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 cancer model 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)2,3,4 to the liver that directly target the tumour suppressor genes Pten (ref. 5) and p53 (also known as TP53 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–loxP-mediated deletion of Pten and p53. DNA sequencing of liver and tumour tissue revealed insertion or deletion mutations of the tumour suppressor genes, including bi-allelic mutations of both Pten and p53 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 of tumour 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.
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Data generated during the work are deposited at NCBI BioProject under accession code PRJNA252101.
We thank D. McFadden, N. Dimitrova, E. Snyder, A. Farago, M. Muzumdar, F. Sanchez-Rivera, J. Doench, L. Cong and S. Levine for discussions and for sharing reagents. We thank the Koch Institute Swanson Biotechnology Center (SBC) for technical support, specifically the Hope Babette Tang (1983) Histology Facility and K. Cormier. This work was supported by grants 2-PO1-CA42063, RO1-EB000244, RO1-CA115527 and RO1-CA132091 from the National Institutes of Health and supported in part by Cancer Center Support (core) grant P30-CA14051 from the National Cancer Institute. This work was supported, in part, by NIH Grant R01-CA133404 and Casimir-Lambert Fund to P.A.S. H.Y. is supported by 5-U54-CA151884-04 NIH Centers for Cancer Nanotechnology Excellence and the Harvard-MIT Center of Cancer Nanotechnology Excellence. S.C. is a Damon Runyon Fellow (DRG-2117-12). W.X. was supported by fellowships from the American Association for Cancer Research and the Leukemia Lymphoma Society and is currently supported by grant 1K99CA169512. T.J. is a Howard Hughes Medical Institute (HHMI) Investigator, the David H. Koch Professor of Biology, and a Daniel K. Ludwig Scholar.
Extended data figures
This file shows deep sequencing data for Pten and p53 indels in the liver.
This file shows deep sequencing data for Pten and p53 indels in 3T3 cells.
This file shows deep sequencing data for sgPten off-target analysis.
This file shows sequences of Pten and p53 alleles in sgPten+sgp53 liver tumors.
This file shows deep sequencing data for β-Catenin indels in the liver.
About this article
Nature Reviews Clinical Oncology (2019)