CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein-9) system, discovered in 2012 by the 2020 Nobel Prize-winning Laureates Jennifer Doudna and Emmanuelle Charpentier is a bacterial defensive mechanism that exhibits the cleavage of genomic DNA at the desired location, resulting in the exit of the old genes and the induction of a new set of genes. The accuracy, precision or fidelity of the genetic cut depends on the target and the proto-spacer adjacent motif (PAM) sequences. The Cas9 protein recognises the PAM sequence (5'-NGG-3') by selecting the correct location of base-pair bonds within the target sequence on the host genome. Assembling the nucleotide sequence related to PAM and target sequence into a plasmid and then transfecting the plasmid into a cell shows that Cas9 with the help of a crRNA detected the correct sequence within a host cell. This results in a single or double-stranded break at the appropriate location in the DNA, thereby working as a molecular scissor and performing a genetic cut. We choreographed this tool in achieving the information related to the generation of the PAM sequence and the off-target sites associated with the EGFR gene. We have identified the top 4 best gRNA sequences based on the highest SYNTHEGO scores range 0.98 to 0.99. Furthermore, we used CCTop to identify the 4 best targets and guide RNA sequences with the highest efficacy score. Our manuscript is aimed at showcasing the best target sequence utilizing model software like that of SYNTHEGO and CCTop.

Keywords: CRISPR/Cas9; gRNA; molecular scissor; EGFR gene; SYNTHEGO and CCTop computational tool.
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