Project Summary Treatments to improve liver regeneration could help patients survive otherwise fatal liver diseases. We have previously identified important regulators of liver regeneration by conducting a screen of 43 candidate genes for effect on liver repopulation after injury in the mouse model of hereditary tyrosinemia, the Fah-/- mouse. We found that overexpression of TNFR1 led to a block in liver repopulation and activated the cell cycle regulator p21. This is congruent with clinical observations that elevations in TNF correlate with worse outcomes from alcoholic liver diseases and cirrhosis. Unfortunately, blocking TNF directly leads to systemic immunosuppression and life-threating infections. Ideally, treatment targeted to TNF signaling within hepatocytes could be targeted to improve outcomes in liver injuries without affecting immune cells. We hypothesize that TNFR1 effector genes within hepatocytes regulate liver injury and repopulation, and that targeting these factors will improve outcomes in liver diseases. In order to uncover the key intracellular targets that lie downstream of TNFR1, and to expand our genetic screening capabilities, we have now adapted the CRISPR-Cas9 system for a high content genetic screening in vivo. We have designed a CRISPR inhibition (CRISPRi) approach, which we have incorporated into the Fah-/- liver repopulation model. We previously derived a mouse Rosa26 gene knock-in allele with a nuclease-deficient dCas9 gene. We designed a plasmid library which expresses FAH together with linked guide RNAs (gRNAs) for CRISPRi, which blocks target gene transcription. We have already completed a pilot experiment using gRNAs targeted to Myc and p53 expression in vitro in cultured mouse embryonic fibroblasts, and in vivo in hepatocytes, and we found robust CRISPRi activity on growth and gene expression using these assays. This has provided ample evidence that CRISPRi can be used for screening. In this proposal, we aim to elucidate the key effectors of TNFR1 signaling in repopulating hepatocytes by performing a genetic rescue experiment. We will build a library of plasmids that will express FAH, TNFR1, and gRNAs targeting TNFR1-pathway genes. The gRNAs that block effectors of TNFR1 will `rescue' the hepatocytes, allowing them to proceed with liver repopulation. We will validate our findings with additional assays in Fah-/- mice and in other models of liver injury. Our experiments are designed to identify the mechanisms of TNFR1-associated blockage in hepatocyte repopulation in the setting of liver diseases. In addition, the results will form a basis for future proposals to develop therapies to target these genes. This R03 award will also help the PI and his nascent independent laboratory to generate publications and to obtain R01 grant funding.