PT - JOURNAL ARTICLE AU - Wakana Sato AU - Melanie Rasmussen AU - Nathaniel Gaut AU - Mahima Devarajan AU - Kaitlin Stokes AU - Christopher Deich AU - Aaron E. Engelhart AU - Katarzyna P. Adamala TI - A gene expression control technology for cell-free systems and synthetic cells via targeted gene silencing and transfection AID - 10.1101/2022.07.28.501919 DP - 2022 Jan 01 TA - bioRxiv PG - 2022.07.28.501919 4099 - http://biorxiv.org/content/early/2022/07/28/2022.07.28.501919.short 4100 - http://biorxiv.org/content/early/2022/07/28/2022.07.28.501919.full AB - 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 oligo’s 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.Competing Interest StatementThe authors have declared no competing interest.