Figure 2.

The key base substitutions in BCL11A-XL-specific ZnF domains reactivate HbF expression in adult erythroid cells

(A) A diagrammatic representation of each of the target sites to induce nucleobase modifications by using ABE. BCL11A-gRNA targeting initiation codon of BCL11A, Enh-gRNA targeting the BCL11A erythroid-enhancer region, 2 gRNAs each targeting ZnF4 (ZnF4-1 and -2) and ZnF5 (ZnF5-1 and -2), respectively, and 1 gRNA targeting ZnF6 to produce missense mutations. BS gRNA to alter the BCL11A-XL BS in the −115 cluster of HBG promoter. AAVS1-gRNA targeting the safe harbor site at the AAVS1 locus for negative control. (B) Base conversion efficiency at all of the target sites on day 8 of expansion was analyzed using the EditR in silico tool after Sanger sequencing. Mean ± SEM of n = 3 independent experiments. (C) Close-up views of the BCL11A-XL-specific ZnF (ZnF4, ZnF5, and ZnF6) structures, illustrating the amino acids targeted in each of the indicated mutants. Yellow dashed lines indicate hydrogen bonds, and bases are named according to the HBG1/2 promoter numbering. (D) The protein expression analysis by western blot confirms no significant change in BCL11A expression on ZnF editing compared to the reduction of BCL11A protein observed on disrupting the initiation codon and the BCL11A-enhancer; 35 μg of protein used for loading. (E) Analysis of the erythroid differentiation profile (CD71 and CD235a) of the base-edited HUDEP2 cells on the end stage of differentiation (day 8) showed no significant variation among the edited samples. Mean ± SEM of n = 3 independent experiments. (F) Increase in percentage of intracellular HbF levels measured at the end stage of differentiation (day 8) by flow cytometry (HbF⁺ cells) on generated BCL11A ZnF mutants. Mean ± SEM of n = 3 independent experiments. (G) The elevation in the total HbF tetramer formation in adult erythroid cells in the BCL11A-modified cells was analyzed at the end stage of differentiation (day 8). Mean ± SEM of n = 3 independent experiments.