The study investigates the mechanism by which CRISPR-Cas9 variants enhance targeting accuracy. Using single-molecule Förster resonance energy transfer (smFRET) experiments, the authors show that high-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants of the Cas9 nuclease are trapped in an inactive state when bound to mismatched targets. They identify a non-catalytic domain within Cas9, REC3, which recognizes target complementarity and governs the HNH nuclease to regulate catalytic competence. By designing a new hyper-accurate Cas9 variant (HypaCas9), they demonstrate high genome-wide specificity without compromising on-target activity in human cells. The findings provide a comprehensive model for rationalizing and modifying the balance between target recognition and nuclease activation to improve precision genome editing.The study investigates the mechanism by which CRISPR-Cas9 variants enhance targeting accuracy. Using single-molecule Förster resonance energy transfer (smFRET) experiments, the authors show that high-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants of the Cas9 nuclease are trapped in an inactive state when bound to mismatched targets. They identify a non-catalytic domain within Cas9, REC3, which recognizes target complementarity and governs the HNH nuclease to regulate catalytic competence. By designing a new hyper-accurate Cas9 variant (HypaCas9), they demonstrate high genome-wide specificity without compromising on-target activity in human cells. The findings provide a comprehensive model for rationalizing and modifying the balance between target recognition and nuclease activation to improve precision genome editing.