Targeted gene correction of the mutant RUNX1 allele in FPD/AML iPSCs. (A) Schematic of RUNX1 protein and genomic locus, as well as the gene-targeting strategy to correct the FPD mutation. (Green bolt) ZFN recognition/cleavage site. (Red vertical line) location of the Y260X mutation. Schematic of RUNX1 correction vector is also shown, and proper targeting introduces a new BglII site to the RUNX1 locus. (Gray box) location of the DNA probe for Southern blot hybridization. (B) Screening of selected iPSC clones from gene targeting with Southern blot hybridization using the probe in (A). Clones A to F contain integrations of the targeting vector in the RUNX1 locus. III-5: The FPD iPSC line derived from FPD patient III-5, which was used for targeted mutation correction. “WT” and “Targeted” denote germline and gene-targeted bands (4.0 kb and 3.2 kb) detected by the probe, respectively. (C) Detection of single nucleotide polymorphisms (SNP) by genomic polymerase chain reaction (PCR) to determine which RUNX1 allele was targeted. (Red arrows) locations of primers used in PCR. The combination of AG alleles at the indicated SNPs is associated with the FPD mutant chromosome, and GA allele combination is for the wild-type chromosome. Three of the 7 targeted iPSC lines had insertion of the targeting vector into the FPD chromosome. (D) Sequencing of bulk (sequencing traces) and individual reverse transcription (RT)-PCR products shows only the production of wild-type RUNX1 transcripts (blue peak for C) in corrected clones (clones E and F), whereas the original FPD iPSCs (III-5) produced both mutant (green peak for A) and wild-type (8/28 and 20/28 of the sequenced clones, respectively) RUNX1 transcripts.