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，有可能影响实验室科学，生物工程和精准医学。该技术核心的大型多结构域Cas9蛋白由一种复杂的信号传导机制控制，该机制指导DNA的靶向切割。然而，Cas9机制的许多基本细节在分子水平上知之甚少，阻碍了利用其尖端功能潜力的直观化学工具的开发。该项目将在原子水平上研究Cas9，以确定这种蛋白质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。这种见解将导致开发具有增强的空间和时间特异性的CRISPR-Cas9蛋白。这个项目将创造互动学习经验，教蛋白质结构功能的关系，研究生，本科生，主要是代表性不足的普罗维登斯高中学生。 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）光谱，分子模拟，体外和体内生物化学，以进一步了解Cas9中的分子串扰，该分子串扰被提议驱动复杂的变构机制。建立这种机制的努力对于开发增强的Cas9变体是有吸引力的，因为变构调节提供了对蛋白质功能的上级空间和时间控制，这两者目前都阻碍了Cas9的应用。该项目将通过NMR自旋弛豫和计算网络分析探测的差分运动分析来剖析变构途径，以绘制负责传输促进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内在蛋白质动力学对变构的贡献，以合理化促进多结构域蛋白质结构通讯的氨基酸“途径”的作用。具体而言，该项目将阐明变构信号传导耦合远端Cas9结构域的途径，确定变构网络如何在配体结合后重新布线，表征活性或特异性改变突变对变构信号传导的影响，并建立嗜温和嗜热Cas9物种之间的生物物理比较。还提出了对MIF和Cas9变构网络进行残差水平去卷积的进一步途径，并需要更直观地将空间或时间控制细化和设计到这些系统中。该项目由分子和细胞生物科学部的分子生物物理学小组资助。该奖项反映了NSF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.