Genetic Circuits Research Articles

CRISPR Tools for Engineering Prokaryotic Systems: Recent Advances and New Applications.

In the past decades, the broad selection of CRISPR-Cas systems has revolutionized biotechnology by enabling multimodal genetic manipulation in diverse organisms. Rooted in a molecular engineering perspective, we recapitulate the different CRISPR components and how they can be designed for specific genetic engineering applications. We first introduce the repertoire of Cas proteins and tethered effectors used to program new biological functions through gene editing and gene regulation. We review current guide RNA (gRNA) design strategies and computational tools and how CRISPR-based genetic circuits can be constructed through regulated gRNA expression. Then, we present recent advances in CRISPR-based biosensing, bioproduction, and biotherapeutics across in vitro and in vivo prokaryotic systems. Finally, we discuss forthcoming applications in prokaryotic CRISPR technology that will transform synthetic biology principles in the near future.

Effects of mRNA conformational switching on translational noise in gene circuits.

Intragenic translational heterogeneity describes the variation in translation at the level of transcripts for an individual gene. A factor that contributes to this source of variation is the mRNA structure. Both the composition of the thermodynamic ensemble, i.e., the stationary distribution of mRNA structures, and the switching dynamics between those play a role. The effect of the switching dynamics on intragenic translational heterogeneity remains poorly understood. We present a stochastic translation model that accounts for mRNA structure switching and is derived from a Markov model via approximate stochastic filtering. We assess the approximation on various timescales and provide a method to quantify how mRNA structure dynamics contributes to translational heterogeneity. With our approach, we allow quantitative information on mRNA switching from biophysical experiments or coarse-grain molecular dynamics simulations of mRNA structures to be included in gene regulatory chemical reaction network models without an increase in the number of species. Thereby, our model bridges a gap between mRNA structure kinetics and gene expression models, which we hope will further improve our understanding of gene regulatory networks and facilitate genetic circuit design.

Towards Autonomous Materials–Challenges in Chemical Communication

AbstractCoordination of functions in multi‐body or multi‐component systems requires communication among its parts and is a prerequisite for achieving complex tasks. While electromagnetic (network) communication provides a key ingredient for modern robotics, its molecular equivalent is largely missing for soft robots. With advancements in programmed cargo release, DNA strand‐displacement reactions, enzymatic cascades, and genetic circuits, budding solutions to such chemical network communication have emerged with the potential to drive function in soft machines. In order for such chemical communication to be useful, however, new control concepts, (orthogonal) communication protocols, and molecular solutions should be found. Herein, we provide a critical perspective on the current state‐of‐the‐art chemical communication and identify key challenges towards autonomous soft materials, including signal processing, dealing with noise, switching signaling modalities, and limitations in time and length scales that determine material design. Building on an emerging body of examples, we illustrate gaps, synergies, and new concepts that may provide possible solutions to achieve standardized and reliable molecular information exchange for regulating (soft) robotic function.

Automatic design of gene regulatory mechanisms for spatial pattern formation

Gene regulatory mechanisms (GRMs) control the formation of spatial and temporal expression patterns that can serve as regulatory signals for the development of complex shapes. Synthetic developmental biology aims to engineer such genetic circuits for understanding and producing desired multicellular spatial patterns. However, designing synthetic GRMs for complex, multi-dimensional spatial patterns is a current challenge due to the nonlinear interactions and feedback loops in genetic circuits. Here we present a methodology to automatically design GRMs that can produce any given two-dimensional spatial pattern. The proposed approach uses two orthogonal morphogen gradients acting as positional information signals in a multicellular tissue area or culture, which constitutes a continuous field of engineered cells implementing the same designed GRM. To efficiently design both the circuit network and the interaction mechanisms—including the number of genes necessary for the formation of the target spatial pattern—we developed an automated algorithm based on high-performance evolutionary computation. The tolerance of the algorithm can be configured to design GRMs that are either simple to produce approximate patterns or complex to produce precise patterns. We demonstrate the approach by automatically designing GRMs that can produce a diverse set of synthetic spatial expression patterns by interpreting just two orthogonal morphogen gradients. The proposed framework offers a versatile approach to systematically design and discover complex genetic circuits producing spatial patterns.

Abstract 6666: Leveraging an obligate anaerobic salmonella strain for targeted cargo delivery in the tumor microenvironment to restore immunotherapy sensitivity

Abstract Tumor immunotherapy is often hindered by unresponsive cold tumor microenvironments (TME). Although Salmonella-based immunotherapy has shown potential in overcoming these cold immune environments, safety concerns remain. Our study introduces a genetically reprogrammed Salmonella typhimurium strain (OASL) with a dual safety lock to enhance safety and modify immunosuppressive TME, sensitizing tumors to immunotherapies. We propose using OASL to alter the cold TME and locally release therapeutic cargos such as PDL1 nanobodies and tumor-specific neoantigen peptides identified through bioinformatic algorithms, activating immune cells within the tumor while minimizing side effects. We selected YB1, an obligate anaerobic Salmonella strain that thrives in hypoxic environments, as our base strain and incorporated a self-lysis genetic circuit for controlled bacterial growth and cargo release upon reaching a specific population density within the tumor core. We assembled and evaluated the self-lysis gene circuit in vitro, documenting the event using an optical density (OD) reader and confirming OASL's ability to express genetic cargos through western blot analysis. We then investigated OASL's therapeutic effect in vivo and characterized TME changes using flow cytometry and immunohistochemistry staining. Our results indicate that OASL undergoes lysis and releases cargos upon reaching a specific population density in vitro. Additionally, OASL-delivered neoantigen peptides elicited a tumor-specific immune response in vivo. OASL also demonstrated the capacity to modify the TME and restore sensitivity to PDL1 in previously unresponsive tumors, enhancing therapeutic effects. Citation Format: Yige He, Jiandong Huang. Leveraging an obligate anaerobic salmonella strain for targeted cargo delivery in the tumor microenvironment to restore immunotherapy sensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6666.

Transport-controlled growth decoupling for self-induced protein expression with a glycerol-repressible genetic circuit.

Decoupling cell formation from recombinant protein synthesis is a potent strategy to intensify bioprocesses. Escherichia coli strains with mutations in the glucose uptake components lack catabolite repression, display low growth rate, no overflow metabolism, and high recombinant protein yields. Fast growth rates were promoted by the simultaneous consumption of glucose and glycerol, and this was followed by a phase of slow growth, when only glucose remained in the medium. A glycerol-repressible genetic circuit was designed to autonomously induce recombinant protein expression. The engineered strain bearing the genetic circuit was cultured in 3.9 g L-1 glycerol + 18 g L-1 glucose in microbioreactors with online oxygen transfer rate monitoring. The growth was fast during the simultaneous consumption of both carbon sources (C-sources), while expression of the recombinant protein was low. When glycerol was depleted, the growth rate decreased, and the specific fluorescence reached values 17% higher than those obtained with a strong constitutive promoter. Despite the relatively high amount of C-sourceused, no oxygen limitation was observed. The proposed approach eliminates the need for the substrate feeding or inducers addition and is set as a simple batch culture while mimicking fed-batch performance.

Engineering pluripotent stem cells with synthetic biology for regenerative medicine

Abstract Pluripotent stem cells (PSCs), characterized by self-renewal and capacity of differentiating into three germ layers, are the programmable building blocks of life. PSC-derived cells and multicellular systems, particularly organoids, exhibit great potential for regenerative medicine. However, this field is still in its infancy, partly due to limited strategies to robustly and precisely control stem cell behaviors, which are tightly regulated by inner gene regulatory networks in response to stimuli from the extracellular environment. Synthetic receptors and genetic circuits are powerful tools to customize the cellular sense-and-response process, suggesting their underlying roles in precise control of cell fate decision and function reconstruction. Herein, we review the progress and challenges needed to be overcome in the fields of PSC-based cell therapy and multicellular system generation, respectively. Furthermore, we summarize several well-established synthetic biology tools and their applications in PSC engineering. Finally, we highlight the challenges and perspectives of harnessing synthetic biology to PSC engineering for regenerative medicine.

Development and utilization of new O2-independent bioreporters.

Fluorescent proteins have revolutionized science since their discovery in 1962. They have enabled imaging experiments to decipher the function of proteins, cells, and organisms, as well as gene regulation. Green fluorescent protein and all its derivatives are now standard tools in cell biology, immunology, molecular biology, and microbiology laboratories around the world. A common feature of these proteins is their dioxygen (O2)-dependent maturation allowing fluorescence, which precludes their use in anoxic contexts. In this work, we report the development and in cellulo characterization of genetic circuits encoding the O2-independent KOFP-7 protein, a flavin-binding fluorescent protein. We have optimized the genetic circuit for high bacterial fluorescence at population and single-cell level, implemented this circuit in various plasmids differing in host range, and quantified their fluorescence under both aerobic and anaerobic conditions. Finally, we showed that KOFP-7-based constructions can be used to produce fluorescing cells of Vibrio diazotrophicus, a facultative anaerobe, demonstrating the usefulness of the genetic circuits for various anaerobic bacteria. These genetic circuits can thus be modified at will, both to solve basic and applied research questions, opening a highway to shed light on the obscure anaerobic world.IMPORTANCEFluorescent proteins are used for decades, and have allowed major discoveries in biology in a wide variety of fields, and are used in environmental as well as clinical contexts. Green fluorescent protein (GFP) and all its derivatives share a common feature: they rely on the presence of dioxygen (O2) for protein maturation and fluorescence. This dependency precludes their use in anoxic environments. Here, we constructed a series of genetic circuits allowing production of KOFP-7, an O2-independant flavin-binding fluorescent protein. We demonstrated that Escherichia coli cells producing KOFP-7 are fluorescent, both at the population and single-cell levels. Importantly, we showed that, unlike cells producing GFP, cells producing KOFP-7 are fluorescent in anoxia. Finally, we demonstrated that Vibrio diazotrophicus NS1, a facultative anaerobe, is fluorescent in the absence of O2 when KOFP-7 is produced. Altogether, the development of new genetic circuits allowing O2-independent fluorescence will open new perspective to study anaerobic processes.

Enzymatic Assembly of Small Synthetic Genes with Repetitive Elements.

Gene synthesis efficiency has greatly improved in recent years but is limited when it comes to repetitive sequences, which results in synthesis failure or delays by DNA synthesis vendors. This represents a major obstacle for the development of synthetic biology since repetitive elements are increasingly being used in the design of genetic circuits and design of biomolecular nanostructures. Here, we describe a method for the assembly of small synthetic genes with repetitive elements: First, a gene of interest is split in silico into small synthons of up to 80 base pairs flanked by Golden-Gate-compatible overhangs. Then, synthons are made by oligo extension and finally assembled into a synthetic gene by Golden Gate Assembly. We demonstrate the method by constructing eight challenging genes with repetitive elements, e.g., multiple repeats of RNA aptamers and RNA origami scaffolds with multiple identical aptamers. The genes range in size from 133 to 456 base pairs and are assembled with fidelities of up to 87.5%. The method was developed to facilitate our own specific research but may be of general use for constructing challenging and repetitive genes and, thus, a valuable addition to the molecular cloning toolbox.

Assessing electrogenetic activation via a network model of biological signal propagation

Introduction: Molecular communication is the transfer of information encoded by molecular structure and activity. We examine molecular communication within bacterial consortia as cells with diverse biosynthetic capabilities can be assembled for enhanced function. Their coordination, both in terms of engineered genetic circuits within individual cells as well as their population-scale functions, is needed to ensure robust performance. We have suggested that “electrogenetics,” the use of electronics to activate specific genetic circuits, is a means by which electronic devices can mediate molecular communication, ultimately enabling programmable control.Methods: Here, we have developed a graphical network model for dynamically assessing electronic and molecular signal propagation schemes wherein nodes represent individual cells, and their edges represent communication channels by which signaling molecules are transferred. We utilize graph properties such as edge dynamics and graph topology to interrogate the signaling dynamics of specific engineered bacterial consortia.Results: We were able to recapitulate previous experimental systems with our model. In addition, we found that networks with more distinct subpopulations (high network modularity) propagated signals more slowly than randomized networks, while strategic arrangement of subpopulations with respect to the inducer source (an electrode) can increase signal output and outperform otherwise homogeneous networks.Discussion: We developed this model to better understand our previous experimental results, but also to enable future designs wherein subpopulation composition, genetic circuits, and spatial configurations can be varied to tune performance. We suggest that this work may provide insight into the signaling which occurs in synthetically assembled systems as well as native microbial communities.

Polyamines: Their Role in Plant Development and Stress.

This review focuses on the intricate relationship between plant polyamines and the genetic circuits and signaling pathways that regulate various developmental programs and the defense responses of plants when faced with biotic and abiotic aggressions. Particular emphasis is placed on genetic evidence supporting the involvement of polyamines in specific processes, such as the pivotal role of thermospermine in regulating xylem cell differentiation and the significant contribution of polyamine metabolism in enhancing plant resilience to drought. Based on the numerous studies describing effects of the manipulation of plant polyamine levels, two conceptually different mechanisms for polyamine activity are discussed: direct participation of polyamines in translational regulation and the indirect production of hydrogen peroxide as a defensive mechanism against pathogens. By describing the multifaceted functions of polyamines, this review underscores the profound significance of these compounds in enabling plants to adapt and thrive in challenging environments. Expected final online publication date for the Annual Review of Plant Biology, Volume 75 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Harnessing Catalytic RNA Circuits for Construction of Artificial Signaling Pathways in Mammalian Cells

AbstractEngineering of genetic networks with artificial signaling pathways (ASPs) can reprogram cellular responses and phenotypes under different circumstances for a variety of diagnostic and therapeutic purposes. However, construction of ASPs between originally independent endogenous genes in mammalian cells is highly challenging. Here we report an amplifiable RNA circuit that can theoretically build regulatory connections between any endogenous genes in mammalian cells. We harness the system of catalytic hairpin assembly with combination of controllable CRISPR‐Cas9 function to transduce the signals from distinct messenger RNA expression of trigger genes into manipulation of target genes. Through introduction of these RNA‐based genetic circuits, mammalian cells are endowed with autonomous capabilities to sense the changes of RNA expression either induced by ligand stimuli or from various cell types and control the cellular responses and fates via apoptosis‐related ASPs. Our design provides a generalized platform for construction of ASPs inside the genetic networks of mammalian cells based on differentiated RNA expression.

Harnessing Catalytic RNA Circuits for Construction of Artificial Signaling Pathways in Mammalian Cells.

Engineering of genetic networks with artificial signaling pathways (ASPs) can reprogram cellular responses and phenotypes under different circumstances for a variety of diagnostic and therapeutic purposes. However, construction of ASPs between originally independent endogenous genes in mammalian cells is highly challenging. Here we report an amplifiable RNA circuit that can theoretically build regulatory connections between any endogenous genes in mammalian cells. We harness the system of catalytic hairpin assembly with combination of controllable CRISPR-Cas9 function to transduce the signals from distinct messenger RNA expression of trigger genes into manipulation of target genes. Through introduction of these RNA-based genetic circuits, mammalian cells are endowed with autonomous capabilities to sense the changes of RNA expression either induced by ligand stimuli or from various cell types and control the cellular responses and fates via apoptosis-related ASPs. Our design provides a generalized platform for construction of ASPs inside the genetic networks of mammalian cells based on differentiated RNA expression.

Improving Microbial Cell Factory Performance by Engineering SAM Availability.

Methylated natural products are widely spread in nature. S-Adenosyl-l-methionine (SAM) is the secondary abundant cofactor and the primary methyl donor, which confer natural products with structural and functional diversification. The increasing demand for SAM-dependent natural products (SdNPs) has motivated the development of microbial cell factories (MCFs) for sustainable and efficient SdNP production. Insufficient and unsustainable SAM availability hinders the improvement of SdNP MCF performance. From the perspective of developing MCF, this review summarized recent understanding of de novo SAM biosynthesis and its regulatory mechanism. SAM is just the methyl mediator but not the original methyl source. Effective and sustainable methyl source supply is critical for efficient SdNP production. We compared and discussed the innate and relatively less explored alternative methyl sources and identified the one involving cheap one-carbon compound as more promising. The SAM biosynthesis is synergistically regulated on multilevels and is tightly connected with ATP and NAD(P)H pools. We also covered the recent advancement of metabolic engineering in improving intracellular SAM availability and SdNP production. Dynamic regulation is a promising strategy to achieve accurate and dynamic fine-tuning of intracellular SAM pool size. Finally, we discussed the design and engineering constraints underlying construction of SAM-responsive genetic circuits and envisioned their future applications in developing SdNP MCFs.

Orthogonal LoxPsym sites allow multiplexed site-specific recombination in prokaryotic and eukaryotic hosts

Site-specific recombinases such as the Cre-LoxP system are routinely used for genome engineering in both prokaryotes and eukaryotes. Importantly, recombinases complement the CRISPR-Cas toolbox and provide the additional benefit of high-efficiency DNA editing without generating toxic DNA double-strand breaks, allowing multiple recombination events at the same time. However, only a handful of independent, orthogonal recombination systems are available, limiting their use in more complex applications that require multiple specific recombination events, such as metabolic engineering and genetic circuits. To address this shortcoming, we develop 63 symmetrical LoxP variants and test 1192 pairwise combinations to determine their cross-reactivity and specificity upon Cre activation. Ultimately, we establish a set of 16 orthogonal LoxPsym variants and demonstrate their use for multiplexed genome engineering in both prokaryotes (E. coli) and eukaryotes (S. cerevisiae and Z. mays). Together, this work yields a significant expansion of the Cre-LoxP toolbox for genome editing, metabolic engineering and other controlled recombination events, and provides insights into the Cre-LoxP recombination process.

Engineering mammalian cell growth dynamics for biomanufacturing

Precise control over mammalian cell growth dynamics poses a major challenge in biopharmaceutical manufacturing. Here, we present a multi-level cell engineering strategy for the tunable regulation of growth phases in mammalian cells. Initially, we engineered mammalian death phase by employing CRISPR/Cas9 to knockout pro-apoptotic proteins Bax and Bak, resulting in a substantial attenuation of apoptosis by improving cell viability and extending culture lifespan. The second phase introduced a growth acceleration system, akin to a “gas pedal”, based on an abscidic acid inducible system regulating cMYC gene expression, enabling rapid cell density increase and cell cycle control. The third phase focused on a stationary phase inducing system, comparable to a “brake pedal”. A tetracycline inducible genetic circuit based on BLIMP1 gene led to cell growth cessation and arrested cell cycle upon activation. Finally, we developed a dual controllable system, combining the “gas and brake pedals”, enabling for dynamic and precise orchestration of mammalian cell growth dynamics. This work exemplifies the application of synthetic biology tools and combinatorial cell engineering, offering a sophisticated framework for manipulating mammalian cell growth and providing a unique paradigm for reprogramming cell behaviour for enhancing biopharmaceutical manufacturing and further biomedical applications.

A genetic circuit on a single DNA molecule as an autonomous dissipative nanodevice

Realizing genetic circuits on single DNA molecules as self-encoded dissipative nanodevices is a major step toward miniaturization of autonomous biological systems. A circuit operating on a single DNA implies that genetically encoded proteins localize during coupled transcription-translation to DNA, but a single-molecule measurement demonstrating this has remained a challenge. Here, we use a genetically encoded fluorescent reporter system with improved temporal resolution and observe the synthesis of individual proteins tethered to a DNA molecule by transient complexes of RNA polymerase, messenger RNA, and ribosome. Against expectations in dilute cell-free conditions where equilibrium considerations favor dispersion, these nascent proteins linger long enough to regulate cascaded reactions on the same DNA. We rationally design a pulsatile genetic circuit by encoding an activator and repressor in feedback on the same DNA molecule. Driven by the local synthesis of only several proteins per hour and gene, the circuit dynamics exhibit enhanced variability between individual DNA molecules, and fluctuations with a broad power spectrum. Our results demonstrate that co-expressional localization, as a nonequilibrium process, facilitates single-DNA genetic circuits as dissipative nanodevices, with implications for nanobiotechnology applications and artificial cell design.

Enhancing tumor-specific recognition of programmable synthetic bacterial consortium for precision therapy of colorectal cancer

Probiotics hold promise as a potential therapy for colorectal cancer (CRC), but encounter obstacles related to tumor specificity, drug penetration, and dosage adjustability. In this study, genetic circuits based on the E. coli Nissle 1917 (EcN) chassis were developed to sense indicators of tumor microenvironment and control the expression of therapeutic payloads. Integration of XOR gate amplify gene switch into EcN biosensors resulted in a 1.8-2.3-fold increase in signal output, as confirmed by mathematical model fitting. Co-culturing programmable EcNs with CRC cells demonstrated a significant reduction in cellular viability ranging from 30% to 50%. This approach was further validated in a mouse subcutaneous tumor model, revealing 47%-52% inhibition of tumor growth upon administration of therapeutic strains. Additionally, in a mouse tumorigenesis model induced by AOM and DSS, the use of synthetic bacterial consortium (SynCon) equipped with multiple sensing modules led to approximately 1.2-fold increased colon length and 2.4-fold decreased polyp count. Gut microbiota analysis suggested that SynCon maintained the abundance of butyrate-producing bacteria Lactobacillaceae NK4A136, whereas reducing the level of gut inflammation-related bacteria Bacteroides. Taken together, engineered EcNs confer the advantage of specific recognition of CRC, while SynCon serves to augment the synergistic effect of this approach.

Application of (bio) chemical engineering concepts and tools to model genetic regulatory circuits, and some essential central carbon metabolism pathways in living cells. Part 4. Applications in the design of some Genetically Modified Micro-Organisms (GMOs)

In the first part of this work, the general Chemical and Biochemical Engineering (CBE) concepts and rules are briefly reviewed, together with the rules of the control theory of Nonlinear Systems (NSCT), all in the context of (i) deriving deterministic Modular Structured Kinetic Models (MSDKM) to describe the dynamics of metabolic processes in living cells, and (ii) of Hybrid Structured Modular Dynamic Models (HSMDM) (with continuous variables, linking the cell-nano-scale MSDKM state variables to the macro-scale state variables of the bioreactor dynamic model). Thus, in the HSMDM model, both prediction quality and its validity range are improved. By contrast, the current (classical/default) approach in bioengineering practice for solving design, optimization, and control problems based on the math models of industrial biological reactors is to use unstructured Monod (for cell culture reactor) or simple Michaelis-Menten (if only enzymatic reactions are retained) global kinetic models by ignoring detailed representations of metabolic cellular processes. By contrast, as reviewed, and exemplified in the second part of this work, an accurate and realistic math modelling of the dynamic individual GERMs (gene expression regulatory module), or genetic regulatory circuits (GRC), and cell-scale CCM (central carbon metabolism) key-modules can be done by only using the novel holistic ’Whole-Cell Of Variable-Volume’ (WCVV) modelling framework, under isotonic/homeostatic conditions/constraints introduced and promoted by the author. An example was given in the same Part 2 for the case study of a dynamic model for the oscillating glycolysis coupled with the Tryptophan (TRP) oscillating synthesis in the E. coli cells.

The naringenin-dependent regulator FdeR can be applied as a NIMPLY gate controlled by naringenin and arabinose.

The FdeR regulator has been reported as a transcriptional activator dependent on the interaction with naringenin. Previously, FdeR and its cognate promoter were used to construct naringenin-sensitive sensors, though no correlation was associated between the FdeR level of expression and outputs. Therefore, to understand this correlation, we constructed a circuit with FdeR expression adjusted by the arabinose concentration through an AraC-PBAD system and the FdeR-regulated promoter controlling the expression of GFP. We observed a significant reduction in the activity of the target promoter by increasing FdeR expression, indicating that although FdeR has been primarily classified as a transcriptional activator, it also represses transcription. Leveraging the bifunctional feature of FdeR, acting as both transcriptional activator and repressor, we demonstrated that this genetic circuit, when previously switched on by naringenin, can be switched off by inducing an increased FdeR expression level. This engineered system functioned as a NIMPLY gate, effectively decreasing GFP expression by 50% when arabinose was added without removing naringenin from the medium. Exploiting FdeR versatility, this study demonstrates an innovative application of this transcriptional factor for developing novel NIMPLY gates activated by a molecule with low toxicity and nutraceutical properties that may be important for several applications. Graphical Abstract.