@article {pmid41606292,
year = {2026},
author = {Qin, W and Lin, SJ and Zhang, Y and Huang, K and Petree, C and Varshney, P and Varshney, GK},
title = {High-efficiency TadA cytosine base editors for precise modelling of human disease variants.},
journal = {Nature biomedical engineering},
volume = {10},
number = {11},
pages = {2363-2378},
pmid = {41606292},
issn = {2157-846X},
support = {R24 OD034438/OD/NIH HHS/United States ; R24OD034438P//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Animals ; Zebrafish/genetics ; Humans ; *Cytosine/metabolism ; CRISPR-Cas Systems/genetics ; Mutation, Missense/genetics ; Hearing Loss/genetics ; Genetic Variation ; },
abstract = {Many missense mutations identified in genetic testing are variants of uncertain significance (VUS), not yet classified as either benign or pathogenic. Systematic determination of their functional relevance is a pressing clinical need. CRISPR-mediated base editing can precisely introduce precise variants into model organisms for functional testing, but current editors face efficiency and targeting constraints. We developed TCBE-Umax, a family of TadA-derived cytosine base editors optimized for zebrafish. Engineering the TadA deaminase domain improved editing efficiency and reduced sequence-context bias, expanded PAM compatibility, and minimized bystander edits and indel formation. Our editors achieved efficient biallelic editing, enabling rapid functional assessment of genetic variants in the F0 (founding) zebrafish. As a proof of concept, we evaluated 15 VUS linked to hereditary hearing loss, determining pathogenicity through phenotypic analysis. With high efficiency and versatility, TCBE-Umax base editors provide a powerful platform for studying genetic variants and disease in vivo.},
}

Animals
Zebrafish/genetics
Humans
*Cytosine/metabolism
CRISPR-Cas Systems/genetics
Mutation, Missense/genetics
Hearing Loss/genetics
Genetic Variation

@article {pmid42749924,
year = {2026},
author = {Yu, T and Ji, M and Yu, D and Guan, Z and Zhu, R and Jiang, Y and Yang, Z and Qiu, L and Zhang, Z and Mu, J and Mao, F and Xiang, K and Bai, L and Li, K},
title = {Structure-guided discovery and engineering of miniature CRISPR-Cas12m for epigenome editing.},
journal = {Nature structural & molecular biology},
volume = {33},
number = {11},
pages = {1624-1639},
pmid = {42749924},
issn = {1545-9985},
mesh = {*Epigenome Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; Humans ; Mice ; Dependovirus/genetics ; Cryoelectron Microscopy ; Protein Engineering/methods ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; DNA/metabolism ; Genetic Vectors ; HEK293 Cells ; },
abstract = {CRISPR-based epigenome editing represents a programmable strategy to precisely modulate gene expression, holding promise for therapeutic applications. However, the large size of dCas proteins substantially impedes delivery using adeno-associated virus (AAV) vectors. Here, through iterative bioinformatics analysis, structure-guided predictions and functional assays, we identified and characterized a miniature subtype V-M CRISPR-Cas12m from Pelomicrobium methylotrophicum. PmCas12m exhibited flexible 5'-YTN-3' PAM-dependent recognition and robust double-stranded DNA-binding properties while lacking DNA cleavage activity, thus rendering it a valuable tool for epigenome editing. Cryo-electron microscopy structures of PmCas12m unveiled its molecular mechanism of target DNA binding. Guided by these structural insights, we used deep mutational scanning and protein engineering to develop xCas12m, a hypercompact variant with highly potent and specific epigenome-editing capabilities in human cells. We further constructed the xCas12m-CRISPRoff platform in a single AAV vector, which achieved durable epigenetic silencing and effective inhibition of hepatitis B virus infection in a mouse model. Collectively, these findings establish xCas12m as a versatile epigenome-editing platform with notable potential for treating diseases, paving the way for clinical translation of epigenetic therapies.},
}

*Epigenome Editing/methods
*CRISPR-Cas Systems/genetics
Animals
Humans
Mice
Dependovirus/genetics
Cryoelectron Microscopy
Protein Engineering/methods
*CRISPR-Associated Proteins/genetics/metabolism/chemistry
DNA/metabolism
Genetic Vectors
HEK293 Cells

@article {pmid42782284,
year = {2026},
author = {Freed, G and Quijada-Álamo, M and Lee, L and Wang, K and Poddar, N and Autar, S and Carcamo, S and Fiore, P and Kim, EK and Martinez, IG and Zhang, M and Saniei, S and Chao, C and Mekerishvili, L and Diaz, Z and Ma, S and Hasson, D and Wagenblast, E},
title = {A CRISPR-based humanized model reveals a cooperative role of STAG2 loss in familial GATA2-deficient MDS progression.},
journal = {Blood advances},
volume = {10},
number = {22},
pages = {7690-7701},
doi = {10.1182/bloodadvances.2026019911},
pmid = {42782284},
issn = {2473-9537},
mesh = {Humans ; Animals ; Mice ; *Myelodysplastic Syndromes/genetics/pathology/etiology/metabolism ; *GATA2 Deficiency/genetics ; Disease Models, Animal ; *GATA2 Transcription Factor/deficiency/genetics ; Disease Progression ; Hematopoietic Stem Cells/metabolism ; *CRISPR-Cas Systems ; },
abstract = {Myelodysplastic syndrome (MDS) is a heterogeneous myeloid malignant neoplasm driven by hematopoietic stem cell dysfunction, leading to ineffective hematopoiesis and cytopenias. Familial GATA2 deficiency is the most common cause of MDS in adolescents, with progression often accelerated by co-occurring mutations, notably STAG2 loss of function. Using CRISPR/Cas9-mediated genome engineering in primary human fetal liver-derived hematopoietic stem cells and xenotransplantation in mice, we modeled GATA2-deficient MDS with acquired STAG2 loss to investigate disease initiation and progression. Although GATA2 deficiency alone had minimal short-term impact in our model, combined GATA2 deficiency and STAG2 loss increased hematopoietic stem cell maintenance and self-renewal, induced a myeloid-lineage bias, and expanded primitive progenitors. Single-cell transcriptional profiling revealed upregulation of stemness-associated genes and inflammatory pathways. This humanized model faithfully recapitulates high-risk GATA2-deficient MDS, providing mechanistic insights into how cooperative mutations drive stem cell expansion, inflammatory signaling, and myeloid skewing.},
}

Humans
Animals
Mice
*Myelodysplastic Syndromes/genetics/pathology/etiology/metabolism
*GATA2 Deficiency/genetics
Disease Models, Animal
*GATA2 Transcription Factor/deficiency/genetics
Disease Progression
Hematopoietic Stem Cells/metabolism
*CRISPR-Cas Systems

@article {pmid42843117,
year = {2027},
author = {Nagahara, M and Namula, Z and Hirata, M and Nguyen, ST and Nakayama, Y and Nakai, A and Kashiwakura, Y and Tsukida, K and Ohmori, T and Muramatsu, K and Otoi, T and Tanihara, F},
title = {Laparoscopic transfer of in vitro fertilized gene-edited embryos in pigs.},
journal = {Theriogenology},
volume = {268},
number = {},
pages = {118244},
doi = {10.1016/j.theriogenology.2026.118244},
pmid = {42843117},
issn = {1879-3231},
mesh = {Animals ; *Embryo Transfer/veterinary/methods ; Female ; Swine/genetics/embryology ; *Laparoscopy/veterinary/methods ; Pregnancy ; *Fertilization in Vitro/veterinary/methods ; *Gene Editing/veterinary ; CRISPR-Cas Systems ; *Animals, Genetically Modified ; },
abstract = {Laparoscopic embryo transfer in pigs has been explored as a minimally invasive alternative to conventional laparotomic methods, which often lead to surgical risks such as adhesions, prolonged recovery, and postoperative complications. This study is the first to report the generation of genetically modified pigs via embryo transfer using a laparoscopic approach. Gene-edited embryos were prepared by delivering the CRISPR/Cas9 system via electroporation to target the Coagulation Factor VIII, WDR45, and IL2RG genes and then transferred into the oviducts of five recipient gilts using a laparoscopic technique. Embryos putatively carrying modifications in the Coagulation Factor VIII and WDR45 genes were each transferred into a single recipient gilt, and both recipients became pregnant. In contrast, embryos putatively carrying a modification in the IL2RG gene were transferred into three recipient gilts, of which two became pregnant. In total, four recipient gilts were carried to full term, resulting in the birth of 9 live piglets. Genotyping confirmed that all of the offspring carried the genetic modifications, demonstrating this method may have potential for producing genetically modified pigs. The successful production of live gene-edited offspring demonstrates the feasibility of laparoscopic embryo transfer for genetically modified embryos. Although further research, including a large number of recipient pigs, as well as direct comparisons with conventional laparotomy, is needed to evaluate embryo survival, implantation efficiency, and animal welfare, laparoscopic embryo transfer represents a feasible approach for generating genetically modified pigs.},
}

Animals
*Embryo Transfer/veterinary/methods
Female
Swine/genetics/embryology
*Laparoscopy/veterinary/methods
Pregnancy
*Fertilization in Vitro/veterinary/methods
*Gene Editing/veterinary
CRISPR-Cas Systems
*Animals, Genetically Modified

@article {pmid42951937,
year = {2026},
author = {Komatsu, D and Hiraizumi, M and Fell, CW and Fujimatsu, K and Nagahata, N and Yamada, S and Sapkota, S and Ramani, H and Arya, B and Shibata, M and Yamashita, K and Gootenberg, JS and Abudayyeh, OO and Nishimasu, H},
title = {Structural mechanism of programmable gene integration by the R2 retrotransposon coupled with CRISPR-Cas9.},
journal = {Nature structural & molecular biology},
volume = {33},
number = {11},
pages = {1640-1653},
pmid = {42951937},
issn = {1545-9985},
mesh = {Cryoelectron Microscopy ; *Retroelements/genetics ; *CRISPR-Cas Systems ; Models, Molecular ; Humans ; Animals ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry ; DNA/chemistry ; Reverse Transcription ; },
abstract = {R2 retroelements are non-long-terminal-repeat retrotransposons that primarily insert into 28S ribosomal DNA loci across diverse eukaryotes. R2 elements home to their target sites through target-primed reverse transcription (TPRT), in which the R2 protein recognizes and nicks the target DNA to prime reverse transcription using an R2-encoding RNA as the template. The avian R2 system from Taeniopygia guttata (R2Tg) can insert a payload sequence within an RNA template into 28S ribosomal DNA loci in human cells. Here, we present cryo-electron microscopy structures of the R2Tg-template RNA-target DNA complex in the TPRT initiation state, both alone and bound to a Cas9-guide RNA complex. These structures provide mechanistic insights into R2Tg-mediated TPRT in its natural context and in the programmable gene integration system STITCHR (site-specific target-primed insertion through targeted CRISPR homing of retroelements). Furthermore, we combine these structural insights with computational protein design methods to engineer an enhanced STITCHR system.},
}

Cryoelectron Microscopy
*Retroelements/genetics
*CRISPR-Cas Systems
Models, Molecular
Humans
Animals
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry
DNA/chemistry
Reverse Transcription

@article {pmid43010433,
year = {2026},
author = {Capelli, L and Hao, Y and Shi, Q and Li, Q and Yan, Q and Li, J and Zhu, Y and Fan, C and Bertucci, A and Ge, Z},
title = {Programming CRISPR Compartmentalization on DNA Origami for Multiplexed Biosensing.},
journal = {Analytical chemistry},
volume = {98},
number = {44},
pages = {31052-31062},
doi = {10.1021/acs.analchem.6c04535},
pmid = {43010433},
issn = {1520-6882},
support = {22304114//National Natural Science Foundation of China (NSFC)/ ; 22374099//National Natural Science Foundation of China (NSFC)/ ; 22574100//National Natural Science Foundation of China (NSFC)/ ; U24A20497//National Natural Science Foundation of China (NSFC)/ ; //National Research Foundation Singapore/ ; //Shanghai Municipal Science and Technology Major Project/ ; //New Cornerstone Science Foundation/ ; 2023YFB3208200//National Basic Research Program of China (973 Program)/ ; 31108//Associazione Italiana per la Ricerca sul Cancro/ ; 31710//Associazione Italiana per la Ricerca sul Cancro/ ; 21TQ1400222//Shanghai Jiao Tong University/ ; JYB2025XDXM602//Ministry of Education of the People's Republic of China/ ; //National Center for Translational Medicine (Shanghai)/ ; },
mesh = {*DNA Nanostructures/chemistry ; *Biosensing Techniques/methods ; *DNA/chemistry/genetics ; Molecular Dynamics Simulation ; *CRISPR-Cas Systems ; CRISPR-Associated Proteins/metabolism ; },
abstract = {CRISPR-Cas12a has emerged as a transformative tool for nucleic acid detection and molecular diagnostics. However, achieving one-pot, orthogonal, multiplexed sensing remains challenging due to the nonspecific trans-cleavage activity of Cas12a. Here, we present a modular DNA origami-based platform that spatially confines and independently controls Cas12a activity, enabling simultaneous detection of multiple DNA targets within a single reaction. This spatial programming approach enables orthogonal one-pot detection of three DNA targets (EGFR L858R/Del19 mutations and HPV16) with minimal cross-talk, demonstrating 60% cleavage efficiency within 120 min at 100 nM. Through integrated molecular dynamics simulations and experimental validation, we identify an optimal ∼6 nm spatial window that maximizes cleavage efficiency, while the platform maintains robust performance in complex biological environments with a 10 nM detection limit in 20% serum. This compartmentalization strategy establishes a framework for spatially programming multi-enzyme systems on DNA nanostructures for synthetic biological circuits.},
}

*DNA Nanostructures/chemistry
*Biosensing Techniques/methods
*DNA/chemistry/genetics
Molecular Dynamics Simulation
*CRISPR-Cas Systems
CRISPR-Associated Proteins/metabolism

@article {pmid43010442,
year = {2026},
author = {Li, H and Zhou, Y and Yan, Y and Ye, J and Dong, X and Chen, M and Meng, T and Jiao, D and Wang, D and Zhu, L and Yang, X},
title = {Decoupled Signal Transduction via a Non-Copper Laccase Mimic for CRISPR-Based Electrochemical Detection of Hepatocellular Carcinoma.},
journal = {Analytical chemistry},
volume = {98},
number = {44},
pages = {31469-31484},
doi = {10.1021/acs.analchem.6c05197},
pmid = {43010442},
issn = {1520-6882},
support = {22404119//National Natural Science Foundation of China/ ; 32501984//National Natural Science Foundation of China/ ; 2024NSFSC1129//Sichuan Science and Technology Program/ ; 2025ZB395//Jiangsu Funding Program for Excellent Postdoctoral Talent/ ; },
mesh = {*Laccase/chemistry/metabolism ; Humans ; *Electrochemical Techniques/methods ; *Liver Neoplasms/diagnosis ; *Biosensing Techniques/methods ; *Carcinoma, Hepatocellular/diagnosis ; *CRISPR-Cas Systems/genetics ; alpha-Fetoproteins/analysis ; MicroRNAs/blood/analysis ; Signal Transduction ; Manganese/chemistry ; Tryptophan/chemistry ; Biomarkers, Tumor/blood/analysis ; Copper ; *Biomimetic Materials/chemistry ; },
abstract = {Laccase-mimicking nanozymes have been overwhelmingly copper-dependent, and largely confined to environmental contexts, with clinical translation remaining unrealized. Here, we depart from this paradigm by designing a non-copper nanozyme, Mn-Trp NR, assembled through manganese-tryptophan coordination. Spectroscopic and theoretical analyses show that tryptophan coordination finely modulates the Mn electronic structure, strengthening O2 adsorption and lowering the reaction barrier, thereby conferring laccase-like activity, substrate affinity, and stability superior to the native enzyme. To overcome broad-substrate promiscuity that precludes specific biomarker recognition, we decoupled signal transduction from target identification: Mn-Trp NR serves as a universal amplifier, whereas CRISPR/Cas12a confers high specificity. Accordingly, a cascaded electrochemical biosensor was built on a DNA triangular prism integrating catalytic hairpin assembly, hybridization chain reaction, and CRISPR/Cas12a activation. The platform detects hepatocellular carcinoma (HCC) biomarkers microRNA-122 (miRNA-122) and alpha-fetoprotein (AFP) with limits of 11.98 aM and 10.31 fg/mL, respectively, in a streamlined 160 min, two-step procedure. In clinical sera, the biosensor perfectly distinguishes HCC patients from healthy donors (AUC = 1.00) and agrees strongly with benchmark methods (RT-qPCR for miRNA-122, ELISA for AFP, R2 > 0.97). By expanding laccase-mimic synthesis beyond copper via ligand-field engineering and establishing a decoupled architecture that reconciles broad-spectrum catalysis with high-specificity recognition, this work provides a generalizable strategy for deploying nanozymes in precision diagnostics.},
}

*Laccase/chemistry/metabolism
Humans
*Electrochemical Techniques/methods
*Liver Neoplasms/diagnosis
*Biosensing Techniques/methods
*Carcinoma, Hepatocellular/diagnosis
*CRISPR-Cas Systems/genetics
alpha-Fetoproteins/analysis
MicroRNAs/blood/analysis
Signal Transduction
Manganese/chemistry
Tryptophan/chemistry
Biomarkers, Tumor/blood/analysis
Copper
*Biomimetic Materials/chemistry

@article {pmid43010510,
year = {2026},
author = {Zhou, S and Xiang, T and Cha, Z and Song, J and Xiang, Y and Li, X and Huang, C and Mei, Q and Huang, X and Zhang, C and Wu, J},
title = {A Two-Step Strategy Based on Fpg Recognition and RCA-CRISPR/Cas12a for Ultrasensitive Detection of Gene Mutation.},
journal = {Analytical chemistry},
volume = {98},
number = {44},
pages = {30873-30882},
doi = {10.1021/acs.analchem.6c04145},
pmid = {43010510},
issn = {1520-6882},
support = {2026NSFSC2079//Sichuan Science and Technology Program/ ; 2025ZYD0025//Sichuan Provincial Central Government-Guided Local Scientific Research Project/ ; 2025JYJ034, 2024LZXNYDJ008//Luzhou Science and Technology Program/ ; 2025JBH004, 2025LZXNYDJC17//The Science and Technology Strategic Cooperation Programs of Luzhou Municipal People's Government and Southwest Medical University/ ; 2026NSFSC1552//Natural Science Foundation of Sichuan Province/ ; 82374073//National Natural Science Foundation of China (NSFC)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Mutation ; *Tumor Suppressor Protein p53/genetics ; *DNA-Formamidopyrimidine Glycosylase/metabolism ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Accurate detection of gene mutations is crucial for acute myeloid leukaemia (AML) diagnosis and treatment. Although CRISPR/Cas systems enable sensitive mutation detection, their adaptability is constrained by dependence on restriction endonucleases. Herein, we propose a two-step strategy, termed Fpg-RCA-CRISPR/Cas12a for ultrasensitive detection of the p53 R175H, which integrates formamidopyrimidine DNA glycosylase (Fpg)-mediated recognition with rolling circle amplification (RCA)-coupled CRISPR/Cas12a technology. Validation confirmed that Fpg exhibits minimal activity when adenine (A) is opposite the damaged site. Based on this finding, we designed a padlock probe with a site-specific 8-oxoguanine (8-oxoG) modification. This modification enables selective recognition of G > A mutations. For the target TP53 c.524G > A (p53 R175H), the G > A substitution opposite the 8-oxoG site blocks Fpg cleavage, facilitating probe circularization and subsequent RCA. The resulting RCA products then activate the trans-cleavage activity of Cas12a, enabling cascade signal amplification and generating a strong fluorescence output. For wild-type p53, Fpg-mediated cleavage prevents circularization and amplification, yielding weak fluorescence. Taking advantage of the high specificity of the Fpg enzyme and the cascade amplification effect of the RCA-CRISPR/Cas12a system, this method achieves a limit of detection (LOD) of 0.29 fM and covers a linear range from 0.8 fM to 800 pM, along with high reproducibility, excellent selectivity, and negligible background interference. Notably, the assay can detect mutation frequencies as low as 0.1% in excess wild-type p53. Crucially, its clinical feasibility was confirmed by analyzing human serum samples, yielding results consistent with standard assays and thus demonstrating its practical reliability and translational potential.},
}

*CRISPR-Cas Systems/genetics
Humans
*Mutation
*Tumor Suppressor Protein p53/genetics
*DNA-Formamidopyrimidine Glycosylase/metabolism
*Nucleic Acid Amplification Techniques/methods
Limit of Detection
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid43010518,
year = {2026},
author = {Hou, XY and Li, MY and Sun, WB and Wu, SL and Tan, Y and Yang, QF and Li, KD and Li, QN and Zhu, LN and Kong, DM},
title = {Modular Cell-Surface-to-Solution CRISPR-Cas12a Platform for Wash-Free Quantification of Cell-Surface Proteins on Living Cells.},
journal = {Analytical chemistry},
volume = {98},
number = {44},
pages = {31528-31541},
doi = {10.1021/acs.analchem.6c05289},
pmid = {43010518},
issn = {1520-6882},
support = {22293030//National Natural Science Foundation of China/ ; 22474063//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Aptamers, Nucleotide/chemistry ; *Epithelial Cell Adhesion Molecule/analysis ; *Membrane Proteins/analysis ; },
abstract = {Cell-surface proteins are important biomarkers for cell phenotyping, disease diagnosis, therapeutic evaluation, and drug screening. However, their quantitative analysis on intact living cells remains challenging because conventional methods often require cell lysis, fixation, repeated washing, extensive sample processing, or bulky instrumentation. Herein, we developed a modular and reconfigurable cell-surface-to-solution CRISPR-Cas12a platform for wash-free quantitative analysis of cell-surface proteins on intact living cells. Aptamer recognition of extracellularly accessible targets deblocks a DNA activator, thereby activating Cas12a trans-cleavage in solution and converting localized cell-surface recognition into amplified fluorescence while maintaining a low background in the absence of targets. Using EpCAM as a model target, we engineered a split dual-aptamer architecture that enabled cooperative target recognition, efficient activator deblocking, and low-background signal amplification. The platform achieved sensitive EpCAM detection with a limit of detection of 32 fM and enabled live-cell analysis down to 2 cells/μL. It distinguished cell populations with different surface EpCAM levels and quantitatively monitored epithelial-mesenchymal transition-associated changes in cell-surface EpCAM abundance. Coordinated redesign of the target-responsive nucleic acid components extended the platform to PTK7 detection and the analysis of drug-induced cell-surface PTK7 remodeling. Coupling the system to a lateral flow readout enabled portable visual detection. This work establishes a modular and reconfigurable CRISPR-Cas12a sensing strategy for wash-free amplified quantification of cell-surface proteins on living cells, providing a convenient tool for investigating their dynamic remodeling during physiological and pathological processes and therapeutic responses, as well as for drug screening.},
}

Humans
*CRISPR-Cas Systems/genetics
Aptamers, Nucleotide/chemistry
*Epithelial Cell Adhesion Molecule/analysis
*Membrane Proteins/analysis

@article {pmid43010997,
year = {2026},
author = {Mao, W and Zhang, L},
title = {Microfluidic Biosensors for the Detection of Foodborne Pathogens: A Comprehensive Review.},
journal = {Food science & nutrition},
volume = {14},
number = {11},
pages = {e72588},
pmid = {43010997},
issn = {2048-7177},
abstract = {Foodborne pathogen detection in complex foods remains challenging because sample preparation and signal readout can be affected by lipids, proteins, and background microorganisms. This review compares microfluidic biosensors by substrate, fabrication route, sample handling, and optical, electrochemical, or nucleic-acid readout. Published studies demonstrate rapid detection in selected food samples, but reported detection limits cannot be compared without their matrices, units, enrichment steps, and assay times. Laboratory performance also does not establish field readiness. Reproducible manufacture, reagent stability, integrated sample-to-answer operation, and validation in authentic foods remain major translational barriers. Orthogonal readouts, CRISPR-assisted assays, and AI-supported interpretation are promising, but their benefit over simpler methods requires direct comparison and independent validation. Future studies should report matrix-specific recovery and performance, test robustness across sites, and address usability, cost, and regulatory requirements before claiming commercial readiness.},
}

@article {pmid43011237,
year = {2026},
author = {Ma, X and Li, L and Sun, Y},
title = {CRISPR Technology: From Bench to Bedside Applications.},
journal = {MedComm},
volume = {7},
number = {11},
pages = {e71011},
pmid = {43011237},
issn = {2688-2663},
abstract = {CRISPR-Cas genome editing represents one of the most transformative advances in modern molecular biology, enabling programmable site-specific genetic modification that has revolutionized functional genomics and precision therapeutic development. Beyond canonical Cas9 nucleases, the CRISPR toolkit has diversified into precision base editors, prime editors, CRISPR-associated transposases, and Cas12/Cas13 RNA-targeting effectors, each with distinct mechanistic properties. Clinically, ex vivo CRISPR therapies for hemoglobinopathies have secured regulatory approval, while base editing, prime editing, and in vivo Chimeric Antigen Receptor (CAR)-T candidates are advancing through trials for a broad spectrum of hematologic, oncologic, and rare genetic disorders. However, existing reviews largely address technical innovation or clinical translation in isolation, lacking a holistic, up-to-date synthesis of the full bench-to-bedside trajectory of CRISPR technology. This review systematically dissects the molecular mechanisms, advantages, and limitations of core CRISPR editing platforms; summarizes pivotal clinical data across hemoglobinopathies, CAR-T immunotherapy, muscular dystrophy, cystic fibrosis, cardiometabolic diseases and rare monogenic diseases; evaluates delivery systems including lipid nanoparticles and viral vectors; and discusses key translational barriers such as off-target genotoxicity and immunogenicity alongside emerging optimization strategies. This work provides a holistic bench-to-bedside framework for CRISPR research, offering critical insights to advance next-generation precision genetic medicine.},
}

@article {pmid43012140,
year = {2026},
author = {Gomase, VS and Nimje, HM and Kale, S and Midathada, A and Dhamane, SP},
title = {Next-Generation Mycobacterium tuberculosis Diagnostics: A Systematic Review of Molecular, CRISPR, and Biosensor Technologies.},
journal = {Recent patents on biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.2174/0118722083512070261026092440},
pmid = {43012140},
issn = {2212-4012},
abstract = {INTRODUCTION: TB (tuberculosis) due to Mycobacterium tuberculosis continues to be a significant worldwide health issue. Timely and reliable diagnostic tests will improve treatment outcomes and reduce the spread of disease. Traditional TB diagnostics currently available, such as smear microscopy and culture, have significant limitations with regard to sensitivity and turnaround times, especially for smear-negative cases and for tuberculosis in extrapulmonary locations.

OBJECTIVES: The purpose of this review is to analyze the evolution of TB diagnostics from traditional microbiology to new molecular diagnostic methods (including CRISPR, biosensor technology, and microfluidic methods), including an assessment of their diagnostic testing performance, feasibility, and potential for the future.

METHODS: The systematic narrative review aimed to systematically assess literature published from 2020 to 2025 retrieved from PubMed, Scopus, Web of Science, IEEE Xplore, ScienceDirect, and Google Scholar. The review was carried out according to the PRISMA guidelines and included 89 studies after screening and assessing for eligibility. The data were analysed qualitatively (i.e., through thematic analysis) by comparing data from conventional, molecular, and next-generation diagnostic methods.

RESULTS: Molecular tests (e.g., GeneXpert, Line Probe Assays, PCR, and Whole Genome Sequencing) provide greater sensitivity and specificity than traditional methods and allow for faster diagnosis. Additionally, newer CRISPR-based tests, biosensors, and microfluidic devices hold promise for rapid, portable, point-of-care diagnosis of tuberculosis.

DISCUSSION: The findings indicate that molecular diagnostics improve rapid detection and drug-resistance profiling, while CRISPR and biosensor platforms offer potential for rapid point-of-care testing. However, affordability, infrastructure, clinical validation, and scalability remain important barriers.

CONCLUSION: The use of molecular diagnostic testing, CRISPR technology, biosensors, artificial intelligence, and portable platforms will dramatically enhance existing TB surveillance systems and strategies for controlling the disease globally.},
}

@article {pmid43012455,
year = {2026},
author = {El-Sehrawy, AAMA and Sapaev, I and Nematov, O and Turakulov, R and Bayramdurdi, S and Turaeva, NO and Bainsal, N and Bansal, P and Smerat, A},
title = {Mapping Osteosarcoma Biology With CRISPR/Cas: From Gene Function to Therapeutic Targets.},
journal = {Cell biology international},
volume = {50},
number = {11},
pages = {},
doi = {10.1002/cbin.70233},
pmid = {43012455},
issn = {1095-8355},
mesh = {*Osteosarcoma/genetics/therapy/metabolism ; Humans ; *CRISPR-Cas Systems/genetics ; *Bone Neoplasms/genetics ; Drug Resistance, Neoplasm/genetics ; Gene Editing/methods ; Animals ; Oncogenes ; Autophagy ; },
abstract = {Osteosarcoma is a highly aggressive primary bone malignancy characterized by rapid proliferation, chemoresistance, and early metastasis. Recent advances in CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) genome editing have enabled precise targeting of oncogenes, tumor suppressors, and regulators of epigenetic, metabolic, and signaling pathways, providing new insights into osteosarcoma biology. CRISPR-mediated knockout of cell-cycle regulators, including CDK11, impairs proliferation, while restoration of TP53 and suppression of TERT promoter activity inhibits tumor growth. Other line of studies has revealed that chemoresistance mechanisms mediated by ABCB1/MDR1, CD44, ATR, RAD18, and autophagy-related genes have been effectively reversed through CRISPR interventions, restoring sensitivity to cisplatin and doxorubicin. Furthermore, CRISPR applications in immune modulation, such as PD-L1 knockout, may enhance antitumor immunity. Importantly, high-throughput CRISPR screens also may identify essential genes, novel oncogenic drivers, and synthetic lethal targets, thus informing potential precision therapeutic strategies. This review aims to critically summarize CRISPR/Cas-mediated studies in osteosarcoma, focusing on targeting oncogenes and tumor suppressors, overcoming chemoresistance, modulating immune responses, regulating metabolism and autophagy, and identifying essential genes and vulnerabilities, providing a translational framework for precision therapy.},
}

*Osteosarcoma/genetics/therapy/metabolism
Humans
*CRISPR-Cas Systems/genetics
*Bone Neoplasms/genetics
Drug Resistance, Neoplasm/genetics
Gene Editing/methods
Animals
Oncogenes
Autophagy

@article {pmid43012712,
year = {2026},
author = {Du, R and Wang, W and Xie, J and Li, Y and Wu, N and Xu, Q},
title = {Clustered Regularly Interspaced Palindromic Repeats-Cas Genome-Editing Systems in Filamentous Fungi: Current Tools, Optimization Strategies, and Future Perspectives.},
journal = {Journal of agricultural and food chemistry},
volume = {74},
number = {44},
pages = {34662-34682},
doi = {10.1021/acs.jafc.6c12748},
pmid = {43012712},
issn = {1520-5118},
support = {22378210//National Natural Science Foundation of China/ ; 32603244//National Natural Science Foundation of China/ ; 32603627//National Natural Science Foundation of China/ ; BK20233003//Natural Science Foundation of Jiangsu Province/ ; 23KJA180003//Major Basic Research Project of the Natural Science Foundation of the Jiangsu Higher Education Institutions/ ; },
mesh = {*Fungi/genetics/metabolism ; *Gene Editing/methods/trends ; *CRISPR-Cas Systems ; Genome, Fungal ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Filamentous fungi are important microbial cell factories for producing antibiotics, organic acids, enzymes, and other valuable products, with broad applications in food, agriculture, and medicine. However, inefficient and imprecise genome editing remains a major bottleneck limiting rational strain engineering. This review summarizes major genome-editing tools in filamentous fungi, including Cre-loxP, TALENs, and CRISPR-Cas systems, with emphasis on their mechanisms, advantages, and limitations. We highlight CRISPR-Cas9 as the predominant platform and discuss optimization strategies involving sgRNA design, engineered Cas9 variants, promoter engineering, AMA1-based replicative plasmids, and ribonucleoprotein delivery. Emerging technologies, including base editing, prime editing, Cas12a, and Mad7, are also discussed for their potential to broaden editing capabilities. Finally, we outline future perspectives focusing on extending the application of existing genome-editing systems in filamentous fungi and discovering novel genome-editing tools from large-scale omics data sets. This review will accelerate the development of filamentous fungi as versatile platforms for biomanufacturing.},
}

*Fungi/genetics/metabolism
*Gene Editing/methods/trends
*CRISPR-Cas Systems
Genome, Fungal
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid43013067,
year = {2026},
author = {Yang, H and Zhang, H and Zhao, Z and Ren, Y and Xu, Y and Cai, K and Cheng, J and Liu, X and Chen, Y and Yin, L},
title = {Tumor-Specific Gene Editing Enhances Cancer Immunogenicity through Succinate Metabolism-Mediated MHC-I Reprogramming.},
journal = {Journal of the American Chemical Society},
volume = {148},
number = {44},
pages = {48152-48167},
doi = {10.1021/jacs.6c14840},
pmid = {43013067},
issn = {1520-5126},
support = {52303200//National Natural Science Foundation of China/ ; 52325305//National Natural Science Foundation of China/ ; 52573340//National Natural Science Foundation of China/ ; NA//Collaborative Innovation Center of Suzhou Nano Science and Technology/ ; NA//Higher Education Discipline Innovation Project/ ; NA//Joint International Research Laboratory of Carbon-Based Functional Materials and Devices/ ; },
mesh = {Animals ; Mice ; Humans ; *Gene Editing ; *Succinic Acid/metabolism ; CRISPR-Cas Systems ; *Histocompatibility Antigens Class I/immunology/genetics/metabolism ; Cell Line, Tumor ; },
abstract = {Impaired expression of major histocompatibility complex class I (MHC-I) greatly compromises cytotoxic T-cell-mediated anticancer immunotherapy. Existing MHC-I activation strategies are greatly challenged by poor tumor specificity and off-target toxicity. Herein, we report a dual tumor-specific CRISPR/Cas9 gene editing system that can precisely knock out succinate dehydrogenase subunit C (Sdhc) to activate the MHC-I pathway via epigenetic and transcriptional regulation. A tumor-specific gene editor is first engineered via reversible shielding of sgRNA using an apurinic/apyrimidinic endonuclease 1 (APE1)-responsive, complementary ssDNA, yielding AP-sgRNA with blocked editing activity in normal cells yet completely restored activity in APE1-overexpressing tumor cells. Subsequently, tumor-targeted delivery of AP-sgRNA and Cas9 mRNA (mCas9) is achieved using dibenzocyclooctyne-decorated lipid nanoparticle (DLNP), which upon bioorthogonal click reaction can anchor cancer cells that are prelabeled with azido groups using the unnatural sugar DCL-AAM. Thus, AP-sgSdhc/mCas9@DLNP enables efficient and selective Sdhc editing in tumor cells, leading to epigenetic and transcriptional activation of MHC-antigen processing and presentation genes and thereby provoking robust antitumor efficacy in the B16-OVA melanoma model through enhanced, T cell-mediated immune responses. Moreover, it completely suppressed pulmonary metastasis after combination with aPD-L1 in the 4T1 lung metastasis model. This study provides an enlightened paradigm for precision gene editing and renders a promising mechanism for potentiating cancer immunotherapy.},
}

Animals
Mice
Humans
*Gene Editing
*Succinic Acid/metabolism
CRISPR-Cas Systems
*Histocompatibility Antigens Class I/immunology/genetics/metabolism
Cell Line, Tumor

@article {pmid43015537,
year = {2027},
author = {Berry, AM and Costa, BLD and Quinn, PMJ},
title = {Prime Editing: Mechanism and Developments.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3099},
number = {},
pages = {1-7},
pmid = {43015537},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; Animals ; },
abstract = {Prime editing is a "search and replace" based genome editing method that can introduce point mutations, insertions, and deletions into DNA at desired sites, making it the editing technique capable of making the most diverse range of edits. Prime editing utilizes a Cas9 nickase fused to a reverse transcriptase to introduce edits encoded by a specially designed DNA oligonucleotide called a prime editing guide RNA (pegRNA). While prime editing has been shown to achieve robust editing in different genomic loci and in different cell types, this technology still has the potential for further optimization and improvement. Many groups have been successful in improving prime editing outcomes by optimizing the design of prime editors and pegRNAs. Additionally, modulating key cellular pathways has also been an effective tool for achieving high levels of editing. Due to its high efficiency and relative safety, prime editing has the potential to transform disease modeling and genomic medicine.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
Animals

@article {pmid42749803,
year = {2026},
author = {Levesque, S and Kawashima, N and Hwang, GH and Zeng, J and Toskov, V and Barry, T and Mannherz, W and Homfeldt, L and Becerra, B and Schoonenberg, VAC and Pinello, L and Agarwal, S and Bauer, DE},
title = {Targeted genomic integration and rearrangement using prime assembly.},
journal = {Nature},
volume = {659},
number = {8140},
pages = {532-541},
pmid = {42749803},
issn = {1476-4687},
support = {R01 HG013618/HG/NHGRI NIH HHS/United States ; R01 HL165061/HL/NHLBI NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Gene Rearrangement/genetics ; Exons/genetics ; DNA/genetics/biosynthesis ; Transgenes/genetics ; *Genome, Human/genetics ; *Genomics ; },
abstract = {Although therapeutic genome editing holds great potential to remedy diverse inherited and acquired disorders, targeted installation of medium-to-large genomic modifications in therapeutically relevant cells remains challenging[1]. Here we develop prime assembly, an approach that permits DNA sequence assembly and integration in human cells leveraging CRISPR-targeted dual flap synthesis. This method enables RNA-programmable site-specific integration of single or double-stranded DNA fragments. Unlike homology-directed repair, prime assembly is similarly active in dividing and non-dividing cells. We applied prime assembly to perform targeted exon recoding, transgene integration and megabase-scale rearrangements, including at therapeutically relevant loci in primary human cells. Prime assembly expands the capabilities of genome engineering by enabling the targeted integration of medium to large-sized DNA sequences without relying on double-stranded DNA donors, nuclease-driven double-strand breaks or cell cycle progression.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Gene Rearrangement/genetics
Exons/genetics
DNA/genetics/biosynthesis
Transgenes/genetics
*Genome, Human/genetics
*Genomics

@article {pmid42799341,
year = {2026},
author = {Zhou, H and Wang, YZ and Zhang, SM and Yan, X and Chen, H and Jin, SX and Chen, WH and Zhang, XZ},
title = {Targeting FAK-YAP Signaling Axis Inhibits Tumor Stemness and Overcomes Tumor Resistance to Cuproptosis.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {38},
number = {63},
pages = {},
doi = {10.1002/adma.75155},
pmid = {42799341},
issn = {1521-4095},
support = {52273301//National Natural Science Foundation of China/ ; 52473145//National Natural Science Foundation of China/ ; 52333004//National Natural Science Foundation of China/ ; 52131302//National Natural Science Foundation of China/ ; 2024DJA011//Hubei Province Science and Technology Innovation Talent Program/ ; //Institutional Animal Care and Use Committee/ ; WP20240086//Animal Experiment Center of Wuhan University/ ; },
mesh = {*Cuproptosis ; *Signal Transduction/drug effects ; Humans ; *Neoplastic Stem Cells/drug effects/metabolism/pathology ; Cell Line, Tumor ; YAP-Signaling Proteins ; Animals ; *Focal Adhesion Kinase 1/metabolism/genetics ; *Transcription Factors/metabolism ; Drug Resistance, Neoplasm/drug effects ; *Adaptor Proteins, Signal Transducing/metabolism ; CRISPR-Cas Systems/genetics ; },
abstract = {Cuproptosis has recently been identified as a novel metabolic cell death pathway that holds great promise for cancer therapy. However, cuproptosis resistance developed by tumor cells severely impairs its anticancer efficacy. Here, our study uncovers that the FAK-YAP signaling axis in tumor cells plays important roles in the development of cuproptosis resistance and the promotion of tumor stemness. To therapeutically target this signaling axis, a CRISPR-cuproptosis synergistic nanoplatform (CPLNP@HA) is elaborately designed for targeted co-delivery of a Ptk2 (encoding FAK) CRISPR-Cas9 knockout plasmid to block FAK-YAP signaling axis and Cu-elesclomol (Cu-ES, a cuproptosis inducer) into tumor cells. Mechanistically, CPLNP@HA efficiently silences FAK expression, which subsequently blocks the cuproptosis-induced activation of the FAK-YAP signaling cascade. This intervention not only sustains intracellular copper accumulation to reinforce cuproptosis but also reduces tumor stemness, thereby significantly potentiating the therapeutic outcomes of cuproptosis and activating tumoricidal immunity as evidenced by transcriptomic analysis. In combination with immune checkpoint blockade therapy, CPLNP@HA markedly inhibits tumor development upon rechallenge after surgical resection. Our work establishes FAK-YAP signaling axis as a new therapeutic target to overcome cuproptosis resistance and inhibit tumor stemness, providing a promising combinatorial paradigm to sensitize tumor to cuproptosis and immunotherapy.},
}

*Cuproptosis
*Signal Transduction/drug effects
Humans
*Neoplastic Stem Cells/drug effects/metabolism/pathology
Cell Line, Tumor
YAP-Signaling Proteins
Animals
*Focal Adhesion Kinase 1/metabolism/genetics
*Transcription Factors/metabolism
Drug Resistance, Neoplasm/drug effects
*Adaptor Proteins, Signal Transducing/metabolism
CRISPR-Cas Systems/genetics

@article {pmid42945784,
year = {2027},
author = {Zhang, Y and Deng, F and Sang, R and Liu, L and Barber, TJ and Goldys, EM and Li, M},
title = {Functional double-emulsion droplets enable multiplexed CRISPR quantification of cancer-related miRNAs.},
journal = {Biosensors & bioelectronics},
volume = {317},
number = {},
pages = {119361},
doi = {10.1016/j.bios.2026.119361},
pmid = {42945784},
issn = {1873-4235},
mesh = {Humans ; *MicroRNAs/genetics/blood/isolation & purification ; *Biomarkers, Tumor/genetics/blood ; *Biosensing Techniques/instrumentation ; *CRISPR-Cas Systems/genetics ; *Colorectal Neoplasms/genetics/blood/diagnosis ; Emulsions/chemistry ; Equipment Design ; },
abstract = {Accurate, multiplexed microRNA detection is critical for early cancer diagnosis. We present a droplet-based digital CRISPR/Cas13a platform for simultaneous, amplification-free detection of multiple miRNAs. By integrating double emulsion (DE) microfluidics with programmable crRNAs, the system achieves orthogonal, interference-free readouts, enabling precise absolute quantification in complex biological samples. The assay demonstrates high sequence specificity, robust orthogonality, and consistent performance across target combinations and matrices. Fluorescence-positive droplet counts correlate linearly with input concentrations, allowing quantification of miRNA-21, miRNA-29a, and miRNA-92a with 10 copies/μL representing the lowest concentration experimentally evaluated in this study. The applicability of the platform is further evaluated in a small proof-of-concept cohort of plasma samples from healthy controls and colorectal cancer (CRC) patients. This platform provides an amplification-free approach for multiplexed miRNA quantification and supports further development for clinical biomarker analysis.},
}

Humans
*MicroRNAs/genetics/blood/isolation & purification
*Biomarkers, Tumor/genetics/blood
*Biosensing Techniques/instrumentation
*CRISPR-Cas Systems/genetics
*Colorectal Neoplasms/genetics/blood/diagnosis
Emulsions/chemistry
Equipment Design

@article {pmid42988560,
year = {2027},
author = {Alam, S and Matvienko, D and Boström, J and Pires, MJ and Papadimitropoulou, A and Eshtad, S and Sanjiv, K and Qian, H and Valerie, NC and Brisken, C and Altun, M},
title = {A modular lentiviral system for multiplexed gene perturbation and functional analysis suggests interdependence of hormone receptors in breast cancer growth in vivo.},
journal = {Life science alliance},
volume = {10},
number = {1},
pages = {},
pmid = {42988560},
issn = {2575-1077},
mesh = {Humans ; Female ; *Lentivirus/genetics ; *Breast Neoplasms/genetics/metabolism/pathology ; Animals ; CRISPR-Cas Systems/genetics ; Receptors, Estrogen/metabolism/genetics ; Mice ; Genetic Vectors/genetics ; MCF-7 Cells ; Receptors, Progesterone/metabolism/genetics ; Gene Expression Regulation, Neoplastic ; Cell Line, Tumor ; Receptors, Androgen/metabolism/genetics ; Cell Proliferation/genetics ; },
abstract = {Precise and flexible control of gene expression is essential for dissecting gene function in complex biological systems. Although recent developments in genetic engineering and CRISPR/Cas9 technology have expanded tools for gene activation, suppression, and editing, their application in physiologically relevant models remains challenging, time-consuming, and expensive. Here, we present a modular, doxycycline-inducible vector system that integrates gene overexpression, shRNA-mediated knockdown, and CRISPR/Cas9-mediated regulation within a single, lentivirus-compatible system. The modular design allows rapid exchange of selection markers, epitope tags, and reporters via Gateway cloning, providing broad adaptability across experimental settings. In addition to standard fluorescent and luminescent reporters, the system includes advanced sensors, such as Fucci cell cycle reporters, to enable monitoring of cellular processes. By combining fluorescence barcoding with combinatorial genetic perturbations, the platform supports multiplexed analysis of gene function and genetic interactions through phenotypic characterization by fluorescence imaging or flow cytometry. We demonstrate its utility in vivo with breast cancer intraductal xenografts, which suggest that ER+ breast cancer cells (MCF7) rely on androgen, estrogen, and progesterone receptors for in vivo growth. This versatile gene perturbation system provides tight temporal control, streamlined implementation, and high-content phenotyping capacity facilitating efficient in vitro and in vivo studies while reducing the use of animals in in vivo validation experiments. It thus expands the experimental repertoire for dynamic, multigene interrogation in complex systems.},
}

Humans
Female
*Lentivirus/genetics
*Breast Neoplasms/genetics/metabolism/pathology
Animals
CRISPR-Cas Systems/genetics
Receptors, Estrogen/metabolism/genetics
Mice
Genetic Vectors/genetics
MCF-7 Cells
Receptors, Progesterone/metabolism/genetics
Gene Expression Regulation, Neoplastic
Cell Line, Tumor
Receptors, Androgen/metabolism/genetics
Cell Proliferation/genetics

@article {pmid42989096,
year = {2026},
author = {Xie, Q and Ma, L and Wang, Z and Duan, Y and Cao, Q},
title = {From immunity to identity to exaptation: RNA-based self/non-self discrimination across cellular life.},
journal = {BMC immunology},
volume = {27},
number = {1},
pages = {},
pmid = {42989096},
issn = {1471-2172},
support = {2026TC089//Chinese Universities Scientific Fund/ ; },
mesh = {Animals ; Humans ; RNA Editing ; RNA Interference ; RNA Methylation ; *RNA, Messenger/genetics ; CRISPR-Cas Systems ; Host-Pathogen Interactions/immunology ; *RNA/genetics/immunology ; Epitranscriptome ; },
abstract = {Distinguishing self from non-self is a universal imperative of life, where such discrimination at RNA level is both evolutionarily rational and urgently required to maintain homeostasis. Across kingdoms, organisms have evolved distinct yet convergent strategies to achieve this goal. Animals broadly employ A-to-I mRNA editing and other modifications as self-tags; plants and fungi mainly rely on RNA interference, spatial segregation, and m[6]A methylation; and prokaryotes utilize CRISPR-Cas immunity as a programmable analog of self-recognition. Despite their efficacy, these systems harbor intrinsic vulnerabilities that viruses might exploit through mimicry and modification theft, perpetuating a dynamic host-pathogen arms race. During evolution, these defense mechanisms might be exapted for new roles in hosts: A-to-I editing diversified proteomes, and m[6]A methylation became a global regulator of mRNA fate. Together, these observations reveal that mechanisms born from conflict might be repurposed to drive molecular innovation, linking self-recognition, adaptation, and the evolution of complexity.},
}

Animals
Humans
RNA Editing
RNA Interference
RNA Methylation
*RNA, Messenger/genetics
CRISPR-Cas Systems
Host-Pathogen Interactions/immunology
*RNA/genetics/immunology
Epitranscriptome

@article {pmid42992074,
year = {2026},
author = {Guo, J and Wu, T and Qiao, Q and Zhu, X and Zhao, K and Cui, L and Huo, X and Ge, Y},
title = {Development of a One-Step RT-LAMP-CRISPR/Cas12b Assay for Rapid On-Site Detection of Dabie Bandavirus.},
journal = {ACS omega},
volume = {11},
number = {43},
pages = {66547-66556},
pmid = {42992074},
issn = {2470-1343},
abstract = {Severe fever with thrombocytopenia syndrome (SFTS), caused by Dabie bandavirus (DBV), has rapidly spread to multiple countries worldwide since its discovery in 2009, posing a serious threat to global public health security. SFTS is a tick-borne disease, and people living in mountainous and hilly areas are at increased risk of infection, which highlights the urgent need for rapid, accurate, and field-applicable diagnostic strategies for DBV. In this study, LAMP primers and CRISPR sgRNA were designed based on the conserved region of the DBV L segment. A one-pot, one-step molecular assay integrating reverse transcription loop-mediated isothermal amplification (RT-LAMP) with CRISPR/Cas12b technology was established after systematic optimization of key parameters. The assay operates under isothermal conditions at 60 °C, delivering results within 40 min. Analytical evaluation demonstrated a limit of detection of 25.6 RNA copies per reaction. No cross-reactivity was observed with Hantaan virus, Seoul virus, Chikungunya virus, Japanese encephalitis virus, Dengue virus, and Zika virus. Clinical validation using 42 specimens showed complete concordance with gold-standard qRT-PCR, achieving 100% sensitivity (30/30) and 100% specificity (12/12). For point-of-care application, fluorescence visualization was incorporated into the CRISPR/Cas-based detection workflow, enabling straightforward result interpretation. This integrated RT-LAMP-CRISPR/Cas12b platform provides rapid and specific detection of DBV through a simplified workflow that does not require complex instrumentation, supporting its potential application in resource-limited environments.},
}

@article {pmid42992156,
year = {2026},
author = {Khan, A and Iqbal, A and Athar, H and Sheikh, AS},
title = {Mutation-induced penicillin binding protein 2A inactivation through CRISPR-dCas9 gene editing in methicillin-resistant S. aureus.},
journal = {Iranian journal of microbiology},
volume = {18},
number = {5},
pages = {741-751},
pmid = {42992156},
issn = {2008-3289},
abstract = {BACKGROUND AND OBJECTIVES: The global rise of antimicrobial resistance threatens effective treatment of bacterial infections. Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen whose resistance to β-lactam antibiotics is primarily mediated by the mecA gene, encoding the low-affinity penicillin-binding protein PBP2a. This study aimed to investigate whether CRISPR-dCas9-mediated targeting of mecA could suppress resistance-associated gene expression and restore β-lactam susceptibility in MRSA.

MATERIALS AND METHODS: Seven target sites within mecA, comprising two non-coding and five coding regions, were selected for CRISPR-dCas9 targeting. The effects of gene interference were evaluated using antibiotic disk diffusion assays, minimum inhibitory concentration (MIC) testing with gradient E-strips, RT-PCR, and Sanger sequencing.

RESULTS: Targeting non-coding regions suppressed mecA transcription and reduced PBP2a production, whereas targeting coding regions generated mutations associated with dysfunctional PBP2a. CRISPR-dCas9 treatment significantly reduced mecA expression and increased MRSA susceptibility to β-lactam antibiotics, particularly ampicillin and cefixime. MIC values decreased by up to 12-fold and 6-fold, respectively (p<0.05).

CONCLUSION: CRISPR-dCas9-mediated targeting of mecA effectively reduced β-lactam resistance in MRSA. This approach demonstrates potential for precision-based antimicrobial resistance management and warrants further investigation as a strategy for combating drug-resistant bacterial infections.},
}

@article {pmid42993843,
year = {2026},
author = {Moyer, SM and Ilic, N and Gang, S and Dailey, L and Oh, S and Stavridi, J and Taig, G and Rodarte, KE and DeAngelo, JD and Jiang, A and Shim, BH and Rennhack, JP and Jacob, JR and Akinci, MA and So, J and Wang, H and Piccioni, F and Talamas, JA and Widlund, HR and Root, DE and Clish, CB and Danial, NN and Hahn, WC},
title = {MYC primes a paralog synthetic lethality involving the mitochondrial translocase subunits TIMM17A and TIMM17B and N-Acetylaspartate.},
journal = {Science advances},
volume = {12},
number = {45},
pages = {eaej2662},
pmid = {42993843},
issn = {2375-2548},
support = {U01 CA176058/CA/NCI NIH HHS/United States ; },
mesh = {*Aspartic Acid/analogs & derivatives/metabolism ; Humans ; *Proto-Oncogene Proteins c-myc/metabolism/genetics ; *Synthetic Lethal Mutations ; *Mitochondrial Membrane Transport Proteins/metabolism/genetics ; Mitochondrial Precursor Protein Import Complex Proteins ; Mitochondria/metabolism/genetics ; CRISPR-Cas Systems ; Cell Line, Tumor ; },
abstract = {To identify the genes and pathways required for the survival of MYC-amplified cancers, we engineered an isogenic cell system in which transformation is driven by oncogenic MYC. Using a genome-scale CRISPR-Cas9 library, we identified the mitochondrial membrane translocase TIMM17A as uniquely essential for the survival of MYC-transformed cells. This dependency arises from MYC-driven suppression of TIMM17B, the paralog of TIMM17A. TIMM17A/B is an essential component of the TIM23 mitochondrial protein import complex, and the MYC-induced suppression of TIMM17B creates a strong dependency on TIMM17A. N-acetylaspartate supplementation rescued TIMM17A dependency in MYC-amplified cell lines, highlighting the unique and crucial role of this metabolite in the cell fitness of MYC-amplified cancers. These observations identify a paralog dependency required for the survival of a subset of MYC-driven cancers.},
}

*Aspartic Acid/analogs & derivatives/metabolism
Humans
*Proto-Oncogene Proteins c-myc/metabolism/genetics
*Synthetic Lethal Mutations
*Mitochondrial Membrane Transport Proteins/metabolism/genetics
Mitochondrial Precursor Protein Import Complex Proteins
Mitochondria/metabolism/genetics
CRISPR-Cas Systems
Cell Line, Tumor

@article {pmid42995122,
year = {2026},
author = {Marti, E and González-Villalobos, E and Balcázar, JL},
title = {Editorial: Bacteriophages and CRISPR-Cas for combating multidrug-resistant bacteria.},
journal = {Microbiological research},
volume = {},
number = {},
pages = {128815},
doi = {10.1016/j.micres.2026.128815},
pmid = {42995122},
issn = {1618-0623},
}

@article {pmid42995533,
year = {2026},
author = {Gao, S and Ullah, U and Jin, J and Shi, Y and Zhang, P and Zhang, X and Wang, H and Wen, J and Chen, KM and Liu, WT},
title = {Comprehensive molecular characterization and biosafety assessment of TaDOF7.6 transgenic and gene-edited wheat.},
journal = {BMC plant biology},
volume = {26},
number = {1},
pages = {},
pmid = {42995533},
issn = {1471-2229},
support = {2025CYFZ-02//Construction of a non-variety-dependent gene editing system in wheat and creation of elite germplasm/ ; 32572241//Molecular mechanism of autophagy protein TaATG8c regulating wheat pre-harvest sprouting/ ; },
mesh = {*Triticum/genetics/growth & development ; Plants, Genetically Modified/genetics ; *Gene Editing ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; RNA Interference ; },
abstract = {BACKGROUND: Wheat is one of the major staple food crops worldwide, and stable wheat yield is crucial for ensuring global food security. Transgenic and gene-editing technologies serve as important approaches for wheat genetic improvement, yet further application of related materials requires systematic evaluation of their molecular characteristics, genetic stability and environmental biosafety. In this study, TaDOF7.6-RNAi transgenic wheat and TaDOF7.6-CRISPR/Cas9 gene-edited wheat previously constructed in our laboratory were used as experimental materials to evaluate their yield-related traits, molecular profiles and partial biosafety indicators.

RESULTS: Both RNAi-mediated silencing and CRISPR/Cas9-mediated knockout of TaDOF7.6 significantly improved wheat grain traits and yield-related agronomic traits, further confirming that TaDOF7.6 acts as a negative regulator in wheat yield regulation. For TaDOF7.6-RNAi transgenic wheat, the integration site of T-DNA on wheat chromosome 3B was identified via thermal asymmetric interlaced PCR (TAIL-PCR). Vector-derived fragments including Hyg, GUS and dsRNA were consistently detectable across T3 to T5 generations. Moreover, these RNAi lines maintained stable histochemical GUS activity and sustained silencing efficiency of TaDOF7.6. No significant differences were observed in seed germination, seedling emergence and pollen-related traits between RNAi-25, RNAi-31 lines and wild-type wheat. Within the sampling scope and conventional PCR detection conditions adopted in this research, no target exogenous gene fragments were detected in 24 surrounding plant species. Additionally, there were no obvious disparities in field insect community and weed distribution between transgenic lines and wild-type controls. For TaDOF7.6-CRISPR/Cas9 gene-edited wheat, the insertion locus of exogenous vector on wheat chromosome 4D was successfully mapped. Transgene-free homozygous mutants were acquired through Hi-TOM high-throughput sequencing, Sanger sequencing and progeny screening. No unintended sequence variations were detected at all predicted off-target sites compared with wild-type plants. Meanwhile, the gene-edited lines exhibited consistent performance with wild-type wheat in seed germination, seedling emergence and pollen-associated phenotypes.

CONCLUSION: This study provides fundamental data for molecular characterization and comprehensive biosafety assessment of TaDOF7.6-RNAi transgenic wheat and TaDOF7.6-CRISPR/Cas9 gene-edited wheat, and verifies the application potential of TaDOF7.6 as a promising target gene for wheat yield improvement. The transgene-free gene-edited mutants obtained in this work also supply elite new germplasm resources for subsequent wheat molecular breeding programs.},
}

*Triticum/genetics/growth & development
Plants, Genetically Modified/genetics
*Gene Editing
*Plant Proteins/genetics/metabolism
CRISPR-Cas Systems
RNA Interference

@article {pmid42996482,
year = {2026},
author = {Li, Y and Liu, L and Zhang, J and Wei, G and Zhang, L and Lu, Y and Qin, P and Liu, Z},
title = {Reprogramming secretion systems in Klebsiella quasipneumoniae: from pathogenic functions to candidate industrial secretion chassis.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {14},
number = {},
pages = {1940225},
pmid = {42996482},
issn = {2296-4185},
abstract = {Efficient extracellular secretion remains a major bottleneck in microbial cell factories, motivating the search for chassis organisms with native high-throughput transport capacity. Klebsiella quasipneumoniae (Kqp), a recently delineated member of the Klebsiella pneumoniae species complex, encodes diverse secretion-related systems, including RND-type efflux pumps, type II and type VI secretion systems (T2SS/T6SS), outer membrane vesicle (OMV) pathways, and capsular polysaccharide (CPS) export machinery. In clinical settings, these systems contribute to opportunistic pathogenicity, antimicrobial resistance, and host adaptation; in biotechnology, they may provide transport modules that can be detoxified and redirected for extracellular production. This review examines the feasibility of reprogramming Kqp from a pathogen-related bacterium into a safe industrial secretion chassis. We summarize the molecular architecture, substrate-recognition logic, and regulatory networks of major secretion pathways, then discuss CRISPR-Cas-driven removal of virulence and resistance determinants, biocontainment, and synthetic-biology strategies for reallocating secretory capacity. We further survey potential applications in bulk chemicals, human milk oligosaccharides, recombinant proteins, and industrial enzymes. Finally, we identify key barriers to industrial translation, including growth-secretion energetic trade-offs, periplasmic protein misfolding, endotoxin-associated downstream processing, OMV heterogeneity, scale-up robustness, and quantitative safety validation. Establishing Kqp as an industrial chassis will require evidence-tiered strain selection, quantitative secretion-capacity measurement, regulatory decoupling of resistance-associated pumps, and One Health-aligned safety assessment. Direct evidence in K. quasipneumoniae remains limited; therefore, this review distinguishes direct Kqp observations from studies in other KpSC members or K. oxytoca, evidence from non-Klebsiella hosts, and forward-looking proposals. OMV yield is considered separately from lipid A/endotoxin-related properties.},
}

@article {pmid42996656,
year = {2026},
author = {Nagar, P and Islam, MR and Rahman, NA and Heeamoni, SA and Hasan, MM and Huq, S and Bushra, FA and Hu, M and Ali, R and Hossain, M and Rahman, MA},
title = {CRISPR-mediated correction of oncogenic AS-NMD in splicing factor mutant cancer.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1958532},
pmid = {42996656},
issn = {1664-3224},
support = {R35 GM154991/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *Nonsense Mediated mRNA Decay/genetics ; Enhancer of Zeste Homolog 2 Protein/genetics/metabolism ; *Serine-Arginine Splicing Factors/genetics ; *Mutation ; *CRISPR-Cas Systems ; Exons/genetics ; Alternative Splicing ; Polycomb Repressive Complex 2/metabolism ; Gene Expression Regulation, Neoplastic ; Cell Line, Tumor ; },
abstract = {Alternative splicing coupled to nonsense-mediated mRNA decay (AS-NMD) evolved as a master regulator of gene expression. Dysregulated AS-NMD has been identified as the root of many human maladies, from developmental defects to deadly cancer. Poison exons (PEs) are highly conserved alternative exons that contain a premature termination codon and elicit AS-NMD when included in a transcript. Cancer cells often exploit the inclusion of PEs to downregulate tumor suppressors or the exclusion of PEs to upregulate oncoproteins. Therefore, PEs have drawn significant attention as a novel therapeutic avenue for cancer and other diseases. It was previously reported that an oncogenic mutation in the splicing factor SRSF2 promotes downregulation of the tumor suppressor EZH2 by including a PE, resulting in altered chromatin regulation, defective hematopoiesis, and cancer development. Here, we examine a therapeutic proof-of-concept for manipulating PE-mediated oncogenic AS-NMD using a CRISPR-based approach. Using paired guide RNA, we successfully deleted the PE of EZH2 from the genome of SRSF2-mutated leukemia. This editing resulted in EZH2 mRNAs without a PE, and restored EZH2 protein expression. This subsequently restored Polycomb Repressive Complex 2 (PRC2) association, and global H3K27 histone methylation. The proposed CRISPR strategy provides compelling evidence for correcting oncogenic PE inclusion, highlighting its potential for therapeutic application against PE in cancer and other human diseases.},
}

Humans
*Nonsense Mediated mRNA Decay/genetics
Enhancer of Zeste Homolog 2 Protein/genetics/metabolism
*Serine-Arginine Splicing Factors/genetics
*Mutation
*CRISPR-Cas Systems
Exons/genetics
Alternative Splicing
Polycomb Repressive Complex 2/metabolism
Gene Expression Regulation, Neoplastic
Cell Line, Tumor

@article {pmid42997561,
year = {2026},
author = {Dangelmaier, CA and Elzoheiry, M and Vajipayajula, DN and Wright, MN and Kim, S and Tsygankov, AY and Kunapuli, SP},
title = {Phosphorylation of tyrosine 519 and 520 on Syk is essential for ITAM and hemITAM signaling and function in platelets.},
journal = {Blood vessels, thrombosis & hemostasis},
volume = {3},
number = {4},
pages = {100225},
pmid = {42997561},
issn = {2950-3272},
support = {R35 HL155694/HL/NHLBI NIH HHS/United States ; },
abstract = {The protein spleen tyrosine kinase (Syk) plays a central role in immunoreceptor tyrosine-based activation motif (ITAM) and hemi-ITAM signal transduction in numerous hematopoietic cell systems. To assess the mechanism of Syk regulation, the tandem tyrosines 519 and 520 in the kinase domain of mouse Syk were mutated to phenylalanines by CRISPR-Cas. The breeding of these mice did not follow the expected Mendelian ratio, as genotype analysis of pups from heterozygous intercrosses revealed that less than one-tenth were the mutant form of Syk lacking phosphorylation at Y519,Y520 (Syk Y519,520F). On gross examination, the intestines of Syk Y519,520F mice were diffusely reddened, raising suspicion for vascular inflammation. Lymphocytes were reduced by ∼50% in the Syk Y519,520F mice. Aggregation and secretion induced by 2-methyl-thio-ADP or AYPGKF were unaffected in the Syk 519,520F mouse platelets. There was no aggregation or dense-/α-granule secretion when Syk Y519,520F platelets were challenged with any concentration of collagen-related peptide or C-type lectin receptor-2 (CLEC-2) cross-linking. No phosphorylation of the downstream signaling proteins linker of activation of T cells Y220 or phospholipase Cγ2 Y1217 was detected in the mutated platelets. The Syk Y519,520F mutation had a clear effect on hemostasis as evidenced by a significant increase in tail bleeding time and the time to thrombus formation using the ferric chloride injury model. These data reveal that phosphorylation of Y519 and Y520 on Syk after stimulation of either glycoprotein VI or CLEC-2 receptors was crucial for the ability of Syk to transduce a downstream signal to regulate in vivo and ex vivo platelet functions.},
}

@article {pmid42999773,
year = {2026},
author = {Raji, S and Soltaninejad, H and Hosseinkhani, S and Hamidieh, AA},
title = {CRISPR/Cas9-Mediated Targeting of the Integrin Beta-2 Gene as a Gene-Editing Strategy for Leukocyte Adhesion Deficiency Type I.},
journal = {Iranian journal of medical sciences},
volume = {51},
number = {10},
pages = {720-726},
pmid = {42999773},
issn = {1735-3688},
mesh = {Humans ; *Leukocyte-Adhesion Deficiency Syndrome/genetics/therapy ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *CD18 Antigens/genetics ; HEK293 Cells ; Genetic Therapy/methods ; },
abstract = {BACKGROUND: Leukocyte adhesion deficiency (LAD) is a genetic immunodeficiency that severely impairs the leukocyte adhesion cascade and migration to the sites of infection and inflammation. As a result, the main consequence is recurrent and severe infections with bacteria and fungi, which can lead to death. The most common variant, LAD-I, is due to mutations in the integrin beta 2 (ITGB2) gene responsible for the β2 integrin (CD18) subunit. Corrective strategies based on gene therapy and genome editing are the most promising methods for treating genetic defects.

METHODS: In the present experimental in vitro study conducted at Tarbiat Modares University, Tehran, Iran, in 2024, we designed a single-guide RNA (sgRNA) for the CRISPR/Cas9 system to target exon 6 of ITGB2 and performed validation experiments. We also investigated peptide nanocarriers as a potential strategy for delivering the CRISPR components. The PX458 plasmid encoding Cas9 and the designed sgRNA was delivered into HEK293T cells, followed by genome-editing analysis.

RESULTS: Genome-editing analysis confirmed a measurable level of indel formation at the target locus. Furthermore, the results of MPG nanopeptide production and purification demonstrate the potential of peptide nanocarriers for genome-editing systems.

CONCLUSION: Overall, this work establishes a foundation for subsequent knock-in and gene-correction studies in clinically relevant immune cell models, paving the way for potential LAD-I gene therapy applications.},
}

Humans
*Leukocyte-Adhesion Deficiency Syndrome/genetics/therapy
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*CD18 Antigens/genetics
HEK293 Cells
Genetic Therapy/methods

@article {pmid43000033,
year = {2026},
author = {Piao, CL and Zhang, Y and Cui, ML},
title = {Optimized Genetic Transformation Method Facilitates Gene Functional Analysis and Genome Editing in the Asteraceae Model Plant Senecio vulgaris.},
journal = {Physiologia plantarum},
volume = {178},
number = {6},
pages = {},
doi = {10.1111/ppl.71177},
pmid = {43000033},
issn = {1399-3054},
support = {2018FR004//the scientific research funds of Zhejiang A&F University/ ; },
mesh = {*Transformation, Genetic ; *Senecio/genetics ; *Gene Editing/methods ; Plants, Genetically Modified ; Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; },
abstract = {Senecio vulgaris belongs to the Asteraceae and serves as an important model for studying ecological adaptation, evolutionary processes, and capitulum development. However, our understanding of the molecular regulatory mechanisms of capitulum development has been hindered by low transformation efficiency and the lack of reliable genome editing tools. Here, we report an optimized Agrobacterium-mediated transformation and genome editing method for S. vulgaris using leaf explants, with selection carried out for 6 weeks. Approximately 88% of kanamycin-resistant shoots exhibited normal morphology, and more than 80% of transformants showed strong GFP expression, which was confirmed by Southern blot. Furthermore, we used this method to successfully silence the SnAPETALA1-6, a MADS-box gene associated with inflorescence development, and to edit the phytoene desaturase gene (SvPDS) in S. vulgaris. Taken together, these improved methods overcome earlier technical barriers, enabling in-depth investigation of the molecular evolutionary processes that underpin flower-head formation in Senecio and other Asteraceae.},
}

*Transformation, Genetic
*Senecio/genetics
*Gene Editing/methods
Plants, Genetically Modified
Plant Proteins/genetics/metabolism
CRISPR-Cas Systems/genetics

@article {pmid43000447,
year = {2026},
author = {Niu, W and Qian, Y and Wang, D and Hao, Y and Yu, H and Gao, X and Zheng, W and He, G and Lai, L and Li, Z},
title = {Reconstruction of PAM-interacting domains to broad PAM compatibility.},
journal = {Genome biology},
volume = {27},
number = {1},
pages = {},
pmid = {43000447},
issn = {1474-760X},
support = {2022YFA1105404//National Key Research and Development Program of China/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; HEK293 Cells ; *Gene Editing/methods ; Mutation ; *Nucleotide Motifs ; },
abstract = {The protospacer adjacent motif (PAM) requirement limits CRISPR-Cas9 targetability. Here, we develop REPAM, an ancestral sequence reconstruction-based strategy that re-engineers PAM-interacting domains to expand PAM recognition. REPAM broadens the PAM specificities of Nme1Cas9 and Nme2Cas9 from N4GATT/N4CC to N4CNH, while additional mutations further extend Nme2Cas9 recognition to N4VHH. Applied to SpaCas9, REPAM expands PAM compatibility from NNGYRA to NNHH, increasing theoretical target coverage by approximately 36-fold. The engineered SpaCas9 achieves up to 76.6% editing efficiency in HEK293T cells, outperforming SpRY. Corresponding base editors efficiently generate protective variants and precise splice-site mutations, highlighting REPAM as a versatile platform for CRISPR engineering.},
}

Humans
*CRISPR-Cas Systems
HEK293 Cells
*Gene Editing/methods
Mutation
*Nucleotide Motifs

@article {pmid43000506,
year = {2026},
author = {Mao, Y and Yang, N and Zhao, Y and Wang, L and Xu, K and Chen, Y and Ding, Y and Zhong, B and Ma, S and Liu, J and Zhang, Y and Ni, Q and Feng, G and Zhou, Q and Zhou, Q and Li, W},
title = {CROSS: A cross-species chromosome substitution platform for dissecting chromosome architecture and activity.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {43000506},
issn = {2041-1723},
mesh = {Animals ; *X Chromosome/genetics ; Rats ; Mice ; CRISPR-Cas Systems ; Male ; Histone-Lysine N-Methyltransferase/metabolism/genetics ; Mouse Embryonic Stem Cells/metabolism ; Histones/metabolism ; Long Interspersed Nucleotide Elements/genetics ; Species Specificity ; },
abstract = {The functional integrity of a mammalian chromosome depends on its long-term co-evolution with species-specific nuclear environment. How chromosomes co-adapt with their native environment to define 3D architecture and transcription remains unclear, largely due to a lack of experimental models that dissect this co-evolution. Here, we report cross-species chromosome substitution (CROSS), a method enabling stable, scarless replacement of host chromosomes with divergent orthologs. We integrate microcell-mediated chromosome transfer with CRISPR/Cas9 to import the intact 152-Mb rat X chromosome into male mouse embryonic stem cells, and subsequently replace the mouse X chromosome, maintaining its stability and integrity. Rat-specific LINE1 and RatSatRep2 repeats recruit mouse SETDB1 inefficiently, causing local H3K9me3 loss. This triggers 3D remodeling, characterized by de novo topologically associating domain (TAD) boundaries that aberrantly activate adjacent genes, including Rhox5, impairing differentiation. We provide a platform for dissecting sequence and epigenome cooperation in chromosome regulation, and evaluating fidelity of large-scale heterologous DNA.},
}

Animals
*X Chromosome/genetics
Rats
Mice
CRISPR-Cas Systems
Male
Histone-Lysine N-Methyltransferase/metabolism/genetics
Mouse Embryonic Stem Cells/metabolism
Histones/metabolism
Long Interspersed Nucleotide Elements/genetics
Species Specificity

@article {pmid43001262,
year = {2026},
author = {Raudstein, M and Datsomor, AK and Stewart, R and McGrew, M and Fraslin, C and Crespo, D and Kleppe, L},
title = {The potential for large-scale genome editing in finfish aquaculture via germline stem cell culture and transplantation.},
journal = {Frontiers in genetics},
volume = {17},
number = {},
pages = {1924271},
pmid = {43001262},
issn = {1664-8021},
abstract = {Genome editing has emerged as a promising tool to complement selective breeding programmes and accelerate genetic gain in aquaculture. Although CRISPR-based genome editing has been successfully applied in several economically important fish species, its application at an industry-relevant scale remains challenging due to limitations of current delivery methods, which primarily rely on manual embryo microinjection. This approach is labour-intensive, technically demanding, and frequently results in high levels of mosaicism in founder individuals, necessitating additional generations of breeding to establish stable genotypes. In this mini review, we discuss the potential of combining germline stem cell (GSC) culture and transplantation approaches as an alternative platform for further improved genome editing in fish. By enabling genetic changes to be introduced, validated, and expanded in vitro prior to germline transmission, this approach has the potential to improve scalability, reduce mosaicism, and shorten breeding timelines. Although several technical challenges remain, continued progress in GSC culture and transplantation methodologies could facilitate the practical implementation of tailored genome editing for improved genetic gain, fish health, and welfare in aquaculture.},
}

@article {pmid43003575,
year = {2026},
author = {Chen, K and Gao, T and Yu, L and Zhang, Q and Lin, Y and Wang, Y and Xiong, W and Wang, Y and Li, J and Sun, X and Xue, J and Chen, Q and Niu, B},
title = {RPA-assisted three-channel Cas13a-Cas13b-Cas12a fluorescence architecture for three-channel viral nucleic acid detection.},
journal = {Analytica chimica acta},
volume = {1427},
number = {},
pages = {346423},
doi = {10.1016/j.aca.2026.346423},
pmid = {43003575},
issn = {1873-4324},
mesh = {Fluorescence ; *CRISPR-Associated Proteins/genetics/chemistry/metabolism ; *Encephalitis Viruses, Tick-Borne/genetics/isolation & purification ; *Encephalitis Virus, Venezuelan Equine/genetics/isolation & purification ; *RNA, Viral/analysis/genetics ; CRISPR-Cas Systems ; Limit of Detection ; Animals ; *Bacterial Proteins/genetics/chemistry ; *Nucleic Acid Amplification Techniques/methods ; Endodeoxyribonucleases ; },
abstract = {Multiplex nucleic-acid analysis requires orthogonal signal channels capable of resolving multiple targets without substantial cross-interference. Here, an RPA-assisted three-channel CRISPR fluorescence readout was developed by coupling the distinct collateral-cleavage activities of LwaCas13a, PsmCas13b, and LbaCas12a to FAM-, ROX-, and HEX-based reporters, respectively, for detection of Venezuelan equine encephalitis virus (VEEV), tick-borne encephalitis virus (TBEV), and an eastern/western equine encephalitis virus (EEEV/WEEV) group target. The corresponding singleplex assays showed no detectable cross-reactivity with the tested non-target viral templates and achieved operational analytical detection limits of 10 copies/μL for VEEV and TBEV and 100 copies/μL for the EEEV/WEEV group assay. Integration of the three Cas-crRNA-reporter modules into one downstream reaction enabled discrimination of single-, dual-, and three-target combinations, with an analytical detection limit of 100 copies/μL for each target in the equal-concentration three-target mixture. Reporter optimization provided balanced channel responses, while inter-day repeatability and interlaboratory reproducibility yielded coefficients of variation below 10%. In a preliminary matrix evaluation using contrived equine nasal-swab samples, plasmid-spiked samples and unspiked negative controls showed concordant classification between the CRISPR readout and RT-qPCR, indicating compatibility of the assay workflow with the tested sample matrix. The method provides a three-channel analytical architecture that consolidates three downstream CRISPR readouts into one reaction while retaining target-assigned fluorescence interpretation.},
}

Fluorescence
*CRISPR-Associated Proteins/genetics/chemistry/metabolism
*Encephalitis Viruses, Tick-Borne/genetics/isolation & purification
*Encephalitis Virus, Venezuelan Equine/genetics/isolation & purification
*RNA, Viral/analysis/genetics
CRISPR-Cas Systems
Limit of Detection
Animals
*Bacterial Proteins/genetics/chemistry
*Nucleic Acid Amplification Techniques/methods
Endodeoxyribonucleases

@article {pmid43004607,
year = {2026},
author = {Su, F and Vipasha, V and Dong, Y and Huang, S and Jiang, X and Mei, F and Zhang, J},
title = {Differential Impact of CRISPR/Cas9-Mediated OsMIR396ef Mutagenesis on Yield Components Across Eight Diverse Rice Varieties.},
journal = {Physiologia plantarum},
volume = {178},
number = {6},
pages = {},
doi = {10.1111/ppl.71185},
pmid = {43004607},
issn = {1399-3054},
support = {//Ji'nan Haiyou Talent to J.S./ ; 2023ZD04074//Biological Breeding-Major Projects/ ; },
mesh = {*Oryza/genetics/growth & development ; *CRISPR-Cas Systems/genetics ; Mutagenesis/genetics ; Plants, Genetically Modified ; *MicroRNAs/genetics/metabolism ; Plant Proteins/genetics/metabolism ; Seeds/genetics ; Mutation ; Genes, Plant/genetics ; },
abstract = {Substantial gains in rice productivity are a continual demand for breeders, farmers, as well as consumers. Identifying and introducing favorable yield-related genes in elite cultivars is the most efficient strategy for increasing yield. So far, many yield-related genes in rice have been cloned. However, rice varieties with a vast diversity of genetic constitutions differ tremendously in the levels of grain yield. Here, we edited yield-related MIR396e and MIR396f genes in eight japonica rice varieties and found that mutation efficiency varied with genetic background in the T0 generation. Notably, the knockout of MIR396e and MIR396f genes resulted in increased leaf, panicle, and grain size in all eight genetic backgrounds. However, plant height, culm strength, grain number per panicle, and seed setting rate varied with different genetic backgrounds. Results also showed that among the eight varieties, grain yield increased in five different rice varieties compared to the corresponding wild type, while three of them had decreased yield due to the reduced number of grains per panicle or the seed-setting rate. Thus, our findings broaden the understanding that OsmiR396 can have diverse, even opposing effects on grain yield and plant architecture among genetically distinct rice varieties.},
}

*Oryza/genetics/growth & development
*CRISPR-Cas Systems/genetics
Mutagenesis/genetics
Plants, Genetically Modified
*MicroRNAs/genetics/metabolism
Plant Proteins/genetics/metabolism
Seeds/genetics
Mutation
Genes, Plant/genetics

@article {pmid43006236,
year = {2026},
author = {Patel, P and Treat, AR and Gonzalez, R and Rajaraman, S and Cruga, BN and Chinea, EB and Onwuka, A and Nguyen, C and Terns, MP and Santiago-Frangos, A},
title = {Archaeal histones coordinate conserved DNA sequence motifs to promote CRISPR adaptive immunity.},
journal = {Nucleic acids research},
volume = {54},
number = {21},
pages = {},
pmid = {43006236},
issn = {1362-4962},
support = {//Lyrasis Consortium/ ; R35GM162543/GM/NIGMS NIH HHS/United States ; R35 GM162543/GM/NIGMS NIH HHS/United States ; //Roy & Diana Vagelos Scholars Program in the Molecular Life Sciences/ ; /NH/NIH HHS/United States ; //Institute for RNA Innovation of the Perelman School of Medicine/ ; //Burroughs Wellcome Fund/ ; //CURF's Ruth Marcus Kanter College Alumni Society Undergraduate Research/ ; //Trustees of the University of Pennsylvania/ ; //University of Pennsylvania/ ; R00 GM147842/GM/NIGMS NIH HHS/United States ; //University of Pennsylvania's Center for Undergraduate Research & Fellowships's (CURF) Penn Undergraduate Research Mentoring Program/ ; R00GM147842/GM/NIGMS NIH HHS/United States ; },
mesh = {*Histones/metabolism ; Nucleotide Motifs ; Conserved Sequence ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *DNA, Archaeal/chemistry ; Pyrococcus furiosus/genetics/immunology ; Base Sequence ; Mutation ; Binding Sites ; Archaeal Proteins/metabolism ; },
abstract = {Archaea and bacteria acquire resistance to viruses by integrating fragments of foreign DNA at the beginning of a CRISPR locus. In the archaeon Pyrococcus furiosus, histones bind the leader to promote specific integration at the first CRISPR repeat. However, it remains unknown whether archaeal CRISPR leaders contain conserved DNA sequence motifs required for integration. Here, we analyze 542 archaeal CRISPR leaders found across Thermococcales and Methanococcales. We identify 36 motifs found up to 500 bps upstream of archaeal CRISPR loci. Scrambling or truncating novel motifs or histone binding sites found 100-400 bps upstream of the P. furiosus CRISPR modestly decreases integration efficiency. However, mutation of motif T2, which overlaps with the proximal histone site and is adjacent to the CRISPR, ablates both histone-mediated and histone-independent integration. Mutations tiled across T2 reveal that a pyrimidine at position -3 of the leader is required for histone-independent integration, and basepairs -4 to -19 are required for histone-mediated integration. T2 is found in most Thermococcales, and a similar sequence is found in most Methanococcales leaders, suggesting this motif may be a broadly conserved element that facilitates CRISPR adaptive immunity across taxonomic orders. These data shed light on the sequence requirements for histone-mediated DNA integration.},
}

*Histones/metabolism
Nucleotide Motifs
Conserved Sequence
*Clustered Regularly Interspaced Short Palindromic Repeats
*DNA, Archaeal/chemistry
Pyrococcus furiosus/genetics/immunology
Base Sequence
Mutation
Binding Sites
Archaeal Proteins/metabolism

@article {pmid43007365,
year = {2026},
author = {Saini, A and Sharma, N and Sharma, N and Kumari, N and Singh, B and Thakur, AK},
title = {CRISPR interference-based gene silencing in insects: from functional genomics to prospective insect-pest management applications.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {43007365},
issn = {1573-4978},
mesh = {Animals ; *Gene Silencing ; *CRISPR-Cas Systems/genetics ; *Insecta/genetics ; Genomics/methods ; RNA Interference ; Pest Control, Biological/methods ; Gene Editing/methods ; *Insect Control/methods ; },
abstract = {Insect-pests remain a major constraint to global agriculture, causing yield losses through direct feeding and transmission of phytopathogens. Chemical pesticides and transgenic crops expressing Bacillus thuringiensis toxins have shown initial success; however, their use is increasingly constrained by resistance, non-target effects, environmental contamination and regulatory restrictions. Although RNA interference (RNAi) presents a targeted and eco-friendly alternative, it is limited by species variability, rapid double-stranded RNA (dsRNA) degradation, inconsistent silencing and off-target effects. Nuclease-active Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) enables genetic editing, but its irreversible genome modifications raise ethical, ecological and regulatory concerns. CRISPR interference (CRISPRi) offers a programmable approach for sequence-specific, reversible transcriptional suppression without inducing double-strand DNA breaks. Using catalytically inactive Cas9 (dead Cas9 or dCas9) -guided by single-guide RNA (sgRNA), CRISPRi inhibits transcription initiation or elongation while maintaining genomic integrity. Experimental studies in insect models, including disease-vector mosquitoes, have demonstrated effective suppression of coding and non-coding genes, supporting CRISPRi as a functional genomics tool. This review discusses CRISPRi mechanisms, applications, advantages and limitations in insects, comparing it with RNAi and CRISPR/Cas9. Overall, current evidences indicate that CRISPRi is a precise and non-mutagenic platform for insect-pest management, particularly where controlled, reversible and species-specific gene regulation is desirable. However, its application in agricultural pest management remains an extrapolation from functional-genomics studies and requires validation in pest species under whole-organism and field-relevant conditions.},
}

Animals
*Gene Silencing
*CRISPR-Cas Systems/genetics
*Insecta/genetics
Genomics/methods
RNA Interference
Pest Control, Biological/methods
Gene Editing/methods
*Insect Control/methods

@article {pmid43008001,
year = {2026},
author = {Sheet, S and Kim, HJ and Kim, WI and Kim, S and Lee, P and Cho, A and Kim, D},
title = {CRISPR/Cas9-mediated PD-L1 knockout reshapes temporal transcriptomic responses to PRRSV infection in porcine alveolar macrophages.},
journal = {PloS one},
volume = {21},
number = {11},
pages = {e0355142},
pmid = {43008001},
issn = {1932-6203},
mesh = {Animals ; *Macrophages, Alveolar/virology/metabolism/immunology ; Swine ; *Porcine respiratory and reproductive syndrome virus ; *CRISPR-Cas Systems ; *Transcriptome ; *B7-H1 Antigen/genetics ; *Porcine Reproductive and Respiratory Syndrome/genetics/virology/immunology ; Gene Knockout Techniques ; },
abstract = {Porcine reproductive and respiratory syndrome virus (PRRSV) suppresses innate antiviral immunity in porcine alveolar macrophages, and the PD-1/PD-L1 axis is thought to contribute to this immune dysregulation. This study investigated how PD-L1 knockout (KO) alters the host transcriptional response to PRRSV infection compared to wild-type (WT) immortalized porcine alveolar macrophages (iPAMs). PD-L1 KO iPAMs were generated by CRISPR/Cas9 targeting exon 2 of porcine CD274 locus, and knockout was confirmed in clone #35. After PRRSV-2 infection, KO#35 cells exhibited approximately 5-fold reduced viral RNA accumulation at 48 h post-infection compared with WT cells. Time-course RNA sequencing at 0, 6, and 48 h post-infection was then performed to characterize the transcriptional consequences of PD-L1 KO during acute PRRSV-2 infection. PD-L1 KO iPAMs exhibited downregulation of immune checkpoint and exhaustion-related genes (LAG3, PDCD1, TIGIT, TOX family) and NF-κB inhibitors (SOCS4, NFKBIA), alongside upregulation of antigen presentation (MHC I/II genes like SLA-2, SLA-5, B2M, SLA-DMA, SLA-DMB, HLA-DRA) and co-stimulatory molecules (CD80, CD86, CD40). Cytokine profiling indicated a reprogrammed response favoring antiviral signaling, including sustained elevation of CXCL10/11, ISGs (MX1/2, ISG15, IFIT1-3), and select interleukins (IL15, IL6), while suppressing pro-inflammatory TNF and CXCL8 across time points. Pathway enrichment analysis revealed significant enrichment of the cytokine-receptor interaction pathway. Weighted gene co-expression network analysis (WGCNA) further identified a key co-expression module containing 1,763 DEGs that was associated with innate immune activation. Moreover, protein-protein interaction (PPI) network analysis detected PXDN as a key hub gene connected to the PI3K-Akt signaling and natural killer cytotoxicity pathways. These findings provide transcriptomic insight into the association between PD-L1 deficiency and altered macrophage transcriptional responses to PRRSV infection in an in-vitro iPAM model and provide a basis for future mechanistic investigation of PD-L1 in PRRSV pathogenesis.},
}

Animals
*Macrophages, Alveolar/virology/metabolism/immunology
Swine
*Porcine respiratory and reproductive syndrome virus
*CRISPR-Cas Systems
*Transcriptome
*B7-H1 Antigen/genetics
*Porcine Reproductive and Respiratory Syndrome/genetics/virology/immunology
Gene Knockout Techniques

@article {pmid43009953,
year = {2026},
author = {Li, K and Tang, W and Shi, B and Zhao, X and Luo, R and Li, Y and Wang, T and Huang, F},
title = {Visual and rapid detection of Fasciola spp. in fecal samples using a one-pot RPA-CRISPR/Cas12a assay.},
journal = {BMC veterinary research},
volume = {22},
number = {1},
pages = {},
pmid = {43009953},
issn = {1746-6148},
support = {XZ202501ZY0122//Science and Technology Projects of Xizang Autonomous Region, China/ ; XZ202401JD0012//Base and Talent Program of Science and Technology Plan in Xizang Autonomous Region/ ; },
mesh = {Animals ; *Feces/parasitology ; CRISPR-Cas Systems ; Sheep ; *Fasciola/isolation & purification/genetics ; *Nucleic Acid Amplification Techniques/veterinary/methods ; *Fascioliasis/veterinary/diagnosis/parasitology ; Cattle ; DNA, Helminth ; Sensitivity and Specificity ; *Sheep Diseases/diagnosis/parasitology ; *Cattle Diseases/diagnosis/parasitology ; Rapid Diagnostic Tests ; },
abstract = {BACKGROUND: Fasciolosis caused by Fasciola spp. has substantial veterinary and public-health importance, while conventional fecal microscopy has limited sensitivity and PCR depends on laboratory equipment. This study aimed to develop a rapid, visual one-pot recombinase polymerase amplification (RPA)-CRISPR/Cas12a assay for detecting Fasciola DNA in fecal samples.

RESULTS: RPA primers and CRISPR RNAs targeting the conserved internal transcribed spacer 1 region were screened and optimized. The final assay was completed at 37 °C within 30 min, and results were read using a fluorescence detector or visualized under blue light. The analytical limit of detection was 10 copies/µL, and DNA corresponding to 0.1 egg equivalents was detectable. No cross-reactivity was observed with six non-target parasite species. Among 54 field fecal samples (42 yak and 12 Tibetan sheep samples) from Xizang, three were positive, with complete qualitative agreement between the one-pot assay and conventional PCR in this limited sample set. In a preliminary mixed-DNA interference evaluation using 24 artificially spiked fecal samples, all 23 samples containing Fasciola DNA, including samples containing DNA from both Fasciola species or Fasciola DNA combined with selected non-target parasite DNA, were positive, whereas the non-target-only control was negative.

CONCLUSIONS: The one-pot RPA-CRISPR/Cas12a assay provides rapid and analytically sensitive detection of Fasciola DNA, with no cross-reactivity observed within the tested parasite panel. The method shows potential for surveillance and resource-limited testing, but broader multi-region validation and simplified sample preparation are required before routine field or point-of-care deployment.},
}

Animals
*Feces/parasitology
CRISPR-Cas Systems
Sheep
*Fasciola/isolation & purification/genetics
*Nucleic Acid Amplification Techniques/veterinary/methods
*Fascioliasis/veterinary/diagnosis/parasitology
Cattle
DNA, Helminth
Sensitivity and Specificity
*Sheep Diseases/diagnosis/parasitology
*Cattle Diseases/diagnosis/parasitology
Rapid Diagnostic Tests

@article {pmid43009987,
year = {2026},
author = {Ma, Y and Li, J and Jin, X and Liu, S and Chang, Y and Lin, T and Zhang, C and Zhu, L},
title = {A quantitative and visual detection probe based on isothermal nucleic acid amplification coupled with CRISPR/Cas12a system (INACS) for miR155 detection.},
journal = {Journal of nanobiotechnology},
volume = {24},
number = {1},
pages = {},
pmid = {43009987},
issn = {1477-3155},
support = {RG/R1/241228//Royal Society of UK/ ; 2024JY039//Nantong University Special Research Fund for Clinical Medicine/ ; },
mesh = {*MicroRNAs/analysis/genetics/blood ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; Biosensing Techniques/methods ; Rapid Diagnostic Tests ; },
abstract = {MicroRNAs (miRNAs) have emerged as ideal biomarkers for early clinical diagnosis. Achieving the accurate detection of miRNAs plays a critical role for assuring its implementation in their clinical applications. In this study, we developed a dual-signal amplified ultrasensitive fluorescent sensing platform for miR155 detection by coupling isothermal nucleic acid amplification with the CRISPR/Cas12a system (INACS). Using a hairpin nucleic acid probe (HP) as the substrate, the system specifically recognizes the target miR155, triggering the opening of the hairpin loop structure. Subsequently, under the catalysis of Bst DNA polymerase, target gene amplification produces dsDNA with a specific sequence. This dsDNA activates the trans-cleavage activity of Cas12a, which cleaves reporter strands labeled with quencher and fluorescent groups, restoring the fluorescence signal. The fluorescence intensity is proportional to miR155 concentration, exhibiting a strong linear relationship between fluorescence intensity ratio and the logarithm of miR155 concentration over the range of 0.5-50 pM (r = 0.9961). By integrating nucleic acid amplification with CRISPR/Cas12a amplification, INACS achieves high sensitivity, excellent selectivity, and a detection limit as low as 34.46 fM. INACS was successfully applied to human serum samples, revealing elevated miR155 levels in breast cancer patients compared to non-cancer controls. Moreover, INACS was adapted for semi-quantitative detection using lateral flow assay (LFA) test strips. Results were assessed by naked-eye color comparison, facilitating rapid onsite auxiliary diagnosis of breast cancer. The LFA outcomes were consistent with serum fluorescence measurements, enabling efficient and user-friendly visual detection. This work thus provides a promising tool for early clinical diagnosis.},
}

*MicroRNAs/analysis/genetics/blood
*Nucleic Acid Amplification Techniques/methods
Humans
*CRISPR-Cas Systems/genetics
Limit of Detection
Biosensing Techniques/methods
Rapid Diagnostic Tests

@article {pmid43010390,
year = {2026},
author = {Chen, Q and Chen, Z and Peng, Y and Wang, H and Xu, H and Yao, B and Fatima, N and Chen, W},
title = {PAM-Free Programmable Enzymatic Cascade Design Enabling Highly Sensitive CRISPR/Cas12a Lateral Flow Assay for Foodborne Pathogen Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {44},
pages = {30560-30570},
doi = {10.1021/acs.analchem.6c03699},
pmid = {43010390},
issn = {1520-6882},
support = {32172295//National Natural Science Foundation of China/ ; ZDYF2022XDNY248//Hainan Provincial Department of Science and Technology/ ; 2022YFF1102900//National Key Research and Development Program of China/ ; 2024YFF0618100//National Key Research and Development Program of China/ ; 22N31900500//Science and Technology Commission of Shanghai Municipality/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Food Microbiology ; *CRISPR-Associated Proteins/metabolism ; *Bacterial Proteins/metabolism ; Rapid Diagnostic Tests ; *Endodeoxyribonucleases/metabolism ; },
abstract = {CRISPR/Cas12a-assisted nucleic acid assays have shown great potential for rapid pathogen detection. However, their implementation in lateral flow assays remains constrained by strict protospacer adjacent motif (PAM) requirements and low-contrast, signal-off readouts. To effectively address these limitations, we present a new PAM-free programmable enzymatic cascade-enabled CRISPR/Cas12a lateral flow assay (PAEC-LFA) for highly sensitive foodborne pathogen detection. In this strategy, target amplification products are enzymatically digested to generate programmable single-stranded DNA activators, enabling PAM-independent Cas12a activation and flexible target recognition. Upon activation, Cas12a exhibits efficient trans-cleavage activity, which subsequently triggers terminal deoxynucleotidyl transferase (TdT)-mediated extension, forming an enzymatic cascade that amplifies signal output and generates a high-contrast signal-on readout on lateral flow assays. Integrating RPA and enzymatic digestion with PAM-free Cas12a-TdT signal amplification into two sequential one-pot modules simplifies the workflow, minimizes contamination risk, and maintains effective amplification performance. Under optimized conditions, PAEC-LFA achieved detection limits of 1.05 CFU/mL for Salmonella and Staphylococcus aureus, and 3.13 CFU/mL for Pseudomonas aeruginosa. Visual detection limits in spiked food samples reached 10 CFU/mL for all three pathogens. This work establishes a generalizable PAM-free enzymatic cascade framework for highly sensitive CRISPR/Cas12a lateral flow assays, advancing practical pathogen detection in food safety applications.},
}

*CRISPR-Cas Systems/genetics
*Food Microbiology
*CRISPR-Associated Proteins/metabolism
*Bacterial Proteins/metabolism
Rapid Diagnostic Tests
*Endodeoxyribonucleases/metabolism

@article {pmid40972176,
year = {2025},
author = {Liu, G and Meng, Z and Zheng, W and Qiu, A and Tian, Y and Zhang, Z and Zhang, T and Wu, P and Ding, P},
title = {Dual-modal aptamer/CRISPR-Cas12a biosensor with Fe-C-N@Au nanozyme for simultaneous detection of azithromycin and macrolide resistance genes and degradation of azithromycin.},
journal = {Journal of hazardous materials},
volume = {498},
number = {},
pages = {139850},
doi = {10.1016/j.jhazmat.2025.139850},
pmid = {40972176},
issn = {1873-3336},
mesh = {Gold/chemistry ; *Biosensing Techniques/methods ; *Aptamers, Nucleotide/chemistry ; *Metal Nanoparticles/chemistry/radiation effects ; *Anti-Bacterial Agents/metabolism ; CRISPR-Cas Systems ; Iron/chemistry ; *Macrolides/pharmacology ; *Drug Resistance, Bacterial/genetics ; Colorimetry ; },
abstract = {The pervasive contamination of aquatic systems by azithromycin (AZM) and its role in proliferating macrolide resistance genes, particularly ermB, constitutes a critical threat to ecological and public health. Current methodologies lack integrated on-site detection and efficient degradation. We engineered a multifunctional single-atom iron nanozyme decorated with gold nanoparticles (Fe-C-N@Au) platform integrating aptamer-mediated recognition, CRISPR-Cas12a detection, colorimetric-photothermal dual-modal sensing, and nanozyme catalysis. For detection, AZM binding liberates Fe-C-N@Au-aptamer conjugates from magnetic complexes, enabling quantification through nanozyme-catalyzed 3,3',5,5'-tetramethylbenzidine (TMB) oxidation and oxTMB-derived photothermal signals under near-infrared radiation (NIR) irradiation. Parallel ermB identification occurs via Cas12a trans-cleavage-triggered Fe-C-N@Au release. This approach achieves exceptional sensitivity with limit of detection (LOD) of 2.34 pM (colorimetric) and 9.17 pM (photothermal) for AZM, alongside 3.77 pM (colorimetric) and 8.46 pM (photothermal) for ermB. Synchronous quantification is facilitated through smartphone colorimetry and portable infrared thermometry. Crucially, the identical Fe-C-N@Au nanozyme efficiently activates hydrogen peroxide (H2O2) to generate reactive oxygen species (ROS), accomplishing > 90 % AZM degradation within 60 min. The integrated "detect-and-degrade" system creates a closed-loop process that couples real-time monitoring with immediate remediation. This field-deployable strategy combats antibiotic resistance by simultaneously eliminating selective pressure and genetic determinants, providing a transformative approach for controlling the dissemination of antimicrobial resistance at the source.},
}

Gold/chemistry
*Biosensing Techniques/methods
*Aptamers, Nucleotide/chemistry
*Metal Nanoparticles/chemistry/radiation effects
*Anti-Bacterial Agents/metabolism
CRISPR-Cas Systems
Iron/chemistry
*Macrolides/pharmacology
*Drug Resistance, Bacterial/genetics
Colorimetry

@article {pmid41199023,
year = {2026},
author = {Liu, M and Huang, K and Zhang, J and Li, Q and Wang, X and Gu, B and Tang, H and Du, Z and Hu, L and Qi, S and Ma, Y and Yu, H and Xie, W and Fang, X and Wang, H},
title = {CRISPR live-cell imaging reveals chromatin dynamics and enhancer interactions at multiple non-repetitive loci.},
journal = {Nature biotechnology},
volume = {44},
number = {11},
pages = {1906-1919},
pmid = {41199023},
issn = {1546-1696},
mesh = {*Chromatin/genetics/metabolism ; *Enhancer Elements, Genetic/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Transcription Factors/genetics/metabolism ; Promoter Regions, Genetic/genetics ; Animals ; RNA, Guide, CRISPR-Cas Systems/genetics ; Bromodomain Containing Proteins ; Epigenesis, Genetic ; Cell Cycle Proteins ; },
abstract = {Existing methods to visualize dynamic changes in the three-dimensional genome, promoter-enhancer interactions and the influence of epigenetic modifications in non-repetitive loci are limited. Here we introduce CRISPR PRO-LiveFISH (Pooled gRNAs with Orthogonal bases LiveFISH), which combines orthogonal bases from expanded genetic alphabet technology and rational single guide RNA (sgRNA) design to efficiently label multiple non-repetitive loci in living cells. The optimized method allows simultaneous imaging of up to six genomic loci and uses as few as 10 sgRNAs for non-repetitive loci imaging without signal amplification. We demonstrate the method in diverse cell types, including primary cells, and apply it to reveal enhancer-promoter dynamics and a correlation between genomic dynamics and epigenetic states. We also show that PCDHα-enhancer interactions may persist despite spatial mobility and that BRD4 maintains super-enhancer contacts regulating MYC oncogene expression in cancer cells. CRISPR PRO-LiveFISH can be applied to diverse studies of chromatin dynamics and genome organization in living cells.},
}

*Chromatin/genetics/metabolism
*Enhancer Elements, Genetic/genetics
Humans
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Transcription Factors/genetics/metabolism
Promoter Regions, Genetic/genetics
Animals
RNA, Guide, CRISPR-Cas Systems/genetics
Bromodomain Containing Proteins
Epigenesis, Genetic
Cell Cycle Proteins

@article {pmid41219485,
year = {2026},
author = {McDiarmid, TA and Taylor, ML and Chen, W and Chardon, FM and Choi, J and Liao, H and Li, X and Kim, H and Lalanne, JB and Li, T and Nathans, JF and Martin, BK and Knuth, J and Coradini, ALV and Gray, JM and Pinglay, S and Shendure, J},
title = {A parts list of promoters and gRNA scaffolds for mammalian genome engineering and molecular recording.},
journal = {Nature biotechnology},
volume = {44},
number = {11},
pages = {1894-1905},
pmid = {41219485},
issn = {1546-1696},
support = {R01HG010632//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; K99 HG012973/HG/NHGRI NIH HHS/United States ; RM1 HG009491/HG/NHGRI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R00 HG012973/HG/NHGRI NIH HHS/United States ; R01 HG010632/HG/NHGRI NIH HHS/United States ; DP5 OD036167/OD/NIH HHS/United States ; },
mesh = {Animals ; Humans ; *Genetic Engineering/methods ; *Promoter Regions, Genetic/genetics ; Mice ; *Genome/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Synthetic Biology/methods ; },
abstract = {A standardized 'parts list' of sequences for genetic engineering of microbes has been indispensable to progress in synthetic biology, but few analogous parts exist for mammalian systems. Here we design libraries of extant, ancestral, mutagenized or miniaturized variants of polymerase III promoters and guide RNA (gRNA) scaffolds and quantify their abilities to mediate precise edits to the mammalian genome through multiplex prime editing. We identify thousands of parts for reproducible editing in human and mouse cell lines, including hundreds with greater activity than commonly used sequences. Saturation mutagenesis screens identify tolerated sequence variants that further enhance sequence diversity. In an application to molecular recording, we design a 'ten key' array that, in mammalian cells, achieves balanced activity of pegRNAs as predicted by the activity of the component parts. The data reported here will aid the design of synthetic loci encoding arrays of gRNAs exhibiting predictable, differentiated levels of activity for applications in multiplexed perturbation, biological recorders and complex genetic circuits.},
}

Animals
Humans
*Genetic Engineering/methods
*Promoter Regions, Genetic/genetics
Mice
*Genome/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics
Synthetic Biology/methods

@article {pmid42978934,
year = {2026},
author = {Syombua, ED and Venado, RE and Achary, VMM and Kaur, S and Palacios Rojas, N and Ibba, MI and Lewis, L and Hearne, SJ and Bandyopadhyay, A},
title = {Genome editing for nutritional biofortification in maize: bridging molecular innovation and food security in the Global South.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1868062},
pmid = {42978934},
issn = {1664-462X},
abstract = {Micronutrient deficiency is a prevalent challenge in Sub-Saharan Africa and other geographies of the Global South where populations rely on maize as the primary calorie source. Globally, the most widespread micronutrient deficiencies include iron, zinc, and vitamin A, which are inherently low in maize. Besides, maize grain protein percentage of the essential amino acids falls below the recommended daily allowance. Traditional plant breeding practices have enabled the generation of hybrid plant varieties with superior nutritional value, such as Quality Protein Maize (QPM). However, obtaining novel varieties from existing gene pools often results in linkage drag drawbacks, yield penalties, poor-quality kernels, and requires multiple backcrossing cycles. Thus, genome editing offers a precise strategy for enhancing nutritional value, without the associated trade-offs. CRISPR/Cas, base editors and prime editors reduce breeding timelines and enable precise targeting of nutrient-accumulation pathways. In this review, we explore how genome editing technology could serve as the next-generation approach for maize biofortification. The review outlines various ways CRISPR/Cas allow precise and targeted modification of genes responsible for micronutrient biosynthesis, transport, and storage. We describe the application of CRISPR in disrupting negative regulators of nutrient transport, up-regulating nutrient biosynthesis pathways, and knocking out anti-nutritional factor genes. We further highlight that CRISPR-enabled biofortification is not just a technological innovation but a public health intervention for mitigating nutrient deficiencies. However, fully unlocking the potential of gene-edited biofortified maize will require coordinated efforts in regulatory policy and alignment with farmer/consumer preference.},
}

@article {pmid42979205,
year = {2026},
author = {Fang, Q and Lin, Y and Hu, G and Hu, J and Cheng, J and Dai, Y and Yan, L and Wu, X},
title = {Point-of-care testing in the diagnosis of infectious diseases.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1949318},
pmid = {42979205},
issn = {1664-302X},
abstract = {Point-of-care testing (POCT) has emerged as an indispensable diagnostic strategy for addressing the increasing global burden of infectious diseases and the worsening threat of antimicrobial resistance. Owing to prominent advantages such as rapid detection of infectious agents, portability, and ease of accessibility, POCT can effectively address the inherent limitations of traditional laboratory diagnostic workflows. This review summarizes the core technical platforms that underlie current POCT technologies, including lateral flow assays, chemiluminescence immunoassays, isothermal amplification systems, CRISPR/Cas-based diagnostic systems, and microfluidic chip technologies. Furthermore, we describe the key clinical scenarios in which POCT can be applied, including respiratory tract, bloodstream, gastrointestinal, urinary tract, and sexually transmitted infections. We also comprehensively discuss the technical characteristics and clinical applicability of mainstream POCT modalities, including immunochromatography and molecular diagnostics. Despite limitations associated with detection sensitivity, quality control, cost efficiency, and clinical integration, POCT technology continues to undergo consistent improvements. Advances in nanomaterials, microfluidic engineering, and artificial intelligence and continuous improvements in industry standardization have led to increased performance, systematic integration, and intelligent detection in POCT. Given these technological advancements and optimizations, POCT shows promising applicability and is therefore expected to become a core diagnostic tool for both precision clinical management and epidemic prevention and control in public health.},
}

@article {pmid42979655,
year = {2026},
author = {Dionisio, G and Tiwari, S and Jogam, P},
title = {Editorial: CRISPR/CAS innovations: advancing precision and specificity in plant genetic engineering.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {2012559},
pmid = {42979655},
issn = {1664-462X},
}

@article {pmid42980178,
year = {2026},
author = {Mogila, I and Smalakyte, D and Tamulaitis, G},
title = {Expanding arsenal of type III CRISPR-Cas antiviral defense.},
journal = {FEMS microbiology reviews},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsre/fuag057},
pmid = {42980178},
issn = {1574-6976},
abstract = {CRISPR-Cas systems are adaptive immune systems that protect prokaryotes from invading genetic elements through acquisition of foreign nucleic acid sequences and RNA-guided recognition of target molecules. Among the diverse CRISPR-Cas families, type III systems represent a highly complex and versatile branch that couples sequence-specific interference with nucleotide-based signaling and activation of diverse downstream immune responses. Recent discoveries have substantially expanded the functional diversity of type III CRISPR-Cas systems, revealing new subtypes, ancillary effectors, nucleotide second messengers, signal-degrading enzymes, and viral counter-defense strategies. Here, we summarize current mechanistic and structural insights into type III CRISPR-Cas biology, from adaptation and target recognition to immune signaling, ancillary effector-mediated responses, and defense regulation. We further discuss emerging biotechnological applications of type III systems and highlight key unanswered questions shaping future research in this rapidly evolving field.},
}

@article {pmid42980409,
year = {2026},
author = {Borah, M and Gautron, N and Courdavault, V and Naseri, G},
title = {FASTOP─Fast Editing Toolkit for Top Expression Sites in Yeast.},
journal = {Journal of agricultural and food chemistry},
volume = {74},
number = {43},
pages = {34106-34115},
pmid = {42980409},
issn = {1520-5118},
support = {NA 1650/4-1//German Research Council (DFG)/ ; },
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; Flavanones/biosynthesis ; *Gene Editing/methods ; CRISPR-Cas Systems ; Saccharomyces cerevisiae Proteins/metabolism/genetics ; },
abstract = {Budding yeast Saccharomyces cerevisiae is a widely used chassis for producing food- and agriculture-related compounds, but construction of multienzyme pathways often requires repeated genomic integration. We developed FASTOP, a genome-editing toolkit for rapid engineering of eight transcriptionally active genomic hot-spot loci. FASTOP combines selectable markers, extended homology arms, and Cas9/gRNA-assisted integration, and enables PCR-free release of donor cassettes by restriction digestion to reduce sequence errors introduced during amplification. After characterizing the sites, we integrated a five-gene naringenin biosynthetic pathway into the hot spots as a proof of concept. Our results demonstrated that, upon expressing the five essential genes in simple shake-flask culture, naringenin production reached 505.7 mg/L, 6-fold higher than an episomal control expressing the same pathway genes and 4.5-fold higher than the previously reported strain using additional precursor engineering and controlled bioreactor cultivation. FASTOP therefore provides a practical platform for the rapid assembly of multienzyme pathways in yeast for stable heterologous gene expression.},
}

*Saccharomyces cerevisiae/genetics/metabolism
Flavanones/biosynthesis
*Gene Editing/methods
CRISPR-Cas Systems
Saccharomyces cerevisiae Proteins/metabolism/genetics

@article {pmid42982292,
year = {2026},
author = {Ghoreyshizadeh, E and Namaki, M and Naeimi, F and Sedaghat, FR and Rezaee, MA and Kafil, HS},
title = {The plasmid arsenal within: Type IV-A CRISPR-Cas systems in Enterobacteriaceae as modulators of resistance and virulence.},
journal = {Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases},
volume = {146},
number = {},
pages = {106053},
doi = {10.1016/j.meegid.2026.106053},
pmid = {42982292},
issn = {1567-7257},
abstract = {The Enterobacteriaceae family, which includes prominent pathogens such as K. pneumoniae, E. coli, and S. enterica, poses a significant global health threat due to the rapid dissemination of multidrug resistance and virulence determinants. Central to this dissemination are conjugative plasmids, which act as vehicles for horizontal gene transfer. Among the myriad defense systems that interact with these mobile genetic elements, the type IV-A CRISPR-Cas system stands out due to its enigmatic nature and unique plasmid-centric biology. Unlike canonical CRISPR-Cas systems that are chromosomal and target bacteriophages, type IV-A systems are predominantly encoded on large, conjugative plasmids, particularly those bearing IncHI1B/IncFIB replicons. This review synthesizes current evidence on the biology of type IV-A systems within Enterobacteriaceae, elucidating their role as mediators of inter-plasmid competition, their paradoxical association with the spread of antibiotic resistance and virulence genes, and their emerging applications as programmable tools. We explore the mechanistic basis for their function as a natural CRISPR interference (CRISPRi) system, the epidemiological landscape of their distribution, and the dual role they play in both constraining and facilitating the dissemination of clinically relevant traits. Finally, we propose ambitious research avenues, including the development of "plasmid-destroying" probiotics, the use of type IV-A systems as epidemiological biosensors, and the exploration of hypotheses concerning their role in mediating bacterial "altruism" or serving as a "genetic memory" of the plasmid "secretoome."},
}

@article {pmid42982423,
year = {2026},
author = {He, Z and Zhang, J and Kuang, S and Li, S and Chen, J and Yang, J and Ma, Z and Ding, J and Zhang, B and Zhang, Y},
title = {Ecological and Molecular Basis of Capripoxvirus Cross-Host Transmission: From Transmission Networks to Adaptive Evolution.},
journal = {Veterinary dermatology},
volume = {},
number = {},
pages = {},
doi = {10.1111/vde.70138},
pmid = {42982423},
issn = {1365-3164},
support = {2022D01C697//Xinjiang Tianchi Doctoral Project/ ; },
abstract = {BACKGROUND: Capripoxvirus (CaPV), comprising sheeppox virus (SPPV), goatpox virus (GTPV) and lumpy skin disease virus (LSDV), represents a growing transboundary threat to global livestock, with expanding geographic distribution and increasingly prominent cross-species transmission in mixed farming systems.

OBJECTIVES: This review systematically examines the multifaceted drivers of CaPV spillover from ecological, molecular and evolutionary perspectives.

METHODS: This review integrates ecological, molecular and evolutionary analyses, incorporating mathematical modelling and summarising advances in CRISPR/Cas-based diagnostics.

RESULTS: Ecologically, a tripartite network involving habitat sharing, landscape permeability (e.g., forest farmland edges) and arthropod vectors is proposed; mathematical modelling identifies vector-to-host ratio and vaccination coverage as critical modulators of outbreak severity. Molecularly, genetic plasticity in terminal genomic regions (e.g., ankyrin family) and host range genes (HRGs) such as M062R, M159, and LSDV012 enable antagonism of host restriction factors including SAMD9, IFIT1 and PKR, with the C7L superfamily playing a pivotal role in breaching species barriers. Evolutionarily, recombination-driven diversification, immune selection pressure and genetic bottlenecks collectively shape adaptive radiation and frequent host jumping events. Advances in CRISPR/Cas-based diagnostics are summarised. However, live attenuated vaccines show limitations in safety and efficacy.

Persistent challenges include ecological surveillance, molecular discrimination of the three species and vaccine-wild type recombination risks. By integrating these disciplines, this review offers a comprehensive framework for understanding CaPV cross-species transmission and supports the design of precision control strategies targeting ecological and molecular vulnerabilities.},
}

@article {pmid42983524,
year = {2026},
author = {Matvere, A and Menchon, G and Martinetti, L and Menant, A and Callac, L and Ferrari, H and Jarry, U and Gourdel, M and Bore, A and Chassignolle, M and Le Bars, V and Cadieu, E and Derrien, T and Aubry, M and Gilot, D and Pedeux, R},
title = {CRISPR screens reveal MCT1-driven metabolic escape from SMAD3 inhibition as a therapeutic vulnerability.},
journal = {Molecular cancer},
volume = {25},
number = {1},
pages = {},
pmid = {42983524},
issn = {1476-4598},
support = {Marie Skłodowska-Curie grant agreement No 899546//European Union's Horizon 2020 research and innovation program/ ; Campustart fellowship from Campus Innovation//Campus Innovation, University of Rennes/ ; ANR-10-EQPX-03 and ANR-10-INBS-09-08//Agence Nationale de la Recherche/ ; INCa-DGOS-465 and INCa-DGOS-Inserm_12554//SiRIC/ ; Plan Cancer III//ITMO-Cancer Aviesan/ ; lncRESIST//Canceropole Grand Ouest grant/ ; C20013NS//Inserm, Centre Eugene Marquis, University of Rennes, Region Bretagne with financial support from ITMO Cancer of Aviesan within the framework of the 2021-2030 Cancer Control Strategy/ ; },
mesh = {*Monocarboxylic Acid Transporters/metabolism/genetics/antagonists & inhibitors ; Monocarboxylate Transport Protein 1 ; Humans ; *Smad3 Protein/antagonists & inhibitors/metabolism/genetics ; Animals ; *Symporters/genetics/metabolism/antagonists & inhibitors ; Mice ; Metabolic Reprogramming ; Cell Line, Tumor ; Drug Resistance, Neoplasm/genetics ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Proto-Oncogene Proteins B-raf/genetics ; Melanoma/genetics/metabolism/drug therapy/pathology ; },
abstract = {Resistance to therapy remains a critical challenge in cancer. This is exemplified by TGF-β pathway inhibitors which, despite broad clinical testing, have failed to deliver survival benefit. More precise SMAD3 targeting has shown preclinical promise, yet resistance mechanisms to SMAD3 inhibition remain entirely unknown. Using complementary genome-wide CRISPR activation and knockout screens, we discover that cancer cells escape SMAD3 inhibition through metabolic reprogramming. Strikingly, this escape is driven not by transcriptional bypass but by the lactate transporter MCT1, with the effect preserved across BRAF-mutated melanoma, KRAS-mutated lung adenocarcinoma, and mouse melanoma. Mechanistically, SMAD3 inhibition creates energetic stress that cancer cells exploit through MCT1. By conferring metabolic flexibility and switching toward glycolysis under drug pressure, MCT1 increases anabolic activity to drive lipid and cholesterol synthesis. Pharmacological and genetic MCT1 inhibition synergizes strongly with SMAD3 blockade to impair tumor viability in BRAF-mutated melanoma, while suppressing drug-tolerant cell emergence in BRAF inhibitor-resistant cells. Critically, this synergy extends to preclinical models, where co-inhibition of SMAD3 and MCT1 potently reduces tumor growth in vivo. Given the additional and well-established role of MCT1 in shaping the tumor immune microenvironment via lactate transport, these results position MCT1 as both a metabolic and immunological target and provide a strong rationale for the repurposing of TGF-β/SMAD3 pathway inhibitors in combination.},
}

*Monocarboxylic Acid Transporters/metabolism/genetics/antagonists & inhibitors
Monocarboxylate Transport Protein 1
Humans
*Smad3 Protein/antagonists & inhibitors/metabolism/genetics
Animals
*Symporters/genetics/metabolism/antagonists & inhibitors
Mice
Metabolic Reprogramming
Cell Line, Tumor
Drug Resistance, Neoplasm/genetics
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Proto-Oncogene Proteins B-raf/genetics
Melanoma/genetics/metabolism/drug therapy/pathology

@article {pmid42983748,
year = {2026},
author = {Liu, H and Zhang, Y and Wen, H and Lu, J and Gao, W and Wang, W},
title = {RPA-CRISPR/Cas12-based rapid visual on-site detection method for bovine Cryptosporidium.},
journal = {BMC veterinary research},
volume = {22},
number = {1},
pages = {},
pmid = {42983748},
issn = {1746-6148},
support = {No. 2023-JSGC-5)//The National Center of Technology Innovation for Dairy/ ; Study on Genome Breeding of Cashmere Goat and Prevention and Control Technology of Pathogenic Diarrhea in Lambs//Ejin Horo Banner Unveiled Project/ ; },
mesh = {Animals ; Cattle ; *Cryptosporidium/isolation & purification/genetics ; *Cattle Diseases/diagnosis/parasitology ; *CRISPR-Cas Systems ; *Cryptosporidiosis/diagnosis/parasitology ; Sensitivity and Specificity ; Rapid Diagnostic Tests ; *Nucleic Acid Amplification Techniques/veterinary/methods ; RNA, Ribosomal, 18S/genetics ; },
abstract = {BACKGROUND: Four types of Cryptosporidium (C. parvum, C. bovis, C. ryanae, and C. andersoni) and mixed infections were common among cattle in the Inner Mongolia Autonomous Region of China, causing severe harm. For grassroots farms and pastures, the prevention and control of bovine Cryptosporidium requires simultaneous detection of the four major Cryptosporidium species, and a one-time diagnosis of bovine Cryptosporidium without distinguishing between species is necessary to achieve more efficient prevention and treatment. In this study, recombinant polymerase amplification (RPA) was combined with the CRISPR/Cas system to design crRNAs and screen RPA primers targeting the 18S rRNA genes of four bovine Cryptosporidium species that primarily infect cattle.

RESULTS: Detection results were presented using two distinct single-stranded DNA reporter molecules via fluorescence quantification and lateral flow immunochromatography assay (LFIA) test strips. Using specificity and sensitivity assessments on clinical samples, the RPA-CRISPR/Cas12 visual on-site rapid detection method for bovine Cryptosporidium established in this study was completed within 60 min, with a minimum detection limit of 0.1 copies/µL. There was no cross-reactivity with the six pathogens: Haemonchus contortus, coccidia, Cysticercus tenuicollis, trematode, Caenorhabditis elegans, and tapeworm. Four types of bovine Cryptosporidium were specifically detected.

CONCLUSION: Clinical detection showed an 86% positivity rate for bovine Cryptosporidium, consistent with the Cryptosporidium nested PCR method (National Standard). Therefore, the RPA-CRISPR/Cas12a rapid on-site detection method established in this study has high specificity and sensitivity, convenience, and speed, without requiring specialized operational steps or expensive equipment.},
}

Animals
Cattle
*Cryptosporidium/isolation & purification/genetics
*Cattle Diseases/diagnosis/parasitology
*CRISPR-Cas Systems
*Cryptosporidiosis/diagnosis/parasitology
Sensitivity and Specificity
Rapid Diagnostic Tests
*Nucleic Acid Amplification Techniques/veterinary/methods
RNA, Ribosomal, 18S/genetics

@article {pmid42985255,
year = {2026},
author = {Malitha, MD and Jain, H and Wolobah, Z and Munzeiwa, WA and Singh, L and Sharma, NK},
title = {CRISPR-integrated nanobiosensors for rapid and ultra-sensitive food allergen detection: a comprehensive review.},
journal = {Food science and biotechnology},
volume = {35},
number = {13},
pages = {3521-3538},
pmid = {42985255},
issn = {2092-6456},
abstract = {UNLABELLED: Reliable allergen detection in increasingly complex and globally distributed food systems demands analytical platforms that overcome the limitations of ELISA, PCR, and traditional biosensors. CRISPR-based diagnostics provide exceptional specificity and programmability, yet their practical performance depends heavily on signal amplification efficiency, enzyme stability, and tolerance to inhibitors commonly present in processed food matrices. Integrating nanomaterials with CRISPR systems offers powerful enhancements in sensitivity, kinetics, and matrix robustness, but a unified evaluation of this convergence is still lacking. This review critically examines CRISPR-nanotechnology-enabled allergen detection by integrating insights on biomarker selection, Cas enzyme architectures, nanomaterial-mediated signal engineering, and emerging device formats including microfluidics, lateral flow assays, and smartphone-based platforms. Persistent challenges such as quantification, multiplexing, reagent stability, and regulatory validation are highlighted. By mapping these technological dependencies and bottlenecks, this review provides a clear roadmap for advancing CRISPR-integrated nanobiosensors toward rapid, ultra-sensitive, and field-deployable allergen monitoring solutions.

SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at https://doi.org/10.1007/s10068-026-02206-9.},
}

@article {pmid42985595,
year = {2026},
author = {, },
title = {Correction: Antimicrobial peptides in the era of resistance: integrating AI-driven design, host-microbe interactions, and nanotechnological delivery.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {2021107},
doi = {10.3389/fmicb.2026.2021107},
pmid = {42985595},
issn = {1664-302X},
abstract = {[This corrects the article DOI: 10.3389/fmicb.2026.1946449.].},
}

@article {pmid42985920,
year = {2026},
author = {Dong, S and Zhao, S and Deng, J and Zhang, L and Sun, J},
title = {CRISPR-Cas Assays for Protein Detection: Strategies and Clinical Applications.},
journal = {Small methods},
volume = {},
number = {},
pages = {},
doi = {10.1002/smtd.71186},
pmid = {42985920},
issn = {2366-9608},
support = {2021YFA0909400//National Key R&D Program of China/ ; 2025YFF0518300//National Key R&D Program of China/ ; 22227805//National Natural Science Foundation of China/ ; 22534001//National Natural Science Foundation of China/ ; 22574035//National Natural Science Foundation of China/ ; YSBR-036//CAS Project for Young Scientists in Basic Research/ ; XDA0580000//the Strategic Priority Research Program of the Chinese Academy of Sciences/ ; 2025M780886//China Postdoctoral Science Foundation/ ; 20250673//Young Elite Scientists Sponsorship Program of the Beijing High Innovation Plan/ ; },
abstract = {Measurement of protein biomarkers with high accuracy is critical for disease diagnostics and prognostic monitoring. However, conventional analytical platforms for protein detection suffer from insufficient sensitivity for low-abundance targets and require time-consuming, multi-step workflows. The CRISPR-Cas system is emerging as a powerful tool for protein analysis, with advantages including high programmability, excellent sensitivity, and compatibility with diverse readouts. This review presents a systematic overview of recent advances in CRISPR-based protein assays. We first summarize the design principles of aptamer-, antibody-, and enzyme-mediated systems that convert protein recognition events into CRISPR-activatable nucleic acid signals. We next examine the signal amplification and readout strategies, such as direct CRISPR activation, enzyme-mediated nucleic acid amplification, and enzyme-free DNA nanotechnology, along with colorimetric, fluorescent, or electrochemical signal outputs. We also review applications of these assays in infectious diseases, cancer, and Alzheimer's disease. Finally, we discuss challenges and perspectives for next-generation CRISPR-based protein detection platforms in clinical translation.},
}

@article {pmid42986779,
year = {2026},
author = {Lilić, B and Cianciulli Sesso, A and Berghoff, B and Moll, I and Sonnleitner, E and Bläsi, U},
title = {The small protein PhrS-P, encoded by the dual-function small RNA PhrS, augments fitness of Pseudomonas aeruginosa in the presence of aminoglycosides.},
journal = {Microbiology (Reading, England)},
volume = {172},
number = {11},
pages = {},
doi = {10.1099/mic.0.001742},
pmid = {42986779},
issn = {1465-2080},
mesh = {*Pseudomonas aeruginosa/genetics/drug effects/metabolism/growth & development/physiology ; *Aminoglycosides/pharmacology ; *Bacterial Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; *Anti-Bacterial Agents/pharmacology ; *RNA, Bacterial/genetics/metabolism ; *RNA, Small Untranslated/genetics/metabolism ; Quorum Sensing/genetics ; Drug Resistance, Bacterial/genetics ; Biofilms/growth & development ; },
abstract = {PhrS (Pseudomonas Hfq-binding RNA S) is a dual-function small RNA of Pseudomonas aeruginosa. The base-pairing small regulatory RNA PhrS-R positively regulates pyocyanin biosynthesis. PhrS-R stimulates the production of the Pseudomonas quinolone signal (PQS) by indirectly activating the translation of pqsR mRNA, encoding the quorum sensing regulator PqsR (MvfR), and by inhibiting the synthesis of the anthranilate degradation regulator AntR. Furthermore, PhrS-R was shown to promote biofilm formation, swarming motility and CRISPR-Cas immunity. In addition, PhrS encodes a 37 amino acid long and conserved small protein (PhrS-P), the function of which remained unknown. Here, we provide evidence that PhrS-P is an inner membrane protein which does not contribute to the known physiological roles of its RNA counterpart. RNA-Seq-based transcriptome analyses revealed that multiple aminoglycoside resistance genes, including those of the AmgR regulon, are differentially expressed depending on PhrS-P. AmgR is the response regulator of the AmgRS two-component system (TCS), which affects aminoglycoside and zinc resistance in P. aeruginosa. A PhrS-P-deficient mutant strain exhibited reduced fitness when exposed to sub-inhibitory concentrations of the aminoglycosides tobramycin and amikacin as well as zinc. Based on our findings, we present a working model wherein PhrS-P positively regulates the AmgRS TCS activity through an unknown mechanism, leading to increased bacterial fitness in the presence of aminoglycosides.},
}

*Pseudomonas aeruginosa/genetics/drug effects/metabolism/growth & development/physiology
*Aminoglycosides/pharmacology
*Bacterial Proteins/genetics/metabolism
Gene Expression Regulation, Bacterial
*Anti-Bacterial Agents/pharmacology
*RNA, Bacterial/genetics/metabolism
*RNA, Small Untranslated/genetics/metabolism
Quorum Sensing/genetics
Drug Resistance, Bacterial/genetics
Biofilms/growth & development

@article {pmid42987788,
year = {2026},
author = {Xu, L and Bao, Z},
title = {A VIPR that wraps: A triplex biochemistry for next-gen biotechnology.},
journal = {Molecular cell},
volume = {86},
number = {21},
pages = {4320-4322},
doi = {10.1016/j.molcel.2026.09.038},
pmid = {42987788},
issn = {1097-4164},
mesh = {*Biotechnology/methods ; *CRISPR-Cas Systems ; *Bacterial Proteins/genetics/chemistry/metabolism ; *DNA/genetics/metabolism/chemistry ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; },
abstract = {A putative ancestor to CRISPR systems, VIPR, was recently discovered.[1][,][2] VIPR adopts a distinct non-contiguous code for PAM-free RNA-guided DNA recognition and is ultracompact and reprogrammable, making it a promising candidate for various genome-manipulation applications. Many open questions remain and their answers will foster exciting opportunities for next-generation biotechnology.},
}

*Biotechnology/methods
*CRISPR-Cas Systems
*Bacterial Proteins/genetics/chemistry/metabolism
*DNA/genetics/metabolism/chemistry
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism

@article {pmid41027168,
year = {2026},
author = {Sarac, B and Yücer, S and Ciftci, F},
title = {Synthetic biology-driven biosensors for healthcare applications: A roadmap toward programmable and intelligent diagnostics.},
journal = {Biosensors & bioelectronics},
volume = {291},
number = {},
pages = {118036},
doi = {10.1016/j.bios.2025.118036},
pmid = {41027168},
issn = {1873-4235},
mesh = {*Biosensing Techniques/instrumentation/methods ; *Synthetic Biology/methods/instrumentation ; Humans ; Environmental Monitoring/instrumentation/methods ; Point-of-Care Systems ; Artificial Intelligence ; CRISPR-Cas Systems ; },
abstract = {Synthetic biology has revolutionized biosensor design by enabling programmable, modular systems that integrate biological components with engineered logic. This review explores recent innovations in synthetic biology-driven biosensors, highlighting applications in healthcare, environmental monitoring, and point-of-care diagnostics. Central to these advancements are synthetic gene circuits, CRISPR-based control systems, RNA regulators, and logic gate architectures enabling high specificity, multiplexed detection, and memory-enabled response. Both whole-cell and cell-free platforms have been developed for detecting pathogens, cancer biomarkers, metabolic imbalances, and environmental contaminants. Notably, wearable and paper-based devices now offer real-time monitoring with minimal infrastructure. In medical applications, engineered biosensors show promise for early diagnosis, personalized treatment monitoring, and integrated theranostics, including systems that trigger therapeutic responses upon biomarker detection. Environmental applications include the detection of heavy metals like arsenic and cadmium via genetically modified microbial platforms. Despite significant advances, challenges remain in circuit stability, biosafety, and large-scale deployment. Future directions involve integrating artificial intelligence, feedback-regulated systems, and hybrid materials to enhance sensor adaptability and performance. This review provides a comprehensive framework for developing next-generation biosensors that merge biological intelligence with engineering precision to meet the growing demand for smart, responsive, and scalable diagnostic tools.},
}

*Biosensing Techniques/instrumentation/methods
*Synthetic Biology/methods/instrumentation
Humans
Environmental Monitoring/instrumentation/methods
Point-of-Care Systems
Artificial Intelligence
CRISPR-Cas Systems

@article {pmid41038010,
year = {2026},
author = {Fan, Y and Pan, W and Xiu, Q and Qiao, Y and Zhang, P and Liao, T and Feng, J and Nie, C and Chu, S and Li, N and Wu, X and Zheng, L and Cai, Z and Li, B},
title = {Cationic LIposome capsulated CRISPR-Cas13a Kit (CLICK) for in situ detection of circulating PD-L1 mRNA in single EV towards monitoring of immunotherapy efficacy.},
journal = {Biosensors & bioelectronics},
volume = {291},
number = {},
pages = {118032},
doi = {10.1016/j.bios.2025.118032},
pmid = {41038010},
issn = {1873-4235},
mesh = {Humans ; *RNA, Messenger/blood/genetics/isolation & purification ; *B7-H1 Antigen/genetics/blood ; Immunotherapy ; Liposomes/chemistry ; *Extracellular Vesicles/chemistry/genetics ; CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Biomarkers, Tumor/blood/genetics ; *Neoplasms/blood/therapy/genetics ; Cations/chemistry ; },
abstract = {Tumor immunotherapy has significantly improved cancer treatment, yet immune checkpoint inhibitors only benefit a subset of patients. A critical challenge is non-invasive detection of tumor PD-L1 mRNA, a key biomarker that dynamically reflects PD-L1 expression and correlates with therapeutic outcomes. Current methods are hindered by the instability of circulating mRNA and the need for invasive tissue biopsies. Herein, we introduce the Cationic LIposome capsulated CRISPR-Cas13a Kit (CLICK) for in-situ detection of circulating PD-L1 mRNA in extracellular vesicles (EVs). CLICK achieves direct target identification in 10 μL plasma within 2 h without EV purification or RNA extraction, demonstrating a detection limit of 10[3] EV/mL for PD-L1 mRNA. By integrating membrane fusion-mediated CRISPR delivery with nanoflow cytometry, CLICK resolves PD-L1 mRNA (+) EV subpopulations at single-vesicle resolution, revealing heterogeneous distributions. Clinically validated in 53 immunotherapy patients, CLICK accurately stratifies progressive disease (PD) from stable disease (SD) patients (AUC = 0.8236; 95 % CI: 0.7054-0.9417; p = 0.0006) based on PD-L1 mRNA (+) EV percentages. This work establishes a rapid, blood-based paradigm for therapy response monitoring, advancing liquid biopsy through precise EV RNA characterization.},
}

Humans
*RNA, Messenger/blood/genetics/isolation & purification
*B7-H1 Antigen/genetics/blood
Immunotherapy
Liposomes/chemistry
*Extracellular Vesicles/chemistry/genetics
CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
Biomarkers, Tumor/blood/genetics
*Neoplasms/blood/therapy/genetics
Cations/chemistry

@article {pmid41794221,
year = {2026},
author = {Zhu, K and Liu, H and Zhou, K and Cheng, T and Mo, L and Xu, PF and Li, Y},
title = {A novel zebrafish model unveils folate deficiency disrupts embryonic development via p53-driven cell cycle arrest.},
journal = {Journal of advanced research},
volume = {89},
number = {},
pages = {363-377},
doi = {10.1016/j.jare.2026.02.054},
pmid = {41794221},
issn = {2090-1224},
mesh = {Animals ; *Zebrafish/embryology/genetics/metabolism ; *Folic Acid Deficiency/metabolism/genetics ; *Tumor Suppressor Protein p53/metabolism/genetics ; *Embryonic Development/genetics ; *Cell Cycle Checkpoints/genetics ; Female ; Disease Models, Animal ; *Zebrafish Proteins/metabolism/genetics ; Folic Acid/metabolism ; Mitochondria/metabolism ; CRISPR-Cas Systems ; DNA Damage ; },
abstract = {INTRODUCTION: Folate (vitamin B9) is a fundamental cofactor in one-carbon metabolism, essential for embryonic development. While maternal folate deficiency is a well-established risk factor for neural tube and congenital heart defects, its specific impact on early embryogenesis, particularly during early post-blastula stages, and the underlying molecular mechanisms remain insufficiently characterized.

OBJECTIVES: This study aimed to establish a vertebrate model to facilitate real-time visualization and mechanistic dissection of how folate deficiency drives diverse developmental defects.

METHODS AND RESULTS: We used CRISPR/Cas9 to generate a folrΔ1 zebrafish line harboring a one-nucleotide deletion in the folate receptor gene (folr), resulting in maternal folate deficiency. Embryos derived from folrΔ1 homozygote females exhibited embryonic lethality and defective dorsoventral patterning. Untargeted metabolomics analysis revealed that folate deficiency disrupts nucleotide biosynthesis and mitochondrial homeostasis. Specifically, maternal folate deficiency impaired embryonic DNA synthesis, exacerbated DNA damage, and induced G1/S phase cell cycle arrest. It also compromised mitochondrial integrity, triggering compensatory mitophagy. Notably, suppression of p53 activation in folr mutants improved dorsoventral patterning and alleviated cell cycle arrest. However, it did not mitigate the extent of DNA damage or mitophagy, suggesting p53 acts downstream of these metabolic stresses.

CONCLUSION: We have established a robust zebrafish model of maternal folate deficiency that recapitulates key metabolic and developmental features of the human condition. Our findings demonstrated that folate deficiency disrupts metabolism, leading to DNA damage and mitochondrial dysfunction. Crucially, this pathology activates p53, which drives G1/S cell cycle arrest and severe embryonic defects, including impaired body axis formation. This model provides a powerful platform for further delineating the precise roles of folate in vertebrate development and in the pathogenesis of congenital anomalies.},
}

Animals
*Zebrafish/embryology/genetics/metabolism
*Folic Acid Deficiency/metabolism/genetics
*Tumor Suppressor Protein p53/metabolism/genetics
*Embryonic Development/genetics
*Cell Cycle Checkpoints/genetics
Female
Disease Models, Animal
*Zebrafish Proteins/metabolism/genetics
Folic Acid/metabolism
Mitochondria/metabolism
CRISPR-Cas Systems
DNA Damage

@article {pmid42800597,
year = {2026},
author = {Melo, DM and Sarquis, SR and Luz, PM and Barcellos Bevitori, RC and de Almeida Barbosa, Y and Pagotto, RF and Trancozo, M and de Vargas Wolfgramm Dos Santos, E and de Paula, F},
title = {In vitro validation of a CRISPR/Cas9 guide RNA targeting COL1A1 gene by cleavage assay.},
journal = {Methods (San Diego, Calif.)},
volume = {256},
number = {},
pages = {102-108},
doi = {10.1016/j.ymeth.2026.09.004},
pmid = {42800597},
issn = {1095-9130},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Collagen Type I/genetics ; Collagen Type I, alpha 1 Chain ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Exons ; Osteogenesis Imperfecta/genetics ; },
abstract = {The CRISPR/Cas9 system has become a widely used tool for genome editing; however, editing efficiency is largely determined by the design and functional activity of the guide RNA (gRNA). Experimental validation prior to cellular applications is particularly important when targeting genes associated with genetic disorders, such as COL1A1, which is frequently mutated in Osteogenesis Imperfecta. This study performed an in vitro functional validation of a CRISPR/Cas9 gRNA designed to target exon 2 of the COL1A1 gene, using a cell-free cleavage assay as a preliminary step before cellular genome editing applications. Three gRNAs targeting distinct COL1A1 exons were designed using bioinformatics tools; the exon 2 candidate was selected for experimental validation based on predicted efficiency, favorable off-target profile, and successful PCR amplification of its target region. The selected gRNA was assembled with recombinant Cas9 protein into a ribonucleoprotein complex, which was incubated with PCR-amplified DNA substrates derived from five distinct human cell lines. Cleavage products were resolved by polyacrylamide gel electrophoresis and quantified by densitometric analysis. The gRNA directed reproducible, specific Cas9-mediated cleavage at the intended COL1A1 exon 2 target site across all cell lines tested, with mean cleavage efficiencies ranging from 58.79% to 68.84%. These results confirm functional validation of the gRNA through in vitro cleavage activity and support its candidate selection for subsequent cellular studies targeting COL1A1, representing an initial step toward future knockout-style cellular models of Osteogenesis Imperfecta.},
}

Humans
*CRISPR-Cas Systems/genetics
*Collagen Type I/genetics
Collagen Type I, alpha 1 Chain
*RNA, Guide, CRISPR-Cas Systems/genetics
*Gene Editing/methods
Exons
Osteogenesis Imperfecta/genetics

@article {pmid42880244,
year = {2026},
author = {Thapa, C and Oh, EK and Sirkin, D and Lahey, J and Díaz de León Guerrerro, S and McCarroll, A and Gowda, P and Zhang, H and Barishman, A and Peyton, L and Zhang, S and Pollak, RM and Hart, RP and Pato, CN and Kreimer, A and Mulle, JG and Sanders, AR and Pang, ZP and Duan, J},
title = {Female iPSC X chromosome inactivation erosion and its transcriptomic effects during CRISPR gene editing and neural differentiation.},
journal = {Stem cell reports},
volume = {21},
number = {11},
pages = {103120},
doi = {10.1016/j.stemcr.2026.103120},
pmid = {42880244},
issn = {2213-6711},
mesh = {*X Chromosome Inactivation/genetics ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; Female ; *Cell Differentiation/genetics ; *Neurons/cytology/metabolism ; *Gene Editing ; *Transcriptome/genetics ; RNA, Long Noncoding/genetics ; *CRISPR-Cas Systems/genetics ; Genes, X-Linked ; Epigenesis, Genetic ; },
abstract = {Human induced pluripotent stem cells (hiPSCs), together with CRISPR editing, are commonly used for studying neurodevelopmental disorders. Female iPSCs gradually lose the expression of the noncoding X chromosome inactive-specific transcript (XIST), leading to random X chromosome inactivation (XCI) erosion that affects both X-linked and autosomal gene expression. However, the effects of CRISPR editing and neural differentiation on XCI erosion and how this may confound a real-world analysis of differentially expressed genes (DEGs) are poorly understood. Here, we analyzed hundreds of RNA sequencing (RNA-seq) samples of CRISPR-edited iPSC lines for 66 genes and the matched single-neuron RNA-seq data. We found variable XCI in CRISPR-edited female iPSCs, which was largely preserved in iPSC-derived neurons and predominantly influenced X-linked genes. Mechanistically, XIST mediates allele-specific epigenetic regulation of both X-linked and autosomal genes, with the former showing stronger allele-specific expression (ASE) bias that affects neurodevelopmental genes. These results have significant implications in hiPSC-based modeling of neurodevelopmental disorders.},
}

*X Chromosome Inactivation/genetics
*Induced Pluripotent Stem Cells/metabolism/cytology
Humans
Female
*Cell Differentiation/genetics
*Neurons/cytology/metabolism
*Gene Editing
*Transcriptome/genetics
RNA, Long Noncoding/genetics
*CRISPR-Cas Systems/genetics
Genes, X-Linked
Epigenesis, Genetic

@article {pmid42971974,
year = {2026},
author = {Xu, T and Zhang, Y and Tang, X and Wei, H and Kang, L and Zhou, Q and Li, S and Zhao, X and Li, J and Zhang, Z and Wan, C and Zhang, Y and Chen, D and Tian, Y and Liu, BF and Chen, P},
title = {Reframing decentralized diagnostics with a programmable multi-dimensional microfluidic system.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42971974},
issn = {2041-1723},
support = {22574058//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32201179//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32571640//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; Smartphone ; Rapid Diagnostic Tests ; Point-of-Care Systems ; *Microfluidic Analytical Techniques/methods/instrumentation ; Immunoassay/methods/instrumentation ; *Microfluidics/methods/instrumentation ; CRISPR-Cas Systems ; Sensitivity and Specificity ; Nucleic Acid Amplification Techniques/methods/instrumentation ; *Lab-On-A-Chip Devices ; },
abstract = {Decentralized diagnostics are vital for infectious disease control, yet current point-of-care tools remain largely one-dimensional, failing to capture the multidimensional profiles required for precision management. Here, we show SID-WAVES (Smartphone-Integrated Diagnostics with Wax-encoded Amplified and Versatile Evaluation System), an intelligent, multimodal microfluidic platform. Integrating low-melting-point phase-change and dual-siphon valves with RPA-CRISPR/Cas12a nucleic acid detection and protein immunoassays, the platform enables automated, one-step, sample-to-answer operation via an AI-assisted smartphone readout that reduces operator-dependent variability. Spatial-temporal-signal encoding supports multiplexed, multi-sample, and multimodal analysis, achieving attomolar-level sensitivity (10[-18] M) and >95% diagnostic accuracy across 225 clinical and self-collected molecular assays, demonstrating robust usability. For proteins, a triple-amplification strategy and hierarchical capture enhance sensitivity 125-fold, improving accuracy from 87.6% to 100% in 54 simulated samples. Functioning as a reconfigurable analytical framework where panels are changed by swapping targets and probes, this work helps mitigate the Sensitivity-Multiplexing-Accessibility trade-off, reframing decentralized diagnostics from single-target screening to multi-dimensional health profiling.},
}

Humans
Smartphone
Rapid Diagnostic Tests
Point-of-Care Systems
*Microfluidic Analytical Techniques/methods/instrumentation
Immunoassay/methods/instrumentation
*Microfluidics/methods/instrumentation
CRISPR-Cas Systems
Sensitivity and Specificity
Nucleic Acid Amplification Techniques/methods/instrumentation
*Lab-On-A-Chip Devices

@article {pmid42972957,
year = {2026},
author = {Brüssow, H},
title = {The Arms Race Between Bacteria and Their Viruses: Update on Phage Countermeasures Against Bacterial Defence Systems.},
journal = {Microbial biotechnology},
volume = {19},
number = {11},
pages = {},
pmid = {42972957},
issn = {1751-7915},
mesh = {*Bacteriophages/genetics/physiology ; *Bacteria/virology/genetics/immunology/metabolism ; Viral Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Bacterial Proteins/metabolism/genetics ; },
abstract = {In this editorial, I summarize recent progress on phage-encoded anti-defence against bacterial anti-phage systems (APS). Computational approaches based on structural predictions of co-folding bacterial and phage proteins identified many new PAD sponge proteins. New anti-CRISPR-Cas systems were characterized that destroy cas12a mRNA, inactivate Cas12a by acetylation, engage the seed region of the crRNA, or prevent Cas3 from cleaving DNA. The phage defence protein Gad1 forms an octamer web that wraps around the complex of the Gabija APS. Tad1 and 2 are phage sponge proteins that sequester signalling nucleotides of the Thoeris APS. Tad3 to 7 interact directly with the ThsB sensor and Tad8 with the ThsA effector of the Thoeris APS. Phage sponge proteins were also described inactivating the CBASS and Pycsar APS. Still other phages use phosphodiesterases that cleave cyclic nucleotide signals to inactivate APS. Many bacterial APS effectors are NADase, destroying the essential metabolite NAD[+]. Phages evolved enzymes that resynthesize NAD[+] from its cleavage products or proteins directly inactivating the NADase. Many phages degrade the bacterial genome which creates signals activating the Metis or the Kongming APS. Phages counteracted these APS by suppressing pathways leading to the creation of particular mononucleotides degradation products detected by these APS. Several APS cleave specific tRNA, phages responded by mutating cleavage sites in tRNA (anti-PARIS) or increasing tRNA expression to compensate for their destruction (anti-Septu). Phages counteract toxin-antitoxin APS by evolving their own antitoxin or by blocking the toxin protein. Researchers described cases where phages escaped detection by mutating phage proteins detected by bacterial sensors. Against the stringent response phages responded by destroying alarmones or inactivating enzymes producing these stress signals. The arms race between bacteria and phages can involve complicated layers of defence and anti-defences and both bacteria and phages encode multiple defence systems.},
}

*Bacteriophages/genetics/physiology
*Bacteria/virology/genetics/immunology/metabolism
Viral Proteins/metabolism/genetics
CRISPR-Cas Systems
Bacterial Proteins/metabolism/genetics

@article {pmid42976630,
year = {2026},
author = {Naderi, M and Sadidi, R and Besharati, R and Kebriae, A and Azimian, A},
title = {Nucleic acid amplification testing for obligate intracellular and cell wall-deficient bacteria: from assay performance to clinical interpretation, a structured narrative review.},
journal = {Diagnostic microbiology and infectious disease},
volume = {117},
number = {1},
pages = {117721},
doi = {10.1016/j.diagmicrobio.2026.117721},
pmid = {42976630},
issn = {1879-0070},
abstract = {Obligate intracellular bacteria (Rickettsia, Orientia, Ehrlichia, Anaplasma, Coxiella, Chlamydia) and cell wall-deficient Mycoplasma species present profound diagnostic challenges due to their fastidious growth, low circulating burden, and genomic instability. Nucleic Acid Amplification Tests (NAATs) have revolutionized detection, yet critical gaps persist in distinguishing active infection from persistent DNA, standardizing cycle threshold interpretation, and integrating host-response biomarkers. This structured narrative review provides a comprehensive analysis of the complete diagnostic armamentarium for these pathogens. It is based on a reproducible, non-systematic literature survey and is not a PRISMA-compliant systematic review. We cover conventional methods (culture, serology), NAAT platforms (qPCR, digital PCR, isothermal amplification), and emerging technologies including CRISPR-Cas diagnostics, metagenomic sequencing, nanopore sequencing, biosensors, and host-response biomarker profiling. We introduce four conceptual frameworks: the "biological noise" paradigm reframing low-copy detection as a signal-to-noise challenge; a temporal-dynamic algorithm accounting for pathogen-specific detection windows; the "detection-disease disconnect" emphasizing clinical contextualization of NAAT positivity; and multi-analyte integration combining NAAT with viability markers and host inflammatory signatures. We also position these frameworks relative to prior diagnostic-interpretation theories, including STARD, QUADAS-2, MIQE, Bayesian pretest-posttest reasoning, Ct-value interpretation guidance, and host-response classifier frameworks. We compare ten clinically significant pathogens across technical and biological parameters, evaluate point-of-care deployment strategies, and address antimicrobial resistance testing. Performance metrics are presented as context-dependent estimates rather than fixed constants. The next frontier lies not in incremental assay improvements but in system-level integration of pathogen detection, viability assessment, host-response profiling, and clinical phenotyping, supported by artificial intelligence-enhanced interpretation and global standardization efforts.},
}

@article {pmid42615363,
year = {2026},
author = {Xiao, Q and Tian, Z and Weng, L and Xu, K and Han, D and Wu, Z and Li, J and Li, S and Zhang, Q and Zhao, X and Li, G and Wei, Y and Gu, Z},
title = {Compact 9dBEs Enable Efficient and Precise Genome Editing in Mammalian Cells and In Vivo.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {62},
pages = {},
pmid = {42615363},
issn = {2198-3844},
support = {32501335//National Natural Science Foundation of China/ ; 52233013//National Natural Science Foundation of China/ ; 3250120657//National Natural Science Foundation of China/ ; 32441080//National Natural Science Foundation of China/ ; 2021YFA0909900//National Key R&D Program of China/ ; LQN26C050001//Zhejiang Provincial Natural Science Foundation of China/ ; 2026-3-097//Jinhua Science and Technology Plan Projects/ ; },
mesh = {*Gene Editing/methods ; Animals ; Humans ; Mice ; *CRISPR-Cas Systems/genetics ; *Proprotein Convertase 9/genetics ; Dependovirus/genetics ; },
abstract = {Despite the promise of DNA base editors for diverse genome editing applications, their utility remains constrained by off-target effects, which are exacerbated by short spacers in miniature systems and the large size of Cas9-derived editors, which impedes adeno-associated virus (AAV) delivery. Here, guided by structural insights into a compact Cas9d nuclease from Deltaproteobacteria, we developed an efficient Cas9d system (Cas9d[Ultra]) through gRNA and protein engineering, and further developed its base editors (9dBEs). Cas9d[Ultra] and 9dBEs enabled efficient and precise genome editing in human cells. Notably, 9dCBE induced premature termination codons in 89% of mouse pups by microinjection, facilitating robust disease modeling. Furthermore, a single AAV delivering 9dCBE achieved efficient Pcsk9 editing and a concomitant reduction in serum LDL-C levels in mice. Collectively, this study establishes a series of compact, potent genome editing tools poised to advance biological and biomedical translational research.},
}

*Gene Editing/methods
Animals
Humans
Mice
*CRISPR-Cas Systems/genetics
*Proprotein Convertase 9/genetics
Dependovirus/genetics

@article {pmid42735456,
year = {2026},
author = {Taha, BA and Addie, AJ and Haider, AJ and Ibnaouf, K and Apsari, R and Arsad, N},
title = {Light-activated CRISPR/dCas9 nanomedicine for programmable control of renal fibrosis.},
journal = {Journal of photochemistry and photobiology. B, Biology},
volume = {284},
number = {},
pages = {113563},
doi = {10.1016/j.jphotobiol.2026.113563},
pmid = {42735456},
issn = {1873-2682},
mesh = {Humans ; Fibrosis ; *Nanomedicine ; Animals ; *Light ; *Kidney Diseases/pathology/therapy ; *CRISPR-Cas Systems/genetics ; Kidney/pathology/metabolism ; },
abstract = {Renal fibrosis is the final common pathway of progressive chronic kidney disease and is maintained by spatially heterogeneous interactions among injured epithelial cells, activated fibroblasts, immune cells, extracellular matrix remodeling, metabolic stress, and persistent profibrotic transcriptional programs. Current therapies slow renal functional decline but do not directly control the regulatory circuits that stabilize maladaptive repair. Photoresponsive renal nanomedicine offers a potential strategy to add external control to anti-fibrotic intervention by combining kidney-directed delivery with light-gated release or activation of molecular payloads. This review examines the emerging interface between photoresponsive nanomaterials and CRISPR/dCas9-based gene regulation for renal fibrosis, with emphasis on upconversion nanoparticles, photoresponsive polymers, ROS- and pH-responsive matrices, optogenetic switches, and renal-compartment-directed carrier design. We argue that the most defensible therapeutic objective is not permanent genome editing or autonomous organ regeneration, but spatially confined, temporally limited, and reversible regulation of validated fibrotic or protective gene programs using CRISPRa, CRISPRi, or dCas9-based epigenome editors. The review therefore evaluates material requirements, optical-dosimetry constraints, payload architecture, renal biodistribution, target-selection logic, safety risks, and preclinical validation criteria. By defining the engineering and biological conditions required for controlled anti-fibrotic regulation, this framework positions photoresponsive renal nanomedicine as a translationally testable route toward localized modulation of fibrotic cell states rather than an overextended claim of kidney regeneration.},
}

Humans
Fibrosis
*Nanomedicine
Animals
*Light
*Kidney Diseases/pathology/therapy
*CRISPR-Cas Systems/genetics
Kidney/pathology/metabolism

@article {pmid42755348,
year = {2026},
author = {Nicholls, SJ},
title = {CRISPR therapy for dyslipidemia.},
journal = {Current opinion in lipidology},
volume = {37},
number = {6},
pages = {222-227},
doi = {10.1097/MOL.0000000000001062},
pmid = {42755348},
issn = {1473-6535},
mesh = {Humans ; *Dyslipidemias/genetics/therapy ; *Genetic Therapy/methods ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Animals ; },
abstract = {PURPOSE OF REVIEW: Several lipid and lipoprotein risk factors play an important role in the causality of atherosclerotic cardiovascular disease. Many therapies are limited by suboptimal long-term adherence. This narrative review summarizes the potential for gene editing and epigenetic therapy for the management of dyslipidemia.

RECENT FINDINGS: Recent advances in genomic engineering permit the ability to develop gene editing, base editing and epigenomic editing of specific regions of DNA in humans. These therapies have advanced to early phase clinical evaluation with initial reports demonstrating favorable reductions in ANGPTL3 and proprotein convertase subtilisin kexin type 9, with consequent decreases in levels of low-density lipoprotein cholesterol and triglycerides. Apart from mild-moderate infusion reactions, the early experience with these therapies appears to be well tolerated.

SUMMARY: Advances in genomic engineering have developed a number of agents with the potential for robust and durable changes to atherosclerotic lipid and lipoprotein risk factors. How these agents will enter clinical practice will require further evaluation in clinical trials.},
}

Humans
*Dyslipidemias/genetics/therapy
*Genetic Therapy/methods
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Animals

@article {pmid42826647,
year = {2026},
author = {Esmaeili, H and Das, S and Rytel, K and Sidransky, E and Chen, Y},
title = {CRISPR screening in neurodegeneration and lysosomal biology: Uncovering disease mechanisms and genetic modifiers.},
journal = {Molecular genetics and metabolism},
volume = {149},
number = {3},
pages = {110265},
pmid = {42826647},
issn = {1096-7206},
support = {Z01 HG200336/ImNIH/Intramural NIH HHS/United States ; ZIA HG200336/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Humans ; *Lysosomes/genetics/metabolism ; *Neurodegenerative Diseases/genetics ; Animals ; *Lysosomal Storage Diseases/genetics ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Genes, Modifier ; },
abstract = {Growing genetic and mechanistic evidence has highlighted substantial convergence between neurodegenerative disorders and lysosomal biology. Variants in lysosomal protein-encoding genes associated with lysosomal storage disorders (LSDs) have been implicated in common neurodegenerative diseases, suggesting that perturbations of lysosomal function represent a shared pathogenic mechanism. However, the low penetrance of neurodegenerative diseases in individuals carrying variants in these lysosomal genes suggests that additional genetic modifiers are involved as well. Advances in functional genomics, particularly clustered regularly interspaced short palindromic repeat (CRISPR)-based high-throughput screens, have opened new avenues to identify such modifiers and elucidate disease-associated pathways. These unbiased approaches have accelerated the discovery of molecular mechanisms underlying neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Although to date few studies have applied CRISPR-based functional screens directly to LSDs, findings from neurodegenerative disease models provide a valuable framework for investigating lysosomal pathways and identifying genetic factors that influence phenotypic variability. In this review, we summarize recent advances in in vitro CRISPR-based screening approaches in neurodegenerative disorders and discuss their implications for understanding lysosomal biology and LSD-related mechanisms.},
}

Humans
*Lysosomes/genetics/metabolism
*Neurodegenerative Diseases/genetics
Animals
*Lysosomal Storage Diseases/genetics
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
*Genes, Modifier

@article {pmid42838451,
year = {2026},
author = {Diener Blake, A and Zhang, Z and Alexandre, C and Gorayeb, A and Jain, M and Liu, Q and Pattali, R and Bednarska, A and Kaur Pandher, P and Zhang, S and Yue, X and Nuñez, J and Vaisse, C},
title = {Cell-type-specific repression reveals MC4R neurons as a site of action for ciliary obesity genes.},
journal = {Molecular metabolism},
volume = {114},
number = {},
pages = {102454},
pmid = {42838451},
issn = {2212-8778},
support = {F31 DK137538/DK/NIDDK NIH HHS/United States ; R01 DK124769/DK/NIDDK NIH HHS/United States ; R35 GM155044/GM/NIGMS NIH HHS/United States ; S10 OD028611/OD/NIH HHS/United States ; P30 DK098722/DK/NIDDK NIH HHS/United States ; R01 DK060540/DK/NIDDK NIH HHS/United States ; R01 DK106404/DK/NIDDK NIH HHS/United States ; },
mesh = {*Receptor, Melanocortin, Type 4/metabolism/genetics ; Animals ; *Neurons/metabolism ; *Obesity/genetics/metabolism ; Mice ; Adenylyl Cyclases/metabolism/genetics ; Cilia/metabolism/genetics ; Male ; Mice, Inbred C57BL ; CRISPR-Cas Systems ; Energy Metabolism ; Body Weight/genetics ; Humans ; },
abstract = {OBJECTIVES: Understanding how obesity-associated genes function within defined neuronal subpopulations remains a major barrier to translating human genetics into pathophysiological mechanisms. Melanocortin-4 receptor-expressing (MC4R) neurons are key regulators of energy homeostasis and represent a potential site of action for these genes. Here, we investigated whether obesity-associated ciliary genes regulate body weight through MC4R neurons in vivo, using a Cre-dependent CRISPR interference (CRISPRi) strategy that enables temporal, spatial, and cell-type-specific gene repression without permanent DNA modification.

METHODS: Gene repression was achieved in MC4R neurons using a Cre-dependent CRISPRi system with AAV-delivered sgRNAs, and physiological outcomes, including body weight and body composition, were assessed following gene downregulation.

RESULTS: CRISPRi-mediated repression of Mc4r in adult PVN[MC4R] neurons reduced transcript and protein levels and induced progressive obesity, validating the approach. Downregulation of the ciliary adenylyl cyclase Adcy3 specifically in adult PVN[MC4R] neurons was sufficient to drive weight gain, establishing a cell-type-specific requirement for ADCY3 in body-weight regulation. An in vivo CRISPRi screen of obesity-associated ciliopathy genes (Bbs10, Inpp5e, Ankrd26) demonstrated that their repression in MC4R neurons is sufficient to promote obesity, identifying these neurons as a convergent site linking human ciliopathies to obesity.

CONCLUSIONS: These findings define MC4R neurons as a critical neuronal population through which multiple obesity-associated ciliary genes act and establish a direct link between ciliary function in defined neuronal populations and organismal energy balance. This work provides insight into the neural mechanisms underlying obesity and a framework for linking human genetic variations to specific neuronal populations in vivo.},
}

*Receptor, Melanocortin, Type 4/metabolism/genetics
Animals
*Neurons/metabolism
*Obesity/genetics/metabolism
Mice
Adenylyl Cyclases/metabolism/genetics
Cilia/metabolism/genetics
Male
Mice, Inbred C57BL
CRISPR-Cas Systems
Energy Metabolism
Body Weight/genetics
Humans

@article {pmid42852500,
year = {2026},
author = {Chen, X and Qin, S and Yang, C and Xing, Z and Liu, C and Zhang, X and Wu, X and Li, X and Gong, Y and Liu, Z},
title = {Chromatin Modifications Orchestrate DNA Repair Pathway Choice: Epigenetics Strategies for Enhancing CRISPR-Cas9-Mediated Knock-In Efficiency.},
journal = {Cell proliferation},
volume = {59},
number = {11},
pages = {},
pmid = {42852500},
issn = {1365-2184},
support = {2024YFA0916600//the National Key R&D Program of China/ ; C1-2024-010//Tianjin University Key Project of the Graduate Top-notch Innovation Award Program in Arts and Sciences/ ; JNYYKY-202301//Jinnan Hospital Research program/ ; 2022GKF-0269//the Funding of Thinkingbiomed Biotechnology (Beijing) Co., Ltd/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; *Epigenesis, Genetic ; Animals ; *DNA Repair/genetics ; *Chromatin/metabolism/genetics ; Histones/metabolism ; Gene Editing/methods ; DNA End-Joining Repair ; Epigenome Editing ; },
abstract = {CRISPR-Cas9-mediated precise gene knock-in relies on homology-directed repair (HDR) at low frequencies in most mammalian cells, limiting precise gene editing in both research and therapeutic applications. Although multiple strategies have been developed to increase knock-in efficiency, their application can be restricted by technical complexity, limited applicability, or potential safety concerns. In this review, we propose a shift in focus from conventional strategies toward epigenetic methods to increase knock-in efficiency. It has been proved that histone modifications such as H3K36me3, H3K9me3, H4K20me2, and H2AK15ub are involved in DNA damage signallings and the recruitment of key HDR and non-homologous end joining (NHEJ) factors. We further discuss conceptual strategies for transient chromatin modulation to bias repair pathway choice and highlight associated technical challenges and safety considerations. Together, these findings and insights highlight the essential role of histone modification in shaping chromatin states and orchestrating DNA repair dynamics. Accordingly, epigenetic regulation emerges as a compelling strategy to facilitate high-fidelity gene knock-in, offering valuable mechanistic clues and actionable epigenetic targets to gene editing for basic research and therapeutic translation.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Knock-In Techniques/methods
*Epigenesis, Genetic
Animals
*DNA Repair/genetics
*Chromatin/metabolism/genetics
Histones/metabolism
Gene Editing/methods
DNA End-Joining Repair
Epigenome Editing

@article {pmid42937111,
year = {2026},
author = {Luo, W and Fan, L and Wei, D and Xiang, C and Xu, H and Liu, Y and Zhang, W},
title = {A one-step LAMP-CRISPR/Cas12a platform targeting lytA and cpsA for rapid parallel identification and virulence stratification of Streptococcus pneumoniae.},
journal = {The Analyst},
volume = {151},
number = {22},
pages = {6756-6766},
doi = {10.1039/d6an01151a},
pmid = {42937111},
issn = {1364-5528},
mesh = {*Streptococcus pneumoniae/pathogenicity/genetics/isolation & purification ; *Nucleic Acid Amplification Techniques/methods ; *Bacterial Proteins/genetics ; Virulence ; *CRISPR-Cas Systems/genetics ; *Molecular Diagnostic Techniques/methods ; Humans ; *N-Acetylmuramoyl-L-alanine Amidase/genetics ; Rapid Diagnostic Tests ; },
abstract = {Streptococcus pneumoniae (S. pneumoniae, SPN) is the primary pathogen causing community-acquired pneumonia (CAP), purulent meningitis, sepsis, and acute otitis media. Accurate species identification and rapid differentiation of invasive strains are critical for clinical management and infection control. Based on loop-mediated isothermal amplification (LAMP) and CRISPR/Cas12a technologies, this study established a parallel dual-target detection platform targeting the S. pneumoniae-specific lytA gene and the capsular polysaccharide synthesis regulatory gene cpsA. A dual-readout mode combining fluorescence detection (FD) and lateral flow test strip (LFTS) was adopted to meet diverse laboratory testing requirements. For each target-specific reaction, LAMP amplification and CRISPR/Cas12a cleavage were sequentially performed in a single closed tube using paraffin-oil stratification to suppress solvent evaporation, effectively preventing aerosol contamination. Range-finding serial-dilution assays revealed that the lowest gDNA concentrations producing positive signals in all three technical replicates reached 1 fg μL[-1] (FD) and 10 fg μL[-1] (LFTS). This assay specifically distinguished S. pneumoniae from other common clinical pathogens and preliminarily identified encapsulated invasive strains, with 100% sensitivity and specificity consistent with conventional PCR. This LAMP-CRISPR/Cas12a platform, with its closed-tube design, avoids contamination, while FD and LFTS dual readouts cater to diverse testing needs. Featuring high sensitivity, excellent specificity, simplicity, and speed, it provides an innovative and practical solution for the rapid diagnosis of S. pneumoniae and holds great clinical potential.},
}

*Streptococcus pneumoniae/pathogenicity/genetics/isolation & purification
*Nucleic Acid Amplification Techniques/methods
*Bacterial Proteins/genetics
Virulence
*CRISPR-Cas Systems/genetics
*Molecular Diagnostic Techniques/methods
Humans
*N-Acetylmuramoyl-L-alanine Amidase/genetics
Rapid Diagnostic Tests

@article {pmid42970595,
year = {2026},
author = {Wright, RCT and Lovell, SC and Richmond, A and Harrison, C and Ashworth, E and Kadioglu, A and Fothergill, JL and Friman, VP and Westra, ER and Brockhurst, MA},
title = {Resistance mutation supply modulates the benefit of CRISPR immunity against virulent phages in Pseudomonas aeruginosa.},
journal = {PLoS biology},
volume = {24},
number = {11},
pages = {e3004094},
doi = {10.1371/journal.pbio.3004094},
pmid = {42970595},
issn = {1545-7885},
abstract = {Only a fraction of bacterial genomes encode CRISPR-Cas systems, but the selective causes of this variation are unexplained. How naturally virulent bacteriophages (phages) select for CRISPR immunity has rarely been tested experimentally. Here, we show against a panel of genetically and functionally diverse virulent phages that CRISPR immunity was not universally beneficial to Pseudomonas aeruginosa, and its fitness effect varied strongly between phages in predictable ways. In addition to mechanisms known to alter the effectiveness of CRISPR immunity, such as encoding a matching spacer or a protective nuclear shell, we show that the fitness effect of CRISPR immunity negatively associated with the probability of evolving receptor-based resistance to the phage via spontaneous mutation. The supply of resistance mutations differed strongly between very closely related lipopolysaccharide-binding phages and was associated with variation at the C-terminus of the tail fibre protein altering residues involved in hydrogen bonding and the predicted binding site. Our results show that CRISPR immunity is more beneficial against virulent phages that are harder to evolve resistance to via receptor mutations, suggesting that virulent phage community composition and diversity will be important drivers of the prevalence of CRISPR immunity.},
}

@article {pmid42971139,
year = {2026},
author = {Amankwah, S and Wicke, L and Kałuski, Ł and Stefańczyk, E and Łobocka, M},
title = {Molecular insights into the long terminal repeat regions of therapeutic staphylococcal Twortvirinae phages.},
journal = {Microbiological research},
volume = {315},
number = {},
pages = {128803},
doi = {10.1016/j.micres.2026.128803},
pmid = {42971139},
issn = {1618-0623},
abstract = {Staphylococcal bacteriophages with proven therapeutic efficacy in anti-staphylococcal therapy have large (~140 kb) genomes and belong to subfamily Twortvirinae. Representatives of four genera (Kayvirus, Silviavirus, Sepunavirus, and Twortvirus) are the most intensively studied and are advancing toward clinical applications. However, the functions of many of their genes remain unknown and cannot be predicted from sequence homology alone. The least explored and most diverse genomic regions are the long terminal repeats (LTRs), which comprise ~10% of the virion DNA. These regions were often omitted from early genome assemblies or misplaced in genomic maps. Only recent studies have shown that LTRs encode genes expressed immediately upon infection and involved in host takeover, genome establishment, and interactions with bacterial defense systems, highlighting their critical role in successful infection during phage therapy. This review synthesizes current knowledge on the genomic architecture, transcriptional organization, and molecular functions of LTR regions in Twortvirinae phages. By integrating comparative genomics with recent transcriptomic and phage-host interaction studies, we identify LTRs as key regulatory and evolutionary hubs in Twortvirinae biology and highlight major knowledge gaps concerning their roles in bacterial immunity and phage genome evolution. The enrichment of LTRs in targets of bacterial anti-phage defense systems, together with their dynamic evolution through gene gain, loss, and diversification, suggests a central role in phage adaptation to diverse hosts. Finally, our synthesis of available data allows us, for the first time, to infer which end of the phage genome enters the host cell first during infection.},
}

@article {pmid42971416,
year = {2026},
author = {Wang, X and Wu, Y and Zhang, X and Du, Y and Liu, N and Wang, Z and Zhang, H and Gao, X and Zheng, A},
title = {Research progress in the production, purification, and applications of long single-stranded DNA.},
journal = {Biotechnology advances},
volume = {94},
number = {},
pages = {109089},
doi = {10.1016/j.biotechadv.2026.109089},
pmid = {42971416},
issn = {1873-1899},
abstract = {Long single-stranded DNA (long ssDNA) plays important roles in cutting-edge fields such as DNA origami, gene therapy, and synthetic biology. However, the production, preparation, and purification of long ssDNA remain technically challenging. This review systematically summarizes current strategies for long ssDNA production and preparation, including phage-E. coli-based biological production, template-dependent enzymatic amplification, and viral-vector-based generation of ssDNA genomes. CRISPR-Cas-assisted approaches are discussed separately as complementary tools for post-synthetic processing, sequence-selective cleavage, enrichment, and quality control, whereas other enzymatic approaches are considered for post-synthetic strand conversion. We compare the characteristics and limitations of these production and preparation strategies, discuss purification techniques for long ssDNA, and summarize its applications in DNA origami nanotechnology and gene therapy. This review comprehensively synthesizes the bottlenecks in long ssDNA production, preparation, and purification, and outlines further development trends. We highlight template-independent de novo enzymatic DNA synthesis, automated DNA manufacturing, synthetic biology-based production systems, and AI-assisted sequence optimization. These developments are expected to accelerate the transition of long ssDNA technologies from laboratory research toward scalable manufacturing and practical applications.},
}

@article {pmid42971796,
year = {2026},
author = {Lipsick, J},
title = {A History of Cancer Research: Mouse Models of Cancer Genetics.},
journal = {Cold Spring Harbor perspectives in medicine},
volume = {16},
number = {11},
pages = {},
pmid = {42971796},
issn = {2157-1422},
mesh = {Animals ; History, 20th Century ; *Disease Models, Animal ; Mice ; *Neoplasms/genetics/history ; Humans ; History, 21st Century ; Mice, Transgenic ; Mice, Knockout ; },
abstract = {Studies of genetically engineered mice have contributed significantly to our understanding of tumor biology, and there are now countless mouse models of different human cancers. Breeding of mice for cancer research began more than 100 years ago. This provided early insights into cancer genetics, but it was the development of techniques for generating transgenic and knockout mice that transformed the field in the 1980s. The advent of CRISPR-Cas technology 30 years later made mouse models even easier to generate. In this excerpt from his forthcoming book on the history of cancer research, Joe Lipsick looks back at a century of advances in mouse cancer genetics, from the early pioneering work of Abbie Lanthrop to the Nobel-winning discoveries that now allow researchers to manipulate potential cancer genes in mice and explore their roles in tumorigenesis.},
}

Animals
History, 20th Century
*Disease Models, Animal
Mice
*Neoplasms/genetics/history
Humans
History, 21st Century
Mice, Transgenic
Mice, Knockout

@article {pmid42967794,
year = {2026},
author = {Wang, Y and Wu, Y and Huang, X and Tian, Y and Liu, Q},
title = {A CRISPR/Cas12a-based digital microfluidic platform for parallel detection of foodborne pathogens.},
journal = {Analytica chimica acta},
volume = {1426},
number = {},
pages = {346361},
doi = {10.1016/j.aca.2026.346361},
pmid = {42967794},
issn = {1873-4324},
mesh = {*Food Microbiology ; *CRISPR-Cas Systems/genetics ; *Microfluidic Analytical Techniques/instrumentation ; Escherichia coli O157/isolation & purification ; Animals ; *Lab-On-A-Chip Devices ; Listeria monocytogenes/isolation & purification/genetics ; },
abstract = {BACKGROUND: Rapid and automated on-chip detection of multiple foodborne bacterial pathogens remains challenging because conventional molecular assays require multistep liquid handling and are difficult to integrate for parallel analysis. Here, we developed an EWOD-based DMF platform integrating RPA with CRISPR/Cas12a for parallel detection of S. aureus, E. coli O157:H7, L. monocytogenes, and C. sakazakii. The 16-site DMF chip was organized into four pathogen-specific detection modules, each containing a positive control, a negative control, and two replicate sample sites, enabling programmable droplet manipulation and sequential on-chip amplification and fluorescence detection without external pumps or complex valves.

RESULTS: Assay conditions were systematically optimized for fluidic compatibility and CRISPR/Cas12a performance. Following off-chip sample preparation and genomic DNA extraction, the automated on-chip RPA amplification and CRISPR/Cas12a detection workflow was completed within approximately 50 min. Under these conditions, the platform achieved detection limits of 10[3], 10[2], 10, and 10[2] CFU/mL for S. aureus, E. coli O157:H7, L. monocytogenes, and C. sakazakii, respectively. No apparent cross-reactivity was observed among 35 food-associated bacterial strains. In artificially contaminated milk, milk powder, and chicken samples, comparable detection limits were obtained after off-chip sample processing, despite matrix-dependent variations in fluorescence intensity.

SIGNIFICANCE: These results demonstrate that integrating EWOD-based digital microfluidics with RPA and CRISPR/Cas12a enables rapid, parallelized, and programmable molecular detection of multiple foodborne bacterial pathogens on a compact platform. By integrating droplet manipulation, amplification, and fluorescence detection on-chip, the proposed system reduces reliance on external fluidic components and provides a promising basis for further development toward automated and integrated food-safety testing.},
}

*Food Microbiology
*CRISPR-Cas Systems/genetics
*Microfluidic Analytical Techniques/instrumentation
Escherichia coli O157/isolation & purification
Animals
*Lab-On-A-Chip Devices
Listeria monocytogenes/isolation & purification/genetics

@article {pmid42969371,
year = {2026},
author = {Cai, J and Fan, A and Bai, Z and Zhao, B and Chen, Y and Wu, X},
title = {SFRP2 Editing Enhances Dermal Papilla Cell Function via Activation of the PI3K-Akt Signaling Pathway.},
journal = {Journal of cellular physiology},
volume = {241},
number = {11},
pages = {},
doi = {10.1002/jcp.70248},
pmid = {42969371},
issn = {1097-4652},
support = {32072724//National Natural Science Foundation of China/ ; CARS-43-A-1//China Agriculture Research System of MOF and MARA/ ; KYCX25_4054//Graduate Research and Innovation Projects in the Jiangsu Province/ ; },
mesh = {*Proto-Oncogene Proteins c-akt/metabolism/genetics ; Secreted Frizzled-Related Proteins ; Signal Transduction ; *Hair Follicle/metabolism/growth & development/cytology ; *Phosphatidylinositol 3-Kinases/metabolism/genetics ; Cell Proliferation/genetics ; *Membrane Proteins/genetics/metabolism ; Humans ; *Gene Editing/methods ; Animals ; Apoptosis/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Secreted frizzled-related protein 2 (SFRP2) is an important molecule that regulates cell growth and development and participates in hair follicle (HF) development and homeostasis. The present study seeks to determine whether SFRP2 directly targets dermal papilla cells (DPCs), the pivotal functional cell population within HF, and characterize the intracellular signaling regulatory network triggered downstream. In this study, a CRISPR/Cas9-mediated SFRP2 gene editing DPC model was established in vitro. Multi-omics analyses, including transcriptomics and metabolomics, were performed alongside functional assays to investigate the regulatory mechanisms of SFRP2 in DPCs. Among three designed single guide RNAs (sgRNAs), sgRNA3 showed the highest editing efficiency (67.0%) without off-target effects. Genetic editing of SFRP2 markedly increased the transcript levels of pivotal hair follicle‑development‑related genes (LEF1, CCND1 and Wnt2). Meanwhile, it suppressed TGF‑β1 transcription, which resulted in elevated proliferation capacity and diminished apoptotic activity within DPCs. Transcriptomic profiling identified 2288 differentially expressed genes (DEGs) enriched in PI3K-Akt, MAPK and TNF signaling pathways, while metabolomic analysis identified 185 differential metabolites (DMs) associated with apoptosis, ABC transporters, and PI3K-Akt signaling. Integrated multi-omics analysis confirmed PI3K-Akt signaling as a central regulatory axis. Western blot analysis revealed increased phosphorylation of PI3K and Akt following SFRP2 editing, indicating pathway activation. Treatment of SFRP2-edited DPCs with the PI3K inhibitor LY294002 reduced the elevated phosphorylation and reversed the functional phenotypes, confirming PI3K-Akt pathway involvement. Collectively, these findings indicate that SFRP2 mediates DPC biological activities by means of PI3K‑Akt‑driven transcriptional‑metabolic networks, yielding novel perspectives on the molecular regulatory machinery controlling HF physiology.},
}

*Proto-Oncogene Proteins c-akt/metabolism/genetics
Secreted Frizzled-Related Proteins
Signal Transduction
*Hair Follicle/metabolism/growth & development/cytology
*Phosphatidylinositol 3-Kinases/metabolism/genetics
Cell Proliferation/genetics
*Membrane Proteins/genetics/metabolism
Humans
*Gene Editing/methods
Animals
Apoptosis/genetics
CRISPR-Cas Systems/genetics

@article {pmid42554239,
year = {2026},
author = {Walker, MWG and Margolis, C and Wiegand, T and Gavin, N and Loewy, E},
title = {Targeted Genetic Knockout of Can f 1, the Major Allergen in Dogs.},
journal = {The CRISPR journal},
volume = {9},
number = {5-6},
pages = {254-262},
doi = {10.1177/25731599261473124},
pmid = {42554239},
issn = {2573-1602},
mesh = {Animals ; Dogs ; *Allergens/genetics/immunology ; Immunoglobulin E/immunology ; *Gene Knockout Techniques/methods ; CRISPR-Cas Systems ; *Hypersensitivity/immunology/genetics/veterinary ; *Lipocalins/genetics/immunology ; Frameshift Mutation ; Animals, Genetically Modified ; Skin Tests ; },
abstract = {Dog allergy is a prevalent IgE-mediated condition, with the salivary lipocalin Can f 1 accounting for the majority of dog-specific IgE reactivity in sensitized individuals. Existing strategies for managing dog allergy primarily rely on modulating host immune responses and do not address allergen production at its source. Here, we report the generation of genetically engineered dogs lacking Can f 1. Using CRISPR-Cas9 editing and somatic cell nuclear transfer, we produced two healthy beagle puppies carrying a frameshift mutation in exon 1 of the Can f 1 gene. Salivary and hair/dander analysis demonstrated the absence of Can f 1 protein in the edited dogs. Skin prick testing in a sensitized individual demonstrated robust IgE-mediated reactivity to control dog extracts but no detectable response to extracts from the edited dogs. Together, these findings demonstrate that targeted genetic knockout of the major dog allergen is compatible with canine development and can abolish the IgE-mediated allergic response, supporting the feasibility of a gene-based approach to reducing canine allergenicity.},
}

Animals
Dogs
*Allergens/genetics/immunology
Immunoglobulin E/immunology
*Gene Knockout Techniques/methods
CRISPR-Cas Systems
*Hypersensitivity/immunology/genetics/veterinary
*Lipocalins/genetics/immunology
Frameshift Mutation
Animals, Genetically Modified
Skin Tests

@article {pmid42680037,
year = {2026},
author = {Bulle, M and Kondi, RKR and Rahman, MM and Sharma, M and Rana, P and Chaudhary, C and Subudhi, PK},
title = {Beyond the salt barrier: CRISPR-mediated DNA reprogramming to uncouple yield from tolerance in Rice: A review.},
journal = {International journal of biological macromolecules},
volume = {382},
number = {Pt 1},
pages = {154254},
doi = {10.1016/j.ijbiomac.2026.154254},
pmid = {42680037},
issn = {1879-0003},
mesh = {*Oryza/genetics/growth & development/physiology ; *Salt Tolerance/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing ; },
abstract = {Rice (Oryza sativa L.) feeds half of humanity, yet its cultivation is increasingly threatened by soil salinization, which now affects 1.4 billion hectares globally. Decades of breeding and engineering have focused on Na[+] exclusion, principally through the Saltol QTL and the xylem-unloading transporter OsHKT1;5, yet this strategy has reached a physiological ceiling. Excluder genotypes survive salinity but fail to fill grain, because the ATP-intensive cost of continuous ion extrusion starves reproductive sinks, while ABA-mediated stomatal closure imposes chronic carbon limitation. The resulting "survival-yield gap" exposes a fundamental flaw in single-trait approaches to a polygenic stress. In this review, we argue that durable, yield-stable salt tolerance requires a coordinated systems-level intervention spanning five mechanistic tiers: (i) CRISPR/Cas9-mediated removal of negative regulatory brakes (OsRR22, RST1, PC1) that suppress plant's latent stress-adaptive capacity; (ii) reinforcement of actin-myosin cytoskeletal transport to sustain SOS1, NHX1, and HKT1;5 delivery under ionic stress; (iii) importation of halophyte design principles from Oryza coarctata, including salt gland architecture and superior Na[+] compartmentalization; (iv) recalibration of the ROS-photosynthesis axis via the DHHC09-STRK1-CatC molecular switch and stomatal density engineering; and (v) pyramiding these modules into a "Salt-Shield Rice" genotype through multiplex editing, marker-assisted introgression, speed breeding, and genomic selection. We propose a phased ten-year roadmap that integrates synthetic biology circuit design with conventional breeding to deliver field-ready, multi-module varieties with greater than 70% yield stability at 8-10 dS m[-1]. This remains an aspirational design target rather than a demonstrated outcome, as three of the five tiers-halophyte-derived structural traits, cytoskeletal reinforcement, and full multi-module pyramiding-remain unvalidated in rice.},
}

*Oryza/genetics/growth & development/physiology
*Salt Tolerance/genetics
*CRISPR-Cas Systems/genetics
Gene Editing

@article {pmid42722248,
year = {2026},
author = {Jang, S and Shin, S and Jeong, Y and An, Y and Han, Y and Kim, H and Lim, D},
title = {Spacer-engineered donor DNA enhances CRISPR-Cas9-mediated knockin to establish a chemical knockdown platform for endogenous proteins.},
journal = {International journal of biological macromolecules},
volume = {382},
number = {Pt 2},
pages = {154428},
doi = {10.1016/j.ijbiomac.2026.154428},
pmid = {42722248},
issn = {1879-0003},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; Humans ; *DNA/genetics ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Knockdown Techniques ; HEK293 Cells ; },
abstract = {Precise installation of functional protein domains at endogenous loci is a powerful approach for interrogating protein functions, but its broad application is limited by the low efficiency of homology-directed repair (HDR)-mediated knockin during Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 gene editing. Here, we investigated a simple donor DNA engineering strategy that enhances HDR-mediated gene knockin by appending additional gRNA-recognizable spacer sequences to donor templates. Systematic analysis of linear dsDNA and plasmid donors showed that spacer position, length, and orientation influenced HDR efficiency, and that spacer-containing donors improved knockin across multiple genomic loci, insertion sizes, cell types and delivery modalities. Mechanistic analyses revealed that spacer-containing donors formed stable complexes with Cas9/gRNA and showed increased nuclear localization, supporting nuclear delivery as a key contributor to improved editing outcomes. We then applied this gene-editing strategy to establish a chemical knockdown platform by installing drug-responsive degrons at endogenous loci, generating cell lines in which GSK3β or Lin28A protein could be rapidly, potently and reversibly depleted by drug treatment. These platforms enable selective modulation of endogenous proteins and reveal cellular responses that may differ from those obtained using conventional genetic perturbation. Together, this work establishes a readily implementable framework that integrates improved gene editing with on-demand chemical knockdown of endogenous proteins.},
}

*CRISPR-Cas Systems/genetics
*Gene Knock-In Techniques/methods
Humans
*DNA/genetics
Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
*Gene Knockdown Techniques
HEK293 Cells

@article {pmid42802908,
year = {2026},
author = {Vykhlyantseva, I and Ramachandran, H and Morales-Juarez, DA and Rossi, A and Corn, J and Kontarakis, Z},
title = {PARP1 Constrains Insertional Prime Editing in a Context-Dependent Manner.},
journal = {The CRISPR journal},
volume = {9},
number = {5-6},
pages = {263-273},
doi = {10.1177/25731599261491273},
pmid = {42802908},
issn = {2573-1602},
mesh = {Humans ; *Poly (ADP-Ribose) Polymerase-1/genetics/metabolism ; *Gene Editing/methods ; K562 Cells ; Poly(ADP-ribose) Polymerase Inhibitors/pharmacology ; *Mutagenesis, Insertional ; CRISPR-Cas Systems/genetics ; DNA Damage ; DNA Repair ; High-Throughput Nucleotide Sequencing ; },
abstract = {Prime editing (PE) enables precise insertions, deletions, and substitutions without double-strand breaks, but editing efficiency remains highly variable. To identify modulators of PE outcomes, we screened 569 compounds targeting DNA damage and repair pathways in K562 cells using targeted next-generation sequencing. PARP inhibitors selectively enhanced PE-mediated insertions at the HEK3 locus by more than 2-fold, without improving substitution or deletion editing. Genetic loss of PARP1 reproduced this phenotype, identifying PARP1 as the principal mediator. PARP1 inhibition enhanced insertion efficiency across multiple PE architectures, insertion sizes, and cellular backgrounds, including primary human T cells, and promoted more complete incorporation of pegRNA-encoded sequence changes without increasing indels or scaffold incorporation. However, this effect was dependent on genomic locus and reverse transcription template configuration. Together, these findings identify PARP1 as a context-dependent constraint on insertional PE.},
}

Humans
*Poly (ADP-Ribose) Polymerase-1/genetics/metabolism
*Gene Editing/methods
K562 Cells
Poly(ADP-ribose) Polymerase Inhibitors/pharmacology
*Mutagenesis, Insertional
CRISPR-Cas Systems/genetics
DNA Damage
DNA Repair
High-Throughput Nucleotide Sequencing

@article {pmid42817219,
year = {2026},
author = {Tubalova, M and Koksharova, G and Costa, SSD and Krepischi, ACV and Fishman, V and Gridina, M},
title = {Hidden Outcomes of CRISPR-Cas9 Genome Editing Revealed by Targeted Sequencing in Isogenic Human iPSC Clones.},
journal = {The CRISPR journal},
volume = {9},
number = {5-6},
pages = {274-284},
doi = {10.1177/25731599261492640},
pmid = {42817219},
issn = {2573-1602},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; High-Throughput Nucleotide Sequencing/methods ; Genotype ; Genome, Human ; },
abstract = {Advances in CRISPR-Cas9-based genome editing have simplified the generation of isogenic induced pluripotent stem cell (iPSC) pairs differing by a single genetic perturbation, minimizing interindividual variability in disease modeling. However, imprecise editing can cause unintended mutations that distort genotype-phenotype interpretation. Here, we investigated the molecular consequences of CRISPR-Cas9 editing in human iPSCs carrying a tandem duplication of the STAG2 locus. Using hybridization capture-based targeted next-generation sequencing of genomic regions surrounding Cas9-induced double-strand breaks, we identified inversions, plasmid DNA integrations, and complex structural rearrangements that had been missed by conventional PCR-based genotyping. The main advance of our study is the application of a practical hybridization capture-based workflow for resolving such hidden editing outcomes during the generation of isogenic iPSC models. In the selected clones subjected to detailed analysis, targeted sequencing enabled us to revise the initial interpretation of editing outcomes and resolve discrepancies between genotypic and transcriptomic data. Our results demonstrate that complex on-target alterations can remain undetected by conventional PCR-based genotyping and show that hybridization capture-based targeted sequencing can serve as a practical quality-control approach for the characterization of edited iPSC clones.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
High-Throughput Nucleotide Sequencing/methods
Genotype
Genome, Human

@article {pmid42965525,
year = {2026},
author = {Ernste, K and Zhang, Q and Cazares, O and Lu, K and Palencia, S and Hatzi, K and Eidenshenk, C and Tong, Z and Jiang, Y and Jin, HY},
title = {Scalable Arrayed T-cell CRISPR Screening in Autologous Tumor Co-cultures to Prioritize Combination Partners for T-cell Engagers.},
journal = {Current protocols},
volume = {6},
number = {11},
pages = {},
pmid = {42965525},
issn = {2691-1299},
mesh = {Humans ; *T-Lymphocytes/immunology ; *Leukemia, Lymphocytic, Chronic, B-Cell/immunology/therapy ; Coculture Techniques/methods ; Flow Cytometry/methods ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {T-cell-engaging therapies (TCE) have shown clinical promise in hematologic malignancies, but the T-cell-intrinsic determinants of TCE-mediated cytotoxicity remain incompletely defined. This is in part because functional studies in patient-derived primary samples have been technically challenging. Here, we describe a flow cytometry-based arrayed CRISPR knockout screening platform to prioritize regulators of TCE-mediated cytotoxicity in suspension tumor cells using patient-derived, autologous chronic lymphocytic leukemia (CLL) samples. The principal advantage of this system is the use of matched T cells and tumor cells from the same patient, retaining patient-matched effector-target pairings that are not captured in donor-mismatched or cell line-based assays. In this workflow, CD3+ T cells isolated from viably frozen CLL peripheral blood mononuclear cells (PBMC) are expanded and edited for 2 days using arrayed Cas9 ribonucleoprotein nucleofection. TCE-mediated cytotoxicity was assessed in a 72-hr, feeder-supported co-culture assay using patient-matched CLL tumor cells in the presence of CD20×CD3 TCE. In the current implementation, the workflow is organized into modules that accommodate up to 96 arrayed perturbations, with technical replicates evaluated under TCE and control conditions. Because perturbations remain physically indexed in an arrayed format, the workflow is amenable to parallelization and laboratory automation, providing a path toward substantially larger targeted screens. This platform therefore provides a scalable framework for mechanistic studies and combination-target discovery for TCE therapies. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: CD3+ T cell isolation from CLL patient PBMC (Day 0) Alternate Protocol 1: Sequential label-free isolation of CD19+ and CD3+ T cells from healthy donor PBMC Alternate Protocol 2: Sequential label-free isolation of CD3+ T cells and B-CLL cells from CLL donor PBMC Basic Protocol 2: Transfer activated T cells to G-Rex for large-scale expansion (Day 4) Basic Protocol 3: Harvest and CRISPR Editing of Expanded T Cells (Day 11) Support Protocol 1: CRISPR knockout optimization and validation in T cells Basic Protocol 4: CLL co-culture with CRISPR-edited T cells (Day 13) Basic Protocol 5: Flow cytometry analysis of tumor viability and T cell phenotype (Day 16) Support Protocol 2: Selecting suitable co-culture conditions for E:T ratio and TCE concentration for large-scale screening.},
}

Humans
*T-Lymphocytes/immunology
*Leukemia, Lymphocytic, Chronic, B-Cell/immunology/therapy
Coculture Techniques/methods
Flow Cytometry/methods
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42965688,
year = {2026},
author = {Thakur, SK and Malviya, R and Chandra, P and Sridhar, SB and Shareef, J and Wadhwa, T},
title = {Dual-vector nanoengineered mRNA-CRISPR delivery systems for precision gene therapy in choroidal melanoma.},
journal = {Nanomedicine (London, England)},
volume = {21},
number = {21},
pages = {3013-3032},
pmid = {42965688},
issn = {1748-6963},
mesh = {Humans ; *Genetic Therapy/methods ; *Choroid Neoplasms/therapy/genetics ; *Melanoma/therapy/genetics ; *RNA, Messenger/genetics ; Animals ; Gene Transfer Techniques ; *CRISPR-Cas Systems/genetics ; Genetic Vectors/genetics ; Gene Editing/methods ; Gene Therapy Agents ; },
abstract = {Choroidal melanoma is the most common primary intraocular malignancy, and effective gene therapy remains limited by ocular barriers, tumor heterogeneity, and inefficient nucleic acid delivery. This review critically analyses the molecular rationale, engineering principles, emerging nanoarchitectures, biological validation, and translational prospects of dual-vector nanoengineered mRNA-CRISPR delivery systems for precision gene therapy in choroidal melanoma. The manuscript also highlights key challenges and future strategies for clinical implementation. Current evidence indicates that dual-vector platforms enable independent optimization of mRNA and CRISPR cargos while maintaining coordinated spatiotemporal therapeutic activity. We conducted a literature search in PubMed, Scopus, and Web of Science for relevant studies published from January 2015 to September 2026. Functional cargo partitioning, vector modularity, programmable intracellular trafficking, and ocular-targeted nanoengineering improve delivery precision and reduce biological compromise associated with co-encapsulation. Lipid, polymeric, hybrid, biomimetic, and viral-nonviral architectures each provide distinct engineering advantages depending on therapeutic objectives. Preclinical studies support enhanced genome editing, transient protein restoration, improved tumor suppression, and reduced off-target effects. Nevertheless, manufacturing scalability, regulatory standardization, long-term safety, and clinical validation remain major barriers to translation. Dual-vector nanoengineered mRNA-CRISPR systems represent a promising next-generation platform for precision gene therapy in choroidal melanoma by enabling coordinated and programmable therapeutic delivery. Continued advances in modular nanoengineering, personalized cargo selection, and clinically scalable manufacturing will be essential for successful translation into precision ocular oncology.},
}

Humans
*Genetic Therapy/methods
*Choroid Neoplasms/therapy/genetics
*Melanoma/therapy/genetics
*RNA, Messenger/genetics
Animals
Gene Transfer Techniques
*CRISPR-Cas Systems/genetics
Genetic Vectors/genetics
Gene Editing/methods
Gene Therapy Agents

@article {pmid42967748,
year = {2026},
author = {Wen, J and Zhu, Z and Zhang, J and Wang, Z and Chen, J and Chen, Y},
title = {Asymmetric V-shaped amplification-enhanced and PAM-relaxed one-tube dual-mode CRISPR/Cas12a platform for rapid and ultrasensitive detection of foodborne pathogens.},
journal = {Food research international (Ottawa, Ont.)},
volume = {245},
number = {Pt 4},
pages = {120941},
doi = {10.1016/j.foodres.2026.120941},
pmid = {42967748},
issn = {1873-7145},
mesh = {*Food Microbiology/methods ; *CRISPR-Cas Systems/genetics ; *Foodborne Diseases/microbiology/diagnosis ; DNA, Bacterial/genetics ; DNA, Single-Stranded/genetics ; Rapid Diagnostic Tests ; *Nucleic Acid Amplification Techniques/methods ; Polymerase Chain Reaction/methods ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Foodborne pathogen infections pose a serious threat to food safety and public health. As a low-cost method with strong potential for widespread use in most laboratories, conventional PCR and integrated PCR-CRISPR assays are still constrained by prolonged assay times, dedicated instrumentation, limited closed-tube naked-eye visualization, tube-opening-associated contamination risks, and PAM-dependent target recognition, thereby restricting their practical applicability. To address these challenges, we developed an asymmetric V-shaped (rapid thermal cycling) amplification-enhanced CRISPR/Cas12a one-tube dual-mode detection platform. Asymmetric amplification enabled rapid ssDNA enrichment by tuning primer concentrations, reducing amplification time from ∼1 h to <30 min while enhancing Cas12a cleavage efficiency and reducing dependence on canonical PAM sites. Moreover, one-tube dual-mode highly sensitive visual pathogen detection (naked-eye: 10 CFU/mL, fluorescence excitation: 1 CFU/mL) was achieved simply by adjusting the probe concentration in the reaction system. By eliminating post-amplification tube opening and reagent transfer while avoiding reliance on nanomaterials or lateral-flow strips, this platform provides a one-tube strategy for rapid, sensitive, and simplified visual detection of foodborne pathogens in resource-limited laboratories.},
}

*Food Microbiology/methods
*CRISPR-Cas Systems/genetics
*Foodborne Diseases/microbiology/diagnosis
DNA, Bacterial/genetics
DNA, Single-Stranded/genetics
Rapid Diagnostic Tests
*Nucleic Acid Amplification Techniques/methods
Polymerase Chain Reaction/methods
CRISPR-Associated Proteins/genetics
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid42967779,
year = {2026},
author = {Shi, H and Chen, J and Lin, X and Zhang, Y and Wu, H and Zeng, B and Zhang, P and Luo, Q and Liu, T and Zeng, T},
title = {An extraction-free one-pot immunomagnetic-RPA-CRISPR/Cas13a platform for ultrasensitive detection of exosomal PD-L1.},
journal = {Analytica chimica acta},
volume = {1426},
number = {},
pages = {346335},
doi = {10.1016/j.aca.2026.346335},
pmid = {42967779},
issn = {1873-4324},
mesh = {*B7-H1 Antigen/blood/analysis/immunology ; *Exosomes/chemistry/metabolism ; Humans ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; *Nucleic Acid Amplification Techniques ; Carcinoma, Hepatocellular/diagnosis ; Liver Neoplasms/diagnosis ; *Immunomagnetic Separation ; },
abstract = {Programmed death-ligand 1 (PD-L1) expressed on the surface of exosomes (exo) is a promising liquid biopsy biomarker for monitoring response to immunotherapy in hepatocellular carcinoma (HCC). However, existing detection methods are limited by insufficient sensitivity and complicated operation. To address this, we developed an extraction-free immunomagnetic recombinase polymerase amplification (RPA)-based one-pot high-sensitivity CRISPR/Cas13a platform (iMRO-HCRISPR) employing DNA-conjugated antibodies. Exosomes in blood were enriched with anti-CD63/CD81/CD9 antibody-conjugated magnetic beads and recognized by PD-L1-specific DNA-antibody probes; the probe DNA then underwent RPA amplification, in vitro transcription (IVT), and Cas13a trans-cleavage, achieving multi-level signal amplification with fluorescent readout. Toehold-mediated DNA affinity pull-down effectively reduced the detection background. The platform achieved a limit of detection (LOD) as low as 10[1] particles/mL (positive detection at the single-particle level), a limit of quantification (LOQ) of 10[3] particles/mL, and a quantitative range of 10[3]-10[11] particles/mL, with the entire process completed in a single closed tube without exosome isolation. Clinical validation demonstrated that the platform effectively distinguished HCC patients from healthy controls (P < 0.0001), with an area under the receiver operating characteristic (ROC) curve (AUC) of 0.964. This study provides robust technical support for the ultrasensitive detection of exosomal PD-L1 in HCC and for dynamic monitoring of immunotherapy response.},
}

*B7-H1 Antigen/blood/analysis/immunology
*Exosomes/chemistry/metabolism
Humans
*CRISPR-Cas Systems/genetics
Limit of Detection
*Nucleic Acid Amplification Techniques
Carcinoma, Hepatocellular/diagnosis
Liver Neoplasms/diagnosis
*Immunomagnetic Separation

@article {pmid42087678,
year = {2026},
author = {Shen, Y and Wang, H and Gao, W and Luo, WF and Fan, T and Shi, Y and Xie, X and Li, L and Xiang, S and Chai, A and Li, B},
title = {On-Site, Highly Sensitive, and Multisignal Detection of Cucumber Bacteria via CRISPR/Cas12a Coupled with Duplex Recombinase Polymerase Amplification.},
journal = {Plant disease},
volume = {110},
number = {10},
pages = {4540-4552},
doi = {10.1094/PDIS-11-25-2416-RE},
pmid = {42087678},
issn = {0191-2917},
mesh = {*Cucumis sativus/microbiology ; *CRISPR-Cas Systems/genetics ; *Plant Diseases/microbiology ; Recombinases/metabolism ; *Nucleic Acid Amplification Techniques/methods ; *Pseudomonas/isolation & purification/genetics ; *Pectobacterium/isolation & purification/genetics ; Rapid Diagnostic Tests ; Sensitivity and Specificity ; },
abstract = {Pseudomonas amygdali pv. lachrymans (Pal) and Pectobacterium brasiliense (Pb) are highly destructive bacterial pathogens that cause severe oozy diseases in cucumber crops, often resulting in significant yield losses. Rapid and accurate detection is essential for effective disease management, yet current molecular detection methods are limited by their reliance on sophisticated instrumentation, time-consuming procedures, and laboratory-dependent operations, significantly limiting their practicality in field settings. To address these limitations, we developed a portable detection platform that integrates dual recombinase polymerase amplification with CRISPR/Cas12a technology. This system demonstrated exceptional sensitivity with a detection limit of 10[-5] ng/μl for both Pal and Pb while exhibiting high specificity and enabling multisignal output capabilities. For rapid on-site detection, we further developed a portable device integrating a heating unit and a blue light-excited fluorescence detection system, coupled with a smartphone-based readout for high-throughput field testing. The entire process was completed within 1 h. This platform not only provides a powerful tool for rapid field detection of plant pathogens but also pioneers a novel universal nucleic acid detection strategy with broad application prospects in point-of-care molecular diagnostics across healthcare, agriculture, and other fields. By bridging the gap between laboratory precision and field practicality, this technology opens new avenues for decentralized diagnostics in crop protection, microbial ecology, and public health.},
}

*Cucumis sativus/microbiology
*CRISPR-Cas Systems/genetics
*Plant Diseases/microbiology
Recombinases/metabolism
*Nucleic Acid Amplification Techniques/methods
*Pseudomonas/isolation & purification/genetics
*Pectobacterium/isolation & purification/genetics
Rapid Diagnostic Tests
Sensitivity and Specificity

@article {pmid42624887,
year = {2026},
author = {Hanif, SZ and Kutz, C and Au, CC and Torregroza, I and Palikhe, S and Jannath, SY and Fabiha, T and Bhinder, B and Washburn, MP and Devost, D and Liu, S and Bhardwaj, P and Evans, T and Liang, X and Anand, PK and Tarran, R and Elemento, O and Dow, LE and Blenis, J and Hébert, TE and Brown, KA},
title = {Role of orphan G protein-coupled receptor GPR52 in breast cancer cell multicellular organization and collective invasion.},
journal = {British journal of cancer},
volume = {135},
number = {10},
pages = {1715-1730},
pmid = {42624887},
issn = {1532-1827},
support = {T2025-007//V Foundation for Cancer Research (V Foundation)/ ; T32GM152349//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Humans ; *Receptors, G-Protein-Coupled/genetics/metabolism ; Animals ; Female ; Zebrafish ; MDA-MB-231 Cells ; Neoplasm Invasiveness ; *Triple Negative Breast Neoplasms/pathology/genetics/metabolism ; Cyclic AMP/metabolism ; Cell Line, Tumor ; *Breast Neoplasms/pathology/genetics ; CRISPR-Cas Systems ; Spheroids, Cellular/pathology ; Gene Knockout Techniques ; },
abstract = {BACKGROUND: G protein-coupled receptors (GPCRs) are the largest class of membrane-bound receptors and are emerging as targets for the effective treatment of cancer. The role of orphan GPCR GPR52 in cancer has not been characterized. Low mRNA expression of GPR52 in breast tumours correlates with reduced overall survival, leading to the hypothesis that loss of GPR52 supports breast cancer progression.

METHODS: CRISPR-Cas9 was used to knock out GPR52 in the human triple-negative breast cancer cell lines MDA-MB-468 and MDA-MB-231. 2D and 3D in vitro studies, electron microscopy, and a zebrafish xenograft model were used to assess the morphology and behaviour of GPR52 KO cells.

RESULTS: Loss of GPR52 was associated with elevated levels of cAMP, increased cell-cell interaction in 2D cultures, more spindle-like morphology on collagen, altered 3D spheroid morphology, and increased propensity to organize and invade collectively. Zebrafish injected with GPR52 KO cells developed a greater total cancer area than control. RNA sequencing and proteomic analyses of GPR52-null cells revealed an increased cAMP signalling signature. Re-expression of GPR52 and inhibition of cAMP production rescued some GPR52 KO phenotypes.

CONCLUSIONS: GPR52 loss is a potential mechanism by which breast cancer progression may occur and supports the investigation of GPR52 agonism as a therapeutic option for breast cancer.

STATEMENT OF SIGNIFICANCE: Loss of the orphan GPCR GPR52 in human breast cell lines leads to increased cell clustering, hybrid/partial EMT, and increased tumour burden in zebrafish, further expanding our understanding of mechanisms driving cancer progression and opening the door to novel therapeutic approaches.},
}

Humans
*Receptors, G-Protein-Coupled/genetics/metabolism
Animals
Female
Zebrafish
MDA-MB-231 Cells
Neoplasm Invasiveness
*Triple Negative Breast Neoplasms/pathology/genetics/metabolism
Cyclic AMP/metabolism
Cell Line, Tumor
*Breast Neoplasms/pathology/genetics
CRISPR-Cas Systems
Spheroids, Cellular/pathology
Gene Knockout Techniques

@article {pmid42876085,
year = {2026},
author = {Singh, S and Panchal, OV and Ivics, Z},
title = {Beyond Viruses: Transposons and CRISPR-Programmable Nonviral Systems for Genome Engineering.},
journal = {Human gene therapy},
volume = {37},
number = {23-24},
pages = {1094-1107},
doi = {10.1177/10430342261493722},
pmid = {42876085},
issn = {1557-7422},
mesh = {Humans ; *DNA Transposable Elements/genetics ; *CRISPR-Cas Systems ; *Genetic Engineering/methods ; Animals ; *Genetic Therapy/methods ; *Gene Editing/methods ; Gene Transfer Techniques ; Genetic Vectors/genetics ; },
abstract = {Human gene therapy has been transformed by repurposing mobile genetic elements as tools for efficient gene delivery and precise genome modification. Viruses, transposons, and retrotransposons mobilize genetic material through distinct mechanisms with distinct advantages for genome engineering platforms with therapeutic potential. This review describes the mechanisms, performance characteristics, and translational relevance of nonviral methods of gene transfer and genome engineering, with emphasis on engineered systems such as the Sleeping Beauty (SB) and piggyBac (PB) DNA transposons, R2 retrotransposon-based platforms, CRISPR-associated transposons (CASTs), and prime editing (PE). Clinical trials using both SB- and PB-engineered cells have shown feasibility by demonstrating sustained therapeutic effect through stable transgene integration, such as CD19-directed CAR-T cell therapy for hematological malignancies. These transposon-based vectors enable robust gene transfer for cell engineering, but are fairly promiscuous with respect to where in the genome they insert their genetic cargo. On the contrary, R2-derived systems and CASTs represent promising technologies for programmable insertion, but are still under preclinical development. CRISPR-based PE provides a distinct route to precise sequence correction without double-strand breaks and is particularly attractive for the correction of monogenic disorders. Taken together, these technologies define a continuum from natural genome mobility to clinically relevant genome engineering. Continued optimization of delivery, targeting, and editing fidelity will determine how rapidly these approaches move from experimental innovation into routine clinical use.},
}

Humans
*DNA Transposable Elements/genetics
*CRISPR-Cas Systems
*Genetic Engineering/methods
Animals
*Genetic Therapy/methods
*Gene Editing/methods
Gene Transfer Techniques
Genetic Vectors/genetics

@article {pmid42922027,
year = {2026},
author = {Chen, B and Nong, B and Lin, S and Yang, Y and Yu, Y and Tan, W and Zhu, T and Pei, Y and Xu, W and Wu, Y and Ma, X and Yu, J and Huang, W and Hu, Y and Songyang, Z},
title = {De novo structural mining and engineering of TadA deaminases for high efficiency adenine base editors.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2026.10.022},
pmid = {42922027},
issn = {1525-0024},
abstract = {Conventional enzyme discovery relies primarily on sequence homology and phylogenetic relationships, limiting the identification of functionally related but sequence-divergent proteins. Here, we developed a structure-guided mining framework integrating AlphaFold2-predicted structures with Foldseek-based three-dimensional alignment to identify previously uncharacterized TadA scaffolds with conserved folds and divergent sequences. Two candidates, eMaTadA and eErTadA, were engineered into functional adenine base editors (ABEs) by fusion to Cas9 nickase. The resulting editors mediated robust A-to-G editing with high product purity and restricted bystander editing. Under expression-stratified and activity-matched conditions, eMa-ABE and eEr-ABE showed reduced guide-dependent DNA off-target editing relative to ABE8e, while orthogonal R-loop assays revealed lower SpCas9-independent deamination of exposed single-stranded DNA (ssDNA). In human CD34[+] hematopoietic stem and progenitor cells, eMa-ABE achieved up to approximately 80% editing at established HBG1/2 promoter targets and induced fetal hemoglobin expression following erythroid differentiation. Lipid nanoparticle delivery corrected approximately 50% of ATP7B P992L alleles in mouse liver, with no detectable increase in editing at the examined computationally predicted off-target loci. Furthermore, embedding eMaTadA within Cas9 expanded the editing window from A3-A9 to A3-A14 and enabled efficient start-codon-mediated gene inactivation. Collectively, this study establishes a structure-guided strategy for discovering and engineering sequence-divergent deaminases and expands the molecular diversity and therapeutic potential of the ABE toolkit.},
}

@article {pmid42957916,
year = {2026},
author = {Shuran, Z and Yuzhen, H and Zili, Z and Wenming, H and Baosheng, Z and Ying, C},
title = {Research progress of gene correction for genetic diseases via in vivo prime editing.},
journal = {Molecular genetics and genomics : MGG},
volume = {301},
number = {1},
pages = {},
pmid = {42957916},
issn = {1617-4623},
support = {82060039//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Gene Editing/methods ; *Genetic Therapy/methods ; *Genetic Diseases, Inborn/therapy/genetics ; *CRISPR-Cas Systems/genetics ; Animals ; Genetic Vectors/genetics ; },
abstract = {Curative therapies for genetic diseases caused by pathogenic mutations demand precise and safe genome editing technologies. Prime editing (PE), a CRISPR-Cas9-derived technology that enables precise base conversions without double-strand breaks (DSBs), has emerged as a transformative tool with the ability to correct 89% of human pathogenic mutations. However, transitioning PE from preclinical models to clinical applications faces critical challenges, particularly in the development of in vivo delivery systems and scalable manufacturing. This review systematically summarizes advances of in vivo PE therapeutics, focusing on the therapeutic outcomes across some retinal, metabolic, and hematologic genetic diseases. We further dissect barriers to clinical translation, including in the immunogenicity of viral vectors, off-target risks in nondividing cells, and large-scale production bottlenecks. By highlighting the unique advantages of in vivo PE and proposing actionable solutions for scalable manufacturing, the work provides a roadmap for accelerating in vivo PE-based therapies toward clinical reality.},
}

Humans
*Gene Editing/methods
*Genetic Therapy/methods
*Genetic Diseases, Inborn/therapy/genetics
*CRISPR-Cas Systems/genetics
Animals
Genetic Vectors/genetics

@article {pmid42958025,
year = {2026},
author = {Yuan, Y and Liu, M and Wang, B and Wang, X and Xing, Q and Liang, W and Wang, G and Zhang, W and Yan, J},
title = {Rapid visual detection technologies for Botrytis cinerea resistance to QoI and SDHI fungicides based on RPA-CRISPR/Cas12a.},
journal = {BMC plant biology},
volume = {26},
number = {1},
pages = {},
pmid = {42958025},
issn = {1471-2229},
support = {CARS-29-bc-2//China Agriculture Research System of MOF and MARA/ ; JKTD2025002//Outstanding Scientist Project of Beijing Academy of Agriculture and Forestry Sciences/ ; BAIC08-2022//Beijing Innovation Consortium of Agriculture Research System/ ; },
mesh = {*Botrytis/drug effects/genetics ; *Drug Resistance, Fungal/genetics ; *Fungicides, Industrial/pharmacology ; CRISPR-Cas Systems ; Succinate Dehydrogenase/antagonists & inhibitors ; Plant Diseases/microbiology ; Vitis/microbiology ; Nucleic Acid Amplification Techniques/methods ; Rapid Diagnostic Tests ; },
abstract = {Grape gray mold caused by Botrytis cinerea is one of the most important fungal diseases affecting grape production and postharvest quality. The extensive use of fungicides has led to the emergence of resistance in B. cinerea, particularly to quinone outside inhibitors (QoIs) and succinate dehydrogenase inhibitors (SDHI), which compromises the efficacy of disease control. Therefore, detection techniques for fungicide resistance in B. cinerea are needed to provide scientific guidance for disease management. Based on recombinase polymerase amplification (RPA)-CRISPR/Cas12a technology, this study designed specific RPA primer pairs and crRNA targeting resistance-related mutation sites in fungicide target genes cytb and SdhB, incorporated single-stranded fluorescent reporter molecules, and optimized key reaction components. This study achieved rapid fluorescence-based resistance detection within 1 h, with detection sensitivities of 450 and 430 pg/µL for the two fungicide genes.},
}

*Botrytis/drug effects/genetics
*Drug Resistance, Fungal/genetics
*Fungicides, Industrial/pharmacology
CRISPR-Cas Systems
Succinate Dehydrogenase/antagonists & inhibitors
Plant Diseases/microbiology
Vitis/microbiology
Nucleic Acid Amplification Techniques/methods
Rapid Diagnostic Tests

@article {pmid42958909,
year = {2026},
author = {Zhou, Y and Wang, Y and Zhao, G and Liu, C and Zeng, R and Liang, W and Han, R and Kong, H and Cao, X and Li, M and Chen, H and Tan, B and Liu, H and Xu, Z},
title = {Natural variation in the evolutionarily conserved F-box gene SDEF promotes seed dormancy in rice.},
journal = {The Plant journal : for cell and molecular biology},
volume = {128},
number = {3},
pages = {},
pmid = {42958909},
issn = {1365-313X},
support = {32472032//National Natural Science Foundation of China/ ; 31771888//National Natural Science Foundation of China/ ; 31271804//National Natural Science Foundation of China/ ; 2021B10564001//The Double First-Class Discipline Promotion Project/ ; 2022A1515010843//Natural Science Foundation of Guangdong Province/ ; },
mesh = {*Oryza/genetics/physiology ; *Plant Dormancy/genetics/physiology ; *Plant Proteins/genetics/metabolism ; Abscisic Acid/metabolism ; *F-Box Proteins/genetics/metabolism ; Seeds/genetics/physiology ; Germination/genetics ; Gene Expression Regulation, Plant ; Plants, Genetically Modified ; CRISPR-Cas Systems ; Genetic Variation ; },
abstract = {Insufficient seed dormancy causes pre-harvest sprouting (PHS) and severe yield losses in rice; however, genetic resources with practical breeding value remain limited. Here, we isolated a major dormancy gene, SDEF, via map-based cloning using chromosomal single segment substitution lines (SSSLs), identifying a functional SNP (Arg-to-Trp) as the causal mutation. CRISPR/Cas9-mediated knockout of SDEF led to PHS susceptibility, while overexpression significantly enhanced dormancy. Importantly, SDEF enhanced seed dormancy without compromising grain yield in SSSLs, confirming it as a promising breeding target. Moreover, SDEF encodes an angiosperm-conserved, nuclear-localized F-box protein and is preferentially expressed in the embryo, with expression increasing during seed maturation and declining upon germination. Mechanistically, protein interaction assays confirmed that SDEF recruits OSK1, with the Trp-type SDEF[K] exhibiting a more stable interaction than the Arg-type SDEF[H]. SDEF is induced by abscisic acid (ABA) and appears to promote seed dormancy through the ABA pathway by activating ABA signaling genes and repressing α-amylase genes, as evidenced by the reduced ABA sensitivity of sdef mutants. Additionally, haplotype analysis revealed that natural variation in SDEF is associated with divergent dormancy levels, and reduced nucleotide diversity in cultivated rice indicates that SDEF was selected during domestication. Collectively, our results provide insights into the molecular basis of SDEF-mediated dormancy, providing an ideal target for breeding PHS-resistant rice.},
}

*Oryza/genetics/physiology
*Plant Dormancy/genetics/physiology
*Plant Proteins/genetics/metabolism
Abscisic Acid/metabolism
*F-Box Proteins/genetics/metabolism
Seeds/genetics/physiology
Germination/genetics
Gene Expression Regulation, Plant
Plants, Genetically Modified
CRISPR-Cas Systems
Genetic Variation

@article {pmid42961522,
year = {2026},
author = {Nazari, M and Kardan, R and Ahmadi, A and Abolhasani, FS and Hemmati, J},
title = {A comprehensive review of mixed-species biofilm infections and innovative therapeutic strategies.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42961522},
issn = {1573-4978},
mesh = {*Biofilms/drug effects/growth & development ; Humans ; Quorum Sensing/drug effects ; *Coinfection/microbiology/therapy ; Animals ; Bacteria ; CRISPR-Cas Systems ; Phage Therapy/methods ; },
abstract = {Mixed-species biofilm infections are important causes of persistent and difficult-to-treat infections. Compared with monospecies biofilms, mixed-species communities contain microorganisms with different physiological and genetic characteristics that interact through metabolic cooperation, chemical signaling, competition, and modification of the local environment. These interactions, together with the EPS matrix, physiological heterogeneity, and HGT, can increase antimicrobial tolerance, contributing to treatment failure and infection recurrence. Polymicrobial biofilms are associated with chronic wounds, respiratory infections, catheter- and device-associated infections, and other persistent infections. However, treatment of polymicrobial biofilms remains challenging because their complex structure and interspecies interactions can reduce antimicrobial efficacy. This review summarizes the clinical significance, formation, and persistence of mixed-species biofilms and discusses recent therapeutic strategies for their control. We also discuss recent approaches for controlling polymicrobial biofilms, including natural compounds, QS inhibition, CRISPR-Cas-based approaches, biofilm matrix targeting, metabolic and nutrient disruption, nanotechnology, and phage therapy. Recent studies show that these approaches can inhibit biofilm formation, disrupt established biofilms, enhance antimicrobial activity, and selectively target specific microbial populations. However, their effectiveness varies with microbial composition, biofilm maturity, matrix characteristics, and treatment conditions. Overall, current evidence supports the development of targeted and combination-based strategies for mixed-species biofilms. Further studies using clinically relevant models are needed to exactly define treatment efficacy and support the translation of these approaches into clinical practice.},
}

*Biofilms/drug effects/growth & development
Humans
Quorum Sensing/drug effects
*Coinfection/microbiology/therapy
Animals
Bacteria
CRISPR-Cas Systems
Phage Therapy/methods

@article {pmid42961748,
year = {2027},
author = {Bryson, JW},
title = {Array Assembler: A Constraint-Aware Pipeline for Cas12 and Cas13 crRNA Array Design and Oligonucleotide Decomposition.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3083},
number = {},
pages = {237-253},
pmid = {42961748},
issn = {1940-6029},
mesh = {*Oligonucleotides/genetics ; *CRISPR-Cas Systems/genetics ; *Software ; *CRISPR-Associated Proteins/genetics ; Algorithms ; *Computational Biology/methods ; *Oligonucleotide Array Sequence Analysis/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Array-based CRISPR systems such as Cas12 and Cas13 enable compact multiplexed perturbations by encoding multiple spacers within a single precursor transcript that is autonomously processed into individual targeting crRNAs. In practice, however, crRNA arrays are difficult to synthesize directly because conserved direct repeats introduce extensive sequence repetitiveness, and reliable cloning requires that ligation junctions avoid repeated sequences while remaining compatible with a chosen plasmid context. Here, we describe Array Assembler, an interactive, browser-based computational workflow that formalizes crRNA array design as a constrained sequence construction and oligonucleotide decomposition problem. Given a CRISPR variant, an ordered list of spacer sequences, and basic cloning parameters (including direct-repeat context and overhang sequences), the pipeline constructs the full in silico array insert and algorithmically decomposes it into paired forward and reverse oligonucleotides that satisfy junction placement rules and practical synthesis limits. Output oligonucleotides are exported as a vendor-ready spreadsheet to enable rapid ordering and downstream assembly. The chapter provides step-by-step instructions for running the tool, outlines the underlying segmentation logic for developers, and includes a worked example illustrating design-to-assembly for a multiplexed Cas12 crRNA array.},
}

*Oligonucleotides/genetics
*CRISPR-Cas Systems/genetics
*Software
*CRISPR-Associated Proteins/genetics
Algorithms
*Computational Biology/methods
*Oligonucleotide Array Sequence Analysis/methods
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42383490,
year = {2026},
author = {Nam, O and Grouneva, I and Mackinder, LCM},
title = {A versatile tool for gene editing in the diatom Thalassiosira pseudonana.},
journal = {Plant physiology},
volume = {202},
number = {3},
pages = {},
doi = {10.1093/plphys/kiag385},
pmid = {42383490},
issn = {1532-2548},
support = {MR/T020679/1//United Kingdom Research and Innovation Future Leaders Fellowship/ ; BB/T017589/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/S015337/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/R001014/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; EP/W024063/1//Engineering and Physical Research Council/ ; },
mesh = {*Diatoms/genetics ; CRISPR-Cas Systems/genetics ; Luminescent Proteins/metabolism/genetics ; Green Fluorescent Proteins/genetics/metabolism ; *Gene Editing/methods ; Plasmids/genetics ; },
abstract = {Diatoms are major contributors to marine primary production, with the centric diatom Thalassiosira pseudonana a powerful model for understanding biogeochemical processes including carbon fixation and silicification. While there are molecular tools available for fluorescent protein (FP) tagging and CRISPR/Cas9 genome editing in T. pseudonana, these require the delivery of multiple vectors or have limited versatility. Additionally, scarless endogenous tagging, that results in a FP fusion expressed from its native genomic location, has yet to be developed. Here, we describe a versatile modular Golden Gate-based toolkit for T. pseudonana that through the delivery of a single-episome via bacterial conjugation enables: (i) FP tagging, (ii) dual FP tagging, (iii) CRISPR/Cas9 genome editing, (iv) simultaneous FP tagging with gene editing, and (v) scarless endogenous FP tagging. We further expand the available parts for T. pseudonana by validating 3 additional FPs and 2 untested promoter/terminator pairs. We demonstrate the versatility of our system by knocking out diatom pyrenoid component 1 (DPC1) while simultaneously GFP tagging the rbcS (Rubisco small subunit), and by endogenously GFP tagging the bestrophin-like protein BST2. While DPC1 knockout does not result in a major pyrenoid structural defect due to unperturbed rbcS-GFP localization to the pyrenoid, we confirm that BST2 localizes to the pyrenoid and exhibits increased fluorescence under low CO2-supporting a role in diatom carbon fixation. Our developed genetic tools provide a robust framework for exploring cellular processes in diatoms, accelerating routine studies and enabling systematic, quantitative and large-scale studies.},
}

*Diatoms/genetics
CRISPR-Cas Systems/genetics
Luminescent Proteins/metabolism/genetics
Green Fluorescent Proteins/genetics/metabolism
*Gene Editing/methods
Plasmids/genetics

@article {pmid42861929,
year = {2026},
author = {Pozzi, B and Lucero, AM and Buggiano, V and Pelaez, MA and García, CC and Srebrow, A},
title = {Targeting CLK1 by CRISPR tools and pharmacological inhibition modulates the innate immune response and dengue virus replication.},
journal = {The Biochemical journal},
volume = {483},
number = {12},
pages = {2307-2325},
doi = {10.1042/BCJ20260367},
pmid = {42861929},
issn = {1470-8728},
support = {Impacto-CONVE-2023-100766162-APN-MCT//Secretaria de Innovacion, Ciencia y Tecnologia (ex Ministerio de Ciencia, Tecnologia e Innovacion) de Argentina/ ; 20020220200041BA//UBA | Secretaría de Ciencia y Técnica, Universidad de Buenos Aires (UBACyT)/ ; PICT 2019-00263//Agencia Nacional de Investigaciones Cientificas y Tecnologicas de Argentina/ ; PICT 2020-00198//Agencia Nacional de Investigaciones Cientificas y Tecnologicas de Argentina/ ; },
mesh = {*Dengue Virus/physiology/drug effects ; *Protein Serine-Threonine Kinases/genetics/antagonists & inhibitors/metabolism ; *Virus Replication/drug effects ; *Immunity, Innate/drug effects/genetics ; Humans ; *Dengue/immunology/genetics ; *CRISPR-Cas Systems ; Animals ; Alternative Splicing ; Interferons/pharmacology ; Protein-Tyrosine Kinases ; },
abstract = {Alternative splicing is a key regulatory mechanism known to be altered upon viral infection. These alterations can arise both from direct viral interference with the splicing machinery and from cellular responses such as activation of innate immunity. Here, we investigated splicing changes shared between dengue virus (DENV) infection and interferon (IFN) treatment in cultured cells, the latter serving as a model for virus-independent innate immune activation. Among the common events, we identified an increased production of non-coding mRNA isoforms from the CLK1 gene, which gives rise to a kinase that phosphorylates splicing factors including SR proteins and spliceosomal components. Consistent with this finding, IFN treatment led to a reduction in CLK1 protein levels. Using stable cell lines with CRISPR/dCas9-mediated modulation of CLK1 expression, we found that silencing CLK1 enhanced the induction of immune response genes, while its overexpression attenuated it. Inhibition of CLK1 kinase activity with the pharmacological inhibitor TG003 further potentiated IFN-induced gene expression and reduced DENV replication. Altogether, these results identify CLK1 as a proviral negative regulator of IFN-stimulated gene expression and suggest that its inhibition could enhance antiviral defenses and become a target for therapeutic strategies.},
}

*Dengue Virus/physiology/drug effects
*Protein Serine-Threonine Kinases/genetics/antagonists & inhibitors/metabolism
*Virus Replication/drug effects
*Immunity, Innate/drug effects/genetics
Humans
*Dengue/immunology/genetics
*CRISPR-Cas Systems
Animals
Alternative Splicing
Interferons/pharmacology
Protein-Tyrosine Kinases