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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.

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


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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