Fig. 3: Treatment of HUDEP2 cells with TETv4 causes localized promoter demethylation and HBG activation.

a Workflow for TETv4 treatments in the HUDEP2 cell line. The TETv4 construct contains a TET1 catalytic domain (CD) to remove CpG methylation linked to catalytically dead Cas9 (dCas9) and three nuclear localization signals (NLS), with blue fluorescent protein (BFP) attached by a P2A linker. dTETv4 used as a negative control, contains a mutation to depress catalytic activity. Editor and guide plasmids were introduced into HUDEP2 cells by electroporation and enriched by fluorescence-activated cell sorting (FACS). Cells were cultured for at least 12 days to eliminate plasmid expression before subsequent analysis. Created in BioRender. Bell, H. (2025) https://BioRender.com/ztcmiqi. b CpG methylation in the HBG distal promoter (left), and HBG proximal promoter and 5′ UTR (right) in TETv4-treated and mock transfected HUDEP2 cells, determined by amplicon bisulfite sequencing. Points indicate mean CpG methylation of two technical replicates, except for TETv4 EPX, where only one replicate was analyzed. Numbered black bars indicate the positions of sgRNAs used to target HBG. NG = no guide. c HBG mRNA expression in HUDEP2 cells treated with TETv4 or controls, normalized to total HBG and HBB expression (HBG/(HBG + HBB)) measured by qPCR. Points represent measurements from two technical replicates. Bars represent mean values. d Serial measurements of HBG mRNA expression in TETv4 HBG-treated and control HUDEP2 lines from (c). at 35 days, 65 days, and 92 days post-nucleofection. Two technical replicates were analyzed across three time points. e Flow cytometry plots showing HbF staining (F-cells) in mock and TETv4 HBG-treated HUDEP2 lines at day 37 (top) and day 92 (bottom). f Relative HBG mRNA expression in HUDEP2 cells treated with TETv4 following subsequent treatment with D3AL and controls. Expression normalized to total HBG and HBB expression (HBG/(HBG + HBB)) measured by qPCR. Points represent measurements from three technical replicates. Bars represent mean values. Source data are provided as a Source Data file.