Background: - The root-knot nematode Meloidogyne incognita, is a highly destructive parasite that manipulates host plant processes through effector proteins, affecting agriculture globally. Despite advances in genomic and transcriptomic studies, the regulatory mechanisms controlling effector gene expression, especially at the chromatin level, are still poorly understood. Gene regulation studies in plant-parasitic nematodes (PPN) face several challenges, including the absence of transformation systems and technical barriers in chromatin preparation, particularly for transcription factors (TFs) expressed in secretory gland cells. Conventional methods like Chromatin Immunoprecipitation (ChIP) are limited in PPN due to low chromatin yields, the impermeability of nematode cuticles, and difficulties in producing antibodies for low-abundance TFs. These issues call for alternative approaches, such as dCas9-based CAPTURE (CRISPR Affinity Purification in siTU of Regulatory Elements) that allows studying chromatin interactions by using a catalytically inactive dCas9 protein to target specific genomic loci without relying on antibodies. Results - This study presents an optimized in vitro dCas9-based CAPTURE for M. incognita that addresses key challenges in chromatin extraction and stability. The protocol focuses on the promoter region of the effector gene 6F06, a critical gene for parasitism. Several optimizations were made, including improvements in nematode disruption, chromatin extraction, and protein-DNA complex stability. This method successfully isolated chromatin-protein complexes and identified four putative chromatin-associated proteins, including BANF1, linked to chromatin remodelling complexes like SWI/SNF. Conclusion - The optimized in vitro dCas9-based CAPTURE protocol offers a new tool for investigating chromatin dynamics and regulatory proteins in non-transformable nematodes. This method expands the scope of effector gene regulation research and provides new insights into parasitism in M. incognita. Future research will aim to validate these regulatory proteins and extend the method to other effector loci, potentially guiding the development of novel nematode control strategies.

Apomixis, a process of clonal reproduction through seed, has the potential to significantly change agriculture by enabling a clonal seed propagation system for hybrid crops. Here, we demonstrate that hybrid seed from synthetically induced apomictic sorghum hybrids can be generated and maintained across multiple seed generations. This was achieved through the combination of avoidance of meiosis and induced parthenogenesis. Avoidance of meiosis was generated by the CRISPR/Cas9 knockout of the sorghum meiosis genes Spo11, Rec8, and OsdL1 and OsdL3. Parthenogenesis was induced in the resultant diploid egg cell using a maize egg cell promoter to express the Cenchrus ASGR-BBML2 gene coding sequence. Two strategies incorporating these components were used to induce synthetic apomixis in two different sorghum hybrids. Each hybrid used Tx623 as a female parent and either Tx430 or the African landrace Macia as a male parent. Seed yields in the induced apomictic hybrids were consistent and stable for multiple generations following self-pollination but reduced relative to the sexual hybrids. Sorghum contains two copies of the Osd1 gene that function in meiotic non-reduction. CRISPR/Cas9 knockout of both OsdL1 and OsdL3 loci was sufficient to produce clonal hybrid progeny in conjunction with the other apomixis induction components, but this led to a significant reduction in seed set. By contrast, a single in-frame edit of either OsdL1 or OsdL3 significantly improved seed set of clonal hybrid progeny. Fine-tuning OsdL activity appears to be essential to optimizing fertility. As the efficiency of seed set in the induced synthetic sorghum apomicts was lower than that of the sexual hybrid control, additional improvements are required to unlock the agronomic potential of synthetically induced apomictic sorghum in the field.

Heterochromatin is a repressive epigenetic state that suppresses transcription and safeguards genomic integrity. However, the full mechanism of how it is regulated remains elusive. Here, we focus on a previously described Pol II variant called rpb2-N44Y, which has a single substitution mutation within the Rpb2 subunit of Pol II that reduces RNAi-dependent heterochromatin. Through CRISPR-mediated site-directed mutagenesis, we find that rpb2-N44Y is a gain-of-function mutation. Furthermore, the heterochromatin defects of the rpb2-N44Y mutant requires a subunit of the Elongator complex called Elongator Protein 1 (Elp1), a protein that canonically promotes in mcm5s2U34 tRNA modifications. Intriguingly, we find that loss of Elp1, but not of other Elongator subunits such as Elp3, can robustly suppress heterochromatin defects in the rpb2-N44Y mutant. Elp1 acts independently of the mcm5s2 U34 tRNA modification to suppress RNAi-dependent heterochromatin at the pericentromere and the levels of small interfering RNAs (siRNAs) at affected heterochromatin. Overall, our study reveals two distinct Rpb2-centric pathways, via RNAi or Elp1, that can positively or negatively regulate heterochromatin, respectively. Furthermore, our findings reveal the first evidence of a chromatin function for Elp1 that is distinct from its canonical role in tRNA modifications. This work expands our understanding of how Elp1 can influence chromatin biology.

Long-read sequencing enables the incorporation of isoform-level expression into single-cell transcriptomic studies, offering detail beyond those accessible with short-read methods. Although insightful, these approaches have typically been costly and yielded limited data for each individual cell. Recent advances in library preparation approaches and sequencing throughput have brought long-read single-cell studies closer to the mainstream. Here, we present a comparative analysis of commercial approaches for single-cell long-read sequencing. We have performed parallel analyses of the same cDNA material, generated using the 10X genomics platform, on Illumina short-read, and PacBio and Oxford Nanopore long-read platforms. We also demonstrate the impact of CRISPR-based depletion of libraries, to remove highly expressed transcripts, prior to long-read sequencing in these experiments. By analysing single-source cDNA libraries in parallel, we enable a direct comparison of each platform, evaluating standard metrics alongside concordance in clustering and cell type identification. While each approach generates usable gene and isoform expression data, we identify limitations common across platforms, primarily linked to cDNA synthesis inefficiencies and read filtering strategies. Our work demonstrates the increasing utility of single-cell long-read sequencing for isoform-resolved analyses, such as direct immunoglobulin chain reconstruction without additional amplification, and the detection of alternative splicing patterns across immune cell subtypes in CD45, a key gene for immune cell activation and differentiation. Our benchmarking of current platform options provides a foundation for researchers looking to adopt single-cell long-read sequencing into their transcriptomic studies, providing a framework for its integration into diverse biological questions.

Maintenance of T cell population size, which is important for immune homeostasis, is controlled by interleukin-7 (IL-7) and low-affinity TCR/MHC interactions that provide limited survival cues. Using arrayed CRISPR screening of miR-17~92 targets, Bio-ID proximity labeling and proteomics we identified Tmem127 as an essential regulator of the T cell surface proteome. We validated interaction with the common gamma chain (IL-2Rγ) in a multi-protein complex. Tmem127 reduces IL-7 receptor surface expression to restrict homeostatic proliferation, thereby controlling naive and central memory T cell population sizes. Tmem127 germline knockout (KO) mice display splenomegaly, accelerated experimental autoimmune encephalomyelitis and Tmem127-deficient bone marrow displays a competitive advantage over wildtype cells. Thus, we identified Tmem127 as an important regulator of the common gamma chain and immune homeostasis.

Selenocysteine (Sec), the 21st amino acid, is co-translationally inserted at UGA codons via a specialized machinery requiring SECIS elements, Sec-tRNA^Sec, eEFSec, and SECIS-binding protein 2 (SBP2). While SBP2 is essential for Sec incorporation in vitro and in vivo, the function of its paralog, SECISBP2L, remains incompletely defined. In this study, we investigated the distinct roles of SBP2 and SECISBP2L in the human hepatocellular carcinoma cell line HepG2, which expresses a broad selenoproteome. Using CRISPR-Cas9 genome editing, we generated SBP2 and SECISBP2L edited cell lines. Consistent with previous findings, SBP2 targeting impaired selenoprotein mRNA and protein expression, whereas SECISBP2L targeting did not. However, transcriptomic profiling by RNA-seq revealed that SECISBP2L targeting induced differential expression of over 800 genes, with significant enrichment in pathways related to extracellular matrix organization and cell adhesion. In contrast, SBP2 targeting produced a distinct transcriptomic signature enriched for metabolic and ion transport processes. Notably, only limited overlap in differentially expressed genes was observed between the two knockout models. Mass spectrometry and immunoblot data indicated that CRISPR-targeted SECISBP2L cells produce a truncated protein via internal translation initiation, suggesting that observed gene expression changes may be attributable to loss of a portion of the SECISBP2L N-terminus. These findings support a model in which SECISBP2L plays a noncanonical role in regulating gene expression independent of selenoprotein synthesis. Given prior associations between SECISBP2L downregulation or mutation and cancer progression, our data raise the possibility that SECISBP2L modulates cell adhesion and extracellular matrix gene networks relevant to metastatic potential. This work establishes a foundation for further mechanistic studies into the role of SECISBP2L in gene regulation and disease.

Abstract Synthetic lethality (SL) underlies the success of PARP1 inhibitors (PARPi) in treating homologous recombination (HR) deficient cancers, but extending this paradigm to other DNA damage response (DDR) deficiencies has proven challenging. We performed an in vivo CRISPR screen to identify DDR gene mutations that both enhance tumorigenesis and confer sensitivity to PARPi. Our screen identified FANCA deficiency as a driver of PARPi SL that was validated across diverse human cancer models. FANCA deficiency does not impair HR but disrupts Okazaki fragment maturation (OFM), causing lagging strand gaps and RPA exhaustion upon PARPi treatment. These effects require FANCA interaction with FEN1, independently of its canonical role in interstrand crosslink repair. We find FANCA-mediated FEN1 recruitment is required for OFM at oncogene-associated R loops during PARPi treatment. These findings establish a novel and non-canonical function for FANCA in FEN1-mediated OFM that can be leveraged for PARPi synthetic lethality in FANCA-mutant cancers.

During zygotic genome activation (ZGA) in Drosophila, broad domains of Polycomb-modified chromatin are rapidly established across the genome. Here, we investigate the spatial and temporal dynamics by which Polycomb group (PcG) histone modifications, H3K27me3 and H2Aub, emerge during early embryogenesis. Using ChIP-seq and live imaging of CRISPR-engineered GFP-tagged PcG components, we show that PRC2-dependent H3K27me3 accumulates adjacent to a subset of E(z)-bound prospective Polycomb Response Elements (PREs) beginning in nuclear cycle 14 (NC14), with patterns indicative of nucleation followed by spreading. Surprisingly, PRE-binding factors Pho, Combgap, and GAGA-factor are excluded from interphase nuclei prior to NC10 despite nuclear localization of E(z) throughout early interphases. Loss-of-function studies further demonstrate that GAGA-factor is largely dispensable for PcG domain establishment, whereas the pioneer factor Zelda is required for proper deposition of H3K27me3 and H2Aub at a subset of Polycomb domains. The role of Zelda at Polycomb domains is context-dependent; a large subset of targets requires Zelda not for PcG factor recruitment, but instead to license a loaded PRE to deposit H3K27me3 and H2Aub. Our findings support a model where licensing of PcG domains is an initial step in the regulatory processes governing Polycomb-regulated developmental genes.

Transcriptome profiling of bladder cancer has revealed distinct basal-like and luminal-like molecular subtypes, which may be correlated with pathological subtypes of different patient outcomes. However, whether these molecular subtypes originate from the corresponding cell types in the normal urothelium and whether different cells of origin influence bladder cancer progression remain unclear. Here, we conducted cell-type-specific lineage tracing in CRISPR/Cas9-induced mouse bladder cancer models of Pten and Trp53 targeting. We show that although basal, intermediate, and superficial umbrella cells can all serve as the cell of origin for bladder cancer, transformed umbrella cells were gradually displaced by tumor cells from inner layers, particularly transformed basal cells, which had highest stemness. Histological and single cell RNA-sequencing data comparing basal- and intermediate-cell-induced bladder tumors revealed that basal-induced tumors displayed higher heterogeneity, and contained unique cell clusters including Krt14+Ki67+ highly proliferative basal cells, squamous cell carcinoma, and transitioning cells towards the Gata3+ luminal subtype. Trajectory analysis confirmed the cell lineage differentiation hierarchy uncovered in lineage tracing. Moreover, human bladder cancer molecular subtype signatures were highly enriched in mouse tumor cell clusters of the corresponding cell of origin, and a gene signature derived from the unique basal-induced clusters is predictive of worse patient outcome. Overall, our results support that the basal and luminal molecular subtypes of bladder cancer have the corresponding cells of origin as their basis, and that urothelial basal cells are intrinsically more competitive than intermediate and umbrella cells in generating aggressive bladder cancer subtypes.

Extrachromosomal circular DNA (eccDNA) of chromosomal origin is commonly present in all eukaryotic organisms and tissue tested so far. EccDNA populations exhibit immense diversity and a characteristically low degree of overlap between samples, suggesting low inherence of eccDNA between cells or a deficiency the methods by which eccDNA is detected. This study revisits the Circle-seq approach for enrichment of eccDNA to address if these limitations, hypothesizing that experimental procedures significantly contribute to the observed low eccDNA overlap. We optimized the protocol by reducing the time. Linear DNA is digested by increasing exonuclease V activity. We employed CRISPR-Cas9 for mitochondrial linearization, which proved superior to restriction enzymes. A key finding is the critical role of random hexamer primer concentration and genomic DNA input in Rolling Circle Amplification (RCA) for generating high-quality long amplicons from eccDNA (concatemeric tandem copy, CTC), essential for confident de novo eccDNA construction from long-read sequencing data. Lower primer concentrations substantially increased the percentage of CTC-derived eccDNA and improved the overlap of identified eccDNAs in technical replicates. Applying this revisited approach to human myeloma and breast cancer cell lines, as well as xenograft models, demonstrated that the optimized conditions enhanced the overlap of detected eccDNA up to over 50% overlap which substantially improved over previous studies (less than 1%). These findings provide guidelines for developing standardized procedures for eccDNA profiling, advancing our understanding of eccDNA biology and its potential clinical applications.

Abstract Deficiency of the Monocarboxylate Transporter 8 (MCT8) severely impairs thyroid hormone (TH) transport into the brain, disrupting brain development as well as peripheral TH homeostasis. Studies assessing MCT8 expression patterns and tissue-specific pathologies induced by local TH-deficiency are often inconclusive due to unreliable antibody staining and the lack of functional tools to specifically target MCT8-expressing cells. For this purpose, we generated non-inducible Mct8-Cre and tamoxifen-inducible Mct8-CreERT2 mice. Mct8-Cre;Sun1-sfGFP mice demonstrated ubiquitous Sun1-sfGFP expression, due to early recombination driven by Mct8 gene expression at the stage of trophoblast implantation. Tamoxifen injection in 6-week-old Mct8-CreERT2 mice induced reporter expression specifically in Mct8-expressing cells in the brain and peripherally in liver, kidney, and thyroid, without leaky reporter expression in vehicle controls. Using vDISCO tissue clearing and 3D-imaging of GFP-nanobody-boosted mice, we further identified the sublingual salivary gland and the prostate as prominent Mct8-expressing organs. Nuclei from Mct8-expressing cells could selectively be enriched using fluorescence-activated nuclei sorting on Mct8-CreERT2;Sun1-sfGFP mice and characterized as choroid plexus cells and tanycytes. Our new inducible Mct8-CreERT2 line provides researchers with a tool to reliably mark, enrich, and characterize Mct8-expressing cells and to genetically modify genes specifically in these cells to study thyroid hormone transport and function.

Abstract Primary aldosteronism (PA), also known as Conn’s syndrome or adrenal aldosterone producing adenoma (APA), is predominantly caused by functional adrenal tumors and is a common cause of secondary hypertension. The KCNJ5 gene mutations are frequently associated with APA, leading to increased aldosterone production. This study investigates the effect of KCNJ5 mutations on CYP11A1 and the aldosterone biosynthesis pathway. We created two model cell lines by introducing homozygous p.L168R and p.G151R mutations in the KCNJ5 gene to SW13 cell line using CRISPR/Cas9 technology. The mutations were verified succeed through Sanger sequencing and multi-omics analysis. Aldosterone and its biosynthesis intermediates were quantitatively analyzed, and the expression of CYP11A1 mRNA in wild-type and mutant cell lines was detected through qPCR. Increased concentrations of aldosterone, pregnenolone, progesterone, and corticosterone in the KCNJ5 mutated cell lines were observed compared to the wild type, while cholesterol levels remained unchanged. qPCR results showed increased CYP11A1 mRNA expression in KCNJ5 mutant cells. Therefore, KCNJ5 mutations promote aldosterone synthesis by enhancing CYP11A1 activity, which catalyzes the conversion of cholesterol to pregnenolone, a critical step in the aldosterone biosynthesis pathway. This study highlights the potential of CYP11A1 as a therapeutic target for treating APA-induced secondary hypertension.

Corynebacterium ulcerans is an emerging zoonotic pathogen that can cause diphtheria-like infections in humans. In this study, we report the novel detection and comprehensive phenotypic and genomic characterization of three atoxigenic C. ulcerans strains isolated from domestic animals in Brazil. Notably, all isolates belonged to the multilocus sequence type ST-339, which has been previously identified in both human and animal hosts from geographically distant regions, suggesting the potential for international dissemination. Whole-genome sequencing confirmed species identity and revealed high genetic simila-rity among isolates, although distinct phylogenetic subclades were observed. Genomic analyses identified conserved virulence-associated determinants, including incomplete pilus gene clusters, iron acquisition systems, and the pld gene encoding phospholipase D. In contrast, the tox gene was absent in all strains. Notably, one isolate exhibited ciproflo-xacin resistance associated with double mutations (S89L and D93G) in the quinolone- resistance-determining region of GyrA. Molecular modeling and dynamics simulations demonstrated that these mutations impair key interactions within the ciprofloxacin– magnesium–water complex, thereby compromising the stability of drug binding. Additionally, the presence of diverse mobile genetic elements, prophages, and CRISPR-Cas systems highlighted the genomic plasticity of these isolates. Our findings provide new insights into the zoonotic potential, antimicrobial resistance mechanisms, and genomic diversity of C. ulcerans, underscoring the need for strengthened surveillance and mole-cular monitoring of this emerging pathogen in both veterinary and public health contexts.

Summary Polyamines are essential and evolutionarily conserved metabolites present at millimolar concentrations in mammalian cells. Cells tightly regulate polyamine homeostasis through complex feedback mechanisms, yet the precise role necessitating this regulation remains unclear. Here, we show that polyamines function as endogenous buffers of redox-active iron, providing a molecular link between polyamine metabolism and ferroptosis. Using genome-wide CRISPR screens, we identified a synthetic lethal dependency between polyamine depletion and the key ferroptosis suppressor, GPX4. Mechanistically, we show that polyamine deficiency triggers a redistribution of cellular iron, increasing the labile iron pool and upregulating ferritin. To directly visualize this iron buffering in living cells, we developed a genetically encoded fluorescent reporter for redox-active iron. Live-cell analysis revealed a striking inverse correlation between intracellular polyamine levels and redox-active iron at single-cell resolution. These findings reposition polyamines as key regulators of iron homeostasis, with implications for ferroptosis-linked disease states and cellular redox balance.

ABSTRACT Mosaic analysis has been instrumental in advancing developmental and cell biology. Most current mosaic techniques rely on exogenous site-specific recombination sequences that need to be introduced into the genome, limiting their application. Mosaic analysis by gRNA-induced crossing-over (MAGIC) was recently developed in Drosophila to eliminate this requirement by inducing somatic recombination through CRISPR/Cas9-generated DNA double-strand breaks. However, MAGIC has not been widely adopted because gRNA-markers, a required component for this technique, are not yet available for most chromosomes. Here, we present a complete, genome-wide gRNA-marker kit that incorporates optimized designs for enhanced clone induction and more effective clone labeling in both positive MAGIC (pMAGIC) and negative MAGIC (nMAGIC). With this kit, we demonstrate clonal analysis in a broad range of Drosophila tissues, including cell types that have been difficult to analyze using recombinase-based systems. Notably, MAGIC enables clonal analysis of pericentromeric genes and deficiency chromosomes and in interspecific hybrid animals, opening new avenues for gene function study, rapid gene discovery, and understanding cellular basis of speciation. This MAGIC kit complements existing systems and makes mosaic analysis accessible to address a wider range of biological questions.

Against the backdrop of global population growth and the continuous escalation of food demand, the acceleration of agricultural modernization has emerged as the core pathway to safeguard food security. As the world’s fourth-largest food crop, soybean (Glycine max) possesses multiple strategic values for food security, livestock feed, and industrial raw materials, thanks to its high protein content (accounting for over 40% of the dry weight of seeds) and oil resource attributes. However, the soybean industry is confronted with multiple challenges: the long cycle of genetic breeding (8-10 years required by traditional methods), annual losses from diseases and pests reaching 40% (data from FAO 2023), and the reliance on empirical decision-making in field management, all of which urgently call for intelligent solutions.At present, agricultural knowledge is experiencing explosive growth - more than 4,000 new soybean-related literatures are added annually in PubMed, and agricultural technology Q&A platforms (Zhihu, Baidu Tieba, etc.) generate over 3,000 daily questions. However, this multi-source heterogeneous knowledge is scattered in books, literatures, Q&A communities, and gene databases, lacking systematic integration, resulting in a knowledge utilization rate of less than 30%. In response to this, this paper proposes the “Fengshu-Agri” large model, an agricultural knowledge integration and innovation engine based on the collaboration of Retrieval-Augmented Generation (RAG) and Knowledge Graph (KG). The model achieves breakthroughs through the following core technological innovations: Deep integration of multi-source heterogeneous data: For the first time, a systematic integration of 256 agricultural monographs (covering standardized knowledge such as planting techniques and pest control), 66,772 cutting-edge research literatures (genomics, agronomic trait analysis, etc.), 880,000 production practice Q&A (covering scenarios such as pest diagnosis and environmental stress response), and 120,000 gene annotation data (functional genes, expression regulation mechanisms) has been carried out to construct the largest knowledge graph in the agricultural field - containing 2 million entity nodes, realizing full-chain knowledge modeling from molecular-level gene regulation to field management. Double-layer collaborative retrieval mechanism: Innovatively integrating RAG semantic vector retrieval with KG structured reasoning to solve problems such as low recall rate (<65%) and semantic ambiguity in traditional systems. Specifically, it achieves dual matching through local keywords (entity-level) and global keywords (relationship-level); Optimization of domain-specific generation engine: Based on the QWEN-QWQ32B large language model, a three-stage fine-tuning is carried out (pre-training, agricultural instruction fine-tuning, and reinforcement learning from human feedback) [15], which improves the professionalism of generated texts by 35% (manual evaluation index) and significantly reduces the LLM hallucination problem [12]. Experimental results show that Fengshu-Agri achieves an accuracy rate of 89.6% in soybean knowledge retrieval tasks (a 25.4% improvement over traditional RAG models), a recall rate of 87.3% (20.5% higher than pure KG reasoning models), and an F1 score of 88.4%. It can efficiently answer complex questions involving multi-entity associations (such as “the impact mechanism of CRISPR-editing the GmSWEET gene on lepidopteran pest resistance”). Analysis of typical cases indicates that the model’s responses not only cover technical principles (sgRNA design, Agrobacterium transformation procedures) but also integrate metabolomics data (70% improvement in insect resistance with no significant yield reduction), demonstrating cross-modal knowledge fusion capabilities. In the future, this model will be expanded to crops such as maize and potato to construct an agricultural intelligent ecosystem covering the entire process of cultivation management and breeding improvement, promoting the upgrading of precision agriculture toward a data-driven model.

The CRISPR–Cas9 system has become a widely used tool for genome engineering. Here we present a new method for small-molecule control of CRISPR-Cas9 using bio-orthogonal chemistry between tetrazine (Tz) and trans -cyclooctene (TCO). We carried out molecular modeling studies and identified a unique position on single guide RNA (sgRNA) that can be site-specifically tagged with Tz without disrupting its activity. We also synthesized a series of TCO-modified CRISPR suppressors. When exogenously added, they click to the Tz-tagged sgRNA, perturb the system and drastically reduce the nuclease activity. The most successful suppressor is a TCO-modified six amino acid long cell-penetrating peptide, which shows excellent cell permeability. We showed that out method to control CRISPR-Cas9 nuclease activity is general by applying it to three different sgRNAs. We also showed that our method works in solution, as well as live HEL293 cells. We utilized flow cytometry to demonstrate temporal control of CRISPR-Cas9 targeting GFP. Lastly, we showed the therapeutic potential of our method by targeting vascular endothelial growth factor A (VEGFA).

Type III CRISPR systems typically generate cyclic oligoadenylate (cOA) second messengers such as cyclic tetra-adenylate (cA 4 ) on detection of foreign RNA, activating ancillary effector proteins which elicit a diverse range of immune responses. The CalpLTS system elicits a transcriptional response to infection when CalpL binds cA 4 in its SAVED (SMODS associated and fused to various effectors domain) sensor domain, resulting in filament formation and activation of the Lon protease domain, which cleaves the anti-Sigma factor CalpT, releasing the CalpS Sigma factor for transcriptional remodelling. Here, we show that thermophilic viruses have appropriated the SAVED domain of CalpL as an anti-CRISPR, AcrIII-2, which they use to degrade cA 4 . AcrIII-2 dimers sandwich cA 4 , degrading it in a shared active site to short linear products, using a mechanism highly reminiscent of CalpL. This results in inhibition of a range of cA 4 activated effectors in vitro . This is the first example of a virally-encoded SAVED domain with ring nuclease activity, highlighting the complex interplay between viruses and cellular defences.

Extracellular vesicles (EVs) are emerging as versatile mediators of intercellular communication and promising tools for drug discovery and targeted therapies. These lipid bilayer-bound nanovesicles facilitate the transfer of functional proteins, RNAs, lipids, and other biomolecules between cells, thereby influencing various physiological and pathological processes. This review outlines the molecular mechanisms governing EV biogenesis and cargo sorting, emphasizing the role of regulators, such as ubiquitin-like 3 (UBL3), in modulating protein packaging. We explored the critical involvement of EVs in various disease microenvironments, including cancer progression, neurodegeneration, and immune modulation. Their ability to cross biological barriers and deliver bio-active cargo renders them highly attractive for precise drug delivery systems, especially in neurological and oncological disorders. Moreover, this review highlights advances in engineering EVs for delivering RNA therapeutics, CRISPR-Cas systems, and targeted small molecules. The utility of EVs as diagnostic tools in liquid biopsies and their integration into personalized medicine and companion diagnostics were also discussed. Patient-derived EVs offer dynamic insights into disease state and enable real-time treatment stratification. Despite their potential, challenges such as scalable isolation, cargo heterogeneity, and regulatory ambiguity remain significant hurdles. We also reported novel pharmacological approaches targeting EV biogenesis, secretion, and uptake pathways, and considered UBL3 as a promising drug target for EV cargo modulation. Future directions include the standardization of EV analytics, scalable biomanufacturing, and classification of EV-based therapeutics under evolving regulatory frameworks. This review emphasizes the multifaceted roles of EVs and their transformative potential as therapeutic platforms and biomarker reservoirs in next-generation precision medicine.

Gliomas, particularly aggressive glioblastoma (GBM), pose significant therapeutic challenges due to limited understanding of their single-cell drivers. Here, we integrate large glioma genetic data with brain multi-omics (bulk and single-cell) to identify causal genes and their cell-type-specific roles. We prioritize 11 high-confidence and 47 potential causal genes; 41 are novel associations. Analyses suggest most of these 58 genes are druggable, supported by CRISPR/RNAi screens showing essentiality/dependency for 53.7% novel candidates in glioma cell lines. Single-cell data identifies astrocytes and oligodendrocyte precursor cells (OPCs) as likely GBM cells-of-origin and reveals increased tumor microenvironment (TME) communication involving neurons. We uncover 14 cell-type-specific causal gene effects, including EGFR in astrocytes, CDKN2A in OPCs, and JAK1 in excitatory neurons. Notably, 85.7% effects occur in non-risk populations (glial and neural), highlighting complex interplays. This study provides critical cell-resolved insights into glioma susceptibility mechanisms and identifies potential therapeutic targets within complex intratumoral interactions, advancing targeted precision therapies.

The "dark genome," comprising pseudogenes and various non-coding DNA elements, has historically been overlooked due to the assumption of its non-functionality. Recent advances in genomics and epigenetics have overturned this view, revealing that these sequences play crucial roles in genetic regulation, development, disease, and evolution. Pseudogenes, once dismissed as evolutionary relics, are now recognized for their regulatory potential via RNA interference, decoy functions, and epigenetic modulation. Non-coding regions such as long non-coding RNAs (lncRNAs), enhancer RNAs (eRNAs), and other untranslated elements contribute to transcriptional control and chromatin architecture. This review explores the biological functions of these components, their implications in health and disease, and their growing relevance in biomedical research. Furthermore, we examine how emerging technologies such as single-cell sequencing, CRISPR-based editing, and integrative multi-omics are shedding light on the regulatory functions of the dark genome. Despite significant progress, many challenges persist, including functional validation, annotation inconsistency, and interpretation of non-coding variants. This paper aims to synthesize current findings, highlight biomedical applications, discuss limitations, and propose future research directions, emphasizing the need to embrace the dark genome for a more comprehensive understanding of gene regulation and genome complexity.

Genetic interaction (GI) networks in model organisms have revealed how combinations of genome variants can impact phenotypes and underscored the value of GI maps for functional genomics. To advance efforts toward a reference human GI network, we developed the q uantitative G enetic I nteraction (qGI) score, a method for precise GI measurement from genome-wide CRISPR-Cas9 screens in isogenic human cell lines. We found surprising systematic variation unrelated to genetic interactions in CRISPR screen data, including both genomically linked effects and functionally coherent covariation. Leveraging ∼40 control screens and half a billion differential fitness effect measurements, we developed a CRISPR screen data processing and normalization pipeline to correct these artifacts and measure accurate, quantitative GIs. Finally, we comprehensively characterized GI reproducibility by recording 4 – 5 biological replicates for ∼125,000 unique gene pairs. The qGI framework enables systematic identification of human GIs and provides broadly applicable strategies for analyzing context-specific CRISPR screen data.

Modern gene-synthesis platforms let us probe protein function and genome biology at unprecedented scale. Yet in large, diverse gene libraries the proportion of error-free constructs decreases with length due to the propagation of oligo synthesis errors. To rescue these rare, error-free molecules we developed BAR-CAT (Barcode-Assisted Retrieval CRISPR-Activated Targeting), an in-vitro enrichment method that couples unique PAM-adjacent 20-nt barcodes to each library member and uses multiplexed dCas9-sgRNA complexes to fish out the barcodes corresponding to perfect assemblies. After a single 15-min reaction and optimized wash regime (BAR-CAT v1.0), three low-abundance targets in a 300, 000-member test library were enriched 600-fold, greatly reducing downstream requirements. When applied to 384x and 1, 536x member DropSynth gene libraries, BAR-CAT retrieved up to 122-fold enrichment for 12 targets and revealed practical limits imposed by sgRNA competition and library complexity, which now guide ongoing protocol scaling. By eliminating laborious clone-by-clone validation and working directly on plasmid libraries, BAR-CAT provides a versatile platform for recovering perfect synthetic genes, subsetting large libraries, and ultimately lowering the cost of functional genomics at scale.

The impact of genotype on gene expression can depend on both cellular and organismal context. Here, we leverage an extensive blood atlas of genotyped patients with varying severity of infection produced by the COVID-19 Multi-omics Blood ATlas (COMBAT) Consortium to study the role of genetic regulation on gene expression in a context-specific manner. We analyzed single-cell transcriptomic and genome-wide genetic data from ∼500,000 cells and 76 donors of European ancestry. Across 15 cell types, we identified 2,607 independent cis-eQTLs in high linkage disequilibrium (R2>0.8) with 48 infectious and 386 inflammatory disease-associated risk variants, including rheumatoid arthritis (RA) and inflammatory bowel disease (IBD). Notably, we found infection-specific eQTLs absent from a general population dataset (OneK1K), such as REL , IRF5 and TRAF , all of which were differentially regulated by infection and whose variants are associated with RA and/or IBD. We also identified infection-modified eQTLs, including RPS26 and ADAM10 , implying that the regulatory sequences context of these genes may play a role in specific immune cell subsets in infection. Our work demonstrates that the overriding effect of genetics on gene expression in blood immune cells is independent of infection status or severity. However, small numbers of eQTLs are modified by infection, and these differences can illustrate potentially important immune biology.

CRISPR homing gene drive holds great potential for pest control, but its success is challenged by the generation of resistance alleles. To mitigate the impact of resistance, multiplexed gRNA strategies have been demonstrated. However, unless both outmost sites are cleaved simultaneously, poor homology during DNA repair may compromise efficiency, leading to decline in drive conversion efficiency when the number of gRNAs is higher than two. Here, to better estimate the rate of drive efficiency decline, we designed and assessed the efficiency of single gRNA drives with imperfect homologous arms, refining a detailed gRNA multiplexing model. To mitigate the greater than expected efficiency loss, we further evaluated two new strategies: (1) extended homology arms to span all target sites with mutations in the PAMs and (2) a population-level multiplexing gRNAs system involving two or more drives, each carrying two gRNAs. Specifically, the population-level multiplexing system has four adjacent gRNA target sites, and the drives have small mutations in their homologous arms to prevent cleavage by the other drive. Mutations in both strategies did not impair efficiency, but they were not consistently inherited, and undesired cutting in the homologous arms decreased drive efficiency. We simulated homing suppression drive using a dual 2-gRNA population-level gRNA multiple xing strategy based on our experimental evaluation. Despite being somewhat more vulnerable to functional resistance than a standard 4-gRNA drive, the higher individual drive efficiency of the population-level multiplexing system increased successful population elimination outcomes. Thus, population-level multiplexing can be a useful for improving suppression drive power.