Identifying Synthetic Lethal Interactions in A375 Cells

A375 cells stably integrated with dCas9-VP64 and MS2-p65-HSF1 were transduced with individual guides from the top screening hits of the Zeocin and Puromycin screens (13 genes total, 3 sgRNAs per gene) as well as available cDNA at an MOI of <0.2 as described above. Cells were selected for guide expression with Zeocin (Life Technologies) for 5 days and replated at low density (3 × 10³ cells per well in a 96-well plate). A375 cells and A375 cells expressing dCas9-VP64 and MS2-p65-HSF1 were plated as controls. Different concentrations of PLX-4720 (2µM, 0.5µM, 0.15µM) or vehicle (DMSO) were added 3h after plating. Cells were treated with PLX-4720 for 4 days before cell viability was measured using CellTiter-Glo Luminescent Cell Viability Assay (Promega). For qPCR quantification of target gene upregulation, cells were also plated at 5 DPI (3 × 10⁴ cells per well in a 96-well plate) and harvested for mRNA 24h after plating.