Diverse Mechanisms of CRISPR-Cas9 Inhibition by Type IIC Anti-CRISPR Proteins
Diverse Mechanisms of CRISPR-Cas9 Inhibition by Type IIC Anti-CRISPR Proteins
复制标题
IIC 型抗 CRISPR 蛋白抑制 CRISPR-Cas9 的多种机制
DOI：
10.1016/j.molcel.2019.01.038
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发表时间：
2019
期刊：
影响因子：
16
通讯作者：
Gao Pu
中科院分区：
文献类型：
--
作者：
Zhu Yalan;Gao Ang;Zhan Qi;Wang Yong;Feng Han;Liu Songqing;Gao Guangxia;Serganov Alex;er;Gao Pu
靶向CRISPR-Cas9系统的抗CRISPR蛋白（Acr）代表了基于Cas9的应用的天然“关闭开关”。最近，发现AcrIIC 1、AcrIIC 2和AcrIIC 3蛋白在细菌和人类细胞中抑制脑膜炎奈瑟氏球菌Cas9（NmeCas 9）活性。在这里，我们报告了生化和结构数据，表明AcrIIC 2和AcrIIC 3介导的Cas9抑制的分子机制。AcrIIC 2二聚体与Cas9的桥螺旋相互作用，干扰RNA结合，并阻止DNA加载到Cas9中。AcrIIC 3通过结合Cas9的HNH结构域的非保守表面来阻断DNA加载步骤。AcrIIC 3还与Cas9的REC叶形成额外的相互作用，并诱导AcrIIC 3-Cas9复合物的二聚化。虽然AcrIIC 2靶向来自不同亚型的Cas9直系同源物，但效率不同，但AcrIIC 3特异性抑制NmeCas 9。NmeCas 9直系同源物的结构引导变化将其转化为抗CRISPR敏感蛋白。我们的研究为抗CRISPR介导的抑制机制提供了见解，并为在基于Cas9的应用中设计调控工具提供了指导。
Anti-CRISPR proteins (Acrs) targeting CRISPR-Cas9 systems represent natural "off switches" for Cas9-based applications. Recently, AcrIIC1, AcrIIC2, and AcrIIC3 proteins were found to inhibitNeisseria meningitidisCas9 (NmeCas9) activity in bacterial and human cells. Here we report biochemical and structural data that suggest molecular mechanisms of AcrIIC2- and AcrIIC3-mediated Cas9 inhibition. AcrIIC2 dimer interacts with the bridge helix of Cas9, interferes with RNA binding, and prevents DNA loading into Cas9. AcrIIC3 blocks the DNA loading step through binding to a non-conserved surface of the HNH domain of Cas9. AcrIIC3 also forms additional interactions with the REC lobe of Cas9 and induces the dimerization of the AcrIIC3-Cas9 complex. While AcrIIC2 targets Cas9 orthologs from different subtypes, albeit with different efficiency, AcrIIC3 specifically inhibits NmeCas9. Structure-guided changes in NmeCas9 orthologs convert them into anti-CRISPR-sensitive proteins. Our studies provide insights into anti-CRISPR-mediated suppression mechanisms and guidelines for designing regulatory tools in Cas9-based applications.