Unbiased Forward Genetic Screening with Chemical Mutagenesis to Uncover Drug–Target Interactions

The steadily increasing throughput in next-generation sequencing technologies is revolutionizing a number of fields in biology. One application requiring massive parallel sequencing is forward genetic screening based on chemical mutagenesis. Such screens interrogate the entire genome in an entirely unbiased fashion and can be applied to a number of research questions. CRISPR/Cas9-based screens, which are largely limited to a gene’s loss of function, have already been very successful in identifying drug targets and pathways related to the drug’s mode of action. By inducing single nucleotide changes using an alkylating reagent, it is possible to generate amino acid changes that perturb the interaction between a drug and its direct target, resulting in drug resistance. This chemogenomic approach combined with latest sequencing technologies allows deconvolution of drug targets and characterization of drug–target binding interfaces at amino acid resolution, therefore nicely complementing existing biochemical approaches. Here we describe a general protocol for a chemical mutagenesis-based forward genetic screen applicable for drug–target deconvolution.