Disabling Cas9 by an anti-CRISPR DNA mimic

• DOI: 10.1126/sciadv.1701620
• Publication Date: 2017-07-13T01:00:34Z
• Authors: Jiyung Shin; Fuguo Jiang; Jun-Jie Liu; Nicolas L. Bray; Benjamin J. Rauch; Seung Hyun Baik; Eva Nogales; Joseph Bondy-Denomy; Jacob E. Corn; Jennifer A. Doudna
• Keywords: Models, Molecular; 0301 basic medicine; 570; 1.1 Normal biological development and functioning; Molecular Conformation; Cell Line; Structure-Activity Relationship; 03 medical and health sciences; Models; CRISPR-Associated Protein 9; Genetics; 2.1 Biological and endogenous factors; Humans; Clustered Regularly Interspaced Short Palindromic Repeats; Gene Silencing; Research Articles; Gene Editing; Molecular; High-Throughput Nucleotide Sequencing; DNA; Biological Sciences; 3. Good health; 5.1 Pharmaceuticals; RNA; Biochemistry and Cell Biology; Generic health relevance; CRISPR-Cas Systems; Guide; Biotechnology; RNA, Guide, Kinetoplastida

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 human cells. However, mechanism inhibition not known, potential applications for Cas9 inhibitor proteins mammalian cells have been fully established. We that anti-CRISPR protein AcrIIA4 binds only assembled Cas9-single-guide RNA (sgRNA) complexes alone. A 3.9 Å resolution cryo-electron microscopy structure Cas9-sgRNA-AcrIIA4 complex revealed surface highly acidic with 1:1 stoichiometry region normally engages DNA protospacer adjacent motif. Consistent this binding mode, order-of-addition experiments showed interferes recognition but has no effect on preformed Cas9-sgRNA-DNA complexes. Timed delivery into as either or expression plasmid allows on-target while reducing off-target edits. These results provide mechanistic understanding function demonstrate can modulate extent outcomes editing.