Probing Electrostatic Interactions in DNA-Bound CRISPR/Cas9 Complexes by Molecular Dynamics Simulations

Engineered protein mutations may be exploited to tune molecular interactions in the cellular environment. Here, we have explored the structural consequences of different Cas9 mutations in genome-editing CRISPR/Cas9 systems by means of Molecular Dynamics simulations. We have characterized mutation-induced structural changes and their implications for changes in protein–DNA, DNA–RNA, and DNA–DNA interactions. We present the analysis of multiple trajectories over the cumulative time scale of 7.7 μs, focusing on triple mutations that have been associated with enhancement of genome editing specificity, as well as control mutations. We find that the structural changes induced by the protein mutations are consistent with decreasing the strength of the interaction between Cas9 and the nontarget DNA strand. We discuss the implications of this finding for genome editing specificity.