Figure 1 Screening of single-cell clones positive for GTKO/hCD55 gene editing
Note： A， Design of sgRNA for GGTA1 gene knockout and schematic diagram of the vector for hCD55 gene overexpression； B， Genotype of the sgRNA targeted region of GGTA1 in positive single-cell clones 35# and 45#； C， PCR identification of the hCD55 gene from 48 single-cell clones； PC， Positive controls； NC， Negative controls； -， Indicates the knockout bases； +， Indicates the bases insertion； M， DNA ladder； WT， Wild type； WT （2/8） indicates among 8 single colonies sequencing after TA cloning， four genotypes were identified： two WT， three -134 bp， two -26/+2 bp and one -3 bp.

Figure 2 Identification of GTKO/hCD55 gene-edited cloned fetal pigs
Note： A， Two cloned fetal pigs at 33 days of gestation； B，PCR results of sex identification of fetal pig heart tissue； C， PCR results of blood group gene identification of fetal pig heart tissue； D， PCR identification of hCD55 gene in fetal pig heart tissue； E， PCR results of the target region of GGTA1 gene sgRNA in fetal pig heart tissue； F， Genotype results of the target region of GGTA1 gene sgRNA in fetal pig heart tissue； G， hCD55 mRNA expression levels in fetal pig F01 and F02 heart tissues； H， Flow cytometric analysis results of α-Gal and hCD55 expression in fetal porcine F02 fibroblasts. M， DNA ladder； WT， Wild type； PC， Positive controls； NC， Negative controls； ♂， Male； ♀， Female； AO， AO blood type； AA， AA blood type； OO， OO blood type； Blank， Control without antibody； F02， Fetal number； ^**P＜0.01.

Figure 3 Identification of GTKO/hCD55 gene-edited cloned piglets
Note： A， Photo of cloned piglets with GTKO/hCD55 gene editing； B， PCR identification results of hCD55 gene in 11 piglets； C， PCR results of the sgRNA target region of the piglet GGTA1 gene； D， GGTA1 genotype of the sgRNA target region in piglet P20； E， Flow cytometry analysis results of α-Gal and hCD55 expression in P20 piglet-derived fibroblasts； F， Immunofluorescence results of piglet kidney tissues，the scale bar indicates 50 μm. M， DNA ladder； WT， wild type.

Figure 4 Methylation levels of hCD55 gene in mixed single-cell clones， fetal pigs， and cloned piglets
Note： A， Schematic representation of CpG island in the hCD55 gene； B， Methylation levels of hCD55 gene in mixed single-cell clones of C35&C45， cloned fetal pig F02 fibroblasts and cloned piglets （P01， P13， P12， P14， P17） kidney tissues. Solid black circles indicate methylation sites， and hollow circles indicate unmethylated sites.

Figure 5 Expression levels of hCD55 in fibroblasts before and after optimization of CpG island region codon
Note： A， Sanger sequencing results of CpG island region in hCD55 gene before and after codon optimization； B， Porcine fibroblasts were transfected with the codon unoptimized and codon optimized vectors （represents by hCD55CG 81% and hCD55CG 69% respectively）， and the expression level of hCD55 in the cells was detected by flow cytometry （triplicate experimental results）. Blank， control without antibody； WT， wild type.

Figure 6 Evaluation of anti-immune rejection efficacy of GTKO/hCD55 gene-edited pigs
Note： A， Binding extent analysis of human IgG and IgM antibodies to porcine fibroblasts after incubation with mixed human serum； B， Porcine cell death analysis induced by complement after incubation of porcine fibroblasts with mixed human serum； C， The statistical analysis results of figures A and B. WT， wild-type； GTKO/hCD55， experimental group； Blank， control without antibody； MFI， mean fluorescence intensity； CDC， complement-dependent cytotoxicity； ^**P＜0.01； ^nsP>0.05.