轉座子編碼CRISPR-Cas系統靶向DNA的結構基礎
作者：
小柯機器人發布時間：2019/12/19 16:04:46
研究人員使用冷凍電鏡描述了由霍亂弧菌Tn6677轉座子編碼的TniQ–Cascade複合物結構，並揭示了這種功能性偶聯的機理基礎。冷凍電鏡圖可以從頭建模和完善轉座蛋白TniQ，該蛋白在Casscade和Cas7形成的界面（臨近CRISPR RNA （crRNA）3'末端附近）以頭尾相接的形式與Cascade複合物結合形成二聚體。天然Cas8–Cas5融合蛋白結合5'crRNA手柄，並通過靈活的插入域與TniQ二聚體接觸。靶DNA結合結構揭示了原間隔物相鄰基序識別和R環形成所必需的關鍵相互作用。這項工作為從結構上了解TniQ–Cascade的DNA靶向如何導致下遊轉座酶蛋白質的下遊募集奠定了基礎，並將指導蛋白質工程工作以利用該系統在基因組工程應用中進行可編程DNA插入。
據悉，細菌利用CRISPR和Cas基因編碼的適應性免疫系統在病原體和移動遺傳元件攻擊時保持基因組完整性。I型CRISPR-Cas系統通常通過核糖核蛋白複合體Cascade和解旋酶核酸酶Cas3的聯合作用靶向外源DNA降解，但是缺少Cas3的核酸酶缺陷型I系統被細菌重新用於RNA引導的轉座（通過類似於Tn7-RNA的轉座子）。但CRISPR和轉座子相關的機器如何在DNA靶向和插入過程中協同工作仍然未知。
附：英文原文
Title: Structural basis of DNA targeting by a transposon-encoded CRISPR–Cas system
Author: Tyler S. Halpin-Healy, Sanne E. Klompe, Samuel H. Sternberg & Israel S. Fernández
Issue&Volume: 2019-12-18
Abstract: Bacteria use adaptive immune systems encoded by CRISPR and Cas genes to maintain genomic integrity when challenged by pathogens and mobile genetic elements13. Type I CRISPRCas systems typically target foreign DNA for degradation via joint action of the ribonucleoprotein complex Cascade and the helicasenuclease Cas34,5, but nuclease-deficient type I systems lacking Cas3 have been repurposed for RNA-guided transposition by bacterial Tn7-like transposons6,7. How CRISPR- and transposon-associated machineries collaborate during DNA targeting and insertion remains unknown. Here we describe structures of a TniQCascade 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 Cas8Cas5 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 TniQCascade 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. Cryo-electron microscopy structures of the TniQCascade complex encoded by the Vibrio cholerae Tn6677 transposon reveal the mechanistic basis of the functional association of CRISPR- and transposon-associated machineries.
DOI: 10.1038/s41586-019-1849-0
Source:https://www.nature.com/articles/s41586-019-1849-0