The growing threat of antimicrobial resistance has driven the search for new bioactive compounds in extreme environments such as Antarctica. Streptomyces fildesensis So13.3, isolated from Antarctic soil, has been shown to contain a biosynthetic gene cluster associated with producing actinomycin D, an antibiotic with therapeutic potential. In this study, we analysed the regulatory role of TetR/AcrR family transcription factors present within this BGC, focusing on their activation under different nutritional conditions and their structural scharacterisation using bioinformatics tools and molecular dynamics simulations. The results showed that TetR/AcrR expression increased significantly in ISP4 and IMA media, suggesting their involvement in nutrient-dependent regulation of the cluster. At the structural level, two TetR proteins (TetR-206 and TetR-279) were modelled, with the latter standing out due to a C-terminal tetracycline repressor-like domain. A 200 ns molecular dynamics simulation was performed in GROMACS to evaluate the stability and flexibility of TetR-279, including analysis of point mutations (S166P, V167A, V167I). The S166P mutation significantly impacted structural flexibility, while V167A and V167I caused only minor alterations. This work demonstrates the value of integrating omics approaches, structural modelling, and gene editing with CRISPR to study and potentially activate silent BGCs in non-model bacteria such as Antarctic Streptomyces. In particular, the targeted inhibition of TetR-279 may trigger metabolic rewiring and facilitate the expression of novel antibiotics encoded in cryptic biosynthetic gene clusters.

Neuron cell culture stands at the forefront of neuroscience innovation, offering unparalleled insights into neuronal development, pathology, and regeneration. This review critically examines advances and persistent obstacles in culturing neurons, with a focus on axon and dendrite growth—a pivotal yet underexplored frontier for regenerative medicine. We synthesize breakthroughs in extracellular matrix (ECM) engineering, 3D biomimetic microenvironments, and molecular interventions while highlighting intrinsic challenges such as limited neuronal longevity, tumorigenicity risks in stem cell approaches, and reproducibility gaps. Introducing a biomimetic engineering framework, we liken neuronal regeneration to a multidimensional optimization problem, where balancing mechanical, biochemical, and epigenetic variables dictates functional outcomes. Key findings include: 1. 3D hydrogels mimicking brain ECM enhance neurite outgrowth by 40–60% compared to 2D systems. 2. Secretory pathway disparities between axons and dendrites reveal evolutionarily conserved growth mechanisms. 3. Tumorigenicity remains a critical barrier, with CRISPR-Lin28-edited iPSCs reducing teratoma formation by 65% in preclinical models. We advocate for standardized, scalable protocols and CRISPR-epigenetic tools to silence inhibitory pathways (e.g., Nogo-A). By bridging in vitro models with clinical translation, this work charts a roadmap for overcoming regenerative bottlenecks in neurodegenerative diseases and CNS injuries.

Prokaryotes carry clusters of phage defense systems in “defense islands” that have been extensively exploited bioinformatically and experimentally for discovery of immune functions. However, little effort has been dedicated to determining which specific system(s) within defense islands limit lytic phage reproduction in clinical bacterial strains. Here, we employed the CRISPR-based Cascade-Cas3 system to delete defense islands in a Pseudomonas aeruginosa clinical isolate to identify mechanisms of lytic phage antagonism. Deletion of one island in a cystic fibrosis-derived clinical isolate sensitized the strain to phages from the Pbunavirus family, which are commonly used as therapeutics. The causal defense system is a Type IIS restriction endonuclease-like protein (END PaCF1 ), common in Pseudomonads, however it lacks an associated methyltransferase typical Type IIS R-M systems. END PaCF1 protects bacteria against phages with hypermodified DNA and is surprisingly agnostic to the specific structure of the modification, which is unlike typical type IV restriction endonucleases. In END PaCF1 , the endonuclease domain is fused to a catalytically inactive Endonuclease III (iEndoIII), a domain that recognizes non-canonical bases to repair DNA in prokaryotes and eukaryotes. We therefore propose that nucleases containing an i En doIII d omain ( END nucleases) can sense diverse DNA hypermodifications. Our findings reveal modularity of the sensing and cleavage domains, as expected of a modification-dependent endonucleases. We further show that some hypermodified phages, including Pbunavirus family members and Wrowclawvirus family (Pa5oct-like) of jumbo phages, encode END nuclease inhibitors that directly bind to the nuclease, likely via the iEndoIII domain. These inhibitors are necessary for Pbunavirus to plaque on clinical isolates and sufficient to enable other hypermodified phages to plaque in the presence of this defense system.

Prairie voles ( Microtus ochrogaster ) are a powerful model for studying the neurobiology of social bonding, yet tools for region- and cell type-specific gene regulation remain underdeveloped in this species. Here, we present a lentivirus-mediated CRISPR activation and interference (CRISPRa/i) platform for somatic gene modulation in the prairie vole brain. This system enables non-mutagenic, titratable regulation of gene expression in the adult brain without germline modification. Our dual-vector system includes one construct expressing dCas9-VPR (CRISPRa) or dCas9-KRAB-MeCP2 (CRISPRi) under a neuron-specific promoter, and a second construct delivering a U6-driven sgRNA alongside an EF1α-driven mCherry reporter. We detail the design, production, and stereotaxic delivery of these tools and demonstrate their application by targeting four genes implicated in social behavior ( Oxtr, Avpr1a, Drd1, Drd2 ) across two mesolimbic brain regions: the nucleus accumbens and ventral pallidum. Gene expression analyses confirmed robust, bidirectional transcriptional modulation for select targets, establishing proof of concept for CRISPRa/i in this non-traditional model. The dual-vector design is readily adaptable to other gene targets, cell types, and brain regions, and can be multiplexed to provide a flexible and scalable framework for investigating gene function in behaviorally relevant circuits. These advances represent the first successful implementation of somatic CRISPRa/i in prairie voles and expand the genetic toolkit available for this species.

Effective and scalable sex separation remains a critical challenge for mosquito genetic control strategies. Genetic sexing strains (GSS) address this by genetically linking maleness with selectable traits, enabling efficient removal of females before release. Here, we describe a robust platform for the development of GSSs in the invasive Aedes albopictus mosquito by integrating a CRISPR-engineered selectable phenotype with sex conversion via nix , the male-determining factor. As a proof-of-concept, we disrupt the yellow gene to generate a vivid pigmentation marker, then rescue its function in males using nix -containing transgenes, creating a stable strain where all females are yellow and all engineered males are dark. The resulting GSS males are fertile, robust, and despite lacking the ancestral M locus, exhibit gene expression profiles closely resembling wild-type males. We benchmark sex separation based on pigmentation and discover that yellow mutant females exhibit slower larval development, enhancing protandry-based sorting. The GSS strain is compatible with existing size-based sex sorting systems, allowing for improved separation accuracy through the integration of natural and engineered sexually dimorphic traits. Additionally, we find that GSS females lay desiccation-sensitive eggs, reducing the risk of accidental female releases. Our approach is the first to engineer a sex-linked selectable trait by precisely targeting an endogenous gene and restoring its function in males, establishing a versatile platform for GSS development in Aedes mosquitoes.

A major challenge in human evolutionary biology is to pinpoint genetic differences that underlie human-specific traits, such as increased neuron number and differences in cognitive behaviors. We used human-chimpanzee tetraploid cells to distinguish gene expression changes due to cis -acting sequence variants that change local gene regulation, from trans expression changes due to species differences in the cellular environment. In neural progenitor cells, examination of both cis and trans changes — combined with CRISPR inhibition and transcription factor motif analyses — identified cis -acting, species-specific gene regulatory changes, including to TNIK, FOSL2 , and MAZ , with widespread trans effects on neurogenesis-related gene programs. In excitatory neurons, we identified POU3F2 as a key cis -regulated gene with trans effects on synaptic gene expression and neuronal firing. This study identifies cis -acting genomic changes that cause cascading trans gene regulatory effects to contribute to human neural specializations, and provides a general framework for discovering genetic differences underlying human traits.

Abstract LIMD1 is a tumour suppressor gene frequently lost in non-small cell lung cancer (NSCLC), but its role in cancer-immune cell interactions remains unexplored. Here, we demonstrate that LIMD1 loss results in upregulation of the key immune checkpoint protein PD-L1. Using multi-region sequencing from the TRACERx dataset, we identify that LIMD1 loss is clonal in over 80% of squamous cell carcinoma (LUSC) and 40% of lung adenocarcinoma (LUAD) cases, correlating with increased PD-L1 expression. LIMD1 deficiency results in upregulation of basal and IFNγ-induced PD-L1 expression in NSCLC cells and, consistent with its early loss during oncogenesis, in primary human small airway epithelial cells. Mechanistically, we demonstrate that LIMD1 interacts with the E3 ubiquitin ligase ARIH1 to mediate efficient PD-L1 ubiquitination and degradation, a process that is significantly impaired in LIMD1-deficient cells, resulting in increased PD-L1 stability. As a consequence, LIMD1-deficient tumour cells suppressed CD8+ T cell activation in vitro, and blockade of PD-L1 reversed this suppression. Clinically, we show that LIMD1 loss is associated with enhanced response to immune checkpoint inhibitors (ICIs) in NSCLC patient cohorts, revealing a novel cancer cell-intrinsic correlation of ICI efficacy. Our results uncover a tumour suppressor-mediated mechanism of PD-L1 expression and pave the way for stratified immunotherapy approaches in LIMD1 -/- NSCLC.

Genetic defects in glycine decarboxylase (GLDC) cause non-ketotic hyperglycinemia (NKH), a rare and frequently fatal neurometabolic disease, which lacks FDA-approved therapies. We characterized CRISPR Cas9-edited humanized mice expressing a prevalent clinical mutation after administration with a single intraperitoneal dose of a novel recombinant of adeno-associated viral vector 9 expressing GLDC (rAAV9-GLDC). Long term biological activity of rAAV9-GLDC was first validated by assessment of its systemic efficacy over five and ten months in mice. Access of rAAV9 to the brain was confirmed by tracking green fluorescent protein (GFP) after a single intraperitoneal dose of rAAV9-GFP. Over five months, control ‘mock’ treated GFP-mice showed reduction in astrocytes but not microglia, oligodendrocytes or neurons in the brain. 37% of these animals suffered long term neurological disease and/or death. rAAV9-GLDC boosts astrogenesis without triggering an inflammatory response and confers 100% protection against disease progression and fatality due to NKH.

Microbial and viral co-evolution has created immunity mechanisms involving oligonucleotide signaling that share mechanistic features with human anti-viral systems 1 . In these pathways, including CBASS and type III CRISPR systems in bacteria and cGAS-STING in humans, oligonucleotide synthesis occurs upon detection of virus or foreign genetic material in the cell, triggering the antiviral response 2–4 . In a surprising inversion of this process, we show here that the CRISPR-related enzyme mCpol synthesizes cyclic oligonucleotides constitutively as part of an active mechanism that maintains cell health. Cell-based experiments demonstrated that the absence or loss of mCpol-produced cyclic oligonucleotides triggers cell death, preventing spread of viruses that attempt immune evasion by depleting host cyclic nucleotides. Structural and mechanistic investigation revealed mCpol to be a di-adenylate cyclase whose product, c-di-AMP, prevents toxic oligomerization of the effector protein 2TMβ. Analysis of cells by fluorescence microscopy showed that lack of mCpol allows 2TMβ-mediated cell death due to inner membrane collapse. These findings unveil a powerful new defense strategy against virus-mediated immune suppression, expanding our understanding of oligonucleotides in cell health and disease. These results raise the possibility of similar protective roles for cyclic oligonucleotides in other organisms including humans.

The novel duck reovirus (NDRV) disease presents a significant threat to the poultry industry due to the absence of effective therapeutic measures. As a result, there is an urgent need to develop innovative rapid diagnostic methods for early virus detection. In this study, we developed a Rapid Visual CRISPR Assay to detect the NDRV S3 gene using novel Cas12a orthologs. Specifically, we compared the performance of two candidates, Gs12-16 and Gs12-18, in detecting the NDRV S3 gene to identify a highly sensitive and efficient CRISPR-based diagnostic method. Our results demonstrated that both Gs12-16 and Gs12-18 exhibited strong cis - and trans -cleavage activities for classical “TTTV” protospacer adjacent motif (PAM)-containing targets in vitro , although they required different reaction temperatures. Notably, Gs12-18 showed relatively higher activity for dsDNA targets compared to Gs12-16, indicating that Gs12-18 is more suitable for CRISPR-based nucleic acid detection applications. To leverage these properties, we integrated Gs12-18 with loop-mediated isothermal amplification (LAMP) technology to establish a LAMP-CRISPR/Gs12-18-mediated method for detecting the NDRV S3 gene. This approach enables highly sensitive and visually detectable on-site identification of the NDRV S3 gene, achieving a sensitivity of 38 copies per reaction. Our LAMP-CRISPR/Gs12-18-based method can be utilized for highly sensitive detection of NDRV nucleic acids.

Bacteria use antiphage systems to combat phages, their ubiquitous competitors, and evolve new defenses through repeated reshuffling of basic functional units into novel reformulations. A common theme is generating a nucleotide-derived second messenger in response to phage that activates an effector protein to halt virion production. Phages respond with counter-defenses that deplete these second messengers, leading to an escalating arms race with the host. Here we discover a novel antiphage system we call Panoptes that detects phage infection by surveying the cytosol for phage proteins that antagonize the nucleotide-derived second messenger pool. Panoptes is a two-gene operon, optSE . OptS is predicted to synthesize a second messenger using a minimal CRISPR polymerase (mCpol) domain, a version of the polymerase domain found in Type III CRISPR systems (Cas10) that is distantly related to GGDEF and Thg1 tRNA repair polymerase domains. OptE is predicted to be a transmembrane effector protein that binds cyclic nucleotides. optSE potently restricted phage replication but mutant phages that had loss-of-function mutations in anti-CBASS protein 2 (Acb2) escaped defense. These findings were unexpected because Acb2 is a nucleotide “sponge” that antagonizes second messenger signaling. Using genetic and biochemical assays, we found that Acb2 bound the OptS-synthesized nucleotide, 2′,3′-cyclic adenosine monophosphate (2′,3′-c-di-AMP); however, 2′,3′-c-di-AMP was synthesized constitutively by OptS and inhibited OptE. Nucleotide depletion by Acb2 released OptE toxicity thereby initiating abortive infection to halt phage replication. These data demonstrate a sophisticated immune strategy that hosts use to guard their second messenger pool and turn immune evasion against the virus.

ABSTRACT Animal models with a clinically relevant phenotype remain important for robust evaluation of novel therapeutics for the fatal, X-linked genetic disorder, Duchenne Muscular Dystrophy (DMD). Demonstration of functional improvement is crucial for both patients and regulatory authorities. DMD is associated with a decline in musculoskeletal function with progressive paresis, muscle atrophy and fibrosis: phenotypic features that are also seen in the DE50-MD canine model of DMD. Here we investigate non-invasive methods to quantify changes in activity and behaviour in DE50-MD dogs, using collar-based, tri-axial accelerometers. We measured activity in affected DE50-MD male dogs (3-8 per age point) and littermate wild-type (WT) male controls (3-13 per age point) at monthly intervals from 3 to 18 months of age using Axivity-AX3 accelerometers attached ventrally on each dog’s collar. Data were recorded for 48 hours while dogs remained in their kennels with outside runs following their normal routine. Acceleration vector magnitudes were used to derive various activity indicators over a 24-hour period. Mixed model analyses were used to examine differences between affected and WT groups at different ages. DE50-MD dogs’ activity indicators were significantly higher for % time spent at rest (p<0.001) and significantly lower for all other activity indicators (all p<0.05), when compared to age-matched WT dogs. Sample size calculations reveal that these non-invasive and objective biomarkers offer significant promise for preclinical testing of therapeutics in this model of DMD. Our approach reveals opportunities for cross-model standardisation of activity monitoring methods, applicable to both research and companion animal settings. Summary statement The DE50-MD dog model of Duchenne muscular dystrophy shows significant age-associated reduction in activity quantified through non-invasive, wearable accelerometers. Activity metrics tested show promise for objective assessment of activity patterns for preclinical trials.

Single-cell RNA sequencing and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) screening facilitate the high-throughput study of genetic perturbations at a single-cell level. Characterising combinatorial perturbation effects, such as the subset of genes affected by a specific perturbation, is crucial yet computationally challenging in the analysis of single-cell CRISPR screening datasets due to the sparse and complex structure of unknown biological mechanisms. We propose Gaussian process based sparse perturbation regression (GPerturb) to identify and estimate interpretable gene-level perturbation effects for such data. GPerturb uses an additive structure to disentangle perturbation-induced variation from background noise, and can learn sparse, gene-level perturbation-specific effects from either discrete or continuous responses of perturbed samples. Crucially, GPerturb provides uncertainty estimates for both the presence and magnitude of perturbation effects on individual genes. We validate the efficacy of GPerturb on both simulated and real-world datasets, demonstrating that its prediction and generalisation performance is competitive with existing state-of-the-art methods. Using real-world datasets, we also show that the model reveals interesting gene-perturbation interactions and identifies perturbation effects consistent with known biological mechanisms. Our findings confirm the utility of GPerturb in revealing new insights into the complex dependency structure between gene expressions and perturbations.

TDP-43 is an RNA-binding protein constituting the pathological inclusions observed in ∼95% of ALS and ∼50% of FTD patients. In ALS and FTD, TDP-43 mislocalises to the cytoplasm and forms insoluble, hyperphosphorylated and ubiquitinated aggregates that enhance cytotoxicity and contribute to neurodegeneration. Despite its primary role as an RNA/DNA-binding protein, how RNA-binding deficiencies contribute to disease onset and progression are little understood. Among many identified familial mutations in TDP-43 causing ALS/FTD, only two mutations cause an RNA-binding deficiency, K181E and K263E. In this study, we used CRISPR/Cas9 to knock-in the two disease-linked RNA-binding deficient mutations in SH-SY5Y cells, generating both homozygous and heterozygous versions of the mutant TDP-43 to investigate TDP-43-mediated neuronal disruption. Significant changes were identified in the transcriptomic profiles of these cells, in particular, between K181E homozygous and heterozygous cells, with the most affected genes involved in neuronal differentiation and synaptic pathways. This result was validated in cell studies where the neuronal differentiation efficiency and neurite morphology were compromised in TDP-43 cells compared to unmodified control. Interestingly, divergent neuronal regulation was observed in K181E-TDP-43 homozygous and heterozygous cells, suggesting a more complex signalling network associated with TDP-43 genotypes and expression level which warrants further study. Overall, our data using cell models expressing the ALS/FTD disease-causing RNA-binding deficient TDP-43 mutations at endogenous levels show a robust impact on transcriptomic profiles at the whole gene and transcript isoform level that compromise neuronal differentiation and processing, providing further insights on TDP-43-mediated neurodegeneration.

SUMMARY Spiral ganglion neurons (SGNs) are crucial for hearing, and the loss of SGNs causes hearing loss. Stem cell-based therapies offer a promising approach for SGN regeneration and require understanding the mechanisms governing SGN differentiation. We investigated the chromatin remodeler CHD7 in neuronal differentiation using immortalized multipotent otic progenitor (iMOP) cells. We demonstrated that CHD7 knockdown impaired neuronal differentiation. Genome-wide analysis revealed CHD7 binding at diverse cis -regulatory elements, with notable enrichment at sites marked by the insulator-binding protein CTCF between topologically associating domains (TADs). Insulators marked by the enrichment of CHD7 and CTCF resided near genes critical for neuronal differentiation, including Mir9-2 . Targeting these regulatory regions in iMOPs with CRISPR interference (CRISPRi) and activation (CRISPRa) increased miR-9 transcription, irrespective of the method. Blocking the CHD7 and CTCF marked sites suggested that the elements function as insulators to regulate gene expression. The study highlights CHD7 activity at insulators and underscores an unreported mechanism for promoting neuronal differentiation.

ABSTRACT Insulin-like growth factor 1 (IGF1) is produced primarily in the placenta in utero and is an essential hormone for neurodevelopment. Specifically, how placental IGF1 production persistently influences the brain is unclear. This study evaluated the effects of placental Igf1 overexpression on embryonic and postnatal brain development, particularly for striatum, a region highly linked to neurodevelopmental disorders. Placental Igf1 was overexpressed via placental-targeted CRISPR manipulation. This overexpression altered placenta structure and function distinctly in females and males. Early differences in placental function altered the trajectory of striatal development, as adult females showed persistent changes in striatal cell composition and striatal dependent behavior while males were less affected in brain and behavior outcomes. Overall, these results demonstrate that placental Igf1 expression alters striatal development and behavior in ways relevant to neurodevelopmental disorders. These findings expand our understanding of placental influence on neurodevelopment and will aid in identifying placental-targeted preventive interventions. GRAPHICAL ABSTRACT

Droplet-based organoid culture offers several advantages over conventional bulk organoid culture, such as improved yield, reproducibility, and throughput. However, organoids grown in droplets typically display only a spherical geometry and lack the intricate structural complexity found in native tissue. By incorporating singularized pancreatic ductal adenocarcinoma cells into collagen droplets, we achieve the growth of branched structures, indicating a more complex interaction with the surrounding hydrogel. A comparison of organoid growth in droplets of different diameters showed that while geometrical confinement improves organoid homogeneity, it also impairs the formation of more complex organoid morphologies. Thus, only in 750 µ m diameter collagen droplets did we achieve the consistent growth of highly branched structures with a morphology closely resembling the structural complexity achieved in traditional bulk organoid culture. Moreover, our analysis of organoid morphology and transcriptomic data suggests an accelerated maturation of organoids cultured in collagen droplets, highlighting a shift in developmental timing compared to traditional systems.

Cancers, especially fusion oncoprotein (FO)-driven hematological cancers and sarcomas, often develop from a low number of key mutations. Solitary Fibrous Tumor (SFT) is a rare mesenchymal tumor driven by the NAB2-STAT6 oncofusion gene. Currently, the treatment options for SFT remain limited, with anti-angiogenic drugs providing only partial responses and an average survival of two years. To address this challenge, we constructed SFT cell models harboring specific NAB2-STAT6 fusion transcripts using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology. High-throughput drug screens demonstrated that the BET inhibitor Mivebresib can differentially reduce proliferation in SFT cell models. Subsequently, BET inhibitors Mivebresib and BMS-986158 efficiently reduced tumor growth in an SFT patient-derived xenograft (PDX) animal model. Furthermore, our data showed that NAB2-STAT6 fusions may lead to higher levels of DNA damage in SFTs. Consequently, combining BET inhibitors with PARP (Poly (ADP-ribose) polymerase) or ATR inhibitors significantly enhanced anti-proliferative effects in SFT cells. Taken together, our study established BET inhibitors Mivebresib and BMS-986158 as promising anti-SFT agents.

The increasing prevalence of bacteria resistant to many or all types of antibiotics poses a major health crisis. Novel classes of antibiotics are only slowly being developed and alternative strategies are needed to tackle the issue. Mobile genetic elements and class 1 integrons are important facilitators for antibiotic resistance genes, with the latter being highly conserved in human pathogens. The growing prevalence of multidrug-resistant bacteria and the paucity in the development of new antibiotics underscore the urgent need for innovative approaches in the treatment of pathogens. Among these, CRISPR-Cas nucleases can be used to cleave acquired resistance genes, leading to either plasmid curing or cell death if the target is on a chromosome. In this study, we investigate the feasibility of using class 1 integrons as a target for Cas9-based cleavage leading to re-sensitizing antibiotic-resistant bacteria. We analyze the conserved and widespread integrase gene intI1 and conclude that it is a suitable target for Cas-based re-sensitization due to its high sequence conservation and its occurrence largely limited to human pathogens, alleviating the risk of targeting benign bacteria. We developed a broad host range conjugative plasmid encoding a class 1 integron-targeting Cas9 system that leads to removal of resistance plasmids in target bacteria with subsequent re-sensitization towards antibiotics. We find that 290 distinct ARGs co-occur on int1 -harboring plasmids, showing the potential for re-sensitization towards a very broad range of antibiotics.

DNA double-strand breaks (DSBs) pose a critical threat to cellular proliferation and genomic integrity. Upon genotoxic stress, the DNA damage response (DDR) rapidly activates repair pathways and halts cell cycle progression through checkpoint activation. Previously, we demonstrated that DDR-activated c-Abl tyrosine kinase (ABL1) attenuates error-prone late-phase DSB repair. However, the broader functional implications of c-Abl in DDR regulation, and the fate of any residual unrepaired DNA fragments remained poorly understood. Here, we show that c-Abl regulates G2/M checkpoint release by targeting Polo-like kinase1 (Plk1). Depletion or inhibition of c-Abl resulted in increased G2-M accumulation and impaired checkpoint exit. We identified Y217 as a c-Abl phosphorylation site on Plk1, important for Plk1-mediated Claspin destabilization, a key step in G2/M checkpoint release. CRISPR-mediated introduction of the phopsho-silencing Plk1 Y217F mutation or the phospho-mimicking Y217E mutation into cells resulted in impaired and enhanced G2/M checkpoint release, respectively. Intriguingly, c-Abl-mediated G2/M checkpoint release correlated with elevated DNA damage-induced micronuclei (MNi) formation. Depletion or inhibition of c-Abl reduced MNi formation, whereas induction of c-Abl expression increased it, implicating c-Abl as an active effector in both processes. We propose a trade-off model whereby, following the rapid initial repair phase, c-Abl shifts cellular priorities from prolonged, potentially error-prone DSB repair toward cell cycle resumption, thereby promoting G2-M checkpoint exit and DDR deactivation at the cost of increased MNi formation. Our findings describe a novel regulatory DDR axis involving c-Abl and Plk1 and provide mechanistic insights into how DDR termination is orchestrated.

Summary ▪ In strawberry, the axillary bud (AXB) can produce either an elongated stem called stolon giving a daughter-plant (asexual reproduction) or an inflorescence-bearing branch crown (BC) (sexual reproduction). The fate of the AXB depends on node position on the axis and on genetic and environmental factors. Here, in Fragaria vesca , we addressed the largely unanswered question of how molecular factors determine AXB fate. ▪ To get insights into the mechanisms already at play in a morphologically indistinguishable (undifferentiated) AXB, depending on its fate, we combined (1) the phenotypic characterization of AXB development throughout plant growth with (2) the RNA-seq analysis of undifferentiated AXBs, using three different genotypes producing either BCs or stolons ( fvetfl1 and fvega20ox mutants, FveFT3 overexpressor). ▪ Results: allowed the identification of genes regulating AXB fate and outgrowth, among which FveBRC1 . The analysis of FveBRC1 expression in genotypes combining various traits (perpetual/seasonal flowering; runnering/runneless) and the generation of CRISPR/Cas9 brc1 mutants further demonstrated that FveBRC1 plays a central role in the determination of AXB fate in strawberry, in addition to its well-known function in BC outgrowth. ▪ These original results provide new insights into the determination of AXB fate and, consequently, the control of fruit yield in strawberry.

Although lifespan has long been the focus of ageing research, the need to enhance healthspan - the fraction of life spent in good health - is a more pressing societal need. Caloric restriction improves healthspan across eukaryotes but is unrealistic as a societal intervention. Here, we describe the rewiring of a highly conserved nutrient sensing system to prevent senescence onset and declining fitness in budding yeast even when aged on an unrestricted high glucose diet. We show that AMPK activation can prevent the onset of senescence by activating two pathways that remove excess acetyl coenzyme A from the cytoplasm into the mitochondria - the glyoxylate cycle and the carnitine shuttle. However, AMPK represses fatty acid synthesis from acetyl coenzyme A, which is critical for normal cellular function and growth. AMPK activation therefore has positive and negative effects during ageing. Combining AMPK activation with a point mutation in fatty acid synthesis enzyme Acc1 that prevents inhibition by AMPK (the A2A mutant) allows cells to maintain fitness late in life without reducing the mortality associated with advanced age. Our research shows that ageing in yeast is not intrinsically associated with loss of fitness, and that metabolic re-engineering allows high fitness to be preserved to the end of life.

Abstract Background: Engineers seeking to generate natural product analogs through altering modular polyketide synthases (PKSs) face significant challenges when genomically editing large stretches of DNA. Results: We describe a CRISPR-Cas9 system that was employed to reprogram the PKS in Streptomyces venezuelae ATCC 15439 that helps biosynthesize the macrolide antibiotic pikromycin. We first demonstrate its precise editing ability by generating strains that lack megasynthase genes pikAI - pikAIV or the entire pikromycin biosynthetic gene cluster but produce pikromycin upon complementation. We then employ it to replace 4.4-kb modules in the pikromycin synthase with those of other synthases to yield two new macrolide antibiotics with activities similar to pikromycin. Conclusion: Our gene-editing tool has enabled the efficient replacement of extensive and repetitive DNA regions within streptomycetes.

ABSTRACT Deubiquitinases (DUBs) form a specific class of proteases removing ubiquitin from target proteins. They are involved in the regulation of many cellular processes including cell growth and proliferation. Among them, USP36 is a key regulator of the oncogenic transcription factor c-Myc, preventing its degradation by the proteasome. These two proteins form an evolutionary conserved complex providing the opportunity to investigate USP36 mechanisms of action in vivo in a genetically tractable model such as Drosophila melanogaster . Null mutants of dUsp36 die early during larval development and exhibit severe growth defects. Strikingly, we report here that flies carrying a CRISPR/Cas9-induced catalytic mutation of dUsp36 survive to adulthood with only minor growth defects, yet males are infertile. This finding indicates that dUSP36 deubiquitinating activity is dispensable for cell growth but essential for spermatogenesis. Our results thus reveal that dUSP36 functions through both catalytic-dependent and catalytic-independent mechanisms, highlighting a dual mode of action with implications for the understanding of DUBs mechanism of action.

Understanding and manipulating the complexity of microbial community diversity, including the mobile genetic elements contained within, represents a great challenge that has wide-ranging potential benefits across synthetic biology, agriculture and medicine. An important component to this complexity is the acquisition of genetic material via conjugative plasmids, which can encode new functions such as antibiotic resistance, and the degradation of those plasmids by CRISPR interference. In this work we use single-cell tracking of E. coli populations in microfluidic devices coupled with high resolution fluorescence microscopy to characterize plasmid dynamics at the single-cell and population level. On a population level we find that the ability of cells to clear plasmids is highly dependent on both the number of spacer targets present and the defense expression level. Additionally we assess the impact that counter-defense mechanisms such as plasmid addiction have on plasmid population dynamics and show that CRISPR may be an ineffective method to counter plasmids with such mechanisms. By leveraging single-cell tracking, we were also able to report conjugation rate per neighboring donor cell, estimate the latent period between plasmid uptake and subsequent onward transmission, and corroborate existing estimates of cascade search time. This synthesis of population and single-cell measurements suggests that plasmids are the subject of a dynamic tug-of-war between defense expression, spacer distribution, neighboring cell identity and plasmid cost-benefit tradeoffs. The use of imaging and analysis techniques used here and subsequent multi-scale measurements will facilitate the disentanglement of how these factors coordinate to realize community-wide plasmid dynamics in diverse contexts.