@article {pmid40545778,
year = {2025},
author = {Li, Y and Jin, L and Li, W and Wang, K and Su, H and Mao, H and Chen, W and Lan, C and Li, Q and Kaufmann, K and Yan, W},
title = {Modulation of an evolutionarily conserved epigenetic regulon controlling abscisic acid catabolism enhances drought tolerance in wheat.},
journal = {The New phytologist},
volume = {},
number = {},
pages = {},
doi = {10.1111/nph.70302},
pmid = {40545778},
issn = {1469-8137},
support = {32272127//National Natural Science Foundation of China/ ; },
abstract = {Drought stress significantly reduces crop yield by triggering abscisic acid (ABA) accumulation in plants. It involves the suppression of CYP707A genes, which encode enzymes that catalyze ABA. However, little is known about epigenetic control in the CYP707A gene-mediated drought stress response in wheat. In this study, we reported that TaCYP707A-6A/6B/6D but not TaCYP707A-5A/5B/5D participates in drought response in common wheat. Disruption of TaCYP707A-6B showed enhanced drought tolerance but also decreased fertility. Expression of TaCYP707A-6B is negatively associated with H3K27me3 level. An evolutionarily conserved CTCTGYTY motif cluster (binding site for a Jumonji H3K27me3 demethylase) was found in the intron of TaCYP707A-6B as well as the intron of CYP707A homologs in other plant species. Blocking the CTCTGYTY motif by dead Cas9 (dCas9) maintained a high level of H3K27me3 on the CYP707A gene, while decreasing its expression level leading to enhanced drought tolerance in both wheat and Arabidopsis. In particular, the mutant in which the intron bound by H3K27me3 demethylase was cut out without change of splicing pattern showed enhanced drought tolerance. Therefore, our study provides a novel approach to improve plant drought tolerance by manipulating an evolutionarily conserved cis-element bound by histone demethylases in the intron of CYP707A genes.},
}

@article {pmid40545632,
year = {2025},
author = {Swinnen, G and Lizé, E and Loera Sánchez, M and Stolz, S and Soyk, S},
title = {Application of a GRF-GIF chimera enhances plant regeneration for genome editing in tomato.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.70212},
pmid = {40545632},
issn = {1467-7652},
support = {802008//European Research Council/International ; 310030_212218//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung/ ; },
abstract = {Genome editing has become a routine tool for functionally characterizing plant and animal genomes. However, stable genome editing in plants remains limited by the time- and labour-intensive process of generating transgenic plants, as well as by the efficient isolation of desired heritable edits. In this study, we evaluated the impact of the morphogenic regulator GRF-GIF on plant regeneration and genome editing outcomes in tomato. We demonstrate that expressing a tomato GRF-GIF chimera reliably accelerates the onset of shoot regeneration from callus tissue culture by approximately one month and nearly doubles the number of recovered transgenic plants. Consequently, the GRF-GIF chimera enables the recovery of a broader range of edited haplotypes and simplifies the isolation of mutants harbouring heritable edits, but without markedly interfering with plant growth and development. Based on these findings, we outline strategies that employ basic or advanced diagnostic pipelines for efficient isolation of single- and higher-order mutants in tomato. Our work represents a technical advantage for tomato transformation and genome editing, with potential applications across other Solanaceae species.},
}

@article {pmid40545185,
year = {2025},
author = {Mondal, AR and Misra, A},
title = {'Emerging cell-specific therapies in cardiovascular disease'.},
journal = {Vascular pharmacology},
volume = {},
number = {},
pages = {107516},
doi = {10.1016/j.vph.2025.107516},
pmid = {40545185},
issn = {1879-3649},
abstract = {Atherosclerosis is a leading cause of cardiovascular morbidity and mortality worldwide, driven by complex interactions among various plaque cell types, including endothelial cells, macrophages, and smooth muscle cells. Traditional therapies targeting systemic risk factors such as cholesterol and blood pressure fail to directly address the underlying mechanisms governing plaque formation and progression. Recent advances in cell-specific therapies offer new avenues for targeting the cellular and molecular processes driving atherosclerosis. This Review explores innovative strategies including nanoparticles, viral vectors and CRISPR-Cas9 technology, which have the potential to modulate gene expression and behaviour within plaques cells to alleviate disease. By focusing on the specific roles of key cell types in atherosclerosis, these emerging therapies promise to provide more precise, effective, and personalised treatment options without inducing off-target effects. Moreover, insights gained from successful applications of these technologies in oncology are considered for potential repurposing in atherosclerosis-related disease. As these cell-specific approaches advance through preclinical and clinical development, they may significantly enhance our ability to treat atherosclerosis at its cellular roots, offering new hope for reducing the burden of cardiovascular disease.},
}

@article {pmid40544537,
year = {2025},
author = {Sinha, S and Upadhyay, LSB},
title = {Understanding antimicrobial resistance (AMR) mechanisms and advancements in AMR diagnostics.},
journal = {Diagnostic microbiology and infectious disease},
volume = {113},
number = {2},
pages = {116949},
doi = {10.1016/j.diagmicrobio.2025.116949},
pmid = {40544537},
issn = {1879-0070},
abstract = {The overuse and abuse of antibiotics, which results in the evolution of resistant microorganisms, is the primary cause of the global health catastrophe known as antimicrobial resistance (AMR). The enzymatic breakdown of antibiotics, target site modification, efflux pump overexpression, and the formation of biofilm are some of the mechanisms responsible for acquiring antimicrobial resistance (AMR). These mechanisms enable bacteria to evade or neutralize the effects of antimicrobial agents, complicating treatment options and increasing mortality rates. The rapid dissemination of resistance genes via horizontal gene transfer further exacerbates the problem, necessitating urgent intervention. Advanced AMR diagnostics are transforming the fight against antimicrobial resistance. Biosensors enable rapid, point-of-care detection; Cluster regularly interspaced short palindromic repeat (CRISPR) technologies offer precise identification of resistance genes; and mass spectrometry provides fast, accurate profiling. Automated systems streamline workflows and boost throughput, while flow cytometry delivers real-time, single-cell analysis of phenotypic resistance. Together, these innovations accelerate detection and support targeted antimicrobial stewardship, essential for combating the global AMR threat. This review covers the mechanisms underlying antimicrobial resistance (AMR) and recent advancements in AMR diagnostic technologies.},
}

@article {pmid40544408,
year = {2025},
author = {Yu, D and Xie, Z and Zhang, Y and Xie, Z and Fan, Q and Luo, S and Xie, L and Li, M and Zeng, T and Zhang, M and Li, X and Wei, Y and Wu, A and Wan, L},
title = {A dual fluorescence channel RAA-based CRISPR-Cas12a/Cas13a system for highly sensitive detection of Gyrovirus galga1 and Gyrovirus homsa1.},
journal = {Virulence},
volume = {16},
number = {1},
pages = {2521012},
doi = {10.1080/21505594.2025.2521012},
pmid = {40544408},
issn = {2150-5608},
abstract = {Gyrovirus galga1 (GyG1) and Gyrovirus homsa1 (GyH1) are the second and third most common gyroviruses identified, respectively, after chicken anaemia virus. They were first reported in 2011 and are currently prevalent worldwide. However, limited research on these pathogens and a lack of prevention and control strategies have necessitated the establishment of a rapid diagnostic technique to address new challenges in infectious diseases. Recombinase acid amplification (RAA) combined with CRISPR - Cas12a or CRISPR - Cas13a technology has major advantages for highly sensitive and rapid diagnosis. Specific targets can activate CRISPR-Cas trans-cleavage activity, resulting in non-specific cleavage of single-stranded DNA by the CRISPR - Cas12a complex and RNA cleavage by the CRISPR - Cas13a complex. In this study, for the first time, we combined RAA-based CRISPR - Cas12a and CRISPR - Cas13a systems for simultaneous differential diagnosis of GyG1 and GyH1 infection. The results showed that dual fluorescence channel RAA-based CRISPR - Cas12a/Cas13a technology could detect GyG1 and GyH1 within one hour, with a minimum detection limit of 1.5 copies of the target DNA standard/µL and no cross-reactivity with other avian pathogens. In addition, this method could be used for clinical detection, with the results exhibiting high consistency with those obtained by qPCR. These findings demonstrate that our RAA-based CRISPR - Cas12a/Cas13a dual-channel detection system can detect two different subtypes of gyrovirus in a sample with good specificity and high sensitivity, improving the detection efficiency and providing a new technique for the study of viral infection dynamics.},
}

@article {pmid40544221,
year = {2025},
author = {Ajibode, ET and Bender, AR and Yehl, K},
title = {Profiling crRNA architectures for enhanced Cas12 biosensing.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {947},
pmid = {40544221},
issn = {2399-3642},
abstract = {CRISPR-Cas diagnostics are revolutionizing point-of-care molecular testing due to the programmability, simplicity, and sensitivity of Cas systems with trans-cleavage activity. CRISPR-Cas12 assays are promising for detecting single nucleotide polymorphisms (SNPs). However, reports vary widely describing Cas12 SNP sensitivity, and an underlying mechanism is lacking. We systematically varied crRNA length and valency to investigate the role of crRNA architectures on Cas12 biosensing in the context of speed-of-detection, sensitivity, and selectivity. Our results demonstrate that crRNAs complementary to 20 base pairs of the target DNA is optimal for rapid and sensitive detection, while a complementary length of 15 base pairs is ideal for robust SNP detection. Additionally, we uncovered a unique periodicity in SNP sensitivity based on nucleotide position and developed a structural model explaining what drives Cas12 SNP sensitivity. Lastly, we showed that bivalent CRISPR-Cas sensors have synergistic and enhanced activity that is distance dependent.},
}

@article {pmid40542569,
year = {2025},
author = {Han, R and Gao, X and Qi, Y and Lu, X and Wang, X and Tang, X},
title = {Spacer-Complementary Single-Stranded DNA Oligonucleotides Can Serve as Target-Specific Inhibitors in CRISPR/Cas9 Systems.},
journal = {Cell biochemistry and function},
volume = {43},
number = {6},
pages = {e70088},
doi = {10.1002/cbf.70088},
pmid = {40542569},
issn = {1099-0844},
support = {//This research was supported by Guangxi Natural Science Foundation Project (2025GXNSFAA069363, 2023GXNSFAA026260)./ ; },
abstract = {The continuous expression of the CRISPR/Cas system in organisms can lead to various potential issues. Some anti-CRISPR strategies have been developed to achieve precise control over CRISPR/Cas, yet these strategies are predominantly protein-based, with the most commonly used anti-CRISPR (Acr) proteins lacking sufficient target specificity. However, in this study, we designed a single-stranded DNA (ssDNA) inhibitor that was complementary to the spacer region on the guide RNA, operating at the nucleic acid level. We demonstrated that this method effectively inhibits the cleavage activity of Cas9-sgRNA ribonucleoprotein (RNP) in a target-specific manner through in vitro cleavage experiments. Furthermore, we explored the binding position and effective length of this inhibitory ssDNA, finding that its inhibitory effect was significantly reduced when the length of continuous complementarity with the 5' end of the spacer was less than 7nt. The truncated ssDNA also showed potential in reducing off-target effects. Moreover, we applied nucleic acid inhibitors to embryos via microinjection, and gene editing activity was significantly reduced, as evidenced by a decrease in the mosaicism rate of mouse embryos undergoing normal gene editing from (84.4 ± 4.4) % to 0%. Our study introduces a convenient and target-specific nucleic acid inhibitor capable of achieving precise regulation of gene editing.},
}

@article {pmid40540289,
year = {2025},
author = {Han, JH and Lee, SY and Park, HH},
title = {AcrVIA6 Is a Monomeric DNA-Binding Protein That Does Not Directly Inhibit Cas13a.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {39},
number = {12},
pages = {e70753},
pmid = {40540289},
issn = {1530-6860},
support = {RS-(2025-02316334)//National Research Foundation of Korea (NRF)/ ; },
abstract = {The CRISPR-Cas system is a crucial adaptive immune mechanism in prokaryotes, providing defense against invading genetic elements. Among various CRISPR-Cas systems, the type VI-A system, employing RNA-guided RNase Cas13a, has been extensively studied for its ability to target and degrade single-stranded RNA. Anti-CRISPR (Acr) proteins have evolved as natural inhibitors of these systems, with AcrVIA proteins specifically targeting the Cas13a enzyme. However, there is currently conflicting debate regarding the anti-CRISPR function of AcrVIA6. This study reveals that AcrVIA6 functions as a DNA-binding protein rather than a Cas13a inhibitor, as it does not block RNA cleavage. These findings challenge its role in CRISPR-Cas regulation.},
}

@article {pmid40540136,
year = {2025},
author = {Ponnurangan, V and Namachivayam, R and Pradeep, RKM and Jesudoss, D and Eswaran, K and Loganathan, A and Kumar, KK and Vaikuntavasan, P and Maduraimuthu, D and Shanmugam, V},
title = {Biotechnological breakthroughs in rice disease management: an overview from transgenics to CRISPR.},
journal = {Molecular biology reports},
volume = {52},
number = {1},
pages = {616},
pmid = {40540136},
issn = {1573-4978},
support = {BT/PR40456/ AGIII/103/1248/2020//Department of Biotechnology, New Delhi, India/ ; },
abstract = {Rice production faces persistent challenges due to a plethora of infectious agents, which cause substantial economic losses and pose significant threats to food security. Effective management of rice diseases is hindered by the lack of resistant sources and the slow pace of conventional breeding, which often fails to keep up with the rapid evolution of pathogens. Although transgenic approaches have contributed to disease resistance, they are limited by regulatory hurdles and public concern over the use of genetically modified organisms. Emerging genome-editing technologies, particularly CRISPR/Cas systems, offer promising alternatives by enabling the precise modification of native genes involved in plant susceptibility. CRISPR/Cas9-mediated inactivation of negative defense regulators, such as SWEET and ERF family genes, conferred broad-spectrum disease resistance. This review discusses the use of CRISPR/Cas technologies in developing disease-resistant rice varieties, along with emerging approaches and databases relevant to genome editing in plant research. Furthermore, it also highlights recent developments in CRISPR-based diagnostics for pathogen detection and the regulatory frameworks for the commercialization of edited crops. Overall, it underscores the transformative potential of CRISPR technologies in developing elite, climate-resilient rice cultivars, thereby supporting sustainable disease management and global food security.},
}

@article {pmid40539514,
year = {2025},
author = {Li, Y and Li, J and Pei, X and Wei, J and Gan, J and Lin, J},
title = {Catalytic-state structure of Candidatus Hydrogenedentes Cas12b revealed by cryo-EM studies.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf519},
pmid = {40539514},
issn = {1362-4962},
support = {2017YFA0504602//National Key Research and Development Program of China/ ; 32130063//National Key Research and Development Program of China/ ; 31770784//National Natural Science Foundation of China/ ; 32371252//National Natural Science Foundation of China/ ; 2021-01-07-00-07-E00074//Shanghai Municipal Education Commission/ ; SN-ZJU-SIAS009//Zhejiang University/ ; 31770784//National Natural Science Foundation of China/ ; 32371252//National Natural Science Foundation of China/ ; },
abstract = {The CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein) systems are adaptive immune mechanisms that play critical roles in defending archaea and bacteria against invading entities. These systems can be divided into two classes, with class 2 comprising three types (II, V, and VI). Because of their ability to cleave double-stranded DNA, many class 2 CRISPR-Cas proteins have been harnessed as genome editing tools. Unlike the well-studied type II Cas9 proteins, the structural studies of the type V-B Cas12b proteins are limited, hindering their engineering and broader application. Here, we report four complex structures of ChCas12b, which reveal many unique structural features. The folding of the single guide RNA (sgRNA) of ChCas12b is distinct from that of AacCas12b and BthCas12b. Notably, many of these unique features are involved in ChCas12b-sgRNA interaction, suggesting that they are co-evolved. While ChCas12b shares a conserved two-cation-assisted catalytic mechanism with its homologs, it recognizes a longer guide:target heteroduplex, potentially offering higher fidelity in DNA editing. Altogether, our studies suggested that Cas12b family proteins exhibit significant diversity in their folding, sgRNA and target DNA binding. In the future, it is worth characterizing more representative proteins to identify CRISPR-Cas proteins with higher gene editing ability and fidelity.},
}

@article {pmid40539280,
year = {2025},
author = {Bak, RO and Holm, M and Møller, B and Mikkelsen, JG and Mogensen, TH},
title = {[CRISPR/Cas gene editing of haematopoietic stem cells for curing primary immunodeficiency].},
journal = {Ugeskrift for laeger},
volume = {187},
number = {22},
pages = {},
doi = {10.61409/V02250083},
pmid = {40539280},
issn = {1603-6824},
abstract = {Primary immunodeficiencies are rare monogenic inborn errors of immunity and can involve any combination of infection, autoimmunity, inflammation, and malignancy. While increased use of whole genome sequencing has vastly improved diagnosis, curative treatment options beyond haematopoietic stem cell transplantation are still lacking behind. In this review, we present and discuss the promising avenues of CRISPR/Cas gene editing of patient stem cells for curing these diseases through homology-directed repair, base- or prime editing and delivery by nanoparticles or viral derivatives. However, technological, regulatory, and economic challenges exist on the road to safe and broad implementation of this technology for personalized medicine in the clinic.},
}

@article {pmid40538711,
year = {2025},
author = {Lathakumari, RH and Vajravelu, LK and Gopinathan, A and Vimala, PB and Panneerselvam, V and Ravi, SSS and Thulukanam, J},
title = {The gut virome and human health: From diversity to personalized medicine.},
journal = {Engineering microbiology},
volume = {5},
number = {1},
pages = {100191},
pmid = {40538711},
issn = {2667-3703},
abstract = {The human gut virome plays a crucial role in the gut and overall health; its diversity and regulatory functions influence bacterial populations, metabolism, and immune responses. Bacteriophages (phages) and eukaryotic viruses within the gut microbiome contribute to these processes, and recent advancements in sequencing technologies and bioinformatics have greatly expanded our understanding of the gut virome. These advances have led to the development of phage-based therapeutics, diagnostics, and artificial intelligence-driven precision medicine. The emerging field of phageomics shows promise for delivering personalized phage therapies that combat antimicrobial resistance by specifically targeting pathogenic bacteria while preserving beneficial microbes. Moreover, CRISPR-Cas systems delivered via phages have shown success in selectively targeting antibiotic resistance genes and enhancing treatment effectiveness. Phage-based diagnostics are highly sensitive in detecting bacterial pathogens, offering significant benefits for human health and zoonotic disease surveillance. This synthesis of the current knowledge highlights the pivotal role of the gut virome in regulating microbial communities and its transformative potential in personalized medicine, emphasizing its importance in advancing therapeutic and diagnostic strategies for improving health outcomes.},
}

@article {pmid40536889,
year = {2025},
author = {Wall, LA and Wall, D},
title = {Acquired CRISPR spacers and rhamnose-glucose polysaccharide defects confer resistance to Streptococcus mutans phage ɸAPCM01.},
journal = {Microbiology (Reading, England)},
volume = {171},
number = {6},
pages = {},
pmid = {40536889},
issn = {1465-2080},
mesh = {*Streptococcus mutans/virology/genetics/metabolism ; *Rhamnose/metabolism/genetics ; Biofilms/growth & development ; *Streptococcus Phages/genetics/physiology ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Glucose/metabolism ; Mutation ; *Polysaccharides/metabolism ; Bacterial Proteins/genetics/metabolism ; *Polysaccharides, Bacterial/genetics ; },
abstract = {Streptococcus mutans is commonly associated with the development of dental caries worldwide. Due to their specificity for S. mutans, phage represents a promising avenue for future targeted therapeutic strategies. In this study, we investigated how phage resistance develops in S. mutans. As a model phage, we used ɸAPCM01, which is known to infect a serotype e strain. We isolated and sequenced the genomes of 15 spontaneous resistant mutants and found that 10 had acquired novel clustered regularly interspaced short palindromic repeats (CRIPSR) spacers targeting the phage, with a total of 18 new spacers identified. Additionally, eight strains contained mutations in rhamnose-glucose polysaccharide biosynthetic genes, three of which also acquired spacers. Only the rgp mutants exhibited defects in phage adsorption, supporting the role of these cell surface glycans as the phage receptor. Mutations in rgpF and the newly identified gene rgpX led to severe cell division defects and impaired biofilm formation, the latter of which was also shared by an rgpD mutant. Thus, rgp mutations confer phage resistance but impose severe fitness costs, limiting pathogenic potential. Surprisingly, we found that ɸAPCM01 was capable of binding to and injecting its genome into UA159, a model serotype c strain. However, UA159 was resistant to infection due to an unknown post-entry defence mechanism. Consequently, ɸAPCM01 has the potential to infect both major serotypes associated with dental caries.},
}

*Streptococcus mutans/virology/genetics/metabolism
*Rhamnose/metabolism/genetics
Biofilms/growth & development
*Streptococcus Phages/genetics/physiology
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Glucose/metabolism
Mutation
*Polysaccharides/metabolism
Bacterial Proteins/genetics/metabolism
*Polysaccharides, Bacterial/genetics

@article {pmid40536816,
year = {2025},
author = {Liao, X and Liu, Q and Chuai, G},
title = {PrimeNet: rational design of Prime editing pegRNAs by deep learning.},
journal = {Briefings in bioinformatics},
volume = {26},
number = {3},
pages = {},
doi = {10.1093/bib/bbaf293},
pmid = {40536816},
issn = {1477-4054},
support = {T24250193//National Natural Science Foundation of China/ ; 32341008//National Natural Science Foundation of China/ ; 62002265//National Natural Science Foundation of China/ ; 2021YFF1201200//National Key Research and Development Program of China/ ; 2021YFF1200900//National Key Research and Development Program of China/ ; //Shanghai Pilot Program for Basic Research/ ; //Shanghai Science and Technology Innovation Action Plan-Key Specialization in Computational Biology/ ; //Shanghai Shuguang Scholars Project/ ; //Shanghai Excellent Academic Leader Project/ ; 2021SHZDZX0100//Shanghai Municipal Science and Technology Major Project/ ; //Fundamental Research Funds for the Central Universities/ ; 2025080107//Tongji University "Medicine + X" Cross Research Program/ ; },
mesh = {Humans ; *Gene Editing/methods ; *Deep Learning ; HEK293 Cells ; K562 Cells ; CRISPR-Cas Systems ; DNA Methylation ; Epigenesis, Genetic ; },
abstract = {The rapid development of gene editing technology has revolutionized life science research and biotechnology applications. Prime editing, a precise gene editing tool, has shown promise in various applications, including disease research and therapeutic interventions. However, its suboptimal editing efficiency for extensive fragments and lack of predictive models have hindered its widespread adoption. Existing models exhibit low prediction accuracy and limitations, such as neglecting epigenetic factors that impact gene editing effects. To address these challenges, we developed PrimeNet, a novel prediction model that integrates significant epigenetic factors, including chromatin accessibility and DNA methylation. By incorporating data from multiple cell lines and introducing multiscale convolution and attention mechanisms, PrimeNet enhances the accuracy of predictions and generalization performance. Our results show that PrimeNet achieves a Spearman correlation coefficient of 0.94 and 0.82 on two datasets originated from HEK293T and K562 cell lines, respectively, outperforming existing models. This novel model has the potential to guide experimental design, enhance the success rate of gene editing, and reduce unnecessary experimental costs, thereby advancing the application of gene editing technology in genetic disease treatment and related fields.},
}

Humans
*Gene Editing/methods
*Deep Learning
HEK293 Cells
K562 Cells
CRISPR-Cas Systems
DNA Methylation
Epigenesis, Genetic

@article {pmid40536049,
year = {2025},
author = {Ullah, M and Naeem, M and Andoh, V and Khan, MN and Han, J and Rizwan, M and Hussain, N and Saeed, M and Chen, Y and Chen, H},
title = {Lactic Acid Bacteria as Biofactories: Mechanistic Insights, Engineering Strategies, and Future Horizons for Heterologous Enzyme Expression.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c02311},
pmid = {40536049},
issn = {1520-5118},
abstract = {Lactic acid bacteria (LAB) constitute a genetically heterogeneous group that is uniquely capable of converting soluble carbohydrates into lactic acid. Such LAB, with a long history of safe consumption in fermented foods, are considered food-grade microorganisms and are highly sought after for a variety of biotechnological applications. Due to their unique properties, LAB can be genetically engineered to produce industrially significant enzymes. LAB act as an expression host for these enzymes by combining already existing engineering systems with techniques such as CRISPR-Cas. This review outlines the progress achieved to date on genome manipulation methods for LAB engineering and future perspectives of genetic tools. These strategies contribute greatly to fully unleashing the potential of LAB, and we further elaborate on how genome editing tools can enhance the capacity of heterologous expression in LAB.},
}

@article {pmid40533429,
year = {2025},
author = {Avaro, AS and Griffiths, AD and Santiago, JG},
title = {Degradation of Reporter Molecules Imposes a Fundamental Limit of Detection on CRISPR Diagnostics.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c01060},
pmid = {40533429},
issn = {1520-6882},
abstract = {The sensitivity of CRISPR-Cas systems used for molecular diagnostics remains a major bottleneck in the adoption of this technology. The vast majority of CRISPR-based assays use dually labeled, single-stranded reporters and fluorescence signal readouts to infer enzymatic activity and the presence (or absence) of target nucleic acid. The limit of detection of such assays is set by the kinetics of the Cas enzymes and a slow yet measurable increase in the fluorescence signal. We demonstrate here that the background signal and limits of detection of most assays are very likely limited by the degradation of reporter molecules. This degradation is dynamic and is not associated with enzymatic activity. We present theory and experiments to design and calibrate CRISPR assays. We introduce a new kinetic framework to account for the degradation of reporter molecules and derive a fundamental limit of detection for CRISPR-based assays. Our data show that Michaelis-Menten kinetics alone are insufficient to describe reporter (substrate) cleavage rates. The framework and techniques presented here should help reduce the frequency and magnitude of errors currently routinely made in quantifying CRISPR kinetics and interpreting CRISPR diagnostic fluorescence signals.},
}

@article {pmid40531937,
year = {2025},
author = {Poudel, B and Sathe, A and Bede, JC and Kushalappa, AC},
title = {Editing metacaspase (StMC7) gene enhances late blight resistance in Russet Burbank potato.},
journal = {PloS one},
volume = {20},
number = {6},
pages = {e0325702},
pmid = {40531937},
issn = {1932-6203},
mesh = {*Solanum tuberosum/genetics/microbiology ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics ; Phytophthora infestans/pathogenicity ; CRISPR-Cas Systems ; *Gene Editing ; *Plant Proteins/genetics ; *Caspases/genetics ; Gene Expression Regulation, Plant ; Gene Silencing ; Alternaria/pathogenicity ; Plants, Genetically Modified ; },
abstract = {Plants induce hypersensitive response programmed cell death (HR-PCD), upon biotrophic pathogen infection, to contain the pathogen to the point of infection. Apoptotic-like PCD (AL-PCD) has been reported upon prolonged hemibiotrophic and necrotrophic pathogen infection in potato, to feed on the dead cells for their growth. In potato, silencing of the gene StHRC lead to the suppression of AL-PCD, thus increasing resistance to blights in potato. This was also associated with a significant reduction in the expression of the metacaspase gene StMC7. Accordingly, the gene StMC7 was silenced in potato cultivar 'Russet Burbank' using CRISPR-Cas9 to improve disease resistance against late blight of potato caused by Phytophthora infestans. Following pathogen infection, the disease severity, pathogen biomass and StMC7 gene expression was lower in Stmc7 mutants as compared to wild type. Disease severity was also decreased in Alternaria solani inoculated Stmc7 mutants, compared to the wild type, suggesting possible multiple disease resistance in the Stmc7 knockdown mutants. This confirms that the silencing of StMC7 improves late blight disease resistance in potato.},
}

*Solanum tuberosum/genetics/microbiology
*Disease Resistance/genetics
*Plant Diseases/microbiology/genetics
Phytophthora infestans/pathogenicity
CRISPR-Cas Systems
*Gene Editing
*Plant Proteins/genetics
*Caspases/genetics
Gene Expression Regulation, Plant
Gene Silencing
Alternaria/pathogenicity
Plants, Genetically Modified

@article {pmid40531391,
year = {2025},
author = {Wang, Y and Li, H and Luo, S and Zhong, M and Liu, J and Li, B},
title = {Research Progress on Signal Conversion Based on Aptamer Combined CRISPR/Cas System in Biosensors.},
journal = {Molecular diagnosis & therapy},
volume = {},
number = {},
pages = {},
pmid = {40531391},
issn = {1179-2000},
support = {2025YFHZ0329//Sichuan Province Science and Technology Support Program/ ; 2024JYJ014//Sichuan Province Science and Technology Support Program/ ; 24CGZH10//Sichuan Province Science and Technology Support Program/ ; },
abstract = {The CRISPR/Cas system has been extensively used in the fields of biology, food safety, and environmental monitoring. This is in part because its can to be used in combination with isothermal amplification-mediated signal amplification technology along with its extraordinary trans-cleavage ability, which has initiated a new era of biosensing applications. The popularity of functional nucleic acids has enabled aptamers to convert non-nucleic acid substances into programmable nucleic acid sequences through methods such as direct detection, lock activation, sandwich design, induction of conformations, and split aptamers. Additionally, CRISPR/Cas systems have been extended beyond nucleic acid detection to include ions, small molecules, proteins, cells, bacteria, viruses, and other non-nucleic acid-based target substances. This article provides a brief overview of the mechanisms of action of four Cas proteins, the generation of aptamers, and their combined applications. Moreover, we focus on the research progress of biosensors based on aptamer-based signal conversion combined with the CRISPR/Cas system.},
}

@article {pmid40531235,
year = {2025},
author = {Wang, Y and Yang, H and Wang, K and Zhu, W and Wang, X},
title = {Establishment of a method for the rapid detection of carbapenem-resistant Pseudomonas aeruginosa based on bla_NDM with one-tube RPA-CRISPR/Cas12a visualization.},
journal = {Archives of microbiology},
volume = {207},
number = {8},
pages = {179},
pmid = {40531235},
issn = {1432-072X},
support = {No. LYG06521202376//the Lianyungang City Sixth 521 High-level Talent Training Project Scientific Research Project/ ; No. Z2022070//the 2022 Medical Research guiding Project of Jiangsu Provincial Health Commission/ ; No. SJCX23_1838//the 2023 Jiangsu Graduate Research Innovation Plan project/ ; },
mesh = {*Pseudomonas aeruginosa/genetics/drug effects/isolation & purification/enzymology ; *Carbapenems/pharmacology ; *Pseudomonas Infections/microbiology/diagnosis ; Humans ; *Nucleic Acid Amplification Techniques/methods ; CRISPR-Cas Systems ; Anti-Bacterial Agents/pharmacology ; *beta-Lactamases/genetics ; Bacterial Proteins/genetics ; China ; Drug Resistance, Bacterial/genetics ; Microbial Sensitivity Tests ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Pseudomonas aeruginosa (PA), which is a common Gram-negative bacterium, can become carbapenem-resistant Pseudomonas aeruginosa (CRPA) upon the development of antibiotic resistance, making clinical treatment difficult. CRPA with antibiotic resistance genes (ARGs), such as bla_NDM and bla_KPC, is common in China. The development of tests for ARGs would facilitate the more rapid identification of CRPA in China. Isothermal amplification research has improved, but limitations remain, including a lack of specialized equipment, the difficulty of developing sophisticated primers, and aerosol pollution. Thus, clinical examination methods must improve. We successfully integrated RPA with CRISPR/Cas12a, and we identified bla_NDM as our institution's primary CRPA. RPA-CRISPR/Cas12a could accurately detect target DNA within 40 min without cross-reacting with other bacteria. The results showed high concordance with conventional culture-based methods, including 100% agreement in clinical sample validation. The method reliably identified standard PA strains and 29 clinical isolates, detecting PA at concentrations as low as 10[-1] CFU. In addition, the closed-tube format effectively minimized the risk of aerosol contamination. This platform offers a rapid and user-friendly tool for identifying bla_NDM-positive CRPA, and this tool is particularly suitable for early screening and clinical management in resource-limited settings. The simplicity and accuracy of this approach make it a promising option for infection control and public health surveillance.},
}

*Pseudomonas aeruginosa/genetics/drug effects/isolation & purification/enzymology
*Carbapenems/pharmacology
*Pseudomonas Infections/microbiology/diagnosis
Humans
*Nucleic Acid Amplification Techniques/methods
CRISPR-Cas Systems
Anti-Bacterial Agents/pharmacology
*beta-Lactamases/genetics
Bacterial Proteins/genetics
China
Drug Resistance, Bacterial/genetics
Microbial Sensitivity Tests
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40530883,
year = {2025},
author = {Ganote, CL and Caesar, L and Rice, DW and Whitaker, RJ and Newton, ILG},
title = {Evolutionary trends in Bombella apis CRISPR-Cas systems.},
journal = {mSystems},
volume = {},
number = {},
pages = {e0016625},
doi = {10.1128/msystems.00166-25},
pmid = {40530883},
issn = {2379-5077},
abstract = {UNLABELLED: Bacteria and archaea employ a rudimentary immune system, CRISPR-Cas, to protect against foreign genetic elements such as bacteriophage. CRISPR-Cas systems are found in Bombella apis. B. apis is an important honey bee symbiont, found primarily in larvae, queens, and hive compartments. B. apis is found in the worker bee gut but is not considered a core member of the bee microbiome and has therefore been understudied with regard to its importance in the honey bee colony. However, B. apis appears to play beneficial roles in the colony, by protecting developing brood from fungal pathogens and by bolstering their development under nutritional stress. Previously, we identified CRISPR-Cas systems as being acquired by B. apis in its transition to bee association, as they are absent in a sister clade. Here, we assess the variation and distribution of CRISPR-Cas types across B. apis strains. We found multiple CRISPR-Cas types, some of which have multiple arrays, within the same B. apis genomes and also in the honey bee queen gut metagenomes. We analyzed the spacers between strains to identify the history of mobile element interaction for each B. apis strain. Finally, we predict interactions between viral sequences and CRISPR systems from different honey bee microbiome members. Our analyses show that the B. apis CRISPR-Cas systems are dynamic; that microbes in the same niche have unique spacers, which supports the functionality of these CRISPR-Cas systems; and that acquisition of new spacers may be occurring in multiple locations in the genome, allowing for a flexible antiviral arsenal for the microbe.

IMPORTANCE: Honey bee worker gut microbes have been implicated in everything from protection from pathogens to breakdown of complex polysaccharides in the diet. However, there are multiple niches within a honey bee colony that host different groups of microbes, including the acetic acid bacterium Bombella apis. B. apis is found in the colony food stores, in association with brood, in worker hypopharyngeal glands, and in the queen's digestive tract. The roles that B. apis may serve in these environments are just beginning to be discovered and include the production of a potent antifungal that protects developing bees and supplementation of dietary lysine to young larvae, bolstering their nutrition. Niche specificity in B. apis may be affected by the pressures of bacteriophage and other mobile elements, which may target different strains in each specific bee environment. Studying the interplay between B. apis and its mobile genetic elements (MGEs) may help us better understand microbial community dynamics within the colony and the potential ramifications for the honey bee host.},
}

@article {pmid40530826,
year = {2025},
author = {Carolak, E and Czajkowska, J and Stypułkowska, A and Waszczuk, W and Dutkiewicz, A and Grzymajlo, K},
title = {Being a better version of yourself: genetically engineered probiotic bacteria as host defense enhancers in the control of intestinal pathogens.},
journal = {Gut microbes},
volume = {17},
number = {1},
pages = {2519696},
doi = {10.1080/19490976.2025.2519696},
pmid = {40530826},
issn = {1949-0984},
mesh = {*Probiotics ; Humans ; *Gastrointestinal Microbiome ; Genetic Engineering ; Animals ; *Microorganisms, Genetically-Modified/genetics ; *Bacteria/genetics ; },
abstract = {Intestinal pathogens pose a significant global health burden, and traditional antibiotic treatments often disrupt the beneficial gut microbiota that plays a crucial role in maintaining host health through pathogen prevention and immune regulation. Although probiotics have emerged as promising therapeutic agents, their efficacy is limited by strain-dependent variations, survival challenges in the gastrointestinal tract, and inconsistent immune responses. Recent advances in genetic engineering, particularly CRISPR-Cas systems and their combinations with complementary technologies, such as Cre-lox and RecE/T, have enabled the precise modification of probiotic strains to enhance their therapeutic potential. These enhanced probiotics demonstrate improved functionality through multiple mechanisms, including increased adhesion via the expression of specific proteins (InlA, FnBPA, and LAP), targeted antimicrobial activity through engineered sensing systems (Lactococcus lactis detecting Vibrio cholerae CAI-1), and enhanced immunomodulation through cytokine production. Results have demonstrated the potential of genetically modified probiotics in preventing and treating gastrointestinal infections through mechanisms that include competitive exclusion, bacteriocin production, intestinal barrier reinforcement, and immune modulation. However, challenges remain in ensuring genetic stability and preventing horizontal gene transfer. Future research should focus on optimizing probiotic strains for targeted applications while addressing biosafety concerns. By understanding the complex interplay between probiotics, pathogens, and host immunity, innovative strategies can be developed to harness the full therapeutic potential of probiotic interventions in maintaining gut health.},
}

*Probiotics
Humans
*Gastrointestinal Microbiome
Genetic Engineering
Animals
*Microorganisms, Genetically-Modified/genetics
*Bacteria/genetics

@article {pmid40530689,
year = {2025},
author = {Budzak, J and Siegel, TN},
title = {Next generation genetic screens in kinetoplastids.},
journal = {Nucleic acids research},
volume = {53},
number = {11},
pages = {},
doi = {10.1093/nar/gkaf515},
pmid = {40530689},
issn = {1362-4962},
support = {ALTF 764-2022//EMBO Postdoctoral Fellowship/ ; 101105761 - VSG-SWITCH//Marie Skłodowska-Curie Actions Postdoctoral Fellowship/ ; SI 1610/2-2//German Research Foundation/ ; SI 1610/4-1//German Research Foundation/ ; 101044320//ERC-CoG/ ; },
mesh = {*Kinetoplastida/genetics ; *Genetic Testing/methods ; Single-Cell Analysis/methods ; Genome, Protozoan ; CRISPR-Cas Systems ; Genomics/methods ; High-Throughput Nucleotide Sequencing ; },
abstract = {The genomes of all organisms encode diverse functional elements, including thousands of genes and essential noncoding regions for gene regulation and genome organization. Systematic perturbation of these elements is crucial to understanding their roles and how their disruption impacts cellular function. Genetic perturbation approaches, which disrupt gene expression or function, provide valuable insights by linking genetic changes to observable phenotypes. However, perturbing individual genomic elements one at a time is impractical. Genetic screens overcome this limitation by enabling the simultaneous perturbation of numerous genomic elements within a single experiment. Traditionally, these screens relied on simple, high-throughput readouts such as cell fitness, differentiation, or one-dimensional fluorescence. However, recent advancements have introduced powerful technologies that combine genetic screens with image-based and single-cell sequencing readouts, allowing researchers to study how perturbations affect complex cellular phenotypes on a genome-wide scale. These innovations, alongside the development of CRISPR-Cas technologies, have significantly enhanced the precision, efficiency, and scalability of genetic screening approaches. In this review, we discuss the genetic screens performed in kinetoplastid parasites to date, emphasizing their application to both coding and noncoding regions of the genome. Furthermore, we explore how integrating image-based and single-cell sequencing technologies with genetic screens holds the potential to deliver unprecedented insights into cellular function and regulatory mechanisms.},
}

*Kinetoplastida/genetics
*Genetic Testing/methods
Single-Cell Analysis/methods
Genome, Protozoan
CRISPR-Cas Systems
Genomics/methods
High-Throughput Nucleotide Sequencing

@article {pmid40529587,
year = {2025},
author = {Lee, CK and Lee, HJ and Jeong, SH and Lee, SJ},
title = {Precision targeting of genetic variations in mixed bacterial cultures using CRISPR-Cas12a-programmed λ phages.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1575339},
pmid = {40529587},
issn = {1664-302X},
abstract = {The CRISPR-Cas system, an adaptive immune mechanism in prokaryotes against bacteriophages, has been developed into a versatile tool for recognizing and cleaving target nucleic acid sequences. In this study, we developed a model system by integrating CRISPR-Cas12a into the genome of temperate bacteriophage λ, enabling precise regulation of lysogeny and lysis in Escherichia coli. We confirmed that λ phage, armed with Cas12a nuclease and CRISPR RNA (crRNA) targeting specific sequences, could inhibit the lysogenic cycle of E. coli cells. We demonstrated that the CRISPR-Cas12a-loaded temperate λ phage mimicked a lytic phage by selectively killing cells carrying the target genomic sequence. Furthermore, by employing truncated crRNA to enhance target recognition specificity, we found that the synthetic phage could distinguish single nucleotide variations in the genomic target DNA, enabling precise targeting and selective elimination of target cells in homogeneous bacterial cultures. To further validate its specificity, we tested this system in mixed bacterial cultures, wherein Cas12a nuclease and truncated crRNA-loaded bacteriophages selectively eliminated only those cells carrying the target sequences perfectly matching the crRNA. These results highlight the potential of this approach for advancing precision microbiome modulation.},
}

@article {pmid40528015,
year = {2025},
author = {Ottaviani, L and Lefeuvre, R and Montes, E and Widiez, T and Giorni, P and Mithöfer, A and Marocco, A and Lanubile, A},
title = {A loss-of-function of ZmWRKY125 induced by CRISPR/Cas9 improves resistance against Fusarium verticillioides in maize kernels.},
journal = {Plant cell reports},
volume = {44},
number = {7},
pages = {144},
pmid = {40528015},
issn = {1432-203X},
mesh = {*Zea mays/microbiology/genetics/immunology/metabolism ; *Fusarium/physiology/pathogenicity ; *Plant Diseases/microbiology/genetics/immunology ; *Disease Resistance/genetics ; *Plant Proteins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Plant ; Cyclopentanes/metabolism ; Oxylipins/metabolism ; Abscisic Acid/metabolism ; Plant Growth Regulators/metabolism ; Loss of Function Mutation ; Transcription Factors/genetics/metabolism ; },
abstract = {ZmWRKY125 negatively regulates maize resistance to Fusarium verticillioides infection through modulating phytohormone, ROS scavenging and secondary metabolite gene expression as well as jasmonic and abscisic acid biosynthetic pathway activity. Fusarium verticillioides causes heavy damage to maize growth and yield and is responsible for mycotoxin contamination. Despite its widespread occurrence, few resistant genes have been identified and functionally validated for their role in the defense mechanisms against this fungus in maize. WRKY transcription factors are known to be crucial in regulating the expression of defense-responsive genes towards pathogen attack. In this context, in our previous genome-wide association study one SNP in the gene ZmWRKY125 was found significantly associated with the responses to F. verticillioides infection in maize seedlings. Here, loss-of-function mutant lines of ZmWRKY125 were obtained by the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system. The zmwrky125 edited lines were phenotypically evaluated showing a decrease by about 5 and 4 times of Fusarium ear rot (FER) severity and fumonisin contamination, respectively, compared to the wild-type genotype. The transient expression of ZmWRKY125 in maize protoplasts confirmed a nuclear localization as expected for a transcription factor. RNA-seq analysis comparison using two zmwrky125 edited lines and the wild-type genotype highlighted an enhanced modulation of the jasmonic acid (JA) and abscisic acid (ABA) hormones, redox state, cell wall modification, and secondary metabolism-associated genes after fungal infection. Moreover, the increased expression of JA- and ABA-related genes correlated with a wider accumulation of these two phytohormones in the mutant background in contrast to wild-type. This data provided new information for understanding the function of ZmWRKY125, despite further field evaluations will be required for validation of the resistance against FER.},
}

*Zea mays/microbiology/genetics/immunology/metabolism
*Fusarium/physiology/pathogenicity
*Plant Diseases/microbiology/genetics/immunology
*Disease Resistance/genetics
*Plant Proteins/genetics/metabolism
*CRISPR-Cas Systems/genetics
Gene Expression Regulation, Plant
Cyclopentanes/metabolism
Oxylipins/metabolism
Abscisic Acid/metabolism
Plant Growth Regulators/metabolism
Loss of Function Mutation
Transcription Factors/genetics/metabolism

@article {pmid40527733,
year = {2025},
author = {Obuchi, W and Zargani-Piccardi, A and Leandro, K and Rufino-Ramos, D and Di Lanni, E and Frederick, DM and Maalouf, K and Nieland, L and Xiao, T and Repiton, P and Vaine, CA and Kleinstiver, BP and Bragg, DC and Lee, H and Miller, MA and Breakefield, XO and Breyne, K},
title = {Engineering of CD63 Enables Selective Extracellular Vesicle Cargo Loading and Enhanced Payload Delivery.},
journal = {Journal of extracellular vesicles},
volume = {14},
number = {6},
pages = {e70094},
pmid = {40527733},
issn = {2001-3078},
support = {//Ionis Pharmaceuticals/ ; NCI-CA232103/BC/NCI NIH HHS/United States ; //Friedreich's Ataxia Research Alliance Australia/ ; //Daiichi Sankyo Company/ ; NIH DP2-CA259675//National Science Foundation/ ; NIH DP2-CA281401//National Science Foundation/ ; NIH K22-CA2802019-01//National Science Foundation/ ; NIH R01-GM138790//National Science Foundation/ ; //Kayden-Lambert MGH Research Scholar Award/ ; },
mesh = {*Extracellular Vesicles/metabolism ; *Tetraspanin 30/metabolism/genetics ; Animals ; Humans ; Mice ; HEK293 Cells ; Protein Engineering/methods ; },
abstract = {Extracellular vesicles (EVs) are mediators of intercellular communication through the transfer of nucleic acids, lipids and proteins between cells. This property makes bioengineered EVs promising therapeutic vectors. However, it remains challenging to isolate EVs with a therapeutic payload due to the heterogeneous nature of cargo loading into EVs. In this study, enrichment of EVs with a desired cargo was possible through engineering of the hallmark CD63 transmembrane protein. E-NoMi refers to engineered CD63 with mCherry on the inside of the EV membrane and a tag (3xFLAG) exposed on the outside of the EV membrane. To facilitate EV loading during biogenesis, cargo proteins, such as EGFP, Cre recombinase and the CRISPR-Cas nuclease (SaCas9), were fused to a nanobody (Nb) protein with a high affinity for mCherry. FLAG-tag-based immunocapture from cell conditioned media allowed selection of cargo-loaded E-NoMi-EVs, and tobacco etch virus (TEV) protease cleavage sites were used to remove the 3xFLAG-tag from the surface of E-NoMi-EVs after capture. For functional payload delivery to recipient cells, the vesicular stomatitis virus G (VSV-G) fusogenic protein was incorporated into E-NoMi-EVs to form fusogenic EV-based vectors (EVVs) and proved to be 10-fold more effective at cargo delivery than EVs generated by size-exclusion chromatography. Functional delivery of cargo with E-NoMi-EVVs was validated in two mouse brain models in vivo.},
}

*Extracellular Vesicles/metabolism
*Tetraspanin 30/metabolism/genetics
Animals
Humans
Mice
HEK293 Cells
Protein Engineering/methods

@article {pmid40527417,
year = {2025},
author = {Yaikhan, T and Singkhamanan, K and Suwannasin, S and Dechathai, T and Yingkajorn, M and Chusri, S and Surachat, K},
title = {Genomic insights into Metapseudomonas otitidis PA-NS83: The first clinical isolate from Thailand and its comparative genomic analysis.},
journal = {Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases},
volume = {},
number = {},
pages = {105786},
doi = {10.1016/j.meegid.2025.105786},
pmid = {40527417},
issn = {1567-7257},
abstract = {Metapseudomonas otitidis was first isolated from human middle ear fluid and has since been detected in both environmental and clinical samples, emerging as an opportunistic pathogen linked to chronic otitis media and other infections. This study reports the first clinical isolate of M. otitidis from Thailand, PA-NS83, and presents a comprehensive genomic characterization. Whole-genome sequencing and comparative analysis with 37 publicly available M. otitidis genomes revealed a diverse antimicrobial resistance (AMR) profile, with PA-NS83 carrying AMR genes commonly found in environmental isolates. Virulence gene analysis identified key determinants associated with biofilm formation, motility, secretion systems, and iron acquisition, highlighting its potential pathogenicity. Pan-genome analysis demonstrated substantial genomic diversity, with PA-NS83 clustering closely with M. otitidis CSMC7, an environmental isolate from polystyrene waste. However, PA-NS83 harbored 419 unique genes, including virulence-associated genes and a CRISPR-Cas system, suggesting adaptation to clinical settings. These findings underscore the genetic plasticity of M. otitidis and its potential role in human infections. Continued genomic surveillance and functional studies are essential to further assess its clinical significance and antimicrobial resistance mechanisms.},
}

@article {pmid40527291,
year = {2025},
author = {Wang, J and Zhang, W and Li, W and Xie, Q and Zang, Z and Liu, C},
title = {Enhancement of CRISPR-Cas12a system through universal circular RNA design.},
journal = {Cell reports methods},
volume = {5},
number = {6},
pages = {101076},
doi = {10.1016/j.crmeth.2025.101076},
pmid = {40527291},
issn = {2667-2375},
mesh = {*RNA, Circular/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Humans ; Gene Editing/methods ; *Endodeoxyribonucleases/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; DNA Repair ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Precise control of Cas12a activity is crucial to address incompatibility in isothermal amplification-CRISPR-Cas12a one-pot nucleic acid detection. We developed a light-triggerable circular RNA system for dynamic LbCas12a regulation. By employing circular CRISPR guide RNA (crRNA) or a split circular universal direct repeat region with a replaceable spacer, we resolved the incompatibility between isothermal amplification and CRISPR detection. This system demonstrated robust performance in detecting trace nucleic acids in clinical samples. Furthermore, DNA modifications on circular crRNA enabled CRISPR-Cas12a regulation via base excision repair (BER) enzymes, offering potential for BER enzyme detection and modulation of LbCas12a cleavage activity by BER enzymes. This programmable strategy holds promise for selective gene editing in cells with elevated BER enzyme expression, such as uracil DNA glycosylase (UDG) in colon cancer cells. The circular RNA-assisted approach represents a resource-efficient method with significant potential for medical diagnostics and future clinical gene therapy applications.},
}

*RNA, Circular/genetics/metabolism
*CRISPR-Cas Systems/genetics
Humans
Gene Editing/methods
*Endodeoxyribonucleases/metabolism/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
DNA Repair
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid40526416,
year = {2025},
author = {Ortega-Sanz, I and Rubio, A and Pérez-Pulido, AJ},
title = {Bacteriophages in Pseudomonas aeruginosa evade the CRISPR-Cas I-F system by depletion of PAM sequences.},
journal = {Microbial genomics},
volume = {11},
number = {6},
pages = {},
pmid = {40526416},
issn = {2057-5858},
mesh = {*Pseudomonas aeruginosa/virology/genetics ; *CRISPR-Cas Systems/genetics ; *Bacteriophages/genetics ; Genome, Viral ; Base Composition ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins systems provide bacteria with an adaptive immune system against exogenous sequences, such as plasmids and bacteriophages (viruses of prokaryotes). To avoid autoimmunity, the recognition of a very short sequence called the protospacer adjacent motif (PAM) is essential for efficient immune response triggering. This raises the question of whether viruses targeted by certain CRISPR-Cas systems have a higher or lower frequency of their PAM sequences than non-targeted viruses. This was tested here in the opportunistic human pathogen Pseudomonas aeruginosa that presents five different types of CRISPR-Cas systems. The most frequent of them is the subtype I-F (present in 36% of the strains), which has the PAM 5'-CC sequence. When the viral genomes targeted by this system were analysed, their PAM frequency was found to be lower than that of non-targeted viruses. Remarkably, targeted viruses have comparatively lower G+C content. All this could be the result of selection pressure on the genome of these viruses to escape from the CRISPR-Cas I-F system.},
}

*Pseudomonas aeruginosa/virology/genetics
*CRISPR-Cas Systems/genetics
*Bacteriophages/genetics
Genome, Viral
Base Composition
*Clustered Regularly Interspaced Short Palindromic Repeats
Humans

@article {pmid40526281,
year = {2025},
author = {Bulle, M and Rahman, MM and Islam, MR and Abbagani, S},
title = {Strategies to develop climate-resilient chili peppers: transcription factor optimization through genome editing.},
journal = {Planta},
volume = {262},
number = {2},
pages = {30},
pmid = {40526281},
issn = {1432-2048},
mesh = {*Capsicum/genetics/physiology ; *Gene Editing/methods ; *Transcription Factors/genetics/metabolism ; Stress, Physiological ; Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Climate Change ; Genome, Plant ; },
abstract = {Chili peppers (Capsicum spp.), a globally significant crop revered for their nutritional, economic, and cultural importance, are increasingly imperiled by the converging burdens of climate-induced abiotic stresses, including drought, heat, and salinity, and relentless biotic assaults from pathogens and insect herbivores. These overlapping stressors not only destabilize yield but also compromise the metabolic intricacy responsible for the accumulation of health-promoting secondary metabolites. Although Capsicum exhibits remarkable genetic and phytochemical diversity, the integrated transcriptional, metabolic, and epigenetic frameworks that underpin its stress resilience remain poorly delineated. This review synthesizes recent advances in decoding core transcription factor families, such as CaNAC, CaWRKY, and CaMYB, that serve as pivotal regulators of osmotic adjustment, reactive oxygen species detoxification, hormonal crosstalk, and secondary metabolite biosynthesis under stress conditions. We further highlight how multi-omics-guided gene discovery, when paired with CRISPR/Cas-mediated genome editing, enables precise reprogramming of key regulatory loci to enhance adaptive responses. Emerging innovations, including base editing, prime editing, and novel nucleases like Cas12a and Cas13d, are expanding the functional genome-editing landscape, while the integration of morphogenic regulators and genotype-independent transformation platforms is beginning to circumvent long-standing obstacles in Capsicum genetic engineering. Lastly, we propose a transformative framework that converges transcription factor modulation, multi-omics strategies, precision phenotyping, and next-generation genome editing to accelerate the development of climate-resilient Capsicum cultivars with optimized metabolic traits. This strategic convergence of molecular insight and biotechnological innovation offers a robust foundation for building next-generation chili pepper varieties capable of withstanding intensifying environmental and pathogenic pressures, ultimately safeguarding yield, nutritional quality, and agricultural sustainability in the face of global climate change.},
}

*Capsicum/genetics/physiology
*Gene Editing/methods
*Transcription Factors/genetics/metabolism
Stress, Physiological
Plant Proteins/genetics/metabolism
CRISPR-Cas Systems
Climate Change
Genome, Plant

@article {pmid40525624,
year = {2025},
author = {Zhang, Y and Lin, Y and Li, R and Jiang, D and Cai, R and Su, G and Yu, Y and Qu, G},
title = {Split G-Quadruplex-Integrated CRISPR-Cas Biosensor for One-Pot, Signal-On Visual Detection of Pathogen Nucleic Acids.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c02037},
pmid = {40525624},
issn = {1520-6882},
abstract = {Pathogen nucleic acid analysis has emerged as an indispensable component of contemporary healthcare systems, serving dual roles in personalized clinical management and population-level disease surveillance. Herein, we present a novel G-quadruplex-integrated CRISPR-Cas biosensing platform that performs in a signal-on mechanism for colorimetric detection of pathogen nucleic acids in one-pot. By harnessing the unique properties of split G4 structures, we develop a universal visual probe that generates a distinct green colorimetric signal upon target recognition, which effectively couples with both Cas12 and Cas13 systems. Using the monkeypox virus (MPXV) B7R gene and a conserved respiratory syncytial virus (RSV) sequence as model targets for Cas12a and Cas13, respectively, we establish a detection workflow combining recombinase polymerase amplification (RPA) with CRISPR-mediated cleavage, visualized through enzymatic mediated color conversion. The tube-in-tube cartridge architecture adopted in this work enables seamless integration of RPA and CRISPR-based detection within a single closed-tube system, effectively eliminating cross-contamination risks. We successfully validate the platform for detection of MPXV in environmental samples and RSV in clinical specimens, achieving a detection limit of 1 copy per test and perfect concordance with PCR methods (40/40 agreement). The colorimetric biosensing platform developed herein demonstrates rapid (<60 min) and facial performance, establishing a novel molecular diagnostic paradigm that achieves laboratory-comparable accuracy for real-time surveillance and point-of-care applications.},
}

@article {pmid40524239,
year = {2025},
author = {Villora, SA and Zhao, Y and Silva, PC and Hahn, AA and Olanin, V and Groll, D and Maurer, S and Roetzer, V and Szymanski, W and Procida-Kowalski, T and Philipp, N and Koch, A and Bartkuhn, M and Graumann, J and Volckmann, R and Koster, J and Rossbach, O and Salzig, D and Dammann, R and Sigges, C and Halbritter, J and Haerteis, S and Richter, AM},
title = {Epigenetic silencing and CRISPR-mediated reactivation of tight junction protein claudin10b (CLDN10B) in renal cancer.},
journal = {Clinical epigenetics},
volume = {17},
number = {1},
pages = {102},
pmid = {40524239},
issn = {1868-7083},
support = {2023_1_01//FCMH Forschungscampus Mittelhessen/ ; 2023_1_01//FCMH Forschungscampus Mittelhessen/ ; 2023_1_01//FCMH Forschungscampus Mittelhessen/ ; 2024_1_02//FCMH Forschungscampus Mittelhessen,/ ; 2024_1_02//FCMH Forschungscampus Mittelhessen,/ ; 2024_1_02//FCMH Forschungscampus Mittelhessen,/ ; 031B1232B//BMBF/ ; 031B1232B//BMBF/ ; 031B1232B//BMBF/ ; },
mesh = {Humans ; *Claudins/genetics ; DNA Methylation ; *Kidney Neoplasms/genetics ; *Carcinoma, Renal Cell/genetics ; Epigenesis, Genetic ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Gene Silencing ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; Male ; Female ; Tight Junctions ; Mixed Function Oxygenases ; Proto-Oncogene Proteins ; },
abstract = {BACKGROUND: The kidney's tubular system relies on cell polarity and tight junctions to maintain structure and function and disruptions contribute to diseases like cancer. Loss of tight junction proteins such as Claudins can actively contribute to tumorigenesis.

RESULTS: We aimed to identify biomarkers for renal carcinoma, after kidney transplantation and conventional kidney tumors. We identified the epigenetic silencing of the Claudin 10 gene isoform B (CLDN10B) through DNA hypermethylation in renal cancers, including clear cell (ccRCC), papillary (pRCC) and post-transplantation renal carcinoma (PT-ccRCC). In contrast, CLDN10A was hypomethylated in ccRCC and pRCC. Differential methylation of the isoforms discriminates RCC from other malignancies. The epigenetic alteration of CLDN10B significantly correlated with reduced patient survival and advanced tumor staging. CLDN10B overexpression or induction significantly inhibited migration, cell cycle progression, and cellular growth. Using a CRISPR-based epigenetic editing tool reactivated CLDN10B to its endogenous level using VP160 and TET1 by promoter demethylation and significantly demonstrated its tumor-suppressive effects in 2D and 3D cell models.

CONCLUSION: Our findings suggest that CLDN10B acts as a tumor suppressor, and its epigenetic regulation may represent a therapeutic target for RCC. Ultimately, understanding CLDN10B's regulation and function could provide new insights into renal cancer treatment.},
}

Humans
*Claudins/genetics
DNA Methylation
*Kidney Neoplasms/genetics
*Carcinoma, Renal Cell/genetics
Epigenesis, Genetic
Cell Line, Tumor
Gene Expression Regulation, Neoplastic
Gene Silencing
Promoter Regions, Genetic
CRISPR-Cas Systems
Male
Female
Tight Junctions
Mixed Function Oxygenases
Proto-Oncogene Proteins

@article {pmid40524010,
year = {2025},
author = {Levengood, H and Smith, L and Gillis, S and Zhou, Y and Zhang, C},
title = {Plantago species are emerging model organisms for functional genomics and stress biology.},
journal = {Plant cell reports},
volume = {44},
number = {7},
pages = {142},
pmid = {40524010},
issn = {1432-203X},
support = {1923557//National Science Foundation/ ; },
mesh = {*Genomics/methods ; *Plantago/genetics/physiology ; *Stress, Physiological/genetics ; Genome, Plant/genetics ; CRISPR-Cas Systems ; },
abstract = {Species in the Plantago genus are emerging model organisms to multiple research disciplines. The genus Plantago has long been recognized for its significance in various research fields, yet it remains underutilized as a model organism in scientific studies. Several Plantago species possess unique traits, including easily accessible vascular tissues, medicinal properties, gynodieocity, and remarkable adaptability to diverse environmental conditions. These characteristics position Plantago as a promising model for research in areas such as plant vascular biology, stress physiology, reproductive biology, ecology, and medicinal biochemistry. Recent advancements, including the development of genetic transformation systems, the availability of sequenced genomes, and the application of CRISPR-Cas9 technology, have significantly enhanced the capability of using Plantago as a model system. This review discusses the research potential of Plantago species, highlighting key historical discoveries and recent breakthroughs that demonstrate their value across multiple scientific disciplines.},
}

*Genomics/methods
*Plantago/genetics/physiology
*Stress, Physiological/genetics
Genome, Plant/genetics
CRISPR-Cas Systems

@article {pmid40523955,
year = {2025},
author = {Rong, C and Zhang, R and Xie, J and Li, J and Yan, T and Liu, Z and Liu, Y and Xu, R and Shi, X and Zhao, X and Song, J and Meng, Y and Chang, Z and Ding, Y and Ding, C},
title = {Type-A and -C response regulator genes positively impact rice plant height and panicle architecture.},
journal = {Plant cell reports},
volume = {44},
number = {7},
pages = {141},
pmid = {40523955},
issn = {1432-203X},
support = {No BK20231470//Basic Research Program of Jiangsu Province/ ; },
mesh = {*Oryza/genetics/growth & development/metabolism/anatomy & histology ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; Cytokinins/metabolism ; Signal Transduction/genetics ; Mutation ; Plant Growth Regulators/metabolism ; CRISPR-Cas Systems ; Genes, Plant ; },
abstract = {Thirteen type -A and two type -C RR genes exhibited distinct expression patterns and unique functions. Notably, RR2 and RR4 had the most significant positive effects impact on panicle development. Cytokinin signal transduction occurs through a "two-component system". Type-A and -C response regulators (RRs) are groups of proteins of similar structures constituting significant components of cytokinin signal transduction. In rice, 13 (Type-A) and 2 (Type-C) RRs have been identified to date; however, their functions remain partially known. In this study, we examined the expression patterns of Type-A and Type-C RRs in rice using RNA-Seq and confirmed their functions by constructing mutants of the 15 genes with CRISPR/Cas9. Almost all Type-A RRs played positive roles in the development of secondary branches and secondary spikelets, particularly RR2 and RR4. Notably, rr1 rr2 and rr8 rr12 rr13 higher-order mutants displayed small panicle sizes and decreased plant height. Additionally, both Type-C RRs played positive roles in regulating heading date. RNA-seq revealed several genes with significantly altered expression in the rr2 and rr4 mutants, with almost half of the differentially expressed genes (DEGs) overlapping between the two mutants. Many of the DEGs were associated with the cytokinin and abscisic acid pathways. Our findings provide new insights into the functions of Type-A and -C RRs in rice growth and may serve as a foundation for future studies focusing on cytokinin signaling.},
}

*Oryza/genetics/growth & development/metabolism/anatomy & histology
Gene Expression Regulation, Plant
*Plant Proteins/genetics/metabolism
Cytokinins/metabolism
Signal Transduction/genetics
Mutation
Plant Growth Regulators/metabolism
CRISPR-Cas Systems
Genes, Plant

@article {pmid40522908,
year = {2025},
author = {Schloßhauer, JL and Shamloo, S and Schamrin, K and Imig, J},
title = {Dual CRISPR-Interference Strategy for Targeting Synthetic Lethal Interactions Between Non-Coding RNAs in Cancer Cells.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {219},
pages = {},
doi = {10.3791/67752},
pmid = {40522908},
issn = {1940-087X},
mesh = {Humans ; *RNA, Long Noncoding/genetics ; Cell Line, Tumor ; *CRISPR-Cas Systems ; Streptococcus pyogenes/genetics/enzymology ; *Melanoma/genetics/pathology ; Staphylococcus aureus/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Long non-coding RNAs (lncRNAs) represent a vast and functionally diverse class of RNA molecules, with over 100,000 predicted in the human genome. Although lncRNAs are less conserved across species compared to protein-coding genes, they play critical roles in gene regulation, chromatin interactions, and cancer progression. Their involvement in cancer makes them promising therapeutic targets. CRISPR interference (CRISPRi), utilizing catalytically inactive Cas9 fused with a transcriptional repressor such as KRAB-MeCP2, offers a precise method for targeting nuclear lncRNAs and assessing their functions. This study introduces a dual CRISPRi system using orthogonal CRISPRi technologies from Staphylococcus aureus and Streptococcus pyogenes based on dCas9-KRAB-MeCP2, optimized for combinatorial targeting of lncRNAs in human melanoma cells. The protocol facilitates combinatorial gene knockdown or synthetic lethal screening of lncRNA pairs, providing a novel tool for cancer research. By exploring synthetic lethality between lncRNAs, this approach can help identify lncRNA interactions critical for cancer cell survival, offering new therapeutic strategies. The dual system's functionality is demonstrated, highlighting its potential in identifying critical cancer-specific lncRNA interactions.},
}

Humans
*RNA, Long Noncoding/genetics
Cell Line, Tumor
*CRISPR-Cas Systems
Streptococcus pyogenes/genetics/enzymology
*Melanoma/genetics/pathology
Staphylococcus aureus/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40522884,
year = {2025},
author = {Christenson, AE and Divekar, NS and Hernández Zamora, MG and Nguyen, CD and Nuñez, JK},
title = {CRISPR Epigenome Editing in Human Cells using Plasmid DNA Transfection and mRNA Nucleofection Delivery.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {219},
pages = {},
doi = {10.3791/68089},
pmid = {40522884},
issn = {1940-087X},
mesh = {Humans ; *Plasmids/genetics/administration & dosage ; *RNA, Messenger/genetics/administration & dosage ; *Transfection/methods ; *DNA/genetics/administration & dosage ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Epigenome/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Epigenome Editing ; },
abstract = {Epigenetics refers to chemical modifications of histone proteins and DNA that can regulate the expression of genes. The human epigenome is altered dynamically during cell differentiation and aging, and many diseases are associated with aberrant epigenome patterning. Recent advances in CRISPR have led to the development of programmable tools to edit epigenetic modifications at targeted genomic loci, enabling precise rewriting of epigenetic modifications in human cells. CRISPR-based epigenome editors rely on catalytically dead Cas9 coupled with epigenetic modifiers that ultimately result in programmed repression or activation of targeted genes in mammalian genomes. Unlike traditional genome editing methods, epigenome editing does not require DNA breaks or changes in the human genome sequence and thus serves as a safer alternative to control gene expression. In this protocol, we highlight two different methods to perform dCas9-mediated epigenome editing in human cell lines using plasmid DNA transfections and nucleofection of mRNAs encoding CRISPR epigenome editors. We demonstrate programmable epigenome editing to transiently repress genes using CRISPR interference (CRISPRi) and for silencing genes durably for many weeks using CRISPRoff, a fusion of dCas9 to the KRAB domain and de novo DNA methyltransferase complex. We also provide guidance on quantitative methods to measure successful epigenome editing of target genes and key considerations on which epigenome editing tool to use, depending on experimental criteria.},
}

Humans
*Plasmids/genetics/administration & dosage
*RNA, Messenger/genetics/administration & dosage
*Transfection/methods
*DNA/genetics/administration & dosage
*Gene Editing/methods
*CRISPR-Cas Systems
*Epigenome/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
Epigenome Editing

@article {pmid40522066,
year = {2025},
author = {Zhang, J and Zhu, A and Wei, S and Shi, Y and Chen, C and Huang, T and Tang, T and Zhao, J and Cai, Y and Han, C and Zhao, J and Wang, Y},
title = {Identification of Pan-Coronavirus Neutralizing Aptamers Through CRISmers Targeting the Conserved Fusion Peptide.},
journal = {Advanced healthcare materials},
volume = {},
number = {},
pages = {e2501869},
doi = {10.1002/adhm.202501869},
pmid = {40522066},
issn = {2192-2659},
support = {2023YFA0915000//National Key Research and Development Program of China/ ; 2022YFC2303700//National Key Research and Development Program of China/ ; 82273967//National Natural Science Foundation of China/ ; 82025001//National Natural Science Foundation of China/ ; 2021QN020576//Department of Science and Technology of Guangdong Province/ ; 2021B1515130005//Guangdong Basic and Applied Research Projects/ ; JSGG20200807171801013//Shenzhen Science and Technology Innovation Committee/ ; //Shenzhen Institute of Synthetic Biology Scientific Research Program/ ; 2023M742397//China Postdoctoral Science Foundation/ ; GZC20231724//Postdoctoral Fellowship Program of CPSF/ ; },
abstract = {A series of coronavirus outbreaks emphasize the necessity to develop pan-coronavirus therapeutics. Previously, the CRISmers system is developed, a Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated proteins (CRISPR/Cas)-mediated cellular screening platform for the identification of RNA aptamers. In this study, CRISmers are applied to target the fusion peptide (FP) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), a conserved region across coronavirus genera, with E.coli serving as the screening host. From this approach, a lead aptamer, #FP-10 is identified, which demonstrates potent pan-coronavirus neutralization activity against multiple SARS-CoV-2 variants and alphacoronaviruses (HCoV-229E and HCoV-NL63), demonstrating its potential for development into a broad-spectrum therapeutic candidate.},
}

@article {pmid40521185,
year = {2025},
author = {Wu, D and Shu, H and Zhang, M and Wei, X and Ji, J and Shen, H and Zhang, H and Xie, L and Zhou, L and Yang, L and Jiang, J and Chen, C and Tian, S and Zhang, X and Long, X and He, X and Wang, H},
title = {Mannosylated neutrophil vesicles targeting macrophages alleviate liver inflammation by delivering CRISPR/Cas9 RNPs.},
journal = {Theranostics},
volume = {15},
number = {13},
pages = {6221-6235},
pmid = {40521185},
issn = {1838-7640},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Mice ; *Macrophages/metabolism ; *Neutrophils/metabolism ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics/metabolism ; *Ribonucleoproteins/genetics/administration & dosage/metabolism ; Gene Editing/methods ; Liver/pathology ; Disease Models, Animal ; Mice, Inbred C57BL ; Mannose/metabolism/chemistry ; Inflammation/therapy ; Humans ; CRISPR-Associated Protein 9 ; Male ; Hepatitis/therapy ; Inflammasomes/metabolism ; },
abstract = {Background: Inflammation is a key driver of various liver diseases. NLRP3 inflammasome in hepatic macrophages is a key mediator of inflammation and has emerged as a promising target. Genome editing presents a powerful approach to modulate inflammation by directly disrupting genes such as NLRP3 directly. However, efficient and cell-specific delivery of CRISPR/Cas9 ribonucleoproteins (RNPs) remains challenging. Methods: We developed a novel delivery system by encapsulating CRISPR/Cas9 RNPs within mannosylated neutrophil membranes vesicles (Cas9/gNLRP3@M-N) to enhance targeting hepatic macrophages. Results: Cas9/gNLRP3@M-N selectively accumulated in hepatic macrophages, effectively disrupted the NLRP3 gene, attenuated inflammation in acute fulminant hepatitis, and improved disease outcomes in chronic steatohepatitis model. Conclusions: Cas9/gNLRP3@M-N represents a promising targeted gene-editing approach for the treatment of inflammatory liver diseases.},
}

Animals
*CRISPR-Cas Systems/genetics
Mice
*Macrophages/metabolism
*Neutrophils/metabolism
NLR Family, Pyrin Domain-Containing 3 Protein/genetics/metabolism
*Ribonucleoproteins/genetics/administration & dosage/metabolism
Gene Editing/methods
Liver/pathology
Disease Models, Animal
Mice, Inbred C57BL
Mannose/metabolism/chemistry
Inflammation/therapy
Humans
CRISPR-Associated Protein 9
Male
Hepatitis/therapy
Inflammasomes/metabolism

@article {pmid40521034,
year = {2025},
author = {Al-Ouqaili, MTS and Ahmad, A and Jwair, NA and Al-Marzooq, F},
title = {Harnessing bacterial immunity: CRISPR-Cas system as a versatile tool in combating pathogens and revolutionizing medicine.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1588446},
pmid = {40521034},
issn = {2235-2988},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; *Bacteria/genetics/immunology ; *Bacterial Infections/therapy/immunology ; Animals ; Genetic Therapy/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has emerged as an adaptable instrument for several uses. The CRISPR-Cas system employs Cas proteins and programmable RNA molecules to guide the recognition and cleavage of specific DNA regions, permitting accurate genome editing. It is derived from the bacterial immune system and allows for accurate and efficient modification of DNA sequences. This technique provides unparalleled gene editing, control, and precise alteration opportunities. This review aims to offer a comprehensive update of the core concepts of the CRISPR-Cas system and recent progress, while also providing an overview of the significant applications in diverse fields such as microbiology and medicine. The CRISPR-Cas9 gene editing technique has facilitated substantial advancements in comprehending gene function, simulating diseases, and creating innovative therapeutics. CRISPR-based therapeutics present a hopeful prospect for addressing intricate ailments, including genetic disorders, malignancies, and infectious diseases, as they serve as viable substitutes for conventional pharmaceuticals. In microbiology, this method serves as a diagnostic and therapeutic tool that proves highly efficient in eliminating bacteria that have developed resistance to various antibiotics. Despite its significant potential, CRISPR encounters ethical, safety, and regulatory obstacles that necessitate meticulous deliberation. Concerns regarding off-target effects, poor delivery to target tissues, and unwanted side effects emphasize the necessity to thoroughly examine the technology. It is necessary to balance the advantages and difficulties CRISPR presents. Consequently, more rigorous preclinical and clinical experiments are essential before using it in humans.},
}

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

@article {pmid40520364,
year = {2025},
author = {Nitulescu, AM and Du, W and Glaser, V and Kath, J and Aird, EJ and Cullot, G and Greensmith, R and Mikkelsen, NS and Stein, M and Bak, RO and Kaminski, M and Corn, JE and Wagner, DL},
title = {Single-stranded HDR templates with truncated Cas12a-binding sequences improve knock-in efficiencies in primary human T cells.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {2},
pages = {102568},
pmid = {40520364},
issn = {2162-2531},
abstract = {CRISPR-Cas12a gene editing offers an alternative to Cas9-based methods, providing better targeting of AT-rich regions, simplified guide RNA manufacturing, and high specificity. However, the efficacy of donor-based editing is subject to various factors, with template format playing a crucial role. Currently, the predominant non-viral template format for homology-directed repair (HDR) after nuclease-induced DNA breaks is double-stranded DNA, which is toxic when transfected at high doses. Others have demonstrated that using single-stranded DNA (ssDNA) with flanking double-stranded Cas-target-sequences (CTS) as a template for Cas9-mediated gene editing can mitigate this toxicity and increase knock-in efficiency. Here, we investigate CTS design for AsCas12a Ultra by exploring PAM orientation and binding requirements. Additionally, we rule out ssDNase activity of AsCas12a under cell-physiological Mg[2+] conditions. Finally, we showcase the advantage of ssDNA donors with CTS (ssCTS) at high doses for delivering clinically relevant transgenes of varying sizes into three TCR-CD3 complex genes (TRAC, CD3ζ, CD3ε), achieving up to 90% knock-in rates for a 0.8kb-insert at the CD3ε locus. Long-read sequencing confirmed higher HDR rates and revealed that CTS reduced partial integration events compared to unmodified ssDNA. Overall, AsCas12a and ssCTS represent a platform for highly efficient knock-in in primary human T cells with minimal toxicity.},
}

@article {pmid40518326,
year = {2025},
author = {Zhao, Y and Tan, J and Fang, L and Jiang, L},
title = {Leveraging gene editing to combat methane emissions in ruminant agriculture.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2025.05.020},
pmid = {40518326},
issn = {1879-3096},
abstract = {Methane emissions from ruminants represent a major contributor to global greenhouse gases (GHGs), posing challenges for sustainable agriculture and climate change mitigation. Recent advances in clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas)-based gene editing offer transformative approaches to address this issue by targeting both forage crops and rumen methanogens. Enhancing lipid content and secondary metabolites in forage crops can suppress methanogenesis and improve feed efficiency, while precise genome editing in methanogenic Archaea can disrupt pathways critical to methane production. However, challenges remain regarding delivery methods, gene targeting specificity, ecological impacts, and regulatory acceptance. In this review, we explore current progress, identify key knowledge gaps, and highlight the need for interdisciplinary strategies that integrate biotechnology, synthetic biology, and regulatory frameworks to develop effective and scalable methane mitigation solutions.},
}

@article {pmid40517395,
year = {2025},
author = {Marín-Sanz, M and Sánchez-León, S and Guzmán-López, MH and Starker, CG and Voytas, DF and Barro, F},
title = {Cas9- and Cas12a-mediated excision and replacement of the celiac disease-related α-gliadin immunogenic complex in hexaploid wheat.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.70200},
pmid = {40517395},
issn = {1467-7652},
support = {//European Union "NextGenerationEU"/PRTR/ ; QUAL21_023 IAS//Junta de Andalucía/ ; //Conexión TRIGO/WheatNet/ ; PID2022-142139OB-I00//Ministerio de Ciencia, Innovación y Universidades/ ; TED2021-129733B-I00//Ministerio de Ciencia, Innovación y Universidades/ ; },
abstract = {Celiac disease (CD) is a chronic enteropathy affecting approximately 1% of the global population. Wheat α-gliadins are a major contributor to the autoimmune response, as they contain one of the most immunogenic peptides, the 33-mer, along with numerous variants. In this study, we used CRISPR/Cas technology to mutate genes encoding α-gliadins. This approach employed paired sgRNAs to precisely excise immunogenic regions while preserving non-immunogenic sequences within the α-gliadins. Furthermore, we replaced the excised region with an α-gliadin-based double-stranded oligodeoxynucleotide (dsODN) designed with nucleotide changes to reduce immunoreactivity and increase peptidase cleavage sites. Two different CRISPR systems, Cas9 and Cas12a, were applied to generate wheat protoplasts and plants with non-immunogenic regions. Cas9 demonstrated superior performance in terms of editing frequency, excision and replacement of immunogenic fragments. However, the Cas12a nuclease (Cpf1) showed promising editing efficiency, offering the potential for future wheat editing applications. Using the Cas9 system, we achieved a 74.2% excision rate of the 33-mer in wheat plants. Subsequent analyses showed a significant reduction in the reactivity to the G12 monoclonal antibody, capable of identifying the 33-mer peptide and a decrease in the prolamin levels compared to the wild-type. Additionally, we developed a high-throughput sequencing-based software specifically designed to identify mutations in multi-copy gene families. This innovative tool enabled fast, parallel screening of the samples in this study and facilitated the identification of the specific editing patterns produced by the designed constructs.},
}

@article {pmid40516885,
year = {2025},
author = {Cunha da Silva, G and Rossi, CC},
title = {Defense systems and mobile elements in Staphylococcus haemolyticus: a genomic view of resistance dissemination.},
journal = {Microbial pathogenesis},
volume = {206},
number = {},
pages = {107808},
doi = {10.1016/j.micpath.2025.107808},
pmid = {40516885},
issn = {1096-1208},
abstract = {Staphylococcus haemolyticus is a multidrug-resistant opportunistic pathogen and a major reservoir of antimicrobial resistance (AMR) genes within the Staphylococcaceae family. Its high genomic plasticity, frequent association with mobile genetic elements (MGEs), and prevalence in clinical settings underscore its relevance as both a threat and a conduit for resistance dissemination. In this study, we performed a comprehensive pan-genomic analysis of the S. haemolyticus defensome - including restriction-modification (RM), abortive infection (Abi), and CRISPR-Cas systems - across 692 high-quality genomes. Our results reveal a highly diverse and modular repertoire of immune systems, often organized in physical clusters and frequently associated with MGEs. We identified evidence of antagonistic interactions, with both defense and anti-defense elements encoded on plasmids and prophages. CRISPR spacer analysis showed a predominant targeting of phages, and genomes encoding CRISPR-Cas systems exhibited a lower abundance of MGEs and AMR genes, suggesting a trade-off between defense and gene acquisition. RNA-seq data from one reference strain indicate that only a fraction of the defensome is actively transcribed under standard conditions, hinting at environment-responsive regulation. Together, these findings provide new insights into the genomic strategies sustaining the persistence and adaptability of S. haemolyticus in clinical environments. The interplay between its immune systems and mobilome likely contributes not only to its evolutionary trajectory, but also to its role in the horizontal transfer of resistance determinants among pathogenic staphylococci. A deeper understanding of this immune-mobilome interface may help inform future strategies to limit the spread of resistance.},
}

@article {pmid40516718,
year = {2025},
author = {Bu, G and Ma, X and Jin, H and Wang, L},
title = {CRISPR-Cas and RNA sequencing reveal nutrient enhancement pathways in quinoa for plant-based athlete recovery diets and future food security.},
journal = {International journal of biological macromolecules},
volume = {318},
number = {Pt 4},
pages = {144785},
doi = {10.1016/j.ijbiomac.2025.144785},
pmid = {40516718},
issn = {1879-0003},
abstract = {Quinoa (Chenopodium quinoa Willd.) is a nutrient-dense, climate-resilient pseudocereal with growing relevance to food and nutritional security. However, its potential remains underutilized due to suboptimal levels of key micronutrients. In this study, to apply CRISPR-Cas9 for the simultaneous enhancement of multiple nutrients in quinoa, marking a significant advancement in crop biofortification, we employed multiplexed CRISPR-Cas9 genome editing to target five genes involved in lysine transport, phytic acid biosynthesis, and vitamin C and E biosynthetic pathways. Both knockout and homology-directed knock-in strategies were applied to induce heritable mutations in genes such as CqAAP1, CqIPK1, CqGGP, and CqHPT. Homology-directed knock-in refers to a precise gene-editing method that uses a template to insert specific genetic changes at targeted sites in the genome. Edited lines exhibited significant improvements in seed nutrient concentrations, including lysine (+35 %), zinc (+43 %), vitamin C (+114 %), and vitamin E (+45 %), without yield or growth penalties. Whole-transcriptome profiling via RNA sequencing (RNA-Seq) identified 1284 differentially expressed genes (FDR < 0.05), predominantly associated with amino acid metabolism, redox regulation, and vitamin biosynthesis. Gene Ontology (GO) and KEGG enrichment analyses confirmed the transcriptional activation of nutrient assimilation and antioxidant pathways. Integration of qRT-PCR with RNA-Seq confirmed the reliability of expression data. Structural validations of edits were performed using PCR, gel electrophoresis, and Sanger sequencing. Phenotypic assessments, including seed weight, morphology, and exploratory herbivory assays, confirmed agronomic stability. This study presents a technically validated framework for simultaneous nutritional trait stacking in quinoa using nucleic acid engineering tools. Our findings offer critical insights into the transcriptional plasticity of quinoa and establish a functional genomics platform for developing multi-nutrient biofortified crops to address hidden hunger and advance sustainable food systems.},
}

@article {pmid40515942,
year = {2025},
author = {Saha, T and Saha, RP and Singh, MK and Priya, K and Singh, S and Rajeev, M and Bhattacharya, D and Nag, M and Lahiri, D},
title = {An overview on in-vivo generation of CAR-T cells using CRISPR-loaded functionalized nanocarriers for treating B-cell lineage acute lymphoblastic leukemia.},
journal = {Molecular biology reports},
volume = {52},
number = {1},
pages = {596},
pmid = {40515942},
issn = {1573-4978},
mesh = {Humans ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Immunotherapy, Adoptive/methods ; Animals ; *Receptors, Chimeric Antigen/genetics/metabolism/immunology ; T-Lymphocytes/immunology/metabolism ; Mice ; Nanoparticles/chemistry ; *Precursor Cell Lymphoblastic Leukemia-Lymphoma/therapy/immunology/genetics ; *Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/therapy/immunology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; B-Lymphocytes ; },
abstract = {Chimeric antigen receptor T (CAR-T) cell therapy has become a milestone in the management of B cell lineage acute lymphoblastic leukemia. Yet, the traditional method-dependent on ex vivo manipulation, amplification, and reinfusion of autologous T cells-is high-cost, low-scalability, and severely immune-related toxicity. Here, we report a new nano-immunoengineering platform that allows in vivo production of chimeric antigen receptor T cells through the use of functionalized nanoparticles carrying clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) gene editing elements. These nanoparticles are engineered to specifically target blood circulating T lymphocytes and deliver CRISPR/Cas9 complexes that have the ability to integrate chimeric antigen receptor constructs into the TRAC locus and knock out immune checkpoint genes like programmed cell death protein 1 (PD-1) simultaneously. Targeted delivery, endosomal escape, and efficient genome editing with minimal off-target effects are ensured through gold-based and DNA nanostructure-based carriers. Preclinical models show effective in vivo programming of functional chimeric antigen receptor T cells with vigorous antitumor efficacy, improved persistence, and decreased cytokine release syndrome. This method is a revolutionary breakthrough in cancer immunotherapy that provides a scalable, economical, and clinically flexible replacement for conventional chimeric antigen receptor T cell production.},
}

Humans
Gene Editing/methods
CRISPR-Cas Systems/genetics
*Immunotherapy, Adoptive/methods
Animals
*Receptors, Chimeric Antigen/genetics/metabolism/immunology
T-Lymphocytes/immunology/metabolism
Mice
Nanoparticles/chemistry
*Precursor Cell Lymphoblastic Leukemia-Lymphoma/therapy/immunology/genetics
*Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/therapy/immunology
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
B-Lymphocytes

@article {pmid40515919,
year = {2025},
author = {Ye, Y and Zhang, M and Nie, H and Duan, Y and Ye, Z and Zheng, C},
title = {Screening for Host Proteins with Pro- and Antiviral Activity via High-Throughput CRISPR.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2940},
number = {},
pages = {273-281},
pmid = {40515919},
issn = {1940-6029},
mesh = {Humans ; *CRISPR-Cas Systems ; Antiviral Agents/pharmacology ; *High-Throughput Screening Assays/methods ; *Host-Pathogen Interactions/genetics ; Animals ; Cell Line ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Virus Diseases/virology/genetics ; Viruses/genetics ; Virus Replication ; },
abstract = {The whole-genome library for CRISPR screening serves as an important biotechnological tool aimed at probing gene function in mammalian cells, providing a foundation for the systematic discovery of essential genes corresponding to biological effects. Research indicates that whole-genome library cell lines can be used to identify host factors and potential drug targets associated with viral infections effectively at high throughput, providing crucial evidence for the development of novel antiviral drugs. Here, we primarily discuss the methods for constructing genome-scale CRISPR screens in cell lines and their applications in virology research. By cloning and expressing the whole genomic DNA of specific organisms, stable cell lines, which can be utilized for functional validation, drug screening, and gene function studies, can be established. Through gene knockout or overexpression techniques, in-depth analyses of the key roles that genes play in the viral life cycle could be conducted, revealing how viruses exploit the biological mechanisms of host cells for replication and evasion of immune responses. These findings not only enhance our understanding of the interactions between viruses and host cells but also yield new targets for the development of antiviral drugs and vaccines.},
}

Humans
*CRISPR-Cas Systems
Antiviral Agents/pharmacology
*High-Throughput Screening Assays/methods
*Host-Pathogen Interactions/genetics
Animals
Cell Line
*Clustered Regularly Interspaced Short Palindromic Repeats
Virus Diseases/virology/genetics
Viruses/genetics
Virus Replication

@article {pmid40515918,
year = {2025},
author = {Wang, J and Zheng, C and Xiao, W},
title = {Zebrafish as a Model for Studying Virus-Host Interactions.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2940},
number = {},
pages = {261-272},
pmid = {40515918},
issn = {1940-6029},
mesh = {Animals ; *Zebrafish/virology/genetics ; *Host-Pathogen Interactions/genetics ; *Rhabdoviridae/physiology/genetics ; Disease Models, Animal ; CRISPR-Cas Systems ; *Rhabdoviridae Infections/virology/genetics ; *Fish Diseases/virology/genetics ; },
abstract = {During the long evolutionary process, viruses and hosts have engaged in a game of immune escape and viral clearance, resulting in an exquisite model of virus-host interaction. An appropriate animal model can help unravel the mysteries of this process. Owing to their susceptibility to multiple viruses, small size, high fecundity, low cost, ease of visualization, and efficient gene editing, zebrafish (Danio rerio) have recently emerged as a powerful vertebrate model system for studying virus-host interactions. In this chapter, we describe in detail the experimental protocols for analyzing virus-host interactions in a zebrafish model in which spring viremia of carp virus (SVCV) is used as an example. These protocols include the generation of knockout zebrafish via CRISPR/Cas9 technology, methods for viral infection of zebrafish larvae or adults, analysis of in vivo viral resistance of host genes, analysis of host and viral gene expression in zebrafish larvae via qRT-PCR and Western blotting, and detection of host protein binding to viral proteins via immunoprecipitation. These experimental protocols provide an effective reference for studying virus-host interactions in a zebrafish model.},
}

Animals
*Zebrafish/virology/genetics
*Host-Pathogen Interactions/genetics
*Rhabdoviridae/physiology/genetics
Disease Models, Animal
CRISPR-Cas Systems
*Rhabdoviridae Infections/virology/genetics
*Fish Diseases/virology/genetics

@article {pmid40515917,
year = {2025},
author = {Zhou, Q and Wu, T and Zheng, C and Zou, X},
title = {The Application of Base-Editing Technology to Investigate Virus-Host Interactions and Antiviral Therapeutic Strategies.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2940},
number = {},
pages = {251-260},
pmid = {40515917},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems ; *Host-Pathogen Interactions/genetics ; *Antiviral Agents/therapeutic use/pharmacology ; *Virus Diseases/virology/genetics/drug therapy/therapy ; *Viruses/genetics/drug effects ; Animals ; },
abstract = {Understanding viral pathogenesis and developing effective countermeasures hinge on deciphering the complex relationships between viruses and their hosts. While conventional approaches have shown limitations in unraveling these intricate interactions, researchers are now turning to cutting-edge genetic tools, particularly base-editing technology. This section explores how base editing can be innovatively applied to investigate virus-host dynamics and to create novel antiviral therapeutic approaches.},
}

*Gene Editing/methods
Humans
CRISPR-Cas Systems
*Host-Pathogen Interactions/genetics
*Antiviral Agents/therapeutic use/pharmacology
*Virus Diseases/virology/genetics/drug therapy/therapy
*Viruses/genetics/drug effects
Animals

@article {pmid40515905,
year = {2025},
author = {Huang, W and Zhao, X and Zheng, C and Shen, Y},
title = {CRISPR-Mediated Viral Gene Knock-In for Studying Viral-Host Interactions.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2940},
number = {},
pages = {109-119},
pmid = {40515905},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Herpesvirus 1, Human/genetics/physiology ; Red Fluorescent Protein ; Humans ; Luminescent Proteins/genetics/metabolism ; Virus Replication/genetics ; *Host-Pathogen Interactions/genetics ; *Gene Knock-In Techniques/methods ; Animals ; Capsid Proteins/genetics/metabolism ; },
abstract = {CRISPR-mediated gene knock-in is a powerful tool for studying virus-host interactions. In this protocol, we describe the methodology for the knock-in of a red fluorescent protein, mCherry, fused to the viral protein VP26, a component of the herpesvirus capsid. By integrating the mCherry gene into the HSV-1 genome, we enable detailed studies of viral protein dynamics, viral replication, and virus-host interactions in living cells. In particular, this approach allows real-time monitoring of the subcellular localization of the viral capsid. The protocol provides a step-by-step guide for designing the gene knock-in vectors, transfection, selection, and validation of the mCherry fusion to the VP26 protein in the HSV-1 genome.},
}

*CRISPR-Cas Systems
*Herpesvirus 1, Human/genetics/physiology
Red Fluorescent Protein
Humans
Luminescent Proteins/genetics/metabolism
Virus Replication/genetics
*Host-Pathogen Interactions/genetics
*Gene Knock-In Techniques/methods
Animals
Capsid Proteins/genetics/metabolism

@article {pmid40514654,
year = {2025},
author = {Li, M and Zhou, S and Zhang, S and Xie, X and Nie, J and Wang, Q and Ma, L and Cheng, Y and Luo, J},
title = {Transdermal delivery of CRISPR/Cas9-mediated melanoma gene therapy via polyamines-modified thermosensitive hydrogels.},
journal = {Journal of nanobiotechnology},
volume = {23},
number = {1},
pages = {441},
pmid = {40514654},
issn = {1477-3155},
support = {2024BCA001//Project of Technological Innovation Plan in Hubei Province/ ; 2024BCA001//Project of Technological Innovation Plan in Hubei Province/ ; 2024040701010046//Natural Science Foundation of Wuhan City/ ; 2025AFB729//Hubei Provincial Natural Science Foundation of China/ ; },
mesh = {*Hydrogels/chemistry ; Animals ; *CRISPR-Cas Systems/genetics ; Doxorubicin/pharmacology/administration & dosage/chemistry ; *Melanoma/therapy/genetics/drug therapy ; Humans ; Cell Line, Tumor ; *Genetic Therapy/methods ; *Polyamines/chemistry ; Mice ; Y-Box-Binding Protein 1/genetics/metabolism ; Gene Editing ; Administration, Cutaneous ; Drug Delivery Systems ; Mice, Nude ; },
abstract = {The main obstacles to the clinical application of the CRISPR/Cas9 system are off-target effects and low delivery efficiency. There is an urgent need to develop new delivery strategies and technologies. Three types of in situ injectable hydrogels with different electrical properties were created to find the most secure and efficient sustained-release drug delivery system. After in vitro and in vivo comparisons, we found that the positively charged hydrogels had higher cellular uptake, stronger gene editing efficiency, greater cytotoxicity, longer tumor accumulation, and better anti-tumor efficacy than negatively charged and neutral hydrogels. We designed single guide RNA targeting the Y-box binding protein 1 (YB-1) gene and then used it to create a ribonucleoprotein complex with Cas9 protein. Doxorubicin was co-encapsulated into this positively charged hydrogel to create a co-delivery system. By knocking down YB-1, the expression of YB-1 was reduced, inhibiting the growth and migration of melanoma cells. The strategy of combining YB-1 gene editing and intratumoral injection enhanced the therapeutic effect of doxorubicin while reducing side effects.},
}

*Hydrogels/chemistry
Animals
*CRISPR-Cas Systems/genetics
Doxorubicin/pharmacology/administration & dosage/chemistry
*Melanoma/therapy/genetics/drug therapy
Humans
Cell Line, Tumor
*Genetic Therapy/methods
*Polyamines/chemistry
Mice
Y-Box-Binding Protein 1/genetics/metabolism
Gene Editing
Administration, Cutaneous
Drug Delivery Systems
Mice, Nude

@article {pmid40514251,
year = {2025},
author = {Qurtam, AA},
title = {Epigenetic reprogramming in breast cancer: The role of CRISPR-Cas 9.},
journal = {Bulletin du cancer},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.bulcan.2025.03.007},
pmid = {40514251},
issn = {1769-6917},
abstract = {Epigenetic alterations are known to be a significant factor in the development and advancement of breast cancer (BC), which continues to be a substantial cause of illness and death in women globally. The emergence of CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9) technology has fundamentally transformed our capacity to edit the genome with unparalleled accuracy, providing novel opportunities for therapeutic intervention. This review examines the utilization of CRISPR-Cas9 to alter epigenetic landscapes to combat BC. We examine the fundamental processes of CRISPR-Cas9 and its derivatives, including dCas9, in their ability to specifically target DNA methylation and histone alterations. The highlighted review showcases the potential of CRISPR-Cas9 in reactivating silenced tumor suppressor genes and inhibiting oncogenes. In addition, we analyze the incorporation of CRISPR-based epigenetic editing into current medicines, offering valuable knowledge on the use of combination therapies to improve treatment effectiveness and overcome resistance. This review intends to highlight the revolutionary potential of CRISPR-Cas9 in generating targeted, personalized therapeutics for BC by explaining the present advancements and future applications. The incorporation of this state-of-the-art technology with conventional and developing therapies holds the potential to establish more efficient and long-lasting remedies in the battle against BC, ultimately enhancing patient results and rates of survival.},
}

@article {pmid40512855,
year = {2025},
author = {Boardman, DA and Mangat, S and Gillies, JK and Leon, L and Fung, VCW and Haque, M and Mojibian, M and Halvorson, T and Huang, Q and Tuomela, K and Wardell, CM and Brown, A and Lam, AJ and Levings, MK},
title = {Armored human CAR Treg cells with PD1 promoter-driven IL-10 have enhanced suppressive function.},
journal = {Science advances},
volume = {11},
number = {24},
pages = {eadx7845},
pmid = {40512855},
issn = {2375-2548},
mesh = {Humans ; *Interleukin-10/genetics/metabolism ; *T-Lymphocytes, Regulatory/immunology/metabolism ; *Programmed Cell Death 1 Receptor/genetics ; *Promoter Regions, Genetic ; Animals ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; Mice ; Dendritic Cells/immunology ; CRISPR-Cas Systems ; Immunotherapy, Adoptive/methods ; },
abstract = {Regulatory T cell (Treg cell) therapy has been transformed through the use of chimeric antigen receptors (CARs). We previously found that human Treg cells minimally produce IL-10 and have a limited capacity to control innate immunity compared to type 1 regulatory T cells (Tr1 cells). To create "hybrid" CAR Treg cells with Tr1 cell-like properties, we examined whether the PDCD1 locus could be exploited to endow Treg cells with CAR-regulated IL-10 expression. CRISPR-mediated PD1 deletion increased CAR Treg cell activation, and knock-in of IL10 under control of the PD1 promoter resulted in CAR-induced IL-10 secretion. IL10 knock-in improved CAR Treg cell function, as determined by increased suppression of dendritic cells and alloantigen- and islet autoantigen-specific T cells. In vivo, IL10 knock-in CAR Treg cells were stable, safe, and suppressed dendritic cells and xenogeneic graft-versus-host disease. CRISPR-mediated engineering to simultaneously remove an inhibitory signal and enhance a suppressive mechanism is a previously unexplored approach to improve CAR Treg cell potency.},
}

Humans
*Interleukin-10/genetics/metabolism
*T-Lymphocytes, Regulatory/immunology/metabolism
*Programmed Cell Death 1 Receptor/genetics
*Promoter Regions, Genetic
Animals
*Receptors, Chimeric Antigen/genetics/immunology/metabolism
Mice
Dendritic Cells/immunology
CRISPR-Cas Systems
Immunotherapy, Adoptive/methods

@article {pmid40510793,
year = {2025},
author = {Zhang, Y and Lv, L and Xu, S and Chen, Y},
title = {Innovative nucleic acid detection of Clostridioides difficile utilizing the PAM-unconventional, one-step LAMP/CRISPR-Cas12b detection platforms.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1594271},
pmid = {40510793},
issn = {2235-2988},
mesh = {*Clostridioides difficile/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; *Clostridium Infections/diagnosis/microbiology ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Humans ; Bacterial Proteins/genetics ; Feces/microbiology ; Bacterial Toxins/genetics ; },
abstract = {INTRODUCTION: Clostridioides difficile (C. difficile), a human pathogen that causes diarrhea and colon lesions, has garnered widespread attention. Rapid and accurate detection of bacterial virulence factors is essential for the diagnosis of C. difficile infection (CDI). To date, numerous laboratory tests have been developed; however, none fully meet the combined requirements of speed, cost-effectiveness, portability, sensitivity, and specificity. Molecular diagnostic technologies based on CRISPR-Cas systems have provided a promising solution to this challenge. Nonetheless, the limited compatibility between pre-amplification and CRISPR cleavage, coupled with the inherent selectivity of CRISPR systems for protospacer adjacent motif (PAM) sequences near the target site, poses additional constraints on the broader adoption of this approach.

METHODS: Here, we developed PAM-unconventional, One-step LAMP/CRISPR-Cas12b (POLC) detection platforms for the toxin-encoding genes tcdA and tcdB of C. difficile.

RESULTS: The POLC platforms operated at 60 °C, enabling result interpretation either through fluorescence intensity measurements or direct visualization under UV light. The limits of detection (LoDs) ranged from 3 to 14 copies/μL using a fluorescence reader and from 6 to 18 copies/μL via direct observation. Compared to qPCR, which typically requires over an hour, the POLC platforms reduced the detection time to approximately 40 minutes. Each reaction cost approximately USD 6.5, offering a substantial cost saving compared to qPCR-based commercial kits (over USD 10 per test). In clinical validation with 55 fecal samples, the tcdA POLC assay achieved 86.4% sensitivity and 84.8% specificity, while the tcdB POLC assay demonstrated 96.6% sensitivity and 100% specificity, using qPCR as the reference standard.

DISCUSSION: Our research presents innovative CRISPR-based one-step nucleic acid detection platforms that eliminate canonical PAM sequence requirements. These platforms exhibit high sensitivity and specificity while achieving rapid detection under simple conditions, making them promising candidates for clinical diagnostics and point-of-care testing (POCT).},
}

*Clostridioides difficile/genetics/isolation & purification
*CRISPR-Cas Systems
*Molecular Diagnostic Techniques/methods
*Clostridium Infections/diagnosis/microbiology
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
Humans
Bacterial Proteins/genetics
Feces/microbiology
Bacterial Toxins/genetics

@article {pmid40507794,
year = {2025},
author = {Ho, CH and Tsai, CY and Chang, CC and Hu, CJ and Huang, CY and Lu, YC and Lin, PH and Lin, CH and Lin, HI and OuYang, CH and Hsu, CJ and Liu, TC and Chen, YT and Chan, YH and Cheng, YF and Wu, CC},
title = {Low Efficiency of Homology-Independent Targeted Integration for CRISPR/Cas9 Correction in the Vicinity of the SLC26A4 c.919-2A>G Variant.},
journal = {International journal of molecular sciences},
volume = {26},
number = {11},
pages = {},
pmid = {40507794},
issn = {1422-0067},
support = {106-T04//National Taiwan University Hospital/ ; 107-2622-B-002-008-CC2//National Science and Technology Council of Taiwan/ ; },
mesh = {*Sulfate Transporters/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Hearing Loss, Sensorineural/genetics ; Goiter, Nodular/genetics ; *Membrane Transport Proteins/genetics ; },
abstract = {Recessive variants of SLC26A4 are a common cause of hereditary hearing impairment and are responsible for non-syndromic enlarged vestibular aqueducts and Pendred syndrome. Patients with bi-allelic SLC26A4 variants often suffer from fluctuating hearing loss and recurrent vertigo, ultimately leading to severe to profound hearing impairment. However, there are currently no satisfactory prevention or treatment options for this condition. The CRISPR/Cas9 genome-editing technique is a well-known tool for correcting point mutations or manipulating genes and shows potential therapeutic applications for hereditary disorders. In this study, we used the homology-independent targeted integration (HITI) strategy to correct the SLC26A4 c.919-2A>G variant, the most common SLC26A4 variant in the Han Chinese population. Next-generation sequencing was performed to evaluate the editing efficiency of the HITI strategy. The results showed that only 0.15% of the reads successfully exhibited HITI integration, indicating that the c.919-2 region may not be a suitable region for HITI selection. This suggests that other site selection or insertion strategies may be needed to improve the efficiency of correcting the SLC26A4 c.919-2A>G variant. This experience may serve as a valuable reference for other researchers considering CRISPR target design in this region.},
}

*Sulfate Transporters/genetics
Humans
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Hearing Loss, Sensorineural/genetics
Goiter, Nodular/genetics
*Membrane Transport Proteins/genetics

@article {pmid40506744,
year = {2025},
author = {Kristof, A and Karunakaran, K and Allen, C and Mizote, P and Briggs, S and Jian, Z and Nash, P and Blazeck, J},
title = {Engineering novel CRISPRi repressors for highly efficient mammalian gene regulation.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {164},
pmid = {40506744},
issn = {1474-760X},
support = {DP2 CA280622/CA/NCI NIH HHS/United States ; NSF GRFP//National Science Foundation/ ; 1DP2CA280622-01//NIH Office of the Director/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Expression Regulation ; *Repressor Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; Animals ; Gene Knockdown Techniques ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Associated Protein 9 ; },
abstract = {BACKGROUND: CRISPR interference (CRISPRi), the repurposing of the RNA-guided endonuclease dCas9 as a programmable transcriptional repressor, allows highly specific repression (knockdown) of gene expression. CRISPRi platforms can often have incomplete knockdown, performance variability across cell lines and gene targets, and inconsistencies dependent on the guide RNA sequence employed.

RESULTS: Here, we explore the combination of novel repressor domains with strong Krüppel-associated box (KRAB) repressors, screening > 100 bipartite and tripartite fusion proteins for their ability to reduce gene expression as CRISPRi effectors. We show that these novel repressor fusions have reduced dependence on guide RNA sequences, better slow cell growth when used to knock down expression of essential genes, and function in either fusion or scaffold modalities. Furthermore, we isolate and characterize a particularly effective CRISPRi platform, dCas9-ZIM3(KRAB)-MeCP2(t), which shows improved gene repression of endogenous targets both at the transcript and protein level across several cell lines and when deployed in genome-wide screens.

CONCLUSIONS: We posit that these novel repressor fusions can enhance the reproducibility and utility of CRISPRi in mammalian cells.},
}

Humans
*CRISPR-Cas Systems
*Gene Expression Regulation
*Repressor Proteins/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
HEK293 Cells
Animals
Gene Knockdown Techniques
Cell Line
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Associated Protein 9

@article {pmid40506725,
year = {2025},
author = {Wang, T and Jiang, W and Huang, Z and Yuan, Z and Chen, Z and Lin, J},
title = {Multiplex detection of respiratory RNA viruses without amplification based on CRISPR-Cas13a immunochromatographic test strips.},
journal = {Virology journal},
volume = {22},
number = {1},
pages = {192},
pmid = {40506725},
issn = {1743-422X},
support = {2024T021//Fuzhou University Testing Fund of Precious Apparatus/ ; 2022-S-wr4//Fuzhou Health Science and Technology Plan Soft Science Research Project/ ; 2022-S-rc5//Young and Middle-aged Talent Research Project of Fuzhou City/ ; 2020J05279//Fujian Provincial Natural Science Foundation of China/ ; 2020Y9140//Joint Funds for the Innovation of Sience and Technology, Fujian Province/ ; 2021Y9107//Joint Funds for the Innovation of Science and Technology, Fujian Province/ ; 2021S263//Fuzhou Science and Technology Project/ ; 2021ZQNZD010//Major Research Projects for Young and Middle-aged Researchers of Fujian Provincial Health Commission/ ; 2022J01521//Natural Science Foundation of Fujian Province/ ; 2022Y4003//Fujian Provincial Science and Technology Plan Project/ ; 2022ZD01001//Fujian Provincial Major Health Research Project/ ; 2021GGA053//Fujian Provincial Health Technology Project/ ; 2023YFC3304304//National Key Research & Development Plan/ ; },
mesh = {Humans ; *SARS-CoV-2/isolation & purification/genetics/immunology ; Sensitivity and Specificity ; *Chromatography, Affinity/methods ; *COVID-19/diagnosis/virology ; *RNA Viruses/isolation & purification/genetics ; CRISPR-Cas Systems ; *Respiratory Tract Infections/diagnosis/virology ; Reagent Strips ; Influenza, Human/diagnosis/virology ; },
abstract = {Acute respiratory infections, caused by RNA viruses like respiratory syncytial virus, influenza, rhinovirus, and coronavirus, are major global health threats. Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) is the gold standard for detecting these viruses but is time-consuming, complex, and requires specialized equipment. There is a need for rapid, convenient, and multi-target detection methods to improve disease prevention and control. This study developed a multi-target immunochromatographic detection method using LbuCas13a protein and "band elimination" test strips for detecting SARS-CoV-2 and influenza virus. The method's performance was evaluated by testing known 5 positive and 4 negative samples for SARS-CoV-2 and comparing results with fluorescent PCR and colloidal gold methods. Detection sensitivity was quantified using digital PCR and qPCR. The immunochromatographic test strips showed 100% concordance with fluorescent PCR and colloidal gold methods in initial clinical SARS-CoV-2 detection. Subsequently, we used dual-target immunochromatographic test strips to detect 9 SARS-CoV-2 positive samples and 9 H3N2 positive samples. However, false negatives were observed in dual-target detection of SARS-CoV-2 and H3N2 samples, likely due to low sample concentration or sample degradation. The method had a minimum detection limit of 381.75 copies/µL, as determined by digital PCR and qPCR. The developed multi-target immunochromatographic detection method offers a rapid, low-cost, and simple approach for detecting both SARS-CoV-2 and influenza viruses. With high sensitivity, specificity, and reliability, this method holds promise as a practical tool for RNA virus diagnosis and improving public health response to respiratory infections.},
}

Humans
*SARS-CoV-2/isolation & purification/genetics/immunology
Sensitivity and Specificity
*Chromatography, Affinity/methods
*COVID-19/diagnosis/virology
*RNA Viruses/isolation & purification/genetics
CRISPR-Cas Systems
*Respiratory Tract Infections/diagnosis/virology
Reagent Strips
Influenza, Human/diagnosis/virology

@article {pmid40504161,
year = {2025},
author = {Tien, JC and Zhai, Y and Wu, R and Zhang, Y and Chang, Y and Cheng, Y and Todd, AJ and Wheeler, CE and Li, S and Mannan, R and Cheng, C and Magnuson, B and Cruz, G and Cao, Y and Mahapatra, S and Stolfi, C and Cao, X and Su, F and Wang, R and Yang, J and Zhou, L and Qiao, Y and Xiao, L and Cieslik, M and Wang, X and Wang, Z and Chou, J and Fearon, ER and Ding, K and Cho, KR and Chinnaiyan, AM},
title = {Defining CDK12 as a tumor suppressor and therapeutic target in mouse models of tubo-ovarian high-grade serous carcinoma.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {24},
pages = {e2426909122},
doi = {10.1073/pnas.2426909122},
pmid = {40504161},
issn = {1091-6490},
support = {P50-CA186786//HHS | NIH | National Cancer Institute (NCI)/ ; R35-CA231996//HHS | NIH | National Cancer Institute (NCI)/ ; U2C-CA271854//HHS | NIH | National Cancer Institute (NCI)/ ; 22037003//MOST | National Natural Science Foundation of China (NSFC)/ ; W81XWH-21-1-0458//U.S. Department of Defense (DOD)/ ; },
mesh = {Animals ; Female ; Mice ; *Ovarian Neoplasms/genetics/pathology/drug therapy/metabolism ; *Cyclin-Dependent Kinases/genetics/metabolism ; *Cystadenocarcinoma, Serous/genetics/pathology/drug therapy/metabolism ; Disease Models, Animal ; Humans ; CRISPR-Cas Systems ; Tumor Suppressor Protein p53/genetics/metabolism ; Mice, Knockout ; Cell Line, Tumor ; },
abstract = {Ovarian cancer is the sixth leading cause of cancer death among American women, with most fatalities attributable to tubo-ovarian high-grade serous carcinoma (HGSC). This malignancy usually develops resistance to conventional chemotherapy, underscoring the need for robust preclinical models to guide the development of novel therapies. Here, we introduce an HGSC mouse model generated via Ovgp1-driven Cre recombinase effecting CRISPR/Cas9-mediated deletion of Trp53, Rb1, and Nf1 tumor suppressors in mouse oviductal epithelium (m-sgPRN model). Cyclin-dependent kinase 12 (CDK12) inactivation-frequently observed in human HGSC-is associated with poorer outcomes, DNA damage accumulation (including tandem duplications), and increased tumor immunogenicity. In our system, coablation of Cdk12 (m-sgPRN;Cdk12KO) recapitulated hallmark features of HGSC, while accelerating tumor progression and reducing survival. In a conventional (Cre-lox-mediated) Trp53/Nf1/Rb1 triple knockout model with concurrent Cdk12 ablation (PRN;Cdk12KO mice), we observed T cell-rich immune infiltrates mirroring those seen clinically. We established both models as subcutaneous or intraperitoneal syngeneic allografts of CDK12-inactivated HGSC that exhibited sensitivity to immune checkpoint blockade. Furthermore, a CRISPR/Cas9 synthetic lethality screen in PRN;Cdk12KO-derived cell lines identified CDK13-an essential paralog of CDK12-as the most depleted candidate, confirming a previously reported synthetic lethal interaction. Pharmacologic CDK13/12 degradation (employing YJ1206) demonstrated enhanced efficacy in cell lines derived from both m-sgPRN;Cdk12KO and PRN;Cdk12KO models. Our results define CDK12 as a key tumor suppressor in tubo-ovarian HGSC and highlight CDK13 targeting as a promising therapeutic approach in CDK12-inactive disease. Additionally, we have established valuable in vivo resources to facilitate further investigation and drug development in this challenging malignancy.},
}

Animals
Female
Mice
*Ovarian Neoplasms/genetics/pathology/drug therapy/metabolism
*Cyclin-Dependent Kinases/genetics/metabolism
*Cystadenocarcinoma, Serous/genetics/pathology/drug therapy/metabolism
Disease Models, Animal
Humans
CRISPR-Cas Systems
Tumor Suppressor Protein p53/genetics/metabolism
Mice, Knockout
Cell Line, Tumor

@article {pmid40503054,
year = {2025},
author = {Nawaz, A and Ariffin, NS and Wong, TW},
title = {Functionalized chitosan as nano-delivery platform for CRISPR-Cas9 in cancer treatment.},
journal = {Asian journal of pharmaceutical sciences},
volume = {20},
number = {3},
pages = {101041},
pmid = {40503054},
issn = {2221-285X},
abstract = {CRISPR-Cas system permanently deletes any harmful gene-of-interest to combat cancer growth. Chitosan (CS) is a potential cancer therapeutic that mediates via PI3K/Akt/mTOR, MAPK and NF-kβ signaling pathway modulation. CS and its covalent derivatives have been designed as nanocarrier of CRISPR-Cas9 alone (plasmid or ribonucleoprotein) or in combination with chemical drug for cancer treatment. The nanocarrier was functionalized with polyethylene glycol (PEG), targeting ligand, cell penetrating ligand and its inherent positive zeta potential to mitigate premature clearance and particulate aggregation, and promote cancer cell/nucleus targeting and permeabilization to enable CRISPR-Cas9 acting on the host DNA. Different physicochemical attributes are required for the CS-based nanocarrier to survive from the administration site, through the systemic circulation-extracellular matrix-mucus-mucosa axis, to the nucleus target. CRISPR-Cas9 delivery is met with heterogeneous uptake by the cancer cells. Choice of excipients such as targeting ligand and PEG may be inappropriate due to lacking overexpressed cancer receptor or availability of excessive metabolizing enzyme and immunoglobulin that defies the survival and action of these excipients rendering nanocarrier fails to reach the target site. Cancer omics analysis should be implied to select excipients which meet the pathophysiological needs, and chitosan nanocarrier with a "transformative physicochemical behavior" is essential to succeed CRISPR-Cas9 delivery.},
}

@article {pmid40501577,
year = {2025},
author = {Babajanyan, SG and Garushyants, SK and Wolf, YI and Koonin, EV},
title = {Evolution of antivirus defense in prokaryotes depending on the environmental virus prevalence and virome dynamics.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2025.05.27.656525},
pmid = {40501577},
issn = {2692-8205},
abstract = {UNLABELLED: Prokaryotes can acquire antivirus immunity via two fundamentally distinct types of processes: direct interaction with the virus as in CRISPR-Cas adaptive immunity systems and horizontal gene transfer (HGT) which is the main route of transmission of innate immunity systems. These routes of defense evolution are not mutually exclusive and can operate simultaneously, but empirical observations suggest that at least in some bacterial and archaeal species, one or the other route dominates the defense landscape. We hypothesized that the observed dichotomy stems from different life-history tradeoffs characteristic of these organisms. To test this hypothesis, we analyzed a mathematical model of a well-mixed prokaryote population under a stochastically changing viral prevalence. Optimization of the long-term population growth rate reveals two contrasting modes of defense evolution. In stable, predictable and fluctuating, unpredictable environments with a moderate viral prevalence, direct interaction with the virus and horizontal transfer of defense genes become the optimal routes of immunity acquisition, respectively. In the HGT-dominant mode, we observed a universal distribution of the fraction of microbes with different immune repertoires. Under very low virus prevalence, the cost of immunity exceeds the benefits such that the optimal state of a prokaryote is complete defense systems. By contrast, under very high virus prevalence, horizontal spread of defense systems dominates regardless of the stability of the virome. These findings might explain consistent but enigmatic patterns in the spread of antivirus defense systems among prokaryotes such as the ubiquity of adaptive immunity in hyperthermophiles contrasting their patchy distribution among mesophiles.

IMPORTANCE: The virus-host arms race is a major component of the evolutionary process in all organisms that drove the evolution of a broad variety of immune mechanisms. In the last few years, over 200 distinct antivirus defense systems have been discovered in prokaryotes. There are two major modes of immunity acquisition: innate immune systems spread through microbial populations via horizontal gene transfer (HGT) whereas adaptive-type immune systems acquire immunity via direct interaction with the virus. We developed a mathematical model to explore the short term evolution of prokaryotic immunity and show that in stable environments with predictable viral repertoires, adaptive-type immunity is the optimal defense strategy whereas in fluctuating environments with unpredictable virus composition, HGT dominates the immune landscape.},
}

@article {pmid40501453,
year = {2025},
author = {Kamoen, L and de Bruin, DA and Kralemann, LEM and Roos, K and Wildhagen, MMDA and van Schendel, R and Hooykaas, PJJ and de Pater, S and Tijsterman, M},
title = {Division of labor within polymerase theta in repair of CRISPR-induced DNA breaks in Arabidopsis thaliana.},
journal = {PNAS nexus},
volume = {4},
number = {6},
pages = {pgaf183},
pmid = {40501453},
issn = {2752-6542},
abstract = {To develop efficient strategies for precise mutagenesis in plants, it is crucial to characterize the mechanisms involved in the repair of CRISPR-induced double strand breaks (DSBs). Polymerase theta (Polθ)-mediated end joining (TMEJ) and classical nonhomologous end joining are key pathways that generate a wide array of mutations during DSB repair. To direct repair towards more predictable outcomes, we examined the impact of direct repeats flanking DSBs, which may trigger extended microhomology-mediated end joining (eMMEJ). Unexpectedly, we found that eMMEJ in Arabidopsis thaliana requires Polθ, in contrast to eMMEJ in animals. By reintroducing mutated versions of Polθ into Polθ-deficient plants we discovered that only the helicase activity of Polθ is needed for eMMEJ; we demonstrate that plants lacking Polθ's polymerase domain are incapable of TMEJ and are resistant to TMEJ-dependent T-DNA integration but still support extended microhomology-guided DSB repair at genomic sites with direct repeats. These findings reveal species-specific functionality of Polθ and point to functional divergence in TMEJ across species. Additionally, these insights provide new opportunities to direct targeted mutagenesis in plants toward single, predictable outcomes, paving the way for more efficient crop engineering. Classification: Biological, Health, and Medical Sciences.},
}

@article {pmid40500518,
year = {2025},
author = {Song, D and Xu, W and Zhuo, Y and Zhu, A and Long, F},
title = {Orthogonal CRISPR/Cas system facilitated dual-color fluorescence fiber-embedded optofluidic nano-biochip for parallel amplification-free on-site detection of bacterium and virus.},
journal = {Mikrochimica acta},
volume = {192},
number = {7},
pages = {417},
pmid = {40500518},
issn = {1436-5073},
support = {8242031//Natural Science Foundation of Beijing/ ; 2022YFF0609102//National Key R&D Program of China/ ; },
mesh = {*CRISPR-Cas Systems ; *Escherichia coli O157/isolation & purification/genetics ; *SARS-CoV-2/isolation & purification/genetics ; Limit of Detection ; *Lab-On-A-Chip Devices ; COVID-19/diagnosis/virology ; Humans ; Optical Fibers ; Fluorescence ; Biosensing Techniques/methods ; },
abstract = {Bacterial and viral co-infections significantly exacerbate morbidity and mortality. Rapid, sensitive, and parallel detection of these pathogens remains a critical challenge. Here, an orthogonal CRISPR/Cas system facilitated dual-color fluorescence fiber-embedded optofluidic nano-biochip (CD-FOB) was fabricated. Leveraging the time-resolved effect, the CD-FOB achieved ultrasensitive parallel detection of Escherichia coli O157:H7 (E. coli O157:H7) and SARS-CoV-2 based on a multiple signal enhancement strategy, including the collateral cleavage activity of CRISPR/Cas, evanescent wave fluorescence enhancement, DNA-mediated signal amplification, and air-displacement fluorescence enhancement. Without the need for amplification, the CD-FOB system has a detection limit of 643 CFU/mL for E. coli O157:H7 and 3.48 copies/μL for SARS-CoV-2 within 50 min analysis time. To enable rapid on-site detection, a lyophilized CRISPR/Cas assay was prepared using stabilized freeze-dried reagents for detecting E. coli O157:H7 and SARS-CoV-2 in actual samples, achieving recoveries ranging from 70.5% to 200.5%. The unique combination of technical simplicity, multiplexing capability, and operational robustness positions CD-FOB as a versatile solution for combating current and future pathogen threats.},
}

*CRISPR-Cas Systems
*Escherichia coli O157/isolation & purification/genetics
*SARS-CoV-2/isolation & purification/genetics
Limit of Detection
*Lab-On-A-Chip Devices
COVID-19/diagnosis/virology
Humans
Optical Fibers
Fluorescence
Biosensing Techniques/methods

@article {pmid40500390,
year = {2025},
author = {Chen, S and Chen, Q and You, X and Zhou, Z and Kong, N and Ambrosio, F and Cao, Y and Abdi, R and Tao, W},
title = {Using RNA therapeutics to promote healthy aging.},
journal = {Nature aging},
volume = {5},
number = {6},
pages = {968-983},
pmid = {40500390},
issn = {2662-8465},
mesh = {Humans ; *Healthy Aging/genetics ; Animals ; *Aging/genetics ; *RNA/therapeutic use ; RNA Interference ; Aptamers, Nucleotide/therapeutic use ; *Genetic Therapy/methods ; Neurodegenerative Diseases/therapy/genetics ; },
abstract = {Aging is characterized by a gradual decline of cellular and physiological functions over time and an increased risk of different diseases. RNA therapeutics constitute an emerging approach to target the molecular mechanisms of aging and age-related diseases via rational design and have several advantages over traditional drug therapies, including high specificity, low toxicity and the potential for rapid development and production. Here, we discuss the latest developments in RNA therapeutics designed to promote healthy aging, including RNA activation, messenger RNA therapy, RNA interference, antisense oligonucleotides, aptamers and CRISPR-Cas-mediated RNA editing. We also review the latest preclinical and clinical studies of RNA technology for treating age-related diseases, including neurodegenerative, cardiovascular and musculoskeletal diseases. Finally, we discuss the challenges of RNA technology aimed at supporting healthy aging. We anticipate that the fusion of RNA therapeutics and aging biology will have an important effect on the development of new medicines and maximization of their efficacy.},
}

Humans
*Healthy Aging/genetics
Animals
*Aging/genetics
*RNA/therapeutic use
RNA Interference
Aptamers, Nucleotide/therapeutic use
*Genetic Therapy/methods
Neurodegenerative Diseases/therapy/genetics

@article {pmid40500074,
year = {2025},
author = {Lv, J and Yang, Y and Fabrick, JA and Wu, Y},
title = {Gene editing to enhance pesticide resistance in a beneficial predatory mite.},
journal = {Pesticide biochemistry and physiology},
volume = {212},
number = {},
pages = {106466},
doi = {10.1016/j.pestbp.2025.106466},
pmid = {40500074},
issn = {1095-9939},
mesh = {Animals ; *Gene Editing ; *Mites/genetics/drug effects ; CRISPR-Cas Systems ; Receptors, Nicotinic/genetics ; Drug Resistance/genetics ; Female ; Pest Control, Biological ; *Pesticides/pharmacology ; Acaricides/pharmacology ; Benzamides/pharmacology ; Predatory Behavior ; },
abstract = {Successful integrated pest management (IPM) often depends on a suite of control strategies that are compatible with one another. The predatory mite Neoseiulus californicus (Acari: Phytoseiidae) is a key biological control agent of spider mites (especially Tetranychus spp.) and other diminutive, yet important arthropod pests of agriculture. However, like many natural enemies, N. californicus is highly sensitive to chemical pesticides, limiting its overall effectiveness in the field. Here, we used CRISPR/Cas9 gene editing and Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) to create N. californicus harboring loss-of-function mutations in the nicotinic acetylcholine receptor α6 subunit (nAChRα6), the target of the pesticide, spinetoram. The resulting knockout strain (FZ-α6KO) exhibited a 23-fold increase in resistance to spinetoram compared to its parental strain. Inheritance of resistance to spinetoram in FZ-α6KO was autosomal, recessive, and tightly linked with the nAChRα6 gene. We demonstrate that by pairing gene editing with ReMOT Control, extremely small and fragile beneficial arthropods can be genetically manipulated. These results suggest that genetic modification to enhance pesticide resistance in beneficial predatory mites could improve the compatibility between the use of pesticides and biological control organisms.},
}

Animals
*Gene Editing
*Mites/genetics/drug effects
CRISPR-Cas Systems
Receptors, Nicotinic/genetics
Drug Resistance/genetics
Female
Pest Control, Biological
*Pesticides/pharmacology
Acaricides/pharmacology
Benzamides/pharmacology
Predatory Behavior

@article {pmid40500063,
year = {2025},
author = {Mocchetti, A and Nikoloudi, AA and Vontas, J and De Rouck, S and Van Leeuwen, T},
title = {CRISPR/Cas9 knock-out of nAChR α6 confers resistance to spinosyns in Frankliniella occidentalis and is associated with a higher fitness cost than target-site mutation G275E.},
journal = {Pesticide biochemistry and physiology},
volume = {212},
number = {},
pages = {106455},
doi = {10.1016/j.pestbp.2025.106455},
pmid = {40500063},
issn = {1095-9939},
mesh = {Animals ; *Receptors, Nicotinic/genetics ; *Insecticides/pharmacology ; *CRISPR-Cas Systems/genetics ; *Insecticide Resistance/genetics ; *Macrolides/pharmacology/toxicity ; *Thysanoptera/genetics/drug effects ; Drug Combinations ; Mutation ; Gene Knockout Techniques ; },
abstract = {Thrips are major agricultural pests globally and spinosyn insecticides like spinosad and spinetoram are commonly used for their control. However, numerous cases of resistance have emerged, often linked to mutations in the nicotinic acetylcholine receptor (nAChR) α6 subunit, the main molecular target of spinosyns. In this study, toxicological data for spinosad and spinetoram were obtained from a susceptible strain of Frankliniella occidentalis, as well as two field-collected resistant strains carrying the G275E resistance mutation. Notably, a new candidate resistance mutation never reported before, T202A, was identified in one of the field collected populations and its possible role in resistance is discussed. Further, CRISPR/Cas9-mediated knockout (KO) of α6 was performed in the susceptible strain to shed light on the phenotypic strength of this resistance mechanism previously observed in the field. The KO conferred complete insensitivity to spinosad and significant resistance to spinetoram, although higher doses of spinetoram remained lethal, suggesting potential interaction with a secondary target. Finally, in an experimental evolution approach, the α6 KO allele rapidly disappeared, indicating a substantial fitness cost. In contrast, G275E alleles persisted.},
}

Animals
*Receptors, Nicotinic/genetics
*Insecticides/pharmacology
*CRISPR-Cas Systems/genetics
*Insecticide Resistance/genetics
*Macrolides/pharmacology/toxicity
*Thysanoptera/genetics/drug effects
Drug Combinations
Mutation
Gene Knockout Techniques

@article {pmid40499559,
year = {2025},
author = {Vaz, M and Watson, KL and Moorehead, RA},
title = {Reduced JAG1 Expression Through miR-200 Overexpression or Crispr-Cas Mediated Knockout Impairs TNBC Growth and Metastasis.},
journal = {Molecular carcinogenesis},
volume = {},
number = {},
pages = {},
doi = {10.1002/mc.23937},
pmid = {40499559},
issn = {1098-2744},
support = {//This study was funded by a CIHR project grant PJT-162218 and a CIHR Priority grant PLL-192130/ ; },
abstract = {Studies from our lab demonstrated that increasing miR-200 expression in human triple negative breast cancer (TNBC) reduced tumor growth and metastasis In Vivo. In this study, we found that overexpression of miR-200s in TNBC cells significantly reduced the expression of JAG1. When JAG1 was knocked out in MDA-MB-231 cells proliferation and invasion were significantly reduced In Vitro. Moreover, loss of JAG1 inhibited mammary tumor growth and metastasis In Vivo. RNA sequencing revealed that loss of JAG1 altered the expression of genes associated with the ECM, angiogenesis, and EMT. These results imply that miR-200s may mediate some of their antitumor actions through reducing JAG1 expression and suggest that agents targeting JAG1 should be further evaluated as a therapeutic strategy for TNBC.},
}

@article {pmid40499557,
year = {2025},
author = {Kang, B and Lee, S and Ko, DH and Venkatesh, J and Kwon, JK and Kim, H and Kang, BC},
title = {Virus-induced systemic and heritable gene editing in pepper (Capsicum annuum L.).},
journal = {The Plant journal : for cell and molecular biology},
volume = {122},
number = {5},
pages = {e70257},
pmid = {40499557},
issn = {1365-313X},
support = {RS-2024-00322053//Rural Development Administration, Republic of Korea/ ; RS-2021-NR059217//the National Research Foundation of Korea/ ; },
mesh = {*Capsicum/genetics/virology ; *Gene Editing/methods ; Plants, Genetically Modified/genetics ; *Plant Viruses/genetics ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Oxidoreductases/genetics ; },
abstract = {Genome editing using the CRISPR/Cas system enables rapid and efficient plant breeding by directly introducing desired traits into elite lines within a short time frame. However, challenges associated with conventional Agrobacterium tumefaciens-mediated transformation and regeneration have limited gene editing in pepper (Capsicum annuum L.). In this study, we applied and optimized a virus-induced gene editing (VIGE) system to overcome these limitations. We inoculated transgenic pepper seedlings already expressing Cas9 with vectors based on tobacco rattle virus 2 (TRV2) expressing single guide RNAs (sgRNAs) targeting Phytoene desaturase (PDS); shoots regenerated from inoculated cotyledons displayed photobleaching phenotypes. To promote sgRNA mobility and maintain its integrity, we modified the pTRV2-sgRNA vector by incorporating a self-cleaving hammerhead ribozyme (HH) sequence to produce an intact sgRNA fused to part of the mobile RNA of FLOWERING LOCUS T. Additionally, we tested alternative mobile elements, such as tRNA[Ile] and tRNA[Met]. Furthermore, we cultivated plants at the low temperature of 20°C following TRV inoculation to increase TRV persistence and spread. These optimizations, including vector modifications and cultivation conditions, resulted in a systemic editing efficiency of 36.3%, as evidenced by systemic leaves showing photobleaching phenotypes. We determined that 8.5% of progeny from plants inoculated with the pTRV-HH-CaPDS-sgRNA-FT construct were mutated at the CaPDS locus. In addition, we used our VIGE system to successfully edit FASCICULATE, producing mutants whose inflorescences showed a fasciculate phenotype. Direct inoculation with a TRV-based vector expressing a mobile sgRNA to bypass tissue culture, therefore, offers an effective tool for molecular studies and breeding in pepper.},
}

*Capsicum/genetics/virology
*Gene Editing/methods
Plants, Genetically Modified/genetics
*Plant Viruses/genetics
CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Oxidoreductases/genetics

@article {pmid40499551,
year = {2025},
author = {Stadager, J and Bernardini, C and Hartmann, L and May, H and Wiepcke, J and Kuban, M and Najafova, Z and Johnsen, SA and Legewie, S and Traube, FR and Jude, J and Rathert, P},
title = {CRISPR GENome and epigenome engineering improves loss-of-function genetic-screening approaches.},
journal = {Cell reports methods},
volume = {5},
number = {6},
pages = {101078},
doi = {10.1016/j.crmeth.2025.101078},
pmid = {40499551},
issn = {2667-2375},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Induced Pluripotent Stem Cells/metabolism/cytology ; *Epigenome/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Genetic Testing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Epithelial-Mesenchymal Transition/genetics ; *Loss of Function Mutation/genetics ; Gene Knockout Techniques ; Cell Differentiation/genetics ; Epigenome Editing ; },
abstract = {CRISPR-Cas9 technology has revolutionized genotype-to-phenotype assignments through large-scale loss-of-function (LOF) screens. However, limitations like editing inefficiencies and unperturbed genes cause significant noise in data collection. To address this, we introduce CRISPR gene and epigenome engineering (CRISPRgenee), which uses two specific single guide RNAs (sgRNAs) to simultaneously repress and cleave the target gene within the same cell, increasing LOF efficiencies and reproducibility. CRISPRgenee outperforms conventional CRISPR knockout (CRISPRko), CRISPR interference (CRISPRi), and CRISPRoff systems in suppressing challenging targets and regulators of cell proliferation. Additionally, it efficiently suppresses modulators of epithelial-to-mesenchymal transition (EMT) and impairs neuronal differentiation in a human induced pluripotent stem cell (iPSC) model. CRISPRgenee exhibits improved depletion efficiency, reduced sgRNA performance variance, and accelerated gene depletion compared to individual CRISPRi or CRISPRko screens, ensuring consistency in phenotypic effects and identifying more significant gene hits. By combining CRISPRko and CRISPRi, CRISPRgenee increases LOF rates without increasing genotoxic stress, facilitating library size reduction for advanced LOF screens.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Induced Pluripotent Stem Cells/metabolism/cytology
*Epigenome/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
*Genetic Testing/methods
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Epithelial-Mesenchymal Transition/genetics
*Loss of Function Mutation/genetics
Gene Knockout Techniques
Cell Differentiation/genetics
Epigenome Editing

@article {pmid40499189,
year = {2025},
author = {Fernandes, PMB and Fernandes, AAR and Maurastoni, M and Rodrigues, SP},
title = {Lab Legends and Field Phantoms: The Tale of Virus-Resistant Plants.},
journal = {Annual review of virology},
volume = {},
number = {},
pages = {},
doi = {10.1146/annurev-virology-092623-101850},
pmid = {40499189},
issn = {2327-0578},
abstract = {Plant viruses present significant challenges to global agriculture, causing crop losses, threatening food security, and imposing economic burdens. Advances in biotechnology have revolutionized strategies to attack these threats, with genetically modified and genome-edited virus-resistant plants, developed using precision tools such as RNA interference and CRISPR/Cas technology, playing pivotal roles. Despite these breakthroughs, fragmented regulatory frameworks and divergent policies across regions including the European Union and the Global South hinder the global adoption of such innovations. Multifaceted approaches, including gene pyramiding, microbiome-based strategies, and pathogen-targeted defenses, show promise for enhancing plant resilience. This review explores the biological, regulatory, and ethical dimensions of deploying virus-resistant crops, emphasizing the need for harmonization of international regulation to maximize biotechnological benefits. By addressing these challenges, biotechnology can advance sustainable agriculture, secure food systems, and mitigate the effect of plant viral diseases.},
}

@article {pmid40499044,
year = {2025},
author = {Zhan, X and Jiang, Y and Li, Z and Hu, X and Lan, F and Ying, B and Wu, Y},
title = {Split DNA Tetrahedron-Mediated Spatiotemporal-Hierarchy CRISPR Cascade Integrated with Au@Pt Nanolabels and Artificial Intelligence for a Cervical Cancer MicroRNA Bioassay.},
journal = {ACS nano},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsnano.5c01376},
pmid = {40499044},
issn = {1936-086X},
abstract = {The screening and monitoring of microRNAs as cancer molecular biomarkers is clinically significant, but traditional methods lack sufficient sensitivity, accuracy, and convenience. The CRISPR-colorimetric lateral flow assay (CLFA) integration offers a promising and efficient solution; however, cumbersome preamplification and poor quantification hinder clinical adoption. In this study, we developed a one-step isothermal CRISPR-Cas cascaded sensing system that is preamplification-free. At its core is a designed and selected split DNA tetrahedron activator, employing spatiotemporal-hierarchy mechanisms to precisely bidirectionally drive the kinetics of two Cas enzymes, accelerating the activation of Cas13a while delaying the initiation of Cas12a, to achieve optimal balance. This system enables ultrasensitive, single-step, single-tube, and rapid detection of a cervical cancer relative biomarker, microRNA-21, achieving a limit of detection of 38 aM with a broad linear range. The CRISPR system is further integrated with CLFA enhanced by ultrathin platinum-protected gold nanolabels (Au@Pt, also named Au@s-Pt), along with a smartphone equipped with dual convolutional neural network models (YOLO v5 and MobileNet v3), enabling more precise, rapid quantification of target miRNA. Using this integrated platform, miRNA-21 levels in cervical cancer and precancerous samples can be accurately quantified with approximately 30 min at low cost and without the need for large, sophisticated instruments, with results showing good concordance with quantitative real-time polymerase chain reaction. This platform provides an efficient, highly sensitive, user-friendly, and quantifiable point-of-care testing solution.},
}

@article {pmid40498336,
year = {2025},
author = {Patel, E and Das, P and Hazra, S and Sharma, M and Chhabra, G and Gill, BS and Sharma, S and Kaur, A and Singla, D and Sandhu, JS},
title = {Mutation in soybean Lox-2 PLAT/LH2 domain through CRISPR/Cas9 reduces seed lipoxygenase activity: responsible for undesirable flavour.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {29},
pmid = {40498336},
issn = {1573-9368},
mesh = {*Glycine max/genetics/enzymology/growth & development ; *Seeds/genetics/enzymology ; *CRISPR-Cas Systems/genetics ; *Lipoxygenase/genetics/metabolism ; Plants, Genetically Modified/genetics/growth & development ; Mutation ; *Plant Proteins/genetics/metabolism ; },
abstract = {Soybean, a protein and oil rich legume is primarily used as livestock feed and to a lesser extent for human consumption due to undesirable flavour in the seeds caused by L-2 isozyme of lipoxygenase. Herein, soybean with reduced isozyme activity was developed through CRISPR/Cas9 targeted mutation in L-2 encoding Lox-2 gene. sgRNA designed from PLAT/LH2 domain in second exon of Lox-2 (Lox-2 E2) was validated by in vitro cleavage assay; inserted in CRISPR/Cas9 binary vector and used for genetic transformation of SL1074 cultivar hypocotyl segments. A total of 12 T0 putative plants were identified through PCR. Amongst these, four revealed mutation at the target sgRNA site by CEL1 assay and substitution of a base A with G six bp upstream of PAM converting lysine to glutamic acid at 119 position. T1 and T2 seeds derived from mutant T0-37 plant showed upto 25.49% reduction in isozyme activity as compared to SL1074. The base substitution was confirmed in T1 progeny; segregation analysis revealed homozygosity and heritability of mutation in T2 plants. The interaction between structural models of SL1074, mutant domains and negatively charged substrates revealed strong binding affinity of the substrates with positively charged lysine in SL1074 domain due to formation of two hydrogen bonds. On the contrary, weak binding of the substrates with negatively charged glutamic acid in mutant domain and absence of hydrogen bond explained reduction of isozyme activity in T2 seeds. The mutant soybean with reduced isozyme activity is an important source for introgressing the trait in plant breeding programs.},
}

*Glycine max/genetics/enzymology/growth & development
*Seeds/genetics/enzymology
*CRISPR-Cas Systems/genetics
*Lipoxygenase/genetics/metabolism
Plants, Genetically Modified/genetics/growth & development
Mutation
*Plant Proteins/genetics/metabolism

@article {pmid40498069,
year = {2025},
author = {Chen, P and Li, X and Zhou, Q and Chen, J and Lu, L and Wang, P and Zhang, G and Sun, D and Huang, X and Liu, J and Wang, X},
title = {Configuration of adaptable template RNA architectures to unfold the editable space of a nuclease prime editor.},
journal = {Nucleic acids research},
volume = {53},
number = {11},
pages = {},
pmid = {40498069},
issn = {1362-4962},
support = {2021YFF1000700//National Key R&D Program of China/ ; 32272848//National Natural Science Foundation of China/ ; CARS-39-03//China Agricultural Research System/ ; 2022GDTSLD-46//China Agricultural Research System/ ; },
mesh = {*Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; *RNA/chemistry/genetics ; Humans ; DNA Breaks, Double-Stranded ; CRISPR-Cas Systems ; },
abstract = {The nuclease prime editor (PEn) combines double-strand break (DSB) induction with reverse transcription for editing. Recently, high-activity PEn forms (e.g. uPEn) have been developed via the concomitant application of DNA repair regulator(s). While the standard uPEn introduces edits only downstream of the nuclease-induced DNA break, we seek innovative designs to enable upstream-directed editing by re-configuring guide/template RNAs to drive prime edits into the target strand (TS), instead of the conventional non-TS. We first devise a dual-RNA uPEn strategy by supplementing a cleavage-competent sgRNA with an accessory template RNA for modifying target strand (ActRNA:t). Characterization of the dual-RNA system allows us to next develop a bifunctional target strand-programming pegRNA (tsp-pegRNA). Both the dual- and single-RNA upstream-modifying uPEn forms (versions 3.1/3.2) successfully drive diverse types of accurate edits into a panel of locations refractory to the standard uPEn and the latest nickase PE. Moreover, we provide insights on the role of uPEn's helper module (i.e. i53) in driving TS prime edits. Additional co-administration of a DNA-dependent protein kinase inhibitor with uPEn3.2 leads to further optimization of editing purities. Together, these advances transform uPEn into a highly applicable tool with much-expanded editable space, and lay a strong foundation for future development of PEn/PE platforms.},
}

*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics/chemistry
*RNA/chemistry/genetics
Humans
DNA Breaks, Double-Stranded
CRISPR-Cas Systems

@article {pmid40498068,
year = {2025},
author = {Qiao, J and Zhang, J and Jiang, Q and Jin, S and He, R and Qiao, B and Liu, Y},
title = {Boosting CRISPR/Cas12a intrinsic RNA detection capability through pseudo hybrid DNA-RNA substrate design.},
journal = {Nucleic acids research},
volume = {53},
number = {11},
pages = {},
pmid = {40498068},
issn = {1362-4962},
support = {2022YFC2304304//National Key Research and Development Program of China/ ; 2023DJC136//Science and Technology Innovation Talent Plan of Hubei Province/ ; 2025AFB825//Natural Science Foundation of Hubei Province/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *CRISPR-Associated Proteins/metabolism/genetics ; MicroRNAs/genetics/analysis ; *DNA/chemistry/genetics ; *RNA/analysis/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; Nucleic Acid Hybridization ; Limit of Detection ; },
abstract = {The CRISPR/Cas12a [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 12a] system is known for its intrinsic RNA-guided trans-cleavage activity; however, its RNA detection sensitivity is limited, with conventional methods typically achieving detection limits in the nanomolar range. Here, we report the development of a "pseudo hybrid DNA-RNA" (PHD) assay that significantly enhances the RNA detection capability of Cas12a. The PHD assay achieves a striking detection limit of 7.7 pM using single CRISPR RNA (crRNA) and 33.8 fM using pooled crRNAs. Importantly, this assay exhibits ultra-high specificity, capable of distinguishing mutated RNA target sequences at the protospacer adjacent motif (PAM)-distal region. It can also detect ultrashort RNA sequences as short as 6-8 nt and long RNAs with complex secondary structures. Additionally, the PHD assay enables PAM-free attomolar-level DNA detection. We further demonstrate the practical utility of the PHD assay by successfully detecting miR-155 biomarkers and human pappilloma virus 16 DNA in clinical samples. We anticipate that the design principles established in this study can be extended to other CRISPR/Cas enzymes, thereby accelerating the development of powerful nucleic acid testing tools for various applications.},
}

*CRISPR-Cas Systems/genetics
Humans
*CRISPR-Associated Proteins/metabolism/genetics
MicroRNAs/genetics/analysis
*DNA/chemistry/genetics
*RNA/analysis/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
*Endodeoxyribonucleases/metabolism/genetics
*Bacterial Proteins/metabolism/genetics
Nucleic Acid Hybridization
Limit of Detection

@article {pmid40497031,
year = {2025},
author = {Galli, C},
title = {Current Techniques of Gene Editing in Pigs for Xenotransplantation.},
journal = {Transplant international : official journal of the European Society for Organ Transplantation},
volume = {38},
number = {},
pages = {13807},
pmid = {40497031},
issn = {1432-2277},
mesh = {Animals ; *Transplantation, Heterologous/methods ; *Gene Editing/methods ; Swine/genetics ; Humans ; CRISPR-Cas Systems ; Animals, Genetically Modified ; Nuclear Transfer Techniques ; Endogenous Retroviruses ; },
abstract = {Shortage of human organs for transplantation has created a demand for alternative solutions of which xenotransplantation is amongst the most promising one in the short term. However, the immune reaction following transplantation of a pig organ is greater than the one elicited during allotransplantation. Genetic engineering of the pig is required so that pig organs or tissues are made less immunogenic to humans by eliminating some antigens and by expressing human proteins that can reduce the damage by the host immune system. To generate founder animals with the desired mutations genetic engineering of somatic cells with multiplexed mutations combined with somatic cell nuclear transfer (SCNT) is the best solution with the technology available today. Safety concerns include potential zoonosis, primarily porcine endogenous retroviruses (PERVs). Ethical considerations might arise from the use animals involved in research. Genome editing techniques based CRISPR-Cas9, have greatly facilitated the modification of pig's genome to address coagulation and inflammation issues, to mention just a few, arising after the pig organ is transplanted into a human. However, further research is needed to ensure safety and efficacy of the genome edits introduced in the pig genome are compatible with the health and welfare of the pigs.},
}

Animals
*Transplantation, Heterologous/methods
*Gene Editing/methods
Swine/genetics
Humans
CRISPR-Cas Systems
Animals, Genetically Modified
Nuclear Transfer Techniques
Endogenous Retroviruses

@article {pmid40496602,
year = {2025},
author = {Jangra, S and Seal, DR and Ghosh, A},
title = {Biotechnological advancements for sustainable management of thrips.},
journal = {3 Biotech},
volume = {15},
number = {7},
pages = {204},
pmid = {40496602},
issn = {2190-572X},
abstract = {Thrips are minute, slender, polyphagous, thysanopteran insect pests that cause huge economic losses to crops by directly feeding on the phloem sap and transmitting several plant viruses. Thrips have emerged as a major threat to global agriculture and food security. Current management options mainly rely on chemical insecticides; however, thrips have evolved resistance to most commonly used insecticides, making management extremely difficult. The availability of host-plant resistance is limited in the case of thrips. Biotechnological approaches such as genetic engineering, RNA interference (RNAi), antisense oligonucleotides (ASOs), artificial microRNA (amiRNA), and genome editing have paved the way for the development of environmentally sustainable thrips management options. However, the adoption of these biotechnological approaches needs further refinement and validation. Transgenic plants with anti-herbivory proteins hold promise to be successful in managing thrips. Spray-induced gene silencing (SIGS) and gene editing would be novel alternatives to hazardous pesticides. This review discusses the progress made towards using modern biotechnological interventions in functional genomics, emphasizing their application in sustainable thrips management.},
}

@article {pmid40494869,
year = {2025},
author = {Basu, M and Xiao, JF and Kailasam Mani, SK and Qu, F and Lin, Y and Duex, J and Ye, H and Neang, V and Theodorescu, D},
title = {Genes driving three-dimensional growth of immortalized cells and cancer.},
journal = {Cell death & disease},
volume = {16},
number = {1},
pages = {442},
pmid = {40494869},
issn = {2041-4889},
support = {R01 CA075115/CA/NCI NIH HHS/United States ; R29 CA075115/CA/NCI NIH HHS/United States ; CA075115//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Humans ; Animals ; *Urinary Bladder Neoplasms/genetics/pathology ; Mice ; Cell Proliferation/genetics ; Mitogen-Activated Protein Kinase 1/genetics/metabolism/antagonists & inhibitors ; Cell Line, Tumor ; Cell Transformation, Neoplastic/genetics ; CRISPR-Cas Systems ; },
abstract = {Unchecked growth in three-dimensions (3D) in culture is a key feature of immortalized cells on the path to malignant transformation and hence a potential target phenotype for prevention. Also, expression of genes driving this process, but not that of 2D growth, would likely be more specific to cancer development and their inhibition would be less toxic to normal cells, many of which can grow in 2D but rarely in 3D culture. To define such genes, we compared CRISPR depletion screens performed in HBLAK, a spontaneously immortalized, non-tumorigenic human urothelial cell line, grown in 2D to those in 3D. Using the CRISPR Bassik DTKP (drug target kinase phosphatase) deletion library targeting 2,333 genes, we identified 85 genes which were specifically lost in 3D cultures. Prioritizing hits to those associated with bladder cancer in patients provided us with a set of 11 genes. Only one gene, MAPK1 remained relevant if a human pan-cancer criteria was applied. Single gene in vitro validation confirmed that MAPK1 inhibition was specific to 3D growth. We also found that MAPK1 depletion led to significant growth reductions in human tumor xenografts in vivo. Inhibition of MAPK1 by Ulixertinib, an orally active MAPK1 inhibitor, led to human bladder cancer growth inhibition in both 3D in vitro and in vivo models. In summary, screening for genes specifically driving 3D growth in immortalized cells may provide targets for both prevention and early therapy in bladder and other cancers while potentially limiting therapeutic toxicity.},
}

Humans
Animals
*Urinary Bladder Neoplasms/genetics/pathology
Mice
Cell Proliferation/genetics
Mitogen-Activated Protein Kinase 1/genetics/metabolism/antagonists & inhibitors
Cell Line, Tumor
Cell Transformation, Neoplastic/genetics
CRISPR-Cas Systems

@article {pmid40494841,
year = {2025},
author = {Liu, H and Zhang, M and Sun, L and Peng, Y and Sun, Y and Fan, Y and Li, H and Liu, D and Lu, H},
title = {Synergistic optimization enhancing the precision and efficiency of cytosine base editors in poplar.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {904},
pmid = {40494841},
issn = {2399-3642},
mesh = {*Populus/genetics/drug effects ; *Gene Editing/methods ; *Cytosine/metabolism ; *CRISPR-Cas Systems ; Genome, Plant ; Plants, Genetically Modified/genetics ; },
abstract = {CRISPR/Cas9 genome editing technology, particularly cytosine base editing (CBE) systems, emerges as a powerful tool for precise genomic modification in plants, offering transformative applications across agricultural and forestry research and breeding programs. However, current CBE systems in poplar exhibit low efficiency and imprecise base substitutions, and optimization of base editing systems specifically for poplar remains a significant challenge. To address these limitations, we engineer a high-efficiency poplar CBE system (hyPopCBE) by integrating the MS2-UGI system, fusing Rad51 DNA-binding domain, and modifying the nuclear localization signal. Through stepwise optimization, we develop hyPopCBE-V4, which exhibits a synergistic effect in woody plants. Compared to the original hyPopCBE-V1, hyPopCBE-V4 improves C to T editing efficiency while reducing byproducts and exhibiting a narrower editing window. The proportion of plants with clean C to T edits (without byproducts) increases from 20.93% to 40.48%, and the efficiency of clean homozygous C to T editing rises from 4.65% to 21.43%. Using hyPopCBE-V1 and its variants, we induce Pro197Leu mutation in the herbicide target gene PagALS. Poplar lines with edits in all four PagALS homologues exhibit high resistance to tribenuron and nicosulfuron. This study employs a multi-component synergistic optimization strategy that specifically enhances the efficiency and precision of CBE editing in poplar while improving synchronous editing of alleles. Through editing the herbicide resistance gene PagALS, we obtain the herbicide-resistant poplar germplasm. Our research provides a more precise and efficient CBE tool for genetic modification in poplar that can also be applied to other forestry species, demonstrating its potential for advancing forestry research and breeding programs.},
}

*Populus/genetics/drug effects
*Gene Editing/methods
*Cytosine/metabolism
*CRISPR-Cas Systems
Genome, Plant
Plants, Genetically Modified/genetics

@article {pmid40493278,
year = {2025},
author = {Kaur, J and Viswanathan P, A and Bari, VK},
title = {CRISPR/Cas9-mediated editing of jasmonic acid pathways to enhance biotic & abiotic stress tolerance: An overview & prospects.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {125},
pmid = {40493278},
issn = {1438-7948},
mesh = {*Cyclopentanes/metabolism ; *Oxylipins/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Stress, Physiological/genetics ; Crops, Agricultural/genetics ; Plant Growth Regulators/metabolism ; Signal Transduction ; },
abstract = {Food security is becoming increasingly important as the world's population grows, and the likelihood that climate change could impair agricultural supply complicates matters further. However, plants actively suppress growth as an adaptation strategy to enhance survival under stress conditions. Phytohormone jasmonates (JAs) regulate various physiological processes, including plant growth, development, and senescence. Plant biotic and abiotic stress responses cause dynamic shifts in the metabolism and signaling of JAs, suggesting that JAs response impacts plant development and resistance to various stresses. The JAs-associated responses depend on core components of JAs -signaling, including the transcriptional repressors protein JAZ and the transcription factor MYC2. While traditional breeding has greatly benefited the world, this approach has several disadvantages, such as the emergence of undesirable traits and species barriers. Genome editing technology has revolutionized plant biology research and has significant ramifications for agriculture and global food security, particularly in light of climate change and population growth. CRISPR/Cas9 and its derivative tools have been used for genome editing in numerous crops to improve or alter desired plant phenotypes. This review summarizes JA's role in plant stress and defense and how CRISPR/Cas9-editing technology modifies plant JA's responses, especially against biotic and abiotic stress.},
}

*Cyclopentanes/metabolism
*Oxylipins/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems
*Stress, Physiological/genetics
Crops, Agricultural/genetics
Plant Growth Regulators/metabolism
Signal Transduction

@article {pmid40493189,
year = {2025},
author = {Gorelik, A and Paulo, JA and Schroeter, CB and Lad, M and Shurr, A and Mastrokalou, C and Siddiqi, S and Suyari, O and Brognard, J and Walter, D and Matthews, J and Palmer, TM and Gygi, SP and Ahel, I},
title = {CRISPR screens and quantitative proteomics reveal remodeling of the aryl hydrocarbon receptor-driven proteome through PARP7 activity.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {24},
pages = {e2424985122},
doi = {10.1073/pnas.2424985122},
pmid = {40493189},
issn = {1091-6490},
support = {224095/Z/21/Z//Wellcome Trust (WT)/ ; 210634 223107 302632//Wellcome Trust (WT)/ ; BB/R007195/1 BB/W016613/1//UKRI | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; C35050/A22284//Cancer Research UK (CRUK)/ ; GM67945//HHS | NIH (NIH)/ ; GM132129//HHS | NIH (NIH)/ ; },
mesh = {*Receptors, Aryl Hydrocarbon/metabolism/genetics ; Humans ; *Proteome/metabolism/genetics ; *Poly(ADP-ribose) Polymerases/metabolism/genetics ; Proteomics/methods ; Suppressor of Cytokine Signaling 3 Protein/metabolism/genetics ; Cell Line, Tumor ; Signal Transduction ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Lung Neoplasms/metabolism/genetics ; Nucleoside Transport Proteins ; Basic Helix-Loop-Helix Transcription Factors ; },
abstract = {PARP7 is an enzyme that uses donor substrate NAD[+] to attach a single ADP-ribose moiety onto proteins related to immunity, transcription, and cell growth and motility. Despite the importance of PARP7 in these processes, PARP7 signaling networks remain underresearched. Here, we used genome-wide CRISPR screens and multiplex quantitative proteomics in distinct lung cancer cell lines treated with a PARP7 inhibitor to better understand PARP7 molecular functions. We find that manipulating the aryl hydrocarbon receptor (AHR) transcriptional activity mediates PARP7 inhibitor sensitivity and triggers robust changes to the AHR-controlled proteome (AHR-ome). One of the striking features of such AHR-ome remodeling was the downregulation of filamins A and B concurrent with the induction of the corresponding E3 ubiquitin ligase ASB2. We also show that suppressor of cytokine signaling 3 (SOCS3) crosstalks to AHR. Inhibition of PARP7 in SOCS3 knockout cells leads to reduced viability compared to wild-type cells treated with a PARP7 inhibitor. Our results reveal signaling interplay between PARP7, AHR, and SOCS3 and establish an invaluable resource to study the role of PARP7 in the regulation of AHR signaling and innate immunity through its ADP-ribosyl transferase activity.},
}

*Receptors, Aryl Hydrocarbon/metabolism/genetics
Humans
*Proteome/metabolism/genetics
*Poly(ADP-ribose) Polymerases/metabolism/genetics
Proteomics/methods
Suppressor of Cytokine Signaling 3 Protein/metabolism/genetics
Cell Line, Tumor
Signal Transduction
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Lung Neoplasms/metabolism/genetics
Nucleoside Transport Proteins
Basic Helix-Loop-Helix Transcription Factors

@article {pmid40491435,
year = {2025},
author = {Santarossa, BA and Mariani, É and Corrêa, ADP and Costa, FC and Taylor, MC and Kelly, JM and Elias, MC and Calderano, SG},
title = {Stage-specific MCM protein expression in Trypanosoma cruzi: insights into metacyclogenesis and G1 arrested epimastigotes.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1584812},
pmid = {40491435},
issn = {2235-2988},
mesh = {*Trypanosoma cruzi/growth & development/genetics/metabolism ; *Protozoan Proteins/genetics/metabolism ; *Life Cycle Stages ; *Minichromosome Maintenance Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *G1 Phase Cell Cycle Checkpoints ; },
abstract = {Trypanosoma cruzi is a protozoan parasite that is the etiological agent of Chagas disease, which is endemic to Latin America with reported cases in non-endemic regions such as Europe, Asia, and Oceania due to migration. During its lifecycle, T. cruzi alternates between replicative and non-replicative infective lifeforms. Metacyclogenesis is the most studied transition of the T. cruzi life cycle, where replicative epimastigotes differentiate into infective metacyclic trypomastigotes inside the gut of the triatomine vector. This early-branching organism expresses a divergent pre-replication complex (pre-RC) where the only conserved component is the MCM2-7 protein family. Given the role of pre-RC components in cell cycle regulation, we investigated whether MCM expression and location could be involved in proliferation control in epimastigotes and during metacyclogenesis. Using CRISPR/Cas9, we tagged MCM subunits and tracked their expression and subcellular localization. Our findings reveal that MCM subunits are consistently expressed and localized to the nucleus throughout the epimastigote cell cycle, including in G1/G0-arrested cells. However, MCM subunits are degraded during metacyclogenesis as cells enter the G0 state, marking the transition to replication arrest. Therefore, epimastigotes arrested in G1/G0 can either maintain MCM complex expression and resume the cell cycle when conditions become favorable, or they can undergo metacyclogenesis, exiting the cell cycle and entering a G0 state, where MCM subunits are degraded as part of the replication repression mechanism.},
}

*Trypanosoma cruzi/growth & development/genetics/metabolism
*Protozoan Proteins/genetics/metabolism
*Life Cycle Stages
*Minichromosome Maintenance Proteins/genetics/metabolism
CRISPR-Cas Systems
*G1 Phase Cell Cycle Checkpoints

@article {pmid40491004,
year = {2025},
author = {Capelli, L and Marzari, S and Spezzani, E and Bertucci, A},
title = {Synthetic CRISPR Networks Driven by Transcription Factors via Structure-Switching DNA Translators.},
journal = {Journal of the American Chemical Society},
volume = {147},
number = {24},
pages = {21184-21193},
doi = {10.1021/jacs.5c06913},
pmid = {40491004},
issn = {1520-5126},
mesh = {*CRISPR-Cas Systems ; *Transcription Factors/metabolism/chemistry ; *DNA/chemistry/metabolism/genetics ; Synthetic Biology ; CRISPR-Associated Proteins/metabolism/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; },
abstract = {CRISPR-Cas systems have advanced many domains in life sciences, enabling diverse applications in gene editing, diagnostics, and biosensing. Here, we introduce a platform that leverages transcription factors (TFs) to regulate CRISPR-Cas12a trans-cleavage activity via engineered DNA translators. These dynamic DNA structures respond to TF binding by switching conformations, modulating Cas12a activity. Using TATA-binding protein and Myc-Max as TF models, we optimized DNA translators for precise and tunable control with rapid response kinetics. We demonstrated the platform's specificity and versatility by integrating TF-induced regulation into synthetic biology networks, including the activation of a fluorogenic RNA aptamer (Mango III) and the creation of an artificial multimolecular communication pathway between Cas12a and Cas13a. This work establishes TFs as effective regulators of CRISPR-Cas systems, enabling novel protein-nucleic acid communication channels, showing potential for novel synthetic biology applications.},
}

*CRISPR-Cas Systems
*Transcription Factors/metabolism/chemistry
*DNA/chemistry/metabolism/genetics
Synthetic Biology
CRISPR-Associated Proteins/metabolism/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
Gene Editing

@article {pmid40490981,
year = {2025},
author = {Zheng, Y and Du, F and Hang, Y and Ma, W and Yang, G and Wu, N and Sun, X},
title = {Recent Advances in Genome Base Editing Technology and Its Applications in Industrial Microorganism.},
journal = {Biotechnology journal},
volume = {20},
number = {6},
pages = {e70052},
doi = {10.1002/biot.70052},
pmid = {40490981},
issn = {1860-7314},
support = {2023YFA0914400//National Key Research and Development Program of China/ ; BK20230059//Natural Science Foundation of Jiangsu Province/ ; 32422068//National Natural Science Foundation of China/ ; 22378209//National Natural Science Foundation of China/ ; 22038007//National Natural Science Foundation of China/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Industrial Microbiology/methods ; Metabolic Engineering ; Synthetic Biology ; Saccharomyces cerevisiae/genetics ; },
abstract = {Base editing technology is a novel gene-editing approach derived from the CRISPR/Cas9 system, enabling precise and efficient base conversion. Due to operational simplicity, strong target specificity, high editing efficiency, and minimal editing byproducts, base editing has been widely applied in gene therapy, crop breeding, construction of model organisms, and microbial metabolic engineering. In this review, we systematically summarize the development history and recent advancements of several major base editors, including cytosine base editors (CBEs), adenine base editors (ABEs), CRISPR-free base editors, C-to-G base editors (CGBEs), glycosylase base editors (GBEs), and IscB-derived base editors. Furthermore, we comprehensively summarize optimization strategies for base editors and its application in constructing efficient industrial microorganism, such as Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, Yarrowia lipolytica, and Aspergillus niger. This review aims to facilitate the broader application of base editing technologies in synthetic biology and accelerate their translational potential.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Industrial Microbiology/methods
Metabolic Engineering
Synthetic Biology
Saccharomyces cerevisiae/genetics

@article {pmid40490955,
year = {2025},
author = {Ruan, S and Wang, A and Zou, H and Lin, Y and Ye, L and Liang, S},
title = {Synthetic Genetic Circuits Enabled in Komagataella phaffii Through T7 RNAP/CRISPRa System.},
journal = {Biotechnology journal},
volume = {20},
number = {6},
pages = {e70036},
doi = {10.1002/biot.70036},
pmid = {40490955},
issn = {1860-7314},
support = {2021YFC2104000//National Key Research and Development Program/ ; 32272276//National Natural Science Foundation of China/ ; },
mesh = {*Gene Regulatory Networks/genetics ; *DNA-Directed RNA Polymerases/genetics/metabolism ; *Synthetic Biology/methods ; *Saccharomycetales/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Viral Proteins/genetics/metabolism ; Promoter Regions, Genetic ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The CRISPR activation (CRISPRa) transcriptional system has become a powerful synthetic biology tool for the regulation of endogenous gene expression, allowing for precise fine-tuning of target genes through the simple modification of sgRNA sequences. In this study, we demonstrate that sgRNAs can be effectively expressed using the T7 transcription system. The insertion of tRNA sequences between PT7 and sgRNAs significantly enhances the efficiency of transcriptional activation. Furthermore, the design of PT7-tRNA-sgRNA arrays facilitates the multiplexed activation of genes. sgRNA expression was regulated by the Tet-on induction system, split-T7 system, and RNA cleavage processing by HH-HDV, resulting in the creation of a Boolean logic gene circuit capable of performing both AND and OR logic operations. Finally, we developed a UPR self-responsive system by utilizing endogenous promoters that are responsive to UPR signals to control the expression of T7 RNAP. This system dynamically regulates the expression of the endogenous HAC1 transcription factor, thus enhancing the secretion of heterologous proteins. The findings from this study highlight the potential of utilizing the T7 transcription system for the construction of genetic circuits, providing a practical toolkit for gene regulation in the industrial Komagataella phaffii strain.},
}

*Gene Regulatory Networks/genetics
*DNA-Directed RNA Polymerases/genetics/metabolism
*Synthetic Biology/methods
*Saccharomycetales/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Viral Proteins/genetics/metabolism
Promoter Regions, Genetic
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40490752,
year = {2025},
author = {Masson, JD and Taglietti, V and Ruby, F and Ono, H and Mouri, N and Jorge, A and Guillaud, L and Tiret, L and Relaix, F},
title = {Extensive striated muscle damage in a rat model of Duchenne muscular dystrophy with Dmd exons 10-17 duplication.},
journal = {Skeletal muscle},
volume = {15},
number = {1},
pages = {16},
pmid = {40490752},
issn = {2044-5040},
support = {19507, 22946//AFM-Téléthon/ ; EQU20200301021//Fondation pour la Recherche Médicale/ ; 101080690//European MAGIC Project consortium/ ; },
mesh = {Animals ; *Muscular Dystrophy, Duchenne/genetics/pathology ; Exons ; Disease Models, Animal ; Rats ; *Dystrophin/genetics ; Rats, Sprague-Dawley ; *Gene Duplication ; Male ; *Muscle, Striated/pathology ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {BACKGROUND: Duchenne muscular dystrophy (DMD) mainly affects young boys with out-of-frame mutations in the DMD gene, leading to dystrophin deficiency. This loss disrupts the assembly of the sarcolemmal dystrophin-associated glycoprotein complex, resulting in membrane fragility and damage during muscle contraction-relaxation cycles. Consequently, patients experience progressive muscle weakness, loss of ambulation and cardiorespiratory failure. Gene therapy represents one of the most promising therapeutic approaches, requiring rigorous preclinical validation of candidate strategies. While several preclinical models of dystrophin deficiency mimic point mutations or exon deletions, no existing rat model accurately replicates DMD gene duplications, which account for approximately 10% of DMD cases.

METHODS: Using CRISPR/Cas9 genome editing, we generated a ~ 125 kbp duplication encompassing exons 10-17 of the Dmd gene in Sprague Dawley rats. To characterise disease progression in these rats, we assessed biochemical, histological and functional biomarkers at 6 and 10 months of age, comparing them to their healthy littermates.

RESULTS: We established the R-DMDdup10-17 line. The microstructure of limb, diaphragm and cardiac muscles of R-DMDdup10-17 (DMD) rats exhibited dystrophic changes at 6 and 10 months, including loss of myofibres and fibrosis. These alterations led to a significant body mass reduction, muscle weakness (including diaphragm deficiency) and cardiac electrical defects. Premature lethality was observed between 10 and 13 months.

CONCLUSION: Duplication of the Dmd genomic region encompassing exons 10 to 17 in rats results in dystrophin deficiency, severe striated muscle dystrophy, and premature death. The R-DMDdup10-17 line represents the first reported genetic model of a severe and early lethal duplication variant in the Dmd gene. It provides a critical tool for assessing targeted gene therapies aimed to correct such mutations.},
}

Animals
*Muscular Dystrophy, Duchenne/genetics/pathology
Exons
Disease Models, Animal
Rats
*Dystrophin/genetics
Rats, Sprague-Dawley
*Gene Duplication
Male
*Muscle, Striated/pathology
CRISPR-Cas Systems
Gene Editing

@article {pmid40489621,
year = {2025},
author = {Zheng, X and Wu, S and Qiu, J and Li, A and Li, L and Yan, G and Li, M and Meng, F and Zhang, K and Lin, S},
title = {Induction of a neurotoxin in diatoms by iron limitation via cysteine synthase.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {24},
pages = {e2424843122},
doi = {10.1073/pnas.2424843122},
pmid = {40489621},
issn = {1091-6490},
support = {U2106205//MOST | NSFC | National Natural Science Foundation of China-Shandong Joint Fund (-)/ ; tstp20231216//Special Foundation for Taishan Scholar of Shandong Province/ ; },
mesh = {*Iron/metabolism ; *Diatoms/metabolism/genetics ; *Neurotoxins/biosynthesis/metabolism ; *Amino Acids, Diamino/biosynthesis/metabolism ; *Cysteine Synthase/metabolism/genetics ; Humans ; Cyanobacteria Toxins ; CRISPR-Cas Systems ; },
abstract = {The β-N-methylamino-L-alanine (BMAA) is an emerging neurotoxin associated with human neurodegenerative diseases such as Alzheimer's disease. Here, we report the prevalence of BMAA synthesis in protein forms by marine diatoms and reconstruct its tentative biosynthesis pathway. Remarkably, the BMAA production is strongly induced by iron limitation. Transcriptomic analyses suggest that cysteine synthase (CysK) is involved in BMAA synthesis. This is verified as CRISPR/Cas9-based CysK knockout abolished BMAA production and addition of the recombinant CysK to the mutant restored BMAA synthesis. As diatoms are the most abundant primary producers in ocean, the prevalence of BMAA in diatoms has significant public health implications. The biosynthesis pathway provides biomarkers for further investigation of BMAA production in marine diatoms and insights for understanding the pathological mechanism for human neurodegenerative diseases.},
}

*Iron/metabolism
*Diatoms/metabolism/genetics
*Neurotoxins/biosynthesis/metabolism
*Amino Acids, Diamino/biosynthesis/metabolism
*Cysteine Synthase/metabolism/genetics
Humans
Cyanobacteria Toxins
CRISPR-Cas Systems

@article {pmid40489409,
year = {2025},
author = {Yuan, Y and Jiang, Z and Zeng, Y and Tang, J and Luo, J and Xie, C and Gong, Y},
title = {Genome-Wide CRISPR Screen for Unveiling Radiosensitive and Radioresistant Genes.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {219},
pages = {},
doi = {10.3791/67982},
pmid = {40489409},
issn = {1940-087X},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Radiation Tolerance/genetics ; Lung Neoplasms/genetics/radiotherapy ; Gene Editing/methods ; Cell Line, Tumor ; },
abstract = {The CRISPR-Cas9 system has been harnessed and repurposed into a powerful genome editing tool. By leveraging this technology, researchers can precisely cut, paste, and even rewrite DNA sequences within living cells. Nevertheless, the application of CRISPR screen technology goes far beyond mere experimentation. It serves as a pivotal tool in the fight against genetic diseases, systematically dissecting complex genetic landscapes, empowering researchers to unravel the molecular mechanisms underlying biological phenomena, and enabling scientists to identify and target the root causes of illnesses such as cancer, cystic fibrosis, and sickle cell anemia. Among all, cancer poses a formidable challenge for medicine, spurring eradication efforts. Radiotherapy, as a traditional treatment, yields results but has limitations. It eradicates cancer cells but also damages healthy tissues, causing adverse effects that reduce quality of life. Additionally, not all cancer cells respond to radiotherapy, and some may develop resistance, worsening the condition. To address this, a comprehensive whole-genome CRISPR screen technology is introduced, as it enables the efficient identification of radiosensitive and radioresistant genes, thereby advancing the field of cancer research and treatment. A genome-wide CRISPR screen was conducted in lung adenocarcinoma cells exposed to irradiation following the described protocol, through which both radioresistance- and radiosensitivity-associated genes were identified.},
}

Humans
*CRISPR-Cas Systems/genetics
*Radiation Tolerance/genetics
Lung Neoplasms/genetics/radiotherapy
Gene Editing/methods
Cell Line, Tumor

@article {pmid40487915,
year = {2025},
author = {Wu, X and Liu, Y and He, Z and Zhou, X and Liesack, W and Peng, J},
title = {Coevolution and cross-infection patterns between viruses and their host methanogens in paddy soils.},
journal = {ISME communications},
volume = {5},
number = {1},
pages = {ycaf088},
pmid = {40487915},
issn = {2730-6151},
abstract = {Methanogens play a critical role in global methane (CH4) emissions from rice paddy ecosystems. Through the integration of metagenomic analysis and meta-analysis, we constructed a CRISPR spacer database comprising 14 475 spacers derived from 351 methanogenic genomes. This enabled the identification of viruses targeting key methanogenic families prevalent in rice paddies, including Methanosarcinaceae, Methanotrichaceae, Methanobacteriaceae, Methanocellaceae, and Methanomassiliicoccaceae. We identified 419 virus-host linkages involving 56 methanogenic host species and 189 viruses, spanning the families Straboviridae, Salasmaviridae, Kyanoviridae, Herelleviridae, and Demerecviridae, along with 126 unclassified viral entities. These findings highlight a virome composition that is markedly distinct from those observed in gut environments. Cross-infection patterns were supported by the presence of specific viruses predicted to infect multiple closely related methanogenic species. Evidence for potential virus-host coevolution was observed in 24 viruses encoding anti-CRISPR proteins, likely facilitating evasion of host CRISPR-mediated immunity. Collectively, this study reveals a complex and dynamic network of virus-host interactions shaping methanogen communities in rice paddy ecosystems.},
}

@article {pmid40483517,
year = {2025},
author = {Takahashi, G and Maeda, M and Shinozaki, K and Harada, G and Ito, S and Miyaoka, Y},
title = {High-throughput robotic isolation of human iPS cell clones reveals frequent homozygous induction of identical genetic manipulations by CRISPR-Cas9.},
journal = {Stem cell research & therapy},
volume = {16},
number = {1},
pages = {295},
pmid = {40483517},
issn = {1757-6512},
support = {20K21409//Japan Society for the Promotion of Science/ ; 20H03442//Japan Society for the Promotion of Science/ ; 24K02028//Japan Society for the Promotion of Science/ ; 18K15054//Japan Society for the Promotion of Science/ ; 22K15386//Japan Society for the Promotion of Science/ ; 23jm0610092h0001//Japan Agency for Medical Research and Development (AMED)/ ; Takeda Science Foundation//Takeda Science Foundation/ ; Sumitomo Foundation//Sumitomo Foundation/ ; Ichiro Kanehara Foundation//Ichiro Kanehara Foundation/ ; },
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Homozygote ; Clone Cells/cytology ; *Robotics ; },
abstract = {BACKGROUND: Genome editing in human iPS cells is a powerful approach in regenerative medicine. CRISPR-Cas9 is the most common genome editing tool, but it often induces byproduct insertions and deletions in addition to the desired edits. Therefore, genome editing of iPS cells produces diverse genotypes. Existing assays mostly analyze genome editing results in cell populations, but not in single cells. However, systematic profiling of genome editing outcomes in single iPS cells was lacking. Due to the high mortality of human iPS cells as isolated single cells, it has been difficult to analyze genome-edited iPS cell clones in a high-throughput manner.

METHODS: In this study, we developed a method for high-throughput iPS cell clone isolation based on the precise robotic picking of cell clumps derived from single cells grown in extracellular matrices. We first introduced point mutations into human iPS cell pools by CRISPR-Cas9. These genome-edited human iPS cells were dissociated and cultured as single cells in extracellular matrices to form cell clumps, which were then isolated using a cell-handling robot to establish genome-edited human iPS cell clones. Genome editing outcomes in these clones were analyzed by amplicon sequencing to determine the genotypes of individual iPS cell clones. We identified and distinguished the sequences of different insertions and deletions induced by CRISPR-Cas9 while determining their genotypes. We also cryopreserved the established iPS cell clones and recovered them after determining their genotypes.

RESULTS: We analyzed over 1,000 genome-edited iPS cell clones and found that homozygous editing was much more frequent than heterozygous editing. We also observed frequent homozygous induction of identical genetic manipulations, including insertions and deletions, such as 1-bp insertions and 8-bp deletions. Moreover, we successfully cryopreserved and then recovered genome-edited iPS cell clones, demonstrating that our cell-handling robot-based method is valuable in establishing genome-edited iPS cell clones.

CONCLUSIONS: This study revealed a previously unknown property of genome editing in human iPS cells that identical sequence manipulations tend to be induced in both copies of the target sequence in individual cells. Our new cloning method and findings will facilitate the application of genome editing to human iPS cells.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Homozygote
Clone Cells/cytology
*Robotics

@article {pmid40481308,
year = {2025},
author = {Du, W and Zhao, L and Diao, K and Zheng, Y and Yang, Q and Zhu, Z and Zhu, X and Tang, D},
title = {A versatile CRISPR/Cas9 system off-target prediction tool using language model.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {882},
pmid = {40481308},
issn = {2399-3642},
support = {82070199//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; Deep Learning ; Software ; Computational Biology/methods ; },
abstract = {Genome editing with the CRISPR/Cas9 system has revolutionized life and medical sciences, particularly in treating monogenic genetic diseases by enabling long-term therapeutic effects from a single intervention. However, the CRISPR/Cas9 system can tolerate mismatches and DNA/RNA bulges at target sites, leading to unintended off-target effects that pose challenges for gene-editing therapy development. Existing high-throughput detection and in silico prediction methods are often limited to specifically designed single guide RNAs (sgRNAs) and perform poorly on unseen sequences. To address these limitations, we introduce CCLMoff, a deep learning framework for off-target prediction that incorporates a pretrained RNA language model from RNAcentral. CCLMoff captures mutual sequence information between sgRNAs and target sites and is trained on a comprehensive, updated dataset. This approach enables accurate off-target identification and strong generalization across diverse NGS-based detection datasets. Model interpretation reveals the biological importance of the seed region, underscoring CCLMoff's analytical capabilities. The development of CCLMoff lays the foundation for a comprehensive, end-to-end sgRNA design platform, enhancing both the precision and efficiency of CRISPR/Cas9-based therapeutics. CCLMoff is a versatile tool and is publicly available at github.com/duwa2/CCLMoff .},
}

*CRISPR-Cas Systems
*Gene Editing/methods
Humans
RNA, Guide, CRISPR-Cas Systems/genetics
Deep Learning
Software
Computational Biology/methods

@article {pmid40480713,
year = {2025},
author = {Zhou, C and Zhang, Y and Yang, X and Zhao, Z and Xia, M and Wei, C and Wu, Q and Chang, Z and Ma, L and Yin, L},
title = {A magnetic CRISPR/Cas12a-SERS nanobiosensor for amplification-free and ultrasensitive detection of norovirus in water and food samples.},
journal = {Analytica chimica acta},
volume = {1363},
number = {},
pages = {344133},
doi = {10.1016/j.aca.2025.344133},
pmid = {40480713},
issn = {1873-4324},
mesh = {*Norovirus/isolation & purification/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Spectrum Analysis, Raman/methods ; *Food Contamination/analysis ; Limit of Detection ; DNA, Single-Stranded/chemistry ; *Water Microbiology ; *Food Microbiology ; Magnetic Phenomena ; *CRISPR-Associated Proteins/metabolism ; },
abstract = {BACKGROUND: Norovirus (NoV) is the leading cause of foodborne disease outbreaks worldwide, typically spreading via contaminated food and water. Rapid, sensitive, and portable detection of NoV is crucial.

RESULTS: Here, we presented a magnetic CRISPR/Cas12a-SERS nanobiosensor capable of detecting NoV with high sensitivity, accuracy, speed, and portability. In this nanobiosensor, SERS nanoprobes linked to magnetic nanoprobes via linker single-stranded DNAs (ssDNAs). The presence of NoV nucleic acid triggered Cas12a's trans-cleavage activity, degrading the linker ssDNA. After magnetic separation, the dissociated SERS nanoprobes were efficiently separated from the magnetic nanoprobes. This enhanced the SERS signal in the supernatant, detectable using a portable Raman spectrometer. The detection limit for NoV is 100 copies/mL within 60 min. The nanobiosensor was further assessed in real-world settings, demonstrating excellent sensitivity and selectivity for detecting trace NoV in complex food samples.

SIGNIFICANCE: This approach not only broadens CRISPR-based pathogen detection but also provides a reliable tool for monitoring foodborne viruses. Its potential extends beyond NoV, promising enhanced surveillance of various pathogens in food safety, environmental monitoring, and public health sectors.},
}

*Norovirus/isolation & purification/genetics
*Biosensing Techniques/methods
*CRISPR-Cas Systems
Spectrum Analysis, Raman/methods
*Food Contamination/analysis
Limit of Detection
DNA, Single-Stranded/chemistry
*Water Microbiology
*Food Microbiology
Magnetic Phenomena
*CRISPR-Associated Proteins/metabolism

@article {pmid40480203,
year = {2025},
author = {McNamara, HM and Sozen, B},
title = {From genes to geometry: Controlling embryo models by programming genomic activation.},
journal = {Cell stem cell},
volume = {32},
number = {6},
pages = {857-858},
doi = {10.1016/j.stem.2025.04.013},
pmid = {40480203},
issn = {1875-9777},
mesh = {Animals ; Mice ; *Embryo, Mammalian/metabolism/cytology ; Mouse Embryonic Stem Cells/metabolism/cytology ; CRISPR-Cas Systems/genetics ; Embryonic Stem Cells/metabolism/cytology ; *Genome ; *Models, Biological ; },
abstract = {Embryo-like models derived from stem cells have emerged as powerful tools to study early development. In this issue, Lodewijk et al.[1] demonstrate that activating just two enhancers via CRISPR activation (CRISPRa) in mouse embryonic stem cells (ESCs) can drive self-organization into structured embryo-like models, offering a genome-driven approach in stem cell and developmental biology.},
}

Animals
Mice
*Embryo, Mammalian/metabolism/cytology
Mouse Embryonic Stem Cells/metabolism/cytology
CRISPR-Cas Systems/genetics
Embryonic Stem Cells/metabolism/cytology
*Genome
*Models, Biological

@article {pmid40478881,
year = {2025},
author = {Ratajczyk, EJ and Bath, J and Šulc, P and Doye, JPK and Louis, AA and Turberfield, AJ},
title = {Controlling DNA-RNA strand displacement kinetics with base distribution.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {23},
pages = {e2416988122},
pmid = {40478881},
issn = {1091-6490},
support = {EP/W524311/1//UKRI | Engineering and Physical Sciences Research Council (EPSRC)/ ; CCF 2211794//National Science Foundation (NSF)/ ; },
mesh = {Kinetics ; *DNA/chemistry/genetics/metabolism ; *RNA/chemistry/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Thermodynamics ; Gene Editing ; },
abstract = {DNA-RNA hybrid strand displacement underpins the function of many natural and engineered systems. Understanding and controlling factors affecting DNA-RNA strand displacement reactions is necessary to enable control of processes such as CRISPR-Cas9 gene editing. By combining multiscale modeling with strand displacement experiments, we show that the distribution of bases within the displacement domain has a very strong effect on reaction kinetics, a feature unique to DNA-RNA hybrid strand displacement. Merely by redistributing bases within a displacement domain of fixed base composition, we are able to design sequences whose reaction rates span more than four orders of magnitude. We extensively characterize this effect in reactions involving the invasion of dsDNA by an RNA strand, as well as the invasion of a hybrid duplex by a DNA strand. In all-DNA strand displacement reactions, we find a predictable but relatively weak sequence dependence, confirming that DNA-RNA strand displacement permits far more thermodynamic and kinetic control than its all-DNA counterpart. We show that oxNA, a recently introduced coarse-grained model of DNA-RNA hybrids, can reproduce trends in experimentally observed reaction rates. We also develop a simple kinetic model for predicting strand displacement rates. On the basis of these results, we argue that base distribution effects may play an important role in natural R-loop formation and in the function of the guide RNAs that direct CRISPR-Cas systems.},
}

Kinetics
*DNA/chemistry/genetics/metabolism
*RNA/chemistry/genetics/metabolism
CRISPR-Cas Systems/genetics
Thermodynamics
Gene Editing

@article {pmid40478482,
year = {2025},
author = {Syahrani, RA and Wanandi, SI and Nihayah, S and Arumsari, S and Watanabe, Y and Mizuno, S and Louisa, M and Wuyung, PE},
title = {Survivin knockout attenuates the progressiveness of BT549 triple negative-breast cancer cells: an in vitro study highlighting stemness and cellular stress response mechanisms.},
journal = {Molecular biology reports},
volume = {52},
number = {1},
pages = {560},
pmid = {40478482},
issn = {1573-4978},
mesh = {Humans ; *Survivin/genetics/metabolism ; *Triple Negative Breast Neoplasms/genetics/metabolism/pathology ; Cell Line, Tumor ; Cell Proliferation/genetics ; *Neoplastic Stem Cells/metabolism/pathology ; Apoptosis/genetics ; Female ; Gene Knockout Techniques ; Gene Expression Regulation, Neoplastic/genetics ; CRISPR-Cas Systems ; Stress, Physiological/genetics ; Cell Cycle/genetics ; },
abstract = {BACKGROUND: Survivin, an inhibitor of apoptosis proteins (IAPs), is more strongly expressed in triple negative-breast cancer (TNBC) than other subtypes of breast cancer and closely associated with aggressiveness characterized by rapid progression and poor prognosis. The main function of survivin is to regulate cell division and prevent apoptosis. However, other survivin mechanisms are complex and not fully understood. The aim of this study was to evaluate the effects of survivin knockout on TNBC progressiveness relating to proliferation, apoptosis, stemness, cellular stress response, and metastasis mechanisms.

METHODS AND RESULTS: The CRISPR/Cas9 (clustered regularly interspaced short palindrom repeat-associated Cas9) system was utilized to establish survivin knockout in the BT549 TNBC cell line. Both knockout and wild-type cells were used to study the role of survivin in various biological mechanisms. Apoptosis-, pluripotency-, and cellular stress-related proteins were examined via proteomic arrays. The cell cycle, apoptosis, and expression of breast cancer stem cell markers were analyzed via flow cytometry. Metastasis-related markers were evaluated via qRT‒PCR. Here, we report that survivin knockout inhibits proliferation and induces apoptosis in TNBC cells. Moreover, survivin knockout suppressed the expression of pluripotent markers and promoted a shift toward activation response to cellular stress, such as genotoxic, hypoxic, and oxidative stress. Additionally, survivin knockout suppressed metastasis.

CONCLUSION: The loss of survivin leads to attenuation of TNBC progression by altering various mechanisms, particularly by suppressing stemness and altering the cellular stress response. We propose that knocking out survivin could be a potential strategy for breast cancer therapy, especially TNBC.},
}

Humans
*Survivin/genetics/metabolism
*Triple Negative Breast Neoplasms/genetics/metabolism/pathology
Cell Line, Tumor
Cell Proliferation/genetics
*Neoplastic Stem Cells/metabolism/pathology
Apoptosis/genetics
Female
Gene Knockout Techniques
Gene Expression Regulation, Neoplastic/genetics
CRISPR-Cas Systems
Stress, Physiological/genetics
Cell Cycle/genetics

@article {pmid40474742,
year = {2025},
author = {Hung, YH and Wang, E and Agtarap, T and Klaas, G and Slotkin, RK},
title = {Engineering an RNA/protein-binding module for higher transgene protein production and improved long-term durability.},
journal = {The Plant journal : for cell and molecular biology},
volume = {122},
number = {5},
pages = {e70254},
doi = {10.1111/tpj.70254},
pmid = {40474742},
issn = {1365-313X},
support = {DBI-2050394//National Science Foundation/ ; MCB-1904326//National Science Foundation/ ; MCB-2230587//National Science Foundation/ ; },
mesh = {*Arabidopsis/genetics/metabolism ; *Transgenes/genetics ; Plants, Genetically Modified/genetics/metabolism ; *RNA-Binding Proteins/metabolism/genetics ; *Arabidopsis Proteins/genetics/metabolism ; Gene Editing ; RNA Interference ; Gene Expression Regulation, Plant ; RNA, Plant/genetics/metabolism ; CRISPR-Cas Systems ; DNA Methylation ; RNA, Messenger/metabolism/genetics ; },
abstract = {Improvement and research of plants depends on the long-term expression of transgenes. However, the durability of transgene expression is routinely hampered by silencing pathways that start as the post-transcriptional process of mRNA degradation by RNA interference (RNAi). To avoid transgene silencing, we aimed to inhibit the sorting of transgene mRNAs into RNAi. We manipulated a well-studied protein/RNA-binding module from Arabidopsis into a transgene transcript, where the transcript is now bound by an engineered RNA-binding protein that preferentially sorts the RNA into translation. We used the Cas9 transcript as a proof-of-principle and demonstrated higher Cas9 protein production and gene editing rates. In addition, transgenes with the engineered protein/RNA-binding module had improved long-term durability of transgene expression, as after several inbred generations these plants had higher Cas9 protein accumulation and lower levels of DNA methylation, a hallmark of transgene silencing. Our engineered system represents a successful manipulation of post-transcriptional RNA sorting for improved transgene performance, and could be applied to any transgene transcript.},
}

*Arabidopsis/genetics/metabolism
*Transgenes/genetics
Plants, Genetically Modified/genetics/metabolism
*RNA-Binding Proteins/metabolism/genetics
*Arabidopsis Proteins/genetics/metabolism
Gene Editing
RNA Interference
Gene Expression Regulation, Plant
RNA, Plant/genetics/metabolism
CRISPR-Cas Systems
DNA Methylation
RNA, Messenger/metabolism/genetics

@article {pmid40473912,
year = {2025},
author = {Omura, SN and Alfonse, LE and Ornstein, A and Morinaga, H and Hirano, H and Itoh, Y and Munoz, G and Garrity, AJ and Hoffman, GR and DiTommaso, T and Yan, WX and Cheng, DR and Scott, DA and Maben, Z and Nureki, O},
title = {Structural basis for target DNA cleavage and guide RNA processing by CRISPR-Casλ2.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {876},
pmid = {40473912},
issn = {2399-3642},
support = {JP23fa627001//Japan Agency for Medical Research and Development (AMED)/ ; },
mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; Cryoelectron Microscopy ; *DNA Cleavage ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; Bacteriophages/genetics/enzymology ; Gene Editing ; },
abstract = {RNA-guided CRISPR-Cas nucleases are widely used as versatile genome-engineering tools. Among the diverse CRISPR-Cas effectors, CRISPR-Casλ-also referred to as Cas12n-is a recently identified miniature type V nuclease encoded in phage genomes. Given its demonstrated nuclease activity in both mammalian and plant cells, Casλ has emerged as a promising candidate for genome-editing applications. However, the precise molecular mechanisms of Casλ family enzymes remain poorly understood. In this study, we report the identification and detailed biochemical and structural characterizations of CRISPR-Casλ2. The cryo-electron microscopy structures of Casλ2 in five different functional states unveiled the dynamic domain rearrangements during its activation. Our biochemical analyses indicated that Casλ2 processes its precursor crRNA to a mature crRNA using the RuvC active site through a unique ruler mechanism, in which Casλ2 defines the spacer length of the mature crRNA. Furthermore, structural comparisons of Casλ2 with Casλ1 and CasΦ highlighted the diversity and conservation of phage-encoded type V CRISPR-Cas enzymes. Collectively, our findings augment the mechanistic understanding of diverse CRISPR-Cas nucleases and establish a framework for rational engineering of the CRISPR-Casλ-based genome-editing platform.},
}

*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/metabolism/genetics
Cryoelectron Microscopy
*DNA Cleavage
*CRISPR-Associated Proteins/metabolism/genetics/chemistry
Bacteriophages/genetics/enzymology
Gene Editing

@article {pmid40473600,
year = {2025},
author = {Zhou, J and Hepperla, A and Simon, JM and Kim, K and Hu, Q and Zhang, C and Dong, L and Hu, L and Zhang, C and Liao, C and Fang, A and Adachi, Y and Fu, H and Wang, T and Liang, Q and Zhao, F and Liu, H and Takeda, M and Fang, J and Zhong, H and Ly, P and Wang, L and Kapur, P and Xu, L and Jia, L and Malladi, S and Brugarolas, J and Simon, MC and Li, B and Zhang, Q},
title = {SWI/SNF ATPase silenced HLF potentiates lung metastasis in solid cancers.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5226},
pmid = {40473600},
issn = {2041-1723},
support = {R35 CA220483/CA/NCI NIH HHS/United States ; R01 CA294133/CA/NCI NIH HHS/United States ; RR190058//Cancer Prevention and Research Institute of Texas (Cancer Prevention Research Institute of Texas)/ ; P30 CA142543/CA/NCI NIH HHS/United States ; R01CA211732//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; R35 GM146979/GM/NIGMS NIH HHS/United States ; R01 CA211732/CA/NCI NIH HHS/United States ; P50 CA196516/CA/NCI NIH HHS/United States ; P50CA196516//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Humans ; *Lung Neoplasms/secondary/genetics/metabolism ; *Transcription Factors/genetics/metabolism ; Animals ; *DNA Helicases/metabolism/genetics ; Cell Line, Tumor ; *Nuclear Proteins/metabolism/genetics ; Mice ; Cell Movement/genetics ; Gene Expression Regulation, Neoplastic ; *Carcinoma, Renal Cell/genetics/pathology/metabolism ; *Kidney Neoplasms/pathology/genetics/metabolism ; Female ; Gene Silencing ; CRISPR-Cas Systems ; },
abstract = {Metastasis is the main cause of cancer-related deaths, yet the underlying mechanisms remain elusive. Here, using clear cell renal cell carcinoma (ccRCC), a tumor type with frequent lung metastases, we conduct an in vivo genome-wide CRISPR-Cas9 screen and identify HLF as a potent suppressor of lung metastasis. HLF depletion enhances ccRCC cell migration and lung metastasis, whereas HLF overexpression abrogates these effects. In ccRCC patients, HLF expression is reduced at metastatic sites and associates with epigenetic silencing mediated by the SWI/SNF ATPase subunit BRG1. HLF levels negatively correlate with migration potential in collagen. Mechanistically, HLF regulates LPXN expression, modulating the integration of collagen's mechanical cues with the actin cytoskeleton through Paxillin, thereby suppressing cancer cell migration and lung metastasis. Overexpression of HLF or pharmacological inhibition of BRG1 reduces cell invasion across multiple cancer types. Our findings suggest that targeting the BRG1-HLF axis offers a promising therapeutic strategy for combating metastatic cancers.},
}

Humans
*Lung Neoplasms/secondary/genetics/metabolism
*Transcription Factors/genetics/metabolism
Animals
*DNA Helicases/metabolism/genetics
Cell Line, Tumor
*Nuclear Proteins/metabolism/genetics
Mice
Cell Movement/genetics
Gene Expression Regulation, Neoplastic
*Carcinoma, Renal Cell/genetics/pathology/metabolism
*Kidney Neoplasms/pathology/genetics/metabolism
Female
Gene Silencing
CRISPR-Cas Systems

@article {pmid40472130,
year = {2025},
author = {Ghdayer Al Kaabi, NA and Kandhan, K and Hayat, F and Matar Al Blooshi, SA and Sheteiwy, MS and Alyafei, M},
title = {Shaping the future of date palm (Phoenix dactylifera) through new genetic improvement strategies.},
journal = {Functional plant biology : FPB},
volume = {52},
number = {},
pages = {},
doi = {10.1071/FP25021},
pmid = {40472130},
issn = {1445-4416},
mesh = {*Phoeniceae/genetics/metabolism ; *Plant Breeding/methods ; Gene Editing ; CRISPR-Cas Systems ; Genome-Wide Association Study ; Polymorphism, Single Nucleotide ; },
abstract = {Conventional breeding of date palm (Phoenix dactylifera) is inherently challenging due to its long generation time, dioecious nature, and high genetic heterogeneity. However, current developments in genomics and molecular biology offer promising avenues for accelerating breeding programs, particularly through high-throughput technologies including functional genomics. This article reviews genomic tools such as like CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated protein 9) that may bring significant changes in date palm breeding. The CRISPR-Cas9 system enables scientists to accurately target genomic regions, which helps enhance breeding accuracy by adding advantageous traits and eliminating unfavorable genes through precision editing. Transcriptome and metabolome analyses have also explained the regulation of thousands of differentially expressed genes (DEGs) and metabolic pathways under environmental stress. These studies contribute to enhance the knowledge of stress tolerance mechanisms, which include the secondary metabolic process of flavonoids. Genomic studies illustrating single nucleotide polymorphism (SNP)-based diversity between cultivars from north African and the Arabian Gulf provide new genetic resources for selective breeding. The work relates genome-wide association studies (GWAS) and miRNA profiling to elucidate key regulatory networks involved in fruit development and stress resilience. The integration of such advanced technologies, especially the CRISPR-Cas9 system, is revolutionizing the landscape of date palm breeding, opening new avenues for accelerated development of superior cultivars that meet the needs of modern agriculture.},
}

*Phoeniceae/genetics/metabolism
*Plant Breeding/methods
Gene Editing
CRISPR-Cas Systems
Genome-Wide Association Study
Polymorphism, Single Nucleotide

@article {pmid40472129,
year = {2025},
author = {Sarfraz, Z and Zarlashat, Y and Ambreen, A and Mujahid, M and Iqbal, MS},
title = {Advanced gene editing techniques for enhancing disease resistance and climate resilience in crops.},
journal = {Functional plant biology : FPB},
volume = {52},
number = {},
pages = {},
doi = {10.1071/FP24357},
pmid = {40472129},
issn = {1445-4416},
mesh = {*Gene Editing/methods ; *Crops, Agricultural/genetics ; *Disease Resistance/genetics ; Climate Change ; CRISPR-Cas Systems ; Plants, Genetically Modified ; },
abstract = {Ensuring food security and solving the issues brought on by climate change require breeding and engineering of climate-resilient crops. Despite its contributions to reducing agricultural diseases, genetic engineering has several limitations, including high labor costs, lengthy processing times, and poor productivity. Genome editing has become a potential method to provide notable opportunities to explain complex biological processes, genetically solve the causes of diseases, and improve crops for disease resistance by effectively modifying multiple traits. Genome editing techniques including TALENs, ZFNs, and CRISPR/Cas9 increase agricultural productivity by developing climate-resistant crops and promoting climate-resilient agriculture. Among these approaches, CRISPR/Cas9 shows exceptional efficacy, minimal chance of off-target effects, and improved traits such as drought tolerance and disease resistance. This study explores advanced gene editing techniques for improving disease resistance in crops and developing climate-resilient varieties to reduce food insecurity and hunger. It demonstrates that these techniques have enhanced the nutritional content and resilience of many crops by fighting abiotic and biotic stresses. Future agricultural practices could alter the genes and improve disease-resistant crops by genome editing techniques.},
}

*Gene Editing/methods
*Crops, Agricultural/genetics
*Disease Resistance/genetics
Climate Change
CRISPR-Cas Systems
Plants, Genetically Modified

@article {pmid40471011,
year = {2025},
author = {Li, H and Shen, J and Zhao, X and Ji, J and Wang, Y and Yang, L and Zhuang, M and Liu, L and Zhang, Y and Lv, H},
title = {Rapid design of transgene-free cabbage with desired anthocyanin contents via HI-Edit.},
journal = {Journal of integrative plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1111/jipb.13943},
pmid = {40471011},
issn = {1744-7909},
support = {CAAS-ASTIP-IVFCAAS//the Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences/ ; CARS-23//the China Agriculture Research System of MOF and MARA/ ; 2023YFD1201501//The National Key R&D Program of China/ ; BE2023366//The Key Technology R&D Program of Jiangsu Province/ ; },
abstract = {The HI-Edit system combines haploid induction and CRISPR/Cas-based genome editing to provide a promising way to design crops with desired traits in a rapid, precise and transgene-free manner. HI-Edit was applied to produce cabbages with desired anthocyanin contents.},
}

@article {pmid40470110,
year = {2025},
author = {Bao, H and Chen, Y and Zong, Y and Jin, K and Lan, H},
title = {Recent Advances in the Delivery of Bone Morphogenetic Proteins for Targeting Glioma: An Updated Review.},
journal = {International journal of nanomedicine},
volume = {20},
number = {},
pages = {7093-7112},
pmid = {40470110},
issn = {1178-2013},
mesh = {Humans ; *Glioma/drug therapy/therapy ; Animals ; *Brain Neoplasms/drug therapy ; *Bone Morphogenetic Proteins/administration & dosage/therapeutic use/pharmacokinetics ; Genetic Therapy/methods ; *Drug Delivery Systems/methods ; Nanoparticles/chemistry ; Blood-Brain Barrier/metabolism ; Hydrogels/chemistry ; },
abstract = {Bone Morphogenetic Proteins might be the most prospective in glioma treatment because of the facts that they can differentiate glioma cells, inhibit tumor growth and manage glioma stem cells. Its clinical application is hindered by several challenges, including limited permeability across the blood-brain barrier, which impedes effective delivery to the central nervous system; high susceptibility to enzymatic degradation, which compromises stability and therapeutic efficacy; and nonselective binding, which reduces specificity and may result in unintended off-target effects. This review systematically covers the advanced BMP delivery systems such as nanoparticles, smart carriers, gene therapy, and exosome-based system. Hydrogels, scaffolds, and microspheres' local delivery methods are also discussed as prospective options. The in vitro studies reveal that BMPs are effective and using in vivo glioma models there is also evidence of the effectiveness of BMPs. In addition, new clinical trials reveal concern with safety, tolerability, and therapeutic effects of BMPs, especially their combination with chemotherapy and immunotherapy. BMP specificity and therapeutic performance are further optimized by Personalized medicine and CRISPR/Cas engineering. However, regulatory barriers and product commercialization are challenging issues. This review highlights the need for novel approaches and advanced technologies to address the challenges associated with BMP delivery, aiming to establish BMP-based therapies as an effective treatment strategy for glioma.},
}

Humans
*Glioma/drug therapy/therapy
Animals
*Brain Neoplasms/drug therapy
*Bone Morphogenetic Proteins/administration & dosage/therapeutic use/pharmacokinetics
Genetic Therapy/methods
*Drug Delivery Systems/methods
Nanoparticles/chemistry
Blood-Brain Barrier/metabolism
Hydrogels/chemistry

@article {pmid40469526,
year = {2025},
author = {Isaacson, J and Bhanap, P and Putnam, N and Padilla, J and Fatima, N and Dotson, M and Hayoun, D and Ahmadi, M and Nonterah, G and Ji, Y},
title = {Enhancing CAR T-cell therapy manufacturing efficiency through semi-automated bioprocessing.},
journal = {Clinical & translational immunology},
volume = {14},
number = {6},
pages = {e70025},
pmid = {40469526},
issn = {2050-0068},
abstract = {OBJECTIVES: Chimeric antigen receptor (CAR) T-cell therapies have revolutionised the treatment of blood-based malignancies. The use of manual CAR T-cell manufacturing methods is one of the challenges that contributes to these delays. As CAR T therapy emerges as a potential first- or second-line treatment option for these cancers, the demand for these therapies continues to rise. However, challenges persist in ensuring that the patients who need these therapies receive them in a timely manner. Automated CAR T-cell manufacturing methods that use software to control the equipment used in the process can help overcome the roadblocks associated with manual manufacturing, ultimately enabling a reduction in variability, increased efficiency, improved product quality and better data management. This paper aims to present an end-to-end semi-automated methodology for manufacturing CAR T cells using the Cell Therapy Systems (CTS™) Cellmation software - an off-the-shelf software solution - to control physically connected modular cell therapy instruments that eliminates the roadblocks associated with manual manufacturing.

METHODS: T cells from healthy donors were isolated and processed into CAR T cells using a semi-automated, connected, multi-instrument setup that leveraged electroporation and a CRISPR/Cas system for delivering the CD19-CAR construct to the T cells. Flow cytometry was used to assess cell type composition, cell viability and expression of T-cell activation markers throughout the process. We also measured exhaustion marker expression on T cells, T-cell receptor (TCR) knock-out, CAR knock-in and cytotoxic activity against NALM6 cells.

RESULTS: The results demonstrated the successful generation of functional CAR T cells using a semi-automated instrument workflow. The results were similar to the results from CAR T cells manufactured using non-automated processes; however, the successful connection and control of the instruments using automated software present an exciting opportunity for process developers and manufacturers who want to reduce manual touchpoints in their cell therapy manufacturing process.

CONCLUSION: The method that we describe in this paper could be beneficial to process development and manufacturing teams that might require flexibility in their CAR T cell manufacturing workflow and want to take advantage of modular systems that can be automated using the Cellmation software to reduce the problems associated with manual handling.},
}

@article {pmid40469284,
year = {2025},
author = {Lin, Z and Song, Z and Yu, H and Zhou, Y and Liu, D and Zhang, P and Wei, L and Dai, G and Liang, G and He, Z and Hu, X and Chen, Y and Zhao, P and Lu, H and Zheng, M},
title = {Ultra-sensitive in situ detection of intracellular Mycobacterium tuberculosis with CRISPR/Cas12a.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1597654},
pmid = {40469284},
issn = {1664-3224},
mesh = {*Mycobacterium tuberculosis/genetics/isolation & purification ; Humans ; Animals ; *CRISPR-Cas Systems ; Mice ; *Macrophages/microbiology ; RAW 264.7 Cells ; *Tuberculosis/diagnosis/microbiology ; Bronchoalveolar Lavage Fluid/microbiology ; Sensitivity and Specificity ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Mycobacterium tuberculosis (Mtb) invades and survives inside macrophages, evading detection and resisting antibiotic treatment, which results in severe clinical consequences such as fatal respiratory failure and systemic inflammation. Rapid and specific detection of intracellular Mtb is crucial for accurate diagnosis and optimizing treatment strategies. In this study, we developed a one-step CRISPR/Cas12a assay targeting the IS6110 gene for the specific and rapid detection of intracellular Mtb. Upon efficient delivery into RAW264.7 macrophages, the assay enabled direct visualization of Mtb IS6110 nucleic acid, generating detectable fluorescence signals. The diagnostic performance was further validated using bronchoalveolar lavage fluid (BALF) samples from clinical participants, achieving a sensitivity of 94%, which surpassed conventional methods such as culture (67%) and Xpert (78%), while maintaining a specificity of 100%. This CRISPR/Cas12a-based assay offers a highly sensitive, rapid, and innovative approach for intracellular Mtb detection, with significant potential to enhance tuberculosis diagnostic methodologies and improve clinical outcomes.},
}

*Mycobacterium tuberculosis/genetics/isolation & purification
Humans
Animals
*CRISPR-Cas Systems
Mice
*Macrophages/microbiology
RAW 264.7 Cells
*Tuberculosis/diagnosis/microbiology
Bronchoalveolar Lavage Fluid/microbiology
Sensitivity and Specificity
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins