Centrioles are microtubule-based organelles critical for signaling, motility and division. The microtubule-binding protein SAS-1 is homologous to the human ciliopathy component C2CD3 and contributes to centriole integrity in C. elegans , but how this function is exerted is incompletely understood. Here, through the generation of a null allele and analysis with U-Ex-STED, we establish that SAS-1 is dispensable for the onset of centriole assembly, but essential for organelle integrity during oogenesis, spermatogenesis and in the early embryo. Additionally, we uncover that SAS-1 is present at the transition zone of sensory neurons, where it contributes in a partially redundant manner to ciliary function. Furthermore, we investigate the relationship between SAS-1 and the C. elegans Sjögren Syndrome Nuclear Antigen 1 protein SSNA-1, establishing that SSNA-1 localizes next to the SAS-1 C-terminus in the centriole architecture. Moreover, through molecular epistasis experiments with null alleles of both components, we reveal that SAS-1 is essential for SSNA-1 localization to centrioles during oogenesis and to the transition zone during ciliogenesis. Moreover, using a heterologous human cell assay, we establish that SAS-1 recruits SSNA-1 to microtubules. Overall, our findings help clarify how SAS-1, together with SSNA-1, ensures centriole integrity and reveals that it contributes to cilium function.

Bactrocera zonata is a highly invasive agricultural pest that causes extensive damage to fruit crops. The Sterile Insect Technique (SIT), a species-specific and environmentally friendly pest control method, depends on the availability of Genetic Sexing Strains (GSSs) to enable efficient mass production of males for sterile release. However, no GSS currently exists for B. zonata limiting SIT applications targeting this important invasive pest. Here, we report two key advancements toward GSS development in this species. First, we present a high-quality, chromosome-level genome assembly from male B. zonata , identifying two scaffolds derived from the Y chromosome, which represent potential targets for future male-specific genetic engineering. Second, we demonstrate the feasibility of CRISPR/Cas9 genome editing in B. zonata by generating stable, homozygous white-eye mutants through targeted disruption of the conserved white-eye gene. This visible, recessive phenotype serves as a proof-of-concept for developing selectable markers in this species. Together, these results provide foundational genomic and genetic tools to support the development of GSSs in B. zonata , advancing the potential for sustainable, genetics-based pest control strategies.

ABSTRACT FBXO42 is a poorly characterized F-box protein that is essential in 15% of cancer cell lines from diverse tissue types. FBXO42 has been implicated in the regulation of mitosis and p53 signaling. High-throughput approaches indicate that FBXO42 function correlates with that of CCDC6, and that the two proteins interact physically, but the relationship between these proteins is not understood. Through a genome-wide CRISPR knockout screen, we found that mutation of FBXO42 is synthetically lethal with mutations in the γ- tubulin ring complex proteins MZT1 and MZT2B, suggesting that cells with centrosome and/or mitotic spindle assembly dysfunction are more sensitive to FBXO42 loss. Furthermore, we found that FBXO42 and CCDC6 contribute to p53 activation in response to centrosome depletion. Using mass spectrometry-based proteomics, we found that FBXO42 binds, is required for the ubiquitination of, and negatively regulates the expression of PPP4C (protein phosphatase 4 catalytic subunit). FBXO42’s interaction with PPP4C was independent of CCDC6. Similarly, we found that CCDC6 physically interacts with PPP4C independently of FBXO42 and does not affect PPP4C ubiquitination. Knockdown of PPP4C reduced FBXO42-CCDC6 interactions, suggesting that FBXO42 and CCDC6 may bind to and regulate PPP4C through separate mechanisms. Using gene knockdown rescue experiments, we confirmed that aberrant expression of PPP4C is a major driver of cell death in an FBXO42-essential Neuroblastoma cell line. These findings shed light on the function of two poorly understood proteins in regulating PP4 activity, p53 signaling, mitosis and cancer cell survival. A better understanding of FBXO42 and CCDC6 could inform the development of targeted cancer therapeutics.

Levels of receptors on the cell surface control the sensitivity of immune cells, which is central to achieving effective responses while avoiding inflammatory diseases. The chemoattractant receptor FPR1 activates strong chemotactic and cytotoxic responses in neutrophils. While its precise regulation is critical for appropriate responses, the mechanisms controlling its cell surface levels remain unclear. Here, we investigated the roles of both classic and unknown regulators. First, we found that multiple G protein-coupled receptor kinases (GRK2, GRK3, and GRK6), and both β-arrestin1 and 2 are important for FPR1 internalization. However, FPR1 uses multiple endocytic pathways, as cells lacking β-arrestins have strong, but incomplete defects in FPR1 internalization. Moreover, we performed two parallel genome-wide CRISPR/Cas9 screens for the regulators of FPR1 surface expression and internalization in a neutrophil-like cell line. These screens identified regulators of FPR1 surface expression, recycling, and endocytosis. We identified the formin mDia1 and the small GTPase Arf6 as specific regulators of FPR1 internalization, and we confirmed these phenotypes using chemical inhibitors in primary human neutrophils. Furthermore, our data indicates that Arf6 contributes to the β-arrestin-independent pathway. Collectively, our results clarify the contributions of GRKs and β-arrestins in FPR1 internalization, indicate that internalization involves multiple compensatory routes, and uncover previously unidentified regulators of FPR1 biogenesis and trafficking, providing new mechanistic insight into these processes.

CRISPR/Cas9-mediated genome editing has rapidly become a popular tool for studying gene functions and generating genetically modified organisms. However, using this system, stochastic integration of random insertions and deletions restricts precise genome manipulation. Advanced CRISPR/Cas9 technologies using Prime Editors (PEs), Cas9 proteins fused with reverse transcriptase, enable programmed integration of short DNA modifications into the genome. However, its application in precise genome editing in animal models is challenging. Here, we utilise a nickase- and a nuclease-based PE to perform programmed short DNA substitution and insertion in various loci in the zebrafish genome. Whereas the nickase-based PE is advantageous for nucleotide substitutions, we find that the nuclease-based PE can be used to insert short DNA fragments precisely with high efficiency. To further evaluate our approach, we inserted a nuclear localisation signal into a reporter transgene to incorporate longer fragments by prime editing. These gene modifications were transmitted to the next generation. We demonstrate that PE-mediated prime editing can efficiently manipulate genome information in zebrafish without using exogenous donor DNA.

ABSTRACT Kinetoplastids, such as Trypansoma brucei , are eukaryotes that likely separated from the main lineage at an exceptionally early point in evolution. Consequently, many aspects of kinetoplastid biology differ significantly from other eukaryotic model systems, including yeasts, plants, worms, flies and mammals. As in many eukaryotes the T. brucei genome contains repetitive elements at various chromosomal locations including centromere- and telomere-associated repeats and interspersed retrotransposon elements. T. brucei also contains intermediate-sized and mini- chromosomes that harbor abundant 177 bp repeat arrays, and 70 bp repeat elements implicated in Variable Surface Glycoprotein (VSG) gene switching. In many eukaryotes repetitive elements are assembled in specialised chromatin such as heterochromatin, however, apart from centromere- and telomere-associated repeats, little is known about chromatin-associated proteins that decorate these and other repetitive elements in kinetoplastids. Here we utilize affinity selection of synthetic TALE DNA binding proteins designed to target specific repeat elements to identify enriched proteins by proteomics. Validating the approach, a telomere repeat binding TelR-TALE identifies many proteins previously implicated in telomere function. Furthermore, the 70R-TALE designed to bind 70 bp repeats indicates that proteins involved in DNA repair are enriched on these elements that reside adjacent to VSG genes. Interestingly, the 177 bp repeat binding 177R-TALE enriches for many kinetochore proteins suggesting that intermediate-sized and mini- chromosomes assemble kinetochores related in composition to those located on the main megabase chromosomes. This provides a first insight into the chromatin landscape of repetitive regions of the trypanosome genome with relevance for their mechanisms of chromosome integrity, immune evasion and cell replication.

Cardiac fibrosis is mediated by the persistent activity of myofibroblasts, which differentiate from resident cardiac fibroblasts in response to tissue damage and stress signals. The signaling pathways and transcription factors regulating fibrotic transformation have been thoroughly studied. In contrast, the roles of chromatin factors in myofibroblast differentiation and their contribution to pathogenic cardiac fibrosis remain poorly understood. Here, we combined bulk and single-cell CRISPR screens to characterize the roles of chromatin factors in the fibrotic transformation of primary cardiac fibroblasts. We uncover strong regulators of fibrotic states including Srcap and Kat5 chromatin remodelers. We confirm that these factors are required for functional processes underlying fibrosis including collagen synthesis and cell contractility. Using chromatin profiling in perturbed cardiac fibroblasts, we demonstrate that pro-fibrotic chromatin complexes facilitate the activity of well-characterized pro-fibrotic transcription factors. Finally, we show that KAT5 inhibition alleviates fibrotic responses in patient-derived human fibroblasts.

Abstract The gastrointestinal parasitic nematode Strongyloides spp. has a unique life cycle that alternates between a parasitic generation that reproduces through mitotic parthenogenesis and a dioecious free-living sexually reproducing generation. Adult females from these two generations are genetically identical, making them an informative model to identify molecular differences between parasitic and free-living lifestyles and understand different reproductive strategies. We investigated the expression of small RNAs (sRNAs) that are either enriched for a 5’ monophosphate modification (5’pN) or are 5’ modification-independent, across five life cycle stages of the rodent parasite Strongyloides venezuelensis . We identified miRNAs and small-interfering RNAs expressed by S. venezuelensis that are predicted to target and regulate the expression of protein-coding genes and transposable elements (TEs). Three previously unreported classes of sRNA were identified: (i) 25Gs with a putative role in reproduction in adult females, (ii) tRNA-derived 24–28 nt sRNAs (tsRNAs) which are predicted to target TEs in free-living females, and (iii) 5’pN-enriched 26-29Cs with 5’ CGAATCC and 3’ TTT motifs expressed in parasitic females. We also confirmed that S. venezuelensis expresses the 27G class of sRNAs involved in TE regulation, which was previously identified in the rodent parasite Strongyloides ratti . Taken together, these results provide new insights into the role of sRNAs in reproductive biology and parasitism.

The global emergence of gyrovirus galga1 (GyVg1) across diverse regions and species un-derscores an urgent demand for rapid diagnostics. This study aimed to engineer a field-deployable diagnostic platform for rapid pathogen detection. We established two visual detection methods by integrating recombinase-aided amplification (RAA) and CRISPR/Cas12a technologies: RAA‒CRISPR/Cas12a combined with fluorescence and RAA‒CRISPR/Cas12a combined with lateral flow strips. By systematically optimizing the reaction conditions, designed primers and crRNA enabled target recognition within 1 hour, and demonstrated no cross-reactivity with other relevant avian pathogens. RAA‒CRISPR/Cas12a combined with fluorescence achieved a detection limit of 2 copies/µL (10 copies/µL visually under UV), and RAA-CRISPR/Cas12a-lateral flow strips demonstrated a detection limit of 5×10² copies/µL. Clinical validation using 192 samples revealed ~10% positivity rates across both novel methods and fluorescence quantitative PCR, with high concordance in positive identifications. The results suggest that the two RAA‒CRISPR/Cas12a visual detection methods established in this study are highly efficient, specific and sensitive, and can be used for the rapid field detection of GyVg1, providing a cost-effective and powerful diagnostic tool for grassroot workers.

Summary Decelerated translation elongation caused by non-optimal codons can reduce mRNA stability through codon optimality-mediated mRNA degradation . A key element of this process is the coupling of sensing the mRNA codon usage with the regulation of translation efficiency and stability. We report that two paralog RNA-binding proteins (ZC3H7A and ZC3H7B), which are only found in Chordates, preferentially bind to and reduce the stability and translation of mRNAs enriched in non-optimal codons with A/U at their wobble sites (A/U3 codons). ZC3H7A/B engage with ribosomes that lack elongation factors and induce mRNA degradation or block translation initiation through their interactions with the CCR4-NOT and the GIGYF2/4EHP translation repressor complex, respectively. Depletion of ZC3H7A/B or 4EHP impairs the repression of non-optimal A/U3-rich mRNAs. This study provides insights into a unique mechanism in higher eukaryotes that couples codon usage with the regulation of translation efficiency and mRNA stability.

Biallelic loss-of-function (LoF) variants in the BTRR complex members BLM , TOP3A, RMI1, and RMI2 cause Bloom syndrome (BS). The BTRR complex mainly acts on DNA replication and DNA repair processes, and dysfunction of this complex underlies, e.g., increased genomic instability and cancer predisposition associated with the BS phenotype. Here, we report CRISPR/Cas9-based genome-edited isogenic induced pluripotent stem cell (iPSC) models with compound heterozygous LoF variants in BLM, TOP3A , and RMI1 . The cellular phenotype of all three knockout (KO) iPSC lines included chromosome segregation defects, increased sister chromatid exchange rates, and impaired homologous recombination repair. Using single-cell whole genome sequencing, we showed that BTRR complex deficiency causes increased copy number alterations (CNAs) in the genomes and, therefore, represents a driver for genomic instability. CNA load was further induced by applying replication stress, and we observed that BTRR KO iPSCs acquired fewer de novo CNA events compared to wild-type cells, suggesting a possible limitation of genomic instability induction. Importantly, induced and non-induced CNAs in single-cell genomes were not stochastically distributed throughout the genome, but instead enriched at fragile sites. This finding might offer an opportunity for the development of novel NGS-based approaches to measure rates of genomic instability in disease conditions.

RNA-protein interactions (RPIs) are as important as protein-protein interactions (PPIs) for the formation of membraneless organelles (MLOs) and play a vital role in various biological processes. Despite remarkable advances in PPI analysis technologies in recent years, the development of RPI analysis tools has lagged behind. To advance RPI analysis, we integrated three established PPI tools—bimolecular fluorescence complementation (BiFC), NanoBiT, and split-TurboID—with the RNA-targeting CRISPR/Cas13. We applied these tools to analyze paraspeckles, one of the best known MLOs formed by interactions between the long non-coding RNA NEAT1 and the RNA-binding protein NONO. The optimized BiFC-dCas13 allows live cell imaging and quantitative detection of the NEAT1-NONO interaction. The NanoBiT-dCas13 detects dynamic changes in the NEAT1-NONO interaction in an immediate and reversible manner. As a proximity labeling tool, the Split-TurboID-dCas13 induces biotinylation of proteins surrounding paraspeckles, leading to the identification of the N6-methyladenosine reader protein YTHDC1 as a novel paraspeckles-associated protein. The BiFC-dCas13, NanoBiT-dCas13, and Split-TurboID-dCas13 systems have a broad utility for the analysis of RPIs and MLOs.

The success of CRISPR genome editing studies depends critically on the precision of guide RNA (gRNA) design. Sequence polymorphisms in outcrossing tree species pose design hazards that can render CRISPR genome editing ineffective. Despite recent advances in tree genome sequencing with haplotype resolution, sequence polymorphism information remains largely inaccessible to various functional genomics research efforts. The Populus VariantDB v3.2 addresses these challenges by providing a user-friendly search engine to query sequence polymorphisms of heterozygous genomes. The database accepts short sequences, such as gRNAs and primers, as input for searching against multiple poplar genomes, including hybrids, with customizable parameters. We provide examples to showcase the utilities of VariantDB in improving the precision of gRNA or primer design. The platform-agnostic nature of the probe search design makes Populus VariantDB v3.2 a versatile tool for the rapidly evolving CRISPR field and other sequence-sensitive functional genomics applications. The database schema is expandable and can accommodate additional tree genomes to broaden its user base.

Transcription factors (TFs) are key effectors of enhancer activity. MYB is a critical hematopoietic TF that is frequently dysregulated in cancer. Despite its well-established role, the exact mechanisms by which MYB influences enhancer function—and the specific stages of enhancer activation at which it operates—remain poorly understood. Using high resolution Micro-Capture-C, we show that upon MYB degradation, highly defined enhancer-promoter interactions at specific MYB binding sites are lost. Loss of these interactions, together with other hallmarks of enhancer activity—reduced H3 lysine-27 acetylation and enhancer RNA transcription—correlates with significant downregulation of target gene expression in leukemia, indicating that MYB mediates transcription activation via maintenance of enhancer function. When anchored to DNA within a gene desert region that is devoid of histone marks and active transcription, the MYB transactivation domain is sufficient and necessary for the nucleation of an enhancer-like region. This results in the activation of transcription from distal cryptic elements and the establishment of long-range chromatin interactions up to 400 kb away from the anchor point. Together, these results indicate that MYB activity alone is sufficient to induce long-range interactions and transcription, achieving this through highly precise enhancer-promoter crosstalk.

CRISPR/Cas9 genome editing is a powerful tool in genetic engineering and gene therapy; however, off-target effects pose significant challenges for clinical applications. Accurate prediction of these unintended edits is crucial for ensuring safety and efficacy. In this study, we propose a novel approach that integrates DNABERT, a pre-trained DNA language model, with epigenetic features to improve off-target effect prediction. We evaluated DNABERT-based models against five state-of-the-art baseline models (GRU-Emb, CRISPR-BERT, CRISPR-HW, CRISPR-DIPOFF, and CrisprBERT) using four key performance metrics (F1-score, MCC, ROC-AUC, and PR-AUC). Additionally, we conducted ablation studies to assess the impact of DNABERT’s pre-training and epigenetic features, demonstrating that both significantly enhance predictive performance. Furthermore, we explored an ensemble modeling approach, which achieves superior prediction accuracy compared to individual models. Finally, we visualized DNABERT’s attention weights to gain insights into its decision-making process, revealing biologically relevant patterns in off-target recognition. The source codes used in this study are available at github.com/kimatakai/CRISPR_DNABERT.

Abstract Transposable elements (TEs) constitute a major portion of plant genomes and play key roles in shaping genome architecture, regulating gene expression, and driving genome evolution. In this study, we generated a comprehensive and curated TE library for the woodland strawberry ( Fragaria vesca ) by integrating two bioinformatic pipelines (EDTA and DeepTE). Our annotation revealed that TEs account for approximately 37% of the F. vesca genome. Analysis of TE-derived inverted repeats (IRs) and miniature inverted-repeat transposable elements (MITEs) demonstrated their association with 24-nt small interfering RNA (siRNA) production and differential DNA methylation patterns across tissues, suggesting a role in the epigenetic regulation of gene expression, particularly during fruit ripening. This MITE-mediated epigenetic regulatory mechanism was confirmed by evaluating gene expression and chromatin organization at FvH4_7g18570, which encodes the alcohol acyl transferase ( FvAAT1 ). Three MITEs located upstream or downstream of the FvAAT1 coding sequence were shown to influence epigenetically this gene expression. Furthermore, we analyzed 210 re-sequenced accessions from the F. vesca European germplasm collection to identify and annotate TE insertion and deletion polymorphisms. A principal component analysis (PCA) based on these polymorphisms revealed subpopulation structures that reflect geographic origins. A genome-wide association study (GWAS) uncovered significant associations between specific TE polymorphisms and economically important fruit traits, including aroma-related volatile compounds and fruit size. Among them, the insertion of a hAT MITE near FvH4_2g00610 correlated with increased levels of γ-decalactone, a desirable aroma compound in strawberries. These findings underscore the functional significance of TE-derived elements as key contributors to phenotypic diversity through novel regulatory functions. By integrating TE polymorphisms into population-genomic and functional studies, this work provides valuable insights into strawberry fruit development and quality traits. It also highlights the potential of harnessing TE-mediated variation in breeding initiatives and genome editing strategies to improve fruit quality.

CRISPR-Cas13 RNA nucleases have emerged as powerful tools for programmable RNA targeting. A light-controlled RNA nuclease could be transformative by enabling researchers to selectively knock down transcripts at desired positions in a cell or tissue or at timepoints of interest. Here, we develop a set of multimodal RfxCas13d tools that can be controlled by either light or small molecule addition. Screening an RfxCas13d library containing insertions of the AsLOV2 photoswitchable domain revealed an OptoCas13d-off variant that induced target RNA cleavage in the dark and switched to an inactive state under blue light. Insertion at this same allosteric hotspot could be further exploited to generate an OptoCas13d-on with the opposite light dependence and a ChemoCas13d that is activated upon the addition of rapamycin analogs. Through biochemical assays, we showed that AsLOV2 domain switching did not substantially affect Cas13d-RNA complex formation, indicating allosteric control over Cas13d catalytic activity. We applied the OptoCas13d-on system to target several endogenous transcripts and showed that it exhibited efficient mRNA knockdown only upon blue light illumination. Overall, our results demonstrate that engineered OptoCas13d can achieve cellular RNA modulation with high spatial and temporal precision.

ABSTRACT The ALMS1 gene plays a crucial role in maintaining cellular homeostasis through its involvement in primary cilium assembly, cytoskeletal regulation, and signalling pathways such as NOTCH and TGF-β. Pathogenic variants in ALMS1 are associated with Alström Syndrome (ALMS), a multi-systemic ciliopathy characterised by neurosensory deficits, metabolic disorders, and multi-organ fibrosis. To better understand the tissue-dependent role of ALMS1 , we utilised CRISPR/Cas9 technology to develop a zebrafish model with alms1 depletion. Multi-tissue transcriptomic profiling revealed that alms1 depletion has pleiotropic effects on gene expression, with the brain and eyes displaying the most pronounced transcriptomic alterations, including disrupted ciliary function and immune dysregulation. Inflammatory and innate immune pathways along with glutamatergic synapse-related processes were significantly affected in the brain and eyes but with different gene expression signatures. The analysis further highlights tissue-specific processes, primarily associated with organ dysfunction. Additionally, our findings underscore the role of alms1 in regulating age-associated gene expression profiles in the brain, suggesting a link between ciliary dysfunction and accelerated brain ageing. Comparative analyses with Bardet-Biedl Syndrome iPSC models revealed shared pathways, reinforcing the potential of ciliopathies as models for ageing-related disorders. This study provides novel insights into the tissue-specific functions of alms1 and the molecular mechanisms underlying ALMS, paving the way for the development of targeted therapeutic strategies.

Adipose tissues exhibit a remarkable capacity to expand, regress, and remodel in response to energy status. The cellular mechanisms underlying adipose remodelling are central to metabolic health. Hypertrophic remodelling - characterised by the enlargement of existing adipocytes - is associated with insulin resistance, type 2 diabetes, and cardiovascular disease. In contrast, hyperplastic remodelling – in which new adipocytes are generated - is linked to improved metabolic outcomes. Despite its clinical importance, the regulation of hypertrophic and hyperplastic adipose remodelling remains poorly understood. In this study, we first leveraged human genetic and transcriptomic data to identify candidate genes involved in adipose remodelling. We then developed a quantitative imaging pipeline to assess hyperplastic and hypertrophic morphology in zebrafish subcutaneous adipose tissue, and applied it in an F0 CRISPR mutagenesis screen targeting 25 candidate genes. This screen identified six genes that significantly altered adipose morphology; including Sushi Repeat Containing Protein (Srpx) - a gene with previously unknown roles in adipose. Among the identified genes, foxp1b mutants were notable for inducing hypertrophic morphology. To investigate further, we generated stable loss-of-function alleles for both zebrafish foxp1 genes. We found that foxp1b mutants display a developmental bias towards hypertrophic adipose growth but fail to undergo further hypertrophic remodelling in response to a high-fat diet - suggesting that early developmental patterning constrains later adaptability to diet. Together, these findings establish a scalable and tractable in vivo screening platform for identifying regulators of adipose remodelling, and reveal a potential developmental influence on the capacity for diet-induced adipose expansion.

Abstract Insect pest population control via sterile insect technique severely benefits from separation by sex prior to release. To simplify this process, traditional genetics has been deployed to develop genetic sexing strains (GSSs) for several disease vectors and agricultural pests of vast economic significance, although very few are applied in the field due to associated fitness costs and instability. In this study, we generated a method to engineer cisgenic GSS (CGSS) in insects. We use CRISPR/Cas9-mediated homology-directed repair to seamlessly translocate a sex-specific alternatively spliced intron into a dominant phenotypic gene generating a genetically stable strain that enables sex-sorting by eye. To achieve this feat, we use Ceratitis capitata as our model and relied on the sex-specifically spliced intron of the endogenous transformer gene, which we seamlessly inserted into the pupal colouration white pupae gene. This minimal modification resulted in the generation of a homozygous strain we term IMPERIAL that was phenotypically stable where all female pupae are brown while male pupae are white with overall good fitness. By minimally editing the genome, our CGSS approach can be applied to other pests that may aid more efficient and economically suitable pest control.

Abstract New Breeding Techniques (NBTs), such as CRISPR-Cas9, TALENs, and ODM, are reshaping the way we develop plant-based products for food and cosmetic applications by allowing for more precise and efficient trait improvements. However, despite their scientific potential, global acceptance and regulation of NBT-derived products continue to vary widely. This study provides a comparative analysis of public perceptions across six countries—Spain, France, China, Japan, Brazil, and the United States—focusing on consumer attitudes toward NBT use in food and skincare products. Based on survey data from 724 participants, we found clear regional differences in familiarity, risk perception, and acceptance of NBTs. Participants from China and Japan showed relatively higher awareness and openness toward these technologies. In contrast, respondents from Spain and France were more skeptical, especially when it came to environmental risks. In Brazil, opinions were more evenly split, reflecting ongoing national discussions. Despite the U.S. having a product-based regulatory framework, trust in NBT safety and willingness to consume NBT-derived goods was notably low. One of the most consistent findings was strong support for mandatory labeling across all countries, signaling a shared expectation for transparency. Claims framing NBT products as “green” or sustainable received limited agreement, particularly in Western countries. Statistical analyses confirmed significant differences in national response patterns, although average acceptance scores did not vary significantly. These results point to the importance of considering social and cultural context in the development and communication of NBT innovations. While awareness is gradually increasing, broader adoption will depend on transparent regulation, locally adapted communication strategies, and genuine public engagement. Building trust through clear, evidence-based messaging—and respecting regional values and concerns—will be essential to ensuring responsible and widely accepted use of genome-edited technologies.

Abstract Cas9 can process poly(T) single-stranded DNA molecules upon activation in an RNA-guided manner. Here, we uncover key structural determinants underlying this function. First, we show that open R-loops in the PAM-distal region favor trans -cleavage activity, which occur when targeting short double-stranded or single-stranded DNA molecules. Second, we show that elongated guide RNA spacers beyond the canonical 20 bases, even by a few bases, severely impairs this collateral activity. Third, although trans -cleavage is mediated by the RuvC domain, we show that a catalytically active HNH domain contributes to an efficient process. Structural analyses of domain rearrangements provide mechanistic insight. Together, these findings illustrate a fine modulation of Cas9 function.

The liverwort Marchantia polymorpha is a widely used model organism for studying land plant biology, which has also proven to be a promising testbed for bioengineering. CRISPR/Cas9 technology has emerged as a transformative tool for precise genome modifications in M. polymorpha . However, a robust method for the simultaneous expression of multiple gRNAs, which is crucial for enhancing the efficiency and versatility of CRISPR/Cas9-based genome editing, has yet to be fully developed. In this study, we introduce an adaptation from the OpenPlant kit CRISPR/Cas9 tools, that facilitates expression of multiple gRNAs from a single transcript through incorporation of tRNA sequences. This approach significantly improves the efficiency and scalability of genome editing in M. polymorpha . Additionally, by combining this vector system with a simplified and optimized protocol for thallus transformation, we further streamline the generation of CRISPR/Cas9 mutants in M. polymorpha . The resulting gene- editing system offers a versatile, time-saving and straightforward tool for advancing functional genomics in M. polymorpha , enabling more comprehensive genetic modifications and genome engineering.

Background Understanding the interplay between genome variation and epigenomic structure is fundamental to the study of the development and mechanisms of disease. Previous studies have leveraged population-scale genotype surveys to associate alleles with epigenomic states in heterogenous tissue types. However, epigenomes are inherently cell type-specific, giving rise to unique genome-epigenome interactions that can influence distinct functional states and susceptibility to disease. Moreover, the extent of individual variation in cell type-specific epigenotypes remains poorly understood, posing additional challenges to accurately link genotypes with epigenomic features. Results We generated comprehensive genomic and epigenomic measurements in four functionally defined human breast cell types across eight individuals. We developed a method to measure histone modification variance, discovering significantly higher variation in repressive chromatin states marked by H3K27me3 compared to the active states marked by H3K27ac and H3K4me3. Genetic variation linked to variation in chromatin state was highly cell type-specific, with nearly 90% occurring uniquely in a single cell type, and active histone modifications were enriched in these variants relative to repressive modifications. Association with gene transcription allowed for the prioritization of functional candidates, and the regulatory impact of an ANXA1 -linked variant, rs75071948, was validated in vitro with CRISPR/Cas9-mediated HDR. Conclusions We define structures of epigenomic variability among breast cell types and present evidence of extensive cell type-specific genome-epigenome interactions, highlighting the critical role of cell type in mediating these associations in the breast.

SUMMARY Mitochondrial function is critical for neural progenitor regulation, yet its dysregulation during early human brain development remains poorly defined. Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a neurodevelopmental disorder caused by MLC1 mutations, previously attributed to postnatal astrocyte dysfunction. Using patient-derived human cortical organoids, we show that MLC1 is expressed in early neuroepithelial cells. To assess mitochondrial state in live organoids, we developed the MAGO (Matrigel-coated gold nanostructure) platform for real-time, label-free detection of redox activity. MLC1 mutant organoids showed mitochondrial hyperactivation, increased ATP and ROS, reduced membrane potential, and altered fusion protein expression. These changes were accompanied by enhanced BrdU incorporation and expansion of PAX6⁺/SOX2⁺ progenitors. To assess the causal role of MLC1 mutation, we generated isogenic organoids using CRISPR prime editing, which recapitulated redox hyperactivation and increased proliferation. Our findings redefine MLC as a disorder of early mitochondrial and progenitor dysregulation and establish a tractable platform to study metabolic mechanisms in neurodevelopmental disease.