III-E 型 CRISPR-Cas7-11 效应物复合物的结构与工程化。
Structure and engineering of the type III-E CRISPR-Cas7-11 effector complex.
机构信息
Structural Biology Division, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
出版信息
Cell. 2022 Jun 23;185(13):2324-2337.e16. doi: 10.1016/j.cell.2022.05.003. Epub 2022 May 27.
The type III-E CRISPR-Cas effector Cas7-11, with dual RNase activities for precursor CRISPR RNA (pre-crRNA) processing and crRNA-guided target RNA cleavage, is a new platform for bacterial and mammalian RNA targeting. We report the 2.5-Å resolution cryoelectron microscopy structure of Cas7-11 in complex with a crRNA and its target RNA. Cas7-11 adopts a modular architecture comprising seven domains (Cas7.1-Cas7.4, Cas11, INS, and CTE) and four interdomain linkers. The crRNA 5' tag is recognized and processed by Cas7.1, whereas the crRNA spacer hybridizes with the target RNA. Consistent with our biochemical data, the catalytic residues for programmable cleavage in Cas7.2 and Cas7.3 neighbor the scissile phosphates before the flipped-out fourth and tenth nucleotides in the target RNA, respectively. Using structural insights, we rationally engineered a compact Cas7-11 variant (Cas7-11S) for single-vector AAV packaging for transcript knockdown in human cells, enabling in vivo Cas7-11 applications.
III-E 型 CRISPR-Cas 效应因子 Cas7-11 具有双重 RNase 活性，可用于前体 CRISPR RNA（pre-crRNA）加工和 crRNA 引导的靶 RNA 切割，是细菌和哺乳动物 RNA 靶向的新平台。我们报告了 Cas7-11 与 crRNA 和其靶 RNA 复合物的 2.5 Å 分辨率冷冻电镜结构。Cas7-11 采用由七个结构域（Cas7.1-Cas7.4、Cas11、INS 和 CTE）和四个结构域间连接子组成的模块化结构。crRNA 5' 标签由 Cas7.1 识别和加工，而 crRNA 间隔区与靶 RNA 杂交。与我们的生化数据一致，Cas7.2 和 Cas7.3 中的催化残基分别位于靶 RNA 中翻转的第四个和第十个核苷酸之前的可切割磷酸酯的附近，用于可编程切割。利用结构见解，我们合理设计了紧凑型 Cas7-11 变体（Cas7-11S），用于单载体 AAV 包装以在人类细胞中进行转录敲低，从而实现体内 Cas7-11 的应用。
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