Pigs are important hosts in the ecology and cross-species transmission of influenza A viruses because their respiratory tract can contain both human-type α2,6-linked and avian-type α2,3-linked sialylated glycans. These glycan structures may allow pigs to support infection by influenza viruses of different host origins and can facilitate viral reassortment when coinfection occurs. Here, we used CRISPR/Cas9-mediated gene editing to disrupt the porcine ST6GAL1 and ST3GAL4 genes, which encode sialyltransferases involved in the biosynthesis of α2,6-linked and α2,3-linked sialylated glycans, respectively. We generated ST6GAL1/ST3GAL4 double-knockout Landrace pigs and observed markedly reduced Sambucus nigra agglutinin (SNA)- and Maackia amurensis lectin Ⅱ (MAL Ⅱ)-reactive sialylated glycans in the respiratory tract. Fibroblasts derived from the double-knockout pigs showed reduced susceptibility to H1N1, H3N2, and H9N2 influenza A virus infection in vitro. Transcriptomic analysis further revealed distinct gene-expression profiles between knockout and wild-type fibroblasts before and after H1N1 infection. Complementation of ST6GAL1 and ST3GAL4 restored sialylated glycan expression and increased viral susceptibility in cultured cells, supporting a causal contribution of these sialyltransferases to influenza A virus infection. Further in vivo challenge, transmission, safety, and independent-founder studies are required to determine whether this strategy can reduce influenza A virus infection or transmission in live pigs.
