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Proc Natl Acad Sci U S A. 2015 Nov 10;112(45):13982-7. doi: 10.1073/pnas.1512392112. Epub 2015 Oct 27.

CRISPR/Cas9 somatic multiplex-mutagenesis for high-throughput functional cancer genomics in mice.

Author information

1
Department of Medicine II, Klinikum Rechts der Isar, Technische Universität München, 81675 Munich, Germany; German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany;
2
Department of Medicine II, Klinikum Rechts der Isar, Technische Universität München, 81675 Munich, Germany;
3
The Wellcome Trust Sanger Institute, CB10 1SA Hinxton/Cambridge, United Kingdom;
4
Department of Pathology, Klinikum Rechts der Isar, Technische Universität München, 81675 Munich, Germany;
5
Institute of Radiology, Klinikum Rechts der Isar, Technische Universität München, 81675 Munich, Germany;
6
Institute of Virology, Technische Universität München and Helmholtz Zentrum München, 81675 Munich, Germany;
7
Institute of Pathology, Heinrich-Heine-University Düsseldorf, 40225 Düsseldorf, Germany;
8
Instituto de Medicina Oncológica y Molecular de Asturias, 33193 Oviedo, Spain;
9
Research Unit Radiation Cytogenetics, Helmholtz Zentrum München, 85764 Neuherberg, Germany;
10
Institute of Virology, Technische Universität München and Helmholtz Zentrum München, 81675 Munich, Germany; Division of Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany;
11
Instituto de Biomedicina y Biotecnología de Cantabria, 39011 Santander, Spain.
12
Department of Medicine II, Klinikum Rechts der Isar, Technische Universität München, 81675 Munich, Germany; German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany; roland.rad@tum.de.

Abstract

Here, we show CRISPR/Cas9-based targeted somatic multiplex-mutagenesis and its application for high-throughput analysis of gene function in mice. Using hepatic single guide RNA (sgRNA) delivery, we targeted large gene sets to induce hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). We observed Darwinian selection of target genes, which suppress tumorigenesis in the respective cellular/tissue context, such as Pten or Cdkn2a, and conversely found low frequency of Brca1/2 alterations, explaining mutational spectra in human ICC/HCC. Our studies show that multiplexed CRISPR/Cas9 can be used for recessive genetic screening or high-throughput cancer gene validation in mice. The analysis of CRISPR/Cas9-induced tumors provided support for a major role of chromatin modifiers in hepatobiliary tumorigenesis, including that of ARID family proteins, which have recently been reported to be mutated in ICC/HCC. We have also comprehensively characterized the frequency and size of chromosomal alterations induced by combinatorial sgRNA delivery and describe related limitations of CRISPR/Cas9 multiplexing, as well as opportunities for chromosome engineering in the context of hepatobiliary tumorigenesis. Our study describes novel approaches to model and study cancer in a high-throughput multiplexed format that will facilitate the functional annotation of cancer genomes.

KEYWORDS:

chromosome engineering; hepatocellular carcinoma; in vivo CRISPR/Cas9; intrahepatic cholangiocarcinoma; somatic multiplex-mutagenesis

PMID:
26508638
PMCID:
PMC4653208
DOI:
10.1073/pnas.1512392112
[Indexed for MEDLINE]
Free PMC Article

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