The aim of this study is to construct CRISPR/Cas9 vectors,which targets the key region of human ezrin enhancer,and as-says knockout function of the vectors.Two gRNAs,aiming at upstream and downstream of human ezrin enhancer key region respective -ly,were designed,synthesized and ligated into plasmid pX459 to construct pX459-sgRNA-EL and pX459-sgRNA-ER.Then,the cor-rect CRISPR/Cas9 recombinant plasmids were co-transfected into human esophageal carcinoma EC 109 cells.Genomic DNA was extrac-ted for PCR amplification of a region flanking the CRISPR target sites and subclonal sequencing analysis.By restriction enzyme diges-tion 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.%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.
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