@article {pmid40465692,
year = {2025},
author = {Cai, Y and Zhao, P and Wu, F and Zhao, H and Shao, H and Marra, A and Patel, P and O'Connell, E and Fink, E and Miele, MM and Li, Z and De Stanchina, E and Cocco, E and Razavi, P and Toska, E and Fanning, SW and Xu, G and Sablina, AA and Scaltriti, M and Chandarlapaty, S},
title = {Inhibition of NR2F2 restores hormone therapy response to endocrine refractory breast cancers.},
journal = {Science translational medicine},
volume = {17},
number = {801},
pages = {eadk7786},
doi = {10.1126/scitranslmed.adk7786},
pmid = {40465692},
issn = {1946-6242},
mesh = {Humans ; *Breast Neoplasms/drug therapy/genetics/metabolism/pathology ; Female ; *COUP Transcription Factor II/metabolism/antagonists & inhibitors/genetics ; Animals ; Cell Line, Tumor ; *Drug Resistance, Neoplasm/drug effects/genetics ; Neurofibromin 1/metabolism/genetics ; Gene Expression Regulation, Neoplastic/drug effects ; Mice ; Receptors, Estrogen/metabolism ; CRISPR-Cas Systems/genetics ; },
abstract = {Endocrine resistance is frequently encountered in estrogen receptor-positive (ER+) breast cancer, often because of somatic mutations such as neurofibromin 1 (NF1) loss. The mechanisms by which ER-directed proliferation is lost in such cases are unknown, limiting the potential use of additional endocrine treatments. Here, we performed CRISPR-Cas9 knockout (KO) screens and found that nuclear receptor subfamily 2 group F member 2 (NR2F2), an orphan nuclear receptor, was essential for NF1 loss-induced endocrine resistance. Induction of NR2F2 was observed in ER+ cell line models and patient samples and occurred via activation of the mitogen-activated protein kinase (MAPK) pathway upon NF1 loss or other MAPK pathway genetic alterations. Mechanistically, increased NR2F2 orchestrated a repressed ER transcriptional program by repartitioning the ER cistrome, altering the balance of its associated transcriptional coregulators, and modifying global chromatin accessibility. Accordingly, genetic depletion or pharmacologic inhibition of NR2F2 restored sensitivity to hormone therapies in multiple models, including ER+ cell lines, patient-derived xenografts, and patient-derived organoid-based xenografts harboring diverse endocrine-resistance mechanisms such as NF1, AT-rich interactive domain-containing protein 1A (ARID1A), phoshatase and tensin homolog (PTEN) loss, or Kirsten rat sarcoma virus (KRAS) overexpression. Together, these findings underscore NR2F2 as a critical modulator of the hormone response pathway and suggest its inhibition as a promising strategy to overcome endocrine resistance in breast cancer.},
}

Humans
*Breast Neoplasms/drug therapy/genetics/metabolism/pathology
Female
*COUP Transcription Factor II/metabolism/antagonists & inhibitors/genetics
Animals
Cell Line, Tumor
*Drug Resistance, Neoplasm/drug effects/genetics
Neurofibromin 1/metabolism/genetics
Gene Expression Regulation, Neoplastic/drug effects
Mice
Receptors, Estrogen/metabolism
CRISPR-Cas Systems/genetics

@article {pmid40465008,
year = {2025},
author = {Jin, Y and Liang, X and Wang, X},
title = {Alternative splicing in stem cells and development: research progress and emerging technologies.},
journal = {Cell regeneration (London, England)},
volume = {14},
number = {1},
pages = {20},
pmid = {40465008},
issn = {2045-9769},
support = {YD9110002078//USTC Research Funds of the Double First-Class Initiative/ ; 32170557//National Natural Science Foundation of China/ ; 32400436//National Natural Science Foundation of China/ ; 2023IHM01034//Research Funds of Centre for Leading Medicine and Advanced Technologies of IHM/ ; 2024C879//Anhui Postdoctoral Scientific Research Program Foundation/ ; },
abstract = {Alternative splicing is a key regulatory mechanism that generates transcriptomic diversity by selectively splicing pre-RNA molecules in different ways, leading to the production of multiple RNA isoforms from a single gene. This process is crucial for the fine-tuning of gene expression and is tightly regulated during various biological processes. Recent studies have highlighted how alternative splicing contributes to stem cells self-renewal and differentiation, as well as how dysregulation of splicing factors can impact stem cells behavior and lead to developmental abnormalities or diseases. This review summarizes the current understanding of alternative splicing in stem cells and development, focusing on the molecular mechanisms that govern alternative splicing regulation, the role of splicing factors, and the impact of splicing isoforms on stem cell fate determination and developmental processes. We also discuss emerging technologies, such as CRISPR/Cas-based tools, single-cell long-read RNA sequencing, imaging technologies and 3D culture systems, which are advancing our ability to study alternative splicing in vitro and in vivo. Overall, this field is rapidly evolving, revealing new insights into how alternative splicing shapes the molecular landscape and functions of stem cells and developmental processes.},
}

@article {pmid40464988,
year = {2025},
author = {Waqas, MAB and Awan, MJA and Amin, I and Arif, M and Mukhtar, Z and Mansoor, S},
title = {Engineering high yield basmati rice by editing multiple negative regulators of yield.},
journal = {Molecular biology reports},
volume = {52},
number = {1},
pages = {545},
pmid = {40464988},
issn = {1573-4978},
mesh = {*Oryza/genetics/growth & development ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; Plant Proteins/genetics ; Genes, Plant/genetics ; Quantitative Trait Loci/genetics ; Edible Grain/genetics ; Gene Expression Regulation, Plant ; },
abstract = {BACKGROUND: Yield improvement in Basmati rice is important to meet the growing demand of Basmati and pivotal to world food security. Yield in rice is a complex quantitative trait controlled by many genes and yield-determining factors. Although dominant genes introgression through breeding have been extensively used but it is a laborious and time-consuming process. Yield genes OsD27, OsGW2, OsTGW6 and OsGN1a have been shown to negatively regulate an increase in the number of tillers, grain width, grain weight, and number of grains per panicle respectively in different genetic backgrounds of rice separately. The aim of our study is to establish a multiplex system in Super Basmati to check if it acts similarly and what is their combinatorial effect on yield enhancement.

METHODS: Here we generated Super Basmati quadruple edited lines for all four genes OsD27, OsGW2, OsTGW6 and OsGN1a through CRISPR-Cas9 polycistronic tRNA-gRNA (PTG) endogenous processing efficient multiplex editing system. Quadruple edited lines were characterized for key yield parameters such as number of tillers, number of grains, grain weight, overall yield increase, lodging resistance and cooking quality related traits.

RESULTS: We found that all quadruple edited plants produced more tillers, a greater number of grains with high grain weight in field evaluations along with decreased plant height. Overall, the contribution of all four genes was additive resulting in boosting the yield in Super Basmati remarkably. Our data hence demonstrated a promising multiplex genome editing approach for rapid generation of superior alleles for all four yield contributing factor genes in elite Basmati variety with 30% co-editing efficiency.

CONCLUSIONS: We conclude that multiplex CRISPR-Cas9 genome editing of yield-negative regulators may boost the yield of elite Basmati rice in a short time without causing any drastic effect on other agronomic and quality traits such as aroma, chalkiness, and cooking qualities. Data suggests that the tRNA-gRNA multiplexing CRISPR-Cas9 system is efficient in the Basmati rice background for simultaneously editing multiple genes. Due to its capability of rapid generation of beneficial alleles, this multiplex system is well suited for pyramiding of multiple alleles in Basmati rice and may prove a promising approach to enhance yield in Basmati rice while it may provide useful germplasm resource for high-yielding future rice breeding programs.},
}

*Oryza/genetics/growth & development
*Gene Editing/methods
CRISPR-Cas Systems/genetics
Plant Breeding/methods
Plants, Genetically Modified/genetics
Plant Proteins/genetics
Genes, Plant/genetics
Quantitative Trait Loci/genetics
Edible Grain/genetics
Gene Expression Regulation, Plant

@article {pmid40464976,
year = {2025},
author = {Sharma, SS and Pandey, A and Kashyap, A and Goyal, L and Garg, P and Kushwaha, R and Sharma, J and Tripathi, S and Kumari, S and Thomas, G and Verma, M and Gupta, NC and Gupta, AK and Bhattacharya, R and Sharma, S and Rao, M},
title = {CRISPR/Cas9: efficient and emerging scope for Brassica crop improvement.},
journal = {Planta},
volume = {262},
number = {1},
pages = {14},
pmid = {40464976},
issn = {1432-2048},
support = {NASF/BGAM-9021/2022-23/NIPB//ICAR - National Agricultural Science Fund/ ; 2001-2019//Indian Council of Agricultural Research/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Brassica/genetics ; *Gene Editing/methods ; *Crops, Agricultural/genetics ; Genome, Plant ; Plant Breeding/methods ; Plants, Genetically Modified ; },
abstract = {CRISPR/Cas9 revolutionizes Brassica crop improvement by enhancing yield, quality, and stress resistance, providing a precise and versatile tool for genetic and agronomic advancements. The rapidly advancing CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) technologies are being employed in both diploid and polyploid species of Brassica for gene functions and precise genetic improvements. CRISPR/Cas technology has sparked significant attention among the scientific community due to its affordability, precision, and effectiveness compared to other genome editing techniques. The recent discoveries highlight the diverse applications of the CRISPR/Cas9 genome editing tool in enhancing agriculturally important traits in Brassica species. This technology has been utilized to improve yield, quality, and resistance to both biotic and abiotic stresses globally. Here, we present an overview that encourages researchers to explore and improve the functionality and genetic progress of Brassica U-triangle species utilizing genome editing technologies. In addition, ethical considerations and concerns associated with CRISPR technologies are addressed, providing valuable insight into how CRISPR/Cas9 tools and have revolutionized crop improvement with special emphasis on Brassica for various agronomically and nutritionally important traits.},
}

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

@article {pmid40464068,
year = {2025},
author = {Liu, Y and Zhang, S and Hu, C},
title = {Cas7 meets Cas14: a strategic partnership in the type VII CRISPR-Cas.},
journal = {Protein & cell},
volume = {16},
number = {2},
pages = {79-82},
pmid = {40464068},
issn = {1674-8018},
support = {23-0178-A0001//National University of Singapore Presidential Young Professorship (PYP)/ ; 23-1065-P0001//Singapore Ministry of Education Tier 1/ ; },
}

@article {pmid40461565,
year = {2025},
author = {Yang, C and Fang, Q and Li, M and Zhang, J and Li, R and Zhou, T and Wang, K and Deng, J and Wang, X and Huang, C and Feng, Y and Zhang, X and Shi, L and Bi, C and Zhang, X and Yu, J and Hao, J},
title = {Prime editor with rational design and AI-driven optimization for reverse editing window and enhanced fidelity.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5144},
pmid = {40461565},
issn = {2041-1723},
support = {32471478//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82203238//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; *Artificial Intelligence ; },
abstract = {Prime editing (PE) is a precise tool for introducing genetic mutations in eukaryotes. Extending the efficient editing scope and mitigating undesired byproducts are possible. We introduce reverse PE (rPE), a SpCas9-directed variant that enabled DNA editing at the 3' direction of HNH-mediated nick site. The rPE leveraging nCas9-D10A and rPE gRNA targeting the 5' direction of HNH-mediated nick site inscribes genetic alterations, achieving a reverse editing window and potentially high fidelity. HNH and reverse transcriptase engineered using protein language models in conjunction with La facilitate circular erPEmax and erPE7max, achieving editing efficiency up to 44.41% without nick gRNA or positive selection. Furthermore, our findings underscore the capability of rPE in inserting functionally enhanced variant (PIK3CD[E527G]) for cell therapy. By expanding the editing scope and enhancing genomic manipulability, rPE represents a meaningful advancement in prime editing, improving its utility for research and therapeutic applications.},
}

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

@article {pmid40461259,
year = {2025},
author = {Esser, SP and Turzynski, V and Plewka, J and Nuy, J and Moore, CJ and Banas, I and Soares, AR and Lee, J and Woyke, T and Probst, AJ},
title = {Differential Expression of Core Metabolic Functions in Candidatus Altiarchaeum Inhabiting Distinct Subsurface Ecosystems.},
journal = {Environmental microbiology reports},
volume = {17},
number = {3},
pages = {e70096},
pmid = {40461259},
issn = {1758-2229},
support = {DFG PR1603/2-1//Ministerium für Kultur und Wissenschaft des Landes Nordrhein- Westfalen ("Nachwuchsgruppe Dr. Alexander Probst") and the German Research Foundation/ ; //Aker B.P.: GeneOil Project/ ; //U.S. Department of Energy Joint Genome Institute/ ; //DOE Office of Science User Facility/ ; //the Office of Science of the U.S. Department of Energy operated/ ; },
mesh = {*Ecosystem ; Germany ; Groundwater/microbiology ; Symbiosis ; Transcriptome ; Gene Expression Profiling ; Genome, Archaeal ; Multigene Family ; },
abstract = {Candidatus Altiarchaea are widespread across aquatic subsurface ecosystems and possess a highly conserved core genome, yet adaptations of this core genome to different biotic and abiotic factors based on gene expression remain unknown. Here, we investigated the metatranscriptome of two Ca. Altiarchaeum populations that thrive in two substantially different subsurface ecosystems. In Crystal Geyser, a high-CO2 groundwater system in the USA, Ca. Altiarchaeum crystalense co-occurs with the symbiont Ca. Huberiarchaeum crystalense, while in the Muehlbacher sulfidic spring in Germany, an artesian spring high in sulfide concentration, Ca. A. hamiconexum is heavily infected with viruses. We here mapped metatranscriptome reads against their genomes to analyse the in situ expression profile of their core genomes. Out of 537 shared gene clusters, 331 were functionally annotated and 130 differed significantly in expression between the two sites. Main differences were related to genes involved in cell defence like CRISPR-Cas, virus defence, replication, transcription and energy and carbon metabolism. Our results demonstrate that altiarchaeal populations in the subsurface are likely adapted to their environment while influenced by other biological entities that tamper with their core metabolism. We consequently posit that viruses and symbiotic interactions can be major energy sinks for organisms in the deep biosphere.},
}

*Ecosystem
Germany
Groundwater/microbiology
Symbiosis
Transcriptome
Gene Expression Profiling
Genome, Archaeal
Multigene Family

@article {pmid40460129,
year = {2025},
author = {Jiang, W and Wang, S and Ahlheit, D and Fumagalli, T and Yang, Z and Ramanathan, S and Jiang, X and Weber, T and Dahlin, J and Borodina, I},
title = {High-throughput metabolic engineering of Yarrowia lipolytica through gene expression tuning.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {23},
pages = {e2426686122},
doi = {10.1073/pnas.2426686122},
pmid = {40460129},
issn = {1091-6490},
support = {NNF20CC0035580//Novo Nordisk Fonden (NNF)/ ; NNF20OC0060809//Novo Nordisk Fonden (NNF)/ ; NNF21OC0072559//Novo Nordisk Fonden (NNF)/ ; No. 101123257//EC | H2020 | PRIORITY 'Excellent science' | H2020 European Research Council (ERC)/ ; },
mesh = {*Yarrowia/genetics/metabolism ; *Metabolic Engineering/methods ; Transcription Factors/genetics/metabolism ; Promoter Regions, Genetic ; *Gene Expression Regulation, Fungal ; CRISPR-Cas Systems ; Plasmids/genetics ; Gene Library ; Fungal Proteins/genetics/metabolism ; },
abstract = {The challenge of accurately predicting which genetic alternations lead to the desired phenotype necessitates high-throughput metabolic engineering approaches where numerous hypotheses can be tested simultaneously. We describe the CRISPR-Cas9-based method TUNE[YALI] that enables high-throughput tuning of gene expression in the common industrial yeast Yarrowia lipolytica. The method is based on replacing the promoters of the target genes with native Y. lipolytica promoters of varying strengths or removing the promoters entirely. To demonstrate the method's capabilities, we created a plasmid library that targets 56 transcription factors (TFs) and changes the expression of each TF to seven different levels. We transformed this library into reference and betanin-producing strains of Y. lipolytica and screened the resulting clones for changes in morphology, thermotolerance, or improved betanin production. The genetic markup of the yeast clones with the desired phenotypic changes was determined by sequencing the inserted plasmids. We identified multiple TFs whose regulatory changes increased thermotolerance, two TFs that eliminated pseudohyphal growth, and several TFs that increased betanin production. Analogous libraries can be designed to target any chosen group of genes and even all the genes. The libraries can be shared and reused, accelerating applied strain development projects and fundamental functional genomics research (TUNE[YALI]-TF kit and TUNE[YALI]-TF library are available via AddGene under catalog numbers #1000000255 and #217744).},
}

*Yarrowia/genetics/metabolism
*Metabolic Engineering/methods
Transcription Factors/genetics/metabolism
Promoter Regions, Genetic
*Gene Expression Regulation, Fungal
CRISPR-Cas Systems
Plasmids/genetics
Gene Library
Fungal Proteins/genetics/metabolism

@article {pmid40411798,
year = {2025},
author = {Shi, K and Zhang, Y and Tao, Y and Wang, Y and Yang, J and Deng, R and Yang, H},
title = {Preamplification-Free Detection of RNA N6-Methyladenosine Modification at Single-Base Resolution Using the CRISPR Tandem Assay.},
journal = {Analytical chemistry},
volume = {97},
number = {22},
pages = {11454-11461},
doi = {10.1021/acs.analchem.4c06782},
pmid = {40411798},
issn = {1520-6882},
mesh = {*Adenosine/analogs & derivatives/analysis/metabolism ; Humans ; *CRISPR-Cas Systems ; RNA, Long Noncoding/genetics ; *RNA ; Lung Neoplasms/diagnosis/genetics ; },
abstract = {N6-Methyladenosine (m[6]A) ranks among the most prevalent modifications in RNA, which serves as a biomarker for diseases, such as lung cancer. Herein, we developed a CRISPR/Cas13a-Csm6 tandem assay (termed CRISPRm[6]A assay) allowing for preamplification-free, sensitive, and rapid detection of RNA m[6]A modifications. The coupling of Cas13a-Csm6 tandem with MazF endoribonuclease enables the assay to identify m[6]A RNA with single-base resolution. Compared to the CRISPRm[6]A assay using Cas13a alone, the tandem CRISPRm[6]A assay yielded an improved sensitivity for RNA detection by ∼22 times, thus enabling preamplification-free detection of RNA m[6]A. Particularly, the proposed assay enabled quantification of m[6]A abundance down to 0.5% at the picomole level in lncRNA MALAT1 and demonstrated a 100% correlation in diagnosing nonsmall cell lung cancer. In summary, the CRISPRm[6]A assay supports two key applications in biological samples: (1) precise determination of m[6]A sites and (2) quantitative measurement of m[6]A fractions. Therefore, the CRISPR tandem method presents a promising tool for RNA epigenetics-based diagnostics.},
}

*Adenosine/analogs & derivatives/analysis/metabolism
Humans
*CRISPR-Cas Systems
RNA, Long Noncoding/genetics
*RNA
Lung Neoplasms/diagnosis/genetics

@article {pmid40411467,
year = {2025},
author = {Wang, X and Zhong, L and Zhang, W and Wu, P and Wang, M and Li, D and Dong, L and Wang, G},
title = {CRISPR Digital Sensing: From Micronano-Collaborative Chip to Biomolecular Detection.},
journal = {ACS nano},
volume = {19},
number = {22},
pages = {20427-20451},
doi = {10.1021/acsnano.5c03474},
pmid = {40411467},
issn = {1936-086X},
mesh = {Humans ; *COVID-19/diagnosis/genetics/virology ; *CRISPR-Cas Systems/genetics ; SARS-CoV-2/genetics/isolation & purification ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Biosensing Techniques/methods ; *Lab-On-A-Chip Devices ; },
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) sensing technology proved to be valuable during the COVID-19 pandemic through its sensitivity, specificity, robustness, and versatility. However, issues such as overreliance on amplification, susceptibility to false positives, lack of quantification strategies, and complex operation procedures have hindered its broader application in bioanalysis and clinical diagnostics. The collision between micronano-collaborative chips and CRISPR technology has effectively addressed these bottlenecks, offering innovative solutions for diagnosis and treatment. Unlike conventional micronano chips, micronano digital chips enhance CRISPR's response to trace amounts of target molecules by leveraging highly controllable local environments and compartmentalized microreactors. This advancement improves detection efficiency and revolutionizes traditional in vitro bioanalytical processes. First, the working principles, fabrication techniques, and performance metrics of CRISPR-based digital droplet microfluidics and microarray chips are examined. Then, the applications of CRISPR digital sensing chips in bioassays are reviewed, emphasizing their importance in advancing in vitro detection systems for gene editing. Finally, the prospects of CRISPR digital sensing technology are explored, particularly its potential for body surface biomonitoring and its broader development opportunities in the biomedical field.},
}

Humans
*COVID-19/diagnosis/genetics/virology
*CRISPR-Cas Systems/genetics
SARS-CoV-2/genetics/isolation & purification
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Biosensing Techniques/methods
*Lab-On-A-Chip Devices

@article {pmid40390640,
year = {2025},
author = {Zhang, C and Chen, Y and Chen, X and Lin, X and Huang, Z and Zhang, L and Liu, R and Lv, Y},
title = {Single nanoparticle analysis-based CRISPR/Cas12 bioassay for amplification-free HIV detection.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {48},
pages = {8759-8762},
doi = {10.1039/d5cc01716e},
pmid = {40390640},
issn = {1364-548X},
mesh = {Humans ; *CRISPR-Cas Systems ; Limit of Detection ; *Nanoparticles/chemistry ; *Biological Assay/methods ; *HIV/isolation & purification ; *Biosensing Techniques/methods ; *HIV-1/isolation & purification ; *HIV Infections/diagnosis ; *Metal Nanoparticles/chemistry ; },
abstract = {To reduce the "window period" in HIV detection, most analytical methods require additional enzymes for signal amplification. Exempting challenges like primer interference and false positives in amplification strategies, we developed an amplification-free bioassay that uses CRISPR's potent cleavage activity and the competent sensitivity of single-nanoparticle analysis. An attomolar detection limit was achieved with adequate selectivity. Serum and cell tests confirm the bioassay's accurate and sensitive HIV detection.},
}

Humans
*CRISPR-Cas Systems
Limit of Detection
*Nanoparticles/chemistry
*Biological Assay/methods
*HIV/isolation & purification
*Biosensing Techniques/methods
*HIV-1/isolation & purification
*HIV Infections/diagnosis
*Metal Nanoparticles/chemistry

@article {pmid40326747,
year = {2025},
author = {Chen, Y and Yu, K and Jiang, Z and Yang, G},
title = {CRISPR-based genetically modified scaffold-free biomaterials for tissue engineering and regenerative medicine.},
journal = {Biomaterials science},
volume = {13},
number = {12},
pages = {3149-3175},
doi = {10.1039/d5bm00194c},
pmid = {40326747},
issn = {2047-4849},
mesh = {*Tissue Engineering/methods ; Humans ; *Regenerative Medicine/methods ; *Biocompatible Materials/chemistry ; Animals ; Gene Editing ; *CRISPR-Cas Systems ; Tissue Scaffolds ; },
abstract = {CRISPR-based genetically modified scaffold-free biomaterials, including extracellular vehicles, cell sheets, cell aggregates, organoids and organs, have attracted significant attention in the fields of regenerative medicine and tissue engineering in recent years. With a wide range of applications in gene therapy, modeling disease, tissue regeneration, organ xenotransplantation, modeling organogenesis as well as gene and drug screening, they are at a critical juncture from clinical trials to therapeutic applications. Xenografts have already been tested on non-human primates and humans. However, we have to admit that a series of obstacles still need to be addressed, such as immune response, viral infection, off-target effects, difficulty in mass production, and ethical issues. Therefore, future research should pay more attention to improving their safety, accuracy of gene editing, flexibility of production, and ethical rationality. This review summarizes various types of CRISPR-based genetically modified scaffold-free biomaterials, including their preparation procedures, applications, and possible improvements.},
}

*Tissue Engineering/methods
Humans
*Regenerative Medicine/methods
*Biocompatible Materials/chemistry
Animals
Gene Editing
*CRISPR-Cas Systems
Tissue Scaffolds

@article {pmid40287111,
year = {2025},
author = {Yang, Q and Zhang, Y and Chen, L and Fang, L and Xiao, S},
title = {Isolation, pathogenicity, and an infectious cDNA clone of the prevalent G2c variant of porcine epidemic diarrhea virus in China.},
journal = {Microbial pathogenesis},
volume = {205},
number = {},
pages = {107637},
doi = {10.1016/j.micpath.2025.107637},
pmid = {40287111},
issn = {1096-1208},
mesh = {Animals ; Swine ; *Porcine epidemic diarrhea virus/genetics/pathogenicity/isolation & purification ; China/epidemiology ; *Swine Diseases/virology/epidemiology ; Chlorocebus aethiops ; Vero Cells ; *Coronavirus Infections/veterinary/virology/epidemiology ; *DNA, Complementary/genetics ; Virulence ; Diarrhea/veterinary/virology/epidemiology ; Spike Glycoprotein, Coronavirus/genetics ; Disease Outbreaks ; Mutation ; Phylogeny ; CRISPR-Cas Systems ; },
abstract = {Porcine epidemic diarrhea virus (PEDV) is a major enteric coronavirus causing diarrhea in piglets, and the recent prevalence of its G2c variant poses a significant threat to the pig industry in China. In this study, we successfully isolated a G2c variant strain (designated EHuB4) from a pig farm experiencing an outbreak of severe diarrhea in China, and constructed its full-length cDNA infectious clone using CRISPR/Cas9 technology. The parental EHuB4 strain and its rescued virus, rEHuB4, exhibited similar biological characteristics in Vero cells, including rapid cytopathic effects and the formation of large syncytia, suggesting strong cell fusion ability and replication efficiency. Pathogenicity experiments in piglets demonstrated that both EHuB4 and rEHuB4 caused severe diarrhea and mortality, further confirming that the prevalence of the G2c variant may be the primary reason for the current high incidence of PED outbreaks. We also analyzed the mutation frequency and variation tendency in the amino acids encoded by the EHuB4 Spike (S) gene and identified multiple high-frequency mutation sites located not only in the S1 subunit but also in the intermediate region between heptad repeat 1 (HR1) and HR2. Furthermore, these mutations have persisted since 2011 and may be closely related to virus prevalence, host adaptation, and pathogenicity. In summary, this study not only characterizes the genetic evolution and pathogenicity of the prevalent G2c strain but also provides a valuable molecular tool for developing novel vaccines through the established full-length cDNA infectious clone.},
}

Animals
Swine
*Porcine epidemic diarrhea virus/genetics/pathogenicity/isolation & purification
China/epidemiology
*Swine Diseases/virology/epidemiology
Chlorocebus aethiops
Vero Cells
*Coronavirus Infections/veterinary/virology/epidemiology
*DNA, Complementary/genetics
Virulence
Diarrhea/veterinary/virology/epidemiology
Spike Glycoprotein, Coronavirus/genetics
Disease Outbreaks
Mutation
Phylogeny
CRISPR-Cas Systems

@article {pmid40459995,
year = {2025},
author = {Wu, JL and Zheng, SS and Wang, L and Xiang, X and Li, F and Lv, ML and Wu, Q and Huang, ZX and Miao, HB},
title = {CRISPR-Cas System-Mediated Genetic Modification in Bacillus spp.: Current Status and Future.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c03140},
pmid = {40459995},
issn = {1520-5118},
abstract = {Bacillus spp. are a group of Gram-positive bacteria that have shown significant potential for development in recent years. It is capable of utilizing low-cost substrates to produce various high-value-added compounds, making it widely applicable in fields such as feed, pharmaceuticals, and food. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system-mediated genetic modification is recognized as one of the most efficient technologies. The application of this technology for the genetic improvement of Bacillus spp. greatly enhanced the production performance of these strains. In this review, we summarize the various CRISPR-Cas systems that have been applied to Bacillus spp., with a particular focus on systematically outlining the strategies for implementing CRISPR-Cas-mediated genetic modification in these bacteria. Notably, homologous recombination is the most widely used strategy, while base editing is emerging as a novel and precise approach. Additionally, we discuss the importance of expression regulation strategies in establishing Bacillus spp. as a cell factory. Finally, we propose potential solutions to current technical challenges, providing insights for the development of high-performance genetically modified Bacillus spp. production strains.},
}

@article {pmid40459840,
year = {2025},
author = {Keerthana, R and Rakshana, P and Salunkhe, SR and Sakthi, AR and Kokiladevi, E and Saraswathi, T and Pushpam, R and Raveendran, M and Sudha, M},
title = {CRISPR-Cas9 mediated enhancement of abiotic stress resilience in tomato: a comprehensive review of target genes.},
journal = {Molecular biology reports},
volume = {52},
number = {1},
pages = {538},
pmid = {40459840},
issn = {1573-4978},
mesh = {*Solanum lycopersicum/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Stress, Physiological/genetics ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; },
abstract = {Tomato (Solanum lycopersicum L.), a major vegetable crop grown worldwide, is consumed as both fresh and processed products. Concerns about the impact of abiotic stresses on tomato production are growing worldwide as climate change alters global weather patterns, adversely affecting crop yield and produce quality due to stresses like salt, heat, cold, and drought. Conventional breeding approaches such as hybridization, marker-assisted selection (MAS), and mutation breeding, have long been utilized to improve tomato resilience against abiotic stresses. These approaches are often hampered by extensive field trials, and require multiple generations limiting their efficiency in rapidly developing stress-tolerant cultivars. The efforts of traditional breeding systems are hindered by the narrow genetic base of tomatoes which poses a major bottleneck. Researchers have utilized CRISPR-Cas genome-editing technology to address this challenge to offer a precise and accelerated alternative for enhancing stress resilience in tomato. This versatile tool has gained attention for its simple, precise, and effective gene-editing capabilities. CRISPR-Cas based genome editing has successfully modified key genes related to stress-response pathways, enhancing abiotic stress resilience. Developing resistant cultivars help mitigate the impact of abiotic stress, thereby contributing to increased food production and food security. This review highlights recent progress in use of CRISPR-Cas9 gene editing to enhance tomato resilience to abiotic stresses.},
}

*Solanum lycopersicum/genetics
*CRISPR-Cas Systems/genetics
Gene Editing/methods
*Stress, Physiological/genetics
Plant Breeding/methods
Plants, Genetically Modified/genetics

@article {pmid40459690,
year = {2025},
author = {Lakhani, H and Kumar, N and Jangra, A and Negi, S and Dholariya, T and Tiwari, S},
title = {Streamlined protoplast transfection system for in-vivo validation and transgene-free genome editing in Banana.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {28},
pmid = {40459690},
issn = {1573-9368},
support = {BT/PR25789/GET/119/97/2017//Department of Biotechnology, Ministry of Science and Technology, India/ ; BIRAC/Tech Transfer/08/I2/QUT-BBF//Biotechnology Industry Research Assistance Council/ ; },
mesh = {*Musa/genetics/growth & development ; *Protoplasts/metabolism ; *Gene Editing/methods ; *Transfection/methods ; *Plants, Genetically Modified/genetics/growth & development ; CRISPR-Cas Systems/genetics ; Transgenes/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome, Plant ; },
abstract = {The advancement in the CRISPR/Cas system has significantly streamlined genome editing in plants, rendering it simple, reliable, and efficient. However, the development of transgene-free crops is a challenging task for vegetatively propagated plants like banana. In the present study, we established banana protoplasts-based versatile and efficient platform for genome editing to overcome this limitation. Herein, a protocol has been optimized for protoplast isolation by considering leaf and embryogenic cell suspension (ECS) of banana cultivar Grand Naine. Freshly prepared ECS was identified as the best source for protoplast isolation. The protoplast viability and competency were checked by transfection with plasmid and RNP complex. Polyethylene glycol (PEG)-mediated protoplast transfection using pCAMBIA1302 and pJL50TRBO vectors showed GFP expression with 30 and 70% efficiency, respectively, eventually proving the protocol's efficacy. Further, gRNAs targeting banana β-carotene hydroxylase gene are validated by in-vitro cleavage test and subsequently used for RNP complex formation with varied ratios (1:1, 1:2, 1:5, and 1:10) of SpCas9 to gRNA1. Among these, a 1:2 molar ratio proved best to generate indel frequency with 7%. Sequencing analysis of the target amplicon revealed mutations upstream of the PAM region, specifically with gRNA1, among the three in-vitro validated gRNAs. This study evaluated the effectiveness of gRNAs in-vitro and in-vivo, yielding inconsistent results that highlight the need for comprehensive in-vivo validation of their functionality. Conclusively, the optimized protocol for banana transfection has the potential to be harnessed for the generation of transgene-free genetically improved banana.},
}

*Musa/genetics/growth & development
*Protoplasts/metabolism
*Gene Editing/methods
*Transfection/methods
*Plants, Genetically Modified/genetics/growth & development
CRISPR-Cas Systems/genetics
Transgenes/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Genome, Plant

@article {pmid40457455,
year = {2025},
author = {Zhang, T and Meng, Z and Yu, H and Zhang, Z and Liu, G and Qiu, A and Zheng, W and Ding, P and Kai, T},
title = {An integrated multi-mode detection platform based on CRISPR/Cas 12a and aptamers for ultra-sensitive identification of sulfamethazine and genes associated with sulfonamide resistance.},
journal = {Journal of nanobiotechnology},
volume = {23},
number = {1},
pages = {408},
pmid = {40457455},
issn = {1477-3155},
support = {2025ZZTS0167//Graduate Research Innovation Program of Central South University/ ; 82373635//the National Natural Science Foundation of China/ ; 2022RC1206//the Science and Technology Innovation Program of Hunan Province/ ; 2023QYJC013//the Central South University Research Program of Advanced Interdisciplinary Studies/ ; },
mesh = {*Sulfamethazine/analysis/pharmacology ; *Aptamers, Nucleotide/chemistry ; *CRISPR-Cas Systems/genetics ; *Sulfonamides/pharmacology ; Biosensing Techniques/methods ; *Anti-Bacterial Agents/analysis/pharmacology ; Electrochemical Techniques ; *Drug Resistance, Bacterial/genetics ; Metal-Organic Frameworks/chemistry ; Benzidines/chemistry ; Limit of Detection ; },
abstract = {The production and buildup of sulfamethazine (SMZ) and resistance genes for sulfonamide antibiotics (sul1) pose a serious risk to environmental and public health safety. Creating advanced sensing systems that are both highly sensitive and selective for the prolonged observation of SMZ concentrations in the environment, along with the quantification of sul1 gene prevalence, aims to identify trends in resistance, posing a considerable challenge. Here, we devised a platform (SMZ-sul1 multi-mode detection platform) that allows for the fluorescence detection of SMZ in environmental samples. This is achieved through the competition for the aptamer between the complementary base and SMZ, along with the colorimetric, photothermal, and electrochemical tracking of sul1, using a magnetic separation unit (FP@cDNA). MOF-818@PtPd (MPP) nanozymes with high peroxide mimetic enzyme activity were linked to FP@cDNA through Zr-O-P bond and employed as a catalyst for the 3,3',5,5'-tetramethylbenzidine (TMB) oxidation, as well as for electrocatalytic hydrogen peroxide (H2O2) reduction. The ability of Cas12a to perform trans cleavage was activated by its precise identification of the sul1, leading to the non-selective cutting of single-stranded DNA (ssDNA). Thereafter, the MPP nanoparticles were released into the supernatant, where they catalyzed the oxidation of TMB. Alternatively, the functioning CRISPR/Cas12a system specifically targeted and cleaved ssDNA present on the electrode, resulting in altered loading of MPP nanozymes and a decrease in the current associated with the catalytic reduction of H2O2. The remarkable magnetic separation capabilities of FP@cDNA, combined with the superior target recognition features of CRISPR/Cas12a and aptamer, facilitated the creation of a highly sensitive detection system, achieving detection limits of 0.67 pM for SMZ and 7.6 fM for sul1, and exhibit great potential for monitoring and prediction in the field of public health.},
}

*Sulfamethazine/analysis/pharmacology
*Aptamers, Nucleotide/chemistry
*CRISPR-Cas Systems/genetics
*Sulfonamides/pharmacology
Biosensing Techniques/methods
*Anti-Bacterial Agents/analysis/pharmacology
Electrochemical Techniques
*Drug Resistance, Bacterial/genetics
Metal-Organic Frameworks/chemistry
Benzidines/chemistry
Limit of Detection

@article {pmid40456823,
year = {2025},
author = {Hong, D and Shu, M and Liu, J and Liu, L and Cheng, H and Zhu, M and Du, Y and Xu, B and Hu, D and Liu, Z and Zhao, Y and Dai, J and Lu, F and Huang, J},
title = {Divergent combinations of enhancers encode spatial gene expression.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5091},
pmid = {40456823},
issn = {2041-1723},
support = {92474104//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32370586//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; *Enhancer Elements, Genetic/genetics ; Mice ; Humans ; Brain/metabolism/embryology ; Chromatin/metabolism/genetics ; Basic Helix-Loop-Helix Transcription Factors/genetics/metabolism ; Breast Neoplasms/genetics ; Spinal Cord/metabolism/embryology ; Gene Expression Regulation, Developmental ; Melanoma/genetics ; CRISPR-Cas Systems ; },
abstract = {Spatial transcriptomics and epigenomics have enabled mapping gene regulation in the tissue context. However, it remains poorly understood how spatial gene expression patterns are orchestrated by enhancers. Here we build eSpatial, a computational framework that deciphers spatially resolved enhancer regulation of gene expression by integrating spatial profiles of gene expression and chromatin accessibility. Applying eSpatial to diverse spatial datasets, including mouse embryo and brain, as well as human melanoma and breast cancer, we reveal a "spatial enhancer code", in which divergent combinations of enhancers regulate the same gene in spatially segregated domains. We validate the spatial enhancer code using public spatial datasets such as VISTA, Allen in situ hybridization (ISH), and H3K27ac MERFISH. Moreover, we conduct transgenic reporter assays and in vivo CRISPR/Cas9-mediated perturbation experiments to confirm the Atoh1 spatial enhancer code in determining Atoh1 spatial expression in mouse embryonic spinal cord and brain. Our study establishes the spatial enhancer code concept, revealing how combinations of enhancers dynamically shape gene expression across diverse biological contexts, providing insights into tissue-specific regulatory mechanisms and tumor heterogeneity.},
}

Animals
*Enhancer Elements, Genetic/genetics
Mice
Humans
Brain/metabolism/embryology
Chromatin/metabolism/genetics
Basic Helix-Loop-Helix Transcription Factors/genetics/metabolism
Breast Neoplasms/genetics
Spinal Cord/metabolism/embryology
Gene Expression Regulation, Developmental
Melanoma/genetics
CRISPR-Cas Systems

@article {pmid40425072,
year = {2025},
author = {Nair, A and Rao, AS and Surabhi, MA and Gnanika, M and More, SS},
title = {Unravelling fungal pathogenesis: Advances in CRISPR-Cas9 for understanding virulence and adaptation.},
journal = {Fungal genetics and biology : FG & B},
volume = {179},
number = {},
pages = {104006},
doi = {10.1016/j.fgb.2025.104006},
pmid = {40425072},
issn = {1096-0937},
mesh = {*CRISPR-Cas Systems ; *Fungi/pathogenicity/genetics ; Virulence/genetics ; Gene Editing ; Genome, Fungal ; *Adaptation, Physiological/genetics ; Humans ; Mycoses/microbiology ; Virulence Factors/genetics ; },
abstract = {Fungi, with their billion-year evolutionary history, have adapted to diverse ecological niches, including pathogenic roles that threaten global health, agriculture, and ecosystems. Fungal pathogenicity is shaped by the dynamic evolution of genetic traits that enable fungi to infect hosts, evade immune defenses, and develop resistance to antifungal treatments. Despite their significant clinical and ecological impact, the evolutionary processes underlying fungal virulence and adaptation remain incompletely understood. This review emphasizes the transformative role of CRISPR-Cas9 genome editing in revealing these mechanisms. By allowing precise manipulation of fungal genomes, CRISPR technologies have provided key insights into virulence factors, stress response mechanisms, immune evasion, and antifungal resistance pathways. These advances demonstrate how fungi adapt to selective pressures, repurpose conserved genetic pathways, and exploit genomic plasticity to thrive in host environments. This review explores the intersection of CRISPR technology and fungal biology, shedding light on its implications for understanding fungal pathogenesis and the potential to develop innovative therapeutic strategies against fungal infections. The integration of CRISPR applications into mycology holds promise for furthering our understanding of fungal evolutionary trajectories and enhancing the development of novel therapeutic approaches.},
}

*CRISPR-Cas Systems
*Fungi/pathogenicity/genetics
Virulence/genetics
Gene Editing
Genome, Fungal
*Adaptation, Physiological/genetics
Humans
Mycoses/microbiology
Virulence Factors/genetics

@article {pmid40413994,
year = {2025},
author = {Liu, Y and Liu, Y and Wu, S and Cao, R and Pan, Y and Zhou, F},
title = {Engineered Cas12a-based one-tube detection of DNMT3A R882 H/C mutation in acute myeloid leukemia.},
journal = {Biosensors & bioelectronics},
volume = {286},
number = {},
pages = {117609},
doi = {10.1016/j.bios.2025.117609},
pmid = {40413994},
issn = {1873-4235},
mesh = {DNA Methyltransferase 3A ; *Leukemia, Myeloid, Acute/genetics/diagnosis ; Humans ; *DNA (Cytosine-5-)-Methyltransferases/genetics ; *CRISPR-Cas Systems/genetics ; Mutation ; *Biosensing Techniques/methods ; *Endodeoxyribonucleases/genetics/chemistry ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Advances in sequencing technologies have identified numerous genetic alterations associated with acute myeloid leukemia (AML), many of which play critical roles in diagnosis, classification, and prognosis. Among these, mutations in the DNA methyltransferase 3 alpha (DNMT3A) gene are particularly prevalent, with the R882H and R882C variants being the most common. Accurate and sensitive detection of DNMT3A mutations is crucial for prognosis, treatment guidance, and early intervention in AML. However, existing detection methods often fail to achieve an optimal balance among sensitivity, turnaround time, and operational simplicity. To address this limitation, we aimed to develop a rapid and highly sensitive method for detecting DNMT3A mutations. The CRISPR/Cas12a system shows promise for genetic detection due to its high sensitivity and single-base specificity. Here, we established a Cas12a-based one-tube assay for the detection of DNMT3A R882 H/C mutations. We utilized the mismatch tolerance of enAsU-R Cas12a to design crRNA for DNMT3A R882 H/C mutation and integrated CRISPR/Cas12a system with ERA. The entire detection process can be completed within 1 h at 37 °C. The optimized detection system demonstrated a sensitivity of 0.1 % when analyzing genomic DNA. To validate its clinical applicability, we tested samples from 49 AML patients and successfully identified all DNMT3A R882H/C-positive cases, including one with a mutation rate as low as 0.24 %. These results highlight the potential of our Cas12a-based one-tube detection system as a rapid, sensitive, and cost-effective method for detecting DNMT3A R882 H/C mutation. This approach could serve as a valuable tool for both diagnostic and therapeutic monitoring.},
}

DNA Methyltransferase 3A
*Leukemia, Myeloid, Acute/genetics/diagnosis
Humans
*DNA (Cytosine-5-)-Methyltransferases/genetics
*CRISPR-Cas Systems/genetics
Mutation
*Biosensing Techniques/methods
*Endodeoxyribonucleases/genetics/chemistry
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid40408898,
year = {2025},
author = {Li, M and Tao, C and Tang, Z and Li, K and Wang, Y and Zhao, C and Tao, C and Geng, J and Sun, K},
title = {Ultrasensitive clinical identification of hepatitis B surface antigen (HBsAg) by CRISPR-assisted nanopore sensing.},
journal = {Biosensors & bioelectronics},
volume = {286},
number = {},
pages = {117579},
doi = {10.1016/j.bios.2025.117579},
pmid = {40408898},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Humans ; *Hepatitis B Surface Antigens/blood/isolation & purification/genetics ; *Nanopores/ultrastructure ; *Hepatitis B virus/isolation & purification/genetics ; *CRISPR-Cas Systems/genetics ; *Hepatitis B/diagnosis/virology/blood ; Limit of Detection ; Aptamers, Nucleotide/chemistry ; },
abstract = {Hepatitis B virus (HBV) infection is a major global health issue. The underdiagnosis of HBV contributes to the increasing mortality from hepatitis B-related complications. Hepatitis B surface antigen is a biomarker guiding the clinical management of chronic hepatitis B, and its disappearance from the blood is a key sign of functional cure. There is a need for highly sensitive detection methods for early intervention and prevention of disease recurrence. We presented a new CRISPR-assisted nanopore sensing method for ultrasensitive detection of hepatitis B surface antigen. It uses the high specificity and turnover efficiency of the CRISPR-Cas12a system. The system is activated by the competitive binding between hepatitis B surface antigen and its aptamer, followed by restriction enzyme digestion. The products are detected by a nanopore for precise quantification at very low concentrations. The result achieves the limit of quantification (LOQ) of 10 fM, outperforming conventional assays. Clinical validation with patient samples confirms its superiority. This integrated technology is a powerful tool for HBV early diagnosis, treatment monitoring, and disease assessment, and paves the way for nanopore technology in clinical diagnostics.},
}

*Biosensing Techniques/methods
Humans
*Hepatitis B Surface Antigens/blood/isolation & purification/genetics
*Nanopores/ultrastructure
*Hepatitis B virus/isolation & purification/genetics
*CRISPR-Cas Systems/genetics
*Hepatitis B/diagnosis/virology/blood
Limit of Detection
Aptamers, Nucleotide/chemistry

@article {pmid40408895,
year = {2025},
author = {Xu, Y and Deng, R and Liu, X and Zhou, Y},
title = {CRISPR/Cas12a-triggered electrochemiluminescence biosensor to ultrasensitive detect herpes simplex virus via self-enhanced near-infrared selenium-based polymer dots.},
journal = {Biosensors & bioelectronics},
volume = {286},
number = {},
pages = {117597},
doi = {10.1016/j.bios.2025.117597},
pmid = {40408895},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; *Polymers/chemistry ; Humans ; Limit of Detection ; *Simplexvirus/isolation & purification/genetics ; *Selenium/chemistry ; Luminescent Measurements/methods ; *Herpes Simplex/virology/diagnosis ; Electrochemical Techniques/methods ; *Quantum Dots/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Herpes simplex virus (HSV) as one of the most popular herpesviruses exhibits nearly identical clinical symptoms among the family of herpesvirus species and it is urgent to develop high specific biosensors to realize accurate detection of HSV. CRISPR/Cas12a systems with programmability and high specificity could serve an essential role in accurate diagnosis of HSV in complex samples. Herein, a novel near-infrared selenium-based polymer dots with self-enhanced effect were unprecedentedly designed and successfully synthesized in this work, and an ultrasensitive biosensor has been constructed together with the CRISPR/Cas12a system for the specific detection of HSV. Notably, the as-prepared selenium-based polymer dots exhibited near-infrared emission with a peak at 760 nm, which could significantly minimize background noise and achieve high sensitivity. The proposed biosensor demonstrated a wide linear range from 1 fM to 1 nM and the limit of detection as low as 0.1 fM (S/N = 3). Undoubtedly, the CRISPR/Cas12a-triggered ECL biosensor proposed in this work could provide a simple, sensitive, and environmentally friendly approach to detect HSV in clinical applications.},
}

*Biosensing Techniques/methods
CRISPR-Cas Systems/genetics
*Polymers/chemistry
Humans
Limit of Detection
*Simplexvirus/isolation & purification/genetics
*Selenium/chemistry
Luminescent Measurements/methods
*Herpes Simplex/virology/diagnosis
Electrochemical Techniques/methods
*Quantum Dots/chemistry
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40354126,
year = {2025},
author = {Lv, B and Liang, P and Chang, C and Li, D},
title = {Sensitive aptasensing of tobramycin through a rational design of catalytic hairpin assembly and hybridization chain reaction amplification monomers for CRISPR/Cas12a activation.},
journal = {The Analyst},
volume = {150},
number = {12},
pages = {2580-2590},
doi = {10.1039/d5an00267b},
pmid = {40354126},
issn = {1364-5528},
mesh = {*CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Limit of Detection ; *Aptamers, Nucleotide/chemistry/genetics ; *Nucleic Acid Amplification Techniques/methods ; Milk/chemistry ; *Tobramycin/analysis ; Nucleic Acid Hybridization ; Animals ; *Anti-Bacterial Agents/analysis ; DNA/chemistry/genetics ; Inverted Repeat Sequences ; *CRISPR-Associated Proteins/metabolism/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {The catalytic hairpin assembly (CHA) and hybridization chain reaction amplification (HCR) are enzyme-free isothermal DNA amplification methods based on the self-assembly of hairpin monomers. Recently, CRISPR/Cas12a-based biosensors in combination with CHA or HCR signal amplification have shown promising performance. Herein, several design strategies for hairpin monomers in CHA and HCR were evaluated in the context of CRISPR/Cas12a-based biosensor construction. The SL-HCR strategy, in which the CRISPR/Cas12a target strand is blocked in the loop of one hairpin monomer DNA and released in the duplex HCR products, demonstrated superior performance in terms of a low background signal, wide linear detection range, and high signal-to-noise ratio. With the assistance of an aptamer-containing probe, a highly sensitive aptasensor was constructed for tobramycin detection, whereby the SL-HCR served the function of signal amplification, whereas the CRISPR/Cas12a system acted to cleave the FQ probes, thereby resulting in the production of a fluorescent signal. After optimization, the aptasensor enables linear detection of tobramycin concentrations ranging from 125 pM to 2500 nM, with a limit of detection (LOD) of 92.87 pM. Moreover, the aptasensor was utilized to detect tobramycin in beef and milk samples, yielding satisfactory results. The assay is concise and cost-effective due to the absence of nanomaterial DNA labeling and magnetic separation procedures. Furthermore, the entire detection workflow operates under isothermal conditions, which makes it suitable for use in food safety control and environmental monitoring. In addition, the results presented here may shed new light on the design of CRISPR/Cas12a-based biosensors in combination with CHA or HCR.},
}

*CRISPR-Cas Systems
*Biosensing Techniques/methods
Limit of Detection
*Aptamers, Nucleotide/chemistry/genetics
*Nucleic Acid Amplification Techniques/methods
Milk/chemistry
*Tobramycin/analysis
Nucleic Acid Hybridization
Animals
*Anti-Bacterial Agents/analysis
DNA/chemistry/genetics
Inverted Repeat Sequences
*CRISPR-Associated Proteins/metabolism/genetics
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid40456709,
year = {2025},
author = {Hanlon, MB and Shohet, JM and Wolfe, SA},
title = {Selective targeting of genome amplifications and repeat elements by CRISPR-Cas9 nickases to promote cancer cell death.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5126},
pmid = {40456709},
issn = {2041-1723},
support = {R01 CA275945/CA/NCI NIH HHS/United States ; R01CA275945//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Amplification ; *Neuroblastoma/genetics/pathology ; Cell Line, Tumor ; N-Myc Proto-Oncogene Protein/genetics ; Cell Death/genetics ; *Deoxyribonuclease I/metabolism/genetics ; DNA Breaks, Double-Stranded ; Neoplasms/genetics ; },
abstract = {Focal gene amplification serves as an oncogenic driver during tumorigenesis and is a hallmark of many forms of cancer. Oncogene amplifications promote genomic instability, which is integral to cancer cell survival and evolution. However, focal gene amplification potentially affords an opportunity for therapeutic exploitation. As a proof-of-concept, we leverage CRISPR-Cas9 nickase to selectively promote cancer cell death in MYCN-amplified neuroblastoma in a gene amplification-dependent manner. Our analysis demonstrates that CRISPR-Cas9 nickase can generate a lethal number of highly toxic, replication-dependent double-strand breaks in cells harboring amplified loci. Furthermore, we demonstrate that Cas9 nickase-mediated toxicity can be modulated in combination with small molecule inhibitors targeting key regulators of the DNA-damage response or cell death pathways. Importantly, our findings in MYCN-amplified neuroblastoma translate to other cancer types with distinct oncogene amplifications.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Amplification
*Neuroblastoma/genetics/pathology
Cell Line, Tumor
N-Myc Proto-Oncogene Protein/genetics
Cell Death/genetics
*Deoxyribonuclease I/metabolism/genetics
DNA Breaks, Double-Stranded
Neoplasms/genetics

@article {pmid40412122,
year = {2025},
author = {Wang, R and Yang, Y and Wang, Z and Ma, C and Wu, M and Du, Y and Zhang, X and Cao, M and Xu, H},
title = {Stimuli-responsive peptide nanocarriers for tumor-specific CRISPR/Cas9 delivery and precision genome editing.},
journal = {Journal of colloid and interface science},
volume = {697},
number = {},
pages = {137932},
doi = {10.1016/j.jcis.2025.137932},
pmid = {40412122},
issn = {1095-7103},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; *Nanoparticles/chemistry ; *Peptides/chemistry ; HeLa Cells ; *Drug Carriers/chemistry ; Particle Size ; *Ribonucleoproteins/chemistry/genetics ; Cell Survival/drug effects ; *Neoplasms/genetics ; Surface Properties ; },
abstract = {CRISPR/Cas9 ribonucleoprotein (RNP) delivery remains a critical challenge due to its large size, instability, and off-target effects. Here, we report a stimuli-responsive cationic amphiphilic peptide, (CR3)3C, designed for cancer-targeted delivery of CRISPR/Cas9 RNP. The peptide integrates three functional domains: (1) a naphthyl-diphenylalanine (Nap-FF) motif enabling self-assembly into stable nanoparticles via aromatic interactions, (2) a matrix metalloproteinase-7 (MMP7)-cleavable linker (GPLGLA) for tumor microenvironment-specific activation, and (3) a redox-responsive cationic domain ((CRRR)3-C) for electrostatic RNP binding and glutathione (GSH)-triggered intracellular release. The (CR3)3C/RNP nanocomplexes (108.8 nm diameter, ζ = +10.89 mV) demonstrate exceptional stability and cellular uptake efficiency. Mechanistic studies reveal caveolae-mediated endocytosis and lipid raft-associated pathways, proton sponge effect-driven endosomal escape, and nuclear localization facilitated by Cas9's nuclear localization signal. In HeLa-EGFP cells, (CR3)3C/RNP shows 33.8 % gene editing efficiency at 100 nM RNP with >90 % cell viability. This work establishes a programmable, non-viral platform that synergizes enzymatic and redox responsiveness for tumor-targeted genome editing, addressing critical barriers in CRISPR therapeutics.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Editing
*Nanoparticles/chemistry
*Peptides/chemistry
HeLa Cells
*Drug Carriers/chemistry
Particle Size
*Ribonucleoproteins/chemistry/genetics
Cell Survival/drug effects
*Neoplasms/genetics
Surface Properties

@article {pmid40403777,
year = {2025},
author = {Wu, HJ and Yang, JP and Ma, WJ and Li, ZH and Feng, HY and Yang, ZN and Xu, HJ},
title = {A CRISPR/Cas9-induced point mutation on the GABA receptor subunit RDL confers high resistance to phenylpyrazole insecticides in the rice planthopper Laodelphax striatellus.},
journal = {Insect biochemistry and molecular biology},
volume = {181},
number = {},
pages = {104327},
doi = {10.1016/j.ibmb.2025.104327},
pmid = {40403777},
issn = {1879-0240},
mesh = {Animals ; *Hemiptera/genetics/drug effects/metabolism ; *Insecticide Resistance/genetics ; *Insecticides/pharmacology ; *Pyrazoles/pharmacology ; CRISPR-Cas Systems ; *Receptors, GABA/genetics/metabolism ; *Point Mutation ; *Insect Proteins/genetics/metabolism ; Female ; },
abstract = {The Delphacidae planthopper Laodelphax striatellus (Order: Hemiptera) is one of the most damaging insect pests of rice crops in Asia. The phenylpyrazole insecticide fipronil was introduced in China in the mid-1990s to control these pests, but its widespread use has led to the development of high levels of resistance. Field sampling coupled with in vitro assays indicated that an A2'N-point mutation in the gamma-aminobutyric acid receptor RDL has been linked to fipronil resistance; however, genetic evidence supporting this association has been lacking. Here, we generated an A2'N-point mutation (LsRDL[A/N]) in L. striatellus using CRISPR/Cas9-mediated homologous recombination. Bioassay revealed that the LsRDL[A/N] mutants conferred a 1211-fold higher resistance to fipronil compared to wild-type planthoppers. Furthermore, these mutants also showed significant resistance (>1000-fold) to ethiprole but not to imidacloprid, demonstrating that the A2'N mutation confers resistance specific to phenylpyrazole insecticides. However, the LsRDL[A/N] mutants displayed reduced fecundity, lower egg hatching rates, and prolonged egg and nymph stages, indicating that fipronil resistance carries a substantial fitness cost. These findings enhance our understanding of the genetic mechanisms underlying phenylpyrazole resistance and provide valuable insights for developing effective resistance management strategies for planthoppers.},
}

Animals
*Hemiptera/genetics/drug effects/metabolism
*Insecticide Resistance/genetics
*Insecticides/pharmacology
*Pyrazoles/pharmacology
CRISPR-Cas Systems
*Receptors, GABA/genetics/metabolism
*Point Mutation
*Insect Proteins/genetics/metabolism
Female

@article {pmid40368251,
year = {2025},
author = {Ramadan, NK and Gaber, N and Ali, NM and Amer, OSO and Soliman, H},
title = {SHERLOCK, a novel CRISPR-Cas13a-based assay for detection of infectious bursal disease virus.},
journal = {Journal of virological methods},
volume = {337},
number = {},
pages = {115185},
doi = {10.1016/j.jviromet.2025.115185},
pmid = {40368251},
issn = {1879-0984},
mesh = {*Infectious bursal disease virus/isolation & purification/genetics ; Animals ; Chickens/virology ; *Birnaviridae Infections/diagnosis/veterinary/virology ; *Poultry Diseases/diagnosis/virology ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; RNA, Viral/genetics ; },
abstract = {Infectious bursal disease (IBD) is an extremely contagious viral infection that primarily affects young chicks, leading to significant economic losses in the poultry industry. The disease is caused by a double-stranded RNA virus of the genus Avibirnavirus, family Birnaviridae, namely, the infectious bursal disease virus (IBDV). Unfortunately, current methods for detecting IBDV lack adequate sensitivity. Accordingly, the advantages of the Specific High Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) assay were employed to develop an ultrasensitive assay (IBD-SHERLOCK assay) for the detection of IBDV in clinical chicken tissues. The assay comprises two steps: isothermal preamplification of the target RNA through reverse transcription recombinase polymerase amplification (RT-RPA) and a subsequent detection step, which is based on the CRISPR-Cas13a system. The integration of lateral flow (LFD) visual detection of the IBD-SHERLOCK products strengthens the feasibility of the assay for use as a point-of-care test in chicken farms. Compared with RT-qPCR, this method exhibited ultra-analytical and clinical sensitivity. The assay has a lower detection limit of 5 aM, which is equivalent to three IBDV-RNA molecules. The assay demonstrated the ability to detect IBDV-RNA in 70 clinical field samples, 15 of which tested negative by RT-qPCR. This evidence highlights its superior sensitivity and potential for early detection of IBDV in chicken tissues. This study effectively established and verified a CRISPR-based diagnostic test for the early detection of IBDV in clinical chicken tissues, demonstrating remarkable specificity and sensitivity. The IBD-SHERLOCK assay can be used as a highly sensitive point-of-care diagnostic tool in chicken farms.},
}

*Infectious bursal disease virus/isolation & purification/genetics
Animals
Chickens/virology
*Birnaviridae Infections/diagnosis/veterinary/virology
*Poultry Diseases/diagnosis/virology
Sensitivity and Specificity
*CRISPR-Cas Systems
*Molecular Diagnostic Techniques/methods
*Nucleic Acid Amplification Techniques/methods
RNA, Viral/genetics

@article {pmid40456230,
year = {2025},
author = {Huda, NU and Hasan, KA and Saleem, F and Naz, S and Khan, S and Mirani, ZA and Syed, MN and Alswat, AS and Sohail, M},
title = {Coriander borne Salmonella superbug: genomic assessments of chromosomal and plasmid-associated resistance, virulence and MGEs of XDR Salmonella enterica Typhi NH1.},
journal = {Pathogens and global health},
volume = {},
number = {},
pages = {1-15},
doi = {10.1080/20477724.2025.2513769},
pmid = {40456230},
issn = {2047-7732},
abstract = {The drug-resistant strains of Salmonella enterica serovar Typhi (S. Typhi) are serious threats to health sectors worldwide. This study focuses on genomic characterization of environmentally derived an extensively drug resistant (XDR) strain of Salmonella Typhi, highlighting its potential to cause significant outbreak. The XDR strain (labeled as NH1) was isolated from fresh coriander, and characterized through whole-genome sequencing to investigate its lineage diversity, niche adaptation, sources and resistant mechanism. The NH1 strain exhibits phylogenomic association with clinical lineages S. Typhi NCTC8385 and ASM3025476. Its genome, assembled in 67 contigs, exhibited expected coverage and GC content, harboring a mega-plasmid, virulence factors, and intrinsic and extrinsic antimicrobial resistance genes. Plasmid annotation revealed IncQ1 and IncY plasmids responsible for the acquired resistance. SPIFinder identified SPI1-10 pathogenicity islands, and the CRISPR-Cas locus, associated with bacterial defense, bile salt resistance and biofilm formation, was present. NH1 strain also possesses numerous mobile elements (proteins and transposases), 12 prophages, and secretion systems (types I-IV, VI), aiding its survival in environment like vegetables. The genomic characterization of S. Typhi NH1 highlights the emergence of coriander-borne XDR strain, underscoring the need for targeted prevention to mitigate its public health impact.},
}

@article {pmid40455764,
year = {2025},
author = {De Pablo-Moreno, JA and González-Brusi, L and Miguel-Batuecas, A and Bermejo-Álvarez, P and Revuelta, L and Liras, A},
title = {Development of a novel and viable knock-in factor V deficiency murine model: Utility for an ultra-rare disease.},
journal = {PloS one},
volume = {20},
number = {6},
pages = {e0321864},
pmid = {40455764},
issn = {1932-6203},
mesh = {Animals ; Mice ; *Disease Models, Animal ; *Factor V Deficiency/genetics/pathology ; *Factor V/genetics/metabolism/chemistry ; *Gene Knock-In Techniques ; Humans ; Female ; Male ; CRISPR-Cas Systems ; Phenotype ; Homozygote ; Mutation, Missense ; *Rare Diseases/genetics ; Partial Thromboplastin Time ; Prothrombin Time ; },
abstract = {Factor V deficiency is a congenital coagulation disorder characterized by the absence or malfunction of factor V (FV). The purpose of this study was to develop a viable FV-deficient mouse model using CRISPR/Cas9 technology. A viable pathological model of the disease was not available to develop new therapies. A previous in silico study was performed to select a mutation causing a mild disease phenotype in humans (Thr1898Met missense). Such mutation was replicated in mice by CRISPR-mediated homology directed repair. Following crossing, homozygous individuals were subjected to coagulometry assays, including FV levels, prothrombin time (PT), and activated partial thromboplastin time (aPTT). The in silico study suggested that the mutation destabilizes FV structure of both mouse and human variants, putatively producing a mild phenotype of the disease in mice. Mendelian inheritance was observed in the offspring. No spontaneous signs of blood clotting disturbances, premature deaths or gestational dysfunctions were observed. FV levels in homozygous animals were 24.5% ± 5.1; 39.7 sec ± 2.8; PT was 61.8% ± 6.3; 23.4 sec ± 1.6 (INR = 1.47 ± 0.12); and aPTT was 46.9 sec ± 3.2. A viable FV-deficient mouse model was generated by introducing a missense mutation in FV. The model exhibits a mild phenotype of the disease, akin to that observed in humans.},
}

Animals
Mice
*Disease Models, Animal
*Factor V Deficiency/genetics/pathology
*Factor V/genetics/metabolism/chemistry
*Gene Knock-In Techniques
Humans
Female
Male
CRISPR-Cas Systems
Phenotype
Homozygote
Mutation, Missense
*Rare Diseases/genetics
Partial Thromboplastin Time
Prothrombin Time

@article {pmid40455288,
year = {2025},
author = {Hu, J and Yu, W and Cui, J and Zhang, L and Yu, W},
title = {Recent advances in diagnostic technologies for postoperative central nervous system infections: a review.},
journal = {Neurological sciences : official journal of the Italian Neurological Society and of the Italian Society of Clinical Neurophysiology},
volume = {},
number = {},
pages = {},
pmid = {40455288},
issn = {1590-3478},
abstract = {Postoperative central nervous system infections (PCNSIs), including meningitis, cerebral abscesses, and implant-associated infections, represent critical complications following neurosurgical procedures. These infections pose significant risks to patient outcomes due to delayed diagnosis, escalating antimicrobial resistance, and limited therapeutic efficacy. Conventional diagnostic approaches, such as cerebrospinal fluid (CSF) analysis, microbial cultures, and neuroimaging, exhibit notable limitations in sensitivity, specificity, and rapidity. This review highlights transformative technologies reshaping PCNSI diagnostics, including molecular assays (e.g., quantitative PCR, digital droplet PCR), metagenomic next-generation sequencing (mNGS), CRISPR-based pathogen detection platforms, metabolomics, and advanced molecular imaging modalities. Furthermore, we address translational challenges in clinical adoption, including cost barriers, standardization gaps, and the need for interdisciplinary collaboration. Emerging artificial intelligence (AI)-driven strategies are proposed to optimize pathogen identification, predict antimicrobial resistance profiles, and tailor personalized therapeutic regimens.},
}

@article {pmid40398759,
year = {2025},
author = {Zhao, S and Zhang, J and Yu, S and He, D and Li, B and Fan, Y and Liu, G and Tang, Y and Diao, Y},
title = {Integration of RPA and CRISPR-Cas13a collateral activity for one-step detection of DHAV-3: A biological macromolecule-enabled diagnostic platform.},
journal = {International journal of biological macromolecules},
volume = {314},
number = {},
pages = {144400},
doi = {10.1016/j.ijbiomac.2025.144400},
pmid = {40398759},
issn = {1879-0003},
mesh = {*CRISPR-Cas Systems/genetics ; Animals ; *Recombinases/metabolism ; RNA, Viral/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Picornaviridae/genetics/isolation & purification ; Ducks/virology ; },
abstract = {Duck hepatitis A virus type 3 (DHAV-3), which is families of the Picornaviridae, poses severe threats to waterfowl industries due to rapid antigenic evolution and limitations in conventional diagnostics. Herein, we engineered a CRISPR-Cas13a-mediated RNA detection system by leveraging the intrinsic HEPN domain-dependent collateral cleavage activity of Cas13a, synergistically integrated with recombinase polymerase amplification (RPA) to target DHAV-3 RNA. This biological macromolecule-driven platform achieved ultrasensitive detection (1 copies/μL) within 35 min through sequence-specific crRNA guidance and Cas13a-triggered fluorescent/lateral flow signal amplification. Rigorous validation against four avian pathogens (ARV, H9N2 AIV, TMUV, AstV) confirmed 100 % specificity, highlighting the precise macromolecular interactions between Cas13a and target RNA. Clinical evaluation of 30 field samples demonstrated complete concordance with RT-qPCR. By harnessing the programmable functionality of Cas13a and the thermostable enzymes in RPA, this study provides a novel paradigm for RNA-guided biological macromolecule applications in point-of-care diagnostics, bridging molecular mechanisms with agricultural biosecurity needs.},
}

*CRISPR-Cas Systems/genetics
Animals
*Recombinases/metabolism
RNA, Viral/genetics
*Nucleic Acid Amplification Techniques/methods
*Picornaviridae/genetics/isolation & purification
Ducks/virology

@article {pmid40347696,
year = {2025},
author = {Martin, OL and Lynch, CRH and Fleming, R},
title = {Advancing forensic body fluid identification: A comparative analysis of RT-LAMP+CRISPR-Cas12a and established mRNA-based methods.},
journal = {Forensic science international. Genetics},
volume = {78},
number = {},
pages = {103297},
doi = {10.1016/j.fsigen.2025.103297},
pmid = {40347696},
issn = {1878-0326},
mesh = {Humans ; *RNA, Messenger/genetics ; Female ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Associated Proteins/genetics ; Sensitivity and Specificity ; Cervix Mucus/chemistry ; Male ; Forensic Genetics/methods ; Semen/chemistry ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {In forensic science, the analysis of body fluid evidence determines the cellular origin of a sample, aiding in the reconstruction of a potential crime. Messenger ribonucleic acid (mRNA) based confirmatory tests address limitations of current conventional methods, providing increased specificity and sensitivity, minimal sample consumption, and the detection of a broader range of body fluids. However, they require expensive instrumentation, longer reaction times, and lack portability. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) coupled with clustered regular interspaced short palindromic repeats (CRISPR) with CRISPR-associated protein 12a (Cas12a) has the potential to overcome these challenges. This approach offers reduced testing time and cost, while potentially providing equivalent sensitivity and specificity, as observed in the field of viral diagnostics. Visual detection capabilities enable the development of rapid, portable screening tests suitable for testing at the crime scene. In the context of a sexual assault investigation, RT-LAMP+CRISPR-Cas12a could potentially increase the efficiency and detection rate. This study compares this novel method to two other mRNA-based methods, endpoint reverse transcription polymerase chain reaction (RT-PCR) multiplex assay CellTyper 2, and a real-time reverse transcription quantitative PCR (RT-qPCR) multiplex assay. The tests' sensitivity and specificity were evaluated on single-source and mixed body fluid samples, including rectal mucosa, a fluid which is minimally explored in forensic literature. The RT-qPCR assay demonstrated the highest sensitivity, specificity, and precision in mixed samples. In addition, RT-qPCR offers a greater linear dynamic range, faster processing time and easier methodology compared to CellTyper 2, only limited by its expensive nature. Notably, rectal mucosa samples exhibited non-specific marker expression of CellTyper 2 markers and expression of CYP2B7P (vaginal fluid) for all methods. This emphasises the need for a dedicated rectal mucosa marker. RT-LAMP+CRISPR-Cas12a exhibited a high specificity, displaying off-target expression of CYP2B7P in two fluid types. However, the method lacked sensitivity and precision for most markers except MMP3 (menstrual blood), demonstrating detection down to 1:10,000 with 100 % specificity. RT-LAMP+CRISPR requires further development, but its quick, inexpensive nature and high specificity suggest it has potential as a confirmatory test that could reduce the limitations of existing methods.},
}

Humans
*RNA, Messenger/genetics
Female
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Associated Proteins/genetics
Sensitivity and Specificity
Cervix Mucus/chemistry
Male
Forensic Genetics/methods
Semen/chemistry
*Clustered Regularly Interspaced Short Palindromic Repeats
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid40294708,
year = {2025},
author = {Gao, X and Zhou, C and Feng, Y and Ye, B and Zhao, Z and Qi, L and Hu, L and Deng, Y and Lin, C and Ding, Q and Liu, G and Wang, C and Song, C and Qian, B and Wu, T and Wang, X and Liu, Z and Lin, Z and Zhang, M},
title = {Research progress of gene editing technology in neurological diseases.},
journal = {Gene},
volume = {962},
number = {},
pages = {149534},
doi = {10.1016/j.gene.2025.149534},
pmid = {40294708},
issn = {1879-0038},
mesh = {*Gene Editing/methods/trends ; Humans ; *Nervous System Diseases/therapy/genetics ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; Animals ; },
abstract = {Gene editing (GE) technology is a genetic manipulation technique based on artificial nucleases that enables the precise modification of DNA or RNA. With the development of technology, GE in disease treatment is becoming increasingly widespread, playing an essential role in haematology, cancer, and neurological disorders (ND). This review describes the principles, advantages, and limitations of four GE technologies, focusing on the fourth generation of GE (next-generation GE). The next-generation GE technology breaks the limitations of traditional GE technology, makes GE more precise and stable, and broadens the scope of gene technology applications. Additionally, this review explores the latest gene therapy strategies for ND, focusing on the application of next-generation GE technologies to examine the prospects for the application of GE technologies. This study discusses and analyses the great advantages and potential of GE technology for treating ND and elucidates the shortcomings of GE in this field.},
}

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

@article {pmid40286513,
year = {2025},
author = {Tang, Q and Liao, Q and Huang, X and Huang, H and Tang, Q and Zhang, K and Liao, X},
title = {A highly sensitive MiRNA detection sensor powered by CRISPR/Cas13a and entropy-driven amplification.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {165},
number = {},
pages = {108992},
doi = {10.1016/j.bioelechem.2025.108992},
pmid = {40286513},
issn = {1878-562X},
mesh = {*MicroRNAs/analysis/genetics ; *CRISPR-Cas Systems ; *Entropy ; Humans ; *Biosensing Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Electrochemical Techniques/methods ; Luminescent Measurements/methods ; },
abstract = {MicroRNAs (miRNAs) are critical regulators of numerous physiological and pathological processes, influencing gene expression and playing essential roles in cellular development, differentiation, and disease progression. Their sensitive and specific detection is vital for advancing biomedical research and clinical diagnostics, particularly for early disease detection and biomarker discovery. However, traditional miRNA detection methods often face significant challenges, such as limited sensitivity, insufficient specificity, and the inability to detect low-abundance miRNAs in complex biological samples. To overcome these limitations, we present a novel miRNA detection electrochemiluminescence (ECL) platform that integrates entropy-driven amplification with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a-mediated RNA cleavage. The entropy-driven amplification strategy exploits the thermodynamic advantages of nucleic acid hybridization, driving spontaneous molecular reorganization to amplify detection signals and achieve ultralow detection limits. CRISPR/Cas13a, an RNA-targeting nuclease, provides exceptional sequence specificity by recognizing and binding to target miRNA sequences, activating a collateral cleavage mechanism. This activity cleaves hairpin (HP) structure, generating a signal that further triggers EDA over DNA tetrahedron (DT) to induce a vigorous ECL response. Based on this strategy, we achieve rapid and precise quantification of miR-17 at femtomolar levels. Experimental results demonstrate high sensitivity, specificity, and the ability to analyze complex biological samples in the laboratory. This innovative approach holds great promise for advancing molecular diagnostics and personalized medicine.},
}

*MicroRNAs/analysis/genetics
*CRISPR-Cas Systems
*Entropy
Humans
*Biosensing Techniques/methods
*Nucleic Acid Amplification Techniques/methods
Limit of Detection
Electrochemical Techniques/methods
Luminescent Measurements/methods

@article {pmid40273641,
year = {2025},
author = {Bhoir, K and Prakash, G and Odaneth, A},
title = {Genetic Engineering of Yarrowia lipolytica for 1,8-cineole production: A sustainable approach.},
journal = {Enzyme and microbial technology},
volume = {189},
number = {},
pages = {110659},
doi = {10.1016/j.enzmictec.2025.110659},
pmid = {40273641},
issn = {1879-0909},
mesh = {*Yarrowia/genetics/metabolism ; *Eucalyptol/metabolism ; *Metabolic Engineering/methods ; Streptomyces/enzymology/genetics ; CRISPR-Cas Systems ; Mevalonic Acid/metabolism ; Genetic Engineering ; Intramolecular Lyases/genetics/metabolism ; Squalene/metabolism ; Diterpenes ; Diphosphates ; },
abstract = {1,8-Cineole, a monoterpene with diverse industrial and pharmaceutical applications, has garnered significant interest due to its unique properties. This study aims to achieve sustainable production of 1,8-cineole from Yarrowia lipolytica through metabolic and media engineering strategies. The heterologous 1,8-cineole synthase from Streptomyces clavuligerus was integrated through CRISPR-Cas9, along with overexpression of key genes in the mevalonate pathway and a double mutation in the Erg20p to enhance flux towards geranyl pyrophosphate. The modified strain was further investigated for varying carbon and nitrogen sources with MgSO4 addition. The above approaches achieved a titer of 4.68 mg/L of 1,8-cineole along with 1108.53 mg/L of intracellular squalene when grown on 5 % WCO, marking the first report of genetic engineering of Y. lipolytica for 1,8-cineole production. Further studies are in progress to redirect internal fluxes to 1,8-cineole for improvising yields and productivities. This work shows a sustainable and innovative approach to biotechnology improvements in terpene biosynthesis and waste valorization.},
}

*Yarrowia/genetics/metabolism
*Eucalyptol/metabolism
*Metabolic Engineering/methods
Streptomyces/enzymology/genetics
CRISPR-Cas Systems
Mevalonic Acid/metabolism
Genetic Engineering
Intramolecular Lyases/genetics/metabolism
Squalene/metabolism
Diterpenes
Diphosphates

@article {pmid40221070,
year = {2025},
author = {Khademi, Z and Mottaghi-Dastjerdi, N and Morad, H and Sahebkar, A},
title = {The role of CRISPR-Cas9 and CRISPR interference technologies in the treatment of autoimmune diseases.},
journal = {Autoimmunity reviews},
volume = {24},
number = {7},
pages = {103816},
doi = {10.1016/j.autrev.2025.103816},
pmid = {40221070},
issn = {1873-0183},
mesh = {Humans ; *Autoimmune Diseases/therapy/genetics/immunology ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genetic Therapy/methods ; Animals ; },
abstract = {Autoimmune disorders can be described as inappropriate immune responses directed against self-antigens, which account for substantial healthcare concerns around the world. Immunosuppression or immune modulation are the main therapeutic modalities for autoimmune disorders. These modalities, however, impair the ability of the immune system to fight against infections, thereby predisposing to opportunistic diseases. This review explores existing therapies for autoimmune disorders, highlighting their limitations and challenges. Additionally, it describes the potential of CRISPR-Cas9 technology as a novel therapeutic approach to address these challenges.},
}

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

@article {pmid40175650,
year = {2025},
author = {Guo, P and Song, S and Niu, Y and Kuang, X and Zhou, D and Zhou, Z and Zhang, Y and Ma, X},
title = {Alternative splicing of bunched confers a dual role in hippo pathway-dependent growth and tumorigenesis.},
journal = {Oncogene},
volume = {44},
number = {24},
pages = {1949-1960},
pmid = {40175650},
issn = {1476-5594},
support = {32322027//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32170824//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32370710//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; *Alternative Splicing/genetics ; *Drosophila Proteins/genetics/metabolism ; *Protein Serine-Threonine Kinases/metabolism/genetics ; *Carcinogenesis/genetics ; Signal Transduction/genetics ; Protein Isoforms/genetics ; *Intracellular Signaling Peptides and Proteins/metabolism/genetics ; Drosophila melanogaster/genetics ; YAP-Signaling Proteins ; Hippo Signaling Pathway ; Nuclear Proteins/genetics/metabolism ; Trans-Activators/genetics/metabolism ; Transcription Factors/genetics/metabolism ; Cell Proliferation/genetics ; CRISPR-Cas Systems ; },
abstract = {Alternative splicing is a fundamental mechanism that generates functionally distinct proteins from individual genes, contributing to gene regulation and proteomic diversity. In Drosophila, the bunched (bun) gene, a member of the TSC-22 domain gene family, undergoes alternative splicing, yielding diverse protein isoforms involved in crucial biological processes. Nevertheless, the specific roles and regulatory mechanisms of each isoform remain elusive. Here, we employed CRISPR/Cas9 technology to introduce targeted deletions within the endogenous locus of the bun gene, resulting in the removal of either long or short isoforms. We discovered that the short isoforms demonstrated a growth-suppressive role, whereas the long isoforms exhibited a growth-promoting effect. Surprisingly, the long isoforms exhibited a remarkable dual functionality, as both deletion and amplification of long isoform expression impede the excess growth induced by Hippo pathway inactivation. Mechanistically, ectopically expressed Bun long isoforms act as the transcriptional suppressor by competitively binding to targets' promoter regions in conjunction with Yorkie/Scalloped (Yki/Sd), thereby inhibiting its transcriptional outputs and ultimately leading to the growth suppression. These findings unveil the intricate interaction between distinct spliced isoforms of Bun and oncogenic outcomes, highlighting Bun long isoforms as the critical transcription suppressor regulating Hippo pathway inactivation-mediated growth and tumorigenesis in Drosophila.},
}

Animals
*Alternative Splicing/genetics
*Drosophila Proteins/genetics/metabolism
*Protein Serine-Threonine Kinases/metabolism/genetics
*Carcinogenesis/genetics
Signal Transduction/genetics
Protein Isoforms/genetics
*Intracellular Signaling Peptides and Proteins/metabolism/genetics
Drosophila melanogaster/genetics
YAP-Signaling Proteins
Hippo Signaling Pathway
Nuclear Proteins/genetics/metabolism
Trans-Activators/genetics/metabolism
Transcription Factors/genetics/metabolism
Cell Proliferation/genetics
CRISPR-Cas Systems

@article {pmid40156991,
year = {2025},
author = {Tang, Q and Zhang, J and Pang, J and Huang, Y and Guan, Y and Gong, Y and Tang, Q and Zhang, K and Liao, X},
title = {Hybridization chain reaction and CRISPR/Cas12a-integrated biosensor for precise Ago2 detection.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {165},
number = {},
pages = {108975},
doi = {10.1016/j.bioelechem.2025.108975},
pmid = {40156991},
issn = {1878-562X},
mesh = {*Argonaute Proteins/analysis/genetics/metabolism ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Humans ; *Nucleic Acid Hybridization ; Luminescent Measurements/methods ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {This study introduces an innovative electrochemiluminescence (ECL) biosensor for the highly sensitive and specific detection of Argonaute 2 (Ago2) activity. Ago2, a key enzyme in the RNA interference (RNAi) pathway, plays a crucial role in gene regulation, and its dysregulation is associated with diseases such as cancer and viral infections. The biosensor integrates hybridization chain reaction (HCR) amplification and the CRISPR/Cas12a system, leveraging a multi-stage signal amplification strategy. The detection mechanism begins with Ago2-mediated cleavage of a designed hairpin RNA (HP-RNA), releasing single-stranded RNA (ssRNA) that triggers HCR. This amplification step generates long DNA polymers, which serve as activators for the CRISPR/Cas12a system. Cas12a's collateral cleavage activity amplifies the signal further by cleaving a DNA reporter labeled with a ruthenium-based luminophore, enhancing the ECL output. This dual amplification strategy achieves exceptional sensitivity, with a detection limit of 0.126 aM. The biosensor demonstrates excellent specificity, distinguishing Ago2 from other Argonaute proteins, and maintains high reproducibility and stability, retaining 94 % of its signal after two weeks of storage. Real-world applicability was confirmed by accurately detecting Ago2 in spiked cell lysates, with recovery rates exceeding 100 %. The combination of HCR, CRISPR/Cas12a, and ECL establishes a robust platform for biomarker detection, offering superior sensitivity and adaptability for clinical diagnostics, disease monitoring, and therapeutic evaluation. This biosensor represents a significant advancement in the development of next-generation diagnostic tools.},
}

*Argonaute Proteins/analysis/genetics/metabolism
*Biosensing Techniques/methods
*CRISPR-Cas Systems
Humans
*Nucleic Acid Hybridization
Luminescent Measurements/methods
Limit of Detection
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40118066,
year = {2025},
author = {Lodewijk, GA and Kozuki, S and Han, CJ and Topacio, BR and Lee, S and Nixon, L and Zargari, A and Knight, G and Ashton, R and Qi, LS and Shariati, SA},
title = {Self-organization of mouse embryonic stem cells into reproducible pre-gastrulation embryo models via CRISPRa programming.},
journal = {Cell stem cell},
volume = {32},
number = {6},
pages = {895-913.e8},
doi = {10.1016/j.stem.2025.02.015},
pmid = {40118066},
issn = {1875-9777},
mesh = {Animals ; Mice ; *Mouse Embryonic Stem Cells/cytology/metabolism ; Cell Differentiation/genetics ; CDX2 Transcription Factor/genetics/metabolism ; *Embryo, Mammalian/cytology/metabolism ; GATA6 Transcription Factor/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Models, Biological ; Cell Lineage ; },
abstract = {Embryonic stem cells (ESCs) can self-organize into structures with spatial and molecular similarities to natural embryos. During development, embryonic and extraembryonic cells differentiate through activation of endogenous regulatory elements while co-developing via cell-cell interactions. However, engineering regulatory elements to self-organize ESCs into embryo models remains underexplored. Here, we demonstrate that CRISPR activation (CRISPRa) of two regulatory elements near Gata6 and Cdx2 generates embryonic patterns resembling pre-gastrulation mouse embryos. Live single-cell imaging revealed that self-patterning occurs through orchestrated collective movement driven by cell-intrinsic fate induction. In 3D, CRISPRa-programmed embryo models (CPEMs) exhibit morphological and transcriptomic similarity to pre-gastrulation mouse embryos. CPEMs allow versatile perturbations, including dual Cdx2-Elf5 activation to enhance trophoblast differentiation and lineage-specific activation of laminin and matrix metalloproteinases, uncovering their roles in basement membrane remodeling and embryo model morphology. Our findings demonstrate that minimal intrinsic epigenome editing can self-organize ESCs into programmable pre-gastrulation embryo models with robust lineage-specific perturbation capabilities.},
}

Animals
Mice
*Mouse Embryonic Stem Cells/cytology/metabolism
Cell Differentiation/genetics
CDX2 Transcription Factor/genetics/metabolism
*Embryo, Mammalian/cytology/metabolism
GATA6 Transcription Factor/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Models, Biological
Cell Lineage

@article {pmid38997889,
year = {2025},
author = {Yin, E and Esbin, MN},
title = {Optimized CRISPR-based knockout in BeWo cells.},
journal = {Placenta},
volume = {166},
number = {},
pages = {71-76},
doi = {10.1016/j.placenta.2024.07.005},
pmid = {38997889},
issn = {1532-3102},
mesh = {Humans ; *Gene Knockout Techniques/methods ; *CRISPR-Cas Systems ; Female ; *Trophoblasts/metabolism ; *Gene Editing/methods ; Pregnancy ; Cell Line, Tumor ; Ribonucleoproteins/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Electroporation ; Pregnancy Proteins/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {CRISPR genome editing is a widely used tool to perturb genes of interest within cells and tissues and can be used as a research tool to study the connection between genotypes and cellular phenotypes. Highly efficient genome editing is limited in certain cell types due to low transfection efficiency or single-cell survivability. This is true for BeWo cells, an in vitro model of placental syncytiotrophoblast cell-cell fusion and hormone secretion. Here we describe an optimized and easy-to-use protocol for knockout in BeWo cells using CRISPR Cas9 ribonucleoprotein (RNP) complexes delivered via electroporation. Further, we describe parameters for successful guide RNA design and how to assess genetic knockouts in BeWo cells so that users can apply this technique to their own genes of interest. We provide a positive control for inducing highly efficient knockout of the cell-cell fusion protein Syncytin-2 (ERVFRD-1) and assessing editing efficiency at this locus. We anticipate that efficient RNP-mediated genetic knockouts in BeWo cells will facilitate the study of new genes involved in cell-cell fusion and hormone secretion in this important cellular model system. Furthermore, this strategy of optimized nucleofection and RNP delivery may be of use in other difficult-to-edit trophoblast cells or could be applied to efficiently deliver transgenes to BeWo cells.},
}

Humans
*Gene Knockout Techniques/methods
*CRISPR-Cas Systems
Female
*Trophoblasts/metabolism
*Gene Editing/methods
Pregnancy
Cell Line, Tumor
Ribonucleoproteins/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Electroporation
Pregnancy Proteins/genetics
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40455261,
year = {2025},
author = {Zhang, M and Zhou, L and Afridi, M and Guo, H and Cheng, H},
title = {An efficient hairy root system for validation of CRISPR/Cas system activities in cotton.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {116},
pmid = {40455261},
issn = {1438-7948},
support = {NK2022010303//the Ministry of Agriculture/ ; B23CJ0208//Hainan Seed Industry Laboratory/ ; YSPTZX202502//Hainan Province Academician Innovation Platform/ ; },
mesh = {*Gossypium/genetics/growth & development/microbiology ; *Plant Roots/genetics/growth & development ; *CRISPR-Cas Systems ; Agrobacterium/genetics ; *Gene Editing/methods ; Plants, Genetically Modified/genetics ; },
abstract = {The hairy root induction system has been widely applied in studying gene expression and function in plant species due to its rapidity and efficiency. The hairy root system is an efficient tool for evaluating the activities of CRISPR/Cas systems. Cotton hairy roots were primarily induced through cotton tissue culture under aseptic conditions and by injecting cotton stem tips under non-aseptic conditions. However, both methods are lab-intensive and time-consuming. In this study, an efficient cotton hairy root induction procedure was established via infecting cotton hypocotyls with Agrobacterium rhizogenes under non-sterile conditions. Cotton seedlings with expanded cotyledons were decapitated with a slanted cut, and the residual hypocotyl (maintained 1 cm apical portion) was inoculated with A. rhizogenes. Over 90% of the infected explants from all three tested varieties could produce hairy roots after 8 days of inoculation. The effictiveness of the method was tested by overexpressing two reporter genes (eGFP and GUS). The transformation efficiency of the GUS and eGFP were ranged from50-68.18% and 40.9-68.18%. In addition, the editing efficiency of target sites in different CRISPR/Cas systems were also tested in hairy root. This method provided technical support for screening suitable target sites for cotton gene editing.},
}

*Gossypium/genetics/growth & development/microbiology
*Plant Roots/genetics/growth & development
*CRISPR-Cas Systems
Agrobacterium/genetics
*Gene Editing/methods
Plants, Genetically Modified/genetics

@article {pmid40454797,
year = {2025},
author = {Wu, J and Yan, Q and Qiu, H and Gao, EB},
title = {Integrating Enzyme-DNA Complex and CRISPR/Cas12a for Robust Norovirus Detection.},
journal = {Journal of medical virology},
volume = {97},
number = {6},
pages = {e70426},
doi = {10.1002/jmv.70426},
pmid = {40454797},
issn = {1096-9071},
support = {//The Ningbo Clinical Research Center for Children's Health and Diseases (no. 2019A21002), the Ningbo public welfare project (no. 2022S127), the Ningbo key discipline Pediatrics (no. 2022-B17), the Innovation Project of Distinguished Medical Team in Ningbo (no. 2022020405). Ningbo Medical and Health Brand Discipline (PPXK2024-06)./ ; },
mesh = {*Norovirus/isolation & purification/genetics ; Humans ; *Caliciviridae Infections/diagnosis/virology ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Gastroenteritis/virology/diagnosis ; *Nucleic Acid Amplification Techniques/methods ; RNA, Viral/genetics ; *Molecular Diagnostic Techniques/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Human norovirus (NoV) is a primary cause of acute gastroenteritis in children, making accurate and rapid detection essential for effective disease prevention and control. In this study, we developed a sensitive and efficient platform for pathogen nucleic acid detection by integrating asymmetric nucleic acid sequence-based amplification (asymmetric NASBA), enzyme-DNA molecular complex, and the clustered regularly interspaced short palindromic repeats (CRISPR) system, namely an A-enDMC platform. The target recognition capability of the enzyme-DNA complex operates independently from the signal amplification function of the CRISPR system. By decoupling the CRISPR reaction from the dependence on specific target sequences, the platform's universality and modularity are enhanced. The assay is fast (< 1.5 h), highly sensitive (< 5 copies/µL), and demonstrates no cross-reactivity with other common viruses. Compared to the widely used RT-qPCR method, the platform demonstrates high consistency in detection results, with the detection coincidence rate of 96.77% and a kappa value of 0.87. This platform provides a versatile technological tool for highly sensitive and specific RNA detection, demonstrating its extensive potential in real sample analysis.},
}

*Norovirus/isolation & purification/genetics
Humans
*Caliciviridae Infections/diagnosis/virology
*CRISPR-Cas Systems
Sensitivity and Specificity
*Gastroenteritis/virology/diagnosis
*Nucleic Acid Amplification Techniques/methods
RNA, Viral/genetics
*Molecular Diagnostic Techniques/methods
Clustered Regularly Interspaced Short Palindromic Repeats
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40453079,
year = {2025},
author = {Lund, S and Gong, C and Yu, X and Staudt, LM and Hodson, DJ and Scheich, S},
title = {Strategies for CRISPR-based knock-ins in primary human B cells and lymphoma cell lines.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1589729},
pmid = {40453079},
issn = {1664-3224},
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Knock-In Techniques/methods ; *Gene Editing/methods ; *B-Lymphocytes/metabolism/immunology ; Cell Line, Tumor ; *Lymphoma, Large B-Cell, Diffuse/genetics ; },
abstract = {Since its advent about ten years ago, the CRISPR-Cas9 system has been frequently used in biomedical applications. It has advanced various fields, and CRISPR-Cas9-based therapeutics have shown promising results in the treatment of specific hematological diseases. Furthermore, CRISPR gene editing technologies have revolutionized cancer research by enabling a broad range of genetic perturbations, including genetic knockouts and precise single nucleotide changes. This perspective focuses on the state-of-the-art methodology of CRISPR knock-ins to engineer immune cells. Since this technique relies on homology-directed repair (HDR) of double-strand breaks (DSBs) induced by the Cas9 enzyme, it can be used to introduce specific mutations into the target genome. Therefore, this methodology offers a valuable opportunity to functionally study specific mutations and to uncover their impacts not only on overall cell functions but also on the mechanisms behind cancer-related alterations in common signaling pathways. This article highlights CRISPR knock-in strategies, protocols, and applications in cancer and immune research, with a focus on diffuse large B cell lymphoma.},
}

Humans
*CRISPR-Cas Systems
*Gene Knock-In Techniques/methods
*Gene Editing/methods
*B-Lymphocytes/metabolism/immunology
Cell Line, Tumor
*Lymphoma, Large B-Cell, Diffuse/genetics

@article {pmid40451886,
year = {2025},
author = {Okamoto, M and Sasamoto, K and Takahashi-Nakaguchi, A and Zhao, F and Yamaguchi, M and Chibana, H},
title = {CRISPR-Cas9 RNP-Mediated Deletion of ERG25 in Non-albicans Candida Species, Including Candida auris.},
journal = {Medical mycology journal},
volume = {66},
number = {2},
pages = {35-43},
doi = {10.3314/mmj.24-00023},
pmid = {40451886},
issn = {2186-165X},
mesh = {*CRISPR-Cas Systems ; Candida glabrata/genetics ; *Candida auris/genetics ; *Fungal Proteins/genetics ; *Gene Deletion ; *Candida/genetics ; Candidiasis/microbiology ; Antifungal Agents/pharmacology ; },
abstract = {The incidence of infections caused by non-albicans Candida (NAC) species, including Candida glabrata and Candida tropicalis, has recently increased. Furthermore, Candida auris, a multidrug-resistant yeast, poses a serious threat to global health. The development of novel antifungal agents with alternative mechanisms of action is necessary to combat these fungi. However, genetic analyses of the virulence factors in these NAC species are insufficient for this purpose. Recent advancements in the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system have facilitated enhanced the genetic analysis of NAC species. The RNP-based system, in which the Cas9-gRNA complex is assembled in vitro and introduced into cells, offers a simplified approach to genetic modification, eliminating the need for species-specific plasmids. Previous our research identified the ERG25 gene, which encodes C-4 sterol methyl oxidase, as a promising antifungal target in C. glabrata. This study demonstrated deletion of the ERG25 homolog in C. glabrata and C. auris using an RNP-based CRISPR-Cas9 system. The deletion of ERG25 in C. auris and C. glabrata indicated that Erg25 is crucial for the survival of these pathogenic yeasts within the host. Furthermore, we have successfully deleted the ERG25 alleles in C. tropicalis and Candida parapsilosis, demonstrating the effectiveness of using both the CRISPR-Cas9 and Cre-loxP systems in these species for the first time.},
}

*CRISPR-Cas Systems
Candida glabrata/genetics
*Candida auris/genetics
*Fungal Proteins/genetics
*Gene Deletion
*Candida/genetics
Candidiasis/microbiology
Antifungal Agents/pharmacology

@article {pmid40347811,
year = {2025},
author = {Wang, R and He, B and Liang, Z and Liu, Y and Yang, J and Jin, H and Wei, M and Ren, W and Suo, Z and Xu, Y},
title = {A dual-mode sensor for rapid detection of procymidone: "Dark box" qualitative analysis and electrochemical quantification mediated by PdHPCN-222/PEI-rGO and CRISPR/Cas12a.},
journal = {Food chemistry},
volume = {486},
number = {},
pages = {144586},
doi = {10.1016/j.foodchem.2025.144586},
pmid = {40347811},
issn = {1873-7072},
mesh = {*Electrochemical Techniques/methods/instrumentation ; *Biosensing Techniques/methods/instrumentation ; CRISPR-Cas Systems ; Graphite/chemistry ; Limit of Detection ; Polyethyleneimine/chemistry ; Fluorescence ; Food Contamination/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {This study presents an integrated dual-mode sensing strategy, in which qualitative fluorescence screening is followed by quantitative electrochemical detection, improving detection efficiency and enabling rapid PCM analysis. It develops a novel fluorescence electrochemical aptasensor that combines in vitro "Dark-box" applications with CRISPR/Cas12a system electrode surface sensing technology. PCM activates the DNA walker, and the DNAzyme induces cyclic cleavage of DNA strands bearing the Carboxyfluorescein (FMA) group. After magnetic separation, the fluorescence reaction combined with the "Dark box" enables the preliminary qualitative analysis of procymidone (PCM). Following the preliminary qualitative detection, the solution is introduced to the electrochemical aptasensor platform integrated with the CRISPR/Cas12 system. The Cas12a system triggers cyclic cleavage, producing a signal change that enables the electrochemical quantitative detection of PCM. An fluorescence (FL) response occurs when the PCM concentration in spiked samples is at or above 1 pg·mL[-1], allowing for qualitative fluorescence detection. The EC platform's detection limit is 8.51 × 10[-6] ng·mL[-1], with a range of 1 × 10[-2] ng·mL[-1] to 1 × 10[4] ng·mL[-1]. The designed dual-mode sensor provides reliable monitoring of PCM in real samples.},
}

*Electrochemical Techniques/methods/instrumentation
*Biosensing Techniques/methods/instrumentation
CRISPR-Cas Systems
Graphite/chemistry
Limit of Detection
Polyethyleneimine/chemistry
Fluorescence
Food Contamination/analysis
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid36847138,
year = {2025},
author = {Baerwald, MR and Funk, EC and Goodbla, AM and Campbell, MA and Thompson, T and Meek, MH and Schreier, AD},
title = {Rapid CRISPR-Cas13a genetic identification enables new opportunities for listed Chinook salmon management.},
journal = {Molecular ecology resources},
volume = {25},
number = {5},
pages = {e13777},
doi = {10.1111/1755-0998.13777},
pmid = {36847138},
issn = {1755-0998},
support = {4600012328//Department of Water Resources/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Salmon/genetics/classification ; California ; *Genotyping Techniques/methods ; },
abstract = {Accurate taxonomic identification is foundational for effective species monitoring and management. When visual identifications are infeasible or inaccurate, genetic approaches provide a reliable alternative. However, these approaches are sometimes less viable (e.g., need for near real-time results, remote locations, funding concerns, molecular inexperience). In these situations, CRISPR-based genetic tools can fill an unoccupied niche between real-time, inexpensive, but error-prone visual identification and more expensive or time-consuming, but accurate genetic identification for taxonomic units that are difficult or impossible to visually identify. Herein, we use genomic data to develop CRISPR-based SHERLOCK assays capable of rapidly (<1 h), accurately (94%-98% concordance between phenotypic and genotypic assignments), and sensitively (detects 1-10 DNA copies/reaction) distinguishing ESA-listed Chinook salmon runs (winter- and spring-run) from each other and from unlisted runs (fall- and late fall-run) in California's Central Valley. The assays can be field deployable with minimally invasive mucus swabbing negating the need for DNA extraction (decreasing costs and labour), minimal and inexpensive equipment needs, and minimal training to conduct following assay development. This study provides a powerful genetic approach for a species of conservation concern that benefits from near real-time management decision-making but also serves as a precedent for transforming how conservation scientists and managers view genetic identification going forward. Once developed, CRISPR-based tools can provide accurate, sensitive, and rapid results, potentially without the prohibitive need for expensive specialty equipment or extensive molecular training. Further adoption of this technology will have widespread value for the monitoring and protection of our natural resources.},
}

Animals
*CRISPR-Cas Systems
*Salmon/genetics/classification
California
*Genotyping Techniques/methods

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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