Excessive reactive aldehydes caused by ADH5 or ALDH9A1 deficiency cause synthetic lethality in FANCD2-deficient cells by increased DNA damage. (A) Comparison of genomic sequences in FANCD2^−/− clones and the WT FANCD2. (B) Breakdown of indels in FANCD2 gene as inferred by Tracking of Indels by DEcomposition (TIDE) analysis (http://shinyapps.datacurators.nl/tide/). (C) A summary of indels in the FANCD2^−/− clones. (D) A growth curve of WT and four FANCD2^−/− clones. Error bars, SEM. N = 3. (E–G) Population doublings over time in WT and FANCD2^−/− Jurkat cells expressing indicated sgRNAs. Cells were infected by lentivirus containing Cas9 and sgRNA. After puromycin selection, cumulative population doublings were calculated. WT cells showed no differences in population doubling time regardless of sgRNA used. FANCD2^−/− cells showed lower population doubling time when sgADH5 or sgALDH9A1 were used, but not when sgALDH2 or nontargeting control (sgCTRL) were used. One-way ANOVA followed by Tukey’s multiple comparison test was performed for the last time point. (ns, not significant; *, P < 0.05; ****, P < 0.0001). Error bars, SEM. N = 3. (H) A competition assay as introduced in Fig. 1 G using mCherry-sgSLC7A11. Cells infected with mCherry-sgSLC7A11 were significantly outcompeted by cells infected with GFP-sgCTRL in all cell lines tested. Such differences were not observed when mCherry-sgCTRL was used instead. One-way ANOVA test was performed for the last time point for WT-sgSCL7A11, FANCD2^−/− c#1-sgSLC7A11, and FANCD2^−/− c#2-sgSLC7A11 (ns, not significant). Error bars, SEM. N = 3. (I) GO term analysis of genes depleted in FANCD2^−/− cells. Multiple genes in the GPI anchor biosynthetic process were depleted in FANCD2^−/− cells, suggesting disruption of this pathway is detrimental to the survival of FANCD2^−/− cells. (J) Examples of 53BP1 and γ-H2AX immunofluorescence staining in the indicated cells Quantification of these experiments is shown in Fig. 2, E and F. Scale bar = 10 µm. 630× magnification.