CRISPR/Cas9 somatic multiplex-mutagenesis for high-throughput functional cancer genomics in mice.
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.
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;
Julia
J
Weber
Department of Medicine II, Klinikum Rechts der Isar, Technische Universität München, 81675 Munich, Germany;
Rupert
R
Öllinger
The Wellcome Trust Sanger Institute, CB10 1SA Hinxton/Cambridge, United Kingdom;
Mathias
M
Friedrich
Department of Medicine II, Klinikum Rechts der Isar, Technische Universität München, 81675 Munich, Germany;
Ursula
U
Ehmer
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;
Maxim
M
Barenboim
Department of Pathology, Klinikum Rechts der Isar, Technische Universität München, 81675 Munich, Germany;
Katja
K
Steiger
Institute of Radiology, Klinikum Rechts der Isar, Technische Universität München, 81675 Munich, Germany;
Irina
I
Heid
Department of Medicine II, Klinikum Rechts der Isar, Technische Universität München, 81675 Munich, Germany;
Sebastian
S
Mueller
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;
Roman
R
Maresch
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;
Thomas
T
Engleitner
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;
Nina
N
Gross
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;
Ulf
U
Geumann
The Wellcome Trust Sanger Institute, CB10 1SA Hinxton/Cambridge, United Kingdom;
Beiyuan
B
Fu
Department of Pathology, Klinikum Rechts der Isar, Technische Universität München, 81675 Munich, Germany;
Angela
A
Segler
Institute of Virology, Technische Universität München and Helmholtz Zentrum München, 81675 Munich, Germany;
Detian
D
Yuan
Department of Medicine II, Klinikum Rechts der Isar, Technische Universität München, 81675 Munich, Germany;
Sebastian
S
Lange
The Wellcome Trust Sanger Institute, CB10 1SA Hinxton/Cambridge, United Kingdom;
Alexander
A
Strong
The Wellcome Trust Sanger Institute, CB10 1SA Hinxton/Cambridge, United Kingdom;
Jorge
J
de la Rosa
Institute of Pathology, Heinrich-Heine-University Düsseldorf, 40225 Düsseldorf, Germany;
Irene
I
Esposito
The Wellcome Trust Sanger Institute, CB10 1SA Hinxton/Cambridge, United Kingdom;
Pentao
P
Liu
Instituto de Medicina Oncológica y Molecular de Asturias, 33193 Oviedo, Spain;
Juan
J
Cadiñanos
The Wellcome Trust Sanger Institute, CB10 1SA Hinxton/Cambridge, United Kingdom;
George S
GS
Vassiliou
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 M
RM
Schmid
Department of Medicine II, Klinikum Rechts der Isar, Technische Universität München, 81675 Munich, Germany;
Günter
G
Schneider
Research Unit Radiation Cytogenetics, Helmholtz Zentrum München, 85764 Neuherberg, Germany;
Kristian
K
Unger
The Wellcome Trust Sanger Institute, CB10 1SA Hinxton/Cambridge, United Kingdom;
Fengtang
F
Yang
Institute of Radiology, Klinikum Rechts der Isar, Technische Universität München, 81675 Munich, Germany;
Rickmer
R
Braren
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;
Mathias
M
Heikenwälder
Instituto de Biomedicina y Biotecnología de Cantabria, 39011 Santander, Spain.
Ignacio
I
Varela
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;
Dieter
D
Saur
The Wellcome Trust Sanger Institute, CB10 1SA Hinxton/Cambridge, United Kingdom;
Allan
A
Bradley
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.
Roland
R
Rad
D000818:Animals; D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D006528:Carcinoma, Hepatocellular / Q000235:genetics*; D004195:Disease Models, Animal; D018390:Gene Targeting; D023281:Genomics / Q000379:methods*; D057166:High-Throughput Screening Assays; D006652:Histological Techniques; D008099:Liver / Q000378:metabolism; D008113:Liver Neoplasms / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics*; D012641:Selection, Genetic / Q000235:genetics
chromosome engineering; hepatocellular carcinoma; in vivo CRISPR/Cas9; intrahepatic cholangiocarcinoma; somatic multiplex-mutagenesis