SUMMARY CRISPR-Cas loci provide bacteria with adaptive immunity against phages and plasmids. By remembering and destroying foreign genetic elements, CRISPR-Cas systems also restrict horizontal gene transfer, a frequent route for the dissemination of antibiotic resistance genes and toxins. To balance defense with genetic exchange, CRISPR-Cas systems likely undergo spatiotemporal regulation; however, little is known about the ways in which CRISPR-Cas loci interact with their bacterial hosts or changing environments. My laboratory studies type II CRISPR-Cas systems, which encode the gene-editing tool cas9. CRISPR-Cas9 systems are frequently found in human pathogens like S. pyogenes, and we are focused on understanding how CRISPR-Cas9 activities are intertwined with and often defined by the biology of their bacterial host cell. We recently discovered that S. pyogenes Cas9 performs a novel autoregulatory function. A non-canonical guide RNA repurposes Cas9 from a nuclease into a transcriptional repressor that silences its own promoter. This finding helps explain how CRISPR-Cas9 systems prevent autoimmunity against the bacterial chromosome, but it remains unclear how CRISPR-Cas expression can be induced as needed, for instance during or preceding a phage infection. In this proposal, we explore two new directions in CRISPR-Cas biology. First, we investigate the non-canonical regulatory roles of Cas9. Specifically, we characterize the conditions and mechanisms that allow Cas9 to transiently relieve its repression and induce CRISPR-Cas expression, and we ask whether Cas9 evolved to regulate other bacterial genes outside the CRISPR-Cas locus. These studies will establish a new foundation for understanding the role of Cas9 in the physiology of commensal and pathogenic bacteria. Furthermore, our studies on non-canonical guide RNAs will lead to new strategies for the development of controllable Cas9 technologies and therapies. In our second project, we establish an innovative imaging platform to observe live CRISPR-Cas immunization events for the first time. This project will illuminate a fundamental unanswered question in CRISPR-Cas biology: why are new memories successfully formed in only one in a million infected cells? The answers will provide new clues into the ways in which heterogeneity within bacterial populations enables survival. Furthermore, these insights and tools will be valuable for the development of phage therapies, which are offering promise in combatting the growing threat of antibiotic-resistant pathogens.