Abstract The yeast Saccharomyces cerevisiae is widely employed in industrial biotechnology for chemical and pharmaceutical production. However, engineering yeast for high product titers remains challenging due to metabolic imbalances and competition for cellular resources. To address this, we developed an orthogonal quorum sensing (QS) system based on N -acyl-homoserine lactones (AHLs) for cell density-dependent regulation in yeast. Using metabolic engineering, we established AHL production in yeast. Next, we improved AHL-biosensors via directed evolution and a novel growth-based screening strategy with amdS as a counter-selectable marker. We identified LuxR variants with enhanced sensitivity, which were engineered for QS-controlled expression of a reporter gene. Additionally, we engineered LuxR to function as a repressor, achieving QS-dependent repression. The QS system was applied to enhance aloesone production, a plant-derived metabolite with cosmetic and pharmaceutical applications. The established system showed 51% increased production through QS-controlled repression of FAS1 . This work establishes a versatile QS-based regulatory platform to support dynamic pathway regulation for metabolic engineering in yeast.

Abstract Stargardt’s Disease (STGD1), Retinitis pigmentosa (RP), and Leber’s congenital amaurosis (LCA) inherited eye conditions that are among the main causes of blindness and visual impairment. The primary cause of these diseases, which lead to gradual vision loss, is genetic mutations affecting retinal cells. Gene-editing technologies, especially the CRISPR-Cas9 system, are emerging as promising ways to correct harmful genetic mutations. This review focuses on how effectively CRISPR-Cas9 can be used to treat certain inherited eye conditions. In experiments with patient-derived human induced pluripotent stem cells (hiPSCs), researchers were able to fix STGD1 mutations in the ABCA4 gene using CRISPR-Cas9. The corrected cells retained their ability to differentiate into different cell types and showed minimal off-target effects. This suggests the approach could offer a safe and precise way of treating these conditions. In RP, CRISPR-Cas9 was used to target the RPGR gene in mice models, successfully preserving the shape and function of the photoreceptor cells. The CEP290 mutation in LCA10 was corrected in human patients using the CRISPR-Cas9 based EDIT-101 in the BRILLIANCE clinical trial, which demonstrated a notable improvement in visual outcomes with minimal adverse effects. Even though the results so far are promising, challenges remain including long-term safety, the administration of these treatments, and the risk of unintended effects. This review emphasizes the need for further research and clinical trials to improve these therapies. It also highlights the exciting potential of CRISPR-Cas9 as a curative treatment for inherited retinal disorders.

ABSTRACT Mutations in the dystrophin (DMD) gene can cause a spectrum of muscle-wasting disorders ranging from the milder Becker muscular dystrophy (BMD) to the more severe Duchenne muscular dystrophy (DMD). Among these, exon 45 deletion is the most frequently reported single exon deletion in DMD patients worldwide. In this study, we generated a novel rat model with an exon 45 deletion using CRISPR/Cas9 technology. The Dmd Δ45 rat recapitulate key clinical and molecular features of DMD, including progressive skeletal muscle degeneration, cardiac dysfunction, cognitive deficits, elevated circulating muscle damage biomarkers, impaired muscle function, and overall reduced lifespan. Transcriptomics analyses confirmed the deletion of exon 45 and revealed gene expression patterns consistent with dystrophin deficiency. In the skeletal muscle, RNA-seq profiles demonstrated a transition from early stress responses and regenerative activity at 6 months to chronic inflammation, fibrosis, and metabolic dysfunction by 12 months. Similarly, the cardiac transcriptomic shifted from an early inflammatory and stress-responsive state to one characterized by fibrotic remodelling and metabolic impairment. Despite these pathological features, the Dmd Δ45 rats exhibited a milder phenotype than other DMD rat models. This attenuation may be attributed to spontaneous exon 44 skipping, which partially restores the reading frame and results in an age-dependent increase in revertant dystrophin-positive fibres. Further analysis indicated downregulation of spliceosome-related genes, suggesting a potential mechanism driving exon skipping in this model. In summary, the Dmd Δ45 rat represents a valuable model for investigating both the molecular determinants of phenotypic variability and the endogenous mechanisms of exon skipping. These findings offer important insights for the development of personalized exon-skipping therapies, particularly for DMD patients with exon 45 deletions.

A GGGGCC repeat expansion in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The repeat expansion is translated into five different dipeptide repeat proteins: polyGA, polyGP, polyGR, polyAP and polyPR. To investigate the effect of polyGA, which is the most abundant dipeptide repeat protein in patient brains, we used CRISPR/Cas9 to insert 400 codon-optimized polyGA repeats immediately downstream of the mouse C9orf72 start codon. This generated (GA)400 knock-in mice driven by the endogenous mouse C9orf72 promoter, coupled with heterozygous C9orf72 reduction. (GA)400 mice develop subtle pathology including mild motor dysfunction characterized by impaired rotarod performance. Quantitative proteomics revealed polyGA expression caused protein alterations in the spinal cord, including changes in previously identified polyGA interactors. Our findings show that (GA)400 mice are a complementary in vivo model to better understand C9ALS/FTD pathology and determine the specific role of single DPRs in disease.

ABSTRACT Enhancers are key gene regulatory elements that ensure the precise spatiotemporal execution of developmental gene expression programmes. However recent findings indicate that approaches to identify enhancers may not capture the full repertoire of active enhancers in mammalian genomes. Here, we combine massively parallel enhancer assays with chromatin structure and transcriptome profiling to functionally annotate enhancers genome-wide in human induced pluripotent stem cells. We find that a substantial fraction (∼40%) of accessible chromatin regions with enhancer function lack key features associated with active enhancers, including the active enhancer mark histone H3 lysine 27 acetylation and enhancer-associated RNAs. Perturbation of this class of non-canonical enhancers by CRISPR-mediated epigenome editing results in decreased levels of target gene expression and, in one instance, loss of pluripotent stem cell characteristics. Collectively, our data demonstrate enhancer activity for a class of gene regulatory elements that had until now only been associated with a neutral or inactive status, challenging current models of enhancer function.

The laboratory contaminant Noodlococcus was named for its coccoid cells and unusual colony morphology, which resembled a pile of noodles. Along with laboratory characterisation and electron microscopy, we generated a complete Noodlococcus genome sequence using Illumina and Oxford Nanopore data. The genome consisted of a single, circular, 2732108 bp chromosome that shared 97.5% Average Nucleotide Identity (ANI) with the Kocuria rhizophila type strain TA68. We identified genomic features involved in replication ( oriC ), carotenoid synthesis ( crt ), and genome defence (CRISPR-Cas), and discovered four novel mobile elements (IS Krh4-7 ). Despite its environmental ubiquity and relevance to food production, bioremediation, and human medicine, there have been few genomic studies of the Kocuria genus. We conducted a comparative, phylogenetic, and pangenomic examination of all 257 publicly available Kocuria genomes, with a particular focus on the 56 that were identified as K. rhizophila . We found that there are two phylogenetically distinct clades of K. rhizophila , with within-clade ANI values of 96.7-100.0% and between-clade values of 89.5-90.4%. The second clade, which we refer to as K. pseudorhizophila , exhibited ANI values of <95% relative to TA68 and constitutes a separate species. Delineation of the two clades would be consistent with the rest of the genus, where all other species satisfy the 95% ANI threshold criteria. Differences in the K. rhizophila and K. pseudorhizophila pangenomes likely reflect phenotypic as well as evolutionary divergence. This distinction is relevant to clinical and industrial settings, as strains and genomes from both clades are currently used interchangeably, which may lead to reproducibility issues and phenotype-genotype discordance. Investigating an innocuous laboratory contaminant has therefore provided useful insights into the understudied species K. rhizophila , prompting an unexpected reassessment of its taxonomy. Impact statement Bacterial genome sequence databases are dominated by a relatively small number of medically relevant genera, while most of the global bacterial population’s diversity is largely uncharacterised. Kocuria is a widespread bacterial genus with industrial and medical relevance. Despite its ubiquity, only 22 complete and 235 draft Kocuria genomes were publicly available at the outset of this study. Our phylogenetic and pangenomic examination of all available Kocuria genomes was the first for this genus, providing insights into its diversity and taxonomy. Most notably, we found that Kocuria rhizophila is comprised of two clades that are sufficiently divergent to constitute different species, but are frequently used interchangeably in experimental and genomic research. The complete, high-quality Noodlococcus genome generated and characterised here can serve as a reference for true K. rhizophila , particularly while there is only a draft genome sequence available for type strain TA68. Data summary Sequencing reads and the assembled Noodlococcus genome are available from NBCI BioProject accession PRJNA835814 and BioSample accession SAMN28111796. The complete sequence of the Noodlococcus chromosome can be found in the GenBank nucleotide database under accession number CP097204.1 . Entries for the novel insertion sequences IS Krh4 to IS Krh7 can be found in the ISFinder database ( https://isfinder.biotoul.fr ).

The high cost and attrition rate of drug development underscore the need for more effective strategies for therapeutic target discovery. Here, we present a network medicine-based machine learning framework that integrates single-cell transcriptomics, bulk multi-omic profiles, genome-wide CRISPR perturbation screens, and protein-protein interaction networks to systematically prioritise disease-specific targets. Applied to clear cell renal cell carcinoma, the framework successfully recovered established targets and predicted five therapeutic candidates, with subsequent in vitro validation demonstrating that among these, ENO2 inhibition had the strongest anti-tumour effect, followed by LRRK2, a repurposing candidate with phase III Parkinson’s disease inhibitors. The proposed approach advances target discovery by moving beyond single-feature, single-modality heuristics to a scalable, machine learning-driven strategy that is generalisable across diseases.

Summary We previously found that specific exhausted T cell subsets defined response, but not resistance, to donor lymphocyte infusions (DLI), a curative immunotherapy for leukemic relapse following allogeneic stem cell transplant (SCT). To identify leukemia molecular pathways that drive resistance, we analyzed whole exome and targeted mutation panel sequencing in two independent cohorts of DLI-treated patients, nominating oncogenic, truncating mutations in ASXL1 ( ASXL1 MUT ) as the genetic basis for DLI resistance. Deep interrogation of 138,152 bone marrow single myeloid cell transcriptomes (scRNA-seq) from this cohort linked DLI resistance to a transcriptional state notable for leukemic stem cell identity and HLA-I suppression. In silico analysis of publicly available scRNA- and ATAC-seq data in acute myeloid and chronic myelomonocytic leukemias, respectively, confirmed an association between ASXL1 MUT and HLA-I suppression across myeloid malignancies. CRISPR correction of the endogenous ASXL1 MUT in the K562 leukemic cell line increased HLA-I, but not HLA-II, surface protein expression through increased deposition of the activating H3K4Me3 mark with only modest effect on the repressive H3K27Me3 mark, suggesting a Polycomb-independent mechanism of action. Indeed, inhibitors of EZH2, a critical component of the PRC2 complex, significantly upregulated HLA-I surface protein expression independently of ASXL1 MUT , suggesting that EZH2 inhibition could bypass ASXL1 MUT -mediated HLA-1 suppression. Importantly, ASXL1 CORRECTION significantly increased CD8+ T cell recognition, activation and killing, and ASXL1 MUT -mediated T cell suppression could be overcome by EZH2 inhibition. Thus, by integrating molecular analyses with immuno-functional studies, we define a novel oncogene-driven pathway of immune evasion and propose a therapeutic strategy to re-engage T cell killing in ASXL1 MUT tumors.

ABSTRACT Missense variants in EXOSC3, an RNA exosome subunit, have been identified in patients with PCH1b. We investigated three missense variants in the S1 domain of EXOSC3, including one variant of uncertain significance (VUS) and two pathogenic variants (hence S1 variants). EXOSC3 S1 variant cell lines were generated using CRISPR-Cas9 resulting in widespread proteome changes including decreases in some RNA exosome subunits paired with increases in the catalytic subunit DIS3. Thermal stability, analyzed by PISA, revealed extensive destabilization of RNA exosome cap subunits and the cap-associated exonuclease EXOSC10. Functionally, S1 variants altered rRNA processing with corresponding protein compensation observed in rRNA processing proteins outside the RNA exosome. Exogenous overexpression of EXOSC3 rescues many molecular defects caused by S1 variants suggesting that protein destabilization and turnover strongly contribute to molecular defects. Overall, our findings define the mechanisms through which cells respond to EXOSC3 S1 variant disruption of RNA processing homeostasis.

ABSTRACT Cardiac arrhythmias afflict tens of millions of people, causing one-fifth of all deaths 1 . Although mouse models have aided understanding of some pacemaker genes and arrhythmias 2,3 , mice are not known to naturally acquire arrhythmias, and the substantial differences between mouse and human cardiac anatomy and physiology have limited their utility in preclinical studies and pharmacological testing 2,4 . To establish a primate genetic model organism for arrhythmias, we carried out an electrocardiographic (ECG) screen of over 350 lab and wild mouse lemurs ( Microcebus spp. ), an emerging model organism that is among the smallest, fastest-reproducing, and most abundant primates 5 . Twenty-two lemurs (6.2%) were identified with eight different naturally-occurring arrhythmias resembling human ECG pathologies (SSS, PACs, Afib, PVCs, NSVT, STD, iTWs, STE). Pedigree construction showed two were familial, premature atrial contractions (PACs)/atrial fibrillation (Afib) and sick sinus syndrome (SSS), an episodic bradycardia. Genome sequencing of the SSS pedigree mapped the disease locus to a 1.4 Mb interval on chromosome 7 and supported autosomal recessive Mendelian inheritance. The most appealing candidate gene in the interval was SLC41A2 , a little studied magnesium transporter 6,7 . SLC41A2 is expressed in human iPSC-derived sinoatrial node cells (iSANC) and localizes to the sarcoplasmic reticulum. Although mouse SLC41A2 knockouts do not show a cardiac pacemaker phenotype 8 , CRISPR-mediated SLC41A2 knockout altered human iSANC magnesium dynamics and slowed their calcium transient firing rate. The results suggest SLC41A2 functions cell autonomously and primate-specifically in cardiac pacemaker cells, and that intracellular magnesium dynamics have a crucial but previously unappreciated role in setting pacemaker rate. Thus, mouse lemur is a valuable model for discovering new genes, molecules, and mechanisms of the primate pacemaker, and for identifying novel candidate genes and therapeutic targets for human arrhythmias. The approach can be used to elucidate other primate diseases and traits.

Pseudoalteromonas has been used as a model system to study cold adaptation and is of widespread interest in biotechnology and ecology. To explore its physiological responses to extreme cold, uncover functional genes, and clarify their ecological roles, efficient genetic tools are essential. However, existing genetic manipulation methods in Pseudoalteromonas rely on traditional homology-based recombination, which is inefficient and time-consuming. Consequently, improving editing efficiency is crucial for advancing both basic research and applied potential. Here, we introduced the CRISPR/Cas9 system into Pseudoalteromonas for the first time, and conducted an extensive investigation into the application of the Type II CRISPR/Cas9 system for gene editing in Pseudoalteromonas fuliginea , a representative species thriving in the frigid polar oceans. To validate the feasibility of the CRISPR/Cas system in P. fuliginea , multiple genes were selected as targets and confirmed the gene editing effects through phenotypic changes or gene expression. We have successfully achieved both gene knockouts and insertions in P. fuliginea , encompassing the deletion of genes such as fliJ , indA , and genes encoding Pf sRNAs, as well as the in vivo insertion of 3×flag and the gfp gene. The average CRISPR/Cas9 gene editing efficiency in P. fuliginea exceeded 70% (range: 73.3%-95.8%), which is significantly higher than the traditional homology-based approach (less than 0.1%). In summary, we developed an efficient CRISPR/Cas9-based editing system in P. fuliginea , which can be utilized to accelerate the development of Pseudoalteromonas as a model system for addressing fundamental questions related to extreme environmental adaptation and to fulfill its potential biotechnological applications. IMPORTANCE Pseudoalteromonas fuliginea is a marine bacterium with great potential for ecological and biotechnological research, yet its genetic manipulation has long been a technical challenge. In this study, we developed a gene editing system based on CRISPR technology that enables efficient and precise genome modification in this organism. Using this system, we successfully deleted, inserted, and tagged multiple genes, including regulatory and non-coding elements, with high success rates. Notably, several of these genes are linked to key traits such as motility and stress response, which contribute to microbial adaptation in polar environments. This tool allows researchers to directly test gene function and study microbial adaptation in cold marine environments. The ability to perform reliable genetic edits in Pseudoalteromonas fuliginea opens new possibilities for its use as a model organism and will support future advances in microbial ecology, environmental microbiology, and marine biotechnology.

ABSTRACT The spatial organization of RNA condensates is fundamental for understanding of basic cellular functions, but may also provide pivotal insights into diseases. One of the major challenges to understanding the role of condensates is the lack of technologies to map condensate-scale protein architecture at subcompartmental or nanoscale resolution. To address this, we introduce HCR-Proxy, a proximity labelling technique that couples Hybridization Chain Reaction (HCR)-based signal amplification with in situ proximity biotinylation (Proxy), enabling proteomic profiling of RNA-proximal proteomes at subcompartmental resolution. We benchmarked HCR-Proxy using nascent pre-rRNA targets to investigate the distinct proteomic signatures of the nucleolar subcompartments and to uncover a spatial logic of protein partitioning shaped by RNA sequence. Our results demonstrate HCR-Proxy’s ability to provide spatially-resolved maps of RNA interactomes within the nucleolus, offering new insights into the molecular organisation and compartmentalisation of condensates. This subcompartment-specific nucleolar proteome profiling enabled integration with deep learning frameworks, which effectively confirmed a sequence-encoded basis for protein partitioning across nested condensate subcompartments, characterised by antagonistic gradients in charge, length, and RNA-binding domains. HCR-Proxy thus provides a scalable platform for spatially resolved RNA interactome discovery, bridging transcript localisation with proteomic context in native cellular environments. GRAPHICAL ABSTRACT Bullet points - HCR-Proxy enables the first nanoscale-resolution mapping of RNA-proximal proteomes in situ . - HCR-Proxy establishes a broadly applicable modular platform for spatially resolved RNA– interactomics. - Subcompartmental proteomes are resolved across nucleolar subdomains by targeting specific pre-rRNA regions. - Deep learning confirms a sequence-encoded logic of protein partitioning within condensate subcompartments.

ABSTRACT The androgen receptor (AR) is a critical driver of prostate cancer (PCa). To study regulators of AR protein levels and oncogenic activity, we created the first live cell quantitative endogenous AR fluorescent reporters. Leveraging this novel AR reporter, we performed genome-scale CRISPRi flow cytometry sorting screens to systematically identify genes that modulate AR protein levels. We identified and validated known AR protein regulators including HOXB13 and GATA2 and also unexpected top hits including PTGES3, a poorly characterized gene in PCa. PTGES3 repression resulted in loss of AR protein, cell cycle arrest, and cell death in AR-driven PCa models. PTGES3 is not a commonly essential gene, and our data nominate it as a prime PCa therapeutic target. Clinically, analysis of PCa data demonstrate that PTGES3 expression is associated with AR-directed therapy resistance. Mechanistically, we show PTGES3 binds directly to AR, forms a protein complex with AR in the nucleus, regulates AR protein stability in vitro and in vivo and modulates AR function in the nucleus at AR target genes. PTGES3 represents a novel therapeutic target for overcoming known mechanisms of resistance to existing AR-directed therapies in PCa.

Abstract Background The small molecule SR8278 was initially identified as an antagonist of the REV-ERB (reverse c-ERBAa) nuclear receptor proteins, which play an important role in metabolism and circadian rhythms. Though SR8278 has been shown to have beneficial physiological effects in a variety of preclinical disease contexts, its impact on gene expression and cell proliferation in keratinocytes has not previously been examined. Methods An RNA-seq analysis was used to identify genes differentially impacted by SR8278 treatment in human HaCaT keratinocytes, which was confirmed by RT-qPCR and western blotting. Cell growth and viability assays were further used to examine cell proliferation in HaCaT and other cell lines. CRISPR/Cas9 genome editing was used to generate cells lacking REV-ERBα and β. Results RNA-seq analysis indicated genes involved in the G1/S transition of the cell cycle were significantly impacted by SR8278 treatment, which was confirmed via RT-qPCR and western blotting. Cell proliferation assays showed that SR8278 slowed cell growth but did not induce apoptosis. Finally, the knockout of the REV-ERBs did not impact the effect of SR8278 on gene expression and cell proliferation. Conclusions We conclude that the anti-proliferative effects of SR8278 are not mediated by the REV-ERB proteins, and thus care should be taken when interpreting studies involving this compound unless complementary genetic approaches are also shown.

Genome replication start sites, called origins, begin to be specified by Origin Recognition Complex (ORC) proteins prior to replication through a process called origin licensing. Once licensed, origins become active and initiate DNA synthesis with varying efficiencies influenced by local chromatin environment, transcription, and 3D genome organization. ORC proteins have also been implicated in regulating chromatin state and nuclear organization. However, it is unclear if there is interplay between ORC and the chromatin architectures underlying origin activation, as we lack a systems-level understanding of how ORC proteins interact, post-licensing, with the nuclear environments conducive to genome synthesis. To infer this context-specific ORC interactome, I used data from genome-wide CRISPR fitness screens, a cell-wide proximity labeling study, and proteomic profiling of nascent DNA-associated proteins to identify 17 novel factors that genetically and proteomically interact with ORC subunits and genome replication. Unexpectedly, the candidate pool was significantly enriched for factors involved in the homeostasis of RNA Polymerase III (Pol III) transcripts, particularly 5S ribosomal RNA (rRNA) and transfer RNA (tRNA). Follow-up protein-protein structure predictions by AlphaFold 3 (AF3) proposed direct interactions between ORC subunits and Pol III transcript biogenesis factors, as well as epigenetic regulators and a cyclin-dependent kinase. Given the prominence of 5S rRNA and tRNA biogenesis factors in my results, and prior reports of ORC subunits binding RNA, I also used protein-RNA structure prediction to identify candidate ORC3 interactions with 5S rRNA and tRNA. Altogether, my analysis integrates biological process, molecular proximity in human cells, and structural prediction to nominate novel protein and RNA interactions for involvement in the human replication program. These results augment and expand current models for ORC function and origin activation, particularly those involving chromatin state and transcriptional activity, and generate testable hypotheses to explore the interdependencies of replication patterning, histone modification, and nuclear RNA homeostasis.

Bioluminescence monitoring techniques have greatly contributed to revealing a variety of biological regulatory systems in living organisms, including circadian clocks. In plant science, these techniques are applied to long-term quantitative analyses of gene expression behavior. Transient transfection with a luciferase reporter using the particle bombardment method has been used for bioluminescence observations at the single-cell level. This allows for capturing heterogeneity and temporal fluctuations in cellular gene expression. We developed a novel CRISPR/Cas9-induced restoration of bioluminescence reporter system, CiRBS, to monitor cellular bioluminescence from a reporter gene in the genome of transgenic Arabidopsis . In this method, the enzymatic activity of an inactive luciferase mutant , LUC40Ins26bp , which has a 26-bp insertion at the 40th codon, was restored by introducing an indel at the insertion site using CRISPR/Cas9. We succeeded in long-term monitoring of the cellular bioluminescence of Arabidopsis plants expressing LUC40Ins26bp , which was restored by transient transfection with CRISPR/Cas9-inducible constructs using particle bombardment. Recombination events via indels were mostly complete within 24 h of CRISPR/Cas9 induction, and 7.2% of CRISPR/Cas9-transfected cells restored bioluminescence. It was estimated that 94% of the bioluminescence-restored cells carried only one chromosome having the optimal recombination construction. Thus, CiRBS allows for reliable single-cell gene expression analysis of cell-to-cell heterogeneity and temporal fluctuations from a single locus.

ABSTRACT CRISPR-Cas9–mediated gene editing was used to generate specific mutants of the bantam gene in Drosophila melanogaster . To drive non-homologous end joining (NHEJ) and achieve a precise deletion of most of the bantam locus, two guide RNAs targeting sites 90 base pairs apart were expressed in the germline using the UAS/GAL4 system. Thirty lethal and eight viable lines were established and analyzed. One lethal line exhibited the expected 90 bp deletion, while the others carried diverse indels at one or both cleavage sites. Among the viable lines, seven harbored a single-nucleotide deletion that did not disrupt bantam function. Notably, one viable line, ban d1-44 , carried a hypomorphic allele that reduced organismal size without affecting viability. To generate precisely edited bantam variants, CRISPR-Cas9–mediated homology-directed repair (HDR) was used using donor plasmids containing engineered mutations in the miRNA seed region, along with a scarless dsRED fluorescent marker. Approximately 40% of the resulting fluorescent lines were correctly edited, demonstrating the efficiency of this strategy for producing specific bantam variants. The remaining lines exhibited unexpected outcomes, among which partial HDR events, where the dsRED marker was integrated but not the bantam mutations, and full donor plasmid integrations, which led to duplication of the bantam locus. These findings reveal the complexity of CRISPR-Cas9 outcomes, emphasizing the need for thorough screening and characterization of individual candidates in gene-editing experiments. They also provide valuable insights for optimizing genome editing strategies. Article summary The authors used Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) gene editing to mutate the bantam microRNA in Drosophila melanogaster . They guided Non-Homologous End Joining to induce a precise 90 bp deletion. It was the rarest occurrence, while small inactivating indels occurred frequently. They employed Homology-Directed Repair using a fluorescent marker to specifically target the bantam seed region. This efficiently produced the intended mutations but also led to unexpected outcomes, including partial sequence replacements and full donor plasmid integrations. These results reveal the complexity of gene editing outcomes and highlight the importance of thorough molecular characterization in genome engineering experiments.

TBLR1 is a subunit of the NCoR corepressor complex that is mutated in a range of neurodevelopmental disorders. Here, we report that TBLR1 functions as a molecular scaffold that physically connects ANKRD11 and SETD5 – two of the most frequently mutated proteins in neurodevelopmental disorders – and links them to the rest of the NCoR complex. The resulting assembly resembles the yeast SET3 complex (SET3C) – a transcriptional regulator. Pathogenic missense mutations in TBLR1, ANKRD11 and SETD5 disrupt this assembly, and an engineered mutation that specifically abolishes SETD5 incorporation into SET3C causes severe developmental impairments in mice. Disruptions of mammalian SET3C components cause highly correlated changes in gene expression – including upregulation of already highly transcribed genes. Together, our results reveal that failure of transcriptional regulation by SET3C is a convergent molecular basis for a family of neurodevelopmental disorders.

Summary We report the development of REMEDY (REpair of heterozygous Mutations independent of Exogenous Donor template with high efficiencY), a genome editing strategy that allows efficient repair of heterozygous mutations in human and mouse cells without necessitating an exogenous donor DNA template. Here, we used in-PAM or near-PAM CRISPR strategies to induce a double-strand break (DSB) in mutant alleles. Following the DSB, the wild-type homologous chromosome itself serves as an endogenous DNA donor template and initiates the correction of the mutant allele. Concurrently treating the cells with HDR enhancers, such as AZD7648, further improved the efficiency of the correction. We demonstrated the utility of REMEDY in the context of six different diseases with heterozygous mutations such as IBMPFD, cystic fibrosis, progeria, ITPR3-associated combined immunodeficiency, ACTA1, and TBCD in human patient derived primary cells and complementary mouse model cell lines. Abstract Figure Graphical Abstract

Groundwater ecosystems harbor diverse microbial communities adapted to energy-limited, light-deprived conditions, yet the role of viruses in these environments remains poorly understood. Here, we analyzed 1.26 terabases of metagenomic and metatranscriptomic data from seven wells in the Hainich Critical Zone Exploratory (CZE) to characterize groundwater viromes. We identified 257,252 viral operational taxonomic units (vOTUs) (>=5 kb), with 99% classified as novel, highlighting extensive uncharacterized viral diversity. Viruses exhibited a distinct host range, primarily targeting Proteobacteria, Candidate Phyla Radiation (CPR) bacteria, and DPANN archaea. Notably, CPR lineages displayed low virus-host ratios and viral CRISPR targeting multiple hosts, suggesting a virus decoy mechanism where they may absorb viral pressure, protecting bacteria hosts. Additionally, 3,378 vOTUs encoded auxiliary metabolic genes (AMGs) linked to carbon, nitrogen, and sulfur cycling, with viruses targeting 31.5% of host metabolic modules. These findings demonstrate viruses influence on microbial metabolic reprogramming and nutrient cycling in groundwater, shaping subsurface biogeochemistry.

The increasing global prevalence of Mycobacterium abscessus infections presents a significant clinical challenge due to the pathogen’s intrinsic resistance to multiple antibiotics and poor treatment outcomes. Despite the necessity of genetic tools for studying its physiology, pathogenesis, and drug resistance, efficient methods for large-fragment deletions remain underdeveloped. Here, we report a CRISPR/Cas9-based dual-sgRNA system employing Streptococcus thermophilus CRISPR1-Cas9 (Sth1Cas9), enabling efficient large-fragment knockout in M. abscessus with deletion efficiencies exceeding 90% at certain loci and spanning up to 16.7 kb. Furthermore, we systematically optimized the modular arrangement of genetic components in Cas9/dual-sgRNA expression plasmids and refined their construction workflow, achieving a significant reduction in cassette loss rates while enabling single-step plasmid assembly. Notably, deletion efficiency was position-dependent rather than correlated with target size, suggesting an influence of chromatin structure on editing outcomes. As the first CRISPR/Cas9-based platform capable of kilobase-scale deletions in M. abscessus , this system advances functional genomics studies and facilitates targeted investigations into virulence and antibiotic resistance mechanisms.

CRISPR/Cas12a-based assays, when integrated with lateral flow tests (LFTs), provide highly specific nucleic acid detection in a simple, rapid, and equipment-free format. Nevertheless, traditional DNA probes utilized for cleavage by Cas12a have notable limitations as the cleaved probe only has one label. To overcome this challenge, we engineered a novel type of DNA probe with multiple fluorescein (FAM) labels and a biotin-labeled single-stranded DNA fragment (polyFAM probe). The cleaved polyFAM parts of probes were detected using a specially designed sandwich LFT, where FAM-specific antibodies were immobilized in the test zone and conjugated with gold nanoparticles. The LFT ensured accurate recognition of the cleaved polyFAM fragments within 10 minutes. A comparison of five distinct polyFAM probes revealed that the highest signal-to-noise ratio was achieved with a tripod-branched probe synthesized via trebler phosphoramidite modification. Each arm of the tripod probe consists of a hexaethylene glycol spacer ending in a FAM label. Upon Cas12a cleavage, the tripod structure carrying three FAMs is released and detected by LFT. A rapid magnetic separation strategy was subsequently implemented, facilitating the efficient removal of uncleaved probes via biotin–streptavidin capture within 5 minutes. The CRISPR/Cas12a–tripod–LFT strategy demonstrated excellent sensitivity without preamplification, with a detection limit of 1.4 pM for DNA target of Salmonella Typhimurium. The CRISPR/Cas12a-tripod-LFT with preliminary loop-mediated isothermal amplification enabled the detection of as few as 0.3 cells per reaction. This innovative tripod probe with corresponding LFT creates a universal, sensitive, rapid, and equipment-free biosensing platform for CRISPR/Cas12a-based diagnostics in point-of-care applications.

Host cells contest invasion by intracellular bacterial pathogens with multiple strategies that recognise and / or damage the bacterial surface. To identify novel host defence factors targeted to intracellular bacteria, we developed a versatile proximity biotinylation approach coupled to quantitative mass spectrometry that maps the host-bacterial interface during infection. Using this method, we discovered that intracellular Shigella and Salmonella become targeted by UFM1-protein ligase 1 (UFL1), an E3 ligase that catalyses the covalent attachment of Ubiquitin-fold modifier 1 (UFM1) to target substrates in a process called UFMylation. We show that Shigella antagonises UFMylation in a dual manner: first, using its lipopolysaccharide (LPS) to shield from UFL1 recruitment; second, preventing UFM1 decoration by the bacterial effector IpaH9.8. Absence of UFMylation leads to an increase of bacterial burden in both human cells and zebrafish larvae, suggesting that UFMylation is a highly conserved antibacterial pathway. Contrary to canonical ubiquitylation, the protective role of UFMylation is independent of autophagy. Altogether, our proximity mapping of the host-bacterial interface identifies UFMylation as an ancient antibacterial pathway and holds great promise to reveal other cell-autonomous immunity mechanisms.

ABSTRACT The fungal pathogen Candida albicans colonises the human gut where short-chain fatty acids (SCFAs) offer sources of carbon. This fungus harbours one of the largest microbial families of ATO (Acetate Transport Ortholog) genes, which encode putative SCFA transport proteins. Here, we generate C. albicans null mutants lacking individual or all known putative SCFA transporter genes and compare their phenotypes in vitro and in vivo . We show that blocking ATO function in C. albicans impairs SCFA uptake and growth, particularly on acetate. The uptake of acetate is largely dependent on a functional Ato1 (also known as Frp3/Ato3) and it is effectively abolished upon deletion of all ATO genes. We further demonstrate that deletion of the entire ATO gene family, but not inactivation of ATO1 alone, compromises the stable colonisation of C. albicans in the murine gastrointestinal tract following bacterial disruption by broad-spectrum antibiotics. Our data suggest that the ATO gene family has expanded and diversified during the evolution of C. albicans to promote the fitness of this fungal commensal during gut colonisation, in part through SCFA utilisation. IMPORTANCE The human gut is rich in microbial fermentation products such as SCFAs, which serve as key nutrients for both bacteria and fungi. C. albicans , a common fungal resident of the gut and a cause of opportunistic infections, carries an unusually large family of ATO genes. This study reveals that this ATO gene family is required for the efficient uptake of acetate, the most abundant SCFA in the gut, and for stable colonisation of the gut. These findings uncover a new layer of metabolic adaptation in fungal commensals of humans and suggest that transporter gene expansion can shape microbial fitness in response to environmental nutrient signals.

Abstract Aim ; The study was conducted to establish the association between CRISPR-Cas system in Pseudomonas aeruginosa clinical and environmental strains to the sensitivity to phages and antibiotics. Method; In this study, the occurrence and distribution of CRISPR-Cas system was analyzed across the 50 Pseudomonas aeruginosa strains. Further, to evaluate the role of CRISPR-Cas system as inhibitor of HGT, the role of CRISPR-Cas system in relation to susceptibility of Pseudomonas aeruginosa to various antibiotics and phages was studied. Result : A total of 5 different types of Type I CRISPR- Cas systems i.e. Type IF, Type IE, Type IC, Type IV and Type IU were identified in P. aeruginosa strains. A total of 5 (17%) clinical strains and 8 (40%) environmental strains were positive for both CRISPR and cas3 gene. The analysis of phage as well as antibiotic susceptibility of both clinical and environmental strains revealed 36% clinical strains and75% environmental strains were found to be resistant to phages. Antibiotic susceptibility results were in contrast to phage susceptibility as 50% clinical strains were resistant to at least 3 antibiotics i.e. they were multidrug resistant (MDR) while only 15% environmental strains were classified as MDR. Conclusion : The phage susceptibility and antibiotic resistance data was found to be correlating to presence of CRISPR-Ca system in the P. aeruginosa .