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.

项目概要 最近，人们发现了新的大分子系统，它结合了 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 CRISPR-Cas9 等工具的强大功能。该提案旨在揭示分子 控制 CRISPR 转座酶 (CRISPR-TNP) 中两种尚未知之甚少的现象的机制 系统：靶位点免疫和程序化 DNA 插入。被认为负责的中心蛋白质 在多组件 shCAST 系统中观察到的这两种行为都是 shTnsC。我们建议利用高 分辨率冷冻电镜以确定 DNA 结合 shTnsC 的结构。此外，shTnsC 是一种 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 不同的核苷酸结合状态，以揭示 ATP 水解在转座中的作用。最后，我们的目标是 揭示通过 Cas12k 和 shTniQ 控制 shTnsC 招募到目标位点的机制，以及如何 这些因素之间的关联最终引发 shCAST 转座。我们强大的初步数据 表明这些目标很可能会成功。此外，凯洛格实验室在以下领域得到了良好的支持： 康奈尔社区实现本提案中概述的目标。