A study led by Janice S. Chen and colleagues reveals that the CRISPR-Cas9 system's targeting accuracy is governed by a conformational proofreading mechanism. The research focuses on SpCas9 variants, including SpCas9-HF1 and eSpCas9(1.1), which exhibit reduced off-target cleavage in human cells. Using single-molecule FRET experiments, the team found that these variants become trapped in an inactive state when bound to mismatched targets. A non-catalytic domain, REC3, within Cas9 recognizes target complementarity and regulates the HNH nuclease activity. This discovery led to the development of a new hyper-accurate Cas9 variant, HypaCas9, which demonstrates high genome-wide specificity without compromising on-target activity. The study shows that the HNH nuclease domain undergoes a conformational change upon target binding, which activates the RuvC nuclease for DNA cleavage. The HNH domain becomes trapped in an inactive conformation when bound to mismatched targets, suggesting that high-fidelity variants of Cas9 raise the threshold for HNH conformational activation to improve off-target discrimination. Structural studies indicate that the REC3 domain interacts with the RNA/DNA heteroduplex and undergoes conformational changes upon target binding. The researchers identified five clusters of residues within the REC3 domain that, when mutated, enhance Cas9 specificity. The study also demonstrates that the REC2 domain regulates access of the HNH nuclease to the target strand scissile phosphate. The findings suggest that the conformational proofreading mechanism is essential for accurate targeting by Cas9. The researchers developed HypaCas9, a hyper-accurate Cas9 variant, which exhibits improved specificity compared to existing variants. The study provides a comprehensive model for understanding the balance between target recognition and nuclease activation for precision genome editing. The results highlight the importance of conformational changes in ensuring accurate targeting by Cas9 and offer a strategy for improving Cas9 specificity through rational design.A study led by Janice S. Chen and colleagues reveals that the CRISPR-Cas9 system's targeting accuracy is governed by a conformational proofreading mechanism. The research focuses on SpCas9 variants, including SpCas9-HF1 and eSpCas9(1.1), which exhibit reduced off-target cleavage in human cells. Using single-molecule FRET experiments, the team found that these variants become trapped in an inactive state when bound to mismatched targets. A non-catalytic domain, REC3, within Cas9 recognizes target complementarity and regulates the HNH nuclease activity. This discovery led to the development of a new hyper-accurate Cas9 variant, HypaCas9, which demonstrates high genome-wide specificity without compromising on-target activity. The study shows that the HNH nuclease domain undergoes a conformational change upon target binding, which activates the RuvC nuclease for DNA cleavage. The HNH domain becomes trapped in an inactive conformation when bound to mismatched targets, suggesting that high-fidelity variants of Cas9 raise the threshold for HNH conformational activation to improve off-target discrimination. Structural studies indicate that the REC3 domain interacts with the RNA/DNA heteroduplex and undergoes conformational changes upon target binding. The researchers identified five clusters of residues within the REC3 domain that, when mutated, enhance Cas9 specificity. The study also demonstrates that the REC2 domain regulates access of the HNH nuclease to the target strand scissile phosphate. The findings suggest that the conformational proofreading mechanism is essential for accurate targeting by Cas9. The researchers developed HypaCas9, a hyper-accurate Cas9 variant, which exhibits improved specificity compared to existing variants. The study provides a comprehensive model for understanding the balance between target recognition and nuclease activation for precision genome editing. The results highlight the importance of conformational changes in ensuring accurate targeting by Cas9 and offer a strategy for improving Cas9 specificity through rational design.