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Stengl, C.

Publications and source records attributed to Stengl, C..

3 recordsLinked to original sources

Experimental investigation of oxygen diffusion in the peak and valley region of minibeam patterns during X-Ray irradiation

BackgroundMinibeam radiotherapy has demonstrated its potential to reduce normal tissue toxicity, while maintaining tumor control. However, the underlying mechanisms behind this phenomenon remain unknown. Recent theoretical studies suggest a dose surrogate by diffusion of H2O2 into the valley regions. PurposeThe aim of this study is to experimentally investigate oxygen depletion and diffusion upon minibeam (MB) irradiation. MethodsA 3D-printed water phantom with four sensors was developed to enable the real-time, simultaneous measurement of oxygen concentration in the peak and valley. Water with 0 % - 11 % O2 and 0.1 % / 5 % CO2 was irradiated with broad beam (BB) and MB characterized by peak and valley widths of 2 mm x 2 mm and 0.5 mm x 2 mm. The depletion was further compared in other chemical environments. ResultsThe oxygen depletion rates per dose in hypoxic water in the valley regions were found to be 3 to 7 times higher compared to the peaks or BB. This observation was found to be independent of oxygen concentration above 2 %, indicating oxygen depletion saturation. For MB, diffusion between peaks and valleys was observed. After a certain period, an equilibrium between diffusion and dose rate differences was established. Glutathione and HEPES as medium increased the depletion further and distinguished MB from BB. ConclusionsA novel way of simultaneously measuring oxygen in the peak and valley of the MB dose pattern was introduced. The observed oxygen depletion saturation and diffusion between the peaks and valleys suggest the importance of oxygen in spatially fractionated radiotherapy studies, which is even greater for 5 mM glutathione compared to water.

biochemistry↗

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↗

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↗