Restoration of F8 mRNA expression by correction of the intronic variant in patient-derived iPSCs. The patient-derived iPSCs with the insertion of the EF1α promoter before the transcriptional start site of F8 were transduced with SpCas9 (RNP) and an 83-nucleotide single oligonucleotide to correct the variant; then a single clone was isolated. (A) Confirmation of the gene correction by DNA sequencing at the variant in the isolated clone. The color blue indicates the replaced nucleotides derived from the single oligonucleotide. (B) Disappearance of aberrant F8 transcripts between exons 14 and 15 by gene correction was assessed with RT-PCR. The red arrows indicate the size of the amplicons. (C-D) Relative expression of the aberrant transcript (C) and F8 mRNA between exons 25 and 26 (D). Values represent the mean ± standard deviation (n = 3). Statistical analysis was performed using one-way analysis of variance with Tukey post hoc multiple comparison test. (E) Representative results of the long-read RNA-seq of the F8 full-length transcript. (F) Magnified image of intron 14. The gray boxes represent the depth of exon coverage, and the blue lines indicate the splicing junctions of F8 mRNA. The exons in the F8 gene are shown at the bottom. Unrepaired, patient-derived iPSCs with insertion of the EF1α promoter; repaired, gene-corrected patient-derived iPSCs with insertion of the EF1α promoter; healthy, healthy donor-derived iPSCs with insertion of the EF1α promoter.