Hematopoietic reconstitution of gene-edited HSPCs in immunodeficient mice. (A) Human engraftment measured by the presence of human CD45⁺ cells by flow cytometry at week 16 in the BM, PB, spleen, and thymus of NSGS mice transplanted with HD, naive (n = 15 mice), or gene-edited XMEN CD34⁺ HSPCs (n = 3 mice [–i53, 2 experiments], 12 mice [+i53, 5 experiments], and 24 mice [+i53+hGSE, 4 experiments]). The dotted line indicates the threshold for engraftment at 0.2% hCD45⁺ cells in the BM. (B) Immunophenotypic analysis of BM at 16 weeks posttransplant by flow cytometry showing the percentages of CD34⁺ cells, myeloid (CD33⁺), lymphoid B (CD19⁺), and T (CD3⁺) after gating on the hCD45⁺ population. (C-D) Percentage of NKG2D expression in NK cells from the spleen (C) and CD8⁺ T cells from the PB (D), spleen, and thymus at week 16 posttransplant. (E) Targeted insertion quantified by ddPCR in human CD45⁺ cells isolated from the BM after GE without enhancers (red bar), with i53 (blue bar), or with i53+hGSE (green bar); BM from the mice injected with the same cells were pooled. (F) Targeted insertion quantified by ddPCR in the lymphoid T (CD3), myeloid (CD33/CD14), and lymphoid B (CD19) cells sorted from the spleen after GE with i53+hGSE. Data are shown as mean ± SD.