FIGURE 3 from Genome-wide CRISPR Screen Reveals RAB10 as a Synthetic Lethal Gene in Colorectal and Pancreatic Cancers Carrying SMAD4 Loss

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Abstract
Construction and isolation of Cas9-expressing clones and workflow of our CRISPR screens. A, Levels of SMAD4 protein detected by the Sally Sue Simple Western system in the four cell lines HT29, SW620, DLD1, and MIAPaCa-2. B, Levels of Cas9 protein detected by the Sally Sue Simple Western system, in response to doxycycline treatment (exposure at 1 μg/mL for 3 days) in the four clones HT29, SW620, DLD1, and MIAPaCa-2 used in this study. C, Schematic representation of genome-wide CRISPR viability screens performed in HT29, SW620, DLD1, and MIAPaCa-2. Cells were infected at low MOI with a genome-wide library of sgRNAs. After selection, doxycycline was added to induce Cas9 and the KOs. Cells were regularly split and harvested, while keeping at least 200 million cells in culture to ensure a 1,000x library coverage. Cell growth curves of transduced cell lines following puromycin selection are plotted over time, for both replicates (circle and square). Arrows indicate samples that were harvested for sequencing at PD0 and PD15. sgRNA sequences were amplified and identified by NGS (NovaSeq). Enrichment/depletion of sgRNAS were assessed by comparing sgRNA counts at PD15 and PD0 using the Mageck analysis tool and by removing published core essential genes (Hart et al. 2017). PD, population doublings.