The −113A>G HPFH mutation elevates γ-globin expression and creates a de novo binding site for GATA1 in vivo. (A) The CRISPR-Cas9 sgRNA target sites used to introduce the −113A>G (HPFH) and −113A>C mutations and the “artificial GATA” site into HUDEP-2(Δ^Gγ) WT cells. The protospacer adjacent motif (PAM) site is indicated along with the various modifications introduced. (B) γ-globin mRNA expression levels, expressed as a percentage (γ/γ+β) in HUDEP-2(Δ^Gγ) WT, −113A>G, −113A>C, and “artificial GATA” clonal populations. Eleven technical replicates are shown for the HUDEP-2(Δ^Gγ) WT parental clone, 10 clonal populations for −113A>G HPFH mutation, and 11 clonal populations for the −113A>C and “artificial GATA” motif. Shown is the mean plus or minus standard error of the mean (SEM). A nonparametric Mann-Whitney statistical test was used to determine statistical significance: P < .0001 (****), P = .0001 to .001 (***), P = .001 to .01 (**). (C) The percentage of HbF protein levels [(HbF/(HbF + HbA)] in HUDEP-2(Δ^Gγ) WT (n = 6), −113A>G (n = 10), −113A>C (n = 11) and “artificial GATA” (n = 11) clonal populations as determined by HPLC. The mean plus or minus SEM is represented. A nonparametric Mann-Whitney statistical test was used to determine statistical significance: P = .0001 to .001 (***). (D) GATA1 ChIP-qPCR in HUDEP-2(Δ^Gγ) WT (n = 7), −113A>G (n = 7), −113A>C (n = 5), and “artificial GATA” site (n = 5) clonal populations. The relative fraction of input, normalized to the positive control (+ctrl) locus ZFPM1 (mean plus or minus SEM), is represented. A nonparametric Mann-Whitney statistical test was used to determine statistical significance: P = .01 to .05 (*). Target loci include the −115 site of the γ-globin promoter, the KLF1 promoter as a +ctrl, and the −4.5 kb upstream region of the IFNB gene as negative control (-ctrl). n.s., nonsignificant. HbA, adult hemoglobin.