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Sci Adv. 2017 Jul 12;3(7):e1701620. doi: 10.1126/sciadv.1701620. eCollection 2017 Jul.

Disabling Cas9 by an anti-CRISPR DNA mimic.

Shin J1,2, Jiang F2,3, Liu JJ2,4, Bray NL1,2, Rauch BJ5, Baik SH1,2, Nogales E2,4,6, Bondy-Denomy J5, Corn JE1,2, Doudna JA1,2,3,4,6,7.

Author information

1
Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
2
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
3
California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720, USA.
4
Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
5
Department of Microbiology and Immunology and Quantitative Biosciences Institute, University of California, San Francisco, San Francisco, CA 94158, USA.
6
Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
7
Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA.

Abstract

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. Natural inhibitors of CRISPR-Cas9 enable phages to evade immunity and show promise in controlling Cas9-mediated gene editing in human cells. However, the mechanism of CRISPR-Cas9 inhibition is not known, and the potential applications for Cas9 inhibitor proteins in mammalian cells have not been fully established. We show that the anti-CRISPR protein AcrIIA4 binds only to assembled Cas9-single-guide RNA (sgRNA) complexes and not to Cas9 protein alone. A 3.9 Å resolution cryo-electron microscopy structure of the Cas9-sgRNA-AcrIIA4 complex revealed that the surface of AcrIIA4 is highly acidic and binds with a 1:1 stoichiometry to a region of Cas9 that normally engages the DNA protospacer adjacent motif. Consistent with this binding mode, order-of-addition experiments showed that AcrIIA4 interferes with DNA recognition but has no effect on preformed Cas9-sgRNA-DNA complexes. Timed delivery of AcrIIA4 into human cells as either protein or expression plasmid allows on-target Cas9-mediated gene editing while reducing off-target edits. These results provide a mechanistic understanding of AcrIIA4 function and demonstrate that inhibitors can modulate the extent and outcomes of Cas9-mediated gene editing.

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