Dysfunction of Complementarity Determining Region 1 Encoded by T Cell Receptor Beta Variable Gene Is Potentially Associated with African Swine Fever Virus Infection in Pigs.

The beta T-cell receptor (TRB) expressed by beta T cells is essential for foreign antigen recognition. The TRB locus contains a TRBV family that encodes three complementarity determining regions (CDRs). CDR1 is associated with antigen recognition and interactions with MHC molecules. In contrast to domestic pigs, African suids lack a 284-bp segment spanning exons 1 and 2 of the TRBV27 gene that contains a sequence encoding CDR1. In this study, we used the African swine fever virus (ASFV) as an example to investigate the effect of deleting the TRBV27-encoded CDR1 on the resistance of domestic pigs to exotic pathogens. We first successfully generated TRBV27-edited fibroblasts with disruption of the CDR1 sequence using CRISPR/Cas9 technology and used them as donor cells to generate gene-edited pigs via somatic cell nuclear transfer. The TRBV-edited and wild-type pigs were selected for synchronous ASFV infection. White blood cells were significantly reduced in the genetically modified pigs before ASFV infection. The genetically modified and wild-type pigs were susceptible to ASFV and exhibited typical fevers (>40 °C). However, the TRBV27-edited pigs had a higher viral load than the wild-type pigs. Consistent with this, the gene-edited pigs showed more clinical signs than the wild-type pigs. In addition, both groups of pigs died within 10 days and showed similar severe lesions in organs and tissues. Future studies using lower virulence ASFV isolates are needed to determine the relationship between the TRBV27 gene and ASFV infection in pigs over a relatively long period.

Efficient Generation of P53 Biallelic Mutations in Diannan Miniature Pigs Using RNA-Guided Base Editing.

The base editing 3 (BE3) system, a single-base gene editing technology developed using CRISPR/Cas9n, has a broad range of applications for human disease model construction and gene therapy, as it is highly efficient, accurate, and non-destructive. P53 mutations are present in more than 50% of human malignancies. Due to the similarities between humans and pigs at the molecular level, pig models carrying P53 mutations can be used to research the mechanism of tumorigenesis and improve tumor diagnosis and treatment. According to pathogenic mutations of the human P53 gene at W146* and Q100*, sgRNAs were designed to target exon 4 and exon 5 of the porcine P53 gene. The target editing efficiencies of the two sgRNAs were 61.9% and 50.0%, respectively. The editing efficiency of the BE3 system was highest (about 60%) when C (or G) was at the 5th base. Puromycin screening revealed that 75.0% (21/28) and 68.7% (22/32) of cell colonies contained a P53 mutation at sgRNA-Exon5 and sgRNA-Exon4, respectively. The reconstructed embryos from sgRNA-Exon5-5# were transferred into six recipient gilts, all of which aborted. The reconstructed embryos from sgRNA-Exon4-7# were transferred into 6 recipient gilts, 3 of which became pregnant, resulting in 14 live and 3 dead piglets. Sequencing analyses of the target site confirmed 1 P53 monoallelic mutation and 16 biallelic mutations. The qPCR analysis showed that the P53 mRNA expression level was significantly decreased in different tissues of the P53 mutant piglets (p < 0.05). Additionally, confocal microscopy and western blot analysis revealed an absence of P53 expression in the P53 mutant fibroblasts, livers, and lung tissues. In conclusion, a porcine cancer model with a P53 point mutation can be obtained via the BE3 system and somatic cell nuclear transfer (SCNT).