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 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 Characterization, modelling and mitigation of gene expression burden in mammalian cells

2019 ◽  
Author(s):  
T Frei ◽  
F Cella ◽  
F Tedeschi ◽  
J Gutierrez ◽  
GB Stan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Genome Engineering ◽  
Host Cells ◽  
Genetic Circuits ◽  
Circuit Performance ◽  
Resource Requirements ◽  
Synthetic Circuit ◽  
Resource Aware

AbstractDespite recent advances in genome engineering, the design of genetic circuits in mammalian cells is still painstakingly slow and fraught with inexplicable failures. Here we demonstrate that competition for limited transcriptional and translational resources dynamically couples otherwise independent co-expressed exogenous genes, leading to diminished performance and contributing to the divergence between intended and actual function. We also show that the expression of endogenous genes is likewise impacted when genetic payloads are expressed in the host cells. Guided by a resource-aware mathematical model and our experimental finding that post-transcriptional regulators have a large capacity for resource redistribution, we identify and engineer natural and synthetic miRNA-based incoherent feedforward loop (iFFL) circuits that mitigate gene expression burden. The implementation of these circuits features the novel use of endogenous miRNAs as integral components of the engineered iFFL device, a versatile hybrid design that allows burden mitigation to be achieved across different cell-lines with minimal resource requirements. This study establishes the foundations for context-aware prediction and improvement of in vivo synthetic circuit performance, paving the way towards more rational synthetic construct design in mammalian cells.

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

2019 ◽  
Vol 47 (14) ◽  
pp. 7703-7714 ◽  
Author(s):  
Michael Tyler Guinn ◽  
Gábor Balázsi
Keyword(s):  
Gene Expression ◽  
Noise Reduction ◽  
Negative Feedback ◽  
Mammalian Cells ◽  
Cellular Proliferation ◽  
Protein Domain ◽  
Gene Circuits ◽  
Expression Control ◽  
Gene Expression Control

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.

scholarly journals The Many Faces of Gene Regulation in Cancer: A Computational Oncogenomics Outlook

Genes ◽  
2019 ◽  
Vol 10 (11) ◽  
pp. 865 ◽  
Author(s):  
Enrique Hernández-Lemus ◽  
Helena Reyes-Gopar ◽  
Jesús Espinal-Enríquez ◽  
Soledad Ochoa
Keyword(s):  
Gene Expression ◽  
Complex Disease ◽  
Genomic Analysis ◽  
Regulatory Control ◽  
Control Of Gene Expression ◽  
Factors Affecting ◽  
Expression Control ◽  
Downstream Effects ◽  
Gene Expression Control ◽  
The Many

Cancer is a complex disease at many different levels. The molecular phenomenology of cancer is also quite rich. The mutational and genomic origins of cancer and their downstream effects on processes such as the reprogramming of the gene regulatory control and the molecular pathways depending on such control have been recognized as central to the characterization of the disease. More important though is the understanding of their causes, prognosis, and therapeutics. There is a multitude of factors associated with anomalous control of gene expression in cancer. Many of these factors are now amenable to be studied comprehensively by means of experiments based on diverse omic technologies. However, characterizing each dimension of the phenomenon individually has proven to fall short in presenting a clear picture of expression regulation as a whole. In this review article, we discuss some of the more relevant factors affecting gene expression control both, under normal conditions and in tumor settings. We describe the different omic approaches that we can use as well as the computational genomic analysis needed to track down these factors. Then we present theoretical and computational frameworks developed to integrate the amount of diverse information provided by such single-omic analyses. We contextualize this within a systems biology-based multi-omic regulation setting, aimed at better understanding the complex interplay of gene expression deregulation in cancer.

scholarly journals A yeast optogenetic toolkit (yOTK) for gene expression control in Saccharomyces cerevisiae

2019 ◽  
Author(s):  
Jidapas (My) An-adirekkun ◽  
Cameron J. Stewart ◽  
Stephanie H. Geller ◽  
Michael T. Patel ◽  
Justin Melendez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Biological Networks ◽  
Modular Assembly ◽  
Expression Control ◽  
Control Gene Expression ◽  
Gene Expression Control ◽  
Rapid Generation ◽  
Spatiotemporal Control ◽  
Different Levels

AbstractOptogenetic tools for controlling gene expression are ideal for tuning synthetic biological networks due to the exquisite spatiotemporal control available with light. Here we develop an optogenetic system for gene expression control and integrate it with an existing yeast toolkit allowing for rapid, modular assembly of light-controlled circuits in the important chassis organism Saccharomyces cerevisiae. We reconstitute activity of a split synthetic zinc-finger transcription factor (TF) using light-induced dimerization. We optimize function of this split TF and demonstrate the utility of the toolkit workflow by assembling cassettes expressing the TF activation domain and DNA-binding domain at different levels. Utilizing this TF and a synthetic promoter we demonstrate that light-intensity and duty-cycle can be used to modulate gene expression over the range currently available from natural yeast promoters. This work allows for rapid generation and prototyping of optogenetic circuits to control gene expression in Saccharomyces cerevisiae.

scholarly journals Chemical control of a CRISPR-Cas9 acetyltransferase

2017 ◽  
Author(s):  
Jonathan H. Shrimp ◽  
Carissa Grose ◽  
Stephanie R. T. Widmeyer ◽  
Ajit Jadhav ◽  
Jordan L. Meier
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Critical Role ◽  
Regulation Of Transcription ◽  
Transcriptional Activators ◽  
Guide Rna ◽  
Transcriptional Reporter ◽  
Expression Control ◽  
Gene Expression Control ◽  
Spatiotemporal Control

AbstractLysine acetyltransferases (KATs) play a critical role in the regulation of transcription and other genomic functions. However, a persistent challenge is the development of assays capable of defining KAT activity directly in living cells. Towards this goal, here we report the application of a previously reported dCas9-p300 fusion as a transcriptional reporter of KAT activity. First we benchmark the activity of dCas9-p300 relative to other dCas9-based transcriptional activators, and demonstrate its compatibility with second generation short guide RNA architectures. Next, we repurpose this technology to rapidly identify small molecule inhibitors of acetylation-dependent gene expression. These studies validate a recently reported p300 inhibitor chemotype, and reveal a role for p300’s bromodomain in dCas9-p300-mediated transcriptional activation. Comparison with other CRISPR-Cas9 transcriptional activators highlights the inherent ligand tuneable nature of dCas9-p300 fusions, suggesting new opportunities for orthogonal gene expression control. Overall, our studies highlight dCas9-p300 as a powerful tool for studying gene expression mechanisms in which acetylation plays a causal role, and provide a foundation for future applications requiring spatiotemporal control over acetylation at specific genomic loci.

scholarly journals A tunable dual-input system for on-demand dynamic gene expression regulation

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Elisa Pedone ◽  
Lorena Postiglione ◽  
Francesco Aulicino ◽  
Dan L. Rocca ◽  
Sandra Montes-Olivas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Stability ◽  
Mammalian Cells ◽  
Dynamic Range ◽  
Embryonic Stem ◽  
Cellular Systems ◽  
Control Of Gene Expression ◽  
Cell Functions ◽  
Input System ◽  
Gene Expression Control

Abstract Cellular systems have evolved numerous mechanisms to adapt to environmental stimuli, underpinned by dynamic patterns of gene expression. In addition to gene transcription regulation, modulation of protein levels, dynamics and localization are essential checkpoints governing cell functions. The introduction of inducible promoters has allowed gene expression control using orthogonal molecules, facilitating its rapid and reversible manipulation to study gene function. However, differing protein stabilities hinder the generation of protein temporal profiles seen in vivo. Here, we improve the Tet-On system integrating conditional destabilising elements at the post-translational level and permitting simultaneous control of gene expression and protein stability. We show, in mammalian cells, that adding protein stability control allows faster response times, fully tunable and enhanced dynamic range, and improved in silico feedback control of gene expression. Finally, we highlight the effectiveness of our dual-input system to modulate levels of signalling pathway components in mouse Embryonic Stem Cells.

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