Fig. 2: LRF BS disruption and KLF1 BS creation in the HBG1/2 promoters of SCD HSPC-derived erythroblasts.

a Experimental protocol used for base-editing experiments in non-mobilized SCD HSPCs. A BE-, a sgRNA- and a GFP- (optional for enzyme plasmids that do not contain a GFP cassette) expressing plasmid were co-transfected in SCD HSPCs and 18 h post-transfection GFP⁺ cells were FACS-sorted based on GFP medium (med) and high (high) expression. b C-G to T-A or A-T to G-C base-editing efficiency, calculated by the EditR software in samples subjected to Sanger sequencing. The LRF 4C editing profile was obtained by pooling data from CBE-NRCH-, CBE-SpG- and CBE-SpRY- treated samples. Data are expressed as mean ± SEM (n = 12 (LRF 4C), n = 4 (LRF 8C), n = 4 (LRF 2T med), n = 4 (LRF 2T high), n = 3 (KLF1 med), n = 4 (KLF1 high) biologically independent experiments, 4 donors). c Frequency of InDels, measured by TIDE analysis for control, base- and Cas9-edited samples subjected to Sanger sequencing. The insertion or deletion of a C (±1 nt) in the homopoly-C stretch of the LRF 2T profile was separated from the overall frequency of InDels, as it was considered a sequencing error (Supplementary Note 2). Data are expressed as mean ± SEM (n = 12 (control), n = 12 (LRF 4C), n = 4 (LRF 8C), n = 4 (LRF 2T med), n = 4 (LRF 2T high), n = 3 (KLF1 med), n = 3 (KLF1 high, n = 3 (Cas9 med), n = 3 (Cas9 high) biologically independent experiments). ****P ≤ 0.0001 (ordinary one-way ANOVA with Dunnett correction for multiple comparisons). d Frequency of the 4.9-kb deletion, measured by ddPCR, for base- and Cas9-edited samples. Data are expressed as mean ± SEM (n = 8 (control), n = 12 (LRF 4C), n = 5 (LRF 8C), n = 3 (LRF 2T med), n = 3 (LRF 2T high), n = 3 (KLF1 med), n = 3 (KLF1 high), n = 3 (Cas9 med), n = 4 (Cas9 high) biologically independent experiments). *P = 0.0125 (ordinary one-way ANOVA with Dunnett correction for multiple comparisons). e 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. The base-editing efficiency is indicated for each sample in the lower part of the panel. Data are expressed as single values or as mean ± SEM (n = 4 (control), n = 6 (LRF 4C), n = 2 (LRF 8C), n = 2 (LRF 2T med), n = 1 (LRF 2T high), n = 1 (KLF1 med), n = 1 (KLF1 high), n = 1 (Cas9 med), n = 2 (Cas9 high) biologically independent experiments, 2 donors). *P = 0.0141 for LRF 4C, or P = 0.0380 for LRF 8C; ****P ≤ 0.0001 (two-way ANOVA with Dunnett correction for multiple comparisons). f Flow cytometry histograms showing the percentage of HbF- and HbS- expressing cells in GYPA^+high population for unstained (GYPA stained only), control (transfected with TE buffer for donor 1 and transfected with TE buffer or with CBE-SpRY plasmid and a sgRNA targeting the unrelated AAVS1 locus for donor 2) and edited samples. g Time-course measurement of the frequency of non-sickle cells upon O₂ deprivation in control (transfected with TE buffer for donor 1 and transfected with TE buffer or with CBE-SpRY plasmid and a sgRNA targeting the unrelated AAVS1 locus for donor 2) and edited samples. Data are expressed as single values or as mean ± SEM (n = 2 (control), n = 3 (LRF 4C), n = 1 (LRF 8C), n = 1 (LRF 2T med), n = 1 (LRF 2T high), n = 1 (KLF1 med), n = 1 (KLF1 high), n = 1 (Cas9 high) biologically independent experiments, 2 donors). h Correlation between HBG mRNA relative expression and base-editing efficiency in single BFU-E colonies (1 donor). HBG mRNA expression was normalized to HBA1/2 mRNA and expressed as a percentage of the total HBB + HBG mRNA. Base-editing efficiency was calculated by the CRISPRESSO 2 software in samples subjected to NGS. Colonies highlighted by a black outline carried InDels. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001 (KLF1: R² = 0.7299, Y = 0.8896*X + 31.37, P < 0.0001 non-zero slope significance; LRF 2T: R² = 0.8574, Y = 0.5977*X + 25.07, P < 0.0001 non-zero slope significance; LRF 8C: R² = 0.6102, Y = 0.4824*X + 23.77, P < 0.0001 non-zero slope significance; LRF 4C: R² = 0.1678, Y = 0.2529*X + 30.52, P = 0.0522 non-zero slope significance; Multiple t test). BFU-Es edited at the AAVS1 locus were used as negative controls. i Frequency of InDels, measured by the CRISPRESSO 2 software, for edited or control (AAVS1) single BFU-E colonies (KLF1 n = 17; LRF 2T n = 17; LRF 8C n = 11; LRF 4C n = 15; AAVS1 n = 8; 1 donor). j Frequency of the 4.9-kb deletion, measured by ddPCR, for edited or control (AAVS1) single BFU-E colonies (KLF1 n = 11; LRF 2T n = 11; LRF 8C n = 9; LRF 4C n = 15; AAVS n = 8; 1 donor). k Frequency of chromosome 11 loss, as indicated by the ratio of CARS (p arm) and PODL1 (q arm), measured by ddPCR, for edited or control (AAVS1) single BFU-E colonies (KLF1 n = 16; LRF 2T n = 15; LRF 8C n = 10; LRF 4C n = 14; AAVS1 n = 9; 1 donor). Source data are provided as a Source Data file.