Chen, Janice S.Dagdas, Yavuz S.Kleinstiver, BenjaminWelch, Moira M.Sousa, AlexanderHarrington, Lucas B.Sternberg, Samuel H.Joung, J. KeithYildiz, AhmetDoudna, Jennifer A.2018-05-302017Chen, Janice S., Yavuz S. Dagdas, Benjamin P. Kleinstiver, Moira M. Welch, Alexander A. Sousa, Lucas B. Harrington, Samuel H. Sternberg, J. Keith Joung, Ahmet Yildiz, and Jennifer A. Doudna. 2017. “Enhanced proofreading governs CRISPR-Cas9 targeting accuracy.” Nature 550 (7676): 407-410. doi:10.1038/nature24268. http://dx.doi.org/10.1038/nature24268.http://nrs.harvard.edu/urn-3:HUL.InstRepos:37068176The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing1–4. High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity were unknown5–9. Using single-molecule Förster resonance energy transfer (smFRET) experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state10 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.en-USJournal Article2018-05-3010.1038/nature24268