Detection of anti-HPA-9b alloantibodies using genetically edited iPSC-derived MKs. (A) Schematic illustration of HDR template and targeting strategy for converting HPA-9a to HPA-9b in HPA-3b iPSC clone. The gRNA binding site (orange bar) and the PAM sequence (magenta) will guide Cas9 to its cleavage site (red arrow head) next to the HPA-9 allele. A 199-bp HPA-9b HDR template was designed to introduce the Val→Met amino acid polymorphism. The G>A mutation responsible for the HPA-9a/HPA-9b polymorphism (highlighted in red) is flanked by 99 nucleotide homology arms. Silent mutations (highlighted in blue) were introduced to prevent re-cleavage by Cas9 and create a PstI site at the target locus that can be used for genotyping. (B) Genomic DNA, isolated from puromycin-resistant iPSC clones was amplified by PCR and digested with PstI, which differentiates the HPA-9b allelic isoform from HPA-9a. Red arrow indicates the expected fragment sizes of a typical clone that had been converted to HPA-9b. (C) Sequencing data confirmed the G>A 13790 point mutation in CRISPR-edited HPA-9b iPSCs. The red arrow indicates the target G>A mutation. (D) Reactions of anti-HPA-9b patient sera with allele-specific iPSC-derived MKs in flow cytometric analysis. All of the HPA-3a (gray), HPA-3b (blue) and HPA-9b (red) iPSC lines were differentiated into CD41⁺/CD42b⁺ MKs. The MKs were incubated with patient sera followed by PE-conjugated donkey anti-human IgG. Anti-HPA-9b P1-P3 sera were clinically confirmed with either an MACE or an MAIPA assay. Anti-HPA-9b P4-P6 were HPA-9b suspected patient samples from clinically unresolved FNAIT cases.