Deciphering the QR Code of the CRISPR-Cas9 System: Synergy between Gln768 (Q) and Arg976 (R).

Markov state models (MSMs) and machine learning (ML) algorithms can extrapolate the long-time-scale behavior of large biomolecules from molecular dynamics (MD) trajectories. In this study, an MD-MSM-ML scheme has been applied to probe the large endonuclease (Cas9) in the bacterial adaptive immunity CRISPR-Cas9 system. CRISPR has become a programmable and state-of-the-art powerful genome editing tool that has already revolutionized life sciences. CRISPR-Cas9 is programmed to process specific DNA sequences in the genome. However, human/biomedical applications are compromised by off-target DNA damage. Characterization of Cas9 at the structural and biophysical levels is a prerequisite for the development of efficient and high-fidelity Cas9 variants. The Cas9 wild type and two variants (R63A-R66A-R70A, R69A-R71A-R74A-R78A) are studied herein. The configurational space of Cas9 is provided with a focus on the conformations of the side chains of two residues (Gln768 and Arg976). A model for the synergy between those two residues is proposed. The results are discussed within the context of experimental literature. The results and methodology can be exploited for the study of large biomolecules in general and for the engineering of more efficient and safer Cas9 variants for applications.