CAREER: Mechanistic Investigations of Conformational Activation and Catalysis in Emerging CRISPR-Cas Systems

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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).With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Giulia Palermo from the University of California, Riverside, to investigate the molecular mechanisms underlying the catalytic activities of newly emerging CRISPR (clustered regularly interspaced short palindromic repeat)-Cas systems. Inspired by the initial CRISPR-Cas9 gene editing tool, several novel CRISPR-Cas systems have recently been developed that precisely manipulate DNA and RNA in a cell. State-of-the-art of computational methods will be used to determine how these new systems change the chemical composition of DNA bases, cut both strands of the DNA double-helix, and target RNA. The results from this study are expected to aid in the development of novel CRISPR-Cas-based biotechnologies, such as nucleic acid detection and imaging. In addition, the applications of methods development aspects of this project have the potential to extend from the biosciences to fuel production and to the development of drought-resistant crops. This project will integrate with an educational plan that mentors a large cohort of pre-college and undergraduate students, including women and those from underrepresented minority groups. Students will receive their first hands-on experience in program coding and computation, building the technical skills to succeed in the 21st century technological workplace. CRISPR-Cas is an integral part of a bacterial adaptive immune response that has revolutionized life sciences as a genome editing tool. This project will employ advanced computational methods to characterize the mechanisms of conformational activation and catalysis in novel CRISPR-Cas systems, including Cas12a and Cas13a and emerging Cas9-based complexes. Long timescale molecular dynamics (MD) simulations will be combined with a variety of enhanced sampling techniques and free-energy methods to thoroughly investigate the conformational changes of the Cas proteins that prompt nucleic acid cleavages. Mixed quantum mechanics/molecular mechanics (QM/MM) approaches and ab-initio MD will be used to elucidate details of the chemical mechanisms driving the catalysis of nucleic acids, with the potential to enhance catalytic efficiency and guide enzyme design. The computational studies will be coordinated in collaboration with experimentalists to assist with the interpretation of experimental data and enable testable predictions. Overall, this study will help elucidate the biomolecular function of these emergent CRISPR-Cas systems, bridging the gap between understanding molecular mechanisms and engineering applications.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.