@article {pmid40653316,
year = {2025},
author = {Kouvela, A and Jaramillo Ponce, JR and Giarimoglou, N and Chicher, J and Marzi, S and Stathopoulos, C and Stamatopoulou, V},
title = {Coupling tRNAGly gene redundancy with staphylococcal cell wall integrity, antibiotic susceptibility, and virulence potential.},
journal = {Nucleic acids research},
volume = {53},
number = {13},
pages = {},
doi = {10.1093/nar/gkaf599},
pmid = {40653316},
issn = {1362-4962},
support = {//University of Patras/ ; },
mesh = {*Cell Wall/genetics/drug effects/metabolism ; *Staphylococcus aureus/genetics/pathogenicity/drug effects ; Humans ; Anti-Bacterial Agents/pharmacology ; Virulence/genetics ; CRISPR-Cas Systems ; Biofilms/growth & development/drug effects ; Gene Expression Regulation, Bacterial ; Riboswitch ; Staphylococcal Infections/microbiology ; Protein Biosynthesis ; Gene Editing ; },
abstract = {Redundancy in transfer RNA (tRNA) gene copies across species remains poorly understood and, in many cases, largely unexplored. In Staphylococcus aureus, multiple tRNAGly genes encode isoacceptors involved in protein synthesis and cell wall formation, aminoacylated by a sole glycyl-tRNA synthetase (GlyRS) which is under the transcription regulation of a species-specific glyS T-box riboswitch. The T-box can interact with all tRNAGly isoacceptors to adopt species-specific conformations and affect both pathways. Using CRISPR/Cas9 editing, we ablated a gene copy corresponding to the proteinogenic P1 tRNAGlyGCC. Surprisingly, the growth and the overall translational activity of the edited strain were found unaffected, suggesting functional compensation by the remaining tRNAGly genes. On the other hand, transcriptomics and proteomics analyses combined with functional assays revealed nutrient-dependent stress responses with surprisingly impaired cell wall integrity and increased susceptibility to cell wall-targeting antibiotics. Additionally, the edited strain displayed reduced biofilm formation but retained the ability to invade human cells in vitro. Overall, the present study underscores the critical role of tRNA gene redundancy in the physiology of S. aureus and highlights tRNAs as regulators of metabolic homeostasis in pathogenic bacteria.},
}

*Cell Wall/genetics/drug effects/metabolism
*Staphylococcus aureus/genetics/pathogenicity/drug effects
Humans
Anti-Bacterial Agents/pharmacology
Virulence/genetics
CRISPR-Cas Systems
Biofilms/growth & development/drug effects
Gene Expression Regulation, Bacterial
Riboswitch
Staphylococcal Infections/microbiology
Protein Biosynthesis
Gene Editing

@article {pmid40652883,
year = {2025},
author = {Zhang, S and Kim, JC and Ahn, J},
title = {Phage engineering strategies to expand host range for controlling antibiotic-resistant pathogens.},
journal = {Microbiological research},
volume = {300},
number = {},
pages = {128278},
doi = {10.1016/j.micres.2025.128278},
pmid = {40652883},
issn = {1618-0623},
abstract = {Bacteriophages are known as a promising alternative to control rising bacterial resistance. The adsorption phase is critical for the successful infection of phages, as it determines their ability to recognize and attach to specific bacterial host cells. However, their limited host ranges due to narrow host specificity significantly limit their potential applications and overall effectiveness. Receptor-binding proteins (RBPs) are crucial in the recognition process, and modifying these proteins provides a valuable opportunity to broaden host ranges and enhance adsorption rates. Therefore, gaining a more comprehensive understanding of the interactions between phages and their bacterial hosts is essential. To overcome this challenge, various in vivo and in vitro engineering platforms have been developed, including recombineering, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins (Cas) systems, yeast-based technologies, and cell-free systems. These methods provide diverse strategies and flexibility for constructing customized phage genomes with desired characteristics, ultimately enhancing phage application efficiency. This review discusses different types of RBPs in phages and their mechanisms of adsorption, highlighting their relevance for adaptable engineering strategies. We also summarize various phage engineering platforms and explore the design of synthetic phages with expanded host ranges. Finally, we highlight the advantages and limitations of current engineering methods, providing insights to guide future research efforts.},
}

@article {pmid40652308,
year = {2025},
author = {Oliver-Caldes, A and Mañe Pujol, J and Battram, AM and Perez-Amill, L and Bachiller, M and Calderon, H and Castella, M and Carpio, J and Salsench, SV and Tovar, N and Cardus, O and Urbano-Ispizua, A and Moreno, DF and Rodríguez-Lobato, LG and Lozano, E and Rosiñol, L and Juan, M and Martín-Antonio, B and Fernández de Larrea, C},
title = {TIGIT blockade in the context of BCMA-CART cell therapy does not augment efficacy in a multiple myeloma mouse model.},
journal = {Oncoimmunology},
volume = {14},
number = {1},
pages = {2529632},
doi = {10.1080/2162402X.2025.2529632},
pmid = {40652308},
issn = {2162-402X},
mesh = {Animals ; *Multiple Myeloma/therapy/immunology/pathology ; *Receptors, Immunologic/antagonists & inhibitors/immunology/genetics ; Mice ; *Immunotherapy, Adoptive/methods ; Humans ; Disease Models, Animal ; *Receptors, Chimeric Antigen/immunology ; *B-Cell Maturation Antigen/immunology ; Cell Line, Tumor ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; Female ; },
abstract = {BCMA-directed CAR-T therapies have shown promising results in multiple myeloma (MM). However, patients continue to relapse. T cell exhaustion with increased TIGIT expression is a resistance mechanism which was confirmed in CAR-T cells from ARI0002h trial, an academic CAR-T developed in our institution. We aimed to analyze the impact of blocking TIGIT on the efficacy of ARI0002h. We used three different strategies to block TIGIT: (1) Addition of an external blocking anti-TIGIT-antibody (Ab), (2) Modify ARI0002h into a 4[th] generation CAR-T, named ARITIGIT, capable of secreting a soluble TIGIT-blocking scFv and (3) TIGIT knock-out in ARI0002h using CRISPR/Cas9. Each strategy was evaluated in vitro and in vivo. Adding a TIGIT-blocking Ab to ARI0002h improved in vitro cytotoxicity, but failed to enhance mice survival. The new 4[th] generation CAR-T, ARITIGIT, was also unable to achieve better survival outcomes despite favoring the in vivo model by using a myeloma cell line with high expression of the TIGIT ligand PVR. Interestingly, when mice were challenged with a second infusion of tumor cells, mimicking a relapse model, a trend for improved survival with ARITIGIT was observed (p = 0.11). Finally, TIGIT-knock-out on ARI0002h (KO-ARI0002h) using CRISPR/Cas9 showed similar in vitro activity to ARI0002h. In an in vivo stress model, TIGIT KO-ARI0002h prolonged survival (p = 0.02). However, this improvement was not significant compared to ARI0002h (p = 0.07). This study failed to demonstrate a significant benefit of TIGIT-blockade on ARI0002h cells despite using three different approaches, suggesting that targeting a single immune checkpoint may be insufficient.},
}

Animals
*Multiple Myeloma/therapy/immunology/pathology
*Receptors, Immunologic/antagonists & inhibitors/immunology/genetics
Mice
*Immunotherapy, Adoptive/methods
Humans
Disease Models, Animal
*Receptors, Chimeric Antigen/immunology
*B-Cell Maturation Antigen/immunology
Cell Line, Tumor
Xenograft Model Antitumor Assays
CRISPR-Cas Systems
Female

@article {pmid40490045,
year = {2025},
author = {Ye, X and Jia, H and Zu, Y},
title = {lmod2a mutations affect F-actin and SRF pathway leading to cardiac dysfunction in zebrafish.},
journal = {Developmental biology},
volume = {525},
number = {},
pages = {306-316},
doi = {10.1016/j.ydbio.2025.06.002},
pmid = {40490045},
issn = {1095-564X},
mesh = {Animals ; *Zebrafish/genetics/embryology/metabolism ; *Actins/metabolism/genetics ; *Serum Response Factor/metabolism/genetics ; *Zebrafish Proteins/genetics/metabolism ; Mutation/genetics ; Signal Transduction/genetics ; Cardiomyopathy, Dilated/genetics ; *Heart/embryology/physiopathology ; *Muscle Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Microfilament Proteins/genetics/metabolism ; Disease Models, Animal ; },
abstract = {Leiomodin 2 (LMOD2), a critical pathogenic gene associated with human dilated cardiomyopathy (DCM), is essential in regulating thin filament length during cardiac development. This study generated a homozygous knockout zebrafish line (lmod2a[-/-]) using CRISPR/Cas9 genome editing. lmod2a[-/-] embryos exhibited impaired locomotor activity alongside irregular heart rhythms, reduced cardiac output, compromised contractility, and delayed calcium transients, as revealed by high-speed imaging and calcium optical mapping. Immunofluorescence staining demonstrated a marked reduction in filamentous actin (F-actin), corroborated by QPCR data showing downregulation of the F-actin marker gene acta1b. Moreover, expression levels of key downstream targets of the serum response factor (SRF) signaling pathway were markedly reduced in mutants. These findings indicate that lmod2a deficiency disrupts F-actin homeostasis and SRF-mediated gene regulation, ultimately leading to defective cardiac performance. This study establishes a novel zebrafish model for investigating LMOD-associated cardiomyopathies and provides valuable insights for future therapeutic interventions targeting actin-related cardiac disorders.},
}

Animals
*Zebrafish/genetics/embryology/metabolism
*Actins/metabolism/genetics
*Serum Response Factor/metabolism/genetics
*Zebrafish Proteins/genetics/metabolism
Mutation/genetics
Signal Transduction/genetics
Cardiomyopathy, Dilated/genetics
*Heart/embryology/physiopathology
*Muscle Proteins/genetics/metabolism
CRISPR-Cas Systems
*Microfilament Proteins/genetics/metabolism
Disease Models, Animal

@article {pmid40404079,
year = {2025},
author = {Ferrari, K and Gurung, S and Loges, LN and Batta, SPR and Hammond, MA and Griciunaite, M and DeMoya, R and Restrepo, NK and Sumanas, S},
title = {Zebrafish Kelch-like family member 4 is required for vasculogenesis and hematopoiesis.},
journal = {Developmental biology},
volume = {525},
number = {},
pages = {1-12},
pmid = {40404079},
issn = {1095-564X},
mesh = {Animals ; *Zebrafish/embryology/genetics/metabolism ; *Hematopoiesis/physiology/genetics ; *Zebrafish Proteins/genetics/metabolism ; *Neovascularization, Physiologic/genetics/physiology ; Gene Expression Regulation, Developmental ; Hematopoietic Stem Cells/metabolism/cytology ; Endothelial Cells/metabolism ; Apoptosis ; Embryo, Nonmammalian/metabolism ; CRISPR-Cas Systems ; Cell Proliferation ; Transcription Factors/metabolism/genetics ; Mutation ; },
abstract = {Molecular mechanisms regulating vascular development and hematopoiesis are still incompletely understood. The KLHL (Kelch-like) family of proteins function as adapters to target proteins for ubiquitination. However, their role in vascular development has not been previously analyzed. Here we have characterized a novel regulator of vascular development, kelch-like family member 4 (klhl4) in zebrafish. We show that zebrafish klhl4 is expressed in early vascular endothelial and hematopoietic progenitors, while its expression is restricted to vascular endothelial cells during later developmental stages. To determine the functional role of klhl4, we generated loss-of-function zebrafish mutants using CRISPR/Cas9 genome editing. klhl4 mutant embryos were viable, yet they exhibited delayed sprouting of intersegmental vessels (ISVs), which correlated with reduced expression of vascular endothelial and erythroid specific molecular markers. Time-lapse imaging showed that vascular endothelial and hematopoietic progenitor cells exhibit delayed migration towards the midline and undergo increased apoptosis and reduced proliferation in klhl4 mutants. Expression of npas4l and etv2/etsrp, two master regulators of endothelial and hematopoietic development, was reduced in klhl4 mutants, suggesting that some vascular defects could be caused by the reduction of npas4l and etv2 expression. However, npas4l or etv2 overexpression failed to rescue ISV sprouting defects in klhl4 mutants, suggesting that klhl4 may promote vasculogenesis by additional mechanisms. In summary, our findings demonstrate a novel role for zebrafish klhl4 in regulating vascular endothelial and hematopoietic development during embryogenesis. Because the Klhl4 protein sequence is highly conserved between different vertebrates, it is likely that it may play a similar role in other organisms.},
}

Animals
*Zebrafish/embryology/genetics/metabolism
*Hematopoiesis/physiology/genetics
*Zebrafish Proteins/genetics/metabolism
*Neovascularization, Physiologic/genetics/physiology
Gene Expression Regulation, Developmental
Hematopoietic Stem Cells/metabolism/cytology
Endothelial Cells/metabolism
Apoptosis
Embryo, Nonmammalian/metabolism
CRISPR-Cas Systems
Cell Proliferation
Transcription Factors/metabolism/genetics
Mutation

@article {pmid40651052,
year = {2025},
author = {Arutselvan, R and Kumar, S and Akash, AU and Greeshma, K and Sinha, SS and Khan, AS and Pati, K and Chauhan, VBS and Gowda, KH and Pradhan, S and Jeeva, ML and Veena, SS and Makeshkumar, T and Meena, M and Sangeetha, BG and Laxminarayana, K and Nedunchezhiyan, M},
title = {Deciphering the complex signaling networks in phytophthora infected plants: Insights into microbiome interactions and plant defense mechanisms.},
journal = {Plant physiology and biochemistry : PPB},
volume = {228},
number = {},
pages = {110222},
doi = {10.1016/j.plaphy.2025.110222},
pmid = {40651052},
issn = {1873-2690},
abstract = {Phytophthora species are destructive plant pathogens that cause severe economic losses in agriculture and natural ecosystems, known for their rapid spread through soil and water and resistance to conventional control methods. Understanding the complex signaling networks activated in plants during Phytophthora infection is crucial for developing effective management strategies. This review summarizes research findings on Phytophthora-plant interactions, with special emphasis on Phytophthora-plant microbiome interactions. Initially, molecular mechanisms involved in the plant response to Phytophthora infection are discussed, further emphasizing key signaling pathways activated by Phytophthora in host plants. The role of phytohormones in imparting resistance to Phytophthora infections is explored in depth. Additionally, the interaction and effects of Phytophthora and the plant immune system with the plant microbiome are examined, highlighting how these interactions facilitate disease and/or aid in plant defense. Various biotechnological approaches for enhancing plant resistance to Phytophthora, including recent technologies like CRISPR-Cas systems, are also reviewed. The conclusion addresses the need for further research into signaling networks within Phytophthora-plant-microbiome interactions and their future implications for crop protection.},
}

@article {pmid40650977,
year = {2025},
author = {Faber, A and Politan, RJ and Stukenberg, D and Morris, KM and Kim, R and Jeon, E and Inckemann, R and Becker, A and Thuronyi, B and Fritz, G},
title = {Expanding genetic engineering capabilities in Vibrio natriegens with the Vnat Collection.},
journal = {Nucleic acids research},
volume = {53},
number = {13},
pages = {},
doi = {10.1093/nar/gkaf580},
pmid = {40650977},
issn = {1362-4962},
support = {FT230100283//Australian Research Council/ ; //Forrest Research Foundation/ ; //Forrest Research Foundation Scholarship/ ; GRK 2937//Deutsche Forschungsgemeinschaft/ ; //Williams College/ ; //Australian Research Council/ ; },
mesh = {*Vibrio/genetics/metabolism ; *Genetic Engineering/methods ; Promoter Regions, Genetic ; Synthetic Biology/methods ; Metabolic Engineering/methods ; Gene Editing/methods ; CRISPR-Cas Systems ; Operon ; Cloning, Molecular ; },
abstract = {Vibrio natriegens, with its exceptionally fast growth rate, has great promise as a revolutionary chassis for synthetic biology, yet the realization of its full potential has been limited by the lack of robust, standardized genetic tools. Here, we present the Vnat Collection, a comprehensive, modular toolkit specifically engineered to overcome these limitations. Leveraging optimized Golden Gate cloning strategies, we introduce improved junction sequences and a highly efficient dropout part system, achieving up to a 300-fold increase in assembly efficiency. Our toolkit significantly expands the synthetic biology toolbox by providing a wide array of characterized inducible promoters, enabling precise, orthogonal gene regulation, and novel operon connectors to streamline the construction of multi-gene pathways critical for metabolic engineering. Furthermore, we enhance genome editing workflows through refined NT-CRISPR methods, incorporating homology-flanked targeting constructs and demonstrating a simplified protocol that eliminates intermediate purification steps. With over 220 rigorously validated modular components, the Vnat Collection establishes an advanced standard for genetic engineering of V. natriegens, empowering researchers to efficiently harness this organism's unparalleled potential for diverse biotechnology applications.},
}

*Vibrio/genetics/metabolism
*Genetic Engineering/methods
Promoter Regions, Genetic
Synthetic Biology/methods
Metabolic Engineering/methods
Gene Editing/methods
CRISPR-Cas Systems
Operon
Cloning, Molecular

@article {pmid40650973,
year = {2025},
author = {Chi, H and McMahon, S and Daniel-Pedersen, L and Graham, S and Gloster, TM and White, MF},
title = {SAM-AMP lyases in type III CRISPR defence.},
journal = {Nucleic acids research},
volume = {53},
number = {13},
pages = {},
doi = {10.1093/nar/gkaf655},
pmid = {40650973},
issn = {1362-4962},
support = {BB/T004789/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; 101018608//European Research Council Advanced Grant/ ; //University of St Andrews/ ; },
mesh = {S-Adenosylmethionine/metabolism ; *CRISPR-Cas Systems ; Escherichia coli/genetics ; *Bacterial Proteins/chemistry/metabolism/genetics ; Thionucleosides/metabolism/chemistry ; Models, Molecular ; *Lyases/chemistry/metabolism/genetics ; Deoxyadenosines/metabolism/chemistry ; Crystallography, X-Ray ; },
abstract = {Type III CRISPR systems detect non-self RNA and activate the enzymatic Cas10 subunit, which generates nucleotide second messengers for activation of ancillary effectors. Although most signal via cyclic oligoadenylate, an alternative class of signalling molecule SAM-AMP, formed by conjugating ATP and S-adenosylmethionine, was described recently. SAM-AMP activates a trans-membrane effector of the CorA magnesium transporter family to provide anti-phage defence. Intriguingly, immunity also requires SAM-AMP degradation by means of a specialized CRISPR-encoded NrN family phosphodiesterase in Bacteroides fragilis. In Clostridium botulinum, the nrn gene is replaced by a gene encoding a SAM-AMP lyase. Here, we investigate the structure and activity of C. botulinum SAM-AMP lyase, which can substitute for the nrn gene to provide CorA-mediated immunity in Escherichia coli. The structure of SAM-AMP lyase bound to its reaction product 5'-methylthioadenosine-AMP reveals key details of substrate binding and turnover by this PII superfamily protein. Bioinformatic analysis revealed a phage-encoded SAM-AMP lyase that degrades SAM-AMP efficiently in vitro, consistent with an anti-CRISPR function.},
}

S-Adenosylmethionine/metabolism
*CRISPR-Cas Systems
Escherichia coli/genetics
*Bacterial Proteins/chemistry/metabolism/genetics
Thionucleosides/metabolism/chemistry
Models, Molecular
*Lyases/chemistry/metabolism/genetics
Deoxyadenosines/metabolism/chemistry
Crystallography, X-Ray

@article {pmid40650969,
year = {2025},
author = {Madariaga-Marcos, J and Baltramonaitis, M and Henkel-Heinecke, S and Kauert, DJ and Irmisch, P and Bigelyte-Stankeviciene, G and Silanskas, A and Karvelis, T and Siksnys, V and Sasnauskas, G and Seidel, R},
title = {Structural and mechanistic insights into the sequential dsDNA cleavage by SpCas12f1.},
journal = {Nucleic acids research},
volume = {53},
number = {13},
pages = {},
doi = {10.1093/nar/gkaf588},
pmid = {40650969},
issn = {1362-4962},
support = {S-MIP-19-32//Research Council of Lithuania/ ; 02-002-P-0001//Mission-driven Implementation of Science and Innovation Programmes/ ; //New Generation Lithuania/ ; GA 724863/ERC_/European Research Council/International ; SPP2141//Deutsche Forschungsgemeinschaft/ ; SE 1646/8-1//Deutsche Forschungsgemeinschaft/ ; //Alexander von Humboldt Foundation/ ; //Leipzig University/ ; //German Research Foundation/ ; },
mesh = {*DNA Cleavage ; *DNA/metabolism/chemistry/genetics ; *CRISPR-Associated Proteins/chemistry/metabolism/genetics ; CRISPR-Cas Systems ; Models, Molecular ; Kinetics ; Catalytic Domain ; R-Loop Structures ; Protein Binding ; *Bacterial Proteins/chemistry/metabolism/genetics ; },
abstract = {Miniature CRISPR-Cas12f1 effector complexes have recently attracted considerable interest for genome engineering applications due to their compact size. Unlike other Class 2 effectors, Cas12f1 functions as a homodimer bound to a single ∼200 nt RNA. While the basic biochemical properties of Cas12f1, such as its use of a single catalytic center for catalysis, have been characterized, the orchestration of the different events occurring during Cas12f1 reactions remained little explored. To gain insights into the dynamics and mechanisms involved in DNA recognition and cleavage by Cas12f1 from Syntrophomonas palmitatica (SpCas12f1), we solved the structure of SpCas12f1 bound to target DNA and employed single-molecule magnetic tweezers measurements in combination with ensemble kinetic measurements. Our data indicate that SpCas12f1 forms 18 bp R-loops, in which local contacts of the protein to the R-loop stabilize R-loop intermediates. DNA cleavage is catalyzed by a single SpCas12f1 catalytic center, which first rapidly degrades a ∼11 bp region on the nontarget strand by cutting at random sites. Subsequent target strand cleavage is slower and requires at least a nick in the nontarget strand.},
}

*DNA Cleavage
*DNA/metabolism/chemistry/genetics
*CRISPR-Associated Proteins/chemistry/metabolism/genetics
CRISPR-Cas Systems
Models, Molecular
Kinetics
Catalytic Domain
R-Loop Structures
Protein Binding
*Bacterial Proteins/chemistry/metabolism/genetics

@article {pmid40650785,
year = {2025},
author = {Samuels, M and Besta, S and Betrán, AL and Nia, RS and Xie, X and Gu, X and Shu, Q and Giamas, G},
title = {CRISPR screening approaches in breast cancer research.},
journal = {Cancer metastasis reviews},
volume = {44},
number = {3},
pages = {59},
pmid = {40650785},
issn = {1573-7233},
mesh = {Humans ; *Breast Neoplasms/genetics/diagnosis/pathology/therapy ; Female ; *CRISPR-Cas Systems ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The emergence of CRISPR-Cas9 technology has transformed functional genomics, offering unmatched opportunities to dissect and understand biological pathways and identify novel therapeutic targets in cancer. Breast cancer is a complex, heterogeneous disease and remains a major cause of morbidity and mortality in women, particularly when diagnosed at advanced or metastatic stages where effective treatments are limited. High-throughput CRISPR screening is undoubtedly a powerful tool to discover novel drug targets, uncover synthetic lethal interactions, and identify vulnerabilities in cancer. This review focuses on advances in our understanding of breast cancer developed through CRISPR-based screening technology, particularly in identifying drivers of breast cancer progression, growth, and metastasis, as well as in identifying potential new therapeutic targets and combination therapies. We discuss recent discoveries, current challenges, and limitations of this approach and explore how advancements in CRISPR technology could have a profound impact on the future of breast cancer treatment.},
}

Humans
*Breast Neoplasms/genetics/diagnosis/pathology/therapy
Female
*CRISPR-Cas Systems
Animals
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40650287,
year = {2025},
author = {Gao, Z and Long, T and Guo, P and Luo, J and Nie, X and Xie, Q and Chen, G and Hu, Z},
title = {The Nuclear Transcription Factor SlNF-YC9 Regulates the Protrusion of Tomato Fruit Tip.},
journal = {International journal of molecular sciences},
volume = {26},
number = {13},
pages = {},
doi = {10.3390/ijms26136511},
pmid = {40650287},
issn = {1422-0067},
support = {31872121//National Natural Science Foundation of China/ ; },
mesh = {*Solanum lycopersicum/genetics/metabolism/growth & development ; *Fruit/genetics/growth & development/metabolism ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; *Transcription Factors/genetics/metabolism ; Promoter Regions, Genetic ; *CCAAT-Binding Factor/genetics/metabolism ; Indoleacetic Acids/metabolism ; CRISPR-Cas Systems ; },
abstract = {NF-Y transcriptional regulators play crucial roles in diverse biological processes in plants, primarily through the formation of NF-Y complexes that bind to specific DNA motifs. These complexes modulate the expression of downstream genes, which influence plant development and growth. In our research, the function of the NF-Y family C subunit member SlNF-YC9 gene in tomato was investigated with the CRISPR/Cas9 method. In contrast to the WT (wild type), the mutant CR-SlNF-YC9 exhibited a prominent protrusion at the fruit tip. The quantitative PCR analysis displayed that the transcription levels of genes associated with auxin transport (PIN4, PIN5, and PIN9) as well as auxin response genes (ARF7 and LAX3) were enhanced in the CR-SlNF-YC9 fruits than in the WT. Analysis of dual-luciferase reporter and EMSA assays showed that the SlNF-YC9-YB13b-YA7a trimer specifically binds the FUL2 promoter and represses its expression. In conclusion, our results suggest that SlNF-YC9 is crucial in influencing tomato fruit shape by the formation of NF-Y heterotrimeric complexes.},
}

*Solanum lycopersicum/genetics/metabolism/growth & development
*Fruit/genetics/growth & development/metabolism
Gene Expression Regulation, Plant
*Plant Proteins/genetics/metabolism
*Transcription Factors/genetics/metabolism
Promoter Regions, Genetic
*CCAAT-Binding Factor/genetics/metabolism
Indoleacetic Acids/metabolism
CRISPR-Cas Systems

@article {pmid40650152,
year = {2025},
author = {Kovalev, MA and Mamaeva, NY and Kristovskiy, NV and Feskin, PG and Vinnikov, RS and Oleinikov, PD and Sosnovtseva, AO and Yakovlev, VA and Glukhov, GS and Shaytan, AK},
title = {Epigenome Engineering Using dCas Systems for Biomedical Applications and Biotechnology: Current Achievements, Opportunities and Challenges.},
journal = {International journal of molecular sciences},
volume = {26},
number = {13},
pages = {},
doi = {10.3390/ijms26136371},
pmid = {40650152},
issn = {1422-0067},
support = {075-15-2024-539//Russian Ministry of Science and Higher Education/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Epigenome/genetics ; *Biotechnology/methods ; Animals ; *Epigenesis, Genetic ; *Epigenomics/methods ; Epigenome Editing ; },
abstract = {Epigenome engineering, particularly utilizing CRISPR/dCas-based systems, is a powerful strategy to modulate gene expression and genome functioning without altering the DNA sequence. In this review we summarized current achievements and prospects in dCas-mediated epigenome editing, primarily focusing on its applications in biomedicine, but also providing a wider context for its applications in biotechnology. The diversity of CRISPR/dCas architectures is outlined, recent innovations in the design of epigenetic editors and delivery methods are highlighted, and the therapeutic potential across a wide range of diseases, including hereditary, neurodegenerative, and metabolic disorders, is examined. Opportunities for the application of dCas-based tools in animal, agricultural, and industrial biotechnology are also discussed. Despite substantial progress, challenges, such as delivery efficiency, specificity, stability of induced epigenetic modifications, and clinical translation, are emphasized. Future directions aimed at enhancing the efficacy, safety, and practical applicability of epigenome engineering technologies are proposed.},
}

Humans
*CRISPR-Cas Systems
*Gene Editing/methods
*Epigenome/genetics
*Biotechnology/methods
Animals
*Epigenesis, Genetic
*Epigenomics/methods
Epigenome Editing

@article {pmid40650151,
year = {2025},
author = {Jung, YJ and Kim, JY and Cho, YG and Kang, KK},
title = {CRISPR/Cas9-Mediated Knockout of OsbZIP76 Reveals Its Role in ABA-Associated Immune Signaling in Rice.},
journal = {International journal of molecular sciences},
volume = {26},
number = {13},
pages = {},
doi = {10.3390/ijms26136374},
pmid = {40650151},
issn = {1422-0067},
support = {RS-2024-00322378//New Breeding Technologies Development Program/ ; },
mesh = {*Oryza/genetics/immunology/microbiology/metabolism ; *Abscisic Acid/metabolism/pharmacology ; *CRISPR-Cas Systems ; *Plant Proteins/genetics/metabolism ; Signal Transduction ; Plant Diseases/microbiology/genetics/immunology ; Gene Expression Regulation, Plant ; Gene Knockout Techniques ; Xanthomonas/pathogenicity ; Disease Resistance/genetics ; *Basic-Leucine Zipper Transcription Factors/genetics/metabolism ; *Plant Immunity/genetics ; Ascomycota ; },
abstract = {The basic leucine zipper (bZIP) transcription factors are involved in a wide range of physiological processes in plants, including hormone signaling, stress responses, and growth and development regulation. They play a key role in abscisic acid (ABA)-mediated immune regulation. However, the immune-related function of OsbZIP76 in rice remains poorly understood. In this study, we generated OsbZIP76 knockout (KO) lines using CRISPR/Cas9-mediated genome editing and examined their phenotypic responses to the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo) and the fungal pathogen Magnaporthe oryzae. The KO lines showed increased susceptibility to both pathogens compared to wild-type (WT) plants. Furthermore, qRT-PCR analysis revealed that, upon pathogen infection, the expression of pathogenesis-related genes such as PR1a, PR5, and NPR1 was significantly suppressed in the KO lines. ABA treatment experiments showed that KO lines were hypersensitive to exogenous ABA, indicating a role for OsbZIP76 in ABA perception and signaling. Notably, the expression of the OsbZIP76 gene itself was strongly induced by both ABA treatment and pathogen infection, supporting its role as a positive regulator in ABA-associated immune signaling. Overall, this study demonstrates that OsbZIP76 functions as an important immune regulator by integrating defense gene expression with ABA signaling, providing new insights into the molecular crosstalk between hormonal signaling and pathogen defense mechanisms.},
}

*Oryza/genetics/immunology/microbiology/metabolism
*Abscisic Acid/metabolism/pharmacology
*CRISPR-Cas Systems
*Plant Proteins/genetics/metabolism
Signal Transduction
Plant Diseases/microbiology/genetics/immunology
Gene Expression Regulation, Plant
Gene Knockout Techniques
Xanthomonas/pathogenicity
Disease Resistance/genetics
*Basic-Leucine Zipper Transcription Factors/genetics/metabolism
*Plant Immunity/genetics
Ascomycota

@article {pmid40650142,
year = {2025},
author = {Becchi, G and Whitehead, M and Harvey, JP and Sladen, PE and Dushti, M and Chapple, JP and Yu-Wai-Man, P and Cheetham, ME},
title = {CRISPRa-Mediated Increase of OPA1 Expression in Dominant Optic Atrophy.},
journal = {International journal of molecular sciences},
volume = {26},
number = {13},
pages = {},
doi = {10.3390/ijms26136364},
pmid = {40650142},
issn = {1422-0067},
support = {5167/5168//Fight for Sight/ ; },
mesh = {*GTP Phosphohydrolases/genetics/metabolism ; Humans ; *Optic Atrophy, Autosomal Dominant/genetics/metabolism/therapy/pathology ; HEK293 Cells ; *CRISPR-Cas Systems ; Mitochondria/metabolism/genetics ; Gene Editing/methods ; Gene Expression Regulation ; Retinal Ganglion Cells/metabolism/pathology ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Dominant Optic Atrophy (DOA) is the most common inherited optic neuropathy and presents as gradual visual loss caused by the loss of retinal ganglion cells (RGCs). Over 60% of DOA cases are caused by pathogenic variants in the OPA1 gene, which encodes a mitochondrial GTPase essential in mitochondrial fusion. Currently, there are no treatments for DOA. Here, we tested the therapeutic potential of an approach to DOA using CRISPR activation (CRISPRa). Homology directed repair was used to introduce a common OPA1 pathogenic variant (c.2708_2711TTAGdel) into HEK293T cells as an in vitro model of DOA. Heterozygous c.2708_2711TTAGdel cells had reduced levels of OPA1 mRNA transcript, OPA1 protein, and mitochondrial network alterations. The effect of inactivated Cas9 fused to an activator (dCas9-VPR) was tested with a range of guide RNAs (gRNA) targeted to the promotor region of OPA1. gRNA3 and dCas9-VPR increased OPA1 expression at the RNA and protein level towards control levels. Importantly, the correct ratio of OPA1 isoform transcripts was maintained by CRISPRa. CRISPRa-treated cells showed an improvement in mitochondrial networks compared to untreated cells, indicating partial rescue of a disease-associated phenotype. Collectively, these data support the potential application of CRISPRa as a therapeutic intervention in DOA.},
}

*GTP Phosphohydrolases/genetics/metabolism
Humans
*Optic Atrophy, Autosomal Dominant/genetics/metabolism/therapy/pathology
HEK293 Cells
*CRISPR-Cas Systems
Mitochondria/metabolism/genetics
Gene Editing/methods
Gene Expression Regulation
Retinal Ganglion Cells/metabolism/pathology
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40650127,
year = {2025},
author = {El Hazzouri, S and Al-Rifai, R and Surges, N and Rath, M and Singer, H and Oldenburg, J and El-Maarri, O},
title = {FVIII Trafficking Dynamics Across Subcellular Organelles Using CRISPR/Cas9 Specific Gene Knockouts.},
journal = {International journal of molecular sciences},
volume = {26},
number = {13},
pages = {},
doi = {10.3390/ijms26136349},
pmid = {40650127},
issn = {1422-0067},
support = {2019-7-05; 2020-7-05; 2022-7-02//University Hospital Bonn/ ; IISR-2023-DE-200366//Takeda (Germany)/ ; 91724372//German Academic Exchange Service/ ; Internal Funding//Institute of Experimental Hematology and Transfusion Medicine - University Hospital Bonn/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; HEK293 Cells ; Protein Transport ; Gene Knockout Techniques ; *Factor VIII/metabolism/genetics ; Calnexin/metabolism/genetics ; Endoplasmic Reticulum/metabolism ; rab GTP-Binding Proteins/metabolism ; *Organelles/metabolism ; rab7 GTP-Binding Proteins ; Calreticulin/metabolism/genetics ; Golgi Apparatus/metabolism ; },
abstract = {Factor VIII (FVIII) interacts with Endoplasmic Reticulum (ER) chaperones Calnexin (CANX) and Calreticulin (CALR) and with ER-Golgi Intermediate Compartment (ERGIC) transporters, Lectin, mannose-binding 1 (LMAN1) and Multiple Coagulation Deficiency 2 (MCFD2). We previously reported that the Gamma-aminobutyric Acid Receptor-associated proteins (GABARAPs) also influence FVIII secretion. Here, we further investigated the intracellular dynamics of FVIII using single and double CRISPR/Cas9 Knockout (KO) models of the abovementioned chaperones as well as the GABARAP proteins in HEK293 cells expressing FVIII. Cellular pathways were manipulated by Brefeldin A (BFA), Chloroquine (CQ), a Rab7 inhibitor, and subjected to glucose starvation. The effect of each KO on FVIII secretion and organelle distribution was assessed by a two-stage chromogenic assay and immunofluorescence (IF) microscopy, prior and upon cell treatments. Using these approaches, we first observed distinct effects of each studied protein on FVIII trafficking. Notably, intracellular localization patterns revealed clustering of FVIII phenotypes in GABARAP[KO], CANX[KO], and CALR[KO] cells together under both basal and treated conditions, an observation that was also reflected in their respective double KO combinations. Besides, a clear involvement of additional components of the endomembrane system was evident, specifically at the trans-Golgi space, as marked by FVIII colocalization with the Ras-like proteins in brain (Rab8 and Rab7) and with the Vesicle-Associated Membrane Protein (VAMP8), along with the observed impact of the selected cell treatments on FVIII phenotypes. These outcomes enhance our understanding of the molecular mechanisms regulating FVIII and pave the way for new perspectives, which could be further projected into FVIII replacement, cell and gene therapies.},
}

Humans
*CRISPR-Cas Systems
HEK293 Cells
Protein Transport
Gene Knockout Techniques
*Factor VIII/metabolism/genetics
Calnexin/metabolism/genetics
Endoplasmic Reticulum/metabolism
rab GTP-Binding Proteins/metabolism
*Organelles/metabolism
rab7 GTP-Binding Proteins
Calreticulin/metabolism/genetics
Golgi Apparatus/metabolism

@article {pmid40650046,
year = {2025},
author = {Cattaneo, M and Giagnorio, E and Lauria, G and Marcuzzo, S},
title = {Therapeutic Approaches for C9ORF72-Related ALS: Current Strategies and Future Horizons.},
journal = {International journal of molecular sciences},
volume = {26},
number = {13},
pages = {},
doi = {10.3390/ijms26136268},
pmid = {40650046},
issn = {1422-0067},
support = {//Italian Ministry of Health (RRC)/ ; T4-AN-09 prog. ZRPOS2//CALabria HUB per Ricerca Innovativa ed Avanzata- CALHUB.RIA "Creazione di Hub delle Sci-enze della Vita"/ ; prog. ZRA124//AriSLA foundation, "Bulb-Omics"/ ; PNRR-MCNT2-2023-12377336//the European Union - Next Generation EU - NRRP M6C2 - Investment 2.1 Enhancement and strengthening of biomedical research in the NHS,/ ; },
mesh = {Humans ; *C9orf72 Protein/genetics/metabolism ; *Amyotrophic Lateral Sclerosis/genetics/therapy/metabolism/pathology ; Animals ; DNA Repeat Expansion ; Gene Editing ; Genetic Therapy/methods ; Mutation ; Oligonucleotides, Antisense/therapeutic use ; },
abstract = {Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of upper and lower motor neurons. One of its major genetic causes is C9ORF72, where mutations lead to hexanucleotide repeat expansions in the C9ORF72 gene. These expansions drive disease progression through mechanisms, including the formation of toxic RNAs and the accumulation of damaged proteins such as dipeptide repeats (DPRs). This review highlights these pathogenic mechanisms, focusing on RNA foci formation and the accumulation of toxic DPRs, which contribute to neuronal damage. It also discusses promising targeted therapies, including small molecules and biological drugs, designed to counteract these specific molecular events. Small molecules such as G-quadruplex stabilizers, proteasome and autophagy modulators, and RNase-targeting chimeras show potential in reducing RNA foci and DPR accumulation. Furthermore, targeting enzymes involved in repeat-associated non-AUG (RAN) translation and nucleocytoplasmic transport, which are crucial for disease pathogenesis, opens new therapeutic avenues. Even some anti-viral drugs show encouraging results in preclinical studies. Biological drugs, such as antisense oligonucleotides and gene-editing technologies like CRISPR-Cas, were explored for their potential to specifically target C9ORF72 mutations and modify the disease's molecular foundations. While preclinical and early clinical data show promise, challenges remain in optimizing delivery methods, ensuring long-term safety, and improving efficacy. This review concludes by emphasizing the importance of continued research and the potential for these therapies to alter the disease trajectory and improve patient outcomes.},
}

Humans
*C9orf72 Protein/genetics/metabolism
*Amyotrophic Lateral Sclerosis/genetics/therapy/metabolism/pathology
Animals
DNA Repeat Expansion
Gene Editing
Genetic Therapy/methods
Mutation
Oligonucleotides, Antisense/therapeutic use

@article {pmid40649992,
year = {2025},
author = {Reddy, KD and Rathnayake, SNH and Idrees, S and Boedijono, F and Xenaki, D and Padula, MP and Berge, MVD and Faiz, A and Oliver, BGG},
title = {A Novel Regulatory Role for RPS4Y1 in Inflammatory and Fibrotic Processes.},
journal = {International journal of molecular sciences},
volume = {26},
number = {13},
pages = {},
doi = {10.3390/ijms26136213},
pmid = {40649992},
issn = {1422-0067},
support = {GNT1158186//National Health and Research Council, Australia (NHMRC)/ ; },
mesh = {Humans ; *Asthma/genetics/metabolism/pathology ; *Ribosomal Proteins/genetics/metabolism ; Female ; Male ; *Inflammation/genetics/metabolism/pathology ; Fibrosis ; Interleukin-6/genetics/metabolism ; Tenascin/genetics/metabolism ; Animals ; CRISPR-Cas Systems ; },
abstract = {Asthma is a chronic inflammatory respiratory disease well-known to demonstrate sexual dimorphism in incidence and severity, although the mechanisms causing these differences remain incompletely understood. RPS4X and RPS4Y1 are X and Y-chromosome-linked genes coding ribosomal subunits previously associated with inflammation, airway remodelling and asthma medication efficacy. Particularly, RPS4Y1 has been under-investigated within the context of disease, with little examination of molecular mechanisms and pathways regulated by this gene. The ribosome, a vital cellular machinery, facilitates the translation of mRNA into peptides and then proteins. Imbalance or dysfunction in ribosomal components may lead to malfunctioning proteins. Using CRISPR-Cas9 knockout cellular models for RPS4Y1 and RPS4X, we characterised the function of RPS4Y1 in the context of the asthma-relevant processes, inflammation and fibrosis. No viable RPS4X knockouts could be generated. We highlight novel molecular mechanisms such as specific translation of IL6 and tenascin-C mRNA by RPS4Y1 containing ribosomes. Furthermore, an RPS4Y1-centric gene signature correlates with clinical lung function measurements, specifically in adult male asthma patients. These findings inform the current understanding of sex differences in asthma, as females do not produce the RPS4Y1 protein. Therefore, the pathologically relevant functions of RPS4Y1 may contribute to the complex sexually dimorphic pattern of asthma susceptibility and progression.},
}

Humans
*Asthma/genetics/metabolism/pathology
*Ribosomal Proteins/genetics/metabolism
Female
Male
*Inflammation/genetics/metabolism/pathology
Fibrosis
Interleukin-6/genetics/metabolism
Tenascin/genetics/metabolism
Animals
CRISPR-Cas Systems

@article {pmid40649886,
year = {2025},
author = {Chang, TY and Lin, LC and Kao, CY and Lu, JJ},
title = {Study of lug Operon, SCCmec Elements, Antimicrobial Resistance, MGEs, and STs of Staphylococcus lugdunensis Clinical Isolates Through Whole-Genome Sequencing.},
journal = {International journal of molecular sciences},
volume = {26},
number = {13},
pages = {},
doi = {10.3390/ijms26136106},
pmid = {40649886},
issn = {1422-0067},
support = {TCRD-TPE-NSTC-113-18 and TCRD-TPE-114-04(1/3)//Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation/ ; NSTC 113-2320-B-303-006//National Science and Technology Council, Taiwan/ ; },
mesh = {*Staphylococcus lugdunensis/genetics/drug effects/isolation & purification ; Whole Genome Sequencing ; *Operon ; Humans ; *Staphylococcal Infections/microbiology ; Multilocus Sequence Typing ; Genome, Bacterial ; Anti-Bacterial Agents/pharmacology ; *Drug Resistance, Bacterial/genetics ; Drug Resistance, Multiple, Bacterial/genetics ; Interspersed Repetitive Sequences ; Phylogeny ; Microbial Sensitivity Tests ; *Bacterial Proteins/genetics ; CRISPR-Cas Systems ; },
abstract = {Staphylococcus lugdunensis is a coagulase-negative staphylococcus known for its significant pathogenic potential, often causing severe infections such as endocarditis and bacteremia, with virulence comparable to S. aureus. Despite general susceptibility to most antibiotics, the emergence of oxacillin-resistant strains is increasingly concerning. This study conducted whole-genome sequencing on 20 S. lugdunensis isolates from Chang Gung Memorial Hospital to explore their genetic diversity, antimicrobial resistance mechanisms, and mobile genetic elements. The lugdunin biosynthetic operon, essential for antimicrobial peptide production, was present in multilocus sequence typing (MLST) types 1, 3, and 6 but absent in STs 4, 27, and 29. Additionally, IS256 insertion elements, ranging from 7 to 17 copies, were identified in four strains and linked to multidrug resistance. CRISPR-Cas systems varied across STs, with type III-A predominant in ST1 and ST6 and type IIC in ST4, ST27, and ST29; notably, ST3 lacked CRISPR systems, correlating with a higher diversity of SCCmec elements and an increased potential for horizontal gene transfer. Phage analysis revealed stable phage-host associations in ST1, ST6, and ST29, whereas ST4 displayed a varied prophage profile. Phenotypic resistance profiles generally aligned with genomic predictions, although discrepancies were observed for aminoglycosides and clindamycin. These findings highlight the complex genetic landscape and evolutionary dynamics of S. lugdunensis, emphasizing the need for genomic surveillance to inform clinical management and prevent the spread of resistant strains.},
}

*Staphylococcus lugdunensis/genetics/drug effects/isolation & purification
Whole Genome Sequencing
*Operon
Humans
*Staphylococcal Infections/microbiology
Multilocus Sequence Typing
Genome, Bacterial
Anti-Bacterial Agents/pharmacology
*Drug Resistance, Bacterial/genetics
Drug Resistance, Multiple, Bacterial/genetics
Interspersed Repetitive Sequences
Phylogeny
Microbial Sensitivity Tests
*Bacterial Proteins/genetics
CRISPR-Cas Systems

@article {pmid40648011,
year = {2025},
author = {Fan, X and Zhang, Y and Gu, P and Naz, M},
title = {Epitranscriptomic Control of Drought Tolerance in Rice: The Role of RNA Methylation.},
journal = {Plants (Basel, Switzerland)},
volume = {14},
number = {13},
pages = {},
doi = {10.3390/plants14132002},
pmid = {40648011},
issn = {2223-7747},
support = {(2023-BS-209 and 2024-BS-281)//Liaoning Provincial Department of Science and Technology Doctoral Research Initiation Fund Program/ ; K20241057//Wuxi's "Taihu Light" Scientific and Technological Innovation Initiative/ ; JC2024019//Natural science foundation of Nantong/ ; },
abstract = {Drought stress is a predominant abiotic constraint adversely affecting global rice (Oryza sativa) production and threatening food security. While the transcriptional and post-transcriptional regulation of drought-responsive pathways has been widely investigated, the emerging field of epitranscriptomics, particularly RNA chemical modifications such as N6-methyladenosine (m[6]A), adds a new dimension to gene regulation under stress. The most prevalent internal modification in eukaryotic messenger RNA influences RNA metabolism by interacting dynamically with enzymes that add, remove, or recognize the modification. Recent studies in rice reveal that m[6]A deposition is not static but dynamically regulated in response to water-deficit conditions, influencing transcript stability, splicing, nuclear export, and translation efficiency of key drought-responsive genes. This review critically synthesizes current findings on the distribution and functional implications of m[6]A and other epitranscriptomic marks (e.g., 5-methylcytosine [m[5]C], pseudouridine [Ψ]) in modulating rice responses to drought. We discuss the regulatory circuitry involving m[6]A effectors such as OsMTA, OsFIP37, and YTH domain proteins and their integration with known drought-signaling pathways including ABA and reactive oxygen species (ROS) cascades. We also highlight emerging high-resolution technologies such as m[6]A-seq, direct RNA sequencing, and nanopore-based detection that facilitate epitranscriptomic profiling in rice. Finally, we propose future directions for translating epitranscriptomic knowledge into crop improvement, including CRISPR/Cas-based modulation of RNA modification machinery to enhance drought tolerance.},
}

@article {pmid40647992,
year = {2025},
author = {Sutula, M and Tussipkan, D and Kali, B and Manabayeva, S},
title = {Molecular Mechanisms Underlying Defense Responses of Potato (Solanum tuberosum L.) to Environmental Stress and CRISPR/Cas-Mediated Engineering of Stress Tolerance.},
journal = {Plants (Basel, Switzerland)},
volume = {14},
number = {13},
pages = {},
doi = {10.3390/plants14131983},
pmid = {40647992},
issn = {2223-7747},
support = {BR21882180//Ministry of Science and Higher Education of the Republic of Kazakhstan/ ; },
abstract = {Environmental stresses, such as drought, salinity, and pathogen attacks, significantly affect potato growth, development, and yield by disrupting key physiological and biochemical processes. Plant responses to these stresses are mediated by changes in gene expression, transcriptional regulation, and the activity of various functional proteins, all of which contribute to the molecular mechanisms of stress tolerance. Genome editing using the CRISPR/Cas9 system has been effectively used to enhance the resistance of potato to environmental stresses and to improve its nutritional value. This article provides a comprehensive review of recent studies retrieved from academic databases focusing on the effects of various environmental stressors on potato growth, yield, and postharvest storage. It also examines the influence of these stresses on the production of secondary metabolites and their associated molecular pathways. Finally, the review highlights advancements in the application of CRISPR/Cas-based genome editing technologies between 2021 and 2025 to improve stress tolerance and nutritional traits in potato plants.},
}

@article {pmid40647524,
year = {2025},
author = {Khan, R and Phely, L and Ehrenfeld, S and Schmitz, T and Veratti, P and Wolfes, J and Shoumariyeh, K and Andrieux, G and Martens, US and Bra, S and Auer, M and Schilling, O and Boerries, M and Speicher, M and Illert, AL and Duyster, J and Miething, C},
title = {Modeling the t(2;5) Translocation of Anaplastic Large Cell Lymphoma Using CRISPR-Mediated Chromosomal Engineering.},
journal = {Cancers},
volume = {17},
number = {13},
pages = {},
doi = {10.3390/cancers17132226},
pmid = {40647524},
issn = {2072-6694},
abstract = {BACKGROUND/OBJECTIVES: ALK+ Anaplastic Large Cell Lymphoma (ALCL) is an aggressive T-cell lymphoma that is characterized by expression of the Anaplastic Lymphoma Kinase (ALK), which is induced by the t(2;5) chromosomal rearrangement, leading to the expression of the NPM-ALK fusion oncogene. Most previous preclinical models of ALK+ ALCL were based on overexpression of the NPM-ALK cDNA from heterologous promoters. Due to the enforced expression, this approach is prone to artifacts arising from synthetic overexpression, promoter competition and insertional variation.

METHODS: To improve the existing ALCL models and more closely recapitulate the oncogenic events in ALK+ ALCL, we employed CRISPR/Cas-based chromosomal engineering to selectively introduce translocations between the Npm1 and Alk gene loci in murine cells.

RESULTS: By inducing precise DNA cleavage at the syntenic loci on chromosome 11 and 17 in a murine IL-3-dependent Ba/F3 reporter cell line, we generated de novo Npm-Alk translocations in vivo, leading to IL-3-independent cell growth. To verify efficient recombination, we analyzed the expression of the NPM-ALK fusion protein in the recombined cells and could also show the t(11;17) in the IL-3 independent Ba/F3 cells. Subsequent functional testing of these cells using an Alk-inhibitor showed exquisite responsiveness towards Crizotinib, demonstrating strong dependence on the newly generated ALK fusion oncoprotein. Furthermore, a comparison of the gene expression pattern between Ba/F3 cells overexpressing the Npm-Alk cDNA with Ba/F3 cells transformed by CRISPR-mediated Npm-Alk translocation indicated that, while broadly overlapping, a set of pathways including the unfolded protein response pathway was increased in the Npm-Alk overexpression model, suggesting increased reactive changes induced by exogenous overexpression of Npm-Alk. Furthermore, we observed clustered expression changes in genes located in chromosomal regions close to the breakpoint in the new CRISPR-based model, indicating positional effects on gene expression mediated by the translocation event, which are not part of the older models.

CONCLUSIONS: Thus, CRISPR-mediated recombination provides a novel and more faithful approach to model oncogenic translocations, which may lead to an improved understanding of the molecular pathogenesis of ALCL and enable more accurate therapeutic models of malignancies driven by oncogenic fusion proteins.},
}

@article {pmid40647454,
year = {2025},
author = {Sidabraite, A and Mosert, PL and Ahmed, U and Jones, SK and Gulla, A},
title = {Advancing Cholangiocarcinoma Diagnosis: The Role of Liquid Biopsy and CRISPR/Cas Systems in Biomarker Detection.},
journal = {Cancers},
volume = {17},
number = {13},
pages = {},
doi = {10.3390/cancers17132155},
pmid = {40647454},
issn = {2072-6694},
support = {101078247/ERC_/European Research Council/International ; IG 5728-2024//European Molecular Biology Organization (EMBO)/ ; P-SV-24-447//Research Council of Lithuania/ ; P-LT-TW-24-21//Research Council of Lithuania/ ; },
abstract = {Background/Objectives: Cholangiocarcinoma (CCA) is a highly heterogeneous malignancy of the biliary tract with limited diagnostic tools for early detection. Current serum markers, such as CA19-9, lack specificity and sensitivity, particularly in early-stage disease, which hinders the effectiveness of curative interventions. This narrative review evaluates the limitations of existing diagnostic approaches and explores the potential of combining liquid biopsy (LB) technologies with CRISPR/Cas-based systems for precise, minimally invasive biomarker detection. Methods: A narrative review was conducted, synthesizing literature from 2018 to 2025 across PubMed, MDPI, Web of Science, Google Scholar, and Embase using MeSH terms such as "cholangiocarcinoma," "liquid biopsy," "miRNA," and "CRISPR/Cas." Results: Circulating microRNAs (e.g., miR-21, miR-16, miR-877) exhibit high diagnostic accuracy. The RACE (Rolling Circle Amplification-assisted CRISPR/Cas9 Cleavage) platform shows promise for detecting extracellular vesicle (EV)-derived miRNAs with high sensitivity and single-nucleotide specificity. When paired with liquid biopsy, CRISPR-based assays enable real-time, cost-effective, and multiplexed detection of tumor-specific biomarkers. Conclusions: The introduction of LB combined with CRISPR/Cas systems could potentially revolutionize the early and accurate diagnosis of CCA, thereby advancing the overall treatment strategy. However, this method is still under development and requires further testing before it can be incorporated into routine diagnostics.},
}

@article {pmid40647071,
year = {2025},
author = {Nascimento, APS and Barros, AN},
title = {Sustainable Innovations in Food Microbiology: Fermentation, Biocontrol, and Functional Foods.},
journal = {Foods (Basel, Switzerland)},
volume = {14},
number = {13},
pages = {},
doi = {10.3390/foods14132320},
pmid = {40647071},
issn = {2304-8158},
abstract = {The growing demand for more sustainable food systems has driven the development of solutions based on food microbiology, capable of integrating safety, functionality, and environmental responsibility. This paper presents a critical and up-to-date review of the most relevant advances at the interface between microbiology, sustainability, and food innovation. The analysis is structured around three main axes: (i) microbial fermentation, with a focus on traditional practices and precision technologies aimed at valorizing agro-industrial waste and producing functional foods; (ii) microbial biocontrol, including the use of bacteriocins, protective cultures, bacteriophages, and CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated)-based tools as alternatives to synthetic preservatives; and (iii) the development of functional foods containing probiotics, prebiotics, synbiotics, and postbiotics, with the potential to modulate the gut microbiota and promote metabolic, immune, and cognitive health. In addition to reviewing the microbiological and technological mechanisms involved, the paper discusses international regulatory milestones, scalability challenges, and market trends related to consumer acceptance and clean labeling. Finally, emerging trends and research gaps are addressed, including the use of omics technologies, artificial intelligence, and unexplored microbial resources. Food microbiology, by incorporating sustainable practices and advanced technologies, is positioned as a strategic pillar for building a healthy, circular, science-based food model.},
}

@article {pmid40646930,
year = {2025},
author = {Han, P and Wang, Y and Sun, H},
title = {Impact of Temperature Stresses on Wheat Quality: A Focus on Starch and Protein Composition.},
journal = {Foods (Basel, Switzerland)},
volume = {14},
number = {13},
pages = {},
doi = {10.3390/foods14132178},
pmid = {40646930},
issn = {2304-8158},
abstract = {With climate change, maintaining wheat quality has become essential for the functional properties, end-use, commodity value, and nutritional benefits of wheat flour. Temperature indirectly influences wheat quality by modulating grain size, starch and protein content, and the balance between these components. This review systematically analyzes temperature-mediated alterations in wheat grain quality, with particular emphasis on the two core components: starch and protein. Specifically, daytime warming generally increases protein content while reducing starch accumulation; however, temperatures exceeding 30 °C diminish key protein quality parameters (UPP%, Glu/Gli ratio, HMW-GS/LMW-GS ratio). Nighttime warming enhances protein quality but compromises starch content and yield potential. Conversely, under low-temperature conditions, starch content declines, whereas protein content is primarily influenced by genotypes and treated temperatures. Furthermore, the underlying mechanisms driving temperature-induced changes in wheat quality traits are discussed. However, the mechanisms of temperature effects have not been fully elucidated, and the results often vary between regions or over years. Thus, identifying conserved high/low-temperature resistance genes, QTLs, epialleles, and epiQTL, as well as developing corresponding molecular markers and epi-markers, is an urgent priority. Meanwhile, genome-editing tools such as CRISPR/Cas could serve as a powerful approach for creating new wheat germplasm with durable high/low-temperature resistance.},
}

@article {pmid40646801,
year = {2025},
author = {Han, H and Zhang, D and Hao, W and Liu, A and Xia, N and Cui, M and Luo, J and Jiang, S and Zheng, W and Chen, N and Gu, J and Bai, J and Zhu, J},
title = {Parallel and Visual Detections of ASFV by CRISPR-Cas12a and CRISPR-Cas13a Systems Targeting the Viral S273R Gene.},
journal = {Animals : an open access journal from MDPI},
volume = {15},
number = {13},
pages = {},
doi = {10.3390/ani15131902},
pmid = {40646801},
issn = {2076-2615},
support = {32473040; 32172867//Natural Science Foundation of China/ ; },
abstract = {African swine fever virus (ASFV) causes a highly contagious and lethal hemorrhagic disease and significantly threatens the pig industry. There is no commercially effective vaccine available currently, making the detection of ASFV critical for control and prevention. Previously, we established the CRISPR-LbCas12a and LwCRSIRP-Cas13a visual detections of ASFV, separately, targeting the structural p17 gene D117L. In this study, we performed the parallel detections of ASFV based on the conserved viral protease gene S273R using CRISPR-LbCas12a and CRISPR-LbuCas13a systems. Our results showed that both systems are able to specifically detect ASFV as low as two copies of the S273R gene, and effectively detect clinical samples with minimal DNA purification. The work promotes CRISPR-Cas systems for the application of on-site detection in the field.},
}

@article {pmid40646370,
year = {2025},
author = {Liu, Y and Liu, H and Wang, G and Jia, X and Zhou, J and Dong, X and Li, H and Chang, H and Li, Z and Jin, Z and Li, K},
title = {Dual-Check CRISPR-SERS strategy for sensitively detecting Monkeypox DNA and its single-base mutated DNA.},
journal = {Mikrochimica acta},
volume = {192},
number = {8},
pages = {497},
pmid = {40646370},
issn = {1436-5073},
support = {2025GXNSFHA069082//Guangxi Natural Science Foundation Project/ ; AD22035215//Guangxi Science and Technology Project/ ; 2022YFE0134600//National Key Research and Development Program of China/ ; AB22080055//Key Research and Development Program of Guangxi/ ; },
mesh = {*Spectrum Analysis, Raman/methods ; Metal Nanoparticles/chemistry ; Gold/chemistry ; *CRISPR-Cas Systems ; *DNA, Viral/genetics/analysis ; DNA, Single-Stranded/chemistry/genetics ; Limit of Detection ; Plasmids/genetics ; CRISPR-Associated Proteins/metabolism ; Endodeoxyribonucleases/metabolism ; Carbocyanines/chemistry ; Silver/chemistry ; Bacterial Proteins/metabolism ; },
abstract = {This study presents a convenient and efficient dual-check strategy for detecting Monkeypox DNA utilizing the SERS (Surface-enhanced Raman Spectroscopy)-CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system. The Monkeypox plasmid DNA (mpDNA) is recognized by the CRISPR RNA (crRNA)-Cas12a protein complex, where crRNA encompasses a targeting complementary sequence. Upon recognition, the trans-cleavage activity of Cas12a is activated and trans-cleaves the probe DNA (Cy3-ssDNA) which is modified on Au nanoparticles (AuNPs). As the ssDNA strand is cleaved, Cy3 molecules are released in the solution, while the amount of Cy3 modified on the AuNPs decreases. The free Cy3 molecules are collected from the supernatant, and their SERS intensities are measured using the silver nanopillar (AgNRs) substrate. The mpDNA with varying concentration from 5 nM to 0.5 fM can be quantitatively determined based on the SERS signals of free Cy3 and the collected nanotag. This strategy allows the detection of mpDNA with a concentration of 50 fM within 60 min. Moreover, the strategy can successfully detect single-base mutated mpDNA. Owing to the non-specific trans-cleavage activity of the protein, this strategy can be adapted to various nucleic acid detection scenarios by designing complementary RNA and DNA sequences.},
}

*Spectrum Analysis, Raman/methods
Metal Nanoparticles/chemistry
Gold/chemistry
*CRISPR-Cas Systems
*DNA, Viral/genetics/analysis
DNA, Single-Stranded/chemistry/genetics
Limit of Detection
Plasmids/genetics
CRISPR-Associated Proteins/metabolism
Endodeoxyribonucleases/metabolism
Carbocyanines/chemistry
Silver/chemistry
Bacterial Proteins/metabolism

@article {pmid40646080,
year = {2025},
author = {Jung, Y and Seo, E and Yang, S and Bae, S and Kim, JH and Jang, HK and Jo, DH},
title = {Establishment and rescue of fibroblast cell lines carrying a nonsense mutation of RB1 by CRISPR-based base editing.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {25074},
pmid = {40646080},
issn = {2045-2322},
support = {18-2023-0010//Seoul National University Hospital/ ; GTL24021-000//Ministry of Science and ICT, South Korea/ ; RS-2024-00400829//Ministry of Science and ICT, South Korea/ ; RS-2023-00260351//Ministry of Science and ICT, South Korea/ ; RS-2023-00301850//Ministry of Education/ ; RS-2024-00438476//Ministry of Health & Welfare/ ; },
mesh = {*Codon, Nonsense/genetics ; Humans ; *Gene Editing/methods ; *Fibroblasts/metabolism/cytology ; *CRISPR-Cas Systems ; *Retinoblastoma Binding Proteins/genetics/metabolism ; Cell Line ; Cell Proliferation/genetics ; Retinoblastoma/genetics/pathology ; Ubiquitin-Protein Ligases ; },
abstract = {Pathogenic variants of the RB1 gene have commonly been found in many cancer types, including retinoblastoma. Nonsense mutations are the most common mutation type in retinoblastoma; however, few cell lines mimic nonsense mutations in the RB1 gene that are commonly observed in patients. Here, we established retinoblastoma-like cell lines carrying mono- and bi-allelic nonsense mutations in the RB1 gene. We introduced the R552X mutation using target activation-induced cytidine deaminase base editing and successfully constructed cell lines carrying mono- and bi-allelic mutations. The model cell lines showed decreased RB1 expression at both the mRNA and protein levels, and increased cell proliferation. Furthermore, we rescued the nonsense mutation in the RB1 gene in model cell lines by converting stop codon 552 to tryptophan using an adenine base editor. This approach may be applicable for establishing cell lines with pathogenic variants found in patients and suggests a strong potential for the application of gene editing as a therapeutic strategy.},
}

*Codon, Nonsense/genetics
Humans
*Gene Editing/methods
*Fibroblasts/metabolism/cytology
*CRISPR-Cas Systems
*Retinoblastoma Binding Proteins/genetics/metabolism
Cell Line
Cell Proliferation/genetics
Retinoblastoma/genetics/pathology
Ubiquitin-Protein Ligases

@article {pmid40645983,
year = {2025},
author = {Ching, RW and Świst-Rosowska, KM and Erikson, G and Koschorz, B and Engist, B and Jenuwein, T},
title = {Forced expression of MSR repeat transcripts above a threshold limit breaks heterochromatin organisation.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6420},
pmid = {40645983},
issn = {2041-1723},
support = {CRC992 'MEDEP'//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {*Heterochromatin/metabolism/genetics ; Animals ; Mice ; *DNA, Satellite/genetics/metabolism ; Fibroblasts/metabolism ; Transcription, Genetic ; CRISPR-Cas Systems ; Chromosome Segregation/genetics ; },
abstract = {Mouse heterochromatin is characterised by transcriptionally competent major satellite repeat (MSR) sequences and it has been proposed that MSR RNA contributes to the integrity of heterochromatin. We establish an inducible dCas9-effector system in mouse embryonic fibroblasts, where we can modulate MSR transcription through the targeting of a dCas9-Repressor or a dCas9-Activator. With this system, we can define a threshold limit of >300-fold deregulation of MSR transcript levels, above which the structural organisation of heterochromatin becomes disrupted. MEF cells expressing MSR RNA above this threshold limit are not viable and the defects in heterochromatin organisation and chromosome segregation cannot be reverted. This study highlights the importance of restricting MSR RNA output to maintain heterochromatin integrity and relates MSR transcript levels to either physiological or pathological conditions. It also reveals that the structural organisation of heterochromatin is governed by the transcriptional chromatin state and associated MSR RNA of the MSR repeats.},
}

*Heterochromatin/metabolism/genetics
Animals
Mice
*DNA, Satellite/genetics/metabolism
Fibroblasts/metabolism
Transcription, Genetic
CRISPR-Cas Systems
Chromosome Segregation/genetics

@article {pmid40600900,
year = {2025},
author = {Hou, Y and Li, Y and Zheng, R and Zhang, F and Guo, F and Li, M and Zeng, M},
title = {Leveraging protein language models for cross-variant CRISPR/Cas9 sgRNA activity prediction.},
journal = {Bioinformatics (Oxford, England)},
volume = {41},
number = {7},
pages = {},
doi = {10.1093/bioinformatics/btaf385},
pmid = {40600900},
issn = {1367-4811},
support = {2022YFC3400300//National Key Research and Development Program of China/ ; 62472445//National Natural Science Foundation of China/ ; 2023JJ40763//Hunan Provincial Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Associated Protein 9/genetics/metabolism ; Deep Learning ; Computational Biology/methods ; },
abstract = {MOTIVATION: Accurate prediction of single-guide RNA (sgRNA) activity is crucial for optimizing the CRISPR/Cas9 gene-editing system, as it directly influences the efficiency and accuracy of genome modifications. However, existing prediction methods mainly rely on large-scale experimental data of a single Cas9 variant to construct Cas9 protein (variants)-specific sgRNA activity prediction models, which limits their generalization ability and prediction performance across different Cas9 protein (variants), as well as their scalability to the continuously discovered new variants.

RESULTS: In this study, we proposed PLM-CRISPR, a novel deep learning-based model that leverages protein language models to capture Cas9 protein (variants) representations for cross-variant sgRNA activity prediction. PLM-CRISPR uses tailored feature extraction modules for both sgRNA and protein sequences, incorporating a cross-variant training strategy and a dynamic feature fusion mechanism to effectively model their interactions. Extensive experiments demonstrate that PLM-CRISPR outperforms existing methods across datasets spanning seven Cas9 protein (variants) in three real-world scenarios, demonstrating its superior performance in handling data-scarce situations, including cases with few or no samples for novel variants. Comparative analyses with traditional machine learning and deep learning models further confirm the effectiveness of PLM-CRISPR. Additionally, motif analysis reveals that PLM-CRISPR accurately identifies high-activity sgRNA sequence patterns across diverse Cas9 protein (variants). Overall, PLM-CRISPR provides a robust, scalable, and generalizable solution for sgRNA activity prediction across diverse Cas9 protein (variants).

The source code can be obtained from https://github.com/CSUBioGroup/PLM-CRISPR.},
}

*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*Gene Editing/methods
*CRISPR-Associated Protein 9/genetics/metabolism
Deep Learning
Computational Biology/methods

@article {pmid40480611,
year = {2025},
author = {Huang, L and Song, Y and Li, N and Gao, J and Zhang, B and Liu, Z and Zheng, Y},
title = {Deletion of bikaverin and fusarubin biosynthesis gene clusters via CRISPR/Cas9 system in Fusarium fujikuroi and its effect on GA3 biosynthesis.},
journal = {Journal of biotechnology},
volume = {405},
number = {},
pages = {229-237},
doi = {10.1016/j.jbiotec.2025.06.004},
pmid = {40480611},
issn = {1873-4863},
mesh = {*Fusarium/genetics/metabolism ; *Gibberellins/metabolism ; *CRISPR-Cas Systems/genetics ; Multigene Family/genetics ; Gene Deletion ; Fungal Proteins/genetics/metabolism ; Xanthones ; },
abstract = {Gibberellic acid (GA3) is a critical plant hormone with significant agricultural applications, yet its production in Fusarium fujikuroi is constrained by competition for metabolic precursors, particularly acetyl-CoA, which is essential for GA3 biosynthesis. The genome of F. fujikuroi harbors numerous secondary metabolite biosynthetic gene clusters that divert acetyl-CoA away from the GA3 pathway, thereby limiting its yield. To address this challenge, we employed the CRISPR/Cas9 system to delete the bikaverin and fusarubin biosynthesis gene clusters, which are known to compete with GA3 biosynthesis for acetyl-CoA. This genetic intervention resulted in a substantial increase in GA3 production, with the ΔBIKΔFSR strain yielding 31.67 % more GA3 compared to the wild-type strain. Notably, the deletion of these gene clusters not only enhanced GA3 biosynthesis but also improved mycelial growth and carbon assimilation, as evidenced by increased consumption of reducing sugars during fermentation. We further employed qRT-PCR to assess comparative expression levels of genes associated with the glycohydrolysis, glycolysis, and the TCA pathway in engineered strain. Results indicated that removing by-product gene clusters enhances the glycohydrolase system, accelerating carbon assimilation. Given the presence of dozens of secondary metabolite biosynthetic gene clusters in the F. fujikuroi genome, the strategy reported here offers a promising avenue for further enhancing GA3 production by targeting additional non-essential gene clusters.},
}

*Fusarium/genetics/metabolism
*Gibberellins/metabolism
*CRISPR-Cas Systems/genetics
Multigene Family/genetics
Gene Deletion
Fungal Proteins/genetics/metabolism
Xanthones

@article {pmid40472724,
year = {2025},
author = {Shimon, O and Dean, AM and Cohen, S and Moser, AL and Dacso, CC and Gilad, Y and Lonard, DM and O'Malley, BW},
title = {CRISPR-Cas9 engineering of human T regulatory cells - Design and optimization of a manufacturing process.},
journal = {Molecular immunology},
volume = {184},
number = {},
pages = {13-21},
doi = {10.1016/j.molimm.2025.05.019},
pmid = {40472724},
issn = {1872-9142},
mesh = {Humans ; *T-Lymphocytes, Regulatory/immunology ; *CRISPR-Cas Systems/genetics/immunology ; Animals ; *Gene Editing/methods ; Mice ; Immunotherapy, Adoptive/methods ; },
abstract = {Regulatory T cells (Tregs) are a subset of CD4 + T cells that comprise 5-10 % of the total CD4 + T cell population. Tregs, which are critically important for the maintenance of immune tolerance and immune homeostasis, are distinguished from other subtypes of CD4 + T cells by the expression of the transcription factor FOXP3. Because of the centrality to immunoregulation, Tregs have gained increasing attention as promising targets for clinical applications in autoimmune diseases, transplant rejection and graft-versus-host disease (GvHD). However, the essential role of Tregs in the complex network of the immune system implies their targeting as a promising therapeutic approach also in other medical indications, such as neurodegenerative diseases and cancer. Our group recently published a study showing that genetically modified Tregs are capable of clearing solid malignancies in various mice models, including an aggressive triple negative breast cancer (TNBC) and prostate cancer, which provides the impetus to develop an adoptive cell therapy using Steroid Receptor Coactivator 3 (SRC-3) knock out (KO) Tregs. It is well known that isolation, genetic editing and the expansion of Tregs as a homogenous and healthy population present specific technical challenges. In this context, here we outline the development of a process for the production of SRC-3 KO human Tregs (hTregs), which can subsequently be adapted for Current Good Manufacturing Practice (cGMP) settings to facilitate clinical-scale production.},
}

Humans
*T-Lymphocytes, Regulatory/immunology
*CRISPR-Cas Systems/genetics/immunology
Animals
*Gene Editing/methods
Mice
Immunotherapy, Adoptive/methods

@article {pmid40449665,
year = {2025},
author = {Gomes, D and Rodrigues, JL and Scrutton, NS and Rodrigues, LR},
title = {De novo production of prenylnaringenin compounds by a metabolically engineered Escherichia coli.},
journal = {Journal of biotechnology},
volume = {405},
number = {},
pages = {215-228},
doi = {10.1016/j.jbiotec.2025.05.017},
pmid = {40449665},
issn = {1873-4863},
mesh = {*Escherichia coli/genetics/metabolism ; *Metabolic Engineering/methods ; *Flavanones/metabolism ; Dimethylallyltranstransferase/metabolism/genetics ; Biosynthetic Pathways/genetics ; CRISPR-Cas Systems ; Organophosphorus Compounds/metabolism ; Hemiterpenes ; },
abstract = {Prenylnaringenin (PN) compounds, namely 8-prenylnaringenin (8-PN), 3'-prenylnaringenin (3'-PN), and 6-prenylnaringenin (6-PN), are reported to have several interesting bioactivities. This study aimed to validate a biosynthetic pathway for de novo production of PN in Escherichia coli. A previously optimized E. coli chassis capable of efficiently de novo producing naringenin was used to evaluate eleven prenyltransferases (PTs) for the production of PN compounds. As PT reaction requires dimethylallyl pyrophosphate (DMAPP) as extended substrate that has limited availability inside the cells, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 12a (Cas12a) (CRISPR-Cas12a) was used to construct ten boosted DMAPP-E. coli strains. All the PTs, in combination with the naringenin biosynthetic pathway, were tested in these strains. Experiments in 96-well deep well plates identified twelve strains capable of producing PN. E. coli M-PAR-121 with the integration of the 1-deoxy-D-xylulose-5-phosphate synthase (DXS) gene from E. coli (EcDXS) into the lacZ locus of the genome (E. coli M-PAR-121:EcDXS) expressing the soluble aromatic PT from Streptomyces roseochromogenes (CloQ) and the naringenin biosynthetic pathway was selected as the best producer strain. After optimizing the production media in shake flasks, 160.57 µM of 3'-PN, 4.4 µM of 6-PN, and 2.66 µM of 8-PN were obtained. The production was then evaluated at the bioreactor scale and 397.57 µM of 3'-PN (135.33 mg/L) and 25.61 µM of 6-PN (8.72 mg/L) were obtained. To the best of our knowledge, this work represents the first report of de novo production of PN compounds using E. coli as a chassis.},
}

*Escherichia coli/genetics/metabolism
*Metabolic Engineering/methods
*Flavanones/metabolism
Dimethylallyltranstransferase/metabolism/genetics
Biosynthetic Pathways/genetics
CRISPR-Cas Systems
Organophosphorus Compounds/metabolism
Hemiterpenes

@article {pmid40393529,
year = {2025},
author = {Hegeman, CV and Elsharkasy, OM and Driedonks, TAP and Friesen, KRJ and Vader, P and de Jong, OG},
title = {Modulating binding affinity of aptamer-based loading constructs enhances extracellular vesicle-mediated CRISPR/Cas9 delivery.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {384},
number = {},
pages = {113853},
doi = {10.1016/j.jconrel.2025.113853},
pmid = {40393529},
issn = {1873-4995},
mesh = {*Extracellular Vesicles/metabolism ; *CRISPR-Cas Systems ; *Aptamers, Nucleotide/administration & dosage/genetics/metabolism ; Humans ; *Ribonucleoproteins/administration & dosage ; RNA, Guide, CRISPR-Cas Systems/genetics/administration & dosage ; *CRISPR-Associated Protein 9 ; HEK293 Cells ; Gene Editing ; },
abstract = {The CRISPR/Cas9 toolbox consists of modular nucleases that can be employed to efficiently modify genomic sequences with high specificity. However, delivery of the large Cas9-sgRNA ribonucleoprotein (RNP) complexes remains challenging due to their immunogenicity, size, and overall negative charge. An approach to overcome these limitations is the use of extracellular vesicles (EVs) as intracellular delivery vehicles. EVs exhibit the natural ability to carry and deliver RNA and proteins across biological barriers, and can be engineered to load and deliver a variety of biotherapeutic molecules. Previous studies have shown that efficient EV-mediated cargo delivery does not only require active loading strategies, but also benefits from strategies to release cargo from the EV membrane. Here, we load Cas9 RNP complexes into EVs by expressing sgRNAs containing MS2 aptamers (MS2-sgRNAs), alongside Cas9 and a fusion protein of CD63 and tandem MS2 coat proteins (MCPs). We demonstrate that efficient Cas9 RNP delivery can also be facilitated by modulating the binding affinity between MS2 aptamers and the MCPs. To study the effect of altering the binding affinity between the MS2 hairpin and the MCP on Cas9 RNP delivery, various mutations affecting the binding affinity were made in both the interacting MS2-hairpin and the RNA-binding domain of the MCPs. Comparing Cas9 RNP delivery of the modulated MS2-sgRNAs revealed that adapting binding affinity highly affects functional RNP delivery. Mutations resulting in high affinity did not facilitate efficient RNP delivery unless combined with a photo-inducible release strategy, showing that cargo release was a limiting factor in RNP delivery. Mutations that decreased affinity resolved this issue, resulting in Cas9 RNP delivery without the requirement of additional release strategies. However, further decreasing affinity resulted in decreased Cas9 gene-editing efficiency due to decreased levels of Cas9 RNP loading into EVs. A similar effect on functional delivery was seen after modification of the RNA-binding domain of the MCPs. Our results demonstrate that EVs are capable of functional Cas9-sgRNA complex delivery, and that modulation of binding affinity can be used to increase efficient functional delivery with non-covalent loading constructs, without the need for additional engineering strategies for cargo release.},
}

*Extracellular Vesicles/metabolism
*CRISPR-Cas Systems
*Aptamers, Nucleotide/administration & dosage/genetics/metabolism
Humans
*Ribonucleoproteins/administration & dosage
RNA, Guide, CRISPR-Cas Systems/genetics/administration & dosage
*CRISPR-Associated Protein 9
HEK293 Cells
Gene Editing

@article {pmid40644543,
year = {2025},
author = {Cao, D and Zhu, J and Guo, Y and Zhou, Y and Zeng, J and Tu, Y and Zhao, Z and Xie, L and Song, E and Zhu, M and Yin, L},
title = {Dynamically covalent lipid nanoparticles mediate CRISPR-Cas9 genome editing against choroidal neovascularization in mice.},
journal = {Science advances},
volume = {11},
number = {28},
pages = {eadj0006},
doi = {10.1126/sciadv.adj0006},
pmid = {40644543},
issn = {2375-2548},
mesh = {Animals ; *Choroidal Neovascularization/genetics/therapy/pathology ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Mice ; *Nanoparticles/chemistry ; Vascular Endothelial Growth Factor A/genetics/antagonists & inhibitors ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Lipids/chemistry ; Disease Models, Animal ; Humans ; RNA, Messenger/genetics ; Retinal Pigment Epithelium/metabolism ; CRISPR-Associated Protein 9 ; Liposomes ; },
abstract = {As an important modality for choroidal neovascularization (CNV) treatment, intravitreal injection of vascular endothelial growth factor A (VEGFA) inhibitors suffers from undesired response rate, low patient compliance, and ocular damage. Here, dynamically covalent lipid nanoparticles (LNPs) were engineered to mediate VEGFA gene editing and CNV treatment by codelivering Cas9 mRNA (mCas9) and single guide RNA (sgRNA) targeting VEGFA (sgVEGFA). A library of lipidoids bearing iminoboronate ester linkage was developed via facile "one-pot" synthesis, and the top-performing lipidoid-A4B3C7 was formulated into LNP-A4B3C7 with the highest mRNA transfection efficiency. Inside the diseased retinal pigment epithelial cells, LNPs were dissociated upon H2O2-triggered lipidoid degradation, facilitating mRNA/sgRNA release to potentiate the gene editing efficiency. In laser-induced CNV mice, mCas9/sgVEGFA@LNP-A4B3C7 after single intravitreal injection led to pronounced VEGFA disruption and CNV area reduction, outperforming the clinical anti-VEGF drug in eliciting sustained therapeutic effect. This study establishes a robust nonviral platform for mRNA delivery and genome editing and renders a promising strategy for CNV treatment.},
}

Animals
*Choroidal Neovascularization/genetics/therapy/pathology
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Mice
*Nanoparticles/chemistry
Vascular Endothelial Growth Factor A/genetics/antagonists & inhibitors
RNA, Guide, CRISPR-Cas Systems/genetics
*Lipids/chemistry
Disease Models, Animal
Humans
RNA, Messenger/genetics
Retinal Pigment Epithelium/metabolism
CRISPR-Associated Protein 9
Liposomes

@article {pmid40644365,
year = {2025},
author = {Yan, J and Dong, H and Tian, T and Xiao, C and Sun, Y and Gong, J and Xia, Q and Hou, Y},
title = {Doublesex and GATAβ4 synergistically regulate the sex-dimorphic expression of storage protein 1 in Bombyx mori.},
journal = {PLoS genetics},
volume = {21},
number = {7},
pages = {e1011762},
doi = {10.1371/journal.pgen.1011762},
pmid = {40644365},
issn = {1553-7404},
mesh = {Animals ; *Bombyx/genetics ; Female ; Male ; *Insect Proteins/genetics/metabolism ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; Sex Characteristics ; *GATA Transcription Factors/genetics/metabolism ; Gene Expression Regulation ; Transcription Factors/genetics ; DNA-Binding Proteins ; },
abstract = {Sexually dimorphic traits are widespread in organisms and are crucial for reproduction and behavior. These traits are typically controlled by sex-specific genes. However, their regulatory mechanisms are complex and incompletely understood. In Bombyx mori, a group of sex-differential storage proteins (SPs) exists, with storage protein 1 (SP1) expressed exclusively in females. In this study, we used the CRISPR/Cas9 system to knock out the doublesex gene and found that SP1 expression was sharply upregulated in male doublesex mutants and downregulated in female doublesex mutants, which suggests that doublesex is a key factor in the sex-differential expression of SP1. Then, we revealed that the female-specific doublesex isoform (dsxF) bound to and activated the SP1 promoter more strongly than the male-specific isoform (dsxM). Meanwhile, a transcription factor named GATAβ4 was found to be involved in the regulation by doublesex. Overexpression of GATAβ4 in Bombyx mori larvae affected adult reproductive behavior and dramatically upregulated SP1 expression in males. Furthermore, GATAβ4 interacted with both dsxF and dsxM, promoting nuclear translocation of dsxM, which in turn inhibited GATAβ4 binding to the SP1 promoter. In total, we found that dsxM did not directly repress SP1 expression in males but instead cooperated with other transcription factors to regulate downstream gene expression. These findings provide new insights into the regulation of sex-specific genes and the mechanisms controlling dimorphic traits.},
}

Animals
*Bombyx/genetics
Female
Male
*Insect Proteins/genetics/metabolism
Promoter Regions, Genetic
CRISPR-Cas Systems
Sex Characteristics
*GATA Transcription Factors/genetics/metabolism
Gene Expression Regulation
Transcription Factors/genetics
DNA-Binding Proteins

@article {pmid40643795,
year = {2025},
author = {Kim, YM and Na, HJ and Kwon, DH and Lee, JH and Park, BM and Lee, S and Nam, TW and Park, MY and Park, SH and Kim, SJ and Choi, B and Lee, HW},
title = {Generation of NOD SCID mice with near-complete deletions of Il2rg and Prkdc for human cancer and HSC engraftment.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {35},
pmid = {40643795},
issn = {1573-9368},
support = {RS-2024-00359509//National Research Foundation of Republic of Korea and Ministry of Science and ICT/ ; RS-2023-00224201//National Research Foundation of Republic of Korea and Ministry of Science and ICT/ ; RS-2023-00224201//National Research Foundation of Republic of Korea and Ministry of Science and ICT/ ; RS-2023-00224201//National Research Foundation of Republic of Korea and Ministry of Science and ICT/ ; RS-2023-00224201//National Research Foundation of Republic of Korea and Ministry of Science and ICT/ ; 20012378//the Ministry of Trade, Industry & Energy (MOTIE, Korea)/ ; 20012378//the Ministry of Trade, Industry & Energy (MOTIE, Korea)/ ; 20012378//the Ministry of Trade, Industry & Energy (MOTIE, Korea)/ ; 20012378//the Ministry of Trade, Industry & Energy (MOTIE, Korea)/ ; 20012378//the Ministry of Trade, Industry & Energy (MOTIE, Korea)/ ; 20012378//the Ministry of Trade, Industry & Energy (MOTIE, Korea)/ ; S3142545//the Ministry of SMEs and Startups (MSS, Korea)/ ; S3142545//the Ministry of SMEs and Startups (MSS, Korea)/ ; S3142545//the Ministry of SMEs and Startups (MSS, Korea)/ ; S3142545//the Ministry of SMEs and Startups (MSS, Korea)/ ; S3142545//the Ministry of SMEs and Startups (MSS, Korea)/ ; },
mesh = {Animals ; Humans ; Mice ; *Interleukin Receptor Common gamma Subunit/genetics ; Mice, Inbred NOD ; Mice, SCID ; CRISPR-Cas Systems ; *DNA-Activated Protein Kinase/genetics ; *Neoplasms/genetics/therapy/pathology ; *Hematopoietic Stem Cell Transplantation ; Disease Models, Animal ; Mice, Knockout ; },
abstract = {Immunodeficient mouse models are invaluable tools for preclinical research, particularly for cancer therapies and studies of the human immune system. Notably, strains with combined Prkdc (scid) and Il2rg (null) mutations-such as NOG and NSG mice- are widely used due to their profound immunodeficiency, allowing efficient engraftment of various human cells. However, these models were generated by disrupting the Il2rg gene through replacement with a neomycin resistance (Neo) cassette in embryonic stem cells. Incomplete excision of this cassette can inadvertently alter the expression of neighboring genes, thereby introducing potential confounding variables. In addition, they may still express mutant mRNAs that escape nonsense-mediated decay (NMD) and/or produce truncated proteins with residual activity, potentially compromising the interpretation of experimental outcomes. To address this, we developed the N2G mouse strain (NOD-2-Genes KO) where almost all genomic loci of both Prkdc and Il2rg genes are deleted via CRISPR/Cas9 genome editing. N2G mice exhibited tumor growth comparable to NOG mice following the transplantation with several human cancer cell lines. Moreover, human CD34[+] cord blood (CB) cells engrafted into N2G mice showed robust reconstitution of human immune cells, especially T cells in peripheral blood, spleen and bone marrow, compared to NSG mice. These results suggest that N2G mice, lacking residual mutant mRNA and the exogenous Neo resistant gene, offer an advanced model for preclinical studies.},
}

Animals
Humans
Mice
*Interleukin Receptor Common gamma Subunit/genetics
Mice, Inbred NOD
Mice, SCID
CRISPR-Cas Systems
*DNA-Activated Protein Kinase/genetics
*Neoplasms/genetics/therapy/pathology
*Hematopoietic Stem Cell Transplantation
Disease Models, Animal
Mice, Knockout

@article {pmid40643739,
year = {2025},
author = {Friberg, M and Sharma, S and Sitbon, F and Andersson, M and Hofvander, P},
title = {Modifying the potato tuber storage protein patatin targeting improved thermal stability.},
journal = {Planta},
volume = {262},
number = {2},
pages = {46},
pmid = {40643739},
issn = {1432-2048},
support = {2018-01846//Svenska Forskningsrådet Formas/ ; "Breeding of starch potatoes"//SLU Grogrund/ ; "Potato tuber//C4F/ ; sink//C4F/ ; starch development"//C4F/ ; },
mesh = {*Solanum tuberosum/genetics/metabolism ; *Plant Proteins/genetics/metabolism/chemistry ; *Plant Tubers/genetics/metabolism ; Gene Editing/methods ; Protein Stability ; CRISPR-Cas Systems ; Multigene Family ; Carboxylic Ester Hydrolases ; },
abstract = {Gene editing of the patatin gene cluster using a single-guide RNA sequence consistently modifies over 10% of the targeted genes in modified individuals. Patatins have gained recent attention, as a group of highly nutritious proteins with excellent functional properties. Some techniques have been suggested for industrial-scale patatin purification, mostly as a by-product from potato starch processing. The purification process has proved to be a challenge due to the low thermostability of patatins, especially under acidic conditions. One strategy to make patatin more accessible for extraction would be to stabilize the protein structure through the introduction of point mutations. Here, we show that the tuber expression of patatin genes is dominated by a few genes from the extended gene family, most of which were predicted to be catalytically inactive. We have further evaluated the suitability of the patatin gene cluster as a target for clustered regularly interspaced repeat (CRISPR)/Cas9-based mutagenesis. In the mutation study, we show that targeting using a single single-stranded guide RNA (sgRNA) can lead to mutations in over 10% of all alleles. Finally, four patatin variants with amino acid substitutions were designed based on in silico analysis of patatin protein structure. These modified patatins were then heterologously expressed in bacteria and evaluated for increased thermostability. While none of the mutant proteins performed better than a wild-type variant, with regard to their thermal properties, one candidate proved to be less sensitive to shifting pH, making it an interesting candidate for further optimizations.},
}

*Solanum tuberosum/genetics/metabolism
*Plant Proteins/genetics/metabolism/chemistry
*Plant Tubers/genetics/metabolism
Gene Editing/methods
Protein Stability
CRISPR-Cas Systems
Multigene Family
Carboxylic Ester Hydrolases

@article {pmid40642869,
year = {2025},
author = {Li, Y and Tang, Y and Li, X and Høyland-Kroghsbo, NM and Ingmer, H and Jiao, X and Li, Q},
title = {Quorum Sensing Inhibits Type III-A CRISPR-Cas System Activity Through Repressing Positive Regulators SarA and ArcR in Staphylococcus Aureus.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e06049},
doi = {10.1002/advs.202506049},
pmid = {40642869},
issn = {2198-3844},
support = {2024YFE0198800//National Key Research and Development Program of China/ ; 2023YFD1800503//National Key Research and Development Program of China/ ; 2022YFC2604200//National Key Research and Development Program of China/ ; BK20190883//Natural Science Foundation of Jiangsu Province/ ; 32072821//National Natural Science Foundation of China/ ; 31730094//National Natural Science Foundation of China/ ; KYCX21_3267//Postgraduate Research & Practice Innovation Program of Jiangsu Province/ ; R264-2017-3936//Lundbeck Foundation/ ; 2024ZB284//Jiangsu Funding Program for Excellent Postdoctoral Talent/ ; JX103SYL2024060102//Open Co-operation Project of Center for Global Health/ ; //Priority Academic Program Development of Jiangsu Higher Education Institutions/ ; },
abstract = {CRISPR-Cas is an adaptive immune system that protects prokaryotes from the invasion of foreign genetic elements. The components and immunity mechanisms of CRISPR-Cas have been extensively studied, but the regulation of this system in Staphylococci remains unclear. Here, it is shown that the cell-cell communication, known as quorum sensing (QS), inhibits the expression and activity of the type III-A CRISPR-Cas system in S. aureus. The QS regulator, AgrA, directly binds to the promoters of two transcriptional regulators encoding the genes sarA and arcR to inhibit their expression. However, both SarA and ArcR act as positive regulators that promote the transcription of cas genes by directly binding to a novel promoter Pcas. Furthermore, the Pcas of 300 bp located in cas1 displays as a critical regulatory node to initiate the transcription of cas10 and csm3. Our data reveal a new regulatory mechanism for QS-mediated repression of the Type III-A CRISPR-Cas system, which may allow S. aureus to acquire foreign genetic elements encoding antibiotic resistance or virulence factors specifically at high cell density.},
}

@article {pmid40641638,
year = {2025},
author = {Hua, G and He, C and Zuo, E},
title = {SuperDecode: A versatile toolkit for mutation analysis in genome editing.},
journal = {aBIOTECH},
volume = {6},
number = {2},
pages = {377-380},
pmid = {40641638},
issn = {2662-1738},
abstract = {The CRISPR-Cas system has revolutionized modern life sciences, enabling groundbreaking applications ranging from functional genomics to therapeutic development. Despite its transformative potential, significant technical limitations persist in current computational tools for quantifying editing efficiency - particularly concerning data processing capabilities, analytical throughput, and operational flexibility. This research presents SuperDecode, a novel computational framework designed to address these methodological constraints. The SuperDecode offers key advantages, including local processing capabilities, large-size sequencing files, batch-processing, and diversified operational functions.},
}

@article {pmid40641119,
year = {2025},
author = {Du, J and Gong, X and Huang, R and Zheng, B and Chen, C and Yang, Z},
title = {Harnessing CRISPR/Cas9 to overcome targeted therapy resistance in non‑small cell lung cancer: Advances and challenges (Review).},
journal = {Oncology reports},
volume = {54},
number = {3},
pages = {},
doi = {10.3892/or.2025.8944},
pmid = {40641119},
issn = {1791-2431},
mesh = {Humans ; *Carcinoma, Non-Small-Cell Lung/genetics/drug therapy/therapy ; *CRISPR-Cas Systems/genetics ; *Drug Resistance, Neoplasm/genetics ; *Lung Neoplasms/genetics/drug therapy/therapy ; *Gene Editing/methods ; Molecular Targeted Therapy/methods ; Genetic Therapy/methods ; },
abstract = {Targeted therapy has markedly improved outcomes for patients with non‑small cell lung cancer (NSCLC). However, the emergence of drug resistance remains a major clinical challenge, limiting long‑term treatment efficacy. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, a revolutionary gene‑editing technology, offers precise and efficient genetic modifications, providing new insights into the mechanisms of drug resistance in NSCLC. The present review explored the application of CRISPR/Cas9 in overcoming resistance associated with key oncogenic drivers, including EGFR, KRAS, ALK, ROS1, MET and BRAF. It summarized recent advances in CRISPR‑based strategies to reverse resistance, enhance targeted therapy effectiveness and develop potential therapeutic interventions. Additionally, it discussed current limitations, including off‑target effects, delivery challenges and ethical concerns, while highlighting future directions for clinical translation. Using CRISPR/Cas9 technology may pave the way for novel, personalized treatment approaches in NSCLC, ultimately improving patient outcome.},
}

Humans
*Carcinoma, Non-Small-Cell Lung/genetics/drug therapy/therapy
*CRISPR-Cas Systems/genetics
*Drug Resistance, Neoplasm/genetics
*Lung Neoplasms/genetics/drug therapy/therapy
*Gene Editing/methods
Molecular Targeted Therapy/methods
Genetic Therapy/methods

@article {pmid40457715,
year = {2025},
author = {Bourel, C and Souza-Fonseca-Guimaraes, F and Lesage, S},
title = {Highlights of 2024: unleashing the power of NK cells-cancer's worst nightmare.},
journal = {Immunology and cell biology},
volume = {103},
number = {6},
pages = {526-529},
doi = {10.1111/imcb.70037},
pmid = {40457715},
issn = {1440-1711},
mesh = {Humans ; *Killer Cells, Natural/immunology ; *Neoplasms/therapy/immunology ; *Immunotherapy, Adoptive/methods ; Animals ; CRISPR-Cas Systems ; Cytokines/metabolism ; Lymphocyte Activation ; Cytotoxicity, Immunologic ; },
abstract = {In this article for the Highlights of 2024 Series, we discuss strategies to enhance NK cell-based cancer therapies. These include (1) cytokine expression on bacterial membranes to boost NK cell activation in tumors, (2) optimizing CAR-NK cell manufacturing for improved efficacy, (3) using CRISPR-Cas9 to identify and target inhibitory genes, and (4) using tetraspecific engagers to enhance cytotoxicity and cytokine memory-like NK cells strengthening anti-tumor responses. This year's progress holds much promise for cancer treatments exploiting NK cells.},
}

Humans
*Killer Cells, Natural/immunology
*Neoplasms/therapy/immunology
*Immunotherapy, Adoptive/methods
Animals
CRISPR-Cas Systems
Cytokines/metabolism
Lymphocyte Activation
Cytotoxicity, Immunologic

@article {pmid40640739,
year = {2025},
author = {Dai, P and Huang, T and Ye, X and Mi, S and Zhang, J and Luo, X and Hu, D and Zhang, H},
title = {K1 Klebsiella pneumoniae is more conserved than K2 for both virulence plasmid and chromosome.},
journal = {BMC genomics},
volume = {26},
number = {1},
pages = {652},
pmid = {40640739},
issn = {1471-2164},
support = {2023KY1326//Zhejiang Provincial Health Commission/ ; 82172332//National Natural Science Foundation of China/ ; GSWS2019039//Gusu Health Youth Talent of Suzhou/ ; SKY2021007//Science and Technology Program of Suzhou/ ; XKTJ-TD202001//Discipline Construction of the Second Affiliated Hospital of Soochow University/ ; },
abstract = {OBJECTIVE: To illustrate the differences between K1 and K2 Klebsiella pneumoniae strains.

METHODS: Totally 68 K1 and 99 K2 K. pneumoniae strains from GenBank were analyzed for virulence genes, sequence types (STs), restriction-modification (R-M) systems, and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems. Phylogenetic trees of the virulence plasmids and chromosomes in the strains were built using kSNP4.

RESULTS: Virulence genes peg-344, allS, p-rmpA, p-rmpA2, c-rmpA, iroN, and iucA were more prevalent in K1 strains than K2. K1 strains were categorized into 7 STs with 79.41% being ST23 while K2 strains were categorized into 14 STs with 38.38% being ST14. K1 strains showed higher rates of CRISPR-Cas systems than K2 while lower rates of Type I and II R-M systems were found in K1 strains than K2. More rates of virulence plasmids (52/68 vs. 24/99) were found in K1 strains than K2. Based upon the phylogenetic tree of virulence plasmids, 46 in K1 strains belonged to the same clade while 11 and 7 virulence plasmids in K2 strains constituted the 2 major clades. For the chromosomes, 61 K1 strains belonged to the same clade while 99 K2 strains could be categorized into 4 major clades.

CONCLUSIONS: K1 K. pneumoniae strains are more conserved than K2 for both virulence plasmids and chromosomes. K1 strains are deficient in R-M systems but rich in CRISPR-Cas, which is contrary to K2.},
}

@article {pmid40640156,
year = {2025},
author = {Imburgia, C and Organick, L and Zhang, K and Cardozo, N and McBride, J and Bee, C and Wilde, D and Roote, G and Jorgensen, S and Ward, D and Anderson, C and Strauss, K and Ceze, L and Nivala, J},
title = {Random access and semantic search in DNA data storage enabled by Cas9 and machine-guided design.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6388},
pmid = {40640156},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; *DNA/genetics ; *Information Storage and Retrieval/methods ; *CRISPR-Associated Protein 9/metabolism/genetics ; Machine Learning ; Semantics ; Sequence Analysis, DNA/methods ; },
abstract = {DNA is a promising medium for digital data storage due to its exceptional data density and longevity. Practical DNA-based storage systems require selective data retrieval to minimize decoding time and costs. In this work, we introduce CRISPR-Cas9 as a user-friendly tool for multiplexed, low-latency molecular data extraction. We first present a one-pot, multiplexed random access method in which specific data files are selectively cleaved using a CRISPR-Cas9 addressing system and then sequenced via nanopore technology. This approach was validated on a pool of 1.6 million DNA sequences, comprising 25 unique data files. We then developed a molecular similarity-search approach combining machine learning with Cas9-based retrieval. Using a deep neural network, we mapped a database of 1.74 million images into a reduced-dimensional embedding, encoding each embedding as a Cas9 target sequence. These target sequences act as molecular addresses, capturing clusters of semantically related images. By leveraging Cas9's off-target cleavage activity, query sequences cleave both exact and closely related targets, enabling high-fidelity retrieval of molecular addresses corresponding to in silico image clusters similar to the query. These approaches move towards addressing key challenges in molecular data retrieval by offering simplified, rapid isothermal protocols and new DNA data access capabilities.},
}

*CRISPR-Cas Systems/genetics
*DNA/genetics
*Information Storage and Retrieval/methods
*CRISPR-Associated Protein 9/metabolism/genetics
Machine Learning
Semantics
Sequence Analysis, DNA/methods

@article {pmid40640155,
year = {2025},
author = {Wang, J and Ye, X and Liu, Y and Li, W and Zhang, X and Zhang, W and Yi, C and Liu, C},
title = {Regulating cleavage activity and enabling microRNA detection with split sgRNA in Cas12b.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6392},
pmid = {40640155},
issn = {2041-1723},
support = {22307150//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; *MicroRNAs/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Herpesvirus 4, Human/genetics/isolation & purification ; Colorectal Neoplasms/blood/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; HEK293 Cells ; },
abstract = {The CRISPR-Cas12b system has revolutionized molecular diagnostics, yet its reliance on single guide RNAs (sgRNAs) exceeding 100 nt limits precise regulation and applications. We present a split sgRNA strategy for Cas12b, utilizing universal components with customizable Spacer to detect various nucleic acid targets by simply replacing Spacer. Glyoxal labeling of the universal split direct repeat (DR) region represses Cas12b activity, which is restored by elevated temperatures or prolonged incubation, enabling dynamic regulation. Incorporating a photo-cleavable linker into the DR allows UV-mediated modulation, ensuring compatibility with recombinase polymerase amplification. Successful detection of Epstein-Barr virus in clinical plasma samples matched the sensitivity of traditional qPCR. Importantly, microRNAs can replace the Spacer, enabling direct detection without reverse transcription or amplification. Supported by evidence from cultured cells and plasma from healthy individuals and colorectal cancer patients, this method yields consistent results with RT-qPCR while simplifying protocols. This split strategy enhances Cas12b systems, offering a promising approach for clinical nucleic acid analysis.},
}

Humans
*MicroRNAs/genetics/metabolism
*CRISPR-Cas Systems/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Herpesvirus 4, Human/genetics/isolation & purification
Colorectal Neoplasms/blood/genetics
*CRISPR-Associated Proteins/genetics/metabolism
HEK293 Cells

@article {pmid40639408,
year = {2025},
author = {Ille, K and Melzer, S},
title = {Efficient and versatile rapeseed transformation for new breeding technologies.},
journal = {The Plant journal : for cell and molecular biology},
volume = {123},
number = {1},
pages = {e70330},
doi = {10.1111/tpj.70330},
pmid = {40639408},
issn = {1365-313X},
support = {Enable FKZ: 031B0801D//Bundesministerium für Bildung und Forschung/ ; Epibrass FKZ:031B1223B//Bundesministerium für Bildung und Forschung/ ; },
mesh = {*Brassica napus/genetics ; *Transformation, Genetic ; Plants, Genetically Modified/genetics ; *Plant Breeding/methods ; CRISPR-Cas Systems ; Gene Editing ; Plant Proteins/genetics/metabolism ; Phenotype ; },
abstract = {Many gene functions are widely studied and understood in Arabidopsis; however, the lack of efficient transformation systems often limits the application and verification of this knowledge in crop plants. Brassica napus L., a member of the Brassicaceae family, is usually transformed by Agrobacterium-mediated hypocotyl transformation, but not all growth types are equally amenable to transformation. In particular, winter rapeseed, which requires vernalization to initiate flowering, is recalcitrant to in vitro regeneration and transformation. The analysis of gene functions in rapeseed is further complicated by the allotetraploid nature of its genome and the genome triplication within the Brassica genus, which has led to the presence of a large number of gene homologs for each Arabidopsis ortholog. We have established a transformation method that facilitates the regeneration of winter rapeseed by using the WUSCHEL gene from Beta vulgaris. This allowed us to efficiently transform a winter and spring rapeseed genotype in small-scale experiments. As proof of principle, we targeted BnCLV3 and BnSPL9/15 with CRISPR/Cas9 and showed that entire gene families are effectively edited using this transformation protocol. This allowed us to simultaneously study many redundantly acting homologous genes in rapeseed. We observed mutant phenotypes for BnCLV3 and BnSPL9/15 in primary transformants, indicating that biallelic knockouts were obtained for up to eight genes. This allowed an initial phenotypic characterization to be performed already a few months after starting the experiment.},
}

*Brassica napus/genetics
*Transformation, Genetic
Plants, Genetically Modified/genetics
*Plant Breeding/methods
CRISPR-Cas Systems
Gene Editing
Plant Proteins/genetics/metabolism
Phenotype

@article {pmid40637794,
year = {2025},
author = {Hajian, M and Pirali, A and Moghaddam, SHH and Moradi-Hajidavaloo, R and Varnosfaderani, SR and Jozi, M and Izadi, T and Tanhaie-Vash, N and Kues, W and Nasr-Esfahani, MH and Jafarpour, F and Eghbalsaied, S},
title = {Efficient gene editing of BMP15, GDF9, and MSTN-but not the imprinted CLPG gene-in goat embryos via electrotransfection and handmade cloning.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {150},
pmid = {40637794},
issn = {1438-7948},
support = {CT1402091612//Iranian Vice President for Science/ ; Ref 3.4-IRN-1191261-GF-E//Alexander von Humboldt-Stiftung/ ; },
mesh = {Animals ; *Gene Editing/methods ; *Myostatin/genetics ; *Bone Morphogenetic Protein 15/genetics ; *Goats/genetics/embryology ; *Growth Differentiation Factor 9/genetics/metabolism ; CRISPR-Cas Systems ; Electroporation ; Nuclear Transfer Techniques ; Genomic Imprinting ; Female ; Cloning, Organism ; Embryo, Mammalian/metabolism ; },
abstract = {CRISPR/Cas9 technology represents a powerful tool for advancing livestock breeding by enabling precise, on-target gene edits without the genomic mixing associated with traditional introgression methods. In this study, we employed a dual gRNA-based CRISPR/Cas9 strategy to induce targeted deletions and indel mutations in both reproductive and growth-related genes. These included the metacentric genes bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9), which are associated with increased ovulation rate and litter size, as well as the telomeric genes myostatin (MSTN) and callipyge (CLPG), which are linked to muscle development and enhanced meat production. We employed an optimized electrotransfection protocol consisting of 10-20 µg of each plasmid DNA, 250 µL OptiMEM-GlutaMAX, and one million goat fibroblast cells. The electroporation was performed using a Bio-Rad system in a 4-mm cuvette, with two 10-millisecond pulses at 270 volts, separated by a 10-second interval. This protocol enabled efficient genome editing of goat embryonic fibroblast cells, which were subsequently used to generate cloned embryos via handmade somatic cell nuclear transfer (SCNT), involving manual enucleation and cell-oocyte fusion steps. Sequencing revealed high mutation rates (78-97%) and a predominance of biallelic edits in BMP15, GDF9, and MSTN. Notably, MSTN gRNAs with a 7-bp overlapping sequence at their 3' ends showed a high editing efficiency. In contrast, the imprinted CLPG gene exhibited a significantly lower mutation rate (~ 30%), likely due to epigenetic constraints. While overall mutation rates did not differ significantly between metacentric and telomeric genes, on-target deletions were more frequent in metacentric genes (43%) than in telomeric ones (20%). Embryo development rates from gene-edited cells were comparable to those from non-edited controls. These findings underscore the utility of combining electrotransfection with SCNT for efficient editing of non-imprinted genes and highlight the need for improved strategies to overcome barriers in editing imprinted loci.},
}

Animals
*Gene Editing/methods
*Myostatin/genetics
*Bone Morphogenetic Protein 15/genetics
*Goats/genetics/embryology
*Growth Differentiation Factor 9/genetics/metabolism
CRISPR-Cas Systems
Electroporation
Nuclear Transfer Techniques
Genomic Imprinting
Female
Cloning, Organism
Embryo, Mammalian/metabolism

@article {pmid40637230,
year = {2025},
author = {Amistadi, S and Fontana, L and Magnoni, C and Felix, T and Charvin, MK and Martinucci, P and Gautier, C and Greau, L and Bessières, B and Antoniou, P and Romano, O and Allemand, E and Mussolino, C and Miccio, A},
title = {Dissecting the epigenetic regulation of the fetal hemoglobin genes to unravel a novel therapeutic approach for β-hemoglobinopathies.},
journal = {Nucleic acids research},
volume = {53},
number = {13},
pages = {},
doi = {10.1093/nar/gkaf637},
pmid = {40637230},
issn = {1362-4962},
support = {//French National Research Agency/ ; ANR-10-IAHU-01//Agence Nationale de la Recherches/ ; ANR-21-CE18-0066-01//Agence Nationale de la Recherches/ ; CA21113//European Cooperation in Science and Technology/ ; //Institut Imagine/ ; PLP202110014595//Fondation pour la Recherche Médicale/ ; },
mesh = {Humans ; *Fetal Hemoglobin/genetics/metabolism ; *Epigenesis, Genetic ; DNA Methylation ; gamma-Globins/genetics ; *beta-Globins/genetics ; *Anemia, Sickle Cell/genetics/therapy ; Promoter Regions, Genetic ; Gene Editing/methods ; *Hemoglobinopathies/genetics/therapy ; CRISPR-Cas Systems ; Erythroid Cells/metabolism ; },
abstract = {Beta-hemoglobinopathies are severe genetic diseases caused by mutations affecting the production of the adult β-globin chain. The clinical severity is mitigated by the co-inheritance of mutations that reactivate the production of the fetal β-like γ-globin in adults. However, the epigenetic mechanisms underlying the adult-to-fetal hemoglobin (HbA-to-HbF) switching are still not fully understood. Here, we used epigenome editing technologies to dissect the molecular mechanisms underlying γ- and β-globin gene regulation and to develop novel potential therapeutics for β-hemoglobinopathies. Targeted removal of DNA methylation by dCas9-Tet1 (alone or together with the deposition of histone acetylation by CBP-dCas9) at the fetal promoters led to efficient and durable γ-globin reactivation, demonstrating that DNA methylation is a driver for HbF repression. This strategy, characterized by high specificity and a good safety profile, led to a substantial correction of the pathological phenotype in erythroid cells from patients with sickle cell disease.},
}

Humans
*Fetal Hemoglobin/genetics/metabolism
*Epigenesis, Genetic
DNA Methylation
gamma-Globins/genetics
*beta-Globins/genetics
*Anemia, Sickle Cell/genetics/therapy
Promoter Regions, Genetic
Gene Editing/methods
*Hemoglobinopathies/genetics/therapy
CRISPR-Cas Systems
Erythroid Cells/metabolism

@article {pmid40636259,
year = {2025},
author = {Zheng, H and Wang, B and Dong, X and Wu, J and Shi, L and Zhang, J and Chen, H and Zhou, A},
title = {SIRT7 deletion inhibits Glaesserella parasuis-mediated inflammatory responses in porcine alveolar macrophages.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1589199},
pmid = {40636259},
issn = {2235-2988},
mesh = {Animals ; Swine ; *Sirtuins/genetics/metabolism/deficiency ; *Macrophages, Alveolar/microbiology/immunology ; *Haemophilus parasuis/immunology/pathogenicity ; *Inflammation/microbiology ; *Haemophilus Infections/veterinary/immunology/microbiology/pathology ; Signal Transduction ; *Swine Diseases/microbiology/immunology ; Gene Deletion ; CRISPR-Cas Systems ; },
abstract = {Glaesserella parasuis (GPS) infection causes severe inflammatory disorder, resulting in lung injury. SIRT7 is an NAD[+]-dependent deacetylase known to regulate inflammatory responses, but its role in GPS infection remains unclear. Here we found that GPS infection increased SIRT7 expression and induced inflammatory responses. Deficiency of SIRT7 by CRISPR/Cas9 technology significantly inhibited GPS-induced cytopathic effects and inflammatory responses. In addition, RNA-seq analysis showed that differentially expressed genes(DEGs) induced by SIRT7 deficiency were enriched in biological processes such as cell proliferation, actin cytoskeleton formation, lipid synthesis, protein kinase activation regulation, and GTPase activity regulation. Functional enrichment analysis further indicated the involvement of these DEGs in tight junction pathway, PI3K-Akt signaling pathway, actin cytoskeleton regulation, cGMP-PKG signaling pathway, Hippo signaling pathway, and TNF signaling pathway. Finally, we identified some hub genes (GNAI3, GNAI1, JAK1, NDUFS8, CYC1) related to oxidative phosphorylation. In summary, our results demonstrate that SIRT7 is pivotal for GPS-induced inflammatory responses, which represents a promising target resistant to GPS infection.},
}

Animals
Swine
*Sirtuins/genetics/metabolism/deficiency
*Macrophages, Alveolar/microbiology/immunology
*Haemophilus parasuis/immunology/pathogenicity
*Inflammation/microbiology
*Haemophilus Infections/veterinary/immunology/microbiology/pathology
Signal Transduction
*Swine Diseases/microbiology/immunology
Gene Deletion
CRISPR-Cas Systems

@article {pmid40635088,
year = {2025},
author = {Wulff, JP and Laminack, RK and Scott, MJ},
title = {Genetic and behavioral analyses suggest that larval and adult stages of Lucilia cuprina employ different sensory systems to detect rotten beef.},
journal = {Parasites & vectors},
volume = {18},
number = {1},
pages = {270},
pmid = {40635088},
issn = {1756-3305},
support = {DEB-2030345//National Science Foundation/ ; },
mesh = {Animals ; Larva/genetics/physiology/growth & development ; Female ; Cattle ; Receptors, Odorant/genetics ; *Calliphoridae/genetics/physiology/growth & development ; Insect Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Lucilia cuprina ; },
abstract = {BACKGROUND: The blowfly Lucilia cuprina is a destructive parasite of sheep that causes flystrike or myiasis. Larvae consume the animal's living flesh, producing large wounds that can lead to death. The main aim of this study was to identify genes that may play important roles in the behavior and physiology of L. cuprina larvae.

METHODS: An RNA-Seq analysis of RNA from whole larvae at different developmental stages and third-instar head and gut tissues was used to identify sensory receptors and other genes relevant to the physiology of L. cuprina larvae. In addition, CRISPR/Cas9 gene editing was used to obtain a loss-of-function mutation for the L. cuprina odorant coreceptor gene (LcupOrco). The response of mutant larvae and adult females to fresh and rotten meat at different temperatures was evaluated.

RESULTS: The RNA-Seq analysis suggested that odorant (OR), gustatory, ionotropic, and Pickpocket receptors may not play a central role in the L. cuprina larval sensory signaling and digestive systems. Rather, ATP-binding cassettes (ABCs) were highly enriched in head and gut RNA, and odorant-binding proteins (OBPs) only in the head. To confirm that ORs are not essential for larval detection of rotten beef, diet-choice assays were performed including larvae and adults homozygous for a null mutation in LcupOrco. While the attraction of adult females to rotten beef was disrupted, LcupOrco mutant larvae showed no change in diet preference.

CONCLUSIONS: The expression pattern of the ABC and OBP gene families suggests a central role in the sensory system of the L. cuprina larva for these receptors. Behavioral assays showed that ORs are essential for the adult female response to rotten beef, but not for larval behavior. These findings are consistent with high levels of expression of LcupOrco in the adult female antenna but very low expression in larvae.},
}

Animals
Larva/genetics/physiology/growth & development
Female
Cattle
Receptors, Odorant/genetics
*Calliphoridae/genetics/physiology/growth & development
Insect Proteins/genetics/metabolism
CRISPR-Cas Systems
Lucilia cuprina

@article {pmid40634958,
year = {2025},
author = {Tian, Y and Bao, X and Lei, S and Huang, Y and Wang, X and Tu, Y and He, Q and Zhang, F and Xu, H and Ashrafizadeh, M and Sethi, G and Wang, F and Zeng, Z},
title = {In vivo CRISPR screening identifies POU3F3 as a novel regulator of ferroptosis resistance in hepatocellular carcinoma via retinoic acid signaling.},
journal = {Cell communication and signaling : CCS},
volume = {23},
number = {1},
pages = {329},
pmid = {40634958},
issn = {1478-811X},
support = {32260169//National Natural Science Foundation of China/ ; 82303903//National Natural Science Foundation of China/ ; BSH-Q-2023-06//the Affiliated Hospital of Guizhou Medical University Postdoctoral fellow Start-up fund/ ; },
mesh = {*Ferroptosis/drug effects/genetics ; Humans ; *Carcinoma, Hepatocellular/genetics/pathology/metabolism/drug therapy ; *Liver Neoplasms/genetics/pathology/metabolism/drug therapy ; Animals ; *Drug Resistance, Neoplasm/genetics/drug effects ; *Signal Transduction/drug effects ; *Tretinoin/metabolism ; Sorafenib/pharmacology ; Mice ; *Octamer Transcription Factor-3/metabolism/genetics ; Cell Line, Tumor ; Mice, Nude ; *CRISPR-Cas Systems/genetics ; },
abstract = {BACKGROUND: Sorafenib, a ferroptosis agonist, is a first-line treatment for advanced hepatocellular carcinoma (HCC). However, its clinical efficacy is limited due to drug resistance, resulting in modest improvements in patient survival. Hence, the present study has been designed to identify critical molecular targets associated with sorafenib resistance and investigate the potential inhibitors in overcoming this therapeutic challenge.

METHODS: In vivo whole-genome CRISPR/Cas9 library screens were conducted to identify resistance factors to ferroptosis agonists, such as RSL3 and sorafenib, in HCC. The effects and underlying molecular mechanisms of these resistance factors were investigated in HCC cells using ferroptosis detection assays, xenograft tumor models, chromatin immunoprecipitation (ChIP), and dual-luciferase reporter assays. Potential inhibitors targeting these factors were evaluated through computer-aided virtual screening, molecular dynamics simulations, surface plasmon resonance analysis, and functional evaluations.

RESULTS: A retinoic acid metabolism gene cluster, including ADH4, ALDH1A1, ALDH1A3, FABP5, RBP1, and RDH10, was found demonstrating upregulation in HCC cells treated with ferroptosis agonist, sorafenib. This gene cluster contributes to the ferroptosis resistance by producing the strong reducing agent retinoic acid. The transcription factor POU3F3 was identified as a key regulator for the retinoic acid metabolism gene cluster, which simultaneously binds to their promoters, increasing their transcription and promoting retinoic acid production. Knockdown of POU3F3 significantly enhanced the pro-ferroptotic and inhibitory effects of sorafenib on HCC cells by suppressing retinoic acid metabolism. Furthermore, rosarin was identified as a POU3F3 inhibitor, with an equilibrium dissociation constant of 7.57 µM, and demonstrated a synergistic effect with sorafenib against HCC cells both in vitro and in vivo.

CONCLUSIONS: According to the results, POU3F3 acts as a protective regulator against sorafenib-induced ferroptosis in HCC cells by enhancing the transcription of multiple retinoic acid metabolism genes and promoting retinoic acid production. The POU3F3 inhibitor, rosarin, shows potential as an ideal candidate for overcoming sorafenib resistance in HCC.},
}

*Ferroptosis/drug effects/genetics
Humans
*Carcinoma, Hepatocellular/genetics/pathology/metabolism/drug therapy
*Liver Neoplasms/genetics/pathology/metabolism/drug therapy
Animals
*Drug Resistance, Neoplasm/genetics/drug effects
*Signal Transduction/drug effects
*Tretinoin/metabolism
Sorafenib/pharmacology
Mice
*Octamer Transcription Factor-3/metabolism/genetics
Cell Line, Tumor
Mice, Nude
*CRISPR-Cas Systems/genetics

@article {pmid40516842,
year = {2025},
author = {Sun, M and Ni, C and Li, A and Liu, J and Guo, H and Xu, F and Li, K and Cao, X and Shi, X and Guo, R},
title = {A biomimetic nanoplatform mediates hypoxia-adenosine axis disruption and PD-L1 knockout for enhanced MRI-guided chemodynamic-immunotherapy.},
journal = {Acta biomaterialia},
volume = {201},
number = {},
pages = {618-632},
doi = {10.1016/j.actbio.2025.06.021},
pmid = {40516842},
issn = {1878-7568},
mesh = {Animals ; *Immunotherapy ; *B7-H1 Antigen/genetics/metabolism/deficiency ; *Nanoparticles/chemistry/therapeutic use ; *Magnetic Resonance Imaging ; Mice ; Cell Line, Tumor ; Manganese Compounds/chemistry/pharmacology ; *Adenosine/metabolism ; Humans ; *Biomimetic Materials/chemistry/pharmacology ; *Gene Knockout Techniques ; CRISPR-Cas Systems ; Oxides/chemistry/pharmacology ; Tumor Microenvironment ; Melanoma, Experimental/therapy/pathology/diagnostic imaging ; Biomimetics ; },
abstract = {Malignant melanoma is an extremely aggressive and fatal form of skin cancer due to the limited efficacy of conventional therapies. While immune checkpoint blockade therapy and chemodynamic therapy (CDT) have emerged as promising strategies for melanoma treatment, their effectiveness is compromised by the immunosuppressive and complex tumor microenvironment (TME). Here, cancer cell membrane-camouflaged nanoplatforms (PPMC@CM) were developed to co-deliver the CRISPR/Cas9-PD-L1 system and manganese dioxide nanoparticles (MnO2 NPs) for magnetic resonance imaging (MRI)-guided CDT and enhanced immunotherapy. The formed PPMC@CM could efficiently accumulate at tumor sites by homologous targeting, generate O2 to relieve hypoxia, and deplete glutathione (GSH) to enhance Mn[2+]-mediated Fenton-like reactions for enhanced CDT. Meanwhile, CRISPR/Cas9-mediated PD-L1 knockout effectively suppressed the PD-L1 expression, while hypoxia relief attenuated the immunosuppressive hypoxia-CD39/CD73-adenosine (ADO) pathway, thereby boosting the PD-L1-mediated immunotherapy. In vivo experimental results demonstrated that PPMC@CM nanoplatform could efficiently inhibit the growth and metastasis of melanoma by enhanced CDT and amplified immunotherapy, and provide targeted MRI of tumors. This work presents a novelty strategy to design biomimetic theranostic nanoplatform for melanoma by the combination of CDT and improved immunotherapy with CRISPR/Cas9-PD-L1 system and hypoxia-ADO axis inhibition. STATEMENT OF SIGNIFICANCE: Malignant melanoma is a highly aggressive and treatment-refractory skin cancer, where conventional therapies exhibit limited efficacy and immune checkpoint blockade (ICB) is often compromised by the immunosuppressive tumor microenvironment (TME). To address these challenges, we developed a biomimetic nanoplatform (PPMC@CM) to codeliver MnO2 nanoparticles and the CRISPR/Cas9-PD-L1 gene-editing system for MRI-guided chemodynamic therapy and enhanced immunotherapy. The PPMC@CM nanoplatform could efficiently accumulate at tumor sites by homologous targeting and relieve hypoxia to suppress the hypoxia-CD39/CD73-adenosine immunosuppressive axis. Additionally, the CRISPR/Cas9-mediated PD-L1 knockout significantly suppresses PD-L1 expression, thereby boosting ICB efficacy. Moreover, PPMC@CM could deplete glutathione in the TME to amplify Mn[2+]-mediated Fenton-like reactions for enhanced chemodynamic therapy. This research represents a promising theranostic nanoplatform for melanoma by combining chemodynamic therapy and immunotherapy.},
}

Animals
*Immunotherapy
*B7-H1 Antigen/genetics/metabolism/deficiency
*Nanoparticles/chemistry/therapeutic use
*Magnetic Resonance Imaging
Mice
Cell Line, Tumor
Manganese Compounds/chemistry/pharmacology
*Adenosine/metabolism
Humans
*Biomimetic Materials/chemistry/pharmacology
*Gene Knockout Techniques
CRISPR-Cas Systems
Oxides/chemistry/pharmacology
Tumor Microenvironment
Melanoma, Experimental/therapy/pathology/diagnostic imaging
Biomimetics

@article {pmid40499611,
year = {2025},
author = {Wang, C and Wang, Q and Jin, Y and Li, C and Xin, M and Jiang, X and Wan, J},
title = {Lambda exonuclease assisted helicase-dependent amplification CRISPR/Cas12a detection of Listeria monocytogenes.},
journal = {Biochimie},
volume = {235},
number = {},
pages = {106-112},
doi = {10.1016/j.biochi.2025.06.002},
pmid = {40499611},
issn = {1638-6183},
mesh = {*Listeria monocytogenes/genetics/isolation & purification ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *DNA Helicases/metabolism ; DNA, Single-Stranded/genetics ; *Bacterial Proteins/genetics/metabolism ; *Nucleic Acid Amplification Techniques/methods ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {We describe the construction of a protospacer adjacent motif-free CRISPR/Cas12a fluorescent biosensor based on lambda exonuclease (λ-exo) and helicase-dependent amplification (HDA) to detect Listeria monocytogenes(L. monocytogenes). The hlyA gene of L. monocytogenes was amplified by HDA. After λ-exo catalyzed cleavage of 5' phosphorylated single-stranded DNA of amplification product double-stranded DNA, the double-stranded DNA formed single-stranded DNA (ssDNA). The ssDNA as a substrate activated the trans-cleavage capability of CRISPR/Cas12a to cleave the reporter gene to produce fluorescence signals. Under optimized experimental conditions, the lower limit of L. monocytogenes detection by the fluorescent biosensor was 11.5 CFU/mL, with a linear range of detection from 10[1] to 10[7] CFU/mL. The fluorescent biosensor permits simple and sensitive detection of L. monocytogenes and provides a promising analysis platform for clinical diagnosis and biomedical research without protospacer adjacent motif sequence ssDNA.},
}

*Listeria monocytogenes/genetics/isolation & purification
*Biosensing Techniques/methods
*CRISPR-Cas Systems
*DNA Helicases/metabolism
DNA, Single-Stranded/genetics
*Bacterial Proteins/genetics/metabolism
*Nucleic Acid Amplification Techniques/methods
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40634673,
year = {2025},
author = {Asumadu, P and Guo, Z and Qi, S and Liu, C and Li, Y and Shi, Q and Kong, D and Ye, H and Fu, C and Wang, Z},
title = {Programmable DNA aptamer logic gates: from structural design to integrated systems for intelligent nanoscale biosensors.},
journal = {Analytical and bioanalytical chemistry},
volume = {},
number = {},
pages = {},
pmid = {40634673},
issn = {1618-2650},
support = {BZ2022056//Caili Fu/ ; Science//Caili Fu/ ; Technology Project of Jiangsu Province//Caili Fu/ ; 32372417//Hua Ye/ ; National Natural Science Foundation of China//Hua Ye/ ; 1182921902//Dezhao Kong/ ; 32001804//Dezhao Kong/ ; Emerging Science//Dezhao Kong/ ; Technology Innovation Team Fu//Dezhao Kong/ ; National Natural Science Foundation of China//Dezhao Kong/ ; 22406069//Chang Liu/ ; National Natural Science Foundation of China//Chang Liu/ ; 1184882301//Qiaoqiao Shi/ ; Natural Science Foundation of the Jiangsu Higher E//Qiaoqiao Shi/ ; },
abstract = {DNA aptamer-based logic gates represent significant advances in molecular computing, enabling complex biological computations at the nanoscale. These systems leverage the unique programmable properties of DNA aptamers-short, single-stranded oligonucleotides with high specificity and binding affinity for diverse applications across fields such as clinical diagnostics, food/environmental monitoring, and targeted therapeutic delivery, garnering significant research interest in the past few decades. In this review, we first expand on the fundamentals of aptamers, including its SELEX process and post-SELEX modifications. We systematically examine the design principles and operation mechanisms of DNA aptamer-based logic gates, mainly AND, OR, INHIBIT and NOT as reported by researchers. Then, we highlight various logic gates based on different oligonucleotides spanning from intact and split aptamers to DNA origami architectures, DNA nanorobots, and G-quadruplex structural switches, bringing to light their applications across various fields. Recent innovations in multi-input/output gate cascades, CRISPR-Cas-integrated systems and signal amplification approaches are highlighted as key developments in DNA aptamer-based logic gates. Finally, we elucidate challenges relating to DNA aptamer-based systems such as aptamer performance, cross-reactivity in complex multi-input systems and the complexities of merging other systems to amplify output readability, among others, to the end that in addressing these challenges, we will be able to unlock the full potential of this system.},
}

@article {pmid40632881,
year = {2025},
author = {Thi Pham, N and Wang, CH and Chen, CH and Tseng, YT and Guthula, LS and Chau, LK and Yu, CY and Chen, YL},
title = {Integration of CRISPR/Cas12a and a Fiber Optic Particle Plasmon Resonance Sensor for Single Nucleotide Polymorphism Detection in an Aldehyde Dehydrogenase 2 Gene.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.5c01035},
pmid = {40632881},
issn = {2379-3694},
abstract = {The highly prevalent single nucleotide polymorphism (SNP, rs671) of the aldehyde dehydrogenase (ALDH2) gene in Asian populations instigates various human pathologies and thus accentuates the urgent need for effective diagnostic tools. In this study, we present an ultrasensitive biosensing method by a combination of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a with the fiber optic nanogold-linked sorbent assay (FONLISA) for precise SNP identification. This method leverages the sequence-specific recognition capability of the CRISPR/Cas system and the ultrahigh sensitivity via the dual signal enhancement mechanisms by integrating the trans-cleavage mechanism of Cas12a to amplify the signal from an activity reporter and the subsequent waveguide-enhanced nanoplasmonic absorption by a signaling reporter. In this method, Cas12a targets a double-stranded DNA from the ALDH2 SNP and then activates the degradation of the activity reporter, a free biotin-labeled single-stranded DNA probe (ssDNA[b]), by trans-cleavage. An unhybridized complementary single-stranded DNA probe (ssDNA[c]) labeled with a gold nanoparticle (AuNP) as the signaling reporter (AuNP@ssDNA[c]) is subsequently released and captured by the immobilized ssDNA[b] on the fiber core surface, resulting in a detectable nanoplasmonic absorption signal. The method also utilized an indispensable nanoplasmonic signal generator, carboxymethyl dextran-coated AuNP, to improve the preparation and bioconjugation processes. The CRISPR-FONLISA system demonstrates the ability to analyze the ALDH2 rs671 SNP from double-stranded DNA with a limit of detection of 71 aM. Furthermore, both cell lines and unamplified DNA extracted from blood samples were conducted to verify the system accuracy for ALDH2 rs671 SNP detection.},
}

@article {pmid40632864,
year = {2025},
author = {Yi, JY and Choi, H and Kim, M and Jeong, Y and Hahn, JS and Son, B and Park, HH and Sung, C},
title = {High-throughput multiplexed gene and cell doping analysis through CRISPR-Cas12a system integrated with blood direct PCR.},
journal = {Science advances},
volume = {11},
number = {28},
pages = {eadv7234},
doi = {10.1126/sciadv.adv7234},
pmid = {40632864},
issn = {2375-2548},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Doping in Sports ; *Polymerase Chain Reaction/methods ; *High-Throughput Screening Assays ; },
abstract = {Advancements in gene and cell therapies introduce "gene and cell doping," requiring efficient and sensitive detection methods. Here, we report a high-throughput multiplexed gene and cell doping analysis (HiMDA) using CRISPR-Cas12a system integrated with blood direct polymerase chain reaction (PCR). Blood direct PCR enables simultaneous amplification of multiple exogenous genes directly from whole-blood samples. Coupled with sequence-specific DNA recognition and fluorescence reporter system, HiMDA achieves multiplexed, on-target detection of doping genes and cells. Our results demonstrate HiMDA's feasibility with only 5 microliters of blood required for the entire 90-minute process. HiMDA exhibits exceptional sensitivity, detecting as few as 2.5 copies of doping target genes from blood-four times more sensitive than current anti-doping standards-and identifying in vivo doping up to 10 days. These findings highlight HiMDA's robust high-throughput, multiplexed capabilities, satisfying the sensitivity and selectivity demands of anti-doping research. HiMDA offers a flexible solution to meet future doping detection challenges.},
}

*CRISPR-Cas Systems/genetics
Humans
*Doping in Sports
*Polymerase Chain Reaction/methods
*High-Throughput Screening Assays

@article {pmid40632850,
year = {2025},
author = {Pal, P and Gao, S and Gao, H and Qin, X and Cella, M and Wang, Q and Shan, L},
title = {Establishment of a reverse genetics system for studying human immune functions in mice.},
journal = {Science advances},
volume = {11},
number = {28},
pages = {eadu1561},
doi = {10.1126/sciadv.adu1561},
pmid = {40632850},
issn = {2375-2548},
mesh = {Animals ; Humans ; Mice ; *Reverse Genetics/methods ; Antigens, CD34/metabolism/genetics ; CRISPR-Cas Systems ; Gene Editing ; Fetal Blood/cytology ; Disease Models, Animal ; },
abstract = {Reverse genetics approaches in mice are widely used to understand gene functions and their aberrations in diseases. However, limitations exist in translating findings from animal models to human physiology. Humanized mice provide a powerful bridge to understanding human physiology and mechanisms of disease pathogenesis while maintaining the feasibility of working with small animals. Methods for generating humanized mouse models that allow scientists to probe contributions of particular genes have been rudimentary. Here, we established an efficient method for generating genetically modified human cord blood-derived CD34[+] cells for transplantation, resulting in humanized mice with near-complete loss of specific gene expression by the human immune system. Mice transplanted with Cas9-edited human CD34[+] cells recapitulate functional consequences of specific gene losses in the human immune system. Our approach enables targeted gene knockouts in humanized mice, offering a valuable tool for human gene function studies in vivo.},
}

Animals
Humans
Mice
*Reverse Genetics/methods
Antigens, CD34/metabolism/genetics
CRISPR-Cas Systems
Gene Editing
Fetal Blood/cytology
Disease Models, Animal

@article {pmid40632760,
year = {2025},
author = {Chao, A and Wang, J and Xiu, L and Bold, B and Ghonaim, AH and Chen, J and Hu, Q and Yin, K},
title = {CRISPR/Cas-Based Biosensing Strategies for Non-Nucleic Acid Contaminants in Food Safety: Status, Challenges, and Perspectives.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c04162},
pmid = {40632760},
issn = {1520-5118},
abstract = {Non-nucleic acid targets (non-NATs), such as heavy metals, toxins, and pesticide residues, pose critical threats to food safety. Although CRISPR/Cas systems were initially developed for nucleic acid detection, recent advances have enabled their adaptation to non-NATs analysis by transducing target recognition into nucleic acid signals. Unlike previous reviews categorized by target type, this work establishes a mechanism-centric framework, systematically classifying non-NAT-to-nucleic acid signal conversion methodologies into three paradigms: (1) aptamer-based systems, (2) catalytic nucleic acid-based methods (e.g., DNAzymes), and (3) protein-mediated strategies (e.g., antibodies, transcription factors). When integrated with CRISPR/Cas, these systems achieve rapid, sensitive detection at picomolar (pM) levels without relying on chromatographic or spectroscopic instruments. Furthermore, we critically discuss challenges, including the limited diversity of recognition elements, inefficient signal conversion, and inflexible signal outputs, proposing solutions including synthetic-biology-driven bioreceptor design and artificial-intelligence-based data analysis. By bridging mechanistic principles with applications in complex food matrices, this review provides actionable insights to advance CRISPR-based tools for rapid, on-site, food safety monitoring.},
}

@article {pmid40632661,
year = {2025},
author = {Wang, S and Zheng, J and Zhang, X and Zhao, M and Li, J and Su, M and Qiu, W},
title = {Genome-Wide CRISPR-Cas9 Knockout Screening Identifies Genes Modulating Cisplatin-Induced Cytotoxicity in Renal Proximal Tubule Epithelial Cells.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {39},
number = {13},
pages = {e70780},
doi = {10.1096/fj.202402401RR},
pmid = {40632661},
issn = {1530-6860},
support = {5202009//Natural Science Foundation of Beijing Municipality/ ; 5192022//Natural Science Foundation of Beijing Municipality/ ; 81870196//MOST | National Natural Science Foundation of China (NSFC)/ ; },
mesh = {*Cisplatin/toxicity/pharmacology/adverse effects ; *Kidney Tubules, Proximal/drug effects/metabolism/cytology/pathology ; *CRISPR-Cas Systems/genetics ; *Epithelial Cells/drug effects/metabolism/pathology ; Humans ; *Antineoplastic Agents/toxicity ; Animals ; Gene Knockout Techniques ; },
abstract = {Cisplatin is widely used as a first-line chemotherapy drug for various cancers. However, cisplatin-induced nephrotoxicity (CIN) greatly restricts its application. Renal proximal tubular epithelial cells (RPTECs) can be extensively damaged during CIN. However, it still lacks an ideal method to prevent CIN, because the mechanism and therapeutic targets of CIN remain largely unclear. In the present study, we used a genome-scale CRISPR-Cas9 knock-out method to functionally screen key genes of cisplatin-induced RPTEC injury. We found 815 genes significantly enriched (p < 0.05) from positive selection screening strategy, which may synergistically enhance cisplatin cytotoxicity in RPTECs. Importantly, we identified ERAP2 as a novel molecule associated with CIN. We found that the expression of ERAP2 in RPTECs was significantly up-regulated by cisplatin. Data from CCK-8 assay and flow cytometry showed that inhibition of ERAP2 alleviated cisplatin-induced RPTEC injury. Furthermore, RNA-seq and qPCR results revealed that three necroptosis-associated genes, PLA2G4C, HIST1H2AC, and HIST1H2AM, were downregulated following ERAP2 inhibition, suggesting that ERAP2 may be a novel therapeutic target of CIN through the modulation of necroptosis pathway.},
}

*Cisplatin/toxicity/pharmacology/adverse effects
*Kidney Tubules, Proximal/drug effects/metabolism/cytology/pathology
*CRISPR-Cas Systems/genetics
*Epithelial Cells/drug effects/metabolism/pathology
Humans
*Antineoplastic Agents/toxicity
Animals
Gene Knockout Techniques

@article {pmid40632351,
year = {2025},
author = {Lokya, V and Singh, S and Chaudhary, R and Jangra, A and Tiwari, S},
title = {Emerging trends in transgene-free crop development: insights into genome editing and its regulatory overview.},
journal = {Plant molecular biology},
volume = {115},
number = {4},
pages = {84},
pmid = {40632351},
issn = {1573-5028},
support = {BIRAC/TechTransfer/08/I2/QUT-BBF//Biotechnology Industry Research Assistance Council/ ; Core research grant//BRIC-National Agri-Food and Biomanufacturing Institute (BRIC-NABI)/ ; },
mesh = {*Gene Editing/methods/trends ; *Crops, Agricultural/genetics/growth & development ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; *Genome, Plant ; Transgenes ; },
abstract = {Genome editing tools have revolutionized plant biology research offering unparalleled applications for genome manipulation and trait improvement in crops. Adopting such advanced biotechnological tools is inevitable to meet increasing global food demand and address challenges in food production, including (a)biotic stresses and inadequate nutritional value. Despite reliance on conventional genetic manipulation methods, the CRISPR-Cas-mediated genome editing toolbox allows precise modification of DNA/RNA in a target organism's genome. So far, CRISPR-Cas has been widely used to enhance yield, quality, stress tolerance, and nutritional value in various food crops. However, challenges such as reagent delivery in suitable explants, precise editing with minimal off-target effect, and generating transgene-free plants persist as major bottlenecks in most plant species. Components of CRISPR-Cas construct mainly Cas, guide RNA (gRNA), and selectable marker genes are often integrated into the host genome, which raises regulatory concerns. However, adapting advanced gene-editing strategies, including high-efficiency Cas endonucleases, DNA-independent RNP delivery, morphogenetic regulators, and grafting-mediated editing, are paving the way for transgene-free crop improvement while easing biosafety regulations. Further, regulatory frameworks for genome-edited crops vary globally, with several countries accepting them and others debating their legal status. Hence, the disparity in global regulatory guidelines of genome editing curbs commercialization. The current review highlights the emerging CRISPR-mediated tools or methods and their applications in developing transgene-free designer crops to harness the benefits of advanced genome manipulation.},
}

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

@article {pmid40632173,
year = {2025},
author = {Thomas, L and Abraham, T},
title = {Disabling iron uptake and pilus assembly in uropathogenic Escherichia coli using CRISPR-Cas9: a step towards antivirulence therapy.},
journal = {Antonie van Leeuwenhoek},
volume = {118},
number = {8},
pages = {110},
pmid = {40632173},
issn = {1572-9699},
support = {363661//University Grants Commission/ ; },
mesh = {*Uropathogenic Escherichia coli/genetics/metabolism/pathogenicity ; *CRISPR-Cas Systems ; *Iron/metabolism ; *Fimbriae, Bacterial/metabolism/genetics ; Escherichia coli Proteins/genetics/metabolism ; Escherichia coli Infections/microbiology ; Virulence Factors/genetics/metabolism ; Virulence/genetics ; Molecular Docking Simulation ; Humans ; Fimbriae Proteins/genetics/metabolism ; Gene Editing ; },
abstract = {Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs), driven by virulence factors such as iron acquisition systems and adhesive pili. In this study, we employed CRISPR-Cas9-mediated genome editing to functionally inactivate two critical virulence genes-iucD, involved in aerobactin-mediated iron uptake, and papC, encoding the outer membrane usher protein essential for P pilus assembly. Using a clinical UPEC isolate, we introduced premature stop codons via homologous repair templates guided by gene-specific single-guide RNAs. Colony PCR and Sanger sequencing confirmed precise site-specific editing, leading to truncated protein variants. In silico analyses using InterPro and Swiss-Model revealed a complete loss of essential domains in both proteins. Molecular docking studies demonstrated a marked reduction in binding affinities of truncated iucD for NAD(P)H and impaired protein-protein interaction between truncated PapC and PapG. This study highlights the utility of CRISPR-Cas9 as a powerful tool for dissecting bacterial pathogenesis and supports the potential of targeting virulence determinants like iucD and papC as part of an antivirulence strategy for managing UPEC infections.},
}

*Uropathogenic Escherichia coli/genetics/metabolism/pathogenicity
*CRISPR-Cas Systems
*Iron/metabolism
*Fimbriae, Bacterial/metabolism/genetics
Escherichia coli Proteins/genetics/metabolism
Escherichia coli Infections/microbiology
Virulence Factors/genetics/metabolism
Virulence/genetics
Molecular Docking Simulation
Humans
Fimbriae Proteins/genetics/metabolism
Gene Editing

@article {pmid40631558,
year = {2025},
author = {He, C and Zhu, W and Zhang, X and Wu, W and Tan, J and Li, B and Huang, D and Chen, Y and Xiang, Z and Huang, L and Gong, L},
title = {Sensitive and Visualized Detection of Hantavirus Using CRISPR/Cas12a Based on AutoCORDSv2 Design.},
journal = {Journal of medical virology},
volume = {97},
number = {7},
pages = {e70460},
doi = {10.1002/jmv.70460},
pmid = {40631558},
issn = {1096-9071},
support = {//This study is financially supported by The President Foundation of Nanfang Hospital, Southern Medical University (grant number 2023B036); National Science and Technology Infrastructure of China (Project No. National Pathogen Resource Center-NPRC-32); The CAMS Innovation Fund for Medical Sciences (grant number 2021-I2M-1-039); National Key R&D Program of China, grant number (grant number 2022YFC2303400); The Guangdong Basic and Applied Basic Research Foundation (grant number 2023A1515140080); Dongguan Social development technology projects Science and Technology Planning Project (grant number 20231800940792)./ ; },
mesh = {*CRISPR-Cas Systems ; Sensitivity and Specificity ; Humans ; *Molecular Diagnostic Techniques/methods ; *Orthohantavirus/isolation & purification/genetics ; RNA, Viral/genetics ; *Hantavirus Infections/diagnosis/virology ; *Hantaan virus/genetics/isolation & purification ; DNA Primers/genetics ; },
abstract = {In recent years, detection technologies based on the CRISPR/Cas12a method have been extensively utilized in the fields of nucleic acid, enzyme, and macromolecule detection, thereby reinforcing their significant role in the detection landscape. Enhancing the simplicity of design, efficiency, and automation of the CRISPR/Cas12a detection system is essential for advancing its application in diagnostics. Recently, we developed an automated CRISPR/Cas12a design system named AutoCORDSv2. This system can process published genomic sequences of pathogenic bacteria in a high-throughput manner and automatically generate conserved and highly specific crRNA sequences, along with primer sequences for target amplification. This capability facilitates the specific and precise design of the CRISPR/Cas12a detection system. In this study, crRNAs targeting the Hantaan virus (HTNV) and Seoul virus (SEOV), as well as RT-PCR primers and RT-RPA primers, were designed using AutoCORDSv2. The experimental results demonstrated that the CRISPR/Cas12a system, automatically designed by AutoCORDSv2, was specific for the detection of both the HTNV and SEOV, with no cross-reactivity observed with other pathogens. The detection sensitivity reached 6 copies/μL (equivalent to 111 copies per amplification reaction), whether measured by a microplate reader or directly observed with the naked eye. The detection results for 50 samples were consistent with those obtained from commercial RT-qPCR kits, indicating high precision. Furthermore, the CRISPR/Cas12a system designed by AutoCORDSv2 can also be utilized for the development of a single-tube detection system with a sensitivity of 42 copies per reaction. This system combined with a 5-min extraction step and RT-RPA, further underscoring its potential for application.},
}

*CRISPR-Cas Systems
Sensitivity and Specificity
Humans
*Molecular Diagnostic Techniques/methods
*Orthohantavirus/isolation & purification/genetics
RNA, Viral/genetics
*Hantavirus Infections/diagnosis/virology
*Hantaan virus/genetics/isolation & purification
DNA Primers/genetics

@article {pmid40630641,
year = {2025},
author = {Pan, K and Zeng, A and Ruan, X and Mo, X and Shen, B and Zhao, J and Zhou, Y},
title = {The apicoplast localized isocitrate dehydrogenase is needed for de novo fatty acid synthesis in the apicoplast of Toxoplasma gondii.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1542122},
pmid = {40630641},
issn = {2235-2988},
mesh = {*Toxoplasma/enzymology/growth & development/genetics/metabolism ; *Isocitrate Dehydrogenase/metabolism/genetics ; Animals ; *Fatty Acids/biosynthesis ; *Apicoplasts/enzymology/metabolism/genetics ; Mice ; Virulence ; CRISPR-Cas Systems ; Gene Deletion ; NADP/metabolism ; Glucose/metabolism ; },
abstract = {Toxoplasma gondii (T. gondii), an apicomplexan parasite, infects a wide range of warm-blooded animals and poses significant risks to human health. The fatty acid synthesis II (FASII) pathway in the apicoplast, which is the major source of fatty acids in parasites, is considered a potential drug target. The apicoplast also harbors some enzymes of central carbon metabolism, which are crucial for its survival, but their biological roles remain unclear. In this study, we focused on apicoplast-localized isocitrate dehydrogenase 1 (ICDH1) and deleted it using CRISPR-Cas9 technology. The Δicdh1 mutant tachyzoites displayed markedly impaired growth kinetics, with further suppression under serum-deprived conditions. However, this deletion did not affect the viability or virulence of the Δicdh1 mutant in mice. NADPH, a product of ICDH1-mediated decarboxylation of isocitrate, is an essential cofactor for fatty acid synthesis. Using [13]C6 glucose as a metabolic carbon source, we showed that the mutant strains had reduced incorporation of glucose-derived carbons into medium-chain length fatty acids (C14:0 and C16:0). Additionally, the growth of the mutant was partially restored by supplementation with exogenous C14:0 and C16:0 fatty acids. These results indicate that ICDH1 deletion affects the FASII pathway and parasite growth. Consistent with previous studies, this study confirms that T. gondii has metabolic flexibility in the apicoplast that allows it to acquire fatty acids through various pathways.},
}

*Toxoplasma/enzymology/growth & development/genetics/metabolism
*Isocitrate Dehydrogenase/metabolism/genetics
Animals
*Fatty Acids/biosynthesis
*Apicoplasts/enzymology/metabolism/genetics
Mice
Virulence
CRISPR-Cas Systems
Gene Deletion
NADP/metabolism
Glucose/metabolism

@article {pmid40629383,
year = {2025},
author = {Gou, F and Liu, D and Gong, C and Wang, K and Wang, X and Chen, Y and Liu, Q and Tian, C},
title = {Development of an efficient heterologous protein expression platform in Aspergillus niger through genetic modification of a glucoamylase hyperproducing industrial strain.},
journal = {Microbial cell factories},
volume = {24},
number = {1},
pages = {160},
pmid = {40629383},
issn = {1475-2859},
support = {2023YFC3402300//National Key Research & Developmental Program of China/ ; U22A20441//National Natural Science Foundation of China/ ; XDA0510300//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; },
mesh = {*Aspergillus niger/genetics/metabolism/enzymology ; *Glucan 1,4-alpha-Glucosidase/genetics/biosynthesis/metabolism ; Recombinant Proteins/genetics/biosynthesis/metabolism ; CRISPR-Cas Systems ; Industrial Microbiology ; Genetic Engineering ; },
abstract = {BACKGROUND: Aspergillus niger is widely used in industrial enzyme production due to its strong secretion capacity and the status of generally recognized as safe (GRAS). However, heterologous protein expression in A. niger is frequently constrained by high levels of background endogenous protein secretion, limited access to native high transcription loci, and limitations in the efficiency of the secretory machinery. To address these limitations, this study genetically engineered a chassis strain based on an industrial glucoamylase-producing A. niger strain AnN1 for constructing the improved heterologous protein expression.

RESULTS: In this study, by using CRISPR/Cas9-assisted marker recycling, we deleted 13 of the 20 copies of the heterologous glucoamylase TeGlaA gene and disrupted the major extracellular protease gene PepA, resulting in the low-background strain AnN2. Compared to the parental strain AnN1, AnN2 exhibited 61% less extracellular protein and significantly reduced glucoamylase activity, while retaining multiple transcriptionally active integration loci. Four diverse proteins were integrated into the high-expression loci originally occupied by the TeGlaA gene in the chassis AnN2. These recombinant protein included a homologous glucose oxidase (AnGoxM), a thermostable pectate lyase A (MtPlyA), a bacterial triose phosphate isomerase (TPI), and a medical protein Lingzhi-8 (LZ8). All target proteins were successfully expressed and secreted within 48-72 h, with yields ranging from 110.8 to 416.8 mg/L in 50 mL shake-flasks cultivation. The enzyme activities of AnGoxM, MtPlyA and TPI reached ~ 1276 - 1328 U/mL, ~ 1627. 43 - 2105.69 U/mL, and ~ 1751.02 to 1906.81 U/mg after 48 h, respectively. Additionally, Overexpression of Cvc2, a COPI vesicle trafficking component, further enhanced MtPlyA production by 18%, highlighting the benefit of combining transcriptional and secretory pathway engineering.

CONCLUSIONS: Our results demonstrated that the chassis AnN2 served as a robust, modular, and time-efficient platform for heterologous protein expression in A. niger. Through site-specific integration of target genes into native high-expression loci and strategic modulation of the secretory pathway, we successfully enabled the rapid production of functional enzymes and bioactive proteins from diverse origins. This dual-level optimization strategy, which integrates rational genomic engineering with targeted enhancement of the secretory pathway, enabled high-yield expression while minimizing background interference. Together, these findings offer a practical framework for constructing versatile fungal expression systems and highlight the potential of combining genetic and cellular engineering to improve recombinant protein production in filamentous fungi.},
}

*Aspergillus niger/genetics/metabolism/enzymology
*Glucan 1,4-alpha-Glucosidase/genetics/biosynthesis/metabolism
Recombinant Proteins/genetics/biosynthesis/metabolism
CRISPR-Cas Systems
Industrial Microbiology
Genetic Engineering

@article {pmid40586242,
year = {2025},
author = {Sreekanth, V and Jan, M and Zhao, KT and Lim, D and Siriwongsup, S and Davis, JR and McConkey, M and Kovalcik, V and Barkal, S and Law, BK and Fife, J and Tian, R and Vinyard, ME and Becerra, B and Kampmann, M and Sherwood, RI and Pinello, L and Liu, DR and Ebert, BL and Choudhary, A},
title = {A Molecular Glue Approach to Control the Half-Life of CRISPR-Based Technologies.},
journal = {Journal of the American Chemical Society},
volume = {147},
number = {27},
pages = {23844-23856},
doi = {10.1021/jacs.5c06230},
pmid = {40586242},
issn = {1520-5126},
mesh = {Half-Life ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Thalidomide/analogs & derivatives/chemistry/pharmacology/metabolism ; Ubiquitination ; },
abstract = {Cas9 is a programmable nuclease that has furnished transformative technologies, including base editors and transcription modulators (e.g., CRISPRi/a), but several applications of these technologies, including therapeutics, mandatorily require precision control of their half-life. For example, such control can help avert any potential immunological and adverse events in clinical trials. Current genome editing technologies to control the half-life of Cas9 are slow, have lower activity, involve fusion of large response elements (>230 amino acids), utilize expensive controllers with poor pharmacological attributes, and cannot be implemented in vivo on several CRISPR-based technologies. We report a general platform for half-life control using the molecular glue, pomalidomide, that binds to a ubiquitin ligase complex and a response-element bearing CRISPR-based technology, thereby causing the latter's rapid ubiquitination and degradation. Using pomalidomide, we were able to control the half-life of large CRISPR-based technologies (e.g., base editors and CRISPRi) and small anti-CRISPRs that inhibit such technologies, allowing us to build the first examples of on-switch for base editors. The ability to switch on, fine-tune, and switch-off CRISPR-based technologies with pomalidomide allowed complete control over their activity, specificity, and genome editing outcome. Importantly, the miniature size of the response element and favorable pharmacological attributes of the drug pomalidomide allowed control of activity of base editor in vivo using AAV as the delivery vehicle. These studies provide methods and reagents to precisely control the dosage and half-life of CRISPR-based technologies, propelling their therapeutic development.},
}

Half-Life
Humans
*Gene Editing/methods
*CRISPR-Cas Systems
*Thalidomide/analogs & derivatives/chemistry/pharmacology/metabolism
Ubiquitination

@article {pmid40577080,
year = {2025},
author = {Butti, P and Bellusci, F and Brambilla, E and Branduardi, P},
title = {Genomically integrated cassettes swapping: bringing modularity to the strain level in Saccharomyces cerevisiae.},
journal = {FEMS yeast research},
volume = {25},
number = {},
pages = {},
doi = {10.1093/femsyr/foaf032},
pmid = {40577080},
issn = {1567-1364},
support = {//University of Milano-Bicocca/ ; CN_00000033//Italian Ministry of University and Research/ ; H43C22000530001//Italian Ministry of University and Research/ ; },
mesh = {*Saccharomyces cerevisiae/genetics ; CRISPR-Cas Systems ; *Synthetic Biology/methods ; *Gene Editing/methods ; *Genome, Fungal ; },
abstract = {A large variety of synthetic biology toolkits for the introduction of multiple expression cassettes is available for Saccharomyces cerevisiae. Unfortunately, none of these tools is designed to allow the modification - exchange or removal - of the cassettes already integrated into the genome in a standardized way. The application of the modularity principle therefore ends to the steps preceding the final host engineering, making microbial cell factories construction stiff and strictly sequential. In this work, we describe a system that easily allows CRISPR-mediated swapping or removal of previously integrated cassettes, thus bringing the modularity to the strain level, enhancing the possibility of modifying existing strains with a reduced number of steps. In the system, each cassette is tagged with specific barcodes, which can be used as targets for CRISPR nucleases (Cas9 and Cas12a), allowing the excision of the construct from the genome and its substitution with another expression cassette or the restoration of the wild type locus in one single standardized step. The system has been applied to the previously developed Easy-MISE toolkit and tested by swapping fluorescent protein expression cassettes with an efficiency of ∼90% quantified by PCR and flow cytometry.},
}

*Saccharomyces cerevisiae/genetics
CRISPR-Cas Systems
*Synthetic Biology/methods
*Gene Editing/methods
*Genome, Fungal

@article {pmid40552742,
year = {2025},
author = {Petroulia, S and Hockemeyer, K and Tiwari, S and Berico, P and Shamloo, S and Banijamali, SE and Vega-Saenz de Miera, E and Gong, Y and Thandapani, P and Wang, E and Schloßhauer, JL and Tsirigos, A and Osman, I and Aifantis, I and Imig, J},
title = {Uncovering Novel lncRNAs Linked to Melanoma Growth and Migration with CRISPR Inhibition Screening.},
journal = {Cancer research communications},
volume = {5},
number = {7},
pages = {1102-1118},
doi = {10.1158/2767-9764.CRC-24-0416},
pmid = {40552742},
issn = {2767-9764},
mesh = {Humans ; *Melanoma/genetics/pathology ; *RNA, Long Noncoding/genetics ; Cell Movement/genetics ; Cell Line, Tumor ; Cell Proliferation/genetics ; *Skin Neoplasms/genetics/pathology ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; Drug Resistance, Neoplasm/genetics ; Apoptosis/genetics ; },
abstract = {UNLABELLED: Melanoma being one of the most common and deadliest skin cancers has been increasing since the past decade. Patients at advanced stages of the disease have very poor prognoses, as opposed to at the earlier stages. Nowadays, the standard of care of advanced melanoma is resection, followed by immune checkpoint inhibition-based immunotherapy. However, a substantial proportion of patients either do not respond or develop resistance. This underscores a need for novel approaches and therapeutic targets as well as a better understanding of the mechanisms of melanoma pathogenesis. Long noncoding RNAs (lncRNA) comprise a poorly characterized class of functional players and promising targets in promoting malignancy. Certain lncRNAs have been identified to play integral roles in melanoma progression and drug resistance; however, systematic screens to uncover novel functional lncRNAs are scarce. In this study, we profile differentially expressed lncRNAs in patient-derived short-term metastatic cultures and BRAF-MEK inhibition-resistant cells. We conduct a focused growth-related CRISPR inhibition screen of overexpressed lncRNAs, validate, and functionally characterize lncRNA hits with respect to cellular growth, invasive capacities, and apoptosis in vitro as well as the transcriptomic impact of our lead candidate the novel lncRNA XLOC_030781. In sum, we extend the current knowledge of ncRNAs and their potential relevance in melanoma.

SIGNIFICANCE: LncRNAs have emerged as novel players in regulating many cellular aspects also in melanoma. The number of functional significances of most lncRNAs remains elusive. We provide a comprehensive strategy to identify functionally relevant lncRNAs in melanoma by combining expression profiling with CRISPR inhibition growths screens. Our results broaden the characterized lncRNAs as potential targets for future therapeutic applications.},
}

Humans
*Melanoma/genetics/pathology
*RNA, Long Noncoding/genetics
Cell Movement/genetics
Cell Line, Tumor
Cell Proliferation/genetics
*Skin Neoplasms/genetics/pathology
Gene Expression Regulation, Neoplastic
CRISPR-Cas Systems
Drug Resistance, Neoplasm/genetics
Apoptosis/genetics

@article {pmid40482807,
year = {2025},
author = {Geiger, AB and Kennedy, JG and Staker, LG and Wensel, TG and Casson, RJ and Thomas, PQ},
title = {Shining light on CRISPR/Cas9 therapeutics for inherited retinal diseases.},
journal = {Progress in retinal and eye research},
volume = {107},
number = {},
pages = {101376},
doi = {10.1016/j.preteyeres.2025.101376},
pmid = {40482807},
issn = {1873-1635},
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genetic Therapy/methods ; *Retinal Diseases/therapy/genetics ; Animals ; },
abstract = {Inherited retinal diseases (IRDs), such as retinitis pigmentosa, are a heterogenous group of genetic eye diseases characterized by degeneration of photoreceptors. They are the leading cause of blindness in the working age population in high-income countries and are an ideal target for the expanding gene editing tool kit, including rapidly evolving CRISPR/Cas9 technology. In this review, we provide a comprehensive analysis of CRISPR/Cas9 technologies currently being explored as therapeutic interventions for IRDs. Given the challenges posed by the growing complexity and size of gene editing systems, the delivery of these therapeutics to the retina has necessitated innovative approaches. We review current delivery methods, including nanoparticles, virus-like particles and traditional viral vectors, highlighting their advantages and limitations. This review underscores the potential transformative impact of gene editing on genetic disease management, emphasising that advancements in these technologies, coupled with improved pre-clinical models, bring clinically safe and effective treatments for IRDs within view.},
}

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

@article {pmid40434115,
year = {2025},
author = {Wiechert, J and Badia Roigé, B and Dohmen-Olma, D and Hindra, and Zhang, X and Stella, RG and Elliot, MA and Frunzke, J},
title = {CRISPR/dCas-mediated counter-silencing: reprogramming dCas proteins into antagonists of xenogeneic silencers.},
journal = {mBio},
volume = {16},
number = {7},
pages = {e0038225},
doi = {10.1128/mbio.00382-25},
pmid = {40434115},
issn = {2150-7511},
support = {458090666//Deutsche Forschungsgemeinschaft/ ; 162340/CAPMC/CIHR/Canada ; //Jürgen Manchot Stiftung/ ; },
mesh = {*CRISPR-Cas Systems ; Promoter Regions, Genetic ; *Bacterial Proteins/genetics/metabolism ; *Streptomyces/genetics ; *Gene Silencing ; *Gene Expression Regulation, Bacterial ; *Corynebacterium glutamicum/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {UNLABELLED: Lsr2-like nucleoid-associated proteins function as xenogeneic silencers (XSs) inhibiting expression of horizontally acquired, adenine-thymine-rich DNA in actinobacteria. Interference by transcription factors can lead to counter-silencing of XS target promoters, but relief of this repression typically requires promoter engineering. In this study, we developed a novel clustered regularly interspaced short palindromic repeats (CRISPR)/dCas-mediated counter-silencing (CRISPRcosi) approach by using nuclease-deficient dCas enzymes to counteract the Lsr2-like XS protein CgpS in Corynebacterium glutamicum or Lsr2 in Streptomyces venezuelae. Systematic in vivo reporter studies with dCas9 and dCas12a and various guide RNAs revealed effective counter-silencing of different CgpS target promoters in response to guide RNA/dCas DNA binding, independent of promoter sequence modifications. The most prominent CRISPRcosi effect was observed when targeting the CgpS nucleation site, an effect that was also seen in S. venezuelae when targeting a known Lsr2 nucleation site within the chloramphenicol biosynthesis gene cluster. Analyzing the system in C. glutamicum strains lacking the XS protein CgpS revealed varying strengths of counteracting CRISPR interference effects based on the target position and strand. Genome-wide transcriptome profiling in single-guide RNA/dCas9 co-expressing C. glutamicum wild-type strains revealed high counter-silencing specificity with minimal off-target effects. Thus, CRISPRcosi provides a promising strategy for the precise upregulation of XS target genes with significant potential for studying gene networks as well as for developing applications in biotechnology and synthetic biology.

IMPORTANCE: Lsr2-like nucleoid-associated proteins act as xenogeneic silencers (XSs), repressing the expression of horizontally acquired, adenine-thymine-rich DNA in actinobacteria. The targets of Lsr2-like proteins are very diverse, including prophage elements, virulence gene clusters, and biosynthetic gene clusters. Consequently, the targeted activation of XS target genes is of interest for fundamental research and biotechnological applications. Traditional methods for counter-silencing typically require promoter modifications. In this study, we developed a novel clustered regularly interspaced short palindromic repeats (CRISPR)/dCas-mediated counter-silencing (CRISPRcosi) approach, utilizing nuclease-deficient dCas enzymes to counteract repression by Lsr2-like proteins in Corynebacterium glutamicum and Streptomyces venezuelae. The strongest effect was observed when targeting the Lsr2 nucleation site. Genome-wide transcriptome profiling revealed high specificity with minimal off-target effects. Overall, CRISPRcosi emerges as a powerful tool for the precise induction of genes silenced by xenogeneic silencers, offering new opportunities for exploring gene networks and advancing biotechnological applications.},
}

*CRISPR-Cas Systems
Promoter Regions, Genetic
*Bacterial Proteins/genetics/metabolism
*Streptomyces/genetics
*Gene Silencing
*Gene Expression Regulation, Bacterial
*Corynebacterium glutamicum/genetics/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid39915620,
year = {2025},
author = {Pagliaro, A and Andreatta, F and Finger, R and Artegiani, B and Hendriks, D},
title = {Generation of human fetal brain organoids and their CRISPR engineering for brain tumor modeling.},
journal = {Nature protocols},
volume = {20},
number = {7},
pages = {1846-1883},
pmid = {39915620},
issn = {1750-2799},
mesh = {Humans ; *Organoids/cytology/metabolism ; *Brain Neoplasms/genetics/pathology ; *Brain/embryology/cytology ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Fetus/cytology ; },
abstract = {The developing human brain displays unique features that are difficult to study in animal models. Current in vitro models based on human brain tissue face several challenges, including the limited cellular heterogeneity in two- or three-dimensional neural stem cell cultures, while tissue slice cultures suffer from short survival. We recently established culture conditions to derive organoid cultures directly from human fetal brain tissue by preserving tissue integrity, which can be long-term expanded and display cellular heterogeneity and complex organization. In this Protocol, we describe detailed procedures to establish human fetal brain organoids (FeBOs) that broadly retain regional characteristics, along with procedures for their passaging and characterization. In addition, we describe genome engineering approaches applied to FeBOs to generate mutant FeBO lines that serve as versatile bottom-up brain cancer models. Lastly, we exemplify various downstream applications applicable to both healthy and mutant FeBOs. Scientists with experience in tissue culture can expect the establishment of human FeBO cultures to take 2-3 weeks, while genome engineering of FeBOs takes 2-4 months.},
}

Humans
*Organoids/cytology/metabolism
*Brain Neoplasms/genetics/pathology
*Brain/embryology/cytology
*CRISPR-Cas Systems
*Gene Editing/methods
Fetus/cytology

@article {pmid40627177,
year = {2025},
author = {Azani, A and Sharafi, M and Doachi, R and Akbarzadeh, S and Lorestani, P and Haji Kamanaj Olia, A and Zahed, Z and Gharedaghi, H and Ghasrsaz, H and Foroozand, H and Rahimi, H and Tahmasebi, S and Behfar, Q},
title = {Applications of CRISPR-Cas9 in mitigating cellular senescence and age-related disease progression.},
journal = {Clinical and experimental medicine},
volume = {25},
number = {1},
pages = {237},
pmid = {40627177},
issn = {1591-9528},
mesh = {Humans ; *Cellular Senescence/genetics ; *CRISPR-Cas Systems ; *Aging/genetics ; Epigenesis, Genetic ; *Gene Editing/methods ; Disease Progression ; Animals ; Telomerase/genetics/metabolism ; },
abstract = {Aging is a multifaceted process influenced by many elements. During cell division, the repetitive DNA sequences at the ends of chromosomes called telomeres protect them from degradation. Telomeres shorten alongside each cell division, eventually contributing to cellular senescence and aging. Telomerase as an enzyme has a role in the maintenance of telomere length. Reduced function of telomerase is linked to acceleration of aging and age-related diseases. By affecting cellular function, mutations in particular genes can cause aging. Genes involved in DNA repair, cellular metabolism, and inflammation play the key roles in this process. Accumulated mutations result in cellular dysfunction and age-related diseases over time. Epigenetic changes are the modifications that impact gene expression without altering the DNA sequence. Lifestyle factors (diet, exercise, stress) and environmental influences (toxins, trauma) can cause epigenetic alterations. DNA methylation as well as histone modifications are examples of epigenetic alterations. They influence how cells work and are essential to the aging process. Understanding these molecular mechanisms is essential for developing interventions to promote healthy aging and prevent age-related diseases. This paper explores the potential of CRISPR/Cas9 as a gene-editing tool to target these mechanisms and mitigate age-related conditions, ultimately enhancing longevity and quality of life.},
}

Humans
*Cellular Senescence/genetics
*CRISPR-Cas Systems
*Aging/genetics
Epigenesis, Genetic
*Gene Editing/methods
Disease Progression
Animals
Telomerase/genetics/metabolism

@article {pmid40627029,
year = {2025},
author = {M, KR and C, SD},
title = {Recent insights into actinobacteria research in antimicrobial resistance: a review.},
journal = {Molecular biology reports},
volume = {52},
number = {1},
pages = {683},
pmid = {40627029},
issn = {1573-4978},
mesh = {*Actinobacteria/genetics/metabolism/drug effects ; *Anti-Bacterial Agents/pharmacology ; Multigene Family ; Humans ; *Drug Resistance, Multiple, Bacterial/genetics ; Streptomyces/genetics/metabolism ; *Drug Resistance, Bacterial/genetics ; Biosynthetic Pathways/genetics ; },
abstract = {Antimicrobial resistance (AMR) has emerged as a global health crisis, taking 4.71 million lives in the year 2021 and posing significant challenges to healthcare systems. Actinobacteria, particularly Streptomyces sp., are a well-established source of bioactive secondary metabolites, including antibiotics such as polyketides, aminoglycosides, and macrolides with activity against multidrug-resistant (MDR) bacteria. However, only 10% of the antibiotic genes are expressed, and others are silent in cryptic biosynthetic gene clusters (BGCs) that remain inactive under standard laboratory conditions. Advances in genome mining, bioinformatics tools like antiSMASH, and molecular techniques such as CRISPR-Cas have facilitated the identification of these clusters. Furthermore, innovative strategies such as co-culturing and HDAC inhibitors have shown promise in activating cryptic biosynthetic pathways to combat emerging antimicrobial resistance. Despite these advancements, the rapid evolution of resistance requires continuous research and global collaboration to ensure a sustainable pipeline of effective antibiotics. This review provides insight into actinobacteria-derived antibiotics, resistance mechanisms, and emerging biotechnological interventions to address the AMR crisis, underscoring the urgent need for multidisciplinary antibiotic discovery and stewardship efforts.},
}

*Actinobacteria/genetics/metabolism/drug effects
*Anti-Bacterial Agents/pharmacology
Multigene Family
Humans
*Drug Resistance, Multiple, Bacterial/genetics
Streptomyces/genetics/metabolism
*Drug Resistance, Bacterial/genetics
Biosynthetic Pathways/genetics

@article {pmid40626824,
year = {2025},
author = {Özdemir, BH},
title = {Navigating Immunological Barriers in Xenotransplantation: Recent Advances and Promising Strides.},
journal = {Experimental and clinical transplantation : official journal of the Middle East Society for Organ Transplantation},
volume = {23},
number = {6},
pages = {421-430},
doi = {10.6002/ect.2023.0351},
pmid = {40626824},
issn = {2146-8427},
mesh = {Humans ; Animals ; *Transplantation, Heterologous/adverse effects ; *Graft Rejection/immunology/prevention & control/genetics ; Animals, Genetically Modified ; Swine ; *Immunity, Humoral ; *Graft Survival ; *Antibodies, Heterophile/immunology/genetics/blood ; *Antigens, Heterophile/immunology/genetics ; Immunity, Cellular ; Treatment Outcome ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {The review introduces the challenges and potential solutions in xenotransplantation, focusing on pig-to-human organ transplant. Xenotransplantation, mainly with the use of pig organs, is a promising solution because of the reproductive capacity, size, and physiological resemblance of pigs to humans. However, immunological barriers, especially humoral and cellular immune responses, pose substantial challenges. The humoral immune response, involving antibodies targeting xenoantigens, is a substantial barrier. Anti-α-galactose antibodies, targeting α-Gal epitopes, are crucial in hyperacute rejection and acute humoral xenograft rejection. Genetic modifications, including CRISPR/Cas9 technology, aim to eliminate xenoantigens like α-Gal, potentially overcoming these challenges. This review discusses the use of genetically modified pigs for xenotransplantation, emphasizing the removal of xenoantigens, expression of human complement regulatory proteins, and transgenic expres-sion of human regulatory factors. Recent advancements, such as the world's first porcine-to-human heart transplant, highlight the potential of genetic manipu-lation in overcoming immune rejection barriers.},
}

Humans
Animals
*Transplantation, Heterologous/adverse effects
*Graft Rejection/immunology/prevention & control/genetics
Animals, Genetically Modified
Swine
*Immunity, Humoral
*Graft Survival
*Antibodies, Heterophile/immunology/genetics/blood
*Antigens, Heterophile/immunology/genetics
Immunity, Cellular
Treatment Outcome
CRISPR-Cas Systems
Gene Editing

@article {pmid40626243,
year = {2025},
author = {Mitousis, L and Musiol-Kroll, E and Wohlleben, W},
title = {CRISPR-Cas in actinomycetes: still a lot to be discovered.},
journal = {microLife},
volume = {6},
number = {},
pages = {uqaf010},
pmid = {40626243},
issn = {2633-6693},
abstract = {Actinomycetes are important producers of valuable natural products that are applied in medicine or industry. The enzymes necessary for the synthesis of those compounds are encoded in biosynthetic gene clusters (BGCs) in the genome. However, the discovery of new natural products or the improvement of production levels can be hindered by difficulties in genetic manipulation, since standard methods often do not or not efficiently work in actinomycetes. One possible explanation for this could be the presence of nucleic acid defense systems such as CRISPR-Cas. Even though there is a lot of research published about CRISPR-Cas systems in general, the knowledge about the function of CRISPR-Cas in actinomycetes is very limited. Based on sequence data it is known that CRISPR-Cas systems occur in around half of all sequenced actinobacterial genomes. Moreover, in silico analyses of those systems have led to the discovery of new subtypes. The few examples of experimental evidence of CRISPR-Cas activity in vivo or in vitro, however, point to some special features, regarding crRNA maturation or life-cycle dependent CRISPR-Cas activity. This short review draws attention to this neglected research area and highlights the available data about CRISPR-Cas in actinomycetes.},
}

@article {pmid40364716,
year = {2025},
author = {Cahn, JKB and Ludwicki, H and Shingler, J and Gulvin, S and Zhang, Y and Kristopeit, A and Ton, C and Winters, MA and Wagner, JM and Moore, J},
title = {CRISPR-Editing of the Vero Cell Line Improves Processability of Live Virus Vaccines by Enabling Targeted Proteolysis of Fibronectin.},
journal = {Biotechnology and bioengineering},
volume = {122},
number = {8},
pages = {2082-2092},
doi = {10.1002/bit.29028},
pmid = {40364716},
issn = {1097-0290},
mesh = {Animals ; Chlorocebus aethiops ; Vero Cells ; *Gene Editing/methods ; *Fibronectins/genetics/metabolism ; *CRISPR-Cas Systems ; *Viral Vaccines/genetics/metabolism ; Proteolysis ; Vaccines, Attenuated/genetics ; },
abstract = {Removal of host cell components is a significant cost driver in the production of live virus vaccines. Filtration processes such as tangential flow filtration can be effective in this capacity by leveraging the relative size difference between viral particles and host proteins; however, filtration membranes can be fouled by larger proteins, particularly those of the extracellular matrix. In this study, we used CRISPR editing to insert the recognition element of the highly-selective TEV protease into various positions of the gene encoding fibronectin in the genome of the Vero cell line, a common platform for viral production. By screening edited cell lines, we identified a promising candidate line in which fibronectin could be effectively removed by treating with the protease during processing, eliminating filter fouling and allowing for viral purification without the need for costly chromatography steps.},
}

Animals
Chlorocebus aethiops
Vero Cells
*Gene Editing/methods
*Fibronectins/genetics/metabolism
*CRISPR-Cas Systems
*Viral Vaccines/genetics/metabolism
Proteolysis
Vaccines, Attenuated/genetics

@article {pmid40345583,
year = {2025},
author = {Flores-Arenas, C and Malekos, E and Montano, C and Covarrubias, S and Sudek, L and Dempsey, V and Huynh, V and Katzman, S and Carpenter, S},
title = {CRISPRi screen uncovers lncRNA regulators of human monocyte growth.},
journal = {The Journal of biological chemistry},
volume = {301},
number = {6},
pages = {110204},
pmid = {40345583},
issn = {1083-351X},
mesh = {Humans ; *RNA, Long Noncoding/genetics/metabolism ; *Monocytes/cytology/metabolism ; *Cell Proliferation ; *CRISPR-Cas Systems ; THP-1 Cells ; Interferon Regulatory Factors/genetics/metabolism ; Apoptosis ; },
abstract = {Long noncoding RNAs are emerging as critical regulators of biological processes. While there are over 36,000 lncRNAs annotated in the human genome, we do not know the function of the majority. Here, we performed a high-throughput CRISPRi screen to identify those lncRNAs that are important for viability in human monocytes using the cell line THP1. We identified a total of 38 hits from the screen and validated and characterized two of the top intergenic hits. The first is a lncRNA neighboring the macrophage viability transcription factor IRF8 (RP11-542M13.2 hereafter referred to as long noncoding RNA regulator of monocyte proliferation, LNCRMP), and the second is a lncRNA called OLMALINC (oligodendrocyte maturation-associated long intervening non-coding RNA) that was previously found to be important in oligodendrocyte maturation. Transcriptional repression of LNCRMP and OLMALINC from monocytes severely limited their proliferation capabilities. RNA-seq analysis of knockdown lines showed that LNCRMP regulated proapoptotic pathways, while knockdown of OLMALINC impacted genes associated with the cell cycle. Data support both LNCRMP and OLMALINC functioning in cis to regulate their neighboring proteins that are also essential for THP1 cell growth. This research highlights the importance of high-throughput screening as a powerful tool for quickly discovering functional long non-coding RNAs (lncRNAs) that play a vital role in regulating monocyte viability.},
}

Humans
*RNA, Long Noncoding/genetics/metabolism
*Monocytes/cytology/metabolism
*Cell Proliferation
*CRISPR-Cas Systems
THP-1 Cells
Interferon Regulatory Factors/genetics/metabolism
Apoptosis

@article {pmid40193304,
year = {2025},
author = {Florindi, C and Jang, Y and Shani, K and Moretti, P and Bertarelli, C and Lanzani, G and Parker, KK and Lodola, F and Vurro, V},
title = {A Cardiac Microphysiological System for Studying Ca2+ Propagation via Non-genetic Optical Stimulation.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {217},
pages = {},
doi = {10.3791/67823},
pmid = {40193304},
issn = {1940-087X},
mesh = {*Photic Stimulation ; *Light Signal Transduction ; *Calcium/metabolism ; Induced Pluripotent Stem Cells ; Humans ; *Myocytes, Cardiac/metabolism ; CRISPR-Cas Systems ; CRISPR-Associated Protein 9 ; Cations, Divalent/metabolism ; },
abstract = {In vitro cardiac microphysiological models are highly reliable for scientific research, drug development, and medical applications. Although widely accepted by the scientific community, these systems are still limited in longevity due to the absence of non-invasive stimulation techniques. Phototransducers provide an efficient stimulation method, offering a wireless approach with high temporal and spatial resolution while minimizing invasiveness in stimulation processes. In this manuscript, we present a fully optical method for stimulating and detecting the activity of an in vitro cardiac microphysiological model. Specifically, we fabricated engineered laminar anisotropic tissues by seeding human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) generated in a 3D bioreactor suspension culture. We employed a phototransducer, an amphiphilic azobenzene derivative, named Ziapin2, for stimulation and a Ca[2+] dye (X-Rhod 1) for monitoring the system's response. The results demonstrate that Ziapin2 can photomodulate Ca[2+] responses in the employed system without compromising tissue integrity, viability, or behavior. Furthermore, we showed that the light-based stimulation approach offers a similar resolution compared to electrical stimulation, the current gold standard. Overall, this protocol opens promising perspectives for the application of Ziapin2 and material-based photostimulation in cardiac research.},
}

*Photic Stimulation
*Light Signal Transduction
*Calcium/metabolism
Induced Pluripotent Stem Cells
Humans
*Myocytes, Cardiac/metabolism
CRISPR-Cas Systems
CRISPR-Associated Protein 9
Cations, Divalent/metabolism

@article {pmid40624545,
year = {2025},
author = {Ahmed, HMM and Zheng, L and Hunnekuhl, VS},
title = {Transferable approaches to CRISPR-Cas9 induced genome editing in non-model insects: a brief guide.},
journal = {Frontiers in zoology},
volume = {22},
number = {1},
pages = {13},
pmid = {40624545},
issn = {1742-9994},
support = {TE1380/1//Deutsche Forschungsgemeinschaft/ ; },
abstract = {Despite the large variety of insect species with divergent morphological, developmental and physiological features questions on gene function could for a long time only be addressed in few model species. The adaption of the bacterial CRISPR-Cas system for genome editing in eukaryotic cells widened the scope of the field of functional genetics: for the first time the creation of heritable genetic changes had become possible in a very broad range of organisms. Since then, targeted genome editing using the CRISPR-Cas technology has greatly increased the possibilities for genetic manipulation in non-model insects where molecular genetic tools were little established. The technology allows for site-specific mutagenesis and germline transformation. Importantly, it can be used for the generation of gene knock-outs, and for the knock-in of transgenes and generation of gene-reporter fusions. CRISPR-Cas induced genome editing can thus be applied to address questions in basic research in various insect species and other study organisms. Notably, it can also be used in applied insect biotechnology to design new pest and vector control strategies such as gene drives and precision guided Sterile Insect Technique. However, establishing CRISPR in a new model requires several practical considerations that depend on the scientific questions and on the characteristics of the respective study organism. Therefore, this review is intended to give a literature overview on different CRISPR-Cas9 based methods that have already been established in diverse insects. After discussing some required pre-conditions of the study organism, we provide a guide through experimental considerations when planning to conduct CRISPR-Cas9 genome editing, such as the design and delivery of guide RNAs, and of Cas9 endonuclease. We discuss the use of different repair mechanisms including homology directed repair (HDR) for a defined insertion of genetic elements. Furthermore, we describe different molecular methods for genetic screening and the use of visible markers. We focus our review on experimental work in insects, but due to the ubiquitous functionality of the CRISPR-Cas system many considerations are transferable to other non-model organisms.},
}

@article {pmid40623912,
year = {2025},
author = {Wang, YW and Tang, YM},
title = {[Advances in the application strategies of CRISPR/Cas9 technology in chimeric antigen receptor T cell therapy for hematological malignancies].},
journal = {Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi},
volume = {46},
number = {5},
pages = {481-488},
doi = {10.3760/cma.j.cn121090-20240911-00343},
pmid = {40623912},
issn = {0253-2727},
mesh = {Humans ; *CRISPR-Cas Systems ; *Hematologic Neoplasms/therapy ; *Receptors, Chimeric Antigen ; *Immunotherapy, Adoptive/methods ; T-Lymphocytes/immunology ; Gene Editing ; },
abstract = {Chimeric antigen receptor (CAR) T-cell therapy has achieved breakthroughs in treating relapsed/refractory B-cell malignancies. However, it still faces challenges, including complex manufacturing processes, limited indications, T-cell exhaustion, and insufficient durability of therapeutic efficacy. CRISPR/Cas9, a highly efficient and relatively simple gene-editing technology, offers new avenues for overcoming these limitations. This review briefly outlines the working mechanism of CRISPR/Cas9 and focuses on its recent applications and clinical practices in developing universal CAR T-cells, enhancing T-cell function, and extending CAR T-cell therapy to T-cell and myeloid leukemias. Furthermore, this review highlights optimization strategies developed over the past two years to enhance the editing precision, delivery efficiency, and safety of the CRISPR/Cas9 system, aiming to provide insights for the optimal design and clinical application of CAR T-cell therapy.},
}

Humans
*CRISPR-Cas Systems
*Hematologic Neoplasms/therapy
*Receptors, Chimeric Antigen
*Immunotherapy, Adoptive/methods
T-Lymphocytes/immunology
Gene Editing

@article {pmid40623181,
year = {2025},
author = {Aumann, RA and Gouvi, G and Gregoriou, ME and Rehling, T and Sollazzo, G and Bourtzis, K and Schetelig, MF},
title = {Decoding and engineering temperature-sensitive lethality in Ceratitis capitata for pest control.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {28},
pages = {e2503604122},
doi = {10.1073/pnas.2503604122},
pmid = {40623181},
issn = {1091-6490},
support = {FF17000//SITplus, Hort Innovation/ ; SCHE 1833/7-1//Deutsche Forschungsgemeinschaft (DFG)/ ; 101059523//Horizon Europe Research and Innovation Action/ ; },
mesh = {Animals ; *Ceratitis capitata/genetics ; *Pest Control, Biological/methods ; Male ; Female ; Temperature ; Mutation ; Phenotype ; },
abstract = {The Sterile Insect Technique (SIT) is a species-specific and environmentally friendly method for effectively controlling pest insect populations based on releasing reared, sterile insects into infested areas. Sex sorting in rearing facilities, enabling male-only releases, is necessary to ensure SIT programs are efficient, cost-effective and, in case of mosquito control, also safe. This can be greatly facilitated by genetic sexing strains (GSS), exhibiting sex-specific phenotypic markers. However, the development of GSS remains challenging. The construction of a temperature-sensitive lethal (tsl)-based GSS in the Mediterranean fruit fly (Ceratitis capitata) over three decades ago was considered a major breakthrough for SIT programs but was never successfully replicated in other pests. After over 30 y of research, we have pinpointed a specific mutation in the C. capitata lysine--tRNA ligase (Lysyl-tRNA synthetase, LysRS) gene responsible for the tsl phenotype. Introducing this specific mutation into a wild-type strain produced full embryonic lethality under heat stress, replicating the original mutant phenotype. The random integration of a LysRS minigene reversed this effect. The high conservation of LysRS among insects suggests that tsl-based GSS could be expanded to multiple pest species and extend applications of SIT programs for disease prevention and the protection of agriculture.},
}

Animals
*Ceratitis capitata/genetics
*Pest Control, Biological/methods
Male
Female
Temperature
Mutation
Phenotype

@article {pmid40622878,
year = {2025},
author = {Perry, JK and Schwartzberg, PL and Golec, DP},
title = {Investigating Murine CD4 T Cell Differentiation Using CRISPR-Cas9 Ribonucleoprotein Complex-mediated Gene Ablation.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {220},
pages = {},
doi = {10.3791/67380},
pmid = {40622878},
issn = {1940-087X},
mesh = {Animals ; Mice ; *Ribonucleoproteins/genetics ; *CRISPR-Cas Systems ; *CD4-Positive T-Lymphocytes/cytology/immunology ; Cell Differentiation/genetics ; *Gene Knockout Techniques/methods ; Mice, Knockout ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The widespread accessibility of clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 technology has made gene targeting in primary cells a routine method for evaluating gene function in T cells. Given the cost and limited availability of knockout (KO) mouse strains, testing preliminary hypotheses involving gene function in T cells can be prohibitive using gene-targeted animal models. However, using commercially available resources, including predesigned guide RNAs (gRNAs), researchers can conveniently generate gene-targeted naïve T cells that can be used for T cell activation and differentiation studies. Here we outline a protocol for using nucleofection-delivered CRISPR-Cas9 ribonucleoprotein complexes (RNPs) to efficiently generate gene KO murine naïve CD4 T cells that can be used to evaluate gene function in CD4 T cell differentiation, in vitro. Isolation of naïve CD4 T cells from mouse secondary lymphoid organs, followed by nucleofection with Cas9-gRNA complexes ensures gene KO is initiated before downstream T cell activation, offering a strategic advantage over retroviral-mediated gRNA delivery, which typically requires preactivation of T cells, preventing the evaluation of effects in naïve T cells. Furthermore, this nucleofection-based method bypasses potential developmental issues associated with gene KO animals. Following Cas9-gRNA delivery, we describe protocols for studying CD4 T cell differentiation into Th1, Th2, Th17, and Treg lineages using in vitro polarization. In addition, this protocol is adaptable to using gene-targeted CD4 or CD8 T cells for numerous downstream applications, including other T cell activation studies in vitro and adoptive transfer studies in vivo. The use of CRISPR-Cas9 methods has streamlined our ability to evaluate gene function in T cells and allows for the routine KO of many genes of interest, freeing researchers from limitations associated with studying gene KO animals.},
}

Animals
Mice
*Ribonucleoproteins/genetics
*CRISPR-Cas Systems
*CD4-Positive T-Lymphocytes/cytology/immunology
Cell Differentiation/genetics
*Gene Knockout Techniques/methods
Mice, Knockout
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40622492,
year = {2025},
author = {Parmar, H and Goel, A and Gelaw, TA and Reddy, MK},
title = {Enhancing drought resilience in crops: mechanistic approaches in the face of climate challenge.},
journal = {Plant molecular biology},
volume = {115},
number = {4},
pages = {82},
pmid = {40622492},
issn = {1573-5028},
support = {09/512(0244)/2019-EMR-I//Human Resource Development Centre, Council of Scientific And Industrial Research/ ; },
mesh = {*Crops, Agricultural/genetics/physiology ; *Droughts ; Climate Change ; Quantitative Trait Loci/genetics ; Stress, Physiological ; Plant Breeding ; Gene Editing ; Gene Expression Regulation, Plant ; },
abstract = {Enhancing drought resilience in crops has become a critical challenge in the face of global climate change, which is exacerbating the frequency and severity of drought events. This review explores mechanistic approaches aimed to improve crop drought tolerance, focusing on physiological, biochemical, and molecular mechanisms. We examine the key molecular pathways involved in drought stress responses, including the Mitogen-Activated Protein Kinase (MAPKs) signaling pathway, hormonal regulation, transcriptional control, and post-translational modifications such as ubiquitination-mediated protein degradation, and plant-microbe interaction. The review also delves into the mechanisms of drought stress tolerance, including drought escape, avoidance, and tolerance. It highlights significant traits contributing to drought resilience, such as stomatal regulation and root architecture. Furthermore, we discuss genomics and breeding approaches, including quantitative trait loci (QTL) mapping, marker-assisted selection (MAS), and cutting-edge CRISPR-Cas-based genome editing technologies. These advanced techniques, such as base editing, prime editing, and multiplexing, transform crop improvement strategies by facilitating precise and efficient modifications for enhanced drought resilience, with the success stories in crops such as rice, maize, wheat, and others. Integrating these mechanistic and technological approaches offers promising avenues for developing drought-resilient crops, ensuring food security under increasingly unpredictable climate conditions.},
}

*Crops, Agricultural/genetics/physiology
*Droughts
Climate Change
Quantitative Trait Loci/genetics
Stress, Physiological
Plant Breeding
Gene Editing
Gene Expression Regulation, Plant

@article {pmid40619763,
year = {2025},
author = {Fritz, B and Lapp, CJ and Gescher, J},
title = {Influence of Different Transposon Families on Genomic Stability of Shewanella oneidensis MR1.},
journal = {Microbial biotechnology},
volume = {18},
number = {7},
pages = {e70188},
doi = {10.1111/1751-7915.70188},
pmid = {40619763},
issn = {1751-7915},
support = {031B0847A//Bundesministerium für Bildung und Forschung/ ; },
mesh = {*Shewanella/genetics ; *DNA Transposable Elements/genetics ; *Genomic Instability ; Transposases/genetics/metabolism ; Genome, Bacterial ; Plasmids/genetics ; Whole Genome Sequencing ; },
abstract = {Shewanella oneidensis, recognised as an important model organism for exoelectrogenic electron transport, has been extensively studied for its potential applications in bioelectrochemical systems. To date, the activity of transposable elements in this organism has not been conclusively investigated. This study focused on transposases, specifically insertion sequences (IS), which make up approximately 4.7% of the organism's genome, and evaluated their impact on genome stability under stress conditions. Using whole genome sequencing, two IS families, ISSOD1 and ISSOD2, were identified as the most active, both showing similar transposition patterns across all tested stressors. A CRISPR/dCas9 cytosine deaminase system was used to introduce stop codons in the ISSOD2 transposase genes, resulting in a significant reduction of transposition events under stress conditions. Analysis of transposition patterns revealed a high frequency of insertions occurring on the megaplasmid, which predominantly carries non-essential genes. Experiments performed here to delete the megaplasmid resulted in the elimination of approximately 35% of its sequence, including an unexpected complete loss of the ori/repA region. Therefore, it was hypothesised that the megaplasmid either exists in a metastable state, possibly representing a cointegrated intermediate within the ISSOD9 (Tn3 member) transposition mechanism, or consists of two replicons that have been combined in previous assemblies due to long overlapping homologies resulting from the presence of ISSOD9. These findings highlight the dynamics of transposable elements in S. oneidensis and suggest strategies to improve strain stability by inactivating these elements and at least reducing megaplasmid sequences. Such approaches could improve the suitability of the organism for industrial applications.},
}

*Shewanella/genetics
*DNA Transposable Elements/genetics
*Genomic Instability
Transposases/genetics/metabolism
Genome, Bacterial
Plasmids/genetics
Whole Genome Sequencing

@article {pmid40384567,
year = {2025},
author = {Schneider, PG and Liu, S and Bullinger, L and Ostendorf, BN},
title = {BEscreen: a versatile toolkit to design base editing libraries.},
journal = {Nucleic acids research},
volume = {53},
number = {W1},
pages = {W68-W72},
doi = {10.1093/nar/gkaf406},
pmid = {40384567},
issn = {1362-4962},
support = {70114327//German Cancer Aid/ ; 10.24.1.010MN//Fritz Thyssen/ ; //Guangzhou/ ; //Charité-BIH Digital Clinician/ ; //German Research Foundation/ ; },
mesh = {*Software ; *Gene Editing/methods ; *Gene Library ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Internet ; CRISPR-Cas Systems ; },
abstract = {Base editing enables the high-throughput screening of genetic variants for phenotypic effects. Base editing screens require the design of single guide RNA (sgRNA) libraries to enable either gene- or variant-centric approaches. While computational tools supporting the design of sgRNAs exist, no solution offers versatile and scalable library design enabling all major use cases. Here, we introduce BEscreen, a comprehensive base editing guide design tool provided as a web server (bescreen.ostendorflab.org) and as a command line tool. BEscreen provides variant-, gene-, and region-centric modes to accommodate various screening approaches. The variant mode accepts genomic coordinates, amino acid changes, or rsIDs as input. The gene mode designs near-saturation libraries covering the entire coding sequence of given genes or transcripts, and the region mode designs all possible guides for given genomic regions. BEscreen enables selection of guides by biological consequence, it features comprehensive customization of base editor characteristics, and it offers optional annotation using Ensembl's Variant Effect Predictor. In sum, BEscreen is a highly versatile tool to design base editing screens for a wide range of use cases with seamless scalability from individual variants to large, near-saturation libraries.},
}

*Software
*Gene Editing/methods
*Gene Library
*RNA, Guide, CRISPR-Cas Systems/genetics
Humans
Internet
CRISPR-Cas Systems

@article {pmid40377102,
year = {2025},
author = {Chapdelaine-Trépanier, V and Shenoy, S and Masud, W and Minju-Op, A and Bérubé, MA and Schönherr, S and Forer, L and Fradet-Turcotte, A and Taliun, D and Cuella-Martin, R},
title = {CRISPR-BEasy: a free web-based service for designing sgRNA tiling libraries for CRISPR-dependent base editing screens.},
journal = {Nucleic acids research},
volume = {53},
number = {W1},
pages = {W193-W202},
doi = {10.1093/nar/gkaf382},
pmid = {40377102},
issn = {1362-4962},
support = {FRN:DV2-197674/CAPMC/CIHR/Canada ; //Canada Excellence Chair in Genomic Medicine/ ; //McGill University/ ; //Fonds de Recherche du Québec-Santé/ ; //Doctoral Training Scholarships/ ; //Canada Research Chair Tier II in Molecular Biology and Genomic Instability/ ; FRN:DV2-197674/CAPMC/CIHR/Canada ; },
mesh = {Internet ; *Gene Editing/methods ; *Software ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Library ; Humans ; },
abstract = {CRISPR-dependent base editing (BE) enables the modeling and correction of genetic mutations at single-base resolution. Base editing screens, where point mutations are queried en masse, are powerful tools to systematically draw genotype-phenotype associations and characterise the function of genes and other genomic elements. However, the lack of user-friendly web-based tools for designing base editing screens can hinder broad technology adoption. Here, we introduce CRISPR-BEasy (https://crispr-beasy.cerc-genomic-medicine.ca), a free, automated web-based server that streamlines the creation of single guide (sg)RNA tiling libraries for base editing screens. Researchers can provide their genes or genomic features of interest, their base editors of choice, and target sequences to act as positive and negative controls. The server designs and annotates sgRNA libraries by integrating custom code with publicly available tools such as crisprVerse and Ensembl's Variant Effect Predictor. CRISPR-BEasy provides downloadable results, including sgRNA on/off-target scores, predicted mutational outcomes per base editor, and intuitive interactive visualizations for data quality assessment. CRISPR-BEasy also provides a separate tool that assembles sgRNA libraries into oligonucleotides for cloning following the detailed protocol documented in the searchable web server manual. Together, CRISPR-BEasy ensures the seamless design of cloning-ready sgRNA libraries, seeking to democratise access to base editing screening technologies.},
}

Internet
*Gene Editing/methods
*Software
*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/genetics
Gene Library
Humans

@article {pmid40337925,
year = {2025},
author = {Mekonnen, AM and Seong, K and Kim, H and Park, J},
title = {Variant-aware Cas-OFFinder: web-based in silico variant-aware potential off-target site identification for genome editing applications.},
journal = {Nucleic acids research},
volume = {53},
number = {W1},
pages = {W118-W124},
doi = {10.1093/nar/gkaf389},
pmid = {40337925},
issn = {1362-4962},
support = {RS-2024-00322053//Rural Development Administration/ ; //National Research Foundation of Korea/ ; //Korean government/ ; RS-2023-00278658//MSIT/ ; //Bio & Medical Technology Development/ ; RS-2024-00439078//Korean government (MSIT)/ ; BK21 Four//Korean government (MSIT)/ ; },
mesh = {*Gene Editing/methods ; *Software ; Humans ; Internet ; *CRISPR-Cas Systems ; Genome, Human ; Genetic Variation ; Haplotypes ; Computer Simulation ; },
abstract = {Genome editing based on CRISPR systems has been widely used in the vast areas of biomedical and agricultural applications. However, identifying the potential off-target sites remains challenging, particularly in individuals with diverse genetic variations. Several in silico tools have been developed to predict potential off-target sites, but they have limitations on their performance and scalability. In this paper, we present "Variant-aware Cas-OFFinder," a novel pipeline based on Cas-OFFinder for identifying potential off-target sites by accounting for individual genetic variants. We benchmarked the pipeline's improved scalability and performance with the human genome and pepper cultivars, having unique potential off-target sites on each allele at the haplotype level. The web tool is open to all users without a login requirement and is freely available online at https://rgetoolkit.com/var-cas-offinder.},
}

*Gene Editing/methods
*Software
Humans
Internet
*CRISPR-Cas Systems
Genome, Human
Genetic Variation
Haplotypes
Computer Simulation

@article {pmid40334276,
year = {2025},
author = {Liu, H and Dong, J and Wu, R and Dai, J and Lou, X and Xia, F and Willner, I and Huang, F},
title = {Light-Triggered CRISPR/Cas12a for Genomic Editing and Tumor Regression.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {64},
number = {28},
pages = {e202502892},
doi = {10.1002/anie.202502892},
pmid = {40334276},
issn = {1521-3773},
support = {22377112//National Natural Science Foundation of China/ ; 22090050//National Natural Science Foundation of China/ ; 2021YFA1200400//National Key R&D Program of China/ ; 22161142020//Joint NSFC-ISF Research Grant Program/ ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Light ; Animals ; Hep G2 Cells ; Mice ; Hepatocyte Growth Factor/genetics ; Cell Proliferation ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {A photo-triggered CRISPR/Cas12a machinery for in vitro and in vivo gene editing is introduced. The system consists of a caged, inactive ortho-nitrobenzyl phosphate ester photo-responsive crRNA, which, upon light-induced deprotection, yields the active CRISPR/Cas12a gene editing machinery (LAC12aGE). The LAC12aGE system induces specific thymidine-rich (TTTN) protospacer-adjacent motif (PAM)-guided double-stranded breaks in genomic DNA, which upon non-homologous end-joining lead to gene repair. The LAC12aGE machinery is applied for gene editing of an exogenous dual fluorescent reporter gene in living cells, as well as the endogenous gene encoding DNA methyltransferase 1. In addition, the LAC12aGE is applied for in vitro gene editing and disruption of the hepatocyte growth factor (HGF) gene in HepG2 cells, where knockout of the HGF gene results in inhibited cell proliferation and migration, as well as enhanced apoptosis. Moreover, the in vivo knockout and disruption of the HGF gene in HepG2 tumors by the LAC12aGE machinery is demonstrated. The cyclic temporal development of the LAC12aGE system in tumors shows effective inhibition of tumor growth and enhanced apoptosis/necrosis of tumor tissues compared to control systems.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems/genetics
*Light
Animals
Hep G2 Cells
Mice
Hepatocyte Growth Factor/genetics
Cell Proliferation
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40308216,
year = {2025},
author = {Marino, GB and Evangelista, JE and Clarke, DJB and Ma'ayan, A},
title = {L2S2: chemical perturbation and CRISPR KO LINCS L1000 signature search engine.},
journal = {Nucleic acids research},
volume = {53},
number = {W1},
pages = {W338-W350},
doi = {10.1093/nar/gkaf373},
pmid = {40308216},
issn = {1362-4962},
support = {OT2OD036435/GF/NIH HHS/United States ; OT2OD030160/GF/NIH HHS/United States ; U24CA264250/GF/NIH HHS/United States ; U24CA271114/GF/NIH HHS/United States ; R01DK131525/GF/NIH HHS/United States ; RC2DK131995/GF/NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; Gene Knockout Techniques ; *Software ; Internet ; *Search Engine ; Gene Expression Profiling ; Small Molecule Libraries/pharmacology ; Cell Line ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Transcriptome ; },
abstract = {As part of the Library of Integrated Network-Based Cellular Signatures (LINCS) NIH initiative, 248 human cell lines were profiled with the L1000 assay to measure the effect of 33 621 small molecules and 7508 single-gene CRISPR knockouts. From this massive dataset, we computed 1.678 million sets of up- and down-regulated genes. These gene sets are served for search by the LINCS L1000 Signature Search (L2S2) web server application. With L2S2, users can identify small molecules and single gene CRISPR KOs that produce gene expression profiles similar or opposite to their submitted single or up/down gene sets. L2S2 also includes a consensus search feature that ranks perturbations across all cellular contexts, time points, and concentrations. To demonstrate the utility of L2S2, we crossed the L2S2 gene sets with gene sets collected for the RummaGEO resource. The analysis identified clusters of differentially expressed genes that match drug classes, tissues, and diseases, pointing to many opportunities for drug repurposing and drug discovery. Overall, the L2S2 web server application can be used to further the development of personalized therapeutics while expanding our understanding of complex human diseases. The L2S2 web server application is available at https://l2s2.maayanlab.cloud.},
}

Humans
*CRISPR-Cas Systems
Gene Knockout Techniques
*Software
Internet
*Search Engine
Gene Expression Profiling
Small Molecule Libraries/pharmacology
Cell Line
*Clustered Regularly Interspaced Short Palindromic Repeats
Transcriptome

@article {pmid39729054,
year = {2025},
author = {Ballantine, J and Tisdale, JF},
title = {Gene therapy for sickle cell disease: recent advances, clinical trials and future directions.},
journal = {Cytotherapy},
volume = {27},
number = {7},
pages = {826-834},
pmid = {39729054},
issn = {1477-2566},
support = {T32 CA060441/CA/NCI NIH HHS/United States ; ZIA HL006008/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Humans ; *Anemia, Sickle Cell/therapy/genetics ; *Genetic Therapy/methods ; Gene Editing ; Clinical Trials as Topic ; Hematopoietic Stem Cell Transplantation ; CRISPR-Cas Systems/genetics ; Genetic Vectors/genetics ; Animals ; beta-Thalassemia/therapy/genetics ; },
abstract = {Sickle cell disease (SCD) is the most common inherited blood disorder worldwide, impacting millions and imposing severe healthcare challenges, particularly in resource-limited regions. Current treatments have variable efficacy and require lifelong adherence. Allogeneic Hematopoietic Stem Cell Transplantation can be curative but comes with significant side effects and limited donor availability limits its widespread applicability. Gene therapy, by addressing the root genetic causes, offers a revolutionary alternative. This article discusses the molecular mechanisms of SCD and β-thalassemia and highlights advancements in gene therapy, such as gene addition via lentiviral vectors and gene editing with CRISPR/Cas9 technology. Clinical trials have brought about significant progress but challenges remain, including leukemogenesis, delivery efficiency and cost. Future efforts must focus on enhancing efficiency, reducing costs, developing nongenotoxic conditioning regimens and methods for in vivo application.},
}

Humans
*Anemia, Sickle Cell/therapy/genetics
*Genetic Therapy/methods
Gene Editing
Clinical Trials as Topic
Hematopoietic Stem Cell Transplantation
CRISPR-Cas Systems/genetics
Genetic Vectors/genetics
Animals
beta-Thalassemia/therapy/genetics

@article {pmid40619482,
year = {2025},
author = {Bodmer, N and Uth, K and Mehmeti, R and Demir, CS and Stroka, D and Ghaffari-Tabrizi-Wizsy, N and Lugli, A and de Wever, O and Zlobec, I and Tschan, MP},
title = {CDX2 loss in colorectal cancer cells is associated with invasive properties and tumor budding.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {24113},
pmid = {40619482},
issn = {2045-2322},
support = {ESKAS, nr. 2021.0019 / Kosovo / OP//Swiss Government Excellence Scholarship/ ; 31003A_166578/1//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung/ ; 31003A_166578/1//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung/ ; },
mesh = {*CDX2 Transcription Factor/genetics/metabolism ; *Colorectal Neoplasms/pathology/genetics/metabolism ; Humans ; Animals ; Cell Movement/genetics ; Cell Line, Tumor ; Neoplasm Invasiveness ; Mice ; Cell Proliferation ; Epithelial-Mesenchymal Transition/genetics ; Cadherins/metabolism ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; },
abstract = {In colorectal cancer (CRC), tumor buds (TB) are observed histologically as single tumor cell or small tumor cell clusters located mainly at the advancing tumor edge. TB are a marker of poor prognosis and correlate with metastatic disease in CRC patients. They often lack expression of CDX2 and overexpress markers involved in epithelial-mesenchymal transition (EMT). We evaluated the function of CDX2 in CRC proliferation and migration using CRISPR/Cas9 technology and demonstrated a possible link to tumor dissociation and tumor budding. Knocking out CDX2 in CRC cell lines significantly increased migration. Importantly, the observed phenotypes could be rescued by re-expressing CDX2 and by specific CRISPR synergistic activation mediator (SAM) of endogenous CDX2 in CDX2 low expressing CRC cell lines. Multiplex immunofluorescence (mIF) analysis of primary tumor regions compared to TB in a CDX2-positive CRC patient sample as well as patient derived xenografts (PDX) revealed significantly lower CDX2 expression and correlating E-cadherin levels in TB compared to primary tumor regions, in both models. Accordingly, increased invasiveness of CRC CDX2 knockout cells was seen in ex ovo xenografts. Taken together, our results provide further insight into the function of CDX2 in preventing CRC cell migration, tumor budding and tumor aggressiveness.},
}

*CDX2 Transcription Factor/genetics/metabolism
*Colorectal Neoplasms/pathology/genetics/metabolism
Humans
Animals
Cell Movement/genetics
Cell Line, Tumor
Neoplasm Invasiveness
Mice
Cell Proliferation
Epithelial-Mesenchymal Transition/genetics
Cadherins/metabolism
Gene Expression Regulation, Neoplastic
CRISPR-Cas Systems

@article {pmid40617436,
year = {2025},
author = {Marpaung, DSS and Chen, YY and Singuru, MMR and Chuang, MC},
title = {Structure-transformable poly (thymine) activators of CRISPR/Cas12a for highly sensitive detection of mercury (II) ions.},
journal = {International journal of biological macromolecules},
volume = {319},
number = {Pt 4},
pages = {145748},
doi = {10.1016/j.ijbiomac.2025.145748},
pmid = {40617436},
issn = {1879-0003},
abstract = {Off-target effects of the CRISPR/Cas system refer to suboptimal activity triggered by activators containing unintended modifications beyond the intended target, which can lead to unwanted genetic changes-particularly problematic in therapeutic applications. In this study, the off-target effects of Cas12a are strategically repurposed for advanced Hg[2+] detection by leveraging a structure-transformable activator. The proposed approach integrates a two-segment poly-T DNA activator with the CRISPR/Cas12a system, where the activator forms hairpin structures through thymine-Hg[2+]-thymine base pairing in response to mercury concentrations. Increasing Hg[2+] concentrations promote the formation and stability of hairpin DNA, significantly suppressing Cas12a activity and resulting in an ON-OFF fluorescence signal modulated by trans-cleavage activity. The effect of varying thymine segment lengths in the poly-T activator was investigated to increase the hybridization burden for crRNA, ultimately enhancing detection sensitivity. The Hg[2+]-dependent structural transformation of the activator was characterized using circular dichroism (CD), while urea and native PAGE analyses confirmed the catalytic behavior of both cis- and trans-cleavage activities, respectively, as a function of Hg[2+] concentration. The trans-cleavage efficiency (kcat/KM) decreased by 3.03-fold as Hg[2+] concentration increased from 0 to 40 nM. Under optimized conditions, the biosensor demonstrated a linear detection range of 0-30 nM, a detection limit as low as 0.372 nM, and high selectivity for Hg[2+] over other metal ions. Furthermore, the biosensor was successfully applied for Hg[2+] detection in field water samples. These findings establish a robust foundation for exploiting the off-target effects of the CRISPR/Cas12a system for the efficient detection of heavy metals in environmental monitoring.},
}

@article {pmid40617152,
year = {2025},
author = {Rangu, SS and Misra, CS and Shaikh, S and Singh, M and Bindal, G and Singh, S and Purageri, S and Shettigar, S and Warke, R and Cherian, S and Rath, D},
title = {RNA extraction-free CRISPR-based SARS-CoV-2 detection in viral transport medium and dry swab-a comparative analysis: Short title: Extraction-free CRISPR-based Covid detection.},
journal = {Diagnostic microbiology and infectious disease},
volume = {113},
number = {3},
pages = {116982},
doi = {10.1016/j.diagmicrobio.2025.116982},
pmid = {40617152},
issn = {1879-0070},
abstract = {CRISPR-Cas-based methods have shown high efficacy in detecting SARS-CoV-2. Amidst the Covid-19 pandemic, numerous studies have explored SARS-CoV-2 detection methods without the need for RNA extraction, aiming to reduce cost and processing time. Here, we assessed a CRISPR-based SARS-CoV-2 detection method's ability to detect the virus in viral transport medium (VTM). Swabs obtained from Covid-19 positive patients and stored in two Indian brands of VTM were examined alongside dry swab samples. The samples underwent proteinase -K treatment followed by heat incubation. The released nucleic acids were tested by RT-LAMP and CRISPR-based detection. We conclude that SARS-CoV-2 can be detected in VTM of two commercial preparations as well as dry swab samples without RNA extraction and purification. COVID detection was found to be more efficient for dry swab samples compared to VTM samples. The work flow described in this paper can be extended to other respiratory diseases.},
}

@article {pmid40057303,
year = {2025},
author = {Wang, L and Hu, Y and Qiu, Y and Lin, H and Li, X and Fu, S and Zeng, YY and Ghouse, M and Long, C and Liu, Y and Fei, JF},
title = {Establishing a semi-homology-directed recombination method for precision gene integration in axolotls.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {52},
number = {7},
pages = {942-953},
doi = {10.1016/j.jgg.2025.03.001},
pmid = {40057303},
issn = {1673-8527},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; *Ambystoma mexicanum/genetics ; Gene Editing/methods ; SOXB1 Transcription Factors/genetics ; *Homologous Recombination ; },
abstract = {The axolotl is broadly used in regenerative, developmental, and evolutionary biology research. Targeted gene knock-in is crucial for precision transgenesis, enabling disease modeling, visualization, tracking, and functional manipulation of specific cells or genes of interest (GOIs). Existing CRISPR/Cas9-mediated homology-independent method for gene knock-in often causes "scars/indels" at integration junctions. Here, we develop a CRISPR/Cas9-mediated semi-homology-directed recombination (HDR) knock-in method using a donor construct containing a single homology arm for the precise integration of GOIs. This semi-HDR approach achieves seamless single-end integration of the Cherry reporter gene and a large inducible Cre cassette into intronless genes like Sox2 and Neurod6 in axolotls, which are challenging to modify with the homology-independent method. Additionally, we integrate the inducible Cre cassette into intron-containing loci (e.g., Nkx2.2 and FoxA2) without introducing indels via semi-HDR. GOIs are properly expressed in F0 founders, with approximately 5%-10% showing precise integration confirmed by genotyping. Furthermore, using the Nkx2.2:CreER[T2] line, we fate-map spinal cord p3 neural progenitor cells, revealing that Nkx2.2[+] cells adopt different lineages in development and regeneration, preferentially generating motoneurons over oligodendrocytes during regeneration. Overall, this semi-HDR method balances efficiency and precision in the integration of GOIs, providing a valuable tool for generating knock-in axolotls and potentially extending to other species.},
}

Animals
CRISPR-Cas Systems/genetics
*Gene Knock-In Techniques/methods
*Ambystoma mexicanum/genetics
Gene Editing/methods
SOXB1 Transcription Factors/genetics
*Homologous Recombination

@article {pmid40616579,
year = {2025},
author = {Urnov, F and Kassim, S and Musunuru, K and Liu, D and Lee, A and Barrera, L and Stetkiewicz, P and Bruno, J and Hewitt, M and Lister, T and Malech, H and Gasch, L and Diver, M and Gertler, N and Grignon, F and Le, A and Lehmicke, M and Almendro-Navarro, V and Lembong, J},
title = {Advancing gene-editing platforms to improve the viability of rare-disease therapeutics: key insights from a 2024 Scientific Exchange hosted by ARM, ISCT, and Danaher.},
journal = {Cytotherapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jcyt.2025.06.010},
pmid = {40616579},
issn = {1477-2566},
abstract = {Rare-disease therapeutics face viability challenges due to small patient populations and drug-development and regulatory frameworks that were not developed to address rapidly progressive or quickly fatal conditions. Because the majority of rare diseases are genetic in nature, gene-editing modalities offer substantial promise. This Scientific Exchange, co-hosted by the Alliance for Regenerative Medicine, the International Society for Cell and Gene Therapy, and Danaher Corporation in November 2024, set out to address the challenge of realizing the full promise of gene editing for rare-disease therapies by advancing platforms that leverage stable and reusable processes or components to develop multiple therapies. Through multi-stakeholder engagement and discussions of case studies in CRISPR/Cas nuclease, base, and prime editing, 4 key opportunities emerged that deliver value by holding platform elements constant and/or streamlining development steps: (1) consistent delivery vehicle; (2) consistent manufacturing; (3) benefit-risk appropriate quality requirements; and (4) expansive clinical trial designs. Together, these opportunities could yield up to 5-fold efficiency gains and result in substantial value creation for patients, regulators, and developers, potentially decreasing the time required to dose patients with a new gene-editing therapy from years down to 6 months.},
}

@article {pmid40615444,
year = {2025},
author = {Novotná, M and Tinti, M and Faria, JRC and Horn, D},
title = {Precision-edited histone tails disrupt polycistronic gene expression controls in trypanosomes.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6194},
pmid = {40615444},
issn = {2041-1723},
support = {217105/Z/19/Z//Wellcome Trust (Wellcome)/ ; },
mesh = {*Histones/genetics/metabolism ; *Trypanosoma brucei brucei/genetics/metabolism ; Protozoan Proteins/genetics/metabolism ; RNA Polymerase II/metabolism/genetics ; Promoter Regions, Genetic/genetics ; CRISPR-Cas Systems ; *Gene Expression Regulation ; Gene Editing ; Mutation ; Acetylation ; Lysine/metabolism ; Protein Processing, Post-Translational ; Methylation ; Gene Expression Profiling ; Chromatin/metabolism ; },
abstract = {Transcription of protein coding genes in trypanosomatids is atypical and almost exclusively polycistronic. In Trypanosoma brucei, for example, approximately 150 polycistrons, and 8000 genes, are constitutively transcribed by RNA polymerase II. The RNA pol-II promoters are also unconventional and characterised by regions of chromatin enriched for histones with specific patterns of post-translational modification on their divergent N-terminal tails. To investigate the roles of histone tail-residues in gene expression control in T. brucei, we engineered strains exclusively expressing mutant histones. We used an inducible CRISPR-Cas9 system to delete >40 histone H4 genes, complementing the defect with a single ectopic H4 gene. The resulting "hist[one]H4" strains were validated using whole-genome sequencing and transcriptome analysis. We then performed saturation mutagenesis of six histone H4 N-terminal tail lysine residues, that are either acetylated or methylated, and profiled relative fitness of 384 distinct precision-edited mutants. H4[lys10] mutations were not tolerated, but we derived nineteen strains exclusively expressing distinct H4[lys4] or H4[lys14] mutants. Proteomic and transcriptomic analysis of H4[lys4] glutamine mutants revealed significantly reduced expression of genes adjacent to RNA pol-II promoters, where glutamine mimics abnormally elevated acetylation. Thus, we present direct evidence for polycistronic expression control by modified histone H4 N-terminal tail residues in trypanosomes.},
}

*Histones/genetics/metabolism
*Trypanosoma brucei brucei/genetics/metabolism
Protozoan Proteins/genetics/metabolism
RNA Polymerase II/metabolism/genetics
Promoter Regions, Genetic/genetics
CRISPR-Cas Systems
*Gene Expression Regulation
Gene Editing
Mutation
Acetylation
Lysine/metabolism
Protein Processing, Post-Translational
Methylation
Gene Expression Profiling
Chromatin/metabolism

@article {pmid40615387,
year = {2025},
author = {Lou, S and DJiake Tihagam, R and Wasko, UN and Equbal, Z and Venkatesan, S and Braczyk, K and Przanowski, P and Il Koo, B and Saltani, I and Singh, AT and Likhite, S and Powers, S and Souza, GMPR and Maxwell, RA and Yu, J and Zhu, LJ and Beenhakker, M and Abbott, SBG and Lu, Z and Green, MR and Meyer, KC and Tushir-Singh, J and Bhatnagar, S},
title = {Targeting microRNA-dependent control of X chromosome inactivation improves the Rett Syndrome phenotype.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6169},
pmid = {40615387},
issn = {2041-1723},
mesh = {*Rett Syndrome/genetics/therapy ; *MicroRNAs/genetics/metabolism ; *X Chromosome Inactivation/genetics ; Animals ; Female ; Methyl-CpG-Binding Protein 2/genetics/metabolism ; Humans ; Mice ; RNA, Long Noncoding/genetics/metabolism ; Phenotype ; Fibroblasts/metabolism ; CRISPR-Cas Systems ; Disease Models, Animal ; Male ; },
abstract = {X chromosome inactivation (XCI) is induced by Xist long non-coding RNA and protein-coding genes. However, the role of small non-coding RNA function in XCI remains unidentified. Our genome-wide, loss-of-function CRISPR/Cas9 screen in female fibroblasts identified microRNAs (miRNAs) as regulators of XCI. A striking finding is the identification of miR106a among the top candidates from the screen. Loss of miR106a is accompanied by altered Xist interactome, leading to dissociation and destabilization of Xist. XCI interference via miR106a inhibition has therapeutic implications for Rett syndrome (RTT) girls with a defective X-linked MECP2 gene. Here, we discovered that the inhibition of miR106a significantly improves several facets of RTT pathology: it increases the life span, enhances locomotor activity and exploratory behavior, and diminishes breathing variabilities. Our results suggest that miR106a targeting offers a feasible therapeutic strategy for RTT and other monogenic X-linked neurodevelopmental disorders.},
}

*Rett Syndrome/genetics/therapy
*MicroRNAs/genetics/metabolism
*X Chromosome Inactivation/genetics
Animals
Female
Methyl-CpG-Binding Protein 2/genetics/metabolism
Humans
Mice
RNA, Long Noncoding/genetics/metabolism
Phenotype
Fibroblasts/metabolism
CRISPR-Cas Systems
Disease Models, Animal
Male

@article {pmid40613815,
year = {2025},
author = {Pinto, D and Mendes, I and Cunha, MV},
title = {Genomic Survey Reveals no Detectable Bacteriophage Activity in Mycobacterium bovis Across a Large Population.},
journal = {FEMS microbiology ecology},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsec/fiaf072},
pmid = {40613815},
issn = {1574-6941},
abstract = {Phages are major drivers of bacterial evolution, yet their ecological and evolutionary interactions with Mycobacterium bovis, a key member of the Mycobacterium tuberculosis complex (MTBC), remain understudied. In this work, we investigate the elusive phage-bacterium interface in M. bovis by integrating comparative genomics of 200 isolates from infected animals with molecular analyses of M. bovis-positive environmental samples. Despite employing diverse and complementary approaches, we found no evidence of active or recent phage infections: no novel prophages beyond the conserved phiRv1, no expansion of CRISPR arrays, and no co-occurrence of M. bovis and mycobacteriophages in host tissues or environmental matrices. Intriguingly, we identified multiple independent excision events of phiRv1 across closely related lineages, suggesting recent prophage mobilization driven by unidentified ecological or genomic triggers. These findings echo previous observations in M. tuberculosis and point toward a stable, phage-scarce landscape across MTBC members. Our results raise compelling questions about the barriers to phage predation in M. bovis, the functionality of its CRISPR-Cas system, and the selective pressures underlying prophage retention and loss. By shedding light on these underexplored dynamics, our study reveals critical gaps in the ecological understanding of M. bovis and highlights opportunities for phage-based innovation in TB control.},
}

@article {pmid40613714,
year = {2025},
author = {Wu, D and Snead, S and Ganguly, C and Babu, K and Li, Y and Kathiresan, V and Shen, R and Zhang, X and Rajan, R and Qin, PZ},
title = {Structural integrity and side-chain interaction at the loop region of the bridge helix modulate Cas9 substrate discrimination.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf557},
pmid = {40613714},
issn = {1362-4962},
support = {MCB-1716744//National Science Foundation/ ; MCB-1818107//National Science Foundation/ ; MCB-1716423//National Science Foundation/ ; MCB-2424888//National Science Foundation/ ; R35-GM145341/NH/NIH HHS/United States ; //Arnold and Mabel Beckman Foundation/ ; },
mesh = {*CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry/genetics ; DNA/metabolism/chemistry/genetics ; Substrate Specificity ; CRISPR-Cas Systems ; Models, Molecular ; Protein Binding ; DNA Cleavage ; Protein Conformation ; },
abstract = {CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) has been revolutionizing genome engineering, and in-depth understanding of mechanisms governing its DNA discrimination is critical for continuing technology advances. An arginine-rich bridge helix (BH) connecting the nuclease lobe and the recognition lobe, which is conserved across the Cas9 family, exists in a helix-loop-helix conformation in the apo wild-type protein but converts to a long contiguous helix in the Cas9/RNA binary complex. In this work, distances measured with spin labels were utilized to investigate BH's conformational transitions in the solution state upon single-guide RNA (sgRNA) binding, which is a critical early event preceding DNA binding and cleavage. Analyses show that sgRNA binding drives BH conformational changes in the wild-type SpyCas9 (SpyCas9WT) as well as in two BH-loop variants, SpyCas92Pro and SpyCas92Ala. Each Cas9-sgRNA binary complex, however, exhibits distinct BH features that reveal mutation-specific effects on helical integrity versus side-chain interactions. In addition, the BH conformational variations can be correlated to the observed changes in the mismatch cleavage profiles of the Cas9 variants. The work represents the first use of distances measured by site-directed spin labeling to investigate Cas9 protein conformational changes in the solution state and advances our understanding on the structure-dynamic-function relationship governing DNA target discrimination by Cas9.},
}

*CRISPR-Associated Protein 9/chemistry/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry/genetics
DNA/metabolism/chemistry/genetics
Substrate Specificity
CRISPR-Cas Systems
Models, Molecular
Protein Binding
DNA Cleavage
Protein Conformation

@article {pmid40613710,
year = {2025},
author = {Hibshman, GN and Taylor, DW},
title = {Structural basis of a dual-function type II-B CRISPR-Cas9.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf585},
pmid = {40613710},
issn = {1362-4962},
support = {R35GM138348/NH/NIH HHS/United States ; F-1938//Welch Foundation/ ; },
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; *Francisella/genetics/enzymology ; Gene Editing ; *Bacterial Proteins/chemistry/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Streptococcus pyogenes/genetics/enzymology ; DNA/genetics/chemistry/metabolism ; DNA Cleavage ; },
abstract = {Cas9 from Streptococcus pyogenes (SpCas9) revolutionized genome editing by enabling programmable DNA cleavage guided by an RNA. However, SpCas9 tolerates mismatches in the DNA-RNA duplex, which can lead to deleterious off-target editing. Here, we reveal that Cas9 from Francisella novicida (FnCas9) possesses a unique structural feature-the REC3 clamp-that underlies its intrinsic high-fidelity DNA targeting. Through kinetic and structural analyses, we show that the REC3 clamp forms critical contacts with the PAM-distal region of the R-loop, thereby imposing a novel checkpoint during enzyme activation. Notably, F. novicida encodes a noncanonical small CRISPR-associated RNA (scaRNA) that enables FnCas9 to repress an endogenous bacterial lipoprotein gene, subverting host immune detection. Structures of FnCas9 with scaRNA illustrate how partial R-loop complementarity hinders REC3 clamp docking and prevents cleavage in favor of transcriptional repression. The REC3 clamp is conserved across type II-B CRISPR-Cas9 systems, pointing to a potential path for engineering precise genome editors or developing novel antibacterial strategies. These findings reveal the molecular basis of heightened specificity and virulence enabled by FnCas9, with broad implications for biotechnology and therapeutic development.},
}

*CRISPR-Cas Systems
*CRISPR-Associated Protein 9/chemistry/genetics/metabolism
*Francisella/genetics/enzymology
Gene Editing
*Bacterial Proteins/chemistry/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Streptococcus pyogenes/genetics/enzymology
DNA/genetics/chemistry/metabolism
DNA Cleavage

@article {pmid40613705,
year = {2025},
author = {Schwartz, CI and Abell, NS and Li, A and Aradhana, and Tycko, J and Truong, A and Montgomery, SB and Hess, GT},
title = {Toward optimizing diversifying base editors for high-throughput mutational scanning studies.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf620},
pmid = {40613705},
issn = {1362-4962},
support = {R35GM150462/NH/NIH HHS/United States ; NIH-1F99DK126120-01/NH/NIH HHS/United States ; NIH-4K00DK126120-03/NH/NIH HHS/United States ; S10RR025518-01/NH/NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems ; Cytidine Deaminase/genetics/metabolism ; HEK293 Cells ; CRISPR-Associated Protein 9/genetics/metabolism ; Point Mutation ; RNA, Guide, CRISPR-Cas Systems/genetics ; Mutation ; DNA Mutational Analysis/methods ; AICDA (Activation-Induced Cytidine Deaminase) ; },
abstract = {Base editors, including diversifying base editors that create C>N mutations, are potent tools for systematically installing point mutations in mammalian genomes and studying their effect on cellular function. Numerous base editor options are available for such studies, but little information exists on how the composition of the editor (deaminase, recruitment method, and fusion architecture) affects editing. To address this knowledge gap, the effect of various design features, such as deaminase recruitment and delivery method (electroporation or lentiviral transduction), on editing was assessed across ∼200 synthetic target sites. The direct fusion of a hyperactive variant of activation-induced cytidine deaminase to the N-terminus of dCas9 (DivA-BE) produced the highest editing efficiency, ∼4-fold better than the previous CRISPR-X method. Additionally, DivA-BE mutagenized the DNA strand that anneals to the targeting sgRNA (target strand) to create complementary C>N mutations, which were absent when the deaminase was fused to the C-terminus of dCas9. Based on these studies that comprehensively analyze the editing patterns of several popular base editors, DivA-BE editors efficiently diversified their target sites, albeit with increased indel frequencies. Overall, the improved editing efficiency makes the DivA-BE editors ideal for discovering functional variants in mutational scanning assays.},
}

*Gene Editing/methods
Humans
CRISPR-Cas Systems
Cytidine Deaminase/genetics/metabolism
HEK293 Cells
CRISPR-Associated Protein 9/genetics/metabolism
Point Mutation
RNA, Guide, CRISPR-Cas Systems/genetics
Mutation
DNA Mutational Analysis/methods
AICDA (Activation-Induced Cytidine Deaminase)

@article {pmid40613203,
year = {2025},
author = {Criollo Delgado, L and Zamalutdinov, A and Potokina, E},
title = {Identification of Soybean E1-E4 Gene Orthologs in the Guar Genome Using Comprehensive Transcriptome Assembly and Annotation.},
journal = {Frontiers in bioscience (Scholar edition)},
volume = {17},
number = {2},
pages = {26548},
doi = {10.31083/FBS26548},
pmid = {40613203},
issn = {1945-0524},
support = {24-26-00073//Russian Science Foundation/ ; },
mesh = {*Glycine max/genetics ; *Cyamopsis/genetics ; *Transcriptome/genetics ; *Genome, Plant ; Molecular Sequence Annotation ; Photoperiod ; Gene Expression Regulation, Plant ; Plant Proteins/genetics ; *Genes, Plant ; Gene Expression Profiling ; Galactans ; Mannans ; Plant Gums ; },
abstract = {BACKGROUND: We publish the first available transcriptome assembly of guar (Cyamopsis tetragonoloba (L.) Taub.), a well-known source of guar gum (food additive E 412). At high latitudes, e.g., in Russia, the main challenge for guar cultivation is the long photoperiod during summer, which delays flowering and maturation of guar plants. Meanwhile, identifying of genes affecting the photoperiod sensitivity of guar would have a major impact on the development of marker-assisted breeding of this valuable food crop.

METHODS: RNA isolated from leaves of early and late flowering guar plants grown under long-day conditions were used to generate de novo transcriptome assembly. A similarity search was conducted using BLASTN 2.2.31+ with default settings to identify homologous sequences of soybean maturity genes E1-E4 in guar transcriptome and genome assembly. Gene prediction tools such as AUGUSTUS and FGENESH+ were used to predict the exon-intron structure of the candidate genes. Functional annotation of the amino acid sequence was performed using InterProScan v. 5.68-100.

RESULTS: The transcriptome assembly contained sequences of 96,447 clustered transcript isoforms in the leaves of guar plants grown under long-day conditions. The transcriptome assembly was annotated using BLAST against the Glycine max genome, and 42,615 guar transcripts (44.2%) were found to be similar to soybean genes. We used the developed transcriptome assembly to discover orthologs of the E1-E4 soybean loci in the guar genome that have the greatest impact on the flowering and maturation of this closely related, short-day legume crop. A high level of identity was detected between peptide sequences encoding by orthologous genes E1 and CtE1 (80%), E2 and CtE2 (93%), E3 and CtE3 (83%), and E4 and CtE4 (91%). The sequences and the intron-exon structure of the genes in soybean and guar were similar, suggesting that the genetic pathways underlying basic flowering mechanisms are conserved between these two legume crops.

CONCLUSIONS: The revealed intron-exon structure of the guar genes CtE1-CtE4 creates possibilities for their targeted mutagenesis, e.g., using CRISPR-Cas and developing new guar germplasm with low sensitivity to photoperiod.},
}

*Glycine max/genetics
*Cyamopsis/genetics
*Transcriptome/genetics
*Genome, Plant
Molecular Sequence Annotation
Photoperiod
Gene Expression Regulation, Plant
Plant Proteins/genetics
*Genes, Plant
Gene Expression Profiling
Galactans
Mannans
Plant Gums