Abstract

CRISPR tiling screens are a powerful methodology whereby one or multiple target genes are saturated with single guide RNAs (sgRNAs) tiled along the coding region. Inhibitors targeting mitogen-activated protein kinase kinase 1 (MAP2K1/MEK1) have been developed as cancer treatments to counter the frequently aberrant mitogen activated protein kinase (MAPK) pathway. Here, we carried out drop-out and enrichment Cas9 tiling screens against the MAP2K1 gene to identify regions of biological relevance. In a novel drop-out screen, functional regions important for MAP2K1 ATP binding, kinase activation and protein-protein interaction were identified. In addition, surface regions including known cancer driver hotspots were also identified. In an enrichment screen, a novel region responsible for conferring drug resistance was observed. Interestingly, this novel region was identified in screens with three out of four inhibitors (selumetinib, trametinib, cobimetinib and binimetinib), the exception being trametinib. This may be relevant to the unique binding mode and mechanism of action of trametinib, with further validation ongoing. In addition, regions at the N-terminus inhibitory helix and at the R201 loop, where mutations are known to confer resistance, were identified in the screens with all four drugs. All these regions were mapped to primary protein sequence and to PDB 3D structures. Gene editing outcomes at the resistance regions were deconvoluted. Our data demonstrate that CRISPR tiling drop-out screens can identify protein functional regions for cancer cell viability, including surface regions potentially useful for fragment or structure-based drug discovery. In enrichment screens, regions and mutations which modulate drug resistance can be identified to increase our understanding of the underlying mechanisms of action.