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。

• 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
• 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

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-提供了极其通用的基因组编辑技术 指导性核酸内切酶，CAS9。该系统最初是从链球菌中分离出来的，已适应 用于在广泛的生物中使用，可以编程以操纵​​基因组中的任何位点。 虽然CAS9易于重新编程，但仔细的CAS9介导的编辑品种的效率 基因组靶标。与其他常见的细菌不同，CAS9表现出单转动力学 形成稳定的产品配合物，需要外力释放切割的DNA。这个持久的产品 状态会损害通过修复机械的访问双链断裂的机会，并有助于减少基因组编辑 效率。尽管CAS9结构是抽象的，但产品络合物的结构仍保持 在预防DNA裂解的条件下进行了以前的研究。因此， 我们对整个CAS9反应周期的结构理解仍然不完整，不足以解释为什么 CAS9表现出单位动力学。我们的实验室最近使用冷冻EM来确定 催化活性的Cas9-SgrNA-DSDNA三元复合物，并捕获了三个不同的构象状态（前 和催化后和产品状态）。这些结构为CAS9的协调提供了新的见解 整个催化和揭示了产品状态中持续的cas9-核酸相互作用。指导 通过这种新见解，我们将扩展这些研究，以定义有助于 CAS9催化和单位动力学。使用创新的体外动力学测定并建立结构 分析，我们将研究控制CAS9削减速率并释放速率的结构特征 靶向DNA。使用合理设计与随机诱变结合使用，我们将设计多变态 能够释放其切割的DNA产物的酶。使用体外基因组编辑测定法 和一个体内半乳糖苷酶测定法，我们将筛选Cas9突变体以增加DNA裂解速率。我们 预期所提出的研究不仅会提高我们对Cas9反应周期的分子理解 但也支持开发更有效的CRISPR/CAS9技术。