Fig 2: Process of human IAPP precursor processing to form mature IAPP and homology comparison of IAPP mature proteins in different species.a Human IAPP was processed to form mature IAPP. The signal peptide consisting of 22 amino acid residues is represented by blue, the flanking regions of the original N-terminal and C-terminal regions of IAPP are indicated by green, and the mature peptide sequences are indicated by red. b Comparison and analysis of amino acid sequence homology of mature IAPP proteins between different species. c Unrooted tree of IAPP mature protein between different species

Fig 3: Sequence analysis of mature pig-to-human IAPP protein and construction of the humanized IAPP gene CRISPR/Cas9 system.a Sequence homology comparison between pig and human IAPP mature protein. The red box marks the different nucleotide sequences of the porcine and human IAPP mature proteins. The blue box marks the nucleotide sequence that does not cause amino acid changes. b A pie chart of amino acid differences between the pig and human mature IAPP proteins. The red part indicates the percentage of differential amino acids (33.64%); the black part indicates the percentage of the same amino acids. c A nucleotide difference pie chart corresponding to the mature IAPP protein of pigs and humans. The red part indicates the percentage of differential nucleotides (15.32%); the black part indicates the percentage of the same nucleotides (84.68%). d Schematic representation of sgRNAs to porcine IAPP exon 3. sgRNA targeting sequences are shown, and PAMs are highlighted in blue. e Genomic sequences of CRISPR target regions in wild-type PFFs and transfected PFFs, as indicated. Cleavage sites are labeled with red arrows. f Off-target detection. The sequencing peaks showed that both sgRNAs were not cleaved at other sites

Fig 4: The preparation of hIAPP miniature pigs.a Experimental flow of IAPP-humanized pig construction. ① Isolation and culture of miniature PFFs; ② Transfection of miniature PFFs; ③ Inoculate cells into 10 cm culture dishes using the limiting dilution; ④ Monoclonal cell picking; ⑤ Repeat step ④; ⑥ Positive colonies were screened by restriction enzyme digestion assay; ⑦ Expansion of culture-positive clones; ⑧ Somatic cell nuclear transfer; ⑨ Embryo transfer; ⑩ Identification of hIAPP cloned pigs. b, c Positive cloned cells were identified by restriction enzyme digestion assay and verified by Sanger sequencing. d Statistics of blastocyst development were performed on 4 positive clones. 8.5 days after nuclear transfer, the cells that could develop normally into blastocysts, and the results of nuclear staining also prove this conclusion. e Statistical analysis of the blastocyst development rate of positive cloned cells. No significant differences were found compared to the control group