Department of Biological Sciences, Purdue University, 915 W. State Street, West Lafayette, IN 47907, USA.
Purdue University Center for Cancer Research, Purdue University, 915 W. State Street, West Lafayette, IN 47907, USA.
Nucleic Acids Res. 2021 Jan 11;49(1):584-594. doi: 10.1093/nar/gkaa1199.
CRISPR-Cas systems are adaptive immune systems in bacteria and archaea to defend against mobile genetic elements (MGEs) and have been repurposed as genome editing tools. Anti-CRISPR (Acr) proteins are produced by MGEs to counteract CRISPR-Cas systems and can be used to regulate genome editing by CRISPR techniques. Here, we report the cryo-EM structures of three type I-F Acr proteins, AcrIF4, AcrIF7 and AcrIF14, bound to the type I-F CRISPR-Cas surveillance complex (the Csy complex) from Pseudomonas aeruginosa. AcrIF4 binds to an unprecedented site on the C-terminal helical bundle of Cas8f subunit, precluding conformational changes required for activation of the Csy complex. AcrIF7 mimics the PAM duplex of target DNA and is bound to the N-terminal DNA vise of Cas8f. Two copies of AcrIF14 bind to the thumb domains of Cas7.4f and Cas7.6f, preventing hybridization between target DNA and the crRNA. Our results reveal structural detail of three AcrIF proteins, each binding to a different site on the Csy complex for inhibiting degradation of MGEs.
CRISPR-Cas 系统是细菌和古菌中的一种适应性免疫系统，用于防御移动遗传元件（MGEs），并被重新用作基因组编辑工具。抗 CRISPR（Acr）蛋白由 MGEs 产生，以对抗 CRISPR-Cas 系统，并可用于调节 CRISPR 技术的基因组编辑。在这里，我们报告了三种 I 型 F Acr 蛋白（AcrIF4、AcrIF7 和 AcrIF14）与铜绿假单胞菌 I 型 F CRISPR-Cas 监测复合物（Csy 复合物）结合的冷冻电镜结构。AcrIF4 结合到 Cas8f 亚基 C 末端螺旋束上一个前所未有的位点，阻止了 Csy 复合物激活所需的构象变化。AcrIF7 模拟靶 DNA 的 PAM 双链体，并与 Cas8f 的 N 端 DNA 钳结合。两个 AcrIF14 拷贝结合到 Cas7.4f 和 Cas7.6f 的拇指结构域上，防止靶 DNA 与 crRNA 杂交。我们的结果揭示了三种 AcrIF 蛋白的结构细节，每种蛋白都结合到 Csy 复合物的不同位点，以抑制 MGEs 的降解。
