Jiangsu Key Laboratory of Xenotransplantation,Nanjing Medical University
目的：利用CRISPR/Cas9基因编辑技术构建ACE2(angiotensin converting enzyme 2, ACE2)基因编辑的长白猪胎儿成纤维(porcine fetal fibroblasts, PFFs)细胞系,为构建新冠病毒不易感猪模型提供实验材料。方法：首先,利用生物信息学方法对大鼠和鸡的ACE2氨基酸序列进行比对,将大鼠ACE2与SARS-CoV-2 S蛋白相互作用的关键氨基酸残基替换鸡ACE2的氨基酸残基,设计嵌合的ACE2基因并构建ACE2基因敲入供体。其次,设计并合成靶向猪ACE2基因的sgRNA,克隆到pX330载体上构建ACE2基因打靶载体。最后,将ACE2打靶载体和ACE2基因敲入供体以及Neomycin抗性质粒共转染至长白猪胎儿成纤维细胞,筛选G418抗性单细胞克隆并进行测序鉴定。结果：根据氨基酸序列比对结果,将大鼠ACE2蛋白的第19,31,34,35,42,353,354位氨基酸残基替换为鸡ACE2的氨基酸残基,设计合成了嵌合的ACE2基因。对G418抗性单细胞克隆进行基因型分析的结果显示获得了阳性的单细胞克隆。结论：利用CRISPR/Cas9技术成功获得了内源ACE2基因敲除、嵌合ACE2敲入的长白公猪PFFs细胞系,为构建新冠病毒不易感猪模型奠定了基础。
Objective: To establish ACE2 (angiotensin converting enzyme 2, ACE2) gene-modified porcine fetal fibroblast (PFF) cell lines via CRISPR/Cas9 system-mediated gene modification, which could serve as donor cells for the subsequent construction of a SARS-CoV-2 insensitive pig model. Methods: Firstly, the ACE2 amino acid sequences of rats and chickens were compared by bioinformatic analysis. The ACE2 amino acid residues, which play a crucial role in the interaction between ACE2 and SARS-CoV-2 S protein, were replaced by the amino acid residues of ACE2 chickens at the corresponding sites. The chimeric ACE2 gene was synthesized and cloned into the knock-in vector. Secondly, sgRNA targeting the porcine ACE2 gene was designed, synthesized, and cloned into the pX330 vector to construct the ACE2 gene targeting vector. Finally, the ACE2 targeting vector, the ACE2 gene knock-in donor vector, and a neomycin-resistant plasmid were co-transfected into porcine fetal fibroblasts. G418 was used to screen the resistant single-cell colonies, and sequencing was performed to identify the correct ones. Results: According to the amino acid sequence alignment, the rat ACE2 amino acid residues at positions 19th, 31st, 34th, 35th, 42nd, 353rd, and 354th were replaced by the amino acid residues of chicken ACE2. The chimeric ACE2 gene was synthesized, and the ACE2 knock-in vector was successfully constructed. Genotype analysis of G418 resistant colonies showed that single-cell colonies with expected modification were successfully obtained. Conclusion: The PFFs with endogenous ACE2 knock-out and chimeric ACE2 knock-in were successfully generated using CRISPR/Cas9-mediated gene modification, which will lay a foundation for the construction of SARS-CoV-2 insusceptible pig models.