Improved genome editing of the HBG1 and HBG2 promoters in HSCs by Cas9-3xNLS. (A) Schematic overview of Cas9-2xNLS and Cas9-3xNLS proteins showing locations of nuclear localization signals. (B) Normal human CD34⁺ cells from 2 different donors (#1 and #2 represented by blue and red dots, respectively) were edited using 4 different delivery methods with the Lonza 4D-Nucleofector platform (x-axis) with RNPs composed of sgRNA-1 and either Cas9-3xNLS (red bar) or Cas9-2xNLS (blue bar), then cultured in CD34⁺ cell maintenance medium (supplemental Table 2). Nonedited donor-cell controls (C) were processed in parallel, but not electroporated. Indels were analyzed at day 3 by NGS. (C-F) Normal CD34⁺ cells from 2 different donors were edited with the Lonza 4D-Nucleofector (EO-100 program) and RNP composed of sgRNA-1:Cas9-3xNL and then transplanted into NBSGW mice. (C) Normalized chimerism of human CD45⁺ donor cells in recipient bone marrow at 16 weeks after xenotransplantation. (D) Chimerism for different human lineages, labeled as in Figure 3B. (E) Frequencies of small indels detected by TIDE assay and large (4.9 kb) deletions detected by the Δ366⁺ assay (Figure 4A) in donor CD34⁺ cells 5 days after editing (Pre) and 16 weeks after bone marrow transplantation. The 13-nt HPFH mutation is indicated in dark blue; all other mutations are represented by light blue. (F) Donor CD34⁺ cells were purified from recipient bone marrow and grown in culture for 18 days under erythroid differentiation conditions. The bar chart shows %HbF as determined by ion-exchange HPLC. Each dot represents a biological replicate experiment with 2 different CD34⁺ cell donors (blue and red). **P < .01 and ****P < .0001 (unpaired Student t test).