CD44 is dispensable for erythroid differentiation of primary human HSPCs. (A) Schematic of genome editing strategy to generate CD44-null cRBCs from primary human CD34⁺ HSPCs using CRISPR/Cas9. Ribonucleoprotein (RNP) complexes consisting of rCas9 and 2 CD44-targeting sgRNAs were introduced by nucleofection on day 2 of culture. Differentiating erythroblasts were plated on a murine stromal cell layer on day ∼13 to 14 to facilitate recovery of enucleated cRBCs. (B) Flow cytometry analysis of CD44 surface expression in unmodified (WT) cRBCs, or an isogenic population that was nucleofected with RNPs targeting CD44 (CD44-CRISPR). Anti-CD44 antibody BRIC 222 (IBGRL) was used at a ratio of 1:10 000 followed by goat anti-mouse IgG-PE (1:2000). (C) Representative growth curves of primary human CD34⁺ HSPCs during ex vivo erythropoiesis. Nucleofection was performed on day 2 to generate CD44-CRISPR cells, Cas9 control cells (WT cells nucleofected with rCas9 only), or mock control cells (nontransfected). (D) Cytospin images of CD44-CRISPR vs isogenic WT erythroid cells during the time course of ex vivo erythropoiesis. Cas9, nucleofected with Cas9 only; Mock, nontransfected; scale bar, 10 μm. (E) Representative experiment showing enucleation rate of CD44-null cRBCs upon terminal differentiation, as detected by flow cytometry using the cell-permeable DNA stain Vybrant DyeCycle Violet.