Institute for Cell and Molecular Biology, University of Texas at Austin, Austin, TX, 78712, USA.
Department of Chemistry, University of Texas at Austin, Austin, TX, 78712, USA.
Nat Commun. 2020 Nov 23;11(1):5931. doi: 10.1038/s41467-020-19785-8.
Bacteria and archaea employ CRISPR (clustered, regularly, interspaced, short palindromic repeats)-Cas (CRISPR-associated) systems as a type of adaptive immunity to target and degrade foreign nucleic acids. While a myriad of CRISPR-Cas systems have been identified to date, type I-C is one of the most commonly found subtypes in nature. Interestingly, the type I-C system employs a minimal Cascade effector complex, which encodes only three unique subunits in its operon. Here, we present a 3.1 Å resolution cryo-EM structure of the Desulfovibrio vulgaris type I-C Cascade, revealing the molecular mechanisms that underlie RNA-directed complex assembly. We demonstrate how this minimal Cascade utilizes previously overlooked, non-canonical small subunits to stabilize R-loop formation. Furthermore, we describe putative PAM and Cas3 binding sites. These findings provide the structural basis for harnessing the type I-C Cascade as a genome-engineering tool.
细菌和古菌利用 CRISPR（成簇、规律、间隔、短回文重复）-Cas（CRISPR 相关）系统作为一种适应性免疫来靶向和降解外来核酸。虽然迄今为止已经鉴定出了无数种 CRISPR-Cas 系统，但 I-C 型是自然界中最常见的亚型之一。有趣的是，I-C 型系统采用了最小的 Cascade 效应复合物，其操纵子中仅编码三个独特的亚基。在这里，我们呈现了来自脱硫弧菌的 I-C 型 Cascade 的 3.1Å 分辨率冷冻电镜结构，揭示了 RNA 指导的复合物组装所依赖的分子机制。我们展示了这种最小的 Cascade 如何利用以前被忽视的非典型小亚基来稳定 R 环的形成。此外，我们还描述了潜在的 PAM 和 Cas3 结合位点。这些发现为利用 I-C 型 Cascade 作为基因组工程工具提供了结构基础。
