Extended Data Fig. 5: 3′ HBG1 enhancer–edited clones in HUDEP-2 cells.
From: Single-nucleotide-level mapping of DNA regulatory elements that control fetal hemoglobin expression
(a) Genome browser screenshot of ZBTB7A occupancy profiles in HBG1 locus. gRNA track showing the location of the gRNA. Wild type HUDEP-2 ChIP-seq was downloaded from GSE103445. Two mutated clones (designated H2_mut_C1 and H2_mut_C2) were generated using ABEmax and gRNAs targeting 3′ HBG1 CRE. The position of the CRE was highlighted in blue. (b) Amplicon sequencing confirming the mutations in HUDEP-2 cells derived from single clones after treatment with the Chr11-3 gRNA. Edited adenines are marked in red box. (c–e) Validation studies of two HUDEP-2 single clones with mutations in the 3′ HBG1 enhancer. (c) The percentage of γ-globin mRNA as determined by real-time RT-qPCR. The error bars represent the ± S.E.M from three independent experiments. **** P = 4X10−7; unpaired t-test, two side. (d) The hemoglobin F fraction measured by IE-HPLC. The values represent the mean ± S.E.M from three independent experiments. ****P = 6X10−7; unpaired t-test, two side. (e) F-cell fractions measured by immuno-flow cytometry (left). The bar chart (right) shows the values from two independent experiments.
