Enhanced Proofreading Governs CRISPR-Cas9 Targeting Accuracy

Abstract

The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing. DNA cleavage of Cas9 is controlled by the conformational state of the HNH nuclease domain, but the mechanism that governs HNH activation at on-target DNA while reducing cleavage activity at off-target sites remains poorly understood. Using single-molecule Förster resonance energy transfer (smFRET), we identified an intermediate state of Streptococcus pyogenes Cas9, representing a conformational checkpoint between DNA binding and cleavage. Upon DNA binding, the HNH domain transitions between multiple conformations before docking into its active state. We showed that the high-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) Cas9 variants are trapped in an inactive state when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target complementarity and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we designed a new hyper-accurate Cas9 variant (HypaCas9) that demonstrates high genome-wide specificity without compromising on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.