Catching CRISPR-Cas9 in Action.

CRISPR-Cas9 has revolutionized genome editing, yet its structural dynamics and functional properties remain incompletely understood, partly due to limited atomic-level characterization of its active conformation with a full R-loop. Capitalizing on recent advances in Cas9 structural determination, we constructed a catalytic-state Cas9 model bound to a R-loop and performed an integrated computational investigation. Our molecular dynamics simulations reveal substantial conformational heterogeneity in the PAM (protospacer-adjacent motif)-distal nontarget DNA strand and adjacent Cas9 regions, leading to dynamically fluctuating interactions, thereby challenging experimental resolution of the full R-loop complex. Comparative analysis highlights a conformational barrier restricting final activation of the HNH nuclease domain, suggesting that strategic modulation of HNH interactions on its two sides could enhance cleavage efficiency. Furthermore, quantum mechanics/molecular mechanics simulations indicate that with H983 protonated at Nε, the RuvC domain favors a phosphate-mediated over a histidine-mediated pathway for nontarget strand cleavage. Additionally, we identify an alternative HNH-mediated target strand cleavage pathway, involving a water nucleophile aligned at the 5' side of the scissile phosphate. Inspired by the basic residue ladder observed in RuvC, we propose extending a similar ladder in HNH to strengthen DNA binding and catalytic activity. Our study provides critical insights into Cas9 structure, dynamics, and catalysis, laying a foundation for the rational design of next-generation CRISPR-Cas9 systems with optimized specificity-efficiency balance.