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. 2022 Apr 7;139(14):2107–2118. doi: 10.1182/blood.2021014205

Figure 3.

Figure 3.

Disruption of known transcription factor binding sites or intervening sequence in the adult HBB promoter is sufficient to elevate fetal HBG expression. (A) CRISPR-Cas9 sgRNA cut sites (black arrows) and transcription factor binding sites (white boxes). (B) HBG and HBB mRNA levels in WT HUDEP-2 (n = 18) and cells cut with −21 (n = 9), −84 (n = 26), −90 (n = 7), and −98 (n = 27) guides that resulted in >1bp deletions. (C) HBG and HBB mRNA levels in WT HUDEP-2 (n = 18) and cells cut with the −21, −84, −90, and −98 guides: no known binding site (n = 15), weak KLF1 site (n = 8), strong KLF1 site (n = 5), 2 KLF1 sites (n = 8), NFY site (n = 3), NFY and strong KLF1 sites (n = 7), 2 KLF1 sites and NFY site (n = 5), TATA box (n = 9), and whole HBB promoter (n = 3). For panels B-C, values are relative to a mean WT level of 1 and 100 for HBG and HBB, respectively (mean plus or minus SEM). Significance determined by a Mann-Whitney U test (ns P > .05, *P < .05, **P < .01, ***P < .001, ****P < .0001). (D) Hemoglobin protein levels (as determined by HPLC) for WT HUDEP-2 (n = 3) and TATA box mutation (n = 3), with peaks for HbF, HbA and HbA2 indicated. A mean trace is shown with mean percent value plus or minus SEM for each peak.