CRISPR/Cas9

Introduction: Breakthrough research in this field has earned Jennifer Doudna and Emmanuelle Charpentier with Noble prize in Chemistry for the year 2020. Their findings in 2012 that CRISPR/Cas9 could be programmed with RNA to edit the genomic DNA brought a significant milestone in the advancement of molecular biological research. Briefly, CRISPR/Cas9 is a bacterial defensive mechanism that exhibits the cutting of the genomic DNA at its desired location resulting in to the exit of the old ones with the induction of a new set of genes. Fidelity or accuracy or precision of the genetic cut depends on the target and the protospacer adjacent motif (PAM) sequence. The target sequence is 20 bases long which belong to a particular CRISPR locus on a crRNA array.Cas9 protein selects the correct location of base pair bonding with in the target sequence. The PAM sequence (5'-NGG-3') on the host genome is recognized by Cas9. Assembling the sequence related to PAM, target sequence into plasmid and transfecting the plasmid into a cell shows that Cas9 with the help of crRNA was able to detect the correct sequence with in a host cell resulting to a single or double stranded break at the appropriate location in the DNA thereby addressing the issue of genetic cut and working as a molecular scissor. We orchestrated this tool in achieving the information related to the generation of PAM sequence and the off target sites related to Neu genes responsible for breast cancer prognosis. To achieve the same we analyzed the set of the said genes on the preformed online software tool available like that of ccTop, and CRISTA in order to generate the best possible target sequence along with their appropriate guide RNA's. Furthermore, approach has also been done to establish the protein characteristics related to generation of hydropathy index, the alpha helix, beta-turns, helix wheel, and the transmembrane tendency. Experimental Design: The amino acid sequence of the Neu protein with its accession number was obtained from Expassy. The FASTA file of the nucleotide sequence of each amino acid was scanned batch mode in CRISTA and CCTop computational tools respectively. Result: We have identified the top 4 best gRNA sequences based on the highest CRISTA scores range 0.98 to 0.99. Furthermore, we used CCTop to break down the entire sequence into several target sequences and also to find out a guideRNA corresponding to each target sequence and accordingly we have identified the 4 best target and guide RNA sequences with highest efficacy score. Conclusion: Our manuscript is aimed at showcasing the best target sequence and guideRNA sequence complimentary to the target sequence utilizing the model software like that of CRISTA