Gut microbiome and metabolome profiling in Framingham heart study reveals cholesterol-metabolizing bacteria.

Accumulating evidence suggests that cardiovascular disease (CVD) is associated with an altered gut microbiome. Our understanding of the underlying mechanisms has been hindered by lack of matched multi-omic data with diagnostic biomarkers. To comprehensively profile gut microbiome contributions to CVD, we generated stool metagenomics and metabolomics from 1,429 Framingham Heart Study participants. We identified blood lipids and cardiovascular health measurements associated with microbiome and metabolome composition. Integrated analysis revealed microbial pathways implicated in CVD, including flavonoid, γ-butyrobetaine, and cholesterol metabolism. Species from the Oscillibacter genus were associated with decreased fecal and plasma cholesterol levels. Using functional prediction and in vitro characterization of multiple representative human gut Oscillibacter isolates, we uncovered conserved cholesterol-metabolizing capabilities, including glycosylation and dehydrogenation. These findings suggest that cholesterol metabolism is a broad property of phylogenetically diverse Oscillibacter spp., with potential benefits for lipid homeostasis and cardiovascular health.

Subunits of an E3 Ligase Complex as Degrons for Efficient Degradation of Cytosolic, Nuclear, and Membrane Proteins.

Protein degradation is a highly regulated cellular process crucial to enable the high dynamic range of the response to external and internal stimuli and to balance protein biosynthesis to maintain cell homeostasis. Within mammalian cells, hundreds of E3 ubiquitin ligases target specific protein substrates and could be repurposed for synthetic biology. Here, we present a systematic analysis of the four protein subunits of the multiprotein E3 ligase complex as scaffolds for the designed degrons. While all of them were functional, the fusion of a fragment of Skp1 with the target protein enabled the most effective degradation. Combination with heterodimerizing peptides, protease substrate sites, and chemically inducible dimerizers enabled the regulation of protein degradation. While the investigated subunits of E3 ligases showed variable degradation efficiency of the membrane and cytosolic and nuclear proteins, the bipartite SSD (SOCSbox-Skp1(ΔC111)) degron enabled fast degradation of protein targets in all tested cellular compartments, including the nucleus and plasma membrane, in different cell lines and could be chemically regulated. These subunits could be employed for research as well as for diverse applications, as demonstrated in the regulation of Cas9 and chimeric antigen receptor proteins.

Chemically inducible split protein regulators for mammalian cells.

Chemically inducible systems represent valuable synthetic biology tools that enable the external control of biological processes. However, their translation to therapeutic applications has been limited because of unfavorable ligand characteristics or the immunogenicity of xenogeneic protein domains. To address these issues, we present a strategy for engineering inducible split protein regulators (INSPIRE) in which ligand-binding proteins of human origin are split into two fragments that reassemble in the presence of a cognate physiological ligand or clinically approved drug. We show that the INSPIRE platform can be used for dynamic, orthogonal and multiplex control of gene expression in mammalian cells. Furthermore, we demonstrate the functionality of a glucocorticoid-responsive INSPIRE platform in vivo and apply it for perturbing an endogenous regulatory network. INSPIRE presents a generalizable approach toward designing small-molecule responsive systems that can be implemented for the construction of new sensors, regulatory networks and therapeutic applications.

Concatenated Coiled-Coil Tag for Highly Efficient, Small Molecule-Inducible Upregulation of Endogenous Mammalian Genes.

Regulation of epigenomic functions requires controlled site-specific alteration of epigenetic information. This can be achieved by using designed DNA-binding domains, associated with effector domains, that function as targeted transcription factors or epigenetic modifiers. These effectors have been employed to study the implications of epigenetic modifications, and sequence-specific targeting has been instrumental in understanding the effect of these modification on gene regulation. Ultimately, these tools could be used for therapeutic applications to revert the epigenetic aberrations that have been linked to various diseases. The ability to spatiotemporally control gene expression is especially important for precise regulation of the epigenomic state. In this chapter, we describe the protocol for achieving highly efficient small molecule-inducible transcriptional activation of endogenous mammalian genes, mediated by a dCas9-based system that recruits transcriptional activation domains binding to a chain of concatenated coiled-coil peptides.

helixCAM: A platform for programmable cellular assembly in bacteria and human cells.

Interactions between cells are indispensable for signaling and creating structure. The ability to direct precise cell-cell interactions would be powerful for engineering tissues, understanding signaling pathways, and directing immune cell targeting. In humans, intercellular interactions are mediated by cell adhesion molecules (CAMs). However, endogenous CAMs are natively expressed by many cells and tend to have cross-reactivity, making them unsuitable for programming specific interactions. Here, we showcase "helixCAM," a platform for engineering synthetic CAMs by presenting coiled-coil peptides on the cell surface. helixCAMs were able to create specific cell-cell interactions and direct patterned aggregate formation in bacteria and human cells. Based on coiled-coil interaction principles, we built a set of rationally designed helixCAM libraries, which led to the discovery of additional high-performance helixCAM pairs. We applied this helixCAM toolkit for various multicellular engineering applications, such as spherical layering, adherent cell targeting, and surface patterning.

Binding of the transcription activator-like effector augments transcriptional regulation by another transcription factor.

DNA transcription is regulated by a range of diverse mechanisms and primarily by transcription factors that recruit the RNA polymerase complex to the promoter region on the DNA. Protein binding to DNA at nearby or distant sites can synergistically affect this process in a variety of ways, but mainly through direct interactions between DNA-binding proteins. Here we show that a Transcription Activator-Like Effector (TALE), which lacks an activation domain, can enhance transcription in mammalian cells when it binds in the vicinity of and without direct interaction with several different dimeric or monomeric transcription factors. This effect was observed for several TALEs regardless of the recognition sequences and their DNA-bound orientation. TALEs can exert an effect over the distance of tens of nucleotides and it also potentiated KRAB-mediated repression. The augmentation of transcriptional regulation of another transcription factor is characteristic of TALEs, as it was not observed for dCas9/gRNA, zinc finger, or Gal4 DNA-binding domains. We propose that this mechanism involves an allosteric effect exerted on DNA structure or dynamics. This mechanism could be used to modulate transcription but may also play a role in the natural context of TALEs.

Engineering and Rewiring of a Calcium-Dependent Signaling Pathway.

An important feature of synthetic biological circuits is their response to physicochemical signals, which enables the external control of cellular processes. Calcium-dependent regulation is an attractive approach for achieving such control, as diverse stimuli induce calcium influx by activating membrane channel receptors. Most calcium-dependent gene circuits use the endogenous nuclear factor of activated T-cells (NFAT) signaling pathway. Here, we employed engineered NFAT transcription factors to induce the potent and robust activation of exogenous gene expression in HEK293T cells. Furthermore, we designed a calcium-dependent transcription factor that does not interfere with NFAT-regulated promoters and potently activates transcription in several mammalian cell types. Additionally, we demonstrate that coupling the circuit to a calcium-selective ion channel resulted in capsaicin- and temperature-controlled gene expression. This engineered calcium-dependent signaling pathway enables tightly controlled regulation of gene expression through different stimuli in mammalian cells and is versatile, adaptable, and useful for a wide range of therapeutic and diagnostic applications.

A tunable orthogonal coiled-coil interaction toolbox for engineering mammalian cells.

Protein interactions guide most cellular processes. Orthogonal hetero-specific protein-protein interaction domains may facilitate better control of engineered biological systems. Here, we report a tunable de novo designed set of orthogonal coiled-coil (CC) peptide heterodimers (called the NICP set) and its application for the regulation of diverse cellular processes, from cellular localization to transcriptional regulation. We demonstrate the application of CC pairs for multiplex localization in single cells and exploit the interaction strength and variable stoichiometry of CC peptides for tuning of gene transcription strength. A concatenated CC peptide tag (CCC-tag) was used to construct highly potent CRISPR-dCas9-based transcriptional activators and to amplify the response of light and small molecule-inducible transcription in cell culture as well as in vivo. The NICP set and its implementations represent a valuable toolbox of minimally disruptive modules for the recruitment of versatile functional domains and regulation of cellular processes for synthetic biology.

CRISPRa-mediated FOXP3 gene upregulation in mammalian cells.

BACKGROUND: Forkhead box P3+ (FOXP3 +) regulatory T cells (Tregs) are a subset of lymphocytes, critical for the maintenance of immune homeostasis. Loss-of-function mutations of the FOXP3 gene in animal models and humans results in loss of differentiation potential into Treg cells and are responsible for several immune-mediated inflammatory diseases. Strategies of increasing FOXP3 expression represent a potential approach to increase the pool of Tregs within the lymphocyte population and may be employed in therapies of diverse autoimmune conditions. In the present study, a dCas9 CRISPR-based method was systematically employed to achieve upregulation and sustained high expression of endogenous FOXP3 in HEK293 and human Jurkat T cell lines through targeting of the core promotor, three known regulatory regions of the FOXP3 gene (CNS1-3), and two additional regions selected through extensive bioinformatics analysis (Cage1 and Cage2). RESULTS: Using an activator-domain fusion based dCas9 transcription activator, robust upregulation of FOXP3 was achieved, and an optimal combination of single guide RNAs was selected, which exerted an additive effect on FOXP3 gene upregulation. Simultaneous targeting of FOXP3 and EOS, a transcription factor known to act in concert with FOXP3 in initiating a Treg phenotype, resulted in upregulation of FOXP3 downstream genes CD25 and TNFR2. When compared to ectopic expression of FOXP3 via plasmid electroporation, upregulation of endogenous FOXP3 via the Cas9-based method resulted in prolonged expression of FOXP3 in Jurkat cells. CONCLUSIONS: Transfection of both HEK293 and Jurkat cells with dCas9-activators showed that regulatory regions downstream and upstream of FOXP3 promoter can be very potent transcription inducers in comparison to targeting the core promoter. While introduction of genes by conventional methods of gene therapy may involve a risk of insertional mutagenesis due to viral integration into the genome, transient up- or down-regulation of transcription by a CRISPR-dCas9 approach may resolve this safety concern. dCas9-based systems provide great promise in DNA footprint-free phenotype perturbations (perturbation without the risk of DNA damage) to drive development of transcription modulation-based therapies.

Design of fast proteolysis-based signaling and logic circuits in mammalian cells.

Cellular signal transduction is predominantly based on protein interactions and their post-translational modifications, which enable a fast response to input signals. Owing to difficulties in designing new unique protein-protein interactions, designed cellular logic has focused on transcriptional regulation; however, that process has a substantially slower response, because it requires transcription and translation. Here, we present de novo design of modular, scalable signaling pathways based on proteolysis and designed coiled coils (CC) and implemented in mammalian cells. A set of split proteases with highly specific orthogonal cleavage motifs was constructed and combined with strategically positioned cleavage sites and designed orthogonal CC dimerizing domains with tunable affinity for competitive displacement after proteolytic cleavage. This framework enabled the implementation of Boolean logic functions and signaling cascades in mammalian cells. The designed split-protease-cleavable orthogonal-CC-based (SPOC) logic circuits enable response to chemical or biological signals within minutes rather than hours and should be useful for diverse medical and nonmedical applications.

Polarized displacement by transcription activator-like effectors for regulatory circuits.

The interplay between DNA-binding proteins plays an important role in transcriptional regulation and could increase the precision and complexity of designed regulatory circuits. Here we show that a transcription activator-like effector (TALE) can displace another TALE protein from DNA in a highly polarized manner, displacing only the 3'- but not 5'-bound overlapping or adjacent TALE. We propose that the polarized displacement by TALEs is based on its multipartite nature of binding to DNA. The polarized TALE displacement provides strategies for the specific regulation of gene expression, for construction of all two-input Boolean genetic logic circuits based on the robust propagation of the displacement across multiple neighboring sites, for displacement of zinc finger-based transcription factors and for suppression of Cas9-gRNA-mediated genome cleavage, enriching the synthetic biology toolbox and contributing to the understanding of the underlying principles of the facilitated displacement.

Designed Transcriptional Regulation in Mammalian Cells Based on TALE- and CRISPR/dCas9.

Transcriptional regulation lies at the center of many cellular processes and is the result of cellular response to different external and internal signals. Control of transcription of selected genes enables an unprecedented access to shape the cellular response. While orthogonal transcription factors from bacteria, yeast, plants, or other cells have been used to introduce new cellular logic into mammalian cells, the discovery of designable modular DNA binding domains, such as Transcription Activator-Like Effectors (TALEs) and the CRISPR system, enable targeting of almost any selected DNA sequence. Fusion or conditional association of DNA targeting domain with transcriptional effector domains enables controlled regulation of almost any endogenous or ectopic gene. Moreover, the designed regulators can be linked into genetic circuits to implement complex responses, such as different types of Boolean functions and switches. In this chapter, we describe the protocols for achieving efficient transcriptional regulation with TALE- and CRISPR-based designed transcription factors in mammalian cells.

Transcription activator-like effector-mediated regulation of gene expression based on the inducible packaging and delivery via designed extracellular vesicles.

Transcription activator-like effector (TALE) proteins present a powerful tool for genome editing and engineering, enabling introduction of site-specific mutations, gene knockouts or regulation of the transcription levels of selected genes. TALE nucleases or TALE-based transcription regulators are introduced into mammalian cells mainly via delivery of the coding genes. Here we report an extracellular vesicle-mediated delivery of TALE transcription regulators and their ability to upregulate the reporter gene in target cells. Designed transcriptional activator TALE-VP16 fused to the appropriate dimerization domain was enriched as a cargo protein within extracellular vesicles produced by mammalian HEK293 cells stimulated by Ca-ionophore and using blue light- or rapamycin-inducible dimerization systems. Blue light illumination or rapamycin increased the amount of the TALE-VP16 activator in extracellular vesicles and their addition to the target cells resulted in an increased expression of the reporter gene upon addition of extracellular vesicles to the target cells. This technology therefore represents an efficient delivery for the TALE-based transcriptional regulators.

Benchmarking of TALE- and CRISPR/dCas9-Based Transcriptional Regulators in Mammalian Cells for the Construction of Synthetic Genetic Circuits.

Transcriptional activator-like effector (TALE)- and CRISPR/Cas9-based designable recognition domains represent a technological breakthrough not only for genome editing but also for building designed genetic circuits. Both platforms are able to target rarely occurring DNA segments, even within complex genomes. TALE and dCas9 domains, genetically fused to transcriptional regulatory domains, can be used for the construction of engineered logic circuits. Here we benchmarked the performance of the two platforms, targeting the same DNA sequences, to compare their advantages for the construction of designed circuits in mammalian cells. Optimal targeting strands for repression and activation of dCas9-based designed transcription factors were identified; both platforms exhibited good orthogonality and were used to construct functionally complete NOR gates. Although the CRISPR/dCas9 system is clearly easier to construct, TALE-based activators were significantly stronger, and the TALE-based platform performed better, especially for the construction of layered circuits.

Locked and proteolysis-based transcription activator-like effector (TALE) regulation.

Development of orthogonal, designable and adjustable transcriptional regulators is an important goal of synthetic biology. Their activity has been typically modulated through stimulus-induced oligomerization or interaction between the DNA-binding and activation/repression domain. We exploited a feature of the designable Transcription activator-like effector (TALE) DNA-binding domain that it winds around the DNA which allows to topologically prevent it from binding by intramolecular cyclization. This new approach was investigated through noncovalent ligand-induced cyclization or through a covalent split intein cyclization strategy, where the topological inhibition of DNA binding by cyclization and its restoration by a proteolytic release of the topologic constraint was expected. We show that locked TALEs indeed have diminished DNA binding and regain full transcriptional activity by stimulation with the rapamycin ligand or site-specific proteolysis of the peptide linker, with much higher level of activation than rapamycin-induced heterodimerization. Additionally, we demonstrated reversibility, activation of genomic targets and implemented logic gates based on combinations of protein cyclization, proteolytic cleavage and ligand-induced dimerization, where the strongest fold induction was achieved by the proteolytic cleavage of a repression domain from a linear TALE.

A bistable genetic switch based on designable DNA-binding domains.

Bistable switches are fundamental regulatory elements of complex systems, ranging from electronics to living cells. Designed genetic toggle switches have been constructed from pairs of natural transcriptional repressors wired to inhibit one another. The complexity of the engineered regulatory circuits can be increased using orthogonal transcriptional regulators based on designed DNA-binding domains. However, a mutual repressor-based toggle switch comprising DNA-binding domains of transcription-activator-like effectors (TALEs) did not support bistability in mammalian cells. Here, the challenge of engineering a bistable switch based on monomeric DNA-binding domains is solved via the introduction of a positive feedback loop composed of activators based on the same TALE domains as their opposing repressors and competition for the same DNA operator site. This design introduces nonlinearity and results in epigenetic bistability. This principle could be used to employ other monomeric DNA-binding domains such as CRISPR for applications ranging from reprogramming cells to building digital biological memory.

Designable DNA-binding domains enable construction of logic circuits in mammalian cells.

Electronic computer circuits consisting of a large number of connected logic gates of the same type, such as NOR, can be easily fabricated and can implement any logic function. In contrast, designed genetic circuits must employ orthogonal information mediators owing to free diffusion within the cell. Combinatorial diversity and orthogonality can be provided by designable DNA- binding domains. Here, we employed the transcription activator-like repressors to optimize the construction of orthogonal functionally complete NOR gates to construct logic circuits. We used transient transfection to implement all 16 two-input logic functions from combinations of the same type of NOR gates within mammalian cells. Additionally, we present a genetic logic circuit where one input is used to select between an AND and OR function to process the data input using the same circuit. This demonstrates the potential of designable modular transcription factors for the construction of complex biological information-processing devices.

DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency.

Synthetic scaffolds that permit spatial and temporal organization of enzymes in living cells are a promising post-translational strategy for controlling the flow of information in both metabolic and signaling pathways. Here, we describe the use of plasmid DNA as a stable, robust and configurable scaffold for arranging biosynthetic enzymes in the cytoplasm of Escherichia coli. This involved conversion of individual enzymes into custom DNA-binding proteins by genetic fusion to zinc-finger domains that specifically bind unique DNA sequences. When expressed in cells that carried a rationally designed DNA scaffold comprising corresponding zinc finger binding sites, the titers of diverse metabolic products, including resveratrol, 1,2-propanediol and mevalonate were increased as a function of the scaffold architecture. These results highlight the utility of DNA scaffolds for assembling biosynthetic enzymes into functional metabolic structures. Beyond metabolism, we anticipate that DNA scaffolds may be useful in sequestering different types of enzymes for specifying the output of biological signaling pathways or for coordinating other assembly-line processes such as protein folding, degradation and post-translational modifications.