Jiangsu Key Laboratory of Xenotransplantation, NMU, Nanjing
The National Natural Science Foundation of China (General Program, Key Program, Major Research Plan)
目的：利用 CRISPR/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 fibroblasts, PFFs）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PFFs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RMSD值为0.149。成功构建打靶载体并转染PPFs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PFFs 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 provide 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 FⅨ by 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 gRNAs. 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 FIX and the 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 PPFs lays an essential foundation for the construction of Bama minipig model of hemophilia B.