A cryptic binding pocket regulates the metal-dependent activity of Cas9

Cas9 is a metal-dependent nuclease used for genome editing across diverse cells and organisms exhibiting distinct ionic environments, yet how metal ions regulate its catalytic function, and consequently its editing efficiency remains unclear. Here, molecular simulations, Markov state models, and mixed quantum–classical approaches reveal that divalent metals promote activation of the catalytic HNH domain through formation of a divalent metal-binding pocket (DBP) at the HNH–RuvC interface. This cryptic pocket emerges dynamically during HNH activation and is supported by NMR measurements revealing metal-dependent structural perturbations within the HNH–L2 region. Mutations targeting the DBP disrupt HNH activation and impair the coupled catalytic activity of both nucleases, identifying the pocket as a key regulator of Cas9’s metal-dependent activity. Together with quantum–classical simulations revealing distinct catalytic behaviours among divalent metals, these findings uncover an ion-dependent regulatory mechanism in Cas9 with insights for genome editing across diverse biological environments.