I 型 C 蛋白通过多种机制抑制 CRISPR-Cas9。
Diverse Mechanisms of CRISPR-Cas9 Inhibition by Type IIC Anti-CRISPR Proteins.
机构信息
CAS Key Laboratory of Infection and Immunity, CAS Center for Excellence in Biomacromolecules, National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016, USA.
出版信息
Mol Cell. 2019 Apr 18;74(2):296-309.e7. doi: 10.1016/j.molcel.2019.01.038. Epub 2019 Mar 5.
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 inhibit Neisseria meningitidis Cas9 (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.
抗 CRISPR 蛋白（Acr）针对 CRISPR-Cas9 系统代表了 Cas9 为基础的应用的天然“关闭开关”。最近，发现 AcrIIC1、AcrIIC2 和 AcrIIC3 蛋白抑制脑膜炎奈瑟菌 Cas9（NmeCas9）在细菌和人类细胞中的活性。这里我们报告了生化和结构数据，这些数据表明 AcrIIC2 和 AcrIIC3 介导的 Cas9 抑制的分子机制。AcrIIC2 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。AcrIIC3 通过与 Cas9 的 HNH 结构域的非保守表面结合来阻止 DNA 加载步骤。AcrIIC3 还与 Cas9 的 REC 叶形成额外的相互作用，并诱导 AcrIIC3-Cas9 复合物的二聚化。虽然 AcrIIC2 靶向来自不同亚型的 Cas9 同源物，但效率不同，AcrIIC3 特异性抑制 NmeCas9。基于结构的 NmeCas9 同源物的变化将它们转化为抗 CRISPR 敏感蛋白。我们的研究提供了对抗 CRISPR 介导的抑制机制的深入了解，并为 Cas9 为基础的应用中设计调控工具提供了指导。
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