PROJECT SUMMARY We previously developed a suite of tools for modulating and studying RNA based on the RNA-targeting CRISPR- Cas13 system, which has been adopted or extended by many researchers in the life sciences. This proposal seeks to discover and characterize additional programmable RNA binding proteins and develop them for use as molecular technologies. In particular, we are focusing on identifying ultra-small Cas13 proteins, which can be fused to RNA editing effectors to create compact platforms for precision, single-base transcript editing. RNA editing has significant therapeutic potential across a spectrum of conditions, including genetic diseases where it is not possible or too risky to edit the genome as well as acute insults where transient genetic changes are desirable. Thus, another aim is to demonstrate the feasibility of using RNA editing therapeutically. Beyond RNA editors, we will also use the new proteins we identify to develop transcriptional state sensors, which can be used to mark specific cell sub-types within a heterogenous population, either for imaging, isolation, or functional outcomes. To achieve these goals, we will leverage our previous experience to discover and characterize new RNA targeting CRISPR systems, with a focus on identifying small enzymes that support RNA editing activity. We will also explore the possibility of using novel RNA deaminase enzymes in our RNA editing constructs. In addition to creating RNA editing constructs, we will also fuse the RNA targeting enzymes to GFP or Cre to create transcriptional sensors. A critical aspect of our work will be protein engineering. Candidate enzymes (or their RNA components) may need to be modified for efficient, specific activity in mammalian cells. We will use protein engineering to increase the specificity and activity of RNA deaminases, as well as extend the substrate base preference of these enzymes. For our transcriptional state sensors, protein engineering will be central to successfully generating sensors that afford high specificity and high signal-to-noise ratios. All of these efforts will be guided by structural and biochemical studies of the relevant enzymes. Finally, we will apply the compact, high-specificity RNA editors in a mouse model of acute liver damage to demonstrate their therapeutic potential as short-lived, reversible treatments. In parallel, we will demonstrate the feasibility of using RNA editing to correct a mutation that causes the neurodevelopmental disorder Rett syndrome using a previously established mouse model of this disease. This work will substantially advance RNA editing toward clinical use, as well as uncover new biology about CRISPR systems and other microbial defense systems.