Abstract CRISPR Base Editors enable precise single-nucleotide modifications, offering advantages over CRISPR-Cas9 knock-out in programming the desired genetic effect. However, in pooled screens targeting essential genes, discrepancies between expected genetic and phenotypic outcomes are frequent: single guide RNAs (sgRNAs), expected to be disruptive, often appear “phenotypically silent” likely due to inefficient editing rather than absence of functional impact. Here, we investigate if Cas9-based gene-level sgRNA depletion data can help to predict the probability that an sgRNA used in base editing will yield the expected fitness effect in pooled proliferation screening. We analysed proliferative effects (z-scores) from high-throughput CRISPR screens using cytosine Base Editors (BEs) and trained machine learning models to predict fitness effects. Our models integrate sequence features, edited strand, mutation type, predicted editing efficiencies and Cas9 gene essentiality scores. Our models discriminate BE sgRNAs that generate a strong phenotypic effect (depletion) in pooled screening, with AUC-ROC greater than 93% in different cell lines. We provide exhaustive analysis of feature importance highlighting the significant impact of sequence features for predicting BE-associated fitness effects. We found that editor-associated fitness predictions are primarily driven by sgRNA sequence features rather than predicted editing efficiency. Moreover, Cas9-derived gene essentiality partially contributes to predictions.

Abstract Precise chromosomal integration of large DNA fragments remains a significant challenge in metabolic engineering of Escherichia coli, largely due to the unpredictable and often low cleavage efficiency of individual single-guide RNAs (sgRNAs). To overcome this issue, we have developed a modular and versatile "multi-cutters" platform comprising plasmids designed to express up to four distinct sgRNAs simultaneously from a single vector. Building upon the HoSeI (Homologous Sequence Integration) method, this platform synergizes CRISPR-Cas9-mediated cleavage with λ-Red-mediated repair to enable one-step, scarless editing. Our construction strategy uses a modular assembly approach with specific linkers and restriction sites to enable the rapid and flexible generation of double, triple and quadruple cutters. We demonstrate the utility of this platform by successfully 'rewriting' genes—the one-step replacement of approximately 1 kbp chromosomal regions—by replacing the high-expression ompC and ompF loci with fluorescent reporter genes (mCherry and gfp). The resulting dual-reporter strain, RHA00802, functioned as an effective osmotic biosensor, exhibiting concentration-dependent fluorescence responses to sucrose levels (0–10%) in accordance with the regulation of the EnvZ–OmpR two-component system. This multi-cutter platform and gene rewriting strategy provides a powerful, generalized toolkit for complex genomic design and the rapid construction of functional microbial devices. (198 words)

Abstract This study was conducted to determine the effects of knocking out the myostatin gene ( mstnb1 ), which restricts muscle development in rainbow trout ( Oncorhynchus mykiss ), using CRISPR/Cas9 technology on growth performance and feed efficiency. Within the scope of this research, which aims to increase sectoral productivity in aquaculture, two different gRNAs targeting the first exon of the mstnb1 gene were designed. Sanger sequencing analyses confirmed the successful creation of mutations in the F0 generation, involving a 5 bp deletion with gRNA1 and a 1 bp insertion and 6 bp deletion with gRNA2. During a 10-month follow-up period between May 2024 and March 2025, the growth parameters of mutant individuals were compared with a wild-type (WT) control group. At the end of the experiment, mutant fish reached an average weight of 825.0 ± 12.3 g, while the WT group remained at 464.1 ± 9.2 g. These results demonstrate that CRISPR/Cas9-mediated mstnb1 disruption successfully induces the "double muscularity" phenotype and has high biotechnological potential in rainbow trout farming, potentially shortening production time and reducing unit costs.

Abstract Background Streptococcus agalactiae (GBS) causes severe tilapia streptococcosis with heavy aquaculture losses; existing vaccines have administration or efficacy limitations. This study used CRISPR-Cas9 to construct recombinant E. coli DH5α-ORF4-GFP (targeting GBS ScpB gene ORF4 fragment), optimized tilapia immersion immunization doses/frequencies, and evaluated the vaccine’s protective efficacy, biosafety and regulatory effects via multi-dimensional assays. Results The optimal regimen was single immersion at 1.5×10⁴ CFU/mL, with a maximum RPS of 73.12% and stable 65.26% in validation. Immunized tilapia showed elevated immune indices (161.40% higher platelets) and enhanced globulin synthesis, normal liver/kidney function, and improved oxidative stress resistance with no tissue damage. The vaccine did not change intestinal microbial richness but optimized its structure, enriching beneficial taxa like Alphaproteobacteria via the microbiota-immunity axis, enhancing host’s defense capacity via the "microbiota-immunity" axis. Conclusions In conclusion, this study successfully developed a highly effective and safe genetically engineered vaccine against GBS in tilapia. The precise CRISPR-Cas9-mediated construction strategy and confirmed immune protective effect provide a novel technical approach for controlling this disease in aquaculture and offer important references for the development of related genetically engineered vaccines.

Abstract 1. Conservation faces a paradox. As urban expansion and industrial-scale agriculture erode relational values between people and nature, a privileged minority dictates global biodiversity narratives. This shift is reinforced by media and technological interventions that frequently override lived, local experiences. For instance, gene editing tools like CRISPR are incorporated for de-extinction projects to resurrect the dire wolf, signaling a shift toward technology-mediated conservation spectacle. Such efforts limit species as genomic artifacts and not ecosystems of constituent community processes and self-sustaining populations. This raises urgent questions about ecological coherence of power and priorities: whose desires drive restoration/resurrection biology, and what ongoing extinctions are sidelined in the process? 2. Drawing on ecological theory, field insights from South Asia, and critical engagement with de-extinction discourse, this article examines the ecological and ethical implications of engineering genomes to produce lookalikes of the extinct. Using the dire wolf as a case study, I contrast individual-level pseudo-mimicry with population-level processes that sustain species through trophic interactions, microbiomes, and landscape contexts. I argue that conservation anchored in functional ecology must prioritise living systems over nostalgic reconstructions of the past. 3. I evaluated fictional ecosystem depictions, e.g., Jurassic Park, reflecting de-extinction conservation politics. Like the Mirror of Erised in Harry Potter, such resurrections reveal collective longing minus an ecological backdrop. In contrast, emerging artificial intelligence (AI) technologies offer powerful, non-invasive alternatives: immersive visualisation, holography, and digital reconstruction can democratise public engagement with extinct species at a fraction of the ecological and financial costs. AI can enhance storytelling, education, and historical understanding without diverting scarce resources from urgent conservation crises. The challenge is not technological capacity, but ethical direction. 4. Conservationists must resist the seductive appeal of ecologically perilous Species Ghosts. Vultures—not dire wolves—embody the appropriate species for resurrection. Their catastrophic, human-driven global declines illustrate how ecosystem services, rural livelihoods, and public health intertwine. Investing in vulture recovery foregrounds ecological function, social justice, and coexistence, rather than spectacle. We must aim to secure species’ viable populations, habitats, and people–nature relationships. In the Anthropocene, the priority is not to resurrect the irrecoverable past, but to prevent the imminent Sixth mass extinction unfolding before us.

Abstract Triple-negative breast cancer (TNBC) remains a therapeutic challenge due to its aggressive nature and limited treatment options. Through integrated genomic and proteomic profiling, we identified the RNA-binding protein LSM4 as a key driver of TNBC progression. LSM4 was significantly upregulated in TNBC tissues and showed progressively increasing expression from normal breast epithelial cells to highly aggressive cancer models. Functional genomics using CRISPR screening nominated LSM4 as a functionally essential spliceosomal gene among nine critical candidates. Genetic suppression of LSM4 in TNBC cell lines (MDA-MB-231 and CA1a) markedly inhibited malignant phenotypes including proliferation, migration, invasion, and colony formation. RNA-sequencing analysis demonstrated that LSM4 knockdown alters 1,593 alternative splicing events, predominantly through exon skipping, and disrupts genes involved in key cancer pathways including RNA degradation. We established an LSM4-associated gene signature that correlated with an immunosuppressive microenvironment characterized by altered expression of immune markers and checkpoint molecules. Clinical validation using the FUSCC cohort revealed that this signature strongly predicted poor overall survival (p = 0.00067), recurrence-free survival (p = 0.05), and distant metastasis-free survival (p = 0.0092). Our findings establish LSM4 as an important splicing regulator in TNBC, linking spliceosomal dysfunction to tumor progression and immune evasion, and nominate LSM4 as a promising prognostic biomarker and therapeutic target.

Rapid developments in biotechnology, rDNA, and gene editing technologies have transformed both biomedical sciences and environmental biotechnology, especially genome editing, synthetic biology, and reproductive biotechnology, etc., and have revolutionized medicine, agriculture, and environmental sciences, etc. However, such biotechnologies also pose serious bioethical, biosafety, and biosecurity risks and challenges, etc. This review critically discusses and analyzes the current bioethical issues and dilemmas associated with biotechnologies such as CRISPR-Cas9, in vitro fertilization (IVF), cloning, xenotransplantation, PGD, transgenic organisms, etc. The study adopted a literature-based methodology to critically examine the current bioethical debates and discussions on biotechnologies, etc. The study findings revealed serious bioethical issues and dilemmas associated with biotechnologies, such as risk-benefit analysis, justice, human dignity, and eugenics, etc. This review also aims to incorporate Islamic bioethics, focusing on maqasid al-shariah, such as maintaining life, lineage, principle of necessity and dignity, etc. This study concluded that biotechnology has tremendous potential, and its use and development require global governance, ethical literacy, and culturally sensitive approaches, etc.

Abstract Aims/hypothesis. Type 1 diabetes (T1D) is driven by destruction of the pancreatic beta cells by autoreactive T cells, which occurs as a result of failed immune tolerance. The disruptions to the molecular mechanisms that maintain this tolerance are complex, and the balance between conventional CD4⁺ T cells (Tconv) and regulatory T cells (Treg) in T1D remain poorly defined. We hypothesised that by integrating chromatin accessibility, 3D chromatin organisation, transcriptomes and functional perturbation we can reveal the key T cell-centred networks altered in T1D. Methods. We performed parallel ATAC-seq and RNA-seq on sorted stimulated Tconv and Treg from children with T1D and age-matched autoantibody-negative controls. We mapped differentially accessible (DA) regions to putative target genes in human Treg and activated CD4⁺ T cells using Hi-C and asked whether 3D contacts assigned enhancers to distal genes not captured by nearest-gene annotation. To interrogate rare T cell subsets and age effects, we analysed single-cell RNA-seq (scRNA-seq) data from peripheral blood mononuclear cells (PBMCs) of adults with T1D and controls. Finally, we used CRISPR–Cas13d to perform multiplex knockdown of 7 candidate transcription factors (TFs) from a TNFα/NF-κB–linked module (FOS, FOSL1, FOSL2, MAFF, EGR1, EGR2 and NR4A3) in primary human Treg, followed by RNA-seq to functionally test the impacts. Results. Hundreds of differentially accessible regions and expressed genes were detected in paediatric Treg and Tconv cells in T1D, with changes enriched for TNFα signalling via NF-κB, interferon responses and IL-2/STAT signalling. TF footprinting highlighted altered occupancy at AP-1 motifs and other immune regulators, consistent with subtle rewiring of regulatory circuits. Integration with T cell Hi-C revealed that a large fraction of T1D-altered enhancers contacts genes other than the nearest transcription start site and uncovered new altered enhancer-gene pairs. Cas13d-mediated 7-TF knockdown induced transcriptional changes strongly overlapping those seen in paediatric T1D Treg. Conclusions. By combining paediatric case–control T-cell ATAC-seq and RNA-seq with T cell Hi-C, adult single-cell transcriptomes and CRISPR–Cas13d perturbation, we describe a multi-layered, Treg-centred network in T1D. This integrative framework provides a blueprint for moving from non-coding association signals to mechanistic models of T-cell dysregulation in T1D and suggests candidate pathways for therapeutic intervention.

Abstract Human immunodeficiency virus (HIV) persists in long-lived latent reservoirs that are not eliminated by antiretroviral therapy (ART), highlighting the need for curative strategies such as CRISPR/Cas9-mediated gene editing delivered through safe non-viral vectors. This study evaluated the short-term in vivo safety, physiological tolerance, hepatic effects, inflammatory responses, and apoptotic gene activation associated with silver nanoparticles (AgNPs) and AgNPs-CRISPR in BALB/c mice. Thirty mice were assigned to control, AgNPs (3–6 mg/kg), or CRISPR/Cas9–AgNPs groups and received a single intraperitoneal injection, followed by a 14-day monitoring period. Behavioural parameters, body weight, hepatic histopathology, apoptosis (Annexin V–FITC/PI), intracellular IL-6 expression, and hepatic TNF-α and Bax gene expression were assessed. No mortality or behavioural abnormalities were observed. Minimal physiological changes occurred at 3 mg/kg, while dose-dependent reductions in body weight appeared at higher concentrations. Histopathological analysis revealed normal hepatic morphology at 3 mg/kg, early inflammatory alterations at 4 mg/kg, and progressive hepatocellular degeneration and fibrosis at 5–6 mg/kg. Flow cytometry demonstrated a dose-dependent increase in apoptosis, accompanied by elevated IL-6, TNF-α, and Bax expression at higher concentrations. Across comparable doses, AgNPs-CRISPR consistently produced milder pathological effects than AgNPs alone. These findings identify 3–4 mg/kg as a short-term safe dose range for AgNPs-mediated CRISPR delivery and provide foundational safety evidence supporting further investigation of this platform for HIV gene-therapy applications.

Abstract Background: Chimeric antigen receptor (CAR)-T cells are therapeutic breakthroughs against advanced non-Hodgkin lymphomas and myelomas. On the other hand, no CAR-T cell product has been so far clinically approved for therapy of Hodgkin Lymphoma (HL), T cell lymphoma (TCL), or Epstein-Barr-Virus (EBV)-associated lymphoproliferative diseases (EBV-LPDs). CD30 (TNFRSF8) is commonly expressed on HL and on subsets of TCL and EBV-LPDs. CD30CAR-T cells generated via transduction with viral vectors have been tested in clinical trials, showing overall good responses against HL. CAR-T cells produced entirely with locus-specific gene editing methods are emerging as attractive next-generation engineered cell products for ease of multiple seamless cell modifications. Methods: Using CRISPR/Cas9-mediated techniques, we optimized homology-directed repair templates (HDRTs) and performed all-in-one multiplex editing to knock-in (KI) CD30CAR within the TCRα constant ( TRAC ) locus and to simultaneously knock-out (KO) PD-1 or/and β2M. CD30CAR-T cells were tested in CD30 + cell models of HL, TCL, and EBV-LPDs. Results: We compared mouse versus human anti-CD30 scFv designs in HDRTs incorporating TRAC homology arms, FcIg spacer/detection domain, and CD28 / CD3z signaling domains. We obtained an average of 30% TRAC KI CD30CAR-T cells and efficient in vitro cytotoxicity with CD30 + cell targets. CARs incorporating the high-affinity humanized 5F11 scFv showed the highest CAR expression, and the editing templates were further modified to incorporate a truncated CD34 (tCD34) spacer/detection domain. 5F11-CD30CAR-tCD34-T cells showed high CAR-KI rates (approx. 50-80% 12-14 days after editing) and potency in vitro and in vivo . Subsequently, we tested all-in-one CAR KI with additional KOs by co-electroporation of guide RNAs (gRNAs) targeting the genes encoding PD-1 or /and β2M to improve function and allow for improved cell persistence in allogeneic recipients, respectively. Compared with CD30CAR-T cells, CD30CAR-β2M KO -T cells were similarly viable and functional and showed low risk of translocations. PD1 KO enabled CD30CAR-T cells to produce higher levels of cytotoxic features upon exposure to targets. However, simultaneous β2M KO and PD-1 KO compromised the expansion capacity of CD30CAR-T cells and resulted in detectable translocations. Conclusions: Non-virally engineered 5F11-CD30CAR-T cells represent a novel cell therapy modality against CD30 + lymphomas. Multiplex editing remains to be optimized to avoid unwanted genomic alterations and chromosomal translocations.

Abstract Despite recent advances in metagenomics, recovering the strain-level diversity of viral communities remains a challenging and laborious process. Metagenomic sequencing approaches are limited by incomplete genome assembly and low coverage for all but the most abundant viral species in a sample. Here, we extend droplet microfluidic cultivation techniques facilitate targeted enrichment of the archaeon Sulfolobus (now Saccharolobus) islandicus and its associated lytic virus S. islandicus rod-shaped virus (SIRV) to enhance viral metagenomics. By compartmentalizing the natural diversity of a Yellowstone hot spring sample into single virus droplets, we eliminate inter-strain competition and preserve population-level diversity during enrichment of the focal viral species. Sequence analysis of enriched metagenomes confirmed that selective enrichment recovered mostly SIRVs, while drop-compartmentalization outperformed a traditional liquid culture to recover the deep local diversity of a lytic viral population from a natural hot spring. Analysis of SIRV variants revealed selective forces shaping the viral population, including local adaptation in response to host CRISPR-immune selection for viral escape mutations.

Abstract Monoclonal antibodies represent half of the top ten selling drugs. Their proven efficacy, however, generally requires repeated administration for prolonged periods of time. In contrast, cell-based therapies offer a different set of pharmacokinetics and pharmacodynamics than traditional medicines, including the potential to have lifetime durability after a single infusion. Here, we describe a genome-engineered stem cell-based platform for continuous antibody production from a single dose. Using CRISPR/Cas9 homology-directed repair mediated editing, we precisely integrated therapeutic antibody expression cassettes into a safe-harbor locus of hematopoietic stem and progenitor cells (HSPCs). Upon differentiation, these gene-targeted HSPCs generate B cells that secrete monoclonal antibodies. We validated this platform using two clinically approved antibodies, achieving efficient targeted integration of the gene-targeted antibodies (GT-Ab) in human HSPCs that successfully engraft in immunodeficient mice. Direct engineering of human B cells demonstrated robust secretion of therapeutic antibodies. To evaluate in vivo antibody production, we transplanted engineered GT-Ab murine HSPCs into immunocompetent mice, achieving durable serum antibody concentrations within the therapeutic range over several months. Lastly, by fusing the antibody to a destabilization domain, we enabled tunable antibody secretion via small molecule regulation. This modular platform establishes a potentially curative approach for chronic diseases currently reliant on repeated antibody administration, offering durable antibody production from a single treatment.

The ability to precisely edit genetic characteristics with a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) immunity complex is a revolutionary advance in science. Originally discovered in bacteria as part of a natural defense mechanism against viruses, CRISPR/Cas provides a precise, efficient, and relatively simple method for editing genes in microbes, plants, animals, and humans. The process relies on the Cas protein, an enzyme that cleaves and unwinds DNA at targeted locations. This process is guided by RNA sequences complementary to the DNA or RNA sequence of interest, allowing for changes to the genome through innate non-homologous end joining (NHEJ) and homology-directed repair (HDR). The potential applications of CRISPR/Cas are immense and in agriculture, is facilitating crop development with resistance to abiotic, biotic, and agronomic characteristics that improve yield, quality, and food security. Gene editing also facilitates the relatively rapid modification of regulatory and complex pathways that enable studies to advance our understanding of gene function. This review provides an update of the fast-evolving CRISPR/Cas modification of important crops to address emerging global population, environmental and climate challenges.

Rice chalkiness is a key constraint on high-quality rice breeding, and unbalanced sucrose transport and starch metabolism are its primary causes. To clarify the molecular mechanism by which OsSUT2 regulates rice grain chalkiness formation, the rice cultivar TP309 was used as material, and ossut2 homozygous mutants were generated via CRISPR/Cas9. Systematic studies were performed using gene overexpression complementation, phenotypic identification, cytological observation, transcriptome sequencing and haplotype analysis. Results showed that loss of OsSUT2 function significantly increased grain chalkiness, deteriorated agronomic traits, induced carbon assimilate accumulation in leaves, blocked sugar transport and starch synthesis in grains, and destroyed starch fine structure; normal phenotype was fully restored by OsSUT2 overexpression. OsSUT2 was expressed in both source and sink organs, with the most obvious inhibition detected in panicles. Mutation of OsSUT2 disordered sucrose and starch metabolic pathways. Three main haplotypes of OsSUT2 were identified in natural populations, with significant indica–japonica differentiation. OsSUT2 is confirmed as a key regulator of rice chalkiness, providing gene resources and theoretical support for rice quality improvement.

Abstract Dihydroflavonol 4-reductase ( DFR ) occupies a critical branch point in flavonoid metabolism, channeling dihydroflavonol substrates toward anthocyanin biosynthesis in competition with flavonol synthase. While DFR's role in floral pigmentation is well established, the broader physiological and transcriptional consequences of its disruption remain poorly characterized, particularly in commercially important ornamental species. Here, we report the generation and comprehensive phenotyping of five independent CRISPR/Cas9-mediated DFR knockout alleles in the commercial Petunia × hybrida cultivar 'Carmine Velour'. The edited lines showed an allele-associated spectrum of loss of floral pigmentation that was broadly consistent with mutation severity, confirming DFR-A as the dominant isoform governing corolla anthocyanin accumulation. Beyond pigmentation, dfr mutants exhibited unexpected reductions in floral dimensions (20–40%), leaf biomass (30–50%), and plastidial pigment content, with chlorophyll and carotenoid levels declining 35–60% in petals despite unchanged leaf anthocyanins. Stem anatomy remained unaffected, revealing organ-specific pleiotropic effects. Transcriptional profiling uncovered feedback reprogramming within the flavonoid pathway: chalcone synthase A ( CHSA ) and chalcone isomerase A ( CHIA ) were downregulated while the competing branch enzyme flavonol synthase ( FLS ) was upregulated almost 2-fold, consistent with the possibility of altered flux partitioning toward flavonol biosynthesis. Strikingly, protochlorophyllide oxidoreductase A ( PORA ), encoding a key chlorophyll biosynthetic enzyme, was severely suppressedby 60–75%, suggesting a possible connection between flavonoid disruption and tetrapyrrole metabolism. Correlation analyses suggested coordinated variation, with floral anthocyanin content positively associated with leaf chlorophyll and carotenoid levels across genotypes. These findings support the view that DFR acts as a functionally important metabolic node whose disruption is associated with effects across pigment classes and organ types, with implications for precision trait engineering in floriculture.

Abstract The transport mechanisms for substrates and nutrients within the heme pathway of the Toxoplasma gondii apicoplast remain poorly understood. This research involved screening potential apicoplast proteins in Toxoplasma gondii through cross-referencing and analyzing protein-protein interaction networks. Within the heme enzyme pathway of the apicoplast, eight enzymes were found to be predominantly conserved in the Sarcocystiade family. Utilizing the CRISPR-Cas9 system alongside a U1 snRNP-mediated gene silencing approach, we developed inducible knockdown strains: ikD-PBGD, iKD-UROS, and iKD-UROD, targeting three key metabolic enzymes crucial for the parasite lytic cycle, as demonstrated through replication experiments. To investigate the transport mechanisms for heme-related nutrients or substrates, we knocked down these three enzymes, using TgGRA12 as an initial marker. Continuous fluorescence signals highlighted the parasitophorous vacuole (PV) membrane surrounding tachyzoites during both early and late replication stages, particularly at 48 h post-rapamycin treatment, indicating a transformation of the PV resembling Toxoplasma gondii cysts. The slowly replicating cyst-like PV appeared to delay the death of membrane apicoplast proteins during the lytic cycle. This study explored the functional roles of the three intermediate metabolic enzymes, offering a novel viewpoint on the gradual demise of Toxoplasma gondii as a potential target for drug development.

Abstract Background Chemoresistance to 5-fluorouracil (5-FU) remains a formidable barrier to the successful treatment of colorectal cancer (CRC). This resistance is largely driven by the metabolic rewiring of the pyrimidine synthesis pathway, specifically failure to suppress key enzymes. We aimed to investigate the role of the ubiquitin-conjugating enzyme E2S (UBE2S) as a master regulator of 5-FU resistance in colorectal cancer. Methods A 5-FU-resistant HCT116 cell model (HCT116R) was established through stepwise dose escalation. Cell viability was assessed via MTT assays following 5-FU treatment. Molecular mechanisms were explored using Western blot, qPCR, and cell proliferation assays. Functional validation was performed through overexpression, siRNA, and CRISPRi (CRISPR interference)-mediated knocldown assays. In vivo efficacy was validated using a zebrafish xenograft model, and pharmacological inhibition was tested using the IKK inhibitor BMS-345541 and NF-κB inhibitor JSH-23. Results HCT116R cells exhibited significantly higher viability than parental HCT116 cells following 24 h of 80 µM 5-FU treatment, as well as 48 h of treatment across a range of 10 to 80 µM. UBE2S was significantly upregulated in HCT116R cells at both the mRNA (9.45-fold) and protein levels. Furthermore, thymidylate synthase (TYMS) and thymidine kinase 1 (TK1), which bridge the de novo and salvage nucleotide synthesis pathways, showed significantly increased mRNA and protein expression in HCT116R cells. siRNA-mediated knockdown of UBE2S in HCT116R cells and UBE2S overexpression in HCT116 cells significantly regulated the mRNA and protein levels of both TYMS and TK1. Additionally, the use of CRISPRi-mediated UBE2S knockdown of HCT116R stable cell lines, or treatment with the inhibitors BMS-345541 and JSH-23, disrupted this metabolic defense and synergistically enhanced 5-FU-induced cytotoxicity. In zebrafish xenografts, UBE2S depletion markedly suppressed tumor growth and restored 5-FU sensitivity. Conclusions Our findings reveal a novel UBE2S-dependent non-canonical NF-κB signaling that governs CRC chemoresistance. By coordinating the TYMS/TK1 "double-lock" defense through NF-κB signaling, UBE2S serves as a potent prognostic biomarker and a high-value therapeutic target for overcoming fluoropyrimidine resistance in clinical settings.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major global health threat responsible for significant morbidity and mortality, accounting for approximately 19,000 deaths annually in the United States. MRSA resistance is primarily mediated by the mecA and mecC genes, which are carried on the staphylococcal cassette chromosome mec (SCCmec) integrated at the OrfX locus of Staphylococcus aureus, resulting in reduced susceptibility to β-lactam antibiotics. Rapid and accurate diagnostic methods are therefore essential to improve clinical outcomes and limit disease transmission. This mini-review evaluates current MRSA diagnostic approaches, including polymerase chain reaction (PCR) and its variants, isothermal amplification techniques (LAMP and RPA), CRISPR-based diagnostics, and electrochemical biosensors. These methods are compared in terms of diagnostic accuracy, clinical utility, cost-effectiveness, and practical limitations. Overall, isothermal amplification demonstrated a more favorable balance in cost-effectiveness and practical limitations compared to other methods. However, when considering clinical utility and diagnostic accuracy, the results were context dependent. No single method was universally optimal, and the choice of diagnostic approach depends on the clinical context and resource availability.

The increasing prevalence of the plasmid-mediated tigecycline resistance gene tet(X4) has severely limited clinical treatment options. Isothermal amplification techniques, which do not require thermal cycling equipment, are designed for rapid clinical detection. Therefore, this study aims to establish and comparatively evaluate two rapid and visual detection technologies for tet(X4): Recombinase polymerase amplification (RPA)-CRISPR/Cas12a and Loop-mediated isothermal amplification (LAMP)-CRISPR/Cas12a nucleic acid detection system. The two methods were compared based on amplification temperature, amplification time, sensitivity, and specificity. The results demonstrated that the LAMP amplification required a reaction time of 50 min, with an optimal temperature of 65°C and a detection limit of 5 copies. The amplification products were further detected using CRISPR/Cas12a, and optimal results were obtained after a 30 min reaction at 35°C. In comparison, RPA exhibited a shorter reaction duration (30 min) and a lower reaction temperature (37°C), while maintaining the same detection limit of 5 copies. Detection using the CRISPR/Cas12a system at 37°C for 30 min produced a clearly visible fluorescence signal. Both methods demonstrated excellent specificity when tested against various bacterial strains. Furthermore, a one-step detection method combining RPA and CRISPR was developed, a mere 30-min reaction at 37°C is sufficient. In summary, the method combining RPA amplification with CRISPR/Cas12a detection offers better compatibility, and the one-step detection method effectively reduces the risk of aerosol contamination. It is well suited for rapid detection.

Abstract The clinical approval of direct KRAS inhibitors represents a milestone in precision oncology. Nonetheless, resistance heterogeneity limits durable clinical responses and impedes the nomination of a ‘‘one-size-fits-all’’ strategy for combination therapies. We circumvent this impasse by uncovering a core vulnerability elicited by KRAS blockade which transcends tissue origin, KRAS mutation, intrinsic KRASi sensitivity, and clinically relevant concurrent mutations. Specifically, we show that KRAS inhibition elicits a functional deficit in DNA damage repair by transcriptionally repressing homologous recombination. This BRCAness phenotype constitutes a conditional synthetic lethality to PARP inhibitors and radiotherapy, which synergize with single KRAS blockade and significantly delay the onset of secondary resistance. Integrative analyses identify the transcription factor FOSL1 as an upstream regulator of homologous recombination whose depletion sensitizes to PARP inhibition and radiotherapy. Collectively, we define a translational trajectory for the combination of KRAS inhibitors with clinically approved DNA-damaging agents as a therapeutic strategy treatment potentially expandable to most, if not all, mutant KRAS cancers.

Abstract Background Limb-Girdle Muscular Dystrophy Type 2E (LGMD2E) is one of the most prevalent phenotypes within the limb-girdle muscular dystrophies (LGMDs). This myopathy is caused by pathogenic mutations in the SGCB gene, which encodes β-type sarcoglycan. LGMD2E is recognized as the most prevalent sarcoglycanopathy among the Iranian population specially within the Baloch ethnic group. To develop innovative gene therapy strategies based on accessible gene delivery and gene editing techniques, it is essential to have a cell line harboring genomic mutations in the SGCB gene. These cell models are crucial for the preliminary evaluation of the efficacy of gene-editing-based methods. In this study, we utilized the clustered regularly interspaced short palindromic repeats (CRISPR) system and the approach of inducing indel mutations to generate an HEK-293T cell model harboring a frameshift mutation in SGCB gene. Methods Two distinct SGCB exon 2 targeting single guide RNAs (sgRNAs) were cloned into PX458 plasmids containing spCas9, and recombinant plasmids were transduced into HEK293T cells. The mutagenesis efficiency was evaluated using the TIDE program on Sanger sequencing data of transduced cells. Mutated cell clones were obtained through serial dilution techniques. Results Our results demonstrated the substantial efficacy of the CRISPR-Cas9 system in inducing mutations within the SGCB gene. This capability presents significant potential for precise editing of the SGCB gene in muscular cells, thereby establishing a robust cellular model for LGMD2E gene-editing strategies. Conclusions The application of CRISPR-Cas9 technology in this study highlights its potential for targeted gene editing in LGMD2E research. These findings pave the way for further investigations into the development of precision therapy for LGMD2E, ultimately contributing to the development of effective therapeutic strategies.

Abstract Over 40% of human cancers harbor extrachromosomal DNA (ecDNA), which correlates with therapy resistance and poor prognosis. Yet research is limited by a lack of robust de novo ecDNA models. We present a CRISPR-Cas9 based approach for generation of ecDNA in cell lines and mouse liver. In HEK293 cells, CRISPR-induced MYC-bearing ecDNA ([MYCcircle]) promoted cancer hallmarks, including enhanced proliferation, increased migration, and reduced apoptosis. To model ecDNA in mice, we combined Cas9 knock in mice with lipid-nanoparticle delivery of sgRNAs in the liver targeting the Myc–Pvt1 locus. When combined with Trp53 or Pten loss, the resultant [Myccircle] caused hepatocellular carcinomas with amplified levels of [Myccircle] and elevated, heterogenous Myc expression, supporting evidence of ecDNA involvement in tumorigenesis. Our approach offers a fast and versatile tool, where target-sequence flexibility allows rapid generation of animal and cell models with any investigator-specified ecDNA, thereby enabling study of the direct role of ecDNA in cancer progression.

Host-directed therapy (HDT) has emerged as a promising strategy for managing infectious diseases by targeting host immune pathways rather than pathogens directly. However, the rational design of HDT approaches remains challenging due to the complexity of host–pathogen interactions, limitations in gene-editing delivery, and constraints in computational modeling. This review presents a conceptual framework integrating three advanced technological domains: quantum-inspired computation, CRISPR-Cas9 gene editing, and nanotechnology-based delivery systems. We propose a functional pipeline in which computational approaches, including quantum molecular simulation and machine learning, support the design of guide RNAs and optimization of nanoparticle formulations. These are coupled with CRISPR-based modulation of key host immune targets, particularly the NLRP3 inflammasome and IL-1 signaling pathways, followed by targeted delivery using lipid nanoparticles and nanoemulsion systems.The review synthesizes current knowledge across CRISPR biology, nanomedicine, immunology, and emerging quantum computational approaches, while critically addressing existing limitations, technological gaps, and translational challenges. We highlight key contradictions in the literature, identify priority research directions, and outline a structured roadmap for future investigation.Although the complete integration of these technologies remains aspirational, each component is individually supported by strong experimental evidence. This framework therefore represents a realistic and forward-looking strategy for advancing host-directed therapies in infectious diseases within a One Health context.

Abstract Background Salt stress is a key abiotic stress factor limiting rice growth and development. Previous studies have shown that OsWRKY53 , OsARF18 , and OsRR22 not only serve as important negative regulators of salt tolerance in rice but are also crucial for growth and development. However, the relative strengths of salt tolerance among these three genes and their combined effects within the same rice variety have not yet been reported. Results In this study, we employed CRISPR/Cas9-mediated genome editing to simultaneously disrupt OsWRKY53 , OsARF18 , and OsRR22 in the rice cultivar Shuanghui 459. Salt tolerance increases sequentially in single-gene, double-gene, and triple-gene mutants. Under 1.0% NaCl stress, the triple mutant exhibited approximately 80% survival versus complete lethality in the wild type (WT). Physiologically, ROS accumulation progressively declined, while key antioxidant enzyme activities (CAT, SOD, POD) significantly increased. More importantly, molecular analyses revealed that the OsWRKY53 WRKY domain binds W-box elements in the OsRR22 and OsARF18 promoters, repressing their transcription. Loss function of OsWRKY53 depresses both genes. Reciprocally, OsARF18 and OsRR22 knockout downregulated OsWRKY53 . Furthermore, OsWRKY53 and OsRR22 directly interact at the protein level. Conclusions Taken together, our results reveal that OsWRKY53 , OsARF18 , and OsRR22 constitute a reciprocal negative-feedback loop, wherein these three transcriptional regulators mutually antagonize each other. Disruption of this antagonistic network likely represents a core mechanism responsible for the robust salt tolerance observed in the triple mutant. Importantly, the polygenic aggregation of OsWRKY53 - OsARF18 - OsRR22 not only significantly enhances rice salt tolerance but also does not affect normal plant growth and development. These findings provide new strategies for polygenic aggregation-based genetic improvement of salt-tolerant rice varieties.

Abstract Single-nucleotide polymorphisms (SNPs) are crucial for dissecting genotype-phenotype associations, clinical diagnosis, and agricultural traits prediction. While CRISPR-based diagnostic tools enable rapid nucleic acid detection, their SNP discrimination ability is constrained by protospacer adjacent motif (PAM) requirements and inherent mismatch tolerance. Here, we found that PCR-generated single-stranded DNA (ssDNA) triggers trans-cleavage activity of Cas12a in a PAM‑free manner, and together with split crRNA enables effective SNP discrimination. Based on these, we developed SNIPER (Single Nucleotide Identification and Precision Evaluation Reporter), a PAM-free strategy optimized specifically for SNP discrimination. SNIPER successfully detects functional SNPs across 106 genetic loci spanning viruses, prokaryotes, and eukaryotes. Compared to previously reported method, SNIPER achieves robust discrimination even at low target concentrations, while SHERLOCK and SUREST failed to efficiently distinguish the given SNPs. SNIPER is compatible with both DNA and RNA targets, and can be formulated as lyophilized pellets of Cas12a and crRNA, thus can served as a versatile and reliable tool for disease control and prevention, clinical diagnosis, and crop germplasm trait prediction.