Gene editing induces HbF expression in cultured erythroid cells. G-CSF–mobilized CD34⁺ cells were edited with Cas9:sgRNA-1 RNP using the Neon Transfection System (supplemental Methods). They were then grown in culture for 21 days under conditions that support erythroid differentiation. Data show studies from 2 different CD34⁺ cell donors (blue or red), with each dot representing separate experiments. (A) Indel frequencies in gene-edited (RNP) and control (C) groups determined 4 days after editing. Dark blue indicates the 13-nt human HPFH mutation; light blue represents all other indels. (B) Cells immunostaining for hemoglobin F (F-cells) measured by flow cytometry at culture day 21. (C) The %HbF in day 21 erythroid cell lysates determined by ion-exchange high-performance liquid chromatography (HPLC). The bar charts in panels A-C show the results as the mean ± SD. **** P < .0001 (unpaired Student t test). (D) Erythroid maturation kinetics at culture days 7, 14, and 21, as determined by immunoflow cytometry measurement of CD49d and Band3 expression on CD235a⁺ erythroid cells. (E) Enucleated cell fractions at culture days 14, 17, and 21, as determined by flow cytometry for CD235a and the cell-permeable DNA dye Hoechst 333412. The numbers in each quadrant show the mean percentages ± SDs. The data reflect studies of CD34⁺ cells from 2 different donors in 7 independent experiments.