Primary erythropoiesis is not affected by GLUT1 KO. (A) Schematic diagram of human CD34⁺ 3-step culture method. PBMCs are isolated from apheresis cones, followed by CD34⁺ magnetic separation. Cells are then nucleofected on day 3 with NT or GLUT1–specific sgRNAs. (B) Flow cytometry histograms show GLUT1 expression of 3 donors and nucleofected with NT or SLC2A1 targeting sgRNAs on days 5, 6, 14, and 21 of differentiation. For days 14 and 21, Hoechst stain was used to identify the reticulocytes. Cells were stained with anti–GLUT1 FITC conjugate (n = 3) or a no-stain control (black). (C) Day-21 filtered reticulocytes stained with anti–GLUT1 FITC conjugate (n = 3) or a no-stain control (black). (D) Percentage of GLUT1–negative population on GLUT1–targeted KO (n = 3) on days 6, 14, and 21 of differentiation. (E) Percentage of reticulocytes (Hoechst stain negative) at day 21 of differentiation on NT control and negative and positive GLUT1 populations of the GLUT1–targeted KO. (F) Percentage of GLUT1–negative population on GLUT1–targeted KO on reticulocytes before and after filtration. (G) Bar graph illustrates GLUT1 expression on reticulocytes from NT and GLUT1–negative and –positive populations of the GLUT1–targeted KO. Data represent the median fluorescence intensity (n = 3). Individual data points are shown. Multiple Mann-Whitney U tests were used to test for differences between groups. ∗P < .05. Error bars indicate standard deviation.