Anti-CRISPR (Acr) proteins are encoded by phages to inactivate CRISPR–Cas systems of bacteria and archaea and are used to enhance the CRISPR toolbox for genome editing. Here we report the structure and mechanism of AcrIF24, an Acr protein that inhibits the type I-F CRISPR–Cas system from Pseudomonas aeruginosa. AcrIF24 is a homodimer that associates with two copies of the surveillance complex (Csy) and prevents the hybridization between CRISPR RNA and target DNA. Furthermore, AcrIF24 functions as an anti-CRISPR-associated (Aca) protein to repress the transcription of the acrIF23-acrIF24 operon. Alone or in complex with Csy, AcrIF24 is capable of binding to the acrIF23-acrIF24 promoter DNA with nanomolar affinity. The structure of a Csy–AcrIF24–promoter DNA complex at 2.7 Å reveals the mechanism for transcriptional suppression. Our results reveal that AcrIF24 functions as an Acr-Aca fusion protein, and they extend understanding of the diverse mechanisms used by Acr proteins.

抗 CRISPR (Acr) 蛋白由噬菌体编码，可灭活细菌和古细菌的 CRISPR-Cas 系统，并用于增强用于基因组编辑的 CRISPR 工具箱。在这里，我们报道了 AcrIF24 的结构和机制，AcrIF24 是一种抑制铜绿假单胞菌IF 型 CRISPR-Cas 系统的 Acr 蛋白。AcrIF24 是一种同二聚体，与监视复合物 (Csy) 的两个拷贝相关联，并阻止 CRISPR RNA 与靶 DNA 之间的杂交。此外，AcrIF24 作为抗 CRISPR 相关 (Aca) 蛋白发挥作用，抑制acrIF23-acrIF24操纵子的转录。单独或与 Csy 复合时，AcrIF24 能够以纳摩尔亲和力结合acrIF23-acrIF24启动子 DNA。2.7 Å 的 Csy-AcrIF24-启动子 DNA 复合物的结构揭示了转录抑制的机制。我们的结果揭示了 AcrIF24 作为 Acr-Aca 融合蛋白发挥作用，并且扩展了对 Acr 蛋白使用的多种机制的理解。