Generation Of ?4GalNT2 And ZBED6 Gene Knockout Pigs Via CRISPR/Cas9

Part one Generation of ?1,4 N-acetylgalactosaminyl transferase(?4Gal NT2)gene knockout pigsPorcine ?4Gal NT2 and its products are one of the important non-Gal antigens causing xenograft rejection.To further reduce the xenograft rejection,?4Gal NT2 knockout pigs were generated by clustered regularly interspaced short palindromic repeats/Cas9(CRISPR/Cas9)in ?-1,3 galactosyltransferase(GGTA1)gene knockout pigs.Designing single guide RNA(sg RNA)targeting porcine ?4Gal NT2 gene,constructing sg RNA expression vector and it was electrically transferred to fibroblasts from GGTA1 knockout pigs.Cells were individually cultured.The engineered mutations of colonies in the ?4Gal NT2 gene were identified by PCR,TA cloning and sequencing.The colonies of ?4Gal NT2 gene mutations were selected as the nuclear donor,and the ?4Gal NT2 knockout pig was generated by somatic cell nuclear transfer technique.Using the ways as same as the method for identification of colonies to identify the ?4Gal NT2 gene mutation of piglet.Peripheral blood mononuclear cells(PBMCs)were isolated to co-incubation with fluorescein isothiocyanate conjugated Dolichos biflorus 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 results of this study were as follows:1.The result showed that 14 of the 25 colonies were mutated.2.Reconstructed embryos were transplanted into 2 estrous recipient gilts.One recipient gilt was pregnant to the end and produced one piglet,and the genotype of piglet in the ?4Gal NT2 gene was-6 bp/-13 bp/WT that suggested the ?4Gal NT2 may be a multi-copy gene.3.No green fluorescence was detected in PBMC of ?4Gal NT2 knockout piglet after coincubation with FITC-DBA,indicating that ?4Gal NT2 gene of piglet was inactivated.4.No potential off-target sites were detected in the live pig via analysis.Thus,this study used CRISPR/ Cas9 technology to generate ?4Gal NT2 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.Part two Generation of zinc finger BED domain-containing protein 6(ZBED6)gene knockout pigsPork is one of the main sources of human intake of protein,how to improve the pig meat rate is a major issue in the field of animal genetic improvement.ZBED6 gene is closely related to muscle growth.At present,there are no reports about ZBED6 inactivation or overexpression pigs in the world.This study used CRISPR/Cas9 technology to generate ZBED6 gene knockout pigs,in order to provide favorable materials for researches of intramuscular growth and development.Designing sg RNA targeting porcine ZBED6 gene,to construct 4 sg RNA expression vectors,and it was electrically transferred to fibroblasts from pigs.Screening effective sg RNA expression vector to co-transfected with enhanced green fluorescent protein(EGFP)into wild type Bama mini pig ear fibroblast.Cells with green fluorescence via screening by flow cytometry were individually cultured.Using the methods of PCR and sequencing to identify mutations of colonies.3 colonies of mutations in ZBED6 gene were be the donor,and ZBED6 gene knockout pigs were be generated through somatic cell nuclear transfer technology.ZBED6 gene mutations in piglets were identified by PCR and sequencing.Selecting 10 high risk potential off-target sites of sg RNA predicted by CRISPR designing software to design the primers.Healthy wild type Bama mini pigs,the same ages as ZBED6 knockout piglets,were be selected as the control group.ZBED6 gene knockout piglets were be as experimental group.Observing the appearances of the control group and experimental group,and weighing the pigs.The concentrations of insulin and IGF2,four items of blood coagulation and physiological indicators about the control group and experimental group were be detected.Using software to analyse the data.The results of this study were as follows:1.The effective sg RNA expression vector Zg3 was be screened out.A total of 57 ZBED6 colonies including 39 mutations were be obtained,the gene mutation rate was 68%,and there were 28 double mutant alleles,the rate was 49%.2.Cloned embryos were be transfered into 3 recipient gilts.3 of them was pregnant and maintained to term resulting in 16 piglets,and 6 piglets were survived.All of the live piglets' ZBED6 gene mutations were-1 bp/-1 bp.3.There were no potential off-target sites detected via PCR and analysis.4.The experimental group and control group had no significant differences in appearances,weight and physiological indicators.The insulin concentration in the experimental group was lower than the control group,but the IGF2 concentration in the experimental group was higher than the control group.Fibrinogen(FIR)in the serum of the experimental group had lower concentration,and speculating the ZBED6 gene might be related to the blood coagulation system.Thus,this study used CRISPR/ Cas9 technology to generate ZBED6 gene knockout pigs.It is expected to provide material for study of ZBED6 gene and the application of swine production performance in our country.