Figure 1. Establishment of an ABE-based system to perturb CREs.
(a) A schematic representation of the overall strategy: 1) Predicting CREs regulating HbF levels. 2) Combining a high-throughput ABEmax screen with computational deconvolution to identify CREs with base-pair resolution. 3) Applying high-resolution CRE mapping to understand functional variants and to identify potential novel therapeutic targets.
(b) ABEmax protein level in wild-type HUDEP-2 (WT) cells and HUDEP-2–ABEmax (ABEmax) cells measured by Western blot analysis. The result is representative of two independent experiments (image was cropped from source data Fig. 1).
(c) Heatmap showing the Pearson correlation of WT and ABEmax cell transcriptomes as measured by RNA-seq. The number in each cell is the correlation coefficient.
(d–i) HUDEP-2–ABEmax cells were transduced with a non-targeting control gRNA (Ctrl) or with a gRNA targeting the BCL11A enhancer (BCL11A–ENH).
(d) Flow-cytometry plots showing the expression of the RBC maturation markers Band3 and CD49d after 5 days of induced differentiation. The result is representative of three independent experiments.
(e) On-target DNA base-editing efficiencies measured by next-generation sequencing. P = 1.8 × 10−4.
(f) BCL11A mRNA measured by real-time RT-qPCR. P = 2.7 × 10−5. (g) The percentage of γ-globin mRNA (γ/γ+β) or β-globin mRNA (β/γ+β) as measured by real-time RT-qPCR.
(h) The hemoglobin (Hb) protein content measured by IE-HPLC after 5 additional days of induced erythroid maturation. The result is representative of three independent experiments.
(i) Flow-cytometry plots showing HbF immunostaining (F-cells). The result is representative of three independent experiments.
*P values were determined using two-tailed unpaired t-test. Bar plots show mean ± S.E.M. of three independent experiments.