Ultrasound-mediated spatial and temporal control of engineered cells in vivo.

Remote regulation of cells in deep tissue remains a significant challenge. Low-intensity pulsed ultrasound offers promise for in vivo therapies due to its non-invasive nature and precise control. This study uses pulsed ultrasound to control calcium influx in mammalian cells and engineers a therapeutic cellular device responsive to acoustic stimulation in deep tissue without overexpressing calcium channels or gas vesicles.

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.

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.

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.

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.

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).

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.

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.

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.

CRISPRa-mediated gene upregulation in mammalian cells.

Background: Forkhead box P3 ( ) regulatory T cells (Tregs) are a subset of lymphocytes, critical for the maintenance of immune homeostasis. Loss-of-function mutations of the 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 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.