CRISPR/Cas9 RNA delivery using LNP improves HSPC tolerance to gene editing. (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 mPB HSPCs. (B) Percentage of B2M^– HSPCs within subpopulations (n = 6). Cells were electroporated with 25 or 50 pmol of RNP or transfected with 1, 1.5, or 2 μg of LNPs. mPB HSPC phenotype (from committed to primitive progenitors) is defined as follows: CD34⁻; CD34⁺CD133⁻; CD34⁺CD133⁺; and CD34⁺CD133⁺CD90⁺. Friedman test with Dunn multiple comparisons performed on the latter subpopulation; median ± IQR. (C) Number of edited cells from panel B, 24 hours after editing (n = 6); Friedman test with Dunn multiple comparisons; median ± IQR. (D) Percentage of live, early/late apoptotic, and necrotic CD34⁺CD133⁺CD90⁺ HSPCs, 24 hours after B2M editing in experiments from panel B (n = 6). Mock electros and empty LNPs were added as controls (n = 6). Friedman test with Dunn multiple comparisons performed on live cells; mean ± SEM. (E) Growth curve of HSPCs from panel D (n = 6); median ± IQR. (F) Cell population composition from panel D, 4 days after treatment; mean ± SEM. (G) Number of colonies generated using mPB HPSCs from panel D (n = 6). Friedman test with Dunn multiple comparisons; median ± IQR. (H) Fold change expression of p21 (left) and APOBEC3H (right) relative to UT 24 hours after treatment from experiments in panel D (n = 6). Friedman test with Dunn multiple comparisons; median ± IQR. (I) Heatmap showing enrichment results from different comparisons between conditions from supplemental Figure 6P against the hallmark gene set (Molecular Signatures Database). Color intensity reflects statistical significance of the test (adjusted P values), whereas signs (positive or negative) correspond to the set of DEGs used in the statistical test (upregulated or downregulated, respectively). (J) Percentage of circulating human (h)CD45⁺ cells over time in mice that underwent transplant with mPB HSPCs edited with 50 pmol of RNP, 1.5 μg of mRNA, or 1.5 μg of LNPs (n = 5,5,6). Kruskal-Wallis test; median ± IQR. (K) Percentage of hCD45⁺ cells in the bone marrow (BM) and spleen of mice from panel J. Kruskal-Wallis test; median. (L) Percentage of B2M^– cells over time, within the human graft in the PB of mice from panel J. Kruskal-Wallis test performed at the last time point; median ± IQR. (M) Percentage of B2M⁻ cells within human graft in BM and spleen of mice from panel J. Kruskal-Wallis test; median. (N) Percentage of circulating human (h)CD45⁺ cells over time in mice that underwent transplantation with CB HSPCs edited with 50 pmol of RNP or 1.5 μg of LNPs (n = 6 and 7); Mann-Whitney test; median ± IQR. (O) Percentage of hCD45⁺ cells in BM and spleen of mice from panel N; Mann-Whitney test; median. (P) Percentage of B2M⁻ cells over time, within the human graft in the PB of mice from panel N. Mann-Whitney test performed at the last time point; median ± IQR. (Q) Percentage of B2M^– cells within human graft in spleen of mice from panel N; Mann-Whitney test; median.