Biology subjects

Bruegmann, T.

Publications and source records attributed to Bruegmann, T..

8 recordsLinked to original sources

bioRxiv2026

A heat-assisted ttLbCas12a workflow incorporating crRNA 3'-tailing enables efficient multiplex biallelic genome editing in hybrid poplar

In forest trees, high genome-editing efficiency in primary transformants is particularly important for functional genomics and trait improvement, as long generation times severely limit the use of subsequent generations to recover fully edited genotypes. This challenge is further compounded in hybrid poplar, where paralogous gene copies and allelic variation make efficient multiplex editing particularly difficult. We optimized LbCas12a-mediated editing in Populus x canescens (Populus tremula x Populus alba), clone INRA 717-1B4, by targeting four paralogous PtaABA8ox4 loci using temperature-tolerant ttLbCas12a, a two-stage heat treatment, and crRNA 3'-tailing. The heat-treatment regime preserved transgenic recovery, while ttLbCas12a-based constructs and heat treatment substantially increased biallelic editing. All seven lines in the highest-performing group, combining ttLbCas12a, 3'-tailed crRNAs, and heat treatment, were biallelically edited at all four targets. crRNA 3'-tailing altered predicted secondary structures and showed a consistent numerical tendency toward improved editing, but its independent effect was not statistically resolved. Mutations were dominated by small deletions, and biallelic out-of-frame recovery increased in ttLbCas12a-based contexts without scaling proportionally with biallelic editing. Thus, heat-assisted ttLbCas12a-mediated approaches enable efficient multiplex editing in hybrid poplar, whereas genotype quality remains dependent on repair outcome and somatic uniformity.

plant biology↗

bioRxiv2026

CRISPR/Cas9 knockout of PtaAGL16 improves post-drought recovery and shoot vitality in hybrid poplar

Drought stress severely limits tree growth and survival, a problem intensifying under climate change due to the rising frequency of extreme weather events. Identifying genetic variants that modulate drought stress responses and enhance resilience is therefore of great interest for developing climate-adapted trees. While the MADS-box transcription factor AGL16 has been implicated in drought responses and stomatal regulation in herbaceous species, the function of its homolog in woody perennials remains largely unexplored. Here, we investigate the role of PtaAGL16 in the poplar hybrid Populus tremula x P. alba. We generated CRISPR/Cas9 knockout lines of PtaAGL16 in the poplar clone INRA 717-1B4. Plants were subjected to a severe drought stress regime followed by a re-watering recovery phase. Morphological and physiological traits, including growth, stomatal conductance, shoot vitality, and stomatal density, together with stress-associated metabolites and drought-responsive gene expression, were analyzed. All agl16 knockout lines exhibited significantly reduced drought-induced damage compared to the wild-type. While the wild-type suffered extensive defoliation and persistent stem necrosis, the knockout lines retained higher shoot vitality and resumed growth after re-watering, including the formation of new leaves. Consequently, vitality in the knockout lines reached up to 86%, compared to only 34% in the wild-type. This improved resilience was accompanied by reduced persistent oxidative membrane damage, as indicated by lower MDA levels after recovery. These findings indicate that the disruption of PtaAGL16 is associated with enhanced drought resilience in a woody perennial species and highlight its potential as a genetic target for improving climate adaptation in fast-growing forest trees.

plant biology↗

bioRxiv2026

First report of genetic transformation and CRISPR/Cas12a-mediated gene editing of European beech (Fagus sylvatica L.)

Fagus sylvatica L. (European beech) is a dominant hardwood forest tree species across Central Europe, supporting diverse ecosystem services and forming the basis of a significant market for high-value timber. However, climate change increasingly threatens beech vitality and productivity, making molecular insights into its stress resilience and functional validation of underlying genes urgently needed. Here, we report a protocol for protoplast isolation from seedling leaves and demonstrate, for the first time, transient genetic transformation and CRISPR/Cas-mediated genome editing in F. sylvatica. PEG-mediated transformation was sequentially optimized, achieving efficiencies of 59 {+/-} 6.19% within distinct seasonal windows. Protoplast yield and transformation efficiency showed pronounced temporal variation throughout the year, indicating a strong seasonal influence on reproducibility of the workflow despite controlled growth conditions. A basic molecular toolkit for functional genomics and future biotechnological applications was established by testing a set of promoters and reporters. For proof-of-concept genome editing, we achieved 4.75 to 32.69% editing efficiencies in the PHYTOENE DESATURASE gene (FsPDS) using temperature-tolerant LbCas12a (ttLbCas12a). Although further optimization of transformation reproducibility and regeneration systems remains necessary, the presented protoplast platform provides a valuable foundation for transient functional assays and genome editing studies in this non-model tree species.

plant biology↗

bioRxiv2026

Overexpression of PtaHDG11 enhances drought tolerance and suppresses trichome formation in Populus tremula x Populus alba

Extended drought periods are increasingly impacting the growth and survival of forest trees in Central Europe, underscoring the need for innovative approaches to adapt trees to drought. Understanding the functions of genes involved in drought tolerance can contribute to the development of effective strategies to improve drought resilience. Although the homeobox-leucine zipper protein HDG11 has been identified as a promising target for enhancing drought stress tolerance in plants, the functional role of the native HDG11 gene in tree species remained uncharacterized. To address this gap, we identified the homolog of the AtHDG11 gene in the poplar hybrid Populus tremula x Populus alba, clone INRA 717-1B4, and subsequently transformed the PtaHDG11 gene into this clone for constitutive overexpression. Drought stress experiments revealed significantly enhanced drought stress tolerance in PtaHDG11-overexpressing poplars compared with the wild type. This improved tolerance was characterized by higher leaf relative water content, reduced leaf shedding, lower malondialdehyde accumulation and higher expression of antioxidant-related genes, including SOD and CAT. Improved tolerance resulted in significantly increased dry biomass after the recovery phase. Notably, the transgenic poplars exhibited altered basal expression of the cell wall-related genes PtaXTH32-like and PtaEXPA15-like under control conditions, indicating a modified cell wall-associated regulatory state before stress exposure. In addition, PtaHDG11 overexpression was associated with a glabrous phenotype lacking trichomes, pointing to functions of PtaHDG11 beyond drought stress responses in a woody perennial. Together, these findings suggest that PtaHDG11 can contribute to improving drought resilience in trees, with implications for sustainable forest management and breeding programs.

plant biology↗

bioRxiv2025

Peroxisome protein import deficiency causes heart failure in mouse and human

Peroxisomes are ubiquitous cellular organelles with potentially vital roles in lipid and reactive oxygen metabolism. The metabolic demands of the heart are substantial; however, the contribution of peroxisomes to cardiac development, health, and their role in heart failure (HF) remain largely unexplored. We developed and examined a mouse and an engineered human myocardium (EHM) model with a deficiency in cardiac peroxisome biogenesis to investigate the role of peroxisomes in cardiac function and pathology. In the EHM, loss of peroxisome protein import and subsequent peroxisomal metabolic impairment trigger mitochondrial damage and compromise cellular respiration and energy production. Peroxisome dysfunction results in incoherent electrical conduction, defective Ca2+-handling, and ultimately presentation of a HF phenotype with pathological force generation. These phenotypes are mirrored in an orthogonal murine model system with defective cardiac peroxisome biogenesis. Preload-dependent deficits in force generation due to insufficient energy supply are eventually fatal. Thus, peroxisomes play an important role in sustaining normal heart operations. Vice versa, peroxisome maintenance is compromised in pressure overload-induced HF, establishing peroxisomes as potential modulators of pathology and targets of therapy.

cell biology↗

bioRxiv2025

Guiding G protein signaling by target enhancement of GPCRs

Activation of G protein coupled receptors coupling to the Gi/o pathway leads to the activation of G protein-activated inward rectifier potassium channels (GIRK) in a fast membrane-delimited manner in excitable cells. Activation of GIRK causes the hyperpolarization of the cell membrane, where hyperpolarization is dependent on te availability of Gi/o coupled GPCRs and GIRK. In particular, in optogenetic and chemogenetic experiments neuronal silencing depends on downstream targets of Gi/o-coupled GPCRs. To selectively enhance Gi/o mediated GIRK currents, we created expression cassettes consisting of a homomer forming GIRK subunit and various light-activated Gi/o-coupled GPCRs (Melanopsin, Long-wave-sensitive opsin 1, Parapinopsin or Opsin 7b). We demonstrate that light-activation of the GIRK/GPCR constructs induces robust GIRK currents in human embryonic kidney 293 cells, cardiomyocytes and cerebellar Purkinje cells and changes the net effect of G protein signaling of the promiscuous Opn4L from a Gq/11 mediated excitation towards an Gi/o mediated inhibition. Thus, our tools enhance target selectivity and improve optogenetic control of the Gi/o pathway by light in excitable cells.

neuroscience↗

bioRxiv2024

Thermogenetics for cardiac pacing

Cardiac arrhythmias are common disorders that can be fatal. Modern methods of treating bradyarrhythmias include the implantation of pacemakers and cardioverter-defibrillators. However, implantable devices can cause various complications related to the electrodes installed inside the heart, including infection. Less invasive heart rhythm modulation could be beneficial for some cohorts of patients. Here, we demonstrate an alternative approach to heart pacing based on thermogenetics. We used adeno-associated viruses to deliver genetic human transient receptor potential subfamily V member 1 (TRPV1), a heat-sensitive cation channel, into isolated cardiomyocytes and the mouse heart. This allowed us to induce action potentials and control contractility using short heat pulses delivered by infrared laser illumination. Using this approach, we demonstrated the thermogenetic pacing of isolated cardiomyocytes in vitro and in the mouse heart in vivo. Our results demonstrate the potential of thermogenetics for developing therapeutic strategies for heart rhythm modulation.

synthetic biology↗

bioRxiv2023

Efficient and sustained optogenetic control of nervous and cardiac systems

Optogenetic control is used to manipulate the activity of specific cell types in vivo for a variety of biological and clinical applications. Here we report ChReef, an improved variant of the channelrhodopsin ChRmine. ChReef offers minimal photocurrent desensitization, a unitary conductance of 80 fS and closing kinetics of 30 ms, which together enable reliable optogenetic control of cells at low light levels with good temporal fidelity and sustained stimulation. We demonstrate efficient and reliable red-light pacing and depolarization-block of ChReef-expressing cardiomyocyte clusters. We used AAV-based gene transfer to express ChReef in retinal ganglion cells, where it restores visual function in blind mice with light sources as weak as an iPad screen. Toward optogenetic hearing restoration, ChReef enables stimulation of the auditory pathway in rodents and non-human primates with nano-Joule threshold, enabling efficient and frequency-specific stimulation by LED-based optical cochlear implants.

biophysics↗