CRISPR-Cas13 immunity in Listeria seeligeri

ABSTRACT CRISPR systems are prokaryotic adaptive immune systems that use RNA-guided Cas nucleases to recognize and destroy bacteriophage nucleic acids containing sequence complementarity to the guide RNA. CRISPR systems harbored by different bacteria can be extremely diverse and use different strategies to neutralize infecting phages. CRISPR systems are not well-represented in traditional model bacteria. As such, their function has typically been studied by heterologous overexpression in E. coli. Accordingly, we have limited knowledge of the complex interactions that arose from the co-evolution of CRISPR-Cas systems with their natural hosts and the phages that infect them. Research in my laboratory focuses on establishing natural models to investigate the interfaces between CRISPR-Cas immunity, bacterial host physiology, and phage infection. While most of the six CRISPR types use Cas DNases to recognize and cleave phage DNA, the type VI CRISPR system uses the nuclease Cas13 to cut RNA instead. I have developed a natural bacterial host of the type VI CRISPR system, Listeria seeligeri, and a collection of its phages as a tractable model for studying how this system protects against infection. Once Cas13 engages target viral RNA, it becomes activated as a non-specific RNase, resulting in widespread cleavage of both phage and bacterial RNA and the abortion of the phage lifecycle. Thus, infected cells with type VI immunity do not lyse, and fail to produce viral progeny, but stop growing and become dormant. The goals of this proposal are to (i) determine which transcripts cleaved by Cas13 trigger entry into dormancy; (ii) understand how L. seeligeri cells survive the dormant state, and resuscitate themselves once the phage has been eliminated, and (iii) discover and characterize endogenous regulatory mechanisms controlling Cas13 activity in L. seeligeri and its phages. The results generated by this research will provide fundamental insights into the molecular biology of RNA-targeting CRISPR systems and aid in their development as biotechnology tools. Finally, Cas13-induced cellular dormancy bears similarity to a phenomenon termed persistence, in which subpopulations of pathogenic bacteria stop growing and become transiently tolerant of bactericidal antibiotics during human infection. Therefore, the studies proposed here could reveal general mechanisms by which persistent bacteria survive antibiotic exposure and re-enter the growth cycle, which would represent attractive targets for therapeutic intervention.