IN SILICO DESIGN OF MAMMALIAN PROMOTERS WITH USER-DEFINED FUNCTIONALITY

摘要

Simplified models of gene regulation are a fundamental requirement for mammalian synthetic biology. However, at the transcriptional level this has been impeded by the complex design rules governing promoter activity dynamics, preventing de novo-design of regulatory elements with user-defined functionalities. As demonstrated herein, when composite transcription factor binding sites do not function synergistically, mammalian promoters can be constructed according to simple design rules. Host-cell transcriptional machinery components were analyzed in silico to identify transcription factors with desired expression dynamics. Cognate binding sites were then comprehensively tested in homotypic and heterotypic architectures to assess modularity and determine the transcriptional activity exhibited by a single copy of each site. When elements were specifically selected to prevent combinatorial interactions, heterotypic promoter activities could be accurately modeled simply as a function of constituent binding site copy numbers. As binding site order, spacing, and orientation had minimal effect on promoter activity, blocks could be optimally combined and arranged in silico according to context-specific design-criteria. To demonstrate this, CHO cell promoters were created de-novo that exhibited designed activity levels and long-term expression stability in vitro. The findings reveal new insights into eukaryotic transcriptional regulatory mechanisms, and provide novel tools for mammalian synthetic biology.