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.

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.

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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.The detailed characterization of bacterial CRISPR-Cas systems 1 and their adaptation for precise genome engineering in mammalian cells 2, 3 has revolutionized the life sciences and enabled novel applications in biotechnology and medicine. The recent discovery of phage-derived anti-CRISPR proteins 4-6 , i.e. potent inhibitors of Cas effectors, provides a shut-off mechanism that can keep this powerful technology in check 7 and enhance the precision at which genome perturbations can be made [8][9][10][11] . While mining of sequence databases and screening of phage libraries proved to be powerful strategies to discover Acrs targeting a variety of Cas effectors 5, 6, 12-18 , these approaches are inherently limited to the naturally occurring protein sequence space. Moreover, for various Cas effectors of major biotechnological interest, nature might be lacking (efficient) anti-CRISPR counterparts.

Availability of oxygen plays an important role in tissue organization and cell-type specific metabolism. It is, however, difficult to analyze hypoxia-related adaptations in vitro because of inherent limitations of experimental model systems. In this study, we establish a microfluidic tissue culture protocol to generate hypoxic gradients in vitro, mimicking the conditions found in the liver acinus. To accomplish this, four microfluidic chips, each containing two chambers, were serially connected to obtain eight interconnected chambers. HepG2 hepatocytes were uniformly seeded in each chamber and cultivated under a constant media flow of 50 µL/h for 72 h. HepG2 oxygen consumption under flowing media conditions established a normoxia to hypoxia gradient within the chambers, which was confirmed by oxygen sensors located at the inlet and outlet of the connected microfluidic chips. Expression of Hif1α mRNA and protein was used to indicate hypoxic conditions in the cells and albumin mRNA and protein expression served as a marker for liver acinus-like zonation. Oxygen measurements performed over 72 h showed a change from 17.5% to 15.9% of atmospheric oxygen, which corresponded with a 9.2% oxygen reduction in the medium between chamber1 (inlet) and 8 (outlet) in the connected microfluidic chips after 72 h. Analysis of Hif1α expression and nuclear translocation in HepG2 cells additionally confirmed the hypoxic gradient from chamber1 to chamber8. Moreover, albumin mRNA and protein levels were significantly reduced from chamber1 to chamber8, indicating liver acinus zonation along the oxygen gradient. Taken together, microfluidic cultivation in interconnected chambers provides a new model for analyzing cells in a normoxic to hypoxic gradient in vitro. By using a well-characterized cancer cell line as a homogenous hepatocyte population, we also demonstrate that an approximate 10% reduction in oxygen triggers translocation of Hif1α to the nucleus and reduces albumin production.

Over recent decades, stereotactic body radiotherapy has garnered increasing popularity. Unfortunately, conventional preclinical 2D in vitro models are often insufficient for studying radiotherapy effects. Therefore, in this study, we developed a novel anthropomorphic in vitro liver phantom, which simulates the relevant hepatocellular carcinoma (HCC) tumor microenvironment and spatial organization. The liver phantom was 3D printed, filled with tissue-mimicking agarose mixture, and designed to fit ten microfluidic chips (MCs), in which HepG2 cells were seeded. Airtight MCs induced hypoxic conditions, as verified by Hif1α staining. Irradiation was conducted with 20 Gy in one fraction using a CyberKnife, in either a 2D setup, or by irradiating MCs arranged in the 3D-printed liver model using an individually calculated treatment plan. Post-irradiation cellular damage was determined via γH2AX staining. Here, we demonstrate a new physiologically relevant approach to model HCC pathology following radiotherapy. Comparing γH2AX staining in normoxic conditions to cells grown in MCs (hypoxic conditions) revealed a reduction in cellular damage of 30.24% (p = 0.0001) in the hypoxic environment. Moreover, we compared the scattering effect of radiation on a conventional 2D in vitro model to our new 3D anthropomorphic liver phantom and observed a significant γH2AX intensity reduction of 9.6% (p = 0.0294) in HepG2 cells irradiated in the phantom. Our approach of utilizing a liver phantom takes into account the hypoxic tumor microenvironment and 3D scattering effects of tissue irradiation, thereby modeling both physical and biological parameters of HCC tumors. The use of tissue phantoms lays the groundwork for future examination of other hypoxic tumors and offers a more comprehensive approach for screening and analysis of novel cancer therapeutics.

Objective: This work investigates the use of Al2O3:C and Al2O3:C,Mg optically stimulated luminescence (OSL) detectors to determine both the dose and the radiation quality in light ion beams. The radiation quality is here expressed through either the linear energy transfer (LET) or the closely related metric Qeff, which depends on the particle's speed and effective charge. The derived LET and Qeff values are applied to improve the dosimetry in light ion beams.

Approach: OSL detectors were irradiated in mono-energetic 1H−, 4He−, 12C−, and 16O−ion beams. The OSL signal is associated with two emission bands that were separated using a pulsed stimulation technique and subjected to automatic corrections based on reference irradiations. Each emission band was investigated independently for dosimetry, and the ratio of the two emission intensities was parameterized as a function of fluence- and dose-averaged LET, as well as Qeff. The determined radiation quality was subsequently applied to correct the dose for ionization quenching.

Main results: For both materials, the Qeff determinations in 1H− and 4He−ion beams are within 5 % of the Monte Carlo simulated values. Using the determined radiation quality metrics to correct the non-linear (ionization quenched) detector response leads to doses within 2 % of the reference doses. 

Significance: Al2O3:C and Al2O3:C,Mg OSL detectors are applicable for dosimetry and radiation quality estimations in 1H- and 4He-ions. Only Al2O3:C,Mg shows promising results for dosimetry in 12C-ions. Across both materials and the investigated ions, the estimated Qeff values were less sensitive to the ion type than the estimated LET values. The reduced uncertainties suggest new possibilities for simultaneously estimating the physical and biological dose in particle therapy with OSL detectors.

BackgroundInterest in spatial fractionation radiotherapy has exponentially increased over the last decade as a significant reduction of healthy tissue toxicity was observed by mini‐beam irradiation. Published studies, however, mostly use rigid mini‐beam collimators dedicated to their exact experimental arrangement such that changing the setup or testing new mini‐beam collimator configurations becomes challenging and expensive.PurposeIn this work, a versatile, low‐cost mini‐beam collimator was designed and manufactured for pre‐clinical applications with X‐ray beams. The mini‐beam collimator enables variability of the full width at half maximum (FWHM), the center‐to‐center distance (ctc), the peak‐to‐valley dose ratio (PVDR), and the source‐to‐collimator distance (SCD).MethodsThe mini‐beam collimator is an in‐house development, which was constructed of 10 ×  40 mm2 tungsten or brass plates. These metal plates were combined with 3D‐printed plastic plates that can be stacked together in the desired order. A standard X‐ray source was used for the dosimetric characterization of four different configurations of the collimator, including a combination of plastic plates of 0.5, 1, or 2 mm width, assembled with 1 or 2 mm thick metal plates. Irradiations were done at three different SCDs for characterizing the performance of the collimator. For the SCDs closer to the radiation source, the plastic plates were 3D‐printed with a dedicated angle to compensate for the X‐ray beam divergence, making it possible to study ultra‐high dose rates of around 40 Gy/s. All dosimetric quantifications were performed using EBT‐XD films. Additionally, in vitro studies with H460 cells were carried out.ResultsCharacteristic mini‐beam dose distributions were obtained with the developed collimator using a conventional X‐ray source. With the exchangeable 3D‐printed plates, FWHM and ctc from 0.52 to 2.11 mm, and from 1.77 to 4.61 mm were achieved, with uncertainties ranging from 0.01% to 8.98%, respectively. The FWHM and ctc obtained with the EBT‐XD films are in agreement with the design of each mini‐beam collimator configuration. For dose rates in the order of several Gy/min, the highest PVDR of 10.09 ± 1.08 was achieved with a collimator configuration of 0.5 mm thick plastic plates and 2 mm thick metal plates. Exchanging the tungsten plates with the lower‐density metal brass reduced the PVDR by approximately 50%. Also, increasing the dose rate to ultra‐high dose rates was feasible with the mini‐beam collimator, where a PVDR of 24.26 ± 2.10 was achieved. Finally, it was possible to deliver and quantify mini‐beam dose distribution patterns in vitro.ConclusionsWith the developed collimator, we achieved various mini‐beam dose distributions that can be adjusted according to the needs of the user in regards to FWHM, ctc, PVDR and SCD, while accounting for beam divergence. Therefore, the designed mini‐beam collimator may enable low‐cost and versatile pre‐clinical research on mini‐beam irradiation.