Noise-reducing optogenetic negative-feedback gene circuits in human cells.

Michael Tyler Guinn,Gábor Balázsi

doi:10.1093/nar/gkz556

Abstract

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 developed for controlling gene expression levels in mammalian cells, but most have utilized activator-based proteins, neglecting negative feedback except for in silico control. Here, we engineer optogenetic gene circuits into mammalian cells to achieve noise-reduction for precise gene expression control by genetic, in vitro negative feedback. We build a toolset of these noise-reducing Light-Inducible Tuner (LITer) gene circuits using the TetR repressor fused with a Tet-inhibiting peptide (TIP) or a degradation tag through the light-sensitive LOV2 protein domain. These LITers provide a range of nearly 4-fold gene expression control and up to 5-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.

Highlights

Gene expression levels and variability dictate transcript and protein production that define the properties of living cells in health and disease [1,2]
We turned to the recent light plate apparatus (LPA) system, which is customizable for in vitro assays, allowing parameter scans for light intensities, pulses and complex light patterns [42]
To characterize the response of VVD and LITer1.0 gene circuits to discontinuous light-induction regimes, we investigated by fluorescence microscopy and flow cytometry measurements how single light pulses of variable duration and fixed LPA intensity (1000 g.s.) affect gene expression (Figure 2A and Supplementary Figure S7)

Summary

Introduction

Gene expression levels and variability (noise) dictate transcript and protein production that define the properties of living cells in health and disease [1,2]. Engineering gene circuits that control gene expression levels and noise simultaneously can reveal important thresholds and sensitivities for broad biological phenomena such as metastasis, epithelial-to-mesenchymal (EMT) transition and drug resistance [18]. A few decades ago, bacterial regulator-based systems emerged capable of controlling intermediate gene expression levels [19,20,21]. Despite this advancement, these systems often suffered from high noise since they lacked feedback regulation, leaving gene expression variability as an uncontrolled parameter [22]. Adjusting noise has been neglected by traditional methods of gene expression control, which tend to focus on extreme gene expression changes (e.g. knockout and overexpression) in cells or organisms

Methods

Results

Conclusion