Disabling Cas9 by an anti-CRISPR DNA mimic

Shing, J.; Jiang, F.; Liu, J.-J.; Bray, N. L.; Rauch, B. J.; Baik, S. H.; Nogales, E.; Bondy-Denomy, J.; Corn, J. E.; Doudna, J. A.

doi:10.1101/129627

bioRxiv · 10.1101/129627

Disabling Cas9 by an anti-CRISPR DNA mimic

Shing, J.·Jiang, F.·Liu, J.-J.·Bray, N. L.·Rauch, B. J.·Baik, S. H.·Nogales, E.·Bondy-Denomy, J.·Corn, J. E.·Doudna, J. A.

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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 [A] 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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Shing, J., Jiang, F., Liu, J.-J., Bray, N. L., Rauch, B. J., Baik, S. H., Nogales, E., Bondy-Denomy, J., Corn, J. E., Doudna, J. A.. 2017-04-22. Disabling Cas9 by an anti-CRISPR DNA mimic. https://doi.org/10.1101/129627

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