Engineered protein switches for exogenous control of gene expression

2020 ◽  
Vol 48 (5) ◽  
pp. 2205-2212
Author(s):  
Shaun Spisak ◽  
Marc Ostermeier
Keyword(s):  
Gene Expression ◽  
Domain Swapping ◽  
Signaling Proteins ◽  
Genetic Circuits ◽  
Control Of Gene Expression ◽  
Regulate Gene Expression ◽  
Substrate Specificities ◽  
Large Pool ◽  
Domain Insertion ◽  
Exogenous Control

There is an ongoing need in the synthetic biology community for novel ways to regulate gene expression. Protein switches, which sense biological inputs and respond with functional outputs, represent one way to meet this need. Despite the fact that there is already a large pool of transcription factors and signaling proteins available, the pool of existing switches lacks the substrate specificities and activities required for certain applications. Therefore, a large number of techniques have been applied to engineer switches with novel properties. Here we discuss some of these techniques by broadly organizing them into three approaches. We show how novel switches can be created through mutagenesis, domain swapping, or domain insertion. We then briefly discuss their use as biosensors and in complex genetic circuits.

scholarly journals High-performance chemical- and light-inducible recombinases in mammalian cells and mice

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Benjamin H. Weinberg ◽  
Jang Hwan Cho ◽  
Yash Agarwal ◽  
N. T. Hang Pham ◽  
Leidy D. Caraballo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
High Performance ◽  
Genome Engineering ◽  
Genetic Circuits ◽  
Control Of Gene Expression ◽  
Expression Control ◽  
Gene Expression Control ◽  
High Performance Systems ◽  
Spatiotemporal Control

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.

scholarly journals A modular toolset for electrogenetics

2021 ◽  
Author(s):  
Joshua M Lawrence ◽  
Yutong Yin ◽  
Paolo Bombelli ◽  
Alberto Scarampi ◽  
Marko Storch ◽  
...  
Keyword(s):  
Gene Expression ◽  
Industrial Applications ◽  
Electrochemical Activation ◽  
Genetic Circuits ◽  
Control Of Gene Expression ◽  
Promoter Library ◽  
Redox Responsive ◽  
Electrochemical Control ◽  
Redox Molecules ◽  
New Applications

Synthetic biology research and its industrial applications rely on the deterministic spatiotemporal control of gene expression. Recently, electrochemical control of gene expression has been demonstrated in electrogenetic systems (redox-responsive promoters used alongside redox inducers and an electrode), allowing for the direct integration of electronics with complex biological processes for a variety of new applications. However, the use of electrogenetic systems is limited by poor activity, tunability and standardisation. Here, we have developed a variety of genetic and electrochemical tools that facilitate the design and vastly improve the performance of electrogenetic systems. We developed a strong, unidirectional, redox-responsive promoter before deriving a mutant promoter library with a spectrum of strengths. We then constructed genetic circuits with these parts and demonstrated their activation by multiple classes of redox molecules. Finally, we demonstrated electrochemical activation of gene expression in aerobic conditions utilising a novel, modular bioelectrochemical device. This toolset provides researchers with all the elements needed to design and build optimised electrogenetic systems for specific applications.

Engineering Gene Control Circuits with Allosteric Ribozymes in Human Cells as a Medicine of the Future

2013 ◽  
pp. 860-883
Author(s):  
Robert Penchovsky
Keyword(s):  
Gene Expression ◽  
Cell Fate ◽  
Regulatory Networks ◽  
Control Of Gene Expression ◽  
Practical Applications ◽  
Gene Therapies ◽  
Sequencing Technologies ◽  
The Future ◽  
Control Circuits ◽  
Exogenous Control

Systems and synthetic biology promise to develop new approaches for analysis and design of complex gene expression regulatory networks in living cells with many practical applications to the pharmaceutical and biotech industries. In this chapter the development of novel universal strategies for exogenous control of gene expression is discussed. They are based on designer allosteric ribozymes that can function in the cell. The synthetic riboswitches are obtained by a patented computational procedure that provides fast and accurate modular designs with various Boolean logic functions. The riboswitches can be designed to sense in the cell either the presence or the absence of disease indicative RNA(s) or small molecules, and to switch on or off the gene expression of any exogenous protein. In addition, the riboswitches can be engineered to induce RNA interference or microRNA pathways that can conditionally down regulate the expression of key proteins in the cell. That can prevent a disease’s development. Therefore, the presented synthetic riboswitches can be used as truly universal cellular biosensors. Nowadays, disease indicative RNA(s) can be precisely identified by employing next-generation sequencing technologies with high accuracy . The methods can be employed not only for exogenous control of gene expression but also for re-programming the cell fate, anticancer, and antiviral gene therapies. Such approaches may be employed as potent molecular medicines of the future.

scholarly journals Effective CRISPRa-Mediated Control of Gene Expression in Bacteria Must Overcome Strict Target Site Requirements

2019 ◽  
Author(s):  
Jason Fontana ◽  
Chen Dong ◽  
Cholpisit Kiattisewee ◽  
Venkata P. Chavali ◽  
Benjamin I. Tickman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Target Site ◽  
Multiple Features ◽  
Control Of Gene Expression ◽  
Regulate Gene Expression ◽  
Guide Rna ◽  
Target Sites ◽  
Helical Turn ◽  
The Right

AbstractIn bacterial systems, CRISPR-Cas transcriptional activation (CRISPRa) has the potential to dramatically expand our ability to regulate gene expression, but we currently lack a complete understanding of the rules for designing effective guide RNA target sites. We have identified multiple features of bacterial promoters that impose stringent requirements on bacterial CRISPRa target sites. Most importantly, we found that shifting a gRNA target site by 2-4 bases along the DNA target can cause a nearly complete loss in activity. The loss in activity can be rescued by shifting the target site 10-11 bases, corresponding to one full helical turn. Practically, our results suggest that it will be challenging to find a gRNA target site with an appropriate PAM sequence at precisely the right position at arbitrary genes of interest. To overcome this limitation, we demonstrate that a dCas9 variant with expanded PAM specificity allows activation of promoters that cannot be activated by S. pyogenes dCas9. These results provide a roadmap for future engineering efforts to further expand and generalize the scope of bacterial CRISPRa.

scholarly journals A Synthetic Transcription Platform for Programmable Gene Expression in Mammalian Cells

2020 ◽  
Author(s):  
William C.W. Chen ◽  
Leonid Gaidukov ◽  
Ming-Ru Wu ◽  
Jicong Cao ◽  
Gigi C.G. Choi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Interferon Gamma ◽  
Mammalian Cells ◽  
Cell Types ◽  
Regulatory Elements ◽  
Genetic Circuits ◽  
Control Of Gene Expression ◽  
Transcription System ◽  
Multiple Cell ◽  
Cellular Phenotypes

Precise, scalable, and sustainable control of genetic and cellular activities in mammalian cells is key to developing precision therapeutics and smart biomanufacturing. We created a highly tunable, modular, versatile CRISPR-based synthetic transcription system for the programmable control of gene expression and cellular phenotypes in mammalian cells. Genetic circuits consisting of well-characterized libraries of guide RNAs, binding motifs of synthetic operators, transcriptional activators, and additional genetic regulatory elements expressed mammalian genes in a highly predictable and tunable manner. We demonstrated the programmable control of reporter genes episomally and chromosomally, with up to 25-fold more EF1[alpha]; promoter activity, in multiple cell types. We used these circuits to program secretion of human monoclonal antibodies and to control T cell effector function marked by interferon-[gamma] production. Antibody titers and interferon-[gamma]; concentrations were significantly correlated with synthetic promoter strengths, providing a platform for programming gene expression and cellular function for biological, biomanufacturing, and biomedical applications.

scholarly journals High-performance chemical and light-inducible recombinases in mammalian cells and mice

2019 ◽  
Author(s):  
Benjamin H. Weinberg ◽  
Jang Hwan Cho ◽  
Yash Agarwal ◽  
N. T. Hang Pham ◽  
Leidy D. Caraballo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
High Performance ◽  
Genome Engineering ◽  
Dynamic Range ◽  
Signal To Noise Ratio ◽  
Genetic Circuits ◽  
Control Of Gene Expression ◽  
Gene Expression Control ◽  
High Performance Systems

ABSTRACTSite-specific DNA recombinases are some of the most powerful genome engineering tools in biology. Chemical and light-inducible recombinases, in particular, enable spatiotemporal control of gene expression. However, the availability of inducible recombinases is scarce due to the challenge of engineering high performance systems with low basal activity and sufficient dynamic range. This limitation constrains the sophistication of genetic circuits and animal models that can be created. To expand the number of available inducible recombinases, here we present a library of >20 orthogonal split recombinases that can be inducibly dimerized and activated by various small molecules, light, and temperature in mammalian cells and mice.Furthermore, we have engineered inducible split Cre systems with better performance than existing inducible Cre systems. Using our orthogonal inducible recombinases, we created a “genetic switchboard” that can independently regulate the expression of 3 different cytokines in the same cell. To demonstrate novel capability with our split recombinases, we created a tripartite inducible Flp and a 4-Input AND gate. We have performed extensive quantitative characterization of the inducible recombinases for benchmarking their performances, including computation of distinguishability of outputs in terms of signal-to-noise ratio (SNR). To facilitate sharing of this set of reagents, we have deposited our library to Addgene. This library thus significantly expands capabilities for precise and multiplexed mammalian gene expression control.

scholarly journals Control of Gene Expression With Quercetin-Responsive Modular Circuits

2021 ◽  
Vol 9 ◽  
Author(s):  
Fernanda Miyuki Kashiwagi ◽  
Brenno Wendler Miranda ◽  
Fabio de Oliveira Pedrosa ◽  
Emanuel Maltempi de Souza ◽  
Marcelo Müller-Santos
Keyword(s):  
Gene Expression ◽  
Regulatory System ◽  
Genetic Circuits ◽  
Plant Metabolites ◽  
Control Of Gene Expression ◽  
E Coli ◽  
Ribosome Binding Sites ◽  
Flavonoid Quercetin ◽  
Quercetin Concentration ◽  
Different Levels

Control of gene expression is crucial for several biotechnological applications, especially for implementing predictable and controllable genetic circuits. Such circuits are often implemented with a transcriptional regulator activated by a specific signal. These regulators should work independently of the host machinery, with low gratuitous induction or crosstalk with host components. Moreover, the signal should also be orthogonal, recognized only by the regulator with minimal interference with the host operation. In this context, transcriptional regulators activated by plant metabolites as flavonoids emerge as candidates to control gene expression in bacteria. However, engineering novel circuits requires the characterization of the genetic parts (e.g., genes, promoters, ribosome binding sites, and terminators) in the host of interest. Therefore, we decomposed the QdoR regulatory system of B. subtilis, responsive to the flavonoid quercetin, and reassembled its parts into genetic circuits programmed to have different levels of gene expression and noise dependent on the concentration of quercetin. We showed that only one of the promoters regulated by QdoR worked well in E. coli, enabling the construction of other circuits induced by quercetin. The QdoR expression was modulated with constitutive promoters of different transcriptional strengths, leading to low expression levels when QdoR was highly expressed and vice versa. E. coli strains expressing high and low levels of QdoR were mixed and induced with the same quercetin concentration, resulting in two stable populations expressing different levels of their gene reporters. Besides, we demonstrated that the level of QdoR repression generated different noise levels in gene expression dependent on the concentration of quercetin. The circuits presented here can be exploited in applications requiring adjustment of gene expression and noise using a highly available and natural inducer as quercetin.

Nucleic Acids-Based Nanotechnology

2018 ◽  
pp. 155-171
Author(s):  
Robert Penchovsky
Keyword(s):  
Gene Expression ◽  
Drug Discovery ◽  
Nucleic Acids ◽  
Gene Silencing ◽  
Self Assembly ◽  
Pharmaceutical Research ◽  
Molecular Computing ◽  
Antibacterial Drug ◽  
Control Of Gene Expression ◽  
Exogenous Control

Nanobiotechnology is emerging as a valuable field that integrates research from science and technology to create novel nanodevices and nanostructures with various applications in modern nanotechnology. Applications of nanobiotechnology are employed in biomedical and pharmaceutical research, biosensoring, nanofluidics, self-assembly of nanostructures, nanopharmaceutics, molecular computing, and others. It has been proven that nucleic acids are a very suitable medium for self-assembly of diverse nanostructures and catalytic nanodevices for various applications. In this chapter, the authors discuss various applications of nucleic-based nanotechnology. The areas discussed here include building nanostructures using DNA oligonucleodite, self-assembly of integrated RNA-based nanodevices for molecular computing and diagnostics, antibacterial drug discovery, exogenous control of gene expression, and gene silencing.

Engineering Gene Control Circuits with Allosteric Ribozymes in Human Cells as a Medicine of the Future

2012 ◽  
pp. 71-92 ◽  
Author(s):  
Robert Penchovsky
Keyword(s):  
Gene Expression ◽  
Cell Fate ◽  
Regulatory Networks ◽  
Control Of Gene Expression ◽  
Practical Applications ◽  
Gene Therapies ◽  
Sequencing Technologies ◽  
The Future ◽  
Control Circuits ◽  
Exogenous Control

Systems and synthetic biology promise to develop new approaches for analysis and design of complex gene expression regulatory networks in living cells with many practical applications to the pharmaceutical and biotech industries. In this chapter the development of novel universal strategies for exogenous control of gene expression is discussed. They are based on designer allosteric ribozymes that can function in the cell. The synthetic riboswitches are obtained by a patented computational procedure that provides fast and accurate modular designs with various Boolean logic functions. The riboswitches can be designed to sense in the cell either the presence or the absence of disease indicative RNA(s) or small molecules, and to switch on or off the gene expression of any exogenous protein. In addition, the riboswitches can be engineered to induce RNA interference or microRNA pathways that can conditionally down regulate the expression of key proteins in the cell. That can prevent a disease’s development. Therefore, the presented synthetic riboswitches can be used as truly universal cellular biosensors. Nowadays, disease indicative RNA(s) can be precisely identified by employing next-generation sequencing technologies with high accuracy . The methods can be employed not only for exogenous control of gene expression but also for re-programming the cell fate, anticancer, and antiviral gene therapies. Such approaches may be employed as potent molecular medicines of the future.

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