Structural Basis of Programmable DNA-Insertion via Cryo-EM Studies of CRISPR-Associated TnsC

项目摘要 最近，人们发现了结合了CRISPR和CRISPR的优点的新的大分子体系 和TNP系统，并显示出作为基因组编辑的可编程DNA插入工具的巨大前景， 补充CRISPR-CAS9等工具的功能。这项提议的目的是揭示分子 CRISPR转座酶(CRISPR-TNP)中两种尚不清楚的现象的机制 系统：靶点免疫和程序化DNA插入。中央蛋白质被认为是负责 在多组分shCAST系统中观察到的这两种行为都是shTnsC。我们建议利用高效率的 分离冷冻-EM以确定DNA结合的shTnsC的结构。此外，shTnsC是一种AAA+ATPase 其核苷酸水解性与转座有关。我们建议确定shTnsC的结构 在不同的核苷酸结合状态下，以揭示ATP-水解酶在转座中的作用。最后，我们的目标是 揭示通过Cas12k和shTniQ将shTnsC招募到目标站点的机制，以及如何 这些因素之间的关联最终启动了shCAST转座。我们强劲的初步数据 预示着这些目标很可能会成功。此外，凯洛格实验室在 康奈尔社区以实现这项提案中概述的目标。

Project Summary Recently, new macromolecular systems have been discovered which marries the benefits of both CRISPR and TNP systems and shows tremendous promise as programmable DNA-insertion tools for genome-editing, complementing the power of tools such as CRISPR-Cas9. This proposal aims to uncover the molecular mechanisms governing two as-yet poorly understood phenomena in CRISPR-Transposase (CRISPR-TNP) systems: target-site immunity and programmed-DNA insertion. The central protein thought to be responsible for both of these observed behaviors in the multi-component shCAST system is shTnsC. We propose to utilize high- resolution cryo-EM to determine the structure of DNA-bound shTnsC. In addition, shTnsC is a AAA+ ATPase whose nucleotide-hydrolysis activity is linked to transposition. We propose to determine the structure of shTnsC in different nucleotide-bound states in order to reveal the role of ATP-hydrolysis in transposition. Finally, we aim to uncover the mechanisms governing shTnsC recruitment to the target-site via Cas12k and shTniQ, and how the association between these factors ultimately initiates shCAST transposition. Our strong preliminary data indicates that these aims are likely to be successful. In addition, the Kellogg lab is well-supported within the Cornell community to achieve the goals outlined in this proposal.