At the core of the CRISPR–Cas9 genome-editing technology, the endonuclease Cas9 introduces site-specific breaks in DNA. However, precise mechanistic information to ameliorate Cas9 function is still missing. Here, multimicrosecond molecular dynamics, free energy and multiscale simulations are combined with solution NMR and DNA cleavage experiments to resolve the catalytic mechanism of target DNA cleavage. We show that the conformation of an active HNH nuclease is tightly dependent on the catalytic Mg²⁺, unveiling its cardinal structural role. This activated Mg²⁺-bound HNH is consistently described through molecular simulations, nuclear magnetic resonance (NMR) and DNA cleavage assays, revealing also that the protonation state of the catalytic H840 is strongly affected by active site mutations. Finally, ab initio quantum mechanics (density functional theory)/molecular mechanics simulations and metadynamics establish the catalytic mechanism, showing that the catalysis is activated by H840 and completed by K866, thus rationalizing DNA cleavage experiments. This information is critical to enhancing the enzymatic function of CRISPR–Cas9 towards improved genome editing.