CRISPR-mediated direct mutation of cancer genes in the mouse liver.
CRISPR-mediated direct mutation of cancer genes in the mouse liver.
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DOI：
10.1038/nature13589
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发表时间：
2014-10-16
期刊：
影响因子：
64.8
通讯作者：
Jacks, Tyler
中科院分区：
文献类型：
--
作者：
Xue, Wen;Chen, Sidi;Yin, Hao;Tammela, Tuomas;Papagiannakopoulos, Thales;Joshi, Nikhil S.;Cai, Wenxin;Yang, Gillian;Bronson, Roderick;Crowley, Denise G.;Zhang, Feng;Anderson, Daniel G.;Sharp, Phillip A.;Jacks, Tyler
在小鼠模型中研究癌症基因传统上依赖于通过转基因或胚胎干细胞（ES）中的基因靶向产生的基因工程菌株。在这里，我们描述了一种在野生型小鼠体内使用CRISPR/Cas系统生成癌症模型的新方法。我们已经使用流体动力学注射将表达Cas9和单向导RNA（sgRNA）的CRISPR质粒DNA递送到肝脏，并直接靶向肿瘤抑制基因Pten和p53（单独和组合）。CRISPR介导的Pten突变导致肝细胞中Akt磷酸化和脂质积累升高，使用Cre-LoxP技术模拟基因缺失的影响。同时靶向Pten和p53诱导的肝脏肿瘤模拟了Cre-loXP介导的Pten和p53缺失引起的肝脏肿瘤。肝脏和肿瘤组织的DNA测序显示了肿瘤抑制基因的插入或缺失（indel）突变，包括肿瘤中Pten和p53的双等位基因突变。此外，共注射携带靶向β-连环蛋白基因（Ctnnbl）的sgRNA的Cas9质粒和携带激活点突变的单链DNA（ssDNA）寡核苷酸供体导致产生具有β-连环蛋白的核定位的肝细胞。本研究证明了利用CRISPR/Cas系统直接突变肝脏中抑癌基因和癌基因的可行性，为肝癌模型和功能基因组学的快速发展提供了新的途径。
The study of cancer genes in mouse models has traditionally relied on genetically-engineered strains made via transgenesis or gene targeting in embryonic stem (ES) cells. Here we describe a new method of cancer model generation using the CRISPR/Cas system in vivo in wild-type mice. We have used hydrodynamic injection to deliver a CRISPR plasmid DNA expressing Cas9 and single guide RNAs (sgRNAs) to the liver and directly target the tumor suppressor genes Pten and p53, 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 tumors that mimicked those caused by Cre-loxP-mediated deletion of Pten and p53. DNA sequencing of liver and tumor tissue revealed insertion or deletion (indel) mutations of the tumor suppressor genes, including bi-allelic mutations of both Pten and p53 in tumors. Furthermore, co-injection of Cas9 plasmids harboring sgRNAs targeting the β-Catenin gene (Ctnnb1) and a single-stranded DNA (ssDNA) 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 tumor 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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影响因子：
48
作者：
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通讯作者：
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