Metabolism-focused CRISPR screen reveals novel endogenous sources of DNA damage. (A) Schematic of the metabolism-focused CRISPR KO screen. WT and FANCD2^−/− Jurkat cells were infected with lentivirus-containing metabolism-focused sgRNA library in biological triplicate at MOI of 0.3. After 72 h of puromycin selection, 40 million cells were harvested for baseline sequencing to achieve 1000x representation per sgRNA. At least 40 million cells were passaged every 4 days for 14 population doublings, after which at least 40 million cells were harvested for final time point sequencing. (B) FANCD2 western blot of Jurkat clones used in the experiments. “c” stands for clone.(C) MMC sensitivity assay of Jurkat clones. The experiment was performed in triplicate. Three independent experiments were performed using WT and FANCD2^−/− clone #1 (c#1). The third experiment included FANCD2^−/− clone #2 (c#2), which is shown in this figure. Error bars, SD. N = 3. (D) FANCD2 western blot of Jurkat clones treated with 1 µM MMC 24 h before harvest. (E) Adjusted P (P adj) values for the top 35 genes that were differentially depleted in the FANCD2^−/− setting were calculated by the DESeq2 package. Values for all data points in this graph are in Table S1. (F) A volcano plot showing log₂ fold change (log₂FC) value and P adj between FANCD2^−/− and WT at the sgRNA level. Only sgRNAs with P adj < 0.01 were used. Eight significantly depleted sgALDH9A1s are indicated. Values for all data points in this graph are in Table S2. (G) A competition assay using WT and FANCD2^−/− clones. The ratio of mCherry to GFP-expressing cells normalized to the starting ratio was decreased in two FANCD2^−/− clones with mCherry-sgALDH9A1 compared with FANCD2^−/− clones with mCherry-sgCTRL (nontargeting). There were no differences between mCherry-sgALDH9A1 and mCherry-sgCTRL in WT cells. One-way ANOVA followed by Tukey’s multiple comparison test was performed for the last time point between FANCD2^−/− c#1 sgCTRL and sgALDH9A1, and between FANCD2^−/−c#2 sgCTRL and sgALDH9A1. (****, P < 0.0001). Two independent experiments were performed. A representative figure is shown. Error bar, SEM. N = 3. Source data are available for this figure: SourceData F1.