1 tRNAs have been recently demonstrated to be processed by ribonucleases into smaller regulatory fragments 2 named tRNA-derived small RNAs (tDRs) with their own distinct function including mRNA stability and silencing, 3 stress granule formation and epigenetic regulation. The function of these tDRs are dependent on their 4 sequence conservation and base modification that in turn determine their structure and protein-binding ability. 5 We have previously detailed the dynamic regulation of tDRs in response to cellular stress and have in the 6 process identified Asp-GTC-3’tDR that plays a critical role in stress-dependent induction of autophagy, is highly 7 expressed in the kidney and appears to be reno-protective in vitro. In several murine in vivo models, cellular 8 levels of Asp-GTC-3’tDR initially increases in the acute phase of renal injury, and subsequently decreases in 9 chronic kidney disease models. Notably in vivo silencing of Asp-GTC-3’tDR in the acute phase of renal injury 10 accelerates cell death and disease progression suggesting a compensatory response. However, tools to 11 manipulate the expression of this and other tDRs in vivo that preserve their cellular modifications presents a 12 significant hurdle in the field. Our preliminary data suggest that the RNA-targeting CRISPR/Cas13, guided by 13 gRNAs, can induce programmable cleavage(s) on tRNAs and generate functional tDRs without disturbing the 14 parent tRNA pool. Notably, we built a functional and smaller Cas13/ANG chimeric protein by replacing two 15 HEPN domains of pspCas13b with Angiogenin (ANG), a small RNase responsible for tDR biogenesis that 16 permits packaging into viral gene delivery vectors. The long-term objective of this proposal is to is to develop a 17 programmable Cas13/ANG platform packaged in AAVs for delivery to kidneys for the biogenesis of 18 endogenous functional tDRs. To achieve our objectives, we plan to: 1) Test the hypothesis that the engineered 19 CAS13/ANG platform, customized with suitable gRNAs, generates endogenous functional tDRs. As a proof-of- 20 concept, we focus on three tDRs with clear readouts: stress granule-inducing Ala-AGC-5’tDRs and Cys-GCA- 21 5’tDRs, and autophagy-inducing Asp-GTC-3’tDRs. These will be tested in HEK cells for their ability to induce 22 robust tDR generation without disturbing the parent tRNA pools; assays checking for stress granule formation 23 and autophagy flux will be used to evaluate the functionality of Cas13/ANG-generated tDRs; and 2) Use AAV- 24 delivered Cas13/ANG machinery to generate functional Asp-GTC-3’tDR in kidneys and determine if this 25 attenuates CKD progression in our mouse CKD model. The functional effects of induction of Asp-GTC-3’tDR 26 will be assessed by northern blotting, histology, immunostaining, western blotting, and RNA-seq to determine 27 effects on autophagy and the progression of CKD. We expect this platform which we aim to make widely 28 available to the scientific community could be used in many different systems to both study the function of 29 tDRs in vivo, and also provide a possible therapeutic avenue for tDR-based therapeutics.

A new class of small RNAs called tRNA-derived small RNAs play an important role in regulating many disease processes. However, tools that allow for generating these RNAs in cells or animals are lacking. Here we develop new tools and techniques, to be shared with the scientific community, that facilitate generation of these RNAs in the kidney as a precursor to future RNA therapies to mitigate kidney disease.