Journal: Science Advances

Article Title: Genome-scale CRISPR-Cas9 screen of Wnt/β-catenin signaling identifies therapeutic targets for colorectal cancer

doi: 10.1126/sciadv.abf2567

Figure Lengend Snippet: ( A ) Schematic diagram of the artificial TOP-GC reporter. The fluorescence of dGFP and mCherry in the reporter cells is shown in the bottom. ( B ) Validation of TOP-GC reporter responsiveness following β-catenin interference using flow cytometric analysis. Left: GFP expression was examined in parental DLD cells or TOP-GC reporter cells stably expressing nontargeting control (NTC) short hairpin–mediated RNA (shRNA) or β-catenin shRNAs. Right: FACS analysis of mCherry expression in DLD1 parental, NTC-ablated, and β-catenin–ablated DLD1 TOP-GC reporter cells. ( C ) Workflow of the screening strategy. ( D ) Volcano plot for the GFP-low hits of TOP-GC reporter screening. The x axis denotes the Z score of gene-level FC [median log 2 (fold change) for all sgRNAs per gene]. The P values in y axis were generated by MAGeCK-VISPR. ( E ) Venn diagram shows the overlaps between the hits of DLD1 TOP-GC reporter (GFP-low) and proliferation screens. ( F ) The distribution of log 2 (fold change) of sgRNAs of the entire library (top) and individual genes (bottom) in TOP-GC reporter and proliferation screenings. MAGeCK P values per gene were indicated. ( G ) Real-time quantitative polymerase chain reaction (RT-qPCR) analysis of the expression of β-catenin targets in DLD1 and SW480 cells at day 7 after infection of lentiviruses harboring β-catenin, TCF4, LEF1, and BCL9L sgRNAs.

Article Snippet: The β-catenin shRNA construct was obtained from Addgene (#18803).

Techniques: Fluorescence, Biomarker Discovery, Expressing, Stable Transfection, Control, shRNA, Generated, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Infection