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

项目摘要 最近，已经发现了新的大分子系统，其结合了CRISPR和CRISPR的优点。 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， 补充了CRISPR-Cas9等工具的力量。这项提案旨在揭示 CRISPR转座酶（CRISPR-TNP）中两种尚未被充分理解的现象的机制 系统：靶点免疫和程序化DNA插入。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- 分辨率cryo-EM以确定DNA结合的shTnsC的结构。此外，shTnsC是AAA+ ATP酶 其核苷酸水解活性与转座有关。我们建议确定shTnsC的结构 在不同的核苷酸结合状态，以揭示在转座ATP水解的作用。最后，我们的目标 揭示通过Cas 12 k和shTniQ将shTnsC募集到靶位点的机制，以及如何 这些因子之间的关联最终启动shCAST转座。我们强大的初步数据 这表明这些目标很可能会成功。此外，凯洛格实验室得到了 康奈尔社区实现本提案中概述的目标。

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.