稳定表达Cas9蛋白的BHK-21单克隆细胞系的建立与鉴定

Establishment and Identification of BHK-21 Monoclona Cell Lines Stably Expressing Cas9 Protein

为建立基于CRISPR/Cas9技术的基因敲除细胞文库,本研究采用慢病毒转导构建稳定表达Cas9蛋白的BHK-21-Cas9单克隆细胞系。将pMD2.G、pSPAX2和lentiCas9-Blast共转染HEK-293T细胞包装慢病毒,感染BHK-21细胞后经Blasticidin S进行阳性筛选,筛选5代后通过有限稀释法培养Cas9单克隆细胞,利用Western blot检测Cas9蛋白的表达。为获得Cas9活性强的BHK-21-Cas9单克隆细胞系,根据紧密连接蛋白(OCLN)基因序列,设计针对OCLN基因的sgRNAs,构建lentiGuide-Puro-OCLN sgRNA重组质粒,测序鉴定后将lentiGuide-Puro-OCLN sgRNA重组载体转染BHK-21-Cas9单克隆细胞系。使用嘌呤霉素进行阳性细胞筛选,经过Western blot检测OCLN蛋白的表达。Western blot检测Cas9蛋白结果显示,共获得了17株BHK-21-Cas9单克隆细胞系;测序结果显示,lentiGuide-Puro-OCLN sgRNA载体构建成功,通过Western blot鉴定,有2株Cas9编辑效果好、具有高敲除效率的BHK-21单克隆细胞系。结果表明,本研究构建了稳定表达Cas9蛋白的BHK-21-Cas9单克隆细胞系,可以用于CRISPR/Cas9敲除文库的转导,建立基因敲除细胞文库,为筛选影响病毒感染和复制的功能基因提供了科学依据。

To establish a gene knockout cell library based on CRISPR/Cas9 technology, lentiviral transduction was used to construct a BHK-21-Cas9 monoclonal cell line stably expressing Cas9 protein. pMD2.G,pSPAX2 and lentiCas9-Blast were cotransfected into HEK-293 T cells to package lentivirus, and infected BHK-21 cells were positively selected by Blasticidin S, Cas9 monoclonal cells were cultured by limiting dilution after 5 passages and Cas9 protein expression was assessed by Western blot. To obtain a BHK-21-Cas9 monoclonal cell line with strong Cas9 activity, sgRNAs against OCLN were designed according to the tight junction protein(OCLN) gene sequence, the lentiGuide-Puro-OCLN sgRNA recombinant plasmid was constructed, and after sequencing identification, the lentiGuide-Puro-OCLN sgRNA recombinant vector was transfected into the BHK-21-Cas9 monoclonal cell line. Puromycin was used for positive cell screening, and the expression of OCLN protein was examined by Western blot. The results of Cas9 protein detection by Western blot showed that a total of 17 BHK-21-Cas9 monoclonal cell lines were obtained;sequencing results showed that the lentiGuide-Puro-OCLN sgRNA vector was successfully constructed, and 2 BHK-21 monoclonal cell lines with good Cas9 editing and high knockdown efficiency were identified by Western blot. Thus, this study provides evidence that BHK-21-Cas9 monoclonal cell line stably expressing Cas9 protein can be used to transduce CRISPR/Cas9 knockout library, to establish gene knockout cell library, and to provide scientific basis for screening functional genes that affect viral infection and replication.