利用CRISPR/Cas9技术高效构建巴马小型猪F9基因敲除细胞系  

Efficient construction of Bama minipig F9 gene knockout cell line using CRISPR/Cas9 technology

作　　者：朱晓晗 刘晓蕊[1] 李琳[1] 王盈[1] 杨海元[1] 戴一凡[1] ZHU Xiaohan;LIU Xiaorui;LI Lin;WANG Ying;YANG Haiyuan;DAI Yifan(Jiangsu Key Laboratory of Xenotransplantation,Nanjing Medical University,Nanjing 211166,China)

机构地区：[1]南京医科大学江苏省异种移植重点实验室,江苏南京211166

出　　处：《南京医科大学学报（自然科学版）》2022年第1期1-7,共7页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金(81970164,81874144)。

摘　　要：目的:利用CRISP/Cas9基因编辑技术建立巴马小型猪F9基因敲除的胚胎成纤维(porcine fetal fibroblast,PFF)细胞系,为后续构建血友病乙巴马小型猪模型提供原材料。方法:首先通过生物信息学方法对人和猪F9基因同源性进行分析,模拟并分析人和猪FⅨ二级、三级结构的相似性;其次选取猪F9基因的第2外显子为敲除靶点,利用CRISPR在线靶点设计工具(http://www.e-crisp.org/E-CRISP/designcrispr.html)设计并合成单链向导RNA(single guide RNA,sgRNA),以pX330质粒为骨架构建打靶载体,同G418抗性质粒一同转染至野生巴马小型猪的PFF中,经药物G418筛选获得抗性单细胞克隆,测序并鉴定其基因型。结果:生物信息学分析表明人和猪的F9基因具有较近的遗传距离,FⅨ的氨基酸序列相似值为83.33%,三维结构的均方根偏差(root mean square deviation,RMSD)值为0.149。成功构建打靶载体并转染PFF细胞,药筛后共获得55个单细胞克隆,经过测序显示有25个单细胞克隆的F9基因发生突变,计算敲除率为45.5%。结论:人和猪F9基因具有高度同源性;构建的Cas9/sgRNA表达载体可以高效编辑PFF中的F9基因。最终获取F9基因敲除的单细胞克隆,为构建乙型血友病巴马小型猪模型奠定了重要的前期工作基础。Objective:Establishing the F9 gene knockout cell lines of porcine fetal fibroblasts(PFFs)of Bama minipig by CRISPR/Cas9 gene-editing technology provides raw materials for the subsequent construction of Bama minipig models of hemophilia B.Methods:Firstly,we analyzed the homology of human and pig F9 gene and the similarity of the secondary and tertiary structure of human and pig FⅨby bioinformatics method.Secondly,we selected the second exon of pig F9 gene as the targeting region and used online tools(http://www.e-crisp.org/E-CRISP/designcrispr.html)to design sgRNA.The pX330 plasmid is used as the backbone to construct targeting vectors,which were then co-transfected with a G418 resistant plasmid into the PFFs of wild Bama miniature pigs.The G418 resistant single-cell colonies were obtained and sequenced.Results:Bioinformatics analysis showed that the F9 gene of human and pig had a close phylogenetic distance.The similarity value of the amino acid sequence of FⅨand the root mean square deviation(RMSD)value of the three-dimensional structure was 83.33%and 0.149,respectively.A total of 55 resistant single-cell colonies were obtained by G418 screening.Twenty-five single-cell colonies bearing mutations in the F9 gene targeting region have been identified by sanger sequencing,indicating the knockout efficiency was 45.5%.Conclusion:The human and pig F9 genes have a high degree of homology.The constructed Cas9/sgRNA expression vectors can efficiently induce mutagenesis in the pig F9 gene.The F9 gene knockout PFF lays an essential foundation for the construction of Bama minipig model of hemophilia B.

关 键 词：血友病乙 F9 CRISPR/Cas9 FⅨ 

分 类 号：Q785[生物学—分子生物学]