In Silico Modeling of a Mutant Cas9 Protein for High-Fidelity Genome Editing

CRISPR/Cas system is a bacterial-acquired immune system against viruses. spCas9 protein-derived from Streptococcus pyogenes is the most common Cas protein used in genome editing. The Cas9 proteins suffer from some problems like unwanted off-target cleavage, which is the major limitation in the application of the CRISPR system, especially for therapeutic issues. The present study aimed to in silico design of a mutant Cas protein to enhance the fidelity of the enzyme.

In this study, a new Cas9 protein was designed in silico via the selection of some mutations. The interaction of the mutant and wild-type Cas9 proteins with DNA was determined by molecular docking. Finally, the stability of the mutant and wild-type proteins was evaluated using molecular dynamics.

The outcome of this study resulted in designing a mutant Cas9 protein bioinformatically containing 5 amino acid substitutions. Molecular docking scores for the wild-type and mutant proteins were -1073.86 and -349.41, respectively.

Molecular docking indicated that the number of hydrogen bonds between protein and DNA reduced in the mutant state. Moreover, based on the molecular dynamic findings, the stability of both mutant and wild-type Cas9 proteins was similar.