Transcriptomic profiling of IDH-wt-hGICs-MGT#4-dEGFP cells upon overexpression of CRISPRa-screen predicted pro-mesenchymal phenotypic drivers.
Functional validation of the genome-scale CRISPR-activation screen by transcriptomic profiling of MGT#4-reporter expressing IDH-wt human glioma initiating cells (hGICs) upon CRISPRa-mediated overexpression of the predicted pro-mesenchymal drivers. Overall design: IDH-wt-hGICs, harboring a constitutively active dCas9-VPR system and the mesenchymal MGT#4-dEGFP reporter, were lentivirally transduced with constructs bearing four individual multiplexed sgRNAs targeting the respective screen hits (RELA, FOSL1, RAC1). Levels of MGT#4-reporter activation upon CRISPRa-mediated transcriptional amplification of the target genes were assessed via FACS at 6 and 14 days post-transduction. At the experimental end-point, guide-bearing reporter-high expressing cells were FACS-purified for subsequent whole transcriptome profiling alongside the unsorted parental IDH-wt-hGICs-MGT#4-dEGFP controls.