Synthetic tunable promoters for flexible control of multi-gene expression in mammalian cells

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. Here, we designed and constructed 24,960 gRNA-based synthetic tunable promoters (STPs). Each STP incorporates universal activation site (UAS) and specific activation site (SAS), allowing multi-level expression control via the CRISPR activation (CRISPRa) system. Using Massively Parallel Reporter Assays (MPRA), we observed that our STP library contains a range of activatable promoters with varying basal strengths, each exhibiting different levels of increased activity upon activation. We validated that our STPs can enhance single-gene expression level by targeting UAS. More importantly, STPs exhibited orthogonality when regulated by targeting SAS, allowing multilevel and stepwise control of single-gene expression within multi-gene system without cross-interference between genes. Furthermore, the combinatorial activation of STPs in a multi-gene system can enlarge the scope of expression levels. 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.