Microbes employ diverse defense systems to provide protection from viruses, and studying these systems is important for understanding immune system evolution, phage therapy, and the discovery of new molecular tools such as CRISPR-Cas systems. While it’s broadly thought that CRISPR-Cas systems work exclusively by degrading nucleic acids, we’ve recently shown that membrane perturbation by accessory proteins (e.g., Csx28) are an unappreciated aspect of CRISPR-Cas’s mechanism of action. However, the molecular mechanisms of how proteins like Csx28 function are unknown. The goal of my research is to understand how CRISPR-Cas systems and their membrane-associated proteins function together to enhance anti-viral defense with an eye towards the discovery of new tools. This proposal focuses on addressing the following gaps: 1) What is the mechanistic and structural details of Csx28 activation by Cas13? We recently discovered that the RNA-targeting Cas enzyme Cas13 is communicating with membrane pore protein Csx28, to mount a robust anti-viral immune response, however we still don’t understand what the precise molecular mechanism involved. Our preliminary data indicate that Cas13 generated RNA cleavage products are acting as ligands to gate Csx28 channel activity. We will use RNA sequencing and membrane biophysics approaches to determine the precise RNA ligands modulating Csx28 activity and how these ligands affect gating of the Csx28 pore, respectively. These experiments will shed light on the molecular details of fascinating anti-phage synergy between Cas13 and Csx28 and provide a clear path for technology development. 2) Do Csx28-like proteins have roles in anti-viral defense outside of CRISPR systems? With our structural studies of Csx28 coupled with the recent advances in structure prediction databases, we discovered that Csx28 shares very close structural similarity to a family of hypothetical proteins that reside in potentially new immune systems. We hypothesize that these Csx28-like proteins are employed to modulate membrane function during viral infection, however no experimental studies have been done. We will use similar genetic, biochemical, and structural approaches to our previous work on Csx28 to study these putative membrane proteins. 3) How does Cas13 associated protein Csx27 modulate antiphage defense? Csx27 is a predicted transmembrane CRISPR accessory gene that resides within a subset of Cas13b containing loci. Csx27 has recently been shown to work with Cas13b from to modulate anti-phage defense, however the mechanism is not understood. Our preliminary data suggest that Csx27, while bearing no similarity to Csx28, also acts as a membrane pore and activation by Cas13b results in a similar loss of membrane potential, and a muted metabolism, however more work needs to be done to understand the details these of effects. We will use a similar set of genetic, biochemical, and structural approaches that were successful previously for Csx28 with...