生物学杂志

生物学杂志

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靶向ezrin增强子关键区的CRISPR/Cas9载体的构建

  

  1. 遵义医学院珠海校区 生物工程系， 珠海 519041
  • 出版日期:2017-12-18 发布日期:2017-12-18
  • 通讯作者: 高书颖，博士，教授，研究方向基因表达调控机制，E-mail：shuyinggao@163.com
  • 作者简介:郭晓龙，专业方向为基因表达调控机制，E-mail: 1406049005@qq.com
  • 基金资助:
    国家自然科学基金（31360212）；贵州省科技厅联合基金（LKZ\[2013\]04）；贵州省科学技术基金（黔科合J字\[2014\]2180号）

Construction of CRISPR/Cas9 vectors targeting to ezrin enhancer key region

  1. Department of Bioengineering, Zhuhai Campus of Zunyi Medical University, Zhuhai 519041, China
  • Online:2017-12-18 Published:2017-12-18

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摘要： 构建靶向人ezrin增强子关键区的CRISPR/Cas9载体并检测其基因敲除功能。设计2个gRNA，分别靶向人ezrin增强子关键区的上、下游。合成gRNA寡核苷酸序列，连接至质粒pX459构建重组质粒pX459-sgRNA-EL和pX459-sgRNA-ER。将鉴定正确的CRISPR/Cas9重组质粒共转染至食管癌EC109细胞中，提取细胞基因组DNA，针对gRNA靶位点两侧进行PCR扩增和亚克隆测序分析。经限制性内切酶酶切和测序鉴定表明，CRISPR/Cas9重组质粒构建正确。在共转染重组质粒的细胞基因组DNA中检测到ezrin增强子关键区的缺失。成功构建了靶向人ezrin增强子关键区的CRISPR/Cas9载体，能够实现目标序列的定向敲除。

关键词: 靶向敲除, ezrin增强子, CRISPR/Cas9

Abstract: The aim of this study is to construct CRISPR/Cas9 vectors, which targets the key region of human ezrin enhancer, and assays knockout function of the vectors. Two gRNAs, aiming at upstream and downstream of human ezrin enhancer key region respectively, were designed, synthesized and ligated into plasmid pX459 to construct pX459-sgRNA-EL and pX459-sgRNA-ER. Then, the correct CRISPR/Cas9 recombinant plasmids were co-transfected into human esophageal carcinoma EC109 cells. Genomic DNA was extracted for PCR amplification of a region flanking the CRISPR target sites and subclonal sequencing analysis. By restriction enzyme digestion and DNA sequencing assay, it was confirmed that CRISPR/Cas9 recombinant plasmids were constructed successfully. Deletion of the ezrin enhancer key region was found in co-transfection cells genomic DNA. Therefore, CRISPR/Cas9 vectors targeting human gene enhancer key region were constructed and could create fixed sequence knockout.

Key words: target knockout, ezrin enhancer, CRISPR/Cas9