CRISPR/Cas9-based strategies for pig host resistance to Influenza A virus

Influenza circulates in different mammalian and avian species, causing epidemics and occasional pandemics. This poses a substantial threat to agricultural productions, animal welfare, human public health, and economy. The causative agent of the disease is Influenza A virus (IAV), whose entry depends on its preference on the receptor molecules since the preference determines whether virus glycoproteins can employ the host cell surface sialic acid (SA) as ligands. There are two major types of sialylated glycans as receptors for viral recognition: one is Neu5Ac α 2,6-Gal (SA α 2,6-Gal for short) preferentially recognised by human IAVs, and the other one is Neu5Ac α 2,3-Gal (SA α 2,3-Gal for short) predominantly recognised by avian IAVs. Pigs harbouring both types of SA-containing receptors have the potential to play a role of ‘intermediate hosts’ or ‘mixing vessels’. Therefore, it is of great interest to develop methods aiming at reducing α 2,6-SA-containing receptors in pig cells, so that they are less susceptible to human IAV infections. β-galactoside α 2,6-sialyltransferase 1 (ST6Gal1) mediates N-linked α 2,6- sialylation on cell surfaces by catalysing the addition of α 2,6-SA to the terminal N-glycans. ST6Gal1 is involved in a wide range of biological events, such as the generation of carbohydrate determinants on the cell surfaces, the immune regulation, and in various carcinomas. ST6Gal1 is encoded by the ST6GAL1 gene, which expression has been reported to display a tissue-specific pattern as a result of the regulations of multiple promoter regions and the differential combinations of 5’- untranslated exons. Driven by the concern that inactivating the ST6GAL1 gene may have deleterious phenotypic effects given the widespread expression profile and the diversity of its biological functions, I pursued a subtle approach to engineering the ST6GAL1 gene — that of removing a single promoter region to alter the expression profile. I hypothesised that reducing the biosynthesis of α 2,6-sialylated glycan structure exclusively on the respiratory tract could potentially block IAV entry without compromising humoral immune responses. To this end, I identified 5’ transcription starting sites (TSSs) and 5’ untranslated region (UTR) exons of transcripts expressed in nine pig tissues. Then we employed the CRISPR/Cas9 system to precisely engineer pig ST6GAL1 gene instead of the whole gene deletion. The consequence of deleting the region surrounding the 5’ TSS of ST6GAL1 transcripts predominantly expressed in airway was assessed (the resulting model was termed as ST6GAL1ΔP). Moreover, I generated a ST6GAL1 functional knockout model by inducing a frameshift mutation in pig trachea cells (the resulting model was termed as ST6GALΔCD). Additionally, human IAV had reduced infectivity in ST6GAL1ΔP relative to non-edited cells, suggesting that a strategy to reduce the biosynthesis of α 2,6-sialylated glycan structure exclusively on the airway could offer an antiviral strategy, independent of inducing a humoral immune response. This work lays a solid foundation in generating engineered pigs for IAV host resistance modelling, and helps us to achieve the genetic improvement in swine herds; also, it provides a good understanding of the fundamental molecular basis of the IAV-host interactions, and develop novel antiviral and therapeutics strategies.