Transcriptome analysis reveals no obvious unintended effects in the spleen of CRISPR/Cas9-mediated CD163 and pAPN double-knockout pigs.

Gene editing provides powerful tools for farm animal breeding. Our group previously obtained CD163/pAPN double-knockout (DKO) pigs via CRISPR/Cas9 and somatic cell nuclear transfer. These pigs are not only resistant to three infectious viruses but also maintain normal production performance. However, unintended effects of CRISPR/Cas9 tools may pose potential risks to animal well-being or safety. This study aimed to characterize the differences in splenic gene expression between wild-type (WT) and DKO pigs, providing a basis for safety evaluation of gene-edited animals. A comprehensive transcriptional panorama reflected considerable congruence in the aggregate gene expression profiles of the DKO and WT pigs. Comparisons between 35-day-old and 11-month-old DKO pigs and their WT equivalents revealed 225 and 242 differentially expressed genes (DEGs), respectively, a count significantly lower than that of the DEGs in the disparate developmental stage comparison groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that the majority of DEGs between DKO and WT pigs correlated with the biological functions of CD163 and pAPN, without any alterations in the expression of tumor suppressor genes in DKO pigs. This revealed a less pronounced effect of dual gene editing on the gene expression profile of porcine spleens than the effect of animal maturation, with no evident unanticipated consequences observed in DKO pigs.