Characterization of structural elements controlling Cas9-mediated DNA cleavage and single-turnover enzyme kinetics.

控制 Cas9 介导的 DNA 切割和单周转酶动力学的结构元件的表征。

基本信息

批准号：
10461615
负责人：
Kaitlyn Kiernan
金额：
$ 3.08万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-05-16 至 2023-11-19
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10461615
关键词：
Address Affinity Binding Biochemical Biological Assay Biomedical Research Biophysics CRISPR screen Catalysis Clustered Regularly Interspaced Short Palindromic Repeats Complex Cryoelectron Microscopy DNA DNA Repair Development Disease Disease model Dissociation Distal Double Strand Break Repair Elements Engineering Enzymatic Biochemistry Enzyme Kinetics Enzymes Event Exhibits Galactosidase Gene Expression Genes Genetic Transcription Genome Genome engineering Genomics Goals Guide RNA Hydrolysis Immune system Impairment In Vitro Kinetics Length Libraries Measures Mediating Methods Molecular Molecular Conformation Monitor Mutagenesis Mutagens Nucleic Acids Organism Proteins Reaction Research Resolution Screening Result Site Site-Directed Mutagenesis Streptococcus pyogenes Structure System Techniques Technology Testing Time Variant Visualization Work beta-Galactosidase chromatin remodeling ds-DNA endonuclease field study flexibility genome editing in vivo innovation insight mutant novel nuclease particle prevent programs rational design success tool

项目摘要

ABSTRACT The CRISPR/Cas9 system offers an extremely versatile genome editing technology by employing an RNA- guided endonuclease, Cas9. Originally isolated from Streptococcus pyogenes, this system has been adapted for use in a wide range of organisms and can be programmed to manipulate almost any site in the genome. While Cas9 is easy to reprogram, the efficiency of Cas9-mediated editing varies considerably across different genomic targets. Unlike other common bacterial endonucleases, Cas9 exhibits single-turnover kinetics where it forms a stable product complex and requires an external force to release the cut DNA. This persistent product state impairs access to the double-strand break by repair machinery and contributes to reduced genome editing efficiency. Despite an abundance of Cas9 structures, the structure of the product complex remains uncharacterized as previous studies were carried out under conditions that prevent DNA cleavage. As a result, our structural understanding of the entire Cas9 reaction cycle remains incomplete and insufficient to explain why Cas9 exhibits single-turnover kinetics. Our lab recently used cryo-EM to determine the structure of the catalytically active Cas9-sgRNA-dsDNA ternary complex and captured three distinct conformational states (pre- and post-catalytic, and product states). These structures provide new insight into the coordination of Cas9 domains throughout catalysis and reveal persistent Cas9-nucleic acid interactions in the product state. Guided by this new insight, we will expand upon these studies to define the major structural elements contributing to Cas9 catalysis and single-turnover kinetics. Using innovative in vitro kinetic assays and established structural analyses, we will investigate the structural features that control the rate at which Cas9 cuts and releases the targeted DNA. Using rational design in conjunction with random mutagenesis, we will engineer a multi-turnover enzyme capable of spontaneously releasing its cleaved DNA product. Using an in vitro genome editing assay and an in vivo -galactosidase assay, we will screen Cas9 mutants for an increased rate of DNA cleavage. We anticipate the proposed studies will not only advance our molecular understanding of the Cas9 reaction cycle, but also support the development of more efficient CRISPR/Cas9 technologies.

抽象的 CRISPR/Cas9 系统通过采用 RNA- 引导核酸内切酶 Cas9 最初是从化脓性链球菌中分离出来的，现已对该系统进行了改造。可用于多种生物体，并且可以通过编程来操纵基因组中的几乎任何位点。虽然 Cas9 很容易重新编程，但 Cas9 介导的编辑效率在不同的环境中差异很大。与其他常见细菌核酸内切酶不同，Cas9 表现出单周转动力学。形成稳定的产物复合物，需要外力来释放切割的DNA，这种持久的产物。状态通过修复机制损害双链断裂，并有助于减少基因组编辑尽管有丰富的 Cas9 结构，但产物复合物的结构仍然存在。由于之前的研究是在防止 DNA 裂解的条件下进行的，因此没有被表征。我们对整个 Cas9 反应循环的结构理解仍然不完整，不足以解释原因 Cas9 表现出单周转动力学。我们的实验室最近使用冷冻电镜来确定该结构。催化活性 Cas9-sgRNA-dsDNA 三元复合物并捕获三种不同的构象状态（预这些结构为 Cas9 的协调提供了新的见解。整个催化过程中的域，并揭示了产物状态下持续的 Cas9 核酸相互作用。通过这一新的见解，我们将扩展这些研究，以定义有助于 Cas9 催化和单周转动力学。使用创新的体外动力学测定和已建立的结构。分析后，我们将研究控制 Cas9 切割和释放速率的结构特征结合随机诱变的合理设计，我们将设计出多周转的DNA。使用体外基因组测定能够自发释放其切割的 DNA 产物的酶。和体内 -半乳糖苷酶测定，我们将筛选 Cas9 突变体以提高 DNA 切割率。预计拟议的研究不仅会增进我们对 Cas9 反应循环的分子理解，还支持开发更高效的 CRISPR/Cas9 技术。