Journal Article

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

Vassiliou, George S., Saur, Dieter, Friedrich, Mathias, Schmid, Roland M., Schneider, Günter, Geumann, Ulf, Engleitner, Thomas, Yang, Fengtang, Fu, Beiyuan, Lange, Sebastian, Braren, Rickmer, Esposito, Irene, Bradley, Allan, Strong, Alexander, Steiger, Katja, Heikenwälder, Mathias, Ehmer, Ursula, Yuan, Detian, Unger, Kristian, Öllinger, Rupert, Barenboim, Maxim, Mueller, Sebastian, Rad, Roland, Weber, Julia, Liu, Pentao, Gross, Nina, Varela, Ignacio, Heid, Irina, Maresch, Roman, de la Rosa, Jorge, Segler, Angela, Cadiñanos, Juan

2015

Overview

Here, we show CRISPR/Cas9-based targeted somatic multiplexmutagenesis 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 asPtenorCdkn2a,and conversely found low frequency ofBrca1/2alterations, 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.

• Publisher: National Academy of Sciences
• Subject: Animals / Base Sequence / Biological Sciences / Cancer / Carcinoma, Hepatocellular - genetics / Chromatin / CRISPR-Cas Systems - genetics / Disease Models, Animal / Gene Targeting / Genes / Genetic screening / Genetics / Genomics / Genomics - methods / High-Throughput Screening Assays / Histological Techniques / Liver - metabolism / Liver Neoplasms - genetics / Mice / Molecular Sequence Data / Mutagenesis / Mutagenesis - genetics / Rodents / Selection, Genetic - genetics