5VW1

Crystal structure of SpyCas9-sgRNA-AcrIIA4 ternary complex

5VW1 の概要

• エントリーDOI: 10.2210/pdb5vw1/pdb
• 分子名称: CRISPR-associated endonuclease Cas9/Csn1, sgRNA, anti-CRISPR protein AcrIIA4, ... (8 entities in total)
• 機能のキーワード: type ii crispr-cas endonculease: cas9: structure: endonuclease: anti-crispr protein: inhibition of cas9: ruvc catalytic pocket: sequence-specific pam recognition: genome editing tool, hydrolase
• 由来する生物種: Streptococcus pyogenes serotype M1; 詳細
• タンパク質・核酸の鎖数: 3
• 化学式量合計: 197146.49

構造登録者

Yang, H.,Patel, D.J. (登録日: 2017-05-21, 公開日: 2017-06-28, 最終更新日: 2023-10-04)

主引用文献

Yang, H.,Patel, D.J.
Inhibition Mechanism of an Anti-CRISPR Suppressor AcrIIA4 Targeting SpyCas9.
Mol. Cell, 67:117-127.e5, 2017

PubMed Abstract: Prokaryotic CRISPR-Cas adaptive immune systems utilize sequence-specific RNA-guided endonucleases to defend against infection by viruses, bacteriophages, and mobile elements, while these foreign genetic elements evolve diverse anti-CRISPR proteins to overcome the CRISPR-Cas-mediated defense of the host. Recently, AcrIIA2 and AcrIIA4, encoded by Listeria monocytogene prophages, were shown to block the endonuclease activity of type II-A Streptococcus pyogene Cas9 (SpyCas9). We now report the crystal structure of AcrIIA4 in complex with single-guide RNA-bound SpyCas9, thereby establishing that AcrIIA4 preferentially targets critical residues essential for PAM duplex recognition, as well as blocks target DNA access to key catalytic residues lining the RuvC pocket. These structural insights, validated by biochemical assays on key mutants, demonstrate that AcrIIA4 competitively occupies both PAM-interacting and non-target DNA strand cleavage catalytic pockets. Our studies provide insights into anti-CRISPR-mediated suppression mechanisms for inactivating SpyCas9, thereby broadening the applicability of CRISPR-Cas regulatory tools for genome editing.

PubMed: 28602637
DOI: 10.1016/j.molcel.2017.05.024

実験手法

X-RAY DIFFRACTION (2.598 Å)