An Engineer’s Guide to Genetic Circuits

Gene therapy and transgenic research have advanced quickly in recent years due to the development of CRISPR technology. The rapid development of CRISPR technology has been largely benefited by chemical engineering. Firstly, chemical or synthetic substance enables spatiotemporal and conditional control of Cas9 or dCas9 activities. It prevents the leaky expression of CRISPR components, as well as minimizes toxicity and off-target effects. Multi-input logic operations and complex genetic circuits can also be implemented via multiplexed and orthogonal regulation of target genes. Secondly, rational chemical modifications to the sgRNA enhance gene editing efficiency and specificity by improving sgRNA stability and binding affinity to on-target genomic loci, and hence reducing off-target mismatches and systemic immunogenicity. Chemically-modified Cas9 mRNA is also more active and less immunogenic than the native mRNA. Thirdly, nonviral vehicles can circumvent the challenges associated with viral packaging and production through the delivery of Cas9-sgRNA ribonucleoprotein complex or large Cas9 expression plasmids. Multi-functional nanovectors enhance genome editing in vivo by overcoming multiple physiological barriers, enabling ligand-targeted cellular uptake, and blood-brain barrier crossing. Chemical engineering can also facilitate viral-based delivery by improving vector internalization, allowing tissue-specific transgene expression, and preventing inactivation of the viral vectors in vivo. This review aims to discuss how chemical engineering has helped improve existing CRISPR applications and enable new technologies for biomedical research. The usefulness, advantages, and molecular action for each chemical engineering approach are also highlighted.

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Stability and Robustness of Unbalanced Genetic Toggle Switches in the Presence of Scarce Resources

Life ◽

10.3390/life11040271 ◽

2021 ◽

Vol 11 (4) ◽

pp. 271

Author(s):

Chentao Yong ◽

Andras Gyorgy

Keyword(s):

Cell Fate ◽

Rational Design ◽

Resource Competition ◽

Dynamic Properties ◽

Mechanistic Model ◽

Robustness Analysis ◽

System Level ◽

Genetic Circuits ◽

Scarce Resources ◽

Genetic Systems

While the vision of synthetic biology is to create complex genetic systems in a rational fashion, system-level behaviors are often perplexing due to the context-dependent dynamics of modules. One major source of context-dependence emerges due to the limited availability of shared resources, coupling the behavior of disconnected components. Motivated by the ubiquitous role of toggle switches in genetic circuits ranging from controlling cell fate differentiation to optimizing cellular performance, here we reveal how their fundamental dynamic properties are affected by competition for scarce resources. Combining a mechanistic model with nullcline-based stability analysis and potential landscape-based robustness analysis, we uncover not only the detrimental impacts of resource competition, but also how the unbalancedness of the switch further exacerbates them. While in general both of these factors undermine the performance of the switch (by pushing the dynamics toward monostability and increased sensitivity to noise), we also demonstrate that some of the unwanted effects can be alleviated by strategically optimized resource competition. Our results provide explicit guidelines for the context-aware rational design of toggle switches to mitigate our reliance on lengthy and expensive trial-and-error processes, and can be seamlessly integrated into the computer-aided synthesis of complex genetic systems.

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Model-guided design of mammalian genetic programs

Science Advances ◽

10.1126/sciadv.abe9375 ◽

2021 ◽

Vol 7 (8) ◽

pp. eabe9375

Author(s):

J. J. Muldoon ◽

V. Kandula ◽

M. Hong ◽

P. S. Donahue ◽

J. D. Boucher ◽

...

Keyword(s):

Mammalian Cells ◽

Computational Models ◽

Biological Complexity ◽

Genetic Circuits ◽

Cellular Functions ◽

High Performing ◽

Design Objective ◽

Complex Functions ◽

Mammalian Genetic ◽

Analog Processing

Genetically engineering cells to perform customizable functions is an emerging frontier with numerous technological and translational applications. However, it remains challenging to systematically engineer mammalian cells to execute complex functions. To address this need, we developed a method enabling accurate genetic program design using high-performing genetic parts and predictive computational models. We built multifunctional proteins integrating both transcriptional and posttranslational control, validated models for describing these mechanisms, implemented digital and analog processing, and effectively linked genetic circuits with sensors for multi-input evaluations. The functional modularity and compositional versatility of these parts enable one to satisfy a given design objective via multiple synonymous programs. Our approach empowers bioengineers to predictively design mammalian cellular functions that perform as expected even at high levels of biological complexity.

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Cell-Free Characterization of Coherent Feed-Forward Loop-Based Synthetic Genetic Circuits

ACS Synthetic Biology ◽

10.1021/acssynbio.1c00024 ◽

2021 ◽

Author(s):

Pascal A. Pieters ◽

Bryan L. Nathalia ◽

Ardjan J. van der Linden ◽

Peng Yin ◽

Jongmin Kim ◽

...

Keyword(s):

Genetic Circuits ◽

Feed Forward ◽

Feed Forward Loop

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

Nature Communications ◽

10.1038/s41467-019-12800-7 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 9

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.

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CRIMoClo plasmids for modular assembly and orthogonal chromosomal integration of synthetic circuits in Escherichia coli

Journal of Biological Engineering ◽

10.1186/s13036-019-0218-8 ◽

2019 ◽

Vol 13 (1) ◽

Cited By ~ 1

Author(s):

Stefano Vecchione ◽

Georg Fritz

Keyword(s):

Escherichia Coli ◽

Synthetic Biology ◽

Integrated Circuits ◽

High Efficiency ◽

Genetic Circuit ◽

Dna Assembly ◽

Chromosomal Integration ◽

Golden Gate ◽

Genetic Circuits ◽

E Coli

Abstract Background Synthetic biology heavily depends on rapid and simple techniques for DNA engineering, such as Ligase Cycling Reaction (LCR), Gibson assembly and Golden Gate assembly, all of which allow for fast, multi-fragment DNA assembly. A major enhancement of Golden Gate assembly is represented by the Modular Cloning (MoClo) system that allows for simple library propagation and combinatorial construction of genetic circuits from reusable parts. Yet, one limitation of the MoClo system is that all circuits are assembled in low- and medium copy plasmids, while a rapid route to chromosomal integration is lacking. To overcome this bottleneck, here we took advantage of the conditional-replication, integration, and modular (CRIM) plasmids, which can be integrated in single copies into the chromosome of Escherichia coli and related bacteria by site-specific recombination at different phage attachment (att) sites. Results By combining the modularity of the MoClo system with the CRIM plasmids features we created a set of 32 novel CRIMoClo plasmids and benchmarked their suitability for synthetic biology applications. Using CRIMoClo plasmids we assembled and integrated a given genetic circuit into four selected phage attachment sites. Analyzing the behavior of these circuits we found essentially identical expression levels, indicating orthogonality of the loci. Using CRIMoClo plasmids and four different reporter systems, we illustrated a framework that allows for a fast and reliable sequential integration at the four selected att sites. Taking advantage of four resistance cassettes the procedure did not require recombination events between each round of integration. Finally, we assembled and genomically integrated synthetic ECF σ factor/anti-σ switches with high efficiency, showing that the growth defects observed for circuits encoded on medium-copy plasmids were alleviated. Conclusions The CRIMoClo system enables the generation of genetic circuits from reusable, MoClo-compatible parts and their integration into 4 orthogonal att sites into the genome of E. coli. Utilizing four different resistance modules the CRIMoClo system allows for easy, fast, and reliable multiple integrations. Moreover, utilizing CRIMoClo plasmids and MoClo reusable parts, we efficiently integrated and alleviated the toxicity of plasmid-borne circuits. Finally, since CRIMoClo framework allows for high flexibility, it is possible to utilize plasmid-borne and chromosomally integrated circuits simultaneously. This increases our ability to permute multiple genetic modules and allows for an easier design of complex synthetic metabolic pathways in E. coli.

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