Abstract:

Objective To obtain α-1，3-galactosyltransferase/β-1，4 N-acetylgalactosaminyltransferase（GGTA1/β4GalNT2）double gene knockout porcine fetal fibroblasts（PFFs）by CRISPR/Cas9 system and lay the foundation forestablishing Bama miniature pigs with α-Gal and SD（a）antigen deficiency. MethodsSingle-guide RNA（sgRNA）targeting the third exon of pig GGTA1 and the eighth exon of pig β4GalNT2 weredesigned using online tools（http://crispr.mit.edu），respectively. The synthesized sgRNA were cloned into pX330 plasmid containing Cas9 skeleton. The cleavage efficiency of the Cas9/sgRNA plasmids was assessed by the T7EN1 enzyme digestion assay. The Cas9/sgRNA vectors wereco-transfected withaneomycin-expression plasmid（tdTomato）into the PFFs. G418 was used to screen the positive monoclonal cells and sanger sequencing was used to determine the genotypes of monoclonal cells. Results Cas9/sgRNA expression vectors targeting GGTA1 and β4GalNT2 gene were successfully constructed and transfected into PFFs. Among the 48 G418-resistant colonies obtained，five hadbiallelic modifications in both GGTA1 and β4GalNT2 loci. Conclusion The GGTA1/β4GalNT2 double gene knockout PFFs were successfully obtained by the highly efficient CRISPR/Cas9 targeting，which could contribute tothe generation of GGTA1/β4GalNT2-deficient Bama miniature pig models.

Key words:

Bama miniature pigs ；CRISPR/Cas9 ；porcine fetal fibroblasts ；α-1, 3-galactosyltransferase/β-1,

4 Nacetylgalactosaminyltransferase

摘要：

目的 利用 CRISPR/Cas9 基因编辑技术建立巴马小型猪胚胎成纤维细胞（porcine fetalfibroblasts, PFFs）α-1，3- 半乳糖基转移酶（α-1，3-galactosyltransferase，GGTA1）/β-1，4 N- 乙酸氨基半乳糖转移酶（β-1，4 N-acetylgalactosaminyltransferase，β4GalNT2）双基因敲除细胞系，为构建 α-1，3- 半乳糖（α-1，3-galactose，α-Gal）和 SD（a）抗原缺失的巴马小型猪模型奠定基础。方法 分别选取猪 GGTA1 基因的第三外显子和 β4GalNT2 基因的第八外显子为敲除靶点，利用在线工具（http://crispr.mit.edu）设计并合成单导向 RNA（single guide RNA，sgRNA），以含有 Cas9 基因的 pX330 质粒为骨架构建打靶载体，转染野生巴马小型猪的 PFFs，用 T7EN1 酶切验证打靶载体敲除效率。将打靶载体与 G418 抗性质粒（tdTomato）共转染巴马小型猪 PFFs，经药物筛选获得阳性单细胞克隆后测序鉴定基因型。结果 成功构建靶向 GGTA1 和 β4GalNT2 基因的Cas9/sgRNA 表达载体。转染 PFFs 后用药物筛选得到 31 个单克隆细胞系，其中 5 个为 GGTA1/β4GalNT2 双基因敲除的细胞系。结论 Cas9/sgRNA 表达载体可以高效编辑PFFs 的 GGTA1/β4GalNT2 基因并获得了双基因敲除的单细胞克隆，为构建 GGTA1/β4GalNT2 敲除的巴马小型猪提供必需的实验材料。

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