Conversion of Pl^A1-homozygous DAMI cells to Pl^A2 using CRISPR/Cas9n-directed gene editing. (A) GFP⁺ (∼40%) DAMI cells, transfected with px461-gRNA1, px461-gRNA2, and Pl^A2-encoding HDR template, were enriched with fluorescence-activated cell sorting and expanded. (B) Surveyor assay detected the insertions/deletions (indels), indicative of Cas9n-mediated cleavage at the Pl^A locus, in GFP⁺ CRISPR-edited cells. The red bracket indicates the range of expected fragment sizes. (C) Genomic DNA from single-cell GFP⁺ DAMI clones was PCR-amplified and digested with NciI to identify those clones encoding the Pl^A2 allelic isoform of GPIIIa. Red arrows indicate the expected NciI digestion products. Red asterisks indicate Pl^A2+ clones #22 and #24. (D) DNA sequence analysis showed the presence of the HDR-introduced T>C 29523 point mutation in clones #22 and #24. The red arrow highlights the heterozygous partial allelic substitution expected in the multiploid DAMI cell line. (E) Cell lysates from wild-type and clone #24 DAMI cells were immunoprecipitated using the GPIIIa-specific mAb, AP3, followed by immunoblotting with human maternal anti-Pl^A2 antiserum. The relative equivalence of antigen loading was determined by immunoblotting whole-cell lysates (WCL) with AP3 and anti-β-actin antibodies. Note that clone #24, but not wild-type DAMI cells, has a Pl^A2-reactive band (red asterisk).