The production of UL16-binding protein 1 targeted pigs using CRISPR technology

Abstract

Two sgRNAs were designed to target the region of exon 2 of the pULBP1 gene by microinjection. The co-injection of modified Cas9-D10A nickase with a pair of sgRNAs into the zygote's cytoplasm easily and efficiently generated biallelic modification of the pULBP1 gene in one step. Five out of nine F0 generation piglets showed insertions or deletions in the targeting site of the pULBP1 gene, indicating that pULBP1 mutation efficiency reached about 56% (5/9). Quantitative determination of pULBP1 showed approximately a 1.53-fold reduction in the amount of protein ULBP1 on the cell surface (ELISA). A human NK-cell cytotoxicity test leads to the conclusion that higher cell viability is observed for -/- ULBP1 (survival rate 85.36%) compared to +/+ ULBP1 (69.58%). ULBP1-KO pigs will provide a more progressive xenograft source for further research studies, especially those measuring the effects of abolishing the gene function in terms of the complexity of the immunological interactions.

Keywords: CRISPR-Cas9; KO pigs; Microinjection; Xenotransplantation.

Fig. 1

Generation of UL16-binding protein 1 targeted pigs using CRISPR-Cas9 technology. a Schematic representation of nucleotide sequences between the target locus (exon 2 of ULBP1) and ULBP1—targeting sgRNAs. The black arrows indicate the putative cleavage site. The primers used are shown above the sequence of ULBP1. b Direct sequencing of PCR products from +/+ ULBP1, −/− ULBP1 pig #184 and alleles separated by cloning. c List of mutations. The inserted base is marked in red. Black bars indicate deletion of bases. PAM-1 and PAM-2, protospacer adjacent motifs

Fig. 2

Survival rate analysis for cells from −/− ULBP1 pig #184 (blue) and +/+ ULBP1 pig (yellow) in the medium with human NK cells. The human NK cells (effector cells) were added to endothelial cells (target cells) in an E:T ratio 5:1. After co-culture for 4 h, CCK-8 was used to detect the remaining vital cells. The survival rate was measured by the average number of living cells. The percentage of surviving cells in each tested group was marked on Y-axis. Means were compared by Welch two-sample t test (p value = 0.0565)

Fig. 3

ULBP1 concentration level in the culture medium was detected using an ELISA assay—the competitive enzyme immunoassay technique utilizing a monoclonal anti-ULBP1 antibody and an ULBP1-HRP conjugate. Analysis was conducted for cells from −/− ULBP1 genetically modified pig #184 and +/+ ULBP1 pig. The ULBP1 concentration was marked on the Y-axis. Presented results are from one of three independent experiments. Means were compared by Welch two-sample t test (p value = 0.00376)