Vemurafenib

Cell lines expressing dCas9-VP64 and dCas9 were made by transducing cells with the lentiviral vector pXPR_109, which expresses blasticidin resistance from a 2A site and dCas9-VP64 from the EF1α promoter, or pXPR_118, which expresses hygromycin resistance from a 2A site and dCas9 from the EF1α promoter, respectively. Prior to screening-scale transduction, dCas9-VP64 and dCas9 cell lines were selected with blasticidin and hygromycin, respectively.
For the dCas9-VP64 screens, cell lines expressing dCas9-VP64 were transduced with the Calabrese library in two biological replicates at a low MOI (~0.5). Transductions were performed with enough cells to achieve a representation of at least 500 cells per sgRNA per replicate, taking into account a 30–50% transduction efficiency. Throughout the screen, cells were split at a density to maintain a representation of at least 500 cells per sgRNA, and cell counts were taken at each passage to monitor growth. Puromycin selection was added 2 days post-transduction and was maintained for 5–7 days. After puromycin selection was complete, each replicate was split to no drug and drug treatment arms, each at a representation of at least 500 cells per sgRNA. A375 screens were performed with 2 μM vemurafenib; MelJuSo screens were performed with 1.5 μM selumetinib. 14 days after the initiation of drug treatment, cells were pelleted by centrifugation, resuspended in PBS, and frozen promptly for genomic DNA isolation.
For the dCas9-only screens, A375 cells expressing dCas9 were transduced with the Calabrese library in two biological replicates at a low MOI (~0.5) via a low-representation, no-spin method. Transductions were performed to achieve a representation of at least 300 sgRNAs per replicate, taking into account a 30–50% transduction efficiency. Cells were seeded into T175 flasks in a total volume of 20 mL of virus-containing media with polybrene at 0.5 μg mL⁻¹. Flasks were then transferred to an incubator overnight. 16–18 h after seeding, the virus-containing media was replaced with fresh media and cells were expanded to achieve a representation of at least 500 transduced cells per sgRNA. Puromycin selection was added 2 days post-transduction and was maintained for 5–7 days. After puromycin selection was complete, each replicate was split to no drug and drug treatment arms, each at a representation of at least 500 cells per sgRNA. 14 days after the initiation of drug treatment, cells were pelleted by centrifugation, resuspended in PBS, and frozen promptly for genomic DNA isolation.
For secondary screens, cells expressing either dCas9 or dCas9-VP64 were transduced with the secondary pool in three biological replicates at a low MOI (~0.5). Transductions were performed with enough cells to achieve a representation of at least 500 cells per sgRNA per replicate, taking into account a 30–50% transduction efficiency. Throughout the screen, cells were split at a density to maintain a representation of at least 2000 cells per sgRNA, and cell counts were taken at each passage to monitor growth. Puromycin selection was added 2 days post-transduction and was maintained for 5–7 days. After puromycin selection was complete, each replicate was split to no drug and drug treatment arms, each at a representation of at least 2000 cells per sgRNA. A375 secondary screens were performed with 2 μM vemurafenib; MelJuSo secondary screens were performed with 10 nM trametinib or 1.5 μM selumetinib. 14 days after the initiation of drug treatment, cells were pelleted by centrifugation, resuspended in PBS, and frozen promptly for genomic DNA isolation.

HEK293T cell line, melanoma A375, SK-MEL-2, SK-MEL-28 cell lines and the CRC cell line HCT116 were obtained from American Type Culture Collection. Normal Human Epidermal Melanocytes (NHEM) was purchased from PromoCell. All cells were incubated according to the recommended protocol. Erianin (purity ≥98%) was provided by Sichuan Weikeqi Biological Technology, Co., Ltd. (Chengdu, China). Vemurafenib (purity ≥99.80%), Dabrafenib (purity ≥99.97%), Encorafenib (purity ≥99.63%), Cobimetinib (purity ≥99.71%), Trametinib (purity ≥99.59%), Binimetinib (purity ≥99.55%), and LY3009120 (purity ≥99.01%) were purchased from MCE. C₁₈H₁₈O₅ (purity ≥98%) was purchased from Sigma. Active BRAF (#B08-11BG), active BRAF V600E (#B08-12G), active RAF1 (EE) (#R01-13G), corresponding RAF substrate inactive MEK1 (#M02-14BG), as well as active MEK1 (#M02-10G), active MEK2 (#M03-10G), inactive ERK1 protein (#M29-14G), and inactive ERK2 protein (#M28-14U) were obtained from Signal Chem. Human RAF-1 protein (His & GST Tag) (#10657-H20B) was purchased from Sino Biological.