Protein homeostasis is tightly controlled by the coordinated actions of E3 ubiquitin ligases and deubiquitinases (DUBs). We identify Spindlin-4 (SPIN4), a histone H3K4me3 reader, as a substrate regulated by opposing pathways: degradation mediated by the Cullin-RING E3 ligase DCAF16 and stabilization by the DUB BAP1. Through CRISPR-Cas9 knockout screens and biochemical analyses, we demonstrate that DCAF16 promotes SPIN4 degradation, while BAP1 interacts with and stabilizes SPIN4 through its catalytic activity. Inhibition or loss of BAP1 reduces SPIN4 levels, highlighting its critical role in maintaining SPIN4 homeostasis. Proteomics and interactome analyses further support this regulatory axis. These findings reveal a dynamic balance controlling SPIN4 stability, with potential implications for epigenetic regulation and disease processes.

ABSTRACT Functional genomics screens have illuminated genetic dependencies in cancer, but conventional in vitro approaches fail to capture vulnerabilities shaped by the tumor microenvironment. Here, we implement CRISPR-StAR (Stochastic Activation by Recombination), a next-generation inducible CRISPR screening platform for large-scale in vivo applications. The system uses a dual lox-based recombination system to enable guide-level normalization and clonal knockout phenotyping. To analyze the rich (barcode-embedded) sequencing output, we developed UMIBB, a superior Bayesian statistical framework for quantifying gene-level dropout and enrichment compared to conventional software packages. Screening a 30,000-sgRNA library in A549 xenografts, followed by clone representation and dropout correlation analyses, showed high fidelity and reproducibility with dropout phenotypes resolvable using as few as 30 tumors for this size library. Validation across multiple tumor models demonstrated that a single tumor can provide reliable, functional annotation for ∼1,000 genes leveraging intra-tumor library controls for normalization. Comparing in vivo and in vitro screens revealed that a substantial subset of tumor suppressor genes exerts strong phenotypic effects only observable in vivo . For example, single-gene knockout and transcriptomic profiling confirmed that KMT2C and KMT2D have contrasting impacts on tumor growth - an insight that would have been overlooked in standard cell culture. Looking ahead, CRISPR-StAR screening, combined with our user-friendly analysis pipeline available on GitHub (R-package), offer an integrated framework for creating in vivo dependency maps that can complement existing vitro datasets like DepMap and Achilles. Critically, our approach reduces animal use by up to 7-fold compared to conventional in vivo dropout screens. This represents a significant ethical and methodological advancement - achieving genome-scale resolution with far fewer animals and greater reproducibility.

Miniature CRISPR-Cas12f nucleases are attractive candidates for therapeutic genome editing owing to their compact size and compatibility with adeno-associated virus (AAV) delivery. However, editing efficiencies in mammalian cells are lower than those of larger systems such as Cas12a and SpCas9. The extensive phylogenetic diversity of Cas12f suggests unexplored mechanistic variation with the potential for optimization. Here, we characterize a naturally occurring Cas12f ortholog discovered through metagenomics, Cas12f-MG119-28, which supports robust genome editing in human cells. Through structural, biochemical, and kinetic analyses, we compare Cas12f-MG119-28 with two recently described orthologs, Oscillibacter sp. Cas12f (OsCas12f) and Ruminiclostridium herbifermentans Cas12f (RhCas12f). These orthologs present divergent architectures and regulatory features governing PAM recognition, gRNA binding, dimerization, and DNA cleavage. Notably, Cas12f-MG119-28 achieves efficient R-loop formation via a stable dimer interface and a naturally optimized guide RNA. These discoveries elucidate key mechanistic determinants of Cas12f activity and may offer a framework for engineering compact genome editors with therapeutic potential.

Identifying new genes responsible for non-syndromic hearing loss remains a critical goal, as many individuals with hereditary deafness still lack a molecular diagnosis despite comprehensive genetic testing. The tectorial membrane (TM) is a specialized, collagen-rich, acellular matrix of the inner ear, essential for stimulating mechanosensitive hair cell bundles during sound transduction, and its structural integrity is critical for frequency tuning and auditory sensitivity. Although mutations in genes encoding a number of non-collagenous proteins found in the TM (TECTA, CEACAM16, OTOG, OTOGL) have been identified as deafness genes, definitive evidence implicating β-tectorin (TECTB) in human hearing loss has been lacking. Here, we present multiple lines of genetic and experimental evidence linking a missense variant in TECTB (c.674G>A, p.Cys225Tyr) to autosomal dominant, non-syndromic hearing loss in a multigenerational family. The variant alters one of eight highly conserved cysteines present within the zona pellucida (ZP) domain of TECTB and is predicted to disrupt protein folding and matrix assembly. Using a Tectb-C225Y knock-in mouse model, we show that homozygous animals exhibit severe hearing loss and profound disruption of TM morphology, while heterozygote animals display decreased matrix content within the TM and increased susceptibility to noise-induced hearing loss—despite normal auditory thresholds. These findings identify TECTB as a novel human deafness gene, further elucidate its structural role in maintaining TM integrity, and highlight its contribution to resilience against environmental and age-related auditory decline.

Summary A pause in DNA synthesis that occurs when the replisome encounters an obstacle could lead to genome instability. Although important, systematic identification of replication pause sites is challenging due to their low frequency and delocalized nature. Here we present the first single-molecule identification of sites of replisome perturbation across a eukaryotic genome using long-read nanopore sequencing. For each single-molecule replication pause we determine the direction of replication, leading/lagging-strand identity, location and approximate duration, and whether the replisome resumed synthesis. Although pauses are largely diffuse over the genome, they are significantly enriched over transcribed features and correlate with transcription and R-loop levels. Transcription-replication conflicts are more numerous when head-on than co-directional. Finally, we identified genomic loci with a strong bias towards leading over lagging strand pauses, consistent with uncoupling of the helicase from polymerase epsilon. Our data support helicase-polymerase uncoupling resulting from replication pausing as the molecular trigger behind epigenetic switching. Graphical abstract

Background The homozygous ADA2 c.506G>A (p.Arg169Gln; p.R169Q) variant accounts for majority of Deficiency in Adenosine Deaminase 2 (DADA2). This monogenic disorder may be amenable to ex vivo gene therapy by correcting the pathogenic mutation in CD34+ hematopoietic stem and progenitor cells (HSPCs). Objective To apply CRISPR-Cas9 and homology-directed repair (HDR) as a surrogate strategy to model correction of the pathogenic ADA2 c.506G>A variant in healthy cord blood HSPCs. Methods HSPCs were electroporated with optimised CRISPR-Cas9 editing reagents, and editing outcomes, including HDR and on-target deletions, were quantified by ddPCR. Cell functionality was assessed through colony-forming unit (CFU) assays and by xenotransplantation into NOD SCID Gamma (NSG) mice. Two HDR enhancement strategies were tested: (1) genetic inhibitors of p53 and non-homologous end joining (NHEJ) pathways, and (2) pharmacological NHEJ inhibition. Results Small-molecule NHEJ inhibitors increased HDR efficiency approximately two-fold (from ∼40 % to ∼80 %). Edited HSPCs retained normal CFU capacity and successfully engrafted in NSG mice. However, up to 8 % of edited cells exhibited on-target chromosome loss, though this declined over time. Up to 40 % of T cells and fibroblasts demonstrated similar losses under NHEJ inhibitors treatment. In contrast, genetically encoded inhibitors did not improve HDR. Conclusion The ADA2 p. c.506G>A variant can be effectively edited employing surrogate strategy in HSPCs without impairing functionality. Although pharmacological inhibition of NHEJ enhances HDR efficiency, it also increases the risk of on-target chromosome aberrations, highlighting the need for careful consideration of the associated risks and benefits in therapeutic gene editing. Key messages 1) The ADA2 p.R169Q variant can be efficiently corrected via HDR, and the edited CD34+ HSPCs retain their engraftment capability in NSG mice. 2) Pharmacological inhibition of NHEJ using small-molecule inhibitors increases HDR efficiency but is associated with significant on-target deletions and chromosomal arm loss, particularly in differentiated cell types, and in a donor-dependent manner. Capsule summary The ADA2 p.R169Q variant is a viable target for precision gene editing in hematopoietic stem cells. Although inhibition of NHEJ improves HDR efficiency, it concomitantly increases the risk of large on-target deletions, particularly in differentiated cells.

Fusarium wilt, caused by Fusarium oxysporum f. sp. vasinfectum (Fov), is one of the most destructive early-season cotton diseases worldwide. The recent emergence of the highly virulent Fov race 4 (Fov4) and its aggressiveness have raised significant concerns for the U.S. cotton industry. Unlike predominant Fov races in US cotton production, which require root-knot nematodes to cause damage, Fov4 is known to infect cotton independent of nematodes. However, molecular mechanisms of Fov4 virulence in cotton are not clearly understood. Secondary metabolites are often identified as the culprits in pathogen virulence toward plant hosts. To investigate these factors in Fov4, we analyzed the genomes of Fov1 and Fov4 using Fungal antiSMASH and identified a Fov4-specific nonribosomal peptide synthetase (NRPS) gene FNP1 . To investigate its function, we generated FNP1 knock-out mutant using CRISPR-Cas9 approach. Growth assays revealed that the mutants exhibit significantly attenuated hyphal production on media containing cotton roots as the sole carbon source, increased sensitivity to cell stress agents, as well as lagged spore germination. Furthermore, the mutant exhibited defect in cotton root rot virulence and significant decrease in Fusaric acid production. Microscopic observation of GFP-labeled FNP1 deletion mutant showed impeded infection progression in cotton roots compared to the wild type (WT), which further explained the impeded virulence in FNP1 mutant. Gene complementation restored the observed defects, confirming that FNP1 is critical for Fov4 virulence, hyphal development, Fusaric acid production, and stress responses. Highlights Comparative genomic analysis between Fov1 and Fov4 identified FNP1 as a gene specific to Fov4. CRISPR/Cas9 system was employed in Fov4 to generate gene deletion mutants and GFP labeling. FNP1 plays a critical role in Fov4 hyphal development, virulence, fusaric acid production, and stress responses. This study is the first report to identify and functionally characterize a virulence gene in Fusarium oxysporum f. sp. vasinfectum (Fov) race 4 (Fov4) in cotton wilt pathogenesis.

SUMMARY Cas9 nucleases are the effectors of the class 2 type II CRISPR system in bacteria and function to restrict invading DNA. They can also be used with single guide RNAs (sgRNAs) as antimicrobials and genome engineering tools in bacteria, yet applications are hindered by an incomplete understanding of Cas9-target interactions. Here, we generate large-scale SaCas9/sgRNA in vivo bacterial activity datasets and train a machine learning model (crisprHAL) to predict SaCas9 activity. The highest predictive performance was found when downstream sequence flanking the canonical NNGRRN PAM motif at positions [+1] and [+2] was included in model training, correlating with high in vivo activity on sites that included T-rich di-nucleotides in the [+1] and [+2] flanking positions. Strikingly, model predictions and experimentally determined activity in pooled sgRNA experiments in Escherichia coli and Citrobacter rodentium showed an ∼10-fold reduced SaCas9 activity at sites with 5′-NNGGAT[C]-3 ′ PAM [+1] sequences. Cleavage assays using plasmid DNA isolated from E. coli inactivated for DNA adenine methyltransferase (DAM) and SaCas9/sgRNA combinations targeting sites with NNGGAT[C] PAM sequences confirmed that adenine methylation impacts SaCas9 cleavage. Moreover, ablation of a GATC DAM site in a PAM sequence enhanced SaCas9 in vitro activity, whereas creation of a DAM site reduced activity, providing a mechanistic link between adenine methylation and SaCas9 activity. Our results show that a general purpose machine learning architecture can provide biologically relevant insights into SaCas9-PAM interactions that can better inform activity predictions for bacterial applications. Avoidance of adenine methylated PAM sites by SaCas9 may be a mechanism of self versus non-self discrimination or reflect an evolutionary adaptation to counter methylation as an anti-restriction strategy by phage or plasmids.

Conditional sex transformation systems are promising tools in the fight against insect pests. In this study, we developed and tested CRISPR-based, tetracycline-repressible sex transformation strains in the Australian sheep blowfly, Lucilia cuprina . Two CRISPR effector molecules, Cas9 and dCas9, were employed to target the sex-determining gene transformer with the goal of turning female blowflies into males. The Cas9 version of the system induced robust knockout of a visual marker gene but failed to trigger sex transformation without external provision of transformer -targeting sgRNAs. Furthermore, we found that dCas9 expression was linked to several deleterious phenotypes, including developmental delays, reduced body weight, and death. Our study provides the first proof-of-concept conditional CRISPR systems in L. cuprina , and suggests that while dCas9 is toxic at high levels in this species, Cas9 is well-tolerated and may be able to induce sex transformation with minor modifications to the system.

The efficiency and specificity of guide RNAs continue to be crucial obstacles for successful experimental design, despite the fact that CRISPR/Cas9 has transformed genome editing. In this work, we introduce a computational method for optimizing CRISPR/Cas9 guide RNA that combines PAM diversity, local efficiency penalties, and weighted off-target scoring to find high-performing guides across a range of genome compositions. To capture a variety of natural genomic complexity, we simulated five sample genomes: AT-rich, GC-rich, balanced GC content, and high-repeat variations. All twenty-nucleotide target sequences were scanned for each genome, and off-target potential was assessed by permitting up to two mismatches with weighted penalties for seed region sites. To accommodate for any secondary structure impacts, efficiency assessment included both local sliding window penalties and global GC content. Furthermore, we looked at several PAM sequences that were pertinent to various Cas9 variations in order to assess how they affected guide selection. The findings show that efficiency scores vary by genome composition, with the highest scoring guides consistently displaying zero anticipated off-target events. While balanced genomes showed intermediate tendencies, GC-rich genomes tended to choose slightly higher efficiency guides than AT-rich genomes. PAM type affects guide efficiency, according to analysis across several genomes, and the combination of efficiency and off-target score consistently indicates guides with good expected performance. Three-dimensional scatter plots of efficiency and off-target counts versus genomic position, violin plots of off-target distributions, and genome-wide heatmaps emphasizing the best guide positions were used to illustrate these findings. In addition to offering a generalizable computational method for choosing CRISPR/Cas9 guides that optimize specificity and efficiency, our study gives fresh insights into the interactions among genome composition, PAM selection, and guide design criteria. By taking into account weighted off-target penalties, genome complexity, and local efficiency effects, this in silico framework overcomes some of the main drawbacks of earlier simulations. It is also easily applicable to direct selection for experimental research on a variety of organisms. The results provide the groundwork for future advancements in genome editing techniques by establishing a predictive computational framework that can expedite CRISPR/Cas9 research and minimize trial and error in guide selection.

Abstract CDKL5 deficiency disorder (CDD) is a severe X-linked neurodevelopmental condition characterized by early-onset epilepsy, intellectual disability, and motor dysfunction. Here, we present a dual-effector CRISPR-based epigenome editing platform that enables targeted reactivation of the silenced CDKL5 allele. Using a split dCas9 system compatible with AAV9 delivery, we achieved simultaneous transcriptional activation and DNA demethylation of the CDKL5 promoter in both murine and human models of disease. In heterozygous Cdkl5 E6del mice, intracerebroventricular delivery of the editor restored Cdkl5 protein expression, re-engaged downstream signaling, and rescued motor and cognitive deficits. In patient-derived neural stem cells and cortical organoids, CDKL5 reactivation normalized gene expression and restored neuronal network activity. These findings establish a scalable, mutation-independent strategy for treating CDD and highlight the therapeutic potential of epigenetic reprogramming for X-linked disorders.

Abstract While the combination of endocrine therapy (ET) and CDK4/6 inhibitors (CDK4/6i) improves progression-free survival (PFS) in HR+/HER2- metastatic breast cancer, resistance remains a major challenge. BRCA2 pathogenic variants have been linked to reduced PFS, potentially due to co-deletion of the neighboring RB1 gene on chromosome 13q. As RB1 is a key target of CDK4/6, its loss drives resistance. Using CRISPR/Cas9, we generated cell lines with single and combined BRCA2 and RB1 deletions. Loss of RB1 but not BRCA2 increased proliferation and conferred resistance to the CDK4/6i palbociclib and abemaciclib. Dual loss reduced proliferation but increased resistance to CDK4/6i in vitro . However, sensitivity to the PARP inhibitor olaparib was maintained. Finally, analysis of real-world clinical data revealed that RB1 mutations were more frequent in tumors exhibiting homologous recombination deficiency signatures and 13q loss. These genomic features were associated with shorter treatment duration on CDK4/6i plus ET. In conclusion, our findings suggest that RB1 loss, alone or with BRCA2 deletion, contributes to CDK4/6 inhibitor resistance and may help explain reduced efficacy in patients with BRCA2 mutations. Importantly, despite this resistance, sensitivity to PARP inhibition is retained, highlighting a potential therapeutic vulnerability in this molecular context.

Abstract Background: Small cell lung cancer (SCLC) is a highly aggressive malignancy characterized by rapid progression and the frequent emergence of resistance to standard chemotherapeutic agents such as cisplatin (DDP) and etoposide (VP16), resulting in poor clinical outcomes. Methods and Results: To elucidate mechanisms underlying chemoresistance, we conducted a genome-wide CRISPR/Cas9 knockout screen, which identified the histone demethylase KDM6B as a critical mediator of drug resistance in SCLC. Pharmacological inhibition of KDM6B using GSKJ1 markedly enhanced the sensitivity of drug-resistant SCLC cells to DDP and VP16. GSKJ1 treatment significantly suppressed cell proliferation and augmented chemotherapy-induced apoptosis, while exhibiting minimal cytotoxic effects when used as monotherapy. To explore the downstream regulatory pathways, we performed transcriptome analysis via RNA-seq followed by KEGG pathway enrichment analysis, which revealed that GSKJ1 treatment modulates key oncogenic signaling pathways. Integration of ChIP-seq data for H3K27me3 with transcriptomic profiles led to the identification of ERG3 as a potential downstream target. Protein interaction network analysis suggested that c-FOS is co-expressed with both KDM6B and ERG3. Co-immunoprecipitation (Co-IP) and Western blot (WB) assays confirmed the formation of a functional KDM6B/ERG3/c-FOS axis. Mechanistically, this axis regulates chemotherapy resistance by modulating apoptotic and ferroptotic pathways. Conclusion: Finally, in vivo experiments using patient-derived xenograft (PDX) models demonstrated that GSKJ1 effectively enhances the antitumor efficacy of chemotherapy in SCLC, providing compelling evidence for the clinical potential of targeting KDM6B to overcome chemoresistance.

Background Epithelial cells in the renal medulla are continuously exposed to hyperosmolality, hypoxia, and oxidative stress, yet they display remarkable resilience. The transcriptional programs that endow this intrinsic stress tolerance remain incompletely defined. Methods We integrated single-nucleus RNA sequencing of mouse kidneys, computational transcription factor (TF) prioritization, and single-cell CRISPR interference screening (Perturb-seq) in inner medullary collecting duct (IMCD3) cells to systematically identify TFs mediating epithelial stress adaptation. The role of Ilf2 (Interleukin Enhancer Binding Factor 2) was further evaluated in IMCD3 cells by bulk RNA-seq, splicing analysis, functional assays under hyperosmotic stress, as well as in a mouse model of kidney ischemia–reperfusion injury (IRI) and kidney tissues from patients with early and advanced chronic kidney disease (CKD). Results Computational prediction and Perturb-seq identified known and novel TFs, that regulate gene expression programs in kidney medullary tubules. Among them, Ilf2 (also called NF45) emerged as a previously unrecognized regulator: Ilf2 knockdown in IMCD3 cells disrupted transcriptional and splicing programs linked to cell proliferation, cytoskeletal organization, and stress adaptation. Ilf2-deficient cells exhibited reduced proliferation, impaired nuclear integrity, and increased sensitivity to hyperosmotic stress. In mouse kidneys, Ilf2 expression increased during tubular repair after IRI, accompanied by induction of Ilf2-dependent transcripts and splicing events. Human kidneys with advanced CKD displayed elevated expression and cytoplasmic translocation of ILF2, suggesting a conserved stress-adaptive response. Conclusions ILF2 orchestrates both transcriptional and post-transcriptional regulatory mechanisms to sustain kidney epithelial stress resilience. Our findings highlight ILF2 as a potential tubular stress biomarker and therapeutic target for enhancing renoprotection. Key Points Ilf2 has significant and profound impact on gene expression programs in cultured kidney medullary epithelial cells (IMCD3). Ilf2 confers cellular resilience, nuclear integrity, RNA splicing, proliferative capacity, and osmotic resistance in IMCD3 cells. Injured kidneys display increased levels of Ilf2 and up-regulation of genes and splicing events related to Ilf2 function.

Background: Neuroblastoma, the most common extracranial solid tumor in children, exhibits considerable clinical heterogeneity influenced by genetic predisposition. While genome-wide association studies (GWAS) in European populations have identified eight susceptibility loci, the genetic basis of neuroblastoma in East Asian populations remains poorly understood. Methods: We conducted the first GWAS in a Chinese cohort comprising 235 neuroblastoma patients and 3,100 controls, followed by multi-omics analyses of gene expression. The novel risk loci were further validated in an independent East Asian cohort (76 cases/269 controls). Functional characterization of a novel locus was carried out in neuroblastoma cell lines using CRISPR/Cas9-mediated deletion and overexpression assays to evaluate its regulatory effects on candidate genes. Findings: We replicated six of eight known loci including genome-wide significant associations at CASC15 (6p22.3; P = 1.55 E-09) and BARD1 (2q35;P = 3.44E-07), and identified 11 novel risk loci. These novel associations implicate genes involved in DNA repair (MUTYH at 1p34.1), neurodevelopment (BASP1 at 4q13.2 and SLC22A4/SLC22A5 at 5q31.1), and immune regulation (HLA at 6p21 and IDO1/IDO2 at 5q31.1). Multi-omics integration revealed that lead variants modulate gene expression (cis-eQTLs) and DNA methylation (mQTLs) in neural crest-derived tissues and immune cells. Two loci (rs2631372 at 5q31.1: P= 0.045; rs2956095 at 11p13: P= 0.027) showed consistent associations in the replication cohort. Functional studies demonstrated that deletion of the 5q31.1 risk interval reduced expression of SLC22A4, SLC22A5, and LOC553103, while their overexpression promoted neuroblastoma cell proliferation. Interpretation: These findings highlight both shared and population-specific genetic contributions to neuroblastoma susceptibility, underscoring the importance of diversifying GWAS efforts to advance ancestry-informed risk assessment and therapeutic strategies.

Resistance to endocrine therapy (ET) remains a major clinical challenge in the treatment of estrogen receptor–positive (ER⁺) breast cancer, underscoring the need for novel therapeutic targets. To identify genetic drivers of ET resistance, we conducted an in vivo genome-wide CRISPR-Cas9 screen in MCF7 cells implanted into ovariectomized nude mice under estrogen-deprived conditions. NFKB1 emerged as a top candidate whose loss promoted estrogen-independent tumor growth and recurrence. Functional studies confirmed that NFKB1 deficiency enhanced tumorigenicity and conferred resistance to tamoxifen and fulvestrant both in vitro and in vivo. Mechanistically, transcriptomic and biochemical analyses revealed that NFKB1 loss activated canonical NF-κB signaling, leading to inflammatory gene induction and hyperactivation of ER signaling. Importantly, pharmacologic inhibition of NF-κB signaling restored ET sensitivity in NFKB1-deficient cells. Clinically, NFKB1 downregulation was enriched in ER⁺ breast tumors and associated with poor patient outcomes. Collectively, these findings establish NFKB1 as a key suppressor of ET resistance, uncover a mechanistic link between inflammation and ER reactivation, and highlight NF-κB signaling as a therapeutic vulnerability in NFKB1-deficient ER⁺ breast cancer.

ABSTRACT Antimicrobial resistance is a major global health threat, with disproportionate impact in regions with limited diagnostic infrastructure. To address this challenge, we developed BADLOCK (Bacterial and AMR Detection by SHERLOCK), a rapid, low-cost molecular diagnostic platform for direct detection of bacterial pathogens and resistance genes from clinical samples. BADLOCK operates as a one-pot CRISPR-Cas13a reaction capable of detecting nine bacterial species and four major resistance genes directly from positive blood culture. It requires only a heat block and supports both fluorescence and paper-based lateral flow readouts. We validated BADLOCK on a prospectively collected clinical cohort of 194 blood culture specimens, supplemented with 69 mock samples generated from banked isolates enriched for targeted resistance genes. Across all cohorts, we conducted 2,224 individual reactions, achieving 97.6% accuracy (2,171/2,224) at the reaction level. At the assay level, 89.5% (274/306) showed perfect or partial concordance with gold-standard species and resistance gene detection, including 255 assays with perfect concordance and 19 with partial concordance (correct detection of at least one pathogen). This included an evaluation of BADLOCK as a potential culture-free diagnostic for urinary tract infections (UTIs), achieving 98.0% reaction-level accuracy. At the assay level, 90.7% (41/43) were perfectly concordant with gold-standard detection of both species and resistance genes, with 2 additional assays showing partial concordance. To our knowledge, this represents the first demonstration of the CRISPR-Cas13a diagnostic platform on clinical bloodstream infections to date and supports BADLOCK’s potential as a practical and scalable solution for rapid pathogen and resistance gene detection in resource-constrained settings.

Recognition of protospacer adjacent motifs (PAMs) is crucial for target site recognition by CRISPR–Cas systems. In genome editing applications, the requirement for specific PAM sequences at the target locus imposes substantial constraints, driving efforts to search for novel Cas9 orthologs with extended or alternative PAM compatibilities. Here, we present CRISPR-PAMdb, a comprehensive and publicly accessible database compiling Cas9 protein sequences from 3.8 million bacterial and archaeal genomes and PAM profiles from 7.4 million phage and plasmid sequences. Through spacer–protospacer alignment, we inferred consensus PAM preferences for 8,003 unique Cas9 clusters. To extend PAM discovery beyond traditional alignment-based approaches, we developed CICERO, a machine learning model predicting PAM preferences directly from Cas9 protein sequences. Built on the ESM2 protein language model and trained on the CRISPR–PAMdb database, CICERO achieved an average accuracy of 0.68 on test data and 0.75 on experimentally validated Cas9 orthologs. For Cas9 clusters where alignment-based predictions were infeasible, CICERO generated PAM profiles for an additional 50,308 Cas9 proteins, including 17,453 high-confidence predictions with accuracies above 0.86. CRISPR–PAMdb, alongside CICERO models, enables large-scale exploration of PAM diversity across Cas9 proteins, accelerating design of next-generation CRISPR-Cas9 tools for precise genome engineering.

Drug resistance in infectious diseases present a major global health concern, reducing treatment efficacy and increasing morbidity and mortality. Resistance arises from different molecular mechanisms including genetic mutations, enzymatic degradation of drugs, alterations in target sites, efflux pump overexpression, and reduced membrane permeability. These mechanisms contribute to the development of multidrug-resistant and extensively drug-resistant pathogens across bacterial, viral, and mycobacterial species. Scientist have developed therapeutic strategies to control these mechanisms of multidrug-resistant. These include the development of novel antimicrobials such as teixobactin and pretomanid, the application of β-lactamase inhibitors, rational drug combinations, host-directed therapies, and antimicrobial peptides. Advances in biotechnology have enabled precise-targeted approaches, including phage therapy, CRISPR-based antimicrobials, and nanocarrier-mediated drug delivery. Public health interventions are important in reducing the burden of AMR. These include global surveillance systems, antimicrobial stewardship programs, vaccination, infection control protocols, and regulatory policies governing antimicrobial use in humans and animals. Future directions emphasize the integration of precision medicine, artificial intelligence, environmental monitoring, and international governance to strengthen AMR control. A sustained and coordinated global response is essential to preserve the efficacy of current therapies and promote the development of new interventions.

Summary In most legume-rhizobium symbioses, rhizobial colonization occurs through host-derived intracellular infection threads, which enable recruitment of compatible rhizobia while presumably modulating the host immune system to prevent rejection. To investigate how legumes regulate immune responses through post-translational mechanisms during the infection, we focused on Cyclophilin A (CyPA), a peptidyl-prolyl cis/trans isomerase. The model legume Lotus japonicus encodes three canonical CyPA genes. Among them, LjCyPA1 was characterized through CRISPR/Cas9-mediated knockout analysis and shown to be important for normal intracellular infection of compatible rhizobia. A gain-of-function LjCyPA1 variant in a soybean cultivar was able to promote symbiosis with not only compatible but also incompatible rhizobia. Structural modeling followed by genetic analysis demonstrated a functional interaction between LjCyPA1 and the immune hub protein LjRIN4. The cis conformation of LjRIN4 promoted intracellular rhizobial infection, while the trans conformation suppressed it. LjCyPA1 acted with the rhizobial type III secretion system (T3SS) which exhibited a cooperative role between host and symbiont in facilitating infection. Phylogenomic analysis showed that conservation of the CyPA1 orthologue is correlated with the trait of intracellular infection in legumes. Our results contribute to the understanding of how legumes accept symbiotic partners while balancing immune responses.

Trehalose-6-phosphate (Tre6P) is the intermediate in the two-step pathway of trehalose biosynthesis mediated by Tre6P-synthases (TPSs) and Tre6P-phosphatases (TPPs). Plants harbor small families of TPS and TPP genes, however most plant TPSs lack enzymatic activity, suggesting they have regulatory functions. The classical mutant ramosa3 (ra3) increases inflorescence branching in maize, and RA3 encodes a catalytic TPP. We found that RA3 interacts with maize ZmTPS1, a non-catalytic TPS. Mutants in ZmTPS1 and its close paralog ZmTPS12 enhance ra3 phenotypes, suggesting their physical interaction is biologically significant. ZmTPS1 also interacts with the two catalytically active maize TPSs, ZmTPS11 and ZmTPS14, however zmtps11;zmtps14 double mutants fail to complete embryogenesis, suggesting that they are essential, as in arabidopsis. Interestingly, the non-catalytic ZmTPS1 protein stimulated the coupled activity of RA3 and ZmTPS14, suggesting that RA3, ZmTPS1, and ZmTPS14 form a complex, and we confirmed this by expressing and purifying the three proteins and by Alphafold predictions. Our results suggest that non-catalytic TPSs form a complex with catalytic TPSs and TPPs to stimulate catalytic activity and regulate plant development.

The basic helix-loop helix transcription factor Twist plays diverse roles in mesodermal development across bilaterians, but its function in cnidarians remains unclear. Here, we investigate the role of Twist in tentacle morphogenesis and tissue homeostasis in the sea anemone Nematostella vectensis . Using a CRISPR/Cas9 generated knockout, we show that twist mutants exhibit impaired secondary tentacle formation, reduced proliferation in budding tentacles. Cross-sections reveal that mutants also lack micronemes, which are incomplete mesenteries that demarcate tentacle boundaries-suggesting defects in spatial patterning. We demonstrate that twist expression is regulated by Wnt, BMP, and Notch signalling but is independent of MAPK and Hedgehog pathways. Loss of Twist disrupts expression of mesodermal transcription factors paraxis and tbx15 and perturbs the TOR-FGF signalling feedback loop necessary for normal tentacle growth. In addition to the impaired tentacle formation phenotype, juvenile or adult mutants develop epithelial neoplasms at the level of the pharynx, with tentacle-like molecular and morphological profiles, indicating a role for Twist in maintaining tissue homeostasis at the oral pole. Together, our findings reveal that Twist integrates major signalling pathways to regulate secondary tentacle patterning and maintain spatial tissue organisation in the diploblastic Nematostella vectensis .

Gene family expansions are critical for functional diversification, yet paralog contributions to metabolic pathways are often unclear. In Caenorhabditis, the expanded O-acyltransferase (OAC) family, enzymes that transfer acyl groups to hydroxylated substrates, remains poorly characterized despite having been implicated in lipid metabolism. Using CRISPR-Cas9 mutagenesis, behavioral assays, gas chromatographic-mass spectral (GC-MS) analyses, and metabolomics, we systematically analyzed 59 OAC-family protein-coding genes to define their roles in regulating signaling molecules. We found that four adjacent paralogs (oac-13, oac-16, oac-25, and oac-28) on chromosome I are required for synthesizing volatile sex pheromones (VSPs), airborne signals critical for male mate-searching. Specifically, oac-13 and oac-16 are necessary for producing both major pheromone components, while the identical tandem paralogs oac-25 and oac-28 regulate the production of the later-eluting component in gas chromatography. Disruption of these genes reduced production of key pheromone components and impaired male attraction. Metabolomics revealed that oac-16 and other OACs also modulate synthesis and secretion of non-volatile ascaroside pheromones, indicating dual roles in chemical signaling. This work uncovers functional specialization within an expanded gene family, illustrating how redundancy and divergence enable adaptive evolution of communication systems.

Polyglutamine (polyQ) diseases, including Huntington's disease and several spinocerebellar ataxias, are caused by abnormally expanded CAG nucleotide repeats, which encode aggregation-prone polyQ tracts. Substantial prior evidence supports a pathogenic role for polyQ protein misfolding and aggregation, with molecular chaperones showing promise in suppressing disease phenotypes in cellular and animal models. In this study, we developed a FRET-based reporter system that models polyQ aggregation in human cells and used it to perform a high-throughput CRISPR interference screen targeting all known molecular chaperones. This screen identified as a strong suppressor of polyQ aggregation the Hsp40 co-chaperone DNAJC7, which has previously been shown to modify aggregation of other disease proteins (tau and TDP-43) and has mutations causative for amyotrophic lateral sclerosis. We validated this phenotype and further established a physical interaction between DNAJC7 and polyQ-expanded protein. In contrast, DNAJC7 did not modify aggregation of polyglycine (polyG) in a FRET-based model of neuronal intranuclear inclusion disease. In addition to establishing new inducible, scalable cellular models for polyQ and polyG aggregation, this work expands the role of DNAJC7 in regulating folding of disease-associated proteins.

Transcriptional regulation is tightly linked to chromatin organization, with H3K4me3 commonly marking both active and bivalent promoters. In embryonic stem cells (ESC), MLL2 is essential for H3K4me3 deposition at bivalent promoters, which has been proposed to facilitate the induction of major developmental genes during pluripotent cell differentiation. However, prior studies point to a functional discrepancy between the loss of H3K4me3 at bivalent promoters and the largely unaltered transcription of major developmental genes in Mll2 -/- cells. In this study, we investigated MLL2-dependent gene regulation in mouse ESC and during their differentiation. Contrary to the prevailing view, we show that MLL2’s primary role is not to oppose Polycomb-mediated repression at the bivalent promoters of developmental genes. Instead, we identify a previously unrecognized regulatory function for MLL2 at the CG-rich 5’ untranslated regions (5’UTR) of evolutionarily young LINE-1 (L1) transposable elements (TE). We found that MLL2 binds to the 5’UTR of L1 elements and is critical for maintaining their active state (H3K4me3 and H3K27ac), while preventing the accumulation of repressive H3K9me3. Using both global genomic approaches (i.e. RNA-seq, ChIP-seq and Micro-C) as well as targeted L1 deletions, we demonstrate that these MLL2-bound L1 elements act as enhancers, modulating the expression of neighboring genes in ESC and, more prominently, during differentiation. Together, our findings illuminate novel aspects of MLL2 regulatory function during early developmental transitions and highlight the emerging role of TE as key components of long-range gene expression control.