Gene editing for resistance to influenza A virus in swine

Influenza A Virus (IAV) presents a major threat to human health and animal welfare. As pigs are susceptible to infection from avian and mammalian origin IAVs, they can be an intermediate host for onwards transmission and act as a mixing vessel in which novel IAVs are generated. ANP32 family proteins have been identified in humans and chickens as host proteins critical to the efficiency of viral genome replication and host factors involved in IAVs adaptation. Host factors recruited by IAV present potential gene-editing targets for controlling IAV transmission and the editing of ANP32 genes in swine represents a potential method of IAV control. Using CRISPR/Cas technology, ANP32A and ANP32B were disrupted in a porcine tracheal cell line (NPTr) to determine whether they are recruited in the same manner as in humans and chickens by IAV polymerase to support viral genome replication. Our results show that human, avian and swine adapted IAVs can recruit ANP32 family proteins in NPTr, and that ANP32A and ANP32B are functionally redundant for IAV and must both be functionally knocked out to reduce the capacity for IAV to propagate. To consider industrial applicability, we have modelled the introgression of IAV resistance alleles into a commercial pig breeding herd by one-step zygote gene-editing. Our model results show that more efficient gene-editing methods will reach fixation quicker, even with greater rates of zygote death, and that the level of germline transmission for the gene-edited alleles will have the largest effect on the flow of alleles to commercial breeders. Together, these results have identified genes for further consideration regarding IAV resistance in swine and that gene-editing will need optimisation in porcine zygotes for implementation in the near-term.