Abstract Transparent model organisms are invaluable for live imaging, yet generating them remains challenging. Here, we present a robust strategy to produce translucent Xenopus laevis , enabling non-invasive, deep-tissue imaging in intact organisms. Using CRISPR/Cas9 technology, we generated quadruple knockouts of slc2a7 , hps6 , and the two tyrosinase homeologs. Instead of in vitro -transcribed single guide RNAs, we employed chemically modified, commercially synthesized two-part guide RNAs, which enabled efficient multiplex genome editing. We produced a high proportion of translucent frogs directly in F0 founder tadpoles, eliminating the need for multi-generational breeding. We validated in vivo live imaging using two transgenic reporter lines with GFP expression in the eye, brain, and heart. Loss of both eumelanin and iridescent pigments in tyr ; hps6 knockouts markedly improved optical clarity and fluorescence visibility. Overall, this multiplexed genome-editing strategy enables the rapid generation of translucent transgenic X. laevis suitable for live imaging, while also providing a simple and efficient protocol for simultaneous multi-gene targeting in X. laevis .

Abstract Colorectal cancer (CRC) is the third most spread cancer globally, commonly arising from activation of the Wnt/β-catenin signaling pathway and microsatellite instability (MSI). Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides and serve as crucial regulators in CRC progression. Depending on their subcellular localization, lncRNAs can be nuclear (e.g., MALAT1), cytoplasmic (e.g., H19), or exosomal, and are genomically classified as sense, antisense, divergent, intergenic, and intronic. They modify gene expression through cis- and trans-regulation functions, regulate mRNA stability through RNA-binding proteins, and function as competitive endogenous RNAs (ceRNAs). LncRNAs control the function of proteins using scaffolding or decoy mechanisms. While exosomal RPPH1 and CAF-derived H19 enhance metastasis by activating β-catenin signaling, oncogenic lncRNAs like HOTAIR and CCAT1-L promote the growth of colorectal cancer. New RNA-based therapies, including RNA interference (RNAi), antisense oligonucleotides (ASOs), and CRISPR technologies (CRISPRi, CRISPR-Cas13, and CRISPR/Cas9), show promising methods for downregulating oncogenic miRNAs (miR-21), restoring tumor-suppressive miRNAs (miR-145), and suppressing carcinogenic lncRNAs like EPIC1, CCAT1, and CCAT2.

Abstract The efficiency of CRISPR interference (CRISPRi) depends on functional dCas9 activity, yet practical and reproducible validation of dCas9-expressing cell lines remains limited. Here, we describe a simple and reproducible assay to assess dCas9 functionality using a single sgRNA targeting the ubiquitously expressed surface protein CD81. We evaluated this approach in multiple hematological and solid tumor cell lines expressing the dCas9-KRAB-MeCP2 repressor complex. In all tested models, CD81 targeting resulted in a consistent reduction of surface protein levels, quantified by flow cytometry. This assay provides a rapid and quantitative functional readout of dCas9 activity without the need for reporter constructs or transcriptional assays. The CD81-targeting sgRNA and validated cell lines are made available to support reproducibility and technical standardization in CRISPRi experiments. This strategy can be readily implemented in any laboratory using CRISPRi-based approaches.

The Solanaceae family includes some of the most economically and agronomically im-portant crops, such as tomato, potato, pepper and eggplant. Recently, CRISPR/Cas-based genome editing has emerged as a powerful tool for functional genomics and crop improvement, enabling precise and efficient genetic modifications. This review provides an overview of CRISPR/Cas-mediated genome editing technologies and their applications in the major cultivated Solanaceae crops. The use of systems for targeted gene knockout and knock-in approaches is described, together with advances in precision editing strategies such as base editing and prime editing, which allow precise nucleotide substitutions and small sequence changes. The expanding CRISPR toolbox is further explored through alternative Cas proteins, such as Cas12a and Cas13 with distinct targeting features and potential applications. Emerging delivery strategies, including ribonucleoprotein-mediated editing in protoplasts, virus-induced gene editing (VIGE), and de novo induction of meristems, represent promising approaches to generate transgene-free edited plants. In addition, the current status of field trials involving genome-edited Solanaceae crops in Europe is outlined, considering the regulatory landscape and legislative requirements for their release in the environment. Despite regulatory constraints, some ge-nomeedited crops have reached the market, highlighting their potential to contribute to sustainable agriculture and crop improvement.

Abstract Phages have evolved various anti-CRISPR(Acr) proteins evade hosts’ immunity by direct CRISPR interference. However, whether other counter-CRISPR mechanisms exist remains unexplored. Here, we report a phage-encoded two-protein system, Healer, which neutralizes CRISPR immunity via a post-cleavage DNA repair mechanism. This phage replication-associated system comprises two proteins: Gp63 (a DUF669 domain-containing protein), and Gp64 (a AAA domain). Mechanistic investigations elucidate that Gp63 acts as a rapid-response effector of CRISPR-induced DNA break, following the binding of ssDNA allows the Gp64 to mediate homologous recombination, repairing CRISPR-induced phage DNA break and enabling phage survival. Notably, co-expression Healer system with CRISPR-Cas9/Cas12 in E. coli, P. aeruginosa, and A. baumannii, demonstrated higher phage genome-editing efficiency. Conclusively, our findings represent a vital anti-CRISPR complementary strategy, providing a promising tool for genome manipulation.

Abstract Background. Bladder cancer (BC) can be characterized clinically as either non-muscle-invasive (NMIBC) or muscle-invasive (MIBC). While NMIBC generally has a favorable prognosis, MIBC is characterized by high morbidity and mortality. Understanding the molecular determinants of tumor invasion is critical, yet research is hampered by the limitations of current experimental models. Standard assays like the Boyden chamber lack physiological complexity, while porcine bladder models suffer from tissue contamination and genetic variability. There is an urgent need for reliable models that mimic the intact tissue architecture. Methods. We established a unique Ex vivo Tissue Model (EXTIM) to evaluate the invasive capacity of BC cells within a largely intact tissue context, using freshly prepared bladders from mice. The invasiveness of human BC cells (RT4, T24, UMUC3) and the immortal urothelial cell strain (Y235T) was comparably evaluated using EXTIM, the Boyden chamber and porcine models. Gene knockdown or ectopic expression of GJB3 or ORP3 indicated suitability of EXTIM to investigate the impact of specific factors on tumor cell invasion. To identify novel genetic regulators of cell invasion, we combined EXTIM with a genome-wide CRISPR-Cas9 knockout screen. Additionally, we utilized the EXTIM to perform a pharmacological screen of a small molecule library comprising 90 substances to identify compounds capable of suppressing BC cell dissemination. Results. Importantly, by combining EXTIM with genome-wide CRISPR-Cas9 screening, we identified several candidate genes involved in BC progression. Notably, discoidin domain receptor tyrosine kinase 1 (DDR1) was identified as a functional inhibitor of tumor cell invasion. Furthermore, the small molecule screen revealed that PD-156707, a selective antagonist of the endothelin receptor A (ETA), significantly suppresses cancer cell invasion within the EXTIM environment. Conclusions. EXTIM serves as a robust and physiologically relevant tool for assessing tumor cell invasion and migration under ex vivo conditions. EXTIM can be used to identify factors involved in the progression of invasive BC by high-troughput genetic screenings in an ex post organ culture system, by culturing cells after transmigration through the bladder tissue. Moreover, the impact of specific genetic factors in the process of tumor cell dissemination can be assessed by placing bladders from genetically modified mice into the EXTIM.

Abstract The critical need for highly sensitive, rapid, low-cost and equipment-free pathogens diagnostics is acutely felt in resource-limited settings, such as in low-income region and at home, where existing methods force a compromise among these key metrics. We break this trade-off with a nucleic acid chip that leverages a novel “dam-break drainage” readout model and a CRISPR-Cas13a-mediated interfacial wettability switching sensing mechanism. This design unlocks both single-plex and duplex visual detection, delivering attomolar sensitivity (10 and 100 aM), rapid results (2 and 5 min) and low cost (0.20$ and 0.30$ per test), respectively. In a 40 clinical-samples validation for SARS-CoV-2, Influenza A virus and Influenza B virus, our platform demonstrated robust clinical concordance and successfully detected samples with ambiguous RT-PCR results (Ct = 35). This work provides a powerful and accessible solution for next-generation point-of-care molecular testing.

Abstract Tumor-associated macrophages (TAMs) are central drivers of resistance to conventional and immune-based therapies. Recent single-cell studies have uncovered an unexpected heterogeneity of macrophage states in human cancers, moving beyond the simplistic “M1–M2” paradigm. However, progress in understanding human TAM biology has been hampered by the lack of physiologically relevant in vivo models. Here, we leverage next-generation humanized mouse models implanted with cell line xenografts (CDX) to dissect the transcriptional and epigenetic programs that shape monocyte-to-macrophage differentiation within the tumor microenvironment. We show that these immuno-CDX models faithfully reproduce the phenotypic and molecular hallmarks of TAMs observed in patient tumors. By integrating single-cell transcriptomic and chromatin accessibility profiling with high-dimensional co-expression and enhancer-driven regulatory network analyses, we reconstruct a comprehensive framework of human TAM states. Furthermore, CRISPR-mediated gene editing of hematopoietic stem cells prior to humanization identifies MAFB as a key regulator of TAM plasticity. Our findings establish humanized models as a powerful platform to study TAM biology in vivo, uncover fundamental regulators of macrophage plasticity, and highlight new therapeutic targets for next-generation cancer immunotherapies.

Abstract Alternative splicing is a fundamental mechanism underlying protein diversity. The microtubule-associated protein tau (MAPT) undergoes age-associated alternative splicing of exon 10 to generate 3R and 4R isoforms, and disruption of the 4R:3R ratio is a central feature of tauopathies. However, the molecular mechanisms regulating tau exon 10 splicing remain incompletely understood. Here, we identify the RNA-binding protein CELF2 as a key promoter of tau exon 10 inclusion. Loss of CELF2 in the mouse brain reduces exon 10 inclusion, resulting in a decreased 4R:3R ratio. We show that an intrinsically disordered region (IDR) within the CELF2 hinge domain drives protein condensation and is essential for its splicing activity. This IDR can be functionally substituted by those of FUS or TAF15. CRISPR-based imaging reveals colocalization of CELF2 condensates with tau RNA. Proteomic analyses identify NOVA2 and SFPQ as CELF2 interactors, which co-condense with CELF2 to cooperatively regulate tau exon 10 splicing. A conserved negatively charged residue (D388) within the IDR is critical for condensate formation, protein interactions, and splicing function. Finally, CELF2 condensation capacity correlates with 4R tau expression in vivo and influences locomotor and cognitive performance. These findings uncover a condensate-based mechanism for tau splicing regulation with implications for tau-related neurodegeneration.

Abstract Background Computational prediction of CRISPR-Cas9 off-target activity is essential for safe guide-RNA design, yet models trained on large proxy datasets often fail to generalize to experimentally validated sites. Methods We present a modular two-stage deep learning framework that separates sequence representation learning from off-target classification. In Stage 1, guide RNA sequences are encoded using frozen, pretrained DNABERT embeddings learned from large genomic corpora. In Stage 2, these embeddings are integrated with mismatch-level and pairwise sequence features within a hybrid CNN-Transformer classifier trained exclusively on a high-throughput proxy dataset. Results On the external TrueOT benchmark, a curated collection of low-throughput, experimentally confirmed off-target sites, the full model achieved a mean ROC-AUC of 0 . 70   ±   0 . 03 and a PR-AUC of 0 . 30   ±   0 . 03 , markedly surpassing the proxy-only baseline (ROC-AUC = 0.64, PR-AUC = 0.22). Ablation studies confirmed that the performance gain arises from the pretrained sequence representations rather than architectural complexity. Conclusions Decoupling representation learning from downstream classification and leveraging frozen transformer-based embeddings substantially improves generalization to biologically relevant off-target predictions. The proposed framework provides a reproducible baseline for the assessment of CRISPR-Cas9 risk and underscores the importance of transfer learning in the integration of proxy test data and experimental results in the real-world.

Abstract Base editing, an extension of CRISPR-Cas9 gene editing, presents a promising strategy for correcting genetic mutations underlying inherited bone marrow failure syndromes. This approach enables ex vivo editing of haematopoietic stem cells, which can be reintroduced into patients without the risk of immune rejection. We have identified pathogenic single nucleotide mutations associated with bone marrow failure syndromes that have a nearby protospacer adjacent motif site and were amenable to adenine base editing, and designed guideRNAs to target. We performed a pooled CRISPR screen to simultaneously target these mutations in haematopoietic cell lines. This pooled strategy addresses the limitation of focusing solely on more prevalent mutations, thereby enabling the inclusion of rarer variants. Our comprehensive analysis revealed correction at 48 loci, with editing efficiencies of up to 26.22% in Jurkat cells and 19.75% in K562 cells. These findings highlight the potential of adenine base editing to correct a broad range of pathogenic variants and accelerate the development of targeted therapies for bone marrow failure syndromes.

Abstract Sex chromosome evolution is traditionally viewed as a unidirectional process of recombination suppression followed by progressive Y degeneration, yet many vertebrates deviate from this canonical trajectory. Frogs represent a striking exception: they retain homomorphic sex chromosomes, exhibit extreme heterochiasmy, and undergo X-Y recombination in sex-reversed females, providing a powerful system to explore alternative evolutionary pathways. Here, we investigate a Swiss Alpine population of the European common frog ( Rana temporaria ) with homomorphic XY chromosomes and three coexisting male genotypes, using whole genome sequencing of pooled samples carrying distinct Y haplotypes, whole genome sequencing of doubled haploid YY individuals, and transcriptomic (RNA-seq) data. We uncover extreme heterogeneity in X–Y divergence: two fully differentiated Y haplotypes span nearly 90% of the sex chromosome (~618–625 Mb), forming the largest non-recombining region (large NRR) described in a vertebrate to date, whereas a semi-differentiated Y harbors only a NRR of 4.64 Mb (small NRR), and XX males display no detectable X-Y divergence. Despite extensive recombination suppression, Y degeneration is minimal even within the large NRR: we detect elevated transposable element insertions but no gene loss, Y-linked gene copy decay, faster-X effects, or enrichment of sex-biased genes. All Y haplotypes share a small NRR encompassing the candidate master sex-determining gene Dmrt1 , defining a common sex-determining locus. Our data support a dynamic model in which extreme heterochiasmy and recurrent X–Y recombination via sex reversal repeatedly reset X-Y divergence, generating and potentially maintaining multiple Y haplotypes with distinct evolutionary histories. The long-term coexistence of these haplotypes across the species range is compatible with a dynamic equilibrium between drift-driven loss and mutation- and recombination-driven renewal of Y chromosome variation. Together, our findings reveal a non-canonical, reversible pathway of sex chromosome evolution shaped by sex-specific recombination patterns and sex reversal, challenging the universality of classical models and highlighting the value of non-model vertebrates for understanding sex chromosome diversity.

Abstract Viruses systematically exploit numerous host immune regulatory factors to establish infections, yet endogenous suppression mechanisms remain inadequately characterized. This review examines RNA interference (RNAi) and CRISPR interference (CRISPRi) screening methodologies for systematically identifying host factors that negatively regulate immune signaling pathways, addressing fundamental questions regarding the molecular mechanisms of immune suppression through ubiquitin regulation, phosphorylation control, and transcriptional repression, Secondly, the discovery of non-canonical regulatory proteins through unbiased functional genomics. This systematic review employed PRISMA-guided literature analysis of peer-reviewed publications from PubMed, Embase, Web of Science, and Cochrane databases (inception-2024), utilizing structured Boolean search strategies with immune-specific MeSH terms, followed by quantitative meta-analysis of screening efficiency data and qualitative synthesis of mechanistic findings through thematic coding and comparative framework analysis. Rigorous comparative analysis demonstrates CRISPRi's better performance with enhanced knockdown efficiency (85% ± 12%) versus RNAi's (60% ± 25%), reduced off-target effects (0.3-0.8% versus 15-30%), and improved temporal stability via direct transcriptional interference. Comprehensive screening reveals diverse immunosuppressants including ubiquitin-editing enzymes (A20, CYLD), transcriptional repressors (BCL6), and metabolic mediators (CH25H), with robust cross-platform validation. Clinical evidence demonstrates that immune suppressor dysregulation correlates with altered viral susceptibility patterns, validating therapeutic potential. This systematic mapping of regulatory networks through advanced functional genomics enables development of innovative host-directed interventions for infectious diseases and cancer immunotherapy applications.

Abstract Background Chinese hamster ovary (CHO) cells are pivotal in biopharmaceutical production, yet balancing high recombinant protein yield with cell survival remains challenging. While previous studies have targeted single apoptotic regulators, the synergistic effects of multi-gene ablation on protein stability are unknown. Results This study presents a multi-knockout strategy for CHO cells. Herewithin, CHO-K1 knockout lines were engineered via CRISPR-Cas9 targeting of key triple (TKO, BAK/BAX/CASP3), double (DKO, BAK/BAX), and single (SKO, CASP3) apoptotic nodes. Subsequently, comprehensive analyses of apoptosis, cell viability, doubling time, cell cycle and mitochondrial membrane potential were conducted for isolated clones. The triple knockout cell lines exhibited the highest overall levels of cell viability, a prolonged doubling time, and enhanced resistance to apoptosis. These characteristics directly translated to improved expression of recombinant blue fluorescent protein, with triple knockouts outperforming WT and single/double knockout lines. Conclusions These findings establish a robust foundation for engineering apoptosis-resistant CHO cell lines with enhanced protein production capacity, offering a promising approach for improved efficiency and reduced costs in biopharmaceutical manufacturing.

Abstract Enhancing methionine and protein content in Saccharomyces cerevisiae is essential for its use as single-cell protein. Here, we applied ethionine resistance–mediated adaptive laboratory evolution (ALE) to generate strains with improved resistance to this toxic methionine analog. Stepwise adaptation enabled growth at ethionine concentrations of up to 0.50 mM and yielded strains with progressively higher intracellular methionine levels and improved protein production efficiency. Whole-genome sequencing identified nonsynonymous SNPs in 32 genes, of which nine candidates were functionally validated. CRISPR/Cas9-based editing demonstrated that mutations in MDE1 and JJJ1 directly elevated free methionine levels, whereas most other mutations increased overall protein accumulation. Functional annotations linked these genes to RNA processing, protein degradation, methionine salvage, and amino acid uptake, highlighting RNA processing as a major target for global protein enhancement. These findings reveal that ethionine resistance–mediated ALE induces multifactorial adaptations. They also provide new insights into protein biosynthesis regulation and lay a foundation for future engineering of high-performance yeast strains.

Abstract Background BAP1 is a tumor-suppressive deubiquitinase essential for DNA repair, and missense mutations in BAP1 are common in clear cell renal cell carcinoma (ccRCC). We previously showed that precise correction of the inactivating Glu31Lys mutation in KMRC-20 ccRCC cells using CRISPR/Cas9 base editing restored BAP1 activity, reinstated anchorage-dependent growth, and re-sensitized cells to anoikis. Here, we asked the converse question: whether disrupting Glu31 is sufficient to induce anchorage-independent growth and anoikis resistance in normal kidney epithelial cells. Methods Using the same adenine base-editing strategy, we introduced an inactivating Glu31Gly mutation into HK-2 normal kidney epithelial cells, generating two independent isogenic BAP1-mutant clones. As an additional control, we created a BAP1-knockout HK-2 clone via CRISPR/Cas9. Parental, mutant, and knockout cells were assessed for BAP1 enzymatic activity, DNA repair capacity, viability, proliferation, cell cycle status, anchorage-independent growth, and anoikis resistance. Migration and invasion of HK-2 mutants and knockouts were compared with KMRC-20 revertant clones in which endogenous Glu31Lys had been corrected. Results The Glu31Gly HK-2 mutants exhibited complete loss of BAP1 deubiquitinase activity and impaired UV-induced DNA damage repair—phenotypes comparable to BAP1-knockout cells—confirming successful functional inactivation. Despite this, both mutant and knockout HK-2 cells maintained parental-like morphology, viability, and proliferation. Surprisingly, Glu31Gly did not confer anchorage-independent growth or anoikis resistance: upon detachment, both mutant and knockout cells showed increased apoptosis. In contrast, in KMRC-20 cells, restoration of BAP1 activity enhanced both migration and invasion. Conversely, BAP1 inactivation or loss in HK-2 cells increased invasion but paradoxically reduced migration. These opposite outcomes indicate that BAP1 regulates motility through distinct mechanisms in normal versus malignant renal cells, likely reflecting differences in lineage state, cytoskeletal organization, and downstream signaling. Conclusions Although BAP1 restoration suppresses anchorage-independent growth and anoikis resistance in KMRC-20 ccRCC cells, BAP1 inactivation alone is insufficient to induce these oncogenic traits in normal HK-2 epithelial cells, implying that additional oncogenic alterations are required for anchorage-independent survival during kidney tumorigenesis. The divergent effects of BAP1 gain versus loss on migration and invasion further underscore the context-dependent nature of BAP1 function. These base-editing studies demonstrate that BAP1 differentially regulates adhesion, anoikis, and motility in normal and malignant renal cells and highlight the utility of precise base editing for dissecting clinically relevant mutations.

Abstract Microbial homeostasis is crucial for host health and ecosystem function, yet the molecular and ecological mechanisms underlying community assembly and stability remain elusive. Here, we uncover a conserved yeast-oomycete metabolic mutualism that promotes their coexistence in the leaf microbiome. Using a continental-scale microbiome survey, we identified a mutualistic interaction between two eukaryotic hub microbes: the yeast Dioszegia hungarica and the obligate oomycete Albugo laibachii. We show that Dioszegia facilitates Albugo colonization by supplying thiamine via a dedicated membrane permease, alleviating Albugo’s auxotrophy. Genomic and transcriptomic analyses reveal that natural selection has acted on thiamine production in D. hungarica, shaping this mutualistic interaction. In planta assays further demonstrate that cross-feeding enhances Albugo colonization and promotes Dioszegia persistence. Our study illustrates how the evolution of nutrient cross-feeding mediates microbial coexistence and microbiome stability. Targeting microbial nutrient flows offers new strategies for engineering microbiomes and enhancing plant resilience in natural and agricultural systems.

Neospora caninum, the causative agent of abortion in cattle, has a major economic impact worldwide. This review aims to provide an overview of key advances of the last 5-8 years in understanding host-pathogen interactions, molecular mechanisms, and emerging control strategies. Epidemiological studies have revealed the influence of environmental, genetic, and ecological factors on parasite transmission dynamics, and emphasized the importance of integrated "One Health" strategies. Characteristics of different Neospora strains have been elucidated through animal models and molecular tools such as clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9)-based gene editing, high-throughput sequencing and advanced proteomics, aiming to shed light on stage-specific gene regulation and virulence factors, contributing to the development of interventions against neosporosis. Insights into immune modulation, immune evasion and parasite persistence contributed to the efforts towards vaccine development. In terms of therapeutics, repurposed drugs but also more targeted inhibitors have shown promising efficacy in reducing parasite burden and mitigating vertical transmission in laboratory models. Here, more recent innovations in nanoparticle-based drug delivery systems and immunomodulatory strategies are prone to enhance therapeutic outcomes. However, a significant challenge remains the integration of molecular and immunological insights into practical applications.

Abstract Genetic inactivation of SKP2 has been shown to effectively prevent cancer initiation and block tumorigenesis. However, direct in vivo evidence for SKP2 on cancer initiation and prostatic microenvironment is still lacking and a SKP2 humanized mouse model is critical for developing prostate cancer immunoprevention approaches through targeting SKP2. We therefore have established a prostate-specific human SKP2 knock-in mouse model driven by an endogenous mouse probasin promoter. Overexpression of hSKP2 induces PIN and low-grade carcinoma. RNA-sequencing analysis revealed significant gene expression alterations in EMT, extracellular matrix, and interferon signaling. Single cell deconvolution showed an increase of fibroblast population and a decrease of CD8+ T cell and B cell populations. Consistently with these results from the SKP2 humanized mouse, SKP2 protein is overexpressed in human prostatic hyperplasia, PIN and prostate adenocarcinoma compared to normal prostate tissues. Overexpression of SKP2 markedly increased cell migration and invasion and induced the gene expression of EMT and interferon pathways. In addition, paired prostate organoids were derived from SKP2 humanized and wild-type mice for drug screening and validated by known SKP2 inhibitors, Flavokawain A and C1. Both of which selectively decreased viability and altered the morphologies of organoids of hSKP2 knock-in rather than wild-type mice. Our studies provide a well-characterized prostate-specific hSKP2 knock-in mouse model and offer new mechanistic insights for understanding the oncogenic role of SKP2 in shaping the prostatic microenvironment during early carcinogenesis.

As an emerging threat to global food security, wheat blast necessitates the development of a rapid and field-deployable detection system to facilitate early diagnosis, enable effective management, and prevent its further spread to new regions. In this study, we aimed to validate and improve an Recombinase Polymerase Amplification coupled with PCRD lateral flow detection (RPA-PCRD strip assay) kit for the rapid and specific identification of Magnaporthe oryzae pathotype Triticum (MoT) in field samples. The assay demonstrated exceptional sensitivity, detecting as low as 10 pg/µL of target DNA, and exhibited no cross-reactivity with M. oryzae Oryzae (MoO) isolates and other major fungal phytopathogens under the genera of Fusarium, Bipolaris, Colletotrichum and Botrydiplodia. The method successfully detected MoT in wheat leaves as early as 4 days post-infection (DPI) (asymptomatic plants), as well as in infected spikes, seeds, and alternate hosts. Furthermore, by combining a simplified polyethylene glycol-NaOH method for extracting DNA from plant samples, the entire RPA-PCRD strip assay enabled the detection of MoT within 30 min with no specialized equipment and high technical skills at ambient temperature (37-39 °C). When applied to field samples, it successfully detected MoT in naturally infected diseased wheat plants from seven different fields in wheat blast hotspot district, Meherpur in Bangladesh. This method offers a practical, low-cost, and portable point-of-care diagnostic tool suitable for on-site surveillance, integrated management, seed health testing, and quarantine screening of wheat blast in resource-limited settings. Furthermore, the RPA-PCRD platform serves as a modular diagnostic template that can be readily adapted to detect a wide array of phytopathogens by integrating target-specific genomic primers.

Abstract Myotonic dystrophy type 1 (DM1) is caused by toxic CTG repeat expansions in the 3′UTR of the DMPK gene, leading to pathogenic RNA gain-of-function effects and widespread splicing abnormalities. RNA-targeting strategies such as antisense oligonucleotides (ASOs) and CRISPR-Cas13 hold strong therapeutic promise, but require reproducible design frameworks that balance specificity with potency. Here, we present a transparent computational pipeline for candidate identification and evaluation at the DMPK locus. The pipeline integrates off-target searches, RNA structure predictions, and composite scoring metrics to generate 50 ASO and 50 Cas13 candidates. ASOs achieved absolute specificity with zero off-targets, clustering tightly around moderate composite scores (mean 57.71), while Cas13 guides consistently carried a single off-target that mapped uniquely to the DMPK gene, with no additional genes affected yet delivered superior thermodynamic properties and higher corrected scores (62.12 vs. 57.71). By anchoring design to the pathogenic 3′UTR region and releasing complete candidate listings, this framework ensures methodological rigor, reproducibility, and translational relevance. Overall, it advances readiness for DM1 therapy by combining ASO safety with Cas13 potency, and establishes a reproducible foundation for precision RNA therapeutics across both monogenic and complex diseases.

Abstract Background Fulfilling the promise of human genetics in elucidating disease requires identifying causal variants and genes underlying genetic association signals. Molecular quantitative trait locus (molQTL) analyses, e.g. expression QTL (eQTL) and splicing QTL (sQTL), link genetic variants to intermediate molecular phenotypes, but pinpointing causal variants and their regulatory effects remains challenging. Here, we integrate sQTL analysis with deep-learning-based splicing effect annotation to identify causal genetic variants and elucidate their functional mechanisms affecting human phenotypes. Results Using a single-cell GWAS method (scHi-HOST) on 96 lymphoblastoid cell lines (LCLs) with and without influenza A virus (IAV) infection, we discovered ~ 43,000 sQTLs associated with 217 genes after IAV infection. Integrating sQTLs with AI splice prediction, we uncovered 76 likely causal variants that affect cis-acting molecular splicing components (5’ donor, 3’ acceptor), supported by further computational analysis. Among these, we experimentally validated a causal sQTL signal affecting poly (ADP-ribose) polymerase 2 (PARP2). The causal variant, rs2297616, alters the 5’ splice donor site in the second intron of PARP2 , resulting in two protein isoforms differing by 13 amino acids. The derived A allele was associated with the longer protein isoform and increased IAV levels in LCLs. CRISPR editing validated the causal effect of this variant on both protein length and IAV infection. Lastly, these 76 putative causal sQTLs were further linked to over a hundred GWAS traits, including many variants associated with autoimmune diseases. Conclusions Our work provides a catalog of causal sQTL with direct splicing impacts, providing causal mechanistic insights from genotype to disease susceptibility.

Spinal cord regeneration requires a transformative strategy capable of rewriting inhibitory genetic programs while orchestrating real-time electrical communication with regenerating neural tissues. Recent advancements in precision CRISPR genome editing effectively silence or activate crucial molecular gatekeepers such as PTEN, SOCS3, and various epigenetic repressors, thereby reactivating dormant intrinsic regenerative pathways and enabling robust axonal growth. Concurrently, cutting-edge bioelectronic technologies utilizing piezoelectric, triboelectric, and magnetoelectric scaffolds have emerged, adeptly harnessing the body's inherent biomechanical energy. These innovative materials convert subtle physiological micromotions into finely tuned electrical stimuli, precisely guiding neuronal regeneration without external power sources, addressing limitations associated with traditional implants such as infection risks and mechanical incompatibility.Integrating these genetic modifications with bioelectric innovations creates a potent synergy. Genome-level reprogramming amplifies neuronal responsiveness to bioelectrical signals, markedly enhancing axonal regeneration. Simultaneously, autonomous electrical stimulation sustains and stabilizes cellular, metabolic, and synaptic improvements induced by genomic interventions, forming a closed-loop, self-sustaining therapeutic platform. This advanced system significantly transcends conventional transient recovery approaches, moving toward durable, personalized outcomes. Such convergence of advanced genetic engineering and intelligent biomaterial design represents a groundbreaking shift in regenerative neurology.Despite promising preclinical outcomes, significant translational challenges remain. Critical hurdles include ensuring precise delivery of CRISPR tools, mitigating off-target genomic effects, enhancing biocompatibility and scaffold stability, and navigating rigorous regulatory pathways. Addressing these challenges necessitates integrating next-generation gene-editing technologies, comprehensive genomic surveillance, advanced biomaterial sciences, and meticulous preclinical evaluations. Future directions in spinal cord injury research encompass multiplex genome editing, AI-driven scaffold optimization via digital twins, and tailored immune-evasive biomaterials. Collectively, this innovative approach has the potential to redefine regenerative medicine's boundaries, offering unprecedented hope for sustained, personalized recovery and dramatically improving quality of life for individuals affected by spinal cord injuries.

Abstract Background Heterozygous variants in CTNND2 , encoding the brain-specific protein δ-catenin, are associated with a broad spectrum of neurodevelopmental disorders, including dyslexia, attention deficit hyperactivity disorder, intellectual disability, and autism. Despite its clinical significance, the full phenotypic spectrum of CTNND2 -associated disorders and the neurodevelopmental role of δ-catenin, a key component of the cadherin-catenin cell adhesion complex, remain poorly defined. Methods Through international collaboration, we assembled the phenotypic and molecular information for 57 individuals, 42 previously unpublished, carrying heterozygous CTNND2 variants. All individuals were evaluated by local clinicians, and the variants were identified through exome or genome sequencing, clinical microarray, or karyotyping. To investigate the effects of δ-catenin loss on early neurogenesis, we performed neural differentiation and transcriptomic profiling in three patient-derived neural stem cell lines and three CRISPR-Cas9-generated CTNND2 knockout lines. In one patient-derived line, we further analyzed cerebral organoid development and performed pathway modulation to assess phenotypic rescue. Results The 41 CTNND2 variants included 12 previously reported loss-of-function- and one missense variant, and 28 novel variants comprising 10 missense and 18 predicted loss-of-function changes. Eight of the novel variants occurred de novo , and 12 were inherited from a parent with a neurodevelopmental phenotype. The most common clinical features were developmental delay (90%), intellectual disability (74%), and behavioral abnormalities (79%). Functional studies revealed impaired early neurogenesis in one patient-derived line, characterized by aberrant neural rosette formation. Transcriptome analysis showed dysregulated WNT signaling, and partial rescue of these defects was achieved by modulating the WNT pathway, highlighting δ-catenin's role in early neural development. Conclusions This study defines the clinical symptoms of CTNND2 -related neurodevelopmental disorders, outlining a recognizable yet variable phenotype that overlaps with other forms of intellectual disability and autism. Our findings provide preliminary evidence of genotype–phenotype correlations and highlight δ-catenin's critical role in modulating WNT signaling during early neural development. These insights advance our understanding of CTNND2 -associated disorders and support the importance of mechanistic studies to inform personalized diagnostics and therapies.

Background: Aging brains are shaped by a persistent dialogue between declining neurogenesis and rising neuroinflammation. Neural stem cells progressively lose regenerative capacity, while microglia and astrocytes shift toward maladaptive states that erode synaptic plasticity and cognition. This convergence defines inflammaging, a slow yet relentless process that undermines resilience. However, the field remains hampered by critical gaps: incomplete mapping of microglial heterogeneity, poorly understood epigenetic scars from inflammasome signaling, lack of longitudinal data, unclear niche-specific immune mechanisms, and uncertain cross-species relevance. This review addresses these pressing barriers, aiming to transform fragmented insights into actionable strategies. Summary: I chart how neurogenesis and neuroinflammation operate in continuous dialogue, identify five major knowledge gaps, and evaluate strategies to reprogram this interaction. Approaches include longitudinal imaging, niche-focused immunomodulation, glial subtype reprogramming, brain-penetrant inflammasome inhibitors, and CRISPR-based epigenetic editing. Each strategy is mapped against translational potential, short-term feasibility, and long-term vision, with emphasis on how mechanistic precision can guide clinical innovation. Conclusion: Here I highlight that neurogenic potential is not entirely lost with age but may be preserved or restored by tuning immune and epigenetic environments. This review proposes a roadmap for reshaping the aging brain’s fate, offering mechanistically grounded strategies to delay cognitive decline. Beyond neurology, the work underscores a broader principle: by integrating cellular plasticity with immune modulation, science edges closer to re-engineering resilience across the lifespan.