Search Results (1,833)

Toxoplasma gondii is a widespread intracellular parasite posing serious risks to immunocompromised individuals and pregnant women, and causing significant economic losses to the livestock industry. Current clinical drugs mainly target acute-stage tachyzoites but are ineffective against chronic cysts, necessitating new interventions. The parasite’s sexual stage in the feline intestine remains poorly understood but is critical for transmission. Here, based on our unpublished weighted gene co-expression network analysis of single-cell transcriptomic data of cat intestine after infection with T. gondii, we selected five previously uncharacterized genes, including three CCCH-type zinc finger proteins (TGME49_257130, TGME49_500318, TGME49_313740) and two subtilisin-like proteases (TGME49_248760/sub7, TGME49_235950/sub8). Using the CRISPR-Cas9 system, individual knockout strains were generated from the type II Pru strain. And, we performed phenotypic assessments at this stage as an essential validation to confirm neutrality before feline infection experiments. Phenotypic analyses revealed that none of the knockout strains showed significant differences in vitro growth, replication, egress, in vivo virulence, or cyst formation from the wild-type strain, corroborating the transcriptional prediction. Publicly available ToxoDB transcriptomic data confirmed that four of these genes were highly expressed at the oocyst or feline enteroepithelial stages, but barely expressed in tachyzoites and bradyzoites, consistent with our phenotypic observations. Collectively, these findings indicate that these genes are dispensable for asexual development, while their expression pattern suggests potential involvement in sexual reproduction, a hypothesis that warrants direct experimental validation. This study provides validated knockout strains and candidate genes, establishing an experimental foundation for future functional studies on Toxoplasma sexual development and transmission-blocking strategies. Full article

N⁶-methyladenosine (m⁶A) is a vital epitranscriptomic modification that regulates plant development and stress responses. In rice (Oryza sativa L.), the identity of the catalytic subunit (MTA) of the core m⁶A writer complex has remained controversial because of inconsistent annotations based primarily on sequence homology. Here, phylogenetic analysis identified LOC_Os02g45110 as the putative catalytic subunit OsMTA, whereas LOC_Os01g16180 and LOC_Os03g05420 were classified as the structural subunits OsMTB1 and OsMTB2, respectively. All three proteins showed nuclear enrichment. Yeast two-hybrid, luciferase complementation imaging, and bimolecular fluorescence complementation assays demonstrated that OsMTA interacts independently with OsMTB1 and OsMTB2. Moreover, transient co-expression of OsMTA with either OsMTB1 or OsMTB2 increased global m⁶A levels in a heterologous system. No homozygous plants carrying frameshift mutations in OsMTA were obtained, suggesting that complete loss of OsMTA function is lethal. Knockdown of OsMTA or CRISPR/Cas9-mediated knockout of OsMTB1 and OsMTB2 significantly delayed heading date and reduced 1000-grain weight. Conversely, overexpressing OsMTA accelerated heading, whereas overexpressing OsMTB1 or OsMTB2 generally accelerated heading but significantly reduced 1000-grain weight. Together, these findings define the core composition of the rice m⁶A writer complex and reveal its effects on heading date and grain weight. Full article

Huanglongbing (HLB), caused by Candidatus Liberibacter spp., remains the most destructive disease affecting citrus worldwide. To support host-directed genome-editing strategies aimed at reducing susceptibility, we optimized key regeneration steps in Citrus sinensis and validated a dual-gRNA CRISPR/Cas9 approach targeting the susceptibility gene CsDMR6. Juvenile explants of ‘Valencia’ and hybrid genotypes (CsH1–CsH3) were successfully established in vitro, and shoot elongation was markedly improved by supplementing Citrus Shoot Multiplication (CiSM) medium with 1 mg L⁻¹ GA3. Callus induction was most efficient in Citrus Callus Induction (CiCM) medium under dark conditions, while a 48 h NAA pulse (100 µM) significantly enhanced rooting, increasing efficiencies to 37.1% in ‘Valencia’ and 52.9% in CsH1. Two guide RNAs targeting conserved regions of CsDMR6 were designed and shown to be identical across all evaluated genotypes. The dual-gRNA cassette was assembled into a CRISPR/Cas9 geminivirus-based vector and transiently delivered into sweet orange leaf tissue via Agrobacterium. GFP fluorescence verified construct expression, and PCR amplification across the target region produced a diagnostic ~447 bp fragment corresponding to the expected ~5.8 kb deletion. Sanger sequencing confirmed precise junction formation between the two cut sites. These results demonstrate efficient large-fragment deletion of CsDMR6 in sweet orange and establish an experimentally validated, genotype-compatible regeneration and editing platform. This study provides a transient validation of the dual-gRNA system and establishes the technical foundation required for future stable, non-transgenic edited lines. Together, these advances support the downstream functional evaluation of CsDMR6 loss-of-function alleles under HLB pressure. Full article

This review systematically summarizes recent advances in CRISPR-Cas12b-based pathogen nucleic acid detection. Starting with an overview of conventional nucleic acid detection methods and core CRISPR-Cas mechanisms, we highlight the unique properties that distinguish Cas12b from other Cas effectors. We elaborate on the working principles of the CRISPR-Cas12b system and present comparative structural and functional analyses with other Cas variants to underscore its distinctive molecular architecture and enzymatic properties. A comprehensive evaluation of current applications demonstrates the efficacy of CRISPR-Cas12b-based diagnostics across diverse pathogens, including viruses, bacteria, and parasites, with a specific focus on its integration with isothermal amplification techniques. We further examine the translational potential of CRISPR-based diagnostics in clinical settings, while critically analyzing persistent technical challenges including off-target effects, signal amplification limitations, and sample preparation requirements. Targeted strategic recommendations are proposed to optimize detection sensitivity, develop multiplexed detection platforms, and implement point-of-care testing configurations. This review aims to systematically correlate the unique characteristics of Cas12b with its broad diagnostic applications, thereby addressing key research gaps in its progression toward clinical validation and field deployment. It also provides a targeted framework to accelerate the translation of this technology from laboratory platforms to practical diagnostic solutions. Full article

Soil salinization affects more than 954 Mha of arable land globally, with approximately 10–20 Mha abandoned annually. Conventional engineering and chemical remediation suffer from high water demand, salt re-accumulation, and secondary pollution risks. While this review draws primarily on the extensive body of research from China—where saline–alkali land covers approximately 99.13 Mha—it also incorporates key international case studies for comparative analysis. The review synthesizes biological and physical technologies for saline–alkali land reclamation, identifies critical challenges, and proposes an integrated remediation framework. A systematic search of Web of Science, Scopus, and CNKI databases (2000–2026) yielded 2847 records, of which 41 studies formed the systematic evidence base for the five technology clusters and 41 were retained as background references following the PRISMA framework. Because the search included the Chinese CNKI database and China contains one of the world’s largest saline–alkali land areas, particular emphasis is placed on Chinese case studies, complemented by representative international examples. Data were extracted on technology type, salt removal efficiency, crop yield, and application stage, and synthesized through quantitative cross-technology comparison. Five dominant technical clusters were summarized: (i) gene-based breeding (CRISPR/Cas, MAS) achieving 20–28% yield gains on sodic soils; (ii) halophyte phytoremediation removing 83–91% of soil salts over three growing seasons; (iii) microbial inoculants improving crop salt tolerance by 15–35% under controlled experimental conditions; (iv) agronomic rotations and straw amendment reducing topsoil salinity by 30–50%; and (v) solar-driven interfacial evaporation achieving 91.4% salt removal in a single proof-of-concept field trial at a material cost of approximately USD 0.004 per straw unit. Integrated bio-physical deployment, however, remains at the experimental scale. Combining rapid physical desalination with long-term biological remediation represents a promising research direction that requires systematic field validation before practical deployment. Key knowledge gaps include the long-term edaphic consequences of solar desalination, field-scale reliability of microbial consortia, and absence of regionally validated integrated protocols. We propose a structured roadmap with explicit timelines and policy recommendations to accelerate translation from research to practice. Full article

The strict requirement for a canonical TTTV protospacer adjacent motif (PAM) restricts target-site selection in clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a (CRISPR/Cas12a)-based diagnostics and limits the flexibility of assay design. Whether suboptimal PAMs can be effectively incorporated into one-pot detection systems for Clostridium perfringens has not been systematically examined. Here, we developed two one-pot recombinase polymerase amplification (RPA)–CRISPR/Cas12a assays targeting the plc and cpe toxin genes of C. perfringens and systematically evaluated 28 crRNAs spanning canonical (TTTV) and suboptimal (VTTV, TCTV) PAMs in both trans-cleavage and one-pot formats. Several suboptimal-PAM crRNAs, including crRNA 4 for plc (PAM: GTTG) and crRNA 7 for cpe (PAM: CTTA), maintained efficient detection in the one-pot system, whereas crRNA performance between the two formats was not predictable, underscoring the need for direct one-pot screening. The optimized assays achieved limits of detection of 50 copies (plc) and 10 copies (cpe), showed no cross-reactivity against four non-target bacterial species. The plc assay was successfully validated in pork, chicken, and beef samples spiked with C. perfringens, with results readable within 40 min using a simple isothermal device and naked-eye readout. The cpe assay was confirmed as a proof-of-concept at the genomic DNA level. These results demonstrate that suboptimal PAMs can serve as a deliberate design strategy to expand target accessibility in one-pot CRISPR/Cas12a diagnostics. Full article

The phenoloxidase gene Laccase2 (Lac2) is an indispensable survival gene in insects, playing key roles in cuticle tanning, exoskeleton development and pigment deposition. However, in lepidopteran insects, the roles of Lac2 in mandibular development and feeding behavior remain largely unexplored. In this study, we demonstrated that Lac2 is essential for mandibular development in early larvae of the model lepidopteran, the silkworm, Bombyx mori. We cloned and characterized BmLac2 and found that it is evolutionarily conserved in lepidopteran insects. Disruption of BmLac2 via CRISPR/Cas9 system induced mandibular defects, which directly impaired larval feeding behavior and culminated in developmental arrest and larval mortality. Moreover, knockout of BmLac2 led to dopamine accumulation during the dormancy stage, but the dopamine level returned to normal after molting, without altering feeding motivation in mutant larvae. Furthermore, RNA sequencing (RNA-seq) and quantitative real-time polymerase chain reaction (qRT-PCR) analyses revealed that loss of BmLac2 affected the expression of genes associated with cuticular formation and immunity, also contributing to larval mortality alongside feeding defects. Altogether, BmLac2-mediated mandibular development and cuticular melanization play key roles in regulating feeding behavior and immunity in early-stage silkworm larvae, making this gene a potential target for early intervention against lepidopteran pests. Full article

Breast cancer is a highly heterogeneous malignancy that remains one of the leading causes of cancer-related mortality among women worldwide. Despite significant advances in breast cancer research and therapy, disease heterogeneity, treatment resistance, and metastatic progression remain major obstacles to effective disease management. Among the molecular regulators involved in breast cancer, microRNAs (miRNAs) have been recognized as critical post-transcriptional regulators of gene expression, functioning as either oncogenes or tumor suppressors. By modulating the expression of target RNAs, miRNAs control key biological processes involved in tumor initiation and progression, including cell proliferation, apoptosis, angiogenesis, epithelial–mesenchymal transition (EMT), invasion, and metastasis. To investigate miRNA function and explore their therapeutic potential, a wide range of approaches have been developed to modulate miRNA expression. These include gain-of-function strategies, like miRNA mimics, miRNA expression vectors, and CRISPR activation (CRISPRa), as well as loss-of-function approaches, including anti-miRNA oligonucleotides (AMOs), miRNA sponges, CRISPR-Cas9-mediated gene knockout, and CRISPR interference (CRISPRi). This review provides a comprehensive overview of the biological roles of miRNAs in breast cancer and discusses current technologies for miRNA modulation, their molecular mechanisms, experimental and therapeutic applications, and associated limitations. In addition, it summarizes recent advances in miRNA delivery systems, including viral vectors, organic nanoparticles, and inorganic nanocarriers, highlighting their potential to improve delivery efficiency, target specificity, and facilitate clinical translation. Finally, the review discusses future perspectives, emphasizing the transition from single-target interventions toward network-level regulation and the integration of miRNA-based strategies into precision oncology to support the development of more effective breast cancer therapies. Full article

The functions of noncoding variants associated with complex traits in livestock remain poorly understood. In this study, we investigated two candidate noncoding variants within the XKR4-CHCHD7 locus identified from our previous analysis. Dual-luciferase reporter assays demonstrated allele-specific regulatory activity of these two regions in bovine muscle satellite cells (BMSCs), 293T cells, and C2C12 cells. Endogenous deletion of the candidate regions using a clustered regularly interspaced short palindromic repeats (CRISPR)-based high-fidelity Cas12Max (hfCas12Max) system revealed that the region containing chr14:22840845 (SNP-0845) exerted broader effects on BMSC function including reduced proliferation and migration, altered cell-cycle progression, and enhanced myogenic differentiation. Expression screening of candidate effector genes further identified PENK and TMEM68 as downstream candidate genes for SNP-0845. Rescue experiments further showed that PENK exerted stronger recovery effects than TMEM68 on the proliferation and migration defects caused by deletion of this region, supporting PENK as a major candidate effector downstream of the SNP-0845. Functional assays showed that PENK knockdown impaired BMSC proliferation and migration while promoting myogenic differentiation, whereas PENK overexpression partially reversed these effects. In vivo Penk knockdown reduced quadriceps femoris weight and altered muscle fiber composition in mice. Collectively, our findings suggest that a noncoding regulatory region modulates BMSC fate and muscle growth-related processes through PENK. Full article

Dwarf cereal cultivars were crucial for the Green Revolution. Dwarfed, lodging-resistant varieties remain essential today, as climate change brings more storms and downpours. In barley, the dwarfing gene HvDEP1 has been widely used in breeding. Although its pleiotropic effects on agronomic traits have been examined, previous studies relied on cultivars developed via random mutagenesis, which carry background mutations that may influence phenotypes. Moreover, its impact on root traits remains underexplored. We used CRISPR/Cas9 to generate precise HvDEP1 mutants and introduce dwarfism into the barley cultivar ‘Maythorpe.’ We assessed the effects on above-ground morphology, yield-related traits, root architecture, biomass via ¹³C labelling, and the root metabolome. HvDEP1 mutations significantly reduced plant height, straw, spike, and awn length, as well as thousand-grain weight. An in-frame mutant showed intermediate height, straw, and awn phenotypes. Belowground, in a root experiment restricted to knockout line #12, specific root length and the length of the finest (0–0.25 mm) roots were reduced, while total root length was lower but not significantly so; (p = 0.062). Root metabolomic profiling detected no genotype-associated differences. These results provide new insights into HvDEP1′s role in both shoot and root systems and demonstrate that precise CRISPR/Cas9-mediated editing can rapidly introduce dwarfism while revealing trade-offs in other agronomic traits. Full article

Lichen-forming fungi establish intimate symbiotic associations with photosynthetic partners and play important roles in diverse ecosystems, but functional genetic studies in these organisms remain limited by the lack of efficient genome-editing tools. In this study, we established an efficient CRISPR-Cas9-mediated gene disruption system in Umbilicaria muhlenbergii. Using this system, we achieved the targeted disruption of six candidate transcription factors with a high replacement efficiency of up to 65.0%. No off-target mutations were detected in any of the three independent mutants examined for each target gene. Preliminary phenotypic characterization of the resulting mutants revealed that disruption of UmSOM1 markedly impaired fungal growth, induced pseudohyphal development, and altered colony morphology and pigmentation. Compared with conventional homologous recombination, the CRISPR-Cas9 system substantially improved gene disruption efficiency, thereby overcoming a major limitation in the genetic manipulation of lichen-forming fungi. This system provides a robust platform for functional genomic studies and will accelerate investigations into the molecular mechanisms underlying fungal–algal symbiosis and morphological transitions in lichen-forming fungi. Full article

The high mortality associated with largemouth bass ranavirus (LMBRaV) disease requires rapid and sensitive diagnosis methods to prevent virus spread and subsequent outbreak. The recombinase polymerase amplification combined with the CRISPR/Cas12a system (RPA-CRISPR/Cas12a) assay and RPA-CRISPR/Cas12a, coupled with lateral flow dipstick (RPA-CRISPR/Cas12a-LFD) assay for LMBRaV, were established in this study for low viral load surveillance. The major capsid protein (mcp) gene is highly conserved and is widely used as the target sequence in LMBRaV detection. First, based on the mcp gene sequence of LMBRaV, three candidate crRNAs were designed. Among them, the crRNA-2, which exhibited the highest cleavage activity, was selected through dual evaluation of fluorescence signal intensity and LFD color development. Then, the key reaction conditions for both assays were optimized as follows: LbCas12a protein concentration of 150 nM, crRNA-2 concentration of 200 nM, and ssDNA reporters’ concentration of 200 nM. Moreover, specificity evaluation showed that both combined assays specifically recognized LMBRaV, with no cross-reactivity detected against other aquatic pathogens, such as CyHV-2, GSIV, ISKNV, WSSV, GCRV II or CrERV. The detection limit was 1 copy/μL of DNA sample for both RPA-CRISPR/Cas12a and RPA-CRISPR/Cas12a-LFD assays. Finally, validation using 24 clinical samples (16 positive, eight negative) showed that both LMBRaV RPA-CRISPR/Cas12a assays achieved 100% detection rate, whereas conventional PCR detected 13 positive samples (detection rate 81.26%). ddPCR served as the reference method for clinical validation. Therefore, the LMBRaV RPA-CRISPR/Cas12a assay and the RPA-CRISPR/Cas12a-LFD assay provide sensitive, specific, and easy-to-operate methods for the rapid detection of LMBRaV. Full article

Predicting CRISPR-Cas9 guide RNA efficiency and off-target activity is a precondition for precise genome editing. Computational models have progressively incorporated chromatin accessibility and epigenetic descriptors into their feature sets, yet synthesising findings from independently published studies—especially when those studies contradict one another—remains an unresolved methodological gap. Large Language Models (LLMs) have been proposed as a route to automate cross-study synthesis, but their utility depends on a constraint that receives less attention than model architecture: how much of the source text actually reaches the model at inference time. Cloud-based models process 48,000-token corpora without hardware limitations, but at the cost of data leaving the local environment and with limited reproducibility across API versions. Local RAG systems avoid the cloud dependency while fragmenting the input, discarding the global context needed to link biological arguments that are distributed across separate papers. We benchmark these strategies using a corpus of four CRISPR-Cas9 efficiency prediction studies and apply the Reduced Interaction Sampling (RIS) engine—a local sparse attention method—to retain the full sequence within the memory envelope of a laboratory server. Preserving that context uncovers three latent inconsistencies. The static epigenetic markers used in DeepCRISPR (CTCF, DNase I) show near-zero Spearman correlations with off-target cleavage ($\rho \le 0.07$), while nucleosome positioning scores from the Block Decomposition Method reach $\rho =0.388$–$0.423$. The sequence-only Apindel model was published in June 2022 without incorporating nucleosome descriptors reported in the concurrent literature. The benchmark review by Konstantakos et al. attributed 10–20% of rank correlation to epigenetics—a figure that reflects the weak feature subset evaluated, not a ceiling on chromatin influence. These discrepancies are invisible when papers are read individually or retrieved as chunks; they become traceable only when the full corpus is processed as a single context window. An independent empirical analysis of 2000 CRISPR-Cas9 off-target cleavage events provides evidence consistent with this pattern: static epigenetic markers yield $\left|\rho \right|\le 0.11$, whereas computed NuPoP Affinity descriptors reach $r=-0.622$ ($p<{10}^{-210}$). On a 30-question cross-study synthesis benchmark (5 independent seeds), baseline accuracy is 53.33%, RAG 60.00%, and RIS (30 seeds, 3% density) 70.00% ($p<0.0001$, t-test vs. RAG, $\sigma =0.00\%$ for all configurations). Full article

The twin-tail phenotype of goldfish represents a striking domestication-associated remodeling of the vertebrate caudal axial system and is classically linked to disruption of Chordin/BMP-mediated dorsal–ventral patterning. Although previous knockdown studies implicated sizzled (szl) in this process, genetic evidence from targeted disruption of endogenous szl loci remains limited. Here, we used CRISPR/Cas9 to mutate conserved coding regions shared by the duplicated goldfish paralogues szlA and szlB in single-tail embryos. Sanger sequencing and ICE analysis showed that szl-sgRNA2 and szl-sgRNA3 efficiently induced indels at both loci, whereas szl-sgRNA1 was ineffective. Across three independent biological replicates, twin-tail-like caudal bifurcation was observed in 44.63–48.19% of szl-sgRNA2-injected larvae, 69.47–79.61% of szl-sgRNA3-injected larvae, and 64.29–76.19% of larvae injected with the sgRNA mixture; szl-sgRNA1-injected larvae remained single-tailed. Calcein staining further revealed separation of distal caudal fin rays and partial splitting of the caudal skeletal complex in szl-edited larvae. qRT-PCR showed selective remodeling of dorsal–ventral patterning genes, including reduced chdA and eve1 expression and increased bmp2 and nog1 expression. These findings provide direct functional evidence that szl regulates median caudal patterning in goldfish and suggest that szl-dependent modulation of the Chordin/BMP network can generate twin-tail-like caudal morphology. Full article

Wolfram syndrome (WS) is a neurodegenerative disorder caused by mutations in the endoplasmic reticulum (ER) transmembrane protein WFS1. Mutations in WFS1 lead to ER stress and dysregulated calcium signaling, resulting in progressive neurological dysfunction. Myelination is a core pathological process in various central nervous system (CNS) diseases. However, the role of WFS1 in oligodendrocyte development and myelination remains unknown. Here, using CRISPR-Cas9-generated wfs1a/wfs1b double-knockout (wfs1^−/−) zebrafish, we demonstrated that wfs1 deficiency significantly delayed the dorsal distribution of oligodendrocyte precursor cells (OPCs) along Mauthner axons. Furthermore, wfs1 mutation was associated with early hypomyelination of axons at 5 dpf, with partial recovery observed by 8 dpf. Mechanistically, combined transcriptomic and pharmacological analysis revealed that wfs1 mutation led to the suppression of the steroid biosynthesis pathway through downregulation of hmgcs1, which encodes the upstream condensation enzyme of the mevalonate pathway and supplies substrate for the downstream rate-limiting enzyme HMGCR, thereby potentially reducing isoprenoid precursor availability. Pharmacological supplementation with geranylgeraniol (GGOH) specifically rescued dorsal distribution defects, supporting involvement of the isoprenylation pathway, although direct regulation of hmgcs1 by wfs1 and complete rescue of myelin structure require further mechanistic validation. Taken together, our study supports a novel wfs1-hmgcs1 axis, which may regulate dorsal distribution and myelination through the isoprenylation pathway; notably, classical ER stress markers were also concurrently upregulated in wfs1^−/− larvae, although whether the isoprenylation deficiency and ER stress act independently or synergistically remains to be determined. These findings provide new insights into WS-associated early hypomyelination and suggest hmgcs1 as a potential therapeutic target for myelin defects caused by WS. Full article