CRISPR-Cas9 proteins function within bacterial immune systems to target and destroy invasive DNA and have been harnessed as a robust technology for genome editing. Small bacteriophage-encoded anti-CRISPR proteins (Acrs) can inactivate Cas9, providing an efficient off switch for Cas9-based applications. Here, we show that two Acrs, AcrIIC1 and AcrIIC3, inhibit Cas9 by distinct strategies. AcrIIC1 is a broad-spectrum Cas9 inhibitor that prevents DNA cutting by multiple divergent Cas9 orthologs through direct binding to the conserved HNH catalytic domain of Cas9. A crystal structure of an AcrIIC1-Cas9 HNH domain complex shows how AcrIIC1 traps Cas9 in a DNA-bound but catalytically inactive state. By contrast, AcrIIC3 blocks activity of a single Cas9 ortholog and induces Cas9 dimerization while preventing binding to the target DNA. These two orthogonal mechanisms allow for separate control of Cas9 target binding and cleavage and suggest applications to allow DNA binding while preventing DNA cutting by Cas9.

中文翻译：

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CRISPR-Cas9 的广谱抑制剂。

CRISPR-Cas9 蛋白在细菌免疫系统中发挥作用，可以靶向和破坏侵入性 DNA，并已被用作基因组编辑的强大技术。小型噬菌体编码的抗 CRISPR 蛋白 (Acrs) 可以使 Cas9 失活，为基于 Cas9 的应用提供有效的关闭开关。在这里，我们展示了两个 Acr，AcrIIC1 和 AcrIIC3，通过不同的策略抑制 Cas9。AcrIIC1 是一种广谱 Cas9 抑制剂，可通过直接结合 Cas9 的保守 HNH 催化结构域来防止 DNA 被多种不同的 Cas9 同源物切割。AcrIIC1-Cas9 HNH 结构域复合物的晶体结构显示 AcrIIC1 如何将 Cas9 捕获在 DNA 结合但催化失活状态。相比之下，AcrIIC3 阻断单个 Cas9 同源物的活性并诱导 Cas9 二聚化，同时阻止与靶 DNA 的结合。