CRISPR-mediated direct mutation of cancer genes in the mouse liver

Wen Xue, Sidi Chen, Hao Yin, Tuomas Tammela, Thales Papagiannakopoulos, Nikhil S. Joshi, Wenxin Cai, Gillian Yang, Roderick Bronson, Denise G. Crowley, Feng Zhang, Daniel G. Anderson, Phillip A. Sharp, Tyler Jacks

Research output: Contribution to journalArticlepeer-review

521 Scopus citations

Abstract

The study of cancer genes in mouse models has traditionally relied on genetically-engineered strains made via transgenesis or gene targeting in embryonic stem cells1. Here we describe a new method of cancermodel generation using the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system in vivo in wild-type mice. We used hydrodynamic injection to deliver a CRISPR plasmid DNA expressing Cas9 and single guide RNAs (sgRNAs)2-4 to the liver that directly target the tumour suppressor genesPten (ref. 5) and p53 (also known asTP53 and Trp53) (ref. 6), alone and in combination. CRISPR-mediated Pten mutation led to elevated Akt phosphorylation and lipid accumulation in hepatocytes, phenocopying the effects of deletion of the gene using Cre-LoxP technology7,8. Simultaneous targeting of Pten and p53 induced liver tumours that mimicked those caused by Cre-lox Pmediated deletion of Pten and p53. DNA sequencing of liver and tumour tissue revealed insertion or deletionmutationsof the tumour suppressor genes, including bi-allelicmutationsof bothPten andp53 in tumours. Furthermore, co-injection of Cas9 plasmids harbouring sgRNAs targeting the β-catenin gene and a single-stranded DNA oligonucleotide donor carrying activating point mutations led to the generation of hepatocytes with nuclear localization of β-catenin. This study demonstrates the feasibility of direct mutation oftumour suppressor genes and oncogenes in the liver using the CRISPR/Cas system, which presents a new avenue for rapid development of liver cancer models and functional genomics.

Original languageEnglish
Pages (from-to)380-384
Number of pages5
JournalNature
Volume514
Issue number7522
DOIs
StatePublished - 16 Oct 2014
Externally publishedYes

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