Abstract The U6 promoter plays a pivotal role in the CRISPR/Cas9 system by driving the transcription of single guide RNA (sgRNA), which directs Cas9 to achieve precise genome editing. Endogenous U6 promoters typically exhibit superior transcriptional activation efficiency compared to exogenous counterparts, thereby enhancing the efficacy of genome editing. However, the endogenous U6 promoter in kenaf ( Hibiscus cannabinus L.) remains uncharacterized. In this study, we conducted a homologous search of the kenaf genome using the Arabidopsis U6 (AtU6-26) RNA sequence as a reference, identifying two candidate promoters, HcU6-1 and HcU6-14. Promoter fragments were amplified from the genomic DNA of kenaf cultivar 'Fuhong 952' and subsequently cloned into a GUS fusion expression vector. Histochemical staining revealed transcriptional activity for both promoters, with HcU6-14 demonstrating significantly stronger activity. To evaluate editing efficiency, we constructed a CRISPR/Cas9 vector containing HcALS sgRNA, driven by either the kenaf U6-14P promoter or the cotton U6-9P (GbU6-9P) promoter. Kenaf hairy roots were regenerated via Agrobacterium rhizogenes K599-mediated transformation. Sequencing analysis of ALS gene fragments from these hairy roots confirmed successful targeted editing when using the kenaf U6-14P promoter, whereas no base mutations were detected with the cotton U6 promoter. These findings highlight the superior editing efficiency of the kenaf U6 promoter and provide a critical foundation for advancing functional genomics research in kenaf.

Abstract Apoptosis is a highly conserved form of programmed cell death controlled by a core molecular pathway that was first defined in Caenorhabditis elegans and is conserved in mammals. This pathway is composed of egl-1/ BH3-only, ced-9 /Bcl-2, ced-4 /Apaf-1, and ced-3/ Caspase. Despite being discovered more than 20 years ago, tissue-specific apoptosis induction as well as endogenous expression pattern and dynamic subcellular localization of apoptosis proteins remain incompletely defined. Here, we generated a complete set of CRISPR/Cas9-engineered transcriptional and translational reporters for all four apoptosis genes and systematically analyzed their expression and subcellular localization in the C. elegans germline and embryo. We show that somatic apoptosis is driven by precise, lineage-specific activation of egl-1 , whereas ced-9 , ced-4 , and ced-3 are ubiquitously expressed. In contrast, DNA-damage triggers a robust CEP-1/p53-dependent-induction of egl-1 throughout the germline, yet apoptosis occurs only in late pachytene cells. We also identify intron1 of egl-1 as essential for CEP-1–dependent transcriptional activation. Analysis of brc-1 and syp-2 mutants demonstrates that distinct meiotic surveillance pathways converge on egl-1 induction. Analysis of the subcellular localization of the downstream regulators CED-9, CED-4, and CED-3 reveals dynamic, tissue-specific localizations that refine the classical apoptosis model. CED-4 transitions from a perinuclear distribution in the germline and early embryos to a predominantly mitochondrial localization later in embryogenesis, while CED-3 changes its subcellular localization depending on developmental stage and apoptotic status. CED-9 localizes to distinct mitochondrial foci in both embryo and germline. Together, these reporters reveal that C. elegans apoptosis is governed by two mechanistically distinct programs: (1) lineage-specific egl-1 activation in embryos and (2) checkpoint-mediated activation of egl-1 in the germline, where additional, yet unidentified pathways restrict apoptotic execution. These reporters also provide a comprehensive toolbox for dissecting apoptotic and non-apoptotic functions of the conserved apoptotic machinery in vivo .

The integration of 3D bioprinting technology and CRISPR-Cas9 genome editing has become a game-changing method for creating complex organotypic cancer models. This integrated platform overcomes the drawbacks of traditional 2D culture systems by enabling precise genetic modifications within physiologically relevant, biomimetic tumor microenvironments. Researchers can more precisely recreate tumor progression, oncogenic mutations, cellular heterogeneity, and drug resistance mechanisms by utilizing the structural complexity provided by 3D bioprinting and the specificity of CRISPR-Cas9-mediated gene editing. CRISPR-Cas9 enables specific gene modifications, including oncogene knockout (e.g., MYC, KRAS) or immune checkpoint genes (e.g., PD-1, PD-L1), in 3D-bioprinted structures made from tumorigenic or patient-specific cell populations. It has been demonstrated that these modified models maintain important histopathological and molecular characteristics of original tumors, allowing for accurate high-throughput screening of immunotherapeutics and anticancer drugs. Significantly speeding up the modeling of tumorigenesis, studies using prostate cancer organoids showed gene correction efficiencies ranging from 50 to 90 %. Additionally, in 3D cultures, combinatorial CRISPR-Cas9 editing has demonstrated synergistic drug responses in models of lung and breast cancer, underscoring the platform's potential for discovering new therapeutic targets. Biomaterial-based vectors, like hydrogels and nanocarriers, are being improved to reduce off-target effects and increase intracellular uptake to increase the accuracy and safety of CRISPR delivery. However, issues with scalability, reproducibility, and standardization still exist, requiring ongoing interdisciplinary cooperation to improve downstream validation procedures, gene-editing tactics, and bioink formulations. The potential of CRISPR-Cas9-integrated 3D bioprinting as a state-of-the-art technique for drug discovery and cancer modeling is highlighted in this review. It emphasizes how the platform can speed up translational research in oncology, lessen dependency on animal models, and customize treatment plans. The goal of this review is to present a thorough summary of current developments, technical difficulties, and potential paths forward in this quickly developing field.

Abstract Background To investigate the distribution of CRISPR-Cas systems in Escherichia coli ( E. coli ) isolates and evaluate their associations with multilocus sequence types (MLST), antimicrobial resistance genes (ARGs), and plasmid features. Results ST1193 (16/65, 24.6%) and ST95 (11/65, 16.9%) were the predominant lineages. ST1193 showed a higher resistance gene burden than ST95, and bla TEM−1 was detected in 87.5% of ST1193 isolates. CRISPR-Cas systems were detected in 22 isolates (33.8%), including 11 with type I-F (50.0%), 10 with type I-E (45.5%), and one with both types. Spacer sequences were primarily directed against plasmid DNA. Plasmid replicons were frequently detected, and plasmid burden varied across lineages. All the ST1193 isolates lacked detectable CRISPR-Cas systems, whereas 90.9% (10/11) of ST95 isolates harbored type I-F systems. Conclusions CRISPR-Cas carriage was strongly lineage-dependent and showed an inverse association with predicted resistance gene burden in this cohort; this pattern should be interpreted as a lineage-structured correlation rather than mechanistic evidence of CRISPR-mediated restriction of ARG acquisition.

Abstract A growing body of evidence supports that targeting the tumour extracellular matrix (ECM) in solid cancers holds great promises to reactivate T cell migration in immune-excluded patients. By matrisome profiling of triple-negative breast cancer (TNBC) patients, we identified two core ECM proteins enriched in fibrotic immune-excluded tumours and associated with reduced CD8+ T cell stromal infiltration, versican (VCAN) and fibronectin (FN1). Both cancer-associated fibroblasts and aggressive cancer cell lines were found to deposit these two proteins in vitro, conferring resistance to T-cell-mediating cytotoxicity. Characterisation of in vivo murine breast cancer models 4T1 and EMT6 revealed significant differences in tumour ECM deposition and immune cell composition. Accordingly, targeting Fn1 and Vcan in both models induced opposite effects on tumour growth. While it appeared unfavourable in inflamed non fibrotic tumours, deletion of Fn1 in cancer cells was beneficial in immune-excluded tumours by promoting TCF7+ T cells and restoring anti-PD1 response.

Abstract Methods Based on two corolla tube types(L-T,C-T), 256 F1 individuals from a complete diallel cross involving, 5000 F2 individuals and 100 S6 (selfed for six generations) populations. Phenotypic traits, cytology, qRT-PCR, and CRISPR/Cas9 assays were conducted to examine genetic differential expression between the two corolla types. Results The order of dominance in hybrids was labiate > campanulate; For shared typical traits, the dominance order was angled > pubescent > veined. Dorsal and ventral cells of the L-T differed significantly, exhibiting thin walls, fewer stomata, and more trichomes; Whereas the C-T displayed uniformly arranged cells, thicker walls, and more stomata. Gene analysis revealed that 20 genes from five classes (A, B, C, E, and AGL6 subfamily) of the MIKCC-type MADS-box family, 30–32 genes from the TCP family, 8 genes from the YABBY family, and 12 genes from the WOX family transcription factors were involved in regulating typical corolla tube traits. Continuous selfing for six generations did not cause significant changes in the related regulatory genes, with the L-T exhibiting higher stability than the C-T. Both corolla tube types underwent six distinct developmental stages. The L-T was primarily activated by CYC/DICH genes, while the C-T was activated by RAD genes, leading to global expression of MADS-box identity transcription factors. The regulatory patterns, genes involved, and formation mechanisms differed significantly Conclusion The corolla tube of Sinningia speciosa exhibits high genetic stability, and the formation of its typical traits demonstrates significant representativeness and classification characteristics.

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.