Long-term quantitative tracking of PIG-A–mutated clones by flow cytometry and deep sequencing. (A) Fraction of FLAER^– granulocytes, monocytes, T cells, and B cells over time posttransplantation in ZI35 and ZL19 monitored by flow cytometry. (B) Deep sequencing of gRNA target sites within PIG-A exon 2 was performed to quantitate indel frequency and identify individual indel types in total granulocytes and sorted FLAER^– granulocytes. Heatmap shows the actual sequences retrieved from ZI35 (top) and ZL19 (bottom), with each row representing a different allele and each column representing a time point. The fractional contribution of individual indels to each sample is represented as a color gradient. Indels are indicated by a red font, and expected Cas9 cut site and protospacer adjacent motif (PAM) are shown in the bottom wild-type (WT) sequence. The fraction of alleles with indels in the 20-bp window surrounding the gRNA target site at the PIG-A exon 2 in total granulocytes (C) and sorted FLAER^– granulocytes (D) from ZI35 and ZL19 plotted over time. (E) Targeted deep sequencing of PIG-A exon 2 in ZI35 and ZL19 was performed in FLAER^– blood cells from the following lineages over time; monocytes, CD3⁺ T cells, and CD20⁺ B cells. The percentage of indels at the target site is shown. IP, infusion product.