Fig. 6: Functional correction of patient-specific endothelial cells differentiated from iPSCs.

a RecF8 expression corrects the int1h inversion. Overview of primer arrangement around the first and second loxF8 site to detect the orientation of the 140 kb fragment (top panel). Agarose gel of PCR products obtained on genomic DNA from patient-specific ECs with or without treatment with RecF8 (lower panel). Marker (M) lanes at 1 kb and 3 kb are indicated. WT EC = ECs differentiated from a donor that does not carry the exon 1 inversion. F8 EC = ECs differentiated from a hemophilic donor carrying the exon 1 inversion. ‘+RecF8’ = cells were transfected with RecF8 mRNA. Neg. = water control. b qPCR-based quantification of the genomic inversion efficiencies in patient-specific ECs with and without RecF8 treatment. Inversion quantification of WT ECs is shown as reference (n = 3, replicates are shown as dots). Error bars represent standard deviation of the mean (SD). c qPCR-based quantification of Factor VIII mRNA transcript. The relative expression of the Factor VIII mRNA was measured after treating patient-specific ECs with or without RecF8 mRNA (n = 3, replicates are shown as dots). Error bars represent standard deviation of the mean (SD). Factor VIII mRNA expression in untreated WT ECs was used for normalization. d Schematic depiction of the first three exons of the Factor VIII mRNA and the primers used for detecting the transcript. A gel image and sequencing read of Factor VIII cDNA of RecF8 treated patient-derived ECs is shown. The exon1–exon2 boundary can only occur, if the inversion is corrected to the WT orientation, the gene is transcribed and the pre-mRNA spliced correctly. e Quantification of indels introduced by CRISPR/Cas9 or RecF8 after inverting the loxF8 genomic locus. Counts of clones harboring different kinds of indels are shown in the pie charts. 22 clones were analyzed for each condition. Parts of the figure were created with BioRender.com. Source data are provided as a Source data file.