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.

CRISPR homing drives can be used to suppress a population by targeting female fertility genes. They convert wild-type alleles to drive alleles in the germline of drive heterozygotes by homology-directed repair after DNA cleavage. However, resistance alleles produced by end-joining pose a great threat to homing drive. They prevent further recognition by Cas9, and therefore weaken suppressive power, or even stop suppression if they preserve the function of the target gene. We used multiplexed gRNAs targeting doublesex in Drosophila to avoid functional resistance and create resistance alleles that were dominant female-sterile. This occurred because the male dsx transcript was generated in females by disruption of the female-specific splicing acceptor site. We rescued dominant sterility of the drive by providing an alternate splicing site. As desired, the drive was recessive female sterile and yielded high drive inheritance among the progeny of both male and female drive heterozygotes. The dominant-sterile resistance alleles enabled stronger suppression in computational models, even in the face of modest drive efficiency and fitness costs. However, we found that male drive homozygotes were also sterile because they used the rescue splice site. Attempts to rescue males with alternate expression arrangements were not successful, though some male homozygotes had less severe intersex phenotypes. Though this negatively impacted the drive, models showed that it still had significantly improved suppressive power. Therefore, this design may have wide applicability to dsx -based suppression gene drives in a variety of organisms with intermediate homing drive performance.

Multi-trait QTL (xQTL) colocalization has shown great promises in identifying causal variants with shared genetic etiology across multiple molecular modalities, contexts, and complex diseases. However, the lack of scalable and efficient methods to integrate large-scale multi-omics data limits deeper insights into xQTL regulation. Here, we propose ColocBoost , a multi-task learning colocalization method that can scale to hundreds of traits, while accounting for multiple causal variants within a genomic region of interest. ColocBoost employs a specialized gradient boosting framework that can adaptively couple colocalized traits while performing causal variant selection, thereby enhancing the detection of weaker shared signals compared to existing pairwise and multi-trait colocalization methods. We applied ColocBoost genome-wide to 17 gene-level single-nucleus and bulk xQTL data from the aging brain cortex of ROSMAP individuals (average N = 595), encompassing 6 cell types, 3 brain regions and 3 molecular modalities (expression, splicing, and protein abundance). Across molecular xQTLs, ColocBoost identified 16,503 distinct colocalization events, exhibiting 10.7(± 0.74)-fold enrichment for heritability across 57 complex diseases/traits and showing strong concordance with element-gene pairs validated by CRISPR screening assays. When colocalized against Alzheimer’s disease (AD) GWAS, ColocBoost identified up to 2.5-fold more distinct colocalized loci, explaining twice the AD disease heritability compared to fine-mapping without xQTL integration. This improvement is largely attributable to ColocBoost ’s enhanced sensitivity in detecting gene-distal colocalizations, as supported by strong concordance with known enhancer-gene links, highlighting its ability to identify biologically plausible AD susceptibility loci with underlying regulatory mechanisms. Notably, several genes including BLNK and CTSH showed sub-threshold associations in GWAS, but were identified through multi-omics colocalizations which provide new functional support for their involvement in AD pathogenesis.

Neural stem cell (NSC) transplantation is a promising therapeutic approach for spinal cord repair, but poor graft survival remains a critical challenge. Here, we demonstrate that the mechanical properties of the transplantation microenvironment play a crucial role in NSC survival in the injured spinal cord. While our previously engineered imidazole-poly(organophosphazene) (I-5) hydrogel effectively prevented cavity formation by promoting extracellular matrix remodeling, NSCs transplanted with 10% hydrogel exhibited poor survival. Remarkably, increasing the hydrogel concentration to 16%, which created a 5-fold stiffer matrix, significantly enhanced NSC graft survival and synaptic integration. Using in vitro models with controlled substrate stiffness, we found that NSCs on stiffer substrates displayed enhanced adhesion, complex morphology, and increased viability. Importantly, we identified the mechanosensitive ion channel Piezo1 as the key molecular mediator of these stiffness-dependent behaviors. CRISPR/Cas9-mediated Piezo1 gene editing in NSCs significantly reduced graft survival in vivo when transplanted with 16% hydrogel, confirming that Piezo1-mediated mechanotransduction is essential for NSC survival in the injured spinal cord. Our findings reveal a previously unrecognized mechanism governing graft survival in the injured spinal cord and suggest that optimizing the mechanical properties of biomaterial scaffolds or targeting Piezo1-dependent mechanotransduction could substantially improve outcomes of cell-based therapies for neurological disorders.

Summary We investigated the roles of Rac guanine-nucleotide factor (Rac-GEF) Prex1 in glucose homeostasis using Prex1 −/− and catalytically-inactive Prex1 GD mice. Prex1 maintains fasting blood glucose levels and insulin sensitivity through its Rac-GEF activity but limits glucose clearance independently of its catalytic activity, throughout ageing. Prex1 −/− mice on high-fat diet are protected from developing diabetes. The increased glucose clearance in Prex1 −/− mice stems from constitutively enhanced hepatic glucose uptake. Prex1 limits Glut2 surface levels, mitochondrial membrane potential and mitochondrial ATP production, and controls mitochondrial morphology in hepatocytes, independently of its catalytic activity. Prex1 limits GPCR trafficking through an adaptor function, and we identify here the inhibitory orphan GPCR Gpr21 as a Prex1 target. The Gpr21-mediated blockade of glucose uptake and mitochondrial ATP production in hepatocytes requires Prex1. We propose that Prex1 limits glucose clearance by maintaining Gpr21 at the hepatocyte surface, thus limiting hepatic glucose uptake and metabolism. Graphical abstract

Intermittent fasting and fasting-refeeding regimens can slow biological aging across taxa 1 . Shifts between fed and fasted states activate ancient nutrient-sensing pathways which alter cellular and epigenetic states to promote longevity 2–4 . Yet how biological age trajectories progress during fasting-refeeding, and how nutrient-sensing pathways reprogram epigenetic state remain largely unknown. Here we observe increases in predicted biological age of Caenorhabditis elegans during prolonged fasting in adult reproductive diapause, followed by extraordinary reduction of biological age during refeeding. We identify hil-1 / H1-0 as an evolutionarily conserved nutrient-regulated linker histone which mediates adaptations to fasting and refeeding downstream of FOXO and TFEB transcription factors. In C. elegans and human cell culture, hil-1 / H1-0 upregulation during low-nutrient states promotes long-term survival and subsequent refeeding-induced recovery. Restoration of C. elegans after prolonged fasting is improved by enhancing the natural downregulation of hil-1 specifically during refeeding. Our study identifies HIL-1/H1.0 as part of an ancestral epigenetic switch during fasting-refeeding that reprograms metabolic and cellular states underlying resilience and restoration.

Accumulation of misfolded α-synuclein protein in intracellular inclusion bodies of dopaminergic neurons underlies the pathogenesis of Synucleinopathies, which include Parkinson’s Disease (PD), Dementia with Lewy Bodies (DLB) and Multiple System Atrophy (MSA). Therefore, clearance of misfolded α-synuclein from dopaminergic neurons could in principle offer a therapeutic window for Synucleinopathies, which currently remain untreatable. In this study, we employ the Affinity-directed PROtein Missile (AdPROM) system consisting of the substrate receptor of the CUL2-E3 ligase complex VHL and a nanobody selectively recognising the human α-synuclein protein and demonstrate targeted degradation of endogenous α-synuclein from human cell lines with remarkable selectivity. We further demonstrate that targeted degradation of α-synuclein prevents the pre-formed fibril (PFF)-induced aggregation of α-synuclein in primary neurons derived from rats expressing human α-synuclein. This approach represents the first demonstration of nanobody-guided proteasomal degradation of all clinically relevant α-synuclein variants, highlighting its potential as a therapeutic strategy against Synucleinopathies.

Leukaemias, driven by mutations in hematopoietic stem cells (HSCs), rely on interactions with the bone marrow (BM) niche and other cell populations such as mesenchymal stromal cells (MSCs) for growth and survival. While chimeric antigen receptor (CAR) T-cell therapy shows promise for other hematological malignancies, its application to acute myeloid leukaemia (AML) is hindered by tumour heterogeneity and off-target toxicity. Combining CRISPR-Cas9 gene editing with CAR T-cell therapy has potential for selectively targeting AML cells while sparing healthy tissue. However, validating the efficacy of these treatments prior to clinical trial is hampered by the differences between humans and animal models typically used for pre-clinical testing. Furthermore, traditional in vitro models fail to replicate the complexity of the BM niche and often overestimate treatments’ efficacy. Here, we present a bioengineered human-cell containing BM niche model combining a fibronectin-presenting polymeric surface and a synthetic peptide hydrogel (PeptiGel) that mimics native BM tissue’s mechanical properties. This platform supports niche phenotypes in MSCs and HSCs and enables the evaluation of combined CRISPR-CAR T-cell therapy, demonstrating potential as a preclinical human model for testing novel therapies.

Epithelial cancers such as stomach and ovary cancer tend to metastasize to the peritoneum, often leading to intractable disease and poor survival. Currently, the mechanisms that enable gastric cancer cells to penetrate the mesothelium, and to implant, invade, and survive in the peritoneal niche are poorly understood. To investigate these mechanisms, we developed a novel human peritoneal explant model. Briefly, fresh peritoneal tissue samples from abdominal surgery patients were cultured on top of a layer of GFP-labeled human gastric adenocarcinoma cells (AGS); 2% of these cells implanted into the peritoneum. The transcriptomic profile of the implanted AGS cells was compared to the profile of AGS cells that failed to implant using RNA sequencing. Differentially expressed genes were enriched significantly (fold change>2) for genes that enable cell adhesion, motility, and membrane depolarization. We compared this list of genes with a previously identified peritoneal metastasis whole exome sequencing dataset (SRP043661). Upon further analysis based on subcellular localization, cell adhesion and cytoskeletal organization, we found nine core “peritoneal implantation” genes. We functionally validated these genes with CRISPR knockout and assessed peritoneal implantation and invasion using the human peritoneal explant model described above. From these data we identified ADAM12 as a key player of peritoneal metastasis. Knock out of ADAM12 significantly impaired peritoneal metastasis in vivo and ex vivo . Exploration of three publicly available independent datasets indicated that ADAM12 is indeed clinically relevant in peritoneal metastasis. To explore the role of ITGAβ1 in mediating cell-matrix interactions in the presence of ADAM12, we performed ITGAβ1 pull-down assays followed by mass spectrometry analysis in ADAM12 WT cells. ADAM12 KO cells show a marked disruption of the ITGAβ1 interactome in GCa cells. Key cytoskeletal proteins such as MYH14, MYH10, MYH9, ACTA2, SPTN1 and TPM1–TPM4 were found to be interactors with ITGAβ1 in ADAM12 WT cells but not ADAM12 KO cells. Our approach and the new data identify a distinct peritoneal metastasis gene set that facilitates implantation and invasion of gastric cancer cells within the peritoneum. Disruption of these pathways with peritoneal-directed therapies has the potential to improve survival in patients with high-risk primary gastric cancer.

Summary Cancer immunotherapy is only effective in a subset of patients, highlighting the need for effective biomarkers and combination therapies. Here we systematically identify genetic determinants of cancer cell sensitivity to anti-tumor immunity by performing whole-genome CRISPR/Cas9 knock-out screens in autologous tumoroid-T cell co-cultures, isogenic cancer cell models deficient in interferon signaling, and in the context of four cytokines. We discover that loss of CHD1 and MAP3K7 potentiates the transcriptional response to IFN-γ, thereby creating an acquired vulnerability through sensitizing cancer cells to tumor-reactive T cells. Immune checkpoint blockade was more effective in a syngeneic mouse model of melanoma deficient in Chd1 and Map3k7 and was associated with elevated intra-tumoral CD8 + T cell numbers and activation. CHD1 and MAP3K7 are recurrently mutated in cancer and reduced expression in tumors correlates with response to immune checkpoint inhibitors in patients, nominating these genes as potential biomarkers of immunotherapy response.

ABSTRACT The development of traditional protein-targeted cancer therapies is a slow and arduous process, often taking years or even decades. In contrast, RNA-based therapies targeting crucial microRNAs (miRNAs) offer a faster alternative due to the sequence specific nature of miRNA inhibitor binding. This, combined with the capacity of individual miRNAs to influence multiple cellular pathways, makes these small RNAs attractive targets for cancer therapy. While miRNA are known to be dysregulated in prostate cancer (PCa), identifying their individual contributions to disease progression and the identification of therapeutically actionable miRNA targets in PCa has been challenging due to limited screening tools. To overcome this, we developed miRKOv2, a miRNA-only CRISPR knockout library enabling systematic, genome-wide loss-of-function screens to identify miRNAs essential for PCa cell survival. Our screens uncovered 69 potential essential miRNA candidates, with miR-483 demonstrating the most significant impact on PCa cell viability. Functional characterization demonstrated that miR-483 disruption significantly potentiated apoptosis in PCa cell lines. Mechanistically, we uncovered a novel regulatory axis wherein miR-483-3p directly modulates a BCLAF1/PUMA/BAK1 apoptotic signaling network, highlighting its critical role in maintaining PCa cell survival. Our findings provide novel insights into the complex regulatory role of miRNA in PCa progression and offer a potential therapeutic strategy for targeting miRNA-mediated pathways in metastatic disease.

DELLA proteins, members of the GRAS-domain family of transcriptional regulators, are critical for plant growth and development. They modulate transcription indirectly via interactions with hundreds of transcription factors. The phytohormone gibberellin (GA) triggers DELLA degradation, providing a mechanism by which plants can integrate developmental and environmental signals to regulate gene expression and optimize growth responses. In agriculture, DELLA mutations have been instrumental in improving crop performance. Most modern wheat ( Triticum aestivum L.) varieties carry Rht-B1b or Rht-D1b alleles that encode DELLA proteins resistant to GA-mediated degradation, resulting in constitutive partial suppression of stem growth, a semi-dwarf stature and lodging resistance. However, these alleles also reduce nitrogen use efficiency and early vigour, limiting their utility in some environments. Understanding how DELLA proteins regulate growth and development is, therefore, critical for refining breeding strategies. In this study, we identified the orthologous C2H2 zinc-finger transcription factors INDETERMINATE DOMAIN 5 ( IDD5 ) in wheat and SEMI-DWARF 3 ( SDW3 ) in barley ( Hordeum vulgare ) as positive regulators of stem and leaf expansion. Both IDD5 and SDW3 physically interact with, and act downstream of, DELLA proteins as key components of GA-mediated growth responses. Altered expression levels of GA biosynthesis genes suggest that IDD5 helps maintain GA homeostasis in addition to growth regulation. Loss-of-function mutations in IDD5 and SDW3 confer a GA-insensitive semi-dwarf phenotype comparable to that of the Rht-D1b ‘Green Revolution’ allele, highlighting their potential as novel dwarfing alleles for cereal improvement. Significance statement Our study identifies homologous wheat and barley transcription factors (IDD5 and SDW3) that interact with DELLA proteins to regulate plant height. Unlike conventional ‘Green Revolution’ DELLA mutations, which can reduce height but also have drawbacks such as lower nitrogen-use efficiency, these IDD genes may provide more targeted approaches to manage plant growth. By showing how IDD proteins promote stem and leaf expansion and by revealing their potential as alternative dwarfing alleles, our research opens new avenues both for fundamental research into plant growth pathways and for applications in cereal breeding. Ultimately, it could help produce crops with improved lodging resistance and fewer negative side effects than current dwarfing alleles.

Pest management has entered a new era with the emergence of three innovative antisense technologies: RNAi, CUAD, and CRISPR/Cas. These technologies, which operate through sequence-specific nucleic acid duplex formation and guided nuclease activity, offer unprecedented potential for targeted pest control. While RNA-guided systems such as RNAi and CRISPR/Cas were initially discovered in non-insect models as fundamental biological mechanisms (primarily in antiviral defense), the DNA-guided CUAD system was first identified in insect pests as a practical tool for pest control, while its broader role in ribosomal RNA (rRNA) biogenesis only recently recognized. These surprising discoveries have unveiled an entirely new dimension of gene regulation, with profound implications for sustainable pest management. Despite certain similarities of these technologies, RNAi, CUAD, and CRISPR/Cas differ in their mode of action, specificity, and applicability. No single approach provides a universal solution for all insect pests; instead, each is likely to be most effective against specific pest groups. Moreover, these technologies enable the rapid adaptation of pest management strategies by countering target-site resistance, ensuring long-term efficacy. This review provides a critical synthesis of the unique advantages and limitations of each antisense technology, highlighting their complementary roles in eco-friendly, nucleic acid-guided insect pest control. By bridging fundamental discoveries with applied research, we offer new perspectives on their practical implementation, underscoring the urgent need for their integration into modern pest management strategies.

The phospholipid scramblases Xkr8 and TMEM16F externalize phosphatidylserine (PS) on cells by distinct molecular mechanisms. Xkr8, a caspase-activated scramblase, is activated by caspase-mediated proteolytic cleavage, and in synergy with caspase-mediated inactivation of P4-type ATP-dependent flippases, results in the irreversible externalization of PS on the dying cells and an “eat-me” signal for efferocytosis. In contrast, TMEM16F is a calcium activated scramblase that reversibly externalizes PS on viable cells via the transient increase in intracellular calcium on activated or growth factor stimulated cells. By contrast to the abovementioned homeostatic mechanisms of PS externalization under physiological conditions, PS becomes constitutively externalized in the tumor microenvironment (TME) in many solid tumor types by a complex mechanistic, posited both via the high apoptotic indexes of tumors, but also by the prolonged oncogenic and metabolic stresses that occur in the TME. Such chronic and persistent PS externalization in the TME has been linked to host immune evasion and the tonic interactions of PS with inhibitory PS receptors such as TAM (Tyro3, Axl, Mertk) and TIM (T cell/transmembrane, immunoglobulin, and mucin) family receptors. Here, in an effort to better understand the contributions of apoptotic vs live cell PS-externalization with respect to tumorigenesis and immune evasion, we employed an E0771 luminal B breast cancer orthotopic in vivo model and genetically ablated Xkr8 and TMEM16F using CRISPR/Cas9. While neither the knockout of Xkr8 nor TMEM16F showed defects in cell intrinsic properties related to cell growth, tumor sphere formation, cell migration, and growth factor signaling, both knockouts suppressed tumorigenicity in immune-competent mice, but not in NOD/SCID or RAG deficient immune-deficient strains. Mechanistically, at the cell biological level, Xkr8 knockout suppressed macrophage-mediated efferocytosis, and TMEM16F knockout suppressed ER stress/calcium-induced PS externalization. Our data support an emerging idea in immune-oncology and immunotherapy that constitutive PS externalization, mediated by the activation of scramblases on tumor cells, can support immune evasion in the tumor microenvironment thereby linking a combination of apoptosis/efferocytosis and oncogenic stress involving calcium dysregulation the contribute to PS-mediated immune escape and cancer progression.

Extrachromosomal DNA (ecDNA) is a common source of oncogene amplification across many types of cancer. The non-Mendelian inheritance of ecDNA contributes to heterogeneous tumour genomes that rapidly evolve to resist treatment. Here, using single-cell and live-cell imaging, single-micronucleus sequencing, and computational modelling, we demonstrate that elevated levels of ecDNA predisposes cells to micronucleation. Damage on ecDNA, commonly arising from replication stress, detaches ecDNA from the chromosomes upon which they hitchhike during cell division, thereby causing micronucleus formation in daughter cells. Clusters of oncogene-containing, CIP2A-TOPBP1-associated ecDNA molecules form, and asymmetrically segregate into daughter cell micronuclei during cell division. ecDNA chromatin remains highly active during mitosis, but upon micronucleation, it undergoes suppressive chromatin remodeling, largely ceasing oncogene transcription. These studies provide insight into the fate of damaged ecDNA during cell division.

Summary The function of HSFs, known otherwise as ‘master thermoregulators,’ in plant developmental remains largely uninvestigated. In this study, we strategically analyze SlHSFB3a , a class B sub member of HSF transcription factor family, uniquely expresses in age-dependent tomato roots and improves root architecture by synchronizing auxin homeostasis. This data demonstrates SlHSFB3a overexpressed transgenics display higher lateral root (LR) density and early LR emergence improving root architecture. Generation of CRISPR-Knockout mutants displayed contrasting phenotype, confirming SlHSFB3a ’s vital role in root growth. In SlHSFB3a manipulated roots, concentration gradient auxin responses oscillated with increase in LR number. We highlight the signal transduction of SlHSFB3a mediated auxin activation that enhances tomato LRs. SlHSFB3a directly inhibits auxin repressors, increases auxin flow via ARF7/LOB20 pathway and positively modulates LR growth.

Abstract Intracellular parasites like Toxoplasma gondii scavenge host nutrients, particularly lipids, to support their growth and survival. Although Toxoplasma is known to adjust its metabolism based on nutrient availability, the mechanisms that mediate lipid sensing and metabolic adaptation remain poorly understood. Here, we performed a genome-wide CRISPR screen under lipid-rich (10% Fetal Bovine Serum (FBS)) and lipid-limited (1% FBS) conditions to identify genes critical for lipid-responsive fitness. We identified the Toxoplasma protein GRA38 as a lipid-dependent regulator of parasite fitness. GRA38 exhibits phosphatidic acid (PA) phosphatase (PAP) activity in vitro, which is significantly reduced by mutation of its conserved DxDxT/V catalytic motif. Disruption of GRA38 led to the accumulation of PA species and widespread alterations in lipid composition, consistent with impaired PAP activity. These lipid imbalances correlated with reduced parasite virulence in mice. Our findings identify GRA38 as a metabolic regulator important for maintaining lipid homeostasis and pathogenesis in Toxoplasma gondii. 

Abstract Human papillomavirus (HPV) infection is a major threat to women’s health worldwide. High-risk subtypes, particularly HPV16, require rigorous screening and long-term surveillance to control cervical cancer. However, traditional HPV testing is hampered by the need for nucleic acid extraction, reliance on specialized technicians, and fluorescence detection equipment, limiting its suitability for rapid on-site testing. In this study, we developed a Concanavalin A-assisted extraction-free one-pot recombinase polymerase amplification (RPA) CRISPR/Cas12a assay (ConRCA) for HPV16. Concanavalin A-coated magnetic beads were used for target enrichment and nucleic-acid-extraction-free processing. Suboptimal protospacer-adjacent motifs were used to achieve a one-pot RPA–CRISPR/Cas12a assay. The ConRCA assay can be completed in approximately 25 min under isothermal conditions and can detect at least 1.2 copies/µL of HPV16 genomic DNA using a fluorescence reader or test strip. The feasibility of this detection method was evaluated with 31 unextracted clinical samples. Compared with qPCR, the overall sensitivity was 95% (19/20), and the specificity was 100% (11/11). Our results indicate that the ConRCA assay has great potential utility as a point-of-care testing for the rapid identification of HPV.

Abstract Mild cognitive impairment (MCI) is an intermediate stage between normal cognition and dementia, with a high risk of progression to Alzheimer’s disease (AD). As an important threshold for the intervention and prevention of AD, accurate diagnosis of AD-related MCI based on plasma biomarkers is crucial. However, early detection of AD-related MCI still poses a huge challenge. Herein, we propose an ultrasensitive CRISPR-based multi-protein detection array (UCMDA) that integrates the array device with CRISPR/Cas12a and antibody pair-based multi-recombinase polymerase amplification to facilitate inexpensive, sensitive, and specific detection of multiple markers in plasma. With only one fluorescence probe, UCMDA can simultaneously detect Aβ40, Aβ42, p-tau181, p-tau217, p-tau231, and p-tau396,404 within 1 h with a detection limit of 1 fg/mL. The applicability of UCMDA was demonstrated in 155 clinical plasma samples from normal senile controls (NCs), individuals with AD-related MCI, and patients with AD. We found that the UCMDA platform combined with machine learning algorithms achieved accurate early diagnosis of AD-related MCI with a detection accuracy, sensitivity, and specificity exceeding 90.38%. We anticipate that this inexpensive, ultrasensitive, and multi-detection platform can also be widely used to detect other protein-related biomarkers besides AD.

While prime editing offers improved precision compared to traditional CRISPR-Cas9 systems, concerns remain regarding potential off-target effects, including epigenetic changes such as DNA methylation. In this study, we investigated whether prime editing induces aberrant CpG methylation patterns. Whole-genome bisulfite sequencing revealed overall methylation similarity between Cas9-edited, and PE2-edited cells. However, localized epigenetic changes were observed, particularly in CpG islands and exon regions. The PE2-edited group showed a higher proportion of differentially methylated regions (DMRs) in some coding sequences compared to controls and Cas9-edited samples. Notably, CpG island methylation reached 0.18% in the PE2 vs. Cas9 comparison, indicating a higher susceptibility of these regulatory elements to epigenetic alterations by prime editing. Gene ontology and KEGG pathway analyses further revealed enrichment in molecular functions related to transcriptional regulation and redox activity in PE2-edited cells. These findings suggest that prime editing, while precise, may introduce subtle but functionally relevant methylation changes that could influence gene expression and cellular pathways. In summary, prime editing can induce localized DNA methylation changes in human cells, particularly within regulatory and coding regions. Understanding these epigenetic consequences is critical for the development of safer and more effective therapeutic applications of genome editing technologies.