Abstract：β1,4 N-acetylgalactosaminyl transferase (β4GalNT2) and its products of porcine (Sus scrofa) are one of the important non-Gal antigens causing xenograft rejection. To further reduce the xenograft rejection, β4GalNT2 knockout pigs were generated by clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) in α-1,3 galactosyltransferase (GGTA1) gene knockout pigs. Firstly, single guide RNA (sgRNA) targeting porcine β4GalNT2 gene was designed, sgRNA expression vector was constructed, and then it was electrically transferred to fibroblasts from GGTA1 knockout pigs, cells were individually cultured. The engineered mutation of colonies in the β4GalNT2 gene were identified by PCR, TA cloning and sequencing. The colonies of β4GalNT2 gene mutation were selected as the nuclear donor, and the β4GalNT2 knockout pig was generated by somatic cell nuclear transfer (SCNT). The method of identifying mutations in colonies was used to identify the β4GalNT2 gene mutations in piglet. Peripheral blood mononuclear cells (PBMCs) were isolated to co-incubation with fluorescein isothiocyanate conjugated Dolichos bi?orus agglutinin (FITC-DBA). Potential off-target sites were investigated by software named Optimized CRISPR Design, and PCR products of off-target sites were sequenced. The result showed that 14 of the 25 colonies occurred mutation. Reconstructed embryos were transplanted into 2 estrous recipient gilts. One recipient gilt was pregnant to the end and produced one piglet, the genotype of piglet in the β4GalNT2 gene was -6 bp/-13 bp/WT. No green fluorescence was detected in PBMC of β4GalNT2 knockout piglet after co-incubation with FITC-DBA, which indicated that β4GalNT2 gene of piglet was inactivated. No potential off-target sites was detected in the live pig via analysis. Thus, CRISPR/Cas9 was used to generate GGTA1/β4GalNT2 gene knockout pig domestically for the first time. It is expected to reduce the xenograft antibody mediated rejection and provide a good research material for the clinicalapplication of organ transplantation.
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