Noise-Reducing Negative-Feedback Optogenetic Circuits in Mammalian Cells

Gene autorepression is widely present in nature and is also employed in synthetic biology, partly to reduce gene expression noise in cells. Optogenetic systems have recently been employed for controlling gene expression levels in mammalian cells, but most have utilized activator-based proteins, neglecting negative feedback. Here, we engineer optogenetic negative-feedback gene circuits in mammalian cells to achieve noise-reduction for precise gene expression control. We build a toolset of these noise-reducing Light-Inducible Tuner (LITer) gene circuits using the TetR repressor fused with a Tet-Inhibitory peptide (TIP) or a degradation tag through the light-sensitive LOV2 protein domain. These LITers provide nearly a range of 4-fold gene expression control and up to five-fold noise reduction from existing optogenetic systems. Moreover, we use the LITer gene circuit architecture to control gene expression of the cancer oncogene KRAS(G12V) and study its downstream effects through phospho-ERK levels and cellular proliferation. Overall, these novel LITer optogenetic platforms should enable precise spatiotemporal perturbations for studying multicellular phenotypes in developmental biology, oncology, and other biomedical fields of research.