Extended Data Fig. 8: Comparison of ABE7.10 and ABE8e editing at γ-globin −175A>G in healthy donor CD34+ HSPCs.

Controls included UT and AAVS1 sgRNA. Cells were edited by electroporation with RNPs, incubated in expansion medium for two days, then transferred to erythroid differentiation medium. a, Results of a Design of Experiment (DoE) study to optimize ABE8e concentration and sgRNA molar ratio (see Methods). Red color indicates most efficient editing. An ABE concentration of 8 µM with a 3.5-fold excess of sgRNA was determined to be optimal and used in subsequent experiments. b, AAVS1 editing frequencies six days after electroporation (n = 3). c, Indel frequencies six days after electroporation (n = 9 for UT, ABE7.10 and ABE8e −175; n = 3 for AAVS1, ABE7.10, and ABE8e). d, Cell viability and e, cell recovery two days after electroporation (n = 3). f, %HbF in control edited cells (n = 3). g, Cell number versus days erythroid differentiation (n = 3). h, Representative flow cytometry scatter plots of maturation markers in CD235a⁺ erythroblasts after 7 and 14 days of in vitro erythroid differentiation. i, Summary of multiple experiments to assess cell maturation using gating strategy depicted in panel h. n = 3 replicates from one CD34+ cell donor. j, Representative flow cytometry scatter plot showing enucleated reticulocytes distinguished by loss of staining with the DNA-binding dye Hoechst 33342 (gated). k, Percentage of enucleated CD235a⁺ erythroid cells (reticulocytes) at differentiation day 21 (n = 9 for UT, ABE7.10 and ABE8e −175; n = 3 for AAVS1, ABE7.10 and ABE8e). l, Percentage of HbF versus editing frequencies. A linear regression model was used to correlate %HbF with %−175A > G in panel l (n = 9). Each symbol represents a different donor. Graphs show mean ± SD. The slope (coefficient estimate), coefficient of multiple determination (R²), and P values were calculated based on two-sample t-test (panel k) and a linear regression model (panel l). UT, untreated; ns, not significant.

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