Journal: Nature Genetics

Article Title: Deep CRISPR mutagenesis characterizes the functional diversity of TP53 mutations

doi: 10.1038/s41588-024-02039-4

Figure Lengend Snippet: a , Scheme for CRISPR–Cas9-mediated TP53 mutagenesis via homology-directed repair (HDR) in HCT116 LSL/Δ cell line. b , Editing efficiency as percentage of single-cell clones that contain a targeted integration of the donor and the desired mutation analyzed by PCR and sequencing, respectively. Shown are results for single mutations and the mean across the panel. c , Western blot demonstrating mutant p53 and p21 protein expression in HCT116 clones after Cre-mediated excision of the LSL cassette in absence and presence of 10 µM N3a. d , Principal component analysis based on RNA-seq data of indicated cell clones ±N3a. e , Gene set enrichment analysis for p53-related gene expression signatures comparing indicated N3a- and DMSO-treated cell clones. f , g , Proliferation of TP53 -mutant cell clones in presence of increasing concentrations of N3a analyzed by real-time live-cell imaging. f , Area under the proliferation curve relative to untreated. g , 50% inhibitory concentration (IC 50 , with 95% CI) for N3a with p53-null (LSL, red) and WT (green) as reference. 95% CI, 95% confidence interval; AUC, area under the curve; FDRq, false discovery rate q-value; LSL, LoxP-Stop-LoxP; Puro, puromycin.

Article Snippet: For the generation of R175 libraries in H460 cells, 4 × 10 6 H460 LSL/Δ/Δ cells were transfected with 20 µg of pX459_blast_In5 LSL and 20 µg of HR700 vector library using the Neon Transfection System (Thermo Fisher Scientific, cat. no. MPK10025).

Techniques: CRISPR, Mutagenesis, Clone Assay, Sequencing, Western Blot, Expressing, RNA Sequencing Assay, Live Cell Imaging, Concentration Assay