Modification of the splice acceptor in CD163 exon 7 of pigs is insufficient to confer resistance to PRRSV.

CD163 is the primary receptor for PRRSV, and its SRCR5 domain, encoded by exon 7, is crucial for supporting PRRSV infection. Previous studies have used CRISPR/Cas9 technology to remove exon 7 from the host genome, and the edited pigs were completely resistant to PRRSV infection. In this study, we used CRISPR/Cas9 technology mimicking an adenine base editor (ABE) to edit the splice acceptor site of exon 7, rendering it nonfunctional. This alteration was intended to cause exon 6 to join directly to exon 8 during mRNA processing, resulting in a mature mRNA transcript that lacks exon 7, which encodes the SRCR5 domain. Piglets carrying the exon 7 splice site modification (CD163Ex7-ABE) were successfully generated. However, these pigs remained fully susceptible to infection with a PRRSV-2 isolate. Analysis of CD163 mRNA from the CD163Ex7-ABE pigs revealed that they predominantly expressed a mature CD163 mRNA lacking exon 7. However, due to cryptic splice sites, two additional mRNA isoforms were expressed, including an in-frame variant containing all of exon 7 and an extra 48 base pairs. This likely resulted in the expression of a full-length CD163 with a 16-amino-acid insertion upstream of the SRCR5 domain, which was sufficient to render the animals susceptible to PRRSV. Overall, our results demonstrate that merely modifying the splice acceptor site of CD163 exon 7 is not sufficient to generate PRRSV-resistant pigs.