@article {pmid39836103,
year = {2025},
author = {Mai, Z and Zhou, T and Lin, Z},
title = {Detecting CYP2C19 genes through an integrated CRISPR/Cas13a-assisted system.},
journal = {Analytical methods : advancing methods and applications},
volume = {},
number = {},
pages = {},
doi = {10.1039/d4ay01930j},
pmid = {39836103},
issn = {1759-9679},
abstract = {CYP2C19 gene single nucleotide polymorphisms (SNPs) should be considered in the clinical use of clopidogrel as they have important guiding value for predicting the risk of bleeding and thrombosis after clopidogrel treatment. The CRISPR/Cas system is increasingly used for SNP detection owing to its single-nucleotide mismatch specificity. Simultaneous detection of multiple SNPs for rapid identification of the CYP2C19 genotype is important, but there is no method to detect a wide variety of CYP2C19 SNPs. This study proposes a new integrated system that integrates the PCR reaction and CRISPR/Cas detection of three CYP2C19 genes on a device, achieving rapid, sensitive, and specific detection. In our design, magnetic beads with three different sizes capture target nucleic acid from the sample, which are dragged through different areas by magnetic force, for PCR amplification reaction and CRISPR/Cas13a detection of CYP2C19*2, CYP2C19*3 and CYP2C19*17 genes. Note that magnetic beads were sorted via microporous PC membranes of different apertures. This study exhibits a broad clinical application prospect and provides a favorable tool for clinical clopidogrel administration.},
}

@article {pmid39834176,
year = {2025},
author = {Tang, M},
title = {Research Status of Clustered Regulary Interspaced Short Palindromic Repeats Technology in the Treatment of Human Papillomavirus (HPV) Infection Related Diseases.},
journal = {Cancer control : journal of the Moffitt Cancer Center},
volume = {32},
number = {},
pages = {10732748241300654},
doi = {10.1177/10732748241300654},
pmid = {39834176},
issn = {1526-2359},
mesh = {Humans ; *Papillomavirus Infections/therapy/genetics/virology ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods ; Female ; Papillomaviridae/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Uterine Cervical Neoplasms/therapy/virology/genetics ; Gene Editing/methods ; Human Papillomavirus Viruses ; },
abstract = {Background: CRISPR/Cas9 technology has rapidly advanced as a pivotal tool in cancer research, particularly in the precision targeting required for both detecting and treating malignancies. Its high specificity and low off-target effects make it exceptionally effective in applications involving Human Papillomavirus (HPV) related diseases, most notably cervical cancer. This approach offers a refined methodology for the rapid detection of viral infections and provides a robust platform for the safe and effective treatment of diseases associated with viral infections through gene therapy.Purpose: Gene therapy, within this context, involves the strategic delivery of genetic material into target cells via a vector. This is followed by the meticulous modulation of gene expression, whether through correction, addition, or suppression, specifically honed to target tumor cells while sparing healthy cells. This dual capacity to diagnose and treat at such a precise level underscores the transformative potential of CRISPR/Cas9 in contemporary medical science, particularly in oncology and virology.Research Design: This article provides an overview of the advancements made in utilizing the CRISPR-Cas9 system as a research tool for HPV-related treatments while summarizing its application status in basic research, diagnosis, and treatment of HPV.Data Collection: Furthermore, it discusses the future prospects for this technology within emerging areas of HPV research and precision medicine in clinical practice, while highlighting technical challenges and potential directions for future development.},
}

Humans
*Papillomavirus Infections/therapy/genetics/virology
*CRISPR-Cas Systems/genetics
*Genetic Therapy/methods
Female
Papillomaviridae/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Uterine Cervical Neoplasms/therapy/virology/genetics
Gene Editing/methods
Human Papillomavirus Viruses

@article {pmid39833630,
year = {2025},
author = {Zhang, B and Li, J and Yu, W},
title = {Integration of CRISPR/dCas9-Based methylation editing with guide positioning sequencing identifies dynamic changes of mrDEGs in breast cancer progression.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {82},
number = {1},
pages = {46},
pmid = {39833630},
issn = {1420-9071},
support = {32270645//National Natural Science Foundation of China/ ; 18JC1411101//Major Special Projects of Basic Research of Shanghai Science and Technology Commission/ ; },
mesh = {Humans ; *Breast Neoplasms/genetics/pathology ; *DNA Methylation/genetics ; Female ; *CRISPR-Cas Systems/genetics ; *Disease Progression ; *Gene Editing/methods ; *Gene Expression Regulation, Neoplastic ; Cell Line, Tumor ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Dynamic changes in DNA methylation are prevalent during the progression of breast cancer. However, critical alterations in aberrant methylation and gene expression patterns have not been thoroughly characterized. Here, we utilized guide positioning sequencing (GPS) to conduct whole-genome DNA methylation analysis in a unique human breast cancer progression model: MCF10 series of cell lines (representing benign/normal, atypical hyperplasia, and metastatic carcinoma). By integrating with mRNA-seq and matched clinical expression data from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO), six representative methylation-related differentially expressed genes (mrDEGs) were identified, including CAVIN2, ARL4D, DUSP1, TENT5B, P3H2, and MMP28. To validate our findings, we independently developed and optimized the dCas9-DNMT3L-DNMT3A system, achieving a high efficiency with a 98% increase in methylation at specific sites. DNA methylation levels significantly increased for the six genes, with CAVIN2 at 67.75 ± 1.05%, ARL4D at 53.29 ± 6.32%, DUSP1 at 57.63 ± 8.46%, TENT5B at 44.00 ± 5.09%, P3H2 at 58.50 ± 3.90%, and MMP28 at 49.60 ± 5.84%. RT-qPCR confirmed an inverse correlation between increased DNA methylation and gene expression. Most importantly, we mimicked tumor progression in vitro, demonstrating that transcriptional silencing of the TENT5B promotes cell proliferation in MCF10A cells owing to the crosstalk between hypermethylation and histone deacetylation. This study unveils the practical implications of DNA methylation dynamics of mrDEGs in reshaping epigenomic features during breast cancer malignant progression through integrated data analysis of the methylome and transcriptome. The application of the CRISPR/dCas9-based methylation editing technique elucidates the regulatory mechanisms and functional roles of individual genes within the DNA methylation signature, providing valuable insights for understanding breast cancer pathogenesis and facilitating potential therapeutic approaches in epigenome editing for patients with breast cancer.},
}

Humans
*Breast Neoplasms/genetics/pathology
*DNA Methylation/genetics
Female
*CRISPR-Cas Systems/genetics
*Disease Progression
*Gene Editing/methods
*Gene Expression Regulation, Neoplastic
Cell Line, Tumor
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid39833501,
year = {2025},
author = {Gawlig, C and Hirschberger, R and Hanci, G and Schott, S and Marandi, S and Hesse, IR and Rühl, M},
title = {Full sequencing of 100mer sgRNA via tandem mass spectrometry by targeted RNase H digestion with customized probes.},
journal = {Analytical and bioanalytical chemistry},
volume = {},
number = {},
pages = {},
pmid = {39833501},
issn = {1618-2650},
abstract = {The use of single-guide RNA (sgRNA) for gene editing using the CRISPR Cas9 system has become a powerful technique in various fields, especially with the growing interest in such molecules as therapeutic options in the last years. An important parameter for the use of these molecules is the verification of the correct sgRNA oligonucleotide sequence. Apart from next-generation sequencing protocols, mass spectrometry (MS) has been proven as a powerful technique for this purpose. The protocol and investigations presented in this work show an optimal digestion and 100% sequence coverage of sgRNA, while top-down approaches or other ribonuclease (RNase) digestion strategies obtain a sequence coverage of up to 80-90% utilizing multiple RNases. The results in this publication were obtained by utilizing DNA-RNA hybrid GAPmer-like probes and RNase H, an enzyme which specifically hydrolyzes RNA in DNA-RNA double strands. We assessed the optimal length of the DNA segment of these hybrid probes to maximize the specificity of the RNase H digestion and to achieve complete sequence confirmation by tandem MS analysis of the resulting digestion products. Furthermore, we showed that the approach is applicable for the identification of common synthesis-related impurities, like truncations and elongations. Despite the fact that the accessibility of this approach for highly modified molecules is limited to nucleotides which are not 2'-O-methylated, the optimized sequence coverage makes it a viable method.},
}

@article {pmid39833409,
year = {2025},
author = {Zhang, X and Qiu, M and Han, B and Liao, L and Peng, X and Lin, J and Zhang, N and Hai, L and Liang, L and Ma, Y and Li, W and Liu, M},
title = {Generation and propagation of high fecundity gene edited fine wool sheep by CRISPR/Cas9.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {2557},
pmid = {39833409},
issn = {2045-2322},
support = {2023SNGGGCC010//the Tianshan Talent Training Program-high-level talents for the advancement of modern agriculture and animal husbandry./ ; 2023TSYCTD0007//the Scientific and Technological Innovation Team Project of Xinjiang Uygur Autonomous Region./ ; },
mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Fertility/genetics ; Sheep/genetics ; Female ; Wool ; Bone Morphogenetic Protein Receptors, Type I/genetics ; Litter Size/genetics ; Alleles ; },
abstract = {CRISPR/Cas9 technology has been widely utilized to enhance productive performance, increase disease resistance and generate medical models in livestock. The FecB allele in sheep is a mutation in the BMPRIB gene, recognized as the first major gene responsible for the high fecundity trait in sheep, leading to an increased ovulation rate in ewe. In this study, we employed CRISPR/Cas9-mediated homologous-directed repair (HDR) to introduce a defined point mutation (c.746 A > G) using single-stranded oligonucleotides (ssODN) and the ligase IV inhibitor (SCR7) into the BMPRIB gene of fine wool sheep. A total of nine gene-edited sheep were produced, six of which carried the targeted point mutation, with a precise base substitution efficiency (A > G) of 31.6%. Based on the six targeted founders (F0), we expanded the BMPRIB-targeted population, which included F1 heterozygous (B+) and F2 homozygous(BB) or heterozygous offspring. The average litter size of F1 ewes carrying the B + allele reached 170%, comparable to that of heterozygous native Australian Booroola sheep. Gene-edited ewes with B + and BB genotype produced 0.62 and 0.42 more lambs, respectively, compared to wide-type ewes (p < 0.01). Our results also indicated that the parity signification, our data demonstrate that highly efficient introduction of the intended base mutation into the sheep genome can be achieved by combining the CRISPR/Cas9 system with ssODN and SCR7. The offspring of BMPR/B edited sheep with the defined mutation exhibited high fecundity performance. Compared to conventional sheep breeding strategies, genetic improvement through gene editing offered significant advantages without compromising the fine wool traits of Merino sheep, which are often affected by routine cross-breeding methods.},
}

Animals
*Gene Editing/methods
*CRISPR-Cas Systems
*Fertility/genetics
Sheep/genetics
Female
Wool
Bone Morphogenetic Protein Receptors, Type I/genetics
Litter Size/genetics
Alleles

@article {pmid39833307,
year = {2025},
author = {Di Cristina, G and Dirksen, E and Altenhein, B and Büschges, A and Korsching, SI},
title = {Pioneering genome editing in parthenogenetic stick insects: CRISPR/Cas9-mediated gene knockout in Medauroidea extradentata.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {2584},
pmid = {39833307},
issn = {2045-2322},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Gene Knockout Techniques/methods ; *Parthenogenesis/genetics ; *Insecta/genetics ; Phenotype ; Female ; Pigmentation/genetics ; },
abstract = {The parthenogenetic life cycle of the stick insect Medauroidea extradentata offers unique advantages for the generation of genome-edited strains, as an isogenic and stable mutant line can in principle be achieved already in the first generation (G0). However, genetic tools for the manipulation of their genes had not been developed until now. Here, we successfully implement CRISPR/Cas9 as a technique to modify the genome of the stick insect M. extradentata. As proof-of-concept we targeted two genes involved in the ommochrome pathway of eye pigmentation (cinnabar and white, second and first exon, respectively), to generate knockout (KO) mutants. Microinjections were performed within 24 h after oviposition, to focus on the mononuclear (and haploid) stage of development. The KOs generated resulted in distinct eye and cuticle colour phenotypes for cinnabar and white. Homozygous cinnabar mutants showed pale pigmentation of eyes and cuticle. They develop into adults capable of producing viable eggs. Homozygous white KO resulted in a completely unpigmented phenotype in developing embryos that were unable to hatch. In conclusion, we show that CRISPR/Cas9 can be successfully applied to the genome of M. extradentata by creating phenotypically different and viable insects. This powerful gene editing technique can now be employed to create stable genetically modified lines using a parthenogenetic non-model organism.},
}

Animals
*CRISPR-Cas Systems
*Gene Editing/methods
*Gene Knockout Techniques/methods
*Parthenogenesis/genetics
*Insecta/genetics
Phenotype
Female
Pigmentation/genetics

@article {pmid39833279,
year = {2025},
author = {Bi, C and Yuan, B and Zhang, Y and Wang, M and Tian, Y and Li, M},
title = {Prevalent integration of genomic repetitive and regulatory elements and donor sequences at CRISPR-Cas9-induced breaks.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {94},
pmid = {39833279},
issn = {2399-3642},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; Regulatory Sequences, Nucleic Acid ; },
abstract = {CRISPR-Cas9 genome editing has been extensively applied in both academia and clinical settings, but its genotoxic risks, including large insertions (LgIns), remain poorly studied due to methodological limitations. This study presents the first detailed report of unintended LgIns consistently induced by different Cas9 editing regimes using various types of donors across multiple gene loci. Among these insertions, retrotransposable elements (REs) and host genomic coding and regulatory sequences are prevalent. RE frequencies and 3D genome organization analysis suggest LgIns originate from randomly acquired genomic fragments by DNA repair mechanisms. Additionally, significant unintended full-length and concatemeric double-stranded DNA (dsDNA) donor integrations occur when donor DNA is present. We further demonstrate that phosphorylated dsDNA donors consistently reduce large insertions and deletions by almost two-fold without compromising homology-directed repair (HDR) efficiency. Taken together, our study addresses a ubiquitous and overlooked risk of unintended LgIns in Cas9 editing, contributing valuable insights for the safe use of Cas9 editing tools.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
Humans
Regulatory Sequences, Nucleic Acid

@article {pmid39833194,
year = {2025},
author = {Kim, DG and Gu, B and Cha, Y and Ha, J and Lee, Y and Kim, G and Cho, BK and Oh, MK},
title = {Engineered CRISPR-Cas9 for Streptomyces sp. genome editing to improve specialized metabolite production.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {874},
pmid = {39833194},
issn = {2041-1723},
support = {RS-2024-00509338//National Research Foundation of Korea (NRF)/ ; RS-2024-00357320//National Research Foundation of Korea (NRF)/ ; RS-2021-NR056596//National Research Foundation of Korea (NRF)/ ; },
mesh = {*Streptomyces/genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Multigene Family ; *Genome, Bacterial ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Secondary Metabolism/genetics ; Metabolic Engineering/methods ; CRISPR-Associated Protein 9/metabolism/genetics ; },
abstract = {The CRISPR-Cas9 system has frequently been used for genome editing in Streptomyces; however, cytotoxicity, caused by off-target cleavage, limits its application. In this study, we implement innovative modification to Cas9, strategically addressing challenges encountered during gene manipulation using Cas9 within strains possessing high GC content genome. The Cas9-BD, a modified Cas9 with the addition of polyaspartate to its N- and C-termini, is developed with decreased off-target binding and cytotoxicity compared with wild-type Cas9. Cas9-BD and similarly modified dCas9-BD have been successfully employed for simultaneous biosynthetic gene cluster (BGC) refactoring, multiple BGC deletions, or multiplexed gene expression modulations in Streptomyces. We also demonstrate improved secondary metabolite production using multiplexed genome editing with multiple single guide RNA libraries in several Streptomyces strains. Cas9-BD is also used to capture large BGCs using a developed in vivo cloning method. The modified CRISPR-Cas9 system is successfully applied to many Streptomyces sp., providing versatile and efficient genome editing tools for strain engineering of actinomycetes with high GC content genome.},
}

*Streptomyces/genetics/metabolism
*CRISPR-Cas Systems
*Gene Editing/methods
*Multigene Family
*Genome, Bacterial
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Secondary Metabolism/genetics
Metabolic Engineering/methods
CRISPR-Associated Protein 9/metabolism/genetics

@article {pmid39833175,
year = {2025},
author = {Goold, HD and Kroukamp, H and Erpf, PE and Zhao, Y and Kelso, P and Calame, J and Timmins, JJB and Wightman, ELI and Peng, K and Carpenter, AC and Llorente, B and Hawthorne, C and Clay, S and van Wyk, N and Daniel, EL and Harrison, F and Meier, F and Willows, RD and Cai, Y and Walker, RSK and Xu, X and Espinosa, MI and Stracquadanio, G and Bader, JS and Mitchell, LA and Boeke, JD and Williams, TC and Paulsen, IT and Pretorius, IS},
title = {Construction and iterative redesign of synXVI a 903 kb synthetic Saccharomyces cerevisiae chromosome.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {841},
pmid = {39833175},
issn = {2041-1723},
support = {DP200100717//Department of Education and Training | Australian Research Council (ARC)/ ; CE200100029//Department of Education and Training | Australian Research Council (ARC)/ ; },
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; Genome, Fungal ; Chromosomes, Artificial, Yeast/genetics ; Chromosomes, Fungal/genetics ; Synthetic Biology/methods ; CRISPR-Cas Systems ; Open Reading Frames/genetics ; RNA, Transfer/genetics/metabolism ; },
abstract = {The Sc2.0 global consortium to design and construct a synthetic genome based on the Saccharomyces cerevisiae genome commenced in 2006, comprising 16 synthetic chromosomes and a new-to-nature tRNA neochromosome. In this paper we describe assembly and debugging of the 902,994-bp synthetic Saccharomyces cerevisiae chromosome synXVI of the Sc2.0 project. Application of the CRISPR D-BUGS protocol identified defective loci, which were modified to improve sporulation and recover wild-type like growth when grown on glycerol as a sole carbon source when grown at 37˚C. LoxPsym sites inserted downstream of dubious open reading frames impacted the 5' UTR of genes required for optimal growth and were identified as a systematic cause of defective growth. Based on lessons learned from analysis of Sc2.0 defects and synXVI, an in-silico redesign of the synXVI chromosome was performed, which can be used as a blueprint for future synthetic yeast genome designs. The in-silico redesign of synXVI includes reduced PCR tag frequency, modified chunk and megachunk termini, and adjustments to allocation of loxPsym sites and TAA stop codons to dubious ORFs. This redesign provides a roadmap into applications of Sc2.0 strategies in non-yeast organisms.},
}

*Saccharomyces cerevisiae/genetics/metabolism
Genome, Fungal
Chromosomes, Artificial, Yeast/genetics
Chromosomes, Fungal/genetics
Synthetic Biology/methods
CRISPR-Cas Systems
Open Reading Frames/genetics
RNA, Transfer/genetics/metabolism

@article {pmid39832868,
year = {2025},
author = {Alarcón-Iniesta, H and de Arana, G and López-Valls, M and Pardo, D and Escalona-Noguero, C and Rodríguez, C and Castellanos, M and Cobelo, S and Martínez-Ramírez, I and Camarero, J and Heras, SL and de Vicente, J and Valera, A and Smith, W and Bernardo-Gavito, R and Cantón, R and Galán, JC and Granados, D and Miranda, R and Guerrero, H and Sot, B},
title = {CRISPR-associated Plasmonic Colorimeter Method (Ca-PCM): A real-time RGB detection system for gold nanoparticles-based nucleic acid biosensors.},
journal = {Analytica chimica acta},
volume = {1338},
number = {},
pages = {343601},
doi = {10.1016/j.aca.2024.343601},
pmid = {39832868},
issn = {1873-4324},
mesh = {*Gold/chemistry ; *Metal Nanoparticles/chemistry ; *Colorimetry ; *Biosensing Techniques/methods ; Humans ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {BACKGROUND: The detection of genetic sequences represents the gold standard procedure for species discrimination, genetic characterisation of tumours, and identification of pathogens. The development of new molecular detection methods, accessible and cost effective, is of great relevance. Biosensors based on plasmonic nanoparticles, such as gold nanoparticles (AuNPs), provide a powerful and versatile platform for highly sensitive, economic, user-friendly and label-free sensing. However, the readout techniques typically employed with such sensors lack temporal and kinetic information, which hampers the ability to perform quantitative detection.

RESULTS: In this study, a novel methodology designated the 'CRISPR-associated Plasmonic Colorimeter Method' (Ca-PCM), has been developed. This method combines RNA target recognition by CRISPR LwaCas13a, AuNPs' aggregation, and real-time colorimetric Red-Green-Blue (RGB) analysis. The system registers the AuNP's plasmonic signatures in real-time using a RGB colour sensor with 3-channel silicon photodiodes having blue, green and red sensitivities. The acquired signals are automatically analysed by an algorithm designed to distinguish between positive and negative samples and to correlate the temporal spectral patterns of aggregation with dose-dependent molecular detection of the RNA target. In addition, the combination of Ca-PCM with a previous isothermal amplification allows the target efficient detection in real clinical applications.

SIGNIFICANCE: We have shown that the combination of RGB analysis and continuous temporal measurements is a novel and promising method to characterise the behaviour of gold nanoparticle-based biosensors and to achieve dose-dependent target detection. In addition, the simplicity and cost-effectiveness of this new approach expand the possibilities of other plasmonic-based biosensors and their applicability in low-resources clinical environments.},
}

*Gold/chemistry
*Metal Nanoparticles/chemistry
*Colorimetry
*Biosensing Techniques/methods
Humans
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
CRISPR-Cas Systems/genetics

@article {pmid39832863,
year = {2025},
author = {Zhang, T and Wang, X and Jiang, Y and Zhang, Y and Zhao, S and Hu, J and Hu, F and Peng, N},
title = {A miniaturized RPA-CRISPR/Cas12a-based nucleic acid diagnostic platform for rapid and simple self-testing of SARS-CoV-2.},
journal = {Analytica chimica acta},
volume = {1338},
number = {},
pages = {343593},
doi = {10.1016/j.aca.2024.343593},
pmid = {39832863},
issn = {1873-4324},
mesh = {*SARS-CoV-2/genetics/isolation & purification ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *COVID-19/diagnosis/virology ; CRISPR-Cas Systems/genetics ; Self-Testing ; COVID-19 Nucleic Acid Testing/methods/instrumentation ; RNA, Viral/analysis/genetics ; Limit of Detection ; Molecular Diagnostic Techniques/methods/instrumentation ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Nucleic acid testing is the most effective detection method currently available for the diagnosis of respiratory infectious diseases. However, the conventional real-time fluorescent quantitative PCR technique, which is regarded as the gold standard method for nucleic acid detection, presents significant challenges for implementation in home self-testing and popularization in underdeveloped regions due to its rigorous experimental standards. It is therefore clear that an easy-to-use, miniaturized nucleic acid testing technology and products for nonprofessionals are of great necessity to define the pathogens and assist in controlling disease transmission. (87) RESULTS: In this study, we propose a strategy for self-testing of respiratory pathogen nucleic acid that is oriented towards the public and user-friendly. The proposed system integrates the processes of extraction-free nucleic acid release, RPA isothermal amplification, and CRISPR fluorescence detection into a compact configuration. A microfluidic testing chip actuated by air pouches and a battery/USB-powered reusable device has been developed to enable simultaneous detection of internal reference genes and viral targets in a fully enclosed condition. The system allows for sample-in, and result-out testing in less than 30 min with a detection limit of 2 copies/μL. Additionally, a straightforward signal-light-based result display method has been developed to make it easy and intuitive for users to access the results. Furthermore, freeze-drying reagent is introduced to guarantee the storage and transportation of testing chips in ambient conditions. (135) SIGNIFICANCE: This work presents a miniaturized, portable, and highly sensitive nucleic acid detection system, where simple operating procedures have been designed for unskilled users. It is our belief that the testing system developed in this work is well suited for home-based self-testing and infection diagnosis in resource-limited areas, due to the above-mentioned advantages. (52).},
}

*SARS-CoV-2/genetics/isolation & purification
*Nucleic Acid Amplification Techniques/methods
Humans
*COVID-19/diagnosis/virology
CRISPR-Cas Systems/genetics
Self-Testing
COVID-19 Nucleic Acid Testing/methods/instrumentation
RNA, Viral/analysis/genetics
Limit of Detection
Molecular Diagnostic Techniques/methods/instrumentation
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid39832801,
year = {2025},
author = {Debnath, A and Sengupta, A and Rudrapal, S and Kumar, A and Rani, M},
title = {In-silico study of molecular adaptations in halophilic Cas9.},
journal = {Letters in applied microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1093/lambio/ovaf006},
pmid = {39832801},
issn = {1472-765X},
abstract = {This study explores the structural adaptations of the CRISPR-Cas9 system in halophilic bacteria, focusing on Cas9 protein of halophilic bacterium Salicibibacter cibi. Protein sequences were analysed using different tools such as ExPASy ProtParam for different physicochemical properties, PONDR web server for disordered regions, and InterPro server and WebLogo for domains. Protein structures were generated using the AlphaFold database, and the quality of the modelled structure was checked through PROCHECK. The protein surface's amino acids and electrostatic potential were visualized using PyMOL, APBS server and UCSF chimera. Comparative analysis revealed that halophilic Cas9 proteins possess a higher abundance of acidic residues, resulting in enhanced stability and hydration in saline conditions; halophilic Cas9 proteins also shows higher intrinsically disordered regions. Electrostatic potential maps confirmed that S. cibi Cas9 proteins maintain a highly negative surface charge, crucial for adaptation to salt-rich environments. These findings provide insights into the molecular mechanisms driving the structural and functional adaptations of Cas9 in salty environment, highlighting its potential applications in genome editing-based biotechnological approaches in extreme conditions.},
}

@article {pmid39832424,
year = {2025},
author = {Choudhary, R and Ahmad, F and Kaya, C and Upadhyay, SK and Muneer, S and Kumar, V and Meena, M and Liu, H and Upadhyaya, H and Seth, CS},
title = {Decrypting proteomics, transcriptomics, genomics, and integrated omics for augmenting the abiotic, biotic, and climate change stress resilience in plants.},
journal = {Journal of plant physiology},
volume = {305},
number = {},
pages = {154430},
doi = {10.1016/j.jplph.2025.154430},
pmid = {39832424},
issn = {1618-1328},
abstract = {As our planet faces increasing environmental challenges, such as biotic pressures, abiotic stressors, and climate change, it is crucial to understand the complex mechanisms that underlie stress responses in crop plants. Over past few years, the integration of techniques of proteomics, transcriptomics, and genomics like LC-MS, IT-MS, MALDI-MS, DIGE, ESTs, SAGE, WGS, GWAS, GBS, 2D-PAGE, CRISPR-Cas, cDNA-AFLP, HLS, HRPF, MPSS, CAGE, MAS, IEF, MudPIT, SRM/MRM, SWATH-MS, ESI have significantly enhanced our ability to comprehend the molecular pathways and regulatory networks, involved in balancing the ecosystem/ecology stress adaptation. This review offers thorough synopsis of the current research on utilizing these multi-omics methods (including metabolomics, ionomics) for battling abiotic (salinity, temperature (chilling/freezing/cold/heat), flood (hypoxia), drought, heavy metals/loids), biotic (pathogens like fungi, bacteria, virus, pests, and insects (aphids, caterpillars, moths, mites, nematodes) and climate change stress (ozone, ultraviolet radiation, green house gases, carbon dioxide). These strategies can expedite crop improvement, and act as powerful tools with high throughput and instant database generation rates. They also provide a platform for interpreting intricate, systematic signalling pathways and knowing how different environmental stimuli cause phenotypic responses at cellular and molecular level by changing the expression of stress-responsive genes like RAB18, KIN1, RD29B, OsCIPK03, OsSTL, SIAGL, bZIP, SnRK, ABF. This review discusses various case studies that exemplify the successful implementation of these omics tools to enhance stress tolerance in plants. Finally, it highlights challenges and future prospects of utilizing these approaches in combating stress, emphasizing the need for interdisciplinary collaborations and bio-technological advancements for sustainable agriculture and food security.},
}

@article {pmid39832407,
year = {2025},
author = {Mao, Z and Chen, R and Huang, L and Ren, S and Liu, B and Gao, Z},
title = {CRISPR analysis based on Pt@MOF dual-modal signal for multichannel fluorescence and visual detection of norovirus.},
journal = {Biosensors & bioelectronics},
volume = {273},
number = {},
pages = {117153},
doi = {10.1016/j.bios.2025.117153},
pmid = {39832407},
issn = {1873-4235},
abstract = {Norovirus is a globally prevalent pathogen that causes acute viral gastroenteritis across all age groups, characterized by its high infectivity and low infectious dose. Consequently, the development of rapid, sensitive, and accurate detection technologies for norovirus presents a significant challenge. In this study, we demonstrate a combination of CRISPR-Cas-based reactions with Pt@MOF-linked immunoassay-like assays. This methodology enables both qualitative analysis and colorimetric readouts of Cas12a-mediated DNA/RNA detection at room temperature, as well as the generation of fluorescent signal readout through base deprotonation-induced Pt@MOF cleavage of a fluorogenic substrate. Furthermore, the integration of RPA amplification with noncanonical PAM-designed CRISPR significantly enhances the sensitivity and flexibility of detection, facilitating the extension of this strategy to other targets. Ultimately, the strategy was validated in spiked food samples with a 100% accuracy rate, consistent with RT-qPCR results. Collectively, this work showcases a viable approach for a dual-functional Pt@MOF-based CRISPR biosensing platform for bioanalysis and a flexible, universal strategy based on noncanonical PAM-designed gRNAs.},
}

@article {pmid39764982,
year = {2025},
author = {Yue, Y and Liu, M and Ma, M and Xu, Z and Zhang, H and Wang, Q and Liu, R},
title = {CRISPR/Cas14a integrated with DNA walker based on magnetic self-assembly for human papillomavirus type 16 oncoprotein E7 ultrasensitive detection.},
journal = {Biosensors & bioelectronics},
volume = {272},
number = {},
pages = {117135},
doi = {10.1016/j.bios.2025.117135},
pmid = {39764982},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *Papillomavirus E7 Proteins/genetics ; Animals ; Humans ; Mice ; *Human papillomavirus 16/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Limit of Detection ; Papillomavirus Infections/virology/diagnosis ; Electrochemical Techniques/methods ; Uterine Cervical Neoplasms/virology/diagnosis ; Female ; },
abstract = {To enhance the biomarker diagnostics sensitivity and selectivity of human papillomavirus type 16 oncoprotein E7 (HPV16 E7) in serum, a label/enzyme-free electrochemical detection platform was developed. This platform featured a type of "Super-turn-off" nanobiosensor monitored through differential pulse voltammetry (DPV). It integrated the magnetic self-assembly property of the α-Fe2O3/Fe3O4@Au/Sub/BSA signal transport nano-medium with the high specificity of CRISPR/Cas14a and the amplification capability of the bipedal walker (DNA walker composed of two ssDNA strands), resulting in the enhanced specificity and anti-interference performance while remaining stable at 4 °C for over 30 days. The results demonstrated that the combination of walker and CRISPR yielded superior sensitivity and analytical capability compared with using either technology alone, achieving a detection limit of 67.17 fg mL[-1], a quantification limit of 0.22 pg mL[-1], and serum sample recovery rates of 98.46%-115.78%. Additionally, this platform was applied to detect serum and tissue samples from mouse models at various stages of cervical cancer, significantly improving the accuracy and effectiveness of early diagnosis and prognostic evaluation. This novel approach held promise as an efficient tool for point-of-care clinical detection of HPV16 E7, potentially reducing cervical cancer mortality.},
}

*Biosensing Techniques/methods
*Papillomavirus E7 Proteins/genetics
Animals
Humans
Mice
*Human papillomavirus 16/genetics/isolation & purification
*CRISPR-Cas Systems
*Limit of Detection
Papillomavirus Infections/virology/diagnosis
Electrochemical Techniques/methods
Uterine Cervical Neoplasms/virology/diagnosis
Female

@article {pmid39740588,
year = {2025},
author = {Liu, H and Lv, MM and Li, X and Su, M and Nie, YG and Ying, ZM},
title = {Ligation-recognition triggered RPA-Cas12a cis-cleavage fluorogenic RNA aptamer for one-pot and label-free detection of MicroRNA in breast cancer.},
journal = {Biosensors & bioelectronics},
volume = {272},
number = {},
pages = {117106},
doi = {10.1016/j.bios.2024.117106},
pmid = {39740588},
issn = {1873-4235},
mesh = {*Breast Neoplasms/genetics/diagnosis/blood ; Humans ; *Biosensing Techniques/methods ; *Aptamers, Nucleotide/chemistry ; *MicroRNAs/blood/genetics/analysis ; Female ; Nucleic Acid Amplification Techniques/methods ; Fluorescent Dyes/chemistry ; CRISPR-Cas Systems ; Limit of Detection ; Bacterial Proteins/chemistry/genetics ; CRISPR-Associated Proteins/chemistry/genetics ; Endodeoxyribonucleases/chemistry ; },
abstract = {"One-pot" assays which combine amplification with CRISPR/Cas12a system are in constant attracted for biosensors development. Herein, we present a one-pot isothermal assay that Ligation-recognition triggered Recombinase Polymerase Amplification (RPA)-CRISPR/Cas12a cis-cleavage (LRPA-CRISPR) fluorescent biosensor for sensitive, specific, and label-free miRNA detection. Firstly, we reveal the programmed double-stranded DNA amplicons, which utilized the ligation-recognition and polymerization to form and amplified by the RPA system. Meanwhile, we enabled exponential ligation-recognition triggered recombinase polymerase amplification of miRNA-21 sequences and exploited the cis-cleavage mechanism of Cas12a with transcription to generate functional Mango RNA for signal output. This assay can be completed within 40 min and can allow a limit of detection of 3.43 aM for miRNA-21 detection, owing to the RPA with transcription amplification and enables to product the functional Mango RNA aptamer by in vitro transcription that binds to the TO1-Biotin fluorogenic dye. Moreover, our method exhibits the advantages of self-supply crRNA, label-free, excellent specificity, and universal detection platform via the design of one-pot detection in serum and cell samples, showing tremendous potential in biomarkers diagnostics of breast cancer.},
}

*Breast Neoplasms/genetics/diagnosis/blood
Humans
*Biosensing Techniques/methods
*Aptamers, Nucleotide/chemistry
*MicroRNAs/blood/genetics/analysis
Female
Nucleic Acid Amplification Techniques/methods
Fluorescent Dyes/chemistry
CRISPR-Cas Systems
Limit of Detection
Bacterial Proteins/chemistry/genetics
CRISPR-Associated Proteins/chemistry/genetics
Endodeoxyribonucleases/chemistry

@article {pmid39622311,
year = {2025},
author = {Kim, M and Hutchins, EJ},
title = {CRISPR-Cas13d as a molecular tool to achieve targeted gene expression knockdown in chick embryos.},
journal = {Developmental biology},
volume = {519},
number = {},
pages = {5-12},
doi = {10.1016/j.ydbio.2024.11.013},
pmid = {39622311},
issn = {1095-564X},
support = {R00 DE028592/DE/NIDCR NIH HHS/United States ; R35 GM150763/GM/NIGMS NIH HHS/United States ; T32 DE007306/DE/NIDCR NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Chick Embryo ; *Gene Knockdown Techniques/methods ; *PAX7 Transcription Factor/genetics/metabolism ; Neural Crest/metabolism/embryology ; Gene Expression Regulation, Developmental/genetics ; Morpholinos/genetics ; },
abstract = {The chick embryo is a classical model system commonly used in developmental biology due to its amenability to gene perturbation experiments. Pairing this powerful model organism with cutting-edge technology can significantly expand the range of experiments that can be performed. Recently, the CRISPR-Cas13d system has been successfully adapted for use in zebrafish, medaka, killifish, and mouse embryos to achieve targeted gene expression knockdown. Despite its success in other animal models, no prior study has explored the potential of CRISPR-Cas13d in the chick. Here, we present an adaptation of the CRISPR-Cas13d system to achieve targeted gene expression knockdown in the chick embryo. As proof-of-principle, we demonstrate the knockdown of PAX7, an early neural crest marker. Application of this adapted CRISPR-Cas13d technique resulted in effective knockdown of PAX7 expression and function, comparable to knockdown achieved by translation-blocking morpholino. CRISPR-Cas13d complements preexisting knockdown tools such as CRISPR-Cas9 and morpholinos, thereby expanding the experimental potential and versatility of the chick model system.},
}

Animals
*CRISPR-Cas Systems/genetics
Chick Embryo
*Gene Knockdown Techniques/methods
*PAX7 Transcription Factor/genetics/metabolism
Neural Crest/metabolism/embryology
Gene Expression Regulation, Developmental/genetics
Morpholinos/genetics

@article {pmid39313516,
year = {2025},
author = {Liang, YL and Hu, YX and Li, FF and You, HM and Chen, J and Liang, C and Guo, ZF and Jing, Q},
title = {Adaptor protein Src-homology 2 domain containing E (SH2E) deficiency induces heart defect in zebrafish.},
journal = {Acta pharmacologica Sinica},
volume = {46},
number = {2},
pages = {404-415},
pmid = {39313516},
issn = {1745-7254},
mesh = {Animals ; *Zebrafish ; *Zebrafish Proteins/genetics/metabolism/deficiency ; Heart Defects, Congenital/genetics/metabolism ; NF-kappa B/metabolism ; Adaptor Proteins, Signal Transducing/genetics/metabolism ; Signal Transduction ; CRISPR-Cas Systems ; },
abstract = {Adaptor proteins play crucial roles in signal transduction across diverse signaling pathways. Src-homology 2 domain-containing E (SH2E) is the adaptor protein highly expressed in vascular endothelial cells and myocardium during zebrafish embryogenesis. In this study we investigated the function and mechanisms of SH2E in cardiogenesis. We first analyzed the spatiotemporal expression of SH2E and then constructed zebrafish lines with SH2E deficiency using the CRISPR-Cas9 system. We showed that homozygous mutants developed progressive pericardial edema (PCE), dilated atrium, abnormal atrioventricular looping and thickened atrioventricular wall from 3 days post fertilization (dpf) until death; inducible overexpression of SH2E was able to partially rescue the PCE phenotype. Using transcriptome sequencing analysis, we demonstrated that the MAPK/ERK and NF-κB signaling pathways might be involved in SH2E-deficiency-caused PCE. This study underscores the pivotal role of SH2E in cardiogenesis, and might help to identify innovative diagnostic techniques and therapeutic strategies for congenital heart disease.},
}

Animals
*Zebrafish
*Zebrafish Proteins/genetics/metabolism/deficiency
Heart Defects, Congenital/genetics/metabolism
NF-kappa B/metabolism
Adaptor Proteins, Signal Transducing/genetics/metabolism
Signal Transduction
CRISPR-Cas Systems

@article {pmid39831708,
year = {2025},
author = {Cochrane, RW and Robino, RA and Ferreira, LMR},
title = {Generation of Human Chimeric Antigen Receptor Regulatory T Cells.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {215},
pages = {},
doi = {10.3791/67200},
pmid = {39831708},
issn = {1940-087X},
mesh = {Humans ; *T-Lymphocytes, Regulatory/immunology ; *Receptors, Chimeric Antigen/immunology/genetics ; *CRISPR-Cas Systems ; Lentivirus/genetics ; Dependovirus/genetics/immunology ; },
abstract = {Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable hematological cancers. These successes have spurred interest in adapting the CAR platform to a small yet pivotal subset of CD4[+] T cells primarily responsible for regulating and inhibiting the immune response, regulatory T cells (Tregs). The ability to redirect Tregs' immunosuppressive activity to any extracellular target has enormous implications for creating cell therapies for autoimmune disease, organ transplant rejection, and graft-versus-host disease. Here, we describe in detail methodologies for bona fide Treg isolation from human peripheral blood, genetic modification of human Tregs utilizing either lentivirus or CRISPR/Cas9-aided knock-in using adeno-associated virus-mediated homologous directed repair (HDR) template delivery, and ex vivo expansion of stable human CAR Tregs. Lastly, we describe the assessment of human CAR Treg phenotypic stability and in vitro suppressive function, which provides insights into how the human CAR Tregs will behave in preclinical and clinical applications.},
}

Humans
*T-Lymphocytes, Regulatory/immunology
*Receptors, Chimeric Antigen/immunology/genetics
*CRISPR-Cas Systems
Lentivirus/genetics
Dependovirus/genetics/immunology

@article {pmid39831695,
year = {2025},
author = {Yan, C and Meng, H and Pei, Y and Sun, W and Zhang, J},
title = {Breeding by Design for Functional Rice with Genome Editing Technologies.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {215},
pages = {},
doi = {10.3791/67336},
pmid = {39831695},
issn = {1940-087X},
mesh = {*Oryza/genetics ; *Gene Editing/methods ; *Plant Breeding/methods ; *CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; },
abstract = {The conventional approaches to crop breeding, which rely predominantly on time-consuming and labor-intensive methods such as traditional hybridization and mutation breeding, face challenges in efficiently introducing targeted traits and generating diverse plant populations. Conversely, the emergence of genome editing technologies has ushered in a paradigm shift, enabling the precise and expedited manipulation of plant genomes to intentionally introduce desired characteristics. One of the most widespread editing tools is the CRISPR/Cas system, which has been used by researchers to study important biology-related problems. However, the precise and effective workflow of genome editing has not been well-defined in crop breeding. In this study, we demonstrated the entire process of breeding rice varieties enriched with high levels of resistant starch (RS), a functional trait that plays a crucial role in preventing diseases such as diabetes and obesity. The workflow encompassed several key steps, such as the selection of functional SBEIIb gene, designing the single-guide RNA (sgRNA), selecting an appropriate genome editing vector, determining the vector delivery method, conducting plant tissue culture, genotyping mutation and phenotypic analysis. Additionally, the time frame necessary for each stage of the process has been clearly demonstrated. This protocol not only streamlines the breeding process but also enhances the accuracy and efficiency of trait introduction, thereby accelerating the development of functional rice varieties.},
}

*Oryza/genetics
*Gene Editing/methods
*Plant Breeding/methods
*CRISPR-Cas Systems/genetics
Genome, Plant/genetics

@article {pmid39831307,
year = {2025},
author = {Fei, X and Lei, C and Ren, W and Liu, C},
title = {'Splice-at-will' Cas12a crRNA engineering enabled direct quantification of ultrashort RNAs.},
journal = {Nucleic acids research},
volume = {53},
number = {2},
pages = {},
doi = {10.1093/nar/gkaf002},
pmid = {39831307},
issn = {1362-4962},
support = {22074088//National Natural Science Foundation of China/ ; IRT_15R43//Program for Changjiang Scholars and Innovative Research Team in University/ ; GK202305001//Fundamental Research Funds for the Central Universities/ ; //Shaanxi Normal University/ ; },
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; RNA/genetics/chemistry/metabolism ; RNA Splicing ; Bacterial Proteins/genetics/metabolism ; Endodeoxyribonucleases/metabolism/genetics/chemistry ; DNA/genetics/chemistry/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Engineering/methods ; },
abstract = {We present a robust 'splice-at-will' CRISPR RNA (crRNA) engineering mechanism that overcomes the limitations of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system in directly detecting ultrashort RNAs. In this strategy, an intact Cas12a crRNA can be split from almost any site of the spacer region to obtain a truncated crRNA (tcrRNA) that cannot activate Cas12a even after binding an auxiliary DNA activator. While splicing tcrRNAs with a moiety of ultrashort RNA, the formed combination can work together to activate Cas12a efficiently, enabling 'splice-at-will' crRNA engineering. Importantly, the 'splice-at-will' crRNA exhibits almost the same trans-cleavage activation efficiency as that of a conventional intact crRNA. Therefore, by rationally designing a DNA auxiliary activator with a conserved tcrRNA-complementary sequence and an arbitrary short RNA-of-interest recognition domain, a general sensing system is established that directly utilizes traditional DNA-activated Cas12a to detect ultrashort RNAs. This 'splice-at-will' crRNA engineering strategy could faithfully detect ultrashort RNA sequences as short as 6-8 nt, which cannot be achieved by conventional Cas12a and Cas13a systems. Additionally, through flexible splicing site design, our method can precisely distinguish single-base differences in microRNA and other short RNA sequences. This work has significantly expanded the Cas12a-based diagnostic toolbox and opened new avenues for ultrashort RNA detection.},
}

*CRISPR-Cas Systems
*CRISPR-Associated Proteins/metabolism/chemistry/genetics
RNA/genetics/chemistry/metabolism
RNA Splicing
Bacterial Proteins/genetics/metabolism
Endodeoxyribonucleases/metabolism/genetics/chemistry
DNA/genetics/chemistry/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats
Genetic Engineering/methods

@article {pmid39829748,
year = {2025},
author = {Nayfach, S and Bhatnagar, A and Novichkov, A and Estevam, GO and Kim, N and Hill, E and Ruffolo, JA and Silverstein, R and Gallagher, J and Kleinstiver, B and Meeske, AJ and Cameron, P and Madani, A},
title = {Engineering of CRISPR-Cas PAM recognition using deep learning of vast evolutionary data.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2025.01.06.631536},
pmid = {39829748},
issn = {2692-8205},
abstract = {CRISPR-Cas enzymes must recognize a protospacer-adjacent motif (PAM) to edit a genomic site, significantly limiting the range of targetable sequences in a genome. Machine learning-based protein engineering provides a powerful solution to efficiently generate Cas protein variants tailored to recognize specific PAMs. Here, we present Protein2PAM, an evolution-informed deep learning model trained on a dataset of over 45,000 CRISPR-Cas PAMs. Protein2PAM rapidly and accurately predicts PAM specificity directly from Cas proteins across Type I, II, and V CRISPR-Cas systems. Using in silico deep mutational scanning, we demonstrate that the model can identify residues critical for PAM recognition in Cas9 without utilizing structural information. As a proof of concept for protein engineering, we employ Protein2PAM to computationally evolve Nme1Cas9, generating variants with broadened PAM recognition and up to a 50-fold increase in PAM cleavage rates compared to the wild-type under in vitro conditions. This work represents the first successful application of machine learning to achieve customization of Cas enzymes for alternate PAM recognition, paving the way for personalized genome editing.},
}

@article {pmid39829431,
year = {2025},
author = {Chen, Y and Xue, Y and Jiang, Q and Jin, Y and Chen, W and Hua, M},
title = {Disruption of the FOXM1 Regulatory Region Inhibits Tumor Progression in Ovarian Cancer by CRISPR-Cas9.},
journal = {Drug development research},
volume = {86},
number = {1},
pages = {e70049},
doi = {10.1002/ddr.70049},
pmid = {39829431},
issn = {1098-2299},
support = {//This work was supported by the Nantong Social Science and Technology Plan for People's Livelihood Project (grant No. JCZ2023003) from the Science and Technology Bureau of Nantong, China, and the Research Physician Development Fund (grant No. YJXYY202204-YSC03) from the Affiliated Hospital of Nantong University, China./ ; },
mesh = {Female ; *Forkhead Box Protein M1/genetics ; Humans ; *Ovarian Neoplasms/genetics/drug therapy/pathology ; Animals ; Cell Line, Tumor ; *CRISPR-Cas Systems ; Mice ; *Cell Proliferation/drug effects ; Azepines/pharmacology ; Mice, Nude ; Gene Expression Regulation, Neoplastic/drug effects ; Cell Movement/drug effects ; Triazoles/pharmacology ; Xenograft Model Antitumor Assays ; Disease Progression ; },
abstract = {Ovarian cancer is the seventh most common lethal tumor among women in the world. FOXM1 is a transcription factor implicated in the initiation and progression of ovarian cancer by regulating key oncogenic genes. The role of regulatory regions in regulating the expression of FOXM1 in ovarian cancer is not completely clarified. Treatment with bromodomain and extraterminal (BET) inhibitors JQ-1 and I-BET were explored in ovarian cancer cell lines (OVCAR3, A2780, or SKOV3) to evaluate FOXM1 expression and biological behavior by qPCR, CCK8 assay, colony formation assay, wound-healing, and transwell assays. The regulatory regions (enhancer sequence spanning promoter or exon 1) of FOXM1 were deleted using CRISPR-Cas9 in the OVCAR3 cell line. FOXM1 expression and tumor biological behavior were further assessed in FOXM1 regulatory regions deleted OVCAR3 cell line. The mouse xenograft model was assessed at the indicated time points following subcutaneous injection of enhancer-deleted cells. Treatment with the JQ-1 and I-BET reduced the expression of FOXM1, decreasing cell proliferation, migration, and invasion in a panel of ovarian cancer cell lines including OVCAR3, A2780, and SKOV3 cells. By mining the published ChIP-sequencing data (H3K27Ac) from 12 ovarian cancer cell lines, we identified a potential enhancer and promoter region. Deletion of the spanning enhancer and promoter region of FOXM1 reduced mRNA and protein expression. Similarly, cell proliferation, migration, invasion, and tumorigenesis in both cells and mouse xenograft models were significantly attenuated. Our study demonstrates that JQ-1 and I-BET can regulate the expression of the FOXM1 gene-relating network. These data also indicate that disruption of the span enhancer and promoter region activity of FOXM1 has a vital role in the anti-ovarian cancer effect, hiding a potential opportunity for the evaluation of this non-coding DNA deletion disrupts the FOXM1 transcriptional network in ovarian cancer development.},
}

Female
*Forkhead Box Protein M1/genetics
Humans
*Ovarian Neoplasms/genetics/drug therapy/pathology
Animals
Cell Line, Tumor
*CRISPR-Cas Systems
Mice
*Cell Proliferation/drug effects
Azepines/pharmacology
Mice, Nude
Gene Expression Regulation, Neoplastic/drug effects
Cell Movement/drug effects
Triazoles/pharmacology
Xenograft Model Antitumor Assays
Disease Progression

@article {pmid39828694,
year = {2025},
author = {Matthews, MC and van der Linden, J and Robène, I and Rozsasi, S and Coetzee, B and Campa, M and Burger, J and Akwuruoha, UN and Madufor, NJ and Perold, W and Opara, UL and Viljoen, A and Mostert, D},
title = {A combined recombinase polymerase amplification CRISPR/Cas12a assay for detection of Fusarium oxysporum f. sp. cubense tropical race 4.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {2436},
pmid = {39828694},
issn = {2045-2322},
support = {138109//National Research Foundation/ ; 138109//National Research Foundation/ ; 138109//National Research Foundation/ ; 138109//National Research Foundation/ ; 138109//National Research Foundation/ ; 138109//National Research Foundation/ ; 138109//National Research Foundation/ ; 890856//Horizon 2020 Framework Programme/ ; 890856//Horizon 2020 Framework Programme/ ; 890856//Horizon 2020 Framework Programme/ ; 890856//Horizon 2020 Framework Programme/ ; },
mesh = {*CRISPR-Cas Systems ; *Fusarium/genetics/isolation & purification ; *Musa/microbiology ; Nucleic Acid Amplification Techniques/methods ; Plant Diseases/microbiology ; Recombinases/metabolism/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The soilborne pathogen Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) is currently devastating banana production worldwide. Once introduced, it is not possible to eradicate the pathogen from soils where it can survive for decades. The only management option available then is to replace Foc TR4-susceptible with -resistant varieties. Timely detection of the pathogen, however, is an important strategy to prevent the introduction of Foc TR4 into new areas and prevent its spread from infested sites. In this study, a single-tube detection technique was developed by combining recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a technology (RPA-Cas12a) for detection of Foc TR4. The RPA-Cas12a assay was conducted isothermally, had a sensitivity of up to 10 fg target DNA and did not cross react with any of the 76 non-target isolates included in the specificity testing. The RPA-Cas12a assay detected Foc TR4 from naturally infected banana samples collected in the field and visualization was possible with the naked eye under LED blue light transillumination. The method can be integrated with inexpensive fluorescent or electronic detection devices to accelerate Foc TR4 in-field detection and, thereby, fast-track disease containment strategies.},
}

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

@article {pmid39827180,
year = {2025},
author = {Van Goethem, MW and Bezuidt, OKI and Pierneef, R and Vikram, S and Hopkins, DW and Aspray, T and Hall, G and Woodborne, S and Hogg, ID and Northen, TR and Kong, W and Daffonchio, D and Cowan, DA and Van de Peer, Y and Delgado-Baquerizo, M and Makhalanyane, TP},
title = {Novel adaptive immune systems in pristine Antarctic soils.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {2368},
pmid = {39827180},
issn = {2045-2322},
support = {118981//National Research Foundation/ ; 118981//National Research Foundation/ ; 118981//National Research Foundation/ ; 118981//National Research Foundation/ ; 118981//National Research Foundation/ ; 118981//National Research Foundation/ ; 110717//South African National Antarctic Programme/ ; 110717//South African National Antarctic Programme/ ; 110717//South African National Antarctic Programme/ ; 110717//South African National Antarctic Programme/ ; 110717//South African National Antarctic Programme/ ; 110717//South African National Antarctic Programme/ ; },
mesh = {Antarctic Regions ; *Soil Microbiology ; *Phylogeny ; *CRISPR-Cas Systems ; Microbiota/genetics ; Metagenomics/methods ; Adaptive Immunity/genetics ; Genome, Bacterial ; Bacteria/genetics ; Prophages/genetics ; Metagenome ; },
abstract = {Antarctic environments are dominated by microorganisms, which are vulnerable to viral infection. Although several studies have investigated the phylogenetic repertoire of bacteria and viruses in these poly-extreme environments with freezing temperatures, high ultra violet irradiation levels, low moisture availability and hyper-oligotrophy, the evolutionary mechanisms governing microbial immunity remain poorly understood. Using genome-resolved metagenomics, we test the hypothesis that Antarctic poly-extreme high-latitude microbiomes harbour diverse adaptive immune systems. Our analysis reveals the prevalence of prophages in bacterial genomes (Bacteroidota and Verrucomicrobiota), suggesting the significance of lysogenic infection strategies in Antarctic soils. Furthermore, we demonstrate the presence of diverse CRISPR-Cas arrays, including Class 1 arrays (Types I-B, I-C, and I-E), alongside systems exhibiting novel gene architecture among their effector cas genes. Notably, a Class 2 system featuring type V variants lacks CRISPR arrays, encodes Cas1 and Cas2 adaptation module genes. Phylogenetic analysis of Cas12 effector proteins hints at divergent evolutionary histories compared to classified type V effectors and indicates that TnpB is likely the ancestor of Cas12 nucleases. Our findings suggest substantial novelty in Antarctic cas sequences, likely driven by strong selective pressures. These results underscore the role of viral infection as a key evolutionary driver shaping polar microbiomes.},
}

Antarctic Regions
*Soil Microbiology
*Phylogeny
*CRISPR-Cas Systems
Microbiota/genetics
Metagenomics/methods
Adaptive Immunity/genetics
Genome, Bacterial
Bacteria/genetics
Prophages/genetics
Metagenome

@article {pmid39826761,
year = {2025},
author = {Zhou, Z and Liu, S and Saleem, M and Liu, F and Hu, R and Su, H and Dong, D and Luo, Z and Wu, Y and Zhang, Y and He, Z and Wang, C},
title = {Unraveling phase-dependent variations of viral community, virus-host linkage, and functional potential during manure composting process.},
journal = {Bioresource technology},
volume = {},
number = {},
pages = {132081},
doi = {10.1016/j.biortech.2025.132081},
pmid = {39826761},
issn = {1873-2976},
abstract = {The temporal dynamics of bacterial and fungal communities significantly impact the manure composting process, yet viral communities are often underexplored. Bulk metagenomes, viromes, metatranscriptomes, and metabolomes were integrated to investigate dynamics of double-stranded DNA (dsDNA) virus and virus-host interactions throughout a 63-day composting process. A total of 473 viral operational taxonomic units (vOTUs), predominantly Caudoviricetes, showed distinct phase-dependent differentiation. In phase I (initial-mesophilic), viruses targeted Gammaproteobacteria and Firmicutes, utilizing restriction-modification (RM) systems. In phase II (thermophilic-maturing), viruses infected Alphaproteobacteria, Chloroflexi, and Planctomycetes, employing CRISPR-Cas systems. Lysogenic and lytic viruses exerting differential effects on bacterial pathogens across phases. Additionally, six types of auxiliary metabolic genes (AMGs) related to galactose and cysteine metabolisms were identified. The homologous lineages of AMGs with bacterial genes, along with the significant temporal correlation observed between virus-host-metabolite interactions, underscore the critical yet often overlooked role of viral communities in modulating microbial metabolisms and pathogenesis within composting ecosystems.},
}

@article {pmid39826547,
year = {2025},
author = {Hong, SH and An, SY and Park, C and Kim, Y and Kim, EH and Kim, NK and Suh, JY},
title = {Structural variants of AcrIIC5 inhibit Cas9 via divergent binding interfaces.},
journal = {Structure (London, England : 1993)},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.str.2024.12.014},
pmid = {39826547},
issn = {1878-4186},
abstract = {CRISPR-Cas is a bacterial defense system that employs RNA-guided endonucleases to destroy invading foreign nucleic acids. Bacteriophages produce anti-CRISPR (Acr) proteins to evade CRISPR-Cas defense during the infection. AcrIIC5, a type II-C Cas9 inhibitor, exhibits unusual variations in the local backbone fold between its orthologs. Here we investigated how the folding variations affect the inhibition of target Cas9 using AcrIIC5 orthologs. Structural comparison of free AcrIIC5Smu and AcrIIC5Nch confirmed that the folding variation correlated with characteristic indels in the helical region. Mutagenesis and biochemical assays combined with AlphaFold2 predictions identified key residues of AcrIIC5 orthologs important for Cas9 inhibition. Remarkably, AcrIIC5 orthologs employed divergent binding interfaces via folding variations to inhibit the Cas9 targets. Our study suggests that Acr proteins have evolved structural variants to diversify key interfaces for target Cas9, which could be beneficial for the adaptation of phages to evasive mutations on the Cas9 surface.},
}

@article {pmid39700063,
year = {2025},
author = {Munusamy, S and Zheng, H and Jahani, R and Zhou, S and Chen, J and Kong, J and Guan, X},
title = {DNA-Assisted CRISPR-Cas12a Enhanced Fluorescent Assay for Protein Detection in Complicated Matrices.},
journal = {ACS applied bio materials},
volume = {8},
number = {1},
pages = {754-762},
doi = {10.1021/acsabm.4c01600},
pmid = {39700063},
issn = {2576-6422},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *DNA/chemistry ; Materials Testing ; Biocompatible Materials/chemistry ; Particle Size ; CRISPR-Associated Proteins/genetics/metabolism ; Endodeoxyribonucleases/metabolism/genetics ; Proteins/analysis/metabolism ; Bacterial Proteins ; },
abstract = {Proteins are important biological macromolecules that perform a wide variety of functions in the cell and human body, and can serve as important biomarkers for early diagnosis and prognosis of human diseases as well as monitoring the effectiveness of disease treatment. Hence, sensitive and accurate detection of proteins in human biospecimens is imperative. However, at present, there is no ideal method available for the detection of proteins in clinical samples, many of which are present at ultralow (less than 1 pM) concentrations and in complicated matrices. Herein, we report an ultrasensitive and selective DNA-assisted CRISPR-Cas12a enhanced fluorescent assay (DACEA) for protein detection with detection limits reaching as low as attomolar concentrations. The high assay sensitivity was accomplished through the combined DNA barcode amplification (by using dual-functionalized AuNPs) and CRISPR analysis, while the high selectivity and high resistance to the matrix effects of our method were accomplished via the formation of protein-antibody sandwich structure and the specific recognition of Cas12a (under the guidance of crRNA) toward the designed target ssDNA. Given its ability to accurately and sensitively detect trace amounts of proteins in complicated matrices, the DACEA protein assay platform pioneered in this work has a potential application in routine protein biomarker testing.},
}

*CRISPR-Cas Systems/genetics
Humans
*DNA/chemistry
Materials Testing
Biocompatible Materials/chemistry
Particle Size
CRISPR-Associated Proteins/genetics/metabolism
Endodeoxyribonucleases/metabolism/genetics
Proteins/analysis/metabolism
Bacterial Proteins

@article {pmid39825389,
year = {2025},
author = {Abraham, IC and Aboje, JE and Ukoaka, BM and Tom-Ayegunle, K and Amjad, M and Abdulkader, A and Agbo, CE and Akinruli, OA and Akisanmi, TR and Oyetola, EO and Olatunji, G and Kokori, E and Aderinto, N},
title = {Integrating malaria vaccine and CRISPR/Cas9 gene drive: a comprehensive strategy for accelerated malaria eradication.},
journal = {Malaria journal},
volume = {24},
number = {1},
pages = {17},
pmid = {39825389},
issn = {1475-2875},
mesh = {*Malaria Vaccines/immunology/genetics/administration & dosage ; *CRISPR-Cas Systems ; *Malaria/prevention & control ; *Gene Drive Technology/methods ; Humans ; *Disease Eradication/methods ; Animals ; Anopheles/genetics/parasitology ; Mosquito Vectors/genetics ; Mosquito Control/methods ; },
abstract = {Malaria remains a significant public health challenge, particularly in low- and middle-income countries, despite ongoing efforts to eradicate the disease. Recent advancements, including the rollout of malaria vaccines, such as RTS,S/AS01 and R21/Matrix-M™, offer new avenues for prevention. However, the rise of resistance to anti-malarial medications necessitates innovative strategies. This review explores the potential integration of CRISPR/Cas9 gene drive technology with malaria vaccination efforts to enhance vector control and reduce transmission. By employing gene drive mechanisms for population suppression and replacement of malaria-transmitting Anopheles mosquitoes, combined with the immunogenic properties of vaccines, a synergistic approach can be established. This paper discussed the need for integrated strategies to address the biological complexities of malaria and socio-economic factors influencing its prevalence. Challenges such as regulatory hurdles, community acceptance, ecological impacts, and sustainable funding are examined, alongside strategies for implementation within existing malaria control programmes. This integrated approach could significantly contribute to achieving the World Health Organization's targets for malaria reduction by 2030, ultimately enhancing public health outcomes and supporting broader socio-economic development.},
}

*Malaria Vaccines/immunology/genetics/administration & dosage
*CRISPR-Cas Systems
*Malaria/prevention & control
*Gene Drive Technology/methods
Humans
*Disease Eradication/methods
Animals
Anopheles/genetics/parasitology
Mosquito Vectors/genetics
Mosquito Control/methods

@article {pmid39824586,
year = {2025},
author = {Arivarasan, VK and Diwakar, D and Kamarudheen, N and Loganathan, K},
title = {Current approaches in CRISPR-Cas systems for diabetes.},
journal = {Progress in molecular biology and translational science},
volume = {210},
number = {},
pages = {95-125},
doi = {10.1016/bs.pmbts.2024.08.002},
pmid = {39824586},
issn = {1878-0814},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Diabetes Mellitus/therapy/genetics ; Animals ; Gene Editing ; Genetic Therapy/methods ; },
abstract = {In the face of advancements in health care and a shift towards healthy lifestyle, diabetes mellitus (DM) still presents as a global health challenge. This chapter explores recent advancements in the areas of genetic and molecular underpinnings of DM, addressing the revolutionary potential of CRISPR-based genome editing technologies. We delve into the multifaceted relationship between genes and molecular pathways contributing to both type1 and type 2 diabetes. We highlight the importance of how improved genetic screening and the identification of susceptibility genes are aiding in early diagnosis and risk stratification. The spotlight then shifts to CRISPR-Cas9, a robust genome editing tool capable of various applications including correcting mutations in type 1 diabetes, enhancing insulin production in T2D, modulating genes associated with metabolism of glucose and insulin sensitivity. Delivery methods for CRISPR to targeted tissues and cells are explored, including viral and non-viral vectors, alongside the exciting possibilities offered by nanocarriers. We conclude by discussing the challenges and ethical considerations surrounding CRISPR-based therapies for DM. These include potential off-target effects, ensuring long-term efficacy and safety, and navigating the ethical implications of human genome modification. This chapter offers a comprehensive perspective on how genetic and molecular insights, coupled with the transformative power of CRISPR, are paving the way for potential cures and novel therapeutic approaches for DM.},
}

Humans
*CRISPR-Cas Systems/genetics
*Diabetes Mellitus/therapy/genetics
Animals
Gene Editing
Genetic Therapy/methods

@article {pmid39824585,
year = {2025},
author = {Munshi, ID and Acharya, M and Mukherjee, S and Mani, I},
title = {Recent development in CRISPR-Cas systems for cardiac disease.},
journal = {Progress in molecular biology and translational science},
volume = {210},
number = {},
pages = {47-93},
doi = {10.1016/bs.pmbts.2024.08.004},
pmid = {39824585},
issn = {1878-0814},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Animals ; *Heart Diseases/genetics/therapy ; *Gene Editing ; },
abstract = {The CRISPR-Cas system has emerged as a revolutionary tool in genetic research, enabling highly precise gene editing and significantly advancing the field of cardiovascular science. This chapter provides a comprehensive overview of the latest developments in utilizing CRISPR-Cas technologies to investigate and treat heart diseases. It delves into the application of CRISPR-Cas9 for creating accurate models of complex cardiac conditions, such as hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and various arrhythmias, which are essential for understanding disease mechanisms and testing potential therapies. The therapeutic potential of gene editing is also explored, with a focus on genes like PCSK9 and ANGPTL3 that play critical roles in lipid metabolism and cardiovascular health, offering promising avenues for new treatments. Furthermore, the expanding applications of CRISPR in heart tissue regeneration are examined, which could revolutionize the repair of damaged heart tissue. Cutting-edge techniques such as base editing and prime editing are discussed, highlighting their potential to further refine genetic interventions. The discussion concludes by addressing the challenges associated with delivering CRISPR components efficiently and safely, while also exploring recent innovations that may overcome these hurdles, providing insights into the future directions of CRISPR technology in cardiovascular medicine.},
}

Humans
*CRISPR-Cas Systems/genetics
Animals
*Heart Diseases/genetics/therapy
*Gene Editing

@article {pmid39824584,
year = {2025},
author = {Khoshandam, M and Soltaninejad, H and Bhia, I and Goudarzi, MTH and Hosseinkhani, S},
title = {CRISPR challenges in clinical developments.},
journal = {Progress in molecular biology and translational science},
volume = {210},
number = {},
pages = {263-279},
doi = {10.1016/bs.pmbts.2024.08.001},
pmid = {39824584},
issn = {1878-0814},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Animals ; Genetic Therapy/methods ; Neoplasms/genetics/therapy ; },
abstract = {CRISPR-Cas (clustered regularly interspaced short palindromic repeats and associated proteins) is a novel genome editing technology with potential applications in treating diseases. Currently, its use in humans is restricted to clinical trials, although its growth rate is significant, and some have received initial FDA approval. It is crucial to examine and address the challenges for this technology to be implemented in clinical settings. This review aims to identify and explore new research ideas to increase of CRISPR's efficiency in treating genetic diseases and cancer, as well as its future prospects. Given that a substantial amount of previous research has focused on CRISPR-Cas delivery strategies and materials, this overview introduces specific conditions and strategies. It also discusses some of the challenges and opportunities in this field, offering a unique perspective.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Editing
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Animals
Genetic Therapy/methods
Neoplasms/genetics/therapy

@article {pmid39824583,
year = {2025},
author = {Mishra, S and Banerjee, S and Tiwari, BS and Tiwari, AK},
title = {Recent progress in CRISPR-Cas-system for neurological disorders.},
journal = {Progress in molecular biology and translational science},
volume = {210},
number = {},
pages = {231-261},
doi = {10.1016/bs.pmbts.2024.07.017},
pmid = {39824583},
issn = {1878-0814},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Nervous System Diseases/genetics/therapy ; Animals ; Gene Editing ; Genetic Therapy/methods ; },
abstract = {Different neurological diseases including, Parkinson's, Alzheimer's, and Huntington's diseases extant momentous global disease burdens, affecting millions of lives for imposing a heavy disease burden on the healthcare systems. Despite various treatment strategies aimed at alleviating symptoms, treatments remain elusive and ineffective due to the disease's complexity. However, recent advancements in gene therapy via the CRISPR-Cas system offer ground-breaking and targeted treatment options. Based on a bacterial immune mechanism, the CRISPR-Cas system enables precise genome editing, allowing for the alteration of different genetic mutations and the possible cure of genetic diseases. In the context of neurological disorders, the CRISPR-Cas system shows a promising avenue by allowing researchers to conduct genome-editing which is implicated in neurodegenerative disease therapeutics. This book chapter provides an updated overview of the application of the CRISPR-Cas system for addressing target-specific therapeutic approaches for neurodegenerative disorders. Furthermore, we discuss the principles of the CRISPR-Cas mechanism, its role in modeling neurological disorders, identifying molecular targets, and developing gene-based therapies. Additionally, the chapter explores the recent clinical trials and CRISPR-Cas-mediated treatments for neurological conditions. By leveraging the accuracy and versatility of the CRISPR-Cas system, scientists can more effectively handle the genetic underpinnings of neurodegenerative diseases. Furthermore, the chapter extends the critical viewpoints on ethical considerations and technical limitations related to the clinical deployment of this revolutionizing technique.},
}

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

@article {pmid39824582,
year = {2025},
author = {Gowda, DAA and Birappa, G and Rajkumar, S and Ajaykumar, CB and Srikanth, B and Kim, SL and Singh, V and Jayachandran, A and Lee, J and Ramakrishna, S},
title = {Recent progress in CRISPR/Cas9 system for eye disorders.},
journal = {Progress in molecular biology and translational science},
volume = {210},
number = {},
pages = {21-46},
doi = {10.1016/bs.pmbts.2024.07.018},
pmid = {39824582},
issn = {1878-0814},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Animals ; *Eye Diseases/genetics/therapy ; Gene Editing ; },
abstract = {Ocular disorders encompass a broad spectrum of phenotypic and clinical symptoms resulting from several genetic variants and environmental factors. The unique anatomy and physiology of the eye facilitate validation of cutting-edge gene editing treatments. Genome editing developments have allowed researchers to treat a variety of diseases, including ocular disorders. The clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system holds considerable promise for therapeutic applications in the field of ophthalmology, including repair of aberrant genes and treatment of retinal illnesses related to the genome or epigenome. Application of CRISPR/Cas9 systems to the study of ocular disease and visual sciences have yielded innovations including correction of harmful mutations in patient-derived cells and gene modifications in several mammalian models of eye development and disease. In this study, we discuss the generation of several ocular disease models in mammalian cell lines and in vivo systems using a CRISPR/Cas9 system. We also provide an overview of current uses of CRISPR/Cas9 technologies for the treatment of ocular pathologies, as well as future challenges.},
}

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

@article {pmid39824581,
year = {2025},
author = {Singh, S and Raj, D and Mathur, A and Mani, N and Kumar, D},
title = {Current approaches in CRISPR-Cas systems for hereditary diseases.},
journal = {Progress in molecular biology and translational science},
volume = {210},
number = {},
pages = {205-229},
doi = {10.1016/bs.pmbts.2024.07.015},
pmid = {39824581},
issn = {1878-0814},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Genetic Diseases, Inborn/therapy/genetics ; *Gene Editing/methods ; Animals ; },
abstract = {CRISPR-Cas technologies have drastically revolutionized genetic engineering and also dramatically changed the potential for treating inherited disorders. The potential to correct genetic mutations responsible for numerous hereditary disorders from single-gene disorders to complex polygenic diseases through precise DNA editing is feasible. The tactic now employed in CRISPR-Cas systems for treating inherited disorders is the usage of particular guide RNAs to target and edit disease-causing mutations in the patient's genome. Several methods such as CRISPR-Cas9, CRISPR-Cas12, and CRISPR-Cas13 are being thoroughly researched and optimized to increase effectiveness, accuracy, and safety in gene editing. Additionally, it is predicted that CRISPR-based therapies will be able to treat complex genetic illnesses such as cancer predisposition syndromes, neurological disorders, and cardiovascular conditions in addition to single-gene disorders. The available editing tools and creation of base editing technology facilitate the simultaneous correction of many mutations or accurate nucleotide changes leading to further advances in the development of multiplex editing tools and base editing technology fiction. When combined with other paradigms such as gene therapy using stem cell treatment, CRISPR-Cas promises improved efficacy. Patient treatment and lowering side effects significantly in individual genetic profiles will guide CRISPR-based treatments. These procedures will undoubtedly lead to therapies that are both efficient and curative of a wide range of genetic diseases, ushering in a new era of precision medicine. This chapter discusses about CRISPR Cas9 mechanism and its significance in the treatment of Hereditary disorders.},
}

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

@article {pmid39824580,
year = {2025},
author = {Israr, J and Kumar, A},
title = {Current progress in CRISPR-Cas systems for rare diseases.},
journal = {Progress in molecular biology and translational science},
volume = {210},
number = {},
pages = {163-203},
doi = {10.1016/bs.pmbts.2024.07.019},
pmid = {39824580},
issn = {1878-0814},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Rare Diseases/genetics/therapy ; Animals ; *Gene Editing/methods ; Genetic Therapy/methods ; },
abstract = {The groundbreaking CRISPR-Cas gene editing method permits exact genetic code alteration. The "CRISPR" DNA protects bacteria from viruses. CRISPR-Cas utilizes a guide RNA to steer the Cas enzyme to the genome's gene editing target. After attaching to a sequence, Cas enzymes cleave DNA to insert, delete, or modify genes. The influence of CRISPR-Cas technology on molecular biology and genetics is profound. It allows for gene function research, animal disease models, and patient genetic therapy. Gene editing has transformed biotechnology, agriculture, and customized medicine. CRISPR-Cas could revolutionize genetics and medicine. CRISPR-Cas may accurately correct genetic flaws that underlie rare diseases, improving their therapy. Gene mutations make CRISPR-Cas gene editing a viable cure for uncommon diseases. We can use CRISPR-Cas to correct genetic abnormalities at the molecular level. This strategy offers hope for remedies and disease understanding. CRISPR-Cas genome editing may enable more targeted and effective treatments for rare medical illnesses with few therapy options. By developing base- and prime-editing CRISPR technology, CRISPR-Cas allows for accurate and efficient genome editing and advanced DNA modification. This advanced method provides precise DNA alterations without double-strand breakage. These advances have improved gene editing safety and precision, reducing unfavorable effects. Lipid nanoparticles, which use viral vectors, improve therapeutic cell and tissue targeting. In rare disorders, gene therapy may be possible with CRISPR-Cas clinical trials. CRISPR-Cas research is improving gene editing, delivery, and rare disease treatment.},
}

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

@article {pmid39824579,
year = {2025},
author = {Puri, B and Kulkarni, YA and Gaikwad, AB},
title = {Advances in CRISPR-Cas systems for kidney diseases.},
journal = {Progress in molecular biology and translational science},
volume = {210},
number = {},
pages = {149-162},
doi = {10.1016/bs.pmbts.2024.07.020},
pmid = {39824579},
issn = {1878-0814},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Kidney Diseases/genetics/therapy ; Animals ; Gene Editing ; },
abstract = {Recent advances in CRISPR-Cas systems have revolutionised the study and treatment of kidney diseases, including acute kidney injury (AKI), chronic kidney disease (CKD), diabetic kidney disease (DKD), lupus nephritis (LN), and polycystic kidney disease (PKD). CRISPR-Cas technology offers precise and versatile tools for genetic modification in monogenic kidney disorders such as PKD and Alport syndrome. Recent advances in CRISPR technology have also shown promise in addressing other kidney diseases like AKI, CKD, and DKD. CRISPR-Cas holds promise to edit genetic mutations underlying these conditions, potentially leading to more effective and long-lasting treatments. Furthermore, the adaptability of CRISPR-Cas systems allows for developing tailored therapeutic strategies that specifically target the genetic and molecular mechanisms contributing to different kidney diseases. Beyond DNA modifications, CRISPR-Cas technologies also enable editing noncoding RNA, such as lncRNAs and miRNAs, in kidney diseases. Despite these advancements, significant challenges persist, including delivery efficiency to specific kidney cells and potential off-target effects. However, the rapid progress in CRISPR-Cas technology suggests a transformative impact on the future management of kidney diseases, offering the potential for enhanced patient outcomes through personalised and precise therapeutic approaches. This chapter highlights the recent advancement of CRISPR-Cas systems and their potential applications in various kidney diseases.},
}

Humans
*CRISPR-Cas Systems/genetics
*Kidney Diseases/genetics/therapy
Animals
Gene Editing

@article {pmid39824578,
year = {2025},
author = {Ajaykumar, CB and Rajkumar, S and Suresh, B and Birappa, G and Gowda, DAA and Jayachandran, A and Kim, KS and Hong, SH and Ramakrishna, S},
title = {Advances in applications of the CRISPR/Cas9 system for respiratory diseases.},
journal = {Progress in molecular biology and translational science},
volume = {210},
number = {},
pages = {127-147},
doi = {10.1016/bs.pmbts.2024.07.014},
pmid = {39824578},
issn = {1878-0814},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Animals ; Respiratory Tract Diseases/genetics/therapy ; Gene Editing ; Genetic Therapy/methods ; },
abstract = {Genetic and environmental factors can have an impact on lung and respiratory disorders which are associated with severe symptoms and have high mortality rates. Many respiratory diseases are significantly influenced by genetic or epigenetic factors. Gene therapy offers a powerful approach providing therapeutic treatment for lung diseases. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) are promising gene modifying tool that can edit the genome. The utilization of CRISPR/Cas9 systems in the investigation of respiratory disorders has resulted in advancements such as the rectification of deleterious mutations in patient-derived cells and the alteration of genes in multiple mammalian lung disease models. New avenues of treatment for lung disorders have been opened up by advances in CRISPR/Cas9 research. In this chapter, we discuss the known genes and mutations that cause several common respiratory disorders such as COPD, asthma, IPF, and ARDS. We further review the current research using CRISPR/Cas9 in numerous respiratory disorders and possible therapeutic treatments.},
}

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

@article {pmid39824577,
year = {2025},
author = {Arora, Y and Priya, and Kumar, M and Kumar, D},
title = {Current approaches in CRISPR-Cas system for metabolic disorder.},
journal = {Progress in molecular biology and translational science},
volume = {210},
number = {},
pages = {1-19},
doi = {10.1016/bs.pmbts.2024.07.016},
pmid = {39824577},
issn = {1878-0814},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Metabolic Diseases/therapy/genetics ; Animals ; Gene Editing ; Genetic Therapy/methods ; },
abstract = {A new era in genomic medicine has been brought by the development of CRISPR-Cas technology, which presents hitherto unheard-of possibilities for the treatment of metabolic illnesses. The treatment approaches used in CRISPR/Cas9-mediated gene therapy, emphasize distribution techniques such as viral vectors and their use in preclinical models of metabolic diseases like hypercholesterolemia, glycogen storage diseases, and phenylketonuria. The relevance of high-throughput CRISPR screens for target identification in discovering new genes and pathways associated with metabolic dysfunctions is an important aspect of the discovery of new approaches. With cutting-edge options for genetic correction and cellular regeneration, the combination of CRISPR-Cas technology with stem cell therapy has opened new avenues for the treatment of metabolic illnesses. The integration of stem cell therapy and CRISPR-Cas technology is an important advance in the treatment of metabolic diseases, which are difficult to treat because of their intricate genetic foundations. This chapter addresses the most recent developments in the application of stem cell therapy and CRISPR-Cas systems to treat a variety of metabolic disorders, providing fresh hope for effective and maybe curative therapies. This chapter examines techniques and developments that have been made recently to address a variety of metabolic disorders using CRISPR-Cas systems. Our chapter focuses on the foundational workings of CRISPR-Cas technology and its potential uses in gene editing, gene knockout, and activation/repression-based gene modification.},
}

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

@article {pmid39626660,
year = {2025},
author = {Jensen, CL and Chen, LF and Swigut, T and Crocker, OJ and Yao, D and Bassik, MC and Ferrell, JE and Boettiger, AN and Wysocka, J},
title = {Long-range regulation of transcription scales with genomic distance in a gene-specific manner.},
journal = {Molecular cell},
volume = {85},
number = {2},
pages = {347-361.e7},
doi = {10.1016/j.molcel.2024.10.021},
pmid = {39626660},
issn = {1097-4164},
support = {R35 GM131792/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Mice ; *Promoter Regions, Genetic ; *Mouse Embryonic Stem Cells/metabolism/cytology ; Transcription, Genetic ; Chromatin/genetics/metabolism ; Transcription Factor RelA/genetics/metabolism ; Enhancer Elements, Genetic ; Transcriptional Activation ; Gene Expression Regulation ; Transcription Factors/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; },
abstract = {Although critical for tuning the timing and level of transcription, enhancer communication with distal promoters is not well understood. Here, we bypass the need for sequence-specific transcription factors (TFs) and recruit activators directly using a chimeric array of gRNA oligos to target dCas9 fused to the activator VP64-p65-Rta (CARGO-VPR). We show that this approach achieves effective activator recruitment to arbitrary genomic sites, even those inaccessible when targeted with a single guide. We utilize CARGO-VPR across the Prdm8-Fgf5 locus in mouse embryonic stem cells (mESCs), where neither gene is expressed. Although activator recruitment to any tested region results in the transcriptional induction of at least one gene, the expression level strongly depends on the genomic distance between the promoter and activator recruitment site. However, the expression-distance relationship for each gene scales distinctly in a manner not attributable to differences in 3D contact frequency, promoter DNA sequence, or the presence of repressive chromatin marks at the locus.},
}

Animals
Mice
*Promoter Regions, Genetic
*Mouse Embryonic Stem Cells/metabolism/cytology
Transcription, Genetic
Chromatin/genetics/metabolism
Transcription Factor RelA/genetics/metabolism
Enhancer Elements, Genetic
Transcriptional Activation
Gene Expression Regulation
Transcription Factors/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism

@article {pmid39585363,
year = {2025},
author = {Guo, Y and Zhao, X},
title = {CRISPR-based genetic screens in human pluripotent stem cells derived neurons and brain organoids.},
journal = {Cell and tissue research},
volume = {399},
number = {1},
pages = {1-8},
pmid = {39585363},
issn = {1432-0878},
support = {P50HD105353//National Institute of Child Health and Human Development/ ; R01 MH136152/MH/NIMH NIH HHS/United States ; R01 NS138268/NS/NINDS NIH HHS/United States ; P50 HD105353/HD/NICHD NIH HHS/United States ; R01 MH118827/MH/NIMH NIH HHS/United States ; R01MH118827/MH/NIMH NIH HHS/United States ; R01MH118827/MH/NIMH NIH HHS/United States ; },
mesh = {Humans ; *Organoids/cytology/metabolism ; *Pluripotent Stem Cells/cytology/metabolism ; *Neurons/cytology/metabolism ; *Brain/cytology ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genetic Testing/methods ; },
abstract = {Recent large-scale genome-wide association and single-cell RNA sequencing (scRNA-seq) studies have uncovered disease-associated genetic risk factors and cell type-specific genetic alterations. However, our understanding of how these genetic variants cause diseases and the underlying mechanisms remains largely unknown. Functional genomics screens using CRISPR-based technologies offer an effective tool for studying genes relevant to disease phenotypes. Here, we summarize recent CRISPR-based functional genomics screen approaches applied to human pluripotent stem cell (hPSC)-derived neurons and brain organoids. These screens have identified genes crucial for neurogenesis, neuronal survival, morphological development, and migration. Combining CRISPR-based genetic screens with scRNA-seq, researchers have revealed downstream genes and cellular pathways impacted by these genetic variants in human neural cells, providing new insights into the pathogenesis of neurodevelopmental disorders, such as microcephaly and autism spectrum disorders. Finally, we discuss current challenges and future directions for using CRISPR-based screens in furthering our understanding of neurological diseases and developing potential therapeutic strategies. Despite challenges, CRISPR-based screens have enormous potential for advancing the therapeutic development of many diseases.},
}

Humans
*Organoids/cytology/metabolism
*Pluripotent Stem Cells/cytology/metabolism
*Neurons/cytology/metabolism
*Brain/cytology
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Genetic Testing/methods

@article {pmid39822055,
year = {2025},
author = {Göritzer, K and Melnik, S and Schwestka, J and Arcalis, E and Drapal, M and Fraser, P and Ma, JK and Stoger, E},
title = {Enhancing quality and yield of recombinant secretory IgA antibodies in Nicotiana benthamiana by endoplasmic reticulum engineering.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.14576},
pmid = {39822055},
issn = {1467-7652},
support = {760331//Horizon 2020 Framework Programme/ ; 21EBTA/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; J4583//Austrian Science Fund/ ; W1224//Austrian Science Fund/ ; },
abstract = {The production of complex multimeric secretory immunoglobulins (SIgA) in Nicotiana benthamiana leaves is challenging, with significant reductions in complete protein assembly and consequently yield, being the most important difficulties. Expanding the physical dimensions of the ER to mimic professional antibody-secreting cells can help to increase yields and promote protein folding and assembly. Here, we expanded the ER in N. benthamiana leaves by targeting the enzyme CTP:phosphocholine cytidylyltransferase (CCT), which catalyses the rate-limiting step in the synthesis of the key membrane component phosphatidylcholine (PC). We used CRISPR/Cas to perform site-directed mutagenesis of each of the three endogenous CCT genes in N. benthamiana by introducing frame-shifting indels to remove the auto-inhibitory C-terminal domains. We generated stable homozygous lines of N. benthamiana containing different combinations of the edited genes, including plants where all three isofunctional CCT homologues were modified. Changes in ER morphology in the mutant plants were confirmed by in vivo confocal imaging and substantially increased the yields of two fully assembled SIgAs by prolonging the ER residence time and boosting chaperone accumulation. Through a combination of ER engineering with chaperone overexpression, we increased the yields of fully assembled SIgA by an order of magnitude, reaching almost 1 g/kg fresh leaf weight. This strategy removes a major roadblock to producing SIgA and will likely facilitate the production of other complex multimeric biopharmaceutical proteins in plants.},
}

@article {pmid39821831,
year = {2025},
author = {Xie, Y and Liu, X and Wu, T and Luo, Y},
title = {Harnessing the Streptomyces-originating type I-E CRISPR/Cas system for efficient genome editing in Streptomyces.},
journal = {Science China. Life sciences},
volume = {},
number = {},
pages = {},
pmid = {39821831},
issn = {1869-1889},
abstract = {Since their discovery, CRISPR/Cas systems have significantly expanded the genetic toolbox, aiding in the exploration and enhanced production of natural products across various microbes. Among these, class 2 CRISPR/Cas systems are simpler and more broadly used, but they frequently fail to function effectively in many Streptomyces strains. In this study, we present an engineered class 1 type I CRISPR/Cas system derived from Streptomyces avermitilis, which enables efficient gene editing in phylogenetically distant Streptomyces strains. Through a plasmid interference assay, we identified the effective protospacer adjacent motif as 5'-AAN-3'. Utilizing this system, we achieved targeted chromosomal deletions ranging from 8 bp to 100 kb, with efficiencies exceeding 92%. We further utilized this system to insert DNA fragments into different Streptomyces genomes, facilitating the heterologous expression of exogenous genes and the activation of endogenous natural product biosynthetic gene clusters. Overall, we established a type I CRISPR/Cas-based gene-editing methodology that significantly advances the exploration of Streptomyces, known for their rich natural product resources. This is the first report of a gene editing tool developed based on the endogenous class 1 type I CRISPR/Cas system in Streptomyces spp. Our work enriches the Streptomyces gene manipulation toolbox and advances the discovery of valuable natural products within these organisms.},
}

@article {pmid39821024,
year = {2025},
author = {Bu, W and Li, Y},
title = {Rat Models of Breast Cancer.},
journal = {Advances in experimental medicine and biology},
volume = {1464},
number = {},
pages = {123-148},
pmid = {39821024},
issn = {0065-2598},
mesh = {Animals ; Female ; Rats ; Humans ; *CRISPR-Cas Systems ; Breast Neoplasms/genetics/pathology/therapy ; Disease Models, Animal ; Mammary Neoplasms, Experimental/genetics/pathology ; Gene Editing/methods ; Mammary Glands, Animal/pathology/metabolism ; },
abstract = {As the first mammal to be domesticated for research purposes, rats served as the primary animal model for various branches of biomedical research, including breast cancer studies, up until the late 1990s and early 2000s. During this time, genetic engineering of mice, but not rats, became routine, and mice gradually supplanted rats as the preferred rodent model. But recent advances in creating genetically engineered rat models, especially with the assistance of CRISPR/Cas9 technology, have rekindled the significance of rats as a critical model in exploring various facets of breast cancer research. This is particularly pronounced in the study of the formation and progression of the estrogen receptor-positive subtype, which remains challenging to model in mice. In this chapter, we embark on a historical journey through the evolution of rat models in biomedical research and provide an overview of the general and histological characteristics of rat mammary glands. Next, we critically review major rat models for breast cancer research, including those induced by carcinogens, hormones, radiation, germline transgenes, germline knockouts, and intraductal injection of retrovirus/lentivirus to deliver oncogenic drivers into mature mammary glands. We also discuss the advances in building rat models using somatic genome editing powered by CRISPR/Cas9. This chapter concludes with our forward-looking perspective on future applications of advanced rat models in critical areas of breast cancer research that have continued to challenge the mouse model community.},
}

Animals
Female
Rats
Humans
*CRISPR-Cas Systems
Breast Neoplasms/genetics/pathology/therapy
Disease Models, Animal
Mammary Neoplasms, Experimental/genetics/pathology
Gene Editing/methods
Mammary Glands, Animal/pathology/metabolism

@article {pmid39820420,
year = {2025},
author = {Dong, Y and Zhu, J and Pan, N},
title = {Recent advances in rapid detection of Helicobacter pylori by lateral flow assay.},
journal = {Archives of microbiology},
volume = {207},
number = {2},
pages = {35},
pmid = {39820420},
issn = {1432-072X},
mesh = {*Helicobacter pylori/isolation & purification/genetics ; *Helicobacter Infections/diagnosis/microbiology ; Humans ; Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; Nucleic Acid Amplification Techniques/methods ; Point-of-Care Systems ; Enzyme-Linked Immunosorbent Assay/methods ; },
abstract = {Infection with H. pylori (Helicobacter pylori) is the most prevalent human infection worldwide and is strongly associated with many gastrointestinal disorders, including gastric cancer. Endoscopy is mainly used to diagnose H. pylori infection in gastric biopsies. However, this approach is invasive, time-consuming and expensive. On the other hand, serology-based methods can be considered as a non-invasive approach to detecting H. pylori infection. The LFA (lateral flow assay) serves as a rapid point-of-care diagnostic tool. This paper-based platform facilitates the detection and quantification of analytes within human fluids such as blood, serum and urine. Due to ease of production, rapid results, and low costs, LFAs have a wide application in clinical laboratories and hospitals. In this comprehensive review, we examined LFA-based approaches for detection of H. pylori infection from human fluids and compare them with other high-sensitivity methods like ELISA (Enzyme-linked immunosorbent assay). Furthermore, we reviewed methods to elevate LFA sensitivity during H. pylori infection including, CRISPR/Cas system and isothermal amplification approaches. The development and optimization of novel labeling agents such as nanozyme to enhance the performance of LFA devices in detecting H. pylori were reviewed. These innovations aim to improve signal amplification and stability, thereby increasing the diagnostic accuracy of LFA devices. A combination of advances in LFA technology and molecular insight could significantly improve diagnostic accuracy, resulting in a significant improvement in clinical and remote diagnostic accuracy.},
}

*Helicobacter pylori/isolation & purification/genetics
*Helicobacter Infections/diagnosis/microbiology
Humans
Molecular Diagnostic Techniques/methods
Sensitivity and Specificity
Nucleic Acid Amplification Techniques/methods
Point-of-Care Systems
Enzyme-Linked Immunosorbent Assay/methods

@article {pmid39819332,
year = {2025},
author = {Wang, C and Zhou, Y and Wang, Y and Jiao, P and Liu, S and Guan, S and Ma, Y},
title = {CRISPR-Cas9-mediated editing of ZmPL1 gene improves tolerance to drought stress in maize.},
journal = {GM crops & food},
volume = {16},
number = {1},
pages = {1-16},
doi = {10.1080/21645698.2024.2448869},
pmid = {39819332},
issn = {2164-5701},
mesh = {*Zea mays/genetics/physiology ; *Plants, Genetically Modified/genetics ; *Droughts ; *Plant Proteins/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Stress, Physiological/genetics ; Gene Expression Regulation, Plant ; Germination/genetics ; },
abstract = {Maize (Zea mays L.) is a widely grown food crop around the world. Drought stress seriously affects the growth and development process of plants and causes serious damage to maize yield. In the early stage, our research group conducted transcriptome sequencing analysis on the drought-resistant maize inbred line H8186 and screened out a gene with significantly down-regulated expression, Phylloplanin-like (ZmPL1). The ZmPL1 gee expression pattern was analyzed under various abiotic stresses, and the results showed that this gene was greatly affected by drought stress. Subcellular localization analysis showed that the protein was localized on the cell membrane. In order to verify the role of ZmPL1 in drought stress, we overexpressed ZmPL1 in yeast and found that the expression of ZmPL1 could significantly increase the drought sensitivity of yeast. Next, ZmPL1 transgenic plants were obtained by infecting maize callus using Agrobacterium-mediated method. Under drought stress, compared with overexpression lines, gene-edited lines had higher germination rate and seedling survival rate, lower accumulation of MDA, relative conductivity and ROS, higher antioxidant enzyme activity, and the expression levels of stress-related genes and ROS scavenging-related genes were significantly increased. Exogenous application of ABA to each lines under drought stress attenuated the damage caused by drought stress on ZmPL overexpressing plants. In summary, ZmPL1 negatively regulates drought tolerance in maize.},
}

*Zea mays/genetics/physiology
*Plants, Genetically Modified/genetics
*Droughts
*Plant Proteins/genetics/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems
Stress, Physiological/genetics
Gene Expression Regulation, Plant
Germination/genetics

@article {pmid39818894,
year = {2025},
author = {Kaiser, J},
title = {'Safe harbor' gene therapy approach may have first success.},
journal = {Science (New York, N.Y.)},
volume = {387},
number = {6731},
pages = {234-235},
doi = {10.1126/science.adv9833},
pmid = {39818894},
issn = {1095-9203},
mesh = {Humans ; *Genetic Therapy/methods ; *Gene Editing/methods ; Infant ; Liver ; CRISPR-Cas Systems ; Agammaglobulinemia ; Severe Combined Immunodeficiency ; },
abstract = {An obscure gene editor was used to restore a missing liver enzyme in an infant with a devastating metabolic condition.},
}

Humans
*Genetic Therapy/methods
*Gene Editing/methods
Infant
Liver
CRISPR-Cas Systems
Agammaglobulinemia
Severe Combined Immunodeficiency

@article {pmid39746849,
year = {2025},
author = {Paialunga, E and Bagheri, N and Rossetti, M and Fabiani, L and Micheli, L and Chamorro-Garcia, A and Porchetta, A},
title = {Leveraging Synthetic Antibody-DNA Conjugates to Expand the CRISPR-Cas12a Biosensing Toolbox.},
journal = {ACS synthetic biology},
volume = {14},
number = {1},
pages = {171-178},
doi = {10.1021/acssynbio.4c00541},
pmid = {39746849},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *DNA/genetics ; *SARS-CoV-2/genetics/immunology ; Humans ; *Spike Glycoprotein, Coronavirus/genetics/immunology ; Antibodies/immunology ; CRISPR-Associated Proteins/genetics ; COVID-19/diagnosis/virology/genetics ; Saliva ; Endodeoxyribonucleases/genetics/metabolism ; Immunoassay/methods ; Limit of Detection ; Enzyme-Linked Immunosorbent Assay/methods ; Bacterial Proteins/genetics ; },
abstract = {We report here the use of antibody-DNA conjugates (Ab-DNA) to activate the collateral cleavage activity of the CRISPR-Cas12a enzyme. Our findings demonstrate that Ab-DNA conjugates effectively trigger the collateral cleavage activity of CRISPR-Cas12a, enabling the transduction of antibody-mediated recognition events into fluorescence outputs. We developed two different immunoassays using an Ab-DNA as activator of Cas12a: the CRISPR-based immunosensing assay (CIA) for detecting SARS-CoV-2 spike S protein, which shows superior sensitivity compared with the traditional enzyme-linked immunosorbent assay (ELISA), and the CRISPR-based immunomagnetic assay (CIMA). Notably, CIMA successfully detected the SARS-CoV-2 spike S protein in undiluted saliva with a limit of detection (LOD) of 890 pM in a 2 h assay. Our results underscore the benefits of integrating Cas12a-based signal amplification with antibody detection methods. The potential of Ab-DNA conjugates, combined with CRISPR technology, offers a promising alternative to conventional enzymes used in immunoassays and could facilitate the development of versatile CRISPR analytical platforms for the detection of non-nucleic acid targets.},
}

*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
*DNA/genetics
*SARS-CoV-2/genetics/immunology
Humans
*Spike Glycoprotein, Coronavirus/genetics/immunology
Antibodies/immunology
CRISPR-Associated Proteins/genetics
COVID-19/diagnosis/virology/genetics
Saliva
Endodeoxyribonucleases/genetics/metabolism
Immunoassay/methods
Limit of Detection
Enzyme-Linked Immunosorbent Assay/methods
Bacterial Proteins/genetics

@article {pmid39623867,
year = {2025},
author = {Giorgetti, OB and Haas-Assenbaum, A and Boehm, T},
title = {Probing TCR Specificity Using Artificial In Vivo Diversification of CDR3 Regions.},
journal = {European journal of immunology},
volume = {55},
number = {1},
pages = {e202451434},
doi = {10.1002/eji.202451434},
pmid = {39623867},
issn = {1521-4141},
support = {//Max Planck Society/ ; 256073931-SFB1160//Deutsche Forschungsgemeinschaft/ ; },
mesh = {Animals ; *Complementarity Determining Regions/genetics/immunology ; Mice ; *Receptors, Antigen, T-Cell, alpha-beta/genetics/immunology ; CRISPR-Cas Systems ; T-Cell Antigen Receptor Specificity ; Mice, Transgenic ; T-Lymphocytes/immunology ; Receptors, Antigen, T-Cell/immunology/genetics ; },
abstract = {The T-cell receptor sequences expressed on cells recognizing a specific peptide in the context of a given MHC molecule can be explored for common features that might explain their antigen specificity. However, despite the development of numerous experimental and bioinformatic strategies, the specificity problem remains unresolved. To address the need for additional experimental paradigms, we report here on an in vivo experimental strategy designed to artificially diversify a transgenic TCR by CRISPR/Cas9-mediated mutagenesis of Tcra and Tcrb chain genes. In this system, an initially monoclonal repertoire of known specificity is converted into an oligoclonal pool of TCRs of altered antigen reactivity. Tracking the fate of individual clonotypes during the intrathymic differentiation process illuminates the strong selective pressures that shape the repertoire of naïve T cells. Sequence analyses of the artificially diversified repertoires identify key amino acid residues in the CDR3 regions required for antigen recognition, indicating that artificial diversification of well-characterized TCR transgene sequences helps to reduce the complexities of learning the rules of antigen recognition.},
}

Animals
*Complementarity Determining Regions/genetics/immunology
Mice
*Receptors, Antigen, T-Cell, alpha-beta/genetics/immunology
CRISPR-Cas Systems
T-Cell Antigen Receptor Specificity
Mice, Transgenic
T-Lymphocytes/immunology
Receptors, Antigen, T-Cell/immunology/genetics

@article {pmid39818188,
year = {2025},
author = {Liu, Z and Liu, Y and Zhang, S and Wen, Y and Wang, X and Han, J and Yin, H},
title = {Crystal structure of the anti-CRISPR protein AcrIE7.},
journal = {Biochemical and biophysical research communications},
volume = {748},
number = {},
pages = {151315},
doi = {10.1016/j.bbrc.2025.151315},
pmid = {39818188},
issn = {1090-2104},
abstract = {Bacterial adaptive immunity, driven by CRISPR-Cas systems, protects against foreign nucleic acids from mobile genetic elements (MGEs), like bacteriophages. The type I-E CRISPR-Cas system employs the Cascade (CRISPR-associated complex for antiviral defense) complex for target DNA cleavage, guided by crRNA. Anti-CRISPR (Acr) proteins, such as AcrIE7, counteract this defense by inhibiting Cascade activity. In this study, we characterized and determined the structure of AcrIE7, a unique member of the AcrIE family, using X-ray crystallography under two distinct crystallization conditions, achieving resolutions of 2.05 Å and 2.68 Å, respectively. Topological analysis revealed that AcrIE7 consists of seven α-helices with two distinct charge regions, likely mediating its inhibitory interactions. Structural flexibility analysis revealed notable structural stability differences between the two crystallization conditions, indicating varying rigidity of the AcrIE7 protein under different conditions. Homology searches and AlphaFold predictions reinforced the unique nature of AcrIE7, which exhibits a novel fold, underscoring its distinct role within the AcrIE family. These findings enhance our understanding of Acr proteins and provide a theoretical foundation for developing CRISPR-based gene-editing regulatory tools.},
}

@article {pmid39817514,
year = {2025},
author = {Jungfer, K and Moravčík, Š and Garcia-Doval, C and Knörlein, A and Hall, J and Jinek, M},
title = {Mechanistic determinants and dynamics of cA6 synthesis in type III CRISPR-Cas effector complexes.},
journal = {Nucleic acids research},
volume = {53},
number = {2},
pages = {},
doi = {10.1093/nar/gkae1277},
pmid = {39817514},
issn = {1362-4962},
support = {ERC-CoG-820152/ERC_/European Research Council/International ; 51NF40-182880//SNSF/ ; ETH-24-16-2//ETH Zurich/ ; //University of Zurich/ ; },
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/genetics ; Adenine Nucleotides/metabolism/biosynthesis ; Cryoelectron Microscopy ; Bacterial Proteins/metabolism/genetics ; Binding Sites ; Models, Molecular ; Second Messenger Systems ; },
abstract = {Type III clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems (type III CRISPR-Cas systems) use guide RNAs to recognize RNA transcripts of foreign genetic elements, which triggers the generation of cyclic oligoadenylate (cOA) second messengers by the Cas10 subunit of the type III effector complex. In turn, cOAs bind and activate ancillary effector proteins to reinforce the host immune response. Type III systems utilize distinct cOAs, including cyclic tri- (cA3), tetra- (cA4) and hexa-adenylates (cA6). However, the molecular mechanisms dictating cOA product identity are poorly understood. Here we used cryoelectron microscopy to visualize the mechanism of cA6 biosynthesis by the Csm effector complex from Enterococcus italicus (EiCsm). We show that EiCsm synthesizes oligoadenylate nucleotides in 3'-5' direction using a set of conserved binding sites in the Cas10 Palm domains to determine the size of the nascent oligoadenylate chain. Our data also reveal that conformational dynamics induced by target RNA binding results in allosteric activation of Cas10 to trigger oligoadenylate synthesis. Mutations of a key structural element in Cas10 perturb cOA synthesis to favor cA3 and cA4 formation. Together, these results provide comprehensive insights into the dynamics of cOA synthesis in type III CRISPR-Cas systems and reveal key determinants of second messenger product selectivity, thereby illuminating potential avenues for their engineering.},
}

*CRISPR-Cas Systems
*CRISPR-Associated Proteins/metabolism/genetics
Adenine Nucleotides/metabolism/biosynthesis
Cryoelectron Microscopy
Bacterial Proteins/metabolism/genetics
Binding Sites
Models, Molecular
Second Messenger Systems

@article {pmid39817512,
year = {2025},
author = {Zhang, L and Qiu, X and Zhou, Y and Luo, Z and Zhu, L and Shao, J and Xie, M and Wang, H},
title = {A trigger-inducible split-Csy4 architecture for programmable RNA modulation.},
journal = {Nucleic acids research},
volume = {53},
number = {2},
pages = {},
doi = {10.1093/nar/gkae1319},
pmid = {39817512},
issn = {1362-4962},
support = {2023YFF1205400//Ministry of Science and Technology/ ; 32371498//National Natural Science Foundation of China/ ; 202209009//Westlake Laboratory of Life Sciences and Biomedicine/ ; 2024SSYS0007//'Pioneer' and 'Leading Goose' R&D Program of Zhejiang/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; HEK293 Cells ; RNA/metabolism/chemistry/genetics ; Endoribonucleases/metabolism/genetics ; Transcriptome ; },
abstract = {The CRISPR-derived endoribonuclease Csy4 is a popular tool for controlling transgene expression in various therapeutically relevant settings, but adverse effects potentially arising from non-specific RNA cleavage remains largely unexplored. Here, we report a split-Csy4 architecture that was carefully optimized for in vivo usage. First, we separated Csy4 into two independent protein moieties whose full catalytic activity can be restored via various constitutive or conditional protein dimerization systems. Next, we show that introduction of split-Csy4 into human cells caused a substantially reduced extent in perturbation of the endogenous transcriptome when directly compared to full-length Csy4. Inspired by these results, we went on to use such split-Csy4 module to engineer inducible CRISPR- and translation-level gene switches regulated by the FDA-approved drug grazoprevir. This work provides valuable resource for Csy4-related biomedical research and discusses important issues for the development of clinically eligible regulation tools.},
}

Humans
*CRISPR-Cas Systems
HEK293 Cells
RNA/metabolism/chemistry/genetics
Endoribonucleases/metabolism/genetics
Transcriptome

@article {pmid39817421,
year = {2025},
author = {Debaenst, S and Jarayseh, T and De Saffel, H and Bek, JW and Boone, M and Josipovic, I and Kibleur, P and Kwon, RY and Coucke, PJ and Willaert, A},
title = {Crispant analysis in zebrafish as a tool for rapid functional screening of disease-causing genes for bone fragility.},
journal = {eLife},
volume = {13},
number = {},
pages = {},
pmid = {39817421},
issn = {2050-084X},
support = {FWO.OPR.2020.0023.01//Fonds Wetenschappelijk Onderzoek/ ; },
mesh = {*Zebrafish/genetics ; Animals ; Disease Models, Animal ; Bone Density/genetics ; Osteogenesis Imperfecta/genetics ; CRISPR-Cas Systems ; Phenotype ; Zebrafish Proteins/genetics/metabolism ; High-Throughput Nucleotide Sequencing ; },
abstract = {Heritable fragile bone disorders (FBDs), ranging from multifactorial to rare monogenic conditions, are characterized by an elevated fracture risk. Validating causative genes and understanding their mechanisms remain challenging. We assessed a semi-high throughput zebrafish screening platform for rapid in vivo functional testing of candidate FBD genes. Six genes linked to severe recessive osteogenesis imperfecta (OI) and four associated with bone mineral density (BMD) from genome-wide association studies were analyzed. Using CRISPR/Cas9-based crispant screening in F0 mosaic founder zebrafish, Next-generation sequencing confirmed high indel efficiency (mean 88%), mimicking stable knock-out models. Skeletal phenotyping at 7, 14, and 90 days post-fertilization (dpf) using microscopy, Alizarin Red S staining, and microCT was performed. Larval crispants showed variable osteoblast and mineralization phenotypes, while adult crispants displayed consistent skeletal defects, including malformed neural and haemal arches, vertebral fractures and fusions, and altered bone volume and density. In addition, aldh7a1 and mbtps2 crispants experienced increased mortality due to severe skeletal deformities. RT-qPCR revealed differential expression of osteogenic markers bglap and col1a1a, highlighting their biomarker potential. Our results establish zebrafish crispant screening as a robust tool for FBD gene validation, combining skeletal and molecular analyses across developmental stages to uncover novel insights into gene functions in bone biology.},
}

*Zebrafish/genetics
Animals
Disease Models, Animal
Bone Density/genetics
Osteogenesis Imperfecta/genetics
CRISPR-Cas Systems
Phenotype
Zebrafish Proteins/genetics/metabolism
High-Throughput Nucleotide Sequencing

@article {pmid39817182,
year = {2024},
author = {Xue, M and Gonzalez, DH and Osikpa, E and Gao, X and Lillehoj, PB},
title = {Rapid and automated interpretation of CRISPR-Cas13-based lateral flow assay test results using machine learning.},
journal = {Sensors & diagnostics},
volume = {},
number = {},
pages = {},
pmid = {39817182},
issn = {2635-0998},
abstract = {CRISPR-Cas-based lateral flow assays (LFAs) have emerged as a promising diagnostic tool for ultrasensitive detection of nucleic acids, offering improved speed, simplicity and cost-effectiveness compared to polymerase chain reaction (PCR)-based assays. However, visual interpretation of CRISPR-Cas-based LFA test results is prone to human error, potentially leading to false-positive or false-negative outcomes when analyzing test/control lines. To address this limitation, we have developed two neural network models: one based on a fully convolutional neural network and the other on a lightweight mobile-optimized neural network for automated interpretation of CRISPR-Cas-based LFA test results. To demonstrate proof of concept, these models were applied to interpret results from a CRISPR-Cas13-based LFA for the detection of the SARS-CoV-2 N gene, a key marker for COVID-19 infection. The models were trained, evaluated, and validated using smartphone-captured images of LFA devices in various orientations with different backgrounds, lighting conditions, and image qualities. A total of 3146 images (1569 negative, 1577 positive) captured using an iPhone 13 or Samsung Galaxy A52 Android smartphone were analyzed using the trained models, which classified the LFA results within 0.2 s with 96.5% accuracy compared to the ground truth. These results demonstrate the potential of machine learning to accurately interpret test results of CRISPR-Cas-based LFAs using smartphone-captured images in real-world settings, enabling the practical use of CRISPR-Cas-based diagnostic tools for self- and at-home testing.},
}

@article {pmid39816115,
year = {2024},
author = {Syahrani, RA and Wanandi, SI and Arumsari, S and Nihayah, S and Watanabe, Y and Mizuno, S and Louisa, M and Wuyung, PE},
title = {Dual sgRNA-directed knockout survivin gene expression using CRISPR/Cas9 technology for editing survivin gene in triple-negative breast cancer.},
journal = {Narra J},
volume = {4},
number = {3},
pages = {e1177},
pmid = {39816115},
issn = {2807-2618},
mesh = {*Survivin/genetics/metabolism ; Humans ; *CRISPR-Cas Systems/genetics ; *Triple Negative Breast Neoplasms/genetics/therapy ; Cell Line, Tumor ; Female ; *Gene Editing/methods ; *Gene Knockout Techniques/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease 9 (CRISPR/Cas9) offers a robust approach for genome manipulation, particularly in cancer therapy. Given its high expression in triple-negative breast cancer (TNBC), targeting survivin with CRISPR/Cas9 holds promise as a therapeutic strategy. The aim of this study was to design specific single guide ribonucleic acid (sgRNA) for CRISPR/Cas9 to permanently knock out the survivin gene, exploring its potential as a therapeutic approach in breast cancer while addressing potential off-target effects. Survivin gene knockout was conducted in the TNBC cell line BT549. Intron 1, exon 2, and intron 2 of the survivin gene were selected as sgRNA targets. These sgRNAs were designed in silico and then cloned into a CRISPR/Cas9 expression plasmid. The cleavage activity was assessed using an enhanced green fluorescent protein (EGFP) expression plasmid. The sgRNAs with higher cleavage activity were selected for the establishment of knockout cells. After transfecting the plasmid into the cells, the success of the survivin gene knockout was validated at the deoxyribonucleic acid (DNA) level using polymerase chain reaction (PCR) and sequencing analysis, and at the protein expression level using Western blotting. The study found that sgRNAs survin1A (targeting intron 1), survex2A (targeting intron 2), and survin2A (targeting intron 2) demonstrated higher cleavage activities compared to the other sgRNAs. However, using the single sgRNA, survex2A did not generate mutations in the survivin gene. At the protein level, survivin was still expressed, indicating that a single sgRNA was ineffective in knocking out the survivin gene. In contrast, the combination of sgRNA survin1A and sgRNA survin2A was more effective in generating mutations in the survivin gene, resulting in the deletion of the entire exon 2 and leading to a loss of survivin protein expression. In conclusion, our work provides specific sgRNAs and demonstrates the utilization of dual sgRNAs strategy in the CRISPR/Cas9 technology to knock out the survivin gene, showing potential in breast cancer therapy.},
}

*Survivin/genetics/metabolism
Humans
*CRISPR-Cas Systems/genetics
*Triple Negative Breast Neoplasms/genetics/therapy
Cell Line, Tumor
Female
*Gene Editing/methods
*Gene Knockout Techniques/methods
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid39778581,
year = {2025},
author = {Lin, DW and Zhang, L and Zhang, J and Chandrasekaran, S},
title = {Inferring metabolic objectives and trade-offs in single cells during embryogenesis.},
journal = {Cell systems},
volume = {16},
number = {1},
pages = {101164},
pmid = {39778581},
issn = {2405-4720},
mesh = {*Embryonic Development/physiology/genetics ; Animals ; *Single-Cell Analysis/methods ; Mice ; Embryonic Stem Cells/metabolism ; Machine Learning ; CRISPR-Cas Systems ; Cell Proliferation/physiology/genetics ; Humans ; Cell Cycle/physiology ; },
abstract = {While proliferating cells optimize their metabolism to produce biomass, the metabolic objectives of cells that perform non-proliferative tasks are unclear. The opposing requirements for optimizing each objective result in a trade-off that forces single cells to prioritize their metabolic needs and optimally allocate limited resources. Here, we present single-cell optimization objective and trade-off inference (SCOOTI), which infers metabolic objectives and trade-offs in biological systems by integrating bulk and single-cell omics data, using metabolic modeling and machine learning. We validated SCOOTI by identifying essential genes from CRISPR-Cas9 screens in embryonic stem cells, and by inferring the metabolic objectives of quiescent cells, during different cell-cycle phases. Applying this to embryonic cell states, we observed a decrease in metabolic entropy upon development. We further uncovered a trade-off between glutathione and biosynthetic precursors in one-cell zygote, two-cell embryo, and blastocyst cells, potentially representing a trade-off between pluripotency and proliferation. A record of this paper's transparent peer review process is included in the supplemental information.},
}

*Embryonic Development/physiology/genetics
Animals
*Single-Cell Analysis/methods
Mice
Embryonic Stem Cells/metabolism
Machine Learning
CRISPR-Cas Systems
Cell Proliferation/physiology/genetics
Humans
Cell Cycle/physiology

@article {pmid39689711,
year = {2025},
author = {Metzner, E and Southard, KM and Norman, TM},
title = {Multiome Perturb-seq unlocks scalable discovery of integrated perturbation effects on the transcriptome and epigenome.},
journal = {Cell systems},
volume = {16},
number = {1},
pages = {101161},
pmid = {39689711},
issn = {2405-4720},
mesh = {Humans ; *Transcriptome/genetics ; *Epigenome/genetics ; Transcription Factors/genetics/metabolism ; Chromatin/genetics ; Polycomb Repressive Complex 2/genetics/metabolism ; DNA-Binding Proteins/genetics/metabolism ; Single-Cell Analysis/methods ; CRISPR-Cas Systems/genetics ; Cell Line ; Neoplasm Proteins/genetics ; Chromatin Assembly and Disassembly/genetics ; },
abstract = {Single-cell CRISPR screens link genetic perturbations to transcriptional states, but high-throughput methods connecting these induced changes to their regulatory foundations are limited. Here, we introduce Multiome Perturb-seq, extending single-cell CRISPR screens to simultaneously measure perturbation-induced changes in gene expression and chromatin accessibility. We apply Multiome Perturb-seq in a CRISPRi screen of 13 chromatin remodelers in human RPE-1 cells, achieving efficient assignment of sgRNA identities to single nuclei via an improved method for capturing barcode transcripts from nuclear RNA. We organize expression and accessibility measurements into coherent programs describing the integrated effects of perturbations on cell state, finding that ARID1A and SUZ12 knockdowns induce programs enriched for developmental features. Modeling of perturbation-induced heterogeneity connects accessibility changes to changes in gene expression, highlighting the value of multimodal profiling. Overall, our method provides a scalable and simply implemented system to dissect the regulatory logic underpinning cell state. A record of this paper's transparent peer review process is included in the supplemental information.},
}

Humans
*Transcriptome/genetics
*Epigenome/genetics
Transcription Factors/genetics/metabolism
Chromatin/genetics
Polycomb Repressive Complex 2/genetics/metabolism
DNA-Binding Proteins/genetics/metabolism
Single-Cell Analysis/methods
CRISPR-Cas Systems/genetics
Cell Line
Neoplasm Proteins/genetics
Chromatin Assembly and Disassembly/genetics

@article {pmid39528429,
year = {2025},
author = {Ding, S and Zheng, J and Jia, C},
title = {DeepMEns: an ensemble model for predicting sgRNA on-target activity based on multiple features.},
journal = {Briefings in functional genomics},
volume = {24},
number = {},
pages = {},
pmid = {39528429},
issn = {2041-2657},
support = {62071079//National Natural Science Foundation of China/ ; 3132024197//Fundamental Research Funds for the Central Universities/ ; JSKX202203//Hainan Normal University, Ministry of Education/ ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems/genetics ; Neural Networks, Computer ; Deep Learning ; Gene Editing/methods ; Streptococcus pyogenes/genetics ; Humans ; },
abstract = {The CRISPR/Cas9 system developed from Streptococcus pyogenes (SpCas9) has high potential in gene editing. However, its successful application is hindered by the considerable variability in target efficiencies across different single guide RNAs (sgRNAs). Although several deep learning models have been created to predict sgRNA on-target activity, the intrinsic mechanisms of these models are difficult to explain, and there is still scope for improvement in prediction performance. To overcome these issues, we propose an ensemble interpretable model termed DeepMEns based on deep learning to predict sgRNA on-target activity. By using five different training and validation datasets, we constructed five sub-regressors, each comprising three parts. The first part uses one-hot encoding, wherein 0-1 representation of the secondary structure is used as the input to the convolutional neural network (CNN) with Transformer encoder. The second part uses the DNA shape feature matrix as the input to the CNN with Transformer encoder. The third part uses positional encoding feature matrices as the proposed input into a long short-term memory network with an attention mechanism. These three parts are concatenated through the flattened layer, and the final prediction result is the average of the five sub-regressors. Extensive benchmarking experiments indicated that DeepMEns achieved the highest Spearman correlation coefficient for 6 of 10 independent test datasets as compared to previous predictors, this finding confirmed that DeepMEns can accomplish state-of-the-art performance. Moreover, the ablation analysis also indicated that the ensemble strategy may improve the performance of the prediction model.},
}

*RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Cas Systems/genetics
Neural Networks, Computer
Deep Learning
Gene Editing/methods
Streptococcus pyogenes/genetics
Humans

@article {pmid39814846,
year = {2025},
author = {Shin, SW and Kim, SH and Gasselin, A and Lee, GM and Lee, JS},
title = {Comprehensive genome-scale CRISPR knockout screening of CHO cells.},
journal = {Scientific data},
volume = {12},
number = {1},
pages = {71},
pmid = {39814846},
issn = {2052-4463},
support = {RS-2024-00397714//National Research Foundation of Korea (NRF)/ ; RS-2024-00397714//National Research Foundation of Korea (NRF)/ ; RS-2024-00397714//National Research Foundation of Korea (NRF)/ ; RS-2024-00397714//National Research Foundation of Korea (NRF)/ ; },
mesh = {CHO Cells ; Animals ; *Cricetulus ; *CRISPR-Cas Systems ; Gene Knockout Techniques ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome ; High-Throughput Nucleotide Sequencing ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Chinese hamster ovary (CHO) cells play a pivotal role in the production of recombinant therapeutics. In the present study, we conducted a genome-scale pooled CRISPR knockout (KO) screening using a virus-free, recombinase-mediated cassette exchange-based platform in CHO-K1 host and CHO-K1 derived recombinant cells. Genome-wide guide RNA (gRNA) amplicon sequencing data were generated from cell libraries, as well as short- and long-term KO libraries, and validated through phenotypic assessment and gRNA read count distribution. Additionally, we obtained gRNA amplicon sequencing data from the highly productive recombinant cell populations. By analyzing these datasets, essential genes involved in cell fitness as well as functional target genes associated with therapeutic protein production can be identified. Collectively, our next-generation sequencing datasets, derived from a robust and reliable CRISPR screening method, provide valuable insights into CHO genomic functions, advancing the development of next-generation CHO factories.},
}

CHO Cells
Animals
*Cricetulus
*CRISPR-Cas Systems
Gene Knockout Techniques
RNA, Guide, CRISPR-Cas Systems/genetics
Genome
High-Throughput Nucleotide Sequencing
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid39812375,
year = {2025},
author = {Wang, C and Xu, X and Yao, W and Wang, L and Pang, X and Xu, S and Luo, X},
title = {Programmable DNA Nanoswitch-Regulated Plasmonic CRISPR/Cas12a-Gold Nanostars Reporter Platform for Nucleic Acid and Non-Nucleic Acid Biomarker Analysis Assisted by a Spatial Confinement Effect.},
journal = {Nano letters},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.nanolett.4c05829},
pmid = {39812375},
issn = {1530-6992},
abstract = {CRISPR/Cas 12a system based nucleic acid and non-nucleic acid targets detection faces two challenges including (1) multiple crRNAs are needed for multiple biomarkers detection and (2) insufficient sensitivity resulted from photobleaching of fluorescent dyes and the low kinetic cleavage rate for a traditional single-strand (ssDNA) reporter. To address these limitations, we developed a programmable DNA nanoswitch (NS)-regulated plasmonic CRISPR/Cas12a-gold nanostars (Au NSTs) reporter platform for detection of nucleic acid and non-nucleic acid biomarkers with the assistance of the spatial confinement effect. Through simply programming the target recognition sequence in NS, only one crRNA is required to detect both nucleic acid and non-nucleic acid biomarkers. The detection limit decreased by ∼196-fold for miRNA-375 and 122-fold for prostate-specific antigen (PSA), respectively. Moreover, versatile evaluation of miRNA-375 and PSA in clinical urine samples can also be achieved, according to which prostate cancer and healthy groups can be well identified.},
}

@article {pmid39810563,
year = {2025},
author = {Wang, W and Du, H and Dai, C and Ma, H and Luo, S and Wang, X and Guo, M and Kong, D and Wei, D},
title = {Amplification-free detection of Mycobacterium tuberculosis using CRISPR-Cas12a and graphene field-effect transistors.},
journal = {Nanoscale},
volume = {},
number = {},
pages = {},
doi = {10.1039/d4nr03852e},
pmid = {39810563},
issn = {2040-3372},
abstract = {Current molecular tests for tuberculosis (TB), such as whole genome sequencing and Xpert Mycobacterium tuberculosis/rifampicin resistance assay, exhibit limited sensitivity and necessitate the pre-amplification step of target DNA. This limitation greatly increases detection time and poses an increased risk of infection. Here, we present a graphene field-effect transistor (GFET) based on the CRISPR/Cas system for detecting Mycobacterium tuberculosis. The CRISPR/Cas12a system has the ability to specifically recognize and cleave target DNA. By integrating the system onto the FET platform and utilizing its electrical amplification capability, we achieve rapid and sensitive detection without requiring sample pre-amplification, with a limit of detection (LoD) as low as 2.42 × 10[-18] M. Cas12a-GFET devices can differentiate 30 positive cases from 56 serum samples within 5 minutes. These findings highlight its immense potential in future biological analysis and clinical diagnosis.},
}

@article {pmid39774325,
year = {2025},
author = {Funk, JS and Klimovich, M and Drangenstein, D and Pielhoop, O and Hunold, P and Borowek, A and Noeparast, M and Pavlakis, E and Neumann, M and Balourdas, DI and Kochhan, K and Merle, N and Bullwinkel, I and Wanzel, M and Elmshäuser, S and Teply-Szymanski, J and Nist, A and Procida, T and Bartkuhn, M and Humpert, K and Mernberger, M and Savai, R and Soussi, T and Joerger, AC and Stiewe, T},
title = {Deep CRISPR mutagenesis characterizes the functional diversity of TP53 mutations.},
journal = {Nature genetics},
volume = {57},
number = {1},
pages = {140-153},
pmid = {39774325},
issn = {1546-1718},
support = {STI 182/15-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; STI 182/13-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; GRK2573//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 109546710 Project A10//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; SA 1923/7-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CRC1213 A10N//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; JO 1473/1-3//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 031L0063//Bundesministerium für Bildung und Forschung (Federal Ministry of Education and Research)/ ; DZL//Bundesministerium für Bildung und Forschung (Federal Ministry of Education and Research)/ ; LOEWE iCANx//Hessisches Ministerium für Wissenschaft und Kunst (Hessen State Ministry of Higher Education, Research and the Arts)/ ; 65-0004//Von-Behring-Röntgen-Stiftung (Von Behring-Röntgen-Foundation)/ ; 66-LV06//Von-Behring-Röntgen-Stiftung (Von Behring-Röntgen-Foundation)/ ; 71-0012//Von-Behring-Röntgen-Stiftung (Von Behring-Röntgen-Foundation)/ ; 09 R/2018//José Carreras Leukämie-Stiftung (Deutsche José Carreras Leukämie-Stiftung)/ ; TACTIC//Deutsche Krebshilfe (German Cancer Aid)/ ; },
mesh = {Humans ; *Tumor Suppressor Protein p53/genetics ; *Gene Editing/methods ; *Mutagenesis ; *CRISPR-Cas Systems ; *Neoplasms/genetics ; Mutation, Missense/genetics ; Cell Line, Tumor ; Mutation ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Nonsense Mediated mRNA Decay/genetics ; },
abstract = {The mutational landscape of TP53, a tumor suppressor mutated in about half of all cancers, includes over 2,000 known missense mutations. To fully leverage TP53 mutation status for personalized medicine, a thorough understanding of the functional diversity of these mutations is essential. We conducted a deep mutational scan using saturation genome editing with CRISPR-mediated homology-directed repair to engineer 9,225 TP53 variants in cancer cells. This high-resolution approach, covering 94.5% of all cancer-associated TP53 missense mutations, precisely mapped the impact of individual mutations on tumor cell fitness, surpassing previous deep mutational scan studies in distinguishing benign from pathogenic variants. Our results revealed even subtle loss-of-function phenotypes and identified promising mutants for pharmacological reactivation. Moreover, we uncovered the roles of splicing alterations and nonsense-mediated messenger RNA decay in mutation-driven TP53 dysfunction. These findings underscore the power of saturation genome editing in advancing clinical TP53 variant interpretation for genetic counseling and personalized cancer therapy.},
}

Humans
*Tumor Suppressor Protein p53/genetics
*Gene Editing/methods
*Mutagenesis
*CRISPR-Cas Systems
*Neoplasms/genetics
Mutation, Missense/genetics
Cell Line, Tumor
Mutation
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Nonsense Mediated mRNA Decay/genetics

@article {pmid39754554,
year = {2025},
author = {Hu, F and Liu, K and Zhang, Y and Zhao, S and Zhang, T and Yao, C and Lv, X and Wang, J and Liu, X and Peng, N},
title = {Short-Time Preamplification-Assisted One-Pot CRISPR Nucleic Acid Detection Method with Portable Self-Heating Equipment for Point-of-Care Diagnosis.},
journal = {Analytical chemistry},
volume = {97},
number = {1},
pages = {658-666},
doi = {10.1021/acs.analchem.4c05026},
pmid = {39754554},
issn = {1520-6882},
mesh = {*SARS-CoV-2/genetics/isolation & purification ; Humans ; *Nucleic Acid Amplification Techniques ; *COVID-19/diagnosis/virology ; *RNA, Viral/analysis/genetics ; *CRISPR-Cas Systems/genetics ; Point-of-Care Testing ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Point-of-Care Systems ; COVID-19 Nucleic Acid Testing/methods/instrumentation ; Limit of Detection ; },
abstract = {Infectious diseases, especially respiratory infections, have been significant threats to human health. Therefore, it is essential to develop rapid, portable, and highly sensitive diagnostic methods for their control. Herein, a short-time preamplified, one-pot clustered regularly interspaced short palindromic repeats (CRISPR) nucleic acid detection method (SPOC) is developed by combining the rapid recombinase polymerase amplification (RPA) with CRISPR-Cas12a to reduce the mutual interference and achieve facile and rapid molecular diagnosis. SPOC can reduce the detection time and stably detect up to 1 copy/μL of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA without affecting the detection sensitivity. A highly sensitive one-pot assay integrated with reverse transcription RPA is achieved by wrapping paraffin with a specific melting point on the lyophilized CRISPR reagent surface. A self-heating pack is designed based on thermodynamic principles to melt the paraffin and release CRISPR reagents, enabling low-cost and time-saving detection. Notably, the designed system, coupled with RNA extraction-free technology, can achieve "sample-in-answer-out" detection of the SARS-CoV-2 Orf1ab gene within 22 min using smartphone imaging. The developed assay is validated on 12 clinical samples, and the results 100% correlate with real-time polymerase chain reaction. SPOC is time-saving, is easy to operate, and can eliminate centrifugal and complex hardware devices, satisfying the demand for point-of-care diagnostics in resource-constrained settings.},
}

*SARS-CoV-2/genetics/isolation & purification
Humans
*Nucleic Acid Amplification Techniques
*COVID-19/diagnosis/virology
*RNA, Viral/analysis/genetics
*CRISPR-Cas Systems/genetics
Point-of-Care Testing
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Point-of-Care Systems
COVID-19 Nucleic Acid Testing/methods/instrumentation
Limit of Detection

@article {pmid39725131,
year = {2025},
author = {Compiro, P and Chomta, N and Nimnual, J and Sunantawanit, S and Payungporn, S and Rotcheewaphan, S and Keawsapsak, P},
title = {CRISPR-Cas12a-based detection and differentiation of Mycobacterium spp.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {567},
number = {},
pages = {120101},
doi = {10.1016/j.cca.2024.120101},
pmid = {39725131},
issn = {1873-3492},
mesh = {*Mycobacterium/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; Humans ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Mycobacterium species cause several vital human diseases, including tuberculosis and non-tuberculous mycobacterial infections, which are treated with different drug regimens Therefore, accurate and rapid diagnosis is essential for effective treatment and controlling the spread of these pathogens. This study aims to develop an isothermal method combining RPA and CRISPR-Cas12a techniques, named as MyTRACK, to detect and differentiate major clinical mycobacteria at the species level. The assay has no cross-reactivity with limit of detection of 1 to 100 copies/reaction for various targeted mycobacteria. The results demonstrated 100 % specificity and 92.59 % to 100 % sensitivity in clinical isolates and were consistent with the culture technique with LPA for clinical samples. The MyTRACK assay is an effective, portable, rapid, and accurate screening method for mycobacterial detection and identification, especially in low-resource clinical settings.},
}

*Mycobacterium/genetics/isolation & purification
*CRISPR-Cas Systems/genetics
Humans
Nucleic Acid Amplification Techniques/methods

@article {pmid39720925,
year = {2025},
author = {Xu, Y and Chen, B and He, M and Yuan, G and Hu, B},
title = {Dual-Amplification Single-Particle ICP-MS Strategy Based on Strand Displacement Amplification-CRISPR/Cas12a Amplification for Homogeneous Detection of miRNA.},
journal = {Analytical chemistry},
volume = {97},
number = {1},
pages = {811-817},
doi = {10.1021/acs.analchem.4c05385},
pmid = {39720925},
issn = {1520-6882},
mesh = {*MicroRNAs/analysis/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Gold/chemistry ; *Nucleic Acid Amplification Techniques/methods ; *Metal Nanoparticles/chemistry ; *Mass Spectrometry ; MCF-7 Cells ; Limit of Detection ; },
abstract = {MicroRNAs (miRNAs) regulate a myriad of biological processes and thus have been regarded as useful biomarkers in biomedical research and clinical diagnosis. The specific and highly sensitive detection of miRNAs is of significant importance. Herein, a sensitive and rapid dual-amplification elemental labeling single-particle inductively coupled plasma-mass spectrometry (spICP-MS) analytical method based on strand displacement amplification (SDA) and CRISPR/Cas12a was developed for miRNA-21 detection. Taking gold nanoparticles (AuNPs) as the elemental labels, the Au NP probe initially hybridized with linker DNA, forming large aggregates. In the absence of target miRNA-21, large aggregates of AuNPs will produce high pulse signals in spICP-MS detection. In the presence of the target miRNA-21, it triggered the SDA reaction, and the SDA products activated CRISPR/Cas12a's trans-cleavage activity to cleave the linker DNA, resulting in disassembly of the AuNP aggregates. The AuNP aggregates with smaller size displayed lower pulse signals in spICP-MS detection. Under the optimal conditions, a good relationship between the average pulse signal intensity of AuNP aggregates and the concentration of miRNA-21 was obtained in the range of 0.5 fmol L[-1]-100 pmol L[-1] with a quantification limit as low as 0.5 fmol L[-1]. The developed method was successfully used for determination of miRNA-21 in human breast cancer cell lines (SK-BR-3 and MCF-7) and real blood samples from breast cancer patients. It is versatile, can be adapted to detect other targets by modifying the specific sequence of the SDA template chain that is complementary to the analytes, and offers a promising strategy for detecting various biomarkers with high sensitivity and specificity.},
}

*MicroRNAs/analysis/genetics
Humans
*CRISPR-Cas Systems/genetics
*Gold/chemistry
*Nucleic Acid Amplification Techniques/methods
*Metal Nanoparticles/chemistry
*Mass Spectrometry
MCF-7 Cells
Limit of Detection

@article {pmid39716527,
year = {2025},
author = {Liu, R and Ji, W and Jiang, M and Shen, J},
title = {CRISPR technology combined with isothermal amplification methods for the diagnosis of Candida albicans infection.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {567},
number = {},
pages = {120106},
doi = {10.1016/j.cca.2024.120106},
pmid = {39716527},
issn = {1873-3492},
mesh = {*Candida albicans/genetics/isolation & purification ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *Candidiasis/diagnosis/microbiology ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Since Candida albicans, a type of fungus, causes severe infections that pose a significant threat to human health, its rapid detection is critical in clinical antifungal therapy. Traditional fungal diagnostic approaches are largely based on the culture method. This method is time-consuming and laborious, taking about 48-72 h, and cannot identify emerging species, making it unsuitable for critically ill patients with bloodstream infections, sepsis, and so on. Other antigen or nucleic acid amplification-based methods were also found to be unsuitable for Point-of-Care Testing (POCT) diagnosis due to various limitations. Therefore, establishing a new approach for the rapid diagnosis of Candida spp is imperative. Herein, we proposed a novel diagnostic method for invasive fungi detection. Specifically, we created a new CRISPR diagnostic platform for Candida albicans-specific Internal Transcriptional Spacer 2 (ITS2) gene by combining the DNase cleavage activity of Cas12a with Recombinase Polymerase Amplification (RPA). Furthermore, to achieve rapid on-site detection under low-resource conditions, we used a transverse lateral flow strip with a single target to visualize the Cas12a single enzyme digestion product. We designated the platform as a rapid molecular detection tool that integrates RPA and the CRISPR-Cas12a technology. The entire platform can accurately identify Candida albicans within 50 minwhile remaining unaffected by other fungi or bacteria. Furthermore, the detection limit of the platform could reach 10[2] CFU/ml. Moreover, this approach offers additional benefits, including easy operation, low set-up cost, and broad applicability for Candida albicans detection across medical institutions at all levels, especially in township health centers in resource-poor regions.},
}

*Candida albicans/genetics/isolation & purification
Humans
*Nucleic Acid Amplification Techniques/methods
*Candidiasis/diagnosis/microbiology
CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid39711046,
year = {2025},
author = {Yu, H and Xu, PF and Liu, Y and Jia, ZS and Li, YY and Tang, HW},
title = {LRET-Based Simultaneous Detection of Dual miRNAs via Multitrap Optical Tweezers Assisted Suspension Array Tagged by Two Different Luminescent Quenchable UCNPs Combining CRISPR/Cas12a Amplification.},
journal = {Analytical chemistry},
volume = {97},
number = {1},
pages = {602-612},
doi = {10.1021/acs.analchem.4c04895},
pmid = {39711046},
issn = {1520-6882},
mesh = {*MicroRNAs/analysis/genetics ; Humans ; *Optical Tweezers ; *CRISPR-Cas Systems/genetics ; Nanoparticles/chemistry ; Fluorescence Resonance Energy Transfer ; Luminescence ; Nucleic Acid Amplification Techniques ; Limit of Detection ; MCF-7 Cells ; },
abstract = {Nowadays, optical tweezers play a vital role not only in optical manipulation but also in bioassay. As principal optical trapping objects, microbeads can combine optical tweezers with suspension array technology, with amply focused laser beams and adequately concentrated tags contributing to highly sensitive detection. In view of the inefficiency of conventional single-trap optical tweezers, multitrap systems are developed. Here, green- and blue-emitting core-shell-shell upconversion nanoparticles (UCNPs) are adopted to encode microbeads and determine dual miRNAs, with the internal shells leading the luminescence process to facilitate quenching through luminescence resonance energy transfer (LRET). Utilizing the trans cleavage of CRISPR/Cas12a, quenched luminescence signals are recovered and amplified, causing further enhanced detection sensitivity. Ultimately, limits of detection (LOD) of 17 and 22 aM are obtained with excellent specificities verified. Furthermore, dual miRNAs from MCF-7, A549, and MCF-10A cells are extracted and detected, with results consistent with those obtained by PCR. Notably, miR-155 in MCF-7 and A549 cells is detectable at the single-cell level. Thus, the differences in the measured miRNA levels between MCF-7 and MCF-10A cells imply the potential of this method to discriminate breast cancer cells from epithelial cells despite the difficulty in distinguishing different cancer cells due to similar miRNA levels.},
}

*MicroRNAs/analysis/genetics
Humans
*Optical Tweezers
*CRISPR-Cas Systems/genetics
Nanoparticles/chemistry
Fluorescence Resonance Energy Transfer
Luminescence
Nucleic Acid Amplification Techniques
Limit of Detection
MCF-7 Cells

@article {pmid39704707,
year = {2025},
author = {Deng, R and Sheng, J and Xie, Z and Yang, H and Yang, S and Xie, S and Tang, X and Zhao, S and Dong, H and Chen, M and Chang, K},
title = {miR-Cabiner: A Universal microRNA Sensing Platform Based on Self-Stacking Cascaded Bicyclic DNA Circuit-Mediated CRISPR/Cas12a.},
journal = {Analytical chemistry},
volume = {97},
number = {1},
pages = {799-810},
doi = {10.1021/acs.analchem.4c05370},
pmid = {39704707},
issn = {1520-6882},
mesh = {*MicroRNAs/analysis ; Humans ; *CRISPR-Cas Systems/genetics ; *DNA/chemistry ; Biosensing Techniques/methods ; },
abstract = {CRISPR/Cas12a-based diagnostics have great potential for sensing nucleic acids, but their application is limited by the sequence-dependent property. A platform termed miR-Cabiner (a universal miRNA sensing platform based on self-stacking cascaded bicyclic DNA circuit-mediated CRISPR/Cas12a) is demonstrated herein that is sensitive and universal for analyzing miRNAs. This platform combines catalytic hairpin assembly (CHA) and hybrid chain reaction (HCR) into a unified circuit and finally cascades to CRISPR/Cas12a. Compared with the CHA-Cas12a and HCR-Cas12a systems, miR-Cabiner exhibits a significantly higher reaction rate. Panels of miRNAs (miR-130a, miR-10b, miR-21, and miR-1285), which are associated with diagnosis, staging, and prognosis of breast cancer, are designed to demonstrate the universality of miR-Cabiner. Four miRNAs can be detected to the fM-level by simply tuning the sequence in CHA components. Additionally, miRNA panel analysis also shows high accuracy in practical samples. This universally applicable platform for detecting miRNA may serve as an excellent tool for clinical diagnosis.},
}

*MicroRNAs/analysis
Humans
*CRISPR-Cas Systems/genetics
*DNA/chemistry
Biosensing Techniques/methods

@article {pmid39701943,
year = {2025},
author = {Zhao, Z and Xiong, Q and Zhu, Y and Zhang, C and Li, Z and Chen, Z and Zhang, Y and Deng, X and Tao, Y and Xu, S},
title = {CRISPR/Cas12a-Enabled Amplification-Free Colorimetric Visual Sensing Strategy for Point-of-Care Diagnostics of Biomarkers.},
journal = {Analytical chemistry},
volume = {97},
number = {1},
pages = {1019-1027},
doi = {10.1021/acs.analchem.4c06196},
pmid = {39701943},
issn = {1520-6882},
mesh = {Humans ; *Colorimetry ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques ; *Gold/chemistry ; BRCA1 Protein/genetics ; Metal Nanoparticles/chemistry ; Point-of-Care Testing ; Biomarkers, Tumor/blood/analysis ; Benzidines/chemistry ; Point-of-Care Systems ; Horseradish Peroxidase/chemistry/metabolism ; Biomarkers/blood/analysis ; Hydrogen Peroxide/chemistry ; },
abstract = {CRISPR/Cas12a-based biosensors have garnered significant attention in the field of point-of-care testing (POCT), yet the majority of the CRISPR-based POCT methods employ fluorescent systems as report probes. Herein, we report a new CRISPR/Cas12a-enabled multicolor visual biosensing strategy for the rapid detection of disease biomarkers. The proposed assay provided vivid color responses to enhance the accuracy of visual detection. In the existence of the target, the trans-cleavage activity of CRISPR-Cas12a was activated. The report probe modified with magnetic beads (MBs) and horseradish peroxidase (HRP) was cleaved, and HRP was released in the supernatant. As a result, HRP mediated the etching of gold nanobipyramids (AuNBPs) under hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine and generated a vivid color response. The proposed method has been verified by the detection of the breast cancer 1 gene (BRCA1) as a proof-of-principle target. According to the different colors of AuNBPs, our experimental results have demonstrated that as low as 30 pM BRCA1 can be detected with no more than 60 min. Additionally, the proposed sensor has been successfully applied in the analysis of BRCA1 in human serum samples with satisfactory results, which indicates great potential for the sensitive determination of biomarkers and the POCT area.},
}

Humans
*Colorimetry
*CRISPR-Cas Systems/genetics
*Biosensing Techniques
*Gold/chemistry
BRCA1 Protein/genetics
Metal Nanoparticles/chemistry
Point-of-Care Testing
Biomarkers, Tumor/blood/analysis
Benzidines/chemistry
Point-of-Care Systems
Horseradish Peroxidase/chemistry/metabolism
Biomarkers/blood/analysis
Hydrogen Peroxide/chemistry

@article {pmid39670529,
year = {2025},
author = {Chang, G and Li, Y and Peng, L and Shen, C and Lu, Y and Teng, W and Liu, Y and Wang, Y and Zhu, W and Liu, C and He, X and Tong, Y and Zhao, X},
title = {TavWA1 is critical for wheat growth by modulating cell morphology and arrangement.},
journal = {Journal of integrative plant biology},
volume = {67},
number = {1},
pages = {71-86},
doi = {10.1111/jipb.13807},
pmid = {39670529},
issn = {1744-7909},
support = {2023ZD0406802//Biological Breeding-National Science and Technology Major Project/ ; XDA24010202//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; },
mesh = {*Triticum/genetics/growth & development/metabolism ; *Plant Proteins/metabolism/genetics ; *Mutation/genetics ; Phenotype ; Gene Expression Regulation, Plant ; Gene Editing ; CRISPR-Cas Systems/genetics ; },
abstract = {Plant growth is determined by the production of cells and initiation of new organs. Exploring genes that control cell number and cell size is of great significance for understanding plant growth regulation. In this study, we characterized two wheat mutants, ah and dl, with abnormal growth. The ah mutant is a naturally occurring variant characterized by severe dwarfism, increased tiller number, and reduced grain length, while the dl mutant is derived from an ethyl methane sulfonate (EMS)-mutagenized population and exhibits smaller grain size and slightly reduced plant height. Cytological analyses revealed abnormal cell number, cell morphology and arrangement in the stems and leaves of the ah mutant, along with reduced cell length in the grains of the dl mutant. Map-based cloning identified that both mutants carry mutations in the same gene TavWA1-7D, which encodes a protein with a von Willebrand factor A (vWA) domain. The ah mutant harbors a 174-bp insertion in the 1,402-bp coding sequence (CDS) of TavWA1-7D, causing premature termination of protein translation, while the dl mutant contains a Glu420Lys substitution. Mimicking the TavWA1-7D[ah] through clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9-mediated genome editing leads to a severe dwarfism phenotype. The C-terminus of the protein is crucial for its correct subcellular localization and interaction, supporting its critical role for TavWA1-7D function. Proteomic analysis showed that the dwarf phenotype of the ah mutant is associated with impaired photosynthesis, ribosome function, and nucleosome formation. Additionally, TavWA1-7D interacts with an E3 ligase, TaVIP1-3B, the expression levels of which are elevated in both mutants. Overexpression and knockout studies of TaVIP1-3B demonstrated its negative regulatory role in cell length and grain size. Together, our findings suggest that TavWA1-7D plays a vital role in regulating wheat growth and yield-related traits, with the dl mutant's short grain phenotype being associated with TaVIP1-3B expression levels.},
}

*Triticum/genetics/growth & development/metabolism
*Plant Proteins/metabolism/genetics
*Mutation/genetics
Phenotype
Gene Expression Regulation, Plant
Gene Editing
CRISPR-Cas Systems/genetics

@article {pmid39642875,
year = {2025},
author = {Rottner, AK and Lundin, A and Li, S and Firth, M and Maresca, M and Sienski, G},
title = {Optimized prime editing of the Alzheimer's disease-associated APOE4 mutation.},
journal = {Stem cell reports},
volume = {20},
number = {1},
pages = {102372},
doi = {10.1016/j.stemcr.2024.11.002},
pmid = {39642875},
issn = {2213-6711},
mesh = {Humans ; *Alzheimer Disease/genetics ; *Gene Editing/methods ; *Apolipoprotein E4/genetics ; *Induced Pluripotent Stem Cells/metabolism ; *Mutation ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; },
abstract = {Gene editing strategies to safely and robustly modify the Alzheimer's disease-associated APOE4 isoform are still lacking. Prime editing (PE) enables the precise introduction of genetic variants with minimal unintended editing and without donor templates. However, it requires optimization for each target site and has not yet been applied to APOE4 gene editing. Here, we screened PE guide RNA (pegRNA) parameters and PE systems for introducing the APOE4 variant and applied the optimized PE strategy to generate disease-relevant human induced pluripotent stem cell models. We show that introducing a single-nucleotide difference required for APOE4 correction inhibits PE activity. To advance efficient and robust genome engineering of precise genetic variants, we further present a reliable PE enrichment strategy based on diphtheria toxin co-selection. Our work provides an optimized and reproducible genome engineering pipeline to generate APOE4 disease models and outlines novel strategies to accelerate genome editing in cellular disease model generation.},
}

Humans
*Alzheimer Disease/genetics
*Gene Editing/methods
*Apolipoprotein E4/genetics
*Induced Pluripotent Stem Cells/metabolism
*Mutation
*CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
HEK293 Cells

@article {pmid39642091,
year = {2025},
author = {Basol, M and Ersoz-Gulseven, E and Ozaktas, H and Kalyoncu, S and Utine, CA and Cakan-Akdogan, G},
title = {Loss of carbohydrate sulfotransferase 6 function leads to macular corneal dystrophy phenotypes and skeletal defects in zebrafish.},
journal = {The FEBS journal},
volume = {292},
number = {2},
pages = {373-390},
doi = {10.1111/febs.17337},
pmid = {39642091},
issn = {1742-4658},
support = {219S943//Türkiye Bilimsel ve Teknolojik Araştırma Kurumu/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Zebrafish Proteins/genetics/metabolism ; *Carbohydrate Sulfotransferases ; *Sulfotransferases/genetics/metabolism ; *Corneal Dystrophies, Hereditary/genetics/pathology/metabolism ; *Phenotype ; *Disease Models, Animal ; Cornea/pathology/metabolism ; CRISPR-Cas Systems ; Keratan Sulfate/genetics/metabolism ; Humans ; Gene Editing ; },
abstract = {The carbohydrate sulfotransferase 6 (chst6) gene is linked to macular corneal dystrophy (MCD), a rare disease that leads to bilateral blindness due to the accumulation of opaque aggregates in the corneal stroma. chst6 encodes for a keratan sulfate proteoglycan (KSPG) specific sulfotransferase. MCD patients lose sulfated KSPGs (cKS) in the cornea and the serum. The significance of serum cKS loss has not been understood. Zebrafish cornea structure is similar to that of humans and it contains high levels of sulfated cKS in the stroma. Here, zebrafish chst6 is shown to be expressed in the cornea and head structures of the embryos. An animal model of MCD is developed by generating chst6 mutant animals with CRISPR/Cas9-mediated gene editing. The dramatic decrease in cKS epitopes in the mutants was shown with ELISA and immunofluorescence. Morphological defects or alterations of jaw cartilage were detected in a minor fraction of the mutant larvae. Loss of cKS epitopes and morphological defects was fully rescued with wild-type chst6. Mutant adult zebrafish displayed all clinical manifestations of MCD, while a fraction also displayed jaw and skeleton defects. Opaque accumulations formed in the eye, which were alcian blue positive. Loss of cKS in the corneal stroma and a decrease in corneal thickness were shown. Interestingly, alteration of transforming growth factor beta-induced (BIGH3) expression which was not described in patients was also observed. This is the first report of an MCD model in a genetically tractable organism, providing a preclinical model and insight into the importance of KSPG sulfation for proper skeletal morphogenesis.},
}

Animals
*Zebrafish/genetics
*Zebrafish Proteins/genetics/metabolism
*Carbohydrate Sulfotransferases
*Sulfotransferases/genetics/metabolism
*Corneal Dystrophies, Hereditary/genetics/pathology/metabolism
*Phenotype
*Disease Models, Animal
Cornea/pathology/metabolism
CRISPR-Cas Systems
Keratan Sulfate/genetics/metabolism
Humans
Gene Editing

@article {pmid39471459,
year = {2025},
author = {Hesami, M and Pepe, M and Spitzer-Rimon, B and Eskandari, M and Jones, AMP},
title = {Epigenetic factors related to recalcitrance in plant biotechnology.},
journal = {Genome},
volume = {68},
number = {},
pages = {1-11},
doi = {10.1139/gen-2024-0098},
pmid = {39471459},
issn = {1480-3321},
mesh = {*Epigenesis, Genetic ; *Biotechnology/methods ; *Plant Somatic Embryogenesis Techniques/methods ; *Gene Editing/methods ; Plants/genetics ; CRISPR-Cas Systems ; Plants, Genetically Modified ; },
abstract = {This review explores the challenges and potential solutions in plant micropropagation and biotechnology. While these techniques have proven successful for many species, certain plants or tissues are recalcitrant and do not respond as desired, limiting the application of these technologies due to unattainable or minimal in vitro regeneration rates. Indeed, traditional in vitro culture techniques may fail to induce organogenesis or somatic embryogenesis in some plants, leading to classification as in vitro recalcitrance. This paper focuses on recalcitrance to somatic embryogenesis due to its promise for regenerating juvenile propagules and applications in biotechnology. Specifically, this paper will focus on epigenetic factors that regulate recalcitrance as understanding them may help overcome these barriers. Transformation recalcitrance is also addressed, with strategies proposed to improve transformation frequency. The paper concludes with a review of CRISPR-mediated genome editing's potential in modifying somatic embryogenesis-related epigenetic status and strategies for addressing transformation recalcitrance.},
}

*Epigenesis, Genetic
*Biotechnology/methods
*Plant Somatic Embryogenesis Techniques/methods
*Gene Editing/methods
Plants/genetics
CRISPR-Cas Systems
Plants, Genetically Modified

@article {pmid39451161,
year = {2025},
author = {Pan, W and Gao, C and Niu, D and Cheng, J and Zhang, J and Yan, X and Long, Q and Zhu, Y and Sun, W and Xie, Q and He, Y and Deng, XW and Zhang, H and Li, J},
title = {Efficient gene disruption in polyploid genome by Cas9-Trex2 fusion protein.},
journal = {Journal of integrative plant biology},
volume = {67},
number = {1},
pages = {7-10},
doi = {10.1111/jipb.13797},
pmid = {39451161},
issn = {1744-7909},
support = {TSQN202103160//Taishan Scholar Foundation of Shandong Province/ ; ZR202211070163//the Key R&D Program of Shandong Province/ ; ZR2021ZD30//Natural Science Foundation of Shandong Province/ ; ZR2022ZD22//Natural Science Foundation of Shandong Province/ ; ZR202103010168//the Excellent Youth Foundation of Shandong Scientific Committee/ ; },
mesh = {*Polyploidy ; *Gene Editing/methods ; *Genome, Plant/genetics ; Triticum/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; CRISPR-Cas Systems/genetics ; Exodeoxyribonucleases/genetics/metabolism ; Plant Proteins/genetics/metabolism ; Recombinant Fusion Proteins/genetics/metabolism ; },
abstract = {The fusion of the exonuclease Trex2 with the Cas9 protein significantly enhanced the efficiency of genome editing in hexaploid common wheat, particularly for the simultaneous editing of multiple favorable alleles within a single generation, thereby facilitating genome editing-assisted breeding in polyploid crops.},
}

*Polyploidy
*Gene Editing/methods
*Genome, Plant/genetics
Triticum/genetics
CRISPR-Associated Protein 9/metabolism/genetics
CRISPR-Cas Systems/genetics
Exodeoxyribonucleases/genetics/metabolism
Plant Proteins/genetics/metabolism
Recombinant Fusion Proteins/genetics/metabolism

@article {pmid39240004,
year = {2025},
author = {Zhao, Y and Cheng, P and Liu, Y and Liu, C and Hu, Z and Xin, D and Wu, X and Yang, M and Chen, Q},
title = {A highly efficient soybean transformation system using GRF3-GIF1 chimeric protein.},
journal = {Journal of integrative plant biology},
volume = {67},
number = {1},
pages = {3-6},
doi = {10.1111/jipb.13767},
pmid = {39240004},
issn = {1744-7909},
support = {TD2022C003//Natural Science Foundation of Heilongjiang Province/ ; YQ2022C010//Natural Science Foundation of Heilongjiang Province/ ; 31971899//National Natural Science Foundation of China/ ; 32272072//National Natural Science Foundation of China/ ; 32272093//National Natural Science Foundation of China/ ; U20A2027//National Natural Science Foundation of China/ ; 2021YFF1001202//National Key R&D Program of China/ ; 2023YFD2300101-02//National Key R&D Program of China/ ; },
mesh = {*Glycine max/genetics ; *Plants, Genetically Modified/genetics ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Plant Proteins/genetics/metabolism ; Transformation, Genetic ; Recombinant Fusion Proteins/genetics/metabolism ; },
abstract = {Expression of GRF3-GIF1 chimera significantly enhanced regeneration and transformation efficiency in soybean, increasing the number of transformable cultivars. Moreover, GmGRF3-GIF1 can be combined with CRISPR/Cas9 for highly effective gene editing.},
}

*Glycine max/genetics
*Plants, Genetically Modified/genetics
Gene Editing/methods
CRISPR-Cas Systems/genetics
Plant Proteins/genetics/metabolism
Transformation, Genetic
Recombinant Fusion Proteins/genetics/metabolism

@article {pmid39810095,
year = {2025},
author = {Liu, W and Wang, D and He, Q and Cao, S and Cao, J and Zhao, H and Cui, J and Yang, F},
title = {A strategy for controlling Hypervirulent Klebsiella pneumoniae: inhibition of ClpV expression.},
journal = {BMC microbiology},
volume = {25},
number = {1},
pages = {22},
pmid = {39810095},
issn = {1471-2180},
mesh = {*Klebsiella pneumoniae/genetics/pathogenicity/drug effects ; Virulence/genetics ; *Virulence Factors/genetics ; *Klebsiella Infections/microbiology/prevention & control ; Animals ; *Biofilms/growth & development/drug effects ; *Bacterial Proteins/genetics/metabolism ; *Gene Deletion ; Gene Expression Regulation, Bacterial ; Moths/microbiology ; Larva/microbiology ; CRISPR-Cas Systems ; },
abstract = {The emergence and prevalence of hypervirulent Klebsiella pneumoniae (hvKP) have proposed a great challenge to control this infection. Therefore, exploring some new drugs or strategies for treating hvKP infection is an urgent issue for scientific researchers. In the present study, the clpV gene deletion strain of hvKP (ΔclpV-hvKP) was constructed using CRISPR-Cas9 technology, and the biological characteristics of ΔclpV-hvKP were investigated to explore the new targets for controlling this pathogen. The results showed that clpV gene deletion did not affect the growth ability of hvKP. However, knocking out the clpV gene markedly decreased the mucoid phenotype and the biofilm formation ability of hvKP. It reduced the interspecific competition of hvKP with Escherichia coli, Salmonella, Pseudomonas aeruginosa, and Staphylococcus aureus. The clpV deletion significantly changed the transcriptome profile of hvKP, inhibited the expression of virulence factors, and decreased the lethality of hvKP against Galleria mellonella larvae. In vitro experiments showed that lithocholic acid could inhibit the expression of the clpV gene and reduce the virulence of hvKP. Our data suggested that the clpV gene may be a potential target for decreasing hvKP infection risk.},
}

*Klebsiella pneumoniae/genetics/pathogenicity/drug effects
Virulence/genetics
*Virulence Factors/genetics
*Klebsiella Infections/microbiology/prevention & control
Animals
*Biofilms/growth & development/drug effects
*Bacterial Proteins/genetics/metabolism
*Gene Deletion
Gene Expression Regulation, Bacterial
Moths/microbiology
Larva/microbiology
CRISPR-Cas Systems

@article {pmid39810027,
year = {2025},
author = {Weng, Y and Wang, Y and Wang, K and Wu, F and Wei, Y and Jiang, J and Zhu, Y and Wang, F and Xie, H and Xiao, Y and Cai, Q and Xie, H and Zhang, J},
title = {OsLOX1 positively regulates seed vigor and drought tolerance in rice.},
journal = {Plant molecular biology},
volume = {115},
number = {1},
pages = {16},
pmid = {39810027},
issn = {1573-5028},
support = {CARS-01-20//the National Rice Industry Technology System of Modern Agriculture for China/ ; XTCXGC2021001//he "5511" Collaborative Innovation Project for High-quality Development and Surpasses of Agriculture between the Government of Fujian and Chinese Academy of Agricultural Sciences/ ; 2020R1023008//the Special Foundation of Non-Profit Research Institutes of Fujian Province/ ; },
mesh = {*Oryza/genetics/physiology/enzymology ; *Seeds/genetics/physiology/growth & development ; *Droughts ; *Gene Expression Regulation, Plant ; *Germination/genetics ; *Plant Proteins/genetics/metabolism ; *Lipoxygenase/genetics/metabolism ; *Stress, Physiological ; Hydrogen Peroxide/metabolism ; Malondialdehyde/metabolism ; Plants, Genetically Modified ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Antioxidants/metabolism ; Superoxide Dismutase/metabolism/genetics ; Catalase/metabolism/genetics ; Drought Resistance ; },
abstract = {The lipoxygenase (LOX) gene family is widely distributed in plants, and its activity is closely associated with seed viability and stress tolerance. In this study, we cloned the rice(Oryza sativa)lipoxygenase gene OsLOX1, a key participant in the 13-lipoxygenase metabolic pathway. Our primary focus was to investigate its role in mediating responses to drought stress and seed germination in rice. Histochemical staining and qPCR analysis indicated that the expression level of OsLOX1 was relatively high in leaves and early germinating seeds. Our findings revealed that mutant lines with CRISPR/Cas9-induced knockout of OsLOX1 exhibited reduced tolerance to drought stress compared with the wild-type. This was accompanied by elevated levels of H2O2 and malondialdehyde, and a decrease in the expression levels of genes associated with antioxidant enzymes. Furthermore, knockout of OsLOX1 reduced the longevity of rice seeds increased H2O2 and MDA levels, and decreased the activities of the antioxidant enzymes superoxide dismutase and catalase, compared with the wild-type. These findings demonstrated that OsLOX1 positively regulated rice seed vigor and drought stress.},
}

*Oryza/genetics/physiology/enzymology
*Seeds/genetics/physiology/growth & development
*Droughts
*Gene Expression Regulation, Plant
*Germination/genetics
*Plant Proteins/genetics/metabolism
*Lipoxygenase/genetics/metabolism
*Stress, Physiological
Hydrogen Peroxide/metabolism
Malondialdehyde/metabolism
Plants, Genetically Modified
Gene Knockout Techniques
CRISPR-Cas Systems
Antioxidants/metabolism
Superoxide Dismutase/metabolism/genetics
Catalase/metabolism/genetics
Drought Resistance

@article {pmid39809780,
year = {2025},
author = {Park, SJ and Ju, S and Jung, WJ and Jeong, TY and Yoon, DE and Lee, JH and Yang, J and Lee, H and Choi, J and Kim, HS and Kim, K},
title = {Robust genome editing activity and the applications of enhanced miniature CRISPR-Cas12f1.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {677},
pmid = {39809780},
issn = {2041-1723},
support = {RS-2023-00220894//National Research Foundation of Korea (NRF)/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Humans ; HEK293 Cells ; Apoptosis/genetics ; Genetic Therapy/methods ; Cell Line, Tumor ; CRISPR-Associated Proteins/metabolism/genetics ; },
abstract = {With recent advancements in gene editing technology using the CRISPR/Cas system, there is a demand for more effective gene editors. A key factor facilitating efficient gene editing is effective CRISPR delivery into cells, which is known to be associated with the size of the CRISPR system. Accordingly, compact CRISPR-Cas systems derived from various strains are discovered, among which Un1Cas12f1 is 2.6 times smaller than SpCas9, providing advantages for gene therapy research. Despite extensive engineering efforts to improve Un1Cas12f1, the editing efficiency of Un1Cas12f1 is still shown to be low depending on the target site. To overcome this limitation, we develop enhanced Cas12f1 (eCas12f1), which exhibits gene editing activity similar to SpCas9 and AsCpf1, even in gene targets where previously improved Un1Cas12f1 variants showed low gene editing efficiency. Furthermore, we demonstrate that eCas12f1 efficiently induces apoptosis in cancer cells and is compatible with base editing and regulation of gene expression, verifying its high utility and applicability in gene therapy research.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
Humans
HEK293 Cells
Apoptosis/genetics
Genetic Therapy/methods
Cell Line, Tumor
CRISPR-Associated Proteins/metabolism/genetics

@article {pmid39809757,
year = {2025},
author = {Zou, J and Jiang, M and Xiao, R and Sun, H and Liu, H and Peacock, T and Tu, S and Chen, T and Guo, J and Zhao, Y and Barclay, W and Xie, S and Zhou, H},
title = {GGCX promotes Eurasian avian-like H1N1 swine influenza virus adaption to interspecies receptor binding.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {670},
pmid = {39809757},
issn = {2041-1723},
support = {32025036//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32430104//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2021CFA016//Natural Science Foundation of Hebei Province (Hebei Provincial Natural Science Foundation)/ ; },
mesh = {Animals ; *Influenza A Virus, H1N1 Subtype/genetics/metabolism ; Swine ; Humans ; *Virus Replication ; *Receptors, Virus/metabolism/genetics ; CRISPR-Cas Systems ; Hemagglutinin Glycoproteins, Influenza Virus/metabolism/genetics ; Orthomyxoviridae Infections/virology/metabolism ; Madin Darby Canine Kidney Cells ; Dogs ; HEK293 Cells ; Cell Line ; Receptors, Cell Surface/metabolism/genetics ; Mice ; },
abstract = {The Eurasian avian-like (EA) H1N1 swine influenza virus (SIV) possesses the capacity to instigate the next influenza pandemic, owing to its heightened affinity for the human-type α-2,6 sialic acid (SA) receptor. Nevertheless, the molecular mechanisms underlying the switch in receptor binding preferences of EA H1N1 SIV remain elusive. In this study, we conduct a comprehensive genome-wide CRISPR/Cas9 knockout screen utilizing EA H1N1 SIV in porcine kidney cells. Knocking out the enzyme gamma glutamyl carboxylase (GGCX) reduces virus replication in vitro and in vivo by inhibiting the carboxylation modification of viral haemagglutinin (HA) and the adhesion of progeny viruses, ultimately impeding the replication of EA H1N1 SIV. Furthermore, GGCX is revealed to be the determinant of the D225E substitution of EA H1N1 SIV, and GGCX-medicated carboxylation modification of HA 225E contributes to the receptor binding adaption of EA H1N1 SIV to the α-2,6 SA receptor. Taken together, our CRISPR screen has elucidated a novel function of GGCX in the support of EA H1N1 SIV adaption for binding to α-2,6 SA receptor. Consequently, GGCX emerges as a prospective antiviral target against the infection and transmission of EA H1N1 SIV.},
}

Animals
*Influenza A Virus, H1N1 Subtype/genetics/metabolism
Swine
Humans
*Virus Replication
*Receptors, Virus/metabolism/genetics
CRISPR-Cas Systems
Hemagglutinin Glycoproteins, Influenza Virus/metabolism/genetics
Orthomyxoviridae Infections/virology/metabolism
Madin Darby Canine Kidney Cells
Dogs
HEK293 Cells
Cell Line
Receptors, Cell Surface/metabolism/genetics
Mice

@article {pmid39809734,
year = {2025},
author = {Mormile, BW and Yan, Y and Bauer, T and Wang, L and Rivero, RC and Carpenter, SCD and Danmaigona Clement, C and Cox, KL and Zhang, L and Ma, X and Wheeler, TA and Dever, JK and He, P and Bogdanove, AJ and Shan, L},
title = {Activation of three targets by a TAL effector confers susceptibility to bacterial blight of cotton.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {644},
pmid = {39809734},
issn = {2041-1723},
support = {2018-67013-28513//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 2020-67013-41537//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 18-123TX//Cotton Incorporated (Cotton Inc.)/ ; IOS-2421016//National Science Foundation (NSF)/ ; },
mesh = {*Xanthomonas/pathogenicity/genetics/metabolism ; *Plant Diseases/microbiology ; *Transcription Activator-Like Effectors/metabolism/genetics ; *Gossypium/microbiology ; Virulence/genetics ; *CRISPR-Cas Systems ; *Bacterial Proteins/metabolism/genetics ; Gene Editing ; Host-Pathogen Interactions ; Genome, Bacterial ; Gene Expression Profiling ; },
abstract = {Bacterial transcription activator-like effectors (TALEs) promote pathogenicity by activating host susceptibility (S) genes. To understand the pathogenicity and host adaptation of Xanthomonas citri pv. malvacearum (Xcm), we assemble the genome and the TALE repertoire of three recent Xcm Texas isolates. A newly evolved TALE, Tal7b, activates GhSWEET14a and GhSWEET14b, different from GhSWEET10 targeted by a TALE in an early Xcm isolate. Activation of GhSWEET14a and GhSWEET14b results in water-soaked lesions. Transcriptome profiling coupled with TALE-binding element prediction identify a pectin lyase gene as an additional Tal7b target, quantitatively contributing to Xcm virulence alongside GhSWEET14a/b. CRISPR-Cas9 gene editing supports the function of GhSWEETs in cotton bacterial blight and the promise of disrupting the TALE-binding site in S genes for disease management. Collectively, our findings elucidate the rapid evolution of TALEs in Xanthomonas field isolates and highlight the virulence mechanism wherein TALEs induce multiple S genes to promote pathogenicity.},
}

*Xanthomonas/pathogenicity/genetics/metabolism
*Plant Diseases/microbiology
*Transcription Activator-Like Effectors/metabolism/genetics
*Gossypium/microbiology
Virulence/genetics
*CRISPR-Cas Systems
*Bacterial Proteins/metabolism/genetics
Gene Editing
Host-Pathogen Interactions
Genome, Bacterial
Gene Expression Profiling

@article {pmid39807869,
year = {2025},
author = {Sáenz, JS and Rios-Galicia, B and Seifert, J},
title = {Antiviral defense systems in the rumen microbiome.},
journal = {mSystems},
volume = {},
number = {},
pages = {e0152124},
doi = {10.1128/msystems.01521-24},
pmid = {39807869},
issn = {2379-5077},
abstract = {The continuous interaction between phages and their respective hosts has resulted in the evolution of multiple bacterial immune mechanisms. However, the diversity and prevalence of antiviral defense systems in complex communities are still unknown. We therefore investigated the diversity and abundance of viral defense systems in 3,038 high-quality bacterial and archaeal genomes from the rumen. In total, 14,241 defense systems and 31,948 antiviral-related genes were identified. Those genes represented 114 unique system types grouped into 49 families. We observed a high prevalence of defense systems in the genomes. However, the number of defense systems, defense system families, and system density varied widely from genome to genome. Additionally, the number of defense system per genome correlated positively with the number of defense system families and the genome size. Restriction modification, Abi, and cas system families were the most common, but many rare systems were present in only 1% of the genomes. Antiviral defense systems are prevalent and diverse in the rumen, but only a few are dominant, indicating that most systems are rarely present. However, the collection of systems throughout the rumen may represent a pool of mechanisms that can be shared by different members of the community and modulate the phage-host interaction.IMPORTANCEPhages may act antagonistically at the cell level but have a mutualistic interaction at the microbiome level. This interaction shapes the structure of microbial communities and is mainly driven by the defense mechanism. However, the diversity of such mechanism is larger than previously thought. Because of that, we described the abundance and diversity of the antiviral defense system of a collection of genomes, metagenome-assembled genomes (MAGs) and isolates, from the rumen. While defense mechanisms seem to be prevalent among bacteria and archaea, only a few were common. This suggests that most of these defense mechanisms are not present in many rumen microbes but could be shared among different members of the microbial community. This is consistent with the "pan-immune system" model, which appears to be common across different environments.},
}

@article {pmid39807514,
year = {2025},
author = {Saha, D and Panda, AK and Datta, S},
title = {Critical considerations and computational tools in plant genome editing.},
journal = {Heliyon},
volume = {11},
number = {1},
pages = {e41135},
pmid = {39807514},
issn = {2405-8440},
abstract = {Recent advances in genome editing tools and CRISPR-Cas technologies have enabled plant genome engineering reach new heights. The current regulatory exemptions for certain categories of genome edited products, such as those derived from SDN-1 and SDN-2, which are free of any transgene, have significantly accelerated genome editing research in a number of agricultural crop plants in different countries. Although CRISPR-Cas technology is becoming increasingly popular, it is still important to carefully consider a number of factors before planning and carrying conducting CRISPR-Cas studies. To attempt genome editing in a plant, a high-quality genome sequence and a repeatable tissue culture protocol for in vitro regeneration are essential. One of the most important steps in plant genome editing is the designing of a CRISPR construct, which involves selecting the appropriate Cas protein, sgRNA sequence, and appropriate regulatory sequence to trigger expression. Computational tools and algorithms play a crucial role in construct design and gRNA selection to minimize off-target effects and also to optimize their delivery techniques. Researchers may need to select appropriate software tools capable of analyzing post-editing detection of mutation events and other DNA sequence abnormalities to identify off-target effects. To fully fulfill the potential of plant genome editing, continued advances in computational biology are essential to meet the challenges it faces today.},
}

@article {pmid39806509,
year = {2025},
author = {Allais-Bonnet, A and Richard, C and André, M and Gelin, V and Deloche, MC and Lamadon, A and Morin, G and Mandon-Pépin, B and Canon, E and Thépot, D and Laubier, J and Moazami-Goudarzi, K and Laffont, L and Dubois, O and Fassier, T and Congar, P and Lasserre, O and Aguirre-Lavin, T and Vilotte, JL and Pailhoux, E},
title = {CRISPR/Cas9-editing of PRNP in Alpine goats.},
journal = {Veterinary research},
volume = {56},
number = {1},
pages = {11},
pmid = {39806509},
issn = {1297-9716},
support = {101000226//Horizon 2020 Framework Programme/ ; },
mesh = {Animals ; *Goats/genetics ; *CRISPR-Cas Systems ; *Gene Editing/veterinary ; Female ; Goat Diseases/genetics ; Male ; Prion Proteins/genetics ; Gene Knockout Techniques/veterinary ; },
abstract = {Misfolding of the cellular PrP (PrP[c]) protein causes prion disease, leading to neurodegenerative disorders in numerous mammalian species, including goats. A lack of PrP[c] induces complete resistance to prion disease. The aim of this work was to engineer Alpine goats carrying knockout (KO) alleles of PRNP, the PrP[c]-encoding gene, using CRISPR/Cas9-ribonucleoproteins and single-stranded donor oligonucleotides. The targeted region preceded the PRNP[Ter] mutation previously described in Norwegian goats. Genome editors were injected under the zona pellucida prior to the electroporation of 565 Alpine goat embryos/oocytes. A total of 122 two-cell-stage embryos were transferred to 46 hormonally synchronized recipient goats. Six of the goats remained pregnant and naturally gave birth to 10 offspring. Among the 10 newborns, eight founder animals carrying PRNP genome-edited alleles were obtained. Eight different mutated alleles were observed, including five inducing KO mutations. Three founders carried only genome-edited alleles and were phenotypically indistinguishable from their wild-type counterparts. Among them, one male carrying a one base pair insertion leading to a KO allele is currently used to rapidly extend a PRNP-KO line of Alpine goats for future characterization. In addition to KO alleles, a PRNP[del6] genetic variant has been identified in one-third of founder animals. This new variant will be tested for its potential properties with respect to prion disease. Future studies will also evaluate the effects of genetic background on other characters associated with PRNP KO, as previously described in the Norwegian breed or other species.},
}

Animals
*Goats/genetics
*CRISPR-Cas Systems
*Gene Editing/veterinary
Female
Goat Diseases/genetics
Male
Prion Proteins/genetics
Gene Knockout Techniques/veterinary

@article {pmid39806441,
year = {2025},
author = {Lin, Y and Li, C and Chen, Y and Gao, J and Li, J and Huang, C and Liu, Z and Wang, W and Zheng, X and Song, X and Wu, J and Wu, J and Luo, OJ and Tu, Z and Li, S and Li, XJ and Lai, L and Yan, S},
title = {RNA-Targeting CRISPR/CasRx system relieves disease symptoms in Huntington's disease models.},
journal = {Molecular neurodegeneration},
volume = {20},
number = {1},
pages = {4},
pmid = {39806441},
issn = {1750-1326},
mesh = {Animals ; *Huntington Disease/genetics/therapy ; Humans ; *CRISPR-Cas Systems ; *Disease Models, Animal ; Mice ; *Huntingtin Protein/genetics ; HEK293 Cells ; Swine ; Gene Editing/methods ; Mice, Transgenic ; },
abstract = {BACKGROUND: HD is a devastating neurodegenerative disorder caused by the expansion of CAG repeats in the HTT. Silencing the expression of mutated proteins is a therapeutic direction to rescue HD patients, and recent advances in gene editing technology such as CRISPR/CasRx have opened up new avenues for therapeutic intervention.

METHODS: The CRISPR/CasRx system was employed to target human HTT exon 1, resulting in an efficient knockdown of HTT mRNA. This therapeutic effect was substantiated in various models: HEK 293 T cell, the HD 140Q-KI mouse, and the HD-KI pig model. The efficiency of the knockdown was analyzed through Western blot and RT-qPCR. Additionally, neuropathological changes were examined using Western blot, immunostaining, and RNA sequencing. The impact on motor abilities was assessed via behavioral experiments, providing a comprehensive evaluation of the treatment's effectiveness.

RESULTS: CRISPR/CasRx system can significantly reduce HTT mRNA levels across various models, including HEK 293 T cells, HD 140Q-KI mice at various disease stages, and HD-KI pigs, and resulted in decreased expression of mHTT. Utilizing the CRISPR/CasRx system to knock down HTT RNA has shown to ameliorate gliosis in HD 140Q-KI mice and delay neurodegeneration in HD pigs.

CONCLUSIONS: These findings highlight the effectiveness of the RNA-targeting CRISPR/CasRx as a potential therapeutic strategy for HD. Furthermore, the success of this approach provides valuable insights and novel avenues for the treatment of other genetic disorders caused by gene mutations.},
}

Animals
*Huntington Disease/genetics/therapy
Humans
*CRISPR-Cas Systems
*Disease Models, Animal
Mice
*Huntingtin Protein/genetics
HEK293 Cells
Swine
Gene Editing/methods
Mice, Transgenic

@article {pmid39516652,
year = {2025},
author = {Murphy, R and Martin, KR},
title = {Genetic engineering and the eye.},
journal = {Eye (London, England)},
volume = {39},
number = {1},
pages = {57-68},
pmid = {39516652},
issn = {1476-5454},
mesh = {Humans ; *Genetic Therapy/methods ; *Genetic Engineering ; *Eye Diseases/therapy/genetics ; Gene Editing/methods ; CRISPR-Cas Systems ; Genetic Vectors ; },
abstract = {The transformative potential of genetic engineering in ophthalmology is remarkable, promising new treatments for a wide range of blinding eye diseases. The eye is an attractive target organ for genetic engineering approaches, in part due to its relatively immune-privileged status, its accessibility, and the ease of monitoring of efficacy and safety. Consequently, the eye has been at the forefront of genetic engineering advances in recent years. The development of Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9), base editors, prime editors, and transposases have enabled efficient and specific gene modification. Ocular gene therapy continues to progress, with recent advances in delivery systems using viral / non-viral vectors and novel promoters and enhancers. New strategies to achieve neuroprotection and neuroregeneration are evolving, including direct in-vivo cell reprogramming and optogenetic approaches. In this review, we discuss recent advances in ocular genetic engineering, examine their current therapeutic roles, and explore their potential use in future strategies to reduce the growing burden of vision loss and blindness.},
}

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

@article {pmid39806115,
year = {2025},
author = {Zhang, X and Sun, R and Zheng, H and Qi, Y},
title = {Amplification-free sensitive detection of Staphylococcus aureus by spherical nucleic acid triggered CRISPR/Cas12a and Poly T-Cu reporter.},
journal = {Mikrochimica acta},
volume = {192},
number = {2},
pages = {76},
pmid = {39806115},
issn = {1436-5073},
support = {82073602//National Natural Science Foundation of China/ ; 20200201081JC//Natural Science Foundation of Jilin Province/ ; },
mesh = {*Staphylococcus aureus/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Copper/chemistry ; *Metal Nanoparticles/chemistry ; *Gold/chemistry ; *Limit of Detection ; Aptamers, Nucleotide/chemistry ; CRISPR-Associated Proteins/metabolism/genetics ; Biosensing Techniques/methods ; Bacterial Proteins/genetics ; Endodeoxyribonucleases/chemistry/metabolism ; },
abstract = {A spherical nucleic acid (SNA, AuNPs-aptamer) into CRISPR/Cas12a system combined with poly T-template copper nanoparticles as fluorescence reporter was fabricated to establish an amplification-free sensitive method for Staphylococcus aureus (S. aureus) detection. This method, named PTCas12a, utilizes the concept that the bifunction of SNA recognizes the S. aureus and triggers the Cas12a cleavage activity. Then, the Cas12a enzyme cleaves the Poly T40 to generate a signal change in Poly T-Cu fluorescence, indicating the presence or absence of the target bacteria. The PTCas12a platform demonstrated a detection limit as low as 3.0 CFU/mL (3 N/S) in a wide response range of 1.0 × 10[1]-1.0 × 10[6] CFU/mL for S. aureus detection, which holds significant potential in ensuring food safety and preventing the spread of diseases.},
}

*Staphylococcus aureus/genetics/isolation & purification
*CRISPR-Cas Systems
*Copper/chemistry
*Metal Nanoparticles/chemistry
*Gold/chemistry
*Limit of Detection
Aptamers, Nucleotide/chemistry
CRISPR-Associated Proteins/metabolism/genetics
Biosensing Techniques/methods
Bacterial Proteins/genetics
Endodeoxyribonucleases/chemistry/metabolism

@article {pmid39806080,
year = {2025},
author = {Li, Y and Zeng, Z and Lv, X and Jiang, H and Li, A and Liu, Y and Deng, Y and Li, X},
title = {A POCT assay based on commercial HCG strip for miRNA21 detection by integrating with RCA-HCR cascade amplification and CRISPR/Cas12a.},
journal = {Mikrochimica acta},
volume = {192},
number = {2},
pages = {73},
pmid = {39806080},
issn = {1436-5073},
mesh = {*MicroRNAs/blood/genetics/analysis ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Point-of-Care Testing ; *Limit of Detection ; Chorionic Gonadotropin/blood ; Nucleic Acid Hybridization ; Reagent Strips ; },
abstract = {A point-of-care testing (POCT) assay based on commercial HCG strip was proposed for miRNA21 detection by integrating RCA-HCR cascaded isothermal amplification with CRISPR/Cas12a. Three modules were integrated in the proposed platform: target amplification module composed of rolling circle amplification (RCA) cascaded with hybridization chain reaction (HCR), signal transduction module composed of CRISPR/Cas12a combined with HCG-agarose gel beads probes, and signal readout module composed of commercial HCG strips. The proposed RCA-HCR-CRISPR/Cas12a-HCG strip assay for miRNA21 detection had high sensitivity, and the limit of detection was as low as 37 fM. The proposed assay showed excellent specificity for miRNA21, as other miRNAs did not caused interference for detection. The recoveries of miRNA21 were ranged from 89.0 to 118.0%. The intra-batch and inter-batch coefficient of variation (CV) were 10.1-13.4% and 11.9-14.5%, respectively, which indicated a high accuracy and precision, and the serum matrix did not cause any interference. With the advantages of low-cost, high sensitivity, visualization, and easy popularization, the proposed assay is expected to provide a powerful tool for early diagnosis of tumor disease miRNA, especially in resource-limited areas.},
}

*MicroRNAs/blood/genetics/analysis
*Nucleic Acid Amplification Techniques/methods
Humans
*CRISPR-Cas Systems/genetics
*Point-of-Care Testing
*Limit of Detection
Chorionic Gonadotropin/blood
Nucleic Acid Hybridization
Reagent Strips

@article {pmid39804665,
year = {2025},
author = {Liu, X and Huang, L and Li, M and Fu, Y and Zhang, W and Zhang, S and Liang, X and Shen, Q},
title = {An Efficient and Cost-Effective Novel Strategy for Identifying CRISPR-Cas-Mediated Mutants in Plant Offspring.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1089/crispr.2024.0057},
pmid = {39804665},
issn = {2573-1602},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 system has revolutionized targeted mutagenesis, but screening for mutations in large sample pools can be time-consuming and costly. We present an efficient and cost-effective polymerase chain reaction (PCR)-based strategy for identifying edited mutants in the T1 generation. Unlike previous methods, our approach addresses the challenges of large progeny populations by using T0 generation sequencing results for genotype prediction. The T1 generation plants were then divided into two scenarios: ≥4 bp indels and 1-2 bp indels. Specific primers are designed for these categories, employing dual-primers critical annealing temperature PCR for ≥4 bp indels and the derived cleaved amplified polymorphic sequences (dCAPS) method for 1-2 bp indels. This method is straightforward, cost-effective, and allows rapid and precise identification of T1 editing outcomes, distinguishing between wild-type, heterozygous, and homozygous plants. This strategy accelerates gene functional analysis in plants and beyond.},
}

@article {pmid39804233,
year = {2025},
author = {Wang, H and Zhao, R and Zhang, B and Xiao, Y and Yu, C and Wang, Y and Yu, C and Tang, Y and Li, Y and Lu, B and Li, B},
title = {Accurate Molecular Sensing based on a Modular and Customizable CRISPR/Cas-Assisted Nanopore Operational Nexus (CANON).},
journal = {Angewandte Chemie (International ed. in English)},
volume = {},
number = {},
pages = {e202423473},
doi = {10.1002/anie.202423473},
pmid = {39804233},
issn = {1521-3773},
abstract = {Solid-state nanopore is a promising single molecular detection technique, but is largely limited by relatively low resolution to small-size targets and laborious design of signaling probes. Here we establish a universal, CRISPR/Cas-Assisted Nanopore Operational Nexus (CANON), which can accurately transduce different targeting sources/species into different DNA structural probes via a "Signal-ON" mode. Target recognition activates the cleavage activity of a Cas12a/crRNA system and then completely digest the blocker of an initiator. The unblocked initiator then triggers downstream DNA assembly reaction and generate a large-size structure easy for nanopore detection. Such integration of Cas12a/crRNA with DNA assembly establishes an accurate correspondence among the input targets, output DNA structures, and the ultimate nanopore signals. We demonstrated dsDNA, long RNA (i.e., Flu virus gene), short microRNA (i.e., let-7d) and non-nucleic acids (i.e., Pb2+) as input paradigms. Various structural assembly reactions, such as hybridization chain reaction (HCR), G-HCR and duplex polymerization strategy (DPS), are adapted as outputs for nanopore signaling. Simultaneous assay is also verified via transferring FluA and FluB genes into HCR and G-HCR, respectively. CANON is thus a modular sensing platform holding multiple advantages such as high accuracy, high resolution and high universality, which can be easily customized into various application scenes.},
}

@article {pmid39803585,
year = {2024},
author = {Wei, SC and Cantor, AJ and Walleshauser, J and Mepani, R and Melton, K and Bans, A and Khekare, P and Gupta, S and Wang, J and Soares, C and Kiwan, R and Lee, J and McCawley, S and Jani, V and Leong, WI and Shahi, PK and Chan, J and Boivin, P and Otoupal, P and Pattnaik, BR and Gamm, DM and Saha, K and Gowen, BG and Haak-Frendscho, M and Janatpour, MJ and Silverman, AP},
title = {Evaluation of subretinally delivered Cas9 ribonucleoproteins in murine and porcine animal models highlights key considerations for therapeutic translation of genetic medicines.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {39803585},
issn = {2692-8205},
abstract = {Genetic medicines, including CRISPR/Cas technologies, extend tremendous promise for addressing unmet medical need in inherited retinal disorders and other indications; however, there remain challenges for the development of therapeutics. Herein, we evaluate genome editing by engineered Cas9 ribonucleoproteins (eRNP) in vivo via subretinal administration using mouse and pig animal models. Subretinal administration of adenine base editor and double strand break-inducing Cas9 nuclease eRNPs mediate genome editing in both species. Editing occurs in retinal pigmented epithelium (RPE) and photoreceptor cells, with favorable tolerability in both species. Using transgenic reporter strains, we determine that editing primarily occurs close to the site of administration, within the bleb region associated with subretinal injection. Our results show that subretinal administration of eRNPs in mice mediates base editing of up to 12% of the total neural retina, with an average rate of 7% observed at the highest dose tested. In contrast, a substantially lower editing efficiency was observed in minipigs; even with direct quantification of only the treated region, a maximum base editing rate of 1.5%, with an average rate of <1%, was observed. Our data highlight the importance of species consideration in translational studies for genetic medicines targeting the eye and provide an example of a lack of translation between small and larger animal models in the context of subretinal administration of Cas9 eRNPs.},
}

@article {pmid39803448,
year = {2025},
author = {Hossain, KA and Nierzwicki, L and Orozco, M and Czub, J and Palermo, G},
title = {Flexibility in PAM Recognition Expands DNA Targeting in xCas9.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2024.08.26.609653},
pmid = {39803448},
issn = {2692-8205},
abstract = {xCas9 is an evolved variant of the CRISPR-Cas9 genome editing system, engineered to improve specificity and reduce undesired off-target effects. How xCas9 expands the DNA targeting capability of Cas9 by recognizing a series of alternative Protospacer Adjacent Motif (PAM) sequences while ignoring others is unknown. Here, we elucidate the molecular mechanism underlying xCas9's expanded PAM recognition and provide critical insights for expanding DNA targeting. We demonstrate that while wild-type Cas9 enforces stringent guanine selection through the rigidity of its interacting arginine dyad, xCas9 introduces flexibility in R1335, enabling selective recognition of specific PAM sequences. This increased flexibility confers a pronounced entropic preference, which also improves recognition of the canonical TGG PAM. Furthermore, xCas9 enhances DNA binding to alternative PAM sequences during the early evolution cycles, while favouring binding to the canonical PAM in the final evolution cycle. This dual functionality highlights how xCas9 broadens PAM recognition and underscores the importance of fine-tuning the flexibility of the PAM-interacting cleft as a key strategy for expanding the DNA targeting potential of CRISPR-Cas systems. These findings deepen our understanding of DNA recognition in xCas9 and may apply to other CRISPR-Cas systems with similar PAM recognition requirements.},
}

@article {pmid39801754,
year = {2025},
author = {Guha, S and Jagadeesan, Y and Pandey, MM and Mittal, A and Chitkara, D},
title = {Targeting the epigenome with advanced delivery strategies for epigenetic modulators.},
journal = {Bioengineering & translational medicine},
volume = {10},
number = {1},
pages = {e10710},
pmid = {39801754},
issn = {2380-6761},
abstract = {Epigenetics mechanisms play a significant role in human diseases by altering DNA methylation status, chromatin structure, and/or modifying histone proteins. By modulating the epigenetic status, the expression of genes can be regulated without any change in the DNA sequence itself. Epigenetic drugs exhibit promising therapeutic efficacy against several epigenetically originated diseases including several cancers, neurodegenerative diseases, metabolic disorders, cardiovascular disorders, and so forth. Currently, a considerable amount of research is focused on discovering new drug molecules to combat the existing research gap in epigenetic drug therapy. A novel and efficient delivery system can be established as a promising approach to overcome the drawbacks associated with the current epigenetic modulators. Therefore, formulating the existing epigenetic drugs with distinct encapsulation strategies in nanocarriers, including solid lipid nanoparticles, nanogels, bio-engineered nanocarriers, liposomes, surface modified nanoparticles, and polymer-drug conjugates have been examined for therapeutic efficacy. Nonetheless, several epigenetic modulators are untouched for their therapeutic potential through different delivery strategies. This review provides a comprehensive up to date discussion on the research findings of various epigenetics mechanism, epigenetic modulators, and delivery strategies utilized to improve their therapeutic outcome. Furthermore, this review also highlights the recently emerged CRISPR tool for epigenome editing.},
}

@article {pmid39801562,
year = {2025},
author = {Nguyen, T and Meleski, LWG and Belavatta, MP and Gurumoorthi, S and Zhang, C and Heins, AL and Zeng, AP},
title = {A Consecutive Genome Engineering Method Reveals a New Phenotype and Regulation of Glucose and Glycerol Utilization in Clostridium Pasteurianum.},
journal = {Engineering in life sciences},
volume = {25},
number = {1},
pages = {e202400026},
pmid = {39801562},
issn = {1618-0240},
abstract = {Clostridium pasteurianum is a microorganism for production of 1,3-propanediol (1,3-PDO) and butanol, but suffers from lacking genetic tools for metabolic engineering to improve product titers. Furthermore, previous studies of C. pasteurianum have mainly focused on single genomic modification. The aim of this work is the development and application of a method for modification of multiple gene targets in the genome of C. pasteurianum. To this end, a new approach for consecutive genome engineering is presented for the first time using a method based on endogenous CRISPR-Cas machineries. A total of three genome modifications were consecutively introduced in the same mutant and the effect of combined changes on the genome was observed by 39% decreased specific glycerol consumption rate and 29% increased 1,3-PDO yield in mixed substrate fermentations at laboratory scale in comparison to the wildtype strain. Additionally, examination of the phenotype of the generated mutant strain led to discovery of 2,3-butanediol (2,3-BDO) production of up to 0.48 g L[-1], and this metabolite was not reported to be produced by C. pasteurianum before. The developed procedure expands the genetic toolkit for C. pasteurianum and provides researchers an additional method which contributes to improved genetic accessibility of this strain.},
}

@article {pmid39787667,
year = {2025},
author = {Hall, R and Sikora, T and Suter, A and Kuah, JY and Christodoulou, J and Van Bergen, NJ},
title = {Generation and heterozygous repair of human iPSC lines from two individuals with the neurodevelopmental disorder, TRAPPC4 deficiency.},
journal = {Stem cell research},
volume = {82},
number = {},
pages = {103640},
doi = {10.1016/j.scr.2024.103640},
pmid = {39787667},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; *Neurodevelopmental Disorders/genetics ; *Heterozygote ; Male ; CRISPR-Cas Systems ; Female ; Cell Line ; Cell Differentiation ; },
abstract = {A rare neurodevelopmental disorder has been linked to a well-conserved splice site variant in the TRAPPC4 gene (c.454 + 3A > G), which causes mis-splicing of TRAPPC4 transcripts and reduced levels of TRAPPC4 protein. Patients present with severe progressive neurological symptoms including seizures, microcephaly, intellectual disability and facial dysmorphism. We have generated stem cells from fibroblasts of two individuals with the same homozygous TRAPPC4 c.454 + 3A > G pathogenic variant and used CRISPR/Cas9 editing to generate heterozygous gene-corrected isogenic controls. Clones were tested for pluripotency, differentiation potential, genotyped and karyotyped. These iPSC-based models will be used to understand disease mechanisms of TRAPPC4 disorder.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism
*Neurodevelopmental Disorders/genetics
*Heterozygote
Male
CRISPR-Cas Systems
Female
Cell Line
Cell Differentiation

@article {pmid39742627,
year = {2025},
author = {Wei, S and Zhen, Y and Sun, C and Ma, Y and Li, Q and Wen, L},
title = {Generation of a USP9Y knockout human embryonic stem cell line with CRISPR-Cas9 technology.},
journal = {Stem cell research},
volume = {82},
number = {},
pages = {103646},
doi = {10.1016/j.scr.2024.103646},
pmid = {39742627},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems ; *Human Embryonic Stem Cells/metabolism/cytology ; *Gene Knockout Techniques/methods ; Male ; Ubiquitin Thiolesterase/genetics/metabolism ; Cell Line ; Cell Differentiation ; },
abstract = {Human embryonic stem cell (hESC) lines are vital tools for studying gene function, disease modeling, and therapy. We generated a USP9Y knockout hESC line using CRISPR-Cas9 in the male-derived H1 line. Targeted deletion of the USP9Y gene was confirmed via PCR and sequencing. The modified line retained pluripotency markers, exhibited a normal karyotype, and differentiated into all three germ layers. This model provides a valuable platform for studying USP9Y's role in human development and male infertility, offering insights into related disorders and therapeutic potential.},
}

Humans
*CRISPR-Cas Systems
*Human Embryonic Stem Cells/metabolism/cytology
*Gene Knockout Techniques/methods
Male
Ubiquitin Thiolesterase/genetics/metabolism
Cell Line
Cell Differentiation

@article {pmid39708407,
year = {2025},
author = {Liu, X and Zhang, F and Chen, D and Yin, J and Bi, Z and Chen, L and Yan, J and Dong, Q and Peng, W and Xu, T and Guo, Y and Lin, H and Liu, H},
title = {Generation of INS-jGCaMP7f knock-in Ca[2+] reporter human embryonic stem cell line, GZLe001-C, using CRISPR/Cas9-based gene targeting.},
journal = {Stem cell research},
volume = {82},
number = {},
pages = {103633},
doi = {10.1016/j.scr.2024.103633},
pmid = {39708407},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems ; *Human Embryonic Stem Cells/metabolism/cytology ; *Calcium/metabolism ; Gene Targeting/methods ; Cell Line ; Gene Knock-In Techniques/methods ; Insulin/metabolism ; Genes, Reporter ; },
abstract = {As a member of the single-fluorophore genetically encoded calcium indicators (GECIs), jGCaMP7f is widely applied to investigate intracellular Ca[2+] concentrations. Here, we established an INS-jGCaMP7f knock-in H1 human embryonic stem cell (hESC) line by integrating jGCaMP7f gene into insulin locus via CRISPR/Cas9 system. The reporter cell line not only effectively labelled the insulin-producing cells induced from hESC, but also reflected the cytosolic change of Ca[2+] level in response to different stimuli. This reporter cell line is a valuable research tool for studying functional maturation of hESC-derived insulin-producing cells, conducting drug screenings, and exploring the mechanisms of diabetes.},
}

Humans
*CRISPR-Cas Systems
*Human Embryonic Stem Cells/metabolism/cytology
*Calcium/metabolism
Gene Targeting/methods
Cell Line
Gene Knock-In Techniques/methods
Insulin/metabolism
Genes, Reporter

@article {pmid39667064,
year = {2025},
author = {Lu, S and Chen, M and Liu, X and Li, J and Liu, H and Li, S},
title = {Generation of a BEST1 Pr-EGFP reporter human embryonic stem cell line via CRISPR/Cas9 editing.},
journal = {Stem cell research},
volume = {82},
number = {},
pages = {103625},
doi = {10.1016/j.scr.2024.103625},
pmid = {39667064},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems ; *Human Embryonic Stem Cells/metabolism/cytology ; *Gene Editing/methods ; *Green Fluorescent Proteins/metabolism/genetics ; Cell Line ; Cell Differentiation ; Bestrophins/metabolism/genetics ; Retinal Pigment Epithelium/cytology/metabolism ; Genes, Reporter ; },
abstract = {The retinal pigment epithelium (RPE) cell, located between the neural retina and choriocapillaris, is vital for retinal maintenance and photoreceptor function. Human embryonic stem cells (hESCs) provide a limitless source of RPE cells for transplantation. Using CRISPR/Cas9, we inserted a fusion of the BEST1 promoter (an RPE-specific marker) and the EGFP gene into the AAVS1 locus to track differentiation in hESC-induced RPE (hESC-iRPE). The resulting gene-edited line, WAe009-A-2 M, maintained a normal karyotype, expressed pluripotency markers, and demonstrated differentiation potential, making it invaluable for RPE development and therapeutic research.},
}

Humans
*CRISPR-Cas Systems
*Human Embryonic Stem Cells/metabolism/cytology
*Gene Editing/methods
*Green Fluorescent Proteins/metabolism/genetics
Cell Line
Cell Differentiation
Bestrophins/metabolism/genetics
Retinal Pigment Epithelium/cytology/metabolism
Genes, Reporter

@article {pmid39647515,
year = {2025},
author = {Luo, X and Germer, J and Burghardt, T and Grau, M and Lin, Y and Höhn, M and Lächelt, U and Wagner, E},
title = {Dual pH-responsive CRISPR/Cas9 ribonucleoprotein xenopeptide complexes for genome editing.},
journal = {European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences},
volume = {205},
number = {},
pages = {106983},
doi = {10.1016/j.ejps.2024.106983},
pmid = {39647515},
issn = {1879-0720},
mesh = {*Gene Editing/methods ; Humans ; *Ribonucleoproteins/genetics/metabolism ; *CRISPR-Cas Systems ; HeLa Cells ; Hydrogen-Ion Concentration ; RNA, Guide, CRISPR-Cas Systems ; Peptides/chemistry ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated (Cas) protein has been proved as a powerful tool for the treatment of genetic diseases. The Cas9 protein, when combined with single-guide RNA (sgRNA), forms a Cas9/sgRNA ribonucleoprotein (RNP) capable of targeting and editing the genome. However, the limited availability of effective carriers has restricted the broader application of CRISPR/Cas9 RNP. In this study, we evaluated dual pH-responsive amphiphilic xenopeptides (XPs) for delivering CRISPR/Cas9 RNP. These artificial lipo-XPs contain apolar cationizable lipoamino fatty acid (LAF) and polar cationizable oligoaminoethylene acid units such as succinoyl-tetraethylenepentamine (Stp) in various ratios and U-shaped topologies. The carriers were screened for functional Cas9/sgRNA RNP delivery in four different reporter cell lines, including a Duchenne muscular dystrophy (DMD) exon skipping reporter cell model. Significantly enhanced cellular uptake into HeLa cells, effective endosomal disruption in HeLa gal8-mRuby3 cells, and potent genome editing by several Cas9/sgRNA RNP complexes was observed in four different cell lines in the 5 nM sgRNA range. Comparing Cas9/sgRNA RNP complexes with Cas9 mRNA/sgRNA polyplexes in the DMD reporter cell model demonstrated similar splice site editing and high exon skipping of the two different molecular Cas9 modalities. Based on these studies, analogues of two potent U1 LAF2-Stp and LAF4-Stp2 structures were deployed, tuning the amphiphilicity of the polar Stp group by replacement with the six oligoamino acids dmGtp, chGtp, dGtp, Htp, Stt, or GEIPA. The most potent LAF2-Stp analogues (containing dGtp, chGtp or GEIPA) demonstrated further enhanced gene editing efficiency with EC50 values of 1 nM in the DMD exon skipping reporter cell line. Notably, the EC50 of LAF2-dGtp reached 0.51 nM even upon serum incubation. Another carrier (LAF4-GEIPA2) complexing Cas9/sgRNA RNP and donor DNA, facilitated up to 43 % of homology-directed repair (HDR) in HeLa eGFPd2 cells visualized by the switch from green fluorescent protein (eGFP) to blue fluorescent protein (BFP). This study presents a delivery system tunable for Cas9 RNP complexes or Cas9 RNP/donor DNA polyplexes, offering an effective and easily applicable strategy for gene editing.},
}

*Gene Editing/methods
Humans
*Ribonucleoproteins/genetics/metabolism
*CRISPR-Cas Systems
HeLa Cells
Hydrogen-Ion Concentration
RNA, Guide, CRISPR-Cas Systems
Peptides/chemistry
CRISPR-Associated Protein 9/genetics/metabolism

@article {pmid39644600,
year = {2025},
author = {Xu, Y and Wu, H and Jiang, J and Ye, L and Hao, K and Han, K and Hu, S and Lei, W and Guo, Z},
title = {Generation and characterization of the LINC01405 knockout human embryonic stem cell line.},
journal = {Stem cell research},
volume = {82},
number = {},
pages = {103619},
doi = {10.1016/j.scr.2024.103619},
pmid = {39644600},
issn = {1876-7753},
mesh = {Humans ; *Human Embryonic Stem Cells/metabolism/cytology ; *RNA, Long Noncoding/genetics/metabolism ; *Gene Knockout Techniques ; Cell Differentiation ; Cell Line ; CRISPR-Cas Systems ; },
abstract = {Long Intergenic Non-Protein Coding RNA 1405 (LINC01405), with known elevated expression in muscle, has been linked to a number of musculo-skeletal conditions. By utilizing the CRISPR/Cas9 gene editing system, we generated a LINC01405 knockout human embryonic stem cell (hESC) line. This line remains human stem cell-like morphology and pluripotency, exhibits a normal karyotype, and can differentiate into cells from all three germ layers. This cell line will be an invaluable model for the research on LINC01405's role in normal development of cardiac and skeletal muscle, and their diseases.},
}

Humans
*Human Embryonic Stem Cells/metabolism/cytology
*RNA, Long Noncoding/genetics/metabolism
*Gene Knockout Techniques
Cell Differentiation
Cell Line
CRISPR-Cas Systems

@article {pmid39380247,
year = {2025},
author = {Tsuji-Hosokawa, A and Tsuchiya, I and Shimizu, K and Terao, M and Yasuhara, M and Miyamoto, N and Kikuchi, S and Ogawa, Y and Nakamura, K and Matsubara, Y and Takada, S},
title = {Genetically humanized phenylketonuria mouse model as a testing tool for human genome editing in fertilized eggs.},
journal = {Journal of inherited metabolic disease},
volume = {48},
number = {1},
pages = {e12803},
doi = {10.1002/jimd.12803},
pmid = {39380247},
issn = {1573-2665},
support = {19ek0109290h0003//Japan Agency for Medical Research and Development/ ; 2023C-12//National Research Institute for Child Health and Development/ ; 20K18208//Japan Society for the Promotion of Science/ ; },
mesh = {*Phenylketonurias/genetics ; Animals ; *Gene Editing/methods ; *Disease Models, Animal ; Humans ; Mice ; Phenylalanine Hydroxylase/genetics ; Zygote ; Female ; Phenotype ; Male ; CRISPR-Cas Systems ; Genome, Human ; Phenylalanine/genetics ; },
abstract = {Targeted genome editing has made significant advancements; however, safety and ethical issues have not been fully elucidated, resulting in strict control of the technique. We tested genome editing tools on gametes from a genetically humanized mouse model using a phenylketonuria (PKU) mouse model to gain insights into genome editing in human embryos. The human PKU mouse model Pah[hR111X] mice was generated. The junctional region between exon 3 and intron 3 of Pah was replaced with a 120 bp corresponding human PAH sequence, including the pathogenic common variant c.331C > T in Pah[hR111X] mice. Pah[hR111X] mice successfully recapitulated the PKU phenotype and showed cognitive dysfunction and depressive-like behavior, which are observed in human patients with PKU. Genome editing was applied to fertilized eggs of Pah[hR111X] mice utilizing sgRNA that targets the human sequence. Mice with the corrected allele exhibited normal serum phenylalanine levels. Through genome editing, we validated the utility of sgRNA. The genetically humanized mouse model suggested that germ-line genome editing of the pathogenic variant may be feasible for monogenic disorders by revealing the recovery of the phenotype; however, there are remaining issues with the tool, including its efficiency and accuracy. This genome editing protocol using a genetically humanized mouse model will provide insights for improving current issues and contribute to the establishment of heritable human genome editing protocols.},
}

*Phenylketonurias/genetics
Animals
*Gene Editing/methods
*Disease Models, Animal
Humans
Mice
Phenylalanine Hydroxylase/genetics
Zygote
Female
Phenotype
Male
CRISPR-Cas Systems
Genome, Human
Phenylalanine/genetics