Extended Data Fig. 1: Co-depletion of NFIA and NFIX reactivates γ-globin in HUDEP2 cells.
From: Dual function NFI factors control fetal hemoglobin silencing in adult erythroid cells
a, Diagram depicting the structure of NFI gene products. b, Schematic of multiplex AsCas12a sgRNAs. c, Editing efficiency of NFIA, NFIX, NFIC, and BCL11A +58 intronic enhancer in indicated sgRNA infected HUDEP2 cells by AsCas12a (control, n = 4; sgNFIA, n = 4; sgNFIX, n = 4, sgNFIC, n = 2; sgNFIA&X, n = 3; sgNFIA&X&C, n = 4; sgBCL11A +58, n = 2). n represents biological replicates generated from different experiments. Genomic DNAs were obtained from indicated 5 days post-infected HUDEP2 cells. d, Representative gating strategy and HbF staining results in control and indicated Cas12a sgRNA-infected HUDEP2 cells. The first gate selects the live cells in the population. The second gate distinguishes the HbF+ and HbF– population. Experiments were performed twice with similar results. e–h, HUDEP2 cells expressing CRISPR-Cas9 were infected with pLRG2.1 or pLRCherry2.1 lentivirus carrying control sgRNA or sgRNAs targeting indicated NFI genes or the BCL11A +58 enhancer and analyzed at the end of 5 days differentiation. e, Representative immunoblots of NFIA, NFIX, and γ-globin. β-Actin was used as loading control. Experiments were performed three times with similar results. f–h, RT-qPCR quantification of HBG1/2, HBB, and the ratio of HBG/(HBG + HBB) mRNA. Data were normalized to GAPDH (n = 3) and expressed as means ± SEM. *p < 0.05, **p < 0.01. p values were calculated by comparing indicated samples to control using parametric paired two-tailed Student’s t test. f, sgNFIA #1, p = 0.0422; sgNFIA #2, p = 0.0161; sgNFIX #1, p = 0.0644; sgNFIX #2, p = 0.4230; sgNFIA&NFIX #1, p = 0.0154; sgNFIA&NFIX #2, p = 0.0150; sgBCL11A +58, p = 0.0288. g, sgNFIA #1, p = 0.0033; sgNFIA #2, p = 0.0052; sgNFIX #1, p = 0.0005; sgNFIX #2, p = 0.0265; sgNFIA&NFIX #1, p = 0.0007; sgNFIA&NFIX #2, p = 0.0024; sgBCL11A +58, p = 0.0009.
