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.

Invasion plasticity allows malignant cells to toggle between collective, mesenchymal and amoeboid phenotypes while traversing extracellular matrix (ECM) barriers. Current dogma holds that collective and mesenchymal invasion programs trigger the mobilization of proteinases that digest structural barriers dominated by type I collagen, while amoeboid activity allows cancer cells to marshal mechanical forces to traverse tissues independently of ECM proteolysis. Here, we use cancer spheroid-3-dimensional matrix models, single-cell RNA sequencing, and human tissue explants to identify the mechanisms controlling mesenchymal versus amoeboid invasion. Unexpectedly, collective/mesenchymal- and amoeboid-type invasion programs – though distinct – are each characterized by active tunneling through ECM barriers, with expression of matrix-degradative metalloproteinases. CRISPR/Cas9-mediated targeting of a single membrane-anchored collagenase, MMP14/MT1-MMP, ablates tissue-invasive activity while co-regulating cancer cell transcriptional programs. Though changes in matrix architecture, nuclear rigidity, and metabolic stress as well as the presence of cancer-associated fibroblasts are proposed to support amoeboid activity, none of these changes restore invasive activity of MMP14-targeted cancer cells. While a requirement for MMP14 is bypassed in low-density collagen hydrogels, invasion by the proteinase-deleted cells is associated with nuclear envelope and DNA damage, highlighting a proteolytic requirement for maintaining nuclear integrity. Nevertheless, when cancer cells confront explants of live human breast tissue, MMP14 is again required to support invasive activity. Corroborating these results, spatial transcriptomic and immunohistological analyses of invasive human breast cancers identified clear expression of MMP14 in invasive cells that were further associated with degraded collagen, underlining the pathophysiologic importance of this proteinase in directing invasive activity in vivo .

Lipid abnormalities are emerging as key pathogenic mechanisms in neurodegenerative diseases such as Alzheimer’s, Parkinson’s and Lewy body dementia. Astrocytes in the brain provide APOE proteins and influence neuronal metabolism and health. Using live cell imaging and objective neurite imaging techniques, we show that following induction of cellular lipid (cholesterol and triglycerides) load by inhibiting the lysosomal cholesterol transport protein NPC1 in human neuron-astrocyte co-cultures, that human astrocytes CRISPR edited to be either APOE3 or 4 variants have different effects on rescuing dystrophic neurites, where axons and dendrites of nerve cells become disfigured. APOE3, but not APOE4 or APOEKO, astrocytes prevented cholesterol and lipid induced neurite damage in APOE4 neurons. In the media of APOE3 co-cultured astrocytes with neurons the HDL-like particles were larger and presumably more lipidated than equivalent APOE4 co-cultures. This discovery highlights that living APOE3 astrocytes control key biological mechanisms by physiologically enhancing lipid cellular homeostasis, that can rescue lipid-induced neurite structural abnormalities relevant to Alzheimer’s disease and neurodegenerative diseases. Significance statement Neurodegenerative diseases like Alzheimer’s (AD) are often defined by abnormal protein aggregates, but growing evidence points to lipid dysfunction as a key driver, especially in APOE4 carriers, the strongest genetic risk factor for AD. We developed a live cell imaging based human cell culture model using isogenic iPSC-derived neurons and astrocytes (APOE3, APOE4, or APOE knockout) to study this. By blocking cholesterol export via NPC1 inhibition, we mimicked lysosomal lipid stress and found that APOE3 astrocytes uniquely protected APOE4 neurons from forming abnormal neurite swellings. These APOE3 astrocytes produced larger HDL-like particles than APOE4 that supported neuronal lipid balance. Our results show that APOE3 astrocytes can rescue APOE4-related cellular dysfunction, offering a potential path for therapy and biomarker discovery.

ABSTRACT Female infertility represents a significant challenge in reproductive medicine. Although it is known to be primarily due to oogenic and early embryonic failures, the molecular mechanisms underlying these failures remain elusive. The Piwi-piRNA pathway is crucial for gametogenesis in diverse organisms. Yet, its role in mammalian female fertility is unclear. This is partly because most studies are done in mice, which contain only PIWIL1, PIWIL2, and PIWIL4 that are essential for spermatogenesis but not for oogenesis. PIWIL3 emerges in higher mammals and is highly expressed in human oocytes. Interestingly, female PIWIL3 -knockout golden hamsters exhibit only reduced fertility without detectable defects in oogenesis. This has left the function of PIWIL3 in higher mammals, including humans, unexplored. Here, we discovered that PIWIL3 in the rabbit ( Oryctolagus cuniculus ) shares high homology with human PIWIL3. It is the predominant PIWI protein in oocytes in both species. Using CRISPR–cas9-mediated knockout, we demonstrated that rabbit PIWIL3 is essential for female fertility. Its loss leads to severe defects in oogenesis. Moreover, embryos lacking maternal PIWIL3 arrest developmentally at the 8-cell stage. Mechanistically, rabbit PIWIL3 binds ∼18-nucleotide piRNAs, mirroring the behavior of human PIWIL3, and is critical for piRNA biogenesis. Moreover, it regulates transcriptomic and proteomic landscapes and silences a broad array of transposons during late oogenesis yet activates another set of transposons during early embryogenesis. These findings establish PIWIL3 as a pivotal dual regulator of gene expression and transposon activity, which is essential for oogenesis and early embryogenesis in non-rodent mammals, potentially including humans.

Osteocytes play critical roles in bone, making them attractive targets for therapeutics to improve bone mass and strength. The genes driving osteocyte maturation and function are not fully understood. Here we aimed to identify novel genes responsible for osteocyte differentiation and dendrite development by performing a genome-wide CRISPR-interference (CRISPRi) screen in the Ocy454 osteocyte-like cell line. We identify CD61 (integrin β3) as a marker of osteocyte maturation: surface CD61 expression increases during osteocyte maturation, and CD61 high cells express higher levels of osteocyte marker genes. We then developed a flow cytometry-based assay to quantify surface CD61 protein levels as a phenotypic endpoint for functional genomic screening. In a genome-wide screen, we identified Clip2, which encodes a microtubule binding protein, as one of dozens of genes necessary for CD61 expression. Clip2 inhibition decreased surface CD61 expression, reduced expression of osteocyte-specific genes Dmp1 and Sost , and impaired dendrite morphology in vitro . Together, these results highlight the utility of surface CD61 as a marker of osteocyte maturity and identify a role of the microtubule cytoskeleton for osteocyte differentiation, form, and function.

iNKT cells are emerging as a highly promising immunotherapy platform for the treatment of cancer. To maximise the anti-cancer activity of CAR-iNKT against the blood cancer multiple myeloma we investigated optimal CAR designs and their combination with novel iNKT-specific engagers. We find that amongst five different CAR endodomains, underpinned by increased avidity and a cross talk between Plexin D1 on CAR-iNKT and Semaphorin 4A on myeloma cells, BCMA CD28z CAR-iNKT exert the highest anti-myeloma activity. Notably, CD28z CAR-iNKT outperform their CAR-T counterparts. To expand the anti-myeloma potential of CAR-iNKT, we designed and validated a high efficacy BCMA iNKT-specific engager which exerts significant anti-myeloma activity in conjunction with adoptively transferred iNKT cells. Finally, combined, dual target therapy with FCRL5 CAR-iNKT and BCMA iNKT engagers outperforms FCRL5 CAR-iNKT and limits immune escape of FCRL5-negative myeloma. Thus, optimised iNKT-based, dual-target, dual-modality immunotherapy has enhanced anti-tumor activity against multiple myeloma and potentially other malignancies. Abstract Figure

Background Investigating the subcellular distribution of proteins is crucial for understanding complex cell behaviours and disease mechanisms. Fluorescence microscopy is a key tool for visualising protein localisation. However, its application is often hindered by the lack of high-quality antibodies, and the common approach to overexpress recombinant fusion constructs tagged with fluorescent proteins can introduce artefacts. Endogenous protein tagging, where the sequence for a tag (typically a peptide or fluorescent protein) is integrated into the native genetic sequence encoding a protein of interest, can be used to overcome these limitations, enabling proteins to be visualised without the need for antibodies against the target protein and avoiding complications associated with recombinant protein overexpression. ORANGE (Open Resource for the Application of Neuronal Genome Editing) is a CRISPR-Cas9-based endogenous protein tagging technique which relies on homology-independent targeted integration (HITI)-mediated gene editing. Utilising HITI as the DNA repair pathway of choice gives ORANGE the advantage of being less error-prone than classical homology-directed repair (HDR)-based endogenous protein tagging techniques and additionally, means it can be used in post-mitotic cells. Results We applied the ORANGE system to tag three proteins, CYFIP1, JAKMIP1, and STAT3, and confirmed that the expressed fusion proteins demonstrate expected subcellular localisations through fluorescence microscopy. Unexpectedly, the efficiency of ORANGE editing was less than 1% in HEK293 cells, despite high transfection efficiency. To improve the editing efficiency associated with ORANGE, we combined the ORANGE method with an established Sleeping Beauty transposase/CRISPR-Cas9 fusion technique, which has been shown to enhance HITI-mediated gene editing. Using this new method, which we term Sleeping ORANGE, we successfully tagged CYFIP1 with the fluorescent protein mNeonGreen. Importantly, through fluorescence microscopy and flow cytometry, we demonstrate that Sleeping ORANGE increased gene-editing efficiency by approximately 4- to 6-fold compared to the original ORANGE technique. Conclusions We have developed a method to improve the editing efficiency associated with the ORANGE technique, and with further research to ensure that the fluorescent tags are correctly inserted into their target gene without off-target effects, the Sleeping ORANGE technique may form a valuable tool for researchers to use to better study protein subcellular localisation and dynamics.

Abstract Chimeric antigen receptor (CAR) T cells have transformed cancer therapy, yet many tumors remain refractory. To uncover broadly acting mechanisms of resistance, we performed genome-wide CRISPR activation screens across diverse cancer cell types. These screens converged on RNF19B, an E3 ubiquitin ligase whose high expression correlates with poor patient survival and confers robust CAR-T resistance in mouse xenograft models. Mechanistically, RNF19B destabilizes the interferon-γ receptor subunit IFNGR1, blunting interferon-γ signaling, and simultaneously induces CAMKK2, which mediates resistance through an independent pathway. Pharmacologic inhibition of CAMKK2 synergized with CAR-T therapy in different xenograft mouse models. Our findings identify RNF19B as a previously unrecognized, dual-pathway mediator of CAR-T resistance and reveal CAMKK2 inhibition as a potential strategy to enhance CAR-T efficacy.

Backgrounds The cellular slime mold Dictyostelium discoideum is a widely used model system for studying basic processes in cell and developmental biology. While genetic tools, such as targeted gene disruption by homologous recombination and genome editing using CRISPR/Cas9, are well-established in D. discoideum , efficient methods for conditional loss-of-function studies are limited. Here, we developed a nanobody-based degron system for D. discoideum based on ALFA-tagged protein recruitment to the Skp1-Cullin-F-box (SCF) complex. Results ALFA-tagged Histone H1 was efficiently degraded by expressing anti-ALFA nanobody (NbALFA) fused to the D. discoideum FbxD F-box domain (‘dictyGrad-ALFA’). Cell type-specific targeting was achieved by expressing dictyGrad-ALFA under prestalk- and prespore-specific gene promoters. Furthermore, targeting of adenylyl cyclase A (ACA) resulted in the expected aggregation-deficient phenotype, validating the efficacy of dictyGrad-ALFA-mediated protein depletion. Cell type-specific ACA degradation delayed development but eventually resulted in normal fruiting bodies. Our ALFA-tag approach was further used for conditional knockdown in combination with the auxin-inducible degron 2 (AID2) system, which relies on indole-3-acetic acid (IAA)-dependent binding between NbALFA-mAID and a OsTIR-F-box-Skp1A fusion protein. We obtained efficient IAA-induced degradation in prestalk cells; however, efficiency was low in other cell types. Conclusions Together, these systems pave the way for conditional and cell type-specific protein degradation in D. discoideum , enabling functional analyses of essential genes for development and survival.

SUMMARY The transition zone (TZ) is a selective barrier that maintains ciliary compartmentalization by controlling protein entry and exit. Cilia assembly requires the crossing of this barrier by intraflagellar transport (IFT) trains, scaffolded by IFT-A and IFT-B complexes, which move cargo bidirectionally using kinesin-2 and dynein-2 motors. In Caenorhabditis elegans , IFT-A loss abolishes retrograde transport, resulting in truncated cilia packed with IFT material. Here, we show that blocking TZ assembly prevents dynein-2 and IFT-B accumulation inside IFT-A-deficient cilia and partially rescues axoneme length. Single-particle imaging reveals that this rescue occurs without recovery of retrograde IFT. Instead, IFT particles exit cilia by passively diffusing through the disrupted TZ. Moreover, IFT-A/TZ double mutants shed ciliary extracellular vesicles (EVs) abnormally enriched in IFT components, providing a second clearance route. We conclude that TZ removal alters ciliary responses to retrograde transport defects, promoting diffusion and EV release to clear IFT machinery and facilitate axoneme extension. Highlights - TZ loss provides alternative routes for clearing IFT machinery stalled in IFT-A mutant cilia - Axoneme extension is possible without retrograde IFT when the TZ barrier is removed - Disrupted TZ enables exit of IFT particles by passive diffusion in retrograde IFT-deficient cilia - Excess IFT machinery is discarded in ciliary EVs when retrograde IFT and gating are compromised

ABSTRACT Air sampling is a non-invasive alternative to individual testing for respiratory pathogens. Alternative methods to the “gold standard” quantitative RT-PCR (qRT-PCR) are required to enable higher throughput, lower cost, and more multiplexed detection of pathogens. The multiplexed CRISPR-Cas13 CARMEN Respiratory Viral Panel (RVP) was described previously for high-throughput detection of nine respiratory pathogens from nasal swab samples. Here, we modified and optimized the CARMEN RVP assay to overcome the unique challenges of air samples, including low biomass and environmental inhibitors. We monitored for SARS-CoV-2 and influenza A (Flu A) via qRT-PCR in air samples from 15 schools within Dane County, Wisconsin (USA) during the 2023-2024 school year. SARS-CoV-2 was detectable throughout the entire sampling period, while Flu A detection was seasonal from November 2023 to March 2024. We then analyzed a subset of samples from seven schools using an optimized CARMEN RVP assay for air surveillance (RVP_air) and compared results to qRT-PCR. The RVP_air assay detected several additional pathogens beyond our primary targets. The frequencies and patterns of SARS-CoV-2 positivity, but not Flu A, were similar between qRT-PCR and RVP_air across the 2023-2024 sampling period. We developed a secondary panel (RVP_air_flu) to better detect both H1N1 and H3N2 subtypes. Finally, we compared air sample results to clinical nasal swabs collected from the same school district. For several pathogens (SARS-CoV-2, HCoV-OC43, Flu A), positive air detections coincided with positive nasal swabs. These findings demonstrate that the RVP_air assay can effectively detect airborne pathogens from infected individuals within indoor spaces. IMPORTANCE Air sampling offers a cost-effective alternative to individual testing for respiratory pathogens within congregate settings. Optimization and use of multi-pathogen assays are especially valuable for capturing the breadth of pathogens that may be present simultaneously in the same space. The modified CARMEN RVP assays (RVP_air and RVP_air_flu) detected SARS-CoV-2 and Flu A during similar sampling time periods compared to qRT-PCR, while also detecting several additional respiratory pathogens (seasonal Coronaviruses, Respiratory Syncytial Virus). Importantly, pathogens detected from air samples corresponded to those detected from nasal swabs collected from individuals in the same spaces. Together, these findings highlight the utility of the RVP_air and RVP_air_flu assays as alternatives to qRT-PCR for environmental surveillance, with applications extending to other congregate spaces (hospitals, long-term care facilities) and high-risk settings, better informing communities and improving public health.

ABSTRACT Protein S- acylation is a lipid-based, often reversible post-translational modification that can regulate many aspects of protein behavior, including subcellular localization, protein-interactions, and activity. Emerging evidence has identified roles for individual protein acyltransferases encoded by the ZDHHC in cancers, yet the roles of de- S- acylation enzymes are less clear. Recent evidence suggests that acyl-protein thioesterase (APT1)/ LYPLA1 can impact epithelial-mesenchymal transition and metastasis. This study integrates patient datasets, CRISPR dependency data, and in vitro assays to find APT1 as a context-dependent vulnerability in triple-negative breast cancer (TNBC). Despite the highest protein abundance in luminal MCF7 cells, basal-like MDA-MB-468 cells exhibited the most prominent specific APT1 activity, reflecting subtype-specific regulation. Inhibition of APT1 with ML348 increased S -acylation of nuclear and mitochondrial proteins without altering global acylation. Functionally, APT1 inhibition reduced cell proliferation while inducing minimal apoptosis, consistent with cytostatic growth arrest. Cell-cycle analysis revealed G1 accumulation and reduced S/G2 transition, linking proteomic changes to impaired replication. These findings establish APT1 as a regulator of TNBC proliferation through dynamic de- S- acylation of cell-cycle and mitochondrial proteins, highlighting it as a potential therapeutic vulnerability in aggressive breast cancers.