CAREER: Adaptive and regulatory diversity in natural CRISPR-Cas systems

CRISPR-Cas systems are anti-viral immune systems widespread in microbial genomes. Bacteria are “immunized” during viral infection when a fragment of invader DNA (called a spacer) is captured and used as a sequence-specific immunological memory to recognize and destroy matching sequences. This project will investigate the mechanisms by which diverse CRISPR systems acquire immunological memory, and to discover factors that bacteria use to control this process. The programmable sequence-specific nucleic acid recognition and biochemical activities of CRISPR-Cas nucleases have been exploited for a wide range of biotechnologies. Understanding how these systems are programmed by their natural hosts will better enable researchers to generate diverse CRISPR libraries in vivo, and will benefit CRISPR-based technologies aimed at molecular recording of cellular histories. This education plan of this project is comprised of a course-based research experience in which undergraduate participants will systematically measure interference by diverse CRISPR types targeting different viruses. This course will give students hands-on experience with the concepts of molecular biology and viral infection, and also represents a defined framework for students to rapidly generate and publish novel data. The goal of this research is to understand how diverse CRISPR types acquire new spacers from foreign genetic elements. Listeria seeligeri and its phages is a tractable model for studying four distinct CRISPR types, including the RNA-targeting CRISPR-Cas13 system. This model bacterium has been extremely useful for investigating the mechanisms and consequences of Cas13-mediated interference during type VI CRISPR immunity. However, the processes underlying the initial spacer acquisition events that establish immunity remain unclear. This proposal will employ next generation sequencing to detect rare acquisition events with high sensitivity. CRISPR-associated genes will be systematically mutagenized to assess their contributions to immunization. The CRISPR-Cas13 system uniquely targets RNA rather than DNA, and the acquired spacers will be examined to understand whether and how this system specifies the capture of transcribed target sequences. Finally, spacer acquisition will be biochemically reconstituted to investigate the mechanistic basis of spacer integration site selection. 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.