Downregulation of WT CALR in normal CD34⁺cells increases IL-6–dependent CFU-Mk generation. (A) Immunopurified CD34⁺ cells from donor CB units were transfected with a CALR CRISPR-GFP plasmid targeting exon 1 to generate a KO condition, or a mock CRISPR-GFP plasmid; cells were sorted based on high fluorescence intensity and analyzed for CALR mRNA (by qRT-PCR) and protein (by western blotting) expression levels. CALR mRNA levels are expressed relative to RNAseP mRNA levels, used for relative quantity (RQ) calculation. In these same cell preparations, the levels of IL-6 mRNA were measured by qRT-PCR, and expressed relative to WT cells, set at value 1 (panel B). (C-E) CRISPR-edited CD34⁺ cells were sorted based on their high GFP expression and plated in clonogenic plasma clot cultures for growth of CFU-Mk colonies, in the presence (C) or absence (D) of IL-6 in the culture medium. (E) In these experiments, cells were cultured in the absence of IL-6 but in the presence of TCZ (5 ng/mL). Data for each experimental condition represent the mean plus SD of at least 3 experiments. All P values were determined by Student t test (*P < .05; ***P < .001).