Analysis of OT activity. (A) Manhattan plot showing the results of off-targets for CD34⁺ cells by CHANGE-seq assay using gDNA extracted from HD male donor CD34⁺ cells electroporated with Cas9 mRNA and sgRNA#1. The brown arrow indicates the on-target site (MAGT1 gene) on chromosome X. (B) Table reporting the total number (nb) of cleavage sites (on- and off-targets) and the specificity ratio using male and female HD HSPCs or HD peripheral blood mononuclear cells (PBMCs). (C) Pie charts showing fraction of cleavage sites categorized according to their genomic features after CD34⁺ cells from HD male and female donors were edited. (D) Identity of the nucleotide mismatches at the OT sites. ON indicates the on-target site without mismatch, whereas OT1-OT8 indicate the top 8 OT sites shared by male and female HD CD34⁺ HSPCs and HD PBMCs. (E) Percentage of cutting activity evaluated by sequencing at ON and OT sites detected by CHANGE-Seq in in vitro (CD34⁺ cells 2 days after gene editing) and in vivo (hCD45⁺ cells from the BM of NSGS mice that had received a transplant at week 16 posttransplant) samples. For all the graphs, data are shown as mean ± SD.