The structure of HypaCas9. A non-catalytic domain within Cas9, REC3, recognizes target complementarity and governs the HNH nuclease to regulate overall catalytic competence. SpCas9-HF1 is trapped in an inactive state when bound to mismatched targets. Mutation of residues within REC3 that are involved in nucleic acid recognition prevents transitions by the REC lobe, which more stringently traps the HNH domain in the conformational checkpoint in the presence of mismatches. This new hyper-accurate Cas9 variant (HypaCas9) demonstrates high genome-wide specificity without compromising on-target activity in human cells.