A gene expression control technology for cell-free systems and synthetic cells via targeted gene silencing and transfection

Cell-free transcription-translation (TXTL) is an in vitro protein expression platform. In synthetic biology, TXTL is utilized for a variety of technologies, such as genetic circuit construction, metabolic pathway optimization, and building prototypes of synthetic cells. For all these purposes, the ability to precisely control gene expression is essential. Various strategies to control gene expression in TXTL have been developed; however, further advancements on gene-specific and straightforward regulation methods are still demanded. Here, we designed a novel method to control gene expression in TXTL, called a "silencing oligo." The silencing oligo is a short oligonucleotide that binds to the target mRNA. We demonstrated that addition of the silencing oligo inhibits eGFP expression in TXTL in a sequence-dependent manner. We investigated one of the silencing oligos inhibitory mechanisms and confirmed that silencing is associated with RNase H activity in bacterial TXTL reactions. We also engineered a transfection system that can be used in synthetic cells. We screened two dozen different commercially available transfection reagents to identify the one that works most robustly in our system. Finally, we combined the silencing oligo with the transfection technology, demonstrating that we can control the gene expression by transfecting silencing oligo-containing liposomes into the synthetic cells.