Generation of APN-chimeric gene-edited pigs by CRISPR/Cas9-mediated knock-in strategy.

The prevalence of porcine enteric coronaviruses (PECs), including transmissible gastroenteritis virus (TGEV), swine acute diarrhea syndrome coronavirus (SADS-CoV), porcine delta coronavirus (PDCoV), and porcine epidemic diarrhea virus (PEDV), poses a serious threat to animal and public health. Here, we aimed to further optimize the porcine aminopeptidase N (pAPN) gene editing strategy to explore the balance between individual antiviral properties and the biological functions of pAPN in pigs. Finally, APN-chimeric gene-edited pigs were produced through a CRISPR/Cas9-mediated knock-in strategy. Further reproductive tests indicated that these gene-edited pigs exhibited normal pregnancy rates and viability. Notably, in vitro viral challenge assays further demonstrated that porcine kidney epithelial cells isolated from F1-generation gene-edited pigs could effectively inhibit TGEV infection. This study is the first to report the generation of APN-chimeric pigs, which may provide a natural host animal for characterizing PEC infection with APN and help in the development of better antiviral solutions.