Untangling the Molecular Mechanism of SpCas9 Catalytic Activation: A Gear-and-Wedge Fitting Model

The CRISPR-associated endonuclease Streptococcus pyogenes Cas9 (SpCas9) enables site-specific DNA cleavage by transitioning from a pre-catalytic conformation to a catalytically active state, yet how its HNH catalytic domain undergoes an approximately 40 Å displacement towards the target DNA has remained elusive. Here, we combined extensive unbiased molecular dynamics simulations, spanning a cumulative timescale of 160 µs, with Markov state modeling to map the kinetic pathway of SpCas9 activation. In vitro DNA cleavage assays and a cellular fluorescence reporter system further validated the atomic-level mechanisms revealed by our simulations. We found that the folding of the L1 linker and unfolding of the L2 linker serve as the principal driving force, inducing a “gear-and-wedge” cooperative motion within the HNH domain. Concurrently, the REC2 domain moved outward to accommodate the displaced HNH domain and formed transient stabilizing interactions with the HNH domain along the activation route. Site-directed mutagenesis of key L2 linker residues and REC2 loops markedly reduced SpCas9 cleavage efficiency in both HEK293T cells and biochemical assays, underscoring their critical role in SpCas9 ribonucleoprotein activation. Collectively, this study provides a high-resolution view of SpCas9 catalytic activation and opens up new avenues for the rational design of SpCas9 variants with enhanced performance and specificity.