Selective advantage of epigenetically disrupted cancer cells via phenotypic inertia

CRISPR-based large-scale modeling of loss-of-function mutations to investigate mechanisms of stress resistance in cancer

“…We have recently applied this method to interrogate over 300 epigenetic regulator genes in cancer cells, mimicking the frequent inactivating mutations that affect the epigenetic regulatory network in cancer patients, especially in emerging subclones. 1 After generation of epigenetically-disrupted cells, we illustrate how to assess the impact of this network-level perturbation on cancer cell fitness under various types of stress that are typical of the tumor microenvironment.…”

“… 3 From the available library, constructs targeting 318 genes, encoding stricto-sensu epigenetic regulators, were chosen. 1 , 3 If other functional gene classes need to be targeted alternative sources are available. As an example, Sigma-Aldrich and the Wellcome Trust Sanger Institute have joined forces and created genome-wide arrayed libraries for both mouse and human studies.…”

“…For example, TNP1 , TNP2 , HMGB4 , SMC1B and DNMT3L were determined to be unexpressed in MEXF 2090 cells and were used as negative control in our study. 1 To target 318 epigenetic regulator genes the library is arranged in eight 96-well plates, each containing sgRNAs targeting 40 expressed genes, the 5 non-expressed genes (4 replicates each for a total of 20 negative controls) and one inter-plate standard targeting the same expressed gene, which monitors the consistency in experimental conditions across plates. It is essential to exclude external wells and fill with PBS to avoid edge effects which strongly affect cell fitness (Graphical Abstract – step 3).…”

“…soln., methanol free Alfa Aesar Cat#43368 SYTOX™ Green Nucleic Acid Stain Thermo Fisher Scientific Cat#S7020 SYTOX™ Deep Red Nucleic Acid Stain Thermo Fisher Scientific Cat#S11380 Buparlisib Generon Cat#HY-70063 Polybrene Santa Cruz Cat#sc-134220 Triton™ X-100 Merk Cat#9036-19-5 Lullaby OZBiosciences Cat#LL70500 DharmaFECT Set of 4 Transfection Reagents Horizon Cat# T-2005-01 DharmaFECT Duo Horizon Cat#T-2010-01 Lipofectamine RNAiMAX Thermo Fisher Cat#13778100 Fetal bovine serum Thermo Fisher Scientific Cat#10270106 Penicillin-Streptomycin Merck Cat#P4333 L-Glutamine solution Merck Cat# G7513 DAPI Merck Cat#D9542 Critical commercial assays MultiScreenHTS HV Filter Plate, 0.45 μm, clear, sterile Millipore Cat#MSHVS4510 Nunc™ Seals, Tapes, and Foils Thermo Fisher Scientific Cat#232698 Deposited data Z-score from large-scale fitness assays Loukas et al. 1 Table S3 from Loukas et al. 2 Experimental models: Cell lines PDX MEFX 2090 derived cells Loukas et al.…”

“…2 Experimental models: Cell lines PDX MEFX 2090 derived cells Loukas et al. 1 Table S2 from Loukas et al. 2 Cas9 - expressing MEFX 2090 cells Loukas et al.…”