Journal: bioRxiv

Article Title: Trans-Lesion Synthesis and Mismatch Repair Pathway Crosstalk Defines Chemoresistance and Hypermutation Mechanisms in Glioblastoma

doi: 10.1101/2023.10.16.562506

Figure Lengend Snippet: (A) Workflow of genetic screen. (B) Normalized counts (Log 10 ) of sgRNAs targeting DDR genes and non-targeting control across indicated samples in RAD18 +/+ (Blue) and RAD18 −/− (purple) U373 cells. (C) Volcano plot showing Gene Abundance Change Scores (Sigma FC) vs −Log 10 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. (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. (I) Heatmap showing sigmaFC of sgRNAs targeting Polζ complex genes and MIDAS/BIR pathway genes. Data are representative of three independent experiments. All data points represent mean ± SD; P values were determined by multiple unpaired t test (B) and Tukey HSD test (G).

Article Snippet: The resulting bacterial cultures were collected and DDR-CRISPR library plasmid DNA was purified using a Qiagen plasmid DNA maxiprep kit.

Techniques: Cell Culture, DNA Synthesis, Homologous Recombination, Non-Homologous End Joining, Inhibition