Collaborative Research: Mechanisms of RNA-directed activation of a Cas9 nuclease competent for DNA interrogation.

合作研究：RNA 指导激活能够进行 DNA 询问的 Cas9 核酸酶的机制。

• 批准号: 1716744
• 负责人: Peter Qin
• 金额: $ 27.14万
• 依托单位: University of Southern California
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-15 至 2022-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1716744&HistoricalAwards=false
• 关键词: Collaborative; Research; Mechanisms; RNA; directed

This project will investigate how a complex of an RNA and protein, named CRISPR-Cas9, works as a molecular scissor to change an organism's DNA. CRISPR-Cas9 is natively used by lower organisms such as bacteria to defend against viral infections. The CRISPR-Cas9 system is being adapted as a powerful tool for manipulating genomes of many organisms in a 'by-design and on-demand' fashion. On the horizon are innovations that use CRISPR-Cas9 to produce drought-resistant crops; to combat mosquito transmitted diseases such as malaria; and to cure human illness by fixing an individual's faulty DNA. While studies on CRISPR-Cas9 have advanced rapidly and are poised to revolutionize many fields in biology, details on its mechanism of action remain to be elucidated. This project will fill this knowledge gap by identifying the mechanisms by which RNA molecules convert Cas9's molecular structure into a form suitable for selecting DNA targets. Beyond advancing scientific understanding and methodologies, the project will explore collaborations with biotech companies that may yield new technological applications and promote economic developments. It also includes research training for students at all levels and curriculum innovations such as 'virtual reality' (VR) demonstrations of the Cas9-RNA complex, thus contributing to the development of STEM (Science, Technology, Engineering and Mathematics) workforce.The overall objective of the project is to elucidate the roles of specific domains of Cas9 and guide RNA in initiating and directing a cascade of conformational changes towards the state ready for DNA interrogation. The proposed work will leverage on the expertise of the PI in the functional and structural (X-ray crystallography) characterization of protein-nucleic acid complexes; and that of the Co-PI in site directed spin labeling studies of nucleic acids and protein-nucleic acid complexes. Experiments will focus on Streptococcus pyogenes Cas9, the workhorse in genome engineering, and test an overarching hypothesis that the interactions of the bridge-helix of Cas9 with specific RNA elements, initiate and induce Cas9 domain rearrangements to assemble a DNA-interrogative protein-RNA complex. Specific Aims are: Dissect the role of the bridge helix in initiating conformational changes in Cas9 (Aim 1); Identify the protein and RNA elements that displaces the nuclease lobe (Aim 2) and a helical domain (HD-III, Aim 3) in response to the bridge-helix conformational change. The results from the three aims will identify the sequential steps in the conformational cascade and will be widely applicable to similar CRISPR-Cas systems.

该项目将研究一种名为CRISPR-Cas9的RNA和蛋白质复合物如何作为分子生物学改变生物体的DNA。CRISPR-Cas9被细菌等低等生物天然使用，以抵御病毒感染。CRISPR-Cas9系统正在被改造成一种强大的工具，以“按需设计和按需”的方式操纵许多生物的基因组。即将出现的创新是使用CRISPR-Cas9来生产抗旱作物;对抗蚊子传播的疾病，如疟疾;通过修复个人的错误DNA来治愈人类疾病。虽然CRISPR-Cas9的研究进展迅速，并有望彻底改变生物学的许多领域，但其作用机制的细节仍有待阐明。该项目将通过确定RNA分子将Cas9的分子结构转化为适合选择DNA靶标的形式的机制来填补这一知识空白。除了推进科学理解和方法之外，该项目还将探索与生物技术公司的合作，这些公司可能会产生新的技术应用并促进经济发展。它还包括对各级学生的研究培训和课程创新，例如Cas9-RNA复合物的“虚拟现实”（VR）演示，从而有助于STEM的发展。（科学、技术、工程和数学）该项目的总体目标是阐明Cas9和指导RNA的特定结构域在启动和引导构象变化级联中的作用，准备好进行DNA审讯拟议的工作将利用PI在蛋白质-核酸复合物的功能和结构（X射线晶体学）表征方面的专业知识;以及Co-PI在核酸和蛋白质-核酸复合物的定点自旋标记研究方面的专业知识。实验将集中在化脓性链球菌Cas9，基因组工程的主力，并测试一个总体假设，即Cas9的桥螺旋与特定RNA元件的相互作用，启动和诱导Cas9结构域重排组装DNA-疑问蛋白-RNA复合物。具体目标是：剖析桥螺旋在Cas9（Aim 1）中引发构象变化的作用;鉴定响应于桥螺旋构象变化而置换核酸酶叶（Aim 2）和螺旋结构域（HD-III，Aim 3）的蛋白质和RNA元件。这三个目标的结果将确定构象级联中的顺序步骤，并将广泛适用于类似的CRISPR-Cas系统。

项目成果

Single-DNA electron spin resonance spectroscopy in aqueous solutions

水溶液中的单 DNA 电子自旋共振光谱

• DOI: 10.1038/s41592-018-0084-1
• 发表时间: 2018-09-01
• 期刊: NATURE METHODS
• 影响因子: 48
• 作者: Shi, Fazhan;Kong, Fei;Du, Jiangfeng
• 通讯作者: Du, Jiangfeng

Differential Divalent Metal Binding by SpyCas9's RuvC Active Site Contributes to Nonspecific DNA Cleavage

SpyCas9 的 RuvC 活性位点的差异二价金属结合有助于非特异性 DNA 切割

• DOI: 10.1089/crispr.2023.0022
• 发表时间: 2023
• 期刊: The CRISPR Journal
• 作者: Newsom, Sydney N.;Wang, Duen-Shian;Rostami, Saadi;Schuster, Isabelle;Parameshwaran, Hari Priya;Joseph, Yadin G.;Qin, Peter Z.;Liu, Jin;Rajan, Rakhi
• 通讯作者: Rajan, Rakhi

Bridge Helix of Cas9 Modulates Target DNA Cleavage and Mismatch Tolerance

• DOI: 10.1021/acs.biochem.8b01241
• 发表时间: 2019-04-09
• 期刊: BIOCHEMISTRY
• 影响因子: 2.9
• 作者: Babu, Kesavan;Amrani, Nadia;Rajan, Rakhi
• 通讯作者: Rajan, Rakhi