Abstract
INTRODUCTION: Synthetic biology revolutionizes our ability to decode and recode genetic systems. The capability to reconstruct and flexibly manipulate multi-gene systems is critical for understanding cellular behaviors and has significant applications in therapeutics. OBJECTIVES: This study aims to construct a diverse library of synthetic tunable promoters (STPs) to enable flexible control of multi-gene expression in mammalian cells. METHODS: We designed and constructed synthetic tunable promoters (STPs) that incorporate both a universal activation site (UAS) and a specific activation site (SAS), enabling multi-level expression control via the CRISPR activation (CRISPRa) system. To evaluate promoter activity, we utilized Massively Parallel Reporter Assays (MPRA) to assess the basal strengths of the STPs and their activation responses. Next, we constructed a three-gene reporter system to assess the capacity of the synthetic promoters for achieving multilevel control of single-gene expression within multi-gene systems. RESULTS: The promoter library contains 24,960 unique non-redundant promoters with distinct sequence characteristics. MPRA revealed a wide range of promoter activities, showing different basal strengths and distinct activation levels when activated by the CRISPRa system. When regulated by targeting the SAS, the STPs exhibited orthogonality, allowing multilevel control of single-gene expression within multi-gene systems without cross-interference. Furthermore, the combinatorial activation of STPs in a multi-gene system enlarged the scope of expression levels achievable, providing fine-tuned control over gene expression. CONCLUSION: We provide a diverse collection of synthetic tunable promoters, offering a valuable toolkit for the construction and manipulation of multi-gene systems in mammalian cells, with applications in gene therapy and biotechnology.