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

摘要 CRISPR/Cas9系统通过采用RNA-聚合酶链反应提供了一种极其通用的基因组编辑技术。 引导的核酸内切酶，Cas9。该系统最初分离自化脓性链球菌， 用于广泛的生物体，并且可以编程以操纵基因组中的几乎任何位点。 虽然Cas9易于重编程，但Cas9介导的编辑的效率在不同的基因组中变化很大。 基因组靶点。与其他常见的细菌核酸内切酶不同，Cas9表现出单周转动力学，其中它 形成稳定的产物复合物，并需要外力来释放切割的DNA。这种持久的产品 状态通过修复机制损害对双链断裂的访问，并有助于减少基因组编辑 效率尽管有丰富的Cas9结构，但产物复合物的结构仍然存在。 因为以前的研究是在防止DNA裂解的条件下进行的。因此，在本发明中， 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表现出单转换动力学。我们的实验室最近使用冷冻电镜来确定 催化活性的Cas9-sgRNA-dsDNA三元复合物，并捕获了三种不同的构象状态（前- 和后催化和产物状态）。这些结构为Cas9的协调提供了新的见解。 在整个催化过程中，Cas9与结构域的相互作用，并揭示了产物状态下持续的Cas9-核酸相互作用。指导 通过这一新的认识，我们将在这些研究的基础上进行扩展，以确定有助于 Cas9催化和单转换动力学。使用创新的体外动力学分析和建立的结构 通过分析，我们将研究控制Cas9切割和释放细胞的速率的结构特征。 目标DNA使用合理设计结合随机诱变，我们将工程师多周转 能够自发释放其切割的DNA产物的酶。使用体外基因组编辑分析 和体内β-半乳糖苷酶测定，我们将筛选Cas9突变体的DNA切割速率增加。我们 预计所提出的研究不仅将促进我们对Cas9反应周期的分子理解， 同时也支持更高效的CRISPR/Cas9技术的开发。

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.