Abstract
Unraveling the mechanism of action and molecular target of small molecules remains a major challenge in drug discovery. While many cancer drugs target genetic vulnerabilities, loss-of-function screens fail to identify essential genes in drug mechanism of action. Here, we report CRISPRres, a CRISPR-Cas-based genetic screening approach to rapidly derive and identify drug resistance mutations in essential genes. It exploits the local genetic variation created by CRISPR-Cas-induced non-homologous end-joining (NHEJ) repair to generate a wide variety of functional in-frame mutations. Using large sgRNA tiling libraries and known drug–target pairs, we validate it as a target identification approach. We apply CRISPRres to the anticancer agent KPT-9274 and identify nicotinamide phosphoribosyltransferase (NAMPT) as its main target. These results present a powerful and simple genetic approach to create many protein variants that, in combination with positive selection, can be applied to reveal the cellular target of small-molecule inhibitors.
Highlights
Unraveling the mechanism of action and molecular target of small molecules remains a major challenge in drug discovery
We show that large-scale CRISPR single-guide RNA gene tiling libraries can be applied as a genetic screening approach in cancer cells to identify the molecular target of a chemical inhibitor
The KIF11L132Δ mutation protected cells from monopolar mitotic spindle formation induced by ispinesib treatment (Supplementary Fig. 8f panel c). These results demonstrate that the spontaneous genetic variation generated during non-homologous end-joining (NHEJ) repair at the locus of CRISPR-SpCas9-mediated double-strand breaks (DSBs) can be exploited to significantly accelerate the selection of functional drug resistance mutations; a finding independently confirmed by Ipsaro et al.[23] and Donovan et al.[24]
Summary
Unraveling the mechanism of action and molecular target of small molecules remains a major challenge in drug discovery. The gold standard proof for a drug’s target is the identification of functional mutations that confer resistance in a cellular context For this reason, genetic screens in particular, are very powerful tools for drug mechanism of action studies[4]. Chemical mutagenesis to increase the occurrence of single-nucleotide variants has been described[11] Until now, this chemical mutagenesis approach has only been applied to identify loss-of-function resistance mutations to the prototype acute myeloid leukemia drug 6-thioguanine. We show that large-scale CRISPR single-guide RNA (sgRNA) gene tiling libraries can be applied as a genetic screening approach in cancer cells to identify the molecular target of a chemical inhibitor. We demonstrate that the methodology is compatible with the class 2 type V AsCpf[1] CRISPR system, increasing the resolution of the method
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