Abstract Aspergillus fumigatus ( AF ) is the predominant pathogen implicated in invasive aspergillosis (IA) in humans; therefore, prompt and accurate detection is critical for the effective prevention and management of IA. This study developed a rapid detection system targeting the AF -specific anxC4 gene by integrating enzymatic recombinase amplification (ERA) with CRISPR/Cas12a. The reaction proceeds at a stable temperature of 37°C, with amplification and detection systems separately positioned in the tube lid and bottom, respectively, effectively minimizing aerosol contamination typically associated with product transfers. To enhance sensitivity, the One Pot method was optimized. Consequently, the fluorescence detection limit reached 1 fg/µL, and the sensitivity of the test strip reached 10 fg/µL, with no cross-reactivity observed against other fungi. Detection of AF was completed within 60 min, and results were visually displayed through fluorescence signals and nucleic acid test strips. Clinical practicality was further evaluated using aspergillosis samples, which demonstrated satisfactory performance. Pure culture results confirmed that out of 62 sputum samples, 32 were positive and 30 negative. Evaluation of 62 clinical samples using the One Pot ERA-CRISPR/Cas12a system demonstrated sensitivity and specificity rates of 93.75% and 93.33%, respectively, via fluorescence detection, and 90.63% sensitivity and 96.67% specificity using lateral flow strips.

G protein-coupled receptors (GPCRs), the largest family of transmembrane proteins, transduce extracellular stimuli into intracellular signaling cascades to orchestrate human physiology. The transport of newly synthesized receptors from the endoplasmic reticulum (ER) to the plasma membrane (PM) determines cellular responsiveness to incoming ligands, yet the molecular machinery governing GPCR export remains incompletely defined. Here, we combine a synchronized cargo-release assay with a genome-wide CRISPR/Cas9 screen to systematically map regulators of GPCR ER-to-PM transport. Focusing on the δ-opioid receptor (DOR), a prototypical class A GPCR, we identify CNIH1 as a dedicated export factor. In the absence of CNIH1, DOR is retained intracellularly with immature glycosylation, and drives reduced PM signaling. CNIH1 localizes to both ER exit sites and the Golgi, promoting the anterograde transport of a subset of class A GPCRs. Opioid receptors directly interact with CNIH1 and require its putative COPII-binding site for export. Distinct from other human cornichon homologs, CNIH1 defines a selective GPCR-sorting receptor that couples GPCR biosynthesis to signaling competence.

ABSTRACT One of the seminal discoveries from genetic studies of autism spectrum disorder and related neurodevelopmental disorders (NDD) has been that loss-of-function (LoF) mutations in many genes that impact chromatin and transcriptional regulation confer substantial liability to NDD. Haploinsufficiency of the epigenetic regulator POGZ represents one of the strongest such associations; however, little is known about the direct or indirect regulatory targets of POGZ , or the mechanisms by which loss of this chromatin modifier alters early neuronal development and synaptic functions. Here, we created an allelic series of CRISPR-engineered human induced pluripotent stem cell (hiPSC) clones harboring mono- and biallelic POGZ deletions. In hiPSC-derived neural stem cells (NSC) and Neurogenin 2-induced neurons (iN), POGZ LoF altered the expression of genes associated with critical cellular processes and neuronal functions, including synaptic and intracellular signaling and extracellular matrix organization. Our multiomics profiling also showed altered footprinting of critical transcription factors (e.g., activator protein 1 complexes) that were enriched at promoters of differentially expressed genes associated with synaptic function. To further interrogate the shared molecular changes in neuronal development associated with NDD and POGZ regulation, we compared our results to deletions of the transcription factor MEF2C and the sodium channel gene SCN2A that we generated in these same isogenic iN. These analyses revealed strong enrichment of extracellular matrix and intracellular signaling disruption associated with POGZ and MEF2C deletion, whereas POGZ and SCN2A haploinsufficiency exhibited shared transcriptional effects on gene modules enriched for NDD-associated genes with opposing regulatory effects. Notably, we also observed alterations to synaptic firing rate and neurite extension with biallelic deletions, but not heterozygous lines, suggesting subtle effects in neuronal development associated with haploinsufficiency. Overall, these shared molecular consequences suggest key points of convergence that connect epigenetic regulation to neuronal function in the etiology of neurodevelopmental pathologies.

Spalt-like (SALL) proteins are C2H2 zinc-finger transcription factors important for embryogenesis, with mutations in SALL1 and SALL4 causing rare congenital disorders Townes-Brocks and Okihiro syndromes, respectively. While SALL proteins are known to associate with one another, the biological significance of the resulting complexes is unknown. Here we define a conserved glutamine-rich region that mediates SALL1/4 homo- and heterotetramerisation and find that complex formation is indispensable for DNA binding. Modelling a patient mutation that abolishes SALL4 multimerisation led to gene misregulation and, in mice, embryonic lethality, therefore phenocopying a complete Sall4 knockout. Furthermore, a common disease-causing SALL1 truncation, which retains multimerisation but lacks DNA-binding domains, sequesters SALL4 into heterotetramers that are defective in DNA binding, thereby providing a mechanistic explanation for the dominant-negative effects of many Townes-Brocks mutations. Together, our findings establish tetramerisation as a prerequisite for SALL function, linking complex formation to developmental gene regulation and human disease.

Abstract: Circadian clocks regulate daily rhythms in all eukaryotes through ∼24-hour transcriptional-translational feedback loops driven by clock proteins. However, the molecular mechanisms that set the 24-hour period remain poorly defined. Here, using single-molecule imaging and nascent RNA sequencing, we uncover an unexpected RNA-based molecular timer: a single intron in the Drosophila timeless ( tim ) gene that regulates circadian period length by controlling mRNA localization. Strikingly, we find that ∼50% of tim mRNAs are localized to the nucleus due to inefficient post-transcriptional splicing of a single intron (which we named intron P ), in contrast to other core clock transcripts that localize predominantly to the cytoplasm. CRISPR-mediated removal of intron P abolishes nuclear retention of tim transcripts, leading to accelerated TIM protein accumulation and a shortened ∼22-hour period with reduced rhythmic robustness. Remarkably, insertion of intron P alone into heterologous reporters is sufficient to promote nuclear retention in both Drosophila and human cells, acting as a conserved checkpoint that withholds transcripts in the nucleus until splicing is complete. Finally, we identify three RNA-binding proteins, two repressors (Hrb27C and Squid) and one activator (Qkr58E-2, a Sam68 homolog), that modulate intron P splicing in a rheostat-like manner. Together, these findings establish tim intron P as the first intron-based molecular timer in circadian clocks and reveal splicing kinetics as a critical regulatory layer in temporal gene expression programs, with broad implications for other processes such as development and immunity.

Simian immunodeficiency viruses (SIVs) have crossed from apes to humans at least four times, but only one event gave rise to the AIDS pandemic. The host barriers that pandemic HIV-1 group M ( major ) strains overcame to spread efficiently in humans remain poorly understood. To identify such barriers, we performed CRISPR-Cas9 screens driven by the replication efficiency of SIVcpz, the chimpanzee precursor of HIV-1. Guide RNA libraries targeting more than 500 human genes encoding potential antiviral factors were inserted into the replication-competent SIVcpz MB897 molecular clone, which is phylogenetically closely related to HIV-1 group M strains. Propagation in Cas9-expressing human SupT1 T cells significantly enriched for sgRNAs targeting ADAR, AXIN1, CEACAM3, CD72, EHMT2, GRN, HMOX1, HMGA1, ICAM2, CD72, IFITM2, MEFV, PCED1B, SGOL2, SMARCA4, SUMO1 and TMEM173 . These hits only partially overlapped with those identified in analogous HIV-1–based screens, indicating virus-specific restriction profiles. Functional analyses confirmed that IFITM2 (interferon-induced transmembrane protein 2), PCED1B (PC-esterase domain–containing protein 1B), MEFV (Mediterranean fever protein, pyrin/TRIM20), and AXIN1 (Axis inhibition protein 1), restrict replication of SIVcpz but not of HIV-1 group M strains in primary human CD4⁺ T cells. These findings reveal previously unrecognized host factors that limit SIVcpz replication in human cells and highlight barriers that HIV-1 likely overcame during its adaptation for pandemic spread. One Sentence Summary CRISPR screens with replication-competent SIVcpz identify human antiviral factors limiting efficient viral replication after zoonotic transmission.

Abstract Over the past two decades, considerable progress has been made in cataloguing genes and active chromatin elements in humans. However, despite these efforts, less than a quarter of genes have been assigned a function in the context of human disease, which limits our ability to interpret clinical genome sequencing results. In the field of inherited retinal diseases, 30–40% of cases remain genetically undiagnosed. This may be partially due to our limited understanding of the function of most retina-expressed genes, which prevents the correct interpretation of their sequence variations. In the effort of elucidating retinal gene function, we aimed at developing a protocol for in-vivo CRISPR-based gene knock-out perturbation in the mouse retina. This methodology can be useful to study retinal biology in health and disease, to investigate the effects of ablation of novel uncharacterized genes, and to study possible genetic modifiers of retinal phenotypes.

The advent of CRISPR/Cas9 genome editing technology has revolutionized cancer research by enabling precise, efficient, and versatile manipulation of genetic sequences implicated in oncogenesis and tumor progression. This review highlights the pivotal role of CRISPR/Cas9 in unraveling cancer biology, developing innovative therapeutic strategies, and advancing personalized medicine. Conventional cancer treatments such as chemotherapy, radiotherapy, and surgery, while effective, suffer from significant limitations including non-specific toxicity and resistance, necessitating the exploration of novel targeted approaches. CRISPR/Cas9 offers unprecedented capabilities for targeted gene editing, including correction of oncogenic mutations, silencing of tumor-promoting genes, and restoration of tumor suppressor function. Additionally, it facilitates the generation of patient-specific tumor models such as organoids and xenografts that can guide therapeutic decision-making. Current preclinical studies and early-phase clinical trials demonstrate the feasibility and promise of CRISPR-based therapies, although challenges such as off-target effects, efficient delivery, and ethical considerations must be carefully addressed. Emerging technologies including base and prime editing, improved delivery vectors, and RNA-targeting Cas enzymes are expanding the CRISPR toolbox for cancer therapeutics. Furthermore, novel applications targeting the tumor microenvironment and microbiome are gaining traction. In summary, CRISPR/Cas9 represents a transformative platform driving the future of precision oncology, offering hope for more effective, tailored cancer treatments.

CRISPR-Cas9 sex ratio distortion (SRD) systems can suppress insect populations by biasing progeny toward males, but realizing such systems requires reliable Cas9 expression from insect Y chromosomes. Here, we tested whether the spermatocyte-specific β Tub85D promoter can drive functional Cas9 expression when inserted on the Drosophila melanogaster Y chromosome. Using CRISPR-mediated homology-directed repair, we generated a Y-linked β Tub85D -Cas9-T2A-eGFP construct and compared its activity with an autosomal counterpart. Whereas autosomal β Tub85D -Cas9 induced strong male-biased sex ratios when paired with an X-poisoning gRNA, the Y-linked construct failed to distort sex ratios and exhibited approximately 2,000-fold reduction in Cas9 transcript abundance. Nonetheless, weak but detectable GFP fluorescence and Cas9 transcripts confirmed partial Y-linked promoter activity. These findings provide the first direct experimental evidence of meiotic sex chromosome inactivation (MSCI) acting on the Drosophila Y chromosome, revealing that meiotic promoters can remain weakly active despite strong repression. This work defines transcriptional limits of the Drosophila Y chromosome and informs the design of next-generation Y-linked gene drives for sustainable insect control.

Hepatosplenic T-cell lymphoma (HSTCL) is a rare and aggressive neoplasm associated with poor responses to standard chemotherapy regimens and low survival rates. No targeted therapies are available for HSTCL, and preclinical models to test new treatment options have not been established. The JAK-STAT signaling cascade is a key dysregulated pathway in HSTCL, and STAT5B N642H is the most frequent somatic mutation in the disease. Here, we report on newly established clonal, murine γδ T-cell lymphoma cell lines initiated and driven by oncogenic STAT5B N642H , which recapitulate key immunophenotypic features, gene expression profiles and typically low cytolytic activity of patient-derived human HSTCL cells. CRISPR-Cas9 mediated knockout demonstrated growth dependence on STAT5B N642H . Murine C15 cells were allo-engrafted intravenously into both immunodeficient and immunocompetent mice to model an aggressive HSTCL-like disease at high penetrance, with recipient mice displaying hepatosplenomegaly and destructive γδ T cell organ infiltration, including bone marrow and blood involvement. We identified the potential of JAK inhibition as a targeted treatment strategy for HSTCL, and found the clinically approved JAK inhibitor upadacitinib to display selective anti-tumor efficacy against STAT5B -mutated HSTCL cell lines in vitro, in vivo , and in primary HSTCL patient samples. Overall, we describe the first robust STAT5B-driven preclinical model resembling features of HSTCL in an immune competent setting. This tool is expected to accelerate the study of HSTCL disease mechanisms and the testing of novel therapies. Our data further present the JAK inhibitor upadacitinib as a promising targeted treatment option for STAT5B -mutated HSTCL.

ABSTRACT Sulfur modification of tRNA wobble uridines is an evolutionarily conserved mechanism that ensures efficient protein synthesis. In humans, loss of this anticodon modification due to mutations in CTU2 (cytosolic thiouridylase 2) causes DREAM-PL syndrome, a severe congenital disorder often leading to early postnatal death. However, the mechanisms by which loss of tRNA thiolation drives pathology remain unclear. Here, we show that loss of CTU2 triggers significant cellular proteostasis defects in patient cells and model cell lines. Structural and biochemical analyses reveal that the pathogenic CTU2 L63P mutation destabilizes the CTU1/CTU2 complex and abolishes tRNA binding and thiolation. Acute loss of CTU2 caused codon-specific ribosome pausing at A-ending codons decoded by thiolated tRNAs, and decreased ribosome occupancy of A-rich transcripts in a dosage-dependent manner. Codon-biased mRNAs transcribed from genes critical for ciliogenesis are predicted to be most affected, linking their reduced translation to DREAM-PL etiology in humans. Surprisingly, Ctu2 L63P mice display severe thiolation defects, but develop normally, are viable and fertile. Our findings highlight the importance of functional tRNA thiolation for organismal health in humans and identify species-specific vulnerabilities during embryonic development in mammals.

The trans cleavage activity of type V CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Cas12a system has been widely used for the detection of biomolecules. Different Cas12a orthologues exhibit faster or slower trans cleavage kinetics, making some orthologues more suited for sensitive molecular detection. Ionic strength of reaction buffers, and mutations that change the electrostatic environment near the RuvC active site have also been reported to strongly influence trans cleavage kinetics. Studying three commonly used Cas12a orthologues (FnCas12a, AsCas12a, and LbCas12a), we report that electrostatic interactions near the RuvC active site are critical for their trans cleavage activity. Alanine substitution of arginine and lysine residues in the Nuc domain can abolish trans cleavage while modestly reducing cis cleavage. Substitutions of the RuvC lid and substitutions to introduce positively charged residues in the Nuc could enhance both cis and trans cleavage. These Cas12a variants improved DNA detection and genome editing efficiency. Overall this study provides a blueprint for future rational engineering of Cas12a nucleases for their trans cleavage activities. Graphical abstract

Mucopolysaccharidosis type VII (MPS VII) is characterised by progressive locomotor decline, attributed to musculoskeletal and neurological defects. However, the severity varies according to the extent of β-glucuronidase (β-GUS) deficiency, with musculoskeletal deformities typically preceding neurological manifestations. To investigate the underlying cause of neuromuscular pathology, we employed β-GUS-deficient Drosophila models. In Drosophila , β-GUS is encoded by two genes, CG2135 and CG15117. Previously generated CG2135 -/- flies recapitulated several hallmark features of MPS VII, despite retaining approximately 30% residual β-GUS activity contributed by CG15117. To assess the consequences of complete loss of β-GUS, we now generated CG15117 -/- flies using CRISPR/Cas9 and subsequently established CG15117 -/- ;CG2135 -/- double knockout (DKO) flies. Phenotypic assessment revealed differences in susceptibility to starvation, lifespan, and locomotor function, with DKO flies exhibiting more severe impairments than either single knockout flies. Notably, CG15117 -/- did not show any significant defect in lifespan or locomotion, except under starvation conditions. Consistent with our earlier finding in CG2135 -/- fly brains, ATP depletion in DKO fly brains became evident only after 45 days of age, failing to explain the locomotory defects observed in earlier ages. Interestingly, analysis of muscles of 30-day-old CG2135 -/- and DKO flies revealed abnormal mitochondrial accumulation with autophagy defect and severe ATP depletion. Consequently, these defects led to locomotor impairment driven by apoptotic muscle degeneration. Collectively, this work provides the first evidence of tissue-specific vulnerability in MPS VII models, identifying muscle as an early pathological target and offering new insights into disease progression.

ABSTRACT Size is a fundamental property of cells that influences many aspects of their physiology. This is because cell size sets the scale for all subcellular components and drives changes in the composition of the proteome. Given that large and small cells differ in their biochemical composition, we hypothesize that they should also differ in how they respond to signals and make decisions. Here, we investigated how cell size affects the susceptibility to cell death. We found that large cells are more resistant to ferroptosis induced by system x c- inhibition. Ferroptosis is a type of cell death characterized by the iron-dependent accumulation of toxic lipid peroxides. This process is opposed by cysteine-dependent lipid peroxide detoxification mechanisms. We found that larger cells exhibit higher concentrations of the cysteine-containing metabolite glutathione and lower concentrations of membrane lipid peroxides, compared to smaller cells. Mechanistically, this can be explained by the fact that larger cells had lower concentrations of an enzyme that enriches cellular membranes with peroxidation-prone polyunsaturated fatty acids, ACSL4, and increased concentrations of the iron-chelating protein ferritin and the glutathione-producing enzymes glutamate-cysteine ligase and glutathione synthetase. Taken together, our results highlight the significant impact of cell size on cellular function and survival, revealing a size-dependent vulnerability to ferroptosis that could influence therapeutic strategies based on this cell death pathway.

Abstract Campylobacter species are major contributors to foodborne and waterborne zoonotic gastroenteritis. Several species, including C. jejuni, C. coli, C. fetus, C. concisus, C. lari, C. hyointestinalis, C. upsaliensis , and C. hepaticus , are established pathogens, while the pathogenic potential of other members remains unclear. This study presents a comparative genomics analysis of the fifty reported species of Campylobacter genus, encompassing phylogenomic relationships, functional repertoire profiling, virulence genes, diversity of Cytolethal distending toxin gene ( Cdt ), outer membrane components, genome plasticity, and resistome characterization. Phylogenetic analyses revealed that C. hepaticus, C. taniopygae , and C. iguaniorum , traditionally considered non-pathogenic or minor pathogens, cluster with major pathogenic species, suggesting shared evolutionary features. Functional repertoire profiling indicated metabolic flexibility that supports environmental adaptability, while virulence profiling highlighted both conserved and species-specific determinants. Variation in Cdt genes and outer membrane components emerged as key factors in pathogenicity. Notably, C. helveticus shows potential to emerge as a significant pathogen, whereas C. vicugnae and C. vulpis display close evolutionary relationships with C. jejuni . Genome plasticity analyses identified horizontal gene transfer via genomic islands, prophage insertions, and CRISPR arrays, underscoring the dynamic evolution of virulence traits. Resistome characterization revealed widespread antimicrobial resistance genes, raising concerns about multidrug resistance and clinical management. Overall, this study provides an integrative framework to understand the evolutionary dynamics, virulence potential, and antimicrobial resistance of Campylobacter , offering valuable insights for surveillance and therapeutic strategies.

Type III CRISPR systems generate cyclic oligoadenylate (cOA, 3 to 6 AMPs) messengers upon detecting viral RNA, activating downstream effectors to defend against viral infection. Although cOA-activated effectors have been extensively characterized, the cA 5 -specific effectors remained unexplored despite cA 5 being among the most abundant cOA species produced during phage infection. Here, we report that Actinomyces procaprae Csm6 (ApCsm6) selectively employs cA 5 as its activator. Unlike other characterized Csm6 proteins, ApCsm6 self-limits its ribonuclease activity by degrading cOAs via its HEPN domain, rather than relaying on the CARF domain. Cryo-EM structures of ApCsm6 and its complexes with cA 5 and cA 6 reveal a homotetrameric assembly, where each monomer binds a single cOA within a composite pocket formed by two tandem CARF-HEPN domains. Binding of cA 5 , but not cA 6 , enhances tetramerization and induces large conformational shifts in CARF, which in turn allosterically activates ssRNA cleavage in HEPN. These findings advance our understanding of ligand discrimination and signaling regulation in type III CRISPR immunity. Highlights ApCsm6 preferentially recognizes cA 5 as its activator. Cryo-EM structures of ApCsm6 and its complexes with cA 5 or cA 6 reveal the structural basis for cA 5 -selective recognition and allosteric activation. ApCsm6 acts as a self-limiting ribonuclease by degrading cOAs via its HEPN domain rather than relaying on the CARF domain.

Cell therapy manufacturing of primary T cells often results in heterogeneous cell populations within a final product, with many cells lacking desired of receptor expression or those that have exhausted or other dysfunctional phenotypes. Here, we design a novel cell-intrinsic strategy to genetically reprogram primary human T cells to autonomously detect and eliminate dysfunctional cells. This integrated detection and elimination process, known as directed fratricide, is programmed via nonviral CRISPR genome-editing to eliminate the T cell receptor (TCR) alpha chain ( TRAC gene knockout) and integrate a chimeric antigen receptor (CAR) against the urokinase-type plasminogen activator receptor (uPAR), also known as CD87. Within these cell products, strong T cell stimulation or activation during manufacturing causes a small subset of cells to express uPAR, which subsequently triggers CAR-mediated killing by a separate subset of cells within the product. This fratricide induces proliferation in the desired cells and destroys undesired cells, a process that could be modeled computationally and controlled robustly via supplements to the culture media. The strategy enabled enrichment of anti-uPAR and anti-GD2 CAR T cell products up to ≥99% CAR+/TCR-, favoring a stem cell memory-like phenotype (CD45RA high /CD62L high ). Understanding growth dynamics among T cell subsets and reprogramming them via CRISPR could accelerate the biomanufacturing of potent cell products without extensive selection methods. Abstract Figure

Hallmark gene mutations shape cancer cell vulnerabilities and inform drug discovery 1–3 . A systematic map of hallmark gene mutation-defined cancer dependencies and therapeutic responses is essential to uncover novel targets and refine therapeutic strategies. Here, we present the first pan-cancer blueprint of hallmark vulnerabilities, systematically linking hallmark gene mutation markers to cancer cell dependencies and drug sensitivities across 22 cancer cohorts. We integrated multi-omics data from patient tumours with large-scale CRISPR-Cas9 screens and pharmacologic profiling of over a thousand cancer cell lines. Our analysis revealed the cancer type-specific nature of hallmark gene expression programs, uncovered previously unrecognised mutation-target gene dependencies, and highlighted metabolic programs as a dominant class of functional vulnerabilities. Notably, we identified oxidative phosphorylation (OXPHOS) addiction in CDKN2A -loss lung squamous cell carcinoma (LUSC) and experimentally validated this dependency. Our validation highlights the greater selectivity of CDKN2A -loss LUSC cells to metformin, an FDA-approved antidiabetic drug known for its OXPHOS inhibitory activity. Proteogenomic integration further prioritised targets overexpressed in mutant tumours, constituting therapeutic windows. Pharmacologic profiling identified both oncology and non-oncology agents with selective activity in mutation-defined subgroups, revealing opportunities for drug repurposing. Our machine learning framework, Comet-X, for the first time fully leveraged gene mutation combinations to predict these target dependencies and drug responses. The resulting pan-cancer mutation-dependency map provides a comprehensive resource of hallmark gene targets and candidate therapeutics, stratified by mutation markers, to pave the way for drug development, clinical trial design and discovery research.

ABSTRACT Protein-DNA interactions can be manipulated in vitro by changing buffer conditions. Here, we develop a methodology to map the cleavage preferences of chimeric gene editors that are fusions of the I-TevI nuclease domain to CRIPSR nucleases by manipulating in vitro salt concentrations. We found that DNA cleavage by the I-TevI (Tev) nuclease domain at CNNNG sites was de-coupled from the gRNA-targeted site in low salt buffers. For TevCas12a, this non-targeted cleavage activity was enriched at Tev CNNNG cleavage motifs optimally positioned within a 30-bp window upstream of a Cas12a TTTV PAM site. Non-targeted cleavage did not require Cas12a nuclease activity or specific Cas12a gRNA targeting. Similar non-targeted products were observed in low salt buffer conditions for TevSaCas9, Tev-meganuclease and Tev-zinc finger editors. Cas12a and SaCas9 activity at gRNA-directed sites and sites with multiple mismatches were also sensitive to buffer salt concentration. Oxford Nanopore sequencing revealed a remarkably similar Tev CNNNG cleavage preference at different salt concentrations and in different fusion contexts, emphasizing the robustness and specificity of Tev activity. More generally, our work highlights the sensitivity of gene editors to in vitro reaction conditions and how these conditions can be leveraged to functionally dissect the activity of individual domains of chimeric gene editors.

The coexistence of bacteria and phages is marked by dynamic interactions that determine infection outcomes. However, the mechanisms by which the host allocates its resources to cope with phage infection remain largely unknown. In this study, using longitudinal proteomics, we elucidated these interactions for the temperate staphylococcal phage ϕ NM1 and strains of Staphylococcus aureus either harboring its cognate prophage or lacking it. We demonstrated that infection of non-lysogenic S. aureus with ϕ NM1 induces a dramatic shutdown of host translation, reducing proteome allocation by over 20%. Quantitative analysis of the economics of ϕ NM1 infection revealed that the diversion of these cellular resources toward phage replication imposes a significant metabolic burden, thereby impairing cell growth. In contrast, lysogenic cells cope with phage infection and prevent culture collapse through a coordinated response of prophage-encoded defenses, host-encoded stress effectors, and reprogrammed cellular metabolism, thereby avoiding translation shutdown. Through coinfection with the wildtype phage and an engineered phage-like particle carrying a CRISPR-Cas phagemid, we revealed that synthetic DNA cargos evade host defenses and hijack the transcriptional machinery, altering infection outcomes. Without immunity, coinfection could collapse the non-lysogens more quickly than the native phage by overexpressing the cargo proteins, suppressing carbohydrate metabolism, and accelerating structural phage protein production through increased phage genome replication. Together, these findings provide a systems-level understanding of phage infection in S. aureus , uncovering the mechanisms for host takeover and prophage-mediated defense, with implications for next-generation phage therapy. Significance Multidrug-resistant bacteria pose a major threat to human health. Phage therapy offers a precise therapeutic approach by leveraging the specificity of phage infection and cargo delivery. However, as with conventional antibiotics, phage resistance can develop. Understanding the dynamic interactions between bacterial hosts and phages is therefore essential for predicting infection outcomes and designing precision phage therapies to suppress resistance. Using longitudinal proteomics, we elucidated the dynamics by which bacterial hosts reallocate cellular resources to cope with phage infection at the systems level. Coordinated defenses and reprogrammed metabolism are critical for host survival, but phage-delivered cargos can effectively bypass these barriers. These insights into the intricate interplay between the phage and host are crucial for designing next-generation precision phage therapies with greater lethality and less resistance.

SUMMARY Zika virus (ZIKV) can be vertically transmitted from a pregnant mother to the developing fetus, resulting in microcephaly and/or other congenital malformations. Dengue virus (DENV) cross-reactive antibodies can facilitate ZIKV placental transcytosis and enhance ZIKV infection of placenta macrophage-Hofbauer cells through binding to Fc-γ receptors (FcγRs). To understand the role of individual FcγR in antibody-mediated ZIKV placental infection, we generated a comprehensive panel of Fc-variants spanning a wide range of binding affinities to different FcγRs. We found that mutations with increased affinity to FcγRI strongly correlated with an increased frequency of infected pro-monocytic U937 and Hofbauer cells. Next, we genetically deleted individual FcγR in U937 cells, and found that the knockout of FCGR1A gene completely abolished ZIKV infection. In contrast, the deletion of FCGR2B gene showed no effect on ZIKV infection, and the deletion of FCGR2A gene had only a moderate impact on ZIKV infection. We further observed that FcγRI was involved in both increased ZIKV internalization and replication. Collectively, our results establish FcγRI as the key Fc receptor responsible for antibody-mediated ZIKV infection in both U937 and primary placental macrophages. These mechanistic findings not only provide insight into the importance of FcγRI in ZIKV vertical transmission but also highlight FcγRI as a potential therapeutic target, with significant implications for the development of strategies to prevent ZIKV transmission from mother to fetus.

Gene conversion is a specific form of homologous recombination (HR), involving the unidirectional transfer of genetic information from one genomic locus to another. CRISPR-Cas9-directed double strand breaks (DSBs) induce both interallelic and interlocus gene conversion in early human embryos and somatic cells, suggesting its potential for correcting pathogenic mutations. However, the key features in mitotic gene conversion, including its efficiency, the length of conversion track, and its dependency on specific recombination proteins, remain largely undefined. Here, we show that allele-specific CRISPR-Cas9-induced DSBs, without exogenous donor templates, can efficiently correct a heterozygous pathogenic variant (c.1582C>T; p.Arg528Trp) in the ATAD3A gene of patient-derived induced pluripotent stem cells (iPSCs). Amplicon-based next-generation sequencing (NGS) revealed that approximately 38%~53% of edited iPSCs carried two wild-type ATAD3A alleles. Notably, over 99% of the corrected alleles derived from the homologous chromosome, indicating that the repair occurred mainly via interallelic gene conversion. Long-range amplicon nanopore sequencing coupled with haplotype analysis showed that the majority of gene conversion tracts was less than 2 kilobases in length. Whole-genome sequencing of three corrected iPSC clones showed the absence of large deletions or structural rearrangements at the ATAD3A target site. However, one clone carried a heterozygous deletion in ATAD3B locus, suggesting that CRISPR-Cas9 can introduce off-target genomic alterations. Knockdown of key HR proteins, including RAD51, CtIP, and BRCA1/2, significantly reduced the correction efficiency, indicating that the gene conversion relies on a RAD51-dependent HR pathway. Together, our findings provide compelling evidence that template-free CRISPR-Cas9-mediated interallelic gene conversion can be harnessed to correct disease causing variants in human iPSCs.

Mitotic spindle orientation is tightly controlled in epithelia to preserve polarized tissue architecture. Here, we uncover a previously unrecognized role of the adherens junction protein β-catenin in regulating planar mitotic spindle positioning required for symmetric epithelial cell division. Using CRISPR/Cas9-mediated genome editing in MDCK cells, we found that β-catenin—but not its close homolog γ-catenin (plakoglobin)—is required for proper spindle orientation. Loss of β-catenin disrupts astral microtubule anchorage and impairs the localization of LGN–NuMA spindle orientation machinery. Mechanistically, β-catenin mediates spindle regulation via its N-terminal domain, independently of β-catenin binding. We showed that the cortical recruitment of Afadin during mitosis depends on β-catenin, but surprisingly, overexpression of either Afadin or ZO-1 rescue the spindle orientation in β-catenin deficient cells by restoring cortical LGN. Afadin depletion or dominant-negative Afadin abolished ZO-1-mediated compensation, thereby establishing Afadin as the central cortical integrator, potentiated by β-catenin, and operating through mass-action to recruit cortical LGN. Together, our findings define β-catenin as an upstream regulator of the Afadin-based cortical-spindle linkage that coordinate the crosstalk between adherens junctions and mitotic geometry to ensure epithelial homeostasis.

Stathmin-2 (STMN2) is a microtubule associated protein that plays a role in the stability of microtubules of axons in the nervous system of animals. In this study we generated a novel zebrafish STMN2 knockout (KO) model. STMN2 is represented by two genes in the zebrafish genome: stmn2a and stmn2b . Using the CRISPR/Cas9 mutagenic system we selected founder fish lines harbouring frameshift mutations in both genes and bred these together to generate a double stmn2a and stmn2b KO model. Using these models, we observed increased developmental lethality in our double stmn2a and stmn2b KO model and impaired motor function at larval stages of development. Examination of the larval neuromuscular junction (NMJ) revealed a slight increase in the number of orphaned NMJs in trunk musculature as well as a reduction in amplitude of miniature endplate currents in our double stmn2a and stmn2b KO model. In a final series of experiments, we show impaired ventral root axon regrowth following transection in double stmn2a and stmn2b KO larvae. Our findings suggest that while not essential for motor axon development, loss of stmn2a and stmn2b expression perturbs the assembly of zebrafish NMJs during development resulting in a minor motor phenotype and impairs that ability to regenerate motor axons following injury.

The widespread use of antibiotics promotes both resistance and tolerance. While resistance enables bacterial growth in the presence of drugs, tolerance allows survival during treatment, generating persisters that seed relapse and promote resistance. Despite its clinical relevance, the molecular basis of tolerance remains poorly understood. Using proteomic and metabolomic profiling combined with machine learning, we identified thiol oxidation as a robust predictor of tolerance in the human pathogen Pseudomonas aeruginosa . Single-cell analyses established a direct link between thiol oxidation and drug survival, indicating that redox imbalance drives persistence. Whereas depletion of coenzyme A (CoA), a central thiol-containing metabolite, scaled with tolerance, restoring CoA using engineered catalysts from Staphylococcus aureus abolished tolerance, establishing a causal relation between CoA availability and drug susceptibility. Thiol-based predictors also accurately capture tolerance of clinical P. aeruginosa isolates. These findings establish CoA-centered redox control as a key determinant of tolerance, opening opportunities for diagnostics and therapeutic interventions to prevent infection relapses.