CRISPR-based gene editing technology has shown a great promise for biological and biomedical basic and translational research. The off-target cleavage could be a major barrier as one of the genetic therapeutics since it may generate undesired cleavage in a genome and how protospacer adjacent motif helps the search and cleavage process still remains unknown. The detailed mechanism of the cleavage activity of Cas9 is necessary to find out the steps of which the off-target cleavage happens and how protospacer adjacent motif functions. Moreover, the rationale of how currently available Cas9 proteins with high fidelity works is essential to understand the function of the mutated residues.

Based on the confirmed experimental and computational evidence of allosteric behavior of cas9 p...[ Read more ]

CRISPR-based gene editing technology has shown a great promise for biological and biomedical basic and translational research. The off-target cleavage could be a major barrier as one of the genetic therapeutics since it may generate undesired cleavage in a genome and how protospacer adjacent motif helps the search and cleavage process still remains unknown. The detailed mechanism of the cleavage activity of Cas9 is necessary to find out the steps of which the off-target cleavage happens and how protospacer adjacent motif functions. Moreover, the rationale of how currently available Cas9 proteins with high fidelity works is essential to understand the function of the mutated residues.

Based on the confirmed experimental and computational evidence of allosteric behavior of cas9 protein, in this study we try to employ some statistical methods to study co-variance of cas9 protein residues. Statistical coupling analysis and direct coupling analysis were used to map the most important residues which play a role in allosteric transmission and activity of cas9. In addition, based on the mechanisms and important residues, an overall kinetic mechanism will be explained in detail with specific steps involved in a Cas9 activity to explain how protospacer adjacent motif diversity comes from and what it influences.