Fig 2: CRISPR-Sunspot in combination with SunTag activation system for gene activation and mRNA imaging. (A) Lentiviral vector maps for HBG1 activation and mRNA imaging. Workflow of CRISPR-Sunspot system for imaging activated HBG1 mRNA. Stable TRE3G-dCas9-24 × GCN_v4 U2OS cells were infected with scFv-sfGFP-VP64 lentivirus. The cells were then transfected with 2 sets of 3 different sgRNA plasmid with PAMmers targeting HBG1 mRNA for subsequent imaging. (B) Representative images of HBG1 mRNA labeling using the CRISPR-Sunspot system. Scale bars, 20 µm and 5 µm (right panel). The first group (i) showed the representative images of non-activation cell lines (# Ctrl) transfected with the non-targeting control guide RNA with PAMmer. The second group (ii) showed the representative images of non-activation cell lines (# Ctrl) transfected with the sgRNA and PAMmer targeting HBG1 mRNA. The third group (iii) showed the representative images of HBG1 activation cell lines (# 18) transfected with sgRNA and PAMmer targeting HBG1 mRNA. The fourth group (iv) showed the representative images of non-activation cell lines (# Ctrl) transfected by HBG1 overexpression vector and sgRNA with PAMmer targeting HBG1 mRNA. (C) Schematic plots showing the CRISPR-Sunspot system and SunTag-mediated gene activation strategies. (D) SNR of CRISPR-Sunspot labeling of HBG1 mRNA in (B) (n =60, 84, 74, 60 cells). (E) smFISH confirms HBG1 mRNA signals labeling by the CRISPR-Sunspot system in HBG1 activation (by VP64) and HBG1 overexpression (HBG1 OE) U2OS cells. Plots of the arbitrary units (a.u.) along the line indicated the fluorescence intensity in the high-magnification images. Scale bars, 20 µm and 5 µm (right panel). The data are displayed as the mean ± S.E.M. One-way ANOVA with Dunnett's multiple comparisons test was used. *P < 0.05, ***P < 0.001, n.s. not significant.

Fig 3: A CRISPR/Cas9 screen identifies UHRF1 as a repressor of fetal hemoglobin (HbF) production.a Single guide RNAs (sgRNAs) that were enriched in HUDEP2 cells expressing high levels of HbF (see Methods for details). The y-axis shows the log2 fold-change (FC) in sgRNAs comparing HbFHigh to HbFLow cells. Dots represent the average for four sgRNAs targeting the same gene. Positive controls for repressors of HbF, including BCL11A, ZBTB7A, VHL, and a BCL11A repressor binding element in the HBG (γ-globin) promoter (labeled HFPH) are shown. UHRF1 is indicated as an orange point. b Cas9-expressing HUDEP2 cells were transduced with lentiviral vector encoding one of two different UHRF1 sgRNAs (g1, g2) or non-targeting (NT) sgRNA control, induced to undergo erythroid maturation, and analyzed after 5 days. Graph shows HBG (HBG2/HBG1) mRNA as a fraction of total mRNA (HBG/(HBG + HBB)) determined by quantitative real-time qPCR (Data are presented as mean ± s.d. of three independent experiments). c %HbF protein (HbF/(HbF + HbA)) determined by ion exchange high-performance liquid chromatography (HPLC) analysis in two independent experiments described in panel b. Bars represent mean values. d-f Normal human peripheral blood CD34+ cells from three independent donors were modified by electroporation of ribonucleoprotein (RNP) consisting of Cas9 + sgRNA targeting the erythroid-specific enhancer in BCL11A intron 2 (BCL11A enh) or UHRF1 protein coding regions, cultured in maintenance medium, and analyzed after 12 days. sgRNA targeting Adeno-associated virus integration site 1 (AAVS1) was used as control. d %HBG mRNA determined by qPCR. (Graph shows the mean percentage ± s.d. of three biological replicate experiments). e HPLC analysis of cellular hemoglobin content with %HbF indicated. (Graph shows the mean percentage ± s.d. of three biological replicate experiments) f Representative flow cytometry plots showing HbF immunostaining cells (F-cells). g Density plots showing global methylation status determined by Methylation EPIC 850 K array in UHRF1-disrupted or control HUDEP2 cells. h RNA-seq analysis showing transcripts per million mapped reads (TPM) in UHRF1-disrupted or control HUDEP2 cells. Each dot represents an individual gene (average of three technical replicate experiments). **P < 0.01, ***P < 0.001, ****P < 0.0001, Multiplicity-adjusted P values by one-way analysis of variance (ANOVA) for b,d,e. Source data are provided as a Source Data file.

Fig 4: Mutation of the MBD2 methyl-CpG binding domain impairs HBG repression.a Schematic of the MBD2 and MBD3 protein sequences showing the methyl-CpG binding domains (MBD) and the location of the MBD2 Y178F mutation to create an MBD3-like domain with impaired methyl-CpG binding. Created in BioRender. Bell, H. (2025) https://BioRender.com/ztcmiqi. b Diagram of components used in EMSA analysis. The MBD2 single construct (MBD2sc) contains the MBD (red), intrinsically disordered region (IDR, blue), and coiled-coil (CC, dark green) of MBD2 linked to the CC of GATAD2A (light green). Target probes for the BCAT1 and HBG promoters are shown with CpG sites (gray pins) indicated. Created in BioRender. Bell, H. (2025) https://BioRender.com/ztcmiqi. c Electrophoretic mobility Shift assay (EMSA) of probes representing BCAT1 or HBG promoter CpG sites complexed with WT and Y178F MBD2sc expressed protein from b. + or - signs represent the presence or absence of each component in the lane. Arrows indicate the positions of free probe, MBD2:probe complexes, and antibody:MBD2:probe (ab) complex supershifts. d MA plot of differentially expressed genes in MBD2 Y178F mutant vs. WT HUDEP2 cells (five clones per group). Colored points represent genes identified as upregulated (red, 21 genes) or downregulated (blue, 12 genes) with a false discovery rate (FDR) cutoff of 1% (Benjamini-Hochberg method). HBG represents transcripts mapping to both HBG1 and HBG2. e Principal component (PC) analysis of transcript expression in MBD2 Y178F mutant and WT HUDEP2 lines. Source data are provided as a Source Data file.

Fig 5: HBG expression correlates with CpG demethylation in the proximal promoter.a Relationship between the distance of the TETv4 target site to CpG sites within the HBG proximal promoters and 5′ UTR, and demethylation activity at those sites following treatment with TETv4 and HBG guides 1-6 individually. Methylation (Y-axis) represents the methylation % in each sample divided by the mean of the methylation % in the mock-treated samples at each site. Position (X-axis) indicates the distance between the CpG site and the center of the sgRNA sequence. Trend line was generated using the LOESS method with span = 1. Points indicate three technical replicates measured at each site and colored by the sgRNA used for TETv4 treatment. b HBG mRNA expression in HUDEP2 cells treated with TETv4 + HBG sgRNAs 1-6 individually or all together. Points represent measurements from three technical replicates. Bars represent mean values. c Pearson’s correlation between the mean methylation % from a. at all CpG sites across the HBG proximal promoter and 5′ UTR (positions −256, −162, −53, −50, +6, +17, +50; left), sites downstream of the transcription start site (TSS) only (positions +6, +17, +50; center) and sites upstream of the TSS only (positions −256, −162, −53, −50; right), vs. %HBG mRNA from b. in HUDEP2 cells treated with TETv4 and HBG sgRNAs 1–6 individually. Trendline fit was determined using the least squares method. d MA plot of differentially expressed genes in TETv4 HBG-treated vs. mock-treated HUDEP2 cells (3 replicates per group). Colored points represent genes identified as upregulated (red, 207 genes) or downregulated (blue, 126 genes) with a false discovery rate (FDR) cutoff of 1% (Benjamini-Hochberg method). HBG represents transcripts mapping to both HBG1 and HBG2. e Principal component (PC) analysis of transcript expression in HUDEP2 lines treated with mock or TETv4 + HBG sgRNAs 1–6 individually or all together. Source data are provided as a Source Data file.