小柯机器人

日本东京大学Hiroshi Nishimasu等研究人员合作完成III-E型CRISPR-Cas7-11效应子复合物的结构解析和工程化。这一研究成果于2022年5月27日在线发表在国际学术期刊《细胞》上。

研究人员报告了Cas7-11与crRNA及其靶标RNA复合物的2.5Å分辨率冷冻电镜结构。Cas7-11采用模块化结构，包括七个结构域（Cas7.1-Cas7.4、Cas11、INS和CTE）和四个结构域间连接体。crRNA的5′标签被Cas7.1识别和加工，而crRNA的间隔物与靶标RNA杂交。与生化数据一致，Cas7.2和Cas7.3中可编程切割的催化残基分别与靶标RNA中翻转出来的第四和第十个核苷酸之前的易断磷酸根相邻。利用结构上的见解，研究人员合理地设计了一个紧凑的Cas7-11变体（Cas7-11S），用于单载体AAV包装，这能够用于人类细胞的转录本敲除，使Cas7-11在体内的应用成为可能。

据介绍，III-E型CRISPR-Cas效应子Cas7-11具有前体CRISPR RNA（pre-crRNA）加工和crRNA引导的目标RNA切割的双重RNA酶活性，是细菌和哺乳动物RNA靶向的新平台。

附：英文原文

Title: Structure and engineering of the type III-E CRISPR-Cas7-11 effector complex

Author: Kazuki Kato, Wenyuan Zhou, Sae Okazaki, Yukari Isayama, Tomohiro Nishizawa, Jonathan S. Gootenberg, Omar O. Abudayyeh, Hiroshi Nishimasu

Issue&Volume: 2022-05-27

Abstract: The type III-E CRISPR-Cas effector Cas7-11, with dual RNase activities for precursorCRISPR RNA (pre-crRNA) processing and crRNA-guided target RNA cleavage, is a new platformfor bacterial and mammalian RNA targeting. We report the 2.5- resolution cryoelectronmicroscopy structure of Cas7-11 in complex with a crRNA and its target RNA. Cas7-11adopts 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 processedby Cas7.1, whereas the crRNA spacer hybridizes with the target RNA. Consistent withour biochemical data, the catalytic residues for programmable cleavage in Cas7.2 andCas7.3 neighbor the scissile phosphates before the flipped-out fourth and tenth nucleotidesin the target RNA, respectively. Using structural insights, we rationally engineereda compact Cas7-11 variant (Cas7-11S) for single-vector AAV packaging for transcriptknockdown in human cells, enabling in vivo Cas7-11 applications.

DOI: 10.1016/j.cell.2022.05.003

Source: https://www.cell.com/cell/fulltext/S0092-8674(22)00581-5