Journal: bioRxiv

Article Title: Near completely reversing the γ- to β-globin switch by enhancer release, retargeting and reinforcing

doi: 10.64898/2026.01.30.702713

Figure Lengend Snippet: a The results of the CRISPR–Cas9 screen showing enriched sgRNAs in HUDEP-2 cells expressing high levels of HbF. The y-axis shows the log 2 (fold-change (FC)) in sgRNAs. The sgRNAs targeting the homologous sequences of HBB and HBD (blue dots), the proximal CACCC motif (red dot) and the distal CACCC motif (orange dot) in HBB are highlighted. b HbF levels of individuals with mutations in the CACCC motif in the HBB promoter in the Globin Gene Server. c Cas9-expressing HUDEP-2 cells were transduced with sgRNAs targeting the CACCC motif in HBB . The average editing efficiencies of PM-1, PM-2 and DM were 79.1%, 67.4% and 45.4%, respectively. The charts show β-like globin gene expression relative to β-actin mRNA expression as measured by RT–qPCR (mean ± s.d., n = 3). Multiple comparisons were assessed with one-way ANOVA with Tukey’s MCT. * P < 0.05, ** P < 0.01 and *** P < 0.001. d Chromosome conformation capture analysis of control and CACCC motif edited HUDEP-2 cells (mean ± s.d., n = 3). e The relative frequencies of the transcriptional bursts of HBB and HBG in HUDEP-2 clones were tested by Chromium single cell sequencing. f ATAC-seq signals at the β-globin cluster were analyzed in control and CACCC motif edited HUDEP-2 cells, along with CUT&Tag enrichment for CTD, H3K4me3, H3K9Ac, and H3K27Ac.

Article Snippet: A total of 3,784 sgRNAs were designed targeting the downstream region of HBG (hg38/GRCh38, chr11: 5204054-5248601) , followed by the generation of a pooled lentiviral vector library using the lentiGuide-Puro plasmid (Addgene, plasmid 52963) that contains a puromycin-resistance cassette for selection.

Techniques: CRISPR, Expressing, Transduction, Gene Expression, Quantitative RT-PCR, Control, Clone Assay, Single Cell, Sequencing

Journal: bioRxiv

Article Title: Near completely reversing the γ- to β-globin switch by enhancer release, retargeting and reinforcing

doi: 10.64898/2026.01.30.702713

Figure Lengend Snippet: a Cas9-expressing HUDEP-2 cells were transfected with sgRNAs targeting the TGACCA motif in HBG , followed by subsequent transfection with sgRNAs targeting the CACCC motif in HBB . b β-like globin gene expression relative to β-actin mRNA expression in HUDEP-2 cells from ( a ) as measured by RT–qPCR (mean ± s.d., n = 3). c β-like globin mRNA levels relative to β-actin mRNA levels in HUDEP-2 wild type (WT) clones (n = 7), GM clones (n = 14), PG clones (n = 6) and DG clones (n = 4) on day 5 of erythroid differentiation (mean ± s.d.). All GM, PG and DG clones carried four copies of HBG . d Fetal hemoglobin protein levels (normalized to total protein at 280 nm per 100 mAU*min) in four clonal populations from ( c ) as determined by HPLC. e Fetal Chromosome conformation capture analysis of HUDEP-2 cells from ( a ) (mean ± s.d., n = 3). f The relative frequency of the transcriptional bursts of HBB and HBG in HUDEP-2 clones from ( c ) were assessed by Chromium single cell sequencing. g ATAC-seq signals at the β-globin cluster were analyzed in HUDEP-2 cells from ( a ), along with CUT&Tag enrichment for CTD, H3K4me3, H3K9Ac, and H3K27Ac.

Article Snippet: A total of 3,784 sgRNAs were designed targeting the downstream region of HBG (hg38/GRCh38, chr11: 5204054-5248601) , followed by the generation of a pooled lentiviral vector library using the lentiGuide-Puro plasmid (Addgene, plasmid 52963) that contains a puromycin-resistance cassette for selection.

Techniques: Expressing, Transfection, Gene Expression, Quantitative RT-PCR, Clone Assay, Single Cell, Sequencing

Journal: bioRxiv

Article Title: CircNFATc3 promotes Fetal Hemoglobin induction by regulating let-7b/GATA2 axis

doi: 10.1101/2025.10.19.682992

Figure Lengend Snippet: a. Leishman stain showing morphology of EPO-differentiated K562 cells at 0h, 48h and 72h. b.i. Flow cytometry plot shows CD71+ and CD235a+ populations at 0h, 48h, 72h to assess erythroid differentiation of EPO-treated K562 cells. b.ii. Bar graph representing flow cytometry data of EPO-treated K562 cells undergoing erythroid differentiation. c. RT-qPCR analysis of γ-globin mRNA expression at different concentrations of HU-treated K562 cells. d.i. Immunoblot showing HBG protein expression in HU-treated and control K562 cells. d.ii. Bar graph showing the quantitative expression of HBG against β-actin. e. qRT-PCR analysis of predicted circRNAs in HU-treated cells. f. qRT-PCR analysis of let-7 family of miRNAs in high HbF cells. g. RT-qPCR of GATA2 in EPO (0h, 48h, and 72h) and HU-treated K562 cells. h. Schematic diagram showing the linear and circular isoforms transcribed from NFATc3 gene locus and mapping of the divergent and convergent primers. i.i. PCR validation of circNFATc3 using divergent primers. i.ii. Sanger sequencing showing the backspliced junction of circNFATc3. j. qRT-PCR analysis showing resistance of circNFATc3 against RNase R digestion. k. qRT-PCR showing cytoplasmic abundance of circNFATc3. l. qRT-PCR showing preferential backsplicing of NFATc3 in HU-treated K562 cells. All experiments were performed in three biological replicates and graphical data are represented as Mean ± SEM, *p≤0.05, ***p≤0.01, ***p≤0.001, ****p<0.0001.

Article Snippet: The membrane was incubated overnight at 4°C with specific primary antibodies, which included, anti-HBG antibody (#39386, CST), anti-GATA2 antibody (#4595, CST), anti-β-actin antibody (#ab8227, abcam) and anti-H3 antibody (#A2348, abclonal).

Techniques: Leishman Stain, Flow Cytometry, Quantitative RT-PCR, Expressing, Western Blot, Control, Biomarker Discovery, Sequencing

Journal: bioRxiv

Article Title: CircNFATc3 promotes Fetal Hemoglobin induction by regulating let-7b/GATA2 axis

doi: 10.1101/2025.10.19.682992

Figure Lengend Snippet: a. Schematic diagram showing transfection of miRNA mimics (let-7b mimic and miR-NC) in K562 cell line. b. Cell Titer-Glo assay results showing proliferation of the cells at different time points post miRNA transfection. c. qRT-PCR analysis of i. Let-7b, ii. circNFATc3, iii. GATA2, iv. γ(A+G), and v. β-globin. d.i. Immunoblot analysis showing expression of GATA2, Histone H3, HBG, and β-actin in miRNA transfected K562 cells, d.ii. Relative expression analysis of immunoblot bands of GATA2 and HBG. e.i. Schematic showing GATA2 3’UTR sequence seed matching with let-7b and mutated GATA2 seed region mismatched with let-7b, ii. Schematic showing RNAhybrid output of hsa-let-7b-5p and GATA2 interaction (MFE=-28.9kcal/mol) iii. Relative luciferase activity in HEK293T cells co-transfected with let-7b mimics and luciferase reporter vector. All experiments were performed in three biological replicates and graphical data are represented as Mean ± SEM, *p≤0.05, ***p≤0.01, ***p≤0.001, ****p<0.0001.

Article Snippet: The membrane was incubated overnight at 4°C with specific primary antibodies, which included, anti-HBG antibody (#39386, CST), anti-GATA2 antibody (#4595, CST), anti-β-actin antibody (#ab8227, abcam) and anti-H3 antibody (#A2348, abclonal).

Techniques: Transfection, Glo Assay, Quantitative RT-PCR, Western Blot, Expressing, Sequencing, Luciferase, Activity Assay, Plasmid Preparation

Journal: bioRxiv

Article Title: CircNFATc3 promotes Fetal Hemoglobin induction by regulating let-7b/GATA2 axis

doi: 10.1101/2025.10.19.682992

Figure Lengend Snippet: a. Schematic diagram showing K562 cells stably overexpressed with circNFATc3 and NFATc3 exon 2-3. b. Cell Titer-Glo assay results showing proliferation of the cells at different time points post-transfection of pcDNA3.1(+)-circRNA mini vector-NFATc3 exon 2-3 and pcDNA3.1(+)-NFATc3 exon 2-3 c. qRT-PCR analysis of i. circNFATc3, ii. let-7b, iii. GATA2, iv. γ-globin, v. β-globin. d.i. Immunoblot analysis showing expression of GATA2, H3, HBG, β-actin. ii. Relative expression analysis of immunoblot bands of GATA2 and HBG. e.i. Flow cytometry analysis of F-cells overexpressing circNFATc3 and NFATc3 exon 2-3. ii. Bar graph of representative flow cytometry data showing percentage F-cell population. All experiments were performed in three biological replicates and graphical data are represented as Mean ± SEM, *p≤0.05, ***p≤0.01, ***p≤0.001, ****p<0.0001.

Article Snippet: The membrane was incubated overnight at 4°C with specific primary antibodies, which included, anti-HBG antibody (#39386, CST), anti-GATA2 antibody (#4595, CST), anti-β-actin antibody (#ab8227, abcam) and anti-H3 antibody (#A2348, abclonal).

Techniques: Stable Transfection, Glo Assay, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: CircNFATc3 promotes Fetal Hemoglobin induction by regulating let-7b/GATA2 axis

doi: 10.1101/2025.10.19.682992

Figure Lengend Snippet: a. qRT-PCR analysis of i. GATA2 and ii. HBG in let-7b and miR-NC transfected circNFATc3 and NFATc3 overexpressing cells. b.i. Immunoblot analysis of HBG in overexpressed circNFATc3 and NFATc3 cells co-transfected with let-7b mimics and miR-NC. ii. Bar graph showing relative expression analysis of immunoblot bands of HBG. c.i. Schematic showing circNFATc3 sequence seed matching with let-7b against and mutated circNFATc3 seed region mismatched with let-7b ii. Schematic showing RNAhybrid output of circNFATc3 and hsa-let-7b-5p interaction (MFE=-25.8kcal/mol). iii. Relative luciferase activity in HEK293T cells co-transfected with let-7b mimics and luciferase reporter vector. d.i. Schematic showing let-7b-mediated RNA-pull down assay methodology. ii. RT-qPCR showing enriched circNFATc3 expression. All experiments were performed in three biological replicates and graphical data are represented as Mean ± SEM, *p≤0.05, ***p≤0.01, ***p≤0.001, ****p<0.0001.

Article Snippet: The membrane was incubated overnight at 4°C with specific primary antibodies, which included, anti-HBG antibody (#39386, CST), anti-GATA2 antibody (#4595, CST), anti-β-actin antibody (#ab8227, abcam) and anti-H3 antibody (#A2348, abclonal).

Techniques: Quantitative RT-PCR, Transfection, Western Blot, Expressing, Sequencing, Luciferase, Activity Assay, Plasmid Preparation, Pull Down Assay

Journal: bioRxiv

Article Title: CircNFATc3 promotes Fetal Hemoglobin induction by regulating let-7b/GATA2 axis

doi: 10.1101/2025.10.19.682992

Figure Lengend Snippet: a . Schematic diagram showing transfection of siRNA (si-circNFATc3 and si-NC) in K562 cell line. b. Cell Titer-Glo assay results showing proliferation of the cells at different time points post-transfection of different concentrations of siRNA. c. RT-qPCR analysis shows i. dosage optimisation of siRNA on circNFATc3, ii. γ-globin and iii. GATA2. d.i. Immunoblot analysis of HBG expression upon siRNA-mediated circNFATc3 silencing and ii. Relative expression analysis of immunoblot bands of HBG in si-circNFATc3 and si-NC transfected cells. All experiments were performed in three biological replicates and graphical data are represented as Mean ± SEM, *p≤0.05, ***p≤0.01, ***p≤0.001, ****p<0.0001.

Article Snippet: The membrane was incubated overnight at 4°C with specific primary antibodies, which included, anti-HBG antibody (#39386, CST), anti-GATA2 antibody (#4595, CST), anti-β-actin antibody (#ab8227, abcam) and anti-H3 antibody (#A2348, abclonal).

Techniques: Transfection, Glo Assay, Quantitative RT-PCR, Western Blot, Expressing