system targeted the human genome for the first time in 2013 . To date, CRISPR-Cas9 has been commonly used to create gene editing in plants, animal, and human samples. This technique is widely used in various scientific fields, including medical science and therapeutics, as well as plant and animal sciences (Zahir.2020).