Programmable promoter editing for precise control of transgene expression

Abstract

Subtle changes in gene expression direct cells to distinct cellular states. Identifying and controlling dose-dependent transgenes require tools for precisely titrating expression. To this end, we developed a highly modular, extensible framework called DIAL for building editable promoters that allow for fine-scale, heritable changes in transgene expression. Using DIAL, we increase expression by recombinase-mediated excision of spacers between the binding sites of a synthetic zinc finger transcription factor and the core promoter. By nesting varying numbers and lengths of spacers, DIAL generates a tunable range of unimodal setpoints from a single promoter. Through small-molecule control of transcription factors and recombinases, DIAL supports temporally defined, user-guided control of transgene expression that is extensible to additional transcription factors. Lentiviral delivery of DIAL generates multiple setpoints in primary cells and iPSCs. As promoter editing generates stable states, DIAL setpoints are heritable, facilitating mapping of transgene levels to phenotypes. The DIAL framework opens new opportunities for tailoring transgene expression and improving the predictability and performance of gene circuits across diverse applications. HighlightsPromoter editing generates unimodal setpoints from a single synthetic promoter DIAL setpoints are tuned by the length of an excisable spacer within the promoter DIAL setpoints are uniform and robust to varying levels of the transcription factor DIAL transmits transient inputs into heritable promoter states The TET-DIAL system enables small-molecule activation at defined setpoints DIAL regulates physiologically-relevant transgenes and performs in primary cells and iPSCs One Sentence SummaryDIAL offers an extensible framework for designing synthetic promoters that perform across a range of cell types to generate heritable setpoints of gene expression.