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

摘要 CRISPR/Cas9系统通过使用RNA- 引导型内切酶，Cas9。该系统最初是从化脓性链球菌中分离出来的，现已改用 用于广泛的生物体中，并且可以被编程为操纵基因组中的几乎任何位置。 虽然Cas9很容易重新编程，但Cas9中介的编辑效率在不同的 基因组靶标。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体，需要外力才能释放切割的DNA。这款经久不衰的产品 国家削弱了通过修复机制获得双链断裂的途径，并导致基因组编辑减少 效率。尽管有丰富的Cas9结构，但产物复合体的结构仍然 与之前的研究不同，这些研究是在防止DNA切割的条件下进行的。结果, 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表现为单周转动力学。我们实验室最近使用低温电子显微镜确定了 具有催化活性的Cas9-sgRNA-dsDNA三元络合物，捕获了三种不同的构象状态(前 和后催化，以及产物状态)。这些结构为CAS9的协调提供了新的见解 结构域贯穿整个催化过程，并揭示了产物状态下持续的Cas9-核酸相互作用。引导式 通过这种新的见解，我们将在这些研究的基础上扩展，以确定有助于 Cas9催化和单周转动力学。使用创新的体外动力学分析和已建立的结构 分析，我们将研究控制Cas9削减和释放 靶向DNA。使用合理设计和随机突变相结合的方法，我们将设计一个多周转 能够自发释放其裂解的DNA产物的酶。使用体外基因组编辑试验 以及体内-半乳糖苷酶检测，我们将筛选Cas9突变株，以获得更高的DNA裂解率。我们 预计拟议的研究不仅将促进我们对Cas9反应周期的分子理解， 而且还支持开发更高效的CRISPR/CAS9技术。

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.