State Key Laboratory of Experimental Hematology, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Haihe Laboratory of Cell Ecosystem, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China.
State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Wuhan 430062, China.
Nucleic Acids Res. 2023 Sep 22;51(17):9442-9451. doi: 10.1093/nar/gkad669.
CRISPR-Cas systems act as the adaptive immune systems of bacteria and archaea, targeting and destroying invading foreign mobile genetic elements (MGEs) such as phages. MGEs have also evolved anti-CRISPR (Acr) proteins to inactivate the CRISPR-Cas systems. Recently, AcrIIC4, identified from Haemophilus parainfluenzae phage, has been reported to inhibit the endonuclease activity of Cas9 from Neisseria meningitidis (NmeCas9), but the inhibition mechanism is not clear. Here, we biochemically and structurally investigated the anti-CRISPR activity of AcrIIC4. AcrIIC4 folds into a helix bundle composed of three helices, which associates with the REC lobe of NmeCas9 and sgRNA. The REC2 domain of NmeCas9 is locked by AcrIIC4, perturbing the conformational dynamics required for the target DNA binding and cleavage. Furthermore, mutation of the key residues in the AcrIIC4-NmeCas9 and AcrIIC4-sgRNA interfaces largely abolishes the inhibitory effects of AcrIIC4. Our study offers new insights into the mechanism of AcrIIC4-mediated suppression of NmeCas9 and provides guidelines for the design of regulatory tools for Cas9-based gene editing applications.
CRISPR-Cas 系统作为细菌和古菌的适应性免疫系统，靶向并破坏入侵的外来移动遗传元件（MGE），如噬菌体。MGE 还进化出了抗 CRISPR（Acr）蛋白来使 CRISPR-Cas 系统失活。最近，从副流感嗜血杆菌噬菌体中鉴定出的 AcrIIC4 被报道能抑制脑膜炎奈瑟球菌（NmeCas9）的内切酶活性，但抑制机制尚不清楚。在这里，我们从生化和结构上研究了 AcrIIC4 的抗 CRISPR 活性。AcrIIC4 折叠成由三个螺旋组成的螺旋束，与 NmeCas9 的 REC 结构域和 sgRNA 结合。NmeCas9 的 REC2 结构域被 AcrIIC4 锁定，扰乱了靶 DNA 结合和切割所需的构象动力学。此外，AcrIIC4-NmeCas9 和 AcrIIC4-sgRNA 界面关键残基的突变大大消除了 AcrIIC4 的抑制作用。我们的研究为 AcrIIC4 介导的 NmeCas9 抑制机制提供了新的见解，并为 Cas9 为基础的基因编辑应用的调控工具设计提供了指导。
