Biology subjects

Mathony, J.

Publications and source records attributed to Mathony, J..

9 recordsLinked to original sources

bioRxiv2025

Phage-Assisted Evolution of Allosteric Protein Switches

Allostery, the transmission of locally induced conformational changes to distant functional sites, is a key mechanism for protein regulation1. Artificial allosteric effectors enable remote manipulation of cell function2,3; their engineering, however, is hampered by our limited understanding of allosteric residue networks. Here, we introduce a phage-assisted evolution4 platform for in vivo optimization of allosteric proteins. It applies opposing selection pressures to enhance activity and switchability of phage-encoded effectors and leverages retron-based recombineering5 to broadly explore fitness landscapes, introducing point mutations, insertions, and deletions. Applying this framework to the transcription factor AraC yielded near-binary optogenetic switches, with light-controlled activity spanning [~]1,000-fold dynamic range. Long-read sequencing across selection cycles enabled high-resolution tracking of evolving variant pools, revealing adaptive trajectories and context-dependent residue interactions. Mechanistically, we found that linker mutations promoting -helix extension at the sensor-effector junction enhance conformational coupling between LOV2 and AraC. These variants emerged consistently across independently evolved pools, underscoring their functional relevance. Together, we developed a framework for the directed evolution of programmable allosteric switches in vivo. By coupling dynamic selection with deep mutational scanning and temporal sequencing, it enables both functional optimization and mechanistic insight into allosteric networks.

synthetic biology↗

bioRxiv2025

Modular Engineering of Thermo-Responsive Allosteric Proteins

Thermogenetics enables non-invasive spatiotemporal control over protein activity in living cells and tissues, yet its applications have largely been restricted to transcriptional regulation and membrane recruitment. Here, we present a generalizable strategy for engineering thermosensitive allosteric proteins through the insertion of optimized Avena sativa LOV2 domain variants. Applying this approach to a diverse set of structurally and functionally unrelated proteins in Escherichia coli, we generated potent, thermo-switchable chimeric variants that can be tightly controlled within narrow temperature ranges (37-41{degrees}C). Extending this strategy to mammalian systems, we engineered the first CRISPR-Cas genome editors directly modulated by subtle temperature changes within the physiological range. Finally, we showcase the incorporation of a chemoreceptor domain as an alternative thermosensing module, suggesting thermo-sensitivity to be a widespread feature in receptor domains. This work expands the toolkit of thermogenetics, providing a blueprint for temperature-dependent control of virtually any protein of interest.

synthetic biology↗

bioRxiv2024

Rational engineering of allosteric protein switches by in silico prediction of domain insertion sites

Domain insertion engineering is a powerful approach to juxtapose otherwise separate biological functions, resulting in proteins with new-to-nature activities. A prominent example are switchable protein variants, created by receptor domain insertion into effector proteins. Identifying suitable, allosteric sites for domain insertion, however, typically requires extensive screening and optimization. We present ProDomino, a novel machine learning pipeline to rationalize domain recombination, trained on a semi-synthetic protein sequence dataset derived from naturally occurring intradomain insertion events. ProDomino robustly identifies domain insertion sites in proteins of biotechnological relevance, which we experimentally validated in E. coli and human cells. Finally, we employed light- and chemically regulated receptor domains as inserts and demonstrate the rapid, model-guided creation of potent, single-component opto- and chemogenetic protein switches. These include novel CRISPR-Cas9 and -Cas12a variants for inducible genome engineering in human cells. Our work enables one-shot domain insertion engineering and substantially accelerates the design of customized allosteric proteins.

synthetic biology↗

bioRxiv2024

A Versatile Anti-CRISPR Platform for Opto- and Chemogenetic Control of CRISPR-Cas9 and Cas12 across a Wide Range of Orthologs

CRISPR-Cas technologies have revolutionized life sciences by enabling programmable genome editing across diverse organisms. Achieving dynamic and precise control over CRISPR-Cas activity with exogenous triggers, such as light or chemical ligands, remains an important need. Existing tools for CRISPR-Cas control are often limited to specific Cas orthologs or selected applications, restricting their versatility. Anti-CRISPR (Acr) proteins, natural inhibitors of CRISPR-Cas systems, provide a flexible regulatory layer but are constitutively active in their native forms. In this study, we built on our previously reported concept for optogenetic CRISPR-Cas control with engineered, light-switchable anti-CRISPR proteins and expanded it from ortholog-specific Acrs towards AcrIIA5 and AcrVA1, broad-spectrum inhibitors of CRISPR-Cas9 and -Cas12a, respectively. We then conceived and implemented a novel, chemogenetic anti-CRISPR platform based on engineered, circularly permuted ligand receptor domains of human origin, that together respond to six different, clinically-relevant drugs. The resulting toolbox achieves both optogenetic and chemogenetic control of genome editing in human cells with a wide range of CRISPR-Cas effectors, including type II-A and -C CRISPR-Cas9s, and -Cas12a. In sum, this work establishes a versatile platform for multidimensional control of CRISPR-Cas systems, with immediate applications in basic research and biotechnology and potential for therapeutic use in the future.

synthetic biology↗

bioRxiv2023

A modular toolbox for the optogenetic deactivation of transcription

Light-controlled transcriptional activation is a commonly used optogenetic strategy that allows researchers to regulate gene expression with high spatiotemporal precision. The vast majority of existing tools are, however, limited to light-triggered induction of gene expression. Here, we inverted this mode of action and created two complementary optogenetic systems capable of efficiently terminating transcriptional activation in response to blue light. First, we designed highly compact regulators, by photo-controlling VP16 transactivation peptide exposure. Then, applying a two-hybrid strategy, we engineered LOOMINA (light off-operated modular inductor of transcriptional activation), a versatile transcriptional control platform for mammalian cells that is highly adaptable and compatible with various effector proteins. Leveraging the flexibility of CRISPR systems, we integrated LOOMINA with Cas9 as a DNA-binding domain to control transcription from various endogenous promoters with exceptionally high dynamic ranges in multiple cell lines, including neuron-like cells. Both functionally and mechanistically, LOOMINA represents a valuable addition to the optogenetic repertoire for transcriptional regulation.

synthetic biology↗

bioRxiv2023

Dissecting the Determinants of Domain Insertion Tolerance and Allostery in Proteins

Domain insertion engineering is a promising approach to recombine the functions of evolutionarily unrelated proteins. Insertion of light-switchable receptor domains into a selected effector protein, for instance, can yield allosteric effectors with light-dependent activity. However, the parameters that determine domain insertion tolerance are poorly understood. Here, we used an unbiased screen to systematically assess the domain insertion permissibility of several evolutionary unrelated proteins. Training machine learning models on the resulting data allowed us to dissect features informative for domain insertion tolerance and revealed sequence conservation statistics as the strongest indicators of suitable insertion sites. Finally, extending our experimental pipeline towards the identification of switchable hybrids resulted in opto-chemogenetic derivatives of the transcription factor AraC that function as single-protein Boolean logic gates. Our study reveals determinants of domain insertion tolerance and facilitates the engineering of switchable proteins with unique mechanistic properties.

bioengineering↗

bioRxiv2021

Dynamics of SARS-CoV-2 host cell interactions inferred from transcriptome analyses

The worldwide spread of severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) caused an urgent need for an in-depth understanding of interactions between the virus and its host. Here, we dissected the dynamics of virus replication and the host cell transcriptional response to SARS-CoV-2 infection at a systems level by combining time-resolved RNA sequencing with mathematical modeling. We observed an immediate transcriptional activation of inflammatory pathways linked to the anti-viral response followed by increased expression of genes involved in ribosome and mitochondria function, thus hinting at rapid alterations in protein production and cellular energy supply. At later stages, metabolic processes, in particular those depending on cytochrome P450 enzymes, were downregulated. To gain a deeper understanding of the underlying transcriptional dynamics, we developed an ODE model of SARS-CoV-2 infection and replication. Iterative model reduction and refinement revealed that a negative feedback from virus proteins on the expression of anti-viral response genes was essential to explain our experimental dataset. Our study provides insights into SARS-CoV-2 virus-host interaction dynamics and facilitates the identification of druggable host pathways supporting virus replication.

systems biology↗

bioRxiv2019

Optogenetic control of Neisseria meningitidis Cas9 genome editing using an engineered, light-switchable anti-CRISPR protein

Optogenetic control of CRISPR-Cas9 systems has significantly improved our ability to perform genome perturbations in living cells with high precision in time and space. As new Cas orthologues with advantageous properties are rapidly being discovered and engineered, the need for straightforward strategies to control their activity via exogenous stimuli persists. The Cas9 from Neisseria meningitidis (Nme) is a particularly small and target-specific Cas9 orthologue, and thus of high interest for in vivo genome editing applications. Here, we report the first optogenetic tool to control NmeCas9 activity in mammalian cells via an engineered, light-dependent anti-CRISPR (Acr) protein. Building on our previous Acr engineering work, we created hybrids between the NmeCas9 inhibitor AcrIIC3 and the LOV2 blue light sensory domain from Avena sativa. Two AcrIIC3-LOV2 hybrids from our collection potently blocked NmeCas9 activity in the dark, while permitting robust genome editing at various endogenous loci upon blue light irradiation. Structural analysis revealed that, within these hybrids, the LOV2 domain is located in striking proximity to the Cas9 binding surface. Together, our work demonstrates optogenetic regulation of a type II-C CRISPR effector and might suggest a new route for the design of optogenetic Acrs.

synthetic biology↗

bioRxiv2019

Computational design of anti-CRISPR proteins with improved inhibition potency and expanded specificity

Anti-CRISPR (Acr) proteins are bacteriophage-derived antagonists of CRISPR-Cas systems. To date, Acrs were obtained either by mining sequence databanks or experimentally screening phage collections, both of which yield a limited repertoire of naturally occurring variants. Here, we applied structure-based engineering on AcrIIC1, a broad-spectrum inhibitor of type II-C CRISPR systems, to improve its efficacy and expand its specificity. We first show that fusing exogenous protein domains into AcrIIC1 dramatically enhances inhibition of the natural Neisseria meningitidis Cas9 target. Then, using structure-guided design, we converted AcrIIC1 into AcrX, a potent inhibitor of the type II-A CRISPR-Cas9 from Staphylococcus aureus widely applied for in vivo genome editing. Our work introduces designer Acrs as important biotechnological tools and provides an innovative strategy to safeguard the CRISPR technology.

synthetic biology↗