Hoffmann, K. H. | bioRxiv Science

bioRxiv2025

Automated Image-Based Cell Sorting by Targeted Photopolymerization

We present an automated image-based cell sorting method capable of a through-put of hundreds of cells per second. Our microscopy platform integrates automated image acquisition, machine-learning based classification and sub-sequent depletion of up to 99.98 % of negative cells by spatially controlled photo-encapsulation, preserving target cells for collection. Applied to peripheral blood mononuclear cells, the method achieves the label-free, morphology-based enrichment of activated T cells for downstream applications in biomedicine.

cell 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↗