The RNA-guided CRISPR–Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing1,2,3,4. 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 are unknown5,6,7,8,9. Here, using single-molecule Förster resonance energy transfer 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 design 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.
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Sequence Read Archive
Protein Data Bank
We thank A. V. Wright, S. N. Floor, J. C. Cofsky, D. Burstein, C. Fellman, B. L. Oakes and O. Mavrothalassitis for discussions and reading the manuscript, M. S. Prew for technical assistance, and J. M. Lopez for assistance with GUIDE-seq data processing. J.S.C. and L.B.H. are supported by National Science Foundation Graduate Research Fellowships, and B.P.K. from Banting (Natural Sciences and Engineering Research Council of Canada) and Charles A. King Trust Postdoctoral Fellowships. J.A.D. is an Investigator of the Howard Hughes Medical Institute. This work was supported by the National Institutes of Health (GM094522 and GM118773 (A.Y.), R35 GM118158 (J.K.J.)), National Science Foundation (MCB-1617028 (A.Y.) and MCB-1244557 (J.A.D.)), and the Desmond and Ann Heathwood MGH Research Scholar Award (J.K.J.).
Extended data figures
This file contains GUIDE-seq data.
This file contains DNA plasmids and proteins used in this study. All enhanced specificity, high-fidelity, cluster and hyper-accurate SpCas9 variants tested in this study, with Addgene ID numbers for deposited plasmids. The HNH, REC2 or REC3 subscript designation with an enhanced specificity, high-fidelity or cluster SpCas9 variant denotes combination of residue substitutions with indicated FRET construct.
This file contains a list of nucleic acids used in the study.