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Nature. 2014 Oct 16;514(7522):380-4. doi: 10.1038/nature13589. Epub 2014 Aug 6.

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

Author information

1
1] David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA [2].
2
David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA.
3
Tufts University and Harvard Medical School, Boston, Massachusetts 02115, USA.
4
Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, Massachusetts 02142, USA.
5
1] David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA [2] Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA [3] Harvard-MIT Division of Health Sciences &Technology, Cambridge, Massachusetts 02139, USA [4] Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA.
6
1] David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA [2] Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA.
7
1] David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA [2] Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA [3] Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

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 cells. 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) 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 technology. 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.

PMID:
25119044
PMCID:
PMC4199937
DOI:
10.1038/nature13589
[Indexed for MEDLINE]
Free PMC Article

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