Fig. 8: Characterization of ex vivo base editing of HbSS CD34+ cells to install Makassar hemoglobin variant.

a Protospacer and PAM (blue) sequence used for base editing approach to target the HbS allele to convert sickle valine to alanine with target adenines highlighted. b NGS of non-mobilized (n = 2 donors) and mobilized peripheral blood (n = 1 donor) HbSS CD34+ cells 48 h post-electroporation and d14 in vitro erythroid differentiated (IVED) cells indicating highly efficient Makassar editing and low percentage non-synonymous bystander editing. c Globin quantification of relative abundance of Makassar and HbS globins by UHPLC of bulk IVED cultures from (b). Two independent IVED cultures were generated for the mobilized donor and quantified for globin abundance. d Allelic breakdown of editing outcomes from single IVED clones derived non-mobilized (n = 2 donor) and mobilized (n = 1 donor) shown in (a). For non-mobilized donor (n = 172 and 128 single cell colonies) and for mobilized HbSS donor (n = 83 single cell colonies), single colonies were analyzed respectively. e Globin abundance of bulk (n = 1 donor) and single IVED colonies (n = 18 single cell colonies) of a HbAS donor and mono-allelically edited Makassar HbSS IVED colonies (n = 14 colonies). ND not detected, SC single colonies, bulk d18 IVED editing measured at day 18 following IVED. Source data are provided as a Source Data file. Data is presented as mean ± standard deviation.