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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

@article {pmid42886424,
year = {2026},
author = {Akbarin, MM and Farjami, Z and Álvarez, HR},
title = {Fluorogenic RNA aptamers in cancer: emerging platforms for molecular imaging, biosensing, and precision oncology.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42886424},
issn = {1573-4978},
mesh = {Humans ; *Biosensing Techniques/methods ; *Aptamers, Nucleotide/chemistry/metabolism/genetics ; *Neoplasms/genetics/diagnostic imaging/diagnosis/metabolism ; *Molecular Imaging/methods ; *Precision Medicine/methods ; *Fluorescent Dyes/chemistry ; Biomarkers, Tumor ; Fluorescent Chemosensor Compounds ; Animals ; },
abstract = {Fluorogenic RNA aptamers have emerged as powerful molecular tools for cancer imaging, biosensing, and biomarker detection due to their high specificity, low background fluorescence, and genetic encodability. These short RNA molecules activate fluorescence upon binding specific fluorogens, enabling real-time visualization of intracellular targets and dynamic molecular processes in living systems. Recent advances in RNA structural engineering, fluorophore optimization, and biosensor integration have significantly expanded the translational applications of fluorogenic aptamers in oncology. This mini-review summarizes the structural principles, fluorescence activation mechanisms, and cancer-related applications of fluorogenic RNA aptamers and related nucleic acid biosensors. Foundational studies describing the architecture of fluorogenic aptamer families, including Spinach, Broccoli, Mango, Pepper, Squash, and Beetroot, are discussed with emphasis on G-quadruplex-mediated fluorogen stabilization and fluorescence enhancement. Furthermore, recent developments in CRISPR/Cas-assisted fluorogenic biosensors, dual-color RNA imaging systems, fluorogenic DNAzymes, and amplification-driven aptamer platforms are highlighted. Direct applications across multiple malignancies, including brain, breast, colorectal, liver, lung, prostate, and skin cancers, demonstrate the growing utility of these technologies for tumor-associated RNA detection, epigenetic biomarker analysis, molecular imaging, and therapeutic monitoring. In addition, advances in fluorescence brightness, structural stability, and multiplex imaging strategies have improved the sensitivity and adaptability of fluorogenic RNA systems for complex biological environments. Despite current limitations related to RNA stability, intracellular delivery, and tissue penetration, fluorogenic RNA aptamers represent highly promising tools for next-generation precision oncology. Continued integration with nanotechnology, live-cell imaging, artificial intelligence, and molecular diagnostics is expected to enhance further their clinical and translational potential in cancer research and personalized medicine.},
}

Humans
*Biosensing Techniques/methods
*Aptamers, Nucleotide/chemistry/metabolism/genetics
*Neoplasms/genetics/diagnostic imaging/diagnosis/metabolism
*Molecular Imaging/methods
*Precision Medicine/methods
*Fluorescent Dyes/chemistry
Biomarkers, Tumor
Fluorescent Chemosensor Compounds
Animals

@article {pmid42886777,
year = {2026},
author = {van Overbeek, NK and Clijsters, L and Wirth, M and de Graaf, IJ and de Lint, K and Uruci, S and Teunisse, AFAS and Akimov, V and de Ru, AH and Vukic, M and Kirdök, I and Daxinger, L and van Veelen, PA and Blagoev, B and Luijsterburg, MS and Wolthuis, RMF and Berlin, I and Vertegaal, ACO},
title = {Chemogenetic ubiquitin E1 inhibitor TAK-243 CRISPR screen uncovers HAPSTR1/HUWE1 axis in chromatin proteostasis and cell cycle control.},
journal = {Science advances},
volume = {12},
number = {42},
pages = {eaea3388},
pmid = {42886777},
issn = {2375-2548},
mesh = {Humans ; *Ubiquitin-Protein Ligases/metabolism/genetics/chemistry ; *Chromatin/metabolism/genetics ; *Proteostasis/drug effects/genetics ; *Cell Cycle Checkpoints/drug effects/genetics ; *CRISPR-Cas Systems ; Signal Transduction/drug effects ; DNA Replication ; Ubiquitination/drug effects ; Cell Proliferation/drug effects ; Tumor Suppressor Proteins ; Pyrazoles ; Pyrimidines ; Sulfonamides ; Sulfides ; },
abstract = {In this study, we sought to comprehensively map ubiquitin signaling networks at the crossroads of cellular proliferation and proteostasis, both crucial in cancer. Genome-wide CRISPR knockout screening in the presence of the ubiquitin E1 inhibitor TAK-243 identified 239 genes whose ablation sensitizes cells to ubiquitination blockade. Follow-up studies reveal a role for the nuclear protein, C16orf72/HECT, UBA, and WWE domain containing E3 ubiquitin protein ligase 1 (HUWE1)-Associated Protein modifying Stress Responses (HAPSTR1), in chromatin proteostasis and DNA replication. We show that HAPSTR1 recruits the E3 ligase HUWE1 to the chromatin, keeping the chromatin-associated proteome in check, and is itself turned over by HUWE1 during cell cycle progression. Structural modeling of the HAPSTR1/HUWE1 complex, supported by mutational analysis, demonstrates that loss of HUWE1 binding renders HAPSTR1 unable to support cellular proliferation when ubiquitin signaling is suppressed. Consequently, HAPSTR1 ablation impairs clearance of chromatin-associated proteins, triggering global transcriptional and proteostatic adaptations. Under diminished ubiquitin signaling, these adaptive mechanisms give way to replication stress and premature S phase entry, culminating in cell cycle collapse and DNA damage. Our findings uncover protein networks exhibiting synthetic lethality with ubiquitin signaling blockade and highlight vulnerabilities of actively proliferating cells to proteostatic imbalance at the chromatin.},
}

Humans
*Ubiquitin-Protein Ligases/metabolism/genetics/chemistry
*Chromatin/metabolism/genetics
*Proteostasis/drug effects/genetics
*Cell Cycle Checkpoints/drug effects/genetics
*CRISPR-Cas Systems
Signal Transduction/drug effects
DNA Replication
Ubiquitination/drug effects
Cell Proliferation/drug effects
Tumor Suppressor Proteins
Pyrazoles
Pyrimidines
Sulfonamides
Sulfides

@article {pmid42886793,
year = {2026},
author = {Hu 胡文欣, W and Chen 陈竑霖, H and He, S and Casan, JML and Shembrey, C and Li, S and Brown, LM and Zhao, A and Truong, L and Buchner, I and Vassiliadis, D and Hughes, TP and White, DL and Carmichael, CL and De Weck, A and Voskoboinik, I and Gouil, Q and Trapani, JA and Ekert, PG and Sadras, T and Fareh, M},
title = {Programmable and selective silencing of oncogenic fusion transcripts by CRISPR-Cas13b via breakpoint sequence recognition.},
journal = {Science advances},
volume = {12},
number = {42},
pages = {eady0296},
pmid = {42886793},
issn = {2375-2548},
mesh = {*CRISPR-Cas Systems ; Humans ; *Fusion Proteins, bcr-abl/genetics ; *Gene Silencing ; Drug Resistance, Neoplasm/genetics ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics/pathology ; *Oncogene Proteins, Fusion/genetics ; Cell Line, Tumor ; },
abstract = {Oncogenic gene fusions are key drivers of cancer, yet most remain undruggable by current therapies. Here, we establish CRISPR-PspCas13b as a personalizable platform for systematic silencing of various fusion transcripts. We reveal that recognition and cleavage of the breakpoint sequence by PspCas13b disrupt the fusion transcript, resulting in efficient protein depletion. PspCas13b cleavage produces aberrant, translation-incompetent target RNA species, a subset of which harbors internal deletions proximal to the cleavage site. We demonstrate that targeting the BCR::ABL1 breakpoint sequence efficiently degrades both wild-type and drug-resistant BCR::ABL1 mutants, which are the primary drivers of tyrosine kinase inhibitor resistance and relapse in chronic myeloid leukemia. Molecular profiling revealed that PspCas13b impairs BCR::ABL1 tyrosine phosphokinase activity and triggers extensive transcriptomic and proteomic remodeling, leading to erythroid differentiation and apoptosis in drug-resistant cells. Beyond BCR::ABL1, personalized design of PspCas13b effectively silences other undruggable oncogenic fusions. Collectively, this study establishes a molecular framework for precise targeting of undruggable or drug-resistant oncogenic transcripts.},
}

*CRISPR-Cas Systems
Humans
*Fusion Proteins, bcr-abl/genetics
*Gene Silencing
Drug Resistance, Neoplasm/genetics
Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics/pathology
*Oncogene Proteins, Fusion/genetics
Cell Line, Tumor

@article {pmid42887957,
year = {2026},
author = {Han, Y and Yu, H and Lv, M and Cheng, M and Li, W and Ge, H and Gao, S and Yin, L and Ge, Q and Xu, X and Huang, Y and Sun, L and Lu, Z and Liu, Q},
title = {Primer-programmed circular template generation enables padlock-free rolling circle amplification for low-background CRISPR-based pathogen detection.},
journal = {Analytica chimica acta},
volume = {1424},
number = {},
pages = {346200},
doi = {10.1016/j.aca.2026.346200},
pmid = {42887957},
issn = {1873-4324},
mesh = {*Nucleic Acid Amplification Techniques/methods ; *Staphylococcus aureus/isolation & purification/genetics ; DNA, Single-Stranded/chemistry/genetics ; *DNA Primers/chemistry/genetics ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; Biosensing Techniques/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA, Circular ; Humans ; },
abstract = {Rolling circle amplification (RCA) is a powerful nucleic acid amplification strategy, yet conventional RCA-based biosensing often relies on target-specific padlock probes for circular template construction, which increases probe-design complexity and may introduce nonspecific ligation and background amplification. Here, we develop a padlock-free RCA strategy for CRISPR-based pathogen detection through primer-programmed circular template generation (PPCTG) strategy. Asymmetric recombinase polymerase amplification (RPA) with rationally designed phosphorylated primers generates target-derived single-stranded DNA (ssDNA), which subsequently undergoes universal splint-mediated circularization to form amplification templates without target-specific padlock probes. By integrating target recognition into the amplification-derived ssDNA intermediate, PPCTG couples target amplification with circular template generation while simplifying probe design and reducing nonspecific background. The resulting circular templates support continuous RCA, generating long ssDNA scaffolds that provide abundant activator sites for CRISPR-Cas12a, thereby enabling cascaded signal amplification through trans-cleavage of fluorophore-quencher reporters. The platform achieved a limit of detection of 3.47 aM for Staphylococcus aureus with high analytical specificity. Moreover, extraction-free detection was demonstrated in crude sputum and exhaled breath condensate, with recovery rates of 96.4%-105.5% and good agreement with qPCR results. Overall, PPCTG provides a padlock-free strategy for programmable circular template generation and integrates target amplification, RCA, and CRISPR-Cas12a signal amplification into a streamlined workflow, offering a low-background and ultrasensitive approach for pathogen detection in complex biological matrices.},
}

*Nucleic Acid Amplification Techniques/methods
*Staphylococcus aureus/isolation & purification/genetics
DNA, Single-Stranded/chemistry/genetics
*DNA Primers/chemistry/genetics
*CRISPR-Cas Systems/genetics
Limit of Detection
Biosensing Techniques/methods
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
DNA, Circular
Humans

@article {pmid42888353,
year = {2026},
author = {Quyoom, B and Peer, LA},
title = {The CRISPR-dCas9 epigenetic toolbox: prospects for modulating gene expression in climate-resilient crops.},
journal = {Planta},
volume = {264},
number = {6},
pages = {},
pmid = {42888353},
issn = {1432-2048},
mesh = {*Crops, Agricultural/genetics ; Epigenome Editing ; *CRISPR-Cas Systems/genetics ; *Epigenesis, Genetic/genetics ; Climate Change ; Plants, Genetically Modified/genetics ; Gene Expression Regulation, Plant/genetics ; *Gene Editing/methods ; },
abstract = {CRISPR-dCas9 epigenome editing represents a paradigm shift from static DNA sequence editing to dynamic reprogramming of gene expression, offering a powerful and precise toolbox with the potential to develop stable and climate-resilient crops without altering DNA sequences. The escalating challenges of global food security, driven by climate change and population growth, necessitate innovative crop improvement strategies that extend beyond conventional breeding. Although CRISPR-Cas9 has revolutionized plant biotechnology through direct genome editing, CRISPR-based epigenome editing has emerged as a new frontier. By repurposing nuclease-dead Cas9 as a programmable targeting platform fused to epigenetic effector domains, researchers can precisely modulate gene expression without altering the DNA sequence. This approach has already yielded compelling successes, such as the engineering of durable resistance to cassava bacterial blight through targeted epigenetic silencing of a susceptibility gene. This review focuses primarily on the current state of CRISPR-based epigenome editing systems, including acetyltransferases, demethylases, DNA methyltransferases, and deacetylases, and their applications in crop improvement research. We critically evaluate their mechanisms in rewriting epigenetic regulatory information, their demonstrated efficacy in enhancing yield components, disease resistance, and abiotic stress tolerance, and their potential to create stable meiotically heritable epialleles, although most demonstrations remain at the proof-of-concept stage in model plants, with field validation in major crops still limited. This review provides a balanced analysis of key challenges, including editing specificity, context-dependency, delivery limitations, and regulatory considerations. Finally, we outline future directions focusing on technological refinement, multi-omics integration, and field validation. Epigenome editing shows promise as a conceptually distinct and transformative approach in crop biotechnology, through targeted modulation of a plant's epigenetic landscape. However, its full potential hinges on overcoming challenges related to stability, predictability, and field-scale validation.},
}

*Crops, Agricultural/genetics
Epigenome Editing
*CRISPR-Cas Systems/genetics
*Epigenesis, Genetic/genetics
Climate Change
Plants, Genetically Modified/genetics
Gene Expression Regulation, Plant/genetics
*Gene Editing/methods

@article {pmid42889668,
year = {2026},
author = {Dutta, K and Velieva, KM and Skorb, EV and Shityakov, S},
title = {Bacterial Defense and Phage Counterdefense: Unraveling the Molecular Basis of Phage Resistance.},
journal = {ACS omega},
volume = {11},
number = {40},
pages = {60505-60521},
pmid = {42889668},
issn = {2470-1343},
abstract = {Phage therapy represents a promising strategy against multidrug-resistant bacteria, but its clinical effectiveness is increasingly compromised by the emergence of phage-resistant traits (PRTs). This resistance develops through multiple interconnected mechanisms that operate at different levels of bacterial defense. At the primary level, mutations in phage receptors, cell surface glycoconjugates (lipopolysaccharide, capsule polysaccharide, exopolysaccharide), outer membrane proteins, drug efflux pumps, and surface layer proteins provide the first line of defense by preventing phage adsorption. These receptors are highly diverse across Gram-negative bacteria, Gram-positive bacteria, and acid-fast bacteria, with PRT being particularly dominant in Gram-negative species. At the secondary level, bacteria deploy sophisticated antiphage defense (APD) systems, including CRISPR-Cas, restriction-modification systems, and abortive infection mechanisms. The recent discovery of over 150 distinct APD systems underscores the remarkable complexity of bacterial immunity. Concurrently, bacteriophages have evolved anti-antiphage defense (AAPD) systems to counter bacterial immunity, establishing a dynamic coevolutionary arms race. This continuous interplay between APD diversification and AAPD counteradaptation drives the rapid emergence of PRT, which threatens the long-term viability of phage therapy. This systematic review examines the molecular basis of phage resistance across diverse bacterial taxa, analyzes the evolutionary and coevolutionary forces shaping APD and AAPD systems, and highlights previously overlooked aspects of phage-bacteria interactions. By integrating these perspectives, we aim to inform strategies for advancing modern phage therapy and guiding future research directions.},
}

@article {pmid42890117,
year = {2026},
author = {Kim, D and Huang, CW and Pan, R and Qian, J and Wu, X and Devinsky, O and Tsai, YT and Liu, S},
title = {CasNano: An ultracompact CRISPR-Cas12f with a GC-rich PAM for epigenome editing with therapeutic potential.},
journal = {Molecular therapy. Nucleic acids},
volume = {37},
number = {4},
pages = {103106},
pmid = {42890117},
issn = {2162-2531},
support = {R43 NS139830/NS/NINDS NIH HHS/United States ; },
abstract = {Clinical use of CRISPR-Cas proteins for genome engineering is constrained by their large size, limiting in vivo delivery. To address this, we engineered an ultracompact and versatile platform from a minimally active, 400-amino acid Cas12f protein from Blautia lenta. A multi-stage strategy of structure-guided protein mutagenesis and systematic single guide RNA (sgRNA) optimization transformed BlCas12f into a "CasNano" as a potent genome targeting tool for mammalian cells. When a catalytically dead CasNano (dCasNano) is fused to a VPR domain, it activates endogenous genes with an efficiency and specificity comparable or superior to the benchmark SpCas9 system for the guide RNAs (gRNAs) and loci tested under equal-mass plasmid transfection; as editor expression was not quantified and stoichiometric differences would, in any case, preclude equivalent normalization, these comparisons describe functional output rather than intrinsic activity. We established the platform's versatility for bidirectional epigenetic editing by fusing dCasNano to a DNA methyltransferase (DNMT3A) for gene silencing or a dioxygenase (TET1) for gene activation. TET1-mediated reactivation persisted for 5 months in human embryonic stem cells (hESCs), whereas DNMT3A-deposited methylation was not retained at 2 weeks in a dividing reporter line. CasNano's small size and distinct CCN PAM sequence highlight its potential for future therapeutic applications constrained by viral delivery cargo capacity.},
}

@article {pmid42891742,
year = {2026},
author = {Yuan, Y and Deng, J and Li, X and Zheng, X and Tan, Y and Xu, K},
title = {Utilization of the CRISPR-Cas system for investigating tuberculosis drug resistance.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42891742},
issn = {1573-4978},
support = {82574159, 82173572//the National Natural Science Foundation of China/ ; 2026JK2006, 2026JJ80902, 2023JJ30430//he Science and Technology Program of Hunan Province/ ; 23A0075//the Department of Education of Hunan Province/ ; },
mesh = {*Mycobacterium tuberculosis/genetics/drug effects ; *CRISPR-Cas Systems/genetics ; Humans ; Antitubercular Agents/pharmacology ; *Tuberculosis, Multidrug-Resistant/genetics/microbiology/drug therapy ; *Drug Resistance, Bacterial/genetics ; Gene Editing/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Drug-resistant tuberculosis(TB) presents a significant challenge to global efforts to control the disease. Addressing this issue requires a comprehensive understanding of drug resistance in Mycobacterium tuberculosis (M.tb). The clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (Cas) system are efficient genome-editing tools for various organisms and have facilitated new research directions regarding drug resistance in M.tb. This review summarizes recent progress in applying CRISPR/Cas technology to investigate drug resistance mechanisms in M.tuberculosis and discusses the potential of the CRISPR/Cas system as an integrated platform to transform research paradigms and enhance understanding of M.tuberculosis drug resistance mechanisms.},
}

*Mycobacterium tuberculosis/genetics/drug effects
*CRISPR-Cas Systems/genetics
Humans
Antitubercular Agents/pharmacology
*Tuberculosis, Multidrug-Resistant/genetics/microbiology/drug therapy
*Drug Resistance, Bacterial/genetics
Gene Editing/methods
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid42892010,
year = {2026},
author = {Lau, CH and Huang, Y and Ch'ng, ES and Tang, TH and Zhu, H and Citartan, M},
title = {Aptamer-CRISPR/Cas as a tool in point-of-care testing of infectious diseases.},
journal = {Talanta},
volume = {313},
number = {Pt A},
pages = {130767},
doi = {10.1016/j.talanta.2026.130767},
pmid = {42892010},
issn = {1873-3573},
abstract = {Aptamers and the CRISPR/Cas system have become the cornerstones of target detections due to their numerous advantages. Aptamers enable direct recognition of non-nucleic acid targets, such as pathogens and antigens, while CRISPR/Cas system drives signal amplification and rapid detection. The combined aptamer-CRISPR/Cas system can overcome the limitations of traditional target detection by realizing field-deployable, highly sensitive, specific, versatile, and rapid assays. Given the increasing number of different aptamer-CRISPR/Cas systems developed recently, particularly for infectious diseases, we provide a comprehensive overview of these platforms designed for detecting significant viruses such as SARS-CoV-2, Monkeypox virus, Hepatitis B virus and bacteria that include Mycobacterium tuberculosis, Staphylococcus aureus and Salmonella. Current challenges that impede the deployment of aptamer-CRISPR/Cas for point-of-care pathogen detection were also mapped, including primary barriers such as clinical matrix effect and stability. Several strategies to mitigate these issues were also set forth, predominantly the integration of artificial intelligence and machine learning to accelerate the transition of aptamer-CRISPR/Cas system to the clinical translation and commercialization phase, paving the way towards widespread implementation in global health.},
}

@article {pmid42892437,
year = {2026},
author = {Ruowen, G and Haitao, T and Hongkun, F and Weilin, W and Baosheng, Y and Mengya, X and Jia, X and Chaojie, H and Xiaoyu, C},
title = {Development of a rapid RPA-CRISPR/Cas12b assay for dual detection of Hantaan virus and Seoul virus.},
journal = {Journal of virological methods},
volume = {347},
number = {},
pages = {115483},
doi = {10.1016/j.jviromet.2026.115483},
pmid = {42892437},
issn = {1879-0984},
abstract = {Hantaan virus (HTNV) and Seoul virus (SEOV) are the primary causative agents of hemorrhagic fever with renal syndrome (HFRS), presenting a persistent challenge to global public health. Prompt and precise diagnostic tools are essential for clinical investigation and the containment of HFRS outbreaks. Leveraging the programmable precision of CRISPR/Cas12b system, which has significantly advanced the field of molecular diagnostics, we developed a rapid, high-sensitivity assay for the dual detection of HTNV and SEOV. Each virus was detected in a separate, target-specific single-tube reaction. The assay combines recombinase polymerase amplification (RPA) with CRISPR/Cas12b-mediated fluorescence detection and uses virus-specific sgRNAs and RPA primers targeting conserved genomic regions. Using recombinant plasmid standards, both targets were detected at 1 × 10[1] copies/test in three technical replicates; this is a preliminary analytical detection threshold and does not incorporate extraction losses or matrix-associated inhibition. No cross-reactivity was observed within the evaluated four-organism non-target panel. In a preliminary feasibility assessment, all nine RT-qPCR-positive rodent samples (seven HTNV and two SEOV) were positive by the RPA-CRISPR/Cas12b assay, indicating complete positive agreement in this all-positive sample set. Because no negative samples were included, negative agreement, diagnostic specificity, false-positive rate, and overall clinical diagnostic accuracy could not be assessed. These results support further development of the assay as a potential screening tool; larger studies using well-characterized positive and negative clinical or field specimens are required.},
}

@article {pmid42892439,
year = {2026},
author = {Huang, Y and Duan, C and Deng, X and Zhang, X and Wu, M and Peng, S and Liu, R and Hou, D},
title = {Advances in influenza A and B virus detection: molecular diagnostics, biosensors, and point-of-care applications.},
journal = {Journal of microbiological methods},
volume = {251},
number = {},
pages = {107746},
doi = {10.1016/j.mimet.2026.107746},
pmid = {42892439},
issn = {1872-8359},
abstract = {Influenza A and B viruses remain major causes of seasonal epidemics and continue to pose substantial challenges for rapid clinical diagnosis and public-health surveillance. Conventional methods, including viral culture, immunological assays, and reverse-transcription real-time quantitative PCR (RT-qPCR), provide important diagnostic benchmarks but are constrained by long turnaround times, dependence on specialized equipment, or reduced sensitivity in low-viral-load specimens. This review summarizes recent advances in influenza virus detection, with emphasis on molecular diagnostics, biosensors, point-of-care testing, and high-throughput sequencing. Digital PCR offers precise absolute quantification, whereas isothermal amplification technologies such as reverse transcription loop-mediated isothermal amplification (RT-LAMP) and reverse transcription recombinase polymerase amplification (RT-RPA) reduce reliance on thermal cycling and support portable testing. CRISPR-Cas systems further improve sequence specificity and are increasingly being integrated with isothermal amplification, lateral-flow readouts, and microfluidic platforms. Biosensor strategies based on optical, electrochemical, multimodal, and smartphone-assisted readouts provide promising routes for rapid, multiplexed, and point-of-care detection, although standardization and large-scale clinical validation remain key barriers. High-throughput sequencing complements rapid testing by enabling viral typing, variant monitoring, and emerging-pathogen monitoring. Overall, future influenza diagnostics are expected to move toward integrated, low-cost, "sample-in, answer-out" systems that combine sensitivity, speed, portability, and surveillance connectivity.},
}

@article {pmid42892548,
year = {2026},
author = {Wang, H and Liu, J},
title = {From oleaginous microalga to marine chassis: synthetic biology and process-aware biomanufacturing in Nannochloropsis.},
journal = {Biotechnology advances},
volume = {94},
number = {},
pages = {109079},
doi = {10.1016/j.biotechadv.2026.109079},
pmid = {42892548},
issn = {1873-1899},
abstract = {Nannochloropsis has developed from an oleaginous marine microalga valued mainly for eicosapentaenoic acid (EPA)-rich lipids into a credible candidate for marine photosynthetic biomanufacturing. Its toolkit now spans genome resources, efficient nuclear transformation, homologous recombination, CRISPR/Cas editing, marker recycling, defined integration sites, and modular multigene assembly. Researchers have used these capabilities to alter carbon allocation, triacylglycerol accumulation, EPA synthesis and lipid-class partitioning, carotenoid metabolism, recombinant-protein production, specialty-metabolite formation, and cultivation robustness. This review assesses whether these advances are sufficient to support Nannochloropsis as a predictable, reusable, and process-aware chassis. The evidence shows that the genus has moved beyond basic transformability. Targeted genome modification and pathway construction are feasible, and native and non-native products have reached biologically relevant and, in some cases, process-relevant levels. However, outcomes remain strongly influenced by genomic locus, strain background, intracellular compartment, physiological state, and cultivation regime. Platform maturation will therefore depend on reusable genomic architectures, reliable organelle-targeting rules, dynamic regulation, and phenotyping suited to iterative design-build-test-learn workflows. Strain design must also be coupled more closely to cultivation, harvesting, product recovery, and deployment. Product-specific chassis configurations are likely to be more practical than a universal strain for EPA-rich oils, pigments, recombinant products, and specialty metabolites. Nannochloropsis thus provides a useful test of how an industrially relevant, non-model photosynthetic eukaryote can become a more designable marine production host.},
}

@article {pmid42893213,
year = {2026},
author = {Kobilov, F and Elova, N and Murodali, B and Gullola, B and Amirsaidova, D and Xidirova, M and Yodgorova, F and Miralimova, S},
title = {Draft genome data of Pediococcus acidilactici strain AP1 isolated from common carp (Cyprinus carpio).},
journal = {Data in brief},
volume = {69},
number = {},
pages = {113296},
pmid = {42893213},
issn = {2352-3409},
abstract = {This data article presents the whole-genome sequencing and associated genomic datasets of Pediococcus acidilactici strain AP1, isolated from the intestinal tract of a healthy common carp (Cyprinus carpio) obtained from a fish farm in the Tashkent region, Uzbekistan. The datasets comprise raw paired-end sequencing reads, a draft genome assembly, genome-quality assessment reports, structural and functional annotations, and phylogenomic outputs. Whole-genome sequencing was performed using the Illumina MiSeq i100 Plus System with paired-end 2 × 151 bp chemistry. De novo assembly generated a draft genome comprising 19 contigs, with a total length of 2,015,983 bp, an N50 of 388,737 bp, an L50 of 2, and a GC content of 42.02%. CheckM2 estimated 99.99% genome completeness and 0.5% contamination, whereas BUSCO recovered 99.2% of the conserved bacterial single-copy orthologs. Annotation with Prokka v1.15.6 identified 1,897 protein-coding sequences, 52 tRNA genes, four rRNA genes, and one tmRNA. Genome-based phylogenetic and digital DNA-DNA hybridization analyses supported the assignment of strain AP1 to P. acidilactici. Functional annotations generated using Prokka, RAST, eggNOG-mapper, and InterProScan indicated genes putatively associated with carbohydrate fermentation, stress adaptation, exopolysaccharide biosynthesis, peptide transport and proteolysis, cell-surface interactions, and CRISPR-Cas defense. Gene-based functional assignments represent predictions and require experimental validation. The publicly available datasets can support comparative genomic investigations of fish-associated Pediococcus strains and future experimental studies of host-associated and probiotic-relevant traits.},
}

@article {pmid42893944,
year = {2026},
author = {Wang, YM and Zang, X},
title = {Advances in tools and strategies for spatiotemporal regulation of gene expression in Caenorhabditis elegans.},
journal = {Yi chuan = Hereditas},
volume = {48},
number = {10},
pages = {973-985},
doi = {10.16288/j.yczz.26-140},
pmid = {42893944},
issn = {0253-9772},
mesh = {Animals ; *Caenorhabditis elegans/genetics/metabolism ; RNA Interference ; *Gene Expression Regulation ; CRISPR-Cas Systems ; },
abstract = {Spatiotemporal manipulation of gene expression is essential for dissecting gene function, elucidating developmental processes, and uncovering complex physiological mechanisms. As a model organism, Caenorhabditis elegans has played an important role in basic biology research and provides an ideal experimental system for achieving such precise control. With the development of single-copy transgenesis, CRISPR/Cas9-mediated genome editing, and conditional recombination and inducible systems, the genetic toolkits for spatiotemporal regulation of gene expression, and even at the single-cell-resolution, have been greatly expanded in C. elegans. In this review, we systematically summarize the major strategies for spatiotemporal regulation of gene expression in C. elegans, with a focus on tissue-specific RNA interference, conditional protein degradation systems, inducible transcriptional regulation systems, and recently developed approaches such as Ribo-on/Ribo-off. These methods are compared in terms of regulatory levels, technical advantages, limitations, and suitable applications. Together, these tools cover multiple layers of regulation, ranging from transcriptional and post-transcriptional control to protein-level manipulation, and provide powerful approaches for developmental biology, aging, inter-tissue signaling, and disease models, while also establishing a methodological foundation for dissecting cell-autonomous and non-cell-autonomous regulatory mechanisms in complex biological processes.},
}

Animals
*Caenorhabditis elegans/genetics/metabolism
RNA Interference
*Gene Expression Regulation
CRISPR-Cas Systems

@article {pmid42897599,
year = {2026},
author = {Jeon, BJ and Moon, B and Jang, HS and Kwon, DH and Jeon, YL and Eom, KH and Choi, JY and Kim, YC and Jang, G},
title = {Efficient genome editing in ASC-derived porcine lung organoids via GFP-guided enrichment.},
journal = {Stem cell research & therapy},
volume = {17},
number = {1},
pages = {},
pmid = {42897599},
issn = {1757-6512},
support = {RS-2024-00402798//Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through High Value-added Food Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA)/ ; RS-2023-00260968//National Research Foundation of Korea/ ; },
mesh = {Animals ; *Organoids/metabolism/cytology ; Swine ; *Lung/cytology/metabolism ; *Gene Editing/methods ; *Green Fluorescent Proteins/metabolism/genetics ; CRISPR-Cas Systems/genetics ; Humans ; *Adult Stem Cells/metabolism/cytology ; },
abstract = {BACKGROUND: Adult stem cell-derived organoids are invaluable tools for disease modeling and translational medicine owing to their tissue-specific correspondence. However, their complex three-dimensional structure and low genome-editing efficiency have posed significant challenges for functional genetic studies. To address these limitations, this study aimed to establish porcine lung organoid (PLOs) and to develop a robust, high-efficiency genome-editing platform by leveraging the high physiological and anatomical similarity between pigs and humans.

METHODS: PLOs were derived from lung tissues and characterized by immunofluorescence and immunohistochemistry analysis. To enhance genome-editing efficiency using CRISPR/Cas9 and prime editing, a GFP-guided enrichment strategy was used by co-targeting a GFP transgene and endogenous loci, followed by the selection of GFP-negative organoids. Clonal expansion from single organoids was performed to generate genetically homogenous lines. Genetic modifications were validated through cDNA sequencing and western blot analysis. Structural integrity was assessed using AlphaFold2 modeling, and functional changes were evaluated using the forskolin-induced swelling assay.

RESULTS: The established PLOs exhibited sustained self-renewal capacity and expression of surfactant protein C alongside both proximal epithelial and mesenchymal markers, reflecting a composite lung organoid identity. GFP-guided enrichment significantly enriched edited cells; the mutation ratio in the GFP-negative group was significantly higher than that in the GFP-positive group. This system successfully generated fully gene-edited clones with high genetic homogeneity, and effectively enriched for precisely edited organoids, even when applying complex modalities such as prime editing.

CONCLUSIONS: The PLOs and genome-editing platform developed in this study provide a powerful resource for translational medicine. By enabling precise genetic manipulation in a model that closely mimics human lung physiology, this system offers an essential platform for recapitulating human lung pathologies, studying zoonotic infectious diseases, and validating the efficacy of gene therapies.},
}

Animals
*Organoids/metabolism/cytology
Swine
*Lung/cytology/metabolism
*Gene Editing/methods
*Green Fluorescent Proteins/metabolism/genetics
CRISPR-Cas Systems/genetics
Humans
*Adult Stem Cells/metabolism/cytology

@article {pmid42898686,
year = {2026},
author = {Yang, LQ and Shu, SL and Zhang, YH and Huang, G},
title = {Molecular mechanisms and application potential of MYC2 transcription factor-mediated plant stress responses.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1943016},
pmid = {42898686},
issn = {1664-462X},
abstract = {Plant adaptation to drought, salinity, temperature extremes, pathogens, and herbivorous insects depends on coordinated hormone signalling, transcriptional control, and metabolic reprogramming. The jasmonic acid (JA) pathway is a major regulator of defence and the growth-defence trade-off, and the bHLH transcription factor MYC2 is a central node in JA output. Mechanistically, MYC2 is controlled through demonstrated protein-protein interactions with JAZ repressors, MED25, and selected transcriptional or chromatin regulators; it binds G-box-related cis-elements to regulate specific promoters, whereas many reported links to LOX/AOS, CBF, and antioxidant pathways are transcriptional or pathway-level rather than physical interactions. This integrative narrative review compares MYC2 orthologues across species, distinguishes MYC2-specific functions from those shared with MYC3/MYC4 or other clade IIIe bHLHs, and organises beneficial and detrimental stress phenotypes around pathogen lifestyle, target-gene polarity, tissue and developmental context, paralogue redundancy, and experimental design. We integrate genome-wide DNA-binding, transcriptomic, metabolomic, and protein-level studies, and critically evaluate demonstrated versus prospective applications of CRISPR/Cas genome editing. The synthesis identifies regulatory strategies that may exploit MYC2 networks whilst limiting growth, defence, and quality trade-offs.},
}

@article {pmid42898821,
year = {2026},
author = {Azhar, MA and Mir, TA and Ahmed, S and Assiri, AM and Broering, DC},
title = {Biofilm-associated genes in Staphylococcus aureus: role in chronic infections and therapeutic challenges.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1915213},
pmid = {42898821},
issn = {1664-302X},
abstract = {One of the major contributors to persistent healthcare and community-associated infections is S. aureus, which has a remarkable ability to form biofilms that protect it against the host immune system and antimicrobial therapy. In recent years, it has become clear that biofilm development is a highly orchestrated and dynamic process, controlled by complex genetic networks, transcriptional regulators, two-component systems (TCSs), and environmental signals. The present review covers the molecular mechanisms involved in the initiation, maturation, maintenance, and dispersal of biofilms in general, and in particular, the coordinated functions of adhesins, the icaADBC operon, components of the extracellular matrix (ECM), global regulators (agr, sarA, and sigB), and two-component regulatory systems, as well as emerging insights from transcriptomic studies on biofilm adaptation. We then review the roles of these regulatory pathways in metabolic heterogeneity, antibiotic tolerance, immune evasion, and persistence in clinically relevant biofilm-associated infections, including infective endocarditis, osteomyelitis, prosthetic joint infection, catheter-associated infection, and diabetic foot infection. The review further outlines the latest developments in antibiofilm therapeutics, including matrix-degrading enzymes, quorum-sensing inhibitors, antimicrobial peptides, bacteriophage-based therapeutics, lysins, nitric oxide donors, nanoparticle-assisted therapeutics, and CRISPR-Cas-based antimicrobials. Incorporating molecular regulation into infection-specific pathogenesis and emerging therapeutic strategies, this review offers a new perspective on the biology of S. aureus biofilms and highlights opportunities to develop mechanism-based interventions to combat chronic biofilm-associated infections.},
}

@article {pmid42899019,
year = {2026},
author = {Bhatti, M and Thalib, HI and Tayyebah, A and Zobairi, A and Dahan, N and Hassan, FE},
title = {The emerging role of CRISPR-Cas systems in helicobacter pylori and the gut microbiota as regulators of gastrointestinal cancer risk.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1889115},
pmid = {42899019},
issn = {1664-3224},
mesh = {Humans ; *CRISPR-Cas Systems ; *Helicobacter pylori/genetics/immunology/pathogenicity ; *Gastrointestinal Neoplasms/microbiology/etiology/diagnosis ; *Helicobacter Infections/microbiology/complications/immunology ; *Gastrointestinal Microbiome/immunology/genetics ; Animals ; Host-Pathogen Interactions ; },
abstract = {Gastrointestinal cancers contribute to 26% of global cancer incidence and 35% of deaths, with major contributors such as Helicobacter pylori (H. pylori) infection, gut dysbiosis, and lifestyle factors. CRISPR-Cas systems, while best known for their role in bacterial adaptive immunity, also shape bacterial physiology and host-microbe interactions, yet their influence on gastrointestinal carcinogenesis is poorly understood. This review brings together current evidence on the functions of CRISPR-Cas systems in H. pylori and the gut microbiota, highlighting their potential impact on cancer risk, diagnostics, and therapy. Key themes synthesized in this review include their role in bacterial immunity, contributions to microbial imbalance and virulence regulation, and emerging applications in cancer detection, microbiome modulation, and precision oncology. CRISPR profiles are being explored as biomarkers, while engineered systems show promise in targeted editing, phage therapy, and highly sensitive diagnostic platforms such as Cas12a-based assays. Despite this progress, challenges remain, including the risk of off-target effects, technical difficulties in detecting CRISPR signatures within complex microbial communities, and a lack of longitudinal human studies. Overall, CRISPR-Cas systems are emerging as natural regulators of microbial ecology and as engineered tools with potential applications in gastrointestinal cancer research, diagnostics, and precision microbiome interventions.},
}

Humans
*CRISPR-Cas Systems
*Helicobacter pylori/genetics/immunology/pathogenicity
*Gastrointestinal Neoplasms/microbiology/etiology/diagnosis
*Helicobacter Infections/microbiology/complications/immunology
*Gastrointestinal Microbiome/immunology/genetics
Animals
Host-Pathogen Interactions

@article {pmid42899205,
year = {2026},
author = {Senthilkumar, R and Palaniswamy, R and Rathnasamy, SA and Rajagopalan, VR and Eswaran, K and Sengodan, R and Govindasamy, AK and Muthurajan, R and Kanagarajan, S and Manickam, S},
title = {Genome editing beyond transgenes: DNA-free technologies in crop improvement.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1942730},
pmid = {42899205},
issn = {1664-462X},
abstract = {Conventional and transgenic approaches to crop improvement are increasingly constrained by biosafety concerns, stringent regulatory frameworks, and limited public acceptance, necessitating new precision breeding methods. DNA-free genome editing using the CRISPR/Cas9 approach has emerged as a transformative approach that enables precise, targeted genetic modifications without the stable integration of a foreign gene (transgene). This strategy transiently introduces CRISPR/Cas9 ribonucleoprotein (RNP) complexes, or in vitro transcribed mRNA, into plant cells. This review focuses primarily on CRISPR/Cas-mediated DNA-free genome editing and its applications in crop improvement. After targeted genome cleavage, the editing elements are rapidly degraded, leaving only the desired genomic modification and resulting in plants indistinguishable from those developed through conventional breeding. To achieve targeted, DNA-free genome editing, researchers have developed numerous molecular platforms and delivery systems, such as protoplast transfection, biolistic bombardment, nanoparticle-based systems, and emerging pollen- and meristem-targeted systems, for major crop species. Researchers have successfully enhanced several notable traits, including disease and pest resistance, nutritional quality, abiotic stress tolerance, and agronomic performance, in cereals, vegetables, oilseeds, and perennial crops. Recent advancements, including base editing, prime editing, and multiplex gene editing, are further expanding the precision, efficiency, and scope of DNA-free genome editing. Overall, CRISPR/Cas-mediated DNA-free genome editing offers a route to crop improvement that avoids stable transgene integration, though its wider deployment depends on progress in regeneration, molecular validation, and regulatory convergence.},
}

@article {pmid42899467,
year = {2026},
author = {Kirk, RW and Sun, L and Xiao, R and Clark, EA and Nelson, SB},
title = {Multiplexed CRISPRi reveals a transcriptional switch between KLF activators and repressors in the maturing neocortex.},
journal = {eLife},
volume = {14},
number = {},
pages = {},
pmid = {42899467},
issn = {2050-084X},
support = {R01 NS109916/NS/NINDS NIH HHS/United States ; R21 NS147284/NS/NINDS NIH HHS/United States ; 2R01NS109916-05A1/NS/NINDS NIH HHS/United States ; 1R21NS147284-01/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; *Neocortex/growth & development/metabolism ; *Kruppel-Like Transcription Factors/genetics/metabolism ; Mice ; *Gene Expression Regulation, Developmental ; *Transcription, Genetic ; Neurodevelopment ; Kruppel-Like Factor 6/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {A critical phase of mammalian brain development takes place after birth. Neurons of the mouse neocortex undergo dramatic changes in their morphology, physiology, and synaptic connections during the first postnatal month, while properties of immature neurons, such as the capacity for robust axon outgrowth, are lost. The genetic and epigenetic programs controlling prenatal development are well studied, but our understanding of the transcriptional mechanisms that regulate postnatal neuronal maturation is comparatively lacking. By integrating chromatin accessibility and gene expression data from two subtypes of neocortical pyramidal neurons in the neonatal and maturing brain, we predicted a role for the Krüppel-like factor (KLF) family of transcription factors in the developmental regulation of neonatally expressed genes. Using a multiplexed CRISPR interference (CRISPRi) knockdown strategy, we found that a shift in expression from KLF activators (Klf6, Klf7) to repressors (Klf9, Klf13) during early postnatal development functions as a transcriptional 'switch' to first activate, then repress a set of shared targets with cytoskeletal functions including Tubb2b and Dpysl3. We demonstrate that this switch is buffered by redundancy between KLF paralogs, which our multiplexed CRISPRi strategy is equipped to overcome and study. Our results indicate that competition between activators and repressors within the KLF family regulates a conserved component of the postnatal maturation program that may underlie the loss of intrinsic axon growth in maturing neurons. This could facilitate the transition from axon growth to synaptic refinement required to stabilize mature circuits.},
}

Animals
*Neocortex/growth & development/metabolism
*Kruppel-Like Transcription Factors/genetics/metabolism
Mice
*Gene Expression Regulation, Developmental
*Transcription, Genetic
Neurodevelopment
Kruppel-Like Factor 6/genetics/metabolism
CRISPR-Cas Systems

@article {pmid42899764,
year = {2026},
author = {Chen, R and Zhou, H and Wu, Q and Gong, C},
title = {Nano Delivery Systems for In Vivo CRISPR/Cas-Based Gene Therapy: From Vectors to Clinical Outcomes.},
journal = {ACS nano},
volume = {20},
number = {41},
pages = {27559-27640},
doi = {10.1021/acsnano.6c12154},
pmid = {42899764},
issn = {1936-086X},
support = {2026NSFSCZY0081//Department of Science and Technology of Sichuan Province/ ; },
mesh = {*Genetic Therapy/methods ; Humans ; *CRISPR-Cas Systems/genetics ; Animals ; *Gene Transfer Techniques ; *Genetic Vectors/genetics ; *Nanoparticles/chemistry ; },
abstract = {As a powerful gene-editing tool, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system features simple construction, precise targeting, and higher editing efficiency, bringing prospects of radical cures for numerous critical illnesses at the genomic level. To date, ex vivo CRISPR/Cas-based gene therapies represented by Casgevy have achieved significant clinical advances. Nevertheless, cumbersome operational workflows and prohibitive treatment costs severely hinder their widespread clinical accessibility. In contrast, in vivo CRISPR/Cas-based gene therapies stand out with convenient administration procedures and reduced overall costs, endowing them with enormous translational potential. Although it holds great promise, the clinical translation of in vivo CRISPR/Cas-based gene therapies remains hampered by a number of factors. Among those multiple obstacles restricting the clinical translation of in vivo CRISPR/Cas-based gene therapies, delivery systems constitute the core bottleneck dominating in vivo editing efficiency and therapeutic potency. Centered on delivery platforms for in vivo CRISPR/Cas-based gene therapies, this review systematically summarizes prevailing in vivo CRISPR/Cas nano delivery systems, recapitulates relevant preclinical and clinical outcomes, discusses translational hurdles, and prospects the evolution of next-generation delivery systems, aiming to facilitate further development of in vivo CRISPR/Cas-based gene therapies.},
}

*Genetic Therapy/methods
Humans
*CRISPR-Cas Systems/genetics
Animals
*Gene Transfer Techniques
*Genetic Vectors/genetics
*Nanoparticles/chemistry

@article {pmid42901163,
year = {2026},
author = {Custodio, R and Martignoni, MM and Zhu, W and Kuske, R and Alseth, EO and Moura-Alves, P and Brown, SP and Westra, ER},
title = {Interbacterial competition triggers stress-induced CRISPR immunity against bacteriophages.},
journal = {Current biology : CB},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.cub.2026.09.078},
pmid = {42901163},
issn = {1879-0445},
abstract = {Bacteria-phage interactions are often studied in relatively simple experimental systems involving a single bacterial strain infected by a single phage.[1] In natural environments, however, bacteria typically inhabit complex multispecies communities where species compete for limited resources and ecological niches.[2][,][3][,][4] Recent studies show that the microbial community context can have profound effects on the evolution of phage resistance.[5] For example, Pseudomonas aeruginosa evolves phage resistance primarily through receptor loss when grown in isolation but switches to CRISPR-based immunity in the presence of bacterial competitors.[6][,][7] Previous work showed that interspecies competition can favor CRISPR-immune bacteria over surface mutants after phage resistance has evolved.[6] However, it remains unknown whether competition also alters the initial generation of CRISPR immunity by changing CRISPR-Cas expression and spacer acquisition before selection among resistance phenotypes occurs. Here, we show that interbacterial competition can trigger CRISPR-Cas activation and adaptation in P. aeruginosa. When co-cultured with Acinetobacter baumannii, P. aeruginosa exhibited markedly increased expression of CRISPR-Cas genes and a higher frequency of new spacer acquisition, whereas no such induction occurred in monoculture or mixed communities lacking A. baumannii. This upregulation was associated with markers of DNA damage and stress signaling, indicating that competition-induced stress primes the bacterial adaptive immune response. These findings identify interbacterial antagonism as an ecological and physiological trigger for CRISPR activation, linking competitive interactions to enhanced antiphage defense. More broadly, our results suggest that bacteria sense and respond to competitive stress in complex microbial communities by increasing the generation of more adapted genotypes.},
}

@article {pmid42901363,
year = {2026},
author = {Basit, A and Hanif, M},
title = {Next-generation CRISPR-based molecular diagnostics for veterinary parasitology: From laboratory innovation to one health surveillance.},
journal = {Molecular and biochemical parasitology},
volume = {268},
number = {},
pages = {111789},
doi = {10.1016/j.molbiopara.2026.111789},
pmid = {42901363},
issn = {1872-9428},
abstract = {Veterinary parasitic diseases remain a major threat to livestock production, companion animal health, wildlife conservation, and public health worldwide. Conventional diagnostic methods, including microscopy, serology, and culture-based techniques, often suffer from limited sensitivity, poor specificity, prolonged turnaround time, and dependence on specialized laboratory facilities. Recent advances in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based diagnostics have introduced highly sensitive, rapid, and field-deployable platforms for parasite detection. This review critically summarizes the principles and recent applications of CRISPR/Cas12, Cas13, and emerging Cas14-based diagnostic approaches in veterinary parasitology, covering protozoan, helminthic, and arthropod-borne parasites. It further compares CRISPR platforms and discusses their integration with isothermal amplification, lateral flow assays, fluorescence detection, and portable biosensors to improve diagnostic performance. Moreover, current challenges, including sample preparation, assay standardization, multiplex detection, regulatory approval, and commercialization, are highlighted. Finally, future perspectives on amplification-free CRISPR assays, artificial intelligence-assisted diagnostics, and point-of-care platforms are discussed. By providing a translational perspective from laboratory innovation to field application, this review highlights the potential of CRISPR-based diagnostics to transform veterinary parasite surveillance and disease control.},
}

@article {pmid42901663,
year = {2027},
author = {Zhang, Y and Zhang, X and Zheng, H and Wang, H and Yu, M and Jia, W and Jia, Z and Cui, J and Xiong, H and Ma, Y and Wang, X},
title = {The application progress of detection technology for Bacillus cereus in food: Challenges, innovations and future prospects.},
journal = {Food microbiology},
volume = {142},
number = {},
pages = {105310},
doi = {10.1016/j.fm.2026.105310},
pmid = {42901663},
issn = {1095-9998},
mesh = {*Bacillus cereus/isolation & purification/genetics ; *Food Microbiology/methods ; *Food Contamination/analysis ; Humans ; Foodborne Diseases/microbiology/prevention & control ; Animals ; CRISPR-Cas Systems ; Food Safety ; },
abstract = {Food safety is a key issue of global public health. As a foodborne pathogen - Bacillus cereus (B. cereus), it is widely distributed in nature and can cause diseases in both humans and animals. The rapid detection of B. cereus is of great significance for the prevention and control of such diseases. This article provides a detailed overview of the development of B. cereus detection techniques in food, transitioning from traditional methods to a series of current technologies such as immunology, molecular biology and MALDI-TOF-MS. The principles, advantages and disadvantages of various detection techniques, as well as their application progress, were outlined. Furthermore, the application and advantages of combining different techniques in the detection of B. cereus in food were demonstrated. The CRISPR/Cas system possesses the characteristics of high specificity, high sensitivity and ease of operation, and shows great potential in the field of foodborne pathogen detection. This article also explains that the combination of CRISPR/Cas technology with RPA, LFA and AI technologies can further enhance the detection rate and accuracy. The aforementioned technology can enable the early detection and prevention of B. cereus in food, ensuring food safety and preventing the outbreak of foodborne diseases. This article has certain reference value in the research on the detection techniques of B. cereus and other foodborne pathogens in food, and provides strong support for safeguarding food safety and maintaining human health.},
}

*Bacillus cereus/isolation & purification/genetics
*Food Microbiology/methods
*Food Contamination/analysis
Humans
Foodborne Diseases/microbiology/prevention & control
Animals
CRISPR-Cas Systems
Food Safety

@article {pmid42905297,
year = {2026},
author = {Abuden, AV and Çolak, ÇY},
title = {Genomic Exploration of CRISPR-Cas Systems in Raoultella species: Diversity and Architecture.},
journal = {Current microbiology},
volume = {83},
number = {12},
pages = {},
pmid = {42905297},
issn = {1432-0991},
mesh = {Phylogeny ; *CRISPR-Cas Systems ; *Enterobacteriaceae/genetics/classification ; *Genome, Bacterial ; *Genetic Variation ; Clustered Regularly Interspaced Short Palindromic Repeats ; Genomics ; },
abstract = {Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein (CRISPR-Cas) systems are adaptive immune mechanisms that protect bacteria against invading mobile genetic elements. They can influence horizontal gene transfer, phage resistance, and antimicrobial resistance (AMR) dynamics. Although these systems have been investigated in several Klebsiella species, information on historically defined Raoultella species remains limited. In this study, 103 complete genomes representing Raoultella species, currently placed within the broader Klebsiella lineage, were analyzed to characterize the distribution, architecture, and diversity of CRISPR-Cas systems. CRISPR-Cas loci were identified using CRISPRCasFinder and CRISPRone, followed by subtype classification, direct repeat and leader sequence analyses, spacer matching, Cas1-based phylogenetic analysis, and AMR determinant screening. Complete CRISPR-Cas systems were detected in 23 genomes, predominantly in R. ornithinolytica strains. All complete systems were chromosomally located, whereas plasmid-associated orphan CRISPR arrays were observed in selected R. ornithinolytica strains. Two major architectures were identified, with Type I-E being dominant and Type I-F restricted to fewer strains. Direct repeat analysis revealed six consensus sequences, while leader regions displayed conserved AT-rich motifs and strain-specific divergence. Spacer analyses suggested historical interactions with phages and plasmids, particularly mobile genetic elements associated with Klebsiella and other Enterobacteriaceae. Cas1 phylogeny showed subtype-dependent clustering, indicating that CRISPR-Cas evolution does not strictly follow core genome phylogeny. The coexistence of predicted CRISPR-Cas loci and AMR determinants indicates that the presence of these loci does not necessarily preclude the carriage of resistance determinants. Overall, this study expands current knowledge of CRISPR-Cas diversity among the analyzed Raoultella genomes and provides a genomic framework for future functional studies.},
}

Phylogeny
*CRISPR-Cas Systems
*Enterobacteriaceae/genetics/classification
*Genome, Bacterial
*Genetic Variation
Clustered Regularly Interspaced Short Palindromic Repeats
Genomics

@article {pmid42749169,
year = {2026},
author = {Ishikawa, Y and Maeda, M and Taga, Y and Komatsu, Y and Mizuno, K and Saito, K},
title = {TANGO1L is required for collagen secretion in a type-dependent manner and shapes collagen proteostasis in the endoplasmic reticulum.},
journal = {Matrix biology : journal of the International Society for Matrix Biology},
volume = {149},
number = {},
pages = {102052},
doi = {10.1016/j.matbio.2026.102052},
pmid = {42749169},
issn = {1569-1802},
mesh = {Humans ; *Endoplasmic Reticulum/metabolism ; *Proteostasis ; *Collagen/metabolism/genetics ; Fibroblasts/metabolism ; *Aryl Hydrocarbon Receptor Nuclear Translocator/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Collagens are synthesized in the endoplasmic reticulum (ER), where their biosynthesis, quality control, and secretion are tightly coordinated to maintain collagen proteostasis. TANGO1L is an ER exit site protein required for efficient collagen secretion; however, whether it contributes equally to the secretion and intracellular homeostasis of different collagen types remains unclear. Here, we systematically examined the consequences of TANGO1L deficiency using CRISPR/Cas9-generated TANGO1L knockout human fibroblasts. Loss of TANGO1L reduced the secretion of multiple major collagen types and delayed secretion of newly synthesized collagen I. Intracellular responses, however, differed markedly among collagen types: collagen V accumulated intracellularly, whereas collagens I, III, and IV avoided intracellular retention despite markedly reduced extracellular secretion. This differential response was accompanied by decreased collagen gene expression and selective remodeling of the ER collagen biosynthetic machinery, suggesting that these cells adjust collagen synthesis to a reduced secretory capacity. TANGO1S protein was consistently upregulated through a post-transcriptional mechanism, suggesting an adaptive response to chronic TANGO1L deficiency. Biochemical analysis of collagen purified from the culture medium revealed a trend toward modestly increased lysine post-translational modifications, including hydroxylysine glycosylation, without altered triple-helical structure or thermal stability, indicating that TANGO1L deficiency compromises the quantity rather than the quality of secreted collagen. Together, these findings establish that the requirement for TANGO1L differs among collagen types and that its loss induces adaptive remodeling of intracellular collagen proteostasis rather than simply impairing collagen secretion, providing a framework for understanding how secretory cells maintain collagen homeostasis when secretion is compromised.},
}

Humans
*Endoplasmic Reticulum/metabolism
*Proteostasis
*Collagen/metabolism/genetics
Fibroblasts/metabolism
*Aryl Hydrocarbon Receptor Nuclear Translocator/genetics/metabolism
CRISPR-Cas Systems

@article {pmid42883761,
year = {2026},
author = {Liu, F and Liu, Y and Jin, S and Du, Q},
title = {Molecular Diagnostics of Pathogenic Vibrio: Target Selection, Detection Platforms, and Challenges in Field Applications.},
journal = {International journal of molecular sciences},
volume = {27},
number = {19},
pages = {},
pmid = {42883761},
issn = {1422-0067},
support = {No. 2025TSGCCZZB0313//Shandong Provincial Key Research and Development Program Project/ ; No. 42276133//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Vibrio Infections/diagnosis/microbiology ; *Vibrio/genetics/pathogenicity/isolation & purification ; *Molecular Diagnostic Techniques/methods ; CRISPR-Cas Systems ; Biosensing Techniques/methods ; Vibrio parahaemolyticus/genetics/pathogenicity ; Nucleic Acid Amplification Techniques/methods ; Vibrio cholerae/genetics/pathogenicity ; High-Throughput Nucleotide Sequencing/methods ; },
abstract = {Pathogenic Vibrio species, including Vibrio parahaemolyticus, Vibrio cholerae, and Vibrio vulnificus, are major foodborne and environmental pathogens that threaten public health. Rapid and accurate detection is crucial for clinical diagnostics, food safety monitoring, and environmental surveillance. This review provides a comprehensive overview of molecular detection strategies for pathogenic Vibrio, integrating three key aspects: (i) selection of molecular targets, including species-specific, virulence-associated, and regulatory genes; (ii) analytical performance and limitations of major detection platforms, such as polymerase chain reaction (PCR)-based methods, isothermal amplification, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems, high-throughput sequencing (HTS), and biosensor-based approaches; and (iii) applicability across clinical, food, and environmental scenarios. We highlight the influence of genetic variability, sample matrix complexity, and viability on detection accuracy and discuss the advantages and limitations of single- and multi-target approaches. Emerging trends toward next-generation diagnostics, including integrated microfluidic platforms, CRISPR-Cas-based detection, multiplex assays, and artificial intelligence(AI)-assisted data analysis, are also examined. By identifying gaps and challenges in the routine deployment of these technologies, this review offers practical insights for the development of rapid, field-deployable diagnostic strategies for Vibrio pathogens.},
}

Humans
*Vibrio Infections/diagnosis/microbiology
*Vibrio/genetics/pathogenicity/isolation & purification
*Molecular Diagnostic Techniques/methods
CRISPR-Cas Systems
Biosensing Techniques/methods
Vibrio parahaemolyticus/genetics/pathogenicity
Nucleic Acid Amplification Techniques/methods
Vibrio cholerae/genetics/pathogenicity
High-Throughput Nucleotide Sequencing/methods

@article {pmid42883816,
year = {2026},
author = {Yu, X and Zhu, Y and Wang, X and Sun, Y and Gao, L and Zhou, S and Xin, M and Zhang, P},
title = {Molecular Mechanisms Underlying Cucumber Resistance to Cucumber Green Mottle Mosaic Virus: From Host-Virus Interactions to Precision Breeding Strategies.},
journal = {International journal of molecular sciences},
volume = {27},
number = {19},
pages = {},
pmid = {42883816},
issn = {1422-0067},
support = {2025C01118//Science and Technology Department of Zhejiang Province/ ; No. 2025SNJF031//Department of Agriculture and Rural Affairs of Zhejiang Province/ ; },
mesh = {*Cucumis sativus/virology/genetics ; *Tobamovirus/pathogenicity/physiology ; *Disease Resistance/genetics ; *Plant Diseases/virology/genetics ; *Host-Pathogen Interactions/genetics ; *Plant Breeding/methods ; },
abstract = {Cucumber (Cucumis sativus L.) is an economically important vegetable crop cultivated worldwide, but its sustainable production is increasingly threatened by Cucumber green mottle mosaic virus (CGMMV). CGMMV is a positive-sense single-stranded RNA virus belonging to the genus Tobamovirus and represents a major constraint on cucurbit production because of its efficient seed transmission, mechanical spread, environmental persistence, and broad host range. Infection can cause leaf mosaic and mottling, growth suppression, and severe fruit symptoms, including internal discoloration, water-soaked tissues, and flesh deterioration, resulting in substantial losses in yield and commercial quality. Recent advances in cucumber genomics, resistance-germplasm screening, host-virus interaction studies, and molecular breeding have provided new opportunities for improving CGMMV resistance. However, progress remains constrained by the scarcity of well-characterized resistance donors, the limited functional validation of cucumber-specific resistance genes, isolate-dependent resistance responses, and the relatively low efficiency of cucumber transformation and genome editing. This review summarizes current knowledge of CGMMV infection processes, host susceptibility factors, antiviral defense pathways, resistance resources, and resistance-associated loci in cucumber and related cucurbits. Evidence is interpreted according to host species and the level of experimental validation. It further evaluates the potential of marker-assisted selection, genomic selection, genome editing, and RNA-based engineering and outlines an integrated framework for translating molecular findings into durable CGMMV-resistant cucumber cultivars.},
}

*Cucumis sativus/virology/genetics
*Tobamovirus/pathogenicity/physiology
*Disease Resistance/genetics
*Plant Diseases/virology/genetics
*Host-Pathogen Interactions/genetics
*Plant Breeding/methods

@article {pmid42883863,
year = {2026},
author = {Volodina, O and Demchenko, A and Kondrateva, E and Anuchina, A and Ryzhkova, O and Kovalskaia, V and Tabakov, V and Amelina, E and Lavrov, A and Kutsev, S and Smirnikhina, S},
title = {Comparison of Prime Editing and CRISPR-Cas Mediated HDR in Correcting CFTR-F508del Mutation in Patient iPSC-Derived Airway Basal Cells.},
journal = {International journal of molecular sciences},
volume = {27},
number = {19},
pages = {},
pmid = {42883863},
issn = {1422-0067},
support = {none//The Ministry of Education and Science of the Russian Federation/ ; 220724//LLC "MK Development"/ ; },
mesh = {Humans ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; *CRISPR-Cas Systems/genetics ; *Cystic Fibrosis/genetics/therapy ; *Gene Editing/methods ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Mutation ; *Recombinational DNA Repair ; Quinolones/pharmacology ; Genetic Therapy/methods ; Benzodioxoles/pharmacology ; },
abstract = {Gene editing therapy represents a promising strategy to permanently cure cystic fibrosis (CF). This paper presents the results of correcting the most common CF mutation, F508del, in the CFTR gene in airway basal cells (BCs) obtained from patients, using both classical CRISPR-Cas9-mediated homology-directed repair (HDR) and prime editing techniques. Our findings demonstrate that both methods achieve mutation correction with efficiencies reaching up to 18.9% of alleles in successfully transfected cells. However, the CRISPR-Cas9 approach results in insertions and deletions (indels) in approximately 15% of alleles at the editing site, whereas prime editing induced only minimal indel events. Functional analysis evaluating the effectiveness of gene editing revealed that prime editing of the F508del mutation in the CFTR gene-using the pegRNA4 combined with the PE2-NG variant-restores CFTR channel conductance, comparable to triple therapy with CFTR modulators (ivacaftor/tezacaftor/elexacaftor). While both prime editing and CRISPR-HDR resulted in successful correction of the CFTR-F508del mutation in patient BCs, prime editing exhibited a low frequency of unintended modifications both within and outside the editing locus, making this approach a promising candidate for the development of gene therapy strategies for CF.},
}

Humans
*Cystic Fibrosis Transmembrane Conductance Regulator/genetics
*CRISPR-Cas Systems/genetics
*Cystic Fibrosis/genetics/therapy
*Gene Editing/methods
*Induced Pluripotent Stem Cells/metabolism/cytology
*Mutation
*Recombinational DNA Repair
Quinolones/pharmacology
Genetic Therapy/methods
Benzodioxoles/pharmacology

@article {pmid42883970,
year = {2026},
author = {Karakkat, BB and Yasuhara-Bell, J},
title = {Development and Validation of a Real-Time One-Pot RPA-CRISPR/Cas-Based Assay for Detection of 'Candidatus Liberibacter Asiaticus,' the Agent Associated with Citrus Greening Disease or Huanglongbing (HLB).},
journal = {International journal of molecular sciences},
volume = {27},
number = {19},
pages = {},
pmid = {42883970},
issn = {1422-0067},
support = {//Citrus Health Response Program/ ; },
mesh = {*Citrus/microbiology ; *Plant Diseases/microbiology ; *CRISPR-Cas Systems ; *Rhizobiaceae/genetics/isolation & purification ; *Liberibacter/genetics/isolation & purification ; Sensitivity and Specificity ; },
abstract = {Huanglongbing (aka: citrus greening) associated with 'Candidatus Liberibacter asiaticus' (CLas) is one of the most impactful citrus diseases throughout the world. The aim of this project was to support the sensitive and rapid detection of CLas to prevent the continued spread and destruction of US citrus crops by this psyllid vector-borne disease. Two multicopy targets specific to CLas were selected for assay development: the five-copy nrdB (ribonucleotide reductase-RNR) gene and a four-copy locus 4cp (4CP). Published RNR RPA primers were used to develop a one-pot RPA-CRISPR assay, and RPA primers for 4CP were designed. crRNAs for both RNR- and 4CP-RPA-CRISPR/Cas assays were designed, evaluated through preliminary testing, and the best candidate primers and crRNA combinations were selected. The 4CP assay was selected and subjected to a battery of validation tests and determined fit-for-purpose. A field extraction protocol was also developed using commercial reagents, syringes and filters to bring kit sensitivity to the field. When combined with the assay, the complete workflow was able to detect CLas in infected citrus, down to a single infected leaf per subsample; the field workflow showed similar sensitivity to the laboratory-based workflow. While the extraction and assay proved useful, particularly for symptomatic leaves, its performance with asymptomatic leaves for early detection was not evaluated. The complexity of the assay in its current form hinders widespread practical applicability. In the future, revalidation of lyophilized reagents in a convenient kit can be performed for practical deployment; preliminary testing has shown success.},
}

*Citrus/microbiology
*Plant Diseases/microbiology
*CRISPR-Cas Systems
*Rhizobiaceae/genetics/isolation & purification
*Liberibacter/genetics/isolation & purification
Sensitivity and Specificity

@article {pmid42884858,
year = {2026},
author = {Chae, J and Han, SJ and Kim, HJ and Kim, JH and Nam, HJ and Chung, YS and Karthik, S and Heo, JB},
title = {Managing Trait Trade-Offs in Tomato Breeding Through Genome Editing.},
journal = {Plants (Basel, Switzerland)},
volume = {15},
number = {19},
pages = {},
pmid = {42884858},
issn = {2223-7747},
abstract = {Tomato improvement often involves trade-offs between fruit yield and quality, sweetness and fruit size, disease resistance and growth, reproductive performance and stress adaptation, and shelf life and flavour. Although CRISPR-Cas-mediated genome editing enables targeted modification of tomato genes, DNA-level precision does not guarantee an agronomically favourable phenotype because pleiotropy, functional redundancy, allele dosage, linkage, epistasis, genetic background, and environmental conditions can alter editing outcomes. This review evaluates genome editing as a strategy to manage, rather than merely document, these trade-offs. We examine how knockout, multiplex, cis-regulatory, base, prime, and direct allele-repair approaches can match the causal architecture of a breeding constraint. We critically assess representative studies involving plant architecture, reproductive development, fruit size, sugar accumulation, ripening, nutritional composition, disease resistance, and heat resilience, with an emphasis on heritability, molecular identity, multi-trait effects, and agronomic penalties. We further consider domestication-associated variation, structural variation, transformation and regeneration limitations, genotype-by-environment interactions, breeding deployment, and regulatory translation. We propose a trade-off-aware framework that links causal-allele identification, mechanism-matched editing, quantitative allele design, multidimensional phenotyping, and validation across generations, genetic backgrounds, and production-relevant environments. This framework can guide the development of balanced, breeding-ready tomato cultivars rather than lines optimized for a single trait.},
}

@article {pmid42884921,
year = {2026},
author = {Zhao, J and Qian, X and Zhong, X and Xun, H and Guo, D},
title = {Advances and Applications of Genome Editing in Precision Crop Breeding.},
journal = {Plants (Basel, Switzerland)},
volume = {15},
number = {19},
pages = {},
pmid = {42884921},
issn = {2223-7747},
support = {20240303012NC//Jilin Province Science and Technology Department/ ; 2024C002//Jilin Province Development and Reform Commission/ ; },
abstract = {Genome editing is becoming an important tool for crop improvement by enabling precise modification of genetic variation. CRISPR-Cas technologies have expanded beyond nuclease-induced mutations to include base and prime editing, transcriptional and epigenetic regulation, multiplex editing, organelle editing and targeted DNA insertion. These advances support the development of agronomically valuable alleles and extend crop improvement beyond conventional selection, recombination and random mutagenesis. Applications now include plant architecture, yield-related traits, resistance to biotic and abiotic stresses, seed composition and nutritional quality, and extend to de novo domestication, haploid induction and hybrid breeding. Current research is increasingly moving from single-gene modification toward the engineering of promoters, cis-regulatory elements, quantitative alleles and multiple-gene editing, particularly in polyploid crops. Wider breeding applications will depend on efficient delivery and regeneration, recovery of transgene-free and heritable edited germplasm, reliable targeted insertion and validation across relevant environments. Integration with pangenomics, multi-omics, phenomics, artificial intelligence and predictive breeding may further improve target identification, allele design and prediction of editing outcomes, thereby supporting the development of more precise and efficient crop-breeding strategies.},
}

@article {pmid42706427,
year = {2026},
author = {Wang, FZ and Bao, Y and Qiu, J and Chen, MX and Jiang, H and Li, Z and Xiong, X and Jiang, X and Kou, Y and Li, JF},
title = {Cell-based CRISPRa screen identifies a papillae-specific receptor kinase mediating broad-spectrum fungal resistance in rice.},
journal = {Nature plants},
volume = {12},
number = {10},
pages = {1913-1922},
pmid = {42706427},
issn = {2055-0278},
support = {32125004//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32370294//National Natural Science Foundation of China (National Science Foundation of China)/ ; LR24C140001//Natural Science Foundation of Zhejiang Province (Zhejiang Provincial Natural Science Foundation)/ ; },
mesh = {*Oryza/genetics/microbiology/enzymology/immunology ; *Plant Diseases/microbiology/immunology/genetics ; *Disease Resistance/genetics ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; *Protein Kinases/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Gain-of-function screens enable the discovery of gene functions and candidate targets for molecular breeding. Here we developed a protoplast-based dCas9-TV-mediated CRISPR activation screen in rice and identified OsTV1, an epidermal papillae-specific receptor kinase, as a positive regulator of fungal resistance. OsTV1 overexpression enhanced silicon deposition, leaf rigidity and basal defence gene expression, conferring broad-spectrum fungal resistance without compromising growth. This study establishes a high-throughput gain-of-function screen adaptable for investigating diverse cellular processes in plants and provides insights into the roles of silicon in rice defence.},
}

*Oryza/genetics/microbiology/enzymology/immunology
*Plant Diseases/microbiology/immunology/genetics
*Disease Resistance/genetics
*Plant Proteins/genetics/metabolism
Plants, Genetically Modified
*Protein Kinases/genetics/metabolism
CRISPR-Cas Systems

@article {pmid42734356,
year = {2026},
author = {Chen, Y and He, Z and Pan, Y and Luo, Y and Huang, B and Zhang, Y and Li, G and Guo, C},
title = {CRISPR and yeast two-hybrid screens identify CD81 as a proviral host factor for PRRSV.},
journal = {Journal of virology},
volume = {100},
number = {10},
pages = {e0103326},
doi = {10.1128/jvi.01033-26},
pmid = {42734356},
issn = {1098-5514},
support = {2023YFD1801500//National Key Research and Development Program of China/ ; 2024A1515012991//Basic and Applied Basic Research Foundation of Guangdong Province/ ; 2025D04J0072//Science and Technology Planning Project of Guangdong Province/ ; NG2022003//Laboratory of Lingnan Modern Agricultural Project/ ; },
mesh = {Animals ; *Porcine respiratory and reproductive syndrome virus/physiology/genetics/pathogenicity ; *Tetraspanin 28/metabolism/genetics ; Swine ; Two-Hybrid System Techniques ; Virus Internalization ; *Host-Pathogen Interactions ; Viral Envelope Proteins/metabolism/genetics ; *Porcine Reproductive and Respiratory Syndrome/virology/metabolism/genetics ; Macrophages, Alveolar/virology/metabolism ; CRISPR-Cas Systems ; Cell Line ; Virus Replication ; },
abstract = {Porcine reproductive and respiratory syndrome virus (PRRSV) remains a major economic burden on the global swine industry. Identifying host factors that mediate viral invasion is critical for developing antiviral strategies. Here, using a CRISPR screen targeting genes encoding cell surface CD proteins combined with yeast two-hybrid screening, we identified the tetraspanin CD81 as a novel host factor that promoted PRRSV infection by directly interacting with viral glycoprotein 5 (GP5). CD81 knockdown reduced PRRSV infection, whereas CD81 overexpression enhanced it. Consistently, antibody-mediated blockade of CD81 restricted infection in both Marc-145 cells and primary porcine alveolar macrophages. Mechanistic analysis showed that CD81 mainly facilitated viral internalization, and its C-terminal transmembrane region was required for GP5 binding and efficient infection. During infection, CD81 underwent autophagic degradation mediated by the selective autophagy receptor NDP52, suggesting a host restriction response that reduced CD81 availability. PRRSV countered this process by promoting NDP52 degradation, thereby stabilizing CD81 and favoring infection. These findings identify CD81 as both a key factor in PRRSV pathogenesis and a potential target for antiviral intervention.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) remains one of the most economically important swine pathogens worldwide, yet effective antiviral strategies are lacking because of its extensive genetic diversity and immune evasion. Here, we identified the tetraspanin CD81 as a previously unrecognized host factor that promoted efficient PRRSV infection. CD81 directly interacted with the viral glycoprotein GP5 to facilitate viral internalization, whereas disruption of CD81 by genetic depletion or antibody blockade markedly restricted infection. We further demonstrated that CD81 abundance was controlled by NDP52-mediated selective autophagy as an intrinsic antiviral mechanism, which PRRSV overcame by degrading NDP52 to stabilize CD81 and enhance viral replication. These findings reveal a previously unrecognized virus-host interaction that governs PRRSV entry and identify CD81 as a promising host-directed therapeutic target.},
}

Animals
*Porcine respiratory and reproductive syndrome virus/physiology/genetics/pathogenicity
*Tetraspanin 28/metabolism/genetics
Swine
Two-Hybrid System Techniques
Virus Internalization
*Host-Pathogen Interactions
Viral Envelope Proteins/metabolism/genetics
*Porcine Reproductive and Respiratory Syndrome/virology/metabolism/genetics
Macrophages, Alveolar/virology/metabolism
CRISPR-Cas Systems
Cell Line
Virus Replication