Extrachromosomal arrays are unique chromosome-like structures created from DNA injected into the C. elegans germline. Arrays are easy to create and allow for high expression of multiple transgenes. They are, however, unstable unless integrated into a chromosome. Current methods for integration, such as X-rays and CRISPR, damage DNA and are low-efficiency. Here, we demonstrate that the viral integrase PhiC31, which mediates a non-mutagenic recombination between short attB and attP sequences, can be used for extremely efficient and targeted integration of arrays. In this method, a transgene, a selectable marker, and attP sites are injected into the gonad of a strain that (1) has an attB site in its genome, and (2) expresses PhiC31 in its germline. F1 extrachromosomal arrays are cloned, grown for multiple generations with selection, and then screened for homozygous array integrations. The procedure is simple, requires less time than screening for extrachromosomal arrays, and arrays can be screened for transgene function after stable integration. Arrays that transmit are integrated by PhiC31 with 50-95% efficiency, allowing for the isolation of many unique integrants from a single injection. Arrays can also be integrated at fluorescent landing pads and arbitrary sites in the genome. Using nanopore sequencing, we show that three new integrated arrays are between 1.6 and 18 megabases in length, assemble with large repeats, and can contain hundreds of copies of injected transgenes. We have built a collection of strains and plasmids to enable array integration at multiple sites in the genome using various selections. PhiC1-mediated Integration of Arrays of Transgenes (PhiAT) will allow C. elegans researchers to shift from using unstable extrachromosomal arrays to directly integrating arrays.

Abstract Syphilis, caused by Treponema pallidum , is a sexually transmitted infection that has re-emerged globally over the past decade, posing significant public health challenges. Conventional diagnostic methods are limited by lengthy processing times, operational complexity, and moderate sensitivity, highlighting the urgent need for rapid, sensitive, and user-friendly detection strategies. In this study, we developed a visual detection platform for T. pallidum DNA by integrating recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology. The assay can be completed within one hour, with results directly interpreted via fluorescence readout. It demonstrated a detection limit as low as 11.34 copies/µL and high specificity, accurately distinguishing T. pallidum without cross-reactivity with common blood-borne pathogens, including HIV, HBV, HCV, and DENV. Validation with clinical samples showed complete concordance with standard diagnostic outcomes. To enhance suitability for point-of-care applications, the RPA-CRISPR/Cas12a system was further adapted to a lateral flow assay (LFA) format, achieving a detection sensitivity of 5.56×10² copies/µL while minimizing reliance on specialized instrumentation. Overall, this platform provides a rapid, sensitive, and robust approach for point-of-care syphilis diagnosis and offers a reference framework for detecting other pathogenic organisms.

Activation and proliferation of parietal epithelial cells (PECs), located along the inner rim of Bowman’s capsule, drives disease progression in subtypes of glomerulonephritis and focal segmental glomerulosclerosis. In examining the mechanisms contributing to PEC activation two established mouse models were utilized in this study, nephrotoxic serum nephritis (transient model) and podocyte-specific Klf4 knockout (progressive model). A role for transcription factor FRA2 ( Fosl2 ) was uncovered through single nuclear multiomic approaches relating to the regulation of PEC transcriptional/chromatin dynamics. Co-immunoprecipitation followed by mass spectrometry assessed the FRA2 protein interactome in cultured PECs, revealing a potential role for FRA2 in alternative splicing. Fosl2 expression was then blunted through CRISPR-Cas9 gene editing in cultured PECs, revealing reduced proliferative capacity and the downregulation of myofibroblast markers. In-vivo genetic lineage tracing of PECs after nephrotoxic serum revealed PEC-to-myofibroblast trans-differentiation events. Finally, immunostaining of human kidney biopsies with various subtypes of glomerulonephritis confirmed Fosl2 expression in crescentic lesions of activated PECs, with single cell deconvolution strategies assigning PEC-skewed proportion ratios to bulk RNA-seq patient data from the NEPTUNE consortium. These results suggest that FRA2 ( Fosl2 ) directs a conserved molecular program of PEC-specific responses in subtypes of glomerulonephritis and focal segmental glomerulosclerosis.

CRISPR-mediated gene activation (CRISPRa) is among the most efficient and reliable strategies for mimicking sustained activation of endogenous promoters and their corresponding genes at physiological levels. By leveraging guide-RNA (gRNA) library design, CRISPRa screens can be applied on a whole-genome scale and are compatible with both arrayed and pooled formats, depending on assay requirements. Compared with conventional arrayed CRISPRa libraries that use single or dual gRNAs and often require multiple gRNA candidates per target, a recently developed CRISPRa library (termed T. gonfio) incorporates four tandem gRNAs per lentivector per target, thereby reducing library complexity and representing the smallest arrayed genome-wide CRISPRa library. To streamline genome-wide arrayed CRISPRa screening, this study developed a high-throughput automated workflow using the Biomek i7 Hybrid liquid-handling platform, integrated with multiple peripheral instruments. The workflow comprises three pipelines: lentiviral library transduction, cell library passaging, and assay processing. These pipelines together establish and maintain the transduced cell library for extended screening times. This enables assay processing at desired extended time points and improves the likelihood of identifying phenotypes that require longer time to develop, making the workflow suitable even for rapidly proliferating cell models. In a pilot arrayed screen using a T. gonfio mini-library targeting kinases and phosphatases, activation of the EPHA2 receptor promoter induced a growth reduction phenotype in the HEK293 cell model. This phenotype was recapitulated in a parallel pooled CRISPRa screen using the same mini-library and further validated in a co-culture assay.

Cotton (Gossypium hirsutum) is globally cultivated for its high-quality fiber; yet, its seed, rich in oil and protein, offers untapped potential to support various applications, including food, feed, and industry. With cottonseed oil gaining renewed attention as a valuable co-product, efforts to enhance oil content must contend with longstanding breeding priorities focused on lint yield and fiber quality. A central challenge lies in the complex and often antagonistic genetic relationships between oil accumulation and key agronomic traits. Notably, negative correlations between seed oil content and fiber yield, as well as the pleiotropic nature of several regulatory genes and Quantitative Trait Loci (QTLs), present significant barriers to dual-trait improvement. This review synthesizes current knowledge on the genetic and molecular interplay between cottonseed oil content and other agronomic traits. We examine the architecture of oil-related QTLs and pleiotropic loci, co-expression patterns of shared transcriptional regulators, and metabolic trade-offs influencing carbon allocation between seed and fiber. Recent advances in genomics, transcriptomics, and systems biology are explored as tools to disentangle these trait interactions. We highlight strategies such as multi-trait genomic selection, CRISPR-based uncoupling of antagonistic loci, and the use of wild and exotic germplasm to overcome linkage drag. By providing an integrative overview of the constraints and opportunities at the intersection of oil and agronomic trait improvement, this review lays the groundwork for the development of dual-purpose cotton ideotypes. We propose a conceptual framework for breeding programs to simultaneously enhance fiber yield and oil productivity in a sustainable and climate-resilient manner.

Precise genome editing in Enterobacteriaceae is essential for studying gene function, pathogenesis, and antimicrobial resistance, yet many current systems face host-specific and efficiency limitations. We developed pGGTOX, a modular plasmid platform that enables efficient homologous recombination–mediated genome editing across diverse Enterobacteriaceae , including Escherichia coli , Klebsiella pneumoniae , Salmonella enterica , and Enterobacter intestinihominis . The system integrates a rhamnose-inducible toxin (MqsR) for stringent counterselection, a sfGFP reporter for visual tracking of recombination events, Golden Gate cloning for rapid assembly of homologous arms, an FRT-flanked resistance cassette for marker removal, and an oriT sequence for conjugative transfer. Together with the companion plasmid pCP20-oriT, pGGTOX supports precise, marker-free genomic modification. Using pGGTOX, we achieved targeted deletions of dapA in E. coli and mrkCD in carbapenem-resistant K. pneumoniae , both with 100% efficiency. The dapA mutant exhibited diaminopimelate auxotrophy, while mrkCD deletion markedly reduced biofilm formation, consistent with the loss of function associated with these genes. pGGTOX also enabled deletion of a 43.1-kb type IV secretion gene cluster ( tra ) from an IncN/FII plasmid in E. intestinihominis and insertion of a 10-kb CRISPR–Cas9 plasmid-curing module (pCasCure) into an S. enterica IncX1 plasmid. Deletion of the tra gene cluster resulted in a substantial reduction in plasmid conjugation efficiency. Conjugative transfer of the engineered IncX1-pCasCure plasmid into K. pneumoniae facilitated CRISPR-mediated curing of bla KPC , sensitizing carbapenem resistance to susceptibility. In summary, pGGTOX provides a versatile, efficient, and broadly applicable platform for genome engineering and CRISPR delivery in Enterobacteriaceae , expanding the toolkit for bacterial genetics and translational antimicrobial research. Importance Precise genetic manipulation in Enterobacteriaceae remains a major technical challenge, particularly for non-model or multidrug-resistant strains. We developed pGGTOX, a versatile and broadly applicable plasmid platform that enables efficient, marker-free genome editing through homologous recombination. By integrating stringent counterselection, visual screening, modular cloning, and conjugative transfer, pGGTOX simplifies construction and streamlines editing across multiple clinically relevant species. We demonstrate its utility in deleting chromosomal and plasmid-borne loci, inserting large genetic modules, and delivering CRISPR–Cas9 systems for targeted elimination of antibiotic resistance genes. This platform expands the molecular toolkit for functional genomics and provides a powerful new strategy for dissecting bacterial virulence, resistance, and plasmid biology.

Programmable nucleic-acid therapeutics, including lipid nanoparticle (LNP)–formulated mRNA vaccines, genome editors delivered as mRNA, and viral vectors, are transforming precision medicine but remain constrained by innate reactogenicity despite the introduction of chemical modifications into mRNA. Here, we show that mRNA/LNPs and viral vectors elicit a transient inflammatory burst that peaks ∼6 h after dosing in vivo. Co-administration or prophylaxis with hydroxychloroquine (HCQ), a clinically established 4-aminoquinoline, potently attenuated cytokine and chemokine induction across modalities, including strong responses to unmodified mRNA, without compromising therapeutic efficacy. Humoral and cellular immunity to SARS-CoV-2 Spike mRNA vaccines (BNT162b2 and non-modified mRNA/LNP) were preserved, as was live-virus neutralization. Transcriptomic profiling indicated selective dampening of TLR/cGAS–STING pathways with retention of type-I interferon elements compatible with effective vaccination. HCQ further mitigated AAV9-associated blood–brain barrier (BBB) disruption after stereotactic delivery to the mouse brain and reduced mRNA/LNP-associated hepatotoxicity and thrombocytopenia while maintaining therapeutic transgene expression and CRISPR base-editing in vivo. These findings identify HCQ as an anti-reactogenic adjunct that widens the safety window of nucleic-acid therapeutics without sacrificing performance.

Acute myeloid leukemia (AML) is characterized by differentiation arrest and uncontrolled proliferation. Differentiation therapy aims to treat AML by de-repressing latent myeloid maturation programs to induce cell cycle arrest and subsequent cell death. This approach is curative in the promyelocytic AML subtype, but has met with limited success in other subtypes. Genes such as LSD1 have emerged as intriguing non-APL AML differentiation therapy targets, but results as monoagents in clinical trials have been mixed. Here, we performed differentiation-specific CRISPR screens to identify targets whose inhibition synergizes with LSD1 inhibition to induce terminal differentiation of non-APL AML cells. Intriguingly, the MLL co-factor Menin scored as the top hit. Using cell lines, primary patient samples, and mouse AML models, we find that dual inhibition of LSD1 and Menin is a highly promising approach for differentiation therapy. Mechanistically, we determine that inhibition of Menin downregulates drivers of proliferation and stemness such as MEIS1, and inhibition of LSD1 induces inflammatory and interferon-related pro-myeloid differentiation expression programs. Surprisingly, we find that this combination is effective in selected AML models without mutations in MLL or NPM1, thus nominating dual inhibition of LSD1 and Menin as an attractive therapeutic approach for a mutationally diverse set of non-APL AMLs. Highlights Inhibition of LSD1 and Menin synergizes to induce differentiation of MLL-r and MLL-WT AMLs. Inhibition of Menin downregulates drivers of proliferation and stemness. Inhibition of LSD1 induces differentiation-associated inflammatory and interferon responses. LSD1 and Menin occupy different areas of the genome.

The wide variety of protocols and applications for DNA and RNA sequencing makes flexible tools for read processing an important step in sequence analysis. Beyond trimming and demultiplexing, custom read-level processing is commonly required for data exploration, QC and analysis. Existing tools are often task-specific and don’t generalise to new bioinformatic problems. Thus, there is a need for a tool flexible enough to handle the full variety of read processing tasks, and fast and scalable enough to retain high performance on growing sequencing datasets. We introduce matchbox , a read processor that enables fluent manipulation and analysis of FASTA/FASTQ/SAM/BAM files. With a lightweight scripting language designed around error-tolerant pattern-matching, users can write their own matchbox scripts to tackle a wide variety of bioinformatic problems, and incorporate them into existing pipelines and work-flows. We demonstrate matchbox ’s versatility in a number of contexts: demultiplexing long-read scRNA-seq data with 10X or SPLiT-seq barcodes; restranding RNA-seq reads; assessing CRISPR editing efficiency; and haplotyping macrosatellite repeat regions. matchbox achieves a computational performance comparable to existing tools, while addressing a broader range of bioinformatic needs, representing a new state-of-the-art in sequence processing. matchbox is implemented in Rust and available open-source at https://github.com/jakob-schuster/matchbox .

ABSTRACT Imaging-based CRISPR screens enable high-content functional genomics by capturing phenotypic changes in cells after genetic perturbation. Protein barcodes provide cost-effective, easy-to-implement, and imaging-compatible barcoding for pooled perturbations, yet their scalability has been constrained by the need for arrayed cloning, lentiviral recombination between barcodes and guides, and difficulties in decoding barcodes with high confidence. Here, we introduce poolVis and cellPool, an integrated experimental and computational platform designed to address these limitations. poolVis uses Cre-lox-mediated reconfiguration to position barcode-sgRNA pairs in proximity during viral integration, which greatly reduces barcode shuffling during pooled cloning and delivery. cellPool leverages a scalable computational workflow and the unique aspects of protein barcodes to produce unpooled image galleries from multi-terabyte scale datasets. Applying this platform to single- and double-CRISPRi profiling of cell-cycle genes and chromokinesins in the MCF10A cells uncovered established and previously unrecognized phenotypes, including nuclear morphology changes and reciprocal sign epistasis in DNA damage.

ABSTRACT The global rise of convergent carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-hvKp) represents a major clinical challenge, yet the role of virulence plasmids (pVirs) in shaping bacterial physiology and pathogenicity remains incompletely understood. Using a CRISPR-Cas9–based curing system, we precisely eliminated pVirs from two clinical CR-hvKp strains with distinct genetic backgrounds, ST23-KL1 ( bla NDM-1 ) and ST11-KL64 ( bla KPC-2 ). Loss of pVir conferred fitness advantages in vitro, reduced capsule production and hypermucoviscosity, and promoted biofilm formation, while markedly attenuating virulence in murine sepsis models. Despite this reduction, the pVir-cured ST23-KL1 strain retained higher virulence than the pVir-cured ST11-KL64 strain, underscoring the contribution of chromosomal background to pathogenic potential. Transcriptomic profiling revealed both shared and strain-specific transcriptional responses to pVir deletion, with broader perturbations observed in the ST11-KL64 strain. pVir removal had limited effects on antibiotic MICs. Complementation experiments further demonstrated differential regulatory roles of the rmpADC and rmpA2D2 operons in capsule expression and hypermucoviscosity across the two strains. Together, these findings establish pVirs as central determinants of CR-hvKp virulence and highlight complex host–plasmid interactions that influence bacterial adaptation and pathogenicity. IMPORTANCE The emergence of carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-hvKp) poses a critical threat to global health, yet the contribution of virulence plasmids (pVirs) to bacterial fitness and pathogenicity remains poorly defined. By employing a CRISPR-Cas9–based curing strategy, we dissected the role of pVirs in two genetically distinct CR-hvKp strains and uncovered their multifaceted impact on capsule production, hypermucoviscosity, biofilm formation, and virulence. Our findings reveal that pVir loss confers fitness advantages in vitro while attenuating virulence in vivo, with strain-specific transcriptional responses and differential regulation by rmp operons. These results underscore the complex interplay between plasmid-encoded and chromosomal determinants in shaping CR-hvKp pathogenicity and adaptation, offering mechanistic insights that may inform future therapeutic strategies targeting plasmid-mediated virulence.

Triple pistil (TP) wheat is a historical genetic resource capable of producing up to three grains in a single floret and bolstering the current stagnant grain yield potential. TP phenotype is speculated to be the result of a spontaneous mutation; however, the exact underling genetic mechanism remains elusive, with lack of functional markers for early generation trait selection in hybrid wheat breeding programs. Here, scanning electron microscopy highlighted clear developmental differences between single and triple pistil plants started to arise during 1-2 cm long young spike stages. Using a forward genetics approach, we identified consistent TP-associated mutations in two genes ( TraesCS2D02G490900 and TraesCS2D02G491600 ) exhibiting a nearly complete co-segregation with TP phenotype and developed functional markers for early generation trait selection. CRISPR-Cas9 mediated gene-editing of TraesCS2D02G490900 shifted grain set toward single-grain florets in one edited line in transgenic wheat plants. Furthermore, grain yield evaluation exhibited a significant increase in grains per spike, with no statistically significant reduction in grain weight per spike. Hybrids between common and TP wheat exhibited relatively higher yields, highlighting TP wheat as a significant donor to fortify grain yield potential. This study provides co-dominant functional markers for early generation TP trait selection and valuable targets for hybrid wheat breeding programs.

ABSTRACT Tissues exhibit metabolic heterogeneity that tailors metabolism to their physiological demands. How the conserved pathways of metabolism achieve metabolic heterogeneity is not well understood, particularly in vivo. We established a system in Caenorhabditis elegans to investigate tissue-specific requirements for glucose 6-phosphate isomerase (GPI-1), a conserved glycolytic enzyme that also regulates the pentose phosphate pathway (PPP). Using CRISPR-Cas9 genome editing, we found that gpi-1 knockout animals display germline defects consistent with impaired PPP, and somatic defects consistent with impaired glycolysis. We discovered that two GPI-1 isoforms are differentially expressed and localized: GPI-1A is expressed in most tissues, where it displays cytosolic localization, whereas GPI-1B is primarily expressed in the germline, where it localizes to subcellular foci near the endoplasmic reticulum. GPI-1B expression alone is sufficient to maintain wild type levels of reproductive fitness, but insufficient to reconstitute wild-type glycolytic dynamics. Our findings uncover isoform-specific, spatially-compartmentalized functions of GPI-1 that underpin tissue-specific anabolic and catabolic metabolism in vivo , underscoring roles for subcellular localization in achieving tissue-specific metabolic flux.

Hair cells of the zebrafish lateral line have proven to be a good model for studying hair cell function in a system that is easily genetically manipulated, rapidly develops and is experimentally accessible. However, characterization of potential developmental changes, and possible differences along lateral line position are lacking. Here, we used in vivo patch clamp to investigate the electrophysiological and exocytic properties of neuromast hair cells over early development across body location. Long depolarizations led to steady increases in membrane capacitance, presumably due to exocytosis of vesicles localized to ribbon synapses. The magnitude and kinetics of capacitance changes did not vary significantly across the L1 to L6 position of neuromasts along lateral line, but the magnitudes were found to be significantly smaller in hair cells found in the tail region across all developmental time points. For each region, we found no significant changes in capacitance responses between 3 and 7 days after fertilization. Hair cell capacitance responses were greatly reduced in animals injected with CRISPR/Cas9 with gRNAs targeted to otoferlin b. These results confirm the essential role of otoferlin b in neuromast hair cell function, and they establish the fidelity of CRISPR/Cas9 to rapidly mediate genetic removal of critical genes to study their impact on synaptic release.

Gene editing, especially the CRISPR/Cas9 (Clustered Regulatory Interspaced Short Palindromic Repeats/CRISPR associated protein 9) system, has revolutionized trait development in crops. However, large parts of the world are missing out on applying CRISPR in planta. There is an obvious lack of gene editing applications in locally relevant crops in the Global South which tend to be neglected by mainstream agricultural research and development. Access barriers to these new breeding technologies need to be removed to allow the potential impact of these technologies on food security to happen. Here, we present the ENABLE® Gene Editing in planta toolkit, a minimal molecular toolbox allowing users to create a CRISPR knockout vector for transient or stable plant transformation in two simple cloning steps. We validate the toolkit in rice (Oryza sativa) protoplasts and in Arabidopsis thaliana plants. The ENABLE® kit is designed to be utilized specifically by users in the Global South who are new to CRISPR technology by providing a simple workflow, extensive accompanying protocols as well as options for low cost methods for cloning verification and gene editing verification in planta. We hope that our toolkit helps bridge the gap between the recent biotechnological advancements in plant breeding that high income countries can access and the lack of those technologies in low and middle income countries.

Understanding how individual genetic backgrounds shape the effects of disease-associated mutations is central to elucidating the biology of complex psychiatric disorders. We developed a scalable ‘village editing’ strategy that enables simultaneous genome editing across multiple induced pluripotent stem cell (iPSC) lines, allowing systematic assessment of how polygenic context modulates the impact of specific mutations. Using pooled CRISPR editing in 15 iPSC lines spanning a range of schizophrenia (SCZ) polygenic risk scores, we generated homozygous and heterozygous knockouts in two known SCZ-associated genes: LRP1 , involved in cholesterol import, and NRXN1 , a presynaptic adhesion molecule. By mixing all lines prior to editing and de-multiplexing them afterward, we efficiently produced multi-donor knockout neurons at scale. Transcriptomic profiling revealed that LRP1 and NRXN1 loss produce both shared and donor-specific effects on neuronal gene expression, with variable perturbation of neurotransmitter transport and cholesterol biosynthesis pathways across genetic backgrounds. These results demonstrate that village editing enables systematic dissection of gene-background interactions in human neurons, offering a powerful framework for studying the polygenic architecture of psychiatric disease.

ABSTRACT Leishmania donovani (Ld), the etiological agent of visceral leishmaniasis, poses a significant global health burden due to its complex dixenous lifecycle involving both insect vectors and mammalian hosts. Successful infection in mammals requires the coordinated activity of stage-specific virulence factors. The zinc metalloprotease glycoprotease 63 (GP63) is a well-established virulence determinant critical for host cell attachment and invasion by insect-stage promastigotes. For humans, subsequent parasite propagation depends exclusively on intracellular amastigotes arising from lysed macrophages. Classical GP63 expression and function in Ld amastigotes remain poorly understood, and GP63 null mutants reportedly retain infectivity in mice, raising fundamental questions about virulence factor complementation during mammalian infection. By employing comparative transcriptomics, CRISPR-based mutagenesis, complemented with cell biology, and biochemical assays, this work identifies and characterizes multiple Ld GP63 paralogues with distinct roles in mammalian infection. While both copies of GP63 encoded on chromosome 10 (LdGP63_10.51 and 10.52) were functionally redundant, LdGP63_28 encoded on chromosome 28 proved essential for intracellular amastigote survival by suppressing host cell pyroptosis. Moreover, LdGP63_31 (chromosome 31) was found to primarily mediate promastigote attachment to the host macrophages with minimal contribution from LdGP63_28, facilitating initial infection establishment and amastigote genesis. Importantly, the absence of LdGP63_28 impacted amastigote infection more severely as compared to LdGP63_31. Structural and enzymatic analyses revealed divergent localization and substrate specificities to fulfil functional requirement of these divergent proteases, which have evolved independently to carry out diverse function in establishing infection. Collectively, this study indicates evolutionary divergence and functional specialization among GP63 isoforms in Ld by demonstrating that amastigote-specific and promastigote-specific GP63 isoforms synergistically mediate infection establishment and persistence.

ABSTRACT N 6 -methyladenosine (m 6 A) is the most prevalent internal modification of cellular and viral RNA and is critical to the regulation of its localization, stability, and translation. Previous studies on the role of m 6 A during HIV-1 replication have produced conflicting results. Since m 6 A function can vary dramatically by cell type and state, here we aimed to clarify the role of the m 6 A machinery during HIV-1 replication in primary CD4+ T cells. Using CRISPR-Cas9 we targeted 46 cellular genes implicated in m 6 A or 5-methylcytosine (m 5 C) regulation and measured subsequent HIV-1 replication in primary CD4+ T cells. Only knockout of the m 6 A writer complex auxiliary proteins VIRMA and WTAP, and the m 6 A reader YTHDF2 were validated as significantly decreasing HIV-1 replication. In contrast, knockout of METTL3 or METTL14, which form the catalytic core of the writer complex, resulted in only marginal changes in HIV-1 infection, despite significant decreases in total cellular m 6 A levels. Chemical inhibition of METTL3 led to a dose-dependent decrease in HIV-1 infection, coupled with an increase in protein levels of METTL3 and other writer complex members. Expression of writer proteins was also co-dependent, revealing complex regulatory feedback mechanisms. Overall, these results clarify the role of epitranscriptomic machinery during HIV-1 replication in primary CD4+ T cells and suggest regulation by auxiliary members of the m 6 A writer complex is more influential than the function of the catalytic core itself on HIV-1 infection in primary CD4+ T cells. IMPORTANCE m 6 A is the most common chemical modification on cellular and viral RNA and regulates its stability, localization, and translation. m 6 A modification and its regulation varies dramatically between cell types and cell states. In this study, we investigated the role of m 6 A factors during HIV-1 infection of physiologically relevant primary CD4+ T cells. Using CRISPR-Cas9 to knockout 46 cellular genes implicated in RNA modification, we found only the m 6 A writer complex auxiliary members WTAP and VIRMA, and the reader YTHDF2, significantly affected HIV-1 replication in these cells. In contrast, knockout of METTL3 or METTL14, which form the catalytic core of the writer complex, resulted in marginal changes in HIV-1 infection, despite larger reductions in total cellular m 6 A levels. Our findings suggest regulation by auxiliary members of the m 6 A writer complex is more influential than the function of the catalytic core itself on HIV-1 infection in primary CD4+ T cells.

Summary Phosphorus (P) is a vital macronutrient essential for plant growth, and its deficiency significantly hampers agricultural productivity. The PHOSPHATE 1 (PHO1) protein family, characterized by an N-terminal SPX domain, four transmembrane (4TM) domains, and a C-terminal EXS domain, is pivotal in transporting phosphate (Pi) from roots to shoots. Rice, PHO1;2 plays a crucial role in the Pi export process, and defects in this gene cause severe growth retardation and Pi deficiency symptoms even when external Pi levels are adequate. This study examined the roles of the EXS domain and the combined 4TM+EXS domains of OsPHO1;2 in supporting plant growth responses, independent of Pi transport activity, as well as their influence on hormone signaling and gene regulation. Using CRISPR/Cas9, rice lines expressing specific OsPHO1;2 domains (EXS or 4TM+EXS) were created by targeted deletion of particular domain-coding regions. Phenotypic analysis under Pi-sufficient and deficient conditions, as well as phosphate profiling, revealed that EXS lines exhibited notably better growth than loss-of-function mutants, ospho1;2, during early development, despite having similar shoot Pi levels to the null mutant. These lines showed lower levels of defense hormones (jasmonic acid) than ospho1;2 but were comparable to those in the wild type. Conversely, 4TM+EXS lines exhibited growth patterns similar to ospho1;2 mutants. RNA sequencing indicated that the phosphate starvation response (PSR) and defense pathways were less pronounced in the EXS lines compared to ospho1;2 mutants. However, both EXS and 4TM+EXS lines showed seed development defects and reduced total phosphorus content in seeds, mirroring the ospho1;2 phenotype. Heterozygous plants carrying one functional OsPHO1;2 allele displayed normal growth and seed development, indicating the mutation’s recessive nature. The findings suggest that the EXS domain of OsPHO1;2 can promote plant growth independently of Pi transport by decreasing PSR and modulating defense hormone pathways. This further suggests a signaling role for PHO1 domains beyond direct Pi translocation. Overall, these results enhance our understanding of Pi homeostasis and may help form strategies for breeding P-efficient crops.

Neuronopathic Gaucher disease (nGD) is a lysosomal storage disorder caused by GBA1 mutations, leading to defective acid β-glucosidase (GCase) and accumulation of glycosphingolipid substrates, causing inflammation and neurodegeneration. Patients with nGD manifests severe neurological symptoms, but current animal models fail to fully recapitulate human condition, posing a major barrier to the development of effective therapies targeting the brain. To bridge this gap, we have developed midbrain-like organoids (MLOs) from human induced pluripotent stem cells (hiPSCs) of nGD patients with GBA1 L444P/P415R and GBA1 L444P/RecNcil mutations to model nGD brain pathogenesis. These nGD MLOs exhibited GCase deficiency, resulting in diminished enzymatic function, accumulation of lipid substrates, widespread transcriptomic changes, and impaired dopaminergic neuron differentiation, mirroring nGD pathology. GBA1 mutation correction mediated by CRISPR/Cas9 restored GCase activity, normalized lipid substrate levels, and rescued dopaminergic neuron function, confirming the causal role of GBA1 mutations during early brain development. Using this novel platform, we further evaluated therapeutic strategies, including SapC-DOPS nanovesicles delivering GCase, AAV9-GBA1 gene therapy, and substrate reduction therapy with GZ452, a glucosylceramide synthase inhibitor currently under clinical investigation. These treatments either restored GCase activity, reduced lipid substrate accumulation, improved autophagic and lysosomal abnormalities, or ameliorated dysregulated genes involved in neural development. These patient-specific, 3D neural models offer a transformative, physiologically relevant platform for unravelling disease mechanisms and accelerating the discovery of therapies for patients with nGD.

Prion diseases are transmissible neurodegenerative disorders caused by the spread of misfolded prion protein between cells and individuals, yet the paths by which prions colonize cells are undefined. Here we map the determinants of prion uptake with a genome-wide quadruple-guide CRISPR activation screen. Uptake of prions was measured by flow cytometry in PRNP -ablated human SHSY-5Y cells exposed to synthetic ovine prions. We identified 43 genes modulating prion uptake, 6 of which belonged to the core components of the Bone Morphogenetic Protein (BMP) signaling axis. Prion internalization was increased by overexpression of BMP receptors (BMPR1B, BMPR2, ACVRL1) and the Small Mothers Against Decapentaplegic (SMAD1 and SMAD5) intracellular BMP effectors, whereas the inhibitory SMAD6 reduced it. The internalization of other proteopathic seeds (α-synuclein, tau K18, amyloid-β) and other endocytic probes (transferrin, dextran, E. coli bioparticles) was unaffected. Transcriptional profiles of a panel of persistently prion-infected cell lines showed broad dysregulation of BMP-related signaling. Hence, BMP signaling is a gatekeeper of prion entry mechanistically distinct from bulk endocytosis and amyloid uptake. Chronic prion propagation induced SMAD1/5 phosphorylation, suggesting a positive-feedback loop during prion infection. The connection of prion uptake to a developmentally conserved morphogen pathway with rich pharmacology suggests that BMP signaling nodes may serve as tractable levers to modulate early events in prion transmission.

The Maternal Embryonic Leucine Zipper Kinase (MELK) gene is a part of the Snf1/AMPK of serine/threonine kinase family. MELK has recently attracted considerable interest in the fields of stem cell and cancer biology. Furthermore, MELK is expressed normally during embryogenesis and in proliferative tissues; however, its aberrant overexpression has been observed in various malignancies, including glioma, breast, lung, colorectal, gastric, and hematological cancers. Higher MELK levels are often correlated with unfavorable prognosis, aggressive tumor manifestations, resistance to treatment, and stem-like tumor morphologies. Preclinical studies utilizing RNA interference and small-molecule inhibitors such as OTSSP167 demonstrate that MELK promotes cancer cell proliferation, survival, and metastasis. However, contrasting evidence from CRISPR/Cas9-based knockout studies indicates that MELK may not be essential for tumor growth, raising concerns that the observed anti-tumor effects of MELK inhibitors could partly result from off-target activity. This review aims to summarize the current understanding of MELK biology, including its functions in cell cycle regulation, apoptosis, oncogenic signaling pathways, and tumor stemness. In this review, we discuss the therapeutic potential and limitations of MELK inhibitors, the controversy regarding MELK dependency, and the implications for cancer diagnosis and treatment. MELK may not be a universal driver oncogene; nonetheless, it is consistently linked to aggressive disease, underscoring its potential as a prognostic biomarker and a candidate for therapeutic co-targeting in combination treatments.

Human norovirus (HNoV) is a leading cause of acute gastroenteritis, yet the mechanisms by which it interfaces with host innate immunity remain elusive. Here, we demonstrate that the HNoV NS7 protein, an RNA-dependent RNA polymerase, acts as a direct activator of canonical inflammasomes. Using reconstituted cell systems and human intestinal enteroids (HIEs), we found that NS7 interacts with both NLRP3 and NLRP6, promoting ASC speck formation, caspase-1 cleavage, and secretion of IL-1β and IL-18. HNoV infection of HIEs recapitulated these events, including gasdermin D processing and robust IL-18 release. Importantly, CRISPR/Cas9-mediated NLRP6 deficiency abrogated inflammasome activation and markedly enhanced viral replication, underscoring the essential role of NLRP6 in epithelial antiviral defense. These findings identify NS7 as a novel inflammasome activator and establish NLRP6 as a key determinant of innate immune control of HNoV. Our study highlights inflammasome signaling as a potential therapeutic target for norovirus infection. Author summary In this study, we used human intestinal organoid models to explore how norovirus infection triggers a specific immune response known as inflammasome, which helps protect the gut from viral invaders. We focused on a viral protein called NS7 and discovered that it directly activates two types of inflammasome sensors, NLRP3 and NLRP6. We found that NLRP6, which is abundant in the gut lining, is especially important for detecting norovirus and launching an immune response. When we removed NLRP6 from intestinal cells, the virus was able to replicate more easily, and normal immune activation was lost. Our results reveal that norovirus uses its NS7 protein to interact with the body’s immune machinery in the intestine, and that NLRP6 plays a key role in controlling infection. This work highlights a new way in which the gut senses and responds to norovirus and may help guide future efforts to develop treatments that target these immune pathways.

Eukaryotic flagella, or motile cilia, are iconic molecular machines whose beating drives cell propulsion and fluid transport across diverse organisms. Beat type and waveform are tailored to function, differing between species and cell types, and individual flagella can switch between beat types. Aberrant beating causes ciliopathies and infertility in humans 1 and prevents unicellular parasite transmission 2 . Eight distinct dynein motor protein complexes bind to axonemal doublet microtubules (DMTs) within flagella and drive beating, yet despite extensive structural analysis 3–5 , how this machinery achieves different beat types is unknown. Here, using the flagellate unicellular parasite Leishmania , we show a division of labour where specific dyneins drive specific beat types. Using cryo-EM, we determined the structure of the 96-nm repeat unit of the DMT and identified its dynein composition. We used CRISPR–Cas9 to systematically delete all 96-nm repeat proteins, comprehensively mapping necessity for swimming, and determined the contribution of each dynein to incidence and waveform of the preferred beat types. Outer dynein arms (ODAs) were required for symmetric tip-to-base beats, specific single-headed inner dynein arms (IDAs) were important for asymmetric base-to-tip beats (IDA d ), and double-headed IDA f important for both. This systematic analysis indicates that the prevailing dogma that ODAs drive and IDAs shape the beat 6–9 is either incomplete or not universal, and establishes new hypotheses for how different species, cell types and individual flagella achieve their necessary beat types.

Abstract Background Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine delta-coronavirus (PDCoV), and porcine rotavirus-A (PoRV) G9 are major swine pathogens primarily responsible for gastrointestinal diseases, particularly affecting lactating piglets and resulting in significant economic losses, especially in China. This study introduces a novel CRISPR-based nucleic acid detection method that integrates the high specificity of huLbCas12a with the sensitivity of loop-mediated isothermal amplification (LAMP) technology. Central to this method, the crRNA/Cas12a complex, with a molecular weight of approximately 144 kDa, enhances diagnostic accuracy through targeted gene editing. Incorporating fluorescence report probes and a lateral flow dipstick assay, this approach establishes a visual detection system capable of simultaneously identifying all four viruses. Results It enables the visualization of viral genomes from as low as 1 to 10 copies/µL without cross-reactivity. In comparative testing of 95 clinical samples, our quadruplex LAMP-CRISPR assay demonstrated 100% concordance with RT-qPCR for the three porcine coronaviruses and 98.94% concordance with RT-qPCR for PoRV G9. Conclusions Offering a robust and reliable tool for on-site virus detection, this method significantly aids in the timely prevention of virus spread and mitigates its impact on the pig farming industry, demonstrating its critical role in enhancing biosecurity and disease management in veterinary contexts.