Extended Data Figure 7 |. Editing of BCL11A enhancer in SCD patient (β^Sβ^S) HSPCs.

NBSGW mice were transplanted with 3xNLS-Cas9 RNP with MS-sgRNA-1617 edited β^Sβ^S_#1 CD34⁺ HSPCs 24 h (day 1) or 48 h (day 2) after electroporation. BM were collected 16 weeks after transplantation and analyzed by flow cytometry for human cell chimerism (a), multilineage reconstitution (b) or human erythroid cells (c) in BM, as well as the indel frequencies determined by TIDE analysis (d). Error bars indicate standard deviation. e, Editing efficiency of 3xNLS-Cas9 coupled with MS-sgRNA-AAVS1 for control and −1617 for BCL11A enhancer editing in β^Sβ^S_#2 CD34⁺ HSPCs as measured by TIDE analysis. f, β-like globin expression by RT-qPCR analysis in erythroid cells in vitro differentiated from RNP edited β^Sβ^S_#2 CD34⁺ HSPCs. Error bars indicate standard deviation (n = 3 replicates). g, Multilineage reconstitution analysis of BM collected from mice engrafted with control or edited CD34⁺ HSPCs (from donor β^Sβ^S_#2). h, Analysis of in vitro sickling of unedited control or edited enucleated β^Sβ^S_#2 erythroid cells. Images were taken every 1 minute after MBS treatment. Result shown as percent sickled cells at each time point. Data are plotted as mean ± SD for (b, e, f, g). Median of each group with 1 to 3 mice is shown as line for the other panels.