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

该项目将研究一种名为CRISPR-Cas9的RNA和蛋白质复合物如何像分子剪刀一样改变生物体的DNA。CRISPR-Cas9被细菌等低等生物用于抵御病毒感染。CRISPR-Cas9系统正被改造成一种强大的工具，以“设计和按需”的方式操纵许多生物体的基因组。即将出现的创新是利用CRISPR-Cas9培育抗旱作物；防治蚊子传播的疾病，如疟疾；通过修复个体有缺陷的DNA来治愈人类疾病。虽然对CRISPR-Cas9的研究进展迅速，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。除了促进科学理解和方法，该项目还将探索与生物技术公司的合作，以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（科学，技术，工程和数学）劳动力的发展。该项目的总体目标是阐明Cas9的特定结构域和引导RNA在启动和指导一系列构象变化的作用，从而为DNA审讯做好准备。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中的应用。实验将集中在化脓性链球菌Cas9上，这是基因组工程中的主力，并测试一个总体假设，即Cas9的桥式螺旋与特定RNA元件的相互作用，启动并诱导Cas9结构域重排，以组装dna -疑问蛋白-RNA复合物。具体目的是：剖析桥螺旋在启动Cas9构象变化中的作用（目的1）；鉴定取代核酸酶叶（Aim 2）和螺旋结构域（HD-III, Aim 3）以响应桥-螺旋构象变化的蛋白质和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。

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.