Directed molecular evolution of DREADDs: a generic approach to creating next-generation RASSLs

Nat Protoc. 2010 Mar;5(3):561-73. doi: 10.1038/nprot.2009.239. Epub 2010 Feb 25.


G protein-coupled receptors (GPCRs) and their downstream signaling cascades contribute to most physiological processes and a variety of human diseases. Isolating the effects of GPCR activation in an in vivo experimental setting is challenging as exogenous ligands have off-target effects and endogenous ligands constantly modulate the activity of native receptors. Highly specific designer drug-designer receptor complexes are a valuable tool for elucidating the effects of activating particular receptors and signaling pathways within selected cell types in vivo. In this study, we describe a generic protocol for the directed molecular evolution of designer receptors exclusively activated by designer drugs (DREADDs). First, the yeast system is validated with the template receptor. Second, a mutant library is generated by error-prone PCR. Third, the library is screened by drug-dependent yeast growth assays. Mutants exhibiting the desired properties are selected for further rounds of mutagenesis or for characterization in mammalian systems. In total, these steps should take 6-8 weeks of experimentation and should result in the evolution of a receptor to be activated by the chosen ligand. This protocol should help improve the experimental targeting of select cell populations.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Designer Drugs
  • Directed Molecular Evolution / methods*
  • Drug Design
  • Humans
  • Ligands
  • Pheromones / genetics
  • Pheromones / metabolism
  • Protein Engineering
  • Receptors, G-Protein-Coupled / genetics*
  • Receptors, G-Protein-Coupled / metabolism*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Signal Transduction


  • Designer Drugs
  • Ligands
  • Pheromones
  • Receptors, G-Protein-Coupled