Fig. 5: Results of CRISPR screen for RAD18-interacting DDR genes. | Nature Communications

Fig. 5: Results of CRISPR screen for RAD18-interacting DDR genes.

From: Trans-lesion synthesis and mismatch repair pathway crosstalk defines chemoresistance and hypermutation mechanisms in glioblastoma

Fig. 5

a Workflow of genetic screen. MOI Multiplicity of infection, BSD Blasticidin, PD Proliferation doubling, LD Lethal dose. b Normalized counts (Log10) of sgRNAs targeting DNA Damage Response (DDR) genes and non-targeting control across indicated samples in RAD18+/+ (Blue) and RAD18−/− (purple) U373 cells. ***p = 0.001. c Volcano plot showing Gene Abundance Change Scores (Sigma FC) vs -Log10 adjusted p-value for sgRNA depletion or enrichment in PD20 groups when compared with PD0. The −Log10 p value was calculated using a permutation test for DDR gene-targeting sgRNAs relative to non-targeting control sgRNAs. Black dashed lines indicate thresholds for statistical significance. Enriched sgRNAs targeting MMR genes, and depleted sgRNAs targeting POLD3, CHEK2 and PRKDC are highlighted. d Heatmap showing relative dropout of sgRNAs grouped by DDR pathway in RAD18+/+ and RAD18−/− cells cultured with or without TMZ for 20 PD. The numbers on the scale indicate −Log10 of paired t test p value (up) and Log2 fold change (down) of pooled sgRNA counts. BER Base excision repair; TLS Trans-lesion DNA synthesis; FA Fanconi Anemia; HR Homologous recombination; NHEJ Non-homologous end joining; NER Nucleotide excision repair; CS Checkpoint signaling; M/ASC Mitosis/spindle assembly checkpoint; PARP Poly ADP ribose polymerases; NM Nucleotide metabolism; TS Template switch. e Radar plot showing relative dropout (up: p value; down: Log2 fold change) of sgRNAs in DDR pathway between TMZ and DMSO control at PD20 in RAD18+/+ and RAD18−/− cells. f, g Dose response matrices and synergy heatmaps showing effects of pairwise combinations of TMZ with CHK2i (e) or DNA-PKi (f) on inhibition of viability in RAD18+/+ and RAD18−/− cells. Mean value of triplicates used to generate heatmaps. h Clonogenic survival assays showing TMZ sensitivity of WT, RAD18−/−, POLD3−/−, and RAD18−/−POLD3−/− U373 cells. Cultures received a single treatment with TMZ daily for 5 days. All data points represent the mean of triplicates ± SD. ***p = 0.001, *p = 0.036 Experiment had been repeated twice with similar results. i Heatmap showing sigmaFC of sgRNAs targeting Polζ complex genes and MIDAS/BIR pathway genes. p values were determined by two-sided t test with Tukey HSD adjust (b, h). Source data are provided as a Source Data file. a was created with BioRender.com.

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