A massively parallel reporter assay library to screen short synthetic promoters in mammalian cells

Cellular transcription enables cells to adapt to various stimuli and maintain homeostasis. Transcription factors bind to transcription response elements (TREs) in gene promoters, initiating transcription. Synthetic promoters, derived from natural TREs, can be engineered to control exogenous gene expression using endogenous transcription machinery. This technology has found extensive use in biological research for applications including reporter gene assays, biomarker development, and programming synthetic circuits in living cells. However, a reliable and precise method for selecting minimally-sized synthetic promoters with desired background, amplitude, and stimulation response profiles has been elusive. In this study, we introduce a massively parallel reporter assay library containing 6184 synthetic promoters, each less than 250 bp in length. This comprehensive library allows for rapid identification of promoters with optimal transcriptional output parameters across multiple cell lines and stimuli. We showcase this library's utility to identify promoters activated in unique cell types, and in response to metabolites, mitogens, cellular toxins, and agonism of both aminergic and non-aminergic GPCRs. We further show these promoters can be used in luciferase reporter assays, eliciting 50-100 fold dynamic ranges in response to stimuli. Our platform is effective, easily implemented, and provides a solution for selecting short-length promoters with precise performance for a multitude of applications. Overall design: Multiple mammalian cell lines were transfected with the TRE-MPRA library for 24 hours and were then treated with growth factors or specific compounds for six hours. For some experiments, cells were co-transfected with specific G protein-coupled receptors and treated with receptor agonsists for six hours. Some treatments contained multiple technical replicates, while others were performed once. RNA and DNA barcode abundance was determined via Illumina next-generation sequencing.