@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

@article {pmid40902823,
year = {2025},
author = {Zhou, X and Diao, R and Li, X and Ziegler, CA and Gramelspacher, MJ and Freddolino, L and Hou, Z and Zhang, Y},
title = {Cas9 senses CRISPR RNA abundance to regulate CRISPR spacer acquisition.},
journal = {Nature},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41586-025-09577-9},
pmid = {40902823},
issn = {1476-4687},
abstract = {Prokaryotes create adaptive immune memories by acquiring foreign DNA snippets, known as spacers, into the CRISPR array[1]. In type II CRISPR-Cas systems, the RNA-guided effector Cas9 also assists the acquisition machinery by selecting spacers from protospacer adjacent motif (PAM)-flanked DNA[2,3]. Here, we uncover the first biological role for Cas9 that is independent of its dual RNA partners. Following depletion of crRNA and/or tracrRNA, Neisseria apoCas9 stimulates spacer acquisition efficiency. Physiologically, Cas9 senses low levels of crRNA in cells with short CRISPR arrays - such as those undergoing array neogenesis or natural array contractions - and dynamically upregulates acquisition to quickly expand the small immune memory banks. As the CRISPR array expands, rising crRNA abundance in turn reduces apoCas9 availability, thereby dampening acquisition to mitigate autoimmunity risks associate with elevated acquisition. While apoCas9's nuclease lobe alone suffices for stimulating acquisition, only full-length Cas9 responses to crRNA levels to boost acquisition in cells with low immunity depth. Finally, we show that this activity is evolutionarily conserved across multiple type II-C Cas9 orthologs. Altogether, we establish an auto-replenishing feedback mechanism in which apoCas9 safeguards CRISPR immunity depth by acting as both a crRNA sensor and a regulator of spacer acquisition.},
}

@article {pmid40902040,
year = {2025},
author = {Li, L and Dai, H and Sun, R and Zhang, Z and Zhang, B},
title = {MicroRNAs as Biotechnological Targets for Future Food Security and Agricultural Sustainability.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c07100},
pmid = {40902040},
issn = {1520-5118},
abstract = {MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate gene expression, playing key roles in plant growth, development, and stress responses. Their regulatory functions make miRNAs ideal targets for enhancing crop yield, quality, and stress tolerance using biotechnologies, such as transgenic overexpression and CRISPR/Cas genome editing. By targeting multiple genes, miRNAs address complex agricultural challenges effectively. This review focuses on the diverse roles of miRNAs in enhancing crop productivity and resilience; miRNAs are an important biotechnological target for ensuring food security and agricultural sustainability. We also highlight transgenic and CRISPR/Cas genome editing approaches to demonstrate miRNA-driven trait improvements, such as drought/salinity tolerance, pest resistance, and nutrient use efficiency. Due to the quick development of advanced biotechnology tools, both upregulated and downregulated miRNAs can be manipulated for optimizing agronomic traits. Challenges including off-target effects, regulatory barriers, and environmental concerns are analyzed with strategies proposed to overcome them. By leveraging miRNA technologies, this perspective emphasizes their transformative potentials in achieving sustainable agriculture and global food security.},
}

@article {pmid40901825,
year = {2025},
author = {Thiam, R and Ceballos, MS and Beneke, T and Kuk, N and Pasquier, G and Crobu, L and Jeffares, DC and Vergnes, B and Barckmann, B and Sterkers, Y},
title = {A novel Leishmania infantum reference strain for gene editing and the study of visceral leishmaniasis.},
journal = {PloS one},
volume = {20},
number = {9},
pages = {e0327390},
pmid = {40901825},
issn = {1932-6203},
mesh = {*Leishmania infantum/genetics/growth & development/pathogenicity ; *Leishmaniasis, Visceral/parasitology ; *Gene Editing/methods ; Animals ; Humans ; CRISPR-Cas Systems ; Mice ; Macrophages/parasitology ; Genome, Protozoan ; },
abstract = {Parasites of the Leishmania donovani complex are responsible for visceral leishmaniasis, a vector-borne disease transmitted through the bite of female phlebotomine sand flies. As well as the human hosts, these parasites infect many mammals which can serve as reservoirs. Dogs are particularly important reservoirs. Transmission is widespread across Asia, Africa, the Americas, and the Mediterranean basin, including South of France. Visceral leishmaniasis poses a fatal threat if left untreated. Research into the pathophysiology of this neglected disease is of prime importance, as is the development of new drugs. In this study, we evaluated the growth, differentiation, and macrophage infectivity of four L. donovani complex strains and identified L. infantum S9F1 (MHOM/MA/67/ITMAP263, clone S9F1) as a well-adapted strain for genetic engineering studies. We present here the genome sequence and annotation of L infantum S9F1 T7 Cas9, providing the scientific community with easy access to its genomic information. The data has been integrated into the LeishGEdit online resource to support primer design for CRISPR-Cas9 experiments. We now aim to make this strain widely available to foster studies of visceral leishmaniasis.},
}

*Leishmania infantum/genetics/growth & development/pathogenicity
*Leishmaniasis, Visceral/parasitology
*Gene Editing/methods
Animals
Humans
CRISPR-Cas Systems
Mice
Macrophages/parasitology
Genome, Protozoan

@article {pmid40788276,
year = {2025},
author = {Gao, H and Zhang, Y and Wang, Y and Wu, Y and Zheng, Z and Quan, F and Han, Q and Li, Y and Zhang, K},
title = {Integration of CRISPR/Cas12a and Cas13a in one pot for ratiometric calibration of single-nucleotide variations.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {72},
pages = {13675-13678},
doi = {10.1039/d5cc03931b},
pmid = {40788276},
issn = {1364-548X},
mesh = {*Polymorphism, Single Nucleotide ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; Calibration ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Humans ; },
abstract = {Accurate detection of single-nucleotide variations (SNVs) plays a pivotal role in medical diagnosis. Herein, by integrating Cas12a and Cas13a to simultaneously detect wild-type and mutated sites on a single RNA strand, we developed an innovative one-pot SNV analysis method, namely iCasdrop, which is capable of reducing non-specific signals induced by the wild type sequence and achieving ratiometric calibration of SNVs.},
}

*Polymorphism, Single Nucleotide
*CRISPR-Cas Systems/genetics
*CRISPR-Associated Proteins/genetics/metabolism
Calibration
*Endodeoxyribonucleases/genetics/metabolism
*Bacterial Proteins/genetics/metabolism
Humans

@article {pmid40599075,
year = {2025},
author = {Zhang, T and Cai, L and Chu, Z and She, A and Yang, J and Su, X},
title = {Rapid and Sensitive Detection of miRNA by Single-Molecule Fluorescence Dequenching Assay with Target Recycled CRISPR/Cas12a Amplification System.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {21},
number = {35},
pages = {e2412228},
doi = {10.1002/smll.202412228},
pmid = {40599075},
issn = {1613-6829},
support = {32271521//National Natural Science Foundation of China/ ; 31971361//National Natural Science Foundation of China/ ; 2022YFC2603902//State Key Research Development Program of China/ ; PT2406//Fundamental Research Funds for the Central Universities/ ; 2023-NHLHCRF-YXHZ-ZRMS-05//National High Level Hospital Clinical Research Funding/ ; },
mesh = {*MicroRNAs/genetics/metabolism/analysis ; Humans ; *CRISPR-Cas Systems/genetics ; Fluorescence ; Carcinoma, Non-Small-Cell Lung/genetics ; Lung Neoplasms/genetics ; Female ; Cell Line, Tumor ; },
abstract = {Dysregulated miRNAs play a critical role in the development of cancers. A rapid and sensitive single-molecule fluorescence dequenching assay combined with a CRISPR/Cas12a-based target recycling amplification system for miRNA detection is developed. This single-molecule assay detects miRNAs down to ≈10 fM within 10 min. An automated single-molecule fluorescent puncta analysis procedure is also created, improving the signal-to-noise ratio by 3.76-fold compared to traditional hidden Markov model (HMM)based methods. The clinical applicability of this technique is demonstrated. Two key miRNA targets associated with non-small cell lung cancer (NSCLC) and ovarian cancer (OC) from 2867 datasets of the TCGA database are screened. Validation is initially conducted at the cell line level, followed by testing with tissue and blood samples from 10 patients with NSCLC and OC. The assay demonstrated high diagnostic accuracy, with receiver operating characteristic curves (area under the curve (AUC) > 0.93) and significant statistical differentiation (p < 0.001) between cancer and healthy samples. This method's exceptional sensitivity and speed highlight its potential for early cancer diagnostics and personalized medicine.},
}

*MicroRNAs/genetics/metabolism/analysis
Humans
*CRISPR-Cas Systems/genetics
Fluorescence
Carcinoma, Non-Small-Cell Lung/genetics
Lung Neoplasms/genetics
Female
Cell Line, Tumor

@article {pmid40536333,
year = {2025},
author = {Zhao, G and Li, Z and Zhao, MJ and Li, SY and Xia, Q and Xu, S and Zhang, Y and Wang, Y and Li, F and Liu, YL and Guo, YH and Xu, RX and Zhou, H and Zhou, H and Ding, WW and Wang, YC and Miao, Y and Wang, Z},
title = {A High-Fidelity RNA-Targeting Cas13X Downregulates Connexin43 in Macroglia: A Novel Neuroprotective Strategy for Glaucoma.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {33},
pages = {e15856},
doi = {10.1002/advs.202415856},
pmid = {40536333},
issn = {2198-3844},
support = {82301215//National Natural Science Foundation of China/ ; 32271043//National Natural Science Foundation of China/ ; 32471057//National Natural Science Foundation of China/ ; 82171047//National Natural Science Foundation of China/ ; 2018SHZDZX01//Shanghai Municipal Science and Technology Major Project/ ; },
mesh = {*Connexin 43/genetics/metabolism ; *Glaucoma/genetics/metabolism/therapy ; Animals ; Mice ; Retinal Ganglion Cells/metabolism ; Disease Models, Animal ; CRISPR-Cas Systems/genetics ; Down-Regulation ; Intraocular Pressure ; Mice, Inbred C57BL ; Humans ; Optic Nerve/metabolism ; },
abstract = {Glaucoma is a neurodegenerative disease characterized by the progressive degeneration of retinal ganglion cells (RGCs) and their axons, ultimately leading to irreversible vision loss. Elevated intraocular pressure (IOP) is one of the significant risk factors in glaucoma; however, neurodegeneration continues even after effective IOP management, underscoring the need for neuroprotective therapies. This study investigates the role of connexin43 (Cx43), which is extensively expressed in retinal macroglia, in regulating microglial activation and optic nerve degeneration in glaucoma. A high-fidelity CRISPR-Cas13 (hfCas13X) system is employed to selectively target and knock down Cx43 expression in macroglia. The findings reveal that Cx43-mediated ATP release through hemichannels exacerbates microglial activation and neuroinflammation, thereby contributing to RGC loss. Notably, in a mouse model of chronic ocular hypertension (COH) glaucoma, knocking down Cx43 in macroglia using the hfCas13X system significantly promoted the survival of RGCs and the integrity of the optic nerve, and improved visual function. The hfCas13X system, which offers high-fidelity RNA editing with minimal off-target effects, represents a novel and promising therapeutic strategy for glaucoma, highlighting the potential of gene editing technologies in the management of neurodegenerative diseases.},
}

*Connexin 43/genetics/metabolism
*Glaucoma/genetics/metabolism/therapy
Animals
Mice
Retinal Ganglion Cells/metabolism
Disease Models, Animal
CRISPR-Cas Systems/genetics
Down-Regulation
Intraocular Pressure
Mice, Inbred C57BL
Humans
Optic Nerve/metabolism

@article {pmid40534129,
year = {2025},
author = {Nicosia, L and Harrison, PT},
title = {CRISPR for cystic fibrosis: Advances and insights from a systematic review.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {9},
pages = {4091-4112},
doi = {10.1016/j.ymthe.2025.06.021},
pmid = {40534129},
issn = {1525-0024},
mesh = {*Cystic Fibrosis/genetics/therapy ; Humans ; *Gene Editing/methods ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; *CRISPR-Cas Systems ; *Genetic Therapy/methods ; Mutation ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Cystic fibrosis (CF) is a severe genetic disorder caused by loss-of-function mutations in the CFTR gene. Gene-editing approaches have the potential to correct such mutations. This systematic review outlines the mechanisms of the main CRISPR-based technologies, and, through cross-study comparisons, analyzes 27 research articles that applied them to target CF-causing variants. We report and discuss the strategy design, target cell selection, editing efficiency, prevalence of editing byproducts, and levels of CFTR functional restoration achieved in each work, with the aim of providing technical insights for further exploration of CRISPR-based gene-editing approaches. Our findings show that the F508del and W1282X mutations were the most extensively studied CF-causing variants, though over fifteen mutations were targeted overall. The majority of works under review explored the use of homology-directed repair or base editing, with a growing number of studies reporting efficient prime editing. Some studies tackled multiple individual mutations, compared different editors, or tested strategies across various models, while others focused on approaches that rescue CFTR function without directly correcting a mutation. Several works also proposed strategies that could address multiple variants with a single approach, while others highlighted technical difficulties in editing certain regions of the CFTR gene. This cross-study comparison also emphasizes the need for standardized reporting of editing efficiency and functional recovery, and stresses the importance of further single-cell RNA sequencing and in vivo studies to reach clinically relevant conclusions. As gene-editing techniques continue to evolve, and with over 60 ongoing CRISPR-based clinical trials, there is growing optimism for meaningful advancements in CF gene-editing therapeutics.},
}

*Cystic Fibrosis/genetics/therapy
Humans
*Gene Editing/methods
*Cystic Fibrosis Transmembrane Conductance Regulator/genetics
*CRISPR-Cas Systems
*Genetic Therapy/methods
Mutation
Animals
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40518666,
year = {2025},
author = {Yang, Y and Fu, Z and Deng, S and Wu, G and Wang, C and Luo, X and Kang, R and Chen, Y and Peng, C and Zhang, P and Cui, K and Wan, F and Wang, J and Zhou, Q and Chen, W and Xiong, Y and Ma, W and Songyang, Z and Liang, P},
title = {RNA-DNA hybrid binding domain broadens the editing window of base editors.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {9},
pages = {4431-4446},
doi = {10.1016/j.ymthe.2025.06.024},
pmid = {40518666},
issn = {1525-0024},
mesh = {*Gene Editing/methods ; Humans ; Animals ; Mice ; *DNA/genetics/metabolism/chemistry ; CRISPR-Cas Systems ; Ribonuclease H/genetics/metabolism ; *RNA/genetics/metabolism/chemistry ; },
abstract = {Adenine base editors (ABEs) and cytosine base editors (CBEs) are prominent tools for precise genome editing but are hindered by limited editing activity at positions proximal to the protospacer adjacent motif (PAM). This study investigates the potential of enhancing base editors editing activity by fusing them with RNA-DNA hybrid binding domains (RHBDs). Specifically, fusing ABE8e with the RHBD of Homo sapiens RNaseH1 (RHBD1) significantly increased A-to-G editing efficiency in the PAM-proximal region (A9-A15) by up to 3.5-fold, while reducing off-target cytosine editing. Additionally, RHBD1 is compatible with ABEmax, BE4max, and dual base editor (eA&C-BEmax), enhancing their editing activity at the PAM-proximal bases. Notably, RHBD1-fused BE4max led to a 3.1-fold improvement in C-to-T editing efficiency at PAM-proximal region (C9-C12). Furthermore, we demonstrated that RHBD1-fused ABE8e could effectively edit disease-related single nucleotide variations (SNVs) in human cells and validated its efficacy in adult mouse liver. These findings highlight the significance of the RHBD in expanding editing window and the applicability of base editors for gene therapy and disease modeling.},
}

*Gene Editing/methods
Humans
Animals
Mice
*DNA/genetics/metabolism/chemistry
CRISPR-Cas Systems
Ribonuclease H/genetics/metabolism
*RNA/genetics/metabolism/chemistry

@article {pmid40493884,
year = {2025},
author = {Loke, J and Kim, PG and Nguyen, TTP and Boileau, M and McConkey, M and Miller, A and Shin, W and Hergott, CB and Ericsson, M and Nordstrom, A and Llopis, PM and Armstrong, SA and Mancias, JD and Ebert, BL},
title = {An in vivo barcoded CRISPR-Cas9 screen identifies Ncoa4-mediated ferritinophagy as a dependence in Tet2-deficient hematopoiesis.},
journal = {Blood},
volume = {146},
number = {10},
pages = {1174-1186},
doi = {10.1182/blood.2024028033},
pmid = {40493884},
issn = {1528-0020},
mesh = {*CRISPR-Cas Systems ; *Hematopoiesis/genetics ; *Ferritins/metabolism/genetics ; *DNA-Binding Proteins/genetics/deficiency/metabolism ; Animals ; *Nuclear Receptor Coactivators/metabolism/genetics ; Dioxygenases ; Mice ; *Proto-Oncogene Proteins/genetics/deficiency ; Hematopoietic Stem Cells/metabolism ; Mice, Knockout ; *Autophagy ; Iron/metabolism ; Humans ; },
abstract = {TET2 is among the most commonly mutated genes in both clonal hematopoiesis and myeloid malignancies; thus, the ability to identify selective dependencies in TET2-deficient cells has broad translational significance. Here, we identify regulators of Tet2 knockout (KO) hematopoietic stem and progenitor cell (HSPC) expansion using an in vivo CRISPR-Cas9 KO screen, in which nucleotide barcoding enabled large-scale clonal tracing of Tet2-deficient HSPCs in a physiologic setting. Our screen identified candidate genes, including Ncoa4, that are selectively required for Tet2 KO clonal outgrowth compared with wild type. Ncoa4 targets ferritin for lysosomal degradation (ferritinophagy), maintaining intracellular iron homeostasis by releasing labile iron in response to cellular demands. In Tet2-deficient HSPCs, increased mitochondrial adenosine triphosphate production correlates with increased cellular iron requirements and, in turn, promotes Ncoa4-dependent ferritinophagy. Restricting iron availability reduces Tet2 KO stem cell numbers, revealing a dependency in TET2-mutated myeloid neoplasms.},
}

*CRISPR-Cas Systems
*Hematopoiesis/genetics
*Ferritins/metabolism/genetics
*DNA-Binding Proteins/genetics/deficiency/metabolism
Animals
*Nuclear Receptor Coactivators/metabolism/genetics
Dioxygenases
Mice
*Proto-Oncogene Proteins/genetics/deficiency
Hematopoietic Stem Cells/metabolism
Mice, Knockout
*Autophagy
Iron/metabolism
Humans

@article {pmid40492429,
year = {2025},
author = {Meng, R and Li, J and Wang, W and Liang, D and Li, Z and Mao, C and Li, Q and Zhang, Y and Chen, H and Tang, J and Hu, P and Niu, Q and Huang, X and Shen, B and Zhang, J},
title = {Engineered Cas12j-8 is a Versatile Platform for Multiplexed Genome Modulation in Mammalian Cells.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {33},
pages = {e02593},
doi = {10.1002/advs.202502593},
pmid = {40492429},
issn = {2198-3844},
support = {2022YFC2702705//National Key Research and Development Project of China/ ; 82221005//Creative Research Groups of China/ ; 32371546//National Natural Science Foundation of China/ ; 82071434//National Natural Science Foundation of China/ ; 81971398//National Natural Science Foundation of China/ ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; Animals ; HEK293 Cells ; *CRISPR-Associated Proteins/genetics ; },
abstract = {Cas12j-8 is a compact Cas nuclease discovered from the metagenome of giant bacteriophages, consisting of only 717 amino acids and recognizing the '5-TTN-3' protospacer adjacent motif (PAM) sequence. However, its low gene editing efficiency in mammalian cells limits its application in therapeutic gene editing. To address this limitation, structure-guided mutagenesis is employed to replace key negatively charged residues with arginine, strengthening DNA binding. The resulting quintuple mutant, engineered Cas12j-8 (enCas12j-8), demonstrates robust on-target editing efficiency comparable to LbCas12a while maintaining low off-target effects. Cytosine base editors (CBEs) and adenine base editors (ABEs) are developed using enCas12j-8, achieving up to 29.54-fold C-to-T and 36.57-fold A-to-G conversion efficiency compared with the wild-type at the dominated sites, respectively. Notably, enCas12j-8 enables multiplexed editing of three genomic loci simultaneously via a single crRNA array, achieving efficiencies comparable to single-guide approaches. Additionally, enCas12j-8-ABE facilitates the disruption of splice acceptor sites, effectively inducing exon skipping in the SOD1 gene. This strategy holds potential significance for therapeutic genome modulation. These findings establish enCas12j-8 as a versatile, high-precision tool for genome engineering, combining efficient delivery, multiplexing capability, and compatibility with diverse editing modalities.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems/genetics
Animals
HEK293 Cells
*CRISPR-Associated Proteins/genetics

@article {pmid40901634,
year = {2025},
author = {Jiang, X and Wang, X and Shen, S and Hou, S and Yu, C},
title = {3D Genome Engineering: Current Advances and Therapeutic Opportunities in Human Diseases.},
journal = {Research (Washington, D.C.)},
volume = {8},
number = {},
pages = {0865},
doi = {10.34133/research.0865},
pmid = {40901634},
issn = {2639-5274},
abstract = {Dynamic chromatin 3-dimensional (3D) conformation is a key mechanism regulating gene expression and cellular function during development and disease. Elucidating the structure, functional dynamics, and spatiotemporal organization of the 3D genome requires integrating multiple experimental approaches, including chromatin conformation capture techniques, precise genome manipulation tools, and advanced imaging technologies. Notably, CRISPR/Cas systems have emerged as a revolutionary genome-editing platform, offering unprecedented opportunities for manipulating 3D genome organization and investigating disease mechanisms. This review systematically examines recent advances in CRISPR-based mammalian 3D genome engineering and explores the therapeutic potential of 3D genome engineering strategies in disease intervention.},
}

@article {pmid40900077,
year = {2025},
author = {Yu, Y and Zhang, Z and Zhai, Z and Sun, B and Yang, D and Wang, Z and Lin, Q and Zhou, X and Zhao, J},
title = {A Novel Mouse Model of Granular Corneal Dystrophy Type II Reveals Impaired Autophagy and Recapitulates Human Pathogenesis.},
journal = {Investigative ophthalmology & visual science},
volume = {66},
number = {12},
pages = {7},
doi = {10.1167/iovs.66.12.7},
pmid = {40900077},
issn = {1552-5783},
mesh = {Animals ; *Corneal Dystrophies, Hereditary/genetics/pathology/metabolism ; *Autophagy/physiology ; *Disease Models, Animal ; Mice ; Microscopy, Electron, Transmission ; Tomography, Optical Coherence ; Transforming Growth Factor beta/genetics/metabolism ; Blotting, Western ; Extracellular Matrix Proteins/genetics/metabolism ; Humans ; *Mutation ; Mice, Inbred C57BL ; Slit Lamp Microscopy ; CRISPR-Cas Systems ; Phenotype ; *Cornea/pathology ; },
abstract = {PURPOSE: To develop and characterize a novel mouse model of granular corneal dystrophy type II (GCD2) using CRISPR/Cas9 technology and explore the underlying pathogenesis of transforming growth factor-beta-induced protein (TGFBIp) aggregation.

METHODS: CRISPR/Cas9 technology was employed to introduce the R124H mutation in the TGFBI gene of mice. Genomic sequencing and polymerase chain reaction confirmed the mutation. Phenotypic characteristics were evaluated through slit-lamp examination, optical coherence tomography, histological analysis, electron microscopy, and immunofluorescence, comparing wild-type (WT), heterozygous (HE), and homozygous (HO) mice. Transcriptome sequencing was conducted to identify the pathogenesis of GCD2. The findings were further validated through western blotting and transmission electron microscopy.

RESULTS: The R124H mutation in TGFBI was successfully introduced, with breadcrumb-like deposits observed in the corneas of mutant mice, with HO mice displaying more severe phenotypes than HE mice. TGFBIp levels were elevated in HE and HO mice (both P < 0.001). Histological and electron microscopy analyses revealed abnormal collagen arrangement and TGFBIp deposits in the corneal stroma of the HE and HO mice. Transcriptome analysis indicated that the TGFBI-R124H mutation was associated with impaired autophagy, endocytosis, and extracellular matrix signaling. Additional experiments confirmed autophagy-related markers LC3 and SQSTM1 were upregulated in the corneas of mutant mice, accompanied by increased autophagosome formation in corneal keratocytes, indicating impaired autophagy flux in HE and HO mice.

CONCLUSIONS: We established a GCD2 mouse model caused by the R124H mutation using CRISPR/Cas9, providing a reliable platform for understanding pathogenesis for GCD2.},
}

Animals
*Corneal Dystrophies, Hereditary/genetics/pathology/metabolism
*Autophagy/physiology
*Disease Models, Animal
Mice
Microscopy, Electron, Transmission
Tomography, Optical Coherence
Transforming Growth Factor beta/genetics/metabolism
Blotting, Western
Extracellular Matrix Proteins/genetics/metabolism
Humans
*Mutation
Mice, Inbred C57BL
Slit Lamp Microscopy
CRISPR-Cas Systems
Phenotype
*Cornea/pathology

@article {pmid40897812,
year = {2025},
author = {Wolter, JM and James, LM and Boeshore, SL and Mao, H and McCoy, ES and Ryan, DF and Fragola, G and Taylor-Blake, B and Stein, JL and Zylka, MJ},
title = {AAV-dCas9 vector unsilences paternal Ube3a in neurons by impeding Ube3a-ATS transcription.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1332},
pmid = {40897812},
issn = {2399-3642},
support = {631904//Simons Foundation/ ; 1R01NS109304//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; T32HD040127//U.S. Department of Health & Human Services | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; },
mesh = {Animals ; *Dependovirus/genetics ; Mice ; *Neurons/metabolism ; Humans ; *Ubiquitin-Protein Ligases/genetics/metabolism ; Genetic Vectors/genetics ; *Angelman Syndrome/genetics/therapy ; *Transcription, Genetic ; *CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Male ; *RNA, Long Noncoding/genetics ; },
abstract = {Angelman syndrome (AS) is a debilitating neurodevelopmental disorder caused by loss of maternally-inherited UBE3A. In neurons, paternally-inherited UBE3A is silenced in cis by a long non-coding RNA called Ube3a-ATS. Here, we found that Neisseria meningitidis Cas9 with two mutations (D15A and H587A) in the nuclease domains (dNmCas9) can unsilence the dormant paternal Ube3a allele in mouse and human neurons when targeted to Snord115 snoRNA genes located in introns of Ube3a-ATS. Importantly, dNmCas9 disrupted Ube3a-ATS with a non-template bias and in the absence of a chromatin modifying domain, supporting a transcriptional interference mechanism. When packaged into an adeno-associated virus (AAV) vector, dNmCas9 exhibited dose-dependent Ube3a-ATS knock-down and paternal Ube3a unsilencing in vitro and in vivo. This vector also partially rescued the hind limb clasp phenotype when delivered to neonatal AS model mice. Collectively, our study underscores the potential of dCas9-based therapeutics without chromatin repression domains to mediate transcriptional downregulation.},
}

Animals
*Dependovirus/genetics
Mice
*Neurons/metabolism
Humans
*Ubiquitin-Protein Ligases/genetics/metabolism
Genetic Vectors/genetics
*Angelman Syndrome/genetics/therapy
*Transcription, Genetic
*CRISPR-Associated Protein 9/genetics/metabolism
CRISPR-Cas Systems
Male
*RNA, Long Noncoding/genetics

@article {pmid40897312,
year = {2025},
author = {Bircheneder, M and Parniske, M},
title = {Engineering and comparison of cas12a-based genome editing systems in plants.},
journal = {The Plant journal : for cell and molecular biology},
volume = {123},
number = {5},
pages = {e70410},
pmid = {40897312},
issn = {1365-313X},
support = {401867691//Deutsche Forschungsgemeinschaft/ ; //Bayerisches Staatsministerium für Unterricht und Kultus/ ; },
mesh = {*Gene Editing/methods ; *Arabidopsis/genetics ; Nicotiana/genetics ; CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; *Lotus/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Plants, Genetically Modified ; },
abstract = {While Cas9 and Cas12a are both RNA-guided endonucleases used for genome editing, only Cas12a is able to process pre-crRNA via its additional ribonuclease activity. This feature reduces the complexity of Cas12a versus Cas9-based genome editing systems thus providing an attractive alternative for generating site-specific mutations in plants. Here we aimed to improve the efficiency of the cas12a-based generation of two double-strand breaks flanking the open reading frame of a target gene, leading to its full deletion. To this end, we compared the relative impact of different components on cas12a-based gene deletion efficiency in three different eudicotyledons, Arabidopsis thaliana, Lotus japonicus, and Nicotiana benthamiana. We detected the highest cas12a-based editing efficiency with a combination of suitable promoters for crRNA and cas12a expression, a tandem terminator to control cas12a expression, a re-coded cas12a, adapted to the codon usage of Arabidopsis and engineered to carry introns, and encoding a Cas12a flanked by a nuclear localization signal at both ends. Our work revealed the high potential for improving cas12a-based genome editing systems for plant genetic research.},
}

*Gene Editing/methods
*Arabidopsis/genetics
Nicotiana/genetics
CRISPR-Cas Systems/genetics
Genome, Plant/genetics
*Lotus/genetics
*CRISPR-Associated Proteins/genetics/metabolism
*Endodeoxyribonucleases/genetics/metabolism
*Bacterial Proteins/genetics/metabolism
Plants, Genetically Modified

@article {pmid40896718,
year = {2025},
author = {Madsen, CK and Hanak, T and Aronsson, H and Brinch-Pedersen, H},
title = {Rapid one-step CRISPR-cas vector assembly by isothermal spacer removal linearization and sequence-ligation independent cloning (ISRL-SLIC).},
journal = {MethodsX},
volume = {15},
number = {},
pages = {103567},
pmid = {40896718},
issn = {2215-0161},
abstract = {CRISPR-Cas genome editing is a powerful tool in various fields, but current cloning methods can be time-consuming due to the frequent use of intermediate entry vectors and multiple steps involving restriction enzymes and ligases. These multiple steps can create a bottleneck in CRISPR-Cas experiments. In response to this challenge, we propose a highly efficient streamlined approach, which enables simultaneous linearization of the acceptor plasmid and protospacer cloning in a single isothermal reaction. This eliminates the need for entry vectors, pre-linearization of vectors, and in vitro ligation, thus significantly simplifying the cloning process. The method can be applied to clone short synthetic oligos for single protospacer constructs or multiple amplicons for multiplex genome editing designs. Either way, researchers can proceed directly to Escherichia coli transformation after a one-hour isothermal reaction and recover the final construct within two days. By combining the advantages of sequence-ligation independent cloning (SLIC) cloning with a streamlined workflow, our approach facilitates rapid and efficient construction of CRISPR-Cas vectors and holds the promise of accelerating research and development in genome editing and related fields. To expedite the cloning of constructs, we propose a rapid one-step CRISPR-Cas vector assembly method that combines isothermal spacer removal with a sequence-ligation-independent cloning reaction. We could show that Isothermal Spacer Removal Linearization and Sequence-Ligation Independent Cloning (ISRL-SLIC) can create single, double and triple protospacer constructs in one reaction with scalability. The ISRL-SLIC reaction delivers clones under a broad range of oligo concentration making it a robust and time saving alternative to other methods for constructing CRISPR-Cas vectors.},
}

@article {pmid40895203,
year = {2025},
author = {Kesarwani, P and Sundar, D},
title = {Conformational changes induced by K949A mutation in the CRISPR-Cas12a complex drives an effective target-binding mechanism.},
journal = {Current research in structural biology},
volume = {10},
number = {},
pages = {100173},
pmid = {40895203},
issn = {2665-928X},
abstract = {The CRISPR/Cas system is a potential tool for genome editing, yet it faces challenges due to off-target activity caused by mismatches at specific positions. However, Off-target activity can be minimized by optimal design of guide RNA (gRNA) but there remains a possibility of unintended cleavage, highlighting the role of the Cas nuclease in off-target recognition and binding the target site. This study focuses on comparing the conformational dynamics and stability of Wildtype, RR, RVR, RRm and RVRm variants of AsCas12a with gRNA-DNA bound complexes. It was found that the cross-correlation coefficient between His1167 of the NUC domain and Thr384 of the REC II domain significantly increased after the K949A mutation compared to other variants. The extensive spread of principal components also revealed flexibility in both Cas nuclease and gRNA-DNA hybrid of RVR variant and wildtype AsCas12a whereas the confined clusters in PCA plot suggests increased stability in both the variants after mutation. This study shows the role of K949A mutation in improving stability of PAM variants and predicted critical residues such as His1167, Thr384 and Ser959, in inducing stability in mutants of PAM variants.},
}

@article {pmid40893972,
year = {2025},
author = {},
title = {Correction to "High-efficiency genome editing of an extreme thermophile Thermus thermophilus using endogenous type I and type III CRISPR-Cas systems".},
journal = {mLife},
volume = {4},
number = {4},
pages = {470},
doi = {10.1002/mlf2.70010},
pmid = {40893972},
issn = {2770-100X},
abstract = {[This corrects the article DOI: 10.1002/mlf2.12045.].},
}

@article {pmid40891977,
year = {2025},
author = {Li, X and Zhong, Y and Jin, C and Chen, X and Cui, X and Xu, Y and Fan, Y and Song, F and Cen, P and Dong, L and Yu, K and He, Q and Wang, J and Hu, S and Zhang, XY and Li, C and Tian, M and Zhang, H},
title = {CRISPR/Cas9-Engineered Triple-Fusion Reporter Gene Imaging System for Monitoring Transplanted Neural Progenitor Cells in Ischemic Stroke.},
journal = {Radiology},
volume = {316},
number = {3},
pages = {e250305},
doi = {10.1148/radiol.250305},
pmid = {40891977},
issn = {1527-1315},
mesh = {Animals ; *Neural Stem Cells/transplantation ; Rats ; Male ; *Ischemic Stroke/diagnostic imaging/therapy ; *CRISPR-Cas Systems/genetics ; *Genes, Reporter/genetics ; Magnetic Resonance Imaging/methods ; Humans ; Disease Models, Animal ; Rats, Sprague-Dawley ; Positron Emission Tomography Computed Tomography/methods ; },
abstract = {Background Neural progenitor cell therapy holds great potential for repairing brain damage induced by ischemic stroke, and molecular imaging plays a crucial role in evaluating the therapeutic efficacy of neural progenitor cell transplantation. However, the presence of the blood-brain barrier significantly limits the effectiveness of such imaging methods. Purpose To enable long-term monitoring of transplanted human neural progenitor cells (hNPCs) in a rat model of ischemic stroke by combining a clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9)-engineered triple-fusion (TF) reporter gene system with a noninvasive adenosine agonistic micelle (AM)-based probe delivery strategy. Materials and Methods Between January 2021 and May 2025, 60 male rats that were 2 months old were included. Thirty-seven rats with stroke were administered either TF human NPCs (hNPCs) or vehicle (culture media) and underwent MRI, bioluminescence imaging, PET/CT, and neurologic assessments at weeks 1, 2, 4, and 8 after transplantation. Comparisons between groups were determined by t tests, one-way analysis of variance, linear regression, and linear mixed-effects model. Results TF-hNPCs proliferated within the ischemic rat brain (week 8 vs week 1, bioluminescence imaging and PET: P < .001 and P = .02, respectively) and exhibited progressive migration and maturation by 8 weeks after transplantation (proportion of microtubule-associated protein 2-positive TF-hNPCs at week 8 vs week 4: 94.08% ± 3.02 vs 85.47% ± 6.54, respectively [P = .04]; proportion of doublecortin-positive TF-hNPCs at week 4 vs week 2: 83.90% ± 2.84 vs 59.74% ± 0.55, respectively [P = .02]). Moreover, TF-hNPC transplantation increased glucose (fluorine 18 fluorodeoxyglucose) uptake in the ischemic brain (TF-hNPCs vs vehicle at week 4, 0.58 ± 0.04 vs 0.37 ± 0.05, respectively [P = .008]; TF-hNPCs vs vehicle at week 8, 0.52 ± 0.06 vs 0.29 ± 0.02, respectively [P = .01]) and attenuated neurologic deficits compared with the vehicle group (neurologic score, TF-hNPCs vs vehicle at week 8: 9.6 ± 0.25 vs 7.6 ± 0.3, respectively; P = .003). Conclusion A CRISPR/Cas9-engineered TF reporter gene imaging system combined with a noninvasive AM-based approach enabled in vivo monitoring of transplanted human NPCs in a rat model of ischemic stroke. © RSNA, 2025 Supplemental material is available for this article. See also the editorial by Chapelin in this issue.},
}

Animals
*Neural Stem Cells/transplantation
Rats
Male
*Ischemic Stroke/diagnostic imaging/therapy
*CRISPR-Cas Systems/genetics
*Genes, Reporter/genetics
Magnetic Resonance Imaging/methods
Humans
Disease Models, Animal
Rats, Sprague-Dawley
Positron Emission Tomography Computed Tomography/methods

@article {pmid40857331,
year = {2025},
author = {Farr, GH and Reid, W and Hasegawa, EH and Azzam, A and Young, I and Li, ML and Olson, AK and Beier, DR and Maves, L},
title = {A systems genetics approach identifies roles for proteasome factors in heart development and congenital heart defects.},
journal = {PLoS genetics},
volume = {21},
number = {8},
pages = {e1011579},
pmid = {40857331},
issn = {1553-7404},
mesh = {Animals ; Zebrafish/genetics/embryology ; *Proteasome Endopeptidase Complex/genetics/metabolism ; *Heart Defects, Congenital/genetics/pathology ; *Heart/embryology/growth & development ; Humans ; *Zebrafish Proteins/genetics/metabolism ; Protein Interaction Maps/genetics ; Gene Expression Regulation, Developmental ; Transcriptome/genetics ; Mutation ; CRISPR-Cas Systems ; },
abstract = {Congenital heart defects (CHDs) occur in about 1% of live births and are the leading cause of infant death due to birth defects. While there have been remarkable efforts to pursue large-scale whole-exome and genome sequencing studies on CHD patient cohorts, it is estimated that these approaches have thus far accounted for only about 50% of the genetic contribution to CHDs. We sought to take a new approach to identify genetic causes of CHDs. By combining analyses of genes that are under strong selective constraint along with published embryonic heart transcriptomes, we identified over 200 new candidate genes for CHDs. We utilized protein-protein interaction (PPI) network analysis to identify a functionally-related subnetwork consisting of known CHD genes as well as genes encoding proteasome factors, in particular POMP, PSMA6, PSMA7, PSMD3, and PSMD6. We used CRISPR targeting in zebrafish embryos to preliminarily identify roles for the PPI subnetwork genes in heart development. We then used CRISPR to create new mutant zebrafish strains for two of the proteasome genes in the subnetwork: pomp and psmd6. We show that loss of proteasome gene functions leads to defects in zebrafish heart development, including dysmorphic hearts, myocardial cell blebbing, and reduced outflow tracts. We also identified deficits in cardiac function in pomp and psmd6 mutants. These heart defects resemble those seen in zebrafish mutants for known CHD genes and other critical heart development genes. Our study provides a novel systems genetics approach to further our understanding of the genetic causes of human CHDs.},
}

Animals
Zebrafish/genetics/embryology
*Proteasome Endopeptidase Complex/genetics/metabolism
*Heart Defects, Congenital/genetics/pathology
*Heart/embryology/growth & development
Humans
*Zebrafish Proteins/genetics/metabolism
Protein Interaction Maps/genetics
Gene Expression Regulation, Developmental
Transcriptome/genetics
Mutation
CRISPR-Cas Systems

@article {pmid40779935,
year = {2025},
author = {Wu, J and Li, X and Yin, X and Hu, J and Zhou, P and Zhong, X and Wu, M},
title = {Rapid detection of plasma exosomal LncRNA CASC9 for HCC using RT-RPA-CRISPR/Cas12a assay.},
journal = {Journal of pharmaceutical and biomedical analysis},
volume = {266},
number = {},
pages = {117085},
doi = {10.1016/j.jpba.2025.117085},
pmid = {40779935},
issn = {1873-264X},
mesh = {Humans ; *Carcinoma, Hepatocellular/blood/diagnosis/genetics ; *Liver Neoplasms/blood/diagnosis/genetics ; *Exosomes/genetics/metabolism ; *RNA, Long Noncoding/blood/genetics ; Biomarkers, Tumor/blood/genetics ; Male ; Female ; Middle Aged ; ROC Curve ; CRISPR-Cas Systems/genetics ; alpha-Fetoproteins ; Real-Time Polymerase Chain Reaction/methods ; Aged ; },
abstract = {Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide. Early detection is essential for improving patient outcomes. Long non-coding RNAs (lncRNAs) in plasma exosomes have emerged as promising non-invasive biomarkers. However, sensitive detection methods remain limited. Plasma exosomes were isolated and validated using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot (WB). RNA sequencing identified CASC9 as the most significantly upregulated exosomal lncRNA in HCC patients. Its diagnostic value was evaluated using real-time quantitative PCR (RT-qPCR) and a novel RT-RPA-CRISPR/Cas12a fluorescence assay. Diagnostic performance was assessed through receiver operating characteristic (ROC) curve analysis and compared with alpha-fetoprotein (AFP). Exosomal CASC9 levels were significantly elevated in HCC patients and correlated with tumor size, stage, and number (P < 0.001). ROC analysis demonstrated that CASC9 had superior diagnostic accuracy (area under the curve [AUC] = 0.822) compared to AFP (AUC = 0.795), with further improvement when combined (AUC = 0.875). The RT-RPA-CRISPR/Cas12a assay achieved a detection limit of 0.1 copies/μL, outperforming RT-qPCR. When combined with RT-qPCR and AFP, the method achieved an AUC of 0.987 against normal controls and 0.975 against benign cases. Plasma exosomal CASC9 is a promising diagnostic biomarker for HCC. The RT-RPA-CRISPR/Cas12a assay offers a rapid, ultra-sensitive, and clinically feasible detection strategy.},
}

Humans
*Carcinoma, Hepatocellular/blood/diagnosis/genetics
*Liver Neoplasms/blood/diagnosis/genetics
*Exosomes/genetics/metabolism
*RNA, Long Noncoding/blood/genetics
Biomarkers, Tumor/blood/genetics
Male
Female
Middle Aged
ROC Curve
CRISPR-Cas Systems/genetics
alpha-Fetoproteins
Real-Time Polymerase Chain Reaction/methods
Aged

@article {pmid40764055,
year = {2025},
author = {Stewart-Ornstein, J and Irby, MJ and Lilieholm, MK and Laprise, D and Collier, MD and Aunins, T and Harjanto, D and Chang, AN and Reyon, D and Duffield, JS},
title = {3'-end ligation sequencing is a sensitive method to detect DNA nicks at potential sites of off-target activity induced by prime editors.},
journal = {Genome research},
volume = {35},
number = {9},
pages = {2064-2075},
pmid = {40764055},
issn = {1549-5469},
mesh = {Humans ; *Gene Editing/methods ; *Sequence Analysis, DNA/methods ; Genome, Human ; CRISPR-Cas Systems ; *DNA/genetics ; DNA Breaks, Double-Stranded ; },
abstract = {Gene editing makes precise changes in DNA to restore normal function or expression of genes; however, the advancement of gene editing to the clinic is limited by the potential genotoxicity of off-target editing. To comprehensively identify potential sites in the genome that may be recognized by gene editing agents, in vitro approaches, in which the editor is combined with human genomic DNA and sites where editing may occur are identified biochemically, are important tools. Existing biochemical approaches for off-target discovery recognize double-stranded breaks generated by nuclease-based gene editors such as SpCas9, but novel approaches are needed for new editing modalities, such as prime editing, that nick one strand of DNA. To fill this gap, we have developed 3'-end ligation sequencing (PEG-seq), which can identify prime editor-induced nicks throughout the genome on in vitro digested human genomic DNA to identify potential off-target sites. Here we show that PEG-seq is an important addition to the off-target detection toolkit, enabling off-target discovery for DNA nicking gene editors such as prime editors.},
}

Humans
*Gene Editing/methods
*Sequence Analysis, DNA/methods
Genome, Human
CRISPR-Cas Systems
*DNA/genetics
DNA Breaks, Double-Stranded

@article {pmid40757665,
year = {2025},
author = {Carlsson, PO and Hu, X and Scholz, H and Ingvast, S and Lundgren, T and Scholz, T and Eriksson, O and Liss, P and Yu, D and Deuse, T and Korsgren, O and Schrepfer, S},
title = {Survival of Transplanted Allogeneic Beta Cells with No Immunosuppression.},
journal = {The New England journal of medicine},
volume = {393},
number = {9},
pages = {887-894},
doi = {10.1056/NEJMoa2503822},
pmid = {40757665},
issn = {1533-4406},
mesh = {Humans ; Male ; *Diabetes Mellitus, Type 1/therapy/surgery ; *Islets of Langerhans Transplantation/methods ; Transplantation, Homologous ; Gene Editing ; Adult ; *Graft Survival ; CRISPR-Cas Systems ; Insulin/metabolism ; *Insulin-Secreting Cells/transplantation ; },
abstract = {The need to suppress a patient's immune system after the transplantation of allogeneic cells is associated with wide-ranging side effects. We report the outcomes of transplantation of genetically modified allogeneic donor islet cells into a man with long-standing type 1 diabetes. We used clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 12b (Cas12b) editing and lentiviral transduction to genetically edit the cells to avoid rejection; the cells were then transplanted into the participant's forearm muscle. He did not receive any immunosuppressive drugs and, at 12 weeks after transplantation, showed no immune response against the gene-edited cells. C-peptide measurements showed stable and glucose-responsive insulin secretion. A total of four adverse events occurred, none of which were serious or related to the study drug. (Funded by the Leona M. and Harry B. Helmsley Charitable Trust; EudraCT number, 2023-507988-19-00; ClinicalTrials.gov number, NCT06239636.).},
}

Humans
Male
*Diabetes Mellitus, Type 1/therapy/surgery
*Islets of Langerhans Transplantation/methods
Transplantation, Homologous
Gene Editing
Adult
*Graft Survival
CRISPR-Cas Systems
Insulin/metabolism
*Insulin-Secreting Cells/transplantation

@article {pmid40744866,
year = {2025},
author = {Schmal, M and Kramer, LTS and Mach, RL and Mach-Aigner, AR and Zimmermann, C},
title = {Providing a toolbox for genomic engineering of Trichoderma aggressivum.},
journal = {Microbiology spectrum},
volume = {13},
number = {9},
pages = {e0096625},
pmid = {40744866},
issn = {2165-0497},
support = {P 34036//Austrian Science Fund/ ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; Genome, Fungal ; Transformation, Genetic ; *Trichoderma/genetics ; *Genetic Engineering/methods ; Hypocreales/genetics ; Agaricus ; Plasmids/genetics ; Fungal Proteins/genetics ; Plant Diseases/microbiology ; Genomics/methods ; },
abstract = {Fungi belonging to the genus of Trichoderma have a long history of infecting crops of edible fungi and reducing the yield. Trichoderma aggressivum is the main causal agent of green mold disease in Agaricus bisporus. Despite its infamous role as a persistent and aggressive contamination in A. bisporus crops, T. aggressivum can also be used as a biocontrol agent or as a promoter of plant growth. In order to work efficiently with T. aggressivum on a molecular biology level, a transformation protocol is required. This study provides a detailed protocol on how to perform a transformation in T. aggressivum using plasmid DNA for ectopic integration. In addition, a Cas9-RNP-based approach has been established for genome editing. We performed two transformations to confirm the usability of the hph gene as well as the pyr4 gene from Trichoderma reesei as selection markers. First, we integrated the hph gene ectopically and determined the overall transformation efficiency. In the second transformation experiment, we disrupted the ornithine-5' phosphate decarboxylase gene of T. aggressivum by using the CRISPR-Cas9 approach. In total, four candidates showed the expected uridine auxotrophy and resistance to 5-FOA. Additionally, the genomic locus around the CRISPR target sites was sequenced to determine the exact circumstances around the gene disruption. To complete the genetic toolbox for T. aggressivum, the pyr4 gene of T. reesei was tested as a suitable selection marker in one of the generated uridine auxotrophic strains.IMPORTANCEResearchers need an efficient tool for genomic manipulation to investigate the fundamental biology of mycoparasitism of T. aggressivum and its correlation to secondary metabolites. We provide a protocol for transformation of T. aggressivum and successfully demonstrated transformation of T. aggressivum using a plasmid and genome editing applying a Cas9-RNP-based strategy. Simultaneously, we established two selection markers, the hph gene and pyr4 gene from T. reesei. By applying these methods, we give researchers the tools needed to investigate T. aggressivum on a deeper level. Possible applications include activation of biosynthetic gene clusters of secondary metabolites to determine the biosynthetic pathway and biotechnological applications of these compounds.},
}

*Gene Editing/methods
CRISPR-Cas Systems
Genome, Fungal
Transformation, Genetic
*Trichoderma/genetics
*Genetic Engineering/methods
Hypocreales/genetics
Agaricus
Plasmids/genetics
Fungal Proteins/genetics
Plant Diseases/microbiology
Genomics/methods

@article {pmid40646716,
year = {2025},
author = {Coirry, C and Manessier, J and Clot, C and Mortier, M and Fauvarque, MO and Taillebourg, E},
title = {The deubiquitinase USP36 funtions through catalytic-dependent and catalytic-independent mechanisms in Drosophila.},
journal = {Genetics},
volume = {231},
number = {1},
pages = {},
pmid = {40646716},
issn = {1943-2631},
support = {ANR-17-EURE-0003//French National Research Agency/ ; //IDEX Université Grenoble Alpes/ ; //GRAL PhD Operating Costs/ ; },
mesh = {Animals ; *Drosophila Proteins/genetics/metabolism ; Male ; *Drosophila melanogaster/genetics/growth & development ; *Ubiquitin Thiolesterase/genetics/metabolism ; Spermatogenesis/genetics ; CRISPR-Cas Systems ; Female ; Proto-Oncogene Proteins c-myc/metabolism/genetics ; Deubiquitinating Enzymes/metabolism/genetics ; },
abstract = {Deubiquitinases (DUBs) form a specific class of proteases removing ubiquitin from target proteins. They are involved in the regulation of many cellular processes including cell growth and proliferation. Among them, USP36 is a key regulator of the oncogenic transcription factor c-Myc, preventing its degradation by the proteasome. These 2 proteins form an evolutionary conserved complex providing the opportunity to investigate USP36 mechanisms of action in vivo in a genetically tractable model such as Drosophila melanogaster. Null mutants of dUsp36 die early during larval development and exhibit severe growth defects. Strikingly, we report here that flies expressing a catalytically inactive version of dUSP36 produced by CRISPR/Cas9 gene editing survive to adulthood with only minor growth defects, yet males are infertile. This finding indicates that dUSP36 deubiquitinating activity is dispensable for cell growth but essential for spermatogenesis. Our results thus reveal that dUSP36 functions through both catalytic-dependent and catalytic-independent mechanisms, highlighting a dual mode of action with implications for the understanding of DUBs mechanism of action.},
}

Animals
*Drosophila Proteins/genetics/metabolism
Male
*Drosophila melanogaster/genetics/growth & development
*Ubiquitin Thiolesterase/genetics/metabolism
Spermatogenesis/genetics
CRISPR-Cas Systems
Female
Proto-Oncogene Proteins c-myc/metabolism/genetics
Deubiquitinating Enzymes/metabolism/genetics

@article {pmid40555854,
year = {2025},
author = {Basit, A and Liu, A and Zheng, W and Zhu, J},
title = {A review on the mechanism and potential diagnostic application of CRISPR/Cas13a system.},
journal = {Mammalian genome : official journal of the International Mammalian Genome Society},
volume = {36},
number = {3},
pages = {709-726},
pmid = {40555854},
issn = {1432-1777},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Bacteria/genetics ; CRISPR-Associated Proteins/genetics ; },
abstract = {Clustered regularly Interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins form a natural immune defense system in prokaryotic species, with approximately 90% of archaea and 40% of bacteria possessing these systems, highlighting their widespread role in microbial immunity. Among these, the CRISPR/Cas13a system, guided by a single-stranded RNA (crRNA), selectively targets RNA sequences and has shown immense potential in developing sensitive diagnostic tools. Recent advancements have combined Cas13a with amplification methods and lateral flow detection (CRISPR/Cas13a-LFD), improving its application for rapid and accurate RNA detection. In this review, we explore the history, structure, and functional mechanism of the CRISPR/Cas13a system, focusing on its diagnostic capabilities. We compare CRISPR/Cas13a to conventional diagnostic approaches, highlighting their advantages in sensitivity, specificity, speed, and flexibility for point-of-care application. Given the rapid development of CRISPR-based diagnostics in recent years, the Cas13a system shows great potential as a next-generation platform for accurate, portable, and cost-effective detection of viral and bacterial diseases. Furthermore, we address the existing challenges, including reliance upon amplification and off-target effects, and highlight the need for ongoing research to develop amplification-free systems suitable for clinical application.},
}

*CRISPR-Cas Systems/genetics
Humans
Animals
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Bacteria/genetics
CRISPR-Associated Proteins/genetics

@article {pmid40541099,
year = {2025},
author = {Tian, J and Wang, Y and Zhou, Z and Li, Y and Chang, C and Meng, F and Shi, W and Li, H and Wang, W and Wei, Q and Liu, H and Yang, Z and Xiao, S},
title = {Genome-wide CRISPR/Cas9 screen identifies host factors for Newcastle disease virus replication.},
journal = {Poultry science},
volume = {104},
number = {9},
pages = {105421},
pmid = {40541099},
issn = {1525-3171},
mesh = {Animals ; *Newcastle disease virus/physiology ; *Virus Replication/genetics ; *CRISPR-Cas Systems ; Mice ; *Newcastle Disease/virology/genetics ; Cell Line ; },
abstract = {Viruses rely strictly on host factors to determine their susceptibility. Newcastle disease virus (NDV), an avian pathogen, can infect a wide range of avian, mammalian and human cell lines. However, NDV can only infect certain mammals and humans, and it does not cause disease in mammals or mice. A comprehensive identification of NDV-susceptible host genes that promote NDV infection is lacking. Here, we performed a mouse genome-wide CRISPR knockout (GeCKO) screening in murine fibroblast L929 cells infected with NDV. Fifty host genes were highly enriched in the screening. Using a viral minigenome assay and gene overexpression as well as single guide RNA (sgRNA) knockout, one of the genes was shown to predominantly promote viral replication. This gene is the solute carrier family 35 member A1 (SLC35A1), which is a cytidine monophosphate (CMP)-Sia transporter involved in the synthesis of sialic acid (SA). Knockout of SLC35A1 in L929 cells decreased the expression of the NDV receptors α2,3-SA and α2,6-SA on the cell surface, resulting in significant reductions in viral adsorption, internalization and replication. Furthermore, the knockout of a six-residue stretch, [82]LGSPKE[87], of SLC35A1 in cells specifically reduced the expression of the α2,6-SA receptor but not the α2,3-SA receptor, which decreased viral adsorption and replication. The reconstitution of SLC35A1 in the knockout cells completely recovered the α2,3-SA receptor, partially recovered the α2,6-SA receptor and almost completely recovered viral replication. In chicken fibroblast DF-1 cells, siRNA-mediated knockdown of chicken SLC35A1 reduced the expression levels of both the α2,6-SA and α2,3-SA receptors, decreasing viral replication. Our research indicated that SLC35A1 is a key host factor that promotes NDV replication. The CRISPR/Cas9 system can be used to identify essential host factors for the replication of intracellular pathogens. This study provides valuable insights into host susceptibility to NDV.},
}

Animals
*Newcastle disease virus/physiology
*Virus Replication/genetics
*CRISPR-Cas Systems
Mice
*Newcastle Disease/virology/genetics
Cell Line

@article {pmid40527130,
year = {2025},
author = {Jin, S and Wang, X and Liu, X and Xu, Y and Wang, W and Chen, W and Chang, H and Li, Z and Geng, Z},
title = {Effective IHH gene knockout by CRISPR/Cas9 system in chicken DF-1 cells.},
journal = {Poultry science},
volume = {104},
number = {9},
pages = {105433},
pmid = {40527130},
issn = {1525-3171},
mesh = {Animals ; *Chickens/genetics ; *CRISPR-Cas Systems ; *Hedgehog Proteins/genetics/metabolism ; *Gene Knockout Techniques/veterinary ; *Avian Proteins/genetics/metabolism ; Cell Line ; },
abstract = {Indian hedgehog (IHH) gene codes an important signal molecule mediating chondrogenesis and bone development in chickens, which are key factors that affect body weight and several other significant economic traits. The aim of this study was to construct an IHH knockout cell model using CRISPR-associated protein 9 (CRISPR/Cas9) technology to further analyze the function of IHH. TA cloning was used to screen the single-guide RNA (sgRNA1) [45 %] and sgRNA3 (30.8 %) with the highest targeting efficiency. Monoclonal cells were selected by flow cytometry for TA cloning sequencing to construct the IHH knockout cell model. Quantitative PCR (qPCR) was used to detect the changes in downstream gene expression levels after IHH knockout. TA cloning sequencing results showed that the IHH knockout cell model was successfully constructed, and two mutation types were generated with a 100 % mutation rate. In addition, qPCR results revealed that the expression of patched 1 (PTCH1), smoothened, frizzled class receptor (Smo), glioma-associated oncogene homolog 1 (Gli1), glioma-associated oncogene homolog 2 (Gli2), and osteopontin (OPN) was significantly lower in the IHH knockout group, while that of type II collagen (Col Ⅱ) was significantly higher. These results lay a theoretical foundation for the successful application of knockout technology in poultry functional genomics research and provide a stable knockout cell line model for further study of chicken IHH gene function.},
}

Animals
*Chickens/genetics
*CRISPR-Cas Systems
*Hedgehog Proteins/genetics/metabolism
*Gene Knockout Techniques/veterinary
*Avian Proteins/genetics/metabolism
Cell Line

@article {pmid40891974,
year = {2025},
author = {Chapelin, F},
title = {CRISPR-based Triple-Modality Imaging Ushers a New Era for Stem Cell Tracking in Stroke.},
journal = {Radiology},
volume = {316},
number = {3},
pages = {e252546},
doi = {10.1148/radiol.252546},
pmid = {40891974},
issn = {1527-1315},
mesh = {Humans ; *Cell Tracking/methods ; *Stroke/diagnostic imaging/therapy ; *Multimodal Imaging/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Stem Cells ; *CRISPR-Cas Systems ; },
abstract = {"Just Accepted" papers have undergone full peer review and have been accepted for publication in Radiology. This article will undergo copyediting, layout, and proof review before it is published in its final version. Please note that during production of the final copyedited article, errors may be discovered which could affect the content.},
}

Humans
*Cell Tracking/methods
*Stroke/diagnostic imaging/therapy
*Multimodal Imaging/methods
*Clustered Regularly Interspaced Short Palindromic Repeats
*Stem Cells
*CRISPR-Cas Systems

@article {pmid40891659,
year = {2025},
author = {Takizawa, A and Foeckler, J and Knapp, E and Grzybowski, M and Geurts, AM and Carroll, J and Merriman, DK},
title = {Successful Generation of Germline Tyrosinase Gene Edited Thirteen-Lined Ground Squirrels (Ictidomys tridecemlineatus Mitchill 1821).},
journal = {Molecular reproduction and development},
volume = {92},
number = {9},
pages = {e70055},
doi = {10.1002/mrd.70055},
pmid = {40891659},
issn = {1098-2795},
support = {//This study was supported by the National Eye Institute of the National Institutes of Health (NIH) under award number T32EY014537, U24EY029891 and by the National Center for Advancing Translational Sciences, National Institutes of Health, Award Number UL1TR001436./ ; },
mesh = {Animals ; *Monophenol Monooxygenase/genetics/metabolism ; *Sciuridae/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems ; Female ; Male ; *Germ-Line Mutation ; *Germ Cells/metabolism ; },
abstract = {Thirteen-lined ground squirrels (Ictidomys tridecemlineatus Mitchill 1821; 13-LGS) are useful diurnal rodent models of human cone-mediated vision due to their cone photoreceptor-dominant retinas. To develop the 13-LGS as a better model of inherited human visual disorders, we report a gene-editing protocol targeting the 13-LGS tyrosinase (Tyr) gene. CRISPR/Cas9 microinjection into donor embryos, followed by transfer to pseudo-pregnant recipients, yielded two Tyr-mutated founders. Mating these two to wild-type 13-LGS resulted in 22 offspring, of which five were genotyped with either a 17-bp deletion, 1-bp insertion, or 7-bp deletion Tyr mutation. These results demonstrated that this valuable mammalian model is amenable to germline gene editing by conventional methods.},
}

Animals
*Monophenol Monooxygenase/genetics/metabolism
*Sciuridae/genetics
*Gene Editing/methods
CRISPR-Cas Systems
Female
Male
*Germ-Line Mutation
*Germ Cells/metabolism

@article {pmid40891290,
year = {2025},
author = {Deng, Z and Sha, R and Qin, H and Shang, Y and Yuan, A and Xie, W and Peng, H},
title = {A CRISPR Cas protein coronated AuNP nanostructure for enhanced uptake efficiency into cells.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5cc03658e},
pmid = {40891290},
issn = {1364-548X},
abstract = {The effectiveness of nanotechnologies is often limited by their non-specific aggregation in biological environments. We developed a protein coronated nanostructure by functionalizing AuNPs with nucleic acid scaffolds and CRISPR Cas proteins, significantly enhancing nanoparticle stability and cellular uptake efficiency, making it a promising tool for imaging and biomedical applications.},
}

@article {pmid40890415,
year = {2025},
author = {Laidoudi, Y and Davoust, B and Lepidi, H and Levasseur, A},
title = {Emergence of the zoonotic bacterium Necropsobacter rosorum in nutria Myocastor coypus with implications for wildlife and human health.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {32252},
pmid = {40890415},
issn = {2045-2322},
mesh = {Animals ; Humans ; Phylogeny ; Genome, Bacterial ; France/epidemiology ; *Pasteurellaceae/genetics/isolation & purification/classification/pathogenicity ; Animals, Wild/microbiology ; *Zoonoses/microbiology ; Whole Genome Sequencing ; *Rodentia/microbiology ; *Bacterial Zoonoses/microbiology/epidemiology ; },
abstract = {The nutria (Myocastor coypus), a semi-aquatic rodent native to South America, poses significant ecological and agricultural threats as an invasive species in France, where it continues to proliferate despite sustained control efforts. A fatal case of pneumonia in a nutria from Marseille (France) prompted a microbiological investigation that led to the isolation, taxonomic classification, genomic characterization, and phylogenetic analysis of Necropsobacter rosorum. Whole-genome sequencing of the N. rosorum strain RG01 revealed a genome size of 2,505,657 base pairs and 2303 predicted open reading frames, showing high similarity to other publicly available N. rosorum genomes. Comparative pan-genomic analysis indicated a high level of genomic conservation among N. rosorum strains. The presence of putative virulence factors and a CRISPR-Cas system suggests both pathogenic potential and adaptive defense mechanisms against bacteriophage predation. This study also explored the genetic epidemiology of members of the Pasteurellaceae family, highlighting a considerable overlap between species infecting animals and humans. Among the 408,387 sequence records retrieved from GenBank, 62.1% were deemed suitable for genomic epidemiological analysis. Notably, N. rosorum was underrepresented, with only 13 entries spanning nine countries and three host types, revealing critical gaps in current surveillance and research. Collectively, these findings contribute to a better understanding of the microbiology and epidemiology of N. rosorum and Pasteurellaceae-associated infections, and underscore the importance of integrated, genomics-informed approaches for the monitoring, control, and prevention of zoonotic diseases.},
}

Animals
Humans
Phylogeny
Genome, Bacterial
France/epidemiology
*Pasteurellaceae/genetics/isolation & purification/classification/pathogenicity
Animals, Wild/microbiology
*Zoonoses/microbiology
Whole Genome Sequencing
*Rodentia/microbiology
*Bacterial Zoonoses/microbiology/epidemiology

@article {pmid40890128,
year = {2025},
author = {Álvarez-Pérez, JC and Sanjuán-Hidalgo, J and Arenas, AM and Hernández-Navas, I and Benitez-Cantos, MS and Andrades, A and Calabuig-Fariñas, S and Jantus-Lewintre, E and Paz-Ares, L and Ferrer, I and Medina, PP},
title = {High-fidelity Cas9-mediated targeting of KRAS driver mutations restrains lung cancer in preclinical models.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7080},
pmid = {40890128},
issn = {2041-1723},
support = {LAB-AECC-2018//Fundación Científica Asociación Española Contra el Cáncer (Scientific Foundation, Spanish Association Against Cancer)/ ; B-CTS-480-UGR20, C-EXP-051-UGR23, C-CTS-149-UGR23//Universidad de Granada (University of Granada)/ ; },
mesh = {*Lung Neoplasms/genetics/therapy/pathology ; Animals ; Humans ; *Proto-Oncogene Proteins p21(ras)/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Carcinoma, Non-Small-Cell Lung/genetics/therapy/pathology ; Mice ; Xenograft Model Antitumor Assays ; Cell Line, Tumor ; Gene Editing/methods ; *Genetic Therapy/methods ; Mutation ; Female ; Adenoviridae/genetics ; },
abstract = {Missense mutations in the 12[th] codon of KRAS are key drivers of lung cancer, with glycine-to-cysteine (G12C) and glycine-to-aspartic acid (G12D) substitutions being among the most prevalent. These mutations are strongly associated with poor survival outcomes. Given the critical role of KRAS in lung cancer and other cancers, it remains as a major target for the development of new and complementary treatments. We have developed a CRISPR-High Fidelity (HiFi)-Cas9-based therapy strategy that can effectively and specifically target KRAS[G12C] and KRAS[G12D] mutants, avoiding KRAS[WT] off-targeting and affecting KRAS downstream pathways, thereby significantly reducing tumorgenicity. The delivery of HiFiCas9 components via ribonucleoprotein particles (RNPs) and adenovirus (AdV) effectively abrogates cell viability in KRAS-mutant Non-Small Cell Lung Cancer (NSCLC) preclinical models, including 2D and 3D cell cultures, cell-derived xenografts (CDX), and patient-derived xenograft organoids (PDXO). Our in vitro studies demonstrate that HiFiCas9-based therapy achieves superior KRAS inhibition compared to Sotorasib and effectively circumvents certain resistance mechanisms associated with Sotorasib treatment. Moreover, in vivo delivery using adenoviral particles significantly suppresses tumor growth in preclinical NSCLC models. Collectively, our findings establish HiFiCas9 as an effective therapeutic strategy with promising clinical applications, especially if in vivo delivery methods are further optimized.},
}

*Lung Neoplasms/genetics/therapy/pathology
Animals
Humans
*Proto-Oncogene Proteins p21(ras)/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Carcinoma, Non-Small-Cell Lung/genetics/therapy/pathology
Mice
Xenograft Model Antitumor Assays
Cell Line, Tumor
Gene Editing/methods
*Genetic Therapy/methods
Mutation
Female
Adenoviridae/genetics

@article {pmid40825745,
year = {2025},
author = {Zhuang, S and Huang, X and Diao, M and Dong, R and Dong, Y and Liu, J and Liu, Y},
title = {CONVERT: Dynamic crRNA Reconfiguration for Universal One-Pot CRISPR/Cas12a-Based Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {97},
number = {34},
pages = {18776-18785},
doi = {10.1021/acs.analchem.5c03577},
pmid = {40825745},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; *RNA/genetics ; *Bacterial Proteins/metabolism/genetics ; },
abstract = {Current one-pot CRISPR diagnostics necessitate meticulous control of nucleic acid hybridization parameters or extensive modification of CRISPR components to achieve complete enzymatic silencing, a fundamental bottleneck limiting their robustness and generalizability. Here, we resolve this challenge through dynamic crRNA reconfiguration, a paradigm-shifting strategy that exploits the intrinsic structural duality of CRISPR RNA. We present CONVERT (CRISPR One-Pot Nucleic acid detection Via Engineered crRNA Reconfiguration Technology), a universal platform where nontarget intact crRNA acts as a universal suppressor, achieving complete Cas12a inhibition during RPA (recombinase polymerase amplification) by irreversible enzyme sequestration. Target detection is initiated through programmable conversion to split crRNA activators, bypassing sequence-specific design constraints entirely. This crRNA conformational switching mechanism is implemented via photocleavable linker cleavage and subsequent assembly with presupplied truncated crRNA (tcrRNA), activating trans-cleavage signal amplification. Three transformative advances emerge: First, the endogenous crRNA engineering eliminates dependency on exogenous nucleic acid blockers or chemical modifications, reducing optimization costs and time. Second, near-total enzymatic suppression ensures zero cross-talk between amplification and detection phases. Third, the unified workflow enables contamination-resistant operation through spatiotemporal control, requiring no physical partitioning. Validated for Enterocytozoon bieneusi detection, the system demonstrates attomolar sensitivity of 1 aM, 100% diagnostic concordance with qPCR, and absolute specificity against related pathogens. By effectively decoupling CRISPR regulation from sequence-context constraints through rational crRNA structural plasticity, this work establishes a generalized framework for next-generation molecular diagnostics.},
}

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

@article {pmid40742143,
year = {2025},
author = {Liu, G and Wu, J and Yang, B and Kong, J and Li, Y and Chen, C and Hu, J and Sun, Z and Hou, Y and Sun, R and Li, D},
title = {Ultrasensitive eccDNA Detection for Tumor Diagnostics by Using CRISPR/Cas12a-Coupled Nested PCR.},
journal = {Analytical chemistry},
volume = {97},
number = {34},
pages = {18527-18536},
doi = {10.1021/acs.analchem.5c02316},
pmid = {40742143},
issn = {1520-6882},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Polymerase Chain Reaction/methods ; *DNA, Circular/genetics/blood/analysis ; Limit of Detection ; *Biomarkers, Tumor/genetics/blood ; *Neoplasms/diagnosis/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Extrachromosomal circular DNA (eccDNA) has emerged as a novel biomarker for cancer detection due to its tumor-specific amplification and stable structure in circulation. However, its clinical application is hindered by extremely low abundance in biofluids and the lack of robust detection techniques. To address this, we screened for tumor-associated eccDNA biomarkers and developed NPCC (Nested PCR-CRISPR/Cas12a), a novel method combining nested PCR for ultrasensitive amplification with CRISPR/Cas12a for sequence-specific detection. The assay employs two rounds of junction-specific PCR to enrich eccDNA, followed by CRISPR/Cas12a-mediated cleavage guided by target-specific crRNA. Validation using synthetic circular DNA standards demonstrated a limit of detection (LoD) of 10[-6] fM, representing a >100-fold improvement over conventional PCR, with no cross-reactivity to linear or genomic DNA fragments. In plasma samples from 88 cancer patients, NPCC successfully detected multiple tumor-specific eccDNAs, including the hepatocellular carcinoma marker eccDNA-HCC-1 (AUC = 0.8977). NPCC overcomes key technical barriers in liquid biopsy, offering a cost-effective, highly sensitive, and specific platform for noninvasive cancer diagnostics.},
}

Humans
*CRISPR-Cas Systems/genetics
*Polymerase Chain Reaction/methods
*DNA, Circular/genetics/blood/analysis
Limit of Detection
*Biomarkers, Tumor/genetics/blood
*Neoplasms/diagnosis/genetics
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40614014,
year = {2025},
author = {Shan, L and He, Q and Zhang, W and Zheng, X and Zhao, J and Yang, P and Mao, Q and Xia, H},
title = {CRISPR/Cas9-mediated promoterless gene targeting reduces lysosome storage in MPS VII mice.},
journal = {Science China. Life sciences},
volume = {68},
number = {9},
pages = {2697-2706},
pmid = {40614014},
issn = {1869-1889},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Mice ; Glucuronidase/genetics/metabolism/blood ; *Mucopolysaccharidosis VII/therapy/genetics/metabolism ; *Gene Targeting/methods ; *Lysosomes/metabolism ; Disease Models, Animal ; Genetic Vectors/genetics ; Promoter Regions, Genetic ; Genetic Therapy/methods ; Actins/genetics ; Adenoviridae/genetics ; Liver/metabolism ; },
abstract = {Targeted gene integration mediated by CRISPR/Cas9 is a promising therapeutic strategy for monogenic autosomal recessive diseases. In this study, we established a novel all-in-one high-capacity adenovirus (HCAd) that can pack both CRISPR/Cas9 and donor DNA into the same vector and tested it on a mouse model of mucopolysaccharidosis type VII (MPS VII) caused by mutations in the β-glucuronidase (GUSB) gene. This system allowed targeted integration of promoterless GUSB in the mouse beta-actin gene (mActb) locus and the co-expression of GUSB with the self-cleaving peptide T2A (T2A) controlled by a strong endogenous mActb promoter. The in vivo results indicated that the serum GUSB level of MPS VII mice treated with a single intraperitoneal injection of the HCAd vector achieved 14% of that of wild-type mice, resulting in significant amelioration of lysosomal storage in the liver and spleen. Furthermore, the HCAd was injected intraventricularly in the brain of newborn MPS VII mice, leading to strongly positive GUSB enzyme staining in the choroid plexus and perivascular spaces of the periventricular regions and reduced lysosome storage. In summary, by using an all-in-one vector, the study provides a universal, one-for-all therapeutic for MPSVII, a disease caused by different mutations of the GUSB gene.},
}

Animals
*CRISPR-Cas Systems/genetics
Mice
Glucuronidase/genetics/metabolism/blood
*Mucopolysaccharidosis VII/therapy/genetics/metabolism
*Gene Targeting/methods
*Lysosomes/metabolism
Disease Models, Animal
Genetic Vectors/genetics
Promoter Regions, Genetic
Genetic Therapy/methods
Actins/genetics
Adenoviridae/genetics
Liver/metabolism

@article {pmid40377870,
year = {2025},
author = {Amen, RA and Hassan, YM and Essmat, RA and Ahmed, RH and Azab, MM and Shehata, NR and Elgazzar, MM and El-Sayed, WM},
title = {Harnessing the Microbiome: CRISPR-Based Gene Editing and Antimicrobial Peptides in Combating Antibiotic Resistance and Cancer.},
journal = {Probiotics and antimicrobial proteins},
volume = {17},
number = {4},
pages = {1938-1968},
pmid = {40377870},
issn = {1867-1314},
mesh = {Humans ; *Gene Editing/methods ; *Neoplasms/microbiology/therapy/drug therapy ; *Microbiota ; *Antimicrobial Peptides/pharmacology ; CRISPR-Cas Systems ; Animals ; *Drug Resistance, Microbial ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The growing crisis of antibiotic resistance and the increasing incidence of cancer have prompted the exploration of innovative approaches, such as gene editing and antimicrobial peptides (AMPs). The human microbiome is integral to various aspects of health, disease, and therapeutic development, influencing metabolic pathways, immune function, and pathogen resistance. Recent advances in gene editing technologies, particularly CRISPR (clustered regularly interspaced short palindromic repeats), have opened new avenues for leveraging the microbiome to address complex medical challenges, including combating multidrug-resistant pathogens and cancer. The microbiome plays a crucial role in combating antibiotic resistance by modulating microbial communities, influencing pathogen survival and susceptibility to treatments. This review explores the microbiome's dynamic role in metabolic regulation, its contribution to cancer management, and how AMPs help maintain homeostasis and exhibit emerging anticancer properties, supported by both preclinical findings and clinical evidence. Additionally, CRISPR-based microbiome engineering offers potential to enhance host-microbiome interactions, optimizing therapeutic outcomes. The integration of microbiome metagenomics and proteomics has led to the discovery of novel AMPs with targeted anticancer effects. Innovative strategies, such as engineered probiotics and CRISPR-based microbiome engineering, present exciting prospects for next-generation therapies. Despite these advances, the translation of microbiome-based therapies into clinical settings remains challenging due to ethical, regulatory, and ecological hurdles. This review underscores the transformative potential of microbiome-based interventions, emphasizing the role of personalized medicine in maximizing therapeutic efficacy. Furthermore, we also address critical research gaps, limitations, and future directions, including optimizing AMP stability, delivery, and bioavailability, as well as overcoming the regulatory and ethical challenges in clinical translation.},
}

Humans
*Gene Editing/methods
*Neoplasms/microbiology/therapy/drug therapy
*Microbiota
*Antimicrobial Peptides/pharmacology
CRISPR-Cas Systems
Animals
*Drug Resistance, Microbial
Clustered Regularly Interspaced Short Palindromic Repeats