Biology subjects

Aschenbrenner, S.

Publications and source records attributed to Aschenbrenner, S..

10 recordsLinked to original sources

bioRxiv2026

De novo design of light-controlled conformational protein switches

Light-responsive proteins offer the ability to control biology at an unmatched resolution. Yet, optogenetics is limited to a relatively small number of natural and engineered photoreceptors. The design of light-responsive proteins with native-like complexity is challenging as they must incorporate a light-responsive chromophore, couple chemical changes in the chromophore to structural changes in the protein, and ultimately propagate these changes to downstream effectors. Here, we present the computational design of new light-responsive proteins entirely from the bottom-up. We design de novo proteins that accurately bind a retinal chromophore and further design a protein conformational switching motion linked to retinal isomerisation. This switch unlocks a new protein-peptide interaction, creating light-activated heterodimers with an over 30-fold increase in binding upon illumination. Moreover, we show that the designed heterodimers are customisable, with tuneable wavelengths, sensitivities, and specificities. Further, we use the designed heterodimers to control biological function, regulating intracellular protein-protein interactions and cell-signalling receptors. Finally, we use real-time light-activated NMR to demonstrate that they undergo a defined light-activated conformational change. Overall, we computationally design a new class of light-responsive proteins that may open a new optogenetics toolbox with applications in biology.

biochemistry↗

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↗

bioRxiv2019

Induced aneuploidy disrupts MCF10A acini formation and CCND1 expression

Abnormal karyotypes, namely aneuploidy, can be detected in nearly all cancer entities at different grades. The impact of these altering mutations on epigenetic regulation, especially on promoter-enhancer interactions are not well understood. Here, we applied a 3D model of MCF10A cells in a high-content screen to measure induced aneuploidy by RNA interference of 82 mitotic genes associated with aneuploidy and breast cancer. Perturbation of ESPL1 and TOP2A expression led to increased mitotic instability and subsequent aneuploidy and polylobed nuclei. During acinus formation these polylobed cells disrupted proper acinus rotation inhibiting the development of a hollow lumen and a polarized outer cell layer. Further, gene expression profiling identified upregulated CCND1 among other breast cancer related genes. We show that acquisition of aneuploidy affects the morphogenesis of MCF10A acini and expression of cancer relevant genes. By conducting 4C chromosome capturing experiments we linked the alteration of interactions of the promoter region to CCND1 upregulation.

cancer 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↗

bioRxiv2018

Cell-specific CRISPR/Cas9 activation by microRNA-dependent expression of anti-CRISPR proteins

The rapid development of CRISPR/Cas technologies brought a personalized and targeted treatment of genetic disorders into closer reach. To render CRISPR-based therapies precise and safe, strategies to confine the activity of Cas(9) to selected cells and tissues are highly desired. Here, we developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR (Acr) proteins. We inserted target sites for miR-122 or miR-1, which are abundant specifically in liver and muscle cells, respectively, into the 3UTR of Acr transgenes. Co-expressing these with Cas9 and sgRNAs resulted in Acr knockdown and correspondingly in Cas9 activation solely in hepatocytes or myocytes, while Cas9 was efficiently inhibited in off-target cells. We demonstrate control of genome editing and gene activation using a miR-dependent AcrIIA4 in combination with different Streptococcus pyogenes (Spy)Cas9 variants (full-length Cas9, split-Cas9, dCas9-VP64). Finally, to showcase its modularity, we adapted our Cas-ON system to the smaller and more target-specific Neisseria meningitidis (Nme)Cas9 orthologue and its cognate inhibitors AcrIIC1 and AcrIIC3. Our Cas-ON switch should facilitate cell-specific activation of any CRISPR/Cas orthologue, for which a potent anti-CRISPR protein is known.

synthetic biology↗

bioRxiv2018

CD95 receptor activation by ligand-induced trimerization is independent of its partial pre-ligand assembly

CD95 (Fas, APO-1, TNFRSF6) is a widely expressed single-pass transmembrane protein that is implicated in cell death, inflammatory response, proliferation and cell migration. CD95 ligand (CD95L, FasL, TNFSF6), is a potent apoptotic inducer in the membrane form but not when cleaved into soluble CD95L (sCD95L). Here, we aimed at understanding the relation between ligand-receptor multimerization and receptor activation by correlating the kinetics of ligand binding, receptor oligomerization, FADD (FAS-Associated via Death Domain) recruitment and caspase-8 activation inside living cells. Using single molecule localization microscopy and Forster resonance energy transfer imaging we show that the majority of CD95 receptors on the plasma membrane are monomeric at rest. This was confirmed functionally as the wild-type receptor is not blocked by a receptor mutant that cannot bind ligand. Moreover, using time-resolved fluorescence imaging approaches we demonstrated that receptor multimerization follows instantaneously ligand binding, whereas FADD recruitment is delayed. This process can explain the typical delay time seen with caspase-8 activity reporters. Finally, the low activity of sCD95L, which was caused by inefficient FADD recruitment, was not explained by the low avidity for the receptor but by a receptor clustering mechanism that was different from the one induced by the strong apoptosis inducer IZ-sCD95L. Our results reveal that receptor activation is modulated by the capacity of its ligand to trimerize it.\n\nHighlightsO_LIAt a density of less than 10 receptors per {micro}m2 CD95 exists as monomer (58%) and dimer (42%)\nC_LIO_LIPre-formed dimers do not contribute to ligand-induced CD95 apoptotic signaling\nC_LIO_LIThe PLAD of CD95 attenuates overexpression-induced, ligand-independent cell death\nC_LIO_LIsoluble CD95L can rapidly multimerize CD95 after binding but it is still a poor inducer of apoptosis through inefficient FADD recruitment\nC_LIO_LIFADD recruitment kinetics but not ligand binding kinetics correlates with caspase-8 onset of activity\nC_LI

cell biology↗