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