Loss of both ALDH9A1 and FANCD2 causes increased DNA damage and apoptosis. (A) Schematic of the experimental setup. WT and two independent FANCD2^−/− clones were targeted by sgALDH9A1 or nontargeting control guide (sgCTRL) by electroporation of Cas9–sgRNA–RNP complex. “c” stands for clone. (B) Percentage (%) of ALDH9A1 indel was assessed by Sanger sequencing followed by Synthego ICE analysis. ALDH9A1 indel (%) was decreased over time in the two independent FANCD2^−/− clones, but not in WT cells. At least four independent experiments were performed. One-way ANOVA followed by Dunnett’s multiple comparison test (**, P = 0.0055; ****, P < 0.0001). Error bar, SD. N = 4–6. (C) Numbers of chromosome breakage per cell were significantly elevated in two FANCD2^−/− clones targeted by sgALDH9A1 compared with clones targeted by sgCTRL. There were no differences in WT clones targeted by either sgCTRL or sgALDH9A1. A two-sided Student’s t test was performed between sgCTRL and sgALDH9A1 in each clone (*, P < 0.05; **, P < 0.005). One independent experiment was performed. The red line denotes a mean value for each condition. Number of cells analyzed: WT-sgCTRL n = 37, WT-sgALDH9A1 n = 42, FANCD2^−/−c#1-sgCTRL n = 39, FANCD2^−/−c#1-sgALDH9A1 n = 35, FANCD2^−/−c#2-sgCTRL n = 34, and FANCD2^−/−c#2-sgALDH9A1 n = 31). (D) Representative figure of chromosome breakage analysis. Arrows indicate chromosomal breaks. Scale bar = 10 µm. (E) Numbers of 53BP1 foci per cell as assessed by immunofluorescence staining were elevated in two FANCD2^−/− clones targeted by sgALDH9A1 compared with clones targeted by sgCTRL. Two independent experiments were performed. Statistics were performed on pooled replicates. The red line denotes a mean value for each condition. Number of cells analyzed: WT-sgCTRL n = 638, WT-sgALDH9A1 n = 902, FANCD2^−/−c#1-sgCTRL n = 817, FANCD2^−/−c#1-sgALDH9A1 n = 817, FANCD2^−/−c#2-sgCTRL n = 630, and FANCD2^−/−c#2-sgALDH9A1 n = 735). (F) Numbers of γ-H2AX foci per cell as assessed by immunofluorescence staining were elevated in two FANCD2^−/− clones targeted by sgALDH9A1 compared with clones targeted by sgCTRL. Two independent experiments were performed. Statistics were performed on pooled replicates. The red line denotes a mean value for each condition. Number of cells analyzed: WT-sgCTRL n = 866, WT-sgALDH9A1 n = 990, FANCD2^−/−c#1-sgCTRL n = 738, FANCD2^−/−c#1-sgALDH9A1 n = 736, FANCD2^−/−c#2-sgCTRL n = 506, and FANCD2^−/−c#2-sgALDH9A1 n = 547). (G and H) Percentage of annexin V–positive and propidium iodide–negative (early apoptotic) cells (G) and sub-G0 fraction on cell cycle analysis (H) by flow cytometry were elevated in two FANCD2^−/− clones targeted by sgALDH9A1 compared with clones edited with sgCTRL (non-targeting). Two independent experiments were performed. Statistics were performed on pooled replicates. Error bars, SD. N = 6. (E–H) One-way ANOVA followed by Tukey’s multiple comparison test was performed (ns, not significant; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.0001).