Deficiency of both ALDH9A1 and the FA pathway causes loss of HSPC. (A) Schematic of human CD34⁺ cell editing experiments. Human umbilical cord blood CD34⁺ cells were edited by electroporation of Cas9–sgRNA–ATTO550 RNP complex. 24 h later, ATTO550⁺ cells were single-cell sorted onto methylcellulose. 14 days later, colonies were scored and harvested for sequencing. (B) Baseline editing efficiency at the time of sorting was obtained by Sanger sequencing followed by TIDE analysis. (C) Percentage of hematopoietic colonies observed per editing condition. (D) Proportion of CFU colony subtypes per editing condition. M, CFU (colony-forming unit)-monocyte/macrophage; G, CFU-granulocyte; GM, CFU-granulocytes/macrophage; BFU-E, burst-forming unit-erythroid; CFU-E, CFU-erythroid; GEMM, CFU-granulocyte, erythrocyte, monocyte, megakaryocyte. (E) Sanger sequencing of each colony per editing condition. The top row shows FANCD2 sequences of colonies targeted by sgFANCD2 alone. The middle row shows ALDH9A1 sequences of colonies targeted by sgALDH9A1 alone. The bottom row shows FANCD2 (upper) and ALDH9A1 (lower) sequences of colonies targeted by both sgFANCD2 and sgALDH9A1. The frequency of biallelic double KO colonies was lower than expected based on baseline editing efficiency, assuming FANCD2 and ALDH9A1 editing events are independent from each other (observed-to-expected ratio 0.33). Loss-of-function (LOF) indel was defined as frameshift indels of any size or in-frame indels ≥18 bp. WT sequence is shown in blue, monoallelic LOF indels in yellow, and biallelic LOF indels in green. This experiment was performed once. (F) Schematic of experiments using human CD34⁺ cells. (G) Baseline editing efficiency of the ALDH9A1 gene shown as %frameshift mutation assessed using amplicon next-generation sequencing (NGS). Error bars, SD. N = 2. (H) Number of total colonies from the first and second CFU plating, which was normalized to that of shLUC/sgCTRL condition. The first plating was done after in vitro genetic manipulation as summarized in Fig. 1 F. The second CFU plating was done by harvesting cells from the first plating, and seeding the same number of cells on new CFU plates. One-way ANOVA followed by Tukey’s multiple comparison test was performed (*, P < 0.05; **, P < 0.01). Two independent experiments were performed and combined for the analysis. Error bars, SD. N = 4. dKO, double KO (i.e., targeted by both sgFANCD2 and sgALDH9A1). TIDE, Tracking of Indels by DEcomposition.