Dillard Kaylee E, Zhang Hongshan, Dubbs Lianne Z, Chou Chia-Wei, Terrace Cynthia, Javanmardi Kamyab, Kim Wantae, Forsberg Kevin J, Finkelstein Ilya J
Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, United States.
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75390, United States.
Nucleic Acids Res. 2025 Apr 22;53(8). doi: 10.1093/nar/gkaf318.
Mobile genetic elements evade CRISPR-Cas adaptive immunity by encoding anti-CRISPR proteins (Acrs). Acrs inactivate CRISPR-Cas systems via diverse mechanisms but generally coevolve with a narrow subset of Cas effectors that share high sequence similarity. Here, we demonstrate that AcrIIA11 inhibits Streptococcus pyogenes (Sp), Staphylococcus aureus (Sa), and Francisella novicida (Fn) Cas9s in vitro and in human cells. Single-molecule imaging reveals that AcrIIA11 hinders SaCas9 target search by reducing its diffusion on nonspecific DNA. DNA cleavage is inhibited because the AcrIIA11:SaCas9 complex binds to protospacer adjacent motif (PAM)-rich off-target sites, preventing SaCas9 from reaching its target. AcrIIA11 also greatly slows down DNA cleavage after SaCas9 reaches its target site. A negative-stain electron microscopy reconstruction of an AcrIIA11:SaCas9 RNP complex reveals that the heterodimer assembles with a 1:1 stoichiometry. Physical AcrIIA11-Cas9 interactions across type IIA and IIB Cas9s correlate with nuclease inhibition and support its broad-spectrum activity. These results add a kinetic inhibition mechanism to the phage-CRISPR arms race.
移动遗传元件通过编码抗CRISPR蛋白（Acrs）来逃避CRISPR-Cas适应性免疫。Acrs通过多种机制使CRISPR-Cas系统失活，但通常与一小部分具有高度序列相似性的Cas效应蛋白共同进化。在这里，我们证明AcrIIA11在体外和人类细胞中均可抑制化脓性链球菌（Sp）、金黄色葡萄球菌（Sa）和新凶手弗朗西斯菌（Fn）的Cas9。单分子成像显示，AcrIIA11通过减少SaCas9在非特异性DNA上的扩散来阻碍其靶向搜索。DNA切割受到抑制，因为AcrIIA11:SaCas9复合物与富含原间隔相邻基序（PAM）的脱靶位点结合，阻止SaCas9到达其靶标。AcrIIA11还会在SaCas9到达其靶位点后大大减慢DNA切割速度。AcrIIA11:SaCas9核糖核蛋白复合物的负染电子显微镜重建显示，异二聚体以1:1的化学计量组装。AcrIIA11与IIA和IIB型Cas9之间的物理相互作用与核酸酶抑制相关，并支持其广谱活性。这些结果为噬菌体-CRISPR军备竞赛增加了一种动力学抑制机制。
