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

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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.