Rxivist logo

Disabling Cas9 by an anti-CRISPR DNA mimic

By 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

Posted 22 Apr 2017
bioRxiv DOI: 10.1101/129627 (published DOI: 10.1126/sciadv.1701620)

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.

Download data

  • Downloaded 1,935 times
  • Download rankings, all-time:
    • Site-wide: 6,331 out of 118,180
    • In biochemistry: 120 out of 3,572
  • Year to date:
    • Site-wide: 85,169 out of 118,180
  • Since beginning of last month:
    • Site-wide: 70,148 out of 118,180

Altmetric data

Downloads over time

Distribution of downloads per paper, site-wide


Sign up for the Rxivist weekly newsletter! (Click here for more details.)