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Nature. 2017 Oct 19;550(7676):407-410. doi: 10.1038/nature24268. Epub 2017 Sep 20.

Enhanced proofreading governs CRISPR-Cas9 targeting accuracy.

Author information

1
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
2
Biophysics Graduate Group, University of California, Berkeley, California 94720, USA.
3
Molecular Pathology Unit, Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA.
4
Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA.
5
Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.
6
Department of Chemistry, University of California, Berkeley, California 94720, USA.
7
Department of Physics, University of California, Berkeley, California 94720, USA.
8
Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA.
9
Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.

Abstract

The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing. 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 unknown. Here, using single-molecule Förster resonance energy transfer experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) 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 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.

PMID:
28931002
PMCID:
PMC5918688
DOI:
10.1038/nature24268
[Indexed for MEDLINE]
Free PMC Article

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