Meng Fanrong, Zhao Dan, Zhou Qinghua, Liu Zhe
Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenviroment, Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin 300052, China.
Tianjin Medical University, Tianjin 300070, China.
Zhongguo Fei Ai Za Zhi. 2018 May 20;21(5):358-364. doi: 10.3779/j.issn.1009-3419.2018.05.02.
It has been proven that CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9) system was the modern gene-editing technology through the constitutive expression of nucleases Cas9 in the mammalian, which binds to the specific site in the genome mediated by single-guide RNA (sgRNA) at desired genomic loci. The aim of this study is that the animal model of EZH2 gene knockout was constructed using CRISPR/Cas9 technology.
In this study, we designed two single-guide RNAs targeting the Exon3 and Exon4 of EZH2 gene. Then, their gene-targeting efficiency were detected by SURVEYOR assay. The lentivirus was perfused into the lungs of mice by using a bronchial tube and detected by immunohistochemistry and qRT-PCR.
The experimental results of NIH-3T3 cells verify that the designed sgEZH2 can efficiently effect the cleavage of target DNA by Cas9 in vitro. The immunohistochemistry and qRT-PCR results showed that the EZH2 expression in experimental group was significantly decreased in the mouse lung tissue.
The study successfully designed two sgRNA which can play a knock-out EZH2 function. An EZH2 knockout animal model was successfully constructed by CRISPR/Cas9 system, and it will be an effective animal model for studying the functions and mechanisms of EZH2.
已证实CRISPR/Cas9（成簇规律间隔短回文重复序列/CRISPR相关蛋白9）系统是一种现代基因编辑技术，通过在哺乳动物中组成型表达核酸酶Cas9，其在单导向RNA（sgRNA）介导下与基因组中所需基因组位点的特定位点结合。本研究的目的是利用CRISPR/Cas9技术构建EZH2基因敲除动物模型。
在本研究中，我们设计了两个靶向EZH2基因外显子3和外显子4的单导向RNA。然后，通过SURVEYOR检测法检测它们的基因靶向效率。使用支气管将慢病毒灌注到小鼠肺中，并通过免疫组织化学和qRT-PCR进行检测。
NIH-3T3细胞的实验结果证实，设计的sgEZH2能够在体外有效介导Cas9对靶DNA的切割。免疫组织化学和qRT-PCR结果显示，实验组小鼠肺组织中EZH2表达显著降低。
本研究成功设计了两个具有敲除EZH2功能的sgRNA。通过CRISPR/Cas9系统成功构建了EZH2基因敲除动物模型，它将成为研究EZH2功能和机制的有效动物模型。
