gene disruption causes severe limb deformities in pigs.

In an attempt to generate g.A746G substitution in the gene, we unexpectedly obtained homozygous knockout piglets ( ) and heterogeneous knockout piglets with one copy of the A746G mutation ( ) via CRISPR/Cas9 editing. Polymerase chain reaction (PCR) and sequencing revealed complex genomic rearrangements in the target region. All -disrupted piglets showed an inability to stand and walk normally. Both and piglets exhibited severe skeletal dysplasia characterized by distorted and truncated forearms (ulna, radius) and disordered carpal, metacarpal, and phalangeal bones in the forelimbs. The piglets displayed more severe deformities in the hindlimbs by visual inspection, including fibular hemimelia, enlarged tarsal bone, and disordered toe joint bones. Limb deformities were more profound in piglets than in the piglets. Proteomic analysis identified 139 differentially expressed proteins (DEPs) in the hindlimb fibula of piglets compared to the wild-type (WT) controls. Most DEPs are involved in skeletal or embryonic development and/or the TGF-β pathway and tumor progression. Gene Ontology (GO) and protein domain enrichment analysis suggested alterations in these processes. Of the top 50 DEPs, a large proportion, e.g., C1QA, MYO1H, SRSF1, P3H1, GJA1, TCOF1, RBM10, SPP2, MMP13, and PHAX, were significantly associated with skeletal development. Our study provides novel findings on the role of in mammalian limb development.