Enhanced proofreading governs CRISPR-Cas9 targeting accuracy.

Autor: Chen JS; Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA., Dagdas YS; Biophysics Graduate Group, University of California, Berkeley, California 94720, USA., Kleinstiver BP; Molecular Pathology Unit, Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA.; Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA.; Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA., Welch MM; Molecular Pathology Unit, Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA.; Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA., Sousa AA; Molecular Pathology Unit, Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA.; Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA., Harrington LB; Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA., Sternberg SH; Department of Chemistry, University of California, Berkeley, California 94720, USA., Joung JK; Molecular Pathology Unit, Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA.; Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA.; Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA., Yildiz A; Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.; Department of Physics, University of California, Berkeley, California 94720, USA., Doudna JA; Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.; Department of Chemistry, University of California, Berkeley, California 94720, USA.; Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA.; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
Jazyk: angličtina
Zdroj: Nature [Nature] 2017 Oct 19; Vol. 550 (7676), pp. 407-410. Date of Electronic Publication: 2017 Sep 20.
DOI: 10.1038/nature24268
Abstrakt: 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.
Databáze: MEDLINE
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