Abstract
Target deconvolution of new bioactive agents identified from phenotypic screens remains a challenging task. The discovery of mutations that confer resistance to such agents is regarded as the gold standard proof of target identification. Here, we describe a method that exploits the error-prone repair of CRISPR-induced DNA double-strand breaks to enhance mutagenesis and increase the incidence of drug resistancemutations in essential genes. As each DNA double-strand break is introduced at a targeted genomic site predefined by the presence of a protospacer adjacent motif (PAM) and a particular CRISPR single guide RNA (sgRNA), the genetic location of drug resistance mutations can be easily uncovered through targeted sequencing of CRISPR sgRNAs. Moreover, the method allows for the identification of not only the drug target gene, but also the drug-binding domain within the target gene.
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. 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. 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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