PROJECT SUMMARY/ABSTRACT Bacterial populations, including those that affect human health, are largely controlled by bacteriophages. Phages change the composition of bacterial ecosystems and strongly influence bacterial evolution. Defense mechanisms that protect bacteria from phages are important regulators of these host-pathogen interactions. Defining these mechanisms is crucial to understanding bacterial compositional dynamics and pathogenicity. CRISPR-Cas systems are sophisticated and diverse mechanisms that allow bacteria to memorize infection events and defend themselves upon reinfection. In addition to their important role in mediating bacteria- phage interactions, CRISPR-Cas systems have been harnessed for genome manipulation technologies that have greatly facilitated biomedical research and have enormous potential for human therapies. The goal of our research program is to fully define the mechanisms and specificities of a variety of nucleic acid-protein complexes that direct CRISPR-mediated immunity and have potential for CRISPR technology. Our program is divided between understanding the process of adaptation, during which a bacterial cell is immunized, and interference, during which the CRISPR-Cas system neutralizes an infection. We and others have recently discovered higher-order adaptation complexes containing poorly defined protein subunits that are essential for effective immunization. Our goal is to uncover the molecular steps that enable specificity and precision by higher-order adaptation complexes, ensuring productive immunization events. Following immunization, Cas effector complexes that neutralize infection during interference must quickly recognize pathogens, a task made even more challenging when phages evolve and evade detection. Our goal is to understand how Cas effectors can maintain effective immunity even in the face of pathogen evolution. Through this research program, we will contribute to the overall understanding of how CRISPR-Cas systems impact bacterial populations and help ensure that CRISPR-based research and therapeutic tools are used safely and effectively.