@article {pmid42628215,
year = {2026},
author = {Eom, KH and Gim, GM and Lee, MG and Choi, BY and Kim, DY and Jung, D and Heo, S and Yum, SY and Jang, G},
title = {Evaluation of germline transmission of electroporation-mediated double gene-edited cattle lines.},
journal = {Theriogenology},
volume = {266},
number = {},
pages = {118142},
doi = {10.1016/j.theriogenology.2026.118142},
pmid = {42628215},
issn = {1879-3231},
mesh = {Animals ; Cattle/genetics ; Male ; *Electroporation/veterinary ; Female ; *Gene Editing/veterinary/methods ; CRISPR-Cas Systems ; Gene Knockout Techniques/veterinary ; Germ Cells ; Myostatin/genetics ; Animals, Genetically Modified ; Spermatozoa ; Galactosyltransferases/genetics ; },
abstract = {Gene editing in livestock using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) offers a promising approach for genetic improvement in cattle. This study evaluated germline transmission and mutation stability of double-knockout cattle generated by zygote electroporation. Previously reported myostatin/beta-lactoglobulin (MSTN/BLG) and newly generated α-1,3-galactosyltransferase (GGTA1/BLG) double-knockout cattle were produced using CRISPR/Cas9-mediated genome editing. Targeted deep sequencing demonstrated extensive somatic mosaicism across multiple tissues. Computer-assisted sperm analysis (CASA) demonstrated normal sperm motility in MSTN/BLG double-knockout males. Fertilization of wild-type oocytes produced heterozygous embryos, with mutation frequencies of 37.76 ± 10.74% at the MSTN locus and 54.80 ± 7.73% at the BLG locus, as assessed by T7 endonuclease I (T7E1) assay. MSTN/BLG double-knockout sperm were subsequently used for embryo production and for artificial insemination of GGTA1/BLG double-knockout females. Healthy offspring were successfully obtained (n = 3), alongside one stillborn calf. Targeted deep sequencing of all four progenies revealed highly variable allele frequencies that deviated substantially from the approximately 50% expected for heterozygous germline transmission. In contrast, whole-genome sequencing (WGS) results were consistent with Mendelian expectations, underscoring the limitations of PCR-based targeted sequencing for assessing germline transmission in mosaic founders. These results show that CRISPR/Cas9-edited embryos generated by electroporation can develop into healthy, sexually mature cattle capable of germline transmission. While variable transmission rates were observed owing to founder mosaicism, non-mosaic F1 offspring were successfully generated. However, direct, embryo-mediated gene-editing strategies remain technically and economically challenging for large-scale commercial calf production, and reports in cattle are limited. This study provides a reference for future applications of gene-edited embryos and their germline propagation.},
}

Animals
Cattle/genetics
Male
*Electroporation/veterinary
Female
*Gene Editing/veterinary/methods
CRISPR-Cas Systems
Gene Knockout Techniques/veterinary
Germ Cells
Myostatin/genetics
Animals, Genetically Modified
Spermatozoa
Galactosyltransferases/genetics

@article {pmid42704380,
year = {2026},
author = {Paenkaew, S and Sinthao, O and Thapthim, T and Mahakkaprateep, S and Cheukaw, A and Keawmanee, S and Euppayo, T and Tungtrakanpoung, R and Teapunvong, W and Nganvongpanit, K and Buddhachat, K},
title = {A cost-effective and rapid TE-based DNA extraction for near point-of-care detection of Babesia spp. by RPA/CRISPR-Cas12a.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42704380},
issn = {1573-4978},
support = {R2568E050//Research Grant Agreement for Program Director, Faculty of Science, Naresuan University/ ; N42A650332//the National Research Council of Thailand (NRCT)/ ; R2565A060//the Faculty of Science, Naresuan University, Thailand/ ; R2566C051//the Global and Frontier Research University Fund, Naresuan University, Thailand/ ; },
mesh = {*Babesia/genetics/isolation & purification ; Animals ; *Babesiosis/diagnosis/genetics/parasitology ; *Nucleic Acid Amplification Techniques/methods/economics ; *DNA, Protozoan/genetics/isolation & purification ; CRISPR-Cas Systems/genetics ; Cost-Benefit Analysis ; Rapid Diagnostic Tests ; Point-of-Care Systems ; Dogs ; Humans ; },
abstract = {BACKGROUND: Babesia spp. infects a wide range of mammals, including domestic animals, wildlife, and humans, highlighting the need for rapid diagnostics. This study developed a near point-of-care method combining rapid DNA extraction, recombinase polymerase amplification (RPA), and CRISPR-Cas12a for Babesia detection.

METHODS AND RESULTS: The workflow includes three steps: DNA extraction, RPA amplification, and CRISPR-Cas12a signal detection. Five blood DNA extraction methods were evaluated: DNeasy kit, boiling, Tris-EDTA (TE) buffer, methanol-based, and Chelex®100. The commercial kit, boiling method, and TE buffer extraction yielded positive results using RPA/CRISPR-Cas12a based on the cytochrome c oxidase subunit I (COI) gene. The TE buffer extraction and boiling methods were further optimized by RPA/CRISPR-Cas12a assay using Babesia-specific primers based on the 18S rRNA gene. All these extraction methods produced results comparable to the commercial kit. The TE buffer extraction was selected due to its simplicity and speed. The RPA/CRISPR-Cas12a assay specifically detected Babesia spp., including B. vogeli, B. gibsoni, and B. ovata. The limit of detection using TE-extracted DNA was 104 copies per reaction, lower than that achieved with the commercial kit (103 copies per reaction). Evaluation of rapid DNA extraction for Babesia spp. detection by the RPA/CRISPR-Cas12a assay using seven bovine and eight canine blood samples showed complete concordance between the TE buffer extraction and the commercial kit, with six positive samples detected.

CONCLUSIONS: The TE buffer extraction offered a low-cost, simple, and equipment-minimal approach suitable for combination with RPA/CRISPR-Cas12a diagnostics, enabling rapid detection of Babesia spp. in resource-limited or field settings.},
}

*Babesia/genetics/isolation & purification
Animals
*Babesiosis/diagnosis/genetics/parasitology
*Nucleic Acid Amplification Techniques/methods/economics
*DNA, Protozoan/genetics/isolation & purification
CRISPR-Cas Systems/genetics
Cost-Benefit Analysis
Rapid Diagnostic Tests
Point-of-Care Systems
Dogs
Humans

@article {pmid42704632,
year = {2026},
author = {Guo, Y and Tan, X},
title = {Specificity and Regulatory Mechanism of Metal Ions in Cas12a Enzymatic Cleavage Activity.},
journal = {Metallomics : integrated biometal science},
volume = {},
number = {},
pages = {},
doi = {10.1093/mtomcs/mfag027},
pmid = {42704632},
issn = {1756-591X},
abstract = {Metal ions play key roles in both physiological and pathological processes. The activity of nearly all nucleases depends on metal ions as cofactors. CRISPR-Cas system is widely used in gene editing and nucleic acid detection, and its ongoing development and frontier research have attracted significant attention. This study aims to explore and elucidate the effects of various metal ions on Cas12a-catalysed cleavage reactions including their specificity and regulatory mechanisms. Here, we found that certain metal ions significantly affected both the cleavage activity and the reaction specificity of Cas12a. Metal ions mediate enzyme-substrate binding by increasing the affinity between Cas12a and single-stranded DNA (ssDNA). In particular, unlike other metal ions, Mn2+ can achieve cleavage of ssDNA by only activating the catalytic domain without requiring the entire Cas12a. Structural alignment and site-directed mutagenesis of the active site revealed the structural basis of Cas12a catalysis and elucidated how metal ions influence its catalytic reaction in terms of specificity and regulatory mechanism. These results provide a theoretical foundation and offer innovative perspectives for the application of Cas12a-based enzyme technology.},
}

@article {pmid42283406,
year = {2026},
author = {Zhu, Y and Cao, D and Guo, T and Zhang, X and Luo, Y and Li, X and Yang, H and Zhang, M and Guo, S and Song, F},
title = {RNase III-deficient Bacillus thuringiensis enables stable dsRNA production and enhances HD1 activity against Spodoptera frugiperda.},
journal = {Pest management science},
volume = {82},
number = {10},
pages = {9889-9898},
doi = {10.1002/ps.71023},
pmid = {42283406},
issn = {1526-4998},
support = {CAAS-CSCB-202402//Innovation Program of Chinese Academy of Agricultural Sciences/ ; 024YFD1200203//National Key R&D Program of China/ ; 32372623//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Spodoptera/growth & development/genetics ; *Bacillus thuringiensis/genetics/enzymology ; *RNA, Double-Stranded/metabolism/genetics ; *Ribonuclease III/genetics/metabolism/deficiency ; *Pest Control, Biological/methods ; RNA Interference ; Larva/growth & development/genetics ; *Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Spodoptera frugiperda is one of the most important invasive agricultural pests in China. Integrating RNA interference (RNAi) with Bacillus thuringiensis (Bt)-based biocontrol represents a promising strategy for pest control. However, efficient dsRNA production in Bt remains challenging as endogenous RNase activity limits dsRNA stability and accumulation.

RESULTS: In this study, an RNase III-deficient Bt strain, DBΔrnc, was generated using an optimized CRISPR-Cas9 system. Deficiency of RNase III markedly enhanced intracellular dsRNA accumulation. A hairpin dsRNA construct exhibited higher transcript levels than the dual-promoter construct, especially in the DBΔrnc background. Oral delivery of Bt expressed hairpin dsRNA targeting the endochitinase gene SfCHI of S. frugiperda induced effective gene silencing and caused pupation defects. Moreover, co-application with Bt HD1 strain significantly increased larval mortality in both neonate and 2[nd]-instar larvae.

CONCLUSION: Collectively, our findings highlight that the RNase III-deficient strain DBΔrnc is an efficient chassis for dsRNA expression. Furthermore, the enhanced insecticidal activity from co-application of DBΔrnc(hpdsCHI) and HD1 demonstrates the feasibility of integrating RNAi with Bt-based pest control. © 2026 Society of Chemical Industry.},
}

Animals
*Spodoptera/growth & development/genetics
*Bacillus thuringiensis/genetics/enzymology
*RNA, Double-Stranded/metabolism/genetics
*Ribonuclease III/genetics/metabolism/deficiency
*Pest Control, Biological/methods
RNA Interference
Larva/growth & development/genetics
*Bacterial Proteins/genetics/metabolism
CRISPR-Cas Systems

@article {pmid42537346,
year = {2026},
author = {Yang, G and Qin, H and Wang, Q and Cheng, J and Du, L and Fan, A and Lin, W and Huang, J and Hu, X and Li, Y and Wang, H},
title = {In vivo CRISPR screening identifies LTA4H as a target for potentiating immunotherapy efficacy via the LTB4-neutrophil axis.},
journal = {International immunopharmacology},
volume = {187},
number = {},
pages = {117213},
doi = {10.1016/j.intimp.2026.117213},
pmid = {42537346},
issn = {1878-1705},
mesh = {Animals ; *Epoxide Hydrolases/genetics/metabolism ; *Neutrophils/immunology ; *Leukotriene B4/metabolism ; Mice ; Humans ; Signal Transduction ; *Immunotherapy/methods ; Receptors, Leukotriene B4/antagonists & inhibitors/metabolism ; Mice, Inbred C57BL ; Cell Line, Tumor ; Programmed Cell Death 1 Receptor/antagonists & inhibitors/metabolism ; CRISPR-Cas Systems ; CD8-Positive T-Lymphocytes/immunology ; Killer Cells, Natural/immunology ; },
abstract = {Despite the clinical success of PD-1 blockade, therapeutic resistance remains a major barrier. Using an in vivo CRISPR-Cas9 screen with a metabolism-focused sgRNA library in MC38 and B16 syngeneic tumor models, we identified leukotriene A4 hydrolase (LTA4H) as a candidate regulator of PD-1 resistance. Lta4h ablation was associated with enhanced PD-1 responsiveness, increased CD8+ T and NK-cell infiltration and function, and reduced myeloid accumulation. Single-cell and spatial transcriptomic analyses showed that LTA4H is expressed in both malignant and myeloid compartments, with LTA4H-high tumor regions associated with myeloid-enriched niches and TGF-β related signaling. Mechanistically, hypoxia-induced HIF1α upregulated Lta4h in tumor cells, supporting tumor cells as one relevant source of LTA4H/LTB4. LTA4H-LTB4 signaling was linked to myeloid remodeling and TGF-β1 production in LTB4-conditioned neutrophils. Pharmacological blockade of LTB4 receptor signaling with the BLT1 antagonist CP-105696 improved tumor control in combination with PD-1 blockade. These findings identify intratumoral LTA4H-LTB4 signaling as a metabolic-immune pathway associated with suppressive myeloid remodeling and support further investigation of LTB4 receptor blockade to improve PD-1 blockade responses.},
}

Animals
*Epoxide Hydrolases/genetics/metabolism
*Neutrophils/immunology
*Leukotriene B4/metabolism
Mice
Humans
Signal Transduction
*Immunotherapy/methods
Receptors, Leukotriene B4/antagonists & inhibitors/metabolism
Mice, Inbred C57BL
Cell Line, Tumor
Programmed Cell Death 1 Receptor/antagonists & inhibitors/metabolism
CRISPR-Cas Systems
CD8-Positive T-Lymphocytes/immunology
Killer Cells, Natural/immunology

@article {pmid42659797,
year = {2026},
author = {Fu, R and Hong, J and Qu, Q and Hong, Z and Jiang, Q and Xianyu, Y},
title = {CRISPR-Cas12a-based querying of DNA-stored MRI and PET imaging data.},
journal = {Medical image analysis},
volume = {114},
number = {},
pages = {104272},
doi = {10.1016/j.media.2026.104272},
pmid = {42659797},
issn = {1361-8423},
mesh = {*Positron-Emission Tomography/methods ; *Magnetic Resonance Imaging/methods ; *DNA/genetics ; Sensitivity and Specificity ; Reproducibility of Results ; *Information Storage and Retrieval/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Storing magnetic resonance imaging (MRI) and positron emission tomography (PET) imaging data in DNA offers a promising solution for the long-term archival management of rapidly expanding biomedical image volumes. However, querying these data typically requires polymerase chain reaction and sequencing, which increases readout complexity and retrieval latency. Here, we propose a CRISPR-Cas12a-based querying strategy that leverages the programmability of CRISPR RNA (crRNA) for precise matching with identifier (ID) sequences. In this approach, crRNA functions as a query tool that identifies matching IDs and triggers detectable signals. Through a one-to-one mapping between ID and payload sequences established during encoding, the corresponding image data can be retrieved upon query. Computational simulations demonstrate over 99% query accuracy for both MRI and PET data using crRNA. CRISPR-Cas12a cleavage assays and molecular docking further validate the high specificity of crRNA-ID recognition. This work demonstrates the potential of CRISPR-Cas12a for biochemical querying of DNA-encoded biomedical imaging data, enabling targeted access to indexed sequences.},
}

*Positron-Emission Tomography/methods
*Magnetic Resonance Imaging/methods
*DNA/genetics
Sensitivity and Specificity
Reproducibility of Results
*Information Storage and Retrieval/methods
*CRISPR-Cas Systems/genetics
Humans
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid42700392,
year = {2026},
author = {Pittman, CC and Xu, C and Catchpole, RJ and Garrett, S and Fuchs, R and Chu, X and Makarova, KS and Koonin, EV and Zhao, P and Wells, L and Graveley, BR and Ke, A and Terns, MP},
title = {Type IV-C CRISPR-Cas effector complexes recognize double-stranded DNA and switch on collateral cleavage of ssDNA and RNA.},
journal = {Cell reports},
volume = {45},
number = {9},
pages = {117939},
doi = {10.1016/j.celrep.2026.117939},
pmid = {42700392},
issn = {2211-1247},
abstract = {Type IV-C CRISPR-Cas systems remain enigmatic compared to other class 1 systems. Here, we expand the type IV-C catalog, identifying two phylogenetically distinct clades primarily found in archaea (IV-C1) or bacteria (IV-C2), distinguishable by the Cas10IVc subunit architecture. We functionally and structurally characterize type IV-C1 systems from Thermococcus onnurineus (Ton) and Pyrococcus abyssi (Pab). Type IV-C complexes assemble with crRNAs derived from distinct CRISPR arrays and recognize a 5'-GGG-3' protospacer adjacent motif (PAM) to bind double-stranded DNA targets. Target recognition activates the HD domain of Cas10IVc, triggering metal-dependent collateral cleavage of single-stranded DNA and RNA. This behavior is explained by allosteric alignment of the HD active site, triggered by PAM-dependent R-loop formation, as revealed by cryo-EM. Together, our findings suggest that type IV-C systems provide immunity via non-specific cleavage of nucleic acids generated during mobile genetic element replication or transcription.},
}

@article {pmid42700448,
year = {2026},
author = {Allehyani, N and Alissa, M and Alghamdi, A and Alshehri, MA and Abusalim, GS and Alhegaili, AS and Juraybi, TN and Alsuwat, MA},
title = {CRISPR-Engineered CAR-T Cell Therapy for Epstein-Barr Virus-Associated Nasopharyngeal Carcinoma: A Review of Emerging Therapeutic Prospects.},
journal = {Reviews in medical virology},
volume = {36},
number = {5},
pages = {e70199},
pmid = {42700448},
issn = {1099-1654},
support = {PSAU/2026/R/1448//Prince Sattam bin Abdulaziz University/ ; },
mesh = {Humans ; *Immunotherapy, Adoptive/methods ; *Herpesvirus 4, Human/immunology/physiology ; *Nasopharyngeal Carcinoma/therapy/virology/immunology ; *Epstein-Barr Virus Infections/complications/virology/immunology/therapy ; *CRISPR-Cas Systems ; *Receptors, Chimeric Antigen/genetics/immunology ; Gene Editing ; *Nasopharyngeal Neoplasms/therapy ; Tumor Microenvironment ; },
abstract = {Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) remains a clinically challenging malignancy, particularly in recurrent or metastatic disease where durable responses to chemoradiotherapy and immune checkpoint blockade are limited. The viral aetiology of NPC provides a strong biological rationale for immune-based treatment; however, translation of chimaeric antigen receptor (CAR) T-cell therapy into this solid tumour setting is constrained by poor tumour trafficking, antigen heterogeneity, limited surface accessibility of EBV latent antigens, T-cell exhaustion, and an immunosuppressive tumour microenvironment. This review critically evaluates the emerging therapeutic prospects of CRISPR-engineered CAR-T cell therapy for EBV-associated NPC. It synthesises evidence on EBV latency biology, NPC immune evasion, solid-tumour CAR-T limitations, and genome-engineering strategies including conventional CRISPR-Cas9, base editing, prime editing, and double-strand-break-sparing targeted integration. Particular attention is given to genotoxicity, chromosomal rearrangements, chromosome loss, bystander and off-target editing, manufacturing heterogeneity, and the regulatory and biological barriers that currently separate technical feasibility from NPC-specific clinical implementation. Available clinical evidence from checkpoint blockade, EBV-specific adoptive T-cell therapy, base-edited CAR-T cells in haematologic malignancy, and early CRISPR-edited T-cell trials supports the feasibility of immune and genetic redirection but does not establish efficacy of a clinically validated CRISPR-engineered CAR-T platform for NPC. Future development should prioritise surface-accessible antigen validation, fit-for-purpose selection of editing technology, genomic safety, scalable manufacturing, and biomarker-driven early-phase trials.},
}

Humans
*Immunotherapy, Adoptive/methods
*Herpesvirus 4, Human/immunology/physiology
*Nasopharyngeal Carcinoma/therapy/virology/immunology
*Epstein-Barr Virus Infections/complications/virology/immunology/therapy
*CRISPR-Cas Systems
*Receptors, Chimeric Antigen/genetics/immunology
Gene Editing
*Nasopharyngeal Neoplasms/therapy
Tumor Microenvironment

@article {pmid42700480,
year = {2026},
author = {He, R and Miao, L and Deng, R and Xia, X},
title = {Advances in cascaded CRISPR for preamplification-free nucleic acid assays.},
journal = {Biosensors & bioelectronics},
volume = {314},
number = {},
pages = {119197},
doi = {10.1016/j.bios.2026.119197},
pmid = {42700480},
issn = {1873-4235},
abstract = {The development of rapid, sensitive, and specific nucleic acid assays is pivotal for advancing molecular detection in clinical diagnosis, food safety, and environmental monitoring. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) systems, renowned for their programmable and signal amplification capacity, have emerged as efficient tools for meeting these challenges. However, the intrinsic sensitivity of CRISPR/Cas assays relying on a single Cas effector is typically confined to the picomolar level, often necessitating complex nucleic acid preamplification. Cascaded CRISPR systems, which integrate sequential enzymatic reactions or multiple CRISPR effectors, can address this limitation by achieving nucleic acid preamplification-free signal enhancement. This review starts with the introduction of the design principles and working mechanisms of cascaded CRISPR strategies, encompassing Enzyme-Coupled cascades, Multi-effector class 2 CRISPR cascades, and Type III CRISPR-mediated cascades. Then, we highlight the deployment of these techniques across diverse bio-sensing scenarios, ranging from disease diagnosis to food and environmental surveillance. Finally, critical challenges and emerging frontiers are discussed, including integration with digital detection platforms and AI-assistant algorithms.},
}

@article {pmid42700916,
year = {2026},
author = {Zhang, X and Zhang, L and Guo, T and Du, W and Wang, S and Li, Y},
title = {Application of emerging technologies in the antiviral field.},
journal = {Antiviral research},
volume = {255},
number = {},
pages = {106529},
doi = {10.1016/j.antiviral.2026.106529},
pmid = {42700916},
issn = {1872-9096},
abstract = {Viral diseases pose a serious threat to global public health, agriculture, and biosecurity. Conventional antiviral strategies are often limited by an incomplete understanding of disease mechanisms, poor targeting precision, and slow response times. Emerging technologies are now reshaping the landscape of antiviral research. This review examines the roles of four key frontiers, including organoid models, gene editing, AI-driven molecular design, and synthetic biology. Organoids provide physiologically relevant platforms that model virus-host interactions and disease progression. Viral infections remain a major challenge to human and animal health, agriculture, and biosecurity. Progress in antiviral research is constrained by the complexity of viral pathogenesis, the diversity and rapid evolution of viruses, and the limited translational relevance of some traditional model systems. Recent advances in organoid technology, gene editing, artificial intelligence, and synthetic biology are expanding the toolkit available for antiviral research and development. In this review, we discuss how these four technological frontiers contribute to disease modeling, target discovery, molecular design, and translational innovation. Organoids, in particular, provide physiologically relevant systems for investigating viral infection, tissue tropism, host responses, and pathogenesis. Gene editing tools, such as CRISPR, enable precise manipulation of host and viral genomes, facilitating the development of resistant organisms and next-generation vaccine platforms. AI technologies, including AlphaFold for structure prediction and platforms for de novo protein design, address long-standing bottlenecks in structural biology and offer powerful means to engineer antiviral proteins, antibodies, and vaccine antigens. Synthetic biology, guided by the Design-Build-Test-Learn cycle, integrates computational design, genetic assembly, and functional validation into a cohesive pipeline. Together, these technologies form a synergistic workflow that spans disease modeling, target discovery, molecular design, construction, testing, and iterative optimization. This integrated approach is shifting antiviral development from traditional empirical methods toward more precise, intelligent strategies. The review also highlights ongoing challenges in integration and scalability, stressing that high-quality biological datasets and stronger interdisciplinary collaboration are essential for realizing translational potential. By presenting a cohesive view of these converging methodologies, this review offers a framework to guide the intelligent evolution of antiviral strategies in both human and animal health.},
}

@article {pmid42702425,
year = {2026},
author = {Li, J and Yuan, M and Cui, X and Ding, Y and Yang, M and Liu, R and Li, L},
title = {A cascaded signal amplification strategy combining Au@Cu2O core-shell nanozymes and CRISPR-Cas12a self-cycling for ultrasensitive colorimetric detection of miRNA-21.},
journal = {Analytica chimica acta},
volume = {1421},
number = {},
pages = {345987},
doi = {10.1016/j.aca.2026.345987},
pmid = {42702425},
issn = {1873-4324},
mesh = {*MicroRNAs/blood/analysis ; *Colorimetry/methods ; *Copper/chemistry ; *Gold/chemistry ; Humans ; *CRISPR-Cas Systems ; Limit of Detection ; },
abstract = {As a critical biomarker for early-stage tumor diagnosis, miRNA-21 poses significant challenges for colorimetric detection due to its ultra-low physiological abundance and high sequence homology. In this study, a multi-stage cascade amplification sensing platform was engineered by integrating an entropy-driven molecular circuit with a heterogeneous nanozyme catalytic network. The platform utilizes Catalytic Hairpin Assembly (CHA) for initial signal transduction, coupled with a CRISPR-Cas12a positive feedback self-cycling system to achieve exponential signal amplification. At the signal output interface, Au@Cu2O core-shell nanocomposites, synthesized via epitaxial growth, exhibit significantly enhanced peroxidase-like activity through interfacial electronic coupling. Experimental results demonstrate a limit of detection (LOD) as low as 1.39 fM, with the capacity to precisely discriminate between serum samples from lung cancer patients and healthy individuals.},
}

*MicroRNAs/blood/analysis
*Colorimetry/methods
*Copper/chemistry
*Gold/chemistry
Humans
*CRISPR-Cas Systems
Limit of Detection

@article {pmid42702653,
year = {2026},
author = {Hu, S and Liu, J and Deng, J and Yuan, Y and Peng, S and Le, M and Li, J and Xu, K},
title = {Recent advances in CRISPR/Cas12a-based biosensors for heavy metal analysis: a review of signal readout strategies and analytical performance.},
journal = {Analytical and bioanalytical chemistry},
volume = {},
number = {},
pages = {},
pmid = {42702653},
issn = {1618-2650},
support = {GZC20261162//the China Postdoctoral Science Foundation/ ; 82173572//the National Natural Science Foundation of China/ ; 82574159//the National Natural Science Foundation of China/ ; 2023JJ30430//the Science and Technology Program of Hunan Province/ ; 2026JJ80902//the Science and Technology Program of Hunan Province/ ; 2026JK2006//the Science and Technology Program of Hunan Province/ ; 23A0075//the Department of Education of Hunan Province/ ; },
abstract = {Heavy metals are environmental hazards owing to their toxicity, persistence, and bioaccumulation in living organisms. These metals often accumulate or transform into more toxic compounds, and some accumulate in the human body through the food chain, which is extremely harmful to health. Although conventional detection methods are reliable, they are often hampered by high costs and complex operations, motivating the exploration of alternative sensing strategies. CRISPR/Cas (Clustering Regularly Interspaced Short Palindromic Repeats and Related Proteins) systems have shown potential for developing sensitive assays with significant target specificity, programmability, and signal amplification capabilities. This review discusses research progress on using CRISPR/Cas12a to detect and prevent heavy metal contamination in food. This review introduces methodological innovations in various signal readout methods, including fluorescence, colorimetry, electrochemistry, and microfluidics, and critically evaluates their analytical performance, such as sensitivity, selectivity, and practical applicability in complex food matrices. Finally, this review aims to provide valuable guidance for researchers to develop next-generation diagnostic tools that meet the requirements of rapid screening and precision testing for food safety.},
}

@article {pmid42703025,
year = {2026},
author = {Li, B and Liu, L and Jin, K and Huang, Z and Zhang, T and Gao, R and Chen, H and Niu, L and Fan, C and Zhang, H and Huang, P and Wang, H},
title = {One-Pot RPA-CRISPR/Cas12a Assay With Visual Readout for the Ultra-Specific Detection of Monkeypox Virus Clade I.},
journal = {Microbial biotechnology},
volume = {19},
number = {9},
pages = {e70437},
pmid = {42703025},
issn = {1751-7915},
support = {2021YFF0703600//National Key Research and Development Program of China/ ; 20250203114SF//the science and technology development program of Jilin Province/ ; },
mesh = {*CRISPR-Cas Systems ; *Monkeypox virus/genetics/isolation & purification/classification ; *Nucleic Acid Amplification Techniques/methods ; *Mpox, Monkeypox/diagnosis/virology ; Recombinases/metabolism ; Sensitivity and Specificity ; Rapid Diagnostic Tests ; Humans ; Animals ; Limit of Detection ; },
abstract = {In 2024, Monkeypox virus (MPXV) clade I has triggered outbreaks in several countries world-wide. MPXV clade I demonstrates enhanced virulence and transmissibility, with a case fatality rate reaching 10%. In response, we have developed a one-pot detection assay specifically targeting MPXV clade I, combining recombinase polymerase amplification (RPA) and the CRISPR/Cas12a system. The assay can be completed within 40 min and achieved a 95% limit of detection (LOD95) of 27.16 copies/μL. No cross-reactivity was observed with MPXV clade II or other tested viral templates, including Vaccinia virus (Tiantan strain). A preliminary room-temperature evaluation showed that the assay retained detectable performance at 25°C, supporting its potential use in equipment-limited settings. The assay also showed good intra-assay and inter-assay repeatability for recombinant plasmid templates, with all coefficient of variation (CV) values below 10%. In simulated clinical samples, the RPA-CRISPR/Cas12a assay detected more low-concentration plasmid-spiked samples than quantitative polymerase chain reaction (qPCR). These results indicate that the established assay is specific, sensitive, repeatable, and easy to perform, providing a practical tool for field-based screening and decentralized detection of MPXV clade I.},
}

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

@article {pmid41120666,
year = {2026},
author = {Goudy, L and Ha, A and Borah, AA and Umhoefer, JM and Chow, L and Tran, C and Winters, A and Talbot, A and Hernandez, R and Li, Z and Subramanya, S and Arab, A and Kale, N and Lee, JHJ and Muldoon, JJ and Liu, C and Schmidt, R and Santangelo, P and Carnevale, J and Eyquem, J and Shy, BR and Marson, A and Gilbert, LA},
title = {Integrated epigenetic and genetic programming of primary human T cells.},
journal = {Nature biotechnology},
volume = {44},
number = {9},
pages = {1470-1481},
pmid = {41120666},
issn = {1546-1696},
support = {K08 CA273529/CA/NCI NIH HHS/United States ; L30TR002983//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; UM1HG012660//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; 2021316455)//National Science Foundation (NSF)/ ; 1K08CA252605-01//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; K08CA273529//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Humans ; *T-Lymphocytes/metabolism/cytology ; *Epigenesis, Genetic/genetics ; CRISPR-Cas Systems/genetics ; Epigenome Editing ; *Gene Editing/methods ; Animals ; Receptors, Chimeric Antigen/genetics ; },
abstract = {Targeted epigenetic engineering of gene expression in cell therapies would allow programming of desirable phenotypes without many of the challenges and safety risks associated with double-strand break-based genetic editing approaches. Here, we develop an all-RNA platform for efficient, durable and multiplexed epigenetic programming in primary human T cells, stably turning endogenous genes off or on using CRISPRoff and CRISPRon epigenetic editors. We achieve epigenetic programming of diverse targeted genomic elements without the need for sustained expression of CRISPR systems. CRISPRoff-mediated gene silencing is maintained through numerous cell divisions, T cell stimulations and in vivo adoptive transfer, avoiding cytotoxicity or chromosomal abnormalities inherent to multiplexed Cas9-mediated genome editing. Lastly, we successfully combined genetic and epigenetic engineering using orthogonal CRISPR Cas12a-dCas9 systems for targeted chimeric antigen receptor (CAR) knock-in and CRISPRoff silencing of therapeutically relevant genes to improve preclinical CAR-T cell-mediated in vivo tumor control and survival.},
}

Humans
*T-Lymphocytes/metabolism/cytology
*Epigenesis, Genetic/genetics
CRISPR-Cas Systems/genetics
Epigenome Editing
*Gene Editing/methods
Animals
Receptors, Chimeric Antigen/genetics

@article {pmid42314573,
year = {2026},
author = {Landi, E and van Beusekom, E and Ben-Dor, S and Albert, S and Dickman, MM and LaPointe, VLS and van Bokhoven, H},
title = {Biallelic rescue of CTG18.1 in two Fuchs endothelial corneal dystrophy-derived iPSC lines (SCTCi047-A-2, SCTCi046-A-2) following a two-step gene editing strategy.},
journal = {Stem cell research},
volume = {95},
number = {},
pages = {104032},
doi = {10.1016/j.scr.2026.104032},
pmid = {42314573},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; *Fuchs' Endothelial Dystrophy/genetics/pathology/metabolism ; *Gene Editing/methods ; Cell Line ; CRISPR-Cas Systems/genetics ; *Transcription Factor 4/genetics/metabolism ; *Alleles ; },
abstract = {Fuchs endothelial corneal dystrophy (FECD) is an age-related condition distinguished by the degeneration of the corneal endothelium. An intronic CTG18.1 repeat in the transcription factor 4 (TCF4) gene has been associated with a 78-fold increased risk of developing the disease when at least one copy of the CTG18.1 expands above 50 repeats. Employing patient-derived material, we applied a dual CRISPR/Cas9-mediated editing approach to rescue the expansion. Combining non-homologous end-joining (NHEJ) and homologous direct repair (HDR) events, we generated two FECD-derived [+/+](CTG)8 induced pluripotent stem cell (iPSC) lines, which were then successfully characterized, providing relevant isogenic controls for disease-modelling purposes.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism
*Fuchs' Endothelial Dystrophy/genetics/pathology/metabolism
*Gene Editing/methods
Cell Line
CRISPR-Cas Systems/genetics
*Transcription Factor 4/genetics/metabolism
*Alleles

@article {pmid42349105,
year = {2026},
author = {Hamam-Marawi, G and Papadopoulou, M and Ramachandran, H and Dobner, J and Binder, S and Hildebrandt, B and Krutmann, J and Rossi, A},
title = {CRISPR/Cas9-mediated editing of ERCC6 in iPSCs: A disease model for Cockayne Syndrome type B.},
journal = {Stem cell research},
volume = {95},
number = {},
pages = {104044},
doi = {10.1016/j.scr.2026.104044},
pmid = {42349105},
issn = {1876-7753},
mesh = {Humans ; Poly-ADP-Ribose Binding Proteins/genetics ; *Cockayne Syndrome/genetics/pathology/metabolism ; *DNA Helicases/genetics/metabolism ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *DNA Repair Enzymes/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Cell Line ; Base Sequence ; Cell Differentiation ; Excision Repair ; },
abstract = {Cockayne Syndrome type B (CSB) is caused by mutations in the ERCC6 gene, which encodes a key protein involved in transcription-coupled nucleotide excision repair (TC-NER) and chromatin remodeling. Deficiency in CSB leads to defective transcriptional recovery after DNA damage, oxidative stress accumulation, and progressive neurodegeneration. In this work, we generated a CRISPR/Cas9-engineered human induced pluripotent stem cell (iPSC) line, IUFi004-A-12, carrying a homozygous mutation in ERCC6 causing a premature stop codon in its 10th exon. The modified iPSCs displayed normal morphology, expressed pluripotency markers, and differentiated into all three germ layers. This model enables mechanistic studies of CSB dysfunction and facilitates therapeutic development for Cockayne Syndrome.},
}

Humans
Poly-ADP-Ribose Binding Proteins/genetics
*Cockayne Syndrome/genetics/pathology/metabolism
*DNA Helicases/genetics/metabolism
*Induced Pluripotent Stem Cells/metabolism/cytology
*DNA Repair Enzymes/genetics/metabolism
*CRISPR-Cas Systems/genetics
Cell Line
Base Sequence
Cell Differentiation
Excision Repair

@article {pmid42361764,
year = {2026},
author = {Liu, NN and Baljinnyam, E and Hu, R and Salemi, SE and Webb, BD and Marro, SG},
title = {Dual CRISPR/Cas9 correction of compound heterozygous MARS2 mutations in the iPSC line ISMMSi060-A from a patient with COXPD25.},
journal = {Stem cell research},
volume = {95},
number = {},
pages = {104041},
doi = {10.1016/j.scr.2026.104041},
pmid = {42361764},
issn = {1876-7753},
support = {R21 NS130319/NS/NINDS NIH HHS/United States ; },
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Mutation/genetics ; Heterozygote ; Cell Line ; *Mitochondrial Proteins/genetics ; Base Sequence ; Cell Differentiation ; },
abstract = {We previously described the induced pluripotent stem cell (iPSC) line ISMMSi060-A derived from a patient with Combined Oxidative Phosphorylation Deficiency 25 (COXPD25) carrying compound heterozygous pathogenic variants in the mitochondrial methionyl-tRNA synthetase gene, MARS2. Here, we report the generation of the isogenic control line ISMMSi060-A-1 by CRISPR/Cas9-mediated correction of the MARS2 variants c.424C>T (p.Arg142Trp) and c.550C>T (p.Gln184*). The corrected line retained normal morphology, pluripotency, genomic integrity, and differentiation capacity, providing a valuable resource to study MARS2-related mitochondrial dysfunction and therapeutic strategies for COXPD25.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
*Mutation/genetics
Heterozygote
Cell Line
*Mitochondrial Proteins/genetics
Base Sequence
Cell Differentiation

@article {pmid42361765,
year = {2026},
author = {Fan, J and Zhang, S and Sun, H and Sun, M and Gu, X and He, Y},
title = {Generation of a FRMD5 knockout human embryonic stem cell line by CRISPR/Cas9 editing.},
journal = {Stem cell research},
volume = {95},
number = {},
pages = {104048},
doi = {10.1016/j.scr.2026.104048},
pmid = {42361765},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; Cell Line ; *Gene Editing/methods ; *Membrane Proteins/genetics/metabolism ; *Gene Knockout Techniques ; Cell Differentiation ; },
abstract = {FRMD5 is a protein-coding gene on human chromosome 15q15.3 encoding a FERM domain-containing protein. Its mutations are linked to NEDEMA, a rare autosomal dominant neurodevelopmental disorder characterized by developmental delay, intellectual disability, ataxia, epilepsy, and eye movement abnormalities (Keller Sarmiento et al., 2024).We established a FRMD5 knockout (FRMD5[-]/[-]) human embryonic stem cell line via CRISPR/Cas9. This line shows normal karyotype, expresses pluripotency markers, and differentiates into three germ layers, serving as a valuable tool for studying FRMD5 in neural development.},
}

Humans
*CRISPR-Cas Systems/genetics
*Human Embryonic Stem Cells/metabolism/cytology
Cell Line
*Gene Editing/methods
*Membrane Proteins/genetics/metabolism
*Gene Knockout Techniques
Cell Differentiation

@article {pmid42378908,
year = {2026},
author = {Lorthongpanich, C and Srisook, P and Jiamvoraphong, N and Klaihmon, P and Kumsui, S and Laowtammathron, C and Issaragrisil, S},
title = {A human iPSC model with LATS1/2 knockdown (MUSIi012-A-9) for investigating Hippo signaling in stem cell.},
journal = {Stem cell research},
volume = {95},
number = {},
pages = {104052},
doi = {10.1016/j.scr.2026.104052},
pmid = {42378908},
issn = {1876-7753},
mesh = {Humans ; *Protein Serine-Threonine Kinases/metabolism/genetics ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Signal Transduction ; *Tumor Suppressor Proteins/genetics/metabolism ; Cell Differentiation ; Hippo Signaling Pathway ; Gene Knockdown Techniques ; Cell Line ; CRISPR-Cas Systems ; },
abstract = {This study reports the generation and comprehensive characterization of the LATS1/2 knockdown (KD) induced pluripotent stem cell (iPSC) line, MUSIi012-A-9. Created via CRISPR/Cas9-mediated modification of the LATS2 gene in a parental LATS1-KD line, this resource serves as a crucial human model to investigate the roles of LATS1/2 kinases, key regulators of the Hippo signaling pathway. Comprehensive validation confirmed normal iPSC morphology, pluripotency marker expression (OCT3/4, NANOG, SOX2), genetic stability (46,XX karyotype), and robust multilineage differentiation potential. This well-characterized iPSC line is a vital tool for advancing research into Hippo pathway regulation, lineage specification, and cell fate determination in development and disease.},
}

Humans
*Protein Serine-Threonine Kinases/metabolism/genetics
*Induced Pluripotent Stem Cells/metabolism/cytology
*Signal Transduction
*Tumor Suppressor Proteins/genetics/metabolism
Cell Differentiation
Hippo Signaling Pathway
Gene Knockdown Techniques
Cell Line
CRISPR-Cas Systems

@article {pmid42385351,
year = {2026},
author = {Buchmann, S and Aldinger, A and Klopocki, E and Gerull, B},
title = {Establishment of CRISPR/Cas9-edited LEMD2 knock-in (UKWCHFi001-B-1) and knock-out (UKWCHFi001-B-2) iPSC lines to investigate the mechanisms of LEMD2-associated cardiomyopathy.},
journal = {Stem cell research},
volume = {95},
number = {},
pages = {104049},
doi = {10.1016/j.scr.2026.104049},
pmid = {42385351},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Membrane Proteins/genetics/metabolism ; *Cardiomyopathies/genetics/metabolism/pathology ; Cell Line ; Gene Knockout Techniques ; *Nuclear Proteins/genetics/metabolism ; *Gene Editing ; Cell Differentiation ; *Gene Knock-In Techniques ; },
abstract = {LEMD2 is an inner nuclear membrane protein. The pathogenic LEMD2 variant (NM_181336.4: c.38 T > G, p.L13R) has been associated with an inherited cardiomyopathy characterized by left ventricular dysfunction and severe arrhythmias. To gain more insights into this disease and investigate the role of LEMD2 more broadly, LEMD2 p.L13R knock-in (LEMD2-KI; UKWCHFi001-B-1) and knock-out (LEMD2-KO; UKWCHFi001-B-2) iPSC lines were generated in a healthy control iPSC (UKWCHFi001-B) line using CRISPR/Cas9 gene editing. Both new iPSC lines retained pluripotency, normal karyotypes, and differentiation potential, whereby expression of LEMD2 was successfully disrupted in LEMD2-KO cells. Thus, these lines provide a suitable model for analyzing LEMD2-associated diseases.},
}

Humans
*CRISPR-Cas Systems/genetics
*Induced Pluripotent Stem Cells/metabolism/cytology
*Membrane Proteins/genetics/metabolism
*Cardiomyopathies/genetics/metabolism/pathology
Cell Line
Gene Knockout Techniques
*Nuclear Proteins/genetics/metabolism
*Gene Editing
Cell Differentiation
*Gene Knock-In Techniques

@article {pmid42391322,
year = {2026},
author = {Wiesbeck, M and Alard, EL and Merino, F and Chowdhury, N and Egert, L and Danese, A and Imhof, S and Iraci Borgia, M and Rajan, A and Fernandez-Novel Marx, N and Kepesidis, E and Köferle, A and Cerron-Alvan, LM and Vierl, F and Truong, TT and Thorwirth, M and Bilalli, L and Santos Dias Mourão, A and Ninkovic, J and Schieweck, R and Diefenbacher, M and Hauck, SM and Trainor, PA and Mardakheh, FK and Götz, M and Stricker, SH},
title = {Manipulation of protein translation and stem cell self-renewal by CRISPR activation of rRNA transcription.},
journal = {Science (New York, N.Y.)},
volume = {393},
number = {6816},
pages = {eaeh1348},
doi = {10.1126/science.aeh1348},
pmid = {42391322},
issn = {1095-9203},
mesh = {*Protein Biosynthesis/genetics ; *RNA, Ribosomal/genetics ; Animals ; *Transcription, Genetic ; *Neural Stem Cells/cytology/metabolism/physiology ; *Cell Self Renewal/genetics ; Cell Proliferation ; Humans ; Mice ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cell Nucleolus/metabolism ; },
abstract = {Ribosomal RNA (rRNA) transcription rates vary during development, and their dysregulation is linked to diseases such as cancer and ribosomopathies. Owing to their high abundance and genomic redundancy, the functional significance of rRNA levels remains unclear. We developed TAPIR (Targeted Activation of Protein Translation), a CRISPR-based approach to elevate rRNA levels by inducing 47S ribosomal DNA transcription. TAPIR increased nucleolar size and enhanced protein synthesis, even in rapidly proliferating cells. In neural stem cells, elevated translation promoted self-renewal and proliferation in vitro and in vivo. Furthermore, TAPIR enabled the modeling and partial rescue of associated disease phenotypes. Our findings reveal that rRNA levels directly regulate translational output and that protein synthesis capacity can act as a key determinant of mammalian stem cell behavior.},
}

*Protein Biosynthesis/genetics
*RNA, Ribosomal/genetics
Animals
*Transcription, Genetic
*Neural Stem Cells/cytology/metabolism/physiology
*Cell Self Renewal/genetics
Cell Proliferation
Humans
Mice
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Cell Nucleolus/metabolism

@article {pmid42447733,
year = {2026},
author = {Ntzani, ER and Ramachandran, H and Papadopoulou, M and Binder, S and Hildebrandt, B and Haarmann-Stemmann, T and Rossi, A},
title = {CRISPR/Cpf1-mediated knockout of FLG in human induced pluripotent stem cells generates a model for studying epidermal barrier dysfunction.},
journal = {Stem cell research},
volume = {95},
number = {},
pages = {104056},
doi = {10.1016/j.scr.2026.104056},
pmid = {42447733},
issn = {1876-7753},
mesh = {Humans ; Filaggrin Proteins ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; Cell Differentiation ; *Epidermis/metabolism/pathology ; *Gene Knockout Techniques ; Cell Line ; *Intermediate Filament Proteins/genetics/metabolism ; },
abstract = {Loss of filaggrin (FLG) function impairs skin barrier formation and contributes to common inflammatory skin diseases. In this study, we established a FLG knockout human induced pluripotent stem cell (iPSC) line based on KOLF2.1 J using CRISPR/Cas12a (Cpf1)-mediated genome editing. A guide RNA targeting exon 2 introduced a homozygous mutation, which was confirmed by sequencing. The edited cells maintained typical pluripotent stem cell morphology, expressed key undifferentiated markers, and retained the ability to differentiate into all three germ layers. Karyotype and copy number variation (CNV) analyses confirmed genomic stability and parental origin; the cells were free of mycoplasma. This cell line enables studies of FLG-associated skin biology and pathology.},
}

Humans
Filaggrin Proteins
*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
Cell Differentiation
*Epidermis/metabolism/pathology
*Gene Knockout Techniques
Cell Line
*Intermediate Filament Proteins/genetics/metabolism

@article {pmid42470827,
year = {2026},
author = {Dong, Q and Luo, S and Sun, J and Liu, H},
title = {Generation of FCGR3A-EGFP knock-in reporter human embryonic stem cell line, WAe001-A-3S, using CRISPR/Cas9n-based gene targeting.},
journal = {Stem cell research},
volume = {95},
number = {},
pages = {104061},
doi = {10.1016/j.scr.2026.104061},
pmid = {42470827},
issn = {1876-7753},
mesh = {Humans ; *Receptors, IgG/genetics/metabolism ; *Green Fluorescent Proteins/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; *Gene Knock-In Techniques ; Cell Differentiation ; Cell Line ; *Gene Targeting ; Genes, Reporter ; },
abstract = {Fc gamma receptor IIIA (FCGR3A) encodes CD16a, a key mediator of antibody-dependent cellular cytotoxicity (ADCC) that regulates innate and adaptive immunity, especially in natural killer (NK) cells and monocytes. We generated an FCGR3A-EGFP knock-in human embryonic stem cell (hESC) line via CRISPR/Casn9n. The cell line showed a normal karyotype, maintained expression ofthe pluripotency markers OCT4, SOX2, and NANOG, and retained trilineage differentiation potential. This reporter line enables real-time tracking of FCGR3A expression during immune cell differentiation, serving as a useful tool for studying FCGR3A[+] immune cell development and related immune mechanisms.},
}

Humans
*Receptors, IgG/genetics/metabolism
*Green Fluorescent Proteins/metabolism/genetics
*CRISPR-Cas Systems/genetics
*Human Embryonic Stem Cells/metabolism/cytology
*Gene Knock-In Techniques
Cell Differentiation
Cell Line
*Gene Targeting
Genes, Reporter

@article {pmid42492408,
year = {2026},
author = {Duan, C and Sun, X and Ding, C and Mao, S and Liang, Y and Liang, Y and Zhang, R and Chen, H and Chen, J and Tang, S},
title = {Generation of a human iPSC line (CSUASOi016-A) modeling X-linked retinoschisis by introducing the RS1 c.214G>A mutation using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {95},
number = {},
pages = {104060},
doi = {10.1016/j.scr.2026.104060},
pmid = {42492408},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Retinoschisis/genetics/pathology/metabolism ; *CRISPR-Cas Systems/genetics ; *Eye Proteins/genetics/metabolism ; Cell Line ; Mutation ; Cell Differentiation ; Base Sequence ; },
abstract = {X-linked retinoschisis (XLRS) is an inherited retinal degenerative disease caused by mutations in the RS1 gene, leading to visual impairment. The RS1 c.214G>A mutation is a clinically relevant variant associated with XLRS. In this study, we generated a human induced pluripotent stem cell (iPSC) line (CSUASOi016-A) carrying the RS1 c.214G>A (p.E72K) mutation using CRISPR/Cas9. The edited iPSC line exhibited typical pluripotent stem cell morphology, expressed pluripotency markers (OCT4, SSEA4, SOX2, and NANOG), and retained the ability to differentiate into all three germ layers. This cell line provides a valuable resource for modeling XLRS pathogenesis and developing therapeutic strategies.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*Retinoschisis/genetics/pathology/metabolism
*CRISPR-Cas Systems/genetics
*Eye Proteins/genetics/metabolism
Cell Line
Mutation
Cell Differentiation
Base Sequence

@article {pmid42508498,
year = {2026},
author = {Liu, J and Bian, Z and Chen, J and Kang, H and Li, Q and Lin, J and Gou, H and Li, C},
title = {Rapid and visual detection of Lawsonia intracellularis via an RPA-CRISPR/Cas12a assay.},
journal = {Journal of microbiological methods},
volume = {249},
number = {},
pages = {107645},
doi = {10.1016/j.mimet.2026.107645},
pmid = {42508498},
issn = {1872-8359},
mesh = {*Lawsonia Bacteria/isolation & purification/genetics ; Animals ; *Desulfovibrionaceae Infections/diagnosis/veterinary/microbiology ; Swine ; *Swine Diseases/diagnosis/microbiology ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; Rapid Diagnostic Tests ; *Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins/genetics ; },
abstract = {The swine industry faces a substantial economic threat from Lawsonia intracellularis (LI), the bacterium responsible for porcine proliferative enteropathy. Infection commonly presents with diarrhea and increased enterocyte proliferation in the ileum and colon, ultimately resulting in impaired growth performance in affected pigs. Effective management of porcine proliferative enteropathy requires timely and accurate diagnosis. The method provides significant advantages over existing detection techniques, offering enhanced sensitivity and high accuracy. The assay achieves detection within one hour, with no observed cross-reactivity against a panel of common swine pathogens. Results can be visually interpreted under blue light. Clinical validation using 123 samples demonstrated 100% concordance with qPCR results (14 positive and 109 negative). Characterized by simplicity, rapidity, and high sensitivity, the RPA-CRISPR/Cas12a method represents a novel solution for Lawsonia intracellularis detection.},
}

*Lawsonia Bacteria/isolation & purification/genetics
Animals
*Desulfovibrionaceae Infections/diagnosis/veterinary/microbiology
Swine
*Swine Diseases/diagnosis/microbiology
Sensitivity and Specificity
*CRISPR-Cas Systems
Rapid Diagnostic Tests
*Nucleic Acid Amplification Techniques/methods
Bacterial Proteins/genetics

@article {pmid42544618,
year = {2026},
author = {Li, X and Li, D and Wang, Y and Xiang, C and Tan, S and Fan, R and Shi, K and Zhou, W},
title = {HCR-assisted CRISPR/Cas12a platform enables logic-gate and multimodal detection of kanamycin and bisphenol A.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {34},
pages = {7385-7395},
doi = {10.1039/d6ay01075j},
pmid = {42544618},
issn = {1759-9679},
mesh = {Bisphenol A Compounds ; *Benzhydryl Compounds/analysis ; *Phenols/analysis ; *Biosensing Techniques/methods ; *Kanamycin/analysis ; *CRISPR-Cas Systems/genetics ; Animals ; Nucleic Acid Hybridization/methods ; Milk/chemistry ; Limit of Detection ; Colorimetry/methods ; },
abstract = {A novel triple-modal biosensor integrating hybridization chain reaction (HCR) with CRISPR/Cas12a was developed for sensitive and selective detection of kanamycin (KANA) and bisphenol A (BPA). The system employs a phosphorothioate-modified G-rich hairpin (SHG4-2) as a dual-functional reporter probe, which resists Cas12a trans-cleavage and enables multimodal signal output via SG-quadruplex (SG4) formation. Upon target recognition by aptamers, an initiator strand is released to trigger HCR amplification, generating long double-stranded DNA products that activate Cas12a trans-cleavage. This cleaves the (SHG4-2) probe, releasing SG-rich sequences that self-assemble into SG4 structures, yielding fluorescence (with Thioflavin T), colorimetric (via SG4/hemin-catalyzed TMB oxidation), and smartphone-readable RGB signals. This platform enables parallel detection of a single target analyte, allowing flexible detection modes. Under optimized conditions, the sensor achieved detection limits as low as 20.1 pM for KANA and 6.8 pM for BPA in fluorescence mode, 32.3 pM for KANA and 17.1 pM for BPA in colorimetric mode, and 74.3 pM for KANA and 35.8 pM for BPA in smartphone mode, with excellent selectivity against interfering analogues. Successful application in spiked milk samples demonstrated high recovery rates and good reproducibility. In addition, the platform supports the logical gate operations of OR (single-target detection) and AND (dual-target detection), which allows flexible detection modes. This work presents a versatile, amplification-enhanced multimodal sensing strategy for environmental and food safety monitoring, highlighting its potential for logic-driven biosensing applications.},
}

Bisphenol A Compounds
*Benzhydryl Compounds/analysis
*Phenols/analysis
*Biosensing Techniques/methods
*Kanamycin/analysis
*CRISPR-Cas Systems/genetics
Animals
Nucleic Acid Hybridization/methods
Milk/chemistry
Limit of Detection
Colorimetry/methods

@article {pmid42556251,
year = {2026},
author = {Clémençon, M and Brogard, J and Rozen, M and Hourton, C and Meléndez García, R and Bigou, S and Tsang, SH and Thouvenin, O and Grieve, K and Reichman, S},
title = {Generation of retinitis pigmentosa patient-derived hiPSC lines (IDVi007-A, IDVi007-B) carrying the RHO c.68C > A variant (p.P23H) and CRISPR/Cas9-corrected isogenic hiPSC lines (IDVi007-A-1, IDVi007-A-2, IDVi007-A-3).},
journal = {Stem cell research},
volume = {95},
number = {},
pages = {104042},
doi = {10.1016/j.scr.2026.104042},
pmid = {42556251},
issn = {1876-7753},
mesh = {Humans ; *Retinitis Pigmentosa/genetics/pathology/metabolism ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; Cell Line ; Cell Differentiation ; Mutation ; Fibroblasts/metabolism/cytology ; *rho GTP-Binding Proteins/genetics ; },
abstract = {The p.Pro23His (c.68C > A; P23H) mutation leads to autosomal dominant retinitis pigmentosa (adRP). Here, we reprogrammed adRP patient fibroblasts in human induced pluripotent stem cells (hiPSCs) using Sendai virus. We then generated two mutated hiPSC clones and three isogenic controls using CRISPR/Cas9. All five hiPSC lines express pluripotency genes and are able to differentiate into the three germ layers as well as retinal organoids. Altogether, these hiPSCs constitute unique biological tools to elucidate mechanisms of adRP linked to the RHO-P23H mutation.},
}

Humans
*Retinitis Pigmentosa/genetics/pathology/metabolism
*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
Cell Line
Cell Differentiation
Mutation
Fibroblasts/metabolism/cytology
*rho GTP-Binding Proteins/genetics

@article {pmid42566814,
year = {2026},
author = {Wang, S and Feng, X and Shang, B and Wang, H and Hu, X and Wang, Z and Li, Z},
title = {Generation of an NKX2-1-EGFP reporter iPSC line with inducible Cas9 for lung progenitor cell tracing.},
journal = {Stem cell research},
volume = {95},
number = {},
pages = {104072},
doi = {10.1016/j.scr.2026.104072},
pmid = {42566814},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Thyroid Nuclear Factor 1/metabolism/genetics ; *Lung/cytology/metabolism ; *Green Fluorescent Proteins/metabolism/genetics ; CRISPR-Cas Systems/genetics ; Cell Line ; Cell Differentiation ; Genes, Reporter ; },
abstract = {NK2 homeobox 1 (NKX2-1), a master regulator robustly expressed in lung, thyroid, and forebrain, is indispensable for specifying lung epithelial fate and serves as a definitive marker of lung progenitors. Here, we generated a human induced Pluripotent Stem Cell (iPSC) line harboring a doxycycline (dox)-inducible Cas9 and an NKX2-1-EGFP-puro reporter via CRISPR/Cas9-mediated homology-directed repair. This dual-function line combines inducible genome editing with real-time tracing of early lung progenitors, enabling their prospective isolation and screening for stage-specific maturation regulators. Therefore, this engineered iCas9-NKX2-1 EGFP line is a key resource for dissecting human lung development, modeling pulmonary disease, and advancing regenerative therapies.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*Thyroid Nuclear Factor 1/metabolism/genetics
*Lung/cytology/metabolism
*Green Fluorescent Proteins/metabolism/genetics
CRISPR-Cas Systems/genetics
Cell Line
Cell Differentiation
Genes, Reporter

@article {pmid42566815,
year = {2026},
author = {Zhou, H and Li, Y and Jia, M and Du, J and Zheng, K},
title = {CRISPR/Cas9-mediated generation of a homozygous MT4 knockout mouse embryonic stem cell line.},
journal = {Stem cell research},
volume = {95},
number = {},
pages = {104070},
doi = {10.1016/j.scr.2026.104070},
pmid = {42566815},
issn = {1876-7753},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Mice ; *Mouse Embryonic Stem Cells/metabolism/cytology ; Homozygote ; Cell Line ; Mice, Knockout ; Cell Differentiation ; *Metallothionein/genetics/deficiency/metabolism ; Gene Knockout Techniques ; },
abstract = {Metallothionein 4 (MT4) is a low-molecular-weight, cysteine-rich metal-binding protein belonging to the metallothionein family. It exhibits unique skin developmental and differentiation inhibitory activity when functionally impaired and regulates skin cell growth and disease through multiple mechanisms. However, its exact role in cell fate determination remains unknown. Here, we utilized the CRISPR/Cas9 system to generate a homozygous Mt4 knockout (Mt4[-]/[-]) mouse embryonic stem cell (mESC) line. This cell line maintains normal morphology, pluripotency, and the ability to differentiate into all three germ layers. It provides a valuable resource for investigating the mechanisms underlying skin diseases caused by MT4 gene mutations.},
}

Animals
*CRISPR-Cas Systems/genetics
Mice
*Mouse Embryonic Stem Cells/metabolism/cytology
Homozygote
Cell Line
Mice, Knockout
Cell Differentiation
*Metallothionein/genetics/deficiency/metabolism
Gene Knockout Techniques

@article {pmid42615464,
year = {2026},
author = {Kotler, ED and Ashraf, S and Santerre, JP and Kandel, RA},
title = {Optimization of CRISPR dCas9 Lentiviral Transduction in Nucleus Pulposus Cells for Use in Tissue Engineering.},
journal = {Tissue engineering. Part C, Methods},
volume = {32},
number = {8-9},
pages = {310-320},
doi = {10.1177/19373384261477635},
pmid = {42615464},
issn = {1937-3392},
mesh = {Humans ; *Lentivirus/genetics/metabolism ; *Tissue Engineering/methods ; HEK293 Cells ; *Nucleus Pulposus/cytology/metabolism ; *Transduction, Genetic/methods ; *CRISPR-Cas Systems/genetics ; Transfection ; Hepatocyte Nuclear Factor 3-beta/metabolism/genetics ; },
abstract = {Efficient delivery of large gene-editing plasmids, such as the mCherry-CRISPR dCas9 system, into nucleus pulposus (NP) cells is a key step in generating sufficient cells for tissue-engineered intervertebral disc (IVD) constructs and other regenerative therapies for degenerative disc disease (DDD). However, the transfection and transduction of these environmentally sensitive cells remain challenging. This study aimed to identify the best protocol for delivery with minimal cytotoxicity and greatest efficiency. Transfection conditions in HEK293T cells were evaluated using Lipofectamine 3000, Lipofectamine Classic, and ViaFect at different reagent-to-DNA ratios and DNA amounts. Transfection efficiency was quantified by flow cytometry based on mCherry expression. Lentivirus was produced and concentrated by comparing PEG8000, a commercial Lenti-X concentrator, and ultracentrifugation. For NP cell transduction, polybrene and protamine sulfate were tested at multiple concentrations to maximize efficiency and viability. The optimized protocol was validated by delivering a CRISPR/dCas9 Synergistic Activation Mediator (SAM) system to activate endogenous FOXA2, and by seeding the resulting cells onto membranes to assess in vitro NP-like tissue formation. Lipofectamine 3000 at a 2:1 reagent-to-DNA ratio with 0.5 µg DNA per well yielded the highest transfection efficiency in HEK293T cells while minimizing cytotoxicity. Coprecipitation methods for lentiviral concentration, particularly the in-house PEG8000 concentrator, were better than ultracentrifugation. Protamine sulfate at 30 µg/mL yielded efficient NP cell transduction with higher viability than polybrene, as assessed by survival after antibiotic selection. Application of this protocol upregulated endogenous FOXA2 mRNA and protein expression, demonstrating functional efficacy. SAM-FOXA2 cells produced thicker tissue on membrane inserts than SAM controls, confirming compatibility of the protocol with downstream tissue engineering applications. Therefore, an optimized protocol balancing high transfection/transduction efficiency with minimized cytotoxicity was developed, supporting tissue-engineered IVD constructs and other CRISPR-based regenerative therapies for DDD.},
}

Humans
*Lentivirus/genetics/metabolism
*Tissue Engineering/methods
HEK293 Cells
*Nucleus Pulposus/cytology/metabolism
*Transduction, Genetic/methods
*CRISPR-Cas Systems/genetics
Transfection
Hepatocyte Nuclear Factor 3-beta/metabolism/genetics

@article {pmid42624408,
year = {2026},
author = {Zhang, X and Wang, L and Shi, Y and Wang, J and Liu, L and Sun, X and Liang, B and Wang, J and Xu, X},
title = {Development and preliminary evaluation of real-time fluorescence-based RPA and RPA-CRISPR/Cas12a assays for rapid detection of Coxiella burnetii.},
journal = {Journal of microbiological methods},
volume = {249},
number = {},
pages = {107674},
doi = {10.1016/j.mimet.2026.107674},
pmid = {42624408},
issn = {1872-8359},
mesh = {*Coxiella burnetii/genetics/isolation & purification ; Sensitivity and Specificity ; *Q Fever/diagnosis/microbiology ; *CRISPR-Cas Systems ; Humans ; *Nucleic Acid Amplification Techniques/methods ; Rapid Diagnostic Tests ; Animals ; DNA Primers/genetics ; Fluorescence ; DNA, Bacterial/genetics ; },
abstract = {Coxiella burnetii (C. burnetii) is a ubiquitous zoonotic pathogen that exists widely in nature and is an obligate intracellular parasite of eukaryotic cells, causing Q fever in both humans and animals. Therefore, the surveillance and prevention of Q fever are of great importance for public health and the livestock industry. In this study, specific RPA primers, probes, and crRNAs were designed and synthesized targeting the IS1111 gene of C. burnetii, a real-time fluorescence recombinase polymerase amplification (qRPA) assay and a RPA combined with CRISPR/Cas12a (RPA-CRISPR/Cas12a) assay for the rapid detection of C. burnetii were developed. The qRPA assay completed target gene amplification within 20 min, and the amplification curves were monitored in real time on a portable Genie III instrument at 39 °C. The entire single-tube RPA-CRISPR/Cas12a assay, including RPA amplification and CRISPR/Cas12a detection, was completed within 40 min, and the results could be directly visualized under blue-light illumination. Both assays exhibited high specificity for C. burnetii and showed no cross-reactivity with the tested microorganisms. The LOD of the qRPA assay for C. burnetii DNA standard was 54 copies/μL, whereas the LOD of the RPA-CRISPR/Cas12a assay was 1.3 copies/μL. The assays were further evaluated on 456 clinical samples for the detection of C. burnetii. The postive rates of the qRPA and RPA-CRISPR/Cas12a assays were 3.73% (17/456) and 4.17% (19/456), respectively. Compared with qPCR detection results, the concordance rates were 99.34% and 99.78%, respectively. In conclusion, the qRPA and RPA-CRISPR/Cas12a assays developed in this study are rapid, specific and sensitive, making them suitable for the on-site detection of C. burnetii at primary level.},
}

*Coxiella burnetii/genetics/isolation & purification
Sensitivity and Specificity
*Q Fever/diagnosis/microbiology
*CRISPR-Cas Systems
Humans
*Nucleic Acid Amplification Techniques/methods
Rapid Diagnostic Tests
Animals
DNA Primers/genetics
Fluorescence
DNA, Bacterial/genetics

@article {pmid42673729,
year = {2026},
author = {Jones, BM and Xu, M and Zou, J and Liu, C and Zou, Y and Pathak, P and Lomash, RM and Stan, R and Bönnemann, CG and Chen, CZ},
title = {Establishment of induced pluripotent stem cell line TRNDi045-A-38 carrying homozygous DOK7-related Congenital Myasthenia patient-mutation knock-in variant from parental KOLF2.1J.},
journal = {Stem cell research},
volume = {95},
number = {},
pages = {104090},
doi = {10.1016/j.scr.2026.104090},
pmid = {42673729},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Myasthenic Syndromes, Congenital/genetics/pathology ; *Muscle Proteins/genetics/metabolism ; Cell Line ; Homozygote ; *Mutation ; Gene Knock-In Techniques ; Female ; CRISPR-Cas Systems ; },
abstract = {DOK7-related Congenital Myasthenic Syndrome (CMS) is a rare genetic neuromuscular junction disorder. This is one of the most common of the recessive forms of CMS, often presenting with more static proximal weakness (hence also referred to as limb girdle CMS). Whole-genome sequencing of affected patients implicates frameshift duplication mutations in DOK7 as drivers of impaired neuromuscular-junction signaling. In this study, we generated a human induced pluripotent stem cell (hiPSC) line TRNDi045-A-38 from the KOLF2.1J reference line, engineered to carry homozygous DOK7 c.1124_1127dupTGCC mutation knock-in using CRISPR/Cas9. This iPSC line could be used for in vitro disease modeling to study disease pathophysiology and for therapeutic development.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*Myasthenic Syndromes, Congenital/genetics/pathology
*Muscle Proteins/genetics/metabolism
Cell Line
Homozygote
*Mutation
Gene Knock-In Techniques
Female
CRISPR-Cas Systems

@article {pmid42696150,
year = {2026},
author = {Sharma, NN and Kumari, A and Vashisht, I and Sharma, MK},
title = {Advances in CRISPR multiplex gene editing to map and modify stress-responsive transcription factor networks for crop improvement.},
journal = {Plant cell reports},
volume = {45},
number = {9},
pages = {},
pmid = {42696150},
issn = {1432-203X},
support = {DST/INSPIRE Fellowship/2021/IF210110//Department of Science and Technology, Ministry of Science and Technology, India/ ; CRG/2023/007038//Anusandhan National Research Foundation,India/ ; ANRF/PAIR/2025/000029/PAIR//JNU-PAIR Network of Anusandhan National Research Foundation, India/ ; },
mesh = {*Gene Editing/methods ; *Transcription Factors/genetics/metabolism ; *Crops, Agricultural/genetics ; *Stress, Physiological/genetics ; *CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Plant ; Plants, Genetically Modified/genetics ; Plant Proteins/genetics/metabolism ; Genome, Plant/genetics ; },
abstract = {The development of CRISPR multiplex genome-editing (MGE) tools is rapidly transforming plant functional genomics and accelerating crop improvements. By simultaneously targeting two or more DNA loci, it allows scientists to precisely edit multiple genes at the single-nucleotide level, within the target genome. Simultaneous manipulation of multiple targets has revolutionized the functional elucidation studies, particularly the dissection of complex genetic pathways. Due to its superior precision and feasibility, CRISPR-MGE is widely accepted and has largely replaced alternative editing tools such as TALENs and ZFNs. Several CRISPR-MGE strategies, including the use of individual expression cassettes, tRNA-processing enzymes, Csy4 or ribozymes, have been successfully deployed in plants. Recent advancements, such as Cpf1, transgene-free methods, or ultra-multiplexing approaches, have further refined the technology into a powerful, efficient, and robust toolkit. MGE enables complex genome editing, including multiple-gene knockouts, base alterations, transcriptional regulation, metabolic engineering, or their combinations. Consequently, it is ideal for elucidating the function of transcription factors that are key molecular players in regulating diverse plant responses, especially in stress pathways. Several stress-responsive TFs have been functionally characterized via CRISPR-MGE, and more advanced tools are being employed. This review evaluates multiplexing tools, their diverse applications, and the current progress toward developing advanced MGE tools. Ultimately, we provide evidence to encourage the use of advanced MGE tools for functional characterization studies of stress-responsive TFs, thereby highlighting their potential to accelerate crop improvement.},
}

*Gene Editing/methods
*Transcription Factors/genetics/metabolism
*Crops, Agricultural/genetics
*Stress, Physiological/genetics
*CRISPR-Cas Systems/genetics
Gene Expression Regulation, Plant
Plants, Genetically Modified/genetics
Plant Proteins/genetics/metabolism
Genome, Plant/genetics

@article {pmid42697642,
year = {2026},
author = {Ye, WN and Xing, Q and Chen, RY and Zhang, YC and Wu, SF and Gao, CF},
title = {Field resistance monitoring and nAChR α6 target validation support spinetoram as a candidate insecticide for rotation in Nilaparvata lugens resistance management.},
journal = {Pesticide biochemistry and physiology},
volume = {223},
number = {},
pages = {107264},
doi = {10.1016/j.pestbp.2026.107264},
pmid = {42697642},
issn = {1095-9939},
mesh = {Animals ; *Receptors, Nicotinic/genetics/metabolism ; *Insecticide Resistance/genetics ; *Insecticides/pharmacology ; *Hemiptera/drug effects/genetics ; *Macrolides/pharmacology ; *Insect Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Drug Combinations ; },
abstract = {The brown planthopper (BPH), Nilaparvata lugens (Stål), is a major pest threatening rice production in Asia. Severe resistance to commonly used insecticides has become a major challenge for its effective control, highlighting the need for alternative insecticides. In this study, we evaluated the susceptibility of field populations of N. lugens to spinetoram and functionally validated the nicotinic acetylcholine receptor (nAChR) α6 subunit, Nlα6, as a molecular determinant of spinosyn susceptibility. Resistance monitoring showed that field populations remained highly susceptible to spinetoram despite exhibiting moderate to high resistance to several commonly used insecticides. A homozygous Nlα6 knockout strain generated using CRISPR/Cas9 developed high-level resistance to spinosad and spinetoram, with resistance ratios of 554.1 and 357.1, respectively, but did not show broad cross-resistance to nine other nAChR-targeting insecticides. Co-segregation analysis further confirmed tight linkage between Nlα6 loss of function and spinosyn resistance. Inheritance analysis showed that spinetoram resistance mediated by Nlα6 knockout was autosomal and completely recessive. Transcriptome analysis revealed that Nlα6 deficiency was associated with reduced expression of immune defense genes, enrichment of functions related to axonemes and neural processes, and coordinated activation of energy metabolism pathways, suggesting that Nlα6 disruption may alter neural homeostasis and energy demand. These results demonstrate that Nlα6 is a key subunit mediating spinosyn susceptibility in N. lugens and support spinetoram as a candidate rotation insecticide for resistance management.},
}

Animals
*Receptors, Nicotinic/genetics/metabolism
*Insecticide Resistance/genetics
*Insecticides/pharmacology
*Hemiptera/drug effects/genetics
*Macrolides/pharmacology
*Insect Proteins/genetics/metabolism
CRISPR-Cas Systems
Drug Combinations

@article {pmid42697651,
year = {2026},
author = {Zhou, J and Zang, Z and Gao, H and Sun, J and Xu, L and Gao, Q and He, H and Ding, W and Qiu, L and Li, Y},
title = {CRISPR/Cas9-mediated knockout of CsRDL1 decreases the susceptibility of Chilo suppressalis to cyproflanilide.},
journal = {Pesticide biochemistry and physiology},
volume = {223},
number = {},
pages = {107282},
doi = {10.1016/j.pestbp.2026.107282},
pmid = {42697651},
issn = {1095-9939},
mesh = {Animals ; *CRISPR-Cas Systems ; *Insecticides/pharmacology ; Gene Knockout Techniques ; Insecticide Resistance/genetics ; *Insect Proteins/genetics/metabolism ; *Moths/drug effects/genetics ; Ivermectin/analogs & derivatives ; Female ; *Anilides/pharmacology ; *Receptors, GABA/genetics ; Larva/drug effects ; },
abstract = {The novel meta-diamide insecticide cyproflanilide represents a promising tool for pest control, yet its precise molecular target remains uncharacterized. The RDL1 encodes a subunit of insect ionotropic GABA receptors (GABAr), which is targeted by meta-diamide pesticides. This study investigated the functional role of the resistance to dieldrin 1 (RDL1) subunit in Chilo suppressalis, a major rice pest, by generating a homozygous CsRDL1 knockout strain (CsRDL1[-/-], lacking 101 bp) via CRISPR/Cas9-mediated gene editing. Bioassay results showed that the CsRDL1[-/-] strain exhibited a 2.67-fold increase in tolerance to cyproflanilide and a 3.08-fold increase to broflanilide, whereas its susceptibility to abamectin and emamectin benzoate, which also act on the CsRDL1 subunit, remained unchanged. These results indicate that knockout of CsRDL1 decreased susceptibility to cyproflanilide and broflanilide, and that the mode of action of these meta-diamides differs from that of abamectin and emamectin benzoate. Meanwhile, the CsRDL1[-/-] strain exhibited significant changes in key biological traits: 3rd-6th instar larval duration shortened by 9.45%, fecundity (eggs per female) reduced by 29.49%, and both pupal duration and female adult longevity were significantly shortened relative to the wild-type population. These findings provide in vivo evidence that CsRDL1 contributes to cyproflanilide susceptibility and plays important roles in development and reproduction in C. suppressalis. This study lays a foundation for further mechanistic studies on the interaction between cyproflanilide and CsRDL1 subunit, and offers insights for the development of insecticides targeting this subunit.},
}

Animals
*CRISPR-Cas Systems
*Insecticides/pharmacology
Gene Knockout Techniques
Insecticide Resistance/genetics
*Insect Proteins/genetics/metabolism
*Moths/drug effects/genetics
Ivermectin/analogs & derivatives
Female
*Anilides/pharmacology
*Receptors, GABA/genetics
Larva/drug effects

@article {pmid42697663,
year = {2026},
author = {Pang, X and Zhang, Z and Qin, D and Bachler, A and Yang, Y and Wu, Y},
title = {Knockout of SfVipR2 confers Vip3Aa resistance in Spodoptera frugiperda with evidence of genetic complementation among non-allelic mutations of SfVipR1, SfVipR2, and SfCHS2.},
journal = {Pesticide biochemistry and physiology},
volume = {223},
number = {},
pages = {107294},
doi = {10.1016/j.pestbp.2026.107294},
pmid = {42697663},
issn = {1095-9939},
mesh = {Animals ; *Spodoptera/genetics/drug effects ; *Bacterial Proteins/genetics ; Gene Knockout Techniques ; Mutation ; Genetic Complementation Test ; *Insect Proteins/genetics ; *Insecticide Resistance/genetics ; CRISPR-Cas Systems ; },
abstract = {The Vip3Aa protein from Bacillus thuringiensis is widely deployed in transgenic crops to control lepidopteran pests, including the fall armyworm Spodoptera frugiperda, a highly invasive, polyphagous pest of global importance. Loss-of-function mutations in SfVipR1 (a thyroglobulin-like gene) and SfCHS2 (chitin synthase 2) have previously been confirmed to confer high-level, recessive resistance to Vip3Aa in S. frugiperda. A second thyroglobulin-like gene (HaVipR2) has recently been recognized as an important determinant of Vip3Aa resistance in Helicoverpa armigera. In this study, we first confirmed that knockout of SfVipR2 using CRISPR/Cas9 gene editing also confers high-level, recessive resistance to Vip3Aa. Then, three isogenic knockout strains (SfVipR1-KO established previously; and SfVipR2-KO and SfCHS2-KO, newly generated; all in the YJ-19 genetic background) were used to investigate functional relationships among SfVipR1, SfVipR2, and SfCHS2. Reciprocal crosses between any pair of these resistant strains yielded fully susceptible hybrid progeny, demonstrating complete genetic complementation and no epistasis among the non-allelic mutations. Disruption of any of these genes can result in high-level resistance to Vip3Aa, indicating a substantial resistance risk in S. frugiperda and underscoring the need for resistance management tactics such as establishing an adequate proportion of non-Bt host refuges and deploying pyramided crops expressing Vip proteins and Bt proteins with a different mode of action than Vip3Aa (e.g., Cry proteins).},
}

Animals
*Spodoptera/genetics/drug effects
*Bacterial Proteins/genetics
Gene Knockout Techniques
Mutation
Genetic Complementation Test
*Insect Proteins/genetics
*Insecticide Resistance/genetics
CRISPR-Cas Systems

@article {pmid42697672,
year = {2026},
author = {Kang, R and Qian, Y and Zuo, Y and Li, R and Zhang, L},
title = {Development of an RPA-CRISPR/Cas12a-based rapid visual detection method for two insecticide resistance-associated mutations in Aphis gossypii: A302S in the ace1 gene and M918L in the para gene.},
journal = {Pesticide biochemistry and physiology},
volume = {223},
number = {},
pages = {107303},
doi = {10.1016/j.pestbp.2026.107303},
pmid = {42697672},
issn = {1095-9939},
mesh = {Animals ; *Insecticide Resistance/genetics ; Mutation ; *CRISPR-Cas Systems ; Insecticides/pharmacology ; *Insect Proteins/genetics ; },
abstract = {The resistance of Aphis gossypii to pyrethroids and organophosphate/carbamate insecticides has become increasingly severe. The para M918L and ace1 A302S target-site mutations are the key mechanisms conferring resistance to these two types of insecticides, respectively. However, rapid and field-deployable methods for detecting these mutations are still lacking. In this study, the genotype and allele frequencies of eight field populations collected in 2023 were investigated, and a rapid visual detection method based on RPA-CRISPR/Cas12a was developed. By optimizing the concentration of the reaction components, specific recognition of both mutations was achieved. The optimized assay could be completed within 1 h under a single-temperature condition of 37 °C, with fluorescence signals directly visualized using a portable 440-460 nm blue-light illuminator without PCR thermocyclers, fluorescence readers or sequencing platforms. Validation using the tested field samples showed complete concordance with Sanger sequencing. In addition, Sanger sequencing further showed that no homozygous susceptible genotype was detected at the para M918 locus across all populations, whereas the 302S allele frequency at the ace1 A302S locus exhibited significant geographic variation, ranging from 0% to 100%. The RPA-CRISPR-based visual detection method developed in this study is rapid, accurate, and does not require expensive instrumentation. Together with previously developed neonicotinoid resistance detection techniques, it forms a comprehensive monitoring system for multiple resistance in A. gossypii, providing a practical tool for rapid field diagnosis and more informed insecticide selection.},
}

Animals
*Insecticide Resistance/genetics
Mutation
*CRISPR-Cas Systems
Insecticides/pharmacology
*Insect Proteins/genetics

@article {pmid42699674,
year = {2026},
author = {Mohanty, A and Sahoo, RK and Sanket, AS and Rout, E},
title = {Combating New Delhi metallo-beta-lactamase-1 (NDM-1): an integrated review of inhibitor development and next-generation therapeutic platforms.},
journal = {RSC advances},
volume = {},
number = {},
pages = {},
pmid = {42699674},
issn = {2046-2069},
abstract = {New Delhi metallo-β-lactamase-1 (NDM-1), which has emerged globally, exhibits resistance to almost all β-lactam antibiotics, including carbapenems, posing a major challenge to modern antibiotic therapy. Deep sequencing has revealed the evolution of several new NDM variants (some of the variants are more thermostable than NDM-1) due to mutations that increase their zinc-binding and catalytic efficiency and establish a selective pressure on the enzymes. In addition to enzymatic breakdown, NDM-mediated resistance is exorbitantly exhibited via porin loss, efflux pump activity, biofilm formation, and outer membrane vesicle-mediated dissemination. Despite the urgent need for effective countermeasures, no clinically approved inhibitor of NDM-1 has been developed. This review explores the structural and functional characteristics of NDM-1, together with the epidemiology of bla NDM-1-harbouring bacterial pathogens. It further elaborates the catalytic basis of the broad-spectrum β-lactam hydrolysis mechanism, attributed to the presence of dinuclear Zn(ii); the conserved αβ/βα metallo-β-lactamase fold, and flexible active-site loops that govern substrate recognition are also highlighted. In addition, this review provides details on the known inhibitor classes, including thiol-based compounds, hydroxamates, bicyclic boronates, repurposed drugs, and natural products, and discusses the challenges related to pharmacokinetics, toxicity, and variant-specific effectiveness. Other emerging approaches, such as CRISPR-Cas systems for targeted gene editing, nanoparticle-based delivery platforms, and artificial intelligence-driven drug discovery, are also explored as promising options for future therapy development.},
}

@article {pmid42364585,
year = {2027},
author = {Wang, T and Wen, Y and Ma, H and Wang, Y and Liao, K and Xue, F},
title = {RAA-CRISPR/Cas12a-mediated SERS magnetic biosensor combined with a portable Raman spectrometer for rapid and ultrasensitive detection of Lumpy skin disease virus.},
journal = {Talanta},
volume = {311},
number = {},
pages = {130222},
doi = {10.1016/j.talanta.2026.130222},
pmid = {42364585},
issn = {1873-3573},
mesh = {*Biosensing Techniques/methods ; *Spectrum Analysis, Raman/methods ; Animals ; *CRISPR-Cas Systems ; *Lumpy skin disease virus/isolation & purification/genetics ; Cattle ; Nucleic Acid Amplification Techniques/methods ; DNA, Viral/genetics ; Rapid Diagnostic Tests ; Recombinases/metabolism ; Gold/chemistry ; },
abstract = {Lumpy skin disease virus (LSDV) poses a severe threat to global cattle farming, causing significant economic losses. Current laboratory-based methods such as qPCR are accurate procedures; however, they are time-consuming and require sophisticated instrumentation, thus hindering onsite outbreak control. We report a novel magnetic biosensor integrating recombinase-aided amplification (RAA), clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a, and surface-enhanced Raman scattering (SERS) for ultrasensitive and portable detection of LSDV. The biosensor operates through a synergistic cascade: (i) RAA isothermally amplifies the viral DNA target; (ii) the amplified product activates the trans-cleavage activity of CRISPR/Cas12a, thus generating numerous short ssDNA fragments; and (iii) these fragments cleave linker DNA, thereby releasing AuNS@DTNB SERS nanoprobes from the magnetic bead surface and enabling signal readout via a handheld Raman spectrometer. This triple-amplification strategy yielded an ultrahigh sensitivity at 1.2 copies/μL, with a total assay time of only 90 min. The platform exhibited exceptional specificity, discriminating LSDV from genetically homologous Capripoxviruses (up to 97% similarity). In blind tests on 30 beef samples spiked with LSDV nucleic acid, it achieved 100% concordance with qPCR results, thus demonstrating excellent robustness. By integrating a handheld Raman spectrometer and a portable thermostat, this platform delivers laboratory-grade performance in a field-deployable format. This biosensor represents a powerful new tool for onsite LSDV surveillance; given its modular design, it can be readily adapted for detecting other emerging nucleic acid targets.},
}

*Biosensing Techniques/methods
*Spectrum Analysis, Raman/methods
Animals
*CRISPR-Cas Systems
*Lumpy skin disease virus/isolation & purification/genetics
Cattle
Nucleic Acid Amplification Techniques/methods
DNA, Viral/genetics
Rapid Diagnostic Tests
Recombinases/metabolism
Gold/chemistry

@article {pmid42379036,
year = {2027},
author = {Chen, Y and Liu, S and Yuan, Z and He, X and Zhu, X and Wang, G and Wang, X},
title = {Dual-engine amplification: Integrating catalytic hairpin assembly with CRISPR-Cas12a for ultrasensitive point-of-care testing of Chikungunya virus RNA.},
journal = {Talanta},
volume = {311},
number = {},
pages = {130191},
doi = {10.1016/j.talanta.2026.130191},
pmid = {42379036},
issn = {1873-3573},
mesh = {*RNA, Viral/genetics/analysis ; *Chikungunya virus/genetics/isolation & purification ; *Nucleic Acid Amplification Techniques/methods ; *Point-of-Care Testing ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Humans ; *Chikungunya Fever/diagnosis/virology ; Rapid Diagnostic Tests ; Limit of Detection ; Sensitivity and Specificity ; },
abstract = {Chikungunya virus (CHIKV) has emerged as a globally important arthropod-borne pathogen with rapid geographical expansion, urging high-performance yet simple diagnostic tools. Herein, we develop an isothermal dual-engine amplification biosensor by integrating catalytic hairpin assembly (CHA) with CRISPR-Cas12a for ultrasensitive and rapid detection of CHIKV RNA. This strategy combines the enzyme-free isothermal amplification of CHA and the high-specificity trans-cleavage activity of CRISPR-Cas12a to realize cascade signal enhancement. By systematically optimizing reaction parameters including probe ratio, temperature, buffer, and incubation time, we achieve superior analytical performance. The biosensor exhibits a low limit of detection of 4 fM, a wide linear range from 10 fM to 1 μM (R[2] > 0.98), and excellent specificity against dengue virus, Zika virus, and other common blood-borne viruses. It also shows strong resistance to interference from hemolysis, icterus, and lipemia. The whole assay can be finished within 60 min under isothermal conditions without sophisticated thermal cyclers. In clinical serum samples, this method achieves 97.5% concordance with RT-qPCR, with a sensitivity of 96.8% and a specificity of 100%. This CHA-CRISPR-Cas12a dual-amplification platform provides a sensitive, specific, rapid, and cost-effective approach for CHIKV detection and holds great promise for point-of-care testing in resource-limited settings.},
}

*RNA, Viral/genetics/analysis
*Chikungunya virus/genetics/isolation & purification
*Nucleic Acid Amplification Techniques/methods
*Point-of-Care Testing
*CRISPR-Cas Systems
*Biosensing Techniques/methods
Humans
*Chikungunya Fever/diagnosis/virology
Rapid Diagnostic Tests
Limit of Detection
Sensitivity and Specificity

@article {pmid42402225,
year = {2027},
author = {Jia, X and Wang, J},
title = {Aptamer-CRISPR Glucose Transducer for point-of-care IgE detection.},
journal = {Talanta},
volume = {311},
number = {},
pages = {130221},
doi = {10.1016/j.talanta.2026.130221},
pmid = {42402225},
issn = {1873-3573},
mesh = {*Immunoglobulin E/analysis/blood ; Humans ; *Aptamers, Nucleotide/chemistry ; *Biosensing Techniques/methods ; *Point-of-Care Systems ; *Glucose/analysis ; *CRISPR-Cas Systems ; *Point-of-Care Testing ; Limit of Detection ; Rapid Diagnostic Tests ; },
abstract = {Immunoglobulin E (IgE) is a critical biomarker for the diagnosis and therapeutic monitoring of allergic diseases. Conventional IgE detection methods (e.g., ELISA, ImmunoCAP) generally offer satisfactory sensitivity but require hours to days, rely on specialized equipment and trained personnel, and are unsuitable for point-of-care or home-based testing. Here, we developed a portable point-of-care testing platform termed Aptamer-CRISPR Glucose Transducer (ACGT), based on aptamer competition and CRISPR-Cas12a trans-cleavage. The platform employs the D17.4 aptamer as the molecular recognition element. In the presence of target IgE, the aptamer specifically binds IgE, triggering a strand displacement reaction that releases the blocker as ssDNA. This released blocker (now serving as the trigger) activates the trans-cleavage of Cas12a, which cleaves ssDNA linkers on magnetic beads, thereby releasing invertase into the supernatant. The released invertase hydrolyzes sucrose into glucose, which is quantitatively measured using a personal glucose meter. The method achieves a detection limit as low as 76.5 kU/L, with a total assay time of approximately 80 min. By integrating the signal amplification capability of CRISPR with the accessibility of glucose-based transduction, the ACGT platform provides a sensitive, cost-effective, and user-friendly solution for allergen diagnosis and biomarker monitoring in decentralized settings, offering great potential for primary care and home-based testing.},
}

*Immunoglobulin E/analysis/blood
Humans
*Aptamers, Nucleotide/chemistry
*Biosensing Techniques/methods
*Point-of-Care Systems
*Glucose/analysis
*CRISPR-Cas Systems
*Point-of-Care Testing
Limit of Detection
Rapid Diagnostic Tests

@article {pmid42420522,
year = {2026},
author = {Price, D and Zemlyanskiy, G and Trakarnphornsombat, W and Forne, I and Volkova, N and Dubusse, L and Cooper, AJ and Imhof, A and Radzisheuskaya, A},
title = {Identifying critical lysines in mammalian histone H3 with high-throughput CRISPR prime editing.},
journal = {Nature genetics},
volume = {58},
number = {9},
pages = {2303-2319},
pmid = {42420522},
issn = {1546-1718},
support = {UKRI698//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; EP/Y000331/1//RCUK | Engineering and Physical Sciences Research Council (EPSRC)/ ; RG\R1\241175//Royal Society/ ; CRC1309/325871075 and SPP2191/419067076//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {*Histones/genetics/metabolism/chemistry ; Animals ; *Lysine/genetics/metabolism ; Mice ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Protein Processing, Post-Translational/genetics ; Mouse Embryonic Stem Cells/metabolism ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Mutation ; Genomic Instability ; Saccharomyces cerevisiae/genetics ; },
abstract = {Histone post-translational modifications are fundamental to genome regulation, yet dissecting the functions of individual histone marks in mammals remains challenging due to the presence of multiple histone gene copies. Here we develop a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR) prime editing platform enabling precise, reversible and combinatorial mutagenesis of canonical and noncanonical histone H3 genes within their native genomic context. Using systematic lysine-to-arginine substitutions benchmarked against synonymous controls, we identify key residues, including H3K4, H3K9, H3K14, H3K18 and H3K79, whose mutation compromises fitness in mouse embryonic stem cells. We further show that H3K56, linked to genome stability in yeast and Drosophila, has a conserved role in mammalian cells. Through analysis of selected double mutants, we uncover functional crosstalk across residues, with combinations such as H3K27R + H3K36R impairing stem cell self-renewal and altering transcription. Altogether, this study establishes a functional map of histone H3 lysines in mammals and provides a broadly applicable platform for systematic dissection of chromatin regulation.},
}

*Histones/genetics/metabolism/chemistry
Animals
*Lysine/genetics/metabolism
Mice
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Protein Processing, Post-Translational/genetics
Mouse Embryonic Stem Cells/metabolism
Humans
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Mutation
Genomic Instability
Saccharomyces cerevisiae/genetics

@article {pmid42664856,
year = {2026},
author = {Huang, J and Zhang, Q and Yu, FW and Liu, MQ and Jing, HK and Wang, SY and Wang, HY and Li, JQ and Zheng, L and Shao, H and He, XL and Shen, RF and Zhu, XF},
title = {Disruption of OsGONST3 reduces grain cadmium accumulation without yield penalty in field-grown rice.},
journal = {Journal of hazardous materials},
volume = {516},
number = {},
pages = {143397},
doi = {10.1016/j.jhazmat.2026.143397},
pmid = {42664856},
issn = {1873-3336},
mesh = {*Oryza/metabolism/genetics/growth & development ; *Cadmium/metabolism ; *Plant Proteins/genetics/metabolism ; *Soil Pollutants/metabolism ; Plants, Genetically Modified ; Plant Roots/metabolism ; CRISPR-Cas Systems ; },
abstract = {Cadmium (Cd) pollution in paddy soils threatens global food safety. Identifying molecular gateways controlling grain Cd accumulation is essential for breeding low-Cd rice. A field-based screen of a large-scale CRISPR/Cas9 mutant library identified OsGONST3, a Golgi nucleotide sugar transporter family member localized to the plasma membrane. After 1 μM Cd treatment for 7 d, OsGONST3 transcript abundance increased approximately 3.4-fold in root and 5.7-fold in shoot, accompanied by increased OsGONST3-GFP protein accumulation. Heterologous OsGONST3 expression increased yeast Cd content by approximately 23% relative to the empty-vector control. Under hydroponic exposure to 1 μM Cd for 7 d, two independent osgonst3 mutants contained 22-25% less Cd in root, approximately 13% less in shoot, and 16-21% less in xylem sap than Nipponbare. Conversely, OsGONST3-overexpressing lines contained 40-42%, 38-40%, and 28-37% more Cd in root, shoot, and xylem sap, respectively. Under tested field condition, OsGONST3 disruption reduced grain Cd by approximately 25-26% without a significant yield penalty and was associated with secondary transcriptional adjustments in Cd-homeostasis genes. These findings identify OsGONST3 as a plasma membrane-localized Cd influx-associated protein and a promising target for breeding low-Cd rice.},
}

*Oryza/metabolism/genetics/growth & development
*Cadmium/metabolism
*Plant Proteins/genetics/metabolism
*Soil Pollutants/metabolism
Plants, Genetically Modified
Plant Roots/metabolism
CRISPR-Cas Systems

@article {pmid42690973,
year = {2026},
author = {Qin, Y and Zhang, G and Hu, Y},
title = {CRISPR/Cas- and Argonaute-Based In Vivo Nucleic-Acid Imaging Technologies: Strategies, Challenges, and Perspectives.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.6c01856},
pmid = {42690973},
issn = {2379-3694},
support = {32401261//National Natural Science Foundation of China/ ; },
abstract = {Live-cell monitoring of sequence-specific nucleic acids is essential to understanding genome organization, RNA regulation, and disease progression. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) and Argonaute (Ago) systems provide programmable, guide-directed recognition of DNA or RNA and are increasingly used as platforms for in vivo bioimaging. This review summarizes the structural and mechanistic features of representative CRISPR and Ago effectors and discusses design strategies for sensitive, specific, and multiplexed imaging of genomic loci, extrachromosomal DNA, and endogenous RNA in living cells. We compare the analytical performance and limitations of CRISPR- and Ago-based imaging, with particular emphasis on the major technical and biological challenges affecting their accuracy, applicability, and reliability. Finally, this review offers insights into developing high-resolution and user-friendly bioimaging platforms for fundamental biology and future translational applications.},
}

@article {pmid42692561,
year = {2026},
author = {Zheng, Z and Xu, D},
title = {CRISPR-Cas9 screen to identify genes regulating cell death.},
journal = {Methods in cell biology},
volume = {210},
number = {},
pages = {83-96},
doi = {10.1016/bs.mcb.2026.05.010},
pmid = {42692561},
issn = {0091-679X},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Cell Death/genetics ; Animals ; *Apoptosis/genetics ; },
abstract = {Regulated cell death mediated by dedicated molecular machines, known as programmed cell death, plays important roles in health and disease. Understanding the mechanisms of cell death is crucial for elucidating the control of cellular homeostasis and developing therapies for related diseases. Despite extensive research efforts spanning decades, many aspects of cell death mechanisms remain elusive, highlighting the need for continued exploration. Here, we describe how to identify novel regulators involved in cell death pathways using a genome-wide screening approach.},
}

*CRISPR-Cas Systems/genetics
Humans
*Cell Death/genetics
Animals
*Apoptosis/genetics

@article {pmid42692894,
year = {2026},
author = {Gaizauskaite, U and Songailiene, I and Sasnauskas, G and Siksnys, V},
title = {Building CRISPR immunity: evolution and mechanisms of spacer acquisition.},
journal = {Trends in biochemical sciences},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibs.2026.08.004},
pmid = {42692894},
issn = {0968-0004},
abstract = {CRISPR-Cas systems in prokaryotes serve as adaptive immune systems that neutralize phage infections through RNA-guided nucleases. Immunization is achieved during the adaptation stage through Cas1-Cas2 integrase-mediated insertion of short foreign DNA snippets, termed spacers, into a CRISPR array in the host genome. This review examines the evolutionary origins of Cas1-Cas2 and the mechanisms of spacer acquisition in DNA-targeting CRISPR-Cas systems. Particular emphasis is placed on the recently characterized effector-assisted adaptation pathways, in which CRISPR effector proteins, such as Cascade and Cas9, typically involved in target interference, are repurposed for prespacer capture and integration into a CRISPR array.},
}

@article {pmid42694431,
year = {2026},
author = {Putignani, L and Marsiglia, R and Turco, L and Russo, A and Pane, S and Fusco, A and Lopetuso, L and Trecarichi, EM},
title = {The gut reservoir of carbapenem-resistant Enterobacterales: from dysbiosis and colonization to infection and decolonization, with a focus on patients with hematologic malignancies - a narrative review.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1939690},
pmid = {42694431},
issn = {2235-2988},
mesh = {Humans ; *Hematologic Neoplasms/complications/microbiology ; *Dysbiosis/microbiology ; *Enterobacteriaceae Infections/microbiology/therapy ; *Carbapenem-Resistant Enterobacteriaceae/drug effects ; *Gastrointestinal Microbiome ; Fecal Microbiota Transplantation ; Anti-Bacterial Agents/pharmacology/therapeutic use ; Probiotics ; },
abstract = {Carbapenem-resistant Enterobacterales (CRE) remain among the highest-priority antimicrobial-resistant pathogens worldwide, and intestinal colonization is increasingly recognized as the key precursor of invasive infections, particularly in patients with hematological malignancies. Increasing evidence indicates that disruption of the gut microbial ecosystem, reflected in reduced diversity, depletion of beneficial anaerobic taxa, intestinal barrier dysfunction, immune dysregulation, and expansion of Enterobacterales, plays a central role in the transition from colonization to infection. Consequently, restoring colonization resistance through microbiome-targeted interventions has emerged as a promising preventive strategy. This narrative review summarizes the current evidence on the epidemiology and clinical impact of CRE colonization and infection, with particular emphasis on the ecological alterations of the gut microbiome linking gut dysbiosis to epithelial barrier dysfunction, immune dysregulation, and loss of colonization resistance to CRE persistence and invasive infection. We critically discuss both conventional and emerging decolonization approaches, including selective digestive decontamination, probiotics, prebiotics and synbiotics, fecal microbiota transplantation (FMT), bacteriophage therapy, and CRISPR-Cas-based technologies, highlighting their mechanisms of action, available clinical evidence, and current limitations. Particular attention is given to patients with hematological malignancies, in whom the clinical need for effective decolonization strategies is greatest. Although FMT currently represents the most promising microbiome-based intervention, the available evidence remains heterogeneous and largely derived from small studies. Overall, durable and standardized decolonization strategies have yet to be established, underscoring the need for well-designed multicenter randomized clinical trials to define effective microbiome-directed approaches for preventing CRE-related infections in high-risk populations.},
}

Humans
*Hematologic Neoplasms/complications/microbiology
*Dysbiosis/microbiology
*Enterobacteriaceae Infections/microbiology/therapy
*Carbapenem-Resistant Enterobacteriaceae/drug effects
*Gastrointestinal Microbiome
Fecal Microbiota Transplantation
Anti-Bacterial Agents/pharmacology/therapeutic use
Probiotics

@article {pmid42694879,
year = {2026},
author = {Huang, CW and Chen, HH and Jao, TM and Li, CJ and Sung, JM and Tsai, YS and Chen, JS},
title = {CRISPR/Cas9-Driven Fndc5 Knockout Reveals Augmented Mitochondrial Structural and Dynamic Alterations in Diabetic Nephropathy.},
journal = {International journal of medical sciences},
volume = {23},
number = {9},
pages = {2884-2898},
pmid = {42694879},
issn = {1449-1907},
mesh = {Animals ; Mice ; *Diabetic Nephropathies/pathology/genetics ; *Mitochondria/pathology/metabolism/genetics/ultrastructure ; Mice, Knockout ; *Diabetes Mellitus, Experimental/pathology/genetics/chemically induced/complications ; *Fibronectins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Male ; Diet, High-Fat/adverse effects ; Mitochondrial Dynamics/genetics ; Kidney/pathology ; Mice, Inbred C57BL ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism ; Oxidative Stress/genetics ; Humans ; Streptozocin/toxicity ; Lipid Peroxidation/genetics ; },
abstract = {FNDC5 has been implicated in glucose homeostasis and is associated with mitochondrial function. Its role in diabetes and diabetic nephropathy (DN) remains unclear. This study hypothesizes that FNDC5 deficiency predisposes the kidney to accelerated mitochondrial dysfunction in diabetes and DN. Systemic Fndc5 knockout (KO) C57BL/6 mice were generated using CRISPR/Cas9. DN were induced in six-week-old Fndc5 wild-type (WT) and KO mice using high-fat diet combined with streptozotocin injection. Weekly blood and urine analyses assessed glucose, cholesterol, triglycerides, blood urea nitrogen, creatinine, and proteinuria. At 15 weeks, kidneys and metabolic tissues including pancreas, muscle and adipose were collected for histological and molecular analyses. Results showed that while both Fndc5 WT and KO mice were successfully induced with hyperglycemia, the Fndc5 KO DN group exhibited a slightly lower cumulative glycemic burden compared with the WT DN group. Despite this milder metabolic stress, proteinuria remained comparable between the two groups. Furthermore, histological analysis revealed that Fndc5 KO DN mice displayed more severe mesangial expansion, glomerular basement membrane thickening, and podocyte effacement compared with WT DN mice. The elevated lipid peroxidation, reduced PGC-1α expression, and increased DNA fragmentation were also evident in Fndc5 KO DN. More swollen mitochondria with a significantly higher percentage of disrupted cristae were observed in Fndc5 KO DN mice compared with WT DN mice. This was accompanied by the upregulation of mitochondrial fission-related genes (Dnm1l and Fis1), downregulation of the fusion-related gene (Mfn1), and reduced expression of ATP synthase subunits (ATP5A1 and ATP5B). Systemic analysis of other metabolic tissues, including the pancreas and muscle and adipose tissues, revealed increased lipid peroxidation and decreased PGC-1α expression. These findings underscore FNDC5's role in maintaining mitochondrial integrity and cellular health under diabetic conditions, positioning FNDC5 as a potential therapeutic target.},
}

Animals
Mice
*Diabetic Nephropathies/pathology/genetics
*Mitochondria/pathology/metabolism/genetics/ultrastructure
Mice, Knockout
*Diabetes Mellitus, Experimental/pathology/genetics/chemically induced/complications
*Fibronectins/genetics/metabolism
CRISPR-Cas Systems/genetics
Male
Diet, High-Fat/adverse effects
Mitochondrial Dynamics/genetics
Kidney/pathology
Mice, Inbred C57BL
Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism
Oxidative Stress/genetics
Humans
Streptozocin/toxicity
Lipid Peroxidation/genetics

@article {pmid42695329,
year = {2026},
author = {Chowdhury, S and Nayak, SP and Pattanayak, R and Sardar, S and Majhi, A and Yadav, B and Dasari, A and Mandlik, R and Sonah, H and Deshmukh, R and Gupta, I and Bauer, P and Ram, H},
title = {Establishment of a CRISPR-Cas9 Library for Indica Rice and Identification of OsOPR5 (LOC_Os06g11210) as a Regulator of Root Architecture.},
journal = {Physiologia plantarum},
volume = {178},
number = {5},
pages = {e71097},
pmid = {42695329},
issn = {1399-3054},
support = {SRGJ2021/001495//Department of Biotechnology, Ministry of Science and Technology, Govt of India/ ; BT/PR53626/BSA/33/96/2024//Department of Biotechnology, Ministry of Science and Technology, Govt of India/ ; BT/PR56697/AMRIT/165/21/2025//Department of Biotechnology, Ministry of Science and Technology, Govt of India/ ; //BRIC-National Institute of Plant Genome Research/ ; //Alexander von Humboldt Foundation/ ; },
mesh = {*Oryza/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Plant Roots/genetics/anatomy & histology/growth & development/metabolism ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Gene Library ; Gene Expression Regulation, Plant ; Mutation ; Cyclopentanes/metabolism ; Oxylipins/metabolism ; Gene Editing ; },
abstract = {Functional characterization of a large number of rice genes remains a major challenge despite the availability of genome sequences and large-scale transcriptomic datasets. CRISPR-Cas9 library is a powerful approach for high-throughput targeted mutagenesis; however, its application in indica rice cultivars remains limited due to low transformation and regeneration efficiencies. In this study, we developed a CRISPR-Cas9 library targeting 12,000 rice genes and evaluated its utility for functional genomics in the indica cultivar MTU-1010. Sanger sequencing and NGS analysis of the plasmid library revealed high sgRNA coverage and more than 80% accuracy. Transformation of the developed library into the indica cultivar MTU-1010 resulted in a high target editing efficiency, with 90% of analyzed transgenic plants carrying mutations at the intended target site. Functional analysis of one homozygous mutant identified a previously uncharacterized role for OsOPR5 (LOC_Os06g11210), a member of the 12-oxophytodienoate reductase family in root architecture. The opr5 mutants exhibited significant reductions in lateral root number, seminal and crown root number, and root length, demonstrating that OsOPR5 positively regulates root system architecture in rice. Notably, endogenous jasmonic acid (JA) and JA-isoleucine levels were not significantly altered in the mutant, suggesting potential functional specialization or redundancy among rice OPR family members for JA accumulation. The root system architecture is a key determinant of water and nutrient acquisition; our results suggest that OsOPR5 may play an important role in adaptation under adverse environmental conditions. Collectively, this study establishes an efficient genome-editing platform for indica rice and identifies OsOPR5 as a novel regulator of root development.},
}

*Oryza/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Plant Roots/genetics/anatomy & histology/growth & development/metabolism
*Plant Proteins/genetics/metabolism
Plants, Genetically Modified
Gene Library
Gene Expression Regulation, Plant
Mutation
Cyclopentanes/metabolism
Oxylipins/metabolism
Gene Editing

@article {pmid42695976,
year = {2026},
author = {de Kreek, F and Hertzberger, R and van Eeden, F and Illidge, S and Teunis, EJ and Hanemaaijer, M and Lievens, E and Rienstra, F and Wiedhaup, DE and Lisotto, P and Butler, D and Molenaar, D and Kort, R},
title = {Intra-individual genetic diversity of vaginal Lactobacillus crispatus revealed through citizen science-driven isolation and pangenome analysis.},
journal = {Journal of applied microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1093/jambio/lxag218},
pmid = {42695976},
issn = {1365-2672},
abstract = {AIMS: A vaginal microbiome dominated by Lactobacillus crispatus is associated with positive reproductive and sexual health outcomes, yet intra-individual genetic diversity within this species remains largely unexplored. This study characterised inter- and intra-individual genomic variation in L. crispatus strains isolated through a citizen science initiative and assessed implications for multi-strain probiotic development.

METHODS AND RESULTS: Fifty-three women participated in this citizen science project. Self-sampling resulted in 48 shotgun metagenomes. Twenty-two participants isolated their own L. crispatus strains using selective enrichment and LAMP-based species confirmation, resulting in 53 whole-genome-sequenced isolates. L. crispatus dominated 20 of 48 metagenomes (50.1-99.6% relative abundance). Pangenome analysis revealed 3 456 gene families, of which 43.7% were core and 56.3% accessory. A 14-kb plasmid harbouring a Fic-domain toxin-antitoxin protein, but devoid of antimicrobial resistance genes, was present in 44 of 53 strains. Strains from the same individual clustered closely together yet harboured 1-123 gene differences. Intra-individual variation was observed in the pullulanase type I gene required for glycogen degradation: 40 strains were predicted to grow on glycogen, six showed genetic disruptions with unknown consequences, and seven were predicted to lack this ability entirely. Variation within individuals was also found for bacteriocin classes and CRISPR-Cas genes.

CONCLUSIONS: Substantial functional diversity exists within L. crispatus, even among strains from the same individual, supporting the rationale for multi-strain vaginal probiotics. This citizen science approach enabled discovery of host-specific adaptations while ensuring participant ownership of their strains.},
}

@article {pmid42696079,
year = {2026},
author = {Mitra, S and Damini, and Devi, M and Niyogi, SG and Singh, UP and Lo, D and Bishtania, HC and Thakur, S},
title = {Unlocking the potential of bacteriophage-based therapeutic gene delivery in hepatocellular carcinoma.},
journal = {Journal of the Egyptian National Cancer Institute},
volume = {38},
number = {1},
pages = {},
pmid = {42696079},
issn = {2589-0409},
mesh = {Humans ; *Liver Neoplasms/therapy/genetics ; *Carcinoma, Hepatocellular/therapy/genetics ; *Bacteriophages/genetics ; *Genetic Therapy/methods ; *Gene Transfer Techniques ; Animals ; CRISPR-Cas Systems ; Genetic Vectors/genetics/administration & dosage ; Gene Therapy Agents ; },
abstract = {Liver cancer, mainly hepatocellular carcinoma (HCC), remains a global health burden marked by poor prognosis with limited therapeutic efficacy, and high recurrence rates. HCC remains one of the most lethal malignancies worldwide, with limited therapeutic options and high resistance to conventional treatments. Despite low therapeutic efficacy, molecular heterogeneity, treatment resistance and high recurrence rate, hepatocellular carcinoma (HCC) is still a significant health problem worldwide. These restrictions have stimulated the research of focused methods for delivering therapeutic genetic payload into cancer cells. Bacteriophages have been gaining growing attention as an emerging delivery platform due to their genetic versatility, ease of engineering, ability to be surface modified and payload targeted. In this narrative review, the therapeutic potential of engineered bacteriophages in the context of HCC therapy is critically analyzed focusing on phage display-mediated tumor targeting, phage-mediated intracellular gene delivery, TRAIL gene delivery, and CRISPR/Cas-based therapeutic strategies. It has been previously noted in the literature that phage display can be used to attach tumor-targeting ligands to the surface of a phage, which may aid in the recognition of receptors at the tumor site and promote targeted delivery to the receptor. Therapeutic application is stunted by inefficient trafficking to the cytosol, endosomal degradation, immune recognition and clearance, vector stability, manufacturing scalability and regulatory issues. In conclusion, engineered bacteriophages are a promising and versatile tool for targeted gene delivery in HCC but more mechanistic, preclinical and translational research is needed to prove their therapeutic effectiveness and clinical usefulness for this purpose.},
}

Humans
*Liver Neoplasms/therapy/genetics
*Carcinoma, Hepatocellular/therapy/genetics
*Bacteriophages/genetics
*Genetic Therapy/methods
*Gene Transfer Techniques
Animals
CRISPR-Cas Systems
Genetic Vectors/genetics/administration & dosage
Gene Therapy Agents

@article {pmid40923633,
year = {2026},
author = {Ramakrishnan, M and Kaul, R and Sharma, A and Ahmad, Z and Vijayakanth, V and Keerthana, K and Gao, Z and Zhou, M and Wei, Q},
title = {CRISPR RNP-Mediated Transgene-Free Genome Editing in Plants: Advances, Challenges and Future Directions for Tree Species.},
journal = {Plant, cell & environment},
volume = {49},
number = {10},
pages = {7636-7656},
doi = {10.1111/pce.70176},
pmid = {40923633},
issn = {1365-3040},
support = {//The preparation of this review was supported by grants from the National Natural Science Foundation of China (32471977 and 32071848); a grant from the Natural Science Foundation of Jiangsu Province (BK20231289); the Natural Science Foundation for Distinguished Young Scholars of Nanjing Forestry University (JC2019004); the Project for Groundbreaking Achievements of Nanjing Forestry University (202211); and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. The authors are also grateful for the Young Foreign Talent Program (Y20240114) and the support of Metasequoia Faculty Research Start-up Funding (163100028 and 163100036) at the Bamboo Research Institute, Nanjing Forestry University./ ; },
mesh = {*Gene Editing/methods ; *Ribonucleoproteins/genetics ; *Trees/genetics ; *CRISPR-Cas Systems/genetics ; *Genome, Plant/genetics ; Transgenes ; Plants, Genetically Modified/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {CRISPR ribonucleoprotein (RNP)-mediated genome editing offers a transgene-free platform for precise genetic modification in diverse herbaceous and tree species, including rice, wheat, apple, poplar, oil palm, rubber tree and grapevine. However, its application in woody plants faces distinct challenges, notably inefficient delivery and regeneration difficulties, particularly in species such as bamboo. While some of these issues also occur in herbaceous plants, they are often significantly more complex in woody species due to factors such as intricate cell wall architecture, widespread recalcitrant genotypes and inherent limitations of current delivery platforms. This review presents the first in-depth, critical re-evaluation of recent advancements in RNP-mediated editing in woody plants, highlighting these obstacles that warrant focused attention. Unlike plasmid-based CRISPR systems, RNP editing utilises Cas9/Cas12a protein-guide RNA complexes without integrating foreign DNA. This enables a DNA-free editing strategy that simplifies regulatory approval and minimises off-target effects due to the transient presence and rapid degradation of RNPs within plant cells. While PEG-mediated protoplast transfection and particle bombardment remain the primary reported methods for RNP delivery in trees, we evaluate promising alternative strategies such as lipofection, electroporation, cell-penetrating peptides and nanoparticle-based systems for targeted RNP delivery. Despite their promise, these advanced methods remain largely untested in woody species. Finally, we outline future research directions, including the development of tree-specific RNP delivery systems and regeneration protocols to enhance efficiency and minimise cytotoxicity. These innovations are essential for unlocking the full potential of RNP-mediated genome editing in long-lived tree species. This review provides a focused and timely roadmap for expanding the application of RNP technology across diverse woody plants.},
}

*Gene Editing/methods
*Ribonucleoproteins/genetics
*Trees/genetics
*CRISPR-Cas Systems/genetics
*Genome, Plant/genetics
Transgenes
Plants, Genetically Modified/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid42285341,
year = {2027},
author = {Nejatinejad, M and Maleki-Aram, A and Namavar Abibiglou, A and Vahedi, C and Panahian, A and Ebrahimi, A and Yaghoubi, R and Parsaei, H and Vafaei, S and Jafari, D},
title = {Molecular Landscape and Advanced Diagnostic Technologies for BRAF Mutations in Cancer: From Quantitative PCR and ddPCR to CRISPR-Based Platforms.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {592},
number = {},
pages = {121178},
doi = {10.1016/j.cca.2026.121178},
pmid = {42285341},
issn = {1873-3492},
mesh = {Humans ; *Proto-Oncogene Proteins B-raf/genetics ; *Mutation ; *Neoplasms/genetics/diagnosis ; *Polymerase Chain Reaction/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; },
abstract = {BRAF mutations are key oncogenic alterations across multiple malignancies, including melanoma, thyroid carcinoma, colorectal cancer, non-small cell lung cancer, glioma, and hairy cell leukemia. The most prevalent variant, BRAF-V600E, induces constitutive activation of the MAPK signaling pathway, promoting tumor progression and influencing therapeutic responsiveness. Accurate detection of BRAF alterations is therefore essential for molecular classification, prognostic assessment, treatment selection, and resistance surveillance. This review summarizes the molecular heterogeneity of BRAF mutations and critically evaluates current diagnostic methodologies. Conventional approaches such as allele-specific PCR and Sanger sequencing are compared with advanced quantitative platforms, including high-resolution melting analysis, droplet digital PCR, and next-generation sequencing, with emphasis on analytical sensitivity, mutation coverage, and clinical applicability. Emerging technologies such as CRISPR-based assays, rolling circle amplification systems, and nanoparticle-based biosensors and point-of-care diagnostic platforms are also discussed for their potential to enhance ultra-sensitive detection, particularly in liquid biopsy settings. These emerging tools are highlighted for their potential to enable ultra-sensitive, rapid, and decentralized mutation detection, particularly in liquid biopsy settings. Key challenges, including intratumoral heterogeneity, low allele-frequency variants, FFPE-associated artifacts, and clonal evolution under therapeutic pressure, are examined within a translational framework. In addition, we examine critical barriers to clinical implementation, including standardization, cost, and global accessibility of molecular diagnostics, and outline potential solutions through scalable technologies and decentralized testing strategies. We propose that optimal BRAF testing requires a mutation subclass-informed and clinically integrated strategy combining comprehensive baseline profiling with longitudinal molecular monitoring. Future diagnostic paradigms will likely integrate multi-omics data and artificial intelligence (AI)-assisted interpretation to refine precision oncology implementation. Looking forward, we propose that optimal BRAF testing will require integration of multi-omics profiling with AI-assisted interpretation, enabling automated variant classification, real-time clinical decision support, and improved prediction of therapeutic response and resistance.},
}

Humans
*Proto-Oncogene Proteins B-raf/genetics
*Mutation
*Neoplasms/genetics/diagnosis
*Polymerase Chain Reaction/methods
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*CRISPR-Cas Systems/genetics

@article {pmid42379464,
year = {2027},
author = {Zhang, Y and Lin, X and Shi, H and Chen, Z and Luo, Q and Wu, H and Zeng, T},
title = {CRISPR-Cas12a-based liquid biopsy technology: challenges in large-scale clinical application.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {592},
number = {},
pages = {121210},
doi = {10.1016/j.cca.2026.121210},
pmid = {42379464},
issn = {1873-3492},
mesh = {Humans ; Liquid Biopsy/methods ; *CRISPR-Cas Systems/genetics ; Biomarkers, Tumor/analysis/blood/genetics ; },
abstract = {Liquid biopsy technology, which enables the acquisition of tumor-related biomarkers in a minimally invasive and reproducible manner, has been widely applied in clinical scenarios such as early tumor detection, treatment response assessment, minimal residual disease (MRD) monitoring, and recurrence surveillance. However, its clinical utility is still limited by the extremely low abundance of clinically relevant targets, the inhibitory effects of complex matrices, and the high variability in pre-analytical stages. As a next-generation programmable nucleic acid diagnostic platform, CRISPR-Cas12a combines the unique mechanisms of sequence-specific recognition and trans-cleavage signal amplification, demonstrating significant technical advantages and translational potential in the detection of low-abundance targets in liquid biopsies. Nevertheless, a mere improvement in analytical sensitivity is insufficient to support its large-scale clinical application; the robustness of detection methods, the reproducibility of results, and clinical interpretability remain the key factors currently restricting its clinical translation. This review summarizes the mechanism of CRISPR-Cas12a. Focusing on seven representative clinical biofluids, including blood, urine, and cerebrospinal fluid, it analyzes the technical difficulties and adaptation strategies associated with different matrices, and summarizes the performance differences of detection methods alongside the most clinically rational application scenarios across various biofluids. Furthermore, it explores the primary bottlenecks this technology faces when transitioning from laboratory proof-of-concept to routine clinical application, and provides a preliminary discussion on its future development directions based on existing research. Ultimately, this review aims to provide a reference for promoting the clinical translation of CRISPR-Cas12a liquid biopsy technologies and the broader implementation of precision medicine.},
}

Humans
Liquid Biopsy/methods
*CRISPR-Cas Systems/genetics
Biomarkers, Tumor/analysis/blood/genetics

@article {pmid42538409,
year = {2026},
author = {Song, L and Song, Y and Nguyen, V and Xu, S and Ho, K and Mohammed, A and Shoemaker, RH and Hoang, BH and Yu, J and Uchio, E and Zi, X},
title = {The establishment of prostate-specific, SKP2 humanized mice by CRISPR knock-in method reveals neoplastic initiation and microenvironmental reprogramming.},
journal = {Oncogene},
volume = {45},
number = {37},
pages = {3984-3996},
pmid = {42538409},
issn = {1476-5594},
support = {R01CA255643//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; I01 BX005105/BX/BLRD VA/United States ; UG3 CA290368/CA/NCI NIH HHS/United States ; R01 CA260351/CA/NCI NIH HHS/United States ; I01BX005105//U.S. Department of Veterans Affairs (Department of Veterans Affairs)/ ; P30 CA062203/CA/NCI NIH HHS/United States ; UG3 CA290368//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; R01 CA260351//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Animals ; Male ; *S-Phase Kinase-Associated Proteins/genetics/metabolism ; Humans ; *Prostatic Neoplasms/pathology/genetics/metabolism ; Mice ; *Tumor Microenvironment/genetics ; Gene Knock-In Techniques ; Disease Models, Animal ; Prostate/pathology/metabolism ; Mice, Transgenic ; Gene Expression Regulation, Neoplastic ; Cell Transformation, Neoplastic/genetics ; Epithelial-Mesenchymal Transition/genetics ; CRISPR-Cas Systems ; *Carcinogenesis/genetics ; Cell Movement/genetics ; },
abstract = {Genetic inactivation of SKP2 has been shown to effectively prevent cancer initiation and block tumorigenesis. However, direct in vivo evidence for SKP2 on cancer initiation and prostatic microenvironment is still lacking and a SKP2 humanized mouse model is critical for developing prostate cancer immunoprevention approaches through targeting SKP2. We therefore have established a prostate-specific human SKP2 knock-in mouse model driven by an endogenous mouse probasin promoter. Overexpression of hSKP2 induces PIN and low-grade carcinoma. RNA-sequencing analysis revealed significant gene expression alterations in EMT, extracellular matrix, and interferon signaling. Single-cell deconvolution showed an increase of fibroblast population and a decrease of CD8[+] T cell and B cell populations. Consistent with these results from the SKP2 humanized mouse, SKP2 protein is overexpressed in human prostatic hyperplasia, PIN and prostate adenocarcinoma compared to normal prostate tissues. Overexpression of SKP2 markedly increased cell migration and invasion and induced the gene expression of EMT and interferon pathways. Inhibition of SKP2 signaling by Flavokawain A and C1 reverses EMT and affects EMT and interferon-related gene expression. In addition, paired prostate organoids were derived from SKP2 humanized and wild-type mice for drug screening and validated by known SKP2 inhibitors, Flavokawain A and C1. Both of which selectively decreased viability and altered the morphologies of organoids of hSKP2 knock-in rather than wild-type mice. Our studies provide a well-characterized prostate-specific hSKP2 knock-in mouse model and offer new mechanistic insights for understanding the oncogenic role of SKP2 in shaping the prostatic microenvironment during early carcinogenesis.},
}

Animals
Male
*S-Phase Kinase-Associated Proteins/genetics/metabolism
Humans
*Prostatic Neoplasms/pathology/genetics/metabolism
Mice
*Tumor Microenvironment/genetics
Gene Knock-In Techniques
Disease Models, Animal
Prostate/pathology/metabolism
Mice, Transgenic
Gene Expression Regulation, Neoplastic
Cell Transformation, Neoplastic/genetics
Epithelial-Mesenchymal Transition/genetics
CRISPR-Cas Systems
*Carcinogenesis/genetics
Cell Movement/genetics

@article {pmid42571853,
year = {2026},
author = {Nasir, U and Yasmeen, A and Awais, M and Latif, A and Bakhsh, A and Ahmad, N and Shahid, N and Azam, S and Rao, AQ and Shahid, AA},
title = {Multiplexed CRISPR/Cas9 mediated knockdown of BCH gene in potato enhances beta-carotene to combat vitamin A deficiency.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {372},
number = {},
pages = {113370},
doi = {10.1016/j.plantsci.2026.113370},
pmid = {42571853},
issn = {1873-2259},
mesh = {*Solanum tuberosum/genetics/metabolism ; *beta Carotene/metabolism ; *CRISPR-Cas Systems ; *Mixed Function Oxygenases/genetics/metabolism ; Plants, Genetically Modified/metabolism ; *Vitamin A Deficiency/prevention & control/genetics ; Gene Knockdown Techniques ; *Plant Proteins/genetics/metabolism ; Gene Editing ; },
abstract = {The inadequate amounts of provitamin A carotenoids in crops contribute to the widespread vitamin A deficiency, leading to malnutrition and blindness in humans. Suppression of the β-carotene hydroxylase (BCH) increases β-carotene levels. In the current study, we utilized the multiplexed CRISPR/Cas9 approach by designing three targets against the BCH gene in a local potato cultivar. Transformation efficiency was recorded as 15%, the successful integration of the CRISPR/Cas9-BCH multiplex construct in potatoes was confirmed through PCR. When analysed using TIDE software, Sanger sequencing revealed the highest indel efficacy of 92.1% in plant 7 and 26.6% in plant 1. qRT-PCR (quantitative real-time PCR) analysis indicated a significant 89-fold reduction in BCH transcript levels in genome-edited potato lines compared to control plants. Spectrophotometry demonstrated a notable increase in beta-carotene levels in genome-edited potato plants, ranging from 0.831 µg/mL FW to 4.236 µg/mL FW, compared to the control plant with the lowest beta-carotene concentration (0.344 µg/mL FW). HPLC analysis further confirmed increased beta-carotene levels in genome-edited potato plants, ranging from 0.11 mg/mL FW to 0.36 mg/mL FW, compared to the unmodified control plant with a minimum beta-carotene value of 0.09 mg/mL. Our results revealed that the multiplexed CRISPR-Cas9 approach targeting the BCH gene results in enhanced beta-carotene contents in potato tubers.},
}

*Solanum tuberosum/genetics/metabolism
*beta Carotene/metabolism
*CRISPR-Cas Systems
*Mixed Function Oxygenases/genetics/metabolism
Plants, Genetically Modified/metabolism
*Vitamin A Deficiency/prevention & control/genetics
Gene Knockdown Techniques
*Plant Proteins/genetics/metabolism
Gene Editing

@article {pmid42660118,
year = {2026},
author = {Tong, X and Visscher, M and Riemers, FM and Versluis, D and Geijsen, N and Shang, P and Tryfonidou, MA and Poramba-Liyanage, DW},
title = {CRISPR activation reveals SOX5/6/9 as key transcriptional regulators directing iPSC-derived cells toward a notochordal lineage.},
journal = {Stem cell reports},
volume = {21},
number = {9},
pages = {103060},
doi = {10.1016/j.stemcr.2026.103060},
pmid = {42660118},
issn = {2213-6711},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Notochord/cytology/metabolism ; Cell Differentiation/genetics ; *Cell Lineage/genetics ; *SOXD Transcription Factors/genetics/metabolism ; *SOX9 Transcription Factor/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems ; Gene Expression Regulation ; },
abstract = {Intervertebral disc (IVD) degeneration, a leading cause of chronic lower back pain, is associated with loss of vacuolated notochordal cells (NCs) and fibrotic remodeling of the nucleus pulposus. Emerging therapies increasingly focus on NCs, which are rare but therapeutically relevant cells for regenerating degenerated IVDs. In this study, we used CRISPR-based transactivation (CRISPRa) to direct the differentiation of human induced pluripotent stem cells (iPSCs) into the NC lineage. We tested CRISPRa-mediated activation of NOTO, TBXT, FOXA2, SOX5, SOX6, and SOX9, coupled with single-cell sequencing of Aggrecan-2A-mScarlet reporter iPSCs. This approach identified the SOX5/6/9 combination (SOX-trio) as critical for promoting NC lineage commitment. The SOX-trio yielded the largest cell population expressing a range of genes previously associated with NC identity, including SHH, FOXA1, FOXA2, FOXJ1, FN1, ALCAM, KRT8, and KRT18. Our study demonstrates the integration of CRISPRa with single-cell technologies as a powerful platform for investigating and enriching iPSC-derived NCs, supporting future regenerative strategies across various fields.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*Notochord/cytology/metabolism
Cell Differentiation/genetics
*Cell Lineage/genetics
*SOXD Transcription Factors/genetics/metabolism
*SOX9 Transcription Factor/genetics/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*CRISPR-Cas Systems
Gene Expression Regulation

@article {pmid42687278,
year = {2026},
author = {Han, W and Wei, P and Wang, L and Zhu, G and Xie, L and Zhu, L and He, B and Ji, X and Cao, X},
title = {Tube-Anchored Dual-Atom Nanozyme Hydrogel Microreactors with Dual-Interface Engineering for Portable Biosensing.},
journal = {ACS sensors},
volume = {11},
number = {8},
pages = {7426-7434},
doi = {10.1021/acssensors.6c01841},
pmid = {42687278},
issn = {2379-3694},
support = {2024TXTD11//Henan University of Technology/ ; 232300421080//Natural Science Foundation of Henan Province/ ; 242300421035//Natural Science Foundation of Henan Province/ ; 23ZX008//Key Scientific Research Project of Colleges and Universities in Henan Province/ ; },
mesh = {*Biosensing Techniques/methods/instrumentation ; *Hydrogels/chemistry ; DNA/chemistry ; *Nanostructures/chemistry ; Cobalt/chemistry ; Nickel/chemistry ; Colorimetry/methods ; Electrochemical Techniques/methods/instrumentation ; CRISPR-Cas Systems ; Nucleic Acid Hybridization ; Limit of Detection ; Polymers/chemistry ; Indoles ; },
abstract = {DNA hydrogels are promising platforms for portable biosensing, but their combination with high-activity catalytic nanomaterials remains hindered by interfacial incompatibility and inconsistent device-level retention. Here, a tube-anchored hydrogel microreactor is developed, coupling Co-Ni dual-atom nanozymes (CoNi DANs), interfacial stabilization, and CRISPR-triggered release within a standard centrifuge tube. CoNi DANs are synthesized on a defect-rich nitrogen-doped carbon scaffold, achieving high metal loading and improved peroxidase-like activity relative to those of their single-atom counterparts. Carboxymethyl cellulose-mediated interfacial stabilization then suppresses CoNi DAN aggregation through electrostatic complementarity and polymeric steric shielding, enabling uniform dispersion of CoNi DANs within the DNA hydrogel. A polydopamine-based dual-anchoring strategy further combines covalent grafting with sequence-specific DNA hybridization, providing a chemically reinforced hydrogel-device interface. Upon target recognition, CRISPR/Cas12a trans-cleavage activity induces controlled hydrogel degradation and on-demand release of CoNi DANs for colorimetric and electrochemical dual-mode readout. Using atrazine as a proof-of-concept analyte, the platform achieves limits of detection of 6.1 and 2.1 pg mL-1 for colorimetric and electrochemical modes, respectively, with satisfactory recoveries in real samples. A smartphone-assisted digital readout module further improves the portability of the colorimetric mode. The dual-interface design, uniting material-level compatibilization with device-level dual-anchoring, provides a generalizable framework for embedding high-activity nanozymes into responsive DNA hydrogel microreactors for portable on-site biosensing.},
}

*Biosensing Techniques/methods/instrumentation
*Hydrogels/chemistry
DNA/chemistry
*Nanostructures/chemistry
Cobalt/chemistry
Nickel/chemistry
Colorimetry/methods
Electrochemical Techniques/methods/instrumentation
CRISPR-Cas Systems
Nucleic Acid Hybridization
Limit of Detection
Polymers/chemistry
Indoles

@article {pmid42687281,
year = {2026},
author = {Lu, M and Cheng, J and Guo, J},
title = {Advances in Single-Molecule Immunoassay: From Counting Strategies to CRISPR-Enhanced Biosensing.},
journal = {ACS sensors},
volume = {11},
number = {8},
pages = {6600-6616},
doi = {10.1021/acssensors.6c01523},
pmid = {42687281},
issn = {2379-3694},
support = {CSTB2024NSCQ-JQX0012//Natural Science Foundation of Chongqing Municipality/ ; 2023YFF0724300//National Key Research and Development Program of China/ ; 2025ZD01902700//National Science and Technology Major Project/ ; },
mesh = {Immunoassay/methods ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Single-molecule immunoassays (SMIs) overcome the sensitivity limitations of conventional bulk measurements by enabling a paradigm shift from analog to digital signal readouts, thereby facilitating highly sensitive quantification of ultra-low-abundance biomarkers for precision diagnostics. This review provides a systematic overview of recent advances in SMI technologies and the conceptual framework underlying their evolution. First, discretization strategies for single-molecule counting are classified into hard discretization, based on physical confinement, and soft discretization, based on spatiotemporal isolation, within heterogeneous and homogeneous assay systems, respectively. The fundamental mechanisms by which these strategies mitigate diffusion limitations and enhance signal-to-noise ratios are discussed. Second, the integration of SMIs with CRISPR-based diagnostic systems (CRISPR-dx) is examined, with particular emphasis on their complementary roles in target recognition and signal amplification. Finally, recent applications of SMIs in the diagnosis of oncological, neurological, infectious, and cardiovascular diseases are summarized, along with a critical discussion of current engineering challenges and future directions toward clinical translation.},
}

Immunoassay/methods
*Biosensing Techniques/methods
Humans
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid42688545,
year = {2026},
author = {Song, Z and Li, H and Zhao, Y and Li, M},
title = {[Research Progress on the Application of CRISPR/Cas System in Rapid Testing of Drug-Resistant Bacteria in Clinical Practice].},
journal = {Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition},
volume = {57},
number = {4},
pages = {1212-1220},
pmid = {42688545},
issn = {1672-173X},
mesh = {*CRISPR-Cas Systems ; Humans ; *Drug Resistance, Bacterial/genetics ; *Bacteria/genetics/drug effects/isolation & purification ; *Bacterial Infections/diagnosis/microbiology ; Rapid Diagnostic Tests ; },
abstract = {The misuse of antimicrobial agents has made antimicrobial resistance one of the major threats to global public health. Efficient and rapid detection of drug-resistant bacteria and resistance genes is crucial for controlling infection spread and safeguarding human health. However, traditional detection methods (such as culture isolation, polymerase chain reaction, etc.) exhibit significant limitations in detection speed, cost-effectiveness, and convenience, increasingly failing to meet current clinical testing demands. In this context, the CRISPR/Cas system, as an emerging molecular diagnostic technology, offers innovative solutions for this field. This review details the developmental status of CRISPR/Cas systems and their research progress in rapid clinical detection of drug-resistant bacteria. It systematically explains the working principles and efficacy of various CRISPR/Cas-based detection platforms (e.g., SHERLOCK, DETECTR, HOLMES), highlighting the system's advantages in sensitivity, specificity, efficiency, real-time capability, portability, and low cost. Additionally, the article discusses challenges faced by CRISPR/Cas systems, including direct clinical sample detection, multiplex testing, cost reduction, clinical validation, and standardization. Finally, it outlines future development directions, emphasizing technological innovation and clinical translation to establish CRISPR/Cas systems as efficient tools for drug-resistant bacteria prevention and control.},
}

*CRISPR-Cas Systems
Humans
*Drug Resistance, Bacterial/genetics
*Bacteria/genetics/drug effects/isolation & purification
*Bacterial Infections/diagnosis/microbiology
Rapid Diagnostic Tests

@article {pmid42689566,
year = {2026},
author = {Zhang, R and Wen, Z and Zhao, Y and Cheng, L and Liu, C and Bian, S and Xu, L and Pei, R},
title = {Phosphorothioate-Enabled Cas12a Autocatalytic Network for Amplification-Free Ultrasensitive Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {34},
pages = {25454-25462},
doi = {10.1021/acs.analchem.6c05179},
pmid = {42689566},
issn = {1520-6882},
support = {NA//Yinuosai Biotech Co., Ltd./ ; },
mesh = {*CRISPR-Associated Proteins/metabolism/chemistry ; *DNA, Single-Stranded/chemistry/analysis ; CRISPR-Cas Systems ; *Endodeoxyribonucleases/metabolism/chemistry ; *Bacterial Proteins/metabolism/chemistry ; *DNA/analysis ; Limit of Detection ; *Phosphorothioate Oligonucleotides/chemistry ; *Phosphates/chemistry ; },
abstract = {Autocatalytic CRISPR/Cas systems offer a promising route toward amplification-free nucleic acid detection, yet their performance is limited by indiscriminate transcleavage of ssDNA activators and the reliance on structurally complex, thermodynamically unstable DNA architectures. A phosphorothioate (PS)-enabled Cas12a autocatalytic network (SCAN) is presented to address these challenges by leveraging the unique compatibility of F. novicida Cas12a (FnCas12a) with fully PS-modified ssDNA activators. An ssDNA mediator is engineered with fully PS-modified activator domains flanking a cleavable, unmodified spacer. The intact mediator remains sterically inactive toward Cas12a ribonucleoproteins, whereas trace-target-induced cleavage liberates split activators that trigger a self-propagating amplification network. The SCAN platform achieves ultrasensitive detection with limits of detection of 889 aM and 341 aM for ssDNA and dsDNA targets, respectively. The platform further enables amplification-free detection of Salmonella enterica and Escherichia coli in real samples, with detection limits of 30 and 20 CFU/mL, respectively. This work establishes fully PS-modified ssDNA as a robust and generalizable design element for CRISPR-based autocatalytic systems, providing a simple, flexible, and efficient strategy to enhance stability, suppress background, and improve analytical performance.},
}

*CRISPR-Associated Proteins/metabolism/chemistry
*DNA, Single-Stranded/chemistry/analysis
CRISPR-Cas Systems
*Endodeoxyribonucleases/metabolism/chemistry
*Bacterial Proteins/metabolism/chemistry
*DNA/analysis
Limit of Detection
*Phosphorothioate Oligonucleotides/chemistry
*Phosphates/chemistry

@article {pmid42689567,
year = {2026},
author = {Yan, Y and Chen, M and Li, H and Zhou, Y and Chen, X and Dong, X and Liu, F and Xiao, M and Zhu, L},
title = {AND-Logic Gated Electrochemical Sensor for Separate Detection of Aflatoxin B1 and Ochratoxin A via Split-Activator CRISPR/Cas12a.},
journal = {Analytical chemistry},
volume = {98},
number = {34},
pages = {25286-25298},
doi = {10.1021/acs.analchem.6c03714},
pmid = {42689567},
issn = {1520-6882},
support = {22404119//National Natural Science Foundation of China/ ; JWC20250202//Teaching Reform Project of Sichuan Normal University/ ; },
mesh = {*Ochratoxins/analysis ; *Aflatoxin B1/analysis ; *Electrochemical Techniques/methods ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; DNA, Catalytic/metabolism/chemistry ; Limit of Detection ; Food Contamination/analysis ; G-Quadruplexes ; Electrodes ; },
abstract = {To address the frequent co-occurrence of aflatoxin B1 (AFB1) and ochratoxin A (OTA) in real food matrices, as well as the difficulty of achieving both high-specificity recognition and rapid quantification with existing detection methods, this study developed an AND logic-gated electrochemical sensor based on split-activator CRISPR/Cas12a. The sensor constructed an ordered signaling layer on the electrode interface using a DNA tetrahedron. Through DNAzyme-catalyzed reactions triggered separately by the two targets, two split DNA fragments were generated. Only when both fragments coexisted could they jointly activate the trans-cleavage activity of CRISPR/Cas12a, leading to extensive cleavage of the G-quadruplex sequence supported by the DNA tetrahedron. This cleavage caused the loss of the G-quadruplex/hemin signal-reporting group, resulting in a sharp attenuation of the electrochemical signal. By optimizing the heparin sodium concentration, a one-pot integration of the DNAzyme catalytic cycle and the CRISPR cleavage reaction was successfully achieved. The sensor achieved detection limits of 1.12 pg/mL for AFB1 and 0.26 pg/mL for OTA, which are significantly lower than international regulatory limits. For real corn flour, wheat flour, rice flour, and soybean flour samples, spike recoveries ranged from 90.99% to 107.29%, and the results were highly correlated with those obtained using a commercial enzyme-linked immunosorbent assay method (R2 > 0.996) and high-performance liquid chromatography-tandem mass spectrometry (R2 > 0.998). This work not only provides a new, ultrasensitive, and highly specific tool for copresence reporting of dual mycotoxins, but also offers an innovative paradigm for constructing logic-based biosensors designed for intelligent analysis of complex matrices.},
}

*Ochratoxins/analysis
*Aflatoxin B1/analysis
*Electrochemical Techniques/methods
*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
DNA, Catalytic/metabolism/chemistry
Limit of Detection
Food Contamination/analysis
G-Quadruplexes
Electrodes

@article {pmid42689595,
year = {2026},
author = {Liu, Y and Yu, L and Shen, Y and Gan, Z and Tong, J and Zhao, F and Xiao, Y},
title = {Lanthanide Metal-Organic Frameworks Enable Target-Triggered CRISPR-Cas12a Activator Release for Ultrasensitive Non-nucleic Acid Biomarker Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {34},
pages = {24865-24875},
doi = {10.1021/acs.analchem.6c01959},
pmid = {42689595},
issn = {1520-6882},
support = {82273895//National Natural Science Foundation of China/ ; 82572684//National Natural Science Foundation of China/ ; 2025HBBSHXF006//Hubei Provincial Postdoctoral Pioneer Talent Tracking Program/ ; WHYC202503//Wuhan Top-Notch Talent Program/ ; ZNJY202607//Hubei Provincial Clinical Research Center for Molecular Diagnostics/ ; },
mesh = {*CRISPR-Cas Systems ; *Lanthanoid Series Elements/chemistry ; Humans ; Biomarkers/blood/analysis ; Aptamers, Nucleotide/chemistry ; *Metal-Organic Frameworks/chemistry ; Limit of Detection ; *Biosensing Techniques/methods ; *Troponin I/blood/analysis ; *CRISPR-Associated Proteins/metabolism/chemistry ; *Bacterial Proteins/metabolism/chemistry ; *Endodeoxyribonucleases/metabolism ; },
abstract = {Rapid, ultrasensitive detection of cardiac troponin I (cTnI) is critical for the early diagnosis of acute myocardial infarction (AMI). However, CRISPR-Cas systems, despite their unparalleled nucleic acid detection performance, face inherent bottlenecks in protein sensing, including inefficient signal transduction, high background noise, and insufficient anti-interference capability in complex biological matrices. Herein, we report a modular fluorescence signal transduction platform integrating aptamer-functionalized magnetic beads, lanthanide metal-organic frameworks (Ln-MOFs) with high nucleic acid affinity, and CRISPR-Cas12a-mediated collateral cleavage amplification for sensitive cTnI detection. Target cTnI binding to magnetic bead-immobilized aptamers drives the formation of sandwich complexes with CRISPR activator DNA (act)-functionalized Ln-MOFs, triggering phosphate-mediated release of act. The liberated act initiates robust Cas12a trans-cleavage activity, generating an amplified fluorescence readout. This platform achieves a limit of detection (LOD) of 0.1 pg mL-1 in serum samples, with excellent specificity against off-target interfering proteins. Validation in 28 clinical serum samples demonstrates near-perfect agreement with standard clinical ELISA measurements and yields an area under the curve (AUC) of 0.995, confirming its clinical diagnostic accuracy. Furthermore, the modular design enables facile reconfiguration for diverse protein biomarkers via aptamer substitution, providing a universal strategy to expand CRISPR-Cas systems toward non-nucleic acid target detection.},
}

*CRISPR-Cas Systems
*Lanthanoid Series Elements/chemistry
Humans
Biomarkers/blood/analysis
Aptamers, Nucleotide/chemistry
*Metal-Organic Frameworks/chemistry
Limit of Detection
*Biosensing Techniques/methods
*Troponin I/blood/analysis
*CRISPR-Associated Proteins/metabolism/chemistry
*Bacterial Proteins/metabolism/chemistry
*Endodeoxyribonucleases/metabolism

@article {pmid42689621,
year = {2026},
author = {Fu, H and Wang, Z and Guo, J and Wang, T and Li, X and Su, Y and Chen, Z and Yang, H and Xie, Z and Li, D},
title = {High-Sensitivity Molecular Detection of Viral Bioaerosols Using a Composite Collection System and Optimized RPA-CRISPR/Cas12a.},
journal = {Analytical chemistry},
volume = {98},
number = {34},
pages = {24876-24887},
doi = {10.1021/acs.analchem.6c01983},
pmid = {42689621},
issn = {1520-6882},
support = {12274197//National Natural Science Foundation of China/ ; 12304487//National Natural Science Foundation of China/ ; 2023ZDZX2071//Department of Education of Guangdong Province/ ; GJHZ20220913143207014//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; JCYJ20220818102618040//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; JCYJ20230807093808017//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; JCYJ20241202130558075//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; 2022A0505030024//Science and Technology Planning Project of Guangdong Province/ ; 2022B1515020093//Science and Technology Planning Project of Guangdong Province/ ; 2022B1515120012//Science and Technology Planning Project of Guangdong Province/ ; 2026A1515010034//Science and Technology Planning Project of Guangdong Province/ ; NA//Guangdong Provincial Intelligent Diagnosis Engineering Research Center for Molecular Instant Detection of Children?s Infectious Diseases/ ; },
mesh = {Aerosols/analysis ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; },
abstract = {Highly pathogenic airborne microorganisms pose a significant public health threat, particularly because their small particle size can support prolonged suspension and long-range transport. Here, we present an integrated platform that combines a composite bioaerosol collection system with a three-stage enrichment mechanism and recombinase polymerase amplification-CRISPR/Cas12a (RPA-CRISPR/Cas12a) detection. The platform incorporates model-based constant-temperature control, stacked optical filters, and multichannel fluorescence imaging. The temperature-control model accurately described the thermal response of the reaction module, while the optical and imaging subsystems supported stable fluorescence readout. Using aerosolized monkeypox virus (MPXV) pseudovirus as a controlled test target, the platform detected target nucleic acid in collection liquid down to 10 copies/μL under the tested conditions. These results support the feasibility of integrating aerosol enrichment with isothermal molecular detection in a compact prototype. The main contributions lie in innovations and the engineering integration of bioaerosol collection and enrichment, thermal control of biochemical reactions in molecular detection, and precision fluorescence detection techniques for molecular detection. Regarding molecular detection methods, the biochemical reaction parameters were experimentally optimized rather than introducing new biochemical reaction mechanisms. Validation with authentic clinical or environmental viral aerosols remains necessary before diagnostic or field-use claims can be made.},
}

Aerosols/analysis
*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods

@article {pmid42690503,
year = {2026},
author = {Indurthi, S and Kaur, G and Dutta, R and Madhu, S and Chodasani, B and Yadav, A and Singh, M and Meena, TK and Singh, G},
title = {Integrating genomics, multi-omics, CRISPR and speed breeding for stress-resilient vegetable legume improvement.},
journal = {Functional & integrative genomics},
volume = {26},
number = {1},
pages = {},
pmid = {42690503},
issn = {1438-7948},
mesh = {*Fabaceae/genetics/metabolism ; *Plant Breeding ; Multiomics ; *Stress, Physiological/genetics ; Genomics ; Quantitative Trait Loci ; Genome, Plant ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {Vegetable legumes are nutritionally and ecologically important crops. However, their genetic improvement has not kept pace with the increasing challenges posed by climate change due to the polygenic nature of stress tolerance, narrow genetic diversity, and the persistent gap between molecular discoveries and field-level cultivar development. Although recent reviews have examined individual genomic tools or specific stress responses, a comprehensive synthesis integrating genomics-assisted breeding, multi-omics technologies, genome editing, and speed breeding within a unified crop improvement framework has been lacking. This review addresses that gap by critically evaluating how these complementary approaches can accelerate the development of stress-resilient vegetable legumes, including pea, common bean, cowpea, faba bean, cluster bean, yard-long bean, and hyacinth bean. This review synthesizes advances in QTL mapping, genome-wide association studies, transcriptomics, metabolomics, and CRISPR-based functional genomics that have identified key regulators and pathways underlying resistance to major biotic and abiotic stresses. Rather than considering these technologies independently, the review emphasizes their convergence into a systems-level breeding framework integrating genomic discovery, functional validation, predictive breeding, and accelerated generation advancement to improve breeding efficiency. Speed breeding, enabling up to seven to eight generations annually under optimized controlled-environment experimental conditions in cowpea, is discussed as a complementary strategy with genomic selection and genome editing. The review further identifies major translational bottlenecks, including transformation recalcitrance, limited genomic resources for underutilized vegetable legumes, inadequate multi-environment validation, and fragmented omics integration, and presents an integrated systems-breeding framework to bridge the gap between gene discovery and cultivar development.},
}

*Fabaceae/genetics/metabolism
*Plant Breeding
Multiomics
*Stress, Physiological/genetics
Genomics
Quantitative Trait Loci
Genome, Plant
CRISPR-Cas Systems
Gene Editing

@article {pmid42272442,
year = {2026},
author = {Luo, X and Qu, Y and Ye, Z and Li, Z and Zhang, Y and Luo, L and Li, S and Zhao, W and Wang, M and Bock, R and Wan, J and Tan, J},
title = {Synergistic HMGN1 and VP64 Fusions Potentiate High-Precision and PAM-Flexible Base Editing.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {50},
pages = {e76047},
pmid = {42272442},
issn = {2198-3844},
support = {BF2025302//Frontier Technologies R&D Program of Jiangsu/ ; ZSBBL-KY2023-04//Zhongshan Biological Breeding Laboratory/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Oryza/genetics ; Humans ; *HMGN Proteins/genetics ; },
abstract = {RNA-guided CRISPR-derived base editors (BEs) have revolutionized genome editing by enabling targeted base substitutions. However, their application is frequently constrained by the stringent requirement for PAM sequences and low editing precision (bystander editing). Here, we present a robust strategy to overcome these limitations by coupling SpRY, a near-PAM-less Cas9 variant, with truncated CDA1 cytidine deaminases. While this combination enables precise editing of virtually any cytosine in the genome, it initially exhibited suboptimal efficiency. To address this, we systematically screened a diverse panel of candidate DNA-binding proteins and identified that the synergistic fusion of HMGN1 and VP64 substantially enhances editing activity without compromising precision. Importantly, this enhanced editing efficiency was achieved without markedly increasing off-target effects. Our new BEs demonstrated robust performance not only in yeast but also in rice, suggesting broad applicability in gene therapy, precision breeding, and fundamental research.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Oryza/genetics
Humans
*HMGN Proteins/genetics

@article {pmid42315549,
year = {2026},
author = {Sekine, T and Shinzawa, N and Kubota, R and Kobayashi, D and Okubo, Y and Itokawa, K and Isawa, H and Amino, H and Ishino, T},
title = {Versatile, marker-free platform for life cycle-wide imaging of Plasmodium falciparum by integrating an exogenous gene cassette into a conserved intergenic locus.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42315549},
issn = {2045-2322},
support = {JPMJSP2120//Japan Science and Technology Corporation/ ; JP20wm0325018//Japan Agency for Medical Research and Development/ ; JP24wm0325074//Japan Agency for Medical Research and Development/ ; 24K02272//Japan Society for the Promotion of Science/ ; 24KK0149//Japan Society for the Promotion of Science/ ; },
mesh = {Animals ; *Plasmodium falciparum/genetics/growth & development ; *Life Cycle Stages/genetics ; Mice ; Humans ; Genetic Loci ; Gene Editing ; Sporozoites ; Luminescent Proteins/genetics ; CRISPR-Cas Systems ; *DNA, Intergenic/genetics ; Liver/parasitology ; Malaria, Falciparum/parasitology ; },
abstract = {The creation of transgenic Plasmodium falciparum lines with robust fluorescence across the entire life cycle is essential for advancing our understanding of parasite biology, which in turn informs the development of new drugs and vaccines. In this study, we utilized Plasmodium-optimized genome editing to integrate an mCherry expression cassette into a selected intergenic locus without gene disruption. The resulting marker-free line, NF54-mCh, exhibited intense fluorescence throughout all developmental stages, including asexual and sexual blood stages, as well as mosquito (ookinete, oocyst, and sporozoite) and liver stages. NF54-mCh showed normal proliferation, gametocytogenesis, and efficient transmission to mosquitoes. The ultra-high brightness in salivary gland sporozoites allowed for the non-invasive identification of infected mosquitoes. Sporozoites remained highly infectious to humanized mouse livers, thus enabling the completion of the full life cycle. NF54-mCh serves as a parental line for performing additional genetic modifications, because the CRISPR/Cas9-based genome editing method is free of introduced drug resistance markers. The broader applicability of this strategy was validated by generating similar reporter lines in Plasmodium species utilized in rodent malaria models. In summary, NF54-mCh represents a unique, versatile platform that will accelerate fundamental research and support the future development of malaria control strategies, including new vaccines and drugs.},
}

Animals
*Plasmodium falciparum/genetics/growth & development
*Life Cycle Stages/genetics
Mice
Humans
Genetic Loci
Gene Editing
Sporozoites
Luminescent Proteins/genetics
CRISPR-Cas Systems
*DNA, Intergenic/genetics
Liver/parasitology
Malaria, Falciparum/parasitology

@article {pmid42332291,
year = {2026},
author = {Al Madhoun, A and Malik, MZ and Al-Beloushi, S and Abukhalaf, N and Miranda, L and Jacob, S and Nizam, R and John, S and George, P and Channanath, A and Bahman, F and Liao, M and Ahmad, R and Chen, W and Bekri, A and Drapeau, P and Thanaraj, TA and Al-Mulla, F},
title = {Targeting glucagon signaling in metabolic disorders, functional insights from zebrafish receptor knockouts.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {83},
number = {1},
pages = {},
pmid = {42332291},
issn = {1420-9071},
support = {RA-CB-2021-007//Kuwait Foundation for the Advancement of Sciences/ ; },
mesh = {Animals ; *Zebrafish/metabolism/genetics ; *Receptors, Glucagon/genetics/metabolism ; *Signal Transduction ; *Glucagon/metabolism ; *Metabolic Diseases/metabolism/genetics ; Protein Isoforms/genetics/metabolism ; Lipid Metabolism ; Gene Knockout Techniques ; *Zebrafish Proteins/genetics/metabolism ; Energy Metabolism ; Glucose/metabolism ; Transcriptome ; CRISPR-Cas Systems ; },
abstract = {Glucagon receptor (GCGR) signaling is essential for glucose and lipid homeostasis, making it a potential therapeutic target for metabolic disorders. Zebrafish possess two GCGR co-orthologs, GCGRa and GCGRb, however, their distinct function remain unclear. In this study we employed CRISPR/Cas9 gene editing to generate GCGRa[-]/[-], GCGRb[-]/[-], and double-knockout (GCGR[-]/[-]) zebrafish to dissect isoform-specific functions. RNA-Seq analysis was performed to characterize transcriptomic alterations, while an overfeeding protocol was used to assess metabolic tolerance, and ligand-response assays in cell lines evaluated isoform activation dynamics. Transcriptomic analysis revealed that both isoforms regulate overlapping but distinct metabolic pathways. Functional enrichment analysis linked GCGRa to lipid and energy metabolism, cholesterol biosynthesis and glucose homeostasis, through key signaling cascades such as glucagon, PPARγ and PI3K-AKT. In contrast, GCGRb loss altered fatty acid β-oxidation, GPCR signaling, and oxidative phosphorylation networks, implicating roles in metabolism and cellular stress. The GCGR[-]/[-] primarily impacted core metabolic networks including lipid, gluconeogenesis and energy metabolism, indicating complementary and overlapping functions of both receptors in maintaining hepatic metabolic homeostasis. Ligand-response assays revealed that GCGRb, but not GCGRa, is activated by both glucagon (GCGa) and glucagon like-peptide-1 (GLP1a), supporting the post-duplication receptor diversification theory. Notably, all knockouts exhibited impaired growth under high-nutrient conditions, confirming GCGR's role in diet-responsive development. This study provides the first systematic functional comparison of zebrafish GCGR isoforms, establishing zebrafish as a valuable model for investigating glucagon-based metabolic regulation and therapeutic interventions.},
}

Animals
*Zebrafish/metabolism/genetics
*Receptors, Glucagon/genetics/metabolism
*Signal Transduction
*Glucagon/metabolism
*Metabolic Diseases/metabolism/genetics
Protein Isoforms/genetics/metabolism
Lipid Metabolism
Gene Knockout Techniques
*Zebrafish Proteins/genetics/metabolism
Energy Metabolism
Glucose/metabolism
Transcriptome
CRISPR-Cas Systems

@article {pmid42412303,
year = {2026},
author = {Khatun, M and Lundin, K and Tuuri, T and Piltonen, T and Tapanainen, JS and Salumets, A},
title = {From Gene Function to Precision Intervention: CRISPR/Cas9 and Stem Cell-Based Strategies as Emerging Disease-Modifying Approaches in PMOS.},
journal = {Stem cell reviews and reports},
volume = {22},
number = {7},
pages = {3056-3080},
pmid = {42412303},
issn = {2629-3277},
mesh = {Humans ; Female ; *CRISPR-Cas Systems/genetics ; Animals ; Induced Pluripotent Stem Cells/metabolism ; Gene Editing/methods ; Mesenchymal Stem Cells/metabolism ; *Metabolic Syndrome/therapy/genetics ; *Precision Medicine/methods ; },
abstract = {Polyendocrine metabolic ovarian syndrome (PMOS) is a complex endocrine-metabolic disorder affecting up to 18% of women worldwide and remains the leading cause of anovulatory infertility. Despite extensive research, current treatments primarily target symptoms, including menstrual irregularities, hyperandrogenism, and metabolic dysfunction, without addressing the underlying molecular and tissue-level disturbances. Advances in multi‑omic profiling have identified disruptions across neuroendocrine, metabolic, inflammatory, and extracellular matrix pathways, alongside genetic susceptibility at loci such as DENND1A, CYP17A1, LHCGR, FSHR, IRS1, and PPARG. However, the functional roles of many variants remain unresolved. CRISPR/Cas9 gene editing enables precise interrogation of these pathways, while stem cell-based platforms, including mesenchymal stem cells (MSCs), exosomes, and gene-edited induced pluripotent stem cells (iPSCs), may serve as complementary platforms for regeneration and disease modeling. Preclinical studies demonstrate that MSCs and their derivatives modulate inflammation, restore ovarian structure, and improve metabolic parameters, while iPSC-based models enable patient-specific investigation of steroidogenic and metabolic abnormalities. Translational challenges remain, including targeted delivery, off-target effects, phenotypic heterogeneity, and regulatory considerations. Integrating CRISPR‑based functional genomics with stem cell research may shift PMOS management from symptom‑focused care to targeted, mechanism‑driven interventions that could modify the course of PMOS (Graphical Abstract).},
}

Humans
Female
*CRISPR-Cas Systems/genetics
Animals
Induced Pluripotent Stem Cells/metabolism
Gene Editing/methods
Mesenchymal Stem Cells/metabolism
*Metabolic Syndrome/therapy/genetics
*Precision Medicine/methods

@article {pmid42681397,
year = {2026},
author = {Savash Ishanzadeh, MC and Wells, D},
title = {Clustered Regularly Interspaced Palindromic Repeats (CRISPR) Applied to Gametes and Embryos.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3038},
number = {},
pages = {447-475},
doi = {10.1007/978-1-0716-5292-3_24},
pmid = {42681397},
issn = {1940-6029},
mesh = {Humans ; *Gene Editing/methods ; Female ; *Preimplantation Diagnosis/methods ; *CRISPR-Cas Systems ; *Blastocyst/metabolism ; Fertilization in Vitro/methods ; *Germ Cells/metabolism ; Pregnancy ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Preimplantation genetic testing (PGT) facilitates the identification of embryos affected by specific types of genetic abnormalities. However, PGT does not seek to treat the genetic abnormality, rather it is an embryo selection tool, employing a strategy of detection and exclusion. Until recently, the notion that mutations and aneuploidies could be corrected in gametes, or in embryos produced using in vitro fertilization (IVF), seemed improbable. However, rapid progress in the evolution of gene editing technologies may make this a realistic possibility in the near future. Not only would such an approach help to avoid the discard of human embryos, which some find challenging from ethical or religious perspectives, but it would also increase the number of embryos considered suitable for transfer, potentially leading to higher pregnancy rates than achieved in PGT cycles. This chapter considers the use of genome editing applied to human preimplantation embryos, describing a protocol that can be used to inactivate genes for research purposes, and which might, in the future, allow for the correction of pathogenic mutations.},
}

Humans
*Gene Editing/methods
Female
*Preimplantation Diagnosis/methods
*CRISPR-Cas Systems
*Blastocyst/metabolism
Fertilization in Vitro/methods
*Germ Cells/metabolism
Pregnancy
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42681488,
year = {2026},
author = {Fernandes, LGV and Nally, JE},
title = {Gene Knockout in Leptospira spp. by CRISPR/Cas9-NHEJ (Non-Homologous End-Joining) and CRISPR-Prime Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3068},
number = {},
pages = {139-156},
pmid = {42681488},
issn = {1940-6029},
mesh = {*Leptospira/genetics ; *CRISPR-Cas Systems ; *Gene Knockout Techniques/methods ; *DNA End-Joining Repair/genetics ; *Gene Editing/methods ; DNA Breaks, Double-Stranded ; },
abstract = {Genetic manipulation of Leptospira spp. has progressed significantly in recent years. Like most prokaryotes, leptospires are unable to survive double-strand breaks (DSBs) induced by the Cas9 endonuclease, prompting the development of alternative strategies for gene knockout. We have established two systems for targeted mutagenesis of Leptospira spp.: CRISPR/Cas9-NHEJ and CRISPR-Prime Editing (PE). The CRISPR/Cas9-NHEJ approach involves coexpression of the CRISPR/Cas9 machinery alongside the DNA repair proteins LigD and Ku from Mycobacterium smegmatis, facilitating error-prone repair of DSBs that results in indel mutations. In contrast, CRISPR-PE is a DSB-free strategy that utilizes a Cas9-nickase fused to a reverse transcriptase that facilitates precise single-nucleotide edits in the genome. This expanded toolbox has placed Leptospira spp. at the forefront of bacterial genetic manipulation.},
}

*Leptospira/genetics
*CRISPR-Cas Systems
*Gene Knockout Techniques/methods
*DNA End-Joining Repair/genetics
*Gene Editing/methods
DNA Breaks, Double-Stranded

@article {pmid42683944,
year = {2026},
author = {Schulman, J and Egan, R and Kandari, L and Madayiputhiya, N and Mahon, D and Tao, L and Luo, H and Condon, K and Feder, JN and Huang, D and Khetan, A},
title = {TPST Gene Knock-Out Eliminates Tyrosine Sulfation on a Recombinant Antibody Produced in CHO Cells.},
journal = {Biotechnology journal},
volume = {21},
number = {9},
pages = {e70302},
pmid = {42683944},
issn = {1860-7314},
mesh = {Animals ; CHO Cells ; Cricetulus ; *Tyrosine/metabolism ; *Sulfotransferases/genetics/metabolism ; *Recombinant Proteins/metabolism/genetics ; *Antibodies, Monoclonal/metabolism/genetics ; Gene Knockout Techniques ; Protein Processing, Post-Translational ; CRISPR-Cas Systems ; Cricetinae ; Antibodies, Bispecific/metabolism/genetics ; },
abstract = {Tyrosine sulfation is a post-translational modification that has been reported to occur infrequently on recombinant monoclonal antibodies (mAbs). We recently demonstrated that tyrosine sulfation occurred on a bispecific antibody (bsAb) produced in Chinese hamster ovary (CHO) cells, using a multi-enzymatic approach in combination with intact mass and peptide-based mass spectrometry analysis supplemented with the use of synthetic peptides. Tyrosine sulfation needs to be controlled during the manufacturing process due to potential undesired effects, such as impact on potency and immunogenicity. Here, we report that tyrosine sulfation was not significantly inhibited by the addition of chemical inhibitors, such as sodium chlorate. Individual knockout and double knockout (DKO) of two key genes in the tyrosine sulfation pathway were carried out sequentially. Tyrosyl protein sulfotransferase 1/2 (TPST1/2) DKO by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease)-mediated gene editing eliminated tyrosine sulfation while maintaining cell growth, antibody production, and overall product quality.},
}

Animals
CHO Cells
Cricetulus
*Tyrosine/metabolism
*Sulfotransferases/genetics/metabolism
*Recombinant Proteins/metabolism/genetics
*Antibodies, Monoclonal/metabolism/genetics
Gene Knockout Techniques
Protein Processing, Post-Translational
CRISPR-Cas Systems
Cricetinae
Antibodies, Bispecific/metabolism/genetics

@article {pmid42684483,
year = {2026},
author = {Wang, K and Chen, J and Bai, S and Wang, Q and Zhang, W and Qin, Z and Zhang, Z and Zhang, Z and Ma, Y},
title = {Efficient Microinjection and CRISPR/Cas9 Mediated Genome Editing in Urechis unicinctus.},
journal = {Marine biotechnology (New York, N.Y.)},
volume = {28},
number = {5},
pages = {},
pmid = {42684483},
issn = {1436-2236},
support = {42176122//National Natural Science Foundation of China/ ; 32170373//National Natural Science Foundation of China/ ; ZR2025MS368//Natural Science Foundation of Shandong Province/ ; 2022120090//Fundamental Research Funds for the Central Universities/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Microinjections/methods ; Caveolin 1/genetics/metabolism ; Cilia/genetics/metabolism ; *Gene Editing/methods ; Tubulin/genetics ; Base Sequence ; },
abstract = {Urechis unicinctus is an economically important aquaculture species and an emerging model for developmental and evolutionary research; however, progress in functional genetic studies in this species has remained limited by the absence of effective genome editing approaches. Here, we establish a CRISPR/Cas9-mediated genome editing platform in U. unicinctus based on an optimized microinjection procedure. Targeting the cilia-associated gene Caveolin-1, Cas9/sgRNA ribonucleoprotein complexes were delivered into oocytes, generating mosaic G0 larvae harboring insertion-deletion mutations, as confirmed by ICE analysis and cloning-based Sanger sequencing with mutation detection rates of 68.51% and 64.86%, respectively. Ciliary defects were frequently observed in edited larvae, including shortening of the circumoral ciliary ring and reduction of apical ciliary tufts, accompanied by impaired swimming performance. Whole-mount in situ hybridization further revealed altered spatial expression patterns of Caveolin-1 in edited embryos, with 78% of individuals showing detectable changes in expression patterns. To independently assess the robustness of the platform, the conserved cytoskeletal gene α-tubulin was additionally targeted, resulting in successful mutagenesis and associated ciliary abnormalities. Together, these findings establish the first CRISPR/Cas9 genome editing workflow for U. unicinctus, providing a technical framework for functional genomics in echiurans and facilitating future studies of developmental mechanisms and molecular breeding in this species.},
}

Animals
*CRISPR-Cas Systems
Microinjections/methods
Caveolin 1/genetics/metabolism
Cilia/genetics/metabolism
*Gene Editing/methods
Tubulin/genetics
Base Sequence

@article {pmid42686754,
year = {2026},
author = {Dinesh, RK and Wang, X and Mohammad, IA and Gunasekaran, P and Stiklioraitis, K and Villafuerte, JR and Rao, A and Hernandez-Lopez, RA and Sunwoo, JB and Cong, L},
title = {Surfaceome CRISPR activation screening uncovers ligands regulating tumor sensitivity to NK cell killing.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42686754},
issn = {2041-1723},
support = {R35GM155437//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R35HG011316//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R35DE030054//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R35 GM155437/GM/NIGMS NIH HHS/United States ; R35 HG011316/HG/NHGRI NIH HHS/United States ; R01 GM141627/GM/NIGMS NIH HHS/United States ; 1R01GM141627//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R35 DE030054/DE/NIDCR NIH HHS/United States ; },
mesh = {*Killer Cells, Natural/immunology/metabolism ; Humans ; Animals ; Mice ; Ligands ; Cell Line, Tumor ; *Cytotoxicity, Immunologic ; CRISPR-Cas Systems ; Leukosialin/metabolism/genetics/immunology ; Hyaluronan Receptors/genetics/metabolism/immunology ; Sialic Acid Binding Immunoglobulin-like Lectins/metabolism/genetics/immunology ; Female ; Immunotherapy/methods ; Coculture Techniques ; },
abstract = {Natural killer (NK) cell-based immunotherapies are promising for cancer treatment due to their ability to eliminate cancer cells independently of antigen presentation and "off-the-shelf" utility. However, molecular determinants governing tumor susceptibility to NK cytotoxicity remain incompletely understood. Here we employ CRISPR activation (CRISPRa) screening to identify cancer cell surface regulators of NK killing. Using a surfaceome-focused library, we screen human and murine cancer cell lines co-cultured with NK cells, identifying known and novel ligands modulating NK cytotoxicity. Screens reveal established factors including CD43 and previously uncharacterized regulators CD44, PDPN, and Siglec-1/CD169. Validation with orthogonal approaches confirm that disruption of these factors alters NK killing susceptibility in vitro and in humanized mouse models. Mechanistically, we find that CD43-mediated NK resistance operates independently of its proposed interaction with Siglec-7, and that targeting CD43 on NK cells or CAR T cells substantially enhances cytotoxic activity against leukemia. These results establish gain-of-function surfaceome screening as a powerful tool for identifying therapeutic targets for NK cell-based immunotherapy.},
}

*Killer Cells, Natural/immunology/metabolism
Humans
Animals
Mice
Ligands
Cell Line, Tumor
*Cytotoxicity, Immunologic
CRISPR-Cas Systems
Leukosialin/metabolism/genetics/immunology
Hyaluronan Receptors/genetics/metabolism/immunology
Sialic Acid Binding Immunoglobulin-like Lectins/metabolism/genetics/immunology
Female
Immunotherapy/methods
Coculture Techniques

@article {pmid41945283,
year = {2026},
author = {Chu, NH and Le, HD and Quach, NT and Pham, TKL and Ho, NA and Nguyen, THH and Tran, XK and Pham, BN and Chu, HH},
title = {CRISPR/Cas9-Mediated Knockout of the NAD-Dependent Lactate Dehydrogenases for Altered Stereospecific Lactic Acid Production in Lacticaseibacillus paracasei NC4.},
journal = {Biochemical genetics},
volume = {64},
number = {5},
pages = {7016-7031},
pmid = {41945283},
issn = {1573-4927},
support = {TĐNSH0.06/22-24//Vietnam Academy of Science and Technology/ ; },
mesh = {*Lactic Acid/biosynthesis ; *CRISPR-Cas Systems ; *Lacticaseibacillus paracasei/genetics/enzymology/metabolism ; Gene Knockout Techniques ; *L-Lactate Dehydrogenase/genetics/metabolism ; *NAD/metabolism ; *Lactate Dehydrogenases/genetics/metabolism ; Stereoisomerism ; },
abstract = {The optical purity of lactic acid is a critical parameter for producing high-performance polylactic acid (PLA). To investigate the genetic basis of stereospecific lactic acid biosynthesis, the present study aimed to functionally characterize the roles of ldh1 and ldh2 in Lacticaseibacillus paracasei NC4 through targeted gene disruption. A CRISPR/Cas9 nickase-based system was employed to construct three mutant strains (Δldh1, Δldh2, and Δldh1Δldh2). Fermentation experiments were conducted under identical conditions, and the concentrations of D- and L-lactic acid were quantified using HPLC. Three mutant strains, Δldh1, Δldh2, and Δldh1Δldh2, were successfully constructed from the wild-type NC4 using the CRISPR-Cas9 system. Compared with the wild-type NC4, which produced 89.31 ± 0.21 g/L L-lactic acid and 10.74 ± 0.19 g/L D-lactic acid, the Δldh1 mutant produced 76.31 ± 2.22 g/L L-lactic acid and 7.72 ± 0.36 g/L D-lactic acid, while the Δldh2 mutant yielded 81.73 ± 0.46 g/L L-lactic acid and ND (not detected) D-lactic acid. The Δldh1Δldh2 double mutant generated 75.57 ± 2.96 g/L L-lactic acid and ND (not detected) D-lactic acid. The Δldh1Δldh2 double mutant similarly exhibited no detectable D-lactic acid formation, supporting the role of ldh2 in D-lactate biosynthesis. These results indicate that targeted deletion of ldh genes significantly alters the stereospecificity of lactic acid biosynthesis. In particular, deletion of ldh2 was sufficient to eliminate detectable D-lactic acid formation, whereas deletion of ldh1 alone did not completely abolish D-lactate production. Overall, this study provides functional genetic insight into the roles of ldh1 and ldh2 in controlling lactic acid stereospecificity in L. paracasei NC4 and establishes a genetic basis for future metabolic and process-oriented optimization of optically pure lactic acid production.},
}

*Lactic Acid/biosynthesis
*CRISPR-Cas Systems
*Lacticaseibacillus paracasei/genetics/enzymology/metabolism
Gene Knockout Techniques
*L-Lactate Dehydrogenase/genetics/metabolism
*NAD/metabolism
*Lactate Dehydrogenases/genetics/metabolism
Stereoisomerism

@article {pmid42189436,
year = {2026},
author = {Mendoza, A and Simon, I and Hasan, S},
title = {A User-Friendly Protocol for Microinjection into Teleost Embryos to Study Gene Function.},
journal = {Biochemical genetics},
volume = {64},
number = {5},
pages = {7759-7786},
pmid = {42189436},
issn = {1573-4927},
mesh = {Animals ; *Microinjections/methods ; *Oryzias/genetics/embryology ; *Zebrafish/genetics/embryology ; CRISPR-Cas Systems ; *Embryo, Nonmammalian/metabolism ; Gene Knockdown Techniques/methods ; Female ; Morpholinos/genetics/administration & dosage ; },
abstract = {Zebrafish (Danio rerio) and medaka (Oryzias latipes) are popular teleost models used in developmental biology and functional genomics. To achieve high-quality and reproducible microinjections, it is essential to have robust protocols for breeding, egg collection, and the precise delivery of genetic material. In this protocol, we present a comprehensive and optimized methodology for setting up breeding tanks under controlled photoperiod conditions to maximize egg yield while minimizing contamination. We provide detailed procedures for sex identification, pair selection, the use of grated breeding inserts, and methods to increase egg collection efficiency. We outline procedures for making injection gel beds, pulling needles, and calibration using one-microliter microcapillaries to achieve consistent nanoliter-scale injections. Our protocol outlines settings for the pico-liter injector that are optimized to deliver a precise amount per pulse with minimal variability. Finally, we demonstrate the application of these methods for gene knockdown using morpholino antisense oligonucleotides, gene knockout using CRISPR-Cas9, and gain-of-function mRNA overexpression experiments. Phenotypic assessments conducted at various developmental stages to evaluate gene-specific effects reveal consistent phenotypic outcomes between the morpholino and CRISPR-Cas9 approaches. This easy and comprehensive protocol enables efficient, precise, and scalable genetic manipulation of zebrafish and medaka embryos, thereby supporting advanced functional studies in developmental biology and disease modeling. To our knowledge, this is the first unified protocol for both zebrafish and medaka microinjection systems achieving 97.7% phenotype penetrance in CRISPR-Cas9 knockouts with precision together with a triple validation approach that confirms gene function across multiple techniques.},
}

Animals
*Microinjections/methods
*Oryzias/genetics/embryology
*Zebrafish/genetics/embryology
CRISPR-Cas Systems
*Embryo, Nonmammalian/metabolism
Gene Knockdown Techniques/methods
Female
Morpholinos/genetics/administration & dosage

@article {pmid42537911,
year = {2026},
author = {Liu, K and Fan, X and Tong, Z and Zhang, J and Zhao, M and Chen, Y and Wu, C and Wang, T and Wei, S and Liu, Y and Xue, Z and Zheng, Y},
title = {Reprogrammed Komagataella phaffii for enhanced secretory expression of human lactoferrin.},
journal = {Journal of biotechnology},
volume = {419},
number = {},
pages = {1-10},
doi = {10.1016/j.jbiotec.2026.07.011},
pmid = {42537911},
issn = {1873-4863},
mesh = {*Lactoferrin/genetics/metabolism ; Humans ; *Saccharomycetales/genetics/metabolism ; CRISPR-Cas Systems ; Recombinant Proteins/genetics/metabolism ; Endoplasmic Reticulum/metabolism/genetics ; Bioreactors ; Vacuoles/metabolism/genetics ; },
abstract = {Human lactoferrin (hLF) is a multifunctional glycoprotein of the transferrin family derived from milk and mucosal secretions, which exhibits antibacterial, anti-tumor, and immunomodulatory functions, and is an important component of infant formula. Conventional methods for lactoferrin expression are often inefficient, primarily due to inadequate protein synthesis capabilities and poor stability within microbial hosts. Herein, a Komagataella phaffii yeast strain capable of high-level secretory expression of hLF was constructed by reprogramming the endoplasmic reticulum (ER) and vacuole using CRISPR/Cas9 technology. A dual-expression cassette containing the AOX1 promoter, an α-secretion signal peptide, the hLF gene, and a terminator was integrated into three different sites of the K. phaffii genome. The stepwise strategy combining expansion of the ER membrane involved in protein synthesis with knockout of vacuolar proteases further enhanced hLF production. Subsequently, 0.1 g/L FeCl3 was added to the medium to reduce the toxicity of hLF and improve its stability. After high-density cultivation of K. phaffii through optimization of cultivation conditions in shake flasks and a 5 L bioreactor, the secretory intact hLF titer reached 2214 mg/L, representing a 76.3-fold increase achieved through these engineering strategies. In addition, antibacterial experiments demonstrated that this secretory hLF had a significant inhibitory effect on Escherichia coli, Staphylococcus aureus, and yeast. Overall, the developed K. phaffii protein expression platform enabled efficient production of lactoferrin, demonstrating its potential for expressing other lactoproteins.},
}

*Lactoferrin/genetics/metabolism
Humans
*Saccharomycetales/genetics/metabolism
CRISPR-Cas Systems
Recombinant Proteins/genetics/metabolism
Endoplasmic Reticulum/metabolism/genetics
Bioreactors
Vacuoles/metabolism/genetics

@article {pmid42574952,
year = {2026},
author = {Zhu, X and Zhang, W and Sun, T and Chen, L},
title = {Advances in combinatorial CRISPRi screening and applications: Decoding higher-order interactions for next-generation microbial cell factories in synthetic biology.},
journal = {Microbiological research},
volume = {313},
number = {},
pages = {128663},
doi = {10.1016/j.micres.2026.128663},
pmid = {42574952},
issn = {1618-0623},
mesh = {*Synthetic Biology/methods ; *CRISPR-Cas Systems ; *Metabolic Engineering/methods ; Gene Regulatory Networks ; Bacteria/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Biological systems possess high robustness and intricate genetic redundancy, rendering traditional single-gene perturbations largely inadequate for comprehensively elucidating the true regulatory mechanisms underlying complex phenotypes. To address this, combinatorial CRISPR interference (CRISPRi) has emerged as an essential tool in systems and synthetic biology, offering reversible epigenetic control, multi-target regulation, and an absence of DNA toxicity. Unlike pooled single-gene screens, combinatorial CRISPRi facilitates the systematic dissection of buffering, synergy, and metabolic trade-offs by targeting multiple loci simultaneously. This review explores the latest advancements in pairwise and higher-order combinatorial CRISPRi screening, beginning with design strategies for multiplex guide RNA (gRNA) arrays and orthogonal systems. Computational techniques utilized for analyzing high-dimensional screening data and visualizing complex genetic networks are subsequently examined. Building upon these methodological foundations, crucial applications within microbial engineering are highlighted. Specifically, the review details the optimization of carbon flux in microbial cell factories to circumvent production bottlenecks, alongside the elucidation of protective multigenic networks against severe environmental stress. Furthermore, it addresses current obstacles, such as system noise and library construction challenges, and outlines future research directions. Ultimately, this review provides a comprehensive guide for decoding complex traits and driving rational designs of next-generation cell factories.},
}

*Synthetic Biology/methods
*CRISPR-Cas Systems
*Metabolic Engineering/methods
Gene Regulatory Networks
Bacteria/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41047874,
year = {2026},
author = {Ji, Y and Sun, Y and Zhou, H and Liu, Z and Sun, Z and Xu, G and Wen, H and Zheng, Z and Tu, L and Yang, Z and Zhang, Y and Liu, X and Zhou, S and Dong, X and Wang, Y and Li, C and Wan, J},
title = {Engineer the eukaryotic OMEGA-Fanzor systems for genome editing in plants.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3160-3162},
doi = {10.1111/jipb.70049},
pmid = {41047874},
issn = {1744-7909},
support = {2023ZD04074//the Biological Breeding-Major Projects/ ; 2023YFD1202900//the National Key Research and Development Program/ ; ZSBBL-KY2023-04//the Zhongshan Biological Breeding Laboratory/ ; NAUSY-ZZ03//the Guidance Foundation of the Sanya Institute of Nanjing Agricultural University/ ; BK20230038//the Jiangsu Province Natural Science Foundation/ ; //the Nanjing U35 program/ ; 2023AB006-02//the Bingtuan Key Science and Technology Program of Xinjiang Province/ ; KYT2024005//the Fundamental Research Funds for the Central Universities/ ; 31872806//the National Natural Science Foundation of China/ ; },
mesh = {*Oryza/genetics ; *Genome, Plant/genetics ; *Genetic Engineering/methods ; Plants, Genetically Modified ; CRISPR-Cas Systems/genetics ; },
abstract = {The activity of the eukaryotic OMEGA-Fanzor genome editing system remains limited in plants. We engineered the Fanzor nucleases SpuFz1, GtFz1, NlovFz2, and MmeFz2 in plants, with NlovFz2 being the most efficient, achieving up to 50.0% editing in regenerated rice plants, making it a promising tool for plant genome editing.},
}

*Oryza/genetics
*Genome, Plant/genetics
*Genetic Engineering/methods
Plants, Genetically Modified
CRISPR-Cas Systems/genetics

@article {pmid41566884,
year = {2026},
author = {Zhang, R and Tang, X and He, Y and Wang, W and Ren, Q and Qi, Y and Zhang, Y},
title = {Enhanced exonuclease-Cas9 systems promote multiple nucleotide deletions with higher efficiency and broader targeting scope in plants.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3316-3328},
doi = {10.1111/jipb.70155},
pmid = {41566884},
issn = {1744-7909},
mesh = {*CRISPR-Cas Systems/genetics ; *Oryza/genetics ; *Sequence Deletion/genetics ; Gene Editing/methods ; *Exonucleases/metabolism/genetics ; Plants, Genetically Modified ; },
abstract = {CRISPR-Cas9 is a widely used platform for plant genome editing, but its outcomes are typically dominated by small insertions and deletions (indels). Such limited mutation profiles restrict its utility in functional studies of non-coding RNAs and regulatory elements, such as microRNAs (miRNAs), untranslated regions (UTRs), and promoter sequences, where larger sequence disruptions are often required. Here, we developed enhanced exonuclease-Cas9 platforms, termed multiple nucleotide deletion Cas9 (MND-Cas9) systems, for efficient generation of large deletions in rice. By screening four exonucleases (RecJ, T5, TREX2, and SbcB), we established MND-Cas9v1 systems based on TREX2 or SbcB that produced substantially larger deletions without reducing editing efficiency. Further optimization with an inserted DNA-binding domain (DBD) between Cas9 and exonuclease yielded MND-Cas9v2, which simultaneously enhanced efficiency and deletion size. To expand PAM compatibility, we introduced PAM-relaxed Cas9-NG and SpG variants, generating MND-Cas9-NG/SpGv2 systems with broader targeting scope and superior performance compared to their parental nucleases. Finally, we demonstrated the utility of these systems in two applications: MND-Cas9v2 efficiently knocked out the miRNA gene OsMIR530, producing larger seeds, and generated extended deletions in the 3'UTR of OsGhd2, which upregulated its expression and increased grain size. These results demonstrate that MND-Cas9 systems enable high-efficiency generation of extended deletions and facilitate functional analyses of non-coding RNAs and regulatory sequences. Overall, this work establishes a versatile and expandable exonuclease-Cas9 platform that substantially broadens the mutational spectrum and application potential of CRISPR-Cas9 for plant genome engineering.},
}

*CRISPR-Cas Systems/genetics
*Oryza/genetics
*Sequence Deletion/genetics
Gene Editing/methods
*Exonucleases/metabolism/genetics
Plants, Genetically Modified

@article {pmid41588854,
year = {2026},
author = {Zhang, C and Li, J and Li, Y and Yan, L and Yong, CSY and Li, S and He, Y and Xia, L},
title = {Coupling of both a transactivation module and a double-stranded DNA-binding domain boosts Cas12i3 variant-based cytosine and adenine editing in plants.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3196-3207},
doi = {10.1111/jipb.70154},
pmid = {41588854},
issn = {1744-7909},
mesh = {*Gene Editing/methods ; *Oryza/genetics ; *Cytosine/metabolism ; *Adenine/metabolism ; *Transcriptional Activation/genetics ; CRISPR-Cas Systems/genetics ; Protein Domains ; *DNA/metabolism ; },
abstract = {CRISPR/Cas12i3 belongs to the type V-I Cas system, characterized by its smaller protein size and less restricted canonical "TTN" protospacer adjacent motif. Developments of Cas12i3-mediated base editing systems for either C-to-T or A-to-G transitions will expand the editing scope and enrich the plant base editing toolkits for crop improvement. However, while the Cas12i3-based cytosine base editor (CBE) only shows very low editing efficiency in plants, its adenine base editor (ABE) has not been documented as yet. Here, we engineered a series of Cas12i3 (5M)-based CBEs (V0-V5) and ABEs (V0-V5) by fusing a deactivated dCas12i3 (5M) with a transactivation module VP64, a single-stranded DNA-binding domain Rad51, or a double-stranded DNA-binding domain HMG-D, or in combinations, and systemically evaluated their performance in rice protoplasts. Our results demonstrated that synergistic combinations of both VP64 and HMG-D outperformed other architectures and significantly boosted the efficiencies of Cas12i3 (5M)-based CBE and ABE for C-to-T and A-to-G base editing and expanded the editing window. In stable lines, in comparison to the non-fusion control, the optimized Cas12i3 (5M)-based CBE-V5 and ABE-V5 enabled up to 4.78- and 3.35-fold higher editing efficiencies, with the maximum C-to-T and A-to-G efficiencies reaching 32.35% and 38.24%, respectively, and a higher proportion of homozygous mutants in the T0 generation. Furthermore, we generated herbicide-resistant rice germplasm by using CBE-V5 and ABE-V5, demonstrating their potential for precision breeding in crops. Together, here, we report novel Cas12i3 (5M)-based CBE and ABE that substantially enrich base editing toolkits for improvement of rice and potentially other crops.},
}

*Gene Editing/methods
*Oryza/genetics
*Cytosine/metabolism
*Adenine/metabolism
*Transcriptional Activation/genetics
CRISPR-Cas Systems/genetics
Protein Domains
*DNA/metabolism

@article {pmid41664360,
year = {2026},
author = {Liu, X and Jin, S and Xiao, Z and Ma, C and Wang, Q and Wang, H and Zhou, R and Gu, D and Xu, R and Qin, R and Li, J and Wei, P},
title = {Optimizations of Cas12a- and Cas12i-based adenine base editors for efficient precision editing in the plant genome.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3169-3171},
doi = {10.1111/jipb.70177},
pmid = {41664360},
issn = {1744-7909},
support = {//This work was supported by the Agriculture Science and Technology Major Project, the Science and Technology Major Project of Anhui Province (No. 202423110050063, No. 202423m10050002, and No. 2023n06020020), the Natural Science Foundation of China (No. 32300343, No.32572441, and No. 32570484), and Yangtze River Delta Science and Technology Innovation Community Joint Research (Basic Research) Project (No. 2024CSJZN01100)./ ; },
mesh = {*Adenine/metabolism ; *Genome, Plant/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; },
abstract = {A strategy coupling high-activity nucleases with dimeric TadA-8e optimizes plant Cas12-based adenine base editors, boosts editing efficiency, and provides precise editors for crop breeding and genomics research.},
}

*Adenine/metabolism
*Genome, Plant/genetics
*CRISPR-Cas Systems/genetics
*Gene Editing/methods

@article {pmid41814562,
year = {2026},
author = {Zhong, D and Dong, Y and Pan, H and Fu, Y and Zhao, Y and Ruan, S and Yu, W and Wang, Y and Yin, Q and Zhang, Y and Huang, Y and Shen, J and Zhang, H and Wu, Y and Xu, J and Lu, Y},
title = {CasY7: An optimized Cas12i system for enhanced genome editing in monocot crops.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3208-3219},
doi = {10.1111/jipb.70181},
pmid = {41814562},
issn = {1744-7909},
support = {CAIC (2024) 003//Construction project of Changzhou Modern Agricultural Science and Technology Innovation Center/ ; K2023001//Shanghai Agricultural Science and Technology Innovation Program/ ; 2021YFD1201300//National Key R&D Program of China/ ; },
mesh = {*Zea mays/genetics ; Plants, Genetically Modified ; Oryza/genetics ; *CRISPR-Cas Systems/genetics ; *Genome, Plant/genetics ; *Crops, Agricultural/genetics ; *Gene Editing/methods ; Triticum/genetics ; Mutation/genetics ; *CRISPR-Associated Proteins/metabolism ; Base Sequence ; },
abstract = {The CRISPR-Cas12 family nucleases, particularly the Cas12i subtypes, are considered promising alternatives to Cas9 for genome editing in plants. We previously developed a new Cas12i variant, CasY7, which has been successfully applied in clinical trials; its performance in plants remains to be investigated. Initial testing in stable transgenic maize and rice showed that the codon-optimized CasY7 (pCasY7e1) achieved average editing efficiencies of 58.7% and 62.3% across five target sites, respectively, outperforming the typical Cpf1 (pCpf1) control that targets the same sites. To further enhance activity, we fused T5 exonuclease to CasY7 (pCasY7e2), which shifted mutation profiles toward larger deletions, and subsequently integrated an MS2 aptamer into the crRNA scaffold (pCasY7e3). The optimized pCasY7e3 system increased editing efficiencies to 87.7% in maize and 82.9% in rice-approximately 2.7-fold higher than pCpf1. We further demonstrated multiplexed editing in maize, generating biallelic dwarf mutants, and validated functionality in hexaploid wheat with editing efficiencies up to 58.8%. Overall, our comprehensive validation across 942 transgenic plants confirmed robust editing in maize, rice, and wheat, establishing CasY7 as a high-efficiency addition to the CRISPR toolkit.},
}

*Zea mays/genetics
Plants, Genetically Modified
Oryza/genetics
*CRISPR-Cas Systems/genetics
*Genome, Plant/genetics
*Crops, Agricultural/genetics
*Gene Editing/methods
Triticum/genetics
Mutation/genetics
*CRISPR-Associated Proteins/metabolism
Base Sequence

@article {pmid41834254,
year = {2026},
author = {Ge, F and Peng, C and Du, Y and Chen, Y and Zhao, Z and Yu, M and Feng, H and Xie, Y and Sun, S and Liu, S and Xin, B and Zhao, H and Wu, S and Bian, C and Yang, Z and Lai, J and Chen, J},
title = {Synergistic engineering of Casδ nuclease for robust genome editing.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3231-3242},
doi = {10.1111/jipb.70222},
pmid = {41834254},
issn = {1744-7909},
support = {2024M753551//Project funded by China Postdoctoral Science Foundation/ ; 2022YFF1002800//National Key Research and Development Program of China/ ; 32572347//National Natural Science Foundation of China/ ; Z231100003723004//Agriculture Science and Technology Major Project, the Beijing Rural Revitalization Agricultural Science and Technology Project/ ; },
mesh = {*Gene Editing/methods ; Humans ; Zea mays/genetics ; CRISPR-Cas Systems/genetics ; *Genetic Engineering/methods ; *Endonucleases/metabolism/genetics ; },
abstract = {Casδ is a recently identified evolutionary transitional CRISPR system characterized by its compact size (~900 amino acids), broad temperature tolerance, and guidance by a short crRNA without the requirement of a tracrRNA. However, the low editing efficiency of Casδ in eukaryotic cells limits its application. Here, we have developed a hierarchical engineering strategy to improve the genome editing activity of Casδ-1, with optimization focused on enhancing its interactions with the crRNA, the protospacer adjacent motif (PAM) duplex, the single-stranded DNA substrate, and the RNA-DNA heteroduplex. Through this strategy, we successfully generated an activity-enhanced Casδ-1 variant, designated enCasδ, which harbors 9 amino acid substitutions that synergistically augment its editing efficiency. In human cell lines, enCasδ showed 1.3- to 29.3-fold higher editing activity than the wild-type Casδ-1 across ten tested genomic loci, with an average editing efficiency of 54.6%. In addition, enCasδ also mediated robust genome editing in maize; its editing efficiency increased by an average of 5.3-fold relative to Casδ-1, and reached up to an average of 80% at the TS4 and PSY1 loci in stable transgenic lines. The overall editing performance of enCasδ was comparable to that of Streptococcus pyogenes Cas9 (SpCas9) and other Cas12 nucleases. Collectively, enCasδ represents a highly optimized Casδ-1 variant that broadens the applicability of the Casδ CRISPR system and facilitates robust genome editing in both animal cells and plants.},
}

*Gene Editing/methods
Humans
Zea mays/genetics
CRISPR-Cas Systems/genetics
*Genetic Engineering/methods
*Endonucleases/metabolism/genetics

@article {pmid41840837,
year = {2026},
author = {Lin, W and Wu, H and Kuang, H and Feng, X and Bai, M and He, F and Liang, R and Zeng, Y and Li, M and Kong, F and Liu, B and Guan, Y},
title = {Developing a robust multiplex CRISPR/Cas12i3-5M system for trait stacking in soybean.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3172-3174},
doi = {10.1111/jipb.70233},
pmid = {41840837},
issn = {1744-7909},
support = {2023YFF1000203//National Key Research and Development Program of China/ ; },
mesh = {*Glycine max/genetics ; *CRISPR-Cas Systems/genetics ; Base Sequence ; Plants, Genetically Modified ; *Quantitative Trait, Heritable ; },
abstract = {The high-efficiency multiplex gene editing technology based CRISPR-Cas12i3-5M is capable of simultaneously editing 13 target sites in soybean, and was used to generate germplasm with high oleic acid content and no beany flavor.},
}

*Glycine max/genetics
*CRISPR-Cas Systems/genetics
Base Sequence
Plants, Genetically Modified
*Quantitative Trait, Heritable

@article {pmid41928060,
year = {2026},
author = {Cheng, Y and Li, G and Zhou, M and Mandlik, R and Wang, D and Qi, Y},
title = {Enhancing CRISPR-Cas12a base editing in plants with LbCas12a variants and introns.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3243-3259},
doi = {10.1111/jipb.70249},
pmid = {41928060},
issn = {1744-7909},
support = {MD-PSLA-24014//McIntire Stennis Forest Research/ ; },
mesh = {*Oryza/genetics ; *Introns/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Mutation/genetics ; Base Sequence ; Plants, Genetically Modified ; },
abstract = {Cytosine base editors (CBEs) and adenine base editors (ABEs) are powerful tools for precise genome editing in plants. Conventionally, such base editors are built upon the CRISPR-Cas9 systems where Cas9 nickases are used. To expand the base editing scope and minimize off-target effects, base editors derived from the CRISPR-Cas12a systems are desired. However, the use of deactivated Cas12a (dCas12a) in such base editors constrains the editing activity, preventing the wide use of Cas12a base editors for plant research and trait development. In this study, we demonstrate the use of an ABE based on the efficient LbCas12a-RRV variant to introduce herbicide-resistant mutations in OsACCase in rice. To improve Cas12a CBEs and ABEs, we inserted introns into the coding sequence of dLbCas12a-RRV. This intron-containing Cas12a-CBE shows substantial improvement in editing efficiency in rice, compared to the intron-less counterparts. By contrast, the improvement of ABE with the intron-containing dLbCas12a-RRV is very limited, partly due to the already high baseline editing efficiency of the intron-less dLbCas12a-RRV ABE. Testing of these base editors in poplar shows elevated C-to-T base editing by dLbCas12a-RRV-intron-CBE. For A-to-G editing, ABEs built upon dLbCas12a-RV and dLbCas12a-RRV variants showed significant improvement over ABEs derived from wild-type LbCas12a and the ttLbCas12a variant. The addition of introns to dLbCas12a-RRV does not further improve the base editing efficiency. With whole genome sequencing in rice, we evaluated genome editing specificities with these improved Cas12a base editors. Our analyses show that both intron-containing Cas12a CBE and ABE barely introduce guide RNA-dependent off-target mutations. However, they can generate guide RNA-independent off-target mutations, which are likely attributed to the high enzymatic activities of the deaminases. Collectively, our study demonstrates the successful use of a Cas12a base editor for trait development and reports improved Cas12a CBEs and ABEs for precise base editing in plants.},
}

*Oryza/genetics
*Introns/genetics
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Mutation/genetics
Base Sequence
Plants, Genetically Modified

@article {pmid42021470,
year = {2026},
author = {Liu, M and Wang, Y and Zhang, L and Zhang, X and Wang, X and Liu, X and Fu, Y and Li, X and Song, Z and Liu, Y and Wang, R and Zhao, J},
title = {Optimized Cas-SF01 gene-editing toolbox shortens flowering timing in commercial maize inbred JING724.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3178-3180},
doi = {10.1111/jipb.70264},
pmid = {42021470},
issn = {1744-7909},
support = {2024-zz-087//Beijing Postdoctoral Science Foundation/ ; },
mesh = {*Zea mays/genetics/physiology/growth & development ; *Flowers/physiology/genetics ; *Gene Editing/methods ; Time Factors ; Mutation/genetics ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified ; },
abstract = {The gene-editing tool Cas-SF01 was optimized to maximize its efficiency in maize. The Cas-SF01-TREX2 configuration was superior in enabling high-purity gene mutations. This toolkit enabled commercial maize to flower seven days earlier without yield loss, thereby securing harvests and accelerating crop breeding.},
}

*Zea mays/genetics/physiology/growth & development
*Flowers/physiology/genetics
*Gene Editing/methods
Time Factors
Mutation/genetics
*CRISPR-Cas Systems/genetics
Plants, Genetically Modified

@article {pmid42152501,
year = {2026},
author = {Zhou, J and Li, R and Wang, Z and Liu, S and Shi, L and Fu, X and Li, F and Zhang, J and Li, G and Zhu, J and Qian, Q and Dun, B},
title = {Engineering herbicide-resistant sorghum with CRISPR/Cas9-mediated adenine base editing.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3292-3294},
doi = {10.1111/jipb.70298},
pmid = {42152501},
issn = {1744-7909},
mesh = {*Sorghum/genetics/drug effects ; *CRISPR-Cas Systems/genetics ; *Herbicide Resistance/genetics ; *Adenine/metabolism ; Plants, Genetically Modified ; *Gene Editing/methods ; *Herbicides/pharmacology ; },
abstract = {An adenine base-editing system was established to precisely modify the sorghum SbALS gene, generating transgene-free mutant plants. These plants exhibit strong herbicide resistance, with no significant differences in agronomic traits, providing valuable germplasm for herbicide-resistance breeding in sorghum.},
}

*Sorghum/genetics/drug effects
*CRISPR-Cas Systems/genetics
*Herbicide Resistance/genetics
*Adenine/metabolism
Plants, Genetically Modified
*Gene Editing/methods
*Herbicides/pharmacology

@article {pmid42206623,
year = {2026},
author = {Bai, Y and Li, B and Peng, J and Bai, Y and Liu, S and Lai, J and Song, W},
title = {Development of fragrant broomcorn millet (Panicum miliaceum L.) via CRISPR/Cas12i.3-mediated genome editing.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3184-3186},
doi = {10.1111/jipb.70295},
pmid = {42206623},
issn = {1744-7909},
mesh = {*Panicum/genetics ; *CRISPR-Cas Systems/genetics ; Mutation/genetics ; *Genome, Plant/genetics ; *Gene Editing/methods ; },
abstract = {Co-editing the broomcorn millet PmBADH2a and PmBADH2b genes using CRISPR/Cas12i.3 generated double mutants with significantly increased 2-acetyl-1-pyrroline content, producing fragrant broomcorn millet without compromising major agronomic traits.},
}

*Panicum/genetics
*CRISPR-Cas Systems/genetics
Mutation/genetics
*Genome, Plant/genetics
*Gene Editing/methods

@article {pmid42367085,
year = {2026},
author = {Zhang, R and Tang, X and Yang, X and Zhang, Y},
title = {Enhanced Cas12i3 system enables precise OsAUX3 editing for rice grain improvement.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3187-3189},
doi = {10.1111/jipb.70337},
pmid = {42367085},
issn = {1744-7909},
support = {//Agriculture Science and Technology Major Project/ ; },
mesh = {*Oryza/genetics ; Plants, Genetically Modified ; *CRISPR-Cas Systems/genetics ; *Edible Grain/genetics ; Promoter Regions, Genetic/genetics ; Gene Expression Regulation, Plant ; Plant Proteins/genetics/metabolism ; },
abstract = {An optimized Cas12i3 genome-editing system enables highly efficient and predictable editing of regulatory sequences in rice. Precise promoter engineering fine-tunes gene expression, improves grain size, and enhances production potential, demonstrating a powerful new approach for crop improvement through targeted regulation rather than gene disruption.},
}

*Oryza/genetics
Plants, Genetically Modified
*CRISPR-Cas Systems/genetics
*Edible Grain/genetics
Promoter Regions, Genetic/genetics
Gene Expression Regulation, Plant
Plant Proteins/genetics/metabolism

@article {pmid42446212,
year = {2026},
author = {Cao, X and Liu, H and Bai, S and Wang, R and Xia, L and Sun, Y},
title = {Optimization of a hypercompact Fanzor2 system for improved genome editing performance in plants.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3190-3192},
doi = {10.1111/jipb.70346},
pmid = {42446212},
issn = {1744-7909},
mesh = {*Genome, Plant/genetics ; Plants, Genetically Modified ; CRISPR-Cas Systems/genetics ; },
abstract = {Native Fanzor2 nucleases exhibit weak editing activity in plants. An optimized Fanzor2 system engineered via ωRNA optimization and structure-based protein mutagenesis achieves robust editing at recalcitrant genomic sites and enables cytosine base editing, supporting versatile crop genome modification via transformation or viral delivery.},
}

*Genome, Plant/genetics
Plants, Genetically Modified
CRISPR-Cas Systems/genetics

@article {pmid42619441,
year = {2026},
author = {Chen, Z and Chen, Y and Wan, G and Dong, H and Pan, T and Yang, W and Zhou, Y and Luo, B and Zhu, Y and Han, X and Lu, L and Wang, X and Lei, C and Zhao, Z and Wang, J and Ren, Y and Wan, J},
title = {Development of low-prolamin rice germplasm via CRISPR/Cas9 editing to improve eating and cooking quality.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3313-3315},
doi = {10.1111/jipb.70377},
pmid = {42619441},
issn = {1744-7909},
support = {2021YFF1000200//National Key R&D Program of China/ ; 2026//Agriculture Science and Technology Major Project/ ; Y2021YJ18//Central Public-interest Scientific Institution Basal Research Fund/ ; Y2025YC02//Central Public-interest Scientific Institution Basal Research Fund/ ; CAAS-CSNCB-202302//Basic Research Center, Innovation Program of Chinese Academy of Agricultural Sciences/ ; 2025-2027//Inner Mongolia Innovation Center of Biological Breeding Technology/ ; CARS-01-05//Earmarked Fund for China Agriculture Research System/ ; },
mesh = {*Oryza/genetics/metabolism ; *Cooking ; *CRISPR-Cas Systems/genetics ; *Prolamins/metabolism/genetics ; Plants, Genetically Modified ; *Seeds/genetics/metabolism ; Starch/metabolism ; },
abstract = {Simultaneous editing of two major prolamin-encoding genes in rice using a single-guide RNA suppressed prolamin accumulation and reconfigured seed storage protein composition. This targeted modification markedly improved rice eating and cooking quality while maintaining the levels of total starch, total protein, and key agronomic traits.},
}

*Oryza/genetics/metabolism
*Cooking
*CRISPR-Cas Systems/genetics
*Prolamins/metabolism/genetics
Plants, Genetically Modified
*Seeds/genetics/metabolism
Starch/metabolism

@article {pmid42641742,
year = {2026},
author = {Chandrasekaran, S and Duraisamy, N and Jagadeesan, M and Palukuri, YK and Tamilselvan, S},
title = {Engineering CRISPR nanoplatforms to deplete cancer stem cells: Delivery checkpoints, target plasticity, and clinical viability.},
journal = {Nanomedicine : nanotechnology, biology, and medicine},
volume = {76},
number = {},
pages = {103007},
doi = {10.1016/j.nano.2026.103007},
pmid = {42641742},
issn = {1549-9642},
mesh = {*Neoplastic Stem Cells/pathology/metabolism ; Humans ; Animals ; *Nanomedicine/methods ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Neoplasms/genetics/therapy/pathology ; *Nanoparticles/chemistry ; },
abstract = {Cancer stem cells (CSCs) sustain tumor initiation, therapy resistance, and relapse, yet evade durable control because they switch phenotype, enter quiescence, shelter within protective niches, resist drug efflux, and share markers with normal stem cells. Programmable CRISPR editing can disable intracellular self-renewal dependencies that antibodies and small molecules cannot reach, whereas only nanoscale carriers can confine such editing to intended cells; neither component alone solves the CSC problem. This review reframes CSC-directed CRISPR nanomedicine as an integrated design problem. We examine why target plasticity defeats static single-marker targeting; the sequential delivery checkpoints spanning blood stability, organ selection, tumor penetration, CSC recognition, endosomal escape, and productive editing; and advanced architectures including organ-selective lipid nanoparticles, biomimetic and vesicle carriers, metal-organic frameworks, and logic-gated systems. Genotoxicity, immunogenicity, incomplete depletion, manufacturing reproducibility, and absent CSC-specific clinical evidence remain limiting. Clinical viability, not imminent cure, is the realistic near-term objective.},
}

*Neoplastic Stem Cells/pathology/metabolism
Humans
Animals
*Nanomedicine/methods
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Neoplasms/genetics/therapy/pathology
*Nanoparticles/chemistry

@article {pmid42681183,
year = {2026},
author = {Zhang, S and Hubbard, BP},
title = {Production of Cell Models with Differential Zygosity Using CRISPR Base Editors.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3032},
number = {},
pages = {181-196},
pmid = {42681183},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Animals ; Humans ; Adenine ; },
abstract = {CRISPR base editors have revolutionized the ease with which single-nucleotide gene editing can be performed in cell and organismal models. Unlike conventional CRISPR/Cas9 gene editing, which induces a double-strand DNA break and relies on endogenous homology-directed repair (HDR) or non-homologous end joining (NHEJ) repair, base editors only induce a single-strand nick and exploit the mismatch repair (MMR) system for repair. In many instances, this results in fewer off-target effects and less cytotoxicity. Moreover, because base editors use an alternative repair mechanism, the zygosity of repair is often distinct from that of traditional HDR-based editing. Here, we provide a detailed protocol for performing gene editing using an adenine base editor (ABE) to induce a single A-G transition mutation in a gene of therapeutic relevance. We also illustrate how to sort and isolate cells with differential zygosity state, enabling the production of cell models suited to the study of both dominant and recessive mutations. This workflow can easily be adapted to the use of other variants of base editors (e.g., cytosine base editors) and can be employed in diverse cell lines.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
Animals
Humans
Adenine

@article {pmid42681222,
year = {2026},
author = {Powell, S and Martin, SA},
title = {CRISPR-Mediated Gene Editing: Generating Gene Knockouts or Precision Base Changes in F0 Xenopus.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3049},
number = {},
pages = {141-170},
pmid = {42681222},
issn = {1940-6029},
mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Gene Knockout Techniques/methods ; *Xenopus/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Microinjections ; Xenopus laevis/genetics ; Female ; },
abstract = {CRISPR-mediated gene editing has transformed Xenopus research by enabling targeted, heritable gene disruption in both Xenopus laevis and Xenopus tropicalis. The CRISPR/Cas9 system has allowed efficient loss-of-function analysis within days of injection, overcoming the limitations of transient morpholino knockdowns. Recent advances in CRISPR-mediated base editing technology further expands this toolkit, permitting the precision generation of single-base changes in Xenopus. High editing efficiency, external development, and large clutch size make Xenopus embryos exceptionally suited for genome manipulation and phenotype screening. CRISPR-mediated gene editing in Xenopus has reproduced classic developmental phenotypes and generated robust models of human disease, including ciliopathies, congenital heart defects, skeletal dysplasias, and neurodevelopmental disorders. Disease modeling using Xenopus CRISPR mutants has provided critical insights into conserved vertebrate pathways and the pathogenic mechanisms of human gene variants. Here, we describe the complete process of producing a gene knockout or precision base changes in F0 Xenopus: target selection, design and synthesis of sgRNA, base editor selection and synthesis of base editor mRNA, microinjection into fertilized Xenopus eggs, and genotyping to assess whether gene editing has successfully occurred.},
}

Animals
*Gene Editing/methods
*CRISPR-Cas Systems
*Gene Knockout Techniques/methods
*Xenopus/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Microinjections
Xenopus laevis/genetics
Female

@article {pmid42681238,
year = {2026},
author = {Lun, J and Chesneau, A and Borday, C and Perron, M},
title = {Retinal Degeneration and Regeneration in Xenopus laevis Tadpoles.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3049},
number = {},
pages = {415-431},
pmid = {42681238},
issn = {1940-6029},
mesh = {Animals ; *Retinal Degeneration/genetics/physiopathology/pathology/metabolism ; *Xenopus laevis/genetics/physiology ; Larva/physiology ; *Regeneration ; Nitroreductases/genetics/metabolism ; Metronidazole/pharmacology ; *Retina/physiology ; Rhodopsin/genetics ; Animals, Genetically Modified ; Retinal Rod Photoreceptor Cells/metabolism/pathology ; CRISPR-Cas Systems ; Ependymoglial Cells/metabolism ; Monophenol Monooxygenase/genetics ; Cell Proliferation ; },
abstract = {Xenopus laevis offers unique advantages for studying retinal regeneration due to its strong regenerative capacity and amenability to transgenesis and genome editing. We present a detailed protocol for inducing and monitoring rod photoreceptor degeneration and regeneration in Tg(rho:GFP-NTR) tadpoles, where nitroreductase expression under the rhodopsin promoter enables conditional and specific ablation of rods upon metronidazole exposure. In addition, we describe the generation of albino Tg(rho:GFP-NTR); tyr [ -/-] tadpoles using CRISPR/Cas9-mediated knockout of the tyrosinase gene, providing an alternative to existing albino Tg(rho:GFP-NTR) lines and facilitating real-time fluorescence imaging by reducing pigmentation interference. Finally, we detail methods to assess Müller glial proliferation (BrdU/Sox9 colabeling) and rod neurogenesis (BrdU pulse-chase). This manuscript provides a robust experimental model that enables the investigation of the cellular and molecular mechanisms underlying retinal regeneration in X. laevis.},
}

Animals
*Retinal Degeneration/genetics/physiopathology/pathology/metabolism
*Xenopus laevis/genetics/physiology
Larva/physiology
*Regeneration
Nitroreductases/genetics/metabolism
Metronidazole/pharmacology
*Retina/physiology
Rhodopsin/genetics
Animals, Genetically Modified
Retinal Rod Photoreceptor Cells/metabolism/pathology
CRISPR-Cas Systems
Ependymoglial Cells/metabolism
Monophenol Monooxygenase/genetics
Cell Proliferation

@article {pmid42681252,
year = {2026},
author = {Wan, W and Ning, J and Yan, A},
title = {Alanine Scanning Analysis of MexB in Multidrug-Resistant Pseudomonas aeruginosa Using the Native Type I-F CRISPR-Cas-Mediated Genome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3044},
number = {},
pages = {185-201},
pmid = {42681252},
issn = {1940-6029},
mesh = {*Pseudomonas aeruginosa/genetics/drug effects ; *Drug Resistance, Multiple, Bacterial/genetics ; *Bacterial Outer Membrane Proteins/genetics/metabolism ; *Gene Editing/methods ; *Membrane Transport Proteins/genetics/metabolism/chemistry ; *CRISPR-Cas Systems ; Anti-Bacterial Agents/pharmacology ; },
abstract = {The rising multidrug resistance (MDR) of the Gram-negative opportunistic pathogen Pseudomonas aeruginosa poses a global public health threat. One key resistance mechanism employed by this pathogen is the overexpression of the resistance-nodulation-cell division (RND) multidrug efflux transporters. MexAB-OprM, a prototype RND efflux pump, confers resistance to most conventional antibiotics except aminoglycosides. Molecules capable of inhibiting efflux pumps, i.e., efflux pump inhibitors (EPIs), have shown promise as therapeutic agents capable of restoring antibiotic efficacy against these superbugs by targeting MexB. Nevertheless, the structure-activity relationship governing substrate transport of MexB in the native genetic background of clinical MDR P. aeruginosa isolates is poorly characterized due to the lack of efficient genetic tools, hindering targeted EPIs development.In this chapter, we introduce a native Type I-F CRISPR-mediated precise genome editing technique that enables in situ alanine substitutions in MexB in clinical MDR P. aeruginosa genotypes. We outline detailed procedures to construct MexB alanine-substitution mutations and methods to examine the expression levels and efflux activities of the constructed MexB mutants. Methods to evaluate the effect of constructed alanine substitutions on antibiotic susceptibilities are also described. These methodologies are expected to provide advanced insights into the substrate recognition and selection mechanism of MexB in the native genetic background of clinical MDR P. aeruginosa isolates to facilitate the development of effective EPIs.},
}

*Pseudomonas aeruginosa/genetics/drug effects
*Drug Resistance, Multiple, Bacterial/genetics
*Bacterial Outer Membrane Proteins/genetics/metabolism
*Gene Editing/methods
*Membrane Transport Proteins/genetics/metabolism/chemistry
*CRISPR-Cas Systems
Anti-Bacterial Agents/pharmacology

@article {pmid42681339,
year = {2026},
author = {Dong, X and Weiss, RJ},
title = {Detection and Localization of Type II Membrane Proteins by Endogenous Gene Tagging and Confocal Microscopy.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3020},
number = {},
pages = {49-64},
pmid = {42681339},
issn = {1940-6029},
mesh = {Microscopy, Confocal/methods ; Humans ; CRISPR-Cas Systems ; *Membrane Proteins/metabolism/genetics ; Animals ; Protein Transport ; HEK293 Cells ; },
abstract = {Type II membrane proteins are single-pass transmembrane proteins distinguished by their N-terminus facing the cytoplasmic side and C-terminus oriented toward the extracellular or luminal side. The localization of type II membrane proteins is crucial for their functional roles, molecular interactions, and biological activity. Studying their dynamics remains challenging due to limited availability of highly specific antibodies and potential artifacts introduced by overexpression systems. In this context, we outline a detailed protocol for monitoring the endogenous localization of type II membrane proteins in mammalian cells. Here, we combine CRISPR/Cas9-mediated endogenous protein tagging with confocal microscopy. This method provides a powerful tool for precisely labeling endogenous proteins and investigating the localization and dynamics of membrane proteins under physiological conditions.},
}

Microscopy, Confocal/methods
Humans
CRISPR-Cas Systems
*Membrane Proteins/metabolism/genetics
Animals
Protein Transport
HEK293 Cells

@article {pmid42288725,
year = {2026},
author = {Coleman, S and Tye, J and Furniss, D and Sainsbury, R and Rastogi, A and Xi, X and Murnyak, B and Bell, J and Skeate, J and Wang, M and Webber, B and Moriarity, B and Clement, K},
title = {CRISPRessoSea: streamlined analysis and comparison of pooled amplicon CRISPR screens.},
journal = {BMC bioinformatics},
volume = {27},
number = {1},
pages = {},
pmid = {42288725},
issn = {1471-2105},
support = {T15LM007124/NH/NIH HHS/United States ; T32HL007062/NH/NIH HHS/United States ; R21CA237789, R21AI163731, P01CA254849, P50CA136393, U54CA268069, R01AI146009/NH/NIH HHS/United States ; R01AI146009, R01AI161017, P01CA254849, P50CA136393, U24OD026641, U54CA232561, P30CA077598, U54CA268069/NH/NIH HHS/United States ; R00HG011658/NH/NIH HHS/United States ; T15LM007124/NH/NIH HHS/United States ; T15LM007124/NH/NIH HHS/United States ; T32HL007062/NH/NIH HHS/United States ; R21CA237789, R21AI163731, P01CA254849, P50CA136393, U54CA268069, R01AI146009/NH/NIH HHS/United States ; R01AI146009, R01AI161017, P01CA254849, P50CA136393, U24OD026641, U54CA232561, P30CA077598, U54CA268069/NH/NIH HHS/United States ; R00HG011658/NH/NIH HHS/United States ; },
mesh = {*Software ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; High-Throughput Nucleotide Sequencing/methods ; },
abstract = {BACKGROUND: CRISPR genome editing enables precise modification of genomic targets but may also induce unintended edits at off-target sites with similar sequences. Pooled amplicon sequencing can assess on- and off-target editing across many samples, yet analyzing, aggregating, and visualizing results from multiple pooled experiments remains challenging. Tools to simplify and standardize these analyses are needed to provide reproducible and comparable interpretation of editing data.

RESULTS: We developed CRISPRessoSea, a software package that processes, compares, and visualizes genome editing rates from pooled amplicon sequencing experiments. The tool provides standardized workflows for analyzing editing across multiple targets and samples, supports both nuclease- and base-editing modalities, and generates clear, data-rich summaries suitable for downstream interpretation.

CONCLUSIONS: CRISPRessoSea facilitates reproducible, scalable analysis of CRISPR editing outcomes across diverse experimental designs, enabling more efficient and transparent assessment of genome editing specificity. The software is freely available at https://github.com/clementlab/CRISPRessoSea .},
}

*Software
*CRISPR-Cas Systems
*Gene Editing/methods
*Clustered Regularly Interspaced Short Palindromic Repeats
High-Throughput Nucleotide Sequencing/methods

@article {pmid42310064,
year = {2026},
author = {Davoodi, P and Atapour, M and Shahsavari, A and Kiani, R},
title = {Simulation of CRISPR/Cas9-mediated gene editing for the Vitellogenin gene in Apis mellifera.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42310064},
issn = {2045-2322},
support = {ص/3/9/22600, (~1000$ for one year).//University of Kurdistan/ ; },
mesh = {Animals ; *Vitellogenins/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Bees/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Computer Simulation ; Exons ; },
abstract = {CRISPR/Cas9 genome editing provides a powerful framework for interrogating gene function in Apis mellifera. Yet, empirical application remains challenging due to biological constraints, including haplodiploid genetics, narrow embryonic injection window, and the social rearing requirements that complicate functional validation. These constraints necessitate in silico pre-screening to maximize editing success before resource-intensive wet-lab implementation. Within the omnigenic framework, which distinguishes core regulatory genes from peripheral loci buffered by network effects, vitellogenin (Vg) represents an optimal target which is ancestrally dedicated to yolk provisioning; it has been co-opted to orchestrate diverse non-reproductive functions including longevity, stress resistance, immunity, and social behavior. We developed a computational pipeline to design a list of 57 and 56 candidate guide RNAs (gRNA) for targeted Vg knockout, evaluating candidate sites in both functional exons 2 and 3 based on structural accessibility and frameshift efficiency. Comparative analysis revealed complementary strengths in two top-best candidates from initial target pool of predicted gRNAs. The gRNA targeting exon 2 exhibits weaker secondary structure (ΔG = -0.25 kcal/mol versus -2.10 kcal/mol for exon 3), aligning with empirical evidence that sites with ΔG > -1.0 kcal/mol achieve 2-5 × higher Cas9 binding efficiency. This site yielded moderate frameshift frequency (77.8%; 61.9 percentile). Conversely, the predicted editing outcome for the gRNA targeting exon 3, despite stronger structural constraints, demonstrated superior functional disruption metrics demonstrating very high frameshift frequency (88.3%; 95.2 percentile), high in silico editing precision, minimal microhomology-mediated repair bias, and reproducible outcomes wherein nearly all predicted indels disrupt the coding sequence. Protein structure and domain analyses further predict that frameshift edits will generate a truncated protein missing all downstream functional domains. We recommend parallel empirical validation of both exon 2 and exon 3 targets to resolve the trade-off between structural accessibility (favoring higher editing rates) and frameshift efficacy (favoring complete loss-of-function). This dual-target strategy accommodates uncertainty in in vivo performance while maximizing the probability of generating informative phenotypes. Our in silico framework enables rational CRISPR design in non-model organisms by computationally balancing biophysical accessibility with functional impact, accelerating functional genomics in species where empirical optimization faces substantial biological constraints.},
}

Animals
*Vitellogenins/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
Bees/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Computer Simulation
Exons