Structural basis of DNA targeting by a transposon-encoded CRISPR-Cas system
Structural basis of DNA targeting by a transposon-encoded CRISPR-Cas system
复制标题
DOI：
10.1038/s41586-019-1849-0
复制
发表时间：
2020-01-09
期刊：
影响因子：
64.8
通讯作者：
Fernandez, Israel S.
中科院分区：
文献类型：
--
作者：
Halpin-Healy, Tyler S.;Klompe, Sanne E.;Fernandez, Israel S.
当受到病原体和移动遗传元件的挑战时，细菌使用由 CRISPR 和 Cas 基因编码的适应性免疫系统来维持基因组完整性(1-3)。 I 型 CRISPR-Cas 系统通常通过核糖核蛋白复合物 Cascade 和解旋酶核酸酶 Cas3 (4,5) 的联合作用靶向外源 DNA 进行降解，但缺乏 Cas3 的核酸酶缺陷型 I 型系统已被重新用于细菌 Tn7 样转座子的 RNA 引导转座 (6,7)。 CRISPR 和转座子相关机制在 DNA 靶向和插入过程中如何协作仍然未知。在这里，我们使用冷冻电子显微镜描述了由霍乱弧菌 Tn6677 转座子编码的 TniQ-Cascade 复合物的结构，揭示了这种功能耦合的机制基础。冷冻电子显微镜图谱能够对转座蛋白 TniQ 进行从头建模和细化，该蛋白在 CRISPR RNA (crRNA) 3' 端附近由 Cas6 和 Cas7 形成的界面处以头尾结构的二聚体形式与 Cascade 复合物结合。天然 Cas8-Cas5 融合蛋白结合 5' crRNA 手柄，并通过灵活的插入结构域接触 TniQ 二聚体。目标 DNA 结合结构揭示了原型间隔子相邻基序识别和 R 环形成所需的关键相互作用。这项工作为从结构上理解 TniQ-Cascade 的 DNA 靶向如何导致下游招募额外的转座酶蛋白奠定了基础，并将指导蛋白质工程工作在基因组工程应用中利用该系统进行可编程 DNA 插入。
Bacteria use adaptive immune systems encoded by CRISPR and Cas genes to maintain genomic integrity when challenged by pathogens and mobile genetic elements(1-3). Type I CRISPR-Cas systems typically target foreign DNA for degradation via joint action of the ribonucleoprotein complex Cascade and the helicase-nuclease Cas3(4,5), but nuclease-deficient type I systems lacking Cas3 have been repurposed for RNA-guided transposition by bacterial Tn7-like transposons(6,7). How CRISPR- and transposon-associated machineries collaborate during DNA targeting and insertion remains unknown. Here we describe structures of a TniQ-Cascade complex encoded by the Vibrio cholerae Tn6677 transposon using cryo-electron microscopy, revealing the mechanistic basis of this functional coupling. The cryo-electron microscopy maps enabled de novo modelling and refinement of the transposition protein TniQ, which binds to the Cascade complex as a dimer in a head-to-tail configuration, at the interface formed by Cas6 and Cas7 near the 3' end of the CRISPR RNA (crRNA). The natural Cas8-Cas5 fusion protein binds the 5' crRNA handle and contacts the TniQ dimer via a flexible insertion domain. A target DNA-bound structure reveals critical interactions necessary for protospacer-adjacent motif recognition and R-loop formation. This work lays the foundation for a structural understanding of how DNA targeting by TniQ-Cascade leads to downstream recruitment of additional transposase proteins, and will guide protein engineering efforts to leverage this system for programmable DNA insertions in genome-engineering applications.