@article {pmid40451196,
year = {2025},
author = {Smirnov, AV and Yunusova, AM},
title = {Novel CRISPR/Cas9-Based Approaches for Quantitative Study of DSB Repair Mechanics.},
journal = {Biochemistry. Biokhimiia},
volume = {90},
number = {4},
pages = {437-456},
doi = {10.1134/S0006297924601813},
pmid = {40451196},
issn = {1608-3040},
mesh = {*CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; *DNA Repair ; Humans ; Animals ; },
abstract = {This review examines modern approaches to studying double-strand break (DSB) DNA repair in mammalian cells, employing the CRISPR/Cas9 system. Due to its flexibility and efficacy, the Cas9 nuclease is used in numerous genetic reporters. We discuss various fluorescence-based genetic reporters used to monitor the repair process. In addition, among the innovative Cas9-based methods, special attention is given to the techniques that examine both single and multiple DSBs, including approaches such as DSB-TRIP and ddXR. These methods open new possibilities for investigating structural rearrangements or analyzing random genomic sites. Additionally, the review considers how DSBs induced by Cas9 differ from those made by other nucleases and how these peculiarities could impact DNA repair mechanisms. Understanding these differences is crucial for planning experiments aimed at studying DSB repair.},
}

*CRISPR-Cas Systems
*DNA Breaks, Double-Stranded
*DNA Repair
Humans
Animals

@article {pmid40451118,
year = {2025},
author = {Piñeiro-Silva, C and Gadea, J},
title = {Optimizing gene editing in pigs: The role of electroporation and lipofection.},
journal = {Animal reproduction science},
volume = {278},
number = {},
pages = {107874},
doi = {10.1016/j.anireprosci.2025.107874},
pmid = {40451118},
issn = {1873-2232},
abstract = {The production of genetically modified pigs is becoming increasingly important in both the agricultural and biomedical fields. Optimization of these processes is a key objective to improve the precision, scalability and viability of genetically modified animals for research and commercial applications. Among the available techniques, electroporation and lipofection have emerged as promising alternatives to traditional methods such as microinjection and somatic cell nuclear transfer (SCNT) due to their simplicity, cost-effectiveness, and potential for high-throughput applications. These methods allow the direct delivery of CRISPR/Cas components into zygotes and embryos, reducing the technical expertise required and bypassing some of the challenges associated with cloning. This review examines the application, efficacy, and outcomes of electroporation and lipofection as gene editing techniques in porcine gametes and embryos. We provide a comprehensive synthesis of recent advances, compare their efficacy, and discuss their potential in agricultural and biomedical research. The principles and mechanisms of both methods are reviewed, highlighting their advantages, such as cost-effectiveness and ease of implementation, over traditional approaches such as microinjection. In addition, we address their limitations, including variability in efficiency, and discuss recent protocol optimizations aimed at improving reproducibility and applicability. By analyzing these developments, this review provides valuable insights into the evolving role of electroporation and lipofection in porcine genetic modification strategies.},
}

@article {pmid40450609,
year = {2025},
author = {Pal, S and Krishna, R and Dedhia, L and Panwar, HS and Karkute, SG and Rai, N and Kumar, R and Pandey, S and Singh, AK},
title = {CRISPR mediated gene editing for economically important traits in horticultural crops: progress and prospects.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {26},
pmid = {40450609},
issn = {1573-9368},
mesh = {*Gene Editing/methods ; *Crops, Agricultural/genetics/growth & development ; *CRISPR-Cas Systems/genetics ; *Plants, Genetically Modified/genetics/growth & development ; Plant Breeding ; Genome, Plant ; },
abstract = {Horticultural crops, with their cost-effectiveness, rich mineral and vitamin content, and high yield potential, have become indispensable worldwide for ensuring food and nutritional security. With the world's population on the rise and climate change becoming more prominent, it is crucial to focus on creating resilient, high-yielding crop varieties that can withstand the changing climate. Genetic improvement of different horticultural crops using conventional tools is both time-consuming and labourious. However, the breeding period can be cut short by adopting modern breeding techniques, including CRISPR/Cas-mediated genome editing. In the present review, we discuss the progress made so far through genome editing to improve several horticultural crops for various traits like stress resistance, morphology, nutritional attributes, quality, shelf life, male sterility, architecture and economic yield. We have also discussed the emerging CRISPR technologies like base editing, epigenome editing, CRE editing, transposon-based editing, prime editing etc., along with their pros and cons and the future prospects. The ethical considerations for commercialization and current regulatory frameworks for gene-edited products have also been discussed.},
}

*Gene Editing/methods
*Crops, Agricultural/genetics/growth & development
*CRISPR-Cas Systems/genetics
*Plants, Genetically Modified/genetics/growth & development
Plant Breeding
Genome, Plant

@article {pmid40450056,
year = {2025},
author = {Ortiz-Rodríguez, LA and Cabanzo, R and Jaimes-Dueñez, J and Mendez-Sanchez, SC and Duque, JE},
title = {TropD-detector a CRISPR/LbCas12a-based system for rapid screening of Trypanosoma cruzi in Chagas vectors and reservoirs.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {19107},
pmid = {40450056},
issn = {2045-2322},
mesh = {*Trypanosoma cruzi/genetics/isolation & purification ; *Chagas Disease/parasitology/diagnosis ; Animals ; *CRISPR-Cas Systems ; *Disease Reservoirs/parasitology ; Rhodnius/parasitology ; Cytochromes b/genetics ; Endodeoxyribonucleases/genetics ; Histones/genetics ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; },
abstract = {Chagas disease, also known as American Trypanosomiasis, is a zoonosis with global distribution caused by the parasite Trypanosoma cruzi, primarily transmitted through the feces of infected triatomines. The emergence of new cases highlights the importance of early pathogen detection in vectors and reservoirs to generate effective control strategies and establish preventive policies. The objective of this study was to design and validate a detection system of T. cruzi based on specific DNA cleavage, activation of Cas12a and trans-cleavage, targeting the genes Cytochrome B (Cytb), 18 S ribosomal subunit (SR18 s), and histone (H2 A). This system was validated for their uses in both vectors and reservoirs of the parasite. The initial step involved performing a bioinformatic analysis of the target genes, followed by the design of RNA guides specific to each cleavage site, along with primers for amplifying the target region through PCR and RPA. Subsequently, we sequenced the amplified DNA target and validated the detection system using T. cruzi DNA extracted from naturally infected Rhodnius pallescens in the metropolitan area of Bucaramanga, Colombia. After standardizing the method, we tested the CRISPR/Cas system with Silvio X10 laboratory strain of T. cruzi and scaled up to blood samples of naturally infected Didelphis marsupialis. As a result, we observed DNA cleavage using the CRISPR/Cas system with the Cytb guide, achieving a detection sensitivity of 118 parasite equivalents/mL in PCR and 116 parasite equivalents/mL with RPA amplification. Sequencing of the Cytb gene showed no mutations in the cleavage site. However, point mutations and indels were found in SR18S and H2 A, avoiding the formation of the CRISPR/LbCas12 complex. Furthermore, we introduce the design of a fluorescent detection prototype with CRISPR/LbCas12a called "Tropical Diseases Detector" (TropD-Detector). This device operates with an excitation wavelength of 480 nm emitted by an LED and a high-pass light filter with a cutoff wavelength of 500 nm. We detected positive samples using any photographic camera system. The TropD-Detector provides a visual, viable, and sensitive method for detecting T. cruzi in both vectors and reservoirs from endemic areas.},
}

*Trypanosoma cruzi/genetics/isolation & purification
*Chagas Disease/parasitology/diagnosis
Animals
*CRISPR-Cas Systems
*Disease Reservoirs/parasitology
Rhodnius/parasitology
Cytochromes b/genetics
Endodeoxyribonucleases/genetics
Histones/genetics
CRISPR-Associated Proteins/genetics
Bacterial Proteins

@article {pmid40449999,
year = {2025},
author = {Schweitzer, AY and Adams, EW and Nguyen, MTA and Lek, M and Isaacs, FJ},
title = {Precision multiplexed base editing in human cells using Cas12a-derived base editors.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5061},
pmid = {40449999},
issn = {2041-1723},
support = {R01 GM117230/GM/NIGMS NIH HHS/United States ; GM117230//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; CF22-1046//Carlsbergfondet (Carlsberg Foundation)/ ; },
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/metabolism/genetics ; Genome, Human ; HEK293 Cells ; Cell Line ; Bacterial Proteins ; },
abstract = {Base editors enable the direct conversion of target nucleotides without introducing DNA double strand breaks, making them a powerful tool for creating point mutations in a human genome. However, current Cas9-derived base editing technologies have limited ability to simultaneously edit multiple loci with base-pair level precision, hindering the generation of polygenic phenotypes. Here, we test the ability of six Cas12a-derived base editing systems to process multiple gRNAs from a single transcript. We identify base editor variants capable of multiplexed base editing and improve the design of the respective gRNA array expression cassette, enabling multiplexed editing of 15 target sites in multiple human cell lines, increasing state-of-the-art in multiplexing by three-fold in the field of mammalian genome engineering. To reduce bystander mutations, we also develop a Cas12a gRNA engineering approach that directs editing outcomes towards a single base-pair conversion. We combine these advances to demonstrate that both strategies can be combined to drive multiplex base editing with greater precision and reduced bystander mutation rates. Overcoming these key obstacles of mammalian genome engineering technologies will be critical for their use in studying single nucleotide variant-associated diseases and engineering synthetic mammalian genomes.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*CRISPR-Associated Proteins/genetics/metabolism
*Endodeoxyribonucleases/metabolism/genetics
Genome, Human
HEK293 Cells
Cell Line
Bacterial Proteins

@article {pmid40449852,
year = {2025},
author = {Kolesova, E and Pulone, S and Kostyushev, D and Tasciotti, E},
title = {CRISPR/Cas bioimaging: From whole body biodistribution to single-cell dynamics.},
journal = {Advanced drug delivery reviews},
volume = {},
number = {},
pages = {115619},
doi = {10.1016/j.addr.2025.115619},
pmid = {40449852},
issn = {1872-8294},
abstract = {This review explores the transformative role of CRISPR/Cas systems in optical bioimaging, emphasizing how advancements in nanoparticle (NP) technologies are revolutionizing the visualization of gene-editing processes both in vitro and in vivo. Optical imaging techniques, such as near-infrared (NIR) and fluorescence imaging, have greatly benefited from the integration of nanoformulated contrast agents, improving resolution, sensitivity, and specificity. CRISPR/Cas systems, originally developed just for gene editing, are now being coupled with these imaging modalities to enable real-time monitoring and quantitative measurements of metabolites, vitamins, proteins, nucleic acids and other entities in specific areas of the body, as well as tracking of CRISPR/Cas delivery, editing efficiency, and potential off-target effects. The development of CRISPR/Cas-loaded NPs allows for enhanced imaging and precise monitoring across multiple scales with multiplexed and multicolor imaging in complex settings, including potential in vivo diagnostics. CRISPR/Cas therapeutics as well as diagnostics are hindered by the lack of efficient and targeted delivery tools. Biomimetic NPs have emerged as promising tools for improving biocompatibility, enhancing targeting capabilities, and overcoming biological barriers, facilitating more efficient delivery and bioimaging of CRISPR/Cas systems in vivo. As the design of these NPs and delivery mechanisms improves, alongside advancements in endolysosomal escape, CRISPR/Cas-based bioimaging will continue to advance, offering unprecedented possibilities in precision medicine and theranostic applications.},
}

@article {pmid40448764,
year = {2025},
author = {Jin, H and Sophocleous, A and Azfer, A and Ralston, SH},
title = {Analysis of Transcriptional Regulation in Bone Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2885},
number = {},
pages = {247-269},
pmid = {40448764},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems ; *Transcription, Genetic ; Promoter Regions, Genetic ; *Gene Expression Regulation ; Chromatin Immunoprecipitation/methods ; Humans ; Electrophoretic Mobility Shift Assay/methods ; Animals ; *Bone and Bones/cytology/metabolism ; *Osteoblasts/metabolism ; Mice ; Genes, Reporter ; },
abstract = {Transcription is a process by which the rate of RNA synthesis is regulated. Here we describe the techniques for carrying out promoter-reporter assays: electrophoretic mobility shift assays, chromosome conformation capture (3C) assays, chromatin immunoprecipitation assays, and CRISPR-Cas9 assay-five commonly used methods for studying and altering gene transcription.},
}

CRISPR-Cas Systems
*Transcription, Genetic
Promoter Regions, Genetic
*Gene Expression Regulation
Chromatin Immunoprecipitation/methods
Humans
Electrophoretic Mobility Shift Assay/methods
Animals
*Bone and Bones/cytology/metabolism
*Osteoblasts/metabolism
Mice
Genes, Reporter

@article {pmid40448746,
year = {2025},
author = {Kamli, H and Khan, NU},
title = {Revolutionising cancer intervention: the repercussions of CAR-T cell therapy on modern oncology practices.},
journal = {Medical oncology (Northwood, London, England)},
volume = {42},
number = {7},
pages = {228},
pmid = {40448746},
issn = {1559-131X},
mesh = {Humans ; *Neoplasms/therapy/immunology ; *Immunotherapy, Adoptive/methods ; *Receptors, Chimeric Antigen/immunology ; Gene Editing/methods ; Tumor Microenvironment/immunology ; CRISPR-Cas Systems ; Medical Oncology/methods/trends ; },
abstract = {Chimeric Antigen Receptor T-cell (CAR-T) therapy represents a groundbreaking advance in oncology, leveraging patient-specific immune cells to target malignant tumours precisely. By equipping T cells with synthetic receptors, CAR-T therapy achieves remarkable antitumor effects and offers hope for durable cancer control. However, several limitations persist, including antigen scarcity, immunosuppressive tumour microenvironments, and T-cell exhaustion. CRISPR-Cas9 gene editing has enhanced CAR-T potency by knocking out immune checkpoints (PD-1, CTLA-4) and improving persistence, while RNA interference (RNAi) silences immune-evasion genes (e.g. SOCS1). Nanozyme-based delivery systems enable precise CRISPR-Cas9 delivery (> 70% editing efficiency) and tumour targeting, overcoming instability and off-target effects. Innovations like SUPRA CARs, armoured CAR-T cells (e.g. IL-12/IL-21-secreting TRUCKs), and dual checkpoint inhibition synergize to reprogram the tumour microenvironment, reducing relapse by 40% in trials. Despite progress, high costs, manufacturing hurdles, and ethical concerns (e.g. germline editing risks) remain critical barriers. Emerging solutions include universal off-the-shelf CAR-Ts, hybrid nano-CRISPR systems, and AI-driven design, paving the way for scalable, personalised immunotherapy. This review highlights breakthroughs in CRISPR, RNAi, and nanotechnology, underscoring CAR-T therapy's transformative potential while addressing translational challenges for broader clinical adoption.},
}

Humans
*Neoplasms/therapy/immunology
*Immunotherapy, Adoptive/methods
*Receptors, Chimeric Antigen/immunology
Gene Editing/methods
Tumor Microenvironment/immunology
CRISPR-Cas Systems
Medical Oncology/methods/trends

@article {pmid40447846,
year = {2025},
author = {Blaskovich, MAT and Cooper, MA},
title = {Antibiotics re-booted-time to kick back against drug resistance.},
journal = {npj antimicrobials and resistance},
volume = {3},
number = {1},
pages = {47},
pmid = {40447846},
issn = {2731-8745},
abstract = {After decades of neglect and a decline in antibiotic research and development, we are now finally witnessing the advent of new funding programs dedicated to new therapies. In addition to traditional new chemical entities that directly kill or arrest the growth of bacteria, alternative approaches are being identified and advanced towards proof-of-concept trials in the clinic. We briefly review the current pipeline of conventional new antibiotics and highlight in more depth promising alternatives, including potentiators of antibiotic action, bacteriophage, lysins and microbiome modulation. More innovative approaches, such as adaptive and innate immune modulators, CRISPR-Cas and diagnostic guided 'theranostics' are discussed and contrasted. Such exploratory therapies may require the development of alternative regulatory and clinical development pathways, but represent a potential circuit breaker from the current 'arms race' between bacteria and traditional antibiotics.},
}

@article {pmid40447637,
year = {2025},
author = {Schwaemmle, H and Soldati, H and Lykoskoufis, NMR and Docquier, M and Hainard, A and Braun, SMG},
title = {CRISPR screen decodes SWI/SNF chromatin remodeling complex assembly.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5011},
pmid = {40447637},
issn = {2041-1723},
support = {PCEFP3_194305//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; },
mesh = {Animals ; Mice ; *Chromatin Assembly and Disassembly/genetics ; *Chromosomal Proteins, Non-Histone/metabolism/genetics ; *Transcription Factors/metabolism/genetics ; Chromatin/metabolism/genetics ; RNA-Binding Proteins/metabolism/genetics ; Mouse Embryonic Stem Cells/metabolism ; *CRISPR-Cas Systems ; Humans ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The SWI/SNF (or BAF) complex is an essential chromatin remodeler, which is frequently mutated in cancer and neurodevelopmental disorders. These are often heterozygous loss-of-function mutations, indicating a dosage-sensitive role for SWI/SNF subunits. However, the molecular mechanisms regulating SWI/SNF subunit dosage to ensure complex assembly remain largely unexplored. We performed a CRISPR KO screen, using epigenome editing in mouse embryonic stem cells, and identified Mlf2 and Rbm15 as regulators of SWI/SNF complex activity. First, we show that MLF2, a poorly characterized chaperone protein, promotes SWI/SNF assembly and binding to chromatin. Rapid degradation of MLF2 reduces chromatin accessibility at sites that depend on high levels of SWI/SNF binding to maintain open chromatin. Next, we find that RBM15, part of the m[6]A writer complex, controls m[6]A modifications on specific SWI/SNF mRNAs to regulate subunit protein levels. Misregulation of m[6]A methylation causes overexpression of core SWI/SNF subunits leading to the assembly of incomplete complexes lacking the catalytic ATPase/ARP subunits. These data indicate that targeting modulators of SWI/SNF complex assembly may offer a potent therapeutic strategy for diseases associated with impaired chromatin remodeling.},
}

Animals
Mice
*Chromatin Assembly and Disassembly/genetics
*Chromosomal Proteins, Non-Histone/metabolism/genetics
*Transcription Factors/metabolism/genetics
Chromatin/metabolism/genetics
RNA-Binding Proteins/metabolism/genetics
Mouse Embryonic Stem Cells/metabolism
*CRISPR-Cas Systems
Humans
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40446748,
year = {2025},
author = {Zuo, Z and Liang, R and Fan, S and Shan, H and Zhang, H and Wang, X and Fei, T},
title = {Genome-wide CRISPR screens identify key regulators of adipogenesis and glucose uptake in beige adipocytes.},
journal = {Biochemical and biophysical research communications},
volume = {774},
number = {},
pages = {152093},
doi = {10.1016/j.bbrc.2025.152093},
pmid = {40446748},
issn = {1090-2104},
mesh = {*Adipogenesis/genetics ; *Glucose/metabolism ; *Adipocytes, Beige/metabolism/cytology ; Animals ; Humans ; Mice ; *CRISPR-Cas Systems ; Insulin Resistance ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {The beiging of white adipocytes enhances energy expenditure by utilizing fatty acids and glucose, offering therapeutic potential against obesity and type 2 diabetes. However, the genetic mechanisms driving this process remain unclear. Here, we performed multiple fluorescence-activated cell sorting (FACS)-based genome-wide CRISPR loss-of-function screens in beige adipocytes with or without insulin resistance (IR) induction, and systematically identified functional regulators of beige adipocyte adipogenesis and glucose metabolism. We further integrated transcriptomics and human genetics data to pinpoint key genes for adipogenesis and glucose metabolism in beige adipocytes. Moreover, we validated SULT2B1 and ATP1B2 as key adipogenesis genes for beige adipocytes, and COMMD7 gene as important regulator for glucose uptake against IR. These findings not only provide a comprehensive and valuable resource for cataloguing candidate functional genes underlying lipid and glucose homeostasis in beige adipocytes, but also offer potential therapeutic targets against metabolic disorders.},
}

*Adipogenesis/genetics
*Glucose/metabolism
*Adipocytes, Beige/metabolism/cytology
Animals
Humans
Mice
*CRISPR-Cas Systems
Insulin Resistance
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid40434364,
year = {2025},
author = {Hecht, AD and Igoshin, OA},
title = {Kinetic Mechanism for Fidelity of CRISPR-Cas9 Variants.},
journal = {The journal of physical chemistry letters},
volume = {16},
number = {22},
pages = {5570-5578},
doi = {10.1021/acs.jpclett.5c01154},
pmid = {40434364},
issn = {1948-7185},
mesh = {Kinetics ; *CRISPR-Cas Systems ; Fluorescence Resonance Energy Transfer ; Substrate Specificity ; *CRISPR-Associated Protein 9/metabolism/chemistry/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; DNA/chemistry/metabolism ; },
abstract = {CRISPR-Cas9 is a nuclease creating DNA breaks at sites with sufficient complementarity to the RNA guide. Notably, Cas9 does not require exact RNA-DNA complementarity and can cleave off-target sequences. Various high-accuracy Cas9 variants have been developed, but the precise mechanism of how these variants achieve higher accuracy remains unclear. Here, we develop a kinetic model of Cas9 substrate selection and cleavage parametrized by data from the literature, including single-molecule Förster resonance energy transfer (FRET) measurements. Based on observed FRET transition statistics, we predict that the Cas9 substrate recognition and cleavage mechanism must allow for HNH domain transitions independent of substrate binding. Additionally, we show that the enhancement in Cas9 substrate specificity must be due to changes in kinetics rather than changes in substrate binding affinities. Finally, we use our model to identify kinetic parameters for HNH domain transitions that can be perturbed to enable high-accuracy cleavage while maintaining cleavage speeds.},
}

Kinetics
*CRISPR-Cas Systems
Fluorescence Resonance Energy Transfer
Substrate Specificity
*CRISPR-Associated Protein 9/metabolism/chemistry/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
DNA/chemistry/metabolism

@article {pmid40403622,
year = {2025},
author = {Choi, H and Yi, TG and Gho, YS and Kim, JH and Kim, S and Choi, YJ and Lim, S and Eom, SH and Jung, KH and Ha, SH},
title = {Augmenting carotenoid accumulation by multiplex genome editing of the redundant CCD family in rice.},
journal = {Plant physiology and biochemistry : PPB},
volume = {225},
number = {},
pages = {110008},
doi = {10.1016/j.plaphy.2025.110008},
pmid = {40403622},
issn = {1873-2690},
mesh = {*Oryza/genetics/metabolism ; *Gene Editing/methods ; *Carotenoids/metabolism ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; *Dioxygenases/genetics/metabolism ; Gene Knockout Techniques ; Plants, Genetically Modified ; },
abstract = {The biodegradation of carotenoid is carried out and regulated by a family of carotenoid cleavage dioxygenases (CCDs). In rice, potential redundancy of OsCCD1, OsCCD4a, and OsCCD4b, among multiple CCDs, was predicted through in silico protein-ligand docking simulations, which were based on the interactions of diverse carotene and xanthophyll substrates in the active sites. To elucidate the roles of the three CCDs in planta, we generated single, double, and triple knockout (KO) rice lines using CRISPR-Cas9 technology and confirmed their genetic stabilities. Triple KO (osccd1/osccd4a/osccd4b) lines exhibited enhanced carotenoid contents notably in both leaves after the harvesting stage and under dark-induced senescence, verifying that was caused by a simultaneously successful blocking of OsCCD1/OsCCD4a/OsCCD4b activities in these conditions. Transcriptional profiling of this triple KO line revealed downregulation of key genes involved in carotenoid biosynthesis, suggesting a feedback mechanism to regulate carotenoid levels. In order to explore and increase practical agricultural use of this triple KO line, we performed agronomic assessments that showed no adverse effects on major traits such as photosynthetic rate and seed productivity and then developed a T-DNA-free version of this triple KO rice line with high carotenoid content. Our study highlights the capacity of OsCCD1/OsCCD4a/OsCCD4b as promising targets for genome editing in biofortification strategies aimed at improving the functionality of rice and suggests their applicability to other forage crops.},
}

*Oryza/genetics/metabolism
*Gene Editing/methods
*Carotenoids/metabolism
*Plant Proteins/genetics/metabolism
Gene Expression Regulation, Plant
CRISPR-Cas Systems
*Dioxygenases/genetics/metabolism
Gene Knockout Techniques
Plants, Genetically Modified

@article {pmid40403614,
year = {2025},
author = {Dong, H and Qin, Y and Zhang, P and Lv, L and Yu, C and Jia, P and Zhao, J and Du, F and Guo, Y and Sun, X},
title = {"Turn-on" aptamer-immune lateral flow assays for the detection of small molecule targets based on CHA-assisted and CRISPR/Cas12a mediated signal transduction and amplification.},
journal = {Biosensors & bioelectronics},
volume = {285},
number = {},
pages = {117593},
doi = {10.1016/j.bios.2025.117593},
pmid = {40403614},
issn = {1873-4235},
mesh = {*Aptamers, Nucleotide/chemistry ; CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Limit of Detection ; Signal Transduction ; },
abstract = {Lateral flow assays (LFAs) have emerged as crucial tools for on-site food safety detection due to their simple operation and intuitive detection results. Nevertheless, LFAs for small molecule targets such as pesticides often present a "Turn-off" signal output, which leads to their low sensitivity and the risk of false positives. In this study, a CRISPR/Cas12a system-mediated strategy was employed to convert aptamer signals into the signals of immune LFAs, achieving a "Turn-on" signal output for highly sensitive detection of small molecule targets. The binding of aptamers to targets released the trigger sequence to initiate the catalytic hairpin assembly (CHA) reaction, generating double-stranded DNA, which subsequently activated the CRISPR/Cas12a system to cleave the FAM-labeled Reporter. Eventually, the "Turn-on" visual output of the signal was realized through an anti-6-FAM immune LFAs. The experiment optimized the sample pool preparation, CHA reaction conditions, CRISPR/Cas12a activation parameters, and the assembly process of the LFAs. The limit of detection for procymidone was as low as 0.015 ng/mL, which was 52.67 times more sensitive than those of conventional aptamer-based LFAs without signal amplification strategies. This method exhibits high specificity for procymidone and a recovery rate ranging from 94.00% to 104.20% in vegetable samples, demonstrating excellent stability and practicability.},
}

*Aptamers, Nucleotide/chemistry
CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
Limit of Detection
Signal Transduction

@article {pmid40403612,
year = {2025},
author = {Lin, L and Xue, Y and Tan, L and Jiang, C and Liu, M and Li, X and Qiu, J and Zhang, H and Zhou, J and Shu, B},
title = {Micro-scale thermofluidics enable autonomous and scalable CRISPR diagnostics for sexually transmitted infections screening.},
journal = {Biosensors & bioelectronics},
volume = {285},
number = {},
pages = {117591},
doi = {10.1016/j.bios.2025.117591},
pmid = {40403612},
issn = {1873-4235},
mesh = {Humans ; *Biosensing Techniques/instrumentation ; *Sexually Transmitted Diseases/diagnosis/microbiology ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Nucleic Acid Amplification Techniques/instrumentation ; Equipment Design ; CRISPR-Cas Systems ; Lab-On-A-Chip Devices ; },
abstract = {The development of clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid detection has recently been a center of interest for next-generation molecular diagnostics. Despite considerable advances, simple and effective strategies to harness the isothermal amplification reaction and CRISPR-based detection for maximal performance and minimal complexity are still desirable. Here, a thermofluidic approach leverages the micro-scale chemical and physical mechanism to perform autonomous and scalable CRISPR-based diagnostics (CRISPR-Dx) in a greatly simplified format, which was called "Thermofluidic CRISPR". Originating from the concept of convective PCR, it utilizes looped microchannel reactors to perform approximatively undisturbed isothermal amplification reaction at balanced temperature by virtue of the restricted molecular diffusion across the microchannel, in which the reagents of two reactions are compartmentalized virtually; then it creates circulatory flow within the loop channel to mix the amplificons and CRISPR reagents via Rayleigh-Bénard thermal convection, by simply warming up one side of the loop channel. Due to the simplicity and scalability, a low-cost, battery-powered, portable diagnostic platform, incorporating with smartphone-enabled real-time fluorescence readout, to perform rapid (<30 min), highly sensitive (2 copies per reaction), quantitative and multiplexed CRISPR-Dx was constructed. Its diagnostic performance in rapid screening of multiple pathogens from 196 clinical samples for syndromic testing of sexually transmitted infections was evaluated, exhibiting 97.4 % sensitivity and 100 % specificity benchmarked against the laboratory-based testing. Leveraging the micro-scale chemical and physical mechanism to simplify workflows for CRISPR-Dx may enhance their versatility and facilitate their broader applicability at the point of care.},
}

Humans
*Biosensing Techniques/instrumentation
*Sexually Transmitted Diseases/diagnosis/microbiology
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Nucleic Acid Amplification Techniques/instrumentation
Equipment Design
CRISPR-Cas Systems
Lab-On-A-Chip Devices

@article {pmid40398216,
year = {2025},
author = {Huang, Y and Li, H and Qi, L and Wang, Z and Liu, Z and Wu, R and Chen, Q and Zhu, C and Sun, D and Liu, L and Zhang, L and Feng, G},
title = {NanoCRISPR-assisted biomimetic tissue-equivalent patch regenerates the intervertebral disc by inhibiting endothelial-to-mesenchymal transition.},
journal = {Biomaterials},
volume = {322},
number = {},
pages = {123404},
doi = {10.1016/j.biomaterials.2025.123404},
pmid = {40398216},
issn = {1878-5905},
mesh = {Animals ; Humans ; *Intervertebral Disc/physiology ; *Regeneration ; Intervertebral Disc Degeneration/therapy/pathology ; Tissue Scaffolds/chemistry ; Vascular Endothelial Growth Factor A/metabolism ; *Biomimetic Materials/chemistry/pharmacology ; *Nanoparticles/chemistry ; CRISPR-Cas Systems ; Annulus Fibrosus ; Indoles/chemistry ; Polymers/chemistry ; },
abstract = {The integrity of the intervertebral disc (IVD), an immune-privileged organ protected by the blood-disc barrier, is compromised following annulus fibrosus (AF) injury. This breach facilitates angiogenesis, immune cell infiltration, and inflammation, accelerating intervertebral disc degeneration (IDD) and resulting in various clinical disorders. Current treatments fail to adequately address biological repair of AF defects and angiogenesis. Single-cell RNA sequencing analyses reveal that vascular endothelial growth factor (VEGF), secreted by IDD-associated fibrochondrocytes, is crucial in promoting angiogenesis by inducing endothelial-to-mesenchymal transition (EndoMT). This study proposes a nano-clustered regularly interspaced short palindromic repeats (CRISPR)-assisted AF patch with an aligned, polydopamine-modified nano-lamellae nanofibrous scaffold that replicates the hierarchical structure of natural AF, providing a conducive microenvironment for AF repair. A zeolitic imidazolate framework-8-based nanoCRISPR system encapsulates the CRISPR/CRISPR-associated protein 9 complex to target and eliminate VEGF-mediated angiogenic factors. In vitro studies demonstrate that the nanoCRISPR-assisted patch can enhance AF cell adhesion and migration, promote extracellular matrix deposition, knock out VEGF expression, and inhibit EndoMT. In vivo studies show its significant efficacy in promoting AF repair, inhibiting abnormal angiogenesis, and delaying IDD progression. This study presents a promising approach for structural and biological AF regeneration, addressing physical and angiogenic barriers in IVD regeneration.},
}

Animals
Humans
*Intervertebral Disc/physiology
*Regeneration
Intervertebral Disc Degeneration/therapy/pathology
Tissue Scaffolds/chemistry
Vascular Endothelial Growth Factor A/metabolism
*Biomimetic Materials/chemistry/pharmacology
*Nanoparticles/chemistry
CRISPR-Cas Systems
Annulus Fibrosus
Indoles/chemistry
Polymers/chemistry

@article {pmid40378589,
year = {2025},
author = {Chen, Q and Wang, H and Xu, H and Peng, Y and Yao, B and Chen, Z and Yang, J and Adeloju, S and Chen, W},
title = {One-pot RPA-CRISPR/Cas12a integrated dual-mode electrochemical lateral flow strip for ultrasensitive and precise detection of Salmonella.},
journal = {Biosensors & bioelectronics},
volume = {285},
number = {},
pages = {117529},
doi = {10.1016/j.bios.2025.117529},
pmid = {40378589},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *Salmonella/isolation & purification/genetics ; *Electrochemical Techniques/methods ; Limit of Detection ; CRISPR-Cas Systems/genetics ; Food Microbiology ; Equipment Design ; Salmonella Infections/microbiology/diagnosis ; Humans ; },
abstract = {Rapid and accurate screening of pathogenic contamination is essential for timely intervention and infection prevention. In this work, one-pot RPA-CRISPR/Cas12a strategy combined with an innovative electrochemical lateral flow strip (OPRCC-eLFS) was presented for ultrasensitive and precise detection of Salmonella. Highly sensitive dual-mode detection of Salmonella in various samples has been simultaneously achieved with electrochemical detection limit of 3.84 CFU/mL and visual detection limit of 384 CFU/mL, respectively, with improved detection efficiency and prevention of uncapping-related aerosol-contamination. This dual-mode biosensing platform demonstrates exceptional stability, remarkable sensitivity, and robust on-site quantification capability, emphasizing its potential in food safety monitoring and disease prevention.},
}

*Biosensing Techniques/methods
*Salmonella/isolation & purification/genetics
*Electrochemical Techniques/methods
Limit of Detection
CRISPR-Cas Systems/genetics
Food Microbiology
Equipment Design
Salmonella Infections/microbiology/diagnosis
Humans

@article {pmid40359875,
year = {2025},
author = {Morán Torres, JP and Lyu, J and Chen, X and Klaas, AM and Vonk, PJ and Lugones, LG and de Cock, H and Wösten, HAB},
title = {Single and combinatorial gene inactivation in Aspergillus niger using selected as well as genome-wide gRNA library pools.},
journal = {Microbiological research},
volume = {298},
number = {},
pages = {128204},
doi = {10.1016/j.micres.2025.128204},
pmid = {40359875},
issn = {1618-0623},
mesh = {*Aspergillus niger/genetics ; CRISPR-Cas Systems ; Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Plasmids/genetics ; *Gene Silencing ; Gene Library ; Genome, Fungal ; Genetic Vectors ; Phenotype ; Genes, Fungal ; Transformation, Genetic ; },
abstract = {Aspergillus niger is a saprotroph, a pathogen, an endophyte, a food spoiler and an important cell factory. Only a minor fraction of its genes has been experimentally characterized. We here set up a CRISPR/Cas9 mutagenesis screen for functional gene analysis using co-transformation of a pool of gene editing plasmids that are maintained under selection pressure and that each contain a gRNA. First, a pool of gRNA vectors was introduced in A. niger targeting five genes with easy selectable phenotypes. Transformants were obtained with all possible single, double, triple, quadruple and quintuple gene inactivation phenotypes. Their genotypes were confirmed using the gRNA sequences in the transforming vector as barcodes. Next, a gRNA library was introduced in A. niger targeting > 9600 genes. Gene nsdC was identified as a sporulation gene using co-transformation conditions that favored uptake of one or two gRNA construct(s) from the genome-wide vector pool. Together, CRISPR/Cas9 vectors with a (genome-wide) pool of gRNAs can be used for functional analysis of genes in A. niger with phenotypes that are the result of the inactivation of a single or multiple genes.},
}

*Aspergillus niger/genetics
CRISPR-Cas Systems
Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics
Plasmids/genetics
*Gene Silencing
Gene Library
Genome, Fungal
Genetic Vectors
Phenotype
Genes, Fungal
Transformation, Genetic

@article {pmid40339766,
year = {2025},
author = {Gao, L and Yuan, J and Hong, K and Ma, NL and Liu, S and Wu, X},
title = {Technological advancement spurs Komagataella phaffii as a next-generation platform for sustainable biomanufacturing.},
journal = {Biotechnology advances},
volume = {82},
number = {},
pages = {108593},
doi = {10.1016/j.biotechadv.2025.108593},
pmid = {40339766},
issn = {1873-1899},
mesh = {*Saccharomycetales/genetics/metabolism ; *Metabolic Engineering/methods ; Gene Editing ; CRISPR-Cas Systems ; Methanol/metabolism ; *Biotechnology/methods ; },
abstract = {Biomanufacturing stands as a cornerstone of sustainable industrial development, necessitating a shift toward non-food carbon feedstocks to alleviate agricultural resource competition and advance a circular bioeconomy. Methanol, a renewable one‑carbon substrate, has emerged as a pivotal candidate due to its abundance, cost-effectiveness, and high reduction potential, further bolstered by breakthroughs in CO2 hydrogenation-based synthesis. Capitalizing on this momentum, the methylotrophic yeast Komagataella phaffii has undergone transformative technological upgrades, evolving from a conventional protein expression workhorse into an intelligent bioproduction chassis. This paradigm shift is fundamentally driven by converging innovations across CRISPR-empowered advancement in genome editing and AI-powered metabolic pathway design in K. phaffii. The integration of CRISPR systems with droplet microfluidics high-throughput screening has redefined strain engineering efficiency, achieving much higher editing precision than traditional homologous recombination while compressing the "design-build-test-learn" cycle. Concurrently, machine learning-enhanced genome-scale metabolic models facilitate dynamic flux balancing, enabling simultaneous improvements in product titers, carbon yields, and volumetric productivity. Finally, technological advancement promotes the application of K. phaffii, including directing more efficiently metabolic flux toward nutrient products, and strengthening efficient synthesis of excreted proteins. As DNA synthesis automation and robotic experimentation platforms mature, next-generation breakthroughs in genome modification, cofactor engineering, and AI-guided autonomous evolution will further cement K. phaffii as a next-generation platform for decarbonizing global manufacturing paradigms. This technological trajectory positions methanol-based biomanufacturing as a cornerstone of the low-carbon circular economy.},
}

*Saccharomycetales/genetics/metabolism
*Metabolic Engineering/methods
Gene Editing
CRISPR-Cas Systems
Methanol/metabolism
*Biotechnology/methods

@article {pmid40311721,
year = {2025},
author = {Liberty, JT and Bromage, S and Peter, E and Ihedioha, OC and Alsalman, FB and Odogwu, TS},
title = {CRISPR revolution: Unleashing precision pathogen detection to safeguard public health and food safety.},
journal = {Methods (San Diego, Calif.)},
volume = {240},
number = {},
pages = {180-194},
doi = {10.1016/j.ymeth.2025.04.018},
pmid = {40311721},
issn = {1095-9130},
mesh = {*Food Safety/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Food Microbiology/methods ; *Foodborne Diseases/microbiology/diagnosis/virology ; Public Health/methods ; Biosensing Techniques/methods ; *Bacteria/genetics/isolation & purification/pathogenicity ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Molecular Diagnostic Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; Gene Editing/methods ; },
abstract = {Foodborne pathogens represent a significant challenge to global food safety, causing widespread illnesses and economic losses. The growing complexity of food supply chains and the emergence of antimicrobial resistance necessitate rapid, sensitive, and portable diagnostic tools. CRISPR technology has emerged as a transformative solution, offering unparalleled precision and adaptability in pathogen detection. This review explores CRISPR's role in addressing critical gaps in traditional and modern diagnostic methods, emphasizing its advantages in sensitivity, specificity, and scalability. CRISPR-based diagnostics, such as Cas12 and Cas13 systems, enable rapid detection of bacterial and viral pathogens, as well as toxins and chemical hazards, directly in food matrices. Their integration with isothermal amplification techniques and portable biosensors enhances field applicability, making them ideal for decentralized and real-time testing. Additionally, CRISPR's potential extends beyond food safety, contributing to public health efforts by monitoring antimicrobial resistance and supporting One Health frameworks. Despite these advancements, challenges remain, including issues with performance in complex food matrices, scalability, and regulatory barriers. This review highlights future directions, including AI integration for assay optimization, the development of universal CRISPR platforms, and the adoption of sustainable diagnostic solutions. By tackling these challenges, CRISPR has the potential to redefine global food safety standards and create a more resilient food system. Collaborative research and innovation will be critical to fully unlocking its transformative potential in food safety and public health.},
}

*Food Safety/methods
Humans
*CRISPR-Cas Systems/genetics
*Food Microbiology/methods
*Foodborne Diseases/microbiology/diagnosis/virology
Public Health/methods
Biosensing Techniques/methods
*Bacteria/genetics/isolation & purification/pathogenicity
*Clustered Regularly Interspaced Short Palindromic Repeats
Molecular Diagnostic Techniques/methods
Nucleic Acid Amplification Techniques/methods
Gene Editing/methods

@article {pmid40267240,
year = {2025},
author = {Rosengarten, H and D'Amore, A and Kim, HM and Ebrahimi-Fakhari, D},
title = {ap4b1 -/- zebrafish demonstrate morphological and motor abnormalities.},
journal = {Human molecular genetics},
volume = {34},
number = {12},
pages = {1034-1039},
doi = {10.1093/hmg/ddaf056},
pmid = {40267240},
issn = {1460-2083},
support = {K08NS123552-01//NIH/NINDS/ ; //CureAP4 Foundation/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Zebrafish Proteins/genetics/metabolism ; Disease Models, Animal ; Motor Neurons/metabolism/pathology ; *Spastic Paraplegia, Hereditary/genetics/physiopathology/pathology ; CRISPR-Cas Systems ; Seizures/genetics ; Axons/pathology/metabolism ; Gene Editing ; Humans ; },
abstract = {OBJECTIVE: Hereditary spastic paraplegia type 47 (SPG47) is caused by biallelic loss-of-function variants in the AP4B1 gene, leading to neurodevelopmental and progressive motor impairment. This study aimed to generate and characterize a zebrafish (Danio rerio) model of SPG47 to investigate the role of ap4b1 in neurodevelopment and motor function.

METHODS: We employed CRISPR/Cas9 gene-editing to generate a stable ap4b1-/- zebrafish line. Behavioral, morphological, and motor function analyses were performed, including survival under stress conditions, spontaneous locomotor activity, light-dark transition assays, and coiling behavior. Axonal length was assessed via immunofluorescence targeting spinal motor neurons. Seizure susceptibility was evaluated using a PTZ paradigm.

RESULTS: ap4b1-/- zebrafish exhibited significantly reduced axonal length of spinal motor neurons, impaired motor function, and developmental malformations, including brachycephaly, reduced body length, bent spines, and craniofacial defects. Increased tail coiling and reduced spontaneous activity were observed in larvae, alongside absent habituation to light-dark stimuli. Under stress conditions, survival rates were significantly lower in the knockout group compared to controls. Despite early hyperexcitability, no significant increase in PTZ-induced seizures was observed.

INTERPRETATION: This study characterizes an ap4b1-/- zebrafish model that recapitulates some phenotypes of SPG47, including motor deficits and morphological abnormalities. These findings support the utility of zebrafish for studying AP-4 deficiency and provide a platform for investigating the molecular mechanisms underlying SPG47.},
}

Animals
*Zebrafish/genetics
*Zebrafish Proteins/genetics/metabolism
Disease Models, Animal
Motor Neurons/metabolism/pathology
*Spastic Paraplegia, Hereditary/genetics/physiopathology/pathology
CRISPR-Cas Systems
Seizures/genetics
Axons/pathology/metabolism
Gene Editing
Humans

@article {pmid40263938,
year = {2025},
author = {Thornburg, CD and Pipe, SW and Cantore, A and Unzu, C and Jones, M and Miesbach, WA},
title = {Clinical perspective: Advancing hemophilia treatment through gene therapy approaches.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {6},
pages = {2350-2362},
doi = {10.1016/j.ymthe.2025.04.023},
pmid = {40263938},
issn = {1525-0024},
mesh = {Humans ; *Hemophilia A/therapy/genetics ; *Genetic Therapy/methods ; Gene Editing/methods ; Genetic Vectors/genetics ; Dependovirus/genetics ; Lentivirus/genetics ; Animals ; CRISPR-Cas Systems ; Clinical Trials as Topic ; },
abstract = {Hemophilia, a congenital bleeding disorder, can cause arthropathy, impaired mobility, pain, and life-threatening hemorrhage events, significantly impacting quality of life for patients and caregivers. Current therapies, although effective, necessitate costly lifelong treatment, often in specialized settings. However, as a monogenic disorder caused by loss-of-function genetic variants, hemophilia is amenable to gene therapy. In this article, three primary gene therapy approaches at the forefront of clinical development are reviewed. Adeno-associated virus-based gene therapy, having secured approval in the EU, UK, and US after promising phase 3 trial results, demonstrates clear superiority over standard-of-care treatment. Lentivirus-based approaches capable of transducing dividing and nondividing cells may improve the durability of treatment and have low susceptibility to pre-existing neutralizing antibodies to viral vectors. Finally, gene editing techniques such as zinc finger nucleases and CRISPR aim to correct genetic defects directly, holding promise as novel, effective, and highly durable therapeutic strategies in adults and children with hemophilia. This review provides a comprehensive summary of the current status of these gene therapy approaches, highlighting advantages, limitations, and potential future developments.},
}

Humans
*Hemophilia A/therapy/genetics
*Genetic Therapy/methods
Gene Editing/methods
Genetic Vectors/genetics
Dependovirus/genetics
Lentivirus/genetics
Animals
CRISPR-Cas Systems
Clinical Trials as Topic

@article {pmid40216992,
year = {2025},
author = {Sogabe, S and Nakano, H and Ogasahara, Y and Cha, PC and Ando, Y and Taniguchi-Ikeda, M and Matsumoto, R and Kanagawa, M and Kobayashi, K and Murayama, S and Aoi, T and Toda, T and Satake, W},
title = {Regulation of MCCC1 expression by a Parkinson's disease-associated intronic variant: implications for pathogenesis.},
journal = {Journal of human genetics},
volume = {70},
number = {7},
pages = {371-374},
pmid = {40216992},
issn = {1435-232X},
support = {JP23K21409//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP22K19487//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP22H04923//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP21wm0425019//Japan Agency for Medical Research and Development (AMED)/ ; },
mesh = {Humans ; *Parkinson Disease/genetics/pathology ; *Introns/genetics ; Dopaminergic Neurons/metabolism/pathology ; Polymorphism, Single Nucleotide ; Genetic Predisposition to Disease ; Genome-Wide Association Study ; Quantitative Trait Loci ; Induced Pluripotent Stem Cells/metabolism ; Mitochondria/genetics/metabolism ; Gene Expression Regulation ; Brain/metabolism/pathology ; Alleles ; Male ; RNA, Messenger/genetics ; CRISPR-Cas Systems ; Female ; },
abstract = {Parkinson's disease (PD) is a common neurodegenerative disorder characterized by dopaminergic neuron loss and α-synuclein aggregation. While some familial cases result from single-gene mutations, most are sporadic, involving complex genetic and environmental interactions. Among PD risk loci identified through genome-wide association studies, MCCC1 encodes a mitochondrial enzyme essential for leucine catabolism; however, the causal variant remains unclear. Here, we investigated whether the intronic variant rs12637471 regulates MCCC1 mRNA expression and influences PD risk. Postmortem brain analysis revealed significantly elevated MCCC1 mRNA levels in G-allele carriers, consistent with peripheral tissue eQTL data from GTEx. Using CRISPR/Cas9-edited induced pluripotent stem cells, we generated isogenic lines differing only at rs12637471 and observed increased MCCC1 expression in G-allele dopaminergic neurons. Given MCCC1's mitochondrial role, its dysregulation may impact mitochondrial homeostasis, autophagy, or inflammation, potentially contributing to PD pathogenesis.},
}

Humans
*Parkinson Disease/genetics/pathology
*Introns/genetics
Dopaminergic Neurons/metabolism/pathology
Polymorphism, Single Nucleotide
Genetic Predisposition to Disease
Genome-Wide Association Study
Quantitative Trait Loci
Induced Pluripotent Stem Cells/metabolism
Mitochondria/genetics/metabolism
Gene Expression Regulation
Brain/metabolism/pathology
Alleles
Male
RNA, Messenger/genetics
CRISPR-Cas Systems
Female

@article {pmid40213940,
year = {2025},
author = {Hou, Z and Yi, Q and Wu, M and Wu, L and Li, F and Wang, T and Bian, P},
title = {Assessment and Mitigation of CRISPR-Cas9-Induced Nontargeted Translocations.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {21},
pages = {e2414415},
doi = {10.1002/advs.202414415},
pmid = {40213940},
issn = {2198-3844},
support = {12135016//National Natural Science Foundation of China/ ; 12075275//National Natural Science Foundation of China/ ; 12475335//National Natural Science Foundation of China/ ; HYZHXMH02002//Space Medical Experiment Project of China Manned Space Program/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Translocation, Genetic/genetics ; Escherichia coli/genetics ; },
abstract = {The performance of CRISPR-mediated genome editing near inverted repeats (IRs) potentially results in chromosomal translocations and other catastrophic rearrangements. However, the extent of this risk may be significantly underestimated because current reporter systems focus solely on site-specific translocations. Here, trans-acting reporter systems in Escherichia coli are developed to detect nontargeted translocations. Markedly increased frequency of translocations following CRISPR-Cas9 activation is observed, with the magnitude determined primarily by the length of the IRs and the proximity between Cas9 target sites and IRs. These translocations arise through a combination of intramolecular single-strand annealing and alternative end-joining mechanisms. Furthermore, it is discovered that introducing segments homologous to IR loci can substantially mitigate nontargeted translocations without significantly compromising CRISPR-Cas9-mediated editing. The study provides valuable insights into the genetic risks associated with CRISPR technologies and suggests a viable strategy for developing genetically safer CRISPR systems.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Translocation, Genetic/genetics
Escherichia coli/genetics

@article {pmid40187387,
year = {2025},
author = {Tajer, BJ and Kalu, G and Jay, S and Wynn, E and Decaux, A and Gilbert, P and Singer, HD and Kidd, MD and Nelson, JA and Harake, N and Lopez, NJ and Souchet, NR and Luong, AG and Savage, AM and Min, S and Karabacak, A and Böhm, S and Kim, RT and Froitzheim, T and Sousounis, K and Courtemanche, K and Han, J and Payzin-Dogru, D and Blair, SJ and Roy, S and Fei, JF and Tanaka, EM and Whited, JL},
title = {Optimized toolkit for the manipulation of immortalized axolotl fibroblasts.},
journal = {Methods (San Diego, Calif.)},
volume = {240},
number = {},
pages = {21-34},
doi = {10.1016/j.ymeth.2025.03.019},
pmid = {40187387},
issn = {1095-9130},
mesh = {Animals ; *Fibroblasts/transplantation/cytology/metabolism ; *Ambystoma mexicanum/genetics ; Transfection/methods ; CRISPR-Cas Systems/genetics ; *Cell Culture Techniques/methods ; Cell Line ; Regeneration ; },
abstract = {The axolotl salamander model has broad utility for regeneration studies, but this model is limited by a lack of efficient cell-culture-based tools. The Axolotl Limb-1 (AL-1) fibroblast line, the only available immortalized axolotl cell line, was first published over 20 years ago, but many established molecular biology techniques, such as lipofectamine transfection, CRISPR-Cas9 mutagenesis, and antibiotic selection, work poorly or remain untested in AL-1 cells. Innovating technologies to manipulate AL-1 cells in culture and study their behavior following transplantation into the axolotl will complement in-vivo studies, decrease the number of animals used, and enable the faster, more streamlined investigation of regenerative biology questions. Here, we establish transfection, mutagenesis, antibiotic selection, and in-vivo transplantation techniques in axolotl AL-1 cells. These techniques will enable efficient culture with AL-1 cells and guide future tool development for the culture and manipulation of other salamander cell lines.},
}

Animals
*Fibroblasts/transplantation/cytology/metabolism
*Ambystoma mexicanum/genetics
Transfection/methods
CRISPR-Cas Systems/genetics
*Cell Culture Techniques/methods
Cell Line
Regeneration

@article {pmid40172074,
year = {2025},
author = {Boccacci, Y and Dumont, N and Doyon, Y and Laganière, J},
title = {CRISPR-Cas9-driven antigen conversion of clinically relevant blood group systems.},
journal = {Human molecular genetics},
volume = {34},
number = {12},
pages = {1001-1008},
doi = {10.1093/hmg/ddaf040},
pmid = {40172074},
issn = {1460-2083},
support = {//Centre de recherche du CHU de Québec-Université Laval/ ; //Faculté de médecine de l'Université Laval, Mitacs and Héma-Québec/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Erythrocytes/immunology/metabolism ; *Blood Group Antigens/genetics/immunology ; *ABO Blood-Group System/genetics/immunology ; Rh-Hr Blood-Group System/genetics/immunology ; Hematopoietic Stem Cells/metabolism/immunology ; Blood Transfusion ; },
abstract = {The common practice of blood transfusion entirely relies on blood donations from the population. Ensuring blood group compatibility between a donor and a recipient is paramount to prevent critical adverse reactions. Finding compatible blood can be challenging given the high diversity of blood group antigens, especially for chronically transfused patients at higher risk of alloimmunization owing to repeated exposures to foreign RBCs. In addition, due to the immunogenicity of the ABO blood group and the highly polymorphic nature of the Rhesus (Rh) system, they both remain of prime importance in transfusion medicine. Cultured red blood cells (cRBCs) may eventually provide an alternative for blood donations-at least in some circumstances. Combining cRBCs with blood group gene editing could broaden transfusion accessibility by making antigen expression compatible with rare phenotypes, thus meeting the needs of more patients. Starting from mobilized, erythroid-primed hematopoietic stem and progenitor cells (HSPCs), we used virus- and selection-free, CRISPR-Cas9-mediated knockouts to produce erythroid cells devoid of AB and Rh antigen. The approach yielded almost complete conversion to O- and RhNull phenotypes, as determined by standard hemagglutination and flow cytometry analyses. Combined with robust cRBC protocols, these clinically relevant phenotypic changes could eventually expand the accessibility of blood transfusion for specific and unmet clinical needs.},
}

Humans
*CRISPR-Cas Systems/genetics
Gene Editing/methods
Erythrocytes/immunology/metabolism
*Blood Group Antigens/genetics/immunology
*ABO Blood-Group System/genetics/immunology
Rh-Hr Blood-Group System/genetics/immunology
Hematopoietic Stem Cells/metabolism/immunology
Blood Transfusion

@article {pmid40140585,
year = {2025},
author = {Chen, KY and Kibayashi, T and Giguelay, A and Hata, M and Nakajima, S and Mikami, N and Takeshima, Y and Ichiyama, K and Omiya, R and Ludwig, LS and Hattori, K and Sakaguchi, S},
title = {Genome-wide CRISPR screen in human T cells reveals regulators of FOXP3.},
journal = {Nature},
volume = {642},
number = {8066},
pages = {191-200},
pmid = {40140585},
issn = {1476-4687},
mesh = {Animals ; Female ; Humans ; Male ; Mice ; Acetylation ; Cell Differentiation/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Cas Systems ; *Forkhead Transcription Factors/genetics/metabolism ; Gene Expression Regulation ; *Genome, Human/genetics ; Graft vs Host Disease/immunology/prevention & control ; Histone Deacetylases/metabolism ; Histones/metabolism ; Signal Transduction ; Single-Cell Analysis ; *T-Lymphocytes, Regulatory/metabolism/cytology/immunology ; *Genetic Techniques ; },
abstract = {Regulatory T (Treg) cells, which specifically express the master transcription factor FOXP3, have a pivotal role in maintaining immunological tolerance and homeostasis and have the potential to revolutionize cell therapies for autoimmune diseases[1-3]. Although stimulation of naive CD4[+] T cells in the presence of TGFβ and IL-2 can induce FOXP3[+] Treg cells in vitro (iTreg cells), the resulting cells are often unstable and have thus far hampered translational efforts[4-6]. A systematic approach towards understanding the regulatory networks that dictate Treg differentiation could lead to more effective iTreg cell-based therapies. Here we performed a genome-wide CRISPR loss-of-function screen to catalogue gene regulatory determinants of FOXP3 induction in primary human T cells and characterized their effects at single-cell resolution using Perturb-icCITE-seq. We identify the RBPJ-NCOR repressor complex as a novel, context-specific negative regulator of FOXP3 expression. RBPJ-targeted knockout enhanced iTreg differentiation and function, independent of canonical Notch signalling. Repeated cytokine and T cell receptor signalling stimulation in vitro revealed that RBPJ-deficient iTreg cells exhibit increased phenotypic stability compared with control cells through DNA demethylation of the FOXP3 enhancer CNS2, reinforcing FOXP3 expression. Conversely, overexpression of RBPJ potently suppressed FOXP3 induction through direct modulation of FOXP3 histone acetylation by HDAC3. Finally, RBPJ-ablated human iTreg cells more effectively suppressed xenogeneic graft-versus-host disease than control iTreg cells in a humanized mouse model. Together, our findings reveal novel regulators of FOXP3 and point towards new avenues to improve the efficacy of adoptive cell therapy for autoimmune disease.},
}

Animals
Female
Humans
Male
Mice
Acetylation
Cell Differentiation/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
*CRISPR-Cas Systems
*Forkhead Transcription Factors/genetics/metabolism
Gene Expression Regulation
*Genome, Human/genetics
Graft vs Host Disease/immunology/prevention & control
Histone Deacetylases/metabolism
Histones/metabolism
Signal Transduction
Single-Cell Analysis
*T-Lymphocytes, Regulatory/metabolism/cytology/immunology
*Genetic Techniques

@article {pmid40119516,
year = {2025},
author = {Liu, D and Cao, D and Han, R},
title = {Recent advances in therapeutic gene-editing technologies.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {6},
pages = {2619-2644},
doi = {10.1016/j.ymthe.2025.03.026},
pmid = {40119516},
issn = {1525-0024},
mesh = {*Gene Editing/methods/trends ; Humans ; *CRISPR-Cas Systems ; *Genetic Therapy/methods/trends ; Animals ; Epigenesis, Genetic ; },
abstract = {The advent of gene-editing technologies, particularly CRISPR-based systems, has revolutionized the landscape of biomedical research and gene therapy. Ongoing research in gene editing has led to the rapid iteration of CRISPR technologies, such as base and prime editors, enabling precise nucleotide changes without the need for generating harmful double-strand breaks (DSBs). Furthermore, innovations such as CRISPR fusion systems with DNA recombinases, DNA polymerases, and DNA ligases have expanded the size limitations for edited sequences, opening new avenues for therapeutic development. Beyond the CRISPR system, mobile genetic elements (MGEs) and epigenetic editors are emerging as efficient alternatives for precise large insertions or stable gene manipulation in mammalian cells. These advances collectively set the stage for next-generation gene therapy development. This review highlights recent developments of genetic and epigenetic editing tools and explores preclinical innovations poised to advance the field.},
}

*Gene Editing/methods/trends
Humans
*CRISPR-Cas Systems
*Genetic Therapy/methods/trends
Animals
Epigenesis, Genetic

@article {pmid39890502,
year = {2025},
author = {Kamran, M and Waters, MT},
title = {Engineering crop resilience with synthetic gene circuits.},
journal = {Trends in plant science},
volume = {30},
number = {6},
pages = {582-584},
doi = {10.1016/j.tplants.2025.01.005},
pmid = {39890502},
issn = {1878-4372},
mesh = {*Crops, Agricultural/genetics/physiology ; *Genetic Engineering/methods ; *Gene Regulatory Networks ; CRISPR-Cas Systems ; *Genes, Synthetic ; Gene Expression Regulation, Plant ; Plants, Genetically Modified/genetics ; Stress, Physiological/genetics ; },
abstract = {Engineering crops to withstand environmental stresses is critical for addressing climate change and food insecurity. Recently, Khan et al. developed CRISPR interference (CRISPRi)-based synthetic gene circuits to program gene expression in plants. Their findings highlight the potential of these circuits to advance the development of stress-resilient crops.},
}

*Crops, Agricultural/genetics/physiology
*Genetic Engineering/methods
*Gene Regulatory Networks
CRISPR-Cas Systems
*Genes, Synthetic
Gene Expression Regulation, Plant
Plants, Genetically Modified/genetics
Stress, Physiological/genetics

@article {pmid39537537,
year = {2025},
author = {Liu, W and Pan, Y and Zhang, Y and Dong, C and Huang, L and Lian, J},
title = {Intracellularly synthesized ssDNA for continuous genome engineering.},
journal = {Trends in biotechnology},
volume = {43},
number = {6},
pages = {1356-1370},
doi = {10.1016/j.tibtech.2024.10.011},
pmid = {39537537},
issn = {1879-3096},
mesh = {*DNA, Single-Stranded/genetics/biosynthesis ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; },
abstract = {Despite the prevalence of genome editing tools, there are still some limitations in dynamic and continuous genome editing. In vivo single-stranded DNA (ssDNA)-mediated genome mutation has emerged as a valuable and promising approach for continuous genome editing. In this review, we summarize the various types of intracellular ssDNA production systems and notable achievements in genome engineering in both prokaryotic and eukaryotic cells. We also review progress in the development of applications based on retron-based systems, which have demonstrated significant potential in molecular recording, multiplex genome editing, high-throughput functional variant screening, and gene-specific continuous in vivo evolution. Furthermore, we discuss the major challenges of ssDNA-mediated continuous genome editing and its prospects for future applications.},
}

*DNA, Single-Stranded/genetics/biosynthesis
*Gene Editing/methods
CRISPR-Cas Systems/genetics

@article {pmid40447601,
year = {2025},
author = {Hu, H and Guo, S and Li, Y and Dong, K and Lu, Y and Ye, K and Li, L and Zhou, X and Cheng, L and Xiao, X},
title = {Spatially blocked split CRISPR-Cas12a system for ultra-sensitive and versatile small molecule activation and detection.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5035},
pmid = {40447601},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; Humans ; *CRISPR-Associated Proteins/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; },
abstract = {Detecting small molecules is pivotal across fields like clinical diagnostics, environmental monitoring, and food safety. The CRISPR-Cas12a system, known for its simplicity and sensitivity, offers a promising basis for small molecule detection. However, current CRISPR-based detection methods face challenges, including complex design requirements, high background noise, and limited adaptability to different targets. In our study, we introduce the SBS-Cas system, leveraging a split crRNA mode to induce spatial hindrance on the scaffold strand through molecular binding. This approach prevents the assembly with Cas12a, effectively masking its trans-cleavage activity. By introducing small molecules that competitively bind to the macromolecule, we eliminate this spatial hindrance, activating Cas12a. Our results demonstrate high sensitivity, versatility, and adaptability in small molecule detection across multiple reactions, with successful intracellular imaging and responsive fluctuations in complex environments underscoring the system's robustness. This innovative CRISPR-Cas12a-based approach establishes a low-background, highly sensitive platform for small molecule detection. SBS-Cas promises not only to enhance tools for clinical, environmental, and food safety applications but also to advance CRISPR research, providing insights and expanding possibilities in molecular detection science.},
}

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

@article {pmid40447589,
year = {2025},
author = {Liang, R and Wang, S and Cai, Y and Li, Z and Li, KM and Wei, J and Sun, C and Zhu, H and Chen, K and Gao, C},
title = {Circular RNA-mediated inverse prime editing in human cells.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5057},
pmid = {40447589},
issn = {2041-1723},
mesh = {Humans ; *Gene Editing/methods ; *RNA, Circular/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; HEK293 Cells ; CRISPR-Associated Protein 9/genetics/metabolism ; Genome, Human ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Prime editors are restricted to performing precise edits downstream of cleavage sites, thereby limiting their editing scope. Therefore, we develop inverse prime editors (iPEs) that act upstream of the nickase cleavage site by replacing nCas9-H840A with nCas9-D10A, but the editing efficiencies are limited. To address this limitation, we develop circular RNA-mediated iPEs (ciPEs), achieving editing efficiencies ranging from 0.1% to 24.7%. Further optimization using Rep-X helicase increases editing efficiencies to a range of 2.7%-55.4%. The Rep-X-assisted ciPE system thus expands the scope of editing and improves efficiencies at genomic sites that are previously difficult to target. The Rep-X-assisted ciPE system will complement canonical PE system in enabling more extensive and efficient editing across a wider range of the human genome.},
}

Humans
*Gene Editing/methods
*RNA, Circular/genetics/metabolism
*CRISPR-Cas Systems/genetics
HEK293 Cells
CRISPR-Associated Protein 9/genetics/metabolism
Genome, Human
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40447514,
year = {2025},
author = {Yu, T and Zou, S and Long, Y and Ou, Y and Liu, S and Kang, T and Song, L and Sun, C and Liu, G},
title = {Glass fiber-interfaced CRISPR/Cas biosensing adaptable for diverse biomarker detection.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2025.05.001},
pmid = {40447514},
issn = {1879-3096},
abstract = {Developing a generic sensitive platform for detecting diverse biomarkers is essential for a comprehensive understanding of disease states, guiding precision medicine. Herein, we introduce a versatile platform based on glass fiber interfaced CRISPR/Cas with a universal reagent setting (g-CURS), which used a fixed pair of CRISPR RNA (crRNA) and a single-stranded DNA (ssDNA) activator to enable detection of multiple nucleic acids or proteins with ultrahigh sensitivity. The fixed ssDNA activator was labeled on multiple specific ligation products or detection antibodies conjugated on glass fiber to initiate CRISPR/Cas12a-assisted rapid and exponential cascade amplification through circular reporters (CRs), generating fluorescence signals readable by a portable detector. g-CURS was able to detect viral nucleic acids with attomolar sensitivity within 30 min and multiple low-abundance proteins in extracellular vesicles of Parkinson's disease (PD) serum with subpicomolar sensitivity within 80 min. g-CURS simplifies CRISPR/Cas biosensing using a standard reagent setting, holding promise for biomarker discovery free from bulky instruments.},
}

@article {pmid40447397,
year = {2025},
author = {Gao, Y and Li, Y and Gao, Q and Zhang, T and Zhang, Y and Liu, T and Han, K},
title = {Multi-functional dumbbell DNA probe design and its application in signal amplification cascade -based assay of human immunodeficiency virus.},
journal = {Analytica chimica acta},
volume = {1362},
number = {},
pages = {344141},
doi = {10.1016/j.aca.2025.344141},
pmid = {40447397},
issn = {1873-4324},
mesh = {*DNA Probes/chemistry/genetics ; Humans ; *Biosensing Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; CRISPR-Cas Systems ; *DNA, Viral/analysis/genetics ; Limit of Detection ; *HIV/isolation & purification/genetics ; },
abstract = {Sensitive assay of Human immunodeficiency virus (HIV) is the premise of accurate prevention and con-trol of AIDS. Dumbbell DNA showed promising potential in biosensing, imaging applications. This study presents a highly sensitive biosensing platform integrating dumbbell-shaped DNA probes with rolling circle transcription (RCT)-regulated CRISPR-Cas12a to achieve cascade signal amplification. The arrangement of the functional regions in the same unclosed dumbbell probes (UDPs) might affect final performance in the biosensor. Three different types UDPs (A/B/C) were specifically designed and the performances of the UDPs were evaluated in the RCT-CRISPR based cascade platform. Among these, type A UDP shows the best performance with a detection limit of 44.8 aM targeting HIV-DNA as well as high sensitivity and specificity. Also, type A UDP had the best behavior in the clinical sample assay. The difference of UDPs' performance might attribute to the variation of opening form of the UDPs considering the arrangement of various functional regions. The platform's modular design supports customization for diverse nucleic acid targets, making it adaptable for early disease detection and precision diagnostics.},
}

*DNA Probes/chemistry/genetics
Humans
*Biosensing Techniques/methods
*Nucleic Acid Amplification Techniques/methods
CRISPR-Cas Systems
*DNA, Viral/analysis/genetics
Limit of Detection
*HIV/isolation & purification/genetics

@article {pmid40445760,
year = {2025},
author = {Lamperis, SM and McMahon, KM and Calvert, AE and Rink, JS and Vasan, K and Pandkar, MR and Crentsil, EU and Chalmers, ZR and McDonald, NR and Kosmala, CJ and Bonini, MG and Matei, D and Gordon, LI and Chandel, NS and Thaxton, CS},
title = {CRISPR screen reveals a simultaneous targeted mechanism to reduce cancer cell selenium and increase lipid oxidation to induce ferroptosis.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {22},
pages = {e2502876122},
doi = {10.1073/pnas.2502876122},
pmid = {40445760},
issn = {1091-6490},
support = {T2022-018//V Foundation for Cancer Research (VFCR)/ ; T32GM142604//HHS | NIH (NIH)/ ; R01CA216882//HHS | NIH (NIH)/ ; R01ES035353//HHS | NIH (NIH)/ ; },
mesh = {*Ferroptosis/genetics/drug effects ; Humans ; Cell Line, Tumor ; Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism/genetics ; *Selenium/metabolism ; Oxidation-Reduction ; Female ; CRISPR-Cas Systems ; Lipid Peroxidation ; Scavenger Receptors, Class B/metabolism/genetics ; Lipoproteins, HDL/metabolism ; *Ovarian Neoplasms/metabolism/genetics/pathology ; Lipid Metabolism ; Nanoparticles/chemistry ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Ferroptosis is a cell death mechanism distinguished by its dependence on iron-mediated lipid oxidation. Cancer cells highly resistant to conventional therapies often demonstrate lipid metabolic and redox vulnerabilities that sensitize them to cell death by ferroptosis. These include a unique dependency on the lipid antioxidant selenoenzyme, glutathione peroxidase 4 (GPx4), that acts as a ferroptosis inhibitor. Synthetic high-density lipoprotein-like nanoparticle (HDL NP) targets the high-affinity HDL receptor scavenger receptor class B type 1 (SR-B1) and regulates cell and cell membrane lipid metabolism. Recently, we reported that targeting cancer cell SR-B1 with HDL NP depleted cell GPx4, which is accompanied by increased cell membrane lipid peroxidation and cancer cell death. These data suggest that HDL NP may induce ferroptosis. Thus, we conducted an unbiased CRISPR-based positive selection screen and target validation studies in ovarian clear cell carcinoma (OCCC) cell lines to ascertain the mechanism through which HDL NP regulates GPx4 and kills cancer cells. The screen revealed two genes, acyl-CoA synthetase long chain family member 4 (ACSL4) and thioredoxin reductase 1 (TXNRD1), whose loss conferred resistance to HDL NP. Validation of ACSL4 supports that HDL NP induces ferroptosis as the predominant mechanism of cell death, while validation of TXNRD1 revealed that HDL NP reduces cellular selenium and selenoprotein production, most notably, GPx4. Accordingly, we define cancer cell metabolic targets that can be simultaneously actuated by a multifunctional, synthetic HDL NP ligand of SR-B1 to kill cancer cells by ferroptosis.},
}

*Ferroptosis/genetics/drug effects
Humans
Cell Line, Tumor
Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism/genetics
*Selenium/metabolism
Oxidation-Reduction
Female
CRISPR-Cas Systems
Lipid Peroxidation
Scavenger Receptors, Class B/metabolism/genetics
Lipoproteins, HDL/metabolism
*Ovarian Neoplasms/metabolism/genetics/pathology
Lipid Metabolism
Nanoparticles/chemistry
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40445465,
year = {2025},
author = {Hashemi, M and Khanaghah, XM and Nahand, JS},
title = {The CRISPR-Cas revolution in head and neck cancer: a new era of targeted therapy.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {113},
pmid = {40445465},
issn = {1438-7948},
mesh = {Humans ; *Head and Neck Neoplasms/therapy/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genetic Therapy/methods ; Molecular Targeted Therapy ; Signal Transduction ; },
abstract = {Head and neck cancer (HNC) encompasses a diverse array of malignancies impacting the anatomical structures of the head and neck region, ranking as the seventh most prevalent cancer type globally. The occurrence and advancement of HNC are intricately linked to mutations and disruptions within critical signaling pathways, accentuating the imperative for targeted therapeutic interventions to rectify these genetic aberrations. Traditional treatment modalities, including surgical intervention and adjuvant chemotherapy or radiotherapy, frequently culminate in considerable morbidity and suboptimal prognoses. Recently, the CRISPR-Cas system has emerged as a revolutionary gene-editing platform, poised to redefine therapeutic approaches in gene therapy and oncological research. Despite its potential, CRISPR-Cas faces challenges such as off-target effects, delivery inefficiencies, and immunogenicity, which must be addressed for clinical success. This review meticulously evaluates the progress in CRISPR-Cas technologies aimed at targeting essential signaling pathways implicated in HNC, addressing current challenges while highlighting optimal targets, Cas nucleases, and innovative delivery mechanisms, thereby elucidating the therapeutic potential and clinical applicability of the CRISPR-Cas paradigm in the management of HNC.},
}

Humans
*Head and Neck Neoplasms/therapy/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
*Genetic Therapy/methods
Molecular Targeted Therapy
Signal Transduction

@article {pmid40442121,
year = {2025},
author = {Ma, S and Liao, K and Chen, K and Cheng, T and Yang, X and Chen, P and Li, S and Li, M and Zhang, X and Zhang, Y and Huang, T and Wang, X and Wang, L and Lin, Y and Rong, Z},
title = {hpCasMINI: An engineered hypercompact CRISPR-Cas12f system with boosted gene editing activity.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5001},
pmid = {40442121},
issn = {2041-1723},
support = {82370078//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82200072//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82304008//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82303995//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82370003//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Animals ; Humans ; Mice ; HEK293 Cells ; Fibroblast Growth Factors/genetics/metabolism ; Genetic Therapy/methods ; DNA Cleavage ; Tumor Suppressor Protein p53/genetics ; Dependovirus/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; Liver/metabolism ; PTEN Phosphohydrolase/genetics ; },
abstract = {Compact CRISPR-Cas systems have demonstrated potential for effective packaging into adeno-associated viruses (AAVs) for use in gene therapy. However, their applications are currently limited due to modest gene-editing activity. Here we introduce an engineered compact CRISPR-Cas12f (hpCasMINI, 554 aa), with hyper editing efficiency in mammalian cells via adding an α-helix structure to the N-terminus of an Un1Cas12f1 variant CasMINI (529 aa). The hpCasMINI system boosts gene activation and DNA cleavage activity with about 1.4-3.0-fold and 1.1-19.5-fold, respectively, and maintains the high specificity when compared to CasMINI. In addition, the system can activate luciferase reporter gene and endogenous Fgf21 gene in adult mouse liver, as well as construct liver tumorigenesis model via disrupting Trp53 and Pten genes and inserting oncogenic Kras[G12D] into the Trp53 locus. When compared to SpCas9 and LbCas12a, hpCasMINI displays higher gene activation and exhibits higher DNA cleavage specificity, although with lower activity, at the tested sites. Moreover, with a similar strategy, we engineer compact versions of hpOsCas12f1 (458 aa) from enOsCas12f1 and hpAsCas12f1 (447 aa) from AsCas12f1-HKRA, both of which display increased DNA cleavage activity, with hpAsCas12f1 also showing improved gene activation capability. Therefore, we develop activity-increased miniature hpCasMINI, hpOsCas12f1 and hpAsCas12f1 nucleases, which hold great potential for gene therapy in the future.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Animals
Humans
Mice
HEK293 Cells
Fibroblast Growth Factors/genetics/metabolism
Genetic Therapy/methods
DNA Cleavage
Tumor Suppressor Protein p53/genetics
Dependovirus/genetics
*CRISPR-Associated Proteins/genetics/metabolism
Liver/metabolism
PTEN Phosphohydrolase/genetics

@article {pmid40411834,
year = {2025},
author = {Guo, Y and Guo, W and Li, C and Xu, H and Zhang, X and Zou, X and Sun, Z},
title = {Fe3O4@Au Nanoparticle-Enabled Magnetic Separation Coupled with CRISPR/Cas12a for Ultrasensitive Detection of Foodborne Pathogens.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {22},
pages = {13949-13959},
doi = {10.1021/acs.jafc.5c04580},
pmid = {40411834},
issn = {1520-5118},
mesh = {*Biosensing Techniques/instrumentation/methods ; Gold/chemistry ; CRISPR-Cas Systems ; *Staphylococcus aureus/isolation & purification/genetics ; Animals ; Milk/microbiology ; *Salmonella/isolation & purification/genetics ; Food Contamination/analysis ; Metal Nanoparticles/chemistry ; Food Microbiology ; Electrochemical Techniques/instrumentation/methods ; Limit of Detection ; *Endodeoxyribonucleases/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {The rapid detection of foodborne pathogens, such as Staphylococcus aureus and Salmonella, is critical for ensuring food safety. Herein, we present a magnetically controlled electrochemical biosensor integrating CRISPR/Cas12a with Fe3O4@Au nanoparticles designed to achieve ultrasensitive and multiplexed detection. By utilization of the magnetic separation of CRISPR-cleaved ssDNA from Fe3O4@Au nanoparticles, the sensor circumvents intricate electrode modifications, enabling direct signal readout. This approach expedites the workflow to 65 min while achieving a detection limit of 2 CFU/mL. Additionally, the sensor exhibits signal stability over 45 days and demonstrates its versatility by enabling the separate detection of both Gram-positive (S. aureus) and Gram-negative (Salmonella) pathogens. With validation in milk samples with high interference resistance, this platform bridges CRISPR programmability with practical deployability, offering a robust solution for on-site monitoring. The innovation lies in its simplified design, enhanced stability, and clinical versatility, setting a new benchmark for rapid, low-cost pathogen detection in resource-limited environments.},
}

*Biosensing Techniques/instrumentation/methods
Gold/chemistry
CRISPR-Cas Systems
*Staphylococcus aureus/isolation & purification/genetics
Animals
Milk/microbiology
*Salmonella/isolation & purification/genetics
Food Contamination/analysis
Metal Nanoparticles/chemistry
Food Microbiology
Electrochemical Techniques/instrumentation/methods
Limit of Detection
*Endodeoxyribonucleases/genetics
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid40382983,
year = {2025},
author = {Pulito, C and Vaccarella, S and Palcau, AC and Ganci, F and Brandi, R and Frascolla, C and Sacconi, A and Canu, V and Benedetti, A and De Pascale, V and Donzelli, S and Fisch, AS and Manciocco, V and Covello, R and Pimpinelli, F and Morrone, A and Fazi, F and Pellini, R and Muti, P and Meens, J and Karamboulas, C and Nichols, AC and Strano, S and Klinghammer, K and Tinhofer, I and Ailles, L and Fontemaggi, G and Blandino, G},
title = {MicroRNA-mediated PTEN downregulation as a novel non-genetic mechanism of acquired resistance to PI3Kα inhibitors of head & neck squamous cell carcinoma.},
journal = {Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy},
volume = {81},
number = {},
pages = {101251},
doi = {10.1016/j.drup.2025.101251},
pmid = {40382983},
issn = {1532-2084},
mesh = {Humans ; *PTEN Phosphohydrolase/genetics/metabolism ; *MicroRNAs/genetics ; *Drug Resistance, Neoplasm/genetics/drug effects ; *Squamous Cell Carcinoma of Head and Neck/drug therapy/genetics/pathology ; Down-Regulation ; *Head and Neck Neoplasms/drug therapy/genetics/pathology ; Cell Line, Tumor ; *Class I Phosphatidylinositol 3-Kinases/antagonists & inhibitors/genetics ; Gene Expression Regulation, Neoplastic/drug effects ; Thiazoles/pharmacology/therapeutic use ; Polo-Like Kinase 1 ; Cell Cycle Proteins/antagonists & inhibitors/metabolism ; *Phosphoinositide-3 Kinase Inhibitors/pharmacology ; Protein Serine-Threonine Kinases/antagonists & inhibitors/metabolism ; Proto-Oncogene Proteins/antagonists & inhibitors/metabolism ; Animals ; Signal Transduction/drug effects ; CRISPR-Cas Systems ; Phosphorylation ; RNA, Long Noncoding ; },
abstract = {AIMS: Head and neck squamous cell carcinomas (HNSCCs) frequently harbor alterations in the PI3K signalling axis and, particularly, in the PIK3CA gene. The promising rationale of using PI3K inhibitors for the treatment of HNSCC has, however, clashed with the spontaneous development of resistance over time.

METHODS: To identify valuable targets for overcoming acquired resistance to PI3Kα inhibitors in HNSCC, we performed microRNA profiling on a cohort of HNSCC PDXs that were treated with alpelisib, including both responsive and resistant tumors. Using CRISPR/Cas9, siRNA, and PTEN-/- isogenic and alpelisib-resistant cell models, we examined the role of PTEN in resistance acquisition. Phospho-proteomic analysis identified PTEN-dependent phosphorylation events, while PI3Kα inhibitor-resistant organoids were used to assess PLK1 inhibitor efficacy.

RESULTS: We identified microRNAs altered in resistant PDXs, including members of the miR-17-92 cluster. Mechanistically, we observed that the hyperactive c-Myc was recruited to MIR17HG regulatory regions in alpelisib-resistant cells, sustaining miR-17-5p, miR-19b-3p, and miR-20a-5p expression, which downregulated PTEN. PTEN knockout or depletion conferred alpelisib resistance in HNSCC cells. We identified PTEN-dependent phosphorylation events, such as p-PLK1-T210, involved in resistance. Interestingly, pharmacological inhibition of PLK1 strongly reduced the viability of PI3Kα-resistant organoids derived from HNSCC PDXs and cell line models.

CONCLUSION: Overall, this study unveils a novel, microRNA-driven, non-genetic mechanism contributing to acquired resistance to PI3Kα inhibitors in HNSCC. Indeed, linking hyperactive c-Myc to sustain miR-17-92 expression and consequent PTEN downregulation, we also propose that targeting PTEN-dependent downstream effectors, such as PLK1, may offer a powerful therapeutic strategy for resistant HNSCC.},
}

Humans
*PTEN Phosphohydrolase/genetics/metabolism
*MicroRNAs/genetics
*Drug Resistance, Neoplasm/genetics/drug effects
*Squamous Cell Carcinoma of Head and Neck/drug therapy/genetics/pathology
Down-Regulation
*Head and Neck Neoplasms/drug therapy/genetics/pathology
Cell Line, Tumor
*Class I Phosphatidylinositol 3-Kinases/antagonists & inhibitors/genetics
Gene Expression Regulation, Neoplastic/drug effects
Thiazoles/pharmacology/therapeutic use
Polo-Like Kinase 1
Cell Cycle Proteins/antagonists & inhibitors/metabolism
*Phosphoinositide-3 Kinase Inhibitors/pharmacology
Protein Serine-Threonine Kinases/antagonists & inhibitors/metabolism
Proto-Oncogene Proteins/antagonists & inhibitors/metabolism
Animals
Signal Transduction/drug effects
CRISPR-Cas Systems
Phosphorylation
RNA, Long Noncoding

@article {pmid40345045,
year = {2025},
author = {Li, C and Peng, X and Zhang, Z and Liu, Y and Pedro, GC and Fu, C and Yang, Y and Dong, Q and Duan, Y and Sun, X},
title = {Establishment of Agrobacterium-mediated genetic transformation and CRISPR/Cas9-guided gene editing in Elymus nutans.},
journal = {Journal of plant physiology},
volume = {310},
number = {},
pages = {154513},
doi = {10.1016/j.jplph.2025.154513},
pmid = {40345045},
issn = {1618-1328},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Transformation, Genetic ; *Agrobacterium/genetics ; *Elymus/genetics/physiology ; Plants, Genetically Modified/genetics ; },
abstract = {Elymus nutans, an allohexaploid (2n = 6x = 42) species with a StStHHYY genome, is a native perennial in the alpine grasslands of the Qinghai-Xizang Plateau, and has been widely used for artificial pasture and ecological restoration as a forage grass with highest yield on the plateau. Nevertheless, the lack of a stable transformation system has impeded further efforts to trait improvement of E. nutans. In the present study, we established a reliable Agrobacterium-mediated genetic transformation system for E. nutans, and successfully generated EnTCP4-edited plants using the CRISPR/Cas9 system. The editing efficiency achieved 19.23 % in E. nutans. Knocking out EnTCP4 significantly delayed flowering and enhanced water-deficit stress resistance. This research represents a significant breakthrough in the genetic transformation and gene editing of E. nutans, laying a technological foundation to gain insight into gene functions and molecular breeding in E. nutans.},
}

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

@article {pmid40293254,
year = {2025},
author = {Huang, J and Ding, K and Chen, J and Fan, J and Huang, L and Qiu, S and Wang, L and Du, X and Wang, C and Pan, H and Yuan, Z and Liu, H and Song, H},
title = {Comparison of CRISPR-Cas9, CRISPR-Cas12f1, and CRISPR-Cas3 in eradicating resistance genes KPC-2 and IMP-4.},
journal = {Microbiology spectrum},
volume = {13},
number = {6},
pages = {e0257224},
pmid = {40293254},
issn = {2165-0497},
support = {32141003//National Science Foundation of China/ ; 32300080//National Science Foundation of China/ ; 2021YFC2301102//National Science and Technology Major Project/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics/drug effects ; *Gene Editing/methods ; Plasmids/genetics ; *beta-Lactamases/genetics ; Anti-Bacterial Agents/pharmacology ; Microbial Sensitivity Tests ; *Bacterial Proteins/genetics ; Drug Resistance, Bacterial/genetics ; Carbapenems/pharmacology ; Klebsiella pneumoniae/genetics/drug effects ; },
abstract = {UNLABELLED: Bacterial plasmid encoding antibiotic resistance could be eradicated by various CRISPR systems, such as CRISPR-Cas9, Cas12f1, and Cas3. However, the efficacy of these gene editing tools against bacterial resistance has not been systematically assessed and compared. This study eliminates carbapenem resistance genes KPC-2 and IMP-4 via CRISPR-Cas9, Cas12f1, and Cas3 systems, respectively. The eradication efficiency of the three CRISPR systems was evaluated. First, the target sites for the three CRISPR systems were designed within the regions 542-576 bp of the KPC-2 gene and 213-248 bp of the IMP-4 gene, respectively. The recombinant CRISPR plasmids were transformed into Escherichia coli carrying KPC-2 or IMP-4-encoding plasmid. Colony PCR of transformants showed that KPC-2 and IMP-4 were eradicated by the three different CRISPR systems, and the elimination efficacy was both 100.00%. The drug sensitivity test results showed that the resistant E. coli strain was resensitized to ampicillin. In addition, the three CRISPR plasmids could block the horizontal transfer of drug-resistant plasmids, with a blocking rate as high as 99%. Importantly, a qPCR assay was performed to analyze the copy number changes of drug-resistant plasmids in E. coli cells. The results indicated that CRISPR-Cas3 showed higher eradication efficiency than CRISPR-Cas9 and Cas12f1 systems.

IMPORTANCE: With the continuous development and application of CRISPR-based resistance removal technologies, CRISPR-Cas9, Cas12f1, and Cas3 have gradually come into focus. However, it remains uncertain which system exhibits more potent efficacy in the removal of bacterial resistance. This study verifies that CRISPR-Cas9, Cas12f1, and Cas3 can eradicate the carbapenem-resistant genes KPC-2 and IMP-4 and restore the sensitivity of drug-resistant model bacteria to antibiotics. Among the three CRISPR systems, the CRISPR-Cas3 system showed the highest eradication efficiency. Although each system has its advantages and characteristics, our results provide guidance on the selection of the CRISPR system from the perspective of resistance gene removal efficiency, contributing to the further application of CRISPR-based bacterial resistance removal technologies.},
}

*CRISPR-Cas Systems/genetics
*Escherichia coli/genetics/drug effects
*Gene Editing/methods
Plasmids/genetics
*beta-Lactamases/genetics
Anti-Bacterial Agents/pharmacology
Microbial Sensitivity Tests
*Bacterial Proteins/genetics
Drug Resistance, Bacterial/genetics
Carbapenems/pharmacology
Klebsiella pneumoniae/genetics/drug effects

@article {pmid40254669,
year = {2025},
author = {Yang, X and Luo, Y and Su, C and Huang, Z and Tang, Y and Zhang, L},
title = {Ultra-sensitive biosensor detection of microRNA based on the CRISPR/Cas12a system and exonuclease-assisted target recycling signal amplification.},
journal = {Analytical sciences : the international journal of the Japan Society for Analytical Chemistry},
volume = {41},
number = {6},
pages = {867-876},
pmid = {40254669},
issn = {1348-2246},
support = {2021GXNSFBA 076001//Natural Science Foundation of Guangxi Zhuang Autonomous Region/ ; 2022KY0491//Middle-aged and Young Teachers' Basic Ability Promotion Project of Guangxi/ ; Z-C20240897//Autonomous region health commission self-funded research project/ ; },
mesh = {*MicroRNAs/analysis/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Exodeoxyribonucleases/metabolism ; *Nucleic Acid Amplification Techniques ; Limit of Detection ; *Exonucleases/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {MicroRNAs (miRNAs) are essential regulators of gene expression and are significantly involved in both preventing and treating a range of diseases. To that end, we developed an ultra-sensitive detection method for miRNA-141 by integrating exonuclease-assisted target recycling signal amplification with the CRISPR/Cas12a system. This method employs a variable hairpin probe (HP) designed to hybridize with miRNA, which, under the action of exonuclease III (ExoIII), cleaves the hairpin probe and triggers target recycling signal amplification. This results in the formation of output DNAs (ODs) containing multiple repeat sequences. The CRISPR/Cas12a system identifies these repeated sequences in ODs through its crRNA component, which in turn triggers the trans-cleavage function of the Cas12a/crRNA complex. It leads to the cleavage of a fluorescently quenched reporter probe. Consequently, this process restores fluorescence, producing a significantly enhanced fluorescent signal that facilitates the detection of miRNA-141, achieving a detection threshold down to 62 fM. This detection approach can specifically differentiate miRNA-141 from other confounding substances and has effectively identified low concentrations of miRNA-141 in actual sample human serum and diverse cancer cell lysates, showcasing its capability for tracing various nucleic acid biomarkers at minimal levels.},
}

*MicroRNAs/analysis/genetics
*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
Humans
*Exodeoxyribonucleases/metabolism
*Nucleic Acid Amplification Techniques
Limit of Detection
*Exonucleases/metabolism
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40252747,
year = {2025},
author = {Zou, W and Huo, B and Tu, Y and Zhu, Y and Hu, Y and Li, Q and Yu, X and Liu, B and Tang, W and Tan, S and Xiao, H},
title = {Metabolic reprogramming by chemo-gene co-delivery nanoparticles for chemo-immunotherapy in head and neck squamous cell carcinoma.},
journal = {Acta biomaterialia},
volume = {199},
number = {},
pages = {361-373},
doi = {10.1016/j.actbio.2025.04.031},
pmid = {40252747},
issn = {1878-7568},
mesh = {*Squamous Cell Carcinoma of Head and Neck/therapy/pathology/metabolism/drug therapy/genetics ; Humans ; Animals ; *Immunotherapy ; *Nanoparticles/chemistry/therapeutic use ; Cell Line, Tumor ; *Cisplatin/pharmacology/chemistry/therapeutic use ; *Head and Neck Neoplasms/therapy/pathology/metabolism/drug therapy ; Prodrugs/pharmacology/chemistry ; Mice ; *Gene Transfer Techniques ; CRISPR-Cas Systems ; Tumor Microenvironment/drug effects ; Metabolic Reprogramming ; },
abstract = {The therapeutic effects of platinum-based drugs are closely linked to the dysregulation of tumor metabolic-immune microenvironment, particularly aberrant lactate accumulation. Herein, we engineered multifunctional nanoparticles (PPPt[IV] NPs) through electrostatic self-assembly of poly(β-amino ester) to co-encapsulate a cisplatin prodrug (Pt[IV]) and CRISPR/Cas9-PKM2 plasmids. Mechanistically, PPPt[IV] NPs efficiently entered cells via endocytosis, followed by escape from lysosomal degradation and cargo release. The reduction of Pt[IV] prodrug to active Pt[II] via GSH depletion induced DNA damage and ROS upregulation, thereby triggering apoptosis. Concurrently, CRISPR/Cas9-mediated PKM2 knockdown suppressed the Warburg effect, resulting in reduced lactate production and downregulated expression of HIF-1α and PD-L1. These alterations drove immune microenvironment remodeling through enhanced dendritic cell maturation, polarized M1 macrophages, and altered cytokine profiles (characterized by upregulation of IFN-γ, TNF-α, and IL-12 alongside suppression of IL-10), ultimately activating T cell-mediated antitumor immunity. Compared to conventional cisplatin, PPPt[IV] NPs demonstrated superior efficacy against both primary and recurrent tumors while reducing nephrotoxicity through synergistic chemo-immunotherapeutic effects, offering a valuable strategy for HNSCC treatment. STATEMENT OF SIGNIFICANCE: This study engineered an innovative nanoplatform (PPPt[IV]) that synergistically integrates a Pt[IV] prodrug with a CRISPR/Cas9-PKM2 plasmid for treating head and neck squamous cell carcinoma. By simultaneously enhancing DNA damage and reversing lactate-mediated immunosuppression, PPPt[IV] nanoplatform achieved chemo-immunotherapy that showed greater suppression of primary and recurrent tumors with reduced renal toxicity compared to cisplatin. This nanotechnology-driven strategy provides valuable insights into the combination of platinum-based drugs with immunometabolic interventions.},
}

*Squamous Cell Carcinoma of Head and Neck/therapy/pathology/metabolism/drug therapy/genetics
Humans
Animals
*Immunotherapy
*Nanoparticles/chemistry/therapeutic use
Cell Line, Tumor
*Cisplatin/pharmacology/chemistry/therapeutic use
*Head and Neck Neoplasms/therapy/pathology/metabolism/drug therapy
Prodrugs/pharmacology/chemistry
Mice
*Gene Transfer Techniques
CRISPR-Cas Systems
Tumor Microenvironment/drug effects
Metabolic Reprogramming

@article {pmid40184725,
year = {2025},
author = {Rahimian, E and Koochak, M and Traikov, S and Schroeder, M and Brilloff, S and Schäfer, S and Kufrin, V and Küchler, S and Krüger, A and Mirtschink, P and Baretton, G and Schröck, E and Schewe, DM and Ball, CR and Bornhäuser, M and Glimm, H and Bill, M and Wurm, AA},
title = {A quiescence-like/TGF-β1-specific CRISPRi screen reveals drug uptake transporters as secondary targets of kinase inhibitors in AML.},
journal = {Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy},
volume = {81},
number = {},
pages = {101242},
doi = {10.1016/j.drup.2025.101242},
pmid = {40184725},
issn = {1532-2084},
mesh = {Humans ; *Leukemia, Myeloid, Acute/drug therapy/genetics/pathology/mortality ; Cytarabine/pharmacology/therapeutic use ; *Transforming Growth Factor beta1/metabolism/genetics ; *Protein Kinase Inhibitors/pharmacology/therapeutic use ; Drug Resistance, Neoplasm/drug effects/genetics ; Receptor, Transforming Growth Factor-beta Type I/antagonists & inhibitors/genetics ; *Equilibrative Nucleoside Transporter 1/antagonists & inhibitors/genetics/metabolism ; Signal Transduction/drug effects ; Hematopoietic Stem Cells/drug effects/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Prognosis ; },
abstract = {Relapse in acute myeloid leukemia (AML) is driven by resistant subclones that survive chemotherapy. It is assumed that these resilient leukemic cells can modify their proliferative behavior by entering a quiescent-like state, similar to healthy hematopoietic stem cells (HSCs). These dormant cells can evade the effects of cytostatic drugs that primarily target actively dividing cells. Although quiescence has been extensively studied in healthy hematopoiesis and various solid cancers, its role in AML has remained unexplored. In this study, we applied an HSC-derived quiescence-associated gene signature to an AML patient cohort and found it to be strongly correlated with poor prognosis and active TGF-β signaling. In vitro treatment with TGF-β1 induces a quiescence-like phenotype, resulting in a G0 shift and reduced sensitivity to cytarabine. To find potential therapeutic targets that prevent AML-associated quiescence and improve response to cytarabine, we conducted a comprehensive CRISPR interference (CRISPRi) screen combined with TGF-β1 stimulation. This approach identified TGFBR1 inhibitors, like vactosertib, as effective agents for preventing the G0 shift in AML cell models. However, pretreatment with vactosertib unexpectedly induced complete resistance to cytarabine. To elucidate the underlying mechanism, we performed a multi-faceted approach combining a second CRISPRi screen, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and in silico analysis. Our findings revealed that TGFBR1 inhibitors unintentionally target the nucleoside transporter SLC29A1 (ENT1), leading to reduced intracellular cytarabine levels. Importantly, we found that this drug interaction is not unique to TGFBR1 inhibitors, but extends to other clinically significant kinase inhibitors, such as the FLT3 inhibitor midostaurin. These findings may have important implications for optimizing combination therapies in AML treatment.},
}

Humans
*Leukemia, Myeloid, Acute/drug therapy/genetics/pathology/mortality
Cytarabine/pharmacology/therapeutic use
*Transforming Growth Factor beta1/metabolism/genetics
*Protein Kinase Inhibitors/pharmacology/therapeutic use
Drug Resistance, Neoplasm/drug effects/genetics
Receptor, Transforming Growth Factor-beta Type I/antagonists & inhibitors/genetics
*Equilibrative Nucleoside Transporter 1/antagonists & inhibitors/genetics/metabolism
Signal Transduction/drug effects
Hematopoietic Stem Cells/drug effects/metabolism
CRISPR-Cas Systems
Cell Line, Tumor
Prognosis

@article {pmid40172925,
year = {2025},
author = {Grubben, J and Bijsterbosch, G and Aktürk, B and Visser, RGF and Schouten, HJ},
title = {CRISPR/Cas9-induced breaks are insufficient to break linkage drag surrounding the ToMV locus of Solanum lycopersicum.},
journal = {G3 (Bethesda, Md.)},
volume = {15},
number = {6},
pages = {},
doi = {10.1093/g3journal/jkaf068},
pmid = {40172925},
issn = {2160-1836},
support = {GSGT.2019.016//NWO Graduate School Groene Topsectoren/ ; TU18048//Dutch Topsector Horticulture & Starting Materials/ ; //BASF Nunhems/ ; //Bejo Zaden/ ; //HZPC/ ; //KWS/ ; //SESVanderHave/ ; //Syngenta/ ; //Wageningen University & Research, Plant Breeding/ ; },
mesh = {*CRISPR-Cas Systems ; *Solanum lycopersicum/genetics/virology ; Gene Editing ; *Genetic Linkage ; Polymorphism, Single Nucleotide ; DNA Breaks, Double-Stranded ; *Genetic Loci ; Recombination, Genetic ; Alleles ; },
abstract = {Despite the success of CRISPR/Cas9 in inducing DNA double-strand breaks for genome editing, achieving targeted recombination in somatic cells remains challenging, particularly at recombination cold spots like the tomato mosaic virus (ToMV) resistance locus in Solanum lycopersicum. We investigated the potential of CRISPR/Cas9-induced targeted recombination in somatic cells to overcome linkage drag surrounding the ToMV locus. We employed two strategies: first, inducing double-strand breaks in both alleles of F1 tomato seedlings to promote nonhomologous end joining and homology-directed repair; second, targeting a single allele in a heterozygous background to induce homology-directed repair in seedlings. CRISPR/Cas9 activity was confirmed in F1 seedlings by detecting nonhomologous end joining-mediated mutations at the target sites in ToMV. We developed a bioinformatics pipeline to identify targeted recombinants by analyzing SNPs between parental haplotypes, allowing precise tracking of SNP variations. A two-dimensional pooling strategy was employed to distinguish genuine recombination events from PCR artifacts. Despite these advances and the active CRISPR/Cas9 system in F1 progeny, no reliable targeted recombinations were found. We extended our research to protoplasts to assess whether CRISPR/Cas9 could induce targeted recombination under different cellular conditions at the same locus. Consistent with our findings in F1 plants, we observed no increase in recombinant patterns compared to wild-type controls in protoplasts. Our findings suggest that CRISPR/Cas9-induced DSBs were insufficient to break the genetic linkage at the ToMV locus on chromosome 9 at a detectable level.},
}

*CRISPR-Cas Systems
*Solanum lycopersicum/genetics/virology
Gene Editing
*Genetic Linkage
Polymorphism, Single Nucleotide
DNA Breaks, Double-Stranded
*Genetic Loci
Recombination, Genetic
Alleles

@article {pmid40147870,
year = {2025},
author = {Dunipace, L and McGehee, JM and Irizarry, J and Stathopoulos, A},
title = {The proximal enhancer of the snail gene mediates negative autoregulatory feedback in Drosophila melanogaster.},
journal = {Genetics},
volume = {230},
number = {2},
pages = {},
doi = {10.1093/genetics/iyaf058},
pmid = {40147870},
issn = {1943-2631},
support = {R35GM118146//National Institute of Health/ ; },
mesh = {Animals ; *Drosophila melanogaster/genetics/embryology ; *Enhancer Elements, Genetic ; *Drosophila Proteins/genetics/metabolism ; *Snail Family Transcription Factors/genetics/metabolism ; Gene Expression Regulation, Developmental ; *Transcription Factors/genetics/metabolism ; Homeostasis ; Feedback, Physiological ; Embryonic Development/genetics ; CRISPR-Cas Systems ; Nuclear Proteins ; Phosphoproteins ; },
abstract = {Autoregulatory feedback is a mechanism in which a gene product regulates its own expression, stabilizing gene activity amid noise and environmental changes. In Drosophila melanogaster, the gene snail encodes a key transcriptional repressor that regulates the expression of many genes during early embryogenesis, including its own expression. This study focuses on Snail occupancy at both distal and proximal enhancers of the snail gene to understand the cis-regulatory mechanisms involved in autoregulatory control. The coordinated action of these enhancers results in precisely constrained levels of snail expression during early embryogenesis. Using genome editing by CRISPR/Cas9, we found that deletion of each enhancer individually is compatible with embryonic viability under normal conditions. However, the double mutant is lethal, suggesting a functional interplay between the 2 enhancers. To gain further insight, we assayed snail gene expression levels in fixed embryos. Our results revealed that negative autoregulation of snail relies on the proximal enhancer. Moreover, increasing the affinity of binding sites for Dorsal, a transcriptional activator, in the proximal enhancer impaired this autoregulation, suggesting that Snail acts locally to counterbalance Dorsal's input. A mathematical model of snail autoregulatory control further supports our findings, reinforcing the view that the proximal enhancer mediates negative autoregulatory feedback, and implicating the distal enhancer in positive autoregulatory feedback. In summary, Snail's role at the proximal enhancer is pivotal for negative autoregulatory control and essential for balancing the activation mediated by the distal enhancer.},
}

Animals
*Drosophila melanogaster/genetics/embryology
*Enhancer Elements, Genetic
*Drosophila Proteins/genetics/metabolism
*Snail Family Transcription Factors/genetics/metabolism
Gene Expression Regulation, Developmental
*Transcription Factors/genetics/metabolism
Homeostasis
Feedback, Physiological
Embryonic Development/genetics
CRISPR-Cas Systems
Nuclear Proteins
Phosphoproteins

@article {pmid39898499,
year = {2025},
author = {Deivarajan, HR and Chelliah, D and Sethupathy Ramkumar, P and Nandhakumar, D and Chacko, BM and Raghavan, A and Pandian, J and Prajna, L and Prajna, VN and Narendran, S},
title = {Clinical Utility of CRISPR-Based RID-MyC Assay in Smear and Culture-Negative Fungal Keratitis: A Case Series.},
journal = {Cornea},
volume = {44},
number = {7},
pages = {921-924},
pmid = {39898499},
issn = {1536-4798},
support = {1465//Velux Stiftung/ ; },
mesh = {Humans ; *Eye Infections, Fungal/diagnosis/microbiology/drug therapy ; Retrospective Studies ; Male ; Female ; Middle Aged ; Microscopy, Confocal ; Adult ; *Keratitis/microbiology/diagnosis ; *Fungi/genetics/isolation & purification ; *DNA, Fungal/analysis/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Cas Systems ; *Corneal Ulcer/microbiology/diagnosis ; Antifungal Agents/therapeutic use ; Aged ; },
abstract = {PURPOSE: To assess the clinical utility of the clustered regularly interspaced short palindromic repeats/Cas12a-based RID-MyC assay in diagnosing fungal keratitis (FK) in cases where conventional smear and culture methods fail to identify the causative pathogen.

METHODS: This retrospective case series included 5 patients with clinically suspected FK and negative smear and culture results who were evaluated in the Cornea Department at Aravind Eye Hospital, Coimbatore, India, between January 1, 2024, and March 31, 2024. The primary outcome was the diagnostic performance of the RID-MyC assay in detecting fungal nucleic acids in cases of suspected FK with negative smear and culture results. In vivo confocal microscopy served as a reference standard to validate the RID-MyC assay findings.

RESULTS: The RID-MyC assay successfully detected fungal nucleic acids in 3 cases, corroborated by in vivo confocal microscopy findings suggestive of fungal filaments, leading to targeted antifungal therapy and resolution of the infections. In addition, 2 cases tested negative for fungal nucleic acids, aligning with clinical and confocal evidence of nonfungal etiology, thus guiding appropriate alternative treatments that led to clinical improvement.

CONCLUSIONS: The RID-MyC assay demonstrates clinical utility in diagnosing FK in scenarios where conventional smear and culture methods prove inadequate, such as in cases with prior antifungal therapy or polymicrobial infections. This assay facilitates accurate diagnosis and timely initiation of appropriate antifungal treatment without the need for sophisticated equipment or expertise, making it particularly valuable in resource-limited settings.},
}

Humans
*Eye Infections, Fungal/diagnosis/microbiology/drug therapy
Retrospective Studies
Male
Female
Middle Aged
Microscopy, Confocal
Adult
*Keratitis/microbiology/diagnosis
*Fungi/genetics/isolation & purification
*DNA, Fungal/analysis/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
*CRISPR-Cas Systems
*Corneal Ulcer/microbiology/diagnosis
Antifungal Agents/therapeutic use
Aged

@article {pmid40440869,
year = {2025},
author = {Zhang, R and Zhou, Q and Huang, S and Zhang, N and Sun, D},
title = {Advancements in CRISPR-Cas-based strategies for combating antimicrobial resistance.},
journal = {Microbiological research},
volume = {298},
number = {},
pages = {128232},
doi = {10.1016/j.micres.2025.128232},
pmid = {40440869},
issn = {1618-0623},
abstract = {Multidrug resistance (MDR) in bacteria presents a significant global health threat, driven by the widespread dissemination of antibiotic-resistant genes (ARGs). The CRISPR-Cas system, known for its precision and adaptability, holds promise as a tool to combat antimicrobial resistance (AMR). Although previous studies have explored the use of CRISPR-Cas to target bacterial genomes or plasmids harboring resistance genes, the application of CRISPR-Cas-based antimicrobial therapies is still in its early stages. Challenges such as low efficiency and difficulties in delivering CRISPR to bacterial cells remain. This review provides an overview of the CRISPR-Cas system, highlights recent advancements in CRISPR-Cas-based antimicrobials and delivery strategies for combating AMR. The review also discusses potential challenges for the future development of CRISPR-Cas-based antimicrobials. Addressing these challenges would enable CRISPR therapies to become a practical solution for treating AMR infections in the future.},
}

@article {pmid40440223,
year = {2025},
author = {Bachler, A and Padovan, A and Anderson, CJ and Wei, Y and Wu, Y and Pearce, S and Downes, S and James, B and Tessnow, AE and Sword, GA and Williams, M and Tay, WT and Gordon, KHJ and Walsh, TK},
title = {Disruption of HaVipR1 confers Vip3Aa resistance in the moth crop pest Helicoverpa armigera.},
journal = {PLoS biology},
volume = {23},
number = {5},
pages = {e3003165},
pmid = {40440223},
issn = {1545-7885},
mesh = {Animals ; *Moths/genetics/drug effects ; *Insecticide Resistance/genetics ; *Bacterial Proteins/pharmacology/genetics ; Insect Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Crops, Agricultural/genetics/parasitology ; Pest Control, Biological/methods ; Plants, Genetically Modified/genetics ; Bacillus thuringiensis/genetics ; Helicoverpa armigera ; },
abstract = {The global reliance on Bacillus thuringiensis (Bt) proteins for controlling lepidopteran pests in cotton, corn, and soybean crops underscores the critical need to understand resistance mechanisms. Vip3Aa, one of the most widely deployed and currently effective Bt proteins in genetically modified crops, plays a pivotal role in pest management. This study investigates the molecular basis of Vip3Aa resistance in Australian Helicoverpa armigera through genetic crosses, and integrated genomic and transcriptomic analyses. We identified a previously uncharacterized gene, LOC110373801 (designated HaVipR1), as potentially important in Vip3Aa resistance in two field-derived resistant lines. Functional validation using CRISPR/Cas9 knockout in susceptible lines confirmed the gene's role in conferring high-level resistance to Vip3Aa. Despite extensive laboratory selection of Vip3Aa-resistant colonies in Lepidoptera, the biochemical mechanisms underlying resistance have remained elusive. Our research identifies HaVipR1 as a potential contributor to resistance, adding to our understanding of how insects may develop resistance to this important Bt protein. The identification of HaVipR1 contributes to our understanding of potential resistance mechanisms and may inform future resistance management strategies. Future work should explore the biochemical pathways influenced by HaVipR1 and assess its interactions with other resistance mechanisms. The approach utilized here underscores the value of field-derived resistant lines for understanding resistance in agricultural pests and highlights the need for targeted approaches to manage resistance sustainably.},
}

Animals
*Moths/genetics/drug effects
*Insecticide Resistance/genetics
*Bacterial Proteins/pharmacology/genetics
Insect Proteins/genetics/metabolism
CRISPR-Cas Systems
Crops, Agricultural/genetics/parasitology
Pest Control, Biological/methods
Plants, Genetically Modified/genetics
Bacillus thuringiensis/genetics
Helicoverpa armigera

@article {pmid40439284,
year = {2025},
author = {Ukita, Y and Suzuki, R and Miyoshi, K and Saito, K and Okumura, M and Chihara, T},
title = {Generation of Odorant Receptor-QF2 Knock-In Drivers for Improved Analysis of Olfactory Circuits in Drosophila.},
journal = {Genes to cells : devoted to molecular & cellular mechanisms},
volume = {30},
number = {4},
pages = {e70028},
pmid = {40439284},
issn = {1365-2443},
support = {21H02479//Japan Society for the Promotion of Science/ ; 24K02062//Japan Society for the Promotion of Science/ ; 20K15903//Japan Society for the Promotion of Science/ ; JP23KJ1645//Japan Society for the Promotion of Science/ ; },
mesh = {Animals ; *Receptors, Odorant/genetics/metabolism ; *Drosophila melanogaster/genetics/metabolism ; *Drosophila Proteins/genetics/metabolism ; Gene Knock-In Techniques/methods ; CRISPR-Cas Systems ; Animals, Genetically Modified ; Olfactory Receptor Neurons/metabolism ; *Olfactory Pathways/metabolism ; },
abstract = {Drosophila melanogaster has provided numerous insights into the olfactory system, primarily relying on a series of transgenic Gal4 drivers. The combined use of Gal4/UAS and a second binary expression system, such as the QF/QUAS system, provides the opportunity to manipulate the two distinct cell populations, thereby accelerating the elucidation of the olfactory neural mechanisms. However, resources apart from the Gal4/UAS system have been poorly developed. In this study, we generated a series of odorant receptor (Or)-QF2 knock-in driver (Or-QF2[KI]) lines for 23 Ors using the CRISPR/Cas9 knock-in method. In these lines, the QF2 protein is cotranslated with each Or product. The expression pattern of the Or-QF2[KI] drivers mostly corresponded to that of the Or-Gal4 drivers. In addition, the Or42a-QF2[KI] driver identified the additional expression pattern of Or42a, which is consistent with the data of single-nucleus RNA sequencing and is attributed to the Or-QF2[KI] drivers' ability to reflect the endogenous expression of the Or genes. Thus, these Or-QF2[KI] drivers can be used as valuable genetic tools for olfactory research in Drosophila.},
}

Animals
*Receptors, Odorant/genetics/metabolism
*Drosophila melanogaster/genetics/metabolism
*Drosophila Proteins/genetics/metabolism
Gene Knock-In Techniques/methods
CRISPR-Cas Systems
Animals, Genetically Modified
Olfactory Receptor Neurons/metabolism
*Olfactory Pathways/metabolism

@article {pmid40438494,
year = {2025},
author = {Shao, Q and Ndzie Noah, ML and Golubnitschaja, O and Zhan, X},
title = {Mitochondrial medicine: "from bench to bedside" 3PM-guided concept.},
journal = {The EPMA journal},
volume = {16},
number = {2},
pages = {239-264},
pmid = {40438494},
issn = {1878-5077},
abstract = {Mitochondria are the primary sites for aerobic respiration and play a vital role in maintaining physiologic function at the cellular and organismal levels. Physiologic mitochondrial homeostasis, functions, health, and any kind of mitochondrial impairments are associated with systemic effects that are linked to the human health and pathologies. Contextually, mitochondria are acting as a natural vital biosensor in humans controlling status of physical and mental health in a holistic manner. So far, no any disorder is known as happening to humans independently from a compromised mitochondrial health as the cause (primary mitochondrial dysfunction) or a target of collateral damage (secondary mitochondrial injury). This certainty makes mitochondrial medicine be the superior instrument to reach highly ambitious objectives of predictive, preventive, and personalized medicine (PPPM/3PM). 3PM effectively implements the paradigm change from the economically ineffective reactive medical services to a predictive approach, targeted prevention and treatments tailored to individualized patient profiles in primary (protection against health-to-disease transition) and secondary (protection against disease progression) healthcare. Mitochondrial DNA (mtDNA) properties differ significantly from those of nuclear DNA (nDNA). For example, mtDNA as the cell-free DNA molecule is much more stable compared to nDNA, which makes mtDNA be an attractive diagnostic target circulating in human body fluids such as blood and tear fluid. Further, genetic variations in mtDNA contribute to substantial individual differences in disease susceptibility and treatment response. To this end, the current gene editing technologies, such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas, are still immature in mtDNA modification, and cannot be effectively applied in clinical practice posing a challenge for mtDNA-based therapies. In contrast, comprehensive multiomics technologies offer new insights into mitochondrial homeostasis, health, and functions, which enables to develop more effective multi-level diagnostics and targeted treatment strategies. This review article highlights health- and disease-relevant mitochondrial particularities and assesses involvement of mitochondrial medicine into implementing the 3PM objectives. By discussing the interrelationship between 3PM and mitochondrial medicine, we aim to provide a foundation for advancing early and predictive diagnostics, cost-effective targeted prevention in primary and secondary care, and exemplify personalized treatments creating proof-of-concept approaches for 3PM-guided clinical applications.},
}

@article {pmid40437542,
year = {2025},
author = {Li, C and Zha, H and Jiao, Z and Wei, K and Gao, H and Lai, F and Zhou, Z and Luo, H and Li, P},
title = {Genetic engineering of E. coli K-12 for heterologous carbohydrate antigen production.},
journal = {Microbial cell factories},
volume = {24},
number = {1},
pages = {126},
pmid = {40437542},
issn = {1475-2859},
support = {CSTB2024NSCQ-MSX1157//Natural Science Foundation of Chongqing, China/ ; SWU-KT23010//Fundamental Research Funds for the Central Universities/ ; 32300736//National Natural Science Foundation of China/ ; },
mesh = {*Genetic Engineering/methods ; Animals ; *Escherichia coli K12/genetics/metabolism ; Mice ; Multigene Family ; Mice, Inbred BALB C ; },
abstract = {BACKGROUND: Carbohydrate-based vaccines have made a remarkable impact on public health over the past three decades. Efficient production of carbohydrate antigens is a crucial prerequisite for the development of such vaccines. The enzymes involved in the synthesis of bacterial surface carbohydrate antigens are usually encoded by large, uninterrupted gene clusters. Non-pathogenic E. coli glycoengineering starts with the genetic manipulation of these clusters. Heterologous gene cluster recombination through an expression plasmid has several drawbacks, including continuous antibiotic selection pressure, genetic instability, and metabolic burdens. In contrast, chromosome-level gene cluster expression can minimize the metabolic effects on the host and reduce industrial costs.

RESULTS: In this study, we employed the suicide vector-mediated allelic exchange method to directly replace the native polysaccharide gene clusters in E. coli with heterologous ones. Unlike previously strategies, this method does not rely on I-SceI endonuclease or CRISPR/Cas system to release the linearized DNA insert and λ-red recombinase to promote its homologous recombination. Meanwhile, the vectors could be conveniently constructed by assembling multiple large DNA fragments in order in vitro. The scarless chromosomal insertions were confirmed by whole-genome sequencing and the polysaccharide phenotypes of all glycoengineered E. coli mutants were evaluated through growth curves, silver staining, western blot, and flow cytometry. The data indicated that there was no obvious metabolic burden associated with the insertion of large gene clusters into the E. coli W3110 O-antigen locus, and the glycoengineered E. coli can produce LPS with a recovery rate around 1% of the bacterial dry weight. Moreover, the immunogenicity of the heterologously expressed carbohydrate antigens was analyzed by mice immunization experiments. The ELISA data demonstrated the successful induction of anti-polysaccharide IgM or IgG antibodies.

CONCLUSIONS: We have provided a convenient and reliable genomic glycoengineering method to produce efficacious, durable, and cost-effective carbohydrate antigens in non-pathogenic E. coli. Non-pathogenic E. coli glycoengineering has great potential for the highly efficient synthesis of heterologous polysaccharides and can serve as a versatile platform to produce next-generation biomedical agents, including glycoconjugate vaccines, glycoengineered minicells or outer membrane vesicles (OMVs), polysaccharide-based diagnostic reagents, and more.},
}

*Genetic Engineering/methods
Animals
*Escherichia coli K12/genetics/metabolism
Mice
Multigene Family
Mice, Inbred BALB C

@article {pmid40437234,
year = {2025},
author = {Vanhooydonck, M and De Neef, E and De Saffel, H and Boel, A and Willaert, A and Callewaert, B and Claes, KBM},
title = {Prime editing outperforms homology-directed repair as a tool for CRISPR-mediated variant knock-in in zebrafish.},
journal = {Lab animal},
volume = {54},
number = {6},
pages = {165-172},
pmid = {40437234},
issn = {1548-4475},
support = {BOF21/DOC/242//Universiteit Gent (UGent)/ ; BOF GOA019-21//Universiteit Gent (UGent)/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Gene Knock-In Techniques/methods ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Recombinational DNA Repair ; Animals, Genetically Modified ; },
abstract = {Zebrafish serve as a valuable model organism for studying human genetic diseases. While generating knockout lines is relatively straightforward, introducing precise disease-specific genetic variants by knock-in (KI) remains challenging. KI lines, however, enable more accurate studies of molecular and physiological consequences of genetic diseases. Their generation is often hampered by low editing efficiency (EE) and potential off-target effects. Here, we optimized conventional CRISPR-Cas9-mediated homology-directed repair (HDR) strategies for precise KI of genetic variants in zebrafish and compared their efficacy with prime editing, a recently developed technique that is not yet commonly used. Using next-generation sequencing, we determined KI EE by HDR for six unique base-pair substitutions in three different zebrafish genes. We assessed the effect of (1) varying Cas9 amounts, (2) HDR templates with chemical modifications to improve integration efficiency, (3) different microinjection procedures and (4) introduction of additional synonymous guide-blocking variants in the HDR template. Increasing Cas9 amounts augmented KI EE, with optimal injected amounts of Cas9 between 200 pg and 800 pg. The use of Alt-R HDR templates further increased KI EE, while guide-blocking modifications did not. Injecting components directly into the cell was not superior to injections into the yolk. Prime editing, however, increased EE up to fourfold and expanded the F0 founder pool for four targets compared with conventional HDR editing, with fewer off-target effects. Therefore, prime editing is a very promising methodology for improving the creation of precise genomic edits in zebrafish, facilitating the modeling of human diseases.},
}

Animals
*Zebrafish/genetics
*Gene Knock-In Techniques/methods
*Gene Editing/methods
*CRISPR-Cas Systems
*Recombinational DNA Repair
Animals, Genetically Modified

@article {pmid40318489,
year = {2025},
author = {Sun, J and Bai, J and Huang, Y and Langford, PR and Zhang, Y and Li, G},
title = {A CRISPR/Cas12a-based DNAzyme visualization platform for rapid discrimination of Streptococcus suis serotype 2 versus 1/2 and serotype 1 versus 14.},
journal = {Talanta},
volume = {294},
number = {},
pages = {128241},
doi = {10.1016/j.talanta.2025.128241},
pmid = {40318489},
issn = {1873-3573},
mesh = {*Streptococcus suis/genetics/classification/isolation & purification ; *CRISPR-Cas Systems/genetics ; Serogroup ; *DNA, Catalytic/metabolism/chemistry/genetics ; Polymorphism, Single Nucleotide ; Nucleic Acid Amplification Techniques ; Animals ; Swine ; Colorimetry ; G-Quadruplexes ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Streptococcus suis is a major swine pathogen with serotypes 2 and 14 posing zoonotic risks. However, distinguishing serotypes 1/2 from 2 or 1 from 14 remains challenging due to high similarity in their capsule polysaccharide (CPS) loci. Here, we developed a rapid, equipment-free discriminating platform targeting a single nucleotide polymorphism (SNP) at position 483 of the cpsK gene (G in serotypes 2/14 vs. T/C in 1/2/1). The method integrates recombinase polymerase amplification (RPA) with CRISPR/Cas12a and a G-quadruplex-hemin DNAzyme visualization system. RPA enables isothermal amplification, while CRISPR/Cas12a ensures single-nucleotide specificity by cleaving target DNA. Subsequent DNAzyme catalysis converts colorimetric substrates, enabling naked-eye differentiation via distinct color changes (blue for serotypes 1/2/1 vs. colorless for 2/14). This approach achieved a sensitivity of 10[1]-10[2] copies per reaction and demonstrated 100 % specificity across 29 S. suis serotypes and related strains. Compared to PCR-based or sequencing methods, our platform eliminates reliance on thermocyclers or fluorescence detectors, reducing costs and operational complexity. The entire workflow, completed within 70 min, offers a practical solution for point-of-care testing in resource-limited settings. By enabling rapid, accurate discrimination, this tool will become a complementary tool for resolving ambiguous serotypes and enhances outbreak management in swine populations and mitigates zoonotic transmission.},
}

*Streptococcus suis/genetics/classification/isolation & purification
*CRISPR-Cas Systems/genetics
Serogroup
*DNA, Catalytic/metabolism/chemistry/genetics
Polymorphism, Single Nucleotide
Nucleic Acid Amplification Techniques
Animals
Swine
Colorimetry
G-Quadruplexes
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40300474,
year = {2025},
author = {Zhu, T and Jiang, W and Wu, Y and Fang, R and Deng, F and Yang, D},
title = {Advances in CRISPR/Cas13a-based biosensors for non-coding RNA detection.},
journal = {Talanta},
volume = {294},
number = {},
pages = {128223},
doi = {10.1016/j.talanta.2025.128223},
pmid = {40300474},
issn = {1873-3573},
mesh = {*Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *RNA, Untranslated/analysis/genetics ; },
abstract = {Non-coding RNAs play crucial roles in disease initiation and progression, making them promising biomarkers for early diagnosis and treatment monitoring. Conventional nucleic acid diagnostic methods, including polymerase chain reaction (PCR), next-generation sequencing (NGS), and enzyme-linked immunosorbent assay (ELISA), alongside emerging techniques such as single-molecule fluorescence in situ hybridization (smFISH), nanopore sequencing, and single-cell RNA sequencing (scRNA-seq), face inherent limitations in detecting regulatory non-coding RNAs. These challenges include laborious workflows, prolonged processing times, and technical complexities, hindering their broad applicability in rapid and high-throughput RNA analysis. CRISPR/Cas13a-based biosensors, integrated with various signal transduction systems-such as fluorescence, electrochemistry, colorimetry, surface-enhanced Raman spectroscopy (SERS)-show great promise for real-world diagnostic applications. This review provides a comprehensive overview of the CRISPR/Cas13a-mediated RNA detection mechanism, the development of CRISPR/Cas13a-based biosensors, and their integration with innovative signal detection methods. Additionally, we highlight the progress in portable detection devices, including lateral flow assay strips and smartphone-based platforms. Finally, the review discusses the current challenges and future prospects of CRISPR/Cas13a-based biosensors, particularly in the context of clinical diagnostics and personalized medicine.},
}

*Biosensing Techniques/methods
Humans
*CRISPR-Cas Systems/genetics
*RNA, Untranslated/analysis/genetics

@article {pmid40262340,
year = {2025},
author = {Li, X and Li, Y and Wang, C and Chai, J and Liu, H and Jiang, X and Li, Y},
title = {Structure-switchable dsDNA promoter regulates the activity of CRISPR-Cas12a for APE1 detection.},
journal = {Talanta},
volume = {294},
number = {},
pages = {128161},
doi = {10.1016/j.talanta.2025.128161},
pmid = {40262340},
issn = {1873-3573},
mesh = {*DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics/analysis/metabolism/antagonists & inhibitors ; Humans ; *CRISPR-Cas Systems/genetics ; *Promoter Regions, Genetic ; *DNA/genetics/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Apurinic/apyrimidinic endonuclease 1 (APE1) has been considered as a promising biomarker since it is associated with numerous human diseases, involving neurodegenerative diseases and cancer. However, current APE1 detection methods mainly rely on immunology-based methods, which are burdened by time-consuming and procedural complexity. To overcome these shortcomings, we have developed an innovative all-in-one technique that simplifies APE1 detection by integrating enzyme-responsive elements structure-switchable dsDNA promoter with CRISPR/AsCas12a methodology, namely EDC. In this work, the structure-switchable dsDNA promoter has been well-designed to trigger the site-directed incision of APE1 and then release the split activator to illumine the CRISPR/AsCas12a catalyst system by coupling it with another truncated activator. Under optimal circumstances, the proposed strategy enables sensitive detection of the target APE1 with a detection limit of 4.8 × 10[-5] U/mL and a wide linear range from 5.0 × 10[-5] to 1.0 × 10[-1] U/mL. Moreover, this strategy could be gratifyingly applied to screen APE1 inhibitors and monitor APE1 in lysates from cell extractions or clinical serum samples. Overall, this study presents a novel approach that utilizes dsDNA promoter as programmable switching components, effectively enhancing CRISPR/Cas12a-based diagnostic platforms and demonstrating the significant potential for clinical translation.},
}

*DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics/analysis/metabolism/antagonists & inhibitors
Humans
*CRISPR-Cas Systems/genetics
*Promoter Regions, Genetic
*DNA/genetics/chemistry
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40167233,
year = {2025},
author = {Kim, EJ and Hong, WJ and Kim, YJ and Kim, EY and Yun, SD and Moon, S and Lee, SK and Park, SK and Jung, KH},
title = {Global identification of key genes for pollen germination in rice through high-throughput screening and gene editing.},
journal = {Journal of integrative plant biology},
volume = {67},
number = {6},
pages = {1665-1684},
doi = {10.1111/jipb.13900},
pmid = {40167233},
issn = {1744-7909},
support = {RS-2024-00322278//Rural Development Administration/ ; RS-2021-NR060084//National Research Foundation of Korea/ ; RS-2021-NR062086//National Research Foundation of Korea/ ; NRF-2021R1A2C2010124//National Research Foundation of Korea/ ; RS-2023-00217064//National Research Foundation of Korea/ ; RS-2024-00344197//National Research Foundation of Korea/ ; },
mesh = {*Oryza/genetics ; *Pollen/genetics/growth & development ; *Germination/genetics ; *Gene Editing/methods ; *Genes, Plant/genetics ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems/genetics ; *High-Throughput Screening Assays ; Plant Infertility/genetics ; Mutation/genetics ; DNA, Bacterial ; },
abstract = {Successful reproduction depends on the stable germination and growth of the pollen tubes (PT). However, the molecular mechanisms involved in rice PT growth and development remain largely unknown. In a previous study, microarray transcriptome analysis identified 627 genes preferentially expressed in the tricellular and germinating pollen of rice (i.e., Oryza sativa ssp. japonica). To elucidate key genes involved in the gene transfer process facilitated by male gametophytes, we systematically screened T-DNA lines containing disrupted sequences that corresponded to these 627 genes and analyzed the genotypes of heterozygote progeny from 107 T-DNA-indexed lines covering 105 genes. We found that 42 lines exhibited a distorted segregation ratio among the wild-type (WT), heterozygote (HT), and homozygote (HM) genotypes, which deviated from the expected Mendelian ratio of 1:2:1 (WT:HT:HM). Further characterization using CRISPR/Cas9 mutants revealed that knockout mutants of certain genes that exhibited segregation distortion in the T-DNA insertion region were completely sterile. Moreover, even when T-DNA insertion lines followed Mendelian segregation patterns, sterility could be induced by simultaneously mutating functionally redundant genes, thereby overcoming genetic compensation. Interestingly, although some T-DNA insertion lines exhibited segregation ratios approximating 1:1:0, the corresponding CRISPR/Cas9 mutants produced homozygous seeds and showed partial sterility. Partial sterility suggests that despite mutant pollen grains being less competitive than WT pollen, they retain their fertilization potential under relaxed competition from WT pollen. Beyond mutant-based analysis, transcriptomic profiling of sterile mutant lines provided additional insight into the regulatory relationship between key germination regulators and the 105 target genes studied here. Overall, this study demonstrates the effectiveness of a multi-pronged strategy to accelerate the identification of defective phenotypes using mutant studies and provides valuable genetic resources for inducing novel male sterility in rice.},
}

*Oryza/genetics
*Pollen/genetics/growth & development
*Germination/genetics
*Gene Editing/methods
*Genes, Plant/genetics
Gene Expression Regulation, Plant
CRISPR-Cas Systems/genetics
*High-Throughput Screening Assays
Plant Infertility/genetics
Mutation/genetics
DNA, Bacterial

@article {pmid40052456,
year = {2025},
author = {Yin, T and Tai, Y and Sun, Y and Cheng, Z and Wu, C and Sui, Y},
title = {Precise tiller angle control by manipulating TAC1 expression in rice.},
journal = {Journal of integrative plant biology},
volume = {67},
number = {6},
pages = {1444-1446},
doi = {10.1111/jipb.13877},
pmid = {40052456},
issn = {1744-7909},
support = {2024ZD04077//Biological Breeding-National Science and Technology Major Project/ ; 31801323//National Natural Science Foundation of China/ ; //Innovation Program of the Chinese Academy of Agricultural Sciences/ ; },
mesh = {*Oryza/genetics/metabolism/anatomy & histology ; *Plant Proteins/metabolism/genetics ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems/genetics ; Gene Editing ; Phenotype ; },
abstract = {Tiller angle shapes plant architecture, and is one of the top traits in plant breeding. A compact plant type reduces shading between plants, especially at high planting density, but also creates a humid microenvironment often associated with a higher incidence of pathogen and pest attacks, especially under highly humid climates. However, how to precisely manipulate the tiller angle to achieve a desirable plant type has been under-approached. Here we report the creation of gradient tiller angles in indica rice by fine tuning the expression of TILLER ANGLE CONTROL1 (TAC1), a domesticated gene in cultivated rice. We edited the regions upstream and downstream of the TAC1 coding sequence using multiplex CRISPR-Cas9 technology and developed homozygous allelic lines carrying deletions/inversions of various sizes at different positions. Those lines displayed smooth gradient changes in tiller angle that aligned well with TAC1 expression levels. Additionally, changes in the TAC1 expression level had no impact on other agronomic traits examined. TAC1 is well conserved across species, including perennial fruit trees in which mutation of TAC1 orthologs leads to a broomy plant type. Thus, our results provide a guide to creating tiller angles for selection according to climate zones in rice breeding programs, this approach can be extended to diverse species for improving plant architecture.},
}

*Oryza/genetics/metabolism/anatomy & histology
*Plant Proteins/metabolism/genetics
Gene Expression Regulation, Plant
CRISPR-Cas Systems/genetics
Gene Editing
Phenotype

@article {pmid39950533,
year = {2025},
author = {Xue, L and Qu, P and Zhao, H and Liu, H and Huang, B and Wang, X and Zhang, Z and Dai, X and Qin, L and Dong, W and Shi, L and Zhang, X},
title = {Creation of fragrant peanut using CRISPR/Cas9.},
journal = {Journal of integrative plant biology},
volume = {67},
number = {6},
pages = {1438-1440},
doi = {10.1111/jipb.13864},
pmid = {39950533},
issn = {1744-7909},
support = {2022YFD1200400//National Key Research and Development Program of China/ ; 201300111000//Key Project of Science and Technology of Henan Province/ ; 221100110300//Key Project of Science and Technology of Henan Province/ ; CARS-13//the Earmarked Fund/ ; S2012-5//Fund of Henan Province Agriculture Research System/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Arachis/genetics/metabolism ; Mutation/genetics ; Betaine-Aldehyde Dehydrogenase/genetics/metabolism ; },
abstract = {Targeted knockout of the betaine aldehyde dehydrogenase genes AhBADH1 and AhBADH2 using CRISPR/Cas9 produced peanut mutant lines with significantly elevated 2-acetyl-1-pyrroline levels and a strong aroma, marking the first creation of fragrant peanut lines.},
}

*CRISPR-Cas Systems/genetics
*Arachis/genetics/metabolism
Mutation/genetics
Betaine-Aldehyde Dehydrogenase/genetics/metabolism

@article {pmid40436684,
year = {2025},
author = {Jin, YY and Zhang, P and Liu, DP},
title = {Optimizing homology-directed repair for gene editing: the potential of single-stranded DNA donors.},
journal = {Trends in genetics : TIG},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tig.2025.04.014},
pmid = {40436684},
issn = {0168-9525},
abstract = {CRISPR (clustered regularly interspaced short palindromic repeat) system-based precise genome editing remarkably impacts both scientific investigation and therapeutic practices. Among various techniques, DNA donor-mediated homology-directed repair (HDR) represents a promising method for precise gene editing. Although efficiency constraints have previously limited HDR, recent advancements have significantly enhanced its effectiveness. Therefore, it is essential to highlight the progress made in this field and to reassess the potential of the HDR approach. In this review, we explore the fundamental principles of HDR-dependent gene editing and evaluate current strategies to enhance HDR efficiency, with particular emphasis on single-stranded DNA (ssDNA) donor-mediated HDR. Finally, we discuss the prospects of high-efficiency ssDNA donor-mediated precise gene editing in laboratory research and clinical therapies.},
}

@article {pmid40435438,
year = {2025},
author = {Shu, F and Huang, Y and Yang, F and Guo, Y and Xu, R and Xiao, L and Feng, Y and Li, N},
title = {Calcium-dependent protein kinases 2A involved in the growth of both asexual and sexual stages of Cryptosporidium parvum.},
journal = {PLoS neglected tropical diseases},
volume = {19},
number = {5},
pages = {e0013107},
pmid = {40435438},
issn = {1935-2735},
mesh = {*Cryptosporidium parvum/growth & development/enzymology/genetics ; Animals ; Cryptosporidiosis/parasitology ; Mice ; *Protein Kinases/genetics/metabolism ; CRISPR-Cas Systems ; *Protozoan Proteins/genetics/metabolism ; Oocysts/growth & development ; Female ; Humans ; Life Cycle Stages ; Sporozoites/growth & development ; },
abstract = {BACKGROUND: Cryptosporidium parvum is a protozoan pathogen that causes moderate to severe diarrhea in both humans and animals. Calcium-dependent protein kinases (CDPKs) are attractive drug targets against cryptosporidiosis given their critical role in the life cycle of Cryptosporidium spp. and their absence in human and animal hosts.

We used CRISPR-Cas9 technology to endogenously tag the CpCDPK2A gene in C. parvum IIdA20G1-HLJ strain with the hemagglutinin (HA) epitope and to delete the CpCDPK2A gene. An immunofluorescence assay was performed to localize the CpCDPK2A expression in the tagged strain and a luciferase assay was performed to compare growth rates of the tagged and deletion strains in vitro. Oocyst shedding, parasite load, villus length/crypt height ratio and survival of infected mice were used to evaluate the function of CpCDPK2A in vivo. The results revealed that CpCDPK2A was expressed in all the intracellular developmental stages, especially in the motile stages of sporozoites and merozoites. While CpCDPK2A is dispensable, deletion of the gene significantly reduced the growth of late asexual and sexual stages in vitro. In an interferon-γ knockout mouse model, gene deletion of CpCDPK2A reduced oocyst shedding by 25-fold and increased survival of infected mice.

CONCLUSIONS/SIGNIFICANCE: These observations suggest that CpCDPK2A may contribute to both asexual and sexual replication of C. parvum and may be a potential target to block the transmission of this important zoonotic pathogen.},
}

*Cryptosporidium parvum/growth & development/enzymology/genetics
Animals
Cryptosporidiosis/parasitology
Mice
*Protein Kinases/genetics/metabolism
CRISPR-Cas Systems
*Protozoan Proteins/genetics/metabolism
Oocysts/growth & development
Female
Humans
Life Cycle Stages
Sporozoites/growth & development

@article {pmid40432879,
year = {2025},
author = {Melchior, F and Angelidou, IA and Chorianopoulou, M and Teichmann, B},
title = {The genetic technologies questionnaire in the Greek-speaking population: the moral judgement of the lay public.},
journal = {Frontiers in genetics},
volume = {16},
number = {},
pages = {1594724},
pmid = {40432879},
issn = {1664-8021},
abstract = {INTRODUCTION: Advancements in life sciences have significantly boosted biomedical capabilities. Genetic testing forecasts hereditary traits and disease susceptibility, while CRISPR/Cas allows permanent genome alterations. However, ethical considerations arise regarding the morality of these capabilities, particularly concerning the moral status, autonomy, and privacy of living beings. The lack of valid instruments to assess moral judgment in genetic technologies highlights the need for this study, aiming to translate and validate the "Genetic Technologies Questionnaire" (GTQ) and the short version of the "Conventional Technologies Questionnaire" (CTQ5) into Greek. As the full version of the GTQ with 30 questions could be too extensive for some studies, we also tested other versions: The short versions GTQ20-GR and GTQ5-GR which were already presented in the original study, as well as a version which included questions solely about humans (GTQ-H-GR) and is intended for use in human research and therapy, and the GTQ-Moral Status (GTQ-MS-GR), which included questions about genetic testing and gene editing in different living beings to investigate differences in moral status.

METHODS: A cross-sectional study involved 250 participants who completed an online questionnaire, assessing internal consistency, structural validity, known-groups validity, floor/ceiling effects, and retest reliability (subset of 50 participants). Correlational analyses explored relationships with education, age, genetic knowledge, religiosity, and genetic testing experience. The study followed the STROBE checklist for reporting.

RESULTS: The GTQ-GR (Cronbach's α = 0.929) and GTQ20-GR (α = 0.935) exhibit high reliability and stability in assessing moral judgment among lay people, whereas the GTQ5-GR (α = 0.866) and CTQ5-GR (α = 0.758) displayed some weaknesses. Participants tended to rate conventional technologies more favorably than genetic technologies, with genetic testing perceived more positively than genome editing. The two additional derived versions, GTQ-H-GR (α = 0.859) and GTQ-MS-GR (α = 0.787), also demonstrated solid psychometric characteristics.

CONCLUSION: The GTQ-GR is a valid and reliable questionnaire with strong psychometric properties and is now available in Greek.},
}

@article {pmid40431732,
year = {2025},
author = {Meng, P and Ni, B and Li, C and Sha, Z and Liu, C and Ren, W and Wei, R and Liu, F and Li, J and Wang, Z},
title = {Establishment and Implementation of the Point-of-Care RT-RAA-CRISPR/Cas13a Diagnostic Test for Foot-And-Mouth Disease Virus Serotype O in Pigs.},
journal = {Viruses},
volume = {17},
number = {5},
pages = {},
pmid = {40431732},
issn = {1999-4915},
support = {2021YFD1800300//National Key R&D Program of China/ ; },
mesh = {Animals ; Swine ; *Foot-and-Mouth Disease Virus/genetics/isolation & purification/classification ; *Swine Diseases/diagnosis/virology ; *Foot-and-Mouth Disease/diagnosis/virology ; Serogroup ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Point-of-Care Systems ; RNA, Viral/genetics ; *Molecular Diagnostic Techniques/methods ; },
abstract = {Foot and mouth disease virus (FMDV) is a highly pathogenic virus that mainly infects cloven hooved animals, such as pigs. The establishment of a rapid, sensitive and accurate point-of-care detection method is critical for the timely identification and elimination of infected pigs for controlling this disease. In this study, a RT-RAA-CRISPR/Cas13a method was developed for the detection of FMDV serotype O in pigs. Six pairs of RT-RAA primers were designed based on the conserved gene sequence of FMDV serotype O, and the optimal amplification primers and reaction temperatures were screened. The CRISPR-derived RNA (crRNA) was further designed based on the optimal target band sequence and the most efficient crRNA was screened. The results revealed that FMDV-O-F4/R4 was the optimal primer set, and the optimal temperature for the RT-RAA reaction was 37 °C. Moreover, crRNA4 exhibited the strongest detection signal among the six crRNAs. The established RT-RAA-CRISPR/Cas13a method demonstrated high specificity and no cross-reactivity with other common swine pathogens such as Senecavirus A (SVA), porcine reproductive and respiratory virus (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV), and pseudorabies virus (PRV), additionally, it was observed to be highly sensitive, with a detection limit of 19.1 copies/µL. The repeatability of this method was also observed to be good. This method could produce stable fluorescence and exhibited good repeatability when three independent experiments yielded the same results. A validation test using three types of simulated clinical samples (including swab, tissue, and serum samples) revealed a 100% concordance rate. The detection results could be visualized via a fluorescence reader or lateral flow strips (LFSs). Thus, a highly specific and sensitive RT-RAA-CRISPR/Cas13a detection method was developed and is expected to be applied for the rapid detection of FMDV serotype O in situ.},
}

Animals
Swine
*Foot-and-Mouth Disease Virus/genetics/isolation & purification/classification
*Swine Diseases/diagnosis/virology
*Foot-and-Mouth Disease/diagnosis/virology
Serogroup
*CRISPR-Cas Systems
Sensitivity and Specificity
*Point-of-Care Systems
RNA, Viral/genetics
*Molecular Diagnostic Techniques/methods

@article {pmid40429982,
year = {2025},
author = {Leal, K and Machuca, J and Gajardo, H and Palma, M and Contreras, MJ and Nuñez-Montero, K and Gutiérrez, Á and Barrientos, L},
title = {Structural Characterisation of TetR/AcrR Regulators in Streptomyces fildesensis So13.3: An In Silico CRISPR-Based Strategy to Influence the Suppression of Actinomycin D Production.},
journal = {International journal of molecular sciences},
volume = {26},
number = {10},
pages = {},
pmid = {40429982},
issn = {1422-0067},
support = {1210563//Fondecyt Regular/ ; 11230475//Fondecyt Iniciación/ ; 11240632//Fondecyt Iniciación/ ; },
mesh = {*Streptomyces/genetics/metabolism ; *Dactinomycin/biosynthesis ; *Bacterial Proteins/genetics/metabolism/chemistry ; Gene Expression Regulation, Bacterial ; Multigene Family ; Molecular Dynamics Simulation ; *Transcription Factors/genetics/metabolism/chemistry ; Anti-Bacterial Agents/biosynthesis ; *CRISPR-Cas Systems ; Biosynthetic Pathways/genetics ; },
abstract = {The growing threat of antimicrobial resistance has intensified the search for new bioactive compounds, particularly in extreme environments such as Antarctica. Streptomyces fildesensis So13.3, isolated from Antarctic soil, harbours a biosynthetic gene cluster (BGC) associated with actinomycin D production, an antibiotic with biomedical relevance. This study investigates the regulatory role of TetR/AcrR transcription factors encoded within this biosynthetic gene cluster (BGC), focusing on their structural features and expression under different nutritional conditions. Additionally, we propose that repressing an active pathway could lead to the activation of silent biosynthetic routes, and our in-silico analysis provides a foundation for selecting key mutations and experimentally validating this strategy. Expression analysis revealed that TetR-279, in particular, was upregulated in ISP4 and IMA media, suggesting its participation in nutrient-dependent BGC regulation. Structural modelling identified key differences between TetR-206 and TetR-279, with the latter containing a tetracycline-repressor-like domain. Molecular dynamics simulations confirmed TetR-279's structural stability but showed that the S166P CRISPy-web-guided mutation considerably affected its flexibility, while V167A and V167I had modest effects. These results underscore the importance of integrating omics, structural prediction, and gene editing to evaluate and manipulate transcriptional regulation in non-model bacteria. Targeted disruption of TetR-279 may derepress actinomycin biosynthesis, enabling access to silent or cryptic secondary metabolites with potential pharmaceutical applications.},
}

*Streptomyces/genetics/metabolism
*Dactinomycin/biosynthesis
*Bacterial Proteins/genetics/metabolism/chemistry
Gene Expression Regulation, Bacterial
Multigene Family
Molecular Dynamics Simulation
*Transcription Factors/genetics/metabolism/chemistry
Anti-Bacterial Agents/biosynthesis
*CRISPR-Cas Systems
Biosynthetic Pathways/genetics

@article {pmid40429922,
year = {2025},
author = {Sheng, H and Gao, P and Yang, C and Quilichini, TD and Kochian, LV and Datla, R and Xiang, D},
title = {Advances in Genome Editing Through Haploid Induction Systems.},
journal = {International journal of molecular sciences},
volume = {26},
number = {10},
pages = {},
pmid = {40429922},
issn = {1422-0067},
mesh = {*Gene Editing/methods ; *Haploidy ; CRISPR-Cas Systems ; *Crops, Agricultural/genetics ; Plant Breeding/methods ; *Genome, Plant ; Diploidy ; Plants, Genetically Modified/genetics ; },
abstract = {Groundbreaking advances in gene editing technologies are transforming modern plant breeding by enabling precise genetic modifications that dramatically accelerate crop improvement. Haploid and diploid induction systems have emerged as particularly powerful tools in this landscape, offering both efficient gene editing capabilities and rapid production of homozygous lines while seamlessly integrating with the advanced genome-editing platforms such as CRISPR-Cas systems. This review synthesizes the current state of knowledge regarding the mechanisms, applications, and recent progress in haploid and diploid induction systems for gene editing. We examine their transformative potential for enhancing genetic gains and compressing breeding timelines, with significant implications for global food security. Additionally, we provide a critical analysis of emerging challenges of genome editing in crops and outline promising future directions for research and development.},
}

*Gene Editing/methods
*Haploidy
CRISPR-Cas Systems
*Crops, Agricultural/genetics
Plant Breeding/methods
*Genome, Plant
Diploidy
Plants, Genetically Modified/genetics

@article {pmid40429745,
year = {2025},
author = {Nilsri, N and Mekchaaum, R and Kalasin, S and Jongjitwimol, J and Daowtak, K},
title = {CRISPR/Cas9-Based Modeling of JAK2 V617F Mutation in K562 Cells Reveals Enhanced Proliferation and Sensitivity to Therapeutic Agents.},
journal = {International journal of molecular sciences},
volume = {26},
number = {10},
pages = {},
pmid = {40429745},
issn = {1422-0067},
support = {R2564C032 and AH-64-01-002//Naresuan University research funding and Faculty of Allied Health Sciences, Naresuan University research funding/ ; },
mesh = {Humans ; *Janus Kinase 2/genetics/metabolism ; Cell Proliferation/drug effects/genetics ; *CRISPR-Cas Systems/genetics ; K562 Cells ; Interferon-alpha/pharmacology ; *Mutation ; Arsenic Trioxide/pharmacology ; Myeloproliferative Disorders/genetics/drug therapy ; Antineoplastic Agents/pharmacology ; Point Mutation ; },
abstract = {The Janus kinase 2 (JAK2) protein fulfills an important role in hematopoiesis via the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, as it provides the genetic driver of BCR::ABL1-negative myeloproliferative neoplasms (MPNs), which are clinically manifested as polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The most common cause of MPNs is the mutation of JAK2 V617F in the JAK2 gene, which results in increased cell proliferation. However, both the pathogenesis and treatment regimen of BCR::ABL1-negative MPNs remain poorly understood. The aim of the present study was to establish K562 cell lines with a point mutation in exon 14 (JAK2p.V617F) using CRISPR/Cas9 technology. The modified JAK2 V617F cell lines were examined for the gene mutation using droplet digital PCR (DDPCR), and the presence of the mutation was confirmed by DNA sequencing. Modified cells were characterized by measuring JAK2 gene expression and the extent of cell proliferation. Interferon α2a (IFN-α2a) and arsenic trioxide were also administered to the cells to explore their potential effects. The JAK2 V617F-mutated cells were found to exhibit a higher level of JAK2 gene expression compared with the wild type. Interestingly, a significant increase in the proliferation rate was observed with the modified cells compared with the wild type cells (p < 0.001), as assessed from the JAK2 gene expression levels. Furthermore, the treatments with IFN-α2a and arsenic trioxide led to the preferential suppression of the cell proliferation rate of the K562 expressing mutant JAK2 cells compared with the wild type cells, and this suppression occurred in a dose-dependent manner(p < 0.01). Moreover, the modified cells were able to differentiate into megakaryocyte-like cells following stimulation with phorbol 12 myristate 13 acetate (PMA). Taken together, the results of the present study have shown that the CRISPR/Cas9-modified JAK2 V617F model may be used as a disease model in the search of novel therapies for MPNs.},
}

Humans
*Janus Kinase 2/genetics/metabolism
Cell Proliferation/drug effects/genetics
*CRISPR-Cas Systems/genetics
K562 Cells
Interferon-alpha/pharmacology
*Mutation
Arsenic Trioxide/pharmacology
Myeloproliferative Disorders/genetics/drug therapy
Antineoplastic Agents/pharmacology
Point Mutation

@article {pmid40429744,
year = {2025},
author = {Mikhaylova, E},
title = {Virus-Induced Genome Editing (VIGE): One Step Away from an Agricultural Revolution.},
journal = {International journal of molecular sciences},
volume = {26},
number = {10},
pages = {},
pmid = {40429744},
issn = {1422-0067},
support = {24-76-10065//Russian Science Foundation/ ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *Plant Viruses/genetics ; *Genome, Plant ; Agriculture/methods ; *Crops, Agricultural/genetics/virology ; Plants, Genetically Modified/genetics ; Genetic Vectors/genetics ; },
abstract = {There is currently a worldwide trend towards deregulating the use of genome-edited plants. Virus-induced genome editing (VIGE) is a novel technique that utilizes viral vectors to transiently deliver clustered regularly interspaced short palindromic repeat (CRISPR) components into plant cells. It potentially allows us to obtain transgene-free events in any plant species in a single generation without in vitro tissue culture. This technology has great potential for agriculture and is already being applied to more than 14 plant species using more than 20 viruses. The main limitations of VIGE include insufficient vector capacity, unstable expression of CRISPR-associated (Cas) protein, plant immune reaction, host specificity, and reduced viral activity in meristem. Various solutions to these problems have been proposed, such as fusion of mobile elements, RNAi suppressors, novel miniature Cas proteins, and seed-borne viruses, but the final goal has not yet been achieved. In this review, the mechanism underlying the ability of different classes of plant viruses to transiently edit genomes is explained. It not only focuses on the latest achievements in virus-induced editing of crops but also provides suggestions for improving the technology. This review may serve as a source of new ideas for those planning to develop new approaches in VIGE.},
}

*Gene Editing/methods
CRISPR-Cas Systems
*Plant Viruses/genetics
*Genome, Plant
Agriculture/methods
*Crops, Agricultural/genetics/virology
Plants, Genetically Modified/genetics
Genetic Vectors/genetics

@article {pmid40429730,
year = {2025},
author = {Fu, L and Li, P and Rui, Z and Sun, J and Yang, J and Wang, Y and Jia, D and Hu, J and Li, X and Ma, R},
title = {CRISPR/Cas9-Mediated Knockout of the White Gene in Agasicles hygrophila.},
journal = {International journal of molecular sciences},
volume = {26},
number = {10},
pages = {},
pmid = {40429730},
issn = {1422-0067},
support = {202304051001006//Science and Technology Innovation Teams of Shanxi Province/ ; 20210302123386//Fundamental Research Program of Shanxi Province/ ; 2016YJ03//Science and Technology Innovation Fund of Shanxi Agricultural University/ ; ZBXY23A-10//Cultivation and Innovation Program for Scientific research, College of Plant Protection, Shanxi Agricultural University/ ; },
mesh = {*CRISPR-Cas Systems ; Gene Editing/methods ; *Gene Knockout Techniques ; *Amaranthaceae/genetics ; },
abstract = {Agasicles hygrophila is the most effective natural enemy for the control of the invasive weed Alternanthera philoxeroides (Mart.) Griseb. However, research on the gene function and potential genetic improvement of A. hygrophila is limited due to a lack of effective genetic tools. In this study, we employed the A. hygrophila white (AhW) gene as a target gene to develop a CRISPR/Cas9-based gene editing method applicable to A. hygrophila. We showed that injection of Cas9/sgRNA ribonucleoprotein complexes (RNPs) of the AhW gene into pre-blastoderm eggs induced genetic insertion and deletion mutations, leading to white eyes. Our results demonstrate that CRISPR/Cas9-mediated gene editing is possible in A. hygrophila, offering a valuable tool for studies of functional genomics and genetic improvement of A. hygrophila, which could potentially lead to more effective control of invasive weeds through the development of improved strains of this biocontrol agent. In addition, the white-eyed mutant strain we developed could potentially be useful for other transgenic research studies on this species.},
}

*CRISPR-Cas Systems
Gene Editing/methods
*Gene Knockout Techniques
*Amaranthaceae/genetics

@article {pmid40428391,
year = {2025},
author = {Ziółkowska-Suchanek, I and Rozwadowska, N},
title = {Advancements in Gene Therapy for Non-Small Cell Lung Cancer: Current Approaches and Future Prospects.},
journal = {Genes},
volume = {16},
number = {5},
pages = {},
pmid = {40428391},
issn = {2073-4425},
mesh = {Humans ; *Carcinoma, Non-Small-Cell Lung/therapy/genetics ; *Genetic Therapy/methods/trends ; *Lung Neoplasms/therapy/genetics ; Gene Editing/methods ; CRISPR-Cas Systems ; Immunotherapy, Adoptive/methods ; },
abstract = {Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide, characterized by late diagnosis and resistance to conventional therapies. Gene therapy has emerged as a promising alternative for NSCLC therapy, especially for patients with advanced disease who have exhausted conventional treatments. This article delved into the current developments in gene therapy for NSCLC, including gene replacement and tumor suppressor gene therapy, gene silencing, CRISPR/Cas9 gene editing, and immune modulation with CAR-T cell therapy. In addition, the challenges and future prospects of gene-based therapies for NSCLC were discussed.},
}

Humans
*Carcinoma, Non-Small-Cell Lung/therapy/genetics
*Genetic Therapy/methods/trends
*Lung Neoplasms/therapy/genetics
Gene Editing/methods
CRISPR-Cas Systems
Immunotherapy, Adoptive/methods

@article {pmid40355755,
year = {2025},
author = {Wolf, G and Leippe, P and Onstein, S and Goldmann, U and Frommelt, F and Teoh, ST and Girardi, E and Wiedmer, T and Superti-Furga, G},
title = {The genetic interaction map of the human solute carrier superfamily.},
journal = {Molecular systems biology},
volume = {21},
number = {6},
pages = {531-559},
pmid = {40355755},
issn = {1744-4292},
support = {777372//Innovative Medicines Initiative 2/ ; },
mesh = {Humans ; CRISPR-Cas Systems ; *Solute Carrier Proteins/genetics/metabolism ; Gene Knockout Techniques ; Mitochondrial Membrane Transport Proteins/genetics/metabolism ; Cation Transport Proteins/genetics/metabolism ; Cell Line, Tumor ; *Epistasis, Genetic ; Gene Regulatory Networks ; Mitochondrial Proteins ; Organic Anion Transporters ; },
abstract = {Solute carriers (SLCs), the largest superfamily of transporter proteins in humans with about 450 members, control the movement of molecules across membranes. A typical human cell expresses over 200 different SLCs, yet their collective influence on cell phenotypes is not well understood due to overlapping substrate specificities and expression patterns. To address this, we performed systematic pairwise gene double knockouts using CRISPR-Cas12a and -Cas9 in human colon carcinoma cells. A total of 1,088,605 guide combinations were used to interrogate 35,421 SLC-SLC and SLC-enzyme double knockout combinations across multiple growth conditions, uncovering 1236 genetic interactions with a growth phenotype. Further exploration of an interaction between the mitochondrial citrate/malate exchanger SLC25A1 and the zinc transporter SLC39A1 revealed an unexpected role for SLC39A1 in metabolic reprogramming and anti-apoptotic signaling. This full-scale genetic interaction map of human SLC transporters is the backbone for understanding the intricate functional network of SLCs in cellular systems and generates hypotheses for pharmacological target exploitation in cancer and other diseases. The results are available at https://re-solute.eu/resources/dashboards/genomics/ .},
}

Humans
CRISPR-Cas Systems
*Solute Carrier Proteins/genetics/metabolism
Gene Knockout Techniques
Mitochondrial Membrane Transport Proteins/genetics/metabolism
Cation Transport Proteins/genetics/metabolism
Cell Line, Tumor
*Epistasis, Genetic
Gene Regulatory Networks
Mitochondrial Proteins
Organic Anion Transporters

@article {pmid39804466,
year = {2025},
author = {Birling, MC and Hérault, Y and Pavlovic, G},
title = {Genome engineering with Cas9 and AAV repair templates, successes and pitfalls.},
journal = {Mammalian genome : official journal of the International Mammalian Genome Society},
volume = {36},
number = {2},
pages = {376-383},
pmid = {39804466},
issn = {1432-1777},
mesh = {*Dependovirus/genetics ; Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genetic Engineering/methods ; Genetic Vectors/genetics ; Humans ; Genome ; },
abstract = {Genome editing, in particular the CRISPR/Cas9 system, is widely used to generate new animal models. However, the generation of mutations, such as conditional knock-out or knock-in, can remain complex and inefficient, in particular because of the difficulty to deliver the donor DNA (single or double stranded) into the nucleus of fertilized oocytes. The use of recombinant adeno-associated viruses (rAAV) as donor DNA is a rapidly developing approach that promises to improve the efficiency of creation of animal models. In this mini-review, we explore the progress and challenges of using CRISPR/Cas9 in combination with rAAV for precise genome editing. We will summarise the current knowledge of rAAV transduction, data on its use in rodent embryos in combination with CRISPR/Cas9 to easily generate sequence replacements or insertions, the limitations of rAAV and the unexpected events observed to date, and the protocol optimisations already in place to facilitate its use in the generation of animal models.},
}

*Dependovirus/genetics
Animals
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Genetic Engineering/methods
Genetic Vectors/genetics
Humans
Genome

@article {pmid40427606,
year = {2025},
author = {Gutiérrez-Hurtado, IA and García-Acéves, ME and Puga-Carrillo, Y and Guardado-Estrada, M and Becerra-Loaiza, DS and Carrillo-Rodríguez, VD and Plazola-Zamora, R and Godínez-Rubí, JM and Rangel-Villalobos, H and Aguilar-Velázquez, JA},
title = {Past, Present and Future Perspectives of Forensic Genetics.},
journal = {Biomolecules},
volume = {15},
number = {5},
pages = {},
pmid = {40427606},
issn = {2218-273X},
mesh = {Humans ; *Forensic Genetics/trends/methods/history ; Machine Learning ; CRISPR-Cas Systems ; },
abstract = {Forensic genetics has experienced remarkable advancements over the past decades, evolving from the analysis of a limited number of DNA segments to comprehensive genome-wide investigations. This progression has significantly improved the ability to establish genetic profiles under diverse conditions and scenarios. Beyond individual identification, forensic genetics now enables the inference of physical traits (e.g., eye, hair, and skin color, as well as body composition), biogeographic ancestry, lifestyle habits such as alcohol and tobacco use, and even the transfer of genital microbiomes post-coitus, among other characteristics. Emerging trends point to a future shaped by the integration of cutting-edge technologies, including CRISPR-Cas systems, artificial intelligence, and machine learning, which promise to further revolutionize the field. This review provides a thorough exploration of forensic genetics, tracing its evolution from its foundational methods (past) to its diverse modern applications (present) and offering insights into its potential future directions.},
}

Humans
*Forensic Genetics/trends/methods/history
Machine Learning
CRISPR-Cas Systems

@article {pmid40427601,
year = {2025},
author = {Marin-Quilez, A and García-Tuñón, I and Benito, R and Ordoñez, JL and Díaz-Ajenjo, L and Lama-Villanueva, A and Guerrero, C and Pérez-Losada, J and González-Porras, JR and Hernández-Rivas, JM and Del Rey, M and Bastida, JM},
title = {Examining the Effects of the RUNX1 p.Leu43Ser Variant on FPD/AML Phenotypes Using a CRISPR/Cas9-Generated Knock-In Murine Model.},
journal = {Biomolecules},
volume = {15},
number = {5},
pages = {},
pmid = {40427601},
issn = {2218-273X},
mesh = {Animals ; *Core Binding Factor Alpha 2 Subunit/genetics/metabolism ; Mice ; *CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Gene Knock-In Techniques ; *Leukemia, Myeloid, Acute/genetics/pathology ; Phenotype ; *Blood Platelet Disorders/genetics/pathology ; Male ; Homozygote ; },
abstract = {Germline heterozygous variants in RUNX1 lead to Familial Platelet Disorder with Myeloid Leukemia Predisposition (FPD/AML). Cellular and/or animal models are helpful to uncovering the role of a variant in disease progression. Twenty-five mice per genotype (RUNX1[WT/WT], RUNX1[WT/L43S], RUNX1[L43S/L43S]), previously generated by CRISPR/Cas9, and nine sub-lethally irradiated mice per genotype were investigated. Peripheral blood (PB), bone marrow (BM), and spleen samples were analyzed by flow cytometry and histopathology. Deregulated genes were analyzed by RNA-seq in BM. An aberrant myeloid Mac1[+]Sca1[+]ckit[-] population in the PB, BM, and spleen of two homozygous and one heterozygous mouse was observed, as well as BM hypercellularity. No Mac1[+]Sca1[+]ckit[-] cells were detected in any RUNX1[WT/WT] mice. Moreover, the spleen of both homozygous mice showed destruction of the white/red pulp and the presence of apoptotic cells. The aberrant population was also detected in four irradiated mice, two heterozygous and two homozygous, in their PB, BM, and spleen. RNA-seq studies showed 698 genes significantly deregulated in the three non-irradiated Mac1[+]Sca1[+]ckit[-] mice vs. six healthy mice, highlighting the alteration of genes involved in apoptosis and DNA repair. These results indicate that the homozygous form of the variant p.Leu43Ser may contribute to the pathogenesis of aberrant cells.},
}

Animals
*Core Binding Factor Alpha 2 Subunit/genetics/metabolism
Mice
*CRISPR-Cas Systems/genetics
Disease Models, Animal
Gene Knock-In Techniques
*Leukemia, Myeloid, Acute/genetics/pathology
Phenotype
*Blood Platelet Disorders/genetics/pathology
Male
Homozygote

@article {pmid40427577,
year = {2025},
author = {Palit, P and Minkara, M and Abida, M and Marwa, S and Sen, C and Roy, A and Pasha, MR and Mosae, PS and Saha, A and Ferdoush, J},
title = {PlastiCRISPR: Genome Editing-Based Plastic Waste Management with Implications in Polyethylene Terephthalate (PET) Degradation.},
journal = {Biomolecules},
volume = {15},
number = {5},
pages = {},
pmid = {40427577},
issn = {2218-273X},
support = {Start-up fund//University of Tennessee at Chattanooga/ ; },
mesh = {*Polyethylene Terephthalates/metabolism/chemistry ; *Gene Editing/methods ; *Waste Management/methods ; *Plastics/metabolism/chemistry ; Biodegradation, Environmental ; CRISPR-Cas Systems ; Humans ; },
abstract = {Plastic pollution has become a significant environmental issue worldwide, with global plastic production expected to reach 1800 million tons by 2050. One of the most commonly used plastics in the world is polyethylene terephthalate (PET), a synthetic polymer that is extremely durable but difficult to degrade. Thus, PET is dangerous to human health. To address this crisis, innovative approaches are being developed, including genome editing technologies. One of the recently advanced genome editing technologies is PlastiCRISPR, a novel concept that applies CRISPR-based genome editing to transform plastic waste management. PlastiCRISPR utilizes microorganisms to degrade plastic, generating valuable bioproducts like biofuels and biochemicals. Thus, this technology offers a sustainable solution because of its simple design, adequacy, and low cost, which can be integrated into existing waste management systems. Importantly, this review focuses on the PlastiCRISPR-based management of PET because it could drastically lower plastic waste, sustain natural resources by decreasing the requirement for plastic production, minimize energy intake, etc. Overall, this review provides an overview of the principles, applications, challenges, and future prospects of PlastiCRISPR in combating plastic pollution and shaping a more sustainable future.},
}

*Polyethylene Terephthalates/metabolism/chemistry
*Gene Editing/methods
*Waste Management/methods
*Plastics/metabolism/chemistry
Biodegradation, Environmental
CRISPR-Cas Systems
Humans

@article {pmid40426207,
year = {2025},
author = {Wang, ZC and Stegall, H and Miyazawa, T and Keatinge-Clay, AT},
title = {A CRISPR-Cas9 system for knock-out and knock-in of high molecular weight DNA enables module-swapping of the pikromycin synthase in its native host.},
journal = {Microbial cell factories},
volume = {24},
number = {1},
pages = {125},
pmid = {40426207},
issn = {1475-2859},
support = {R01 GM145992/GM/NIGMS NIH HHS/United States ; GM145992/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Streptomyces/genetics/metabolism/enzymology ; *Polyketide Synthases/genetics/metabolism ; Macrolides/metabolism ; Gene Editing/methods ; Gene Knockout Techniques ; Anti-Bacterial Agents/biosynthesis ; *Gene Knock-In Techniques ; Molecular Weight ; },
abstract = {BACKGROUND: Engineers seeking to generate natural product analogs through altering modular polyketide synthases (PKSs) face significant challenges when genomically editing large stretches of DNA.

RESULTS: We describe a CRISPR-Cas9 system that was employed to reprogram the PKS in Streptomyces venezuelae ATCC 15439 that helps biosynthesize the macrolide antibiotic pikromycin. We first demonstrate its precise editing ability by generating strains that lack megasynthase genes pikAI-pikAIV or the entire pikromycin biosynthetic gene cluster but produce pikromycin upon complementation. We then employ it to replace 4.4-kb modules in the pikromycin synthase with those of other synthases to yield two new macrolide antibiotics with activities similar to pikromycin.

CONCLUSION: Our gene-editing tool has enabled the efficient replacement of extensive and repetitive DNA regions within streptomycetes.},
}

*CRISPR-Cas Systems
*Streptomyces/genetics/metabolism/enzymology
*Polyketide Synthases/genetics/metabolism
Macrolides/metabolism
Gene Editing/methods
Gene Knockout Techniques
Anti-Bacterial Agents/biosynthesis
*Gene Knock-In Techniques
Molecular Weight

@article {pmid40425955,
year = {2025},
author = {Chen, Y and Zhang, X and Huang, W and Jin, Z and Wei, X and Li, J},
title = {Ultrasensitive detection of atrazine by Schottky junction photoelectrochemical aptamer sensor based on signal amplification by cascade catalysis of CRISPR/Cas12a and G-quadruplex/hemin DNAzyme.},
journal = {Mikrochimica acta},
volume = {192},
number = {6},
pages = {376},
pmid = {40425955},
issn = {1436-5073},
mesh = {*G-Quadruplexes ; *DNA, Catalytic/chemistry/metabolism ; *Aptamers, Nucleotide/chemistry ; Hemin/chemistry/metabolism ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; Limit of Detection ; *Atrazine/analysis/chemistry ; CRISPR-Cas Systems ; Electrodes ; CRISPR-Associated Proteins/metabolism/chemistry ; Photochemical Processes ; Catalysis ; Indoles/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Atrazine (ATZ) is used extensively, resulting in residues in food and the environment, posing a serious threat to human health. Herein, Cd0.5Zn0.5S/Ti3C2 photoelectric material was synthesized and immobilized on a FTO electrode as a photoanode. A photoelectrochemical (PEC) aptamer sensor was constructed for the highly sensitive and selective determination of ATZ based on signal amplification via cascade catalysis of CRISPR/Cas12a and G-quadruplex/hemin DNAzyme (G4/hemin DNAzyme). G4/hemin DNAzyme catalyses the oxidation reaction between H2O2 and dopamine (DA) to form polydopamine (PDA) deposit. This process, in turn, inhibits the photocurrent at the photoanode, leading to a decrease in photocurrent. Concurrently, the depletion of DA as an electron donor for the PEC reaction at the photoelectrode further contributes to the decrease in photocurrent. ATZ can hybridize with ATZ aptamer (Apt) in Apt/cDNA to release activation strand (cDNA), which activates the activity of CRISPR/Cas12a and triggers cleavage of G4, causing the cleaving of G4/hemin DNAzyme immobilized on the electrode surface. This process leads to a decrease of G4/hemin DNAzymes amount on the electrode, consequently reducing both the PDA generation and the DA consumption. As a result, the photocurrent is restored. The cascade catalysis of CRISPR/Cas12a and G4/hemin DNAzyme has been demonstrated to result in photocurrent amplification. The photocurrent change was linear with the logarithmic value of ATZ concentration in the range 1.00 × 10[-12] to 1.00 × 10[-5] mol/L. The limit of detection was 3.47 × 10[-13] mol/L. The sensor has been successfully applied to the determination of trace ATZ in environmental and food samples.},
}

*G-Quadruplexes
*DNA, Catalytic/chemistry/metabolism
*Aptamers, Nucleotide/chemistry
Hemin/chemistry/metabolism
*Biosensing Techniques/methods
*Electrochemical Techniques/methods
Limit of Detection
*Atrazine/analysis/chemistry
CRISPR-Cas Systems
Electrodes
CRISPR-Associated Proteins/metabolism/chemistry
Photochemical Processes
Catalysis
Indoles/chemistry
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid40398569,
year = {2025},
author = {Zhou, Y and Duan, Y and Chen, L and Yang, Y and Ma, L and Chen, W and Liao, Z and Cai, J and Li, D},
title = {Engineering dispersed mycelium morphology in Aspergillus niger for enhanced mycoprotein production via CRISPR/Cas9-mediated genome editing.},
journal = {Bioresource technology},
volume = {432},
number = {},
pages = {132652},
doi = {10.1016/j.biortech.2025.132652},
pmid = {40398569},
issn = {1873-2976},
mesh = {*Aspergillus niger/genetics/metabolism/cytology ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Mycelium/genetics/metabolism/cytology ; Fermentation ; *Fungal Proteins/biosynthesis/genetics/metabolism ; Biomass ; },
abstract = {Filamentous fungi are widely utilized in industrial fermentation processes due to their high productivity, with mycelial morphology directly influencing fermentation broth viscosity and target product yield, which is a critical parameter for process optimization. Aspergillus niger, an FDA-approved safe filamentous fungus, typically forms tightly packed mycelial pellets in submerged cultures, which severely restricts its industrial application potential by limiting mass transfer efficiency. To address this challenge, CRISPR/Cas9 mediated genome editing coupled with fermentation optimization enhanced microbial protein production in A. niger. Endogenous α-1,3-glucan synthase genes (agsA, agsB) and galactosaminogalactan (GAG) synthase genes (sph3, uge3) were disrupted using CRISPR/Cas9, achieving complete dispersion of filamentous pellets in liquid media. This morphological engineering strategy resulted in a 77.52 % increase in biomass and 39.98 % enhancement in mycelial protein content compared to the wild-type strain (A. niger Li2). Transcriptomic analysis revealed that the engineered strain (A. niger AnΔABSU) exhibited upregulated transporter proteins (ABC transporters, MFS transporters, sugar transporters), accelerating nutrient uptake and energy metabolism; altered cell wall integrity pathways, including activation of the MAPK signaling cascade and increased sensitivity to cell wall stressors; enhanced amino acid biosynthesis, driven by upregulated gene expression in key metabolic pathways. Furthermore, response surface methodology (RSM) with Box-Behnken design optimized the fermentation medium, yielding 16.67 g/L biomass and 45.91 % protein content, representing 115.37 % and 67.01 % improvements over the unoptimized wild-type control. This study establishes a novel paradigm for constructing high-efficiency microbial protein cell factories via integrated morphological-engineering and fermentation optimization.},
}

*Aspergillus niger/genetics/metabolism/cytology
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Mycelium/genetics/metabolism/cytology
Fermentation
*Fungal Proteins/biosynthesis/genetics/metabolism
Biomass

@article {pmid40300704,
year = {2025},
author = {Tarin, M and Saljooghi, AS},
title = {Gasdermin E as a potential target and biomarker for CRISPR-Cas9-based cancer therapy.},
journal = {Biochemical pharmacology},
volume = {237},
number = {},
pages = {116961},
doi = {10.1016/j.bcp.2025.116961},
pmid = {40300704},
issn = {1873-2968},
mesh = {Humans ; *CRISPR-Cas Systems/physiology/genetics ; *Neoplasms/therapy/genetics/metabolism ; *Biomarkers, Tumor/metabolism/genetics/antagonists & inhibitors ; Animals ; Pyroptosis/physiology/drug effects ; Gene Editing/methods ; Gasdermins ; },
abstract = {Gasdermin E (GSDME), a protein pivotal in mediating pyroptosis, has gained significant attention due to its role in cancer pathogenesis and its potential as a therapeutic target. The advent of CRISPR-Cas9, a precise genome editing tool, has revolutionized cancer therapy by enabling the manipulation of GSDME expression and function. This review explores the interplay of GSDME and CRISPR-Cas9 in cancer, emphasizing GSDME's unique mechanism of cleavage-dependent pore formation in the cell membrane and its emerging applications as both a therapeutic target and a diagnostic biomarker. We discuss the potential and challenges of using GSDME-induced pyroptosis as a therapeutic strategy and how can enhance its efficacy and specificity. We conclude by highlighting promising future research directions in this emerging field.},
}

Humans
*CRISPR-Cas Systems/physiology/genetics
*Neoplasms/therapy/genetics/metabolism
*Biomarkers, Tumor/metabolism/genetics/antagonists & inhibitors
Animals
Pyroptosis/physiology/drug effects
Gene Editing/methods
Gasdermins

@article {pmid40425635,
year = {2025},
author = {Imai, Y and Ozaki, S and Noda, T and Kobayashi, I and Sugitani, K and Kasashima, S and Morishita, E and Araiso, Y},
title = {Real-time imaging of blood coagulation and angiogenesis during development in a zebrafish model of type I antithrombin deficiency.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {18538},
pmid = {40425635},
issn = {2045-2322},
support = {JPMJSP2135//Japan Science and Technology Agency/ ; JPMJFR200F//Japan Science and Technology Agency/ ; },
mesh = {Animals ; *Zebrafish/genetics/embryology ; *Blood Coagulation/genetics ; Disease Models, Animal ; *Neovascularization, Physiologic ; Zebrafish Proteins/genetics ; Animals, Genetically Modified ; Thrombosis/genetics ; CRISPR-Cas Systems ; Angiogenesis ; },
abstract = {Severe type I antithrombin (AT) deficiency is considered to cause embryonic lethality. Although several pathological analyses using mice or zebrafish have been attempted, the previous studies did not unveil the detailed mechanism leading to lethality in the early developmental stage. In order to solve this problem, we established type I AT deficient zebrafish by the CRISPR/Cas9 system into Tg(gata1:dsRed) and Tg(fli1a:GFP) lines, so that we could conduct real-time imaging of thrombosis and angiogenesis using fluorescence stereo zoom microscopy. The established zebrafish AT (zAT) mutants harbored frameshift mutations which resulted to be type I AT deficient, unable to secrete zAT protein into blood. Both heterozygous (zAT[+/-]) and homozygous (zAT[-/-]) mutants showed reduced survival rate and diverse thrombosis up to 9 days post fertilization. In addition, blood vessel formation was delayed at 30 hpf in zAT[-/-], which was recovered normally by 5 dpf and had little effect on survival. Notably, we analyzed the differences in gene expression profiles under AT-depleted conditions by real-time quantitative PCR, and zAT[-/-] juvenile zebrafish showed increased PLG gene expression and decreased F2 gene expression. Our in vivo study revealed the effects of AT deficiency on embryos during development from the aspects of coagulation and vascular formation.},
}

Animals
*Zebrafish/genetics/embryology
*Blood Coagulation/genetics
Disease Models, Animal
*Neovascularization, Physiologic
Zebrafish Proteins/genetics
Animals, Genetically Modified
Thrombosis/genetics
CRISPR-Cas Systems
Angiogenesis

@article {pmid40425580,
year = {2025},
author = {Sharma, P and Kim, CY and Keys, HR and Imada, S and Joseph, AB and Ferro, L and Kunchok, T and Anderson, R and Sun, Y and Yilmaz, ÖH and Weng, JK and Jain, A},
title = {Genetically encoded fluorescent reporter for polyamines.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4921},
pmid = {40425580},
issn = {2041-1723},
support = {R35 GM151111/GM/NIGMS NIH HHS/United States ; R35GM151111//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
mesh = {*Polyamines/metabolism/analysis ; Humans ; *Genes, Reporter ; Mitochondria/metabolism ; Parkinson Disease/metabolism/genetics ; CRISPR-Cas Systems ; HEK293 Cells ; },
abstract = {Polyamines are abundant and evolutionarily conserved metabolites that are essential for life. Dietary polyamine supplementation extends life-span and health-span. Dysregulation of polyamine homeostasis is linked to Parkinson's disease and cancer, driving interest in therapeutically targeting this pathway. However, measuring cellular polyamine levels, which vary across cell types and states, remains challenging. We introduce a genetically encoded polyamine reporter for real-time measurement of polyamine concentrations in single living cells. This reporter utilizes the polyamine-responsive ribosomal frameshift motif from the OAZ1 gene. We demonstrate broad applicability of this approach and reveal dynamic changes in polyamine levels in response to genetic and pharmacological perturbations. Using this reporter, we conduct a genome-wide CRISPR screen and uncover an unexpected link between mitochondrial respiration and polyamine import, which are both risk factors for Parkinson's disease. By offering a lens to examine polyamine biology, this reporter may advance our understanding of these ubiquitous metabolites and accelerate therapy development.},
}

*Polyamines/metabolism/analysis
Humans
*Genes, Reporter
Mitochondria/metabolism
Parkinson Disease/metabolism/genetics
CRISPR-Cas Systems
HEK293 Cells

@article {pmid40425554,
year = {2025},
author = {Borot, F and Humbert, O and Ehmsen, JT and Fields, E and Kohli, S and Radtke, S and Swing, K and Pande, D and Enstrom, MR and Laszlo, GS and Mayuranathan, T and Ali, AM and Weiss, MJ and Yen, JS and Newby, GA and Walter, RB and Liu, DR and Mukherjee, S and Kiem, HP},
title = {Multiplex base editing to protect from CD33 directed drugs for immune and gene therapy.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4899},
pmid = {40425554},
issn = {2041-1723},
support = {R01 HL156647/HL/NHLBI NIH HHS/United States ; K99 HL163805/HL/NHLBI NIH HHS/United States ; P51 OD010425/OD/NIH HHS/United States ; R00 HL163805/HL/NHLBI NIH HHS/United States ; R50 CA274319/CA/NCI NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; R01 CA266556/CA/NCI NIH HHS/United States ; P01 HL053749/HL/NHLBI NIH HHS/United States ; R01 HL136135/HL/NHLBI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; R01 HL151765/HL/NHLBI NIH HHS/United States ; U42 OD011123/OD/NIH HHS/United States ; },
mesh = {Humans ; Animals ; *Gene Editing/methods ; *Sialic Acid Binding Ig-like Lectin 3/genetics/metabolism/immunology ; *Genetic Therapy/methods ; Hematopoietic Stem Cells/metabolism/drug effects ; Gemtuzumab/pharmacology ; *Immunotherapy/methods ; Polymorphism, Single Nucleotide ; CRISPR-Cas Systems ; Mice ; },
abstract = {The selection of genetically engineered immune or hematopoietic cells in vivo after gene editing remains a clinical problem and requires a method to spare on-target toxicity to normal cells. Here, we develop a base editing approach exploiting a naturally occurring CD33 single nucleotide polymorphism leading to removal of full-length CD33 surface expression on edited cells. CD33 editing in human and nonhuman primate hematopoietic stem and progenitor cells protects myeloid progeny from CD33-targeted therapeutics without affecting normal hematopoiesis in vivo, thus demonstrating potential for improved immunotherapies with reduced off-leukemia toxicity. For broader application to gene therapies, we demonstrate highly efficient (>70%) multiplexed adenine base editing of the CD33 and gamma globin genes, resulting in long-term persistence of dual gene-edited cells with HbF reactivation in nonhuman primates. Using the CD33 antibody-drug conjugate Gemtuzumab Ozogamicin, we show resistance of engrafted, multiplex edited human cells in vivo, and a 2-fold enrichment for edited cells in vitro. Together, our results highlight the potential of adenine base editors for improved immune and gene therapies.},
}

Humans
Animals
*Gene Editing/methods
*Sialic Acid Binding Ig-like Lectin 3/genetics/metabolism/immunology
*Genetic Therapy/methods
Hematopoietic Stem Cells/metabolism/drug effects
Gemtuzumab/pharmacology
*Immunotherapy/methods
Polymorphism, Single Nucleotide
CRISPR-Cas Systems
Mice

@article {pmid40424180,
year = {2025},
author = {Krishnaprasad, VH and Nayak, V and Kumar, S},
title = {World Health Organisation's Bacterial Pathogen Priority List (BPPL) 2017 and BPPL 2024 to combat global antimicrobial resistance crisis: 'challenges and opportunities'.},
journal = {The Journal of antimicrobial chemotherapy},
volume = {},
number = {},
pages = {},
doi = {10.1093/jac/dkaf167},
pmid = {40424180},
issn = {1460-2091},
abstract = {Antibiotic resistance, in a broader perspective, antimicrobial resistance (AMR) presents a formidable global health challenge, threatening the effectiveness of antibiotics and other antimicrobial agents. As a result, AMR has become more challenging or even impossible to treat, leading to increased morbidity and mortality. The World Health Organisation (WHO) has been at the forefront of international efforts to combat AMR by sensitizing the world about the pressing need to tackle AMR to save the future of the human race. This article analyses WHO's efforts to combat AMR, including creating the Bacterial Pathogen Priority List (BPPL), developing a global action plan to address AMR and promoting surveillance and stewardship programmes. This article also examines the progress achieved by BPPL 2017 and the challenges ahead for BPPL 2024. Additionally, this article explores various efforts to combat AMR through two major approaches, like 'research and development' and 'the policy and regulation-based' approach. This article underscores various emerging strategies to tackle AMR, for example, biofilm disruption, nanotechnology, antibiotic resistance breakers, antibody-antibiotic conjugates, rapid detection tools and alternative therapies like phage therapy, antimicrobial peptides, CRISPR-Cas system, probiotics and microbiota modulations. This article also highlights the importance of coordinated actions and sustained commitment to safeguarding public health and ensuring the continued effectiveness of antimicrobial therapies.},
}

@article {pmid40424009,
year = {2025},
author = {Wei, L and Wang, Z and She, Y and Fu, H},
title = {CRISPR/Cas Multiplexed Biosensing: Advances, Challenges, and Perspectives.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.4c04428},
pmid = {40424009},
issn = {1520-6882},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein systems are renowned for their high sensitivity and specificity, enabling them as a powerful diagnostic toolbox. Multiplexed detection of panels of targets, as opposed to single targets, is imperative for reliable and conclusive disease diagnostics. However, multiplex application of the CRISPR/Cas system has long been hindered by indistinguishable signals from specific targets due to nonspecific chaotic trans-cleavage. To make a breakthrough, substantial efforts have been devoted to CRISPR/Cas-powered multiplexed biosensing strategies, which consequently experienced rapid development over the past five years. This review systematically summarizes recent advances in CRISPR/Cas multiplexed detection encompassing Cas9, Cas12, and Cas13. Key focus issues include multiplex biosensing strategies and their respective advantages and limitations, sensing mechanisms, and detection performance of novel validated examples. Finally, the status and challenges of CRISPR/Cas multiplexed biosensing are critically discussed, and future outlooks are proposed for their potential practical application. This Perspective aims to inspire significant research and promote the development of the next generation of CRISPR/Cas multiplexed biosensing.},
}

@article {pmid40422684,
year = {2025},
author = {Ma, B and Li, Y and Wang, T and Li, D and Jia, S},
title = {Advances in CRISPR/Cas9-Based Gene Editing in Filamentous Fungi.},
journal = {Journal of fungi (Basel, Switzerland)},
volume = {11},
number = {5},
pages = {},
pmid = {40422684},
issn = {2309-608X},
support = {32160143//National Science Foundation of China/ ; },
abstract = {As an important class of microorganisms, filamentous fungi have crucial roles in protein secretion, secondary metabolite production and environmental pollution control. However, characteristics such as apical growth, heterokaryon, low homologous recombination (HR) efficiency and the scarcity of genetic markers mean that the application of traditional gene editing technology in filamentous fungi faces great challenges. The introduction of the RNA-mediated CRISPR/Cas (clustered regularly interspaced short palindromic repeat/CRlSPR-associated protein) system in filamentous fungi in recent years has revolutionized gene editing in filamentous fungi. In addition, the continuously expressed CRISPR system has significantly improved the editing efficiency, while the optimized sgRNA design and reduced cas9 concentration have effectively reduced the off-target effect, further enhancing the safety and reliability of the technology. In this review, we systematically analyze the molecular mechanism and regulatory factors of CRISPR/Cas9, focus on the optimization of its expression system and the improvement of the transformation efficiency in filamentous fungi, and reveal the core regulatory roles of HR and non-homologous end-joining (NHEJ) pathways in gene editing. Based on the analysis of various filamentous fungi applications, this review reveals the outstanding advantages of CRISPR/Cas9 in the enhancement of protein secretion, addresses the reconstruction of secondary metabolic pathways and pollutant degradation in the past decade, and provides a theoretical basis and practical guidance for the optimization of the technology and engineering applications.},
}

@article {pmid40422214,
year = {2025},
author = {Gibson, J and Dhungana, A and Pokhrel, M and Arthur, B and Suresh, P and Adebayo, O and Cottle, RN},
title = {Validation of Clinical-Grade Electroporation Systems for CRISPR-Cas9-Mediated Gene Therapy in Primary Hepatocytes for the Correction of Inherited Metabolic Liver Disease.},
journal = {Cells},
volume = {14},
number = {10},
pages = {},
pmid = {40422214},
issn = {2073-4409},
support = {1R01HL168093-01A1/GF/NIH HHS/United States ; 2021000920//American Association for the Study of Liver Diseases Foundation/ ; R01 HL168093/HL/NHLBI NIH HHS/United States ; },
mesh = {*Hepatocytes/transplantation/metabolism ; *Electroporation/methods ; Animals ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods ; Mice ; Gene Editing/methods ; *Tyrosinemias/therapy/genetics ; Humans ; *Liver Diseases/therapy/genetics ; Disease Models, Animal ; Male ; Hydrolases ; },
abstract = {Hepatocyte transplantation (HTx) combined with ex vivo gene therapy has garnered significant interest due to its potential for treating many inherited metabolic liver diseases. The biggest obstacle for HTx is achieving sufficient engraftment levels to rescue diseased phenotypes, which becomes more challenging when combined with ex vivo gene editing techniques. However, recent technological advancements have improved electroporation delivery efficiency, cell viability, and scalability for cell therapy. We recently demonstrated the impacts of electroporation for cell-based gene therapy in a mouse model of hereditary tyrosinemia type 1 (HT1). Here, we explore the use of the clinical-grade electroporator, the MaxCyte ExPERT GTx, utilized in the first FDA-approved CRISPR therapy, Casgevy, and evaluate its potential in primary hepatocytes in terms of delivery efficiency and cell viability. We assessed the gene editing efficiency and post-transplantation engraftment of hepatocytes from mTmG mice electroporated with CRISPR-Cas9-ribonucleoproteins (RNPs) targeting 4-hydroxyphenylpyruvate dioxygenase (Hpd) in a fumarylacetoacetate hydrolase (Fah)-deficient mouse model of HT1. After surgery, Fah[-/-] graft recipients were cycled off and on nitisinone to achieve independence from drug-induced Hpd inhibition, an indicator of HT1 disease correction. Transplanted hepatocytes subjected to electroporation using the GTx system had a cell viability of 89.9% and 100% on-target gene editing efficiency. Recipients transplanted with GTx-electroporated cells showed a smaller weight reduction than controls transplanted with untransfected cells (7.9% and 13.8%, respectively). Further, there were no mortalities in the GTx-recipient mice, whereas there was 25% mortality in the control recipients. Mean donor cell engraftment was significantly higher in GTx-recipient mice compared to untransfected control recipients (97.9% and 81.6%, respectively). Our results indicate that the GTx system does not negatively impact hepatocyte functionality and engraftment potential, thereby demonstrating the promise of GTx electroporation in hepatocytes as a viable cell therapy for treating genetic diseases that affect the liver.},
}

*Hepatocytes/transplantation/metabolism
*Electroporation/methods
Animals
*CRISPR-Cas Systems/genetics
*Genetic Therapy/methods
Mice
Gene Editing/methods
*Tyrosinemias/therapy/genetics
Humans
*Liver Diseases/therapy/genetics
Disease Models, Animal
Male
Hydrolases

@article {pmid40422195,
year = {2025},
author = {Orlova, NA and Sinegubova, MV and Kolesov, DE and Khodak, YA and Tatarskiy, VV and Vorobiev, II},
title = {Genomic and Phenotypic Characterization of CHO 4BGD Cells with Quad Knockout and Overexpression of Two Housekeeping Genes That Allow for Metabolic Selection and Extended Fed-Batch Culturing.},
journal = {Cells},
volume = {14},
number = {10},
pages = {},
pmid = {40422195},
issn = {2073-4409},
support = {25-24-00140//Russian Science Foundation/ ; },
mesh = {CHO Cells ; Animals ; Cricetulus ; *Batch Cell Culture Techniques/methods ; CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques ; Gene Editing ; Apoptosis/genetics ; *Genes, Essential/genetics ; Phenotype ; *Genomics ; },
abstract = {Re-engineering of CHO cells using genome editing and the overexpression of multiple helper genes is the central track for obtaining better cell lines for the production of biopharmaceuticals. Using two subsequent rounds of genome editing of the CHO S cells, we have developed the cell line CHO 4BGD with four knockouts of two pro-apoptotic genes bak1 and bax, and two common selection markers genes-glul (GS) and dhfr, and additional copies of genes bcl-2 and beclin-1 used for enhancement of macroautophagy. The NGS sequencing of 4BGD cells revealed that all eight targeted alleles were successfully disrupted. Two edited loci out of eight contained large inserts of non-relevant DNA. Further data analysis shows that cells have no off-target DNA editing events, and all known CHO genes are preserved. The cells obtained are completely resistant to the induction of apoptosis, and they are suitable for the generation of stably transfected cell lines with the dhfr selection marker. They also properly undergo the target gene amplification. The 4BGD-derived clonal cell line that secretes the monoclonal antibody retains the ability for prolonged fed-batch culturing. The method of obtaining multiply edited CHO cells using the multiplex CRISPR/Cas9 editing and simultaneous stable transfection of plasmids, coding for the housekeeping genes, is suitable for the rapid generation of massively edited CHO cells.},
}

CHO Cells
Animals
Cricetulus
*Batch Cell Culture Techniques/methods
CRISPR-Cas Systems/genetics
*Gene Knockout Techniques
Gene Editing
Apoptosis/genetics
*Genes, Essential/genetics
Phenotype
*Genomics

@article {pmid40419487,
year = {2025},
author = {Byrne, SM and Burleigh, SM and Fragoza, R and Jiang, Y and Savva, Y and Pabon, R and Kania, E and Rainaldi, J and Portell, A and Mali, P and Briggs, AW},
title = {An engineered U7 small nuclear RNA scaffold greatly increases ADAR-mediated programmable RNA base editing.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4860},
pmid = {40419487},
issn = {2041-1723},
mesh = {*Adenosine Deaminase/metabolism/genetics ; Animals ; *RNA Editing/genetics ; Humans ; Mice ; *RNA, Small Nuclear/genetics/metabolism/chemistry ; *Gene Editing/methods ; *RNA-Binding Proteins/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; },
abstract = {Custom RNA base editing exploiting the human Adenosine Deaminase Acting on RNA (ADAR) enzyme may enable therapeutic gene editing without DNA damage or use of foreign proteins. ADAR's adenosine-to-inosine (effectively A-to-G) deamination activity can be targeted to transcripts using an antisense guide RNA (gRNA), but efficacy is challenged by limits of in vivo delivery. Embedding gRNAs into a U7 small nuclear RNA (snRNA) framework greatly enhances RNA editing with endogenous ADAR, and a 750-plex single-cell mutagenesis screen further improved the framework. An optimized scaffold with a stronger synthetic U7 promoter enables 76% RNA editing in vitro from a single DNA construct per cell, and 75% editing in a Hurler syndrome mouse brain after one systemic AAV injection, surpassing circular gRNA approaches. The technology also improves published DMD exon-skipping designs 25-fold in differentiated myoblasts. Our engineered U7 framework represents a universal scaffold for ADAR-based RNA editing and other antisense RNA therapies.},
}

*Adenosine Deaminase/metabolism/genetics
Animals
*RNA Editing/genetics
Humans
Mice
*RNA, Small Nuclear/genetics/metabolism/chemistry
*Gene Editing/methods
*RNA-Binding Proteins/metabolism/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
HEK293 Cells

@article {pmid40419416,
year = {2025},
author = {Wachholz Junior, D and Pontes, RG and Hryniewicz, BM and Kubota, LT},
title = {Exploring a CRISPR/Cas12a-powered impedimetric biosensor for amplification-free detection of a pathogenic bacterial DNA.},
journal = {Biosensors & bioelectronics},
volume = {},
number = {},
pages = {117607},
doi = {10.1016/j.bios.2025.117607},
pmid = {40419416},
issn = {1873-4235},
abstract = {Timely and precise detection of bacterial infections is essential for improving patient outcomes and reducing healthcare costs, especially for sepsis, where delayed diagnosis increases mortality. Traditional culture- and PCR-based methods are time consuming and require complex sample processing, making them unsuitable for rapid diagnostics in resource-limited settings. CRISPR/Cas-based methods, particularly when combined with electrochemical sensing, offer a promising alternative for rapid point-of-care (POC) diagnostics of bacterial infections due to their simplicity and specificity. This study proposes a label-free impedimetric biosensor using the CRISPR/Cas12a system for rapid and amplification-free detection of Staphylococcus aureus DNA, a primary pathogen responsible for sepsis. By leveraging CRISPR/Cas12a's target-activated collateral cleavage on non-specific DNA reporters we investigated the impact of using a protospacer adjacent motif (PAM) sequence on detection sensitivity and specificity. Our biosensor demonstrated ultra-sensitive detection, with limit of detection as low as 20 aM for dsDNA targets in buffer and without any pre-amplification steps. The study also confirmed CRISPR specificity's dependence on the PAM sequence, showing that mismatches on targeting sequences reduces cleavage efficiency, with a drastic reduction in trans-cleavage activity for single mismatch in PAM-containing sequences. Additionally, we examined how the DNA reporter affects performance, noting reduced cleavage efficiency when a ssDNA target was paired with a dsDNA reporter. Furthermore, validation experiments using human serum samples confirmed the biosensor's accuracy for bacterial DNA detection in clinical settings. This work advances CRISPR-powered electrochemical biosensors, providing a detailed discussion on developing a highly sensitive, fast and amplification-free tool for early detection of sepsis-causing bacteria.},
}

@article {pmid40418657,
year = {2025},
author = {Johnson, MJ and DeFeo, AP and Slipek, NJ and Folsom, TD and Henley, T and Choudhry, MS and Webber, BR and Moriarity, BS},
title = {Non-Viral Engineering of Primary Human T Cells via Homology-Mediated End-Joining Targeted Integration of Large DNA Templates.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {219},
pages = {},
doi = {10.3791/68150},
pmid = {40418657},
issn = {1940-087X},
mesh = {Humans ; *T-Lymphocytes/cytology/immunology ; CRISPR-Cas Systems ; *DNA/genetics ; Animals ; *Cell Engineering/methods ; Mice ; *DNA End-Joining Repair ; *Genetic Engineering/methods ; },
abstract = {Many current adoptive cellular therapies rely on lenti- or retroviral vectors to engineer T cells for the expression of a chimeric antigen receptor (CAR) or exogenous T cell receptor (TCR) to target a specific tumor-associated antigen. Reliance on viral vectors for the production of therapeutic T cells significantly increases the timeline, cost, and complexity of manufacturing while limiting the translation of new therapies, particularly in the academic setting. A process is presented for efficient non-viral engineering of T cells using CRISPR/Cas9 and homology-mediated end joining to achieve targeted integration of large, multicistronic DNA cargo. This approach has achieved integration frequencies comparable to those of viral vectors while yielding highly functional T cells capable of potent anti-tumor efficacy both in vitro and in vivo. Notably, this method is rapidly adaptable to current good manufacturing practices (cGMP) and clinical scale-up, providing a near-term option for the manufacturing of therapeutic T cells for use in clinical trials.},
}

Humans
*T-Lymphocytes/cytology/immunology
CRISPR-Cas Systems
*DNA/genetics
Animals
*Cell Engineering/methods
Mice
*DNA End-Joining Repair
*Genetic Engineering/methods

@article {pmid40417002,
year = {2025},
author = {Gallala, M},
title = {Application of CRISPR/Cas gene editing for infectious disease control in poultry.},
journal = {Open life sciences},
volume = {20},
number = {1},
pages = {20251095},
pmid = {40417002},
issn = {2391-5412},
abstract = {The poultry industry faces multifaceted challenges, including escalating demand for poultry products, climate change impacting feed availability, emergence of novel avian pathogens, and antimicrobial resistance. Traditional disease control measures are costly and not always effective, prompting the need for complementary methods. Gene editing (GE, also called genome editing) technologies, particularly CRISPR/Cas9, offer promising solutions. This article summarizes recent advancements in utilizing CRISPR/Cas GE to enhance infectious disease control in poultry. It begins with an overview of modern GE techniques, highlighting CRISPR/Cas9's advantages over other methods. The potential applications of CRISPR/Cas in poultry infectious disease prevention and control are explored, including the engineering of innovative vaccines, the generation of disease-resilient birds, and in vivo pathogen targeting. Additionally, insights are provided regarding regulatory frameworks and future perspectives in this rapidly evolving field.},
}

@article {pmid40315964,
year = {2025},
author = {Gupta, MK and Gouda, G and Moazzam-Jazi, M and Vadde, R and Nagaraju, GP and El-Rayes, BF},
title = {CRISPR/Cas9-directed epigenetic editing in colorectal cancer.},
journal = {Biochimica et biophysica acta. Reviews on cancer},
volume = {1880},
number = {3},
pages = {189338},
doi = {10.1016/j.bbcan.2025.189338},
pmid = {40315964},
issn = {1879-2561},
mesh = {Humans ; *Colorectal Neoplasms/genetics/therapy/pathology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Epigenesis, Genetic ; Animals ; DNA Methylation ; Gene Expression Regulation, Neoplastic ; Epigenome Editing ; },
abstract = {Colorectal cancer (CRC) remains a leading cause of cancer-related illness and death worldwide, arising from a complex interplay of genetic predisposition, environmental influences, and epigenetic dysregulation. Among these factors, epigenetic modifications-reversible and heritable changes in gene expression-serve as crucial regulators of CRC progression. Understanding these modifications is essential for identifying potential biomarkers for early diagnosis and developing targeted therapeutic strategies. Epigenetic drugs (epidrugs) such as DNA methyltransferase inhibitors (e.g., decitabine) and bromodomain inhibitors (e.g., JQ1) have shown promise in modulating aberrant epigenetic changes in CRC. However, challenges such as drug specificity, delivery, and safety concerns limit their clinical application. Advances in CRISPR-Cas9-based epigenetic editing offer a more precise approach to modifying specific epigenetic markers, presenting a potential breakthrough in CRC treatment. Despite its promise, CRISPR-based epigenome editing may result in unintended genetic modifications, necessitating stringent regulations and safety assessments. Beyond pharmacological interventions, lifestyle factors-including diet and gut microbiome composition-play a significant role in shaping the epigenetic landscape of CRC. Nutritional and microbiome-based interventions have shown potential in preventing CRC development by maintaining intestinal homeostasis and reducing tumor-promoting epigenetic changes. This review provides a comprehensive overview of epigenetic alterations in CRC, exploring their implications for diagnosis, prevention, and treatment. By integrating multi-omics approaches, single-cell technologies, and model organism studies, future research can enhance the specificity and efficacy of epigenetic-based therapies. Shortly, a combination of advanced gene-editing technologies, targeted epidrugs, and lifestyle interventions may pave the way for more effective and personalized CRC treatment strategies.},
}

Humans
*Colorectal Neoplasms/genetics/therapy/pathology
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Epigenesis, Genetic
Animals
DNA Methylation
Gene Expression Regulation, Neoplastic
Epigenome Editing

@article {pmid39568260,
year = {2025},
author = {Wang, B and Fan, A and Liu, M and Zhou, Y and Zhang, W and Yan, J},
title = {An Integrated Rapid Detection of Botryosphaeriaceae Species in Grapevine Based on Recombinase Polymerase Amplification, CRISPR/Cas12a, and Lateral Flow Dipstick.},
journal = {Plant disease},
volume = {109},
number = {5},
pages = {1102-1110},
doi = {10.1094/PDIS-08-24-1615-RE},
pmid = {39568260},
issn = {0191-2917},
mesh = {*Vitis/microbiology ; *Ascomycota/genetics/isolation & purification ; *Plant Diseases/microbiology ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Recombinases/genetics/metabolism ; Sensitivity and Specificity ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Grapevine Botryosphaeria dieback (GBD), caused by Botryosphaeriaceae species, is an important grapevine trunk disease that poses a threat to grape yield and quality in global viticultural regions. Pathogen diagnosis at the species level using morphological methods is difficult and time-consuming. Therefore, this study aimed to develop a rapid and accurate detection method for the pathogens causing GBD. Recombinase polymerase amplification (RPA) with CRISPR/Cas12a cleavage was combined for detecting pathogens associated with GBD, and lateral flow dipsticks were employed to monitor the outcomes. Based on the β-tubulin sequences of Botryosphaeriaceae and their related species, specific RPA primers and CRISPR/Cas12a CrRNA were designed and subsequently selected for specifically detecting pathogens associated with GBD. Under optimized reaction conditions and systems, the developed RPA/CRISPR-Cas12a detection system specifically detected Botryosphaeriaceae species within 30 min of RPA and 25 min of CRISPR/Cas12a reactions at 37°C. Specificity tests showed that specific fragments were amplified with the RPA primers in the DNA of six Botryosphaeriaceae species found in China, while none of the fragments were amplified in the other 22 nontarget fungal pathogen species of grapevine. The detection sensitivity of this method was 1 pg/μl, which is equal to that of real-time PCR. In summary, our method is simple to perform, produces visual results, does not rely on expensive equipment, and therefore possesses high practical value, providing an efficient and robust detection platform to accelerate the field detection of pathogens associated with GBD.},
}

*Vitis/microbiology
*Ascomycota/genetics/isolation & purification
*Plant Diseases/microbiology
*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
Recombinases/genetics/metabolism
Sensitivity and Specificity
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40415627,
year = {2025},
author = {Li, H and Zhu, Q and Zhu, J and Min, J},
title = {[Research progress in the developmental process of non-viral CAR-T technology].},
journal = {Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology},
volume = {41},
number = {5},
pages = {461-467},
pmid = {40415627},
issn = {1007-8738},
mesh = {Humans ; *Immunotherapy, Adoptive/methods ; *Receptors, Chimeric Antigen/genetics/immunology ; Animals ; Gene Transfer Techniques ; Genetic Vectors/genetics ; Gene Editing ; CRISPR-Cas Systems/genetics ; DNA Transposable Elements/genetics ; *T-Lymphocytes/immunology ; Neoplasms/therapy/immunology ; },
abstract = {Chimeric antigen receptor T (CAR-T) lymphocytes are at the forefront of adoptive immunotherapy research, and this technology has significantly advanced the prospects of tumor immunotherapy. CAR-T therapy has demonstrated remarkable efficacy in haematological tumours of lymphoid origin and provided therapeutic possibility for solid tumours. Currently, CAR-T cell preparation predominantly involves transfection of T cells with viral vectors. However, the production of viral vectors is time-consuming, expensive, and the vectors have low loading capacity, along with insertion instability. Consequently, there is a pressing need to develop more convenient and precise non-viral gene delivery methods. This paper reviews the most promising non-viral gene delivery technologies, including CRISPR/Cas9 gene editing, transposon systems such as Sleeping Beauty (SB) and PiggyBac (PB), and mRNA, and anticipates the future development of non-viral vector-based CAR-T therapies.},
}

Humans
*Immunotherapy, Adoptive/methods
*Receptors, Chimeric Antigen/genetics/immunology
Animals
Gene Transfer Techniques
Genetic Vectors/genetics
Gene Editing
CRISPR-Cas Systems/genetics
DNA Transposable Elements/genetics
*T-Lymphocytes/immunology
Neoplasms/therapy/immunology

@article {pmid40414878,
year = {2025},
author = {Lobel, JH and Ingolia, NT},
title = {Precise measurement of molecular phenotypes with barcode-based CRISPRi systems.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {142},
pmid = {40414878},
issn = {1474-760X},
support = {F32 GM148044/GM/NIGMS NIH HHS/United States ; R01 GM135233/GM/NIGMS NIH HHS/United States ; R21 HG012991/HG/NHGRI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Phenotype ; Humans ; Promoter Regions, Genetic ; },
abstract = {Genome-wide CRISPR-Cas9 screens have untangled regulatory networks driving diverse biological processes. Their success relies on interrogating specific molecular phenotypes and distinguishing key regulators from background effects. Here, we realize these goals by optimizing CRISPR interference with barcoded expression reporter sequencing (CiBER-seq) to dramatically improve the sensitivity and scope of genome-wide screens. We systematically address technical factors that distort phenotypic measurements by normalizing expression reporters against closely matched promoters. We use our improved CiBER-seq to accurately capture known components of well-studied RNA and protein quality control systems. These results demonstrate the precision and versatility of CiBER-seq for dissecting cellular pathways.},
}

*CRISPR-Cas Systems
Phenotype
Humans
Promoter Regions, Genetic

@article {pmid40414010,
year = {2025},
author = {Cao, X and Gao, Z and Yin, P and Wang, H and Yang, L},
title = {Crystal structure and inhibition mechanism of AcrIIA11.},
journal = {Biochemical and biophysical research communications},
volume = {772},
number = {},
pages = {152073},
doi = {10.1016/j.bbrc.2025.152073},
pmid = {40414010},
issn = {1090-2104},
abstract = {Anti-CRISPR (Acr) proteins are naturally evolved inhibitors that precisely target and suppress CRISPR-Cas systems, representing a sophisticated molecular arms race between bacteriophages and their bacterial hosts. While Class 1 systems dominate among sequenced prokaryotic genomes, Class 2 systems remain primary sources of editing tools. Here, we report the structural and mechanistic characterization of AcrIIA11, an anti-CRISPR protein that simultaneously inhibits Streptococcus pyogenes (SpyCas9) and Staphylococcus aureus Cas9 (SauCas9). The 3.2 Å crystal structure reveals a compact α/β fold with distinct electropositive clefts implicated in DNA binding. While DALI analysis identified structural homology to transcriptional regulators and the RecA inhibitor PsiB (RMSD 3.3 Å), functional studies established that AcrIIA11 forms stable ternary complexes with both Cas9 orthologs and sgRNA. Biochemical assays demonstrated stronger inhibition of SauCas9 compared to SpyCas9, with EMSA revealing a critical dichotomy: AcrIIA11 maintains SauCas9-sgRNA binding to specific target DNA while completely blocking cleavage activity. Computational docking localizes AcrIIA11 at the HNH-RuvC interface without obstructing DNA-binding channels in SauCas9, suggesting allosteric inhibition through HNH domain displacement. This work establishes AcrIIA11 as a dual-purpose Cas9 inhibitor that preserves target recognition while inactivating nuclease function-a mechanism with potential applications in precision CRISPR control.},
}

@article {pmid40413996,
year = {2025},
author = {Duan, M and Li, G and Shen, J and Dai, R and Li, X and Liu, Z and Jia, F},
title = {A CRISPR/Cas12a biosensor for portable and accessible detection of Salmonella typhimurium via multi-indicator pH millidisc colorimetry and smartphone imaging platform.},
journal = {Biosensors & bioelectronics},
volume = {286},
number = {},
pages = {117611},
doi = {10.1016/j.bios.2025.117611},
pmid = {40413996},
issn = {1873-4235},
abstract = {Conventional colorimetric CRISPR/Cas methods rely on a single chromogenic substrate and bulky and specialized signal detection instrument, which hinder their practical application. Herein, a portable and accessible CRISPR/Cas12a biosensor was for the first time reported to sensitively quantify Salmonella enterica serovar typhimurium (S. typhimurium), utilizing a multi-indicator pH millimeter disc (millidisc) for signal visualization, combined with a smartphone-based imaging platform for signal readout. The pH millidisc, composed of multiple indicators, possessed sensitive pH responsiveness and exhibited diverse color changes. The self-developed RGB mini-program, named DeepFood, was designed for portable smartphone use, featuring user-friendly operation and trend visualization for preliminary result analysis. Based on the RGB signal variation pattern with S. typhimurium, a distinct Senh signal type was designed, enhancing the signal-to-noise ratio from 3.38 to 7.11. Compared to the R signal type, the Senh signal type improved detection sensitivity by 36.23-fold (7.26 CFU/mL) in 0.01 M PBS buffer and 15.53-fold (1.41 × 10[2] CFU/mL) in chicken. The proposed biosensor offers significant improvements in detection sensitivity and practical applicability, with potential in food safety and environmental protection.},
}

@article {pmid40413357,
year = {2025},
author = {Jiang, B and An, Z and Niu, L and Qin, D},
title = {Precise genome editing process and its applications in plants driven by AI.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {109},
pmid = {40413357},
issn = {1438-7948},
support = {BLX202316//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Genome, Plant ; *Plants/genetics ; *Artificial Intelligence ; Plant Breeding ; },
abstract = {Genome editing technologies have emerged as the keystone of biotechnological research, enabling precise gene modification. The field has evolved rapidly through revolutionary advancements, transitioning from early explorations to the breakthrough of the CRISPR-Cas system. The emergence of the CRISPR-Cas system represents a huge leap in genome editing, prompting the development of advanced tools such as base and prime editors, thereby enhancing precise genomic engineering capabilities. The rapid integration of AI across disciplines is now driving another transformative phase in genome editing, streamlining workflows and enhancing precision. The application prospects of genome editing technology are extensive, particularly in plant breeding, where it has already presented unparalleled opportunities for improving plant traits. Here, we review early genome editing technologies, including meganucleases, ZFNs, TALENs, and CRISPR-Cas systems. We also provide a detailed introduction to next-generation editing tools-such as base editors and prime editors-and their latest applications in plants. At the same time, we summarize and prospect the cutting-edge developments and future trends of genome editing technologies in combination with the rapidly rising AI technology, including optimizing editing systems, predicting the efficiency of editing sites and designing editing strategies. We are convinced that as these technologies progress and their utilization expands, they will provide pioneering solutions to global challenges, ushering in an era of health, prosperity, and sustainability.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*Genome, Plant
*Plants/genetics
*Artificial Intelligence
Plant Breeding

@article {pmid40316115,
year = {2025},
author = {Tao, Z and Tian, C and Zhong, C and Ji, B and Li, W and Zhao, Y},
title = {The role of NhaA protein in modulating antibiotic tolerance in Escherichia coli.},
journal = {International journal of biological macromolecules},
volume = {311},
number = {Pt 2},
pages = {143721},
doi = {10.1016/j.ijbiomac.2025.143721},
pmid = {40316115},
issn = {1879-0003},
mesh = {*Escherichia coli/drug effects/genetics/metabolism ; *Anti-Bacterial Agents/pharmacology ; *Escherichia coli Proteins/genetics/metabolism ; *Drug Resistance, Bacterial/genetics ; Adenosine Triphosphate/metabolism ; Proton-Motive Force/drug effects ; Cell Membrane Permeability/drug effects ; Membrane Fluidity/drug effects ; Microbial Sensitivity Tests ; CRISPR-Cas Systems ; },
abstract = {As microbial resistance and recurrent bacterial infections escalate, the growing understanding of the interplay between antibiotic resistance and tolerance has sparked significant interest in the latter. Previous studies have demonstrated that the deletion of cation/proton antiporters (CPAs) induces bacterial phenotypes, such as slow growth and prolonged lag phases, which contribute to the development of tolerance. This study investigates the role of the NhaA protein in antibiotic tolerance in Escherichia coli using CRISPR/Cas9 gene editing to delete the NhaA protein. Our results suggest that the NhaA protein plays a key role in modulating antibiotic tolerance. In response to NhaA deletion, E. coli adapts through multiple mechanisms, including changes in membrane permeability, enhanced efflux activity, increased membrane fluidity, disruption of the proton motive force (PMF), and a reduction in intracellular ATP levels. These adaptive changes collectively promote the development of antibiotic tolerance. Understanding these tolerance mechanisms could uncover new therapeutic targets, help prevent the emergence of tolerance, or sustain bacteria cells in a tolerant state, providing crucial strategies to combat the rise of antibiotic-resistant bacteria.},
}

*Escherichia coli/drug effects/genetics/metabolism
*Anti-Bacterial Agents/pharmacology
*Escherichia coli Proteins/genetics/metabolism
*Drug Resistance, Bacterial/genetics
Adenosine Triphosphate/metabolism
Proton-Motive Force/drug effects
Cell Membrane Permeability/drug effects
Membrane Fluidity/drug effects
Microbial Sensitivity Tests
CRISPR-Cas Systems

@article {pmid40158793,
year = {2025},
author = {Sheng, T and Meng, XZ and Yu, Q and Lv, W and Chen, Y and Cong, Q and Li, W and Gui, L and Li, J and Xu, X},
title = {Transcriptome analysis and CRISPR-Cas9-mediated mutagenesis identify gpr116 as a candidate gene for growth promotion in grass carp (Ctenopharyngodon idella).},
journal = {Comparative biochemistry and physiology. Part A, Molecular & integrative physiology},
volume = {305},
number = {},
pages = {111850},
doi = {10.1016/j.cbpa.2025.111850},
pmid = {40158793},
issn = {1531-4332},
mesh = {Animals ; *Carps/genetics/growth & development ; *CRISPR-Cas Systems ; Zebrafish/genetics/growth & development ; *Receptors, G-Protein-Coupled/genetics/metabolism ; Gene Expression Profiling ; *Fish Proteins/genetics/metabolism ; *Transcriptome ; Mutagenesis ; Brain/metabolism ; },
abstract = {Grass carp (Ctenopharyngodon idella) is an economically important aquaculture species known for its considerable variability in growth performance. In this study, we investigated the growth phenotype by comparing fast-growing and slow-growing groups. Microstructural analyses revealed that slow-growing fish exhibited significantly larger myofibrillar gaps and lower muscle fiber density. To elucidate the underlying molecular basis, we performed transcriptome (RNA-Seq) analysis of brain and dorsal muscle tissues. 328 differentially expressed genes (DEGs) were identified in dorsal muscle tissue (33 up and 295 down-regulated) and 228 in brain tissue (17 up and 211 down-regulated). Gene Ontology and KEGG enrichment analyses indicated that the DEGs were closely associated with apoptosis and angiogenesis pathways. Among the candidate genes, gpr116 was significantly up-regulated in the brain and dorsal muscle tissue of the fast-growing group. Finally, CRISPR-Cas9-mediated knockout in a zebrafish model confirmed that gpr116 deletion significantly restricted growth, underscoring its pivotal role in the growth regulation of grass carp. These discoveries lay significant groundwork for deeper exploration of growth regulation mechanisms in grass carp and offer important clues for selective breeding of key growth marker genes in this species.},
}

Animals
*Carps/genetics/growth & development
*CRISPR-Cas Systems
Zebrafish/genetics/growth & development
*Receptors, G-Protein-Coupled/genetics/metabolism
Gene Expression Profiling
*Fish Proteins/genetics/metabolism
*Transcriptome
Mutagenesis
Brain/metabolism

@article {pmid40147495,
year = {2025},
author = {Panting, M and Holme, IB and Dionisio, G and Brinch-Pedersen, H},
title = {Simplex and multiplex CRISPR/Cas9-mediated knockout of grain protease inhibitors in model and commercial barley improves hydrolysis of barley and soy storage proteins.},
journal = {Plant biotechnology journal},
volume = {23},
number = {6},
pages = {2418-2428},
doi = {10.1111/pbi.70065},
pmid = {40147495},
issn = {1467-7652},
support = {8055-00038B//Innovationsfonden/ ; NNF19OC005658//Novo Nordisk Fonden/ ; 101060057//Horizon Europe/ ; },
mesh = {*Hordeum/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Protease Inhibitors/metabolism ; *Soybean Proteins/metabolism ; *Seed Storage Proteins/metabolism ; *Plant Proteins/metabolism/genetics ; Hydrolysis ; Edible Grain/genetics/metabolism ; Seeds/metabolism/genetics ; Gene Knockout Techniques ; Glycine max/metabolism ; Proteolysis ; },
abstract = {Anti-nutritional factors in plant seeds diminish the utilization of nutrients in feed and food. Among these, protease inhibitors inhibit protein degradation by exogenous proteases during digestion. Through conventional and selection-gene-free genome editing using ovules as explants, we used simplex and multiplex CRISPR/Cas9 for studying the impact of chymotrypsin inhibitor CI-1A, CI-1B and CI-2, Bowman-Birk trypsin inhibitor, Serpin-Z4, and barley ɑ-amylase/subtilisin inhibitor on barley and soybean storage protein degradation. Mutants were generated in the commercial cultivar Stairway, having a high level of protease inhibition, and the barley model cultivar Golden Promise, having a lower inhibition level. In Golden Promise, all individual knockouts decreased the inhibition of the three proteases α-chymotrypsin, trypsin and the commercial feed protease Ronozyme ProAct significantly. The triple knockout of all chymotrypsin inhibitors further decreased the inhibition of α-chymotrypsin and Ronozyme ProAct proteases. Degradations of recombinant barley storage proteins B- and C-hordeins were significantly improved following mutagenesis. In Stairway, a single knockout of CI-1A almost compares to the effect on the proteases achieved for the triple knockout in Golden promise, uncovering CI-1A as the major protease inhibitor in that cultivar. The Stairway mutant demonstrated significantly improved degradation of recombinant barley hordeins and in the soybean storage proteins glycinin and β-conglycinin. The results of this study provide insights into cereal protease inhibitor genes and their negative effects on the degradation of barley storage protein and the most important plant protein from soybeans. The study suggests a future focus on plant protease inhibitors as a major target for improving feed and food protein digestibility.},
}

*Hordeum/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Protease Inhibitors/metabolism
*Soybean Proteins/metabolism
*Seed Storage Proteins/metabolism
*Plant Proteins/metabolism/genetics
Hydrolysis
Edible Grain/genetics/metabolism
Seeds/metabolism/genetics
Gene Knockout Techniques
Glycine max/metabolism
Proteolysis