Generation Of GGTA1/ASGR1 Biallelic Knockout Pigs Via Programmable Nucleases

Porcine liver as donors for xenotransplantation were considered to be a promising solution to the shortage of liver in clinical. In particular, as "transitional liver" for the treatment of acute liver failure which patients would die in 24 h, will reduce the mortality of patients and gain time for follow-up treatment.However, there are several barriers of immune rejection in pig-to-human xenotransplantation. Hyperacute rejection and thrombocytopenia that accompanies liver xenotransplantation are two major barriers. ?1,3-galactosyltransferase(GGTA1), the causative agent of hyperacute rejection in pig-to-human xenotransplantation. Asialoglycoprotein receptor(ASGR) on pig sinusoidal endothelial cells can bound and phagocytose the human platelets and lead thrombocytopenia. This study was to generate GGTA1/ASGR1 knockout Wuzhishan minipig models via programmable nucleases.GGTA1 and ASGR1 knockout Wuzhishan minipigs were producted by transcription activator-like effector nucleases(TALEN) and clustered regularly interspaced short palindromic repeats/Cas9(CRISPR/Cas9) with somatic cell nuclear transfer(SCNT). The results are as follows:Study?: Two TALEN pairs were constructed to target the porcine GGTA1 gene locus. TALEN mRNA was transfected into the ear ?broblasts followed by the enrichment of a-Gal null cells of minipigs using IB4 lectin and magnetic beads. A total of 47 cell colonies were formed and validated to be GGTA1knockout(KO) cells by sequencing and 6 biallelic KO cell colonies were used as nuclear donors for SCNT.Twenty-three GGTA1 biallelic KO piglets were successfully delivered and grew normally. Seventeen potential off-target sites were investigated, and no off-target events were detected in the live piglets. Flow cytometry analysis determined that ?-Gal epitope of cloned pigs had been successfully removed. Serum lysis assay determined that lacking of ?-Gal epitopes could effectively against the rejection of normal human serum, and the survival rate was equivalent compared with fetal bovine serum and heat-inactivated human serum.Study?: ASGR1 knockout pigs were generated by CRISPR/Cas9 in GGTA1 gene knockout pigs,which could avoid hyperacute rejection and diminish platelet destruction in pig-to-humanxenotransplantation. Single guide RNA1(sgRNA1) were assembled and transferred to ?broblasts with enhanced green fluorescent protein(EGFP) plasmid. Forty-one colonies were finally selected, and the targeting efficiency reached 90%(37/41), more remarkably 73%(30/41) of colonies were biallelic knockout, among which four colonies with different genotypes were selected as nuclear donors for SCNT.Cloned embryos were co-transferred into four recipient gilts. Pregnancy was established in two of four transfers. Two pregnancies were maintained to term, resulted in six live-born piglets with biallelic mutations of the ASGR1 gene. Western blot results showed that the ASGR1 gene expression was abrogate in the liver. Thirteen potential off- target sites were investigated, but no off- target event was detected in the live piglets. Taken together, the use of the CRISPR/Cas9 system of fibroblasts followed by SCNT enabled the production of ASGR1 biallelic knockout pigs. Thus, deletion of the ASGR1 gene is a viable strategy to diminish platelet destruction in pig-to-human xenotransplantation.Taken together, the use of programmable nucleases in ?broblasts followed by SCNT enabled the production of GGTA1/ASGR1 biallelic knockout Wuzhishan minipig models. These will be a viable strategy to diminish hyperacute rejection and thrombocytopenia in pig-to-human xenotransplantation.