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

通过冷冻电镜研究 CRISPR 相关 TnsC 的可编程 DNA 插入的结构基础

• 批准号: 10543118
• 负责人: Elizabeth Kellogg
• 金额: $ 32.86万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2023-08-10
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10543118
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Adoption; Area; Base Pairing; Behavior; Binding; Binding Sites; Biological Models; CRISPR/Cas technology; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Complement; Complex; Cryoelectron Microscopy; DNA; DNA Binding; DNA Integration; DNA Structure; Data; Elements; Engineering; Environment; Event; Exclusion; Exhibits; Filament; Genes; Genome engineering; Goals; Guide RNA; Health; Human; Hydrolysis; Immunity; Link; Literature; Modeling; Molecular; Molecular Conformation; Nucleotides; Outcome; Process; Proteins; RNA Binding; Recruitment Activity; Resolution; Role; Site; Structure; System; Transposase; ds-DNA; genome editing; insight; molecular assembly/self assembly; mu transposase; particle; preference; reconstruction; recruit; tool

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.

项目摘要 最近，已经发现了新的大分子系统，它嫁给了两个CRISPR的好处 和TNP系统，并显示出可编程的DNA插入工具的巨大希望，用于基因组编辑， 补充CRISPR-CAS9等工具的功能。该建议旨在发现分子 在CRISPR-转移酶（CRISPR-TNP）中管理两个尚未了解的现象的机制 系统：靶位点免疫和编程DNA插入。被认为是负责的中心蛋白 在多组分Shcast系统中，这两种观察到的行为都是SHTNSC。我们建议利用高 分辨率的冷冻EM确定结合DNA结合的SHTNSC的结构。此外，SHTNSC是AAA+ ATPase 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中，以揭示ATP - 溶解度在转座中的作用。最后，我们的目标 揭示有关通过CAS12K和SHTNIQ招募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换位。我们强大的初步数据 表明这些目标可能会成功。此外，凯洛格实验室在 康奈尔社区以实现该提案中概述的目标。