Fig. 3: RNA-mediated base editing in SCD HPSCs.

a Experimental protocol used for base-editing experiments using BE mRNAs in SCD HSPCs (2 plerixafor-mobilized donors and 1 non-mobilized donor). A BE mRNA and a chemically modified sgRNA were co-transfected in SCD HSPCs. Cells were differentiated into mature RBCs or underwent a CFC assay. b–e C-G to T-A (b, c) or A-T to G-C (d, e) base-editing efficiency, calculated by the EditR software in samples subjected to Sanger sequencing at early (Day 6) or late (Day 13) time points during the in vitro erythroid differentiation protocol. Data are expressed as mean ± SEM (n = 3 biologically independent experiments, 3 donors). f Frequency of InDels, measured by TIDE analysis, for control, base- and Cas9- edited samples. Data are expressed as mean ± SEM (n = 3 biologically independent experiments). ****P ≤ 0.0001 (ordinary one-way ANOVA with Dunnett correction for multiple comparisons). g Frequency of the 4.9-kb deletion, measured by ddPCR, for control, base- and Cas9- edited samples. Data are expressed as mean ± SEM (n = 3 biologically independent experiments). ****P ≤ 0.0001 (ordinary one-way ANOVA with Dunnett correction for multiple comparisons). h CFC frequency for control and edited samples. Data are expressed as mean ± SEM (n = 3 biologically independent experiments, 3 donors). No statistical differences were observed between control and edited samples (two-way ANOVA). i RT-qPCR analysis of β^S- and γ-globin mRNA levels in SCD patient erythroblasts at day 13 of erythroid differentiation. β^S- and γ-globin mRNA expression was normalized to α-globin mRNA and expressed as a percentage of the β^S-+γ- globins mRNA. Data are expressed as mean ± SEM (n = 3 biologically independent experiments, 3 donors). *P = 0.0058; ***P = 0.0003; ****P ≤ 0.0001 (two-way ANOVA with Dunnett correction for multiple comparisons). j Expression of ^Gγ-, ^Aγ-, γ(^Gγ + ^Aγ)-globin chains measured by RP-HPLC in SCD patient RBCs. γ-globin expression was normalized to α-globin. Data are expressed as mean ± SEM (n = 3 biologically independent experiments, 3 donors). *P = 0.0133 for LRF 4C, or P = 0.0257 for LRF 8C; ***P = 0.0002; ****P ≤ 0.0001 (two-way ANOVA with Dunnett correction for multiple comparisons). k Analysis of HbF and HbS by cation-exchange HPLC in SCD patient RBCs. We calculated the percentage of each Hb type over the total Hb tetramers. Data are expressed as mean ± SEM (n = 3 biologically independent experiments, 3 donors). **P = 0.0013; ***P = 0.0003; ****P ≤ 0.0001 (two-way ANOVA with Dunnett correction for multiple comparisons). l Flow cytometry histograms showing the percentage of HbF- and HbS-expressing cells in GYPA⁺ population for unstained (GYPA stained only), control (untreated, or transfected with TE buffer, or transfected with a BE mRNA only, or transfected with a BE mRNA and a sgRNA targeting the unrelated AAVS1 locus) and edited samples. m Frequency of HbF- and HbS- expressing cells in GYPA⁺ population for unstained, control and edited samples. Data are expressed as mean ± SEM (n = 3 biologically independent experiments, 3 donors). *P = 0.0105; **P = 0.0046 for LRF 8C and KLF1, or P = 0.0011 for LRF 2T; ***P = 0.0006 (two-way ANOVA with Dunnett correction for multiple comparisons). n Frequency of sickling cells upon O₂ deprivation in control and edited samples. Data are expressed as single values or as mean ± SEM (n = 3 biologically independent experiments, 3 donors). o Representative photomicrographs of SCD patient RBCs under hypoxia conditions. Red arrows indicate sickling RBCs, and green arrows indicate normal RBCs. Source data are provided as a Source Data file.