PROJECT SUMMARY/ABSTRACT Staphylococcus aureus is a major cause of both community-acquired and nosocomial infections, which have become increasingly challenging to treat due to the widespread evolution of antimicrobial resistance. There is a critical need for the development of alternative therapeutic approaches against multidrug-resistant bacteria that also spare the protective commensal microbiota, which often provide colonization resistance against pathogens. Bacterial disease is driven by S. aureus toxins and other virulence factors, which are mainly encoded by mobile genetic elements (MGEs). In particular, numerous enterotoxins and the superantigen toxin causing Toxic Shock Syndrome are all carried by a class of MGEs called the S. aureus Pathogenicity Islands (SaPIs), which spread between bacteria by hijacking the reproductive machinery of bacteriophages. Staphylococci also possess CRISPR-Cas systems, which provide adaptive immunity by blocking invading MGEs like phages and plasmids. In this proposal, building upon preliminary data, I will test the central hypothesis that CRISPR-Cas systems also prevent the transmission of SaPI elements and their associated virulence genes. In Aim 1, I will define the complex tripartite interplay between staphylococcal CRISPR systems, SaPIs, and their helper phages using various molecular and genetic approaches. In Aim 2, I will investigate the mechanisms by which SaPIs manage to overcome CRISPR-mediated restriction and disseminate throughout bacterial populations. I anticipate that these studies will elucidate both the molecular basis and biological consequences for CRISPR-SaPI interactions. In Aim 3, I will evaluate whether CRISPR can be used to selectively kill SaPI-harboring S. aureus and establish a proof-of-concept for CRISPR-based antimicrobials directed against virulence-encoding MGEs. The proposed experiments will contribute to the long-term goal of designing alternative therapeutic approaches in an effort to overcome the shortcomings of antibiotics in treating multidrug-resistant infections. This fellowship will support my training in the Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD-PhD Program, including my doctoral work in the laboratory of Dr. Luciano Marraffini at Rockefeller and the remainder of my medical training at Weill Cornell. The training plan outlined in this fellowship project is designed to optimally prepare me for a research career as an independent principal investigator and physician-scientist.