Figure 1: Chemogenomic profiling in mammalian cells by CRISPR/Cas9 mutagenesis.
From: Identification of a novel NAMPT inhibitor by CRISPR/Cas9 chemogenomic profiling in mammalian cells
(a) Structure of LB-60-OF61, ZA-87-IW08 and ND-37-YO30 used in this study. (b) Schematic representation of the workflow. A HCT116-Cas9 clone was expanded to 200 million cells and transduced using a sgRNA library with genome-wide coverage (5 sgRNAs/gene). Cells were selected for successful transduction and at day 5 split into three branches: compound treatment at IC30, IC50 and DMSO control. Samples were collected at day 14, 18 and 21 and analyzed for relative sgRNA abundance by NGS. Profiles were then calculated as described in the methods section. (c) Results from the chemogenomic profiling experiment of LB-60-OF61 with special focus on optimal experimental parameters including timepoint and dosage. At the lower compound dose, we consistently identified the nicotinamide phosphoribosyl transferase NAMPT as the strongest hit following by NMNAT1, the next enzyme in the NAD biosynthesis pathway. At the higher dose these hits collapsed back into the pool over time. Resistant hits included major NAD consumers or key enzymes in energy metabolism (SIRT1, PFKP, ACO2, PARG, discussed in the text). Higher compound dose or longer testing appeared to favor identification of resistant hits whereas lower dose and shorter experiments yielded cleaner profiles for hypersensitive hits.
