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

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 prot...

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Bibliographic Details
Published in:Nucleic acids research 2019-08, Vol.47 (14), p.7703-7714
Main Authors: Guinn, Michael Tyler, Balázsi, Gábor
Format: Article
Language:English
Subjects:
Algorithms
Animals
Cells, Cultured
Feedback, Physiological
Gene Expression Regulation - genetics
Gene Expression Regulation - radiation effects
Gene Regulatory Networks - genetics
Gene Regulatory Networks - radiation effects
HEK293 Cells
Humans
Light
Models, Genetic
Optogenetics - methods
Proto-Oncogene Proteins p21(ras) - genetics
Reproducibility of Results
Synthetic Biology - methods
Synthetic Biology and Bioengineering
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Summary: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.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkz556