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Disabling Cas9 by an anti-CRISPR DNA mimic

Jiyung Shing, Fuguo Jiang, Jun-Jie Liu, Nicholas L. Bray, Benjamin J. Rauch, Seung Hyun Baik, Eva Nogales, Joseph Bondy-Denomy, View ORCID ProfileJacob E. Corn, Jennifer A. Doudna
doi: https://doi.org/10.1101/129627
Jiyung Shing
1Innovative Genomics Institute, Berkeley, California 94720
2Department of Molecular and Cell Biology, University of California, Berkeley, California 94720
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Fuguo Jiang
2Department of Molecular and Cell Biology, University of California, Berkeley, California 94720
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Jun-Jie Liu
2Department of Molecular and Cell Biology, University of California, Berkeley, California 94720
5Department of Chemistry, University of California, Berkeley, California 94720
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Nicholas L. Bray
1Innovative Genomics Institute, Berkeley, California 94720
2Department of Molecular and Cell Biology, University of California, Berkeley, California 94720
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Benjamin J. Rauch
3Department of Microbiology and Immunology, and Quantitative Biosciences Institute, University of California, San Francisco, CA 94158
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Seung Hyun Baik
1Innovative Genomics Institute, Berkeley, California 94720
2Department of Molecular and Cell Biology, University of California, Berkeley, California 94720
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Eva Nogales
2Department of Molecular and Cell Biology, University of California, Berkeley, California 94720
5Department of Chemistry, University of California, Berkeley, California 94720
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Joseph Bondy-Denomy
3Department of Microbiology and Immunology, and Quantitative Biosciences Institute, University of California, San Francisco, CA 94158
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Jacob E. Corn
1Innovative Genomics Institute, Berkeley, California 94720
2Department of Molecular and Cell Biology, University of California, Berkeley, California 94720
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  • ORCID record for Jacob E. Corn
  • For correspondence: jcorn@berkeley.edu doudna@berkeley.edu
Jennifer A. Doudna
1Innovative Genomics Institute, Berkeley, California 94720
2Department of Molecular and Cell Biology, University of California, Berkeley, California 94720
4Howard Hughes Medical Institute, Berkeley, California 94720
5Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720
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  • For correspondence: jcorn@berkeley.edu doudna@berkeley.edu
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Abstract

CRISPR-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 has not fully been established. We show here 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-EM structure of the Cas9-sgRNA-AcrIIA4 complex revealed that the surface of AcrIIA4 is highly acidic and binds with 1:1 stoichiometry to a region of Cas9 that normally engages the DNA protospacer adjacent motif (PAM). Consistent with this binding mode, order-of-addition experiments showed that AcrIIA4 interferes with DNA recognition but has no effect on pre-formed 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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Posted April 23, 2017.
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Disabling Cas9 by an anti-CRISPR DNA mimic
Jiyung Shing, Fuguo Jiang, Jun-Jie Liu, Nicholas L. Bray, Benjamin J. Rauch, Seung Hyun Baik, Eva Nogales, Joseph Bondy-Denomy, Jacob E. Corn, Jennifer A. Doudna
bioRxiv 129627; doi: https://doi.org/10.1101/129627
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Disabling Cas9 by an anti-CRISPR DNA mimic
Jiyung Shing, Fuguo Jiang, Jun-Jie Liu, Nicholas L. Bray, Benjamin J. Rauch, Seung Hyun Baik, Eva Nogales, Joseph Bondy-Denomy, Jacob E. Corn, Jennifer A. Doudna
bioRxiv 129627; doi: https://doi.org/10.1101/129627

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