Detection of anti-HPA-3a and HPA-3b alloantibodies using genetically edited iPSC-derived MKs. (A) Schematic illustration of donor plasmid and targeting strategy for converting HPA-3a to HPA-3b in B2MKO iPSCs. Red triangles flanking exon 26 of the ITGA2B gene indicate the 2 gRNA binding sites that will guide Cas9 to remove the entire exon encoding HPA-3a. HDR donor plasmid contains the removed sequence by Cas9 cleavage (orange box) with targeted T>G mutation responsible for HPA-3b conversion, flanked by 600-bp homology arms on each side (orange line). The recognition sequence and the PAM sequence of guide 2 (green line) are added to both ends of the homology arms for linearizing the donor templates in the transfected cells. Donor plasmid also contains silent mutations (blue X) to prevent re-cleavage by Cas9 and to generate an MfeI site for genotyping. (B) Genomic DNA, isolated from puromycin-resistant iPSC clones was amplified by PCR and digested with MfeI, which differentiates the HPA-3b allelic isoform from WT HPA-3a. Red arrows indicate the expected fragment sizes of a typical clone that had been converted to HPA-3b. (C) Sequencing data confirmed the T>G 13809 point mutation in CRISPR-edited HPA-3b iPSCs. The red arrow indicates the target T>G mutation. (D) Reactions of anti-HPA-3a and anti-HPA-3b patient sera with allele-specific iPSC-derived MKs in flow cytometric analysis. Both HPA-3a (gray) and HPA-3b (blue) iPSC lines were differentiated into CD41⁺/CD42b⁺ MKs. The MKs were incubated with patient sera followed by phycoerythrin (PE)-conjugated donkey anti-human immunoglobulin G (IgG). Anti-HPA-3a P3 patient serum did not contain anti-HLA class I antibody and was detectable only by using a whole-platelet assay in a clinical diagnostic laboratory. Other anti-HPA-3a and anti-HPA-3b patient sera were all clinically confirmed with MACE or MAIPA assays. For, forward; Rev, reverse.