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

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

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

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

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

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

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

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

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

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

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

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

@article {pmid40318489,
year = {2025},
author = {Sun, J and Bai, J and Huang, Y and Langford, PR and Zhang, Y and Li, G},
title = {A CRISPR/Cas12a-based DNAzyme visualization platform for rapid discrimination of Streptococcus suis serotype 2 versus 1/2 and serotype 1 versus 14.},
journal = {Talanta},
volume = {294},
number = {},
pages = {128241},
doi = {10.1016/j.talanta.2025.128241},
pmid = {40318489},
issn = {1873-3573},
mesh = {*Streptococcus suis/genetics/classification/isolation & purification ; *CRISPR-Cas Systems/genetics ; Serogroup ; *DNA, Catalytic/metabolism/chemistry/genetics ; Polymorphism, Single Nucleotide ; Nucleic Acid Amplification Techniques ; Animals ; Swine ; Colorimetry ; G-Quadruplexes ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Streptococcus suis is a major swine pathogen with serotypes 2 and 14 posing zoonotic risks. However, distinguishing serotypes 1/2 from 2 or 1 from 14 remains challenging due to high similarity in their capsule polysaccharide (CPS) loci. Here, we developed a rapid, equipment-free discriminating platform targeting a single nucleotide polymorphism (SNP) at position 483 of the cpsK gene (G in serotypes 2/14 vs. T/C in 1/2/1). The method integrates recombinase polymerase amplification (RPA) with CRISPR/Cas12a and a G-quadruplex-hemin DNAzyme visualization system. RPA enables isothermal amplification, while CRISPR/Cas12a ensures single-nucleotide specificity by cleaving target DNA. Subsequent DNAzyme catalysis converts colorimetric substrates, enabling naked-eye differentiation via distinct color changes (blue for serotypes 1/2/1 vs. colorless for 2/14). This approach achieved a sensitivity of 10[1]-10[2] copies per reaction and demonstrated 100 % specificity across 29 S. suis serotypes and related strains. Compared to PCR-based or sequencing methods, our platform eliminates reliance on thermocyclers or fluorescence detectors, reducing costs and operational complexity. The entire workflow, completed within 70 min, offers a practical solution for point-of-care testing in resource-limited settings. By enabling rapid, accurate discrimination, this tool will become a complementary tool for resolving ambiguous serotypes and enhances outbreak management in swine populations and mitigates zoonotic transmission.},
}

*Streptococcus suis/genetics/classification/isolation & purification
*CRISPR-Cas Systems/genetics
Serogroup
*DNA, Catalytic/metabolism/chemistry/genetics
Polymorphism, Single Nucleotide
Nucleic Acid Amplification Techniques
Animals
Swine
Colorimetry
G-Quadruplexes
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40300474,
year = {2025},
author = {Zhu, T and Jiang, W and Wu, Y and Fang, R and Deng, F and Yang, D},
title = {Advances in CRISPR/Cas13a-based biosensors for non-coding RNA detection.},
journal = {Talanta},
volume = {294},
number = {},
pages = {128223},
doi = {10.1016/j.talanta.2025.128223},
pmid = {40300474},
issn = {1873-3573},
mesh = {*Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *RNA, Untranslated/analysis/genetics ; },
abstract = {Non-coding RNAs play crucial roles in disease initiation and progression, making them promising biomarkers for early diagnosis and treatment monitoring. Conventional nucleic acid diagnostic methods, including polymerase chain reaction (PCR), next-generation sequencing (NGS), and enzyme-linked immunosorbent assay (ELISA), alongside emerging techniques such as single-molecule fluorescence in situ hybridization (smFISH), nanopore sequencing, and single-cell RNA sequencing (scRNA-seq), face inherent limitations in detecting regulatory non-coding RNAs. These challenges include laborious workflows, prolonged processing times, and technical complexities, hindering their broad applicability in rapid and high-throughput RNA analysis. CRISPR/Cas13a-based biosensors, integrated with various signal transduction systems-such as fluorescence, electrochemistry, colorimetry, surface-enhanced Raman spectroscopy (SERS)-show great promise for real-world diagnostic applications. This review provides a comprehensive overview of the CRISPR/Cas13a-mediated RNA detection mechanism, the development of CRISPR/Cas13a-based biosensors, and their integration with innovative signal detection methods. Additionally, we highlight the progress in portable detection devices, including lateral flow assay strips and smartphone-based platforms. Finally, the review discusses the current challenges and future prospects of CRISPR/Cas13a-based biosensors, particularly in the context of clinical diagnostics and personalized medicine.},
}

*Biosensing Techniques/methods
Humans
*CRISPR-Cas Systems/genetics
*RNA, Untranslated/analysis/genetics

@article {pmid40262340,
year = {2025},
author = {Li, X and Li, Y and Wang, C and Chai, J and Liu, H and Jiang, X and Li, Y},
title = {Structure-switchable dsDNA promoter regulates the activity of CRISPR-Cas12a for APE1 detection.},
journal = {Talanta},
volume = {294},
number = {},
pages = {128161},
doi = {10.1016/j.talanta.2025.128161},
pmid = {40262340},
issn = {1873-3573},
mesh = {*DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics/analysis/metabolism/antagonists & inhibitors ; Humans ; *CRISPR-Cas Systems/genetics ; *Promoter Regions, Genetic ; *DNA/genetics/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Apurinic/apyrimidinic endonuclease 1 (APE1) has been considered as a promising biomarker since it is associated with numerous human diseases, involving neurodegenerative diseases and cancer. However, current APE1 detection methods mainly rely on immunology-based methods, which are burdened by time-consuming and procedural complexity. To overcome these shortcomings, we have developed an innovative all-in-one technique that simplifies APE1 detection by integrating enzyme-responsive elements structure-switchable dsDNA promoter with CRISPR/AsCas12a methodology, namely EDC. In this work, the structure-switchable dsDNA promoter has been well-designed to trigger the site-directed incision of APE1 and then release the split activator to illumine the CRISPR/AsCas12a catalyst system by coupling it with another truncated activator. Under optimal circumstances, the proposed strategy enables sensitive detection of the target APE1 with a detection limit of 4.8 × 10[-5] U/mL and a wide linear range from 5.0 × 10[-5] to 1.0 × 10[-1] U/mL. Moreover, this strategy could be gratifyingly applied to screen APE1 inhibitors and monitor APE1 in lysates from cell extractions or clinical serum samples. Overall, this study presents a novel approach that utilizes dsDNA promoter as programmable switching components, effectively enhancing CRISPR/Cas12a-based diagnostic platforms and demonstrating the significant potential for clinical translation.},
}

*DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics/analysis/metabolism/antagonists & inhibitors
Humans
*CRISPR-Cas Systems/genetics
*Promoter Regions, Genetic
*DNA/genetics/chemistry
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40167233,
year = {2025},
author = {Kim, EJ and Hong, WJ and Kim, YJ and Kim, EY and Yun, SD and Moon, S and Lee, SK and Park, SK and Jung, KH},
title = {Global identification of key genes for pollen germination in rice through high-throughput screening and gene editing.},
journal = {Journal of integrative plant biology},
volume = {67},
number = {6},
pages = {1665-1684},
doi = {10.1111/jipb.13900},
pmid = {40167233},
issn = {1744-7909},
support = {RS-2024-00322278//Rural Development Administration/ ; RS-2021-NR060084//National Research Foundation of Korea/ ; RS-2021-NR062086//National Research Foundation of Korea/ ; NRF-2021R1A2C2010124//National Research Foundation of Korea/ ; RS-2023-00217064//National Research Foundation of Korea/ ; RS-2024-00344197//National Research Foundation of Korea/ ; },
mesh = {*Oryza/genetics ; *Pollen/genetics/growth & development ; *Germination/genetics ; *Gene Editing/methods ; *Genes, Plant/genetics ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems/genetics ; *High-Throughput Screening Assays ; Plant Infertility/genetics ; Mutation/genetics ; DNA, Bacterial ; },
abstract = {Successful reproduction depends on the stable germination and growth of the pollen tubes (PT). However, the molecular mechanisms involved in rice PT growth and development remain largely unknown. In a previous study, microarray transcriptome analysis identified 627 genes preferentially expressed in the tricellular and germinating pollen of rice (i.e., Oryza sativa ssp. japonica). To elucidate key genes involved in the gene transfer process facilitated by male gametophytes, we systematically screened T-DNA lines containing disrupted sequences that corresponded to these 627 genes and analyzed the genotypes of heterozygote progeny from 107 T-DNA-indexed lines covering 105 genes. We found that 42 lines exhibited a distorted segregation ratio among the wild-type (WT), heterozygote (HT), and homozygote (HM) genotypes, which deviated from the expected Mendelian ratio of 1:2:1 (WT:HT:HM). Further characterization using CRISPR/Cas9 mutants revealed that knockout mutants of certain genes that exhibited segregation distortion in the T-DNA insertion region were completely sterile. Moreover, even when T-DNA insertion lines followed Mendelian segregation patterns, sterility could be induced by simultaneously mutating functionally redundant genes, thereby overcoming genetic compensation. Interestingly, although some T-DNA insertion lines exhibited segregation ratios approximating 1:1:0, the corresponding CRISPR/Cas9 mutants produced homozygous seeds and showed partial sterility. Partial sterility suggests that despite mutant pollen grains being less competitive than WT pollen, they retain their fertilization potential under relaxed competition from WT pollen. Beyond mutant-based analysis, transcriptomic profiling of sterile mutant lines provided additional insight into the regulatory relationship between key germination regulators and the 105 target genes studied here. Overall, this study demonstrates the effectiveness of a multi-pronged strategy to accelerate the identification of defective phenotypes using mutant studies and provides valuable genetic resources for inducing novel male sterility in rice.},
}

*Oryza/genetics
*Pollen/genetics/growth & development
*Germination/genetics
*Gene Editing/methods
*Genes, Plant/genetics
Gene Expression Regulation, Plant
CRISPR-Cas Systems/genetics
*High-Throughput Screening Assays
Plant Infertility/genetics
Mutation/genetics
DNA, Bacterial

@article {pmid40052456,
year = {2025},
author = {Yin, T and Tai, Y and Sun, Y and Cheng, Z and Wu, C and Sui, Y},
title = {Precise tiller angle control by manipulating TAC1 expression in rice.},
journal = {Journal of integrative plant biology},
volume = {67},
number = {6},
pages = {1444-1446},
doi = {10.1111/jipb.13877},
pmid = {40052456},
issn = {1744-7909},
support = {2024ZD04077//Biological Breeding-National Science and Technology Major Project/ ; 31801323//National Natural Science Foundation of China/ ; //Innovation Program of the Chinese Academy of Agricultural Sciences/ ; },
mesh = {*Oryza/genetics/metabolism/anatomy & histology ; *Plant Proteins/metabolism/genetics ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems/genetics ; Gene Editing ; Phenotype ; },
abstract = {Tiller angle shapes plant architecture, and is one of the top traits in plant breeding. A compact plant type reduces shading between plants, especially at high planting density, but also creates a humid microenvironment often associated with a higher incidence of pathogen and pest attacks, especially under highly humid climates. However, how to precisely manipulate the tiller angle to achieve a desirable plant type has been under-approached. Here we report the creation of gradient tiller angles in indica rice by fine tuning the expression of TILLER ANGLE CONTROL1 (TAC1), a domesticated gene in cultivated rice. We edited the regions upstream and downstream of the TAC1 coding sequence using multiplex CRISPR-Cas9 technology and developed homozygous allelic lines carrying deletions/inversions of various sizes at different positions. Those lines displayed smooth gradient changes in tiller angle that aligned well with TAC1 expression levels. Additionally, changes in the TAC1 expression level had no impact on other agronomic traits examined. TAC1 is well conserved across species, including perennial fruit trees in which mutation of TAC1 orthologs leads to a broomy plant type. Thus, our results provide a guide to creating tiller angles for selection according to climate zones in rice breeding programs, this approach can be extended to diverse species for improving plant architecture.},
}

*Oryza/genetics/metabolism/anatomy & histology
*Plant Proteins/metabolism/genetics
Gene Expression Regulation, Plant
CRISPR-Cas Systems/genetics
Gene Editing
Phenotype

@article {pmid39950533,
year = {2025},
author = {Xue, L and Qu, P and Zhao, H and Liu, H and Huang, B and Wang, X and Zhang, Z and Dai, X and Qin, L and Dong, W and Shi, L and Zhang, X},
title = {Creation of fragrant peanut using CRISPR/Cas9.},
journal = {Journal of integrative plant biology},
volume = {67},
number = {6},
pages = {1438-1440},
doi = {10.1111/jipb.13864},
pmid = {39950533},
issn = {1744-7909},
support = {2022YFD1200400//National Key Research and Development Program of China/ ; 201300111000//Key Project of Science and Technology of Henan Province/ ; 221100110300//Key Project of Science and Technology of Henan Province/ ; CARS-13//the Earmarked Fund/ ; S2012-5//Fund of Henan Province Agriculture Research System/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Arachis/genetics/metabolism ; Mutation/genetics ; Betaine-Aldehyde Dehydrogenase/genetics/metabolism ; },
abstract = {Targeted knockout of the betaine aldehyde dehydrogenase genes AhBADH1 and AhBADH2 using CRISPR/Cas9 produced peanut mutant lines with significantly elevated 2-acetyl-1-pyrroline levels and a strong aroma, marking the first creation of fragrant peanut lines.},
}

*CRISPR-Cas Systems/genetics
*Arachis/genetics/metabolism
Mutation/genetics
Betaine-Aldehyde Dehydrogenase/genetics/metabolism

@article {pmid40436684,
year = {2025},
author = {Jin, YY and Zhang, P and Liu, DP},
title = {Optimizing homology-directed repair for gene editing: the potential of single-stranded DNA donors.},
journal = {Trends in genetics : TIG},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tig.2025.04.014},
pmid = {40436684},
issn = {0168-9525},
abstract = {CRISPR (clustered regularly interspaced short palindromic repeat) system-based precise genome editing remarkably impacts both scientific investigation and therapeutic practices. Among various techniques, DNA donor-mediated homology-directed repair (HDR) represents a promising method for precise gene editing. Although efficiency constraints have previously limited HDR, recent advancements have significantly enhanced its effectiveness. Therefore, it is essential to highlight the progress made in this field and to reassess the potential of the HDR approach. In this review, we explore the fundamental principles of HDR-dependent gene editing and evaluate current strategies to enhance HDR efficiency, with particular emphasis on single-stranded DNA (ssDNA) donor-mediated HDR. Finally, we discuss the prospects of high-efficiency ssDNA donor-mediated precise gene editing in laboratory research and clinical therapies.},
}

@article {pmid40435438,
year = {2025},
author = {Shu, F and Huang, Y and Yang, F and Guo, Y and Xu, R and Xiao, L and Feng, Y and Li, N},
title = {Calcium-dependent protein kinases 2A involved in the growth of both asexual and sexual stages of Cryptosporidium parvum.},
journal = {PLoS neglected tropical diseases},
volume = {19},
number = {5},
pages = {e0013107},
pmid = {40435438},
issn = {1935-2735},
mesh = {*Cryptosporidium parvum/growth & development/enzymology/genetics ; Animals ; Cryptosporidiosis/parasitology ; Mice ; *Protein Kinases/genetics/metabolism ; CRISPR-Cas Systems ; *Protozoan Proteins/genetics/metabolism ; Oocysts/growth & development ; Female ; Humans ; Life Cycle Stages ; Sporozoites/growth & development ; },
abstract = {BACKGROUND: Cryptosporidium parvum is a protozoan pathogen that causes moderate to severe diarrhea in both humans and animals. Calcium-dependent protein kinases (CDPKs) are attractive drug targets against cryptosporidiosis given their critical role in the life cycle of Cryptosporidium spp. and their absence in human and animal hosts.

We used CRISPR-Cas9 technology to endogenously tag the CpCDPK2A gene in C. parvum IIdA20G1-HLJ strain with the hemagglutinin (HA) epitope and to delete the CpCDPK2A gene. An immunofluorescence assay was performed to localize the CpCDPK2A expression in the tagged strain and a luciferase assay was performed to compare growth rates of the tagged and deletion strains in vitro. Oocyst shedding, parasite load, villus length/crypt height ratio and survival of infected mice were used to evaluate the function of CpCDPK2A in vivo. The results revealed that CpCDPK2A was expressed in all the intracellular developmental stages, especially in the motile stages of sporozoites and merozoites. While CpCDPK2A is dispensable, deletion of the gene significantly reduced the growth of late asexual and sexual stages in vitro. In an interferon-γ knockout mouse model, gene deletion of CpCDPK2A reduced oocyst shedding by 25-fold and increased survival of infected mice.

CONCLUSIONS/SIGNIFICANCE: These observations suggest that CpCDPK2A may contribute to both asexual and sexual replication of C. parvum and may be a potential target to block the transmission of this important zoonotic pathogen.},
}

*Cryptosporidium parvum/growth & development/enzymology/genetics
Animals
Cryptosporidiosis/parasitology
Mice
*Protein Kinases/genetics/metabolism
CRISPR-Cas Systems
*Protozoan Proteins/genetics/metabolism
Oocysts/growth & development
Female
Humans
Life Cycle Stages
Sporozoites/growth & development

@article {pmid40432879,
year = {2025},
author = {Melchior, F and Angelidou, IA and Chorianopoulou, M and Teichmann, B},
title = {The genetic technologies questionnaire in the Greek-speaking population: the moral judgement of the lay public.},
journal = {Frontiers in genetics},
volume = {16},
number = {},
pages = {1594724},
pmid = {40432879},
issn = {1664-8021},
abstract = {INTRODUCTION: Advancements in life sciences have significantly boosted biomedical capabilities. Genetic testing forecasts hereditary traits and disease susceptibility, while CRISPR/Cas allows permanent genome alterations. However, ethical considerations arise regarding the morality of these capabilities, particularly concerning the moral status, autonomy, and privacy of living beings. The lack of valid instruments to assess moral judgment in genetic technologies highlights the need for this study, aiming to translate and validate the "Genetic Technologies Questionnaire" (GTQ) and the short version of the "Conventional Technologies Questionnaire" (CTQ5) into Greek. As the full version of the GTQ with 30 questions could be too extensive for some studies, we also tested other versions: The short versions GTQ20-GR and GTQ5-GR which were already presented in the original study, as well as a version which included questions solely about humans (GTQ-H-GR) and is intended for use in human research and therapy, and the GTQ-Moral Status (GTQ-MS-GR), which included questions about genetic testing and gene editing in different living beings to investigate differences in moral status.

METHODS: A cross-sectional study involved 250 participants who completed an online questionnaire, assessing internal consistency, structural validity, known-groups validity, floor/ceiling effects, and retest reliability (subset of 50 participants). Correlational analyses explored relationships with education, age, genetic knowledge, religiosity, and genetic testing experience. The study followed the STROBE checklist for reporting.

RESULTS: The GTQ-GR (Cronbach's α = 0.929) and GTQ20-GR (α = 0.935) exhibit high reliability and stability in assessing moral judgment among lay people, whereas the GTQ5-GR (α = 0.866) and CTQ5-GR (α = 0.758) displayed some weaknesses. Participants tended to rate conventional technologies more favorably than genetic technologies, with genetic testing perceived more positively than genome editing. The two additional derived versions, GTQ-H-GR (α = 0.859) and GTQ-MS-GR (α = 0.787), also demonstrated solid psychometric characteristics.

CONCLUSION: The GTQ-GR is a valid and reliable questionnaire with strong psychometric properties and is now available in Greek.},
}

@article {pmid40431732,
year = {2025},
author = {Meng, P and Ni, B and Li, C and Sha, Z and Liu, C and Ren, W and Wei, R and Liu, F and Li, J and Wang, Z},
title = {Establishment and Implementation of the Point-of-Care RT-RAA-CRISPR/Cas13a Diagnostic Test for Foot-And-Mouth Disease Virus Serotype O in Pigs.},
journal = {Viruses},
volume = {17},
number = {5},
pages = {},
pmid = {40431732},
issn = {1999-4915},
support = {2021YFD1800300//National Key R&D Program of China/ ; },
mesh = {Animals ; Swine ; *Foot-and-Mouth Disease Virus/genetics/isolation & purification/classification ; *Swine Diseases/diagnosis/virology ; *Foot-and-Mouth Disease/diagnosis/virology ; Serogroup ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Point-of-Care Systems ; RNA, Viral/genetics ; *Molecular Diagnostic Techniques/methods ; },
abstract = {Foot and mouth disease virus (FMDV) is a highly pathogenic virus that mainly infects cloven hooved animals, such as pigs. The establishment of a rapid, sensitive and accurate point-of-care detection method is critical for the timely identification and elimination of infected pigs for controlling this disease. In this study, a RT-RAA-CRISPR/Cas13a method was developed for the detection of FMDV serotype O in pigs. Six pairs of RT-RAA primers were designed based on the conserved gene sequence of FMDV serotype O, and the optimal amplification primers and reaction temperatures were screened. The CRISPR-derived RNA (crRNA) was further designed based on the optimal target band sequence and the most efficient crRNA was screened. The results revealed that FMDV-O-F4/R4 was the optimal primer set, and the optimal temperature for the RT-RAA reaction was 37 °C. Moreover, crRNA4 exhibited the strongest detection signal among the six crRNAs. The established RT-RAA-CRISPR/Cas13a method demonstrated high specificity and no cross-reactivity with other common swine pathogens such as Senecavirus A (SVA), porcine reproductive and respiratory virus (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV), and pseudorabies virus (PRV), additionally, it was observed to be highly sensitive, with a detection limit of 19.1 copies/µL. The repeatability of this method was also observed to be good. This method could produce stable fluorescence and exhibited good repeatability when three independent experiments yielded the same results. A validation test using three types of simulated clinical samples (including swab, tissue, and serum samples) revealed a 100% concordance rate. The detection results could be visualized via a fluorescence reader or lateral flow strips (LFSs). Thus, a highly specific and sensitive RT-RAA-CRISPR/Cas13a detection method was developed and is expected to be applied for the rapid detection of FMDV serotype O in situ.},
}

Animals
Swine
*Foot-and-Mouth Disease Virus/genetics/isolation & purification/classification
*Swine Diseases/diagnosis/virology
*Foot-and-Mouth Disease/diagnosis/virology
Serogroup
*CRISPR-Cas Systems
Sensitivity and Specificity
*Point-of-Care Systems
RNA, Viral/genetics
*Molecular Diagnostic Techniques/methods

@article {pmid40429982,
year = {2025},
author = {Leal, K and Machuca, J and Gajardo, H and Palma, M and Contreras, MJ and Nuñez-Montero, K and Gutiérrez, Á and Barrientos, L},
title = {Structural Characterisation of TetR/AcrR Regulators in Streptomyces fildesensis So13.3: An In Silico CRISPR-Based Strategy to Influence the Suppression of Actinomycin D Production.},
journal = {International journal of molecular sciences},
volume = {26},
number = {10},
pages = {},
pmid = {40429982},
issn = {1422-0067},
support = {1210563//Fondecyt Regular/ ; 11230475//Fondecyt Iniciación/ ; 11240632//Fondecyt Iniciación/ ; },
mesh = {*Streptomyces/genetics/metabolism ; *Dactinomycin/biosynthesis ; *Bacterial Proteins/genetics/metabolism/chemistry ; Gene Expression Regulation, Bacterial ; Multigene Family ; Molecular Dynamics Simulation ; *Transcription Factors/genetics/metabolism/chemistry ; Anti-Bacterial Agents/biosynthesis ; *CRISPR-Cas Systems ; Biosynthetic Pathways/genetics ; },
abstract = {The growing threat of antimicrobial resistance has intensified the search for new bioactive compounds, particularly in extreme environments such as Antarctica. Streptomyces fildesensis So13.3, isolated from Antarctic soil, harbours a biosynthetic gene cluster (BGC) associated with actinomycin D production, an antibiotic with biomedical relevance. This study investigates the regulatory role of TetR/AcrR transcription factors encoded within this biosynthetic gene cluster (BGC), focusing on their structural features and expression under different nutritional conditions. Additionally, we propose that repressing an active pathway could lead to the activation of silent biosynthetic routes, and our in-silico analysis provides a foundation for selecting key mutations and experimentally validating this strategy. Expression analysis revealed that TetR-279, in particular, was upregulated in ISP4 and IMA media, suggesting its participation in nutrient-dependent BGC regulation. Structural modelling identified key differences between TetR-206 and TetR-279, with the latter containing a tetracycline-repressor-like domain. Molecular dynamics simulations confirmed TetR-279's structural stability but showed that the S166P CRISPy-web-guided mutation considerably affected its flexibility, while V167A and V167I had modest effects. These results underscore the importance of integrating omics, structural prediction, and gene editing to evaluate and manipulate transcriptional regulation in non-model bacteria. Targeted disruption of TetR-279 may derepress actinomycin biosynthesis, enabling access to silent or cryptic secondary metabolites with potential pharmaceutical applications.},
}

*Streptomyces/genetics/metabolism
*Dactinomycin/biosynthesis
*Bacterial Proteins/genetics/metabolism/chemistry
Gene Expression Regulation, Bacterial
Multigene Family
Molecular Dynamics Simulation
*Transcription Factors/genetics/metabolism/chemistry
Anti-Bacterial Agents/biosynthesis
*CRISPR-Cas Systems
Biosynthetic Pathways/genetics

@article {pmid40429922,
year = {2025},
author = {Sheng, H and Gao, P and Yang, C and Quilichini, TD and Kochian, LV and Datla, R and Xiang, D},
title = {Advances in Genome Editing Through Haploid Induction Systems.},
journal = {International journal of molecular sciences},
volume = {26},
number = {10},
pages = {},
pmid = {40429922},
issn = {1422-0067},
mesh = {*Gene Editing/methods ; *Haploidy ; CRISPR-Cas Systems ; *Crops, Agricultural/genetics ; Plant Breeding/methods ; *Genome, Plant ; Diploidy ; Plants, Genetically Modified/genetics ; },
abstract = {Groundbreaking advances in gene editing technologies are transforming modern plant breeding by enabling precise genetic modifications that dramatically accelerate crop improvement. Haploid and diploid induction systems have emerged as particularly powerful tools in this landscape, offering both efficient gene editing capabilities and rapid production of homozygous lines while seamlessly integrating with the advanced genome-editing platforms such as CRISPR-Cas systems. This review synthesizes the current state of knowledge regarding the mechanisms, applications, and recent progress in haploid and diploid induction systems for gene editing. We examine their transformative potential for enhancing genetic gains and compressing breeding timelines, with significant implications for global food security. Additionally, we provide a critical analysis of emerging challenges of genome editing in crops and outline promising future directions for research and development.},
}

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

@article {pmid40429745,
year = {2025},
author = {Nilsri, N and Mekchaaum, R and Kalasin, S and Jongjitwimol, J and Daowtak, K},
title = {CRISPR/Cas9-Based Modeling of JAK2 V617F Mutation in K562 Cells Reveals Enhanced Proliferation and Sensitivity to Therapeutic Agents.},
journal = {International journal of molecular sciences},
volume = {26},
number = {10},
pages = {},
pmid = {40429745},
issn = {1422-0067},
support = {R2564C032 and AH-64-01-002//Naresuan University research funding and Faculty of Allied Health Sciences, Naresuan University research funding/ ; },
mesh = {Humans ; *Janus Kinase 2/genetics/metabolism ; Cell Proliferation/drug effects/genetics ; *CRISPR-Cas Systems/genetics ; K562 Cells ; Interferon-alpha/pharmacology ; *Mutation ; Arsenic Trioxide/pharmacology ; Myeloproliferative Disorders/genetics/drug therapy ; Antineoplastic Agents/pharmacology ; Point Mutation ; },
abstract = {The Janus kinase 2 (JAK2) protein fulfills an important role in hematopoiesis via the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, as it provides the genetic driver of BCR::ABL1-negative myeloproliferative neoplasms (MPNs), which are clinically manifested as polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The most common cause of MPNs is the mutation of JAK2 V617F in the JAK2 gene, which results in increased cell proliferation. However, both the pathogenesis and treatment regimen of BCR::ABL1-negative MPNs remain poorly understood. The aim of the present study was to establish K562 cell lines with a point mutation in exon 14 (JAK2p.V617F) using CRISPR/Cas9 technology. The modified JAK2 V617F cell lines were examined for the gene mutation using droplet digital PCR (DDPCR), and the presence of the mutation was confirmed by DNA sequencing. Modified cells were characterized by measuring JAK2 gene expression and the extent of cell proliferation. Interferon α2a (IFN-α2a) and arsenic trioxide were also administered to the cells to explore their potential effects. The JAK2 V617F-mutated cells were found to exhibit a higher level of JAK2 gene expression compared with the wild type. Interestingly, a significant increase in the proliferation rate was observed with the modified cells compared with the wild type cells (p < 0.001), as assessed from the JAK2 gene expression levels. Furthermore, the treatments with IFN-α2a and arsenic trioxide led to the preferential suppression of the cell proliferation rate of the K562 expressing mutant JAK2 cells compared with the wild type cells, and this suppression occurred in a dose-dependent manner(p < 0.01). Moreover, the modified cells were able to differentiate into megakaryocyte-like cells following stimulation with phorbol 12 myristate 13 acetate (PMA). Taken together, the results of the present study have shown that the CRISPR/Cas9-modified JAK2 V617F model may be used as a disease model in the search of novel therapies for MPNs.},
}

Humans
*Janus Kinase 2/genetics/metabolism
Cell Proliferation/drug effects/genetics
*CRISPR-Cas Systems/genetics
K562 Cells
Interferon-alpha/pharmacology
*Mutation
Arsenic Trioxide/pharmacology
Myeloproliferative Disorders/genetics/drug therapy
Antineoplastic Agents/pharmacology
Point Mutation

@article {pmid40429744,
year = {2025},
author = {Mikhaylova, E},
title = {Virus-Induced Genome Editing (VIGE): One Step Away from an Agricultural Revolution.},
journal = {International journal of molecular sciences},
volume = {26},
number = {10},
pages = {},
pmid = {40429744},
issn = {1422-0067},
support = {24-76-10065//Russian Science Foundation/ ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *Plant Viruses/genetics ; *Genome, Plant ; Agriculture/methods ; *Crops, Agricultural/genetics/virology ; Plants, Genetically Modified/genetics ; Genetic Vectors/genetics ; },
abstract = {There is currently a worldwide trend towards deregulating the use of genome-edited plants. Virus-induced genome editing (VIGE) is a novel technique that utilizes viral vectors to transiently deliver clustered regularly interspaced short palindromic repeat (CRISPR) components into plant cells. It potentially allows us to obtain transgene-free events in any plant species in a single generation without in vitro tissue culture. This technology has great potential for agriculture and is already being applied to more than 14 plant species using more than 20 viruses. The main limitations of VIGE include insufficient vector capacity, unstable expression of CRISPR-associated (Cas) protein, plant immune reaction, host specificity, and reduced viral activity in meristem. Various solutions to these problems have been proposed, such as fusion of mobile elements, RNAi suppressors, novel miniature Cas proteins, and seed-borne viruses, but the final goal has not yet been achieved. In this review, the mechanism underlying the ability of different classes of plant viruses to transiently edit genomes is explained. It not only focuses on the latest achievements in virus-induced editing of crops but also provides suggestions for improving the technology. This review may serve as a source of new ideas for those planning to develop new approaches in VIGE.},
}

*Gene Editing/methods
CRISPR-Cas Systems
*Plant Viruses/genetics
*Genome, Plant
Agriculture/methods
*Crops, Agricultural/genetics/virology
Plants, Genetically Modified/genetics
Genetic Vectors/genetics

@article {pmid40429730,
year = {2025},
author = {Fu, L and Li, P and Rui, Z and Sun, J and Yang, J and Wang, Y and Jia, D and Hu, J and Li, X and Ma, R},
title = {CRISPR/Cas9-Mediated Knockout of the White Gene in Agasicles hygrophila.},
journal = {International journal of molecular sciences},
volume = {26},
number = {10},
pages = {},
pmid = {40429730},
issn = {1422-0067},
support = {202304051001006//Science and Technology Innovation Teams of Shanxi Province/ ; 20210302123386//Fundamental Research Program of Shanxi Province/ ; 2016YJ03//Science and Technology Innovation Fund of Shanxi Agricultural University/ ; ZBXY23A-10//Cultivation and Innovation Program for Scientific research, College of Plant Protection, Shanxi Agricultural University/ ; },
mesh = {*CRISPR-Cas Systems ; Gene Editing/methods ; *Gene Knockout Techniques ; *Amaranthaceae/genetics ; },
abstract = {Agasicles hygrophila is the most effective natural enemy for the control of the invasive weed Alternanthera philoxeroides (Mart.) Griseb. However, research on the gene function and potential genetic improvement of A. hygrophila is limited due to a lack of effective genetic tools. In this study, we employed the A. hygrophila white (AhW) gene as a target gene to develop a CRISPR/Cas9-based gene editing method applicable to A. hygrophila. We showed that injection of Cas9/sgRNA ribonucleoprotein complexes (RNPs) of the AhW gene into pre-blastoderm eggs induced genetic insertion and deletion mutations, leading to white eyes. Our results demonstrate that CRISPR/Cas9-mediated gene editing is possible in A. hygrophila, offering a valuable tool for studies of functional genomics and genetic improvement of A. hygrophila, which could potentially lead to more effective control of invasive weeds through the development of improved strains of this biocontrol agent. In addition, the white-eyed mutant strain we developed could potentially be useful for other transgenic research studies on this species.},
}

*CRISPR-Cas Systems
Gene Editing/methods
*Gene Knockout Techniques
*Amaranthaceae/genetics

@article {pmid40428391,
year = {2025},
author = {Ziółkowska-Suchanek, I and Rozwadowska, N},
title = {Advancements in Gene Therapy for Non-Small Cell Lung Cancer: Current Approaches and Future Prospects.},
journal = {Genes},
volume = {16},
number = {5},
pages = {},
pmid = {40428391},
issn = {2073-4425},
mesh = {Humans ; *Carcinoma, Non-Small-Cell Lung/therapy/genetics ; *Genetic Therapy/methods/trends ; *Lung Neoplasms/therapy/genetics ; Gene Editing/methods ; CRISPR-Cas Systems ; Immunotherapy, Adoptive/methods ; },
abstract = {Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide, characterized by late diagnosis and resistance to conventional therapies. Gene therapy has emerged as a promising alternative for NSCLC therapy, especially for patients with advanced disease who have exhausted conventional treatments. This article delved into the current developments in gene therapy for NSCLC, including gene replacement and tumor suppressor gene therapy, gene silencing, CRISPR/Cas9 gene editing, and immune modulation with CAR-T cell therapy. In addition, the challenges and future prospects of gene-based therapies for NSCLC were discussed.},
}

Humans
*Carcinoma, Non-Small-Cell Lung/therapy/genetics
*Genetic Therapy/methods/trends
*Lung Neoplasms/therapy/genetics
Gene Editing/methods
CRISPR-Cas Systems
Immunotherapy, Adoptive/methods

@article {pmid40355755,
year = {2025},
author = {Wolf, G and Leippe, P and Onstein, S and Goldmann, U and Frommelt, F and Teoh, ST and Girardi, E and Wiedmer, T and Superti-Furga, G},
title = {The genetic interaction map of the human solute carrier superfamily.},
journal = {Molecular systems biology},
volume = {21},
number = {6},
pages = {531-559},
pmid = {40355755},
issn = {1744-4292},
support = {777372//Innovative Medicines Initiative 2/ ; },
mesh = {Humans ; CRISPR-Cas Systems ; *Solute Carrier Proteins/genetics/metabolism ; Gene Knockout Techniques ; Mitochondrial Membrane Transport Proteins/genetics/metabolism ; Cation Transport Proteins/genetics/metabolism ; Cell Line, Tumor ; *Epistasis, Genetic ; Gene Regulatory Networks ; Mitochondrial Proteins ; Organic Anion Transporters ; },
abstract = {Solute carriers (SLCs), the largest superfamily of transporter proteins in humans with about 450 members, control the movement of molecules across membranes. A typical human cell expresses over 200 different SLCs, yet their collective influence on cell phenotypes is not well understood due to overlapping substrate specificities and expression patterns. To address this, we performed systematic pairwise gene double knockouts using CRISPR-Cas12a and -Cas9 in human colon carcinoma cells. A total of 1,088,605 guide combinations were used to interrogate 35,421 SLC-SLC and SLC-enzyme double knockout combinations across multiple growth conditions, uncovering 1236 genetic interactions with a growth phenotype. Further exploration of an interaction between the mitochondrial citrate/malate exchanger SLC25A1 and the zinc transporter SLC39A1 revealed an unexpected role for SLC39A1 in metabolic reprogramming and anti-apoptotic signaling. This full-scale genetic interaction map of human SLC transporters is the backbone for understanding the intricate functional network of SLCs in cellular systems and generates hypotheses for pharmacological target exploitation in cancer and other diseases. The results are available at https://re-solute.eu/resources/dashboards/genomics/ .},
}

Humans
CRISPR-Cas Systems
*Solute Carrier Proteins/genetics/metabolism
Gene Knockout Techniques
Mitochondrial Membrane Transport Proteins/genetics/metabolism
Cation Transport Proteins/genetics/metabolism
Cell Line, Tumor
*Epistasis, Genetic
Gene Regulatory Networks
Mitochondrial Proteins
Organic Anion Transporters

@article {pmid39804466,
year = {2025},
author = {Birling, MC and Hérault, Y and Pavlovic, G},
title = {Genome engineering with Cas9 and AAV repair templates, successes and pitfalls.},
journal = {Mammalian genome : official journal of the International Mammalian Genome Society},
volume = {36},
number = {2},
pages = {376-383},
pmid = {39804466},
issn = {1432-1777},
mesh = {*Dependovirus/genetics ; Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genetic Engineering/methods ; Genetic Vectors/genetics ; Humans ; Genome ; },
abstract = {Genome editing, in particular the CRISPR/Cas9 system, is widely used to generate new animal models. However, the generation of mutations, such as conditional knock-out or knock-in, can remain complex and inefficient, in particular because of the difficulty to deliver the donor DNA (single or double stranded) into the nucleus of fertilized oocytes. The use of recombinant adeno-associated viruses (rAAV) as donor DNA is a rapidly developing approach that promises to improve the efficiency of creation of animal models. In this mini-review, we explore the progress and challenges of using CRISPR/Cas9 in combination with rAAV for precise genome editing. We will summarise the current knowledge of rAAV transduction, data on its use in rodent embryos in combination with CRISPR/Cas9 to easily generate sequence replacements or insertions, the limitations of rAAV and the unexpected events observed to date, and the protocol optimisations already in place to facilitate its use in the generation of animal models.},
}

*Dependovirus/genetics
Animals
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Genetic Engineering/methods
Genetic Vectors/genetics
Humans
Genome

@article {pmid40427606,
year = {2025},
author = {Gutiérrez-Hurtado, IA and García-Acéves, ME and Puga-Carrillo, Y and Guardado-Estrada, M and Becerra-Loaiza, DS and Carrillo-Rodríguez, VD and Plazola-Zamora, R and Godínez-Rubí, JM and Rangel-Villalobos, H and Aguilar-Velázquez, JA},
title = {Past, Present and Future Perspectives of Forensic Genetics.},
journal = {Biomolecules},
volume = {15},
number = {5},
pages = {},
pmid = {40427606},
issn = {2218-273X},
mesh = {Humans ; *Forensic Genetics/trends/methods/history ; Machine Learning ; CRISPR-Cas Systems ; },
abstract = {Forensic genetics has experienced remarkable advancements over the past decades, evolving from the analysis of a limited number of DNA segments to comprehensive genome-wide investigations. This progression has significantly improved the ability to establish genetic profiles under diverse conditions and scenarios. Beyond individual identification, forensic genetics now enables the inference of physical traits (e.g., eye, hair, and skin color, as well as body composition), biogeographic ancestry, lifestyle habits such as alcohol and tobacco use, and even the transfer of genital microbiomes post-coitus, among other characteristics. Emerging trends point to a future shaped by the integration of cutting-edge technologies, including CRISPR-Cas systems, artificial intelligence, and machine learning, which promise to further revolutionize the field. This review provides a thorough exploration of forensic genetics, tracing its evolution from its foundational methods (past) to its diverse modern applications (present) and offering insights into its potential future directions.},
}

Humans
*Forensic Genetics/trends/methods/history
Machine Learning
CRISPR-Cas Systems

@article {pmid40427601,
year = {2025},
author = {Marin-Quilez, A and García-Tuñón, I and Benito, R and Ordoñez, JL and Díaz-Ajenjo, L and Lama-Villanueva, A and Guerrero, C and Pérez-Losada, J and González-Porras, JR and Hernández-Rivas, JM and Del Rey, M and Bastida, JM},
title = {Examining the Effects of the RUNX1 p.Leu43Ser Variant on FPD/AML Phenotypes Using a CRISPR/Cas9-Generated Knock-In Murine Model.},
journal = {Biomolecules},
volume = {15},
number = {5},
pages = {},
pmid = {40427601},
issn = {2218-273X},
mesh = {Animals ; *Core Binding Factor Alpha 2 Subunit/genetics/metabolism ; Mice ; *CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Gene Knock-In Techniques ; *Leukemia, Myeloid, Acute/genetics/pathology ; Phenotype ; *Blood Platelet Disorders/genetics/pathology ; Male ; Homozygote ; },
abstract = {Germline heterozygous variants in RUNX1 lead to Familial Platelet Disorder with Myeloid Leukemia Predisposition (FPD/AML). Cellular and/or animal models are helpful to uncovering the role of a variant in disease progression. Twenty-five mice per genotype (RUNX1[WT/WT], RUNX1[WT/L43S], RUNX1[L43S/L43S]), previously generated by CRISPR/Cas9, and nine sub-lethally irradiated mice per genotype were investigated. Peripheral blood (PB), bone marrow (BM), and spleen samples were analyzed by flow cytometry and histopathology. Deregulated genes were analyzed by RNA-seq in BM. An aberrant myeloid Mac1[+]Sca1[+]ckit[-] population in the PB, BM, and spleen of two homozygous and one heterozygous mouse was observed, as well as BM hypercellularity. No Mac1[+]Sca1[+]ckit[-] cells were detected in any RUNX1[WT/WT] mice. Moreover, the spleen of both homozygous mice showed destruction of the white/red pulp and the presence of apoptotic cells. The aberrant population was also detected in four irradiated mice, two heterozygous and two homozygous, in their PB, BM, and spleen. RNA-seq studies showed 698 genes significantly deregulated in the three non-irradiated Mac1[+]Sca1[+]ckit[-] mice vs. six healthy mice, highlighting the alteration of genes involved in apoptosis and DNA repair. These results indicate that the homozygous form of the variant p.Leu43Ser may contribute to the pathogenesis of aberrant cells.},
}

Animals
*Core Binding Factor Alpha 2 Subunit/genetics/metabolism
Mice
*CRISPR-Cas Systems/genetics
Disease Models, Animal
Gene Knock-In Techniques
*Leukemia, Myeloid, Acute/genetics/pathology
Phenotype
*Blood Platelet Disorders/genetics/pathology
Male
Homozygote

@article {pmid40427577,
year = {2025},
author = {Palit, P and Minkara, M and Abida, M and Marwa, S and Sen, C and Roy, A and Pasha, MR and Mosae, PS and Saha, A and Ferdoush, J},
title = {PlastiCRISPR: Genome Editing-Based Plastic Waste Management with Implications in Polyethylene Terephthalate (PET) Degradation.},
journal = {Biomolecules},
volume = {15},
number = {5},
pages = {},
pmid = {40427577},
issn = {2218-273X},
support = {Start-up fund//University of Tennessee at Chattanooga/ ; },
mesh = {*Polyethylene Terephthalates/metabolism/chemistry ; *Gene Editing/methods ; *Waste Management/methods ; *Plastics/metabolism/chemistry ; Biodegradation, Environmental ; CRISPR-Cas Systems ; Humans ; },
abstract = {Plastic pollution has become a significant environmental issue worldwide, with global plastic production expected to reach 1800 million tons by 2050. One of the most commonly used plastics in the world is polyethylene terephthalate (PET), a synthetic polymer that is extremely durable but difficult to degrade. Thus, PET is dangerous to human health. To address this crisis, innovative approaches are being developed, including genome editing technologies. One of the recently advanced genome editing technologies is PlastiCRISPR, a novel concept that applies CRISPR-based genome editing to transform plastic waste management. PlastiCRISPR utilizes microorganisms to degrade plastic, generating valuable bioproducts like biofuels and biochemicals. Thus, this technology offers a sustainable solution because of its simple design, adequacy, and low cost, which can be integrated into existing waste management systems. Importantly, this review focuses on the PlastiCRISPR-based management of PET because it could drastically lower plastic waste, sustain natural resources by decreasing the requirement for plastic production, minimize energy intake, etc. Overall, this review provides an overview of the principles, applications, challenges, and future prospects of PlastiCRISPR in combating plastic pollution and shaping a more sustainable future.},
}

*Polyethylene Terephthalates/metabolism/chemistry
*Gene Editing/methods
*Waste Management/methods
*Plastics/metabolism/chemistry
Biodegradation, Environmental
CRISPR-Cas Systems
Humans

@article {pmid40426207,
year = {2025},
author = {Wang, ZC and Stegall, H and Miyazawa, T and Keatinge-Clay, AT},
title = {A CRISPR-Cas9 system for knock-out and knock-in of high molecular weight DNA enables module-swapping of the pikromycin synthase in its native host.},
journal = {Microbial cell factories},
volume = {24},
number = {1},
pages = {125},
pmid = {40426207},
issn = {1475-2859},
support = {R01 GM145992/GM/NIGMS NIH HHS/United States ; GM145992/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Streptomyces/genetics/metabolism/enzymology ; *Polyketide Synthases/genetics/metabolism ; Macrolides/metabolism ; Gene Editing/methods ; Gene Knockout Techniques ; Anti-Bacterial Agents/biosynthesis ; *Gene Knock-In Techniques ; Molecular Weight ; },
abstract = {BACKGROUND: Engineers seeking to generate natural product analogs through altering modular polyketide synthases (PKSs) face significant challenges when genomically editing large stretches of DNA.

RESULTS: We describe a CRISPR-Cas9 system that was employed to reprogram the PKS in Streptomyces venezuelae ATCC 15439 that helps biosynthesize the macrolide antibiotic pikromycin. We first demonstrate its precise editing ability by generating strains that lack megasynthase genes pikAI-pikAIV or the entire pikromycin biosynthetic gene cluster but produce pikromycin upon complementation. We then employ it to replace 4.4-kb modules in the pikromycin synthase with those of other synthases to yield two new macrolide antibiotics with activities similar to pikromycin.

CONCLUSION: Our gene-editing tool has enabled the efficient replacement of extensive and repetitive DNA regions within streptomycetes.},
}

*CRISPR-Cas Systems
*Streptomyces/genetics/metabolism/enzymology
*Polyketide Synthases/genetics/metabolism
Macrolides/metabolism
Gene Editing/methods
Gene Knockout Techniques
Anti-Bacterial Agents/biosynthesis
*Gene Knock-In Techniques
Molecular Weight

@article {pmid40425955,
year = {2025},
author = {Chen, Y and Zhang, X and Huang, W and Jin, Z and Wei, X and Li, J},
title = {Ultrasensitive detection of atrazine by Schottky junction photoelectrochemical aptamer sensor based on signal amplification by cascade catalysis of CRISPR/Cas12a and G-quadruplex/hemin DNAzyme.},
journal = {Mikrochimica acta},
volume = {192},
number = {6},
pages = {376},
pmid = {40425955},
issn = {1436-5073},
mesh = {*G-Quadruplexes ; *DNA, Catalytic/chemistry/metabolism ; *Aptamers, Nucleotide/chemistry ; Hemin/chemistry/metabolism ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; Limit of Detection ; *Atrazine/analysis/chemistry ; CRISPR-Cas Systems ; Electrodes ; CRISPR-Associated Proteins/metabolism/chemistry ; Photochemical Processes ; Catalysis ; Indoles/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Atrazine (ATZ) is used extensively, resulting in residues in food and the environment, posing a serious threat to human health. Herein, Cd0.5Zn0.5S/Ti3C2 photoelectric material was synthesized and immobilized on a FTO electrode as a photoanode. A photoelectrochemical (PEC) aptamer sensor was constructed for the highly sensitive and selective determination of ATZ based on signal amplification via cascade catalysis of CRISPR/Cas12a and G-quadruplex/hemin DNAzyme (G4/hemin DNAzyme). G4/hemin DNAzyme catalyses the oxidation reaction between H2O2 and dopamine (DA) to form polydopamine (PDA) deposit. This process, in turn, inhibits the photocurrent at the photoanode, leading to a decrease in photocurrent. Concurrently, the depletion of DA as an electron donor for the PEC reaction at the photoelectrode further contributes to the decrease in photocurrent. ATZ can hybridize with ATZ aptamer (Apt) in Apt/cDNA to release activation strand (cDNA), which activates the activity of CRISPR/Cas12a and triggers cleavage of G4, causing the cleaving of G4/hemin DNAzyme immobilized on the electrode surface. This process leads to a decrease of G4/hemin DNAzymes amount on the electrode, consequently reducing both the PDA generation and the DA consumption. As a result, the photocurrent is restored. The cascade catalysis of CRISPR/Cas12a and G4/hemin DNAzyme has been demonstrated to result in photocurrent amplification. The photocurrent change was linear with the logarithmic value of ATZ concentration in the range 1.00 × 10[-12] to 1.00 × 10[-5] mol/L. The limit of detection was 3.47 × 10[-13] mol/L. The sensor has been successfully applied to the determination of trace ATZ in environmental and food samples.},
}

*G-Quadruplexes
*DNA, Catalytic/chemistry/metabolism
*Aptamers, Nucleotide/chemistry
Hemin/chemistry/metabolism
*Biosensing Techniques/methods
*Electrochemical Techniques/methods
Limit of Detection
*Atrazine/analysis/chemistry
CRISPR-Cas Systems
Electrodes
CRISPR-Associated Proteins/metabolism/chemistry
Photochemical Processes
Catalysis
Indoles/chemistry
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid40398569,
year = {2025},
author = {Zhou, Y and Duan, Y and Chen, L and Yang, Y and Ma, L and Chen, W and Liao, Z and Cai, J and Li, D},
title = {Engineering dispersed mycelium morphology in Aspergillus niger for enhanced mycoprotein production via CRISPR/Cas9-mediated genome editing.},
journal = {Bioresource technology},
volume = {432},
number = {},
pages = {132652},
doi = {10.1016/j.biortech.2025.132652},
pmid = {40398569},
issn = {1873-2976},
mesh = {*Aspergillus niger/genetics/metabolism/cytology ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Mycelium/genetics/metabolism/cytology ; Fermentation ; *Fungal Proteins/biosynthesis/genetics/metabolism ; Biomass ; },
abstract = {Filamentous fungi are widely utilized in industrial fermentation processes due to their high productivity, with mycelial morphology directly influencing fermentation broth viscosity and target product yield, which is a critical parameter for process optimization. Aspergillus niger, an FDA-approved safe filamentous fungus, typically forms tightly packed mycelial pellets in submerged cultures, which severely restricts its industrial application potential by limiting mass transfer efficiency. To address this challenge, CRISPR/Cas9 mediated genome editing coupled with fermentation optimization enhanced microbial protein production in A. niger. Endogenous α-1,3-glucan synthase genes (agsA, agsB) and galactosaminogalactan (GAG) synthase genes (sph3, uge3) were disrupted using CRISPR/Cas9, achieving complete dispersion of filamentous pellets in liquid media. This morphological engineering strategy resulted in a 77.52 % increase in biomass and 39.98 % enhancement in mycelial protein content compared to the wild-type strain (A. niger Li2). Transcriptomic analysis revealed that the engineered strain (A. niger AnΔABSU) exhibited upregulated transporter proteins (ABC transporters, MFS transporters, sugar transporters), accelerating nutrient uptake and energy metabolism; altered cell wall integrity pathways, including activation of the MAPK signaling cascade and increased sensitivity to cell wall stressors; enhanced amino acid biosynthesis, driven by upregulated gene expression in key metabolic pathways. Furthermore, response surface methodology (RSM) with Box-Behnken design optimized the fermentation medium, yielding 16.67 g/L biomass and 45.91 % protein content, representing 115.37 % and 67.01 % improvements over the unoptimized wild-type control. This study establishes a novel paradigm for constructing high-efficiency microbial protein cell factories via integrated morphological-engineering and fermentation optimization.},
}

*Aspergillus niger/genetics/metabolism/cytology
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Mycelium/genetics/metabolism/cytology
Fermentation
*Fungal Proteins/biosynthesis/genetics/metabolism
Biomass

@article {pmid40300704,
year = {2025},
author = {Tarin, M and Saljooghi, AS},
title = {Gasdermin E as a potential target and biomarker for CRISPR-Cas9-based cancer therapy.},
journal = {Biochemical pharmacology},
volume = {237},
number = {},
pages = {116961},
doi = {10.1016/j.bcp.2025.116961},
pmid = {40300704},
issn = {1873-2968},
mesh = {Humans ; *CRISPR-Cas Systems/physiology/genetics ; *Neoplasms/therapy/genetics/metabolism ; *Biomarkers, Tumor/metabolism/genetics/antagonists & inhibitors ; Animals ; Pyroptosis/physiology/drug effects ; Gene Editing/methods ; Gasdermins ; },
abstract = {Gasdermin E (GSDME), a protein pivotal in mediating pyroptosis, has gained significant attention due to its role in cancer pathogenesis and its potential as a therapeutic target. The advent of CRISPR-Cas9, a precise genome editing tool, has revolutionized cancer therapy by enabling the manipulation of GSDME expression and function. This review explores the interplay of GSDME and CRISPR-Cas9 in cancer, emphasizing GSDME's unique mechanism of cleavage-dependent pore formation in the cell membrane and its emerging applications as both a therapeutic target and a diagnostic biomarker. We discuss the potential and challenges of using GSDME-induced pyroptosis as a therapeutic strategy and how can enhance its efficacy and specificity. We conclude by highlighting promising future research directions in this emerging field.},
}

Humans
*CRISPR-Cas Systems/physiology/genetics
*Neoplasms/therapy/genetics/metabolism
*Biomarkers, Tumor/metabolism/genetics/antagonists & inhibitors
Animals
Pyroptosis/physiology/drug effects
Gene Editing/methods
Gasdermins

@article {pmid40425635,
year = {2025},
author = {Imai, Y and Ozaki, S and Noda, T and Kobayashi, I and Sugitani, K and Kasashima, S and Morishita, E and Araiso, Y},
title = {Real-time imaging of blood coagulation and angiogenesis during development in a zebrafish model of type I antithrombin deficiency.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {18538},
pmid = {40425635},
issn = {2045-2322},
support = {JPMJSP2135//Japan Science and Technology Agency/ ; JPMJFR200F//Japan Science and Technology Agency/ ; },
mesh = {Animals ; *Zebrafish/genetics/embryology ; *Blood Coagulation/genetics ; Disease Models, Animal ; *Neovascularization, Physiologic ; Zebrafish Proteins/genetics ; Animals, Genetically Modified ; Thrombosis/genetics ; CRISPR-Cas Systems ; Angiogenesis ; },
abstract = {Severe type I antithrombin (AT) deficiency is considered to cause embryonic lethality. Although several pathological analyses using mice or zebrafish have been attempted, the previous studies did not unveil the detailed mechanism leading to lethality in the early developmental stage. In order to solve this problem, we established type I AT deficient zebrafish by the CRISPR/Cas9 system into Tg(gata1:dsRed) and Tg(fli1a:GFP) lines, so that we could conduct real-time imaging of thrombosis and angiogenesis using fluorescence stereo zoom microscopy. The established zebrafish AT (zAT) mutants harbored frameshift mutations which resulted to be type I AT deficient, unable to secrete zAT protein into blood. Both heterozygous (zAT[+/-]) and homozygous (zAT[-/-]) mutants showed reduced survival rate and diverse thrombosis up to 9 days post fertilization. In addition, blood vessel formation was delayed at 30 hpf in zAT[-/-], which was recovered normally by 5 dpf and had little effect on survival. Notably, we analyzed the differences in gene expression profiles under AT-depleted conditions by real-time quantitative PCR, and zAT[-/-] juvenile zebrafish showed increased PLG gene expression and decreased F2 gene expression. Our in vivo study revealed the effects of AT deficiency on embryos during development from the aspects of coagulation and vascular formation.},
}

Animals
*Zebrafish/genetics/embryology
*Blood Coagulation/genetics
Disease Models, Animal
*Neovascularization, Physiologic
Zebrafish Proteins/genetics
Animals, Genetically Modified
Thrombosis/genetics
CRISPR-Cas Systems
Angiogenesis

@article {pmid40425580,
year = {2025},
author = {Sharma, P and Kim, CY and Keys, HR and Imada, S and Joseph, AB and Ferro, L and Kunchok, T and Anderson, R and Sun, Y and Yilmaz, ÖH and Weng, JK and Jain, A},
title = {Genetically encoded fluorescent reporter for polyamines.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4921},
pmid = {40425580},
issn = {2041-1723},
support = {R35 GM151111/GM/NIGMS NIH HHS/United States ; R35GM151111//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
mesh = {*Polyamines/metabolism/analysis ; Humans ; *Genes, Reporter ; Mitochondria/metabolism ; Parkinson Disease/metabolism/genetics ; CRISPR-Cas Systems ; HEK293 Cells ; },
abstract = {Polyamines are abundant and evolutionarily conserved metabolites that are essential for life. Dietary polyamine supplementation extends life-span and health-span. Dysregulation of polyamine homeostasis is linked to Parkinson's disease and cancer, driving interest in therapeutically targeting this pathway. However, measuring cellular polyamine levels, which vary across cell types and states, remains challenging. We introduce a genetically encoded polyamine reporter for real-time measurement of polyamine concentrations in single living cells. This reporter utilizes the polyamine-responsive ribosomal frameshift motif from the OAZ1 gene. We demonstrate broad applicability of this approach and reveal dynamic changes in polyamine levels in response to genetic and pharmacological perturbations. Using this reporter, we conduct a genome-wide CRISPR screen and uncover an unexpected link between mitochondrial respiration and polyamine import, which are both risk factors for Parkinson's disease. By offering a lens to examine polyamine biology, this reporter may advance our understanding of these ubiquitous metabolites and accelerate therapy development.},
}

*Polyamines/metabolism/analysis
Humans
*Genes, Reporter
Mitochondria/metabolism
Parkinson Disease/metabolism/genetics
CRISPR-Cas Systems
HEK293 Cells

@article {pmid40425554,
year = {2025},
author = {Borot, F and Humbert, O and Ehmsen, JT and Fields, E and Kohli, S and Radtke, S and Swing, K and Pande, D and Enstrom, MR and Laszlo, GS and Mayuranathan, T and Ali, AM and Weiss, MJ and Yen, JS and Newby, GA and Walter, RB and Liu, DR and Mukherjee, S and Kiem, HP},
title = {Multiplex base editing to protect from CD33 directed drugs for immune and gene therapy.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4899},
pmid = {40425554},
issn = {2041-1723},
support = {R01 HL156647/HL/NHLBI NIH HHS/United States ; K99 HL163805/HL/NHLBI NIH HHS/United States ; P51 OD010425/OD/NIH HHS/United States ; R00 HL163805/HL/NHLBI NIH HHS/United States ; R50 CA274319/CA/NCI NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; R01 CA266556/CA/NCI NIH HHS/United States ; P01 HL053749/HL/NHLBI NIH HHS/United States ; R01 HL136135/HL/NHLBI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; R01 HL151765/HL/NHLBI NIH HHS/United States ; U42 OD011123/OD/NIH HHS/United States ; },
mesh = {Humans ; Animals ; *Gene Editing/methods ; *Sialic Acid Binding Ig-like Lectin 3/genetics/metabolism/immunology ; *Genetic Therapy/methods ; Hematopoietic Stem Cells/metabolism/drug effects ; Gemtuzumab/pharmacology ; *Immunotherapy/methods ; Polymorphism, Single Nucleotide ; CRISPR-Cas Systems ; Mice ; },
abstract = {The selection of genetically engineered immune or hematopoietic cells in vivo after gene editing remains a clinical problem and requires a method to spare on-target toxicity to normal cells. Here, we develop a base editing approach exploiting a naturally occurring CD33 single nucleotide polymorphism leading to removal of full-length CD33 surface expression on edited cells. CD33 editing in human and nonhuman primate hematopoietic stem and progenitor cells protects myeloid progeny from CD33-targeted therapeutics without affecting normal hematopoiesis in vivo, thus demonstrating potential for improved immunotherapies with reduced off-leukemia toxicity. For broader application to gene therapies, we demonstrate highly efficient (>70%) multiplexed adenine base editing of the CD33 and gamma globin genes, resulting in long-term persistence of dual gene-edited cells with HbF reactivation in nonhuman primates. Using the CD33 antibody-drug conjugate Gemtuzumab Ozogamicin, we show resistance of engrafted, multiplex edited human cells in vivo, and a 2-fold enrichment for edited cells in vitro. Together, our results highlight the potential of adenine base editors for improved immune and gene therapies.},
}

Humans
Animals
*Gene Editing/methods
*Sialic Acid Binding Ig-like Lectin 3/genetics/metabolism/immunology
*Genetic Therapy/methods
Hematopoietic Stem Cells/metabolism/drug effects
Gemtuzumab/pharmacology
*Immunotherapy/methods
Polymorphism, Single Nucleotide
CRISPR-Cas Systems
Mice

@article {pmid40424180,
year = {2025},
author = {Krishnaprasad, VH and Nayak, V and Kumar, S},
title = {World Health Organisation's Bacterial Pathogen Priority List (BPPL) 2017 and BPPL 2024 to combat global antimicrobial resistance crisis: 'challenges and opportunities'.},
journal = {The Journal of antimicrobial chemotherapy},
volume = {},
number = {},
pages = {},
doi = {10.1093/jac/dkaf167},
pmid = {40424180},
issn = {1460-2091},
abstract = {Antibiotic resistance, in a broader perspective, antimicrobial resistance (AMR) presents a formidable global health challenge, threatening the effectiveness of antibiotics and other antimicrobial agents. As a result, AMR has become more challenging or even impossible to treat, leading to increased morbidity and mortality. The World Health Organisation (WHO) has been at the forefront of international efforts to combat AMR by sensitizing the world about the pressing need to tackle AMR to save the future of the human race. This article analyses WHO's efforts to combat AMR, including creating the Bacterial Pathogen Priority List (BPPL), developing a global action plan to address AMR and promoting surveillance and stewardship programmes. This article also examines the progress achieved by BPPL 2017 and the challenges ahead for BPPL 2024. Additionally, this article explores various efforts to combat AMR through two major approaches, like 'research and development' and 'the policy and regulation-based' approach. This article underscores various emerging strategies to tackle AMR, for example, biofilm disruption, nanotechnology, antibiotic resistance breakers, antibody-antibiotic conjugates, rapid detection tools and alternative therapies like phage therapy, antimicrobial peptides, CRISPR-Cas system, probiotics and microbiota modulations. This article also highlights the importance of coordinated actions and sustained commitment to safeguarding public health and ensuring the continued effectiveness of antimicrobial therapies.},
}

@article {pmid40424009,
year = {2025},
author = {Wei, L and Wang, Z and She, Y and Fu, H},
title = {CRISPR/Cas Multiplexed Biosensing: Advances, Challenges, and Perspectives.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.4c04428},
pmid = {40424009},
issn = {1520-6882},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein systems are renowned for their high sensitivity and specificity, enabling them as a powerful diagnostic toolbox. Multiplexed detection of panels of targets, as opposed to single targets, is imperative for reliable and conclusive disease diagnostics. However, multiplex application of the CRISPR/Cas system has long been hindered by indistinguishable signals from specific targets due to nonspecific chaotic trans-cleavage. To make a breakthrough, substantial efforts have been devoted to CRISPR/Cas-powered multiplexed biosensing strategies, which consequently experienced rapid development over the past five years. This review systematically summarizes recent advances in CRISPR/Cas multiplexed detection encompassing Cas9, Cas12, and Cas13. Key focus issues include multiplex biosensing strategies and their respective advantages and limitations, sensing mechanisms, and detection performance of novel validated examples. Finally, the status and challenges of CRISPR/Cas multiplexed biosensing are critically discussed, and future outlooks are proposed for their potential practical application. This Perspective aims to inspire significant research and promote the development of the next generation of CRISPR/Cas multiplexed biosensing.},
}

@article {pmid40422684,
year = {2025},
author = {Ma, B and Li, Y and Wang, T and Li, D and Jia, S},
title = {Advances in CRISPR/Cas9-Based Gene Editing in Filamentous Fungi.},
journal = {Journal of fungi (Basel, Switzerland)},
volume = {11},
number = {5},
pages = {},
pmid = {40422684},
issn = {2309-608X},
support = {32160143//National Science Foundation of China/ ; },
abstract = {As an important class of microorganisms, filamentous fungi have crucial roles in protein secretion, secondary metabolite production and environmental pollution control. However, characteristics such as apical growth, heterokaryon, low homologous recombination (HR) efficiency and the scarcity of genetic markers mean that the application of traditional gene editing technology in filamentous fungi faces great challenges. The introduction of the RNA-mediated CRISPR/Cas (clustered regularly interspaced short palindromic repeat/CRlSPR-associated protein) system in filamentous fungi in recent years has revolutionized gene editing in filamentous fungi. In addition, the continuously expressed CRISPR system has significantly improved the editing efficiency, while the optimized sgRNA design and reduced cas9 concentration have effectively reduced the off-target effect, further enhancing the safety and reliability of the technology. In this review, we systematically analyze the molecular mechanism and regulatory factors of CRISPR/Cas9, focus on the optimization of its expression system and the improvement of the transformation efficiency in filamentous fungi, and reveal the core regulatory roles of HR and non-homologous end-joining (NHEJ) pathways in gene editing. Based on the analysis of various filamentous fungi applications, this review reveals the outstanding advantages of CRISPR/Cas9 in the enhancement of protein secretion, addresses the reconstruction of secondary metabolic pathways and pollutant degradation in the past decade, and provides a theoretical basis and practical guidance for the optimization of the technology and engineering applications.},
}

@article {pmid40422214,
year = {2025},
author = {Gibson, J and Dhungana, A and Pokhrel, M and Arthur, B and Suresh, P and Adebayo, O and Cottle, RN},
title = {Validation of Clinical-Grade Electroporation Systems for CRISPR-Cas9-Mediated Gene Therapy in Primary Hepatocytes for the Correction of Inherited Metabolic Liver Disease.},
journal = {Cells},
volume = {14},
number = {10},
pages = {},
pmid = {40422214},
issn = {2073-4409},
support = {1R01HL168093-01A1/GF/NIH HHS/United States ; 2021000920//American Association for the Study of Liver Diseases Foundation/ ; R01 HL168093/HL/NHLBI NIH HHS/United States ; },
mesh = {*Hepatocytes/transplantation/metabolism ; *Electroporation/methods ; Animals ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods ; Mice ; Gene Editing/methods ; *Tyrosinemias/therapy/genetics ; Humans ; *Liver Diseases/therapy/genetics ; Disease Models, Animal ; Male ; Hydrolases ; },
abstract = {Hepatocyte transplantation (HTx) combined with ex vivo gene therapy has garnered significant interest due to its potential for treating many inherited metabolic liver diseases. The biggest obstacle for HTx is achieving sufficient engraftment levels to rescue diseased phenotypes, which becomes more challenging when combined with ex vivo gene editing techniques. However, recent technological advancements have improved electroporation delivery efficiency, cell viability, and scalability for cell therapy. We recently demonstrated the impacts of electroporation for cell-based gene therapy in a mouse model of hereditary tyrosinemia type 1 (HT1). Here, we explore the use of the clinical-grade electroporator, the MaxCyte ExPERT GTx, utilized in the first FDA-approved CRISPR therapy, Casgevy, and evaluate its potential in primary hepatocytes in terms of delivery efficiency and cell viability. We assessed the gene editing efficiency and post-transplantation engraftment of hepatocytes from mTmG mice electroporated with CRISPR-Cas9-ribonucleoproteins (RNPs) targeting 4-hydroxyphenylpyruvate dioxygenase (Hpd) in a fumarylacetoacetate hydrolase (Fah)-deficient mouse model of HT1. After surgery, Fah[-/-] graft recipients were cycled off and on nitisinone to achieve independence from drug-induced Hpd inhibition, an indicator of HT1 disease correction. Transplanted hepatocytes subjected to electroporation using the GTx system had a cell viability of 89.9% and 100% on-target gene editing efficiency. Recipients transplanted with GTx-electroporated cells showed a smaller weight reduction than controls transplanted with untransfected cells (7.9% and 13.8%, respectively). Further, there were no mortalities in the GTx-recipient mice, whereas there was 25% mortality in the control recipients. Mean donor cell engraftment was significantly higher in GTx-recipient mice compared to untransfected control recipients (97.9% and 81.6%, respectively). Our results indicate that the GTx system does not negatively impact hepatocyte functionality and engraftment potential, thereby demonstrating the promise of GTx electroporation in hepatocytes as a viable cell therapy for treating genetic diseases that affect the liver.},
}

*Hepatocytes/transplantation/metabolism
*Electroporation/methods
Animals
*CRISPR-Cas Systems/genetics
*Genetic Therapy/methods
Mice
Gene Editing/methods
*Tyrosinemias/therapy/genetics
Humans
*Liver Diseases/therapy/genetics
Disease Models, Animal
Male
Hydrolases

@article {pmid40422195,
year = {2025},
author = {Orlova, NA and Sinegubova, MV and Kolesov, DE and Khodak, YA and Tatarskiy, VV and Vorobiev, II},
title = {Genomic and Phenotypic Characterization of CHO 4BGD Cells with Quad Knockout and Overexpression of Two Housekeeping Genes That Allow for Metabolic Selection and Extended Fed-Batch Culturing.},
journal = {Cells},
volume = {14},
number = {10},
pages = {},
pmid = {40422195},
issn = {2073-4409},
support = {25-24-00140//Russian Science Foundation/ ; },
mesh = {CHO Cells ; Animals ; Cricetulus ; *Batch Cell Culture Techniques/methods ; CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques ; Gene Editing ; Apoptosis/genetics ; *Genes, Essential/genetics ; Phenotype ; *Genomics ; },
abstract = {Re-engineering of CHO cells using genome editing and the overexpression of multiple helper genes is the central track for obtaining better cell lines for the production of biopharmaceuticals. Using two subsequent rounds of genome editing of the CHO S cells, we have developed the cell line CHO 4BGD with four knockouts of two pro-apoptotic genes bak1 and bax, and two common selection markers genes-glul (GS) and dhfr, and additional copies of genes bcl-2 and beclin-1 used for enhancement of macroautophagy. The NGS sequencing of 4BGD cells revealed that all eight targeted alleles were successfully disrupted. Two edited loci out of eight contained large inserts of non-relevant DNA. Further data analysis shows that cells have no off-target DNA editing events, and all known CHO genes are preserved. The cells obtained are completely resistant to the induction of apoptosis, and they are suitable for the generation of stably transfected cell lines with the dhfr selection marker. They also properly undergo the target gene amplification. The 4BGD-derived clonal cell line that secretes the monoclonal antibody retains the ability for prolonged fed-batch culturing. The method of obtaining multiply edited CHO cells using the multiplex CRISPR/Cas9 editing and simultaneous stable transfection of plasmids, coding for the housekeeping genes, is suitable for the rapid generation of massively edited CHO cells.},
}

CHO Cells
Animals
Cricetulus
*Batch Cell Culture Techniques/methods
CRISPR-Cas Systems/genetics
*Gene Knockout Techniques
Gene Editing
Apoptosis/genetics
*Genes, Essential/genetics
Phenotype
*Genomics

@article {pmid40419487,
year = {2025},
author = {Byrne, SM and Burleigh, SM and Fragoza, R and Jiang, Y and Savva, Y and Pabon, R and Kania, E and Rainaldi, J and Portell, A and Mali, P and Briggs, AW},
title = {An engineered U7 small nuclear RNA scaffold greatly increases ADAR-mediated programmable RNA base editing.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4860},
pmid = {40419487},
issn = {2041-1723},
mesh = {*Adenosine Deaminase/metabolism/genetics ; Animals ; *RNA Editing/genetics ; Humans ; Mice ; *RNA, Small Nuclear/genetics/metabolism/chemistry ; *Gene Editing/methods ; *RNA-Binding Proteins/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; },
abstract = {Custom RNA base editing exploiting the human Adenosine Deaminase Acting on RNA (ADAR) enzyme may enable therapeutic gene editing without DNA damage or use of foreign proteins. ADAR's adenosine-to-inosine (effectively A-to-G) deamination activity can be targeted to transcripts using an antisense guide RNA (gRNA), but efficacy is challenged by limits of in vivo delivery. Embedding gRNAs into a U7 small nuclear RNA (snRNA) framework greatly enhances RNA editing with endogenous ADAR, and a 750-plex single-cell mutagenesis screen further improved the framework. An optimized scaffold with a stronger synthetic U7 promoter enables 76% RNA editing in vitro from a single DNA construct per cell, and 75% editing in a Hurler syndrome mouse brain after one systemic AAV injection, surpassing circular gRNA approaches. The technology also improves published DMD exon-skipping designs 25-fold in differentiated myoblasts. Our engineered U7 framework represents a universal scaffold for ADAR-based RNA editing and other antisense RNA therapies.},
}

*Adenosine Deaminase/metabolism/genetics
Animals
*RNA Editing/genetics
Humans
Mice
*RNA, Small Nuclear/genetics/metabolism/chemistry
*Gene Editing/methods
*RNA-Binding Proteins/metabolism/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
HEK293 Cells

@article {pmid40419416,
year = {2025},
author = {Wachholz Junior, D and Pontes, RG and Hryniewicz, BM and Kubota, LT},
title = {Exploring a CRISPR/Cas12a-powered impedimetric biosensor for amplification-free detection of a pathogenic bacterial DNA.},
journal = {Biosensors & bioelectronics},
volume = {},
number = {},
pages = {117607},
doi = {10.1016/j.bios.2025.117607},
pmid = {40419416},
issn = {1873-4235},
abstract = {Timely and precise detection of bacterial infections is essential for improving patient outcomes and reducing healthcare costs, especially for sepsis, where delayed diagnosis increases mortality. Traditional culture- and PCR-based methods are time consuming and require complex sample processing, making them unsuitable for rapid diagnostics in resource-limited settings. CRISPR/Cas-based methods, particularly when combined with electrochemical sensing, offer a promising alternative for rapid point-of-care (POC) diagnostics of bacterial infections due to their simplicity and specificity. This study proposes a label-free impedimetric biosensor using the CRISPR/Cas12a system for rapid and amplification-free detection of Staphylococcus aureus DNA, a primary pathogen responsible for sepsis. By leveraging CRISPR/Cas12a's target-activated collateral cleavage on non-specific DNA reporters we investigated the impact of using a protospacer adjacent motif (PAM) sequence on detection sensitivity and specificity. Our biosensor demonstrated ultra-sensitive detection, with limit of detection as low as 20 aM for dsDNA targets in buffer and without any pre-amplification steps. The study also confirmed CRISPR specificity's dependence on the PAM sequence, showing that mismatches on targeting sequences reduces cleavage efficiency, with a drastic reduction in trans-cleavage activity for single mismatch in PAM-containing sequences. Additionally, we examined how the DNA reporter affects performance, noting reduced cleavage efficiency when a ssDNA target was paired with a dsDNA reporter. Furthermore, validation experiments using human serum samples confirmed the biosensor's accuracy for bacterial DNA detection in clinical settings. This work advances CRISPR-powered electrochemical biosensors, providing a detailed discussion on developing a highly sensitive, fast and amplification-free tool for early detection of sepsis-causing bacteria.},
}

@article {pmid40418657,
year = {2025},
author = {Johnson, MJ and DeFeo, AP and Slipek, NJ and Folsom, TD and Henley, T and Choudhry, MS and Webber, BR and Moriarity, BS},
title = {Non-Viral Engineering of Primary Human T Cells via Homology-Mediated End-Joining Targeted Integration of Large DNA Templates.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {219},
pages = {},
doi = {10.3791/68150},
pmid = {40418657},
issn = {1940-087X},
mesh = {Humans ; *T-Lymphocytes/cytology/immunology ; CRISPR-Cas Systems ; *DNA/genetics ; Animals ; *Cell Engineering/methods ; Mice ; *DNA End-Joining Repair ; *Genetic Engineering/methods ; },
abstract = {Many current adoptive cellular therapies rely on lenti- or retroviral vectors to engineer T cells for the expression of a chimeric antigen receptor (CAR) or exogenous T cell receptor (TCR) to target a specific tumor-associated antigen. Reliance on viral vectors for the production of therapeutic T cells significantly increases the timeline, cost, and complexity of manufacturing while limiting the translation of new therapies, particularly in the academic setting. A process is presented for efficient non-viral engineering of T cells using CRISPR/Cas9 and homology-mediated end joining to achieve targeted integration of large, multicistronic DNA cargo. This approach has achieved integration frequencies comparable to those of viral vectors while yielding highly functional T cells capable of potent anti-tumor efficacy both in vitro and in vivo. Notably, this method is rapidly adaptable to current good manufacturing practices (cGMP) and clinical scale-up, providing a near-term option for the manufacturing of therapeutic T cells for use in clinical trials.},
}

Humans
*T-Lymphocytes/cytology/immunology
CRISPR-Cas Systems
*DNA/genetics
Animals
*Cell Engineering/methods
Mice
*DNA End-Joining Repair
*Genetic Engineering/methods

@article {pmid40417002,
year = {2025},
author = {Gallala, M},
title = {Application of CRISPR/Cas gene editing for infectious disease control in poultry.},
journal = {Open life sciences},
volume = {20},
number = {1},
pages = {20251095},
pmid = {40417002},
issn = {2391-5412},
abstract = {The poultry industry faces multifaceted challenges, including escalating demand for poultry products, climate change impacting feed availability, emergence of novel avian pathogens, and antimicrobial resistance. Traditional disease control measures are costly and not always effective, prompting the need for complementary methods. Gene editing (GE, also called genome editing) technologies, particularly CRISPR/Cas9, offer promising solutions. This article summarizes recent advancements in utilizing CRISPR/Cas GE to enhance infectious disease control in poultry. It begins with an overview of modern GE techniques, highlighting CRISPR/Cas9's advantages over other methods. The potential applications of CRISPR/Cas in poultry infectious disease prevention and control are explored, including the engineering of innovative vaccines, the generation of disease-resilient birds, and in vivo pathogen targeting. Additionally, insights are provided regarding regulatory frameworks and future perspectives in this rapidly evolving field.},
}

@article {pmid40315964,
year = {2025},
author = {Gupta, MK and Gouda, G and Moazzam-Jazi, M and Vadde, R and Nagaraju, GP and El-Rayes, BF},
title = {CRISPR/Cas9-directed epigenetic editing in colorectal cancer.},
journal = {Biochimica et biophysica acta. Reviews on cancer},
volume = {1880},
number = {3},
pages = {189338},
doi = {10.1016/j.bbcan.2025.189338},
pmid = {40315964},
issn = {1879-2561},
mesh = {Humans ; *Colorectal Neoplasms/genetics/therapy/pathology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Epigenesis, Genetic ; Animals ; DNA Methylation ; Gene Expression Regulation, Neoplastic ; Epigenome Editing ; },
abstract = {Colorectal cancer (CRC) remains a leading cause of cancer-related illness and death worldwide, arising from a complex interplay of genetic predisposition, environmental influences, and epigenetic dysregulation. Among these factors, epigenetic modifications-reversible and heritable changes in gene expression-serve as crucial regulators of CRC progression. Understanding these modifications is essential for identifying potential biomarkers for early diagnosis and developing targeted therapeutic strategies. Epigenetic drugs (epidrugs) such as DNA methyltransferase inhibitors (e.g., decitabine) and bromodomain inhibitors (e.g., JQ1) have shown promise in modulating aberrant epigenetic changes in CRC. However, challenges such as drug specificity, delivery, and safety concerns limit their clinical application. Advances in CRISPR-Cas9-based epigenetic editing offer a more precise approach to modifying specific epigenetic markers, presenting a potential breakthrough in CRC treatment. Despite its promise, CRISPR-based epigenome editing may result in unintended genetic modifications, necessitating stringent regulations and safety assessments. Beyond pharmacological interventions, lifestyle factors-including diet and gut microbiome composition-play a significant role in shaping the epigenetic landscape of CRC. Nutritional and microbiome-based interventions have shown potential in preventing CRC development by maintaining intestinal homeostasis and reducing tumor-promoting epigenetic changes. This review provides a comprehensive overview of epigenetic alterations in CRC, exploring their implications for diagnosis, prevention, and treatment. By integrating multi-omics approaches, single-cell technologies, and model organism studies, future research can enhance the specificity and efficacy of epigenetic-based therapies. Shortly, a combination of advanced gene-editing technologies, targeted epidrugs, and lifestyle interventions may pave the way for more effective and personalized CRC treatment strategies.},
}

Humans
*Colorectal Neoplasms/genetics/therapy/pathology
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Epigenesis, Genetic
Animals
DNA Methylation
Gene Expression Regulation, Neoplastic
Epigenome Editing

@article {pmid39568260,
year = {2025},
author = {Wang, B and Fan, A and Liu, M and Zhou, Y and Zhang, W and Yan, J},
title = {An Integrated Rapid Detection of Botryosphaeriaceae Species in Grapevine Based on Recombinase Polymerase Amplification, CRISPR/Cas12a, and Lateral Flow Dipstick.},
journal = {Plant disease},
volume = {109},
number = {5},
pages = {1102-1110},
doi = {10.1094/PDIS-08-24-1615-RE},
pmid = {39568260},
issn = {0191-2917},
mesh = {*Vitis/microbiology ; *Ascomycota/genetics/isolation & purification ; *Plant Diseases/microbiology ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Recombinases/genetics/metabolism ; Sensitivity and Specificity ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Grapevine Botryosphaeria dieback (GBD), caused by Botryosphaeriaceae species, is an important grapevine trunk disease that poses a threat to grape yield and quality in global viticultural regions. Pathogen diagnosis at the species level using morphological methods is difficult and time-consuming. Therefore, this study aimed to develop a rapid and accurate detection method for the pathogens causing GBD. Recombinase polymerase amplification (RPA) with CRISPR/Cas12a cleavage was combined for detecting pathogens associated with GBD, and lateral flow dipsticks were employed to monitor the outcomes. Based on the β-tubulin sequences of Botryosphaeriaceae and their related species, specific RPA primers and CRISPR/Cas12a CrRNA were designed and subsequently selected for specifically detecting pathogens associated with GBD. Under optimized reaction conditions and systems, the developed RPA/CRISPR-Cas12a detection system specifically detected Botryosphaeriaceae species within 30 min of RPA and 25 min of CRISPR/Cas12a reactions at 37°C. Specificity tests showed that specific fragments were amplified with the RPA primers in the DNA of six Botryosphaeriaceae species found in China, while none of the fragments were amplified in the other 22 nontarget fungal pathogen species of grapevine. The detection sensitivity of this method was 1 pg/μl, which is equal to that of real-time PCR. In summary, our method is simple to perform, produces visual results, does not rely on expensive equipment, and therefore possesses high practical value, providing an efficient and robust detection platform to accelerate the field detection of pathogens associated with GBD.},
}

*Vitis/microbiology
*Ascomycota/genetics/isolation & purification
*Plant Diseases/microbiology
*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
Recombinases/genetics/metabolism
Sensitivity and Specificity
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40415627,
year = {2025},
author = {Li, H and Zhu, Q and Zhu, J and Min, J},
title = {[Research progress in the developmental process of non-viral CAR-T technology].},
journal = {Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology},
volume = {41},
number = {5},
pages = {461-467},
pmid = {40415627},
issn = {1007-8738},
mesh = {Humans ; *Immunotherapy, Adoptive/methods ; *Receptors, Chimeric Antigen/genetics/immunology ; Animals ; Gene Transfer Techniques ; Genetic Vectors/genetics ; Gene Editing ; CRISPR-Cas Systems/genetics ; DNA Transposable Elements/genetics ; *T-Lymphocytes/immunology ; Neoplasms/therapy/immunology ; },
abstract = {Chimeric antigen receptor T (CAR-T) lymphocytes are at the forefront of adoptive immunotherapy research, and this technology has significantly advanced the prospects of tumor immunotherapy. CAR-T therapy has demonstrated remarkable efficacy in haematological tumours of lymphoid origin and provided therapeutic possibility for solid tumours. Currently, CAR-T cell preparation predominantly involves transfection of T cells with viral vectors. However, the production of viral vectors is time-consuming, expensive, and the vectors have low loading capacity, along with insertion instability. Consequently, there is a pressing need to develop more convenient and precise non-viral gene delivery methods. This paper reviews the most promising non-viral gene delivery technologies, including CRISPR/Cas9 gene editing, transposon systems such as Sleeping Beauty (SB) and PiggyBac (PB), and mRNA, and anticipates the future development of non-viral vector-based CAR-T therapies.},
}

Humans
*Immunotherapy, Adoptive/methods
*Receptors, Chimeric Antigen/genetics/immunology
Animals
Gene Transfer Techniques
Genetic Vectors/genetics
Gene Editing
CRISPR-Cas Systems/genetics
DNA Transposable Elements/genetics
*T-Lymphocytes/immunology
Neoplasms/therapy/immunology

@article {pmid40414878,
year = {2025},
author = {Lobel, JH and Ingolia, NT},
title = {Precise measurement of molecular phenotypes with barcode-based CRISPRi systems.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {142},
pmid = {40414878},
issn = {1474-760X},
support = {F32 GM148044/GM/NIGMS NIH HHS/United States ; R01 GM135233/GM/NIGMS NIH HHS/United States ; R21 HG012991/HG/NHGRI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Phenotype ; Humans ; Promoter Regions, Genetic ; },
abstract = {Genome-wide CRISPR-Cas9 screens have untangled regulatory networks driving diverse biological processes. Their success relies on interrogating specific molecular phenotypes and distinguishing key regulators from background effects. Here, we realize these goals by optimizing CRISPR interference with barcoded expression reporter sequencing (CiBER-seq) to dramatically improve the sensitivity and scope of genome-wide screens. We systematically address technical factors that distort phenotypic measurements by normalizing expression reporters against closely matched promoters. We use our improved CiBER-seq to accurately capture known components of well-studied RNA and protein quality control systems. These results demonstrate the precision and versatility of CiBER-seq for dissecting cellular pathways.},
}

*CRISPR-Cas Systems
Phenotype
Humans
Promoter Regions, Genetic

@article {pmid40414010,
year = {2025},
author = {Cao, X and Gao, Z and Yin, P and Wang, H and Yang, L},
title = {Crystal structure and inhibition mechanism of AcrIIA11.},
journal = {Biochemical and biophysical research communications},
volume = {772},
number = {},
pages = {152073},
doi = {10.1016/j.bbrc.2025.152073},
pmid = {40414010},
issn = {1090-2104},
abstract = {Anti-CRISPR (Acr) proteins are naturally evolved inhibitors that precisely target and suppress CRISPR-Cas systems, representing a sophisticated molecular arms race between bacteriophages and their bacterial hosts. While Class 1 systems dominate among sequenced prokaryotic genomes, Class 2 systems remain primary sources of editing tools. Here, we report the structural and mechanistic characterization of AcrIIA11, an anti-CRISPR protein that simultaneously inhibits Streptococcus pyogenes (SpyCas9) and Staphylococcus aureus Cas9 (SauCas9). The 3.2 Å crystal structure reveals a compact α/β fold with distinct electropositive clefts implicated in DNA binding. While DALI analysis identified structural homology to transcriptional regulators and the RecA inhibitor PsiB (RMSD 3.3 Å), functional studies established that AcrIIA11 forms stable ternary complexes with both Cas9 orthologs and sgRNA. Biochemical assays demonstrated stronger inhibition of SauCas9 compared to SpyCas9, with EMSA revealing a critical dichotomy: AcrIIA11 maintains SauCas9-sgRNA binding to specific target DNA while completely blocking cleavage activity. Computational docking localizes AcrIIA11 at the HNH-RuvC interface without obstructing DNA-binding channels in SauCas9, suggesting allosteric inhibition through HNH domain displacement. This work establishes AcrIIA11 as a dual-purpose Cas9 inhibitor that preserves target recognition while inactivating nuclease function-a mechanism with potential applications in precision CRISPR control.},
}

@article {pmid40413996,
year = {2025},
author = {Duan, M and Li, G and Shen, J and Dai, R and Li, X and Liu, Z and Jia, F},
title = {A CRISPR/Cas12a biosensor for portable and accessible detection of Salmonella typhimurium via multi-indicator pH millidisc colorimetry and smartphone imaging platform.},
journal = {Biosensors & bioelectronics},
volume = {286},
number = {},
pages = {117611},
doi = {10.1016/j.bios.2025.117611},
pmid = {40413996},
issn = {1873-4235},
abstract = {Conventional colorimetric CRISPR/Cas methods rely on a single chromogenic substrate and bulky and specialized signal detection instrument, which hinder their practical application. Herein, a portable and accessible CRISPR/Cas12a biosensor was for the first time reported to sensitively quantify Salmonella enterica serovar typhimurium (S. typhimurium), utilizing a multi-indicator pH millimeter disc (millidisc) for signal visualization, combined with a smartphone-based imaging platform for signal readout. The pH millidisc, composed of multiple indicators, possessed sensitive pH responsiveness and exhibited diverse color changes. The self-developed RGB mini-program, named DeepFood, was designed for portable smartphone use, featuring user-friendly operation and trend visualization for preliminary result analysis. Based on the RGB signal variation pattern with S. typhimurium, a distinct Senh signal type was designed, enhancing the signal-to-noise ratio from 3.38 to 7.11. Compared to the R signal type, the Senh signal type improved detection sensitivity by 36.23-fold (7.26 CFU/mL) in 0.01 M PBS buffer and 15.53-fold (1.41 × 10[2] CFU/mL) in chicken. The proposed biosensor offers significant improvements in detection sensitivity and practical applicability, with potential in food safety and environmental protection.},
}

@article {pmid40413357,
year = {2025},
author = {Jiang, B and An, Z and Niu, L and Qin, D},
title = {Precise genome editing process and its applications in plants driven by AI.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {109},
pmid = {40413357},
issn = {1438-7948},
support = {BLX202316//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Genome, Plant ; *Plants/genetics ; *Artificial Intelligence ; Plant Breeding ; },
abstract = {Genome editing technologies have emerged as the keystone of biotechnological research, enabling precise gene modification. The field has evolved rapidly through revolutionary advancements, transitioning from early explorations to the breakthrough of the CRISPR-Cas system. The emergence of the CRISPR-Cas system represents a huge leap in genome editing, prompting the development of advanced tools such as base and prime editors, thereby enhancing precise genomic engineering capabilities. The rapid integration of AI across disciplines is now driving another transformative phase in genome editing, streamlining workflows and enhancing precision. The application prospects of genome editing technology are extensive, particularly in plant breeding, where it has already presented unparalleled opportunities for improving plant traits. Here, we review early genome editing technologies, including meganucleases, ZFNs, TALENs, and CRISPR-Cas systems. We also provide a detailed introduction to next-generation editing tools-such as base editors and prime editors-and their latest applications in plants. At the same time, we summarize and prospect the cutting-edge developments and future trends of genome editing technologies in combination with the rapidly rising AI technology, including optimizing editing systems, predicting the efficiency of editing sites and designing editing strategies. We are convinced that as these technologies progress and their utilization expands, they will provide pioneering solutions to global challenges, ushering in an era of health, prosperity, and sustainability.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*Genome, Plant
*Plants/genetics
*Artificial Intelligence
Plant Breeding

@article {pmid40316115,
year = {2025},
author = {Tao, Z and Tian, C and Zhong, C and Ji, B and Li, W and Zhao, Y},
title = {The role of NhaA protein in modulating antibiotic tolerance in Escherichia coli.},
journal = {International journal of biological macromolecules},
volume = {311},
number = {Pt 2},
pages = {143721},
doi = {10.1016/j.ijbiomac.2025.143721},
pmid = {40316115},
issn = {1879-0003},
mesh = {*Escherichia coli/drug effects/genetics/metabolism ; *Anti-Bacterial Agents/pharmacology ; *Escherichia coli Proteins/genetics/metabolism ; *Drug Resistance, Bacterial/genetics ; Adenosine Triphosphate/metabolism ; Proton-Motive Force/drug effects ; Cell Membrane Permeability/drug effects ; Membrane Fluidity/drug effects ; Microbial Sensitivity Tests ; CRISPR-Cas Systems ; },
abstract = {As microbial resistance and recurrent bacterial infections escalate, the growing understanding of the interplay between antibiotic resistance and tolerance has sparked significant interest in the latter. Previous studies have demonstrated that the deletion of cation/proton antiporters (CPAs) induces bacterial phenotypes, such as slow growth and prolonged lag phases, which contribute to the development of tolerance. This study investigates the role of the NhaA protein in antibiotic tolerance in Escherichia coli using CRISPR/Cas9 gene editing to delete the NhaA protein. Our results suggest that the NhaA protein plays a key role in modulating antibiotic tolerance. In response to NhaA deletion, E. coli adapts through multiple mechanisms, including changes in membrane permeability, enhanced efflux activity, increased membrane fluidity, disruption of the proton motive force (PMF), and a reduction in intracellular ATP levels. These adaptive changes collectively promote the development of antibiotic tolerance. Understanding these tolerance mechanisms could uncover new therapeutic targets, help prevent the emergence of tolerance, or sustain bacteria cells in a tolerant state, providing crucial strategies to combat the rise of antibiotic-resistant bacteria.},
}

*Escherichia coli/drug effects/genetics/metabolism
*Anti-Bacterial Agents/pharmacology
*Escherichia coli Proteins/genetics/metabolism
*Drug Resistance, Bacterial/genetics
Adenosine Triphosphate/metabolism
Proton-Motive Force/drug effects
Cell Membrane Permeability/drug effects
Membrane Fluidity/drug effects
Microbial Sensitivity Tests
CRISPR-Cas Systems

@article {pmid40158793,
year = {2025},
author = {Sheng, T and Meng, XZ and Yu, Q and Lv, W and Chen, Y and Cong, Q and Li, W and Gui, L and Li, J and Xu, X},
title = {Transcriptome analysis and CRISPR-Cas9-mediated mutagenesis identify gpr116 as a candidate gene for growth promotion in grass carp (Ctenopharyngodon idella).},
journal = {Comparative biochemistry and physiology. Part A, Molecular & integrative physiology},
volume = {305},
number = {},
pages = {111850},
doi = {10.1016/j.cbpa.2025.111850},
pmid = {40158793},
issn = {1531-4332},
mesh = {Animals ; *Carps/genetics/growth & development ; *CRISPR-Cas Systems ; Zebrafish/genetics/growth & development ; *Receptors, G-Protein-Coupled/genetics/metabolism ; Gene Expression Profiling ; *Fish Proteins/genetics/metabolism ; *Transcriptome ; Mutagenesis ; Brain/metabolism ; },
abstract = {Grass carp (Ctenopharyngodon idella) is an economically important aquaculture species known for its considerable variability in growth performance. In this study, we investigated the growth phenotype by comparing fast-growing and slow-growing groups. Microstructural analyses revealed that slow-growing fish exhibited significantly larger myofibrillar gaps and lower muscle fiber density. To elucidate the underlying molecular basis, we performed transcriptome (RNA-Seq) analysis of brain and dorsal muscle tissues. 328 differentially expressed genes (DEGs) were identified in dorsal muscle tissue (33 up and 295 down-regulated) and 228 in brain tissue (17 up and 211 down-regulated). Gene Ontology and KEGG enrichment analyses indicated that the DEGs were closely associated with apoptosis and angiogenesis pathways. Among the candidate genes, gpr116 was significantly up-regulated in the brain and dorsal muscle tissue of the fast-growing group. Finally, CRISPR-Cas9-mediated knockout in a zebrafish model confirmed that gpr116 deletion significantly restricted growth, underscoring its pivotal role in the growth regulation of grass carp. These discoveries lay significant groundwork for deeper exploration of growth regulation mechanisms in grass carp and offer important clues for selective breeding of key growth marker genes in this species.},
}

Animals
*Carps/genetics/growth & development
*CRISPR-Cas Systems
Zebrafish/genetics/growth & development
*Receptors, G-Protein-Coupled/genetics/metabolism
Gene Expression Profiling
*Fish Proteins/genetics/metabolism
*Transcriptome
Mutagenesis
Brain/metabolism

@article {pmid40147495,
year = {2025},
author = {Panting, M and Holme, IB and Dionisio, G and Brinch-Pedersen, H},
title = {Simplex and multiplex CRISPR/Cas9-mediated knockout of grain protease inhibitors in model and commercial barley improves hydrolysis of barley and soy storage proteins.},
journal = {Plant biotechnology journal},
volume = {23},
number = {6},
pages = {2418-2428},
doi = {10.1111/pbi.70065},
pmid = {40147495},
issn = {1467-7652},
support = {8055-00038B//Innovationsfonden/ ; NNF19OC005658//Novo Nordisk Fonden/ ; 101060057//Horizon Europe/ ; },
mesh = {*Hordeum/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Protease Inhibitors/metabolism ; *Soybean Proteins/metabolism ; *Seed Storage Proteins/metabolism ; *Plant Proteins/metabolism/genetics ; Hydrolysis ; Edible Grain/genetics/metabolism ; Seeds/metabolism/genetics ; Gene Knockout Techniques ; Glycine max/metabolism ; Proteolysis ; },
abstract = {Anti-nutritional factors in plant seeds diminish the utilization of nutrients in feed and food. Among these, protease inhibitors inhibit protein degradation by exogenous proteases during digestion. Through conventional and selection-gene-free genome editing using ovules as explants, we used simplex and multiplex CRISPR/Cas9 for studying the impact of chymotrypsin inhibitor CI-1A, CI-1B and CI-2, Bowman-Birk trypsin inhibitor, Serpin-Z4, and barley ɑ-amylase/subtilisin inhibitor on barley and soybean storage protein degradation. Mutants were generated in the commercial cultivar Stairway, having a high level of protease inhibition, and the barley model cultivar Golden Promise, having a lower inhibition level. In Golden Promise, all individual knockouts decreased the inhibition of the three proteases α-chymotrypsin, trypsin and the commercial feed protease Ronozyme ProAct significantly. The triple knockout of all chymotrypsin inhibitors further decreased the inhibition of α-chymotrypsin and Ronozyme ProAct proteases. Degradations of recombinant barley storage proteins B- and C-hordeins were significantly improved following mutagenesis. In Stairway, a single knockout of CI-1A almost compares to the effect on the proteases achieved for the triple knockout in Golden promise, uncovering CI-1A as the major protease inhibitor in that cultivar. The Stairway mutant demonstrated significantly improved degradation of recombinant barley hordeins and in the soybean storage proteins glycinin and β-conglycinin. The results of this study provide insights into cereal protease inhibitor genes and their negative effects on the degradation of barley storage protein and the most important plant protein from soybeans. The study suggests a future focus on plant protease inhibitors as a major target for improving feed and food protein digestibility.},
}

*Hordeum/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Protease Inhibitors/metabolism
*Soybean Proteins/metabolism
*Seed Storage Proteins/metabolism
*Plant Proteins/metabolism/genetics
Hydrolysis
Edible Grain/genetics/metabolism
Seeds/metabolism/genetics
Gene Knockout Techniques
Glycine max/metabolism
Proteolysis

@article {pmid40139718,
year = {2025},
author = {Tian, M and Luo, L and Jin, B and Liu, J and Chen, T and Tang, J and Shen, Y and Zhang, H and Guo, J and Zhang, H and Cui, G and Huang, L},
title = {Highly efficient Agrobacterium rhizogenes-mediated gene editing system in Salvia miltiorrhiza inbred line bh2-7.},
journal = {Plant biotechnology journal},
volume = {23},
number = {6},
pages = {2406-2417},
doi = {10.1111/pbi.70029},
pmid = {40139718},
issn = {1467-7652},
support = {CI2021A04109//CACMS Innovation Fund/ ; ZZ14-YQ-049//The Fundamental Research Funds for the Central public welfare research institutes/ ; 2023YFC3503900//National Key Research and Development Program of China/ ; 82274054//National Natural Science Foundation of China/ ; 2060302//Key project at central government level: the ability to establish sustainable use of valuable Chinese Medicine Resources/ ; TSQN202103160//Supported by the Taishan Scholars Program/ ; },
mesh = {*Agrobacterium/genetics ; *Gene Editing/methods ; *Salvia miltiorrhiza/genetics ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Genome, Plant/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The CRISPR/Cas9 system is a powerful tool for genomic editing with significant potential for gene function validation and molecular breeding in medicinal plants. Salvia miltiorrhiza, a model medicinal plant, was among the pioneers to utilize CRISPR/Cas9 technology, though achieving high-efficiency homozygous knockout mutants has been challenging. In this study, the analysis of variations at 241 single-guide RNA (sgRNA) across different reference genomes and experimental materials was conducted first, leading to the identification of the six-generation inbred line bh2-7 as the most suitable reference genome and experimental material for gene editing research in S. miltiorrhiza. Next, five Agrobacterium rhizogenes strains were evaluated for hairy root induction, editing efficiency, and mutation patterns, with C58C1 and K599 emerging as the most effective delivery systems. Using the CRISPR/Cas9 vector pZKD672, 53 target sites were successfully edited, with K599 achieving 71.07% editing efficiency and 36.74% homozygous or biallelic (HOM) efficiency and C58C1 showing 62.27% editing efficiency and 23.61% HOM efficiency. We thus constructed a large-scale mutant library targeting 121 genes with 170 sgRNAs, yielding 1664 homozygous or biallelic mutants. Analysis of 65 low-efficiency target sites revealed that sgRNA mismatches and secondary structures were key factors reducing HOM efficiency, offering insights for future target design. This study establishes an efficient CRISPR/Cas9 system, advancing precision breeding and metabolic engineering research in medicinal plants.},
}

*Agrobacterium/genetics
*Gene Editing/methods
*Salvia miltiorrhiza/genetics
*CRISPR-Cas Systems/genetics
Plants, Genetically Modified/genetics
Genome, Plant/genetics
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40112038,
year = {2025},
author = {Xu, T and Li, Y and Liu, X and Yang, X and Huang, Z and Xing, J and Liang, C and Li, J and Tan, Y and Zhang, S and Qi, J and Ye, D and Li, Z and Cao, J and Tang, C and Liu, K},
title = {Rubber biosynthesis drives the biogenesis and development of rubber particles, the rubber-producing organelles.},
journal = {Plant biotechnology journal},
volume = {23},
number = {6},
pages = {2303-2316},
doi = {10.1111/pbi.70052},
pmid = {40112038},
issn = {1467-7652},
support = {32201450//National Natural Science Foundation of China/ ; 32160383//National Natural Science Foundation of China/ ; 31825007//National Natural Science Foundation of China/ ; 32101543//National Natural Science Foundation of China/ ; },
mesh = {*Latex/metabolism/biosynthesis ; *Rubber/metabolism ; *Taraxacum/metabolism/genetics ; Plant Proteins/metabolism/genetics ; *Organelles/metabolism ; Transferases/metabolism/genetics ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; },
abstract = {Rubber particles (RPs) are specialized organelles for the biosynthesis and storage of natural rubber in rubber-producing plants. However, the mechanisms underlying the biogenesis and development of RPs remain unclear. In this study, two latex-specific cis-prenyltransferases (CPTs), TkCPT1 and TkCPT2, were identified in Taraxacum kok-saghyz, with almost identical orthologues retained across other Taraxacum species. For the first time, Tkcpt1 single and Tkcpt1/2 double mutants were successfully generated using the CRISPR/Cas9 system. Rubber biosynthesis was significantly depressed in Tkcpt1 mutants and completely blocked in Tkcpt1/2 mutants. The absence of RPs in the Tkcpt1/2 was confirmed using oil red O and Nile red staining, high-speed centrifugal stratification, cryo-SEM and TEM on fresh latex or laticifer cells. Transcriptomic and proteomic analyses revealed that, in the latex of Tkcpt1/2, rubber biosynthesis was blocked at the protein level, while metabolomic profiling indicated an enrichment of lipids and terpenoids. Furthermore, knockout of TkCPTL1, a latex-specific CPT-like gene that encodes a rubber transferase activator, resulted in outright disruption of rubber biosynthesis and RP ontogeny, a phenotype similar to that of Tkcpt1/2 mutants. These findings indicate that rubber biosynthesis is a driving force for the biogenesis and development of RPs, providing new insights into rubber production mechanisms.},
}

*Latex/metabolism/biosynthesis
*Rubber/metabolism
*Taraxacum/metabolism/genetics
Plant Proteins/metabolism/genetics
*Organelles/metabolism
Transferases/metabolism/genetics
Gene Expression Regulation, Plant
CRISPR-Cas Systems

@article {pmid40029794,
year = {2025},
author = {Livneh, Y and Leor-Librach, E and Agmon, D and Makov-Bouaniche, T and Tiwari, V and Shor, E and Yeselson, Y and Masci, T and Schaffer, A and Charuvi, D and Hirschberg, J and Vainstein, A},
title = {Combined enhancement of ascorbic acid, β-carotene and zeaxanthin in gene-edited lettuce.},
journal = {Plant biotechnology journal},
volume = {23},
number = {6},
pages = {1954-1967},
doi = {10.1111/pbi.70018},
pmid = {40029794},
issn = {1467-7652},
support = {20-01-0209//The National Center for Genome Editing in Agriculture, Israel/ ; 7500158//Ministry of Science and Technology, Israel/ ; 12-01-0037//Ministry of Agriculture and Rural Development/ ; },
mesh = {*Zeaxanthins/metabolism ; *beta Carotene/metabolism ; *Lactuca/genetics/metabolism ; *Ascorbic Acid/metabolism ; *Gene Editing ; Intramolecular Lyases/genetics/metabolism ; CRISPR-Cas Systems ; Plants, Genetically Modified ; },
abstract = {Lettuce is widely grown and consumed but provides lower nutritional value compared to other leafy greens, particularly in the essential vitamins A and C. To address this, major control points in carotenoid and ascorbic acid (AsA) production were targeted using a viral-based CRISPR/Cas9 system in the commercial lettuce cultivar 'Noga'. Knockout of lycopene ε-cyclase (LCY-ε), the enzymatic gatekeeper opposing production of β-branch carotenoids, increased β-carotene (provitamin A) levels up to 2.7-fold and facilitated zeaxanthin accumulation up to 4.3 μg/g fresh weight. Chlorophyll fluorescence measurements revealed that photosystem II efficiency was unaffected in LCY-ε mutants, though their non-photochemical quenching (NPQ) capacity decreased at light intensities above 400 μmol m[2] s[-1]. However, the gene-edited plants exhibited normal growth and comparable plant mass, despite the absence of two major lettuce xanthophylls, lutein and lactucaxanthin. Modifications in a regulatory region in the upstream ORF of GDP-L-galactose phosphorylase 1 and 2 (uGGP1 and uGGP2), the rate-limiting enzyme in AsA production, resulted in an average 6.9-fold increase in AsA levels. The mutation in uGGP2 was found to dominantly influence AsA over-accumulation. Knockout lines that combined the mutations in LCY-ε, uGGP1, uGGP2 and in carotenoid cleavage dioxygenase 4a (CCD4a), an isozyme involved in β-carotene degradation in lettuce, exhibited significantly enhanced content of AsA, β-carotene and zeaxanthin. Our results demonstrate the potential of multi-pathway gene editing to 'supercharge' economically important crops such as lettuce as a means to address micronutrient deficiencies in modern diets.},
}

*Zeaxanthins/metabolism
*beta Carotene/metabolism
*Lactuca/genetics/metabolism
*Ascorbic Acid/metabolism
*Gene Editing
Intramolecular Lyases/genetics/metabolism
CRISPR-Cas Systems
Plants, Genetically Modified

@article {pmid39779901,
year = {2025},
author = {Son, HI and Hamrick, GS and Shende, AR and Kim, K and Yang, K and Huang, TJ and You, L},
title = {Population-level amplification of gene regulation by programmable gene transfer.},
journal = {Nature chemical biology},
volume = {21},
number = {6},
pages = {939-948},
pmid = {39779901},
issn = {1552-4469},
support = {R01EB031869//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Plasmids/genetics ; *Gene Expression Regulation, Bacterial ; *Gene Transfer Techniques ; *Escherichia coli/genetics ; CRISPR-Cas Systems ; },
abstract = {Engineering cells to sense and respond to environmental cues often focuses on maximizing gene regulation at the single-cell level. Inspired by population-level control mechanisms like the immune response, we demonstrate dynamic control and amplification of gene regulation in bacterial populations using programmable plasmid-mediated gene transfer. By regulating plasmid loss rate, transfer rate and fitness effects via Cas9 endonuclease, F conjugation machinery and antibiotic selection, we modulate the fraction of plasmid-carrying cells, serving as an amplification factor for single-cell-level regulation. This approach expands the dynamic range of gene expression and allows orthogonal control across populations. Our platform offers a versatile strategy for dynamically regulating gene expression in engineered microbial communities.},
}

Plasmids/genetics
*Gene Expression Regulation, Bacterial
*Gene Transfer Techniques
*Escherichia coli/genetics
CRISPR-Cas Systems

@article {pmid40412199,
year = {2025},
author = {Shen, Y and Tang, X and Wang, J and Dai, H and Cui, Y and Hu, Q and Wu, Y and Jia, F and Hao, G},
title = {Dual nuclease-amplified sensitive biosensor for enrofloxacin detection using a DNase I-assisted CRISPR/Cas12a (CRISPR-DNase I) system.},
journal = {Talanta},
volume = {295},
number = {},
pages = {128367},
doi = {10.1016/j.talanta.2025.128367},
pmid = {40412199},
issn = {1873-3573},
abstract = {Recent years have witnessed the flourishing of CRISPR/Cas-based biosensors in various fields. However, most of them were developed for nucleic acid detection because non-nucleic acid targets are unable to unleash the cleavage activity of the CRISPR/Cas system directly. To circumvent this problem, activator DNA and deoxyribonuclease I (DNase I) were introduced in this research to render the CRISPR/Cas12a system as a new powerful tool for the detection of enrofloxacin (ENR), a common veterinary drug. In this biosensor, target ENR competed with DNase I- and bovine serum albumin-ENR composite-modified gold nanoparticles (DNase I-AuNPs-BSA-ENR) for the binding sites on the surface of antibody-modified magnetic nanoparticles (immuno-MNPs). Then, the captured DNase I-AuNPs-BSA-ENR degraded the activator DNA in the solution, which inhibited the activation of the CRISPR/Cas12a system. Finally, the fluorescence released by the activated CRISPR/Cas12a system was measured for the quantitative detection of ENR. The ingenious use of activator DNA and DNase I helped transduce the target recognition event into the cleavage activity of the CRISPR/Cas12a system. Moreover, the dual enzymatic amplification from DNase I and the CRISPR/Cas12a system guaranteed the sensitivity of this method with a low detection limit of 0.04 ng/mL. The developed biosensor extended the application of the CRISPR/Cas12a system for the sensitive detection of non-nucleic acid targets, providing a powerful tool in various fields such as environmental monitoring, food safety and clinical diagnosis.},
}

@article {pmid40411669,
year = {2025},
author = {Hassan, HM and Zubair, A and Helal, MH and Almagharbeh, WT and Elmagzoub, RM},
title = {New hope and promise with CRISPR-Cas9 technology for the treatment of HIV.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {108},
pmid = {40411669},
issn = {1438-7948},
mesh = {*CRISPR-Cas Systems ; Humans ; *HIV Infections/therapy/genetics/virology ; *Gene Editing/methods ; *HIV-1/genetics ; Genetic Therapy/methods ; },
abstract = {The commencement of Highly Active Antiretroviral Therapy almost completely stopped viral replication, enabling the immune system to restore its full functionality. The rise in life expectancy has resulted in a decrease in the incidence of classical infections and HIV-associated cancers. HAART has raised concerns, including its exorbitant cost (which hinders its implementation in developing nations), the need for strict adherence, and the potential for both immediate and prolonged ill effects. Lipodystrophy is a significant long-term consequence of HIV that may result in central fat accumulation and severe peripheral fat depletion. Current initiatives to tackle these difficulties include the global expansion of access to HAART, the development of novel drugs that mitigate early side effects, and the introduction of once-daily drug combinations that enhance adherence. The CRISPR-Cas9 system has facilitated the creation of a powerful instrument for precise gene editing. This method has lately established itself as the gold standard for efficient HIV-1 genome editing in HIV therapy, owing to progress in related disciplines. CRISPR may be customized to cleave specific sequences by altering Cas9. This article offers a concise overview of promising CRISPR-Cas9 technology. This technique has the potential to halt the transmission of HIV-1 and alleviate its symptoms. CRISPR-Cas9 technology will be significant in the fight against HIV-1 in the future.},
}

*CRISPR-Cas Systems
Humans
*HIV Infections/therapy/genetics/virology
*Gene Editing/methods
*HIV-1/genetics
Genetic Therapy/methods

@article {pmid40410287,
year = {2025},
author = {Wei, J and Shao, Y and Liang, Y and Bu, X and Zhou, W and Liang, Y and Li, Y},
title = {A type III-associated Cas6 functions as a negative regulator of type I-B CRISPR-Cas system in Thermus thermophilus.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {793},
pmid = {40410287},
issn = {2399-3642},
support = {32170096//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Thermus thermophilus/genetics/metabolism/enzymology ; *CRISPR-Cas Systems ; *Bacterial Proteins/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; RNA, Bacterial/metabolism/genetics ; Gene Expression Regulation, Bacterial ; },
abstract = {CRISPR-Cas systems are small RNA-guided immune systems in prokaryotes. CRISPR RNA (crRNA) provides sequence specificity and programmability, guiding the effector complex to cleave target nucleic acids. Cas6 family ribonucleases can cleave precursor crRNA to generate functional crRNAs in most type I and type III CRISPR-Cas systems. Most existing studies of Cas6 functions are mainly focused on nuclease activity in vitro and Cas6-processed product characterization in vivo. However, in hosts harboring multiple CRISPR systems, the biological functions of the co-occurrence of various Cas6 proteins and their cross-cleavage activity toward different types of crRNAs remain largely unexplored. In this study, we biochemically characterized the cross-cleavage activity of two Cas6 proteins in Thermus thermophilus HB27 and first found that Cas6 could anchor the mature crRNA and interact with Cas5 subunit of type I-B system, revealing the functions of Cas6 to mediate the assembly of type I Cascade complex. We further demonstrated that the type III-associated Cas6 protein could act as a negative regulator by competing with the I-B Cas6 protein during the assembly of type I-B Cascade complex, significantly suppressing the interference activity of type I-B system. Our findings provide an insight into the functional coupling and regulation mechanisms underlying multiple CRISPR-Cas systems.},
}

*Thermus thermophilus/genetics/metabolism/enzymology
*CRISPR-Cas Systems
*Bacterial Proteins/metabolism/genetics
*CRISPR-Associated Proteins/metabolism/genetics
RNA, Bacterial/metabolism/genetics
Gene Expression Regulation, Bacterial

@article {pmid40409866,
year = {2025},
author = {Wang, L and Wu, Y and Pan, S and Pan, F and Chen, J},
title = {Rapid and sensitive detection of Karlodinium veneficum using RAA and CRISPR-Cas12a technologies.},
journal = {Harmful algae},
volume = {146},
number = {},
pages = {102864},
doi = {10.1016/j.hal.2025.102864},
pmid = {40409866},
issn = {1878-1470},
mesh = {*Dinoflagellida/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Harmful Algal Bloom ; },
abstract = {The harmful algal species Karlodinium veneficum (K. veneficum) poses a significant threat to aquatic ecosystems, economic stability, and human health due to its toxin production and widespread occurrence. Rapid climatic changes and eutrophication have intensified harmful algal blooms (HABs), making the timely detection of K. veneficum critical. To address this need, we developed a rapid and accurate detection method of K. veneficum by combining Recombinase Aided Amplification (RAA) with CRISPR/LbCas12a. This method targets the internal transcribed spacer (ITS) sequence of K. veneficum and utilizes the "collateral activity" of CRISPR/LbCas12a for visualization. Our method can detect plasmid DNA as low as 5.9 × 10[2] copies/µL and genomic DNA as low as 3.6 × 10[-2] ng/µL, achieving a detection limit of 10 cells of K. veneficum through a simplified DNA extraction process. The entire detection process, from DNA crude extract to result visualization, can be completed in as fast as 90 min, making it suitable for field applications requiring a rapid response. In addition, our method was validated against a wide range of non-target microalgae species, confirming its specificity to K. veneficum and eliminating the risk of cross-reactivity. Overall, the RAA-CRISPR/LbCas12a system is simple, accurate, and sensitive, showing great potential for field applications in monitoring K. veneficum.},
}

*Dinoflagellida/genetics/isolation & purification
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
Harmful Algal Bloom

@article {pmid40409549,
year = {2025},
author = {Okada, R and Sakaguchi, R and Komaki, T and Nonaka, R and Polat, OK and Kihara, T and Asanuma, K and Yamamoto, T and Isaka, Y and Mori, Y and Mori, MX},
title = {Delayed inactivation of TRPC6 as a determinative characteristic of FSGS-associated variants.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {110256},
doi = {10.1016/j.jbc.2025.110256},
pmid = {40409549},
issn = {1083-351X},
abstract = {Transient receptor potential canonical 6 (TRPC6) is a receptor-operated nonspecific cation channel. To date, more than 30 TRPC6 variants have been reported to focal segmental glomerulosclerosis (FSGS), which can present from infancy to adulthood and is characterized by proteinuria and often nephrotic syndrome leading to kidney failure. These variants may exhibit gain-of-function (e.g. K874X) or loss-of-function (e.g. L395A, G757D) phenotypes, making the role of TRPC6 in FSGS controversial. Here, we characterized Ca[2+]-dependent inactivation (CDI) of TRPC6 after the receptor activation and found that >85% of TRPC6 variants exhibit delayed CDI. Thus, prolonged TRPC6 channel opening due to impaired inactivation may be a common feature of FSGS-associated variants. This effect was confirmed in immortalized mouse podocytes (MPC-5) in which the coiled-coil (CC) domain was deleted from the channel (C6ΔCC). Podocytes expressing C6ΔCC exhibited delayed CDI and increased basal Ca[2+] levels as well as disruption of the F-actin cytoskeleton. Moreover, transcriptomic data from C6ΔCC-expressing podocytes showed weak expression of the podocyte markers Synpo and Magi2. These results indicate that CDI of TRPC6 is critical for maintaining proper podocyte function. Notably, we observed a correlation between the magnitude of the prolongation of TRPC6 channel activity and the age diagnosed with FSGS. Our findings thus demonstrate that delayed inactivation due to lack of CDI is a determinative characteristic of FSGS-associated TRPC6 variants, affecting both the structure and function of glomerular podocytes.},
}

@article {pmid40409480,
year = {2025},
author = {Yi, B and Zhou, B and Zhou, D and Yang, L and Xu, H},
title = {CRISPR/Cas-powered nucleic acid amplification and amplification-free biosensors for public safety detection: Principles, advances and prospects.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108609},
doi = {10.1016/j.biotechadv.2025.108609},
pmid = {40409480},
issn = {1873-1899},
abstract = {Rapid, accurate, cost-effective, and efficient ultrasensitive detection strategies are essential for public health safety (including food safety, disease prevention and environmental governance). The CRISPR/CRISPR-associated (Cas) detection is a cutting-edge technology that has been widely used in the detection of public health safety due to its targeted cleavage properties (signal amplification), attomolar level sensitivity, high specificity (recognizing single-base mismatches), and rapid turnover time. However, the current research about CRISPR/Cas-based biosensors is not clear, such as mechanism problem and application differences of integrating CRISPR/Cas system with other technologies, and how to further innovate and develop in the future. Therefore, further detailed analysis and comparative discussion of CRISPR/Cas-based biosensors is needed. Currently, CRISPR/Cas system powered biosensors can be mainly categorized into two types: CRISPR/Cas system powered nucleic acid amplification biosensors and CRISPR/Cas system powered nucleic acid amplification-free biosensors. The two biosensors have different characteristics and advantages. This paper first provides an in-depth investigation of the enzymatic mechanism of CRISPR/Cas system at the molecular level. Then, this paper summarizes the principles and recent advances of CRISPR/Cas system powered nucleic acid amplification biosensors and CRISPR/Cas system powered nucleic acid amplification-free biosensors and discusses their integration mechanisms in depth. More, the differences and application-oriented between the two biosensors are further discussed. Finally, the application orientation and future perspectives of the two biosensors are discussed, and unique insights into the future development of CRISPR/Cas system are provided.},
}

@article {pmid40408893,
year = {2025},
author = {Kong, H and Yi, K and Zhu, X and Chen, L and Wang, H and Ju, E and Lv, S and Lao, YH and Shao, D and Xie, X and Cheng, D and Zhang, Y and Tao, Y and Li, M},
title = {Antifouling fusion-mediated diagnostic platform to detect viral DNA-positive extracellular vesicles for in situ blood-based liquid biopsy.},
journal = {Biosensors & bioelectronics},
volume = {286},
number = {},
pages = {117568},
doi = {10.1016/j.bios.2025.117568},
pmid = {40408893},
issn = {1873-4235},
abstract = {In liquid biopsy, extracellular vesicles (EVs) have emerged as promising biomarkers due to their ability to carry protected nucleic acids. In particular, DNA enclosed within these vesicles shows great diagnostic potential for monitoring oncovirus-related disease progression. However, current methods still require labor-intensive procedures and bulk analysis. Additionally, in situ detection from blood is hindered by abundant serum proteins, interfering with the accuracy of diagnosis. To address these limitations, we developed an antifouling fusion-mediated CRISPR/Cas detector (AFFECTOR) as a user-friendly and efficient diagnostic platform for directly detecting EV-contained viral DNA in serum samples. Leveraging zwitterionic phosphatidylcholine to resist protein interference, the platform enables stable membrane fusion with intact EVs even in serum-containing environments, allowing highly specific and sensitive detection of internal DNA via the CRISPR/Cas12a sensing system, lasting just 2 h at 37 °C. In clinical samples from oncovirus-infected patients and healthy donors, the platform achieved one-step detection of viral DNA-positive EVs. Notably, viral DNA in circulating EVs was found for the first time to correlate with oncovirus infection stages. Overall, this platform provides a practical tool for diagnostic applications and expands the detection window in liquid biopsy.},
}

@article {pmid40405306,
year = {2025},
author = {Marei, HE},
title = {Stem cell therapy: a revolutionary cure or a pandora's box.},
journal = {Stem cell research & therapy},
volume = {16},
number = {1},
pages = {255},
pmid = {40405306},
issn = {1757-6512},
mesh = {Humans ; *Stem Cell Transplantation/ethics ; *Stem Cell Research/ethics/legislation & jurisprudence ; Gene Editing ; *Cell- and Tissue-Based Therapy/ethics ; Embryonic Stem Cells ; Animals ; CRISPR-Cas Systems ; },
abstract = {This review article examines how stem cell therapies can cure various diseases and injuries while also discussing the difficulties and moral conundrums that come with their application. The article focuses on the revolutionary developments in stem cell research, especially the introduction of gene editing tools like CRISPR-Cas9, which can potentially improve the safety and effectiveness of stem cell-based treatments. To guarantee the responsible use of stem cells in clinical applications, it is also argued that standardizing clinical procedures and fortifying ethical and regulatory frameworks are essential first steps. The assessment also highlights the substantial obstacles that still need to be addressed, such as the moral dilemmas raised by the use of embryonic stem cells, the dangers of unlicensed stem cell clinics, and the difficulties in obtaining and paying for care for patients. The study emphasizes how critical it is to address these problems to stop exploitation, guarantee patient safety, and increase the accessibility of stem cell therapy. The review also addresses the significance of thorough clinical trials, public education, and policy development to guarantee that stem cell research may fulfill its full potential. The review concludes by describing stem cell research as a promising but complicated topic that necessitates a thorough evaluation of both the hazards and the benefits. To overcome the ethical, legal, and accessibility obstacles and eventually guarantee that stem cell treatments may be safely and fairly included in conventional healthcare, it urges cooperation between the scientific community, legislators, and the general public.},
}

Humans
*Stem Cell Transplantation/ethics
*Stem Cell Research/ethics/legislation & jurisprudence
Gene Editing
*Cell- and Tissue-Based Therapy/ethics
Embryonic Stem Cells
Animals
CRISPR-Cas Systems

@article {pmid40404851,
year = {2025},
author = {Syed, M and Khan, RS and Nazir, S and Khan, S and Ul Islam, Z and Khan, S and Ikuo, N},
title = {Silencing of disease susceptibility genes: an effective disease resistance strategy against fungal pathogens.},
journal = {Plant cell reports},
volume = {44},
number = {6},
pages = {127},
pmid = {40404851},
issn = {1432-203X},
mesh = {*Plant Diseases/microbiology/genetics/immunology ; *Disease Resistance/genetics ; *Gene Silencing ; *Fungi/pathogenicity/physiology ; RNA Interference ; Host-Pathogen Interactions/genetics ; Genes, Plant ; CRISPR-Cas Systems ; },
abstract = {Silencing of target susceptibility (S) genes in plants exhibits a promising and durable strategy for enhanced resistance to fungal pathogens by causing disruption in the host mechanisms that the pathogens exploit, offering an alternative to the traditional resistance gene-based approaches. Devastating fungal diseases have significantly reduced crop productivity, posing a potential threat to global food security. Producing disease-resistant cultivars is the most effective strategy for protecting crops against these fungal pathogens. Typically, susceptibility (S) genes in host plants facilitate the penetration and proliferation of phytopathogens. Perturbation of these S genes can potentially impede the compatibility between the host and the fungal pathogens, thereby providing broad-spectrum and lasting resistance. Consequently, the identification and targeting of S-genes have gained increasing interest in enhancing disease resistance in plants. In this review, we describe three distinct categories of S genes that function during different stages of the infection process. We focus on various gene silencing technologies, including RNA interference (RNAi), virus-induced gene silencing (VIGS), and CRISPR-Cas9, to improve plant disease resistance against fungal pathogens. The numerous examples discussed here illustrate the potential of S-genes for use in plant disease-resistance breeding.},
}

*Plant Diseases/microbiology/genetics/immunology
*Disease Resistance/genetics
*Gene Silencing
*Fungi/pathogenicity/physiology
RNA Interference
Host-Pathogen Interactions/genetics
Genes, Plant
CRISPR-Cas Systems

@article {pmid40403646,
year = {2025},
author = {Wu, Y and Kilgore, R and Hetzler, Z and Chu, W and Moore, B and Barbieri, E and Wei, Q and Menegatti, S},
title = {Purification of CRISPR Cas12a from E. coli cell lysates using peptide affinity ligands.},
journal = {Journal of chromatography. A},
volume = {1755},
number = {},
pages = {466065},
doi = {10.1016/j.chroma.2025.466065},
pmid = {40403646},
issn = {1873-3778},
abstract = {CRISPR Cas nucleases are revolutionizing gene therapy by providing a precise and efficient tool for editing the human genome, and are increasingly applied for engineering microorganisms for bioremediation, drought-resistant crops, and livestock with higher productivity. While Cas9 is currently the most widely utilized member of the CRISPR family, Cas12a stands is gaining prominence for its ability to produce staggered cuts in the target DNA while requiring a shorter guide RNA (crRNA). Current methods of Cas purification such as affinity tag, immunoaffinity, and ion exchange chromatography do not provide either the productivity or the purity needed to meet the demand of clinics and biotechnology industries. Responding to this need, this study presents the first affinity ligands for Cas12a purification via affinity chromatography. The ligands were initially designed in silico as peptide mimetics of anti-CRISPR protein inhibitors AcrVA1 and AcrVA4, and ranked experimentally by Cas12a dynamic binding. Selected ligands P5 and P9 were utilized for purifying Cas12a derived from Acidaminococcus sp. (AsCas12a) and Lachnospiraceae sp. (LbCas12a) from clarified Escherichia coli cell lysates. P5-functionalized resin afforded high yield (up to 80 %), purity (> 93 %), and DNA editing activity (∼72 %) of Cas12a from E. coli lysates featuring different Cas12a and host cell protein titers. The characterization of ligand P5 by surface plasmon resonance (SPR) indicated adsorption kinetics (ka ∼ 1.21·10[5] M[-1]s[-1]) and dissociation constant (KD ∼ 1.76·10[-6] M) that confirmed the ligand design criteria and are characteristic of peptide affinity ligands.},
}

@article {pmid40403178,
year = {2025},
author = {Lee, Y and Lee, JH and Lee, T and Shin, M and Yoon, J},
title = {Amplification-Free CRISPR/Cas12a-Based Electrochemical Biosensor with Enhanced Sensitivity for Viral Detection.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.5c00576},
pmid = {40403178},
issn = {2379-3694},
abstract = {To detect contagious viral nucleic acids, traditional biosensors often require target amplification steps or use fluorescence and Raman probes tagged on nucleic acids, which are time-consuming, complex, and expensive. Recently, the CRISPR/Cas12a has received the attraction for development of nucleic acid biosensors, beyond its conventional role-like gene editing, but the enhancement of the sensitivity of CRISPR/Cas-based biosensors is still required to simplify the biosensing steps. Here, we develop a CRISPR/Cas12a-based electrochemical biosensor for the detection of viral nucleic acids in a simple manner. The novel mismatch Ag probe (MAP), as a sensing probe, and the highly conductive gold electrode on indium tin oxide with a nano array (GELITION) are introduced that enable the amplification-free and ultrasensitive detection of nucleic acids using a CRISPR/Cas12a system. The biosensing ability of the developed biosensor is validated using human papillomavirus type 16 and 18 viral DNAs (HPV16 and HPV18), achieving a limit of detection (LOD) of 1 fM without amplification and complex steps. Our developed biosensor is expected to be applicable in detecting various viruses and could contribute to the early detection of future pandemics.},
}

@article {pmid40402294,
year = {2025},
author = {Hassane, AMA and Obiedallah, M and Karimi, J and Khattab, SMR and Hussein, HR and Abo-Dahab, Y and Eltoukhy, A and Abo-Dahab, NF and Abouelela, ME},
title = {Unravelling fungal genome editing revolution: pathological and biotechnological application aspects.},
journal = {Archives of microbiology},
volume = {207},
number = {7},
pages = {150},
pmid = {40402294},
issn = {1432-072X},
mesh = {*Gene Editing/methods ; *Genome, Fungal ; CRISPR-Cas Systems ; *Biotechnology/methods ; *Fungi/genetics/pathogenicity ; Humans ; Candida albicans/genetics/pathogenicity ; Aspergillus fumigatus/genetics/pathogenicity ; },
abstract = {Fungi represent a broad and evolutionarily unique group within the eukaryotic domain, characterized by extensive ecological adaptability and metabolic versatility. Their inherent biological intricacy is evident in the diverse and dynamic relationships they establish with various hosts and environmental niches. Notably, fungi are integral to disease processes and a wide array of biotechnological innovations, highlighting their significance in medical, agricultural, and industrial domains. Recent advances in genetic engineering have revolutionized fungal research, with CRISPR/Cas emerging as the most potent and versatile genome editing platform. This technology enables precise manipulation of fungal genomes, from silencing efflux pump genes in Candida albicans (enhancing antifungal susceptibility) to targeting virulence-associated sirtuins in Aspergillus fumigatus (attenuating pathogenicity). Its applications span gene overexpression, multiplexed mutagenesis, and secondary metabolite induction, proving transformative for disease management and biotechnological innovation. CRISPR/Cas9's advantages-unmatched precision, cost-effectiveness, and therapeutic potential-are tempered by challenges like off-target effects, ethical dilemmas, and regulatory gaps. Integrating nanoparticle delivery systems and multi-omics approaches may overcome technical barriers, but responsible innovation requires addressing these limitations. CRISPR-driven fungal genome editing promises to redefine solutions for drug-resistant infections, sustainable bioproduction, and beyond as the field evolves. In conclusion, genome editing technologies have enhanced our capacity to dissect fungal biology and expanded fungi's practical applications across various scientific and industrial domains. Continued innovation in this field promises to unlock the vast potential of fungal systems further, enabling more profound understanding and transformative biotechnological progress.},
}

*Gene Editing/methods
*Genome, Fungal
CRISPR-Cas Systems
*Biotechnology/methods
*Fungi/genetics/pathogenicity
Humans
Candida albicans/genetics/pathogenicity
Aspergillus fumigatus/genetics/pathogenicity

@article {pmid40401996,
year = {2025},
author = {Pandey, N and Misra, C and Rath, D},
title = {Cas11 augments Cascade functions in type I-E CRISPR system but is redundant for gene silencing and plasmid interference.},
journal = {The Biochemical journal},
volume = {},
number = {},
pages = {},
doi = {10.1042/BCJ20253056},
pmid = {40401996},
issn = {1470-8728},
abstract = {The structural and mechanistic complexity of Escherichia coli's type I CRISPR-Cas system compared to the multidomain, single effector protein-based type II systems, limits its application in genome editing and silencing. Despite higher prevalence of the type I endogenous systems in bacteria, significant research has focused on improving the type II systems. While the type-I CRISPR system possesses several advantages over others, it may benefit from further studies to simplify the system for ease of use. To enable this, the dispensability of the type-I Cascade components (Cas8, Cas11, Cas7, Cas5, Cas6) for genome editing and silencing applications was evaluated in vivo. We created deletion variants of each of the Cascade components and investigated their effects on gene silencing and plasmid interference in two genetically distinct Escherichia coli lineages, BW25113, a K-12 strain that bears an endogenous, albeit repressed type I-E CRISPR system and BL21, a natural mutant lacking the type I-E CRISPR-Cascade system. Cas8, Cas7 and Cas5 were found to be indispensable for gene silencing and plasmid interference. Dispensability of Cas6, which is involved in crRNA maturation, was strain-dependent. Notably, Cas11 which has no definitive function assigned to it, was found to be dispensable for gene silencing and plasmid interference.},
}

@article {pmid40401794,
year = {2025},
author = {Jiao, L and Zhou, Q and Sun, D},
title = {CRISPR-Based Regulation for High-Throughput Screening.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00076},
pmid = {40401794},
issn = {2161-5063},
abstract = {CRISPR technology has revolutionized genome editing by enabling precise, permanent modifications to genetic material. To circumvent the irreversible alterations associated with traditional CRISPR methods and facilitate research on both essential and nonessential genes, CRISPR interference or inhibition (CRISPRi) and CRISPR activation (CRISPRa) were developed. The gene-silencing approach leverages an inactivated Cas effector protein paired with guide RNA to obstruct transcription initiation or elongation, while the gene-activation approach exploits the programmability of CRISPR to activate gene expression. Recent advances in CRISPRi technology, in combination with other technologies (e.g., biosensing, sequencing), have significantly expanded its applications, allowing for genome-wide high-throughput screening (HTS) to identify genetic determinants of phenotypes. These screening strategies have been applied in biomedicine, industry, and basic research. This review explores the CRISPR regulation mechanisms, offers an overview of the workflow for genome-wide CRISPR-based regulation for screens, and highlights its superior suitability for HTS across biomedical and industrial applications. Finally, we discuss the limitations of current CRISPRi/a HTS screening methods and envision future directions in CRISPR-mediated HTS research, considering its potential for broader application across diverse fields.},
}

@article {pmid40401239,
year = {2025},
author = {Mak, JK and Bendandi, A and Salim, JA and Mazoni, I and de Moraes, FR and Borro, L and Störtz, F and Rocchia, W and Neshich, G and Minary, P},
title = {Learning to utilize internal protein 3D nanoenvironment descriptors in predicting CRISPR-Cas9 off-target activity.},
journal = {NAR genomics and bioinformatics},
volume = {7},
number = {2},
pages = {lqaf054},
pmid = {40401239},
issn = {2631-9268},
mesh = {*CRISPR-Cas Systems ; *Machine Learning ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; *CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; Gene Editing ; Mutation ; },
abstract = {Despite advances in determining the factors influencing cleavage activity of a CRISPR-Cas9 single guide RNA (sgRNA) at an (off-)target DNA sequence, a comprehensive assessment of pertinent physico-chemical/structural descriptors is missing. In particular, studies have not yet directly exploited the information-rich internal protein 3D nanoenvironment of the sgRNA-(off-)target strand DNA pair, which we obtain by harvesting 634 980 residue-level features for CRISPR-Cas9 complexes. As a proof-of-concept study, we simulated the internal protein 3D nanoenvironment for all experimentally available single-base protospacer-adjacent motif-distal mutations for a given sgRNA-target strand pair. By determining the most relevant residue-level features for CRISPR-Cas9 off-target cleavage activity, we developed STING_CRISPR, a machine learning model delivering accurate predictive performance of off-target cleavage activity for the type of single-base mutations considered in this study. By interpreting STING_CRISPR, we identified four important Cas9 residue spatial hotspots and associated structural/physico-chemical descriptor classes influencing CRISPR-Cas9 (off-)target cleavage activity for the sgRNA-target strand pairs covered in this study.},
}

*CRISPR-Cas Systems
*Machine Learning
*RNA, Guide, CRISPR-Cas Systems/genetics/chemistry
*CRISPR-Associated Protein 9/chemistry/genetics/metabolism
Gene Editing
Mutation

@article {pmid40400348,
year = {2025},
author = {Khatri, M and Shanmugam, NRS and Zhang, X and Patel, RSKR and Yin, Y},
title = {AcrDB update: Predicted 3D structures of anti-CRISPRs in human gut viromes.},
journal = {Protein science : a publication of the Protein Society},
volume = {34},
number = {6},
pages = {e70177},
pmid = {40400348},
issn = {1469-896X},
support = {R01GM140370/NH/NIH HHS/United States ; R21AI171952/NH/NIH HHS/United States ; 58-8042-9-089//U.S. Department of Agriculture (USDA)/ ; },
mesh = {Humans ; *Bacteriophages/chemistry/genetics ; Protein Conformation ; Databases, Protein ; *Viral Proteins/chemistry/genetics/metabolism ; *Gastrointestinal Microbiome ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Models, Molecular ; },
abstract = {Anti-CRISPR (Acr) proteins play a key role in phage-host interactions and hold great promise for advancing genome-editing technologies. However, finding new Acrs has been challenging due to their low sequence similarity. Recent advances in protein structure prediction have opened new pathways for Acr discovery by using 3D structure similarity. This study presents an updated AcrDB, with the following new features not available in other databases: (1) predicted Acrs from human gut virome databases, (2) Acr structures predicted by AlphaFold2, (3) a structural similarity search function to allow users to submit new sequences and structures to search against 3D structures of experimentally known Acrs. The updated AcrDB contains predicted 3D structures of 795 candidate Acrs with structural similarity (TM-score ≥0.7) to known Acrs supported by at least two of the three non-sequence similarity-based tools (TM-Vec, Foldseek, AcrPred). Among these candidate Acrs, 121 are supported by all three tools. AcrDB also includes 3D structures of 122 experimentally characterized Acr proteins. The 121 most confident candidate Acrs were combined with the 122 known Acrs and clustered into 163 sequence similarity-based Acr families. The 163 families were further subject to a structure similarity-based hierarchical clustering, revealing structural similarity between 44 candidate Acr (cAcr) families and 119 known Acr families. The bacterial hosts of these 163 Acr families are mainly from Bacillota, Pseudomonadota, and Bacteroidota, which are all dominant gut bacterial phyla. Many of these 163 Acr families are also co-localized in Acr operons. All the data and visualization are provided on our website: https://pro.unl.edu/AcrDB.},
}

Humans
*Bacteriophages/chemistry/genetics
Protein Conformation
Databases, Protein
*Viral Proteins/chemistry/genetics/metabolism
*Gastrointestinal Microbiome
*Clustered Regularly Interspaced Short Palindromic Repeats
Models, Molecular

@article {pmid40288547,
year = {2025},
author = {Goméz-Quintero, OS and Morales-Moreno, MD and Valdés-Galindo, EG and Cárdenas-Guerra, RE and Hernandez-Garcia, A},
title = {Enhanced production of functional CRISPR-AsCas12a protein in Escherichia coli.},
journal = {Protein expression and purification},
volume = {232},
number = {},
pages = {106722},
doi = {10.1016/j.pep.2025.106722},
pmid = {40288547},
issn = {1096-0279},
mesh = {*Escherichia coli/genetics/metabolism ; Recombinant Proteins/genetics/biosynthesis/isolation & purification ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/genetics/biosynthesis/isolation & purification ; *Bacterial Proteins/genetics/biosynthesis/isolation & purification ; *Acidaminococcus/genetics/enzymology ; *Endodeoxyribonucleases/genetics/biosynthesis/isolation & purification ; Gene Editing ; },
abstract = {The CRISPR-Cas12a system is a groundbreaking tool widely used for genome editing and diagnostics in biotechnology and biomedicine research laboratories. Despite its growing application, studies optimizing Cas12a protein production at the laboratory scale using straightforward protocols remains scarce. This study aimed to enhance the lab-scale recombinant production of Acidaminococcus sp Cas12a protein (AsCas12a) in E. coli. Through targeted adjustments of simple parameters, AsCas12a production was significantly increased. The optimized conditions included the use of E. coli BL21(DE3), TB medium supplemented with 1 % glucose, induction with 0.3 mM IPTG for at least 6-9 h, and incubation at 30 °C. Notably, these conditions differ from conventional protocols typically used for Cas12a and related proteins, such as Streptococcus pyogenes Cas9. Upon combining all optimized parameters, AsCas12a production increased approximately 3-fold, from 0.95 mg/mL of bacterial lysate under non-optimized conditions to 3.73 mg/mL under optimized ones. After chromatographic purification, the final protein yield rose approximately 4.5-fold, from 5.2 to 23.4 mg/L of culture volume, without compromising functional activity. The purified AsCas12a retained full activity for programmable in vitro DNA cis-cleavage and collateral trans-cleavage, which was successfully applied to detect the N gene of SARS-CoV-2. This optimized method provide an efficient and high-yield approach for producing functional AsCas12a protein using accessible materials and conditions available to many research laboratories worldwide.},
}

*Escherichia coli/genetics/metabolism
Recombinant Proteins/genetics/biosynthesis/isolation & purification
*CRISPR-Cas Systems
*CRISPR-Associated Proteins/genetics/biosynthesis/isolation & purification
*Bacterial Proteins/genetics/biosynthesis/isolation & purification
*Acidaminococcus/genetics/enzymology
*Endodeoxyribonucleases/genetics/biosynthesis/isolation & purification
Gene Editing

@article {pmid40254211,
year = {2025},
author = {Wei, J and Chen, F and Lu, X and Fan, J and Li, M and Huang, J and Liu, N and Zhang, J and Chai, Z and Lu, S},
title = {In silico identification and experimental validation of long-range allosteric inhibition of Staphylococcus aureus Cas9 catalytic activity by an anti-CRISPR protein AcrIIA14.},
journal = {International journal of biological macromolecules},
volume = {310},
number = {Pt 2},
pages = {143324},
doi = {10.1016/j.ijbiomac.2025.143324},
pmid = {40254211},
issn = {1879-0003},
mesh = {*Staphylococcus aureus/enzymology/genetics ; Allosteric Regulation ; Molecular Dynamics Simulation ; *CRISPR-Associated Protein 9/antagonists & inhibitors/chemistry/metabolism/genetics ; *Bacterial Proteins/chemistry/genetics/metabolism/antagonists & inhibitors ; *CRISPR-Cas Systems ; Allosteric Site ; Mutagenesis, Site-Directed ; Computer Simulation ; },
abstract = {Effective temporal and spatial regulation of CRISPR-Cas9 catalytic activity remains a key challenge, limiting the clinical application of CRISPR-Cas9 gene-editing. Here, we investigated the long-range allosteric inhibition of Staphylococcus aureus Cas9 (SauCas9) catalytic activity by its anti-CRISPR (Acr) protein, AcrIIA14, aiming to uncover remote allosteric mechanisms in large protein complexes and identify potential allosteric sites for the design of SauCas9 inhibitors. Through a combined computational-experimental framework integrating extensive molecular dynamics simulations, Markov state models, network community modeling, and site-directed mutagenesis, we identified canonical and non-canonical inhibitory states of SauCas9 regulated by AcrIIA14. Key domains, including REC, L1, HNH, L2, and PI, play crucial roles in transmitting the AcrIIA14-meidated inhibitory signal. Introducing point mutations on the routes of allosteric communication and analyzing these mutants using in vitro DNA cleavage assays and surface plasmon resonance analysis revealed that SauCas9 escaped AcrIIA14's inhibition owing to the disruption of AcrIIA14-meidated allosteric communication. Moreover, two cryptic allosteric sites on SauCas9 were identified as mutations of these sites prevented inhibition of SauCas9 by AcrIIA14. Overall, our results provide a dynamic understanding of CRISPR-Cas9 regulation and an avenue to design SauCas9 inhibitors with a broad range of applications in Cas9 enzyme catalysis, biophysics, and gene-editing.},
}

*Staphylococcus aureus/enzymology/genetics
Allosteric Regulation
Molecular Dynamics Simulation
*CRISPR-Associated Protein 9/antagonists & inhibitors/chemistry/metabolism/genetics
*Bacterial Proteins/chemistry/genetics/metabolism/antagonists & inhibitors
*CRISPR-Cas Systems
Allosteric Site
Mutagenesis, Site-Directed
Computer Simulation

@article {pmid40400168,
year = {2025},
author = {Jiang, M and Zhao, X and Zhang, C and Liu, R and Hu, J and Lv, Y},
title = {Thermus thermophilus Argonaute-Mediated Single Particle Counting Platform for Multiplex Cancer-Related Biomarkers Detection.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c02118},
pmid = {40400168},
issn = {1520-6882},
abstract = {The clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) system has achieved remarkable success in the field of nucleic acid detection, while its Achilles' heel lies in the difficulties encountered in flexibility regarding the multiplex detection. As a sister system of CRISPR-Cas, prokaryotic Argonautes (pAgos) have precise recognition, multiturnover, and more importantly multiple specific cleavage characteristics, which is a potential candidate for the next generation of multiplex detection. Herein, a single particle counting platform was developed for the simultaneous detection of three colorectal cancer-related miRNAs (miR-141, miR-31, and miR-21) by combining single particle inductively coupled plasma mass spectrometry (SP-ICPMS) with the Thermus thermophilus Argonaute protein (TtAgo), with nanoparticles as signal probes for cleavage. The platform demonstrated high sensitivity (aM level) and specificity due to the dual-cycle mechanism of exponential isothermal amplification (EXPAR) and TtAgo cleavage, as well as the combination of TtAgo's specific cleavage capability and the multiplex detection advantages of metal stable isotope tagging. Additionally, the platform showed good robustness in human serum and cell extracts, indicating significant potential in clinical applications.},
}

@article {pmid40400140,
year = {2025},
author = {Jordt, LM and Gellert, M and Zelms, F and Bekeschus, S and Lillig, CH},
title = {The thioredoxin-like and one glutaredoxin domain are required to rescue the iron-starvation phenotype of HeLa GLRX3 knock out cells.},
journal = {FEBS letters},
volume = {},
number = {},
pages = {},
doi = {10.1002/1873-3468.70072},
pmid = {40400140},
issn = {1873-3468},
support = {Li984/3-1//Deutsche Forschungsgemeinschaft/ ; Li984/4-1//Deutsche Forschungsgemeinschaft/ ; },
abstract = {Glutaredoxin 3 (Grx3) is a multidomain protein (Trx-GrxA-GrxB) with a Trx-like domain and two Grx domains containing a CGFS motif for binding Fe2S2 clusters. To study the function of these domains, HeLa cells with GLRX3 knockout were generated via CRISPR/Cas. The knockout activated iron-regulatory protein 1, indicating iron starvation due to impaired iron metabolism. Transfection with constructs encoding wild-type or individual domains showed that only the Trx-GrxA construct could rescue the phenotype, matching the effect of full-length Grx3. The specific role of the second Grx domain in human Grx3, absent in simpler eukaryotes such as yeast, remains unclear. While the individual domains are insufficient to rescue the knockout of full-length Grx3, the Trx-GrxA module is functionally critical. Impact statement Glutaredoxin 3 (Grx3) contains a Trx-like domain and two Grx domains. The importance of the domains in higher eukaryotes has not previously been addressed in physiological or cellular contexts. Here, we report GLRX3 knockout results in activation of iron regulatory protein 1, and a Trx-GrxA construct could rescue the phenotype.},
}

@article {pmid40400023,
year = {2025},
author = {Kermanshahi, AZ and Ebrahimi, F and Taherpoor, A and Eslami, N and Baghi, HB},
title = {HPV-driven cancers: a looming threat and the potential of CRISPR/Cas9 for targeted therapy.},
journal = {Virology journal},
volume = {22},
number = {1},
pages = {156},
pmid = {40400023},
issn = {1743-422X},
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Papillomavirus Infections/therapy/virology/complications ; *Neoplasms/therapy/virology ; *Papillomaviridae/genetics ; *Genetic Therapy/methods ; Oncogene Proteins, Viral/genetics ; Female ; *Uterine Cervical Neoplasms/therapy/virology ; },
abstract = {Cervical and other anogenital malignancies are largely caused by E6 and E7 oncogenes of high-risk human papillomaviruses (HPVs), which inhibit important tumor suppressors like p53 and pRb when they are persistently activated. The main goal of traditional treatments is to physically or chemically kill cancer cells, but they frequently only offer temporary relief, have serious side effects, and have a high risk of recurrence. Exploring the efficacy and accuracy of CRISPR-Cas9 gene editing in both inducing death in HPV-infected cancer cells and restoring the activity of tumor suppressors is our main goal. In this study, we propose a novel precision oncology strategy that targets and inhibits the detrimental effects of the E6 and E7 oncogenes using the CRISPR-Cas9 gene editing system. In order to do this, we create unique guide RNAs that target the integrated HPV DNA and reactivate p53 and pRb. Reactivation is meant to halt aberrant cell development and restart the cell's natural dying pathways. This review discusses the potential of CRISPR/Cas9 in targeting HPV oncogenes, with a focus on studies that have demonstrated its promise in cancer treatment. Given the absence of a definitive treatment for papillomavirus infection and its subsequent association with various cancers, future clinical trials and experimental investigations appear essential to establish and evaluate the therapeutic potential of CRISPR-based approaches. This approach provides a less invasive alternative to conventional treatments and opens the door to personalized care that considers the genetic makeup of each patient's tumor.},
}

Humans
*CRISPR-Cas Systems
*Gene Editing/methods
*Papillomavirus Infections/therapy/virology/complications
*Neoplasms/therapy/virology
*Papillomaviridae/genetics
*Genetic Therapy/methods
Oncogene Proteins, Viral/genetics
Female
*Uterine Cervical Neoplasms/therapy/virology

@article {pmid40399557,
year = {2025},
author = {Bourgade, B and Xie, H and Lindblad, P and Stensjö, K},
title = {Development of a CRISPR activation system for targeted gene upregulation in Synechocystis sp. PCC 6803.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {772},
pmid = {40399557},
issn = {2399-3642},
support = {2021-01669//Svenska Forskningsrådet Formas (Swedish Research Council Formas)/ ; },
mesh = {*Synechocystis/genetics/metabolism ; *Up-Regulation ; *Gene Expression Regulation, Bacterial ; Metabolic Engineering/methods ; Butanols/metabolism ; Biofuels ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The photosynthetic cyanobacterium Synechocystis sp. PCC 6803 offers a promising sustainable solution for simultaneous CO2 fixation and compound bioproduction. While various heterologous products have now been synthesised in Synechocystis, limited genetic tools hinder further strain engineering for efficient production. Here, we present a versatile CRISPR activation (CRISPRa) system for Synechocystis, enabling robust multiplexed activation of both heterologous and endogenous targets. Following tool characterisation, we applied CRISPRa to explore targets influencing biofuel production, specifically isobutanol (IB) and 3-methyl-1-butanol (3M1B), demonstrating a proof-of-concept approach to identify key reactions constraining compound biosynthesis. Notably, individual upregulation of target genes, such as pyk1, resulted in up to 4-fold increase in IB/3M1B formation while synergetic effects from multiplexed targeting further enhanced compound production, highlighting the value of this tool for rapid metabolic mapping. Interestingly, activation efficacy did not consistently predict increases in compound formation, suggesting complex regulatory interactions influencing bioproduction. This work establishes a CRISPRa system for targeted upregulation in cyanobacteria, providing an adaptable platform for high-throughput screening, metabolic pathway optimisation and functional genomics. Our CRISPRa system provides a crucial advance in the genetic toolbox available for Synechocystis and will facilitate innovative applications in both fundamental research and metabolic engineering in cyanobacteria.},
}

*Synechocystis/genetics/metabolism
*Up-Regulation
*Gene Expression Regulation, Bacterial
Metabolic Engineering/methods
Butanols/metabolism
Biofuels
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40399309,
year = {2025},
author = {Schmerer, N and Janga, H and Aillaud, M and Hoffmann, J and Aznaourova, M and Wende, S and Steding, H and Halder, LD and Uhl, M and Boldt, F and Stiewe, T and Nist, A and Jerrentrup, L and Kirschbaum, A and Ruppert, C and Rossbach, O and Ntini, E and Marsico, A and Valasarajan, C and Backofen, R and Linne, U and Pullamsetti, SS and Schmeck, B and Schulte, LN},
title = {A searchable atlas of pathogen-sensitive lncRNA networks in human macrophages.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4733},
pmid = {40399309},
issn = {2041-1723},
support = {LOEWE/2/13/519/03/06.001(0002)/74//Hessisches Ministerium für Wissenschaft und Kunst (Hessen State Ministry of Higher Education, Research and the Arts)/ ; LOEWE/5/A004/519/06/00.005(0008)/E31//Hessisches Ministerium für Wissenschaft und Kunst (Hessen State Ministry of Higher Education, Research and the Arts)/ ; LOEWE/2/13/519/03/06.001(0002)/74//Hessisches Ministerium für Wissenschaft und Kunst (Hessen State Ministry of Higher Education, Research and the Arts)/ ; 10.21.2.024MN//Fritz Thyssen Stiftung (Fritz Thyssen Foundation)/ ; SFB-TR-84 (subproject C10)//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; BA 2168/25-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; TRR 167/2 NeuroMac//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CIBSS - EXC-2189 - Project ID 390939984//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; SFB-1213 (Project A01)//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; SFB-TR-84 (subproject C1)//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; PerMed-COPD (01EK2203A)//Bundesministerium für Bildung und Forschung (Federal Ministry of Education and Research)/ ; Deep Legion (031L0288A)//Bundesministerium für Bildung und Forschung (Federal Ministry of Education and Research)/ ; },
mesh = {Humans ; *RNA, Long Noncoding/genetics/metabolism ; *Macrophages/metabolism/immunology ; *Gene Regulatory Networks ; GATA2 Transcription Factor/genetics/metabolism ; NF-kappa B/metabolism ; Macrophage Activation/genetics ; Gene Expression Regulation ; CRISPR-Cas Systems ; },
abstract = {Long noncoding RNAs (lncRNA) are crucial yet underexplored regulators of human immunity. Here we develop GRADR, a method integrating gradient profiling with RNA-binding proteome analysis, to map the protein interactomes of all expressed RNAs in a single experiment to study mechanisms of lncRNA-mediated regulation of human primary macrophages. Applying GRADR alongside CRISPR-multiomics, we reveal a network of NFκB-dependent lncRNAs, including LINC01215, AC022816.1 and ROCKI, which modulate distinct aspects of macrophage immunity, particularly through interactions with mRNA-processing factors, such as hnRNP proteins. We further uncover the function of ROCKI in repressing the messenger of the anti-inflammatory GATA2 transcription factor, thus promoting macrophage activation. Lastly, all data are consolidated in the SMyLR web interface, a searchable reference catalog for exploring lncRNA functions and pathway-dependencies in immune cells. Our results thus not only highlight the important functions of lncRNAs in immune regulation, but also provide a rich resource for lncRNA studies.},
}

Humans
*RNA, Long Noncoding/genetics/metabolism
*Macrophages/metabolism/immunology
*Gene Regulatory Networks
GATA2 Transcription Factor/genetics/metabolism
NF-kappa B/metabolism
Macrophage Activation/genetics
Gene Expression Regulation
CRISPR-Cas Systems

@article {pmid40399255,
year = {2025},
author = {Riesenberg, S and Kanis, P and Karlic, R and Maricic, T},
title = {Robust prediction of synthetic gRNA activity and cryptic DNA repair by disentangling cellular CRISPR cleavage outcomes.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4717},
pmid = {40399255},
issn = {2041-1723},
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Humans ; *DNA Repair/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; INDEL Mutation ; CRISPR-Associated Protein 9/metabolism/genetics ; DNA Cleavage ; CRISPR-Associated Proteins/metabolism/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; HEK293 Cells ; Endodeoxyribonucleases/metabolism/genetics ; Bacterial Proteins/metabolism/genetics ; },
abstract = {The ability to robustly predict guide RNA (gRNA) activity is a long-standing goal for CRISPR applications, as it would reduce the need to pre-screen gRNAs. Quantification of formation of short insertions and deletions (indels) after DNA cleavage by transcribed gRNAs has been typically used to measure and predict gRNA activity. We evaluate the effect of chemically synthesized Cas9 gRNAs on different cellular DNA cleavage outcomes and find that the activity of different gRNAs is largely similar and often underestimated when only indels are scored. We provide a simple linear model that reliably predicts synthetic gRNA activity across cell lines, robustly identifies inefficient gRNAs across different published datasets, and is easily accessible via online genome browser tracks. In addition, we develop a homology-directed repair efficiency prediction tool and show that unintended large-scale repair events are common for Cas9 but not for Cas12a, which may be relevant for safety in gene therapy applications.},
}

*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Humans
*DNA Repair/genetics
*CRISPR-Cas Systems/genetics
Gene Editing/methods
INDEL Mutation
CRISPR-Associated Protein 9/metabolism/genetics
DNA Cleavage
CRISPR-Associated Proteins/metabolism/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
HEK293 Cells
Endodeoxyribonucleases/metabolism/genetics
Bacterial Proteins/metabolism/genetics

@article {pmid40397713,
year = {2025},
author = {Teng, H and Hang, Q and Zheng, C and Yan, Y and Liu, S and Zhao, Y and Deng, Y and Nie, L and Wu, W and Sheldon, M and Yu, Z and Shi, W and Gao, J and Meng, C and Martinez, C and Zhang, J and Yao, F and Sun, Y and Zhao, D and Gan, B and Meng, T and Ma, L},
title = {In vivo CRISPR activation screen identifies acyl-CoA-binding protein as a driver of bone metastasis.},
journal = {Science translational medicine},
volume = {17},
number = {799},
pages = {eado7225},
doi = {10.1126/scitranslmed.ado7225},
pmid = {40397713},
issn = {1946-6242},
mesh = {Animals ; *Bone Neoplasms/secondary/genetics/metabolism/pathology ; Humans ; Cell Line, Tumor ; Mice ; *Diazepam Binding Inhibitor/metabolism/genetics ; Lipid Metabolism ; Fatty Acids/metabolism ; Reactive Oxygen Species/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Ferroptosis ; Oxidation-Reduction ; Neoplasm Metastasis ; Female ; *CRISPR-Cas Systems/genetics ; },
abstract = {One of the most common sites of cancer metastasis is to the bone. Bone metastasis is associated with substantial morbidity and mortality, and current therapeutic interventions remain largely palliative. Metastasizing tumor cells need to reprogram their metabolic states to adapt to the nutrient environment of distant organs; however, the role and translational relevance of lipid metabolism in bone metastasis remain unclear. Here, we used an in vivo CRISPR activation screening system coupled with positive selection to identify acyl-coenzyme A (CoA) binding protein (ACBP) as a bone metastasis driver. In nonmetastatic and weakly metastatic cancer cells, overexpression of wild-type ACBP, but not the acyl-CoA-binding deficient mutant, stimulated fatty acid oxidation (FAO) and bone metastasis. Conversely, knockout of ACBP in highly bone metastatic cancer cells abrogated metastatic bone colonization. Mechanistically, ACBP-mediated FAO increased ATP and NADPH production, reduced reactive oxygen species, and inhibited lipid peroxidation and ferroptosis. We found that ACBP expression correlated with metabolic signaling, bone metastatic ability, and poor clinical outcomes. In mouse models, pharmacological blockade of FAO or treatment with a ferroptosis inducer inhibited bone metastasis. Together, our findings reveal the role of lipid metabolism in tumor cells adapting and thriving in the bone and identify ACBP as a key regulator of this process. Agents that target FAO or induce ferroptosis represent a promising therapeutic approach for treating bone metastases.},
}

Animals
*Bone Neoplasms/secondary/genetics/metabolism/pathology
Humans
Cell Line, Tumor
Mice
*Diazepam Binding Inhibitor/metabolism/genetics
Lipid Metabolism
Fatty Acids/metabolism
Reactive Oxygen Species/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Ferroptosis
Oxidation-Reduction
Neoplasm Metastasis
Female
*CRISPR-Cas Systems/genetics

@article {pmid40397663,
year = {2025},
author = {Gast, K and Baker, S and Borges, AL and Ward, S and Banfield, JF and Barrangou, R},
title = {Metagenome-Derived CRISPR-Cas12a Mining and Characterization.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1089/crispr.2024.0099},
pmid = {40397663},
issn = {2573-1602},
abstract = {The advent of clustered regularly interspaced short palindromic repeats (CRISPR)-based technologies has revolutionized genome editing, with continued interest in expanding the CRISPR-associated proteins (Cas) toolbox with diverse, efficient, and specific effectors. CRISPR-Cas12a is a potent, programmable RNA-guided dual nickase, broadly used for genome editing. Here, we mined dairy cow microbial metagenomes for CRISPR-Cas systems, unraveling novel Cas12a enzymes. Using in silico pipelines, we characterized and predicted key drivers of CRISPR-Cas12a activity, encompassing guides and protospacer adjacent motifs for five systems. We next assessed their functional potential in cell-free transcription-translation assays with GFP-based fluorescence readouts. Lastly, we determined their genome editing potential in vivo in Escherichia coli by generating 1 kb knockouts. Unexpectedly, we observed natural sequence variation in the bridge-helix domain of the best-performing candidate and used mutagenesis to alter the activity of Cas12a orthologs, resulting in increased gene editing capabilities of a relatively inefficient candidate. This study illustrates the potential of underexplored metagenomic sequence diversity for the development and refinement of genome editing effectors.},
}

@article {pmid40370093,
year = {2025},
author = {Qiu, S and Chen, L and Zhuang, D and Cao, Y and Wei, R and Cao, X and Chen, Y and Lai, X and Wang, S and Lin, Y and Lin, Z and Zhang, S},
title = {Fluorescence Aptasensor for sST2 Detection Using In Vitro Selected Aptamers.},
journal = {Analytical chemistry},
volume = {97},
number = {20},
pages = {10910-10918},
doi = {10.1021/acs.analchem.5c01855},
pmid = {40370093},
issn = {1520-6882},
mesh = {*Aptamers, Nucleotide/chemistry ; Humans ; *Interleukin-1 Receptor-Like 1 Protein/blood/analysis ; *Biosensing Techniques/methods ; Limit of Detection ; Fluorescence ; CRISPR-Cas Systems ; },
abstract = {Soluble suppression of tumorigenicity 2 (sST2) is a critical biomarker for heart failure (HF) diagnosis and prognosis, yet conventional antibody-based detection methods suffer from time-consuming protocols and high costs and involve complex detection procedures. To address these challenges, we first screened high-affinity aptamers under clinically relevant conditions and then coupled with the CRISPR/Cas12a system to develop a fluorescence aptasensor for rapid and sensitive sST2 detection. A serum matrix was introduced during aptamer selection to enhance specificity and anti-interference performance in real biological environments. Three sST2-specific aptamers (Apt-1, Apt-2, and Apt-3) were identified with dissociation constants (KD) of 8.42, 46.08, and 25.02 nM, respectively. Among these, Apt-1 demonstrated superior performance, which was utilized to construct a fluorescence biosensor combining aptamer recognition with CRISPR/Cas12a trans-cleavage signal amplification. The sensor achieved a broad linear detection range (5-120 ng/mL) and an ultralow limit of detection (LOD, 0.816 ng/mL) when applied in detecting sST2 in both the buffer and human serum. Notably, the platform exhibited exceptional resistance to interference from HF-related proteins and maintained high accuracy in clinical serum samples, showing a strong correlation (R[2] = 0.9794) with enzyme-linked immunosorbent assay (ELISA) results. By integration of serum-matrix screening and CRISPR-based signal enhancement, this work establishes a robust, cost-effective, and rapid diagnostic tool for sST2 detection.},
}

*Aptamers, Nucleotide/chemistry
Humans
*Interleukin-1 Receptor-Like 1 Protein/blood/analysis
*Biosensing Techniques/methods
Limit of Detection
Fluorescence
CRISPR-Cas Systems

@article {pmid40366179,
year = {2025},
author = {Zhang, Y and Li, W and Chen, S and Zhang, Y and Zhu, Y and Lan, F and Du, H and Fan, R and Zhu, J and Pan, W and Situ, B and Zheng, L and Luo, S and Yan, X},
title = {Layered-Responsive Multivalent Tetrahedral DNA Framework-Decorated CRISPR-Cas12a Nanocapsule Enables Precise and Enhanced Tumor Chemotherapy.},
journal = {ACS nano},
volume = {19},
number = {20},
pages = {19274-19286},
doi = {10.1021/acsnano.5c01747},
pmid = {40366179},
issn = {1936-086X},
mesh = {*Doxorubicin/pharmacology/chemistry/administration & dosage ; Humans ; *DNA/chemistry ; *CRISPR-Cas Systems ; *Nanocapsules/chemistry ; Animals ; Cell Line, Tumor ; Mice ; *Antineoplastic Agents/pharmacology/chemistry ; *Antibiotics, Antineoplastic/pharmacology/chemistry ; Neoplasms/drug therapy ; },
abstract = {The lack of selective tumor targeting and the high toxicity of conventional chemotherapy treatments remain major challenges in cancer therapy. Here, we develop a self-controlled DNA nanostructure-CRISPR-12a system, a triple-locked cascade tumor therapy nanocapsule (Tatna), for efficient and targeted tumor treatment. Tatna integrates structural DNA tetrahedrons (DTs) with high drug-loading capacity, Cas12a/crRNA ribonucleoprotein (Cas12a RNP), and doxorubicin (DOX) to enable multisite response for precise drug delivery and augmented tumor treatment. By incorporation of a nucleolin-targeting aptamer, Tatna achieves selective targeting and efficient tumor cell internalization. Encapsulation in pH-responsive poly l-lactic-co-glycolic acid (PLGA) nanocapsule ensures stable circulation and controlled release of both DOX and Cas12a until tumor-specific activation in the acidic microenvironment. The Cas12a RNP, triggered by APE1 mRNA overexpression in tumor cells, induces trans-cleavage of DTs, releasing DOX and Cas12a to transport into the nucleus and induce enhanced cell apoptosis. This self-regulating and multifunctional approach enhances the efficacy of chemotherapy while reducing off-target effects. Tatna's programmable, tumor-specific delivery system represents a powerful strategy for advancing precision medicine and personalized cancer treatment.},
}

*Doxorubicin/pharmacology/chemistry/administration & dosage
Humans
*DNA/chemistry
*CRISPR-Cas Systems
*Nanocapsules/chemistry
Animals
Cell Line, Tumor
Mice
*Antineoplastic Agents/pharmacology/chemistry
*Antibiotics, Antineoplastic/pharmacology/chemistry
Neoplasms/drug therapy

@article {pmid40360969,
year = {2025},
author = {Dong, J and Ma, W and Zhou, S and Li, X and Deng, L and Hou, C and Huo, D},
title = {Tri-Mode CRISPR-Based Biosensor for miRNA Detection: Enhancing Clinical Diagnostics with Cross-Validation.},
journal = {Analytical chemistry},
volume = {97},
number = {20},
pages = {10938-10946},
doi = {10.1021/acs.analchem.5c02348},
pmid = {40360969},
issn = {1520-6882},
mesh = {*MicroRNAs/analysis/genetics ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques ; Limit of Detection ; Electrochemical Techniques ; Metal-Organic Frameworks/chemistry ; Methylene Blue/chemistry ; },
abstract = {In vitro diagnostics require the accurate detection of disease-associated target biomolecules at ultralow concentrations. A multimode sensing strategy is considered as a potential method for in vitro diagnosis because it allows cross-validation of test results through data complementation and self-calibration, and provides double confirmation. Here, we present a CRISPR/Cas12a-powered trimode biosensor (CPTMB) for ultrasensitive and reliable analysis of miRNA. Briefly, the presence of target miRNA initiates rolling circle extension-driven loop-mediated isothermal amplification (R-LAMP), which subsequently activates the trans-cleavage activity of CRISPR/Cas12a. Then, the hairpin probe (HP) biogate on nucleic acid-functionalized MB@Fe-MOF signal probe was degraded by Cas12a, leading to the release of methylene blue (MB) signal molecules encapsulated within Fe-MOF nanocarriers. Due to the capability of MB to generate output responses across three distinct modes: electrochemical (EC), fluorescence (FL), and ultraviolet-visible spectroscopy (UV-vis), a trimodal sensing system is achieved. Benefiting from the efficient signal amplification capabilities of R-LAMP and CRISPR/Cas12a, this strategy enables rapid detection of target miRNA at femtomolar levels within 70 min. Furthermore, the detection results across the three modes cross-validate one another, thereby enhancing the reliability of the analysis. More importantly, the platform has been successfully applied to miRNA analysis in real samples, and the detection results are in good agreement with those of the standard method RT-qPCR, indicating its great potential in the clinical diagnosis of early-stage cancer.},
}

*MicroRNAs/analysis/genetics
*Biosensing Techniques/methods
Humans
*CRISPR-Cas Systems
Nucleic Acid Amplification Techniques
Limit of Detection
Electrochemical Techniques
Metal-Organic Frameworks/chemistry
Methylene Blue/chemistry

@article {pmid40323736,
year = {2025},
author = {Chen, Y and Li, Y and Li, P and Li, X and Zhao, S and Zuo, Z},
title = {Catching CRISPR-Cas9 in Action.},
journal = {Journal of chemical theory and computation},
volume = {21},
number = {10},
pages = {5023-5036},
doi = {10.1021/acs.jctc.5c00165},
pmid = {40323736},
issn = {1549-9626},
mesh = {Molecular Dynamics Simulation ; *CRISPR-Cas Systems ; DNA/chemistry/metabolism ; *CRISPR-Associated Protein 9/chemistry/metabolism ; Quantum Theory ; },
abstract = {CRISPR-Cas9 has revolutionized genome editing, yet its structural dynamics and functional properties remain incompletely understood, partly due to limited atomic-level characterization of its active conformation with a full R-loop. Capitalizing on recent advances in Cas9 structural determination, we constructed a catalytic-state Cas9 model bound to a bona fide R-loop and performed an integrated computational investigation. Our molecular dynamics simulations reveal substantial conformational heterogeneity in the PAM (protospacer-adjacent motif)-distal nontarget DNA strand and adjacent Cas9 regions, leading to dynamically fluctuating interactions, thereby challenging experimental resolution of the full R-loop complex. Comparative analysis highlights a conformational barrier restricting final activation of the HNH nuclease domain, suggesting that strategic modulation of HNH interactions on its two sides could enhance cleavage efficiency. Furthermore, quantum mechanics/molecular mechanics simulations indicate that with H983 protonated at Nε, the RuvC domain favors a phosphate-mediated over a histidine-mediated pathway for nontarget strand cleavage. Additionally, we identify an alternative HNH-mediated target strand cleavage pathway, involving a water nucleophile aligned at the 5' side of the scissile phosphate. Inspired by the basic residue ladder observed in RuvC, we propose extending a similar ladder in HNH to strengthen DNA binding and catalytic activity. Our study provides critical insights into Cas9 structure, dynamics, and catalysis, laying a foundation for the rational design of next-generation CRISPR-Cas9 systems with optimized specificity-efficiency balance.},
}

Molecular Dynamics Simulation
*CRISPR-Cas Systems
DNA/chemistry/metabolism
*CRISPR-Associated Protein 9/chemistry/metabolism
Quantum Theory

@article {pmid40272173,
year = {2025},
author = {Paz, M and Moratorio, G},
title = {Deep mutational scanning and CRISPR-engineered viruses: tools for evolutionary and functional genomics studies.},
journal = {mSphere},
volume = {10},
number = {5},
pages = {e0050824},
doi = {10.1128/msphere.00508-24},
pmid = {40272173},
issn = {2379-5042},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Genomics/methods ; *Genome, Viral ; *Viruses/genetics ; *Evolution, Molecular ; Humans ; Mutation ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Mutational Analysis/methods ; },
abstract = {Recent advancements in synthetic biology and sequencing technologies have revolutionized the ability to manipulate viral genomes with unparalleled precision. This review focuses on two powerful methodologies: deep mutational scanning and CRISPR-based genome editing, that enable comprehensive mutagenesis and detailed functional characterization of viral proteins. These approaches have significantly deepened our understanding of the molecular determinants driving viral evolution and adaptation. Furthermore, we discuss how these advances provide transformative insights for future vaccine development and therapeutic strategies.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*Genomics/methods
*Genome, Viral
*Viruses/genetics
*Evolution, Molecular
Humans
Mutation
Clustered Regularly Interspaced Short Palindromic Repeats
DNA Mutational Analysis/methods

@article {pmid40138089,
year = {2025},
author = {Chu, Y and Wei, M and Cao, Z and Chen, L and Tan, J and Bao, W and Yang, F and Zhang, Y and Lin, Y and Zhang, Y and Li, S and Lv, C and Zhou, W and Du, H and Shen, L and Huai, C and Wang, Z and Qin, S},
title = {Integrative analysis based on CRISPR screen identifies apilimod as a potential therapeutic agent for cisplatin-induced acute kidney injury treatment.},
journal = {Science China. Life sciences},
volume = {68},
number = {6},
pages = {1770-1785},
pmid = {40138089},
issn = {1869-1889},
mesh = {*Cisplatin/adverse effects ; *Acute Kidney Injury/chemically induced/drug therapy/genetics ; Animals ; Humans ; Mice ; Apoptosis/drug effects ; *CRISPR-Cas Systems ; *Morpholines/pharmacology/therapeutic use ; Reactive Oxygen Species/metabolism ; Lipid Metabolism/drug effects ; Male ; Antineoplastic Agents/adverse effects ; Signal Transduction/drug effects ; Cell Line ; },
abstract = {Acute kidney injury (AKI), a life-threatening side effect of cisplatin therapy, significantly limits the drug's therapeutic potential. In this study, we conducted a genome-wide CRISPR/Cas9 knockout screen in human renal tubular epithelial cells, integrating the results with transcriptome analyses and the Connectivity Map (CMap) database. Apilimod and elacridar emerged as the top two candidates of mitigating cisplatin-induced nephrotoxicity, with apilimod demonstrating superior efficacy in drug matrix experiments. Apilimod reduced cisplatin-induced apoptosis, inflammation and reactive oxygen species (ROS) generation. Transcriptome analyses suggested that apilimod may protect against cisplatin-induced nephrotoxicity via modulating lipid metabolism. In vitro experiments revealed that apilimod significantly ameliorated cisplatin-induced lipotoxicity by enhancing lipid clearance and upregulating PGC1α-mediated fatty acid oxidation. Mechanism experiments showed that apilimod induces the nuclear translocation of TFEB through the inhibition of its target, PIKfyve, thereby enhancing PGC1α expression and ameliorating lipotoxicity. These protective effects of apilimod were simulated by siRNA-mediated PIKfyve knockdown and diminished by the PGC1α inhibitor SR-18292 and siRNA targeting TFEB, confirming the role of the PIKfyve/TFEB/PGC1α signaling axis in apilimod's renoprotective effects. In vivo, apilimod alleviated apoptosis, inflammation, and lipid accumulation in a cisplatin-induced AKI mouse model. Additionally, apilimod treatment did not compromise the antitumor effect of cisplatin in cancer cells or tumor-bearing mice. Overall, our study suggests that apilimod could be a promising therapeutic agent for the treatment of cisplatin-induced AKI and revealed its underlying molecular mechanism.},
}

*Cisplatin/adverse effects
*Acute Kidney Injury/chemically induced/drug therapy/genetics
Animals
Humans
Mice
Apoptosis/drug effects
*CRISPR-Cas Systems
*Morpholines/pharmacology/therapeutic use
Reactive Oxygen Species/metabolism
Lipid Metabolism/drug effects
Male
Antineoplastic Agents/adverse effects
Signal Transduction/drug effects
Cell Line

@article {pmid40133712,
year = {2025},
author = {Wang, C and Jiang, X and Li, HY and Hu, J and Ji, Q and Wang, Q and Liu, X and Huang, D and Yan, K and Zhao, L and Fan, Y and Wang, S and Ma, S and Belmonte, JCI and Qu, J and Liu, GH and Zhang, W},
title = {RIG-I-driven CDKN1A stabilization reinforces cellular senescence.},
journal = {Science China. Life sciences},
volume = {68},
number = {6},
pages = {1646-1661},
pmid = {40133712},
issn = {1869-1889},
mesh = {*Cellular Senescence/genetics ; Humans ; *Cyclin-Dependent Kinase Inhibitor p21/metabolism/genetics ; *DEAD Box Protein 58/metabolism/genetics ; Mesenchymal Stem Cells/metabolism/cytology ; Receptors, Immunologic/metabolism/genetics ; RNA, Messenger/metabolism/genetics ; CRISPR-Cas Systems ; Signal Transduction ; Immunity, Innate ; },
abstract = {The innate immune signaling network follows a canonical format for signal transmission. The innate immune pathway is crucial for defense against pathogens, yet its mechanistic crosstalk with aging processes remains largely unexplored. Retinoic acid-inducible gene-I (RIG-I), a key mediator of antiviral immunity within this pathway, has an enigmatic role in stem cell senescence. Our study reveals that RIG-I levels increase in human genetic and physiological cellular aging models, and its accumulation drives cellular senescence. Conversely, CRISPR/Cas9-mediated RIG-I deletion or pharmacological inhibition in human mesenchymal stem cells (hMSCs) confers resistance to senescence. Mechanistically, RIG-I binds to endogenous mRNAs, with CDKN1A mRNA being a prominent target. Specifically, RIG-I stabilizes CDKN1A mRNA, resulting in elevated CDKN1A transcript levels and increased p21[Cip1] protein expression, which precipitates senescence. Collectively, our findings establish RIG-I as a post-transcriptional regulator of senescence and suggest potential targets for the mitigation of aging-related diseases.},
}

*Cellular Senescence/genetics
Humans
*Cyclin-Dependent Kinase Inhibitor p21/metabolism/genetics
*DEAD Box Protein 58/metabolism/genetics
Mesenchymal Stem Cells/metabolism/cytology
Receptors, Immunologic/metabolism/genetics
RNA, Messenger/metabolism/genetics
CRISPR-Cas Systems
Signal Transduction
Immunity, Innate

@article {pmid39956793,
year = {2025},
author = {Uzun, S and Özcan, Ö and Gök, A and Işık, A and Bakır, S and Günel-Özcan, A and Onbaşılar, İ and Akyol, A},
title = {A new CRISPR-mediated Apc knockout allele leads to pyloric gland adenoma-like gastric polyps in mice with C57BL/6;FVB/N mixed background.},
journal = {Animal models and experimental medicine},
volume = {8},
number = {5},
pages = {922-929},
doi = {10.1002/ame2.70002},
pmid = {39956793},
issn = {2576-2095},
support = {SBAG-215S926//The Scientific and Technological Research Council of Turkey 1001 program/ ; },
mesh = {Animals ; Mice, Inbred C57BL ; Alleles ; Mice ; *CRISPR-Cas Systems/genetics ; Mice, Knockout ; Disease Models, Animal ; *Stomach Neoplasms/genetics/pathology ; *Adenomatous Polyposis Coli Protein/genetics ; Male ; *Adenoma/genetics/pathology ; Adenomatous Polyps ; },
abstract = {Adenomatous polyposis coli (APC) mutations are the most frequently identified genetic alteration in sporadic colorectal cancer (CRC) cases, and a myriad of genetically engineered Apc-mutant CRC mouse models have been developed using various genetic manipulation techniques. The advent of the CRISPR/Cas9 system has revolutionized the field of genetic engineering and facilitated the development of new genetically engineered mouse models. In this study, we aimed to develop a novel Apc knockout allele using the CRISPR/Cas9 system and evaluate the phenotypic effects of this new allele in two different mouse strains. For this purpose, exon 16 of mouse Apc gene was targeted with a single-guide RNA, and the mouse carrying an Apc frameshift mutation at codon 750 (Δ750) was chosen as the founder. The mutant FVB-Apc[Δ750] mice were backcrossed with wild-type C57BL/6 mice, and the phenotypic effects of the knockout allele were evaluated in F8-FVB-Apc[Δ750], F4-B6;FVB-Apc[Δ750], and F1-B6;FVB-Apc[Δ750] by a macroscopic and microscopic examination of the gastrointestinal system. The result showed that the mean polyp number was significantly higher in F4-BL6;FVB-Apc[Δ750] than in F8-FVB-Apc[Δ750]. Intestinal polyposis was more prominent in F4-BL6;FVB-Apc[Δ750], whereas a higher number of colon polyps than intestinal polyps were observed in F8-FVB-Apc[Δ750]. Additionally, F1-BL6;FVB-Apc[Δ750] mixed background mice developed gastric polyps that morphologically resembled the pyloric gland adenoma of humans. In conclusion, we developed a novel CRISPR-mediated Apc knockout allele using two mouse strains. We showed that this allele can exert a strain-specific effect on the phenotype of mice and can cause gastric polyp formation.},
}

Animals
Mice, Inbred C57BL
Alleles
Mice
*CRISPR-Cas Systems/genetics
Mice, Knockout
Disease Models, Animal
*Stomach Neoplasms/genetics/pathology
*Adenomatous Polyposis Coli Protein/genetics
Male
*Adenoma/genetics/pathology
Adenomatous Polyps

@article {pmid39821833,
year = {2025},
author = {Li, Y and Fu, Y and Li, Y and Zhang, R and Yang, J and Ma, H and Min, L and Zhang, X},
title = {Reversing anther thermotolerance by manipulating the cis-elements in the promoter of a high-temperature upregulated gene Casein Kinase I in upland cotton.},
journal = {Science China. Life sciences},
volume = {68},
number = {6},
pages = {1558-1569},
pmid = {39821833},
issn = {1869-1889},
mesh = {*Gossypium/genetics/physiology ; *Promoter Regions, Genetic/genetics ; *Thermotolerance/genetics ; Gene Expression Regulation, Plant ; *Flowers/genetics/physiology ; *Casein Kinase I/genetics/metabolism ; Gene Editing ; CRISPR-Cas Systems ; Plant Proteins/genetics/metabolism ; Hot Temperature ; Plants, Genetically Modified ; Alleles ; Up-Regulation ; Genotype ; Phenotype ; },
abstract = {High temperature (HT) stress causes male sterility, leading to reduced upland cotton yield. Previously, we identified a key gene, Casein Kinase I (GhCKI), that negatively regulates male fertility in upland cotton under HT. However, conventional genetic manipulations of GhCKI would result in male sterility, hindering its utilization in breeding programs. Here, we engineered quantitative variation for anther thermotolerance-related traits in upland cotton by creating weak promoter alleles of GhCKI genes, using CRISPR/Cas9 and CRISPR/Cpf1 genome editing. Then, we screened and identified two new upland cotton plant lines exhibiting a HT-tolerant phenotype with edited GhCKI promoters, and characterized their corresponding heat-tolerant allelic genotypes. Further research revealed that the primary reason for the HT tolerance of the GhCKI promoter editing mutants is that the trans-acting factors GhMYB73 and GhMYB4, which positively regulate GhCKI expression under HT, failed to bind and activate the expression of GhCKI. Overall, our study not only provides a rapid strategy to generate new beneficial alleles but also offers novel germplasm resources and molecular insights for crop HT tolerance breeding.},
}

*Gossypium/genetics/physiology
*Promoter Regions, Genetic/genetics
*Thermotolerance/genetics
Gene Expression Regulation, Plant
*Flowers/genetics/physiology
*Casein Kinase I/genetics/metabolism
Gene Editing
CRISPR-Cas Systems
Plant Proteins/genetics/metabolism
Hot Temperature
Plants, Genetically Modified
Alleles
Up-Regulation
Genotype
Phenotype

@article {pmid40394594,
year = {2025},
author = {Guo, J and Shan, Y and Hu, G and Zhu, X and Zhao, K and Wu, T and Qiao, Q and Chi, Y and Cui, L and Ge, Y},
title = {Rapid visual detection of Monkeypox virus by one-step LAMP-CRISPR/Cas12b assay.},
journal = {Virology journal},
volume = {22},
number = {1},
pages = {151},
pmid = {40394594},
issn = {1743-422X},
support = {2023YFC2605100, 2023YFC2605104,BK20231374, BK20221413//National Key R&D Program of China；Natural science foundation of Jiangsu Province/ ; },
mesh = {*Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Monkeypox virus/isolation & purification/genetics ; Humans ; *Mpox, Monkeypox/diagnosis/virology ; Limit of Detection ; },
abstract = {BACKGROUND: Monkeypox virus (MPXV) infection has garnered significant global attention due to its rising incidence and substantial public health implications. A rapid, sensitive, and accurate diagnostic method is urgently required to enable early intervention and effective management of MPXV outbreaks.

RESULTS: In this study, we developed a novel one-step assay that integrates loop-mediated isothermal amplification (LAMP) with CRISPR/Cas12b in one-pot for the detection of MPXV. The entire detection process did not require opening the lid of the reaction tube and could be completed within 40 min using extracted viral nucleic acids, which is faster than real-time quantitative PCR (qPCR). And the results could be interpreted through either real-time fluorescence or naked-eye visualization. The limit of detection (LOD) of the assay was demonstrated to be 6.5 copies per reaction and no cross-reactivity with other pathogens such as HSV, EBV, CVA16, EV-A71, and MV was found. Furthermore, when evaluated using 113 clinical samples, the assay achieved 100% sensitivity (71/71) and 100% specificity (42/42) compared to the qPCR.

CONCLUSIONS: In resource-limited settings, our method requires only a portable heat block or water bath and a blue light or ultraviolet flashlight for visual detection of MPXV, making it highly accessible. The integration of LAMP and CRISPR/Cas12b provides a robust, user-friendly platform for point-of-care testing, with promising potential for the rapid molecular diagnosis of infectious diseases.},
}

*Nucleic Acid Amplification Techniques/methods
*Molecular Diagnostic Techniques/methods
Sensitivity and Specificity
*CRISPR-Cas Systems
*Monkeypox virus/isolation & purification/genetics
Humans
*Mpox, Monkeypox/diagnosis/virology
Limit of Detection

@article {pmid40394064,
year = {2025},
author = {Gu, S and Bodai, Z and Anderson, RA and So, HYA and Cowan, QT and Komor, AC},
title = {Elucidating the genetic mechanisms governing cytosine base editing outcomes through CRISPRi screens.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4685},
pmid = {40394064},
issn = {2041-1723},
support = {T32 GM146648/GM/NIGMS NIH HHS/United States ; T32GM146648//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; 27975//Research Corporation for Science Advancement (Research Corporation)/ ; T32GM008326//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; MCB-2048207//National Science Foundation (NSF)/ ; T32 GM008326/GM/NIGMS NIH HHS/United States ; },
mesh = {*Cytosine/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; DNA Repair/genetics ; DNA Mismatch Repair/genetics ; HEK293 Cells ; Ubiquitin-Protein Ligases/metabolism/genetics ; },
abstract = {Cytosine base editors enable programmable and efficient genome editing using an intermediate featuring a U•G mismatch across from a DNA nick. This intermediate facilitates two major outcomes, C•G to T•A and C•G to G•C point mutations, and it is not currently well-understood which DNA repair factors are involved. Here, we couple reporters for cytosine base editing activity with knockdown of 2015 DNA processing genes to identify genes involved in these two outcomes. Our data suggest that mismatch repair factors facilitate C•G to T•A outcomes, while C•G to G•C outcomes are mediated by RFWD3, an E3 ubiquitin ligase. We also propose that XPF, a 3'-flap endonuclease, and LIG3, a DNA ligase, are involved in repairing the intermediate back to the original C•G base pair. Our results demonstrate that competition and collaboration among different DNA repair pathways shape cytosine base editing outcomes.},
}

*Cytosine/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Humans
DNA Repair/genetics
DNA Mismatch Repair/genetics
HEK293 Cells
Ubiquitin-Protein Ligases/metabolism/genetics

@article {pmid40393960,
year = {2025},
author = {Liu, Z and Luan, X and Lu, Q and Qin, S and Zeng, F and Li, Z and He, B and Song, Y},
title = {Reactive oxygen species responsive nanomotors for gene edited metabolic disruption and immunotherapy.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4708},
pmid = {40393960},
issn = {2041-1723},
mesh = {*Reactive Oxygen Species/metabolism ; *Immunotherapy/methods ; *Gene Editing/methods ; Humans ; Animals ; Mice ; CRISPR-Cas Systems ; Cell Line, Tumor ; Tumor Microenvironment ; Heme Oxygenase-1/metabolism ; *Neoplasms/therapy/immunology/genetics ; CD8-Positive T-Lymphocytes/immunology ; Glycolysis ; Nanoparticles/chemistry ; Female ; },
abstract = {While gene-editing-based tumor therapy holds promise, conventional passive-diffusion vectors face limited penetration in dense solid tumors. Here, we developed a ROS-driven gene editing nanomotor (RDN@PL), which takes hemin as the core and encapsulates CRISPR/Cas9 plasmids targeting LDHA (A glycolysis key enzyme). In tumor microenvironments, RDN@PL consumes extracellular ROS to fuel self-diffusiophoresis, achieving higher intratumoral accumulation than passive particles. Upon internalization, heme oxygenase-1 (HO-1) degrades RDN@PL, releasing CO and plasmids. LDHA knockout suppresses glycolysis while CO elevates mitochondrial ROS, which triggers apoptosis by disrupting metabolism and enhancing immunity. Simultaneously, extracellular ROS depletion by non-internalized nanomotors reverses immunogenic cell death (ICD) inhibition, enhancing CD8+ T cell infiltration in tumor. The Janus nanomotor enables extracellular ROS scavenging and intracellular ROS increment via HO-1-responsive cargo release and gene editing. This multi-level intervention strategy demonstrates 93.9 % tumor growth suppression in solid tumor models, providing a blueprint for engineering intelligent nanovesicles in precision oncology.},
}

*Reactive Oxygen Species/metabolism
*Immunotherapy/methods
*Gene Editing/methods
Humans
Animals
Mice
CRISPR-Cas Systems
Cell Line, Tumor
Tumor Microenvironment
Heme Oxygenase-1/metabolism
*Neoplasms/therapy/immunology/genetics
CD8-Positive T-Lymphocytes/immunology
Glycolysis
Nanoparticles/chemistry
Female

@article {pmid40393243,
year = {2025},
author = {Li, H and Lian, S and Zhang, Z and Bi, W and Meng, Q and Ding, Q},
title = {The CRISPR/Cas13a-assisted electrochemiluminescence sensing device combined with entropy-driven and hybrid chain reaction nucleic acid amplification techniques for ultra-sensitive analysis of brain natriuretic peptide.},
journal = {Talanta},
volume = {295},
number = {},
pages = {128310},
doi = {10.1016/j.talanta.2025.128310},
pmid = {40393243},
issn = {1873-3573},
abstract = {Brain natriuretic peptide (BNP) is considered a reliable marker of heart failure disease, and its timely detection can provide important pathological information to prevent or treat heart failure. In this article, an electrochemiluminescence (ECL) sensing device based on a boron carbon nitride/gold nanoparticle (BCN/AuNPs) complex is developed to determine BNP. Prominently, the CRISPR/CAS 13a enzyme reverse cleavage mode, the entropy-driven and hammer hybridization chain reaction processes were involved in the entire detection scheme. Ultimately, with multiple reaction methods and amplification reactions of nucleic acids, this ECL sensing device is able to achieve a detection limit as low as of 0.03 pg/mL and linear range from 0.1 pg/mL to 30 ng/mL for BNP. In addition, the ECL sensing device based on BCN/AuNPs complex obtained satisfactory stability and specificity, and can also be extended to the detection of other pathological markers.},
}

@article {pmid40389868,
year = {2025},
author = {Amoroso, CG and Andolfo, G},
title = {Hazelnut allergome overview and Cor a gRNAs identification.},
journal = {BMC plant biology},
volume = {25},
number = {1},
pages = {661},
pmid = {40389868},
issn = {1471-2229},
support = {E53D23020840001P//Ministero dell'Istruzione, dell'Università e della Ricerca/ ; },
mesh = {*Corylus/genetics/immunology ; *Allergens/genetics/immunology ; *Plant Proteins/genetics/immunology ; Genome, Plant ; CRISPR-Cas Systems ; Phylogeny ; *Antigens, Plant/genetics/immunology ; },
abstract = {BACKGROUND: Corylus species (hazelnuts) are a valuable source of nutrients and are widely consumed worldwide. Nevertheless, Corylus avellana (Cor a) contains 13 allergens (Cor a 1, Cor a 2, Cor a 6, Cor a 8, Cor a 9, Cor a 10, Cor a 11, Cor a 12, Cor a 13, Cor a 14, Cor a 15, Cor a 16, and Cor a TLP) that have been deposited into the official database (WHO/IUIS) for allergen nomenclature. The recent availability of several Corylus genomes provided opportunities to explore allergome variability, and thus to develop hypoallergenic varieties using modern biotech approaches. Certainly, the identification of CRISPR-Cas9 guide RNA (gRNA) is a pivotal step in achieving this goal. User-friendly web tools include limited reference genomes to design CRISPR-Cas9 gRNAs, while bioinformatic software for custom analysis require advanced command-line skills.

RESULTS: This work explored the evolutionary trajectories of allergenic Cor a homologs in C. avellana, C. americana, C. heterophylla, and C. mandshurica genome assemblies. 52 Cor a orthologs were found in the analyzed species, and a recent tandem duplication of Cor a 1 was found in C. americana. Three new gene models were predicted in C. avellana and C. mandshurica for Cor a 16 and Cor a 10. Additionally, we identified 56 Cor a isoallergens, of which ten Cor a isoforms. Furthermore, phylogenetic analysis sheds light on the evolutionary dynamics of three hazelnut allergens revealing the evolutionary complexity of Cor a 1, Cor a 2, and Cor a TLP within the Corylus genus. A list of multiple gRNAs designed for the CRISPR-Cas9 system was provided for the singular and multiple silencing of Cor a homologs in each Corylus genome.

CONCLUSIONS: This study enhances our knowledge on the evolutionary path of Cor a allergens among Corylus species and provides highly accurate on-target guides targeting hazelnut allergome.},
}

*Corylus/genetics/immunology
*Allergens/genetics/immunology
*Plant Proteins/genetics/immunology
Genome, Plant
CRISPR-Cas Systems
Phylogeny
*Antigens, Plant/genetics/immunology

@article {pmid39893321,
year = {2025},
author = {Gumusgoz, E and Kasiri, S and Youssef, I and Verma, M and Chopra, R and Villarreal Acha, D and Wu, J and Marriam, U and Alao, E and Chen, X and Guisso, DR and Gray, SJ and Shah, BR and Minassian, BA},
title = {Focused ultrasound widely broadens AAV-delivered Cas9 distribution and activity.},
journal = {Gene therapy},
volume = {32},
number = {3},
pages = {237-245},
pmid = {39893321},
issn = {1476-5462},
support = {R01 NS127900/NS/NINDS NIH HHS/United States ; P01NS097197//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
mesh = {*Dependovirus/genetics ; Animals ; Mice ; *Genetic Therapy/methods ; Blood-Brain Barrier/metabolism ; Hippocampus/metabolism ; Genetic Vectors/administration & dosage/genetics ; *CRISPR-Associated Protein 9/genetics ; Gene Editing/methods ; CRISPR-Cas Systems ; Humans ; Gene Transfer Techniques ; Disease Models, Animal ; },
abstract = {Because children have little temporal exposure to environment and aging, most pediatric neurological diseases are inherent, i.e. genetic. Since postnatal neurons and astrocytes are mostly non-replicating, gene therapy and genome editing present enormous promise in child neurology. Unlike in other organs, which are highly permissive to adeno-associated viruses (AAV), the mature blood-brain barrier (BBB) greatly limits circulating AAV distribution to the brain. Intrathecal administration improves distribution but to no more than 20% of brain cells. Focused ultrasound (FUS) opens the BBB transiently and safely. In the present work we opened the hippocampal BBB and delivered a Cas9 gene via AAV9 intrathecally. This allowed brain first-pass, and subsequent vascular circulation and re-entry through the opened BBB. The mouse model used was of Lafora disease, a neuroinflammatory disease due to accumulations of misshapen overlong-branched glycogen. Cas9 was targeted to the gene of the glycogen branch-elongating enzyme glycogen synthase. We show that FUS dramatically (2000-fold) improved hippocampal Cas9 distribution and greatly reduced the pathogenic glycogen accumulations and hippocampal inflammation. FUS is in regular clinical use for other indications. Our work shows that it has the potential to vastly broaden gene delivery or editing along with clearance of corresponding pathologic basis of brain disease.},
}

*Dependovirus/genetics
Animals
Mice
*Genetic Therapy/methods
Blood-Brain Barrier/metabolism
Hippocampus/metabolism
Genetic Vectors/administration & dosage/genetics
*CRISPR-Associated Protein 9/genetics
Gene Editing/methods
CRISPR-Cas Systems
Humans
Gene Transfer Techniques
Disease Models, Animal

@article {pmid40389577,
year = {2025},
author = {Lv, C and Zhang, F and Ren, L and Zhu, P and Cheng, X and Yang, X and Chen, C and Liu, B},
title = {Rapid visual detection assay for Bactrocera dorsalis (Hendel) using recombinase polymerase amplification and CRISPR/Cas12b.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {17328},
pmid = {40389577},
issn = {2045-2322},
support = {no. 62272060 and 62272061//National Natural Science Foundation of China/ ; no. 62272060 and 62272061//National Natural Science Foundation of China/ ; no. 62272060 and 62272061//National Natural Science Foundation of China/ ; no. 62272060 and 62272061//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Tephritidae/genetics ; *CRISPR-Cas Systems ; *Recombinases/metabolism/genetics ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; },
abstract = {The oriental fruit fly Bactrocera dorsalis (Hendel) is considered as a quarantine pest in many countries and regions. Challenges remain in distinguishing this species with morphological similarities, especially in relevant development stages. In recent years, CRISPR/Cas12b genetic diagnostics has seen rapid advancements and offers an efficient tool for the identification of pathogens, viruses, and other genetic targets. Here we developed a new and rapid visual detection assay of B. dorsalis using recombinase polymerase amplification (RPA) and the CRISPR/Cas12b system. The system can detect different developmental stages of B. dorsalis within 30-35 min at 43 ℃ and the results are easily observed by the naked eye based on the color change in the tube during the reaction. The specificity and high sensitivity of this method was demonstrated, allowing for detection from 3.2 pg µL[- 1] of DNA. With crude DNA, this diagnostic system successfully identified B. dorsalis by holding the reaction tubes in the hand. Our study demonstrates that RPA-CRISPR/Cas12b visualization system is effective to detect B. dorsalis rapidly and accurately. This approach can be applied for monitoring and identification of other pests in border and relevant locations, preventing biological invasions and ensuring pest control.},
}

Animals
*Tephritidae/genetics
*CRISPR-Cas Systems
*Recombinases/metabolism/genetics
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity

@article {pmid40389480,
year = {2025},
author = {Troester, S and Eder, T and Wukowits, N and Piontek, M and Fernández-Pernas, P and Schmoellerl, J and Haladik, B and Manhart, G and Allram, M and Maurer-Granofszky, M and Scheidegger, N and Nebral, K and Superti-Furga, G and Meisel, R and Bornhauser, B and Valent, P and Dworzak, MN and Zuber, J and Boztug, K and Grebien, F},
title = {Transcriptional and epigenetic rewiring by the NUP98::KDM5A fusion oncoprotein directly activates CDK12.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4656},
pmid = {40389480},
issn = {2041-1723},
support = {P35628//Austrian Science Fund (Fonds zur Förderung der Wissenschaftlichen Forschung)/ ; P35298//Austrian Science Fund (Fonds zur Förderung der Wissenschaftlichen Forschung)/ ; TAI490//Austrian Science Fund (Fonds zur Förderung der Wissenschaftlichen Forschung)/ ; },
mesh = {Humans ; *Nuclear Pore Complex Proteins/metabolism/genetics ; *Oncogene Proteins, Fusion/metabolism/genetics ; *Leukemia, Myeloid, Acute/genetics/metabolism/pathology ; *Cyclin-Dependent Kinases/metabolism/genetics ; *Epigenesis, Genetic ; Cell Line, Tumor ; Animals ; Transcription, Genetic ; Mice ; Gene Expression Regulation, Leukemic ; DNA Damage ; CRISPR-Cas Systems ; Retinoblastoma-Binding Protein 2 ; },
abstract = {Nucleoporin 98 (NUP98) fusion oncoproteins are strong drivers of pediatric acute myeloid leukemia (AML) with poor prognosis. Here we show that NUP98 fusion-expressing AML harbors an epigenetic signature that is characterized by increased accessibility of hematopoietic stem cell genes and enrichment of activating histone marks. We employ an AML model for ligand-induced degradation of the NUP98::KDM5A fusion oncoprotein to identify epigenetic programs and transcriptional targets that are directly regulated by NUP98::KDM5A through CUT&Tag and nascent RNA-seq. Orthogonal genome-wide CRISPR/Cas9 screening identifies 12 direct NUP98::KDM5A target genes, which are essential for AML cell growth. Among these, we validate cyclin-dependent kinase 12 (CDK12) as a druggable vulnerability in NUP98::KDM5A-expressing AML. In line with its role in the transcription of DNA damage repair genes, small-molecule-mediated CDK12 inactivation causes increased DNA damage, leading to AML cell death. Altogether, we show that NUP98::KDM5A directly regulates a core set of essential target genes and reveal CDK12 as an actionable vulnerability in AML with oncogenic NUP98 fusions.},
}

Humans
*Nuclear Pore Complex Proteins/metabolism/genetics
*Oncogene Proteins, Fusion/metabolism/genetics
*Leukemia, Myeloid, Acute/genetics/metabolism/pathology
*Cyclin-Dependent Kinases/metabolism/genetics
*Epigenesis, Genetic
Cell Line, Tumor
Animals
Transcription, Genetic
Mice
Gene Expression Regulation, Leukemic
DNA Damage
CRISPR-Cas Systems
Retinoblastoma-Binding Protein 2