5VZL

cryo-EM structure of the Cas9-sgRNA-AcrIIA4 anti-CRISPR complex

5VZL の概要

• エントリーDOI: 10.2210/pdb5vzl/pdb
• EMDBエントリー: 8749
• 分子名称: single guide RNA (116-MER), CRISPR-associated endonuclease Cas9, phage anti-CRISPR AcrIIA4 (3 entities in total)
• 機能のキーワード: anti-crispr, cas9, crispr, gene editing, on-target, off-target, cryo-em, immune system-rna complex, immune system/rna
• 由来する生物種: Streptococcus pyogenes M1 GAS; 詳細
• タンパク質・核酸の鎖数: 3
• 化学式量合計: 207247.61

構造登録者

Jiang, F.,Liu, J.J.,Nogales, E.,Doudna, J.A. (登録日: 2017-05-29, 公開日: 2017-07-26, 最終更新日: 2025-05-14)

主引用文献

Shin, 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.
Disabling Cas9 by an anti-CRISPR DNA mimic.
Sci Adv, 3:e1701620-e1701620, 2017

PubMed Abstract: 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 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 have not been fully established. We show 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-electron microscopy structure of the Cas9-sgRNA-AcrIIA4 complex revealed that the surface of AcrIIA4 is highly acidic and binds with a 1:1 stoichiometry to a region of Cas9 that normally engages the DNA protospacer adjacent motif. Consistent with this binding mode, order-of-addition experiments showed that AcrIIA4 interferes with DNA recognition but has no effect on preformed 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.

PubMed: 28706995
DOI: 10.1126/sciadv.1701620

実験手法

ELECTRON MICROSCOPY (3.9 Å)