DNA damage in and survival of FANCD2 ^−/− ALDH9A1 ^−/− clones. (A) Summary table for single-cell cloning of FANCD2^−/−ALDH9A1^−/− cells. (B) Results of RT-qPCR for ALDH9A1 gene in WT, FANCD2^−/−, and FANCD2^−/−ALDH9A1^−/− (dKO) clones. One experiment was performed with technical triplicate. A mean of technical triplicate was shown in the graph. (C and D) Quantification of 53BP1 foci per cell (C) and γ-H2AX foci per cell (D) by immunofluorescence. Both were elevated in two dKO clones compared with their parental FANCD2^−/− clone. One-way ANOVA followed by Tukey’s multiple comparison test was performed (*, P < 0.05; **, P < 0.005; ***, P < 0.0005;****, P < 0.0001). The red line indicates the median of each replicate. (C) Number of cells analyzed: WT n = 124, FANCD2^−/− c#2 n = 170, dKO c#2-5 n = 63, dKO c#2-18 n = 62. (D) Number of cells analyzed: WT n = 90, FANCD2^−/− c#2 n = 131, dKO c#2-5 n = 65, and dKO c#2-18 n = 48. (E) cDNA-expressing ALDH9A1 C288A (catalytic-dead mutant) and YFP was overexpressed in WT, ALDH9A1^−/−, FANCD2^−/−, and FANCD2^−/−ALDH9A1^−/− cells, and their relative growth was compared with luciferase-GFP–overexpressed counterparts by monitoring YFP/GFP ratio using flow cytometry. One experiment in biological triplicate was performed. One-way ANOVA followed by Dunnett’s multiple comparison test was performed (ns, not significant). Error bars, SD. N = 3. (F) ALDH activity was measured using a fluorometric ALDH activity assay kit (Abcam) in WT, parental ALDH9A1^−/−, ALDH9A1^−/− cells complemented with luciferase (Luc), WT ALDH9A1, or mutant ALDH9A1 (C288A) cDNA. 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). Error bars, SD. N = 3, except for ALDH9A1^−/− complemented with WT (purple bar), in which one outlier was excluded. (G and H)FANCD2^−/− cells were serially transduced with lentivirus carrying mCherry-sgALDH9A1 followed by either GFP-sgCTRL or GFP-sgATP13A3. Two independent experiments were performed. A representative figure is shown. (G) The ratio of mCherry-GFP double-positive and mCherry single-positive cells (collectively, dKO) was compared with that of GFP single-positive and double-negative cells (collectively, FANCD2^−/−). Error bars, SD. N = 3. (H) The ratio of mCherry-GFP double-positive (tKO) cells was compared with that of mCherry single-positive (dKO) cells. Error bars, SD. N = 3. (I) P adj values for genes that were differentially enriched in FANCD2^−/−ALDH9A1^−/− condition (dKO). They were calculated by the DESeq2 package and shown as a minus log 10 graph. Values for all data points in this graph are in Table S5. (J) Distribution of sizes of the second site indels in the ALDH9A1 gene of FANCD2^−/−ALDH9A1^−/− c#1 cells after retargeting ALDH9A1 with a second sgRNA. The parental clone carries compound heterozygous mutations—1-bp deletion and 7-bp insertion. ALDH9A1 c.765insC (1-bp insertion) was the most abundant second site indel, which would restore the reading frame of 1-bp deletion of the parental clone. Note that even though 2-bp insertions were the most frequent when the allelic frequencies of different 2-bp insertions were combined, ALDH9A1 c.765insC (1-bp insertion) was the most abundant mutant allele with the allelic frequency of 5.62%. (K) Predicted protein sequences of indicated alleles of ALDH9A1 showing that the second-site indels can restore the ALDH9A1 reading frame. WT ALDH9A1 protein is 518-amino acid long. FANCD2^−/−ALDH9A1^−/− c#1 carries 1-bp deletion of the seventh base in exon 5 (c.599DelC), which results in a premature stop codon. 1-bp insertion was the most frequent second-site indel after retargeting of ALDH9A1 with a second gRNA, which is predicted to restore the reading frame and result in a full-length protein with 90.5% amino acid sequence homology. Amino acids identical to WT sequences are shown in blue. dKO, double KO.