CRISPR/Cas9 Mediated CMAH,β4GalNT2 Gene Knockout Of Pigs (Sus Scrofa) And Acute Rejection Dectection

Xenotransplantation is an effective way to solve the shortage of donor organs.The pig（Sus scrofa）is considered to be ideal xenotransplantation donor due to the advantages of organ size,physiological anatomical structure,high reproduction rate and low feeding cost.Pig organs could cause hyperacute rejection（HAR）when they are transplantated into human bodies.The knockout ofα-1,3-galactosyltransferase（GGTA1）can avoid HAR.However,there are many non-galactose antigens on the surface of pig cells,which are expressed in pig endothelial cells and various organs.Those non-galactose antigens can induce acute rejection of antibodies such as antibody deposition and complement activation in xenotransplantation.Neu5Gc and Sd^a blood group antigen are two of the non-galactose antigens.N-acetylneuraminic acid（N-glycolylneuraminic acid,Neu5Gc）is a sialic acid which is catalyzed synthesis by cytidine monophospho-N-acetylneuraminic acid hydroxylase（CMAH）.Pig Sd^a blood group antigen is catalyzed synthesis byβ-1,4-N-acetyl-galactosaminyltransferase 2（β4GalNT2）which is a kind of glycosyltransferase in pigs.Baboons and most humans can produce antibodies that bind to the Sd^a blood group antigen.In this study,the CMAH gene knockout Bama minipigs(CMAH^-/-)andβ4GalNT2 gene knockout Bama minipigs(β4GalNT2^-/-)were generated by CRISPR/Cas9 based on the alpha-1,3-galactosyltransferase（GGTA1）gene knockout pigs.The health and reproductive condition of the CMAH^-/-pigs were evaluated.1.Based on the GTKO pigs,SgRNAs were designed to target the porcine CMAH gene.SgRNA/Cas9 expression vector was constructed and its activity was verified.High-efficiency CMAH-g1 expression vector was transfected into the ear fibroblasts of Bama minipigs（BMEF）.Cells identified as positive were used as nuclear donors,and somatic cell nuclear transfer were used to prepare CMAH^-/-pigs.CMAH gene mutations in piglets were identified by PCR and sequencing;Immunofluorescence detection of CMAH^-/-porcine organs with anti-Neu5Gc antibody to determine the expression of Neu5Gc in piglets;The physical condition of the piglets is stable when they grow to 4 months of age.The CMAH^-/-pigs’blood was collected for the detection of its physiological indexes,the CMAH^-/-boars mating with wide type sows,the litter size of sows was computed to evaluate the CMAH^-/-pigs’breeding capacity.The CMAH^-/-piglets have two homozygous genotypes:-2bp/-2bp and-1bp/-1bp.Immunofluorescence detection results showed that the Neu5Gc was not detected on the surface of CMAH^-/-pigs’pancreas and aorta.Compared with the wild type pigs（WT）,there was no significant difference in blood routine and blood biochemical parameters,and the productivity of CMAH^-/-boars was normal.2.Based on the GTKO pigs,SgRNAs were designed to target the exon4 of porcineβ4GalNT2 gene.SgRNA/Cas9 expression vector was constructed and its activity was verified.High-efficiency sgRNA expression vector was transfected into the ear fibroblasts of Bama minipigs（BMEF）.The engineered mutation of colonies were identified by PCR,TA cloning and sequencing.Cells identified as positive were used as nuclear donors,and somatic cell nuclear transfer were used to prepareβ4GalNT2^-/-pigs.β4GalNT2 gene mutations in piglets were identified by the same method.Peripheral blood mononuclear cells（PBMCs）were isolated to co-incubation with fluorescein isothiocyanate conjugated Dolichos biflorus agglutinin（FITC-DBA）.The highest-efficiency bGal-g5 was used to produceβ4GalNT2 gene knockout pigs.Theβ4GalNT2^-/-piglets have a homozygous genotypes:+1bp/-3bp/-17bp.The results ofβ4GalNT2 gene of cell identification and piglet identification all showed that there were three types,indicating that theβ4GalNT2 gene was a multi-copy gene.No green fluorescence was detected in PBMC ofβ4GalNT2 knockout piglet after co-incubation with FITC-DBA,indicating thatβ4GalNT2 gene of piglet was inactivated.3.The blood of WT,GGTA1 gene knockout（GTKO）pigs,CMAH^-/-pigs,β4GalNT2^-/-pigs were collected to isolate PBMC.The PBMC were incubation with inactivated mixed AB human serum and monkey serum.The combination of PBMC and antibody in human and monkey serum was detected under flow cytometry.The results showed that knockout GGTA1gene could effectively reduce the amount of antibody binding to PBMC.On the basis of the knockout of GGTA1 gene,the knockout of CMAH orβ4GalNT2 can continue reduce the binding amount of the antibody on the original basis.In this study,CRISPR/Cas9 technology and Somatic Cell Nuclear Transfer Technique（SCNT）was used to generated GGTA1/CMAH knockout Bama minipigs and GGTA1/β4GalNT2 knockout Bama minipigs,the health and reproductive condition of CMAH^-/-pigs are normal.The results of antigen antibody reaction showed that CMAH^-/-pig andβ4GalNT2^-/-pigs could reduce the antibody binding content with human serum and monkey serum.The pigs can provide basic research materials for further reducing the acute immune rejection of xenotransplantation.