Combinatorial genetics in liver repopulation and carcinogenesis with a in vivo CRISPR activation platform.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 activation (CRISPRa) systems have enabled genetic screens in cultured cell lines to discover and characterize drivers and inhibitors of cancer cell growth. We adapted this system for use in vivo to assess whether modulating endogenous gene expression levels can result in functional outcomes in the native environment of the liver. We engineered the catalytically dead CRISPR-associated 9 (dCas9)-positive mouse, cyclization recombination-inducible (Cre) CRISPRa system for cell type-specific gene activation in vivo. We tested the capacity for genetic screening in live animals by applying CRISPRa in a clinically relevant model of liver injury and repopulation. We targeted promoters of interest in regenerating hepatocytes using multiple single guide RNAs (gRNAs), and employed high-throughput sequencing to assess enrichment of gRNA sequences during liver repopulation and to link specific gRNAs to the initiation of carcinogenesis. All components of the CRISPRa system were expressed in a cell type-specific manner and activated endogenous gene expression in vivo. Multiple gRNA cassettes targeting a proto-oncogene were significantly enriched following liver repopulation, indicative of enhanced division of cells expressing the proto-oncogene. Furthermore, hepatocellular carcinomas developed containing gRNAs that activated this oncogene, indicative of cancer initiation events. Also, we employed our system for combinatorial cancer genetics in vivo as we found that while clonal hepatocellular carcinomas were dependent on the presence of the oncogene-inducing gRNAs, they were depleted for multiple gRNAs activating tumor suppressors. CONCLUSION: The in vivo CRISPRa platform developed here allows for parallel and combinatorial genetic screens in live animals; this approach enables screening for drivers and suppressors of cell replication and tumor initiation. (Hepatology 2017).

Genetic lineage tracing analysis of the cell of origin of hepatotoxin-induced liver tumors in mice.

UNLABELLED: The expression of biliary/progenitor markers by hepatocellular carcinoma (HCC) is often associated with poor prognosis and stem cell-like behaviors of tumor cells. Hepatocellular adenomas (HCAs) also often express biliary/progenitor markers and frequently act as precursor lesions for HCC. However, the cell of origin of HCA and HCC that expresses these markers remains unclear. Therefore, to evaluate if mature hepatocytes give rise to HCA and HCC tumors and to understand the molecular pathways involved in tumorigenesis, we lineage-labeled hepatocytes by injecting adeno-associated virus containing thyroxine-binding globulin promoter-driven causes recombination (AAV-TBG-Cre) into Rosa(YFP) mice. Yellow fluorescent protein (YFP) was present in >96% of hepatocytes before exposure to carcinogens. We treated AAV-TBG-Cre; Rosa(YFP) mice with diethylnitrosamine (DEN), followed by multiple injections of carbon tetrachloride to induce carcinogenesis and fibrosis and found that HCA and HCC nodules were YFP(+) lineage-labeled; positive for osteopontin, SRY (sex determining region Y)-box 9, and epithelial cell adhesion molecule; and enriched for transcripts of biliary/progenitor markers such as prominin 1, Cd44, and delta-like 1 homolog. Next, we performed the converse experiment and lineage-labeled forkhead box protein L1(Foxl1)-positive hepatic progenitor cells simultaneously with exposure to carcinogens. None of the tumor nodules expressed YFP, indicating that Foxl1-expressing cells are not the origin for hepatotoxin-induced liver tumors. We confirmed that HCA and HCC cells are derived from mature hepatocytes and not from Foxl1-Cre-marked cells in a second model of toxin-induced hepatic neoplasia, using DEN and 3,3',5,5'-tetrachloro-1,4-bis(pyridyloxy)benzene (TCPOBOP). CONCLUSION: Hepatocytes are the cell of origin of HCA and HCC in DEN/carbon tetrachloride and DEN/TCPOBOP induced liver tumors. (Hepatology 2016;64:1163-1177).