@article {pmid41905426,
year = {2026},
author = {Jerin, C and Seo, W and Nishikawa, H},
title = {Genetic disruption of Pdcd-1 upstream enhancer boosts T cell function and antitumor responses.},
journal = {Immunology letters},
volume = {280},
number = {},
pages = {107171},
doi = {10.1016/j.imlet.2026.107171},
pmid = {41905426},
issn = {1879-0542},
mesh = {Animals ; *Programmed Cell Death 1 Receptor/genetics/metabolism ; Mice ; *Enhancer Elements, Genetic/genetics ; Mice, Knockout ; CRISPR-Cas Systems ; *T-Lymphocyte Subsets/immunology/metabolism ; Humans ; T-Cell Exhaustion ; *Neoplasms/immunology/genetics ; },
abstract = {Programmed cell death 1 (PD-1) is an inhibitory receptor that drives T cell exhaustion in tumors, limiting antitumor immunity. Current PD-1 blockade therapies have shown limited success. To uncover new strategies for modulating PD-1, we investigated an upstream enhancer (UpEnh) of the Pdcd-1 gene using a CRISPR-Cas9 knockout mouse model. Deletion of the UpEnh reduced PD-1 expression across various T cell subsets. In a tumor setting, this deletion lowered PD-1 levels on intratumoral exhausted CD8[+], conventional CD4[+], Treg, and γδ T cells. This resulted in improved CD8[+] and γδ T cell function and promoted stronger antitumor immunity. Our findings establish UpEnh as a critical regulator of PD-1, presenting a potential therapeutic target.},
}

Animals
*Programmed Cell Death 1 Receptor/genetics/metabolism
Mice
*Enhancer Elements, Genetic/genetics
Mice, Knockout
CRISPR-Cas Systems
*T-Lymphocyte Subsets/immunology/metabolism
Humans
T-Cell Exhaustion
*Neoplasms/immunology/genetics

@article {pmid41963733,
year = {2026},
author = {Le Phan, TH and Buchard, A and Brakebusch, C},
title = {Dispensable players: N-WASP and WASP are not crucial for homology-directed DNA repair.},
journal = {EMBO reports},
volume = {27},
number = {10},
pages = {2798-2822},
pmid = {41963733},
issn = {1469-3178},
support = {R302-A17455//Danish Cancer Society/ ; 101034291//EC | Horizon 2020 Framework Programme (H2020)/ ; },
mesh = {*Wiskott-Aldrich Syndrome Protein, Neuronal/genetics/metabolism ; Humans ; DNA Breaks, Double-Stranded ; *Wiskott-Aldrich Syndrome Protein/genetics/metabolism ; Actin-Related Protein 2-3 Complex/metabolism/antagonists & inhibitors/genetics ; *Recombinational DNA Repair ; Cell Line, Tumor ; DNA End-Joining Repair ; RNA, Small Interfering/genetics ; *DNA Repair ; CRISPR-Cas Systems ; Indoles ; },
abstract = {N-WASP and WASP can induce actin polymerization via Arp2/3 and were reported to be crucial for homology-directed repair (HDR) of DNA double-strand breaks (DSB). The underlying mechanism was suggested to involve nuclear actin polymerization, but the mechanistic details were debated. Unexpectedly, we show now that neither WASP nor N-WASP is required for HDR during CRISPR-mediated genome editing. Using knock-out and overexpression of N-WASP and WASP in U2OS cells, we did not detect alterations in total gene editing, HDR, or the ratio of HDR to non-homologous end joining (NHEJ) as assessed by different methods. Furthermore, we could not observe colocalization of HA-tagged WASP or N-WASP with DSBs. Finally, while the Arp2/3 inhibitor CK-666 and ARPC4 knockdown by siRNA reduced HDR efficiency in U2OS cells, this corresponded with a decreased transfection efficiency and a reduction of the HDR-proficient cell cycle phases S and G2/M. In summary, contrary to expectations, these data do not support a crucial role for N-WASP and WASP in DSB repair.},
}

*Wiskott-Aldrich Syndrome Protein, Neuronal/genetics/metabolism
Humans
DNA Breaks, Double-Stranded
*Wiskott-Aldrich Syndrome Protein/genetics/metabolism
Actin-Related Protein 2-3 Complex/metabolism/antagonists & inhibitors/genetics
*Recombinational DNA Repair
Cell Line, Tumor
DNA End-Joining Repair
RNA, Small Interfering/genetics
*DNA Repair
CRISPR-Cas Systems
Indoles

@article {pmid41985457,
year = {2026},
author = {Kim, J and Hwang, Y and Kim, S and Kwon, D and Park, J and Cho, B and An, S and Kang, S and Kim, Y and Kim, S and Lengner, CJ and Kim, S and Kwon, Y and Sung, JS and Kim, J},
title = {Electromagnetic field-inducible in vivo gene switch for remote spatiotemporal control of gene expression.},
journal = {Cell},
volume = {189},
number = {11},
pages = {3465-3480.e23},
doi = {10.1016/j.cell.2026.03.029},
pmid = {41985457},
issn = {1097-4172},
mesh = {Animals ; Kruppel-Like Factor 4 ; Humans ; Mice ; *Electromagnetic Fields ; Alzheimer Disease/genetics/pathology/metabolism ; Amyloid beta-Protein Precursor/genetics/metabolism ; *Gene Expression Regulation/radiation effects ; *Genes, Switch ; Tryptophan Hydroxylase/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Gaining precise control of gene expression is crucial in biomedical applications. However, spatiotemporal precision remains challenging. Here, we present a remotely controlled in vivo gene switch responsive to electromagnetic fields (EMFs) that enables precise spatiotemporal activation of target genes. We uncovered the EMF-inducible gene switch activation mechanism via a CRISPR-Cas9 screen, identifying cytochrome b5 type B (Cyb5b) as an essential mediator likely acting as an EMF sensor. The EMF-inducible gene switch was activated by rhythmic oscillatory calcium dynamics rather than generic calcium influx, defining a precisely tuned and bio-orthogonal induction mechanism. Functionally, EMF activation of the Oct4-Sox2-Klf4 (OSK) cassette induced in vivo partial reprogramming in aged mice, conditional expression of human mutant amyloid precursor protein (APP) for Alzheimer's disease (AD) modeling recapitulated pathological features, and EMF-mediated Tph2 expression restored serotonergic activity and ameliorated depressive-like behaviors in Tph2-mutant depression mice. Overall, a remotely controlled EMF-inducible gene switch represents a versatile and effective biomedical platform.},
}

Animals
Kruppel-Like Factor 4
Humans
Mice
*Electromagnetic Fields
Alzheimer Disease/genetics/pathology/metabolism
Amyloid beta-Protein Precursor/genetics/metabolism
*Gene Expression Regulation/radiation effects
*Genes, Switch
Tryptophan Hydroxylase/genetics/metabolism
CRISPR-Cas Systems

@article {pmid41992303,
year = {2026},
author = {Wang, Y and Guo, Y and Xiong, Y and Ren, X and Zhao, Y and Song, L and He, L},
title = {The stimuli-responsive CRISPR-Cas12a system for modulating the selective aggregation of cell membrane receptors.},
journal = {Journal of nanobiotechnology},
volume = {24},
number = {1},
pages = {},
pmid = {41992303},
issn = {1477-3155},
support = {82373630//National Natural Science Foundation of China/ ; 2023GGJS009//the training grant of Henan Province for Young Teachers/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Vascular Endothelial Growth Factor A/metabolism ; A549 Cells ; *Receptors, Transferrin/metabolism/genetics ; Proto-Oncogene Proteins c-met/metabolism/genetics ; *Receptors, Cell Surface/metabolism ; },
abstract = {While CRISPR systems exhibit remarkable programmability in the field of nucleic acid editing, their extension to protein engineering faces a fundamental challenge, namely the traditional CRISPR tools lack the design to efficiently convert stimulus signals into the selective clustering of membrane receptors. This study develops a stimulus-responsive membrane-confined CRISPR-Cas12a platform that enhances selective clustering of membrane receptors for functional regulation. Specifically, a membrane-anchored DNA tetrahedral framework (TD-apt) was designed, which leverages vascular endothelial growth factor (VEGF) to activate Cas12a. Compared with unconfined CRISPR-Cas12a, membrane-confined CRISPR-Cas12a exhibits stronger cleavage activity, the interaction between the cellular-mesenchymal epithelial transition factor (c-Met) receptor and transferrin receptor (TfR) on A549 cells was efficiently modulated by nucleic acid assembly. This manipulation selectively inhibited c-Met function through spatial steric hindrance of TfR, modulating cellular behavior. Notably, the system's generality was validated by engineering of c-Met homodimerization for activation. This cascading regulatory paradigm of environmental sensing (VEGF response)-nucleic acid computation (CRISPR-based nucleic acid molecular computation)-protein assembly (receptor topological remodeling) effectively extends CRISPR's application boundaries to the field of non-genetic regulation protein-protein interaction (PPI) and establishes a versatile toolkit for dynamic and precise functional regulation.},
}

*CRISPR-Cas Systems/genetics
Humans
Vascular Endothelial Growth Factor A/metabolism
A549 Cells
*Receptors, Transferrin/metabolism/genetics
Proto-Oncogene Proteins c-met/metabolism/genetics
*Receptors, Cell Surface/metabolism

@article {pmid42001720,
year = {2026},
author = {Li, X and Lu, H and Li, S and Chitrakar, B and Gu, X},
title = {Rapid and simple detection of Pediococcus using ARMS-CRISPR/Cas12a method.},
journal = {Talanta},
volume = {308},
number = {},
pages = {129806},
doi = {10.1016/j.talanta.2026.129806},
pmid = {42001720},
issn = {1873-3573},
mesh = {*Pediococcus/genetics/isolation & purification ; *RNA, Ribosomal, 16S/genetics ; *CRISPR-Cas Systems ; *Polymerase Chain Reaction/methods ; Polymorphism, Single Nucleotide ; },
abstract = {Pediococcus spp. are lactic acid bacteria, which are prevalent in various environments, including plants and animals. Notably, they constitute a significant component of the intestinal microbiota in both humans and animals. Despite this, numerous obstacles remain in developing tools that are both highly sensitive and specific for distinguishing this genus. This study established a fluorescent detection system using an amplification-resistant mutation system-based polymerase chain reaction (ARMS-CRISPR/Cas12a) for identifying 16S rRNA gene containing single nucleotide polymorphism (SNP) in Pediococcus spp. By aligning the sequences of Pediococcus spp. with those of other genera, we performed a comprehensive statistical analysis of SNP sites within Pediococcus spp. and designed specific primers using the 16S rRNA gene sequence of Pediococcus pentosaceus STS-6. The results demonstrated that, under optimised conditions (a Cas12a:crRNA ratio of 1:1 at 37 °C), the dual recognition process combining ARMS-PCR with CRISPR/Cas12a achieved high specificity and sensitivity in the detection of Pediococcus spp. The detection limit for genomic DNA was 8.15 × 10[-5] ng/μL, demonstrating significantly higher sensitivity than gel electrophoresis. The entire detection process took approximately 1.5 h. In summary, the ARMS-CRISPR/Cas12a detection system established in this study provided a rapid and effective method to detect the 16S rRNA gene of clinically relevant Pediococcus spp. probiotics, meeting the requirements for food production detection.},
}

*Pediococcus/genetics/isolation & purification
*RNA, Ribosomal, 16S/genetics
*CRISPR-Cas Systems
*Polymerase Chain Reaction/methods
Polymorphism, Single Nucleotide

@article {pmid42054950,
year = {2026},
author = {Wang, H and Yang, H and Zhong, K and Wang, R and Zhao, J and Zhao, Y and Zhang, G},
title = {Immune efficacy of two recombinant Turkey herpesviruses expressing the fusion protein of Newcastle disease virus genotype VII and hemagglutinin protein of H9N2 avian influenza virus generated by HDR/NHEJ-CRISPR/Cas9 systems.},
journal = {Veterinary microbiology},
volume = {318},
number = {},
pages = {111047},
doi = {10.1016/j.vetmic.2026.111047},
pmid = {42054950},
issn = {1873-2542},
mesh = {Animals ; *Influenza A Virus, H9N2 Subtype/immunology/genetics ; *Newcastle disease virus/genetics/immunology ; *Herpesvirus 1, Meleagrid/genetics/immunology ; *Influenza in Birds/prevention & control/virology/immunology ; CRISPR-Cas Systems ; Chickens ; *Viral Fusion Proteins/immunology/genetics ; Newcastle Disease/prevention & control/virology ; *Poultry Diseases/prevention & control/virology ; Genotype ; *Viral Vaccines/immunology ; Virus Shedding ; *Hemagglutinin Glycoproteins, Influenza Virus/immunology/genetics ; Chick Embryo ; Vaccines, Synthetic/immunology ; Vaccines, Attenuated/immunology ; Gene Editing ; },
abstract = {Newcastle disease viruses (NDV) and H9N2 avian influenza viruses (AIV) are two major threats to poultry farming. Current vaccination programs against two diseases are complex, requiring considerable labor and resources, and repeated immunizations can induce stress in animals. Therefore, developing a simplified, single-dose strategy capable of providing protection against both infections is highly desirable. Recombinant turkey herpesviruses (rHVT)-based live vaccines provide an attractive and effective platform for controlling avian viral diseases. This study generated two rHVTs, rHVT-OHA-OF(U) and rHVT-OHA-OF(H), each co-expressing F protein of NDV strain aSG10 and HA protein of H9N2 strain G, using a homologous directed repair (HDR) and non-homologous end-joining (NHEJ)-dependent CRISPR/Cas9-based gene editing strategy. In vitro, the two rHVTs correctly expressed the F and HA protein. After 10 passages in primary chicken embryonic fibroblasts (CEF) cells, the exogenous proteins remained stable expressed. In vivo, although rHVT-OHA-OF(H) provided strong protection against H9N2 strain G shedding at 3 and 5 days post-challenge (dpc), it offered no significant protection against mortality following challenge with NDV strain SG10. In contrast, rHVT-OHA-OF(U) provided 100% protection against mortality and significantly suppressed viral shedding following challenge with SG10, as well as achieving a significantly reduction of viral shedding at 3 dpc after H9N2 strain G challenge, indicating a promising vaccine candidate against both viral diseases, which requires further optimization. This study provided a reference for developing rHVT-based live vaccines targeting NDV and/or H9N2, thereby establishing a foundation for the design of dual- or multi-insert rHVTs.},
}

Animals
*Influenza A Virus, H9N2 Subtype/immunology/genetics
*Newcastle disease virus/genetics/immunology
*Herpesvirus 1, Meleagrid/genetics/immunology
*Influenza in Birds/prevention & control/virology/immunology
CRISPR-Cas Systems
Chickens
*Viral Fusion Proteins/immunology/genetics
Newcastle Disease/prevention & control/virology
*Poultry Diseases/prevention & control/virology
Genotype
*Viral Vaccines/immunology
Virus Shedding
*Hemagglutinin Glycoproteins, Influenza Virus/immunology/genetics
Chick Embryo
Vaccines, Synthetic/immunology
Vaccines, Attenuated/immunology
Gene Editing

@article {pmid42068913,
year = {2026},
author = {Shen, M and Zhang, P and Ding, L and Yang, X and He, L and Wu, Y and Yu, S},
title = {Direct microRNA detection via topologically engineered CRISPR/Cas12a cascade amplification assay.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {360},
number = {},
pages = {128016},
doi = {10.1016/j.saa.2026.128016},
pmid = {42068913},
issn = {1873-3557},
mesh = {*MicroRNAs/analysis/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Cell Line, Tumor ; CRISPR-Associated Proteins/metabolism ; *Endodeoxyribonucleases/metabolism ; Base Sequence ; Bacterial Proteins ; },
abstract = {The CRISPR/Cas12a system has emerged as a powerful tool for biosensing due to its high specificity, sensitivity and programmability. However, direct RNA detection is hindered by its inherent DNA-targeting trans-cleavage activity, which typically necessitates a reverse transcription amplification to convert RNA into DNA. Herein, we report a one-pot assay that enables direct detection of microRNA-21 without target amplification. The method employs a molecular switch probe (MSP) that recognizes miRNA-21 and activates Cas12a activity, along with an amplifier probe (AMP) that establishes a self-driven cascade signal amplification. This biosensor achieves a detection limit of 2.88 pM, with high accuracy (recovery of 95.5%-108.6%) and good precision (RSD: 2.35%-7.18%), and exhibits excellent specificity against homologous miRNAs. Using this assay, we successfully quantified the elevated levels of miRNA-21 in lung squamous carcinoma (H520) cells compared to normal bronchial epithelial cells (BEAS-2B). Furthermore, a methodological comparison with RT-qPCR revealed a similar trend between the two methods. This study provides a simple and reliable strategy for direct RNA detection using CRISPR/Cas12a.},
}

*MicroRNAs/analysis/genetics
Humans
*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
*Nucleic Acid Amplification Techniques/methods
Limit of Detection
Cell Line, Tumor
CRISPR-Associated Proteins/metabolism
*Endodeoxyribonucleases/metabolism
Base Sequence
Bacterial Proteins

@article {pmid42090855,
year = {2026},
author = {Tian, Y and Chen, J and Chen, F and Song, J and Shi, K and Yi, Z and Wang, S},
title = {A novel fluorescent aptasensor for sensitive and label-free detection of Di(2-ethylhexyl) phthalate based on catalytic hairpin assembly-assisted CRISPR/Cas9 and G-triplex.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {360},
number = {},
pages = {128020},
doi = {10.1016/j.saa.2026.128020},
pmid = {42090855},
issn = {1873-3557},
mesh = {*Diethylhexyl Phthalate/analysis ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Aptamers, Nucleotide/chemistry ; Spectrometry, Fluorescence/methods ; Limit of Detection ; Fluorescent Dyes/chemistry ; Plasticizers/analysis ; DNA/chemistry ; },
abstract = {Di(2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer that poses significant risks to human health and ecosystems, making its sensitive, rapid, and reliable detection critically important for environmental monitoring and food safety assessment. CRISPR-Cas9, beyond its canonical site-specific endonuclease function, has recently been reported to exhibit trans-cleavage activity upon target recognition, enabling its application in nucleic-acid-triggered signal amplification and biosensing. However, the exploration of Cas9-based trans-cleavage strategies for small-molecule targets such as DEHP remains challenging. By effectively integrating catalytic hairpin assembly (CHA) with Cas9-mediated trans-cleavage, a sensing platform termed Cas9/CHA was constructed for DEHP detection. Within this platform, aptamer-specific recognition of DEHP triggers the CHA reaction, producing abundant double-stranded DNA products that activate Cas9's DNase activity. The activated Cas9 system subsequently cleaves the loop region of the G3-based hairpin probe, releasing the G3 sequence. Upon binding to thioflavin T (ThT), the liberated G3 produces a fluorescence signal through the formation of a G3/ThT complex. Quantitative analysis is achieved by establishing a linear relationship between fluorescence intensity and DEHP concentration over the range of 10 pM to 1 μM. Importantly, the Cas9/CHA platform allows selective detection of DEHP down to 3 pM and shows reliable performance in milk, bottled water and tap water samples. The Cas9/CHA system extends CRISPR/Cas9 applications beyond nucleic acid analysis and provides a versatile and robust framework for developing next-generation analytical tools for food safety surveillance and environmental monitoring.},
}

*Diethylhexyl Phthalate/analysis
*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
*Aptamers, Nucleotide/chemistry
Spectrometry, Fluorescence/methods
Limit of Detection
Fluorescent Dyes/chemistry
Plasticizers/analysis
DNA/chemistry

@article {pmid42107569,
year = {2026},
author = {Cora, D and Seijas, A and Al-Soufi, W and Sánchez, L and Arana, ÁJ and Novo, M},
title = {Critical role of Cas9 aggregation on in vitro DNA cleavage.},
journal = {International journal of biological macromolecules},
volume = {366},
number = {},
pages = {152466},
doi = {10.1016/j.ijbiomac.2026.152466},
pmid = {42107569},
issn = {1879-0003},
mesh = {*DNA Cleavage ; *Protein Aggregates ; *CRISPR-Cas Systems ; *DNA/chemistry/metabolism ; *CRISPR-Associated Protein 9/chemistry/metabolism ; Osmolar Concentration ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing ; },
abstract = {The CRISPR/Cas9 system is a powerful genome-editing tool widely used in molecular biology and gene therapy, whose efficiency strongly depends on the physicochemical properties of the Cas9 ribonucleoprotein complex. Optimizing Cas9 activity remains essential for reliable genome-editing applications, yet the factors limiting its in vitro cleavage efficiency are not fully understood. Among these, protein aggregation has been suggested to critically impair Cas9 functionality, although its role has not been systematically analysed. Here, we investigate Cas9 aggregation under different environmental conditions and evaluate its impact on in vitro DNA cleavage efficiency. Using fluorescently labelled Cas9 and single-molecule fluorescence techniques, we quantify aggregation as a function of buffer composition, ionic strength, salt concentration and sgRNA presence, and relate these properties to cleavage activity. Our results show that Cas9 aggregation significantly reduces DNA cleavage efficiency, with higher aggregation levels consistently correlating with lower activity. In contrast, buffers with higher ionic strength or stabilizing components reduce aggregation and enhance Cas9 performance. Overall, this study demonstrates that Cas9 aggregation plays a critical role in determining in vitro cleavage efficiency and highlights the importance of controlling protein aggregation to optimize CRISPR/Cas9-based genome-editing applications and delivery strategies.},
}

*DNA Cleavage
*Protein Aggregates
*CRISPR-Cas Systems
*DNA/chemistry/metabolism
*CRISPR-Associated Protein 9/chemistry/metabolism
Osmolar Concentration
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Editing

@article {pmid42128117,
year = {2026},
author = {Zou, X and Gu, T and Li, X and Xia, X and Yang, M and Huo, D and Hou, C},
title = {One-tube, protospacer adjacent motif-free and AI-enhanced CRISPR/Cas12a platform for ultra-sensitive detection of human papillomavirus 16 double-stranded DNA.},
journal = {International journal of biological macromolecules},
volume = {366},
number = {},
pages = {152525},
doi = {10.1016/j.ijbiomac.2026.152525},
pmid = {42128117},
issn = {1879-0003},
mesh = {*Human papillomavirus 16/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; *DNA, Viral/genetics ; Humans ; Limit of Detection ; CRISPR-Associated Proteins ; },
abstract = {Cervical cancer, primarily caused by HPV16 infection, remains a major global health concern. While PCR is the gold standard for HPV DNA detection, its limitations include lengthy analysis and equipment dependency. To address these challenges, we developed an asymmetric semi-nested RPA combined with Lambda-assisted Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a CRISPR/Cas12a (ASN-RPA-LCRISPR) for protospacer adjacent motif (PAM) independent, ultrasensitive Human Papillomavirus 16 (HPV16) dsDNA detection. This method employs a three-primer asymmetric amplification strategy combined with Lambda nuclease and the CRISPR/Cas12a system to achieve signal amplification. This approach significantly increases single-stranded DNA yield while overcoming PAM restriction issues, achieving a detection limit of 11.8 aM within 35 min. A single-tube system designed in this study prevents aerosol contamination, while a DenseNet121-based image classifier enables 97% accurate fluorescence interpretation under ambient light, eliminating reliance on specialized equipment. This platform offers rapid, ultrasensitive, and portable HPV16 detection, advancing cervical cancer screening and PAM-free nucleic acid diagnostics.},
}

*Human papillomavirus 16/genetics/isolation & purification
*CRISPR-Cas Systems/genetics
*DNA, Viral/genetics
Humans
Limit of Detection
CRISPR-Associated Proteins

@article {pmid42132561,
year = {2026},
author = {Chen, H and Zhang, C},
title = {Simple yet sensitive MicroRNA detection using allosteric probe-initiated triple amplification and Cas13a/crRNA-based amplification reaction.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {20},
pages = {4154-4160},
doi = {10.1039/d6ay00578k},
pmid = {42132561},
issn = {1759-9679},
mesh = {*MicroRNAs/analysis/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Allosteric Regulation ; },
abstract = {MicroRNAs (miRNAs) have emerged as promising biomarkers for early diagnosis and management of degenerative disc disease (DDD); however, their low abundance and high sequence homology pose significant challenges for clinical detection. Herein, we develop a novel, highly sensitive miRNA detection platform by integrating an allosteric probe-initiated triple-cycle amplification strategy with the CRISPR-Cas13a/crRNA system. The designed allosteric probe undergoes a conformational switch upon target miRNA binding, triggering successive enzymatic amplification steps, including polymerase-mediated extension, nicking enzyme-driven recycling, and T7 RNA transcription, to generate numerous single-stranded RNA activators. These activators specifically recruit the Cas13a/crRNA complex, unleashing its collateral cleavage activity to degrade reporter RNAs and produce amplified fluorescence signals. This method demonstrates a wide dynamic range from 1 fM to 100 pM and achieves an ultra-low detection limit of 548 aM. Notably, the approach exhibits excellent specificity, distinguishing target miRNA-155 from closely related variants and non-target miRNAs. Its operational simplicity, rapid turnaround (60 min), and robustness in serum samples highlight strong potential for clinical translation. By combining catalytic allosteric probing with CRISPR-based signal amplification, this work provides a versatile and powerful tool for miRNA quantification, paving the way for early, minimally invasive diagnosis of degenerative disc diseases and other miRNA-associated pathologies.},
}

*MicroRNAs/analysis/genetics
Humans
*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
Limit of Detection
Allosteric Regulation

@article {pmid42142632,
year = {2026},
author = {Yang, Y and Zhang, T and Pan, Y and Wang, D and Lü, P},
title = {Development of a CRISPR-Cas13a assay for mouse hepatitis virus detection.},
journal = {Journal of virological methods},
volume = {344},
number = {},
pages = {115407},
doi = {10.1016/j.jviromet.2026.115407},
pmid = {42142632},
issn = {1879-0984},
mesh = {Animals ; *Murine hepatitis virus/isolation & purification/genetics ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Mice ; *Hepatitis, Viral, Animal/diagnosis/virology ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; },
abstract = {Mouse hepatitis virus (MHV) is a significant pathogen that undermines the health of laboratory animals and the reliability of research data. The development of rapid and sensitive detection methods for MHV is therefore crucial for ensuring laboratory animal quality and maintaining laboratory biosafety. Here, we established a CRISPR-Cas13a-based assay targeting the E, M, and N genes of MHV by constructing a recombinase-aided amplification (RAA)-coupled detection system. Results demonstrated that the N1 target exhibited the highest sensitivity, achieving a detection limit of 0.1 copy/μL for plasmid templates and 1 copy/μL in spiked serum samples. The system also exhibited robust specificity and a strong signal response in spiked serum samples. Furthermore, we developed a multiplex amplification-free CRISPR-Cas13a assay by combining Cas13a/crRNAs targeting different genes, which achieved a detection limit of 3.06 pM. In summary, this study presents two distinct CRISPR-Cas13a-based strategies for MHV detection, demonstrating the considerable application potential of this technology for pathogen detection in laboratory animal settings.},
}

Animals
*Murine hepatitis virus/isolation & purification/genetics
*CRISPR-Cas Systems
Sensitivity and Specificity
Mice
*Hepatitis, Viral, Animal/diagnosis/virology
*Nucleic Acid Amplification Techniques/methods
Limit of Detection

@article {pmid42166810,
year = {2026},
author = {Huang, H and Yang, K and Wang, C and Bu, R and Chen, W},
title = {Heptameric transmembrane assembly of the CRISPR-Cas13b regulator Csx27.},
journal = {Biochemical and biophysical research communications},
volume = {824},
number = {},
pages = {153975},
doi = {10.1016/j.bbrc.2026.153975},
pmid = {42166810},
issn = {1090-2104},
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/chemistry/metabolism ; Protein Multimerization ; *Cell Membrane/metabolism/chemistry ; Cryoelectron Microscopy ; *Bacterial Proteins/chemistry/metabolism ; Models, Molecular ; *Membrane Proteins/chemistry/metabolism ; },
abstract = {The type VI-B CRISPR-Cas13 system is an RNA-guided immune pathway in which the Cas13b effector is negatively regulated by the accessory protein Csx27. However, the structural and biochemical basis of Csx27 function remains poorly defined. Here, we characterize Bergeyella zoohelcum Csx27 as a membrane associated oligomeric protein. Bioinformatic prediction, detergent dependent purification, and membrane fractionation followed by western blotting support its membrane association. Native PAGE of Csx27 and BS3 cross-linking of Csx27 reconstituted in MSP2N2 nanodiscs further provide orthogonal evidence for oligomerization. Consistently, cryo-EM 2D class averages revealed ring-like particles with apparent sevenfold symmetry, and AlphaFold-assisted modeling supported a heptameric arrangement. Together, our results provide the first direct experimental evidence for the oligomeric, membrane-embedded assembly of Csx27, establishing a crucial structural foundation for elucidating its regulatory mechanism within the CRISPR-Cas13b immune response.},
}

*CRISPR-Cas Systems
*CRISPR-Associated Proteins/chemistry/metabolism
Protein Multimerization
*Cell Membrane/metabolism/chemistry
Cryoelectron Microscopy
*Bacterial Proteins/chemistry/metabolism
Models, Molecular
*Membrane Proteins/chemistry/metabolism

@article {pmid42206605,
year = {2026},
author = {Waller, MA and D'Araujo, TY and Denes, CE and Neely, GG},
title = {Dissecting conserved molecular mechanisms of biological toxin activity through CRISPR screening.},
journal = {Biochemical Society transactions},
volume = {54},
number = {6},
pages = {601-620},
doi = {10.1042/BST20250099},
pmid = {42206605},
issn = {1470-8752},
support = {GNT2020532//National Health and Medical Research Council/ ; DP220103530//Australian Research Council/ ; },
mesh = {*CRISPR-Cas Systems ; *Toxins, Biological/genetics/metabolism ; Humans ; Animals ; Intracellular Space/metabolism ; },
abstract = {Toxins, substances that are produced by living organisms with the potential to cause harm, demonstrate great diversity in their structure, function, and origin. Though some toxins have been repurposed for use as novel therapeutics, research tools, or for application in agriculture, the mechanism of action for many toxins remains uncharacterised. Pooled CRISPR screens offer a high-throughput and unbiased method for rapid annotation of the host cell genome and identification of factors mediating or modifying intoxication. In this review, we provide a brief overview of CRISPR screening before detailing how screens have been used to characterise toxins from various biological kingdoms. We highlight certain cell entry factors and intracellular processes as conserved targets of various toxins. Finally, we highlight limitations in the methods of CRISPR screens used thus far and make recommendations as to how screen design can be modified to more completely characterise toxin activity and elucidate systemic effects of intoxication.},
}

*CRISPR-Cas Systems
*Toxins, Biological/genetics/metabolism
Humans
Animals
Intracellular Space/metabolism

@article {pmid42206706,
year = {2026},
author = {Hu, Y and Zhu, H and Diao, Y and Zhou, L and Bai, C},
title = {[Development and preliminary clinical evaluation of a CRISPR-AaCas12b-based nucleic acid detection method for Mycobacterium tuberculosis].},
journal = {Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology},
volume = {42},
number = {5},
pages = {428-434},
pmid = {42206706},
issn = {1007-8738},
mesh = {*Mycobacterium tuberculosis/genetics/isolation & purification ; *Bacterial Proteins/genetics/metabolism ; *Alicyclobacillus/genetics ; Humans ; *Tuberculosis/diagnosis/microbiology ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/genetics/metabolism ; },
abstract = {Objective To express and purify Alicyclobacillus acidiphilus Cas12b (AaCas12b) protein, and establish a rapid clustered regularly interspaced short palindromic repeats (CRISPR)-based assay for Mycobacterium tuberculosis (Mtb) detection, thereby providing a novel tool for clinical diagnosis of tuberculosis (TB). Methods Following construction and transformation of the recombinant expression vector into E. coli BL21 (DE3), soluble expression of the recombinant AaCas12b protein was induced with low-concentration isopropyl β-D-thiogalactopyranoside (IPTG) at low temperature, followed by purification using nickel-affinity chromatography. Subsequently, a specific recombinase polymerase amplification (RPA) assay targeting the IS6110 insertion sequence of Mtb was developed, and the corresponding single guide RNA (sgRNA) was prepared via in vitro transcription. Then a novel CRISPR-AaCas12b detection assay for Mtb was established, and its diagnostic performance was systematically evaluated against conventional clinical methods including acid-fast staining, T-SPOT.TB assay, Mycobacterium culture, and GeneXpert assay. Results A prokaryotic expression and purification system for recombinant AaCas12b was successfully established, yielding AaCas12b protein with activity at a concentration of 13.42 mg/mL and a recovery rate of 61.45%. A specific RPA-CRISPR-AaCas12b detection system was established targeting the IS6110 sequence, with a limit of detection (LOD) of 1.5 CFU/mL for Mtb. In clinical sample validation, the system achieved a sensitivity of 95.83% (95% CI: 79.97%~99.27%), a specificity of 92.31% (95% CI: 75.88%~97.88%), and an accuracy of 94.00% (95% CI: 83.78%~97.93%). Conclusion The established AaCas12b protein preparation system is efficient and stable, enabling the production of low-cost, high-activity tool enzymes. The RPA-CRISPR-AaCas12b TB diagnostic assay targeting the IS6110 sequence exhibits excellent specificity and sensitivity, providing a technical reference and experimental basis for the further development of rapid TB diagnostic platforms.},
}

*Mycobacterium tuberculosis/genetics/isolation & purification
*Bacterial Proteins/genetics/metabolism
*Alicyclobacillus/genetics
Humans
*Tuberculosis/diagnosis/microbiology
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*CRISPR-Cas Systems
*CRISPR-Associated Proteins/genetics/metabolism

@article {pmid42207849,
year = {2026},
author = {Wu, Y and Shen, F and Hu, Q and Yu, Z and Ye, Z and Ding, X},
title = {Recent advances in integrated CRISPR/Cas biosensing for aquatic food safety: overcoming the matrix interference and the difficulties in point-of-care testing.},
journal = {Critical reviews in food science and nutrition},
volume = {},
number = {},
pages = {1-22},
doi = {10.1080/10408398.2026.2678519},
pmid = {42207849},
issn = {1549-7852},
abstract = {Rapid, sensitive, and on-site detection of pathogenic bacteria in aquatic-product and aquaculture-associated samples is critical for food safety. Although CRISPR/Cas has emerged as a powerful biosensing tool, its practical application is hindered by complex aquatic matrices and the difficulty of integrating pretreatment, amplification, and signal readout into point-of-care testing (POCT) workflows. This review examines integrated CRISPR/Cas biosensing strategies for pathogenic bacterial detection in aquatic matrices. It outlines the enzymatic features of core effectors, reviews pretreatment methods for complex samples, and summarizes how CRISPR/Cas is combined with isothermal amplification to improve analytical sensitivity. It also compares biosensing platforms from the perspective of matrix interference and field applicability. The analysis indicates that isothermal amplification is essential for detecting low-abundance targets in complex aquatic matrices. Among current strategies, ratiometric fluorescence, magnetic separation-assisted sensing, and signal-on electrochemical reporting show particular promise because they improve calibration, reduce matrix background, and limit nonspecific signal loss, respectively. Future progress depends on standardized sample processing, quantitative multiplexing, and automated "sample-in, result-out" systems for real-world deployment.},
}

@article {pmid42209465,
year = {2026},
author = {Ghiotto, G and Zampieri, G and Orellana, E and Chatzis, A and Kougias, PG and Camargo, A and Roux, S and Campanaro, S and Kyrpides, NC and Treu, L},
title = {Single nucleotide variants drive evolutionary phage-host arms race in anaerobic carbon dioxide-converting microbiome.},
journal = {Nature communications},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41467-026-73084-2},
pmid = {42209465},
issn = {2041-1723},
abstract = {Microbial bioconversions are shaped by environmental perturbations and the adaptation of resident microbiomes. Prokaryotes coexist with bacteriophages, yet their coevolutionary trajectories remain underexplored. Here, we investigate the effects of a cultivation vessel leak on an anaerobic consortium performing carbon dioxide reduction. Using time-series shotgun metagenomic sequencing, we reconstruct microbial and viral genomes to track community shifts. We further apply single-nucleotide variant profiling and CRISPR array analysis to monitor viral microdiversity and host defense mechanisms. After bioaugmentation restores bioconversion efficiency, the consortium undergoes pronounced restructuring, with new dominant taxa emerging from the rare biosphere. We identify patterns consistent with phage predation selectively removing certain species, while others exhibit resilience to infection. This shift aligns with a widespread viral outbreak and a transient increased frequency of single nucleotide variants in bacterial CRISPR-Cas defense genes. Expansion of CRISPR spacers further supports that CRISPR-mediated processes influence microbial resilience. Concurrently, phages infecting resilient hosts exhibited adaptive evolution, marked by high genetic heterogeneity. Selective pressure varies across their genomes, targeting infectivity genes and protospacer-adjacent motifs. These findings highlight a dynamic evolutionary arms race driven by the selection of beneficial genetic variants, providing a mechanistic framework for multi-omics investigations, and informing biotechnological applications, including phage-based microbiome manipulation.},
}

@article {pmid42211656,
year = {2026},
author = {Nuevo, JJM and Fortaleza, JAG and Cabuhat, KSP and Ong, CJN and Jalova, AC and Mortel, FA and Bacalzo, GD},
title = {CRISPR-driven strategies to disrupt methicillin-resistant Staphylococcus aureus biofilms: a review.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1784529},
pmid = {42211656},
issn = {2235-2988},
mesh = {*Biofilms/drug effects/growth & development ; *Methicillin-Resistant Staphylococcus aureus/genetics/drug effects/physiology ; *CRISPR-Cas Systems ; Humans ; Quorum Sensing ; Anti-Bacterial Agents/pharmacology ; Staphylococcal Infections/microbiology/therapy ; Virulence Factors/genetics ; },
abstract = {Methicillin-resistant Staphylococcus aureus exhibits heightened tolerance to antimicrobial therapy through restricted drug penetration, extracellular polymeric substance-mediated protection, metabolic heterogeneity, and persister cell formation, limiting the effectiveness of current treatment strategies. CRISPR-Cas systems have emerged as programmable antimicrobial tools capable of targeting resistance genes, virulence determinants, and regulatory pathways; however, existing approaches remain largely gene-centric and insufficiently integrated with the biological complexity of biofilm-associated infections. This review aims to provide a comprehensive and integrative analysis of CRISPR-based strategies for targeting MRSA biofilms by linking molecular CRISPR mechanisms with key biofilm processes and evaluating their translational potential. CRISPR-Cas systems have emerged as programmable antimicrobial platforms with the ability to selectively target resistance genes, virulence factors, and regulatory networks. In MRSA biofilms, these systems are increasingly being explored for their potential to disrupt biofilm-associated determinants and weaken the molecular basis of persistence. Recent advances involving Cas9, Cas12a, and Cas13 highlight the potential of CRISPR-based targeting to interfere with resistance mechanisms, quorum sensing pathways, and structural components relevant to biofilm stability. Emerging in vivo studies, particularly those using engineered bacteriophages and localized delivery systems, provide early evidence that CRISPR-based strategies can reduce bacterial burden and impair biofilm integrity under physiologically relevant conditions. Nevertheless, significant barriers remain, including limited penetration into mature biofilms, delivery inefficiency, off-target activity, immunogenicity, resistance evolution, and regulatory uncertainty. Ultimately, CRISPR-based interventions represent a promising but still developing approach for the control of MRSA biofilm-associated infections, requiring further refinement in delivery design, target selection, and translational validation.},
}

*Biofilms/drug effects/growth & development
*Methicillin-Resistant Staphylococcus aureus/genetics/drug effects/physiology
*CRISPR-Cas Systems
Humans
Quorum Sensing
Anti-Bacterial Agents/pharmacology
Staphylococcal Infections/microbiology/therapy
Virulence Factors/genetics

@article {pmid42211740,
year = {2026},
author = {Matsugi, E and Kishi, K and Kishi, A and Nagase, K and Nigorikawa, K and Nomura, W},
title = {SauCas9-based cell cycle-dependent genome editing via AAV delivery.},
journal = {Molecular therapy. Advances},
volume = {34},
number = {2},
pages = {201751},
pmid = {42211740},
issn = {3117-387X},
abstract = {The CRISPR-Cas system is a widely used genome editing technology with diverse applications. Although homology-directed repair (HDR) offers precise gene editing, its efficiency is typically lower than that of non-homologous end joining (NHEJ). Building on our previous cell cycle-dependent genome editing system for Streptococcus pyogenes Cas9 (SpyCas9), we adopted this approach for Staphylococcus aureus Cas9 (SauCas9) to enable efficient adeno-associated virus (AAV) delivery. To enhance HDR efficiency and editing accuracy in the context of AAV delivery, we developed a cell cycle-dependent genome editing system. We screened 10 anti-CRISPR (Acr) candidates and identified AcrIIA5, A13, A14, A15, and C1 as potent inhibitors of SauCas9. The fusion of these Acrs with the Cdt1(30-120) fragment restricted SauCas9 activity to the S/G2 phases, where HDR is predominant. Although AcrIIA11 and AcrIIA16 alone showed weak inhibition, their Cdt1 fusions (AcrIIA11+Cdt1 and AcrIIA16+Cdt1) showed a 2-fold increase in HDR efficiency within the AAV delivery system. This AAV-based, cell cycle-dependent SauCas9 system, which leverages optimized Acr-Cdt1 fusions, holds promise for improving the efficiency and accuracy of in vivo genome editing. Its small size is ideal for AAV packaging and may offer fewer off-target effects.},
}

@article {pmid42212714,
year = {2026},
author = {Zhang, F and Yan, D and Hou, J and Yang, D and Xiong, Y and Wen, M and Zhu, X},
title = {Writing Big in Plant Genomes: Advances, Challenges and Strategies for Targeted Large-Fragment DNA Insertion.},
journal = {Plant, cell & environment},
volume = {},
number = {},
pages = {},
doi = {10.1111/pce.70634},
pmid = {42212714},
issn = {1365-3040},
support = {2023YFA0913500//National Key Research and Development Program of China/ ; 25HHWCSS0007//Haihe Laboratory of Sustainable Chemical Transformations/ ; 32230012//National Natural Science Foundation of China/ ; 32470363//National Natural Science Foundation of China/ ; 32201738//National Natural Science Foundation of China/ ; },
abstract = {Precise genome editing has transformed plant biology and crop improvement by enabling targeted modification of endogenous loci. Beyond gene knockout and base editing, the site-specific insertion of exogenous DNA, particularly large DNA fragments, has become a central goal for engineering complex traits, reconstructing metabolic pathways and constructing plant artificial chromosomes. A diverse toolkit is now available for targeted DNA integration, including nuclease-dependent strategies, serine and tyrosine recombinases, transposon-derived systems, and CRISPR/Cas-coupled insertion platforms. Here, we review the mechanistic principles and recent advances of these four major tools, highlighting their capacities, insertion precision and compatibility with plant systems. We compare their strengths and limitations in terms of insertion-size capacity, integration efficiency, target site flexibility and technical complexity. Emerging innovations such as AI-guided nuclease and recombinase design, fusion of Cas with recombinases or viral replication proteins and RNA-guided transposition offer promising solutions to overcome these constraints. Together, these advances are rapidly expanding the landscape of targeted DNA insertion in plants and will reinforce future applications in molecular breeding, metabolic pathway engineering and the construction of synthetic genomic architectures.},
}

@article {pmid41833124,
year = {2026},
author = {Park, JS and Kim, YM and Lee, HJ and Han, JY},
title = {Research note: Generation of ovalbumin-null chickens and characterization of altered protein compositions in their egg whites.},
journal = {Poultry science},
volume = {105},
number = {6},
pages = {106715},
pmid = {41833124},
issn = {1525-3171},
mesh = {Animals ; *Chickens/genetics/metabolism/physiology ; *Ovalbumin/genetics/metabolism ; Female ; *Egg Proteins/metabolism/genetics ; *Egg White/chemistry ; Chick Embryo ; CRISPR-Cas Systems ; Gene Knockout Techniques/veterinary ; Animals, Genetically Modified/genetics ; },
abstract = {Chickens are considered an efficient bioreactor platform for production of recombinant proteins due to their high egg laying rate and high capacity to produce proteins in egg white. Ovalbumin (OVAL) comprises 54% of egg white proteins, and targeted insertion of a recombinant protein construct into the OVAL locus leads to significant accumulation of the recombinant protein in egg white. However, it was reported that embryos could not develop or hatch from eggs laid by heterozygous OVAL-knockout hens in which OVAL gene was replaced by foreign protein coding sequences, a limitation that restricted the generation of homozygous OVAL-knockout chickens. In this study, we specifically targeted the OVAL locus using CRISPR/Cas9 and successfully generated OVAL-null chickens by mating heterozygous individuals. Both heterozygous and homozygous OVAL knockout embryos developed and hatched, notably, we found that embryos could develop and hatch from OVAL-deficient eggs, although the hatching rate was reduced by 52.30±14.42%. Furthermore, analysis revealed that concentrations of other major egg white proteins increased, indicating a compensatory accumulation of proteins in response to the removal of OVAL. Collectively, this study demonstrates that OVAL-null chicken lines can be established and that these chickens produce eggs with altered egg white protein compositions.},
}

Animals
*Chickens/genetics/metabolism/physiology
*Ovalbumin/genetics/metabolism
Female
*Egg Proteins/metabolism/genetics
*Egg White/chemistry
Chick Embryo
CRISPR-Cas Systems
Gene Knockout Techniques/veterinary
Animals, Genetically Modified/genetics

@article {pmid41846089,
year = {2026},
author = {Zhang, C and Wang, M and He, B and Yang, X and Li, XZ},
title = {High genotoxicity of CRISPR/Cas9 versus limited efficacy of CRISPRi in chicken primordial germ cells.},
journal = {Poultry science},
volume = {105},
number = {6},
pages = {106722},
pmid = {41846089},
issn = {1525-3171},
mesh = {Animals ; *Chickens/genetics ; *CRISPR-Cas Systems ; *Germ Cells ; *Gene Editing/veterinary/methods ; *DNA Damage ; Humans ; Male ; Female ; },
abstract = {CRISPR/Cas9 technology has transformed genome editing across species; however, its application in avian germ cells remains constrained-not only by editing efficiency, but also by limited evaluation of potential genotoxic effects. In this study, we systematically assessed the performance and genomic safety of CRISPR/Cas9 and CRISPR interference (CRISPRi) in chicken primordial germ cells (PGCs). While CRISPR/Cas9 achieved high editing efficiency, it simultaneously induced substantial DNA damage, apoptosis, and sex-specific cell cycle arrest, revealing the pronounced genotoxic sensitivity of PGCs. In contrast, CRISPRi was well tolerated but failed to achieve effective gene repression in chicken cells. Comparative experiments showed that CRISPRi functioned efficiently in human 293T cells but not in chicken PGCs or somatic DF-1 cells, suggesting species-dependent limitations of mammalian-optimized repression systems. Together, these findings reveal a fundamental trade-off-"efficient but toxic" versus "safe but ineffective"-when applying CRISPR tools to avian germ cells. Our results highlight the need for species-adapted, low-toxicity genome-editing platforms in poultry and provide a framework for evaluating editing strategies in developmentally sensitive cell types.},
}

Animals
*Chickens/genetics
*CRISPR-Cas Systems
*Germ Cells
*Gene Editing/veterinary/methods
*DNA Damage
Humans
Male
Female

@article {pmid41850054,
year = {2026},
author = {Jia, W and Wang, A and Wu, Z and Shi, L and Xie, J and Zhou, Q and Cheng, Y and Lei, X and Liu, L and Tian, L and Zhu, S},
title = {Construction and characterization of recombinant duck enteritis virus expressing duck hepatitis A virus 3 immunogenic genes.},
journal = {Poultry science},
volume = {105},
number = {6},
pages = {106780},
pmid = {41850054},
issn = {1525-3171},
mesh = {Animals ; *Ducks ; *Poultry Diseases/prevention & control/virology/immunology ; *Viral Vaccines/immunology ; *Picornaviridae Infections/veterinary/prevention & control/immunology/virology ; *Hepatitis Virus, Duck/immunology ; CRISPR-Cas Systems ; *Hepatitis, Viral, Animal/prevention & control ; },
abstract = {Duck enteritis virus (DEV) is considered an ideal vector for waterfowl vaccine development due to its favorable safety profile and multiple genomic sites that accommodate foreign gene insertion. Duck hepatitis A virus (DHAV) causes acute hepatitis, neurological symptoms, and high mortality in young ducklings, and the predominant circulating serotype has shifted from type 1 to type 3, underscoring the urgent need for improved vaccines. In this study, we employed a CRISPR/Cas9 genome editing platform combined with dual single-guide RNAs (sgRNAs) and a homologous directed repair (HDR) strategy to construct two recombinant DEV strains (rDEV-DHAV) expressing the immunogenic VP0 or VP1 proteins of DHAV-3. The recombinants were purified by plaque selection and validated using PCR, Western blotting, indirect immunofluorescence, and animal experiments. Both recombinant viruses replicated efficiently in chicken embryo fibroblasts and exhibited growth kinetics comparable to the parental DEV vaccine strain. The inserted VP0 and VP1 genes remained genetically stable over at least 15 serial passages. Immunization trials in ducklings demonstrated that both recombinants elicited strong humoral responses against DEV and DHAV-3. Safety evaluation showed that neither recombinant virus induced clinical signs, pathological lesions, or abnormal viral shedding, and both displayed safety profiles equivalent to the parental vaccine strain. Overall, the two rDEV-DHAV strains generated in this study are genetically stable, safe, and exhibit good immunogenicity. The HDR-CRISPR/Cas9 strategy employing dual sgRNAs provides an efficient approach for the rapid construction of multivalent DEV vector vaccines, highlighting its substantial potential in poultry vaccine development.},
}

Animals
*Ducks
*Poultry Diseases/prevention & control/virology/immunology
*Viral Vaccines/immunology
*Picornaviridae Infections/veterinary/prevention & control/immunology/virology
*Hepatitis Virus, Duck/immunology
CRISPR-Cas Systems
*Hepatitis, Viral, Animal/prevention & control

@article {pmid41887170,
year = {2026},
author = {Liao, Y and Liang, L and Liang, R and Ding, J and Hu, D and Si, H and Song, X and Tang, X},
title = {Abnormal expression pattern of knock-in marker in Eimeria tenella using CRISPR/Cas9.},
journal = {Poultry science},
volume = {105},
number = {6},
pages = {106763},
pmid = {41887170},
issn = {1525-3171},
mesh = {*Eimeria tenella/genetics ; *CRISPR-Cas Systems ; *Gene Knock-In Techniques/veterinary ; *Gene Editing/methods ; Animals ; Chickens ; },
abstract = {Gene editing technology has been widely applied in the genetic manipulation of many organisms and is increasingly being utilized in eukaryotic pathogens. However, its efficiency often requires improvement. In our study using CRISPR/Cas9 to genetically manipulate Eimeria tenella, we aimed to insert a tag into a target gene locus via homologous recombination, but observed outcomes inconsistent with expectations. Whole-genome sequencing analysis of the integration sites revealed that the transgenic E. tenella did not exhibit correct targeted integration. These results indicate that creating double-strand breaks (DSB) at specific genomic sites to trigger homology-directed repair (HDR) for gene modification can lead to mislocalized expression. This study provides insights for utilizing CRISPR/Cas9 technology in genetic editing, particularly in E. tenella, and offers suggestions for improving strategies that employ the co-transfection of multiple plasmids, such as Cas9-gRNA and donor plasmids.},
}

*Eimeria tenella/genetics
*CRISPR-Cas Systems
*Gene Knock-In Techniques/veterinary
*Gene Editing/methods
Animals
Chickens

@article {pmid42063341,
year = {2026},
author = {Nelson, C and Ambros, V},
title = {Multi-dimensional regulation of LIN-28 temporal expression dynamics in the C. elegans heterochronic gene cascade.},
journal = {Development (Cambridge, England)},
volume = {153},
number = {10},
pages = {},
doi = {10.1242/dev.205391},
pmid = {42063341},
issn = {1477-9129},
support = {R35GM131741/NH/NIH HHS/United States ; R35GM131741/NH/NIH HHS/United States ; //University of Massachusetts Chan Medical School/ ; },
mesh = {Animals ; *Caenorhabditis elegans/genetics/metabolism ; *Caenorhabditis elegans Proteins/genetics/metabolism ; MicroRNAs/genetics/metabolism ; *Gene Expression Regulation, Developmental ; 3' Untranslated Regions/genetics ; *RNA-Binding Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Repressor Proteins ; },
abstract = {LIN-28 is an evolutionarily conserved RNA-binding protein that is crucial for regulating pluripotency and cell fate determination during animal development. In Caenorhabditis elegans, lin-28 is an integral component of the heterochronic (developmental timing) gene regulatory cascade. Loss-of-function mutations in lin-28 cause precocious cell fate determination during larval development. Previous studies indicate that proper progression of larval stage-specific cell fates relies on the downregulation of LIN-28, which is negatively regulated by the lin-4 microRNA through complementary sequences in the lin-28 3' untranslated region (UTR). This study employs CRISPR/Cas9 editing of the endogenous lin-28 locus to demonstrate that developmental downregulation of LIN-28 involves multiple inputs, including the action of the let-7 family and lin-4 microRNAs via adjacent complementary sites in the lin-28 3' UTR, along with post-translational inhibition of LIN-28 by the lep-5 long non-coding RNA, collectively accounting for nearly all LIN-28 repression. Additionally, systematic testing of truncations of the lin-28 3' UTR identifies three positive regulatory regions that enhance LIN-28 expression, counteracting the negative effects of the let-7 and lin-4 microRNAs and the lep-5 long non-coding RNA.},
}

Animals
*Caenorhabditis elegans/genetics/metabolism
*Caenorhabditis elegans Proteins/genetics/metabolism
MicroRNAs/genetics/metabolism
*Gene Expression Regulation, Developmental
3' Untranslated Regions/genetics
*RNA-Binding Proteins/genetics/metabolism
CRISPR-Cas Systems/genetics
Repressor Proteins

@article {pmid42105555,
year = {2026},
author = {Xiong, Y and Ye, S and Guo, Y and Li, L and Huang, X and Xiong, Y},
title = {Size-dependent AIENPs enhanced RPA-CRISPR/Cas12a mediated lateral flow assay for ultrasensitive detection of Staphylococcus aureus.},
journal = {Food chemistry},
volume = {517},
number = {},
pages = {149477},
doi = {10.1016/j.foodchem.2026.149477},
pmid = {42105555},
issn = {1873-7072},
mesh = {*Staphylococcus aureus/isolation & purification/genetics/chemistry ; Milk/microbiology ; Animals ; CRISPR-Cas Systems ; Particle Size ; Food Contamination/analysis ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Bacterial Proteins/genetics ; *Biosensing Techniques/methods/instrumentation ; Rapid Diagnostic Tests ; Nanoparticles/chemistry ; },
abstract = {In this study, we developed a lateral flow assay (LFA) utilizing aggregation-induced emission nanoparticles (AIENPs) integrated with recombinase polymerase amplification (RPA) and CRISPR/Cas12a for the ultrasensitive detection of Staphylococcus aureus (S. aureus). We systematically evaluated the influence of AIENP particle size (250, 350, 450, and 600 nm) on the performance of LFA detection. Notably, the 450 nm AIENPs significantly enhanced the sensitivity of the strips due to their low background signal and size-dependent luminescent property. Under optimal conditions, the AIENPs-LFA demonstrated a 100-fold increase in sensitivity over conventional AuNPs-LFA. Combined with RPA, the assay achieved a remarkably low LOD of 9.95 CFU/mL for S. aureus, with a broad linear range (2.4-2.4 × 10[4] CFU/mL) and high specificity against other foodborne pathogens. Intra- and inter-assay validations indicated high accuracy (recoveries of 83.13%-106.93%) and precision (RSD < 14.42%) in S. aureus fortified milk samples.},
}

*Staphylococcus aureus/isolation & purification/genetics/chemistry
Milk/microbiology
Animals
CRISPR-Cas Systems
Particle Size
Food Contamination/analysis
*Nucleic Acid Amplification Techniques/methods
Limit of Detection
Bacterial Proteins/genetics
*Biosensing Techniques/methods/instrumentation
Rapid Diagnostic Tests
Nanoparticles/chemistry

@article {pmid42105559,
year = {2026},
author = {Shi, T and Dai, T and Zhou, J and Li, J and Zhang, L and Cui, J and Ma, X and Dai, J and Chen, A and Wang, X},
title = {Rapid identification of binary and ternary adulteration in camellia oil by CRISPR/Cas12a assay.},
journal = {Food chemistry},
volume = {517},
number = {},
pages = {149501},
doi = {10.1016/j.foodchem.2026.149501},
pmid = {42105559},
issn = {1873-7072},
mesh = {*Food Contamination/analysis ; *Plant Oils/chemistry/analysis ; *Camellia/chemistry/genetics ; *CRISPR-Cas Systems ; },
abstract = {Camellia oil adulteration, bring about a serious potential threat to consumer health and food safety, yet current chromatography-based techniques require costly instruments and trained personnel, limiting their on-site detection. In this study, CRISPR/Cas12a was used for rapid identification of binary and ternary adulteration in camellia oil. On the basis of our screened RPA primer and crRNAs from corresponding rbcL genes, the CRISPR/Cas12a reaction, was completed within 40 min at a detection limit of 5% (w/w) in camellia oil adulterated with soybean oil, rapeseed oil, corn oil, and peanut oil, by visual naked-eye readout. Referring to the traditional gas chromatography technology, our constructed CRISPR/Cas12a method achieved 100% accuracy, highlighting its potential for routine market surveillance, especially for those oil samples at adulterated ratio ≤ 10%. This cost-effective and equipment-minimal assay, provide a promising tool for adulterated camellia oil on site detection, thus strengthening food quality control and consumer protection.},
}

*Food Contamination/analysis
*Plant Oils/chemistry/analysis
*Camellia/chemistry/genetics
*CRISPR-Cas Systems

@article {pmid42203510,
year = {2026},
author = {Pirzada, MUR and Powell-Rodgers, G and Richee, J and Norppa, AJ and Jungers, CF and Colijn, S and Frilander, MJ and Stratman, AN and Djuranovic, S},
title = {Loss of U11/U12 spliceosome gene ZCRB1 leads to aberrant ciliogenesis and WNT signaling.},
journal = {Life science alliance},
volume = {9},
number = {8},
pages = {},
doi = {10.26508/lsa.202503607},
pmid = {42203510},
issn = {2575-1077},
mesh = {Humans ; Animals ; *Wnt Signaling Pathway/genetics ; *Spliceosomes/genetics/metabolism ; *Cilia/metabolism/genetics ; Zebrafish/genetics ; RNA Splicing/genetics ; *RNA-Binding Proteins/genetics/metabolism ; *RNA, Small Nuclear/genetics/metabolism ; Introns/genetics ; CRISPR-Cas Systems ; Cell Line ; },
abstract = {The U12-dependent, or minor, spliceosome processes only 0.5% of human introns, and yet, it is known to profoundly influence gene expression and cellular signaling. The ZCRB1 protein is a core component of the U12 mono-snRNP, but its functional significance to minor splicing, gene regulation, and biological signaling cascades remains poorly understood. Using CRISPR-Cas9 and siRNA-targeted knockout and knockdown strategies, we show that human cell lines with a partial reduction in ZCRB1 expression exhibit significant abnormal splicing events and altered expression of minor intron-containing genes. RNA-sequencing and targeted analyses of minor intron-containing genes indicate direct mis-splicing and expression of genes involved in ciliogenesis, with a coinciding up-regulation of WNT signaling pathway components. CRISPR-Cas12a knockdown of zcrb1 in zebrafish embryos leads to developmental patterning and body axis abnormalities, disrupted ciliogenesis, and up-regulated WNT signaling, complementing our human cell studies. This work highlights a conserved and essential biological role of the minor spliceosome, via ZCRB1, in cellular and developmental processes across species, shedding light on the molecular crosstalk that integrates splicing regulation, ciliogenesis, and WNT signaling.},
}

Humans
Animals
*Wnt Signaling Pathway/genetics
*Spliceosomes/genetics/metabolism
*Cilia/metabolism/genetics
Zebrafish/genetics
RNA Splicing/genetics
*RNA-Binding Proteins/genetics/metabolism
*RNA, Small Nuclear/genetics/metabolism
Introns/genetics
CRISPR-Cas Systems
Cell Line

@article {pmid42204342,
year = {2026},
author = {Walker, MWG and Richard, E and Wiegand, T and Wang, J and Yang, Z and Casas-Ciniglio, AA and Hoffmann, FT and Shahnawaz, H and Gaudet, RG and Arpaia, N and Fernández, IS and Sternberg, SH},
title = {Temperate phages enhance bacterial host fitness via RNA-guided flagellar remodelling.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {42204342},
issn = {2058-5276},
support = {CAREER 2239685//National Science Foundation (NSF)/ ; DGE-2036197//National Science Foundation (NSF)/ ; SF349247//Simons Foundation/ ; U24GM129539//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; DP2AI177904//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01CA259634//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
abstract = {Bacterial flagella drive motility and play crucial roles in host-pathogen interactions, as flagellin is recognized by the mammalian immune system and flagellotropic bacteriophages. We recently discovered a family of phage-encoded, RNA-guided transcription factors called TldR that regulate flagellin expression, but the importance of this regulation to host fitness was unclear. Here we use a human clinical Enterobacter isolate encoding a Flagellin Remodeling prophage (FRφ) to show that FRφ exploits TldR and its flagellin isoform to alter the flagellar composition and phenotypic properties of its host. This transformation enhances bacterial motility and mammalian immune evasion, and cryo-EM structures reveal distinct flagellin architectures underlying physiological changes. FRφ also improves colonization in the murine gut, illustrating the beneficial effect of prophage-mediated flagellar remodelling in a host-associated environment. Collectively, our results reveal how RNA-guided transcription factors emerged in a parallel evolutionary path to CRISPR-Cas and were co-opted by phages to remodel the flagellar apparatus and enhance host fitness.},
}

@article {pmid42204874,
year = {2026},
author = {Patra, S and Shand, H and Ghorai, S},
title = {Beyond conventional therapies: immunotherapeutic strategies targeting HPV-associated cervical cancer.},
journal = {Immunotherapy},
volume = {},
number = {},
pages = {1-21},
doi = {10.1080/1750743X.2026.2679883},
pmid = {42204874},
issn = {1750-7448},
abstract = {Cervical cancer continues to represent a significant global health challenge, primarily due to persistent infection with high-risk human papillomavirus (HPV) types. While prophylactic HPV vaccines have substantially reduced infection rates, their inability to address established infections or HPV-driven malignancies highlights a critical therapeutic gap. Conventional treatment modalities, such as chemotherapy, radiotherapy, and surgery remain the cornerstone of cervical cancer management; however, these approaches are often associated with nonspecific toxicity, diminished quality of life, treatment resistance, and elevated recurrence rates. Notably, conventional therapies are not specifically designed to target persistent HPV infection, and viral clearance may not be consistently achieved. This narrative review synthesizes studies retrieved from PubMed, Scopus, Web of Science, and Google Scholar published up to March 2026. It critically evaluates the limitations of current therapeutic strategies and emphasizes emerging non-conventional immunotherapeutic approaches designed to address HPV persistence and tumor immune evasion. Particular attention is given to nanocarrier-based therapeutic platforms, siRNA-mediated E6/E7 silencing and CRISPR-based disruption of the HPV genome, are presented as promising methods for precise molecular intervention. Additionally, advances in therapeutic vaccines, immune checkpoint inhibition, and γδ T-cell-based immunotherapy are also explored as potential strategies to restore HPV-specific immune surveillance and achieve sustained clinical responses.},
}

@article {pmid42206360,
year = {2026},
author = {Ramirez-Chamorro, L and Bonhomme, F and Wolff, ALI and Stouf, M and Lecointe, F and Hollenstein, M and Krupovic, M and De Paepe, M and Bhoobalan-Chitty, Y},
title = {A pair of DNA glucosyltransferases elevate counter-defense in bacteriophage T4.},
journal = {Nucleic acids research},
volume = {54},
number = {10},
pages = {},
doi = {10.1093/nar/gkag531},
pmid = {42206360},
issn = {1362-4962},
support = {ANR-20-CE12-0008-02//ANR/ ; NNF21OC0067491//Novo Nordisk Fonden/ ; },
mesh = {*Bacteriophage T4/enzymology/genetics ; *Glucosyltransferases/metabolism/genetics ; DNA, Viral/metabolism ; Escherichia coli/virology/genetics ; *Viral Proteins/metabolism/genetics ; 5-Methylcytosine/analogs & derivatives/metabolism ; Glycosylation ; },
abstract = {Bacteriophages encode diverse nucleotide-modification pathways to evade host restriction-modification (RM) and CRISPR-Cas systems. On the other hand, modifications can also serve as a target for host defense systems, illustrating the complexity of the defense and counter defense landscape. Bacteriophage T4 encodes two glucosyltransferases (GTs), α-GT and β-GT, that post-replicatively add a glucose moiety to the hydroxymethylated deoxycytosines (5-hmC) on phage DNA in the α- and β-conformation, respectively. Among all fully sequenced phages, only six closely related phages encode both α-GT and β-GT. Here, through biochemical and genetic analysis, we show that β-GT has higher catalytic activity, whereas α-GT is more strongly expressed. During T4 infection, these factors determine the contributions of both GTs, with 66% of all 5-hmC α-glucosylated and 33% β-glucosylated. Encoding a single GT is sufficient to overcome the Escherichia coli RM systems, unless the glucosylation levels decrease below 80%, which constitute a complete protection threshold. However, when encountering a host encoding DNA glycosylase Brig1, in addition to type I and type IV RM systems, a second GT is necessary to enable Brig1 escapers to resist RM systems. These results demonstrate that encoding multiple GTs serves as a counter-defense mechanism when simultaneously confronted with several antiphage defense systems.},
}

*Bacteriophage T4/enzymology/genetics
*Glucosyltransferases/metabolism/genetics
DNA, Viral/metabolism
Escherichia coli/virology/genetics
*Viral Proteins/metabolism/genetics
5-Methylcytosine/analogs & derivatives/metabolism
Glycosylation

@article {pmid41922342,
year = {2026},
author = {Carey, CM and Parvez, S and Brandt, ZJ and Bisgrove, BW and Yates, CJ and Peterson, RT and Gagnon, JA},
title = {MIC-Drop-seq: scalable single-cell phenotyping of mutant vertebrate embryos.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41922342},
issn = {2041-1723},
support = {F32HL156644//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01 GM134069/GM/NIGMS NIH HHS/United States ; R00HG012593//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01GM134069//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R24 OD035409/OD/NIH HHS/United States ; K01HG013682//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01 GM088040/GM/NIGMS NIH HHS/United States ; R35GM142950//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R24OD035409//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Animals ; *Zebrafish/genetics/embryology ; *Single-Cell Analysis/methods ; Phenotype ; Gene Expression Regulation, Developmental ; *Embryo, Nonmammalian/metabolism/cytology ; Transcription Factors/genetics/metabolism ; Single-Cell Gene Expression Analysis ; Gene Regulatory Networks ; Mutation ; CRISPR-Cas Systems ; Zebrafish Proteins/genetics/metabolism ; Sequence Analysis, RNA/methods ; Mesoderm/metabolism/embryology ; },
abstract = {Pooled perturbation screens can reveal cellular regulatory networks, yet scaling these techniques for large-scale screens in animals remains challenging. Here we present MIC-Drop-seq, a technique that addresses these challenges by combining high-throughput CRISPR gene disruption in zebrafish embryos with phenotyping by multiplexed single-cell RNAseq. In one MIC-Drop-seq experiment, we simultaneously identified changes in gene expression and cell abundance across 74 cell types resulting from loss of function of 50 transcription factors. These observations recapitulate many known phenotypes, while also uncovering previously uncharacterized roles for transcription factors in brain and mesoderm development. A key advantage of whole-animal screens is that they reveal how changes in one cell type affect the development of other cell types. Surprisingly, such cell-extrinsic phenotypes are abundant, indicating that transcription factors frequently exert effects beyond the cells where they are expressed to adjacent cells. We propose that MIC-Drop-seq will facilitate efforts to dissect the complete gene regulatory networks that guide animal development.},
}

Animals
*Zebrafish/genetics/embryology
*Single-Cell Analysis/methods
Phenotype
Gene Expression Regulation, Developmental
*Embryo, Nonmammalian/metabolism/cytology
Transcription Factors/genetics/metabolism
Single-Cell Gene Expression Analysis
Gene Regulatory Networks
Mutation
CRISPR-Cas Systems
Zebrafish Proteins/genetics/metabolism
Sequence Analysis, RNA/methods
Mesoderm/metabolism/embryology

@article {pmid41956025,
year = {2026},
author = {Yang, M and Li, D and Wang, A and Zhang, Y and Ouyang, J and Wang, X and Hong, L and Tong, D and Wang, M and Lin, J and Luo, Q and Wang, G},
title = {CRISPR/Cas12a and Au@UiO-66 nanozyme synergistic dual amplification-based electrochemical biosensor for ultrasensitive ctDNA detection.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {171},
number = {},
pages = {109300},
doi = {10.1016/j.bioelechem.2026.109300},
pmid = {41956025},
issn = {1878-562X},
mesh = {*Biosensing Techniques/methods ; Humans ; *Gold/chemistry ; *Electrochemical Techniques/methods ; Limit of Detection ; *Circulating Tumor DNA/blood/analysis/genetics ; *CRISPR-Cas Systems ; Breast Neoplasms/blood/diagnosis/genetics ; Hydrogen Peroxide/chemistry ; Female ; Metal Nanoparticles/chemistry ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Breast cancer, a globally prevalent malignancy in women, requires early diagnosis to improve patient outcomes, where ctDNA serves as a key biomarker. In the present study, an electrochemical biosensor based on CRISPR/Cas12a and Au@UiO-66 nanozyme synergistic dual amplification was developed to achieve ultrasensitive detection of breast cancer marker ctDNA. Au@UiO-66-modified ssDNA probes were anchored to the gold electrode via thiol groups. The intact ssDNA probe anchors Au@UiO-66 nanozyme to the electrode, where its peroxidase-like activity efficiently catalyzes H2O2 reduction to generate amplified reduction peak currents. Target ctDNA activates Cas12a's trans-cleavage capacity by binding specifically to crRNA. Under the presence of the target, Au@UiO-66 nanozymes are released, resulting in a discernible drop in the peak current linked to H2O2 reduction. Benefiting from the optimized experimental conditions, the biosensor demonstrates a broad linear detection range (from 10 fM to 10 nM) for the target ctDNA, along with an exceptionally low detection limit of 6.14 fM. Successful detection in human serum samples demonstrates its practicality. The platform's high specificity is attributed to the programmable crRNA design, enabling detection of specified DNA sequences and showcasing significant adaptability for diagnosing multiple genetic targets, highlighting its potential in clinical cancer diagnosis.},
}

*Biosensing Techniques/methods
Humans
*Gold/chemistry
*Electrochemical Techniques/methods
Limit of Detection
*Circulating Tumor DNA/blood/analysis/genetics
*CRISPR-Cas Systems
Breast Neoplasms/blood/diagnosis/genetics
Hydrogen Peroxide/chemistry
Female
Metal Nanoparticles/chemistry
Nucleic Acid Amplification Techniques/methods

@article {pmid41986708,
year = {2026},
author = {Roth, MO and Shu, Y and Zhao, Y and Trasanidou, D and Hoffman, RD and Südfeld, C and Bouzetos, E and Trasanidis, N and Zawrotny, M and Gelasco, MK and Medina, ML and Das, A and Rai, J and Goswami, HN and Wang, B and van der Oost, J and Li, H},
title = {Molecular basis for methylation-sensitive editing by Cas9.},
journal = {Nature},
volume = {653},
number = {8116},
pages = {1229-1239},
pmid = {41986708},
issn = {1476-4687},
mesh = {Humans ; *DNA Methylation ; Cryoelectron Microscopy ; Models, Molecular ; *CRISPR-Cas Systems/genetics ; Cytosine/metabolism ; Genome, Human/genetics ; DNA Cleavage ; CRISPR-Associated Proteins/metabolism/chemistry/ultrastructure ; Nucleotide Motifs/genetics ; DNA/metabolism/chemistry/genetics ; CRISPR-Associated Protein 9/metabolism/chemistry ; Female ; *Gene Editing ; },
abstract = {The bacterial CRISPR-Cas9 (Cas9) nuclease has become a powerful genome manipulation tool for a wide range of organisms[1-3]. However, it has yet to fully leverage the pervasive presence of DNA methylation in genomes[4-10]. Here, to fill this gap, we report biochemical, structural and human genome-editing characterizations of a methylation-sensitive Cas9 (ThermoCas9). ThermoCas9 efficiently binds to and cleaves DNA upstream of its protospacer adjacent motif (PAM) 5'-NNNNCGA-3' or 5'-NNNNCCA-3' in vitro. Methylation of the fifth cytosine in either PAM sequence ([5m]CpG or [5m]CpC), however, significantly inhibits ThermoCas9 activity. Cryo-electron microscopy structures of ThermoCas9 in pre-cleavage and post-cleavage states at 2.8 Å and 2.2 Å resolution, respectively, reveal the molecular basis for the stringent requirement of the unmethylated cytosine in PAM binding and provide guidance for further enzyme engineering. We demonstrate methylation-sensitive editing by ThermoCas9 in human cell lines with distinct DNA methylation landscapes. Moreover, we demonstrate that a catalytically enhanced ThermoCas9 efficiently targets luminal expression signature genes that are consistently hypomethylated in patients with breast cancer. Owing to its sensitivity to DNA methylation, ThermoCas9 can specifically target cells with disease-related hypomethylation, which adds another layer of precision to genome-editing technologies.},
}

Humans
*DNA Methylation
Cryoelectron Microscopy
Models, Molecular
*CRISPR-Cas Systems/genetics
Cytosine/metabolism
Genome, Human/genetics
DNA Cleavage
CRISPR-Associated Proteins/metabolism/chemistry/ultrastructure
Nucleotide Motifs/genetics
DNA/metabolism/chemistry/genetics
CRISPR-Associated Protein 9/metabolism/chemistry
Female
*Gene Editing

@article {pmid41992329,
year = {2026},
author = {Cho, E and Lee, J and Kim, J and Choi, J and Kang, M and Song, J},
title = {Application of extracellular vesicles in the CRISPR-based diagnosis and treatment: possibilities and challenges.},
journal = {Journal of biological engineering},
volume = {20},
number = {1},
pages = {},
pmid = {41992329},
issn = {1754-1611},
abstract = {The CRISPR–Cas system has revolutionized molecular diagnostics and gene editing, yet its clinical translation is hindered by delivery barriers, off-target activity, immunogenicity, and manufacturing challenges. Compared with viral vectors and synthetic non-viral carriers such as lipid nanoparticles, extracellular vesicles (EVs) offer a biologically derived delivery platform with superior biocompatibility, reduced immunogenicity, intrinsic cargo protection, and natural barrier-crossing capability. Engineered EVs can further achieve cell- or tissue-specific targeting. In diagnostics, endogenous EV proteins and nucleic acids provide disease-informative signatures that can interface with CRISPR nuclease readouts for highly sensitive detection. This review summarizes the therapeutic and diagnostic potential of EV-CRISPR platforms, covering strategies for loading CRISPR cargos (producer-cell engineering, post-isolation methods), cargo formats, and surface targeting approaches. We evaluate preclinical performance with attention to biodistribution, safety, innate and adaptive immune responses, and genomic integrity, as well as analytical assays and scalable manufacturing considerations essential for clinical translation. Finally, we discuss emerging opportunities, including AI-guided optimization of EV-CRISPR design and integrated EV platforms that combine disease detection with therapeutic intervention, highlighting their promise for advancing precision medicine.},
}

@article {pmid42105578,
year = {2026},
author = {Wei, J and Wang, J and Wu, R and Zhang, J and Ren, H and Tang, Q and Huang, L and Zhang, K and Liao, X},
title = {Aptakiss-guided CRISPR/Cas13a signal amplification for ultrasensitive FEN1 detection using CsPbBr3@PDA@AuNPs-based electrochemiluminescence platform.},
journal = {Biosensors & bioelectronics},
volume = {308},
number = {},
pages = {118775},
doi = {10.1016/j.bios.2026.118775},
pmid = {42105578},
issn = {1873-4235},
mesh = {*Flap Endonucleases/isolation & purification/analysis ; *Biosensing Techniques/methods ; Metal Nanoparticles/chemistry ; Gold/chemistry ; *CRISPR-Cas Systems/genetics ; Electrochemical Techniques/methods ; Luminescent Measurements/methods ; Humans ; Polymers/chemistry ; Indoles/chemistry ; Limit of Detection ; Titanium/chemistry ; },
abstract = {Sensitive and reliable detection of DNA repair enzymes is critical for early cancer diagnosis and therapeutic monitoring. Herein, we report a novel electrochemiluminescence (ECL) biosensor for ultrasensitive detection of Flap Endonuclease 1 (FEN1), integrating an aptakiss-assisted CRISPR/Cas13a signal amplification strategy with a perovskite-based nanocomposite sensing interface. Specifically, a hybrid nanomaterial composed of CsPbBr3 nanocrystals coated with polydopamine (PDA) and decorated with gold nanoparticles (AuNPs) was constructed to form a core-shell-satellite structure (CsPbBr3@PDA@AuNPs). The PDA coating enhanced the aqueous stability of CsPbBr3 and introduced functional groups for probe attachment, while the AuNPs facilitated electron transfer and signal amplification. Upon recognition and cleavage of a flap-structured DNA substrate by FEN1, a downstream transcription reaction was initiated to generate RNA triggers, which in turn activated the Cas13a system. Cas13a cleaved a surface-tethered RNA strand required for forming the aptakiss complex, thereby removing ferrocene-based ECL quenching and restoring strong luminescence. The proposed biosensor exhibited an excellent detection limit of 1.73 fM, with high selectivity against non-specific nucleases, and demonstrated remarkable reproducibility and long-term stability. This study not only presents a powerful biosensing platform for FEN1 but also highlights the potential of CsPbBr3@PDA@AuNPs as a versatile ECL-active material. The modular design and tunable functionality of the nanocomposite enable broad applicability in detecting various biomolecules, paving the way for the development of advanced perovskite-based ECL biosensors for clinical and environmental diagnostics.},
}

*Flap Endonucleases/isolation & purification/analysis
*Biosensing Techniques/methods
Metal Nanoparticles/chemistry
Gold/chemistry
*CRISPR-Cas Systems/genetics
Electrochemical Techniques/methods
Luminescent Measurements/methods
Humans
Polymers/chemistry
Indoles/chemistry
Limit of Detection
Titanium/chemistry

@article {pmid42114484,
year = {2026},
author = {Wan, Y and Hu, Q and Zhu, J and Oung, QW and Lee, CH and Han, H and Lee, HL and Chen, X},
title = {PCR-free cascaded CRISPR-Cas13a colorimetric platform (CLAMP) for non-invasive miRNA-based allergen-specific subtyping of allergic rhinitis.},
journal = {Biosensors & bioelectronics},
volume = {308},
number = {},
pages = {118773},
doi = {10.1016/j.bios.2026.118773},
pmid = {42114484},
issn = {1873-4235},
mesh = {*MicroRNAs/genetics/analysis/isolation & purification ; *Colorimetry/methods ; CRISPR-Cas Systems/genetics ; Animals ; *Rhinitis, Allergic/diagnosis/genetics/immunology ; *Allergens/genetics/immunology ; *Biosensing Techniques ; Humans ; Mice ; Limit of Detection ; Nasal Lavage Fluid/chemistry ; },
abstract = {Accurate identification of causative allergens is critical for personalized management of allergic rhinitis (AR), yet current methods often fail to distinguish clinical relevance from sensitization, particularly in polysensitized patients. To address this, we developed CLAMP (Cascaded CRISPR-Cas13a LAMP Colorimetric Assay), a platform that leverages a single Cas13a/crRNA complex to initiate a simplified CRISPR cascade amplification, enabling one-pot two-step detection without tube opening after sample addition. The assay employs a colorimetric readout based on CIELAB ΔE, allowing quantitative biomarker analysis with minimal equipment, requiring only a simple heat source. CLAMP achieves a detection limit of 1.15 × 10[-15] M with excellent specificity and a turnaround time under 60 min. In preclinical validation using nasal lavage fluid from 200 allergen-specific AR mouse models, CLAMP enabled allergen-based AR classification via multiplex miRNA detection, achieving 91.5% accuracy in distinguishing major allergen subtypes. This low-cost, non-invasive platform holds strong potential for rapid allergen classification and future translation toward personalized immunotherapy in AR.},
}

*MicroRNAs/genetics/analysis/isolation & purification
*Colorimetry/methods
CRISPR-Cas Systems/genetics
Animals
*Rhinitis, Allergic/diagnosis/genetics/immunology
*Allergens/genetics/immunology
*Biosensing Techniques
Humans
Mice
Limit of Detection
Nasal Lavage Fluid/chemistry

@article {pmid42142405,
year = {2026},
author = {Zhang, X and Zhang, Z and Chen, K and Zhang, J and Wu, Z and Tang, B and Cheng, Y},
title = {CRISPR/Cas12a-powered tri-mode aptasensor for ultrasensitive and multiplexed detection of microcystin-LR.},
journal = {Biosensors & bioelectronics},
volume = {308},
number = {},
pages = {118786},
doi = {10.1016/j.bios.2026.118786},
pmid = {42142405},
issn = {1873-4235},
mesh = {*Microcystins/analysis/isolation & purification/chemistry ; Marine Toxins ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Aptamers, Nucleotide/chemistry ; Limit of Detection ; *Water Pollutants, Chemical/analysis/isolation & purification ; Metal Nanoparticles/chemistry ; Gold/chemistry ; DNA, Single-Stranded/chemistry ; },
abstract = {Exploiting CRISPR/Cas12a-powered multiple-mode sensing platforms is urgently needed to meet flexible and multi-scenario analytical requirements while improving detection accuracy for target pollutants. Herein, we developed a novel CRISPR/Cas12a-powered tri-mode aptasensor for ultrasensitive and multiplexed detection of microcystin-LR (MC-LR) in environmental water. First, target-specific recognition and the subsequent release of activator DNA were achieved through an aptamer-based competitive displacement reaction. Then, the trans-cleavage activity of Cas12a was activated in the presence of MC-LR, and a fluorescence-quenched ssDNA (FAM-ssDNA-BHQ1) and a dual -functionalized probe (MBs-ssDNA-AuNPs-4MBA) as signal reporters were employed for the tri-signal readouts. In the fluorescence mode, an ultrasensitive digital droplet fluorescence system was employed. The released activator DNA and Cas12a/crRNA complex were co-encapsulated into monodisperse nanoliter droplets, which not only accelerated the reaction but also amplified local fluorescence signals. This approach enabled rapid detection of MC-LR within 35 min, with a detection limit as low as 1.0 aM. For SERS and colorimetric modes, a novel dual-functionalized reporter was designed to provide both Raman and colorimetric readouts following magnetic separation. This extended the dynamic detection range from 1.0 × 10[-13] M to 1.0 × 10[-6] M. The proposed tri-mode aptasensor, using a single workflow, addresses both trace-level warning and high-concentration detection of MC-LR. It demonstrated good analytical performance in real samples, with recoveries ranging from 81.26% to 113.21% (n = 3). This CRISPR/Cas12a-driven strategy thus provides a versatile tool for reliable MC-LR monitoring across diverse water scenarios.},
}

*Microcystins/analysis/isolation & purification/chemistry
Marine Toxins
*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
*Aptamers, Nucleotide/chemistry
Limit of Detection
*Water Pollutants, Chemical/analysis/isolation & purification
Metal Nanoparticles/chemistry
Gold/chemistry
DNA, Single-Stranded/chemistry

@article {pmid42149978,
year = {2026},
author = {Verzier, LH and Hesping, E and Doerflinger, M and Herold, MJ and Boddey, JA},
title = {Cas9-expressing HC-04 hepatocytes facilitate CRISPR-based analysis of Plasmodium falciparum sporozoite-host interactions.},
journal = {PLoS genetics},
volume = {22},
number = {5},
pages = {e1012137},
doi = {10.1371/journal.pgen.1012137},
pmid = {42149978},
issn = {1553-7404},
mesh = {*Plasmodium falciparum/genetics/pathogenicity ; Humans ; *Hepatocytes/parasitology/metabolism ; *Sporozoites/genetics/pathogenicity ; *CRISPR-Cas Systems/genetics ; *Host-Parasite Interactions/genetics ; *Malaria, Falciparum/parasitology/genetics ; Cell Line ; Animals ; Erythrocytes/parasitology ; Liver/parasitology ; },
abstract = {Sporozoites of Plasmodium falciparum, the deadliest malaria parasite, are injected into the skin by infected mosquitoes and must reach the liver to initiate infection. There, they invade hepatocytes and develop into exoerythrocytic merozoites that eventually enter the bloodstream and invade erythrocytes, causing malaria. The sporozoite's journey requires cell traversal, where sporozoites transiently enter and exit host cells, lysing membranes to move deeper into tissue and evade immune cell destruction. After reaching the liver and traversing several hepatocytes, sporozoites productively invade a final hepatocyte to establish an exoerythrocytic form. The molecular mechanisms underlying traversal, invasion, and intracellular development remain incompletely understood, particularly with respect to host factors. To address this, we engineered human HC-04 hepatocytes, the only known cell line supporting P. falciparum liver-stage development, to express Cas9-mCherry, enabling CRISPR-based functional genomics studies. We validated Cas9 activity of HC-04.2B3 and demonstrated successful guide-RNA-directed gene disruption via non-homologous end joining. Optimized traversal and invasion assays led to a robust cytometric readout suitable for screening human genes involved in P. falciparum infection. Disruption of 10 human genes previously implicated in infection by bacterial and viral pathogens confirmed utility of this platform. This study provides the basis for genome-wide CRISPR screens to uncover hepatocyte biology and host determinants of infection.},
}

*Plasmodium falciparum/genetics/pathogenicity
Humans
*Hepatocytes/parasitology/metabolism
*Sporozoites/genetics/pathogenicity
*CRISPR-Cas Systems/genetics
*Host-Parasite Interactions/genetics
*Malaria, Falciparum/parasitology/genetics
Cell Line
Animals
Erythrocytes/parasitology
Liver/parasitology

@article {pmid42190658,
year = {2026},
author = {Hoshino, M and Nehlsen, M and Batista, RA and Raphalen, M and Wakimoto, T and Uwai, S and Kogame, K and Alva, V and Coelho, SM},
title = {PKN is a sex- and species-specific fertilization factor in brown algae.},
journal = {Current biology : CB},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.cub.2026.04.065},
pmid = {42190658},
issn = {1879-0445},
abstract = {Fertilization, the fusion of male and female gametes, is fundamental to sexual reproduction, yet the molecular mechanisms that mediate gamete recognition and enforce species specificity remain poorly understood, and only a handful of proteins are known to act as core fertilization factors across eukaryotes. Here, we identify PICKINESS-ASSOCIATED PROTEIN (PKN), a female gamete-specific transmembrane protein, as an essential determinant of fertilization in brown algae. CRISPR-Cas-mediated knockout of PKN abolishes successful male-female gamete interactions and prevents fertilization without affecting earlier mating behaviors, such as gamete attraction. Remarkably, PKN also enforces reproductive isolation by preventing interspecific fertilization, establishing it as a molecular gatekeeper of species specificity. Structural analyses reveal extracellular β-propeller and mucin-like domains enriched in predicted glycosylation sites and displaying rapid sequence evolution. Functional and comparative analyses suggest that PKN-dependent recognition mechanisms are conserved across diverse brown algal lineages. Because PKN originated within brown algae, its dual role in mediating both male-female gamete recognition and species-specific fertilization reveals a striking conceptual parallel with fertilization factors described in animals, suggesting that evolution repeatedly converges on lineage-specific gamete-expressed membrane proteins as key arbiters of reproductive recognition.},
}

@article {pmid42191275,
year = {2026},
author = {Vuong, HL and Thi Thu Le, H},
title = {Advances in protein engineering.},
journal = {International review of cell and molecular biology},
volume = {402},
number = {},
pages = {61-87},
doi = {10.1016/bs.ircmb.2025.11.002},
pmid = {42191275},
issn = {1937-6448},
mesh = {*Protein Engineering/methods ; Humans ; Animals ; Synthetic Biology ; CRISPR-Cas Systems/genetics ; Proteins/genetics/chemistry ; },
abstract = {Protein engineering (PE) has been applied to various medicines, food, and environments. Contributions of proteins have been reported with remarkable results in protein therapeutics, antibody engineering, enzyme synthesis, and more specific functions in industrial processes. Therefore, this chapter highlights the most recent PE advances in a battle against mainly human diseases and biomedical sciences. The application of PE will be reviewed, focusing on developing innovative techniques, including evolution, rational design, semi-rational design, and hybrid approaches to protein design in applications. In addition, we provide key achievements of PE in CRISPR/Cas systems, high-through data, and synthetic biology with updated results. Current challenges of using PE, ethical considerations, and various approaches for protein therapeutics are also discussed. In this chapter, the updated findings provide a comprehensive overview of the transformative potential of PE for researchers in the application areas of human disease, especially in cancer therapeutics.},
}

*Protein Engineering/methods
Humans
Animals
Synthetic Biology
CRISPR-Cas Systems/genetics
Proteins/genetics/chemistry

@article {pmid42193394,
year = {2026},
author = {Siringan, MJ and Chen, X and Huo, J},
title = {RNA-Loaded Nanoparticles for Targeted Lung Delivery.},
journal = {Biomedicines},
volume = {14},
number = {5},
pages = {},
doi = {10.3390/biomedicines14051069},
pmid = {42193394},
issn = {2227-9059},
abstract = {The lung represents a promising yet underexploited target for RNA therapeutics due to its large surface area and accessibility via non-invasive inhalation delivery. Despite rapid advances in RNA-based modalities, including small interfering RNA (siRNA), microRNA (miRNA), messenger RNA (mRNA), and CRISPR-Cas systems, efficient pulmonary delivery remains a major challenge. Multiple biological barriers, such as mucus and surfactant layers, mucociliary clearance, immune surveillance, and limited cellular uptake of negatively charged nucleic acids, significantly restrict therapeutic efficacy. In addition, aerosolization processes may introduce mechanical stress, compromising RNA integrity. Nanoparticle-based delivery systems have emerged as a central strategy to address these limitations. By protecting RNA cargo, enhancing mucus penetration, and promoting cellular internalization, engineered nanoparticles enable more effective pulmonary delivery. In this review, we adopt a barrier-centered perspective to examine the key biological obstacles to lung-targeted RNA delivery and highlight recent advances in nanoparticle-mediated strategies, with a focus on lipid nanoparticles, polymeric systems, and inorganic nanomaterials. We further discuss design principles that govern RNA stability, transport, and intracellular release and critically compare the strengths, limitations, and translational potential of each platform, including considerations of toxicity, biodegradability, and clinical readiness. Finally, we outline emerging clinical applications of RNA-loaded nanoparticles, using lung cancer as a representative disease model, and discuss remaining challenges and future directions. Continued innovation in nanoparticle engineering and delivery strategies is expected to accelerate the clinical translation of RNA therapeutics for pulmonary diseases.},
}

@article {pmid42193866,
year = {2026},
author = {Siddika, A and Rousseau, J and Veillette, F and Bouchard, C and Lu, Y and Tremblay, JP},
title = {Gene Editing Strategies for Duchenne Muscular Dystrophy: From Molecular Mechanisms to Clinical Translation.},
journal = {Cells},
volume = {15},
number = {10},
pages = {},
doi = {10.3390/cells15100852},
pmid = {42193866},
issn = {2073-4409},
support = {Defeat Duchenne Foundation (53320215); VCGS (492510)//Defeat Duchenne Foundation ; VCGS (CIHR)/ ; },
mesh = {*Muscular Dystrophy, Duchenne/genetics/therapy ; Humans ; *Gene Editing/methods ; *Translational Research, Biomedical ; Animals ; *Genetic Therapy/methods ; Dystrophin/genetics ; CRISPR-Cas Systems/genetics ; Mutation/genetics ; },
abstract = {Duchenne muscular dystrophy (DMD) remains a major challenge in genetic medicine due to the difficulty of achieving durable, body-wide restoration of dystrophin in post-mitotic muscle tissues. Although current therapies-including exon skipping and micro-dystrophin gene replacement-have demonstrated clinical feasibility, their benefits are limited by incomplete efficacy, mutation specificity, and the need for repeated or high-dose interventions. These limitations highlight the need for strategies capable of directly and permanently correcting the underlying genetic defect. Recent advances in genome editing have positioned CRISPR-based technologies as promising candidates for this objective. Rather than functioning as a single approach, gene-editing platforms encompass a spectrum of strategies-including exon deletion, exon reframing, base editing, and prime editing-each with distinct advantages depending on the mutational context. In particular, the emergence of precision editing tools has enabled controlled nucleotide-level modifications, expanding the range of correctable mutations while reducing reliance on double-strand DNA breaks. In this review, we adopt a comparative and translational perspective to evaluate gene-editing strategies for DMD. We examine how different approaches align with specific mutation types, summarize key findings from preclinical studies, and analyze the major barriers to clinical implementation, including delivery efficiency, immune responses, editing durability, and genomic safety. We further discuss emerging innovations in editing technologies and delivery systems that aim to address these limitations. Collectively, this work reframes gene editing as a decision-oriented and application-driven therapeutic framework. Continued integration of advances in genome engineering, delivery platforms, and muscle biology will be essential to translate these technologies into safe, effective, and durable treatments capable of altering the clinical trajectory of DMD.},
}

*Muscular Dystrophy, Duchenne/genetics/therapy
Humans
*Gene Editing/methods
*Translational Research, Biomedical
Animals
*Genetic Therapy/methods
Dystrophin/genetics
CRISPR-Cas Systems/genetics
Mutation/genetics

@article {pmid42193938,
year = {2026},
author = {Silva, FRD and Dias, PRF and Pavan, ICB and Oliveira, AP and Basei, FL and Santos, LED and Sousa, LM and Consonni, SR and Oliveira, AG and Silveira, LR and Kobarg, J},
title = {NEK6 Knockout Causes Defects in Mitochondrial Morphology and Respiration.},
journal = {Cells},
volume = {15},
number = {10},
pages = {},
doi = {10.3390/cells15100924},
pmid = {42193938},
issn = {2073-4409},
mesh = {Humans ; *Mitochondria/metabolism/ultrastructure ; *NIMA-Related Kinases/metabolism/genetics/deficiency ; *Gene Knockout Techniques ; Cell Respiration ; Cell Line, Tumor ; CRISPR-Cas Systems ; },
abstract = {The family of Nek kinases has 11 human members that are conserved in their kinase domains but diverse in their regulatory domains. Functionally, they can be associated with diverse aspects of cell cycle regulation, from mitosis and primary cilia function to centrosome disjunction in the G2 phase and checkpoints of the DNA damage response. However, novel functional contexts have emerged in recent years, including regulatory roles of Neks 1, 4, 5, and 10 in mitochondrial metabolic and morphological homeostasis. We recently generated, by CRISPR-Cas9 technology, a DU-145 prostate cancer cell line, with an NEK6 gene knockout. Here, we focus on a detailed characterization of changes in this cell line, in mitochondrial respiration function and morphology. DU-145 NEK6 knockout cells exhibited reduced mitochondrial respiration and a fragmented phenotype in electron microscopy, with reduced mitochondrial cristae numbers. Alterations in mitochondrial architecture and respiration were correlated with increased expression of anaerobic glycolytic proteins (HK2, PFKP, and LDHA) and decreased expression of PDH, an enzyme of aerobic glycolysis. Molecular analysis by Western blot revealed decreased levels of mitochondrial mass and biogenesis protein markers (TOM20, TFAM), without alterations in other markers such as VDAC1/3 or mtDNA copy number in the NEK6 knockout cells. Furthermore, the regulators of mitochondrial fusion/fission are altered in the knockout cells (decrease in the Long-OPA1:Short-OPA1 ratio and DRP1 total level), which is associated with an increase in endoplasmic reticulum-mitochondria contact at ≤20 nm observed in transmission electron microscopy (TEM) image analysis. Using analysis of TEM micrographs, we found an increase in the autophagic structures (autophagosome, amphisome, and autolysosome), with mitochondria as cargo in some structures, which was correlated with a decrease in LC3A/B and an increase in the BECLIN1 total level, and with an increase in acidic vesicles approximation, suggesting that reduction in TOM20 and TFAM without alterations in VDAC1/3 and mtDNA copy number might be related to mitochondrial degradation through autophagy. Together, our data suggest a new role for NEK6 in regulating mitochondrial homeostasis, where its loss alters mitochondrial morphology and respiration, and could be associated with an increase in the degradation of the dysfunctional mitochondria through autophagy.},
}

Humans
*Mitochondria/metabolism/ultrastructure
*NIMA-Related Kinases/metabolism/genetics/deficiency
*Gene Knockout Techniques
Cell Respiration
Cell Line, Tumor
CRISPR-Cas Systems

@article {pmid42196146,
year = {2026},
author = {Kim, S and Kim, GN and Jeong, YJ and Cho, J and Jang, M and Hong, J and Sung, YH},
title = {CRISPR-Cpf1-Mediated Gene-Editing System Based on a Single Bidirectional Promoter.},
journal = {International journal of molecular sciences},
volume = {27},
number = {10},
pages = {},
doi = {10.3390/ijms27104162},
pmid = {42196146},
issn = {1422-0067},
support = {2018R1A2B6002192//Ministry of Science and ICT/ ; 20012477//Ministry of Trade, Industry and Energy/ ; RS-2023-00283544//Korea Drug Development Fund/ ; },
mesh = {*Promoter Regions, Genetic ; *Gene Editing/methods ; Dependovirus/genetics ; *CRISPR-Cas Systems ; Animals ; Genetic Vectors/genetics ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; Mice ; *Bacterial Proteins/genetics/metabolism ; *CRISPR-Associated Proteins/genetics ; *Endodeoxyribonucleases/genetics/metabolism ; Endonucleases/genetics ; Acidaminococcus/genetics ; HEK293 Cells ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Recent advances in gene therapy have highlighted the potential of CRISPR-based gene-editing systems combined with adeno-associated virus (AAV) vectors. However, the limited packaging capacity of AAV remains a significant challenge for the simultaneous expression of Cas effector proteins and guide RNAs within a single vector. To address this limitation, we developed a compact AAV vector that enables the co-expression of Acidaminococcus sp. Cas12a (AsCpf1) and CRISPR RNAs (crRNAs) using a single bidirectional promoter derived from the mouse H1 promoter. Our single bidirectional H1 promoter supported indel formation comparable to that achieved by dual-promoter systems and facilitated scalable genome editing with single-, dual-, and triple-target configurations. Genome editing was successfully accomplished both in vitro and in vivo following AAV delivery. This study shows that our engineered compact AAV vector platform is capable of simultaneously delivering AsCpf1 and multiplexed crRNAs.},
}

*Promoter Regions, Genetic
*Gene Editing/methods
Dependovirus/genetics
*CRISPR-Cas Systems
Animals
Genetic Vectors/genetics
Humans
RNA, Guide, CRISPR-Cas Systems/genetics
Mice
*Bacterial Proteins/genetics/metabolism
*CRISPR-Associated Proteins/genetics
*Endodeoxyribonucleases/genetics/metabolism
Endonucleases/genetics
Acidaminococcus/genetics
HEK293 Cells
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42196223,
year = {2026},
author = {Ma, S and Li, Y and Fei, T},
title = {CRISPR Screening in Hepatocellular Carcinoma: From Tumor Progression to Immune Evasion and Therapeutic Resistance.},
journal = {International journal of molecular sciences},
volume = {27},
number = {10},
pages = {},
doi = {10.3390/ijms27104241},
pmid = {42196223},
issn = {1422-0067},
support = {2023A1515140084//Guangdong Basic and Applied Basic Research Foundation/ ; 32470673//National Natural Science Foundation of China/ ; B16009//the 111 Project/ ; 2022JH13/10200026//the Construction Project of Liaoning Provincial Key Laboratory, China/ ; },
mesh = {Humans ; *Carcinoma, Hepatocellular/genetics/therapy/immunology/pathology ; *Liver Neoplasms/genetics/immunology/therapy/pathology ; *Drug Resistance, Neoplasm/genetics ; *CRISPR-Cas Systems ; Animals ; Disease Progression ; *Tumor Escape/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Immune Evasion/genetics ; },
abstract = {Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and a leading cause of cancer-related mortality worldwide. Despite advances in targeted therapies and immunotherapies, clinical outcomes remain poor owing to profound molecular heterogeneity, intrinsic therapeutic resistance, and complex immune evasion mechanisms. Although genomic profiling has identified recurrent alterations in HCC, large-scale functional validation of candidate drivers and vulnerabilities remains challenging. CRISPR (clustered regularly interspaced short palindromic repeats)-based screening technologies have transformed this landscape by enabling systematic interrogation of gene function in physiologically relevant contexts. In this review, we summarize recent studies that have applied CRISPR screening approaches in HCC research. These efforts have uncovered multilayered dependency programs that govern ferroptosis resistance, metabolic reprogramming, epigenetic regulation, tumor suppressor networks, immune evasion, and resistance to targeted therapies. We also discuss the major limitations of current studies, including model bias, incomplete representation of HCC heterogeneity, and technical constraints intrinsic to pooled screening. Overall, integration of CRISPR screening with patient-derived models, single-cell readouts, and precision editing technologies is expected to accelerate mechanistic discovery and biomarker-guided therapeutic prioritization for HCC.},
}

Humans
*Carcinoma, Hepatocellular/genetics/therapy/immunology/pathology
*Liver Neoplasms/genetics/immunology/therapy/pathology
*Drug Resistance, Neoplasm/genetics
*CRISPR-Cas Systems
Animals
Disease Progression
*Tumor Escape/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
*Immune Evasion/genetics

@article {pmid42196225,
year = {2026},
author = {Zaman, W and Park, S},
title = {Advances in Functional Genomics and Biotechnology for Enhancing Therapeutic Potential of Medicinal Plants.},
journal = {International journal of molecular sciences},
volume = {27},
number = {10},
pages = {},
doi = {10.3390/ijms27104245},
pmid = {42196225},
issn = {1422-0067},
mesh = {*Plants, Medicinal/genetics/metabolism ; *Biotechnology/methods ; *Genomics/methods ; Metabolic Engineering/methods ; Synthetic Biology/methods ; Gene Editing ; CRISPR-Cas Systems ; Humans ; },
abstract = {Medicinal plants have long served as a primary source of bioactive compounds with essential therapeutic applications. Recent advances in functional genomics and plant biotechnology now enable precise manipulation of metabolic pathways to enhance the production of specialized metabolites with medicinal value. However, an integrative understanding of how genomic discovery can be linked with pathway engineering, scalable production systems, and healthcare applications remains insufficiently developed. This knowledge gap limits the effective translation of molecular insights into the sustainable production of medicinally important compounds. The novelty of this review lies in its integrated framework linking functional genomic discovery with pathway engineering, synthetic biology, artificial intelligence-assisted prediction, and scalable production systems for medicinal plant-derived therapeutics. This review aims to provide a comprehensive overview of cutting-edge approaches in medicinal plant research, emphasizing high-throughput RNA sequencing, CRISPR/Cas9 gene editing, synthetic biology, and metabolic engineering for optimizing the production of key bioactive compounds, including artemisinin, cannabinoids, ginsenosides, and taxol. It further examines how these tools collectively support metabolite discovery, pathway elucidation, yield improvement, and biotechnological production in major medicinal plant systems. We explore the application of genomic and biotechnological approaches in plants such as Artemisia annua, Cannabis sativa, Panax ginseng, and Taxus baccata to enhance metabolite yields and promote sustainable production. The review highlights case studies that demonstrate how genetic modification, metabolic engineering, and synthetic pathway design have been successfully employed to increase the synthesis of key medicinal compounds. Moreover, we discuss the integration of artificial intelligence and machine learning to predict gene-metabolite relationships, support personalized phytochemical therapies, and facilitate sustainable, large-scale production. Finally, the review addresses the implications of these innovations for the pharmaceutical industry, healthcare, and agriculture, while also highlighting sustainable and scalable directions for future medicinal plant biotechnology.},
}

*Plants, Medicinal/genetics/metabolism
*Biotechnology/methods
*Genomics/methods
Metabolic Engineering/methods
Synthetic Biology/methods
Gene Editing
CRISPR-Cas Systems
Humans

@article {pmid42196288,
year = {2026},
author = {De, N and Bhadra, J and Momin, MSA and Mitra, K and Bhunia, D and Sannigrahi, A},
title = {Therapeutic Innovations for Monkeypox Inhibition.},
journal = {International journal of molecular sciences},
volume = {27},
number = {10},
pages = {},
doi = {10.3390/ijms27104307},
pmid = {42196288},
issn = {1422-0067},
support = {EEQ/2022/000548//Science and Engineering Research Board/ ; },
mesh = {Humans ; *Antiviral Agents/therapeutic use/pharmacology ; Peptide Nucleic Acids/therapeutic use ; Animals ; CRISPR-Cas Systems ; Biocompatible Materials/therapeutic use ; },
abstract = {This review investigates biomaterial-based strategies for improved treatment of MPXV. We focus on emerging synthetic biomedical approaches to combating the virus. These include peptide nucleic acids, CRISPR-based systems, and small-molecule therapeutics. These methods work by targeting and blocking viral proteins and enzymes. Such synthetic platforms may help reduce viral transmission and minimize side effects. They also offer potential solutions to challenges such as viral resistance in humans. In addition, biomaterials contribute to the development of more stable and effective vaccines. Combining these biomaterials with mRNA technology provides a promising framework for future vaccine development. Overall, this review underscores biomaterial-driven antiviral systems as a major frontier in translational medicine with profound implications for global health and pandemic awareness.},
}

Humans
*Antiviral Agents/therapeutic use/pharmacology
Peptide Nucleic Acids/therapeutic use
Animals
CRISPR-Cas Systems
Biocompatible Materials/therapeutic use

@article {pmid42198588,
year = {2026},
author = {Camacho-Aguilar, P and Delgado-Nungaray, JA and Reynaga-Delgado, E and Gonzalez-Reynoso, O and Rodriguez-Anaya, LZ and Muñoz Miranda, LA and Rincón Enríquez, G and Higuera-Ciapara, I and Figueroa-Yáñez, LJ},
title = {New Insights into CRISPR-like Arrays in Helicobacter pylori: An Exploratory Analysis from Genomic Data.},
journal = {Pathogens (Basel, Switzerland)},
volume = {15},
number = {5},
pages = {},
doi = {10.3390/pathogens15050461},
pmid = {42198588},
issn = {2076-0817},
support = {1309039//SECIHTI/ ; 1267568//SECIHTI/ ; },
mesh = {*Helicobacter pylori/genetics ; *Genome, Bacterial ; Genomic Islands ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins/genetics ; Genomics/methods ; Antigens, Bacterial/genetics ; Computational Biology/methods ; CRISPR-Cas Systems ; Humans ; Helicobacter Infections/microbiology ; },
abstract = {Helicobacter pylori (H. pylori) is a highly adaptable gastric pathogen with marked genomic plasticity. Whilst functional CRISPR-Cas systems provide adaptive immunity in many bacteria, they have not been identified in H. pylori, unlike CRISPR-like sequences. In this study, eight H. pylori genomes were analysed using the bioinformatics tools CRISPRCasFinder, CRISPRCasTyper, and CRISPRloci. A total of 25 CRISPR-like arrays were identified, showing high conservation (88%) both between and within strains, suggesting that these arrays are not random remnants but rather organised structures possibly involved in cellular processes. Notably, a structural association was observed between the CRISPR-like sequences and the cag pathogenicity island (CagA-PAI). Conversely, CagA-PAI instability in specific strains was observed in the presence of the TnpA and TnpB transposons. Furthermore, in strain 29CaP, CRISPR-like assemblies were located in genomic proximity to the prophage Helico 1961P, leading to the hypothesis of a compensatory or regulatory effect in the absence of CagA-PAI. Taken together, these findings indicate that CRISPR-like arrays in H. pylori characterise a genomic architecture within regions of high plasticity. This study provides a solid exploratory foundation for future functional research on the adaptive and pathogenic evolution of H. pylori.},
}

*Helicobacter pylori/genetics
*Genome, Bacterial
Genomic Islands
*Clustered Regularly Interspaced Short Palindromic Repeats
Bacterial Proteins/genetics
Genomics/methods
Antigens, Bacterial/genetics
Computational Biology/methods
CRISPR-Cas Systems
Humans
Helicobacter Infections/microbiology

@article {pmid42198599,
year = {2026},
author = {Abdulrahman, B and Rahimi Aqdam, S and Mosca, M and Ahmed-Hassan, H and Razcon-Echeagaray, M and Popa, L and Gilch, S and Babelhadj, B and Vaccari, G and Schätzl, HM},
title = {A Neuronal Cell Line Model for Studying Camel Prions.},
journal = {Pathogens (Basel, Switzerland)},
volume = {15},
number = {5},
pages = {},
doi = {10.3390/pathogens15050472},
pmid = {42198599},
issn = {2076-0817},
support = {R01 NS121016/NS/NINDS NIH HHS/United States ; 201900008//Alberta Innovates/ ; 201600009//Alberta Innovates/ ; },
mesh = {Animals ; *Camelus ; *Neurons/metabolism/pathology ; Cell Line ; *Prion Diseases/metabolism/pathology/veterinary ; *Prions/metabolism/genetics ; Mice ; Brain/pathology/metabolism ; Gene Knockout Techniques ; },
abstract = {Prion diseases are fatal neurodegenerative disorders that affect humans and animals, caused by the conformational conversion of the normal cellular prion protein (PrP[C]) into its misfolded, infectious isoform PrP[Sc]. Recently, camel prion disease (CPrD) was identified in dromedary camels (Camelus dromedarius) in Algeria. Due to the potential implications for animal and human health, as well as the possible socio-economic impact in Mediterranean regions where camels play a pivotal role as a source of food, in-depth characterization of camel prions is important to increase our understanding of camel prion disease. We developed a neuronal cell line model for studying the molecular features of camel prion infection. We genetically edited mouse neuronal CAD5 cells to generate CAD5 PrP knockout (KO) cells. We then used lentiviral transduction to generate CAD5 cells expressing camel PrP (CAD5-camel-PrP). Following infection of these cells with a CPrD-positive camel brain homogenate, we observed PrP[Sc] signals at various passages, as indicated by immunoblotting analysis. RT-QuIC (Real-Time Quaking-Induced Conversion) assays further supported these findings, demonstrating transient prion conversion activity in the CPrD-infected CAD5-camel-PrP cells. Taken together, our data describe the first neuronal cell line permissive to camel prion infection, a novel in vitro tool for mechanistic studies of camel prion disease.},
}

Animals
*Camelus
*Neurons/metabolism/pathology
Cell Line
*Prion Diseases/metabolism/pathology/veterinary
*Prions/metabolism/genetics
Mice
Brain/pathology/metabolism
Gene Knockout Techniques

@article {pmid42199442,
year = {2026},
author = {Xia, L and Tang, C and Tong, D and He, Q},
title = {Research advances in the application of microfluidic chip technology for rapid detection of antibiotic-resistant bacteria.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1819856},
pmid = {42199442},
issn = {2235-2988},
mesh = {*Bacteria/drug effects/isolation & purification/genetics ; Humans ; *Drug Resistance, Bacterial ; Anti-Bacterial Agents/pharmacology ; *Microfluidics/methods/instrumentation ; Microbial Sensitivity Tests/methods ; *Microfluidic Analytical Techniques/methods ; Rapid Diagnostic Tests ; *Lab-On-A-Chip Devices ; *Bacterial Infections/diagnosis/microbiology ; Point-of-Care Systems ; },
abstract = {The escalating global burden of antimicrobial resistance (AMR) necessitates diagnostic strategies that can overcome the limitations of conventional culture-based methods, which often require several days to generate clinically actionable results. Such delays are associated with increased mortality, inappropriate antibiotic use, and continued transmission of resistant pathogens. In this context, microfluidic chip technology has emerged as a promising platform for rapid, miniaturized, and increasingly automated point-of-care diagnostics. Recent advances have enabled integrated lab-on-a-chip systems that combine bacterial isolation, phenotypic antimicrobial susceptibility testing, and genotypic resistance detection within closed and self-contained architectures, thereby reducing contamination risk and operator dependence. In addition, these platforms are increasingly capable of operating at single-cell resolution, allowing the detection of heteroresistance and resistant subpopulations that may be overlooked by conventional bulk assays. A major advantage of microfluidic systems is their ability to bridge phenotypic and genotypic diagnostics by enabling real-time monitoring of bacterial growth, metabolic activity, and morphological responses to antibiotics while simultaneously incorporating on-chip nucleic acid amplification for resistance gene detection. This integrated approach improves the interpretation of discrepancies between genetic determinants and functional resistance. Studies to date have demonstrated high sensitivity and specificity in complex clinical matrices, including blood, urine, and sputum, with turnaround times reduced from days to less than one hour in some applications. Furthermore, the integration of CRISPR-Cas systems, nanomaterial-enhanced biosensing, and machine learning has further improved analytical performance and data interpretation. Nevertheless, important translational challenges remain, including scalable manufacturing, regulatory standardization, and integration into routine clinical workflows. Future microfluidic platforms are expected to support multiplexed, intelligent antimicrobial susceptibility testing capable of simultaneous pathogen identification, resistance profiling, and therapeutic guidance, thereby advancing precision diagnostics for AMR management.},
}

*Bacteria/drug effects/isolation & purification/genetics
Humans
*Drug Resistance, Bacterial
Anti-Bacterial Agents/pharmacology
*Microfluidics/methods/instrumentation
Microbial Sensitivity Tests/methods
*Microfluidic Analytical Techniques/methods
Rapid Diagnostic Tests
*Lab-On-A-Chip Devices
*Bacterial Infections/diagnosis/microbiology
Point-of-Care Systems

@article {pmid42199538,
year = {2026},
author = {Wizrah, MSI},
title = {CRISPR-Cas systems as next-generation antimicrobials: a systemic review of mechanisms, delivery strategies, and translational challenges.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1747931},
pmid = {42199538},
issn = {1664-302X},
abstract = {INTRODUCTION: The rapid global increase in multidrug-resistant (MDR) bacteria has compromised the effectiveness of conventional antibiotics, stressing the urgent need for alternative antimicrobial strategies. CRISPR-Cas systems, originally evolved as bacterial adaptive immune mechanisms, provide programmable and highly specific tools for targeting antimicrobial resistance (AMR) determinants.

OBJECTIVE: This systematic review aims to evaluate the antibacterial mechanisms, delivery strategies, preclinical evidence, safety considerations, and translational potential of CRISPR-Cas systems for combating MDR bacterial infections.

METHODS: A systematic literature search was conducted in PubMed, Scopus, Cochrane Library, and Web of Science up to January 2026 in accordance with PRISMA 2020 guidelines. Eligible studies included original in vitro and in vivo experimental or preclinical investigations assessing CRISPR-Cas systems (Cas9, Cas12, Cas13, or related effectors) for antibacterial activity or antibiotic resensitization. Data were extracted on CRISPR effector type, bacterial target, delivery platform, and therapeutic outcome. Due to methodological heterogeneity, results were synthesized narratively.

RESULTS: Most studies reported effective killing or resensitization of MDR bacteria through chromosomal double-strand break induction, resistance plasmid curing, integron disruption, or RNA-targeted cleavage. Cas9 was the most frequently employed effector, followed by Cas12 and Cas13. Delivery strategies included bacteriophages, conjugative plasmids, and nanoparticle-based systems, with phage-mediated delivery demonstrating the most consistent efficacy in complex environments and animal models. Notably, a CRISPR-enhanced engineered bacteriophage cocktail (LBP-EC01) has advanced to clinical evaluation.

DISCUSSION: Overall, the evidence supports CRISPR-Cas antimicrobials as a promising precision-based approach for addressing AMR. However, major barriers remain, including limited host range, instability in physiological environments, emergence of escape mutations, and insufficient data on off-target effects and long-term safety. Addressing these challenges through optimized delivery platforms, multiplex targeting strategies, and standardized safety and regulatory frameworks will be essential for clinical translation.

https://www.crd.york.ac.uk/PROSPERO/view/CRD420261319789, identifier CRD4201319789.},
}

@article {pmid42201448,
year = {2026},
author = {Abuhassan, Q and Al-Ameer, HJ and Gajjar, TB and Hanumanthayya, M and Shukla, SK and Panigrahi, R and Bainsal, N and Khaydarova, D},
title = {CRISPR/Cas‑driven biosensing: molecular mechanisms and advances in diagnostics.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42201448},
issn = {1573-4978},
mesh = {*Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; DNA/genetics ; Electrochemical Techniques/methods ; },
abstract = {The advent of CRISPR-based technologies for DNA diagnostics represents a new epoch in providing technologies for DNA sequence-specific detection of any DNA target. By using the programmable, precise targeting capabilities associated with Cas effector proteins (e.g., Cas9 and Cas12), scientists have been able to repurpose these proteins with their inherent properties: high programmability, single base specificity, and collateral cleavage activity to develop novel, highly capable biosensors for DNA analysis. Unlike previous reviews, this work provides a systematic and mechanism-based classification of CRISPR/Cas biosensors, highlighting recent advances beyond conventional descriptive summaries. This review provides a comprehensive overview of the rapid evolution and application of CRISPR/Cas-based biosensors as a novel strategy for detecting a wide range of human health biomarkers, from nucleic acids to proteins and small molecules. First, we describe the principle of CRISPR/Cas system. Then, we critically analyze and compare the integration of CRISPR/Cas systems with distinct signal transduction strategies, with a dedicated focus on optical (photoelectrochemical, electrochemiluminescence, and fluorescence) and electrochemical readout platforms. Key technological breakthroughs, including ultra-sensitive detection in the attomolar-femtomolar range, advanced amplification strategies (e.g., RCA and EXPAR), and multiplex detection capabilities, are highlighted. Finally, we emphasize the clinical relevance, scalability challenges, and translational potential of these platforms, providing insights into their application in early disease diagnosis, real-time monitoring, and point-of-care testing. Overall, this review offers a critical perspective on current limitations and future directions, positioning CRISPR-based biosensors as promising tools for next-generation precision diagnostics and improved global health outcomes.},
}

*Biosensing Techniques/methods
Humans
*CRISPR-Cas Systems/genetics
DNA/genetics
Electrochemical Techniques/methods

@article {pmid42201536,
year = {2026},
author = {Chen, X and Zhan, B and Shi, R and Chen, J and Lin, Z and Li, Z},
title = {Identification of Closantel as a small-molecule inhibitor of the compact CRISPR-Cas RNA editor Cas13bt3.},
journal = {Molecular diversity},
volume = {},
number = {},
pages = {},
pmid = {42201536},
issn = {1573-501X},
support = {32471255//National Natural Science Foundation of China/ ; 2024J02006//Natural Science Foundation of Fujian Province/ ; },
abstract = {The type VI CRISPR-Cas systems are widely employed for programmable RNA editing, and the ultra-compact Cas13bt3 ribonuclease offers particular advantages for cellular delivery due to its minimal molecular size. However, its therapeutic potential is hindered by nonspecific collateral RNA cleavage activity and the lack of small-molecule inhibitors to enable spatiotemporal regulation of its function. Here, we performed a high-throughput screen for Cas13bt3 inhibitors using a fluorescence resonance energy transfer (FRET)-based RNA cleavage assay. From a library of 17,760 compounds, we identified Closantel as a specific Cas13bt3 inhibitor, with an IC50 of 7.48 µM. Biochemical assays confirmed that Closantel abrogates both on-target and collateral RNA cleavage by Cas13bt3, while exerting negligible inhibitory activity against Cas13a, a closely related Cas13 ortholog. Combined molecular docking and electrophoretic mobility shift assay (EMSA) analyses further revealed that Closantel binds to the cavity of Cas13bt3 that accommodates the direct repeat region of crRNA, thereby competitively interfering with crRNA-Cas13bt3 binding. Finally, to minimize nonspecific RNA cleavage of Cas13bt3, we engineered a K748A mutant that retains robust on-target RNA cleavage activity with reduced collateral activity in vitro. Our findings provide a selective small-molecule chemical probe for Cas13bt3 and an optimized variant with improved targeting precision, collectively advancing the utility of Cas13bt3 for precise RNA editing applications.},
}

@article {pmid42201952,
year = {2026},
author = {Shen, Y and Yeung, AT and Wang, B and Yeh, CT and Ditchfield, P and Korn, E and Han, C},
title = {Tuning mitotic recombination with patterned DNA nicks for precision mosaic analysis.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {22},
pages = {e2531265123},
doi = {10.1073/pnas.2531265123},
pmid = {42201952},
issn = {1091-6490},
support = {R24OD031953//HHS | NIH | NIH Office of the Director (OD)/ ; },
mesh = {Animals ; *Mitosis/genetics ; *Mosaicism ; CRISPR-Cas Systems ; *Recombination, Genetic ; Drosophila/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Deoxyribonuclease I/genetics/metabolism ; Drosophila melanogaster/genetics ; Crossing Over, Genetic ; DNA Breaks, Double-Stranded ; },
abstract = {CRISPR/Cas9-based mosaic analysis is a powerful tool for in vivo genetics but is limited by cytotoxicity and mutagenesis associated with DNA double-strand breaks. Here, we establish Cas9-derived nickases as safer and more reliable alternatives for inducing mitotic recombination in Drosophila. We demonstrate that single-strand nicks are sufficient to generate mosaic clones and systematically dissect the parameters governing this process. We find that clone frequency can be controlled by the gRNA nicking pattern, with two distant nicks on the same DNA strand synergistically enhancing recombination by over ninefold compared to a single nick. Based on these findings, we propose a mechanistic model for nick-induced crossover and provide a versatile toolkit for generating tissue-specific nickases. This work establishes nickase-based mosaic analysis by gRNA-induced crossing-over as a superior method for high-fidelity clonal analysis, enabling more precise investigation of gene function in development and disease.},
}

Animals
*Mitosis/genetics
*Mosaicism
CRISPR-Cas Systems
*Recombination, Genetic
Drosophila/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
*Deoxyribonuclease I/genetics/metabolism
Drosophila melanogaster/genetics
Crossing Over, Genetic
DNA Breaks, Double-Stranded

@article {pmid42201953,
year = {2026},
author = {Hong, Y and Yu, Z and Zhu, W and Sun, J and Zhu, Z and Wang, Z and Cao, M and Lang, Z and Lyu, YX and Liu, P and Zhu, JK},
title = {Multiplex gene editing enables the multibiofortification of essential vitamins and other health-promoting phytonutrients in tomato.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {22},
pages = {e2603937123},
doi = {10.1073/pnas.2603937123},
pmid = {42201953},
issn = {1091-6490},
mesh = {*Solanum lycopersicum/genetics/metabolism/chemistry ; *Gene Editing/methods ; Animals ; *Biofortification/methods ; Humans ; Plants, Genetically Modified/genetics ; *Vitamins/metabolism ; Mice ; *Phytochemicals/metabolism ; CRISPR-Cas Systems ; },
abstract = {Dietary deficiencies in essential micronutrients and other phytonutrients represent a global health and economic burden, contributing to "hidden hunger" and chronic diseases. While genome editing has been employed to improve individual nutritional traits in crops, multibiofortification through simultaneous modification of multiple distinct metabolic pathways is more challenging. Here, we designed a multiplex CRISPR-Cas strategy to edit five key genes in tomato: Sl7-DR2, SlGAD3, SlSGR1, SlGGP1, and SlGGP2. This approach successfully generated quintuple mutant (5m) tomato lines simultaneously biofortified with seven health-promoting compounds: vitamin D3 (from 0 to 0.70 μg/g dry weight), vitamin C (up to 2.53-fold), provitamin A/β-carotene (up to 3.86-fold), α-carotene (up to 2.47-fold), lutein (up to 3.26-fold), lycopene (up to 7.07-fold), and γ-aminobutyric acid (GABA, up to 5.26-fold). Notably, these multibiofortified tomatoes exhibited no significant trade-offs in plant growth or fruit quality. Extracts from 5m tomatoes showed enhanced suppression of colorectal cancer cell proliferation in vitro. This antiproliferative effect was validated in vivo, where dietary supplementation with 5m tomato powder significantly inhibited tumor growth in a mouse xenograft model. Our work demonstrates an effective strategy for developing a next generation of "functional foods" through multibiofortification, creating a single, nutrient-dense crop that combats both micronutrient malnutrition and chronic diseases.},
}

*Solanum lycopersicum/genetics/metabolism/chemistry
*Gene Editing/methods
Animals
*Biofortification/methods
Humans
Plants, Genetically Modified/genetics
*Vitamins/metabolism
Mice
*Phytochemicals/metabolism
CRISPR-Cas Systems

@article {pmid42202045,
year = {2026},
author = {Zhang, J and Chen, L and Zhu, X and Cai, Y and Wei, S and Zhou, X and Shi, Y and Liu, C and Huang, C and Bi, S and Wu, F and Zhou, X and Hong, J and Wang, Y},
title = {Coordinated regulation using small-molecule drugs enables controlled therapeutic genome editing and enhanced genomic precision in situ.},
journal = {Science translational medicine},
volume = {18},
number = {851},
pages = {eadx7857},
doi = {10.1126/scitranslmed.adx7857},
pmid = {42202045},
issn = {1946-6242},
mesh = {Humans ; Animals ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Mice ; *Genomics ; Dependovirus/genetics ; },
abstract = {Achieving precise temporal control over genome editing is essential for safety but remains a challenge, especially when using small-molecule drugs as external regulators over systems like clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated systems). Consequently, controlled therapeutic in situ editing that maintains both precision and efficacy has yet to be demonstrated. Here, we report the PRINCE system, in which nuclease proteins and guide RNAs are both inducible, to deliver programmable nucleases under control more effectively. PRINCE demonstrated temporal precision in human cell cultures over a 2-year period, even after stable genomic integration. The design principles of PRINCE were broadly applicable from CRISPR-Cas9 to a prime editor and also compact programmable nucleases, and the latter platform was named "Little Prince." Upon administration of drug inducers, Little Prince, delivered in a single adeno-associated virus vector in situ to humanized mouse models, ameliorated pathological phenotypes of hypercholesterolemia (average reductions of 45 and 47% in serum total cholesterol and low-density lipoprotein cholesterol, respectively) and neovascular age-related macular degeneration, with significantly reduced lesion size and leakage (P < 0.0001). Last, we demonstrated a consistent and marked reduction in off-target activity across the PRINCE and Little Prince systems in comparison with constitutive editors, with fewer off-target sites and substantially lower editing frequencies, irrespective of nuclease type, delivery method, or genomic target. These results position PRINCE and Little Prince as controlled genome editing platforms with potential for in vivo, particularly in situ, therapeutic applications.},
}

Humans
Animals
*Gene Editing/methods
CRISPR-Cas Systems/genetics
Mice
*Genomics
Dependovirus/genetics

@article {pmid42202049,
year = {2026},
author = {Galtier, M and Krawczyk, A and Fuche, FJ and Charpenay, LH and Stzepourginski, I and Pignotti, S and Arraou, M and Terrasse, R and Brödel, AK and Poquet, C and Prevot, G and Spadoni, D and Buhot, B and Muench, K and Havránek, J and Cárdenas Ramírez, P and Rouquette, M and Decrulle, A and Kerbarh, O and Lieberman, E and Bramorski, C and Grienenberger, A and Hessel, EM and Salzano, G and Garry, DJ and Leveau, A and Duportet, X and Bikard, D and Fernandez-Rodriguez, J},
title = {Treatment of Shiga toxin-producing E. coli infection by CRISPR-Cas-targeted cleavage of the Shiga toxin gene in animal models.},
journal = {Science translational medicine},
volume = {18},
number = {851},
pages = {eadw8114},
doi = {10.1126/scitranslmed.adw8114},
pmid = {42202049},
issn = {1946-6242},
mesh = {Animals ; *Escherichia coli Infections/microbiology/therapy ; *CRISPR-Cas Systems/genetics ; Disease Models, Animal ; *Shiga-Toxigenic Escherichia coli/genetics/pathogenicity ; *Shiga Toxin/genetics ; Mice ; Humans ; Female ; },
abstract = {Escherichia coli is not only a ubiquitous gut commensal but also an opportunistic pathogen responsible for severe intestinal and extraintestinal infections. Shiga toxin-producing E. coli (STEC) poses a notable public health threat, particularly in children, where infections can lead to bloody diarrhea and progress to hemolytic uremic syndrome, a life-threatening condition with long-term complications. Antibiotics are contraindicated in STEC infections because of their potential to induce prophages carrying Shiga toxin (stx) genes, triggering toxin production. Here, we developed a CRISPR-based antimicrobial strategy using a Cas12 nuclease to selectively eliminate O157 STEC clinical isolates, cleaving more than 99% of stx variants, and prevent toxin release. To enable targeted delivery, we engineered a bacteriophage-derived capsid to specifically transfer a nonreplicative DNA payload to E. coli O157, preventing its dissemination. Our therapeutic candidate, EB003, reduced bacterial burden in a murine STEC colonization model. Moreover, EB003 mitigated clinical symptoms, abrogated Stx-mediated toxicity, and accelerated epithelial repair at therapeutically relevant doses in an infant rabbit disease model. These findings demonstrate the potential of CRISPR-based antimicrobials for treating STEC infections and support further clinical development of EB003 as a precision therapeutic against antibiotic-refractory bacterial pathogens.},
}

Animals
*Escherichia coli Infections/microbiology/therapy
*CRISPR-Cas Systems/genetics
Disease Models, Animal
*Shiga-Toxigenic Escherichia coli/genetics/pathogenicity
*Shiga Toxin/genetics
Mice
Humans
Female

@article {pmid40914913,
year = {2026},
author = {McFarlane, GR and Whitaker, K and Plett, KL and O'Rourke, B and Bogema, DR},
title = {Optimising Guide RNA Production for Multiplexed Cas9-Targeted Nanopore Sequencing to Detect Pathogens.},
journal = {Molecular biotechnology},
volume = {68},
number = {5},
pages = {2399-2409},
pmid = {40914913},
issn = {1559-0305},
support = {A-023//Centre for Invasive Species Solutions/ ; },
mesh = {*Nanopore Sequencing/methods ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Triticum/microbiology ; *Fungi/genetics/pathogenicity/isolation & purification ; },
abstract = {In agriculture, biosecurity, and human health, the rapid and accurate detection of pathogens and pests is crucial. Our study investigates the sensitivity and practicality of six guide RNA (gRNA) production methods for use in Nanopore Cas9-targeted sequencing (nCATS), focusing on their implications for multiplexed pathogen and pest detection. Each production method generated a library of eight gRNAs capable of excising ~ 1.6 kb fragments of the 5.8S_rRNA-ITS2-28S_rRNA regions of five economically significant wheat fungal pathogens. Through comparative analysis, we evaluated the efficacy of commercially synthesised and in-house in vitro-transcribed gRNAs, assessing their impact on sequencing enrichment outcomes. Our findings highlight differences amongst the methods in terms of gRNA yield, integrity, performance, and costs. Our best performing gRNA production method was able to successfully identify all target sequences across the 0.96 to 8.4 pg ranges we tested with coverage ranging from 66 to 2037X. This study highlights the challenges and opportunities in implementing nCATS for multiplexed pathogen and pest detection, offering insights into the development of cost-effective and reliable gRNA production strategies for nCATS.},
}

*Nanopore Sequencing/methods
*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/genetics
Triticum/microbiology
*Fungi/genetics/pathogenicity/isolation & purification

@article {pmid41137959,
year = {2026},
author = {Chen, Y and Wang, Z and Lei, X and Liu, Y and Cai, Y and Wang, C and Jin, J and Ding, X},
title = {Efficient Construction of Recombinant CHO Cells for Stable Expression of Therapeutic Proteins Based on New Hotspot 'LOC'.},
journal = {Molecular biotechnology},
volume = {68},
number = {5},
pages = {2527-2537},
pmid = {41137959},
issn = {1559-0305},
mesh = {Animals ; CHO Cells ; Cricetulus ; *Recombinant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Cricetinae ; *Gene Editing/methods ; Humans ; Green Fluorescent Proteins/genetics/metabolism ; Interferon-alpha/genetics/metabolism ; Interferon alpha-2/genetics ; },
abstract = {Efficient generation of stably express therapeutic recombinant proteins in Chinese hamster ovary (CHO) cell lines remains as a major challenge in biologics development. Among available strategies, CRISPR/Cas9-mediated homology-directed repair (HDR) has emerged as a highly efficient method for inserting genes of interest (GOIs) into predefined genomic 'hotspot.' In this study, we report such a 'hotspot' (LOC site) which has discovered previously by our research team, which is a non-coding RNA region within the CHO-K1 genome at position 1689 of NW_003626341.1, located in the LOC103162981 gene. Utilizing this 'hotspot,' the study implemented an integrated platform combining CRISPR/Cas9 genome editing with Bxb1 recombinase-mediated cassette exchange (RMCE) to enable efficient GOIs integration and followed by sustained expression. First, to create a fluorescent landing pad (LP) cell by inserting a Bxb1 attP site, an EGFP cassette and a promoter trap into LOC site via CRISPR/Cas9. Then, LP cell undergoes subculture for 60 consecutive generations, and the fluorescence was consistently observed, illustrating the capacity of LOC site for stable EGFP expression. Donor cassettes harboring GOIs flanked by Bxb1 attB sites were then easily swapped in through RMCE. This platform enabled one‑step insertion and stable expression of therapeutic target proteins ranging from 12 to 190 kDa including human serum albumin (HSA), interferon alpha-2b (INFα2b), recombinant factor VII (rFVII), monoclonal antibodies against the epidermal growth factor receptor (anti-EGFR, AE01), and a disintegrin and metalloproteinase with thrombospondin type 1 motif, 13 (ADAMST13). Here, we efficiently generated HSA CHO cell clones within three weeks; HSA titers reached 220 mg/L in shake‑flask batch culture conditions. Collectively, this study establishes LOC site as a 'hotspot' and shows that the CRISPR/Cas9‑Bxb1 pipeline markedly accelerates CHO cell‑line development for biologics manufacturing.},
}

Animals
CHO Cells
Cricetulus
*Recombinant Proteins/genetics/metabolism
CRISPR-Cas Systems
Cricetinae
*Gene Editing/methods
Humans
Green Fluorescent Proteins/genetics/metabolism
Interferon-alpha/genetics/metabolism
Interferon alpha-2/genetics

@article {pmid41162805,
year = {2026},
author = {Jatwani, S and Aggarwal, F and Jadhav, RR and Yadav, R and Sarma, SJ and Khare, D},
title = {Engineered CRISPR-Cas9 Vector for Efficient Agrobacterium-Mediated Transformation of Phanerochaete chrysosporium.},
journal = {Molecular biotechnology},
volume = {68},
number = {5},
pages = {2549-2559},
pmid = {41162805},
issn = {1559-0305},
mesh = {*Phanerochaete/genetics ; *Transformation, Genetic ; *Genetic Vectors/genetics ; *CRISPR-Cas Systems ; *Agrobacterium/genetics ; Gene Editing/methods ; DNA, Bacterial/genetics ; Luminescent Proteins/genetics/metabolism ; Hygromycin B/pharmacology/analogs & derivatives ; Red Fluorescent Protein ; Promoter Regions, Genetic ; Acetophenones ; },
abstract = {Phanerochaete chrysosporium is a model white-rot-fungus with exceptional lignolytic activity, but its genetic manipulation has remained a major challenge due to low transformation efficiencies. Here, we report the development of an optimized Agrobacterium-mediated transformation (AMT) protocol optimized for this species. A CRISPR-Cas9 compatible binary vector was modified by replacing the mCherry cassette with a strong sGFP reporter under the constitutive 35S promoter, enabling reliable identification of transformants. Using mycelial disks as starting material and applying a sandwich selection method, we achieved stable integration of T-DNA carrying the hygromycin resistance and sGFP genes. Transformation efficiency was influenced by co-cultivation parameters, with low-temperature incubation (23°C) and the addition of acetosyringone significantly improving recovery of resistant colonies. Stable expression of sGFP was observed in transformed mycelia, confirming the reproducibility of the system. Although CRISPR-Cas9-mediated gene editing was not demonstrated in this study, our results provide proof-of-concept that a Cas9-based vector can be stably maintained in P. chrysosporium and drive heterologous gene expression. This work represents the first step toward establishing a functional genome editing platform in this ligninolytic fungus. By expanding the molecular toolbox for P. chrysosporium, the optimized transformation strategy lays the foundation for metabolic engineering aimed at lignin valorization, bioplastics, biofuels, and high-value biochemical production within a sustainable circular bioeconomy framework.},
}

*Phanerochaete/genetics
*Transformation, Genetic
*Genetic Vectors/genetics
*CRISPR-Cas Systems
*Agrobacterium/genetics
Gene Editing/methods
DNA, Bacterial/genetics
Luminescent Proteins/genetics/metabolism
Hygromycin B/pharmacology/analogs & derivatives
Red Fluorescent Protein
Promoter Regions, Genetic
Acetophenones

@article {pmid41264211,
year = {2026},
author = {Kim, S and Park, YG and Choi, JH and Moon, SH},
title = {CRISPR Technology for Livestock Improvement: Advances and Future Directions.},
journal = {Molecular biotechnology},
volume = {68},
number = {5},
pages = {2185-2200},
pmid = {41264211},
issn = {1559-0305},
support = {RS-2023-00220207//Ministry of Science and ICT, South Korea/ ; 25A0203L1//Ministry of Science and ICT, South Korea/ ; Graduate Research Scholarship in 2023//Chung-Ang University/ ; },
mesh = {Animals ; *Livestock/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Breeding ; Animals, Genetically Modified/genetics ; },
abstract = {CRISPR-Cas9 technology is revolutionizing genetic engineering by enabling precise genome modifications across a wide range of organisms, particularly in livestock. This review focuses on livestock improvement and the most recent transformative developments in the application of CRISPR-based genome editing techniques. We analyzed productivity improvements achieved by editing growth factor genes, immunogenic resistance enhancement through the editing of immune loci, and ecological footprint reduction for sustainability. This review also addresses the intricate ethical and regulatory issues posed by the application of CRISPR technology in animal breeding, including concerns about animal welfare violations, unintended off-target effects, and impacts on public perception. Furthermore, we discuss key untapped CRISPR targets in livestock genetics enabled by precision gene editing, the emerging integration of CRISPR with artificial intelligence, and the need for collaboration to address complex ethical and regulatory challenges related to applying CRISPR in animal breeding.},
}

Animals
*Livestock/genetics
*Gene Editing/methods
*CRISPR-Cas Systems
Breeding
Animals, Genetically Modified/genetics

@article {pmid41329461,
year = {2026},
author = {Nguyen, AH and Quang, MT},
title = {CRISPR/Cas9 Genome Editing in Oncology: Mechanisms, Therapeutic Platforms and Translational Challenges.},
journal = {Molecular biotechnology},
volume = {68},
number = {5},
pages = {2201-2229},
pmid = {41329461},
issn = {1559-0305},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Neoplasms/genetics/therapy ; *Gene Editing/methods ; Animals ; Genetic Therapy/methods ; Translational Research, Biomedical ; },
abstract = {The CRISPR/Cas9 genome editing technology has had a significant impact on cancer research and therapeutic development, providing unprecedented precision in manipulating cancer-associated genes. Although this review focuses on Cas9, we situate it within the broader CRISPR landscape that includes DNA-targeting effectors (Cas9/Cas12), RNA-targeting systems such as Cas13, and type III systems with dual DNA and RNA activity, modalities that expand both experimental and therapeutic possibilities. This comprehensive review examines the current applications of CRISPR/Cas9 in oncology, including its mechanisms and the challenges associated with its clinical translation. Knockout, interference, and activation CRISPR screening platforms have transformed functional genomics by systematically interrogating gene function, identifying therapeutic vulnerabilities, and clarifying resistance mechanisms across diverse cancer phenotypes. This technology has also reshaped cancer modeling, enabling precise recapitulation of disease-relevant mutations from engineered cell lines to patient-derived xenografts that capture tumor heterogeneity and microenvironmental interactions. Notably, the integration of CRISPR/Cas9 with CAR-T therapy has enabled multiplex editing to eliminate alloreactivity, overcome checkpoint-mediated exhaustion, and engineer universal CAR-T cells. Emerging in vivo strategies that directly generate or reprogram CAR-T cells in patients via targeted viral and nonviral delivery underscore accelerating translational momentum. However, significant challenges, including off-target mutagenesis, delivery barriers, p53-mediated selective pressure favoring potentially oncogenic populations, and Cas9 immunogenicity, continue to hinder clinical translation. These limitations necessitate high-fidelity nucleases, optimized guide designs, and improved delivery systems. The future of CRISPR/Cas9 in cancer therapy will depend on technological innovation, comprehensive safety frameworks, and rigorous clinical evaluation as next-generation editing modalities advance toward transformative precision oncology.},
}

Humans
*CRISPR-Cas Systems/genetics
*Neoplasms/genetics/therapy
*Gene Editing/methods
Animals
Genetic Therapy/methods
Translational Research, Biomedical

@article {pmid42060320,
year = {2026},
author = {Zhong, N and Wang, M and Jiang, W and Li, G and Miao, J and Yin, H and Vanhnaseng, P and Gong, J and Yu, Z and Han, X},
title = {One-pot CRISPR/Cas12b-LAMP platform for dual-mode detection of Pasteurella multocida.},
journal = {Letters in applied microbiology},
volume = {79},
number = {5},
pages = {},
doi = {10.1093/lambio/ovag044},
pmid = {42060320},
issn = {1472-765X},
support = {2024-02-08-00-12-F00051//Shanghai Agriculture Applied Technology Development Program/ ; 2023YFD1800700//National Key Research and Development Program of China/ ; 2025I0030//External Cooperation Program of Fujian Science and Technology Plan Project/ ; },
mesh = {*Pasteurella multocida/genetics/isolation & purification ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Animals ; *Molecular Diagnostic Techniques/methods ; Milk/microbiology ; *Pasteurella Infections/diagnosis/microbiology ; Sensitivity and Specificity ; Clustered Regularly Interspaced Short Palindromic Repeats ; Limit of Detection ; },
abstract = {Pasteurella multocida is a significant bacterial pathogen that poses a significant threat to public health and causes substantial economic losses. Existing detection methods for P. multocida have limitations, including time-consuming and technically complex methods. Here, we describe a simple and accurate detection platform that combines loop-mediated isothermal amplification (LAMP) with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12b system. By using heparin sodium to modulate the cis-cleavage activity of Cas12b, we developed a one-pot LAMP-CRISPR/Cas12b assay in a single closed tube. The assay achieved a detection limit of 5.0 × 101 CFU ml-1 and showed no cross-reactivity with other bacterial species, indicating high sensitivity and specificity. Furthermore, we validated the clinical utility of the platform using milk samples artificially contaminated with P. multocida, which successfully detected P. multocida in the LAMP-CRISPR/Cas12b results. In summary, this study establishes a novel and robust detection system for P. multocida and highlights its potential for nucleic acid-based diagnostics in practical applications.},
}

*Pasteurella multocida/genetics/isolation & purification
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
Animals
*Molecular Diagnostic Techniques/methods
Milk/microbiology
*Pasteurella Infections/diagnosis/microbiology
Sensitivity and Specificity
Clustered Regularly Interspaced Short Palindromic Repeats
Limit of Detection

@article {pmid42126187,
year = {2026},
author = {Huang, J and Ma, K and Ding, S and Wang, Y and Xiong, J and Yi, J and Zhang, J and He, Z and Huang, L and Ren, X and Zhou, J and Chen, X and Liu, L and Qi, W and Wang, S and Liao, M},
title = {A CRISPR activation screen identifies CH25H as a restriction factor against influenza viruses by targeting accessible cholesterol.},
journal = {Emerging microbes & infections},
volume = {15},
number = {1},
pages = {2651464},
doi = {10.1080/22221751.2026.2651464},
pmid = {42126187},
issn = {2222-1751},
mesh = {*Cholesterol/metabolism ; Animals ; Humans ; *Steroid Hydroxylases/genetics/metabolism ; *Influenza A Virus, H7N9 Subtype/physiology ; Virus Internalization/drug effects ; *Influenza, Human/virology ; Mice ; CRISPR-Cas Systems ; Endoplasmic Reticulum/metabolism ; Cell Membrane/metabolism ; Dogs ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Influenza A viruses (IAVs) cause severe outbreaks with high mortality in birds and humans. A deeper understanding of cell-intrinsic defense mechanisms against influenza viruses is therefore crucial for developing novel antiviral strategies. Herein, we perform a genome-wide CRISPR activation screen to systematically elucidate host restriction factors against influenza A (H7N9) virus. Among multiple candidates, cholesterol 25-hydroxylase (CH25H) is shown to be induced by influenza virus infection and inhibit viral membrane fusion. Notably, our previous work demonstrated that CH25H blocks the entry of plasma membrane-fusing viruses such as coronaviruses. This inhibition occurs by relocating accessible cholesterol from the plasma membrane (PM) to the endoplasmic reticulum (ER). Here, we extend this finding and show that the same mechanism works against endocytosis-dependent viruses such as influenza viruses. The exogenous supplementation of cholesterol can restore depleted accessible cholesterol and reverse the CH25H-mediated restriction. Additionally, we prove that acyl-CoA:cholesterol acyltransferase (ACAT) is required to recruit the accessible cholesterol in this process. However, how hydrophobic accessible cholesterol is transported remains unclear. Here, we demonstrate that GRAMD1/Aster-mediated non-vesicular cholesterol transport is utilized to mobilize accessible cholesterol upon stimulation of CH25H. 25-hydroxycholesterol (25HC), the catalytic product of CH25H, is a natural metabolite that potently inhibits influenza virus infection both in vitro and in vivo. These findings underscore the promising therapeutic potential of 25HC against influenza viruses.},
}

*Cholesterol/metabolism
Animals
Humans
*Steroid Hydroxylases/genetics/metabolism
*Influenza A Virus, H7N9 Subtype/physiology
Virus Internalization/drug effects
*Influenza, Human/virology
Mice
CRISPR-Cas Systems
Endoplasmic Reticulum/metabolism
Cell Membrane/metabolism
Dogs
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42126246,
year = {2026},
author = {Song, R and Yin, C and Chen, B and Qu, B and Qiao, W and Li, R and Gao, Y and Song, X},
title = {Machine Learning-Assisted Portable Ai BOX Based on RPA-CRISPR/Cas12a for Rapid On-Site Detection of Foodborne Pathogens.},
journal = {Analytical chemistry},
volume = {98},
number = {20},
pages = {14913-14927},
doi = {10.1021/acs.analchem.6c00303},
pmid = {42126246},
issn = {1520-6882},
mesh = {*Machine Learning ; *CRISPR-Cas Systems ; *Food Microbiology ; *Listeria monocytogenes/isolation & purification/genetics ; },
abstract = {Foodborne pathogens present a major threat to global public health. However, conventional detection methods and equipment are often unsuitable for the on-site and timely monitoring of these pathogens. To overcome this critical limitation and establish a rapid detection workflow, we developed the portable smart Ai BOX (artificial intelligence BOX). This device is a compact, palm-sized, internet of things (IoT)-enabled instrument that utilizes isothermal fluorescence diagnostics and weighs only 180 g. The Ai BOX features an optimized minimalist industrial design, ultralow power consumption, and a high-sensitivity optical sensing system. The device performs real-time fluorescence detection, with results automatically interpreted and transmitted to a dedicated mobile application (APP) via an integrated smart camera, enabling comprehensive food monitoring. Furthermore, the incorporation of artificial intelligence and machine learning (ML) algorithms significantly enhances the processing capability of the RPA-CRISPR/Cas12a fluorescence signal, thereby ensuring superior detection accuracy. The Ai BOX is ideally suited for on-site point-of-care testing (POCT) of foodborne pathogens. By integrating the one-pot-RPA-CRISPR/Cas12a method, the device achieves an exceptionally low limit of detection (LOD) of 1 × 10[1] CFU/mL for Listeria monocytogenes. In tests using simulated samples, it demonstrated 100% sensitivity and specificity. Consequently, the Ai BOX exhibits promising application potential for diverse public and personal health scenarios, including the detection of meat adulteration, food contamination, and wastewater monitoring.},
}

*Machine Learning
*CRISPR-Cas Systems
*Food Microbiology
*Listeria monocytogenes/isolation & purification/genetics

@article {pmid42133534,
year = {2026},
author = {Liu, C and Feng, M and Yu, H and Zhang, X and Li, Y and Sui, G and Jing, W and Cheng, X},
title = {CRISPR-Cas12a2-Based Multiplexed Diagnostic for Rapid and Highly Sensitive Detection of Respiratory Viruses.},
journal = {Analytical chemistry},
volume = {98},
number = {20},
pages = {14775-14787},
doi = {10.1021/acs.analchem.5c07791},
pmid = {42133534},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Lab-On-A-Chip Devices ; *Respiratory Syncytial Viruses/isolation & purification/genetics ; Molecular Diagnostic Techniques ; *Influenza A virus/isolation & purification/genetics ; },
abstract = {Infectious diseases severely threaten global public health security, necessitating rapid and highly sensitive diagnosis. This study presents a novel multiplex diagnostic platform combining transcription-mediated amplification (TMA) with the CRISPR-Cas12a2 system for rapid and highly sensitive detection of respiratory viruses. The assay uses an integrated microfluidic chip, which can simultaneously identify influenza A/B and respiratory syncytial viruses (RSV-A/B) with optimized CRISPR RNAs and isothermal amplification, achieving detection limits as low as 10[2] copies/μL within 60 min. The detection system showed excellent specificity; nonspecific reactions were not observed in the presence of nucleic acids from other respiratory pathogens. Clinical validation using nasopharyngeal swabs demonstrated high concordance with real-time quantitative reverse transcription polymerase chain reaction, with most positive samples detected within 40 min. The system eliminates DNA amplification steps, reduces contamination risk, and simplifies the workflow. Using two-step reactions on a centrifugal microfluidic chip, the TMA-CRISPR-Cas12a2 platform offers a promising integrated platform for multiplex respiratory pathogen screening, thereby supporting timely diagnosis and outbreak management.},
}

*CRISPR-Cas Systems/genetics
Humans
Nucleic Acid Amplification Techniques/methods
Limit of Detection
Lab-On-A-Chip Devices
*Respiratory Syncytial Viruses/isolation & purification/genetics
Molecular Diagnostic Techniques
*Influenza A virus/isolation & purification/genetics

@article {pmid42184721,
year = {2026},
author = {da Silva, GE and Obst, S and Carvalho, P and Forner, J and Ruf, S and Saibo, NJM and Bock, R},
title = {Generation of a recipient line for Rubisco engineering by multiplex genome editing in tobacco.},
journal = {The Plant journal : for cell and molecular biology},
volume = {126},
number = {4},
pages = {e70930},
doi = {10.1111/tpj.70930},
pmid = {42184721},
issn = {1365-313X},
support = {//Max-Planck-Gesellschaft/ ; UIDB/04551/2020//Fundação para a Ciência e a Tecnologia/ ; PD/BD/138096/2018//Fundação para a Ciência e a Tecnologia/ ; },
mesh = {*Ribulose-Bisphosphate Carboxylase/genetics/metabolism ; *Nicotiana/genetics/enzymology ; *Gene Editing/methods ; CRISPR-Cas Systems ; Photosynthesis/genetics ; Plants, Genetically Modified/genetics ; Plant Proteins/genetics/metabolism ; Genetic Engineering/methods ; },
abstract = {Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase) is the primary CO2-fixing enzyme on our planet. Its slow kinetics and poor discrimination between carbon dioxide and oxygen as substrates severely limit the efficiency of photosynthetic carbon fixation by plants. Attempts to improve Rubisco by genetic engineering have been hampered by the complexity of the Rubisco system, with a gene family for the enzyme's small subunit being encoded in the nuclear genome and the gene for the large subunit residing in the plastid (chloroplast) genome. Another nuclear gene family encodes the enzyme Rubisco activase, which is required to facilitate catalysis in the active site of the enzyme. The Rubisco subunits and the activase have co-evolved, making single-gene replacements largely fruitless and typically resulting in dysfunctional Rubiscos. Here, we have generated a dedicated plant line for Rubisco engineering that lacks nuclear genes for components of the Rubisco system. Using multiplex genome editing by CRISPR-Cas9 in the diploid tobacco species Nicotiana sylvestris, we have knocked out six loci encoding the small subunit of Rubisco and three loci encoding Rubisco activase. The generated mutants are incapable of autotrophic growth, and grafting experiments are underway to obtain transgene-free T1 progeny. The recipient line produced here provides a clean genetic background, in which heterologous Rubisco systems from other organisms can be readily implemented and systematically tested in comparative functional studies. It greatly simplifies Rubisco engineering and creates new opportunities for future efforts to enhance photosynthetic carbon assimilation and increase crop yields.},
}

*Ribulose-Bisphosphate Carboxylase/genetics/metabolism
*Nicotiana/genetics/enzymology
*Gene Editing/methods
CRISPR-Cas Systems
Photosynthesis/genetics
Plants, Genetically Modified/genetics
Plant Proteins/genetics/metabolism
Genetic Engineering/methods

@article {pmid42185623,
year = {2026},
author = {Tsai, FY and Sternberg, SH},
title = {Flying under the radar: immune-evasive DNA for genome engineering.},
journal = {Cell research},
volume = {},
number = {},
pages = {},
pmid = {42185623},
issn = {1748-7838},
support = {RM1HG009490//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; R01EB027793//U.S. Department of Health & Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB)/ ; CAREER 2239685//NSF | BIO | Division of Biological Infrastructure (DBI)/ ; },
}

@article {pmid42187217,
year = {2026},
author = {Awotundun, TA and Samson, OJ and Olanbiwoninu, AA},
title = {Viruses that heal: harnessing bacteriophages in the era of antibiotic resistance.},
journal = {Voprosy virusologii},
volume = {71},
number = {2},
pages = {91-108},
doi = {10.36233/0507-4088-366},
pmid = {42187217},
issn = {2411-2097},
mesh = {*Bacteriophages/genetics/pathogenicity ; Humans ; *Phage Therapy/methods ; *Bacteria/virology/genetics ; *Bacterial Infections/therapy/genetics/microbiology/virology ; Anti-Bacterial Agents/therapeutic use ; Genetic Engineering ; CRISPR-Cas Systems/genetics ; *Drug Resistance, Microbial/genetics ; },
abstract = {The global rise in antimicrobial resistance (AMR) poses an urgent threat to public health, and novel alternatives to traditional antibiotics are needed. One of the most promising options is bacteriophages, viruses that infect and destroy bacteria. Once overshadowed by the discovery of antibiotics, phage therapy is now regaining attention, driven by advances in genomics, synthetic biology, and targeted medicine. This review examines the biology, diversity, and therapeutic use of bacteriophages in treating bacterial infections, especially those caused by multidrug-resistant pathogens. It also discusses how phages act through natural mechanisms, such as lytic enzymes (holins, endolysins, and muralysins), and highlights new genetic engineering techniques, such as CRISPR-Cas systems, phage recombineering, and synthetic genome reboots. In addition to clinical applications, we evaluate phages as biocontrol agents for food safety, environmental sanitation, and biofilm management. Additionally, the article explores key issues in phage therapy, including regulatory frameworks, formulation stability, dynamics of phage-host resistance, and the importance of rapid diagnosis. When properly integrated into modern health and biotechnology practices, bacteriophages offer significant potential and a sustainable solution to the global challenge of antimicrobial resistance.},
}

*Bacteriophages/genetics/pathogenicity
Humans
*Phage Therapy/methods
*Bacteria/virology/genetics
*Bacterial Infections/therapy/genetics/microbiology/virology
Anti-Bacterial Agents/therapeutic use
Genetic Engineering
CRISPR-Cas Systems/genetics
*Drug Resistance, Microbial/genetics

@article {pmid42187489,
year = {2026},
author = {Liu, T and Guo, H and Yu, M and Peng, J and Guan, L and Xie, S and Hao, X and Yang, Y},
title = {A Magnetic-Assisted CRISPR-Cas12a Biosensor Incorporating a Y-DNA Probe for Sensitive Detection of Schistosoma japonicum Eggs.},
journal = {Biosensors},
volume = {16},
number = {5},
pages = {},
doi = {10.3390/bios16050293},
pmid = {42187489},
issn = {2079-6374},
support = {22277047//National Natural Science Foundation of China/ ; 82160631//National Natural Science Foundation of China/ ; 20252BAC250153//Jiangxi Provincial Natural Science Foundation/ ; },
mesh = {Animals ; *Biosensing Techniques ; *Schistosoma japonicum/isolation & purification ; *CRISPR-Cas Systems ; DNA Probes ; Rabbits ; Ovum ; Magnetics ; Aptamers, Nucleotide ; },
abstract = {Schistosomiasis, caused by Schistosoma species, is notoriously difficult to accurately diagnose with conventional methods. In this study, we present an innovative biosensor that integrates CRISPR-Cas12a technology with nucleic acid aptamers for the highly sensitive detection of Schistosoma japonicum eggs. The biosensor leverages a Y-shaped DNA structure (Y-DNA) that incorporates an aptamer specific to S. japonicum eggs, along with an activator DNA and a segment for immobilization on magnetic nanomaterials. Upon target recognition, the Y-DNA releases the activator, which triggers the collateral cleavage activity of Cas12a, enabling the direct detection of eggs. This system demonstrates remarkable sensitivity, being capable of detecting individual eggs in infected rabbit serum and feces. Moreover, it effectively distinguishes the eggs of S. japonicum from those of other parasitic species. The simplicity, high sensitivity, and rapid detection of our biosensor offer significant potential for improving the diagnosis of schistosomiasis, providing a novel, reliable tool for early detection in clinical settings.},
}

Animals
*Biosensing Techniques
*Schistosoma japonicum/isolation & purification
*CRISPR-Cas Systems
DNA Probes
Rabbits
Ovum
Magnetics
Aptamers, Nucleotide

@article {pmid42187493,
year = {2026},
author = {Bao, C and Zhang, H and Jiang, L and Liu, T and Liu, W and Qi, Q and Ren, X and Fu, H and Sun, M},
title = {Synthetic Biology-Enabled Biosensing Platforms for Point-of-Care In Vitro Diagnostics: Programmable Modules, Clinical Applications, and Translational Challenges.},
journal = {Biosensors},
volume = {16},
number = {5},
pages = {},
doi = {10.3390/bios16050297},
pmid = {42187493},
issn = {2079-6374},
support = {JYBS2025011LK//Jilin Medical University Institutional Research Project/ ; No. S202413706005, No. 202113706067, and No. 202513706018).//National Innovation and Entrepreneurship training Program for College Students/ ; 20250204071YY//the Science and Technology Department of Jilin Province/ ; },
mesh = {*Biosensing Techniques ; *Synthetic Biology ; Humans ; *Point-of-Care Systems ; CRISPR-Cas Systems ; Rapid Diagnostic Tests ; },
abstract = {Synthetic biology is reshaping in vitro diagnostics (IVD) by enabling programmable and modular biosensing elements that can be integrated into point-of-care testing (POCT) platforms. Compared with conventional assays that depend on fixed chemistries and centralized instrumentation, synthetic biology-based systems offer adaptable molecular recognition, tunable signal processing, and flexible readout formats for decentralized diagnostics. In this review, we present synthetic biology-enabled IVD as programmable biosensing platforms organized into four functional layers: molecular recognition, signal transduction and amplification, output generation, and system integration. We discuss four major enabling modules, including cell-free protein synthesis (CFPS) systems, aptamer and riboswitch sensors, CRISPR-Cas diagnostic platforms, and microfluidic integration technologies. We summarize representative clinical applications from 2021 to 2025 in infectious disease detection, cancer biomarker analysis, and drug metabolism/toxicity screening. In addition, we examine practical considerations beyond analytical sensitivity, including matrix tolerance, workflow complexity, manufacturability, quantitative capability, and regulatory readiness. Finally, we highlight future directions for programmable diagnostics, including AI-assisted biosensor design, multimodal readouts, interoperable platform architectures, and real-world clinical validation.},
}

*Biosensing Techniques
*Synthetic Biology
Humans
*Point-of-Care Systems
CRISPR-Cas Systems
Rapid Diagnostic Tests

@article {pmid42187704,
year = {2026},
author = {Jiang, C and Yang, D and Sun, C and Ren, X and Li, T and Wu, J and Tian, J and Feng, M and Yao, Y and Song, J and Weng, X and Mu, Y},
title = {An Episomal Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 System for Transgene-Free Multiplex Gene Editing in Pig Cells.},
journal = {Biology},
volume = {15},
number = {10},
pages = {},
doi = {10.3390/biology15100742},
pmid = {42187704},
issn = {2079-7737},
support = {2022YFA1105402//Key Project of Natural Science Foundation of Heilongjiang Province of China/ ; 32272885//National Natural Science Foundation of China/ ; },
abstract = {Despite significant advancements in CRISPR/Cas-based genome editing technology over the past decade, achieving simultaneous homozygous gene editing at multiple targets in primary cells remains a major challenge. In this study, we developed and constructed a CRISPR multi-gene targeting system that integrates episomal vectors with tRNA-sgRNA array technology. This approach leverages scaffold/matrix attachment region (S/MAR) sequences to enable sustained episomal expression of both Cas9 and single-guide RNAs (sgRNAs) without genomic integration, thereby enhancing gene editing efficiency. For simultaneous editing of multiple loci, we used the tRNA-sgRNA architecture to process multiple sgRNAs from a single vector. Using this system in porcine fetal fibroblasts, we achieved concurrent editing of six genes, namely ANXA7, GSK3A, ENTPD6, SIRT3, CYP20A1, and SOCS2, in individual cells. These edited cells supported normal development following somatic cell nuclear transfer, yielding blastocysts with unaltered developmental competence. Collectively, our findings establish a framework for the application of CRISPR/Cas9 in gene-edited pigs, facilitating the generation of multi-gene-edited animals for biomedical and agricultural applications.},
}

@article {pmid42187710,
year = {2026},
author = {Singh, S and Tiwari, H and Singh, M and Gautam, V and Gautam, A and Gautam, HK},
title = {Expanding the Microbial Genomic Landscape and Biotechnological Applications of CRISPR-Cas Systems.},
journal = {Biology},
volume = {15},
number = {10},
pages = {},
doi = {10.3390/biology15100748},
pmid = {42187710},
issn = {2079-7737},
support = {(File No.: ANRF/IRG/2025/000135/LS)//Anusandhan National Research Foundation (ANRF)/ ; CST/D-1187//Council of Science and Technology, Uttar Pradesh, India (CST-UP)/ ; },
abstract = {The CRISPR-Cas systems, identified initially as adaptive immune mechanisms in bacteria and archaea against viral threats, have rapidly evolved into transformative tools in genetic engineering and biotechnology. These RNA-guided systems are broadly classified into Class 1, comprising multi-subunit complexes, and Class 2, characterized by compact single-effector protein, such as Cas9, Cas12, and Cas13. Their remarkable structural and functional diversity enables microorganisms to adapt to diverse ecological niches, offering a vast repertoire of genome-editing strategies. Beyond their natural role in maintaining genome integrity and defense, CRISPR-Cas systems have been extensively repurposed for precise genome modification, transcriptional regulation, epigenetic editing, and nucleic acid detection. Recent advances in computational mining of microbial genomes and metagenomes have uncovered a broad range of novel CRISPR effectors with unique properties, distinct protospacer adjacent motif (PAM) requirements, RNA-targeting capabilities, miniature architectures, and promiscuous cleavage activities that significantly expand the molecular biology toolkit. The development of CRISPR-based technologies such as base editing, prime editing, gene knock-in/out, and live-cell DNA/RNA imaging exemplifies the versatility of these systems. Despite the challenges associated with delivering complex Class 1 systems, both classes are now being actively harnessed across diverse microbial platforms. Concurrently, the CRISPR-Cas research, particularly for guide RNA (gRNA) design and activity prediction, has revolutionized target specificity and editing efficiency. This review presents a comprehensive overview of CRISPR-Cas system diversity, their genomic landscape in microorganisms, and their cutting-edge biotechnological applications. It also emphasizes the transformative potential of CRISPR in synthetic biology, therapeutics, diagnostics, environmental remediation, and agriculture, while also addressing the ethical and biosafety considerations surrounding its deployment. As CRISPR-Cas systems continue to evolve, they stand at the forefront of innovations that bridge natural microbial immunity with engineered precision tools for next-generation biotechnology.},
}

@article {pmid42189993,
year = {2026},
author = {Sabol, AL and Mengiste, AA and Singh, P and Sreekanth, V and Hendel, SJ and Tran, MTN and Barybin, AM and Chaudhary, S and Harris, RM and Liivak, KE and Severance, ZC and Locicero, CM and Kailass, K and Lee, C and Xu, LQ and Butty, VL and Choudhary, A and Shoulders, MD},
title = {Anti-CRISPR-mediated continuous directed evolution of CRISPR-Cas9 in human cells.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {22},
pages = {e2536003123},
doi = {10.1073/pnas.2536003123},
pmid = {42189993},
issn = {1091-6490},
support = {N66001-17-2-4055//DOD | ARPA | Defense Sciences Office, DARPA (DSO)/ ; R35GM136354//HHS | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R01GM132825//HHS | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R01DK132900//HHS | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)/ ; R01GM137606//HHS | NIH | National Institute of General Medical Sciences (NIGMS)/ ; 2330699//National Science Foundation (NSF)/ ; 587836-2024//Natural Sciences and Engineering Research Council of Canada (NSERC)/ ; DRG-2539-24//Damon Runyon Cancer Research Foundation (DRCRF)/ ; P30-CA14051//HHS | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Humans ; *Directed Molecular Evolution/methods ; *CRISPR-Cas Systems/genetics ; Streptococcus pyogenes/genetics ; Mutation ; Adenoviridae/genetics ; HEK293 Cells ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Engineering CRISPR-Cas systems for improved or altered function is critical to both research and therapeutic applications. Unfortunately, most optimization, especially directed evolution in bacterial hosts, fails to capture the functional requirements of the complex mammalian cellular milieu, where activity is usually required. Robust strategies to enable continuous directed evolution of genome-targeting agents directly in human cells remain lacking. Here, we introduce CRISPR-MACE (Mammalian cell-enabled Adenovirus-assisted Continuous Evolution) as a foundational technology to address this need. CRISPR-MACE integrates virus-based continuous evolution with anti-CRISPR-based tunable selection to generate Streptococcus pyogenes Cas9 variants with both increased and decreased DNA binding capacity and nearly 1,000-fold-enhanced resistance to AcrIIA4, the strongest known inhibitor of SpCas9. Notably, across independent evolution campaigns, the same Cas9 gatekeeper mutation reproducibly emerged first, enabling subsequent adaptive steps along two interdependent axes of Cas9 function. In addition to advancing CRISPR technologies, this work establishes key principles and synthetic circuits for continuously evolving CRISPR-Cas systems directly in human cells.},
}

Humans
*Directed Molecular Evolution/methods
*CRISPR-Cas Systems/genetics
Streptococcus pyogenes/genetics
Mutation
Adenoviridae/genetics
HEK293 Cells
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42190501,
year = {2026},
author = {Adams, BG and Wu, J},
title = {Imaging genome dynamics in real time with CRISPR-based technologies.},
journal = {Current opinion in chemical biology},
volume = {93},
number = {},
pages = {102700},
doi = {10.1016/j.cbpa.2026.102700},
pmid = {42190501},
issn = {1879-0402},
abstract = {Gene expression is a fundamental aspect of cellular function, driving diverse biological processes and disease. Dynamic interactions between genomic loci play an essential role in gene regulation. Therefore, visualizing the spatiotemporal dynamics of these interactions is vital to elucidating their function. CRISPR-Cas technology has enabled many powerful techniques for dynamic genome imaging. Recently, new methods for imaging single and multiple loci in live cells have been developed. This review describes the most recent advancements in CRISPR-based genome imaging, covering background reduction, signal amplification, and guide RNA tiling approaches. Fluorescence microscopy techniques complementing CRISPR-based imaging methods are also discussed.},
}

@article {pmid42173700,
year = {2026},
author = {Ramachandran, H and Dobner, J and Nguyen, T and Binder, S and Tolle, I and Vykhlyantseva, I and Krutmann, J and Miccio, A and Staerk, C and Brusson, M and Kontarakis, Z and Prigione, A and Rossi, A},
title = {CleanFinder: a scalable framework for comprehensive genome editing analysis.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2026.04.024},
pmid = {42173700},
issn = {1879-3096},
abstract = {Genome editing often generates complex mixtures of alleles rather than single, predefined outcomes. Resolving these heterogeneous edits across diverse editing modalities, sequencing platforms, and multiplexed designs remains a persistent analytical challenge. To address this, we developed CleanFinder, a browser-native framework for genotyping genome editing outcomes using a constrained semi-global alignment strategy. Context-aware alignment modes support a broad spectrum of editing scenarios, including indels, base substitutions, and complex prime editing modifications across nuclear and mitochondrial targets. Additional modules include an optional turbo mode for high-throughput heuristic alignment in exploratory workflows and an allele-aware module that leverages heterozygous single-nucleotide polymorphisms to detect allelic dropout. To evaluate scalability and practical performance, we applied CleanFinder to a primary small-molecule screen of 1849 compounds in HEK293T cells. The software efficiently processed the dataset, enabling high-throughput comparison of editing outcomes and nomination of candidate compounds for follow-up analysis. Together, CleanFinder provides a flexible and scalable platform for genome editing analysis, enabling detailed genotyping and systematic comparison of editing outcomes across diverse edit types and genomic contexts.},
}

@article {pmid42176206,
year = {2026},
author = {Dias, RG and Freitas, FPM and Barbosa, SL and Assis, JVMG and Entringer, TL and Fonseca, JSC and Romanizio, ME and Pimentel, BBZ and Campos-Galvão, MEM and Vieira, NM and Fietto, LG and Zsögön, A and da Silveira, WB},
title = {10 years of CRISPR/CAS genomic engineering in Yarrowia lipolytica.},
journal = {Bioprocess and biosystems engineering},
volume = {},
number = {},
pages = {},
pmid = {42176206},
issn = {1615-7605},
abstract = {Yarrowia lipolytica is a versatile cell factory widely used in bioprocesses for producing lipids, organic acids, and other high-value compounds. Historically, its genetic engineering was constrained by low homologous recombination (HR) efficiency and the predominance of non-homologous end joining (NHEJ), limiting strain development and metabolic pathway optimization. The advent of CRISPR-Cas technologies has revolutionized genome editing in Y. lipolytica, enabling precise, efficient, and multiplexed modifications. Innovations such as pCAS1yl and pCRISPRyl plasmids, along with genomic Cas9 integration, have enhanced targeted editing efficiency. CRISPR applications now extend to metabolic engineering for lipids, itaconic acid, erythritol, and other compounds. Beyond canonical Cas9, alternative systems, including CRISPRa, Cas12a, base editors, and sgRNA libraries, provide increased flexibility and functional diversity. Central to these approaches is the rational design of guide RNAs (gRNAs), supported by bioinformatics platforms such as CHOPCHOP v3, CRISPOR, CCTop, and Cas-OFFinder, which assist in target site selection, off-target prediction, and editing optimization. This review summarizes the main CRISPR/Cas9 applications in Y. lipolytica, highlighting key engineered strains and emphasizing the critical role of bioinformatics in improving editing strategies. We also propose a pipeline for systematic gRNA design based on published evidence and discuss future perspectives, including the integration of machine learning, artificial intelligence, and emerging CRISPR variants to further advance yeast metabolic engineering.},
}

@article {pmid42178819,
year = {2026},
author = {Yadav, A and Saini, A and Dilbaghi, N and Yadav, N},
title = {Advances in Nano-Enabled Biosensing Technology for Gastric Cancer Diagnosis: Mechanistic Insights and Translational Perspectives.},
journal = {Critical reviews in analytical chemistry},
volume = {},
number = {},
pages = {1-28},
doi = {10.1080/10408347.2026.2672560},
pmid = {42178819},
issn = {1547-6510},
abstract = {Gastric cancer (GC) remains a critical burden on healthcare, affecting millions of people annually. Helicobacter pylori infection is a critical contributor to GC. The diagnostic confirmation of GC is generally obtained at late stages owing to the delayed onset of symptoms. Early detection can significantly improve the disease outcomes. Several approaches, like endoscopy, MRI, and computed tomography, are conventionally employed. But they often have certain drawbacks, such as less accessibility, invasiveness, and potential false results. Delayed diagnosis and poor prognosis by conventional strategies have underlined the need for an efficient and precise solution. These obstacles can be mitigated by implementing advanced biosensing platforms. The amalgamation of nanotechnology, machine learning, and advanced computational intelligence has extensively evolved sensor technology. This review offers a holistic overview of GC pathogenicity and conventional diagnostics with special emphasis on recently fabricated biosensors. Advanced biosensing platforms, like CRISPR-Cas, smartphone-integrated, breath-based, and ingestible biosensors, are also explored. This review further highlights the translational perspectives along with the increasing role of AI and advanced algorithms. With a critical discussion on key challenges, this article provides a future roadmap for the detection of GC biomarkers. Significant innovations are needed to translate biosensors into a state-of-the-art technique in GC diagnostics.},
}

@article {pmid42178964,
year = {2026},
author = {Sha, T and Zhao, D and Zhao, X and Lu, Y and Wang, R and Liu, J and Li, Y and Li, S and Chen, M and Bi, C and Zhang, X},
title = {Mutant-Initiated Structure-Guided Refinement Enables Second-Generation Compact IscB Genome Editors.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {27},
number = {10},
pages = {e70392},
doi = {10.1002/cbic.70392},
pmid = {42178964},
issn = {1439-7633},
support = {XDC0110200//the Strategic Priority Research Program of the Chinese Academy of Sciences/ ; 32225031//National Science Fund for Distinguished Young Scholar/ ; 32488301//the Basic Science Center Project of the National Natural Science Foundation of China/ ; 32271483//National Natural Science Foundation of China/ ; 2022177//Youth Innovation Promotion Association CAS/ ; },
mesh = {*Gene Editing/methods ; Humans ; Mutation ; DNA/chemistry/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; },
abstract = {Compact RNA-guided nucleases such as IscB represent an attractive foundation for next-generation genome editors, yet their application in mammalian cells has been constrained by suboptimal activity. Here, instead of re-engineering enzymes, we establish a mutant-initiated, structure-guided optimization strategy to generate second-generation high-activity IscB editors. Using AlphaFold3 to model the engineered IscB*-ωRNA-DNA complex, we reveal remodeling of the nucleic-acid-binding interface induced by activity-enhancing substitutions. Guided by this predicted structure, we perform a focused mutational scan and identify V367 as an activity hotspot. Saturation mutagenesis at this position yields a single substitution, V367Y (IscB*-Act), which increases mean editing efficiency by 34% and achieves up to 2.1-fold improvement across endogenous targets in mammalian cells. Importantly, the V367Y substitution is transferable to an IscB-based adenine base editor, elevating A-to-G conversion by 68% on average and up to 4.46-fold at individual loci without altering the intrinsic editing window. Targeted off-target profiling at loci suggests that V367Y does not substantially increase off-target indels or A-to-G conversion. Together, our work demonstrates a practical framework for second-generation refinement of compact genome editors, bridging deep-learning-enabled structural prediction with interpretable protein engineering, and expands the functional potential of miniature IscB systems for both nuclease and base editing applications.},
}

*Gene Editing/methods
Humans
Mutation
DNA/chemistry/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism

@article {pmid42179772,
year = {2026},
author = {Vats, P and Baweja, B and Saini, C and Kushwah, AS and Kumar, A and Srivastava, SK and Nema, R},
title = {An overview of CRISPR-artificial intelligence theranostics: Current and emerging applications.},
journal = {Biomaterials translational},
volume = {7},
number = {1},
pages = {79-120},
pmid = {42179772},
issn = {2096-112X},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics are revolutionizing precision medicine by enabling highly sensitive detection of nucleic acid and protein biomarkers. Building on these capabilities, CRISPR-based theranostics now aim to unify real-time disease detection with targeted therapeutic interventions. However, traditional CRISPR diagnostics face several limitations, including restricted multiplexing, off-target effects, and challenges in delivery efficiency. To overcome these issues, artificial intelligence (AI) has significantly enhanced CRISPR platforms by enabling intelligent guide RNA (gRNA) design, interpretation of complex biosensor outputs, and facilitation of rapid clinical decision-making. Machine learning tools such as DeepCRISPR, Azimuth 2.0, DeepHF, and CRISPRpred support the development of highly specific gRNAs, reduce off-target events, and personalize genome-editing strategies based on individual genomic profiles. Recently, by combining CRISPR systems with nanomaterials, fluorescence-based detection, and electrochemical sensing, researchers have developed advanced biosensors capable of detecting a broad spectrum of disease biomarkers, from cancer-associated nucleic acids to viral and genetic disorders. These advances support both diagnostics and gene therapy, enabling accurate, low-cost testing at home, in point-of-care settings, and in resource-limited environments. Together, the integration of AI and CRISPR is accelerating biomarker discovery and the development of intelligent, adaptive therapeutic platforms. New point-of-care diagnostic tests (POCTs) based on CRISPR-AI are essential for early screening of high-mortality diseases, and CRISPR-based diagnostic assays have emerged as powerful, versatile alternatives to traditional nucleic acid tests, offering rapid, programmable, and portable diagnostic solutions. This review explores the evolution of CRISPR-AI theranostic systems, current and emerging POCT applications. It highlights the technological, clinical, and ethical challenges shaping their translation into next-generation precision diagnostics.},
}

@article {pmid42180316,
year = {2026},
author = {Chen, X and Zhang, M and Yang, L and Chen, Y and Chi, Y and Zhao, Y and Ma, Z and Li, Y and Wang, X},
title = {CRISPR spacer profiling and prophage mining reveal diverse bacteriophages associated with Streptococcus Mutans.},
journal = {Journal of oral microbiology},
volume = {18},
number = {1},
pages = {2674332},
pmid = {42180316},
issn = {2000-2297},
abstract = {BACKGROUND: Streptococcus mutans is a key cariogenic bacterium. Current antimicrobials lack species specificity, while phage-based approaches remain experimental and require more S. mutans phage isolates.

OBJECTIVE: To profile the diversity of S. mutans-associated phages and strain-level heterogeneity in phage exposure using genome-informed CRISPR spacer and prophage analyses.

MATERIALS AND METHODS: We compiled 944 publicly available S. mutans genomes and dereplicated them into 735 non-redundant strains. CRISPR-Cas systems, spacers, spacer targets, and putative prophages were identified, quality-assessed, and functionally annotated. Phylogenetic relationships of (pro)phages were evaluated using terminase large subunit proteins, and comparative genomics compared spacer-positive and spacer-negative strains.

RESULTS: CRISPR systems were detected in 548/735 strains, yielding 14,263 spacers, 1,864 phage-targeting spacers mapped to 110 viral genomes, including 41 cultured isolates, 51 metagenome-assembled phages, and 18 uncultured viral genomes. The most frequently targeted cultured phage was phiKSM96, whereas metagenome-assembled Caudoviricetes ctNo011 showed broader targeting. Prophage mining identified 186 regions in 130 strains, including 37 of ≥ medium quality and elements related to ctNo011 and phiKSM96. TerL phylogeny showed that most high-quality endogenous prophages clustered with phiKSM96 and ctNo011.

CONCLUSION: These findings reveal a vast, uncultivated phage repertoire targeting S. mutans, providing a critical genomic roadmap to guide the future isolation of novel phages for caries prevention.},
}

@article {pmid42180538,
year = {2026},
author = {Yang, J and Chen, Y and Chen, X and Ping, Y},
title = {Non-viral delivery of genome-editing tools for treatment of genetic disorders.},
journal = {Acta pharmaceutica Sinica. B},
volume = {16},
number = {5},
pages = {2903-2928},
pmid = {42180538},
issn = {2211-3835},
abstract = {Pathogenic mutations within protein-coding regions of genomic DNA can disrupt protein structure and lead to hereditary disorders. Genome-editing technologies, particularly those based on clustered, regularly interspaced, short palindromic repeats-associated protein (CRISPR-Cas), are promising therapeutic tools for correcting genetic abnormalities. To date, viral delivery vectors for genome-editing biomacromolecules have shown numerous promises in treating genetic disorders. However, safe viral delivery for genome-editing components remains challenging, largely due to the immunogenicity of viruses. As an alternative, non-viral delivery systems are emerging as a safer choice and may offer solutions to address the safety challenges. In this review, we first introduce CRISPR-Cas9-based genome editing tools and their delivery formats. Then, we outline the pathology of major genetic disorders and both preclinical and clinical approaches for these diseases by therapeutic genome editing, and provide an overview of current non-viral delivery strategies and their potential to overcome existing limitations. Finally, we discuss the current challenges and future outlooks of non-viral delivery of gene-editing components in treating genetic diseases.},
}

@article {pmid42182021,
year = {2026},
author = {Li, X and Huang, Y and Zhang, X and Du, L and Qiu, Y and Jiang, L},
title = {Recent advances in rapid multiplex detection of nucleic acid markers using RPA and CRISPR-Cas.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1810544},
pmid = {42182021},
issn = {1664-302X},
abstract = {The integration of recombinase polymerase amplification (RPA) with CRISPR-Cas systems has emerged as a powerful platform for rapid multiplex nucleic acid detection. Compared with quantitative polymerase chain reaction (qPCR) and Next-generation sequencing (NGS), RPA-CRISPR operates isothermally (37 °C-42 °C), requires minimal equipment, and achieves attomolar sensitivity in 20-90 min via collateral cleavage. Recent multiplex strategies, namely two-tube, spatial separation one-tube, and homogeneous one-pot, they have overcome crosstalk and enabled highly multiplexed detection in complex food matrices such as poultry, milk, and lettuce. These approaches are particularly suited for foodborne pathogen screening (e.g., Salmonella, Listeria), antimicrobial resistance profiling, and on-site surveillance, aligning with the scope of research at the frontier of food microbiology diagnostics. Despite advances, challenges persist in standardization, matrix inhibition, and regulatory approval. This mini-review summarizes recent advances (2020-2025) in RPA-CRISPR multiplex detection, outlines future directions for clinical implementation and food safety deployment, and provides guidance for subsequent research on its practical applications in these fields.},
}

@article {pmid42182524,
year = {2026},
author = {Huang, X and Sun, X and Dong, X and Tang, Y and Xu, S and Wu, Y and Hu, S and Ren, Y and Tu, Q and Zhang, Y},
title = {Prospects and challenges in using engineered lactic acid bacteria in aquaculture applications.},
journal = {Engineering microbiology},
volume = {6},
number = {2},
pages = {100275},
pmid = {42182524},
issn = {2667-3703},
abstract = {Despite the considerable potential of lactic acid bacteria (LAB) as probiotics, there is a fundamental gap between the functional limitations of wild-type strains and the complex demands of aquaculture. Modular and intelligent engineering strategies are the primary avenues for bridging this gap. This article systematically reviews the strategies and advances in the application of genetically engineered LAB. Technologies, including clustered regularly interspaced short palindromic repeat (CRISPR)/Cas systems, Red/ET recombination, and functional modifications have significantly enhanced the targeted delivery, environmental tolerance, and multiple probiotic functions of LAB, successfully yielding engineered strains such as oral vaccine strains expressing pathogen antigens, antimicrobial peptide-high-yielding antibacterial strains, and nitrite-degrading water-improving strains. These engineered strains have demonstrated superior performance in disease prevention, growth promotion, and environmental remediation compared to wild-type strains in the farming of tilapia, shrimp, and shellfish. However, challenges, such as plasmid instability, biosafety risks, and regulatory barriers, remain unresolved. Future research should focus on multi-omics-guided precision design, development of environmentally responsive genetic circuits, and full-cycle risk assessment, promoting engineered LAB as a core solution for sustainable aquaculture through collaboration across industries, academia, and research.},
}

@article {pmid42184324,
year = {2026},
author = {Aliev, TI and Imatdinov, AR and Prudnikova, EY and Imatdinov, IR},
title = {[Experimental Selection of Effective sgRNAs for MmCas12m Targeting the Region of the Start Codon of the HIV-1 gag Gene].},
journal = {Molekuliarnaia biologiia},
volume = {60},
number = {1},
pages = {120-131},
doi = {10.7868/S3034555326010073},
pmid = {42184324},
issn = {0026-8984},
mesh = {*HIV-1/genetics/metabolism ; Humans ; *CRISPR-Cas Systems ; *Codon, Initiator/genetics ; *Gene Editing ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *gag Gene Products, Human Immunodeficiency Virus/genetics/metabolism ; *HIV Infections/genetics/therapy ; },
abstract = {HIV-1 remains a threat to global health. There is no effective vaccine or drug for a complete cure of HIV infection. Work continues on the development of gene therapy drugs against HIV-1. The use of DNA base editors delivered to the editing site by CRISPR-Cas systems has shown some success in the field of HIV-1 gene therapy. The MmCas12m isoform obtained from Mycolicibacterium mucogenicum can become a promising platform for this task. MmCas12m has a compact size, the ability to bind strongly to the target DNA sequence, and an absence of nuclease activity. Thus, MmCas12m can act not only as a platform for the delivery of DNA base editors, but also as an inhibitor of transcription from HIV-1 proviral DNA. We experimentally selected in vitro the most effective sgRNAs for MmCas12m to target the start codon of the gag HIV-1, the product of which is important in virion assembly. Of the nine sgRNAs we selected, four showed statistically significant effectiveness in targeting the desired region of the HIV-1 genome. To test the effectiveness of each sgRNA, we have developed a system suitable for evaluating the binding of any Cas protein to the target site of HIV-1 genome editing.},
}

*HIV-1/genetics/metabolism
Humans
*CRISPR-Cas Systems
*Codon, Initiator/genetics
*Gene Editing
*RNA, Guide, CRISPR-Cas Systems/genetics
*gag Gene Products, Human Immunodeficiency Virus/genetics/metabolism
*HIV Infections/genetics/therapy

@article {pmid42184325,
year = {2026},
author = {Kolesnikova, OA and Svetlakova, AV and Furtak, ED and Zvereva, SD and Kukushkin, ID and Komedchikova, EN and Shipunova, VO},
title = {[Establishment and Validation of Cancer Cell Lines with HER2 Receptor Gene Deletion].},
journal = {Molekuliarnaia biologiia},
volume = {60},
number = {1},
pages = {132-143},
doi = {10.7868/S3034555326010086},
pmid = {42184325},
issn = {0026-8984},
mesh = {Humans ; *Erb-b2 Receptor Tyrosine Kinases/genetics/metabolism ; Cell Line, Tumor ; CRISPR-Cas Systems ; *Gene Deletion ; Gene Editing ; Cell Proliferation ; },
abstract = {One of the clinically significant molecular targets in oncotheranostics is the receptor tyrosine kinase HER2 (HER2/neu, ERBB2), which is involved in the activation of various cellular programs. The overexpression of this receptor leads to uncontrolled cell proliferation, the initiation of oncogenesis, and is considered one of the most important oncogenic biomarkers. In vitro studies utilizing various HER2-positive cancer cell lines play a crucial role in the development of anti-HER2 targeted drug formulations. These cell lines differ in their structural and metabolic features, as well as in their sensitivity to hormones and other factors; therefore, the selection of an optimal cellular control is essential for the successful testing of HER2-specific agents. In the present study, a deletion in the ERBB2 gene was generated using CRISPR/Cas9 technology, with the success of the editing confirmed by sequencing of the target locus. A reduction in ERBB2 mRNA levels was demonstrated in three cancer cell lines with varying baseline HER2 receptor levels, alongside an alteration in the receptor's functional activity on the cell surface. Assessment of the binding efficiency of a fluorescently labeled HER2-specific antibody to the generated cell clones revealed a decrease in fluorescence intensity by 80.6-fold, 33.7-fold, and 2-fold in the SK-BR-3, SK-OV-3, and A549 cell lines, respectively. The generated cell lines with ERBB2 deletion represent a key tool for testing targeted therapeutics and can be utilized in the development of treatment modalities aimed at HER2-overexpressing malignant neoplasms.},
}

Humans
*Erb-b2 Receptor Tyrosine Kinases/genetics/metabolism
Cell Line, Tumor
CRISPR-Cas Systems
*Gene Deletion
Gene Editing
Cell Proliferation

@article {pmid42184676,
year = {2026},
author = {Li, P and Hu, Q and Li, Y and Li, S and Ruan, Z and Yin, K and Sun, G},
title = {Amplification-free light-activated CRISPR/Cas12a system with nano-amplifier for quantitative detection of non-nucleic acid targets.},
journal = {Biosensors & bioelectronics},
volume = {309},
number = {},
pages = {118844},
doi = {10.1016/j.bios.2026.118844},
pmid = {42184676},
issn = {1873-4235},
abstract = {Quantitative amplification-free CRISPR/Cas diagnosis to non-nucleic acid clinical biomarkers is limited by inefficient signal conversion and suboptimal sensitivity. Here, we present a light-activated CRISPR/Cas12a biosensing platform (LANA) to response this challenge. The LANA arms with a cascade signal enhancing strategy that integrates immunomagnetic enrichment, aptamer-functionalized gold nanoparticles carrying photocleavable activators, and UV-triggered release of activators to initiate Cas12a trans-cleavage. This light-controlled mechanism overcomes steric hindrance caused by surface confinement, provides precise temporal control of signal initiation, and effectively suppresses background activation. Using cardiac troponin I (cTnI) as a model analyte, LANA achieved a detection limit of 50 pg/mL, a wide dynamic range of 0.05-500 ng/mL, demonstrating high sensitivity and reliable quantitative performance with a simple fluorescence readout. Owing to its modular and amplification-free design, the platform can be readily adapted to other protein or small-molecule biomarkers, offering a generalizable and amplification-free framework for sensitive detection of non-nucleic acid targets.},
}

@article {pmid42173587,
year = {2026},
author = {Lu, X and Wang, Y and Che, Y and Li, Y and Nong, B and Ge, Y and Wang, X and Guo, Y and Li, R and Liu, J and Guo, J and Yao, Y and Geng, M},
title = {CRISPR/Cas9 mediated knockout of MeSSI enhances resistant starch content without compromising yield in cassava.},
journal = {Carbohydrate polymers},
volume = {385},
number = {},
pages = {125382},
doi = {10.1016/j.carbpol.2026.125382},
pmid = {42173587},
issn = {1879-1344},
mesh = {*Manihot/genetics/metabolism/chemistry ; *CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; *Starch/chemistry/metabolism ; *Plant Proteins/genetics/metabolism ; Amylopectin/metabolism/chemistry ; Gene Editing ; Amylose/metabolism ; Gene Expression Regulation, Plant ; *Resistant Starch/metabolism ; },
abstract = {Enhancing resistant starch (RS) content in cassava is vital for developing nutritionally improved, functional food crops. In this study, targeted mutagenesis of the MeSSI gene via CRISPR/Cas9 was conducted to investigate its role in starch biosynthesis and RS accumulation. MeSSI knockout lines exhibited a 6.74-fold increase in RS content and a 16.42% elevation in amylose levels compared to the wild-type, without compromising total starch content or root yield. Starch structural analysis revealed an increased number of smaller granules per amyloplast and a shift in amylopectin chain-length distribution, characterized by reduced short chains (DP 6-12) and enrichment of intermediate and long chains, resulting in a lower branching degree. These modifications were associated with enhanced thermal stability and altered pasting behavior. Transcriptomic profiling indicated compensatory upregulation of AGPase subunits, and glycolytic genes, suggesting a reprogramming of carbon metabolism to sustain starch accumulation. This work identifies MeSSI as a key determinant of amylopectin fine structure and RS formation, providing a precise genome-editing strategy to improve the nutritional profile of cassava.},
}

*Manihot/genetics/metabolism/chemistry
*CRISPR-Cas Systems/genetics
Gene Knockout Techniques
*Starch/chemistry/metabolism
*Plant Proteins/genetics/metabolism
Amylopectin/metabolism/chemistry
Gene Editing
Amylose/metabolism
Gene Expression Regulation, Plant
*Resistant Starch/metabolism

@article {pmid42019459,
year = {2026},
author = {Yun, Q and Gu, M and Li, L and Xia, WH},
title = {Rapid and visual detection of Mycoplasma pneumoniae using a novel ERA-CRISPR/Cas12a-based lateral flow assay.},
journal = {Diagnostic microbiology and infectious disease},
volume = {116},
number = {1},
pages = {117416},
doi = {10.1016/j.diagmicrobio.2026.117416},
pmid = {42019459},
issn = {1879-0070},
mesh = {Humans ; *Mycoplasma pneumoniae/isolation & purification/genetics ; Sensitivity and Specificity ; *Pneumonia, Mycoplasma/diagnosis/microbiology ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Point-of-Care Testing ; Limit of Detection ; *Nucleic Acid Amplification Techniques/methods ; Child ; Adhesins, Bacterial/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {OBJECTIVE: Mycoplasma pneumoniae (MP) is a leading cause of community-acquired pneumonia in children. Conventional detection methods often lack the speed or accessibility required for point-of-care testing (POCT). This study aims to develop a rapid, highly sensitive, and instrument-free diagnostic platform integrating Enzymatic Rapid Amplification (ERA), CRISPR/Cas12a cleavage, and Lateral Flow Assay (LFA) for visual MP detection.

METHODS: Specific primers targeting the conserved P1 adhesin gene of MP were screened for ERA efficiency. A CRISPR/Cas12a system was designed to specifically recognize the ERA amplicons, triggering the trans-cleavage of a reporter probe. The results were visually interpreted using LFA strips. The assay's limit of detection (LoD), specificity against six common respiratory pathogens, and clinical performance on 80 throat swab samples were evaluated.

RESULTS: The optimized ERA-CRISPR/Cas12a-LFA assay can be completed within 40 minutes. The LoD was established at 200 copies/mL. Specificity testing showed no cross-reactivity with S. pneumoniae, H. influenzae, or other tested pathogens. In clinical validation (n = 80), the assay demonstrated a sensitivity of 96.23% and specificity of 100% compared to qPCR, with a Kappa value of 0.945.

CONCLUSION: The established ERA-CRISPR/Cas12a-LFA method offers a rapid, sensitive, and specific alternative for MP screening. Its minimal equipment requirements make it highly suitable for resource-limited settings and primary care clinics.},
}

Humans
*Mycoplasma pneumoniae/isolation & purification/genetics
Sensitivity and Specificity
*Pneumonia, Mycoplasma/diagnosis/microbiology
*CRISPR-Cas Systems
*Molecular Diagnostic Techniques/methods
Point-of-Care Testing
Limit of Detection
*Nucleic Acid Amplification Techniques/methods
Child
Adhesins, Bacterial/genetics
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid42172144,
year = {2026},
author = {Aguilar, G and Sickmann, ME and Bieli, D and Born, G and Affolter, M and Müller, M},
title = {In situ mutational screening and CRISPR interference define apterous cis-regulatory inputs during compartment boundary formation.},
journal = {eLife},
volume = {12},
number = {},
pages = {},
pmid = {42172144},
issn = {2050-084X},
support = {310030_192659/SNSF_/Swiss National Science Foundation/Switzerland ; 310030B_176400/SNSF_/Swiss National Science Foundation/Switzerland ; },
mesh = {Animals ; *Drosophila Proteins/genetics/metabolism ; *Wings, Animal/embryology/growth & development ; *Gene Expression Regulation, Developmental ; *Transcription Factors/genetics/metabolism ; *Drosophila melanogaster/genetics/embryology ; CRISPR-Cas Systems ; Homeodomain Proteins/genetics/metabolism ; Body Patterning/genetics ; Mutation ; },
abstract = {The establishment of tissue axes is fundamental during embryonic development. In the Drosophila wing, the anterior/posterior (AP) and the dorsal/ventral (DV) compartment boundaries provide the basic coordinates around which the tissue develops. These boundaries arise as a result of two lineage decisions, the acquisition of posterior fate by the selector gene engrailed (en) and dorsal fate by the selector gene apterous (ap). While the en expression domain is set up during embryogenesis, ap expression begins only during early wing development. Thus, the correct establishment of the ap expression pattern relative to en must be tightly controlled. Here, we functionally investigate the transcriptional inputs integrated by the early ap enhancer (apE) and their requirement for correct boundary formation. Detailed mutational analyses using CRISPR/Cas revealed a role for apE in positioning the DV boundary relative to the AP boundary, with apE mutants often displaying mirror-image anterior wing duplications. We then designed and applied methods to accomplish tissue-specific enhancer disruption via dCas9 expression. This approach allowed us to dissect the spatiotemporal requirement for apE function, clarifying the mechanism by which apE misregulation leads to AP defects. Base-pair-resolution analyses of apE uncovered a single HOX-binding site essential for wing development that, when mutated, led to wingless flies. We demonstrated that the transcription factors Pointed (Pnt), Homothorax (Hth), and Grain (Grn) are required for apE function, and the HOX gene Antennapedia (Antp) contributes to early wing development. Together, our results provide a comprehensive molecular basis of early ap activation and the developmental consequences of its misregulation, shedding light on how compartmental boundaries are set up during development.},
}

Animals
*Drosophila Proteins/genetics/metabolism
*Wings, Animal/embryology/growth & development
*Gene Expression Regulation, Developmental
*Transcription Factors/genetics/metabolism
*Drosophila melanogaster/genetics/embryology
CRISPR-Cas Systems
Homeodomain Proteins/genetics/metabolism
Body Patterning/genetics
Mutation

@article {pmid42172311,
year = {2026},
author = {Guo, C and Zhang, S and Yerramsetti, R and Zhang, J and Guan, X and Yang, R and Hou, C and Pei, M and Schalper, KT and Liu, X and Li, Z and Perpetua, L and Gan, W and Ibrahim, O and Clark, RA and Liu, C},
title = {Single-nucleotide variant profiling in liquid biopsy with RECO-Cas.},
journal = {Science advances},
volume = {12},
number = {21},
pages = {eaed1757},
pmid = {42172311},
issn = {2375-2548},
mesh = {Humans ; Liquid Biopsy/methods ; *Polymorphism, Single Nucleotide ; *CRISPR-Cas Systems/genetics ; Proto-Oncogene Proteins p21(ras)/genetics ; *Cell-Free Nucleic Acids/genetics/blood ; ErbB Receptors/genetics ; Class I Phosphatidylinositol 3-Kinases/genetics ; *Neoplasms/genetics/diagnosis ; Mutation ; },
abstract = {Mutation detection of cell-free DNA (cfDNA) through liquid biopsy is essential for precision oncology, resistance profiling, and informed clinical decision-making. However, its clinical application has remained limited by the lack of simple, rapid, accurate, and cost-effective detection approaches. Here, we report a recombined DNA construct-activated Cas12a (RECO-Cas) assay for profiling cfDNA mutations. The RECO-Cas assay uses a recombined DNA construct generated from Argonaute-nicked mutant DNA and an artificial DNA activator to selectively trigger CRISPR-Cas12a, enabling 0.01% variant allele frequency sensitivity and single-nucleotide resolution. Using the assay, we detect KRAS, EGFR, and PIK3CA point mutations in cfDNA from clinical plasma samples, demonstrating high sensitivity (90.48%) and excellent specificity (100%). We also apply it to successfully classify and identify KRAS missense variants. RECO-Cas provides a simple, rapid, and affordable solution that is compatible with a compact, wirelessly powered point-of-care diagnostic platform incorporating smartphone-based fluorescence detection. This assay enables highly sensitive and specific detection of low-frequency mutations, facilitating early cancer diagnosis and supporting the development of personalized treatment strategies.},
}

Humans
Liquid Biopsy/methods
*Polymorphism, Single Nucleotide
*CRISPR-Cas Systems/genetics
Proto-Oncogene Proteins p21(ras)/genetics
*Cell-Free Nucleic Acids/genetics/blood
ErbB Receptors/genetics
Class I Phosphatidylinositol 3-Kinases/genetics
*Neoplasms/genetics/diagnosis
Mutation

@article {pmid42173102,
year = {2026},
author = {Peng, S and Xie, W and Zhu, J and Bao, Z},
title = {Multiplex genome engineering: Methodologies and applications.},
journal = {Cell systems},
volume = {},
number = {},
pages = {101614},
doi = {10.1016/j.cels.2026.101614},
pmid = {42173102},
issn = {2405-4720},
abstract = {To facilitate the study of applied genetics and enable a rapid translation of genetic insights, it is highly desirable to concurrently modify many genomic loci in an organism of interest. While single-locus editing is well-established and technically straightforward, multiplex genome engineering (MGE) poses significant technological barriers. With the convergence of low-cost DNA synthesis, advanced genome editing techniques, and laboratory automation, a plethora of MGE methodologies were recently developed and applied in fields ranging from basic research to applied sectors. This review analyzes one-step and iterative MGE methodologies, with an emphasis on recombineering and CRISPR-Cas systems, and showcases emerging paradigm-shifting applications in biomanufacturing, agriculture, and therapeutics. We conclude by analyzing the limitations of existing technologies and discussing future directions for further optimizing MGE to solve system-level problems.},
}

@article {pmid42173386,
year = {2026},
author = {Mishra, S and Rehan, S and Barekzai, AM and Sharma, A and Raghav, A},
title = {Emerging frontiers in genome editing: From CRISPR to next-generation technologies.},
journal = {Methods (San Diego, Calif.)},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymeth.2026.05.011},
pmid = {42173386},
issn = {1095-9130},
abstract = {Genome editing has revolutionized molecular biology. It offers precise modification of genetic material across diverse organisms. This review outlines the evolution of genome editing technologies from homologous recombination to advanced Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (CRISPR-9) based systems that now dominate the field. Early methods, such as Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs), established the foundation for site-specific DNA cleavage. However, they were limited by complexity and cost. The advent of the CRISPR-Cas systems, particularly CRISPR-Cas9, transformed the landscape due to their simplicity, high efficiency, and adaptability. Variants such as CRISPR-Cas12a, base editors, and prime editors enhanced editing precision. They enable single-nucleotide modifications and targeted insertions without double-strand breaks. Emerging tools such as CRISPR-associated transposases, recombinase fusions, and RNA-targeting Cas13 enzymes expand the scope of manipulation beyond DNA to RNA. At the same time, epigenome editing, and gene drives present new therapeutic and ecological applications. Efficient delivery systems, both viral (Adeno-Associated Virus (AAV), lentivirus, adenovirus) and non-viral (lipid nanoparticles, gold nanoparticles, DNA nano clews), remain critical for clinical translation. Future directions emphasize artificial intelligence-guided design, retroelement-based integration, and novel biomimetic delivery vehicles to overcome current efficiency and safety barriers. These innovations can help overcome current efficiency and safety barriers. Together, they are propelling genome editing toward precise, programmable, and ethically responsible therapeutic applications. Despite unresolved challenges involving off-target effects, immunogenicity, and germline ethics, genome editing redefines biomedical research, drug development, and disease correction. CRISPR-derived technologies now stand at the forefront of next-generation genetic medicine.},
}

@article {pmid41652852,
year = {2026},
author = {Li, Y and Xu, B and Gao, X and Wang, Y and Liu, X and Xu, R and Li, J and Wei, P and Qin, R},
title = {Tuning Rice Gene Expression via In Situ Promoter Truncations Using a Prime Editing Library.},
journal = {Plant biotechnology journal},
volume = {24},
number = {6},
pages = {3528-3530},
doi = {10.1111/pbi.70587},
pmid = {41652852},
issn = {1467-7652},
support = {2023ZD04074//Biological Breeding - Major Projects/ ; 32400335//National Natural Science Foundation of China/ ; 32572441//National Natural Science Foundation of China/ ; 32570484//National Natural Science Foundation of China/ ; 2024CSJZN01100//Yangtze River Delta Science and Technology Innovation Community Joint Research (Basic Research) Project/ ; 202423110050063//Science and Technology Major Project of Anhui Province/ ; 202423m10050002//Science and Technology Major Project of Anhui Province/ ; 2023n06020020//Science and Technology Major Project of Anhui Province/ ; },
mesh = {*Oryza/genetics ; *Promoter Regions, Genetic/genetics ; *Gene Editing/methods ; *Gene Expression Regulation, Plant/genetics ; CRISPR-Cas Systems ; Plants, Genetically Modified ; Gene Library ; },
abstract = {Promoter engineering holds immense potential for fine-tuning gene expression and optimising agronomic traits, yet conventional genome-editing tools face limitations in precision, scalability, and risk mitigation. Here, we develop Prime Editing-mediated Promoter Engineering (PEPE), a DSB-free platform integrating bidirectional Protospacer adjacent motif (PAM) recognition (NGG/CCN) with combinatorial duo-pegRNA strategies to achieve tiled, overlapping deletions across entire plant promoters. Applying PEPE to the 1.8-kb rice D53 promoter, we generated a mutant library with stepwise deletions. Edited alleles showed stable inheritance, and dual-method validation confirmed the precision at junctions. Quantitative profiling revealed functional modularity: core-region deletions suppressed D53 expression by 70%-85%, while a distal deletion (D53-G9C10) paradoxically upregulated transcription 2.2-fold, uncovering a cryptic repressor element. Phenotypic variation corresponded with transcriptional changes, establishing a direct link between cis-regulatory diversity and agronomic traits. By circumventing DSBs and enabling kilobase-scale CRE manipulation, PEPE establishes a robust framework for decoding promoter logic and accelerating trait pyramiding in crops. This study advances plant genome editing by merging precision with scalability, offering transformative potential for breeding climate-resilient, high-yield cultivars.},
}

*Oryza/genetics
*Promoter Regions, Genetic/genetics
*Gene Editing/methods
*Gene Expression Regulation, Plant/genetics
CRISPR-Cas Systems
Plants, Genetically Modified
Gene Library

@article {pmid41734974,
year = {2026},
author = {Ding, Y and Chen, X and Wu, K and Hou, H and Wang, Y and Yan, B and Khursheed, MHUR and Shang, C and Zhang, X and Pan, Y and Wu, L and Li, J},
title = {A Truncated WRKY Protein Enhances Drought Resistance in Wild Tomatoes Through the SlWRKY16-CIP2b-SlSYP121 Module.},
journal = {Plant biotechnology journal},
volume = {24},
number = {6},
pages = {3840-3860},
doi = {10.1111/pbi.70596},
pmid = {41734974},
issn = {1467-7652},
support = {Nos.CSTB2023TIAD-KPX0026//Special Key Project of Technological Innovation and Application Development of Chongqing/ ; CSTB2023TIAD-KPX0025//Special Key Project of Technological Innovation and Application Development of Chongqing/ ; 31872123//National Natural Science Foundation of China/ ; 32172597//National Natural Science Foundation of China/ ; SWU-KQ22041//Fundamental Research Funds for the Central Universities/ ; CARS-23-B08//China Agriculture Research System/ ; },
mesh = {*Solanum lycopersicum/genetics/physiology/metabolism ; *Droughts ; *Plant Proteins/genetics/metabolism ; *Transcription Factors/genetics/metabolism ; Gene Expression Regulation, Plant ; Polymorphism, Single Nucleotide/genetics ; Stress, Physiological/genetics ; CRISPR-Cas Systems ; Drought Resistance ; },
abstract = {Drought stress is a major abiotic factor that severely affects plant growth and food production. Identifying drought-resistant genes and their regulatory mechanisms is essential for mitigating the negative impacts of drought on plants. In this study, we identified a natural single nucleotide polymorphism (SNP) mutation in SlWRKY16 that is closely linked to drought tolerance in tomato. This SNP leads to the expression of a truncated SlWRKY16 protein. The CRISPR/Cas9 knockout of SlWRKY16, which produces this truncated SlWRKY16 protein, exhibits enhanced drought tolerance, whereas the overexpression lines demonstrate the opposite effect. Yeast two-hybrid screening demonstrated that SlWRKY16 physically interacted with CONSTANS Interacting Protein 2b (CIP2b). The CIP2b knockout mutants displayed increased sensitivity to drought stress. Importantly, this drought-sensitive phenotype was rescued in double mutants (cip2b/slwrky16). RNA-seq analysis revealed that a syntaxin gene (SlSYP121) co-expressed with both SlWRKY16 and CIP2b. Electrophoretic mobility shift assays confirmed that SlWRKY16 directly binds to the promoter of SlSYP121 and represses its expression, while the truncated SlWRKY16 protein failed to bind. Moreover, SlSYP121 acts as a positive regulator of drought tolerance. Our findings further demonstrate that the interaction between CIP2b and SlWRKY16 reduces the binding affinity of SlWRKY16 to the SlSYP121 promoter. This study identified a key SNP associated with differences in drought tolerance between wild and cultivated tomato, elucidated the regulatory function of the SlWRKY16-CIP2b-SlSYP121 module in the tomato drought response, and enhanced our understanding of the molecular mechanisms underlying plant drought resistance.},
}

*Solanum lycopersicum/genetics/physiology/metabolism
*Droughts
*Plant Proteins/genetics/metabolism
*Transcription Factors/genetics/metabolism
Gene Expression Regulation, Plant
Polymorphism, Single Nucleotide/genetics
Stress, Physiological/genetics
CRISPR-Cas Systems
Drought Resistance

@article {pmid41818620,
year = {2026},
author = {Huang, ZJ and Li, FM and Tu, YF and Feng, KK and Li, CL and Tian, SC and Hu, YS and Shao, JW and Liu, ZH},
title = {CRISPR/Cas9-Based Vanadium MXene-Free Radical Spatiotemporally Controlled Nanoreactor for Photothermal-Induced Multi-Effect Synergistic Antitumor Therapy.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {29},
pages = {e22535},
doi = {10.1002/advs.202522535},
pmid = {41818620},
issn = {2198-3844},
mesh = {*Photothermal Therapy/methods ; Animals ; Mice ; *CRISPR-Cas Systems/genetics ; Humans ; Free Radicals ; Cell Line, Tumor ; *Neoplasms/therapy ; Tumor Microenvironment ; Apoptosis ; Nitrites ; Transition Elements ; },
abstract = {Photothermal therapy (PTT), a non-invasive tumor treatment, shows promise but is limited in solid tumors by restricted tissue penetration, thermotolerance, anti-apoptotic and immunosuppressive effects. In this study, tumor microenvironment-responsive nanoplatform VARH was constructed based on MXene. Under NIR-II laser irradiation, VARH achieves a high photothermal conversion efficiency of 44.21%. Loaded 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride decomposes at high temperatures to generate alkyl radicals, synergizing with hydroxyl radicals from V[4+]-catalyzed endogenous H2O2 decomposition, enabling chemodynamic therapy (CDT) and thermal dynamic therapy to enhance tumor cell oxidative damage. Triggered by high glutathione, VARH releases ribonucleoprotein (RNP) complexes to knockout heat shock protein 90 (HSP90), attenuating cellular heat resistance and promoting apoptosis. It also enhances T cell-mediated anti-tumor immunity and, with free radicals, promotes tumor cell immunogenic cell death (ICD), achieving immunotherapeutic multi-effect synergy. Integrating nanotechnology with precise gene editing, this study develops a novel multimodal synergistic therapy system, providing new insights for multi-modal treatment R&D and advancing PTT and free radical-based cancer therapies.},
}

*Photothermal Therapy/methods
Animals
Mice
*CRISPR-Cas Systems/genetics
Humans
Free Radicals
Cell Line, Tumor
*Neoplasms/therapy
Tumor Microenvironment
Apoptosis
Nitrites
Transition Elements

@article {pmid41834768,
year = {2026},
author = {Hou, Y and Wong, DCJ and Wang, L and Kang, Y and Zhou, H and Kafle, S and Liu, Y and Xu, M and Meng, L and Liang, Z and Yu, G and Wang, Q and Xin, H},
title = {A Hierarchical VvbHLH30-VvERF70-VvACS2 Module Orchestrates Ethylene Biosynthesis and Cold Adaptation in Grapevine.},
journal = {Plant biotechnology journal},
volume = {24},
number = {6},
pages = {4317-4341},
doi = {10.1111/pbi.70640},
pmid = {41834768},
issn = {1467-7652},
support = {32272691//National Natural Science Foundation of China/ ; 202403AP140029//Yunnan Provincial Key Research and Development Program/ ; },
mesh = {*Vitis/genetics/metabolism/physiology ; *Ethylenes/biosynthesis ; Gene Expression Regulation, Plant ; *Plant Proteins/metabolism/genetics ; Plant Roots/metabolism/genetics ; Cold Temperature ; Transcription Factors/metabolism/genetics ; Plant Growth Regulators/biosynthesis/metabolism ; Cold-Shock Response/genetics ; CRISPR-Cas Systems ; },
abstract = {Ethylene is a key gaseous phytohormone that plays crucial roles in regulating plant growth, development and stress responses. However, ethylene-associated biosynthetic and transcriptional regulatory mechanisms governing cold-adaptation responses in plants remain poorly understood. In this work, genome-wide analysis from grapevines (Vitis vinifera) identified nine ACS family members, of which VvACS2, VvACS4 and VvACS6 exhibited the most dynamic transcriptional responses to cold stress and were chosen for functional validation. CRISPR-Cas9-mediated knockout and overexpression experiments revealed that VvACS2 is the major contributor to ethylene biosynthesis during cold stress in grapevine roots. Screening time-course cold treatment data from Vitis vinifera and Vitis amurensis roots identified VvERF70 and VvbHLH30 as the only two TFs, among six candidates, that directly regulate VvACS2 expression. Overexpression and CRISPR-Cas9-mediated knockout of VvERF70 or VvbHLH30 in roots further confirmed their contribution to enhanced ethylene production and cold tolerance under low-temperature treatment. Furthermore, the induction of VvACS2 was greatly enhanced when VvERF70 dimerized with VvbHLH30. Notably, VvbHLH30 further positively regulates ethylene biosynthesis under cold stress by interacting with VvERF70 and binding to its promoter. Taken together, we define a hierarchical transcriptional regulatory network where the VvbHLH30-VvERF70-VvACS2 module is pivotal for ethylene biosynthesis and underpins grapevine cold tolerance. This work provides new mechanistic insights into cold adaptation mechanisms and offers novel strategies to mitigate frost damage in agricultural crops.},
}

*Vitis/genetics/metabolism/physiology
*Ethylenes/biosynthesis
Gene Expression Regulation, Plant
*Plant Proteins/metabolism/genetics
Plant Roots/metabolism/genetics
Cold Temperature
Transcription Factors/metabolism/genetics
Plant Growth Regulators/biosynthesis/metabolism
Cold-Shock Response/genetics
CRISPR-Cas Systems

@article {pmid41928521,
year = {2026},
author = {Diao, K and Duff, SMG and Li, H},
title = {An Idea to Explore: Enhancing the Teaching of Genome Editing Through 3D Printed Models of CRISPR-Cas9 Technology.},
journal = {Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology},
volume = {54},
number = {3},
pages = {292-296},
doi = {10.1002/bmb.70047},
pmid = {41928521},
issn = {1539-3429},
mesh = {*Gene Editing/methods ; *Printing, Three-Dimensional ; *CRISPR-Cas Systems/genetics ; Humans ; *Teaching ; *Molecular Biology/education ; },
abstract = {Innovative biological discoveries are crucial for addressing global challenges, yet teaching these complex concepts poses significant difficulties due to the complexity of the subject matter as well as limited educational resources and methodologies. Genome editing, specifically with CRISPR-Cas9, represents a convergence of technology, molecular biology, and engineering, enabling precise manipulation of DNA sequences in various organisms. It leverages advanced tools and a deep understanding of molecular biology to target specific genes while applying engineering principles to improve editing mechanisms. However, the complexity of this field poses educational challenges due to a scarcity of accessible resources. To enhance the accessibility of genome editing to scientists and students, we propose utilizing 3D modeling and printing to create tangible models of key components in the genome editing process. By visualizing these molecular structures, our goal is to simplify and enrich the educational experience, making the intricate principles of genome editing more comprehensible and engaging for students.},
}

*Gene Editing/methods
*Printing, Three-Dimensional
*CRISPR-Cas Systems/genetics
Humans
*Teaching
*Molecular Biology/education

@article {pmid41942889,
year = {2026},
author = {Qiu, X and Yuan, L and Liu, X and Li, Y and Ma, X and Du, B and Yuan, M and Li, Z},
title = {A one-pot CRISPR/Cas12b assay for extraction-free and visual detection of Haemophilus influenzae.},
journal = {BMC microbiology},
volume = {26},
number = {1},
pages = {},
pmid = {41942889},
issn = {1471-2180},
support = {2024A102//Chinese Center for Disease Control and Prevention/ ; 2021YFC2301105//National Key Research and Development Program of China/ ; },
mesh = {*Haemophilus influenzae/genetics/isolation & purification ; Humans ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Haemophilus Infections/diagnosis/microbiology ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; Sputum/microbiology ; DNA, Bacterial/genetics ; },
abstract = {Haemophilus influenzae is a major respiratory pathogen, particularly in pediatric populations, and its rapid and accurate detection is critical for early diagnosis and targeted treatment. Traditional diagnostic methods, such as bacterial culture and PCR, are often time-consuming and require specialized equipment. In this study, we developed the Hi-ExCad assay, a one-pot, extraction-free CRISPR/Cas12b-based system for the rapid, simple, and accurate detection of H. influenzae. The assay integrates loop-mediated isothermal amplification and CRISPR/Cas12b detection in a single reaction, enabling direct detection from clinical sputum samples without the need for nucleic acid extraction. The Hi-ExCad assay demonstrated high specificity, correctly identifying H. influenzae in clinical samples, with a limit of detection of 1 pg of genomic DNA. The assay also exhibited excellent performance in real-time detection and visual result interpretation under blue light, making it highly suitable for point-of-care testing and resource-limited settings. These findings suggest that the Hi-ExCad assay provides a rapid, reliable, and user-friendly method for the detection of H. influenzae, with significant potential for clinical application.},
}

*Haemophilus influenzae/genetics/isolation & purification
Humans
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
*Haemophilus Infections/diagnosis/microbiology
*Molecular Diagnostic Techniques/methods
Sensitivity and Specificity
Sputum/microbiology
DNA, Bacterial/genetics