The expression of aberrant F8 transcripts in patient-derived iPSCs by insertion of the EF1α promoter. (A) DNA sequencing (a part of F8 intron 14) of iPSCs derived from a healthy volunteer and the patient with hemophilia A. The red nucleotide represents the variant. (B-F) Healthy donor and patient-derived iPSCs were treated with the SpCas9 RNP, and the AAV vector harboring the EF1α promoter with homology arms, and then single clones were isolated. (B) Experimental scheme for inserting the EF1α promoter before the transcriptional start site of F8. The red arrows indicate the primers used to confirm the insertion. (C) The insertion of the EF1α promoter was confirmed by PCR in an isolated clone. (D) Relative F8 mRNA expression assessed by quantitative reverse transcription (RT) PCR in healthy volunteer–derived iPSCs without or with the insertion of the EF1α promoter. Values represent the mean ± standard deviation (n = 3). Statistical analysis was performed by two-tailed Student t test. (E-F) Identification of aberrant F8 transcripts between exons 14 and 15 by RT-PCR in patient-derived iPSCs with insertion of the EF1α promoter. (E) The transcript between exons 14 and 15 assessed by microchip gel electrophoresis. The red arrows indicate the size of the amplicons. (F) Sequencing of the aberrant F8 transcript between exons 14 and 15 from patient-derived iPSCs. Additional sequences (124 bp) were inserted between exons 14 and 15. (G) Hypothesis for insertion of the aberrant mRNA sequence by abnormal splicing in the patient. The variant (red) at intron 14 in the patient’s F8 genome may provide an abnormal splice acceptor site, leading to the insertion of 124 bp of the pseudoexon just after the variant. The gray boxes indicate the pseudoexon. ITR, inverted terminal repeat; RQ, relative quantification; SA, splicing acceptor; SD, splice donor.