Exploring alternative catalytic mechanisms of the Cas9 HNH domain

Understanding the reaction mechanism of CRISPR-associated protein 9 (Cas9) is crucial for the application of programmable gene editing. Despite the availability of the structures of Cas9 in apo- and substrate-bound forms, the catalytically active structure is still unclear. Our first attempt to explore the catalytic mechanism of Cas9 HNH domain has been based on the reasonable assumption that we are dealing with the same mechanism as endonuclease VII, including the assumption that the catalytic water is in the first shell of the Mg²⁺ . Trying this mechanism with the cryo-EM structure forced us to induce significant structural change driven by the movement of K848 (or other positively charged residue) close to the active site to facilitate the proton transfer step. In the present study, we explore a second reaction mechanism where the catalytic water is in the second shell of the Mg²⁺ and assume that the cryo-EM structure by itself is a suitable representation of a catalytic-ready structure. The alternative mechanism indicates that if the active water is from the second shell, then the calculated reaction barrier is lower compared with the corresponding barrier when the water comes from the first shell.