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Hepatology. 2018 Aug;68(2):663-676. doi: 10.1002/hep.29626. Epub 2018 May 14.

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

Author information

1
Department of Medicine, Division of Gastroenterology, University of Pennsylvania, Philadelphia, PA.
2
Department of Genetics, University of Pennsylvania, Philadelphia, PA.
3
Epigenetics Institute, Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA.
4
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA.

Abstract

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).

PMID:
29091290
PMCID:
PMC5930141
[Available on 2019-08-01]
DOI:
10.1002/hep.29626

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