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

• DOI: 10.1002/hep.29626
• Publication Date: 2017-11-01T20:16:46Z
• Authors: Kirk J. Wangensteen; Yue J. Wang; Zhixun Dou; Amber W. Wang; Elham Mosleh‐Shirazi; Max A. Horlbeck; Luke A. Gilbert; Jonathan S. Weissman; Shelley L. Berger; Klaus H. Kaestner
• Keywords: Repopulation

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 inhibitors of cancer growth. We adapted this system for use vivo assess whether modulating endogenous gene expression levels can result functional outcomes the native environment liver. engineered catalytically dead CRISPR-associated (dCas9)-positive mouse, cyclization recombination-inducible (Cre) CRISPRa type-specific vivo. tested capacity screening live animals by applying a clinically relevant model liver injury repopulation. targeted promoters interest regenerating hepatocytes using multiple single guide RNAs (gRNAs), employed high-throughput sequencing enrichment gRNA sequences during repopulation link specific gRNAs initiation carcinogenesis. All components were expressed manner activated Multiple cassettes targeting proto-oncogene significantly enriched following repopulation, indicative enhanced division cells expressing proto-oncogene. Furthermore, hepatocellular carcinomas developed containing that oncogene, events. Also, we our combinatorial genetics as found while clonal dependent on presence oncogene-inducing gRNAs, they depleted activating tumor suppressors.