PROJECT SUMMARY/ABSTRACT Our research group studies human NAFLD using patient-derived induced pluripotent stem cells (iPSCs) for in vitro disease modeling. We recently showed that iPSCs from a cohort of NAFLD patients, when differentiated to hepatocytes (iPSC-Heps), display a spontaneous disease signature in cell culture. This underscores the importance of genetic background to NAFLD disease modeling and offers a unique opportunity to study the impact of NAFLD risk genes on disease phenotype. We theorize that the disease phenotype in NAFLD iPSC- Heps is due in part to established genetic risk factors identified through GWAS and in part to others that are either poorly characterized or unknown. To address the impact of established and emerging genetic risk factors on the NAFLD phenotype in iPSC-derived liver cells, we will leverage our unparalleled collection of 61 disease-specific iPSC lines (41 NAFLD, 19 control) and our ability to differentiate iPSCs along multiple liver cell lineages to create mono- and co-cultures. In the course of three aims we will systematically study these cells and catalogue the resulting resources and information for dissemination to the hepatology community. In Aim 1 we will develop a scorecard comprising the results of 15 transcriptomic, proteomic and functional assays for all 61 iPSC lines. The data will be used to develop individual and aggregate measures distinguishing normal from diseased cellular phenotypes and correlate phenotypic profiles with individual and polygenic risk factors. This aim will generate a large body of multi-omic data in the NAFLD iPSC model system that will be used as the foundation for subsequent gene editing. Aim 2 will constitute a systematic effort to correct 113 variant genes in 33 NAFLD iPSC lines and repeat the full scorecard analysis after each edit. Comparisons will be made between scorecards from individual gene-edited vs. parent lines, as well as in groups of iPSCs with similar edits. Many iPSC lines will be subjected to sequential gene corrections and may revert to normal; iPSC lines whose scorecard does not normalize will be scrutinized for the presence of novel variants with a plausible disease association, followed by direct testing with further gene correction. Aim 3 will employ a complementary but independent strategy involving whole-genome CRISPR screening in a NAFLD iPSC line to identify genes whose inhibition suppresses a NAFLD signature. This aim will make use of fluorescent reporter iPSC lines and high-content imaging to assess NAFLD-related outcomes. The CRISPR screen will enable us to discover novel genes that have a direct impact on cellular phenotype and may be suitable for translation to the clinic. This RC2 project will yield several deliverables: (a) rich, multi-omic datasets from a large cohort of parent iPSC lines and isogenic gene-edited derivatives following multicellular differentiation and NAFLD modeling; (b) > 100 iPSC lines from which the data were generat...