@article {pmid42582274,
year = {2026},
author = {Liu, W and Gu, J and Xie, C and Du, B and Liu, M and Zhang, J},
title = {Rewriting CAR-T cell fate: CRISPR/Cas gene editing for solid tumor therapy.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1910092},
pmid = {42582274},
issn = {1664-3224},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Immunotherapy, Adoptive/methods ; *Neoplasms/therapy/immunology/genetics ; *Receptors, Chimeric Antigen/immunology/genetics ; Animals ; Tumor Microenvironment/immunology ; *T-Lymphocytes/immunology/metabolism ; T-Cell Exhaustion ; },
abstract = {Although chimeric antigen receptor T (CAR-T) cell therapy has achieved remarkable success in hematological malignancies, its therapeutic efficacy in solid tumors remains limited by several challenges, including insufficient tumor infiltration, T cell exhaustion and the immunosuppressive tumor microenvironment (TME). CRISPR/Cas, a third-generation gene editing technology developed in recent years, is characterized by its simplicity and high efficiency. This technology has demonstrated broad application potential across multiple fields and has emerged as a powerful tool for improving CAR-T cell therapy. In this review, we summarize recent advances in the application of CRISPR/Cas gene editing technology to enhance the antitumor activity of CAR-T cells against solid tumors. We also discuss the key challenges currently faced and systematically propose potential strategies for overcoming the limitations.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems
*Immunotherapy, Adoptive/methods
*Neoplasms/therapy/immunology/genetics
*Receptors, Chimeric Antigen/immunology/genetics
Animals
Tumor Microenvironment/immunology
*T-Lymphocytes/immunology/metabolism
T-Cell Exhaustion

@article {pmid42582990,
year = {2026},
author = {Ma, AJ and Brown, BH and Kim, S and Hilton, IB},
title = {Clinical translation of epigenome editing technologies.},
journal = {Current opinion in biomedical engineering},
volume = {38},
number = {},
pages = {},
pmid = {42582990},
issn = {2468-4511},
support = {R01 EB036003/EB/NIBIB NIH HHS/United States ; R35 GM143532/GM/NIGMS NIH HHS/United States ; },
abstract = {CRISPR/Cas-based epigenome editing technologies hold great promise for identifying novel therapeutic targets, improving gene and cell therapies, and directly addressing the underlying issues in many diseases, all while minimizing risks of genotoxicity often associated with conventional genome editing. Exciting recent advances in CRISPR/Cas-based epigenome editing technologies have drastically enhanced the ability to precisely control the timing, levels, and durations of endogenous gene expression and reprogram epigenetic states in human cells. As a result, epigenome editing is now poised to unlock new biomedical discoveries and treatments for diseases driven by transcriptional and epigenetic dysregulation as well as those stemming from aberrantly repetitive genomic regions or complex genomic arrangements that are difficult to target using conventional genome editing. Additionally, the power of epigenome editors is generating new strategies to control cell fate and function, which has direct and important implications for cell therapies and regenerative medicines. Here, as the first wave of CRISPR/Cas-based epigenome editors move into clinical trials, we cover recent advances as the field looks to address pressing hurdles facing widespread clinical deployment of epigenome editing technologies including delivery, performance, and safety. For instance, the discovery of compact Cas chassis, engineering efforts to reduce effector sizes for efficient delivery, and campaigns to tailor the targeting discrimination of epigenome editors are rapidly progressing, as is research into the development of new effector domains with high specificity, robust performance, and a lack of immunogenicity and cytotoxicity. This exciting progress is quickly moving the community closer to fulfilling the promise of CRISPR/Cas-based epigenome editing as a powerful class of platform technologies for biological discoveries, biotechnological innovations, and medicines.},
}

@article {pmid42584708,
year = {2026},
author = {Skoczek, D and Hohendorff, J and Malecki, MT and Roig-Merino, A and Bak, RO and Kachamakova-Trojanowska, N},
title = {CRISPR/Cas9-based repair of a heterozygous HNF1A mutation in patient-derived hiPSCs.},
journal = {Human genetics},
volume = {145},
number = {1},
pages = {},
pmid = {42584708},
issn = {1432-1203},
support = {2020/38/E/NZ3/00516//Narodowe Centrum Nauki/ ; 2020/38/E/NZ3/00516//Narodowe Centrum Nauki/ ; },
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; *Hepatocyte Nuclear Factor 1-alpha/genetics ; *CRISPR-Cas Systems/genetics ; Heterozygote ; Frameshift Mutation ; Mutation ; },
abstract = {Human induced pluripotent stem cells (hiPSCs) represent a powerful platform for disease modeling, especially in monogenic diseases as they preserve the donor's genetic background while enabling directed differentiation into disease-relevant cell types. This makes them highly suitable for studying disease mechanisms in a patient-specific and physiologically relevant context. Although CRISPR/Cas9 is widely applied for genome editing, precise correction of pathogenic variants in hiPSCs remains challenging due to the lack of standardized CRISPR component selection and experimental design. Here, we describe an optimized CRISPR-based strategy for correcting a heterozygous HNF1A frameshift mutation (c.235_236insG; p.Glu79Glyfs*16) in HNF1A-MODY patient-derived hiPSCs. Using electroporation, we efficiently delivered CRISPR components, including a ribonucleoprotein complex of Cas9 and single-guide RNA, along with a single-stranded oligodeoxynucleotide repair template. Corrected hiPSC lines were validated for pluripotency, absence of exogenous reprogramming factors, and off-target effects. Additionally, we discuss key technical challenges encountered during the editing process and provide practical recommendations that may improve the generation of mutation-corrected hiPSC lines. These guidelines could serve as a useful reference for researchers employing CRISPR-based strategies for generation of reliable disease modelling tools.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism
*Hepatocyte Nuclear Factor 1-alpha/genetics
*CRISPR-Cas Systems/genetics
Heterozygote
Frameshift Mutation
Mutation

@article {pmid41820594,
year = {2026},
author = {Lee, SJ and Nam, BG and Hong, SA and Jo, DH and Lee, SM and Bae, S and Kim, JH},
title = {Topical application of Cas9 ribonucleoproteins inhibits corneal neovascularization in a mouse model of alkali burn injury.},
journal = {Gene therapy},
volume = {33},
number = {4},
pages = {435-447},
pmid = {41820594},
issn = {1476-5462},
support = {2022M3A9E4017127//National Research Foundation of Korea (NRF)/ ; RS-2023-00260351//National Research Foundation of Korea (NRF)/ ; RS-2024-00467177//National Research Foundation of Korea (NRF)/ ; 18-2023-0010//Seoul National University Hospital (SNUH)/ ; GTL24021-000//National Research Council of Science and Technology (National Research Council of Science & Technology)/ ; },
mesh = {Animals ; *Corneal Neovascularization/therapy/genetics ; Mice ; *Burns, Chemical/therapy ; *Genetic Therapy/methods ; Disease Models, Animal ; *Eye Burns/therapy/chemically induced ; Vascular Endothelial Growth Factor A/genetics/metabolism ; *CRISPR-Cas Systems ; *Ribonucleoproteins/genetics/administration & dosage ; Gene Therapy Agents ; Gene Editing ; Mice, Inbred C57BL ; Administration, Topical ; *CRISPR-Associated Protein 9/genetics ; },
abstract = {Corneal neovascularization is a sight-threatening condition for which current treatments such as anti-VEGF agents are limited by invasiveness and side effects. We present the first non-viral, CRISPR/Cas9-based gene therapy delivered via topical eye drops that penetrates the cornea and inhibits pathological neovascularization. Cas9 ribonucleoproteins (RNPs) targeting the Vegfa gene were complexed with a liposomal carrier (lipofectamine) and administered to mice after alkali burn injury to the cornea. This approach achieved approximately 2% gene editing at the Vegfa locus in vivo, which significantly reduced local VEGF-A expression. Consequently, treated corneas showed markedly decreased macrophage infiltration and robust suppression of both hemangiogenesis and lymphangiogenesis compared to untreated controls. These findings demonstrate that even modest in vivo gene editing can yield a strong therapeutic effect, highlighting a clinically relevant strategy for controlling corneal angiogenesis. Our study introduces a feasible and safe topical CRISPR therapy for corneal diseases, offering a potential alternative to invasive or virus-based gene delivery methods.},
}

Animals
*Corneal Neovascularization/therapy/genetics
Mice
*Burns, Chemical/therapy
*Genetic Therapy/methods
Disease Models, Animal
*Eye Burns/therapy/chemically induced
Vascular Endothelial Growth Factor A/genetics/metabolism
*CRISPR-Cas Systems
*Ribonucleoproteins/genetics/administration & dosage
Gene Therapy Agents
Gene Editing
Mice, Inbred C57BL
Administration, Topical
*CRISPR-Associated Protein 9/genetics

@article {pmid42174157,
year = {2026},
author = {Li, JX and Zhang, SM and Ma, XY and Deng, DH and Bai, PD and Zhao, JJ and Gong, A and Pang, SC and Dong, F and Wang, SD and Zhang, JP and Zhang, XB},
title = {AviTag-seq unifies nucleotide-resolution maps of CRISPR off-targets and AAV vector integrations.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {42174157},
issn = {2399-3642},
support = {82402188//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; *Genetic Vectors/genetics ; *Dependovirus/genetics ; Mice ; *Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; Liver/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Virus Integration ; },
abstract = {Comprehensive safety assessment of gene-editing therapies requires quantifying both off-target cleavage and vector integration. However, current double-strand break (DSB)-dependent assays are fundamentally limited when evaluating nickase-based editors and are hindered by tag polarity constraints. Here, we present AviTag-seq, a platform repurposing AAV Inverted Terminal Repeats (ITRs) as universal capture tags. By exploiting the ITRs' single-stranded hairpin structure, AviTag-seq overcomes polarity issues, enabling high-sensitivity detection with a single primer pair, particularly in iPSCs. Crucially, it captures off-target events from prime and base editors that evade conventional detection. In vivo, AviTag-seq outperformed DISCOVER-Seq+ in profiling Pcsk9 off-targets in mouse liver while simultaneously mapping AAV integration sites. This dual profiling revealed that, unlike in vitro, AAV vectors in vivo preferentially integrate into active gene promoters, highlighting a specific genotoxic risk for liver-directed therapies. AviTag-seq thus offers a unified, regulatory-grade solution for evaluating diverse genome-editing modalities.},
}

Animals
*Genetic Vectors/genetics
*Dependovirus/genetics
Mice
*Gene Editing/methods
Humans
*CRISPR-Cas Systems
Liver/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats
*Virus Integration

@article {pmid42243947,
year = {2026},
author = {Wei, Z and Xu, X and Qiao, Q and Guo, J and Wu, T and Rong, H and Ning, S and Zhu, X and Zhao, K and Ke, J and He, L and Chi, Y and Ge, Y and Cui, L and Min, X},
title = {A temporally controlled isothermal amplification-CRISPR/Cas12a platform for the rapid detection of monkeypox virus.},
journal = {Virology journal},
volume = {23},
number = {1},
pages = {},
pmid = {42243947},
issn = {1743-422X},
support = {JSJK2026D00033//Science and Technology Program Project of Jiangsu Provincial Bureau of Disease Prevention and Control/ ; MQ2025039//Scientific Research Project of Jiangsu Commission of Health/ ; M2024024//Scientific Research Project of Jiangsu Commission of Health/ ; BK20231374//Natural Science Foundation of Jiangsu Province/ ; 2023YFC2605100, 2023YFC2605104//National Key Research and Development Program of China/ ; },
mesh = {*Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *Monkeypox virus/isolation & purification/genetics ; *CRISPR-Cas Systems ; *Mpox, Monkeypox/diagnosis/virology ; Rapid Diagnostic Tests ; *Molecular Diagnostic Techniques/methods ; Humans ; Animals ; },
abstract = {BACKGROUND: Given the continuous threat posed by emerging and re-emerging infectious diseases worldwide, a rapid, sensitive, and practical molecular detection technology is urgently required for timely point-of-care (POC) diagnosis. Although the quantitative real-time PCR (qPCR) based technologies exhibit high sensitivity and specificity compared with traditional pathogen detection methods, they are not applicable for POC detection scenarios.

METHODS: Based on the photocontrolled principle, this study established a universal photoactivation strategy for LbCas12a by modifying four sites in the repeat region of LbCas12a crRNA with the photocleavable protecting group 6-nitropiperonyloxymethyl (NPOM). This strategy was integrated with multienzyme isothermal rapid amplification (MIRA) to develop a photocontrolled one-pot rapid detection method for monkeypox virus (MPXV), termed the temporally controlled MIRA-CRISPR/Cas12a (TC-MIRA-CRISPR/Cas12a) assay.

RESULTS: The TC-MIRA-CRISPR/Cas12a assay exhibited a 100-fold improvement in detection sensitivity over the conventional one-step assay, achieving a limit of detection (LOD) of 3.9 copies per reaction, which was comparable to stepwise detection assay. The entire assay can be completed within 40 min, faster than the widely used qPCR. In clinical sample detection, this method showed good consistency with qPCR, with a Kappa coefficient of 0.980 (P < 0.001), a sensitivity of 98.4%, and a specificity of 100%.

CONCLUSIONS: This method effectively avoids amplicon contamination while maintaining high sensitivity, and exhibits excellent universality and expandability. It enables detection of other pathogens simply by modifying the spacer sequence of crRNA, providing a novel technical approach and research perspective for POC detection of MPXV and other pathogens.},
}

*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
*Monkeypox virus/isolation & purification/genetics
*CRISPR-Cas Systems
*Mpox, Monkeypox/diagnosis/virology
Rapid Diagnostic Tests
*Molecular Diagnostic Techniques/methods
Humans
Animals

@article {pmid42265600,
year = {2026},
author = {Jiang, S and Chen, F and Ma, H and Wu, S and Tang, X and Pan, X and Li, Q and Tao, A and Xu, J and Qi, J and Fang, P and Chen, J and Zhang, L},
title = {Cloning and functional verification of endogenous U6 promoters for developing an efficient CRISPR/Cas9-mediated genome editing system in kenaf (Hibiscus cannabinus L.).},
journal = {BMC plant biology},
volume = {26},
number = {1},
pages = {},
pmid = {42265600},
issn = {1471-2229},
support = {32472219//the National Natural Science Foundation of China/ ; 2023J01443//Fujian Provincial Natural Science Foundation of China/ ; CARS-16//China Agricultural Research System of MOF and MARA/ ; KFB23001//Science and Technology Innovation Project of Fujian Agriculture and Forestry University/ ; ASTIP-IBFC-01//Agriculture Science and Technology Innovation Program/ ; },
mesh = {*Promoter Regions, Genetic/genetics ; *CRISPR-Cas Systems ; *Hibiscus/genetics ; *Gene Editing/methods ; Cloning, Molecular ; *RNA, Small Nuclear/genetics ; Plants, Genetically Modified ; Genome, Plant ; },
abstract = {BACKGROUND: The U6 promoter is a critical component of the CRISPR/Cas9 system, as it drives the transcription of single-guide RNAs (sgRNAs) to enable precise genome editing. Endogenous promoters typically exhibit higher transcriptional activity than their exogenous counterparts, which can significantly enhance editing efficiency. However, the endogenous U6 promoter in kenaf (Hibiscus cannabinus L.), an important fiber crop, has not yet been characterized.

METHODS: Using the Arabidopsis U6-26 (AtU6-26) promoter as a reference, we performed a homologous sequence search and identified two candidate U6 promoters in kenaf, designated HcU6-1 and HcU6-14. Promoter fragments were amplified from the kenaf cultivar 'Fuhong 952' and cloned into a β-glucuronidase (GUS) reporter vector. Histochemical GUS staining assays revealed that both HcU6 promoters were transcriptionally active, with HcU6-14 showing significantly stronger expression levels compared to HcU6-1.

RESULTS: To further evaluate the utility of these promoters for genome editing, we constructed CRISPR/Cas9 vectors targeting the kenaf acetolactate synthase (ALS) gene, driven by either HcU6-14P or the exogenous cotton GbU6-9P promoter. Agrobacterium rhizogenes K599-mediated transformation was used to induce hairy roots, and mutation analysis of the ALS gene was performed via Sanger sequencing. Notably, targeted mutations in the ALS gene were detected in hairy roots transformed with the HcU6-14P-driven CRISPR/Cas9 vector, whereas no mutations were observed in roots transformed with the exogenous GbU6-9P promoter. These results demonstrate that the endogenous HcU6-14 promoter confers superior genome editing efficiency compared to the heterologous promoter, which facilitates the development of improved varieties with enhanced agronomic traits.},
}

*Promoter Regions, Genetic/genetics
*CRISPR-Cas Systems
*Hibiscus/genetics
*Gene Editing/methods
Cloning, Molecular
*RNA, Small Nuclear/genetics
Plants, Genetically Modified
Genome, Plant

@article {pmid42409798,
year = {2026},
author = {Zhao, C and Shih, M and Ahmed, S and Song, S and Lennon, A and Mayes, JM and Fan, MJ and Lo, PY and Perera, J and Tutar, A and Kang, A and Warren, E and McClure, R and Khan, AA and Kelley, SO and Abdrabou, AM},
title = {AI-guided CRISPR screening reveals therapeutic targets in psoriasis.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42409798},
issn = {2041-1723},
mesh = {Humans ; Animals ; *Psoriasis/genetics/drug therapy/chemically induced ; Keratinocytes/metabolism/drug effects ; Mice ; *Receptors, Interleukin-17/metabolism/genetics ; Arachidonate 5-Lipoxygenase/genetics/metabolism ; Imiquimod ; Receptors, Oxytocin/antagonists & inhibitors/genetics/metabolism ; CRISPR-Cas Systems ; Hydroxyurea/pharmacology/analogs & derivatives ; Clustered Regularly Interspaced Short Palindromic Repeats ; Interleukin-17 ; },
abstract = {Psoriasis affects over 125 million people globally. Biologics targeting the IL-17/IL-17RA axis are effective but require systemic administration, are costly, and are unsuitable for some patients. Developing topical small-molecule alternatives requires a better understanding of how IL-17 receptor A (IL17RA) is regulated in keratinocytes, the principal effector cells of psoriatic lesions. Here, we report a genome-wide CRISPR knockout screen for regulators of surface IL17RA in primary human epidermal keratinocytes. We prioritize hits using experimental enrichment together with VirtualCRISPR, a language-model framework trained on functional-genomics data, and validate two regulators with minimal prior connection to IL17RA: 5-lipoxygenase (ALOX5) and the oxytocin receptor (OXTR), which act through distinct cell-intrinsic mechanisms. Topical zileuton, an ALOX5 inhibitor, and cligosiban, an OXTR antagonist, suppress imiquimod-induced psoriasiform dermatitis in mice, mirroring systemic anti-IL17RA antibody efficacy. By linking AI- guided selection to genetic perturbation screening, this study provides an efficient route from candidate gene nomination to biological validation and therapeutic discovery.},
}

Humans
Animals
*Psoriasis/genetics/drug therapy/chemically induced
Keratinocytes/metabolism/drug effects
Mice
*Receptors, Interleukin-17/metabolism/genetics
Arachidonate 5-Lipoxygenase/genetics/metabolism
Imiquimod
Receptors, Oxytocin/antagonists & inhibitors/genetics/metabolism
CRISPR-Cas Systems
Hydroxyurea/pharmacology/analogs & derivatives
Clustered Regularly Interspaced Short Palindromic Repeats
Interleukin-17

@article {pmid42414323,
year = {2026},
author = {Wu, X and Li, Y and Cao, Y and Zhao, Z and Lu, H and Liang, S and Lui, GCY and Chan, DPC and Hsing, IM},
title = {Thermodynamically programmed one-pot CRISPR platform for point-of-care SNP genotyping.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42414323},
issn = {2041-1723},
support = {16303522//Research Grants Council, University Grants Committee (RGC, UGC)/ ; 16304225//Research Grants Council, University Grants Committee (RGC, UGC)/ ; },
mesh = {*Polymorphism, Single Nucleotide/genetics ; Thermodynamics ; *Point-of-Care Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Genotyping Techniques/methods ; DNA Primers/genetics ; Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Genotype ; },
abstract = {One-pot CRISPR diagnostics face a fundamental incompatibility: isothermal nucleic acid amplification enables rapid target accumulation, whereas CRISPR activation irreversibly consumes those substrates, destabilizing reaction kinetics. Here we show that reaction order can be programmed into DNA primers through thermodynamic design. Differences in primer-binding strength create two sequential amplification stages, delaying CRISPR activation until enough amplicons have accumulated without physical separation or external control. The design also introduces the protospacer adjacent motif (PAM), a short sequence required for CRISPR recognition, through the primer rather than relying on its presence in the native target, expanding target accessibility while retaining single-nucleotide discrimination. An ordinary differential equation model captures the threshold behavior and establishes a predictable framework for primer design. Building on this principle, we develop Thermodynamically Encoded Molecular Programming for One-pot diagnostics (TEMPO), which achieves attomolar sensitivity within 30 min and enables sequencing-concordant SNP genotyping and pathogen detection in a single-step microfluidic format.},
}

*Polymorphism, Single Nucleotide/genetics
Thermodynamics
*Point-of-Care Systems
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Genotyping Techniques/methods
DNA Primers/genetics
Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
Genotype

@article {pmid42574383,
year = {2026},
author = {Xu, A and Cates, K and Kerr, C and Wysong, K and Barna, M},
title = {Isolation of Subcellular Ribosome Subpopulations Based on Recombinant Peptide Tag-Specific Location-Restricted Illumination-Enhanced Biotinylation.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {233},
pages = {},
doi = {10.3791/66881},
pmid = {42574383},
issn = {1940-087X},
mesh = {*Biotinylation/methods ; *Ribosomes/chemistry/metabolism ; Carbon-Nitrogen Ligases/chemistry/metabolism/genetics ; Biotin/chemistry ; Humans ; Ribosomal Proteins/chemistry/genetics ; CRISPR-Cas Systems ; },
abstract = {Growing evidence suggests that mRNA translation is a highly compartmentalized process within a cell, and that subcellular trafficking and localization of specific mRNAs is key to ensuring that proteins with compartment-specific functions are produced in the ideal milieu and in appropriate quantities. However, techniques for subcellular isolation and characterization of the ribosomes that translate these mRNAs have been limited to date, such that much remains unknown about how the composition of translational machinery contributes to regulation of localized mRNA translation. Here, we demonstrate AviTag-specific Location-restricted Illumination-enhanced Biotinylation (ALIBi), a method that combines epitope tagging, a newly developed optogenetically activated split-biotin ligase, and affinity purification to rapidly and specifically label and isolate ribosomes localized to any subcellular compartment of interest. First, CRISPR/Cas9 editing is used to fuse an AviTag peptide, a tobacco etch virus (TEV) protease cleavage site, and a FLAG epitope tag to a ribosomal protein. The split biotin ligase, fused to an organelle-targeting domain, is expressed in this cell line and is inactive under normal biotin concentrations. Upon activation by supplemental biotin and blue light illumination, the ligase biotinylates AviTagged ribosomes in the immediate vicinity, allowing for affinity purification of biotinylated ribosomes and associated proteins and mRNAs on streptavidin-coated beads. Non-denaturing elution via TEV protease cleavage yields samples suitable for downstream characterization of core ribosomal proteins, ribosome-associated proteins, and ribosome-bound mRNAs via RNA sequencing or mass spectrometry proteomics. In this protocol, we review design principles for fusing AviTag to a ribosomal protein and targeting the split biotin ligase enzyme to the organelle of interest. We demonstrate activation of the ALIBi system, cell lysis, affinity purification, and sample elution. Finally, we discuss typical results and troubleshooting.},
}

*Biotinylation/methods
*Ribosomes/chemistry/metabolism
Carbon-Nitrogen Ligases/chemistry/metabolism/genetics
Biotin/chemistry
Humans
Ribosomal Proteins/chemistry/genetics
CRISPR-Cas Systems

@article {pmid42574397,
year = {2026},
author = {Langille, ER and Al-Zahrani, KN and Nurtanto, J and Lee, Y and Chiu, CH and Schramek, D},
title = {Parallel In Vivo Screening of Gene Knockout and Activation in the Mouse Mammary Gland.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {233},
pages = {},
doi = {10.3791/71115},
pmid = {42574397},
issn = {1940-087X},
mesh = {Animals ; Mice ; *Mammary Glands, Animal/physiology/metabolism ; Female ; *Gene Knockout Techniques/methods ; CRISPR-Cas Systems ; },
abstract = {Forward genetics screens are routinely employed to perturb thousands of genetic elements in a pooled fashion with the goal of producing large-scale genotype-to-phenotype maps. While often carried out in cell culture systems, accumulating evidence supports that in vivo screens have the power to unveil new biology that cannot be recapitulated in vitro. However, the widespread application of this approach has been limited by two major challenges: a predominant focus on loss-of-function perturbations rather than gene activation and the significant technical hurdles of delivering complex genetic libraries to specific tissues in vivo. To overcome these challenges, we describe a simple and versatile intraductal injection strategy that enables efficient and rapid functional genomic screening in the mouse mammary gland, by generating tens of thousands of discrete epithelial clones. Furthermore, we provide all the details necessary for library generation, intraductal injection, screen deconvolution, and analysis of CRISPR-Knockout and Activation libraries for comprehensive in vivo screens. Using these tools, which we termed CRISPR-KOALA (Knockout and Activation Linked Assay), we have identified new tumor suppressors and oncogenes within the coding and non-coding genome in pooled libraries ranging from 46 loci to one-fifth of the genome. Importantly, this approach and analysis can be applied to other organs to study the biological function of any gene during homeostasis or disease.},
}

Animals
Mice
*Mammary Glands, Animal/physiology/metabolism
Female
*Gene Knockout Techniques/methods
CRISPR-Cas Systems

@article {pmid42574536,
year = {2026},
author = {Yedier-Bayram, O and Guvener, EA and Bagci-Onder, T},
title = {Epigenome-Wide CRISPR-Cas9-Based Knockout Screens on Chemoresistant Cells.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {233},
pages = {},
doi = {10.3791/71175},
pmid = {42574536},
issn = {1940-087X},
mesh = {Humans ; *CRISPR-Cas Systems ; *Drug Resistance, Neoplasm/genetics ; Cell Line, Tumor ; Paclitaxel/pharmacology ; *Gene Knockout Techniques/methods ; *Epigenome/genetics ; *Triple Negative Breast Neoplasms/genetics/drug therapy ; Female ; },
abstract = {Chemotherapy resistance remains a major challenge in cancer treatment, driven by cancer cells' ability to acquire adaptive properties, rewire signaling pathways, and alter chromatin structure to evade drug-induced cytotoxicity. Because these processes rely heavily on epigenetic mechanisms that regulate chromatin organization and transcriptional plasticity, epigenetic regulators have emerged as key contributors to chemotherapy resistance. To investigate resistance to paclitaxel, one of the most widely used chemotherapeutic agents in triple-negative breast cancer (TNBC), we employed an epigenome-focused knockout library (EPIKOL), a CRISPR-Cas9-based library, designed to systematically disrupt genes involved in chromatin regulation. Chemoresistant cell lines were generated through a stepwise dose-escalation protocol that recapitulates clinically relevant drug adaptation. However, these resistant cells exhibit a multidrug-resistant (MDR) phenotype, posing significant challenges for efficient viral transduction and the selection of stable cell populations. In this study, we describe key methodological steps for achieving high-efficiency lentiviral transduction and selection, enabling the successful application of EPIKOL CRISPR screens in chemoresistant TNBC models. Following the described protocol, an epigenome-wide CRISPR screen was conducted on chemoresistant TNBC cells, and novel epigenetic regulators of chemoresistance were identified. This protocol provides a robust framework for identifying epigenetic regulators that contribute to acquired paclitaxel resistance using a CRISPR-based loss-of-function approach.},
}

Humans
*CRISPR-Cas Systems
*Drug Resistance, Neoplasm/genetics
Cell Line, Tumor
Paclitaxel/pharmacology
*Gene Knockout Techniques/methods
*Epigenome/genetics
*Triple Negative Breast Neoplasms/genetics/drug therapy
Female

@article {pmid42577382,
year = {2026},
author = {Lin, W and Shi, J and Chen, H and Chai, R and Zhu, S and Chen, L and Mo, S and Wang, Z and Li, H and Feng, Y and Zhao, L and Chen, J and Yu, G and Lu, T and Wang, J},
title = {Establishment of a scalable engineered cell-line platform for direct, GMP-grade production of eVLP vectors enabling streamlined generation of gene-edited CAR-T/NK cells.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1878099},
pmid = {42577382},
issn = {1664-3224},
mesh = {Humans ; *Killer Cells, Natural/immunology/metabolism ; *Genetic Vectors/genetics ; *Receptors, Chimeric Antigen/genetics/immunology ; HEK293 Cells ; Animals ; *Gene Editing/methods ; *Immunotherapy, Adoptive/methods ; *Cell Engineering/methods ; CRISPR-Cas Systems ; *T-Lymphocytes/immunology ; Cell Line ; },
abstract = {INTRODUCTION: CRISPR-Cas9 has transformed the engineering of chimeric antigen receptor T (CAR-T) cells and chimeric antigen receptor NK (CAR-NK) cells; however, its clinical translation remains constrained by the high cost, batch-to-batch variability, and stringent regulatory requirements associated with current viral and electroporation-based manufacturing approaches.

METHODS: We report an industrial-grade platform based on monoclonal producer cell lines that enables the continuous and scalable generation of engineered virus-like particles (eVLPs) co-packaging Cas9-gRNA ribonucleoproteins (RNPs). A progenitor cell line was established by stably integrating three core modules-Gag-Pol, Gag-Cas9, and the baboon endogenous virus (BaEV) envelope-into a single HEK293T clone. Introduction of a self-inactivating (SIN) retroviral vector encoding the gRNA cassette (exemplified here by CD7) converted this progenitor into a dedicated eVLP producer within 10 days.

RESULTS: Using this platform, we generated CD7-knockout CAR-T/NK cells that retained robust in vitro cytotoxicity, confirming preserved functional activity. Owing to its modular architecture, the platform is readily extensible. For example, integration with Recombinant Adeno-associated Virus (rAAV) donor templates enables site-specific CAR insertion, while multiplexed eVLP cocktails allow simultaneous disruption of multiple genomic loci.

DISCUSSION: It is worth noting that this workflow eliminates the need for electroporation, reduces serum dependency, and significantly lowers the cost of reagent consumables. Collectively, this system provides a GMP-compliant and broadly adaptable strategy for the streamlined manufacturing of next-generation autologous and allogeneic gene-edited CAR-T/NK therapies.},
}

Humans
*Killer Cells, Natural/immunology/metabolism
*Genetic Vectors/genetics
*Receptors, Chimeric Antigen/genetics/immunology
HEK293 Cells
Animals
*Gene Editing/methods
*Immunotherapy, Adoptive/methods
*Cell Engineering/methods
CRISPR-Cas Systems
*T-Lymphocytes/immunology
Cell Line

@article {pmid42577913,
year = {2026},
author = {Tenkolu, LA and Balcha, F},
title = {Molecular Diagnosis of Bacterial Meningitis in Ethiopia: A Narrative Review of Current Evidence and Implementation Gaps.},
journal = {The Canadian journal of infectious diseases & medical microbiology = Journal canadien des maladies infectieuses et de la microbiologie medicale},
volume = {2026},
number = {},
pages = {4447180},
pmid = {42577913},
issn = {1712-9532},
abstract = {BACKGROUND: Bacterial meningitis (BM) remains a significant public health concern in Ethiopia, particularly due to diagnostic challenges posed by limited laboratory infrastructure, late patient presentation, and technical skills required for cerebrospinal fluid (CSF) sampling. Molecular diagnostic methods offer rapid, sensitive, and specific alternatives to conventional culture.

OBJECTIVE: This narrative review synthesizes available evidence on molecular diagnostics for BM in Ethiopia, evaluates their performance compared to conventional methods, and identifies barriers to implementation.

METHODS: A structured literature search was conducted in PubMed, Google Scholar, Scopus, and Web of Science (August 1977-September 2024) using the terms: Meningitis OR Bacterial meningitis AND Diagnostics OR Molecular diagnostics, including PCR, CRISPR, next-generation sequencing (NGS), and MALDI-TOF. The review used a repeatable research selection procedure, well-defined inclusion/exclusion criteria, and PRISMA reporting requirements. Studies from Ethiopia and other low-resource settings were included. After screening titles/abstracts and full texts, 66 articles were selected.

RESULTS: In Ethiopia, multiplex PCR detected bacterial DNA in 10%-22% of the CSF samples, whereas culture was positive in only 0.5%-1% of the cases, primarily due to pre-admission antibiotic use. Globally, molecular panels (e.g., BioFire FilmArray ME) show > 90% sensitivity and specificity. Advanced techniques (CRISPR-Cas, NGS, and MALDI-TOF) have not yet been implemented in Ethiopian routine diagnostics.

CONCLUSION: Molecular methods vastly improve the detection of BM in Ethiopia, but high costs, infrastructure gaps, and shortage of trained personnel prevent widespread adoption. Molecular assays have outstanding analytical sensitivity, but culture remains the gold standard for confirmation of the viability of live pathogens. Implementation gaps require urgent targeted investment in point-of-care diagnostics and specialized transport networks.},
}

@article {pmid42578365,
year = {2026},
author = {Lv, S and Wang, Q and Gao, S and Wang, L and Zheng, L},
title = {Specific cleavage of 5' overhangs of non-target strand by Cas9 activated by target DNA binding.},
journal = {Nucleic acids research},
volume = {54},
number = {15},
pages = {},
pmid = {42578365},
issn = {1362-4962},
support = {U23A20265//National Natural Science Foundation of China/ ; 32530084//National Natural Science Foundation of China/ ; 2408085MC072//Anhui Provincial Natural Science Foundation/ ; PA2025GDGP0026//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*DNA/metabolism/genetics/chemistry ; *CRISPR-Cas Systems ; DNA Cleavage ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; SARS-CoV-2/genetics ; *CRISPR-Associated Protein 9/metabolism ; Humans ; Base Sequence ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has become a powerful genome-editing tool that uses RNA-DNA pairing to cleave target DNA with protospacer adjacent motif (PAM) sequences. While its primary function is well-studied, secondary activities remain poorly understood, causing unintended off-target effects. This study reports for the first time that Cas9 specifically cleaves the 5' overhang of the non-target strand (NTS) in target double-stranded DNA. This specific cleavage requires an additional PAM element at the NTS 5' region, is mediated by Cas9's RuvC domain, and is regulated by the HNH domain. It depends on the exact positioning of the NTS 5' end, but not on the overhang homopolymer sequence or overhang length. Adequate single-guide RNA-DNA complementarity is also essential. This discovery potentially advances our understanding of Cas9's enzymatic versatility to enhance genome-editing precision and efficacy and offers new nucleic acid detection strategies. Based on this cleavage, we developed a sensitive assay for Severe Acute Respiratory Syndrome Coronavirus 2 pseudovirus down to 2.4 copies μL-1, demonstrated extremely high sensitivity in diagnostic applications.},
}

*DNA/metabolism/genetics/chemistry
*CRISPR-Cas Systems
DNA Cleavage
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
SARS-CoV-2/genetics
*CRISPR-Associated Protein 9/metabolism
Humans
Base Sequence

@article {pmid42578374,
year = {2026},
author = {Zou, S and Ye, T and Fu, L and Zhou, JQ},
title = {A CRISPR/Cas9-induced blunt-end telomere system in S. pombe reveals RNase H2-dependent RNA primer removal at the terminal Okazaki fragment of lagging telomeres.},
journal = {Nucleic acids research},
volume = {54},
number = {15},
pages = {},
pmid = {42578374},
issn = {1362-4962},
support = {2023YFA0913400//National Key Research and Development Program of China/ ; //Shanghai Academy of Natural Sciences/ ; },
mesh = {*Telomere/genetics/metabolism/chemistry ; *Schizosaccharomyces/genetics/metabolism ; *Ribonuclease H/metabolism/genetics ; *CRISPR-Cas Systems ; *RNA/metabolism/genetics ; DNA Replication/genetics ; *Schizosaccharomyces pombe Proteins/genetics/metabolism ; *DNA/genetics/metabolism ; },
abstract = {Studying the fine-scale dynamics of telomere replication has been hindered by the heterogeneity of native telomeres and the limitations of existing tools. Here, we report a highly efficient and inducible CRISPR/Cas9-mediated system for generating de novo telomeres with defined blunt ends in S. pombe. This fine setting allows for the precise dissection of post-replicative telomere end structures at near single-nucleotide resolution. Using this system, we show that the replicated leading-strand telomere is blunt-ended, while the lagging-strand counterpart contains an ∼10-nt 3' overhang resulting from RNA primer removal. By analyzing mutants deficient in ribonucleases, we found that the removal of this terminal RNA primer is specifically dependent on RNase H2, but not RNase H1. This RNase H2-dependent mechanism is essential for defining the mature structure of the lagging-strand telomere with authentic telomeric sequences. Our findings reveal a fundamental asymmetry in telomere end processing after replication and establish RNase H2 as the key enzyme responsible for resolving the terminal RNA primer in lagging-strand telomeres. This mechanism, conserved from budding to fission yeast, underscores the critical and evolutionarily ancient role of RNase H2 in defining eukaryotic telomere architecture.},
}

*Telomere/genetics/metabolism/chemistry
*Schizosaccharomyces/genetics/metabolism
*Ribonuclease H/metabolism/genetics
*CRISPR-Cas Systems
*RNA/metabolism/genetics
DNA Replication/genetics
*Schizosaccharomyces pombe Proteins/genetics/metabolism
*DNA/genetics/metabolism

@article {pmid42579777,
year = {2026},
author = {Ma, Y and Deng, Y and Xu, J and Wang, S and Shi, B and Liang, K and Deng, R and Yang, H},
title = {Engineering CRISPR for Point-of-Care Tests.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.6c01485},
pmid = {42579777},
issn = {2379-3694},
support = {2026YFHZ0080//International S and T Cooperation Program of Sichuan Province/ ; 22504094//National Natural Science Foundation of China/ ; 32271392//National Natural Science Foundation of China/ ; 32571536//National Natural Science Foundation of China/ ; 82270970//National Natural Science Foundation of China/ ; 82470967//National Natural Science Foundation of China/ ; //Sichuan University/ ; 2023NSFSC0333//Natural Science Foundation of Sichuan Province/ ; 2024NSFSC0676//Natural Science Foundation of Sichuan Province/ ; 2025ZNSFSC1041//Natural Science Foundation of Sichuan Province/ ; },
abstract = {CRISPR-based molecular diagnostics have emerged as powerful and programmable platforms that enable sensitive and specific detection for disease management and epidemiological surveillance. Advances in CRISPR engineering and assay design are driving the emergence of next-generation detection platforms that are highly sensitive, rapid, and amenable to field deployment. These engineering breakthroughs have the potential to reshape point-of-care tests (POCT) and transform how emerging and persistent health threats are monitored in decentralized and resource-limited settings. Herein, we systematically review the recent advancements in CRISPR engineering strategies aimed at improving detection sensitivity and specificity, eliminating the dependence on preamplification, and enabling robust POC deployment. The discussed strategies encompass both the rational engineering of CRISPR ribonucleoproteins (RNPs) and the optimization of downstream signaling modules for molecular diagnostic applications. We further highlight key challenges and future perspectives that may inspire impactful research directions and accelerate the advancement of CRISPR engineering strategies toward robust, field-deployable POCT platforms.},
}

@article {pmid42580522,
year = {2026},
author = {Reis, BCC and Georget, C and Meunier, AC and de Andrade Silva, EM and Perin, C and Herbert, L and Dos Santos Lopes, N and Micheli, F and de Oliveira Mendes, TA},
title = {Easy detection of CRISPR/Cas9-Induced Insertions, Deletions, and Substitutions in Rice Genes OsMADS26, OsRAC1 and OsNRT1.1b using SYBR Green qPCR and Robust HRM analysis in R software.},
journal = {New biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.nbt.2026.07.005},
pmid = {42580522},
issn = {1876-4347},
abstract = {Rice is a major cereal crop for global food and nutritional security and a key target for genetic improvement. CRISPR/Cas9 enables precise genetic modification in crops, but mutation screening remains a technical and economic barrier to broader genome-editing applications. Although several detection methods are available, some require labor-intensive procedures, specialized equipment, high costs, or limited sensitivity to specific mutation types. High-resolution melting (HRM) analysis is an established approach for screening CRISPR/Cas-induced mutations in plants, including rice. Here, we evaluated an adapted HRM workflow combining conventional SYBR Green-based qPCR chemistry with downstream computational analysis to detect CRISPR/Cas9-induced mutations at three rice loci: OsMADS26, OsRAC1, and OsNRT1.1b. The workflow detected insertions, deletions, and base substitutions. Across the three loci, the 1% edited-DNA mixtures showed a slight observable deviation from the wild-type melting profile under the conditions evaluated, although this should not be interpreted as a validated detection threshold. Although the assessment of heterozygous samples was limited by their availability, the results support the potential applicability of the approach for individual sample analysis and expanded sample screening. A customizable R script complemented visual analysis by evaluating melting temperature (Tm) and GCP-derived dissimilarity, supporting sample classification. By combining standard SYBR Green chemistry with an adaptable analysis workflow, the method offers an alternative to dedicated HRM reagents and proprietary platforms. This approach provides a practical and potentially lower-cost option for mutation screening at the evaluated rice loci and may be adapted and validated for other targets and plant species.},
}

@article {pmid42580703,
year = {2026},
author = {Yamaji, M and Tabata, H and Nakamura, M and Fuse, R and Okada, T and Ohba, SI and Ohishi, T and Kawada, M and Saito, I and Sato, S and Nakanishi, T},
title = {Adenovirus Vector-Mediated In Vivo Knock-in Treatment of Neonatal Phenylketonuria Mice Using Terminally Cleaved Donor DNA.},
journal = {The journal of gene medicine},
volume = {28},
number = {8},
pages = {e70104},
pmid = {42580703},
issn = {1521-2254},
support = {JP21fk0108560//Japan Agency for Medical Research and Development/ ; JP24fk0310523//Japan Agency for Medical Research and Development/ ; JP19K06476//Japan Society for the Promotion of Science/ ; JP23K27095//Japan Society for the Promotion of Science/ ; JP24K21938//Japan Society for the Promotion of Science/ ; },
mesh = {Animals ; *Genetic Vectors/genetics/administration & dosage ; *Gene Knock-In Techniques/methods ; Mice ; *Adenoviridae/genetics ; *Phenylalanine Hydroxylase/genetics ; *Phenylketonurias/therapy/genetics ; CRISPR-Cas Systems ; Genetic Therapy ; Disease Models, Animal ; Animals, Newborn ; Humans ; Gene Editing ; RNA, Guide, CRISPR-Cas Systems/genetics ; *DNA/genetics ; },
abstract = {BACKGROUND: Adenovirus vectors (AdVs) are widely used and have an advantage of large insert capacity compared with adeno-associated virus vectors. However, AdVs have scarcely been used in genome-editing knock-in strategies because of low efficiency.

METHODS: Novel AdVs possessing a very large, 3.7 kb donor DNA fragment and six or eight multiplex gRNA expression units were developed for CRISPR/Cas9-mediated knock-in to correct a phenylalanine hydroxylase (Pah) gene in a Pah[enu2] phenylketonuria mouse model. These AdVs were co-infected to Hepa1-6 cells or liver cells in vivo together with an AdV expressing either native Cas9 or Cas9 nickase (Cas9n) for double-nicking cleavage.

RESULTS: In vitro knock-in of the AdVs carrying 3.7 kb donor DNA and six gRNA units targeting the cell genome was observed in both cases using Cas9 and Cas9n, though their efficiencies were low. Therefore, we generated AdVs carrying an additional two gRNA units that cleave the donor DNA terminus in the AdV genome via native Cas9 or Cas9 nickase. The knock-in efficiency increased approximately twofold for both vectors and reached a maximum of 8% for native Cas9 without selection. Newborn phenylketonuria model mice were intravenously administered the knock-in AdV together with the native-Cas9 AdV. Although the knock-in efficiency by homologous recombination occurred in only approximately 1% of hepatocytes, blood phenylalanine levels were reduced by up to 30%. Also, unintended fragments produced by nonhomologous end-joining were observed between the cleavage site at the terminus of the donor DNA in the AdV genome and the target site in the cell genome.

CONCLUSIONS: The knock-in efficiency of AdVs can be increased by cleaving the terminus of the donor DNA, although it would be desirable to avoid nonhomologous end-joining between double-strand break termini.},
}

Animals
*Genetic Vectors/genetics/administration & dosage
*Gene Knock-In Techniques/methods
Mice
*Adenoviridae/genetics
*Phenylalanine Hydroxylase/genetics
*Phenylketonurias/therapy/genetics
CRISPR-Cas Systems
Genetic Therapy
Disease Models, Animal
Animals, Newborn
Humans
Gene Editing
RNA, Guide, CRISPR-Cas Systems/genetics
*DNA/genetics

@article {pmid42581041,
year = {2026},
author = {Kinney, KJ and Jia, K and Zhang, H and Schmaljohn, E and Osborne, T and Thommandru, B and Murugan, K and Sánchez-Peña, A and West, S and Chen, S and Codipilly, R and Sturgeon, M and Turk, R and McNeill, MS and Behlke, M and Jacobi, A and Cromer, MK and Rettig, G and Kurgan, GL},
title = {UNCOVERseq enables sensitive and controlled gene editing off-target nomination across CRISPR-Cas modalities and systems.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42581041},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; Hematopoietic Stem Cells/metabolism ; DNA Breaks, Double-Stranded ; },
abstract = {The rapid expansion of CRISPR-Cas gene editing enables new therapeutic strategies but complicates assessment of unintended editing risks due to emerging modalities and unclear analytical standards. We present UNCOVERseq (Unbiased Nomination of CRISPR Off-target Variants using Enhanced RhPCR), an improved in cellulo off-target nomination workflow that sensitively identifies rare off-target events using defined inputs and analytical process controls. Using an inter-method off-target confirmation benchmarking dataset, UNCOVERseq demonstrates high analytical sensitivity (97.6%) and precision (78%), outperforming published nomination methods. We apply UNCOVERseq across 192 guide RNAs and identify six guides spanning a broad specificity range, enabling relative risk assessment across S. pyogenes Cas9, high-fidelity variants, and base editors in hematopoietic stem and progenitor cells. We further show that double-strand break nomination sites retain strong rank-order concordance with single-strand break-mediated base editing. Together, these results establish UNCOVERseq as a robust framework for informed off-target risk assessment in translational gene-editing systems.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Humans
RNA, Guide, CRISPR-Cas Systems/genetics
Hematopoietic Stem Cells/metabolism
DNA Breaks, Double-Stranded

@article {pmid42086878,
year = {2026},
author = {Wang, MM and Li, Y and Ho, CEH and Yu, WSS and Coupland, SE and Park, G and Chan, ASY and Figueiredo, CR},
title = {Novel CRISPR-Cas9 BAP1 knockout pre-clinical tumor model recapitulates human melanoma tumorigenesis and immune evolution.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {42086878},
issn = {2399-3642},
mesh = {Animals ; *Ubiquitin Thiolesterase/genetics ; Humans ; *Tumor Suppressor Proteins/genetics ; *Melanoma/genetics/immunology/pathology/metabolism ; *CRISPR-Cas Systems ; Mice ; *Carcinogenesis/genetics ; Mice, Knockout ; Disease Models, Animal ; Cell Line, Tumor ; Uveal Melanoma ; *Skin Neoplasms/genetics/pathology/immunology ; Uveal Neoplasms/genetics/immunology/pathology ; Female ; },
abstract = {BAP1-deficient melanocytic tumors exhibit strong immunosuppressive features and poor prognosis. Currently, no immune-competent preclinical models exist to study their tumor-immune interactions or test new immunotherapies. This limitation hinders progress in understanding how BAP1 loss drives tumor aggressiveness and immune evasion. To address this, we generate a syngeneic BAP1 knockout melanocyte tumor line using CRISPR-Cas9. We then evaluate its functional and immunological impact in immune-competent mice, including its ability to recapitulate metabolic and immunosuppressive features of human BAP1-deficient melanomas. The selected knockout clone exhibits hallmarks of aggressive skin and intraocular melanomas, including epithelioid morphology, in vivo tumorigenic potential, rapid growth, and key immunosuppressive features, mirroring those observed in human BAP1-deficient melanomas. Cross-species single-cell transcriptome analysis demonstrates strong molecular overlap between BAP1 knockout mouse tumors and high-risk (class 2) human uveal melanomas, highlighting shared pathways in lipid metabolism, transmembrane receptor signaling, and immune modulation. Gene Set Enrichment Analysis confirms that lipid metabolic reprogramming, previously described in human tumors, is also a key feature of our model, validating its ability to recapitulate human disease biology. This study introduces a syngeneic preclinical model that mimics the immunosuppressive landscape of BAP1-deficient melanocytic tumors, enabling the development and optimization of new combination immunotherapies.},
}

Animals
*Ubiquitin Thiolesterase/genetics
Humans
*Tumor Suppressor Proteins/genetics
*Melanoma/genetics/immunology/pathology/metabolism
*CRISPR-Cas Systems
Mice
*Carcinogenesis/genetics
Mice, Knockout
Disease Models, Animal
Cell Line, Tumor
Uveal Melanoma
*Skin Neoplasms/genetics/pathology/immunology
Uveal Neoplasms/genetics/immunology/pathology
Female

@article {pmid42120907,
year = {2026},
author = {Jajarmi, J and Guest, MR and Ma, LJ and Thu, KL and Chari, R and Lockwood, WW},
title = {Pervasive Cas9 expression driven by mammalian promoter activity in E. coli affects representation of CRISPR sgRNA libraries.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {42120907},
issn = {2399-3642},
support = {N/A//Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Santé du Canada)/ ; HHSN261201500003I/CA/NCI NIH HHS/United States ; HHSN261201500003I//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {*Escherichia coli/genetics/metabolism ; *Promoter Regions, Genetic ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Animals ; *CRISPR-Cas Systems ; *Gene Library ; Humans ; *CRISPR-Associated Protein 9/genetics/metabolism ; Mice ; Gene Editing ; Plasmids/genetics ; },
abstract = {CRISPR-Cas9 screening relies on uniform representation of single-guide RNA (sgRNA) libraries to enable accurate gene discovery. However, technical biases during library preparation can compromise screen performance. Here we show that commonly used "all-in-one" CRISPR vectors expressing both Cas9 and sgRNAs drive unintended Cas9 protein expression in Escherichia coli during plasmid amplification. This bacterial Cas9 expression causes guide-specific toxicity, leading to selective loss of sgRNAs and highly skewed library representation. We demonstrate that this effect occurs across multiple bacterial strains and affects both targeted and genome-wide libraries, including widely used human CRISPR libraries. Mechanistically, toxicity is driven by Cas9 expression rather than plasmid size and is only partially alleviated by catalytically inactive Cas9. Importantly, replacing the EF-1α promoter with a mouse phosphoglycerate kinase promoter suppresses Cas9 expression in bacteria while preserving genome editing efficiency in mammalian cells, restoring sgRNA uniformity. These findings identify a previously unrecognized source of bias in CRISPR library preparation and provide a practical solution to improve screening fidelity.},
}

*Escherichia coli/genetics/metabolism
*Promoter Regions, Genetic
*RNA, Guide, CRISPR-Cas Systems/genetics
Animals
*CRISPR-Cas Systems
*Gene Library
Humans
*CRISPR-Associated Protein 9/genetics/metabolism
Mice
Gene Editing
Plasmids/genetics

@article {pmid42213648,
year = {2026},
author = {Kaminski, PJ and Min, K and Traxler, EA and Khandros, E and Abdulmalik, O and Godfrey, B and Keller, CA and Giardine, BM and Hardison, RC and Shi, J and Blobel, GA},
title = {Dissecting polycomb complexes for enhanced fetal hemoglobin production.},
journal = {Blood},
volume = {148},
number = {7},
pages = {882-895},
doi = {10.1182/blood.2026033804},
pmid = {42213648},
issn = {1528-0020},
mesh = {*Fetal Hemoglobin/genetics/biosynthesis/metabolism ; Animals ; Humans ; Mice ; *Enhancer of Zeste Homolog 2 Protein/genetics/metabolism ; Erythroid Cells/metabolism ; CRISPR-Cas Systems ; Polycomb Repressive Complex 2/metabolism/genetics ; Histones/metabolism ; *Polycomb-Group Proteins/metabolism/genetics ; },
abstract = {Polycomb repressive complex 1 (PRC1) and PRC2 regulate diverse developmental processes, including the fetal-to-adult switch in hemoglobin (Hb) production, a process whose reversal is a goal for the treatment of sickle cell disease and β-thalassemia. PRC inhibitors show promise for various disorders, but use is limited because of pleiotropic PRC activities. We explored whether fetal Hb (HbF) can be reactivated in adult erythroid cells by selective perturbations of PRC1 or PRC2 components without complete loss of PRC function. A high-density CRISPR-CRISPR-associated protein 9 (Cas9) mutagenesis screen identified a region in EZH2 in which Cas9 induced exon 14 skipping (EZH2Δ14). EZH2Δ14, which lacks a portion of the CXC domain, relieves HbF repression while largely maintaining cellular fitness. EZH2Δ14 retains H3K27 methylation and repression of a PRC target gene subset. Experiments in cells derived from mice bearing human β-globin genes confirm that pathways mediating EZH2 control of HbF expression can function in a mouse model of HBG switching. These findings demonstrate that partial disruption of PRC can yield selective phenotypes, highlighting the therapeutic potential of targeting nonenzymatic domains within chromatin-modifying complexes.},
}

*Fetal Hemoglobin/genetics/biosynthesis/metabolism
Animals
Humans
Mice
*Enhancer of Zeste Homolog 2 Protein/genetics/metabolism
Erythroid Cells/metabolism
CRISPR-Cas Systems
Polycomb Repressive Complex 2/metabolism/genetics
Histones/metabolism
*Polycomb-Group Proteins/metabolism/genetics

@article {pmid42486984,
year = {2026},
author = {Meng, H and Lei, Z and Yan, Y and Wang, L and Zhang, S and Rao, X and Shao, C and Zhang, X and Chen, K and Yang, L and Liu, R and Yang, G and Shen, R and Gu, R and Wang, X and Wang, Y and Lu, S and Lv, Z and He, B and Wen, H and Li, D and Yi, C},
title = {Precise DNA base editing using AlphaFold3-based contact modelling.},
journal = {Nature},
volume = {656},
number = {8127},
pages = {463-473},
pmid = {42486984},
issn = {1476-4687},
mesh = {*DNA/genetics/chemistry/metabolism ; *Gene Editing/methods ; *Models, Molecular ; CRISPR-Cas Systems/genetics ; Adenine/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry ; Substrate Specificity ; CRISPR-Associated Proteins/metabolism/chemistry ; Base Sequence ; Adenosine Deaminase ; Escherichia coli Proteins ; },
abstract = {Achieving high specificity in biochemical transformations is crucial for research and therapeutics. This is particularly important for genome editing, where enhancing tool specificity ensures effective and precise editing outcomes[1,2]. Current strategies are constrained by activity-specificity trade-offs, high labour intensity and low success rates[3,4]. Here we present ContactSeek, an artificial-intelligence-driven framework that uses AlphaFold3 (AF3)-predicted contact probability[5] to improve the specificity of genome editors. Using Cas9-TadA adenine base editors[6-8] as a demonstration, we mapped their genome-wide off-targets and fed the off-target DNA sequences to AF3. Among AF3 outputs, we found that contact probability was more sensitive than predicted three-dimensional structures for detecting differential interactions between on- and off-target complexes. Correlating contact probability with sequencing-based off-target signals, ContactSeek identified and ranked consensus contact regions, which are neighbouring Cas residues with consistent contact changes to DNA/guide RNA, and pinpointed specificity-determining residues within them. ContactSeek can also be applied modularly and identified key residues in the TadA8e deaminase. Targeted amplicon sequencing, genome-wide profiling, R-loop assay and RNA-sequencing together confirmed the greatly enhanced specificity; our best variant, combining two mutations of Cas9 and TadA8e, outperformed several known high-fidelity adenine base editors. ContactSeek is also generalized to Cas12a-based cytosine base editors. Collectively, our framework represents an AF3-driven model tailored for specificity improvement, establishing a paradigm for improving the precision of genome editing tools through the integration of structural and functional dimensions.},
}

*DNA/genetics/chemistry/metabolism
*Gene Editing/methods
*Models, Molecular
CRISPR-Cas Systems/genetics
Adenine/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry
Substrate Specificity
CRISPR-Associated Proteins/metabolism/chemistry
Base Sequence
Adenosine Deaminase
Escherichia coli Proteins

@article {pmid42570142,
year = {2026},
author = {Ahad, A and Hullon, D and Singh, T and Dabiry, SM and Sakthivel, L and Padaria, J and Rekhraj, AS and Bhattacharjee, A},
title = {The Vascular Genome as a Therapeutic Target: A Systematic Review of CRISPR-based Gene Editing In Vascular Disease.},
journal = {Cardiovascular toxicology},
volume = {26},
number = {8},
pages = {},
pmid = {42570142},
issn = {1559-0259},
mesh = {Humans ; Animals ; *Gene Editing ; *Genetic Therapy/adverse effects/methods ; *Vascular Diseases/genetics/therapy/metabolism/physiopathology ; *CRISPR-Cas Systems ; Vascular Remodeling/genetics ; Genetic Predisposition to Disease ; Phenotype ; },
abstract = {Despite advances in therapy, arterial, venous, and pulmonary vascular diseases remain leading causes of morbidity and mortality. Persistent endothelial dysfunction, inflammation, oxidative stress, and maladaptive vascular remodeling continue to drive disease progression and residual risk. CRISPR/Cas9 technology offers a unique opportunity to modify the molecular pathways underlying vascular pathophysiology directly. The PRISMA 2020 guidelines guided the systematic review. The databases PubMed/MEDLINE, Embase, Web of Science, Cochrane Library, ClinicalTrials.gov, and Google Scholar were searched from their inception until September 2025 for experimental and/or clinical studies evaluating the application of CRISPR/Cas9 on vascular disease. Included were in vitro studies, animal model studies, and early-phase human studies aimed at targeting the endothelial cell regulatory pathways, inflammatory pathways, metabolic remodeling processes, and hereditary causes of vasculopathy. Seventeen studies met the inclusion criteria. CRISPR technologies targeting PCSK9, NOS3, HIF1A, NLRP3, METTL4, BMPR2, and ACTA2 were identified to enhance repair mechanisms in endothelial cells, regulate inflammation, modulate lipid metabolism, and remodel the vascular system. The human studies demonstrated sustained gene silencing effects following a single dose of CRISPR-induced in vivo editing. The use of CRISPR technology to edit cell genomes offers potential to alter disease progression in vascular medicine, with a growing body of translational evidence supporting the feasibility and durability of the approach.},
}

Humans
Animals
*Gene Editing
*Genetic Therapy/adverse effects/methods
*Vascular Diseases/genetics/therapy/metabolism/physiopathology
*CRISPR-Cas Systems
Vascular Remodeling/genetics
Genetic Predisposition to Disease
Phenotype

@article {pmid42570236,
year = {2026},
author = {Jiang, S and Tian, X and Wang, F and Han, W and She, Q and Feng, X},
title = {Recruitment of Cas3 enables DNA cleavage by the type I-A CRISPR-Cas system of Saccharolobus islandicus.},
journal = {Cell reports},
volume = {45},
number = {8},
pages = {117820},
doi = {10.1016/j.celrep.2026.117820},
pmid = {42570236},
issn = {2211-1247},
abstract = {Type I CRISPR-Cas systems constitute the most prevalent prokaryotic adaptive immune pathways and are classified into seven subtypes (I-A to I-G). These antiviral systems typically exploit a Cascade complex for RNA-guided DNA recognition and a Cas3 helicase-nuclease effector for DNA degradation, yet their diverse activation mechanisms remain not fully understood. In this study, we isolate the I-A Cascade from Saccharolobus islandicus, revealing a minimal form of Cascade lacking both Cas3 and the CRISPR-RNA maturase Cas6. Cas3 is recruited to the R-loop structure formed after Cascade binding to target DNA, which activates the effector for both cis- and trans-DNA cleavage. Strikingly, ATP not only enables the processive target degradation by Cas3 but also suppresses the trans-cleavage of the same enzyme. Together, the Sa. islandicus I-A system operates via target-dependent Cas3 recruitment-a mechanism distinct from other characterized I-A systems, thus underscoring the mechanistic diversity within type I CRISPR-Cas immunity.},
}

@article {pmid42571080,
year = {2026},
author = {Ullah, O},
title = {Breaking the growth-defense trade-off in cereal crops: CRISPR/Cas and moonlighting proteins in biotic stress resistance.},
journal = {Molecular breeding : new strategies in plant improvement},
volume = {46},
number = {8},
pages = {80},
pmid = {42571080},
issn = {1572-9788},
abstract = {Recurrent crop disease outbreaks linked to global warming pose challenges to sustainable food production. Conventional plant breeding techniques may become less effective at addressing these threats, as improving disease resistance often causes yield reduction. Amid these challenges, CRISPR/Cas-based gene editing offers targeted and tractable solutions. This review synthesizes recent approaches to uncoupling immunity from productivity in cereals. We show that susceptibility (S) gene disruption can provide resistance without activating costly defense mechanisms. We also discuss the generation of new alleles through targeted modifications that mitigate autoimmunity-associated fitness costs. Here, we propose CRISPR-mediated de-moonlighting, an approach for decoupling multifunctional protein activities. Multiplex editing of minor resistance loci, especially in polyploids such as wheat, offers long-lasting, broad-spectrum protection. These strategies converge on the manipulation of canonical moonlighting proteins, multifunctional signaling hubs, and pleiotropic regulators, which serve as regulatory nodes that link development and immunity. CRISPR-mediated precision modification of these regulators can fine-tune the growth-defense balance. Combining these approaches with systems biology, AI-driven design and advanced breeding pipelines can help develop high-yielding, disease-resistant cereals for sustainable agriculture.},
}

@article {pmid42571242,
year = {2026},
author = {Eken, JA and Havenaar, FRM and de Groen, RAL and Quinten, E and Mei, H and Sepúlveda-Yáñez, JH and Navarrete, MA and Drexler, HG and Vermaat, JSP and van Bergen, CAM and Veelken, H},
title = {Alternative oncogenic drivers and sensitivity to BTK inhibition in CRISPR/Cas gene-edited human DLBCL cell models.},
journal = {Blood neoplasia},
volume = {3},
number = {3},
pages = {100261},
pmid = {42571242},
issn = {2950-3280},
abstract = {Diffuse large B-cell lymphoma (DLBCL) can be subclassified by phenotype into germinal center B-cell-like and activated B-cell-like (ABC) subtypes and by recurrent potentially oncogenic mutations into 5 to 7 genetic clusters. In ABC-DLBCL, potentially oncogenic mutations frequently occur in genes involved in B-cell receptor (BCR) signaling and NF-κB activation. Autonomous BCR signaling acts as an alternative immunologic driver predominantly in ABC-type DLBCL that cannot be captured by either subclassification system. The relative functional contribution and interdependence of these mechanistically diverse oncogenic drivers have not been completely defined. To directly compare the effects of autonomously signaling BCR and signalosome-activating CARD11 mutations on NF-κB activation and survival of ABC-DLBCL, we reciprocally exchanged these driver mechanisms in the MYD88[L265P]-mutated ABC-DLBCL cell lines TMD8 and OCI-Ly3. Only CARD11[L251P] (not CARD11[K215N], CARD11[D230N], and CARD11[R337Q]) compensated TMD8 cells for the loss of autonomous BCR signaling, as indicated by survival of BCR knockout and conversion to complete resistance to acalabrutinib. Transduction of the TMD8 BCR rescued OCI-Ly3 cells from replacing the CARD11[L215P] variant with CARD11[WT]. The autonomous TMD8 BCR signal provided a slight growth advantage over CARD11[L251P]-driven cells in both reciprocal systems. Unsupervised clustering of genetically engineered TMD8 and OCI-Ly3 clones demonstrated tight clustering with their parental cells and only minor alterations of cellular pathways. Only the strongest signalosome-activating mutation has functional near-equivalency to an autonomously signaling BCR for NF-κB activation and growth and survival in ABC-DLBCL. Quantifying the effects of co-occurring potential NF-κB-activating mechanisms is essential to predict Bruton tyrosine kinase (BTK) inhibition sensitivity in individual ABC-DLBCL cases.},
}

@article {pmid42572397,
year = {2026},
author = {Baoling, L and Lina, S and Tong, X and Qinrui, J and Ling, Z and Zhiwen, X},
title = {A Rapid, Field-Deployable Diagnostic Platform for Getah Virus Based on RT-RAA and CRISPR EsCas13d.},
journal = {Microbial biotechnology},
volume = {19},
number = {8},
pages = {e70429},
pmid = {42572397},
issn = {1751-7915},
support = {2024YFD1800102//National Key Research and Development Program of China during the 14th Five-Year Plan: Research and Development of Key Technologies for the Prevention and Control of Important Diseases in Wild Animals/ ; 2024YFD1800500//Research and Application of Integrated Prevention, Control and Purification Technologies for Major Swine Epidemic Diseases/ ; 2021ZDZX0010-3//Sichuan Province Major Science and Technology Project for Sichuan Pigs during the 14th Five-Year Plan Period/ ; sccxtd-2024-08//Sichuan Pig Innovation Team of the National Modern Agricultural Industry Technology System/ ; },
mesh = {Animals ; Rapid Diagnostic Tests ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; Humans ; RNA, Viral/genetics ; Reverse Transcription ; *CRISPR-Cas Systems ; Temperature ; },
abstract = {The emerging zoonotic Getah virus (GETV) poses an increasing threat to both animal and human health, underscoring the need for rapid, sensitive and field-deployable diagnostic tools. In this study, we developed and optimized a rapid, one-step, visual detection (ROSVD) platform for GETV by integrating reverse transcription recombinase-aided amplification (RT-RAA) with CRISPR-EsCas13d-mediated collateral RNA cleavage. Notably, the ROSVD assay uses a simplified sample-preparation strategy based on rapid nucleic acid release, eliminating the need for conventional nucleic acid extraction and purification. The entire workflow, including amplification and detection, is completed within 30 min at 37°C or ambient temperature (25°C) without specialized instrumentation. Detection results can be visualized directly under ultraviolet light or with a lateral flow assay. At 37°C, the ROSVD assay achieved sensitivity comparable to RT-qPCR, and evaluation of clinical specimens showed 100% concordance with RT-qPCR results. Collectively, these findings demonstrate that ROSVD is a rapid, sensitive, cost-effective and instrument-independent diagnostic platform, providing a practical solution for on-site surveillance of GETV and a versatile framework for the detection of other emerging RNA pathogens.},
}

Animals
Rapid Diagnostic Tests
Sensitivity and Specificity
*Nucleic Acid Amplification Techniques/methods
Humans
RNA, Viral/genetics
Reverse Transcription
*CRISPR-Cas Systems
Temperature

@article {pmid42573069,
year = {2026},
author = {Fan, X and Liang, L and Wang, H and Liu, G and Tan, H and Zhang, F and Liu, L and Liu, R},
title = {A self-iterative orthogonal base-editing platform enables multiplex N-to-N diversification and genome-scale functional screening in Escherichia coli.},
journal = {Nucleic acids research},
volume = {54},
number = {15},
pages = {},
pmid = {42573069},
issn = {1362-4962},
support = {2023YFC3402300//National Key Research and Development Program of China/ ; 22278058//National Natural Science Foundation of China/ ; 22578048//National Natural Science Foundation of China/ ; 22208044//National Natural Science Foundation of China/ ; SRICSPYF-ZY2025107//Scientific Research Innovation Capability Support Project for Young Faculty/ ; XLYC2203075//Xingliao Talent Plan/ ; 2024-MSBA-09//Natural Science Foundation of Liaoning Province/ ; 2025JH2/101330156//Natural Science Foundation of Liaoning Province/ ; 2023JJ12SN030//Science and Technology Innovation Foundation of Dalian/ ; DUT24YG131//Fundamental Research Funds for the Central Universities/ ; DUT25LAB105//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Escherichia coli/genetics/metabolism ; *Gene Editing/methods ; *Genome, Bacterial ; Butanols/metabolism ; Escherichia coli Proteins/genetics/metabolism ; DNA Repair/genetics ; Codon/genetics ; Gene Library ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Base editing enables precise genome modification without double-strand breaks but remains limited by narrow editing windows, DNA repair pathway biases, and restricted nucleotide diversity. Here, we report MUTATOR, a MUlTiplexAble and self-iTerative ORthogonal base-editing platform that enables N-to-N diversification in Escherichia coli. MUTATOR combines CWBE and ABE with iterative editing on two complementary DNA strands, thereby overcoming endogenous DNA repair constraints and expanding A-to-N and C-to-N editing outcomes across both strands. This strategy substantially expands accessible nucleotide outcomes, codon variants, and amino-acid diversity within existing editing windows relative to conventional editors. Using four gRNAs, MUTATOR facilitated four-site editing of ompR, generating 84 distinct amino-acid combinations and 252 codon combinations, with the synonymous OmpR_P160P variant increasing isobutanol production by up to 56.2%. We further applied MUTATOR to a 151-gene library encompassing transcriptional regulators, translation factors, DNA repair proteins, ribosomal components, and NAD(P)H-associated metabolic genes, identifying single and combinatorial mutations that markedly enhanced cell growth and ethanol utilization when ethanol was used as the sole carbon source. Together, these results establish MUTATOR as a broadly applicable platform for genome-wide diversification, functional dissection, and rapid engineering of industrial microbial chassis.},
}

*Escherichia coli/genetics/metabolism
*Gene Editing/methods
*Genome, Bacterial
Butanols/metabolism
Escherichia coli Proteins/genetics/metabolism
DNA Repair/genetics
Codon/genetics
Gene Library
CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid42573087,
year = {2026},
author = {Guy, J and Hein, E and Alexander-Howden, B and von Bock Und Polach, T and Mathieson, T and Kleinstiver, BP and Zoghbi, HY and Bird, A},
title = {Translational reading frame predicts the pathogenicity of C-terminal frameshift deletions in MeCP2.},
journal = {eLife},
volume = {14},
number = {},
pages = {},
pmid = {42573087},
issn = {2050-084X},
support = {10.35802/222507//Wellcome/ ; 13669477_13669480//Rett Syndrome Research Trust/ ; DP2CA281401/NH/NIH HHS/United States ; 13800235_1380239//Simons Initiative for the Developing Brain/ ; },
mesh = {*Methyl-CpG-Binding Protein 2/genetics/metabolism ; Animals ; Humans ; *Frameshift Mutation ; Mice ; *Rett Syndrome/genetics/pathology ; Disease Models, Animal ; *Sequence Deletion ; },
abstract = {Mutations in the MECP2 gene cause the severe neurological disorder Rett syndrome. A cluster of frameshift-causing C-terminal deletions (CTDs) removes ~100 amino acids and accounts for approximately 10% of RTT-causing mutations. Their pathogenicity is unexpected because this C-terminal domain is dispensable in mice. Analysis of pathogenic and benign human MECP2 variants reveals that some individuals with apparently typical CTDs do not develop Rett syndrome, confirming that C-terminal truncations are not intrinsically pathogenic. Using human sequence data and mouse models we show that pathogenicity results from a marked reduction in MeCP2 levels and depends on the presence of a proline proline stop motif (-PPX) generated by a shift to the +2 reading frame. CTDs that shift to the +1 frame avoid this motif and are benign. Replacing the stop codon of the PPX motif with tryptophan restores MeCP2 expression and rescues RTT-like phenotypes in a CTD mouse model. An adenine base editor efficiently introduces this substitution in cultured cells. These findings define a reliable prognostic distinction between benign and pathogenic CTDs and establish a potential editing strategy for correcting disease-causing CTD mutations.},
}

*Methyl-CpG-Binding Protein 2/genetics/metabolism
Animals
Humans
*Frameshift Mutation
Mice
*Rett Syndrome/genetics/pathology
Disease Models, Animal
*Sequence Deletion

@article {pmid42573653,
year = {2026},
author = {Nguyen, BD and Kolluri, SK},
title = {Genome-wide CRISPR screen reveals CGS-15943 induced heme-dependent cell death mediated by aryl hydrocarbon receptor in lung cancer cells.},
journal = {Apoptosis : an international journal on programmed cell death},
volume = {31},
number = {8},
pages = {},
pmid = {42573653},
issn = {1573-675X},
mesh = {Humans ; *Receptors, Aryl Hydrocarbon/genetics/metabolism ; *Heme/metabolism ; *Lung Neoplasms/genetics/drug therapy/metabolism/pathology ; Cell Line, Tumor ; *Apoptosis/drug effects ; Cell Death/drug effects ; CRISPR-Cas Systems ; Gene Expression Regulation, Neoplastic/drug effects ; *Basic Helix-Loop-Helix Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Oxidative Stress/drug effects ; },
abstract = {Induction of programmed cancer cell death by selective aryl hydrocarbon receptor (AHR) ligands represents a promising strategy for developing novel anticancer therapeutics. In this study, we characterized the anticancer activity and underlying mechanism of the selective AHR ligand CGS-15943 in lung cancer cells. CGS-15943 potently inhibited the growth of lung cancer cell lines expressing high levels of AHR, whereas CRISPR-mediated knockout of AHR in H460 and H69AR cells markedly rescued cells from CGS-15943-induced cell death, demonstrating an essential role for AHR. To identify additional mediators of this response, we performed a genome-wide CRISPR knockout screen, which revealed eight enzymes involved in the heme biosynthesis pathway, three heme-containing enzymes, as well as AHR and its transcriptional partner ARNT, as critical determinants of CGS-15943-induced cell death. Transcriptomic analyses further showed that CGS-15943 induced AHR-dependent transcriptional programs enriched for oxidative stress and oxidized phospholipid response pathways. Together, these findings identify key components of the AHR signaling network that regulate a programmed heme-dependent cell death pathway and establish CGS-15943 as a promising lead compound for targeting AHR-positive lung cancers.},
}

Humans
*Receptors, Aryl Hydrocarbon/genetics/metabolism
*Heme/metabolism
*Lung Neoplasms/genetics/drug therapy/metabolism/pathology
Cell Line, Tumor
*Apoptosis/drug effects
Cell Death/drug effects
CRISPR-Cas Systems
Gene Expression Regulation, Neoplastic/drug effects
*Basic Helix-Loop-Helix Proteins/genetics/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Oxidative Stress/drug effects

@article {pmid42573697,
year = {2026},
author = {Ward, JD},
title = {Protein Depletion in Caenorhabditis elegans Using the Auxin-Inducible Degradation System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3069},
number = {},
pages = {169-185},
pmid = {42573697},
issn = {1940-6029},
mesh = {Animals ; *Indoleacetic Acids/pharmacology/metabolism ; *Caenorhabditis elegans/genetics/metabolism ; F-Box Proteins/genetics/metabolism ; Degrons ; *Proteolysis/drug effects ; *Caenorhabditis elegans Proteins/genetics/metabolism ; Arabidopsis Proteins/genetics/metabolism ; Receptors, Cell Surface/genetics/metabolism ; CRISPR-Cas Systems ; Gene Knock-In Techniques/methods ; Arabidopsis/genetics ; },
abstract = {The auxin-inducible degradation (AID) system is a powerful tool in modern molecular genetics that allows for conditional protein depletion. In Caenorhabditis elegans, tools exist to allow for rapid, tissue-specific depletion of target proteins. The system requires tagging a gene of interest with an AID degron and a transgene expressing the Arabidopsis thaliana TIR1 F-box protein, which can form a functional SCF ubiquitin ligase with endogenous Skp and Cullin proteins. Here, I describe how to generate degron knock-ins by CRISPR, cross them to TIR1-expressing strains, and perform depletion experiments. I provide a description of our current methods and highlight alternative approaches.},
}

Animals
*Indoleacetic Acids/pharmacology/metabolism
*Caenorhabditis elegans/genetics/metabolism
F-Box Proteins/genetics/metabolism
Degrons
*Proteolysis/drug effects
*Caenorhabditis elegans Proteins/genetics/metabolism
Arabidopsis Proteins/genetics/metabolism
Receptors, Cell Surface/genetics/metabolism
CRISPR-Cas Systems
Gene Knock-In Techniques/methods
Arabidopsis/genetics

@article {pmid42323652,
year = {2026},
author = {Liu, X and Wang, Y and Wong, JCY and Zhang, X and Wu, C and Chen, P and Yang, Y and Liu, P and Lau, CS and Cook, M and Li, PH},
title = {Modeling Hereditary Angioedema With Personalized Expanded Potential Stem Cell-Derived Hepatocytes: A CRISPR-Validated Platform for Mutation-Specific Mechanisms and Therapeutic Innovation.},
journal = {Allergy},
volume = {81},
number = {8},
pages = {2858-2873},
pmid = {42323652},
issn = {1398-9995},
support = {//the InnoHK initiative of the Innovation and Technology Commission of the Hong Kong Special Administrative Region Government/ ; },
mesh = {Humans ; *Hepatocytes/metabolism/cytology ; *Mutation ; *Angioedemas, Hereditary/genetics/therapy/metabolism ; Cell Differentiation ; *Stem Cells/cytology/metabolism ; *CRISPR-Cas Systems ; Complement C1 Inhibitor Protein/genetics/metabolism ; Models, Biological ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Precision Medicine ; },
abstract = {Hereditary angioedema (HAE) with C1 esterase inhibitor (C1INH) deficiency is caused by pathogenic SERPING1 mutations that disrupt production of the plasma protease inhibitor C1INH. However, the molecular mechanisms and consequences of patient-specific mutations remain poorly understood due to the lack of physiologically relevant human models. Here, we established a personalized, isogenic, stem-cell-derived hepatocyte platform to investigate the underlying mutation-specific mechanisms of HAE. Specifically, peripheral blood mononuclear cell (PBMC)-expanded erythroblasts from four representative HAE-C1INH-Type1 patients containing distinct point, insertion, deletion, or large fragment SERPING1 mutations were reprogrammed into expanded potential stem cells (EPSCs) and further differentiated into hepatocyte-like cells (HLCs). These HLCs exhibited appropriate transcriptional transitions, mature hepatic features, and C1INH secretion comparable to that observed in human plasma. All patient-derived HLCs demonstrated impaired C1INH secretion with mutation-specific differences in both SERPING1 transcription and intracellular accumulation. Moreover, to verify that the mutations directly drive the phenotype, we performed CRISPR/Cas9-mediated genome repair, which restored SERPING1 mRNA expression and C1INH secretion. Conversely, identical patient mutations installed into healthy EPSCs showed the same transcriptional and secretory defects, confirming sufficiency. Collectively, we have established a robust human hepatocyte model that accurately recapitulates key hepatocyte-specific aspects of HAE pathophysiology and provides a scalable foundation for investigation of future precision therapies.},
}

Humans
*Hepatocytes/metabolism/cytology
*Mutation
*Angioedemas, Hereditary/genetics/therapy/metabolism
Cell Differentiation
*Stem Cells/cytology/metabolism
*CRISPR-Cas Systems
Complement C1 Inhibitor Protein/genetics/metabolism
Models, Biological
*Clustered Regularly Interspaced Short Palindromic Repeats
Precision Medicine

@article {pmid42389905,
year = {2026},
author = {Minaiyan, G and Aussel, C and Ammann, S and Cathomen, T},
title = {Genome Editing for Familial Hemophagocytic Lymphohistiocytosis: Design Principles, Challenges, and Translational Perspectives.},
journal = {Human gene therapy},
volume = {37},
number = {15-16},
pages = {702-712},
doi = {10.1177/10430342261465555},
pmid = {42389905},
issn = {1557-7422},
mesh = {Humans ; *Lymphohistiocytosis, Hemophagocytic/genetics/therapy ; *Gene Editing/methods ; *Genetic Therapy/methods ; Animals ; Translational Research, Biomedical ; Perforin/genetics ; Membrane Proteins/genetics ; Mutation ; CRISPR-Cas Systems ; Hematopoietic Stem Cell Transplantation ; },
abstract = {Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome caused by genetic defects in cytotoxic lymphocyte function. Current therapies can control disease activity, but transplantation of allogeneic hematopoietic stem and progenitor cells (HSPCs) remains the only curative option and is associated with substantial risks. These limitations have accelerated development of genome editing approaches enabling precise correction of disease-causing mutations in autologous cells. Familial HLH (FHL) represents a compelling target for genome editing, but successful and safe clinical translation has remained challenging. Preclinical studies demonstrate that targeted editing of key genes, such as PRF1 and UNC13D, can restore cytotoxic function in HSPCs and T cells. Translation to the clinic, however, depends on multiple factors, including the choice of target cell population, the level of functional correction required, and gene-specific constraints such as locus complexity and regulation of gene expression. In this review, we synthesize current progress in genome editing for FHL and highlight critical biological and technical barriers to clinical implementation. We propose a conceptual framework for designing genome editing strategies tailored to FHL, emphasizing the alignment of editing platform, gene architecture, and cellular context to enable effective and clinically translatable therapies.},
}

Humans
*Lymphohistiocytosis, Hemophagocytic/genetics/therapy
*Gene Editing/methods
*Genetic Therapy/methods
Animals
Translational Research, Biomedical
Perforin/genetics
Membrane Proteins/genetics
Mutation
CRISPR-Cas Systems
Hematopoietic Stem Cell Transplantation

@article {pmid42393087,
year = {2026},
author = {Weickert, P and Liu, Y and Strecker, J},
title = {Prokaryotic Schlafen proteins cleave tRNAs during type III CRISPR immunity.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42393087},
issn = {2041-1723},
support = {DP2HL185100//U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; },
mesh = {*RNA, Transfer/metabolism/genetics ; *CRISPR-Cas Systems ; *Bacterial Proteins/metabolism/genetics/chemistry ; Cryoelectron Microscopy ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Schlafen nucleases restrict viral infection in mammals by cleaving self RNAs, however, their function and mechanism in prokaryotic immunity is unknown. Here, we uncover CRISPR-associated Schlafen (Cash) proteins containing a Schlafen domain fused to Csx15, an uncharacterized member of Rossmann-like nucleotide-binding sensors. Cash is activated by cyclic tetra-adenylate (cA4) produced during type III CRISPR interference and induces cell toxicity by cleaving tRNAs, primarily in the T-loop. Cryo-electron microscopy structures of Chloroflexi bacterium Cash reveal an inactive dodecamer, the formation of a filament upon cA4 binding to align catalytic interfaces, and the molecular basis of substrate recognition and cleavage in a tRNA-bound complex. We identify numerous families of prokaryotic Schlafen proteins associated with diverse antiviral defense systems and characterized by unique sensor domains. This work highlights tRNA depletion by Schlafen nucleases as an evolutionary recurring antiviral strategy and reveals mechanistic differences between Cash and human Schlafen members.},
}

*RNA, Transfer/metabolism/genetics
*CRISPR-Cas Systems
*Bacterial Proteins/metabolism/genetics/chemistry
Cryoelectron Microscopy
*CRISPR-Associated Proteins/metabolism/genetics/chemistry
Humans
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42424999,
year = {2026},
author = {Dong, J and Li, X and Gu, T and Zhang, Y and Deng, L and Luo, X and Hou, C and Huo, D},
title = {Compartmentalization-inspired dual-chamber CRISPR sensing coupled with single-atom electrocatalysis for crosstalk-free multiplex microRNA detection.},
journal = {Biosensors & bioelectronics},
volume = {312},
number = {},
pages = {119016},
doi = {10.1016/j.bios.2026.119016},
pmid = {42424999},
issn = {1873-4235},
mesh = {*MicroRNAs/genetics/isolation & purification/analysis/blood ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; Electrochemical Techniques ; Nucleic Acid Amplification Techniques ; Catalysis ; Equipment Design ; },
abstract = {Compartmentalization is a hallmark of cells, enabling parallel biochemical processes to proceed with high fidelity and minimal interference. Drawing inspiration from this spatial isolation principle, we developed a compartmentalization-inspired dual-chamber sensing platform for crosstalk-minimized multiplex miRNA analysis. In each physically isolated compartment, the target miRNA directs padlock-probe ligation to form a circular template, followed by rolling-circle-extension-driven loop-mediated isothermal amplification (R-LAMP). The resulting amplicons specifically activate the corresponding CRISPR/Cas12a-crRNA complex, triggering trans-cleavage of a hairpin-DNA biogate that seals Fe-MOF nanocontainers. Gate opening releases distinct electroactive reporters (3,3',5,5'-tetramethylbenzidine, TMB; or methylene blue, MB) from their respective chambers. After the compartmentalized reactions finish, the two supernatants are combined and read out on a screen-printed electrode modified with a Co-N-C single-atom catalyst, producing two well-resolved DPV peaks for simultaneous quantification. The platform achieves femtomolar detection limits in simultaneous assays (0.87 fM for miRNA-21 and 0.72 fM for miRNA-155), a broad linear range (1 fM-100 pM), and high discrimination against non-cognate or mismatched sequences. Accurate recoveries in diluted human serum and consistent trends in cell lysates (validated by RT-qPCR) confirm practical applicability. By integrating bioinspired compartmentalization with CRISPR precision and single-atom electrocatalysis, this platform provides a generalizable route to multiplex nucleic acid diagnostics with enhanced fidelity and sensitivity.},
}

*MicroRNAs/genetics/isolation & purification/analysis/blood
*Biosensing Techniques/methods
Humans
*CRISPR-Cas Systems/genetics
Limit of Detection
Electrochemical Techniques
Nucleic Acid Amplification Techniques
Catalysis
Equipment Design

@article {pmid42431049,
year = {2026},
author = {Ji, S and Wang, B and Yan, Y and Zhang, C and Ren, H and Yang, J and Wei, X and Hou, W and Huang, M and Song, J and Wang, S and Qiu, L and Wang, H},
title = {TOPS-CRISPR: Thermally-regulated and oligonucleotide-mediated one-pot CRISPR-Cas12a assay for ultra-sensitive and rapid on-site diagnostics.},
journal = {Biosensors & bioelectronics},
volume = {312},
number = {},
pages = {118998},
doi = {10.1016/j.bios.2026.118998},
pmid = {42431049},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Biosensing Techniques/methods ; Oligonucleotides/chemistry/genetics ; *Brucella/isolation & purification/genetics/pathogenicity ; *Brucellosis/diagnosis/microbiology ; Rapid Diagnostic Tests ; Temperature ; CRISPR-Associated Proteins/genetics ; Limit of Detection ; },
abstract = {CRISPR-Cas12a has emerged as a powerful tool in molecular diagnostics, owing to its robust signal amplification and compact crRNA design. However, its uncontrolled enzymatic activity often hampers application in streamlined one-pot assays. Although existing temporal or spatial regulation strategies can mitigate this issue, they typically introduce operational complexity or increased cost. Here, we designed a Thermally regulated, Oligonucleotide-mediated one-Pot System for CRISPR-Cas12a (TOPS-CRISPR), which employs a programmable inhibition strategy based on complementary RNA blockers with tunable length and binding sites, enabling efficient and reversible steric inhibition of the LbCas12a-crRNA ribonucleoprotein (RNP) complex, resolving the inherent contradiction between amplification and cleavage in one-pot assay. TOPS-CRISPR not only is operational simple and cost-effective but also achieves over 60-fold higher sensitivity than conventional one-pot platforms. We demonstrated the clinical applicability of TOPS-CRISPR by accurately detecting Brucella and Streptococcus in both spiked and clinical samples. Moreover, the system integrates seamlessly with rapid sample processing, lyophilized reagents, and miniaturized workflows, enabling field-deployable pathogen identification within 50 min.},
}

*CRISPR-Cas Systems/genetics
Humans
*Biosensing Techniques/methods
Oligonucleotides/chemistry/genetics
*Brucella/isolation & purification/genetics/pathogenicity
*Brucellosis/diagnosis/microbiology
Rapid Diagnostic Tests
Temperature
CRISPR-Associated Proteins/genetics
Limit of Detection

@article {pmid42447864,
year = {2026},
author = {Langley, J and Baudrier, L and Curry, J and Narta, K and Todesco, HM and Potts, K and Morrissy, S and Mahoney, DJ and Billon, P},
title = {Compound delivery of eVLPs enhances prime editing for targeted genome engineering and high-throughput screening.},
journal = {Cell genomics},
volume = {6},
number = {8},
pages = {101302},
doi = {10.1016/j.xgen.2026.101302},
pmid = {42447864},
issn = {2666-979X},
mesh = {Humans ; *Gene Editing/methods ; *High-Throughput Screening Assays/methods ; HEK293 Cells ; CRISPR-Cas Systems/genetics ; Lentivirus/genetics ; *Virion/genetics ; Tumor Suppressor Protein p53/genetics ; },
abstract = {Engineered virus-like particles (eVLPs) enable transgene-free ribonucleoprotein delivery for genome editing, yet optimized strategies for high-throughput applications remain unexplored. Prime editing enables precise genomic modifications but suffers from limited efficiency. Here, we present PRIME-VLP (Progressive Repeated Infections for Maximized Editing via Virus-Like Particles), which exploits eVLP-mediated editing kinetics through multiple sequential sub-saturating transductions at optimal intervals. PRIME-VLP achieves 1.5- to 2.9-fold improvements in prime editing efficiency across diverse genomic targets and cell types without increasing off-target editing, compromising cellular viability or causing transcriptional perturbations. By decoupling pegRNA and editor delivery through pegRNA-free eVLPs, PRIME-VLP enables pooled prime editing screens and circumvents transgene silencing limitations. Using a 6,000-pegRNA library targeting TP53, PRIME-VLP achieved 2.8-fold higher editing and improved reproducibility compared to conventional lentiviral delivery, identifying TP53 loss-of-function variants conferring Nutlin-3 resistance. This work expands the versatility of eVLPs beyond their current in vivo therapeutic applications, demonstrating their promise for high-throughput functional genomics.},
}

Humans
*Gene Editing/methods
*High-Throughput Screening Assays/methods
HEK293 Cells
CRISPR-Cas Systems/genetics
Lentivirus/genetics
*Virion/genetics
Tumor Suppressor Protein p53/genetics

@article {pmid42470832,
year = {2026},
author = {Azhar, M and Malviya, R and Chandra, P and Sridhar, SB and Shareef, J and Wadhwa, T},
title = {Precision prime editing of TP53 mutations for functional tumor suppression in colorectal cancer.},
journal = {Biochemical and biophysical research communications},
volume = {831},
number = {},
pages = {154311},
doi = {10.1016/j.bbrc.2026.154311},
pmid = {42470832},
issn = {1090-2104},
mesh = {*Colorectal Neoplasms/genetics/therapy ; Humans ; *Mutation ; *Tumor Suppressor Protein p53/genetics ; CRISPR-Cas Systems ; Animals ; Genetic Therapy/methods ; Precision Medicine/methods ; *Gene Editing/methods ; },
abstract = {BACKGROUND: Colorectal cancer (CRC) is a major global health concern, with high mortality due to genetic heterogeneity and resistance to treatment. Tumor Protein p53 (TP53) mutations are also among the most important molecular changes that can disrupt genomic stability and facilitate tumor progression, so it is a critical target for precision-based interventions.

AIM: This review aims to discuss the future potential of prime editing as a new generation of genome engineering to identify precise approaches to correct TP53 mutations in colorectal cancer.

METHOD: A focused literature review was conducted on PubMed, Scopus, Web of Science, and Google Scholar for articles published between the years of 2010 and 2026. The keywords used in the search were CRC, TP53 mutation, prime editing, Prime Editing Guide RNA (pegRNA), CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9), and precision oncology. Studies were screened for experimental, mechanistic, and translational, and the focus was on mutation-specific editing, delivery platforms, organoid validation, clinically relevant barriers, etc. RESULTS/DISCUSSION: Prime editing is a programmable search-and-replace method that does not involve two single-stranded Deoxyribonucleic Acid (DNA) breaks, resulting in fewer Insertions/deletions (indels) and greater precision compared with traditional CRISPR-Cas9 approaches. Recent systems like Prime Editor Max (PEmax), PE5/PE5max, engineered pegRNAs, twin prime editors, PrimeDel, and PASTE have enhanced the efficiency, range, and flexibility. Hotspot and organoid studies suggest that variants of TP53, particularly R175H, R248Q/W, R273 H/C, and R282W, can be repaired. But cargo size, delivery specificity, tumor heterogeneity, varying cargo editing efficiency, cargo recognition by the immune system, and off-target risk are all barriers to clinical translation.

CONCLUSION: Precision oncology with prime editing has the potential to be a useful tool for CRC, though optimized delivery, thorough preclinical testing, and safety monitoring will be required for therapeutic adoption.

ORIGINALITY: This review combines TP53 hotspot biology, recent breakthroughs in prime editing technology, and CRC-specific translational challenges, and provides a step-by-step approach to its clinical application in a unique way.},
}

*Colorectal Neoplasms/genetics/therapy
Humans
*Mutation
*Tumor Suppressor Protein p53/genetics
CRISPR-Cas Systems
Animals
Genetic Therapy/methods
Precision Medicine/methods
*Gene Editing/methods

@article {pmid42472526,
year = {2026},
author = {Liu, Z and Wang, J and Yang, Z and Huang, H and Zhang, Z and Wu, T},
title = {High-specificity gene point mutation detection by PAM-free Cas12a system with double-stranded substrate positioning-unwinding.},
journal = {Biosensors & bioelectronics},
volume = {312},
number = {},
pages = {119043},
doi = {10.1016/j.bios.2026.119043},
pmid = {42472526},
issn = {1873-4235},
mesh = {*Point Mutation/genetics ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *DNA/genetics/chemistry ; *CRISPR-Associated Proteins/genetics ; DNA Mutational Analysis/methods ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {The CRISPR/Cas12a system holds great promise for nucleic acid detection, but its strict dependence on the protospacer adjacent motif (PAM) severely limits its application in gene point mutation analysis, with fewer than 2% of known mutation sites naturally harboring adjacent PAM sequences. Herein, we developed a PAM-free Cas12a system with double-stranded substrate positioning-unwinding (dsPU-Cas12a), wherein "bubble" structures formed by unpaired base pairs release partial single-stranded target strand as a toehold, and excess auxiliary strands induce local unwinding of double-stranded DNA to facilitate R-loop formation. After optimization, the dsPU-Cas12a system achieved an ultra-low limit of detection of 0.013% for gene point mutations, with excellent linearity over the mutation abundance range of 0-10%. Furthermore, it exhibited robust feasibility and accuracy in detecting the JAK2 V617F mutation in blood samples from patients with myeloproliferative neoplasms. This simple and universal strategy overcomes the sequence limitation of Cas12a, providing a high-performance tool for clinical gene point mutation detection.},
}

*Point Mutation/genetics
*Biosensing Techniques/methods
Humans
*CRISPR-Cas Systems/genetics
*DNA/genetics/chemistry
*CRISPR-Associated Proteins/genetics
DNA Mutational Analysis/methods
Limit of Detection
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid42474386,
year = {2026},
author = {Liao, J and Su, Y and Jiang, F},
title = {CRISPR-Cas12a assay for rapid and specific detection of Shigella flexneri 2a in clinical samples.},
journal = {Journal of clinical microbiology},
volume = {64},
number = {8},
pages = {e0161525},
pmid = {42474386},
issn = {1098-660X},
mesh = {Humans ; Sensitivity and Specificity ; *Shigella flexneri/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Dysentery, Bacillary/diagnosis/microbiology ; Feces/microbiology ; Rapid Diagnostic Tests ; *Molecular Diagnostic Techniques/methods ; Bacterial Proteins/genetics ; CRISPR-Associated Proteins/genetics ; Endodeoxyribonucleases ; },
abstract = {Shigella flexneri 2a is the most common cause of shigellosis, a major public health concern in developing countries. Rapid and reliable diagnostic tools are critical for timely outbreak detection and management. Leveraging clustered regularly interspaced short palindromic repeats (CRISPR) technology, we developed a CRISPR-Cas12a-based assay for the rapid and specific detection of S. flexneri 2a and validated its performance using stool specimens from patients. Two guide RNAs targeting the gtrII and gtrX genes, unique markers of the S. flexneri 2a serotype, were designed to ensure specificity. Recombinase polymerase amplification (RPA) was coupled with Cas12a-mediated collateral cleavage for signal amplification, with detection by fluorescence or lateral flow. Analytical sensitivity, specificity, and clinical accuracy were compared with conventional PCR using purified DNA and 588 clinical stool specimens. The CRISPR-Cas12a assay achieved a detection limit of 10 copies/µL, comparable to PCR, and showed 100% analytical specificity without cross-reactivity to other bacteria. The isothermal reaction operated at room temperature and was completed within 1 h. Both readouts allowed visual interpretation without specialized equipment. Clinical validation of the CRISPR-Cas12a assay demonstrated a diagnostic sensitivity of 95% and specificity of 98%, comparable to PCR when evaluated using the same clinical specimens. This study provides two key advances: it establishes a CRISPR-Cas12a assay specifically targeting S. flexneri 2a, the predominant serotype, and validates it using a large clinical cohort. The assay's simplicity, speed, and high diagnostic accuracy make it a valuable tool for clinical diagnostics and field-based surveillance in resource-limited settings.IMPORTANCERapid and accessible diagnostics are essential for effective management of infectious diseases such as shigellosis. We developed a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a-based assay that specifically detects Shigella flexneri 2a, the predominant serotype responsible for the global disease burden. This assay integrates isothermal amplification with CRISPR-mediated detection to achieve low-copy detection (10 copies/µL) within 1 h, eliminating the need for complex instrumentation. Dual fluorescence and lateral-flow readouts enable flexible use in both clinical laboratories and low-resource settings. The method's simplicity, accuracy, and adaptability demonstrate the practical potential of CRISPR diagnostics for point-of-care applications. By enabling rapid, on-site identification of S. flexneri 2a, this approach can significantly improve clinical diagnosis and strengthen public health responses to enteric pathogen outbreaks.},
}

Humans
Sensitivity and Specificity
*Shigella flexneri/genetics/isolation & purification
*CRISPR-Cas Systems
*Dysentery, Bacillary/diagnosis/microbiology
Feces/microbiology
Rapid Diagnostic Tests
*Molecular Diagnostic Techniques/methods
Bacterial Proteins/genetics
CRISPR-Associated Proteins/genetics
Endodeoxyribonucleases

@article {pmid42485703,
year = {2026},
author = {Tan, Y and He, X and Shao, E and Chen, Q and Zhang, L and Deng, S},
title = {A one-tube autocatalytic transcription-driven CRISPR cascade for ultrasensitive mRNA detection.},
journal = {Biosensors & bioelectronics},
volume = {312},
number = {},
pages = {119049},
doi = {10.1016/j.bios.2026.119049},
pmid = {42485703},
issn = {1873-4235},
mesh = {*RNA, Messenger/genetics/analysis/isolation & purification ; Humans ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Transcription, Genetic ; *Biomarkers, Tumor/genetics/isolation & purification ; *Breast Neoplasms/genetics/diagnosis ; Nucleic Acid Amplification Techniques ; Limit of Detection ; },
abstract = {The rapid identification and precise quantification of cancer biomarkers are essential for the purposes of diagnosis, classification, and therapeutic intervention. Traditional molecular diagnostic methodologies, such as polymerase chain reaction (PCR), provide considerable sensitivity; however, they depend on exponential amplification and advanced instrumentation, thereby constraining their applicability for point-of-care testing. In this study, we developed a Transcription-driven CRISPR Cascade Amplification (TCCA) for one-tube detection of mRNA at the concentration of 0.6 copies/μL within just 30 min. The target-induced assembly of a three-way junction (TWJ) facilitates the generation of a split T7 promoter, which initiates transcription and activates Cas13a collateral cleavage. The activated Cas13a subsequently cleaves cascade probes and releases new trigger strands, forming a transcription-driven cascade amplification circuit. The assay enables multiplex detection of breast cancer-associated mRNA biomarkers (HBB, KRT17, and CD55) in cell lysates, demonstrating robust performance in intricate biological matrices. Furthermore, incorporation of a multiplex OR-gated logic design enables parallel target recognition, thereby enhancing detection reliability for rapid and accurate clinical diagnostics.},
}

*RNA, Messenger/genetics/analysis/isolation & purification
Humans
*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
Transcription, Genetic
*Biomarkers, Tumor/genetics/isolation & purification
*Breast Neoplasms/genetics/diagnosis
Nucleic Acid Amplification Techniques
Limit of Detection

@article {pmid42504876,
year = {2026},
author = {Wang, P and Sayed, S and Buchholz, F},
title = {Restoring Cancer Genomes: Functional Mutation Correction as a Platform for Precision Oncology.},
journal = {Human gene therapy},
volume = {37},
number = {15-16},
pages = {663-669},
doi = {10.1177/10430342261468973},
pmid = {42504876},
issn = {1557-7422},
mesh = {Humans ; *Neoplasms/genetics/therapy/diagnosis ; *Mutation ; *Precision Medicine/methods ; *Gene Editing/methods ; CRISPR-Cas Systems ; *Genetic Therapy/methods ; *Genome, Human ; Animals ; },
abstract = {Cancer genome sequencing has uncovered an extensive landscape of somatic mutations. However, determining which of these alterations are biologically consequential and therapeutically actionable remains a central challenge in oncology. CRISPR-based genome editing now enables precise correction of oncogenic mutations within their endogenous genomic context. Recent work demonstrates that repairing cancer hotspot mutations restores conserved tumor-relevant transcriptional programs across diverse tumor types, revealing tumor-agnostic dependencies. Beyond therapeutic implications, this mutation-correction framework provides a scalable functional platform to stratify drivers, interrogate variants of uncertain significance, and refine precision diagnostics. This perspective discusses how programmable mutation correction advances (i) mechanistic cancer biology, (ii) personalized cancer diagnostics, and (iii) next-generation precision gene therapies. We propose that systematic reversal of cancer mutations represents a conceptual shift from observing mutational landscapes to actively testing their biological necessity.},
}

Humans
*Neoplasms/genetics/therapy/diagnosis
*Mutation
*Precision Medicine/methods
*Gene Editing/methods
CRISPR-Cas Systems
*Genetic Therapy/methods
*Genome, Human
Animals

@article {pmid42508243,
year = {2026},
author = {Wei, K and Heng, H and Wang, T and Liu, Z and Wang, K},
title = {Integration of dual-output TMSD and CRISPR/Cas12a for crosstalk-free electrochemiluminescence detection of aflD gene and AFB1.},
journal = {Biosensors & bioelectronics},
volume = {312},
number = {},
pages = {119062},
doi = {10.1016/j.bios.2026.119062},
pmid = {42508243},
issn = {1873-4235},
mesh = {*Aflatoxin B1/analysis/genetics/isolation & purification ; *Biosensing Techniques/methods ; Luminescent Measurements/methods ; Electrochemical Techniques/methods ; CRISPR-Cas Systems/genetics ; Aptamers, Nucleotide/chemistry ; Humans ; Limit of Detection ; Food Contamination/analysis ; Metallocenes/chemistry ; Ferrous Compounds/chemistry ; },
abstract = {Aflatoxins are among the most toxic mycotoxins and pose a severe threat to food safety and human health. In addition to the direct monitoring of aflatoxin B1 (AFB1), simultaneous detection of its key biosynthesis gene, aflD, can effectively indicate the presence of toxin-producing strains, thereby enhancing the early screening and traceability of AFB1 contamination. Due to the significant functional differences among various biomarkers, performing multi-target analysis on a single detection interface remains challenging. Herein, we constructed a novel dual-target electrochemiluminescence (ECL) biosensor for the sequential and quantitative detection of the aflD gene and AFB1. This sensor innovatively integrates a dual-output toehold-mediated strand displacement (TMSD) and CRISPR/Cas12a trans-cleavage mechanisms to establish a dynamic "signal writing-erasing" regulation on a single ECL emitter. Specifically, aflD triggers the TMSD reaction, driving the enrichment of ferrocene (Fc)-labeled DNA at the electrode interface and quenching the ECL signal, corresponding to signal "write" (signal-off). Subsequently, AFB1 is converted via aptamer recognition into an activator DNA that initiates Cas12a trans-cleavage, leading to the removal of Fc-DNA from the interface and recovery of ECL emission, corresponding to signal "erase" (signal-on). This strategy enables cross-category detection and quantitative analysis of small-molecule toxins and nucleic acid biomarkers within a single luminescence system, effectively avoiding signal crosstalk while offering high sensitivity, high specificity, and high interfacial utilization efficiency. It provides a versatile new approach for the early warning and source tracing of contaminants in complex food matrices.},
}

*Aflatoxin B1/analysis/genetics/isolation & purification
*Biosensing Techniques/methods
Luminescent Measurements/methods
Electrochemical Techniques/methods
CRISPR-Cas Systems/genetics
Aptamers, Nucleotide/chemistry
Humans
Limit of Detection
Food Contamination/analysis
Metallocenes/chemistry
Ferrous Compounds/chemistry

@article {pmid42520408,
year = {2026},
author = {Kumar, J and Alok, A and Fox, J and Srivastava, A and Voytas, DF and Zhang, F and Kianian, SF},
title = {A novel genome editing strategy in plants using broad-host-range viral vectors derived from geminiviruses.},
journal = {Plant physiology},
volume = {201},
number = {4},
pages = {},
doi = {10.1093/plphys/kiag553},
pmid = {42520408},
issn = {1532-2548},
support = {//U.S. Department of Agriculture-Agricultural Research Service (USDA-ARS) National Plant Disease Recovery System (NPDRS)/ ; },
mesh = {*Geminiviridae/genetics ; *Genetic Vectors/genetics ; *Genome, Plant/genetics ; *Host Specificity/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; Plants, Genetically Modified ; },
abstract = {The use of viral vectors offers a promising alternative to traditional transformation methods for creating gene-edited plants. In this study, we developed a novel plant genome editing system by delivering Cas9, Cas12f, and Cas12j nucleases along with their guide RNAs using a broad-host-range geminivirus, Wheat dwarf India virus (WDIV), in combination with Ageratum yellow leaf curl betasatellite (AYLCB). Cas9, Cas12f, and Cas12j nucleases were efficiently expressed along with corresponding guide RNAs under viral promoters. By leveraging tRNA spacers in place of external promoters and terminators, we significantly reduced the overall cargo size, streamlining vector design. Additionally, we compared the traditional AtU6-driven gRNA delivery with a novel spacer:gRNA:spacer format in Cas9-expressing lines and observed comparable editing efficiencies. The broad host range of WDIV and AYLCB, combined with the novel genome-editing platform, opens possibilities for editing across a wide range of plant species.},
}

*Geminiviridae/genetics
*Genetic Vectors/genetics
*Genome, Plant/genetics
*Host Specificity/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Cas Systems
Plants, Genetically Modified

@article {pmid42526210,
year = {2026},
author = {Zhao, J and He, J and Du, H and Dai, C and Ma, X and Kong, D and Yue, Y and Zhang, S and Liu, K and Liu, Y and Zhang, G and Wu, Y and Wei, D},
title = {Photo-switchable CRISPR electrochemical system enables de-interferential biosensing signal output.},
journal = {Biosensors & bioelectronics},
volume = {312},
number = {},
pages = {119067},
doi = {10.1016/j.bios.2026.119067},
pmid = {42526210},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Electrochemical Techniques/methods ; *Carcinoma, Non-Small-Cell Lung/genetics/diagnosis ; *Lung Neoplasms/genetics/diagnosis ; Mutation ; Limit of Detection ; Equipment Design ; Nucleic Acid Amplification Techniques ; Light ; },
abstract = {Most clustered regularly interspaced short palindromic repeats (CRISPR)-based electrochemical biosensors lack controllable switching function and are vulnerable to false signals induced by intrinsic amplification signal crosstalk. Thus, exploring strategies that mitigate intrinsic amplification crosstalk with a precise switch would be extremely useful for reliable and accurate bioanalysis. Herein, we develop a photo-switchable CRISPR/Cas12a electrochemical (PSCE) system by introducing a photocleavable (PC) linker and adopting a light-responsive strategy, which enables output of de-interference electrochemical signals and achieves highly sensitive and accurate detection of gene mutations for non-small cell lung cancer (NSCLC). The system decouples nucleic acid amplification (NAA) from signal transduction and produces unique photo-switchable response signals by light irradiation to activate Cas12a activity. This architectural design further separates signal readout from sample pretreatment, suppressing intrinsic amplification-derived signal crosstalk rather than global biological interference to deliver an ultralow limit of detection (LoD). The PSCE achieves 98.1% sensitivity, 92.7% specificity, and 98.9% overall accuracy when tested with 67 clinical samples. Moreover, the exploration of PSCE system in flexible wearable electronics and machine-learning analysis of clinical patient samples demonstrates significant application potential clinical diagnosis of mutation-associated diseases.},
}

*Biosensing Techniques/methods
Humans
*CRISPR-Cas Systems/genetics
*Electrochemical Techniques/methods
*Carcinoma, Non-Small-Cell Lung/genetics/diagnosis
*Lung Neoplasms/genetics/diagnosis
Mutation
Limit of Detection
Equipment Design
Nucleic Acid Amplification Techniques
Light

@article {pmid42546382,
year = {2026},
author = {Qin, L and Tang, Z and Yu, Y and Liu, T and Zhang, M and Zhu, X and Hu, Q and Zhao, Z and Zhang, F and Tang, BZ and Cen, Y},
title = {DIRECTOR: DNA-guided spatially ordered assembly of split crRNA for activating CRISPR/Cas12a.},
journal = {Biosensors & bioelectronics},
volume = {312},
number = {},
pages = {119075},
doi = {10.1016/j.bios.2026.119075},
pmid = {42546382},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; *DNA/chemistry/genetics ; *CRISPR-Associated Proteins/chemistry/genetics/metabolism ; *Biosensing Techniques/methods ; *Endodeoxyribonucleases/chemistry/genetics ; Molecular Dynamics Simulation ; *MicroRNAs/genetics/analysis ; *Bacterial Proteins/chemistry/genetics ; },
abstract = {Precise regulation of Cas12a activity is crucial for expanding its application in molecular diagnostics. However, existing split crRNA systems exhibit hardly any activation efficiency at low-abundance target and lack a well-defined regulated mechanism, representing a persistent bottleneck for practical application. This work proposes a DNA-guided spatially ordered assembly of split crRNA for activating CRISPR/Cas12a (DIRECTOR) strategy. This work combines artificial intelligence-driven AlphaFold3 structure prediction, computer-powered molecular dynamics simulations with fluorescence analysis to demonstrate that the 3' terminal extension of activator acts as a spatial director, utilizing DNA-guided spatially ordered assembly of split crRNA and stabilizing key Cas12a domains, thereby activating Cas12a. Conversely, the 5' terminal extension serves as a spatial misdirector, inhibiting Cas12a activation by destabilizing the protein structure and introducing the steric hindrance to shield the catalytic center. Furthermore, the structural and energy thresholds required for effective Cas12a activation were identified. Finally, utilizing the spatial director as an energy amplification element, DIRECTOR achieves a limit of detection as low as 42.1 fM for single-target miR-155 and dual-response detection of wide-scope nucleic acids. Owing to its direct activation strategy, DIRECTOR provides mechanistic insights for affordable and programmable CRISPR molecular diagnostics.},
}

*CRISPR-Cas Systems/genetics
*DNA/chemistry/genetics
*CRISPR-Associated Proteins/chemistry/genetics/metabolism
*Biosensing Techniques/methods
*Endodeoxyribonucleases/chemistry/genetics
Molecular Dynamics Simulation
*MicroRNAs/genetics/analysis
*Bacterial Proteins/chemistry/genetics

@article {pmid42546384,
year = {2026},
author = {Li, L and Wei, H and Wei, M and Yang, X and Wang, T and Min, S and Wang, Y and Wang, X and Li, M and Wang, S and Rong, Z},
title = {An integrated centrifugal microfluidic CRISPR-based diagnostics platform for multiplexed point-of-care testing of respiratory pathogens.},
journal = {Biosensors & bioelectronics},
volume = {312},
number = {},
pages = {119083},
doi = {10.1016/j.bios.2026.119083},
pmid = {42546384},
issn = {1873-4235},
mesh = {Humans ; *Biosensing Techniques/instrumentation ; *Point-of-Care Testing ; Equipment Design ; *CRISPR-Cas Systems ; *Respiratory Tract Infections/diagnosis/microbiology/virology ; Lab-On-A-Chip Devices ; Centrifugation/instrumentation ; *Microfluidic Analytical Techniques/instrumentation ; Limit of Detection ; Rapid Diagnostic Tests ; Point-of-Care Systems ; },
abstract = {Rapid, accurate, and multiplexed point-of-care testing (POCT) of respiratory pathogens is critical for clinical triage and infection control. However, existing platforms frequently necessitate trade-offs between sensitivity, throughput, and operational complexity. To address this, we developed the iCARD (integrated Centrifugal Assay on a Rotating Disc) platform, a streamlined microfluidic molecular diagnostic system based on single-step CRISPR kinetics and centrifugal microfluidics. Comprising a polymethyl methacrylate disc pre-loaded with lyophilized CRISPR/Cas13a reagents and a self-developed portable fluorescence analyzer, the platform enables automated multiplexed detection following sample loading. Furthermore, a novel resin-based pretreatment method was engineered to efficiently purify and concentrate nucleic acids from throat swabs. The iCARD system facilitates the parallel screening of six respiratory pathogens across four independent samples within 40 min. Analytical validation confirmed single-copy sensitivity (limit of detection: 0.25-1.0 copies/μL) without cross-reactivity. Clinical validation using 94 retrospective patient throat swabs demonstrated exceptional diagnostic accuracy, achieving a sensitivity of 95.5% (64/67; 95% CI: 87.64%-98.47%) and a specificity of 100.0% (27/27; 95% CI: 87.51%-100.00%). These findings demonstrate that the iCARD platform serves as a robust, high-throughput, and accurate diagnostic tool for decentralized molecular screening and epidemiological surveillance.},
}

Humans
*Biosensing Techniques/instrumentation
*Point-of-Care Testing
Equipment Design
*CRISPR-Cas Systems
*Respiratory Tract Infections/diagnosis/microbiology/virology
Lab-On-A-Chip Devices
Centrifugation/instrumentation
*Microfluidic Analytical Techniques/instrumentation
Limit of Detection
Rapid Diagnostic Tests
Point-of-Care Systems

@article {pmid42557300,
year = {2026},
author = {Du, J and Luo, Z and Xie, D and Chen, Y and Yang, M and Li, Q and Wang, L and Han, L and Zhang, Y and Li, H and Lan, Z and Shi, H and Li, Y and Cheng, Q and Dong, F and Gao, Y and Yao, Y and Cheng, T and Wei, T and Rao, S},
title = {Engineered lipid nanoparticles for in vivo and durable editing of haematopoietic stem cells within humanized mice.},
journal = {Nature biomedical engineering},
volume = {},
number = {},
pages = {},
pmid = {42557300},
issn = {2157-846X},
support = {82370117//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82470240//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82370117//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
abstract = {In vivo genetic engineering of haematopoietic stem and progenitor cells (HSPCs) holds the potential to revolutionize the treatment landscape for numerous diseases. However, despite its transformative potential, it remains hindered by the difficulty in efficiently and specifically targeting quiescent human HSCs while maintaining their long-term functionality. Here, after screening 15 lipid nanoparticles (LNPs), we report an LNP that efficiently delivers reporter mRNA to human HSPCs both in ex vivo and in vivo settings when conjugated with the anti-CD34 antibody (CD34/LNP[DP]). Using CRISPR/Cas editing cargos, CD34/LNP[DP] achieves high editing efficiency in human HSPCs ex vivo. Intrafemoral administration of CD34/LNP[DP] in humanized mice results in efficient editing of the erythroid-specific BCL11A enhancer within human HSPCs, enabling the sustained long-term reactivation of fetal haemoglobin (HbF) expression in erythroid cells. In a humanized neutropaenia model harbouring an ELANE mutation, intrafemoral administration of CD34/LNP[DP] achieves robust editing, targeting exon 2 of ELANE in human HSPCs, partially restoring neutrophil development impairment under long-term observation. Collectively, CD34-targeted delivery enables in vivo HSPC modification without perturbing haematopoiesis, underscoring its suitability for clinical translation.},
}

@article {pmid42558756,
year = {2026},
author = {Sharma, N and Thakur, K and Zinta, R and Shaunak, I and Batta, S and Saini, A and Sehgal, R and Bhagta, S and Singh, B and Thakur, AK},
title = {Transgene-free genome editing in potato, a clonally propagated crop - strategies and future prospects.},
journal = {Physiology and molecular biology of plants : an international journal of functional plant biology},
volume = {32},
number = {8},
pages = {1747-1757},
pmid = {42558756},
issn = {0971-5894},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas)-based genome editing technology has come out as very precise and effective tool for targeted modification in the gene of interest and offers unprecedented potentials in crop improvement. However, in the present regulatory framework for commercialization of genome edited crops, in many countries including India, the edited lines must be transgene-free. In India, only site directed nuclease (SDN) I and SDN II category of genome edited events which are transgene-free are permitted for commercialization. Potato is a vegetatively propagated crop, having autotetraploid genome and is highly heterozygous in nature. Removal of the transgene from potato genome of edited lines through genetic segregation, either by crossing or selfing, is not the appropriate method as the elite background of the genome gets disturbed due to heterozygous nature of the crop. Every individual seed of potato, i.e. true potato seed (TPS) behaves like a different individual than the parental line and is unable to maintain the genetic identity. In this review article, we have discussed several strategies that can be enacted for generation of transgene-free genome edited lines in potato. This article will provide deeper insight and enhance understandings about the optimum use of CRISPR as non-GMO technology in the genetic enhancement of potato and to adopt the best strategies in editing this important tuberous, clonally propagated crop.},
}

@article {pmid42559426,
year = {2026},
author = {Li, A and Cao, C and Yang, C and Liu, Y},
title = {Programmable transcriptional condensates for enhanced CRISPR-based gene regulation.},
journal = {Theranostics},
volume = {16},
number = {14},
pages = {8215-8229},
pmid = {42559426},
issn = {1838-7640},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; HEK293 Cells ; *Gene Expression Regulation ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; HeLa Cells ; Transcription, Genetic ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {RATIONALE: Efficient gene activation or repression through programmable CRISPR-Cas9 has revolutionized molecular biology and drug development. Nonetheless, the currently available CRISPRa/i approaches are modestly potent and require multi-component delivery, which hampers the wide use of the technology in both research and therapy.

METHODS: We developed a modular CRISPR-condensate platform by appending a multivalent RNA nanostar to the 3' end of a single-guide RNA, producing a sgRNA-nanostar chimera that mediates phase separation at Cas9-bound genomic loci. The nanostar scaffold also contains MS2 stem-loops, which recruit MCP-tagged transcriptional effectors (VP64 for activation, KRAB for repression) to the condensate microenvironment at high local concentration. We examined condensate formation, genome targeting, and transcriptional output by using live-cell imaging, RT-qPCR, ChIP-seq, RNA-seq and CUT&Tag in HEK293T, HeLa, U-2 OS, MDA-MB-231, as well as human iPSC cell lines.

RESULTS: The CRISPR-condensate design resulted in up to 50-100-fold target-gene activation, compared with 5-10-fold activation by direct VP64 fusion, and 20-30-fold transcriptional repression, compared with 3-5-fold repression by direct KRAB fusion, with high target specificity (12 versus 28 non-target differentially expressed genes assessed by RNA-seq). Orthogonal kissing-loop (KL) pairings enabled independent condensate systems for simultaneous activation and repression of multiplexed targets. Janus condensates containing both activating and repressive domains enabled bidirectional regulation at a single locus. The system requires delivery of only three independently expressible components-dCas9-NLS, an sgRNA-nanostar chimera bearing MS2 stem-loops (MS2SLs), and an MCP-fused effector (VP64-MCP for activation or KRAB-MCP for repression)-and showed minimal innate immune response and high cell viability.

CONCLUSIONS: The CRISPR-condensate system merges the dramatically enhanced transcriptional efficacy with the reduced complexity of components, providing a modular system for fine-tuned gene expression regulation. This strategy makes biomolecular condensation a general principle for enhancing CRISPR gene regulation, opening up possibilities for functional genomics, cell engineering, and therapy development.},
}

Humans
*CRISPR-Cas Systems/genetics
HEK293 Cells
*Gene Expression Regulation
RNA, Guide, CRISPR-Cas Systems/genetics
*Gene Editing/methods
HeLa Cells
Transcription, Genetic
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42559496,
year = {2026},
author = {Lee, HK and Lim, J and Shepherd, S and Nguyen, MTT and Akin, L and Bacon, A and Sidavi, Y and Shurgalin, M and Cobi, A and Nikolayenko, A and Fuflyigin, V and Bashir, R and Wang, X and Cunningham, BT},
title = {A Dual-Port, Smartphone-Linked, Pocket-Size Fluorimeter for Rapid Molecular Diagnostic Assays at Point of Care.},
journal = {IEEE sensors journal},
volume = {26},
number = {14},
pages = {20538-20555},
pmid = {42559496},
issn = {1530-437X},
support = {U01 AA029348/AA/NIAAA NIH HHS/United States ; },
abstract = {We present the design, testing, and demonstration of a portable, pocket-size, smartphone-linked fluorimeter called the "VPodDuo." The instrument is capable of reading the output of several fluorescence-generating biomolecular detection assays with sensitivity that is similar to larger and more expensive laboratory-based instruments. In this work, we focus on demonstrating the capability for readout of assays used to detect target nucleic acid sequences associated with infectious pathogens and cancer with incubation times of approximately 10 min. The VPodDuo features a dual-port configuration that allows simultaneous measurements of a negative experimental control (CTRL) in parallel with the test sample. We demonstrated compatibility with several assay protocols, including reverse transcription loop-mediated isothermal amplification (RT-LAMP), recombinase polymerase amplification (RPA), clustered regularly interspaced short palindromic repeats (CRISPR)/Cas, and the target recycling amplification process (TRAP). Benchmarking against three commercially available fluorimeters showed comparable detection limits for clinically relevant nucleic acid sequences, including Zika virus (ZIKV, 10[4] copies/μL), methicillin-susceptible Staphylococcus aureus (MSSA, 10[2] copies/μL), human immunodeficiency virus (HIV, 6.83 pM), a lung cancer-associated circulating tumor DNA sequence [L858R point-mutated epidermal growth factor receptor (EGFR) gene, 29.2 pM], and a microRNA biomarker associated with lung cancer (miR-375-3p, 500 pM). We further validated the VPodDuo's performance under varying ambient temperatures through in-lab simulations and real-world outdoor testing using the TRAP assay for miR-375-3p detection, demonstrating cancer-associated biomarker detection at point-of-care (POC) settings. With its compact form-factor, low cost, portability, and real-time data transmission and analytical capabilities, the VPodDuo represents a promising solution for expanding access to rapid, on-site molecular diagnostics in diverse clinical and field settings.},
}

@article {pmid42560391,
year = {2026},
author = {Uc-Chuc, MA and Aguilar-Hernández, V and Jiménez-Ramírez, IA and Brito-Argaez, L and Loyola-Vargas, VM},
title = {In vitro CRISPR/Cas9-RNP cleavage of CcYUC1 in Coffea canephora.},
journal = {Planta},
volume = {264},
number = {3},
pages = {},
pmid = {42560391},
issn = {1432-2048},
support = {1515//Secretaría de Ciencia, Humanidades, Tecnología e Innovación (Secihti)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Coffea/genetics/enzymology/metabolism ; Indoleacetic Acids/metabolism ; *Plant Proteins/genetics/metabolism ; *Ribonucleoproteins/metabolism/genetics ; Base Sequence ; },
abstract = {An in vitro CRISPR/Cas9-RNP system efficiently cleaves CcYUC1 in Coffea canephora, establishing a foundation for DNA-free genome editing in coffee. Somatic embryogenesis depends on auxin biosynthesis and signaling; however, functional validation of candidate genes remains limited. In this study, we identified CcYUC1, a putative flavin monooxygenase gene associated with indole-3-acetic acid biosynthesis, during SE induction in Coffea canephora. CcYUC1 transcripts accumulated during the early stages of SE, suggesting a role in embryogenic induction. To establish a genome editing platform in coffee, we designed a CRISPR/Cas9 ribonucleoprotein (RNP) system targeting exon 4 of CcYUC1. In vitro cleavage assays confirmed specific and efficient digestion of the target, achieving over 80% cleavage under optimized Cas9/sgRNA conditions. These findings establish a proof-of-concept CRISPR/Cas9-RNP platform for DNA-free genome editing in coffee and provide a basis for future functional studies of auxin biosynthesis during somatic embryogenesis.},
}

*CRISPR-Cas Systems/genetics
*Coffea/genetics/enzymology/metabolism
Indoleacetic Acids/metabolism
*Plant Proteins/genetics/metabolism
*Ribonucleoproteins/metabolism/genetics
Base Sequence

@article {pmid42562481,
year = {2026},
author = {Tan, G and Qi, S and Hu, M and Wang, D and Lin, K and Wang, Y and Chen, S and Zhang, Q and Zhao, L},
title = {Understanding phage dynamics and their potential roles during soy sauce fermentation using metagenome-assembled genomes.},
journal = {Food research international (Ottawa, Ont.)},
volume = {241},
number = {},
pages = {119707},
doi = {10.1016/j.foodres.2026.119707},
pmid = {42562481},
issn = {1873-7145},
mesh = {*Fermentation ; *Bacteriophages/genetics/classification/physiology ; *Soy Foods/microbiology/virology ; *Metagenome ; Genome, Viral ; *Food Microbiology ; Metagenomics ; },
abstract = {The composition and functional roles of phages in fermented foods have been gaining increasing attention. However, their ecological functions and underlying mechanisms in high-salt soy sauce fermentation remain largely unexplored. In this study, we investigated phage communities, their potential functions, phage-host interactions, and host defense mechanisms in two different soy sauce fermentation processes (Cantonese-type process, CP; Japanese-type process, JP) using shotgun metagenomics. A total of 823 phage species (viral operational taxonomic units, vOTUs) were identified, with the majority exhibiting a temperate lifestyle (89.19%). The most abundant family was Straboviridae (CP, 9.95%-11.39%; JP, 12.04%-13.73%), followed by Salasmaviridae (CP, 6.92%-7.94%; JP, 5.70%-7.02%). Although the phage composition differed between the two processes, the number of vOTUs was positively correlated with prokaryotic species richness, total acidity, and amino acid nitrogen content, and negatively correlated with pH. A comparative genomic analysis revealed that 91 phages were associated with 26 bacterial genomes (metagenome-assembled genomes, MAGs), with Lactococcus petauri (MAG16) and Halomonas elongata (MAG51) hosting the most phage species. An analysis of host defense mechanisms showed that all 45 bacterial MAGs harbored CRISPR-Cas type I systems, and 95.6% encoded restriction-modification systems. Functional annotation using the KEGG and CAZymes databases indicated that the phages predominantly encoded genes related to cell growth, replication, and metabolism of amino acids, carbohydrates, and nucleotides, with more genes enriched in the JP than in the CP. Additionally, auxiliary metabolic genes (e.g., pfkA, ldh, adhP, ilvE, and arcA) were identified in 29 phages. These genes are potentially involved in metabolic pathways that may be linked to flavor compound production. Together, these findings provide novel insights into the ecological and potential functional roles of phages during soy sauce fermentation.},
}

*Fermentation
*Bacteriophages/genetics/classification/physiology
*Soy Foods/microbiology/virology
*Metagenome
Genome, Viral
*Food Microbiology
Metagenomics

@article {pmid42563405,
year = {2026},
author = {Adil, M and Gul, I and Lu, S and Bashir, S and Razzaq, S and Lu, H and Daud, M and Iqbal, Y and Tao, Y},
title = {Crop Resilience to Combined Drought and Salinity Stress in Drylands: From Soil Processes to Genomic Solutions.},
journal = {Plant, cell & environment},
volume = {},
number = {},
pages = {},
doi = {10.1111/pce.70804},
pmid = {42563405},
issn = {1365-3040},
support = {41871079//National Natural Science Foundation of China/ ; SKLECRA2023//Chinese Research Academy of Environmental Sciences/ ; //Open Foundation of State Key Laboratory of Environmental Criteria and Risk Assessment/ ; },
abstract = {Drought and soil salinization increasingly co‑occur, threatening global food security, particularly in dryland farming systems where these stresses can interact additively, synergistically or antagonistically to reduce crop yields. This review critically distinguishes these interaction types and moves beyond single‑stress frameworks to provide a multiscale synthesis of plant responses, soil water-salt dynamics, root‑system plasticity, rhizosphere microbiome modulation, emerging breeding modelling and agronomic tools. This review provides a detailed overview of the multilevel damage from these co-occurring stresses, including osmotic shock, ionic toxicity and systemic oxidative damage, and examines plant strategies such as ion homoeostasis, osmotic adjustment and antioxidant responses. Beyond plant-centric views, the review explores epigenetic reprogramming for stress memory and the potential of rhizosphere microbiome engineering with plant growth-promoting rhizobacteria as a biological stress-alleviation strategy. It critically examines how high-throughput multiomics techniques, combined with innovative breeding methods, provide a transformative approach to decode complex tolerance traits and accelerate cultivar development. These techniques cover genomics, epigenomics, transcriptomics, proteomics and metabolomics, while the breeding methods include genomic selection and CRISPR-Cas gene editing. Despite progress, key challenges remain: validating combined stress resilience under field conditions, harnessing epigenetic inheritance and integrating microbial solutions into breeding pipelines. We propose a unified framework that merges mechanistic discovery, microbiome-assisted breeding, soil and water management and systems-level predictive modelling. This integrative approach is essential for developing climate-resilient crops to sustain dryland agriculture.},
}

@article {pmid42567578,
year = {2026},
author = {Wang, H and Bao, C and Liu, L and Li, F and He, Y and Liu, X and Yin, Y and Xu, S},
title = {CRISPR/Cas12a and CHA-based SERS platform for ultrasensitive nucleic acid detection.},
journal = {Analytica chimica acta},
volume = {1418},
number = {},
pages = {345840},
doi = {10.1016/j.aca.2026.345840},
pmid = {42567578},
issn = {1873-4324},
mesh = {*Spectrum Analysis, Raman/methods ; *CRISPR-Cas Systems/genetics ; Zea mays/genetics ; Silver/chemistry ; Limit of Detection ; DNA, Single-Stranded/analysis ; Metal Nanoparticles/chemistry ; },
abstract = {BACKGROUND: Highly sensitive nucleic acid detection is essential for analytical applications. Conventional methods often require complex pre-amplification procedures, limiting their practical utility in screening. Developing nucleic acid detection strategies with high sensitivity and selectivity, without target gene pre-amplification, remains a significant challenge.

RESULTS: This study integrates CRISPR/Cas12a recognition, catalytic hairpin assembly (CHA) amplification, and surface-enhanced Raman spectroscopy (SERS). CRISPR/Cas12a recognizes target nucleic acids and cleaves single-stranded DNA (ssDNA), thereby blocking the toehold-mediated strand displacement reaction (TSDR) and triggering CHA. Hairpin probe HP1 with C-Ag[+]-C structures bridges CHA, releasing Ag[+] through cyclic amplification. Ag[+] induces charge transfer and aggregation of AgNPs@4-ABT, generating strong SERS signals. The platform achieved femtomolar sensitivity and high selectivity in detecting pCaMV35S, with 96.4% accuracy in maize seeds and 100% in maize leaves.

SIGNIFICANCE: This strategy eliminates the need for pre-amplification of target genes by combining CRISPR's targeting feature, CHA, and ultrasensitive SERS detection. It demonstrates excellent performance in genetically modified organism screening, seed quality testing, and leaf sample analysis, providing a promising tool for food safety and agricultural regulation.},
}

*Spectrum Analysis, Raman/methods
*CRISPR-Cas Systems/genetics
Zea mays/genetics
Silver/chemistry
Limit of Detection
DNA, Single-Stranded/analysis
Metal Nanoparticles/chemistry

@article {pmid42568984,
year = {2026},
author = {Sahu, A and Kumar, A and Vaidya, A and Mishra, J and Mishra, S and Prajapti, SK},
title = {Decoding the silent conversations: targeting quorum sensing to disarm bacterial pathogens in the age of antimicrobial resistance.},
journal = {RSC medicinal chemistry},
volume = {},
number = {},
pages = {},
pmid = {42568984},
issn = {2632-8682},
abstract = {Antimicrobial resistance (AMR) has emerged as a global health challenge, imposing significant clinical and economic burdens worldwide. The widespread and often indiscriminate use of antibiotics has accelerated resistance, necessitating alternative therapeutic strategies to combat microbial pathogenicity. Quorum sensing, a cell density-dependent signalling system, represents a promising target in this aspect. This review examines the molecular framework of quorum sensing across diverse microbial communities, its signalling cascades, and its role in regulating biofilm formation, efflux pump modulation and horizontal gene transfer with the quorum signalling. It further discusses quorum-sensing inhibition strategies, including natural products, synthetic compounds, quorum-quenching enzymes, and antibody-mediated and vaccine-mediated approaches. Application of CRISPR/Cas, engineered probiotic strains and nanocarrier-mediated delivery systems for quorum signalling disruption has been addressed. A key strength of this review is that it is the first to combine the underlying molecular mechanisms of quorum sensing with future translational tools such as artificial intelligence, CRISPR, engineered probiotics and nanotechnology to develop next-generation anti-virulence solutions for drug-resistant infection. While the preclinical findings have several challenges specific to the specificity and pharmacokinetic properties, strategies to resolve these considerations have been discussed. Overall, quorum signalling as a target is a major paradigm shift that may offer sustainable antimicrobial therapy to fight the global antimicrobial resistance issue. This can be achieved through a multidisciplinary approach to optimise antimicrobial therapy beyond the traditional concept of "killing".},
}

@article {pmid42569449,
year = {2026},
author = {Choi, Y and Yang, J and Kim, J and Enkhtaivan, K and Son, J and Jang, J and Lee, HY and Choi, J},
title = {Exosome-Based Liquid Biopsy in Biliary Tract Cancer: Nanotechnology-Enabled Strategies and Future Perspectives.},
journal = {International journal of nanomedicine},
volume = {21},
number = {},
pages = {613686},
pmid = {42569449},
issn = {1178-2013},
mesh = {Humans ; *Exosomes/chemistry ; *Biliary Tract Neoplasms/pathology/diagnosis ; Liquid Biopsy/methods ; *Nanotechnology/methods ; Biomarkers, Tumor/analysis ; Nanomedicine ; Biosensing Techniques ; },
abstract = {Biliary tract cancer (BTC) remains a formidable clinical challenge owing to its asymptomatic early stages, anatomical complexity, and lack of reliable and noninvasive diagnostic tools. Although traditional tissue biopsy is often limited by invasiveness and sampling bias, liquid biopsy, particularly the analysis of tumor-derived exosomes, has emerged as a promising and clinically relevant alternative for early detection and longitudinal monitoring. Exosomes are specialized extracellular vesicles that sequester diverse molecular cargo including proteins, lipids, and nucleic acids, thereby reflecting the physiological state of their parental tumor cells. However, the clinical translation of exosomal biomarkers is often hindered by technical challenges in achieving high-purity isolation and ultrasensitive detection in complex biological matrices, such as blood and bile. To address these limitations, this review provides a comprehensive overview of recent advancements in nanotechnology-enabled platforms designed to overcome these challenges. First, we examined sophisticated nanostructured systems, such as immuno-magnetic nanoparticles and microfluidic nanoVelcro chips, for high-yield exosome enrichment. Subsequently, we investigated next-generation biosensing modalities with a focus on surface-enhanced Raman scattering for label-free molecular fingerprinting and CRISPR-Cas-integrated nanosensors for amplification-free nucleic acid detection. Significant emphasis has now been placed on the integration of artificial intelligence and deep learning algorithms, which have become indispensable for deciphering complex exosomal signatures to differentiate BTC from benign conditions such as cholangitis. Finally, we discuss the emerging clinical significance of bile-derived exosomes and remaining challenges in standardizing nanomedicine-based liquid biopsies for precision oncology. These integrated platforms could potentially redefine the BTC management paradigm by bridging the gap between advanced nanomaterials and clinical diagnostics.},
}

Humans
*Exosomes/chemistry
*Biliary Tract Neoplasms/pathology/diagnosis
Liquid Biopsy/methods
*Nanotechnology/methods
Biomarkers, Tumor/analysis
Nanomedicine
Biosensing Techniques

@article {pmid42296510,
year = {2026},
author = {Zhang, ZM and Xu, C and Zhu, Y and Wei, Y and Pei, Y and Zhang, H and Pang, J and Xie, ZJ and Xu, K and Wang, J},
title = {Integrated CRISPR-Cas12a-Based Biosensors with Subwavelength Grating Microring Resonators for Ultrasensitive Mutation-Specific Detection.},
journal = {ACS sensors},
volume = {11},
number = {7},
pages = {5674-5682},
doi = {10.1021/acssensors.6c00141},
pmid = {42296510},
issn = {2379-3694},
support = {62422503//National Natural Science Foundation of China/ ; U21A20454//National Natural Science Foundation of China/ ; 2022A0505030024//Natural Science Foundation of Guangdong Province/ ; 2022A1515110756//Natural Science Foundation of Guangdong Province/ ; 2022B1515020093//Natural Science Foundation of Guangdong Province/ ; 2022B1515120012//Natural Science Foundation of Guangdong Province/ ; GJHZ20220913143207014//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; JCYJ20220818102406013//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; JCYJ20220818102618040//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; JCYJ20241202130558075//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; RCYX20210609103707009//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; RCYX20221008092907027//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; 2022YFB3207200//National Key Research and Development Program of China/ ; 2022B1515020057//Science Fund for Distinguished Young Scholars of Guangdong Province/ ; },
mesh = {*Biosensing Techniques/methods/instrumentation ; *CRISPR-Cas Systems/genetics ; Mutation ; Gold/chemistry ; *SARS-CoV-2/genetics/isolation & purification ; Metal Nanoparticles/chemistry ; Humans ; *CRISPR-Associated Proteins ; DNA Probes/chemistry/genetics ; *Endodeoxyribonucleases/genetics ; Bacterial Proteins ; },
abstract = {The rapid and precise detection of nucleic acids is critical for identifying viral mutations, yet it presents formidable difficulties for conventional diagnostics. While established techniques such as quantitative polymerase chain reaction and next-generation sequencing involve complex workflows, emerging on-chip integrated photonic biosensing techniques are often limited by inadequate specificity and sensitivity. Here, we introduce an integrated photonic biosensing platform that synergizes the programmable recognition of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a, with the superior sensitivity of subwavelength grating microring resonators. The sensor surface is functionalized with single-stranded DNA probes conjugated to gold nanoparticles. Upon target recognition, activated Cas12a cleaves the probes, releasing the nanoparticles and generating a quantifiable resonance wavelength shift. In particular, the spectral response gets further amplified by a resonance-enhanced photothermal effect. The detection of SARS-CoV-2 variants enables discrimination between wild-type, Delta, and Omicron strains. The extracted detection limit of 0.7 fM represents a four-order-of-magnitude improvement over conventional fluorescence-based CRISPR assays. Our work establishes a generalizable platform for ultrasensitive, mutation-resolved molecular diagnostics on a CMOS-compatible photonic chip, paving the way for advanced point-of-care testing and genomic surveillance.},
}

*Biosensing Techniques/methods/instrumentation
*CRISPR-Cas Systems/genetics
Mutation
Gold/chemistry
*SARS-CoV-2/genetics/isolation & purification
Metal Nanoparticles/chemistry
Humans
*CRISPR-Associated Proteins
DNA Probes/chemistry/genetics
*Endodeoxyribonucleases/genetics
Bacterial Proteins

@article {pmid42391661,
year = {2026},
author = {Zheng, C and Nong, L and Luo, J and Liang, L and Wei, Y and Luo, Q and Zhang, K and Liao, X},
title = {An electrochemiluminescence biosensor based on the hairpin-mediated exponential amplification and CRISPR/Cas12a amplification for ultrasensitive detection of MMP-2.},
journal = {Colloids and surfaces. B, Biointerfaces},
volume = {267},
number = {},
pages = {115948},
doi = {10.1016/j.colsurfb.2026.115948},
pmid = {42391661},
issn = {1873-4367},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Matrix Metalloproteinase 2/analysis ; *Luminescent Measurements/methods ; *Electrochemical Techniques/methods ; Humans ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Electrodes ; *Nucleic Acid Amplification Techniques/methods ; Inverted Repeat Sequences ; Limit of Detection ; *Bacterial Proteins/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; Endodeoxyribonucleases ; },
abstract = {In this study, we report a novel electrochemiluminescence (ECL) biosensor for the ultrasensitive detection of matrix metalloproteinase-2 (MMP-2), an important biomarker associated with tumor invasion and metastasis. The biosensor integrates hairpin-mediated exponential amplification with CRISPR/Cas12a-based trans-cleavage for dual-stage signal amplification. In this design, MMP-2 specifically cleaves a peptide sequence (GPLG↓VRGK) on the DNA hairpin probe (HP1), releasing an initiator peptide nucleic acid (PNA) that triggers hairpin-mediated exponential amplification reaction. The amplified DNA products then activate the Cas12a/gRNA complex, which induces collateral cleavage of ferrocene (Fc)-labeled probes immobilized on a DNA tetrahedron-modified PEI-Ti3C2Tx/Ru/AuNPs electrode, thereby generating a strong ECL response. The incorporation of the DNA tetrahedron nanostructure provides a well-defined three-dimensional framework that ensures ordered probe orientation, enhanced hybridization efficiency, and reduced steric hindrance on the electrode surface. This structural organization significantly improves electron transfer and signal stability compared with conventional planar immobilization. Under optimized conditions, the biosensor exhibited a broad linear range from 0.01 fM to 10 nM and an ultralow detection limit of 10 aM. It displayed high specificity against interfering proteins (thrombin, IgG, BSA, lysozyme), excellent stability, and satisfactory recoveries (96.9%-105.0%) in LO2 cell culture supernatants. Overall, this enzyme-responsive, DNA-tetrahedron-assisted, CRISPR-amplified ECL biosensor represents a robust and versatile platform for precise and rapid detection of protease activity, showing great promise for biomedical diagnostics and clinical biomarker monitoring.},
}

*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
*Matrix Metalloproteinase 2/analysis
*Luminescent Measurements/methods
*Electrochemical Techniques/methods
Humans
Gold/chemistry
Metal Nanoparticles/chemistry
Electrodes
*Nucleic Acid Amplification Techniques/methods
Inverted Repeat Sequences
Limit of Detection
*Bacterial Proteins/genetics/metabolism
*CRISPR-Associated Proteins/genetics/metabolism
Endodeoxyribonucleases

@article {pmid42399104,
year = {2026},
author = {Liu, Z and He, Y and Lin, X and Pal, T and Pfeilsticker, A and Liu, BM and Chen, J},
title = {Indiscriminate Trans-Cleavage Activity of CRISPR/SuCas12a2 Enables Sensitive Detection of SARS-CoV-2.},
journal = {ACS sensors},
volume = {11},
number = {7},
pages = {5397-5403},
pmid = {42399104},
issn = {2379-3694},
support = {R35 GM147069/GM/NIGMS NIH HHS/United States ; R35GM147069/GM/NIGMS NIH HHS/United States ; },
mesh = {*SARS-CoV-2/genetics/isolation & purification ; Humans ; *CRISPR-Cas Systems ; *COVID-19/diagnosis/virology ; *CRISPR-Associated Proteins/metabolism/genetics ; RNA, Viral/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins/genetics/metabolism ; *COVID-19 Nucleic Acid Testing/methods ; Endodeoxyribonucleases/metabolism/genetics ; Fluorescent Dyes/chemistry ; Rapid Diagnostic Tests ; },
abstract = {Sensitive detection of SARS‑CoV‑2 remains critical for controlling COVID‑19 outbreaks and guiding patient care. Although reverse transcription-polymerase chain reaction (RT‑PCR), the gold standard for detecting SARS-CoV-2, is highly sensitive, the need for specialized equipment and trained personnel limits its widespread application in low or middle-resource settings. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology could overcome these limitations by providing simplicity, low cost, and high specificity. However, current CRISPR-based diagnostics can simultaneously cleave the target and fluorescence probes, as they are the same nucleic acid type (ssDNA or ssRNA), thereby reducing detection sensitivity. Herein, we developed a novel CRISPR-based viral detection method using SuCas12a2 (Cas12a2 from Sulfuricurvum sp. PC08-66), which harnesses its unique broad trans-cleavage activity and offers flexibility in selecting fluorescence probes. Using the conserved SARS‑CoV‑2 envelope gene as the model analyte, the analytical performance of the CRISPR/SuCas12a2 system for viral detection was evaluated. The CRISPR/SuCas12a2 detection workflow achieved a detection limit of 5 × 103 copies/μL for SARS-CoV-2 viral RNA. When detecting nasopharyngeal swab samples from patients, the CRISPR/SuCas12a2 system showed preliminary agreement with RT-qPCR in a set of clinical samples. Our CRISPR/SuCas12a2 system provides a flexible detection platform with simplified probe selection and enhanced compatibility, offering new insights into future portable diagnostic applications and enhancing global public health surveillance.},
}

*SARS-CoV-2/genetics/isolation & purification
Humans
*CRISPR-Cas Systems
*COVID-19/diagnosis/virology
*CRISPR-Associated Proteins/metabolism/genetics
RNA, Viral/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
Bacterial Proteins/genetics/metabolism
*COVID-19 Nucleic Acid Testing/methods
Endodeoxyribonucleases/metabolism/genetics
Fluorescent Dyes/chemistry
Rapid Diagnostic Tests

@article {pmid42402044,
year = {2026},
author = {Kim, T and Scheeres, EC and Fiebig, A and Olive, AJ and Crosson, S},
title = {A genome-wide CRISPR screen defines host determinants of early Brucella infection in human macrophage-like cells.},
journal = {Infection and immunity},
volume = {94},
number = {8},
pages = {e0011726},
doi = {10.1128/iai.00117-26},
pmid = {42402044},
issn = {1098-5522},
support = {R01AI177619/NH/NIH HHS/United States ; R35GM146795/NH/NIH HHS/United States ; },
mesh = {Humans ; *Macrophages/microbiology/metabolism ; *Brucellosis/genetics/microbiology ; *Host-Pathogen Interactions/genetics ; *CRISPR-Cas Systems ; *Brucella ; Brucella abortus ; Cell Line ; },
abstract = {Brucella spp. are widespread intracellular animal pathogens that cause brucellosis, a significant zoonosis. Despite the global impact of brucellosis on animal and human health, the host genes that support Brucella infection remain incompletely defined. To address this knowledge gap, we developed a flow cytometry-based infection assay with fluorescent Brucella and performed a genome-wide CRISPR-Cas9 loss-of-function screen in human macrophage-like cells. Disruption of >150 host genes significantly reduced intracellular B. abortus signal at 3 hours post-infection. In addition to recovering known host factors, the screen revealed previously unappreciated genes linked to endosomal trafficking, cytoskeletal remodeling, and lipid homeostasis. The screen was robust, as validation within these functional categories confirmed that the small GTPase RAB14, the Src-family kinase regulator CSK, and the phospholipid flippase subunit TMEM30A support the B. abortus and B. ovis infection process at a post-entry step. Gene set enrichment analysis further identified positive regulators of mTORC1 signaling as host factors. This result was validated by genetic disruption of LAMTOR2 and AKT1, and pharmacologic inhibition of AKT1. Together, these data indicate that the AKT-Ragulator-mTORC1 axis contributes to establishing a permissive intracellular niche. Finally, to assess whether these host requirements extend beyond Brucella, we examined infection by the unrelated intracellular pathogen Mycobacterium abscessus. CSK, AKT1, and LAMTOR2 were required for efficient M. abscessus infection, whereas RAB14 was dispensable. Together, these results define host genes that impact Brucella infection and distinguish shared versus pathogen-specific host dependencies exploited by intracellular bacteria.},
}

Humans
*Macrophages/microbiology/metabolism
*Brucellosis/genetics/microbiology
*Host-Pathogen Interactions/genetics
*CRISPR-Cas Systems
*Brucella
Brucella abortus
Cell Line

@article {pmid42412687,
year = {2026},
author = {Pan, Y and Yang, Z},
title = {Paper Microfluidic Platform Using Multiplexed Isothermal Amplification and CRISPR/Cas12a for Aquatic Pathogen Detection.},
journal = {ACS sensors},
volume = {11},
number = {7},
pages = {5893-5908},
doi = {10.1021/acssensors.6c00719},
pmid = {42412687},
issn = {2379-3694},
support = {RL-2022-041//Leverhulme Trust/ ; FF\1920\1\36//Royal Academy of Engineering/ ; NE/R013349/2//National Centre for Earth Observation/ ; NE/V010441/1//National Centre for Earth Observation/ ; },
mesh = {*Nucleic Acid Amplification Techniques/methods/instrumentation ; *Paper ; *CRISPR-Cas Systems/genetics ; *Water Microbiology ; *Bacteria/isolation & purification/genetics ; *Microfluidic Analytical Techniques/instrumentation/methods ; Rapid Diagnostic Tests ; Colorimetry ; Limit of Detection ; Molecular Diagnostic Techniques ; },
abstract = {The global health threat posed by microbial contamination of aquatic systems demands feasible pathogen monitoring solutions. However, current detection methods are limited by expensive instrumentation and specialized personnel, which hinders their application in point-of-care testing (POCT). Here, we presented an integrated paper microfluidic platform for spatially multiplexed detection of pathogenic bacteria, including Salmonella, E. coli, C. perfringens, B. cereus, V. parahaemolyticus, S. aureus, and L. monocytogenes, selected due to their epidemiological significance and regulatory relevance in environmental and food safety monitoring. LAMP, RAA-CRISPR, and RPA-CRISPR assays were housed within physically isolated reaction chambers on two-layer chips. An engineered horseradish peroxidase (HRP) cascade-coupled crRNA modification system with DNA-conjugated labels was designed for colorimetric detection. Operation was enabled by solar-powered and portable hardware for incubation and imaging, coupled with a web application for quantitative analysis. Exceptional analytical performance was demonstrated, achieving an LOD of 1 CFU/mL, a dynamic range of 1-107 CFU/mL, high reproducibility (CV <5%), low batch-to-batch variation (<6%), low cost (£2.5 per test), and scalable integration, with a sample-to-answer time of 60 min. Successful field validation in diverse aquatic environments confirmed its practical feasibility, consistent with gold standard PCR (R2 = 0.98). This platform offers a promising POCT solution for public health protection and epidemic monitoring, particularly in resource-limited settings.},
}

*Nucleic Acid Amplification Techniques/methods/instrumentation
*Paper
*CRISPR-Cas Systems/genetics
*Water Microbiology
*Bacteria/isolation & purification/genetics
*Microfluidic Analytical Techniques/instrumentation/methods
Rapid Diagnostic Tests
Colorimetry
Limit of Detection
Molecular Diagnostic Techniques

@article {pmid42425083,
year = {2026},
author = {Nair, U and Akauliya, M and Warner, JE and Parikh, S and Ronsard, L and Ssozi, M and Rays Wahba, L and Esposito, AG and Kelley, B and McCarthy, C and Weldon, SR and Wu, L and Lingwood, D and Batista, FD},
title = {CRISPR-mediated precise large fragment insertion in zygotes enables rapid generation of humanized immunoglobulin heavy-chain mice.},
journal = {Immunity},
volume = {59},
number = {8},
pages = {2334-2350.e8},
pmid = {42425083},
issn = {1097-4180},
support = {R01 AI153098/AI/NIAID NIH HHS/United States ; R01 AI155447/AI/NIAID NIH HHS/United States ; INV-085294/GATES/Gates Foundation/United States ; R01 AI195539/AI/NIAID NIH HHS/United States ; R01 AI168114/AI/NIAID NIH HHS/United States ; INV-046626/GATES/Gates Foundation/United States ; },
mesh = {Animals ; Humans ; Mice ; *Immunoglobulin Heavy Chains/genetics ; *CRISPR-Cas Systems/genetics ; *Zygote ; Chromosomes, Artificial, Bacterial/genetics ; B-Lymphocytes/immunology ; Immunoglobulin Variable Region/genetics ; Mice, Transgenic ; V(D)J Recombination ; Somatic Hypermutation, Immunoglobulin ; },
abstract = {Current CRISPR-Cas9 methods are restricted to small genomic edits. We developed a CRISPR-guided approach that enables direct insertion of large genomic sequences into mouse zygotes. We deleted the murine 2.4-Mb immunoglobulin heavy-chain (IgH) variable (VH) locus and then precisely inserted a bacterial artificial chromosome (BAC) containing a 155-kb human VH DNA fragment flanked by 20-kb homology arms. Full-length, single-copy BAC integration occurred without ectopic recombination. Human sequences were stably transmitted and expressed VH segments that recombined with endogenous mouse sequences, and mice exhibited normal B cell development. Upon immunization, human VH-expressing B cells underwent class-switch recombination and somatic hypermutation, secreting antigen-specific antibodies. We demonstrated modular IgH humanization by replacing endogenous mouse diversity and joining (DH-JH) segments with human sequences, producing V(D)J recombination and diverse antibodies. Unlike traditional methods requiring more than a year, this approach enables the generation and validation of mice carrying large genomic insertions within 8 weeks.},
}

Animals
Humans
Mice
*Immunoglobulin Heavy Chains/genetics
*CRISPR-Cas Systems/genetics
*Zygote
Chromosomes, Artificial, Bacterial/genetics
B-Lymphocytes/immunology
Immunoglobulin Variable Region/genetics
Mice, Transgenic
V(D)J Recombination
Somatic Hypermutation, Immunoglobulin

@article {pmid42433170,
year = {2026},
author = {Qiu, Z and Chen, J and Wu, J and Wang, J and Hu, Y and Feng, W and Zhao, J and Minamoto, T and Chen, X and Zhou, L and Liu, M and Wang, C and Lin, M and Hu, M and Zhou, X and Wang, J},
title = {Optical-Controlled One-Pot RPA-CRISPR Assay for Environmental DNA Detection of a Critically Endangered Species.},
journal = {ACS sensors},
volume = {11},
number = {7},
pages = {5553-5565},
doi = {10.1021/acssensors.5c04638},
pmid = {42433170},
issn = {2379-3694},
support = {ZJZX-06//Ministry of Agriculture and Rural Affairs of the People's Republic of China/ ; },
mesh = {*Endangered Species ; *DNA, Environmental/analysis/genetics ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems/genetics ; Recombinases/metabolism ; Animals ; },
abstract = {Developing a rapid, sensitive, and field-deployable assay for on-site environmental DNA (eDNA) detection of endangered species is crucial, as current PCR-based assays are slow and expensive and rely on laboratory-based thermal cycling. Here, we adapted a previously reported optically controlled one-pot RPA-CRISPR-Cas12a (OORC) assay to detect eDNA of the critically endangered Bahaba taipingensis. The assay combines isothermal recombinase polymerase amplification (RPA) with the high specificity of a CRISPR-Cas12a trans-cleavage reaction in a single tube. The entire process is operated at a constant temperature, avoiding thermal cycling. Low-template replicate experiments conservatively redefined the OORC limit of detection as 6 copies/reaction. To support near-field application, we further integrated the assay with a portable handheld fluorescence detector capable of 365 nm photoactivation and fluorescence readout. This portable and highly sensitive workflow extends the potential of eDNA monitoring, offering a practical tool for the conservation of endangered species.},
}

*Endangered Species
*DNA, Environmental/analysis/genetics
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems/genetics
Recombinases/metabolism
Animals

@article {pmid42441514,
year = {2026},
author = {Wang, X and Zhao, S and Jiang, J and Wang, H and Encarnación, C and Zheng, L and Zhang, L},
title = {5' Dual-Overhang Short PAM-less dsDNA as Switchable Activators of Cas12a trans-Cleavage for Amplification-Free miRNA Detection.},
journal = {ACS sensors},
volume = {11},
number = {7},
pages = {5753-5764},
doi = {10.1021/acssensors.6c00524},
pmid = {42441514},
issn = {2379-3694},
support = {24DX2800100//Science and Technology Innovation Plan Of Shanghai Science and Technology Commission/ ; },
mesh = {*MicroRNAs/blood/analysis/genetics ; Humans ; *DNA/chemistry/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/chemistry ; CRISPR-Cas Systems ; *Bacterial Proteins/metabolism/chemistry ; *Endodeoxyribonucleases/metabolism/chemistry ; },
abstract = {Precise and programmable regulation of CRISPR-Cas12a activity is essential for advancing controllable nucleic acid diagnostics, yet the structural determinants governing Cas12a activation by short PAM-less double-stranded DNA (dsDNA) remain largely unexplored. This study systematically investigates the effects of the terminal architectures of short PAM-less dsDNA on Cas12a trans-cleavage activity. By profiling a series of dsDNA constructs bearing distinct 5'/3' overhang configurations, a 5' dual-overhang motif was identified as a highly effective structural inhibitor that suppresses Cas12a activation. Kinetic fluorescence assays combined with computational structural modeling indicated that this inhibition arises from steric constraints imposed by the 5' terminal architecture. Leveraging this structure-guided regulatory mechanism, an amplification-free CRISPR-Cas12a assay was developed for the direct detection of oncogenic microRNAs miR-155 and miR-21, achieving femtomolar sensitivity without reverse transcription. The assay was further evaluated in human serum samples spiked with target miRNAs, supporting its proof-of-concept performance in a more complex matrix. Collectively, these findings highlight the potential of terminally engineered PAM-less dsDNA as a structural handle for programming Cas12a activity and provide useful insight for the design of CRISPR-based biosensing strategies.},
}

*MicroRNAs/blood/analysis/genetics
Humans
*DNA/chemistry/metabolism/genetics
*CRISPR-Associated Proteins/metabolism/chemistry
CRISPR-Cas Systems
*Bacterial Proteins/metabolism/chemistry
*Endodeoxyribonucleases/metabolism/chemistry

@article {pmid42462715,
year = {2026},
author = {Zhu, Y and Wang, Q and Cao, Y and Hou, L and Yang, L and Cheng, C and Ye, S and Li, W and Zhang, J},
title = {Single cell CRISPR screen identifies antagonism between Nsd1-H3K36me2 and Ezh2-H3K27me3 orchestrates pluripotency transition.},
journal = {Stem cell reports},
volume = {21},
number = {8},
pages = {103016},
doi = {10.1016/j.stemcr.2026.103016},
pmid = {42462715},
issn = {2213-6711},
mesh = {Animals ; *Enhancer of Zeste Homolog 2 Protein/metabolism/genetics ; Mice ; *Histones/metabolism/genetics ; *Histone-Lysine N-Methyltransferase/metabolism/genetics ; Mouse Embryonic Stem Cells/metabolism/cytology ; Epigenesis, Genetic ; DNA Methylation ; CRISPR-Cas Systems ; *Single-Cell Analysis/methods ; DNA Methyltransferase 3A ; DNA (Cytosine-5-)-Methyltransferase 1/genetics/metabolism ; Cell Differentiation/genetics ; *Pluripotent Stem Cells/metabolism/cytology ; DNA (Cytosine-5-)-Methyltransferases/genetics/metabolism ; *Intracellular Signaling Peptides and Proteins/metabolism/genetics ; },
abstract = {The transcriptional and epigenetic landscape imposes constraints on the self-renewal capacity and lineage specification potential of both naive and primed mouse embryonic stem cells (mESCs). CRISPR/Cas9-based functional screening coupled with single-cell RNA-seq (CROP-seq) establishes relationships between gRNA-mediated knockout genotype and transcriptome phenotype, providing a powerful tool to dissect gene regulatory networks. Here, we employed CROP-seq to investigate the epigenetic regulation governing the pluripotency network in mESCs. This highly sensitive method identified key genes essential for the acquisition and exit from pluripotency, and revealed a novel role for H3K36me2 in modulating DNA methylation through regulating the expression of Dnmt1 and Dnmt3a. Specifically, loss of Nsd1-mediated H3K36me2 delayed naive state exit, whereas Ezh2 deficiency accelerated primed entry. Collectively, our findings identify an epigenetic regulatory network critical for determining mESCs' pluripotent state transitions.},
}

Animals
*Enhancer of Zeste Homolog 2 Protein/metabolism/genetics
Mice
*Histones/metabolism/genetics
*Histone-Lysine N-Methyltransferase/metabolism/genetics
Mouse Embryonic Stem Cells/metabolism/cytology
Epigenesis, Genetic
DNA Methylation
CRISPR-Cas Systems
*Single-Cell Analysis/methods
DNA Methyltransferase 3A
DNA (Cytosine-5-)-Methyltransferase 1/genetics/metabolism
Cell Differentiation/genetics
*Pluripotent Stem Cells/metabolism/cytology
DNA (Cytosine-5-)-Methyltransferases/genetics/metabolism
*Intracellular Signaling Peptides and Proteins/metabolism/genetics

@article {pmid42503780,
year = {2026},
author = {Shi, JY and Wu, SL and Tan, Y and Jiang, HX and Liu, SQ and Gan, XQ and Sun, WB and Li, MY and Li, KD and Tang, AN and Zhu, LN and Cai, QL and Kong, DM},
title = {DNA Nanowire-Assisted CRISPR/Cas12a Triple Cascade Amplification for Sensitive Detection of Myeloperoxidase Activity.},
journal = {Analytical chemistry},
volume = {98},
number = {31},
pages = {22931-22942},
doi = {10.1021/acs.analchem.6c02223},
pmid = {42503780},
issn = {1520-6882},
support = {22293030//National Natural Science Foundation of China/ ; 22474063//National Natural Science Foundation of China/ ; 72474154//National Natural Science Foundation of China/ ; 82570985//National Natural Science Foundation of China/ ; 25JCLZJC00250//Tianjin Municipal Science and Technology Program/ ; 25JCLZJC00640//Tianjin Municipal Science and Technology Program/ ; 23JCJQJC00080//Science Fund for Distinguished Young Scholars of Tianjin Municipality/ ; },
mesh = {*Peroxidase/metabolism/blood ; Humans ; *CRISPR-Cas Systems/genetics ; *DNA/chemistry/metabolism ; *Nanowires/chemistry ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Biosensing Techniques/methods ; *CRISPR-Associated Proteins/metabolism ; *Endodeoxyribonucleases/metabolism ; *Bacterial Proteins/metabolism/genetics ; },
abstract = {Myeloperoxidase (MPO) is an inflammation-associated heme enzyme implicated in cardiovascular oxidative stress, but sensitive activity-based detection in complex clinical samples remains challenging. Herein, we report a CRISPR/Cas12a-based triple-cascade amplification platform for rapid and sensitive MPO activity detection. The core sensing element consists of DNA nanowires containing multiple Cas12a activator strands, tethered to magnetic beads via biotin-labeled and phosphorothioate-modified linkers. In the chloride-containing assay system, MPO-catalyzed generation of HOCl oxidatively cleaves these linkers, releasing the nanowires and activating Cas12a, which, in turn, cleaves fluorescent reporters. This design integrates three amplification stages (MPO catalysis, multiactivator release, and Cas12a trans-cleavage), achieving ultrasensitive detection without additional nucleic acid amplification. Under the optimized chloride-containing conditions, the assay achieved a detection limit of 10.20 pg/mL for MPO. A preliminary pilot analysis using human serum samples from acute coronary syndrome patients and healthy individuals showed different signal distributions, supporting the feasibility of applying the platform to complex serum matrices, although contributions from eosinophil peroxidase/HOBr-mediated probe activation cannot be excluded. The platform is readily adaptable to lateral flow assays and portable fluorescence readouts, offering versatile formats for point-of-care-compatible analysis. This work provides a sensitive CRISPR/Cas12a-based strategy for MPO activity-related hypohalous oxidant assessment under defined assay conditions and demonstrates its preliminary applicability in complex serum matrices.},
}

*Peroxidase/metabolism/blood
Humans
*CRISPR-Cas Systems/genetics
*DNA/chemistry/metabolism
*Nanowires/chemistry
*Nucleic Acid Amplification Techniques/methods
Limit of Detection
Biosensing Techniques/methods
*CRISPR-Associated Proteins/metabolism
*Endodeoxyribonucleases/metabolism
*Bacterial Proteins/metabolism/genetics

@article {pmid42550765,
year = {2026},
author = {Torrance, R and Orf, K and White, N and Matti, C and McKenna, AJ and Cosentino, A and Casirati, G and Albuquerque, AS and Thrasher, AJ and Genovese, P and Booth, CA and Fox, TA and Burns, SO and Morris, EC},
title = {Functional restoration of immune defects in STAT1 gain-of-function disease following stem cell gene editing.},
journal = {Blood},
volume = {},
number = {},
pages = {},
doi = {10.1182/blood.2025032242},
pmid = {42550765},
issn = {1528-0020},
abstract = {Germline gain-of-function (GOF) mutations in the signal transducer and activator of transcription 1 (STAT1) gene cause a dominantly inherited inborn error of immunity (IEI) characterized by chronic mucocutaneous candidiasis, autoimmunity, severe opportunistic infections and an increased risk of malignancy. Allogeneic hematopoietic stem cell (HSC) transplantation (HSCT) is curative but is associated with increased risk of morbidity and mortality in STAT1 GOF patients compared to other IEI. To develop a curative, autologous alternative to HSCT, we evaluated gene editing strategies in STAT1 GOF model cell lines, primary T cells, and patient-derived HSCs. Universal and mutation-specific strategies using CRISPR/Cas-mediated homology-directed repair (HDR) were limited by low efficacy (<25%), poor viability, and a lack of allele-specificity. In contrast, adenine base editing corrected the recurrent and highly pathogenic p.T385M mutation with upwards of 90% efficiency in patient T cells and HSCs without significant unintended on- or off-target genomic aberrations. Gene editing functionally restored total STAT1 expression (p<0.0217), STAT1 phosphorylation (p<0.0056), interferon-stimulated gene expression (OAS1; p=0.0005) and improved IL-17 production (p<0.0001). Edited HSCs retained multilineage differentiation capacity and sustained engraftment with persistence of the corrected allele at 16 weeks in humanized immunodeficient mice. These data demonstrate efficient and precise correction of STAT1 GOF mutations by base editing, with maintenance of the correction through long-term engraftment in vivo. This represents the first application of gene editing to correct a dominant gain-of-function mutation causing immunodeficiency, with potential applicability to other genetic disorders associated with heterozygous and gain-of-function mutations.},
}

@article {pmid42551559,
year = {2026},
author = {Khosrojerdi, M and Hashemi, SA and Besharati, R and Haghbin, A and Azimian, A},
title = {CRISPR-Cas systems as precision antimicrobials: Reversing the tide of antimicrobial resistance.},
journal = {Virus research},
volume = {371},
number = {},
pages = {199782},
doi = {10.1016/j.virusres.2026.199782},
pmid = {42551559},
issn = {1872-7492},
abstract = {Antimicrobial resistance (AMR) has escalated into a global health crisis, with resistant pathogens causing over 1.2 million direct deaths annually and threatening to render modern medicine unsustainable. This review provides a comprehensive and updated synthesis of CRISPR-Cas-based antimicrobial strategies with a unique focus on: (i) critical comparison with conventional antibiotics and emerging alternatives; (ii) quantitative evaluation of delivery platforms; (iii) novel strategies including AI-optimized guide design and the ATTACK-CreTA system; (iv) comprehensive analysis of ecological risks; and (v) technology readiness level assessments for clinical translation. The CRISPR-Cas system, originally discovered as a bacterial adaptive immune mechanism, has been repurposed as a programmable precision tool to combat AMR by selectively targeting and eliminating resistance genes. We systematically evaluate the mechanistic diversity of Cas effectors, from DNA-cleaving Cas9 and Cas3 to RNA-targeting Cas13, and their application in reversing resistance phenotypes in WHO priority pathogens. We critically assess emerging delivery platforms, including engineered bacteriophages, conjugative plasmids, nanoparticles, and outer membrane vesicles, quantitatively comparing their delivery efficiency, payload capacity, and biosafety profiles. Novel strategies such as CRISPR interference (CRISPRi) for gene silencing without genomic cleavage, the ATTACK-CreTA system for enhanced bactericidal activity, and AI-driven optimization of guide RNA design are examined with appropriate caveats. We comprehensively address clinical translation challenges including immunogenicity, pharmacokinetics/pharmacodynamics, manufacturing scalability, regulatory pathways, and bacterial resistance mechanisms including anti-CRISPR proteins. No CRISPR-based antimicrobial has yet received regulatory approval, and we critically evaluate the gap between proof-of-concept and clinical utility. A detailed roadmap for clinical development is proposed. By integrating recent advances in Cas protein engineering, delivery technologies, and diagnostic applications, this review positions CRISPR-Cas systems as next-generation precision therapeutics capable of both treating resistant infections and curtailing the spread of AMR across clinical and environmental settings.},
}

@article {pmid42554845,
year = {2026},
author = {Zubair, A and Hemal, MAKP and Ahmed, A and Ahmad, F and Waheed, Y and Afghan, N},
title = {CRISPR/Cas system as a novel therapeutic strategy to combat multi-drug-resistant bacteria.},
journal = {Archives of microbiology},
volume = {208},
number = {11},
pages = {},
pmid = {42554845},
issn = {1432-072X},
mesh = {*Drug Resistance, Multiple, Bacterial/genetics ; *CRISPR-Cas Systems ; *Bacteria/genetics/drug effects ; Anti-Bacterial Agents/pharmacology ; Humans ; *Bacterial Infections/therapy/microbiology ; Gene Editing/methods ; },
abstract = {Antimicrobial resistance (AMR) has diminished the effectiveness of present antibiotics, posing a huge threat to global community health and economic stability. This study investigates the CRISPR-Cas framework's potential as a cutting-edge tactic to fight antimicrobial resistance. Current applications, limitations, and prospective future uses are analyzed. CRISPR antimicrobial strategies, which bring together the latest developments in gene-targeting strategies, engineered delivery platforms, and translational applications to fight multidrug-resistant pathogens. CRISPR technology is different from traditional antimicrobial treatments that target general antimicrobial resistance genes, instead allowing targets to be eliminated specifically by sequence, while retaining beneficial microbial communities, which has the potential to be a transformative precision antimicrobial treatment. Nevertheless, there is still a need for optimization of delivery systems, specificity of targets, biosafety, and regulations to ensure successful clinical translation, especially given their amazing advances. Recent research confirms that CRISPR-based mechanisms also affect different bacterial species, including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species, playing a key function in averting the emergence of resistance genes in these bacteria. Changes to CRISPR loci affect how resistance genes are targeted in ESKAPE pathogens, and CRISPR-Cas9 successfully lowers resistance by focusing on genes like tetM and ermB. A promising application of CRISPR-Cas systems in combating antimicrobial resistance (AMR) is the precise targeting of plasmid-borne mcr-1 resistance genes and other mobile genetic elements that facilitate the dissemination of colistin resistance. But the efficiency of CRISPR-Cas is diminished in some bacterial strains due to variations in their CRISPR loci. Enhancing transformation approaches and minimizing off-target impacts are critical challenges to confirm the precision and safety of CRISPR-based mechanisms in therapeutic applications. Advances in these areas are likely to continue to enable the development of next-generation CRISPR therapeutics for the effective management of multidrug-resistant bacterial infections.},
}

*Drug Resistance, Multiple, Bacterial/genetics
*CRISPR-Cas Systems
*Bacteria/genetics/drug effects
Anti-Bacterial Agents/pharmacology
Humans
*Bacterial Infections/therapy/microbiology
Gene Editing/methods

@article {pmid42556039,
year = {2026},
author = {Li, H and Du, H and Xu, R and Xue, H and Liu, Y and Cao, Y and Guo, Z and Pan, J and Liu, Y},
title = {Point-of-care detection for respiratory diseases: From samples and biomarkers to principles and applications.},
journal = {Talanta},
volume = {312},
number = {Pt A},
pages = {130377},
doi = {10.1016/j.talanta.2026.130377},
pmid = {42556039},
issn = {1873-3573},
abstract = {Early screening can significantly reduce the severe morbidity and mortality of respiratory diseases and alleviate the burden on public healthcare systems. Point-of-care (POC) devices refer to portable instruments that meet the REASSURED criteria and can be conveniently used near the patient without professional laboratory conditions. POC devices played an important role in patient initiated early diagnosis during the COVID-19 pandemic, demonstrating great potential. From a macro-to-micro perspective, this review first comprehensively introduces clinically relevant sample types, including blood, respiratory tract and oral samples, and exhaled breath, along with the clinical significance of corresponding biomarkers (nucleic acids, proteins, gaseous molecules, extracellular vesicles, circulating tumor cells, and pathogen particles) and pre-processing methods. Subsequently, it summarizes the test principles and promising bioreceptors including base pairing-based systems (PCR, isothermal amplification, CRISPR/Cas), antibodies and antibody mimetics, and other affinity-based recognition elements, and innovatively presents portable integration platforms of biosensors from the perspective of bioreceptor compatibility. It then evaluates the application performance of transducers and corresponding optical or electrochemical portable detection devices, including SERS, e-nose, nanopore sensors, and portable GC-MS. Finally, the latest applications of computer technology and artificial intelligence tools in POC detection for respiratory diseases, spanning device design, bioreceptor screening, biomarker discovery, and diagnostic data processing, are presented by functional category. This review aims to provide a reference for the research and application of multiplex biomarker/sample/disease POC testing in respiratory diseases, and to offer perspectives on future directions for technological innovation, intelligentization, and commercial translation.},
}

@article {pmid42556347,
year = {2026},
author = {Zhou, Z and Saffarian-Deemyad, I and Shi, H and Weiss, T and Ur-Rehman, MM and Vohra, K and Skopintsev, P and Yoon, PH and Trinidad, MI and Langeberg, CJ and Kamalu, M and Amerasekera, J and Zhou, Y and Doherty, EE and Aris, KDP and Al-Sayyad, N and Thornton, BW and Weissman, RF and Wasko, KM and Esain-Garcia, I and DeTurk, EC and Savage, DF and Jacobsen, SE and Bryant, Z and Doudna, JA},
title = {Stepwise DNA-unwinding gates TnpB genome-editing activity.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2026.07.015},
pmid = {42556347},
issn = {1097-4164},
abstract = {TnpB is a compact RNA-guided endonuclease and an evolutionary ancestor of CRISPR-Cas12 that offers a promising platform for genome engineering. However, the genome-editing activity of TnpBs remains limited, and its underlying determinants are poorly understood. Here, we used biochemical and single-molecule assays to examine the DNA-unwinding mechanism of Youngiibacter multivorans TnpB (Ymu1 TnpB). DNA unwinding proceeds through a discrete, long-lived, partially unwound intermediate state before reaching a fully unwound open state. The open state forms inefficiently and collapses readily in the absence of negative supercoiling. An optimized variant, H4W-L304F-V305R (Ymu1-WFR), stabilizes formation of both the intermediate and open states, resulting in enhanced DNA cleavage in vitro and increased genome editing in plants. These findings identify the physical basis for the observed minimal activities of natural TnpBs, revealing how stabilizing specific unwinding states enables efficient DNA targeting.},
}

@article {pmid42557080,
year = {2026},
author = {Hu, YW and Zhang, Y and Ren, Y and Qin, H and Zhao, H},
title = {[Advances in phage therapy for pneumonia caused by Klebsiella pneumoniae].},
journal = {Zhonghua jie he he hu xi za zhi = Zhonghua jiehe he huxi zazhi = Chinese journal of tuberculosis and respiratory diseases},
volume = {49},
number = {8},
pages = {902-907},
doi = {10.3760/cma.j.cn112147-20251012-00632},
pmid = {42557080},
issn = {1001-0939},
support = {202304021301063//Shanxi Province scientific and technological achievements transformation guidance project/ ; 2023-190//Research Project Supported by Shanxi Scholarship Council of China/ ; },
mesh = {*Klebsiella pneumoniae/virology ; Humans ; *Phage Therapy/methods ; *Klebsiella Infections/therapy/microbiology ; *Pneumonia, Bacterial/therapy/microbiology ; Bacteriophages ; Animals ; Anti-Bacterial Agents/therapeutic use ; Drug Resistance, Multiple, Bacterial ; },
abstract = {Klebsiella pneumoniae (KP) has emerged as a formidable nosocomial pathogen in the era of antimicrobial resistance, with mortality from pneumonia caused by carbapenem-resistant strains exceeding 50%. Phage therapy has re-emerged as a promising alternative or adjunctive strategy for managing refractory KP infections. This review consolidates the current preclinical and clinical evidence base, outlines the molecular mechanisms of phage-host interactions, and appraises evolving therapeutic approaches. Preclinical investigations in murine pneumonia models have consistently demonstrated that intranasal or nebulization phage administration markedly reduces pulmonary bacterial burden, attenuates inflammatory lung injury, and improves survival, often exhibiting synergistic effects when combined with conventional antibiotics. Clinical case reports and small compassionate-use series have further provided preliminary yet compelling evidence supporting the safety and therapeutic promise of personalized phage formulations in critically ill patients with multidrug-resistant KP pneumonia who have exhausted standard treatment options. Mechanistically, phage tropism is mediated through the specific recognition of bacterial surface receptors-principally capsular polysaccharide and, to a lesser extent, lipopolysaccharide-by phage-encoded receptor-binding proteins, culminating in bacterial lysis. In response, KP has evolved a multilayered defensive arsenal encompassing receptor modification to impede adsorption, nucleic acid interference systems (e.g., CRISPR-Cas and restriction-modification), and abortive infection mechanisms that curtail phage propagation at the population level. To surmount the inherent limitations of narrow host range and the inevitable emergence of phage-resistant mutants, a suite of optimization strategies is under active refinement, including rationally designed phage cocktails, genetically engineered phages with extended tropism, artificial intelligence-assisted host-range prediction, and innovative delivery platforms such as hydrogel encapsulation to enhance pulmonary bioavailability. Despite ongoing challenges in mechanistic complexity, manufacturing standardization, and regulatory uncertainty, current initiatives- such as the establishment of geographically diverse phage libraries, real-time surveillance of phage resistance, and the development of phage-derived enzyme products-hold promise for establishing precision phage therapy as a viable and sustainable component of the antimicrobial stewardship armamentarium.},
}

*Klebsiella pneumoniae/virology
Humans
*Phage Therapy/methods
*Klebsiella Infections/therapy/microbiology
*Pneumonia, Bacterial/therapy/microbiology
Bacteriophages
Animals
Anti-Bacterial Agents/therapeutic use
Drug Resistance, Multiple, Bacterial

@article {pmid42557140,
year = {2026},
author = {Zhou, X and Wang, L and Peng, X and Liu, M and Li, Y and Gui, F and Zhou, L and Deng, Z},
title = {A light-activated one-pot ERA/CRISPR-Cas12a assay for cost-effective dual-mode detection of HBV DNA.},
journal = {Talanta},
volume = {},
number = {},
pages = {130329},
doi = {10.1016/j.talanta.2026.130329},
pmid = {42557140},
issn = {1873-3573},
abstract = {Light-activatable CRISPR-Cas systems offer an effective strategy to overcome the kinetic incompatibility in one-pot nucleic acid assays; however, their practical application remains limited by high reagent cost, the lack of compatible reaction buffers, and reliance on instrument-dependent readout. In this work, we developed a cost-effective light-controlled one-pot ERA/CRISPR-Cas12a platform for rapid hepatitis B virus (HBV) DNA detection. NPOM-caged crRNA was employed to temporarily suppress Cas12a activity during amplification and to trigger target-dependent trans-cleavage by a brief 30 s UV irradiation. By replacing RPA with ERA, the per-reaction reagent cost of the one-pot assay was reduced from approximately US$6.0 to US$2.0, corresponding to a reduction of approximately 66.7%, while maintaining comparable analytical performance. In addition, a PEG-free unified buffer was established through systematic optimization, enabling efficient integration of ERA and Cas12a reactions in a sealed single-tube format. The proposed platform provided dual-mode readout through fluorescence and lateral flow assay (LFA), with a limit of detection of 1 copy μL[-1] and a total assay time of 30 min. Clinical evaluation using 79 serum samples showed a sensitivity of 100% for fluorescence readout and 96.3% for LFA, with excellent agreement with qPCR (Cohen's κ = 0.94 - 1.00). Owing to its low cost, high sensitivity, and operational simplicity, this platform represents a promising tool for rapid HBV molecular diagnosis in both centralized laboratories and resource-limited settings.},
}

@article {pmid42133510,
year = {2026},
author = {Cimolato, C and Petrelli, S and Favaro, D and Frusteri Chiacchiera, A and Del Favero, S and Schenato, L and Pasotti, L and Bellato, M},
title = {Modeling of Conjugative- and Phage-Mediated CRISPR-Based System Against Antimicrobial Resistant Bacteria.},
journal = {IEEE transactions on bio-medical engineering},
volume = {73},
number = {8},
pages = {2961-2971},
doi = {10.1109/TBME.2026.3693402},
pmid = {42133510},
issn = {1558-2531},
mesh = {*Bacteriophages/genetics ; *Drug Resistance, Bacterial/genetics ; *CRISPR-Cas Systems/genetics ; *Models, Biological ; Computer Simulation ; *Bacteria/genetics/virology/drug effects ; *Phage Therapy/methods ; },
abstract = {OBJECTIVE: Antimicrobial resistance (AMR) poses a significant threat to global health by diminishing the effectiveness of conventional antibiotics. This study aims to assess, using a systems biology approach, a potential synthetic biology-based strategy that employs engineered conjugative probiotic bacteria and bacteriophages to combat AMR, examining the implications of implementing targeted gene silencing as an alternative to direct bacterial killing.

METHODS: A comprehensive mathematical model was developed to describe the dynamics of the delivery systems (engineered conjugative probiotic bacteria and engineered phages) and the antimicrobial actuators being studied (genome cutting via CRISPR systems and AMR-gene silencing through CRISPR interference), also compared to traditional phage therapy (selection of phages capable of killing pathogens through bacterial-specific viral infection). The target population includes antibiotic-resistant bacteria competing with other probiotic bacteria for colonizing the host environment. The model explicitly incorporates parameters for mutations that affect actuator functionality and simulates their impact on overall therapeutic performance.

RESULTS: Simulations show how variations in actuator efficiency and emergence of new mutations in target pathogens affect the long-term suppression of resistance genes. Including mutational effects provides insights into system robustness and guides optimal therapeutic design choices.

CONCLUSION: The proposed modeling framework effectively captures key biological and mechanistic aspects of engineered therapies, enabling the prediction and optimization of each intervention against resistant pathogens. It highlights engineered phages and CRISPR interference as the most promising candidates for the design of new engineered biological therapeutics.

SIGNIFICANCE: This study establishes a quantitative foundation for rational design and dosage optimization in engineered phage- and bacterial-based therapies, advancing the use of synthetic biology methods to fight antimicrobial resistance.},
}

*Bacteriophages/genetics
*Drug Resistance, Bacterial/genetics
*CRISPR-Cas Systems/genetics
*Models, Biological
Computer Simulation
*Bacteria/genetics/virology/drug effects
*Phage Therapy/methods

@article {pmid42405454,
year = {2026},
author = {Chen, Z and Wu, H and Chu, LT and Lin, J and Zhang, Y and Lin, X and Zeng, T},
title = {Aptamer-based CRISPR-Cas12a fluorescent biosensors for serum biomarker detection.},
journal = {The Analyst},
volume = {151},
number = {16},
pages = {4516-4534},
doi = {10.1039/d6an00123h},
pmid = {42405454},
issn = {1364-5528},
mesh = {*Biosensing Techniques/methods ; *Aptamers, Nucleotide/chemistry/metabolism/genetics ; *CRISPR-Cas Systems ; Humans ; *Biomarkers/blood ; *CRISPR-Associated Proteins/chemistry/metabolism/genetics ; *Bacterial Proteins/genetics/chemistry/metabolism ; *Endodeoxyribonucleases/genetics/metabolism/chemistry ; Fluorescent Dyes/chemistry ; },
abstract = {The CRISPR-Cas12a system enables sensitive nucleic acid detection due to its programmability, trans-cleavage activity, and biocompatibility. To expand its applications beyond nucleic acid analysis, aptamers have emerged as ideal recognition elements owing to their high specificity, design flexibility, ease of modification and low cost. The integration of Cas12a with aptamers enables the conversion of target-binding signals into nucleic acid recognition signals, thereby combining molecular recognition with signal amplification for the detection of non-nucleic acid targets. This review provides a concise overview of the working mechanism and features of the Cas12a system, with particular emphasis on recent advances in Cas12a-aptamer-based fluorescent biosensors for serum biomarker detection. The advantages and limitations, current challenges, and future prospects are also discussed.},
}

*Biosensing Techniques/methods
*Aptamers, Nucleotide/chemistry/metabolism/genetics
*CRISPR-Cas Systems
Humans
*Biomarkers/blood
*CRISPR-Associated Proteins/chemistry/metabolism/genetics
*Bacterial Proteins/genetics/chemistry/metabolism
*Endodeoxyribonucleases/genetics/metabolism/chemistry
Fluorescent Dyes/chemistry

@article {pmid42547606,
year = {2026},
author = {Akkoul, N and Kumar, T and Sharma, S and Sharma, V and Raja, V and Malik, TG and Wan Mohd Jaafar, WS and Gupta, AK and Hakeem, KR},
title = {CRISPR-Cas-based detection of Mycobacterium tuberculosis: current advances and translational bottlenecks.},
journal = {Protoplasma},
volume = {},
number = {},
pages = {},
pmid = {42547606},
issn = {1615-6102},
abstract = {Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major global health challenge due to persistent diagnostic gaps. CRISPR-Cas-based diagnostics have emerged as highly sensitive and programmable platforms for nucleic acid detection, enabling rapid identification of Mtb targets, including drug-resistance-associated mutations. These systems integrate isothermal amplification, diverse Cas effectors, and multiple signal readout strategies to achieve high analytical performance. This review provides a comparative analysis of clinically evaluated CRISPR-based TB diagnostic platforms, highlighting substantial variability in assay design, performance, and translational readiness. While many platforms demonstrate strong analytical sensitivity, their implementation remains constrained by workflow complexity and limited integration into true point-of-care formats. This highlights that successful clinical translation of CRISPR-based TB diagnostics is determined more by real-world adaptability than by analytical performance alone. The current review presents a comparative analysis of CRISPR-based diagnostic platforms for tuberculosis, evaluating the variability in assay design, analytical and clinical performance, and translational readiness across currently available systems.},
}

@article {pmid42548009,
year = {2026},
author = {Chen, SY and Yang, LH and Liang, ZQ and Li, JD and Li, SD and Deng, YL and Chen, GQ and Ling, JW and Zhou, SS and Chen, G and He, RQ},
title = {CRISPR Screen Reveals Pathways and Factors Driving Tyrosine Kinase Inhibitor Resistance in Hepatocellular Carcinoma.},
journal = {Cancer medicine},
volume = {15},
number = {8},
pages = {e72028},
pmid = {42548009},
issn = {2045-7634},
support = {2025GXNSFAA069130//Joint Project on Regional High-Incidence Diseases Research of Guangxi Natural Science Foundation/ ; NSFC82460783//The National Natural Science Foundation of China/ ; //Creative Research Development Grant from the First Affiliated Hospital of Guangxi Medical University/ ; S202410598060X//The China Undergraduate Innovation and Entrepreneurship Training Program/ ; YCBZ2025127//The Innovation Project of Guangxi Graduate Education/ ; },
mesh = {Humans ; *Carcinoma, Hepatocellular/genetics/drug therapy/pathology/metabolism ; *Liver Neoplasms/genetics/drug therapy/pathology/metabolism ; *Drug Resistance, Neoplasm/genetics ; *Protein Kinase Inhibitors/pharmacology/therapeutic use ; Gene Expression Regulation, Neoplastic ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Signal Transduction ; },
abstract = {Tyrosine kinase inhibitor (TKI) resistance severely limits clinical outcomes in hepatocellular carcinoma (HCC), highlighting the urgent need to elucidate its underlying molecular mechanisms. In this study, an unbiased genome-wide CRISPR/Cas9 screening identified novel key factors related to the therapeutic responsiveness of TKI in HCC. By integrating data from 20 datasets encompassing 322 samples, a comprehensive TKI therapeutic response landscape for HCC was constructed. GO and Reactome enrichment analyses revealed that dysregulated RNA splicing, ubiquitination, endocytosis/exocytosis, and cell cycle pathways modulate TKI sensitivity, with close links to antitumor immunity. This study identified GPATCH4, CCT3, C19orf53, UACA, PPM1M, and LIN37 as key genes mediating TKI resistance in HCC. These six genes were found to be highly expressed in HCC and significantly associated with HCC patient prognosis. Drug sensitivity assays identified a significant association between their expression and responsiveness to TKI agents. In-house quantitative real-time PCR validated their differential expression levels in normal hepatocytes, parental HCC cells, and TKI-resistant HCC sublines. ssGSEA, TIMER2, and ESTIMATE analysis revealed that their expression modulates HCC immune infiltration. Bibliometric analysis revealed a growing focus on immunotherapy-based combination regimens to overcome TKI resistance. Ferroptosis, epithelial-mesenchymal transition and hypoxia were new research directions, which were closely related to the pathways investigated in this study. In conclusion, this study identified RNA splicing, ubiquitination, endocytosis/exocytosis, and cell cycle pathways, as well as GPATCH4, CCT3, C19orf53, UACA, PPM1M, and LIN37, as novel directions and targets for TKI-immunotherapy combination strategies, providing new insights for overcoming TKI resistance in HCC.},
}

Humans
*Carcinoma, Hepatocellular/genetics/drug therapy/pathology/metabolism
*Liver Neoplasms/genetics/drug therapy/pathology/metabolism
*Drug Resistance, Neoplasm/genetics
*Protein Kinase Inhibitors/pharmacology/therapeutic use
Gene Expression Regulation, Neoplastic
*CRISPR-Cas Systems
Cell Line, Tumor
Signal Transduction

@article {pmid42549139,
year = {2026},
author = {Pytlik, D and Gerovac, M and Bischler, T and Schlosser, A and Vogel, J and Schoen, C},
title = {The CRISPR/Cas-associated scaRNA modulates efeUOB expression and stress responses in Neisseria meningitidis.},
journal = {microLife},
volume = {7},
number = {},
pages = {uqag027},
pmid = {42549139},
issn = {2633-6693},
abstract = {Neisseria meningitidis is a human-adapted commensal pathogen that must continuously balance nutrient acquisition with stress tolerance. Here, we identify a type II-C CRISPR/Cas-associated small RNA (scaRNA) as a posttranscriptional regulator of the efeUOB operon and oxidative stress responses. Using in vitro RNA binding and structure probing assays, we show that the scaRNA interacts with the 5' untranslated region of efeO mRNA, leading to reduced translation of this component of the ferrous iron transporter EfeUOB. Consistent with this, efeO translational fusions demonstrate repression by the scaRNA, whereas a ΔscaRNA mutant shows increased reporter expression. We further show that meningococcal Cas9 (Nme1Cas9) is able to cleave scaRNA in vitro, but in vivo phenotypes are primarily scaRNA-dependent, indicating that Nme1Cas9 contributes, at most, indirectly to this regulation. In line with this observation, comparative proteomics revealed overlapping but distinct roles of scaRNA and Nme1Cas9 in oxidative stress adaptation, energy metabolism, and ion transport. While steady-state protein abundances did not capture all scaRNA-dependent effects, functional assays confirmed that scaRNA inactivation reduces survival under oxidative stress. Together, our results identify scaRNA-mediated repression of efeO as a novel posttranscriptional mechanism that contributes to stress adaptation in meningococci. These findings expand the functional repertoire of CRISPR-associated elements and suggest a role for small RNA-based regulation in iron-related stress adaptation in a major human pathogen.},
}

@article {pmid42549572,
year = {2026},
author = {Iwe, IA and Liu, FX and Corsano, A and da Silva, SJR and Doucet, J and Singh, S and Lamothe, G and Zayani, R and Nguyen, J and Matthews, Q and Vigar, JRJ and Bayat, P and Simchi, M and Bozovicar, K and Charania, M and Panfilov, S and Kelly, P and Cai, R and Hubbard, BP and Li, X and Mazzulli, T and Tremblay, JP and Zhao, Y and Green, AA and Li, Z and Yao, S and Pardee, K},
title = {RAPID: evaluation of Cas12a protospacer nicking and chimeric reporters for PAM-independent RNA and DNA diagnostics.},
journal = {Nucleic acids research},
volume = {54},
number = {14},
pages = {},
pmid = {42549572},
issn = {1362-4962},
support = {//Precision Medicine Initiative/ ; PRMUHT2024-001//Canadian Institute of Health Research/ ; 202410MFE-531769-419793//Canadian Institute of Health Research/ ; //Ontario Graduate Scholarship/ ; 201610FDN-375469//CIHR Foundation/ ; 950-231075//Canada Research Chairs Program/ ; 950-233107//Canada Research Chairs Program/ ; RGPIN-2016-06352//NSERC Discovery/ ; N66001-23-2-4042//Defense Advanced Research Projects Agency/ ; PJT-189974//CIHR/ ; },
mesh = {*CRISPR-Associated Proteins/metabolism/genetics ; *RNA/analysis/genetics ; *DNA/analysis/genetics ; *CRISPR-Cas Systems ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; Genes, Reporter ; },
abstract = {CRISPR-Cas nucleases have revolutionized diagnostics and biotechnology by providing programmable specificity. Here, we extend the understanding of Cas12a biology with a screen that, unexpectedly, finds that Cas12a trans-cleavage activity can be modulated by nicks in the protospacer in a position-dependent manner. Wanting to explore the impact of non-conventional trans-cleavage substrates, we subsequently find that non-specific Cas12a cleavage can be significantly reduced with RNA and chimeric (mixed RNA/DNA) reporter sequences. Exploiting these features and building on emerging protospacer adjacent motif (PAM)-independent Cas12a diagnostics that use engineered DNA activators and split-guide architectures, we introduce RAPID (RNA/DNA Advanced chimeric, PAM-independent, Integrated Nicking, Diagnostics), a nick-tuned, PAM-duplex-mediated platform for PAM-independent RNA and DNA detection. By strategically introducing a nick within the spacer region, RAPID expands Cas12a detection to include target RNAs, which can be ligated in situ to create a hybrid protospacer-target with trans-cleavage activity matching conventional Cas12a. We then apply RAPID to detect single-point mutations in ssDNA and RNA substrates, a challenge for traditional Cas12 and Cas13 systems. In combination with RT-LAMP, RAPID is used for PAM-independent RNA detection in clinical samples, achieving sensitivity down to ∼1 aM and 100% concordance with RT-qPCR for samples with Ct ≤ 33.},
}

*CRISPR-Associated Proteins/metabolism/genetics
*RNA/analysis/genetics
*DNA/analysis/genetics
*CRISPR-Cas Systems
*Endodeoxyribonucleases/metabolism/genetics
*Bacterial Proteins/metabolism/genetics
Genes, Reporter

@article {pmid42549575,
year = {2026},
author = {Li, Y and Han, P and Yuan, R and Bai, L and Qing, M},
title = {Cas-regulation-targeting chimera enables selective and tunable control of CRISPR/Cas12a.},
journal = {Nucleic acids research},
volume = {54},
number = {14},
pages = {},
pmid = {42549575},
issn = {1362-4962},
support = {82072378//National Natural Science Foundation of China/ ; 22504009//National Natural Science Foundation of China/ ; CSTB2025NSCQ-LZX0126//Natural Science Foundation of Chongqing/ ; CSTB2025NSCQ-GPX0375//Natural Science Foundation of Chongqing/ ; CQYC202005015//Chongqing Talents: Exceptional Young Talents Project, China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; },
abstract = {Selective and tunable regulation of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a activity enables on-demand control, yet current strategies remain hindered by nonspecific regulation and limited tunability. Inspired by proximity effect, we present a Cas-regulation-targeting chimera (CasTAC) strategy that employs CRISPR RNA (crRNA) as a proximity mediator to carry phosphorothioate regulators to interfere with catalytic or recognition domains of Cas12a and consequently suppress its activity. This crRNA-induced proximity approach can effectively eliminate nonspecific interaction between phosphorothioate regulators and proteins within complex multi-enzyme systems, thereby enabling selective control over CRISPR/Cas12a activity. Furthermore, CRISPR/Cas12a activity can be finely tuned to different inhibitory levels by varying the number of phosphorothioate regulators. The CasTAC strategy also improves nuclease resistance and single-nucleotide discrimination, offering potential advances in the sensitivity of molecular diagnostics and the accuracy of gene editing. Notably, the CasTAC balances the kinetics of nucleic acid amplification and CRISPR cleavage, facilitating efficient product accumulation and resolving compatibility issues in one-pot assays. As a proof of concept, we develop a one-pot, one-step recombinase polymerase amplification-CasTAC assay that achieves over 1000-fold higher detection sensitivity than the conventional one-pot recombinase polymerase amplification-CRISPR/Cas12a assay. The CasTAC strategy provides a versatile framework for fine-tuning Cas activity and advances CRISPR technology toward refined and context-adaptable functionality.},
}

*CRISPR-Cas Systems/genetics
*CRISPR-Associated Proteins/metabolism/genetics
*Endodeoxyribonucleases/metabolism/genetics
*Bacterial Proteins/metabolism/genetics

@article {pmid42549577,
year = {2026},
author = {Winter, E and Emiliani, F and Cook, A and Abderrahim, A and McKenna, A},
title = {BASELINE: a CRISPR base editing platform for mammalian-scale single-cell lineage tracing.},
journal = {Nucleic acids research},
volume = {54},
number = {14},
pages = {},
pmid = {42549577},
issn = {1362-4962},
support = {DP2GM149750/NH/NIH HHS/United States ; //V Foundation/ ; //Pew Biomedical Scholars/ ; },
mesh = {*Cell Lineage/genetics ; *Single-Cell Analysis/methods ; Animals ; *CRISPR-Cas Systems ; Humans ; *Gene Editing/methods ; Pancreatic Neoplasms/genetics/pathology ; },
abstract = {A cell's fate is shaped by its inherited state, or lineage, and the ever-shifting context of its environment. CRISPR-based recording technologies are a promising solution for mapping the lineage of a developing system; however, challenges remain regarding single-cell recovery, engineering complexity, and scale. Here, we introduce BASELINE, which uses base editing to generate high-resolution lineage trees in conjunction with single-cell profiling. BASELINE uses the Cas12a adenine base editor to irreversibly edit nucleotides across target arrays built from 50 synthetic target sites, which are integrated multiple times into a cell's genome. We demonstrate that BASELINE accumulates lineage-specific marks over a wide range of biologically relevant intervals, recording more than 4300 bits of information in a model of pancreatic cancer, a 50-fold increase over existing technologies. Single-cell sequencing reveals high-fidelity capture of these recorders, averaging 29 cell divisions captured per lineage, within the estimated range of mammalian development. We expect BASELINE to apply to a wide range of lineage-tracing projects in development and disease, especially those in which cellular engineering makes small, more distributed systems challenging.},
}

*Cell Lineage/genetics
*Single-Cell Analysis/methods
Animals
*CRISPR-Cas Systems
Humans
*Gene Editing/methods
Pancreatic Neoplasms/genetics/pathology

@article {pmid42549821,
year = {2026},
author = {Jafari, A and Manzari-Tavakoli, A and Manzari Tavakoli, M and Hashemian, SM and Jamaati, H and Akhtari, M and Tabarsi, P and Omrani, M},
title = {Theranostic innovation in infectious lung diseases: integrating biotechnology and nanotechnology for precision medicine.},
journal = {Expert review of molecular diagnostics},
volume = {},
number = {},
pages = {},
doi = {10.1080/14737159.2026.2714060},
pmid = {42549821},
issn = {1744-8352},
abstract = {INTRODUCTION: Introduction: Infectious lung diseases, including pneumonia, tuberculosis (TB), COVID-19, influenza, and emerging fungal infections, are major causes of illness and death worldwide. Traditional methods have serious limitations such as diagnostic delays, antimicrobial resistance, and non-targeted therapy. Theranostics offers a transformative precision medicine paradigm for pulmonary infections.

AREAS COVERED: This review looks closely at how biotechnology and nanotechnology synergistically advance theranostic strategies for infectious lung diseases. We explore biotechnological tools including CRISPR-Cas systems, non-coding RNAs (ncRNAs), and monoclonal antibodies (mAbs) for detecting specific pathogens and intervening directly. We also discuss nanotechnological platforms such as nanosensors, surface-enhanced Raman spectroscopy (SERS), and various nanocarriers (lipid nanoparticles, polymeric nanoparticles, liposomes, metallic nanoparticles, mesoporous silica nanoparticles, and biomimetic systems) for drug, gene, and vaccine delivery with better targeting, controlled release, and imaging capabilities. Integrated case studies across major diseases, including COVID-19, influenza, TB, pneumonia, COPD, and idiopathic pulmonary fibrosis, demonstrate effective theranostic applications. We also address associated challenges like safety, manufacturing, regulatory hurdles, and economic feasibility.

EXPERT OPINION: The combination of biotechnology and nanotechnology represents a paradigm shift toward personalized pulmonary medicine. Future success needs to develop smart, multi-stimuli-responsive nanoplatforms, integrating artificial intelligence for predictive modeling and treatment optimization, and establishing closed-loop theranostic systems that connect real-time diagnostics with adaptive therapies. Key priorities include standardized preclinical models, clear regulations for combination products, and health economic analyses demonstrating cost-effectiveness. Interdisciplinary collaboration among material scientists, molecular biologists, clinicians, and regulatory specialists will be essential to translate these promising platforms from bench to bedside.},
}

@article {pmid42550282,
year = {2026},
author = {Yang, L and Ji, X and Li, Z and Duan, F and Jia, Q and Zhang, S and Hu, B and Zhang, Z},
title = {Ultrasensitive electrochemiluminescence determination of Salmonella based on CRISPR/Cas12a integrated with bimetallic semiconductive metal-organic frameworks.},
journal = {Mikrochimica acta},
volume = {193},
number = {9},
pages = {},
pmid = {42550282},
issn = {1436-5073},
support = {252300420760//Natural Science Foundation of Henan Province/ ; 252300421534//Natural Science Foundation of Henan Province/ ; 254200510025//Zhongyuan Sci-Tech Innovation Leading Talents/ ; 25IRTSTHN003//Program for Innovative Research Team (in Science and Technology) in University of Henan Province/ ; },
mesh = {*Biosensing Techniques/methods ; *Luminescent Measurements/methods ; *Salmonella/isolation & purification/chemistry ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; *Metal-Organic Frameworks/chemistry ; Limit of Detection ; DNA, Single-Stranded/chemistry ; Semiconductors ; *Endodeoxyribonucleases/chemistry/genetics/metabolism ; *CRISPR-Associated Proteins/chemistry/genetics/metabolism ; Bacterial Proteins ; },
abstract = {An ultrasensitive electrochemiluminescence (ECL) biosensor was established by combining CRISPR/Cas12a technique and semiconductive bimetallic-organic framework (scMOF) [[CuxNi3-x(HITP)2] (HITP = 2,3,6,7,10,11-hexaiminotriphenylene)]] emitter and employed to detect Salmonella using the allosteric probe as the recognition component. Given that CuxNi3-x(HITP)2 has demonstrated large specific surface area, both in-plane and out-of-plane charge transfer ability, narrowed band gap, and enhanced separation of holes and electrons, it can be simultaneously employed as the superior ECL emitter and bioplatform for anchoring single-strand DNA (ssDNA), thus improving the detection sensitivity toward Salmonella. The CRISPR/Cas12a-based system can specifically recognize the target sequence of Salmonella and activate the nuclease activity of Cas12a, and the activated Cas12a possesses trans-cleavage ability toward ssDNA. The CuxNi3-x(HITP)2 emitter is then released, resulting in the decline of the ECL response. The developed CuxNi3-x(HITP)2-CRISPR/Cas12a-based ECL biosensor exhibits the ultralow detection limit of 0.25 CFU mL[- 1] in the linear range from 1.0 CFU mL[- 1] to 10[6] CFU mL[- 1], significantly lower than those of reported ones. Furthermore, the developed biosensor exhibits outstanding overall biosensing properties with high selectivity, favorable reproducibility and stability, together with promising practical applicability for the determination of Salmonella in a variety of foodstuffs.},
}

*Biosensing Techniques/methods
*Luminescent Measurements/methods
*Salmonella/isolation & purification/chemistry
*Electrochemical Techniques/methods
*CRISPR-Cas Systems
*Metal-Organic Frameworks/chemistry
Limit of Detection
DNA, Single-Stranded/chemistry
Semiconductors
*Endodeoxyribonucleases/chemistry/genetics/metabolism
*CRISPR-Associated Proteins/chemistry/genetics/metabolism
Bacterial Proteins

@article {pmid42050729,
year = {2026},
author = {Sofianos, G and Petmezas, A and Samaras, A and Karaoglanidis, G},
title = {Resistance of Botrytis cinerea to anilinopyrimidine fungicides: A novel ARMS-PCR method for the detection of Bcpos5 mutations and characterization of resistance using CRISPR/Cas9 editing.},
journal = {Pest management science},
volume = {82},
number = {9},
pages = {8189-8198},
pmid = {42050729},
issn = {1526-4998},
support = {//General Secretariat for Research and Technology/ ; },
mesh = {*Botrytis/drug effects/genetics ; *Fungicides, Industrial/pharmacology ; *Drug Resistance, Fungal/genetics ; *Pyrimidines/pharmacology ; *Polymerase Chain Reaction/methods ; Mutation ; *CRISPR-Cas Systems ; *Fungal Proteins/genetics/metabolism ; Gene Editing ; },
abstract = {BACKGROUND: Anilinopyrimidine (AP) fungicides have been widely used against Botrytis cinerea, yet their resistance mechanisms have only recently been clarified. Resistance is primarily linked to mutations G408V, L412V, and L412F in the Bcpos5 gene, whose encoded protein is localized to the mitochondria. In this study, we developed a detection method and tested the fitness of L412F/V mutants obtained by using the CRISPR/Cas9 editing technique.

RESULTS: For rapid and cost-effective mutation identification, a TETRA-primer amplification refractory mutation system polymerase chain reaction (T-ARMS-PCR) was developed to rapidly detect the nucleotide alterations that lead to L412F and L412V mutations, producing a 702 bp band in all isolates, with additional 470 bp (F) or 252 bp (V) fragments. Isolates harboring only the 702 bp band were further digested with MlyI to confirm the mutation leading to the amino acid substitution G408V mutation (467 bp + 235 bp). Results were validated by Sanger sequencing. Application of the assay to 170 isolates from strawberry and tomato revealed mutation frequencies of 70.2% (L412F), 8.3% (L412V), and 4.7% (G408V) within the resistant fraction of the population (n = 82 resistant isolates). Furthermore, sequencing analysis revealed also a low frequency of the E407K mutation in Bcmdl1, along with evidence of additional, yet undefined, resistance mechanisms. To further characterize the mutations, the B. cinerea reference strain B05.10 was transformed with L412F and L412V alleles via CRISPR/Cas9 and homologous recombination. The resulting mutants displayed resistance to cyprodinil, and potential fitness costs were assessed in both field-derived and CRISPR/Cas9-generated isolates through measurements of mycelial growth and sporulation in vitro, and pathogenicity in planta. The L412F and L412V transformants did not differ significantly from the parental B05.10 strain in any of the evaluated fitness parameters.

CONCLUSION: Overall, the developed ARMS PCR offers a fast, cost-effective tool for resistance monitoring aiming to identify the most common mutations conferring resistance to APs, while CRISPR/Cas9 provides an efficient approach for functional validation of resistance mutations in B. cinerea. Using this approach, we confirmed that L412F and L412V mutations in Bcpos5 confer resistance to APs, while are not associated with fitness cost. © 2026 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.},
}

*Botrytis/drug effects/genetics
*Fungicides, Industrial/pharmacology
*Drug Resistance, Fungal/genetics
*Pyrimidines/pharmacology
*Polymerase Chain Reaction/methods
Mutation
*CRISPR-Cas Systems
*Fungal Proteins/genetics/metabolism
Gene Editing

@article {pmid42546506,
year = {2026},
author = {Pal, P and Anand, U and Saha, SC and Sundaramurthy, S and Okeke, ES and Kumar, M and Radha, and Bontempi, E and Albertini, E and Dey, A and Di Maria, F},
title = {Retraction notice to "Novel CRISPR/Cas technology in the realm of algal bloom biomonitoring: Recent trends and future perspectives" [Environ. Res 231 (2023) 115989].},
journal = {Environmental research},
volume = {306},
number = {Pt 4},
pages = {125335},
doi = {10.1016/j.envres.2026.125335},
pmid = {42546506},
issn = {1096-0953},
}

@article {pmid42547416,
year = {2026},
author = {Zhuang, Q and Wang, F and Zhang, H and Zhang, Q and Gao, Y and Feng, Z and Gao, H and Sun, S and Lin, S and Li, S and Zhao, Q and Huang, X and Li, Q and Sheng, W and Huang, G},
title = {A cardiac-related, promoter-proximal, regulatory element shapes chromatin and JAG1 transcription.},
journal = {Life science alliance},
volume = {9},
number = {10},
pages = {},
pmid = {42547416},
issn = {2575-1077},
mesh = {*Jagged-1 Protein/genetics/metabolism ; *Chromatin/genetics/metabolism ; *Promoter Regions, Genetic/genetics ; Animals ; Transcription, Genetic ; Mice ; Humans ; Enhancer Elements, Genetic ; Gene Expression Regulation ; Apoptosis/genetics ; Cell Movement/genetics ; Cell Proliferation/genetics ; CRISPR-Cas Systems ; Cell Line ; },
abstract = {The Jagged1 (JAG1) gene is essential for cardiac development, yet its tissue-specific transcriptional regulation remains poorly understood. In this study we used an integrative screening approach to identify 19 candidate enhancers within the ±100 kb region flanking the JAG1 locus, among which R7 exhibited the highest activity in dual-luciferase assays. CRISPR/Cas9-mediated deletion of R7 in AC16 cells significantly reduced JAG1 expression, decreased proliferative and migratory capacities, and increased apoptosis. Mechanistically, R7 deletion altered local chromatin contacts and reduced accessibility at CTCF-bound regions near the JAG1 promoter, accompanied by decreased H3K27ac, H3K4me3, RNA polymerase II, and SRF occupancy. These findings identify R7 as a cardiac-associated promoter-proximal regulatory element with enhancer-like activity that contributes to local chromatin organization and transcriptional activity at the JAG1 locus.},
}

*Jagged-1 Protein/genetics/metabolism
*Chromatin/genetics/metabolism
*Promoter Regions, Genetic/genetics
Animals
Transcription, Genetic
Mice
Humans
Enhancer Elements, Genetic
Gene Expression Regulation
Apoptosis/genetics
Cell Movement/genetics
Cell Proliferation/genetics
CRISPR-Cas Systems
Cell Line

@article {pmid42379397,
year = {2026},
author = {Shao, D and Wen, X and Luo, Q and Liu, X and Li, L and Men, S},
title = {Combined T-DNA and CRISPR/Cas9 mutagenesis reveals redundant developmental roles of the Arabidopsis BAG family.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {371},
number = {},
pages = {113305},
doi = {10.1016/j.plantsci.2026.113305},
pmid = {42379397},
issn = {1873-2259},
mesh = {*Arabidopsis/genetics/growth & development/metabolism ; *Arabidopsis Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; DNA, Bacterial/genetics ; Gene Expression Regulation, Plant ; Plant Growth Regulators/metabolism ; Indoleacetic Acids/metabolism ; *Molecular Chaperones/genetics/metabolism ; Germination/genetics ; },
abstract = {BAG (Bcl-2-associated athanogene) genes encode evolutionarily conserved co-chaperones that participate in proteostasis regulation, stress responses, and programmed cell death. However, their collective functions during plant development remain poorly understood. Promoter cis-element analysis revealed multiple hormone-responsive elements in promoters of Arabidopsis thaliana (Arabidopsis) BAG genes, suggesting potential involvement of BAG genes in phytohormone-mediated developmental regulation. To investigate this, we generated a bag-septuple (bag-s) mutant in which all seven Arabidopsis BAG genes were knocked out using a combination of T-DNA insertion alleles and CRISPR/Cas9-mediated mutagenesis. Phenotypic characterization revealed pleiotropic defects, including delayed seed germination, increased seed coat mucilage accumulation, reduced primary root elongation, decreased rosette diameter and plant height, and delayed leaf senescence. Consistent with the delayed leaf senescence phenotype, expression of senescence-associated genes and senescence-promoting transcription factors was downregulated in the bag-s mutant. RT-qPCR analyses further showed that genes involved in auxin biosynthesis and auxin signaling were downregulated in the bag-s mutant. Furthermore, exogenous IAA partially rescued the root elongation defect of the bag-s mutant, supporting a functional association between BAG genes and auxin-dependent root growth. Collectively, these findings indicate that BAG genes redundantly regulate seed germination, vegetative growth, auxin-related root development, and leaf senescence, providing a genetic framework for further dissecting BAG-mediated coordination of proteostasis, hormone signaling, and plant development.},
}

*Arabidopsis/genetics/growth & development/metabolism
*Arabidopsis Proteins/genetics/metabolism
*CRISPR-Cas Systems
DNA, Bacterial/genetics
Gene Expression Regulation, Plant
Plant Growth Regulators/metabolism
Indoleacetic Acids/metabolism
*Molecular Chaperones/genetics/metabolism
Germination/genetics

@article {pmid42391902,
year = {2026},
author = {Liao, W and Li, S and Wu, S and Yang, X and Liu, B and Ling, L and Zeng, Q and Zhang, J},
title = {Sensitive detection of prostate cancer antigen 3 (PCA3) in urine based upon CRISPR/Cas12a and gold nanorods (AuNRs).},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {363},
number = {Pt 2},
pages = {128309},
doi = {10.1016/j.saa.2026.128309},
pmid = {42391902},
issn = {1873-3557},
mesh = {Humans ; Male ; *Gold/chemistry ; *Nanotubes/chemistry/ultrastructure ; *Antigens, Neoplasm/urine ; *Prostatic Neoplasms/urine/diagnosis ; *CRISPR-Cas Systems ; Limit of Detection ; Spectrometry, Fluorescence ; *CRISPR-Associated Proteins/metabolism ; Biosensing Techniques/methods ; Biomarkers, Tumor/urine ; *Bacterial Proteins/metabolism/chemistry ; *Endodeoxyribonucleases/metabolism ; },
abstract = {Prostate Cancer Antigen 3, a type of long non-coding RNA, exhibits outstanding specificity as a biomarker for the diagnosis of prostate cancer, offering a highly effective diagnostic indicator, whereas the currently used prostate specific antigen exhibits low specificity, leading to reduced accuracy in prostate cancer diagnosis. Herein, we designed a novel fluorescent sensing platform for targeted detection of PCA3, which integrates the non-specific trans-cleavage activity of the CRISPR/Cas12a with the remarkable fluorescence quenching effect of Gold Nanorods. The Cas12a recognizes and binds to specific sequences of PCA3, thereby activating nonspecific cleavage activity, which cleaves fluorescent reporter probes adsorbed on AuNRs, thus leading to the recovery of fluorescence signals and enabling sensitive detection. The proposed fluorescent sensor exhibits excellent accuracy and convenience for the detection of PCA3 in urine, and a detection limit as low as 1.65 pM was obtained. This sensing system has achieved effective detection of clinical samples of prostate cancer and is expected to provide significant assistance in the screening and therapeutic feedback of prostate cancer in clinical diagnosis.},
}

Humans
Male
*Gold/chemistry
*Nanotubes/chemistry/ultrastructure
*Antigens, Neoplasm/urine
*Prostatic Neoplasms/urine/diagnosis
*CRISPR-Cas Systems
Limit of Detection
Spectrometry, Fluorescence
*CRISPR-Associated Proteins/metabolism
Biosensing Techniques/methods
Biomarkers, Tumor/urine
*Bacterial Proteins/metabolism/chemistry
*Endodeoxyribonucleases/metabolism

@article {pmid42437612,
year = {2026},
author = {Li, X and Xin, C and Guo, J and Zheng, Y and Huang, F and Li, X and Guo, H and Xu, Y and Shao, W},
title = {Multiplex CRISPR-Cas9 editing of starch branching enzyme II and vacuolar invertase simultaneously enhances resistant starch content and cold-induced sweetening resistance in Solanum chacoense.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {371},
number = {},
pages = {113319},
doi = {10.1016/j.plantsci.2026.113319},
pmid = {42437612},
issn = {1873-2259},
mesh = {*beta-Fructofuranosidase/genetics/metabolism ; *Starch/metabolism ; *1,4-alpha-Glucan Branching Enzyme/genetics/metabolism ; *Solanum/genetics/enzymology/metabolism ; *CRISPR-Cas Systems ; Cold Temperature ; *Gene Editing ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; },
abstract = {Potato processing suffers from a high glycemic index due to amylopectin-rich starch and from undesirable color and acrylamide formation during frying, mainly caused by cold-induced sweetening (CIS). To address both issues simultaneously, we used CRISPR-Cas9 to knock out two key genes in diploid Solanum chacoense: ScSBE II (starch branching enzyme II), which controls amylopectin biosynthesis, and ScVInv (vacuolar invertase), a central regulator of CIS. Knockout of ScSBEⅡ increased tuber fresh weight-based absolute amylose content by ∼5-fold versus wild type. Amylose proportion in total starch elevated from 22% to 54%, while amylopectin abundance declined 1.5-fold, substantially optimizing the amylose/amylopectin mass ratio of tuber starch. These lines also showed a 4-fold reduction in rapidly digestible starch (RDS), 3.5‑fold and 1.2‑fold increases in slowly digestible starch (SDS) and resistant starch (RS), respectively, and markedly improved pasting properties. Enzyme assays confirmed a 2.5‑fold reduction in ScSBE II activity. In wild‑type (WT) tubers, cold storage (4 °C, 7 d) increased ScVInv activity ∼5‑fold (to 48 μg·min[-1]·g[-1]) and reducing sugars 6‑fold (from 11 to 68 mg·g[-1]). Notably, ScVInv single‑knockout and ScSBE II/ScVInv double‑knockout lines produced chips with lighter color and much lower acrylamide than WT or ScSBE II single‑knockout lines. This dual‑gene editing strategy creates novel potato germplasm with enhanced resistant starch (health benefit) and superior processing quality (safer, visually appealing fried products).},
}

*beta-Fructofuranosidase/genetics/metabolism
*Starch/metabolism
*1,4-alpha-Glucan Branching Enzyme/genetics/metabolism
*Solanum/genetics/enzymology/metabolism
*CRISPR-Cas Systems
Cold Temperature
*Gene Editing
*Plant Proteins/genetics/metabolism
Plants, Genetically Modified

@article {pmid42543873,
year = {2026},
author = {Wang, J and Peng, Q},
title = {Innovations, Applications, and Future Trends in Veterinary Diagnostic Technologies.},
journal = {Transboundary and emerging diseases},
volume = {2026},
number = {1},
pages = {e6973879},
pmid = {42543873},
issn = {1865-1682},
support = {32470195//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Veterinary Medicine/trends/methods ; *Animal Diseases/diagnosis ; High-Throughput Nucleotide Sequencing/veterinary ; },
abstract = {Veterinary diagnostics is undergoing a significant transformation driven by technological advancements, extending its scope from the traditional confirmation of specific pathogens to the continuous, dynamic surveillance of animal population's health. This paradigm shift has the potential to enable more timely disease control, precise intervention, and enhanced public health security. Traditional clinical and laboratory diagnostic methods, such as microbial culture, serological assays, and nucleic acid-based polymerase chain reaction, form the cornerstone of the current diagnostic framework and are widely applied based on varying detection needs and practical environments. Nonetheless, the field is experiencing profound innovation. Firstly, novel detection technologies are emerging, such as digital PCR (dPCR), CRISPR-Cas-based molecular diagnostic tools, next-generation sequencing (NGS), and metagenomic sequencing. These technologies have not only achieved breakthroughs in sensitivity and specificity but, more importantly, enable the unbiased discovery of novel pathogens. Secondly, the deep integration of artificial intelligence (AI) and big data is reshaping the diagnostic pipeline. By consolidating and analyzing multimodal information streams from imaging, genomics, wearable devices, and production data, AI algorithms can provide objective, quantitative decision support, facilitating a transition from post-symptomatic diagnosis towards predictive and preventive health management. This scoping review systematically summarizes both mainstream and emerging veterinary diagnostic technologies, elaborates and discusses their advantages and limitations as well as future developmental directions, while highlighting that the combined application of multiple methods represents an optimal diagnostic strategy.},
}

Animals
*Veterinary Medicine/trends/methods
*Animal Diseases/diagnosis
High-Throughput Nucleotide Sequencing/veterinary

@article {pmid42545527,
year = {2026},
author = {Hu, Y and Fang, F and Cui, Z and Chen, S},
title = {A Reproducible Electroporation Strategy for CRISPR-Cas9 RNP and mRNA Delivery in Fish Embryos.},
journal = {Marine biotechnology (New York, N.Y.)},
volume = {28},
number = {4},
pages = {},
pmid = {42545527},
issn = {1436-2236},
support = {2023TD20//Central Public-interest Scientific Institution Basal Research Fund, CAFS/ ; 2023ZD0405503//STI 2030-Major projects/ ; 2022YFD2400101//the National Key R&D Program of China/ ; 32230107//National Natural Science Foundation of China/ ; 2023ZLYS02//the Key Research and Development Project of Shandong Province/ ; },
mesh = {Animals ; *Zebrafish/genetics/embryology ; *Electroporation/methods ; *Ribonucleoproteins/genetics/metabolism ; *CRISPR-Cas Systems ; *RNA, Messenger/genetics/metabolism ; Embryo, Nonmammalian/metabolism ; *Gene Editing/methods ; Gene Knockout Techniques ; Green Fluorescent Proteins/genetics ; Polyglutamic Acid ; Chorion/metabolism ; Zebrafish Proteins/genetics ; },
abstract = {This study presents a streamlined electroporation-based method for efficient macromolecular delivery into zebrafish embryos. We first characterized the physical barrier posed by the chorion using TEM (Transmission Electron Microscope) and established that its removal is prerequisite for effective delivery. A systematic optimization of electroporation parameters for dechorionated embryos identified optimal conditions (e.g., poring pulse: 25 V, 20 ms; transfer pulse: 5 V, 50 ms) that ensured high embryo survival and robust eGFP mRNA transfection. Applying this protocol, we achieved targeted gene knockout by electroporation-mediated delivery of Cas9 ribonucleoproteins (RNPs) against multiple loci. Targeting the tyr locus resulted in a phenotypic albinism rate of (38.6 ± 3.30)%. Furthermore, we incorporated polyglutamic acid (PGA) to modify the RNP complexes (target tyr), which inhibited aggregation and enhanced editing efficiency to (44.45 ± 1.41)%, outperforming a commercial. electroporation system, while targeting the pigmentation-related gene slc24a5 yielded an albinism or hypopigmentation rate of (38.33 ± 2.62)%. In addition, targeting the development-associated gene ddx19b produced developmental defect phenotypes in (38.33 ± 1.88)% of embryos. The successful introduction of indels at the target site was confirmed by sequencing. Our work establishes a highly effective electroporation strategy, augmented by nanotechnology, for the delivery of mRNA and RNP complexes, enabling high-efficiency protein expression and gene editing in zebrafish embryos, with broad potential applications in aquatic biotechnology.},
}

Animals
*Zebrafish/genetics/embryology
*Electroporation/methods
*Ribonucleoproteins/genetics/metabolism
*CRISPR-Cas Systems
*RNA, Messenger/genetics/metabolism
Embryo, Nonmammalian/metabolism
*Gene Editing/methods
Gene Knockout Techniques
Green Fluorescent Proteins/genetics
Polyglutamic Acid
Chorion/metabolism
Zebrafish Proteins/genetics

@article {pmid42545751,
year = {2026},
author = {Haneef, S and Zhou, YJ and Bai, F},
title = {Transcriptional regulation: Efficient genetic engineering tools for non-conventional yeasts.},
journal = {FEMS yeast research},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsyr/foag032},
pmid = {42545751},
issn = {1567-1364},
abstract = {Non-conventional yeasts are recognized as valuable hosts for producing biofuels, pharmaceuticals, and other high-value chemicals, owing to their diverse physiological traits, ability to utilize various substrates, and greater tolerance to environmental stresses compared to conventional model yeast Saccharomyces cerevisiae. To fully optimizing metabolic flux toward desired products, effective genetic engineering tools enabling precise modulation of gene expression and coordinated control of metabolic pathways are essential. In this context, we discussed classical transcriptional regulation tools like promoters, and transcription factors, alongside innovations in synthetic biology that allow metabolic engineering in non-conventional yeasts to produce higher biofuels and other useful products, promoting the development of sustainable resources, and assisting the development of innovative bio-products. It also discussed innovative programmable technologies, such as CRISPR/Cas-mediated transcriptional activation and repression, as well as dynamic regulatory systems that can fine-tune metabolic routes and balance cellular resources. Strategies for promoter engineering, transcription factor manipulation for transcriptional regulation, and metabolic rewiring were highlight as methods to boost pathway efficiency and yields. This review concluded with current challenges and future directions, focusing on integrating synthetic biology and systems biology to create robust, controllable transcriptional frameworks for next-generation yeast cell factories.},
}

@article {pmid42545979,
year = {2026},
author = {Patel, MA and Singh, M and Sinha, H and Vo, PQN and Chin, AB and Ellouzi, A and West, M and Hirukawa, A},
title = {A Digital Microfluidic Electroporation Platform for Low-input CRISPR Genome Editing and mRNA Transfection In Suspension T Cells and 3D Cell Models.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {233},
pages = {},
doi = {10.3791/70573},
pmid = {42545979},
issn = {1940-087X},
mesh = {Humans ; *Electroporation/methods/instrumentation ; *Transfection/methods/instrumentation ; HEK293 Cells ; *RNA, Messenger/genetics/administration & dosage ; *T-Lymphocytes/physiology ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Microfluidic Analytical Techniques/methods/instrumentation ; },
abstract = {Digital microfluidic (DMF) electroporation enables precise, low-volume genetic manipulation of mammalian cells while minimizing cellular input by up to 100x and preserving viability. This study presents a high-throughput DMF-based transfection workflow for CRISPR-mediated knockout of the TRAC locus in primary human suspension T cells and for mRNA transfection of three-dimensional HEK293T spheroids. Using spatially deposited CRISPR guide RNAs and on- cartridge ribonucleoprotein (RNP) assembly, efficient TRAC locus disruption was achieved in both CD4[+] and CD8[+] T-cell populations using only 10,000 cells per condition, with post-editing viabilities exceeding 85%. Biophysical characterization using flow-induced and Taylor dispersion analyses revealed that polymer additives stabilize Cas9-sgRNA complexes under electroporation buffer conditions, supporting reproducible editing at sub microliter volumes. The workflow was further adapted for 3D applications by delivering EGFP mRNA into intact HEK293T spheroids, resulting in robust and spatially uniform fluorescence without impairing spheroid growth or morphology. Together, these results demonstrate that DMF electroporation enables efficient genome editing and mRNA delivery across both suspension immune cells and multicellular spheroids. This platform provides a scalable and low-input solution for applications in CAR-T cell therapy, functional genomics, and advanced 3D cellular models.},
}

Humans
*Electroporation/methods/instrumentation
*Transfection/methods/instrumentation
HEK293 Cells
*RNA, Messenger/genetics/administration & dosage
*T-Lymphocytes/physiology
*Gene Editing/methods
*CRISPR-Cas Systems
*Microfluidic Analytical Techniques/methods/instrumentation

@article {pmid42263565,
year = {2026},
author = {Wang, Q and Wang, Y and Jia, T and Hu, X and Chen, F},
title = {Evaluation of the performance of reverse transcription-recombinase polymerase amplification (RT-RPA) coupled with CRISPR/Cas12a and microfluidics for one-step detection of common HCV genotypes.},
journal = {Diagnostic microbiology and infectious disease},
volume = {116},
number = {3},
pages = {117482},
doi = {10.1016/j.diagmicrobio.2026.117482},
pmid = {42263565},
issn = {1879-0070},
mesh = {Humans ; *Hepacivirus/genetics/isolation & purification/classification ; Genotype ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Hepatitis C/diagnosis/virology ; *Nucleic Acid Amplification Techniques/methods ; *Microfluidics/methods ; Rapid Diagnostic Tests ; *Genotyping Techniques/methods ; },
abstract = {BACKGROUND: Hepatitis C virus (HCV) genotyping is critical for guiding therapy, yet current methods are technically demanding and time-consuming.

AIM: To develop and evaluate a one-step, integrated assay combining reverse transcription-recombinase polymerase amplification (RT-RPA) with CRISPR/Cas12a detection on a microfluidic platform for rapid and accurate HCV genotyping.

METHODS: The assay was validated using 186 clinical samples genotyped by Sanger sequencing. The microfluidic chip enabled sequential RT-RPA amplification and CRISPR/Cas12a detection via centrifugal fluid transfer, with real-time fluorescence monitoring.

RESULTS: The assay demonstrated high concordance with Sanger sequencing (overall accuracy >98%), with sensitivities of 100% for genotypes 1b and 6a, and >97% for 2a and 3a. The limit of detection was 1 IU/mL (5 copies/mL)across major genotypes, with no cross-reactivity against other viruses.

CONCLUSION: The integrated RT-RPA-CRISPR/Cas12a-microfluidics platform offers a rapid, sensitive, and specific one-step assay for HCV genotyping, suitable for point-of-care applications in resource-limited settings.},
}

Humans
*Hepacivirus/genetics/isolation & purification/classification
Genotype
*CRISPR-Cas Systems
Sensitivity and Specificity
*Hepatitis C/diagnosis/virology
*Nucleic Acid Amplification Techniques/methods
*Microfluidics/methods
Rapid Diagnostic Tests
*Genotyping Techniques/methods

@article {pmid42289138,
year = {2026},
author = {Sata, TN and Sah, AK and Ismail, M and Sharma, G and Doloi, R and Nayak, B and Shalimar, and Venugopal, SK},
title = {Development of RPA and nested-RPA based CRISPR/Cas13a diagnostic platform for the identification of HBV DNA and HCV RNA in Indian patient cohort.},
journal = {Diagnostic microbiology and infectious disease},
volume = {116},
number = {3},
pages = {117514},
doi = {10.1016/j.diagmicrobio.2026.117514},
pmid = {42289138},
issn = {1879-0070},
mesh = {Humans ; *Hepatitis B virus/genetics/isolation & purification ; *Hepacivirus/genetics/isolation & purification ; India ; *Hepatitis B/diagnosis/virology ; *Hepatitis C/diagnosis/virology ; *DNA, Viral/genetics/isolation & purification ; *Nucleic Acid Amplification Techniques/methods ; *RNA, Viral/genetics ; Sensitivity and Specificity ; Rapid Diagnostic Tests ; *CRISPR-Cas Systems ; Recombinases/metabolism ; Cohort Studies ; },
abstract = {BACKGROUND: Among the Indian population, hepatitis B virus (HBV) is one of the major burdens and the hepatitis C virus (HCV) chronically infects around 1% Indian population. CRISPR-based detection platforms have shown to be a novel low-cost technology with high sensitivity and specificity. In the presence of target nucleic acids, Cas13a molecule is activated to trans-cleave the fluorophore quencher (FQ)-labeled ssRNA reporter, and illuminate detectable fluorescent signals.

METHODS: Leptotrichia wadei (Lwa) cas13a was expressed and purified. Recombinase Polymerase Amplification (RPA) was implemented to produce T7 RNA polymerase appended amplicons of conserved regions of HBV and HCV at 37°C and 42°C respectively. Corresponding crRNAs have been designed against amplified regions and produced using In-Vitro Transcription (IVT). With T7 RNA polymerase, the RPA-amplified HBV and HCV templates are transcribed into ssRNAs, which are further used in detection assay containing expressed Cas protein, crRNA, and fluorescent probes. This detection was performed in the microplate reader in kinetic format.

RESULTS: LwaCas13a was expressed and was purified using the strep tag. Conserved regions among Indian HBV and HCV genotypes are selected as targets of detection. RPA and Nested-RPA was performed using primers against conjunct region of HBV polymerase and surface antigen and RNA-dependent RNA polymerase (RdRp) region of HCV. The detection assay was performed from 45 HBV and 30 HCV human samples, out of which it could differentiate positive and healthy samples.

CONCLUSION: This approach can be a better alternative to be used in rural India and at the same time with high sensitivity, for a rapid detection of HBV and HCV, which could be used as a novel a low-cost diagnostics platform for identification of HBV DNA and HCV RNA.},
}

Humans
*Hepatitis B virus/genetics/isolation & purification
*Hepacivirus/genetics/isolation & purification
India
*Hepatitis B/diagnosis/virology
*Hepatitis C/diagnosis/virology
*DNA, Viral/genetics/isolation & purification
*Nucleic Acid Amplification Techniques/methods
*RNA, Viral/genetics
Sensitivity and Specificity
Rapid Diagnostic Tests
*CRISPR-Cas Systems
Recombinases/metabolism
Cohort Studies

@article {pmid42328982,
year = {2026},
author = {Mao, Y and Fei, X and Yang, X and Hu, C and Zhu, C and Wang, J and Ge, Y and Ye, C and Yang, S and Cheng, P and Li, Y and He, X and Hu, Z and Qi, Y},
title = {CRISPR-Cas12a-based fluorescent and visual assays for universal detection and clade discrimination of mpox virus.},
journal = {Microbiology spectrum},
volume = {14},
number = {8},
pages = {e0010526},
pmid = {42328982},
issn = {2165-0497},
support = {2025YQZL01//Huadong Research Institute for Medicine and Biotechniques/ ; SKLADCPKFKT202512//State Key Laboratory for Animal Disease Control and Prevention/ ; },
mesh = {Humans ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Orthopoxvirus/genetics/isolation & purification/classification ; Fluorescence ; *Molecular Diagnostic Techniques/methods ; CRISPR-Associated Proteins/genetics ; Polymerase Chain Reaction/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {The global mpox outbreak has highlighted critical gaps in diagnostic capabilities, particularly the need for methods that can distinguish between the high-fatality Clade I and more transmissible Clade II of the mpox virus (MPXV). Current PCR-based approaches remain reliant on laboratory infrastructure, limiting their use in resource-limited settings. There is an urgent need for a versatile diagnostic platform that can provide both accurate clade discrimination and flexible deployment across diverse healthcare environments. We developed a dual-mode detection platform by integrating recombinase-aided amplification with Clustered Regularly Interspaced Short Palindromic Repeats-Cas12a technology, creating two distinct assays: a universal assay targeting OPG034, and a Clade I-discriminatory assay targeting OPG033. The platform achieved detection sensitivities of 1 copy/reaction for both fluorescence and visual readouts with OPG034, and 10 copies (fluorescence) and 1 copy/reaction (visual) with OPG033. Both assays demonstrated high specificity, successfully distinguishing MPXV from related orthopoxviruses and common viruses. Clinical validation using 23 Clade II samples and 10 healthy controls showed that, relative to quantitative PCR (qPCR) (cycle threshold ≤37), the OPG034 fluorescence assay detected all 13 qPCR-positive samples and 2 additional positives (100% sensitivity, 80.0% specificity), while the visual assay detected 12 of 13 positives (92.3% sensitivity, 100% specificity). For Clade I-specific detection, both OPG033 fluorescence and visual assays showed 100% specificity in Clade II samples (23/23). While the clade-discriminatory capability was established through sequence-specific design, further evaluation with authentic Clade I clinical specimens is warranted to confirm typing performance. The dual-mode design provides flexibility for both laboratory and field use, advancing mpox surveillance and outbreak response.IMPORTANCEMpox is a significant zoonosis. Accurate discrimination between its highly lethal Clade I and more transmissible Clade II is critical for clinical management and outbreak control, yet current methods primarily enable only general detection. To address this, we identified novel genetic markers (OPG034 for universal detection and OPG033 for Clade I specificity) and developed a dual-mode detection platform integrating Clustered Regularly Interspaced Short Palindromic Repeats-Cas12a with recombinase-aided amplification. Its key advantage is providing two result readouts: a sensitive fluorescence mode for laboratories and an instrument-free visual colorimetric mode for field use. The demonstrated excellent performance on clinical samples confirms that this platform meets the precision requirements of clinical laboratories while remaining suitable for resource-limited settings like field clinics. Thus, it offers a flexible and practical tool for enhancing mpox surveillance and control globally, particularly in regions with constrained medical resources.},
}

Humans
Sensitivity and Specificity
*CRISPR-Cas Systems
*Orthopoxvirus/genetics/isolation & purification/classification
Fluorescence
*Molecular Diagnostic Techniques/methods
CRISPR-Associated Proteins/genetics
Polymerase Chain Reaction/methods
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid42372673,
year = {2026},
author = {Yang, H and Li, X and Su, Y and Guo, J and Wang, T and Wang, Z and Fu, H and Chen, Z and Xie, Z and Li, D},
title = {Enhanced stability of RPA-CRISPR-Cas12a system for respiratory pathogen detection using Trehalose-Carboxymethyl Chitosan Lyoprotectant.},
journal = {Diagnostic microbiology and infectious disease},
volume = {116},
number = {3},
pages = {117523},
doi = {10.1016/j.diagmicrobio.2026.117523},
pmid = {42372673},
issn = {1879-0070},
mesh = {Humans ; Sensitivity and Specificity ; *Chitosan/analogs & derivatives/chemistry ; *CRISPR-Cas Systems ; Rapid Diagnostic Tests ; *Trehalose/chemistry ; *Respiratory Tract Infections/diagnosis/virology/microbiology ; SARS-CoV-2/genetics/isolation & purification ; Neisseria meningitidis/genetics/isolation & purification ; *Nucleic Acid Amplification Techniques/methods ; Streptococcus pneumoniae/genetics/isolation & purification ; Freeze Drying ; },
abstract = {PURPOSE: Acute respiratory infections caused by bacterial and viral pathogens pose a major global health burden. The recombinase polymerase amplification (RPA)-CRISPR-Cas12a system offers a promising point-of-care testing (POCT) platform, but its field deployment is limited by the instability of lyophilized reagents. This study aims to develop a composite lyoprotectant to enhance the stability of the RPA-CRISPR-Cas12a system for respiratory pathogen detection.

MATERIALS AND METHODS: A composite lyoprotectant composed of trehalose and carboxymethyl chitosan (Tre-CMC) was formulated at various mass ratios. The optimal ratio was identified by evaluating matrix microstructure, enzyme activity retention, and primer-dimer suppression. The lyophilized system was tested for sensitivity, specificity, and long-term stability against six respiratory pathogens (H1N1, IBV, Neisseria meningitidis, SARS-CoV-2, Streptococcus pneumoniae, and human adenovirus) using real-time fluorescence and gel electrophoresis.

RESULTS: This study establishes a promising proof-of-concept framework for an integrated, lyophilized RPA-CRISPR-Cas12a diagnostic system. The optimal Tre: CMC ratio (2:1) produced a porous, non‑hygroscopic matrix that preserved reagent integrity. The lyophilized system achieved a detection limit of 10 copies/reaction within 30 min for all six pathogens, with 100% specificity. After six months of storage at 4°C, RPA enzyme activity remained above 89%, and Cas12a-crRNA complex functionality was fully retained. Tre-CMC significantly reduced primer-dimer formation and nonspecific background fluorescence. While clinical evaluation using 21 nasopharyngeal swab samples demonstrated 100% concordance with RT-qPCR-preliminarily supporting the system's potential diagnostic accuracy-we acknowledge that this initial methodological framework requires broader and more rigorous downstream validation with larger clinical cohorts to formally establish its robust diagnostic performance.

CONCLUSION: The Tre-CMC composite lyoprotectant effectively stabilizes the RPA-CRISPR-Cas12a system, enabling cold-chain-independent storage and reliable POCT for respiratory pathogens in resource-limited settings.},
}

Humans
Sensitivity and Specificity
*Chitosan/analogs & derivatives/chemistry
*CRISPR-Cas Systems
Rapid Diagnostic Tests
*Trehalose/chemistry
*Respiratory Tract Infections/diagnosis/virology/microbiology
SARS-CoV-2/genetics/isolation & purification
Neisseria meningitidis/genetics/isolation & purification
*Nucleic Acid Amplification Techniques/methods
Streptococcus pneumoniae/genetics/isolation & purification
Freeze Drying