Biology subjects

Shen, Y.

Publications and source records attributed to Shen, Y..

At least 73 records · Page 4Linked to original sources

bioRxiv2026

A teichoic acid-like wall modification associated with immune suppression is socially regulated in Streptococcus pyogenes

Streptococcus pyogenes (Group A Streptococcus, GAS) is a human-restricted pathogen with a range of clinical manifestations and worldwide prevalence. The GAS Rgg2/Rgg3 quorum sensing (QS) system, a cell-to-cell communication network, modifies the cell surface resulting in increased lysozyme resistance, biofilm formation, and expression of the qim operon that is responsible for modulation of innate immune responses in macrophages. The operon encodes 10 genes with predicted homology to enzymes involved in bacterial cell surface-associated carbohydrate and teichoic acid biosynthesis pathways. Comparing extracts of GAS cell wall polysaccharides between wildtype and operon mutants determined that the QS-induced genes modify the S. pyogenes cell surface by adding a wall teichoic acid-like moiety of N-acetylglucosamine-linked ribitol (GlcNAc-Rbo). A fluorescently labeled phage receptor-binding protein, RBP-13-GFP, that recognizes GlcNAc-decorated ribitol phosphate repeats, bound to the GAS surface only when qim expression was induced. Deletion of the qim operon eliminated RBP-13-GFP binding, diminished bacterial colonization, and significantly attenuated GAS pathogenesis in a murine skin infection model. These findings indicate that GAS has evolved a strategy to evade innate immune response by presenting a previously unknown carbohydrate moiety upon quorum sensing. IMPORTANCEStreptococcus pyogenes is a major human pathogen, responsible for diverse clinical manifestations of both superficial and invasive infections and can lead to post-infection sequelae like rheumatic heart disease whose prevalence on a global scale rivals the most serious pathogens. Invasive S. pyogenes infections are currently on the rise worldwide, notably correlating with increasing pediatric cases of scarlet fever and enhancing the concern for long term complications. There is much that remains unknown about S. pyogenes virulence and pathogenicity, and studies focused on understanding basic systems regulating virulence factors could lead to better therapeutics and translational research. We show here one such example, where a bacterial communication system regulating a virulence mechanism relevant to in vivo infection confers the ability to alter the host innate immune response. We find that modifications to the cell wall arise when this virulence system is activated that has a direct role in host-pathogen interactions. Further research into this system could provide a mechanism for disruption and serve to treat S. pyogenes infection.

microbiology↗

bioRxiv2025

Genome editing for treatment of JAK2 V617F-driven myeloproliferative neoplasms

JAK2 V617F is a common haematological driver mutation and underlies most cases of myeloproliferative neoplasms (MPNs). Reducing variant allele frequency (VAF) is an important treatment goal, but no current treatment modalities fully and specifically inhibit mutant signalling. Thus, the consequences of V617F inactivation are unclear, including whether selective inhibition of V617F signalling will eradicate mutant cells due to oncogene addiction. Here, we describe an allele-selective CRISPR-Cas genome editing strategy that achieves selective and efficient JAK2 V617F inactivation in patient stem and progenitor cells. We show that JAK2 V617F heterozygous cells are not oncogene addicted and maintain viability and differentiation potential upon loss of their mutant allele. In contrast, homozygous mutant cells are eradicated upon deletion of both mutant alleles. Across in vitro, organoid, xenotransplantation and single-cell assays, selective deletion of V617F alleles reverts MPN hallmarks including erythroid clonogenicity, inflammatory gene expression signatures, as well as myelofibrosis and splenomegaly phenotypes in an in vivo xenograft model. Collectively, our results show that is it possible to revert heterozygous JAK2 V617F mutant cells to a normal phenotype and suggest that ex vivo genome editing of stem and progenitor cells may be a viable treatment option to achieve rapid and deep reductions in VAF.

cancer biology↗

bioRxiv2025

Gasdermin E couples mitochondrial damage to pyroptotic neurodegeneration in Parkinson disease

Parkinsons disease (PD) features progressive loss of nigrostriatal dopamine neurons, but how mitochondrial damage engages programmed cell death pathways remains unresolved. Here, we identify gasdermin E (GSDME), the caspase-3-activated executor of pyroptosis, as a critical mediator of neurodegeneration in toxin-based PD models. In primary neurons and SH-SY5Y cells, the mitochondrial complex I inhibitor MPTP/MPP triggered caspase-3 activation, GSDME cleavage, and lytic membrane rupture. Genetic silencing of Gsdme or its transcriptional regulator SP1 reduced neuronal death. In vivo, Gsdme deficiency preserved substantia nigra pars compacta dopaminergic neurons, improved motor performance, and mitigated anxiety- and depression-like behaviors after MPTP administration. Loss of Gsdme also dampened microglial and astrocytic activation and lowered proinflammatory cytokines in striatum and substantia nigra. Mechanistically, cleaved GSDME localized to mitochondria, disrupted membrane potential, increased reactive oxygen species, and precipitated organelle injury, thereby coupling mitochondrial dysfunction to pyroptotic cell death. These findings identify GSDME-mediated pyroptosis as a mechanistic link between mitochondrial toxicity and neuroinflammation in PD and nominate GSDME as a therapeutic entry point to slow disease progression. SignificanceHow mitochondrial injury kills dopamine neurons in Parkinsons disease is a central unresolved question. We show that the pyroptosis executor GSDME is required for neurodegeneration and neuroinflammation in MPTP models, mechanistically linking caspase-3 activation and mitochondrial damage to lytic cell death. Targeting GSDME may provide a strategy to protect vulnerable neurons in PD. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=191 SRC="FIGDIR/small/694264v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@15364e7org.highwire.dtl.DTLVardef@57b65org.highwire.dtl.DTLVardef@1e85108org.highwire.dtl.DTLVardef@1200d34_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsGSDME drives PD-linked neuronal loss. Silencing GSDME (or its transcriptional factor Sp1) ameliorates MPTP-triggered neuronal cell death. Genetic ablation of Gsdme is protective in the MPTP-induced PD mouse model. Gsdme-/-mice retain nigrostriatal DA neurons, show improved motor and affective behaviors, and exhibit reduced microglial and astrocytic activation and cytokines release upon MPTP treatment. Cleaved GSDME accumulates on mitochondria, collapses {Delta}{Psi}m, raises ROS, and links mitochondrial toxicity to pyroptosis, thus positioning GSDME as a tractable target to slow PD progression.

neuroscience↗

bioRxiv2025

Postnatal increase in MRTF-dependent transcription reduces EGFR activation and proliferation of apical but not basal NSCs

In the ventricular-subventricular zone (V-SVZ), both apical and basal NSCs undergo quiescence and proliferation. The epidermal growth factor receptor (EGFR) and interactions with the extracellular matrix are key regulators of adult NSC proliferation and quiescence, respectively. Here, we show that activation of EGFR significantly declines after the first postnatal weeks in apical NSCs. This decline is accompanied by a shift in serum responsive factor (SRF)-dependent transcription in apical NSCs. Specifically, growth-promoting genes targeted by SRF and ternary complex transcription factors are downregulated, whereas those targeted by SRF and myocardin-related transcription factors (MRTFs), including those involved in the extracellular matrix remodeling, are upregulated. Blocking of MRTFs, whose activity is regulated by the Rho/actin pathway and extracellular matrix interactions, restores EGFR activation and EGFR-dependent proliferation in adult apical NSCs. Thus, transcriptional programs regulated by extracellular cues differentially control EGFR activation and proliferation in neonatal and adult apical NSCs. Graphical abstractGraphical representation of the postnatal changes in transcriptional programs observed in apical and basal NSCs in V-SVZ. O_FIG O_LINKSMALLFIG WIDTH=164 HEIGHT=200 SRC="FIGDIR/small/693930v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@ff3367org.highwire.dtl.DTLVardef@87d18eorg.highwire.dtl.DTLVardef@cec1e7org.highwire.dtl.DTLVardef@1c5030_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

In situ chromatin dynamics and HIV-1 nuclear trafficking

The organization and dynamics of chromatin within intact nuclei remain poorly defined, limiting understanding of how nuclear architecture influences macromolecular transport. HIV-1 provides a striking example of a large complex that must traverse this dynamic landscape to access integration sites within transcriptionally active regions. Here, we combine a functional nuclear import system with correlative cryo-electron tomography to visualize chromatin architecture and HIV-1 cores in situ during nuclear penetration. Native nucleosomes resolved at 5.6 [A] reveal four structural classes, from compact conformations at the nuclear periphery to open, flexible forms in the interior, defining a spatially heterogeneous and dynamic chromatin landscape. Within this environment, HIV-1 cores decorated with nuclear factors exclude nearby nucleosomes and preferentially associate with open chromatin. Disruption of CPSF6-capsid interactions abolishes these associations, confining viral cores to peripheral chromatin. These findings establish an in situ framework linking chromatin structure and dynamics to HIV-1 nuclear trafficking and provide a structural basis for viral navigation within the dynamic nuclear landscape.

microbiology↗

bioRxiv2025

Harnessing anti-CRISPR to suppress, subtract, and segregate Cas9 activity for precision CRISPR in Drosophila

Tissue-specific CRISPR (ts-CRISPR) is a powerful approach for studying cell and developmental biology by restricting mutagenesis to specific tissues. However, the precision of this approach is often compromised by non-specific, "leaky" Cas9 activity that confounds phenotypic analysis and destabilizes Cas9/gRNA stocks. To address these limitations in Drosophila, we developed a toolkit based on the Anti-CRISPR (Acr) protein AcrIIA4. We first identified AcrIIA4 as a potent in vivo Cas9 inhibitor with high stability and established the temporal requirements for its function. Based on these findings, we generated three classes of Acr tools. First, a collection of AcrIIA4-bearing balancers robustly suppresses Cas9 and enables the stable maintenance of complex Cas9/gRNA stocks. Importantly, maternal deposition of AcrIIA4 from these balancers provides a means of temporal control, delaying Cas9 activity until metamorphosis. Second, tissue-specific AcrIIA4 transgenes refine leaky Cas9 drivers in a "tissue-subtraction" strategy. Finally, germline-specific and soma-specific Acr tools efficiently segregate Cas9 activity, solving bidirectional leakiness between these two compartments. This comprehensive AcrIIA4 toolkit provides new levels of precision, versatility, and temporal control for Drosophila CRISPR applications.

genetics↗

bioRxiv2025

Unified Probabilistic Analysis of CyTOF: A Deep Generative Approach using CytoOne

Extracting meaningful biological signals from Cytometry by time-of-flight (CyTOF) data remains challenging due to heterogeneity, data characteristics, and the presence of various technical artifacts. Current analysis workflows typically rely on task-specific tools assembled into pipelines, which often make inconsistent distributional assumptions and fail to fully leverage the structure of the data. We present CytoOne, a unified probabilistic framework tailored for CyTOF data that integrates batch correction, differential analysis, and visualization within a single model. CytoOne is built upon a Bayesian hierarchical architecture inspired by Nouveau Variational Autoencoders (NVAE) and employs a novel quasi zero-inflated softplus-normal (QZIPN) likelihood to flexibly model the sparse and noisy nature of CyTOF measurements. We demonstrate via qualitative and quantitative evaluations that CytoOne effectively approximates both marginal and joint distributions of CyTOF data, removes batch-specific artifacts, enables fine-grained differential expression analysis, and facilitates interpretable embeddings for exploratory analysis.

bioinformatics↗

bioRxiv2025

Comparative evaluation of reference genomes and cell-type annotation frameworks for single-nucleus transcriptomic analysis in apple

Single-nucleus RNA sequencing (snRNA-seq) provides a powerful tool to profile cell-type-specific transcriptional programs in woody fruit trees. Yet its application in Malus domestica has been limited by heterogeneous reference genomes and the lack of validated marker genes. Here, we first established a comprehensive set of marker genes corresponding to 29 plant cell types and identified their orthologs in apple. Using these resources, we systematically evaluated six publicly available apple genome assemblies as references for snRNA-seq analysis. Our results demonstrate that reference genome selection has a major impact on mapping performance and downstream cell type annotation, with the GDDH13 assembly consistently producing the highest transcript counts per nucleus and the greatest number of identifiable transcriptional clusters (15 clusters representing 11 distinct cell types across 35,557 high-quality nuclei). To achieve robust cell type annotation, we integrated classical marker gene-based annotation with two cross-species computational frameworks -- Orthologous Marker Gene Groups (OMGs) and XSpeciesSpanner. These three approaches enabled confident identification of 11 major cell types in apple seedlings and provided cross-validation for ambiguous or previously unannotated clusters. Pseudotime analysis reconstructed the developmental trajectories of both procambial and stomatal lineages, revealing their progressive transcriptional transitions and enabling the delineation of key molecular programs underlying vascular differentiation and early guard-cell formation. Together, this work provides a reference-quality single-nucleus transcriptomic atlas for apple and underlined the critical impact of genome choice and multi-method cell-type annotation on single-cell studies in non-model crops.

bioinformatics↗

bioRxiv2025

An Lrs14 family protein functions as a nucleoid-associated protein regulating cell cycle progression in Sulfolobales

Archaea of the order Sulfolobales possess eukaryotic-like cell cycle. The chromatin organization in these archaea is supposed to rely on nucleoid-associated proteins (NAPs). Genomes of Sulfolobales species encode multiple members of the Lrs14 protein family, which have been postulated to function as NAPs. Here, we show that one of the Lrs14 family protein, named Sul14a, is a potential key player in dynamic chromatin organization in Saccharolobus islandicus. Sul14a is transcribed in a cell cycle-dependent manner and appears to be essential for cell viability. The protein exhibits DNA-binding activity preferring AT-rich sequences and can bridge DNA in vitro. ChIP-seq analysis revealed that Sul14a is enriched at AT-rich regions of the chromatin. Overexpression of Sul14a leads to cell cycle arrest at G2 phase. High-throughput chromosome conformation capture (Hi-C) and transcriptomic analyses indicate that Sul14a overexpression results in chromatin contact reduction and global transcriptional changes. In addition, we observed that the chromatin contact pattern changes markedly between 1h and 5h after release from G2 synchronization, corresponding to the lowest and highest levels of sul14a transcription, respectively, during the cell cycle. Our results establish that Sul14a is an NAP intimately associated with chromatin dynamics and global transcriptional changes during the cell cycle in Sulfolobales.

microbiology↗

bioRxiv2025

Ferrapoptosis: Discovery of a Hybrid Programmed Cell Death

Traditional taxonomies classify ferroptosis and apoptosis as distinct forms of regulated cell death. Here, we challenge this view by defining and validating a previously unrecognized neuronal death entity in traumatic brain injury (TBI), which we term "Ferrapoptosis". This hybrid mode of death is characterized by the coexistence of ferroptotic and apoptotic molecular and ultrastructural features within the same neuron-- ultrastructural alterations that cannot be fully characterized by any known cell death modalities. By integrating single-cell transcriptomics with multi-layered functional assays, we show that Ferrapoptosis dominates in severe injury and the acute phase, and is critically driven by mitochondrial oxidative stress. Over time, its predominance is gradually replaced by death modes in which either ferroptosis or apoptosis alone becomes the major pathway. Genome-wide CRISPR screening further identifies Smg7 as a key regulator that synchronously activates both death programs to drive the hybrid phenotype, whereas genetic or viral inhibition of Smg7 reduces Ferrapoptosis and promotes neurological recovery after TBI in mice. Our work systematically delineates a previously unrecognized form of cell death and its pathogenetic mechanisms, providing experimental evidence to refine cell death classification, and suggesting a conceptual strategy for treating complex diseases such as TBI by targeting shared regulatory nodes. HIGHLIGHTSO_LIDefined and validated a previously unrecognized cell death modality, termed "Ferrapoptosis". C_LIO_LIElucidated the ordered pattern of Ferrapoptosis across key stages of TBI pathogenesis. C_LIO_LIRevealed mitochondrial oxidative stress as the pivotal hub integrating ferroptosis and apoptosis. C_LIO_LIIdentify Smg7 as a core driver of Ferrapoptosis and a promising neuroprotective target. C_LI

cell biology↗

bioRxiv2025

Injury-induced tau pathology promotes aggressive behavior in Drosophila without neurodegeneration.

The microtubule-associated protein tau is implicated in neurodegenerative diseases, but its physiological roles remain poorly understood. Here, we find that pan-neuronal expression of human tau (HsTau) in Drosophila coupled with injury triggers hyper-aggression in male flies, which is absent in flies expressing non-phosphorylatable tau. These behavioral manifestations result from activation of dopaminergic circuits without neurodegeneration. Using in vitro reconstitution assays, we find that phosphorylated HsTau maintains microtubule binding but loses its ability to suppress catastrophes, thereby promoting microtubule dynamicity. In contrast, unphosphorylated HsTau and fly tau (DmTau) stabilize microtubules by reducing catastrophe frequency. Our findings challenge the canonical view of tau as a simple microtubule stabilizer and instead position it as a dynamic regulator of microtubule function and neuronal excitability. These results reveal how acute tau phosphorylation can alter neural circuit function and behavior prior to neurodegeneration, providing new insights into taus physiological and pathological roles.

neuroscience↗

bioRxiv2025

Molecular motors orchestrate pause-and-run dynamics to facilitate intracellular transport

Intracellular transport is essential for cellular organization and function. This process is driven by molecular motors that ferry cargo along microtubules, but is characterized by intermittent motility, where cargoes switch between directed runs and prolonged pauses. The fundamental nature of these pauses has remained a mystery, specifically whether they are periods of motor detachment and passive drifting or states of active motor engagement. By combining single-particle tracking with largescale motion analysis, we discovered that pauses are not passive. Instead, they are active, motor-driven states. We uncovered a unifying quantitative law: the diffusivity of a vesicle during a pause scales with the square of its velocity during a run. This parabolic relationship, Deff {propto} v2, holds true for both kinesin and dynein motors, different cargo types, and a variety of cellular perturbations. We show that this coupling arises because the number of engaged motors governs motility in both states. When we reduce motor engagement, either by severing the motor-cargo link or by chemically modifying the microtubule track, vesicles move slower and become trapped in longer, less mobile pauses, failing to reach their destination. Our work redefines transport pauses as an essential, motor-driven part of the delivery process, revealing a fundamental principle that ensures robust cargo transport through the crowded cellular environment.

biophysics↗

bioRxiv2025

Scalable Human Cellular Models of Parkinson Disease Reveal A Druggable Link Between the Angiotensin Receptor 1 and Alpha-Synuclein Pathology

BackgroundParkinsons disease (PD) involves progressive loss of midbrain dopaminergic (mDA) neurons in the substantia nigra. No disease-modifying treatments exist, only symptomatic relief. Our lab reported an unbiased screen in larval zebrafish identifying renin-angiotensin-aldosterone system (RAAS) inhibitors, including clinically used AGTR1 inhibitors for hypertension, as potent neuroprotective agents. This study aims to investigate the effects of AGTR1 inhibition on human mDA neuron survival using inducible neurodegenerative 2D and 3D models for human mDA neuron degeneration. MethodsWe report a scalable high-content platform, using CRISPR-engineered human induced pluripotent stem cell (hiPSC)-derived mDA neurons expressing a tyrosine hydroxylase (TH) fluorescent reporter, allowing to track mDA neuron survival live "in a dish". We developed chemically inducible neurodegenerative 2D and 3D models for human mDA neuron degeneration, allowing to recapitulate PD pathology in human cells in vitro. ResultsOur model establishes scalable human cellular models of PD well-suited for therapeutic discovery. Using 2D and 3D mono and co-cultures, this study demonstrates that inhibition of AGTR1, via chemical or genetic means, protects against chemically induced mDA neuron degeneration. Transcriptomic analyses show AGTR1 inhibition lowers synuclein transcription, by reducing SNCA and SNCB gene expression. In 3D neuron-glia assembloids, AGTR1 inhibition protects against the accumulation of phosphorylated form of -synuclein (p129-Syn), key PD pathological marker. ConclusionsWe highlight AGTR1 as a key regulator of -synuclein transcription and aggregation in human mDA neurons, and AGTR1 inhibition as pro-survival in human iPSC-derived models. These findings position inhibition of AGTR1 as a promising therapeutic strategy for PD neuroprotection.

neuroscience↗

bioRxiv2025

Multimodal AI Decodes Extreme Environment Functional Dark Matter Beyond Homology

Functional annotation of proteins from extreme environments represents a major bottleneck for bioresource discovery, as a vast reservoir of functional dark matter defies existing homology-based methods. We demonstrate that environmental pressures impart conserved physicochemical energy signatures that co-determine protein function with sequence and structure. Here we developed ACCESS, a multimodal graph neural network employing hierarchical contrastive learning with a tailored label-sample co-embedding to fuse energy, sequence, and structural information and overcome homology scarcity. ACCESS surpasses state-of-the-art methods including BLASTp and CLEAN in annotating low-identity enzymes. Applied to extreme environmental metagenomics, we constructed a function map of extremophile enzymes to expand the biocatalyst library, pinpointed functionally critical residues to guide rational design, and enabled large-scale, function-based macro-evolutionary analyses. This paradigm transcends the limitations of homology, illuminating protein dark matter and accelerating the exploration of the biospheres functional diversity for applications in biotechnology and therapeutic development.

bioinformatics↗

bioRxiv2025

MotifAE Reveals Functional Motifs from Protein Language Model: Unsupervised Discovery and Interpretability Analysis

Protein language models (pLMs) learn sequence patterns at evolutionary scale, but these patterns remain inaccessible within these "black box" models. To discover them, we developed MotifAE, an unsupervised framework based on the sparse autoencoder (SAE) architecture that projects pLM embeddings into an interpretable, sparse latent space. MotifAE introduces an additional smoothness loss to encourage coherent feature activation, which markedly improves the identification of known functional motifs compared to the standard SAE. The sequence patterns captured by MotifAE exhibit rich diversity, align with known functional motifs, and are reflected in the models weight space. Beyond short motifs, MotifAE also captures structural domains, with latent feature activation scores correlating with residue importance for different domain functions. By aligning MotifAE features with experimental data, we further identified features associated with domain folding stability. These features enable the prediction of a stability-specific fitness landscape that improves stability prediction and supports the engineering of domains with enhanced stability. Overall, MotifAE provides a general framework for systematic sequence pattern discovery and interpretation, with the potential to advance protein function analysis, mutation effect interpretation, and rational protein engineering.

bioinformatics↗

bioRxiv2025

Data-Driven Symbolic Higher-Order Epistasis Discovery with Kolmogorov-Arnold Networks

Many human diseases are polygenic conditions that arise from a complex interplay of interactions between multiple genes at different loci, but currently most Genome-Wide Association Studies (GWAS) largely only consider the main additive effects of single nucleotide polymorphisms (SNPs), resulting in a missing heritability problem in some complex traits. Identifying non-additive interactions, or epistasis, at a higher-order could aid in filling this gap, but it is computationally difficult due to the massive search space involved. Current epistasis detection approaches struggle with noncartesian higher order interactions and lack inherent explainability. We present a novel deep learning (DL) approach, EPIstasis Discovery with Kolmogorov-Arnold Networks (EPIK), a data-driven, modular, and symbolically representable framework. We also introduce a novel approach for higher-order XOR (a non-Cartesian type) interaction detection, utilized in EPIKs XOR detection module. EPIK slightly outperforms other DL approaches on simulated pure epistasis interactions benchmark in average F1 score. It outperforms other, general, traditional epistasis detection approaches on simulated mixed epistasis detection datasets and real-world GWAS datasets of Arabidopsis Thaliana. Finally, EPIK recovers a known gene interaction between MAPT and WNT3 for Parkinsons Disease (PD) while also suggesting a more complex interaction between MAPT, WNT3, and another gene, KANSL1.

genomics↗

bioRxiv2025

Tuning Mitotic Recombination with Patterned DNA Nicks for Precision Mosaic Analysis

CRISPR/Cas9-based mosaic analysis is a powerful tool for in vivo genetics but is limited by cytotoxicity and mutagenesis associated with DNA double-strand breaks (DSBs). Here, we establish Cas9-derived nickases as safer and more reliable alternatives for inducing mitotic recombination in Drosophila. We demonstrate that single-strand nicks are sufficient to generate mosaic clones and systematically dissect the parameters governing this process. We find that clone frequency can be controlled by the gRNA nicking pattern, with two distant nicks on the same DNA strand synergistically enhancing recombination by over nine-fold compared to a single nick. Based on these findings, we propose a mechanistic model for nick-induced crossover and provide a versatile toolkit for generating tissue-specific nickases. This work establishes nickase-based MAGIC as a superior method for high-fidelity clonal analysis, enabling more precise investigation of gene function in development and disease. SIGNIFICANCE STATEMENTThe CRISPR/Cas9-based mosaic technique, MAGIC, is a versatile tool for in vivo biological investigations. However, its reliance on DNA double-strand breaks (DSBs) can cause significant, unintended cell damage. Here we establish that Cas9-derived nickases, which create gentler single-strand nicks, are a superior alternative. We show that nickases safely induce genetic mosaics in Drosophila by avoiding this cellular toxicity. By systematically dissecting the process, we discovered principles of gRNA design that allow clone frequencies to be tuned for different experimental needs. This work provides a new mechanistic model for nick-induced genetic exchange, a high-fidelity "nickase-MAGIC" method, and a versatile toolkit for precision clonal analysis.

developmental biology↗

bioRxiv2025

Engineering auxin degradation into root-associated bacteria promotes plant growth

Overproduction of indole-3-acetic acid (IAA) by rhizosphere bacteria disrupts plant auxin homeostasis and induces root growth inhibition (RGI). Variovorax reverses this effect by degrading IAA, but the underlying pathway remains incompletely resolved. Here, using genetics, metabolomics, and isotope tracing, we define a nine-gene region (iadCDEFGHIJK2) required for IAA catabolism in Variovorax paradoxus CL014, identify a previously uncharacterized intermediate (CHNO), and revise the early oxidative steps of the pathway. Although IadDE adopts a Rieske-type oxygenase architecture, our data are consistent with the IadCDE complex functioning as a monooxygenase during IAA degradation. Using these insights, we introduce iad genes into two rhizobacterial chassis, Polaromonas MF047 and Paraburkholderia MF376. These engineered strains degrade IAA and alleviate RGI induced by exogenous IAA, an auxin-producing strain, and a synthetic bacterial community. Engineered Paraburkholderia MF376 delivers the strongest performance, improving plant growth in natural soil. Together, these results establish a framework for engineering auxin-balancing root commensals.

microbiology↗