To identify genetic drivers of CRC resistance to DRAs, we performed a CRISPR-Cas9–based LOF screen, as previously described (15). A brief overview of the procedure follows. A library of viral vectors encoding LOF sgRNA inserts were cloned into a lentiviral expression vector encoding Cas9, packaged with a psPAX2 plasmid, and pseudotyped with VSVG (vesicular stomatitis virus glycoprotein). Once the pooled lentiviral library was produced by transfection of 293T cells, it was titered and used to infect DRA-resistant RKO cancer cells. RKO cells were seeded at 500,000 cells per well in six-well plates, incubated overnight, and transduced at a multiplicity of infection of 0.3 the next day. After puromycin selection, a sample was taken to verify representation of the various knockout genes. The transduced population was maintained under puromycin selection for 1 week, after which the library of cells was then exposed to vehicle, TRAIL, or the DRA (each treatment condition in duplicate) for 2 weeks. Cell samples were obtained, DNA was extracted (DNeasy Blood & Tissue Kit; Qiagen), and sgRNA barcodes were isolated, prepared for sequencing as previously described (49). The samples were sequenced by next-generation Illumina sequencing (HudsonAlpha), and the raw data were processed to identify hits that sensitized RKO cells to each treatment, as evidenced by their depletion in drug versus vehicle treatment conditions. The fractional representation (FR) for a given guide in the final condition after vehicle treatment was compared with its FR final condition after TRAIL or DRA treatment. The depletion level of each sgRNA barcode (drug versus vehicle conditions) was calculated as the FR from treated population normalized to the FR from vehicle control (both at the final time point). Depleted barcodes represent sensitizer genes, as they were specifically depleted in the drug-treated cell populations. Depletion comparisons were used to generate a scoring metric called the “3-score,” which represents the average of the three most depleted sgRNAs for a particular gene (52). The depletion median, mean, and 3-scores for both replicates of all genes are provided in table S1. The genes were ranked by their 3-scores; top hits are those that sensitized the cells to DRA treatment when knocked out by the CRISPR-Cas9 machinery. Data are presented as the depletion metric mean of the 3-score per gene in the library. Hits are genes with a low 3-score, and examples of the genes representing top hits are denoted in table S2. All data extractions and calculations were coded and completed using R. The hits were subsequently filtered to retain genes that encoded proteins for which specific inhibitors are commercially available.

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