Biology subjects

Walter, D.

Publications and source records attributed to Walter, D..

11 recordsLinked to original sources

bioRxiv2026

Comparative Single-Cell Profiling of CRISPR Knockout and Interference Defines Modality-Specific Strengths in Functional Genomics

Pooled CRISPR screens coupled with single-cell RNA sequencing enable high-throughput functional interrogation of gene regulatory networks, yet systematic comparisons of CRISPR knockout (CRISPRko) and CRISPR interference (CRISPRi) remain limited. We established a single-cell CRISPRko workflow and benchmarked it against CRISPRi using 87 sgRNAs targeting 29 unfolded protein response genes. CRISPRko generated transcriptional phenotypes are highly concordant with CRISPRi, and induced comparable pathway-level responses. While CRISPRi allows direct assessment of target gene repression, CRISPRko provides an effective complementary approach for complete loss-of-function studies, expanding the toolkit for single-cell functional genomics.

genomics↗

bioRxiv2026

Scarless conditional sgRNAs via endogenous mascRNA processing enable rapid and temporally controlled genome editing

Precise temporal control of gene editing is essential for studying dynamic biological processes, interrogating essential gene function, and improving the interpretability of pooled perturbation screens. Cre-dependent single guide RNA (sgRNA) switches provide temporal regulation by coupling guide activation to site-specific recombination, but existing designs retain a loxP-derived 5' sequence (scar) on the mature sgRNA that can impair guide function. We developed a scarless conditional sgRNA platform that combines Cre-loxP recombination with endogenous RNA processing to restore the native sgRNA architecture following induction. A MALAT1-associated small cytoplasmic RNA (mascRNA) module was positioned upstream of the guide sequence such that, after Cre-mediated recombination, cellular RNase P and RNase Z remove the residual loxP-derived overhang, generating a mature sgRNA with an authentic 5' terminus. Using guides targeting endogenous cell-surface marker genes, the scarless design maintained stringent OFF-state control while improving ON-state editing performance compared with a conventional Cre-activated sgRNA switch, resulting in faster editing kinetics, greater perturbation penetrance, and more consistent editing efficiency. This modular strategy provides a simple approach for conditional CRISPR genome editing that preserves guide integrity and should be readily adaptable to time-resolved functional genomics and pooled screening applications.

cell biology↗

bioRxiv2026

Photoswitchable optoGPCRs for reversible control of Gs and arrestin signalling

OptoGPCRs are light-activatable G protein-coupled receptors (GPCRs) used for optogenetic control of physiological processes. Most existing optoGPCRs are based on monostable opsins, which are limited by photobleaching and irreversibility. The bistable jumping spider rhodopsin 1 (JSR1) carrying the single point mutant S199F introduces a [~]150 nm spectral separation between the active and inactive states, enabling bidirectional control with distinct wavelengths of light. Here, we show that JSR1-S199F demonstrates robust, light-reversible arrestin recruitment and Gq/i protein activity. We then engineered JSR1-S199F-based optoGPCRs with Gs protein activity, expanding the limited repertoire of bistable Gs-coupled opsins. Specifically, we present optoDRD1, a chimeric optoGPCR that redirects the native Gq/i protein activity of JSR1 towards the Gs pathway of the dopamine D1 receptor (DRD1). Through systematic screening of intracellular domain combinations, we identified an optimal chimeric configuration comprising ICL2, ICL3, helix 8, and the C-terminus from DRD1. The resulting optoGPCR is activated by violet light ({lambda}max = 397 nm) and deactivated by green light ({lambda}max = 531 nm) at physiologically relevant light intensities. A single violet light pulse drives sustained Gs signalling for several hours, while green light deactivation enables precise signal termination at any timepoint. OptoDRD1 closely mimics wild-type DRD1 signalling kinetics and G protein selectivity. Compared to JellyOp, the only previously characterised natively Gs-coupled opsin, optoDRD1 shows higher signal amplitude and reversibility over multiple light cycles. We further demonstrate optoDRD1s utility for optogenetic control of Gs-regulated processes in vitro, including insulin secretion in human {beta}-cells and signalling modulation in a neuronal cell line, supporting its potential for in vivo applications. The biochemical stability and known structure of JSR1 make it a robust scaffold for this rational engineering and for future biophysical characterization. Together, optoDRD1 and JSR1-S199F expand the optoGPCR toolkit and open new opportunities for dissecting dopaminergic signalling, Gs-mediated physiology, and GPCR signalling pharmacology.

synthetic biology↗

bioRxiv2026

Latitudinal diversity gradients and selective microbialexchange at the Atlantic ocean-air interface

Microbial communities at the ocean-atmosphere interface play a vital role in nutrient, aerosol, and water cycling, yet their large-scale biogeography remains scarcely studied. Here we assessed microbial cell abundance and community structure in air and surface ocean samples along a 14,400 km transect from the polar circle to the equator. Air and surface ocean microbiomes were taxonomically distinct across the North East Atlantic. The microbiome in the air had a lower local community richness compared to the surface ocean, yet was more diverse across larger geographical scales. Our results suggest that terrestrial-derived air masses affected air and surface ocean communities. Air microbial communities showed a latitudinal diversity gradient with richness and cell abundance increasing from the polar circle to the equator. We observed an exchange of microbial lineages between ocean and air, however, most lineages were confined to one realm, suggesting lineage-selective aerosolization and deposition of microbial cells.

microbiology↗

bioRxiv2026

Social context modulates cannibalistic attraction in Drosophila larvae

Animals often face conflicting decisions to ensure survival. Under food limitation, cannibalism can provide a nutritional advantage despite substantial costs. In Drosophila melanogaster larvae, feeding is essential for successful metamorphosis, making food source decisions critical. We find that dead conspecifics act as attractive cues, but this attraction is weak for live larval groups. It is enhanced when dead larvae are injured or homogenized, or after repeated exposure. Larvae are also more attracted to dead individuals from other Drosophila or insect species. Testing live larvae individually revealed the strongest attraction, indicating that social context suppresses feeding-related attraction. Attraction to dead conspecifics depends on multiple chemosensory and mechanosensory modalities, highlighting the complex integration of social and nutritional cues in larval decision-making. Our results indicate that fly larvae flexibly balance attraction and avoidance based on social context to optimize resource use under ecological constraints. Short summaryDrosophila melanogaster larvae flexibly balance attraction and social avoidance when encountering dead conspecifics. Attraction depends on carcass condition, prior experience, and social context, and is mediated by chemosensory, gustatory, and mechanosensory pathways. These findings reveal how larvae integrate multimodal social and nutritional cues to guide context-dependent feeding decisions.

neuroscience↗

bioRxiv2025

A rapid ionic liquid-based DNA extraction method for molecular diagnostics of urinary tract infections

Rapid and reliable DNA extraction from urine is a critical bottleneck in advancing molecular diagnostics for urinary tract infections (UTIs) in both centralized and decentralized settings. Here, we present an ionic liquid-based DNA extraction method (IL-DEx) that enables recovery of bacterial DNA from urine samples in under 30 minutes using minimal equipment and no hazardous chemicals. IL-DEx was benchmarked against a widely used commercial kit (QIAamp DNA Mini Kit, QIAGEN) using reference strains, clinical isolates, and spiked urine samples. For gram-negative bacteria, IL-DEx achieved comparable DNA yields (47-102% relative efficiency), while recoveries from gram-positive bacteria were lower (0.7-8%) but sufficient for downstream detection. Quantitative PCR (qPCR) revealed linear DNA recovery across five to six orders of magnitude (108-102 CFU/ml, R2 >0.99), with detection limits of [~]102-103 CFU/ml for gram-negatives and [~]103-104 CFU/ml for gram-positives using 1 ml urine. Clinical evaluation with 13 patient urine samples (ten culture-positive, three culture-negative) demonstrated that IL-DEx reliably enabled pathogen detection by qPCR and full-length 16S rRNA gene sequencing (Oxford Nanopore). Performance was comparable to three other extraction methods tested head-to-head, including the QIAamp DNA Mini Kit (QIAGEN), the MagaZorb DNA Mini-Prep Kit (Promega), and a phenol-chloroform extraction method. These findings establish IL-DEx as the first ionic liquid-based approach evaluated for DNA recovery from clinical urine samples, providing a fast, simple, and low-cost method suitable for integration into molecular workflows for UTI diagnostics across diverse laboratory and clinical settings. ImportanceUrinary tract infections (UTIs) are among the most common infections worldwide and a major driver of antibiotic use. Rapid and accurate diagnosis is critical to guide therapy, reduce inappropriate antibiotic prescriptions, and improve patient outcomes. While molecular diagnostics can drastically reduce time to identify uropathogens, their implementation remains constrained by upstream DNA extraction - a step that is often laborious, cost-intensive, or incompatible with rapid diagnostic workflows. We developed a fast, simple, and low-cost DNA extraction method (IL-DEx) that uses an ionic liquid and magnetic beads to recover bacterial DNA directly from urine. IL-DEx eliminates hazardous reagents and complex equipment while delivering performance comparable to established extraction kits. By streamlining this critical pre-analytical step, IL-DEx enables faster molecular diagnostics and broadens access to modern UTI testing. Its simplicity and robustness position it as a valuable tool for improving diagnostic speed, antimicrobial stewardship, and patient care across healthcare settings.

molecular biology↗

bioRxiv2025

Predicting Personalised Therapeutic Combinations in Non-Small Cell Lung Cancer Using In Silico Modelling

The disease burden from non-small cell lung cancer (NSCLC) adenocarcinoma is substantial, with around a million new cases diagnosed globally each year, and a 5-year survival rate of less than 20%. A lack of therapeutic options personalized to individual patient genetics, and the targeted therapies that exist quickly succumbing to resistance, leads to high variation in survival. Patient stratification combined with greater personalisation of therapies have the potential to improve outcomes, however, the wide variation in mutations found in NSCLC adenocarcinoma patients mean that experimentally determining suitable treatment combinations is time-consuming and expensive. Here we present an in silico model encompassing tumour intrinsic key oncogenic signalling pathways, including EGFR, AKT, JAK/STAT and WNT for efficiently predicting rational drug-drug and drug-radiotherapy combination therapies in NSCLC. Using this model, we simulate diverse genetic profiles and test over 10,000 therapeutic combinations to identify optimal strategies to overcome resistance mechanisms specific to genetic profiles and p53 status. Our in silico model reproduces drug additivity experiments, predicts radio-sensitising genes validated in a CRISPR screen and identifies 53BP1 as a potential drug target that improves the therapeutic window during radiotherapy, as well as potential to use ATM inhibitors to overcome p53 loss-of-function driven radiotherapy resistance. We further use the in silico model to identify a 19-gene signature to stratify patients most likely to benefit from radiotherapy and validated this using TCGA data. These results further demonstrate the utility of in silico mechanistic modelling and present a bespoke computational resource for large-scale screening of personalised therapies applied to NSCLC.

cancer biology↗

bioRxiv2024

Unveiling the Origins and Genetic Makeup of the 'Forgotten People': A Study of the Sarmatian-Period Population in the Carpathian Basin

SO_SCPLOWUMMARYC_SCPLOWThe nomadic Sarmatians dominated the Pontic Steppe from 3rd century BCE and the Great Hungarian Plain from 50 CE until the Huns 4th-century expansion. In this study, we present the first large-scale genetic analysis of 156 genomes from 1st- to 5th-century Hungary and the Carpathian foothills. Our findings reveal minor East Asian ancestry in the Carpathian Basin (CB) Sarmatians, distinguishing them from other regional populations. Using F4-statistics, qpAdm, and IBD analysis, we show that CB Sarmatians descended from Steppe Sarmatians originating in the Ural and Kazakhstan regions, with Romanian Sarmatians serving as a genetic bridge between the two groups. We also identify two previously unknown migration waves during the Sarmatian era and a notable continuity of the Sarmatian population into the Hunnic period, despite a smaller influx of Asian-origin individuals. These results shed new light on Sarmatian migrations and the genetic history of a key population neighbouring the Roman Empire.

genetics↗

bioRxiv2024

A benchmark comparison of CRISPRn guide-RNA design algorithms and generation of small single and dual-targeting libraries to boost screening efficiency

Genome-wide CRISPR sgRNA libraries have emerged as transformative tools to systematically probe gene function. While these libraries have been iterated over time to be more efficient, their large size limits their use in some applications. Here, we benchmarked publicly available genome-wide single-targeting sgRNA libraries and evaluated dual targeting as a strategy for pooled CRISPR loss-of-function screens. We leveraged this data to design two minimal genome-wide human CRISPR-Cas9 libraries that are 50% smaller than other libraries and that preserve specificity and sensitivity, thus enabling broader deployment at scale.

molecular biology↗

bioRxiv2020

Hematopoietic stem cells fail to regenerate following inflammatory challenge.

Hematopoietic stem cells (HSCs) are canonically defined by their capacity to maintain the HSC pool via self-renewal divisions. However, accumulating evidence suggests that HSC function is instead preserved by sustaining long-term quiescence. Here, we study the kinetics of HSC recovery in mice, following an inflammatory challenge that induces HSCs to exit dormancy. Repeated inflammatory challenge resulted in a progressive depletion of functional HSCs, with no sign of later recovery. Underlying this observation, label retention experiments demonstrated that self-renewal divisions were absent or extremely rare during challenge, as well as during any subsequent recovery period. While depletion of functional HSCs held no immediate consequences, young mice exposed to inflammatory challenge developed blood and bone marrow hypocellularity in old age, similar to elderly humans. The progressive, irreversible attrition of HSC function demonstrates that discreet instances of inflammatory stress can have an irreversible and therefore cumulative impact on HSC function, even when separated by several months. These findings have important implications for our understanding of the role of inflammation as a mediator of dysfunctional tissue maintenance and regeneration during ageing.

cell biology↗

bioRxiv2018

A histone H4K20 methylation-mediated chromatin compaction threshold ensures genome integrity by limiting DNA replication licensing

The decompaction and re-establishment of chromatin organization immediately after mitosis is essential for genome regulation. The mechanisms underlying chromatin structure control in daughter cells are not fully understood. Here, we show that a chromatin compaction threshold in cells exiting mitosis ensures genome integrity by limiting replication licensing in G1 phase. Upon mitotic exit, appropriate chromatin relaxation is safeguarded by SET8-dependent methylation of histone H4 on lysine 20. Thus, in the absence of either SET8 or the H4K20 residue, substantial genome-wide chromatin decompaction occurs which allows excessive loading of the Origin Recognition Complex (ORC) in the daughter cells. ORC overloading stimulates aberrant recruitment of the MCM2-7 complex that promotes single-stranded DNA formation and DNA damage. Restoring chromatin compaction restrains excess replication licensing and the loss of genome integrity. Our findings identify a cell cycle-specific mechanism whereby fine-tuned chromatin relaxation suppresses excessive detrimental replication licensing and maintains genome integrity at the cellular transition from mitosis to G1 phase.

molecular biology↗