@article {pmid42501539,
year = {2026},
author = {Qin, C and Jiang, S and Xiao, F and Huang, J and Jin, M and Yang, K and Meng, H},
title = {DAZL-targeted inducible kill switches enable efficient ablation of chicken primordial germ cells.},
journal = {Poultry science},
volume = {105},
number = {11},
pages = {107459},
pmid = {42501539},
issn = {1525-3171},
mesh = {Animals ; *Chickens/genetics ; *Germ Cells ; *RNA-Binding Proteins/genetics/metabolism ; *Avian Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Chick Embryo ; *Genes, Transgenic, Suicide ; },
abstract = {Competition from endogenous primordial germ cells (PGCs) in recipient embryos limits the generation of fully donor-derived offspring in avian surrogate-host systems. An inducible and germline-restricted ablation strategy is therefore needed to create sterile recipients without compromising somatic development. Using CRISPR/Cas9-mediated homology-directed repair, we inserted three inducible suicide-gene cassettes (iCaspase9, RapaCasp9, and CD) into the endogenous DAZL locus of chicken PGCs. All knock-in lines showed stable reporter expression and retained typical PGC morphology. Comparative functional analyses identified iCaspase9 activated by AP20187 as the most sensitive and specific ablation system, achieving near-complete killing at nanomolar concentrations without detectable toxicity in control cells. RapaCasp9 induced with rapamycin also ablated engineered PGCs efficiently, but rapamycin caused marked non-specific growth inhibition in control cells. Replacing rapamycin with the synthetic A/C heterodimerizer AP21967 largely eliminated this off-target toxicity while preserving rapid and robust killing. By contrast, yeast-derived CD variants did not substantially improve 5-fluorocytosine sensitivity in chicken PGCs. DAZL-restricted iCaspase9/AP20187 and the optimized RapaCasp9/AP21967 system provide efficient inducible kill switches for chicken PGCs in vitro. These platforms establish a practical genetic toolkit for generating sterile surrogate hosts to support avian genome editing, germline replacement, and Sire Dam Surrogate breeding.},
}

Animals
*Chickens/genetics
*Germ Cells
*RNA-Binding Proteins/genetics/metabolism
*Avian Proteins/genetics/metabolism
CRISPR-Cas Systems
Chick Embryo
*Genes, Transgenic, Suicide

@article {pmid42580267,
year = {2026},
author = {Farooq, M and Han, D and Zhang, L and Wadood, AA and Geng, Q and Chen, J and Liang, K and Xi, Y and Jiao, X and Hou, J and Zhang, Y},
title = {Clustered regularly interspaced short palindromic repeats‑associated protein 9 (CRISPR-Cas9) based genome editing in avian primordial germ cells: Comparative technologies, translational applications, and regulatory challenges.},
journal = {Poultry science},
volume = {105},
number = {11},
pages = {107469},
pmid = {42580267},
issn = {1525-3171},
mesh = {Animals ; *Gene Editing/veterinary/methods ; *CRISPR-Cas Systems ; *Germ Cells ; *Chickens/genetics ; },
abstract = {Primordial germ cells (PGCs) are a unique platform for heritable gene editing in avian species, because they allow easy isolation, culture, and can then be reintroduced into the host. CRISPR/Cas9 technologies have advanced avian genome editing by enabling targeted editing of genes and traits for production, health, reproduction, and welfare. This review critically evaluates genome-editing tools in avian PGCs, including CRISPR/Cas9, transcription activator-like effector nucleases (TALENs), base editing, and prime editing. Germline transmission efficiency, suitability, precision, and heritability are compared. Efficiency, cytotoxicity, and translational feasibility of delivery strategies, including viral vectors, electroporation and the use of ribonucleoproteins are assessed. Applications of these techniques in chickens are for muscle growth via myostatin (MSTN) gene disruption, viral resistance via editing of the sodium/hydrogen exchanger 1 (NHE1) gene, and control of male and female ratios through modification of sex determination genes. Additionally, applications in biopharmaceutical protein production and animal biodiversity conservation are also explored. Despite advances, some limitations remain, including low efficiency of homology-directed repair, off-target effects, mosaicism, and variability in transmission through the germ line. Current evidence demonstrates a significant lack of germline validation and scalability creating barriers to translate this potential into commercial avian production, especially, poultry breeding. Regulatory frameworks and their implications for food and commercialization are also discussed. Future research should prioritize precision editing, scalable delivery systems, and regulatory alignment to enable practical, ethical, and responsible implementation.},
}

Animals
*Gene Editing/veterinary/methods
*CRISPR-Cas Systems
*Germ Cells
*Chickens/genetics

@article {pmid42671510,
year = {2026},
author = {Roman, G and Lauritzen, KH and Smolkova, B and Almaas, R and Sandset, PM and Stavik, B and Sullivan, GJ and Chollet, ME},
title = {From Design to Single-Cell Cloning: A Complete RNP-Based CRISPR-Cas9 Protocol for Precision Gene Correction in Human iPSCs.},
journal = {Stem cell reviews and reports},
volume = {22},
number = {8},
pages = {3790-3816},
pmid = {42671510},
issn = {2629-3277},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Ribonucleoproteins/metabolism/genetics ; *Single-Cell Analysis/methods ; },
abstract = {Precise genome editing of induced pluripotent stem cells (iPSCs) using clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR-Cas9) has opened unprecedented avenues for advancements in regenerative medicine and disease modelling. However, the establishment of isogenic single-cell-derived iPSC populations, particularly upon CRISPR-Cas9 gene editing modifications, is one of the major challenges still associated with these advanced methodologies relying on low-rate editing events and requiring defined clonogenicity. In response, we have developed a systematic, comprehensive and efficient workflow combining generation, genotyping and expansion of high-quality monoclonal iPSC lines following CRISPR-Cas9 genome editing. In particular, the protocol incorporates optimized single-cell cloning procedures for two commercially available dispensing platforms, one based on microfluidic imaging and the other on impedance technology, together with rapid droplet digital PCR (ddPCR)-based screening of non-homologous end joining (NHEJ) and homology-directed repair (HDR) outcomes. By combining gentle single-cell handling with advanced genotyping methodologies, the protocol enables efficient early assessment of editing outcomes before commitment to labour-intensive clonal derivation, thereby accelerating project timelines, minimising cell stress and loss, preserving genetic fidelity and supporting scalability. Coupled with precisely defined culture conditions tailored for post-seeding recovery, these approaches aim to improve iPSC viability and clonal outgrowth, achieving at least 60% in 96-well plate format within 10 days. Importantly, the protocol goes beyond step-by-step experimental instructions by providing comprehensive design strategies, decision-making criteria, and practical advice for avoiding and addressing the common pitfalls and unintended consequences of these advanced methods. Collectively, this integrated end-to-end approach provides a robust and high-throughput framework for the reliable production of monoclonal genome-edited iPSCs, thereby advancing translational research and the development of iPSC-based regenerative therapies and disease models.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Ribonucleoproteins/metabolism/genetics
*Single-Cell Analysis/methods

@article {pmid42700775,
year = {2027},
author = {Manjunatha Reddy, GB and Vignesh, M and Megha, NV and Brindhaa, R and Uzma, J and Shivasharanappa, N},
title = {Rapid point-of-care lateral flow assay for differentiation of capripoxviruses using RPA-CRISPR-Cas12a.},
journal = {Journal of virological methods},
volume = {347},
number = {},
pages = {115465},
doi = {10.1016/j.jviromet.2026.115465},
pmid = {42700775},
issn = {1879-0984},
mesh = {Animals ; *Capripoxvirus/isolation & purification/genetics/classification ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Rapid Diagnostic Tests ; *Poxviridae Infections/diagnosis/veterinary/virology ; *Nucleic Acid Amplification Techniques/methods ; *Point-of-Care Systems ; Cattle ; Goats ; Sheep ; Goat Diseases/diagnosis/virology ; Sheep Diseases/diagnosis/virology ; },
abstract = {The Capripoxviruses (CaPVs) are highly contagious viruses that includes sheeppox virus, goatpox virus, and lumpy skin disease virus infecting sheep, goats, and cattle respectively resulting in significant morbidity and mortality. This study presents a diagnostic tool based on recombinase polymerase amplification (RPA) assisted with CRISPR-Cas12a platform coupled with a lateral flow assay (LFA) that facilitate timely detection and differentiation of Capripox viruses, and support effective surveillance programs. The assay operates through the cleavage of a dual labelled oligonucleotide reporter molecule (Biotin-Fam), generating a detectable signal. The results can be interpreted either using a fluorescence-based detection system or through a lateral flow assay (LFA), where gold nanoparticles conjugated with anti-FAM antibodies enable visual signal detection. Under optimized assay conditions (50 nM LbCas12a and 1 µM reporter probe with a reaction time of 25 min), the CRISPR-Cas12a platform along with RPA (39°C for 20 min) substantially enhanced analytical sensitivity, enabling reliable detection of as few as 10 copies/µL of viral DNA. The specificity of the assay was rigorously evaluated using individual targets, mixed-template samples, and 90 clinical specimens, demonstrating complete species-specific discrimination without any detectable cross-reactivity. Collectively, the developed single-gene RPA-CRISPR-Cas12a lateral flow assay (LFA) represents a rapid, highly sensitive, and specific point-of-care diagnostic platform, providing visual readout by the naked eye and offering significant potential for decentralized veterinary diagnostics and field-based disease surveillance.},
}

Animals
*Capripoxvirus/isolation & purification/genetics/classification
*CRISPR-Cas Systems
Sensitivity and Specificity
Rapid Diagnostic Tests
*Poxviridae Infections/diagnosis/veterinary/virology
*Nucleic Acid Amplification Techniques/methods
*Point-of-Care Systems
Cattle
Goats
Sheep
Goat Diseases/diagnosis/virology
Sheep Diseases/diagnosis/virology

@article {pmid42742174,
year = {2026},
author = {Jerabek, S and Jung, C and Kappy, M and Zhao, Q and Sung, J and Wang, N and Kim, E and Kim, J and Kulmann, MIR and King, MB and McAndrew, MJ and Li, M and Bhatele, S and Isado, M and Jang, HS and Dolezal, M and Prosser, R and Xu, S and Hwang, GH and Pichova, I and Xu, J and Marin, D and Woo, JS and Bae, S and Treff, N and Lapinaite, A and Egli, D},
title = {Highly efficient base editing at PCSK9 and normal human embryo development.},
journal = {Nature},
volume = {658},
number = {8138},
pages = {1289-1300},
pmid = {42742174},
issn = {1476-4687},
mesh = {Humans ; *Gene Editing/methods ; *Proprotein Convertase 9/genetics ; Female ; *Embryonic Development/genetics ; Blastocyst/metabolism/cytology ; CRISPR-Cas Systems/genetics ; DNA Repair/genetics ; DNA Breaks, Double-Stranded ; Alleles ; Chromosome Aberrations ; *Embryo, Mammalian/metabolism/cytology ; Homozygote ; },
abstract = {Cas9-based tools enable programmable DNA lesions for studying repair outcomes, gene function and genome correction. In human embryos, Cas9-induced DNA double-stranded breaks are genotoxic, causing frequent aneuploidy and large deletions[1,2]. Here, we evaluate DNA repair outcomes at nicks and mismatches introduced by base editors at the PCSK9 and HBG loci in human embryos. Delivering ABE8e(V106W) as a protein at fertilization achieved editing at all PCSK9 alleles, supporting development to the blastocyst stage and the derivation of homozygous edited stem cell lines. No insertions or deletions were detected, although rare on-target chromosome breakages and chromosomal abnormalities occurred. Furthermore, editing at bystander and off-target sites was mosaic, and the introduction of the editor as mRNA caused frequent embryo arrest due to guide-independent deaminase activity. Thus, in contrast to Cas9-induced DNA breaks, base-editor-induced lesions are efficiently repaired. However, undesirable consequences for the genome and development can occur, currently precluding the clinical use of base editing in reproduction.},
}

Humans
*Gene Editing/methods
*Proprotein Convertase 9/genetics
Female
*Embryonic Development/genetics
Blastocyst/metabolism/cytology
CRISPR-Cas Systems/genetics
DNA Repair/genetics
DNA Breaks, Double-Stranded
Alleles
Chromosome Aberrations
*Embryo, Mammalian/metabolism/cytology
Homozygote

@article {pmid42744980,
year = {2026},
author = {Jiao, Z and Lin, L and Tang, R and Zhong, CQ and Wu, C and Li, W and Xu, R and Gao, Y and He, P and Wang, C and Zhang, QQ and Liu, S and Yuan, J and Zhang, Q and Li, J},
title = {Histone H2B monoubiquitination drives sexual commitment in malaria parasites.},
journal = {Nature microbiology},
volume = {11},
number = {10},
pages = {2805-2820},
pmid = {42744980},
issn = {2058-5276},
support = {82472314//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82072302//National Natural Science Foundation of China (National Science Foundation of China)/ ; W2411080//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82230077//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Histones/metabolism/genetics ; *Ubiquitination ; Animals ; *Plasmodium falciparum/genetics/metabolism/growth & development ; *Plasmodium yoelii/genetics/metabolism/growth & development/physiology ; Protozoan Proteins/metabolism/genetics ; Humans ; *Malaria/parasitology/transmission ; Ubiquitin-Conjugating Enzymes/genetics/metabolism ; Ubiquitin-Protein Ligases/metabolism/genetics ; Gametogenesis ; Transcription Factor AP-2/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Transmission of malaria parasites to mosquito vectors relies on the successful conversion from asexual blood-stage forms into sexual gametocytes. Protein ubiquitination regulates many cellular processes, including cell differentiation in other eukaryotes, but whether specific ubiquitination machinery mediates Plasmodium sexual conversion is unclear. Here we conduct CRISPR-Cas9 mutagenesis screens to systematically profile Plasmodium yoelii E2 ubiquitin-conjugating enzymes for their impact on parasite development. We demonstrate that an E2 enzyme (Rad6B), and its partner RING-type E3 ligase (Tex1), play determinant roles in gametocytogenesis and transmission to mosquitoes. Deletion of either Rad6B or Tex1 results in severe defects in gametocyte production and oocyst formation. The Rad6B-Tex1 complex catalyses histone H2B monoubiquitination in Plasmodium yoelii and Plasmodium falciparum. H2B monoubiquitination promotes H3K4 tri-methylation occupancy at the promoter region of the gene encoding transcription factor AP2-G, a master regulator of sexual conversion, thereby inducing ap2-g expression and initiating sexual commitment. These findings reveal a key epigenetic mechanism leading to sexual differentiation in both human and rodent malaria parasites, providing potential targets for transmission-blocking interventions.},
}

*Histones/metabolism/genetics
*Ubiquitination
Animals
*Plasmodium falciparum/genetics/metabolism/growth & development
*Plasmodium yoelii/genetics/metabolism/growth & development/physiology
Protozoan Proteins/metabolism/genetics
Humans
*Malaria/parasitology/transmission
Ubiquitin-Conjugating Enzymes/genetics/metabolism
Ubiquitin-Protein Ligases/metabolism/genetics
Gametogenesis
Transcription Factor AP-2/genetics/metabolism
CRISPR-Cas Systems

@article {pmid42754710,
year = {2026},
author = {Maire, A and Ortelli, M and Tkacz, E and Dehio, C and Chassaing, B and Sokol, H and Rolhion, N and Bikard, D},
title = {In vivo CRISPRi screens reveal Escherichia coli functional adaptations in the mouse gut.},
journal = {Nature microbiology},
volume = {11},
number = {10},
pages = {2911-2924},
pmid = {42754710},
issn = {2058-5276},
support = {101044479//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; ANR-10-LABX-62-IBEID//Agence Nationale de la Recherche (French National Research Agency)/ ; 51NF40_180541//Fondation pour la Recherche Médicale (Foundation for Medical Research in France)/ ; 51NF40_180541//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; },
mesh = {Animals ; *Escherichia coli/genetics/physiology ; Mice ; *Adaptation, Physiological/genetics ; Escherichia coli Infections/microbiology ; Prophages/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Fitness ; Escherichia coli Proteins/genetics ; *CRISPR-Cas Systems ; *Gastrointestinal Tract/microbiology ; },
abstract = {Escherichia coli exhibits remarkable genetic diversity that enables it to adapt to the intestinal environment. Here we establish an in vivo CRISPR interference platform that leverages bacterial gene fitness as a high-resolution functional reporter of E. coli adaptations within mice harbouring a defined minimal microbial community (OligoMM12). The screen revealed that diet profoundly shapes the metabolic landscape of E. coli and the essential gene profile identified cross-feeding interactions. Comparison between a laboratory strain (MG1655), a uropathogenic strain (CFT073) and an adherent-invasive E. coli (AIEC LF82) identified distinct genetic requirements for intestinal colonization, highlighting divergent motility, stress response and respiration strategies. In a host inflammatory environment, we found that AIEC LF82 preferably colonized the small intestine with a mobile genetic element, Gally prophage, playing an important role in modulating fitness. These findings provide a high-resolution genetic atlas of E. coli's functional adaptation and demonstrate the utility of functional genomics to probe the gut environment itself.},
}

Animals
*Escherichia coli/genetics/physiology
Mice
*Adaptation, Physiological/genetics
Escherichia coli Infections/microbiology
Prophages/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
Genetic Fitness
Escherichia coli Proteins/genetics
*CRISPR-Cas Systems
*Gastrointestinal Tract/microbiology

@article {pmid42814570,
year = {2026},
author = {Cohen Yaron, K and Socol, Y and Yedidi, N and Bejerano-Sagie, M and Ravins, M and Fine-Leebhoff, S and Birimberg-Schwartz, L and Grunewald, M and Hullahalli, K and de Sousa Vieira, A and Waldor, MK and Bartfeld, S and Ben-Yehuda, S and Rosenshine, I},
title = {Dual roles of host N-glycosylation in enteropathogenic Escherichia coli infection.},
journal = {Cell reports},
volume = {45},
number = {10},
pages = {118043},
pmid = {42814570},
issn = {2211-1247},
mesh = {Humans ; Glycosylation ; *Enteropathogenic Escherichia coli/pathogenicity/physiology/metabolism ; *Escherichia coli Infections/metabolism/microbiology ; *Host-Pathogen Interactions ; Polysaccharides/metabolism ; Escherichia coli Proteins/metabolism ; Bacterial Adhesion ; Host Tropism ; CRISPR-Cas Systems ; },
abstract = {Host factors determine pathogen tropism but knowledge of host genes facilitating enteropathogenic Escherichia coli (EPEC) infection is limited. To identify host determinants required for infection by EPEC, we performed two independent genome-scale CRISPR-Cas9 screens. Both converged on host N-glycosylation as a critical requirement for infection. Genetic or pharmacological inhibition of N-glycan maturation revealed that N-glycosylation promotes infection through two distinct pathways. First, N-linked poly-N-acetyllactosamine glycans serve as receptors for the major EPEC adhesin, the bundle-forming pilus (BFP). Second, perturbation of N-glycosylation compromises host membrane structure, resulting in reduced type III secretion system activity, independently of host cell attachment. Using epithelial monolayers derived from human colon and jejunum organoids, we further show that EPEC attachment inversely correlates with terminal mannose exposure, suggesting glycan composition may be involved in determining intestinal tropism. Together, these findings establish host N-glycan maturation as a key host pathway exploited during enteric infection.},
}

Humans
Glycosylation
*Enteropathogenic Escherichia coli/pathogenicity/physiology/metabolism
*Escherichia coli Infections/metabolism/microbiology
*Host-Pathogen Interactions
Polysaccharides/metabolism
Escherichia coli Proteins/metabolism
Bacterial Adhesion
Host Tropism
CRISPR-Cas Systems

@article {pmid42837260,
year = {2026},
author = {Ma, W and Liu, B and Gao, J and Nie, R and Liu, Z and Wang, W and Wang, Y and Ning, G and Zhao, C and Wang, Y and Yue, X and Ding, D and Ma, X and He, J and Wang, X and Qi, F and Liu, Y and Wang, F and Wang, X and Zhi, D and Wang, D},
title = {Genome-wide CRISPR screening identified hemojuvelin as a non-neuronal receptor for the botulinum neurotoxin serotypes A and C.},
journal = {Cell reports},
volume = {45},
number = {10},
pages = {118106},
doi = {10.1016/j.celrep.2026.118106},
pmid = {42837260},
issn = {2211-1247},
mesh = {Animals ; *Botulinum Toxins/metabolism ; Mice ; *Botulinum Toxins, Type A/metabolism ; *CRISPR-Cas Systems/genetics ; *GPI-Linked Proteins/metabolism/genetics ; Humans ; Mice, Knockout ; Neuromuscular Junction/metabolism ; Protein Binding ; Sarcolemma/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Botulinum neurotoxins (BoNTs) enter cells primarily through neuronal receptors and gangliosides. However, the mechanism that enables their specific accumulation at neuromuscular junctions (NMJs) remains poorly understood. In this study, a genome-wide CRISPR-Cas9 screen identifies hemojuvelin (HJV), a membrane protein abundant in skeletal muscle, as a non-neuronal receptor for BoNT/A and BoNT/C. Knockout and rescue experiments show that HJV mediates toxin binding by a direct interaction between its C-terminal region (residues 155-393) and the toxin WY loop within the receptor-binding domain (HCR). In mice, HJV knockout markedly reduces toxin enrichment on the sarcolemma and significantly attenuates toxin sensitivity such as digital paralysis and lethality. Thus, HJV captures these toxins on the muscle sarcolemma, establishing a critical prerequisite for efficient recognition and internalization by neuronal receptors at NMJs. These findings further refine the entry mechanism of BoNTs in vivo and offer actionable insights for treating BoNT intoxication and for optimizing their clinical applications.},
}

Animals
*Botulinum Toxins/metabolism
Mice
*Botulinum Toxins, Type A/metabolism
*CRISPR-Cas Systems/genetics
*GPI-Linked Proteins/metabolism/genetics
Humans
Mice, Knockout
Neuromuscular Junction/metabolism
Protein Binding
Sarcolemma/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42849292,
year = {2026},
author = {D'Attilia, C and Bonarrigo, M and Tarkowská, D and Buffagni, V and Hayta, S and Smedley, MA and Masci, S and Palombieri, S and Sestili, F},
title = {CRISPR-Cas9 knockout of IBH1 fine-tunes brassinosteroid metabolism and modulates plant architecture and grain traits in durum wheat.},
journal = {Plant physiology and biochemistry : PPB},
volume = {239},
number = {},
pages = {111831},
doi = {10.1016/j.plaphy.2026.111831},
pmid = {42849292},
issn = {1873-2690},
mesh = {*Triticum/metabolism/genetics/anatomy & histology ; *Brassinosteroids/metabolism ; *Plant Proteins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Plant ; Gene Knockout Techniques ; *Transcription Factors/genetics/metabolism ; *Edible Grain/genetics/metabolism ; Signal Transduction ; },
abstract = {Brassinosteroids (BRs) are essential steroidal plant hormones that regulate cell elongation, vascular differentiation and reproductive development, thereby shaping plant architecture and grain yield. Fine-tuning BR signalling, rather than altering hormone accumulation, has been proposed as a potential strategy to regulate plant development. Here we investigated the role of IBH1 transcription factor, a negative regulator within the ILI1/PRE1-IBH1 module controlling BR-mediated cell elongation in durum wheat (Triticum turgidum ssp. durum), using a CRISPR-Cas9-mediated knockout approach. IBH1 loss of function was associated with transcriptional changes consistent with enhanced BR signalling, including the coordinated upregulation of key signalling components (BRI1 and BZR1) and downstream regulators (ILI1 and PRE1). Metabolic profiling revealed selective remodelling of BR biosynthesis, characterized by differential accumulation of specific pathway intermediates rather than a general increase in BR content. Phenotypic analyses showed that TdIBH1 mutants exhibited increased plant height, internode elongation, leaf inclination, grain size, and thousand grain weight, while no significant differences in fertility were detected. These findings support a role for TdIBH1 as a conserved negative regulator of BR-mediated growth in durum wheat and suggest that the manipulation of the ILI1/PRE1-IBH1 module may provide a potential strategy to modulate plant architecture and grain-related traits in cereal crops.},
}

*Triticum/metabolism/genetics/anatomy & histology
*Brassinosteroids/metabolism
*Plant Proteins/genetics/metabolism
*CRISPR-Cas Systems/genetics
Gene Expression Regulation, Plant
Gene Knockout Techniques
*Transcription Factors/genetics/metabolism
*Edible Grain/genetics/metabolism
Signal Transduction

@article {pmid42867989,
year = {2026},
author = {Das, M and Banerjee, D and Hossain, A and Jensen, M and Mogre, S and Sun, J and Mao, J and Glick, AB and Salis, HM and Girirajan, S},
title = {Quantifying the impact of genetic mutations on enhancer dynamics.},
journal = {Cell reports},
volume = {45},
number = {10},
pages = {118131},
doi = {10.1016/j.celrep.2026.118131},
pmid = {42867989},
issn = {2211-1247},
mesh = {*Enhancer Elements, Genetic/genetics ; Humans ; *Mutation/genetics ; Gene Expression Regulation ; Transcription Factors/metabolism/genetics ; CRISPR-Cas Systems/genetics ; Animals ; },
abstract = {Transcriptional regulation is mediated by enhancers, yet how genetic perturbations alter enhancer activity and gene expression remains unclear. We develop UDI-UMI-STARR-seq, integrating dual indexes and unique molecular identifiers, and combine it with RNA-seq to profile perturbation-associated changes in enhancer activity and gene expression. We apply this approach to 253,632 fragments representing 46,142 cell type-specific candidate enhancers and assess the impact of CRISPR/Cas9-mediated deletion of six transcription factors (ATF2, CTCF, FOXA1, LEF1, TCF7L2, and SCRT1). We identify responsive fragments that are repressed or induced and enriched for p53 family motifs. Enhancer-gene mapping reveals associations between responsive fragments and gene expression changes, including Wnt/p53 targets after ATF2 or LEF1 depletion. A deep learning model recapitulates enhancer grammar, including cooperative motif syntax and flanking sequence context. Applying this framework to 16p12.1 deletion identifies responsive fragments associated with genes involved in neuronal function, providing a scalable readout of enhancer dynamics across genetic perturbations.},
}

*Enhancer Elements, Genetic/genetics
Humans
*Mutation/genetics
Gene Expression Regulation
Transcription Factors/metabolism/genetics
CRISPR-Cas Systems/genetics
Animals

@article {pmid42924445,
year = {2026},
author = {Ono, S and Ono, M and Brettschneider, R and Sauer, D and Müller, K and Balboni, M and van der Heide, M and Schnittger, A},
title = {Reconstructing tissue culture to improve Agrobacterium-mediated transformation of maize.},
journal = {The Plant journal : for cell and molecular biology},
volume = {128},
number = {2},
pages = {},
pmid = {42924445},
issn = {1365-313X},
support = {SCHN 736/20-1//Deutsche Forschungsgemeinschaft/ ; Research Unit 5235//Deutsche Forschungsgemeinschaft/ ; },
mesh = {*Zea mays/genetics ; Plants, Genetically Modified/genetics ; *Transformation, Genetic ; *Tissue Culture Techniques/methods ; DNA, Bacterial/genetics ; *Agrobacterium/genetics ; CRISPR-Cas Systems ; },
abstract = {The ability to insert, delete, or modify genetic information is crucial for mechanistic studies and biotechnological applications. However, efficient genetic transformation remains a major bottleneck for research in maize and many other crops. Here, we report an optimized Agrobacterium-mediated transformation platform based on systematic reconstruction of tissue culture handling in the maize inbred line A188. Refinement of callus induction, selection, and regeneration substantially improved recovery of transgenic plantlets. To distinguish independent T-DNA insertion events, we developed TAFLP (T-DNA amplified fragment length polymorphism), a simple and inexpensive assay that amplifies T-DNA flanking sequences and can be performed using standard laboratory equipment. Our enhanced transformation pipeline was also applicable to the inbred line B104 as well as to hygromycin and G418 selection systems, demonstrating broad utility of our method. We validated the platform for CRISPR/Cas9 mutagenesis and reporter-line generation. Using this approach, we isolated new loss-of-function alleles of the maize-specific MAC1 (MULTIPE ARCHESPORIAL CELLS 1) and F-box protein encoding ACOZ1 (ABNORMAL CHROMOSOME ORGANIZATION IN ZYGOTENE 1), a homolog of the Arabidopsis RMF1/2 (REDUCED MALE FERTILITY 1/2) and rice ZYGO1 (ZYGOTENE 1). The concomitant generation of reporter lines for ACOZ1 and the nuclear lamin-like proteins NCH1 (NMCP/CROWN HOMOLOG1) and NCH2, as well as SUN1 (SUN DOMAIN PROTEIN 1) revealed that the function of this F-box protein class in releasing the somatic constraints of chromosome organization for meiotic recombination is highly conserved in flowering plants.},
}

*Zea mays/genetics
Plants, Genetically Modified/genetics
*Transformation, Genetic
*Tissue Culture Techniques/methods
DNA, Bacterial/genetics
*Agrobacterium/genetics
CRISPR-Cas Systems

@article {pmid42925420,
year = {2026},
author = {Li, Q and Zeng, Z and Zhao, Y and Bi, J and Yan, Q and Wang, C},
title = {TaqW-6BL, a novel gene positively regulates grain WE-AX content in wheat.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {139},
number = {11},
pages = {},
pmid = {42925420},
issn = {1432-2242},
support = {32401870//National Natural Science Foundation of China/ ; 251100110200//Henan Province Major Science and Technology Project/ ; },
mesh = {*Triticum/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; *Genes, Plant ; *Seeds/genetics/chemistry ; Phenotype ; Edible Grain/genetics/chemistry ; Plants, Genetically Modified ; CRISPR-Cas Systems ; Mutation, Missense ; },
abstract = {The TaqW-6BL gene, which encodes a TAXI-type inhibitor, influences the water-extractable arabinoxylan content in wheat grains. Water-extractable arabinoxylans (WE-AX) are major dietary fiber components and play a crucial role in wheat end-use quality, especially its nutritional quality. In this study, TaqW-6BL was cloned from parental varieties of a recombinant inbred line (RIL) population, in which a missense mutation (+ 296 bp, T/C) was detected and found to cause a methionine (Met) to threonine (Thr) substitution. Overexpression of TaqW-6BL positively regulated grain WE-AX content, thousand kernel weight (TKW) and mean grain perimeter (MP), with increases of 30.80%, 14.59% and 14.53%, respectively. Conversely, CRISPR/Cas9-mediated knockout of TaqW-6BL led to significant decreases in WE-AX content, TKW and MP. Cytological observations revealed that the aleurone layer of overexpression lines partially proliferated into two layers, whereas knockout lines showed disordered cell arrangement and abnormal morphology. Subcellular localization analysis confirmed that TaqW-6BL protein localizes in the endoplasmic reticulum (ER). The relative expression level and WE-AX content of the favorable haplotype (TaqW-6BL[Avocet]) were significantly higher than those of the unfavorable haplotype (TaqW-6BL[Chilero]) at 14 days and 21 days after flowering (DAF) (P < 0.01). Meanwhile, we developed a functional cleaved amplified polymorphic sequence (CAPS) marker CAPS-6B-296. The wheat varieties with favorable haplotype TaqW-6BL[Avocet] accounted for 68% of 160 wheat varieties. Therefore, our findings provide a valuable gene and a reliable functional marker for nutritional quality breeding in wheat.},
}

*Triticum/genetics/metabolism
*Plant Proteins/genetics/metabolism
Gene Expression Regulation, Plant
*Genes, Plant
*Seeds/genetics/chemistry
Phenotype
Edible Grain/genetics/chemistry
Plants, Genetically Modified
CRISPR-Cas Systems
Mutation, Missense

@article {pmid42926261,
year = {2027},
author = {Hargadon, KM and Bushhouse, DZ and Johnson, CE and Willams, CJ},
title = {Generation of Functional Gene Knockout Melanoma Cell Lines by CRISPR-Cas9 Gene Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3089},
number = {},
pages = {25-46},
pmid = {42926261},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Animals ; *Gene Knockout Techniques/methods ; Cell Line, Tumor ; Mice ; Forkhead Transcription Factors/genetics ; *Gene Editing/methods ; *Melanoma/genetics/pathology ; Humans ; },
abstract = {Recent advances in the treatment of metastatic melanoma have emerged only from advances in our understanding of melanoma development and progression at the cellular and molecular level. Despite the impact that such advances have made on the clinical management of this cancer over the last decade, additional insights into factors that promote melanoma progression and therapeutic resistance are needed to combat this disease. CRISPR-Cas9 gene editing technology is a powerful tool for studying gene function in a timely and cost-effective manner, enabling the manipulation of specific DNA sequences via a targeted approach. Herein, we describe a protocol for generating functional gene knockouts in melanoma cell lines by CRISPR-Cas9 gene editing, and we present an example application of this protocol for the successful knockout of the Foxc2 transcription factor-encoding gene in the B16-F1 murine melanoma cell line.},
}

*CRISPR-Cas Systems
Animals
*Gene Knockout Techniques/methods
Cell Line, Tumor
Mice
Forkhead Transcription Factors/genetics
*Gene Editing/methods
*Melanoma/genetics/pathology
Humans

@article {pmid42927881,
year = {2026},
author = {Khaskheli, MA and Raza, A and Jueping, S and Hassan, MA and Janiad, S and Sabir, S and Khaskheli, RA and Khan, MA and Zeb, A and Hayyat, Q and Wang, Y},
title = {Revolutionizing the tomato cultivation: advances and frontiers in virus-resistant tomato breeding.},
journal = {Functional plant biology : FPB},
volume = {53},
number = {11},
pages = {},
doi = {10.1071/FP25162},
pmid = {42927881},
issn = {1445-4416},
mesh = {*Solanum lycopersicum/genetics/virology/growth & development ; *Plant Breeding/methods ; *Disease Resistance/genetics ; *Plant Diseases/virology/genetics ; *Plant Viruses ; Plants, Genetically Modified ; RNA Interference ; },
abstract = {Plant viruses are major pathogens that significantly reduce the quality and yield of tomato crops. The most influential viruses belong to the genera Crinivirus, Begomovirus, Tobamovirus, and Potyvirus. Tomato breeders have successfully incorporated virus-resistance genes, such as Ty, Sw, Tm, and Pot 1, from wild germplasms into cultivated varieties. However, the challenge remains that most resistance genes confer only qualitative resistance, making them vulnerable to new viruses, recombination, and mixed infections. To our knowledge, this review is the first to systematically compare the synergy and complementarity among three antiviral breeding strategies-conventional R-gene pyramiding (e.g. Ty, Sw, Tm), RNA interference (RNAi)-based transgenics, and CRISPR/Cas genome editing-in tomato. We also innovatively incorporate the emerging genus Amalgavirus into the antiviral framework and propose targeted countermeasures against its escape mechanisms, including synthetic NLR decoys and Cas13-mediated transcriptional degradation. Using the three breeding methodologies, we concluded by outlining the difficulties and offering recommendations for future improvements in tomato antiviral breeding.},
}

*Solanum lycopersicum/genetics/virology/growth & development
*Plant Breeding/methods
*Disease Resistance/genetics
*Plant Diseases/virology/genetics
*Plant Viruses
Plants, Genetically Modified
RNA Interference

@article {pmid42928967,
year = {2026},
author = {Iqbal, S and Tariq, K and Elatafi, E and Elhendawy, B and Yu, H and Liu, W and Ali, S and Aziz, RB and Nadeem, MY and Fang, J},
title = {Jasmonate-Induced Metabolic Reprogramming Enhances Fruit Quality, Bioactive Compounds, and Stress Tolerance.},
journal = {Physiologia plantarum},
volume = {178},
number = {6},
pages = {},
doi = {10.1111/ppl.71165},
pmid = {42928967},
issn = {1399-3054},
mesh = {*Cyclopentanes/metabolism/pharmacology ; *Oxylipins/metabolism/pharmacology ; Stress, Physiological ; *Fruit/physiology/metabolism ; *Plant Growth Regulators/metabolism ; Gene Expression Regulation, Plant ; Signal Transduction ; Metabolic Reprogramming ; Drought Resistance ; },
abstract = {Jasmonates (JAs), including jasmonic acid (JA), methyl jasmonate (MeJA), jasmonoyl-isoleucine (JA-Ile), and cis-jasmone, have emerged as central integrators of plant stress adaptation, developmental regulation, and fruit quality formation in horticultural crops. This review comprehensively summarizes recent advances in JA biosynthesis, metabolic conversion, signal perception, and downstream regulatory networks governing abiotic stress resilience and quality-associated traits. We highlight the coordinated chloroplast-peroxisome biosynthetic pathway leading to the formation of bioactive jasmonates and discuss the molecular basis of JA signaling mediated through the SCF^COI1-JAZ co-receptor complex and key transcriptional regulators, including MYC, MYB, NAC, ERF, WRKY, and bHLH families. Emerging evidence demonstrates that JA signaling orchestrates reactive oxygen species (ROS) homeostasis, antioxidant defense, osmotic adjustment, secondary metabolism, hormonal crosstalk, and stress-responsive gene expression in response to drought, salinity, temperature extremes, and heavy metal toxicity. Beyond stress adaptation, jasmonates play pivotal roles in regulating fruit ripening, anthocyanin biosynthesis, phenolic accumulation, aroma volatile production, cell wall remodeling, vitamin C metabolism, and postharvest quality maintenance. We also discuss recent progress in multi-omics integration, epigenetic regulation, CRISPR/Cas-based genome editing, systems biology, and precision agriculture approaches that are accelerating understanding and manipulation of JA-mediated pathways in horticultural species. This review provides a mechanistic framework linking JA signaling with metabolic plasticity, stress adaptation, and fruit quality. However, major knowledge gaps remain regarding context-dependent JA responses, hormone crosstalk, metabolic trade-offs, and field-scale validation. Future research should integrate multi-omics, spatial analyses, and standardized field trials to develop precise strategies for improving fruit quality and stress tolerance.},
}

*Cyclopentanes/metabolism/pharmacology
*Oxylipins/metabolism/pharmacology
Stress, Physiological
*Fruit/physiology/metabolism
*Plant Growth Regulators/metabolism
Gene Expression Regulation, Plant
Signal Transduction
Metabolic Reprogramming
Drought Resistance

@article {pmid42929528,
year = {2026},
author = {Colin, MM and Thaventhiran, JED and Willis, AE},
title = {Human-relevant disease models to accelerate the development of nucleic acid-based therapies and other advanced therapeutics.},
journal = {Disease models & mechanisms},
volume = {19},
number = {10},
pages = {},
doi = {10.1242/dmm.053109},
pmid = {42929528},
issn = {1754-8411},
support = {MC_UP_A600_1023/MRC_/Medical Research Council/United Kingdom ; C_UU_00025/12/MRC_/Medical Research Council/United Kingdom ; R/W020564/1/MRC_/Medical Research Council/United Kingdom ; 316658/Z/24/Z/WT_/Wellcome Trust/United Kingdom ; //Lister Institute of Preventive Medicine/ ; },
mesh = {Humans ; Animals ; *Nucleic Acids/therapeutic use ; *Models, Biological ; Genetic Therapy ; *Therapeutics ; Disease Models, Animal ; },
abstract = {Advanced therapeutics, including programmable nucleic acid-based therapies (NATs), such as small interfering RNAs (siRNAs), antisense oligonucleotides (ASOs), CRISPR-Cas 9 editing systems, modified in vitro transcribed messenger RNAs (mRNAs) and Adeno-associated viruses (AAVs), have the potential to transform healthcare. However, while there have been considerable successes, there have also been notable clinical failures in early-stage human trials, which slowed down the progression of these therapeutic platforms. Multiple trials of NATs have been halted or discontinued, or approved products carry black box warnings as unforeseen or underestimated toxicities and poor efficacy have been observed in humans. There are a variety of reasons for failure of NATs, one of which is that many preclinical studies lack disease modelling in a human-relevant manner and, therefore, do not capture disease-specific effects on therapy tolerability and efficacy. This Perspective provides a short overview of why better human disease modelling is required to understand the potential therapeutic liabilities of NATs across modalities.},
}

Humans
Animals
*Nucleic Acids/therapeutic use
*Models, Biological
Genetic Therapy
*Therapeutics
Disease Models, Animal

@article {pmid42930100,
year = {2026},
author = {Bo, X and Li, J and Huang, C and Liu, Q and Qi, M and Li, Q and Dai, L and Zhu, R and Ding, S},
title = {A Collective CRISPR-Cas12a Biosensing Platform for Effect-Based Screening of Pesticide Residues.},
journal = {Biosensors},
volume = {16},
number = {10},
pages = {},
pmid = {42930100},
issn = {2079-6374},
support = {2024YFC3505800//National Key R&D Program of China/ ; ZR2023MH249, ZR2023QE331//Natural Science Foundation of Shandong Province of China/ ; 2025M773938//China Postdoctoral Science Foundation/ ; M20241840//Shandong Provincial Science and Technology Project of Traditional Chinese Medicine/ ; SDACM202204//Shandong Association of Traditional Chinese Medicine Clinical Pharmacy Research Special Fund Project/ ; Z04J2025E170//2025 YaoDong-ShenZhou- Pharmaceutical Research Capacity Building Fund Project/ ; KY2025J02//Shandong University of Traditional Chinese Medicine Scientific Research Fund Project/ ; },
mesh = {*Biosensing Techniques ; *CRISPR-Cas Systems ; *Pesticide Residues/analysis ; Alkaline Phosphatase ; },
abstract = {The simultaneous screening of multicomponent pesticide residues, rather than a single specific compound, remains a critical yet challenging frontier in safety surveillance, largely due to the inherent one-to-one recognition bottleneck in conventional aptamer- or antibody-based biosensors. Herein, we develop a collective CRISPR-Cas12a biosensing platform for effect-based screening of pesticide residues by integrating effect-based enzymatic recognition with PAM-independent CRISPR signal amplification. Rather than employing a dedicated aptamer or antibody for each pesticide, the proposed strategy exploits the inhibitory effects of diverse pesticide residues on alkaline phosphatase (ALP) as a common biochemical response. ALP activity inhibition serves as a common trigger, converting the presence of diverse toxicants into the controlled generation of Cu[+] as a unified biochemical signal via ALP-mediated ascorbic acid production. To transduce this signal into a robust nucleic acid output, we further engineer a CRISPR-Cas12a system that operates completely independent of PAM sequences through click chemistry-mediated covalent ligation of two hairpin fragments. The complete activator strand activated Cas12a trans-cleavage, thereby circumventing the sequence constraints imposed by PAM recognition. The platform exhibits quantitative responses toward TCE, CPF, and Piri, with limits of detection of 22.1 nM, 19.3 nM, and 12.0 nM, respectively. Furthermore, it successfully reports pesticide contamination in a complex real sample matrix, which provides a simple and flexible strategy for preliminary pesticide screening. Importantly, the platform is not intended to convert the collective fluorescence response into the total concentration of diverse pesticide residues; rather, the output reflects the integrated perturbation of the ALP-mediated biochemical pathway under the defined assay conditions. Overall, this work establishes an effect-based collective sensing paradigm that shifts pesticide screening from compound-specific molecular recognition toward integrated biochemical response, providing a flexible strategy for rapid screening and early warning of multicomponent pesticide contamination.},
}

*Biosensing Techniques
*CRISPR-Cas Systems
*Pesticide Residues/analysis
Alkaline Phosphatase

@article {pmid42930118,
year = {2026},
author = {Wang, T and Li, L and Wang, Y and Han, Y and Li, T and Yu, C and Lou, Y and Rong, Z and Lin, K and Wang, S},
title = {Integrated Microfluidic CRISPR-Based Diagnostic Device for Rapid Testing of HPVs.},
journal = {Biosensors},
volume = {16},
number = {10},
pages = {},
pmid = {42930118},
issn = {2079-6374},
support = {82472105//National Natural Science Foundation of China/ ; 20230484317//Beijing Nova Program/ ; },
mesh = {Humans ; CRISPR-Cas Systems ; *Lab-On-A-Chip Devices ; Rapid Diagnostic Tests ; Human papillomavirus 16 ; *Human Papillomavirus Viruses/isolation & purification ; *Biosensing Techniques ; Limit of Detection ; Microfluidics ; Papillomavirus Infections/diagnosis ; },
abstract = {Screening for high-risk human papillomavirus (HR-HPV) is a pivotal strategy for the prevention of invasive cervical cancer, particularly in resource-constrained settings. In this study, we developed a CRISPR-based compact automated real-time detection system, termed Ccard, for rapid testing of HPV16, HPV18, and the internal reference gene GAPDH through a streamlined workflow. A simple room-temperature treatment efficiently releases nucleic acids directly from swab samples, bypassing the need for extraction. The mixture was driven through the channels and loaded into the reaction chambers on the microfluidic chip by a vacuum pump. The lyophilized reagents in the reaction zone of the microfluidic chip were redissolved. The system subsequently performs a single-step RPA-CRISPR/Cas13a reaction, with real-time detection enabled by an Internet of Things device. The assay achieved detection within 30 min, with a limit of detection (LOD) of 5 copies/µL for both HPV16 and HPV18. Validation with clinical samples demonstrated high sensitivity (96.8% for HPV16 and 100% for HPV18) and 100% specificity. These results highlight the reliability, precision, and strong potential of the Ccard system for rapid testing of HR-HPVs.},
}

Humans
CRISPR-Cas Systems
*Lab-On-A-Chip Devices
Rapid Diagnostic Tests
Human papillomavirus 16
*Human Papillomavirus Viruses/isolation & purification
*Biosensing Techniques
Limit of Detection
Microfluidics
Papillomavirus Infections/diagnosis

@article {pmid42930125,
year = {2026},
author = {Huo, S and Zhu, Y and Ji, C and Fan, F and Lin, R and Ye, W and Fu, Y and Hua, J and Feng, G and Chen, L and Yun, T and Zhang, C and Wu, Z and Xu, X and Wu, Y and Ni, Z},
title = {Development of a One-Pot MIRA-CRISPR/Cas12b Fluorescence Biosensing Platform for Rapid Detection of Brucella canis.},
journal = {Biosensors},
volume = {16},
number = {10},
pages = {},
pmid = {42930125},
issn = {2079-6374},
support = {No. 2025R22A77C03//Zhejiang Academy of Agricultural Sciences-Pinghu Institute-Local Government Science and Technology Cooperation Project/ ; No. 2021DG700024-KF202507//the State Key Laboratory for Managing Biotic and Chemical Threats to Quality and Safety of the Agro-products/ ; },
mesh = {*Brucella canis/isolation & purification ; *Biosensing Techniques ; Animals ; Dogs ; CRISPR-Cas Systems ; Fluorescence ; *Brucellosis/diagnosis/veterinary ; Nucleic Acid Amplification Techniques ; },
abstract = {Brucellosis is an important zoonotic disease worldwide, and Brucella canis (B. canis) is an emerging concern in companion animal health. Rapid and reliable detection of B. canis in dogs is essential for disease control. Herein, we developed a one-pot MIRA CRISPR/Cas12b fluorescence biosensing platform targeting the conserved omp31 gene. The platform integrates MIRA mediated isothermal amplification with CRISPR/Cas12b-based target recognition and collateral cleavage. After optimization, the assay achieved a detection limit of 5 copies/reaction within 20 min, comparable to qPCR. No cross reactivity was observed with seven common canine pathogens. Among 128 clinical blood samples, the biosensor showed complete agreement with qPCR, achieving 100% sensitivity and 100% specificity. These findings demonstrate that the biosensor provides a rapid, sensitive, and specific approach for B. canis detection, with potential for decentralized veterinary testing through its endpoint visual fluorescence readout and applications in One Health surveillance.},
}

*Brucella canis/isolation & purification
*Biosensing Techniques
Animals
Dogs
CRISPR-Cas Systems
Fluorescence
*Brucellosis/diagnosis/veterinary
Nucleic Acid Amplification Techniques

@article {pmid42931860,
year = {2026},
author = {Fajariani, W and Hungsaprug, K and Chaidee, A and Lynagh, PG and Purnama, PR and Comai, L and Chadchawan, S},
title = {Promoter editing of OsGTL1 improves instantaneous water use efficiency via targeted stomatal reduction in rice.},
journal = {Plant cell reports},
volume = {45},
number = {11},
pages = {},
pmid = {42931860},
issn = {1432-203X},
support = {BCG_FF_68_261_2300_064//Thailand Science Research and Innovation Fund of Chulalongkorn University/ ; },
mesh = {*Oryza/genetics/metabolism/physiology ; *Plant Stomata/genetics/metabolism/physiology ; *Water/metabolism ; *Promoter Regions, Genetic/genetics ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant/genetics ; *Gene Editing/methods ; Plants, Genetically Modified ; Drought Resistance ; Droughts ; CRISPR-Cas Systems/genetics ; Photosynthesis/genetics ; },
abstract = {OsGTL1 promoter modification decreases stomatal density (SD) and is inherited as a recessive trait compared to the wild type. Lower SD increases water use efficiency but lowers yield components. GT2-LIKE1 (GTL1) is a predicted drought-responsive gene in rice involved in stomatal development, yet its regulatory relationship with putative targets like Stomatal Density and Distribution 1 remains complex. Here, we employed CRISPR/Cas-based editing to alter GTL1 expression by designing a tRNA-based CRISPR to mutagenize its promoter, targeting ~ 1,500 bp 5' of the transcription start site with 7 gRNAs. Two promoter-edited lines with decreased stomatal density were selected and tested for inheritance by crossing with wild type and demonstrated to be recessive alleles, which regulated the lower expression of OsGTL1 in the developing leaves, leading to a tendency of OsSDD1 higher expression. Lower OsGTL1 expression did not significantly affect meristemoid formation, but it prevented some guard mother cells from completing symmetrical division to form mature guard cells. Lower stomatal density did not decrease the photosynthesis rate, but increased water use efficiency in the tillering stage under normal conditions and in the reproductive stage under drought stress. However, yield components in the edited lines were lower than in WT under normal conditions, but drought stress at the reproductive stage reduced yield components in the edited lines less than in WT. These findings indicate that altered OsGTL1 expression can affect stomatal development and water-use efficiency, while also revealing a trade-off between water conservation and yield components under different water regimes.},
}

*Oryza/genetics/metabolism/physiology
*Plant Stomata/genetics/metabolism/physiology
*Water/metabolism
*Promoter Regions, Genetic/genetics
*Plant Proteins/genetics/metabolism
Gene Expression Regulation, Plant/genetics
*Gene Editing/methods
Plants, Genetically Modified
Drought Resistance
Droughts
CRISPR-Cas Systems/genetics
Photosynthesis/genetics

@article {pmid42932357,
year = {2026},
author = {Li, H and Liu, H and Xu, W and Zeng, Y and Huang, P and Guo, J and Cai, B and Chen, Y and Lin, Y and Zhang, C},
title = {Functional impact of PCSK9 variants on LDL uptake in a knockout hepatic model.},
journal = {PloS one},
volume = {21},
number = {10},
pages = {e0345908},
pmid = {42932357},
issn = {1932-6203},
mesh = {*Proprotein Convertase 9/genetics/metabolism ; Humans ; Hep G2 Cells ; Receptors, LDL/metabolism/genetics ; Gene Knockout Techniques ; *Liver/metabolism ; *Lipoproteins, LDL/metabolism ; *Cholesterol, LDL/metabolism ; CRISPR-Cas Systems ; },
abstract = {Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a central regulator of low-density lipoprotein (LDL) cholesterol metabolism, yet the functional consequences of many clinically observed PCSK9 variants remain unknown. To establish a rigorous system for quantitative variant assessment, we generated a PCSK9 knockout (KO) HepG2 cell line through CRISPR/Cas9-mediated deletion of exons 2-8, effectively removing both the prodomain and catalytic regions required for PCSK9 function. This null background enabled systematic functional mapping of wild-type (WT) PCSK9 and multiple clinically relevant variants representing well-characterized, recurrent, and previously understudied alleles. Functional assays revealed pronounced heterogeneity among variant activities. The classical gain-of-function (GOF) variants D374Y and R496W exhibited robust suppression of LDL uptake, whereas A443T-an infrequently reported and previously uncharacterized variant-demonstrated a loss-of-function (LOF)-like phenotype with significantly enhanced LDL uptake. Additional poorly characterized variants, including V4I, R104C/V114A, and R496W/N425S, displayed varied functional profiles, providing novel mechanistic insights. Surface LDL receptor (LDLR) levels generally correlated with LDL uptake but revealed unique patterns for specific variants. This KO-based rescue system provides a high-resolution framework for mechanistic classification of both established and poorly characterized PCSK9 variants, bridging the gap between genetic discovery and functional interpretation while supporting precision lipid-lowering strategies.},
}

*Proprotein Convertase 9/genetics/metabolism
Humans
Hep G2 Cells
Receptors, LDL/metabolism/genetics
Gene Knockout Techniques
*Liver/metabolism
*Lipoproteins, LDL/metabolism
*Cholesterol, LDL/metabolism
CRISPR-Cas Systems

@article {pmid42936235,
year = {2026},
author = {Kumar, A and Kanika, and Rani, S and Warghat, AR},
title = {Advanced soilless cultivation of medicinal plants: integrating dynamic LED lighting, elicitation, and smart technologies for sustainable biomass and phytochemical production.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1970412},
pmid = {42936235},
issn = {1664-462X},
abstract = {Countries with limited water and land resources confront a significant problem due to the growing demand for medicinal plants. These plants are used worldwide to extract and isolate therapeutically important bioactive molecules, known as secondary metabolites. Many medicinal plant species are currently facing significant conservation challenges due to excessive, unregulated harvesting, driven by rapidly growing demand for plant-derived therapies. Soilless farming techniques like hydroponics, aeroponics, and vertical farming offer a sustainable alternative for generating high-quality planting material on a large scale in a considerably shorter period and with less space. Soilless farming revolutionises agriculture and food production, making it particularly well-suited for urban agriculture by reducing maintenance requirements and maximizing crop yields. Soilless farming of numerous medicinal herbs, fruit crops, and vegetables has become increasingly popular in recent years, particularly for its ability to produce large amounts of biomass, high yields, and a wide range of pharmacologically active metabolites. These high-tech systems have significant promise for increased crop yields, cleaner produce, season-independent cultivation, and reduced environmental impact, particularly in water use and disease, compared to traditional soil-based systems. This review article rigorously evaluates recent advancements in soilless agriculture, including LED spectral regulation, nano-elicitation, microbial biostimulants and precursor-feeding strategies, reusable substrates, computational technologies, and CRISPR/Cas-based editing, with respect to their efficacy in generating high-quality plant biomass, improving automation, and optimizing shoot architecture. Implementing soilless farming to produce and synthesize bioactive molecules is expected to be crucial in enhancing sustainability and human well-being.},
}

@article {pmid42190933,
year = {2026},
author = {Delinière, A and Boiteux, C and Blancfuney, C and Hysa, S and Martín, LR and Sechi, S and Nevoret, E and Chevalier, P and Boulin, T and Andrini, O},
title = {In vivo analysis of trafficking and functional impact of hERG pore-domain missense variants in a CRISPR/Cas9-engineered C.elegans model of long QT syndrome.},
journal = {Heart rhythm},
volume = {23},
number = {11},
pages = {e2991-e3002},
doi = {10.1016/j.hrthm.2026.05.031},
pmid = {42190933},
issn = {1556-3871},
mesh = {Animals ; *Long QT Syndrome/genetics/metabolism ; *Mutation, Missense ; Caenorhabditis elegans/genetics ; *ERG1 Potassium Channel/genetics/metabolism ; Disease Models, Animal ; *CRISPR-Cas Systems ; Humans ; *DNA/genetics ; },
abstract = {BACKGROUND: Loss-of-function variants in KCNH2, which encodes hERG, are responsible for long QT syndrome type 2 (LQT2), a major cause of sudden cardiac death. Most hERG pore-domain missense variants are associated with severe LQT2. However, functional heterogeneity exists among these variants, with a small subset associated with milder phenotypes. Only a limited number of these atypical variants have been functionally characterized. Caenorhabditis elegans expresses UNC-103, a voltage-gated potassium channel orthologous to hERG, with high conservation in sequence and channel biogenesis regulators, supporting its use as an in vivo model to investigate hERG variants.

OBJECTIVE: This study aimed to develop a C.elegans model for in vivo characterization of hERG pore-domain missense variants.

METHODS: Using CRISPR/Cas9 genome editing, we generated fluorescent UNC-103 knockin strains. We established an approach combining fluorescence in vivo imaging to assess channel trafficking efficiency, with behavioral readouts of muscle hyperexcitability to evaluate the integrated functional impact of hERG variants, encompassing defects in trafficking, gating, and potassium permeability.

RESULTS: The model faithfully recapitulated the profiles of G628S and G604S, 2 well-characterized hERG variants associated with severe LQT2. Moreover, it enabled in vivo characterization of G603S, a recently identified atypical pore-domain variant associated with a mild LQT2 phenotype in heterozygous carriers and a severe long QT syndrome in the homozygous state. G603S exhibited a trafficking defect responsible for a partial loss of function.

CONCLUSION: This CRISPR/Cas9-engineered C.elegans model complements in vitro assays and complex transgenic animals, enabling in vivo characterization of hERG pore-domain missense variants.},
}

Animals
*Long QT Syndrome/genetics/metabolism
*Mutation, Missense
Caenorhabditis elegans/genetics
*ERG1 Potassium Channel/genetics/metabolism
Disease Models, Animal
*CRISPR-Cas Systems
Humans
*DNA/genetics

@article {pmid42754663,
year = {2026},
author = {Li, L and An, XN and Ruan, YT and Yang, R and Li, P and Shen, PT and Zeng, HY and Wu, XY and Huang, XC and Song, MY and Hao, JL and Gao, P and Dong, XM},
title = {CRISPR/Cas9 screening revealed BIRC6-AS1/BIRC6 mediates abiraterone resistance via NHEJ pathway-dependent A20 degradation in prostate cancer.},
journal = {Oncogene},
volume = {45},
number = {44},
pages = {4723-4742},
pmid = {42754663},
issn = {1476-5594},
support = {2023-JC-YB-716//Natural Science Foundation of Shaanxi Province (Shaanxi Province Natural Science Foundation)/ ; 82472665//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Male ; Humans ; *Prostatic Neoplasms/genetics/drug therapy/pathology/metabolism ; *Drug Resistance, Neoplasm/genetics ; CRISPR-Cas Systems ; Animals ; *Androstenes/pharmacology/therapeutic use ; *DNA End-Joining Repair/genetics ; *Inhibitor of Apoptosis Proteins/genetics/metabolism ; Cell Line, Tumor ; *RNA, Long Noncoding/genetics ; Mice ; Gene Expression Regulation, Neoplastic/drug effects ; },
abstract = {Abiraterone acetate is a standard-of-care therapy for prostate cancer (PCa). However, resistance frequently emerges, often characterized by the progression to AR-independent phenotypes. Employing a genome-wide CRISPR/Cas9 library screening strategy, we identified 523 long non-coding RNAs (lncRNAs) and 2,183 protein-coding genes as potential candidates associated with abiraterone resistance. Notably, a pair of sense-antisense genes, BIRC6-AS1/BIRC6, was identified as a significant contributor to abiraterone resistance, serving as a critical survival factor in AR-independent contexts. BIRC6-AS1 depletion led to a reduction in both the mRNA and protein levels of BIRC6. Moreover, depletion of either BIRC6-AS1 or BIRC6 enhanced the sensitivity of PCa cells to abiraterone in both in vitro and in vivo settings. Further investigation revealed that BIRC6-AS1 stabilized the mRNA of BIRC6 through interaction with ILF2. Suppression of either BIRC6-AS1 or BIRC6 attenuated non-homologous end joining (NHEJ) repair activity, resulting in the disassembly of 53BP1 foci at DNA damage sites and an increased accumulation of DNA damage, thereby exposing a vulnerability in AR-independent resistant cells. Mechanistically, BIRC6 interacted with A20 and facilitated the K48-linked ubiquitination and subsequent degradation of A20 at the K337 residue. Additionally, A20 knockdown effectively reversed the abiraterone sensitivity induced by BIRC6-AS1 depletion. Collectively, our study provides a landscape of lncRNAs and protein-coding genes associated with abiraterone resistance and suggests that targeting the BIRC6-AS1/BIRC6 axis represents a potential therapeutic strategy to eradicate AR-independent resistant tumors in prostate cancer.},
}

Male
Humans
*Prostatic Neoplasms/genetics/drug therapy/pathology/metabolism
*Drug Resistance, Neoplasm/genetics
CRISPR-Cas Systems
Animals
*Androstenes/pharmacology/therapeutic use
*DNA End-Joining Repair/genetics
*Inhibitor of Apoptosis Proteins/genetics/metabolism
Cell Line, Tumor
*RNA, Long Noncoding/genetics
Mice
Gene Expression Regulation, Neoplastic/drug effects

@article {pmid42886223,
year = {2026},
author = {Wang, Y and Haghighat Naeini, A and Kumar, N and Zhao, M},
title = {High-Throughput Screening of a Lipid Nanoparticle (LNP) Library Identifies Lead Formulations for CRISPR-Cas9 Ribonucleoprotein Delivery and Gene Editing.},
journal = {ACS applied materials & interfaces},
volume = {18},
number = {42},
pages = {57043-57056},
doi = {10.1021/acsami.6c11615},
pmid = {42886223},
issn = {1944-8252},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Nanoparticles/chemistry ; Animals ; *Lipids/chemistry ; Mice ; HEK293 Cells ; *Ribonucleoproteins/chemistry/genetics/metabolism ; High-Throughput Screening Assays ; Liposomes ; },
abstract = {Delivery of CRISPR-Cas9 ribonucleoprotein (RNP) using synthetic materials can bypass the host translational machinery and limit the duration of genomic exposure, minimizing the risk of off-target mutations. Despite their advantages, lipid nanoparticle (LNP) platforms for CRISPR-Cas9 RNP delivery that achieve highly efficient editing at low doses remain limited. In the present study, we developed a library of ionizable lipids (ILs) and corresponding LNPs to enhance the delivery and editing efficiencies of CRISPR-Cas9 RNP complexes. Following comprehensive in vitro screening and formulation optimization, an optimal LNP candidate containing the C12A9 IL (denoted as LNPC12A9) was identified that achieved ∼90% EGFP disruption in HEK293T-EGFP cells at an RNP concentration of 30 nM and ∼60% at 15 nM. The editing performance was consistently observed at an RNP concentration of 15 nM when editing the Gcgr and HPRT1 genes across multiple hepatic cell lines. Moreover, coencapsulation of RNP and a single-stranded oligodeoxynucleotide (ssODN) template for homology-directed repair (HDR) within LNPC12A9 induced an HDR rate of 37.8%. Systemic administration of LNPC12A9 into C57BL/6J mice led to liver tropism and a total hepatic Pcsk9 gene disruption rate of 1.39%. In conclusion, this study provides insights into the construction of an LNP platform to improve the delivery efficiency of CRISPR-Cas9 RNP, thereby facilitating the development of LNPs for safe, precise, and tissue-specific Cas9 RNP-based genome editing therapies.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Nanoparticles/chemistry
Animals
*Lipids/chemistry
Mice
HEK293 Cells
*Ribonucleoproteins/chemistry/genetics/metabolism
High-Throughput Screening Assays
Liposomes

@article {pmid42916848,
year = {2026},
author = {Chen, C and Ma, H and Xia, H and Zhang, B and Yin, K and Xie, F and Yue, Y and Bai, L and Wen, J and Yang, Y and Lv, X and Kong, D and Liu, Y and Wei, D},
title = {Mismatch-Engineered CRISPR Transistors for Ultra-Low-Frequency Single-Nucleotide Variant Detection.},
journal = {Nano letters},
volume = {26},
number = {37},
pages = {12500-12509},
doi = {10.1021/acs.nanolett.6c03317},
pmid = {42916848},
issn = {1530-6992},
support = {22304031//National Natural Science Foundation of China/ ; YG2025-KEY-03//Fudan University/ ; 23XD1420200//Science and Technology Commission of Shanghai Municipality/ ; DP2020036//Chongqing Municipal Education Commission/ ; T2425006//National Science Fund for Distinguished Young Scholars/ ; 24CL2901100//Science and Technology Innovation Plan Of Shanghai Science and Technology Commission/ ; NA//Shanghai Academy of Natural Sciences/ ; SIMIS-ID-2025-NC//Shanghai Institute for Mathematics and Interdisciplinary Sciences/ ; },
mesh = {*Polymorphism, Single Nucleotide/genetics ; Humans ; *CRISPR-Cas Systems ; *Transistors, Electronic ; Base Pair Mismatch ; *Proto-Oncogene Proteins B-raf/genetics ; *Biosensing Techniques/instrumentation ; Mutation ; *Clustered Regularly Interspaced Short Palindromic Repeats ; RNA/genetics ; },
abstract = {Accurate detection of low-frequency single-nucleotide variants (SNVs) in a high wild-type (WT) background remains challenging. Here, we report a mismatch-engineered CRISPR transistor (MECT) for rapid, amplification-free detection of mutant RNA. MECT integrates a Cas13a-crRNA recognition interface with a graphene field-effect transistor and employs a spaced mismatch crRNA design centered on the mutation site in a nonseed region. It leverages the synergistic effect between natural and engineered mismatches at the mutation site to weaken WT binding while preserving mutant recognition, thereby amplifying the difference in response between mutants and the WT. Using BRAF V600E RNA as a target, MECT generates a mutant response approximately 11.58 times stronger than the WT background, with a detection limit of 3 × 10-18 mol L-1. MECT can also detect mutants as low as 0.0001% against a WT background. A portable MECT-based prototype is validated using cell-derived RNA and clinical samples, supporting decentralized analysis of low-frequency cancer mutations.},
}

*Polymorphism, Single Nucleotide/genetics
Humans
*CRISPR-Cas Systems
*Transistors, Electronic
Base Pair Mismatch
*Proto-Oncogene Proteins B-raf/genetics
*Biosensing Techniques/instrumentation
Mutation
*Clustered Regularly Interspaced Short Palindromic Repeats
RNA/genetics

@article {pmid42917120,
year = {2026},
author = {Han, R and Xiao, N and Wu, Z and Wang, J and Wang, X and Tang, X},
title = {RPA-CRISPR/Cas12a Assay for ALV-J with Broad-Spectrum Detection and Reduced Residual Amplicon Load.},
journal = {ACS infectious diseases},
volume = {12},
number = {10},
pages = {3408-3418},
doi = {10.1021/acsinfecdis.6c00419},
pmid = {42917120},
issn = {2373-8227},
support = {2023GXNSFAA026260//Guangxi Natural Science Foundation Project/ ; 2025GXNSFAA069363//Guangxi Natural Science Foundation Project/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Avian Leukosis Virus/genetics/isolation & purification/classification ; *Nucleic Acid Amplification Techniques/methods ; Rapid Diagnostic Tests ; *Poultry Diseases/virology/diagnosis ; Recombinases ; Chickens ; },
abstract = {Avian leukosis virus subgroup J (ALV-J) is a retrovirus that poses a serious threat to the poultry industry. It is prone to genetic variation, leading to the emergence of multiple epidemic strains, which creates substantial challenges for clinical detection. Although the recombinase polymerase amplification (RPA)-CRISPR/Cas12a rapid detection assay demonstrates high sensitivity and specificity, it lacks broad applicability across multiple viral strains and is susceptible to amplicon contamination. In this study, a mixed crRNA-based CRISPR/Cas12a detection system was established. Sequence analysis predicts its potential to cover most currently circulating ALV-J variant strains. By optimizing the amount of RPA mix and the buffer conditions, a one-tube RPA-CRISPR/Cas12a assay was developed, with the lowest tested concentration giving 9/9 positive reactions determined to be 1 × 100 copies/μL (equivalent to 15 copies/reaction in a 15 μL reaction). To simplify the sample-handling procedure and reduce detection time, a one-pot RPA-CRISPR/Cas12a assay was established by adding heparin sodium, achieving the lowest consistently positive concentration of 1 × 102 copies/μL (1500 copies/reaction). Furthermore, for lateral flow assay (LFA) that requires exposure to the environment, the concentration of amplicons was reduced by restricting RPA amplification and employing an enhanced Cas12a protein while maintaining a lowest tested concentration of 1 × 101 copies/μL (150 copies/reaction). Specificity evaluation revealed no cross-reactivity with nontarget avian viruses, yielding a positive signal only for ALV-J. When applied to clinical samples, the one-tube RPA-CRISPR/Cas12a assay exhibited complete agreement with RT-qPCR in terms of positive detection rates. The multiple CRISPR/Cas12a-RPA assays developed in this study feature broad strain coverage in principle, high accuracy, and low contamination risk, providing practical tools for rapid ALV-J detection across diverse scenarios.},
}

Animals
*CRISPR-Cas Systems
Sensitivity and Specificity
*Avian Leukosis Virus/genetics/isolation & purification/classification
*Nucleic Acid Amplification Techniques/methods
Rapid Diagnostic Tests
*Poultry Diseases/virology/diagnosis
Recombinases
Chickens

@article {pmid42917132,
year = {2026},
author = {Singha, S and Yigit, MV and Chandrasekaran, AR},
title = {A CRISPR-Cas12a-Responsive DNA Tetrahedron.},
journal = {Biochemistry},
volume = {65},
number = {19},
pages = {2935-2939},
pmid = {42917132},
issn = {1520-4995},
support = {R35 GM150672/GM/NIGMS NIH HHS/United States ; R35 GM156250/GM/NIGMS NIH HHS/United States ; R35GM150672/GM/NIGMS NIH HHS/United States ; R35GM156250/GM/NIGMS NIH HHS/United States ; },
mesh = {DNA Nanostructures/chemistry ; *CRISPR-Cas Systems ; *Bacterial Proteins/metabolism/chemistry/genetics ; *DNA/chemistry/metabolism ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; *Endodeoxyribonucleases/metabolism/chemistry/genetics ; Listeria monocytogenes/genetics ; Campylobacter jejuni/genetics ; },
abstract = {Functionalizing DNA nanostructures for stimuli-responsive reconfiguration has been a key area of development in DNA nanotechnology. Here, we demonstrate programmable control over the structural integrity of a tetrahedral DNA nanostructure using an external DNA stimulus that is modulated through an intermediate CRISPR-Cas12a reaction. We designed toehold-containing DNA tetrahedra that can be disassembled by a DNA or RNA invading strand. Activation of the Cas12a enzyme by double-stranded DNA provides precise control over the disassembly of the DNA tetrahedra by digesting the DNA invading strand but not the RNA, enabling highly responsive and programmable DNA architectures. Further, targeted activation of Cas12a by conserved genomic regions of three bacterial pathogens, namely Listeria monocytogenes, Campylobacter jejuni, and Shiga toxin-producing Escherichia coli, provides biologically relevant responsiveness and demonstrates the platform's potential for future applications. Our results show that the DNA tetrahedra are observed in the presence of the genomic target but not observed in its absence due to disassembly, demonstrating a highly programmable and responsive molecular level architecture.},
}

DNA Nanostructures/chemistry
*CRISPR-Cas Systems
*Bacterial Proteins/metabolism/chemistry/genetics
*DNA/chemistry/metabolism
*CRISPR-Associated Proteins/metabolism/chemistry/genetics
*Endodeoxyribonucleases/metabolism/chemistry/genetics
Listeria monocytogenes/genetics
Campylobacter jejuni/genetics

@article {pmid42917134,
year = {2026},
author = {Lashkari, M and Connell, NE and Hanson, E and Adade, EE and Tharu, RJ and Yigit, MV},
title = {Programmable Genome Sensing with Visual, Fluorescence, and Glucometer Readings: Cell-Free In Vitro Translation Approach Using RNA Switch-CRISPR.},
journal = {Biochemistry},
volume = {65},
number = {19},
pages = {2972-2985},
pmid = {42917134},
issn = {1520-4995},
support = {R35 GM156250/GM/NIGMS NIH HHS/United States ; NA//University at Albany/ ; R35GM156250/GM/NIGMS NIH HHS/United States ; },
mesh = {Cell-Free System ; *CRISPR-Cas Systems ; Protein Biosynthesis ; Fluorescence ; *Genome, Bacterial ; beta-Galactosidase/genetics ; Colorimetry/methods ; Glucose/metabolism ; *Biosensing Techniques/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Rapid and accessible detection of pathogens through diverse detection modalities remains highly desirable. Most current pathogen detection assays rely on a single detection modality. Here, we have used the same RNA switch-CRISPR platform for detection of multiple pathogenic genomes through three different detection modalities. In this study, the RNA switch is repurposed as a reprogrammable platform for genome detection, enabling three orthogonal signal outputs: glucometer-based, fluorescence, and visual. Target pathogen genomes selectively regulate cell-free in vitro translation of β-galactosidase through the RNA switch, producing several hundred-fold increases in glucose following lactose hydrolysis while simultaneously generating fluorescence and colorimetric signals. The platform is readily reprogrammed to recognize conserved genomic regions from five distinct pathogens, with specificity dictated by CRISPR-Cas12a-mediated processing of a DNA regulator that controls RNA switch activation and initiates the downstream cell-free translation cascade. Glucometer-based, fluorescence, and paper-based colorimetric outputs each enabled selective detection of Salmonella typhi, Salmonella enterica, Listeria monocytogenes, Shiga toxin-producing Escherichia coli (STEC), and Campylobacter jejuni. Fluorescence and colorimetric responses were analyzed both qualitatively and quantitatively using image processing and showed strong agreement with glucometer measurements obtained using a handheld glucometer, providing a simple and accessible quantitative readout. The platform was further validated using the complete Listeria monocytogenes genome, achieving a detection sensitivity approaching a single genome copy per microliter. Together, this reprogrammable CRISPR-RNA switch platform enables the same detection setup to be programmed for multiple targets and readouts through three different detection modalities, some of which are well suited for resource-limited environments.},
}

Cell-Free System
*CRISPR-Cas Systems
Protein Biosynthesis
Fluorescence
*Genome, Bacterial
beta-Galactosidase/genetics
Colorimetry/methods
Glucose/metabolism
*Biosensing Techniques/methods
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42920424,
year = {2026},
author = {Sidhartha, E and Pratama, R and Ng, ER and Christiawan, IN and Valentine, Y and Hermantara, R and Artika, IM and Poetri, ON and Safithri, M},
title = {High-Quality Draft Genome Sequence of Endophytic Kluyvera Ascorbata Strain PCL31 Isolated from Piper Crocatum (red betel).},
journal = {F1000Research},
volume = {15},
number = {},
pages = {1073},
pmid = {42920424},
issn = {2046-1402},
mesh = {*Genome, Bacterial ; *Piper/microbiology ; *Endophytes/genetics ; Whole Genome Sequencing ; },
abstract = {Endophytic bacteria within medicinal plants are a largely untapped reservoir of bioactive compounds and adaptive genetic traits. Piper crocatum (red betel), a Southeast Asian medicinal herb rich in flavonoids, alkaloids, and phenolics, offers a chemically selective niche for endophytes, yet the genomes of its microbial associates remain poorly characterised. Kluyvera ascorbata occurs as a plant-associated endophyte and an opportunistic human pathogen, and its chromosomal blaKLUA gene is the recognised progenitor of the clinically important CTX-M-2 β-lactamase sublineage. We report a high-quality draft genome of endophytic strain PCL31, isolated from surface-sterilised Piper crocatum tissues collected in Bogor, Indonesia. Whole-genome sequencing used the Oxford Nanopore PromethION platform, with de novo assembly in Flye, polishing in Medaka, and quality assessment by QUAST and BUSCO. The assembly spans 4,978,299 bp across eight contigs (N50 1.45 Mbp; GC content 54.26%; 100% BUSCO completeness, enterobacterales_odb10, n = 440). Species identity was confirmed by digital DNA-DNA hybridisation (dDDH 88.6% against the type strain K. ascorbata ATCC 33433) and average nucleotide identity (ANI 98.6% against reference genome NZ_CP096201.1). Annotation identified 4,579 protein-coding sequences, 22 ribosomal RNA, 88 transfer RNA, and 81 non-coding RNA genes, and no CRISPR/Cas systems. antiSMASH 8.0 predicted four biosynthetic gene clusters (BGCs): a 53.8 kb non-ribosomal peptide synthetase (NRPS)-metallophore cluster matching the conserved enterobactin locus (MIBiG similarity 0.83), plus three low-similarity clusters (≤ 0.50) of uncertain novelty - a ribosomally synthesised and post-translationally modified peptide (RiPP)-like cluster, a terpene-precursor cluster, and an azole-containing RiPP. CARD-RGI profiling returned one Perfect hit (blaCTX-M-2) and 25 Strict hits, the latter dominated by intrinsic Enterobacteriaceae efflux and target-modification genes; phenotypic confirmation was not performed. This genome provides a resource for bioprospecting secondary metabolites from medicinal-plant endophytes and underscores the value of plant-associated isolates for biosafety and antimicrobial-resistance surveillance in opportunistic pathogens.},
}

*Genome, Bacterial
*Piper/microbiology
*Endophytes/genetics
Whole Genome Sequencing

@article {pmid42921428,
year = {2026},
author = {Liu, L and Bao, C and Wang, H and Li, F and Liu, X and He, Y and Chang, J and Yin, Y and Xu, S},
title = {Catalytic Hairpin Assembly-Aided CRISPR/Cas-SERS for Nucleic Acid Detection Enhanced by Multiple Catalytic Amplification.},
journal = {ACS measurement science au},
volume = {6},
number = {5},
pages = {1656-1664},
pmid = {42921428},
issn = {2694-250X},
abstract = {This study established a surface-enhanced Raman scattering (SERS) detection method based on the catalytic hairpin assembly (CHA) reaction combined with clustered regularly interspaced short palindromic repeats and its associated proteins (CRISPR/Cas), integrated with the guanine-quadruplexes/Hemin (G4/Hemin) catalytic amplification effect, which is available for identifying a typical indicator for genetically modified crops, the 35S promoter from the cauliflower mosaic virus (CaMV35S). Target CaMV35S-derived RNA (CaMV35S RNA) triggers the CHA reaction, yielding a double-stranded DNA product (C1/C2) that is recognized by CRISPR RNA (crRNA), thereby activating the trans-cleavage activity of Cas12a. A magnetic bead-based nanoprobe was fabricated by linking the hybridization chain reaction (HCR) product to the magnetic bead. This probe can fold into repeated G4 units under a specific potassium ion concentration to enable a high loading of Hemin, thereby providing strong peroxidase-like catalytic activity for 3,3',5,5'-tetra-methyl-benz-idine (TMB). In a positive trial, the 35S RNA initiates CHA, and the CHA product activates Cas12a, disrupting the HCR products on the magnetic bead probe, preventing the G4 structure formation, accordingly yielding a weak SERS signal from TMB. In contrast, a negative trial fails to activate Cas12a, and the probe retains its nanoenzyme activity, resulting in a strong SERS signal from the produced oxTMB. Owing to the collateral trans-cleavage activity of Cas12a and the high peroxidase-like nanozyme activity of G4/Hemin, this method achieves the highly sensitive SERS detection of genetically modified samples without target gene preamplification.},
}

@article {pmid42921675,
year = {2026},
author = {Ou, Y and Zhang, Z and Liu, G},
title = {Spatially Resolved Multiplex Detection of Cytokines in Parkinson's Disease Rat Brain via CRISPR/Cas Biosensing.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.6c02709},
pmid = {42921675},
issn = {2379-3694},
support = {2021CX02Y066//Guangdong Provincial Pearl River Talents Program/ ; 22174121//National Natural Science Foundation of China/ ; 22211530067//National Natural Science Foundation of China/ ; 62671539//National Natural Science Foundation of China/ ; T2250710180//National Natural Science Foundation of China/ ; },
abstract = {The ability to accurately quantify localized neuroinflammatory and neurodegenerative biomarkers in vivo is critical for enabling the early diagnosis of Parkinson's disease (PD). Here, we demonstrate a strategy for localized neuroinflammation quantification using an implantable glass-fiber-based CRISPR/Cas immunosensor. When interfaced with a portable fluorescence detector, our platform enables the spatially resolved multiplex detection of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and alpha-synuclein (α-syn) in the PD rat brain. The sensor surface was modified with functionalized liquid metal nanoparticles to enable antibody immobilization and long-term in vivo operation. This method demonstrated high specificity, excellent linearity in artificial cerebrospinal fluid (R2 > 0.91), sustained surface stability over 28 days, good biocompatibility with PC12 cells, and selective antibacterial activity against Staphylococcus aureus. Following cannula implantation into the substantia nigra pars compacta (SNc) of a 6-hydroxydopamine-induced PD rat model, the functionalized glass fiber enabled repeated, minimally invasive biomarker sampling at 6, 12, and 24 h postimplantation. Systemic inflammation induced by lipopolysaccharide (LPS) triggered marked elevations in TNF-α, IL-6, and α-syn, peaking at 6 h post-LPS. This implantable fiber-based biosensing method offers a promising reliable approach for early PD diagnosis, disease progression tracking, and investigation of neuroimmune interactions in neurodegenerative diseases in a point-of-care scenario.},
}

@article {pmid41644849,
year = {2026},
author = {Gu, D and Kim, GD and Park, M and Park, AD and Lee, HS and Eom, S and Weon, H and Park, J and Lee, J and Ahn, SH and Oh, H and Kim, J and Kim, SH and Seong, N and Hur, JK and Jang, ES and Chi, SW},
title = {Abasic CRISPR RNAs inherently harness fidelity of SpCas9 for genome editing.},
journal = {Nature chemical biology},
volume = {22},
number = {11},
pages = {1741-1752},
pmid = {41644849},
issn = {1552-4469},
support = {RS-2023-00221182//National Research Foundation of Korea (NRF)/ ; RS-2025-24683408//National Research Foundation of Korea (NRF)/ ; 2V09840-23-P024//Korea Institute of Science and Technology (KIST)/ ; },
mesh = {*Streptococcus pyogenes/genetics/enzymology ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Base Sequence ; },
abstract = {CRISPR-Cas9, an RNA-guided immune system, functions specifically in bacteria while controlling autoimmunity. However, its application to genome editing often causes deleterious off-target cleavages. Here, by sequencing CRISPR RNAs (crRNAs), we discovered abasic modifications that naturally suppress off-target self-cleavages from activated Cas9 in Streptococcus pyogenes (SpCas9). Bacteriophage infection induces oxidative stress, preferentially oxidizing the 5' end of crRNAs into abasic modifications. Mechanistically, abasic substitutions at the 5' end reduce off-target effects by limiting base pairing while preserving SpCas9-interacting backbones to maintain on-target efficiency. Abasic extensions at the 5' end reduce off-target effects by sterically constraining SpCas9 but retain on-target activity by avoiding extra base pairs. Moreover, these approaches can be combined (abasic substitution and extension), enhancing SpCas9 fidelity by increasing mismatch intolerance at the protospacer-adjacent motif-distal region and outperforming SpCas9 variants. Biologically inspired, we developed abasic chemical modifications for guide RNAs that improve CRISPR-Cas9 genome-editing specificity, demonstrating potential for in vivo application.},
}

*Streptococcus pyogenes/genetics/enzymology
*CRISPR-Cas Systems/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Base Sequence

@article {pmid41813882,
year = {2026},
author = {Hao, K and Liu, Y and Barrett, M and Samadi, Z and Zarezadeh, A and McGrath, Y and Zernicka-Goetz, M and Askary, A},
title = {Genetic recording and in situ readout of single-cell signaling memory.},
journal = {Nature chemical biology},
volume = {22},
number = {11},
pages = {1753-1764},
pmid = {41813882},
issn = {1552-4469},
support = {R00EY031782//U.S. Department of Health & Human Services | NIH | National Eye Institute (NEI)/ ; },
mesh = {Humans ; *Signal Transduction/genetics ; *Single-Cell Analysis/methods ; Bone Morphogenetic Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Wnt Signaling Pathway ; Cell Communication ; Wnt Proteins/metabolism/genetics ; },
abstract = {Intensity and duration of biological signals encode a few pathways to direct diverse cellular behaviors, yet quantifying these features in single cells remains difficult. To address this challenge, we developed INSCRIBE, which uses a CRISPR base editor to mutate genomic targets at rates proportional to signaling activity. Edits are recovered at the endpoint through a new ratiometric readout strategy from images of two fluorescence channels. We engineered human cells to record WNT and BMP activity. Following defined exogenous stimulations, INSCRIBE accurately recovered signal intensity in dose-response experiments and exposure duration in time-course experiments. Applying INSCRIBE revealed a persistent memory in the BMP pathway, where progeny of high-responding cells remained more sensitive to subsequent BMP stimulation for up to 3 weeks. Together, our results establish a scalable platform for genetic recording and in situ readout of signaling activity in single cells, advancing quantitative analysis of cell-cell communication during development and disease.},
}

Humans
*Signal Transduction/genetics
*Single-Cell Analysis/methods
Bone Morphogenetic Proteins/metabolism/genetics
CRISPR-Cas Systems
Wnt Signaling Pathway
Cell Communication
Wnt Proteins/metabolism/genetics

@article {pmid42549590,
year = {2026},
author = {Mojzesova, N and Hladikova, P and Kozovska, Z and Poturnajova, M and Tyciakova, S and Matuskova, M},
title = {CRISPR/Cas9-edited organoids as a platform for tailored research of colorectal cancer.},
journal = {Neoplasma},
volume = {73},
number = {5},
pages = {319-334},
doi = {10.4149/neo_2026_260529N175},
pmid = {42549590},
issn = {0028-2685},
mesh = {Humans ; *Colorectal Neoplasms/genetics/pathology/therapy ; *Organoids/pathology ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Animals ; },
abstract = {Colorectal cancer is one of the most commonly diagnosed cancers worldwide. Mortality rates and limited therapeutic options justify the development of reliable preclinical research models, as their translational value remains limited. Simple in vitro models do not recapitulate tumor heterogeneity, while in vivo research faces ethical concerns and interspecies differences. Patient-derived organoids offer a physiologically more relevant platform that retains the genetic, epigenetic, and phenotypic characteristics of the original tumor. Tumor-derived organoids enable precise investigation of novel treatments, functional genomics, and modeling of cancer development. Integration with CRISPR/Cas9 gene editing further enables accurate manipulation of specific genes to study carcinogenesis and therapeutic resistance. This review highlights recent advances in the use of organoids for colorectal cancer research and explores the potential of gene-edited organoids to discover the genetic and molecular mechanisms underlying colorectal cancer development, progression, and treatment resistance.},
}

Humans
*Colorectal Neoplasms/genetics/pathology/therapy
*Organoids/pathology
*CRISPR-Cas Systems
*Gene Editing/methods
Animals

@article {pmid42791629,
year = {2026},
author = {Maham, S and Sridharan, B and Park, JH and Lee, C and Park, YS and Kim, D and Sung, Y and Kwon, T and Kang, J and Lim, HG},
title = {Ultrasound-Mediated CRISPR Delivery and Sonogenetic Modulation for Precision Therapeutics.},
journal = {Advanced healthcare materials},
volume = {15},
number = {40},
pages = {},
doi = {10.1002/adhm.71753},
pmid = {42791629},
issn = {2192-2659},
support = {RS-2024-00338853//National Research Foundation of Korea/ ; 2022R1A5A8023404//National Research Foundation of Korea/ ; 202506850001//Pukyong National University/ ; },
mesh = {Humans ; Animals ; *Genetic Therapy/methods ; *CRISPR-Cas Systems/genetics ; *Precision Medicine/methods ; *Gene Editing/methods ; Microbubbles ; *Gene Transfer Techniques ; },
abstract = {The development of CRISPR-Cas9 technology has transformed gene editing with precise mutations in DNA targeted to treat genetic disorders yet various challenges such as immunogenicity, off-target editing, and lack of temporal control after delivery, making it difficult for therapeutic agents to reach deep tissues like brain, and cardiac tissues. Ultrasound has emerged as a promising solution by influencing sonogenetic modulation and delivery of CRISPR tools in deep tissues for targeted gene editing. Sonogenetic editing has revolutionized the technological advancement of CRISPR-editing and among multiple strategies, ultrasound-mediated delivery of CRISPR systems has enabled non-invasive and controlled genome editing through sonoporation, and upregulation of stimuli-responsive genes. Focused ultrasound combined with microbubbles has demonstrated CRISPR delivery to brain tissue with ∼80% higher efficiency than traditional strategies, allowing precise and advanced treatment of neurodegenerative diseases. In cancer applications, ultrasound has enabled complete tumor eradication via sonodynamic therapy, achieving 64% tumor regression via CAR T-cell therapy. Significant advances in gene therapy for cardiovascular diseases, obesity, and related diseases were also demonstrated recently. Collectively, the review elaborates potential of ultrasound-CRISPR technologies as a powerful tool that enables targeted, and image-guided genetic interventions, capable of advancing the outcomes of gene therapy as a widespread clinical practice.},
}

Humans
Animals
*Genetic Therapy/methods
*CRISPR-Cas Systems/genetics
*Precision Medicine/methods
*Gene Editing/methods
Microbubbles
*Gene Transfer Techniques

@article {pmid42817051,
year = {2026},
author = {Matsuo, T and Honda, Y and Chino, T and Nomoto, T and Osakabe, Y and Miura, Y and Nishiyama, N},
title = {Sequentially Self-Assembled Supramolecular Nanocomplexes Enable Systemic Cas9 RNP Delivery and In Vivo Tumor Genome Editing.},
journal = {ACS applied materials & interfaces},
volume = {18},
number = {40},
pages = {54618-54635},
doi = {10.1021/acsami.6c14858},
pmid = {42817051},
issn = {1944-8252},
support = {23ak0101173//Japan Agency for Medical Research and Development/ ; 26ama221258s0101//Japan Agency for Medical Research and Development/ ; JP22am0401018//Japan Agency for Medical Research and Development/ ; 21K20513//Japan Society for the Promotion of Science/ ; 22H00581//Japan Society for the Promotion of Science/ ; 22K18193,//Japan Society for the Promotion of Science/ ; 23H04060//Japan Society for the Promotion of Science/ ; 24K03278//Japan Society for the Promotion of Science/ ; 26K24035//Japan Society for the Promotion of Science/ ; 20243017//Ministry of Education, Culture, Sports, Science and Technology/ ; JPMJPF2202//Japan Science and Technology Agency/ ; },
mesh = {Animals ; *Gene Editing/methods ; Boronic Acids/chemistry ; Mice ; Humans ; Tannins/chemistry ; *Ribonucleoproteins/chemistry/genetics/metabolism ; Cell Line, Tumor ; CRISPR-Cas Systems ; Polo-Like Kinase 1 ; Protein Serine-Threonine Kinases/metabolism/genetics ; Cell Cycle Proteins/metabolism/genetics ; *CRISPR-Associated Protein 9/metabolism/genetics ; Proto-Oncogene Proteins/genetics/metabolism ; *Neoplasms/genetics/therapy/pathology ; },
abstract = {In vivo genome editing utilizing Cas9/sgRNA ribonucleoproteins (RNPs) holds substantial therapeutic promise, yet rapid bloodstream clearance and degradation of RNPs have hindered its accumulation within the target site and effective gene editing. Herein, we report a supramolecular ternary complex platform where RNPs are co-assembled with tannic acid (TA) and phenylboronic acid (PBA)-conjugated polymers through sequential self-assembly. This ∼30 nm core-shell structure protects RNPs from enzymatic degradation and dissociates selectively at endosomal pH. Upon intravenous administration in subcutaneous tumor-bearing mice, the ternary complex exhibits prolonged blood circulation and preferential tumor accumulation, achieving 37.2% gene editing at tumor sites compared with only 1.5% for free RNPs. The platform successfully disrupts PLK1 in subcutaneous tumors, accompanied by significant tumor growth suppression. This is further applied to a KRASG13D-mutant tumor model, in which treatment delayed tumor growth. By integrating sequential supramolecular self-assembly with stimuli-responsive cargo release, this strategy presents a promising approach for systemic RNP delivery and tumor genome editing.},
}

Animals
*Gene Editing/methods
Boronic Acids/chemistry
Mice
Humans
Tannins/chemistry
*Ribonucleoproteins/chemistry/genetics/metabolism
Cell Line, Tumor
CRISPR-Cas Systems
Polo-Like Kinase 1
Protein Serine-Threonine Kinases/metabolism/genetics
Cell Cycle Proteins/metabolism/genetics
*CRISPR-Associated Protein 9/metabolism/genetics
Proto-Oncogene Proteins/genetics/metabolism
*Neoplasms/genetics/therapy/pathology

@article {pmid42915618,
year = {2026},
author = {Bräuer, M and Walker, E and Schneider, S and Weiß, L and Geiger, A and Lindner, B and Hädicke, O and Schmidt, M and Fischer, S and Otte, K},
title = {Paving the Way for a Minimal CHO Chassis(?)Exploring the Upper Limits of Megabase-Scale Deletions through DNA Repair Modulation.},
journal = {ACS synthetic biology},
volume = {15},
number = {9},
pages = {3770-3783},
doi = {10.1021/acssynbio.6c00460},
pmid = {42915618},
issn = {2161-5063},
support = {13FH016KX2//Bundesministerium fur Forschung und Technologie/ ; },
mesh = {Animals ; CHO Cells ; Cricetulus ; *CRISPR-Cas Systems/genetics ; *DNA Repair/genetics ; DNA End-Joining Repair/genetics ; *Sequence Deletion/genetics ; *Gene Editing/methods ; Antibodies, Monoclonal/biosynthesis/genetics ; Cricetinae ; },
abstract = {Chinese hamster ovary (CHO) cells are the predominant mammalian host for therapeutic protein production, yet their large genome contains substantial non-essential DNA that may impose metabolic burden and contribute to process variability. Here, we establish a CRISPR/Cas9-based strategy for megabase-scale genomic deletions in CHO cells, targeting regions from 3 to over 100 megabases (Mb). Clones carrying deletions of up to 50 Mb were isolated, while a candidate deletion junction spanning 130 Mb was detected in the edited pool, demonstrating the potential for large-scale genome engineering in mammalian cells. Importantly, removal of genomic regions with minimal coding content did not impair growth, viability, or monoclonal antibody productivity. Mechanistic analysis revealed rapid deletion formation and a size-dependent shift in DNA repair pathway usage. Transient inhibition of non-homologous end joining (NHEJ) significantly improved deletion efficiency and clone recovery without compromising cell fitness. These findings establish large-scale genome reduction as a feasible and controllable strategy for rational CHO genome streamlining and the development of next-generation biopharmaceutical cell factories.},
}

Animals
CHO Cells
Cricetulus
*CRISPR-Cas Systems/genetics
*DNA Repair/genetics
DNA End-Joining Repair/genetics
*Sequence Deletion/genetics
*Gene Editing/methods
Antibodies, Monoclonal/biosynthesis/genetics
Cricetinae

@article {pmid42915619,
year = {2026},
author = {Zhu, W and Liao, J and Liu, Y and Zhang, H},
title = {A Novel, Simple, and Highly Efficient Dual-sgRNA CRISPR/Cas9-Based Editing System in the Filamentous Fungus Fusarium oxysporum.},
journal = {ACS synthetic biology},
volume = {15},
number = {9},
pages = {3676-3685},
doi = {10.1021/acssynbio.6c00299},
pmid = {42915619},
issn = {2161-5063},
support = {2022YFC2804203//National Key Research and Development Program of China Stem Cell and Translational Research/ ; },
mesh = {*Fusarium/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Plasmids/genetics/metabolism ; Promoter Regions, Genetic ; Fungal Proteins/genetics ; },
abstract = {Fusarium oxysporum, as one of the most common filamentous fungi, possesses great biosynthetic potential for natural products. However, the lack of efficient genetic tools has hindered functional genome mining and metabolic engineering in this fungus. In this study, a novel highly efficient CRISPR/Cas9-based dual-sgRNA expression editing system for F. oxysporum was successfully developed through construction of a robust plasmid platform pFRCas9-G418 using incorporation of an endogenous histone H2B nuclear localization signal and a 5S rRNA promoter-driven polycistronic tRNA-sgRNA cassette. This system is suitable not only for single-gene editing but also for large-fragment deletion and multiplex gene editing, although the editing efficiency is somewhat lower. First, this new CRISPR/Cas9 system exhibited a high efficiency of 93.75% ± 6.25% for deletion of the Fusarium cyclin C1 (fcc1) gene (∼1 kb), which was usually selected as the target gene responsible for yellow pigment accumulation. Then, knockout of the core NRPS gene sanB (∼19 kb) and knock-in of the strong promoter gpdA in the N-methylsansalvamide (SA) biosynthetic gene cluster (BGC) in strain F. oxysporum R1 using this system, respectively, led to no SA yield and an increase of 26.4% SA titer, confirming its capacity for large gene deletion and gene knock-in. Furthermore, one-step dual-gene knockout of hat1 (histone acetyltransferase gene, ∼1.5 kb) and pacC (pH-responsive transcription factor, ∼2 kb) was first achieved in Fusarium species. This versatile platform provides a powerful tool for editing gene(s) of various sizes in F. oxysporum.},
}

*Fusarium/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics
Plasmids/genetics/metabolism
Promoter Regions, Genetic
Fungal Proteins/genetics

@article {pmid42915621,
year = {2026},
author = {Wo, J and Yu, H and Yang, Y and Wu, H and Xiong, F and Kong, D},
title = {Reprogramming Enfumafungin Biosynthesis for High-Titer Production in the Endophytic Fungus Hormonema carpetanum ATCC 74360.},
journal = {ACS synthetic biology},
volume = {15},
number = {9},
pages = {3653-3663},
doi = {10.1021/acssynbio.6c00248},
pmid = {42915621},
issn = {2161-5063},
support = {32500050//National Natural Science Foundation of China/ ; 2023YFA0914200//National Key Research and Development Program of China/ ; },
mesh = {*Metabolic Engineering/methods ; CRISPR-Cas Systems/genetics ; Fermentation ; Promoter Regions, Genetic/genetics ; Mevalonic Acid/metabolism ; Antifungal Agents/metabolism ; *Ascomycota/metabolism/genetics ; },
abstract = {Enfumafungin is the essential precursor for the FDA-approved antifungal agent Ibrexafungerp, yet its production is hindered by low yields, restrictive solid-state fermentation, and the genetic intractability of its producer strain, Hormonema carpetanum ATCC 74360. Here, we developed a CRISPR/Cas9-mediated platform that halved the genetic iteration cycle to less than 15 days. By rewiring the biosynthetic cluster through promoter engineering, we successfully transitioned production to scalable liquid fermentation, achieving an initial 21.6 mg/L. Then, the mevalonate pathway genes (tHMGR, ERG9, and ERG1) were overexpressed, which successfully boosted the titer to 250.1 mg/L. Following medium optimization, the engineered strain HC07 reached a record-breaking 592.4 mg/L in 250 mL shake flasks, the highest yield achieved via metabolic engineering to date. This study provides a robust microbial platform and a streamlined genetic toolbox, paving the way for the sustainable biomanufacturing of next-generation triterpenoid antifungals.},
}

*Metabolic Engineering/methods
CRISPR-Cas Systems/genetics
Fermentation
Promoter Regions, Genetic/genetics
Mevalonic Acid/metabolism
Antifungal Agents/metabolism
*Ascomycota/metabolism/genetics

@article {pmid42915626,
year = {2026},
author = {Li, P and Wang, H and Fan, X and Zhang, F and Lv, J and Tan, H and Liang, L and Liu, R},
title = {AI-Guided Engineering of Tunable CcdB Variants for Flexible Selection in Escherichia coli.},
journal = {ACS synthetic biology},
volume = {15},
number = {9},
pages = {3702-3711},
doi = {10.1021/acssynbio.6c00344},
pmid = {42915626},
issn = {2161-5063},
support = {22278058//National Natural Science Foundation of China (NSFC)/ ; 22578048//National Natural Science Foundation of China (NSFC)/ ; 62503077//National Natural Science Foundation of China (NSFC)/ ; XLYC2203075//Xingliao Talent Plan Project/ ; SRICSPYF-ZY2025107//Scientific Research Innovation Capability Support Project for Young Faculty/ ; DUT24YG131//Fundamental Research Funds for the Central Universities/ ; DUT25LAB105//Fundamental Research Funds for the Central Universities/ ; 2023YFC3402300//National Basic Research Program of China (973 Program)/ ; 2024-MSBA-0914//Natural Science Foundation of Liaoning Province/ ; 2025-BS-0025//Natural Science Foundation of Liaoning Province/ ; 2025JH2/101330156//Natural Science Foundation of Liaoning Province/ ; },
mesh = {*Escherichia coli/genetics/metabolism ; *Protein Engineering/methods ; DNA Gyrase/metabolism/genetics ; *Escherichia coli Proteins/genetics/metabolism ; Plasmids/genetics/metabolism ; Mutation ; *Bacterial Proteins/genetics/metabolism ; Succinic Acid/metabolism ; CRISPR-Cas Systems/genetics ; },
abstract = {The strong cytotoxicity of wild-type CcdB limits its use as a counter-selection marker, as leaky expression can cause unintended cell death. Here, we report the rational engineering of a tunable CcdB variant with minimal basal toxicity while retaining inducible lethality. Using a structure-aware deep learning-guided strategy, we prioritized mutations predicted to alter CcdB-DNA gyrase binding energetics and experimentally identified variants with reduced basal toxicity and retained inducible killing. Targeted validation identified CcdB_L96P, which significantly reduces basal toxicity while preserving effective killing upon induction. This variant exhibits minimal leakage and robust inducible lethality across multiple Escherichia coli strains without requiring specialized hosts. It enables antibiotic-free CRISPR gRNA plasmid construction with high accuracy and efficient enrichment of correctly assembled clones. In a succinic acid-producing chassis, CcdB_L96P imposes no detectable metabolic burden and supports effective removal of residual cells using low concentrations of anhydrotetracycline. Together, this work provides a generalizable protein-level strategy for tuning toxin activity in synthetic biology.},
}

*Escherichia coli/genetics/metabolism
*Protein Engineering/methods
DNA Gyrase/metabolism/genetics
*Escherichia coli Proteins/genetics/metabolism
Plasmids/genetics/metabolism
Mutation
*Bacterial Proteins/genetics/metabolism
Succinic Acid/metabolism
CRISPR-Cas Systems/genetics

@article {pmid42915694,
year = {2026},
author = {Chai, H and Ye, W and You, H and Yan, R and Miao, P},
title = {Highly Sensitive Electrochemical Biosensing Strategy Based on Multiple CRISPR/Cas12a Trans-Cleavage Units.},
journal = {Analytical chemistry},
volume = {98},
number = {39},
pages = {28638-28645},
doi = {10.1021/acs.analchem.6c03570},
pmid = {42915694},
issn = {1520-6882},
support = {2024BYGT-003//Gansu Provincial Innovation Center for Functional Genomics and Molecular Diagnosis/ ; 32401205//National Natural Science Foundation of China (NSFC)/ ; SGY2022C03//Science Innovation Fund of the People?s Hospital of Suzhou New District/ ; SSD2024017//Basic Research Program of Suzhou/ ; },
mesh = {*Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; *MicroRNAs/analysis/genetics ; *CRISPR-Cas Systems/genetics ; Nucleic Acid Hybridization ; DNA Nanostructures ; Limit of Detection ; Electrodes ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; Humans ; DNA/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {This study presents the assembly of multiple CRISPR/Cas12a units by target-induced hybridization chain reaction (HCR), which is capable of highly sensitive electrochemical detection of target miRNA. The confinement effect of the HCR/Cas12a system is demonstrated, which shows significantly improved cleavage compared with the single Cas12a unit. The tetrahedral DNA nanostructure on the surface of the electrode supports the substrate for cleavage, which possesses the merits of excellent reactivity and accessibility. Because of the dual signal amplification, a low limit of detection down to 2.1 aM is achieved. This method also exhibits excellent specificity, storage stability, and accuracy, indicating its value for miRNA sensing in real biological samples and related clinical applications.},
}

*Biosensing Techniques/methods
*Electrochemical Techniques/methods
*MicroRNAs/analysis/genetics
*CRISPR-Cas Systems/genetics
Nucleic Acid Hybridization
DNA Nanostructures
Limit of Detection
Electrodes
*CRISPR-Associated Proteins/metabolism/genetics/chemistry
Humans
DNA/chemistry
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid42915703,
year = {2026},
author = {Zhao, W and Luan, X and Long, W and Li, Z and Dong, P and He, B and Gao, Y and Liu, H and Yin, Y and Song, Y},
title = {Cascade-Amplified Microfluidic Detection of Tumor Biomarkers via Metal-DNA Nanospheres Activating CRISPR-Cas12a Assay.},
journal = {Analytical chemistry},
volume = {98},
number = {39},
pages = {28932-28940},
doi = {10.1021/acs.analchem.6c03578},
pmid = {42915703},
issn = {1520-6882},
support = {5431ZZXM2304//State Key Laboratory of Analytical Chemistry for Life Sciences/ ; 2024300315//Fundamental Research Funds for the Central Universities/ ; BE2021373//Jiangsu Provincial Key Research and Development Program/ ; 2019YFA0709200//National Basic Research Program of China (973 Program)/ ; 22477056//National Natural Science Foundation of China (NSFC)/ ; 82572370//National Natural Science Foundation of China (NSFC)/ ; },
mesh = {*Biomarkers, Tumor/analysis ; Humans ; *CRISPR-Cas Systems/genetics ; *Nanospheres/chemistry ; *DNA/chemistry ; Biosensing Techniques ; Gold/chemistry ; Metal Nanoparticles/chemistry ; *Microfluidic Analytical Techniques ; },
abstract = {The development of ultrasensitive protein detection platforms is pivotal for early cancer diagnosis, yet existing methods are often constrained by inefficient "protein-to-signal" conversion and complex instrumentation. While CRISPR-Cas-based biosensors offer higher sensitivity for nucleic acids, their application in protein analysis is fundamentally limited by the low nucleic acid loading capacity and tedious functionalization of traditional inorganic signal carriers such as gold nanoparticles and metal-organic frameworks (MOFs). Herein, we report a highly efficient signal-transduction strategy utilizing Fe-coordinated DNA nanospheres (FDNSs)─a programmable, metal-nucleic acid assembly─as a high-capacity DNA reservoir for amplified biomarker sensing. Unlike conventional surface-modified inorganic nanoparticles, the solid-core FDNSs achieve an unprecedented DNA loading density and exhibit specific stimulus-responsive degradation. In this FACC (FDNS-activated CRISPR chip) platform, the recognition of tumor biomarkers triggers the rapid collapse of FDNSs, releasing a massive flux of barcode DNA that subsequently activates the CRISPR-Cas12a system within a microfluidic device. This dual-amplification architecture enables the ultrasensitive quantification of prostate-specific antigen (PSA) and carcinoembryonic antigen (CEA) with detection limits of 0.028 fg/mL and 0.0265 fg/mL, respectively. By replacing complex inorganic carriers with this programmable DNA assembly, our platform simplifies the sensing workflow and significantly enhances sensitivity, providing a robust and portable solution for cancer screening in resource-limited settings.},
}

*Biomarkers, Tumor/analysis
Humans
*CRISPR-Cas Systems/genetics
*Nanospheres/chemistry
*DNA/chemistry
Biosensing Techniques
Gold/chemistry
Metal Nanoparticles/chemistry
*Microfluidic Analytical Techniques

@article {pmid42915869,
year = {2026},
author = {Li, F and Han, R and Yao, R and Zhou, B and Jiang, X and Zhu, H and Shi, Y and Fan, L and Wu, D and Li, G},
title = {DNA Fragment Fusion-Triggered Label-Free Detection of Listeria monocytogenes Subtypes via Fusion RAA and Hybrid Split G-Quadruplex.},
journal = {Journal of agricultural and food chemistry},
volume = {74},
number = {38},
pages = {30407-30417},
doi = {10.1021/acs.jafc.6c09842},
pmid = {42915869},
issn = {1520-5118},
support = {32302199//National Natural Science Foundation of China/ ; 2024M761896//China Postdoctoral Science Foundation/ ; 20242BAB20333//Natural Science Foundation of Jiangxi Province/ ; 20252BAC240614//Natural Science Foundation of Jiangxi Province/ ; 2025BSHSDZZ336//Shaanxi Province Postdoctoral Science Foundation/ ; },
mesh = {*Listeria monocytogenes/genetics/isolation & purification/classification ; G-Quadruplexes ; *DNA, Bacterial/genetics ; *Nucleic Acid Amplification Techniques/methods ; Recombinases/genetics ; Biosensing Techniques/methods ; CRISPR-Cas Systems ; },
abstract = {Bacterial subtype detection using multiplex amplification with multiple distinguishable labels is complex and costly, whereas recombinant DNA technology via fragment fusion provides a simpler, cheaper alternative. Here, we propose a label-free method for Listeria monocytogenes subtype detection based on a hybrid split G-quadruplex (G4) and fusion recombinase-aided amplification (RAA)-cascaded CRISPR/Cas13a (HGFRC) platform. Fusion RAA co-amplified species-specific and clonal complex (CC)-specific targets into 535 bp fusion products, which activated T7 transcription and Cas13a trans-cleavage activity. Activated Cas13a cleaved the RNA linker, disrupting G4 assembly and reducing fluorescence to generate a signal-off response. This design eliminated labeled probes, enabling single-signal detection. The assay achieved a sensitivity of 1.0 × 101 CFU/mL within 45 min, with good specificity and sample suitability. Overall, this study provides a label-free, sensitive, and specific method for detecting L. monocytogenes CC5, offering new insights for bacterial subtype biosensing.},
}

*Listeria monocytogenes/genetics/isolation & purification/classification
G-Quadruplexes
*DNA, Bacterial/genetics
*Nucleic Acid Amplification Techniques/methods
Recombinases/genetics
Biosensing Techniques/methods
CRISPR-Cas Systems

@article {pmid42915910,
year = {2026},
author = {Zhang, X and Qiu, M and Huang, J and Zhang, Y and Tian, Y and Zhang, W and Jiang, Y and Zhang, X},
title = {Machine Learning-Assisted Multiplexed and Ultrasensitive Biosensing of Pathogenic Bacteria based on a TtAgo-Powered Autocatalytic Nucleic Acid Circuit and Encoded SERS Tags.},
journal = {Analytical chemistry},
volume = {98},
number = {37},
pages = {27495-27509},
doi = {10.1021/acs.analchem.6c03958},
pmid = {42915910},
issn = {1520-6882},
support = {YQ2025C013//Natural Science Foundation of Heilongjiang Province/ ; },
mesh = {*Biosensing Techniques/methods ; *Spectrum Analysis, Raman/methods ; Gold/chemistry ; *Machine Learning ; Metal Nanoparticles/chemistry ; *Salmonella typhimurium/isolation & purification ; Silver/chemistry ; Limit of Detection ; *Staphylococcus aureus/isolation & purification ; },
abstract = {Programmable nucleases have obtained widespread application in the field of pathogen detection, yet they face considerable challenges in multiplex detection. As a sister technology to CRISPR/Cas systems, Argonaute proteins (Ago) possess programmability, sequence specificity, and multi-turnover capability, rendering them promising candidates for next-generation multiplex detection technologies. Herein, a novel machine learning-assisted amplification-free and multiple SERS biosensing platform based on an Ago-driven autocatalytic nucleic acid circuit (SAANC) was developed for the multiplexed detection of pathogenic bacteria. The SAANC platform utilized the cross-catalytic activity of TtAgo to construct an autocatalytic nucleic acid circuit, enabling continuous reactions without pre-amplification and achieving autocatalytic signal amplification. Meanwhile, by precisely modulating the density of reporter DNA on the surface of silver@Raman reporter@gold nanoparticles (Ag@Ra@Au NPs), both cleavage efficiency and Raman response were significantly enhanced. This integrated approach significantly enhanced reaction efficiency, achieving highly sensitive and specific multiplex detection of pathogenic bacteria. The SAANC method exhibited linear detection ranges of 102-108 CFU/mL for Salmonella typhimurium and 103-108 CFU/mL for Staphylococcus aureus, respectively. Moreover, satisfactory recoveries (89%-110%) in food sample detection were obtained. Furthermore, machine learning-assisted quantitative analysis was introduced to effectively address the challenge of overlapping SERS signals from dual targets, substantially improving the robustness of simultaneous multi-target quantification. This platform provides a feasible strategy for developing high-performance Ago-based multiplex pathogen detection systems and offers an innovative solution for efficient multiplex detection of pathogenic bacteria.},
}

*Biosensing Techniques/methods
*Spectrum Analysis, Raman/methods
Gold/chemistry
*Machine Learning
Metal Nanoparticles/chemistry
*Salmonella typhimurium/isolation & purification
Silver/chemistry
Limit of Detection
*Staphylococcus aureus/isolation & purification

@article {pmid42915930,
year = {2026},
author = {Liao, X and Chen, J and Wan, Y and Xu, W and Zhou, J and Liu, Y and Li, X and Yang, C and Wang, J},
title = {An XNA-CRISPR Platform for PAM-Free Detection of the katG Ser315Thr Mutation Associated with Isoniazid Resistance in Tuberculosis.},
journal = {Analytical chemistry},
volume = {98},
number = {37},
pages = {27354-27361},
doi = {10.1021/acs.analchem.6c02379},
pmid = {42915930},
issn = {1520-6882},
support = {JCYJ20220818102014028//Science and Technology Foundation of Shenzhen City/ ; JCYJ20230807111120043//Science and Technology Foundation of Shenzhen City/ ; 2020B1212060077//Guangdong Provincial Key Laboratory of Sensor Technology and Biomedical Instrument/ ; 22174167//National Natural Science Foundation of China/ ; LQ23H160002//Natural Science Foundation of Zhejiang Province/ ; 2023YFC2307305//National Key Research and Development Program of China/ ; 2024A1515011281//Basic and Applied Basic Research Foundation of Guangdong Province/ ; 2024A1515011959//Basic and Applied Basic Research Foundation of Guangdong Province/ ; 2026A1515012722//Basic and Applied Basic Research Foundation of Guangdong Province/ ; B2303003//Shenzhen Medical Research Fund/ ; ILH24001//China Resources Research Institute of Science & Technology/ ; LZZLX23C002//Yangtze Delta Region Institute of Tsinghua University, Zhejiang/ ; },
mesh = {*Mycobacterium tuberculosis/genetics/drug effects ; *Isoniazid/pharmacology ; *Bacterial Proteins/genetics ; Mutation ; *Drug Resistance, Bacterial/genetics ; *Catalase/genetics ; Humans ; *Antitubercular Agents/pharmacology ; *CRISPR-Cas Systems/genetics ; },
abstract = {Drug-resistance Tuberculosis (TB) is a major threat to global public health. Simple and precise detection of the mutations associated with drug resistance is still a significant challenge. CRISPR-based nucleic acid detection has emerged as a transformative technology in molecular diagnostics due to its high specificity and programmability. However, the CRISPR-Cas12a system requires a protospacer adjacent motif (PAM) in the target double-stranded DNA (dsDNA) for efficient recognition and activation, which limits its application for detecting single-nucleotide variants (SNVs). To overcome this limitation, we leveraged high-affinity xenonucleic acids (XNAs) to engineer an XNA-CRISPR platform that combines a helicase and sequence-specific XNAs (such as peptide nucleic acid (PNA), locked nucleic acid (LNA), or 2'-modified RNA), to unwind the dsDNA, enabling them to invade and form stable hybrids, thereby displacing a single strand for PAM-independent Cas12a activation. This recognition mechanism, combining XNA hybridization and CRISPR verification, achieves single-base resolution. To bridge the sensitivity gap for clinical application, we integrated this platform with polydisperse droplet digital technology (PddXNA-CRISPR), which significantly enhanced the sensitivity. Clinical sputum samples were detected by using PddXNA-CRISPR for Mycobacterium tuberculosis (MTB) katG Ser315Thr mutation, which showed 90% concordance with sequencing results. This modular, amplification-free platform expands the CRISPR-based diagnostic toolkit and provides a versatile and sensitive approach for detecting critical SNVs.},
}

*Mycobacterium tuberculosis/genetics/drug effects
*Isoniazid/pharmacology
*Bacterial Proteins/genetics
Mutation
*Drug Resistance, Bacterial/genetics
*Catalase/genetics
Humans
*Antitubercular Agents/pharmacology
*CRISPR-Cas Systems/genetics

@article {pmid42916138,
year = {2026},
author = {Lamothe, G and Veillette, F and Iwe, IA and Corsano, A and Shrestha, S and Singh, S and Bouchard, C and Godbout, K and Lu, Y and Rousseau, J and Pardee, K and Tremblay, JP},
title = {Split Cas12a Protospacer Engineering Enables Ultraspecific Recognition of a PAM-Less DNA Strand via an External PAM-Proximal Duplex.},
journal = {Analytical chemistry},
volume = {98},
number = {40},
pages = {29285-29298},
pmid = {42916138},
issn = {1520-6882},
support = {NA//Sant?/ ; 950-233107//Canada Research Chairs/ ; NA//China Scholarship Council/ ; 202403PJT-520192-BE2-CEAA-129834//Canadian Institutes of Health Research (CIHR)/ ; GA1-177695//Canadian Institutes of Health Research (CIHR)/ ; },
mesh = {*DNA/metabolism/chemistry/genetics ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; Fluorescence Resonance Energy Transfer ; *Endodeoxyribonucleases/metabolism/genetics/chemistry ; *Bacterial Proteins/metabolism/genetics/chemistry ; Base Sequence ; CRISPR-Cas Systems ; },
abstract = {CRISPR-Cas12a is a programmable RNA-guided endonuclease whose activation by double-stranded DNA normally depends on the recognition of an adjacent protospacer-adjacent motif (PAM), which constrains target selection in diagnostic applications. Here, we divide the DNA activator into PAM-proximal (Pp) and PAM-distal (Pd) duplexes, spatially separating the PAM requirement from the interrogation of a PAM-less target sequence. FRET, electrophoretic mobility shift, fluorescence polarization, and functional assays indicate that either fragment can associate with Cas12a, but that robust productive activation requires the PAM-containing Pp duplex together with the Pd duplex. Our data further support a model in which PAM-mediated protein-DNA contacts stabilize an assembly-competent complex and facilitate binding of the PAM-less Pd target capture. Weakening hybridization between the guide and Pp with a chimeric DNA/RNA increased the dependence on PAM-mediated stabilization and improved structural selectivity in the Pp region and single-nucleotide specificity in the Pd region. The resulting split-duplex architecture provides an ultraspecific platform for resolving single-nucleotide polymorphisms in the PAM-less DNA strand.},
}

*DNA/metabolism/chemistry/genetics
*CRISPR-Associated Proteins/metabolism/genetics/chemistry
Fluorescence Resonance Energy Transfer
*Endodeoxyribonucleases/metabolism/genetics/chemistry
*Bacterial Proteins/metabolism/genetics/chemistry
Base Sequence
CRISPR-Cas Systems

@article {pmid42916361,
year = {2026},
author = {Li, X and Li, Y and Sun, S and Cao, J and Li, L and Wu, D and Wu, Y and Sun, F},
title = {Multiplexed Detection of Foodborne Pathogens and the Antibiotic Resistance Genes Based on the CRISPR/Cas System with Engineered LwaCas13a.},
journal = {Analytical chemistry},
volume = {98},
number = {38},
pages = {28519-28529},
doi = {10.1021/acs.analchem.6c04448},
pmid = {42916361},
issn = {1520-6882},
support = {MR/Y015223/1//UK Research and Innovation/ ; 22406001//National Natural Science Foundation of China/ ; 2408085MC067//Natural Science Foundation of Anhui Province/ ; 2024AH050461//University Natural Science Research Project of Anhui Province/ ; 2024YFE0106300//National Key Research and Development Program of China/ ; PA2025GDSK0067//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Food Microbiology ; Listeria monocytogenes/genetics/isolation & purification ; Escherichia coli/genetics/isolation & purification ; Staphylococcus aureus/genetics/isolation & purification ; *Drug Resistance, Bacterial/genetics ; Rapid Diagnostic Tests ; Limit of Detection ; Animals ; Anti-Bacterial Agents/pharmacology ; Salmonella typhimurium/genetics/isolation & purification ; },
abstract = {Foodborne pathogens represent a persistent threat to global public health, necessitating the development of point-of-care testing (POCT) platforms that integrate rapidity, ultrahigh sensitivity, cost effectiveness, and operational simplicity. Herein, we developed a system for high-specificity identification of foodborne pathogens and associated antibiotic resistance genes, which is a dual-modal diagnostic platform integrating recombinase polymerase amplification (RPA) with an engineered LwaCas13a variant. By leveraging the CRISPR/Cas13a system, the platform establishes a multiplex fluorescence array that simultaneously detects four major foodborne pathogens (Salmonella enterica serovar typhimurium, Listeria monocytogenes, Escherichia coli, and Staphylococcus aureus) and their corresponding critical antibiotic resistance genes (ARGs), including mecA, blaCTX-M, mcr-1, ermB, and tet(M). Notably, the system retained robust trans-cleavage activity in complex biological matrices, achieving a remarkable limit of detection (LOD) at 100 copies/mL. Furthermore, the CRISPR/Cas13a system is coupled to an instrument-free lateral flow assay (LFA) for the detection of L. monocytogenes and tet(M), which effectively circumvents matrix-induced viscosity and ionic inhibition in egg and milk samples. Moreover, our developed platform was validated using samples from 40 household refrigerators, which drastically reduces assay time and thereby circumvents the inhibitory effects that constrain qPCR in food matrices and achieves 100% diagnostic agreement compared with the gold-standard methods. Collectively, this dual-modal strategy provides a promising paradigm with point-of-care capability for food safety surveillance and antimicrobial resistance monitoring.},
}

*CRISPR-Cas Systems/genetics
*Food Microbiology
Listeria monocytogenes/genetics/isolation & purification
Escherichia coli/genetics/isolation & purification
Staphylococcus aureus/genetics/isolation & purification
*Drug Resistance, Bacterial/genetics
Rapid Diagnostic Tests
Limit of Detection
Animals
Anti-Bacterial Agents/pharmacology
Salmonella typhimurium/genetics/isolation & purification

@article {pmid42916405,
year = {2026},
author = {Pan, Y and Li, W and Cooper, J and Yang, Z},
title = {RPA-CRISPR/Cas12a and Aptamer-Based Electrochemical Biosensing Duplex Platform for On-Site Monitoring of Antimicrobial Resistance in Water.},
journal = {Analytical chemistry},
volume = {98},
number = {38},
pages = {28259-28271},
doi = {10.1021/acs.analchem.6c03568},
pmid = {42916405},
issn = {1520-6882},
support = {RL-2022-041//Leverhulme Trust/ ; FF\1920\1\36//Royal Academy of Engineering/ ; NE/R013349/2//Natural Environment Research Council (NERC)/ ; },
mesh = {*Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems/genetics ; Gold/chemistry ; *Anti-Bacterial Agents/analysis/pharmacology ; *Aptamers, Nucleotide/chemistry ; Metal Nanoparticles/chemistry ; Electrodes ; Wastewater/microbiology ; Drinking Water ; },
abstract = {Monitoring antimicrobial resistance (AMR) in aquatic systems is essential for tracking resistance mechanisms and identifying microbial sources. Here, we present a dual-module electrochemical sensing platform that integrates a gold-electrode-based RPA-CRISPR/Cas12a biosensor for detecting antibiotic resistance genes (ARGs) with a paper-based electrochemical aptasensor for antibiotic quantification. The system employs thiol-modified CRISPR/Cas12a probes alongside amino-functionalized graphene/thionine/gold nanoparticles (NH2-G/THI/AuNPs) nanocomposites, enabling highly sensitive and specific detection (Limits of detection: 1 copy μL-1 for ARGs and 1 nM for antibiotics). Validation was performed in drinking water, wastewater, and river water samples, demonstrating a rapid sample-to-result time of ∼60 min. Coupled with a portable electrochemical workstation, the platform allows users to introduce samples and acquire electrochemical signals with a smartphone application, giving quantitative outputs displayed through a custom web interface. Field validation showed high stability and recovery rates (95-110%) for antibiotic detection. We believe that this platform addresses critical gaps in aquatic AMR monitoring by providing a rapid, affordable, and field-deployable solution, with the potential for offering on-site surveillance in resource-limited settings.},
}

*Biosensing Techniques/methods
*Electrochemical Techniques/methods
*CRISPR-Cas Systems/genetics
Gold/chemistry
*Anti-Bacterial Agents/analysis/pharmacology
*Aptamers, Nucleotide/chemistry
Metal Nanoparticles/chemistry
Electrodes
Wastewater/microbiology
Drinking Water

@article {pmid42916579,
year = {2026},
author = {Saavedra, MA and Grassi, S and Jespersen, MG and Rocha, C and Kandasamy, V and Nielsen, LK and Nikel, PI and Donati, S},
title = {Automated Mini-Bioreactors Reveal the Temporal Dynamics and Multi-Omics Responses of CRISPRi Knockdowns in Pseudomonas putida.},
journal = {ACS synthetic biology},
volume = {15},
number = {10},
pages = {4023-4038},
doi = {10.1021/acssynbio.6c00205},
pmid = {42916579},
issn = {2161-5063},
support = {NNF20CC0035580//Novo Nordisk Fonden/ ; NNF24SA0100980//Novo Nordisk Fonden/ ; },
mesh = {*Pseudomonas putida/genetics/metabolism ; *Bioreactors ; Multiomics ; *CRISPR-Cas Systems/genetics ; Gene Knockdown Techniques/methods ; Bacterial Proteins/genetics/metabolism ; Gene Silencing ; Arginine/biosynthesis ; Metabolomics ; },
abstract = {Characterizing CRISPR interference (CRISPRi) phenotypes presents a fundamental temporal challenge: pre-existing overabundance of target proteins can mask early silencing, requiring extended growth for dilution, yet prolonged repression rapidly selects for escaper mutants. To resolve this, we integrated a tightly regulated CRISPRi system in Pseudomonas putida with an automated mini bioreactor platform operating in turbidostat mode. By maintaining continuous exponential growth, we mapped the exact temporal dynamics of essential gene silencing. We identified a critical observation window between 7.8 and 26.7 h (corresponding to 6.4-23.5 cumulative cell doublings of the uninduced condition) where repression exerts its maximum physiological impact, directly preceding population takeover by target-site mutated escapers. Applying this workflow to the arginine biosynthesis pathway, multi-omics profiling disentangled transient physiological buffering from long-term mutational events, revealing that argH and argG knockdowns trigger highly diverse metabolomic perturbations. This scalable framework overcomes batch culture limitations for CRISPRi experiments, ensuring precise temporal control for accurate phenotypic characterization and reliable functional genomics.},
}

*Pseudomonas putida/genetics/metabolism
*Bioreactors
Multiomics
*CRISPR-Cas Systems/genetics
Gene Knockdown Techniques/methods
Bacterial Proteins/genetics/metabolism
Gene Silencing
Arginine/biosynthesis
Metabolomics

@article {pmid42916610,
year = {2026},
author = {Chen, Y and Dou, K and Lv, L and Wang, M and Niu, G},
title = {A Blue Light-Activated Sensor System Enables Dynamic and Reversible Control of Gene Expression in Streptomyces.},
journal = {ACS synthetic biology},
volume = {15},
number = {10},
pages = {4345-4359},
doi = {10.1021/acssynbio.6c00563},
pmid = {42916610},
issn = {2161-5063},
support = {2023YFD1700700//National Basic Research Program of China (973 Program)/ ; 32370077//National Natural Science Foundation of China (NSFC)/ ; },
mesh = {Blue Light ; *Streptomyces/genetics/metabolism/radiation effects ; *Gene Expression Regulation, Bacterial/radiation effects/genetics ; CRISPR-Cas Systems/genetics ; Indoles/metabolism ; Benzoisochromanequinones ; Anthraquinones/metabolism ; Bacterial Proteins/genetics/metabolism ; Green Light ; Transcriptional Activation/radiation effects ; Light ; },
abstract = {Programmable control of bacterial gene expression is critical for advancing applications in synthetic biotechnology. This is particularly true for the genus Streptomyces, a well-recognized prolific producer of diverse natural products. Although several inducible regulatory systems have been developed for Streptomyces, there remains an escalating demand to expand the repertoire of high-performance induction tools. Herein, we present Strep-LitRS, a light-sensing system for Streptomyces that exhibits pronounced activation under blue and green light, with only weak induction under yellow light. Moreover, blue light was more effective than green light in triggering transcriptional activation. This system enables tunable, reversible, and spatially controlled gene expression upon blue-light illumination. It demonstrates effective programmable control of morphological differentiation via CRISPR/dCas9-mediated interference. Its utility is further validated through the blue light-inducible production of actinorhodin and violacein in Streptomyces chassis strains. Furthermore, by coupling Strep-LitRS with the Cre-loxP recombination system, we achieve precise and conditional gene activation in response to blue light. A synthetic genetic circuit also allows light-induced control to be gated by rhamnose, enabling multi-input regulatory logic. Collectively, this work establishes a versatile light-sensing module and highlights its broad applicability for the programmable manipulation of diverse target genes across Streptomyces species.},
}

Blue Light
*Streptomyces/genetics/metabolism/radiation effects
*Gene Expression Regulation, Bacterial/radiation effects/genetics
CRISPR-Cas Systems/genetics
Indoles/metabolism
Benzoisochromanequinones
Anthraquinones/metabolism
Bacterial Proteins/genetics/metabolism
Green Light
Transcriptional Activation/radiation effects
Light

@article {pmid42329082,
year = {2026},
author = {Xu, H and Liu, H and Lu, T and Chen, Y and Li, Y and Zhu, Y and Wei, L and Fan, B and Zhang, Y and Zhang, C and Fang, R and Xie, S and Shen, B},
title = {Genome-wide CRISPR screen identifies ELFN2 as a key regulator of host autophagy and lipid metabolism important for Toxoplasma gondii proliferation.},
journal = {Autophagy},
volume = {22},
number = {11},
pages = {3317-3330},
doi = {10.1080/15548627.2026.2693781},
pmid = {42329082},
issn = {1554-8635},
mesh = {Animals ; *Toxoplasma/growth & development/physiology ; *Autophagy/genetics ; *Lipid Metabolism/genetics ; Cell Line ; Mice ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Swine ; Cell Proliferation ; CRISPR-Cas Systems/genetics ; Leucine-Rich Repeat Proteins ; },
abstract = {Toxoplasma gondii is a globally prevalent foodborne zoonotic pathogen that threatens animal production and human health. Consumption of undercooked meat like pork and lamb is a major route of human infection. In this study, we performed a genome-wide CRISPR knockout screen in the porcine cell line PK15 to identify host factors that are critical for T. gondii replication. The results showed that disrupting the ELFN2 (extracellular leucine rich repeat and fibronectin type III domain containing 2) gene in PK15 did not affect host cell growth, but significantly reduced the proliferation of Toxoplasma parasites. Loss of ELFN2 decreased macroautophagy/autophagy in PK15 cells and impaired lipid metabolism, resulting in reduced lipid availability for the parasites and consequent suppression of T. gondii proliferation. Exogenous lipid supplementation or pharmacological activation of autophagy could fully restore the replication of parasites in ΔELFN2 cells. The requirement of host ELFN2 for optimal parasite proliferation in vivo was validated by constructing elfn2[-/-] mice, which showed increased resistance to T. gondii infection and reduced parasite burden, highlighting the value of ELFN2 in breeding Toxoplasma-resistant animals. Notably, naturally occurring loss-of-function mutations in ELFN2 could be found in certain pig breeds, further indicating the feasibility of breeding T. gondii-resistant animals like pigs, to reduce the transmission of Toxoplasma.Abbreviations: 3-MA: 3-methyladenine; ATG5: autophagy related 5; ATG7: autophagy related 7; BODIPY-C12: BODIPY FL C12; CCK-8: Cell Counting Kit-8; ComN2: complemented with an ectopic copy ofELFN2; ELFN2: extracellular leucine rich repeat and fibronectin type III domain containing 2;elfn2[-/-]:elfn2homozygous knockout; FASII: type II synthesis pathway; GFP: green fluorescent protein; KO: knockout; LD: lipid droplets; MG: monoacylglycerol; MOI: multiplicity of infection; MTORC1: mechanistic target of rapamycin kinase complex 1; PV: parasitophorous vacuole; PVM: parasitophorous vacuole membrane; T. gondii: Toxoplasma gondii; WT: wild type.},
}

Animals
*Toxoplasma/growth & development/physiology
*Autophagy/genetics
*Lipid Metabolism/genetics
Cell Line
Mice
Humans
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Swine
Cell Proliferation
CRISPR-Cas Systems/genetics
Leucine-Rich Repeat Proteins

@article {pmid42806622,
year = {2026},
author = {Tharu, RJ and Imtiaz, Y and Singha, S and Hanson, E and Gold, RE and Royzen, P and Yigit, MV},
title = {Split Activator Assembly via Aptamer Binding Enables CRISPR-Cas12a Activation With Non-Nucleic Acid Targets.},
journal = {Small methods},
volume = {10},
number = {20},
pages = {e71075},
pmid = {42806622},
issn = {2366-9608},
support = {R35 GM156250/GM/NIGMS NIH HHS/United States ; R35GM156250//National Institute of General Medical Sciences of the National Institutes of Health/ ; },
mesh = {*Aptamers, Nucleotide/chemistry/metabolism ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism ; Humans ; Adenosine/analysis ; *Endodeoxyribonucleases/metabolism ; Prostate-Specific Antigen ; *Bacterial Proteins/metabolism/genetics ; Biosensing Techniques ; DNA Nanostructures ; DNA/chemistry ; },
abstract = {The programmability of CRISPR-Cas12a has enabled transformative advances in nucleic acid detection; however, extending its activation to non-nucleic-acid targets remains highly desirable. Here, we report a universal split-activator strategy that enables programmable Cas12a activation in response to a small molecule and a protein. In this approach, a canonical activator is divided into two fragments (p1 and p2), such that Cas12a activation occurs only upon their simultaneous assembly. We integrate this mechanism with aptamer-mediated conformation switching, wherein target binding destabilizes an aptamer-p2 duplex, releasing p2 to complete the activator complex and trigger Cas12a nuclease activity. Using this design, we demonstrate selective recognition of the small-molecule metabolite adenosine and the protein biomarker prostate-specific antigen (PSA), with high specificity against structurally related controls. Signal generation is achieved using fluorescent DNA nanoclusters (DFN-1), which undergo fluorescence decrease upon Cas12a-mediated degradation. The system exhibits concentration-dependent responses for both targets. Together, this work establishes a modular framework for coupling aptamer-based molecular recognition to programmable Cas12a activation, expanding the functional scope of CRISPR systems beyond nucleic acids and enabling new opportunities in CRISPR-Cas12a-mediated detection.},
}

*Aptamers, Nucleotide/chemistry/metabolism
*CRISPR-Cas Systems
*CRISPR-Associated Proteins/metabolism
Humans
Adenosine/analysis
*Endodeoxyribonucleases/metabolism
Prostate-Specific Antigen
*Bacterial Proteins/metabolism/genetics
Biosensing Techniques
DNA Nanostructures
DNA/chemistry

@article {pmid42914777,
year = {2026},
author = {Moreno, MM and Zacarias, J and García, I and Garcia, MA and Valdés, EG and Hernandez-Garcia, A},
title = {Leveraging high temperature for enhanced and faster detection of SARS-CoV-2 and its Omicron variant with CRISPR-AsCas12a.},
journal = {Journal of pharmaceutical and biomedical analysis},
volume = {284},
number = {},
pages = {117767},
doi = {10.1016/j.jpba.2026.117767},
pmid = {42914777},
issn = {1873-264X},
abstract = {CRISPR-based methods are effective for detecting SARS-CoV-2, with performance comparable to gold-standard PCR. Developing simpler, faster, and more sensitive CRISPR-based diagnostics will accelerate their adoption in field. Adjusting the temperature at which Cas proteins operate is crucial for enhancing DNA recognition kinetics, thereby improving specificity, sensitivity, and practicality. However, optimization is challenging due to the narrow temperature range for activity of wild-type Cas orthologs. Here, we show that heating the wild-type AsCas12a protein above its optimal temperature, up to 65ºC, accelerates nucleic acid detection, reducing the limit of detection (LoD) by 33-fold without causing false positives. This effect was further enhanced by buffer optimization. We applied these conditions to develop a two-step fluorescence- and lateral flow strip-based method for detecting SARS-CoV-2 in as little as 5 min from LAMP amplicons, and demonstrated its applicability for discriminating the Omicron variant from the wild-type strain using plasmid DNA. Overall, these findings demonstrate that increasing reaction temperature is a simple, effective strategy to enhance CRISPR-Cas12a diagnostics for pathogens, without the need for protein engineering or thermophilic Cas12a orthologs.},
}

@article {pmid42915270,
year = {2026},
author = {Shen, H and Pan, X and Wu, Y and Chen, B and Zhou, X and Qiao, L and Effah, CY and Zhou, J and Yu, J and Zhang, Z and Yang, X and Wu, Y and Ding, L},
title = {CHAI-sEV: A Programmable Hairpin-Driven Cas9 Platform with AI Integration for Dual-Protein Profiling and Intelligent Classification of Tumor-Derived Small Extracellular Vesicles.},
journal = {Analytical chemistry},
volume = {98},
number = {36},
pages = {27244-27257},
doi = {10.1021/acs.analchem.6c05200},
pmid = {42915270},
issn = {1520-6882},
support = {62574188//National Natural Science Foundation of China/ ; 82273685//National Natural Science Foundation of China/ ; 82304194//National Natural Science Foundation of China/ ; 2023M743196//China Postdoctoral Science Foundation/ ; 2023TQ0304//China Postdoctoral Science Foundation/ ; 242102311174//Science and Technology Department of Henan Province/ ; },
mesh = {Humans ; *Extracellular Vesicles/metabolism/chemistry/classification ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/metabolism/genetics ; Aptamers, Nucleotide/chemistry ; },
abstract = {Multiplexed protein profiling of tumor-derived small extracellular vesicles (TsEVs) requires signal-conversion strategies that are sensitive, low-background, and orthogonal. Collateral-cleavage CRISPR assays based on Cas12a or Cas13a provide efficient amplification but can compromise single-pot multitarget detection, whereas the sequence-specific cleavage of Cas9 remains underexplored for converting vesicle-surface protein recognition into orthogonal amplified outputs. Here, we report CHAI-sEV, a programmable Cas9-mediated trigger-release strategy based on PAM-bearing hairpin-locking probes (PHLPs) for dual-protein profiling of TsEVs. Each PHLP integrates Cas9 recognition and signal conversion into a single molecular substrate, in which sgRNA-guided Cas9 cleavage unlocks a sequestered CHA activator to initiate orthogonal signal amplification. After EGFR- or PD-L1-binding aptamers label TsEVs, engineered aptamer tails recruit specific Cas9-sgRNA complexes to the vesicle surface, enabling protein-specific PHLP activation and amplified fluorescence readout. This Cas9-programmed trigger-release mechanism enables dual-channel TsEV protein detection without relying on nonspecific collateral cleavage. Under optimized conditions, CHAI-sEV achieved detection limits of 162 and 328 particles/μL for EGFR and PD-L1. In plasma samples, CHAI-sEV differentiated lung cancer patients from benign-lung-disease patients and healthy controls and showed decreased EGFR and PD-L1 signals in post-treatment samples. As proof-of-concept extensions, the CHAI-sEV was further coupled to an IGZO-FET electrical readout and an exploratory machine-learning analysis combining dual-protein signals with clinical features. Overall, PHLPs provide a substrate-design principle that converts Cas9 cleavage into amplification-compatible and orthogonally multiplexable biosensing outputs, enabling sensitive and multiplexed protein profiling of TsEVs and offering potential for extension to diverse molecular targets.},
}

Humans
*Extracellular Vesicles/metabolism/chemistry/classification
*CRISPR-Cas Systems
*CRISPR-Associated Protein 9/metabolism/genetics
Aptamers, Nucleotide/chemistry

@article {pmid42915288,
year = {2026},
author = {Min, YH and Lee, DG and Yoo, JH and Shin, YB and Byun, JY},
title = {RNase H-Assisted, Split-crRNA-Based CRISPR/Cas12a Lateral Flow Assay for Amplification-Free, Multi-Site Detection of Bacterial 16S rRNA.},
journal = {Analytical chemistry},
volume = {98},
number = {36},
pages = {27195-27202},
doi = {10.1021/acs.analchem.6c04681},
pmid = {42915288},
issn = {1520-6882},
support = {CRC22021-400//National Research Council of Science & Technology (NST)/ ; KGM1032612//Biotechnology (KRIBB) Research Initiative Program/ ; RS-2024-00439931//National Research Foundation of Korea (NRF)/ ; RS-2026-25481370//National Research Foundation of Korea (NRF)/ ; },
mesh = {*RNA, Ribosomal, 16S/analysis/genetics ; *Ribonuclease H/metabolism/chemistry ; *CRISPR-Cas Systems/genetics ; *RNA, Bacterial/analysis/genetics ; Animals ; Limit of Detection ; *Bacteria/genetics ; },
abstract = {With the growing demand for on-site testing of foodborne pathogens, rapid and sensitive detection methods are urgently needed. Here, we developed a split-crRNA-based CRISPR/Cas12a assay integrated with a lateral flow assay (sCR-LFA) for rapid, sensitive detection of bacterial 16S rRNA within 1 h, without pre-amplification. To detect highly structured 16S rRNA, we used RNA fragmentation via DNA-helper-mediated RNase H pretreatment. When short RNA targets interact with corresponding RNA blockers, unblocked RNA spacers hybridize with a DNA activator to form spacer/activator duplexes, which activate split-crRNA/Cas12a complexes. The sCR-LFA achieved a detection limit of 104 CFU/mL for single-site targeting. Using an enhanced sCR-LFA that integrates a multi-site targeting strategy, we achieved a lower detection limit of 102 CFU/mL. Furthermore, the enhanced sCR-LFA was successfully validated in simulated milk and mushroom samples spiked with bacteria. Collectively, this work presents a promising approach for effective food safety monitoring and prevention of infectious diseases.},
}

*RNA, Ribosomal, 16S/analysis/genetics
*Ribonuclease H/metabolism/chemistry
*CRISPR-Cas Systems/genetics
*RNA, Bacterial/analysis/genetics
Animals
Limit of Detection
*Bacteria/genetics

@article {pmid42915300,
year = {2026},
author = {Wu, SL and Shi, JY and Tan, Y and Jiang, HX and Hou, XY and Liu, SQ and Li, KD and Sun, WB and Li, MY and Zhu, LN and Cai, QL and Kong, DM},
title = {Cell-Surface-to-Solution Signal Transduction via a CRISPR/Cas12a-Based Dual-Target Aptamer Sensor for Detecting Urothelial Carcinoma Cells in Urine.},
journal = {Analytical chemistry},
volume = {98},
number = {36},
pages = {26905-26913},
doi = {10.1021/acs.analchem.6c03925},
pmid = {42915300},
issn = {1520-6882},
support = {22293030//National Natural Science Foundation of China/ ; 22474063//National Natural Science Foundation of China/ ; 72474154//National Natural Science Foundation of China/ ; 82570985//National Natural Science Foundation of China/ ; 25JCLZJC00250//Tianjin Municipal Science and Technology Program/ ; 25JCLZJC00640//Tianjin Municipal Science and Technology Program/ ; 23JCJQJC00080//Science Fund for Distinguished Young Scholars of Tianjin Municipality/ ; },
mesh = {Humans ; *Aptamers, Nucleotide/chemistry/metabolism ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Signal Transduction ; Cell Line, Tumor ; *Endodeoxyribonucleases/metabolism/genetics ; Limit of Detection ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Urothelial carcinoma (UC) is a common malignancy of the genitourinary system with high recurrence rates, requiring non-invasive postoperative monitoring. Direct detection of exfoliated UC cells in urine can provide tumor-origin evidence but remains challenging because of low target-cell abundance, complex urine interference, and subjective cytological interpretation. Herein, we developed a CRISPR/Cas12a-based dual-target aptamer sensor for sensitive and specific detection of exfoliated UC cells in urine. The sensing system spatially couples CKAP4-mediated magnetic capture with PD-L1-dependent retention of a Cas12a activator on the same captured cell. Magnetic washing removes unbound activator probes, whereas the cell-retained activator subsequently initiates Cas12a trans-cleavage in the solution-phase reaction. This capture-retain-activate architecture provides an AND-gate-like route for converting spatially coupled cell-surface recognition events into an amplified fluorescence output. The sensor achieved a detection limit of 12 cells per assay and showed good selectivity against interfering cells. A pilot clinical evaluation using 32 urine samples distinguished UC patients from non-UC individuals with an area under the receiver operating characteristic curve (AUC) of 0.996. This work provides a capture-retain-activate strategy for the non-invasive analysis of intact exfoliated UC cells and illustrates a potential design route for coupling cell-surface recognition with solution-phase CRISPR/Cas12a signal amplification.},
}

Humans
*Aptamers, Nucleotide/chemistry/metabolism
*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
Signal Transduction
Cell Line, Tumor
*Endodeoxyribonucleases/metabolism/genetics
Limit of Detection
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid42915359,
year = {2026},
author = {Gold, R and Imtiaz, Y and Morya, V and Halvorsen, K and Hanson, E and Yigit, MV},
title = {Invasive RNA Programs Optical Switching in DNA Nanoclusters for CRISPR Sensing.},
journal = {Nano letters},
volume = {26},
number = {36},
pages = {11988-11995},
pmid = {42915359},
issn = {1530-6992},
support = {R35 GM156250/GM/NIGMS NIH HHS/United States ; R35 GM124720/GM/NIGMS NIH HHS/United States ; 2022-08596//National Institute of Food and Agriculture/ ; R35GM156250/GM/NIGMS NIH HHS/United States ; NA//Albert Mossin Physical and Life Sciences Endowment Scholarship/ ; },
mesh = {*Biosensing Techniques/methods ; Nucleic Acid Hybridization ; *Listeria monocytogenes/genetics/pathogenicity ; *CRISPR-Cas Systems ; Silver/chemistry ; *Metal Nanoparticles/chemistry ; DNA Nanostructures/chemistry ; *RNA/chemistry ; *DNA/chemistry ; CRISPR-Associated Proteins/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Endodeoxyribonucleases/chemistry ; Fluorescence ; Bacterial Proteins ; },
abstract = {DNA-templated silver nanoclusters have regained attention due to recent advances in stability and tunable optical properties. Here, we report a highly regulated DNA-templated silver nanocluster (DFN2) that exhibits strong fluorescence at 561 nm and enables reversible, label-free fluorescence switching governed by nucleic acid hybridization. The fluorescence of DFN2 is efficiently quenched with complementary DNA or RNA and fully restored through invading RNA strand mediated displacement, enabling robust and programmable ON-OFF-ON optical control. We integrated this hybridization-responsive platform with CRISPR-Cas12a to construct a target-activated biosensing system. Upon recognition of a conserved genomic fragment from a foodborne pathogen Listeria monocytogenes, the activated Cas12a cleaves a regulator DNA, initiating a cascade of hybridization and strand-displacement reactions that restore nanocluster fluorescence through an invasive RNA. This strategy exhibits high specificity against conserved genomic regions from other foodborne pathogens. These findings establish DFN2 as programmable, enzyme-responsive optical reporters for rapid, sensitive, and modular point-of-care biosensing.},
}

*Biosensing Techniques/methods
Nucleic Acid Hybridization
*Listeria monocytogenes/genetics/pathogenicity
*CRISPR-Cas Systems
Silver/chemistry
*Metal Nanoparticles/chemistry
DNA Nanostructures/chemistry
*RNA/chemistry
*DNA/chemistry
CRISPR-Associated Proteins/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
Endodeoxyribonucleases/chemistry
Fluorescence
Bacterial Proteins

@article {pmid42915614,
year = {2026},
author = {She, L and Liang, Z and Zou, Q and Huo, YX},
title = {Engineering DNA-Targeting CRISPR and CRISPR-Like Effectors: Advances from Rational Design and High-Throughput Screening to AI-Driven Development.},
journal = {ACS synthetic biology},
volume = {15},
number = {9},
pages = {3518-3542},
doi = {10.1021/acssynbio.6c00386},
pmid = {42915614},
issn = {2161-5063},
support = {32400442//National Natural Science Foundation of China/ ; 2024YFC3407000//National Key Research and Development Program of China/ ; C2023105024//Natural Science Foundation of Hebei Province/ ; ZR2024MC001//Natural Science Foundation of Shandong Province/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Artificial Intelligence ; High-Throughput Screening Assays/methods ; *DNA/genetics ; Protein Engineering/methods ; Synthetic Biology/methods ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute adaptive immune systems in prokaryotes and have transformed life sciences, precision medicine, and synthetic biology as programmable genome-editing tools. Despite their broad utility, naturally occurring DNA-targeting Cas effectors remain constrained by several intrinsic limitations, including large protein size that complicates delivery, stringent protospacer adjacent motif (PAM) requirements that restrict targetable genomic space, and mismatch tolerance that can lead to off-target activity and potential genotoxicity. These challenges have made Cas protein engineering and the discovery of novel CRISPR and CRISPR-like systems from metagenomic resources central to the development of next-generation genome-editing platforms. This Review places recent advances within an integrated synthetic biology engineering continuum that links natural effector discovery, structure-guided hypothesis generation, high-throughput functional screening, machine learning-enabled model construction, and iterative redesign. This Review summarizes progress in the screening, optimization, and functional engineering of DNA-targeting CRISPR and CRISPR-like effectors, with emphasis on structure-guided rational design, directed evolution coupled with high-throughput screening, bioinformatics- and evolution-guided mining of novel systems from large-scale sequence databases, and artificial intelligence-assisted development. By integrating these strategies, we highlight how CRISPR effector engineering is moving toward design-build-test-learn (DBTL)-inspired workflows that expand the functional landscape of genome-editing technologies and advance genome editing toward improved efficiency, safety, and programmability.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Artificial Intelligence
High-Throughput Screening Assays/methods
*DNA/genetics
Protein Engineering/methods
Synthetic Biology/methods

@article {pmid42915617,
year = {2026},
author = {Wen, L and Jing, M and Li, Y and Shang, X and Liao, X},
title = {DeepCRISPR-Typer: Accurate CRISPR-Cas Identification and Subtyping in Metagenomes via Integrated Deep Learning.},
journal = {ACS synthetic biology},
volume = {15},
number = {9},
pages = {3738-3749},
doi = {10.1021/acssynbio.6c00398},
pmid = {42915617},
issn = {2161-5063},
support = {D5000230363//The Fundamental Research Funds for the Central Universities/ ; D5000250279//The Fundamental Research Funds for the Central Universities/ ; 62002388//National Natural Science Foundation of China (NSFC)/ ; 62433016//National Natural Science Foundation of China (NSFC)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Deep Learning ; *Metagenome/genetics ; Gene Editing/methods ; *Metagenomics/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Software ; },
abstract = {Accurate identification and classification of CRISPR-Cas systems are crucial for understanding microbial immune mechanisms and developing novel genome-editing tools. However, traditional homology-based mining methods face severe computational bottlenecks and assembly fragmentation challenges when processing massive metagenomic data. Here, we present DeepCRISPR-Typer, a comprehensive computational framework integrating a large protein language model (TEMC-Cas), a deep sequence feature extractor (CRISPR-RepTyper), and an adaptive targeted HMM profiling strategy. DeepCRISPR-Typer integrates array and Cas evidence and significantly reduces computational overhead by dynamically invoking subtype-specific HMM subsets. In metagenomic dataset evaluations, DeepCRISPR-Typer achieved a classification accuracy of 94.26% and demonstrated a significant acceleration of approximately 1 orders of magnitude compared to existing mainstream tools. This research provides a robust and scalable engine for metagenome-scale CRISPR system discovery, significantly expanding the mining toolbox for genome engineering applications.},
}

*CRISPR-Cas Systems/genetics
*Deep Learning
*Metagenome/genetics
Gene Editing/methods
*Metagenomics/methods
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Software

@article {pmid42869113,
year = {2027},
author = {Batır, MB},
title = {Single-gRNA CRISPR/eCas9-mediated simultaneous targeting of cIAP1 and cIAP2 in colorectal cancer cells: RNA-seq reveals distinct transcriptomic and apoptotic responses.},
journal = {Gene},
volume = {1016},
number = {},
pages = {150445},
doi = {10.1016/j.gene.2026.150445},
pmid = {42869113},
issn = {1879-0038},
mesh = {Humans ; *Baculoviral IAP Repeat-Containing 3 Protein/genetics/metabolism ; *Apoptosis/genetics ; *Colorectal Neoplasms/genetics/pathology ; *Inhibitor of Apoptosis Proteins/genetics ; *CRISPR-Cas Systems ; HCT116 Cells ; *Ubiquitin-Protein Ligases/genetics ; Transcriptome ; Gene Expression Regulation, Neoplastic ; RNA-Seq ; },
abstract = {Cellular inhibitor of apoptosis proteins 1 and 2 (cIAP1 and cIAP2), encoded by BIRC2 and BIRC3, respectively, are important regulators of cellular survival and apoptosis. These proteins contribute to cancer cell survival by suppressing apoptotic signaling. While individual knockouts of BIRC2 or BIRC3 in cancer cells have been investigated, the consequences of simultaneously targeting both paralogs remain poorly understood. Thus, a single-gRNA CRISPR/eCas9 strategy was developed in which one gRNA targeting a shared sequence in BIRC2 and BIRC3 was used to simultaneously disrupt both genes in human colorectal cancer HCT-116 cells, and the resulting cellular and transcriptomic effects were compared with those of the individual knockouts. Individual and dual-targeting CRISPR/eCas9 constructs were experimentally validated by immunofluorescence analysis, RT-PCR, and RT-qPCR, while fluorescence microscopy and T7EI analysis were used to evaluate single-cell-derived clones. Apoptosis was evaluated using flow cytometry, and RNA sequencing was performed for differentially expressed gene (DEG) analysis. The single-gRNA dual-targeting strategy resulted in concurrent loss of cIAP1 and cIAP2 expression and produced a stronger reduction in cell survival and induction of apoptosis than targeting either gene individually. In addition, the expression levels of TNFRSF25, TRAF2, TNFSF15, and MIR663AHG were significantly upregulated following simultaneous BIRC2/BIRC3 targeting. RNA-seq analysis provided detailed insight into the molecular alterations associated with concurrent cIAP1 and cIAP2 depletion. Overall, these findings demonstrate the technical feasibility of targeting two highly homologous genes with a single gRNA and reveal that simultaneous disruption of cIAP1 and cIAP2 produces biological effects that are more pronounced than those observed following individual targeting.},
}

Humans
*Baculoviral IAP Repeat-Containing 3 Protein/genetics/metabolism
*Apoptosis/genetics
*Colorectal Neoplasms/genetics/pathology
*Inhibitor of Apoptosis Proteins/genetics
*CRISPR-Cas Systems
HCT116 Cells
*Ubiquitin-Protein Ligases/genetics
Transcriptome
Gene Expression Regulation, Neoplastic
RNA-Seq

@article {pmid42911498,
year = {2027},
author = {Chao, L and Li, Z and Li, Z and Cheng, X and Fei, T and Li, W},
title = {Cas13d-Mediated Knockdown of Long Noncoding RNAs.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3091},
number = {},
pages = {277-289},
pmid = {42911498},
issn = {1940-6029},
mesh = {*RNA, Long Noncoding/genetics ; *Gene Knockdown Techniques/methods ; *CRISPR-Cas Systems ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; Lentivirus/genetics ; RNA Interference ; Genetic Vectors/genetics ; HEK293 Cells ; },
abstract = {CRISPR-Cas13 systems represent a powerful and programmable platform for RNA-targeted applications, including RNA knockdown. Long noncoding RNA (lncRNA), which lacks an open reading frame and is thereby often refractory to Cas9-mediated indel production, can be successfully knocked down by Cas13 systems. DeepCas13, a deep learning-based model, was developed to predict Cas13d on-target activity for RNA interference with high accuracy. Here, we describe detailed protocols about sgRNA design and experimental procedures for the lentiviral vector-based CRISPR-Cas13 system to knock down individual lncRNAs. Our protocol can be naturally extended to knockdown other types of coding and noncoding RNAs, including messenger RNA (mRNA) circular RNA (circRNA).},
}

*RNA, Long Noncoding/genetics
*Gene Knockdown Techniques/methods
*CRISPR-Cas Systems
Humans
RNA, Guide, CRISPR-Cas Systems/genetics
Lentivirus/genetics
RNA Interference
Genetic Vectors/genetics
HEK293 Cells

@article {pmid42913359,
year = {2026},
author = {Shamlan, A and Khan, MU and Farid, A and Huo, S and Liu, H and Chen, Z},
title = {From detection to prediction: AI-integrated molecular and biosensing approaches for psychrotrophic and thermophilic spoilage bacteria in dairy systems.},
journal = {International journal of food microbiology},
volume = {463},
number = {},
pages = {112130},
doi = {10.1016/j.ijfoodmicro.2026.112130},
pmid = {42913359},
issn = {1879-3460},
abstract = {Psychrotrophic Gram-negative bacteria and thermophilic or thermoduric spore-forming bacteria are major causes of quality deterioration and shelf-life loss in milk and dairy products. Their impact is mediated primarily by heat-stable peptidases and lipases, heat-resistant spores and biofilms that persist in processing environments; with the notable exception of toxigenic members of the Bacillus cereus group, most of these organisms are spoilage rather than safety hazards. Culture-based methods remain the reference standard but are slow and provide single time-point information. This review critically evaluates molecular and sensing methods, including quantitative and digital PCR, isothermal amplification, CRISPR-Cas assays, biosensors, viability assays, spectroscopy and (meta)omics, with explicit attention to whether performance has been demonstrated in naturally contaminated dairy matrices rather than in buffer or spiked samples, and examines how their outputs could inform predictive microbiology and machine learning. A classification framework based on growth-temperature range, heat resistance, sporulation and dairy relevance is provided, and each detection-to-prediction pathway is graded as demonstrated in dairy, partly demonstrated or proposed. Validated dairy applications currently exist mainly for individual detection assays and for mechanistic or stochastic shelf-life models driven by culture-derived data; prospective, validated workflows in which molecular or biosensor outputs are used as model inputs to forecast spoilage have not yet been reported. Because heat-stable enzymes can spoil ultra-high-temperature products after the producing cells have been inactivated, viable counts alone are insufficient predictors of such defects. Key barriers include matrix effects, small and imbalanced datasets, domain shift, limited external validation and poorly defined intended use. Detection-to-prediction integration should therefore be regarded as an emerging research direction rather than an established industrial capability.},
}

@article {pmid40106146,
year = {2027},
author = {Carman, LE and Samulevich, ML and Aneskievich, BJ},
title = {Protocol Development for CRISPR/Cas9 Knockout of the Anti-inflammatory Protein TNIP1 in HaCaT Keratinocytes.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3110},
number = {},
pages = {1-20},
pmid = {40106146},
issn = {1940-6029},
mesh = {Humans ; *CRISPR-Cas Systems ; *Keratinocytes/metabolism/cytology ; *Gene Knockout Techniques/methods ; *DNA-Binding Proteins/genetics/metabolism ; HaCaT Cells ; Gene Editing/methods ; Cell Line ; Transfection ; },
abstract = {Gene editing in cultured cells, including the intent of sequence disruption to achieve a functional knockout of the targeted gene, has been greatly facilitated with the advent of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) technology. Primary cell strains and immortalized cell lines from diverse tissue types have been successfully targeted both for basic research examining the effects of loss of the correlating protein and for modeling select loss-of-function disorders. Such accomplishments have extended to cutaneous cells, especially epidermal keratinocytes given their key structural and functional role in barrier formation and surveillance of and response to surface events such as triggering and processing inflammatory responses. Here we describe disruption of the Tumor Necrosis factor-induced protein 3-Interacting Protein 1 (TNIP1) gene in human HaCaT keratinocytes to generate an ongoing loss of expression as a parallel system to transient knockdown we had previously achieved with siRNA transfection. The TNIP1 protein restricts cytoplasmic progression of inflammatory signals. We cover our CRISPR/Cas9 vector choice, enrichment of transfected cells via positive selection for puromycin resistance, their subsequent cloning, and gene disruption and expression analysis. We also emphasize prior keratinocyte-CRISPR/Cas9 literature as a springboard for other investigators and to illustrate the widespread relevance of such editing to the diverse, yet highly consequentially different, genes expressed in keratinocytes.},
}

Humans
*CRISPR-Cas Systems
*Keratinocytes/metabolism/cytology
*Gene Knockout Techniques/methods
*DNA-Binding Proteins/genetics/metabolism
HaCaT Cells
Gene Editing/methods
Cell Line
Transfection

@article {pmid42668338,
year = {2027},
author = {Coon, A and Ayers, JL and Cole, C and Tiwaa, A and Parihar, A and Vincent, T and Gudjonsson, JE and Sarkar, MK and Simpson, CL},
title = {CRISPR/Cas9 Editing of Human Keratinocytes for Organotypic Modeling of Genetic Epidermal Disorders.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3110},
number = {},
pages = {21-40},
pmid = {42668338},
issn = {1940-6029},
mesh = {Humans ; *Keratinocytes/metabolism/cytology ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Animals ; *Epidermis/metabolism/pathology ; Mice ; Gene Knockout Techniques ; Cell Line ; *Skin Diseases, Genetic/genetics/pathology ; },
abstract = {CRISPR/Cas9-based DNA editing has revolutionized biomedical research by enabling investigators to precisely and permanently inactivate genes in human cells to provide greater insight into the mechanisms that support normal physiology and drive disease pathology. To leverage this technology for cutaneous biology research, we optimized a strategy for efficient gene editing in human keratinocytes, which can be grown into a differentiated organotypic epidermis to extend studies of gene function to a mature 3D tissue model. Here, we describe step-by-step methods for deploying CRISPR/Cas9 to engineer a TERT-immortalized human epidermal keratinocyte line (N/TERT-2G) to generate monoclonal heterozygous and homozygous knockout cell lines. Additionally, we provide a detailed protocol for growing gene-edited keratinocytes into organotypic epidermis, which we have used to model the pathology of monogenic dermatologic disorders that were not replicated in traditional knockout mice.},
}

Humans
*Keratinocytes/metabolism/cytology
*CRISPR-Cas Systems
*Gene Editing/methods
Animals
*Epidermis/metabolism/pathology
Mice
Gene Knockout Techniques
Cell Line
*Skin Diseases, Genetic/genetics/pathology

@article {pmid42680083,
year = {2027},
author = {Han, R and Xiao, N and Wang, J and Wu, Z and Wang, X and Tang, X},
title = {Identification of MDV and REV using RPA-CRISPR/Cas12a combined with fluorescence and lateral flow assays.},
journal = {Analytical biochemistry},
volume = {720},
number = {},
pages = {116240},
doi = {10.1016/j.ab.2026.116240},
pmid = {42680083},
issn = {1096-0309},
mesh = {*CRISPR-Cas Systems/genetics ; Animals ; *Nucleic Acid Amplification Techniques/methods ; *Reticuloendotheliosis virus/genetics/isolation & purification ; Fluorescence ; Rapid Diagnostic Tests ; Chickens ; Recombinases/metabolism ; *Herpesvirus 2, Gallid/genetics/isolation & purification ; Limit of Detection ; },
abstract = {Marek's disease virus (MDV) and reticuloendotheliosis virus (REV) are two major immunosuppressive oncogenic viruses that severely threaten the global poultry industry. Field co-infection of these two pathogens is prevalent, accompanied by highly similar pathological lesions, making conventional diagnostic technologies incapable of rapid on-site differential identification. Recombinase polymerase amplification (RPA) coupled with the CRISPR/Cas12a system represents an innovative platform for rapid nucleic acid detection. In this study, target-specific crRNAs were designed against the conserved unique genes of MDV and REV, respectively, to establish a CRISPR-based diagnostic system capable of differentiating the two viruses for the first time. Comprehensive optimization of RPA reaction composition, buffer formulation and incubation temperature improved the overall detection performance. Three independent visual signal readout modalities were supported by this assay, including real-time fluorescence quantification, naked-eye fluorescence visualization under blue light, and lateral flow assay (LFA). Sensitivity evaluation demonstrated that the limit of detection (LOD) reached 1 × 10° copies/μL for MDV and 1 × 10[1] copies/μL for REV. Specificity assays verified that the system only generated specific positive signals in response to target viruses without cross-reactivity against other common avian oncogenic viruses and their subgroups. Blind testing of 30 clinical chick fecal specimens revealed that the assay achieved a 100% positive detection rate for both viruses when referenced to qPCR and RT-qPCR, with outstanding diagnostic efficacy and perfect detection consistency. Collectively, the developed RPA-CRISPR/Cas12a assay overcomes the limitations of traditional diagnostic methods and holds great promise for on-site routine screening in poultry farms and epidemiological surveillance of MDV and REV.},
}

*CRISPR-Cas Systems/genetics
Animals
*Nucleic Acid Amplification Techniques/methods
*Reticuloendotheliosis virus/genetics/isolation & purification
Fluorescence
Rapid Diagnostic Tests
Chickens
Recombinases/metabolism
*Herpesvirus 2, Gallid/genetics/isolation & purification
Limit of Detection

@article {pmid42697518,
year = {2026},
author = {Parida, L and Swain, RK and Sahoo, PK},
title = {Molecular characterization and functional analysis of septin 8a in rohu (Labeo rohita): insights into immune regulation and developmental roles revealed by CRISPR/Cas9-mediated F0 mutagenesis.},
journal = {Fish & shellfish immunology},
volume = {179},
number = {},
pages = {111706},
doi = {10.1016/j.fsi.2026.111706},
pmid = {42697518},
issn = {1095-9947},
mesh = {Animals ; *Septins/genetics/immunology/chemistry ; *Fish Diseases/immunology ; *Immunity, Innate/genetics ; Fish Proteins/genetics/immunology/chemistry ; Aeromonas hydrophila/physiology ; Gram-Negative Bacterial Infections/immunology/veterinary ; Amino Acid Sequence ; Sequence Alignment/veterinary ; *Carps/immunology/genetics ; CRISPR-Cas Systems ; *Gene Expression Regulation/immunology ; Phylogeny ; Gene Expression Profiling/veterinary ; Poly I-C/pharmacology ; Base Sequence ; },
abstract = {Septins are conserved GTP-binding cytoskeletal proteins that regulate cytokinesis, membrane dynamics, and innate immunity. Here, we report the first molecular and functional characterization of septin 8a in rohu (Labeo rohita), an economically important Indian major carp. The full-length septin 8a cDNA encodes a 420-amino acid protein containing conserved GTP-binding motifs (G1-G5) belonging to the SEPT6 subgroup with a predicted molecular weight of ∼48.98 kDa, hydrophilic character and intracellular localization consistent with cytoskeletal scaffolding. Developmental expression analysis revealed peak transcript abundance at fertilization, suggesting maternal deposition and an early embryogenic role. Tissue profiling showed the highest constitutive expression in gills, kidney, and spleen. The challenge experiments with Aeromonas hydrophila, Argulus siamensis, and the viral mimic poly I:C demonstrated a broad pathogen-induced upregulation, particularly in immune tissues. Administration of generated recombinant septin 8a in vivo activated pro-inflammatory cytokines, antimicrobial enzymes, antioxidant genes, and the MX gene, and conferred 55% relative percent survival against A. hydrophila challenge. Indirect ELISA revealed progressive increase in protein levels within liver and kidney tissues during infection. CRISPR/Cas9-mediated F0 mutagenesis in zebrafish caused developmental delay, morphological abnormalities, and a survival rate of 26%, emphasizing its role during early development. Successful mutagenesis in rohu embryos validated cross-species sgRNA efficacy. Following bacterial challenge, exon 2-edited F0 crispants showed the highest mortality (80%) relative to wild-type controls (53.33%), whereas the mortality in exon 4-edited rohu did not differ from controls. These findings establish septin 8a as a multifunctional immune regulator and developmental factor with potential applications in aquaculture disease management.},
}

Animals
*Septins/genetics/immunology/chemistry
*Fish Diseases/immunology
*Immunity, Innate/genetics
Fish Proteins/genetics/immunology/chemistry
Aeromonas hydrophila/physiology
Gram-Negative Bacterial Infections/immunology/veterinary
Amino Acid Sequence
Sequence Alignment/veterinary
*Carps/immunology/genetics
CRISPR-Cas Systems
*Gene Expression Regulation/immunology
Phylogeny
Gene Expression Profiling/veterinary
Poly I-C/pharmacology
Base Sequence

@article {pmid42744283,
year = {2026},
author = {Scholz, V and Schönrock, V and Erdmann, H and Prokosch, V and Schoedel, M and Almus, M and Sauer, M and Mayer, V and Breithausen, E and van Buren, I and Dineiger, C and Golibrzuch, M and Montero-Hernández, M and Lischka, A and Eggermann, K and Heintz, C and Hallermayr, A and Neuhann, T and Holinski-Feder, E and Abicht, A and Benet-Pagès, A and Lucas, MC},
title = {Parallel Analysis of Repeat Expansions: An Updated Clinical Nanopore Cas9-Targeted Sequencing Workflow for Nanopore R10 Flow Cells.},
journal = {The Journal of molecular diagnostics : JMD},
volume = {28},
number = {11},
pages = {811-824},
doi = {10.1016/j.jmoldx.2026.04.007},
pmid = {42744283},
issn = {1943-7811},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Nanopore Sequencing/methods ; *Nanopores ; *DNA Repeat Expansion/genetics ; Workflow ; *High-Throughput Nucleotide Sequencing/methods ; Sequence Analysis, DNA/methods ; },
abstract = {Hereditary ataxias, caused by expansions of short tandem repeats, are difficult to diagnose using traditional PCR and Southern blot methods, which struggle to detect complex repeat expansions and cannot assess repeat interruptions or methylation. An updated Clinical Nanopore Cas9-Targeted Sequencing workflow is presented for analyzing repeat expansions, now compatible with the Oxford Nanopore Technologies R10 flow cell. The workflow incorporates the Oxford Nanopore Technologies wf-human-variation Epi2Me workflow, including the Straglr tool to analyze base-called reads, ensuring compatibility with past, current, and future sequencing chemistries. It expands the number of genes analyzed from 10 to 27 and introduces new gene panels for ataxia, myopathy, neurodegeneration, and amyotrophic lateral sclerosis/motor neuron disease. Validated with Coriell reference and clinical samples, this method improves the analysis of pathogenic repeat expansions, providing deeper insights into repeat structures while addressing the limitations of traditional approaches. In this work, the use of multiplexing, Flongle flow cells, and single-gene targeting were explored as alternatives to panel-based approaches in the Clinical Nanopore Cas9-Targeted Sequencing workflow, finding that only single-gene targeting provides compatibility and reliable performance.},
}

Humans
*CRISPR-Cas Systems/genetics
*Nanopore Sequencing/methods
*Nanopores
*DNA Repeat Expansion/genetics
Workflow
*High-Throughput Nucleotide Sequencing/methods
Sequence Analysis, DNA/methods

@article {pmid42759923,
year = {2026},
author = {Rocco, MA and Kirvaitytė, M and Lencioni, G and Mariani, S and McDonnell, LA and Hussein, M and Giovannetti, E},
title = {CRISPR-based diagnostics as a specificity layer for the early detection of pancreatic cancer.},
journal = {Cancer letters},
volume = {661},
number = {},
pages = {218851},
doi = {10.1016/j.canlet.2026.218851},
pmid = {42759923},
issn = {1872-7980},
mesh = {Humans ; *Pancreatic Neoplasms/diagnosis/genetics/blood ; *Early Detection of Cancer/methods ; *Biomarkers, Tumor/genetics ; *Carcinoma, Pancreatic Ductal/diagnosis/genetics/blood ; Liquid Biopsy/methods ; *CRISPR-Cas Systems ; High-Throughput Nucleotide Sequencing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Pancreatic ductal adenocarcinoma (PDAC) remains a highly lethal malignancy, largely because early-stage symptoms are non-specific and diagnosis is often delayed. Although liquid biopsies (LBs) offer a promising non-invasive strategy for early detection, their clinical translation is hindered by substantial analytical limitations. This review examines the current landscape of nucleic acid-based liquid biopsy biomarkers for PDAC detection. We evaluate key limitations of established diagnostic methodologies, including next-generation sequencing (NGS) and droplet digital PCR (ddPCR). In complex biofluids, these approaches may be confounded by background signal and amplification artifacts, particularly when applied for the detection of ultra-rare targets. To address this diagnostic gap, we discuss the role of CRISPR-based biosensing and bioassay not as a replacement but as an additional specificity layer within existing diagnostic workflows. In this framework, CRISPR technologies can act as sequence-specific molecular filters that introduce an active post-amplification validation step. Although pre-analytical limitations remain to be fully characterized, integrating CRISPR into the diagnostic workflow may contribute to a rapid, cost-effective, and highly specific framework for the early detection of PDAC.},
}

Humans
*Pancreatic Neoplasms/diagnosis/genetics/blood
*Early Detection of Cancer/methods
*Biomarkers, Tumor/genetics
*Carcinoma, Pancreatic Ductal/diagnosis/genetics/blood
Liquid Biopsy/methods
*CRISPR-Cas Systems
High-Throughput Nucleotide Sequencing/methods
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42825477,
year = {2026},
author = {Shi, Z and Ding, L and Zhou, LA and Liu, Y and Wang, Y and Xu, Y and Cao, Y and Jiang, Z and Liu, Q},
title = {Portable CRISPR/Cas12a-Based Aptasensor System for Point-of-Care Detection of Cytokine Tumor Necrosis Factor-Alpha (TNF-α).},
journal = {ACS sensors},
volume = {11},
number = {10},
pages = {9039-9048},
doi = {10.1021/acssensors.6c01817},
pmid = {42825477},
issn = {2379-3694},
support = {2024M762843//China Postdoctoral Science Foundation/ ; LMS25C100001//Natural Science Foundation of Zhejiang Province/ ; 25gya24//Science and Technology Plan Project of Taizhou/ ; 25nyb05//Science and Technology Plan Project of Taizhou/ ; },
mesh = {*Tumor Necrosis Factor-alpha/analysis/blood ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods/instrumentation ; *Aptamers, Nucleotide/chemistry/genetics ; Humans ; *Point-of-Care Systems ; Limit of Detection ; Rapid Diagnostic Tests ; *Endodeoxyribonucleases/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Tumor necrosis factor-α (TNF-α) is an important inflammatory cytokine whose rapid and quantitative detection is essential for disease diagnosis and monitoring. However, conventional methods are often limited by bulky instrumentation and are not well suited for analysis in complex biological environments. Herein, a portable CRISPR/Cas12a-based aptasensor system was developed for detection of TNF-α by integrating aptamer recognition, Cas12a trans-cleavage signal amplification, and a miniaturized fluorescence detection device. An aptamer for TNF-α was designed to regulate the accessibility of the target sequence to the CRISPR RNA (crRNA), and the resulting change in Cas12a activity was converted into fluorescence signals. The portable device integrated light-emitting diode (LED) excitation and photodiode-based detection, with modulated signals wirelessly transmitted to a smartphone for readout. The aptasensor showed concentration-dependent responses, and the inhibition rate exhibited a linear relationship with TNF-α concentration. The applicability of the system was validated in cell culture supernatants and cervical tissue samples, including tumor and adjacent normal tissues. Distinct TNF-α levels were observed between these samples, showing good agreement with enzyme-linked immunosorbent assay (ELISA) results. This portable CRISPR/Cas12a aptasensor system provides a practical approach for point-of-care TNF-α detection and extends CRISPR-based sensing to protein biomarkers.},
}

*Tumor Necrosis Factor-alpha/analysis/blood
*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods/instrumentation
*Aptamers, Nucleotide/chemistry/genetics
Humans
*Point-of-Care Systems
Limit of Detection
Rapid Diagnostic Tests
*Endodeoxyribonucleases/genetics
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid42825812,
year = {2026},
author = {Wang, J and Tian, Q and Li, Y and Qin, Y and Xiong, X and Ding, S and Xu, W and Shen, M and Liu, C},
title = {Single-Atom Nanozyme Integrated Double-Clasp Transition Probe-Driven CRISPR/Cas12a Cascade for Preamplification-Free Detection of Non-Nucleic Acid Targets.},
journal = {ACS sensors},
volume = {11},
number = {10},
pages = {9527-9541},
doi = {10.1021/acssensors.6c02981},
pmid = {42825812},
issn = {2379-3694},
support = {2024MSXM038//Chongqing Municipal Health Commission/ ; 82302643//National Natural Science Foundation of China/ ; 82502225//National Natural Science Foundation of China/ ; 2024MSXM038//Chongqing Municipal Science and Technology Bureau/ ; CSTB2025NSCQ-GPX1050//Natural Science Foundation of Chongqing Municipality/ ; CSTB2025NSCQ-GPX1151//Natural Science Foundation of Chongqing Municipality/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Erb-b2 Receptor Tyrosine Kinases/blood/genetics ; *Biosensing Techniques/methods ; Luminescent Measurements/methods ; Electrochemical Techniques/methods ; *CRISPR-Associated Proteins/chemistry/genetics ; Reactive Oxygen Species/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {CRISPR/Cas12a diagnostics have greatly advanced nucleic acid testing, yet their extension to non-nucleic acid targets remains constrained by the intricate preamplification process and by inefficient signal transduction. Herein, we present a non-nucleic acid detection platform that integrates a CRISPR/Cas12a positive-feedback cascade with a single-atom nanozyme. In this strategy, cleavage of the designed double-clasp transition probe (DCTP) activates additional Cas12a ribonucleoprotein (RNP) complexes, enabling cascade signal amplification without a preamplification treatment. Meanwhile, the created single-atom Pt-engineered PdCeB nanozyme (Pt1/PdCeB SAN) functions as an efficient electrochemiluminescence (ECL) nanolabel in the luminol/O2 system, converting dissolved oxygen (O2) into reactive oxygen species (ROS) to produce a robust ECL response without an external coreactant. Using human epidermal growth factor receptor 2 (HER2) as a clinically relevant non-nucleic acid target, the DCTP-driven Cas12a positive-feedback system shortened the cleavage time from 33 min in the conventional Cas12a system to 18 min and achieved a wide linear range from 10-3 to 104 ng/mL, with a low detection limit of 0.513 pg/mL. Furthermore, preliminary analysis of 31 clinical serum samples supported the feasibility of the strategy for quantitative serum HER2 (sHER2) determination, although validation in larger independent cohorts is required.},
}

*CRISPR-Cas Systems/genetics
Humans
Erb-b2 Receptor Tyrosine Kinases/blood/genetics
*Biosensing Techniques/methods
Luminescent Measurements/methods
Electrochemical Techniques/methods
*CRISPR-Associated Proteins/chemistry/genetics
Reactive Oxygen Species/chemistry
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid42832371,
year = {2026},
author = {Wang, H and Chen, M and He, Q and Wu, Z and Zheng, J and He, X and Deng, L and Mao, X and Sheng, Y},
title = {Molecular Programming of CRISPR Diagnostics: From Target Recognition to Field Deployment.},
journal = {ACS sensors},
volume = {11},
number = {10},
pages = {8839-8866},
doi = {10.1021/acssensors.6c02636},
pmid = {42832371},
issn = {2379-3694},
support = {32500026//National Natural Science Foundation of China/ ; 82502793//National Natural Science Foundation of China/ ; CSTB2025NSCQ-GPX0622//Natural Science Foundation of Chongqing Municipality/ ; },
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; Biosensing Techniques/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Rapid Diagnostic Tests ; },
abstract = {Rapid and accurate diagnostics are essential for controlling emerging infectious diseases and antimicrobial resistance, yet CRISPR assays with exceptional analytical sensitivity often remain difficult to translate into deployable tests. Existing reviews have commonly organized CRISPR diagnostics by Cas effector, biosensor format, or application, offering limited guidance on how design decisions interact across complete workflows or why promising systems remain at the proof-of-concept stage. We introduce an integrated framework that connects five coupled design layers with five levels of translational evidence. The design layers comprise guide RNA engineering, signal amplification circuits, molecular adaptors, computational design support, and specimen and intended-use implementation. The evidence levels progress from purified-target characterization and spiked-matrix testing to retrospective specimen evaluation, prospective integrated-workflow evaluation, and intended-use operation by target users supported by controls, usability assessment, real-time stability, manufacturing consistency, cost per valid result, and claim-specific regulatory preparation. This dual framework shows how improvements at one layer can shift the dominant bottleneck to another. For example, preamplification can lower the analytical limit of detection while increasing contamination risk and workflow burden, whereas guide designs that improve sequence discrimination may reduce cognate-target activation. The framework supports bottleneck-guided selection of effectors, signal-gain strategies, adaptors, computational tools, and multiplexing formats according to target type and abundance, specimen matrix, intended decision, target users, and deployment setting. High analytical sensitivity or single-nucleotide discrimination under controlled conditions does not establish clinical utility unless such performance improves classification at the intended decision threshold within the complete workflow. Translation requires standardized benchmarking, matrix-aware validation, appropriate controls, stability and manufacturability assessment, prospective intended-use studies, and economic evaluation suited to the intended claim and setting. This framework shifts the evaluation of CRISPR diagnostics from isolated analytical performance toward evidence-based workflow co-design.},
}

Humans
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*CRISPR-Cas Systems/genetics
Biosensing Techniques/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Rapid Diagnostic Tests

@article {pmid42849655,
year = {2026},
author = {Lang, F and Fu, H and Kaur, K and Zhang, M and Song, H and Zhang, W and Ayanlaja, AA and Hossain, MS and Aladjem, MI and Yang, C},
title = {Genome-wide CRISPR screen identifies PKMYT1 inhibition as a synthetic lethal vulnerability in IDH1-mutant glioma.},
journal = {Cancer letters},
volume = {661},
number = {},
pages = {218887},
pmid = {42849655},
issn = {1872-7980},
support = {Z99 CA999999/ImNIH/Intramural NIH HHS/United States ; ZIA BC011684/ImNIH/Intramural NIH HHS/United States ; ZIA BC011760/ImNIH/Intramural NIH HHS/United States ; ZIA BC011761/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Humans ; Animals ; *Glioma/genetics/pathology/drug therapy ; *Isocitrate Dehydrogenase/genetics ; Cell Line, Tumor ; *Protein Serine-Threonine Kinases/genetics/antagonists & inhibitors ; Mutation ; DNA Replication/drug effects ; *Membrane Proteins/genetics/antagonists & inhibitors ; *Synthetic Lethal Mutations ; Mice ; CRISPR-Cas Systems ; *Brain Neoplasms/genetics/pathology/drug therapy ; Xenograft Model Antitumor Assays ; Histones/metabolism ; Jumonji Domain-Containing Histone Demethylases/metabolism ; Protein-Tyrosine Kinases ; },
abstract = {Mutations in isocitrate dehydrogenase (IDH) are frequently observed in several aggressive malignancies, including gliomas, chondrosarcomas, and acute myeloid leukemia. Mutant IDH enzymes exhibit neomorphic activity that produces the oncometabolite D-2-hydroxyglutarate (D-2-HG), leading to widespread DNA and histone hypermethylation. This aberrant epigenetic state remodels chromatin accessibility, thereby compromising both DNA repair and replication. However, the mechanisms underlying IDH mutation-induced DNA replication stress and the therapeutic potential of this vulnerability remain elusive. Through a whole-genome CRISPR screen, we defined differential genetic dependencies between IDH1 wild-type and mutant cells. We identified the G2/M checkpoint kinase PKMYT1 as a selective survival factor for IDH1-mutated cells. Pharmacologic or genetic inhibition of PKMYT1 profoundly suppresses the growth of IDH1-mutated tumor cells in vitro and in vivo. Mechanistically, IDH1-mutated cancer cells exhibit profound replication stress, characterized by reduced nucleotide incorporation, slowed replication fork progression, and delayed S-phase entry. D-2-HG elevates H3K9 trimethylation by inhibiting KDM4A, thereby impeding DNA replication initiation and fork progression. PKMYT1 blockade alters cell cycle timing, forcing unscheduled mitotic entry with incompletely replicated genomes, resulting in elevated DNA damage and mitotic catastrophe. Collectively, our findings demonstrate a mechanistic link between IDH1-driven epigenetic dysregulation, replication stress, and heightened reliance on the G2/M checkpoint. Our findings propose PKMYT1 inhibition as a therapeutic strategy that exploits the intrinsic replication deficiency of IDH1-mutant glioma.},
}

Humans
Animals
*Glioma/genetics/pathology/drug therapy
*Isocitrate Dehydrogenase/genetics
Cell Line, Tumor
*Protein Serine-Threonine Kinases/genetics/antagonists & inhibitors
Mutation
DNA Replication/drug effects
*Membrane Proteins/genetics/antagonists & inhibitors
*Synthetic Lethal Mutations
Mice
CRISPR-Cas Systems
*Brain Neoplasms/genetics/pathology/drug therapy
Xenograft Model Antitumor Assays
Histones/metabolism
Jumonji Domain-Containing Histone Demethylases/metabolism
Protein-Tyrosine Kinases

@article {pmid42859051,
year = {2026},
author = {Tong, Z and Huang, Z and Liu, J and Su, J and Zhao, Q and Zhu, R and Xiong, R and Xie, W and Xiao, R},
title = {CRISPR/Cas13a-Based Nucleic Acid-Functionalized Discrete Floating-Gate Field-Effect Transistor for Amplification-Free Rapid Nipah Virus Detection.},
journal = {ACS sensors},
volume = {11},
number = {10},
pages = {9198-9209},
doi = {10.1021/acssensors.6c02218},
pmid = {42859051},
issn = {2379-3694},
mesh = {*CRISPR-Cas Systems/genetics ; *Nipah Virus/isolation & purification/genetics ; Gold/chemistry ; *Transistors, Electronic ; *Biosensing Techniques/methods/instrumentation ; Metal Nanoparticles/chemistry ; *Nucleic Acids/chemistry/genetics ; Nucleic Acid Amplification Techniques ; },
abstract = {Nipah virus (NiV) is a highly lethal zoonotic pathogen, and existing rapid diagnostic tools are scarce and reliant on nucleic acid amplification. In this study, we designed and fabricated a thin-film field-effect transistor with an IGZO channel and, for the first time, combined a discrete AuNP floating gate with the CRISPR/Cas13a system to achieve amplification-free detection of NiV. A pair of partially complementary nucleic acid reporter probes was used to attach AuNPs onto the channel, forming a nucleic acid-functionalized discrete floating gate. Under positive gate voltage, the AuNPs electrostatically induce mirror charges in the IGZO channel, amplifying the sensing signal. Additionally, the electrons can be injected from AuNPs into the channel via the Fowler-Nordheim tunneling effect under negative gate voltage, as a potential complementary sensing mechanism. The AuNPs are immobilized via nucleic acid strands, ensuring good biocompatibility. CrRNA was designed and the CRISPR system was optimized for compatibility with the FET, achieving amplification-free detection of NiV standards down to 10° copies/μL within 10 min. This study not only provides a reliable and rapid detection tool for NiV but also establishes a biosensing framework applicable to other pathogens.},
}

*CRISPR-Cas Systems/genetics
*Nipah Virus/isolation & purification/genetics
Gold/chemistry
*Transistors, Electronic
*Biosensing Techniques/methods/instrumentation
Metal Nanoparticles/chemistry
*Nucleic Acids/chemistry/genetics
Nucleic Acid Amplification Techniques

@article {pmid42859054,
year = {2026},
author = {Xu, C and Cao, L and Li, S and Ye, W and Zhao, J and Li, X and Luo, S and Zhang, L and Liu, G and Gao, F and Wang, Y},
title = {Centrifugal Microfluidic Soil-to-DNA Preparation for the Portable Detection of Phytophthora infestans Using a CRISPR/Cas12a-Based Quantum Dot Fluorescent Lateral Flow Assay.},
journal = {ACS sensors},
volume = {11},
number = {10},
pages = {9049-9062},
doi = {10.1021/acssensors.6c01885},
pmid = {42859054},
issn = {2379-3694},
support = {2024CXGC010905//Key Technology Research and Development Program of Shandong Province/ ; 32471994//National Natural Science Foundation of China/ ; 62401569//National Natural Science Foundation of China/ ; },
mesh = {*Phytophthora infestans/isolation & purification/genetics ; *CRISPR-Cas Systems/genetics ; *Quantum Dots/chemistry ; Centrifugation ; *Soil/chemistry ; *Microfluidic Analytical Techniques/methods/instrumentation ; Lab-On-A-Chip Devices ; Rapid Diagnostic Tests ; *DNA/genetics ; Limit of Detection ; },
abstract = {Portable detection of soil-borne pathogens is constrained by laborious soil sample preparation and nucleic acid extraction. Here, we report a centrifugal Soil-on-a-Disc (SoaD) microfluidic chip for rapid soil-to-DNA preparation with automated on-chip clarification and DNA purification, combined with a CRISPR/Cas12a-based quantum dot (QD) fluorescent lateral flow assay (LFA) and smartphone-based fluorescence analysis for the portable detection of Phytophthora infestans (P. infestans). The seven-layer PMMA disc integrates cascaded 5 and 0.8 μm membranes for stepwise particle filtration, together with a silica membrane and active puncture valves for sequential DNA capture, washing, and elution under centrifugal control. The SoaD chip reduced the turbidity of soil suspensions significantly from 12,980 NTU to below the detection limit (∼0 NTU), yielding a clarified sample matrix for downstream DNA extraction, and completed DNA extraction within 20 min. Compared with a commercial soil DNA kit, the relative total DNA recovery efficiency for P. infestans-spiked soils was 94.9 ± 3.3%. Coupled with downstream recombinase polymerase amplification and smartphone-assisted fluorescence readout, the system achieved a limit of detection of 0.135 ng/μL for PiNW-derived RPA products and detected P. infestans in soils spiked at levels as low as 1 spore/g. As a secondary validation of extraction compatibility, 16S rRNA/ITS amplicon sequencing showed that chip-prepared DNA yielded bacterial and fungal community profiles highly consistent with those obtained using the commercial soil DNA kit (r = 0.996/0.923). This work advances portable CRISPR-based sensing toward complex soil samples and provides a field-oriented strategy for molecular surveillance of soil-borne plant diseases.},
}

*Phytophthora infestans/isolation & purification/genetics
*CRISPR-Cas Systems/genetics
*Quantum Dots/chemistry
Centrifugation
*Soil/chemistry
*Microfluidic Analytical Techniques/methods/instrumentation
Lab-On-A-Chip Devices
Rapid Diagnostic Tests
*DNA/genetics
Limit of Detection

@article {pmid42862206,
year = {2026},
author = {Jiang, Q and Avaro, AS and Bae, H and Sorensen, A and Santiago, JG},
title = {Experimental Methods for CRISPR Enzyme Assays with Fluorescence Readout.},
journal = {ACS sensors},
volume = {11},
number = {10},
pages = {8780-8797},
doi = {10.1021/acssensors.6c02175},
pmid = {42862206},
issn = {2379-3694},
mesh = {Kinetics ; *Enzyme Assays/methods ; Fluorescence ; *CRISPR-Cas Systems/genetics ; Spectrometry, Fluorescence/methods ; },
abstract = {Fluorescence-based CRISPR diagnostic assays have become a popular platform for nucleic acid detection due to their programmability, configurability, specificity, and compatibility with standard laboratory equipment. However, reported enzymatic kinetic rates and limits of detection for CRISPR trans-cleavage assays vary by several orders of magnitude across the literature. This variation in performance parameters is coupled with, and exacerbated by, inconsistent calibration, incomplete correction of measurement biases, and nonstandardized or incomplete data analysis procedures. We present an experimental protocol and quantitative analysis framework for fluorescence-based enzyme assays using routine laboratory instrumentation, including thermocyclers and fluorescence microplate readers. Building on previous studies of CRISPR enzyme kinetics and fluorescence calibration, we describe procedures for flat-field and background correction; comprehensive fluorescence calibration, including correction for the inner filter effect; quantification and implications of reporter degradation; extraction of Michaelis-Menten kinetic parameters; and determination of assay's limits of detection. We provide step-by-step experimental guidelines and open-source Python implementations for each stage of the workflow. Using representative Cas12 trans-cleavage datasets, we demonstrate that explicit fluorescence calibration and correction procedures substantially reduce systematic bias in measured kinetic rates and improve consistency between experiments. Our framework aims to establish standardized practices for quantitative fluorescence-based CRISPR assays and provides researchers with practical tools for reproducible kinetic characterization and rational assay design.},
}

Kinetics
*Enzyme Assays/methods
Fluorescence
*CRISPR-Cas Systems/genetics
Spectrometry, Fluorescence/methods

@article {pmid42905497,
year = {2026},
author = {Azfer, A and Berg, K and Johnson, K and Thomson, P and Wilson, JF and Wani, S and Tang, JCY and Albagha, OME and Ralston, SH},
title = {Dysregulation of macrophage colony stimulating factor in Paget's disease of bone.},
journal = {Rheumatology (Oxford, England)},
volume = {},
number = {},
pages = {},
doi = {10.1093/rheumatology/keag571},
pmid = {42905497},
issn = {1462-0332},
abstract = {OBJECTIVE: To determine if common genetic variants at the chromosome 1p13 locus which predisposes to Paget's Disease of Bone (PDB) regulate Macrophage Colony stimulating factor (M-CSF) expression in bone cells and to determine if there is dysregulation of M-CSF in patients with PDB.

METHODS: The regions surrounding single nucleotide polymorphisms (SNP) previously associated with PDB at a genome wide significant level were deleted in clones of MG63 osteoblast-like cells using CRISPR/Cas and levels of M-CSF mRNA and protein were evaluated in cells with and without deletions. Serum M-CSF was measured by ELISA in PDB and controls and values in patients were related to clinical features of PDB and biochemical markers of bone remodelling.

RESULTS: Deletion of the region surrounding rs484959 increased expression of M-CSF mRNA by 1.31 fold [95%,1.04-1.58], p = 0.026 and M-CSF protein by 1.91 fold [1.25-2.57], p = 0.013, compared with control cells. Serum M-CSF was significantly elevated in PDB versus controls (mean ± SD = 781.7 ± 678 vs. 541 ± 491 pg/mL, p < 0.001). Furthermore, M-CSF levels were associated with number of affected bones (p = 0.029), bodily pain assessed by SF-36 (p = 0.026) and biochemical markers of bone turnover.

CONCLUSION: The rs484959 SNP is a regulatory variant which modulates M-CSF mRNA and protein expression in osteoblasts. Serum M-CSF is increased in PDB, is associated with pain, disease extent and biochemical markers of bone remodelling. This suggest that there is dysregulation of M-CSF in PDB and that this cytokine may play a role in pathogenesis of the disease.},
}

@article {pmid42905526,
year = {2026},
author = {Benedetti, L and Yi, HA and Wang, L and Lippincott-Schwartz, J},
title = {Endogenous Protein Tagging in Postmitotic Neurons via Lentiviral and Adeno-associated Virus Delivery.},
journal = {Current protocols},
volume = {6},
number = {10},
pages = {},
pmid = {42905526},
issn = {2691-1299},
support = {/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*Lentivirus/genetics ; *Neurons/metabolism/cytology ; Animals ; *Dependovirus/genetics ; Genetic Vectors/genetics ; CRISPR-Cas Systems ; Humans ; Membrane Proteins/metabolism/genetics ; },
abstract = {This protocol describes a method for endogenous protein tagging in postmitotic primary neurons using homology-independent targeted integration (HITI), a CRISPR/Cas9-based approach that exploits non-homologous end joining (NHEJ). Unlike homology-directed repair (HDR), HITI does not require cell division, making it uniquely suited for postmitotic neurons. SpCas9 is delivered by a replication-incompetent simian immunodeficiency lentiviral vector (pLenti SIV), while the sgRNA, donor tag cassette, and a fluorescent plasma membrane marker are co-packaged in a single AAV vector. The two vectors are co-delivered at DIV5, and endogenous tagging is assessed at DIV15-23. The workflow is illustrated using Junctophilin-3 (JPH3) tagged with mEmerald at its endogenous locus. Airyscan microscopy imaging of endogenously tagged JPH3 at native expression levels revealed its localization at endoplasmic reticulum-plasma membrane (ER-PM) contact sites and uncovered a periodic distribution of ER-PM junctions along the dendritic arbor, enabling identification of a previously unrecognized ladder-like organization of the ER in neuronal dendrites. Genomic integration of the tag is validated at both 5' and 3' junctions by next-generation sequencing. The protocol can be adapted to other neuronal proteins by redesigning the guide RNA and donor sequence, and supports fluorescent proteins as well as epitope and self-labeling tags. The plasma membrane marker can also be replaced with other fluorescent reporters or markers for intracellular organelles to assess infection efficiency and verify subcellular localization of the protein of interest. The full workflow takes 5-6 weeks. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Protocols included in this article: Basic Protocol 1: sgRNA design and HITI donor vector cloning Basic Protocol 2: Primary rat neuron culture and viral co-transduction Basic Protocol 3: Live-cell imaging of endogenously tagged proteins Support Protocol 1: Production of lentiviral and AAV particles Support Protocol 2: Validation of knock-in by next-generation sequencing (NGS).},
}

*Lentivirus/genetics
*Neurons/metabolism/cytology
Animals
*Dependovirus/genetics
Genetic Vectors/genetics
CRISPR-Cas Systems
Humans
Membrane Proteins/metabolism/genetics

@article {pmid42905851,
year = {2026},
author = {Murillo, A and Alpaugh, M and Peter Durairaj, RR and Randall, EL and Larin, M and Heraty, L and Aston, AN and Taylor, AS and Monteys, AM and Stöberl, N and Heuchan, AER and Aeschlimann, P and Fears, K and Landles, C and Osborne, GF and Mangin, A and Bhattacharyya, S and Metzakopian, E and Allen, ND and Puymirat, J and Bates, GP and Davidson, BL and Cicchetti, F and Lelos, MJ and Dion, V},
title = {Cas9 nickase-mediated contraction of CAG/CTG repeats is associated with phenotypic improvements in a Huntington's disease mouse model.},
journal = {Science translational medicine},
volume = {18},
number = {872},
pages = {eaef9466},
doi = {10.1126/scitranslmed.aef9466},
pmid = {42905851},
issn = {1946-6242},
mesh = {Animals ; *Huntington Disease/genetics/therapy/pathology/physiopathology ; Disease Models, Animal ; Phenotype ; Humans ; *Trinucleotide Repeat Expansion/genetics ; *Deoxyribonuclease I/metabolism ; Mice ; Neurons/metabolism/pathology ; *CRISPR-Cas Systems/genetics ; Myotonic Dystrophy/genetics/pathology ; Astrocytes/metabolism ; },
abstract = {Expanded CAG/CTG repeats cause more than 15 different diseases that all remain without a disease-modifying treatment. Because repeat length accounts for most of the variation in disease severity, contracting CAG/CTG repeats presents an attractive therapeutic avenue. Here, we show that the CRISPR-Cas9 nickase targeted to CAG/CTG repeats leads to efficient repeat contractions in Huntington's disease (HD) patient-derived neurons and astrocytes and in myotonic dystrophy type 1 patient-derived neurons. The approach was allele selective and free of detectable off-target mutations. Injection of the Cas9 nickase into the striatum of the R6/1 mouse model of HD using adeno-associated viral vectors led to repeat contractions in more than half of striatal neurons. Upon injection, we observed a reduction in the number of inclusion bodies, an improved gene expression profile, and amelioration of locomotion deficits in the open field test. The effects were greater than expected from the contractions induced, suggesting that non-cell autonomous mechanisms may be involved. Our results provide the proof of concept that correction of CAG/CTG repeats can improve HD phenotypes in a mouse disease model.},
}

Animals
*Huntington Disease/genetics/therapy/pathology/physiopathology
Disease Models, Animal
Phenotype
Humans
*Trinucleotide Repeat Expansion/genetics
*Deoxyribonuclease I/metabolism
Mice
Neurons/metabolism/pathology
*CRISPR-Cas Systems/genetics
Myotonic Dystrophy/genetics/pathology
Astrocytes/metabolism

@article {pmid42907433,
year = {2026},
author = {Zhu, H and Dai, S and Deng, H and Hou, S and Li, Y and Tan, X and Jia, N and Li, N and Ma, Y},
title = {A phage DUF669 protein mediates post-cleavage DNA repair against host CRISPR immunity.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42907433},
issn = {2041-1723},
support = {JCYJ2022053011260200//Shenzhen Science and Technology Innovation Commission/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Viral Proteins/metabolism/genetics ; *Bacteriophages/genetics/metabolism ; *DNA Repair ; Escherichia coli/genetics/virology ; Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Pseudomonas aeruginosa/genetics/virology ; DNA, Single-Stranded/metabolism ; Homologous Recombination ; },
abstract = {Phages have evolved various anti-CRISPR (Acr) proteins or RNA-based anti-CRISPR (Racr) to evade host CRISPR immunity by direct interference. However, whether other counter-CRISPR mechanisms exist remains unexplored. Here, we report a phage-encoded two-protein system, termed 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 (an AAA domain-containing protein). Mechanistic investigations elucidate that Gp63 acts as a rapid-response effector of CRISPR-induced DNA breaks. After Gp63 binds ssDNA, it promotes Gp64-mediated homologous recombination, which repairs CRISPR-generated phage DNA breaks and supports phage viability. Notably, co-expression of the Healer system with Cas9/Cas12 in E. coli, P. aeruginosa, and A. baumannii demonstrated high phage genome-editing efficiency. Collectively, our findings reveal an important anti-CRISPR complementary strategy, providing a promising tool for genome engineering.},
}

*CRISPR-Cas Systems/genetics
*Viral Proteins/metabolism/genetics
*Bacteriophages/genetics/metabolism
*DNA Repair
Escherichia coli/genetics/virology
Gene Editing/methods
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Pseudomonas aeruginosa/genetics/virology
DNA, Single-Stranded/metabolism
Homologous Recombination

@article {pmid42907902,
year = {2026},
author = {Zheng, W and Yu, L and Wu, C and Cao, S and Zhao, M and Wang, G and So, KH and Song, J and Cheng, C and Ho, JWK and Liu, X and Wu, M and Liu, Z and Wang, H and Liu, P and Lan, G and Huang, Y},
title = {Accurate prediction of CRISPR editing outcomes in somatic cell lines and zygotes with few-shot learning with inDecay.},
journal = {Genome biology},
volume = {27},
number = {1},
pages = {},
pmid = {42907902},
issn = {1474-760X},
mesh = {Animals ; *Zygote/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Mice ; Cattle ; Cell Line ; Swine ; *Machine Learning ; INDEL Mutation ; Prediction Algorithms ; },
abstract = {Prediction of CRISPR/Cas outcomes remains unsatisfactory in embryos due to distinct DNA repair preferences and the lack of large-scale embryo editing profiles. We introduce inDecay, a flexible system for predicting the proportion of CRISPR-induced indels from the target sequence. Owing to its parameter-efficient and cell-type-aware design, inDecay performs well in both in-sample training and out-of-sample fine-tuning. Starting with cross-cell-line predictions, we observed that inDecay maintains high accuracy in mouse zygote editing and in goat, cattle and porcine embryos. inDecay may accelerate mouse model generation, livestock embryonic editing, and gene therapy applications.},
}

Animals
*Zygote/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems
Mice
Cattle
Cell Line
Swine
*Machine Learning
INDEL Mutation
Prediction Algorithms

@article {pmid42908527,
year = {2026},
author = {Shi, Q and Zhang, Y and Mo, M and Li, G},
title = {Glycogen synthase AfGsy2 regulates mycelial growth, stress response, predatory activity, and chlamydospore development in the nematode-trapping fungus Arthrobotrys flagrans.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1891167},
pmid = {42908527},
issn = {2235-2988},
mesh = {Animals ; *Glycogen Synthase/genetics/metabolism ; Glycogen/metabolism ; Spores, Fungal/growth & development ; *Stress, Physiological ; *Ascomycota/growth & development/genetics/enzymology/physiology ; Caenorhabditis elegans/microbiology ; *Mycelium/growth & development ; *Nematoda/microbiology ; Gene Expression Regulation, Fungal ; Gene Deletion ; Hyphae/growth & development ; CRISPR-Cas Systems ; },
abstract = {INTRODUCTION: Glycogen is a universal carbon and energy reserve that plays crucial roles in fungal stress adaptation, cellular homeostasis, and predatory activity. However, the role of glycogen metabolism in nematode-trapping fungi has largely remained unexplored. Here, we performed a systematic functional characterization of AfGsy2, the glycogen synthase-encoding gene in the biocontrol fungus Arthrobotrys flagrans.

METHODS: Using a CRISPR-Cas9-mediated deletion mutant, together with overexpression and complementation strains, we investigated the involvement of AfGsy2 in mycelial growth, stress tolerance, trap formation, nematicidal activity against Caenorhabditis elegans, chlamydospore production and germination, and glycogen accumulation. Subcellular localization was examined using an AfGsy2-GFP fusion protein.

RESULTS: Deletion of AfGsy2 reduced hyphal growth on both nutrient-rich and nutrient-poor media but enhanced tolerance to osmotic, salt, and oxidative stresses, suggesting that blocked glycogen synthesis may trigger metabolic rerouting toward alternative stress-protective compounds. Notably, the ΔAfGsy2 mutants exhibited reduced trap production and nematicidal activity against C. elegans, whereas overexpression of AfGsy2 accelerated these processes, suggesting a link between glycogen metabolism and predatory activity. Moreover, AfGsy2 positively regulated chlamydospore production, glycogen accumulation, and chlamydospore germination. AfGsy2-GFP predominantly localized to the vacuoles of chlamydospores, and complementation restored the mutant phenotypes to wild-type levels.

DISCUSSION: Collectively, these findings uncover a link between glycogen metabolism and growth, stress resilience, sporulation, and predatory activity in A. flagrans, and establish a molecular foundation for the rational improvement of nematode-trapping fungi. Together, these results identify AfGsy2 as a pleiotropic regulator linking glycogen metabolism to biocontrol-related traits.},
}

Animals
*Glycogen Synthase/genetics/metabolism
Glycogen/metabolism
Spores, Fungal/growth & development
*Stress, Physiological
*Ascomycota/growth & development/genetics/enzymology/physiology
Caenorhabditis elegans/microbiology
*Mycelium/growth & development
*Nematoda/microbiology
Gene Expression Regulation, Fungal
Gene Deletion
Hyphae/growth & development
CRISPR-Cas Systems

@article {pmid42908864,
year = {2026},
author = {Wang, X and Shangguan, B and Zhang, Y and Su, T and Ji, J and Zhao, J and Zhang, C and Li, Y and Hou, X and Liu, T},
title = {Research progress in gene editing of Brassicaceae crops.},
journal = {Biodesign research},
volume = {8},
number = {4},
pages = {100122},
pmid = {42908864},
issn = {2693-1257},
abstract = {Brassicaceae crops are globally important economic plants, whose yield and quality directly affect the supply of edible oil and vegetables. Most of these crops are polyploids with complex genomes and numerous homoeologous gene copies, which demand high efficiency and specificity from gene editing tools, while providing ideal models for studying the rules of polyploid genome editing. Traditional breeding methods, such as crossbreeding and mutation breeding, are constrained by long cycles, low efficiency, and poor specificity, making them insufficient for precise genetic improvement. Gene editing technologies, characterized by precision, efficiency, and targeting specific genomic loci, are core tools for crop breeding. Among these, ZFNs and TALENs rely on protein-DNA recognition and are complicated and costly to design; CRISPR-Cas, which achieves precise recognition through base pairing between guide RNA and target DNA, is simple, efficient, and the most widely used editing tool. Base and prime editors enable single-nucleotide substitutions or small fragment insertions without generating double-strand breaks, making them suitable for directed modification of point mutations. This review summarizes the progress of gene editing in Brassicaceae for bolting/flowering, plant architecture, nutritional quality, tolerance to abiotic stresses (temperature, salinity, drought, heavy metals), and resistance to biotic stresses (diseases, pests). We analyze challenges, including off-target effects, difficulties in polyploid genome editing, and imperfect genetic transformation systems, and discuss future directions integrating gene editing with multi-omics and artificial intelligence. This review aims to provide a theoretical basis and technical support for precise molecular breeding of Brassicaceae crops.},
}

@article {pmid42908989,
year = {2026},
author = {Dong, S and Han, Z and He, Y and Wang, Z and Qi, D and Feng, W},
title = {Generation and Validation of a GnRH-iCreERT2-tdTomato Mouse Line for Genetic Manipulation in GnRH Neurons.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {64},
number = {5},
pages = {},
doi = {10.1002/dvg.70072},
pmid = {42908989},
issn = {1526-968X},
support = {2024-GZL-GD06//Xiamen Municipal Major Project of High-Quality Development of Health and Wellness Technology Program/ ; 82271197//National Natural Science Foundation of China/ ; 81974175//National Natural Science Foundation of China/ ; gect-2025-Z20//Shanghai Key Laboratory of Gene Editing and Cell Therapy for Rare Diseases/ ; },
mesh = {Animals ; *Gonadotropin-Releasing Hormone/genetics/metabolism ; *Neurons/metabolism ; Mice ; Mice, Transgenic ; CRISPR-Cas Systems ; Female ; DNA-Binding Proteins/genetics/metabolism ; Hypothalamic-Pituitary-Gonadal Axis ; Gene Knock-In Techniques ; Male ; DNA Helicases/genetics/metabolism ; Red Fluorescent Protein ; },
abstract = {Gonadotropin-releasing hormone (GnRH) neurons play a key role in the hypothalamic-pituitary-gonadal axis. We generated a new GnRH-P2A-iCreERT2-P2A-tdTomato (hereafter referred to as the GnRH-CT-tdT line) knock-in mouse line utilizing CRISPR/Cas9 technology. Characterization analysis of this line revealed that tdTomato expression precisely recapitulates endogenous GnRH expression and iCreERT2 recombines in GnRH neurons with excellent specificity and sensitivity. Therefore, this newly constructed GnRH-CT-tdT mouse line provides a useful tool to perform genetic manipulation in GnRH neurons. As a proof-of-principle, we used this line to investigate the role of Chd7 (Chromodomain helicase DNA-binding protein 7), a gene with its loss-of-function associated with isolated gonadotropin-releasing hormone deficiency (IGD). Surprisingly, ablation of Chd7 in GnRH neurons does not affect the number of GnRH neurons, pubertal onset, and reproductive organ morphology. Therefore, Chd7 is dispensable in GnRH neurons after they have initiated GnRH expression. In summary, the GnRH-CT-tdT mouse line is a valuable tool to study gene function in GnRH neurons and to dissect the pathogenesis of IGD.},
}

Animals
*Gonadotropin-Releasing Hormone/genetics/metabolism
*Neurons/metabolism
Mice
Mice, Transgenic
CRISPR-Cas Systems
Female
DNA-Binding Proteins/genetics/metabolism
Hypothalamic-Pituitary-Gonadal Axis
Gene Knock-In Techniques
Male
DNA Helicases/genetics/metabolism
Red Fluorescent Protein

@article {pmid42909932,
year = {2026},
author = {Wu, X and Kou, X and Bai, L and Liu, C and Wu, Q and Zhang, M and Wang, N and Gong, C},
title = {Recent advances in CRISPR technologies for biomedical applications and disease treatment.},
journal = {Theranostics},
volume = {16},
number = {16},
pages = {9384-9415},
pmid = {42909932},
issn = {1838-7640},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Genetic Therapy/methods ; Animals ; Gene Transfer Techniques ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Vectors ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR), a precise genome-editing platform, has revolutionized genetic engineering. The highly selective and programmable characteristics of CRISPR have enabled its broad application. Recent advances in CRISPR biotechnology have promoted its widespread use in various biomedical fields, such as CRISPR screening. Ongoing preclinical and clinical trials will help determine the full capabilities of CRISPR in disease therapy. The efficacy of CRISPR-based applications is fundamentally dependent on optimized delivery strategies. Multiple approaches for delivering the CRISPR components have been investigated, primarily relying on physical, viral and non-viral delivery methods. Each method has specific strengths and limitations. Notably, non-viral delivery systems hold significant promise for CRISPR applications, offering distinct advantages such as transient expression, low immunogenicity, and fewer size constraints for cargo delivery. Various biomaterials demonstrate strong potential for delivering CRISPR components to achieve gene editing, including inorganic nanoparticles, polymeric nanoparticles, lipid nanoparticles, metal-organic frameworks, DNA origami, and virus-like particles. Therefore, this review provides an overview of recent research on the molecular principles of CRISPR and its biomedical applications. Recent advances in CRISPR-based treatments and their delivery vectors are also reviewed. Finally, the challenges faced by CRISPR technology are discussed.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems
*Genetic Therapy/methods
Animals
Gene Transfer Techniques
*Clustered Regularly Interspaced Short Palindromic Repeats
Genetic Vectors

@article {pmid42911441,
year = {2026},
author = {Han, S and Shen, P and Ren, H and Xu, J and Chen, F and Huang, J and Zhou, M and Xu, Z},
title = {High efficient production of adenine in Bacillus subtilis with novel metabolic engineering strategies.},
journal = {World journal of microbiology & biotechnology},
volume = {42},
number = {11},
pages = {},
pmid = {42911441},
issn = {1573-0972},
support = {2021YFC2103200//National Key Research and Development Program of China/ ; 22578390//National Natural Science Foundation of China/ ; },
mesh = {*Bacillus subtilis/metabolism/genetics ; *Adenine/biosynthesis/metabolism ; *Metabolic Engineering/methods ; Fermentation ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Adenosine/metabolism ; Purines ; Ribose/metabolism ; },
abstract = {Adenine, serves as a crucial precursor for nucleic acid drugs and food additives, currently suffered from limited availability since its high production costs associated with expensive precursors and enzyme preparations. Here, we developed an effective strategy for high-yield adenine production using Bacillus subtilis as the host. Beginning with an adenosine-producing strain, we first knocked-out the apt and adeC genes, which were involved in product degradation pathways, and deleted the purR gene to alleviate the feedback inhibition on purine operon through CRISPR-Cas9 technology. Then, we overexpressed the nucleoside hydrolase RihC, PRPP amidotransferase PurF, and ribokinase RbsK to enhance the de novo adenine biosynthesis and enable the recycling of D-ribose. The production of adenine reached 18 g/L after the fed-batch fermentation, and the titer continued to reach 78 g/L when adenosine was added as the substrate subsequently, revealing the significant catalytic role of RihC produced during the fermentation. This work innovatively integrated de novo biosynthesis and enzymatic conversion strategies to achieve high yield adenine production in Bacillus subtilis, demonstrating a promising scalable process for industrial adenine manufacturing.},
}

*Bacillus subtilis/metabolism/genetics
*Adenine/biosynthesis/metabolism
*Metabolic Engineering/methods
Fermentation
Bacterial Proteins/genetics/metabolism
CRISPR-Cas Systems
Adenosine/metabolism
Purines
Ribose/metabolism

@article {pmid42911482,
year = {2027},
author = {Savickyte, I and Lapinaite, A},
title = {Role of Chromatin-Associated RNAs in Eye Physiology and Pathology.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3091},
number = {},
pages = {1-46},
pmid = {42911482},
issn = {1940-6029},
mesh = {Humans ; *Chromatin/genetics/metabolism ; Animals ; *Eye Diseases/genetics/metabolism/pathology ; *RNA, Untranslated/genetics ; Gene Expression Regulation ; *Eye/metabolism/pathology/physiopathology ; CRISPR-Cas Systems ; Epigenesis, Genetic ; Retinal Pigment Epithelium/metabolism ; },
abstract = {Noncoding RNAs, including long noncoding RNAs, enhancer RNAs, circular RNAs, and small nuclear and nucleolar RNAs, represent a major, yet still largely uncharacterized, regulatory layer in the eye. A subset of these molecules, chromatin-associated RNAs (caRNAs), physically interacts with DNA and nuclear protein complexes to influence genome organization, transcriptional output, and stress responses. Although caRNAs have emerged as essential regulators of chromatin topology and gene expression in diverse tissues, their identity, dynamics, and mechanisms in the retina and retinal pigment epithelium (RPE) remain elusive. Recent high-resolution genomic studies, disease transcriptomes, and mechanistic studies implicate dysregulated caRNAs in age-related macular degeneration, glaucoma, and diabetic retinopathy, yet causal chromatin-level functions have rarely been demonstrated. In this review, we summarize current understanding of ncRNA-mediated regulation in vision, highlight emerging evidence for caRNA involvement in retinal development, photoreceptor and RPE specialization, and disease-associated epigenetic remodeling, and outline major mechanistic gaps that must be addressed. We then survey the rapidly expanding CRISPR toolbox, including base and prime editors, CRISPRi/a, Cas13 knockdown and editing platforms, and RNA-recruitment and spatial repositioning systems, that now enable targeted perturbation of both caRNA genomic loci and transcripts. These technologies provide the first practical route to move beyond correlative RNA-chromatin maps toward the mechanistic dissection of how caRNAs organize regulatory hubs, modulate enhancer-promoter communication, and shape long-range genome interactions in the eye. As the catalog of pathogenic noncoding variants grows, the ability to decode and engineer caRNA networks will be central to future precision therapeutic strategies for ocular disorders.},
}

Humans
*Chromatin/genetics/metabolism
Animals
*Eye Diseases/genetics/metabolism/pathology
*RNA, Untranslated/genetics
Gene Expression Regulation
*Eye/metabolism/pathology/physiopathology
CRISPR-Cas Systems
Epigenesis, Genetic
Retinal Pigment Epithelium/metabolism

@article {pmid42394280,
year = {2026},
author = {Shi, Q and Wang, J and Pang, X and Zhang, D and Xu, C and Lu, Q and Wang, Y and Wang, X and Zhang, F},
title = {A Triple-Signal Output Lateral Flow Platform Leveraging CRISPR/Cas12a and Nanozyme Cascades for Ultra-Sensitive Aflatoxin B1 Detection.},
journal = {ACS sensors},
volume = {11},
number = {9},
pages = {8110-8122},
doi = {10.1021/acssensors.6c01569},
pmid = {42394280},
issn = {2379-3694},
support = {3002066//Hebei Agricultural University/ ; 3002093//Hebei Agricultural University/ ; BJK2023049//Colleges and Universities in Hebei Province Science and Technology Research Project/ ; },
mesh = {*Aflatoxin B1/analysis ; *Biosensing Techniques/methods ; Aptamers, Nucleotide/chemistry ; *CRISPR-Cas Systems/genetics ; Colorimetry/methods ; Limit of Detection ; Arachis/chemistry ; Endodeoxyribonucleases/chemistry ; Platinum/chemistry ; Food Contamination/analysis ; },
abstract = {The presence of the potent carcinogen aflatoxin B1 (AFB1) in agricultural products poses a grave threat to food safety and human health, underscoring the imperative development of highly sensitive detection methodologies. Despite the considerable efforts that have been dedicated to aptamer-based biosensors, the sensitivity of typical nucleic acid-integrated aptasensors is still limited by inefficient strand displacement. To address this limitation, a "three-in-one" multireadout lateral flow assay for AFB1 was developed, integrating CRISPR/Cas12a single-stranded DNase activity with PtPdRu nanozyme-mediated colorimetric signal amplification. The detection process initiates with a "locked activator" DNA configuration, wherein aptamer-specific recognition of AFB1 activates the nonspecific ssDNA cleavage activity of CRISPR/Cas12a, resulting in a fluorescence output. The integrated PtPdRu functions as both a colorimetric probe and a peroxidase mimic, allowing for catalytic amplification based on 3,3',5,5'-tetramethylbenzidine for improved colorimetric detection. The signal-amplified aptasensor achieves ultrasensitive detection with a limit of detection of 0.22 pg/mL, which is 13-fold and 2.7-fold lower than those of the fluorescence mode and the intrinsic colorimetric mode, respectively. Coupled with a smartphone app, the "samples‑in, signal‑out" strategy exhibits high specificity in moldy corn and peanut samples. The triple-modal outputs provide a versatile tool for sensitive detection of AFB1 in agricultural products.},
}

*Aflatoxin B1/analysis
*Biosensing Techniques/methods
Aptamers, Nucleotide/chemistry
*CRISPR-Cas Systems/genetics
Colorimetry/methods
Limit of Detection
Arachis/chemistry
Endodeoxyribonucleases/chemistry
Platinum/chemistry
Food Contamination/analysis

@article {pmid42414241,
year = {2026},
author = {Yu, L and Liu, T and Cai, Y and Gai, Y and Liu, H and Fang, L and Ma, Y and Zhou, C and Ye, X and Liang, B},
title = {An Interface-Regulated Electrochemical Biosensing Platform Based on the Cascade Amplification of Primer Exchange Reaction and CRISPR/Cas12a for Noninvasive Bladder Cancer Diagnosis.},
journal = {Analytical chemistry},
volume = {98},
number = {36},
pages = {26555-26564},
doi = {10.1021/acs.analchem.6c02591},
pmid = {42414241},
issn = {1520-6882},
support = {2023C03094//Key Research and Development Program of Zhejiang Province/ ; 2024C03008//Key Research and Development Program of Zhejiang Province/ ; 82272120//National Natural Science Foundation of China/ ; LR26H180001//Natural Science Foundation of Zhejiang Province/ ; LTGG23H180001//Natural Science Foundation of Zhejiang Province/ ; ZCLZ24F0101//Natural Science Foundation of Zhejiang Province/ ; 2024YFA0920400//National Key Research and Development Program of China/ ; 226-2025-00036//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Biosensing Techniques/methods ; *Urinary Bladder Neoplasms/diagnosis/urine ; *MicroRNAs/urine/genetics ; *Electrochemical Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; },
abstract = {Early screening and timely treatment of bladder cancer are crucial to improving patient survival rates. However, the lack of sensitive and noninvasive diagnostic approaches remains a major challenge. Herein, we developed an electrochemical biosensor for urinary miRNA-182 in bladder cancer detection based on the primer exchange reaction (PER) and CRISPR/Cas12a one-pot cascade reaction. Specifically, the presence of the target miRNA-182 initiated the PER reaction to generate multiple CRISPR/Cas12a activation sequences, which subsequently activated the trans-cleavage activity of CRISPR/Cas12a and induced the cleavage of methylene-blue-labeled signal probes (SP-MB). The uncleaved SP-MB was captured by the complementary sequence (CS) immobilized on the electrode surface, generating an electrochemical response signal that decreased with increasing target concentration, thereby enabling the quantitative detection of miRNA-182. Notably, the detection performance of biosensors constructed on different DNA nanostructure-modified interfaces (S, H, TDN, and TDR) containing CS was systematically compared to elucidate the regulatory mechanism of the biosensing interface. The optimized interface significantly improved the dynamic detection range, with a low detection limit of 1.60 pM. This study not only provides a promising noninvasive diagnostic strategy for bladder cancer but also provides fundamental insights into the rational design of DNA nanostructure-based biosensing interfaces for high-performance electrochemical biosensors.},
}

*Biosensing Techniques/methods
*Urinary Bladder Neoplasms/diagnosis/urine
*MicroRNAs/urine/genetics
*Electrochemical Techniques/methods
Humans
*CRISPR-Cas Systems/genetics

@article {pmid42485003,
year = {2026},
author = {Kim, KM and Lee, Y and Lee, JM and Kim, CY and Kim, YP},
title = {Cut-and-Bind Colorimetric Detection of Viral and Non-Coding RNAs Using CRISPR/Cas12a with Zinc Finger-Facilitated Gold Nanoparticle Assembly.},
journal = {ACS sensors},
volume = {11},
number = {9},
pages = {8019-8031},
doi = {10.1021/acssensors.6c01324},
pmid = {42485003},
issn = {2379-3694},
support = {//Hanyang University/ ; 2023R1A6C101A009//Korea Basic Science Institute/ ; HY202400000003857//Korea Basic Science Institute/ ; 2020R1A6A1A06046728//National Research Foundation of Korea/ ; RS-2025-00553158//National Research Foundation of Korea/ ; RS-2025-02232977//National Research Foundation of Korea/ ; },
mesh = {*Gold/chemistry ; *Metal Nanoparticles/chemistry ; *Colorimetry/methods ; Zinc Fingers ; *RNA, Viral/analysis/genetics ; *CRISPR-Cas Systems/genetics ; *RNA, Untranslated/analysis/genetics ; Humans ; CRISPR-Associated Proteins/chemistry/genetics ; *Bacterial Proteins/chemistry/genetics ; *Endodeoxyribonucleases/chemistry/genetics/metabolism ; Rapid Diagnostic Tests ; Limit of Detection ; },
abstract = {Accurate and rapid detection of RNA biomarkers is crucial for early disease diagnosis and infectious disease management. Although RT-qPCR remains the gold standard, its utility for point-of-care testing is limited by run-to-run variability and dependence on specialized instrumentation. Conversely, colorimetric assays offer operational simplicity but generally fall short of the sensitivity and specificity demanded for reliable applications. To bridge this gap, we present an innovative cut-and-bind colorimetric RNA detection platform that synergistically integrates the sequence-specific programmable cleavage fidelity of CRISPR/Cas12a with zinc finger protein-mediated gold nanoparticle assembly (termed CasFinger-Gold). Strategically engineered ssDNA-dsDNA hybrid overhang probes sharpened target discrimination while minimizing off-target background, ensuring robust readouts within complex biological matrices. When coupled with reverse transcription-recombinase polymerase amplification, the CasFinger-Gold system achieved visually interpretable RNA detection with a limit of detection of 1.8 × 103 copies μL-1 for HCoV-OC43 viral RNA and 0.4 ng total RNA μL-1 for the long non-coding RNA PCA3. The platform further enabled direct, rapid (<120 min), purification-free PCA3 detection from cell-derived extracellular vesicles (EVs) at clinically relevant vesicle inputs (down to ∼106 EVs μL-1). Notably, no cross-reactivity was observed against four phylogenetically distinct respiratory viruses or in PCA3-negative cell lines. By delivering instrument-free visual readouts comparable to RT-qPCR, CasFinger-Gold represents a versatile diagnostic modality poised to expand the molecular diagnostic toolbox toward accessible point-of-care solutions.},
}

*Gold/chemistry
*Metal Nanoparticles/chemistry
*Colorimetry/methods
Zinc Fingers
*RNA, Viral/analysis/genetics
*CRISPR-Cas Systems/genetics
*RNA, Untranslated/analysis/genetics
Humans
CRISPR-Associated Proteins/chemistry/genetics
*Bacterial Proteins/chemistry/genetics
*Endodeoxyribonucleases/chemistry/genetics/metabolism
Rapid Diagnostic Tests
Limit of Detection

@article {pmid42691481,
year = {2026},
author = {Ni, Q and Jiao, J and Jiao, R and Luo, C and Ye, J and Pan, K and Hang, X and Yao, L and Wu, G and Zhao, Q and Bo, Q and Qin, P},
title = {Development of a Colorimetric and Raman Dual-Mode Biosensor for Point-of-Care Bacterial Detection Based on CRISPR/dCas9-Mediated Self-Assembly.},
journal = {ACS sensors},
volume = {11},
number = {9},
pages = {8100-8109},
doi = {10.1021/acssensors.6c01512},
pmid = {42691481},
issn = {2379-3694},
support = {2022xkjT006//Anhui Medical University/ ; 2308085QC112//Natural Science Foundation of Anhui Province/ ; 2025AHGXZK40750//University Natural Science Research Project of Anhui Province/ ; 2022YFF1102900//National Key Research and Development Program of China/ ; },
mesh = {*Biosensing Techniques/methods ; *Spectrum Analysis, Raman/methods ; *Colorimetry/methods ; Gold/chemistry ; *Point-of-Care Systems ; Metal Nanoparticles/chemistry ; *CRISPR-Cas Systems/genetics ; Humans ; Sulfhydryl Compounds/chemistry ; Aniline Compounds/chemistry ; Rapid Diagnostic Tests ; Escherichia coli/isolation & purification/genetics ; },
abstract = {Timely and reliable identification of bacterial infections in emergency settings remains a major challenge. To address this challenge, here we utilized CRISPR/dCas9-mediated self-assembly to develop a colorimetric and Raman dual-mode biosensor (referred to as dCARD) and applied it to point-of-care bacterial detection. Briefly, dCARD utilized the CRISPR/dCas9-mediated precise recognition of target amplicons to build bridges, attaching gold nanoparticle (GNP)-probe loaded with 4-aminothiophenol (4-ATP) onto the surface of GNP-streptavidin (GNP-SA). The dispersion-to-aggregation change of particles triggered a significant color change from red to purple, along with the formation of Raman hotspots. As a result, the presence of target bacteria was translated into cross-validated color and Raman signals, showcasing high reliability. This biosensor not only maintained the simplicity of visual readout but also integrated the quantitative function of a portable Raman spectrometer, allowing operators to flexibly switch signal modes in different scenarios. The specific primer-based isothermal amplification and CRISPR/dCas9-mediated precise recognition of amplicons together improved the specificity of dCARD, enabling accurate detection of the target bacteria down to about 10 CFU/mL without interference from non-target bacteria. By using a simplified preprocessing strategy, the assay time could be further reduced to less than 30 min. The robustness and programmability of dCARD were also demonstrated by detecting three common bacteria in real urine specimens. We expect this biosensor to evolve into a universal and field-deployable screening tool for bacterial infections.},
}

*Biosensing Techniques/methods
*Spectrum Analysis, Raman/methods
*Colorimetry/methods
Gold/chemistry
*Point-of-Care Systems
Metal Nanoparticles/chemistry
*CRISPR-Cas Systems/genetics
Humans
Sulfhydryl Compounds/chemistry
Aniline Compounds/chemistry
Rapid Diagnostic Tests
Escherichia coli/isolation & purification/genetics

@article {pmid42692324,
year = {2027},
author = {Li, W},
title = {A CRISPR/Cas9 multiplex nucleic acid sensing platform based on rolling circle amplification-mediated monomeric substrate generation.},
journal = {Analytical biochemistry},
volume = {720},
number = {},
pages = {116249},
doi = {10.1016/j.ab.2026.116249},
pmid = {42692324},
issn = {1096-0309},
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *MicroRNAs/blood/genetics ; Humans ; *Biosensing Techniques/methods ; },
abstract = {The multiplex detection of nucleic acid biomarkers, including circulating tumor DNAs (ctDNAs) and microRNAs (miRNAs), holds promise for improving the accuracy of molecular diagnostic techniques. In this study, we developed a CRISPR/Cas9-based sensing platform capable of simultaneously detecting multiple nucleic acid biomarkers. This approach leverages the activation of the CRISPR/Cas9 system through the generation of monomeric substrates mediated by rolling circle amplification (RCA). Specifically, upon the presence of a specific target, the RCA process is initiated, producing concatemeric DNA products. By incorporating an endonuclease and complementary reporter molecules, the RCA products are cleaved into monomeric substrates. Subsequently, Cas9/sgRNA complexes recognize and bind these monomeric substrates, activating the CRISPR/Cas9 systems, which further cleave the substrates to produce amplified fluorescence signals. Compared to conventional RCA methods, which often suffer from product aggregation, the monomeric substrate generation strategy demonstrated enhanced sensing capabilities and analytical performance, with signal intensities nearly double those observed in traditional approaches. To enable multiplex detection, three distinct sets of padlock probes, reporter molecules, and sgRNAs were designed to selectively target three different nucleic acid sequences. The platform exhibited high sensitivity and simultaneous detection of multiple ctDNAs and miRNAs, achieving limits of detection (LODs) ranging from 1.28 fM to 1.50 fM for ctDNAs and 0.61 fM to 0.97 fM for miRNAs. Additionally, analyses of clinical serum samples confirmed the method's accuracy and applicability within complex biological matrices. Collectively, this work introduces a multiplex nucleic acid biomarker detection platform with significant potential for advancing molecular sensing and clinical diagnostic applications.},
}

*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
*MicroRNAs/blood/genetics
Humans
*Biosensing Techniques/methods

@article {pmid42720009,
year = {2026},
author = {Yin, N and Zhang, L and Lu, R and Yu, H and Wang, W and Lv, K and Peng, Y and Han, X and Zhang, Y and Chen, Y and Zhang, Z and Guo, B and Yang, G and Xie, G},
title = {Input-Triggered Allosteric Activation of CRISPR-Cas12a by Modular Loop-Engineered Hairpins.},
journal = {ACS sensors},
volume = {11},
number = {9},
pages = {8440-8451},
doi = {10.1021/acssensors.6c02330},
pmid = {42720009},
issn = {2379-3694},
support = {82372351//National Natural Science Foundation of China/ ; 82572673//National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Associated Proteins/metabolism/genetics/chemistry ; *CRISPR-Cas Systems/genetics ; Allosteric Regulation ; *Endodeoxyribonucleases/metabolism/genetics/chemistry ; DNA, Single-Stranded/genetics/chemistry ; *Bacterial Proteins/metabolism/genetics/chemistry ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Inverted Repeat Sequences ; },
abstract = {Achieving precise control over CRISPR-Cas12a activity remains a fundamental challenge in the development of versatile sensing platforms, particularly for applications in diagnostics. Herein, we report an allosteric strategy that employs modular loop-engineered hairpin (MLEH) to precisely control Cas12a activation, thereby establishing a plug-and-play sensing framework with enhanced versatility. The MLEH comprises both the loop‑embedded ssDNA activator sequence and its complementary blocking domain. The input-triggered linearization of the MLEH via toehold-mediated strand displacement governs the accessibility of the activator, thereby controlling the binding and activation of Cas12a. A distinct advantage of this strategy is its elimination of the reliance on protospacer adjacent motif (PAM) sequences, customized CRISPR RNA (crRNA), or external activators. By merely substituting the responsive region of the MLEH, the system can be flexibly reconfigured to detect a wide range of targets, encompassing nucleic acids, proteins, and small molecules. MLEH-Cas12a broadens the scope of CRISPR technologies, offering a robust tool for diverse diagnostic applications. Collectively, our work delineates a novel allosteric regulatory mechanism for Cas12a activation and highlights the potential of engineered hairpins as key components in next-generation molecular sensing platforms, overcoming current limitations in CRISPR-based diagnostics and enabling broader target detection.},
}

*CRISPR-Associated Proteins/metabolism/genetics/chemistry
*CRISPR-Cas Systems/genetics
Allosteric Regulation
*Endodeoxyribonucleases/metabolism/genetics/chemistry
DNA, Single-Stranded/genetics/chemistry
*Bacterial Proteins/metabolism/genetics/chemistry
Clustered Regularly Interspaced Short Palindromic Repeats
*Inverted Repeat Sequences

@article {pmid42734255,
year = {2026},
author = {Liu, J and Li, L and Tang, Z and Li, Y and Zhou, F and He, Z and Ji, X},
title = {An aptamer-regulated CRISPR/Cas12a system based on split crRNA for theophylline and tobramycin detection.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {40},
pages = {9344-9354},
doi = {10.1039/d6ay01469k},
pmid = {42734255},
issn = {1759-9679},
mesh = {*Aptamers, Nucleotide/chemistry/genetics ; *Tobramycin/analysis ; *Theophylline/analysis/blood ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; *Biosensing Techniques/methods ; *Anti-Bacterial Agents/analysis ; },
abstract = {Most existing CRISPR/Cas12a methods for small molecules detection employ DNA aptamers as an intermediary, while RNA aptamers remain underexplored. The conventional CRISPR/Cas12 system offers limited programmability due to its partially conserved crRNA sequence. In this study, we developed an aptamer-regulated CRISPR/Cas12a system based on split crRNA. We divided the full crRNA into a crRNA handle segment (RH) and a crRNA spacer segment that is identical to the RNA aptamer sequence of target (RS-Apt). RS-Apt was endowed with dual functions: activating the activity of Cas12a and binding to target small molecules, ingeniously linking non-nucleic acid targets with the CRISPR system. The strong affinity between the target and RS-Apt reduces the amount of RS-Apt available to participate in the CRISPR system, resulting in a decrease in the fluorescence signal of the system. Within 1.5 h, the system for theophylline (TP) exhibited a linear range from 50 nM to 200 µM and a detection limit (LOD) of 2 nM, while the system for tobramycin (TOB) showed a linear range of 200 nM to 100 µM with an LOD of 63 nM. Notably, visual semi-quantitative detection of TP and TOB was achieved via lateral flow strips. Moreover, the system achieved satisfactory recovery and visual detection of TP using lateral flow strips in TP-spiked serum samples. This system can detect different targets simply by changing the sequence of the RS-Apt and activator, without the need for complex sequence design of the chains or large-scale instruments, providing a new strategy for CRISPR/Cas12a in the detection of small molecules.},
}

*Aptamers, Nucleotide/chemistry/genetics
*Tobramycin/analysis
*Theophylline/analysis/blood
*CRISPR-Cas Systems/genetics
Limit of Detection
*Biosensing Techniques/methods
*Anti-Bacterial Agents/analysis

@article {pmid42800399,
year = {2027},
author = {Yiğit, İ and Azrak, B and Uygun, ZO},
title = {CRISPR-dCas13a-based electrochemical biosensor for sensitive detection of miR-331-5p as a potential biomarker of Parkinson's disease.},
journal = {Analytical biochemistry},
volume = {720},
number = {},
pages = {116260},
doi = {10.1016/j.ab.2026.116260},
pmid = {42800399},
issn = {1096-0309},
mesh = {*Biosensing Techniques/methods ; *MicroRNAs/analysis/blood/genetics ; Humans ; *Electrochemical Techniques/methods ; *Parkinson Disease/diagnosis/genetics ; Biomarkers/analysis/blood ; Limit of Detection ; *CRISPR-Cas Systems/genetics ; Dielectric Spectroscopy ; Gold/chemistry ; Electrodes ; Dendrimers/chemistry ; },
abstract = {This study aimed to develop a CRISPR-dCas13a-based electrochemical biosensor as a proof-of-concept analytical platform for the sensitive and selective detection of miR-331-5p, which has been reported to be associated with Parkinson's disease. The biosensor was prepared by sequentially forming a self-assembled monolayer with 11-mercaptoundecanoic acid (MUA) on a gold electrode, functionalizing the surface with a generation-4 polyamidoamine (PAMAM-G4) dendrimer, immobilizing dCas13a, and assembling the target-specific sgRNA recognition complex. The electrode-modification steps were evaluated using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). The impedance response increased linearly with miR-331-5p concentration over the experimentally evaluated range of 5-400 pM. The limit of detection and limit of quantification were 7.82 and 23.71 pM, respectively. Responses to the non-target miRNAs miR-146a-5p, miR-19 b-3p, miR-24-3p, and miR-7-5p were substantially lower than the response to miR-331-5p. Triplicate spike-recovery measurements in healthy human serum and storage-stability studies supported the analytical applicability and reproducibility of the platform. These findings demonstrate a CRISPR-dCas13a/EIS-based proof-of-concept analytical platform for miR-331-5p detection. Validation in well-characterized clinical cohorts will be required before any diagnostic application can be established.},
}

*Biosensing Techniques/methods
*MicroRNAs/analysis/blood/genetics
Humans
*Electrochemical Techniques/methods
*Parkinson Disease/diagnosis/genetics
Biomarkers/analysis/blood
Limit of Detection
*CRISPR-Cas Systems/genetics
Dielectric Spectroscopy
Gold/chemistry
Electrodes
Dendrimers/chemistry

@article {pmid42886048,
year = {2026},
author = {Zhao, H and Wu, J and Wei, J and Chen, F},
title = {Triphenyl Phosphate Exposure Is Associated With BRG1-Related Cellular and Metabolic Readouts and EREG-EGFR/ERK Signalling in Glioma Cells.},
journal = {The international journal of medical robotics + computer assisted surgery : MRCAS},
volume = {22},
number = {5},
pages = {e70242},
doi = {10.1002/rcs.70242},
pmid = {42886048},
issn = {1478-596X},
mesh = {*Transcription Factors/metabolism ; Humans ; *Glioma/metabolism ; Cell Line, Tumor ; ErbB Receptors/metabolism ; *DNA Helicases/metabolism ; *Nuclear Proteins/metabolism ; *Organophosphates/toxicity/pharmacology ; Flame Retardants ; Signal Transduction/drug effects ; *MAP Kinase Signaling System/drug effects ; CRISPR-Cas Systems ; Promoter Regions, Genetic ; },
abstract = {BACKGROUND: Triphenyl phosphate (TPP), a ubiquitous organophosphate flame retardant and plasticiser, has emerged as a pervasive environmental contaminant with suspected endocrine-disrupting properties.

METHODS: In this study, we employed an integrated approach combining epigenetic CRISPR-Cas9 knockout screening, spatial transcriptomics, and metabolic flux analysis to explore the response of glioma cells to in vitro exposure to a nominal TPP concentration of 11 μM. Targeted cellular, biochemical, promoter-occupancy and signalling assays were performed.

RESULTS: TPP exposure was associated with changes in several cellular readouts in glioma cells, including a higher CCK-8 signal, differences in MMP secretion, Rac1 activity and matrix adhesion and differences in glucose, lactate and mitochondrial-respiration readouts. TPP exposure was also associated with increased BRG1 occupancy at the assayed epidermal growth factor receptor (EGFR) promoter region, together with higher EGFR- and epiregulin (EREG)-related readouts. This pattern is consistent with a candidate EREG-EGFR/MAPK-ERK pathway association but does not prove a self-sustaining loop or direct chemical recruitment. Spatial transcriptomic analysis described regional heterogeneity in mitochondrial-gene expression within public glioblastoma tissue.

CONCLUSION: Our findings support a candidate BRG1-associated cellular framework in which nominal TPP exposure is associated with BRG1-related chromatin and EGFR/metabolic readouts in glioma cells.},
}

*Transcription Factors/metabolism
Humans
*Glioma/metabolism
Cell Line, Tumor
ErbB Receptors/metabolism
*DNA Helicases/metabolism
*Nuclear Proteins/metabolism
*Organophosphates/toxicity/pharmacology
Flame Retardants
Signal Transduction/drug effects
*MAP Kinase Signaling System/drug effects
CRISPR-Cas Systems
Promoter Regions, Genetic

@article {pmid42886351,
year = {2026},
author = {Amrutha, K and Resmi, MS and Soniya, EV and Vivek, PJ},
title = {Calcium-dependent protein kinase signalling in plant immune responses: current status and future prospects.},
journal = {Planta},
volume = {264},
number = {6},
pages = {},
pmid = {42886351},
issn = {1432-2048},
mesh = {*Plant Immunity/physiology/genetics ; *Protein Kinases/metabolism/genetics ; *Signal Transduction ; Calcium-Calmodulin-Dependent Protein Kinases ; *Plant Proteins/metabolism/genetics ; *Plants/immunology/enzymology ; Calcium/metabolism ; Gene Expression Regulation, Plant ; },
abstract = {This review focuses on the critical role of CDPKs in advancing plant immunity research, highlighting how their functions can uncover key defence mechanisms and bridge critical knowledge gaps in the field. Calcium serves as the cornerstone of immune signalling, propagating pathogen-induced signals through specific sensors and decoders. Among these, calcium-dependent protein kinases (CDPKs) stand out as plant-specific signal transducers, directly converting calcium fluxes into defence activation. By modulating key immune components, CDPKs act as key hubs in plant immunity. Deciphering their role and crosstalks with other signalling networks is crucial for understanding the complexities of plant defence mechanisms. Although functional redundancy and extensive crosstalk among CDPK family members have historically hindered precise functional characterization, these features also highlight the robustness and adaptability of plant immune systems. Recent advances including high-throughput transcriptomics, comprehensive CDPK family-wide analyses, and multiplex genome-editing approaches such as CRISPR-Cas-based multi-gene targeting are now enabling finer functional resolution of CDPK members within complex multigene families. This review synthesizes current insights into CDPK-mediated signal transduction, with particular emphasis on their roles in coordinating defence responses and integrating with major signalling pathways, including mitogen-activated protein kinase (MAPK) cascades, reactive oxygen species (ROS) signalling, and WRKY transcription factor-mediated gene regulation. Importantly, the continued development of integrative and high-resolution approaches is poised to overcome longstanding limitations in the field, facilitating deeper dissection of CDPK-specific functions. Collectively, these advances position CDPK research at the forefront of plant immunity studies, with strong potential to reveal novel regulatory mechanisms.},
}

*Plant Immunity/physiology/genetics
*Protein Kinases/metabolism/genetics
*Signal Transduction
Calcium-Calmodulin-Dependent Protein Kinases
*Plant Proteins/metabolism/genetics
*Plants/immunology/enzymology
Calcium/metabolism
Gene Expression Regulation, Plant

@article {pmid42886411,
year = {2026},
author = {Kaknale, S and Ranjan, A and Enoch, IVMV and Vyas, A and Manikantan, V},
title = {Precision gene editing in infant neuroblastoma: a review of nanoparticle: enabled CRISPR/Cas9 therapies.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42886411},
issn = {1573-4978},
mesh = {Humans ; *Neuroblastoma/genetics/therapy ; *CRISPR-Cas Systems/genetics ; *Nanoparticles/chemistry ; *Gene Editing/methods ; Infant ; *Genetic Therapy/methods ; N-Myc Proto-Oncogene Protein/genetics ; Animals ; Anaplastic Lymphoma Kinase/genetics ; },
abstract = {Infant neuroblastoma (NB), defined by diagnosis during the first year of life, represents a clinically heterogeneous disease spectrum ranging from localized tumours with favourable outcomes and a capacity for spontaneous regression, to high-risk metastatic disease requiring intensive multimodal therapy. In infants, treatment intensity is determined by disease stage, tumor biology, MYCN status, risk classification, symptoms, response to therapy, and metastatic involvement, particularly relevant to high-risk disease, consequently the potential toxicity of intensive treatment is specific to high-risk disease, as compared to favorable risk tumors which can be managed with surgery, reduced frequency techniques, or observations. Important determinations of neuroblastoma biology and prognosis are genetic alterations involving MYCN and ALK, but the predominately investigated field with therapeutic feasibility of targeting these changes with CRISPR/Cas9 is in preclinical neuroblastoma models rather than in infants. An investigational hypothesis is accordingly considered by their potential relevance to infant neuroblastoma rather than an established clinical application. The safe and efficient in vivo delivery of CRISPR/Cas9 components remains a major challenge, owing to cargo size, immunogenicity, and degradation, particularly within the delicate physiological environment of infants. This review emphasises the need for advanced delivery systems, focusing on the potential of nanoparticle-mediated approaches. Emerging preclinical findings support the feasibility of nanoparticle-mediated CRISPR/Cas9 delivery, although direct evidence in infant neuroblastoma remains limited. Importantly, the evidence for nanoparticle-mediated CRISPR/Cas9 therapy remains largely preclinical. Studies performed in general neuroblastoma models, non-cancer models, or adult cancers should not be interpreted as direct evidence of efficacy in infant neuroblastoma.},
}

Humans
*Neuroblastoma/genetics/therapy
*CRISPR-Cas Systems/genetics
*Nanoparticles/chemistry
*Gene Editing/methods
Infant
*Genetic Therapy/methods
N-Myc Proto-Oncogene Protein/genetics
Animals
Anaplastic Lymphoma Kinase/genetics