Conversion of iPSCs from Pl^A1 to Pl^A2. (A) Genomic DNA, isolated from iPSCs that had been transfected with px462-gRNA1, px462-gRNA2, and Pl^A2-ssODN, was PCR amplified and digested with NciI, which differentiates the Pl^A1 allelic isoform from Pl^A2. Red arrows indicate the expected fragment sizes of a typical clone that had been converted to Pl^A2. (B) Sequencing data confirmed the T>C 29523 point mutation in CRISPR-edited Pl^A2 iPSCs. The red arrow indicates the target T>C mutation. Blue arrows indicate silent mutations that were intentionally introduced into the repair oligo to prevent digestion of the final edited genome by Cas9n. (C) Allele-specific expression of GPIIb-IIIa (CD41) on both native and CRISPR-edited iPSC-derived day 8 HPCs. Nonadherent HPCs express abundant levels of the CD41/CD61 complex (integrin αIIb-β3) as well as CD235 (glycophorin A). Note that both cell lines were similarly double-positive. (D) Cell lysates from wild-type, Pl^A1, and CRISPR-edited Pl^A2 iPSC–derived HPCs were immunoprecipitated with AP3, followed by immunoblotting with human maternal anti-Pl^A2 antiserum. Note that the anti-Pl^A2 antiserum is positive for GPIIIa expressed in the gene-edited, but not native, iPSC line (red arrow), whereas the Pl^A1-selective mAb, SZ21, binds GPIIIa from native, but not gene-edited, iPSCs.