CRISPR/Cas9 RNA delivery using LNPs allows HDR-mediated editing in human HSPCs. (A) Schematic representations of gene editing experimental procedure, and related analyses, after CRISPR/Cas9 RNA delivery using LNPs or CRISPR/Cas9 RNP/RNA delivery via electroporation, in combination with the cognate AAV6, in mPB HSPCs. (B) Percentage of GFP⁺ cells within mPB HSPC subpopulations 96 hours after AAVS1 editing. Cells were edited with 25 pmol of RNP or 1 μg of LNPs and transduced with AAV6 immediately after electroporation or 2 hours before transfection, respectively. Wilcoxon test on individual subpopulations. Median ± IQR. (C) Percentage of live, early/late apoptotic, and necrotic CD34⁺CD133⁺CD90⁺ mPB HSPCs 24 hours after AAVS1 editing in experiments from panel B (n = 6). UTs, mock electros, and empty LNPs were added as controls (n = 6). Friedman test with Dunn multiple comparisons performed on live cells. Mean ± SEM. (D) Growth curve from panel C (n = 6). Median ± IQR. (E) Number of edited cells from panel C 24 hours after editing (n = 6). Wilcoxon test. Median ± IQR. (F) Cell population composition from panel C (n = 6), 4 days after treatment. Mean ± SEM. (G) Number of colonies generated by mPB HPSCs from panel C (n = 6). Median ± IQR. Friedman test with Dunn multiple comparisons. (H) Fold change expression of p21 and APOBEC3H relative to UTs 24 hours after treatment from experiments in panel C. Friedman test with Dunn multiple comparisons. Median ± IQR. (I) Percentage of circulating hCD45⁺ cells over time in mice that underwent transplantation with mPB HSPCs (from a pool of donors), edited with 25 pmol RNP or 1 μg of LNPs, in combination with AAV6 (n = 4 and 5). Median ± range. (J) Percentage of hCD45⁺ cells in the BM and spleen of mice from panel I. Median. (K) Percentage of circulating hCD45⁺ cells over time in mice that underwent transplantation with mPB HSPCs edited as in panel I but using a different pool of donors (n = 7). Mann-Whitney test. Median ± range. (L) Percentage of hCD45⁺ cells in BM and spleen of mice from panel K. Mann-Whitney test. Median. (M-P) Percentage of GFP+ cells over time within human graft in PB (M,O) or hematopoietic organs (N,P) of mice from panel I (M-N) and panel K (O-P). Mann-Whitney test. Median or median ± range.