Fig. 3. Spacer-nick leads to precise and efficient HDR with minimal NHEJ events in long-term HSCs.

(A) FACS analysis, pregated on CD34⁺CD38⁻ cells, showing frequencies of HSC (CD90⁺CD45RA⁻, red), MPP1 (CD90⁻CD54RA⁻, black), and MPP2 (CD90⁻CD45RA⁺, blue) subsets 3 days after targeting the B2M locus in human CD34⁺ cells treated with control (Ctrl; no RNPs), Cas9, double-nick, or spacer-nick RNPs and AAV-B2M-mCherry vectors. (B) Summary of frequencies of HSC, MPP1, and MPP2 subsets in (A) based on three independent experiments. (C) FACS analysis showing percentages of mCherry⁺ cells within HSC, MPP1, and MPP2 subsets. (D) Bar graph showing frequencies of mCherry⁺ HSC, MPP1, and MPP2 cells in (C). Data are shown as means ± SD based on three independent experiments. (E) Pie charts of frequencies of WT (gray), NHEJ (blue), and HDR (orange) sequences at the targeted B2M locus in the sorted long-term HSCs (CD34⁺CD38⁻CD45RA⁻CD90⁺EPCR⁺) 3 days after targeting. Summary of the ratio of HDR:NHEJ events at the targeted B2M locus in long-term HSCs treated as indicated. Data are shown as means ± SD and based on three independent experiments. (G) Sal I–mediated RFLP assay showing the gene correction efficiency of the targeted HBB locus in long-term HSCs treated as indicated. (H) Pie charts showing frequencies of WT (gray), NHEJ (blue), and HDR (orange) sequences at the targeted HBB locus in long-term HSCs. (I) Bar graph representing the ratio of HDR:NHEJ events at the targeted HBB locus in long-term HSCs. Data are shown as means ± SD and based on three independent experiments.