RT Journal Article
SR Electronic
T1 Disabling Cas9 by an anti-CRISPR DNA mimic
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 129627
DO 10.1101/129627
A1 Jiyung Shing
A1 Fuguo Jiang
A1 Jun-Jie Liu
A1 Nicholas L. Bray
A1 Benjamin J. Rauch
A1 Seung Hyun Baik
A1 Eva Nogales
A1 Joseph Bondy-Denomy
A1 Jacob E. Corn
A1 Jennifer A. Doudna
YR 2017
UL http://biorxiv.org/content/early/2017/04/23/129627.abstract
AB 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.