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

ABSTRACT

CRISPR/Cas9 activation (CRISPRa) systems have enabled genetic screens in cultured cells 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 dCas9⁺ mouse, a Cre-inducible 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 cell division of cells expressing the proto-oncogene. Furthermore, hepatocellular carcinomas developed containing gRNAs that activated this oncogene, indicative of cancer initiation events. Furthermore, 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. This article is protected by copyright. All rights reserved.

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