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. 2023 Jun 2;12:e85258. doi: 10.7554/eLife.85258

Figure 3. Endogenous editing of the HBD promoter in HSPCs.

(A) Editing efficiencies showing percentages of unmodified, NHEJ, or HDR alleles. Conditions tested were Cas9 and sgRNA RNP with no repair template (no RT), K, DT, and KDT repair templates. The data is presented as independent editing experiments with four different donor samples. (B) qRT-PCR of HBD after pooled editing of HSPCs with no RT, K, DT, and KDT. Cells were expanded in erythroid expansion conditions and differentiated for 5 days. Data is plotted as % of all β-like globins (HBB, HBG1/2, HBD). The data is presented as independent editing experiments with four different donor samples. (C) qRT-PCR of HBD of clonal erythroblast populations after 5 days of differentiation. Genotypes were determined by NGS. Data is plotted as % of β-like globins (HBB, HBG1/2, HBD). The data is presented as independent editing experiments with four different donor samples and each dot denotes an individual clonal population. (D) Editing efficiencies showing percentages of unmodified, NHEJ, or HDR alleles. Conditions tested were Cas9 and sgRNA RNP with no repair template (no RT), KDT repair template, and KDT repair template with AZD-7648 (KDT +enh). The data is presented as one editing experiment with three different donor samples. (E) qRT-PCR of HBD after pooled editing of HSPCs with no RT, KDT, or KDT +enh. Cells were expanded in erythroid expansion conditions and differentiated for 5 days. Data is plotted as % of all β-like globins (HBB, HBG1/2, HBD). The data is presented as one editing experiment with three different donor samples.

Figure 3.

Figure 3—figure supplement 1. Characterization of HBD promoter edited CD34 +HSPCs.

Figure 3—figure supplement 1.

(A) Editing efficiencies showing percentages of indels after pooled editing of HSPCs with no RT. The data is presented as one editing experiments with four different donor samples. (B) qRT-PCR of HBD after pooled editing of HSPCs with no RT. Cells were expanded in erythroid expansion conditions and differentiated for 5 days. Data is plotted as % of all β-like globins (HBB, HBG1/2, HBD). The data is presented as one editing experiment with four different donor samples. P value indicates paired, two-tailed student t test (ns, non-significant; *, p≤0.05; **, p≤0.01). (C) An example subset of genotypes of WT/WT, WT/NHEJ, NHEJ/NHEJ, NHEJ/HDR, and HDR/HDR clonal erythroblasts as analyzed by CRISPResso2 of NGS data. (D) qRT-PCR of HBD of clonal erythroblast populations after 5 days of differentiation. Genotypes were determined by NGS. Data is plotted as normalized to HBA. The data is presented as independent editing experiments with four different donor samples and a dot denotes individual clonal populations.