@article {pmid40950596,
year = {2025},
author = {Hadian, S and Smith, DL and Suproniene, S},
title = {Genomic and phenotypic profiling of an Artemisia endophyte: dual biostimulant and biocontrol activities in pea under Fusarium stress.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1643204},
pmid = {40950596},
issn = {1664-302X},
abstract = {OBJECTIVE: To evaluate the plant growth-promoting and disease-suppressing potential of Serratia sp. AR11, an endophytic bacterium isolated from Artemisia absinthium, through phenotypic assessment and whole-genome analysis in pea (Pisum sativum) under normal and Fusarium-stress conditions.

MATERIALS AND METHODS: Greenhouse experiments were conducted to assess the effects of AR11 inoculation on shoot and root growth, biomass, chlorophyll content, and Fusarium-induced stunting. Whole-genome sequencing was performed using the PacBio SMRT platform, followed by functional annotation to identify genes related to nutrient metabolism, secondary metabolite biosynthesis, and stress adaptation. Biosafety assessment included screening for virulence and antibiotic resistance genes.

RESULTS: AR11 inoculation significantly increased shoot and root length and biomass, while reducing Fusarium-induced stunting by over 70%. Under pathogen stress, treated plants showed a 67% increase in SPAD index compared to controls. Genome analysis revealed a complete 5.49 Mb circular genome with 5,175 protein-coding genes, including those for nitrogen metabolism, phosphate solubilization, siderophore production, and antifungal secondary metabolite biosynthesis. Additional features included stress-responsive regulatory systems, CRISPR-Cas loci, and DNA methylation patterns. No high-risk virulence or acquired antibiotic resistance genes were detected.

CONCLUSION: Serratia sp. AR11 is a safe, multifunctional endophytic bacterium with strong genomic and phenotypic evidence for enhancing pea growth and suppressing Fusarium-related diseases, making it a promising candidate for sustainable low-input agriculture.},
}

@article {pmid40946761,
year = {2025},
author = {Uddin, N and Ullah, MW and Li, K and Liu, F and Xin, X},
title = {Plant cell wall biosynthesis: Immune signaling, genome editing, and physiological implications for biomass valorization.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108714},
doi = {10.1016/j.biotechadv.2025.108714},
pmid = {40946761},
issn = {1873-1899},
abstract = {Plants continuously face biotic stress from pathogens, pests, and environmental challenges that threaten their survival and productivity. In response, plants have developed complex immune systems, with the cell wall playing a central role in defense. The plant cell wall not only provides mechanical strength but also acts as a dynamic barrier against pathogens, influencing both plant growth and immune responses. This review discusses the molecular mechanisms of cell wall biosynthesis, facilitated by multi-omics technologies, particularly the synthesis and regulation of lignin and other polysaccharides, which contribute to cell wall integrity and plant immunity. It explores the interplay between cell wall modifications and immune signaling pathways, highlighting the role of pattern recognition receptors in pathogen detection and defense activation. Additionally, the potential of genome editing, especially CRISPR-Cas, in enhancing cell wall characteristics to improve pathogen resistance and biomass utilization is discussed. With growing interest in lignocellulosic biomass as a renewable resource for biofuels and bioproducts, this review also addresses the challenges of biomass recalcitrance, focusing on biotechnological advancements to improve saccharification efficiency. Finally, the review proposes integrated strategies combining genetic modifications, biotechnological innovations, and sustainable practices to optimize lignocellulosic biomass for a bio-based economy, contributing to both agricultural resilience and sustainable energy production. As climate change accelerates, these technologies hold the promise of developing resilient crops and enhancing the capacity of the bioeconomy to mitigate environmental impacts.},
}

@article {pmid40946164,
year = {2025},
author = {Sun, J and Liu, K and Sheng, Y and Zhang, H and Wang, J and Wang, Y and Tian, R and Zhu, X and Tian, SS and Wang, P and Zhao, X and Liu, Y},
title = {High-Fidelity AaCas12b[Max] Enables the Development of an Engineered T Cell Therapy with Enhanced Safety and Functional Fitness.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2025.09.009},
pmid = {40946164},
issn = {1525-0024},
abstract = {CRISPR-Cas systems have transformed genome editing, yet the commonly used Streptococcus pyogenes Cas9 (SpCas9) is limited by off-target effects and chromosomal instability. Here, we characterize AaCas12b[MAX], an engineered Alicyclobacillus acidiphilus Cas12b variant, as a high-precision editing platform optimized for tumor infiltrating lymphocyte (TIL) therapy. Using an FDA-compliant safety assessment framework, we systemically compared AaCas12b[MAX]- and SpCas9-edited TIL products in terms of on-target efficiency, genome-wide off-target activity, and structural variants (SVs) formation. AaCas12b[MAX] achieved >80% on-target editing efficiency with undetectable off-target events and a 3.3-fold reduction in SVs relative to SpCas9. Mechanistic studies revealed different DNA repair kinetics in AaCas12b[MAX]-edited cells, reducing sustained DNA damage responses and chromosomal instability. Structural modeling suggested a more stable enzyme-sgRNA-DNA ternary complex, enabling stringent PAM specificity and minimal mismatch tolerance. Functionally, AaCas12b[MAX]-edited TILs exhibited superior therapeutic potential, including enhanced cellular fitness, a twofold increase in expansion capacity, and enrichment of stem-like tumor-reactive CD39[-]CD69[-]CD8[+] subsets. Together, these results establish AaCas12b[MAX] as a robust, clinically translatable platform that improves the safety and functional limitations of SpCas9, enabling the development of next-generation T cell therapies.},
}

@article {pmid40945731,
year = {2025},
author = {Jain, M and Pattnayak, AK and Aggarwal, S and Rai, P and Kavya, J and Chandrayan, S and Goel, M and Gaur, V},
title = {Branched DNA processing by a thermostable CAS-Cas4 from Thermococcus onnurineus: expanding biochemical landscape of nuclease activity.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {110701},
doi = {10.1016/j.jbc.2025.110701},
pmid = {40945731},
issn = {1083-351X},
abstract = {The adaptive immune function of CRISPR-Cas systems in bacteria and archaea is mediated through CRISPR-Associated Proteins (Cas). The adaptation module, typically involving Cas1, Cas2, and Cas4, helps integrate viral "spacer" sequences into the host genome. Cas4 proteins are classified into two types based on neighboring genes: CAS-Cas4, flanked by other cas genes, and Solo-Cas4, which exist independently. While CAS-Cas4 proteins are implicated in adaptation, they remain biochemically uncharacterized in archaea, unlike archaeal Solo-Cas4 proteins. This study biochemically characterizes TON_0321, a CAS-Cas4 protein from the Type IV-C CRISPR cassette of Thermococcus onnurineus. TON_0321 exhibits 5' to 3' exonuclease activity and unique structure-dependent endonuclease activity, shedding light on CAS-Cas4 functional diversity. A distinct spatial organization of the catalytic site, angled with the positively charged patch on the protein surface, enables TON_0321 to recognize branching points in DNA substrates. Furthermore, this spatial arrangement facilitates cleavage 2 to 3 nucleotides away from the branch point in the 5' direction, demonstrating structure-specific endonuclease activity.},
}

@article {pmid40943511,
year = {2025},
author = {Ma, Z and Ren, J and Liu, Q and Li, J and Zhao, H and Tibesigwa, DG and Matola, SH and Gulfam, T and Yang, J and Wang, F},
title = {Integrating Traditional Breeding and Modern Biotechnology for Advanced Forest Tree Improvement.},
journal = {International journal of molecular sciences},
volume = {26},
number = {17},
pages = {},
pmid = {40943511},
issn = {1422-0067},
abstract = {In the context of global climate change and efforts toward "carbon peak and carbon neutrality," forest resource protection and restoration have become fundamental to ecological civilization. The genetic improvement of trees, as the primary component of forest ecosystems, holds strategic importance for ecological security, resource supply, and carbon neutrality. Traditional tree breeding techniques, including selective and hybrid breeding, have established robust technical systems through extensive practice. However, these methods face limitations such as extended cycles, reduced efficiency, and constrained genetic gains in meeting contemporary requirements. Modern biotechnologies, including genomic selection (GS), gene editing (CRISPR/Cas9), and marker-assisted selection (MAS), substantially enhance the precision and efficiency of genetic improvement. Nevertheless, exclusive reliance on either traditional or modern methods proves insufficient for addressing complex environmental adaptation and rapid breeding requirements. Consequently, the integration of traditional breeding with modern biotechnology to develop intelligent, sustainable, and efficient breeding strategies has emerged as a central focus in tree genetics and breeding. An integrated "step-by-step" approach warrants promotion, supported by a multi-source data sharing platform, an optimized core germplasm repository, and a "climate-soil-genotype" matching model to facilitate the region-specific deployment of improved varieties.},
}

@article {pmid40943296,
year = {2025},
author = {Sheveleva, O and Butorina, N and Protasova, E and Medvedev, S and Grigor'eva, E and Melnikova, V and Kuziaeva, V and Minzhenkova, M and Tatarenko, Y and Lyadova, I},
title = {The Generation of iPSCs Expressing Interferon-Beta Under Doxycycline-Inducible Control.},
journal = {International journal of molecular sciences},
volume = {26},
number = {17},
pages = {},
pmid = {40943296},
issn = {1422-0067},
support = {№ 0088-2024-0013//government basic research program at the Koltzov Institute of Developmental Biology of the Russian Academy of Sciences in 2024/ ; },
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology/drug effects ; Humans ; *Doxycycline/pharmacology ; *Interferon-beta/genetics/metabolism ; Cell Differentiation/drug effects ; CRISPR-Cas Systems ; Cell Line ; },
abstract = {Type 1 interferons (IFN-Is) exhibit significant antiviral, antitumor, and immunoregulatory properties, demonstrating substantial therapeutic potential. However, IFN-Is are pleiotropic cytokines, and the available data on their effect under specific pathological conditions are inconclusive. Furthermore, the systemic administration of IFN-Is can result in side effects. Generating cells that can migrate to the pathological focus and provide regulated local production of IFN-Is could overcome this limitation and provide a model for an in-depth analysis of the biological and therapeutic effects of IFN-Is. Induced pluripotent stem cells (iPSCs) are a valuable source of various differentiated cell types, including human immune cells. In this study, we describe the generation of genetically modified human iPSCs with doxycycline-controlled overexpression of interferon β (IFNB1). Three IFNB1-overexpressing iPSC lines (IFNB-iPSCs) and one control line expressing the transactivator M2rtTA (TA-iPSCs) were generated using the CRISPR/Cas9 technology. The pluripotency of the generated cell lines has been confirmed by the following: (i) cell morphology; (ii) the expression of the pluripotency markers OCT4, SOX2, TRA 1-60, and NANOG; and (iii) the ability to spontaneously differentiate into the derivatives of the three germ layers. Upon the addition of doxycycline, all IFNB-iPSCs upregulated IFNB1 expression at RNA (depending on the iPSC line, 126-816-fold) and protein levels. The IFNB-iPSCs and TA-iPSCs generated here represent a valuable cellular model for studying the effects of IFN-β on the activity and differentiation trajectories of different cell types, as well as for generating different types of cells with controllable IFN-β expression.},
}

*Induced Pluripotent Stem Cells/metabolism/cytology/drug effects
Humans
*Doxycycline/pharmacology
*Interferon-beta/genetics/metabolism
Cell Differentiation/drug effects
CRISPR-Cas Systems
Cell Line

@article {pmid40938447,
year = {2025},
author = {Kang, X and Tian, F and Liu, X and Xiao, G and Cai, Y},
title = {CRISPR/Cas13 system-based entropy-driven DNAzyme switch powered DNA walking system for sensitive and direct rotavirus detection.},
journal = {Mikrochimica acta},
volume = {192},
number = {10},
pages = {663},
pmid = {40938447},
issn = {1436-5073},
support = {No. 2018QN035//Sichuan Provincial Administration of Traditional Chinese Medicine Medical Research Special 2018/ ; },
mesh = {*DNA, Catalytic/chemistry/metabolism/genetics ; *Rotavirus/isolation & purification/genetics ; Entropy ; *CRISPR-Cas Systems ; Limit of Detection ; Metal Nanoparticles/chemistry ; *Biosensing Techniques/methods ; Gold/chemistry ; DNA Probes/chemistry/genetics ; Nucleic Acid Amplification Techniques/methods ; Humans ; },
abstract = {DNA walker-based strategies are confronted with significant challenges in harmonizing design complexity, sequence dependence, and amplification efficiency. This study describes the innovative design of a double-stranded DNA probe, named the "LW probe," which integrates a locked DNAzyme segment, enabling the coupling of the entropy-driven amplification (EDA) process with a DNAzyme-powered DNA walker. In the absence of the target, the "LW probe" remains in an inactive ("OFF") state. Upon encountering target rotavirus sequences, the LW probe receives the trans-cleavage activity of Cas13a/crRNA and undergoes a conformational change, transforming into an activated structure. This structural transition initiates the EDA process continuously, leading to the release of the DNAzyme segment. Subsequently, the released DNAzyme segment acts on the surface of gold nanoparticles (AuNPs), cleaving the "Substrate probe" and consequently liberating fluorescence signals. Distinct from traditional DNA walkers that rely exclusively on the EDA for product amplification, the proposed approach synergistically combines the high-precision target recognition capacity of the EDA process with the potent signal amplification efficiency of DNA walkers. This integration results in remarkable enhancements in both specificity, demonstrated by the ability to discriminate single-base mismatched sequences, and sensitivity, with a detection limit as low as 2.7 fM. By synergizing EDA with the DNAzyme-driven DNA walker, our method achieves high sensitivity, with a detection limit of 2.7 fM, outperforming or matching the performance of previous DNA walker-based systems. This system enables highly sensitive and specific detection of low-abundance rotavirus with robust stability, offering a promising platform for disease diagnosis and biomedical research.},
}

*DNA, Catalytic/chemistry/metabolism/genetics
*Rotavirus/isolation & purification/genetics
Entropy
*CRISPR-Cas Systems
Limit of Detection
Metal Nanoparticles/chemistry
*Biosensing Techniques/methods
Gold/chemistry
DNA Probes/chemistry/genetics
Nucleic Acid Amplification Techniques/methods
Humans

@article {pmid40938388,
year = {2025},
author = {Higa, L and Blank, M and Hampson, E and Matsuyama, J and Wilkes, K and Uehara, A and Bouwman, T and Lee, K and Wang, K and Muszynski, M and Du, ZY},
title = {Rapid assessment of CRISPR gRNAs with optimized protoplast transformation in Maize.},
journal = {Plant cell reports},
volume = {44},
number = {10},
pages = {212},
pmid = {40938388},
issn = {1432-203X},
support = {2121410//National Science Foundation/ ; },
mesh = {*Zea mays/genetics ; *Protoplasts/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Transformation, Genetic ; Plants, Genetically Modified ; Transfection ; },
abstract = {We developed an optimized CRISPR/Cas9 gene editing system using maize mesophyll protoplasts to enable rapid evaluation of guide RNA (gRNA) activity. Using the tropical inbred line Tzi8, we improved protoplast isolation and transfection protocols, achieving high yields of 17.88 × 10[6] viable protoplasts per gram fresh weight while extending post-transfection viability. Etiolated seedlings and vertical leaf cutting significantly enhanced protoplast recovery and viability. A transfection efficiency of ~ 50% was achieved using 10 µg of plasmid DNA; higher DNA inputs did not result in significant gains, resulting in a more resource-efficient approach. Protoplast viability was maintained for up to seven days post-transfection, allowing for downstream applications that require extended incubation. This optimized system was used to assess the editing efficiency of nine gRNAs targeting three key floral repressors (ZmCCT9, ZmCCT10, and ZmRap2.7) across four maize genotypes (Tzi8, CML277, B73, and B104). These floral repressor genes are involved in the photoperiod sensitivity of tropical maize, a major challenge in the effort to introduce tropical maize germplasm into temperate breeding programs. Editing efficiencies ranged from 0.4% to 23.7%, with some variation observed between gRNAs and genotypes. Although protoplast-based assays do not currently enable plant regeneration, this platform offers a rapid method for in vivo gRNA validation, reducing assay time from months to days. This work expands the gene editing toolkit for tropical maize, supporting efforts to overcome breeding barriers through gene editing.},
}

*Zea mays/genetics
*Protoplasts/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics
*Transformation, Genetic
Plants, Genetically Modified
Transfection

@article {pmid40934206,
year = {2025},
author = {Lima, DA and Costa-Silva, HM and Albergaria, KSS and Ribeiro, JM and Resende, DM and Santarossa, BA and Liarte, DB and Calderano, SG and Murta, SMF},
title = {Glycosomal ABC transporter 3 (GAT3) deletion enhances the oxidative stress responses and reduces the infectivity of Trypanosoma cruzi.},
journal = {PLoS neglected tropical diseases},
volume = {19},
number = {9},
pages = {e0013479},
pmid = {40934206},
issn = {1935-2735},
mesh = {*Trypanosoma cruzi/genetics/pathogenicity/drug effects/metabolism ; *Oxidative Stress ; *ATP-Binding Cassette Transporters/genetics/metabolism ; Animals ; *Protozoan Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Mice ; Chagas Disease/parasitology ; Nitroimidazoles/pharmacology ; *Microbodies/metabolism/genetics ; Trypanocidal Agents/pharmacology ; Gene Knockout Techniques ; Gene Deletion ; },
abstract = {Glycosomes, peroxisome-like organelles in Trypanosoma cruzi, contain enzymes involved in various metabolic processes, including glycolysis. Glycosomal ABC transporters (GATs) play a vital role in maintaining metabolic homeostasis by facilitating metabolite exchange between glycosomes and the cytoplasm. GAT3 is a member of the GAT family, which also includes GAT1 and GAT2. GAT3 transcript levels are downregulated in benznidazole-resistant T. cruzi populations; however, its specific functions remain unknown. Therefore, in this study, we generated GAT3 single-knockout and null mutant lines of the T. cruzi Dm28c strain using the CRISPR/Cas9 system to investigate GAT3 roles in parasite biology. RT-qPCR revealed increased GAT2 transcript levels in the GAT3 null mutant line, without any changes in GAT1 levels. Our findings suggest that GAT3 is not essential for T. cruzi survival, as null mutant parasites showed no growth difference compared to the Cas9-expressing controls. Moreover, the GAT3 single-knockout line exhibited increased resistance to benznidazole, whereas the null mutant line exhibited benznidazole susceptibility similar to the control. Furthermore, both GAT3 single-knockout and null mutant lines showed increased tolerance to hydrogen peroxide-induced oxidative stress. In vitro infection assay of L929 murine fibroblasts revealed that the GAT3 null parasites exhibited a significantly lower infection rate and fewer intracellular amastigotes than the controls. Overall, GAT3 is crucial for T. cruzi infectivity and the regulation of oxidative stress responses, playing key roles in the metabolic regulation and pathogenicity of this parasite.},
}

*Trypanosoma cruzi/genetics/pathogenicity/drug effects/metabolism
*Oxidative Stress
*ATP-Binding Cassette Transporters/genetics/metabolism
Animals
*Protozoan Proteins/genetics/metabolism
CRISPR-Cas Systems
Mice
Chagas Disease/parasitology
Nitroimidazoles/pharmacology
*Microbodies/metabolism/genetics
Trypanocidal Agents/pharmacology
Gene Knockout Techniques
Gene Deletion

@article {pmid40934013,
year = {2025},
author = {Zhang, Y and Liu, T and Zhang, P and Ni, B and Wang, X and Bai, L and Sun, W and Guan, Y and Xia, X and Cao, H and Gu, J},
title = {A rapid and accurate method for Helicobacter pylori detection via integrating LAMP assay with CRISPR/Cas12b detection by one-step in one-pot.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1611134},
pmid = {40934013},
issn = {2235-2988},
mesh = {*Helicobacter pylori/genetics/isolation & purification ; Humans ; *Molecular Diagnostic Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; *Helicobacter Infections/diagnosis/microbiology ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Limit of Detection ; },
abstract = {INTRODUCTION: Accurate and timely detection of Helicobacter pylori (HP) is crucial for the diagnosis and management of gastritis and other HP-associated gastrointestinal disorders. Conventional diagnostic methods, such as PCR and culture, require specialized equipment and expertise, limiting their applicability in resource-limited settings. There is a pressing need for a rapid, cost-effective, and user-friendly diagnostic platform for HP detection, particularly in point-of-care settings.

METHODS: We developed an integrated detection platform combining loop-mediated isothermal amplification (LAMP) with the CRISPR/Cas12b system in a single, one-step, one-pot reaction. The assay was optimized to function at a constant temperature of 58 °C and provides results within 45 minutes. The clinical performance of the system was evaluated using 22 clinical samples, and its diagnostic accuracy was compared with conventional PCR.

RESULTS: The LAMP-CRISPR/Cas12b assay demonstrated a limit of detection (LOD) of 14.77 copies per test, with no cross-reactivity observed against potential interfering nucleic acids, ensuring 100% specificity for HP. Clinical validation revealed a concordance rate of 90.91% (20/22) between the LAMP-CRISPR/Cas12b platform and conventional PCR, supporting the diagnostic reliability of the system.

DISCUSSION: The integrated LAMP-CRISPR/Cas12b platform represents a promising alternative for the rapid and sensitive detection of HP. It combines the simplicity and rapidity of LAMP with the specificity of CRISPR/Cas12b, offering a robust, cost-effective, and high\-sensitivity diagnostic tool without the need for complex instrumentation. The method shows great potential for use in point-of-care testing (POCT) and could significantly enhance clinical practice by facilitating timely diagnosis and treatment of HP-related diseases.},
}

*Helicobacter pylori/genetics/isolation & purification
Humans
*Molecular Diagnostic Techniques/methods
*Nucleic Acid Amplification Techniques/methods
*Helicobacter Infections/diagnosis/microbiology
*CRISPR-Cas Systems
Sensitivity and Specificity
Limit of Detection

@article {pmid40825449,
year = {2025},
author = {Chowdhury, R and Roure, A and Darras, S},
title = {Towards functional genetics in the European amphioxus: Efficient CRISPR/Cas9 editing reveals Ascl1/2.1 requirement for peripheral nervous system development.},
journal = {Developmental biology},
volume = {527},
number = {},
pages = {218-225},
doi = {10.1016/j.ydbio.2025.08.012},
pmid = {40825449},
issn = {1095-564X},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Basic Helix-Loop-Helix Transcription Factors/genetics/metabolism ; *Lancelets/genetics/embryology ; *Peripheral Nervous System/embryology/metabolism ; Gene Expression Regulation, Developmental ; },
abstract = {Amphioxus, or cephalochordates, have a key phylogenetic position among chordates and serve as pivotal invertebrate models for investigating the evolutionary origins of vertebrate traits. Although functional genetic tools have recently been developed, their application has been limited to the Floridian and Asian species, Branchiostoma floridae and Branchiostoma belcheri, respectively. In this study, we established a CRISPR/Cas9-based genome editing protocol to generate F0 mosaic mutants (crispants) in the European amphioxus B. lanceolatum. As a proof of concept, we targeted the Bl-Ascl1/2.1 gene, a putative regulator of epidermal sensory neuron (ESN) development in the peripheral nervous system coding for a bHLH transcription factor. Using a novel microinjection method of the sgRNA/Cas9 complex in fertilized eggs and two-cell stage embryos, we demonstrated the disruption of Bl-Ascl1/2.1 that resulted in a partial to complete loss of ESNs. Importantly, this phenotype could be rescued by Bl-Ascl1/2.1 mRNA microinjection. These findings demonstrate the efficiency of CRISPR/Cas9-mediated gene editing in B. lanceolatum and establish a foundation for future functional studies in this emerging EvoDevo model.},
}

Animals
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Basic Helix-Loop-Helix Transcription Factors/genetics/metabolism
*Lancelets/genetics/embryology
*Peripheral Nervous System/embryology/metabolism
Gene Expression Regulation, Developmental

@article {pmid40770575,
year = {2025},
author = {Southard, KM and Ardy, RC and Tang, A and O'Sullivan, DD and Metzner, E and Guruvayurappan, K and Norman, TM},
title = {Comprehensive transcription factor perturbations recapitulate fibroblast transcriptional states.},
journal = {Nature genetics},
volume = {57},
number = {9},
pages = {2323-2334},
pmid = {40770575},
issn = {1546-1718},
support = {GM132083//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; DRG-2462-22//Damon Runyon Cancer Research Foundation (Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation)/ ; DP2 GM140925/GM/NIGMS NIH HHS/United States ; AWD-GC-259296//Damon Runyon Cancer Research Foundation (Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation)/ ; GM140925//U.S. Department of Health & Human Services | NIH | NIH Office of the Director (OD)/ ; HG012103//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {*Fibroblasts/metabolism ; Humans ; *Transcription Factors/genetics/metabolism ; Kruppel-Like Factor 4 ; *Transcription, Genetic ; Animals ; Mice ; Gene Expression Regulation ; Kruppel-Like Transcription Factors/genetics ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; Chromatin/genetics ; },
abstract = {Cell atlas projects have revealed that common cell types often comprise distinct, recurrent transcriptional states, but the function and regulation of these states remain poorly understood. Here, we show that systematic activation of transcription factors can recreate such states in vitro, providing tractable models for mechanistic and functional studies. Using a scalable CRISPR activation (CRISPRa) Perturb-seq platform, we activated 1,836 transcription factors in two cell types. CRISPRa induced gene expression within physiological ranges, with chromatin features predicting responsiveness. Comparisons with atlas datasets showed that transcription factor perturbations recapitulated key fibroblast states and identified their regulators, including KLF2 and KLF4 for a universal state present in many tissues, and PLAGL1 for a disease-associated inflammatory state. Inducing the universal state suppressed the inflammatory state, suggesting therapeutic potential. These findings position CRISPRa as a nuanced tool for perturbing differentiated cells and establish a general strategy for studying clinically relevant transcriptional states ex vivo.},
}

*Fibroblasts/metabolism
Humans
*Transcription Factors/genetics/metabolism
Kruppel-Like Factor 4
*Transcription, Genetic
Animals
Mice
Gene Expression Regulation
Kruppel-Like Transcription Factors/genetics
CRISPR-Cas Systems
Cell Differentiation/genetics
Chromatin/genetics

@article {pmid40748247,
year = {2025},
author = {Zhang, Y and Yang, X and Wang, Y and Chen, F and Liu, Y and Jiang, H and Wang, Y and Hu, Y and Li, S},
title = {Establishment of a CRISPR/Cas12b-Based Multiple Cross Displacement Amplification Assay for the Rapid, Sensitive, and Specific Detection of Brucella ovis.},
journal = {ACS infectious diseases},
volume = {11},
number = {9},
pages = {2446-2456},
doi = {10.1021/acsinfecdis.5c00287},
pmid = {40748247},
issn = {2373-8227},
mesh = {*Brucella ovis/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Brucellosis/diagnosis/veterinary/microbiology ; Animals ; Sheep ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *Sheep Diseases/diagnosis/microbiology ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Brucella ovis (B. ovis), a major pathogenic species within the Brucella genus, causes ovine epididymitis. Although the isolation and identification of B. ovis remain the gold standard for diagnosis, these methods are unsuitable for early detection. The traditional polymerase chain reaction (PCR) offers faster detection but requires specialized equipment such as PCR thermal cyclers and gel electrophoresis imagers, limiting its use in basic laboratories. Thus, developing rapid, sensitive, and specific diagnostic strategies is vital for preventing and controlling the spread of ovine brucellosis. In this study, we developed a diagnostic assay combining clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12b with multiple cross displacement amplification (MCDA)─termed CRISPR/Cas12b-MCDA─for rapid, sensitive, and specific identification of B. ovis. In the CRISPR/Cas12b-MCDA system, MCDA amplicons containing protospacer adjacent motif (PAM) sites are recognized by the Cas12b/gRNA complex, which binds the target region and triggers trans-cleavage of a single-stranded DNA (ssDNA) reporter. The CRISPR/Cas12b-MCDA assay demonstrated a detection limit of 10 fg/μL for synthetic genomic DNA and exhibited 100% specificity for B. ovis, with no cross-reactivity against other Brucella or non-Brucella species. The preamplification for template extraction takes 20 min, then 5 min for uracil DNA glycosylase (UDG) digestion, and 45 min for MCDA amplification. The total detection time was 75 min using real-time fluorescence analysis and 90 min with a lateral flow biosensor (LFB). Additionally, the results were validated using UV visualization to confirm the CRISPR/Cas12b-MCDA results. Notably, both LFB and UV analyses are instrument-free, enhancing their accessibility. In conclusion, the CRISPR/Cas12b-MCDA assay is a simple, rapid, sensitive, specific, and reliable method for detecting B. ovis.},
}

*Brucella ovis/genetics/isolation & purification
*CRISPR-Cas Systems
*Brucellosis/diagnosis/veterinary/microbiology
Animals
Sheep
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
*Sheep Diseases/diagnosis/microbiology
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40437754,
year = {2025},
author = {Xu, Y and Li, J and Wang, Z and Lu, R and Liu, Y and Wang, M and Li, H and Zhao, R and Feng, W},
title = {Ablation of dysmorphic neurons is a safe and effective treatment for focal cortical dysplasia II.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {9},
pages = {4414-4430},
pmid = {40437754},
issn = {1525-0024},
mesh = {*Epilepsy/genetics/pathology/therapy ; *Malformations of Cortical Development, Group I/genetics/pathology/therapy ; Proto-Oncogene Proteins c-akt/genetics ; Disease Models, Animal ; Animals ; Mice ; *Diphtheria Toxin/therapeutic use ; *Ablation Techniques ; *Heparin-binding EGF-like Growth Factor/genetics ; Humans ; HEK293 Cells ; TOR Serine-Threonine Kinases/genetics ; *Neurons/pathology ; CRISPR-Cas Systems ; *Gene Editing/methods ; },
abstract = {Focal cortical dysplasia type II (FCDII) is a leading cause of refractory epilepsy in children, yet treatment options remain limited. The most frequent genetic cause of FCDII is mosaic and somatic variants in genes of the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT)-mammalian target of rapamycin (mTOR) pathway, leading to hyperactivation of mTOR signaling. The presence of dysmorphic neurons (DNs) resulting from hyperactive mTOR signaling is critical for the development of epilepsy in FCDII. One critical therapeutic challenge and opportunity for FCDII is to selectively eliminate DNs. Here, we developed two strategies to specifically ablate DNs in FCDII mouse models, and the results demonstrate that DN ablation is sufficient to both prevent and eliminate epilepsy in mice. Moreover, the associated neurobehavioral abnormalities were also reversed following treatment. Therefore, our study provides proof-of-concept evidence that DN ablation is a highly promising approach for curing FCDII in the future.},
}

*Epilepsy/genetics/pathology/therapy
*Malformations of Cortical Development, Group I/genetics/pathology/therapy
Proto-Oncogene Proteins c-akt/genetics
Disease Models, Animal
Animals
Mice
*Diphtheria Toxin/therapeutic use
*Ablation Techniques
*Heparin-binding EGF-like Growth Factor/genetics
Humans
HEK293 Cells
TOR Serine-Threonine Kinases/genetics
*Neurons/pathology
CRISPR-Cas Systems
*Gene Editing/methods

@article {pmid40943207,
year = {2025},
author = {Gilyazova, I and Korytina, G and Kochetova, O and Savelieva, O and Mikhaylova, E and Vershinina, Z and Chumakova, A and Markelov, V and Abdeeva, G and Karunas, A and Khusnutdinova, E and Gusev, O},
title = {Advances in Genomics and Postgenomics in Poultry Science: Current Achievements and Future Directions.},
journal = {International journal of molecular sciences},
volume = {26},
number = {17},
pages = {},
pmid = {40943207},
issn = {1422-0067},
support = {№ 075-15-2025-484//Government of Russian Federation/ ; },
abstract = {The poultry industry, a globally fast growing agricultural sector, provides affordable animal protein due to high efficiency. Gallus gallus domesticus are the most common domestic birds. Hybrid chicken breeds (crosses) are widely used to achieve high productivity. Maintaining industry competitiveness requires constant genetic selection of parent stock to improve performance traits. Genetic studies, which are essential in modern breeding programs, help identify genome variants linked to economically important traits and preserve population health. Next-generation sequencing (NGS) has identified millions of single nucleotide polymorphisms (SNPs) and insertions/deletions (INDELs), enabling detection of genome-wide regions associated with selection traits. Recent studies have pinpointed such regions using broiler lines, laying hen lines, or pooled genomic data. This review discusses advances in chicken genomic and transcriptomic research focused on traits enhancing meat breed performance and reproductive abilities. Special attention is given to transcriptome studies revealing regulatory mechanisms and key signaling pathways involved in artificial molting, as well as metagenome studies investigating resistance to infectious diseases and climate adaptation. Finally, a dedicated section highlights CRISPR/Cas genomic editing techniques for targeted genome modification in chicken genomics.},
}

@article {pmid40943047,
year = {2025},
author = {Huang, C and Cheng, L},
title = {Unlocking Casein Bioactivity: Lactic Acid Bacteria and Molecular Strategies for Peptide Release.},
journal = {International journal of molecular sciences},
volume = {26},
number = {17},
pages = {},
pmid = {40943047},
issn = {1422-0067},
abstract = {Bioactive peptides encrypted in bovine β-casein display diverse physiological functions, including antihypertensive, antioxidative, antimicrobial, and immunomodulatory activities. These peptides are normally released during gastrointestinal digestion or microbial fermentation, especially by proteolytic systems of lactic acid bacteria (LAB). However, peptide yields vary widely among LAB strains, reflecting strain-specific protease repertoires. To overcome these limitations, the scientific goal of this study is to provide a comprehensive synthesis of how synthetic biology, molecular biotechnology, and systems-level approaches can be leveraged to enhance the targeted discovery and production of β-casein-derived bioactive peptides. Genome engineering tools such as clustered regularly interspaced short palindromic repeats associated system (CRISPR/Cas) systems have been applied to modulate gene expression and metabolic flux in LAB, while inducible expression platforms allow on-demand peptide production. Additionally, cell-free systems based on LAB lysates further provide rapid prototyping for high-throughput screening. Finally, multi-omics approaches, including genomics, transcriptomics, proteomics, and metabolomics, further help pinpoint regulatory bottlenecks and facilitate rational strain optimization. This review provides a comprehensive overview of bioactive peptides derived from bovine β-casein and highlights recent progress in LAB-based strategies-both natural and engineered-for their efficient release. These advances pave the way for developing next-generation functional fermented foods enriched with targeted bioactivities.},
}

@article {pmid40825952,
year = {2025},
author = {Vats, P and Kumar, R and Kumar, R and Kaushik, JK and Mohanty, AK and Kumar, S},
title = {Deciphering the role of MFGE8 in lactation using CRISPR-CAS9 based gene editing in Buffalo mammary epithelial cells.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {30194},
pmid = {40825952},
issn = {2045-2322},
support = {EMR/2017/000152//DST-SERB/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Female ; *Lactation/genetics ; *Epithelial Cells/metabolism ; *Buffaloes/genetics ; *Mammary Glands, Animal/cytology/metabolism ; *Milk Proteins/genetics/metabolism ; Proteomics ; Gene Knockout Techniques ; *Glycoproteins/genetics/metabolism ; Proteome ; },
abstract = {Milk fat globule EGF factor 8 (MFGE8) is a glycoprotein which plays a crucial role in mammary gland remodeling. Our group previously identified MFGE8 as a marker associated with high milk yielding cows. Here, we generated MFGE8 knock-out buffalo mammary epithelial cells (BuMEC) via CRISPR-cas9 technology to decipher its role in lactation. gRNA3 reduced MFGE8 expression with good efficiency which was confirmed at transcriptomic and proteomic level and the stable knock-out cells obtained were named mfge8-/-/gRNA3. The amplicon sequencing of the edited region using next generation sequencing (NGS) showed that 54% of total reads showed indels, 3-4 bp upstream to PAM site in 2nd exon. A total 4282 proteins were identified when proteome level changes were examined and 178 were found to be differentially expressed above and below a threshold of ≥ 1.5 and ≤ 0.6. Major DEPs were found to be associated with regulation of hydrolase activity, endopeptidase activity and cytoskeletal organization and some DEPs including FABP3, FABP4, FABP5, KNG1, MT2A, CD82, SLC7A1 and SERPINH1 belonged to genes associated with milk synthesis. To the best of our knowledge, this is the first study which provides a comprehensive proteome profile of MFGE8 knockout BuMEC and explores downstream effects of disruption of MFGE8 gene.},
}

Animals
*CRISPR-Cas Systems
*Gene Editing/methods
Female
*Lactation/genetics
*Epithelial Cells/metabolism
*Buffaloes/genetics
*Mammary Glands, Animal/cytology/metabolism
*Milk Proteins/genetics/metabolism
Proteomics
Gene Knockout Techniques
*Glycoproteins/genetics/metabolism
Proteome

@article {pmid40825942,
year = {2025},
author = {Countryman, AD and Doherty, CA and Herrera-Perez, RM and Kasza, KE},
title = {Endogenous OptoRhoGEFs reveal biophysical principles of epithelial tissue furrowing.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7665},
pmid = {40825942},
issn = {2041-1723},
support = {F31 HD118793/HD/NICHD NIH HHS/United States ; F31HD118793//U.S. Department of Health & Human Services | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; P40 OD018537/OD/NIH HHS/United States ; R35GM138380//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R35 GM138380/GM/NIGMS NIH HHS/United States ; Sloan Fellowship//Alfred P. Sloan Foundation/ ; Packard Fellowship//David and Lucile Packard Foundation (David & Lucile Packard Foundation)/ ; CAREER Award//National Science Foundation (NSF)/ ; },
mesh = {Animals ; *Drosophila Proteins/metabolism/genetics ; Epithelium/embryology/metabolism ; *Drosophila melanogaster/embryology/genetics/metabolism ; Optogenetics/methods ; Morphogenesis ; Actomyosin/metabolism ; Animals, Genetically Modified ; *Rho Guanine Nucleotide Exchange Factors/metabolism/genetics ; Gastrulation ; Embryo, Nonmammalian/metabolism ; CRISPR-Cas Systems ; Signal Transduction ; Gene Expression Regulation, Developmental ; },
abstract = {During development, epithelia function as malleable sheets that undergo extensive remodeling to shape developing embryos. Optogenetic control of Rho signaling provides an avenue to investigate mechanisms of epithelial morphogenesis, but transgenic optogenetic tools can be limited by variability in expression levels and deleterious effects of transgenic overexpression on development. Here, we use CRISPR/Cas9 to tag Drosophila RhoGEF2 and Cysts/Dp114RhoGEF with components of the iLID/SspB optogenetic heterodimer, permitting light-dependent control over endogenous protein activities. Using quantitative optogenetic perturbations, we uncover a dose-dependence of tissue furrow depth and bending behavior on RhoGEF recruitment, revealing mechanisms by which developing embryos can shape tissues into particular morphologies. We show that at the onset of gastrulation, furrows formed by cell lateral contraction are oriented and size-constrained by basal actomyosin. Our findings demonstrate the use of quantitative, 3D-patterned perturbations of cell contractility to precisely shape tissue structures and interrogate developmental mechanics.},
}

Animals
*Drosophila Proteins/metabolism/genetics
Epithelium/embryology/metabolism
*Drosophila melanogaster/embryology/genetics/metabolism
Optogenetics/methods
Morphogenesis
Actomyosin/metabolism
Animals, Genetically Modified
*Rho Guanine Nucleotide Exchange Factors/metabolism/genetics
Gastrulation
Embryo, Nonmammalian/metabolism
CRISPR-Cas Systems
Signal Transduction
Gene Expression Regulation, Developmental

@article {pmid40825812,
year = {2025},
author = {Netsawang, C and Tongbaen, M and Jearawiriyapaisarn, N and Leecharoenkiat, K},
title = {Precise correction of G6PD Viangchan mutation in iPSCs by prime editing strategy.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {30192},
pmid = {40825812},
issn = {2045-2322},
support = {B05F640126//The NSRF via the Program Management Unit for Human Resources & Institutional Development, Research and Innovation/ ; 196807//Thailand Science research and Innovation Fund Chulalongkorn University/ ; },
mesh = {Humans ; *Glucosephosphate Dehydrogenase/genetics ; *Induced Pluripotent Stem Cells/metabolism ; *Gene Editing/methods ; HEK293 Cells ; *Glucosephosphate Dehydrogenase Deficiency/genetics/therapy ; *Mutation ; CRISPR-Cas Systems ; },
abstract = {Individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency hold a significant risk of severe hemolytic crises under oxidative stress. Currently, the definitive and curative treatment for the disorder has not been developed. Among over 200 G6PD variants, G6PD Viangchan (c.871 G > A) is the most prevalent and has been extensively studied in Southeast Asia. This study assessed the effectiveness of prime editing for correcting the G6PD Viangchan mutation in an established mutant HEK293T cell line and G6PD-deficient induced pluripotent stem cells (iPSCs). Using optimized modalities, prime editing achieved a high correction efficiency of over 25% in the HEK293T cells. In iPSCs, this gene editing tool yielded satisfactory correction outcomes, with approximately 5% corrected alleles. Our findings indicate that prime editing provides high precision, producing minimal by-products below baseline and showing undetectable off-target effects. Overall, prime editing has the potential to correct the G6PD Viangchan mutation, providing a valuable approach for future therapeutic strategies and the generation of isogenic cell lines to promote extensive studies in drug discovery and the pathogenesis of the G6PD variant.},
}

Humans
*Glucosephosphate Dehydrogenase/genetics
*Induced Pluripotent Stem Cells/metabolism
*Gene Editing/methods
HEK293 Cells
*Glucosephosphate Dehydrogenase Deficiency/genetics/therapy
*Mutation
CRISPR-Cas Systems

@article {pmid40825783,
year = {2025},
author = {Calvo-Villamañán, A and Sastre-Dominguez, J and Barrera-Martín, Á and Costas, C and San Millan, Á},
title = {Dissecting pOXA-48 fitness effects in clinical Enterobacterales using plasmid-wide CRISPRi screens.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7700},
pmid = {40825783},
issn = {2041-1723},
support = {ALTF 322-2022//European Molecular Biology Organization (EMBO)/ ; },
mesh = {*Plasmids/genetics ; *beta-Lactamases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; *Enterobacteriaceae/genetics/drug effects ; Humans ; Enterobacteriaceae Infections/microbiology/drug therapy ; *Genetic Fitness ; CRISPR-Cas Systems/genetics ; Drug Resistance, Multiple, Bacterial/genetics ; Anti-Bacterial Agents/pharmacology ; Carbapenems/pharmacology ; },
abstract = {Conjugative plasmids are the main vehicle for the spread of antimicrobial resistance (AMR) genes in clinical bacteria. AMR plasmids allow bacteria to survive antibiotic treatments, but they also produce physiological alterations in their hosts that commonly translate into fitness costs. Despite the key role of plasmid-associated fitness effects in AMR evolution, their origin and molecular bases remain poorly understood. In this study, we introduce plasmid-wide CRISPR interference (CRISPRi) screens as a tool to dissect plasmid-associated fitness effects. We design and perform CRISPRi screens targeting the globally distributed carbapenem resistance plasmid pOXA-48 in 13 different multidrug resistant clinical Enterobacterales. Our results reveal that pOXA-48 gene-level effects are conserved across clinical strains, and expose the key role of the carbapenemase-encoding gene, blaOXA-48, as the main culprit for pOXA-48 fitness costs. Moreover, our results highlight the relevance of postsegregational killing systems in pOXA-48 vertical transmission, and uncover new genes implicated in pOXA-48 stability (pri, korC, DNDJGHEP_13 and 14 and H-NS). This study sheds new light on the biology and evolution of carbapenem resistant Enterobacterales and endorses CRISPRi screens as a powerful method for studying plasmid-mediated AMR.},
}

*Plasmids/genetics
*beta-Lactamases/genetics/metabolism
*Bacterial Proteins/genetics/metabolism
*Enterobacteriaceae/genetics/drug effects
Humans
Enterobacteriaceae Infections/microbiology/drug therapy
*Genetic Fitness
CRISPR-Cas Systems/genetics
Drug Resistance, Multiple, Bacterial/genetics
Anti-Bacterial Agents/pharmacology
Carbapenems/pharmacology

@article {pmid40824957,
year = {2025},
author = {Naes, SM and Ab-Rahim, S and Mazlan, M and Syafruddin, SE and Mohtar, MA and Abuhamad, AY and Abdul Rahman, A},
title = {CRISPR/Cas9 mediated ENT2 gene knockout altered purine catabolic pathway and induced apoptosis in colorectal cell lines.},
journal = {PloS one},
volume = {20},
number = {8},
pages = {e0329501},
pmid = {40824957},
issn = {1932-6203},
mesh = {Humans ; *Apoptosis/genetics ; *CRISPR-Cas Systems ; *Purines/metabolism ; *Colorectal Neoplasms/genetics/metabolism/pathology ; Gene Knockout Techniques ; Cell Line, Tumor ; *Equilibrative-Nucleoside Transporter 2/genetics/metabolism ; Reactive Oxygen Species/metabolism ; Xanthine Oxidase/metabolism ; HT29 Cells ; Hypoxanthine/metabolism ; Gene Expression Regulation, Neoplastic ; },
abstract = {Although purine metabolism is one of the most impacted pathways in colorectal cancer (CRC), little is known about the role of equilibrative nucleoside transporter 2 (ENT2) in CRC development and its association with the altered purine metabolism pathway. This study aimed to determine the role of ENT2 in altered purine metabolism in the early and late stages of CRC using CRISPR/Cas9 gene editing tools and a variety of functional experiments. The expression of ENT2 was significantly higher (P < 0.001) in all CRC cell lines as compared to the normal colon cells. The two CRC cell lines with the highest ENT2 expression, the early stage HT29 cells and the late stage DLD1 cells, were knocked out (KO) using the CRISPR/Cas9 tool. The hypoxanthine (HPX) level and the xanthine oxidase (XO) activity were significantly higher in both HT29/KO and DLD1/KO single cell-derived clones (P < 0.01). The increase in HPX level and XO activity were associated with an elevation in the reactive oxygen species (ROS) level. These data suggest that the ENT2 KO elevated the ROS levels induced apoptosis and impaired the cell proliferation of the early stage of CRC cell line, i.e., HT29/KO clonal cells. In this context, targeting ENT2 gene might be a potential strategy in CRC treatment by increasing the production of ROS and hence, inducing the apoptosis pathway.},
}

Humans
*Apoptosis/genetics
*CRISPR-Cas Systems
*Purines/metabolism
*Colorectal Neoplasms/genetics/metabolism/pathology
Gene Knockout Techniques
Cell Line, Tumor
*Equilibrative-Nucleoside Transporter 2/genetics/metabolism
Reactive Oxygen Species/metabolism
Xanthine Oxidase/metabolism
HT29 Cells
Hypoxanthine/metabolism
Gene Expression Regulation, Neoplastic

@article {pmid40823809,
year = {2025},
author = {Migliori, V and Bruntraeger, MB and Gyulev, IS and Lichou, F and Burgold, T and Gitterman, DP and Iwama, S and Trinh, AL and Washer, SJ and Jones, CP and Trynka, G and Bassett, AR},
title = {ONE-STEP tagging: a versatile method for rapid site-specific integration by simultaneous reagent delivery.},
journal = {Nucleic acids research},
volume = {53},
number = {15},
pages = {},
pmid = {40823809},
issn = {1362-4962},
support = {220540/Z/20/A/WT_/Wellcome Trust/United Kingdom ; //Sanger Translation Committee/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Induced Pluripotent Stem Cells/metabolism ; T-Lymphocytes/metabolism ; Integrases/genetics/metabolism ; },
abstract = {We present a novel, versatile genome editing method termed ONE-STEP tagging, which combines CRISPR-Cas9-mediated targeting with Bxb1 integrase-based site-specific integration for efficient, precise, and scalable protein tagging. Applied in human-induced pluripotent stem cells (hiPSCs), cancer cells and primary T cells, this system enables rapid generation of endogenously tagged proteins. By enhancing the nuclear localization signal of the catalytically superior eeBxb1 integrase and co-delivering a DNA-PK inhibitor, we achieved up to ∼90% integration efficiency at the ACTR10 locus in hiPSCs. ONE-STEP tagging is robust across loci and cell types and supports large DNA cargo integration, with efficiencies reaching 16.6% for a 14.4 kb construct. The method also enables multiplexed tagging of multiple proteins within the same cell and simultaneous CRISPR-based editing at secondary loci, such as gene knockouts or homology-directed repair. Importantly, we demonstrate successful application in primary T cells by targeting the T cell receptor locus while simultaneously knocking out B2M, a key step towards generating immune-evasive, off-the-shelf chimeric antigen receptor T cells. Additionally, we introduce a dual-cassette version of the method compatible with universal donor plasmids, allowing use of entirely off-the-shelf reagents. Together, these advances establish ONE-STEP tagging as a powerful tool for both basic and therapeutic genome engineering.},
}

Humans
*CRISPR-Cas Systems
*Gene Editing/methods
Induced Pluripotent Stem Cells/metabolism
T-Lymphocytes/metabolism
Integrases/genetics/metabolism

@article {pmid40814254,
year = {2025},
author = {Nevot, G and Güell, M and Santos-Moreno, J},
title = {Critical Analysis of Preprints and Inquiry-Based Lessons Improve the Synthetic Biology Learning Experience.},
journal = {ACS synthetic biology},
volume = {14},
number = {8},
pages = {2878-2884},
pmid = {40814254},
issn = {2161-5063},
mesh = {*Synthetic Biology/education ; Humans ; *Problem-Based Learning/methods ; Students ; Universities ; CRISPR-Cas Systems/genetics ; },
abstract = {Synthetic biology is a transformative field crucial to address global challenges. It is highly interdisciplinary, integrating different subjects beyond biology. Therefore, traditional lecture-based teaching methods often fall short in effectively covering the diverse and rapidly evolving advancements in synthetic biology. We developed active learning workflows for complementing classic theoretical lectures in universities to improve the synthetic biology learning experience. We used preprints as an educational resource for the students to critically analyze differences comparing manuscripts and the final published work. In addition, we designed a practical laboratory session where students had to infer the logic behind CRISPRi-based gene circuits that they assembled, thus engaging with every step of the design-build-test-learn cycle. Following these activities, 90% of the students reported having improved critical analysis skills and 80% felt that they had learned a wide range of synthetic biology concepts. These approaches demonstrate the potential of innovative teaching for synthetic biology, which helps students with both technical and soft skills at the same time and has the potential to be adapted to other fields.},
}

*Synthetic Biology/education
Humans
*Problem-Based Learning/methods
Students
Universities
CRISPR-Cas Systems/genetics

@article {pmid40736249,
year = {2025},
author = {Zhao, Y and Guo, G and Sun, Y and Zhang, M and Yang, G and Liu, Z and Song, Y and Ghonaim, AH and Ma, N and Zhang, M and Jongkaewwattana, A and He, Q and Li, W},
title = {Membrane protein CRISPR screen identifies RPSA as an essential host factor for porcine epidemic diarrhea virus replication.},
journal = {Journal of virology},
volume = {99},
number = {8},
pages = {e0064925},
pmid = {40736249},
issn = {1098-5514},
mesh = {Animals ; *Porcine epidemic diarrhea virus/physiology ; *Virus Replication ; Swine ; *Coronavirus Infections/virology/veterinary/metabolism ; CRISPR-Cas Systems ; *Swine Diseases/virology/metabolism/genetics ; Host-Pathogen Interactions ; *Membrane Proteins/genetics/metabolism ; MAP Kinase Signaling System ; Gene Knockout Techniques ; Chlorocebus aethiops ; },
abstract = {UNLABELLED: Porcine epidemic diarrhea, caused by porcine epidemic diarrhea virus (PEDV), is one of the most devastating diseases in the global pig industry due to its high mortality rate in piglets. The host factors required for PEDV replication, including receptors, remain poorly understood. Here, we developed a porcine membrane-protein-scale CRISPR/Cas9 knockout (PigMpCKO) library and performed two rounds of PEDV infection. Ribosomal protein SA (RPSA), the known receptor of dengue virus, was found to be a potent host factor. Moreover, our research revealed that RPSA is involved in the replication stage of PEDV and not in the entry stage. Inhibitor and activator experiments demonstrated that knockout (KO) of RPSA downregulates the ERK1/2 signaling pathway to impair PEDV infection. Additionally, RNA sequencing data indicated that cellular lipid biosynthesis and lipid transport processes were significantly inhibited in the absence of RPSA during PEDV infection. Mechanistic studies revealed that the reduction in total cholesterol and triglyceride levels, resulting from RPSA KO, was partially mediated by the ERK1/2 pathway, leading to impaired lipid accumulation during PEDV replication. Interestingly, RPSA KO also significantly downregulated the expression of aminopeptidase N (APN) and inhibited infection by transmissible gastroenteritis virus (TGEV) and porcine deltacoronavirus (PDCoV), both of which belong to the swine enteric coronavirus group. In summary, our results establish RPSA as a novel host factor that is critical for coronavirus replication. This provides new insights into the mechanisms of virus-host interactions and paves the way for the development of broad-spectrum antiviral therapies.

IMPORTANCE: Swine enteric coronaviruses (SeCoVs) cause severe economic losses to the global swine industry and pose a potential threat to public health. Identification of receptors required for PEDV infection could develop novel targets for drug therapy and disease-resistant breeding. We conducted a CRISPR/Cas9 screen targeting membrane proteins in porcine kidney cells infected with PEDV to identify possible receptors and discovered numerous novel candidate host factors. Considering RPSA's known role as a receptor for multiple viruses, we focused on investigating its potential in coronavirus infection. Our results revealed that RPSA does not contribute to the entry stage but to the replication stage of coronavirus infection. We first reported the role that RPSA plays in the regulation of APN expression and lipid metabolism. RPSA is essential for PEDV and other SeCoVs replication, providing a novel insight into the search for the receptor of PEDV and identifying potential therapeutic targets for coronaviruses.},
}

Animals
*Porcine epidemic diarrhea virus/physiology
*Virus Replication
Swine
*Coronavirus Infections/virology/veterinary/metabolism
CRISPR-Cas Systems
*Swine Diseases/virology/metabolism/genetics
Host-Pathogen Interactions
*Membrane Proteins/genetics/metabolism
MAP Kinase Signaling System
Gene Knockout Techniques
Chlorocebus aethiops

@article {pmid40707359,
year = {2025},
author = {Campbell, RR and Green, M and Choi, EY and Wulff, AB and Siclair, AN and Khatri, S and Virata, G and Barrett, C and Key, S and Patel, S and Rowell, MB and Franco, D and Ganapathy-Kanniappan, S and Mathur, BN and Lobo, MK},
title = {Dopamine Receptor 1 Specific CRISPRa Mice Exhibit Disrupted Behaviors and Striatal Baseline Cellular Activity.},
journal = {eNeuro},
volume = {12},
number = {8},
pages = {},
pmid = {40707359},
issn = {2373-2822},
support = {R01 DA038613/DA/NIDA NIH HHS/United States ; R33 DA052101/DA/NIDA NIH HHS/United States ; },
mesh = {Animals ; *Receptors, Dopamine D1/genetics/metabolism ; Mice, Transgenic ; *Corpus Striatum/metabolism ; Mice ; Male ; Reward ; *Behavior, Animal/physiology ; CRISPR-Cas Systems ; Mice, Inbred C57BL ; Motor Activity/physiology ; },
abstract = {The two main cell types in the striatum, dopamine receptor 1 and adenosine receptor 2a spiny projection neurons (D1-SPNs and A2A-SPNs), have distinct roles in regulating motor- and reward-related behaviors. Cre-selective CRISPR-dCas9 systems allow for cell-type specific, epigenomic-based manipulation of gene expression with gene-specific single guide RNAs (sgRNAs) and have potential to elucidate molecular mechanisms underlying striatal subtype mediated behaviors. Conditional transgenic Rosa26:LSL-dCas9-p300 mice were recently generated to allow for robust expression of dCas9-p300 expression with Cre-driven cell-type specificity. This system utilizes p300, a histone acetyltransferase which regulates gene expression by unwinding chromatin and making that region of the genome more accessible for transcription. Rosa26-LSL-dCas9-p300 mice were paired with Drd1-Cre and Ador2a-Cre mice to generate Drd1-Cre:dCas9-p300 and Ador2a-Cre:dCas9-p300 mouse lines and underwent behavioral phenotyping when sgRNAs were not present. Both Drd1-Cre:dCas9-p300 and Ador2a-Cre:dCas9-p300 have cell-type-specific expression of spCas9 mRNA. Baseline behavioral assessments revealed that, under a sgRNA absent nontargeted state, Drd1-Cre:dCas9-p300 mice display repetitive spinning behavior, hyperlocomotion, and enhanced acquisition of reward learning in comparison with all genotypic littermates. In contrast, Ador2a-Cre:dCas9-p300 do not exhibit any changes in behavior in comparison with their littermates. Electrophysiological recordings of dorsal striatum D1-SPNs revealed that Drd1-Cre:dCas9-p300 mice have increased input resistance and increased spontaneous excitatory postsynaptic current amplitude, together suggesting greater excitatory drive of D1-SPNs. Overall, these data demonstrate the necessity to validate CRISPR-dCas9 lines for research investigations. Additionally, the Drd1-Cre:dCas9-p300 line has the potential to be used to study underlying mechanisms of stereotypy and reward learning.},
}

Animals
*Receptors, Dopamine D1/genetics/metabolism
Mice, Transgenic
*Corpus Striatum/metabolism
Mice
Male
Reward
*Behavior, Animal/physiology
CRISPR-Cas Systems
Mice, Inbred C57BL
Motor Activity/physiology

@article {pmid40941236,
year = {2025},
author = {Liu, Y and Xie, Y and Wang, Z and Gai, Z and Zhang, X and Chen, J and Lei, H and Xu, Z and Shen, X},
title = {A Simple, Rapid, and Contamination-Free Ultra-Sensitive Cronobacter sakazakii Visual Diagnostic Platform Based on RPA Combined with CRISPR/Cas12a.},
journal = {Foods (Basel, Switzerland)},
volume = {14},
number = {17},
pages = {},
doi = {10.3390/foods14173120},
pmid = {40941236},
issn = {2304-8158},
support = {2023YFF1105103//National Key Research and Development Program of China/ ; },
abstract = {CRISPR/Cas systems have made significant progress in the field of molecular diagnostics in recent years. To overcome the aerosol contamination problem brought on by amplicon transfer in the common two-step procedure, the "one-pot method" has become a major research hotspot in this field. However, these methods usually rely on specially designed devices or additional chemical modifications. In this study, a novel "one-pot" strategy was developed to detect the foodborne pathogen Cronobacter sakazakii (C. sakazakii). A specific sequence was screened out from the virulence gene ompA of C. sakazakii as the detection target. Combining with the recombinase polymerase amplification (RPA), a rapid detection platform for C. sakazakii based on the CRISPR/Cas12a system was established for the first time. The sensitivity of this method was determined from three different levels, which are 10[-4] ng/μL for genomic DNA (gDNA), 1.43 copies/μL for target DNA, and 6 CFU/mL for pure bacterial culture. Without any microbial enrichment, the detection limits for artificially contaminated cow and goat milk powder samples were 4.65 CFU/mL and 4.35 CFU/mL, respectively. To address the problem brought on by aerosol contamination in the common RPA-CRISPR/Cas12a two-step method, a novel pipette tip-in-tube (PTIT) method for simple and sensitive one-pot nucleic acid detection was further developed under the inspiration of the capillary principle. The RPA and CRISPR/Cas systems were isolated from each other by the force balance of the solution in a pipette tip before amplification. The detection limits of the PTIT method in pure bacterial culture and the spiked samples were exactly the same as that of the two-step method, but with no false positive cases caused by aerosol contamination at all. Compared with other existing one-pot methods, the PTIT method requires no additional or specially designed devices, or any chemical modifications on crRNA and nucleic acid probes. Therefore, the PTIT method developed in this study provides a novel strategy for realizing one-pot CRISPR/Cas detection easily and holds significant potential for the rapid point-on-care testing (POCT) application.},
}

@article {pmid40940734,
year = {2025},
author = {Cattin, E and Schena, E and Mattioli, E and Marcuzzo, S and Bonanno, S and Cavalcante, P and Corradi, F and Benati, D and Farinazzo, G and Cattaneo, M and De Sanctis, V and Bertorelli, R and Maggi, L and Giannotta, M and Pini, A and Vattemi, G and Cassandrini, D and Cavallo, M and Manferdini, C and Lisignoli, G and Fontana, B and Pace, I and Bruno, C and Roncarati, R and Fiorillo, C and Ferracin, M and Schirmer, EC and Recchia, A and Lattanzi, G},
title = {Profibrotic Molecules Are Reduced in CRISPR-Edited Emery-Dreifuss Muscular Dystrophy Fibroblasts.},
journal = {Cells},
volume = {14},
number = {17},
pages = {},
doi = {10.3390/cells14171321},
pmid = {40940734},
issn = {2073-4409},
support = {20223WFJJ3//Ministero dell'università e della ricerca/ ; CUP B33C22001640007//Associazione Italiana DIstrofia Muscolare di Emery-Dreifuss - AIDMED/ ; CUP B33C22001640007//Associazione Alessandra Proietti OdV/ ; ECOSISTER Project. cod. ECS_00000033-CUP B89I22000650001//EU-funded PNRR/ ; ECOSISTER project ECS_00000033 - CUP E93C22001100001//EU-funded PNRR/ ; TREAT-LMNA 2019-004426-24//AIFA/ ; T3-AN-03 CUP: B53C22002520006 REGINA//Ministero della Salute/ ; RRC//The Italian Ministry of Health/ ; },
abstract = {Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in EMD, LMNA, SYNE1, SYNE2, and other related genes. The disease is characterized by joint contractures, muscle weakening and wasting, and heart conduction defects associated with dilated cardiomyopathy. Previous studies demonstrated the activation of fibrogenic molecules such as TGFbeta 2 and CTGF in preclinical models of EDMD2 and increased secretion of TGFbeta 2 in patient serum. A wide screening of patient cells suggested fibrosis, metabolism, and myogenic signaling as the most affected pathways in various EDMD forms. In this study, we show that alpha-smooth muscle actin-positive myofibroblasts are overrepresented in patient fibroblast cultures carrying EMD, LMNA, or SYNE2 mutations, and profibrotic miRNA-21 is upregulated. Upon CRISPR/Cas correction of the mutated EMD or LMNA sequence in EDMD1 or EDMD2 fibroblasts, respectively, we observe a reduced expression of fibrogenic molecules. However, in patient myoblasts, neither fibrogenic proteins nor miRNA-21 were upregulated; instead, miRNA-21-5p was downregulated along with muscle-specific miRNA-133b and miRNA-206, which have a crucial role in muscle cell homeostasis. These observations suggest that the conversion of laminopathic fibroblasts into a profibrotic phenotype is a determinant of EDMD-associated muscle fibrosis, while miRNA-206-dependent defects of laminopathic myoblasts, including altered regulation of VEGF levels, contribute to muscle cell deterioration. Notably, our study provides a proof-of-principle for the application of gene correction to EDMD1 and EDMD2 and presents EDMD1 isogenic cells that exhibit an almost complete rescue of a disease-specific miRNA signature. These cells can be used as experimental models for studying muscular laminopathies.},
}

@article {pmid40938972,
year = {2025},
author = {Abdirassilova, AA and Yessimseit, DT and Kassenova, AK and Abdeliyev, BZ and Zhumadilova, ZB and Tokmurziyeva, GZ and Kovaleva, GG and Abdel, ZZ and Meka-Mechenko, TV and Umarova, SK and Begimbayeva, EZ and Agzam, SD and Motin, VL and Reva, ON and Rysbekova, AK},
title = {Whole genome sequencing of Yersinia pestis isolates from Central Asian natural plague foci revealed the role of adaptation to different hosts and environmental conditions in shaping specific genotypes.},
journal = {PLoS neglected tropical diseases},
volume = {19},
number = {9},
pages = {e0013533},
doi = {10.1371/journal.pntd.0013533},
pmid = {40938972},
issn = {1935-2735},
abstract = {The genetic diversity and biovar classification of Yersinia isolates from Central Asia were investigated using whole-genome sequencing. In total, 98 isolates from natural plague foci were sequenced using the MiSeq platform. Computational pipelines were developed for accurate assembly of Y. pestis replicons, including small cryptic plasmids, and for identifying genetic polymorphisms. A panel of 99 diagnostic polymorphisms was established, enabling the distinction of dominant Medievalis isolates derived from desert and upland regions. Evidence of convergent evolution was observed in polymorphic allele distributions across genetically distinct Y. pestis biovars, Y. pseudotuberculosis, and other Y. pestis strains, likely driven by adaptation to similar environmental conditions. Genetic polymorphisms in the napA, araC, ssuA, and rhaS genes, along with transposon and CRISPR-Cas insertion patterns, were confirmed as suitable tools for identifying Y. pestis biovars, although their homoplasy suggests limited utility for phylogenetic inference. Notably, a novel cryptic plasmid, pCKF, previously associated with the strain of the population 2.MED0 from the Central-Caucasus high-altitude autonomous plague focus, was detected in a genetically distinct isolate of 2.MED1 population from the Ural-Embi region, indicating potential plasmid transfer across the 2.MED lineage. These findings emphasize the need for ongoing genomic surveillance to monitor the spread of virulence-associated genetic elements and to improve our understanding of Y. pestis evolution and ecology.},
}

@article {pmid40938680,
year = {2025},
author = {Yang, K and Wang, T and Zhu, Q and Shen, C},
title = {CRISPR/Cas-based detection strategies for tumor biomarker detection.},
journal = {Analytical methods : advancing methods and applications},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5ay01150g},
pmid = {40938680},
issn = {1759-9679},
abstract = {Tumor biomarkers, such as nucleic acids, proteins, extracellular vesicles (EVs) and circulating tumor cells (CTCs), can provide valuable information for tumor risk assessment, diagnosis, prognosis and recurrence monitoring. Currently, polymerase chain reaction (PCR)-based approaches and enzyme linked immunosorbent assay (ELISA) are typically used for detecting tumor biomarkers in clinics. However, PCR-based methods have limits in sensitivity and detection channels. Besides, ELISA suffers from cumbersome operation and limited sensitivity. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems are adopted for developing novel detection strategies due to the characteristics of high sensitivity, high specificity, simple operability and flexible programmability. Besides, CRISPR/Cas systems are amenable to combination with isothermal amplification techniques, primarily attributed to their compatibility and stability. Moreover, the combination of different CRISPR/Cas systems enables multiplex target detection. Therefore, CRISPR/Cas-based detection strategies have emerged as highly promising approaches for the sensitive, specific and multiplex detection of tumor biomarkers. In this review, we at first introduced the classification and working mechanisms of CRISPR/Cas systems. And then, we comprehensively summarized recently developed CRISPR/Cas-based detection strategies for tumor biomarkers. Besides, we reviewed detection strategies based on CRISPR/Cas systems for multiplex tumor biomarker detection. Furthermore, the challenges and prospects of existing CRISPR/Cas-based detection strategies were thoroughly discussed.},
}

@article {pmid40935887,
year = {2025},
author = {Alves, CRR and Das, S and Krishnan, V and Ha, LL and Fox, LR and Stutzman, HE and Shamber, CE and Kalailingam, P and McCarthy, S and Lino Cardenas, CL and Fong, CE and Imai, T and Mitra, S and Yun, S and Wood, RK and Benning, FMC and Roh, K and Lawton, J and Kim, N and Silverstein, RA and Ferreira da Silva, J and de la Cruz, D and Richa, R and Xie, J and Gray-Edwards, HL and Malhotra, R and Chung, DY and Chao, LH and Tsai, SQ and Maguire, CA and Lindsay, ME and Kleinstiver, BP and Musolino, PL},
title = {Treatment of a severe vascular disease using a bespoke CRISPR-Cas9 base editor in mice.},
journal = {Nature biomedical engineering},
volume = {},
number = {},
pages = {},
pmid = {40935887},
issn = {2157-846X},
support = {P01HL142494//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01NS125353//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; DP2CA281401//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; K01NS134784//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01NS125353//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01NS125353//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
abstract = {Pathogenic missense mutations in the alpha actin isotype 2 (ACTA2) gene cause multisystemic smooth muscle dysfunction syndrome (MSMDS), a genetic vasculopathy that is associated with stroke, aortic dissection and death in childhood. Here we perform mutation-specific protein engineering to develop a bespoke CRISPR-Cas9 enzyme with enhanced on-target activity against the most common MSMDS-causative mutation ACTA2 R179H. To directly correct the R179H mutation, we screened dozens of configurations of base editors to develop a highly precise corrective A-to-G edit with minimal deleterious bystander editing that is otherwise prevalent when using wild-type SpCas9 base editors. We create a murine model of MSMDS that shows phenotypes consistent with human patients, including vasculopathy and premature death, to explore the in vivo therapeutic potential of this strategy. Delivery of the customized base editor via an engineered smooth muscle-tropic adeno-associated virus (AAV-PR) vector substantially prolongs survival and rescues systemic phenotypes across the lifespan of MSMDS mice, including in the vasculature, aorta and brain. Our results highlight how bespoke mutant-specific CRISPR-Cas9 enzymes can improve mutation correction with base editors.},
}

@article {pmid40934884,
year = {2025},
author = {Gast, K and Barrangou, R},
title = {All systems go: CRISPR crosstalk for enhanced immunity.},
journal = {Cell host & microbe},
volume = {33},
number = {9},
pages = {1470-1472},
doi = {10.1016/j.chom.2025.08.002},
pmid = {40934884},
issn = {1934-6069},
abstract = {In this issue of Cell Host & Microbe, companion manuscripts from Margolis & Meeske[1] and Smith & Fineran[2] demonstrate that CRISPR-Cas systems have an unprecedented level of cooperative crosstalk between different subtypes, which enables primed spacer acquisition. These studies illustrate how CRISPR-Cas systems cooperate to enhance adaptive immunity in bacteria.},
}

@article {pmid40641152,
year = {2025},
author = {Robertson, NR and Lenert-Mondou, C and Leonard, AC and Tafrishi, A and Carrera, S and Lee, S and Aguilar, Y and Sanchez Zamora, L and Nguyen, T and Beltrán, J and Li, M and Cutler, SR and Whitehead, TA and Wheeldon, I},
title = {PYR1 Biosensor-Driven Genome-Wide CRISPR Screens for Improved Monoterpene Production in Kluyveromyces marxianus.},
journal = {ACS synthetic biology},
volume = {14},
number = {8},
pages = {2972-2978},
pmid = {40641152},
issn = {2161-5063},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Kluyveromyces/genetics/metabolism ; *Monoterpenes/metabolism ; Metabolic Engineering/methods ; *Fungal Proteins/genetics/metabolism ; Acyclic Monoterpenes/metabolism ; Genome, Fungal ; },
abstract = {Monoterpenes are valued for their roles as flavors, fragrances, insecticides, and energy-dense fuels. Microorganisms provide sustainable biosynthesis routes for these important molecules, but production levels remain limited. Here, we introduce a biosensor-driven microbial engineering strategy to enhance monoterpene production, specifically targeting geraniol. Using mutagenized libraries of the PYR1 receptor─a versatile biosensor from plant ABA signaling pathways with a malleable binding pocket─we screened 24 monoterpenes and identified PYR1 variants responsive to eight, including geraniol. A low background, highly selective geraniol-sensitive PYR1 variant was expressed in the thermotolerant yeast Kluyveromyces marxianus as a growth-based biosensor circuit, allowing for rapid strain engineering. By coupling the geraniol-sensitive PYR1 sensor with a genome-wide CRISPR-Cas9 mutagenesis approach, we identified six gene knockouts that enhance geraniol production, achieving up to a 2-fold increase in titer. This study demonstrates the power of the PYR1 biosensor platform to enable rapid strain engineering and the identification of mutants that improve the titer of a desired metabolite.},
}

*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
*Kluyveromyces/genetics/metabolism
*Monoterpenes/metabolism
Metabolic Engineering/methods
*Fungal Proteins/genetics/metabolism
Acyclic Monoterpenes/metabolism
Genome, Fungal

@article {pmid40580745,
year = {2025},
author = {Asadi-Sarabi, P and Rismani, E and Shabanpouremam, M and Hendi, Z and Nikoubin, B and Rahimi, S and Taleb, M and Khosravi, A and Zarrabi, A and Hassan, M and Vosough, M},
title = {Hypoimmunogenic pluripotent stem cells: A game-changer in cell-based regenerative medicine.},
journal = {International immunopharmacology},
volume = {162},
number = {},
pages = {115134},
doi = {10.1016/j.intimp.2025.115134},
pmid = {40580745},
issn = {1878-1705},
mesh = {Humans ; *Regenerative Medicine/methods ; *Pluripotent Stem Cells/immunology/transplantation ; Animals ; Gene Editing ; *Cell- and Tissue-Based Therapy/methods ; CRISPR-Cas Systems ; },
abstract = {Hypoimmunogenic pluripotent stem cells (hPSCs) represent a transformative innovation in regenerative medicine, offering solutions to the longstanding challenge of immune rejection in cell-based therapies. Through advanced gene-editing techniques, particularly CRISPR/Cas9, hPSCs are engineered to downregulate or eliminate the expression of major histocompatibility complex (MHC) molecules while upregulating immunomodulatory proteins such as HLA-G, PD-L1, and CD47. These modifications enhance immune evasion and create the foundation for universal donor cells. Compared to conventional cell therapies that rely on lifelong immunosuppression, hPSC-based strategies offer safer, more sustainable, and patient-friendly solutions by minimizing the risks of infection, malignancy, and drug toxicity. Beyond immune compatibility, critical challenges persist, including the risk of tumorigenicity, off-target genetic alterations, and ethical considerations surrounding genome editing. Recent advances, such as the integration of suicide gene systems and sensitive monitoring assays, offer promising strategies to enhance the safety and functional stability of hPSC-derived therapies. This review comprehensively discusses the molecular engineering of hPSCs, their biomedical applications, safety strategies, ethical implications, and the evolving regulatory frameworks needed for clinical translation. By addressing both the scientific and societal dimensions, hPSCs have the potential to revolutionize personalized and off-the-shelf regenerative treatments, provided that rigorous safeguards are implemented.},
}

Humans
*Regenerative Medicine/methods
*Pluripotent Stem Cells/immunology/transplantation
Animals
Gene Editing
*Cell- and Tissue-Based Therapy/methods
CRISPR-Cas Systems

@article {pmid40931807,
year = {2025},
author = {Wang, Y and Phelps, A and Godbehere, A and Evans, B and Takizawa, C and Chinen, G and Singh, H and Fang, Z and Du, ZY},
title = {Revolutionizing Agriculture With CRISPR Technology: Applications, Challenges, and Future Perspectives.},
journal = {Biotechnology journal},
volume = {20},
number = {9},
pages = {e70113},
doi = {10.1002/biot.70113},
pmid = {40931807},
issn = {1860-7314},
support = {//MBBE Molecular Biotechnology Lab/ ; HAW05047-H//USDA National Institute of Food and Agriculture/ ; 6114549//USDA REEU-Technology/ ; 5605280//CTAHR Agricultural Research and Extension Stations (CARES)/ ; //Undergraduate Research Opportunities Program (UROP)/ ; 5605280//U.S. Department of Agriculture/ ; 6114549//U.S. Department of Agriculture/ ; HATCH project HAW05047-H//U.S. Department of Agriculture/ ; },
mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Agriculture/methods/trends ; Crops, Agricultural/genetics ; Livestock/genetics ; Aquaculture ; },
abstract = {CRISPR technologies are rapidly transforming agriculture by enabling precise and programmable modifications across a wide range of organisms. This review provides an overview of CRISPR applications in crops, livestock, aquaculture, and microbial systems, highlighting key advances in sustainable agriculture. In crops, CRISPR has accelerated the improvement of traits such as drought tolerance, nutrient efficiency, and pathogen resistance. In livestock and aquaculture, CRISPR has enabled disease-resistant pigs and poultry, hornless cattle, and fast-growing, stress-tolerant fish. Engineered microbes are also being leveraged to enhance nitrogen fixation and reduce input reliance. We examine the evolution of CRISPR tools, such as base and prime editing, multiplex editing, and epigenome modulation, that expand precision and control beyond traditional gene knockouts. These innovations offer significant advantages over conventional breeding, yet challenges remain, including off-target effects, delivery efficiency, and regulatory variability across countries. The review also explores emerging directions such as novel Cas variants and AI-integrated breeding platforms for high-throughput trait discovery. Together, these developments demonstrate the transformative potential of CRISPR technology to reshape agriculture, not only by enhancing productivity and resilience but also by reducing environmental impacts. With responsible implementation, CRISPR-enabled innovations are well-positioned to support global food security and sustainability targets by 2050.},
}

Animals
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Agriculture/methods/trends
Crops, Agricultural/genetics
Livestock/genetics
Aquaculture

@article {pmid40931602,
year = {2025},
author = {Pan, L and Wang, P},
title = {DNA nanotechnology-enabled bioanalysis of extracellular vesicles.},
journal = {Nanoscale horizons},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5nh00557d},
pmid = {40931602},
issn = {2055-6764},
abstract = {Extracellular vesicles (EVs) have emerged as valuable sources for liquid biopsy in disease diagnostics, given their protein and nucleic acid cargoes (e.g., miRNA, mRNA, glycoRNA) can serve as critical biomarkers. DNA nanotechnology, leveraging its inherent programmability, high specificity, and powerful signal amplification capability, offers a transformative approach for the bioanalysis of EVs. This review summarizes recent advances in DNA nanotechnology-based analytical methodologies for detecting EV-associated proteins and nucleic acids. We detail the underlying principles, applications, and performance of key strategies, including aptamer-based recognition, enzyme-free catalytic amplification circuits (e.g., HCR, CHA), enzyme catalytic amplification techniques (e.g., RCA, CRISPR-Cas systems), and DNA nanostructures-assisted amplification. The integration of these DNA tools into multiplexed detection platforms is also discussed. Finally, current challenges and future perspectives concerning clinical translation of EV detection are presented.},
}

@article {pmid40114032,
year = {2025},
author = {Tang, K and Zhou, L and Tian, X and Fang, SY and Vandenbulcke, E and Du, A and Shen, J and Cao, H and Zhou, J and Chen, K and Kim, HR and Luo, Z and Xin, S and Lin, SH and Park, D and Yang, L and Zhang, Y and Suzuki, K and Majety, M and Ling, X and Lam, SZ and Chow, RD and Ren, P and Tao, B and Li, K and Codina, A and Dai, X and Shang, X and Bai, S and Nottoli, T and Levchenko, A and Booth, CJ and Liu, C and Fan, R and Dong, MB and Zhou, X and Chen, S},
title = {Cas12a-knock-in mice for multiplexed genome editing, disease modelling and immune-cell engineering.},
journal = {Nature biomedical engineering},
volume = {9},
number = {8},
pages = {1290-1308},
pmid = {40114032},
issn = {2157-846X},
support = {RF1DA048811//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; K99CA282989//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; F30CA250249//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; RF1 DA048811/DA/NIDA NIH HHS/United States ; R01 CA231112/CA/NCI NIH HHS/United States ; DP2 CA238295/CA/NCI NIH HHS/United States ; T32 GM007205/GM/NIGMS NIH HHS/United States ; F30 CA250249/CA/NCI NIH HHS/United States ; U54CA209992//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; DP2CA238295//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; K99 CA282989/CA/NCI NIH HHS/United States ; U54 CA209992/CA/NCI NIH HHS/United States ; R33 CA281702/CA/NCI NIH HHS/United States ; T32GM007205//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R33CA281702//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R33 CA225498/CA/NCI NIH HHS/United States ; R01CA231112//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Animals ; *Gene Editing/methods ; Mice ; Mice, Inbred C57BL ; *Gene Knock-In Techniques/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; Disease Models, Animal ; CRISPR-Cas Systems/genetics ; *Cell Engineering/methods ; Dependovirus/genetics ; Humans ; Dendritic Cells ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {The pleiotropic effects of human disease and the complex nature of gene-interaction networks require knock-in mice allowing for multiplexed gene perturbations. Here we describe a series of knock-in mice with a C57BL/6 background and with the conditional or constitutive expression of LbCas12a or of high-fidelity enhanced AsCas12a, which were inserted at the Rosa26 locus. The constitutive expression of Cas12a in the mice did not lead to discernible pathology and enabled efficient multiplexed genome engineering. We used the mice for the retrovirus-based immune-cell engineering of CD4[+] and CD8[+] T cells, B cells and bone-marrow-derived dendritic cells, for autochthonous cancer modelling through the delivery of multiple CRISPR RNAs as a single array using adeno-associated viruses, and for the targeted genome editing of liver tissue using lipid nanoparticles. We also describe a system for simultaneous dual-gene activation and knockout (DAKO). The Cas12a-knock-in mice and the viral and non-viral delivery vehicles provide a versatile toolkit for ex vivo and in vivo applications in genome editing, disease modelling and immune-cell engineering, and for the deconvolution of complex gene interactions.},
}

Animals
*Gene Editing/methods
Mice
Mice, Inbred C57BL
*Gene Knock-In Techniques/methods
*CRISPR-Associated Proteins/genetics/metabolism
Disease Models, Animal
CRISPR-Cas Systems/genetics
*Cell Engineering/methods
Dependovirus/genetics
Humans
Dendritic Cells
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid40930534,
year = {2025},
author = {Lejars, M and Maeda, T and Guillier, M},
title = {EASY-edit: a toolbox for high-throughput single-step custom genetic editing in bacteria.},
journal = {Nucleic acids research},
volume = {53},
number = {17},
pages = {},
doi = {10.1093/nar/gkaf883},
pmid = {40930534},
issn = {1362-4962},
support = {/ERC_/European Research Council/International ; 818750//European Union's Horizon 2020 research and innovation/ ; //CNRS/ ; ANR-11-LABX-0011//Initiative d'Excellence/ ; },
mesh = {*Gene Editing/methods ; *Escherichia coli/genetics ; *CRISPR-Cas Systems ; Operon ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genes, Reporter ; },
abstract = {Targeted gene editing can be achieved using CRISPR-Cas9-assisted recombineering. However, high-efficiency editing requires careful optimization for each locus to be modified, which can be tedious and time-consuming. In this work, we developed a simple, fast and cheap method: Engineered Assembly of SYnthetic operons for targeted editing (EASY-edit) in Escherichia coli. Highly efficient editing of the different constitutive elements of the operons can be achieved by using a set of optimized guide RNAs and single- or double-stranded DNA repair templates carrying relatively short homology arms. This facilitates the construction of multiple genetic tools, including mutant libraries or reporter genes. EASY-edit is also highly modular, as we provide alternative and complementary versions of the operon inserted in three loci which can be edited iteratively and easily combined. As a proof of concept, we report the construction of several fusions with reporter genes confirming known post-transcriptional regulation mechanisms and the construction of saturated and unbiased mutant libraries. In summary, the EASY-edit system provides a flexible genomic expression platform that can be used both for the understanding of biological processes and as a tool for bioengineering applications.},
}

*Gene Editing/methods
*Escherichia coli/genetics
*CRISPR-Cas Systems
Operon
RNA, Guide, CRISPR-Cas Systems/genetics
Genes, Reporter

@article {pmid40930528,
year = {2025},
author = {Jiang, Q and Jin, S and Qin, Z and Zhang, J and He, R and Chen, Z and Qiao, B and Qiao, J and Liu, Y},
title = {CRISPR/Cas12a DTR system: a topology-guided Cas12a assay for specific dual detection of RNA and DNA targets.},
journal = {Nucleic acids research},
volume = {53},
number = {17},
pages = {},
doi = {10.1093/nar/gkaf893},
pmid = {40930528},
issn = {1362-4962},
support = {2022YFC2304304//National Key Research and Development Program of China/ ; 2023DJC136//Science and Technology Innovation Talent Plan of Hubei Province/ ; 2025AFB825//Natural Science Foundation of Hubei Province/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *MicroRNAs/genetics/analysis ; *DNA/genetics/analysis ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; *RNA/genetics/analysis ; *Bacterial Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The CRISPR/Cas12a technology has revolutionized molecular diagnostics. However, existing Cas12a systems depend on continuous target DNA activation, which limits them to single-target detection. In this study, we developed a novel topology-guided Cas12a system, the double-target responsive (DTR) system, capable of being activated by noncontiguous dual RNA/DNA targets. The DTR system employs two split CRISPR RNA (crRNA) fragments and two Cas12a proteins that cooperatively reconstitute upon recognizing two nucleic acid activators. We demonstrated the DTR system's ability to specifically detect dual nucleic acid substrates in a single readout, achieving a detection limit of 78 fM for RNA and exceptional specificity for single-nucleotide variations. Additionally, we successfully applied the DTR system to clinical samples, enabling simultaneous detection of two oral squamous cell carcinoma-related microRNAs (miR-155 and miR-let-7a), thereby distinguishing healthy individuals from patients. This work establishes an efficient Cas12a-based platform for sensitive, simultaneous, and discriminative detection of RNA and DNA targets, enhancing the versatility of Cas12a in analytical detection and clinical diagnosis.},
}

*CRISPR-Cas Systems/genetics
Humans
*MicroRNAs/genetics/analysis
*DNA/genetics/analysis
*CRISPR-Associated Proteins/genetics/metabolism
*Endodeoxyribonucleases/genetics/metabolism
*RNA/genetics/analysis
*Bacterial Proteins/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40929845,
year = {2025},
author = {Chang, Y and Ding, J},
title = {CircRNA knockout/knockdown tools in molecular biology research.},
journal = {Biochemical and biophysical research communications},
volume = {783},
number = {},
pages = {152607},
doi = {10.1016/j.bbrc.2025.152607},
pmid = {40929845},
issn = {1090-2104},
abstract = {Circular RNAs (circRNAs), characterized by their covalently closed circular architecture, represent a unique class of endogenous RNA molecules that serve as pivotal regulators in post-transcriptional gene regulation in organisms. Accumulating evidence has established their potential as promising diagnostic biomarkers across various human pathologies, including but not limited to malignant neoplasms, neurodegenerative disorders, and metabolic dysregulation.By inhibiting circRNA expression, we can better understand their functions and their impact on related biological processes. Over the past decade, remarkable advancements have emerged in circRNAs manipulation technologies, including siRNA, DNAzyme, and CRISPR-Cas systems emerging as powerful tools for precise circRNAs editing in both experimental models and preclinical studies. In this review, we summarize the advantages and identification of these editing methods and discuss future challenges and prospects.},
}

@article {pmid40927181,
year = {2025},
author = {Ziemann, M and Mitrofanov, A and Stöckl, R and Alkhnbashi, OS and Backofen, R and Hess, WR},
title = {Analysis of tracrRNAs reveals subgroup V2 of type V-K CAST systems.},
journal = {microLife},
volume = {6},
number = {},
pages = {uqaf020},
pmid = {40927181},
issn = {2633-6693},
abstract = {Clustered regularly interspaced palindromic repeats (CRISPR)-associated transposons (CAST) consist of an integration between certain class 1 or class 2 CRISPR-Cas systems and Tn7-like transposons. Class 2 type V-K CAST systems are restricted to cyanobacteria. Here, we identified a unique subgroup of type V-K systems through phylogenetic analysis, classified as V-K_V2. Subgroup V-K_V2 CAST systems are characterized by an alternative tracrRNA, the exclusive use of Arc_2-type transcriptional regulators, and distinct differences in the length of protein domains in TnsB and TnsC. Although the occurrence of V-K_V2 CAST systems is restricted to Nostocales cyanobacteria, it shows signs of horizontal gene transfer, indicating its capability for genetic mobility. The predicted V-K_V2 tracrRNA secondary structure has been integrated into an updated version of the CRISPRtracrRNA program available on GitHub under https://github.com/BackofenLab/CRISPRtracrRNA/releases/tag/2.0.},
}

@article {pmid40678944,
year = {2025},
author = {Zhu, C and Huang, Q and Fu, R and Xun, Z and Ou, Q and Xianyu, Y and Liu, C},
title = {A Triple-Modal Biosensing Strategy for Hepatitis B Virus Based on Mg[2+]-Mediated Modulation of CRISPR/Cas12a and Au@Pt Nanoparticles.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {21},
number = {36},
pages = {e05341},
doi = {10.1002/smll.202505341},
pmid = {40678944},
issn = {1613-6829},
support = {82030063 82372317 82372316//National Natural Science Foundation of China/ ; 2023Y4004//University-Industry Research Cooperation Project of Science and Technology of Fujian Province/ ; 2025C02124//"Pioneer" and "Leading Goose" R&D Program of Zhejiang/ ; 2023QH1102//Startup Fund for Scientific Research of Fujian Medical University/ ; 2024QH2043//Startup Fund for Scientific Research of Fujian Medical University/ ; },
mesh = {*Hepatitis B virus/genetics/isolation & purification ; *Biosensing Techniques/methods ; *Metal Nanoparticles/chemistry/ultrastructure ; *Gold/chemistry ; *CRISPR-Cas Systems/genetics ; *Magnesium/chemistry ; DNA, Viral ; *Platinum/chemistry ; Nucleic Acid Amplification Techniques ; Humans ; },
abstract = {Hepatitis B virus (HBV) infection remains a significant global public health issue, and rapid detection of HBV DNA is crucial for disease prevention and control. However, traditional methods for HBV DNA detection are limited by their reliance on precise instruments and single readout, which can hardly meet the requirements of on-site detection. In this study, the Mg[2+]-enhanced trans-cleavage activity of clustered regularly interspaced short palindromic repeats/associated protein 12a (CRISPR/Cas12a) is reported and coupled with loop-mediated isothermal amplification (LAMP) and Au@Pt nanoparticles as a signaling reporter for on-site detection of HBV DNA. This triple-modal biosensing strategy enables multiple signal readouts including UV-vis spectrum, RGB value, and temperature with high sensitivity and accuracy. The linear detection ranges using UV-vis spectroscopy, RGB color recognition, and photothermal modes are from 50 to 10,000 copies µL[-1], with the limitation of detection of 24.07, 39.65, and 23.33 copies µL[-1], respectively. This biosensing strategy is further employed for the qualitative detection of HBV DNA in 48 serum samples, achieving sensitivities of 100%, 100%, and 95.24% for triple modes. This work offers a promising tool of next-generation LAMP-CRISPR/Cas12a for the rapid and portable detection of nucleic acids.},
}

*Hepatitis B virus/genetics/isolation & purification
*Biosensing Techniques/methods
*Metal Nanoparticles/chemistry/ultrastructure
*Gold/chemistry
*CRISPR-Cas Systems/genetics
*Magnesium/chemistry
DNA, Viral
*Platinum/chemistry
Nucleic Acid Amplification Techniques
Humans

@article {pmid40925176,
year = {2025},
author = {He, Z and Zhang, J and Kuang, S and Li, S and Wang, Y and Ding, J and Ma, Z and Zhang, B},
title = {Colloidal gold technology in viral diagnostics: Recent innovations, clinical applications, and future perspectives.},
journal = {Virology},
volume = {612},
number = {},
pages = {110686},
doi = {10.1016/j.virol.2025.110686},
pmid = {40925176},
issn = {1096-0341},
abstract = {Colloidal gold technology has revolutionized viral diagnostics through its rapid, cost-effective, and user-friendly applications, particularly in point-of-care testing (POCT). This review synthesizes recent advancements, focusing on its role in detecting respiratory viruses, hepatitis viruses, and emerging pathogens. The technology leverages the unique optical and physicochemical properties of gold nanoparticles (AuNPs), including localized surface plasmon resonance (LSPR) and high surface-to-volume ratios, to achieve rapid antigen-antibody recognition with visual readouts within 15 min. Innovations such as CRISPR-Cas-integrated lateral flow immunoassays (LFIAs), dual-mode plasmonic biosensors, and nanomaterials like CeO2-colloidal gold composites have enhanced sensitivity and multiplex capability, enabling simultaneous identification of co-circulating pathogens. Case studies highlight its efficacy in dengue serotyping, SARS-CoV-2 neutralizing antibody quantification, and HBV/HCV co-detection, demonstrating high clinical specificity. However, challenges persist, including the need for improved sensitivity; interference of sample matrix with immunity; false positives caused by cross-reactions; and limitations of semi-quantitative analysis. Recent progress in hybrid nanomaterial synthesis, surface functionalization, and device-level multiplexing-coupled with AI-driven data interpretation- promises to address these gaps. Future trends emphasize integration with surface-enhanced Raman scattering (SERS), microfluidics, and portable sensors to achieve sub-zeptomolar sensitivity and scalable deployment. By bridging nanotechnology with precision diagnostics, colloidal gold platforms are poised to redefine global viral surveillance, particularly in resource-limited settings, underscoring their indispensable role in pandemic preparedness.},
}

@article {pmid40922689,
year = {2025},
author = {Zhang, Q and Ren, J and Wu, S and Tan, Y and Wang, W and Feng, C and Zhao, L and Zhu, Z},
title = {Plasmid-Free CRISPR/Cpf1 Genome Editing With In Vivo T7 RNA Polymerase-Transcribed CRISPR RNA From Short Double-Stranded DNA.},
journal = {Biotechnology and bioengineering},
volume = {},
number = {},
pages = {},
doi = {10.1002/bit.70062},
pmid = {40922689},
issn = {1097-0290},
support = {//This study was funded by the National Key Research and Development Program (Grant Number: 2022YFA0911802); the National Natural Science Foundation of China (Grant Number: 22177018); the Liaoning Revitalization Talents Program (Grant Number: XLYC2203057) and the Fundamental Research Funds for the Central Universities (Grant Number: DUT23LAB104)./ ; },
abstract = {Plasmids are commonly employed in the delivery of clustered regularly interspaced shortpalindromic repeats (CRISPR)/CRISPR-associated (Cas) components for genome editing. However, the absence of heritable plasmids in numerous organisms limits the development of CRISPR/Cas genome editing tools. Moreover, cumbersome procedures for plasmid construction and curing render genome editing time-consuming. In this study, we developed a plasmid-free CRISPR/Cpf1 genome editing system for Saccharomyces cerevisiae and Starmerella bombicola. This system leveraged integrative expression of the Cpf1 nuclease and T7 RNA polymerase (T7RNAP), as well as the delivery of linear fragments including (i) a marker cassette for integration and selection, (ii) short double-stranded DNA (crDNA) for in vivo transcription of crRNA by T7RNAP, and (iii) donor DNA for homology-directed repair. We demonstrated that this editing system enabled efficient multiplexed and iterative genome editing without the need for marker recycling and plasmid curing. The use of short crDNA (87 bp) and donor DNA (≤ 120 bp), both readily prepared from ordered oligonucleotides via annealing or overlap extension, dramatically simplified the editing process. Successful implementation in S. bombicola, which lacks heritable plasmids for genetic engineering, highlighted the potential of this approach especially for genome engineering of genetically intractable organisms in a plasmid-free way.},
}

@article {pmid40920944,
year = {2025},
author = {Mangeot, PE and Ohlmann, T},
title = {[Harnessing retroviral engineering for genome reprogramming].},
journal = {Medecine sciences : M/S},
volume = {41},
number = {8-9},
pages = {647-656},
doi = {10.1051/medsci/2025098},
pmid = {40920944},
issn = {1958-5381},
support = {ANRS0516//ANRS/ ; },
mesh = {Humans ; *Retroviridae/genetics/physiology ; *Genetic Engineering/methods ; Genetic Vectors/genetics ; Animals ; CRISPR-Cas Systems ; Gene Editing/methods ; Genome, Human ; *Cellular Reprogramming/genetics ; },
abstract = {The accumulated knowledge on the biology of the HIV-1 virus has led to the emergence of technologies that exploit the architecture of retroviruses and their integration or vectorization properties. This field of study constitutes retroviral vectorology, democratized in laboratories by the use of lentiviral vectors. By hijacking retroviral assembly, other systems are emerging and are increasingly mentioned in recent literature. In particular, defective retroviral particles are capable of transiently delivering effectors that act on the genome: they thus appear to be more suitable tools for delivering genetic scalpels, whose persistence in the target cell or organism is not required. Since the description of the CRISPR Cas9 system in 2012, genome engineering techniques have continued to evolve in terms of capacity and reliability. Several derivatives of the CRISPR system can now modify the human genome with nucleotide-level precision. Introducing these effectors into the cell or organism remains a major technical challenge that vector scientists are striving to overcome. This review describes the major retroviral systems used for genome manipulation. Following an overview of genetic engineering techniques, we will see how researchers have developed a wide range of genomic tools by manipulating different processes in the retroviral architecture.},
}

Humans
*Retroviridae/genetics/physiology
*Genetic Engineering/methods
Genetic Vectors/genetics
Animals
CRISPR-Cas Systems
Gene Editing/methods
Genome, Human
*Cellular Reprogramming/genetics

@article {pmid40920796,
year = {2025},
author = {Nakagata, N and Nakao, S and Mikoda, N and Yamaga, K and Nakagawa, Y and Sakuma, T and Yamamoto, T and Takeo, T},
title = {Improved protocol for the vitrification and warming of rat zygotes by optimizing the warming solution and oocyte donor age.},
journal = {PloS one},
volume = {20},
number = {9},
pages = {e0328718},
pmid = {40920796},
issn = {1932-6203},
mesh = {Animals ; Female ; *Zygote/physiology/drug effects/cytology ; *Vitrification ; Rats ; *Oocytes/cytology ; *Cryopreservation/methods ; Rats, Sprague-Dawley ; Embryo Transfer ; Fertilization in Vitro/methods ; Rats, Inbred F344 ; Male ; CRISPR-Cas Systems ; Sucrose/pharmacology ; },
abstract = {Zygotes are used to create genetically modified animals by electroporation using the CRISPR-Cas9 system. Such zygotes in rats are obtained from superovulated female rats after mating. Recently, we reported that in vivo-fertilized zygotes had higher cryotolerance and developmental ability than in vitro-fertilized zygotes in Sprague Dawley (SD) and Fischer 344 rats. To apply the in vitro-fertilized zygotes in creating genetically modified rats, we need to address their low cryotolerance and developmental ability. Hence, we evaluated the effects of warming solutions containing different sucrose concentrations (0-0.3 M) and the oocyte donor's age (3-7-week-old SD rats) on the viability of vitrified-warmed zygotes after in vitro fertilization and on developmental ability by embryo transfer in SD rats. A warming solution containing 0.1 M sucrose enhanced the survival rate of vitrified-warmed zygotes and their rate of development to two-cell embryos. Additionally, zygotes derived from 6- and 7-week-old female rats had higher cryotolerance and developmental ability than those from 3-week-old ones. Next, vitrified-warmed rat zygotes produced using the optimized protocol underwent genome editing by electroporation with Cas9 ribonucleoprotein and gRNA introduced to disrupt the Tyr gene. We then found that 86.5% of the pups derived from zygotes demonstrated mutation of the targeted gene. Therefore, the improved protocol for vitrifying and warming rat zygotes is useful for preserving and producing genetically modified rats.},
}

Animals
Female
*Zygote/physiology/drug effects/cytology
*Vitrification
Rats
*Oocytes/cytology
*Cryopreservation/methods
Rats, Sprague-Dawley
Embryo Transfer
Fertilization in Vitro/methods
Rats, Inbred F344
Male
CRISPR-Cas Systems
Sucrose/pharmacology

@article {pmid40891143,
year = {2025},
author = {Zheng, X and Zhai, Y and Chathurika, HAW and Ni, X and Lv, R and Wu, C and Sun, Z and Shen, Y and Zhang, CY and Zheng, P and Sun, J},
title = {A Highly Efficient 5S rRNA-CRISPR/Cas9 Genome Editing Toolkit in Acremonium chrysogenum.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {36},
pages = {22607-22616},
doi = {10.1021/acs.jafc.5c06429},
pmid = {40891143},
issn = {1520-5118},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Acremonium/genetics/metabolism ; *RNA, Ribosomal, 5S/genetics/metabolism ; Cephalosporins/biosynthesis ; Fungal Proteins/genetics/metabolism ; },
abstract = {Acremonium chrysogenum is an important industrial producer of cephalosporin C (CPC), and efficient genome editing tools are critical for its exploitation and metabolic engineering. Currently, CRISPR/Cas9 systems for A. chrysogenum employ heterologous promoters, including Aspergillus nidulans PgpdA or Aspergillus fumigatus AfU6p, to drive sgRNA expression. These systems often required additional sgRNA processing elements such as ribozymes or tRNAs, which increased cloning complexity and experimental workload. Here, we developed a simplified and highly efficient CRISPR/Cas9 genome editing system using the endogenous 5S rRNA promoter for sgRNA transcription in A. chrysogenum. This system obviated the need for processing elements and achieved up to 100% gene disruption efficiency, as demonstrated by targeting the sorB gene. Furthermore, this platform enabled 100% single gene deletion and efficient large-scale chromosomal deletion, up to 66.17 kb within the sorbicillinoid biosynthetic gene cluster, without donor DNA. To our knowledge, this represents the largest chromosomal deletion reported in A. chrysogenum to date. Moreover, the system also facilitated precise and iterative gene editing through homologous recombination-mediated marker replacement at the kusA locus. Overall, this 5S rRNA-CRISPR/Cas9 system provides a versatile, powerful, and efficient genome editing toolkit for functional genomics and strain improvement in A. chrysogenum.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*Acremonium/genetics/metabolism
*RNA, Ribosomal, 5S/genetics/metabolism
Cephalosporins/biosynthesis
Fungal Proteins/genetics/metabolism

@article {pmid40877754,
year = {2025},
author = {Singuru, MMR and Bhattacharyya, P and Sriramakrishnan, HP and You, M},
title = {Sensitive Detection of Intercellular Tensile Forces via Cas12a-Assisted Membrane Molecular Probes.},
journal = {Nano letters},
volume = {25},
number = {36},
pages = {13519-13525},
doi = {10.1021/acs.nanolett.5c02983},
pmid = {40877754},
issn = {1530-6992},
mesh = {Cadherins/metabolism ; Humans ; *Cell Membrane/chemistry/metabolism ; CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; Tensile Strength ; Mechanotransduction, Cellular ; Epithelial-Mesenchymal Transition ; *Endodeoxyribonucleases/metabolism/chemistry/genetics ; *Molecular Probes/chemistry ; *Bacterial Proteins/metabolism/chemistry ; },
abstract = {Intercellular forces are critical for shaping cells, driving migration, and guiding tissue development and morphogenesis. However, these transient and low-intensity forces are still challenging to detect. Here, we developed a Force-Responsive Cas12a-assisted Tension Sensor (FRCTS), which leverages the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a technology to enable more reliable detection of cumulative molecular force events generated at cell-cell junctions. FRCTS incorporates a lipid-modified DNA hairpin to spontaneously anchor onto live-cell membranes. The hairpin unfolds upon molecular tension exerted by neighboring cells through an integrin or cadherin receptor and reveals a hidden strand to activate Cas12a. Cas12a activation leads to an irreversible cleavage of a fluorogenic reporter on the cell surface, causing cumulative cell membrane fluorescence signals for recording intercellular force events. After systematic optimization, we applied FRCTS to quantify E-cadherin/N-cadherin mechanical correlations during the epithelial-mesenchymal transition. This modular and sensitive FRCTS platform can potentially be used for assessing various intercellular mechanotransduction processes.},
}

Cadherins/metabolism
Humans
*Cell Membrane/chemistry/metabolism
CRISPR-Cas Systems
*CRISPR-Associated Proteins/metabolism/chemistry/genetics
Tensile Strength
Mechanotransduction, Cellular
Epithelial-Mesenchymal Transition
*Endodeoxyribonucleases/metabolism/chemistry/genetics
*Molecular Probes/chemistry
*Bacterial Proteins/metabolism/chemistry

@article {pmid40816522,
year = {2025},
author = {Meng, XQ and Xu, XL and Gao, Y and Deng, SL},
title = {Establishment of CRISPR/Cas9 lineage tracking technology for pig embryos.},
journal = {Molecular and cellular probes},
volume = {83},
number = {},
pages = {102046},
doi = {10.1016/j.mcp.2025.102046},
pmid = {40816522},
issn = {1096-1194},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Swine/genetics/embryology ; *Cell Lineage/genetics ; *Embryo, Mammalian/cytology/metabolism ; Single-Cell Analysis/methods ; },
abstract = {Understanding tissue development in pigs is critical for biomedical research and genetic engineering, particularly for modeling human disease. However, tracing developmental origins and reconstructing lineage trees for pig cells remains a significant challenge. Here, we present a high-resolution lineage tracing system that combines molecular barcoding with single-cell transcriptomics in pigs. Our system combines two key components: DNA barcodes (three CRISPR/Cas9 target sites and an 8-base pair intBC) integrated into the genome via piggyBac transposition, and a constitutive Cas9-EGFP cassette stably integrated at the Rosa26 locus using CRISPR/Cas12a. By combining lineage barcodes with single-cell RNA sequencing (scRNA-seq), we constructed an evolutionary lineage recorder that captures distinct cell states across developmental or differentiation trajectories. This system provides an essential tool for the subsequent construction of complete porcine cell fate maps. Our work provides a tool for studying porcine developmental biology, but also helps to optimize regenerative medicine strategies and improve the design of genetically engineered animal models.},
}

Animals
*CRISPR-Cas Systems/genetics
Swine/genetics/embryology
*Cell Lineage/genetics
*Embryo, Mammalian/cytology/metabolism
Single-Cell Analysis/methods

@article {pmid40815167,
year = {2025},
author = {Sunshine, S and Puschnik, A and Retallack, H and Laurie, MT and Liu, J and Peng, D and Knopp, K and Zinter, MS and Ye, CJ and DeRisi, JL},
title = {Defining the host dependencies and the transcriptional landscape of RSV infection.},
journal = {mBio},
volume = {16},
number = {9},
pages = {e0101025},
doi = {10.1128/mbio.01010-25},
pmid = {40815167},
issn = {2150-7511},
support = {//Chan Zuckerberg Biohub San Francisco/ ; F31AI150007//National Institute of Allergy and Infectious Diseases/ ; },
mesh = {Humans ; *Respiratory Syncytial Virus Infections/virology/genetics/immunology ; *Respiratory Syncytial Virus, Human/genetics/physiology ; *Host-Pathogen Interactions/genetics ; Single-Cell Analysis ; CRISPR-Cas Systems ; Gene Expression Profiling ; },
abstract = {Respiratory syncytial virus (RSV) is a globally prevalent pathogen, causes severe disease in older adults, and is the leading cause of bronchiolitis and pneumonia in the United States for children during their first year of life. Despite its prevalence worldwide, RSV-specific treatments remain unavailable for most infected patients. Here, we leveraged a combination of genome-wide CRISPR knockout screening and single-cell RNA sequencing to improve our understanding of the host determinants of RSV infection and the host response in both infected cells and uninfected bystanders. These data reveal temporal transcriptional patterns that are markedly different between RSV-infected and bystander-activated cells. Our data show that expression of interferon-stimulated genes is primarily observed in bystander activated cells, while genes implicated in the unfolded protein response and cellular stress are upregulated specifically in RSV-infected cells. Furthermore, genome-wide CRISPR screens identified multiple host factors important for viral infection, findings which we contextualize relative to 29 previously published screens across 17 additional viruses. These unique data complement and extend prior studies that investigate the proinflammatory response to RSV infection, and juxtaposed to other viral infections, provide a rich resource for further hypothesis testing.IMPORTANCERespiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in infants and the elderly. Despite its substantial global health burden, RSV-targeted treatments remain unavailable for the majority of individuals. While vaccine development is underway, a detailed understanding of the host response to RSV infection and identification of required human host factors for RSV may provide insight into combatting this pathogen. Here, we utilized single-cell RNA sequencing and functional genomics to understand the host response in both RSV-infected and bystander cells, identify what host factors mediate infection, and contextualize these findings relative to dozens of previously reported screens across 17 additional viruses.},
}

Humans
*Respiratory Syncytial Virus Infections/virology/genetics/immunology
*Respiratory Syncytial Virus, Human/genetics/physiology
*Host-Pathogen Interactions/genetics
Single-Cell Analysis
CRISPR-Cas Systems
Gene Expression Profiling

@article {pmid40767522,
year = {2025},
author = {Guo, G and Zhang, M and Xu, Z and Xi, P and Zhu, H and Evers, A and Lebbink, RJ and Lang, Y and He, Q and Huang, Y-W and Li, T and Bosch, BJ and Li, W},
title = {Genome-wide CRISPR screen reveals key role of sialic acids in PEDV and porcine coronavirus infections.},
journal = {mBio},
volume = {16},
number = {9},
pages = {e0162825},
doi = {10.1128/mbio.01628-25},
pmid = {40767522},
issn = {2150-7511},
mesh = {*Porcine epidemic diarrhea virus/physiology/pathogenicity/genetics ; Animals ; *Coronavirus Infections/virology/veterinary/metabolism ; Swine ; Humans ; *CRISPR-Cas Systems ; *Sialic Acids/metabolism ; *Swine Diseases/virology/metabolism ; Virus Internalization ; Sialyltransferases/genetics/metabolism ; *N-Acetylneuraminic Acid/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Host-Pathogen Interactions ; Cell Line ; },
abstract = {Porcine epidemic diarrhea virus (PEDV) is a globally distributed alphacoronavirus with economic importance that can cause severe watery diarrhea and even death in piglets. To identify host factors essential for PEDV infection, we performed a genome-wide CRISPR/Cas9 screen in human hepatocellular carcinoma cells (Huh7) using the highly virulent PEDV GIIb strain GDU. Several genes involved in the sialic acid and heparan sulfate biosynthesis pathway and cholesterol metabolism were highly enriched following PEDV selection. We validated that the host factor ST3 beta-galactoside alpha-2,3-sialyltransferase 4 (ST3GAL4), which catalyzes the transfer of sialic acid to sugar chains via α2,3-linked linkages, is important for PEDV infection. To systematically investigate the role of sialic acid in PEDV infection, we knocked out genes related to sialic acid synthesis. This led to a reduced abundance of sialic acid on the cell surface, which in turn inhibited PEDV adsorption and internalization. Furthermore, we found that both α2,3-linked and α2,6-linked sialic acids can serve as cellular attachment factors for PEDV. We conducted a glycan microarray screen to determine which sialoglycans are preferred by the PEDV spike protein. The results revealed that PEDV favors binding to α2,3-sialoglycans. Additionally, we found that not only current circulating PEDV strains but also other porcine coronaviruses rely on sialic acid for efficient infection. Collectively, our findings provide insights into critical host factors involved in PEDV infection and demonstrate that disrupting genes involved in sialic acid biosynthesis negatively affects the infectivity of multiple porcine enteric coronaviruses.IMPORTANCEA wide range of viruses utilize sialic acid as receptors. Sialic acid binding may serve as a key determinant of viral host range. Different viruses exhibit distinct preferences for specific types of sialic acid linkages. However, it remains unclear which specific subtypes of sialic acid are utilized during PEDV infection. In this study, we performed CRISPR-based genome-wide knockout screening and identified ST3GAL4 as a key host factor for PEDV infection. Furthermore, we found that both α2,3-linked and α2,6-linked sialic acids can function as attachment factors for PEDV infection. A glycan microarray screen revealed that PEDV S1 shows the strongest binding preference for α2,3-linked and α2,8-linked sialosides. Sialic acids were also implicated in infections by other porcine enteric coronaviruses. Overall, our findings advance our understanding of viral entry mechanisms of PEDV and other swine coronaviruses and may provide avenues for designing antiviral strategies.},
}

*Porcine epidemic diarrhea virus/physiology/pathogenicity/genetics
Animals
*Coronavirus Infections/virology/veterinary/metabolism
Swine
Humans
*CRISPR-Cas Systems
*Sialic Acids/metabolism
*Swine Diseases/virology/metabolism
Virus Internalization
Sialyltransferases/genetics/metabolism
*N-Acetylneuraminic Acid/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats
Host-Pathogen Interactions
Cell Line

@article {pmid40744238,
year = {2025},
author = {Wen, Z and Yang, D and Yang, Y and Hu, J and Parviainen, A and Chen, X and Li, Q and VanDeusen, E and Ma, J and Tay, F},
title = {The path to biotechnological singularity: Current breakthroughs and outlook.},
journal = {Biotechnology advances},
volume = {84},
number = {},
pages = {108667},
doi = {10.1016/j.biotechadv.2025.108667},
pmid = {40744238},
issn = {1873-1899},
mesh = {Humans ; *Biotechnology/trends ; Gene Editing ; Artificial Intelligence ; Synthetic Biology ; Regenerative Medicine ; CRISPR-Cas Systems ; Brain-Computer Interfaces ; },
abstract = {Fueled by rapid advances in gene editing, synthetic biology, artificial intelligence, regenerative medicine, and brain-computer interfaces, biotechnology is approaching a transformative era often referred to as biotechnological singularity. CRISPR-based gene editing has revolutionized genetic engineering, enabling precise modifications for treating hereditary diseases and cancer. Synthetic biology facilitates sustainable biomaterial production and innovative therapeutic applications. Artificial intelligence accelerates drug discovery, enhances diagnostic accuracy, and personalizes treatment through deep learning models. Driven by stem cell research, regenerative medicine offers promising avenues for reversing aging and treating degenerative diseases. Brain-computer interfaces merge human cognition with technology, enabling direct neural control of prosthetics and expanding human-machine interactions. These breakthroughs, however, raise ethical, regulatory, and societal concerns, including equitable access, biosecurity risks, and the implications of human enhancement. The convergence of biological and computational technologies challenges traditional boundaries, necessitating comprehensive governance frameworks. By embracing responsible innovation, society can harness these advancements for transformative health interventions, environmental sustainability, and extended longevity. The realization of biotechnological singularity depends on interdisciplinary collaboration among scientists, policymakers, and the public to ensure that progress aligns with the well-being of humanity and ethical considerations.},
}

Humans
*Biotechnology/trends
Gene Editing
Artificial Intelligence
Synthetic Biology
Regenerative Medicine
CRISPR-Cas Systems
Brain-Computer Interfaces

@article {pmid40552689,
year = {2025},
author = {Yao, B and Yang, Q and Snijders Blok, CJB and Daniels, MA and Doevendans, PA and Schiffelers, R and Sluijter, JPG and Lei, Z},
title = {Insights into pegRNA design from editing of the cardiomyopathy-associated phospholamban R14del mutation.},
journal = {FEBS letters},
volume = {599},
number = {17},
pages = {2543-2554},
doi = {10.1002/1873-3468.70097},
pmid = {40552689},
issn = {1873-3468},
support = {10250022110004//The Netherlands Organisation for Health Research and Development (ZonMw) Psider-Heart/ ; 202006170055//China Scholarship Council/ ; #725229//European Research Council (ERC), EVICARE project under H2020/ ; 2021/TTW/01038252//Netherlands Organization for Scientific Research (NWO) - Applied and Engineering Sciences (TTW)/ ; },
mesh = {*Gene Editing/methods ; Humans ; *Calcium-Binding Proteins/genetics ; *Cardiomyopathies/genetics/pathology ; CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Mutation ; HEK293 Cells ; },
abstract = {Prime editing (PE) represents a transformative genome-editing technology and enables precise insertions, deletions, and base substitutions without introducing double-strand breaks, thereby reducing undesired indels and off-target effects. Despite advancements in enhanced prime editors and optimized prime editing guide RNAs (pegRNAs), designing effective pegRNAs remains a major challenge. The phospholamban (PLN) R14del mutation is associated with cardiomyopathies, making it a crucial target for precise gene-editing strategies. In this study, we explored pegRNA features that contribute to high editing efficiency using the FluoPEER.PLN R14del reporter cell line. Through systematic screening, we identified three pegRNAs with significantly enhanced editing efficiency. Our findings underscore the importance of pegRNA secondary structure and stability in optimizing prime editing, providing valuable insights into precise gene correction strategies.},
}

*Gene Editing/methods
Humans
*Calcium-Binding Proteins/genetics
*Cardiomyopathies/genetics/pathology
CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/genetics
Mutation
HEK293 Cells

@article {pmid40920775,
year = {2025},
author = {Tan, Y and Kumagai-Takei, N and Shimizu, Y and Yamasaki, A and Hara-Yamamoto, M and Mitani, S and Ito, T},
title = {Targeting the Exon2 splice cis-element in PD-1 and its effects on lymphocyte function.},
journal = {PloS one},
volume = {20},
number = {9},
pages = {e0331468},
pmid = {40920775},
issn = {1932-6203},
mesh = {*Programmed Cell Death 1 Receptor/genetics/metabolism ; Humans ; *Exons/genetics ; CRISPR-Cas Systems ; *CD8-Positive T-Lymphocytes/metabolism/immunology ; *RNA Splicing ; Cytokines/metabolism ; RNA Precursors/genetics ; Lymphocyte Activation ; Cell Proliferation ; },
abstract = {T-cell therapies have proven to be a promising treatment option for cancer patients in recent years, especially in the case of chimeric antigen receptor (CAR)-T cell therapy. However, the therapy is associated with insufficient activation of T cells or poor persistence in the patient's body, which leads to incomplete elimination of cancer cells, recurrence, and genotoxicity. By extracting the splice element of PD-1 pre-mRNA using biology based on CRISPR/dCas13 in this study, our ultimate goal is to overcome the above-mentioned challenges in the future. PD-1 plays an important role in controlling T cell responses and is expressed at the cell surface of T cells following activation. The receptor PD-1 interferes with T cell receptor (TCR) signaling following interaction with PD-L1. The outcome of stimulation via PD-1 leads to decreases in cytokine secretion and cell proliferation. We extracted the RNA region of PD-1 pre-mRNA using CD8+T cell lines and examined the effect of targeting the Exon2 splice cis-element on the production of cytokines in the present study. In particular, the production of IFN-γ, TNF-α, GM-CSF was lower in RNA-targeted cells than in non-targeted cells, but the cytokine secretion capacity and cell proliferation were maintained in RNA-targeted cells. These results suggested that the use of the RNA editing technology, CRISPR/dCas13 strategy offers a novel approach to mitigate genotoxicity in lymphocytes with cytokine production and cell proliferation.},
}

*Programmed Cell Death 1 Receptor/genetics/metabolism
Humans
*Exons/genetics
CRISPR-Cas Systems
*CD8-Positive T-Lymphocytes/metabolism/immunology
*RNA Splicing
Cytokines/metabolism
RNA Precursors/genetics
Lymphocyte Activation
Cell Proliferation

@article {pmid40919784,
year = {2025},
author = {Leite, VLM and Faria, AR and Guerra, CF and Souza, SdSR and Freitas, AdAR and Morais, JM and Merquior, VLC and Planet, PJ and Teixeira, LM},
title = {Hidden diversity in Enterococcus faecalis revealed by CRISPR2 screening: eco-evolutionary insights into a novel subspecies.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0142825},
doi = {10.1128/spectrum.01428-25},
pmid = {40919784},
issn = {2165-0497},
abstract = {Enterococcus faecalis is a commensal bacterium that colonizes the gut of humans and animals and is a major opportunistic pathogen, known for causing multidrug-resistant healthcare-associated infections (HAIs). Its ability to thrive in diverse environments and disseminate antimicrobial resistance genes (ARGs) across ecological niches highlights the importance of understanding its ecological, evolutionary, and epidemiological dynamics. The CRISPR2 locus has been used as a valuable marker for assessing clonality and phylogenetic relationships in E. faecalis. In this study, we identified a group of E. faecalis strains lacking CRISPR2, forming a distinct, well-supported clade. We demonstrate that this clade meets the genomic criteria for classification as a novel subspecies, here referred to as "subspecies B." Through a comprehensive pangenome analysis and comparative genomics, we explored the adaptive ecological traits underlying this diversification process, identifying clade-specific features and their predicted functional roles. Our findings suggest that the frequent isolation of subspecies B from meat products and processing facilities may reflect dissemination routes involving environmental contamination (e.g., water, plants, soil) from avian species. The absence of key virulence traits required for pathogenicity in mammals, particularly humans, and the lack of clinically relevant resistance determinants indicate that subspecies B currently poses minimal threat to public health compared with the broadly disseminated "subspecies A." Nevertheless, the unclear potential for genetic exchange between these subspecies and the frequent association of subspecies B with food sources calls for continued genomic surveillance of E. faecalis from a One Health perspective to detect and mitigate the emergence of high-risk variants in advance.IMPORTANCEExploring intraspecific genetic variability in generalist bacteria with pathogenic potential, such as Enterococcus faecalis, is a key to uncovering stable evolutionary trends. By screening the CRISPR2 locus across a representative set of genomes from diverse sources, this study reveals a previously unrecognized lineage within the population structure of E. faecalis, associated with underexplored nonhuman and nonhospital reservoirs. These findings broaden our knowledge of the species' genetic landscape and shed light on its adaptive strategies and patterns of ecological dissemination. By bridging phylogenetic patterns with variation in genetic defense systems and accessory traits, the study generates testable hypotheses about the genomic determinants and corresponding selective pressures that shape the species' behavior and long-term dissemination. This work offers new perspectives on the eco-evolutionary dynamics of E. faecalis and highlights the value of genomic surveillance beyond clinical settings, in alignment with One Health principles.},
}

@article {pmid40845666,
year = {2026},
author = {Wang, M and Zhou, W and Wang, M and Zhang, K},
title = {A highly sensitive ECL biosensor for NF-κB p50 detection based on entropy-driven amplification and CRISPR/Cas12a signal enhancement.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {167},
number = {},
pages = {109081},
doi = {10.1016/j.bioelechem.2025.109081},
pmid = {40845666},
issn = {1878-562X},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *NF-kappa B p50 Subunit/analysis ; Humans ; *Entropy ; *Luminescent Measurements/methods ; Electrochemical Techniques/methods ; Limit of Detection ; Exodeoxyribonucleases/metabolism ; Nucleic Acid Amplification Techniques/methods ; *CRISPR-Associated Proteins/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Transcription factors, particularly NF-κB p50, play crucial roles in regulating gene expression and are involved in several diseases such as cancer, autoimmune disorders, and chronic inflammation. The sensitive detection of NF-κB p50 is essential for clinical diagnostics and therapeutic monitoring. In this study, we present an electrochemiluminescence (ECL) biosensor designed for the highly sensitive and specific detection of NF-κB p50. The biosensor integrates entropy-driven amplification and CRISPR/Cas12a-based signal enhancement to detect trace amounts of NF-κB p50. Upon detection of NF-κB p50, a ternary complex forms with a double-stranded DNA (dsDNA) probe, which prevents subsequent cleavage by exonuclease III (Exo III) and inhibits the CRISPR/Cas12a system. In the absence of NF-κB p50, Exo III digestion triggers entropy-driven amplification, which activates CRISPR/Cas12a, leading to enhanced electrochemical signals. The ECL biosensor demonstrated a detection limit of 0.56 pM, high selectivity, and excellent reproducibility. Furthermore, the biosensor successfully detected NF-κB p50 in complex biological samples, such as HeLa cell lysates, showcasing its potential for clinical applications in disease diagnostics.},
}

*Biosensing Techniques/methods
*CRISPR-Cas Systems
*NF-kappa B p50 Subunit/analysis
Humans
*Entropy
*Luminescent Measurements/methods
Electrochemical Techniques/methods
Limit of Detection
Exodeoxyribonucleases/metabolism
Nucleic Acid Amplification Techniques/methods
*CRISPR-Associated Proteins/metabolism
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid40845664,
year = {2026},
author = {Zhou, Y and Lv, Z and Geng, Y and Liu, R},
title = {An electrochemical RNA aptasensor based on sheet-like α-Fe2O3/Fe3O4 magnetic nanocomposites and CRISPR/Cas13a system for supersensitive detection of osteopontin.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {167},
number = {},
pages = {109085},
doi = {10.1016/j.bioelechem.2025.109085},
pmid = {40845664},
issn = {1878-562X},
mesh = {*CRISPR-Cas Systems ; *Aptamers, Nucleotide/chemistry/genetics ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; *Nanocomposites/chemistry ; *Osteopontin/analysis/blood ; Limit of Detection ; Humans ; *Ferric Compounds/chemistry ; },
abstract = {Osteopontin (OPN) exhibits markedly elevated expression in malignant tumor tissues, rendering it a crucial tumor marker for cancer prevention and monitoring-underscoring the significance of its detection. This work proposed an electrochemical RNA aptasensor based on a novel sheet-like α-Fe2O3/Fe3O4 magnetic nanocomposites (MNCs) and CRISPR/Cas13a system to effectively detect OPN. The proposed aptasensor used the sheet-like α-Fe2O3/Fe3O4 MNCs as the conduction matrix and applied their magnetic property to accomplish self-assembly of the sensing element onto the electrode. The current change of this aptasensor depended on the activation degree of the CRISPR/Cas13a system, which correlated with the amount of OPN expression. Double-stranded RNA (Apt/Activator) was compelled disassembly due to the OPN's strong affinity of Apt, while single-stranded RNA (Activator) could be guided by crRNA to combine with Cas13a/crRNA and activated the enzymatic activity of Cas13a. Cas13a enzyme affected the current and electrical resistance by shearing the nucleic acid strands (Reporter) on the electrode, ultimately enabling the quantitative detection of OPN. The aptasensor demonstrated excellent selectivity, reproducibility, and stability, with the detection limit (LOD) of 0.33 pg·mL[-1] and the wide linear detection range of 1 pg·mL[-1] - 10 ng·mL[-1]. These results offer a novel idea for advancing tumor marker electrochemical biosensors.},
}

*CRISPR-Cas Systems
*Aptamers, Nucleotide/chemistry/genetics
*Biosensing Techniques/methods
*Electrochemical Techniques/methods
*Nanocomposites/chemistry
*Osteopontin/analysis/blood
Limit of Detection
Humans
*Ferric Compounds/chemistry

@article {pmid40757822,
year = {2025},
author = {Echavarria Galindo, M and Lai, Y},
title = {CRISPR-based genetic tools for the study of host-microbe interactions.},
journal = {Infection and immunity},
volume = {93},
number = {9},
pages = {e0051024},
doi = {10.1128/iai.00510-24},
pmid = {40757822},
issn = {1098-5522},
support = {26100423//Research Grants Council, University Grants Committee/ ; PF22-69790//Hong Kong PHD Fellowship Scheme/ ; R9829//Hong Kong University of Science and Technology (HKUST)/ ; },
mesh = {Humans ; Animals ; *CRISPR-Cas Systems ; *Host Microbial Interactions/genetics ; Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Microbiota/genetics ; *Host-Pathogen Interactions/genetics ; },
abstract = {CRISPR-based genetic tools have revolutionized our ability to interrogate and manipulate genes. These tools can be applied to both host and microbial cells, and their use can enhance our understanding of the dynamic nature of host-microbe interactions by uncovering their genetic underpinnings. As reviewed here, CRISPR-based tools are being used to explore the microbiome in an efficient, accurate, and high-throughput manner. By employing CRISPR screens, targeted genome editing, and recording systems to the study of host cells and microorganisms, we can gain critical insights into host defense mechanisms, potential vulnerabilities, and microbial pathogenesis, as well as essential or condition-specific genes involved in host-microbe interactions. Additionally, CRISPR-based genetic tools are being used in animal models to study host-microbe interactions in vivo. Recent advancements in CRISPR-derived technology can be combined with emerging techniques, such as single-cell RNA sequencing, to examine the complex interactions between hosts and microbes, shedding light on the role of the microbiome in health and disease. This review aims to provide a comprehensive overview of how these cutting-edge genetic tools are being used to investigate host-microbial systems, as well as their current limitations. Current research is likely to yield even more advanced genetic toolkits than those presently available, and these can serve researchers in identifying and exploring new therapeutic targets for diseases related to host-microbe interactions.},
}

Humans
Animals
*CRISPR-Cas Systems
*Host Microbial Interactions/genetics
Gene Editing/methods
*Clustered Regularly Interspaced Short Palindromic Repeats
*Microbiota/genetics
*Host-Pathogen Interactions/genetics

@article {pmid40749504,
year = {2026},
author = {Ji, Z and Cheng, S and Li, W and Xing, Y and Tang, Z and Zhu, X and Wang, D and Hao, C and Wang, B and Shi, M},
title = {Ultrasensitive detection of miR-31 using a signal-on electrochemiluminescence biosensor based on CRISPR/Cas12a and MXene nanocomposites.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {167},
number = {},
pages = {109059},
doi = {10.1016/j.bioelechem.2025.109059},
pmid = {40749504},
issn = {1878-562X},
mesh = {*MicroRNAs/analysis/blood/genetics ; *Biosensing Techniques/methods ; Humans ; *Nanocomposites/chemistry ; *CRISPR-Cas Systems ; *Electrochemical Techniques/methods ; Limit of Detection ; *Luminescent Measurements/methods ; *Endodeoxyribonucleases/metabolism ; *CRISPR-Associated Proteins/metabolism ; Gold/chemistry ; Electrodes ; Metallocenes ; Bacterial Proteins ; Nitrites ; Transition Elements ; },
abstract = {MicroRNAs (miRNAs) have emerged as critical biomarkers for early cancer diagnosis due to their high specificity and stability in biological fluids. In this study, we report a highly sensitive and specific electrochemiluminescence (ECL) biosensor for the detection of miR-31, a miRNA closely associated with non-small cell lung cancer. The sensing platform integrates a PEI-Ru@Ti3C2@AuNPs-modified electrode with a ferrocene-labeled DNA probe (DNA1-Fc) to construct a target-responsive signal-on system. Upon recognition of miR-31, an isothermal strand displacement amplification (ISDA) reaction is initiated, producing abundant double-stranded DNA (dsDNA) that activates the CRISPR/Cas12a complex. The trans-cleavage activity of Cas12a then cleaves the DNA1-Fc probes on the electrode surface, removing the quenching ferrocene moiety and restoring the Ru-based ECL signal. Under optimized conditions, the biosensor exhibited a wide dynamic range from 10 aM to 100 pM and a remarkably low detection limit of 1.67 aM. The system also showed excellent specificity against homologous miRNAs, and its applicability was successfully validated in spiked human serum samples, achieving high recovery and reproducibility. The synergistic combination of nanomaterial-enhanced ECL emission, isothermal nucleic acid amplification, and CRISPR-based enzymatic cleavage provides a powerful strategy for ultrasensitive nucleic acid detection. This work offers a promising approach for early cancer diagnosis and has great potential for clinical translation and point-of-care testing.},
}

*MicroRNAs/analysis/blood/genetics
*Biosensing Techniques/methods
Humans
*Nanocomposites/chemistry
*CRISPR-Cas Systems
*Electrochemical Techniques/methods
Limit of Detection
*Luminescent Measurements/methods
*Endodeoxyribonucleases/metabolism
*CRISPR-Associated Proteins/metabolism
Gold/chemistry
Electrodes
Metallocenes
Bacterial Proteins
Nitrites
Transition Elements

@article {pmid40719649,
year = {2025},
author = {Chen, M and Choi, HK and Goldston, LL and Hou, Y and Jiang, C and Lee, KB},
title = {Advanced Cancer Liquid Biopsy Platform for miRNA Detection in Extracellular Vesicles Using CRISPR/Cas13a and Gold Nanoarrays.},
journal = {ACS nano},
volume = {19},
number = {35},
pages = {31438-31456},
doi = {10.1021/acsnano.5c06940},
pmid = {40719649},
issn = {1936-086X},
mesh = {*MicroRNAs/genetics/analysis/blood ; Humans ; *Extracellular Vesicles/chemistry/metabolism/genetics ; *Gold/chemistry ; *CRISPR-Cas Systems ; Liquid Biopsy/methods ; *Colorectal Neoplasms/diagnosis/genetics ; Biomarkers, Tumor/genetics/blood ; Epithelial Cell Adhesion Molecule/metabolism ; Cell Line, Tumor ; Metal Nanoparticles/chemistry ; },
abstract = {Liquid biopsy is a transformative, noninvasive tool for cancer diagnosis and monitoring, with the potential to revolutionize personalized medicine. In this study, we introduce an advanced liquid biopsy platform for highly sensitive and selective detection of extracellular vesicle (EV) microRNAs (miRNA-21 and miRNA-23a) as biomarkers for colorectal cancer. The platform combines two innovations: (1) gold nanoarrays with epithelial cell adhesion molecule (EpCAM)-specific aptamers to selectively isolate tumor-derived EVs from plasma and (2) CRISPR/Cas13a-encapsulated liposomes that fuse with EVs to form nanoscale reactors. Upon fusion, the CRISPR/Cas13a complex is activated by target miRNA, triggering trans-cleavage of RNA reporters and generating an amplified fluorescence signal for enhanced detection sensitivity. The assay achieves a linear detection range of 10 to 10[6] EVs/μL and a detection limit of 2.5 × 10[1] EVs/μL on the gold nanoarray. Its performance relies on three strategies: (i) EpCAM-mediated tumor EV enrichment, (ii) CRISPR/Cas13a-based collateral activity for ultrasensitive miRNA detection, and (iii) fluorescence signal enhancement via localized nanoreactors. Validation with a 2D SW480 cell model, a 3D vascularized tumor spheroid (VTS) model, and clinical plasma samples confirms diagnostic accuracy, with miRNA quantification comparable to RT-qPCR but without the need for labor-intensive RNA extraction and amplification. By integrating nanotechnology with CRISPR-based diagnostics, this platform bridges research and clinical translation, improving diagnostic precision and streamlining workflows. Future development will focus on multiplexed biomarker detection and single-EV analysis to reveal insights into EV heterogeneity and function in cancer. This technology supports the application in precision oncology, offering a tool for early detection, treatment monitoring, and therapeutic decision-making.},
}

*MicroRNAs/genetics/analysis/blood
Humans
*Extracellular Vesicles/chemistry/metabolism/genetics
*Gold/chemistry
*CRISPR-Cas Systems
Liquid Biopsy/methods
*Colorectal Neoplasms/diagnosis/genetics
Biomarkers, Tumor/genetics/blood
Epithelial Cell Adhesion Molecule/metabolism
Cell Line, Tumor
Metal Nanoparticles/chemistry

@article {pmid40653285,
year = {2025},
author = {Monteiro, JP and Vacante, F and De Pace, AL and Bennett, M and Rodor, J and O'Carroll, D and Wu, JC and Quertermous, T and Baker, AH},
title = {Targeting Cis-regulatory elements for CRISPR-mediated transcriptional activation of the human MIR503HG locus.},
journal = {Vascular pharmacology},
volume = {160},
number = {},
pages = {107521},
doi = {10.1016/j.vph.2025.107521},
pmid = {40653285},
issn = {1879-3649},
mesh = {Humans ; *MicroRNAs/genetics/metabolism ; *Transcriptional Activation ; *CRISPR-Cas Systems ; *RNA, Long Noncoding/genetics/metabolism ; Lentivirus/genetics ; HEK293 Cells ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Vectors ; *Gene Editing/methods ; *Regulatory Elements, Transcriptional ; Human Umbilical Vein Endothelial Cells/metabolism ; },
abstract = {Advances in genome annotation have revealed a striking increase in the number and complexity of non-coding RNA (ncRNA) genes, particularly multi-transcript loci that harbor long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) within the same genomic region. These loci can often function as coordinated regulatory units, with their transcription modulated by shared cis-acting regulatory elements (CREs). Traditional gene manipulation approaches, which typically target individual transcripts, are insufficient to capture the full regulatory and therapeutic potential of these complex loci. Here, we present "cis-ON" a single-vector lentiviral delivery system based on CRISPR activation (CRISPRa), designed to simultaneously upregulate multiple noncoding RNA transcripts by targeting a single CRE. We focused on the evolutionarily conserved MIR503HG locus, which encodes seven lncRNA isoforms and hosts the miR-424/503 cluster, both implicated in various cellular processes, including proliferation, angiogenesis, and endothelial-to-mesenchymal transition. Using integrative analysis of histone marks (H3K27Ac, H3K4Me3), DNase hypersensitivity, and CAGE-seq data, we identified the primary promoter of the MIR503HG locus. A dual fluorescent reporter screen selected optimal single guide RNAs (sgRNAs) for targeting this region. We then engineered cis-ON, a novel lentiviral system combining dCas9-VPR and sgRNA to enable a streamlined single-vector delivery approach. Transduction of primary human endothelial cells with this system robustly activated the entire locus including the MIR503HG isoforms and co-embedded miRNAs miR-424 and miR-503, demonstrating coordinated transcriptional regulation. Additionally, the neighboring LINC00629 lncRNA locus remained unaffected, highlighting regulatory specificity. This approach demonstrates the feasibility of modulating complex ncRNA loci across a ∼ 10 kb genomic region by targeting a single CRE, bypassing limitations of transcriptspecific strategies. By enabling simultaneous upregulation of lncRNAs and miRNAs, the cis-ON platform provides a streamlined strategy for restoring regulatory networks disrupted in disease.},
}

Humans
*MicroRNAs/genetics/metabolism
*Transcriptional Activation
*CRISPR-Cas Systems
*RNA, Long Noncoding/genetics/metabolism
Lentivirus/genetics
HEK293 Cells
*Clustered Regularly Interspaced Short Palindromic Repeats
Genetic Vectors
*Gene Editing/methods
*Regulatory Elements, Transcriptional
Human Umbilical Vein Endothelial Cells/metabolism

@article {pmid39992014,
year = {2025},
author = {Chen, RH and Bai, Y and Shi, LD and Liu, XY and Li, DL and Di, M and Duan, JX and Zhang, ZH and Xu, JL and He, ZQ and Li, K},
title = {Functional exploration of the Sex combs reduced gene in Gryllus bimaculatus.},
journal = {Insect molecular biology},
volume = {34},
number = {5},
pages = {608-618},
doi = {10.1111/imb.12987},
pmid = {39992014},
issn = {1365-2583},
mesh = {Animals ; *Insect Proteins/genetics/metabolism ; Female ; CRISPR-Cas Systems ; Male ; Gene Knockout Techniques ; *Homeodomain Proteins/genetics/metabolism ; },
abstract = {The Hox gene Sex combs reduced (Scr) is recognized as a key factor in the development of the head and thorax in insects. However, its function in the growth, development and morphogenesis of Gryllus bimaculatus remains poorly understood. This study aimed to explore the function of the Scr gene in G. bimaculatus by using CRISPR/Cas9 technology to generate an Scr gene knock-out strain. Intercrossing the G0 generation knock-out individuals with wild-type individuals yielded the G1 generation to screen the mutant strain. It was found that the knock-out of the Scr gene had a severe impact on the growth and development of G. bimaculatus, resulting in high mortality and making it difficult to obtain Scr[-/-] mutants. Therefore, heterozygous individuals (Scr[+/-]) with 1 bp deleted were obtained for investigation. The results showed that the Scr deletion led to ectopic segment formation in the G0 generation. In the G2 generation, it was observed that stable Scr[-/-] strains displayed abnormal embryonic development, characterized by enlarged, blackened and lethal eggs during embryogenesis. During the post-embryonic stage, Scr[-/-] mutants exhibited abnormalities in body segmentation, particularly in the head-thorax region, resulting in a dorsal ridge structure. Furthermore, some Scr[+/-] individuals exhibited a dorsal ridge during the nymphal stage. Notably, this characteristic did not persist into the adult stage. Our findings highlight the distinct but crucial roles of the Scr gene in both embryonic and post-embryonic growth and development of G. bimaculatus.},
}

Animals
*Insect Proteins/genetics/metabolism
Female
CRISPR-Cas Systems
Male
Gene Knockout Techniques
*Homeodomain Proteins/genetics/metabolism

@article {pmid40918426,
year = {2025},
author = {Senbadejo, TY and Ntiamoah Osei, S and Isawumi, A},
title = {Snapshot of Defense Systems in Multidrug Resistant Klebsiella pneumoniae.},
journal = {microPublication biology},
volume = {2025},
number = {},
pages = {},
pmid = {40918426},
issn = {2578-9430},
abstract = {Bacterial defense mechanisms protect pathogens from host immunity, bacteriophages, and harsh environments. This study investigates defense systems in multidrug-resistant Klebsiella pneumoniae from Ghanaian hospital ICUs, focusing on CRISPR-Cas, restriction-modification (R-M), and toxin-antitoxin (TA) systems. Genomes of environmental (NS2) and clinical (PS4) strains were sequenced and analyzed using PADLOC, defensefinder, and TADB3.0. NS2 carries 12 defense systems, including CRISPR-Cas, while PS4 has five. Both possess diverse RM and TA systems. These strains, resistant to six antibiotic classes, encode clinically significant defense systems, suggesting microbial exchange between fomites and humans, potentially increasing infection risks in ICU environments requiring targeted surveillance.},
}

@article {pmid40918304,
year = {2025},
author = {Dash, B and Bhuyan, SS and Sahoo, RK and Swain, N and Jeughale, KP and Sarkar, S and Verma, RL and Parameswaran, C and Devanna, BN and Samantaray, S},
title = {CRISPR/Cas-mediated genome editing: playing a versatile role in mitigating the challenges of sustainable rice improvement.},
journal = {3 Biotech},
volume = {15},
number = {10},
pages = {327},
pmid = {40918304},
issn = {2190-572X},
abstract = {Just as Gregor Mendel's laws of inheritance laid the foundation for modern genetics, the emergence of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas systems has catalyzed a new era in precision genome engineering. CRISPR/Cas has revolutionized rice (Oryza sativa L.) breeding by enabling precise, transgene-free edits to improve yield, nutrition, and stress tolerance. Advanced tools like base and prime editing further refine these capabilities, offering powerful solutions for climate-resilient agriculture and global food security. The review synthesizes the CRISPR-mediated strategies for improving resistance against major biotic (bacterial blight, blast, sheath blight) and abiotic (drought, salinity, submergence, nutrient deficiency) stresses. Additionally, we explore the critical prerequisites for efficient genome editing in rice, ranging from target site design, PAM specificity, delivery systems (like Agrobacterium, RNPs, and nanoparticle-mediated delivery), to screening and validation of mutants. This review also highlights recent breakthroughs in multiplex genome editing for complex traits, including the development of haploid inducer lines and clonal seed technology. Haploid inducers accelerate breeding by producing homozygous lines without tissue culture, while engineered apomixis enables clonal propagation of elite hybrids. Beyond technical dimensions, this review underscores the broader socio-economic and regulatory implications of genome-edited rice, addressing the emerging ethical concerns, intellectual property issues, farmer access, and equitable technology dissemination in resource-limited agricultural regions. As the global policy landscape transitions to accommodate CRISPR-edited crops, transparent regulatory frameworks, stakeholder engagement, and public perception will play pivotal roles in ensuring sustainable, safe, and inclusive adoption of genome editing in agriculture.},
}

@article {pmid40918251,
year = {2025},
author = {Zu, H and Sun, R and Li, J and Guo, X and Wang, M and Guo, W and Wang, X},
title = {Integrated CRISPR-Cas12a and RAA one-pot visual strategy for the rapid identification of Streptococcus equi subspecies equi.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1526516},
pmid = {40918251},
issn = {2235-2988},
mesh = {*Streptococcus equi/genetics/isolation & purification/classification ; Horses ; *CRISPR-Cas Systems ; Animals ; *Streptococcal Infections/veterinary/diagnosis/microbiology ; *Horse Diseases/diagnosis/microbiology ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases/metabolism/genetics ; *Molecular Diagnostic Techniques/methods ; Iceland ; Bacterial Proteins/genetics ; Endodeoxyribonucleases ; Streptococcus ; CRISPR-Associated Proteins ; },
abstract = {Strangles, a highly contagious disease caused by Streptococcus equi subspecies equi (S.equi), significantly impacts horse populations worldwide, with Iceland as the only exception. This disease poses serious threats to equine health and results in considerable economic losses. Consequently, the accurate, sensitive, and rapid detection of S.equi from clinical samples is essential for early warning and effective disease management. This study introduces a novel detection method that integrates recombinase-aided amplification (RAA) with CRISPR/Cas12a technologies. We specifically designed RAA primers and CRISPR RNA to target the eqbE gene of S.equi, and we have carefully optimized the reaction systems for this purpose. The newly established visual diagnostic method has shown to be highly effective, demonstrating 97.14% specificity and 100% sensitivity, with the capability to detect as few as 5.6×10[0] copies of the target. This is the first study to propose the combined application of RAA and CRISPR/Cas12a for the on-site rapid detection of S.equi. This is the first study to propose the combined application of RAA and CRISPR/Cas12a for the on-site rapid detection of S.equi, which enables visual point-of-care diagnosis of Strangles.},
}

*Streptococcus equi/genetics/isolation & purification/classification
Horses
*CRISPR-Cas Systems
Animals
*Streptococcal Infections/veterinary/diagnosis/microbiology
*Horse Diseases/diagnosis/microbiology
Sensitivity and Specificity
*Nucleic Acid Amplification Techniques/methods
*Recombinases/metabolism/genetics
*Molecular Diagnostic Techniques/methods
Iceland
Bacterial Proteins/genetics
Endodeoxyribonucleases
Streptococcus
CRISPR-Associated Proteins

@article {pmid40918142,
year = {2025},
author = {Golinelli, G and Scholler, J and Roussel-Gervais, A and Šakić, A and Ilmjärv, S and Song, D and Gabunia, K and Ji, M and Fan, TJ and Gupta, A and Deshmukh, M and Berjis, A and Cuoghi Costantini, R and Apodaca, K and Sheppard, NC and Kili, S and Dominici, M and Alessandrini, M and June, CH and Levine, BL},
title = {Multiplex engineering using microRNA-mediated gene silencing in CAR T cells.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1647433},
pmid = {40918142},
issn = {1664-3224},
mesh = {*MicroRNAs/genetics ; Animals ; Humans ; *Receptors, Chimeric Antigen/genetics/immunology ; *Immunotherapy, Adoptive/methods ; Mesothelin ; *Gene Silencing ; Mice ; *T-Lymphocytes/immunology/metabolism ; Cell Line, Tumor ; Gene Editing ; CRISPR-Cas Systems ; Receptors, Antigen, T-Cell/genetics ; *Pancreatic Neoplasms/therapy/immunology/genetics ; Xenograft Model Antitumor Assays ; GPI-Linked Proteins/genetics/immunology ; },
abstract = {BACKGROUND: Multiplex gene-edited chimeric antigen receptor (CAR) T-cell therapies face significant challenges, including potential oncogenic risks associated with double-strand DNA breaks. Targeted microRNAs (miRNAs) may provide a safer, functional, and tunable alternative for gene silencing without the need for DNA editing.

METHODS: As a proof of concept for multiplex gene silencing, we employed an optimized miRNA backbone and gene architecture to silence T-cell receptor (TCR) and major histocompatibility complex class I (MHC-I) in mesothelin-directed CAR (M5CAR) T cells. The efficacy of this approach was compared to CD3ζ and β2-microglobulin (β2M) CRISPR/Cas9 knockout (KO) cells. miRNA-expressing cassettes were incorporated into M5CAR lentiviral vectors, enabling combined gene silencing and CAR expression. Antitumor activity was evaluated using in vitro assays and in vivo pancreatic ductal adenocarcinoma models.

RESULTS: Silenced (S) M5CAR T cells retained antitumor functionality comparable to, and in some cases exceeding, that of KO cells. In vivo, S M5CAR T cells achieved tumor control with higher persistence and superior metastasis prevention. In vitro assays demonstrated enhanced resistance to alloreactive natural killer (NK) cells and peripheral blood mononuclear cells (PBMCs).

CONCLUSIONS: Titratable multiplex gene silencing via targeted miRNAs offers an alternative to gene editing for CAR T cells, with potential advantages in potency, persistence, metastasis prevention, and immune evasion for allogeneic products. This strategy may overcome tumor-induced immunosuppression while avoiding the risks associated with DNA double-strand breaks.},
}

*MicroRNAs/genetics
Animals
Humans
*Receptors, Chimeric Antigen/genetics/immunology
*Immunotherapy, Adoptive/methods
Mesothelin
*Gene Silencing
Mice
*T-Lymphocytes/immunology/metabolism
Cell Line, Tumor
Gene Editing
CRISPR-Cas Systems
Receptors, Antigen, T-Cell/genetics
*Pancreatic Neoplasms/therapy/immunology/genetics
Xenograft Model Antitumor Assays
GPI-Linked Proteins/genetics/immunology

@article {pmid40916896,
year = {2025},
author = {Courtier-Orgogozo, V},
title = {Risks associated with CRISPR homing gene drive.},
journal = {Comptes rendus biologies},
volume = {348},
number = {},
pages = {211-227},
doi = {10.5802/crbiol.182},
pmid = {40916896},
issn = {1768-3238},
mesh = {*Gene Drive Technology/adverse effects ; Humans ; Animals ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Ecosystem ; Gene Editing ; },
abstract = {CRISPR homing gene drive is a disruptive biotechnology developed over the past decade with potential applications in public health, agriculture, and conservation biology. This technology relies on an autonomous selfish genetic element able to spread in natural populations through the release of gene drive individuals. However, it has not yet been deployed in the wild. In this review, we examine the key risks associated with CRISPR homing gene drives. First, we explore technical limitations, where gene drives might not be as efficient as intended, and cases where mitigation strategies may not be able to block a gene drive. Then, we present four main categories of adverse effects: (a) ecological risks, corresponding to unintended consequences on ecosystems and non-target populations; (b) sociological risks, i.e. concerns over public perception, governance, and societal acceptance; (c) risks associated with research activities; and (d) risks associated with malevolent usage. Regulatory aspects are not addressed here. This article provides a foundation for evaluating gene drive risks to ensure responsible and informed decision-making.},
}

*Gene Drive Technology/adverse effects
Humans
Animals
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Ecosystem
Gene Editing

@article {pmid40916704,
year = {2025},
author = {Ficoseco, CMA and Chieffi, D and Montemurro, M and Bavaro, A and Rizzello, CG and Nader-Macias, MEF and Fadda, S and Fanelli, F and Fusco, V and Vignolo, GM},
title = {Genomic Characterisation of Limosilactobacillus fermentum CRL2085 Unveiling Probiotic Traits for Application in Cattle Feed.},
journal = {Environmental microbiology reports},
volume = {17},
number = {5},
pages = {e70176},
doi = {10.1111/1758-2229.70176},
pmid = {40916704},
issn = {1758-2229},
support = {//This work was financially supported by the Joint Bilateral Agreement CNR/CONICET (ITALY-Argentina) "Lactic Acid Bacteria as bioprotective agents against zoonotic pathogens in the meat chain" Biennial Programme 2023-2024/ ; },
mesh = {Animals ; *Probiotics ; Cattle ; *Limosilactobacillus fermentum/genetics/isolation & purification/metabolism ; *Animal Feed/microbiology ; *Genome, Bacterial ; Genomics ; Genomic Islands ; Bacterial Adhesion ; },
abstract = {Limosilactobacillus fermentum CRL2085, isolated from feedlot cattle rations, displayed high efficiency as a probiotic when administered to animals. A comprehensive genomic analysis was performed to elucidate the genetic basis underlying its probiotic potential. Fifteen genomic islands and CRISPR-Cas elements were identified in its genome. Pan-genomic analysis highlighted the dynamic evolution of this species, and clustering based on the nucleotide genomic similarity only partially correlated with the source of isolation or the geographic origin of the strains. Several genes known to confer probiotic properties were identified, including those related to adhesion, resistance to acidic pH and bile salts, tolerance to oxidative stress, metabolism/transport of sugars and other compounds, and genes for exopolysaccharide biosynthesis. In silico analysis of antimicrobial resistance genes and virulence determinants confirmed the safety of this strain. Moreover, genes related to B-group vitamins biosynthesis and feruloyl esterase hydrolase were also found, showing the nutritional contribution of the strain, which also showed moderate adhesion capability, exopolysaccharide production when grown with sucrose, and the capacity to metabolise 42 out of 95 carbon substrates tested. This data provides the genetic basis for deciphering the mechanisms beyond the benefits demonstrated by its use during cattle intensive raising and confirms its promising role as a probiotic.},
}

Animals
*Probiotics
Cattle
*Limosilactobacillus fermentum/genetics/isolation & purification/metabolism
*Animal Feed/microbiology
*Genome, Bacterial
Genomics
Genomic Islands
Bacterial Adhesion

@article {pmid40914328,
year = {2025},
author = {Rathored, J and Budhbaware, T},
title = {Integrative Strategies Against Multidrug-Resistant Bacteria: Synthesizing Novel Antimicrobial Frontiers for Global Health.},
journal = {Microbial pathogenesis},
volume = {},
number = {},
pages = {108018},
doi = {10.1016/j.micpath.2025.108018},
pmid = {40914328},
issn = {1096-1208},
abstract = {Concerningly, multidrug-resistant bacteria have emerged as a prime worldwide trouble, obstructing the treatment of infectious diseases and causing doubts about the therapeutic accidentalness of presently existing drugs. Novel antimicrobial interventions deserve development as conventional antibiotics are incapable of keeping pace with bacteria evolution. Various promising approaches to combat MDR infections are discussed in this review. Antimicrobial peptides are examined for their broad-spectrum efficacy and reduced ability to develop resistance, while phage therapy may be used under extreme situations when antibiotics fail. In addition, the possibility of CRISPR-Cas systems for specifically targeting and eradicating resistance genes from bacterial populations will be explored. Nanotechnology has opened up the route to improve the delivery system of the drug itself, increasing the efficacy and specificity of antimicrobial action while protecting its host. Discovering potential antimicrobial agents is an exciting prospect through developments in synthetic biology and the rediscovery of natural product-based medicines. Moreover, host-directed therapies are now becoming popular as an adjunct to the main strategies of therapeutics without specifically targeting pathogens. Although these developments appear impressive, questions about production scaling, regulatory approvals, safety, and efficacy for clinical employment still loom large. Thus, tackling the MDR burden requires a multi-pronged plan, integrating newer treatment modalities with existing antibiotic regimens, enforcing robust stewardship initiatives, and effecting policy changes at the global level. The international health community can gird itself against the growing menace of antibiotic resistance if collaboration between interdisciplinary bodies and sustained research endeavours is encouraged. In this study, we evaluate the synergistic potential of combining various medicines in addition to summarizing recent advancements. To rethink antimicrobial stewardship in the future, we provide a multi-tiered paradigm that combines pathogen-focused and host-directed strategies.},
}

@article {pmid40912804,
year = {2025},
author = {Qiu, Y and Li, X and Fan, M and Tang, H and Zhang, S and Huang, W and Han, Z and Wang, S and Peng, H and Xiao, Y and Ye, X and Wang, K},
title = {Modification of starch traits in commercial wheat through TaWaxy gene editing.},
journal = {Carbohydrate polymers},
volume = {368},
number = {Pt 1},
pages = {124105},
doi = {10.1016/j.carbpol.2025.124105},
pmid = {40912804},
issn = {1879-1344},
mesh = {*Triticum/genetics/metabolism/chemistry ; *Gene Editing/methods ; Amylose/genetics ; *Starch/genetics/metabolism/chemistry ; *Plant Proteins/genetics/metabolism ; Mutation ; CRISPR-Cas Systems ; Plants, Genetically Modified ; },
abstract = {Amylose content (AC) is a key determinant of wheat quality, and the TaWaxy gene determined amylose synthesis with a dose-dependent effect on AC. In this study, the TaWOX5 gene, which significantly enhances wheat transformation efficiency, was combined with CRISPR/SpCas9 system to generate TaWaxy mutants in a commercial winter wheat Jimai 22. Seven transgene-free mutant types were produced, compared to only three transgene-free mutants in the spring wheat variety Ningchun 4. The TaWaxy mutants from the two varieties showed decreased ACs ranging from 0 to 19.05 %. Results demonstrated that the Waxy-B1 protein has the most significant effect on amylose synthesis. The mutants with TaWaxy-abd, TaWaxy-ab, and TaWaxy-bd alleles showed waxy wheat trait. Interestingly, the TaWaxy-b mutant from Jimai 22 exhibited a waxy trait, unlike the TaWaxy-b mutant from Ningchun 4. Transmission electron microscope and scanning electron microscopy showed increased B-type starch granules in mutant grains. The mutants displayed varying effects on bread, cake, cookie, and noodle quality. All mutants showed decreased quality in bread and cake production, while TaWaxy-ad-JM and TaWaxy-b-NC mutants showed improved noodle and cookie quality. The generated mutants provide optimized optimized amylose content, enhancing noodle and biscuit quality as a practical alternative to blending.},
}

*Triticum/genetics/metabolism/chemistry
*Gene Editing/methods
Amylose/genetics
*Starch/genetics/metabolism/chemistry
*Plant Proteins/genetics/metabolism
Mutation
CRISPR-Cas Systems
Plants, Genetically Modified

@article {pmid40912763,
year = {2025},
author = {Joshi, R and Paliwal, T and Sharma, S and Kapoor, DU and Prajapati, BG},
title = {Chitosan polyplexes for targeted gene delivery: From mechanisms to clinical applications.},
journal = {Carbohydrate polymers},
volume = {368},
number = {Pt 1},
pages = {124080},
doi = {10.1016/j.carbpol.2025.124080},
pmid = {40912763},
issn = {1879-1344},
mesh = {*Chitosan/chemistry ; Humans ; *Gene Transfer Techniques ; Animals ; Genetic Therapy/methods ; DNA/chemistry ; },
abstract = {As a diverse natural polymer called Chitosan, it created ground-breaking advancements in nucleic acid therapeutic delivery techniques for handling essential DNA and RNA delivery hurdles. The article investigates how nucleic acids form stable polyplexes with chitosan through electrostatic bonds, as well as explores their chemical and biological properties. The review explores how molecular weight, combined with the degree of deacetylation, combined with advanced functionalization strategies, help enhance delivery results. Cellular uptake and specific targeting of polyplexes are improved through research into different targeting methods, which include ligand attachment, charge manipulation, and cell-penetrating peptides. Endosomal release of polyplexes is enabled by pH-sensitive and redox-sensitive polyplex architectures, while simultaneous control strategies are achieved through these delivery systems. The field of nucleic acid therapy now includes applications with CRISPR-Cas delivery and mRNA vaccines, and gene silencing therapy. The safety assessment encompasses cytotoxicity testing alongside immunogenicity evaluation and biodegradability assessment, which is conducted alongside direct comparison with alternative polymeric delivery vehicles. The potential for clinical use and targeted delivery, and combined smart transport systems establishes chitosan polyplexes as an innovative solution for nucleic acid drug delivery according to recent research findings.},
}

*Chitosan/chemistry
Humans
*Gene Transfer Techniques
Animals
Genetic Therapy/methods
DNA/chemistry

@article {pmid40911134,
year = {2025},
author = {Jiang, C and Li, Y and Yu, P and Fang, M and Huang, D and Fang, X and Xu, Z},
title = {A rapid and ultrasensitive CRISPR/Cas12a-based assay for the accurate identification of T-even type phages.},
journal = {Biotechnology letters},
volume = {47},
number = {5},
pages = {95},
pmid = {40911134},
issn = {1573-6776},
support = {2021YFC2103200//the National Key Research and Development Program of China/ ; 22404149//the National Natural Science Foundation of China/ ; 2023M742994//the China Postdoctoral Science Foundation/ ; GZC20232319//the Postdoctoral Fellowship Program of CPSF/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *T-Phages/genetics/isolation & purification/classification ; Limit of Detection ; Wastewater/virology ; },
abstract = {Phage contamination poses a significant threat to industrial fermentation, leading to substantial economic losses. Virulent T-even type phages (T2/T4/T6) represent particularly concerning biological hazards in fermentation systems. This paper developed a novel CRISPR/Cas12a-based system integrated with recombinase polymerase amplification (RPA), enabling ultrasensitive identification of T-even type phages. This method targeted the TerL gene of T-even type phages as a detection marker. The optimized RPA-CRISPR assay demonstrated exceptional sensitivity with a limit of detection (LOD) reaching 1 aM for synthetic targets. Besides, this system achieved detection thresholds of 1 and 10 PFU/μL for T2 and T4 phages, respectively. Comparative validation with quantitative PCR (qPCR) confirmed the method's reliability through strong correlation in the detection for both spiked and wastewater samples. The detection platform exhibited remarkable potential for rapid, sensitive monitoring of T-even type phages contamination in fermentation processes, offering promising application prospects for quality control in biochemical industries.},
}

*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
*T-Phages/genetics/isolation & purification/classification
Limit of Detection
Wastewater/virology

@article {pmid40904109,
year = {2025},
author = {Clabby, T and Tesson, F and Gaborieau, B and Bernheim, A},
title = {Why do bacteria accumulate antiphage defence systems?.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {380},
number = {1934},
pages = {20240082},
pmid = {40904109},
issn = {1471-2970},
support = {//MSDAVENIR/ ; //Pasteur Institute/ ; /ERC_/European Research Council/International ; },
mesh = {*Bacteria/classification/genetics/immunology/virology ; *Bacteriophages/genetics/physiology ; Ecology ; CRISPR-Cas Systems ; Gene Transfer, Horizontal ; Interspersed Repetitive Sequences ; *Phage Therapy ; },
abstract = {While it is well established that bacterial genomes encode multiple and diverse antiphage systems, the reasons for their co-occurrence and their heterogeneous distribution remain debated. This review examines why bacteria accumulate antiphage systems and how this influences phage-bacteria interactions, particularly in the context of phage therapy. Two main hypotheses may explain this phenomenon: (i) the pan-immunity hypothesis, which suggests that defence system accumulation provides protection against phage predation at the community level, and (ii) mobile genetic element (MGE) competition, where defence systems primarily protect intra-bacterial MGEs against other ones rather than the bacterial host itself. The ecological context also influences the distribution of antiphage systems, with defencee accumulation shaping phage-bacteria interactions in diverse communities but playing a lesser role at the species level, potentially explaining why multiple defences do not strongly limit phage host range in therapeutic settings. Finally, we address the challenges in understanding the drivers shaping the distribution of defence systems across bacterial genomes (expressions, costs, etc.) and their implications for elucidating the ecological role of defence systems and optimizing phage therapy strategies.This article is part of the discussion meeting issue 'The ecology and evolution of bacterial immune systems'.},
}

*Bacteria/classification/genetics/immunology/virology
*Bacteriophages/genetics/physiology
Ecology
CRISPR-Cas Systems
Gene Transfer, Horizontal
Interspersed Repetitive Sequences
*Phage Therapy

@article {pmid40889469,
year = {2025},
author = {Li, Y and Dou, Y and Lu, Z and Wang, Y and Zhou, H and Li, T},
title = {CRISPR/Cas12a-functionalized silicon nanowires field-effect transistor sensor for ultra-sensitive detection of pathogen nucleic acids.},
journal = {Biosensors & bioelectronics},
volume = {289},
number = {},
pages = {117936},
doi = {10.1016/j.bios.2025.117936},
pmid = {40889469},
issn = {1873-4235},
mesh = {*Biosensing Techniques/instrumentation ; *Nanowires/chemistry/ultrastructure ; *Silicon/chemistry ; *CRISPR-Cas Systems/genetics ; Transistors, Electronic ; *Bacillus anthracis/genetics/isolation & purification/pathogenicity ; Limit of Detection ; *DNA, Bacterial/genetics/isolation & purification/analysis ; *Endodeoxyribonucleases/chemistry ; Bacterial Proteins/chemistry ; CRISPR-Associated Proteins ; },
abstract = {Rapid, sensitive, and accurate detection of pathogen nucleic acids is critical for ensuring public safety and health. Nevertheless, current methods still encounter significant challenges. Field-effect transistor (FET) biosensors are renowned for high sensitivity, rapid response, and label-free detection. However, when employed for the direct detection of long-chain DNA extracted from pathogens, these sensors exhibit low recognition efficiency, poor accuracy, and prolonged reaction times. To address these limitations, we propose a novel silicon nanowires FET sensing strategy functionalized with the CRISPR/Cas12a system. The Cas12a/crRNA complex rapidly scans and precisely cleaves target sequences within long double-stranded DNA (dsDNA). This mechanism effectively mitigates detection performance degradation caused by nucleic acid folding and entanglement, thereby significantly enhancing both sensitivity and accuracy. Additionally, Cas12a/crRNA cleaves long dsDNA into specific-length fragments, thereby ensuring their distribution within the Debye length and enhancing signal consistency. Using this approach, we successfully achieved quantitative detection of Bacillus anthracis dsDNA within 10 min, with a detection limit at the attomolar (aM) level. Furthermore, the correlation coefficient between detection results of real whole-genome samples and digital PCR reached 0.912, validating the reliability of this strategy. In summary, this strategy provides a highly valuable reference for the direct detection of pathogen nucleic acids.},
}

*Biosensing Techniques/instrumentation
*Nanowires/chemistry/ultrastructure
*Silicon/chemistry
*CRISPR-Cas Systems/genetics
Transistors, Electronic
*Bacillus anthracis/genetics/isolation & purification/pathogenicity
Limit of Detection
*DNA, Bacterial/genetics/isolation & purification/analysis
*Endodeoxyribonucleases/chemistry
Bacterial Proteins/chemistry
CRISPR-Associated Proteins

@article {pmid40886437,
year = {2025},
author = {Xu, Q and Wang, X and Gu, Z and Duan, N and Jiang, S and Wu, S and Yuan, W},
title = {An electrochemical/colorimetric sensor for 5-methyltetrahydrofolate based on a screened specific aptamer and DNA walker-driven CRISPR/Cas12a cascade amplification.},
journal = {Biosensors & bioelectronics},
volume = {289},
number = {},
pages = {117931},
doi = {10.1016/j.bios.2025.117931},
pmid = {40886437},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *Colorimetry/methods ; *Aptamers, Nucleotide/chemistry ; Electrochemical Techniques/methods ; Humans ; Limit of Detection ; CRISPR-Cas Systems/genetics ; *Tetrahydrofolates/analysis/isolation & purification/chemistry ; Gold/chemistry ; DNA, Single-Stranded/chemistry ; Endodeoxyribonucleases/chemistry/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {5-Methyltetrahydrofolate (5-MTHF), the primary bioactive form of folate (vitamin B9), played a vital role in human metabolism. In this work, an electrochemical/colorimetric dual-mode aptasensor for 5-MTHF was constructed by combining a DNA Walker-driven CRISPR-Cas12a trans-cleavage system. A 5-MTHF aptamer D1a was obtained through Capture-SELEX with subsequent trimming of non-binding regions, which exhibiting high affinity and specificity. CuMOF@CuO@RuO2@IrO2, serving as a multifunctional indicator with high conductivity and peroxidase-like activity, was immobilized on a gold electrode (AuE) via ssDNA. The DNA Walker and nicking enzyme (Nt.BbvCI) continuously released Cas12a activators, triggering Cas12a-mediated cleavage of ssDNA on the AuE, leading to reduced electrochemical signals. The released materials were collected for colorimetric detection. The detection limits were determined to be 0.044 ng/mL (electrochemical) and 0.114 ng/mL (colorimetric). This dual-mode aptasensor demonstrated high selectivity, sensitivity, and stability for 5-MTHF detection, establishing a novel sensing platform for 5-MTHF analysis in food samples. The proposed aptasensor exhibited excellent sensitivity, selectivity, and reproducibility. This strategy offered a promising platform for 5-MTHF analysis in nutritional evaluation and dietary supplementation guidance.},
}

*Biosensing Techniques/methods
*Colorimetry/methods
*Aptamers, Nucleotide/chemistry
Electrochemical Techniques/methods
Humans
Limit of Detection
CRISPR-Cas Systems/genetics
*Tetrahydrofolates/analysis/isolation & purification/chemistry
Gold/chemistry
DNA, Single-Stranded/chemistry
Endodeoxyribonucleases/chemistry/genetics
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid40886435,
year = {2025},
author = {He, X and Liang, J and Zhang, J and Fang, W and Liu, J and Zhang, M and Wang, L and Song, C},
title = {CRISPR/Cas13a triggered-DNA walker amplified SERS sensor for ultrasensitive detection of cancer-related exosomal miRNA.},
journal = {Biosensors & bioelectronics},
volume = {289},
number = {},
pages = {117924},
doi = {10.1016/j.bios.2025.117924},
pmid = {40886435},
issn = {1873-4235},
mesh = {Humans ; *MicroRNAs/isolation & purification/genetics/blood ; *Biosensing Techniques/methods ; *Exosomes/chemistry/genetics ; Limit of Detection ; *Spectrum Analysis, Raman/methods ; CRISPR-Cas Systems/genetics ; *Neoplasms/genetics/diagnosis/blood ; DNA/chemistry ; DNA, Catalytic/chemistry ; },
abstract = {Accurate quantification of cancer-related miRNA in exosomes offers a promising approach for early and effective cancer diagnosis. However, reliably detecting extremely low-abundance exosomal miRNAs in complex bodily fluids remains a significant challenge. Herein, a CRISPR/Cas13a triggered-DNA walker amplified SERS sensor has been proposed for detection of cancer cell-derived exosomal miRNA-106a. The sensor comprises three main components: SERS Tags, SERS capture substrates and the CRISPR/Cas13a system. In the presence of miRNA-106a, the CRISPR/Cas13a trans-cleavage is activated, and the cleavage product further enables the DNAzymes to 'walk' on the SERS Tags, leading to the linking of the SERS Tags to a SERS capture substrate, ultimately generating significantly amplified SERS signal. The proposed SERS sensor exhibits good detection capability for miRNA-106a, i.e., rapid detection time within 80 min, wide linear response ranging from 100 aM to 1 nM, limit of detection (LOD) as low as 53.16 aM, good specificity, excellent reproducibility, and satisfactory recovery rates in human serum. Furthermore, the exosomal miRNA-106a derived from gastric cancer cells were detected and a LOD as low as 6.1 × 10[3] particles/mL of exosome was achieved, and the sensing results of the clinical serum samples underscore the potential of accurately differentiate between cancer patients and healthy individuals via analyzing cancer-related exosomal miRNAs, which indicates that the proposed SERS sensor can be a powerful tool for exosomal miRNA detection and holds good potential for precise liquid biopsy of tumor-derived exosomes.},
}

Humans
*MicroRNAs/isolation & purification/genetics/blood
*Biosensing Techniques/methods
*Exosomes/chemistry/genetics
Limit of Detection
*Spectrum Analysis, Raman/methods
CRISPR-Cas Systems/genetics
*Neoplasms/genetics/diagnosis/blood
DNA/chemistry
DNA, Catalytic/chemistry

@article {pmid40882515,
year = {2025},
author = {Li, QN and Huang, HR and Li, RY and Hou, XY and Yang, QF and Jiang, HX and Cai, QL and Kong, DM},
title = {Position-independent single-nucleotide polymorphism discrimination by CRISPR/Cas12a via rational activator strand engineering.},
journal = {Biosensors & bioelectronics},
volume = {289},
number = {},
pages = {117929},
doi = {10.1016/j.bios.2025.117929},
pmid = {40882515},
issn = {1873-4235},
mesh = {*Polymorphism, Single Nucleotide/genetics ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Humans ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Single-nucleotide polymorphisms (SNPs) are critical biomarkers for disease diagnosis and genetic research, yet their sensitive and specific detection remains challenging. Here, we report a rational activator strand design strategy that significantly enhances the SNP discrimination capability of CRISPR/Cas12a-based biosensing systems. By systematically optimizing the length of the crRNA-complementary region and the architecture of the 3'-terminal random extension sequence, we developed an engineered CRISPR/Cas12a platform capable of discrimination SNPs with single-nucleotide resolution, regardless of mutation position. Our optimized activator strand (ssAS13+3-X) leverages the "RESET" effect (random extending sequences enhance trans-cleavage activity) enables simple one-pot detection of low-abundance mutations (0.1 %) without target pre-amplification, offering significant advantages over conventional SNP detection methods in clinical settings. The single-stranded flexibility and length tolerance of the 3'-terminal extension further ensure broad applicability across diverse genomic contexts. This work not only deepens our fundamental understanding of CRISPR/Cas12a regulation, but also provides a versatile and streamlined platform for applications in molecular diagnostics, pathogen surveillance, and precision medicine.},
}

*Polymorphism, Single Nucleotide/genetics
*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
Humans
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40865357,
year = {2025},
author = {Wang, H and Wang, Z and Wu, M and Chen, M and He, X and Deng, L and Huang, J and Lin, R and Mao, X and Li, Q and Sheng, Y},
title = {CrispHunter-enabled systematic crRNA design and structure-optimized hairpin probes for enhanced CRISPR-Cas12a detection of Burkholderia pseudomallei.},
journal = {Biosensors & bioelectronics},
volume = {289},
number = {},
pages = {117912},
doi = {10.1016/j.bios.2025.117912},
pmid = {40865357},
issn = {1873-4235},
mesh = {*Burkholderia pseudomallei/genetics/isolation & purification/pathogenicity ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Humans ; Limit of Detection ; *Melioidosis/diagnosis/microbiology ; *RNA, Bacterial/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Effective control of genetically diverse pathogens necessitates rapid and accurate diagnostics, as their genomic variability undermines assay reliability and complicates public health interventions. Herein, we introduce a CRISPR-Cas12a-based molecular diagnostic platform integrating robust in silico crRNA design via our CrispHunter pipeline with a high-performance detection module featuring structure-optimized hairpin probes. We validated this approach using Burkholderia pseudomallei, whose high pathogenicity, genetic diversity, and diagnostic evasion exemplify the challenges of detecting heterogeneous pathogens. Applying CrispHunter to 3245 publicly available B. pseudomallei genomes, we systematically identified 12 highly conserved crRNA candidates and optimized a penta-crRNA strategy that reduced the limit of detection (LOD) from the previously reported 100 pM to 7.5 pM, thereby minimizing the risk of false negatives due to pathogen genomic diversity. Engineered hairpin-structured molecular beacons with iteratively optimized loop and stem lengths further enhanced assay performance, lowering the LOD to 2.1 pM without pre-amplification while improving signal-to-noise ratio by 42-fold, response sensitivity by 47-fold, and reducing time-to-positive from 56 to 3 min with excellent specificity. Employing recombinase polymerase amplification further enhanced sensitivity, lowering the LOD to 14.82 copies/μL, which is significantly below the concentrations typically found in clinical specimens. The modular architecture of CrispHunter, together with the optimized CRISPR-Cas12a detection platform, provides a versatile framework for rapid, sensitive, and specific molecular diagnostics. This strategy enhances detection of B. pseudomallei and can be readily adapted for the diagnosis of other genetically diverse pathogens, supporting broader applications in infectious disease management and public health.},
}

*Burkholderia pseudomallei/genetics/isolation & purification/pathogenicity
*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
Humans
Limit of Detection
*Melioidosis/diagnosis/microbiology
*RNA, Bacterial/genetics
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40848338,
year = {2025},
author = {Hiniduma, K and De Silva, PIT and Canete, R and Vora, P and Gunathillaka, H and Clement, O and Shawky, SM and Rouge, JL and Mosa, IM and Steffens, DC and Manning, K and Breno, D and Rusling, JF},
title = {ECL-CRISPR array for multiplexed detection of miRNAs.},
journal = {Biosensors & bioelectronics},
volume = {289},
number = {},
pages = {117855},
doi = {10.1016/j.bios.2025.117855},
pmid = {40848338},
issn = {1873-4235},
mesh = {*MicroRNAs/genetics/isolation & purification/blood ; Humans ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; Limit of Detection ; *Luminescent Measurements/methods/instrumentation ; *CRISPR-Cas Systems/genetics ; *Alzheimer Disease/genetics/blood/diagnosis ; Equipment Design ; },
abstract = {We describe here an electrochemiluminescent (ECL) array for individually detecting 3 miRNAs utilizing CRISPR/Cas13a. Detection involves binding a target miRNA to Cas 13a protein that includes the RNA complement to the target, This activated Cas13a then cleaves a poly-RNA rich in r-Guanosine to produce electrochemiluminescent (ECL) activators that increases ECL output proportional to target miRNA concentration. Specifically, poly-r-guanosine (poly-r-G) is cleaved by the collateral RNase activity of Cas13a to generate small poly-r-G fragments that are efficient in activating ECL of (bis-2,2'-bipyridyl) ruthenium polyvinylpyridine ([Ru(bpy)2PVP10] (ClO4)2) (RuPVP) films on sensor electrodes at +1.1 V vs. Ag/AgCl. The 3D-printed array was used to detect three Alzheimer's disease (ALZ) miRNA biomarkers (30e-5p, 34c-3p and 200c-5p). ECL is generated in the 3D-printed array designed with reference, counter and four separate RuPVP sensor electrodes. Detection limits for miRNAs were 7.4 fg/mL to 7 pg/mL with high sensitivities in linear dynamic ranges from 70 pg/mL to 70 μg/mL. Limits of detection (LOD) were 42 pg/mL, 0.074 fg/mL, and 0.15 fg/mL for miR30e-5p, miR34c-5p, and miR200c-3p, respectively. Spike recovery studies and patient plasma analyses after RNA extraction gave high accuracy and specificity, and excellent correlation with a referee CRISPR fluorescence method.},
}

*MicroRNAs/genetics/isolation & purification/blood
Humans
*Biosensing Techniques/methods
*Electrochemical Techniques/methods
Limit of Detection
*Luminescent Measurements/methods/instrumentation
*CRISPR-Cas Systems/genetics
*Alzheimer Disease/genetics/blood/diagnosis
Equipment Design

@article {pmid40834995,
year = {2025},
author = {De Marco, F and Sebastian, IR and Napoleone, A and Molin, A and Riedl, M and Bydlinski, N and Motheramgari, K and Hussein, MK and Kramer, L and Kelly, T and Jostock, T and Borth, N},
title = {A genome-scale CRISPR deletion screen in Chinese hamster ovary cells reveals essential regions of the coding and non-coding genome.},
journal = {Metabolic engineering},
volume = {92},
number = {},
pages = {194-207},
doi = {10.1016/j.ymben.2025.08.007},
pmid = {40834995},
issn = {1096-7184},
mesh = {Animals ; CHO Cells ; Cricetulus ; *Genome ; *CRISPR-Cas Systems ; Cricetinae ; *Gene Deletion ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Sequence Deletion ; },
abstract = {The biopharmaceutical sector relies on CHO cells to investigate biological processes and as the preferred host for production of biotherapeutics. Simultaneously, advancements in CHO cell genome assembly have provided insights for developing sophisticated genetic engineering strategies. While the majority of these efforts have focused on coding genes, with some interest in transcribed non-coding RNAs (e.g., microRNAs and lncRNAs), there remains a lack of genome-wide systematic studies that precisely examine the remaining 90 % of the genome and its impact on cellular phenotypes. This unannotated "dark matter" includes regulatory elements and other poorly characterized genomic features that may be potentially critical for cell behaviour. In this study, we deployed a genome-scale CRISPR screening platform with 112,272 paired guide RNAs targeting 14,034 genomic regions for complete deletion of 150 kb long sections. This platform enabled the execution of a negative screen that selectively identified dying cells to determine regions essential for cell survival. By using paired gRNAs, we overcame the intrinsic limitations of traditional frameshift strategies, which will likely have little or no effect on the non-coding genome. This study revealed 427 regions essential for CHO cell survival, many of which currently lack gene annotation or known functions. For these regions, we present their annotation status, transcriptional activity and annotated chromatin states. Selected regions, particularly those lacking all of the above, were individually deleted to confirm their essentiality. This work sheds a novel light on a substantial portion of the mammalian genome that has been traditionally difficult to investigate and therefore neglected. Notably, the fact that the deletion of some of these regions is lethal to cells suggests they encode critical regulatory functions. A better genome-wide understanding of these functions could open new avenues for engineering cells with improved bioprocess relevant properties.},
}

Animals
CHO Cells
Cricetulus
*Genome
*CRISPR-Cas Systems
Cricetinae
*Gene Deletion
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Sequence Deletion

@article {pmid40803011,
year = {2025},
author = {Jiang, Y and Wu, J and Xiang, X and Wei, J and Cheng, K and Cui, L and Xu, H and Li, Z},
title = {A cleavage-gated terminal exposure-driven CRISPR-RCA self-amplifying system for ultra-fast DNA detection.},
journal = {Biosensors & bioelectronics},
volume = {289},
number = {},
pages = {117857},
doi = {10.1016/j.bios.2025.117857},
pmid = {40803011},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *DNA, Viral/genetics/isolation & purification/analysis ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; Humans ; CRISPR-Associated Proteins/chemistry ; Endodeoxyribonucleases/chemistry ; Bacterial Proteins ; },
abstract = {The one-pot detection technology based on nucleic acid isothermal amplification combined with CRISPR has a significant advantage in on-site detection of infectious diseases. It is superior to quantitative polymerase chain reaction (qPCR) due to its lack of temperature variation and significantly faster reaction speed. Nevertheless, Cas proteins compete with amplification enzymes for reaction substrates, which results in the signal amplification effect being less than expected. To overcome this limitation, we have developed a single-tube self-amplifying system driven by cleavage-gated terminal exposure based on CRISPR and rolling circle amplification (AURORA), enabling ultra-fast and sensitive monkeypox virus (MPXV) detection. This method innovatively designs a dual-function probe (DF probe). In the presence of the target, the trans-cleavage activity of Cas12a is activated, only cleaving the single-stranded DNA to expose the 3' terminal of the DF probe. The cleaved DF probe hybridizes with the circular DNA template and is bound by phi29 DNA polymerase to initiate RCA. Here, we utilize the characteristics of Cas12a and phi29 DNA polymerase acting on substrates in different strand states to avoid substrate competition between the two enzymes in a single-tube reaction. This assay can achieve ultra-fast signal amplification of MPXV DNA within 8 min, with a limit of detection (LOD) of 88 aM (53 copies/μL). Combining the viral nucleic acid thermal lysis method, it is possible to achieve results from sample input to output in 10 min. The AURORA detection strategy was further used to detect MPXV in clinical samples (36 MPXV samples), and the results were completely consistent with qPCR. The AURORA system features ultra-fast and precise detection, providing a more efficient tool for the prevention and control of severe infectious diseases.},
}

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

@article {pmid40752104,
year = {2025},
author = {Pavlikova, L and Krepop, J and Sulova, Z and Breier, A and Seres, M},
title = {Differential UVC radiation sensitivity in multidrug-resistant l1210 cells: Insights into p53 and Bcl-XL expression/function.},
journal = {Journal of photochemistry and photobiology. B, Biology},
volume = {271},
number = {},
pages = {113229},
doi = {10.1016/j.jphotobiol.2025.113229},
pmid = {40752104},
issn = {1873-2682},
mesh = {*Ultraviolet Rays ; *Tumor Suppressor Protein p53/genetics/metabolism ; *bcl-X Protein/metabolism/genetics ; Animals ; Mice ; Cell Line, Tumor ; *Drug Resistance, Multiple/radiation effects ; Drug Resistance, Neoplasm/radiation effects ; Doxorubicin/pharmacology ; DNA Damage/radiation effects ; Vincristine/pharmacology ; Apoptosis/radiation effects/drug effects ; CRISPR-Cas Systems ; Radiation Tolerance ; },
abstract = {Multidrug-resistant (MDR) variants of L1210 cells, selected for resistance to vincristine (R) or doxorubicin (D), exhibit elevated ABCB1 (P-glycoprotein) expression but differ in UVC sensitivity. D cells resemble parental L1210 (S) cells, whereas R cells are significantly more UVC vulnerable. To investigate this, we analyzed the expression of genes involved in DNA damage response, Trp53 family members, cyclin-dependent kinase inhibitors (p15, p21), Bcl-2 family genes, and DNA repair genes in S, R, and D cells before and after UVC irradiation. The most striking difference was the absence of Trp53 expression in R cells at both mRNA and protein levels, while S and D cells expressed this gene. Instead, R cells uniquely expressed Trp63. CRISPR/Cas9-generated p53-null mutants of S and D cells showed increased UVC-induced cell death, but their sensitivity did not reach that of R cells. Bcl-XL, a protein linked to resistance against UVC-induced apoptosis, was also reduced at both transcript and protein levels in R cells. Pharmacological inhibition of Bcl-XL in S and D cells with A-1155463 and A-1331852 enhanced UVC-induced cell death but did not replicate the high sensitivity observed in R cells. Our findings suggest that the heightened UVC sensitivity of R cells results from a combined deficiency of p53 and Bcl-XL, impairing DNA damage response and apoptosis. These results reveal distinct molecular adaptations in MDR variants and provide insight into the mechanisms underlying differential UVC sensitivity.},
}

*Ultraviolet Rays
*Tumor Suppressor Protein p53/genetics/metabolism
*bcl-X Protein/metabolism/genetics
Animals
Mice
Cell Line, Tumor
*Drug Resistance, Multiple/radiation effects
Drug Resistance, Neoplasm/radiation effects
Doxorubicin/pharmacology
DNA Damage/radiation effects
Vincristine/pharmacology
Apoptosis/radiation effects/drug effects
CRISPR-Cas Systems
Radiation Tolerance

@article {pmid40835108,
year = {2025},
author = {Shan, X and Zhang, X and Tao, B and Song, Y and Zhu, Z and Hu, W and Chen, J},
title = {CRISPR/Cas9-mediated editing of ptprfb (protein tyrosine phosphatase receptor type fb) reveals its regulatory role in zebrafish spermatogenesis.},
journal = {Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology},
volume = {280},
number = {},
pages = {111146},
doi = {10.1016/j.cbpb.2025.111146},
pmid = {40835108},
issn = {1879-1107},
mesh = {Animals ; *Zebrafish/genetics/physiology ; Male ; *CRISPR-Cas Systems ; *Spermatogenesis/genetics ; *Gene Editing ; *Zebrafish Proteins/genetics/metabolism ; Spermatozoa/metabolism ; },
abstract = {Gonadal development and gamete maturation are essential for fish reproduction. The protein tyrosine phosphatase receptor type Fb (Ptprfb) is a member of the tyrosine phosphatase family. In the present study, we used CRISPR/Cas9 to mutate ptprfb in zebrafish. A significantly reduced natural fertilization rate of sperm from mutant fish was observed. The mutant fish produced fewer sperm with shorter flagella, and a smaller proportion of sperm could be activated. RNA-seq analysis revealed abnormal expression of some genes in testicular cells, such as hemoglobin subunit βA1 (hbba1), myosin heavy chain 11b (myh11b), and transgelin (tagln), as well as some genes involved in focal adhesion formation. These findings demonstrate that ptprfb contributes to spermatogenesis in zebrafish, and its dysfunction can adversely affect both the quantity and quality of sperm.},
}

Animals
*Zebrafish/genetics/physiology
Male
*CRISPR-Cas Systems
*Spermatogenesis/genetics
*Gene Editing
*Zebrafish Proteins/genetics/metabolism
Spermatozoa/metabolism

@article {pmid40190036,
year = {2025},
author = {Wang, J and Xu, K and Liu, T and Zhao, H and Jamal, MA and Chen, G and Huo, X and Yang, C and Jiao, D and Wei, T and Huang, H and Zhao, H and Guo, J and Wang, F and Zhang, X and Liu, K and Qu, S and Wang, G and Guo, H and Chen, G and Zhao, HY and Zeng, Z and Dou, K and Wei, HJ},
title = {Production and Functional Verification of 8-Gene (GGTA1, CMAH, β4GalNT2, hCD46, hCD55, hCD59, hTBM, hCD39)-Edited Donor Pigs for Xenotransplantation.},
journal = {Cell proliferation},
volume = {58},
number = {9},
pages = {e70028},
pmid = {40190036},
issn = {1365-2184},
support = {202102AA310047//Major Science and Technology Project of Yunnan Province/ ; 2019YFA0110700//National Key Research and Development Program of China/ ; 2023-JCJQ-ZD-118-00//173 Basic Strengthening Program/ ; },
mesh = {Animals ; *Transplantation, Heterologous/methods ; *Gene Editing/methods ; Swine ; Humans ; CRISPR-Cas Systems/genetics ; Galactosyltransferases/genetics ; Animals, Genetically Modified ; CD59 Antigens/genetics ; N-Acetylgalactosaminyltransferases/genetics ; Membrane Cofactor Protein/genetics ; Mixed Function Oxygenases ; },
abstract = {Gene-edited (GE) pig-to-human xenotransplantation continues to make breakthroughs, but which kind of gene combination is suitable for organ-specific transplantation remains unclear. In this study, we utilised CRISPR/Cas9 gene editing technology, PiggyBac transposon system, and serial somatic cell cloning technology to develop GTKO/CMAHKO/β4GalNT2KO/hCD46/hCD55/hCD59/hCD39/hTBM 8 gene-edited cloned (GEC) donor pigs and performed pig-to-non-human primate (NHP) transplantation to evaluate the effectiveness of these GEC pigs. The 8-GEC pigs were obtained by recloning with a 33-day-old 8-GEC fetus with O blood type, which was generated after cell transfection, screening of cell colonies, and somatic cell cloning. Molecular identification at DNA, mRNA, and protein levels confirmed successful 8-gene editing. Three copies of transgenes were identified by droplet digital polymerase chain reaction and whole genome sequencing, which were inserted into the introns of pig RFTN1 and MYO10 genes, as well as the intergenic region between PRLR and LOC110257300 genes of these 8-GEC pigs. The 8-GEC pigs also exhibited the ability of germline transmission when mated with our previously generated 4-GEC male pigs. Moreover, antigen-antibody binding assay and complement-dependent cytotoxicity assay demonstrated that 8-gene editing effectively reduced the immune incompatibility and kidney xenograft from 8-GEC pigs survived for 15 and 17 days in two NHPs, respectively. Postoperatively, the recipient serum antibodies IgA, IgG and IgM, complements C3 and C4, coagulation indicators PT, APTT, TT and FIB, as well as most electrolytes and liver function indicators remained relatively stable. Serum creatinine was normal within 10 days post operation. However, the kidney xenograft developed active antibody-mediated rejection at necropsy, characterised by the deposition of antibodies IgG and IgM, as well as complements C4d, C3c and C5b-C9, infiltration of CD68[+] macrophages, and micro-thrombotic embolism of glomerular capillaries, etc. In conclusion, we successfully developed fertile 8-GEC pigs, which effectively alleviated immune rejection and exerted life-supporting kidney function in the recipients.},
}

Animals
*Transplantation, Heterologous/methods
*Gene Editing/methods
Swine
Humans
CRISPR-Cas Systems/genetics
Galactosyltransferases/genetics
Animals, Genetically Modified
CD59 Antigens/genetics
N-Acetylgalactosaminyltransferases/genetics
Membrane Cofactor Protein/genetics
Mixed Function Oxygenases

@article {pmid40909725,
year = {2025},
author = {Hong, A and Liu, M and Truta, A and Talaie, A and Smith, GR and Bondy-Denomy, J},
title = {Gabija restricts phages that antagonize a conserved host DNA repair complex.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2025.08.30.673261},
pmid = {40909725},
issn = {2692-8205},
abstract = {Anti-bacteriophage systems like restriction-modification and CRISPR-Cas have DNA substrate specificity mechanisms that enable identification of invaders. How Gabija, a highly prevalent nuclease-helicase anti-phage system, executes self- vs. non-self-discrimination remains unknown. Here, we propose that phage-encoded DNA end-binding proteins that antagonize host RecBCD sensitize phages to Gabija. When targeting temperate phage D3 in Pseudomonas aeruginosa, Gabija functions early by preventing phage genome circularization in a non-abortive manner. Phage and plasmid DNA-end sensitivity to Gabija is licensed by a phage exonuclease and ssDNA-annealing protein. Unrelated F8 and JBD30 phages are sensitized to Gabija by Gam_Mu, a distinct DNA end-binding protein that antagonizes loading of the host repair complex RecBCD. Escape phages lacking these end-binding proteins become protected from Gabija by RecBCD activities, which also prevent Gabija from targeting self-DNA. Therefore, we propose that Gabija antagonizes circularization of linear DNA devoid of RecBCD as a mechanism to identify foreign invaders.},
}

@article {pmid40543483,
year = {2026},
author = {Wang, H and Yan, Y and Codjia, CR and Dan, Y and Li, L and He, S and Yang, X and Chen, L and Liu, H and Wang, X},
title = {CRISPR-Cas13a coupled with recombinase aided amplification: Development of an ultrasensitive point - of - care detection method for visual diagnosis of egg drop syndrome virus.},
journal = {Talanta},
volume = {296},
number = {},
pages = {128470},
doi = {10.1016/j.talanta.2025.128470},
pmid = {40543483},
issn = {1873-3573},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Recombinases/metabolism ; *Nucleic Acid Amplification Techniques/methods ; *Atadenovirus/isolation & purification/genetics ; *Poultry Diseases/diagnosis/virology ; Chickens/virology ; *Point-of-Care Systems ; Limit of Detection ; },
abstract = {Egg Drop Syndrome Virus (EDSV) infection in poultry causes a significant drop in egg - laying rate, harming poultry farms' economic benefits. A sensitive and specific EDSV detection method is urgently needed in clinical practice. The CRISPR-Cas13a system can detect various targets specifically, and recombinase-aided amplification (RAA) can rapidly amplify nucleic acids. Therefore, in this experiment, the RAA was integrated with the CRISPR-Cas13a system to develop a novel, visual, ultrasensitive and point - of - care detection method for EDSV. In this experiment, the reaction system was optimized, and its sensitivity, specificity, repeatability, and clinical sample validation were conducted and evaluated. The results showed that the optimal concentration of the obtained Cas13a protein was 2.4 mg/mL, and that of crRNA 1 was 100 μg/μL. This method not only showed rapid detection (30-50 min), very high sensitivity, with the detection limit reaching 1 copy/μL, but also showed good specificity with no cross - reaction to Marek's Disease Virus (MDV), Infectious Laryngotracheitis Virus (ILTV), Avian Leukosis Virus (ALV), Chicken Anemia Virus (CAV), Astrovirus (AstV), H9N2 subtype of Avian Influenza Virus (H9N2 AIV), Fowl Adenovirus serotype 4 (FAdV-4), Fowl Adenovirus serotype 8 (FAdV-8) and Fowl Adenovirus serotype 11 (FAdV-11). Furthermore, the method displayed excellent repeatability, with the coefficient of variation of both intra - group and inter - group no more than 4 %. Evaluation of this method through 210 clinical samples found that compared with the traditional polymerase chain reaction (PCR) which is the industry standard, its positive coincidence rate was 100 %, the negative coincidence rate was 98.35 %, the overall coincidence rate was 98.57 %, and the kappa value was 0.94. This assay provides a potential point - of - care testing approach for the clinical detection, virology, and epidemiological studies of EDSV.},
}

Animals
*CRISPR-Cas Systems/genetics
*Recombinases/metabolism
*Nucleic Acid Amplification Techniques/methods
*Atadenovirus/isolation & purification/genetics
*Poultry Diseases/diagnosis/virology
Chickens/virology
*Point-of-Care Systems
Limit of Detection

@article {pmid40499384,
year = {2026},
author = {Yang, Y and Ji, X and Zhou, F and He, Z},
title = {A one-pot assay based on PAM-free recombinase polymerase amplification and CRISPR/Cas12a for rapid detection of SARS-CoV-2 N gene.},
journal = {Talanta},
volume = {296},
number = {},
pages = {128448},
doi = {10.1016/j.talanta.2025.128448},
pmid = {40499384},
issn = {1873-3573},
mesh = {*SARS-CoV-2/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *Recombinases/metabolism/genetics ; *COVID-19/diagnosis/virology ; CRISPR-Associated Proteins/metabolism ; *Phosphoproteins/genetics ; Endodeoxyribonucleases/metabolism ; Coronavirus Nucleocapsid Proteins ; Bacterial Proteins ; },
abstract = {SARS-CoV-2 is a pathogenic virus, which exhibits high contagiousness. Therefore, a rapid and sensitive SARS-CoV-2 detection strategy is imperative. Herein, a one-pot assay by the combination of protospacer adjacent motif (PAM)-free recombinase polymerase amplification with CRISPR/Cas12a for detecting SARS-CoV-2 N gene was reported. To avoid the constraint of the PAM site for double-stranded DNA (dsDNA) in CRISPR/Cas12a system, we designed two individual crRNAs to hybridize with two different regions of the target sequence. The presence of N gene DNA was able to initiate the amplification of RPA, exposing the recognition site of crRNA and activating the Cas12a. Whereafter, the Cas12a activation resulted in the digestion of nontarget DNA reporters to induce significant fluorescence signal. The assay completed the detection of N gene DNA within 30 min. And a high sensitivity of 100 aM was obtained because of RPA amplification and Cas12a trans cleavage activity. Meanwhile, the proposed assay showed excellent specificity due to the site-specific recognition ability of CRISPR/Cas12a. More importantly, analysis of spiked samples verified the excellent practical application of the proposed method. Thus, the assay earned promising potential in molecular diagnostics.},
}

*SARS-CoV-2/genetics/isolation & purification
*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
Humans
*Recombinases/metabolism/genetics
*COVID-19/diagnosis/virology
CRISPR-Associated Proteins/metabolism
*Phosphoproteins/genetics
Endodeoxyribonucleases/metabolism
Coronavirus Nucleocapsid Proteins
Bacterial Proteins

@article {pmid40909132,
year = {2025},
author = {Biswas, I},
title = {Ethical dimensions and societal implications: ensuring the social responsibility of CRISPR technology.},
journal = {Frontiers in genome editing},
volume = {7},
number = {},
pages = {1593172},
pmid = {40909132},
issn = {2673-3439},
abstract = {CRISPR-Cas9 is a breakthrough genome-editing platform that can cut chosen DNA sequences with unprecedented speed, accuracy, and affordability. By reprogramming a single guide RNA, researchers now alter gene function, correct pathogenic variants, or introduce novel traits. Earlier tools such as zinc-finger nucleases and TALENs performed similar tasks but were significantly more complex and costly. Yet CRISPR's very power raises urgent ethical concerns: Who controls its use, and how can society prevent germ-line enhancement, eugenic selection, or unequal access that favors wealthy nations and patients? A well-publicized case of embryo editing already showed how premature, unregulated experiments can erode public trust. This perspective therefore frames CRISPR's scientific promise alongside its social responsibilities, arguing that proactive, globally coordinated governance is essential to unlock benefits while preventing new forms of genetic inequality.},
}

@article {pmid40907709,
year = {2025},
author = {Chugh, P and Soni, S and Ghanghas, N and Kumar, S and Mohan, H},
title = {Comprehensive insights into Japanese encephalitis virus: From molecular characterization to advanced detection and vaccine strategies.},
journal = {Antiviral research},
volume = {},
number = {},
pages = {106268},
doi = {10.1016/j.antiviral.2025.106268},
pmid = {40907709},
issn = {1872-9096},
abstract = {The Japanese encephalitis virus (JEV) remains a major cause of viral encephalitis in Asia, with significant morbidity and mortality. This review offers a comprehensive overview of the current landscape of JEV research, focusing on its genomic structure, protein composition, and global epidemiology. We highlight the complexity of JEV transmission and pathogenesis, examining the interplay of demographic factors and geographic spread. In particular, we assess the evolution of diagnostic methodologies from traditional molecular and serological techniques to emerging biosensor-based approaches, emphasizing advancements in sensitivity and rapidity. The application of CRISPR/Cas systems for JEV detection marks a promising frontier in molecular diagnostics. Additionally, we review the current status of JEV vaccines, discussing recent innovations in vaccine development aimed at enhancing immunogenicity and accessibility. Beyond prevention, a spectrum of antiviral strategies-including direct-acting antivirals, entry inhibitors, host-directed modulators, neuroprotective agents, and steroidal/synthetic compounds-has demonstrated potent in vitro and in vivo efficacy, targeting viral enzymes, structural proteins, and host pathways. This review underscores the critical role of advanced detection strategies and vaccines in controlling JEV, offering insights into ongoing efforts to mitigate its impact in endemic regions.},
}

@article {pmid40906807,
year = {2025},
author = {Han, Z and Huang, C and Luo, T and Mirkin, CA},
title = {A general genome editing strategy using CRISPR lipid nanoparticle spherical nucleic acids.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {36},
pages = {e2426094122},
doi = {10.1073/pnas.2426094122},
pmid = {40906807},
issn = {1091-6490},
support = {FA9550-22-1-0300//DOD | AF | AMC | AFRL | Air Force Office of Scientific Research (AFOSR)/ ; DMR-2428112//National Science Foundation (NSF)/ ; DMR-2308691//National Science Foundation (NSF)/ ; NSF ECCS-2025633//National Science Foundation (NSF)/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Nanoparticles/chemistry ; Humans ; *Lipids/chemistry ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Nucleic Acids/chemistry/genetics ; HEK293 Cells ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Transfection ; Liposomes ; },
abstract = {Genome editing with CRISPR-Cas systems hold promise for treating a wide range of genetic disorders and cancers. However, efficient delivery of genome editors remains challenging due to the requirement for the simultaneous delivery or intracellular generation of Cas proteins, guide RNAs, and, in some applications, donor DNAs. Furthermore, the immunogenicity and toxicity of delivery vehicles can limit the safety and efficacy of genetic medicines. Here, we combine two nucleic acid delivery approaches to create CRISPR lipid nanoparticle-spherical nucleic acids (LNP-SNAs) that are both efficient and biocompatible. Compared to lipid nanoparticles (LNPs) lacking a surface-bound DNA shell, CRISPR LNP-SNAs exhibit two- to three-fold higher cellular uptake, reduced cytotoxicity, and improved gene transfection efficiency. Across multiple cell lines and genomic loci, CRISPR LNP-SNAs induce insertion-deletion mutations at average frequencies two- to three-fold higher than those observed with LNPs. When codelivered with donor templates, CRISPR LNP-SNAs enable homology-directed repair at an average efficiency of 21 ± 7%, a 2.5-fold improvement over LNPs (8 ± 4%). The ease of synthesis and biocompatibility of CRISPR LNP-SNAs highlight their potential as a versatile delivery platform for CRISPR-Cas and other gene therapies.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Nanoparticles/chemistry
Humans
*Lipids/chemistry
RNA, Guide, CRISPR-Cas Systems/genetics
*Nucleic Acids/chemistry/genetics
HEK293 Cells
*Clustered Regularly Interspaced Short Palindromic Repeats
Transfection
Liposomes

@article {pmid40906401,
year = {2025},
author = {Luo, J and Lu, W and Liu, R and Zhang, S and Cao, J and Ma, C},
title = {From Panels to Pathogen Networks: The Expanding Role of Targeted Sequencing in Veterinary Medicine.},
journal = {Biology},
volume = {14},
number = {8},
pages = {},
pmid = {40906401},
issn = {2079-7737},
abstract = {Targeted sequencing, a pivotal branch of next-generation sequencing (NGS), enables the selective enrichment of specific genomic regions and has demonstrated significant advantages in the detection of animal pathogens. This review systematically explores the underlying principles of targeted sequencing, various enrichment strategies-including PCR amplification, probe hybridization, and CRISPR-Cas systems-and their key applications in veterinary pathogen diagnostics. Due to its high throughput, sensitivity, and cost-effectiveness, targeted sequencing has been successfully applied in the multiplex detection of pathogens in economically significant livestock, such as cattle, as well as in the surveillance of antimicrobial resistance (AMR) genes, pathogen typing, and source tracing. It is particularly effective in identifying mixed infections and low-abundance pathogens. Nonetheless, wide application is restricted by some factors, like incomprehensive reference databases, cost-effectiveness, and limited application in primary-level laboratories. Further development directions are AI-based panel design, multimodal diagnostic platform integration, standard workflow construction, and introduction of a multi-omics method. Such progress focuses on enhancing the targeted sequencing scalability and precision consistent with the "One Health" initiative objective.},
}

@article {pmid40905590,
year = {2025},
author = {Cheng, L},
title = {Topology-Engineered Guide RNAs for Programmable Control of CRISPR/Cas Activity.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {},
number = {},
pages = {e202511756},
doi = {10.1002/anie.202511756},
pmid = {40905590},
issn = {1521-3773},
support = {2025YFA0920900//National Key R&D Program of China/ ; XDB0960103//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; BNLMS-CXTD-202401//Beijing National Laboratory for Molecular Sciences/ ; 22537005//National Natural Science Foundation of China/ ; },
abstract = {CRISPR/Cas systems have transformed genome editing, yet achieving precise temporal and conditional control remains challenging. Traditional strategies involving linear guide RNAs (gRNAs) modified with multiple chemical groups throughout their strands often face limitations such as heterogeneous reaction outcomes, irreversibility, and variable editing efficiencies. To overcome these issues, topology-engineered guide RNAs (TE-gRNAs) have emerged, featuring defined structural architectures including polymeric, circular, and dendrimer-like topologies that enable precise spatial control, reversibility, and programmable activation of CRISPR activity. By selectively incorporating physical or chemically responsive linkers and stimuli-sensitive groups at specific sites, TE-gRNAs facilitate dynamic and conditional genome editing that can be activated or deactivated with external triggers such as light or chemical signals. These engineered RNA structures significantly improve synthesis feasibility, stability, reduce off-target effects, and provide unprecedented control over gene editing processes. Recent advancements in TE-gRNAs demonstrate their broad applicability in synthetic biology, functional genomics, and therapeutic interventions, highlighting their potential to achieve precise spatiotemporal modulation of CRISPR systems. This review summarizes the current strategies, benefits, and challenges associated with TE-gRNAs, and discusses future directions for enhancing their performance and utility in complex genome editing applications.},
}

@article {pmid40848498,
year = {2026},
author = {Chen, J and Shi, K},
title = {A fluorescent aptasensor for accurate and sensitive detection of glyphosate based on asymmetrically competitive CRISPR/Cas12a with phosphorothioate-modified G-quadruplex.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {345},
number = {},
pages = {126840},
doi = {10.1016/j.saa.2025.126840},
pmid = {40848498},
issn = {1873-3557},
mesh = {Glyphosate ; *Glycine/analogs & derivatives/analysis ; *G-Quadruplexes ; *Aptamers, Nucleotide/chemistry/genetics ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Limit of Detection ; Spectrometry, Fluorescence/methods ; *Fluorescent Dyes/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Accurate detection of glyphosate (GLY) residues is essential for protecting public health, ensuring food safety, and preventing environmental pollution caused by excessive or improper herbicide application. Based on the asymmetric CRISPR/Cas12a system as a signal amplification technique, with phosphorothioate-modified hairpin G-quadruplex (psHG4) acting as a signal probe, a platform named ACC-GLY is developed for the sensitive and accurate detection of GLY. In the designed ACC-GLY platform, the target GLY specifically binds to the aptamer, and the cascading signal amplification strategy, driven by the DNase activity of a single Cas12a, is initiated by two competitive guide RNAs. Under the influence of Cas12a's DNase activity, the psHG4 probe is cleaved, releasing the psG4 sequence. The released psG4 sequence then binds to Thioflavin T (ThT), forming a complex that generates a fluorescence signal. Under optimal conditions, the detection platform can specifically detect GLY at a concentration as low as 0.3 pM. The detection platform demonstrates significant capability in detecting GLY in tap water and corn samples, highlighting its vast potential for applications in environmental monitoring and food safety. To the best of our knowledge, this is the first study to utilize the CRISPR/Cas12a system for GLY detection.},
}

Glyphosate
*Glycine/analogs & derivatives/analysis
*G-Quadruplexes
*Aptamers, Nucleotide/chemistry/genetics
*CRISPR-Cas Systems
*Biosensing Techniques/methods
Limit of Detection
Spectrometry, Fluorescence/methods
*Fluorescent Dyes/chemistry
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40795705,
year = {2026},
author = {Li, H and Tang, X and Li, F and Yang, M and Jing, H and Li, L and Liu, J and Cao, Z and Yang, X and Wang, J},
title = {Neutrophil-inspired CRISPR/dCas9 nanomedicine to program self-destructing and bystander killing of tumor cell for selective cancer therapy.},
journal = {Biomaterials},
volume = {325},
number = {},
pages = {123619},
doi = {10.1016/j.biomaterials.2025.123619},
pmid = {40795705},
issn = {1878-5905},
mesh = {*Neutrophils/metabolism ; Humans ; Animals ; *CRISPR-Cas Systems/genetics ; *Bystander Effect ; *Nanomedicine/methods ; *Neoplasms/therapy/pathology ; Cell Line, Tumor ; Leukocyte Elastase/metabolism/genetics ; Mice ; Female ; },
abstract = {Achieving high efficiency, selectivity, and durability remains a major challenge in the development of innovative cancer treatments, as current clinical therapies often fall short. Inspired by neutrophil-mediated tumor clearance, we engineered a nanocarrier-mediated CRISPR/dCas9 system to activate endogenous expression of neutrophil elastase (ELANE) for precise cancer therapy. Although ELANE was upregulated in both normal and tumor cells, selective killing occurred only in tumor cells through a histone H1.0-dependent self-destructing mechanism. Additionally, secreted ELANE further efficiently eliminated neighboring tumor cells via bystander killing. ELANE-mediated tumor cell death also induced immunogenic responses, potently enhancing antitumor immunity and synergizing with anti-PD-L1 therapy to inhibit tumor metastasis. This study presents a novel CRISPRa-based therapeutic strategy that mimic neutrophil function to achieve potent, selective, and durable tumor eradication through combined mechanisms of self-destruction, bystander killing and immune activation.},
}

*Neutrophils/metabolism
Humans
Animals
*CRISPR-Cas Systems/genetics
*Bystander Effect
*Nanomedicine/methods
*Neoplasms/therapy/pathology
Cell Line, Tumor
Leukocyte Elastase/metabolism/genetics
Mice
Female

@article {pmid40904932,
year = {2025},
author = {Wang, HM and Xu, SJ and Cai, BY and Qiu, WY and Lu, H and Tang, YD},
title = {Highly efficient gene editing of Feline herpesvirus 1 using CRISPR/Cas9 combined with FACS.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1660446},
pmid = {40904932},
issn = {2235-2988},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Animals ; *Varicellovirus/genetics ; Cats ; *Flow Cytometry/methods ; Thymidine Kinase/genetics ; Genes, Reporter ; Herpesviridae Infections/virology/veterinary ; Green Fluorescent Proteins/genetics ; },
abstract = {Feline herpesvirus 1 (FHV-1) is a major causative agent of feline viral rhinotracheitis and ocular lesions. Due to its large DNA genome, the construction of recombinant FHV-1 viruses presents considerable challenges for conventional methodologies. In this study, we implemented an integrated strategy combining CRISPR/Cas9-mediated gene editing with fluorescence-activated cell sorting (FACS) to enable the rapid and efficient generation of recombinant FHV-1 viruses. Specifically, the thymidine kinase (tk) gene was disrupted by inserting a monomeric Cherry (mCherry) reporter gene, and the glycoprotein E (gE) gene was similarly interrupted through the incorporation of a green fluorescent protein (GFP) reporter. The CRISPR/Cas9 system enables precise, site-specific genomic modifications, while FACS allows for effective enrichment and isolation of the desired recombinant viral populations. This combined approach significantly reduces the time required for recombinant virus generation from weeks to days, thereby offering substantial potential to expedite vaccine development and advance functional genomics research.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
Animals
*Varicellovirus/genetics
Cats
*Flow Cytometry/methods
Thymidine Kinase/genetics
Genes, Reporter
Herpesviridae Infections/virology/veterinary
Green Fluorescent Proteins/genetics

@article {pmid40904117,
year = {2025},
author = {Koonce, KC and Mauritzen, JJ and Hitz, IF and Vangsgaard, EF and Putz, EHM and Wajn, AS and Leth, FH and Høyland-Kroghsbo, NM},
title = {The H-NS homologues MvaT and MvaU repress CRISPR-Cas in Pseudomonas aeruginosa.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {380},
number = {1934},
pages = {20240073},
pmid = {40904117},
issn = {1471-2970},
support = {//Danmarks Frie Forskningsfond/ ; //Lundbeck Foundation/ ; },
mesh = {*Pseudomonas aeruginosa/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Bacterial Proteins/genetics/metabolism ; *DNA-Binding Proteins/genetics/metabolism ; Trans-Activators ; },
abstract = {CRISPR-Cas is an adaptive immune system of bacteria and archaea that protects against foreign genetic elements. In Escherichia coli and Salmonella, CRISPR-Cas is inhibited by the conserved global repressor the histone-like nucleoid structuring protein (H-NS), which blocks the expression of AT-rich horizontally acquired genes. While the opportunistic pathogen Pseudomonas aeruginosa harbours two partially redundant H-NS homologues, MvaT and MvaU, their role in CRISPR-Cas regulation in this bacterium remains unexplored. Here, we demonstrate that in the absence of both MvaT and MvaU, CRISPR-Cas activity increases more than tenfold, as measured by a reduction in the transformation efficiency of a CRISPR-targeted plasmid. Importantly, we find that in the absence of MvaT and MvaU, Cas proteins are already produced at low cell density prior to the onset of quorum sensing-mediated activation of CRISPR-Cas, which occurs at high cell density. Moreover, the ∆mvaT ∆mvaU mutant has a significantly reduced growth rate, known to independently increase CRISPR-Cas activity. In addition to regulating CRISPR-Cas, the absence of MvaT and MvaU affects phage-host interactions, including enhancing the adsorption of the LPS-binding phage JBD44, highlighting their broader role in coordinating bacterial defence mechanisms.This article is part of the discussion meeting issue 'The ecology and evolution of bacterial immune systems'.},
}

*Pseudomonas aeruginosa/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Bacterial Proteins/genetics/metabolism
*DNA-Binding Proteins/genetics/metabolism
Trans-Activators

@article {pmid40904116,
year = {2025},
author = {Hoikkala, V and Chi, H and Grüschow, S and Graham, S and White, MF},
title = {Diversity and abundance of ring nucleases in type III CRISPR-Cas loci.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {380},
number = {1934},
pages = {20240084},
pmid = {40904116},
issn = {1471-2970},
support = {/ERC_/European Research Council/International ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Bacteria/genetics/enzymology ; *Archaea/genetics/enzymology ; *Endonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; },
abstract = {Most type III CRISPR-Cas systems facilitate immune responses against invading mobile genetic elements such as phages by generating cyclic oligoadenylates (cOAs). Downstream effectors activated by cOAs are typically non-specific proteins that induce damage to essential cellular components, thereby preventing phage epidemics. Owing to these toxic effects, it is crucial that the production and concentration of cOAs remain under tight regulatory control during infection-free periods or when deactivating the immune response after clearing an infection. Type III CRISPR loci often encode enzymes known as ring nucleases (RNs) that bind and degrade specific cOAs, while some effectors are auto-deactivating. Despite the discovery of several classes of RNs, a comprehensive bioinformatic analysis of type III CRISPR-Cas loci in this context is lacking. Here, we examined 38 742 prokaryotic genomes to provide a global overview of type III CRISPR loci, focusing on the known and predicted RNs. The candidate RNs Csx16 and Csx20 are confirmed as active enzymes, joining Crn1-3. Distributions and patterns of co-occurrence of RNs and associated effectors are explored, allowing the conclusion that a sizeable majority of type III CRISPR systems regulate cOA levels by degrading the signalling molecules, which has implications for cell fate following viral infection.This article is part of the discussion meeting issue 'The ecology and evolution of bacterial immune systems'.},
}

*CRISPR-Cas Systems/genetics
*Bacteria/genetics/enzymology
*Archaea/genetics/enzymology
*Endonucleases/genetics/metabolism
*Bacterial Proteins/genetics/metabolism

@article {pmid40904105,
year = {2025},
author = {Elliott, JFK and Cozens, K and Cai, Y and Waugh, G and Watson, BN and Westra, E and Taylor, TB},
title = {Phage susceptibility to a minimal, modular synthetic CRISPR-Cas system in Pseudomonas aeruginosa is nutrient dependent.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {380},
number = {1934},
pages = {20240473},
pmid = {40904105},
issn = {1471-2970},
support = {//UK Government's Horizon Europe funding guarantee/ ; //Royal Society/ ; //Philip Leverhulme Prize/ ; /BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Pseudomonas aeruginosa/virology/genetics ; *CRISPR-Cas Systems ; *Pseudomonas Phages/physiology/genetics ; *Nutrients/metabolism ; *Bacteriophages/physiology ; },
abstract = {CRISPR-Cas systems can provide adaptive, heritable immunity to their prokaryotic hosts against invading genetic material such as phages. It is clear that the importance of acquiring CRISPR-Cas immunity to anti-phage defence varies across environments, but it is less clear if and how this varies across different phages. To explore this, we created a synthetic, modular version of the type I-F CRISPR-Cas system of Pseudomonas aeruginosa. We used this synthetic system to test CRISPR-Cas interference against a panel of 13 diverse phages using engineered phage-targeting spacers. We observed complete protection against eight of these phages, both lytic and lysogenic and with a range of infectivity profiles. However, for two phages, CRISPR-Cas interference was only partially protective in high-nutrient conditions, yet completely protective in low-nutrient conditions. This work demonstrates that nutrient conditions modulate the strength of CRISPR-Cas immunity and highlights the importance of environmental conditions when screening defence systems for their efficacy against various phages.This article is part of the discussion meeting issue 'The ecology and evolution of bacterial immune systems'.},
}

*Pseudomonas aeruginosa/virology/genetics
*CRISPR-Cas Systems
*Pseudomonas Phages/physiology/genetics
*Nutrients/metabolism
*Bacteriophages/physiology

@article {pmid40904104,
year = {2025},
author = {David, E and Plantady, C and Poissonnier, S and Elliott, JFK and Kenck, E and Le Boulch, J and Gutierrez, A and Chevallereau, A},
title = {Systematic functional assessment of anti-phage systems in their native host.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {380},
number = {1934},
pages = {20240067},
pmid = {40904104},
issn = {1471-2970},
support = {//ATIP-Avenir/ ; //Agence Nationale de la Recherche/ ; //Idex Paris Cité/ ; //Emergence ville de Paris/ ; },
mesh = {*Escherichia coli/virology/genetics ; *Coliphages/physiology ; *Bacteriophages/physiology ; DNA Restriction-Modification Enzymes/genetics ; },
abstract = {Bacterial resistance to bacteriophages (phages) relies on two primary strategies: preventing phage attachment and blocking post-attachment steps. These post-attachment mechanisms are mediated by diverse defence systems, including DNA-degrading systems such as restriction-modification and CRISPR-Cas, along with abortive infection systems that induce cell death or dormancy. Computational analyses suggest that bacterial genomes encode multiple defence systems, which may act synergistically to enhance phage resistance. However, the regulation, interactions and ecological roles of these systems in native hosts remain poorly understood. This study explored the role of eight predicted defence systems in the clinical isolate NILS69 of Escherichia coli by testing its susceptibility to 93 phages. Infectivity and adsorption assays using mutants defective in these systems revealed that only PD-T4-3 and restriction-modification systems restricted phages that were able to adsorb. The restriction-modification system acted via a predicted type IV endonuclease and was also able to limit plasmid conjugation if the plasmid was transferred from a donor strain lacking a methylase, which is the hallmark of type I, II or III restriction-modification systems. Other defence systems showed no detectable activity, likely owing to phage specificity, environmental regulation or cofactor requirements. These findings underscore the need for further studies to investigate the regulation and ecological roles of bacterial defence systems in their native host contexts.This article is part of the discussion meeting issue 'The ecology and evolution of bacterial immune systems'.},
}

*Escherichia coli/virology/genetics
*Coliphages/physiology
*Bacteriophages/physiology
DNA Restriction-Modification Enzymes/genetics

@article {pmid40904102,
year = {2025},
author = {Pons, BJ and Łapińska, U and Lopes-Domingues, I and Chisnall, MAW and Westra, ER and Pagliara, S and van Houte, S},
title = {Phage provoke growth delays and SOS response induction despite CRISPR-Cas protection.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {380},
number = {1934},
pages = {20240474},
pmid = {40904102},
issn = {1471-2970},
support = {/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; /MRC_/Medical Research Council/United Kingdom ; //UK Government's Horizon Europe funding guarantee/ ; //Leverhulme Trust/ ; },
mesh = {*Pseudomonas aeruginosa/virology/growth & development/genetics ; *CRISPR-Cas Systems ; *Pseudomonas Phages/physiology ; *SOS Response, Genetics ; *Bacteriophages/physiology ; },
abstract = {Bacteria evolve resistance against their phage foes with a wide range of resistance strategies whose costs and benefits depend on the level of protection they confer and on the costs for maintainance. Pseudomonas aeruginosa can evolve resistance against its phage DMS3vir either by surface mutations that prevent phage binding or through CRISPR-Cas immunity. CRISPR immunity carries an inducible cost whose exact origin is still unknown, and previous work suggested it stems from the inability of the CRISPR-Cas system to completely prevent phage DNA injection and subsequent gene expression before clearing the phage infection. However, the bacterial processes involved are still unknown, and we hypothesize that CRISPR-immunity-associated costs could come from increased mortality rate or reduced growth ability compared with surface-resistant bacteria. To tease apart these two mechanisms with divergent ecological consequences, we use a novel microfluidics-based single-cell approach combined with flow cytometry methods to monitor the effects of phage exposure on the survival and growth of its host. We observed that while CRISPR immunity protects from phage-induced lysis, it cannot prevent phage-induced division lag, filamentation and SOS response activation in a subpopulation of the host bacteria. These results suggest that the costs associated with CRISPR immunity at the population level are caused by heterogeneity in phage-induced growth defects.This article is part of the discussion meeting issue 'The ecology and evolution of bacterial immune systems'.},
}

*Pseudomonas aeruginosa/virology/growth & development/genetics
*CRISPR-Cas Systems
*Pseudomonas Phages/physiology
*SOS Response, Genetics
*Bacteriophages/physiology

@article {pmid40903118,
year = {2025},
author = {Zhou, J and Shi, X and He, C and Zheng, X and Yuan, R and Yang, X},
title = {SERS biosensor based on the Cas13a assisted entropy-driven system and lychee-like Fe-TiO2 with excellent exciton capture and separation.},
journal = {Analytica chimica acta},
volume = {1372},
number = {},
pages = {344442},
doi = {10.1016/j.aca.2025.344442},
pmid = {40903118},
issn = {1873-4324},
mesh = {*Titanium/chemistry ; *Spectrum Analysis, Raman/methods ; *Entropy ; *Biosensing Techniques/methods ; *Iron/chemistry ; CRISPR-Cas Systems ; *MicroRNAs/analysis ; Limit of Detection ; },
abstract = {BACKGROUND: Entropy-Driven Circuits (EDC), distinguished by their spontaneous operation and absence of enzymatic reactions, represent a superior strategy for integration with CRISPR/Cas systems, as they obviate the potential for interference among various enzymes during the process of DNA amplification and CRISPR/Cas system integration. Due to the wide band gap of TiO2, its response to visible light is limited, and owing to its high crystallinity and exceptionally stable crystal lattice, the charge transfer (CT) process in TiO2 is suboptimal.

RESULTS: In this study, lychee-like Fe-TiO2 was firstly prepared to serve as Raman enhanced substrate, facilitating exciton capture and separation to exhibit an excellent Surface-enhanced Raman spectroscopy (SERS) performance. It is proven that the incorporation of Fe results in a significantly narrower band gap for TiO2, facilitating exciton resonance. The amount of Fe in TiO2 was optimized to fabricate a SERS biosensor for detection of miRNA-21 based on the Cas13a assisted entropy-driven system. The detection limit of miRNA was 43.88 fmol/L.

SIGNIFICANCE: This work proposes a sensing strategy that integrates the CRISPR/Cas system with EDC, leveraging a semiconductor substrate exhibiting superior SERS performance to provide a stable Raman signal, thereby enabling highly sensitive detection of miRNA-21, which has a potential application in disease early warning and treatment.},
}

*Titanium/chemistry
*Spectrum Analysis, Raman/methods
*Entropy
*Biosensing Techniques/methods
*Iron/chemistry
CRISPR-Cas Systems
*MicroRNAs/analysis
Limit of Detection