Extrachromosomal circular DNA (eccDNA) of chromosomal origin is present in all eukaryotic organisms and tissues that have been tested. Populations of eccDNA exhibit immense diversity and a characteristically low degree of overlap between samples, suggesting low inheritance of eccDNA between cells or a deficiency in the methods by which eccDNA is detected. This study revisits the Circle-seq approach for enrichment of eccDNA to address these limitations, hypothesizing that experimental procedures significantly contribute to the observed low eccDNA overlap. We optimized the protocol by reducing the time needed to complete the procedure. Linear DNA is digested by increasing Exonuclease V activity. We employed CRISPR-Cas9 for mitochondrial linearization, which proved superior to using 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 revised approach to human myeloma and breast cancer cell lines, as well as xenograft models, demonstrated >50% overlap in detected eccDNA, a substantial improvement over the <1% overlap observed in previous studies. Additionally, the oncogenic signature of eccDNAs can be identified across all replicates. These findings provide guidelines for developing standardized procedures for eccDNA profiling, advancing our understanding of eccDNA biology, and its potential clinical applications.

Mutations of EIF2AK4 , which encodes the eIF2α kinase GCN2, cause a severe inherited form of pulmonary hypertension called pulmonary veno-occlusive disease (PVOD). Some pathogenic variants of GCN2 are amenable to pharmacological reactivation by low concentrations of ATP-pocket binding inhibitors. Kinase inhibition at modestly elevated concentrations limits the clinical utility of these drugs against PVOD. We therefore performed an in cellulo chemical screen for GCN2 activators and identified three structurally distinct compounds with low micromolar stimulatory activities. Unlike previously described GCN2 activators, one of these molecules activated GCN2 independently of GCN1. Modelling supported by structure activity screens suggested it binds within the ATP-pocket of GCN2, but unlike existing ligands does not protrude inward into the allosteric pocket or outward into the solvent. This overcomes a key requirement of other GCN2 activators.

ABSTRACT Short interspersed nuclear elements (SINEs) are abundant non-autonomous transposable elements derived from RNA polymerase III (POL III)-transcribed short non-coding RNAs. SINEs retain sequence features recognized by the POL III machinery and constitute a substantial portion of vertebrate genomes. Despite their impact on genome stability and evolution, the mechanisms governing SINE transcription remain poorly understood. Although DNA methylation and heterochromatin formation have been implicated in their repression, we find these pathways play only a minor role in mouse embryonic stem cells. Instead, we identify the ChAHP complex as a key repressor of SINE B2 elements. ChAHP directly inhibits POL III transcription by blocking TFIIIB recruitment without affecting TFIIIC binding. This selective interference prevents transcription initiation and highlights a distinct regulatory mechanism. Our findings establish ChAHP as a non-canonical repressor of POL III-dependent SINE transcription, offering new insights into the control of this pervasive class of non-coding genomic elements.

The sequestration of the malaria parasite Plasmodium falciparum in the microvasculature is a major driver of severe malaria, but its pathogenic mechanisms still remain unknown. Advancements in induced pluripotent stem cell (iPSC) technologies offer unique opportunities to study parasite interactions with blood vessels in a well-defined host environment. However, endothelial iPSC-differentiation methods often result in cells with mixed epithelial identity. Here, we have generated an iPSC line with inducible and simultaneous expression of ETS transcription factors (ETV2, FLI1, ERG), which resulted in improved endothelial cell identity and strong barrier function. These cells display a high affinity to infected red blood cells. Exposure to parasite products caused significant endothelial metabolic changes and splicing alterations. Furthermore, it disrupted the iPSC-endothelial barrier, as a consequence of transcriptional downregulation of key barrier processes, and alteration of severe malaria biomarkers. Our novel iPSC-based approach represents a new in vitro platform to study the pathogenesis of vascular infections.

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.

Genes can be knocked out in model organisms by introducing a single guide RNA and Cas9 into one cell zygotes. Recently, the zebrafish and Xenopus communities have employed this method in genetic screening pipelines that assess phenotypes in founders (F0), referred to as “crispants”. In contrast, phenotyping crispant mice has been avoided as results are believed to be confounded by genetic mosaicism, requiring that only established mouse lines undergo phenotypic assessment. Here, we targeted seven genes associated with visible recessive phenotypes. We observed the expected null phenotype in up to 100% founders per gene. Crucially, we achieved 100% editing efficiency in all but two animals. Genetic mosaicism was common, but did not confound an animal’s phenotype when comprised of mutations that all disrupted the targeted gene. Mosaicism included short in-frame mutations, but these were sufficient to disrupt function of five genes. Several founders were compound heterozygotes carrying a null and a non-null allele (short in-frame mutation or late truncation), enabling functional assessment of the non-null allele to dissect protein function. Our results set the stage for using crispant founders for initial phenotypic assessment in genetic screening, before selecting candidates for further study. This will dramatically reduce animal numbers.

Summary The Target of Rapamycin Complex 2 (TORC2) is a central node in signaling feedback loops serving to maintain biophysical homeostasis of the plasma membrane (PM). How TORC2 is regulated by mechanical perturbation of the PM is not well understood. To address this, we determined the cryo-electron microscopy structure of endogenous yeast TORC2 to up to 2.2 Å resolution. Our model refines the position and interactions of TORC2-specific subunits, providing a structural basis for the differential assembly of Tor2 into TORC2. Furthermore, we observe the insertion of the pleckstrin-homology domain of the Avo1 subunit into the Tor2 active site, providing a regulatory mechanism by phosphoinositides. Structure-guided functional experiments reveal a potential TORC2 membrane binding surface and a positively charged pocket in the Avo3 subunit that is necessary for TORC2 activation. Collectively, our data suggest that signaling phosphoinositides activate TORC2 by membrane-induced structural rearrangements via concerted action of conserved regulatory subunits.

A bstract CDK4/6 inhibitors (CDK4/6i) arrest the cell cycle in G1 leading to cellular overgrowth and p53-dependent senescence. They are used to treat metastatic HR+/HER2-breast cancer, but resistance is common, and this has been associated with TP53 loss and senescence evasion. We show here that enlarged CDK4/6i-treated cells that evade senescence mis-segregate chromosomes due to defective chromosomal alignment and a weakened mitotic checkpoint, leading to aneuploidy and DNA damage. The chromosome alignment errors are associated with impaired Sgo1 localisation to centromeres and defective sister chromatin cohesion during mitosis. Importantly, all these mitotic defects can be rescued by constraining cell size during the CDK4/6i-treatment, and specifically restoring cohesion rescues the chromosome segregation errors. Together, this demonstrates mechanistically how cell enlargement drives genetic and karyotypic change in cells that re-enter the cell cycle following CDK4/6 inhibition. This could help fuel the rapid emergence of chemotherapy-resistant clones, especially in p53-null cells that evade senescence to drive drug-resistance in patients.

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.

Understanding how evolution shapes genetic networks to create new developmental forms is a central question in biology. Flowering shoot (inflorescence) architecture varies significantly across plant families and is a key target of genetic engineering efforts in many crops 1–4 . Asteraceae (sunflower family), comprising 10% of flowering plants, all have capitula, a novel inflorescence that mimics a single flower 5,6 . Asteraceae capitula are highly diverse but are thought to have evolved once via unknown mechanisms 7,8 . During capitulum development, shoot stem cells undergo prolonged proliferation to accommodate the formation of intersecting spirals of flowers (florets) along the disk-shaped head 9,10 . Here we show that capitulum evolution paralleled decreases in CLAVATA3 (CLV3) peptide signaling, a conserved repressor of stem cell proliferation. We trace this to novel amino acid changes in the mature CLV3 peptide which decrease receptor binding and downstream transcriptional outputs. Using genetically tractable Asteraceae models, we show that reversion of CLV3 to a more active form impairs Asteraceae stem cell regulation and capitulum development. Additionally, we trace the evolution of CLV3 and its receptors across the Asterales allowing inferences on capitulum evolution within this lineage. Our findings reveal novel mechanisms driving evolutionary innovation in plant reproduction and suggest new approaches for genetic engineering in crop species.

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.

During photosynthetic development, GOLDEN2-LIKE (GLK) factors drive the expression of photosynthesis-associated nuclear (PhAN) genes. Simultaneous loss of GLK1 and GLK2 in Arabidopsis leads to pale-green plants with small, incompletely developed chloroplasts, revealing involvement of additional components. Following mutagenesis of a glk1 -1 knock-down, glk2 -1 knock-out (KO) mutant we identified suppressor of glks ( sgl ) 2 , sgl2 . The sgl2 glk1 glk2 triple mutant showed increases in chlorophyll, PhAN gene transcripts and thylakoid stacking in mesophyll cell chloroplasts, and enhanced cellular content of chloroplasts in mesophyll and bundle-sheath cells. The suppressor mutation delayed flowering but greening phenotypes preceded flowering. sgl2 was caused by loss of function of AUXIN RESPONSE FACTOR2 , ARF2 . arf2 sgl2 boosted chloroplast content. It dramatically suppressed the developmental and chloroplast delay of the phytochrome-deficient long hypocotyl 1 ( hy1 ) mutant. We observed in glk1 -1 glk2 -1 low but detectable expression of GLK1 , which was elevated in arf2 sgl2 glk1 -1 glk2 -1, and saw reduced suppression by arf2 sgl2 of a glk1 -2 glk2 -2 mutant carrying KO alleles, revealing partial epistasis. Overexpression of Growth Regulating Factor 5 ( GRF5 ), upregulated in arf2 sgl2 , rescued chlorophyll levels in glk1-1 glk2- 1, consistently with ARF2 and GRF5 as a transcription module. In developing cereal leaves presumed orthologs of ARF2 and GRF5 are expressed at early stages of plastid proliferation and assembly, preceding GLK expression during greening stage. ARF2 is therefore a negative switch modulating GRF5 early in chloroplast development in photosynthetic cells, upstream of the GLKs. Our results reveal a hierarchy of transcription factor action and feed-forward regulation in chloroplast biogenesis during leaf development.

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.

Plants rely on specialised sensing systems, including transcriptional regulators, to maintain iron (Fe) homeostasis. Among these, Hemerythrin RING Zinc finger (HRZ) proteins have emerged as key regulators of Fe homeostasis. In this study, six wheat HRZ homoeologs TaHRZ1 and TaHRZ2 were identified from rice HRZ sequences and mapped to chromosomes 1 and 3. These encode proteins with conserved N-terminal Hemerythrin (HHE) domains and C-terminal CHY-RING and Zn-ribbon motifs. Phylogenetic analysis grouped these genes into distinct clades, while expression profiling revealed strong root-specific and Fe-responsive expression patterns, indicating roles in nutrient sensing. Functional conservation was demonstrated by complementation of the Arabidopsis bts-1 mutant, where both wheat genes restored normal Fe regulation. Full-length TaHRZ1 and TaHRZ2 interacted with members of wheat bHLH IVc transcription factors, while truncated versions lacking the RING domain did not, emphasising their conserved role in protein interactions. CRISPR-Cas9 editing of the conserved HHE3 domain in all the TaHRZ1 homoeologs, coupled with GRF4-GIF1 chimeric protein, achieved ∼9% regeneration efficiency in wheat cultivar C306. Grain ICP-MS analysis indicated enhanced iron loading in the edited lines, particularly in the scutellum, suggesting improved iron partitioning compared to the wild type. Additionally, qRT-PCR revealed upregulation of FIT and IRO3 , and downregulation of IDEF1 in edited lines, supporting a central role for TaHRZ1 in Fe homeostasis signalling. These findings position TaHRZ1 as a valuable target for biofortification strategies to enhance Fe content in wheat grains.