Exploring CRISPR-Cas9 HNH-Domain Catalyzed DNA Cleavage Using Accelerated Quantum Mechanical Molecular Mechanical Free Energy Simulation

The target DNA (tDNA) cleavage catalyzed by the CRISPR Cas9 enzyme is a critical step in the Cas9-based genome editing technologies. Previously, the tDNA cleavage from an active SpyCas9 enzyme conformation was modeled by Palermo and coworkers [Nierzwicki et al., Nat. Catal. 5, 912 (2022)] using ab initio quantum mechanical molecular mechanical (ai-QM/MM) free energy simulations, where the free energy barrier was found to be more favorable than that from a pseudoactive enzyme conformation. In this work, we performed ai-QM/MM simulations based on another catalytically active conformation (PDB 7Z4J) of the Cas9 HNH domain from cryo-electron microscopy experiments.For the wildtype enzyme, we acquired a free energy profile for the tDNA cleavage that is largely consistent with the previous report. Furthermore, we explored the role of the active-site K866 residue on the catalytic efficiency by modeling the K866A mutant, and found that the K866A mutation increased the reaction free energy barrier, which is consistent with the experimentally observed reduction in the enzyme activity.