@article {pmid41359128,
year = {2025},
author = {Li, Z and Cheng, Y and Li, C and Wu, Q and Xin, Y},
title = {Harnessing microalgae for bioproducts: innovations in synthetic biology.},
journal = {World journal of microbiology & biotechnology},
volume = {41},
number = {12},
pages = {500},
pmid = {41359128},
issn = {1573-0972},
support = {32560020 and 31600059//National Natural Science Foundation of China/ ; RZ2300002678//Start-Up Funds of Hainan University/ ; DC2300001799//Open Project of State Key Laboratory of Marine Resource Utilization in South China Sea/ ; 2018YFA0902500//National Key Research and Development Program of China/ ; },
mesh = {*Microalgae/metabolism/genetics ; *Synthetic Biology/methods ; Biofuels ; *Metabolic Engineering/methods ; Gene Editing ; Lipids/biosynthesis ; CRISPR-Cas Systems ; Metabolic Networks and Pathways ; Photobioreactors ; },
abstract = {Microalgae are increasingly recognized as versatile platforms for sustainable production of biofuels and high-value bioproducts such as lipids, carotenoids and polyunsaturated fatty acids. Rapid progress in synthetic biology is transforming microalgal engineering by enabling precise rewiring of metabolic pathways and overcoming long-standing technical bottlenecks, particularly those related to transformation efficiency, genetic stability and strain scalability. Recent innovations (including CRISPR/Cas genome editing, modular cloning systems, synthetic promoter libraries and dynamic, environment-responsive regulatory circuits) have greatly expanded the genetic toolset available for both model and recalcitrant species. These advances support targeted control of lipid and pigment biosynthesis, improved flux distribution and more robust performance under industrially relevant conditions. When integrated with progress in photobioreactor design, automated cultivation, and process intensification, synthetic biology unlocks new potential for scalable, economically viable microalgal biomanufacturing. This review summarizes these developments, highlights remaining challenges in strain robustness and bioprocess translation, and outlines future pathways toward high-performance microalgal biofactories that can contribute meaningfully to a low-carbon, bio-based economy.},
}

*Microalgae/metabolism/genetics
*Synthetic Biology/methods
Biofuels
*Metabolic Engineering/methods
Gene Editing
Lipids/biosynthesis
CRISPR-Cas Systems
Metabolic Networks and Pathways
Photobioreactors

@article {pmid41356798,
year = {2026},
author = {Birappa, G and Perumalsamy, H and Hong, SH and Gowda, DAA and Chandrasekaran, AP and Karapurkar, JK and Rajkumar, S and Balusamy, SR and Jayachandran, A and Baek, KH and Lee, J and Matam, V and Kim, WJ and Kim, KS and Ramakrishna, S and Suresh, B},
title = {Single-cell RNA sequence analysis reveals USP32 as a therapeutic target to mitigate PD-L1-driven colorectal tumorigenesis in vitro and in vivo.},
journal = {Theranostics},
volume = {16},
number = {2},
pages = {986-1005},
pmid = {41356798},
issn = {1838-7640},
mesh = {Humans ; Animals ; *B7-H1 Antigen/metabolism/genetics ; *Colorectal Neoplasms/genetics/pathology/metabolism ; Mice ; *Ubiquitin Thiolesterase/genetics/metabolism ; Single-Cell Analysis/methods ; Ubiquitination ; *Carcinogenesis/genetics ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; Sequence Analysis, RNA ; },
abstract = {Background: The expression levels of the programmed death-ligand 1 (PD-L1) protein serves as a prognostic indicator for patients with colorectal cancer (CRC). Advancement of CRC is facilitated by deubiquitinating enzymes (DUBs), which regulate oncoprotein levels via the ubiquitin-proteasomal pathway. The post-translational regulatory mechanisms governing PD-L1 protein abundance on CRC, in relation to different tumor grades and their clinical relevance, remains unknown. Methods: We analyzed single-cell RNA sequencing (scRNA-seq) data to identify DUB genes associated with PD-L1 expression in CRC. We used a loss-of-function-based CRISPR/Cas9 library to identify putative DUB genes that regulate the PD-L1 protein level. Immunoprecipitation was used to confirm the interaction between the USP32 and PD-L1 along with its ubiquitination status. A series of in vitro and in vivo carcinogenesis-related experiments were conducted to determine the clinical relevance between USP32 and PD-L1 expression in CRC progression. Results: In this study, we analyzed scRNA-seq data from extensive cohorts of human and mice at the single-cell level to identify DUB genes associated with PD-L1 expression in CRC. Our analysis identified multiple putative DUBs, including USP32 and USP12, as prognostic markers associated with PD-L1 expression, which was found to be elevated in T cells, macrophages, and classical monocytes cell types in patients with CRC. A secondary screening using CRISPR/Cas9-mediated loss-of-function analysis for DUBs found that USP32 modulates PD-L1 protein levels in CRC. Furthermore, we demonstrated that USP32 interacts with, stabilizes, and extends the half-life of PD-L1 by preventing its K-48-linked polyubiquitination as an underlying mechanism that contributes for tumorigenesis. Conclusion: A combination of scRNA-seq analysis and wet-lab experimental validation confirmed that USP32 mediates PD-L1 protein stabilization in colon cancer, identifying it as a potential therapeutic target for CRC. CRISPR/Cas9-mediated targeted knockout of the USP32 gene reduced PD-L1 protein levels and significantly mitigated colorectal cell proliferation and tumorigenesis, both in vitro and in vivo, in a xenograft mouse model, underscoring a novel and alternative approach to the treatment of CRC.},
}

Humans
Animals
*B7-H1 Antigen/metabolism/genetics
*Colorectal Neoplasms/genetics/pathology/metabolism
Mice
*Ubiquitin Thiolesterase/genetics/metabolism
Single-Cell Analysis/methods
Ubiquitination
*Carcinogenesis/genetics
Cell Line, Tumor
Gene Expression Regulation, Neoplastic
CRISPR-Cas Systems
Sequence Analysis, RNA

@article {pmid41356473,
year = {2025},
author = {Wei, C and Chen, Z},
title = {Comprehensive analysis of phage genomes from diverse environments reveals their diversity, potential applications, and interactions with hosts and other phages.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1686402},
pmid = {41356473},
issn = {1664-302X},
abstract = {Phages are ubiquitous and diverse, playing a key role in maintaining microbial ecosystem balance. However, their diversity, potential applications, and their interactions with hosts and other phages remain largely unexplored. To address this, we collected 59,652,008 putative viral genomes from our laboratory, 45 public viral datasets, and an integrated public viral genome database (IGN), covering seven habitats. We obtained 741,692 phage genomes with completeness ≥50% (PGD50), and most (93.83%, 695,938/741,692) of these phage genomes were classified into the Caudoviricetes class. We found that 158,522 species-level viral clusters that contained 28.96% (214,814/741,692) phage genomes without any known phage genomes in the IGN, indicating substantial novelty. Global phylogenetic trees for five iterations based on complete phage genomes significantly expanded the known diversity of the virosphere. Genome analysis revealed phage potential divergence with habitat types and highlighted the utilization of alternative genetic codes. Furthermore, 3D structural similarity searches demonstrated significant potential for annotating previously uncharacterized viral proteins. Analysis of CRISPR spacer inferred potential hosts of phages and competitive networks among phages, highlighting virulent phages as promising candidates for phage therapy against pathogenic bacteria. Intriguingly, diverse CRISPR-Cas systems were detected within phage genomes themselves, suggesting their enormous potential as novel gene editing tools. Collectively, this study provides a comprehensive phage genome resource, foundational for future research into phage-host and phage-phage interactions, phage therapy development, and the mining of next-generation genetic tools.},
}

@article {pmid41356196,
year = {2026},
author = {Zhang, Y and Deng, Q and Xu, Y and Wu, W and Wu, T and Huang, J and Hu, Y and Lin, W and Xu, X and Wu, J},
title = {ROS-responsive cellular vesicles with ferroptosis-targeting siACMSD delivery for acute kidney injury therapy.},
journal = {Theranostics},
volume = {16},
number = {4},
pages = {1941-1958},
pmid = {41356196},
issn = {1838-7640},
mesh = {*Ferroptosis/drug effects ; Animals ; *Reactive Oxygen Species/metabolism ; *Acute Kidney Injury/therapy/metabolism/drug therapy/pathology ; Mice ; Humans ; Cisplatin/pharmacology ; *Carboxy-Lyases/genetics/metabolism ; Disease Models, Animal ; Cell Line ; Male ; Mice, Inbred C57BL ; CRISPR-Cas Systems ; Mitochondria/metabolism/drug effects ; },
abstract = {Background: Acute kidney injury (AKI) is a severe and prevalent nephrotic syndrome which lack of definitive therapies. Alpha-amino-β-carboxymuconic acid-ε-semialdehyde decarboxylase (ACMSD) is a metabolic enzyme mainly expressed in the kidney which exacerbated AKI injury by promoting TCA cycle and inhibiting nicotinamide adenine dinucleotide (NAD[+]) production, whereas lack of effective intervention strategies for ACMSD-targeted therapy. Methods: Herein, we knocked out ACMSD in vitro through CRISPR-Cas9 method, and developed a reactive oxygen species (ROS)-responsive neutrophil-derived cellular vesicles (CVs) drugs (RNAi@ROS-CVs), which efficiently mediated ACMSD knockdown in vivo, exploring the mechanism of ACMSD-induced ferroptosis process in AKI. Results: ACMSD knockout effectively alleviated cisplatin (CP)-induced mitochondrial damage, suppressed TCA cycle progression, promoted NAD[+] synthesis, and inhibited ferroptosis in HK2 cells. In mice AKI model, RNAi@ROS-CVs effectively targeted the injured kidneys, downregulated ACMSD expression in renal tubular epithelial cells, reduced ROS production and lipid peroxidation, and alleviated CP or ischemia/reperfusion (I/R)-induced ferroptosis. Conclusion: These findings highlight the therapeutic potential of ACMSD-targeted knockout in AKI intervention and introduce a versatile and efficient controlled-release drug delivery platform for AKI-targeted therapy, with potential applicability to other acute renal diseases.},
}

*Ferroptosis/drug effects
Animals
*Reactive Oxygen Species/metabolism
*Acute Kidney Injury/therapy/metabolism/drug therapy/pathology
Mice
Humans
Cisplatin/pharmacology
*Carboxy-Lyases/genetics/metabolism
Disease Models, Animal
Cell Line
Male
Mice, Inbred C57BL
CRISPR-Cas Systems
Mitochondria/metabolism/drug effects

@article {pmid41356190,
year = {2026},
author = {Zhong, XY and Yang, YX and Xiong, YF and Ye, GC and Gong, X and Zhong, ML and He, HD and Wang, SG and Xia, QD},
title = {Programmable molecular microscopy: CRISPR/Cas fluorescent probes revolutionizing spatiotemporal genomic imaging.},
journal = {Theranostics},
volume = {16},
number = {4},
pages = {1877-1904},
pmid = {41356190},
issn = {1838-7640},
mesh = {*CRISPR-Cas Systems/genetics ; *Fluorescent Dyes ; Humans ; Animals ; *Molecular Imaging/methods ; *Genomics/methods ; Gene Editing/methods ; },
abstract = {Bioimaging technologies visually resolve spatiotemporal dynamics of biomolecules, cells, and tissues, enabling essential insights into gene regulation, disease mechanisms, and drug metabolism. CRISPR/Cas-based fluorescent probes transform CRISPR from "genetic scissors" into "molecular microscopes," providing an indispensable tool for in situ decoding of molecular events in living systems. Their high nucleic acid specificity establishes CRISPR/Cas as a pivotal technology for dynamically monitoring genomic and transcriptomic events at live-cell and in vivo levels. This work systematically outlines design strategies and functional mechanisms of mainstream CRISPR/Cas fluorescent probes for bioimaging, encompassing five categories: fluorescent proteins, synthetic dyes, smart gated probes, nanomaterials, and multimodal integrated probes. Recent advances and persistent challenges in achieving high-sensitivity targeted imaging, effective signal amplification, and precise delivery control are comprehensively examined, including analysis of their advantages, limitations, and adaptability in complex biological environments. Building on breakthroughs in in vivo delivery systems, diverse carriers demonstrate significant potential for enhancing CRISPR/Cas transport efficiency, improving tissue penetration, and enabling spatiotemporal controlled release. Continued innovation drives CRISPR/Cas imaging platforms toward higher sensitivity, enhanced biocompatibility, and multifunctional integration, thereby fostering the convergence and broad application of gene editing and molecular diagnostics.},
}

*CRISPR-Cas Systems/genetics
*Fluorescent Dyes
Humans
Animals
*Molecular Imaging/methods
*Genomics/methods
Gene Editing/methods

@article {pmid41354981,
year = {2025},
author = {Das, T and Barman, T and Prasad, A},
title = {Precision editing to improve fruit traits: CRISPR/Cas into the picture.},
journal = {Protoplasma},
volume = {},
number = {},
pages = {},
pmid = {41354981},
issn = {1615-6102},
abstract = {Crop growth, quality, and yield can be adversely affected by various biotic and abiotic stresses. Crop characteristics can be improved with conventional breeding and other variation-based breeding strategies. However, these strategies are time as well as resource consuming and to overcome this, novel approaches are necessary. CRISPR/Cas technique allows to improve desired traits more efficiently and accurately by targeting specific genes. Genome editing has become more versatile with CRISPR/Cas systems and is a valuable tool to protect food security by developing commercial crops optimized for yield and nutritional quality. Researchers are able to target and edit stress response pathway genes to develop crops with increased tolerance to stress. A lack of regeneration protocols and sufficient genome sequencing data has restricted fruit editing to only a few fruits (tomatoes, citrus, apple, kiwi, banana, grapes, strawberries, watermelon, etc.). This review is focused on CRISPR/Cas applications on the nutritional aspects of fruit engineering along with the challenges and opportunities. Another aspect which will be covered is the use of CRISPR/Cas technology to improve fruit resilience to biotic and abiotic stress, but not at the cost of yield. We discuss the pros and cons of using this technology, such as unintended effects on fruit traits or public concerns about GMOs. We conclude that the application of CRISPR/Cas9 technology has the potential to be of great benefit to the agricultural industry not only to improve nutritional aspects but also to help reduce crop losses.},
}

@article {pmid41354953,
year = {2025},
author = {Nguyen, VT and Van, BTT and Uyen, TN and Tong, NX and Pham, TL and Vy, NHT and Thuy, DT and Thuy, NP and Kobayashi, M},
title = {Functional divergence of zebrafish keap1 paralogs revealed by CRISPR/Cas9-mediated gene editing: a specialized role for keap1b in inflammation.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {53},
pmid = {41354953},
issn = {1573-9368},
support = {108.06-2020.19//National Foundation for Science and Technology Development/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Zebrafish Proteins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Oxidative Stress/genetics ; *Inflammation/genetics ; Kelch-Like ECH-Associated Protein 1/genetics ; NF-E2-Related Factor 2/genetics ; Signal Transduction/genetics ; Gene Editing ; Carrier Proteins ; },
abstract = {The Keap1/Nrf2 signaling pathway is a master regulator of cellular defense against oxidative and electrophilic stress. In teleosts like zebrafish (Danio rerio), whole-genome duplication resulted in two keap1 paralogs, keap1a and keap1b, whose functional specificities remain incompletely understood. This study investigates the divergent roles of these paralogs by comparing the responses of established keap1a and novel keap1b knockout larvae to distinct chemical stressors. By comparing the responses of keap1b[dl40], keap1a[dl07], and nfe2l2a[dl703] (Nrf2a) larvae to these stressors, we uncovered a striking functional dichotomy. While loss of either paralog conferred resistance to H2O2-induced oxidative stress, keap1b[dl40] larvae, unlike their keap1a[dl07] counterparts, exhibited extreme sensitivity to the lethal effects of CuSO4 exposure, with survival rates plummeting to ~ 25%. This heightened sensitivity to copper sulfate was associated with a blunted transcriptional response of inflammatory markers tnf-a and c3a, suggesting that Keap1b is critical for modulating the Nrf2a-mediated response to inflammatory stress in orchestrating a viable inflammatory response. This work clarifies the non-redundant, vital function of Keap1b in the response to heavy metal-induced stress and provides a valuable genetic resource (keap1b[dl40] null allele) for future studies.},
}

Animals
*Zebrafish/genetics
*Zebrafish Proteins/genetics/metabolism
*CRISPR-Cas Systems/genetics
Oxidative Stress/genetics
*Inflammation/genetics
Kelch-Like ECH-Associated Protein 1/genetics
NF-E2-Related Factor 2/genetics
Signal Transduction/genetics
Gene Editing
Carrier Proteins

@article {pmid41353974,
year = {2025},
author = {Madny, MA and Yadav, KS},
title = {Biomimetic oral drug delivery: Translating nature's design into therapeutic innovation.},
journal = {Colloids and surfaces. B, Biointerfaces},
volume = {259},
number = {},
pages = {115348},
doi = {10.1016/j.colsurfb.2025.115348},
pmid = {41353974},
issn = {1873-4367},
abstract = {Oral drug delivery, the most patient friendly administration route offers convenience and compliance but faces formidable biological barriers. Enzymatic degradation, mucosal entrapment, efflux transport and extensive first-pass metabolism drastically reduce the effectiveness of sensitive therapeutics including peptides, proteins, nucleic acids and vaccines. Conventional formulations often fail to overcome these challenges highlighting the need for innovative approaches. Biomimetic drug delivery has emerged as a transformative strategy. By emulating structures and functions from cells, membranes, exosomes, viruses and gut microbiota these systems achieve immune evasion, mucus penetration, site-specific targeting and stimulus-responsive release. Such approaches improve formulation stability and in vivo absorption but also promise precise and patient centric therapies. This review provides a comprehensive overview of biomimetic oral systems highlighting their mechanisms, design principles and translational potential. Recent advances include cell membrane-coated nanoparticles for tumor targeting and immune modulation, exosome-inspired carriers for protein and RNA transport, virus-like particles (VLPs) for oral vaccines, and mucoadhesive or mucus-penetrating polymers modeled on pathogen strategies. Complementary pH, enzyme and redox-responsive platforms exploit gastrointestinal (GI) microenvironments to ensure controlled release. Emerging tools such as bioinspired computational modeling, 3D/4D printing, organoid-on-chip models and CRISPR/Cas-based platforms accelerate optimization and clinical translation. Although most technologies remain in preclinical development, early findings demonstrate superior pharmacokinetics, therapeutic efficacy, and safety over conventional systems. This article critically examines biomimetic oral drug delivery addressing advances and underlying mechanisms including regulatory considerations and future directions. They stand poised to form the foundation of next-generation precision therapeutics.},
}

@article {pmid41352919,
year = {2026},
author = {Guan, X and Wang, S and Wang, P and Zhang, J and Sun, S},
title = {Enhanced chemiluminescence aptasensing with triple cascade amplification for sensitive detection of tumor-derived exosomes.},
journal = {Analytica chimica acta},
volume = {1383},
number = {},
pages = {344873},
doi = {10.1016/j.aca.2025.344873},
pmid = {41352919},
issn = {1873-4324},
mesh = {*Exosomes/chemistry/metabolism ; Humans ; *Aptamers, Nucleotide/chemistry/metabolism ; *Luminescent Measurements/methods ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Limit of Detection ; Mucin-1 ; Alkaline Phosphatase/chemistry/metabolism ; Nucleic Acid Amplification Techniques ; CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Biomarkers, Tumor ; Tetraspanin 30 ; },
abstract = {BACKGROUND: Tumor-associated exosomes hold significant clinical promise as liquid biopsy biomarkers. However, the accurate detection of these rare exosome subpopulations in clinical samples demands analytical platforms with exceptionally high sensitivity and specificity. While conventional nucleic acid amplification-based methods provide considerable detection sensitivity, they are often hampered by time-consuming procedures, operational complexity, and susceptibility to contamination. Therefore, it is imperative to develop practical exosome measurement platforms that combine high sensitivity, robustness, and rapid analysis capabilities to provide reliable evidence-based support for precision oncology.

RESULTS: In this work, a triple cascade-amplified aptasensor (TCAA) via functionalized gold nanoparticle (fAuNP), CRISPR/Cas12a, and alkaline phosphatase (ALP) was developed for enhanced chemiluminescence (CL) assay of tumor-derived exosomes without nucleic acid amplification. The target exosomes were initially recognized by CD63 and MUC1 aptamers. fAuNP-conjugated Trigger sequences then activated CRISPR/Cas12a to cleave single-stranded DNA and release ALP. Consequently, the ALP catalyzed substrate to produce CL signals correlating with the concentration of the analyte. By simultaneously integrating the signal amplification capabilities of multiple techniques, this TCAA achieved a limit of detection of 44 particles/μL for MUC1-positive exosomes within 60 min with excellent robustness. Compared with the single- and dual-amplification methods, the sensitivity was increased by 40-fold and 6-fold, respectively. Clinical trials showed that the area under the curve of this approach was 0.96, which was higher than that of the commercialized chemiluminescence immunoassay and effectively distinguished breast cancer-derived specimens.

SIGNIFICANCE: These findings indicate that the TCAA strategy provides a highly sensitive, rapid, and robust tool for the detection of low-abundance tumor exosome subpopulations without nucleic acid amplification. It effectively addresses the limitations of conventional methods and demonstrates high clinical utility. This work offers a reliable and practical platform for non-invasive liquid biopsy, holding great potential for trace-level detection of diverse biomarkers.},
}

*Exosomes/chemistry/metabolism
Humans
*Aptamers, Nucleotide/chemistry/metabolism
*Luminescent Measurements/methods
Gold/chemistry
Metal Nanoparticles/chemistry
Limit of Detection
Mucin-1
Alkaline Phosphatase/chemistry/metabolism
Nucleic Acid Amplification Techniques
CRISPR-Cas Systems
*Biosensing Techniques/methods
Biomarkers, Tumor
Tetraspanin 30

@article {pmid41330004,
year = {2025},
author = {Coşar, B and Kılıç, P and İşeri, ÖD},
title = {The intersection of CAR-T immunotherapy with emerging technologies.},
journal = {Cytokine & growth factor reviews},
volume = {86},
number = {},
pages = {238-259},
doi = {10.1016/j.cytogfr.2025.11.001},
pmid = {41330004},
issn = {1879-0305},
mesh = {Humans ; *Immunotherapy, Adoptive/methods ; *Receptors, Chimeric Antigen/immunology/genetics ; *Neoplasms/therapy/immunology ; Animals ; Cytokines/immunology ; *T-Lymphocytes/immunology ; CRISPR-Cas Systems ; Gene Editing ; Tumor Microenvironment/immunology ; },
abstract = {Chimeric antigen receptor (CAR) T-cell (CAR-T) therapy is a transformative modality in cancer immunotherapy that employs genetically engineered T-cells to eliminate malignant cells selectively. Its efficacy and limitations are governed by cytokine- and growth factor-mediated signaling networks that shape T-cell activation, proliferation, differentiation, and persistence. This review traces the molecular evolution of CAR-T architecture across generations, highlighting how synthetic modulation of cytokine and co-stimulatory pathways enhances potency while reducing exhaustion and toxicity. We discuss strategies that incorporate cytokine engineering, metabolic reprogramming, and logic-gated activation to counteract the immunosuppressive tumor microenvironment. Recent technological advances-such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9)-based cytokine pathway editing, induced pluripotent stem cell (iPSC)-derived "off-the-shelf" CAR-T platforms, and extracellular vesicle (EV)-mediated cytokine delivery-are reshaping adoptive immunotherapy. Framing CAR-T development through the lens of cytokine and growth factor biology, we outline how integrating these pathways enables safer, more durable, and scalable next-generation therapies for hematologic and solid tumors.},
}

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

@article {pmid41292433,
year = {2025},
author = {Lan, F and Chen, A and Ding, Y and Yang, C and Zhang, P and Fang, X},
title = {Sensitive and Specific Analysis of miRNAs in Single Tumor-Derived Extracellular Vesicles Using CRISPR-Based Nanoflow Cytometry.},
journal = {Analytical chemistry},
volume = {97},
number = {48},
pages = {26521-26531},
doi = {10.1021/acs.analchem.5c04700},
pmid = {41292433},
issn = {1520-6882},
mesh = {*MicroRNAs/analysis/genetics ; Humans ; *Extracellular Vesicles/chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; *Prostatic Neoplasms/genetics/diagnosis ; *Flow Cytometry/methods ; Male ; Limit of Detection ; Biomarkers, Tumor/genetics ; *Nanotechnology ; },
abstract = {Tumor-derived extracellular vesicle (TEV) microRNAs (miRNAs) are promising cancer biomarkers but pose detection challenges due to their low abundance and sequence homology. Here, we present a CRISPR/Cas13a-based nanoflow cytometry (nFCM) platform integrated with a DNA-guided orthogonal membrane fusion strategy for ultrasensitive miRNA detection of TEVs at the single particle level. TEVs were identified with aptamers against CD63 and EpCAM markers to create an orthogonal barcode-anchored TEV (Orth-TEV). Meanwhile, liposomes preloaded with CRISPR/Cas13a molecular sensing components were modified with cholesterol-tagged DNA probes to produce Tags-CRISPR/Cas13a@Lipo. The complementary DNA sequences on the Orth-TEV and Tags-CRISPR/Cas13a@Lipo vesicles facilitated zipper-like hybridization, thereby achieving specific membrane fusion to effectively eliminate the interference of nontarget vesicles or free molecules. The resulting TEV-CRISPR/Cas13a@Lipo vesicles allow in situ detection of three prostate cancer (PCa)-associated miRNAs in a single TEV via nFCM with a low detection limit (LOD) of 14.7 (miR-153), 16.0 (miR-183), and 23.7 (miR-940) particles/mL, respectively. The approach was further applied to plasma samples from PCa patients and healthy donors, showing significantly elevated miRNA signals in PCa-derived TEV. ROC analysis yielded AUC values of 0.931, 0.923, and 0.869 for the three target miRNAs, confirming excellent diagnostic performance. To enhance classification accuracy, we conducted a statistical multivariate analysis based on the PCA-LDA model, which achieved perfect group separation and a diagnostic accuracy of 91.3%. Overall, this CRISPR/Cas13a-based nFCM platform offers a robust, accurate, and clinically applicable platform for single-vesicle miRNA profiling with broad potential in liquid biopsy-based cancer diagnosis.},
}

*MicroRNAs/analysis/genetics
Humans
*Extracellular Vesicles/chemistry/genetics/metabolism
*CRISPR-Cas Systems
*Prostatic Neoplasms/genetics/diagnosis
*Flow Cytometry/methods
Male
Limit of Detection
Biomarkers, Tumor/genetics
*Nanotechnology

@article {pmid41292075,
year = {2025},
author = {Du, J and Hu, J and An, J and Li, H and Chen, B and Luo, J and Li, S and Teng, Y and Yuan, T and Zhu, X and Jiang, L and Xiong, E and Yang, R},
title = {Guanine-Quadruplex-Engineered crRNA Enables Light-Activated CRISPR/Cas12a System for Robust One-Pot Viral Assay.},
journal = {Analytical chemistry},
volume = {97},
number = {48},
pages = {26580-26589},
doi = {10.1021/acs.analchem.5c04848},
pmid = {41292075},
issn = {1520-6882},
mesh = {*G-Quadruplexes ; *CRISPR-Cas Systems/genetics ; Nucleic Acid Amplification Techniques/methods ; Humans ; *Guanine/chemistry ; *Light ; CRISPR-Associated Proteins/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Conventional one-pot detection platforms integrating CRISPR/Cas12a with isothermal amplification significantly streamline the nucleic acid detection workflow, while minimizing the risk of aerosol contamination. However, the intrinsic cleavage activity of the CRISPR/Cas12a system can substantially interfere with the nucleic acid amplification efficiency, ultimately compromising detection sensitivity. Herein, we develop a light-activated CRISPR/Cas12a system by engineering the crRNA with a guanine-quadruplex (G4) motif at its 3'-terminal, achieving precise regulation of Cas12a activity via photoswitching G4 structure formation. Through coupling with a recombinase polymerase amplification (RPA) reaction, we establish a one-pot detection platform that demonstrates superior detection performance compared to traditional Cas12a-based one-pot systems. The detection sensitivity has been improved by 2 orders of magnitude, reaching a level of 1 copy/μL. Notably, the platform demonstrated comparable sensitivity and specificity to PCR, the gold standard method, in detecting clinical samples, such as Epstein-Barr virus (EBV) and Influenza A virus (IAV), making it a promising technology for clinical diagnostics.},
}

*G-Quadruplexes
*CRISPR-Cas Systems/genetics
Nucleic Acid Amplification Techniques/methods
Humans
*Guanine/chemistry
*Light
CRISPR-Associated Proteins/metabolism
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid41289351,
year = {2025},
author = {Su, T and Wei, T and Wang, Z and Wu, H and Fan, Y and Su, S and Zhu, D and Wang, L},
title = {A Pre-Amplification-Free Modular Dual-CRISPR System for Enhanced Pathogen Detection Sensitivity.},
journal = {Analytical chemistry},
volume = {97},
number = {48},
pages = {26640-26648},
doi = {10.1021/acs.analchem.5c05145},
pmid = {41289351},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; Nucleic Acid Hybridization ; *African Swine Fever Virus/genetics/isolation & purification ; Limit of Detection ; Humans ; *DNA, Viral/analysis/genetics ; Animals ; Nucleic Acid Amplification Techniques ; },
abstract = {CRISPR/Cas12a is extensively utilized for pathogen detection owing to its high specificity and efficiency. However, traditional single-CRISPR/Cas12a encounters challenges due to its limited sensitivity, requiring pre-amplification of nucleic acids. This increases the complexity of the procedure and the potential for cross-contamination and false positives. Herein, a modular dual-CRISPR approach was developed coupled with hybridization chain reaction (HCR) for the universal and sensitive detection of pathogen nucleic acids without the need for pre-amplification. The system comprises two core modules: the first CRISPR/Cas12a recognition module specifically identifies pathogen targets and releases the activating agent, while the second CRISPR/Cas12a signal module is activated by this agent to initiate the HCR reaction for generating a strong fluorescent signal through DNA nanostructure self-assembly. Through rational design, we demonstrate the ability of this dual-CRISPR system to achieve attomolar (aM) level sensitivity for pathogen nucleic acid detection without pre-amplification, showing over six-order-of-magnitude higher sensitivity than a traditional single-CRISPR/Cas12a system. Additionally, the flexibility and versatility of the modular dual-CRISPR system have been confirmed for diverse pathogen targets, such as African swine fever virus (ASFV), severe fever with thrombocytopenia syndrome virus (SFTSV), and human papillomavirus type 16 (HPV-16) DNA. The system's practicality was demonstrated by examining ASFV quality control samples in complex environments. The exploration of the pre-amplification-free dual-CRISPR system offers a new perspective on enhancing pathogen nucleic acid detection systems.},
}

*CRISPR-Cas Systems/genetics
Nucleic Acid Hybridization
*African Swine Fever Virus/genetics/isolation & purification
Limit of Detection
Humans
*DNA, Viral/analysis/genetics
Animals
Nucleic Acid Amplification Techniques

@article {pmid41265176,
year = {2026},
author = {Mao, S and Guo, Y and Dong, C and Wang, D and Wang, X and Weng, L and Yang, Y and Li, Y and Niu, T and Wu, Q and Zheng, Z and Shan, Z and Tan, X and Gao, Y and Jin, J and Wang, P and Ge, X and Shen, B and Yao, X and Fang, L},
title = {Loss of cyclin C drives resistance to anti-TIGIT therapy by upregulating CD155-mediated immune evasion.},
journal = {Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy},
volume = {84},
number = {},
pages = {101318},
doi = {10.1016/j.drup.2025.101318},
pmid = {41265176},
issn = {1532-2084},
mesh = {Humans ; *Receptors, Immunologic/antagonists & inhibitors/immunology ; Killer Cells, Natural/immunology ; Cell Line, Tumor ; *Drug Resistance, Neoplasm/immunology/genetics ; Up-Regulation ; *Neoplasms/immunology/drug therapy/genetics ; CRISPR-Cas Systems ; T-Lymphocytes/immunology ; Gene Expression Regulation, Neoplastic ; Immune Evasion ; Immune Checkpoint Inhibitors/pharmacology ; Tumor Escape ; Receptors, Virus ; },
abstract = {AIMS: CD155 is an immune checkpoint protein expressed in tumor cells that interacts with its ligand T cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) on natural killer (NK) cells and T cells, mediating inhibitory regulation on immune cells. Blockade of the CD155-TIGIT interaction has demonstrated clinical benefits in patients with advanced cancers. The transcriptional and post-translational mechanisms governing CD155 expression remain largely unknown.

METHODS: To identify regulators of CD155, we conducted a genome-wide CRISPR-Cas9 screen in cancer cells. Surface CD155 protein levels were analyzed via flow cytometry. The role of candidate regulators was validated through loss- and gain-of-function experiments with flow cytometry, Western blot, quantitative PCR, and chromatin immunoprecipitation (ChIP) assays. Additionally, ubiquitination assay was performed to examine post-translational modifications. Functional studies, including NK and T cell cytotoxicity assays, were conducted to assess the immune modulatory effects of CD155 regulation. Clinical relevance was evaluated by analyzing Cyclin C (CCNC) and CD155 expression in datasets of cancer patients who underwent immune checkpoint blockade therapy.

RESULTS: The CRISPR-Cas9 screen identified CCNC as a transcriptional suppressor of CD155. CCNC knockout led to increased surface CD155 expression in cancer cell lines. Mechanistically, CCNC inhibited CD155 transcription by suppressing the activity of the transcription factor FOSL2. Furthermore, CCNC was found to be ubiquitinated and degraded by the E3 ubiquitin ligase FBXO11, suggesting a post-translational regulatory mechanism. Functionally, loss of CCNC promoted CD155 upregulation, thereby enhancing tumor immune evasion from NK and T cell-mediated responses. Clinically, CCNC expression was negatively correlated with CD155 levels in cancer patients, particularly those receiving immune checkpoint blockade therapy.

CONCLUSION: This study identifies a previously unrecognized master regulator CCNC that functions as a suppressor of CD155-mediated cancer immune evasion. The findings of this study suggest that tumors with low CCNC expression may be resistant to monotherapy and highlight a combination immunotherapy (TIGIT/PD-1 co-blockade) as a promising anti-cancer therapeutic strategy to overcome immune evasion in CCNC-deficient tumors.},
}

Humans
*Receptors, Immunologic/antagonists & inhibitors/immunology
Killer Cells, Natural/immunology
Cell Line, Tumor
*Drug Resistance, Neoplasm/immunology/genetics
Up-Regulation
*Neoplasms/immunology/drug therapy/genetics
CRISPR-Cas Systems
T-Lymphocytes/immunology
Gene Expression Regulation, Neoplastic
Immune Evasion
Immune Checkpoint Inhibitors/pharmacology
Tumor Escape
Receptors, Virus

@article {pmid41259748,
year = {2025},
author = {Zheng, L and Zhou, X and Zhang, Y and Wang, W and Chen, C and Lin, X and Zheng, Y and Lou, Y},
title = {Rapid Bacterial Identification and Antimicrobial Susceptibility Testing Directly from Urine Samples via an Asymmetric Polymerase Chain Reaction-Cas12a Platform.},
journal = {Analytical chemistry},
volume = {97},
number = {48},
pages = {26466-26474},
doi = {10.1021/acs.analchem.5c04410},
pmid = {41259748},
issn = {1520-6882},
mesh = {Humans ; Microbial Sensitivity Tests ; *Bacteria/drug effects/isolation & purification/genetics ; *Anti-Bacterial Agents/pharmacology ; *Polymerase Chain Reaction/methods ; CRISPR-Cas Systems ; *CRISPR-Associated Proteins/genetics ; *Endodeoxyribonucleases/genetics/metabolism ; Bacterial Proteins ; },
abstract = {Antimicrobial resistance poses a critical global health challenge, largely due to the prolonged turnaround times of conventional pathogen identification (ID) and antimicrobial susceptibility testing (AST). Here, we present a clinically validated diagnostic platform integrating asymmetric polymerase chain reaction (aPCR) with CRISPR/Cas12a for direct bacterial ID and phenotypic AST from urine samples. Unlike traditional multiplex PCR requiring complex primer sets, our platform employs a singleplex aPCR targeting the V3-V4 region of 16S rDNA to generate single-stranded and double-stranded DNA. This design enables protospacer adjacent motif-free activation of Cas12a when required via the ssDNA fraction generated by aPCR, facilitating species-level multiplex detection of six common uropathogens at 10[3] CFU/mL via programmable CRISPR/Cas12a crRNAs. Phenotypic AST is accomplished within 60 min by quantifying nucleic acid changes following antibiotic exposure, allowing accurate discrimination between susceptible and resistant strains. When validated with 86 clinical urine samples, the aPCR-Cas12a platform achieved complete concordance with culture-based identification among the 45 samples carrying target pathogens and demonstrated high accuracy for AST, confirming its reliability for direct pathogen detection and susceptibility assessment from urine. The complete workflow requires only 5.5 h, significantly reducing the diagnostic time compared to standard methods (>48 h). This rapid, cost-effective, and scalable platform offers a promising solution for infection diagnosis and antimicrobial stewardship, with strong potential for integration into routine clinical microbiology and point-of-care settings.},
}

Humans
Microbial Sensitivity Tests
*Bacteria/drug effects/isolation & purification/genetics
*Anti-Bacterial Agents/pharmacology
*Polymerase Chain Reaction/methods
CRISPR-Cas Systems
*CRISPR-Associated Proteins/genetics
*Endodeoxyribonucleases/genetics/metabolism
Bacterial Proteins

@article {pmid41217936,
year = {2025},
author = {He, X and Deng, L and Zhou, S and Gu, T and Li, X and Zhu, S and Luo, X and Huo, D and Hou, C},
title = {Breaking the PAM Restriction: A Universal Double Stranded DNA Detection Method Based on the Sticky End-Mediated CRISPR/Cas12a Coupled RPA and Its Application to KRAS G12C Single Base Mutations.},
journal = {Analytical chemistry},
volume = {97},
number = {48},
pages = {26886-26896},
doi = {10.1021/acs.analchem.5c05908},
pmid = {41217936},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; *Proto-Oncogene Proteins p21(ras)/genetics ; *DNA/analysis/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; Point Mutation ; *CRISPR-Associated Proteins/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; Limit of Detection ; *Recombinases/metabolism ; Bacterial Proteins ; },
abstract = {The CRISPR/Cas12a system facilitates efficient and specific nucleic acid detection, but its dependence on Protospacer Adjacent Motif (PAM) sequences and the complexity of existing sticky end-based methods pose challenges for stable and portable applications. To address these issues, this study developed a universal dsDNA detection method by integrating the sticky end-mediated CRISPR/Cas12a with recombinase polymerase amplification (RPA). By incorporating NlaIII recognition sites into RPA primers, precise cleavage of amplification products was achieved, generating uniform sticky ends and eliminating reliance on PAM sites. In comparison to flat end dsDNA containing PAM sites, the use of sticky end dsDNA significantly enhanced Cas12a activity. This strategy demonstrated sensitivity and specificity, achieving a detection limit of 40 aM and successfully identifying KRAS G12C mutations at a frequency of 0.1%, with genomic DNA results aligning with those obtained from FastNGS. Furthermore, we preliminarily explored a one-tube detection strategy, which effectively streamlined the operational process and reduced aerosol contamination. In summary, we established a simple, sensitive, and universal PAM-free CRISPR/Cas12a detection platform that integrates the advantages of isothermal amplification with a standardized sticky end design, thereby offering broad application prospects in molecular diagnostics and clinical translation.},
}

*CRISPR-Cas Systems/genetics
*Proto-Oncogene Proteins p21(ras)/genetics
*DNA/analysis/genetics
Humans
*Nucleic Acid Amplification Techniques/methods
Point Mutation
*CRISPR-Associated Proteins/metabolism/genetics
*Endodeoxyribonucleases/metabolism/genetics
Limit of Detection
*Recombinases/metabolism
Bacterial Proteins

@article {pmid40974875,
year = {2026},
author = {Sun, D and Bo, L and Jiang, C and Lan, Y and Zhang, B and Zhang, C and Chen, ZS and Fan, Y},
title = {Beyond the boundary: The emerging roles of ATP-binding cassette transporters in multidrug resistance (MDR) and therapeutic targeting in cancer.},
journal = {Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy},
volume = {84},
number = {},
pages = {101310},
doi = {10.1016/j.drup.2025.101310},
pmid = {40974875},
issn = {1532-2084},
mesh = {Humans ; *Drug Resistance, Neoplasm/drug effects/genetics ; *Neoplasms/drug therapy/genetics/pathology ; *Drug Resistance, Multiple/drug effects/genetics ; *ATP-Binding Cassette Transporters/genetics/metabolism/antagonists & inhibitors ; *Antineoplastic Agents/pharmacology/therapeutic use ; Animals ; Gene Editing/methods ; CRISPR-Cas Systems ; Molecular Targeted Therapy ; Immunoconjugates/pharmacology/therapeutic use ; Immunotherapy/methods ; },
abstract = {Multidrug resistance (MDR) remains a primary obstacle to successful cancer chemotherapy, with the overexpression of ATP-binding cassette (ABC) transporters being a principal cause. These transporters actively efflux a wide range of anticancer drugs, reducing their intracellular efficacy. Consequently, targeting ABC transporters represents a critical strategy for overcoming therapeutic resistance. This comprehensive review details the molecular architecture and functional mechanisms of all seven human ABC transporter subfamilies (ABCA-ABCG), elucidating their distinct roles in both cancer progression and the development of MDR. We trace the evolution of therapeutic interventions, from first, second, and third-generation small molecule inhibitors to the potential of natural products. Furthermore, this review explores advanced and emerging strategies designed to circumvent or neutralize ABC transporter activity. These include genetic approaches such as RNA interference and CRISPR-Cas9 gene editing, immunotherapy-based tactics like monoclonal antibodies and antibody-drug conjugates (ADCs), and the application of sophisticated nanoparticle delivery systems designed to bypass efflux mechanisms. By providing a holistic overview of the entire ABC transporter family and the broad array of strategies being developed to counteract their function, this article aims to equip researchers with a full-scope perspective on the field, identifying current challenges and illuminating future directions for combating MDR in cancer.},
}

Humans
*Drug Resistance, Neoplasm/drug effects/genetics
*Neoplasms/drug therapy/genetics/pathology
*Drug Resistance, Multiple/drug effects/genetics
*ATP-Binding Cassette Transporters/genetics/metabolism/antagonists & inhibitors
*Antineoplastic Agents/pharmacology/therapeutic use
Animals
Gene Editing/methods
CRISPR-Cas Systems
Molecular Targeted Therapy
Immunoconjugates/pharmacology/therapeutic use
Immunotherapy/methods

@article {pmid41352908,
year = {2026},
author = {Li, L and Tang, Z and Xu, H and Zhou, F and Ji, X and He, Z},
title = {Investigation on CRISPR-Cas12a-split crRNA system for successively detecting DNA and RNA in one tube.},
journal = {Analytica chimica acta},
volume = {1383},
number = {},
pages = {344860},
doi = {10.1016/j.aca.2025.344860},
pmid = {41352908},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems ; *DNA, Viral/analysis/genetics ; *RNA, Viral/analysis/genetics ; Hepatitis B virus/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; Humans ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; HIV/genetics ; },
abstract = {Recently, CRISPR/Cas system has been proposed as a novel tool with simplicity and high accuracy. The CRISPR RNA (crRNA) can be divided into spacer crRNA and handle crRNA without losing its original function. In this work, we have investigated CRISPR Cas12a with split crRNA to detect HBV DNA and HIV RNA in a single tube. In the first step, Cas12a can recognize HBV DNA and initiate its trans-cleavage on FAM-BHQ1 reporter, after 1 h incubation, the fluorescence intensity was correlated with the concentration of HBV DNA. In the second step, the TAMRA BHQ2 ds DNA reporter was introduced in the same tube to bind with remained Cas12a proteins, HIV RNA and handle crRNA. The trans-cleavage from the first step would not interfere with HIV RNA and dsDNA reporter. With the incubation for another hour, HIV RNA can be quantified by the cis-cleavage of TAMRA BHQ2 reporter. we can successively identify the two nucleic acids with the limit of detection of 0.70 pM for HBV DNA, and 0.47 nM for HIV RNA, respectively. This special designed split crRNA can simplify detecting procedure and only need Cas12a protein in a single tube. Next, we expand this strategy in semi-quantifying two kinds of DNA in one tube. Overall, this study overcomes the limitation of conventional CRISPR-based methods and provides a new, inexpensive, and low-threshold approach based on Cas12a with split crRNA.},
}

*CRISPR-Cas Systems
*DNA, Viral/analysis/genetics
*RNA, Viral/analysis/genetics
Hepatitis B virus/genetics
*CRISPR-Associated Proteins/metabolism/genetics
Humans
*Endodeoxyribonucleases/metabolism/genetics
*Bacterial Proteins/metabolism/genetics
HIV/genetics

@article {pmid41352906,
year = {2026},
author = {Fakhr, ZA and Xie, W and Zeng, S and Cai, S},
title = {Site accessibility-driven CRISPR/Cas13a activation for amplification-free RNA biosensing.},
journal = {Analytica chimica acta},
volume = {1383},
number = {},
pages = {344858},
doi = {10.1016/j.aca.2025.344858},
pmid = {41352906},
issn = {1873-4324},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *RNA/analysis ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics/chemistry ; Kinetics ; *CRISPR-Associated Proteins/metabolism ; },
abstract = {BACKGROUND: CRISPR-Cas13a biosensing enables rapid, amplification-free RNA diagnostics, yet assay sensitivity varies widely because guide RNAs (gRNAs) differ in their ability to activate the enzyme. Two factors, including the gRNA-target binding affinity and the structural accessibility of the target site, have been proposed to govern activation efficiency, but their relative importance remains unclear. In this study, we systematically disentangle these contributions by measuring binding affinities for gRNAs that span a spectrum of site accessibilities and by comparing their Michaelis-Menten kinetic parameters.

RESULTS: Three ciRS-7-specific gRNAs were designed with high, intermediate, and low spacer accessibility. Isothermal titration calorimetry (ITC) quantified site accessibility through entropy changes (ΔS = -862, -813, and -615 cal/mol/K), confirming greater structural exposure for less structured spacers, and also determined binding affinity for each gRNA-target pair. Michaelis-Menten analysis showed kcat values of 1.39, 1.31, and 1.16 s[-1] for the high, intermediate, and low-accessibility guides, respectively, establishing a clear relationship between structural accessibility and catalytic turnover. Importantly, the most structured gRNA exhibited lower activation efficiency compared with the gRNA that had higher site accessibility and lower binding affinity, demonstrating that site accessibility drives Cas13a activation. Detection-limit experiments also confirmed these results, showing that gRNAs with greater spacer accessibility yielded stronger signals and superior sensitivity.

SIGNIFICANCE: Our data establish site accessibility as a critical determinant of Cas13a activation for amplification-free RNA sensing. Prioritizing unstructured spacer regions enables improved enzyme activation efficiency, providing a clear design rule for next-generation CRISPR diagnostics. This accessibility-driven strategy will facilitate the development of faster, simpler, and more sensitive point-of-care assays for diverse RNA biomarkers.},
}

*Biosensing Techniques/methods
*CRISPR-Cas Systems
*RNA/analysis
*RNA, Guide, CRISPR-Cas Systems/metabolism/genetics/chemistry
Kinetics
*CRISPR-Associated Proteins/metabolism

@article {pmid41351274,
year = {2025},
author = {Hou, H and Li, Y and Su, N and Ding, Y and Shang, C and Li, X and Xiong, Z and Sun, Y and Zhan, W and Wang, Y and Zhang, X and Pan, Y and Wu, L and Li, J},
title = {Slmsh1-induced heritable enhancement of traits for tomato breeding improvement.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {5},
pages = {e70607},
doi = {10.1111/tpj.70607},
pmid = {41351274},
issn = {1365-313X},
support = {CSTB2023TIAD-KPX0026//Special Key Project of Technological Innovation and Application Development of Chongqing/ ; 31872123//National Natural Science Foundation of China/ ; 32172597//National Natural Science Foundation of China/ ; CARS-23-B08//China Agriculture Research System/ ; SWU-KF25027//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Solanum lycopersicum/genetics/physiology/growth & development ; *Plant Breeding/methods ; *Plant Proteins/genetics/metabolism ; Fruit/genetics/growth & development ; Droughts ; Quantitative Trait, Heritable ; Phenotype ; CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; },
abstract = {Vegetable grafting is a horticultural technique employed to develop specialized plant varieties by effectively enhancing resistance to both biotic and abiotic stresses, as well as improving fruit quality and yield. However, these advantageous traits are generally non-heritable. The MSH1 gene induced heritable enhancement-through-grafting (HEG) effect on growth vigor, demonstrating promising application potential. In this study, we employed the msh1 mutant tomato as a rootstock to induce heritable superior traits and combined this approach with hybridization techniques to enhance tomato cultivars. Three Slmsh1 mutants were generated using CRISPR/Cas9 which exhibited a dwarf phenotype with whitened spots. By grafting several distinct inbred lines onto Slmsh1, we observed significant HEG, drought stress tolerance, and fruit quality. Under drought conditions, Slmsh1-grafted tomato seedlings exhibited increased biomass and enhanced drought tolerance through the regulation of antioxidant enzyme activities. Differential expression and methylation analyses of the graft progeny revealed that these heritable enhanced traits (HETs) are likely attributable to epigenetic modifications in the expression of ROS-scavenging- and hormone-related genes. Furthermore, to explore practical applications, we crossed inbred lines with HETs and evaluated the growth, yield, and fruit quality of the resulting hybrid combinations. The results indicated that these hybrid combinations improved fruit yield and quality, enhancing the total soluble solids, soluble sugar, and soluble protein content. These findings suggest that Slmsh1-grafted progenies enhanced plant biomass and drought resistance, while their hybrid combinations positively influenced root growth, yield, and fruit quality, providing new insights into the synergistic integration of genome editing and conventional breeding.},
}

*Solanum lycopersicum/genetics/physiology/growth & development
*Plant Breeding/methods
*Plant Proteins/genetics/metabolism
Fruit/genetics/growth & development
Droughts
Quantitative Trait, Heritable
Phenotype
CRISPR-Cas Systems
Gene Expression Regulation, Plant

@article {pmid41350682,
year = {2025},
author = {Lyu, G and Li, P and Lang, W},
title = {A review of recent studies on CRISPR/Cas9-mediated genome editing in a variety of muscle-related genetic disorders.},
journal = {Journal of translational medicine},
volume = {23},
number = {1},
pages = {1381},
pmid = {41350682},
issn = {1479-5876},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Animals ; *Muscular Diseases/genetics/therapy ; *Genetic Diseases, Inborn/genetics ; Genetic Therapy ; Muscular Dystrophies/genetics ; },
abstract = {The human body is capable of mutating a single gene to produce a wide range of debilitating disorders. Genomic editing for disease prevention via phenotypic reversal was a significant challenge prior to the development of clustered regulatory interspaced short tandem repeats (CRISPR) and CRISPR-associated protein (Cas) systems. Gene therapy-editing a patient's DNA to correct a particular mutation-and treating human diseases that have not responded to conventional medicine are two areas where CRISPR/Cas9 technology shows the most promise as a therapeutic tool. This powerful instrument has shown great promise in muscle-related illnesses, offering new insights into muscle biology and developing more effective treatment techniques. Discoveries about the hereditary causes of the majority of inherited myopathies and muscular dystrophies (MDs) have emerged over the last two decades. Additionally, skeletal muscles weaken and degenerate over time due to a group of hereditary disorders known as MDs. The field of skeletal muscle diseases and associated genetic alterations is seeing remarkable progress in developing therapeutic vectors to fix these mutations. Myopathies, MDs, and neuromuscular disorders are just a few examples of the many genetic abnormalities related to muscles that have sparked renewed interest in the potential of genome editing as a therapeutic tool due to its efficiency, adaptability, and relative ease of use in targeted genome editing. Consequently, CRISPR/Cas9 has garnered much interest and is used more often in therapeutic techniques due to its potential capacity to cure various human ailments. To pave the way for more effective and personalized therapies, this review article provides a thorough overview of the revolutionary role of CRISPR/Cas9 in improving our understanding and treatment of genetic disorders related to muscles by combining present knowledge with future perspectives.},
}

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

@article {pmid41349515,
year = {2025},
author = {Kim, I and Suh, JY},
title = {Capture first, then deliver!.},
journal = {Structure (London, England : 1993)},
volume = {33},
number = {12},
pages = {2008-2009},
doi = {10.1016/j.str.2025.11.001},
pmid = {41349515},
issn = {1878-4186},
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/chemistry/metabolism ; DNA/metabolism/chemistry ; *Integrases/chemistry/metabolism ; Protein Binding ; },
abstract = {In this issue of Structure, Henriques et al.[1] present structural snapshots that capture distinct conformational states of the type I-F Cas1-Cas2/3 integrase complex, illustrating that foreign DNA binding triggers a large-scale domain rearrangement that enables prespacer delivery to the CRISPR array.},
}

*CRISPR-Cas Systems
*CRISPR-Associated Proteins/chemistry/metabolism
DNA/metabolism/chemistry
*Integrases/chemistry/metabolism
Protein Binding

@article {pmid41349512,
year = {2025},
author = {Kosaka, Y and Lopez, B and Kishimoto, N and Jacob, S and Montenont, E and Huallanca, R and Coughenour, G and Di Paola, J and Ross, J and Lee, K and Rondina, MT and Bray, PF and Rowley, JW},
title = {Functional classification of platelet gene variants using CRISPR HDR in CD34[+] cell-derived megakaryocytes.},
journal = {American journal of human genetics},
volume = {112},
number = {12},
pages = {2888-2901},
doi = {10.1016/j.ajhg.2025.11.004},
pmid = {41349512},
issn = {1537-6605},
mesh = {Humans ; *Megakaryocytes/metabolism ; *Blood Platelets/metabolism ; *CRISPR-Cas Systems/genetics ; *Antigens, CD34/metabolism/genetics ; Integrin beta3/genetics ; Gene Editing/methods ; Integrin alpha2/genetics ; *Genetic Variation ; Hematopoietic Stem Cells/metabolism ; Thrombasthenia/genetics ; },
abstract = {The interpretation of genetic variants in inherited diseases, such as inherited platelet disorders (IPDs), remains a major clinical challenge, as most are classified as variants of uncertain significance (VUSs). A key barrier to functional evaluation is the lack of accessible, lineage-appropriate assays that reliably reflect native gene regulation and cell-specific biology. To address this gap, we developed CRIMSON HD (CRISPR-edited megakaryocytes [MKs] for surveying platelet variant functions through homology-directed repair [HDR]), a CRISPR-Cas9 HDR-based genome-editing platform applicable to CD34[+] cell-derived blood lineages and optimized for evaluating platelet-associated variants. Using this system, we modeled known and candidate disease-associated variants in integrin alpha 2b (ITGA2B) and integrin beta 3 (ITGB3), which encode the platelet αIIb/β3 integrin and are causative in Glanzmann thrombasthenia (GT). We introduced precise variants into primary human MKs derived from CD34[+] hematopoietic stem and progenitor cells, achieving >90% editing efficiency. Edited MKs faithfully recapitulated both expression and functional phenotypes of known type I, II, and III GT variants. CRIMSON HD enabled functional evaluation and reclassification of several GT VUSs, including αIIb Gly201Ala, a population variant now shown to cause near-complete loss of αIIb/β3 expression; αIIb Ala777Asp, which results in intermediate αIIb/β3 expression and impaired agonist-induced integrin binding; and β3 Arg119Gln, previously linked to the loss of anti-HPA1a antibody binding in fetal and neonatal alloimmune thrombocytopenia (FNAIT), now shown to impair integrin surface expression. These findings demonstrate the importance of lineage-specific, physiologically relevant assays for the functional classification of platelet-related variants, providing mechanistic information and clinically meaningful insights for individuals with IPDs.},
}

Humans
*Megakaryocytes/metabolism
*Blood Platelets/metabolism
*CRISPR-Cas Systems/genetics
*Antigens, CD34/metabolism/genetics
Integrin beta3/genetics
Gene Editing/methods
Integrin alpha2/genetics
*Genetic Variation
Hematopoietic Stem Cells/metabolism
Thrombasthenia/genetics

@article {pmid41348871,
year = {2025},
author = {Puppala, AK and Nielsen, AC and Regan, M and Mancinelli, GE and De Pooter, RF and Arnovitz, S and Harding, C and McGregor, M and Balanis, NG and Clarke, R and Merrill, BJ},
title = {Programmable multistep CRISPR gene activation via control of RNA polymerase III termination.},
journal = {Science advances},
volume = {11},
number = {49},
pages = {eadt1532},
pmid = {41348871},
issn = {2375-2548},
mesh = {Humans ; *RNA Polymerase III/metabolism/genetics ; *CRISPR-Cas Systems ; *Transcriptional Activation ; Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Induced Pluripotent Stem Cells/metabolism/cytology ; HEK293 Cells ; },
abstract = {Although genomes encode instructions for mammalian cell differentiation with rich syntactic relationships, existing methods for genetically programming cells have only modest capabilities for stepwise gene regulation. Here, we develop a sequential genetic system that transcriptionally activates endogenous genes in a preprogrammed, stepwise manner. This system uses the removal of an RNA polymerase III termination sequence to trigger both the transcriptional activation and DNA endonuclease activities of a Cas9-VPR protein, driving progression through a cascade of gene activation events. The system's functionality in human cells, including iPSCs, enables the development of a path for cellular programming by controlling the sequential order of gene activation to influence cellular states.},
}

Humans
*RNA Polymerase III/metabolism/genetics
*CRISPR-Cas Systems
*Transcriptional Activation
Gene Editing/methods
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Induced Pluripotent Stem Cells/metabolism/cytology
HEK293 Cells

@article {pmid41348151,
year = {2025},
author = {Cheng, Y and Gao, W and Shi, S and Han, F and Dong, H},
title = {Identification of the orange pigment in Nonomuraea gerenzanensis and development of a pigment-free mutant with high yield of A40926.},
journal = {AMB Express},
volume = {},
number = {},
pages = {},
doi = {10.1186/s13568-025-01993-4},
pmid = {41348151},
issn = {2191-0855},
support = {2024TSGC0896//Shandong Province Science and Technology-based Small and Medium-sized Enterprises Innovation Capacity Enhancement Project/ ; },
abstract = {The secondary metabolite A40926, a precursor to the glycopeptide antibiotic dalbavancin, is synthesized by the rare actinomycete Nonomuraea gerenzanensis (N. gerenzanensis) within the pharmaceutical industry. The biosynthesis of A40926 is accompanied by the production of an orange pigment, which poses significant challenges and incurs high costs in the purification process of A40926. To identify this orange pigment, a comprehensive analysis was conducted, including the examination of the biosynthetic gene cluster, potential biosynthetic pathways, purification processes, and structural identification. Additionally, the ispF gene, which encodes the enzyme 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase and is implicated in the biosynthesis of orange pigment, was deleted using the CRISPR/Cas9 system. To enhance A40926 production in the ΔIspF mutant, the overexpression of the cyclic AMP receptor protein (Crp) was implemented to assess its regulatory impact on A40926 biosynthesis. Consequently, the orange pigment produced by N. gerenzanensis was identified as lycopene, synthesized via the methylerythritol phosphate (MEP) pathway. Although the ΔIspF mutant was unable to biosynthesize the orange pigment, its production of A40926 was adversely affected and was lower than that of the original strain. Consequently, the overexpression of the global regulator Crp significantly enhanced A40926 production, achieving a yield of 841.1 mg/L. The investigation of pigment-free mutants presented in this study offers valuable insights for effectively reducing production costs within the microbial pharmaceutical industry.},
}

@article {pmid41271111,
year = {2026},
author = {Huang, Q and Zhao, T and Su, W and Li, S and Liu, J and Ge, Z and Zhang, B and Ren, X and Zhang, X and Wei, J},
title = {Screening of monoclonal vaccine strains based on real-time live-cell imaging technology.},
journal = {Journal of virological methods},
volume = {340},
number = {},
pages = {115305},
doi = {10.1016/j.jviromet.2025.115305},
pmid = {41271111},
issn = {1879-0984},
mesh = {Animals ; *Vaccinia virus/genetics/immunology/isolation & purification ; *Viral Vaccines/immunology/genetics/isolation & purification ; Viral Plaque Assay/methods ; Vaccines, Attenuated/immunology/genetics ; Antibodies, Viral/blood/immunology ; Humans ; Green Fluorescent Proteins/genetics ; CRISPR-Cas Systems ; Vaccines, Synthetic/immunology/genetics ; Antibodies, Neutralizing/blood ; },
abstract = {The plaque purification is a critical step in the screening of traditional live-attenuated vaccines and recombinant viral vaccines, aiming to acquire vaccine clones with homogeneous characteristics and desirable immunogenicity to address outbreaks of emerging diseases such as monkeypox, chikungunya fever, and dengue fever. The traditional plaque purification process to screen out a vaccine strain with genetically consistent stability from a mixed pool of viral clones generally requires laborsome work. We utilized live-cell imaging technique enabling us to isolate monoclonal vaccine strains to simplify and improve the efficiency of this process. Here, we genetically engineered the vaccinia virus TianTan (VTT) using CRISPR/Cas9 system to generate recombinant VTT viruses (VTT-WS01-EGFP) that expressed enhanced green fluorescent protein (EGFP). Initially, we performed 9 rounds of plaque purification using traditional plaque assay, yielding 50 candidate clones. The Incucyte Live-Cell Imaging and Analysis system was subsequently performed to conduct a rigorous, high-resolution screening of these candidates in a more automated, sensitive and high-throughput way. Through this screening process, we ultimately obtained 31 pure viral clones that were free of parental strain contamination, followed by the analysis of plaque formation, fluorescent plaque size, and plaque morphology, and 11 candidate clones were selected for immunological evaluation. Furthermore, we found that clone 49 induced a relatively high titer of anti-VTT neutralizing antibodies and elicited the production of cross-reactive IgG against monkeypox virus antigens, thereby validating its potential as a candidate strain as a monkeypox virus vaccine. Taken together, our data demonstrates that live-cell imaging technique significantly accelerates the screening process for the isolation of monoclonal viral clones as recombinant viral vaccines, and holds considerable potential in attenuated strain selection as well as investigations into biological characteristics of viruses, including viral replication.},
}

Animals
*Vaccinia virus/genetics/immunology/isolation & purification
*Viral Vaccines/immunology/genetics/isolation & purification
Viral Plaque Assay/methods
Vaccines, Attenuated/immunology/genetics
Antibodies, Viral/blood/immunology
Humans
Green Fluorescent Proteins/genetics
CRISPR-Cas Systems
Vaccines, Synthetic/immunology/genetics
Antibodies, Neutralizing/blood

@article {pmid41260396,
year = {2026},
author = {Zhong, Z and Li, G and Liang, G and Ren, T and Teng, C and Xiong, J and Ji, G and Zheng, M and Pan, Y and Qin, Y and Ouyang, K and Yin, Y and Chen, Y and Huang, W and Wei, Z},
title = {Establishment of a nucleic acid detection method for foot-and-mouth disease virus serotype O utilizing RPA-CRISPR/Cas12a technology.},
journal = {Journal of virological methods},
volume = {340},
number = {},
pages = {115304},
doi = {10.1016/j.jviromet.2025.115304},
pmid = {41260396},
issn = {1879-0984},
mesh = {*Foot-and-Mouth Disease Virus/genetics/isolation & purification/classification ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *Foot-and-Mouth Disease/diagnosis/virology ; Animals ; Serogroup ; Recombinases/metabolism/genetics ; DNA Primers/genetics ; RNA, Viral/genetics ; *Molecular Diagnostic Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {This study aimed to develop a rapid and visually interpretable nucleic acid detection assay for Foot-and-Mouth Disease Virus serotype O (FMDV-O) by integrating recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology. Specific RPA primers and CRISPR RNA (crRNA) sequences were designed and optimized based on the conserved 3D gene region of FMDV-O. An assay combining RPA pre-amplification with Cas12a-mediated cleavage was subsequently established. The sensitivity and specificity of the RPA-CRISPR/Cas12a method were systematically evaluated, and its diagnostic utility was further assessed using clinical samples. The results demonstrated that the primer set RPA-F1/R1 paired with crRNA1 constituted the optimal combination, with an ideal reaction system comprising 50 nM Cas12a protein and 200 nM crRNA. This system exhibited a detection limit of 2.60 × 10[2] copies/μL for target plasmid DNA following a 20-minute incubation at 37°C. Specificity analysis confirmed positive detection exclusively for FMDV-O plasmids, with no cross-reactivity observed with other tested pathogens. When applied to clinical samples, the proposed method demonstrated a superior detection rate relative to conventional PCR. In conclusion, a novel diagnostic platform for FMDV-O was successfully developed based on RPA-CRISPR/Cas12a. This method is characterized by its rapidity, operational simplicity, high sensitivity, and excellent specificity, holding significant promise for application in clinical diagnostics, epidemiological surveillance, and field-based testing.},
}

*Foot-and-Mouth Disease Virus/genetics/isolation & purification/classification
Sensitivity and Specificity
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
*Foot-and-Mouth Disease/diagnosis/virology
Animals
Serogroup
Recombinases/metabolism/genetics
DNA Primers/genetics
RNA, Viral/genetics
*Molecular Diagnostic Techniques/methods
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41231539,
year = {2025},
author = {Jia, N and Zhou, YJ and Gao, J},
title = {Engineering recombination machinery facilitates the construction of yeast cell factories.},
journal = {FEMS yeast research},
volume = {25},
number = {},
pages = {},
doi = {10.1093/femsyr/foaf066},
pmid = {41231539},
issn = {1567-1364},
support = {22478382//National Natural Science Foundation of China/ ; E411040705//Energy Revolution S&T Program of Yulin Innovation Institute of Clean Energy/ ; },
mesh = {*Metabolic Engineering/methods ; *Gene Editing/methods ; *DNA End-Joining Repair ; *Saccharomyces cerevisiae/genetics/metabolism ; *Homologous Recombination ; *Recombination, Genetic ; CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; },
abstract = {Advances in genome editing have been promoted by programmable nucleases like CRISPR-Cas9, which triggers endogenous DNA repair mechanisms by inducing double-strand break (DSB). Cellular responses to DSBs are governed by competing repair pathways: error-prone non-homologous end joining (NHEJ) and high-fidelity homologous recombination (HR). This review systematically compares the molecular mechanisms and key regulators of NHEJ and HR, with a focus on recent breakthroughs in recombination engineering in non-conventional yeasts. These advances address challenges in precise genome editing, enabling robust metabolic engineering of yeast cell factories for sustainable bioproduction.},
}

*Metabolic Engineering/methods
*Gene Editing/methods
*DNA End-Joining Repair
*Saccharomyces cerevisiae/genetics/metabolism
*Homologous Recombination
*Recombination, Genetic
CRISPR-Cas Systems
DNA Breaks, Double-Stranded

@article {pmid41347244,
year = {2025},
author = {Daraghmeh, DN and AbuIriban, RW and Nawawreh, N and Abuamro, AM and Alassar, MM and Daraghma, SN and Alhajahmed, NM and Thandar, Y},
title = {Advancements in alternative approaches to address antimicrobial resistance in bacterial pneumonia: a comprehensive review.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1704931},
pmid = {41347244},
issn = {1664-302X},
abstract = {PURPOSE: This review explores both current and emerging alternative treatment approaches to combat AMR specifically in the context of bacterial pneumonia, highlighting therapies that extend beyond conventional antibiotics.

METHODS: PubMed, Embase, and Google Scholar were searched for full-text, English-language articles, with emphasis on publications from 2020 to 2025. Earlier seminal studies were also included when necessary to provide historical, mechanistic, or conceptual context. The review focuses was on alternative strategies that have shown effectiveness in preclinical or clinical settings to combat AMR in relation to bacterial pneumonia.

RESULTS: Emerging strategies to tackle AMR in bacterial pneumonia involve several innovative approaches including stem cells, bacteriophage therapy, metal based nanoparticles (e.g., silver, copper, and gold). The adjunctive use of probiotics and herbal medicine has demonstrated potential in enhancing clinical outcomes and modulating host immunity. Moreover, gene editing technologies like CRISPR-CAS and various vaccination programs are being investigated for their roles in prevention and resistance management. While these methods show promise, many are still in the early stages of development and encounter challenges related to standardization, safety, and regulatory approval.

CONCLUSION: Alternative therapies present exciting possibilities for addressing AMR in bacterial pneumonia. However, to effectively translate these innovations into clinical practice, we need thorough research, international collaboration, and supportive policy frameworks. By combining these strategies with antimicrobial stewardship initiatives, we can help maintain antibiotic effectiveness and enhance patient outcomes.},
}

@article {pmid41346702,
year = {2026},
author = {Patra, C and Hussein, Z and Ace, VD and Misnik, EV and Rybalko, DS and Salimova, AA and Ereshko, DS and Dubovichenko, MV and Nour, MAY and Drozd, VS and Kolpashchikov, DM},
title = {The efficacy of oligonucleotide-based gene therapeutics in gene silencing.},
journal = {Theranostics},
volume = {16},
number = {2},
pages = {599-616},
pmid = {41346702},
issn = {1838-7640},
mesh = {Humans ; *Genetic Therapy/methods ; *Gene Silencing ; *Oligonucleotides, Antisense/therapeutic use/genetics ; RNA, Small Interfering/genetics/therapeutic use ; Animals ; *Oligonucleotides/genetics/therapeutic use ; },
abstract = {Oligonucleotide-based gene therapeutics (OGTs) have emerged as a promising strategy for treating a variety of diseases, offering a tool for gene modulation at the mRNA level. Despite significant progress in OGTs development, their efficacy in both experimental and clinical settings has often fallen short of expectations. Current estimates suggest that less than 1% of transfected OGTs are released into the cytosol, significantly limiting the interaction with target RNA. Moreover, data suggests that only about 2% of the tested siRNAs achieve the expected 70% target gene knockdown in vitro. Clinically approved OGTs appear to be effective only against genetic disorders that lack effective alternative treatment, and even in these cases their therapeutic contribution remains marginal. Notably, the majority of approved OGTs, as well as those currently in clinical trials, are antisense oligonucleotides (ASOs) despite cell culture data showing that small interfering RNAs (siRNAs) exhibit greater potency. The delayed commercialization of siRNAs, despite high research interest, may be attributed to passenger stand-dependent off target effect and the immaturity of their design and modification strategies. This review critically evaluates the factors influencing therapeutic efficacy of OGTs and highlights the persistent gap between theoretical promise and clinical reality.},
}

Humans
*Genetic Therapy/methods
*Gene Silencing
*Oligonucleotides, Antisense/therapeutic use/genetics
RNA, Small Interfering/genetics/therapeutic use
Animals
*Oligonucleotides/genetics/therapeutic use

@article {pmid41275796,
year = {2025},
author = {Gao, Y and Zhao, L and Shi, J and Wang, B},
title = {NtQPT2 plays critical roles in nicotine biosynthesis and development of tobacco plant.},
journal = {Biochemical and biophysical research communications},
volume = {793},
number = {},
pages = {153030},
doi = {10.1016/j.bbrc.2025.153030},
pmid = {41275796},
issn = {1090-2104},
mesh = {*Nicotiana/growth & development/genetics/metabolism/enzymology ; *Nicotine/biosynthesis ; *Pentosyltransferases/genetics/metabolism ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Plants, Genetically Modified ; },
abstract = {The enzyme quinolinate phosphoribosyltransferase (QPT), encoded by a small gene family in tobacco plant, plays a critical role in the biosynthesis of nicotine, a defensive pyridine alkaloid in Nicotiana species, in addition to its vital function in the NAD(P)(H) synthesis. Previous studies have demonstrated that two NtQPT genes (NtQPT1 and NtQPT2) are present in N. tabacum genome, and it has been believed that NtQPT1 is responsible for NAD(P)(H) synthesis and thus essential for primary metabolism, while NtQPT2 is specifically involved in nicotine biosynthesis. In this study, we generated knockout tobacco lines for NtQPT1 and NtQPT2 respectively using the CRISPR/Cas9-based genome-editing technology and found that knockout of NtQPT2 caused both dramatic reduction of nicotine biosynthesis and a retardation of plant development, indicating that NtQPT2 is important not only to nicotine biosynthesis, but also to the development of tobacco plant. Like NtQPT2, NtQPT1 was also found to contribute to nicotine biosynthesis although to a much lesser extent than NtQPT2. Meanwhile, knockout of NtQPT1 did not significantly affect plant growth. Together with the observation that NtQPT2's expression is remarkably higher than that of NtQPT1 in root, leaf, stem and flower of tobacco plant, it is reasonable to infer that their functional diversification on nicotine biosynthesis and tobacco plant growth may be attributed largely to their markedly different transcript abundance.},
}

*Nicotiana/growth & development/genetics/metabolism/enzymology
*Nicotine/biosynthesis
*Pentosyltransferases/genetics/metabolism
Gene Expression Regulation, Plant
*Plant Proteins/genetics/metabolism
CRISPR-Cas Systems
Gene Knockout Techniques
Plants, Genetically Modified

@article {pmid41274245,
year = {2025},
author = {Shimura, R and Yamamoto, K and Chang, YH and Otaki, A and Goyama, S},
title = {Development of a CRISPR/Cas9-degron system that enables in vivo specific gene depletion in leukemia models.},
journal = {Biochemical and biophysical research communications},
volume = {793},
number = {},
pages = {153002},
doi = {10.1016/j.bbrc.2025.153002},
pmid = {41274245},
issn = {1090-2104},
mesh = {*CRISPR-Cas Systems/genetics ; Animals ; Humans ; *Leukemia, Myeloid, Acute/genetics/pathology ; Histone-Lysine N-Methyltransferase/genetics ; Mice ; *Gene Editing/methods ; Cell Line, Tumor ; Disease Models, Animal ; CRISPR-Associated Protein 9 ; Degrons ; },
abstract = {The CRISPR/Cas9 system has transformed genome editing, yet precise temporal control of Cas9 activity remains challenging. We developed a Cas9-degron platform that couples degron-tagged Cas9 with a dTAG-based chemical degradation strategy. In the presence of dTAG, Cas9 is rapidly and near-completely degraded, preventing editing; upon dTAG withdrawal, Cas9 activity is restored, enabling precise temporal control. Using this system, we achieved selective in vivo gene depletion in acute myeloid leukemia (AML) models and confirmed that SETDB1, a histone H3K9 methyltransferase, is essential for the in vivo growth of both human (MOLM13) and murine (cSAM) AML cells. By maintaining SETDB1 intact prior to transplantation and depleting it afterward, we avoided culture-induced pre-selection bias inherent to sgRNA transduction and validated its critical role in AML progression within the in vivo context. The Cas9-degron retains activity and delivery efficiency comparable to conventional Cas9 in the absence of dTAG. Thus, this versatile system provides a superior alternative to conventional Cas9 and a powerful platform for in vivo CRISPR screening, gene function studies, and potentially temporally controlled gene therapy.},
}

*CRISPR-Cas Systems/genetics
Animals
Humans
*Leukemia, Myeloid, Acute/genetics/pathology
Histone-Lysine N-Methyltransferase/genetics
Mice
*Gene Editing/methods
Cell Line, Tumor
Disease Models, Animal
CRISPR-Associated Protein 9
Degrons

@article {pmid41086995,
year = {2025},
author = {Jang, SH and Song, HG and Jung, J and Gee, HY},
title = {Recent preclinical and clinical advances in gene therapy for hereditary hearing loss.},
journal = {Molecules and cells},
volume = {48},
number = {12},
pages = {100285},
pmid = {41086995},
issn = {0219-1032},
mesh = {*Genetic Therapy/methods ; Humans ; *Hearing Loss/therapy/genetics ; Animals ; Gene Editing ; Genetic Vectors/genetics ; CRISPR-Cas Systems ; Dependovirus/genetics ; Gene Transfer Techniques ; Clinical Trials as Topic ; },
abstract = {Hereditary hearing loss is a genetically heterogeneous condition that affects millions of people worldwide and has limited curative treatment options. Recent advancements in gene therapy have opened promising avenues for correcting the underlying genetic defects in the inner ear. This review summarizes the key developments in vector platforms, delivery strategies, target genes, preclinical models, and clinical trials relevant to both gene supplementation and gene editing approaches, as well as future directions. Adeno-associated virus vectors have emerged as the leading platform for inner ear gene transfer, owing to their safety and efficacy. Clinical programs, such as those targeting OTOF variants, are currently underway and are supported by robust preclinical data. Additionally, genome editing technologies, including CRISPR/Cas9-mediated nonhomologous end joining, base editing, and prime editing, offer variant-specific therapeutic potential. Despite these advances, challenges remain in expanding the therapeutic window, ensuring long-term safety, and establishing ethical and regulatory frameworks for their use.},
}

*Genetic Therapy/methods
Humans
*Hearing Loss/therapy/genetics
Animals
Gene Editing
Genetic Vectors/genetics
CRISPR-Cas Systems
Dependovirus/genetics
Gene Transfer Techniques
Clinical Trials as Topic

@article {pmid41346247,
year = {2025},
author = {Singh, V and Mishra, M and Singla-Pareek, SL and Roy, JK and Pareek, A},
title = {Lysine Matters: Genetic and Biotechnological Innovations to Combat Protein Malnutrition.},
journal = {Plant, cell & environment},
volume = {},
number = {},
pages = {},
doi = {10.1111/pce.70316},
pmid = {41346247},
issn = {1365-3040},
support = {//This study was supported by Department of Biotechnology, Ministry of Science and Technology, India./ ; },
abstract = {Lysine deficiency in staple crops like maize, rice, and wheat remains a major cause for global protein malnutrition, underscoring the urgent need for effective biofortification strategies. This review critically examines recent advances in enhancing lysine content, spanning conventional breeding and metabolic engineering to cutting-edge precision genome editing. While conventional breeding, exemplified by Quality Protein Maize, has improved lysine levels, it is often constrained by yield and quality trade-offs. Metabolic engineering strategies, including overexpression of lysine biosynthetic genes, suppression of catabolic genes, and modification of storage proteins, have achieved substantial lysine enrichment but face regulatory and consumer acceptance challenges due to their transgenic nature. The advent of CRISPR/Cas technology now enables precise, transgene-free editing of key enzymes such as DHDPS, AK, and LKR/SDH offering a powerful alternative, though concerns regarding off-target effects and pleiotropy remain. While integrating multi-omics with AI-driven predictive modelling can optimise metabolic flux for higher lysine yield, coupling next-generation genome editing with speed breeding offers a transformative route to develop high-lysine, high-yielding crops for sustainable nutritional security.},
}

@article {pmid41345283,
year = {2025},
author = {Saydam, S and Dinçer, P},
title = {Precision rewriting of muscle genetics: therapeutic horizons of base and prime editing in skeletal muscle disorders.},
journal = {Gene therapy},
volume = {},
number = {},
pages = {},
pmid = {41345283},
issn = {1476-5462},
abstract = {Base Editing (BE) and Prime Editing (PE), novel precision tools of the CRISPR/Cas toolbox, have emerged as transformative technologies that enable highly specific genetic modifications. Their compatibility with post-mitotic cell types makes them invaluable for treating genetic skeletal muscle disorders. Despite their severity and progressive nature, monogenic muscle diseases remain without definitive treatments. They are caused by diverse mutations in critical muscle proteins, for which gene editing offers a promising therapeutic avenue. However, traditional CRISPR/Cas9 applications face challenges such as genotoxicity and inefficiency in post-mitotic tissues. BE and PE technologies overcome these limitations by enabling safe and efficient modifications without causing double-strand breaks or requiring homology-directed repair. Their therapeutic potential comes from two key features: their ability to work in non-dividing cells such as myotubes and cardiomyocytes, and their capacity to target a broad range of mutations found in genetic muscle diseases. In this review, we explore mechanisms of BE and PE and summarize their current applications in monogenic skeletal muscle disorders. We discuss the challenges of in vivo application in skeletal muscle and highlight innovations to bypass them. Collectively, both systems offer flexible precision solutions with immense potential for mutation-specific and personalized gene therapy approaches for monogenic skeletal muscle disorders.},
}

@article {pmid41344769,
year = {2026},
author = {Liu, X and Zheng, Y and Chen, Z and Wang, S and Liao, H and Jia, J and Wang, G and Wang, J and Yuan, C and Guo, X and Yin, Y and Hu, Q},
title = {Rapid and visual detection of Listeria monocytogenes by combining one-pot LAMP-CRISPR/Cas12b with lateral flow assay.},
journal = {Food microbiology},
volume = {135},
number = {},
pages = {104977},
doi = {10.1016/j.fm.2025.104977},
pmid = {41344769},
issn = {1095-9998},
mesh = {*Listeria monocytogenes/isolation & purification/genetics ; *Nucleic Acid Amplification Techniques/methods ; Animals ; Food Contamination/analysis ; Food Microbiology/methods ; Swine ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; Limit of Detection ; },
abstract = {Listeria monocytogenes, the leading cause of fatalities worldwide among foodborne pathogens, poses serious risks to food safety and public health. Therefore, a rapid and accurate detection method is crucial for early interception and effective management. In this study, a one-pot LAMP-CRISPR/Cas12b detection system based on the lmo0753 gene was developed for rapid detection of L. monocytogenes by combining loop-mediated isothermal amplification (LAMP) with a CRISPR/Cas12b assay. Further integration of a lateral flow assay (LFA) to develop a LAMP-CRISPR/Cas12b-LFA assay enabled direct detection of the results on the strips with the naked eye. Nine L. monocytogenes strains belonging to eight serotypes tested positive with both the one-pot LAMP-CRISPR/Cas12b and LAMP-CRISPR/Cas12b-LFA assays. Two assays did not show cross-reactivity with L. innocua and eight other foodborne bacteria. The limits of detection were 10 CFU/mL for pure culture and 20 CFU/g for spiked pork samples. Moreover, the enrichment time was substantially shortened to 3 h for pork samples spiked with only L. monocytogenes F2365, and 4-5 h for pork samples spiked with mixed bacteria. In addition, with one-pot LAMP-CRISPR/Cas12b detection, 5 of 66 fresh pork samples, 1 of 20 ready-to-eat food samples, and 2 of 24 raw milk samples tested positive for L. monocytogenes, in agreement with the results obtained through a culture based standard method. Thus, this study established one-pot LAMP-CRISPR/Cas12b and LAMP-CRISPR/Cas12b-LFA assays for rapid, visual detection of L. monocytogenes in food samples.},
}

*Listeria monocytogenes/isolation & purification/genetics
*Nucleic Acid Amplification Techniques/methods
Animals
Food Contamination/analysis
Food Microbiology/methods
Swine
*CRISPR-Cas Systems
*Molecular Diagnostic Techniques/methods
Sensitivity and Specificity
Limit of Detection

@article {pmid41341583,
year = {2025},
author = {Jiang, Z and Jia, B and Hu, N and Zhang, M and Xiao, H and Chen, G and Yu, J and Li, X and Shen, B and Feng, J and Wang, J},
title = {In Vivo engineering of transgenic mice for systemic human neutralizing antibody production against staphylococcal enterotoxin B.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1679421},
pmid = {41341583},
issn = {1664-3224},
mesh = {Animals ; Mice, Transgenic ; Humans ; Mice ; *Enterotoxins/immunology ; *Antibodies, Neutralizing/immunology/genetics/biosynthesis ; Female ; CRISPR-Cas Systems ; Genetic Engineering ; Antibodies, Monoclonal/immunology/genetics ; Glycosylation ; },
abstract = {Transgenic animal bioreactors provide a complementary strategy to traditional mammalian cell culture systems for the production of therapeutic human monoclonal antibodies (mAbs). Here we present a CRISPR/Cas9-mediated breakthrough in creating two novel genetically engineered (GE) mouse models with species-specific chromosomal integration of human anti-staphylococcal enterotoxin B (SEB) mAb genes at either the ROSA26 or Hipp11 (H11) safe-harbor loci - evolutionarily conserved genomic safe harbors (GSH). These genetically optimized animals demonstrated broad tissue capability for glycosylation-competent human antibodies, achieving exceptional secretion levels reaching 208 mg/L in serum, 43 mg/L in mammary secretions, 24 mg/L in saliva on average. The transgenic lines maintained this antibody production stability for >140 weeks without compromising animal viability, while preserving germline transmission fidelity through six successive generations. Furthermore, the highly glycosylated human antibodies derived from these genetic engineered mice exhibited high binding affinity to SEB (KD=0.108 nM for ROSA26; 0.154 nM for H11), providing comprehensive protection against SEB intoxication in vivo. This study opens avenues for utilizing transgenic animal bioreactors for large-scale production of fully human antibodies or disease-resistant livestock in the foreseeable future.},
}

Animals
Mice, Transgenic
Humans
Mice
*Enterotoxins/immunology
*Antibodies, Neutralizing/immunology/genetics/biosynthesis
Female
CRISPR-Cas Systems
Genetic Engineering
Antibodies, Monoclonal/immunology/genetics
Glycosylation

@article {pmid41330077,
year = {2026},
author = {Fathpour, H and Fouladi, M and Jafarpour, F and Moradi-Hajidavaloo, R and Izadi, T and Shiralian-Esfahani, H and Kues, W and Nasr-Esfahani, MH and Hajian, M and Eghbalsaied, S},
title = {Crosstalk between myostatin and callipyge in CRISPR/Cas9-edited goat fibroblast cells.},
journal = {Research in veterinary science},
volume = {198},
number = {},
pages = {105992},
doi = {10.1016/j.rvsc.2025.105992},
pmid = {41330077},
issn = {1532-2661},
mesh = {Animals ; *Myostatin/genetics/metabolism ; *Goats/genetics ; *Fibroblasts/metabolism ; *CRISPR-Cas Systems ; Gene Editing/veterinary ; },
abstract = {Myostatin (MSTN) and Callipyge (CLPG) genes are key regulators of muscle growth. While MSTN inhibits muscle development, the CLPG mutation induces muscle hypertrophy through a specific imprinted genetic mechanism. The interaction between these genes remains of interest for improving livestock muscle traits. In this study, CRISPR/Cas9 was employed to edit MSTN and CLPG genes in goat fibroblast cells via electrotransfection. Cells were selected using puromycin antibiotic, and gene-editing efficiency was evaluated through Sanger sequencing. Gene expression changes were analyzed using RT-qPCR analysis. MSTN gene knockout resulted in significant downregulation of MSTN and CLPG, while GTL2 expression was upregulated by more than 50-fold. Additionally, myosin heavy chain genes (MYH1, MYH3, MYH4) were strongly upregulated, with MYH3 13-fold and MYH4 30-fold increase in the expression. In CLPG-edited cells, the expression of MSTN, TRIM28, and CLPG was reduced, while GTL2 was upregulated by 6-fold. MYH3 and MYH4 expression increased 4-fold in CLPG-edited cells, though the increase was less pronounced compared to MSTN-edited cells. DLK1 expression was undetectable in both non-edited control and gene-edited fibroblast cells. Our findings support the interaction between MSTN and CLPG, contributing to the regulation of muscle growth. Notably, the study also highlights the challenges associated with editing imprinted genes like CLPG and suggests that TRIM28 may play a role downstream of CLPG regulation. These results provide valuable insights into muscle development regulation, offering potential applications in livestock genetic improvement.},
}

Animals
*Myostatin/genetics/metabolism
*Goats/genetics
*Fibroblasts/metabolism
*CRISPR-Cas Systems
Gene Editing/veterinary

@article {pmid41285661,
year = {2026},
author = {Gu, X and Zhang, T and Yao, H and Guo, F and Yang, C and Xu, H and He, X and Ma, Z and Zhang, X and Yu, S and An, R and Wang, F},
title = {CRISPR-Cas12a-integrated pregnancy test strip biosensors: Visual detection of telomerase and miRNA let-7a in cervical cancer diagnostics.},
journal = {Biosensors & bioelectronics},
volume = {294},
number = {},
pages = {118241},
doi = {10.1016/j.bios.2025.118241},
pmid = {41285661},
issn = {1873-4235},
mesh = {Humans ; Female ; *MicroRNAs/genetics/isolation & purification/analysis ; *Uterine Cervical Neoplasms/diagnosis/genetics ; *Biosensing Techniques ; *Telomerase/isolation & purification/genetics/analysis ; CRISPR-Cas Systems/genetics ; HeLa Cells ; Pregnancy ; Limit of Detection ; Reagent Strips/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Cervical cancer is a leading cause of female cancer-related mortality globally, and early screening based on reliable biomarkers is critical for improving prognosis. Telomerase (a key driver of cellular immortalization) and microRNA let-7a (a tumor suppressor with downregulated expression in cervical cancer) are well-validated diagnostic targets, but existing detection methods are hindered by complex procedures, high instrumentation costs, and reliance on specialized technical expertise-limiting their accessibility in resource-constrained settings. To address these limitations, we developed two novel CRISPR-Cas12a-integrated biosensors using commercially available pregnancy test strips (PTS) for instrument-free, visual readout. Both biosensors leverage a core signal mediator, probe 1 ("MB-ssDNA1-hCG"), which links CRISPR-Cas12a activation to visible color development on the PTS. The first Biosensor CRISPR-PTS-Telo detects telomerase activity in one-step without PCR: telomerase-generated (TTAGGG)n repeats activate Cas12a-crRNA1 complex, cleaving the probe 1 to release hCG, achieving a detection limit of 18 HeLa cells-comparable to sensitive laboratory assays. The second Biosensor CRISPR-PTS-let7a detects miRNA let-7a by first converting miRNA signals to Trigger DNA via Assister DNA and probe 2 ("MB-ssDNA2+Trigger"), activating Cas12a-crRNA2 complex, cleaving the probe 1 and inducing PTS coloration. This achieves a detection limit of 25.1 fM for let-7a. Validation with clinical samples (24 cervical tissues and 26 blood samples) confirmed their concordance with gold-standard methods (ELISA for telomerase, RT-qPCR for let-7a). These versatile tools hold significant potential as point-of-care testing (POCT) solutions to facilitate early, accessible cervical cancer screening.},
}

Humans
Female
*MicroRNAs/genetics/isolation & purification/analysis
*Uterine Cervical Neoplasms/diagnosis/genetics
*Biosensing Techniques
*Telomerase/isolation & purification/genetics/analysis
CRISPR-Cas Systems/genetics
HeLa Cells
Pregnancy
Limit of Detection
Reagent Strips/analysis
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41093250,
year = {2025},
author = {Buck-Wiese, M and Liechocki, S and Erfle, H and Starkuviene, V},
title = {Comparative analysis of antibody-mediated loss-of-function versus gene knock-out and knock-down.},
journal = {SLAS discovery : advancing life sciences R & D},
volume = {37},
number = {},
pages = {100283},
doi = {10.1016/j.slasd.2025.100283},
pmid = {41093250},
issn = {2472-5560},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; *Gene Knockdown Techniques/methods ; *Antibodies ; Talin/genetics/metabolism ; RNA Interference ; RNA, Small Interfering/genetics ; Gene Expression Profiling ; Cell Adhesion/genetics ; },
abstract = {In this study we compare three methods for manipulating cell function: RNA interference (RNAi), CRISPR-Cas9 gene knock-out, and antibody-mediated loss-of-function. We have focused on analyzing changes in cell-matrix adhesion via targeting two key regulators, Talin1 (TLN1) and Kindlin-2 (KD2). Adhesion-relevant phenotypic assays revealed distinct temporal onset dynamics for each method. RNAi and CRISPR-Cas9 effectively reduced target mRNA and protein levels. In contrast, antibody transfection induced phenotypic changes without altering target expression, suggesting direct intracellular antibody-target interaction. Transcriptome analysis demonstrated that antibody transfection and CRISPR-Cas9 induced fewer deregulated mRNAs than RNAi. Furthermore, transfected antibodies and sgRNAs shared 30 % and 70 % of deregulated transcripts to their negative controls, respectively. Whereas only 10 % of overlap was recorded between targeting and control siRNAs. Our findings emphasize the importance of considering method-specific temporal dynamics of on-target phenotype appearance and off-target manifestation. Additionally, they highlight intracellular delivered antibodies as a valuable alternative for validating and complementing genetic approaches.},
}

Humans
CRISPR-Cas Systems/genetics
*Gene Knockout Techniques/methods
*Gene Knockdown Techniques/methods
*Antibodies
Talin/genetics/metabolism
RNA Interference
RNA, Small Interfering/genetics
Gene Expression Profiling
Cell Adhesion/genetics

@article {pmid40392482,
year = {2025},
author = {Huang, S and Wu, J and Yang, Y and Zhu, M and Chen, L and Zhang, S and Yang, Y and Sun, X and Xie, Y},
title = {Investigate the Effect of ZFP64 on mRNA Expression of HBG Based on Bioinformatics and Experimental Validation.},
journal = {Cell biochemistry and biophysics},
volume = {83},
number = {4},
pages = {4427-4437},
pmid = {40392482},
issn = {1559-0283},
support = {2020A0505100062, 2023A1515010872, 2024A1515012233//the Natural Science Foundation of Guangdong Province/ ; 32070582//National Natural Science Foundation of China/ ; 2023HLLH01//the Joint Foundation of He Lin Academical Workstation of the Third Affiliated Hospital of Guangzhou Medical University/ ; 2023A03J0386, 2023A03J0395//Guangdong Municipal Department of Science and Technology, Municipal Schools (Institutes) Jointly Funded Project/ ; .02-408-2203-2059//Guangzhou Medical University for the First-class Professional Construction Project in 2022-Enhancement of Undergraduates' Scientific Research and Innovation Ability Project/ ; 2024SRP119//plan on enhancing scientific research in GMU/ ; },
mesh = {Humans ; *RNA, Messenger/metabolism/genetics ; *Transcription Factors/genetics/metabolism ; *Computational Biology ; K562 Cells ; *gamma-Globins/genetics/metabolism ; Protein Interaction Maps ; *DNA-Binding Proteins/genetics/metabolism ; *Gene Expression Regulation ; CRISPR-Cas Systems ; },
abstract = {γ-globin genes (HBG1 and HBG2) are usually expressed during fetal life, and almost no expression after birth. Therefore, the reactivation of HBG is a key target for the treatment of hemoglobinopathy. ZFP64 is a C2H2 type zinc finger transcription factor, which has been shown to play an important role in the maintenance of gene expression in mixed lineage leukemia, and other C2H2 type zinc finger transcription factors (such as ZFP410 and ZFP644) have been shown to regulate the expression of fetal hemoglobin (HbF) in thalassemia. This study aims to investigate the effect of ZFP64 on mRNA expression of HBG. We performed bioinformatics analyses using the Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and protein-protein interaction (PPI) networks to identify genes and transcription factors associated with ZFP64. ZFP64 was knocked out in K562 and HUDEP-2 cell lines by CRISPR-Cas9 electroporation, and the transcription levels of ZFP64, HBB and HBG were analyzed. In undifferentiated and 7-day differentiated HUDEP-2 cells, knocking down ZFP64 resulted in a 1.5-fold and 2.5-fold increase in HBG mRNA expression, respectively (p < 0.05). These findings suggest that ZFP64 is a potential regulator of HBG expression and warrants further investigation as a therapeutic target in hemoglobinopathies.},
}

Humans
*RNA, Messenger/metabolism/genetics
*Transcription Factors/genetics/metabolism
*Computational Biology
K562 Cells
*gamma-Globins/genetics/metabolism
Protein Interaction Maps
*DNA-Binding Proteins/genetics/metabolism
*Gene Expression Regulation
CRISPR-Cas Systems

@article {pmid41341502,
year = {2025},
author = {Gao, Y and Chen, J},
title = {Fast but accurate: a systematic review and meta-analysis on diagnostic performance of MRSA detection in clinical samples by using CRISPR-based rapid molecular methods.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1703247},
pmid = {41341502},
issn = {1664-302X},
abstract = {BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) poses a significant global health threat due to its multidrug resistance and association with severe infections. Conventional culture methods are time-consuming, usually requiring 48-72 h to obtain results, while conventional molecular methods such as PCR or qPCR, though faster, still require trained personnel and specialized instruments, which may delay timely clinical treatment and infection control. CRISPR-based methods have emerged as promising alternative tools for MRSA detection, but their real-world performance still requires comprehensive assessment. This meta-analysis aimed to systematically evaluate the diagnostic accuracy and timeliness of CRISPR/Cas systems for MRSA detection in clinical samples.

METHODS: A systematic search of PubMed, Embase, Web of Science, and Cochrane Library was conducted using search terms related to MRSA, CRISPR/Cas, diagnostic accuracy, and rapid detection. Studies reporting sensitivity and specificity with extractable 2 × 2 contingency tables were included. Quality was assessed via QUADAS-2. Meta-disc 1.4.0 and Stata 16.0 were used for statistical analysis, including pooled sensitivity, specificity, likelihood ratios, diagnostic odds ratios (DOR) and summary receiver operating characteristic (SROC). Median detection time and subgroup analyses were also conducted.

RESULTS: Twelve studies were included. The results showed that the CRISPR-based methods showed a pooled sensitivity of 99% (95% CI: 97-100%) and specificity of 100% (95% CI: 99-100%), with a PLR of 32.68 (95% CI: 15.45-69.15), NLR of 0.03 (95% CI: 0.02-0.07), and DOR of 664.25 (95% CI: 234.59-1880.84). The median detection time across included studies was 60 min (IQR: 41.25-98.75 min).

CONCLUSION: CRISPR-based molecular assays demonstrated exceptional accuracy and rapid detection capability for MRSA in clinical settings, significantly outperforming conventional methods. However, potential publication bias and methodological limitations warrant cautious interpretation of these results.

PROSPERO ID: CRD420251115439.},
}

@article {pmid41340056,
year = {2025},
author = {Braun, S and Knackfuß, K and Ziesmann, T and Mlinzk, L and Goerg, A and Frankenheim, J and Walter, A and Schneider-Brachert, W and Distler, U and Fritsch, J},
title = {Loss of ADAM15 prevents necroptosis induction by partial RIPK1 degradation due to enhanced TNF-R1 surface expression and basal caspase-8 activation.},
journal = {Cell communication and signaling : CCS},
volume = {23},
number = {1},
pages = {520},
pmid = {41340056},
issn = {1478-811X},
mesh = {Humans ; *Caspase 8/metabolism ; *ADAM Proteins/metabolism/genetics/deficiency ; *Receptor-Interacting Protein Serine-Threonine Kinases/metabolism ; *Proteolysis ; *Membrane Proteins/metabolism/genetics/deficiency ; *Necroptosis ; *Receptors, Tumor Necrosis Factor, Type I/metabolism ; Enzyme Activation ; Jurkat Cells ; U937 Cells ; Signal Transduction ; },
abstract = {BACKGROUND: Cell death and survival processes must be tightly regulated to ensure proper tissue homeostasis and prevent excessive inflammation and tissue damage. Death receptors, including TNF-R1, can induce either immunogenic (necroptosis) or non-immunogenic (apoptosis) cell death and relay proliferative / cell survival signaling by activating NFκB and MAPK cascades. In a recent report, we identified the metalloproteinase ADAM15 as a possible TNF-responding enzyme, leading to the hypothesis that it regulates either cell survival or death cascades.

METHODS: CRISPR/Cas-9 was used to knock out the adam15 gene. Loss of gene expression was validated by Western blot and flow cytometry in U937 and Jurkat cells. NFκB, MAPK signaling, and cell death cascades were monitored by Western blot, flow cytometry, and enzyme assays. A bottom-up proteome analysis was performed to elucidate cellular processes affected by ADAM15 loss. The subcellular localization of ADAM15 was monitored by microscopy and immuno-magnetic fractionation.

RESULTS: We identified ADAM15 as a regulator of necroptosis, leaving apoptosis and cell survival signaling unaffected. Loss of ADAM15 resulted in abrogated necroptosis, as evidenced by the application of death ligands TNF, TRAIL, FasL, and TL1a, as well as the BH3 mimetic Obatoclax. We observed enhanced basal Caspase-8 activity, which was not cytotoxic, and partial RIPK1 proteolysis. The loss of ADAM15 was verified in a proteome screen, which revealed alterations in various molecular pathways, including autophagy, organelle trafficking, and sorting. We observed ADAM15 in intracellular compartments, which in part have a lysosomal protein signature. We observed enhanced surface expression of TNF-R1, proposing it as a possible ADAM15 substrate.

CONCLUSIONS: ADAM15 is a previously unknown regulator of necroptosis, likely due to its role in modulating intracellular organelle sorting processes. Its proteolytic activity and possible scaffolding capacity for recruiting adaptor molecules make it a veritable drug target. The activation or deactivation of ADAM15 may be exploited to modulate various disease conditions.},
}

Humans
*Caspase 8/metabolism
*ADAM Proteins/metabolism/genetics/deficiency
*Receptor-Interacting Protein Serine-Threonine Kinases/metabolism
*Proteolysis
*Membrane Proteins/metabolism/genetics/deficiency
*Necroptosis
*Receptors, Tumor Necrosis Factor, Type I/metabolism
Enzyme Activation
Jurkat Cells
U937 Cells
Signal Transduction

@article {pmid41339642,
year = {2025},
author = {Yang, WJ and Liu, BY and Xue, L},
title = {Knockout of protein arginine methyltransferase 1 inhibited cell growth and promoted cell migration in human bronchial epithelial cells.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {43069},
pmid = {41339642},
issn = {2045-2322},
support = {2018BFC360//Fund for Key Laboratory Construction of Hubei Province/ ; 31101047//National Natural Science Foundation of China/ ; CZQ22013//"the Fundamental Research Funds for the Central Universities", South-Central MinZu University/ ; PTZ24018//i Medical Biology International Science and Technology Cooperation Base/ ; },
mesh = {Humans ; *Cell Movement/genetics ; *Cell Proliferation/genetics ; *Protein-Arginine N-Methyltransferases/genetics/metabolism ; *Epithelial Cells/metabolism/cytology ; *Bronchi/cytology/metabolism ; Cell Line ; Apoptosis/genetics ; Gene Knockout Techniques ; *Repressor Proteins/genetics/metabolism ; Cell Cycle/genetics ; CRISPR-Cas Systems ; },
abstract = {Previous studies have demonstrated that PRMT1 was involved in the progression of multiple lung diseases. However, its specific function within the bronchial epithelium was still limited and needed further exploration. In the present study, human bronchial epithelial cell line 16HBE was chosen to elucidate the biological role of PRMT1 in lung epithelium. Cell proliferation, cell-cycle distribution, cell apoptosis, and cell motility capacity were systematically evaluated following CRISPR/Cas9-mediated knockout of PRMT1. We showed that knockout of PRMT1 in 16HBE inhibited cell proliferation, redistributed cell cycle, promoted cell apoptosis, and accelerated cell migration via a series of regulated cyclins, cyclin-dependent kinase regulators, and EMT markers. Taken together, these findings identify PRMT1 as a potential modulator of epithelial cell proliferation, survival, and motility in the human bronchial epithelium, offering new insights into its possible role in epithelial remodeling during pulmonary disorders.},
}

Humans
*Cell Movement/genetics
*Cell Proliferation/genetics
*Protein-Arginine N-Methyltransferases/genetics/metabolism
*Epithelial Cells/metabolism/cytology
*Bronchi/cytology/metabolism
Cell Line
Apoptosis/genetics
Gene Knockout Techniques
*Repressor Proteins/genetics/metabolism
Cell Cycle/genetics
CRISPR-Cas Systems

@article {pmid41337296,
year = {2025},
author = {Cimolato, C and Letrari, S and Chiacchiera, AF and Del Favero, S and Schenato, L and Pasotti, L and Bellato, M},
title = {Modeling of Phage-Mediated CRISPRi System to Inhibit Antibiotic Resistances in Bacteria.},
journal = {Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual International Conference},
volume = {2025},
number = {},
pages = {1-7},
doi = {10.1109/EMBC58623.2025.11253443},
pmid = {41337296},
issn = {2694-0604},
mesh = {*Bacteriophages/genetics ; *CRISPR-Cas Systems/genetics ; *Bacteria/genetics/drug effects/virology ; *Drug Resistance, Bacterial/genetics ; Anti-Bacterial Agents/pharmacology ; Humans ; },
abstract = {Antimicrobial resistance (AMR) poses a critical threat to global health, rendering traditional antibiotics increasingly ineffective and amplifying the urgency for innovative solutions. Among promising alternatives, synthetic biology emerges as a powerful tool to combat AMR. This work proposes an innovative strategy based on engineering bacteriophages to deliver CRISPR interference (CRISPRi) systems into antibiotic-resistant pathogens to precisely silence target resistance genes. A comprehensive mathematical model is developed and simulated to capture the dynamics of phage-mediated CRISPRi delivery. By explicitly incorporating mutations that affect CRISPRi functionality, the study evaluates system performance and its potential for long-term therapeutic efficacy. This model serves as a critical framework for optimizing future CRISPRi-based interventions and advancing synthetic biology-driven approaches to tackle AMR.Clinical relevance- This paper provides a quantitative modeling framework to evaluate key parameters affecting engineered phage therapy efficiency, supporting rational design and phage posology optimization.},
}

*Bacteriophages/genetics
*CRISPR-Cas Systems/genetics
*Bacteria/genetics/drug effects/virology
*Drug Resistance, Bacterial/genetics
Anti-Bacterial Agents/pharmacology
Humans

@article {pmid41336948,
year = {2025},
author = {Yu, ES and Jang, H and Kwon, J and Jeong, H and Park, J and Kang, T and Jeong, KH},
title = {On-chip Nanoplasmonic RT-RPA and CRISPR/Cas12a Assay for Point-of-care Molecular Diagnostics.},
journal = {Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual International Conference},
volume = {2025},
number = {},
pages = {1-4},
doi = {10.1109/EMBC58623.2025.11253682},
pmid = {41336948},
issn = {2694-0604},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; SARS-CoV-2/genetics ; *COVID-19/diagnosis ; *Point-of-Care Systems ; *Nucleic Acid Amplification Techniques/methods/instrumentation ; *Lab-On-A-Chip Devices ; *Molecular Diagnostic Techniques ; *Pathology, Molecular/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Rapid and accurate nucleic acid detection at point-of-care (POC) is essential for advancing effective disease diagnosis and management. Here, we report a handheld nanoplasmonic all-in-one setup for on-chip recombinase polymerase amplification (RPA) and real-time fluorescence detection by CRISPR/Cas12a reaction. The all-in-one setup consists of AuNIs-based nanoplasmonic cavity (AuNIs-NC), a disposable plastic-on-polymer (PoP) cartridge, and fluorescence microlens array (FMLA) camera. The AuNIs-NC allows uniform and efficient photothermal heating under white LED illumination due to strong broadband light absorption and internal reflection by randomly distributed AuNIs and thin Al film. This setup allows the RPA and CRISPR/Cas 12a reactions in a single chamber of PoP cartridge, with fluorescence signals monitored by a FMLA camera. The experimental result demonstrates rapid SARS-CoV-2 E gene plasmid DNA detection within 20 min, achieving a detection sensitivity of 10 copies/ul. Testing with 16 clinical samples shows a linear trend with RT-qPCR, indicating the platform's reliable sensitivity and specificity. This compact platform offers affordable and reliable molecular diagnosis, facilitating rapid and scalable POC testing for a range of infectious diseases.Clinical Relevance- This on-chip real-time RT-RPA and CRISPR/Cas12a assay provides rapid and precise molecular diagnostics at POC using fully integrated plasmonic system.},
}

Humans
*CRISPR-Cas Systems/genetics
SARS-CoV-2/genetics
*COVID-19/diagnosis
*Point-of-Care Systems
*Nucleic Acid Amplification Techniques/methods/instrumentation
*Lab-On-A-Chip Devices
*Molecular Diagnostic Techniques
*Pathology, Molecular/methods
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41332531,
year = {2025},
author = {Zhang, P and Xue, B and Xie, Y and Li, K and Yang, H and Sun, P and Zhang, L},
title = {OSM-11 modulates salinity-stress tolerance in Caenorhabditis elegans.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2025.11.20.689412},
pmid = {41332531},
issn = {2692-8205},
abstract = {Most terrestrial animals exhibit narrow salinity tolerance compared to their marine counterparts. Previous studies identified osm-11 (which encodes a Notch co-ligand) mutations as a driver of hyper-saline tolerance in Caenorhabditis elegans , but mechanistic insights remained unclear. This study employs RNA sequencing and CRISPR/Cas-9 genome editing to demonstrate that osm-11 mutations enhance salinity stress resistance through up-regulation of fatty acid metabolism (acdh-12 , acs-17) and cytochrome P450 pathways (ugt-15), while suppressing calcium signaling. Furthermore, we demonstrated that acdh-12 mutation impairs salinity-stress tolerance by activating ferroptosis and mitophagy, accompanied by down-regulated oxidative phosphorylation and up-regulated autophagic pathways. Morphological observations show that mitochondrial fragmentation contributes to wild-type nematode mortality under high salinity, while enlarged lipid droplets in wild-types correlate with reduced β-oxidation gene expression (dhs-28 , daf-22), whose knockout disrupts tolerance in mutants. These findings unravel the multi-pathway regulatory network of osm-11 -mediated salinity tolerance, providing mechanistic insights for developing protective strategies against environmental salinity stressors impacting animal survival.},
}

@article {pmid41331925,
year = {2025},
author = {Matsumoto, D and Kubota, K and Sato, Y and Kato-Inui, T and Nigorikawa, K and Miyaoka, Y and Nomura, W},
title = {Screening strategy to identify Cas9 variants with higher HDR activity based on diphtheria toxin.},
journal = {Journal of biomedical science},
volume = {32},
number = {1},
pages = {102},
pmid = {41331925},
issn = {1423-0127},
support = {JP23K13844//KAKENHI/ ; JP24K09445//KAKENHI/ ; JP20H03442//KAKENHI/ ; JP20K21253//KAKENHI/ ; JP22H02201//KAKENHI/ ; JP23K23468//KAKENHI/ ; },
mesh = {Humans ; *Diphtheria Toxin/genetics ; *CRISPR-Associated Protein 9/genetics ; *Gene Editing/methods ; *Recombinational DNA Repair/genetics ; *CRISPR-Cas Systems ; HEK293 Cells ; Mutation ; },
abstract = {BACKGROUND: In gene therapy via genome editing, it is essential to precisely repair disease-associated gene sequences without introducing random mutations. However, achieving highly accurate genome editing remains challenging owing to the low efficiency of homology-directed repair (HDR)-mediated gene repair, which relies on template DNA. Therefore, if Cas9 mutants capable of enhancing HDR can be identified, they could enable more precise gene therapies.

METHOD: In this research project, we developed a screening system that uses the acquisition of diphtheria toxin resistance as an indicator of HDR efficiency in human cells and EGFP disruption as an indicator of off-target effect.

RESULTS: By screening a library of SpCas9 variants with random mutations introduced into its nuclease domain, we identified a novel SpCas9 mutant with higher HDR efficiency than wild-type Cas9.

CONCLUSION: We explored the possibility of obtaining Cas9 mutants with high HDR efficiency via this screening system.},
}

Humans
*Diphtheria Toxin/genetics
*CRISPR-Associated Protein 9/genetics
*Gene Editing/methods
*Recombinational DNA Repair/genetics
*CRISPR-Cas Systems
HEK293 Cells
Mutation

@article {pmid41331675,
year = {2025},
author = {Wang, M and Zhang, Y and Bi, C and Li, M},
title = {CRISPR-Cas9-induced double-strand breaks disrupt maintenance of epigenetic information.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {411},
pmid = {41331675},
issn = {1474-760X},
support = {BAS/1/1080-01-01//KAUST Office of Sponsored Research/ ; 5932//KAUST Center of Excellence for Smart Health/ ; },
mesh = {Humans ; *DNA Breaks, Double-Stranded ; *CRISPR-Cas Systems ; DNA Methylation ; *Epigenesis, Genetic ; Gene Editing ; MutL Protein Homolog 1/genetics ; Human Embryonic Stem Cells/metabolism ; Genomic Imprinting ; DNA Repair ; },
abstract = {BACKGROUND: CRISPR-Cas9 genome editing enables precise genetic modifications by introducing targeted DNA double-strand breaks (DSBs). While Cas9-induced DSBs are known to cause unintended on-target mutations, their impact on the epigenetic landscape remains unexplored.

RESULTS: Here, we investigate how Cas9-induced DSBs affect DNA methylation patterns in human embryonic stem cells (hESCs). We induce DSBs at differentially methylated regions of imprinted genomic loci and perform high-coverage, long-read native DNA sequencing to simultaneously obtain genetic variant and base-resolution methylation data in a haplotype-resolved manner. Our findings reveal that DSBs cause significant changes in DNA methylation at target sites through mechanisms including homologous recombination, large structural variations, or defective methylation maintenance during DNA repair. Notably, these epigenetic changes can occur either together with or independently of genetic alterations. Beyond imprinted loci, Cas9-induced DSBs significantly disrupt DNA methylation patterns of the MLH1 epimutation alleles in colorectal cancer cells, and hypermethylated heterochromatin loci in hESCs. Clonal analysis indicates that the aberrant methylation changes are stable during in vitro passaging. Intriguingly, significant changes in DNA methylation levels are also detected around endogenous deletions in unedited genomic regions, suggesting that methylation alterations are not unique to Cas9 nuclease activity but represent a general outcome of DSB repair in human cells.

CONCLUSIONS: This study underscores the importance of assessing and mitigating unintended epigenetic consequences in genome editing applications, as such changes can profoundly affect gene regulation and cellular function.},
}

Humans
*DNA Breaks, Double-Stranded
*CRISPR-Cas Systems
DNA Methylation
*Epigenesis, Genetic
Gene Editing
MutL Protein Homolog 1/genetics
Human Embryonic Stem Cells/metabolism
Genomic Imprinting
DNA Repair

@article {pmid41277755,
year = {2025},
author = {Wang, Z and Zhang, D and Wu, Y and Ye, X and Qian, Y and Chen, F},
title = {A capillary SERS sensor based on CRISPR/Cas13a and DS Au-AgNRs for detecting miRNA-221 in serum of hepatocellular carcinoma patients.},
journal = {Analytical methods : advancing methods and applications},
volume = {17},
number = {47},
pages = {9627-9637},
doi = {10.1039/d5ay01517k},
pmid = {41277755},
issn = {1759-9679},
mesh = {*MicroRNAs/blood/genetics ; Humans ; *Carcinoma, Hepatocellular/blood/diagnosis/genetics ; *Liver Neoplasms/blood/diagnosis/genetics ; *Spectrum Analysis, Raman/methods ; Gold/chemistry ; Silver/chemistry ; CRISPR-Cas Systems ; Nanotubes/chemistry ; Biomarkers, Tumor/blood ; Limit of Detection ; },
abstract = {The low early diagnosis rate of hepatocellular carcinoma (HCC) severely impacts patient prognosis, making the development of highly sensitive and specific early diagnostic technologies crucial. MicroRNA-221 (miR-221), an aberrantly overexpressed biomarker in HCC, holds significant diagnostic potential. This paper constructed a capillary surface-enhanced Raman scattering (SERS) sensing platform utilizing CRISPR/Cas13a trans-cleavage and double-shell gold-silver nanorods (DS Au-AgNRs) to detect serum miR-221 in HCC patients. DS Au-AgNRs were synthesized and assembled onto aminated capillaries, followed by conjugation of Cy5-labeled single-stranded DNA (ssDNA) to the DS Au-AgNR surface via Au-S bonds. In the presence of miR-221, activated CRISPR/Cas13a trans-cleavage cleaves the ssDNA, releasing Cy5 from the sensor surface and diminishing the SERS signal, enabling miR-221 quantification. The synthesized DS Au-AgNRs exhibit uniform morphology and size, are uniformly distributed on the capillary, and form numerous "hotspots", thereby significantly enhancing the SERS signal. According to the characteristic peak of Cy5 at 1074 cm[-1], a linear relationship is established between the log concentration of miR-221 and the measured SERS intensity (y = -3527.97 × -35369.60, R[2] = 0.97767), with a LOD as low as 4.17 × 10[-17] M. The sensor demonstrated high specificity and high sensitivity, and its capacity to detect miR-221 expression aligned with qRT-PCR results when analyzing serum samples, confirming that hepatocellular carcinoma patients exhibited significantly higher miR-221 levels compared to healthy individuals. The capillary SERS sensor thus provides an accurate and convenient approach for early HCC diagnosis.},
}

*MicroRNAs/blood/genetics
Humans
*Carcinoma, Hepatocellular/blood/diagnosis/genetics
*Liver Neoplasms/blood/diagnosis/genetics
*Spectrum Analysis, Raman/methods
Gold/chemistry
Silver/chemistry
CRISPR-Cas Systems
Nanotubes/chemistry
Biomarkers, Tumor/blood
Limit of Detection

@article {pmid41252908,
year = {2026},
author = {Zhou, F and Zhao, X and Wang, Y and Huang, Y},
title = {Cooperation of CRISPR/Cas12a and exonuclease III-assisted cascade cycling amplification for ultrasensitive electrochemical detection of ciprofloxacin.},
journal = {Talanta},
volume = {299},
number = {},
pages = {129131},
doi = {10.1016/j.talanta.2025.129131},
pmid = {41252908},
issn = {1873-3573},
mesh = {*Ciprofloxacin/analysis ; *Exodeoxyribonucleases/metabolism/chemistry ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; Limit of Detection ; *Nucleic Acid Amplification Techniques ; *Anti-Bacterial Agents/analysis ; Food Contamination/analysis ; *CRISPR-Associated Proteins/metabolism ; DNA Probes/chemistry/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Antibiotic residues have been a serious public health concern worldwide, while sensitive and reliable detection of antibiotic residues is significant to control antibiotic contamination, ensure food safety, and safeguard human health. Herein, an ultrasensitive electrochemical biosensor is engineered for the detection of ciprofloxacin (CIP) based on the cooperation of CRISPR/Cas12a and exonuclease III (Exo III)-assisted cascade cycling amplification. The presence of CIP induces the conformational change of DNA probes and further triggers Exo III to catalyze the cascade cycling amplification, enabling propagation and ongoing accumulation of DNA fragments which act as the target strands to activate the trans-cleavage activity of CRISPR/Cas12a. Consequently, the activated CRISPR/Cas12a initiates its trans-cleavage activity to swiftly cleave the signal probes on the surface of electrode, bringing about remarkable change of electrochemical signal and eventually realizing the ultrasensitive detection of CIP. The exceptional enzymatic cycle amplification of Exo III incorporated with the superior trans-cleavage activity of CRISPR/Cas12a synergistically facilitates considerable improvement of analytical performance, resulting in a limit of detection as low as 0.022 ng mL[-1]. Benefiting from the effective amplification capacity, high fidelity and programmability of the designed detection system, the biosensor shows good precision and specificity along with robust stability for CIP detection. Moreover, the proposed electrochemical biosensor dispensing with complicated probe construction is label-free and convenient-operated, which contributes to the credible application for CIP detection in real food samples with satisfactory results, indicating promising practicability in food safety monitoring.},
}

*Ciprofloxacin/analysis
*Exodeoxyribonucleases/metabolism/chemistry
*Biosensing Techniques/methods
*Electrochemical Techniques/methods
*CRISPR-Cas Systems
Limit of Detection
*Nucleic Acid Amplification Techniques
*Anti-Bacterial Agents/analysis
Food Contamination/analysis
*CRISPR-Associated Proteins/metabolism
DNA Probes/chemistry/genetics
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid41223903,
year = {2025},
author = {Alsultan, A and Karim, SM and Al-Saadi, M and Alsallami, D and Ben Said, M and Belkahia, H},
title = {Rapid and sensitive detection of Theileria equi using a novel integrated RPACRISPR/Cas13a lateral flow assay.},
journal = {Journal of equine veterinary science},
volume = {155},
number = {},
pages = {105732},
doi = {10.1016/j.jevs.2025.105732},
pmid = {41223903},
issn = {0737-0806},
mesh = {Horses ; Animals ; *Theileria/isolation & purification ; *Theileriasis/diagnosis/parasitology ; *Horse Diseases/diagnosis/parasitology ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/veterinary/methods ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Equine piroplasmosis (EP), caused by the intracellular protozoa Theileria equi, Babesia caballi, and Theileria haneyi, represents a major health and economic threat to the equine industry worldwide. Existing diagnostic methods, including PCR, serology, and microscopy, are constrained by their dependence on specialized equipment, lengthy protocols, and the requirement for skilled personnel.

AIM: This study aimed to develop a rapid, accurate, and field-deployable molecular diagnostic assay for T. equi.

METHODS: A nucleic acid-based diagnostic platform combining recombinase polymerase amplification (RPA) with CRISPR/Cas13-mediated detection and lateral flow device (LFD) readout was developed. The assay targets a conserved region of the erythrocyte merozoite antigen 1 (EMA-1) gene of T. equi. Validation was performed using 22 blood samples collected from horses, as well as specificity controls including B. caballi- and Anaplasma phagocytophilum-infected samples, synthetic EMA-1 DNA, and non-template controls. All assay steps were conducted at room temperature.

RESULTS: The integrated RPA-CRISPR/Cas13-LFD assay generated clear visual results within 50 minutes. It demonstrated complete specificity with no false positives across all tested samples. The method effectively differentiated horses infected with T. equi, including both clinically affected and asymptomatic individuals, from healthy, uninfected animals, confirming its high accuracy and reliability.

CONCLUSION: The developed assay provides a rapid, precise, and equipment-free diagnostic platform suitable for both field and clinical environments. Although the current protocol relies on DNA extraction, future optimization will aim to enable direct detection from unprocessed blood samples, thereby further simplifying point-of-care diagnostics for equine piroplasmosis.},
}

Horses
Animals
*Theileria/isolation & purification
*Theileriasis/diagnosis/parasitology
*Horse Diseases/diagnosis/parasitology
Sensitivity and Specificity
*Nucleic Acid Amplification Techniques/veterinary/methods
CRISPR-Cas Systems

@article {pmid41221894,
year = {2025},
author = {Wei, R and Wang, S and Pan, Y and Pan, W and Li, N and Tang, B},
title = {CRISPR-coupled triple cascade amplification for simultaneous lateral flow detection of Mycoplasma pneumoniae and H1N1.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {97},
pages = {19241-19244},
doi = {10.1039/d5cc04993h},
pmid = {41221894},
issn = {1364-548X},
mesh = {*Mycoplasma pneumoniae/isolation & purification/genetics ; *Influenza A Virus, H1N1 Subtype/isolation & purification/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Saliva/microbiology/virology ; Limit of Detection ; },
abstract = {We developed a CRISPR-coupled triple cascade system integrating recombinase polymerase amplification (RPA), CRISPR/Cas12a, and catalytic hairpin assembly (CHA) for simultaneous lateral flow detection of Mycoplasma pneumoniae and H1N1 in saliva samples, achieving a LOD of 10 aM for H1N1 RNA and 25 aM for MP DNA on a single LFA.},
}

*Mycoplasma pneumoniae/isolation & purification/genetics
*Influenza A Virus, H1N1 Subtype/isolation & purification/genetics
Humans
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
Saliva/microbiology/virology
Limit of Detection

@article {pmid41202787,
year = {2026},
author = {Xiao, X and Zhong, Y and Xie, H and Liao, H and Wang, H and Liu, H and Liang, F and Chen, J and Zhong, H and Chen, Z and Yu, L},
title = {A reverse transcription-free CRISPR/Cas12a biosensor for ultrasensitive detection of SARS-CoV-2 variants.},
journal = {Talanta},
volume = {299},
number = {},
pages = {129072},
doi = {10.1016/j.talanta.2025.129072},
pmid = {41202787},
issn = {1873-3573},
mesh = {*SARS-CoV-2/genetics/isolation & purification ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *COVID-19/diagnosis/virology ; Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Mutation ; RNA, Viral/genetics ; Reverse Transcription ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {In this work, we developed a reverse transcription-free self-primer isothermal exponential amplification reaction (RTF-SP-EXPAR) combined with a CRISPR/Cas12a biosensor for the simultaneous detection of single- and double-stranded amplification products of SARS-CoV-2 mutant variants. SP-EXPAR can recognize RNA targets directly, simultaneously generate double-stranded DNA products containing PAM sequences, and single-stranded DNA products without PAM sequences. Both types of SP-EXPAR products can be recognized by the crRNA and produce fluorescent signals, thereby enhancing detection sensitivity. This RTF-SP-EXPAR-CRISPR/Cas12a biosensor enables the detection of SARS-CoV-2 mutations within 1 h, achieving a detection limit of 7.49 aM and a dynamic range of 10 aM to 10 pM. This method shows high specificity in differentiating mutant variants from wild-type sequences. For the detection of 106 clinical samples, this RTF-SP-EXPAR-CRISPR/Cas12a assay demonstrates 100 % sensitivity and 100 % specificity compared with DNA sequencing results. These findings highlight our proposed assay's strong applicability for the application of RNA samples without reverse transcription.},
}

*SARS-CoV-2/genetics/isolation & purification
*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
Humans
*COVID-19/diagnosis/virology
Nucleic Acid Amplification Techniques/methods
Limit of Detection
Mutation
RNA, Viral/genetics
Reverse Transcription
CRISPR-Associated Proteins/genetics
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid41175865,
year = {2025},
author = {Ahrens-Nicklas, RC and Musunuru, K},
title = {How to create personalized gene editing platforms: Next steps toward interventional genetics.},
journal = {American journal of human genetics},
volume = {112},
number = {12},
pages = {2826-2829},
doi = {10.1016/j.ajhg.2025.10.006},
pmid = {41175865},
issn = {1537-6605},
mesh = {Humans ; *Gene Editing/methods/trends ; *Precision Medicine/methods/trends ; *Genetic Therapy/methods/trends ; CRISPR-Cas Systems/genetics ; },
abstract = {How do we go from a single individual receiving a personalized gene-editing therapy to a future of "interventional genetics" in which such therapies are the standard of care? First and foremost: regulatory innovation.},
}

Humans
*Gene Editing/methods/trends
*Precision Medicine/methods/trends
*Genetic Therapy/methods/trends
CRISPR-Cas Systems/genetics

@article {pmid41135356,
year = {2026},
author = {Wang, W and Zheng, Y and Zhang, L and Bao, Y and Liu, J and Zang, J and Qu, Y and Zhang, K and Han, R and Ren, H and Zhu, L and Cao, Y and Wang, B and Shen, Q and Sun, W},
title = {A multiplex RPA-CRISPR/Cas12a platform for rapid and accurate toxinotyping of Clostridium perfringens.},
journal = {Talanta},
volume = {298},
number = {Pt B},
pages = {128998},
doi = {10.1016/j.talanta.2025.128998},
pmid = {41135356},
issn = {1873-3573},
mesh = {*Clostridium perfringens/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Bacterial Toxins/genetics/analysis ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases/metabolism ; Humans ; Limit of Detection ; Bacterial Proteins/genetics ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Clostridium perfringens (C. perfringens) is a leading cause of foodborne disease worldwide, requiring rapid and accurate toxinotyping for effective outbreak control and surveillance. Herein, we developed C. perfringens-multiplex RPA-CRISPR/Cas12a, an integrated detection platform combing multiplex Recombinase Polymerase Amplification (RPA) with Clustered Regularly Interspaced Short Palindromic Repeats-associated protein 12a (CRISPR/Cas12a)-mediated detection for comprehensive toxinotyping. The system simultaneously identifies six key toxin genes (cpa, cpb, etx, iap, cpe, netB) in two reaction tubes, enabling discrimination of all seven C. perfringens toxinotypes (A-G). The C. perfringens-multiplex RPA-CRISPR/Cas12a assay platform exhibited exceptional analytical performance, achieving a detection limit of ≤10 copies/μL for across all targets while maintaining absolute specificity against the human genomic DNA and 5 common foodborne pathogens. In validation testing with 12 naturally contaminated food samples, the C. perfringens-multiplex RPA-CRISPR/Cas12a assay platform demonstrated superior performance to commercial qPCR kits, accurately identifying eight Type A (cpa-gene-positive) and four Type F (cpa-gene and cpe-gene co-positive) strains. When coupled with a portable detection device, the platform completed the entire diagnostic workflow within 50 min while maintaining laboratory-level accuracy under field conditions. The rapid, cost-effective, and equipment-free system is particularly suited for decentralized toxin surveillance in resource-limited settings. By integrating high sensitivity, multiplex capability, and field applicability, this system significantly advances Point-of-care Testing (POCT) capabilities for food safety monitoring, supporting global food safety initiatives.},
}

*Clostridium perfringens/genetics/isolation & purification
*CRISPR-Cas Systems
*Bacterial Toxins/genetics/analysis
*Nucleic Acid Amplification Techniques/methods
*Recombinases/metabolism
Humans
Limit of Detection
Bacterial Proteins/genetics
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41109522,
year = {2025},
author = {Garcia, AFS and Farinati, S and Draga, S and Riommi, D and Gabelli, G and Vannozzi, A and Barcaccia, G and Palumbo, F},
title = {Establishing a cutting-edge protoplast technology platform for applying new genomic techniques in Cichorium spp.},
journal = {New biotechnology},
volume = {90},
number = {},
pages = {206-222},
doi = {10.1016/j.nbt.2025.10.008},
pmid = {41109522},
issn = {1876-4347},
mesh = {*Protoplasts/metabolism ; *Gene Editing/methods ; *Genomics/methods ; CRISPR-Cas Systems ; *Cichorium intybus/genetics ; },
abstract = {Genome editing technologies, especially those based on the CRISPR/Cas9 system, have revolutionized crop breeding by enabling precise genetic modifications. Specifically, delivering preassembled ribonucleoprotein (RNP) complexes-consisting of the Cas9 endonuclease coupled to specific single guide RNAs (sgRNAs)-into protoplasts offers an effective DNA-free method that prevents the integration of foreign genetic material. Despite the availability of detailed protocols, establishing a standardized and efficient in vitro regeneration procedure-from protoplast isolation to whole plant regeneration-remains challenging due to significant variability in regeneration efficiency across different varieties and biotypes. Therefore, optimizing each step is essential to maximize the recovery of successful edited plants. In this study, we developed an efficient protocol for regenerating whole plants from protoplasts isolated from 12 representative Italian varieties of chicory and endive. We focused on leaf chicory and endive biotypes with high horticultural value, including Radicchio types, which are important targets for quality improvement. Our optimized platform supports protoplast isolation, PEG-mediated transfection, and plant regeneration, demonstrating promising potential for future genome editing applications. Notably, the high responsiveness of protoplasts to PEG-mediated transfection suggests that coupling this method with our regeneration procedure could facilitate the use of advanced biotechnological strategies. The combination of high transient transformation efficiency, versatile encapsulation techniques, and successful plant regeneration establishes chicory and endive as promising candidates for DNA-free genome editing via protoplasts, providing a technically precise approach with reduced environmental and economic impacts compared to conventional breeding methods.},
}

*Protoplasts/metabolism
*Gene Editing/methods
*Genomics/methods
CRISPR-Cas Systems
*Cichorium intybus/genetics

@article {pmid41075867,
year = {2025},
author = {Zhang, D and Liu, H and Zhong, Y},
title = {Monoclonal antibodies production in microbial systems: Current status, challenges and perspectives.},
journal = {New biotechnology},
volume = {90},
number = {},
pages = {163-173},
doi = {10.1016/j.nbt.2025.10.005},
pmid = {41075867},
issn = {1876-4347},
mesh = {*Antibodies, Monoclonal/biosynthesis/genetics ; *Metabolic Engineering ; Humans ; CRISPR-Cas Systems ; Animals ; Gene Editing ; Synthetic Biology ; },
abstract = {Monoclonal antibodies (mAbs) serve as indispensable tools in diagnostics, clinical therapeutics, and biomedical research. However, their large-scale production faces significant challenges due to the high costs and lengthy timelines associated with conventional mammalian cell-based expression systems. Microbial expression platforms have emerged as a transformative alternative, offering cost-effectiveness, rapid cultivation cycles, and superior genetic tractability for industrial-scale monoclonal antibodies production. Recent advances in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated gene editing enable precise metabolic engineering of host strains to enhance protein folding, secretion efficiency, and translational accuracy. Synthetic biology approaches facilities the reconstruction of mammalian glycosylation pathways in microbial systems, yielding monoclonal antibodies with near-native structural integrity. Furthermore, AI (artificial intelligence)-driven optimization of expression vectors, promoter systems, and culture conditions, combined with high-throughput screening of engineered strains, significantly accelerates the identification of high-yield production clones. This review comprehensively examines current progress in microbial expression systems, strain engineering strategies, and fermentation optimization for enhanced monoclonal antibodies production, while critically discussing existing limitations and potential solutions to advance the field.},
}

*Antibodies, Monoclonal/biosynthesis/genetics
*Metabolic Engineering
Humans
CRISPR-Cas Systems
Animals
Gene Editing
Synthetic Biology

@article {pmid41072226,
year = {2026},
author = {Li, L and Li, M and Wang, S and Dong, Y},
title = {Development of a CRISPR/Cas12a-assisted fluorescent aptasensor for simultaneous detection of zearalenone and ochratoxin A.},
journal = {Talanta},
volume = {298},
number = {Pt B},
pages = {128937},
doi = {10.1016/j.talanta.2025.128937},
pmid = {41072226},
issn = {1873-3573},
mesh = {*Ochratoxins/analysis ; *Zearalenone/analysis ; *Aptamers, Nucleotide/chemistry/genetics/metabolism ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Limit of Detection ; Food Contamination/analysis ; Spectrometry, Fluorescence ; Fluorescence ; *Fluorescent Dyes/chemistry ; },
abstract = {Mycotoxins, such as zearalenone (ZEN) and ochratoxin A (OTA), represent significant hazards to both human and animal health, necessitating strict monitoring and regulation of mycotoxin levels in food, feed, and environment. In this study, a simple and efficient CRISPR/Cas12a-assisted fluorescent aptasensor is presented for the simultaneous detection of ZEN and OTA. Utilizing a Bifunctional aptamer (B-APT), this biosensor achieves dual recognition of the both targets, subsequently converting their concentrations into observable fluorescent signals through the Cas12a/crRNA cis-cleavage activity. Rational modifications of the complementary strands specific to the two targets enable distinct emission wavelengths under the same excitation light, facilitating simultaneous and independent quantitative determination of ZEN and OTA. Under optimized conditions, the CRISPR/Cas12a-aptasensor demonstrates robust detection capabilities for individual ZEN and OTA targets, as well as their mixture, yielding consistent standard curves. This methodology exhibits reliable detection of ZEN and OTA concentrations spanning from 0.25 nM to 256 nM and 1 nM to 1024 nM, with respective limit of detection (LOD) values of 190 pM and 931 pM. Furthermore, this method showcases exceptional selectivity and considerable recovery rates (89.17 %-109.88 % for ZEN and 101.19 %-106.51 % for OTA) in corn oil samples, underscoring its efficacy as an advanced tool for ZEN and OTA detection and offering valuable insights into the simultaneous detection of diverse targets.},
}

*Ochratoxins/analysis
*Zearalenone/analysis
*Aptamers, Nucleotide/chemistry/genetics/metabolism
*Biosensing Techniques/methods
*CRISPR-Cas Systems
Limit of Detection
Food Contamination/analysis
Spectrometry, Fluorescence
Fluorescence
*Fluorescent Dyes/chemistry

@article {pmid40684770,
year = {2025},
author = {Ali, A and Roy, B and Schott, MB and Grove, BD},
title = {AKAP12 Variant 1 Knockout Enhances Vascular Endothelial Cell Motility.},
journal = {Journal of vascular research},
volume = {62},
number = {6},
pages = {312-329},
pmid = {40684770},
issn = {1423-0135},
support = {P20 GM104360/GM/NIGMS NIH HHS/United States ; P20 GM113123/GM/NIGMS NIH HHS/United States ; P30 GM103329/GM/NIGMS NIH HHS/United States ; U54 GM128729/GM/NIGMS NIH HHS/United States ; },
mesh = {*A Kinase Anchor Proteins/genetics/metabolism ; *Cell Movement ; Humans ; *Cell Cycle Proteins/genetics/metabolism ; *Endothelial Cells/metabolism ; Cells, Cultured ; *Human Umbilical Vein Endothelial Cells/metabolism ; Intercellular Junctions/metabolism/genetics ; Gene Knockout Techniques ; CRISPR-Cas Systems ; },
abstract = {INTRODUCTION: Previous work indicates that AKAP12 is expressed in endothelial cells as two variants and may play a role in cell motility. However, the role of each variant in cell motility is unknown; therefore, this study investigated the role of AKAP12 in endothelial cell motility with a specific focus on AKAP12 variants, AKAP12v1 and AKAP12v2.

METHODS: AKAP12 expression levels in cultured endothelial cells were determined by Western blotting and immunofluorescence microscopy. AKAP12 knockdown and AKAP12 variant knockout were done using antisense oligonucleotide and siRNA treatment and CRISPR/Cas9 knockout, respectively. The effect of AKAP12 variant knockout was further analyzed by RNA-seq.

RESULTS: AKAP12 expression was cell density-dependent, with the highest expression in subconfluent cultures and lowest in confluent cultures. AKAP12 expression was also elevated in cells at the wound edge of wounded endothelial cell monolayers. Knockdown of both variants inhibited cell migration, but CRISPR/Cas9 knockout of AKAP12v1 enhanced migration. RNA-seq revealed that loss of AKAP12v1 affected genes associated with cell migration and intercellular junctions.

CONCLUSION: We propose that AKAP12v1 and AKAP12v2 play distinct yet complementary roles in endothelial cell migration and likely work together in controlling the signaling events associated with vascular repair and development.},
}

*A Kinase Anchor Proteins/genetics/metabolism
*Cell Movement
Humans
*Cell Cycle Proteins/genetics/metabolism
*Endothelial Cells/metabolism
Cells, Cultured
*Human Umbilical Vein Endothelial Cells/metabolism
Intercellular Junctions/metabolism/genetics
Gene Knockout Techniques
CRISPR-Cas Systems

@article {pmid41330932,
year = {2025},
author = {Van Vu, T and Thi Nguyen, N and Kim, J and Sung, YW and Chung, WS and Kim, JY},
title = {The evolving landscape of precise DNA insertion in plants.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10428},
pmid = {41330932},
issn = {2041-1723},
support = {RS-2022-NR070609//National Research Foundation of Korea (NRF)/ ; RS-2025-02263262//National Research Foundation of Korea (NRF)/ ; RS-2021-NR060105//National Research Foundation of Korea (NRF)/ ; RS-2020-NR049590//National Research Foundation of Korea (NRF)/ ; RS-2020-NR049590//National Research Foundation of Korea (NRF)/ ; RS-2025-02263262//National Research Foundation of Korea (NRF)/ ; RS-2022-NR070609//National Research Foundation of Korea (NRF)/ ; RS-2025-02263262//National Research Foundation of Korea (NRF)/ ; RS-2021-NR060105//National Research Foundation of Korea (NRF)/ ; RS-2025-02263262//National Research Foundation of Korea (NRF)/ ; RS-2022-NR070609//National Research Foundation of Korea (NRF)/ ; RS-2020-NR049590//National Research Foundation of Korea (NRF)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genome, Plant ; Plants, Genetically Modified/genetics ; *DNA, Plant/genetics ; *Plants/genetics ; *Mutagenesis, Insertional/methods ; Gene Targeting/methods ; },
abstract = {Precise DNA insertion into plant genomes is central to advancing crop improvement and synthetic biology. CRISPR-Cas systems have enabled programmable DNA integration using tools such as gene targeting (GT), prime editing (PE), and recombinase- or transposase-based platforms. These tools are transitioned from theoretical concepts to practical applications, supporting applications like in-locus protein tagging, regulatory element engineering, and multi-gene stacking. Key challenges persist, such as inefficient large-fragment insertion, delivery barriers, and regulatory hurdles. This review traces the evolution from random to CRISPR-Cas-based systems, analyzes current limitations, and discusses emerging solutions paving the way for predictable DNA insertion in modern plant biotechnology.},
}

*CRISPR-Cas Systems/genetics
Gene Editing/methods
Genome, Plant
Plants, Genetically Modified/genetics
*DNA, Plant/genetics
*Plants/genetics
*Mutagenesis, Insertional/methods
Gene Targeting/methods

@article {pmid41330849,
year = {2025},
author = {Boob, AG and Zhang, C and Pan, Y and Zaidi, A and Whitaker, RJ and Zhao, H},
title = {Discovery, characterization, and application of chromosomal integration sites in the hyperthermophilic archaeon Sulfolobus islandicus.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2025.11.003},
pmid = {41330849},
issn = {1879-3096},
abstract = {Sulfolobus islandicus, an emerging archaeal model organism, offers unique advantages for metabolic engineering and synthetic biology applications owing to its ability to thrive in extreme environments. Although several genetic tools have been established for this organism, the lack of well-characterized chromosomal integration sites has limited its potential as a cellular factory. Here, we systematically identified and characterized 13 artificial CRISPR RNAs targeting eight integration sites in S. islandicus using the CRISPR-COPIES pipeline and a multi-omics-informed computational workflow. We leveraged the endogenous CRISPR-Cas system to integrate the reporter gene lacS and validated heterologous expression through a β-galactosidase assay, revealing significant positional effects. As a proof of concept, we utilized these sites to genetically manipulate lipid ether composition by overexpressing glycerol dibiphytanyl glycerol tetraether (GDGT) ring synthase B (GrsB). This study expands the genetic toolbox for S. islandicus and advances its potential as a robust platform for archaeal synthetic biology and industrial biotechnology.},
}

@article {pmid41330674,
year = {2026},
author = {Hou, L and Ruan, F and Zhao, K and Li, B},
title = {Boosting split-crRNA CRISPR/Cas12a activity by 3'-end extension of DNA activator for direct microRNA sensing.},
journal = {Analytica chimica acta},
volume = {1382},
number = {},
pages = {344841},
doi = {10.1016/j.aca.2025.344841},
pmid = {41330674},
issn = {1873-4324},
mesh = {*MicroRNAs/analysis/genetics/blood ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Humans ; *DNA/chemistry/metabolism/genetics ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BACKGROUND: The unique trans-cleavage activity of CRISPR/Cas12a has been extensively utilized in the domain of biosensing. Nevertheless, the detection of miRNAs using the traditional CRISPR/Cas12a system requires nucleic acid amplification or reverse transcription to convert miRNA into DNA, which increases reaction time and the risk of contamination.

RESULTS: This study presents a split-crRNA CRISPR/Cas12a-based biosensing for direct detection of miRNA. The target miRNA-375 was utilized as the spacer region of the crRNA, facilitating its binding to the truncated scaffold RNA, thereby resulting in the formation of a complete crRNA. More importantly, we discovered that the cleavage activity of split-crRNA CRISPR/Cas12a was significantly enhanced by extending sequences at the 3'-end of the DNA activator. Compared with the conventional split-crRNA CRISPR/Cas12a system, the split-crRNA CRISPR/Cas12a with 24-nucleotide random sequence extension at the 3'-end of the DNA activator exhibited a 6.4-fold increase in activity. The enhancement mechanism of 3'-end extension of DNA activator was discussed. This proposed split-crRNA CRISPR/Cas12a system was applied to detect miRNA-375 with a linear range of 5 pM-1 nM, and the detection limit was estimated to be 0.6 pM (3σ). Furthermore, this system was used to detect miRNA-375 in 10 % diluted human serum, achieving satisfactory recovery rate (98 %-106 %).

SIGNIFICANCE: This finding indicates that it is feasible to enhance the activity of the split-crRNA CRISPR/Cas12a by extending the 3'-end of the DNA activator, thereby achieving highly sensitive direct detection of miRNA. It is a simple yet effective strategy for enhancing the sensitivity of direct miRNA detection.},
}

*MicroRNAs/analysis/genetics/blood
*CRISPR-Cas Systems
*Biosensing Techniques/methods
Humans
*DNA/chemistry/metabolism/genetics
Limit of Detection
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41330665,
year = {2026},
author = {Bao, Y and Ding, W and Zhang, L and Wang, W and Liu, J and Qu, Y and Zhu, L and Zhang, K and Zhong, G and Han, R and Shen, Q and Wang, B and Gu, X and Cao, Y and Sun, W},
title = {A novel multiplex RPA/CRISPR-Cas12a integrated biosensor for on-site detection of high-risk HPV genotypes.},
journal = {Analytica chimica acta},
volume = {1382},
number = {},
pages = {344830},
doi = {10.1016/j.aca.2025.344830},
pmid = {41330665},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Humans ; Genotype ; *Nucleic Acid Amplification Techniques/methods ; *Papillomaviridae/genetics/isolation & purification ; *Recombinases/metabolism ; Papillomavirus Infections/virology/diagnosis ; DNA, Viral/genetics ; },
abstract = {Accurate genotyping of high-risk human papillomavirus (HR-HPV) at the point of care is critical for global cervical cancer elimination, but its application remains limited by the need for complex equipment and specialized procedures, particularly in resource-limited regions. Here, we develop H-MRC12a-an integrated platform based on degenerate primers and type-specific crRNAs that combines multiplex recombinase polymerase amplification (RPA) with CRISPR-Cas12a trans-cleavage activity-for ultrasensitive detection of eight key HR-HPV genotypes (16, 18, 31, 33, 52, 53, 58, 66). By introducing a degenerate primer strategy coupled with type-specific crRNAs, the system overcomes interference from primer dimer formation and achieves single-copy sensitivity within 50 min under isothermal conditions (37 °C). Clinical validation demonstrated 100 % concordance with qPCR and identified three additional low viral-load positives (Ct > 35) that were missed by conventional methods. Crucially, the platform enables visual readout under UV light and eliminates the need for specialized instruments. This 'broad-spectrum capture and precision typing' paradigm establishes a versatile framework for multiplexed pathogen detection, advancing accessible molecular diagnostics for global health equity.},
}

*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
Humans
Genotype
*Nucleic Acid Amplification Techniques/methods
*Papillomaviridae/genetics/isolation & purification
*Recombinases/metabolism
Papillomavirus Infections/virology/diagnosis
DNA, Viral/genetics

@article {pmid41288387,
year = {2025},
author = {Kim, B and Yuk, M and Park, M and Ryu, H and Park, J and Yu, H and Park, S and Hong, JT and Lim, KH and Han, SB and Song, N and Park, H},
title = {CRISPR editing of miR-33 restores ApoE lipidation and amyloid-β metabolism in ApoE4 sporadic Alzheimer's disease.},
journal = {Brain : a journal of neurology},
volume = {148},
number = {12},
pages = {4400-4415},
doi = {10.1093/brain/awaf244},
pmid = {41288387},
issn = {1460-2156},
support = {//National Research Foundation of Korea/ ; 2021R1C1C1006551//Korean government/ ; //Bio & Medical Technology Development Program/ ; //National Research Foundation/ ; RS-2024-00440787//Korean government/ ; },
mesh = {*MicroRNAs/genetics/metabolism ; *Alzheimer Disease/genetics/metabolism/pathology ; Animals ; Humans ; Mice ; *Gene Editing/methods ; *Apolipoprotein E4/genetics/metabolism ; *Lipid Metabolism/genetics ; *Amyloid beta-Peptides/metabolism ; Male ; CRISPR-Cas Systems ; Female ; Astrocytes/metabolism ; Mice, Transgenic ; *Apolipoproteins E/metabolism/genetics ; Aged ; Disease Models, Animal ; },
abstract = {Sporadic Alzheimer's disease (sAD) is marked by dysregulated lipid metabolism, prominently involving apolipoprotein E (ApoE). MicroRNA-33 (miR-33) has emerged as a key regulator of lipid homeostasis, yet its role in sAD remains unclear. This study investigated miR-33 dysregulation in APOE ε4 allele (ApoE4)-associated sAD and explored its therapeutic potential using clustered regulatory interspaced short palindromic repeats (CRISPR)-mediated gene editing. Elevated miR-33 expression was observed in both AD patients, particularly those with ApoE4-associated sAD, and in the ApoE4 mouse model, implicating its role in AD pathology. Using CRISPR/Cas9, we modulated miR-33 expression in astrocytes to regulate ApoE lipidation and ameliorate AD-related pathology. Our results show that targeted miR-33 regulation in astrocytes via CRISPR/Cas9 restores ApoE lipidation and mitigates AD pathology in both in vitro and in vivo AD mice. Additionally, applying this gene therapy approach in ApoE4 sAD patient cell lines highlights its translational potential for therapeutic intervention. In conclusion, our findings elucidate miR-33's role in AD pathogenesis and underscore the therapeutic promise of CRISPR-mediated miR-33 targeting for restoring lipid homeostasis and ameliorating AD pathology. This study provides valuable insights into developing miRNA-based gene therapy strategies for treating sAD.},
}

*MicroRNAs/genetics/metabolism
*Alzheimer Disease/genetics/metabolism/pathology
Animals
Humans
Mice
*Gene Editing/methods
*Apolipoprotein E4/genetics/metabolism
*Lipid Metabolism/genetics
*Amyloid beta-Peptides/metabolism
Male
CRISPR-Cas Systems
Female
Astrocytes/metabolism
Mice, Transgenic
*Apolipoproteins E/metabolism/genetics
Aged
Disease Models, Animal

@article {pmid41284724,
year = {2025},
author = {da Silva, GLA and Damasceno, JD and Black, JA and Lapsley, C and McCulloch, R and Tosi, LRO},
title = {ATR, a DNA damage kinase, modulates DNA replication timing in Leishmania major.},
journal = {PLoS genetics},
volume = {21},
number = {11},
pages = {e1011899},
doi = {10.1371/journal.pgen.1011899},
pmid = {41284724},
issn = {1553-7404},
mesh = {*Leishmania major/genetics ; *Ataxia Telangiectasia Mutated Proteins/genetics/metabolism ; DNA Damage/genetics ; *DNA Replication/genetics ; CRISPR-Cas Systems ; *DNA Replication Timing/genetics ; *Protozoan Proteins/genetics/metabolism ; },
abstract = {All cells possess mechanisms to maintain and replicate their genomes, whose integrity and transmission are constantly challenged by DNA damage and replication impediments. In eukaryotes, the protein kinase Ataxia-Telangiectasia and Rad3-related (ATR), a member of the phosphatidylinositol 3-kinase-like family, acts as a master regulator of the eukaryotic response to DNA injuries, ensuring DNA replication completion and genome stability. Here we aimed to investigate the functional relevance of the ATR homolog in the DNA metabolism of Leishmania major, a protozoan parasite with a remarkably plastic genome. CRISPR/cas9 genome editing was used to generate a Myc-tagged ATR cell line (mycATR), and a Myc-tagged C-terminal knockout of ATR (mycATRΔC-/-). We show that the nuclear localisation of ATR depends upon its C-terminus. Moreover, its deletion results in single-stranded DNA accumulation, impaired cell cycle control, increased levels of DNA damage, and delayed DNA replication re-start after replication stress. In addition, we show that ATR plays a key role in maintaining L. major's unusual DNA replication program, where larger chromosomes duplicate later than smaller chromosomes. Our data reveals loss of the ATR C-terminus promotes the accumulation of DNA replication signal around replicative stress fragile sites, which are enriched in larger chromosomes. Finally, we show that these alterations to the DNA replication program promote chromosome instability. In summary, our work shows that ATR acts to modulate DNA replication timing, limiting the plasticity of the Leishmania genome.},
}

*Leishmania major/genetics
*Ataxia Telangiectasia Mutated Proteins/genetics/metabolism
DNA Damage/genetics
*DNA Replication/genetics
CRISPR-Cas Systems
*DNA Replication Timing/genetics
*Protozoan Proteins/genetics/metabolism

@article {pmid41233591,
year = {2025},
author = {Li, L and Gao, J and Yi, D and Sheft, AP and Schimenti, JC and Ding, X},
title = {A primordial germ cell-like-cell platform enables CRISPRi screen for epigenetic fertility modifiers.},
journal = {EMBO reports},
volume = {26},
number = {23},
pages = {6044-6078},
pmid = {41233591},
issn = {1469-3178},
support = {R01 HD082568/HD/NICHD NIH HHS/United States ; 1P50HD096723//HHS | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; CO29155//New York State Stem Cell Science (NYSTEM)/ ; C30293GG//New York State Stem Cell Science (NYSTEM)/ ; R01 HD082568/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; Mice ; *Germ Cells/metabolism/cytology ; *Epigenesis, Genetic ; Male ; Positive Regulatory Domain I-Binding Factor 1/genetics/metabolism ; Cell Differentiation/genetics ; Nanog Homeobox Protein/genetics/metabolism ; *Fertility/genetics ; Transcription Factor AP-2/genetics/metabolism ; Transcription Factors/genetics/metabolism ; *CRISPR-Cas Systems ; Female ; Repressor Proteins/genetics/metabolism ; RNA-Binding Proteins/genetics ; DNA-Binding Proteins/genetics/metabolism ; },
abstract = {Primordial germ cells (PGCs) are the precursors of gametes, and the ability to derive PGC-like cells (PGCLCs) from pluripotent stem cells has transformed germline research. A key limitation remains producing PGCLCs in sufficient numbers for large-scale applications. Here, we show that overexpression of Nanog plus three PGC master regulators - Prdm1, Prdm14, and Tfap2c - in mouse epiblast-like cells and formative embryonic stem cells yields abundant and highly enriched PGCLCs without costly recombinant cytokines. Nanog enhances the PGC regulatory network, suppresses somatic differentiation, and stabilizes PGCLC fate. Transcriptomically, these PGCLCs are developmentally more advanced than cytokine-induced counterparts and can be sustained long-term or differentiated into spermatogonia-like cells. Using this platform, we conduct a CRISPRi screen of 701 epigenetic genes to identify those needed for PGCLC formation. Downregulation of Ncor2, a histone deacetylase (HDAC) recruiter, has the greatest impact. Additionally, the HDAC inhibitors valproic acid and sodium butyrate suppress PGCLC formation and sperm counts of in utero-exposed animals. This work establishes a scalable system for functional screening of genes that influence germline development.},
}

Animals
Mice
*Germ Cells/metabolism/cytology
*Epigenesis, Genetic
Male
Positive Regulatory Domain I-Binding Factor 1/genetics/metabolism
Cell Differentiation/genetics
Nanog Homeobox Protein/genetics/metabolism
*Fertility/genetics
Transcription Factor AP-2/genetics/metabolism
Transcription Factors/genetics/metabolism
*CRISPR-Cas Systems
Female
Repressor Proteins/genetics/metabolism
RNA-Binding Proteins/genetics
DNA-Binding Proteins/genetics/metabolism

@article {pmid41214358,
year = {2025},
author = {Liu, XY and Li, YF and Zhou, JZ and Huang, JQ and Wang, LJ and Yang, L and Li, CY and Fang, X and Chen, XY},
title = {Gamma-selinene synthase catalyzes the first step of dihydroagarofuran sesquiterpene alkaloid biosynthesis in Tripterygium.},
journal = {Science China. Life sciences},
volume = {68},
number = {12},
pages = {3696-3710},
pmid = {41214358},
issn = {1869-1889},
mesh = {*Tripterygium/genetics/enzymology/metabolism ; *Sesquiterpenes/metabolism/chemistry ; *Alkaloids/biosynthesis ; *Plant Proteins/metabolism/genetics ; Biosynthetic Pathways ; Phylogeny ; CRISPR-Cas Systems ; },
abstract = {Tripterygium wilfordii and T. hypoglaucum, which belong to family Celastraceae, are perennial vine shrubs with medicinal uses in treating rheumatoid arthritis, cancer, and male contraception. Among the bioactive ingredients, the macrocyclic dihydroagarofuran sesquiterpene alkaloids (DASAs), also serving as chemotaxonomic markers of Celastraceae, are well-known for cytotoxicity and insecticidal properties. Despite intensive phytochemical investigations over the last half-century, the DASAs biosynthetic pathway remains unsolved. Here, we mined multi-omics data of Tripterygium species and identified 14 sesquiterpene synthase genes. Detailed characterization revealed that a group of enzymes (TwTPS5, TwTPS16, ThTPS5) catalyzed the key step committed to the biosynthesis of DASAs in Tripterygium, as evidenced by CRISPR/Cas9 knockout of TwTPS5 in T. wilfordii. Our biochemical assay showed that these enzymes converted FPP to gamma-selinene. Homologues in other Celastraceae species (EaTPS3, CaTPS5, and CaTPS6) also produced gamma-selinene, suggesting functional conservation in this DASAs-producing lineage. Notably, knocking out TwTPS5 resulted in a reduction of DASAs and enhanced accumulation of pharmaceutically valuable compounds, generating a safer and innovative medicinal plant resource for future applications.},
}

*Tripterygium/genetics/enzymology/metabolism
*Sesquiterpenes/metabolism/chemistry
*Alkaloids/biosynthesis
*Plant Proteins/metabolism/genetics
Biosynthetic Pathways
Phylogeny
CRISPR-Cas Systems

@article {pmid41139756,
year = {2025},
author = {Kang, CM and Jeong, RD},
title = {Rapid and visual detection of Lentinula edodes spherical virus using CRISPR/Cas12a-based RT-RPA.},
journal = {Virus genes},
volume = {61},
number = {6},
pages = {724-732},
pmid = {41139756},
issn = {1572-994X},
mesh = {*Shiitake Mushrooms/virology/genetics ; *CRISPR-Cas Systems ; *Fungal Viruses/genetics/isolation & purification ; Republic of Korea ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; },
abstract = {Lentinula edodes (shiitake) is a globally important edible mushroom, but viral infections can hinder mycelial growth, reduce yield, and degrade quality, leading to economic losses. This study examined viral infections in L. edodes cultivated in South Korea. We developed a CRISPR/Cas12a-based reverse transcription recombinase polymerase amplification (RT-RPA) assay for the rapid and accurate detection of L. edodes spherical virus (LeSV), the predominant mycovirus-infecting L. edodes in Korea. The optimized RT-RPA-CRISPR/Cas12a assay can be completed within 20 min at 34 °C and demonstrated 100 times higher sensitivity than RT-PCR. Field sample verification further confirmed its superior detection rate over RT-PCR. The RT-RPA-CRISPR/Cas12a method described herein has the potential to facilitate early diagnosis of LeSV infections and support disease management in L. edodes cultivation.},
}

*Shiitake Mushrooms/virology/genetics
*CRISPR-Cas Systems
*Fungal Viruses/genetics/isolation & purification
Republic of Korea
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity

@article {pmid41016102,
year = {2026},
author = {Guo, X and Zhang, J and Han, K and Li, Y and Wang, D},
title = {Aptamer-based PSA biosensing technology: From nanomaterials to CRISPR-diagnostics technology.},
journal = {Talanta},
volume = {298},
number = {Pt A},
pages = {128871},
doi = {10.1016/j.talanta.2025.128871},
pmid = {41016102},
issn = {1873-3573},
mesh = {*Prostate-Specific Antigen/analysis/blood ; Humans ; *Biosensing Techniques/methods ; *Aptamers, Nucleotide/chemistry ; *Nanostructures/chemistry ; *Prostatic Neoplasms/diagnosis ; Male ; *CRISPR-Cas Systems ; },
abstract = {Prostate cancer is a leading cause of cancer-related mortality in men, making early detection crucial for improving patient outcomes. Prostate-specific antigen (PSA) is a key biomarker for prostate cancer. Traditional PSA detection methods, such as ELISA and CLIA, are widely used but have limitations including complex sample preparation and long assay times. Aptamer-based biosensors offer a promising alternative due to their high affinity, specificity, and ease of customization. Recent advancements in aptamer-based PSA biosensors, including the use of novel nanomaterials and dual-modal detection technologies, have significantly enhanced sensitivity and reliability. The integration of CRISPR-Dx technology has further improved the accuracy of PSA detection in complex biological samples. These innovations hold great potential for developing portable point-of-care testing (POCT) devices for early screening and personalized treatment. This review examines the latest developments in aptamer-based biosensing technology for PSA detection from 2019 to 2025, focusing on nanomaterial innovation, CRISPR-Dx integration, and dual-modal sensing. It aims to provide a comprehensive overview of the strengths and limitations of current technologies and to outline future directions for more efficient and clinically valuable PSA detection methods.},
}

*Prostate-Specific Antigen/analysis/blood
Humans
*Biosensing Techniques/methods
*Aptamers, Nucleotide/chemistry
*Nanostructures/chemistry
*Prostatic Neoplasms/diagnosis
Male
*CRISPR-Cas Systems

@article {pmid40985613,
year = {2025},
author = {Chen, S and Chen, X and Peng, Y and Li, Q and Zhou, J and Li, J and Du, G and Chen, J and Zhang, G},
title = {CRISPR-DNA Polymerase Assisted Targeted Mutagenesis for Regulable Laboratory Evolution.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {45},
pages = {e11448},
doi = {10.1002/advs.202511448},
pmid = {40985613},
issn = {2198-3844},
support = {2024YFA0918300//National Key Research and Development Program of China/ ; 32172153//National Natural Science Foundation of China/ ; BK20202002//Natural Science Foundation of Jiangsu Province/ ; },
mesh = {*DNA-Directed DNA Polymerase/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Mutagenesis/genetics ; Escherichia coli/genetics ; *Directed Molecular Evolution/methods ; Mutation/genetics ; },
abstract = {Targeted hypermutation tools are useful for engineering proteins and pathways, and exploring the evolutionary landscapes. However, existing targeted hypermutation tools for genomic loci mostly exhibit restricted mutation windows and limited mutational types. Here, by integrating mutagenic, high-processivity bacteriophage T5 or T7 DNA polymerases (DNAPs) with CRISPR-Cas9, the study develops an in vivo mutagenesis system that enables all possible types of nucleotide substitutions and an expanded mutation window of up to 2 kilobases, achieving a maximum mutation rate 1.1 × 10[6]-fold higher than wild-type Escherichia coli. Through MS2-mediated recruitment of T5 or T7 DNAP for co-localization with nickase nCas9, off-target rate is reduced by up to 96.8% without compromising on-target rate. Further benefiting from the dTnpB-based transcriptional repression system, the mutagenesis process can be properly regulated during continuous evolution. Finally, the CRISPR-TDNAP-assisted targeted mutagenesis for regulable laboratory evolution (CTRLE) confers cellular triple-antibiotic resistance in 8 days, and enhances the efficiency of the twin-arginine translocation pathway by over threefold in 6 days. Furthermore, CTRLE proves effective in Bacillus subtilis and Kluyveromyces lactis, yielding targeted mutation rates 1.2 × 10[5]-fold and 5 × 10[7]-fold higher than host backgrounds, respectively. Collectively, CTRLE provides an efficient and universal way to accelerate the continuous evolution of different microbial cells.},
}

*DNA-Directed DNA Polymerase/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Mutagenesis/genetics
Escherichia coli/genetics
*Directed Molecular Evolution/methods
Mutation/genetics

@article {pmid40961712,
year = {2026},
author = {Zheng, Y and Li, Y and Zhu, J and Yi, J and Li, T and Tang, H},
title = {Synergistic UCNP/CRISPR-Cas12a enhanced near-infrared photoelectrochemical biosensor for quantitative Kanamycin detection.},
journal = {Talanta},
volume = {298},
number = {Pt A},
pages = {128857},
doi = {10.1016/j.talanta.2025.128857},
pmid = {40961712},
issn = {1873-3573},
mesh = {*Kanamycin/analysis ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; Milk/chemistry ; Zinc Oxide/chemistry ; *Food Contamination/analysis ; Animals ; *Anti-Bacterial Agents/analysis ; Infrared Rays ; Aptamers, Nucleotide/chemistry ; Limit of Detection ; Sulfides/chemistry ; *Nanoparticles/chemistry ; Cadmium Compounds/chemistry ; *Bacterial Proteins/metabolism/genetics ; Electrodes ; Ytterbium/chemistry ; Photochemical Processes ; Yttrium/chemistry ; Endodeoxyribonucleases ; Zinc Compounds ; CRISPR-Associated Proteins ; },
abstract = {Antibiotic residues in food pose significant health risks, and sensitive on-site detection remains challenging in complex matrices. We report a near-infrared photoelectrochemical (NIR-PEC) biosensor with CRISPR-Cas12a signal amplification for sensitive detection of kanamycin in food. A ZnO/CdS heterojunction photoanode boosts visible-light charge separation, producing a photocurrent of 4.6 mA-96 % higher than ZnO alone. Upconversion nanoparticles (NaYF4:Yb[3+], Er[3+]) convert 980 nm light into 542 nm emission to drive the heterojunction under NIR illumination. In the presence of kanamycin, activator DNA is displaced from an aptamer, triggering Cas12a to cleave ssDNA probes on the electrode and reduce photocurrent. The sensor shows a linear response from 10 to 1000 nM, a detection limit of 0.284 nM and less than 2.8 % cross-reactivity with related compounds. Average recovery in spiked milk samples averages 97 % (RSD = 2.2 %). This platform offers high specificity and sensitivity for antibiotic residue analysis in complex food matrices.},
}

*Kanamycin/analysis
*Biosensing Techniques/methods
*Electrochemical Techniques/methods
*CRISPR-Cas Systems
Milk/chemistry
Zinc Oxide/chemistry
*Food Contamination/analysis
Animals
*Anti-Bacterial Agents/analysis
Infrared Rays
Aptamers, Nucleotide/chemistry
Limit of Detection
Sulfides/chemistry
*Nanoparticles/chemistry
Cadmium Compounds/chemistry
*Bacterial Proteins/metabolism/genetics
Electrodes
Ytterbium/chemistry
Photochemical Processes
Yttrium/chemistry
Endodeoxyribonucleases
Zinc Compounds
CRISPR-Associated Proteins

@article {pmid40956287,
year = {2025},
author = {Shen, Y and Lu, B and Ma, B and Ding, X},
title = {Enhanced One-Pot Dual-CRISPR-Based Assay Lyophilized on a 3D-Printed Disc for Field-Deployable Multiplex Bacteria Detection.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {45},
pages = {e09355},
doi = {10.1002/advs.202509355},
pmid = {40956287},
issn = {2198-3844},
support = {2023YFD2402800//National Key Research and Development Program of China for Young Scientists/ ; 2022YFF1102600//National Key Research and Development Program of China for Young Scientists/ ; 82373629//National Natural Science Foundation of China/ ; BE2023725//Jiangsu Provincial Key Research and Development Program/ ; 4060692202/023//Medical Foundation of Southeast University/ ; },
mesh = {*Printing, Three-Dimensional ; Freeze Drying/methods ; *Nucleic Acid Amplification Techniques/methods ; *Bacteria/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; },
abstract = {One-pot CRISPR-based detection combining recombinase polymerase amplification (RPA) enables rapid and accurate nucleic acid testing but faces challenges in performance, multiplexing, and field-ready lyophilization. Here, an enhanced one-pot, helicase-assisted RPA (hRPA)-combined, dual-CRISPR/uAsCas12a (EOD-CRISPR) assay is described which can be lyophilized on a 3D-printed microfluidic disc to achieve field-deployable multiplex bacteria detection. In EOD-CRISPR reactions, the reaction speed, sensitivity, and fluorescence signal are significantly enhanced due to the synergistic effect of bovine serum albumin, hRPA, and uAsCas12a nuclease. The 3D-printed disc features four central chambers encircled by eight outer chambers, permitting detecting four targets simultaneously. For stable lyophilization on disc chambers, glassfiber membranes are inserted as substrates to adsorb the EOD-CRISPR reagents containing a protectant of 5% trehalose and 1% glycine. Toward point-of-need testing, EOD-CRISPR-lyophilized discs are applied to build an onsite detection platform. Through detecting synthetic food samples contaminated by four foodborne bacteria (i.e., Bacillus cereus, Salmonella enterica, Staphylococcus aureus, and Escherichia coli O157:H7), the onsite detection platform is validated and the sensitivity (80%-88.9%) and specificity (92.3%-100%) are comparable to those of standard PCR methods. Therefore, the field-deployable multiplex EOD-CRISPR assays holds great potentials for onsite bacteria detection and beyond.},
}

*Printing, Three-Dimensional
Freeze Drying/methods
*Nucleic Acid Amplification Techniques/methods
*Bacteria/genetics/isolation & purification
*CRISPR-Cas Systems/genetics

@article {pmid40946480,
year = {2026},
author = {Zhang, Q and Liu, Z and Sheng, E and Liu, Z and Zhao, R and Li, M},
title = {Glucosemeter-based aptasensor with an efficient and green system driven by CRISPR/Cas12a for ZEN detection.},
journal = {Talanta},
volume = {298},
number = {Pt A},
pages = {128844},
doi = {10.1016/j.talanta.2025.128844},
pmid = {40946480},
issn = {1873-3573},
mesh = {*CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *Zearalenone/analysis ; *Aptamers, Nucleotide/chemistry/genetics/metabolism ; Limit of Detection ; *CRISPR-Associated Proteins/metabolism/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {To address the challenge of readily available and cost-effective monitoring, a novel aptasensor platform was proposed relying on the clustered regularly interspaced short palindromic repeats/associated protein 12a (CRISPR/Cas12a)-driven and glucosemeter-assisted system for zearalenone (ZEN). The magnetic bead (MB)-based probes of MB@Apt-cDNA and MB@invertase were prepared to provide specific recognition and efficient conversion. When ZEN presented, the released cDNA activated the cutting function of CRISPR/Cas12a for ssDNA, releasing numerous invertase to convert the environmentally friendly sucrose. Then, the generated glucose signals were positively related to ZEN levels, which quantified by a glucosemeter. The proposed aptasensor displayed an excellent sensitivity, achieving the limit of detection (LOD) and detection range of 0.218 ng/mL and 0.218-109.89 ng/mL, respectively. The platform also exhibited high specificity (with cross-reactivity values below 6.81 % for other analytes) and high accuracy for ZEN detection. The satisfactory reliability and practicability had been demonstrated through its application in real agricultural byproduct samples. The area under the curve was 0.979, with an optimal operating point being (0.125, 1.000). This study can provide an alternative pathway for improving the detection performance and accessibility to public in limited situation, suggesting the efficient CRISPR/Cas12a and green glucosemeter detection strategies for other pollutant monitoring.},
}

*CRISPR-Cas Systems
*Biosensing Techniques/methods
*Zearalenone/analysis
*Aptamers, Nucleotide/chemistry/genetics/metabolism
Limit of Detection
*CRISPR-Associated Proteins/metabolism/genetics
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid40939440,
year = {2026},
author = {Xiang, Q and Zhou, W and Li, D},
title = {Dumbbell-shaped DNA topology drives self-sustaining CRISPR/Cas12a exponential amplification for ultrasensitive monitoring of DNA methyltransferase activity.},
journal = {Talanta},
volume = {298},
number = {Pt A},
pages = {128841},
doi = {10.1016/j.talanta.2025.128841},
pmid = {40939440},
issn = {1873-3573},
mesh = {*CRISPR-Cas Systems ; *DNA/chemistry/metabolism ; DNA Methylation ; Nucleic Acid Conformation ; Humans ; *Enzyme Assays/methods ; *Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {DNA methylation is an essential epigenetic mechanism, and abnormal methylation has been linked to the onset and progression of many diseases, representing a potential threat to health. Monitoring DNA methyltransferase (MTase) activity is essential for understanding DNA methylation regulation and developing MTase-targeted inhibitors. To address this challenge, we developed a dumbbell-shaped DNA topology that drives self-sustaining (autocatalytic) CRISPR/Cas12a system for exponential signal amplification, enabling ultrasensitive fluorescent detection of DNA MTase activity. In this strategy, a DNA dumbbell topological structure (DDTS) is designed, in which two double-strand DNA (dsDNA) loops effectively block the activity of CRISPR/Cas12a. Upon Dam MTase presence, DpnI endonuclease cleaves the methylated recognition sites in the DNA probe, disrupting the DDTS topology to generate linear dsDNA activators. These activators restore the trans-cleavage of CRISPR/Cas12a, which further cleaves the single-stranded DNA (ssDNA) domain in DDTS probes to produce additional activators, creating an exponential amplification loop through autocatalysis. The system achieves a detection limit of 6.37 × 10[-4] U/mL for Dam MTase, with a linear range of 1 × 10[-3] to 15 U/mL, and shows excellent selectivity over other MTases and nucleases. It also enables inhibitor screening, with the half-maximal inhibitory concentration (IC50) value of 1.84 μM for 5-fluorouracil. Therefore, the method has great potential for application in the early diagnosis of diseases and drug discovery.},
}

*CRISPR-Cas Systems
*DNA/chemistry/metabolism
DNA Methylation
Nucleic Acid Conformation
Humans
*Enzyme Assays/methods
*Nucleic Acid Amplification Techniques/methods
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40898619,
year = {2025},
author = {Noh, B and Gopalappa, R and Lin, H and Gee, HY and Choi, JY and Kim, HH and Jung, J},
title = {Engineered virus-like particles for in vivo gene editing ameliorate hearing loss in murine DFNA2 model.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {12},
pages = {6449-6462},
doi = {10.1016/j.ymthe.2025.08.049},
pmid = {40898619},
issn = {1525-0024},
mesh = {Animals ; Mice ; *Gene Editing/methods ; Disease Models, Animal ; *Genetic Therapy/methods ; *Hearing Loss/therapy/genetics ; CRISPR-Cas Systems ; Hair Cells, Auditory, Outer/metabolism ; Genetic Vectors/genetics ; Gene Transfer Techniques ; KCNQ Potassium Channels/genetics ; *Virion/genetics ; CRISPR-Associated Protein 9/genetics ; },
abstract = {Although gene editing therapy is applicable to human diseases, its efficiency and safety require further investigation. Further, non-virus-mediated gene editor delivery is challenging in the inner ear. Here, engineered virus-like particles (eVLPs) were used for inner ear delivery of SpCas9 and single-guided RNA to delete the Kcnq4 dominant-negative mutant allele, which causes progressive hearing loss in a non-syndromic hearing loss murine model. eVLP-delivered SpCas9 was administered to the inner ears of Kcnq4[W277S/+] mice to target the Kcnq4-expressing outer hair cells (OHCs). Hearing loss was significantly alleviated 7 weeks after eVLP administration. OHC survival improved significantly, and OHC-innervating neurite (connected to type II spiral ganglion neuronal body) loss was ameliorated. Finally, OHC membrane potential was hyperpolarized with eVLP gene editor treatment in Kcnq4-mutant mice, indicating that their OHCs were healthier and more stable than those of uninjected mice. Our findings suggest that eVLPs are feasible inner ear gene editor deliverers to treat hearing loss.},
}

Animals
Mice
*Gene Editing/methods
Disease Models, Animal
*Genetic Therapy/methods
*Hearing Loss/therapy/genetics
CRISPR-Cas Systems
Hair Cells, Auditory, Outer/metabolism
Genetic Vectors/genetics
Gene Transfer Techniques
KCNQ Potassium Channels/genetics
*Virion/genetics
CRISPR-Associated Protein 9/genetics

@article {pmid40885667,
year = {2025},
author = {Liu, W and Duan, W and Peng, Z and Liao, Y and Wang, X and Liu, R and Jing, Q and Jiang, H and Fan, Y and Ge, L and Huang, L and Xing, Y},
title = {Highly efficient prime editors for mammalian genome editing based on porcine retrovirus reverse transcriptase.},
journal = {Trends in biotechnology},
volume = {43},
number = {12},
pages = {3253-3278},
doi = {10.1016/j.tibtech.2025.07.029},
pmid = {40885667},
issn = {1879-3096},
mesh = {Animals ; *Gene Editing/methods ; Swine ; *RNA-Directed DNA Polymerase/genetics/metabolism ; Humans ; *Endogenous Retroviruses/genetics/enzymology ; CRISPR-Cas Systems ; Swine, Miniature ; HEK293 Cells ; },
abstract = {Prime editing is a versatile and precise genome-editing tool. Most prime editors (PEs) rely on reverse transcriptase (RT) derived from Moloney murine leukemia virus (MMLV). Here, we established a PE, pvPE, using a RT derived from a porcine endogenous retrovirus (PERV) from a Bama mini-pig. Through various optimization strategies, including RT engineering, structural modifications, and La protein fusion, we gradually upgraded to pvPE-V4. This version achieved 24.38-101.69-fold higher efficiency compared with pvPE-V1 and up to 2.39-fold higher efficiency than another upgraded PE, PE7, with significantly fewer unintended edits across multiple mammalian cell lines. We further show that nocodazole (Noc) significantly enhanced pvPE efficiency by 2.25-fold on average. Using our pvPE system, we efficiently modified three genes simultaneously in porcine fibroblasts and subsequently generated cloned pigs that could serve as valuable models for Alzheimer's disease (AD) in humans. Our results highlight the broad application prospects of pvPE systems in mammalian genome editing.},
}

Animals
*Gene Editing/methods
Swine
*RNA-Directed DNA Polymerase/genetics/metabolism
Humans
*Endogenous Retroviruses/genetics/enzymology
CRISPR-Cas Systems
Swine, Miniature
HEK293 Cells

@article {pmid40878354,
year = {2025},
author = {Shan, S and Pisias, MT and Zhang, Z and Mavrodiev, EV and Gitzendanner, MA and Hauser, BA and Grover, CE and Barbazuk, WB and Soltis, PS and Yang, B and Soltis, DE},
title = {Development of an efficient CRISPR-mediated genome editing platform in the diploid-polyploid model system Tragopogon (Asteraceae).},
journal = {Journal of experimental botany},
volume = {76},
number = {22},
pages = {6700-6713},
doi = {10.1093/jxb/eraf380},
pmid = {40878354},
issn = {1460-2431},
support = {IOS-1923234//National Science Foundation/ ; DEB-2043478//National Science Foundation/ ; },
mesh = {*Gene Editing/methods ; *Polyploidy ; *CRISPR-Cas Systems ; *Tragopogon/genetics ; *Genome, Plant ; Diploidy ; Alcohol Oxidoreductases/genetics/metabolism ; },
abstract = {Polyploidy or whole-genome duplication (WGD) is a significant evolutionary force. However, the mechanisms governing polyploid genome evolution remain unclear, limited largely by a lack of functional analysis tools in organisms that best exemplify the earliest stages of WGD. Tragopogon (Asteraceae) includes an evolutionary model system for studying the immediate consequences of polyploidy. In this study, we significantly improved the transformation system and obtained genome-edited T. porrifolius (2x) and T. mirus (4x) primary generation (T0) individuals. Using CRISPR/Cas9, we knocked out the dihydroflavonol 4-reductase (DFR) gene, which controls anthocyanin synthesis, in both species. All transgenic allotetraploid T. mirus individuals had at least one mutant DFR allele, and 71.4% had all four DFR alleles edited. The resulting mutants lacked anthocyanin, and these mutations were inherited in the T1 generation. This study demonstrates a highly efficient CRISPR platform, producing genome-edited Tragopogon individuals that have completed the life cycle. The approaches used and challenges faced in building the CRISPR system in Tragopogon provide a framework for building similar systems in other non-genetic models. Genome editing in Tragopogon paves the way for novel functional biology studies of polyploid genome evolution and the consequences of WGD on complex traits, holding enormous potential for both basic and applied research.},
}

*Gene Editing/methods
*Polyploidy
*CRISPR-Cas Systems
*Tragopogon/genetics
*Genome, Plant
Diploidy
Alcohol Oxidoreductases/genetics/metabolism

@article {pmid40744808,
year = {2025},
author = {Luo, G and Trinh, MDL and Falkenberg, MKD and Chiurazzi, MJ and Najafi, J and Nørrevang, AF and Correia, PMP and Palmgren, M},
title = {Unlocking in vitro transformation of recalcitrant plants.},
journal = {Trends in plant science},
volume = {30},
number = {12},
pages = {1306-1321},
doi = {10.1016/j.tplants.2025.07.001},
pmid = {40744808},
issn = {1878-4372},
mesh = {*Gene Editing/methods ; *Transformation, Genetic ; Plants, Genetically Modified/genetics ; CRISPR-Cas Systems ; *Plants/genetics ; },
abstract = {Genome editing offers powerful opportunities for crop improvement by enabling precise and targeted mutagenesis. Tools such as CRISPR-associated protein 9 and single-guide RNAs can be introduced into plant cells via in vitro transformation, which, despite the rise of in planta methods, remains an important method because it is highly effective when successful. However, transformation-induced stress is a critical and underexplored barrier to successful in vitro transformation, especially in recalcitrant plants. In this opinion article, we discuss in vitro methods for transforming recalcitrant plants, the challenges encountered, and potential solutions through the conceptual lens of stress biology. Reducing cellular stress, transiently weakening the immune response and optimizing regeneration protocols may be essential for expanding the transformation capacity across a broader range of plant species.},
}

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

@article {pmid41328758,
year = {2026},
author = {Plewnia, A and Hoenig, BD and Lötters, S and Heine, C and Erens, J and Böning, P and Bending, GD and Krehenwinkel, H and Williams, MA},
title = {The Emergence of a CRISPR-Cas Revolution in Ecology: Applications, Challenges, and an Ecologist's Overview of the Toolbox.},
journal = {Molecular ecology resources},
volume = {26},
number = {1},
pages = {e70086},
pmid = {41328758},
issn = {1755-0998},
support = {//University of Warwick/ ; NE/S010270/1//Natural Environment Research Council/ ; },
mesh = {*CRISPR-Cas Systems ; *Ecology/methods ; *Gene Editing/methods ; },
abstract = {CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated nucleases) systems allow researchers to detect, capture, and even alter parts of an organism's genome. However, while the use of CRISPR-Cas has revolutionised many fields in the life sciences, its full potential remains underutilised in ecology and biodiversity research. Here we outline the emerging applications of CRISPR-Cas in ecological contexts, focusing on three main areas: nucleic acid detection, CRISPR-enhanced sequencing, and genome editing. CRISPR-based nucleic acid detection of environmental DNA samples is already reshaping species monitoring, providing highly sensitive and non-invasive tools for both scientists and the public alike, with reduced costs and minimal experience required. Further, CRISPR-enhanced sequencing, including Cas-mediated target enrichment, enables efficient recovery of ecologically relevant loci and supports diverse applications such as amplification-free metagenomics. Finally, while genome editing on wild species remains largely theoretical in ecology, these tools are already being used in controlled settings to study adaptation and resilience in the face of ongoing global stressors. Together, the applications of CRISPR-Cas are paving the way for more affordable, accessible, and impactful applications for species conservation, and promise to improve our ability to tackle the ongoing global biodiversity crisis.},
}

*CRISPR-Cas Systems
*Ecology/methods
*Gene Editing/methods

@article {pmid41328592,
year = {2025},
author = {Geng, Y and Jiang, C and Zhang, H and Yang, H and Peng, Y and Chen, Y and Hu, C and Liu, H and Li, S and Chen, H and Xie, S and Guo, A},
title = {Genome-scale CRISPR screen identifies host factors associated with bovine parainfluenza virus 3 infection.},
journal = {Virulence},
volume = {16},
number = {1},
pages = {2589554},
doi = {10.1080/21505594.2025.2589554},
pmid = {41328592},
issn = {2150-5608},
mesh = {Animals ; Cattle ; *Parainfluenza Virus 3, Bovine/physiology/genetics ; CRISPR-Cas Systems ; Virus Replication ; *Host-Pathogen Interactions/genetics ; Cell Line ; *Respirovirus Infections/virology/veterinary/genetics ; Wnt-5a Protein/genetics/metabolism ; *Cattle Diseases/virology ; Virus Internalization ; Gene Knockout Techniques ; },
abstract = {Bovine parainfluenza virus type 3 (BPIV-3) is a major pathogen associated with the bovine respiratory disease complex. However, the limited understanding of host factors crucial for BPIV-3 replication has hindered the development of effective preventive and therapeutic strategies. To tackle this critical issue, we constructed a bovine genome-wide CRISPR/Cas9 knockout library in Madin-Darby bovine kidney cells, which was then used to systematically identify and characterize the host genes essential for BPIV-3a replication. Subsequently, 10 genes were validated using both RT-qPCR and viral titration assays. Furthermore, through gene knockout or knockdown and rescue experiments, we identified three key genes required for BPIV-3a replication: Wnt family member 5A (WNT5A), solute carrier family 16 member 13 (SLC16A13), and selenoprotein N (SELENON). However, their effects on viral adhesion and internalization varied. WNT5A was involved in both processes, SLC16A13 participated solely in internalization, while SELENON had no significant impact on either. Beyond BPIV-3a, these three genes were also found to be essential for the infection of BPIV-3c and Bovine enterovirus. In conclusion, this study offers novel insights into the molecular mechanisms governing the replication and pathogenesis of BPIV-3a, BPIV-3c, and bovine enterovirus within host cells, thereby providing a foundation for identifying potential targets in the development of novel antiviral strategies.},
}

Animals
Cattle
*Parainfluenza Virus 3, Bovine/physiology/genetics
CRISPR-Cas Systems
Virus Replication
*Host-Pathogen Interactions/genetics
Cell Line
*Respirovirus Infections/virology/veterinary/genetics
Wnt-5a Protein/genetics/metabolism
*Cattle Diseases/virology
Virus Internalization
Gene Knockout Techniques

@article {pmid41328409,
year = {2026},
author = {Zhang, H and Song, Y and Liu, W and Zheng, X and An, X and Li, C and Chen, W and Wang, H and Zhang, Y},
title = {Defect-complementation homologous recombination: A novel strategy for precise genome engineering of virulent phages.},
journal = {Synthetic and systems biotechnology},
volume = {12},
number = {},
pages = {59-70},
pmid = {41328409},
issn = {2405-805X},
abstract = {Engineered bacteriophages (phages) have been developed to overcome the limitations of natural phage therapies and serve as precision-targeted agents against drug-resistant bacterial infections. However, their application has been constrained by the low efficiency of existing genome-editing tools, largely because of the absence of effective selection markers. This study proposed a novel strategy, termed defect-complementation homologous recombination (DCHR), for precise phage genome editing. In this approach, CRISPR-Cas9 cleaves a donor plasmid in host cells to release a linear donor template carrying homology arms, an essential phage gene used as a selection marker, and two lox sites. The donor template undergoes homologous recombination with the genome of essential gene-deficient phage, thereby enabling targeted genome modifications. Using DCHR, we successfully generated large genomic deletions (1.48-kb gp0.4-0.7 and 1.02-kb gp4.3-4.7), achieved gene insertion (3.08-kb lacZ), and introduced a single-base substitution (TGA to TAA) in the stop codon of gp9 within the same T7 phage genome, all with 100 % accuracy. The significant advantages of DCHR are as follows: (i) High-efficiency screening: Only progeny phages derived from successful homologous recombination retain viability and replicative capacity, thereby greatly simplifying recombinant isolation. (ii) Editing flexibility: Unlike CRISPR-Cas systems, DCHR cannot be constrained by protospacer adjacent motif dependence and allows modifications across diverse genomic loci. (iii) High recombination efficiency: DCHR can achieve a recombinant phage titer of 3.1 × 10[5] PFU mL[-1] (plaque-forming units per mL) without relying on exogenous homologous recombination systems. In summary, DCHR demonstrates potential as a precise and efficient general genome-editing tool that facilitates design of engineered phages and advances functional genomic studies.},
}

@article {pmid41328347,
year = {2026},
author = {Li, Q and Bao, Q and Zhao, S and Wu, F and Li, Y and Wang, K and Li, W and Gao, H},
title = {Advancements in CRISPR-based therapies for ocular pathologies: from disease mechanisms to intervention strategies.},
journal = {Theranostics},
volume = {16},
number = {1},
pages = {156-192},
pmid = {41328347},
issn = {1838-7640},
mesh = {Humans ; *Genetic Therapy/methods ; *Eye Diseases/therapy/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; },
abstract = {Eye diseases caused by genetic mutations affect over 2.2 billion people worldwide. The development of CRISPR technology has opened exciting possibilities for how we diagnose and treat these conditions. However, designing effective CRISPR systems, managing potential risks, and considering the ethical questions around gene therapy in clinical practice are major challenges. To move forward successfully, it's important to evaluate how practical CRISPR-based treatments are for eye diseases from a clinical perspective, while also understanding how CRISPR systems work. In this review, we start by covering the basic principles behind CRISPR technology and explore its different types. Next, we look at various ways CRISPR is being used in eye research and treatments, from early studies to new clinical approaches. Lastly, we address the regulatory environment and ethical issues involved, discussing existing rules, safety concerns, and guidelines for genetic modifications in medical settings. Our goal is to share new insights into innovative treatments for eye diseases and to support the safe use of CRISPR in clinical eye care. This review aims to be a helpful resource for researchers, doctors, and regulators working on CRISPR-based therapies.},
}

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

@article {pmid41326076,
year = {2026},
author = {Niu, RC and Zeng, QH and Wang, WJ and Hu, J and Liu, TX and Zhang, SZ},
title = {Multi-omics analyses identify the modulators COX6A1 and NAL as regulators of silk cocoon formation in Plutella xylostella.},
journal = {Pesticide biochemistry and physiology},
volume = {216},
number = {Pt 1},
pages = {106764},
doi = {10.1016/j.pestbp.2025.106764},
pmid = {41326076},
issn = {1095-9939},
mesh = {Animals ; *Moths/genetics/metabolism/growth & development ; *Silk/biosynthesis/genetics/metabolism ; *Insect Proteins/genetics/metabolism ; Larva/metabolism/genetics/growth & development ; Proteomics ; CRISPR-Cas Systems ; *Electron Transport Complex IV/genetics/metabolism ; Transcriptome ; Multiomics ; },
abstract = {The diamondback moth (DBM) is a major global pest of cruciferous crops. Silk production, essential for DBM larval locomotion and pupal attachment, is governed by fibroin heavy chain (FibH), fibroin light chain (FibL), and fibrohexamerin (P25). However, the regulatory mechanisms and downstream key genes involved in silk production in DBM remain poorly understood. To address this, we integrated transcriptomic and proteomic data from CRISPR/Cas9 generated PxFibH, PxFibL, and PxP25 mutants to investigate the impact of silk gene deletions in the silk gland and identify modulators of silk formation. In the transcriptomic analysis, we identified 1994, 913, and 1266 differentially expressed genes (DEGs) in the three mutant strains, respectively. GO and KEGG enrichment analysis revealed significant involvement in pathways such as oxidation-reduction process, transmembrane transport, enzyme activity, and extracellular matrix (ECM) receptor interaction. At the proteomic level, 604, 210, and 266 differentially expressed proteins (DEPs) were identified in the three mutants, respectively. GO and KEGG enrichment analysis of these DEPs consistently highlighted energy metabolism, hydrolase activity, and catalytic activity pathways. Integrated multi-omics analyses identified three conserved regulator genes: cytochrome c oxidase subunit 6A1 (COX6A1), N-acetylneuraminate lyase (NAL), and protein phosphatase 1 regulatory subunit 14B (PPP1R14B). CRISPR/Cas9 knockout of PxCOX6A1 resulted in incomplete cocoon formation, along with increased larval mortality, prolonged development, and reduced oviposition. PxNAL knockout was lethal, while heterozygotes exhibited decreased cocoon formation, pupal weight, and fecundity. This study reveals FibH/FibL/P25-dependent metabolic networks regulating silk production and identifies COX6A1 and NAL as novel targets for environmentally sustainable pest control strategies.},
}

Animals
*Moths/genetics/metabolism/growth & development
*Silk/biosynthesis/genetics/metabolism
*Insect Proteins/genetics/metabolism
Larva/metabolism/genetics/growth & development
Proteomics
CRISPR-Cas Systems
*Electron Transport Complex IV/genetics/metabolism
Transcriptome
Multiomics

@article {pmid41325825,
year = {2025},
author = {Zhang, Y and Deng, S},
title = {Geminivirus vectors: From gene silencing to synthetic biology.},
journal = {Biotechnology advances},
volume = {87},
number = {},
pages = {108771},
doi = {10.1016/j.biotechadv.2025.108771},
pmid = {41325825},
issn = {1873-1899},
abstract = {Geminiviruses, the largest plant DNA virus family, cause devastating diseases in crops worldwide. These viruses possess distinctive features, such as the stem-loop structure and replication protein (Rep), which enable the creation of functional geminiviral replicons (GVRs) in plants. Over three decades, geminiviruses have been developed into vectors for virus-induced gene silencing (VIGS), high-level protein expression, and genome editing. This review introduces the genomic structure, Rep protein domains and functions, as well as the historical applications of geminiviruses, then highlights their prominent roles in VIGS and synthetic biology. As VIGS vectors, bipartite geminiviruses utilize AV1 gene replacement, while monopartite species rely on satellite DNAs to insert target sequences, enabling gene silencing in diverse plants. In synthetic biology, GVRs facilitate high-level protein expression through autonomous replication and enhance CRISPR/Cas genome editing efficiency in crops. Additionally, gene regulatory elements, including tissue-specific promoters and gene expression enhancement sequences from geminiviral genomes or satellite DNA expand their utility in genetic engineering. Finally, this review provides an outlook on the future development of geminivirus vectors. GVRs can work as plasmid-like DNAs for supporting diverse and creative designs in plant synthetic biology. The stem-loop structure and Rep are not unique to geminiviruses, a fact that suggests potential cross-kingdom applications of GVRs beyond plants. Vast viral resources enable further acceleration of GVR applications through resource mining and optimization. Moreover, attenuated or engineered geminiviral strains hold promise as "plant vaccines" via cross-protection. Collectively, geminivirus vectors bridge fundamental viral research with practical innovations in crop improvement, biomanufacturing, and synthetic biology.},
}

@article {pmid41261131,
year = {2025},
author = {Pierce, SE and Erwood, S and Oye, K and An, M and Krasnow, N and Zhang, E and Raguram, A and Seelig, D and Osborn, MJ and Liu, DR},
title = {Prime editing-installed suppressor tRNAs for disease-agnostic genome editing.},
journal = {Nature},
volume = {648},
number = {8092},
pages = {191-202},
pmid = {41261131},
issn = {1476-4687},
mesh = {Humans ; *Gene Editing/methods ; Animals ; *RNA, Transfer/genetics/metabolism ; Mice ; Codon, Nonsense/genetics ; Cystic Fibrosis/genetics/therapy ; Codon, Terminator/genetics ; Female ; Male ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; HEK293 Cells ; },
abstract = {Precise genome-editing technologies such as base editing[1,2] and prime editing[3] can correct most pathogenic gene variants, but their widespread clinical application is impeded by the need to develop new therapeutic agents for each mutation. For diseases that are caused by premature stop codons, suppressor tRNAs (sup-tRNAs) offer a more general strategy. Existing approaches to use sup-tRNAs therapeutically, however, require lifelong administration[4,5] or show modest potency, necessitating potentially toxic overexpression. Here we present prime editing-mediated readthrough of premature termination codons (PERT), a strategy to rescue nonsense mutations in a disease-agnostic manner by using prime editing to permanently convert a dispensable endogenous tRNA into an optimized sup-tRNA. Iterative screening of thousands of variants of all 418 human tRNAs identified tRNAs with the strongest sup-tRNA potential. We optimized prime editing agents to install an engineered sup-tRNA at a single genomic locus without overexpression and observed efficient readthrough of premature termination codons and protein rescue in human cell models of Batten disease, Tay-Sachs disease and cystic fibrosis. In vivo delivery of a single prime editor that converts an endogenous mouse tRNA into a sup-tRNA extensively rescued disease pathology in a model of Hurler syndrome. PERT did not induce detected readthrough of natural stop codons or cause significant transcriptomic or proteomic changes. Our findings suggest the potential of disease-agnostic therapeutic genome-editing approaches that require only a single composition of matter to treat diverse genetic diseases.},
}

Humans
*Gene Editing/methods
Animals
*RNA, Transfer/genetics/metabolism
Mice
Codon, Nonsense/genetics
Cystic Fibrosis/genetics/therapy
Codon, Terminator/genetics
Female
Male
CRISPR-Cas Systems/genetics
Disease Models, Animal
Cystic Fibrosis Transmembrane Conductance Regulator/genetics
HEK293 Cells

@article {pmid41201289,
year = {2025},
author = {Liu, T and Su, C and Yan, X and Zhang, Y and Wang, Y and Duan, K},
title = {Creating Hypoallergenic and Low Antinutritional Soybeans through CRISPR/Cas12a-Mediated Multiplex Gene Editing.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {48},
pages = {30922-30932},
doi = {10.1021/acs.jafc.5c08935},
pmid = {41201289},
issn = {1520-5118},
mesh = {*Glycine max/genetics/immunology/chemistry/metabolism ; Animals ; *Gene Editing ; *CRISPR-Cas Systems ; Mice ; *Soybean Proteins/genetics/immunology ; *Plants, Genetically Modified/genetics/immunology/metabolism/chemistry ; *Allergens/immunology/genetics ; Humans ; Food Hypersensitivity/immunology ; Female ; },
abstract = {Soybean protein is a valuable plant-based nutrition source for food. However, allergens and antinutritional factors like Gly Bd 30k (P34), Kunitz trypsin inhibitor (KTI), and soybean agglutinin (LE) pose risks. In this study, we employed the CRISPR/Cas12a system to generate hypoallergenic and low antinutritional soybean lines without agronomic traits penalties. The edited lines exhibited markedly reduced levels and enzymatic activity of trypsin inhibitors, as well as soybean agglutinin content. Feeding mice with these edited soybeans, deficient in P34, KTI, and LE proteins, significantly alleviated allergenic responses. This research provides a promising resource of hypoallergenic and low antinutritional soybeans for applications in food production, animal feed, and immunotherapy.},
}

*Glycine max/genetics/immunology/chemistry/metabolism
Animals
*Gene Editing
*CRISPR-Cas Systems
Mice
*Soybean Proteins/genetics/immunology
*Plants, Genetically Modified/genetics/immunology/metabolism/chemistry
*Allergens/immunology/genetics
Humans
Food Hypersensitivity/immunology
Female

@article {pmid40715744,
year = {2025},
author = {Zhang, K and Yuan, X and Lu, S and Shu, Y and Wang, C and Cen, J and Wu, B and Hui, L},
title = {Expansion of human hepatocytes and their application in three-dimensional culture and genetic manipulation.},
journal = {Nature protocols},
volume = {20},
number = {12},
pages = {3722-3754},
pmid = {40715744},
issn = {1750-2799},
support = {92168202//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32370793//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32221002//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; *Hepatocytes/cytology/metabolism ; CRISPR-Cas Systems ; Gene Editing/methods ; *Cell Culture Techniques, Three Dimensional/methods ; Lentivirus/genetics ; *Cell Culture Techniques/methods ; },
abstract = {Hepatocytes are one of the most important cell types in the liver, carrying out key functions. They are essential for hepatocyte-based therapy, disease modeling and drug development. However, the availability of primary human hepatocytes (PHHs) is limited by a shortage of donors. It is therefore of great value to expand PHHs in large quantities. Here we provide a detailed protocol for the large-scale expansion of PHHs (proliferating human hepatocytes, ProliHHs) derived from healthy donors and patients with inherited liver diseases, which can be rematured in a three-dimensional culture system. Moreover, we provide a protocol for the genetic manipulation of ProliHHs, including lentivirus transduction and CRISPR-Cas9-mediated knockout and knock-in. The protocol described here will help to realize the full potential of ProliHH-based therapy, organoid-based liver disease modeling and drug screening. The protocol to expand PHHs takes ~1-2 months, the protocol to establish the 3D-cultured ProliHHs takes ~8 d and the protocol to perform gene editing takes ~3 d. Personnel with basic scientific training can conduct these protocols.},
}

Humans
*Hepatocytes/cytology/metabolism
CRISPR-Cas Systems
Gene Editing/methods
*Cell Culture Techniques, Three Dimensional/methods
Lentivirus/genetics
*Cell Culture Techniques/methods

@article {pmid40715743,
year = {2025},
author = {Guruprasad, P and Ramasubramanian, R and Nason, S and Carturan, A and Liu, S and Paruzzo, L and Hornet, V and Plesset, J and Patel, RP and Bhoj, V and Beatty, GL and Ruella, M},
title = {Manufacturing of CRISPR-edited primary mouse CAR T cells for cancer immunotherapy.},
journal = {Nature protocols},
volume = {20},
number = {12},
pages = {3629-3654},
pmid = {40715743},
issn = {1750-2799},
support = {R00CA212302//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01-37-CA262362-03//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Animals ; Mice ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Receptors, Chimeric Antigen/genetics/immunology ; *T-Lymphocytes/immunology/metabolism ; *Neoplasms/therapy/immunology ; *Immunotherapy, Adoptive/methods ; Humans ; Tumor Microenvironment/immunology ; },
abstract = {Editing chimeric antigen receptor (CAR) T cells by using CRISPR-Cas9 has become a routine strategy to improve their antitumor function or safety profile. Xenograft tumor models in immunodeficient mice are often used to evaluate the function of CRISPR-edited human CAR T cells. These models, however, lack functional immune systems and thus fail to recapitulate barriers such as the immunosuppressive tumor microenvironment (TME) that CAR T cells will encounter in patients. Thus, genetically modifying mouse CAR T cells for use in immune-intact models is an attractive approach to explore the impact of a given gene deletion on CAR T cells within a natural TME. Here, we describe a protocol to perform CRISPR-Cas9 editing in primary mouse T cells, thereby enabling studies of gene-edited CAR T within the TME and in the presence of a functional immune system. This protocol is integrated into a standard mouse CAR T manufacturing workflow, a process that typically spans ~5-6 days. The first stage of this protocol involves isolating mouse T cells, electroporating them with a ribonucleoprotein complex and activating them by using magnetic bead stimulation. The second stage involves transducing the CAR gene and expanding these cells, and the third stage focuses on validating knockout efficiency and the functionality of gene-edited mouse CAR T cells. This procedure requires a proficiency in aseptic cell culture techniques and a basic understanding of T cell biology. We anticipate that efficient and reliable genetic modification of mouse T cells will have wide-ranging applications for cancer immunotherapies and related fields.},
}

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

@article {pmid40640356,
year = {2025},
author = {Ma, Y and Xiong, Y and Xu, J and Xu, H and Fu, Z and Zhao, GR and Wu, Y and Yuan, YJ},
title = {Assembly and delivery of large DNA via chromosome elimination in yeast.},
journal = {Nature protocols},
volume = {20},
number = {12},
pages = {3755-3782},
pmid = {40640356},
issn = {1750-2799},
support = {[32471483]//National Natural Science Foundation of China (National Science Foundation of China)/ ; [23JCYBJC00220]//Natural Science Foundation of Tianjin City (Natural Science Foundation of Tianjin)/ ; },
mesh = {*Saccharomyces cerevisiae/genetics ; CRISPR-Cas Systems ; *Chromosomes, Fungal/genetics ; Haploidy ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *DNA/genetics ; *DNA, Fungal/genetics ; },
abstract = {Manipulation of large-scale genetic information provides a powerful approach to deciphering and engineering complex biological functions. However, the manipulation of large DNA, such as assembly and delivery, remains complex and difficult. Here we describe the experimental design strategy and protocol for a chromosome elimination-mediated large DNA assembly and delivery method (HAnDy), which enables efficient Mb-scale DNA assembly and delivery in yeast conveniently. This protocol is divided into three parts: (1) CRISPR-Cas9-mediated elimination of chromosome, which includes design and integration of a synthetic single-guide RNA (sgRNA) site near the centromere, activation of chromosome elimination by mating, and verification of the chromosome elimination. It can be used to eliminate multiple chromosomes, achieving haploidization in yeast. (2) Haploidization-mediated DNA assembly, which includes the design and construction of initial assembly strains, DNA assembly by programmed haploidization and verification of the assembled clones. (3) Haploidization-mediated DNA delivery, which includes the design and construction of inducible haploidization strains, DNA delivery by programmed haploidization and verification of the delivered clones. Users can utilize this protocol entirely or selectively depending on their needs. With the use of this protocol, it takes 10 d to achieve chromosome elimination and 7-11 d to achieve a standard cycle of haploidization-mediated DNA assembly or delivery. This protocol provides an efficient approach that is useful for the elimination, assembly and delivery of large DNA in yeast, requiring basic molecular biology skills.},
}

*Saccharomyces cerevisiae/genetics
CRISPR-Cas Systems
*Chromosomes, Fungal/genetics
Haploidy
*Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
*DNA/genetics
*DNA, Fungal/genetics

@article {pmid40541508,
year = {2025},
author = {Woodward, MJ and Dallaire, A and Paszkowski, U and Kokkoris, V},
title = {Is genetic manipulation of arbuscular mycorrhizal fungi possible?.},
journal = {Trends in microbiology},
volume = {33},
number = {12},
pages = {1331-1343},
doi = {10.1016/j.tim.2025.06.002},
pmid = {40541508},
issn = {1878-4380},
mesh = {*Mycorrhizae/genetics ; CRISPR-Cas Systems ; Transformation, Genetic ; Gene Editing/methods ; Protoplasts ; },
abstract = {Unlike many fungi, arbuscular mycorrhizal (AM) fungi have proven recalcitrant to genetic manipulation due to their obligate biotrophic lifestyle and multinucleate, coenocytic cellular structure. In this review, we examine past attempts at AM fungal transformation, we identify key biological and technical barriers and explore recent advances to overcome them. We focus on techniques never before applied in AM fungi, including CRISPR/Cas9, microinjection, and protoplast-based transformation, and we explore how they provide viable strategies for achieving this elusive goal. We conclude by outlining guidelines for future research, distinguishing between established approaches that are readily applicable to AM fungi and others that first require addressing key outstanding questions in AM fungal cell biology and genetics to ensure success.},
}

*Mycorrhizae/genetics
CRISPR-Cas Systems
Transformation, Genetic
Gene Editing/methods
Protoplasts

@article {pmid41325824,
year = {2025},
author = {Uddin, N and Ullah, MW and Zhu, D and Li, X and Yang, S and Xin, X},
title = {Engineering lignin pathway and plant cell wall modification and genome editing for advanced renewable bioenergy and material applications.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108772},
doi = {10.1016/j.biotechadv.2025.108772},
pmid = {41325824},
issn = {1873-1899},
abstract = {Lignin biosynthesis and plant cell wall engineering are central to plant structural integrity and biomass utility. Recent advances in molecular and synthetic biology have opened opportunities to tailor lignin contents, composition, and polymer structure for renewable bioenergy and sustainable biomaterial applications. This review provides an integrative perspective on biosynthesis, regulation, and engineering of lignin. It summarizes the current progress in understanding the genetic, transcriptional, epigenetic, and metabolic networks that control lignin formation, with a focus on emerging tools such as CRISPR/Cas genome editing, synthetic promoters, and metabolic rewiring. Beyond cataloguing current knowledge, it critically analyzes the trade-offs involved in lignin modification for biomaterials, addressing unresolved challenges such as monolignol transport, metabolic flux control, and species-specific regulatory divergence. Engineered lignin and modified plant cell walls hold significant potential for biorefineries, advanced polymers, pharmaceuticals, and carbon sequestration, yet their translation from the laboratory to the field remains limited. Engineered lignin offers real-world applications across diverse industries, including bioenergy, bioplastics, carbon fiber composites, pharmaceuticals, and sustainable construction materials, thereby reinforcing its pivotal role in advancing a circular bioeconomy. The review further proposes future research directions that integrate multi-omics, single-cell technologies, machine learning, and field-based validation to enable precision lignin engineering. Strategic advances in this field will support next-generation bioenergy systems, advanced biomaterials, and the transition to a circular bioeconomy.},
}

@article {pmid41325351,
year = {2025},
author = {Jansen van Vuuren, J and Matthews, MC and Robène, I and Rozsasi, S and Campa, M and Burger, J and Viljoen, A and Mostert, D},
title = {Combined Recombinase Polymerase Amplification CRISPR/Cas12a Assay for Detecting Fusarium oxysporum f. sp. cubense Tropical Race 4.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {225},
pages = {},
doi = {10.3791/68841},
pmid = {41325351},
issn = {1940-087X},
mesh = {*Fusarium/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Recombinases/genetics/chemistry/metabolism ; *Nucleic Acid Amplification Techniques/methods ; Plant Diseases/microbiology ; DNA, Fungal/genetics/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Regular and accurate surveillance stands central to the efficient management of plant diseases. It can indicate which course of action is most appropriate, and whether prevention, eradication, or no action is required. Surveillance based on symptomology in host plants alone is often not reliable due to similarities in the symptoms caused by biotic and abiotic stresses. Laboratory-based molecular methods such as polymerase chain reaction (PCR) and quantitative (q)PCR are the most commonly and reliably used for plant pathogen detection, but rely on expensive equipment and skilled operators. Here, we describe a protocol combining a simplified DNA extraction, recombinase polymerase amplification (RPA), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a (RPA-Cas12a) for the detection of the invasive pathogen, Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4). The technique provides a simple single-tube detection alternative that is analytically robust with improved specificity compared to available molecular detection assays and negates the need for expensive and sophisticated laboratory equipment.},
}

*Fusarium/genetics/isolation & purification
*CRISPR-Cas Systems
*Recombinases/genetics/chemistry/metabolism
*Nucleic Acid Amplification Techniques/methods
Plant Diseases/microbiology
DNA, Fungal/genetics/analysis
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41324747,
year = {2025},
author = {Priyanka, SS and Iqbal, G and Nidarshan, NC and Kumari, K and Vanjre, S and Rasal, K and Sonwane, A and Brahmane, M and Goswami, M},
title = {Avenues of genome editing for color trait improvement in ornamental fishes: current status and future perspectives.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {51},
pmid = {41324747},
issn = {1573-9368},
mesh = {Animals ; *Gene Editing/methods ; *Fishes/genetics/growth & development ; *Pigmentation/genetics ; CRISPR-Cas Systems/genetics ; Animals, Genetically Modified/genetics/growth & development ; },
abstract = {Ornamental fish industries are growing sectors contributing significantly to livelihood, trade and export, driven by the worldwide demand for colourful and unique species. Pigmentation is the focal point of the visual appeal of ornamental fish, market value, and species-specific interaction. The pigment cells are called chromatophores, derived from neural crest cells and controlled by sophisticated genetic mechanisms, conferring these fish with distinctive colours and patterns. Historically, selective breeding and dietary pigment supplementation have been applied to enhance colouration. Such traditional practices, however, are prone to disadvantages such as slow development, genetic ambiguity, and unforeseen consequences. With the invention of genome editing, such as CRISPR-Cas9, researchers now have a sensitive and powerful tool to control pigmentation traits at the genetic level. Central pigmentation gene manipulation, such as Tyr, Mc1r, and Slc45a2, can allow researchers to introduce stable and uniform color changes. Such enhancements confer unparalleled control of fish colour, a promising avenue in the ornamental fish industry. This review discusses the genetic nature of fish pigmentation. It reports recent advances in CRISPR-based modifications and describes their possible applications and implications for future ornamental fish breeding.},
}

Animals
*Gene Editing/methods
*Fishes/genetics/growth & development
*Pigmentation/genetics
CRISPR-Cas Systems/genetics
Animals, Genetically Modified/genetics/growth & development

@article {pmid41324601,
year = {2025},
author = {Bhattacharya, S and Goyal, K and Satpati, P},
title = {Thermodynamics of PAM Recognition by Cas9 of Streptococcus pyogenes.},
journal = {Journal of chemical information and modeling},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jcim.5c01934},
pmid = {41324601},
issn = {1549-960X},
abstract = {The CRISPR/Cas9 system from Streptococcus pyogenes (SpCas9) requires a canonical 5'-NGG-3' PAM sequence in target DNA for effective genome editing. Base-specific interactions between the guanines (second and third position) and arginine dyad (R1333 and R1335) ensured specificity. We evaluated the PAM recognition strength of SpCas9 by using alchemical free energy calculations, revealing the energetics that influence genome editing accuracy. SpCas9 does not discriminate at the first position of the NGG sequence, but it penalizes mutations in the second and third positions. SpCas9 imposes a higher penalty for guanine mutation in the third PAM position compared to the second due to the greater conformational rigidity of R1335 in relation to R1333. Conformational rigidity of R1335 prevents side-chain readjustment for new protein-DNA interactions in noncanonical PAMs. A guanine-to-cytosine substitution in either the second or third position of canonical PAM disrupts direct protein-PAM interactions and leads to solvent exposure. This happens due to strong electrostatic repulsion between the arginine dyad's guanidinium groups and the amine group of cytosine. Interestingly, the strength of SpCas9 in disfavoring a single cytosine substitution (by >10 kcal/mol) is comparable to that of disfavoring double base substitutions in the NGG sequence. The ability of SpCas9 to differentiate between noncanonical and canonical PAMs (ΔΔG) is directly related to the number of direct interactions between SpCas9 and the PAM sequence, as well as the degree of solvent exposure. Loss of direct interactions and increased solvent exposure enhance ΔΔG. The calculated ΔΔG adequately explains the observed differences in DNA cleavage activity of SpCas9 across various DNA substrates with different PAM sequences. This study connects thermodynamics, structures, and activity to elucidate PAM selectivity in SpCas9 and may also apply to other CRISPR/Cas systems, offering valuable insights for the rational design of Cas9 variants with modified PAM specificities.},
}

@article {pmid41323291,
year = {2025},
author = {Li, Y and Hall-Ponselè, AM},
title = {Plant Cell Strain Improvement Through Engineering Biology for Industrial Plant Cell Culture.},
journal = {Engineering biology},
volume = {9},
number = {1},
pages = {e70002},
pmid = {41323291},
issn = {2398-6182},
abstract = {Plant cell culture (PCC) presents a promising and sustainable alternative to traditional agricultural methods for producing specialty bioactive compounds. However, its widespread industrial application has been hindered by challenges such as low yields, cell line instability and inconsistent product quality. engineering biology (EB) offers a powerful toolkit to overcome these limitations by systematically improving plant cell lines. This review focuses on the application of EB principles to enhance PCC for the production of high-value bioactives from an industry-oriented perspective. We explore three core pillars of the EB toolkit: (1) Multiomics and in silico design, which leverage comprehensive data integration and predictive modelling for rational target identification; (2) gene manipulation and pathway bioengineering, encompassing precise genome editing (e.g., CRISPR/Cas), synthetic gene circuits and directed evolution for targeted metabolic reprogramming and (3) biosensors for high-throughput screening and real-time monitoring, enabling rapid testing and optimisation of engineered cell lines. The synergistic integration of these tools within the iterative design-build-test-learn (DBTL) cycle is highlighted as a key strategy for accelerating strain improvement. Ultimately, the convergence of these EB approaches is transforming PCC into a robust platform for producing pharmaceuticals, functional foods and green chemicals, contributing to a biobased economy with a minimal ecological footprint.},
}

@article {pmid41321825,
year = {2025},
author = {Senthilraja, G and Sandhya, M and Priyadharshini, E and Anand, T and Kavitha, M and Tharmalingam, N},
title = {Targeting effector proteins of plant pathogens as a strategy for durable plant disease resistance.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1681047},
pmid = {41321825},
issn = {1664-302X},
}

@article {pmid41317788,
year = {2025},
author = {Brogan, DJ and Lin, CP and Benetta, ED and Wang, T and Chen, F and Li, H and Lin, C and Komives, EA and Akbari, OS},
title = {Synthetic type III-E CRISPR-Cas effectors for programmable RNA-targeting.},
journal = {Journal of molecular biology},
volume = {},
number = {},
pages = {169566},
doi = {10.1016/j.jmb.2025.169566},
pmid = {41317788},
issn = {1089-8638},
abstract = {The recent discovery of the type III-E class of CRISPR-Cas effectors has reshaped our fundamental understanding of CRISPR-Cas evolution and classification. Type III-E effectors are composed of several Cas7-like domains and a single Cas11-like domain naturally fused together to create a single polypeptide capable of targeting and degrading RNA. Here we identified a novel type III-E-like effector composed of three Cas7 domains and a Cas1 domain which was not active but could be engineered into an active chimeric RNA-targeting Cas effector by domain additions and swaps from other type III-E effectors. The results reveal that various domains in type III-E effectors can be swapped for the equivalent domain from a different type III-E effector. Remarkably, the Cas1 domain located at the C-terminus of Cas7-1 could be swapped in place of the Cas11 domain located between the Cas7.1 and the Cas7.2 domains of DiCas7-11. The results reveal a new modality for engineering type III-E effectors from the blueprints found in nature.},
}

@article {pmid41315365,
year = {2025},
author = {Wang, Y and Liao, Y and Sun, Y and Mitra, B and Guo, R and Piedras, BI and White, S and Tang, HY and Asara, JM and Tempera, I and Lieberman, PM and Gewurz, BE},
title = {The CTLH ubiquitin ligase substrates ZMYND19 and MKLN1 negatively regulate mTORC1 at the lysosomal membrane.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10731},
pmid = {41315365},
issn = {2041-1723},
support = {R01 AI164709/AI/NIAID NIH HHS/United States ; PF-24-1250090-01-IBCD//American Cancer Society (American Cancer Society, Inc.)/ ; P01CA269043//U.S. Department of Health & Human Services | NIH | NCI | Division of Cancer Epidemiology and Genetics, National Cancer Institute (National Cancer Institute Division of Cancer Epidemiology and Genetics)/ ; P01 CA269043/CA/NCI NIH HHS/United States ; R01AI164709//Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Division of Intramural Research of the NIAID)/ ; PF-23-1144614-01-IBCD//American Cancer Society (American Cancer Society, Inc.)/ ; PF-24-1194768-01-TBE//American Cancer Society (American Cancer Society, Inc.)/ ; PF-24-1308318-01-TBE//American Cancer Society (American Cancer Society, Inc.)/ ; R01 DE033907/DE/NIDCR NIH HHS/United States ; R00 DE031016/DE/NIDCR NIH HHS/United States ; },
mesh = {Humans ; *Lysosomes/metabolism ; *Mechanistic Target of Rapamycin Complex 1/metabolism/genetics ; *Ubiquitin-Protein Ligases/metabolism/genetics ; HEK293 Cells ; Cell Line, Tumor ; CRISPR-Cas Systems ; *Intracellular Membranes/metabolism ; Signal Transduction ; Phosphatidylinositol 3-Kinases/metabolism ; Cell Proliferation ; },
abstract = {Most Epstein-Barr virus-associated gastric carcinoma (EBVaGC) harbor non-silent mutations that activate phosphoinositide 3 kinase (PI3K) to drive downstream metabolic signaling. To gain insights into PI3K/mTOR pathway dysregulation in this context, we perform a human genome-wide CRISPR/Cas9 screen for hits that synergistically blocked EBVaGC proliferation together with the PI3K antagonist alpelisib. Multiple subunits of carboxy terminal to LisH (CTLH) E3 ligase, including the catalytic MAEA subunit, are among top screen hits. CTLH negatively regulates gluconeogenesis in yeast, but not in higher organisms. The CTLH substrates MKLN1 and ZMYND19, which highly accumulated upon MAEA knockout, associate with one another and with lysosome outer membranes to inhibit mTORC1. Rather than perturbing mTORC1 lysosomal recruitment, ZMYND19 and MKLN1 block the interaction between mTORC1 and Rheb and also with mTORC1 substrates S6 and 4E-BP1. Thus, CTLH enables cells to rapidly tune mTORC1 activity at the lysosomal membrane via the ubiquitin/proteasome pathway.},
}

Humans
*Lysosomes/metabolism
*Mechanistic Target of Rapamycin Complex 1/metabolism/genetics
*Ubiquitin-Protein Ligases/metabolism/genetics
HEK293 Cells
Cell Line, Tumor
CRISPR-Cas Systems
*Intracellular Membranes/metabolism
Signal Transduction
Phosphatidylinositol 3-Kinases/metabolism
Cell Proliferation

@article {pmid41261109,
year = {2025},
author = {Zhang, T and Zheng, Y and Sheng, M and Wang, Q and Jin, Z and Li, Z and Chen, W and Huang, J and Yang, X},
title = {Electrochemical Biosensing Platform Based on MOF Carrier Signal Probes and CRISPR/Cas12a Cleavage for Sensitive Detection of MicroRNAs.},
journal = {Analytical chemistry},
volume = {97},
number = {47},
pages = {26265-26273},
doi = {10.1021/acs.analchem.5c05821},
pmid = {41261109},
issn = {1520-6882},
mesh = {*MicroRNAs/analysis ; *Metal-Organic Frameworks/chemistry ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; Humans ; *CRISPR-Cas Systems ; DNA, Single-Stranded/chemistry ; *CRISPR-Associated Proteins/metabolism ; *Endodeoxyribonucleases/metabolism ; Methylene Blue/chemistry ; Limit of Detection ; Bacterial Proteins ; },
abstract = {Dysregulation of microRNA (miRNA) expression is associated with a variety of human diseases, including cancers, and the development of highly sensitive miRNA detection is important for the early diagnosis of cancer. In this study, we developed an electrochemical biosensing platform for miRNA detection through the integration of a DNA-gated metal-organic framework (MOF) signal probe, duplex-specific nuclease (DSN)-assisted signal amplification, and a CRISPR/Cas12a system. The zirconium-based MOF UiO-66-NH2 was engineered as a nanocarrier for methylene blue (MB) entrapment, and single-stranded DNA (ssDNA) was capped on the MOF surface as a "gatekeeper" that allowed the controlled release of MB molecules, resulting in a DNA-gated MOF electrochemical signal probe. In the presence of target miRNA, target-initiated DSN cyclic amplification and digestion of the CRISPR/Cas12a initiator DNA sequence blocked the collateral cleavage activity of Cas12a toward ssDNA, thus preventing the release of MB and generating a high electrochemical signal. Conversely, the CRISPR/Cas12a system would be activated to cleave ssDNA in the absence of the target, leading to the release of a signal molecule and a low response. With the usage of the proposed biosensing strategy, sensitive detection of miRNA let-7a, a biomarker associated with nonsmall cell lung cancer, has been achieved. Therefore, this work expands the application scope of a MOF as a nanocarrier to prepare electrochemical signal probes and provides a valuable biosensing method for clinical diagnosis.},
}

*MicroRNAs/analysis
*Metal-Organic Frameworks/chemistry
*Biosensing Techniques/methods
*Electrochemical Techniques/methods
Humans
*CRISPR-Cas Systems
DNA, Single-Stranded/chemistry
*CRISPR-Associated Proteins/metabolism
*Endodeoxyribonucleases/metabolism
Methylene Blue/chemistry
Limit of Detection
Bacterial Proteins