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Nature DOI:10.1038/nature13589

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

Publication TypeJournal Article
Year of Publication2014
AuthorsXue, W, Chen, S, Yin, H, Tammela, T, Papagiannakopoulos, T, Joshi, NS, Cai, W, Yang, G, Bronson, R, Crowley, DG, Zhang, F, Anderson, DG, Sharp, PA, Jacks, T
JournalNature
Volume514
Issue7522
Pages380-4
Date Published2014 Oct 16
ISSN1476-4687
KeywordsAnimals, Base Sequence, beta Catenin, Cell Transformation, Neoplastic, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems, Female, Genes, p53, Genes, Tumor Suppressor, Genetic Engineering, Hepatocytes, Lipid Metabolism, Liver, Liver Neoplasms, Mice, Molecular Sequence Data, Mutagenesis, Mutation, Oncogenes, Phosphorylation, Proto-Oncogene Proteins c-akt, PTEN Phosphohydrolase
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.

URLhttp://dx.doi.org/10.1038/nature13589
DOI10.1038/nature13589
Pubmed

http://www.ncbi.nlm.nih.gov/pubmed/25119044?dopt=Abstract

Alternate JournalNature
PubMed ID25119044
PubMed Central IDPMC4199937
Grant ListR01 EB000244 / EB / NIBIB NIH HHS / United States
R01-CA133404 / CA / NCI NIH HHS / United States
U54 CA151884 / CA / NCI NIH HHS / United States
2-P01-CA42063 / CA / NCI NIH HHS / United States
R01 CA132091 / CA / NCI NIH HHS / United States
P01 CA042063 / CA / NCI NIH HHS / United States
P30 CA014051 / CA / NCI NIH HHS / United States
P30-CA14051 / CA / NCI NIH HHS / United States
R01-CA115527 / CA / NCI NIH HHS / United States
5-U54-CA151884-04 / CA / NCI NIH HHS / United States
R01 CA115527 / CA / NCI NIH HHS / United States
R01 CA133404 / CA / NCI NIH HHS / United States
DP1 MH100706 / MH / NIMH NIH HHS / United States
K99 CA169512 / CA / NCI NIH HHS / United States
R00 CA169512 / CA / NCI NIH HHS / United States
/ / Howard Hughes Medical Institute / United States
R01 DK097768 / DK / NIDDK NIH HHS / United States
1K99CA169512 / CA / NCI NIH HHS / United States
R01-CA132091 / CA / NCI NIH HHS / United States
R01-EB000244 / EB / NIBIB NIH HHS / United States