Fig. 5: i53 variants increase HDR in phenotypic LT-HSC subpopulations and increase the number of cells with successfully corrected alleles.

A HDR and NHEJ editing outcomes in CD34+ HSPCs that were edited with Cas9 RNP and HBB-SNP AAV6 in 3 medium scale manufacturing runs ( ~ 100-200 M HSPCs per editing condition for each donor). n = 3 different HSPC donors. Analysis by two-tailed paired t-test, with Holm-Šídák multiple comparison correction. n.s. = non-significant (p = 0.1275); *p < 0.05; **p < 0.01 (0.0051 in HDR panel, 0.0083 in NHEJ panel). B Percent cell recovery post editing (harvested 24 h post electroporation, calculated by dividing cell count at 24 h by the number of cells electroporated for each condition). Cell counts and viabilities for each condition are shown in Supplementary Fig. 5.1E and in Supplementary Table S5.1. n = 3 different HSPC donors and mean ± SD depicted. Analysis by one-way ANOVA. n.s. = non-significant; *p < 0.05; **p < 0.01; ***p < 0.001. Exact p-values reported in Source Data file. C Percent of colonies bearing either ≥1 HDR allele (green) or 2 indel alleles (red). Individual colonies were genotyped at HBB cut site locus. n = 3 different HSPC donors and mean ± SD depicted. Analysis by two-tailed paired t-test with Holm-Šídák multiple comparison correction. *p < 0.05; **p < 0.01. Exact p-values reported on Source Data file. D %HDR and %NHEJ in HSPC subpopulation expressing surface markers associated with LT-HSCs (CD34 + CD45RA-CD90 + CD201 + CD49f + CD49c + ), sorted from the bulk edited cells (donor 1 and 3 only). VHH = T12V.T14H.L67H. n = 2. Numbers above bars denote average fold change across two donors. A–D Source data are provided as a Source Data file.