Fig. 1

Seamless introduction of heterozygous and homozygous PS1 F105C knock-in mutation into healthy human iPSCs using a combination of CRISPR/Cas9 system and piggyBac transposon. A Schematic representation of the PS1 F105C protein. Asterisk represents the mutation site in the transmembrane domain 1 of PS1. Cross represents the endoproteolysis cleavage site of PS1. B Sequence alignment and conservation of F105 residue in PS1 of different species. C Strategy for seamless introduction of the heterozygous F105C mutation a–d and the homozygous F105C mutation (a–h). a Location of F105 in the exon 4 of PSEN1 allele 1; b DSB in the exon 4 of allele 1 after Cas9 cleavage; c Targeting construct of the piggyBac transposon carrying the selectable markers puro-TK, flanked by 500 bp of PS1 wild-type genomic sequences; d Insertion of the piggyBac transposons through HDR; e Location of F105 in the exon 4 of PSEN1 allele 2; f DSB in the exon 4 of allele 2 after Cas9 cleavage; g The targeting construct of the piggyBac transposon carrying the selectable markers HygR-TK, flanked by 500 bp of PS1 wild-type genomic sequences. d Insertion of the other piggyBac transposons through HDR. After a–d, the cells were selected and amplified in a medium containing puromycin to generate the heterozygous PS1 F105C mutant. Puromycin-resistant clones with heterozygous PS1 F105C were transfected with transposase expression plasmids and treated with FIAU to remove the piggyBac-containing clones. Second gene editing was performed in the puromycin-resistant clones with desired heterozygous PS1 F105C and piggyBac to generate the homozygous PS1 F105C mutant. After e–h, the cells were selected and amplified in a medium containing hygromycin. Hygromycin-resistant clones with the desired homozygous PS1 F105C were then transfected with transposase expression plasmids and treated with FIAU to remove piggyBac-containing clones for both alleles. CAG CAG promoter, ITR inverted terminal repeats. D PSEN1 sequencing alignment showing Cas9 cleavage site and intended mutation site for modifying F105C. E The percentage of accurate HDR using this combined strategy. F Sanger sequencing was used to identify PS1 with F105C mutation. G Oct4 immunostaining of iPSC lines