CAREER: Molecular Resolution of Long-range Allostery in CRISPR-Cas9

该奖项全部或部分由《2021年美国救援计划法案》（公法117-2）资助。CRISPR-Cas9代表了一种变革性的生物分子工具，具有影响实验室科学、生物工程和精准医学的潜力。这项技术的核心是大的、多结构域的Cas9蛋白，它是由一种复杂的信号机制控制的，这种信号机制引导DNA的靶向切割。然而，在分子水平上对Cas9机制的许多潜在细节知之甚少，这阻碍了利用其尖端功能潜力的直观化学工具的发展。该项目将在原子水平上研究Cas9，以确定该蛋白如何在其复杂结构中传递化学信息以影响生物功能，这被认为是该系统的主要驱动力。这一发现将导致具有增强空间和时间特异性的CRISPR-Cas9蛋白的开发。该项目将创造互动式学习体验，向研究生、本科生和主要代表性不足的普罗维登斯高中学生教授蛋白质结构-功能关系。美国救援计划的资金用于支持这位早期职业调查员在他职业生涯的关键阶段。该项目将整合溶液核磁共振（NMR）波谱、分子模拟、体外和体内生物化学，以进一步了解Cas9分子串扰，该串扰被提出驱动复杂的变构机制。努力建立这一机制对于开发增强的Cas9变体具有吸引力，因为变构调节提供了对蛋白质功能的优越空间和时间控制，这两者目前都阻碍了Cas9的应用。该项目将通过核磁共振自旋松弛和计算网络分析探测的微分运动分析来剖析变构途径，以绘制负责传递促进DNA切割的结构或动态变化的特定氨基酸和相互作用。人们越来越多地研究蛋白质内在动力学对变构的贡献，以使氨基酸“途径”的作用合理化，这些途径促进了通过多结构域蛋白质结构的通信。具体来说，该项目将阐明变构信号耦合远端Cas9结构域的途径，确定变构网络如何在配体结合时重新连接，表征活性或特异性改变突变对变构信号的影响，并建立中温和嗜热Cas9物种之间的生物物理比较。提出了进一步的残差水平反卷积MIF和Cas9变构网络的途径，并需要更直观地改进和设计这些系统的空间或时间控制。该项目由分子和细胞生物科学部的分子生物物理集群资助。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

This award is funded in whole or in part under the American Rescue Plan Act of 2021 (Public Law 117-2).CRISPR-Cas9 represents a transformative biomolecular tool with potential to impact laboratory science, bioengineering, and precision medicine. The large, multidomain Cas9 protein at the heart of this technology is governed by an intricate signaling mechanism that guides the targeted cleavage of DNA. However, many of the underlying details of the Cas9 mechanism are poorly understood at the molecular level, hampering the development of intuitive chemical tools that leverage its cutting-edge functional potential. The project will study Cas9 at the atomic level to establish how this protein transmits chemical information throughout its complex structure to affect biological function, which has been proposed as a major driving force for this system. Such insight will lead to the development of CRISPR-Cas9 proteins with enhanced spatial and temporal specificity. This project will create interactive learning experiences to teach protein structure-function relationships to graduate, undergraduate, and predominantly underrepresented Providence High School students. American Rescue Plan funding is used to support this early career investigator at a critical stage in his career. The project will integrate solution nuclear magnetic resonance (NMR) spectroscopy, molecular simulations, in vitro and in vivo biochemistry to further our understanding of molecular crosstalk in Cas9 that is proposed to drive an intricate allosteric mechanism. Efforts to establish this mechanism are attractive for developing enhanced Cas9 variants, since allosteric regulation provides superior spatial and temporal control over protein function, both of which currently hamper Cas9 applications. The project will dissect allosteric pathways through the analysis of differential motions probed by NMR spin relaxation and computational network analysis to map the specific amino acids and interactions responsible for transmitting structural or dynamic changes that facilitate DNA cleavage. The contribution of intrinsic protein dynamics to allostery has been increasingly studied to rationalize the role of amino acid “pathways” that facilitate communication through multidomain protein structures. Specifically, the project will elucidate the pathway(s) of allosteric signaling coupling distant Cas9 domains, determine how allosteric networks rewire upon ligand binding, characterize the effects of activity or specificity-altering mutations on allosteric signaling, and establish biophysical comparisons between mesophilic and thermophilic Cas9 species. Further avenues toward residue-level deconvolution of the MIF and Cas9 allosteric networks are proposed and required to refine and engineer spatial or temporal control into these systems more intuitively. This project is funded by the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.