Objective：This study aims to establish 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 severe acute respiratory syndrome coronavirus type 2(SARS-CoV-2)insensitive pig model. Methods：①The 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 chicken’s ACE2 at the corresponding sites. The chimeric ACE2 gene was synthesized and cloned into the knock-in vector. ②The sgRNA targeting the porcine ACE2 gene was designed，synthesized，and cloned into the pX330 vector to construct the ACE2 gene targeting vector. ③The ACE2 targeting vector，the ACE2 gene knock-in donor vector，and a neomycin-resistant plasmid were co-transfected into PFP. 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 19 th，31st，34 th，35 th，42 nd，353 rd and 354 th 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 PFF cells with endogenous ACE2 knock-out and chimeric ACE2 knock-in are successfully generated using CRISPR/Cas9-mediated gene modification，which will lay a foundation for the construction of SARS-CoV-2 insusceptible pig models.