利用CRISPR/Cas9技术敲除HeLa细胞中YAF2基因

目的 利用CRISPR/Cas9技术建立YAF2（YY1 associated factor 2）基因敲除的宫颈癌HeLa细胞株，用于YAF2基因在宫颈癌细胞中分子功能的研究。方法 首先利用CRISPR/Cas9序列设计网站，设计两对靶向YAF2基因第2外显子不同位点的向导RNA（single-guide RNA，sgRNA）。化学合成sgRNA寡核苷酸序列，分别克隆至质粒pX335中，测序确认插入片段正确的克隆。先用一对重组载体转染HeLa细胞，7天后，再用另一对转染HeLa细胞，第2对转染3天后，取培养细胞的2/3提取基因组DNA，对敲除位点附近的片段进行PCR扩增后通过DNA测序对敲除效率进行分析，剩余细胞在96孔平板上通过有限稀释法筛选YAF2敲除的细胞株。用蛋白质免疫印迹实验初步鉴定YAF2蛋白质表达缺失的细胞克隆，提取其基因组DNA，对敲除位点附近的序列进行PCR扩增，将扩增产物克隆至pGEM-T easy载体，通过DNA测序确认YAF2基因纯合敲除的细胞株。结果 成功构建两对针对人YAF2基因第二外显子的pX335-sgRNA质粒；PCR产物测序证明两对重组载体均有YAF2基因敲除活性；得到2株YAF2敲除的HeLa细胞株。结论 利用CRISPR/Cas9技术在HeLa细胞中成功敲除YAF2基因，为在HeLa细胞中研究YAF2的分子功能奠定基础。

Establishment of YAF2 Gene Knockout HeLa Cell Line by CRISPR/Cas9

Objective The aim of this study was to establish the HeLa cell line with YAF2 gene knockout by CRISPR/Cas9 technique. Methods Firstly, two pairs of short guide RNA were designed to target two different sites of exon 2 of YAF2, and the sequences were chemically synthesized, and inserted into plasmid pX335. Next, the correct clones were confirmed by DNA sequencing. The first pair of pX335-sgRNA was transfected into HeLa cells, and seven days later, the second pair was transfected. Three days after the second transfection, genomic DNA from a total of 2/3 cultured cells were extracted and the DNA fragment encompassing knockout site was amplified by polymerase chain reaction (PCR) to detect the knockout efficiency. The remaining of the cells was cultured to isolate single cell clones by limited dilution method. The candidate clones were cultured and Western blot was carried out to initially screen YAF2 expression level. Genomic DNAs from YAF2-null clones were extracted to serve as the template to amplify the target fragments by PCR. Then, the products were cloned into pGEM-T easy vectors and YAF2 knockout were confirmed through DNA sequencing. Results Two pairs of recombinant plasmids differentially targeting two sites of exon 2 of YAF2 gene were successfully constructed and the knockout efficiency estimated by PCR was high; two stable YAF2 knockout cell lines were obtained. Conclusion The YAF2 gene was successfully knocked out in HeLa cells by CRISPR/cas9 technique, which laid a foundation for the study of the molecular function of YAF2 in HeLa cells.