科学家利用CRISPR/Cas9系统构建肝癌模型

报道：来自美国麻省理工大学的Tyler Jacks教授领导的团队创新开发了利用CRISPR/Cas9系统在小鼠肝细胞直接引入肿瘤基因突变来诱导肿瘤形成并构建肝癌小鼠模型的新方法。这篇文章刊登在最新一期《Nature》杂志上。

中文翻译

【题目】 CRISPR可以直接介导小鼠肝脏癌基因的突变

【译文】对小鼠模型中肿瘤基因的研究一直依赖于通过在胚胎干细胞中转基因或基因打靶产生的基因改造品系。这里我们介绍了一种使用CRISPR/Cas(clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins)系统在野生型小鼠体内构建癌症模型的新方法。我们尾静脉高压注射CRISPR质粒到小鼠体内表达Cas9和sgRNAs到肝脏中，它们结合后可以靶向肿瘤抑制基因 PTEN和P53（又称TP53和Trp53）。CRISPR介导的Pten突变致使肝细胞内Akt的磷酸化水平提高和脂囤积，这与使用Cre-LoxP技术敲除基因产生的表现型相同。同时靶向Pten和p53，诱发了肝脏肿瘤，也与通过Cre-LoxP介导的Pten和p53敲除引发的效果一致。肝脏和肿瘤组织的DNA测序结果表明抑癌基因发生了插入或缺失突变，包括肿瘤内Pten和p53的双等位基因突变。除此之外，把含有靶向β-catenin基因的sgRNA的Cas9质粒和一条载有激活点突变的单链DNA寡核苷酸供体一起注射，导致产生具有β-catenin核定位的肝细胞。这项研究阐述了用CRISPR/Cas系统直接突变抑癌基因和原癌基因的可行性，这代表了一种肝癌模型和功能基因组学快速发展的新途径。

英文原稿

[Title] CRISPR-mediated direct mutation of cancer genes in the mouse liver

[Author] Wen Xue, Sidi Chen, Hao Yin, Tuomas Tammela, Thales Papagiannakopoulos, Nikhil S. Joshi, Wenxin Cai, Gillian Yang, Roderick

                 Bronson, Denise G. Crowley, Feng Zhang, Daniel G. Anderson, Phillip A. Sharp, Tyler Jacks

[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 and p53(also known as TP53 and Trp53) , alone and in 

                  combination. CRISPR-mediated Ptenmutation 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 Ptenand 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.

原文地址: http://www.nature.com/nature/journal/vaop/ncurrent/full/nature13589.html