Abstract Salmonella is one of the most prevalent and highly transmissible food-borne pathogens, making rapid and accurate screening essential for safeguarding human health and ensuring food safety. This study introduces a one-tube nested PCR mediated CRISPR-Cas12a for ultrasensitive visual screening of Salmonella spp. using fluorescent lateral flow strip. By leveraging the simultaneous dual-segment amplification capability of the designed one-tube nested PCR and the collateral activated trans -cleavage activity of CRISPR-Cas12a, the method achieves a detection limit of ‌10 1 CFU/mL‌, with no cross-reactivity against other common food-borne pathogens. This approach employs the fluorophore-labeled DNA reporters that are cleaved by activated Cas12a, allowing for rapid and on-site visualization of detection results. Validation in different food matrices yields satisfactory results, demonstrating robustness against matrix interference. Comparative analysis revealed a ‌10-fold sensitivity improvement‌ over traditional single-primer PCR protocols, attributed to the dual amplification efficiency of designed one-tube nested PCR and the collateral activated cleavage specificity of CRISPR-Cas12a. The portability, rapid visual readout, and ultrasensitive performance of the method enable real-time, on-site screening of Salmonella in diverse food supply chains, even in resource-limited settings. Its high specificity, robustness against matrix effects, and minimal equipment requirements make it a transformative, user-friendly tool for enhancing global food safety surveillance and preventing outbreaks.

Phytophthora nicotianae is an oomycete pathogen that severely threatens tobacco production worldwide. Though several dominant resistance (R) genes against P. nicotianae are used in tobacco breeding, they often fail due to rapid emergence of new virulent strains. Instead, targeting plant susceptibility (S) genes offers a promising approach for durable and broad resistance. Evidence from various plant species demonstrates that loss of the S gene DMR6 enhances disease resistance without compromising yield, emphasizing its value for resistance breeding. In this study, we identified and functionally characterized two DMR6 orthologs in tobacco (Nicotianan tobaccum), NtDMR6T and NtDMR6S, which were both induced upon P. nicotianae infection. Targeted mutagenesis of NtDMR6T and NtDMR6S using CRISPR/Cas9 demonstrated that single-gene knockouts conferred enhanced resistance to P. nicotianae, while double mutants exhibited an additive resistance effect. Notably, all mutant lines showed no obvious growth or developmental defects under greenhouse or field conditions. RT-qPCR analysis indicated that NtDMR6s negatively regulate tobacco resistance by modulating multiple defense pathways, including the MAPK signaling cascade. Collectively, our findings demonstrate that NtDMR6T and NtDMR6S act as negative regulators of resistance in allotetraploid tobacco and represent promising S gene targets for the development of P. nicotianae resistant cultivars, thereby providing a new strategy for tobacco disease resistance breeding.

The precise insertion of DNA sequences into plant genomes is a fundamental goal of modern biotechnology. This capability has the potential to accelerate crop improvement and expand the possibilities in synthetic biology. Recent advancements, driven by CRISPR-Cas systems, have initiated an era of programmable DNA integration. This progress has resulted in a diverse set of tools, including enhanced gene targeting (GT), prime editing (PE), and innovative platforms using transposases and recombinases. These tools are no longer just theoretical; they enable a wide range of applications, such as in-locus protein tagging, engineering of cis-regulatory elements, and the targeted integration of multi-gene cassettes for stacking complex traits. However, significant challenges remain. The most notable issues include low efficiency in inserting large DNA fragments, ongoing delivery challenges to elite cultivars, and evolving regulatory frameworks. In this review, we critically synthesize the development of these technologies, starting from early random integration methods to the latest CRISPR-based programmable systems. We evaluate their underlying mechanisms and identify the key technical barriers that currently hinder their routine application. Finally, we discuss emerging solutions, including next-generation editors, tissue-culture-free delivery systems, and strategies for regulatory harmonization, which are paving the way for efficient and predictable DNA insertion necessary for next-generation plant breeding and biotechnology.

ABSTRACT Phosphoinositides (PIs) are a family of seven membrane lipids, each playing distinct roles as signaling molecules. PIKfyve is a phosphoinositide kinase responsible for generating two low-abundance lipids: phosphoinositide-3,5-bisphosphate (PI(3,5)P₂) and phosphoinositide-5-phosphate (PI5P), whose functions remain incompletely understood. Emerging evidence implicates PIKfyve in key cellular processes, including melanosome formation, phagocytosis, endosomal trafficking, lysosomal maintenance, and autophagy. Complete loss of PIKfyve function is embryonically lethal in model organisms. In humans, heterozygous mutations in PIKFYVE are associated with Fleck corneal dystrophy and congenital cataracts. In this study, we investigate the role of PIKfyve in photoreceptors and the adjacent retinal pigment epithelium (RPE). Photoreceptors detect light via the outer segment (OS), a specialized sensory structure composed of stacked membranous discs rich in photopigment. The OS is continuously renewed, and a primary function of the RPE is to phagocytose OS tips. To assess PIKfyve function in the retina and RPE in our zebrafish model, we employed CRISPR/Cas9-mediated gene editing and pharmacological inhibition using the specific PIKfyve inhibitor apilimod. Loss of PIKfyve activity leads to RPE expansion characterized by the accumulation of LC3- and LAMP1-positive vacuoles, along with defects in phagosome degradation and minor changes to melanosome biogenesis. Photoreceptors deprived of PIKfyve function develop a single large vacuole in the inner segment, while the OS remains mostly intact over the timespan analyzed. Electroretinogram (ERG) recordings revealed complete visual impairment in pikfyve crispant larvae, and significantly reduced visual function in larvae treated with apilimod post embryogenesis. These findings highlight the critical role of PIKfyve in the development and homeostasis of the RPE and retina.

Glycolipids constitute an important component of the plasma membrane based on both abundance as well as function. Gangliosides, being a class of structurally diverse and functionally varied glycolipids, can act both as a receptor as well as a ligand and therefore is established as a crucial player in several normal cellular processes. In certain diseases, and in particular cancer, select gangliosides are overexpressed often leading to disease manifestation. GM2-synthase, the enzyme responsible for the formation of a pro-tumorigenic ganglioside, GM2 is well reported to be over-expressed across various cancer tissues and cell lines. This over-expression of GM2-synthase has been linked with increased migration, invasion and epithelial to mesenchymal transition (EMT) as well as induction of a local and systemic host immune suppression in cancer. Despite only a handful of studies demonstrating an epigenetic regulation underlying the transcriptional regulation of GM2-synthase (B4GalNT1) gene, the detailed mechanism still remains unclear. Here we identified the total proteome associated with the GM2-synthase promoter through a modified CRISPR-dCas9 based proteome profiling approach by categorizing all the identified proteins leading to a detailed elucidation of the molecular drivers behind GM2-synthase transcription. While the previous study identified an acetylation-dependent de-repression of the transcription factor SP1 causing GM2-synthase activation, the underlying molecular mechanism driving its activation wasn’t clear. This study demonstrated that the histone acetyl transferase (1), p300 acts as a pivotal factor which on one hand cause acetylation-mediated degradation of SP1, and on the other hand activates SMAD2/4 to have a direct positive impact on GM2 synthase gene transcription. We identified p300 to have an activator role in GM2 synthase gene transcription through knock out, knock down and over-expression experiments. Furthermore, SP1 degradation, SMAD activation and their DNA binding patterns show the reciprocal role of p300 on SP1 and SMAD complexes. Altogether we have identified SMAD 2/4 as an activator complex, p300 as a positive regulator and uncovered a critical p300-SMAD-SP1 regulatory axis in GM2-synthase transcriptional regulation.

ABSTRACT Neuron-specific expression of particular gap junction channel components defines the configuration and functional properties of electrical synapses. However, how a neuron utilises multiple, simultaneously expressed channel proteins - connexins or innexins -to make meaningful connections with distinct synaptic partners remains largely unknown. Using the posterior mechanosensory circuit in C. elegans, we discovered that individual electrical synapses can be formed by clustering together molecularly distinct gap junction channel-types made of three different innexin proteins, INX-1, UNC-7, and UNC-9. In this previously unknown configuration, which we term as heterochannel synapses, molecularly distinct gap junction channel types functionally collaborate to regulate posterior touch sensory behaviour, enhancing functional robustness. We show that the synaptic trafficking of the molecularly different channel types within a heterochannel synapse is independently regulated by discrete and conserved kinesin motor proteins, while distinct molecular pathways involving channel-specific retrograde kinesins regulate their turnover. These independent, channel-specific regulations also make individual synapse-level alterations in the composition of heterochannel synapses possible under altered environmental conditions, providing a novel mechanism for electrical synapse plasticity. Finally, we present evidence of heterochannel electrical synapses in C. elegans locomotory circuits and in the cerebellar Purkinje neurons of zebrafish larvae. Altogether, we demonstrate a novel heterochannel organization of electrical synapses, their regulation, and functional importance, which may be a conserved feature of metazoan nervous system.

A major challenge in human genetics is to identify all distal regulatory elements and determine their effects on target gene expression in a given cell type. To this end, large-scale CRISPR screens have been conducted to perturb thousands of candidate enhancers. Using these data, predictive models have been developed that aim to generalize such findings to predict which enhancers regulate which genes across the genome. However, existing CRISPR methods and large-scale datasets have limitations in power, scale, or selection bias, with the potential to skew our understanding of the properties of distal regulatory elements and confound our ability to evaluate predictive models. Here, we develop a new framework for highly powered, unbiased CRISPR screens, including an optimized experimental method (Direct-Capture Targeted Perturb-seq (DC-TAP-seq)), a random design strategy, and a comprehensive analytical pipeline that accounts for statistical power. We applied this framework to survey 1,425 randomly selected candidate regulatory elements across two human cell lines. Our results reveal fundamental properties of distal regulatory elements in the human genome. Most element-gene regulatory interactions are estimated to have small effect sizes (<10%), which previous experiments were not powered to detect. Most cis -regulatory interactions occur over short genomic distances (<100 kb). A large fraction of the discovered regulatory elements bind CTCF but do not show chromatin marks typical of classical enhancers. Housekeeping genes have similar frequencies of distal regulatory elements compared to other genes, but with 2-fold weaker effect sizes. Comparisons to the predictions of the ENCODE-rE2G model suggest that, while performance is similar across two cell types, new models will be needed to detect elements with weaker effect sizes, regulatory effects of CTCF sites, and enhancers for housekeeping genes. Overall, this study describes the first unbiased, perturbation-based survey of thousands of distal regulatory element-gene connections, and provides a framework for expanding such efforts to build more complete maps of distal regulation in the human genome.

SPAtial Cell Exploration ( SPACE ) is a novel subcellular imaging-based method that couples large-scale perturbations with whole transcriptome single-cell multi-omics readout, while preserving spatial context, providing unprecedented insights into tissue organization and microenvironmental interactions at very low cost. As a demonstration, we performed SPACE in hundreds of spheroids and discovered novel biology related to CAF-tumor interactions using unbiased approaches. This scalable, cost-effective technology has broad applications in translational research and drug discovery, offering a transformative approach to high-throughput spatial perturbation studies.

BACKGROUND Reliable detection of huntingtin (HTT) is essential for understanding Huntington’s disease (HD) biology and evaluating therapeutic strategies. However, high-quality monoclonal antibodies (mAbs) against the HTT C-terminal domain remain limited. OBJECTIVE We sought to generate and validate novel monoclonal antibodies targeting the HTT C-terminal HEAT-containing domain to better detect HTT independently of potential effects of polyglutamine length that can impact some N-terminally targeted antibodies. METHODS We immunized mice with a highly purified, well-characterized recombinant protein corresponding to the HTT C-terminal domain. We generated monoclonal antibody-producing hybridoma cell lines and characterized the antibodies using parental and HTT-knockout cell lines in common immuno-applications. RESULTS Three novel, independent hybridoma lines producing anti-HTT monoclonal antibodies were derived. Using CRISPR-edited HTT knockout cell lines we identified one clone, anti-HTT [2F8], that was specific and effective across Western blot, immunofluorescence, and ELISA assays. All antibodies bound full-length HTT irrespective of HAP40 interaction or polyQ length and showed no cross-reactivity to the N-terminal HEAT domain. CONCLUSIONS These C-terminal HTT mAbs are thus valuable additional tools for studying endogenous HTT function in both normal and disease contexts.

Proteasomes reversibly form foci bodies in a liquid-liquid phase separation (LLPS)-dependent manner upon stress. We previously reported that internalized protein aggregates were targeted by proteasome-dense foci 1 , and proposed that such transient aggregate-associated droplets (TAADs) may facilitate aggregate removal 2 . Here we use quantitative imaging to show that TAADs represent a novel type of gel-like proteasome condensate. TAADs are irregular in shape and slow to disperse, sequestering proteasomes in agreement with our observation of confined diffusion 3 . We demonstrate that TAADs co-localize with cytosolic alpha-synuclein aggregates to facilitate their clearance. Inhibition of proteasome- or ubiquitination activity abolishes this aggregate clearance. We identify RAD23B necessary for TAAD formation, amid other co-localizing chaperones and (co-)proteins of the ubiquitin-proteasomes system. TAAD formation is associated with higher proteasomal substrate turnover whilst retaining overall catalytic efficiency, suggestive of altered degradation mechanisms upon aggregate engagement. Proteomics analysis reveals impact on key mitochondrial-associated processes even after TAAD-aggregate disengagement. Similar TAAD-aggregate co-localizations are found in iPSC-differentiated neurons and in disease-relevant samples, with no detection of compromised proteasome activity. Together, our results indicate a model where TAADs concentrate local proteasome activity, which facilitates aggregate clearance in healthy ageing cells. Potentially, should pathological aggregates persist, TAADs may remain engaged and conceivably sequester proteasomes from physiological activities, thus contributing to neurodegenerative disorders.

Abstract Protein acetylation regulates essential processes across eukaryotes. In trypanosomatids, stage-specific acetylation suggests roles in parasite differentiation. Here, we functionally characterized zinc-dependent lysine deacetylases (DAC1, DAC3, DAC4, and DAC5) in Leishmania mexicana . CRISPR-Cas9-mediated disruption revealed that DAC1 and DAC3 are essential for procyclics, while DAC4 and DAC5 are dispensable. DAC1 and DAC5 are localized in the cytoplasm, and DAC3 and DAC4 in the nucleus. Functional analysis implicates DAC1, DAC3, and DAC5 in procyclic proliferation, whereas DAC1 and DAC5 drive promastigote-to-metacyclic differentiation. DAC5 was required for metacyclogenesis in the sand flies, the promastigote–amastigote transition, and amastigote intracellular replication. Notably, DAC5-null parasites failed to induce lesions in mice, displaying an attenuated phenotype. Proteomic profiling uncovered altered acetylation patterns in DAC mutants, linking DAC5 to cytoskeleton regulation and cell cycle control. These findings identify acetylation as a central regulator of Leishmania stage differentiation and highlight DAC5 as a key factor in parasite virulence.

Abstract Grain length is a critical agronomic trait that directly determines rice yield. In this study, we identified OsvWA36, a von Willebrand factor A (VWA) domain protein containing intrinsically disordered regions (IDRs). We showed that it is a novel positive regulator of grain length and that its function is achieved through liquid-liquid phase separation (LLPS) and subsequent modulation of cell wall remodeling.OsvWA36 was discovered through proteomic screening, and its abundance was positively correlated with grain length. It preferentially accumulated in developing panicles and formed liquid-like condensates via IDR-mediated LLPS, as indicated by in vitro/in vivo assays and fluorescence recovery after photobleaching analysis. CRISPR-Cas9-generated osvwa36 mutants developed shorter grains due to reductions in glume cell length and the aberrant accumulation of lignin. Transcriptomic and qRT-PCR analyses revealed that deficiency in OsvWA36 suppressed the expression of genes associated with cell wall dynamics, including those involved in cellulose synthesis ( OsCESA4, OsCESA7 , and OsCSLE1 ), pectin metabolism ( OsPME68, OsPME1 ), and lignin modification ( OsCAD2, OsMYB58 , and OsExo70H3 ). Genetic complementation restored the wild-type phenotype, whereas overexpression of OsvWA36 further elongated grains. Deletion of the IDR domain abolished LLPS and resulted in short grains, phenocopying the osvwa36 mutants and underscoring the functional necessity of phase separation. Furthermore, haplotype analysis revealed that natural variation in OsvWA36 was correlated with grain length diversity in rice cultivars.In conclusion, our findings indicate that OsvWA36 regulates grain length by orchestrating cell wall remodeling through IDR-mediated LLPS and could be a useful target for molecular breeding strategies aimed at improving yield.

High-fat diet (HFD)-induced obesity remains a significant global health challenge. In this study, we show that a global knock-in CRISPR mouse with the Kir2.1 L222I single-point mutation exhibits remarkable resistance to HFD-induced obesity. We identify palmitic acid (PA), a prevalent long-chain fatty acid in obesity, as a novel negative regulator of Kir2.1. Kir2.1 L222I previously shown to protect against cholesterol-mediated inhibition of Kir2.1, also confers protection against PA-induced suppression. Moreover, PA-induced suppression of Kir2.1 results in a significant loss of flow-induced vasodilation (FIV), while the L222I mutation exerts a protective effect. Notably, Kir2.1 L222I mice display significant protection against HFD-induced weight gain and adiposity independent of caloric intake. Specifically, the mutant mice show increased lean mass and decreased fat mass, specifically in both visceral and subcutaneous white adipose tissue (WAT) and intrascapular brown adipose tissue (BAT). Importantly, visceral-to-subcutaneous white adipose ratios decrease while BAT/WAT tissue ratios increase, suggesting a metabolically favorable fat distribution. This protection correlates with enhanced physical activity and increased energy expenditure. Metabolomic analysis reveals elevated TCA cycle metabolites in adipose tissue of Kir2.1 L222I mice, consistent with their enhanced energy expenditure. These findings highlight Kir2.1 channels as potential therapeutic targets for obesity and related metabolic disorders.

Hypoxia sensing via the Cys/Arg branch of the N-degron pathway (Cys-NDP) is central for flooding responses in plants, yet how evolutionary and ecological factors have shaped the core oxygen sensing mechanism remains poorly understood. Leveraging the publication of multiple angiosperm genomes, we systematically analysed known Cys-NDP components in 55 angiosperms spanning aquatic, epiphytic, xerophytic, and mesophytic lineages. We also complemented this survey with hypoxia profiling and transcriptomic analyses in a selected panel of plants. This comparative effort revealed variation in Cys-NDP components, with Plant Cysteine Oxidases (PCOs) and group VII Ethylene Response Factors (ERFVIIs) emerging as major sources of diversification. Aquatic monocots displayed complete loss of A-type PCOs and dramatic expansion of a novel clade of ERFVIIs (HRE aqua ), frequently accompanied by loss or modification of the Cys-degron, uncoupling them from oxygen-dependent turnover. By contrast, xerophytes and epiphytes retained core Cys-NDP elements but showed shared hypoxia-induced gene expression, suggesting endogenous developmental or metabolic pressures for pathway conservation in habitats with limited flooding risk. Across all species, we identified a conserved transcriptional core of 11 orthogroups, including fermentation enzymes and regulatory factors, highlighting the early recruitment of these genes to hypoxia responses. Functional assays confirmed contributions of conserved MYB and LBD transcription factors to hypoxia tolerance in Arabidopsis . Together, our results demonstrate that both habitat and anatomy influence the evolution and deployment of oxygen-sensing networks in angiosperms. While persistent submergence promoted diversification of ERFVIIs and PCOs, retention of the core pathway across lineages points to fundamental roles in coping with endogenous oxygen gradients and fluctuations.

Background The PIWI-interacting RNA (piRNA) pathway is the primary defense against the deleterious activity of transposable elements (TEs), a role classically assigned to the germline. We recently discovered that the retrotransposon Copia is a negative regulator of synaptogenesis at the Drosophila larval neuromuscular junction (LNMJ) [1]. Here, we investigated whether the piRNA pathway regulates Copia in this somatic context. Methods Analysis of existing sequencing data revealed the expression of piRNA pathway components in somatic tissues [2]. We focused on Aubergine ( aub ), a core PIWI-clade Argonaute. We utilized CRISPR generated aub reporter lines and confocal microscopy to confirm the enrichment of AUB at the LNMJ and next generation sequencing coupled with digital PCR to validate the upregulation of TEs in aub knockdown larvae and adult tissues. Results Data from genetic reporters and antibody staining show that AUB is expressed and localized to the LNMJ. Tissue-specific knockdown of aub at the LNMJ resulted in increased TE expression, including Copia . In contrast to the synaptic overgrowth seen with Copia depletion [1], aub reduction caused a decrease in synapse number and impaired motor function and lifespan. These phenotypes are consistent with the upregulation of Copia , a negative regulator of synapse growth. Conclusions Our findings demonstrate that AUB functions somatically at the LNMJ to repress TEs, thereby ensuring proper neuromuscular development and function. This work establishes a physiological role for the piRNA pathway in a somatic tissue, linking TE repression to neuromuscular development.

CRISPR-based therapeutics rely on guide RNAs (gRNAs) and the Cas9 endonuclease for precise gene editing. Ensuring gRNA purity and base-level sequence integrity is essential for clinical translation. While industry-standard practice relies on liquid chromatography-high-resolution mass spectrometry to assess oligonucleotide identity and purity, more recent FDA guidance recommends complementary base-by-base sequence analysis (FDA CBER Webinar, 2024). In this study, we evaluated next-generation sequencing (NGS) strategies for characterizing chemically synthesized gRNAs. We found that the widely used SMARTer assay, while capable of producing sequenceable libraries, introduced substantial artifacts during library preparation. These included truncated scaffold species at oligo(A) stretches in the scaffold region and 5′(n-1) deletions within the spacer sequence. Although absent in the original gRNA, these artifacts accounted for over 10% of the sequencing reads, creating the false appearance of impurities. Through experimental and computational approaches, we traced these artifacts to mispriming by template-switching oligonucleotides (TSOs). Importantly, these artifacts occur during sequencing, and although they do not reflect real gRNA impurities, they compromise assay accuracy and can obscure true sequence impurities. To overcome these limitations, we developed FUSS-seq (Full-length Uncoupled Second-strand Synthesis followed by sequencing), a novel assay that integrates principles from 5′ RACE with a modified TSO bearing a 3′ polymerase-blocking moiety. FUSS-seq markedly reduced artifacts and increased full-length gRNA recovery, providing a more accurate and lower-bias method for gRNA purity assessment. This approach supports improved Chemistry Manufacturing and Controls (CMC) characterization of gRNAs and strengthens the analytical toolkit needed for reliable CRISPR-based therapeutic development.

Predicting cellular responses to genetic perturbations is critical for advancing our understanding of gene regulation. While single-cell CRISPR perturbation assays such as Perturb-seq provide direct measurements of gene function, the scale of these experiments is limited by cost and feasibility. This motivates the development of computational approaches that can accurately infer responses to unmeasured perturbations from related experimental data. We introduce dbDiffusion, a generative framework that integrates diffusion models with classifier-free guidance derived from perturbation information, operating in latent space through a variational autoencoder (VAE). Diffusion models are probabilistic generative models that approximate data distributions by reversing a Markovian diffusion process, progressively denoising Gaussian noise into structured outputs. By exploiting biological similarities in gene expression profiles and relationships among perturbations, dbDiffusion enables the conditional generation of gene expressions for previously unobserved perturbations. In contrast to competing approaches, dbDiffusion does not rely on LLM or foundation models, which have been found to yield unsatisfactory results. Rather, it leverages embeddings derived from measured perturbations to generalize to unseen pertur-bations, effectively transferring information across related experimental conditions. In benchmarking against state-of-the-art methods on Perturb-seq datasets, dbDiffusion demonstrates superior accuracy in predicting perturbation responses. A methodological innovation of dbDiffusion is the integration of prediction-powered inference, which corrects for biases inherent in generative models and enables statistically rigorous downstream tasks, including identification of differentially expressed genes. By combining deep generative modeling with principled inference, dbDiffusion establishes a scalable computational framework for predicting and analyzing transcriptomic perturbation responses, significantly extending the utility of Perturb-seq experiments.

The gut microbiota has emerged as a critical immune-metabolic interface, orchestrating a complex network of interactions that extend well beyond digestion. This highly diverse community of bacteria, viruses, archaea, and eukaryotic microbes modulates host immunometabolism, metabolic reprogramming, and systemic inflammatory responses, thereby shaping human health and disease trajectories. Dysbiosis, or disruption of microbial homeostasis, has been implicated in inflammatory bowel disease, cardiometabolic disorders, neurodegeneration, dermatological conditions, and tumorigenesis. Through the biosynthesis of short-chain fatty acids (SCFAs), bile acid derivatives, tryptophan metabolites, and microbial-derived indoles, the gut microbiota regulates epigenetic programming, barrier integrity, and host–microbe cross-talk, thereby influencing disease onset and progression. In oncology, specific microbial taxa and oncomicrobiotics (cancer-modulating microbes) are increasingly recognized as key determinants of immune checkpoint inhibitor (ICI) responsiveness, chemotherapeutic efficacy, and resistance mechanisms. Microbiota-targeted strategies such as fecal microbiota transplantation (FMT), precision probiotics, prebiotics, synbiotics, and engineered microbial consortia are being explored to recalibrate microbial networks and enhance therapeutic outcomes. At the systems level, the integration of multi-omics platforms (metagenomics, metabolomics, transcriptomics, and proteomics) combined with network analysis and machine learning-based predictive modeling is advancing personalized medicine by linking microbial signatures to clinical phenotypes. Despite remarkable progress, challenges remain, including the standardization of microbiome therapeutics, longitudinal monitoring of host–microbe interactions, and the establishment of robust ethical and regulatory frameworks for clinical translation. Future directions should prioritize understanding the causal mechanisms of microbial metabolites in immunometabolic regulation, exploring microbial niche engineering, and developing precision microbiome editing technologies (CRISPR, synthetic biology).

ABSTRACT Background Duchenne muscular dystrophy (DMD) is a lethal pediatric degenerative muscle disease for which there is no cure. Robust preclinical models that recapitulate major clinical features of DMD are required to investigate efficacy of potential DMD therapeutics. Rat models of DMD have emerged as promising small animal models to accomplish this; however, there have been no comprehensive studies investigating the functional skeletal muscle decrements associated with the modeling of DMD in rats. Methods CRISPR/Cas9 gene editing was used to generate a dystrophin-deficient Sprague-Dawley muscular dystrophy rat (MDR). Biochemical and immunofluorescent analyses were performed to confirm loss of dystrophin in striated muscles of this rat model. In situ and ex vivo muscle function was assessed in wild-type (WT) and MDR muscles at 3, 6, and 12 months of age, followed by histopathological analyses. Results MDR muscle tissues exhibited loss of full-length dystrophin and reduced content of other dystrophin glycoprotein complex members. MDR extensor digitorum longus (EDL) muscles and diaphragms displayed pronounced and progressive muscle weakness beginning at 3 months of age, compared to WT littermates. EDLs also exhibit susceptibility to eccentric contraction-induced damage. Functional deficits in soleus muscles were less severe and were associated with a right shift in force-frequency relationship and a muscle fiber-type shift. MDR muscles display progressive histopathology including degenerative lesions, fibrosis, regenerative foci, and modest adipose deposition. Conclusions MDR is a preclinical model of DMD that exhibits many translational features of the human disease, including a large dynamic range of muscle decrements, that has high utility for the evaluation of potential therapeutics for DMD.

The large size of CRISPR-Cas enzymes limits their delivery for therapeutic applications. Cas12j nucleases offers hypercompact alternative but show moderate editing efficiency. To overcome this limitation, we identified eight novel Cas12j orthologues (Cas12j-11 to Cas12j-18) from viral metagenomes. All showed low editing activity in mammalian cells. We engineered T5 exonuclease-Cas12j fusions (T5Exo-Cas12j), two of which, T5Exo-Cas12j-12, and -18 exhibited up to 42% editing in HEK293T and 9% in K-562 cells, outperforming wild-type Cas12j counterparts and comparable to LbCas12a. Intriguingly, robust in cellula editing in both HEK293T and K-562 cells was strictly dependent on the presence of 5′-TAC trinucleotides within the target DNA sequence. Furthermore, we fused the Cas12j orthologues with the TadA8e deaminase and developed base editors, termed Be-(d)Cas12j. Among these, Be-(d)Cas12j-13 demonstrated efficient A-to-G base conversion in mammalian cells. This study expands the CRISPR toolbox by characterizing and engineering novel Cas12j orthologues into compact, high-efficiency genome editors.

Summary Diatoms are a highly diverse group of phytoplankton that have a large impact on global primary production and carbon sequestration in the ocean 1,2 . However, they are evolutionarily divergent from model phototrophs of the green lineage, and limited screening tools have hampered discovery of unique diatom biology. To address this challenge, we developed a genome-wide CRISPR/Cas9 screen in the model marine diatom, Phaeodactylum tricornutum. The screen was applied to identify genes required for survival in different light regimes, including both high light and fluctuating light. We identified a broad set of uncharacterized genes, providing the foundation for mechanistic studies of diatom adaptation to dynamic light. Among these genes, we demonstrated that the red lineage-exclusive gene STROBE1 is a new potentiator of cyclic electron flow (CEF) required for CEF to generate a trans-thylakoid proton gradient 3,4 . As dynamic light conditions are common in marine environments, STROBE1 and other genes identified in this screen may contribute to the broad ecological success of diatoms 5,6 . This genome-wide genetic screen in P. tricornutum will accelerate the unbiased discovery of novel gene functions in these ecologically important organisms.

ABSTRACT Eukaryotic genomes generate a plethora of polyadenylated (pA + ) RNAs 1,2 , that are packaged into ribonucleoprotein particles (RNPs). To ensure faithful gene expression, functional pA + RNPs, including protein-coding RNPs, are exported to the cytoplasm, while transcripts within non-functional pA + RNPs are degraded in the nucleus 1–4 . How cells distinguish these opposing fates remains unknown. The DExD-box ATPase UAP56/DDX39B is a central component of functional pA + RNPs, promoting their docking to the nuclear pore complex (NPC)-anchored ‘transcription and export complex 2 (TREX-2)’ (ref. 5,6 ), which triggers transcript release from UAP56 to facilitate export (ref. 7,8 ). Here, we uncover that the ‘Poly(A) tail exosome targeting (PAXT)’ connection 9 harbors its own TREX-2-like module, which releases pA + RNAs from UAP56 for decay by the nuclear exosome. The core of this module consists of a LENG8-PCID2-SEM1 (LENG8-PS) trimer, which we show is structurally and functionally equivalent to the central GANP-PCID2-SEM1 (GANP-PS) trimer of TREX-2. Mutagenesis and transcriptomic data demonstrate that the nuclear fate of pA + RNPs is governed by the contending actions of nucleoplasmic PAXT and NPC-associated TREX-2, which interpret RNA-bound UAP56 as a signal for RNA decay or export, respectively. As RNA targets of PAXT are generally short and intron-poor, we propose an overall model for pA + RNP fate determination, whereby the distinct sub-nuclear localizations of PAXT and TREX-2 govern the degradation of short non-functional pA + RNAs while allowing export of their longer and functional counterparts.

Abstract Background: Safety and specificity remain major challenges in viral gene therapy for cancer and tissue repair. The Crucible Virus integrates replication deficient lentivirus, herpes simplex virus type one, and influenza A vectors into a single insulated chassis that enforces multi-input promoter gating and multi-tier kill switches to achieve conditional activation and enhanced safety. Methods: Vectors incorporate heat and cytokine responsive promoters HSP70 and NF kappa B, cHS4 insulators, and unique DNA barcodes. In vitro assays quantified promoter induction, basal leak, and engagement of CRISPR Cas nine and protease cleavable degron kill switches. Seven orthotopic and xenograft tumor models received sequential or combined dosing via intravenous and intralesional routes with assessments of biodistribution, immunogenicity, survival, and tumor volume. Results: The HSP70 and NF kappa B promoters achieved up to 168-fold induction with less than 0.2 percent basal leak. Off target CRISPR excision removed more than 90 percent of the payload within ninety minutes, and degron clearance reduced secreted effectors by half in under one hour. Median survival improved by thirty to sixty percent with probability value less than 0.01, and tumor volumes shrank by forty to fifty two percent compared to controls. Vector genomes cleared to below five thousand copies per microgram DNA by day twenty-eight, with off target activation under 0.5 percent and manageable antibody titers. Conclusions: The Crucible Virus delivers robust antitumor efficacy and regenerative potential under rigorous safety controls. Its modular traceable design supports scalable manufacturing and an investigational new drug ready profile for precision oncology and regenerative medicine applications.

The recent development of long-read sequencing has made it possible to catalog variable number tandem repeats (VNTRs) in the human genome. However, little is known about their functional consequences. Here, we characterized the effect of TRACT, a VNTR that is unique to humans and that has sequence variants linked to risk for bipolar disorder and schizophrenia. By adding or removing this VNTR in both mouse models and human neural organoids, we find that TRACT, which is intronic to the L-type voltage-gated calcium channel gene CACNA1C , increases intracellular calcium after neuronal stimulation and leads to widespread changes in activity-dependent transcription programs in neurons. TRACT-dependent changes are enriched for genes associated with synapse formation and plasticity, and partially recapitulate evolutionary changes in activity-dependent transcription between species. These findings demonstrate that a single, human-specific, non-coding element can strongly affect the neuronal response to stimulation, and motivate the study of VNTRs as a genetic source of phenotypic variation in both evolution and disease.

The human aryl hydrocarbon receptor (AHR) integrates chemical signals derived from the environment, gut microbes, and endogenous sources to regulate processes ranging from intestinal barrier integrity to xenobiotic detoxification. Despite strong evidence that dysregulation of AHR signaling is a causal factor in metabolic and autoimmune disorders, we currently lack a comprehensive understanding of the factors that regulate AHR activity in human cells. Here, we use genome-scale CRISPR screening to systematically identify regulators of AHR signaling in hepatocytes. The resulting datasets recapitulate the core AHR signaling pathway and identify a large network of regulators. Many of these factors have roles beyond AHR signaling, reflecting that AHR signaling is deeply integrated into human cell biology. We further dissect this network to reveal novel modes of regulation of AHR expression, protein levels, and signaling. For example, we find that the E3 ubiquitin ligase UBR5 sustains AHR signaling by counteracting degradation of ligand-bound AHR. Finally, we identify components of the AHR regulatory network that are specific to cell types and ligands as potential nodes to manipulate AHR signaling in a targeted manner for therapeutic benefit. Overall, our results define the regulatory network that underpins AHR activation, with implications for our understanding of host-microbe interactions and integrative chemosensation and the etiology of metabolic and inflammatory disorders.