Deletion of the HBB promoter leads to increased expression of HBG and increased HbF in HUDEP-2 cells, whereas deletion of the HBB coding region does not significantly affect HBG expression. (A) The CRISPR-Cas9 sgRNA cut sites for the introduction of the HBB promoter deletion (above) and the HBB gene deletion (below). The promoter (pink bar) and gene (red bar) are indicated with the intron (IVS) locations provided. (B) HBG and HBB mRNA levels in WT HUDEP-2 clonally sorted (n = 12), HBB promoter deletion (n = 19), and HBB gene deletion clonal populations (n = 5) (mean plus or minus SEM). Significance was determined by a Mann-Whitney U test (**P < .01, ****P < .0001). (C) Hemoglobin protein levels as determined by HPLC for clonally sorted WT HUDEP-2 (n = 3), HBB promoter deletion (n = 3), and HBB gene deletion (n = 2), with peaks for HbF, HbA, HbA2, and free α-globin chains indicated by the arrows above the peaks. A mean trace is shown for each with mean percent value plus or minus SEM for each hemoglobin peak.