利用CRISPR/Cas9技术敲除CHO-K1细胞中GS基因的研究  

Study of GS gene knockout from CHO-K1 cell strain by CRISPR/Cas 9 technique

作　　者：于浩洋 仇松寅[1] 王彩霞[1] 林祥梅[1] 贾红[2] 李昊轩 刘晓飞[1] 冯春燕[1] 吴绍强[1] YU Hao-yang;QIU Song-yin;WANG Cai-xia;LIN Xiang-mei;JIA Hong;LI Hao-xuan;LIU Xiao-fei;FENG Chun-yan;WU Shao-qiang(Institute of Animal Inspection and Quarantine,Chinese Academy of Inspection and Quarantine,Beijing 100176,China;Beijing Institute of Animal Sciences and Veterinary Medicine,Chinese Academy of Agricultural Sciences,Beijing 100193,China)

机构地区：[1]中国检验检疫科学研究院动物检验与检疫研究所,北京100176 [2]中国农业科学院北京畜牧兽医研究所,北京100193

出　　处：《中国兽医科学》2022年第10期1273-1280,共8页Chinese Veterinary Science

基　　金：中国检验检疫科学研究院基本科研业务费项目(2020JK020)

摘　　要：谷氨酰胺合成酶(glutamine synthetase,GS)是合成CHO细胞生长所需要的谷氨酰胺的关键酶。为了获得能快速稳定表达外源蛋白的CHO细胞GS筛选系统,根据CRISPR/Cas9技术原理,针对GS基因设计3对sg RNA,分别将连接sg RNA的pX459质粒转染至CHO-K1细胞中以便定向敲除GS基因;经筛选后利用PCR及测序分析鉴定GS基因敲除情况;将GS基因敲除的CHO细胞株(CHO^(GS-/-))分别在含有外源谷氨酰胺培养基与无谷氨酰胺培养基中进行培养,观察细胞的生长状态,绘制生长曲线,并验证该CHO^(GS-/-)细胞株中外源基因的表达能力。结果显示,sg RNA3与sg RNA4共转染能高效双靶位敲除CHO细胞中的GS基因;生长曲线显示CHO^(GS-/-)细胞在富含谷氨酰胺的培养基中生长速度正常或无差异,但无法在无谷氨酰胺的培养基中存活,这表明GS基因功能被破坏;外源基因表达水平及功能验证显示,敲除GS基因后不影响外源单抗在细胞中的表达。这表明,借助CRISPR/Cas9敲除系统获得了CHO^(GS-/-)细胞,大大提高了稳定转染外源基因阳性细胞株的筛选效率,为后期利用CHO细胞大量发酵表达蛋白技术建立了坚实的分子生物学平台。Glutamine synthetase(GS)is a kind of key enzyme that can synthesize the glutamine,which is required for the growth of CHO cell.To obtain a GS screening system of CHO cell line with rapid and stable expression of the exogenous protein,three pairs of sg RNAs targeting GS gene were designed and cloned into p X459 vector according to the principle of CRISPR/Cas9 technology.The recombinant sg RNA plasmids were transfected into CHO-K1 cells for GS gene knockout.After screening,the GS gene knockout monoclonal CHO cells(CHO^(GS-/-))were identified using PCR assay and DNA sequencing analysis.The CHO^(GS-/-)cells were cultured in glutamine free medium or exogenous glutamine medium,respectively.The growth status of the cells was observed,and the growth curve was measured,and the expression level and function of exogenous genes in the CHO^(GS-/-)cell were detected.The results showed that co-transfer of sg RNA3and sgRNA4 could effectively knockout GS gene by double targets.The growth curve showed that the growth rate of CHO^(GS-/-)cells were unchanged in the glutamine-enriched medium,but could not survive in glutamine free medium,indicating that the GS gene function was destroyed.Expression level and functional validation of the exogenous gene showed that the expression of the exogenous monoclonal antibody was not changed in the CHO^(GS-/-)cells.This showed that the present study obtained CHO^(GS-/-)cells with the help of CRISPR/Cas9 system tools,which greatly improved the screening efficiency of stably transfected exogenous gene positive cell lines.And it provided a solid molecular biology platform for the study of fermentation culture and protein expression technology by CHO cells with GS gene knockout in the future.

关 键 词：CRISPR/Cas9 CHO-K1细胞系 谷氨酰胺合成酶基因 基因敲除 

分 类 号：Q78[生物学—分子生物学]