清脆的
PTEN公司
生物
Cas9
基因
基因靶向
突变
点突变
癌症研究
遗传学
分子生物学
PI3K/AKT/mTOR通路
信号转导
作者
Wen Xue,Sidi Chen,Hao Yin,Tuomas Tammela,Thales Papagiannakopoulos,Nikhil S. Joshi,Wenxin Cai,Gillian R. Yang,Roderick T. Bronson,Denise Crowley,Feng Zhang,Daniel G. Anderson,Phillip A. Sharp,Tyler Jacks
出处
期刊:Nature
[Springer Nature]
日期:2014-08-05
卷期号:514 (7522): 380-384
被引量:709
摘要
CRISPR plasmids targeting Pten and p53, alone and in combination, are delivered by hydrodynamic injection to the liver; the CRISPR-mediated mutations phenocopy the effects of deletions using Cre–LoxP technology, allowing the direct mutation of tumour suppressor genes and oncogenes in the liver using the CRISPR/Cas system, which presents a new approach for rapid development of liver cancer models and functional genomics. Genome editing tools exploiting the prokaryotic CRISPR/Cas immune system have been successfully applied in many organisms, including mouse and human cells. Here Tyler Jacks and colleagues demonstrate the feasibility of using the CRISPR/Cas system in vivo to induce direct mutation of tumour-suppressor genes and oncogenes in mouse liver. Cancer genes are traditionally studied using genetically engineered mouse models through embryonic stem cell targeting; this work highlights the power of the CRISPR/Cas9 system for rapid genome editing and the development of novel cancer models, as well as for functional genomics studies. 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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