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Title: Small-molecule inducible transcriptional control in mammalian cells
Tools for tuning transcription in mammalian cells have broad applications, from basic biological discovery to human gene therapy. While precise control over target gene transcription via dosing with small molecules (drugs) is highly sought, the design of such inducible systems that meets required performance metrics poses a great challenge in mammalian cell synthetic biology. Important characteristics include tight and tunable gene expression with a low background, minimal drug toxicity, and orthogonality. Here, we review small-molecule-inducible transcriptional control devices that have demonstrated success in mammalian cells and mouse models. Most of these systems employ natural or designed ligand-binding protein domains to directly or indirectly communicate with transcription machinery at a target sequence, via carefully constructed fusions. Example fusions include those to transcription activator-like effectors (TALEs), DNA-targeting proteins (e.g. dCas systems) fused to transactivating domains, and recombinases. Similar to the architecture of Type I nuclear receptors, many of the systems are designed such that the transcriptional controller is excluded from the nucleus in the absence of an inducer. Techniques that use ligand-induced proteolysis and antibody-based chemically induced dimerizers are also described. Collectively, these transcriptional control devices take advantage of a variety of recently developed molecular biology tools and cell biology insights and more » represent both proof of concept (e.g. targeting reporter gene expression) and disease-targeting studies. « less
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Critical Reviews in Biotechnology
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1 to 20
Sponsoring Org:
National Science Foundation
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    Traditionally engineered genetic circuits have almost exclusively used naturally occurring transcriptional repressors. Recently, non-natural transcription factors (repressors) have been engineered and employed in synthetic biology with great success. However, transcriptional anti-repressors have largely been absent with regard to the regulation of genes in engineered genetic circuits. Here, we present a workflow for engineering systems of non-natural anti-repressors. In this study, we create 41 inducible anti-repressors. This collection of transcription factors respond to two distinct ligands, fructose (anti-FruR) or D-ribose (anti-RbsR); and were complemented by 14 additional engineered anti-repressors that respond to the ligand isopropyl β-d-1-thiogalactopyranoside (anti-LacI). In turn, we use this collection of anti-repressors and complementary genetic architectures to confer logical control over gene expression. Here, we achieved all NOT oriented logical controls (i.e., NOT, NOR, NAND, and XNOR). The engineered transcription factors and corresponding series, parallel, and series-parallel genetic architectures represent a nascent anti-repressor based transcriptional programming structure.

  3. Abstract

    Site-specific DNA recombinases are important genome engineering tools. Chemical- and light-inducible recombinases, in particular, enable spatiotemporal control of gene expression. However, inducible recombinases are scarce due to the challenge of engineering high performance systems, thus constraining the sophistication of genetic circuits and animal models that can be created. Here we present a library of >20 orthogonal inducible split recombinases that can be activated by small molecules, light and temperature in mammalian cells and mice. Furthermore, we engineer inducible split Cre systems with better performance than existing systems. Using our orthogonal inducible recombinases, we create a genetic switchboard that can independently regulate the expression of 3 different cytokines in the same cell, a tripartite inducible Flp, and a 4-input AND gate. We quantitatively characterize the inducible recombinases for benchmarking their performances, including computation of distinguishability of outputs. This library expands capabilities for multiplexed mammalian gene expression control.

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