@article {pmid40862940,
year = {2025},
author = {Guan, X and Wang, P and Wang, Y and Sun, S},
title = {CRISPR/Cas12a-Chemiluminescence Cascaded Bioassay for Amplification-Free and Sensitive Detection of Nucleic Acids.},
journal = {Biosensors},
volume = {15},
number = {8},
pages = {},
pmid = {40862940},
issn = {2079-6374},
support = {21874082//National Natural Science Foundation of China/ ; RXRC2022004//Fundamental Research Funds for the Central Public Welfare Research Institutes/ ; },
mesh = {Human papillomavirus 16/isolation & purification/genetics ; *CRISPR-Cas Systems ; Humans ; *Biosensing Techniques ; Luminescent Measurements ; Parvovirus B19, Human/genetics/isolation & purification ; Limit of Detection ; *DNA, Viral/analysis ; Biological Assay ; DNA, Single-Stranded ; },
abstract = {The CRISPR/Cas system has attracted increasing attention in accurate nucleic acid detection. Herein, we reported a CRISPR/Cas12a-chemiluminescence cascaded bioassay (CCCB) for the amplification-free and sensitive detection of human papillomavirus type 16 (HPV-16) and parvovirus B19 (PB-19). A magnetic bead (MB)-linking single-stranded DNA (LssDNA)-alkaline phosphatase (ALP) complex was constructed as the core component of the bioassay. During the detection process, the single-stranded target DNA was captured and enriched by LssDNA and then activated the trans-cleavage activity of Cas12a. Due to the Cas12a-mediated cleavage of LssDNA, ALP was released from the MB, subsequently catalyzing the substrate to generate a chemiluminescence (CL) signal. Given the cascade combination of CRISPR/Cas12a with the CL technique, the limits of detection for HPV-16 and PB-19 DNA were determined as 0.14 pM and 0.37 pM, respectively, and the whole detection could be completed within 60 min. The practicality and reliability of the platform were validated through target-spiked clinical specimens, and the recovery rate was 93.4-103.5%. This dual-amplification strategy-operating without target pre-amplification-featured high specificity, low contamination risk, facile preparation, and robust stability. It provides a novel approach for sensitive nucleic acid detection, with the potential for rapid extension to the diagnosis of various infectious diseases.},
}

Human papillomavirus 16/isolation & purification/genetics
*CRISPR-Cas Systems
Humans
*Biosensing Techniques
Luminescent Measurements
Parvovirus B19, Human/genetics/isolation & purification
Limit of Detection
*DNA, Viral/analysis
Biological Assay
DNA, Single-Stranded

@article {pmid40862153,
year = {2025},
author = {Pacheco-Acosta, S and Castro-Toro, G and Rojas-Villalobos, C and Valenzuela, C and Haristoy, JJ and Zapata-Araya, A and Moya-Beltrán, A and Sepúlveda-Rebolledo, P and Pérez-Rueda, E and Ulloa, R and Giaveno, A and Issotta, F and Díez, B and Beard, S and Quatrini, R},
title = {Exploring the eco-evolutionary role of plasmids and defense systems in 'Fervidacidithiobacillus caldus' extreme acidophile.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1610279},
pmid = {40862153},
issn = {1664-302X},
abstract = {Plasmids are major drivers of microbial evolution, enabling horizontal gene transfer (HGT) and facilitating adaptation through the dissemination of relevant functional genes and traits. However, little is known about plasmid diversity and function in extremophiles. 'Fervidacidithiobacillus caldus', a meso-thermo-acidophilic sulfur oxidizer, is a key player in sulfur cycling in natural and industrially engineered acidic environments. Here, we present a bioinformatic analysis of the plasmidome, and associated anti-mobile genetic element (anti-MGE) defense systems (defensome), across genomes of this species and metagenomes from diverse natural and industrial settings harboring 'F. caldus'. We identified >30 distinct plasmids, representing five consistent replication-mobilization families. Plasmids ranged in size between 2.5-65 kb, with gene content and plasmid modularity scaling with element size and copy numbers inversely correlating with size. Plasmids carried variable numbers of hypothetical proteins and transposases, with annotated cargo genes reflecting functional differentiation by habitat. Defensome profiling revealed over 50 anti-MGE systems in sequenced 'F. caldus' isolates, including diverse restriction-modification systems, CRISPR-Cas types IV-A and V-F, and widespread abortive infection and composite defense systems such as Wadjet, Gabija, and Zorya. In environmental populations, an inverse relationship was observed between defensome complexity and plasmidome abundance and diversity, underscoring a pivotal role of the host defensome in modulating persistence, compatibility, and overall plasmid diversity across 'F. caldus' populations. Yet, other plasmids appeared decoupled from both host abundance and defensome complexity, suggesting potential host shifts, environmental persistence, or differential replication under suboptimal growth conditions for the host. Altogether, these findings point to a modular, functionally diverse adaptive plasmidome shaped by environmental pressures, by the interplay with the host's defensome, and likely also by other eco-evolutionary processes at play in natural environments. While these associations are compelling, causal relationships remain to be experimentally validated. These insights broaden our understanding of mobile genetic elements in extreme environments and provide a foundation for plasmid-based vector design and synthetic biology applications in acidophiles, with direct implications to biomining and environmental remediation.},
}

@article {pmid40861495,
year = {2025},
author = {Plöhn, O and Singh, AK and Greger, C and Wolfgramm, H and Baglanova, M and Surmann, K and Völker, U and Bröker, BM and Becker, K and Seifert, U and Cammann, C},
title = {Deletion of the E3 ubiquitin ligase LRSAM1 fosters intracellular Staphylococcus aureus survival.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1597830},
pmid = {40861495},
issn = {2235-2988},
mesh = {Humans ; *Staphylococcus aureus/physiology/growth & development ; *Ubiquitin-Protein Ligases/genetics/metabolism/deficiency ; A549 Cells ; *Host-Pathogen Interactions ; CRISPR-Cas Systems ; *Staphylococcal Infections/microbiology/immunology ; Ubiquitination ; Gene Deletion ; Autophagy ; Epithelial Cells/microbiology ; *Microbial Viability ; Interleukin-6/metabolism ; Cytokines/metabolism ; Bacterial Load ; Signal Transduction ; },
abstract = {BACKGROUND: Intracellular invasion and persistence of Staphylococcus aureus can lead to chronic infection and is an effective strategy for the pathogen to evade the host immune response and antibiotic therapy. Selective ubiquitination of bacterial surfaces via E3 ubiquitin ligases is a mechanism by which host cells combat intracellular bacteria and target them for autophagosomal degradation. However, knowledge of the E3 ligases involved in intracellular recognition of S. aureus is still very limited.

METHODS: We studied A549 lung epithelial cells during S. aureus infection, focusing on the role of the E3 ligase leucine rich repeat and sterile alpha motif containing 1 (LRSAM1). We used the CRISPR-Cas9 system to generate LRSAM1-deficient A549 cells and monitored intracellular bacterial survival, activation of host cellular signalling pathways related to cytokine production, and host cell death during S. aureus infection.

RESULTS: In LRSAM1-deficient host cells we observed a significant increase in intracellular bacterial load, which was accompanied by an increased host cell death and elevated secretion of the pro-inflammatory cytokine IL-6. Despite induced selective autophagy, LRSAM1 knockout host cells were incapable of lowering and eliminating the pathogen, which seems to be caused by the reduced ubiquitination of the bacterial surface.

CONCLUSION: The results indicate a significant role of LRSAM1 in the clearance of intracellular S. aureus. This contributes to a deeper understanding of the host cellular responses to S. aureus infection and will facilitate the development of novel therapeutic strategies to combat intracellularly persistent S. aureus.},
}

Humans
*Staphylococcus aureus/physiology/growth & development
*Ubiquitin-Protein Ligases/genetics/metabolism/deficiency
A549 Cells
*Host-Pathogen Interactions
CRISPR-Cas Systems
*Staphylococcal Infections/microbiology/immunology
Ubiquitination
Gene Deletion
Autophagy
Epithelial Cells/microbiology
*Microbial Viability
Interleukin-6/metabolism
Cytokines/metabolism
Bacterial Load
Signal Transduction

@article {pmid40859134,
year = {2025},
author = {Li, L and Wang, Y and Yang, F and Li, W and Wang, Q and Xu, X and Wu, Y and Ji, X},
title = {Establishment of a CRISPR/Cas9 gene editing system based on growth points transformation method in Fraxinus mandshurica.},
journal = {BMC plant biology},
volume = {25},
number = {1},
pages = {1127},
pmid = {40859134},
issn = {1471-2229},
support = {2021YFD2200303//the National Key R&D Program of China/ ; 2021YFD2200303//the National Key R&D Program of China/ ; 2021YFD2200303//the National Key R&D Program of China/ ; 2021YFD2200303//the National Key R&D Program of China/ ; 2021YFD2200303//the National Key R&D Program of China/ ; 2021YFD2200303//the National Key R&D Program of China/ ; 2021YFD2200303//the National Key R&D Program of China/ ; 2021YFD2200303//the National Key R&D Program of China/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Fraxinus/genetics/growth & development/physiology ; Plants, Genetically Modified/genetics ; Droughts ; Transformation, Genetic ; Agrobacterium tumefaciens/genetics ; },
abstract = {The lack of an effective gene editing technology system for Fraxinus mandshurica makes it challenging to improve its traits through genetic engineering methods. In this study, an effective CRISPR/Cas9 gene editing system targeting plant growth points was established through the optimization of Agrobacterium tumefaciens concentration and infection duration. Furthermore, a tissue culture system for clustered buds was developed by supplementing the media with hormones at different concentrations. FmbHLH1-edited chimeric plants were successfully generated using the developed CRISPR/Cas9 gene editing system. Homozygous plants were induced and screened using the developed clustered bud system. Among 100 randomly transformed growing points, 18% of the induced clustered buds were gene-edited, which confirmed that the established CRISPR/Cas9 gene editing system was effective. Phenotypic analysis and evaluation of drought tolerance-related physiological indicators in FmbHLH1 knockout and wild-type lines revealed that FmbHLH1 positively regulated the drought tolerance of F. mandshurica by adjusting its ability to scavenge reactive oxygen species and to regulate osmotic potential. In summary, we developed an effective CRISPR/Cas9 gene editing system for F. mandshurica, providing an effective method for the molecular breeding of F. mandshurica.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
*Fraxinus/genetics/growth & development/physiology
Plants, Genetically Modified/genetics
Droughts
Transformation, Genetic
Agrobacterium tumefaciens/genetics

@article {pmid40858609,
year = {2025},
author = {Xu, D and Besselink, S and Ramadoss, GN and Dierks, PH and Lubin, JP and Pattali, RK and Brim, JI and Christenson, AE and Colias, PJ and Ornelas, IJ and Nguyen, CD and Chasins, SE and Conklin, BR and Nuñez, JK},
title = {Programmable epigenome editing by transient delivery of CRISPR epigenome editor ribonucleoproteins.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7948},
pmid = {40858609},
issn = {2041-1723},
support = {R35GM155044-01//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; UCB 20240097//Gladstone Institutes (J. David Gladstone Institutes)/ ; },
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Epigenome/genetics ; *Ribonucleoproteins/genetics/metabolism ; *Epigenesis, Genetic ; HEK293 Cells ; DNA Methyltransferase 3A ; tau Proteins/genetics/metabolism ; DNA (Cytosine-5-)-Methyltransferases/genetics/metabolism ; Neurons/metabolism ; Epigenome Editing ; Mixed Function Oxygenases ; Proto-Oncogene Proteins ; },
abstract = {Programmable epigenome editors modify gene expression in mammalian cells by altering the local chromatin environment at target loci without inducing DNA breaks. However, the large size of CRISPR-based epigenome editors poses a challenge to their broad use in biomedical research and as future therapies. Here, we present Robust ENveloped Delivery of Epigenome-editor Ribonucleoproteins (RENDER) for transiently delivering programmable epigenetic repressors (CRISPRi, DNMT3A-3L-dCas9, CRISPRoff) and activator (TET1-dCas9) as ribonucleoprotein complexes into human cells to modulate gene expression. After rational engineering, we show that RENDER induces durable epigenetic silencing of endogenous genes across various human cell types, including primary T cells. Additionally, we apply RENDER to epigenetically repress endogenous genes in human stem cell-derived neurons, including the reduction of the neurodegenerative disease associated V337M-mutated Tau protein. Together, our RENDER platform advances the delivery of CRISPR-based epigenome editors into human cells, broadening the use of epigenome editing in fundamental research and therapeutic applications.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Epigenome/genetics
*Ribonucleoproteins/genetics/metabolism
*Epigenesis, Genetic
HEK293 Cells
DNA Methyltransferase 3A
tau Proteins/genetics/metabolism
DNA (Cytosine-5-)-Methyltransferases/genetics/metabolism
Neurons/metabolism
Epigenome Editing
Mixed Function Oxygenases
Proto-Oncogene Proteins

@article {pmid40857323,
year = {2025},
author = {Luo, J and Chia, N and Qin, Y and Tan, P and Zhang, L and Yang, S and Yuan, Z and Hong, L and Lee, SY and Tong, Y},
title = {STAGE: A compact and versatile TnpB-based genome editing toolkit for Streptomyces.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {35},
pages = {e2509146122},
doi = {10.1073/pnas.2509146122},
pmid = {40857323},
issn = {1091-6490},
support = {2021YFA0909500//MOST | National Key Research and Development Program of China (NKPs)/ ; 21TQ1400204//Shanghai Pilot Program for Basic Research/ ; 24HC2810200//Science and Technology Commission of Shanghai Municipality (STCSM)/ ; MMLKF22-03//State Key Laboratory of Microbial Metabolism (SKLMM)/ ; 2022M3J5A1056117//Ministry of Science and ICT, South Korea (MSIT)/ ; },
mesh = {*Gene Editing/methods ; *Streptomyces/genetics ; CRISPR-Cas Systems/genetics ; *Genome, Bacterial ; *Bacterial Proteins/genetics/metabolism ; Multigene Family ; },
abstract = {Streptomyces are naturally endowed with the capacity to produce a wide array of natural products with biomedical and biotechnological value. They have garnered great interest in synthetic biology applications given the abundance of uncharacterized biosynthetic gene clusters (BGCs). However, progress has been hindered by the limited availability of genetic tools for manipulating these bacteria. Several representative CRISPR-Cas systems have been established in Streptomyces to streamline experimental workflows and improve editing efficiency. Nevertheless, their broader applicability has been constrained by issues such as nuclease activity-related cytotoxicity and the large size of effector proteins. To address these challenges, we present Streptomyces-compatible TnpB-assisted genome editing (STAGE), a genetic toolkit based on ISDra2 TnpB, which is approximately one-third the size of Cas9 and enables precise, site-specific gene editing. We demonstrated that STAGE introduces genetic mutations with high efficiency and minimal off-target effects in two industrially important Streptomyces strains. Building on this platform, we developed STAGE-cBEST and STAGE-McBEST, enabling single and multiplexed C·G-to-T·A base editing, respectively, with editing efficiencies exceeding 75%. To further enhance performance, we engineered the ISDra2 TnpB system using an AI-assisted protein engineering framework, resulting in two variants that achieve nearly 100% genome editing efficiency. Additionally, through sequence homology analysis, we identified a TnpB ortholog from the same biological origin of ISDra2 TnpB, which also functions effectively as a gene editing tool. Our study establishes STAGE as a highly precise, programmable, and versatile genome editing platform for Streptomyces, paving the way for advanced genetic manipulation and synthetic biology applications in these industrially important bacteria.},
}

*Gene Editing/methods
*Streptomyces/genetics
CRISPR-Cas Systems/genetics
*Genome, Bacterial
*Bacterial Proteins/genetics/metabolism
Multigene Family

@article {pmid40854749,
year = {2025},
author = {Ju, CR and Xu, X and Xu, X and Xue, WJ},
title = {[Current status and future prospects of antibacterial treatment for Carbapenem-resistant gram-negative bacterial infections in solid organ transplant recipients].},
journal = {Zhonghua yi xue za zhi},
volume = {105},
number = {32},
pages = {2701-2708},
doi = {10.3760/cma.j.cn112137-20250218-00381},
pmid = {40854749},
issn = {0376-2491},
support = {2023C-TS10//Guangzhou Clinical Characteristic Technology Project/ ; GMUCR2024-01007//Major Clinical Research Project under the Research Capacity Enhancement Program of Guangzhou Medical University/ ; SKLRD-L-202504//Clinical Epidemiology Research Project of the State Key Laboratory of Respiratory Diseases/ ; ZJWYH-2023-YIZHI-003//China Medical Education Association/ ; 82470103//National Natural Science Foundation of China/ ; 320.6750.2025-01-1//Wu Jieping Medical Foundation Special Research Grant/ ; },
mesh = {Humans ; *Anti-Bacterial Agents/therapeutic use/pharmacology ; Carbapenems/pharmacology ; *Gram-Negative Bacterial Infections/drug therapy ; Drug Resistance, Multiple, Bacterial ; *Organ Transplantation ; Gram-Negative Bacteria/drug effects ; Transplant Recipients ; },
abstract = {In recent years, infections caused by carbapenem-resistant Gram-negative bacteria (CR-GNB) and other multidrug-resistant (MDR) pathogens have posed significant challenges in solid organ transplantation (SOT). SOT recipients who develop CR-GNB infections face risks such as graft loss, respiratory failure, leading to a mortality rate exceeding 40%. This review examines the current resistance landscape and the characteristics of MDR infections in SOT recipients, discussing the epidemiological features of CR-GNB infections in SOT patients, the resistance profiles and mechanisms of common CR-GNB (e.g., Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa), and evidence-based principles and strategies for antimicrobial therapy. The article provides an in-depth analysis of the efficacy, pharmacokinetic/pharmacodynamic (PK/PD) properties, and adverse effects of both commonly used antibiotics (such as tigecycline, polymyxins, and ceftazidime/avibactam) and the novel agents (including eravacycline and sulbactam/durlobactam), emphasizing the importance of combination therapy and personalized treatment approaches. Additionally, it explores the clinical potential of emerging rapid diagnostic technologies (e.g., CRISPR/Cas systems, mass spectrometry) and innovative treatments such as phage therapy and immunomodulation. Looking ahead, the review envisions a future where, guided by rapid and precise diagnostics, multidisciplinary management can optimize antimicrobial regimens for MDR infections in transplant recipients. The goal is to achieve individualized, effective treatment strategies, thereby improving outcomes in severe infections and advancing the management of CR-GNB infections in SOT patients in China.},
}

Humans
*Anti-Bacterial Agents/therapeutic use/pharmacology
Carbapenems/pharmacology
*Gram-Negative Bacterial Infections/drug therapy
Drug Resistance, Multiple, Bacterial
*Organ Transplantation
Gram-Negative Bacteria/drug effects
Transplant Recipients

@article {pmid40811645,
year = {2025},
author = {Shi, JY and Li, ZW and Yao, ZL and Han, GM and Li, MY and Cai, QL and Kong, DM},
title = {5'-End Engineering of CRISPR/Cas12a Activators: A Versatile Platform for Multiple Biomarker Analysis and Clinical Cancer Tissue Identification.},
journal = {Analytical chemistry},
volume = {97},
number = {33},
pages = {18188-18198},
doi = {10.1021/acs.analchem.5c03168},
pmid = {40811645},
issn = {1520-6882},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Biomarkers, Tumor/analysis/genetics ; MicroRNAs/analysis ; *Carcinoma, Renal Cell/diagnosis ; *Kidney Neoplasms/diagnosis ; *Endodeoxyribonucleases/metabolism/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {The CRISPR/Cas12a system has emerged as a powerful tool for biosensing due to its unique trans-cleavage activity. However, the fundamental mechanisms governing its activation remain inadequately understood, limiting the design flexibility and application scope of CRISPR/Cas12a-based biosensors. In this study, we investigated the activation behavior of CRISPR/Cas12a, focusing on the 5'-end engineering of the activator strand. We discovered that the activation of CRISPR/Cas12a can be significantly suppressed by incorporating a rigid intramolecular hairpin or intermolecular duplex at the 5'-end of the activator strand designed using our discovered RESET effect. Leveraging this finding, we developed a series of CRISPR/Cas12a-based biosensors capable of sensitive and selective detection, as well as live-cell imaging, for various biomarkers including microRNAs, biological small molecules, enzymes, and reactive oxygen species. Notably, the biosensor designed for miR-210, a biomarker for renal cell carcinoma (RCC), demonstrated exceptional performance in distinguishing between clinical RCC tissues and adjacent healthy tissues, highlighting its potential for cancer diagnosis, prognosis, and intraoperative decision-making. This study not only deepens the understanding of CRISPR/Cas12a activation mechanisms but also provides a versatile platform for developing advanced biosensors in molecular diagnostics and therapeutic monitoring.},
}

Humans
*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
*Biomarkers, Tumor/analysis/genetics
MicroRNAs/analysis
*Carcinoma, Renal Cell/diagnosis
*Kidney Neoplasms/diagnosis
*Endodeoxyribonucleases/metabolism/genetics
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid40808297,
year = {2025},
author = {Eggenschwiler, R and Hoffmann, T and Dmytrenko, O and Opitz, M and Ackel-Zakour, M and Wang, P and McCallan, SA and Fráguas-Eggenschwiler, M and Niemann, H and Patronov, A and Beisel, CL and Cantz, T},
title = {PAM-interacting domain turn-helix 51 motifs can improve Cas9-SpRY activity.},
journal = {Nucleic acids research},
volume = {53},
number = {15},
pages = {},
pmid = {40808297},
issn = {1362-4962},
support = {01GM2204C//German Federal Ministry for Education and Research/ ; 01GP2205C//German Federal Ministry for Education and Research/ ; ZN3440//Ministry of Science and Culture of Lower Saxony/ ; 865973//European Research Council Consolidator/ ; //Institute LOM/ ; },
mesh = {*CRISPR-Associated Protein 9/genetics/chemistry/metabolism ; Streptococcus pyogenes/genetics/enzymology ; *CRISPR-Cas Systems ; Gene Editing/methods ; Lysine/chemistry/genetics ; Humans ; Molecular Dynamics Simulation ; Nucleotide Motifs ; Helix-Turn-Helix Motifs ; DNA/metabolism/chemistry/genetics ; DNA Cleavage ; HEK293 Cells ; *Bacterial Proteins/genetics/chemistry/metabolism ; Protein Engineering ; },
abstract = {Cas9-SpRY is an engineered variant of the Streptococcus pyogenes Cas9 with relaxed PAM recognition, which can technically be utilized at any target in the genome but some targets are addressed with low efficiency. Here, we show that a previously unexplored motif at the turn and beginning of α-helix 51 (TH51) can be engineered to improve both nuclease and prime-editing activity of Cas9-SpRY. Interaction of the lysine-rich PID loop 2 (PL2) with the target DNA downstream of the PAM (post-PAM) mediates initiation of R-loop formation and subsequent cleavage yet it was unclear if other regions of the PID engage with post-PAM as well. To this end, the NAAN-PAM-targeting iSpyMac hybrid nuclease, which lacks all lysine residues in PL2, was compared with Cas9-SpRY at identical targets using molecular dynamics simulation and in cell culture models, uncovering four crucial post-PAM-interacting lysines in TH51 and TH53 of iSpyMac. Ectopic insertion of a lysine-rich PL2 into iSpyMac boosted its nuclease and prime-editing activities and, in turn, Cas9-SpRY benefited from certain lysine-rich TH51 motifs. Specifically, TH51 from an uncultured Abiotrophia Cas9 species boosted overall Cas9-SpRY activity. Together, this study demonstrates that engineering of post-PAM interacting motifs opens new avenues for the design of advanced CRISPR enzymes.},
}

*CRISPR-Associated Protein 9/genetics/chemistry/metabolism
Streptococcus pyogenes/genetics/enzymology
*CRISPR-Cas Systems
Gene Editing/methods
Lysine/chemistry/genetics
Humans
Molecular Dynamics Simulation
Nucleotide Motifs
Helix-Turn-Helix Motifs
DNA/metabolism/chemistry/genetics
DNA Cleavage
HEK293 Cells
*Bacterial Proteins/genetics/chemistry/metabolism
Protein Engineering

@article {pmid40806729,
year = {2025},
author = {Katayama, S and Yamamoto, T},
title = {Efficient Genome Editing Using the T2A-Coupled Co-Expression of Two ZFN Monomers.},
journal = {International journal of molecular sciences},
volume = {26},
number = {15},
pages = {},
pmid = {40806729},
issn = {1422-0067},
support = {COI-NEXT Bio-Digital Transformation//Japan Science and Technology Agency/ ; },
mesh = {*Gene Editing/methods ; Humans ; *Zinc Finger Nucleases/genetics/metabolism ; CRISPR-Cas Systems ; HEK293 Cells ; Genetic Vectors/genetics ; Plasmids/genetics ; },
abstract = {Genome editing is commonly used in biomedical research. Among the genome editing tools, zinc finger nucleases (ZFNs) are smaller in size than transcription activator-like effector nucleases (TALENs) and CRISPR-Cas9. Therefore, ZFNs are easily packed into a viral vector with limited cargo space. However, ZFNs also consist of left and right monomers, which both need to be expressed in the target cells. When each monomer is expressed separately, two expression cassettes are required, thus increasing the size of the DNA. This is a disadvantage for a viral vector with limited cargo space. We herein showed that T2A-coupled ZF-ND1 monomers were co-expressed from a single expression cassette and that the corresponding ZF-ND1s efficiently cleaved the target DNA sequences. Furthermore, the total amount of transfected plasmid DNA was reduced by half, and genome editing efficiency was equivalent to that of two separate ZF-ND1 monomers. This study provides a promising framework for the development of ZFN applications.},
}

*Gene Editing/methods
Humans
*Zinc Finger Nucleases/genetics/metabolism
CRISPR-Cas Systems
HEK293 Cells
Genetic Vectors/genetics
Plasmids/genetics

@article {pmid40802893,
year = {2025},
author = {Hu, M and Wang, Y and Qi, W and Zhang, Y and Sun, J and Cheng, M and Zhou, X},
title = {Photocontrolled Programmable Enzymatic Cascade for Robust CRISPR Diagnostics.},
journal = {Journal of the American Chemical Society},
volume = {147},
number = {34},
pages = {31004-31015},
doi = {10.1021/jacs.5c08704},
pmid = {40802893},
issn = {1520-5126},
mesh = {*CRISPR-Cas Systems ; Mycobacterium tuberculosis/genetics ; Humans ; Photochemical Processes ; DNA, Single-Stranded/genetics/chemistry ; *CRISPR-Associated Proteins/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Nucleic Acid Amplification Techniques ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {CRISPR-Cas12a-based diagnostic technologies have revolutionized nucleic acid detection, but their broader application remains constrained by the protospacer adjacent motif (PAM) requirement and limited multiplexing capabilities due to reliance on trans-cleavage. Here, we present a photocontrolled programmable enzymatic cascade strategy that enables temporal regulation of three sequential reactions─nucleic acid amplification, photoactivated lambda exonuclease (λ-exon)-mediated single-stranded DNA (ssDNA) generation, and PAM-independent Cas12a detection─all within a one-pot system, effectively overcoming the PAM constraint. We further exploit the orthogonal trans-cleavage activity of Cas12a and Cas13a to enable simultaneous dual-gene detection within the one-pot system, thereby circumventing multiplexing limitations. Applied to clinical Mycobacterium tuberculosis (MTB) samples, the method allows detection of both the IS6110 gene of MTB and the human ACTB (β-actin) internal control gene. This photocontrolled one-pot CRISPR diagnostic technology enhances flexibility in target site selection and overcomes the limitations of conventional CRISPR diagnostics, which cannot simultaneously detect both target genes and internal controls. This approach holds promise for advancing the clinical application of CRISPR-based diagnostics.},
}

*CRISPR-Cas Systems
Mycobacterium tuberculosis/genetics
Humans
Photochemical Processes
DNA, Single-Stranded/genetics/chemistry
*CRISPR-Associated Proteins/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats
Nucleic Acid Amplification Techniques
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid40787745,
year = {2025},
author = {Wang, M and Wang, Y and Wen, J and Wang, B and Lu, G and Zhang, XB and Chen, M and Ke, G},
title = {Engineering molecular switches of CRISPR/Cas12a for biosensing applications.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {70},
pages = {13052-13065},
doi = {10.1039/d5cc03652f},
pmid = {40787745},
issn = {1364-548X},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; *Bacterial Proteins/metabolism/genetics/chemistry ; *Endodeoxyribonucleases/metabolism/genetics/chemistry ; Humans ; },
abstract = {Benefiting from high specificity, excellent programmability, and efficient signal amplification capability, the CRISPR/Cas12a system has emerged as a crucial tool in the field of molecular diagnostics. To fully realize the application potential of Cas12a, many molecular switches of CRISPR/Cas12a have been recently developed to convert the activity of Cas12a from an inactive state to an active state in response to external stimuli, which significantly improves the biosensing performance in terms of the sensitivity, selectivity, and accuracy. This review summarizes recent progress in the development and application of Cas12a-based molecular switches, with a focus on their conditional activation mechanisms and biosensing applications. Additionally, challenges and future perspectives for the development of intelligent biosensors are discussed.},
}

*Biosensing Techniques/methods
*CRISPR-Cas Systems
*CRISPR-Associated Proteins/genetics/metabolism/chemistry
*Bacterial Proteins/metabolism/genetics/chemistry
*Endodeoxyribonucleases/metabolism/genetics/chemistry
Humans

@article {pmid40543220,
year = {2025},
author = {Samy, A and Alber, A and Fife, M and Hammond, JA},
title = {IFITM knockout DF1 cells produce higher influenza and newcastle disease viral yields: a proof of concept for avian origin cell-based vaccine production.},
journal = {Vaccine},
volume = {61},
number = {},
pages = {127360},
doi = {10.1016/j.vaccine.2025.127360},
pmid = {40543220},
issn = {1873-2518},
mesh = {Animals ; Chickens ; *Newcastle disease virus/immunology/growth & development ; *Newcastle Disease/prevention & control/immunology ; Cell Line ; *Influenza in Birds/prevention & control/immunology ; *Influenza Vaccines/immunology ; *Viral Vaccines/immunology ; *Influenza A virus/immunology/growth & development ; Chick Embryo ; Fibroblasts/virology ; *RNA-Binding Proteins/genetics ; Gene Knockout Techniques ; *Membrane Proteins/genetics ; *Antigens, Differentiation/genetics ; CRISPR-Cas Systems ; Virus Replication ; },
abstract = {Vaccines remain essential for the control of infectious diseases during poultry production, especially in high density systems. Many of poultry vaccines are currently grown in embryonated chicken eggs (ECE) or egg derived primary cells. These systems can be relatively costly and present a potential risk of supply during pandemics when demand for ECE can be high. Furthermore, the scale up of ECE vaccine production can be challenging at short notice, especially when the safe disposal of biohazardous waste is required. Avian-origin immortalised cell lines have the potential to be an ideal surrogate and remove the need to use ECE due to species match. However, the viral yield is often much lower than that of ECE which is at least partly due to the activation of interferon responses. One such response is driven by the interferon-inducible transmembrane proteins (IFITM) that are potent broad range viral restriction factors inhibiting viral cell entry. Using CRISPR/Cas9 we deleted the entire IFITM locus from the immortalised chicken fibroblast cell line DF1 and examined the impact on viral growth. Multiple DF1-IFITM-KO clones confirmed that removing IFITM restriction not only augmented infectivity and viral surface protein expression but significantly increased the viral yield up to 1.5 log10 PFU/ml and 0.8 log10 PFU/ml for influenza A virus (IAV), and Newcastle disease virus (NDV) LaSota strain, respectively. Expression of IFITM3 but not IFITM1 in DF1-IFITM-KO cells restored AIV restriction, while expression of both IFITM1 and IFITM3 restricted NDV infectivity. Together, these data confirm that IFITM proteins significantly reduce viral infectivity and growth in chicken cells and that removing this barrier has the potential to improve cell- based vaccine production.},
}

Animals
Chickens
*Newcastle disease virus/immunology/growth & development
*Newcastle Disease/prevention & control/immunology
Cell Line
*Influenza in Birds/prevention & control/immunology
*Influenza Vaccines/immunology
*Viral Vaccines/immunology
*Influenza A virus/immunology/growth & development
Chick Embryo
Fibroblasts/virology
*RNA-Binding Proteins/genetics
Gene Knockout Techniques
*Membrane Proteins/genetics
*Antigens, Differentiation/genetics
CRISPR-Cas Systems
Virus Replication

@article {pmid40854655,
year = {2025},
author = {Pearl, S and Kumar, H and Vijayakumar, S and Basu, S and Ramaiah, S and Anbarasu, A},
title = {The evolution of superbugs in space: a genomic perspective on pathogens in the International Space Station environment.},
journal = {Journal, genetic engineering & biotechnology},
volume = {23},
number = {3},
pages = {100536},
doi = {10.1016/j.jgeb.2025.100536},
pmid = {40854655},
issn = {2090-5920},
abstract = {Microgravity, pressure, and temperature variations in the International Space Station (ISS) create conditions leading to the emergence of superbugs. Due to technical issues in spacecraft, astronauts are forced to stay in ISS for extended periods; prolonged stay and exposure in stressful ISS environment weakens their immune systems, increasing susceptibility to infections. The presence of hypervirulent and antibiotic-resistant pathogens in space station is a worrisome feature as these might cause serious life-threatening infections in astronauts staying in high stress environments with weakened immune systems. In the present study, we compared antimicrobial resistance genes (ARGs) and virulence factors (VFs) in bacterial genomes from ISS with Earth counterparts. ISS genomes exhibited elevated counts of defense-related genes, particularly in E. ludwigii and E. cancerogenus. Among genes uniquely found in ISS genomes, CRISPR-Cas system components were notably prevalent. Though Earth genomes harbored higher number of ARGs overall, several species from ISS possessed modestly higher ARG counts. VFs profiling showed a slightly lower count in ISS genomes, but P. conspicua, E. ludwigii, and K. pneumoniae from ISS carried exclusive VFs linked to metal ion uptake and secretion systems, suggesting environment-driven functional adaptations. The adaptation of pathogenic bacteria in ISS is alarming and therefore periodic monitoring of bacterial genomic surveillance is important. Our findings shed light on genomic profiles in bacterial strains from both ISS and Earth, enhancing our understanding of the bacterial pathogens' potential impact on drug resistance and pathogenicity in space-missions and the possible threat of spread from ISS.},
}

@article {pmid40828286,
year = {2025},
author = {Gaul, CR and Vijay, T and Johnson, R and Macias, VM},
title = {Manipulation of a New Non-model Insect Genome Using Targeted CRISPR-Era Approaches.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2935},
number = {},
pages = {335-384},
pmid = {40828286},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Genome, Insect ; *Gene Editing/methods ; *Insecta/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Site-specific genome editing is the most direct way to test gene function. When CRISPR-Cas9 was introduced for the editing of eukaryotic genomes, entomologists were ready with questions but had many methodologies to forge for the approach to be useful. Now, roughly 45 non-model insect genomes have been edited to study processes such as insecticide resistance, olfaction, immunity, and development. A useful first step for gene editing in an insect species of interest is identification and targeted editing of a gene with a visible phenotype. Visible markers increase the efficiency of detection of a genetic change; a wide availability of markers is one reason why model insects are so easy to manipulate and so have been key in understanding many biological processes. Here we will describe with detailed protocols how to approach a new insect species with CRISPR-era approaches by targeting a visual marker with Cas9-editing.},
}

Animals
*CRISPR-Cas Systems/genetics
*Genome, Insect
*Gene Editing/methods
*Insecta/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40828285,
year = {2025},
author = {James, AA and Carballar-Lejarazú, R},
title = {History of Mosquito Transgenesis: A Perspective in Review.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2935},
number = {},
pages = {311-333},
pmid = {40828285},
issn = {1940-6029},
mesh = {Animals ; *Culicidae/genetics ; *Gene Transfer Techniques/history ; DNA Transposable Elements/genetics ; Gene Editing/methods ; CRISPR-Cas Systems ; Animals, Genetically Modified/genetics ; },
abstract = {The development of mosquito transgenesis technologies was driven by the need to make stable and heritable modifications to the genomes of these important insects for a variety of basic and applied objectives. While a number of transient assay systems for gene expression analyses were developed, transposable elements (TEs) were the first tools that allowed the production of genetically manipulated strains for studies that involve the complex biology of these insects and their ability to transmit pathogens. TEs have been replaced more recently for most applications by the adaptation of Cas9/guide RNA techniques, but they are still useful in randomly sampling genomes as enhancer traps and identifying neutral regions in the genome free of insertion site effects. Coupled with the Cas9/guide RNA technologies, precise editing and engineering of the genetic mechanisms responsible for much of the biology of these interesting and important insects is now possible.},
}

Animals
*Culicidae/genetics
*Gene Transfer Techniques/history
DNA Transposable Elements/genetics
Gene Editing/methods
CRISPR-Cas Systems
Animals, Genetically Modified/genetics

@article {pmid40817912,
year = {2025},
author = {Xia, Y and Liang, Z and Du, X and Cao, D and Li, J and Sun, L and Huo, YX and Guo, S},
title = {Design of function-regulating RNA via deep learning and AlphaFold 3.},
journal = {Briefings in bioinformatics},
volume = {26},
number = {4},
pages = {},
pmid = {40817912},
issn = {1477-4054},
support = {32370095//National Natural Science Foundation of China/ ; 32371489//National Natural Science Foundation of China/ ; 2024YFA0917501//National Key Research and Development Program of China/ ; },
mesh = {*Deep Learning ; *RNA/genetics/chemistry ; Molecular Dynamics Simulation ; Gene Editing ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; Humans ; Computational Biology/methods ; },
abstract = {RNAs are programmable macromolecules that play diverse regulatory roles in living organisms. However, the intricate structure-function relationships underlying their regulatory activities pose significant challenges for RNA design. Here, we introduce a computational framework that integrates deep learning and energy-based methods to enhance the sequence diversity of sgRNAs designs. Our approach demonstrates high editing efficiencies of up to 75% for gene knockouts, 100% for large fragment deletions, and 62.5% for multiplex gene editing using the designed sgRNAs. Molecular dynamic simulations suggested the stability of DNA-RNA-protein complex is essential to the functionality of designed RNAs. Moreover, we reveal that the confidence metrics of AlphaFold 3 can effectively distinguish functional sequences, enabling one-shot design of crRNAs. This work presents an efficient strategy for designing regulatory RNAs with complex interactions and establishes the potential of AlphaFold 3 in advancing RNA design.},
}

*Deep Learning
*RNA/genetics/chemistry
Molecular Dynamics Simulation
Gene Editing
*RNA, Guide, CRISPR-Cas Systems/genetics/chemistry
Humans
Computational Biology/methods

@article {pmid40817853,
year = {2025},
author = {Cheng, Y and Guo, W and Wan, Y and Li, S and Zhou, J and Wang, J},
title = {Ultrasensitive Detection of Nucleic Acid and Protein at Ambient Temperature by Using an Engineered CRISPR-Cas12a-Based Digital Assay.},
journal = {Analytical chemistry},
volume = {97},
number = {33},
pages = {18355-18363},
doi = {10.1021/acs.analchem.5c03865},
pmid = {40817853},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; *Temperature ; Humans ; Limit of Detection ; *DNA, Viral/analysis ; Point-of-Care Systems ; Smartphone ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The development of sensitive and convenient molecular detection technologies is crucial for early disease diagnosis and precision medicine. The CRISPR-Cas12a system has garnered significant attention due to its efficient molecular detection capabilities. However, most methods were performed at 37 °C or higher, which require a temperature control system and limit their applicability in point-of-care (POC) settings. In this study, we demonstrate that LbCpf1-Ultra, an engineered CRISPR-Cas12a, exhibits robust trans-cleavage activity at room temperature. Leveraging this finding, we developed a versatile digital CRISPR platform capable of ultrasensitive detection of viral DNA and proteins at ambient temperature, achieving a limit of detection (LOD) of 11.9 copies/μL for DNA and 5 fM for proteins. To further enhance its usability in POC environments, we integrated this platform with a smartphone-based fluorescence imaging device, enabling low-cost and on-site detection of nucleic acids and proteins without the need for external equipment. Clinical validation showcases its potential for reliable diagnostics. This platform provides a new direction for future CRISPR-based molecular diagnostics and holds promise for clinical diagnosis and precision medicine.},
}

*CRISPR-Cas Systems/genetics
*Temperature
Humans
Limit of Detection
*DNA, Viral/analysis
Point-of-Care Systems
Smartphone
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40817118,
year = {2025},
author = {Teng, X and Wang, Z and Zhang, Y and Wang, B and Gong, G and Hu, J and Zhu, Y and Peng, B and Wang, J and Chen, J and Shi, S and Nielsen, J and Liu, Z},
title = {Matrix regulation: a plug-and-tune method for combinatorial regulation in Saccharomyces cerevisiae.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7624},
pmid = {40817118},
issn = {2041-1723},
support = {22078012//National Natural Science Foundation of China (National Science Foundation of China)/ ; 22211530047//National Natural Science Foundation of China (National Science Foundation of China)/ ; NNF10CC1016517//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; },
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Expression Regulation, Fungal ; RNA, Guide, CRISPR-Cas Systems/genetics ; Heme/biosynthesis ; Mevalonic Acid/metabolism ; RNA, Transfer/genetics/metabolism ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; },
abstract = {Transcriptional fine-tuning of long pathways is complex, even in the extensively applied cell factory Saccharomyces cerevisiae. Here, we present Matrix Regulation (MR), a CRISPR-mediated pathway fine-tuning method enabling the construction of 6[8] gRNA combinations and screening for the optimal expression levels across up to eight genes. We first identify multiple tRNAs with efficient gRNA processing capacities to assemble a gRNA regulatory matrix combinatorially. Then, we expand the target recognition of CRISPR regulation from NGG PAM to NG PAM by characterizing dCas9 variants. To increase the dynamic range of modulation, we test 101 candidate activation domains followed by mutagenesis and screening the best one to further enhance its activation capability in S. cerevisiae by 3-fold. The regulations generate combinatorial strain libraries for both the mevalonate pathway and the heme biosynthesis pathway and increase squalene production by 37-fold and heme by 17-fold, respectively, demonstrating the versatility of our method and its applicability in fundamental research.},
}

*Saccharomyces cerevisiae/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Gene Expression Regulation, Fungal
RNA, Guide, CRISPR-Cas Systems/genetics
Heme/biosynthesis
Mevalonic Acid/metabolism
RNA, Transfer/genetics/metabolism
Saccharomyces cerevisiae Proteins/genetics/metabolism

@article {pmid40815833,
year = {2025},
author = {Shan, J and Sheng, Y and Luo, L and Wang, W and Liu, X and Ma, Y and Wang, J},
title = {One-Pot Rlock-Mediated CRISPR/Cas12a-Driven RCA Cycle for Rapid and High-Sensitive APE1 Detection.},
journal = {Analytical chemistry},
volume = {97},
number = {33},
pages = {18208-18216},
doi = {10.1021/acs.analchem.5c03234},
pmid = {40815833},
issn = {1520-6882},
mesh = {*DNA-(Apurinic or Apyrimidinic Site) Lyase/analysis/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *Endodeoxyribonucleases/genetics ; Biosensing Techniques/methods ; Limit of Detection ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Apurinic/apyrimidinic endonuclease 1 (APE1) is a key enzyme involved in DNA repair and cellular redox regulation, and is frequently overexpressed in tumor cells. This highlights the urgent need for a rapid and high-sensitive point-of-care testing (POCT) strategy for APE1 to facilitate early cancer diagnosis. Rolling circle amplification (RCA) is a widely used isothermal DNA amplification method; however, its application in APE1 detection remains rare. Here, we introduce a versatile RCA-Lock (Rlock) conversion platform that enables the transformation of RCA-based nucleic acid detection technologies into APE1-responsive assays. Building upon this platform, we further developed a novel POCT method for APE1 detection─Rlock-mediated, CRISPR/Cas12a-driven RCA cycle (RCRE)─which, for the first time, integrates CRISPR/Cas12a with RCA into a one-pot APE1 detection system. The RCRE assay achieves a limit of detection of 8.86 × 10[-4] U/mL within 30 min, while requiring minimal equipment, low cost, and no complex handling procedures. This Rlock-based conversion strategy represents a transformative advance in the field of APE1 diagnostics and offers conceptual inspiration for the design of programmable nucleic acid-based biosensors. The resulting RCRE assay significantly broadens the technological landscape for early cancer detection and paves the way for future clinical translation.},
}

*DNA-(Apurinic or Apyrimidinic Site) Lyase/analysis/genetics/metabolism
*CRISPR-Cas Systems/genetics
Humans
*Nucleic Acid Amplification Techniques/methods
*Endodeoxyribonucleases/genetics
Biosensing Techniques/methods
Limit of Detection
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid40813572,
year = {2025},
author = {Lo, YH and Horn, HT and Huang, MF and Yu, WC and Young, CM and Liu, Q and Tomaske, M and Towers, M and Dominguez, A and Bassik, MC and Lee, DF and Qi, LS and Weissman, JS and Chen, J and Kuo, CJ},
title = {Large-scale CRISPR screening in primary human 3D gastric organoids enables comprehensive dissection of gene-drug interactions.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7566},
pmid = {40813572},
issn = {2041-1723},
mesh = {Humans ; *Organoids/drug effects/metabolism ; *Cisplatin/pharmacology ; *Stomach Neoplasms/genetics/drug therapy/pathology ; *CRISPR-Cas Systems ; Antineoplastic Agents/pharmacology ; Single-Cell Analysis ; *Stomach/drug effects/cytology ; },
abstract = {Understanding how genes influence drug responses is critical for advancing personalized cancer treatments. However, identifying these gene-drug interactions in a physiologically relevant human system remains a challenge, as it requires a model that reflects the complexity and heterogeneity among individuals. Here we show that large-scale CRISPR-based genetic screens, including knockout, interference (CRISPRi), activation (CRISPRa), and single-cell approaches, can be applied in primary human 3D gastric organoids to systematically identify genes that affect sensitivity to cisplatin. Our screens uncover genes that modulate cisplatin response. By combining CRISPR perturbations with single-cell transcriptomics, we resolve how genetic alterations interact with cisplatin at the level of individual cells and uncover an unexpected link between fucosylation and cisplatin sensitivity. We identify TAF6L as a regulator of cell recovery from cisplatin-induced cytotoxicity. These results highlight the utility of human organoid models for dissecting gene-drug interactions and offer insights into therapeutic vulnerabilities in gastric cancer.},
}

Humans
*Organoids/drug effects/metabolism
*Cisplatin/pharmacology
*Stomach Neoplasms/genetics/drug therapy/pathology
*CRISPR-Cas Systems
Antineoplastic Agents/pharmacology
Single-Cell Analysis
*Stomach/drug effects/cytology

@article {pmid40811447,
year = {2025},
author = {Carreira de Paula, J and Solano Parada, J and Rosel Miñarro, JF and García Olmedo, P and Orantes, FJ and Osuna, A and de Pablos, LM},
title = {Erratum: On-site DNA Detection of Trypanosomatid Parasites and Nosema ceranae Through Alkaline Lysis Coupled to RPA/CRISPR/Cas12a System.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {222},
pages = {},
doi = {10.3791/6641},
pmid = {40811447},
issn = {1940-087X},
mesh = {*Nosema/genetics/isolation & purification ; *CRISPR-Cas Systems ; *DNA, Protozoan/genetics/analysis ; *Trypanosoma/genetics/isolation & purification ; Animals ; *DNA, Fungal/genetics/analysis ; },
abstract = {This corrects the article 10.3791/68874.},
}

*Nosema/genetics/isolation & purification
*CRISPR-Cas Systems
*DNA, Protozoan/genetics/analysis
*Trypanosoma/genetics/isolation & purification
Animals
*DNA, Fungal/genetics/analysis

@article {pmid40806200,
year = {2025},
author = {Li, H and Zhang, R and Wang, X and Liu, L and Yao, Z and Zhu, H},
title = {CRISPR/Cas9-Mediated Disruption of lrp6a Leads to Abnormal Median Fin Development and Somitogenesis in Goldfish (Carassius auratus).},
journal = {International journal of molecular sciences},
volume = {26},
number = {15},
pages = {},
pmid = {40806200},
issn = {1422-0067},
support = {KJCX20230216//Scientific and technological innovation capacity building project of Beijing Academy of Agricultural and Forestry Sciences/ ; QNJJ202238//Young scientists fund of the academy/ ; JBGS-2023-02//Reform and development project/ ; 32403015//National Natural Science Foundation of China (NSFC) Youth Science Foundation Project/ ; NY2401170024//Beijing Rural Revitalization Agricultural Science and Technology Project/ ; },
mesh = {Animals ; *Goldfish/genetics/embryology ; *CRISPR-Cas Systems ; *Animal Fins/abnormalities/metabolism/embryology/growth & development ; *Low Density Lipoprotein Receptor-Related Protein-6/genetics/metabolism ; Gene Expression Regulation, Developmental ; *Somites/metabolism ; *Fish Proteins/genetics/metabolism ; Wnt Signaling Pathway ; },
abstract = {In this study, we demonstrated that lrp6a, a co-receptor in the Wnt signaling pathway, is essential for proper median fin formation and somitogenesis in goldfish. We analyzed the gene's sequence features and expression patterns in both wen-type and egg-type goldfish, uncovering distinct tissue-specific expression differences between the two varieties. To explore the functional role of lrp6a, we performed CRISPR/Cas9-mediated gene knockout using eight designed single-guide RNAs (sgRNAs), of which four showed effective targeting. Three high-efficiency sgRNAs were selected and co-injected into embryos to achieve complete gene disruption. Morphological assessments and X-ray microtomography (μCT) imaging of the resulting mutants revealed various abnormalities, including defects in the dorsal, caudal, and anal fins, as well as skeletal deformities near the caudal peduncle. These results confirm that lrp6a plays a key role in median fin development and axial patterning, offering new insights into the genetic regulation of fin formation in teleost fish.},
}

Animals
*Goldfish/genetics/embryology
*CRISPR-Cas Systems
*Animal Fins/abnormalities/metabolism/embryology/growth & development
*Low Density Lipoprotein Receptor-Related Protein-6/genetics/metabolism
Gene Expression Regulation, Developmental
*Somites/metabolism
*Fish Proteins/genetics/metabolism
Wnt Signaling Pathway

@article {pmid40806166,
year = {2025},
author = {Keil, S and Dittmar, T},
title = {Differential Effects of Snail-KO in Human Breast Epithelial Cells and Human Breast Epithelial × Human Breast Cancer Hybrids.},
journal = {International journal of molecular sciences},
volume = {26},
number = {15},
pages = {},
pmid = {40806166},
issn = {1422-0067},
mesh = {*Snail Family Transcription Factors/genetics/metabolism ; Humans ; Epithelial-Mesenchymal Transition/genetics ; Female ; *Breast Neoplasms/genetics/pathology/metabolism ; *Epithelial Cells/metabolism/pathology ; Cell Movement/genetics ; Cell Line, Tumor ; Gene Knockout Techniques ; Gene Expression Regulation, Neoplastic ; Neoplastic Stem Cells/metabolism/pathology ; Hybrid Cells/metabolism/pathology ; CRISPR-Cas Systems ; Zinc Finger E-box-Binding Homeobox 1/metabolism/genetics ; },
abstract = {Snail and Zeb1 have been suggested as markers for the hybrid/mixed epithelial (E)/mesenchymal (M) state of cancer cells. Such cancer cells co-express E- and M-specific transcripts and possess cancer stem cell properties. M13HS-2/-8 tumor hybrid clones derived from human M13SV1-EGFP-Neo breast epithelial cells and human HS578T-Hyg breast cancer cells exhibited co-expression of Snail and Zeb1. To explore the impact of Snail on stemness/epithelial-to-mesenchymal transition (EMT)-related properties in M13HS-2/-8 tumor hybrid clones, Snail was knocked out (KO) using CRISPR/Cas9. Mammosphere formation, colony formation, Western blot analyses, cell migration, and invasion assays were conducted for the characterization of Snail knockout cells. Interestingly, Snail-KO in M13SV1-EGFP-Neo cells resulted in the up-regulation of vimentin and N-cadherin, suggesting EMT induction, which was associated with a significantly enhanced colony formation capacity. In contrast, EMT marker pattern and colony formation capacities of M13HS-2/-8 Snail-KO tumor hybrid clones remained unchanged. Notably, the mammosphere formation capacities of M13HS-2/-8 Snail-KO tumor hybrid clones were significantly reduced. The migratory behavior of all Snail-KO cells was not altered compared with their wild-type counterparts. In contrast, M13HS-2 hybrids and their M13HS-2 Snail-KO variant exhibited a markedly enhanced invasive capacity. Therefore, Snail plays a role as a mediator of stemness properties rather than mediating EMT.},
}

*Snail Family Transcription Factors/genetics/metabolism
Humans
Epithelial-Mesenchymal Transition/genetics
Female
*Breast Neoplasms/genetics/pathology/metabolism
*Epithelial Cells/metabolism/pathology
Cell Movement/genetics
Cell Line, Tumor
Gene Knockout Techniques
Gene Expression Regulation, Neoplastic
Neoplastic Stem Cells/metabolism/pathology
Hybrid Cells/metabolism/pathology
CRISPR-Cas Systems
Zinc Finger E-box-Binding Homeobox 1/metabolism/genetics

@article {pmid40802750,
year = {2025},
author = {McGivney, GR and Brockman, QR and Borcherding, N and Scherer, A and Rauckhorst, AJ and Gutierrez, WR and Solst, SR and Heer, CD and Warrier, A and Floyd, W and Kirsch, DG and Knepper-Adrian, VL and Laverty, EA and Roughton, GA and Spitz, DR and Taylor, EB and Dodd, RD},
title = {Somatic CRISPR tumorigenesis and multiomic analysis reveal a pentose phosphate pathway disruption vulnerability in MPNSTs.},
journal = {Science advances},
volume = {11},
number = {33},
pages = {eadu2906},
pmid = {40802750},
issn = {2375-2548},
mesh = {*Pentose Phosphate Pathway/genetics ; Animals ; Mice ; NF-E2-Related Factor 2/genetics/metabolism ; Humans ; *CRISPR-Cas Systems ; Glucosephosphate Dehydrogenase/genetics/metabolism ; *Carcinogenesis/genetics ; Cyclin-Dependent Kinase Inhibitor p16/genetics ; Tumor Suppressor Protein p53/genetics ; Gene Expression Regulation, Neoplastic ; NADP/metabolism ; Cell Line, Tumor ; },
abstract = {Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive and chemo-resistant sarcomas with poor survival rates. Loss of CDKN2A or P53 following NF1 disruption is a key event in MPNST development. Here, we used CRISPR-Cas9 somatic tumorigenesis in mice to identify transcriptomic and metabolomic features distinguishing CDKN2A- versus P53-deleted MPNSTs. Convergent, multiomic analyses revealed that CDKN2A-deleted MPNSTs are especially dependent on the pentose phosphate pathway (PPP) and NADPH metabolism for growth and viability. Disruption of glucose-6-phosphate dehydrogenase (G6PD), the PPP rate-limiting enzyme, slowed CDKN2A-deleted MPNST growth and sensitized MPNSTs to standard-of-care chemotherapy. Knockdown of the redox-regulated transcription factor NRF2 slowed MPNST growth and decreased G6PD transcription. Analysis of patient MPNSTs identified a NRF2 gene signature correlating with tumor transformation. Furthermore, G6PD and NRF2 expression in PanCancer TCGA samples correlates with patient survival. This work identifies NRF2-PPP dependency as a targetable vulnerability in these difficult-to-treat MPNSTs, particularly in the NF1/CDKN2A-deleted majority.},
}

*Pentose Phosphate Pathway/genetics
Animals
Mice
NF-E2-Related Factor 2/genetics/metabolism
Humans
*CRISPR-Cas Systems
Glucosephosphate Dehydrogenase/genetics/metabolism
*Carcinogenesis/genetics
Cyclin-Dependent Kinase Inhibitor p16/genetics
Tumor Suppressor Protein p53/genetics
Gene Expression Regulation, Neoplastic
NADP/metabolism
Cell Line, Tumor

@article {pmid40802112,
year = {2025},
author = {Minati, MA and Muneta, LL and Achouri, Y and Pirenne, S and Porada, C and Rochais, F and Jacquemin, P},
title = {KRAS4B is required for placental development.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {82},
number = {1},
pages = {308},
pmid = {40802112},
issn = {1420-9071},
support = {J.0085.19//Fonds De La Recherche Scientifique - FNRS/ ; },
mesh = {Animals ; Female ; Pregnancy ; *Proto-Oncogene Proteins p21(ras)/genetics/metabolism ; Mice ; Mice, Knockout ; *Placenta/metabolism/pathology ; *Placentation/genetics ; CRISPR-Cas Systems ; Trophoblasts/metabolism ; Phenotype ; },
abstract = {Beyond its well-established role in cancer, KRAS is also crucial for embryogenesis, as its absence leads to embryonic lethality. However, the precise mechanisms underlying the developmental functions of KRAS, as well as the respective roles of its two splicing isoforms, KRAS4A and KRAS4B, remain incompletely characterized. To address these issues, we generated Kras4A knock-out (Kras4A[-/-]) and Kras4B[-/-] mouse models using CRISPR/Cas9 technology, and compared their phenotypes to those of a Kras[-/-] model, in which both isoforms are simultaneously inactivated. We observed that Kras[-/-] and Kras4B[-/-] embryos show a lethality that starts around E13.5, while Kras4A[-/-] embryos develop normally, with no detectable abnormalities. In contrast, Kras[-/-] embryos displayed a dual phenotype affecting both the heart and placenta, whereas Kras4B[-/-] embryos exhibited only the placental phenotype. The cardiac phenotype was complex, combining ventricular non-compaction, ventricular septal defects, double outlet right ventricle, and overriding aorta, likely resulting from impaired cardiac precursor proliferation. The placental phenotype was characterized by reduced placental size, and a marked decrease in glycogen trophoblast cells, correlating with hypoglycemia and hypoxia in Kras[-/-] and Kras4B[-/-] embryos. Thus, our findings confirm the predominant role of KRAS4B in KRAS-mediated developmental functions, but also suggest hidden functions of KRAS4A. Importantly, this study is the first to identify KRAS as a key regulator of a specific cell differentiation process and to characterize the biological defects caused by its loss.},
}

Animals
Female
Pregnancy
*Proto-Oncogene Proteins p21(ras)/genetics/metabolism
Mice
Mice, Knockout
*Placenta/metabolism/pathology
*Placentation/genetics
CRISPR-Cas Systems
Trophoblasts/metabolism
Phenotype

@article {pmid40801599,
year = {2025},
author = {Yang, J and Guo, F and Chin, HS and Chen, GB and Zhang, Z and Williams, L and Kueh, AJ and Chow, PKH and Herold, MJ and Fu, NY},
title = {Rapid and Robust Generation of Homozygous Fluorescent Reporter Knock-In Cell Pools by CRISPR-Cas9.},
journal = {Cells},
volume = {14},
number = {15},
pages = {},
pmid = {40801599},
issn = {2073-4409},
support = {2020-Gilead-001//Gilead Research Scholars Liver Disease Program (Asia)/ ; MOH‑000935//NMRC OF‑YIRG/ ; MCRF22013//Victorian Cancer Agency Mid‑Career Research Fellowship/ ; 2037907//NHMRC 2024 Ideas Grants/ ; MOH‑000546//NMRC OF‑YIRG/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; Humans ; *Genes, Reporter/genetics ; HEK293 Cells ; *Homozygote ; Gene Editing/methods ; Lentivirus/genetics ; Genetic Vectors ; },
abstract = {Conventional methods for generating knock-out or knock-in mammalian cell models using CRISPR-Cas9 genome editing often require tedious single-cell clone selection and expansion. In this study, we develop and optimise rapid and robust strategies to engineer homozygous fluorescent reporter knock-in cell pools with precise genome editing, circumventing clonal variability inherent to traditional approaches. To reduce false-positive cells associated with random integration, we optimise the design of donor DNA by removing the start codon of the fluorescent reporter and incorporating a self-cleaving T2A peptide system. Using fluorescence-assisted cell sorting (FACS), we efficiently identify and isolate the desired homozygous fluorescent knock-in clones, establishing stable cell pools that preserve parental cell line heterogeneity and faithfully reflect endogenous transcriptional regulation of the target gene. We evaluate the knock-in efficiency and rate of undesired random integration in the electroporation method with either a dual-plasmid system (sgRNA and donor DNA in two separate vectors) or a single-plasmid system (sgRNA and donor DNA combined in one vector). We further demonstrate that coupling our single-plasmid construct with an integrase-deficient lentivirus vector (IDLV) packaging system efficiently generates fluorescent knock-in reporter cell pools, offering flexibility between electroporation and lentivirus transduction methods. Notably, compared to the electroporation methods, the IDLV system significantly minimises random integration. Moreover, the resulting reporter cell lines are compatible with most of the available genome-wide sgRNA libraries, enabling unbiased CRISPR screens to identify key transcriptional regulators of a gene of interest. Overall, our methodologies provide a powerful genetic tool for rapid and robust generation of fluorescent reporter knock-in cell pools with precise genome editing by CRISPR-Cas9 for various research purposes.},
}

*CRISPR-Cas Systems/genetics
*Gene Knock-In Techniques/methods
Humans
*Genes, Reporter/genetics
HEK293 Cells
*Homozygote
Gene Editing/methods
Lentivirus/genetics
Genetic Vectors

@article {pmid40801569,
year = {2025},
author = {Rauf, MA and Rao, A and Sivasoorian, SS and Iyer, AK},
title = {Nanotechnology-Based Delivery of CRISPR/Cas9 for Cancer Treatment: A Comprehensive Review.},
journal = {Cells},
volume = {14},
number = {15},
pages = {},
pmid = {40801569},
issn = {2073-4409},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Neoplasms/therapy/genetics ; Gene Editing/methods ; *Nanotechnology/methods ; Animals ; Genetic Therapy/methods ; *Gene Transfer Techniques ; },
abstract = {CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-associated protein 9)-mediated genome editing has emerged as a transformative tool in medicine, offering significant potential for cancer therapy because of its capacity to precisely target and alter the genetic modifications associated with the disease. However, a major challenge for its clinical translation is the safe and efficient in vivo delivery of CRISPR/Cas9 components to target cells. Nanotechnology is a promising solution to this problem. Nanocarriers, owing to their tunable physicochemical properties, can encapsulate and protect CRISPR/Cas9 components, enabling targeted delivery and enhanced cellular uptake. This review provides a comprehensive examination of the synergistic potential of CRISPR/Cas9 and nanotechnology in cancer therapy and explores their integrated therapeutic applications in gene editing and immunotherapy. A critical aspect of in vivo CRISPR/Cas9 application is to achieve effective localization at the tumor site while minimizing off-target effects. Nanocarriers can be engineered to overcome biological barriers, thereby augmenting tumor-specific delivery and facilitating intracellular uptake. Furthermore, their design allows for controlled release of the therapeutic payload, ensuring sustained efficacy and reduced systemic toxicity. The optimization of nanocarrier attributes, including size, shape, surface charge, and composition, is crucial for improving the cellular internalization, endosomal escape, and nuclear localization of CRISPR/Cas9. Moreover, surface functionalization with targeting ligands can enhance the specificity of cancer cells, leading to improved gene-editing accuracy. This review thoroughly discusses the challenges associated with in vivo CRISPR/Cas9 delivery and the innovative nanotechnological strategies employed to overcome them, highlighting their combined potential for advancing cancer treatment for clinical application.},
}

Humans
*CRISPR-Cas Systems/genetics
*Neoplasms/therapy/genetics
Gene Editing/methods
*Nanotechnology/methods
Animals
Genetic Therapy/methods
*Gene Transfer Techniques

@article {pmid40795957,
year = {2025},
author = {Li, Z and Zhao, W and Li, S and Luo, X and Wei, Y and Zhang, Y and Ye, Z and Li, S and Wang, M and Tan, J and Bock, R},
title = {Engineering of high-precision C-to-G base editors with expanded site selectivity and target compatibility.},
journal = {Nucleic acids research},
volume = {53},
number = {15},
pages = {},
pmid = {40795957},
issn = {1362-4962},
support = {2023ZD04074//National Science and Technology Major Project/ ; NAUSY-ZZ01//Nanjing Agricultural University/ ; BE2023369//Jiangsu Province Key Research and Development Program/ ; ZSBBL-KY2023-04//Project of Zhongshan Biological Breeding Laboratory/ ; //Innovative and Entrepreneurial Talent/ ; },
mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; Base Sequence ; },
abstract = {Base editors (BEs) are powerful tools for single nucleotide substitutions without requiring DNA double-stranded breaks or donor templates. The development of C-to-G base editors (CGBEs) represents a significant advancement by enabling base transversions, thus expanding the range of genetic modifications beyond traditional transitions and facilitating a broader spectrum of (therapeutic) applications. However, current CGBEs suffer from limitations in their editing range, mostly modifying position 6 relative to the distal end of the PAM, and their editing efficiency depends on the sequence context. In this study, by systematic exploration of deaminases to construct CGBEs, we have identified PmCDA1-based CGBEs that preferentially edit position 3. Furthermore, we report that truncations of the CDA1 C-terminus significantly enhance C-to-G editing efficiency. Our CDA1Δ-CGBEs not only exhibit high precision but also display remarkable compatibility with diverse substrate sequence contexts. We also show that they can substantially reduce, or even eliminate, genome-wide off-target editing. Importantly, we demonstrate that the strategy of using truncated CDA1 variants to improve C-to-G editing is effective not only in yeast but also in human and rice cells. These enhanced C-to-G base editing tools hold great promise for a wide range of applications in gene therapy, precision breeding, and fundamental research.},
}

*Gene Editing/methods
Humans
CRISPR-Cas Systems
Saccharomyces cerevisiae/genetics
Saccharomyces cerevisiae Proteins/genetics/metabolism
Base Sequence

@article {pmid40780174,
year = {2025},
author = {Zhu, Y and Lai, J and Yang, X and Wang, S and Gu, D and Huang, Y and Liu, Y and Liu, C},
title = {CRISPR/Cas13a-driven lateral flow assay for preamplification-free and ultrasensitive miRNA-21 detection.},
journal = {Biosensors & bioelectronics},
volume = {288},
number = {},
pages = {117850},
doi = {10.1016/j.bios.2025.117850},
pmid = {40780174},
issn = {1873-4235},
mesh = {*MicroRNAs/isolation & purification/genetics/blood ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Limit of Detection ; Spectrum Analysis, Raman/methods ; Colorimetry/methods ; },
abstract = {Developing a preamplification-free and sensitive clustered regularly interspaced short palindromic repeats (CRISPR)-based method is significant but still extremely challenging for microRNA (miRNA) detection. Here we present a combination of a CRISPR/Cas13a-based reaction with a lateral flow biosensor, which enables the quantitative and colorimetric readout of preamplification-free miRNA detection at room temperature. In this work, the reaction principle and the structure of the lateral flow strip are well-designed to achieve surface-enhanced Raman scattering (SERS)/colorimetric dual-signal "turn-on" response of target miRNA. The CRISPR/Cas13a Reporter is engineered with a DNA-RNA splicing structure to generate DNA cleavage products and reduce nonspecific collateral cleavage. Without the need for nucleic acid preamplification strategy, the developed CRISPR/Cas13a-driven lateral flow biosensor enables the microRNA-21 (miR-21) detection at room temperature with a readout time of 10 min and a total process time of less than 45 min, achieving an impressive limit of detection of 8.96 aM by SERS and 1 fM by visualization, respectively. Moreover, the platform demonstrated excellent recovery rates in spiked human serum samples. The proposed CRISPR/Cas13a-driven, dual-signal "turn-on"-responded lateral flow platform has the potential to simultaneously meet the requirements of convenient point-of-care visualization detection and more accurate and sensitive SERS detection of miR-21, offering a cost-effective, rapid, and reliable tool for early cancer diagnosis.},
}

*MicroRNAs/isolation & purification/genetics/blood
*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
Humans
Limit of Detection
Spectrum Analysis, Raman/methods
Colorimetry/methods

@article {pmid40780172,
year = {2025},
author = {Feng, J and Wu, Z and Zhu, W and Jin, F and Zhao, M and Zhong, W and Dai, C and He, Y and Yan, L and Wu, S and Wang, Y and Rui, Y and Zheng, L and Fu, Q},
title = {A novel thermo-activated one-pot RPA-CRISPR-Cas12b assay for Mycoplasma pneumoniae POCT.},
journal = {Biosensors & bioelectronics},
volume = {288},
number = {},
pages = {117839},
doi = {10.1016/j.bios.2025.117839},
pmid = {40780172},
issn = {1873-4235},
mesh = {Humans ; *Mycoplasma pneumoniae/isolation & purification/genetics ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; Point-of-Care Testing ; *Pneumonia, Mycoplasma/diagnosis/microbiology ; Limit of Detection ; Temperature ; },
abstract = {Mycoplasma pneumoniae (M. pneumoniae), a major human respiratory pathogen, necessitates the development of rapid point-of-care testing (POCT) platforms for clinical management. However, current two-step workflows suffer from operational complexity and aerosol contamination risks. This limitation stems from CRISPR-Cas12 mediated template degradation in single-reaction systems, which compromises amplification efficiency and detection sensitivity. Here, we combined RPA and CRISPR Cas12b by leveraging the difference in their optimal temperatures to construct a novel TRACER (Thermo-activated RPA Amplification for CRISPR-Cas12b Efficient Recognition) technology. Through precise temperature modulation, TRACER sequentially executes isothermal amplification and CRISPR-mediated detection while preventing premature template cleavage, thereby maintaining optimal reaction efficiency. The platform demonstrates exceptional analytical sensitivity with a detection limit of 1 copy/μL, representing a 100-fold improvement over conventional one-pot RPA-CRISPR-Cas12a systems. Clinical validation using 195 specimens revealed diagnostic performance metrics of 99.2 % sensitivity (119/120), 100.0 % specificity (75/75), and 99.5 % accuracy (194/195). This innovative combination of single-tube reaction, field-deployable instrumentation, and cost-effectiveness establishes TRACER as an ideal POCT solution for M. pneumoniae detection in diverse clinical settings.},
}

Humans
*Mycoplasma pneumoniae/isolation & purification/genetics
*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
*Nucleic Acid Amplification Techniques/methods
Point-of-Care Testing
*Pneumonia, Mycoplasma/diagnosis/microbiology
Limit of Detection
Temperature

@article {pmid40770919,
year = {2025},
author = {Vega-Hernández, G and Duque, J and Klein, BJC and Soueid, DM and Rech, JC and Wang, H and Zhou, W and Garner, AL},
title = {CRISPR RiPCA for Investigating eIF4E-m[7]GpppX Capped mRNA Interactions.},
journal = {ACS chemical biology},
volume = {20},
number = {8},
pages = {2038-2048},
doi = {10.1021/acschembio.5c00471},
pmid = {40770919},
issn = {1554-8937},
support = {R01 GM135252/GM/NIGMS NIH HHS/United States ; R35 GM153185/GM/NIGMS NIH HHS/United States ; },
mesh = {*Eukaryotic Initiation Factor-4E/metabolism/genetics ; Humans ; *RNA, Messenger/metabolism/genetics ; *CRISPR-Cas Systems ; *RNA Caps/metabolism/genetics ; RNA-Binding Proteins/metabolism ; HEK293 Cells ; Protein Binding ; },
abstract = {Post-transcriptional modifications expand the information encoded by an mRNA. These dynamic and reversible modifications are specifically recognized by reader RNA-binding proteins (RBPs), which mediate the regulation of gene expression, RNA processing, localization, stability, and translation. Given their crucial functions, any disruptions in the normal activity of these readers can have significant implications for cellular health. Consequently, the dysregulation of these RBPs has been associated with neurodegenerative disorders, cancers, and viral infections. Therefore, there has been growing interest in targeting reader RBPs as a potential therapeutic strategy since developing molecules that restore proper RNA processing and function may offer a promising avenue for treating diseases. In this work, we coupled our previously established live-cell RNA-protein interaction (RPI) assay, RNA interaction with Protein-mediated Complementation Assay (RiPCA), with CRISPR technology to build a new platform, CRISPR RiPCA. As a model for development, we utilized the interaction of eukaryotic translation initiation factor 4E (eIF4E), a reader RBP that binds to the m[7]GpppX cap present at the 5' terminus of coding mRNAs, with an m[7]G capped RNA substrate. Using eIF4E CRISPR RiPCA, we demonstrate our technology's potential for measuring on-target activity of inhibitors of the eIF4E RPI of relevance to cancer drug discovery.},
}

*Eukaryotic Initiation Factor-4E/metabolism/genetics
Humans
*RNA, Messenger/metabolism/genetics
*CRISPR-Cas Systems
*RNA Caps/metabolism/genetics
RNA-Binding Proteins/metabolism
HEK293 Cells
Protein Binding

@article {pmid40751732,
year = {2025},
author = {Ding, S and Li, H and Li, J and Lu, D and Yang, J and Tang, Z},
title = {One-Pot Detection of Biomarker Apurinic/Apyrimidinic Endonuclease 1 Based on the Modified-crRNA Regulated Trans-Cleavage Activity of CRISPR/Cas12a.},
journal = {ACS synthetic biology},
volume = {14},
number = {8},
pages = {3186-3195},
doi = {10.1021/acssynbio.5c00335},
pmid = {40751732},
issn = {2161-5063},
mesh = {*DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics/metabolism/analysis ; *CRISPR-Cas Systems/genetics ; Humans ; Biomarkers/analysis/metabolism ; *Biosensing Techniques/methods ; *Endodeoxyribonucleases/metabolism/genetics ; DNA/genetics/metabolism ; *CRISPR-Associated Proteins/metabolism/genetics ; Bacterial Proteins ; },
abstract = {Apurinic/apyrimidinic endonuclease 1 (APE1), a critical protein in DNA repair, plays indispensable roles in the maintenance of cellular homeostasis, thereby garnering significant attention as a biomarker and therapeutic target for various disorders. Current APE1 sensing methods always require multiple enzymes or complex signal amplification. The high programmability of the CRISPR/Cas12-based signal amplifier provides a new chance for developing biosensors. In this study, we introduce a novel method for the detection of APE1 by leveraging the discovery that modulating the length of modified DNA within CRISPR RNA (crRNA) enables precise control over the trans-cleavage activity of CRISPR/Cas12a. By designing a specific crRNA, the APE1-mediated activity recovery of Cas12a (ARC) was developed for rapid, specific, and one-pot detection of APE1. ARC presented a detection limit of 1.74 × 10[-6] U/μL with high specificity in detecting APE1 in biological samples. Besides, this simple method was feasible for APE1 inhibition assays, highlighting its potential for inhibitor screening and evaluation. Collectively, our findings present an innovative approach for APE1 activity analysis and expand the CRISPR-based non-nucleic acid target sensing toolbox through a novel crRNA design.},
}

*DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics/metabolism/analysis
*CRISPR-Cas Systems/genetics
Humans
Biomarkers/analysis/metabolism
*Biosensing Techniques/methods
*Endodeoxyribonucleases/metabolism/genetics
DNA/genetics/metabolism
*CRISPR-Associated Proteins/metabolism/genetics
Bacterial Proteins

@article {pmid40677232,
year = {2025},
author = {Chen, Z and Zhu, D and Lai, KS and Chen, Y and Hu, Y and Fang, Y and Yan, Z and Hu, B and Zhang, Z and Zhang, M and Li, F},
title = {VEGFA Stop-Gained Variant Deteriorates Cardiac Remodeling in Myocardial Infarction.},
journal = {Circulation. Genomic and precision medicine},
volume = {18},
number = {4},
pages = {e004879},
doi = {10.1161/CIRCGEN.124.004879},
pmid = {40677232},
issn = {2574-8300},
mesh = {Animals ; *Myocardial Infarction/genetics/pathology/metabolism/physiopathology ; *Vascular Endothelial Growth Factor A/genetics/metabolism ; Mice ; *Ventricular Remodeling/genetics ; Humans ; Disease Models, Animal ; Male ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: A sustained dosage of VEGFA (vascular endothelial growth factor A) is crucial for angiogenesis in both homeostasis and cardiovascular diseases. Start codon CUG-initiated alternative translation is a conserved mechanism for producing mature VEGFA. Genetic surveys have identified stop-gained variants predicted to prematurely terminate CUG-initiated translation without affecting start codon ATG-initiated translation. However, the impacts of these variants on the vasculature in steady-state and disease conditions remain unknown.

METHODS: Using CRISPR/Cas9 genome editing, we established the Vegfa[Q150X/Q150] allele (Q150X), a mouse genetic model that mimics the human VEGFA stop-gained variant. The effects of this variant were tested in both adult homeostatic conditions and the acute myocardial infarction (MI) model. We analyzed and quantified cardiac vasculature structure using immunofluorescence and light-sheet imaging. Furthermore, we characterized cellular heterogeneity, cell-cell interactions, and gene regulation using single-nucleus RNA sequencing, as well as cell type-specific transcriptomics and epigenomics.

RESULTS: Homozygous mice carrying the stop-gained variant were viable. VEGFA dosage was reduced to 70% in the Q150X homeostatic heart, with no significant alteration in cardiac function or vasculature. In the MI model, VEGFA dosage in Q150X was reduced to about 40% within the first week post-infarction, leading to functional deterioration in the post-MI hearts. Significant changes in cellular composition were observed 3 days post-MI. In particular, endothelial cells in Q150X diverged into a state that showed a higher level of hypoxia stress, an elevated inflammatory response, and increased extracellular matrix secretion. In addition, we observed an increase in Nppb[+] stressed cardiomyocytes in both 3 days post-MI and homeostasis. Finally, proinflammatory macrophages, neutrophils, and Cd8[+]T cells were enriched in the ischemic zone of Q150X hearts.

CONCLUSIONS: CUG-initiated translation contributes significantly to the production of mature VEGFA in ischemic hearts. VEGFA dosage is critical in determining the cellular microenvironment during ischemic injury.},
}

Animals
*Myocardial Infarction/genetics/pathology/metabolism/physiopathology
*Vascular Endothelial Growth Factor A/genetics/metabolism
Mice
*Ventricular Remodeling/genetics
Humans
Disease Models, Animal
Male
CRISPR-Cas Systems

@article {pmid40643567,
year = {2025},
author = {Majdi, M and Wahl, NJ and Li, L and King, G and Scott, H and Beal, J and Lenaghan, SC and Occhialini, A},
title = {Development of CRISPRi Orthogonal Repression Systems in Plant Cells Using Synthetic Variants of the Figwort Mosaic Virus 34S Promoter with Two Identical sgRNA Binding Sites.},
journal = {ACS synthetic biology},
volume = {14},
number = {8},
pages = {3219-3231},
doi = {10.1021/acssynbio.5c00356},
pmid = {40643567},
issn = {2161-5063},
mesh = {*Promoter Regions, Genetic/genetics ; Nicotiana/genetics ; Binding Sites/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Synthetic Biology/methods ; *Plant Cells/metabolism ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified ; Plant Leaves/genetics ; },
abstract = {The plant synthetic biology toolbox is rapidly expanding; however, there are still limited options for engineering logic gates for the precise modulation of gene expression. CRISPR interference (CRISPRi) represents a promising strategy for engineering logic into plant cells; however, only a limited number of promoter modules have been characterized for CRISPRi-mediated repression. In this study, the transient transgene expression in agroinfiltrated Nicotiana benthamiana leaves was used to assess the repressibility of a number of promoters with different strengths, including the Figwort Mosaic Virus (FMV) 34S promoter, which showed high repression efficiency using CRISPRi. Using dCas9 fused to the SRDX repressor domain, we employed single and double (identical or heterogeneous) sgRNA strategies for evaluating the repressibility of a library of 33 variants of the 34S promoter. This investigation supported a previous computer simulation predicting that a promoter with identical sgRNA binding sites is more efficiently repressed than a counterpart with heterogeneous sites; however, the repression efficiency varied, depending on the binding site location within the target promoter. In a second step, the top-performing 34S mutant/sgRNA/dCas9-repressor was used in combination with a Cre/loxP RNA scaffold orthogonal system to design a genetic switch, providing a versatile tool for modulation of gene expression. These results provide valuable perspectives on the utilization of 34S promoter modules in plant synthetic biology and the design of valuable CRISPRi genetic tools for precise modulation of transgene expression.},
}

*Promoter Regions, Genetic/genetics
Nicotiana/genetics
Binding Sites/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Synthetic Biology/methods
*Plant Cells/metabolism
*CRISPR-Cas Systems/genetics
Plants, Genetically Modified
Plant Leaves/genetics

@article {pmid40854654,
year = {2025},
author = {Gelaye, Y and Li, J and Luo, H},
title = {Exploring the role of Peanut (Arachis hypogaea L.) root architecture in enhancing adaptation to climate change for sustainable agriculture and resilient crop production: A review.},
journal = {Journal, genetic engineering & biotechnology},
volume = {23},
number = {3},
pages = {100535},
doi = {10.1016/j.jgeb.2025.100535},
pmid = {40854654},
issn = {2090-5920},
abstract = {Peanut (Arachis hypogaea L.) cultivation is increasingly vulnerable to climate change, with drought and heat stress emerging as major constraints to productivity and food security. This review explores the critical role of root architecture in enhancing peanut adaptation to environmental stressors, and evaluates current strategies and future directions for improving root traits through genetic, physiological, and agronomic approaches. Efficient root systems, characterized by deeper rooting and optimized xylem design, significantly improve water and nutrient acquisition under drought conditions. Key regulators such as abscisic acid (ABA), strigolactones, and specific root-related genes modulate root development and stress responses. Root exudates further enhance soil root interactions, while the peanut root microbiome contributes to nutrient cycling and resilience. Biotechnological tools, including quantitative trait loci (QTL) mapping and CRISPR/Cas-based genome editing, are being harnessed to manipulate root traits at the molecular level. Agronomic practices like mulching and cover cropping synergize with genetic improvements by enhancing soil structure and moisture retention. Strengthening peanut root architecture through the integration of modern breeding, biotechnological advances, and sustainable soil management offers a promising path toward climate-resilient peanut production. Future research should prioritize the convergence of these approaches, alongside microbiome exploration, to secure yield stability and food security in a changing climate.},
}

@article {pmid40854411,
year = {2025},
author = {Storz, U},
title = {The CRISPR Cas patent files, part 4: All back to zero, think again!.},
journal = {Journal of biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jbiotec.2025.08.001},
pmid = {40854411},
issn = {1873-4863},
abstract = {The epic patent disputes regarding CRISPR Cas9 have been keeping the Biotech community abuzz over the last 10 years. The unclear situation has created, and keeps creating, serious uncertainties among users of this groundbreaking technology. This article gives an overview of the dispute's history, and explains the actual state of the debate.},
}

@article {pmid40853412,
year = {2025},
author = {Iqbal, K and Mishra, A and Sreedharan, SM},
title = {The quintessence of algal biomass in bioplastic production: insightful advancement and sustainable use.},
journal = {Bioresources and bioprocessing},
volume = {12},
number = {1},
pages = {91},
pmid = {40853412},
issn = {2197-4365},
abstract = {Plastics are essential components of modern life, and their global demand is increasing daily. They are gaining recognition as a sustainable source for bioplastic production due to their rapid growth, carbon fixation ability, and capacity to utilize various waste streams. It seems that landfill, incineration, chemical treatment, and plastic recycling are not the best options for minimizing plastic pollution. A novel approach A new approach is needed to reduce this pollution. Bioplastics are biodegradable and come with less toxicity, a low carbon footprint, and are a better alternative to fossil-based plastics. This review explores recent advances in algal bioplastics, focusing on key polymers like polyhydroxyalkanoates (PHAs) and polylactic acid (PLA). Special attention is given to the use of genetic tools such as CRISPR-Cas systems to improve yield and carbon flux. Challenges related to downstream processing, low biomass productivity, and environmental variability are also discussed. This review highlights the importance of standardized life cycle assessments (LCAs) to evaluate environmental impact across the entire production chain. Additionally, regulatory frameworks from different countries are compared to identify gaps and promote progressive policy development. The review aims to provide an integrated perspective on the technical innovation, economic feasibility, and policy needed to support the future of algae-based bioplastics.},
}

@article {pmid40853001,
year = {2025},
author = {Ma, R and Zhong, H and Zhang, R},
title = {Prevalence, diversity, and parasitism of tailed prophages in Vibrio harveyi.},
journal = {mSphere},
volume = {},
number = {},
pages = {e0022825},
doi = {10.1128/msphere.00228-25},
pmid = {40853001},
issn = {2379-5042},
abstract = {Vibrio harveyi, a pathogenic bacterium, contains prophages that significantly influence its pathogenesis and evolutionary traits. Investigating the prevalence, evolution, and ecological roles of these prophages is of great importance as V. harveyi is responsible for luminous bacteriosis in aquatic organisms. In this study, 13 tailed prophages were identified from 55 globally sourced V. harveyi genomes, with prophage-bacterium junctions precisely annotated. These prophages exhibited distinct parasitic mechanisms, including Mu-type transposition, site-specific recombination, and a plasmid-like non-integrated state, reflecting their adaptive plasticity. Proteome-based phylogenetic analysis classified these prophages into eight subfamilies and nine genera, with half representing novel taxonomic singletons. Network analysis of V. harveyi prophages and a large set of prophages across Vibrio species revealed distinct prophage distribution patterns, including broad cross-species dissemination and clade-specific or strain-specific colonization. Further genomic analysis identified homologs of experimentally validated virulence factors associated with motility and biofilm formation, suggesting a potential role of these prophages in enhancing bacterial pathogenicity and adaptive fitness. CRISPR spacer matching provided the intra-species lytic history for 7 out of 13 identified prophages, underscoring their involvement in horizontal transfer of virulence traits. In summary, this study established a comprehensive genomic database of V. harveyi prophages, shedding light on their diversity, prevalence, and parasitic strategies.IMPORTANCEUnderstanding how prophages parasitize Vibrio harveyi holds significant commercial implications, given the pathogen's notoriety for inducing vibriosis across diverse aquatic species and causing substantial economic losses in the global aquaculture industry. We report here 13 well-curated prophage genomes identified from 55 globally collected V. harveyi genomes. Notably, these prophages exhibited previously unrecognized genomic diversity, along with distinct parasitic strategies and hierarchical distribution patterns. In-depth analysis of their genetic profiles identified multiple homologs of experimentally validated virulence determinants involved in regulating bacterial motility and biofilm formation. Lytic history was detected for over half of these prophages, suggesting their role in driving the dissemination of virulence traits within the species.},
}

@article {pmid40852733,
year = {2025},
author = {Brock, N and Kaur, N and Halford, NG},
title = {Advances in genome editing in plants within an evolving regulatory landscape, with a focus on its application in wheat breeding.},
journal = {Journal of plant biochemistry and biotechnology},
volume = {34},
number = {3},
pages = {599-614},
pmid = {40852733},
issn = {0971-7811},
abstract = {Population growth, diminishing resources and climate change are some of the many challenges that agriculture must address to satisfy the needs of the global population whilst ensuring the safety and nutritional value of our food. Wheat (Triticum aestivum) is tremendously important for human nutrition, providing starch (and, therefore, energy), fibre, protein, vitamins, and micronutrients. It is the second most widely grown crop behind maize (Zea mays), with 808 million tonnes of grain being produced in 2021-2022. In comparison, the production figure for 1961 was 222 million tonnes, and there have been similar increases for maize and rice (Oryza sativa). World population over the same period has increased from just over 3 billion to just over 8 billion, a stark reminder of just how important increased crop production has been in maintaining food security over that period, and for these cereals it has been achieved without additional land use. Plant breeding has played an important part in enabling crop production to keep increasing to meet demand and this will have to continue through the coming decades. Innovative technologies will play a part in that, and here we review how the new technology of genome editing is being applied in crop genetic improvement, with a focus on wheat. We cover oligonucleotide-directed mutagenesis and the use of site-directed nucleases, including meganucleases (MegNs), zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nucleases. We describe established genome editing strategies, mainly involving gene 'knockouts', and the new applications of base and prime editing using CRISPR/Cas. We also discuss how genome editing for crop improvement is developing in the context of an evolving regulatory landscape.},
}

@article {pmid40849498,
year = {2025},
author = {Feng, Y and Shi, J and Li, Z and Li, Y and Yang, J and Huang, S and Zheng, J and Han, W and Qiao, Y and Zhang, J and Liu, Q and Yang, Y and Hu, C and Wu, L and Zhang, X and Tang, J and Huang, X and Ma, P},
title = {Discovery of CRISPR-Cas12a clades using a large language model.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7877},
pmid = {40849498},
issn = {2041-1723},
support = {23HC1400700//Science and Technology Commission of Shanghai Municipality (Shanghai Municipal Science and Technology Commission)/ ; 32161133022//National Natural Science Foundation of China (National Science Foundation of China)/ ; 22177073//National Natural Science Foundation of China (National Science Foundation of China)/ ; 24141901302//Shanghai Science and Technology Development Foundation (Shanghai Science and Technology Development Fund)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry/classification ; *Bacterial Proteins/genetics/metabolism/chemistry ; Machine Learning ; *Endodeoxyribonucleases/genetics/metabolism/chemistry ; Polymorphism, Single Nucleotide ; Cryoelectron Microscopy ; Large Language Models ; },
abstract = {CRISPR-Cas systems revolutionize life science. Metagenomes contain millions of unknown Cas proteins. Traditional mining relies on protein sequence alignments. In this work, we employ an evolutionary scale language model (ESM) to learn the information beyond sequences. Trained with CRISPR-Cas data, ESM accurately identifies Cas proteins without alignment. Limited experimental data restricts feature prediction, but integrating with machine learning enables trans-cleavage activity prediction of uncharacterized Cas12a. We discover 7 undocumented Cas12a subtypes with unique CRISPR loci. Structural analyses reveal 8 subtypes of Cas1, Cas2, and Cas4. Cas12a subtypes display distinct 3D-folds. CryoEM analyses unveil unique RNA interactions with the uncharacterized Cas12a. These proteins show distinct double-strand and single-strand DNA cleavage preferences and broad PAM recognition. Finally, we establish a specific detection strategy for the oncogene SNP without traditional Cas12a PAM. This study highlights the potential of language models in exploring undocumented Cas protein function via gene cluster classification.},
}

*CRISPR-Cas Systems/genetics
*CRISPR-Associated Proteins/genetics/metabolism/chemistry/classification
*Bacterial Proteins/genetics/metabolism/chemistry
Machine Learning
*Endodeoxyribonucleases/genetics/metabolism/chemistry
Polymorphism, Single Nucleotide
Cryoelectron Microscopy
Large Language Models

@article {pmid40849322,
year = {2025},
author = {Lampe, GD and Liang, AR and Zhang, DJ and Fernández, IS and Sternberg, SH},
title = {Structure-guided engineering of type I-F CASTs for targeted gene insertion in human cells.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7891},
pmid = {40849322},
issn = {2041-1723},
support = {DP2HG011650//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {Humans ; *Transposases/genetics/metabolism/chemistry ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; DNA/metabolism/genetics/chemistry ; Cryoelectron Microscopy ; HEK293 Cells ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Protein Engineering/methods ; },
abstract = {Conventional genome editing tools rely on DNA double-strand breaks (DSBs) and host recombination proteins to achieve large insertions, resulting in heterogeneous mixtures of undesirable outcomes. We recently leveraged a type I-F CRISPR-associated transposase, PseCAST, for DSB-free DNA integration in human cells, albeit at low efficiencies; multiple lines of evidence suggest DNA binding may be a bottleneck for higher efficiencies. Here we report structural determinants of DNA recognition by the PseCAST QCascade complex using single-particle cryogenic electron microscopy (cryoEM), revealing subtype-specific interactions and RNA-DNA heteroduplex features. By combining structural data, library screens, and rationally engineered mutants, we uncover variants with increased integration efficiencies and modified PAM stringencies. We further leverage transpososome structural predictions to build hybrid CASTs that combine orthogonal DNA binding and integration modules. Our work provides unique structural insights into type I-F CASTs and showcases diverse strategies to investigate and engineer RNA-guided transposase architectures for human genome editing applications.},
}

Humans
*Transposases/genetics/metabolism/chemistry
*Gene Editing/methods
CRISPR-Cas Systems/genetics
DNA/metabolism/genetics/chemistry
Cryoelectron Microscopy
HEK293 Cells
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Protein Engineering/methods

@article {pmid40828659,
year = {2025},
author = {Wang, X and Chen, Z and Liu, C and Zhang, Z and Deng, Y and Tao, L and Tiedje, JM and Deng, J},
title = {Type I-F CRISPR-associated transposons contribute to genomic plasticity in Shewanella and mediate efficient programmable DNA integration.},
journal = {Microbial genomics},
volume = {11},
number = {8},
pages = {},
doi = {10.1099/mgen.0.001476},
pmid = {40828659},
issn = {2057-5858},
mesh = {*Shewanella/genetics ; *DNA Transposable Elements/genetics ; Genome, Bacterial ; *Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; },
abstract = {The genome plasticity of species and strains in the genus Shewanella is closely associated with the diverse mobile genetic elements embedded in its genomes. One mobile element with potential for accurate and efficient DNA insertion in Shewanella is the type I-F3 CRISPR-associated transposon (I-F3 CAST). However, relatively little is known about the distribution and ecological significance of I-F3 CASTs and whether they could be suitable as a tool for targeted genetic manipulation in situ. To better understand the distribution of I-F3 CASTs in Shewanella, we analysed 602 Shewanella genomes. We found that I-F3 CASTs were present in 12% of all genomes, although differences in both gene arrangement and integration locus were observed. These Shewanella I-F3 CASTs carried up to 89 cargo genes, which were associated with diverse functions, including defence, resistance and electron transfer, demonstrating an important role in genomic diversification and ecological adaptation. We tested whether the I-F3 CAST present in Shewanella sp. ANA-3 enhanced gene insertion, both in situ and in a heterologous host. We observed I-F3 CAST-mediated crRNA-targeted integration of the supplied genes into the pyrF locus in Shewanella sp. ANA-3. Heterologous gene insertion with high integration efficiency in Escherichia coli was also demonstrated using a simplified version of ANA-3 I-F3 CAST. Altogether, this work highlights the important role of I-F3 CASTs in promoting genomic plasticity of the Shewanella genus and demonstrates the gene-editing capability of ANA-3-CAST both endogenously and heterologously.},
}

*Shewanella/genetics
*DNA Transposable Elements/genetics
Genome, Bacterial
*Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Cas Systems

@article {pmid40828287,
year = {2025},
author = {Dave, S and Patel, C and Heu, C and Macias, VM},
title = {Adapting and Testing ReMOT Control for Expanded CRISPR-Era Genome Functions in Non-model Insects.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2935},
number = {},
pages = {385-413},
pmid = {40828287},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; Female ; Oocytes/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Drosophila Proteins/genetics/metabolism ; Egg Proteins/genetics/metabolism ; Gene Knockdown Techniques/methods ; *Genome, Insect ; Microinjections ; },
abstract = {The movement of the Drosophila yolk protein (DmYP) across the mosquito oocyte membrane was both fortuitous and puzzling; the cells that become future offspring--oocytes--are closed off to molecules that are not specifically recognized by a receptor, but there is no obvious ortholog of the yolk protein/receptor for DmYP in mosquitoes. Nonetheless, a small fragment of DmYP was sufficient to move the massive ribonucleoprotein complex of Cas9 and a guide RNA from the open circulatory system of a female mosquito into the mosquito oocyte for targeting of the germline DNA and heritable mutation. This procedure, known as ReMOT Control, is a robust method for CRISPR/Cas9-mediated gene knockdown that has been adapted for many orders of insects, for ticks, and even for several species of crustacean by first identifying a suitable peptide for oocyte uptake, then expressing Cas9 as a fusion protein with the peptide and finally performing adult injections with expressed, purified protein and guide RNA against a gene with a visible marker phenotype. In order to support the adaptation of this procedure widely among entomologists, herein, we provide the protocols to: (a) Identify a suitable peptide for any insect by identifying the receptor-binding region of vitellogenin. (b) Clone a nucleotide coding the peptide and a fluorescent protein into the commercially available Addgene plasmid pET28a/Cas9-cys to generate a fusion-protein encoding gene. (c) Express a fusion protein for specific delivery of ReMOT Cas9 to the ovaries of an insect of interest. (d) Adapt a generalized procedure for adult injection of insects targeting the hemolymph and detecting ovary translocation and heritable gene editing.},
}

Animals
*CRISPR-Cas Systems
Female
Oocytes/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Editing/methods
Drosophila Proteins/genetics/metabolism
Egg Proteins/genetics/metabolism
Gene Knockdown Techniques/methods
*Genome, Insect
Microinjections

@article {pmid40824497,
year = {2025},
author = {Ji, W and Yuan, L and Hu, T and Jiang, M and Shen, J},
title = {Establishment of a rapid Bordetella pertussis detection method based on RPA-CRISPR-Cas12a technology.},
journal = {Archives of microbiology},
volume = {207},
number = {9},
pages = {226},
pmid = {40824497},
issn = {1432-072X},
support = {2024zh-06//Anhui Institute of Translational Medicine/ ; 2021xkj061//the Youth Science Foundation of Anhui Medical University/ ; 2023AH050568//he Key Project of Natural Science in Colleges and Universities of Anhui Province/ ; },
mesh = {*Bordetella pertussis/genetics/isolation & purification ; Humans ; *Whooping Cough/diagnosis/microbiology ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; Recombinases/genetics/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Whooping cough (pertussis) is an acute respiratory infectious disease caused by Bordetella pertussis (BP). It poses a risk to infants and young children. This investigation aimed to construct a simple, rapid, and accurate diagnostic protocol for BP detection that does not depend on complex equipment or large-scale instruments. This study combines Recombinase Polymerase Amplification (RPA) technology with the CRISPR/Cas12a system, utilizing immunochromatographic lateral flow strips (ILFS) test and fluorescence curves to observe data. This diagnostic strategy does not require complex equipment used in traditional diagnostic approaches (such as bacterial culture, pathogen detection, and molecular biology techniques), which has increased its accessibility and ease of use. The validation data indicate that the RPA-CRISPR/Cas12a-ILFS and RPA-CRISPR/Cas12a fluorescence detection analyses had a lower detection threshold of 10[2] copies/µL and did not cross-react with other prevalent infections. Furthermore, 40 clinical samples were evaluated and compared via qPCR, which revealed that the RPA-CRISPR/Cas12a method has 100% sensitivity and specificity. In addition, the RPA-CRISPR/Cas12a diagnostic platform showed significant potential for clinical application, specifically when resources are limited, enabling point-of-care testing. This platform's simplicity, accuracy, and efficiency make it a powerful tool for pertussis diagnosis, which can improve patient care and public health outcomes.},
}

*Bordetella pertussis/genetics/isolation & purification
Humans
*Whooping Cough/diagnosis/microbiology
*CRISPR-Cas Systems
Sensitivity and Specificity
*Nucleic Acid Amplification Techniques/methods
Recombinases/genetics/metabolism
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40814228,
year = {2025},
author = {Jiang, Y and Li, B and Xiong, J and Liu, X},
title = {Graph-CRISPR: a gene editing efficiency prediction model based on graph neural network with integrated sequence and secondary structure feature extraction.},
journal = {Briefings in bioinformatics},
volume = {26},
number = {4},
pages = {},
pmid = {40814228},
issn = {1477-4054},
support = {12472248//National Natural Science Foundation of China/ ; FRF-IDRY-24-024//The Fundamental Research Funds for the Central Universities/ ; GJJ2022-18//The Beijing Advanced Innovation Center for Materials Genome Engineering/ ; },
mesh = {*Gene Editing/methods ; *Neural Networks, Computer ; *CRISPR-Cas Systems ; Nucleic Acid Conformation ; Computational Biology/methods ; RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Graph Neural Networks ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) gene-editing technology has transformed molecular biology. Predicting editing efficiency is crucial for optimization, and numerous computational models have been created. However, many current models struggle to generalize across diverse editing systems, often experiencing performance drops with varying conditions or systems. Additionally, most models focus on ribonucleic acid (RNA) sequence and thermodynamic features, overlooking the importance of secondary structure information. Here, we present the first graph-based model (Graph-CRISPR) that integrates both sequence and secondary structure features of single guide RNA enhancing editing efficiency prediction. Tests show Graph-CRISPR consistently surpasses baseline models across systems like CRISPR-Cas9, prime editing, and base editing. It also demonstrates strong resilience, maintaining robust performance under varying experimental conditions. This work highlights the potential of integrating sequence and structural information through graph-based modeling to enhance predictive accuracy and adaptability in gene editing applications. The datasets and source codes are publicly available at: https://github.com/MoonLBH/Graph-CRISPR.},
}

*Gene Editing/methods
*Neural Networks, Computer
*CRISPR-Cas Systems
Nucleic Acid Conformation
Computational Biology/methods
RNA, Guide, CRISPR-Cas Systems/genetics/chemistry
*Clustered Regularly Interspaced Short Palindromic Repeats
Humans
Graph Neural Networks

@article {pmid40811470,
year = {2025},
author = {Ogo, Y and Kawauchi, T and Mimura, M and Naito, K and Itoh, H and Izawa, T},
title = {A 65-kb deletion survey identifies a distal cis-regulatory region for red-light induction of Ghd7, a key rice floral repressor.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {33},
pages = {e2423119122},
pmid = {40811470},
issn = {1091-6490},
support = {JP17H06246//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP20K22585//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP22H05180//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP22H05172//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP22H00367//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; RGP0011/2019//Human Frontier Science Program (HFSP)/ ; },
mesh = {*Oryza/genetics/metabolism/radiation effects/growth & development ; *Gene Expression Regulation, Plant/radiation effects ; *Flowers/genetics/metabolism/radiation effects/growth & development ; Light ; *Plant Proteins/genetics/metabolism ; Circadian Rhythm/genetics ; Sequence Deletion ; CRISPR-Cas Systems ; Regulatory Sequences, Nucleic Acid ; },
abstract = {The Ghd7 (Grain number, plant height, and heading date 7) gene integrates red light signals and circadian rhythms to control floral repression under long-day conditions in rice. CRISPR/Cas9 systems were employed to create a series of deletion mutant lines in the upstream regions of Ghd7, covering a 65-kb genomic region from its transcription start site (TSS). These deletions ranged from 2 to 25 kb in size. Three deletion lines, those from 0 to -3 kb (0/-3 K), -20 to -40 kb (-20/-40 K), and -26 to -30 kb (-26/-30 K) from the TSS, resulted in early flowering, similar to Ghd7 knockout lines. The -20/-40 and -26/-30 K lines exhibited a loss of acute Ghd7 morning induction. Night-break experiments consistently supported these findings, suggesting that the key cis-regulatory region for red light responses was located within the 3.7-kb region in -26/-30 K. In seedlings of the 0/-3 K deletion line, which retains the -29 to -86 bp region, Ghd7 showed a diurnal pattern similar to wild type. This suggests that the deleted region in 0/-3 K is dispensable for both circadian rhythms and red-light responses. Further analyses of two deletion lines within the -26/-30 K region allowed us to narrow down the core cis-regulatory elements, responsive to morning-light signals, within a 228-bp segment located at 28-kb upstream of the TSS in Ghd7.},
}

*Oryza/genetics/metabolism/radiation effects/growth & development
*Gene Expression Regulation, Plant/radiation effects
*Flowers/genetics/metabolism/radiation effects/growth & development
Light
*Plant Proteins/genetics/metabolism
Circadian Rhythm/genetics
Sequence Deletion
CRISPR-Cas Systems
Regulatory Sequences, Nucleic Acid

@article {pmid40809520,
year = {2025},
author = {Ji, T and Fang, X and Gao, Y and Yu, K and Gao, X},
title = {Research progress on the application of RPA-CRISPR/Cas12a in the rapid visual detection of pathogenic microorganisms.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1640938},
pmid = {40809520},
issn = {2235-2988},
mesh = {*CRISPR-Cas Systems ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; *Recombinases/metabolism ; *Bacteria/genetics/isolation & purification ; Point-of-Care Testing ; Sensitivity and Specificity ; Point-of-Care Systems ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {In an increasingly complex global public health landscape, the continuous emergence of novel pathogens and the growing problem of antibiotic resistance highlight the urgent need for rapid, efficient, and precise detection technologies for pathogenic microorganisms. The innovative combination of Recombinase Polymerase Amplification (RPA) and CRISPR/Cas12a enables the rapid amplification of target gene fragments under isothermal conditions and the precise recognition and cleavage of specific nucleic acid sequences. The integration of RPA and CRISPR/Cas12a significantly enhances the sensitivity and accuracy of detection simplifies operational procedures, and reduces the dependence on specialized equipment for testing personnel. This combination demonstrates great potential for application in clinical diagnostics and point-of-care testing. This article provides a detailed overview of the principles of RPA-CRISPR/Cas12a and its latest research progress in the field of pathogen detection, aiming to promote the widespread application of RPA-CRISPR/Cas12a technology in clinical medicine and public health and to offer theoretical support for its further optimization.},
}

*CRISPR-Cas Systems
Humans
*Nucleic Acid Amplification Techniques/methods
*Molecular Diagnostic Techniques/methods
*Recombinases/metabolism
*Bacteria/genetics/isolation & purification
Point-of-Care Testing
Sensitivity and Specificity
Point-of-Care Systems
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40806519,
year = {2025},
author = {Gao, G and Zhang, L and Tong, P and Yan, G and Wu, X},
title = {Enhancing Oil Content in Oilseed Crops: Genetic Insights, Molecular Mechanisms, and Breeding Approaches.},
journal = {International journal of molecular sciences},
volume = {26},
number = {15},
pages = {},
pmid = {40806519},
issn = {1422-0067},
mesh = {*Plant Breeding/methods ; Quantitative Trait Loci ; *Crops, Agricultural/genetics/metabolism ; *Plant Oils/metabolism ; Gene Editing ; *Seeds/genetics/metabolism ; Genome-Wide Association Study ; CRISPR-Cas Systems ; },
abstract = {Vegetable oils are essential for human nutrition and industrial applications. With growing global demand, increasing oil content in oilseed crops has become a top priority. This review synthesizes recent progress in understanding the genetic, environmental, and molecular mechanisms regulating oil content, and presents biotechnological strategies to enhance oil accumulation in major oilseed crops. Oil biosynthesis is governed by intricate genetic-environmental interactions. Environmental factors and agronomic practices significantly impact oil accumulation dynamics. Quantitative trait loci (QTL) mapping and genome-wide association studies (GWAS) have identified key loci and candidate genes involved in lipid biosynthesis pathways. Transcription factors and epigenetic regulators further fine-tune oil accumulation. Biotechnological approaches, including marker-assisted selection (MAS) and CRISPR/Cas9-mediated genome editing, have successfully generated high-oil-content variants. Future research should integrate multi-omics data, leverage AI-based predictive breeding, and apply precision genome editing to optimize oil yield while maintaining seed quality. This review provides critical references for the genetic improvement and breeding of high- and ultra-high-oil-content varieties in oilseed crops.},
}

*Plant Breeding/methods
Quantitative Trait Loci
*Crops, Agricultural/genetics/metabolism
*Plant Oils/metabolism
Gene Editing
*Seeds/genetics/metabolism
Genome-Wide Association Study
CRISPR-Cas Systems

@article {pmid40806492,
year = {2025},
author = {Șerban, M and Toader, C and Covache-Busuioc, RA},
title = {Precision Neuro-Oncology in Glioblastoma: AI-Guided CRISPR Editing and Real-Time Multi-Omics for Genomic Brain Surgery.},
journal = {International journal of molecular sciences},
volume = {26},
number = {15},
pages = {},
pmid = {40806492},
issn = {1422-0067},
mesh = {Humans ; *Glioblastoma/genetics/surgery ; *Brain Neoplasms/genetics/surgery ; *Precision Medicine/methods ; *Genomics/methods ; *CRISPR-Cas Systems ; *Artificial Intelligence ; *Gene Editing/methods ; Proteomics/methods ; Metabolomics/methods ; Multiomics ; },
abstract = {Precision neurosurgery is rapidly evolving as a medical specialty by merging genomic medicine, multi-omics technologies, and artificial intelligence (AI) technology, while at the same time, society is shifting away from the traditional, anatomic model of care to consider a more precise, molecular model of care. The general purpose of this review is to contemporaneously reflect on how these advances will impact neurosurgical care by providing us with more precise diagnostic and treatment pathways. We hope to provide a relevant review of the recent advances in genomics and multi-omics in the context of clinical practice and highlight their transformational opportunities in the existing models of care, where improved molecular insights can support improvements in clinical care. More specifically, we will highlight how genomic profiling, CRISPR-Cas9, and multi-omics platforms (genomics, transcriptomics, proteomics, and metabolomics) are increasing our understanding of central nervous system (CNS) disorders. Achievements obtained with transformational technologies such as single-cell RNA sequencing and intraoperative mass spectrometry are exemplary of the molecular diagnostic possibilities in real-time molecular diagnostics to enable a more directed approach in surgical options. We will also explore how identifying specific biomarkers (e.g., IDH mutations and MGMT promoter methylation) became a tipping point in the care of glioblastoma and allowed for the establishment of a new taxonomy of tumors that became applicable for surgeons, where a change in practice enjoined a different surgical resection approach and subsequently stratified the adjuvant therapies undertaken after surgery. Furthermore, we reflect on how the novel genomic characterization of mutations like DEPDC5 and SCN1A transformed the pre-surgery selection of surgical candidates for refractory epilepsy when conventional imaging did not define an epileptogenic zone, thus reducing resective surgery occurring in clinical practice. While we are atop the crest of an exciting wave of advances, we recognize that we also must be diligent about the challenges we must navigate to implement genomic medicine in neurosurgery-including ethical and technical challenges that could arise when genomic mutation-based therapies require the concurrent application of multi-omics data collection to be realized in practice for the benefit of patients, as well as the constraints from the blood-brain barrier. The primary challenges also relate to the possible gene privacy implications around genomic medicine and equitable access to technology-based alternative practice disrupting interventions. We hope the contribution from this review will not just be situational consolidation and integration of knowledge but also a stimulus for new lines of research and clinical practice. We also hope to stimulate mindful discussions about future possibilities for conscientious and sustainable progress in our evolution toward a genomic model of precision neurosurgery. In the spirit of providing a critical perspective, we hope that we are also adding to the larger opportunity to embed molecular precision into neuroscience care, striving to promote better practice and better outcomes for patients in a global sense.},
}

Humans
*Glioblastoma/genetics/surgery
*Brain Neoplasms/genetics/surgery
*Precision Medicine/methods
*Genomics/methods
*CRISPR-Cas Systems
*Artificial Intelligence
*Gene Editing/methods
Proteomics/methods
Metabolomics/methods
Multiomics

@article {pmid40806337,
year = {2025},
author = {Carullo, N and Haellman, V and Gutbier, S and Schlicht, S and Nguyen, TT and Blum Marti, R and Hartz, P and Lindemann, L and Schukur, L},
title = {Development and Characterization of a Novel α-Synuclein-PEST H4 Cell Line for Enhanced Drug Screening in α-Synucleinopathies.},
journal = {International journal of molecular sciences},
volume = {26},
number = {15},
pages = {},
pmid = {40806337},
issn = {1422-0067},
support = {71095//Roche Pharma AG (Germany)/ ; },
mesh = {*alpha-Synuclein/metabolism/genetics ; Humans ; Drug Evaluation, Preclinical/methods ; *Synucleinopathies/metabolism/drug therapy/genetics ; Cell Line ; Proteolysis ; CRISPR-Cas Systems ; Parkinson Disease/metabolism/drug therapy ; Half-Life ; },
abstract = {Alpha-Synuclein (α-Syn) is a presynaptic neuronal protein implicated in the pathogenesis of Parkinson's disease (PD) and other synucleinopathies, primarily through its aggregation into insoluble fibrils. The extended α-Syn half-life necessitates treatment durations that are incompatible with efficient high-throughput drug screening, can risk compound stability or cause cellular toxicity. To address this, we inserted a PEST sequence, a motif known to promote rapid protein degradation, at the C-terminus of the SNCA gene using CRISPR/Cas9 to create a novel cell line with reduced α-Syn half-life. This modification accelerates α-Syn turnover, providing a robust model for studying α-Syn dynamics and offering a platform that is applicable to other long-lived proteins. Our results demonstrate a six-fold reduction in α-Syn half-life, enabling the rapid detection of changes in protein levels and facilitating the identification of molecules that modulate α-Syn production and degradation pathways. Using inhibitors of the proteasome, transcription, and translation further validated the model's utility in examining various mechanisms that impact protein levels. This novel cell line represents a significant advancement for studying α-Syn dynamics and offers promising avenues to develop therapeutics for α-synucleinopathies. Future research should focus on validating this model in diverse experimental settings and exploring its potential in high-throughput screening applications.},
}

*alpha-Synuclein/metabolism/genetics
Humans
Drug Evaluation, Preclinical/methods
*Synucleinopathies/metabolism/drug therapy/genetics
Cell Line
Proteolysis
CRISPR-Cas Systems
Parkinson Disease/metabolism/drug therapy
Half-Life

@article {pmid40806299,
year = {2025},
author = {Citterio, CE and Morales-Rodriguez, B and Liao, XH and Vu, C and Nguyen, R and Tsai, J and Le, J and Metawea, I and Liu, M and Olson, DP and Refetoff, S and Arvan, P},
title = {A Genetically-Engineered Thyroid Gland Built for Selective Triiodothyronine Secretion.},
journal = {International journal of molecular sciences},
volume = {26},
number = {15},
pages = {},
pmid = {40806299},
issn = {1422-0067},
support = {K01 DK125448/DK/NIDDK NIH HHS/United States ; R01 DK132017/DK/NIDDK NIH HHS/United States ; DK125448 (to C.E.C.)/NH/NIH HHS/United States ; R01-DK132017 (to P. A.)/NH/NIH HHS/United States ; },
mesh = {Animals ; *Triiodothyronine/metabolism ; Mice ; *Thyroid Gland/metabolism ; CRISPR-Cas Systems ; Gene Knock-In Techniques ; Thyroglobulin/genetics/metabolism ; Thyroxine/metabolism ; Hypothyroidism/genetics/metabolism ; Thyrotropin/metabolism ; *Genetic Engineering/methods ; Humans ; },
abstract = {Thyroid hormones (thyroxine, T4, and triiodothyronine, T3) are indispensable for sustaining vertebrate life, and their deficiency gives rise to a wide range of symptoms characteristic of hypothyroidism, affecting 5-10% of the world's population. The precursor for thyroid hormone synthesis is thyroglobulin (Tg), a large iodoglycoprotein consisting of upstream regions I-II-III (responsible for synthesis of most T4) and the C-terminal CholinEsterase-Like (ChEL) domain (responsible for synthesis of most T3, which can also be generated extrathyroidally by T4 deiodination). Using CRISPR/Cas9-mediated mutagenesis, we engineered a knock-in of secretory ChEL into the endogenous TG locus. Secretory ChEL acquires Golgi-type glycans and is properly delivered to the thyroid follicle lumen, where T3 is first formed. Homozygous knock-in mice are capable of thyroidal T3 synthesis but largely incompetent for T4 synthesis such that T4-to-T3 conversion contributes little. Instead, T3 production is regulated thyroidally by thyrotropin (TSH). Compared to cog/cog mice with conventional hypothyroidism (low serum T4 and T3), the body size of ChEL-knock-in mice is larger; although, these animals with profound T4 deficiency did exhibit a marked elevation of serum TSH and a large goiter, despite normal circulating T3 levels. ChEL knock-in mice exhibited a normal expression of hepatic markers of thyroid hormone action but impaired locomotor activities and increased anxiety-like behavior, highlighting tissue-specific differences in T3 versus T4 action, reflecting key considerations in patients receiving thyroid hormone replacement therapy.},
}

Animals
*Triiodothyronine/metabolism
Mice
*Thyroid Gland/metabolism
CRISPR-Cas Systems
Gene Knock-In Techniques
Thyroglobulin/genetics/metabolism
Thyroxine/metabolism
Hypothyroidism/genetics/metabolism
Thyrotropin/metabolism
*Genetic Engineering/methods
Humans

@article {pmid40804581,
year = {2025},
author = {Li, H and Wang, G and Wu, X and Ying, X and Luo, Z and Lu, C and Zhang, P},
title = {Rapid and Specific Visual Detection of Amanita fuliginea by Combining Recombinase Polymerase Amplification with CRISPR/Cas12b and Lateral Flow Strip.},
journal = {Current microbiology},
volume = {82},
number = {10},
pages = {450},
pmid = {40804581},
issn = {1432-0991},
support = {No. 31670015//National Natural Science Foundation of China/ ; No. 31750001//National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Recombinases/metabolism/genetics ; *Amanita/genetics/isolation & purification/classification ; Sensitivity and Specificity ; },
abstract = {Amanita fuliginea is a common lethal mushroom with high mortality rates due to its ease of ingestion and concealed early symptoms. Rapid and specific identification of this species is crucial for clinical diagnosis and treatment. However, the existing rapid detection methods for nucleic acids of A. fuliginea were still plagued by specificity issues. Herein, we developed a recombinase polymerase amplification (RPA) coupled with CRISPR/Cas12b and lateral flow strip (LFS) assay for the rapid and specific detection of A. fuliginea. The RPA-CRISPR/Cas12b-LFS assay provides visual results within 35 min and does not rely on expensive equipment. High specificity was demonstrated against other Amanita species and non-Amanita species, with a sensitivity of 31 pg of genomic DNA per reaction. In conclusion, the RPA-CRISPR/Cas12b-LFS assay presents a rapid, specific, sensitive, and convenient tool for identifying A. fuliginea, particularly suitable for primary healthcare institutions or remote areas.},
}

*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
Recombinases/metabolism/genetics
*Amanita/genetics/isolation & purification/classification
Sensitivity and Specificity

@article {pmid40796566,
year = {2025},
author = {Saha, S and Haynes, WJ and Seo, J and Del Rio, NM and Young, EE and Zhang, J and Holm, AM and Pimentel, M and Flannagan, L and Huang, L and Blashka, W and Murphy, L and Scholz, MJ and Henrichs, A and Suresh Babu, J and Steill, J and Kratz, J and Stewart, R and Kamp, TJ and Brown, ME},
title = {Diminished immune cell adhesion in hypoimmune ICAM-1 knockout human pluripotent stem cells.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7415},
pmid = {40796566},
issn = {2041-1723},
support = {T32 GM081061/GM/NIGMS NIH HHS/United States ; T32 DC009401/DC/NIDCD NIH HHS/United States ; U01 HL134764/HL/NHLBI NIH HHS/United States ; P30 CA014520/CA/NCI NIH HHS/United States ; U01HL134764//U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; R56HL165189//U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; 75N93021C00004/AI/NIAID NIH HHS/United States ; P50 HD105353/HD/NICHD NIH HHS/United States ; R56 HL165189/HL/NHLBI NIH HHS/United States ; },
mesh = {*Intercellular Adhesion Molecule-1/genetics/immunology/metabolism ; Humans ; Animals ; Mice ; Cell Adhesion/immunology/genetics ; *Pluripotent Stem Cells/immunology/metabolism ; CRISPR-Cas Systems ; Gene Editing ; Gene Knockout Techniques ; T-Lymphocytes/immunology ; Immunity, Innate ; Graft Rejection/immunology ; Cell Proliferation ; },
abstract = {Gene edited human pluripotent stem cells are a promising platform for developing reparative cellular therapies that evade immune rejection. Existing first-generation hypoimmune strategies have used CRISPR/Cas9 editing to modulate genes associated with adaptive immune responses, but have largely not addressed the innate immune cells, such as neutrophils, that mediate inflammation and rejection processes occurring early after graft transplantation. We identify the adhesion molecule ICAM-1 as a hypoimmune target that plays multiple critical roles in both adaptive and innate immune responses post-transplantation. In our experiments, we find that ICAM-1 blocking or knockout in human pluripotent stem cell-derived cardiovascular therapies imparts significantly diminished binding of multiple immune cell types. ICAM-1 knockout results in diminished T cell proliferation and activation responses in vitro and in longer in vivo retention/protection of knockout grafts following immune cell encounter in NeoThy humanized mice. We also introduce the ICAM-1 knockout edit into existing first-generation hypoimmune human pluripotent stem cells and prevent immune cell binding. This promising hypoimmune editing strategy has the potential to improve transplantation outcomes for regenerative therapies in the setting of cardiovascular pathologies and several other diseases.},
}

*Intercellular Adhesion Molecule-1/genetics/immunology/metabolism
Humans
Animals
Mice
Cell Adhesion/immunology/genetics
*Pluripotent Stem Cells/immunology/metabolism
CRISPR-Cas Systems
Gene Editing
Gene Knockout Techniques
T-Lymphocytes/immunology
Immunity, Innate
Graft Rejection/immunology
Cell Proliferation

@article {pmid40778406,
year = {2025},
author = {Yuan, X and Xu, Y and Zhang, G and Wang, Y and Jin, X},
title = {CRISPR/Cas12a-Mediated Electrochemical Aptasensor for Simultaneous Determination of Alzheimer's Disease Biomarkers in Human Blood.},
journal = {Analytical chemistry},
volume = {97},
number = {32},
pages = {17715-17724},
doi = {10.1021/acs.analchem.5c03015},
pmid = {40778406},
issn = {1520-6882},
mesh = {Humans ; *Alzheimer Disease/blood/diagnosis ; *Amyloid beta-Peptides/blood ; *Electrochemical Techniques/methods ; *Aptamers, Nucleotide/chemistry/genetics ; Biomarkers/blood ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Peptide Fragments/blood ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Alzheimer's disease (AD) is an irreversible and progressive neurodegenerative disease, and the ratio of 40 and 42 residue amyloid beta peptides (Aβ40 and Aβ42) plays a crucial role as a biomarker for the early diagnosis of AD. Conventional laboratory-based assays for Aβ proteins analysis typically relies on the collection of cerebrospinal fluid (CSF). In contrast, blood samples offer a less invasive alternative but present challenges due to lower concentrations of Aβ. This study presents a novel strategy that combines aptamer-specific recognition with CRISPR/Cas12a-mediated signal amplification in an electrochemical sensing platform. The proposed electrochemical sensing platform achieved reliable and accurate results when applied to clinical serum samples, validating its effectiveness in practical scenarios. This approach enables highly specific detection of Aβ40 and Aβ42 with detection limits as low as 1.1 and 0.8 pg/mL, respectively. Moreover, the platform demonstrated outstanding repeatability and operational stability, underscoring its potential as a robust and highly sensitive diagnostic tool for the early detection of AD.},
}

Humans
*Alzheimer Disease/blood/diagnosis
*Amyloid beta-Peptides/blood
*Electrochemical Techniques/methods
*Aptamers, Nucleotide/chemistry/genetics
Biomarkers/blood
*Biosensing Techniques/methods
*CRISPR-Cas Systems
*Peptide Fragments/blood
Limit of Detection
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40774925,
year = {2025},
author = {Zhu, L and Hu, Q and Wang, Z and Lin, JM and Zhao, RS},
title = {CRISPR/Cas12a-Mediated Rolling Circle Amplification for the Development of Liquid Crystal-Based Sensors.},
journal = {Analytical chemistry},
volume = {97},
number = {32},
pages = {17825-17832},
doi = {10.1021/acs.analchem.5c03500},
pmid = {40774925},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems ; *Liquid Crystals/chemistry ; *Nucleic Acid Amplification Techniques/methods ; *Aflatoxin B1/analysis ; *Biosensing Techniques/methods ; Aptamers, Nucleotide/chemistry ; Limit of Detection ; Food Contamination/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The development of high-performance liquid crystal (LC)-based sensors with remarkable sensitivity and excellent selectivity is of great importance. Herein, a CRISPR/Cas12a-based LC sensor for detecting mycotoxins in food is first reported, and the detection of aflatoxin B1 (AFB1) is chosen as a model. AFB1 is added to magnetic beads (MBs) functionalized with double-stranded DNA (dsDNA) consisting of AFB1 aptamer and cDNA. As the AFB1 aptamer specifically recognizes AFB1, cDNA is released and activates the CRISPR/Cas12a system to cut ligation DNA, thereby preventing the initiation of the rolling circle amplification (RCA) reaction. As the long-chain single-stranded DNA (ssDNA) cannot be produced to capture myristoylcholine (Myr) in the aqueous solution, a dark LC image is obtained because a Myr monolayer forms at the aqueous/LC interface. In contrast, when the RCA reaction is initiated in the absence of AFB1, Myr in the aqueous solution is captured by long-chain ssDNA generated from the RCA reaction, causing a bright LC image. Notably, the RCA reaction on MBs exponentially amplifies the CRISPR/Cas12a-generated signals, resulting in enhanced sensitivity. The limit of detection (LOD) is about 0.312 ng/mL. Furthermore, the selectivity is greatly enhanced due to the introduction of the AFB1 aptamer and MBs. Furthermore, a portable device is developed for rapid onsite detection. Therefore, the study provides a sensitive, selective, convenient, and promising assay for detecting mycotoxins in food.},
}

*CRISPR-Cas Systems
*Liquid Crystals/chemistry
*Nucleic Acid Amplification Techniques/methods
*Aflatoxin B1/analysis
*Biosensing Techniques/methods
Aptamers, Nucleotide/chemistry
Limit of Detection
Food Contamination/analysis
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40749399,
year = {2025},
author = {Yan, B and Wei, C and Lei, X and Ding, L and Yu, S},
title = {cliCRISPR: crRNA-limited CRISPR/Cas12a system for multiplexed detection.},
journal = {Biosensors & bioelectronics},
volume = {288},
number = {},
pages = {117834},
doi = {10.1016/j.bios.2025.117834},
pmid = {40749399},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Humans ; *RNA/genetics/analysis ; Genotype ; Genotyping Techniques ; Spectrometry, Fluorescence ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The CRISPR/Cas12a system is extensively employed for nucleic acid detection, where crRNA is typically administered in excess to quantify target levels. However, its non-specific trans-cleavage activity poses challenges for achieving multiplexed detection within a single-pot CRISPR/Cas12a assay. In this study, we introduce a crRNA-limited strategy for multiplexed detection based on the CRISPR/Cas12a system, termed cliCRISPR. This approach correlates fluorescence intensity with crRNA concentration rather than target concentration. By precisely controlling crRNA concentrations, distinct low, medium, and high fluorescence intensities can be observed for multiple targets. As proof of concept, distinguishable fluorescence intensities for wild-type, mutant, and heterozygous genotypes of rs4646536 which is associated with vitamin D deficiency could be obtained with 10 nM crRNA1 and 3 nM crRNA2. Subsequently, a logic-gate strategy is utilized for rs4646536 genotyping by comparing fluorescence intensity slope with the cut-off values (V0 = 25.96, V1 = 40.16 and V2 = 1815.56). To validate the practicability, the proposed cliCRISPR method was applied to genotype rs4646536 for 10 samples from 2 families. The results were consistent with those obtained using TaqMan qPCR and adhered to Mendel's laws of inheritance. Therefore, cliCRISPR demonstrates potential for developing multiplexed biosensors based on the CRISPR/Cas12a system.},
}

*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
Humans
*RNA/genetics/analysis
Genotype
Genotyping Techniques
Spectrometry, Fluorescence
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40749158,
year = {2025},
author = {Baek, M and Kweon, S and Kim, Y and Lewis, NE and Lee, JS and Lee, GM},
title = {Recombinase-Mediated Cassette Exchange-Based CRISPR Activation Screening Identifies Hyperosmotic Stress-Resistant Genes in Chinese Hamster Ovary Cells.},
journal = {ACS synthetic biology},
volume = {14},
number = {8},
pages = {3116-3126},
doi = {10.1021/acssynbio.5c00268},
pmid = {40749158},
issn = {2161-5063},
mesh = {Animals ; CHO Cells ; Cricetulus ; *CRISPR-Cas Systems/genetics ; *Osmotic Pressure ; *Recombinases/metabolism/genetics ; Cell Proliferation/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cricetinae ; },
abstract = {Chinese hamster ovary (CHO) cells are ubiquitously used for therapeutic protein production. However, fed-batch culture, typically used for large-scale production, often induces hyperosmotic stress, negatively impacting cell growth and productivity. To identify genes conferring resistance to hyperosmotic stress, we performed genome-wide CRISPRa screening in bispecific antibody (bsAb)-producing CHO (CHO-bsAb) cells. Using a virus-free recombinase-mediated cassette exchange (RMCE) system, we established a CRISPRa library and cultured cells in standard and hyperosmolar media. Next-generation sequencing identified 122 significantly enriched and 171 significantly depleted genes under hyperosmolar conditions, with functional enrichment analysis highlighting pathways related to cell proliferation and transcriptional regulation. Among the enriched genes, CRISPRa-based activation of 24 candidates demonstrated that 23 improved cell growth under hyperosmolar conditions. Notably, stable expression of Siah2 or C2cd4a significantly enhanced cell growth, and optimizing their expression levels increased bsAb production by up to 1.3-fold. Additional knockout of Zfr, previously identified in CRISPR knockout screening, further improved cell growth and bsAb production, demonstrating the synergistic benefits of integrating CRISPR knockout and CRISPRa approaches. Thus, CRISPRa screening is a powerful tool for identifying novel engineering targets, facilitating the development of stress-resistant CHO cell lines, and enhancing therapeutic protein production.},
}

Animals
CHO Cells
Cricetulus
*CRISPR-Cas Systems/genetics
*Osmotic Pressure
*Recombinases/metabolism/genetics
Cell Proliferation/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Cricetinae

@article {pmid40743400,
year = {2025},
author = {Lu, B and Lin, S and Lang, Z and Yin, Q and Cao, H},
title = {Aptamer Probe-Assisted Strand Displacement-CRISPR/Cas12a Biosensor for Ultrasensitive Detection of AFB1.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {32},
pages = {20500-20507},
doi = {10.1021/acs.jafc.5c05775},
pmid = {40743400},
issn = {1520-5118},
mesh = {*Biosensing Techniques/methods/instrumentation ; *Aflatoxin B1/analysis ; *Aptamers, Nucleotide/chemistry/genetics ; Food Contamination/analysis ; Limit of Detection ; CRISPR-Cas Systems ; *CRISPR-Associated Proteins/genetics ; Bacterial Proteins/genetics ; Endodeoxyribonucleases ; },
abstract = {The sensitive and accurate detection of aflatoxin B1 (AFB1) is crucial for public health. Herein, the aptamer (Apt)-lock-key-structure (A-LKS), composed of Apt capable of spontaneous amplification and its complementary ssDNA (cDNA), was designed. Based on the identification of AFB1 in A-LKS, an A-LKS-mediated-SDA-Cas12a signal cascade (ASCC) biosensor was developed for ultrasensitive AFB1 detection. In the absence of AFB1, the Apt initiates amplification using DNA hanging from the 5' end of cDNA as a template, thereby enhancing the stability of A-LKS and reducing nonspecific amplification. When AFB1 is present, Apt binds to it, initiating the SDA reaction and activating Cas12a to generate strong fluorescence signals. The proposed biosensor demonstrates excellent selectivity and high sensitivity, with a low LOD of 3.6 pg/mL and a linear range of 0.01-100 ng/mL. This biosensor was successfully applied in real samples with satisfactory recoveries (88.69-105.48%), indicating its potential application in real samples.},
}

*Biosensing Techniques/methods/instrumentation
*Aflatoxin B1/analysis
*Aptamers, Nucleotide/chemistry/genetics
Food Contamination/analysis
Limit of Detection
CRISPR-Cas Systems
*CRISPR-Associated Proteins/genetics
Bacterial Proteins/genetics
Endodeoxyribonucleases

@article {pmid40736916,
year = {2025},
author = {Huang, Y and Yang, X and Liu, H and Xie, S and Chen, W and Dai, C and Zhao, C},
title = {Enhanced Paramylon Production in Euglena gracilis through CRISPR/Cas9 and Its Antiaging Effects.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {32},
pages = {20163-20171},
doi = {10.1021/acs.jafc.5c03014},
pmid = {40736916},
issn = {1520-5118},
mesh = {*Euglena gracilis/metabolism/genetics ; Animals ; Caenorhabditis elegans/drug effects/metabolism/genetics ; *CRISPR-Cas Systems ; *Glucans/pharmacology/metabolism/biosynthesis ; Reactive Oxygen Species/metabolism ; Oxidative Stress/drug effects ; Mitochondria/metabolism ; Longevity/drug effects ; },
abstract = {Euglena gracilis is a photosynthetic microalga that can synthesize paramylon. This study utilized CRISPR/Cas9 to knock out β-1,3-glucan phosphorylase, resulting in engineered strains to increase paramylon production. The ldp1 mutant strain produced paramylon constituting 68.20% of the cellular dry weight and a yield of 1.49 g/L. Replacing the carbon source in AF-6 medium with glucose resulted in an increase in the paramylon content to 72.92% of the dry cell weight and a yield of 1.51 g/L. The observation of a single distinct peak in the light scattering spectrum suggests a high degree of purity (approaching 100%). Paramylon significantly reduces reactive oxygen species in Caenorhabditis elegans, enhances antioxidant enzyme activity, and improves resistance to oxidative stress and high temperatures. It also reduces DNA damage and extends the lifespan. The antiaging effects of paramylon may be mediated through the regulation of the insulin/insulin-like growth factor signaling pathway. In C. elegans (myo-3:GFP(mit)), paramylon treatment increased mitochondrial signaling (p < 0.01), ATP production, membrane potential, and the expression of longevity-related genes, suggesting enhanced mitochondrial function. This research not only optimizes paramylon production but also highlights its potential as an antioxidative and antiaging agent.},
}

*Euglena gracilis/metabolism/genetics
Animals
Caenorhabditis elegans/drug effects/metabolism/genetics
*CRISPR-Cas Systems
*Glucans/pharmacology/metabolism/biosynthesis
Reactive Oxygen Species/metabolism
Oxidative Stress/drug effects
Mitochondria/metabolism
Longevity/drug effects

@article {pmid40735939,
year = {2025},
author = {Hulleman, JD and Jeon, S and Bali, S and DiCesare, SM and Abbas, A and Daniel, S and Ortega, AJ and Collier, GE and Yang, J and Bhattacharyaa, A and McCoy, MK and Joachimiak, LA and Posner, BA},
title = {Select Azo Compounds Post-translationally Modulate HTRA1 Abundance and Activity Potentially through Interactions at the Trimer Interface.},
journal = {ACS chemical biology},
volume = {20},
number = {8},
pages = {1849-1862},
doi = {10.1021/acschembio.4c00818},
pmid = {40735939},
issn = {1554-8937},
mesh = {*High-Temperature Requirement A Serine Peptidase 1/metabolism/genetics/chemistry ; Humans ; *Azo Compounds/pharmacology/chemistry ; *Protein Processing, Post-Translational/drug effects ; Macular Degeneration/genetics ; Retinal Pigment Epithelium/metabolism ; CRISPR-Cas Systems ; },
abstract = {High-temperature requirement protein A1 (HTRA1) is a secreted serine protease with diverse substrates, including extracellular matrix proteins, proteins involved in amyloid deposition, and growth factors. Accordingly, HTRA1 has been implicated in a variety of neurodegenerative diseases including a leading cause of blindness in the elderly, age-related macular degeneration (AMD). In fact, genomewide association studies have identified that the 10q26 locus that contains HTRA1 confers the strongest genetic risk factor for AMD. A recent study has suggested that AMD-associated risk alleles located in the HTRA1 gene correlate with a significant age-related defect in HTRA1 synthesis in the retinal pigmented epithelium (RPE) within the eye, possibly accounting for AMD susceptibility. Thus, we sought to identify small molecule enhancers of HTRA1 transcription and/or protein abundance using an unbiased high-throughput screening approach. To accomplish this goal, we used CRISPR/Sp.Cas9 engineering to introduce an 11-amino-acid luminescent peptide tag (HiBiT) onto the C-terminus of HTRA1 in immortalized ARPE-19 cells. Editing was very efficient (∼88%), verified by genomic DNA analysis, short interfering RNA (siRNA), and HiBiT blotting. A total of 1920 compounds from two libraries were screened. An azo compound with reported antiamyloidogenic and cardioprotective activity, Chicago Sky Blue 6B (CSB), was identified as an enhancer of endogenous HTRA1 secretion (2.0 ± 0.3 fold) and intracellular levels (1.7 ± 0.2 fold). These results were counter-screened using HiBiT complement factor H (CFH) edited ARPE-19 cells, verified using HiBiT blotting, and were not due to HTRA1 transcriptional changes. Importantly, serine hydrolase activity-based protein profiling (SH-ABPP) demonstrated that CSB does not affect HTRA1's specific activity. However, interestingly, in follow-up studies, Congo Red, another azo compound structurally similar to CSB, also substantially increased intracellular HTRA1 levels (up to 3.6 ± 0.3 fold) but was found to significantly impair HTRA1 enzymatic reactivity (0.45 ± 0.07 fold). Computational modeling of potential azo dye interaction with HTRA1 suggests that CSB and Congo Red can bind to the noncatalytic face of the trimer interface but with different orientation tolerances and interaction energies. These studies identify select azo dyes as HTRA1 chemical probes that may serve as starting points for future HTRA1-centered small molecule therapeutics.},
}

*High-Temperature Requirement A Serine Peptidase 1/metabolism/genetics/chemistry
Humans
*Azo Compounds/pharmacology/chemistry
*Protein Processing, Post-Translational/drug effects
Macular Degeneration/genetics
Retinal Pigment Epithelium/metabolism
CRISPR-Cas Systems

@article {pmid40729556,
year = {2025},
author = {Laforge, N and Calabre, M and Jules, M and Planson, AG},
title = {Multiplex Genome Editing and Regulation in Bacillus subtilis with CRISPR-MAD7.},
journal = {ACS synthetic biology},
volume = {14},
number = {8},
pages = {3142-3153},
doi = {10.1021/acssynbio.5c00274},
pmid = {40729556},
issn = {2161-5063},
mesh = {*Bacillus subtilis/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genome, Bacterial/genetics ; },
abstract = {With the advent of MAD7, a Cpf1-like nuclease, there has been a renewed focus on the development of CRISPR-based genome engineering tools in recent years. To improve genome engineering methodologies in B. subtilis, we revisited the potential of MAD7 for gene modification and expression interference. A key challenge in these endeavors is the limited transformation efficiency. To overcome this, we developed an efficient transformation protocol using strains overexpressing competence genes. Our results showed that although MAD7 together with a B. subtilis chromosome-targeting gRNA is lethal, enabling robust counterselection, we successfully engineered a strain carrying the MAD7-gRNA machinery in a reversibly inactivated state, marking a significant advance in the field. We demonstrated that both MAD7 and its catalytically inactive variant (dMAD7) can be conditionally regulated by inactivation at elevated temperatures. In addition, the MAD7-gRNA complex is effective for multiplex genome editing, allowing for the simultaneous deletion, mutation, or insertion of up to four loci, and enabling the combination of gene deletion, gene insertion, and point mutations. Furthermore, we established a strategy that achieves the simultaneous removal of MAD7 and the gRNA along with the desired genome edits. Altogether, this comprehensive study underscores the versatility of MAD7 for complex, scarless genome engineering and lays a strong foundation for further advancing genetic manipulation in B. subtilis.},
}

*Bacillus subtilis/genetics/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Bacterial Proteins/genetics/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Genome, Bacterial/genetics

@article {pmid40721107,
year = {2025},
author = {Oh, SJ and Lim, G and Han, Y and Kim, H and Kim, YG and Bhatia, SK and Yang, YH},
title = {Development of a DNA endonuclease I-SceI-based scarless genome editing system for Cupriavidus necator.},
journal = {Journal of biotechnology},
volume = {406},
number = {},
pages = {285-295},
doi = {10.1016/j.jbiotec.2025.07.020},
pmid = {40721107},
issn = {1873-4863},
mesh = {*Cupriavidus necator/genetics/metabolism ; *Gene Editing/methods ; Plasmids/genetics ; *Deoxyribonuclease I/genetics/metabolism ; CRISPR-Cas Systems ; Homologous Recombination ; Genome, Bacterial ; *Deoxyribonucleases, Type II Site-Specific/genetics/metabolism ; },
abstract = {Cupriavidus necator is a promising microbial chassis capable of fixing CO2 and producing high polyhydroxyalkanoate yields. Consequently, various genetic engineering methods have been explored. While sacB-based homologous recombination (HR) and CRISPR-Cas9 have shown both advantages and disadvantages in C. necator, alternative tools, including the DNA endonuclease I-SceI-mediated HR system could enable precise, scarless genome editing without requiring a large database. We developed a two-plasmid-based I-SceI HR system for efficient gene deletion and insertion in C. necator by altering origin replication and induction systems. The pOUO-1 plasmid was designed for conjugation-based genome integration via first HR, whereas the pOH-4 plasmid was constructed to express I-SceI, inducing second HR. Unlike conventional I-SceI expression strategies, which fail to trigger second HR in C. necator, transformation with pOH-4 alone was sufficient for recombination. A plasmid-curing strategy was optimized to eliminate the highly stable pOH-4 by increasing the incubation temperature to 37°C. Using this optimized system, the phaC1 gene was successfully knocked out; the phaCBP-M-CPF4 was inserted at the same site, resulting in a novel poly(3-hydroxybutyrate-co-5-hydroxyvalerate)-producing strain. This newly established I-SceI HR technique significantly simplifies genome engineering in C. necator, reducing the timeframe to a few weeks and facilitating its further applications in synthetic biology.},
}

*Cupriavidus necator/genetics/metabolism
*Gene Editing/methods
Plasmids/genetics
*Deoxyribonuclease I/genetics/metabolism
CRISPR-Cas Systems
Homologous Recombination
Genome, Bacterial
*Deoxyribonucleases, Type II Site-Specific/genetics/metabolism

@article {pmid40711424,
year = {2025},
author = {Zou, X and Gu, T and Li, X and Deng, L and Zhu, S and Dong, J and Deng, F and Hou, C and Huo, D},
title = {PAM-free activation of CRISPR/Cas12a via semi-nested asymmetric RPA: highly specific detection of HPV16 dsDNA.},
journal = {The Analyst},
volume = {150},
number = {17},
pages = {3891-3898},
doi = {10.1039/d5an00491h},
pmid = {40711424},
issn = {1364-5528},
mesh = {*Human papillomavirus 16/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; Humans ; *DNA, Viral/analysis/genetics ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; DNA, Single-Stranded/genetics ; *Bacterial Proteins/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; Uterine Cervical Neoplasms/virology/diagnosis ; CRISPR-Associated Proteins ; },
abstract = {Early and accurate detection of HPV16 nucleic acids is therefore critical for the effective screening, diagnosis, and prevention of cervical cancer. Although CRISPR/Cas12a-based molecular diagnostics offer a rapid and sensitive approach for HPV16 detection, their application to double-stranded DNA (dsDNA) targets remains constrained by two major limitations: the strict requirement for a protospacer adjacent motif (PAM) site and the insufficient specificity of current amplification strategies, which can lead to off-target amplification and false-positive results. To address these challenges, we developed a semi-nested asymmetric recombinase polymerase amplification (SNA-RPA) method combined with CRISPR/Cas12a for the detection of HPV16 dsDNA. This strategy employs a semi-nested primer design to significantly enhance target sequence specificity during amplification, while asymmetric primer ratios promote the efficient generation of single-stranded DNA (ssDNA) that directly activates Cas12a without the need for a PAM site. Using this approach, we achieved rapid and highly specific detection of HPV16 dsDNA, with a limit of detection as low as 18 aM. Beyond achieving PAM-free detection, our method also substantially improves amplification fidelity, offering a promising solution for precise and reliable HPV diagnostics and cervical cancer screening.},
}

*Human papillomavirus 16/genetics/isolation & purification
*CRISPR-Cas Systems/genetics
Humans
*DNA, Viral/analysis/genetics
*Nucleic Acid Amplification Techniques/methods
Limit of Detection
DNA, Single-Stranded/genetics
*Bacterial Proteins/metabolism/genetics
*Endodeoxyribonucleases/metabolism/genetics
Uterine Cervical Neoplasms/virology/diagnosis
CRISPR-Associated Proteins

@article {pmid40704803,
year = {2025},
author = {Mao, Y and Lv, R and Shao, H and Zhao, Y and Wang, J and Chen, Q and Yi, H and Ge, Y and Wang, H and Li, Y and Qi, Y},
title = {An ELISA-like sensitive and visual detection system targeting Yersinia pestis based on CRISPR/Cas12a and DNAzyme.},
journal = {Journal of clinical microbiology},
volume = {63},
number = {8},
pages = {e0027425},
pmid = {40704803},
issn = {1098-660X},
support = {JK2023gk002//Medical Science and Technology Project/ ; },
mesh = {*Yersinia pestis/genetics/isolation & purification ; *DNA, Catalytic/genetics ; Sensitivity and Specificity ; Humans ; Enzyme-Linked Immunosorbent Assay/methods ; *CRISPR-Cas Systems ; Bacterial Proteins/genetics ; *Plague/diagnosis/microbiology ; Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Associated Proteins/genetics ; *Endodeoxyribonucleases/genetics ; G-Quadruplexes ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Yersinia pestis is the causative agent of plague, a human disease with potentially devastating consequences. Here, we developed an enzyme-linked immunosorbent assay-like visual detection method based on clustered regularly interspaced short palindromic repeats (CRISPR) detection and DNAzyme for the cost-effective and highly sensitive detection of Y. pestis. A novel specific gene sequence (CH57_3927) was screened for the detection target of Y. pestis. The recombinase-aided amplification (RAA) assay, CRISPR/Cas12a detection assay, and G-quadruplex (G4) DNAzyme-based color development assay were separately established and optimized. These three optimized assays were integrated into an advanced ELISA-like visual detection method-RAA-CRISPR/Cas12a-DNAzyme (RCCD)-by further optimization of their components to improve the compatibility between them. The amplified target sequence binds to crRNA and activates the Cas12a nucleases for trans-cleave G4. As a result, the cleaved G4 is unable to bind with hemin to exert peroxidase activity, thus impeding the catalysis of the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS[2-]) colorimetric reaction. Consequently, negative samples exhibit a dark green coloration, while the positive products appear nearly colorless, facilitating visual differentiation with the naked eye. In addition, the RCCD detection platform effectively distinguished Y. pestis from all other closely related species, with a detection limit of 1 copy/reaction. Evaluated using Y. pestis DNA-spiked blood samples and uninfected samples, both sensitivity and specificity were 100%. The method shows significant potential for detecting targets in clinical samples and is well-suited for use in resource-limited environments. It offers advantages such as visual detection, batch detection, and low cost.IMPORTANCEWe utilized Mauve software to screen Yersinia pestis specific genes and integrated CRISPR-Cas12a, RAA amplification, and G-quadruplex DNAzyme technology to establish an advanced ELISA-like visual detection method. The visual detection method offers a more cost-effective alternative compared to the conventional CRISPR detection method that relies on fluorescence-labeled ssDNA reporter or lateral flow (LF) test strips. With only one thermostatic device required, it enhances the convenience of rapid on-site screening of Y. pestis outbreaks, providing effective support for plague detection, prevention, and control within primary medical and health institutions.},
}

*Yersinia pestis/genetics/isolation & purification
*DNA, Catalytic/genetics
Sensitivity and Specificity
Humans
Enzyme-Linked Immunosorbent Assay/methods
*CRISPR-Cas Systems
Bacterial Proteins/genetics
*Plague/diagnosis/microbiology
Clustered Regularly Interspaced Short Palindromic Repeats
*CRISPR-Associated Proteins/genetics
*Endodeoxyribonucleases/genetics
G-Quadruplexes
Nucleic Acid Amplification Techniques/methods

@article {pmid40701479,
year = {2025},
author = {Duan, X and Wu, S and Song, J and Xiong, G and Luo, J and Lu, Y and Tan, D and Lou, J and Hu, H and Tong, X and Ding, X and Dai, F},
title = {Bicaudal-C plays a critical role in regulating oogenesis in the lepidopteran insect Bombyx mori.},
journal = {International journal of biological macromolecules},
volume = {321},
number = {Pt 2},
pages = {145939},
doi = {10.1016/j.ijbiomac.2025.145939},
pmid = {40701479},
issn = {1879-0003},
mesh = {Animals ; *Bombyx/genetics/metabolism/physiology ; *Oogenesis/genetics ; Female ; *Insect Proteins/genetics/metabolism ; Oocytes/metabolism ; CRISPR-Cas Systems ; },
abstract = {Oogenesis is an intricate cellular specialization process that requires the precise expression of multiple genes. However, the functional genes and mechanisms contributing to lepidopteran oogenesis remain incompletely understood. In silkworm, the eggless mutant (sm[n]) has a typical oogenesis disorder phenotype. In sm[n] females, oocyte development is delayed, causing follicle degeneration and the absence of mature eggs in virgin ovarioles. Here, we revealed that BmBic-C, an ortholog of Drosophila melanogaster Bicaudal-C (Bic-C), is responsible for sm[n]. BmBic-C expression was significantly downregulated in sm[n], likely due to an Organdy transposon insertion upstream of BmBic-C. CRISPR/Cas9-mediated knockout of BmBic-C results in a no-egg phenotype consistent with sm[n]. BmBic-C deficiency reduces the absorption efficiency of vitellogenin, a key factor in egg development. A comparative proteomic analysis between the ΔBmBic-C and control strains revealed 633 differentially expressed proteins (DEPs). Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of the DEPs suggested that BmBic-C might contribute to ovarian cell development and survival through signal transduction, autophagy, and other pathways. Moreover, BmBic-C dysfunction triggers the apoptosis cascade, highlighting the essential role of BmBic-C in regulating ovarian homeostasis and cell fate. Phylogenetic and selection pressure analyses revealed that Bic-C is highly conserved across lepidopteran species and has undergone purifying selection throughout evolution, suggesting that Bic-C is a key component in organism survival and reproductive regulation. This study provides novel insights into oogenesis and lays a foundation for precision lepidopteran pest control against species-specific genomic/coding variable regions.},
}

Animals
*Bombyx/genetics/metabolism/physiology
*Oogenesis/genetics
Female
*Insect Proteins/genetics/metabolism
Oocytes/metabolism
CRISPR-Cas Systems

@article {pmid40693287,
year = {2025},
author = {Kiattisewee, C and Karanjia, AV and Cardiff, RAL and Olander, KE and Leejareon, P and Alvi, SS and Carothers, JM and Zalatan, JG},
title = {Systematic Mapping of Bacterial CRISPRa Systems for Synergistic Gene Activation Reveals Antagonistic Effects.},
journal = {ACS synthetic biology},
volume = {14},
number = {8},
pages = {3232-3244},
doi = {10.1021/acssynbio.5c00358},
pmid = {40693287},
issn = {2161-5063},
mesh = {Promoter Regions, Genetic/genetics ; *Transcriptional Activation/genetics ; *CRISPR-Cas Systems/genetics ; Escherichia coli/genetics ; Bacterial Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; Transcription Initiation Site ; },
abstract = {CRISPR gene activation (CRISPRa) tools have shown great promise for bacterial strain engineering but often require customization for each intended application. Our goal is to create generalizable CRISPRa tools that can overcome previous limitations of gene activation in bacteria. In eukaryotic cells, multiple activators can be combined for synergistic gene activation. To identify potential effectors for synergistic activation in bacteria, we systematically characterized bacterial activator proteins with a set of engineered synthetic promoters. We found that optimal target sites for different activators could vary by up to 200 bases in the region upstream of the transcription start site (TSS). These optimal target sites qualitatively matched previous reports for each activator, but the precise targeting rules varied between different promoters. By characterizing targeting rules in the same promoter context, we were able to test activator combinations with each effector positioned at its optimal target site. We did not find any activator combinations that produced synergistic activation, and we found that many combinations were antagonistic. This systematic investigation highlights fundamental mechanistic differences between bacterial and eukaryotic transcriptional activation systems and suggests that alternative strategies will be necessary for strong bacterial gene activation at arbitrary endogenous targets.},
}

Promoter Regions, Genetic/genetics
*Transcriptional Activation/genetics
*CRISPR-Cas Systems/genetics
Escherichia coli/genetics
Bacterial Proteins/genetics/metabolism
Gene Expression Regulation, Bacterial
Transcription Initiation Site

@article {pmid40684992,
year = {2025},
author = {Tafech, B and Carlaw, T and Sadhnani, G and Schmidt, K and Morin, T and Leung, J and Weiner, J and An, K and Balázs, A and Ross, C and Beule, D and Mall, MA and Fuchs, H and Kulkarni, J and Cullis, PR and Hedtrich, S},
title = {Lung tissue-optimized gene editing in human cystic fibrosis models following topical application of lipid nanoparticles.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {385},
number = {},
pages = {114053},
doi = {10.1016/j.jconrel.2025.114053},
pmid = {40684992},
issn = {1873-4995},
mesh = {Humans ; *Cystic Fibrosis/genetics/therapy ; *Gene Editing/methods ; *Nanoparticles/administration & dosage/chemistry ; Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; *Lung/metabolism ; *Lipids/administration & dosage/chemistry ; Administration, Topical ; Deoxyribonuclease I/administration & dosage ; CRISPR-Cas Systems ; Liposomes ; Recombinant Proteins ; },
abstract = {Cystic fibrosis (CF) is a severe monogenic disease characterized by debilitating lung dysfunction caused by loss-of-function mutations in the CFTR gene. While CRISPR-based gene editing holds promise for correcting these mutations and potentially curing CF, efficient delivery of gene editors to the lung epithelium through the mucosal barrier remains a major challenge. In this study, we developed a lung-optimized gene editing strategy using lipid nanoparticles (LNPs) and evaluated it in increasingly complex, biomimetic human-based and patient-derived models. Systematic optimization of helper lipids, genetic cargo, guide RNA modifications, and gene editor ratios, alongside analysis of innate immune responses, achieved ∼50 % editing efficiency in the model gene HPRT in two-dimensional models. Editing efficiency significantly dropped to ∼5 % in biomimetic three-dimensional CF bronchial epithelial tissue models following topical LNP application. Pretreatment with the approved mucolytic agent dornase alpha increased editing efficiency to ∼12.7 %. Finally, in CF patient-derived cells harboring the CFTR[R1162X] mutation, our optimized LNP formulation achieved ∼12 % correction on gene level, offering a potential treatment avenue for this yet untreatable mutation. Taken together, this study demonstrates that optimizing the genetic cargo as well as the delivery vehicle is key when striving for clinically applicable treatment approaches. It further provides insights into gene editing rates in human-based normal and CF patient-derived bronchial tissue models which express all relevant biological barriers and, thus, can pave the way for topically applicable treatment options for patients with CF and other genetic lung diseases.},
}

Humans
*Cystic Fibrosis/genetics/therapy
*Gene Editing/methods
*Nanoparticles/administration & dosage/chemistry
Cystic Fibrosis Transmembrane Conductance Regulator/genetics
*Lung/metabolism
*Lipids/administration & dosage/chemistry
Administration, Topical
Deoxyribonuclease I/administration & dosage
CRISPR-Cas Systems
Liposomes
Recombinant Proteins

@article {pmid40680952,
year = {2025},
author = {Gao, X and Dong, X and Song, H and Fu, Y and Li, J and Fan, G and Wang, T and Sun, Y and Wang, Y and Qiu, HJ and Luo, Y},
title = {A one-pot CRISPR-Cas12a-based assay for rapid, on-site detection of African swine fever virus.},
journal = {International journal of biological macromolecules},
volume = {321},
number = {Pt 2},
pages = {146109},
doi = {10.1016/j.ijbiomac.2025.146109},
pmid = {40680952},
issn = {1879-0003},
mesh = {*African Swine Fever Virus/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; Animals ; Swine ; *African Swine Fever/diagnosis/virology ; DNA, Viral/genetics ; Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Recombinases ; },
abstract = {African swine fever (ASF), caused by African swine fever virus (ASFV), is a highly contagious and devastating disease threatening global swine production. The disease has caused substantial economic losses, driving the need for efficient diagnostic tools to enhance surveillance and control. Despite various available assays for ASF, field-deployable tools enabling rapid, accurate, and user-friendly detection remain urgently needed. Here, we developed and validated a novel one-pot recombinase polymerase amplification (RPA)-CRISPR-Cas12a assay for rapid and sensitive detection of ASFV by integrating all components into a single sealed tube, which requires only isothermal heating and ultraviolet visualization. The assay demonstrated a detection limit of 56 TCID50/mL and could be completed within 35 min, and without cross-reactivity with non-ASFV porcine viruses. In comparative testing of 150 clinical samples, the one-pot RPA-CRISPR-Cas12a assay exhibited 100 % agreement with gold standard quantitative PCR (qPCR). Notably, the assay identified ASFV genomic DNA in whole blood as early as 3 days post-infection with sensitivity comparable to the qPCR. This early detection capability, combined with a field-deployable format, provides a robust tool for implementing timely containment measures against ASF, especially in resource-limited setting.},
}

*African Swine Fever Virus/genetics/isolation & purification
*CRISPR-Cas Systems/genetics
Animals
Swine
*African Swine Fever/diagnosis/virology
DNA, Viral/genetics
Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
Recombinases

@article {pmid40674851,
year = {2025},
author = {Li, D and Hu, J and Hu, J and Zhang, CY},
title = {Integration of AND logic circuit with CRISPR/Cas12a system for sensitive detection of biomarkers and accurate discrimination of breast cancer cells.},
journal = {Biosensors & bioelectronics},
volume = {288},
number = {},
pages = {117790},
doi = {10.1016/j.bios.2025.117790},
pmid = {40674851},
issn = {1873-4235},
mesh = {Humans ; *Breast Neoplasms/diagnosis/genetics/pathology ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Biomarkers, Tumor/genetics/analysis/isolation & purification ; Female ; *Flap Endonucleases/genetics/isolation & purification/analysis ; Cell Line, Tumor ; Limit of Detection ; },
abstract = {Flap endonuclease 1 (FEN1) is a structure-selective nuclease that is of great significance in maintaining genomic stability. FEN1 is up-regulation in various cancers and is regarded as a new biomarker for cancer diagnosis. Herein, we integrate DNA logic circuit with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system for sensitive detection of FEN1 at the cellular level and accurate discrimination of different cell types. In this design, FEN1 cleaves the flap-containing synthetic substrates, and subsequently magnetic separation separates the primers from the cleaved substrate fragments, inducing the release of the primers. The released primers can initiate strand displacement amplification (SDA) reaction to generate abundant activators that can activate the trans-cleavage activity of the CRISPR/Cas12a system to cleave the signal probes for the recovery of Cy5 fluorescence signal. This circuit displays superior sensitivity with a detection limit of 5.19 × 10[-5] U/μL, and it is capable of screening the FEN1 inhibitors, quantifying the activity of FEN1 with sensitivity at the single-cell level, and discriminating diverse FEN1 levels in clinical breast cancer tissues. We further construct an AND logic circuit based on miR-31 and FEN1 to simultaneously monitor the biomarkers at the cellular level and achieves accurate discrimination of different breast cancer cell types with a P-value of less than 0.05. The integration of DNA logic circuit with CRISPR/Cas12a system provides a new approach for biomarker-related biomedical research and clinical diagnostics.},
}

Humans
*Breast Neoplasms/diagnosis/genetics/pathology
*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
*Biomarkers, Tumor/genetics/analysis/isolation & purification
Female
*Flap Endonucleases/genetics/isolation & purification/analysis
Cell Line, Tumor
Limit of Detection

@article {pmid40619069,
year = {2025},
author = {Gao, S and Wang, X and Yang, R and Bian, Z and Yong, D and Xu, S and Huo, L and Tu, Q and Zhang, Y and Fu, J and Li, R},
title = {Efficient genome engineering in Agrobacterium tumefaciens C58 using recombineering assisted by CRISPR/Cas9.},
journal = {Journal of biotechnology},
volume = {406},
number = {},
pages = {99-104},
doi = {10.1016/j.jbiotec.2025.07.005},
pmid = {40619069},
issn = {1873-4863},
mesh = {*Agrobacterium tumefaciens/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genome, Bacterial/genetics ; *Genetic Engineering/methods ; Homologous Recombination ; },
abstract = {Recombineering, a technique derived from phage-encoded homologous recombination, has emerged as a vital approach for bacterial genome engineering. Agrobacterium tumefaciens is extensively utilized to transfer DNA into the host plant genomes. To facilitate the transformation of various plant species, particularly those of considerable economic value, genetic modifications of Agrobacterium strains are essential. Our previous studies established an Agrobacterium-specific phage-encoded homologous recombination system for Agrobacterium species. Yet, recent investigations have indicated that there is a substantial variability in the recombination efficiency of these recombineering systems for gene editing across different genome loci in A. tumefaciens. In this work, we present the development of an efficient genome engineering tool for A. tumefaciens by integrating recombineering with CRISPR/Cas9 technology. Initially, we found that lengthening the homology arms significantly enhanced genome editing efficiency. Nevertheless, at certain genomic sites, even when the length of the homology arms was increased, the editing efficiency remained suboptimal. Subsequently, combination of the Agrobacterium-specific recombineering system with the CRISPR/Cas9 system markedly enhanced the genome engineering efficiency. This study offers an enhanced and efficient genome engineering tool for A. tumefaciens, which could potentially be applied to other species within the Agrobacterium genus.},
}

*Agrobacterium tumefaciens/genetics
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Genome, Bacterial/genetics
*Genetic Engineering/methods
Homologous Recombination

@article {pmid40607797,
year = {2025},
author = {Kuzmina, A and Wattad, S and Murugavelu, P and Amir, N and Tickotsky, N and Levin, L and Taube, R},
title = {Genome-wide CRISPR knockout screen identifies activating transcription factor (ATF1) as an activator of HIV gene expression.},
journal = {mBio},
volume = {16},
number = {8},
pages = {e0055725},
pmid = {40607797},
issn = {2150-7511},
support = {2021273//United States-Israel Binational Science Foundation/ ; 1884/24//Israel Science Foundation/ ; 508136175//Deutsche Forschungsgemeinschaft/ ; 5R21AI270197//All of Us Research Program (All of Us NIH)/ ; },
mesh = {Humans ; CD4-Positive T-Lymphocytes/virology ; *Gene Expression Regulation, Viral ; Virus Latency/genetics ; *HIV-1/genetics/physiology ; Promoter Regions, Genetic ; *Activating Transcription Factor 1/genetics/metabolism ; CRISPR-Cas Systems ; HIV Infections/virology/genetics ; Gene Knockout Techniques ; Receptors, CCR5/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {UNLABELLED: Antiretroviral therapy against the human immunodeficiency virus (HIV) has significantly prolonged the life span of people living with HIV, transforming viral infection into a latent condition that is characterized with undetectable viral loads. Yet, a complete cure of infection is out of reach, as transcriptionally silent but replication-competent proviruses persist in a long-lived reservoir that is resistant to therapy. The current work follows a genome-wide CRISPR knockout screen in human CD4[+] T cells and defines the activating transcription factor 1 (ATF1) as an activator of HIV gene transcription with elevated expression levels in cells that carry transcriptionally active provirus. Additional gain and loss-of-function experiments show that depletion of ATF1 promotes latency. ATF1 directly occupies the HIV promoter, where it regulates the recruitment of RNA Polymerase II and the levels of H3K9me3 histone repression mark. Genome-wide, ATF1 binds cellular gene promoters. Among its targets, ATF1 modulates the levels of CCR5 antisense lncRNA, thereby regulating the protein expression of the CCR5 HIV co-receptor. We conclude that ATF1 is an activator of gene transcription that dictates HIV gene expression via both direct and indirect mechanisms.

IMPORTANCE: HIV persists in resting CD4[+] primary infected cells, forming a reservoir that is resistant to therapy, and thus a main barrier toward elimination of viral infection. An understanding of the mechanisms that control HIV gene expression and drive viral latency is therefore of high clinical importance. This study identifies activating transcription factor 1 (ATF1) as an activator of HIV gene expression. ATF1 binds the HIV promoter, where it modulates the occupancy of RNA Polymerase II and the levels of H3K9me3 histone repression mark. Genome-wide, ATF1 also occupies cellular promoters. One target of ATF1 is the antisense (AS) lncRNA. Through binding to CCR5-AS lncRNA, ATF1 induces CCR5 mRNA stability, thereby indirectly controlling HIV infection. Overall, we provide an additional understanding of the host transcription pathways that regulate HIV gene expression and potentially open new ways to manipulate its reservoir size.},
}

Humans
CD4-Positive T-Lymphocytes/virology
*Gene Expression Regulation, Viral
Virus Latency/genetics
*HIV-1/genetics/physiology
Promoter Regions, Genetic
*Activating Transcription Factor 1/genetics/metabolism
CRISPR-Cas Systems
HIV Infections/virology/genetics
Gene Knockout Techniques
Receptors, CCR5/genetics/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40592435,
year = {2025},
author = {Liu, Y and Li, Z and Zhou, L and Jia, W and Zhou, Z and Yang, Y and Sun, Y and Lin, P and Shen, G and Liu, Q and Hua, X and Zhao, P},
title = {BmPriS promotes silk gland growth by regulating endoreplication in silkworm.},
journal = {International journal of biological macromolecules},
volume = {320},
number = {Pt 1},
pages = {145640},
doi = {10.1016/j.ijbiomac.2025.145640},
pmid = {40592435},
issn = {1879-0003},
mesh = {Animals ; *Bombyx/genetics/growth & development/metabolism ; *Silk/metabolism/biosynthesis/genetics ; *DNA Replication ; *Insect Proteins/genetics/metabolism ; Cell Cycle/genetics ; CRISPR-Cas Systems ; },
abstract = {Endoreplication, also known as the endocycle, is a variant of the cell cycle that occurs in the silk glands of silkworms. Although the primase α subunit (PriS) has been reported to be involved in the initiation of DNA replication, its role in silk gland cell endoreplication remains unclear. In our study, we observed that BmPriS expression in the posterior silk gland (PSG) progressively increased during the late fifth instar. Using the CRISPR/Cas9 system, we specifically mutated BmPriS in the PSG, resulting in a decrease in cocoon shell weight and thinner silk fibers. Comparison with the wild-type revealed that the PSG was completely absent in ∆BmPriS silkworms. Immunofluorescence staining revealed a significant reduction in the size of silk gland cells. The expression of the fibroin genes (FibH, FibL, and P25) was nearly silent, whereas that of the sericin genes (Ser1, Ser2, and Ser3) was significantly downregulated. Moreover, EdU staining indicated a marked impairment of endoreplication in PSG cells, accompanied by a significant downregulation of endoreplication-associated minichromosome maintenance genes (MCM3, MCM5, MCM6, and MCM7). Cell cycle- and growth-related genes (CDK2, CyclinE, and Yki) were also significantly downregulated, whereas apoptosis-related genes (Fadd, Daxx, and Dredd) were significantly upregulated. Collectively, these findings indicate that BmPriS regulates silk gland growth and reduces silk production by modulating PSG endoreplication, and by interfering with the cell cycle and apoptosis processes. This study offers novel perspectives on the involvement of PriS in silk gland development.},
}

Animals
*Bombyx/genetics/growth & development/metabolism
*Silk/metabolism/biosynthesis/genetics
*DNA Replication
*Insect Proteins/genetics/metabolism
Cell Cycle/genetics
CRISPR-Cas Systems

@article {pmid40479615,
year = {2025},
author = {Koshizuka, K and Wu, X and Sato, K and Vo, PTT and Murawska, GM and Ishikawa, T and Wang, Z and Molinolo, AA and Dennis, EA and Nathan, CO and Mali, P and Gutkind, JS},
title = {Genome-Wide CRISPR Screening Reveals That mTOR Inhibition Initiates Ferritinophagy and Ferroptosis in Head and Neck Cancer.},
journal = {Cancer research},
volume = {85},
number = {16},
pages = {3032-3051},
pmid = {40479615},
issn = {1538-7445},
support = {R35 GM139641/GM/NIGMS NIH HHS/United States ; R01 GM020501/GM/NIGMS NIH HHS/United States ; R01 DE030497/DE/NIDCR NIH HHS/United States ; U54 CA274502/CA/NCI NIH HHS/United States ; S10 OD026929/OD/NIH HHS/United States ; R01 CA247551/CA/NCI NIH HHS/United States ; R01 DE026870/DE/NIDCR NIH HHS/United States ; R01CA247551//National Cancer Institute (NCI)/ ; NCI U54CA274502//National Cancer Institute (NCI)/ ; R01DE030497//National Institute of Dental and Craniofacial Research (NIDR)/ ; R01DE026870//National Institute of Dental and Craniofacial Research (NIDR)/ ; the JSPS Overseas Research Fellowships//Japan Society for the Promotion of Science London (JSPS)/ ; the Uehara Memorial Foundation Research Fellowship//Uehara Memorial Foundation (UMF)/ ; Study abroad support of Mochida Memorial Foundation for Medical and Pharmaceutical Research//Mochida Memorial Foundation for Medical and Pharmaceutical Research ()/ ; GM RO1 GM20501-44//National Institute of General Medical Sciences (NIGMS)/ ; R35 GM139641-03//National Institute of General Medical Sciences (NIGMS)/ ; },
mesh = {*Ferroptosis/drug effects/genetics ; Humans ; *Head and Neck Neoplasms/genetics/drug therapy/pathology/metabolism ; *TOR Serine-Threonine Kinases/antagonists & inhibitors/metabolism/genetics ; *MTOR Inhibitors/pharmacology ; *Ferritins/metabolism ; Autophagy/drug effects/genetics ; Animals ; Mice ; *Squamous Cell Carcinoma of Head and Neck/genetics/drug therapy/pathology/metabolism ; Signal Transduction/drug effects ; Cell Line, Tumor ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; Iron/metabolism ; },
abstract = {UNLABELLED: Genomic alterations converging on persistent activation of the PI3K/mTOR pathway represent one of the most frequently altered signaling circuitries in cancer. However, the clinical efficacy of mTOR inhibitors (mTORi) has been limited. In this study, we took advantage of the widespread activation of PI3K/mTOR signaling in head and neck squamous cell carcinoma (HNSCC) and the promising effects of mTORi in HNSCC experimental models and recent clinical trials to gain a mechanistic understanding of the antitumoral activity of mTORi. A genome-wide CRISPR screen revealed that treatment with mTORi promotes the autophagic degradation of ferritin (ferritinophagy), consequently increasing free intracellular iron, inducing lipid peroxidation, and ultimately driving cancer cell demise by ferroptosis. These findings provide a rationale for synergistic combinations repurposing approved drugs that disable cellular ferroptotic defense mechanisms. Together, this study provides a molecular framework underlying the antitumor activity of mTORi in HNSCC, thereby revealing multimodal precision therapies for HNSCC and many human malignancies displaying overactive PI3K/mTOR signaling.

SIGNIFICANCE: Inhibition of mTOR induces ferritinophagy that increases free iron and stimulates ferroptosis, suggesting that this axis could be harnessed to help predict responses and to develop rational combination therapies to overcome resistance.},
}

*Ferroptosis/drug effects/genetics
Humans
*Head and Neck Neoplasms/genetics/drug therapy/pathology/metabolism
*TOR Serine-Threonine Kinases/antagonists & inhibitors/metabolism/genetics
*MTOR Inhibitors/pharmacology
*Ferritins/metabolism
Autophagy/drug effects/genetics
Animals
Mice
*Squamous Cell Carcinoma of Head and Neck/genetics/drug therapy/pathology/metabolism
Signal Transduction/drug effects
Cell Line, Tumor
Xenograft Model Antitumor Assays
CRISPR-Cas Systems
Iron/metabolism

@article {pmid40466595,
year = {2025},
author = {Xiang, Z and Duan, M and Wang, S and Zhao, H and Zhao, W and Li, X and Li, X and Zeng, W and Wu, Y and Yang, F and Liu, X and Tang, C and Cui, L and Yang, Z},
title = {Three Asparagine insertions in the K13-propeller led to Plasmodiumfalciparum becoming resistant to multiple antimalarial drugs.},
journal = {International journal for parasitology. Drugs and drug resistance},
volume = {28},
number = {},
pages = {100590},
pmid = {40466595},
issn = {2211-3207},
mesh = {*Antimalarials/pharmacology ; *Plasmodium falciparum/drug effects/genetics ; *Asparagine/genetics ; *Protozoan Proteins/genetics ; CRISPR-Cas Systems ; Mutagenesis, Insertional ; Humans ; *Drug Resistance, Multiple/genetics ; Drug Resistance/genetics ; Malaria, Falciparum/parasitology/drug therapy ; Parasitic Sensitivity Tests ; Artemisinins/pharmacology ; },
abstract = {Drug resistance in Plasmodium falciparum represents a significant challenge in malaria treatment. Identifying the molecular markers associated with P. falciparum resistance will effectively detect resistance and enhance treatment efficiency. In this study, we utilized the advanced CRISPR/Cas9 technology to precisely insert one, two, or three asparagine residues into the Kelch 13(K13) gene of the 3D7 strain, positioned after the 142nd amino acid residue, resulting in 1N-3D7, 2N-3D7, and 3N-3D7. Using ring-stage survival assays (RSA), drug sensitivity evaluations, and in vitro developmental assessments, our findings revealed a trend: 1) the insertion of asparagine residues into the parasite genome increased RSA, with more asparagine insertions leading to higher RSA. 2) According to the IC50 values, 1N-3D7 and 2N-3D7 exhibited similar sensitivity profiles across all ten tested drugs, with both demonstrating resistance to Naphthoquine, indicating that the insertions of one or two asparagines played an equivalent role in conferring resistance. However, the insertion of three asparagine residues resulted in significantly higher IC50 values compared to the first two forms when tested with Artesunate, Artemether, Dihydroartemisinin, Pyronaridine Phosphate, and Naphthoquine, showing resistance to all five drugs. Furthermore, 3N-3D7 exhibited a prolonged ring phase and a shortened trophozoite phase within red blood cells; the schizont phase appeared synchronous with the others, yet its mature schizonts contained fewer merozoites. Additionally, 3N-3D7 exhibited a fitness defect, with the proportion decreasing gradually during co-culture with 3D7, its fitness cost calculated as 14.88 ± 2.87. All these results support the opinion that the insertion of three asparagines was a molecular marker of resistance to artemisinin derivatives, Pyronaridine Phosphate, and Naphthoquine in P. falciparum.},
}

*Antimalarials/pharmacology
*Plasmodium falciparum/drug effects/genetics
*Asparagine/genetics
*Protozoan Proteins/genetics
CRISPR-Cas Systems
Mutagenesis, Insertional
Humans
*Drug Resistance, Multiple/genetics
Drug Resistance/genetics
Malaria, Falciparum/parasitology/drug therapy
Parasitic Sensitivity Tests
Artemisinins/pharmacology

@article {pmid40403231,
year = {2025},
author = {Morelli, E and Aktas-Samur, A and Maisano, D and Gao, C and Favasuli, V and Papaioannou, D and De Nola, G and Henninger, JE and Liu, N and Turi, M and Folino, P and Vreux, L and Cumerlato, M and Chen, L and Aifantis, I and Fulciniti, M and Anderson, KC and Lytton-Jean, AKR and Gullà, A and Young, RA and Samur, MK and Munshi, NC},
title = {CRISPR-Cas13d functional transcriptomics reveals widespread isoform-selective cancer dependencies on lncRNAs.},
journal = {Blood},
volume = {146},
number = {7},
pages = {847-860},
doi = {10.1182/blood.2025028746},
pmid = {40403231},
issn = {1528-0020},
support = {P01 CA155258/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *RNA, Long Noncoding/genetics/metabolism ; *CRISPR-Cas Systems ; *Transcriptome ; Animals ; Mice ; *Multiple Myeloma/genetics/pathology/metabolism ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; *Neoplasms/genetics ; RNA Isoforms/genetics ; },
abstract = {Long noncoding RNAs (lncRNAs) are a significant yet largely uncharted component of the cancer transcriptome, with their isoform-specific functions remaining poorly understood. In this study, we used RNA-targeting CRISPR-Cas13d to uncover and characterize hundreds of tumor-essential lncRNA (te-lncRNA) isoforms with clinical relevance. Focusing on multiple myeloma (MM), we targeted the lncRNA transcriptome expressed in tumor cells from patients with MM and revealed both MM-specific and pan-cancer dependencies across diverse cancer cell lines, which we further validated in animal models. Additionally, we mapped the subcellular localization of these te-lncRNAs, identifying >30 cytosolic isoforms that proved essential when targeted by cytosol-localized Cas13d. Notably, a specific isoform of small nucleolar RNA host gene 6, enriched in the endoplasmic reticulum, interacts with heat shock proteins to maintain cellular proteostasis. We also integrated functional and clinical data into the publicly accessible LongDEP Portal, providing a valuable resource for the research community. Our study offers a comprehensive characterization of te-lncRNAs, underscoring their oncogenic roles and therapeutic potential.},
}

Humans
*RNA, Long Noncoding/genetics/metabolism
*CRISPR-Cas Systems
*Transcriptome
Animals
Mice
*Multiple Myeloma/genetics/pathology/metabolism
Cell Line, Tumor
Gene Expression Regulation, Neoplastic
*Neoplasms/genetics
RNA Isoforms/genetics

@article {pmid40378078,
year = {2025},
author = {Ding, Q and Gan, P and Xu, Z and Li, H and Guo, L and MacDonald, C and Tan, W and Sanchez-Ortiz, E and McAnally, JR and Zhang, Y and Karri, D and Xu, L and Liu, N and Olson, EN},
title = {Genomic Editing of a Pathogenic Sequence Variant in ACTA2 Rescues Multisystemic Smooth Muscle Dysfunction Syndrome in Mice.},
journal = {Circulation},
volume = {152},
number = {7},
pages = {465-483},
pmid = {40378078},
issn = {1524-4539},
support = {R01 HL157281/HL/NHLBI NIH HHS/United States ; R01 HL130253/HL/NHLBI NIH HHS/United States ; 25POST1372779/AHA/American Heart Association-American Stroke Association/United States ; U54 HD087351/HD/NICHD NIH HHS/United States ; P50 HD087351/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; *Gene Editing/methods ; Humans ; Mice ; *Actins/genetics/metabolism ; *Muscle, Smooth, Vascular/metabolism/pathology/physiopathology ; *Myocytes, Smooth Muscle/metabolism/pathology ; Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems ; Disease Models, Animal ; *Genetic Therapy/methods ; },
abstract = {BACKGROUND: Vascular smooth muscle cells (SMCs), the predominant cell type in the aortic wall, play a crucial role in maintaining aortic integrity, blood pressure, and cardiovascular function. Vascular SMC contractility and function depend on ACTA2 (smooth muscle α-actin 2). The pathogenic variant ACTA2 c.536G>A (p.R179H) causes multisystemic smooth muscle dysfunction syndrome, a severe disorder marked by widespread smooth muscle abnormalities, resulting in life-threatening aortic disease and high risk of early death from aneurysms or stroke. No effective treatments exist for multisystemic smooth muscle dysfunction syndrome.

METHODS: To develop a comprehensive therapy for multisystemic smooth muscle dysfunction syndrome, we used CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) adenine base editing to correct the ACTA2 R179H sequence variant. We generated isogenic human induced pluripotent stem cell lines and humanized mice carrying this pathogenic missense sequence variant. Induced pluripotent stem cell-derived SMCs were evaluated for key functional characteristics, including proliferation, migration, and contractility. The adenine base editor ABE8e-SpCas9-VRQR under control of either an SMC-specific promoter or a cytomegalovirus promoter, and an optimized single guide RNA under control of a U6 promoter were delivered intravenously to humanized R179H mice using adeno-associated virus serotype 9 and phenotypic outcomes were evaluated.

RESULTS: The R179H sequence variant causes a dramatic phenotypic switch in human induced pluripotent stem cell-derived SMCs from a contractile to a synthetic state, a transition associated with aneurysm formation. Base editing prevented this pathogenic phenotypic switch and restored normal SMC function. In humanized mice, the ACTA2[R179H/+] sequence variant caused widespread smooth muscle dysfunction, manifesting as decreased blood pressure, aortic dilation and dissection, bladder enlargement, gut dilation, and hydronephrosis. In vivo base editing rescued these pathological abnormalities, normalizing smooth muscle function.

CONCLUSIONS: This study demonstrates the effectiveness of adenine base editing to treat multisystemic smooth muscle dysfunction syndrome and restore aortic smooth muscle function. By correcting the ACTA2 R179H sequence variant, the pathogenic phenotypic shift in SMCs was prevented, key aortic smooth muscle functions were restored, and life-threatening aortic dilation and dissection were mitigated in humanized mice. These findings underscore the promise of gene-editing therapies in addressing the underlying genetic causes of smooth muscle disorders and offer a potential transformative treatment for patients facing severe vascular complications.},
}

Animals
*Gene Editing/methods
Humans
Mice
*Actins/genetics/metabolism
*Muscle, Smooth, Vascular/metabolism/pathology/physiopathology
*Myocytes, Smooth Muscle/metabolism/pathology
Induced Pluripotent Stem Cells/metabolism
CRISPR-Cas Systems
Disease Models, Animal
*Genetic Therapy/methods

@article {pmid40289704,
year = {2025},
author = {Sun, M and Gao, J and Tang, H and Wang, H and Zhou, L and Song, C and Tian, Y and Li, Q},
title = {D-CAPS: an efficient CRISPR-Cas9-based phage defense system for E. coli.},
journal = {Acta biochimica et biophysica Sinica},
volume = {57},
number = {8},
pages = {1244-1251},
pmid = {40289704},
issn = {1745-7270},
mesh = {*Escherichia coli/virology/genetics ; *CRISPR-Cas Systems ; *Bacteriophage T7/genetics ; },
abstract = {Escherichia coli is widely used in industrial chemical synthesis but faces significant challenges due to bacteriophage contamination, which reduces product quality and yield. Therefore, developing an efficient antiphage system is essential. In this study, we develop a CRISPR-Cas9-based antiphage system (CAPS) targeting essential genes of the T7 phage (gene 5 and gene 19) with single gRNAs transformed into MG1655 strains expressing Cas9. While CAPS provides limited resistance, with plating efficiencies ranging from 10 [-5] to 10 [-1], further optimization is needed. To enhance efficacy, we design a double-site-targeting CRISPR-Cas9-based antiphage system (D-CAPS). D-CAPS demonstrates complete resistance, with no plaques observed even at a high multiplicity of infection (MOI of 2), and growth curve analysis reveals that antiphage E. coli strains grow normally, similar to the wild-type strain, even at a high multiplicity of infection. Furthermore, D-CAPS is effective against BL21(DE3) strains, showing strong resistance and demonstrating its versatility across different E. coli strains. Protein expression analysis via green fluorescent protein confirms that E. coli carrying D-CAPS could maintain normal protein expression levels even in the presence of phages, comparable to wild-type strains. Overall, D-CAPS offers a robust and versatile approach to enhancing E. coli resistance to phages, providing a practical solution for protecting industrial E. coli strains and improving fermentation processes.},
}

*Escherichia coli/virology/genetics
*CRISPR-Cas Systems
*Bacteriophage T7/genetics

@article {pmid39871553,
year = {2025},
author = {Roy, AA and Pokale, R and Mukharya, A and Nikam, AN and Dua, K and Rao, BSS and Seetharam, RN and Mutalik, S},
title = {Synergizing CRISPR-Cas9 with Advanced Artificial Intelligence and Machine Learning for Precision Drug Delivery: Technological Nexus and Regulatory Insights.},
journal = {Current gene therapy},
volume = {25},
number = {4},
pages = {467-496},
pmid = {39871553},
issn = {1875-5631},
support = {5/13/90/2020/NCD-III//Indian Council for Medical Research (ICMR), Government of India, New Delhi/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing/methods ; *Artificial Intelligence ; *Machine Learning ; *Drug Delivery Systems/methods ; Genetic Therapy/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Precision Medicine ; },
abstract = {The evolution of genetic exploration tools, from laborious methods like radiationinduced mutations to the transformative CRISPR-Cas9 system, has fundamentally reshaped genetic research and gene editing capabilities. This journey, initiated by foundational techniques such as ZFNs and TALENs and culminating in the groundbreaking work of Doudna and Charpentier in 2012, has ushered in an era of precise DNA alteration and profound insights into gene functions. The CRISPR/Cas9 system uses the Cas9 enzyme and guides RNA (gRNA) to precisely target and cleave DNA, with subsequent repair via error-prone NHEJ or precise HDR, enabling versatile gene editing. Complementary computational tools like E-CRISP and Azimuth 2.0, alongside advanced deep learning models like DeepCRISPR, have significantly contributed to refining CRISPR experiments, optimizing gRNA efficiency, and predicting outcomes with greater precision. In clinical applications, CRISPR-Cas9 shows great promise for treating complex genetic disorders like sickle cell disease and β-thalassemia, but faces challenges such as off-target effects, immune responses to viral vectors, and ethical issues in germline editing. Overcoming these challenges requires meticulous experimentation and robust regulatory frameworks to ensure responsible and beneficial utilization of the CRISPR-Cas9 technology across diverse fields, including cancer treatment, genetic disease therapies, agriculture, and synthetic biology, while continually addressing ethical, safety, and legal considerations for its advancement and widespread adoption.},
}

*CRISPR-Cas Systems/genetics
Humans
*Gene Editing/methods
*Artificial Intelligence
*Machine Learning
*Drug Delivery Systems/methods
Genetic Therapy/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Precision Medicine

@article {pmid40849301,
year = {2025},
author = {Buonaiuto, G and Desideri, F and Setti, A and Palma, A and D'Angelo, A and Storari, G and Santini, T and Laneve, P and Trisciuoglio, D and Ballarino, M},
title = {LncRNA HSCHARME is altered in human cardiomyopathies and promotes stem cell-derived cardiomyogenesis via splicing regulation.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7880},
pmid = {40849301},
issn = {2041-1723},
support = {RM123188F6B80CE4//Sapienza Università di Roma (Sapienza University of Rome)/ ; AR223188B40CB2D0//Sapienza Università di Roma (Sapienza University of Rome)/ ; B83C22002870006//Ministero dell'Istruzione, dell'Università e della Ricerca (Ministry of Education, University and Research)/ ; B53D23016090006//Ministero dell'Istruzione, dell'Università e della Ricerca (Ministry of Education, University and Research)/ ; B53D23026140001//Ministero dell'Istruzione, dell'Università e della Ricerca (Ministry of Education, University and Research)/ ; B83C22002860006//Ministero dell'Istruzione, dell'Università e della Ricerca (Ministry of Education, University and Research)/ ; B53D23016090006//Ministero dell'Istruzione, dell'Università e della Ricerca (Ministry of Education, University and Research)/ ; DBA.AD005.225-NUTRAGE-FOE2021//Consiglio Nazionale delle Ricerche (National Research Council)/ ; DSB.AD006.371-InvAt-FOE2022//Consiglio Nazionale delle Ricerche (National Research Council)/ ; },
mesh = {Humans ; *RNA, Long Noncoding/genetics/metabolism ; *Myocytes, Cardiac/metabolism/cytology ; Polypyrimidine Tract-Binding Protein/metabolism/genetics ; Animals ; Cell Differentiation/genetics ; Induced Pluripotent Stem Cells/metabolism/cytology ; Mice ; *Cardiomyopathies/genetics/metabolism/pathology ; *RNA Splicing/genetics ; Heterogeneous-Nuclear Ribonucleoproteins/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {A growing body of evidence suggests that tissue-specific lncRNAs play pivotal roles in the heart. Here, we exploit the synteny between the mouse and human genomes to identify the human lncRNA HSCHARME and combine single-cell transcriptomics, CAGE-seq data, RNA-FISH imaging and CRISPR/Cas9 genome editing to document its role in cardiomyogenesis. By investigating the mechanism of action of HSCHARME in hiPSC-derived cardiomyocytes, we report that the locus produces the major pCHARME isoform that associates with SC35-containing speckles and interacts with the splicing regulator PTBP1. Consistently, the functional inactivation of pCHARME influences the splicing of cardiac-specific pre-mRNAs and impacts their expression, which reflects a decline in cardiomyocyte differentiation and physiology. In line with a possible association with disease, large-scale analysis of the lncRNA expression across cardiomyopathy patients reveals increased levels of pCHARME in hypertrophic and dilated hearts. We also find that HSCHARME dosage can modulate the expression of a subset of disease-associated targets. Our findings provide mechanistic insights into the role of pCHARME in cardiac cells with potential implications for disease.},
}

Humans
*RNA, Long Noncoding/genetics/metabolism
*Myocytes, Cardiac/metabolism/cytology
Polypyrimidine Tract-Binding Protein/metabolism/genetics
Animals
Cell Differentiation/genetics
Induced Pluripotent Stem Cells/metabolism/cytology
Mice
*Cardiomyopathies/genetics/metabolism/pathology
*RNA Splicing/genetics
Heterogeneous-Nuclear Ribonucleoproteins/metabolism/genetics
CRISPR-Cas Systems

@article {pmid40848256,
year = {2025},
author = {Guo, JA and Gong, D and Evans, K and Takahashi, K and Jambhale, AD and Shiau, C and Yu, P and Wang, S and Wu, WW and Chugh, S and Kapner, KS and Dilly, J and Zhao, D and Chen, P and Smith, EL and Mancias, JD and Vazquez, F and Singh, H and Hwang, WL and Aguirre, AJ},
title = {Integrative genomic identification of therapeutic targets for pancreatic cancer.},
journal = {Cell reports},
volume = {44},
number = {9},
pages = {116191},
doi = {10.1016/j.celrep.2025.116191},
pmid = {40848256},
issn = {2211-1247},
abstract = {Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease, and new therapeutic strategies are urgently needed. Here, we conduct an integrative, genome-scale examination of genetic dependencies and cell surface targets using CRISPR-Cas screening and multi-omic data, including single-nucleus and spatial transcriptomic data from patient tumors. We systematically identify clinically tractable and biomarker-linked PDAC dependencies, including CDS2 as a synthetic lethal target in cancer cells expressing signatures of epithelial-to-mesenchymal transition. We examine biomarkers and co-dependencies of the KRAS oncogene, defining gene expression signatures of sensitivity and resistance associated with response to pharmacological inhibition of KRAS. mRNA and protein profiling reveal cell surface protein-encoding genes with robust expression in patient tumors and minimal expression in non-malignant tissues. Furthermore, we define intratumoral and interpatient heterogeneity of target gene expression and identify orthogonal targets that suggest combinatorial strategies. Collectively, this work identifies multiple targets that may inform therapeutic strategies for patients with PDAC.},
}

@article {pmid40845808,
year = {2025},
author = {Yang, KK and Amini, AP},
title = {Simplifying protein engineering with deep learning.},
journal = {Cell},
volume = {188},
number = {17},
pages = {4477-4479},
doi = {10.1016/j.cell.2025.07.037},
pmid = {40845808},
issn = {1097-4172},
mesh = {*Deep Learning ; *Protein Engineering/methods ; Gene Editing/methods ; Humans ; CRISPR-Cas Systems ; Proteins/genetics ; },
abstract = {When it comes to deep learning for protein engineering, there is strength in simplicity. In this issue of Cell, through thoughtful deployment of existing fixed-backbone sequence design models, Caixia Gao and colleagues engineer diverse genome editing systems with improved functionality, enabling powerful capabilities in fine-grained and large-scale genome editing as demonstrated through strong experimental validation.},
}

*Deep Learning
*Protein Engineering/methods
Gene Editing/methods
Humans
CRISPR-Cas Systems
Proteins/genetics

@article {pmid40842235,
year = {2025},
author = {Moon, SY and Kim, MR and An, NY and Noh, MH and Lee, JY},
title = {Dual-mode CRISPRa/i for genome-scale metabolic rewiring in Escherichia coli.},
journal = {Nucleic acids research},
volume = {53},
number = {15},
pages = {},
doi = {10.1093/nar/gkaf818},
pmid = {40842235},
issn = {1362-4962},
support = {//Ministry of Science and ICT/ ; //National Research Foundation/ ; 2022M3A9B6082671//Republic of Korea: Bio & Medical Technology Development Program/ ; 2024M3J5A1045980//Artificial Cellular Organelles Project/ ; //Korea-US Collaborative Research Fund (KUCRF)/ ; RS-2024-00468410//MSIT and the Ministry of Health & Welfare, Republic of Korea/ ; },
mesh = {*Escherichia coli/genetics/metabolism ; Cyclic AMP Receptor Protein/genetics/metabolism ; *CRISPR-Cas Systems ; Gene Expression Regulation, Bacterial ; *Escherichia coli Proteins/genetics/metabolism ; *Metabolic Engineering/methods ; Genome, Bacterial ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {CRISPR (clustered regularly interspaced palindromic repeats)-mediated transcriptional regulation is a powerful and programmable approach for controlling gene expression. While CRISPR-based gene repression is well established in bacteria, simultaneous activation and repression remain challenging due to the limited availability of effective bacterial activation domains. Here, we provide an efficient dual-mode CRISPR activation and interference (CRISPRa/i) system that integrates an evolved protospacer adjacent motif (PAM)-flexible dxCas9 with an engineered Escherichia coli cAMP receptor protein (CRP). Through systematic optimization of the CRP domains and linkers, we developed a versatile effector capable of precise gene expression control when combined with dxCas9. Our dxCas9-CRP system demonstrated robust activation of upstream regulatory regions and effective repression of coding sequences, enabling targeted and programmable gene regulation. Using dual-fluorescent reporters, we validated the ability of this system to concurrently regulate multiple genes. Furthermore, with pooled guide RNA libraries, we applied the dxCas9-CRP system to increase violacein production in E. coli via genome-scale activation and repression in a coordinated manner, successfully identifying key regulatory targets that significantly increase production. Overall, this dual-mode CRISPRa/i system advances the potential for bacterial metabolic pathway rewiring, providing precise and flexible control for a wide range of biotechnological applications.},
}

*Escherichia coli/genetics/metabolism
Cyclic AMP Receptor Protein/genetics/metabolism
*CRISPR-Cas Systems
Gene Expression Regulation, Bacterial
*Escherichia coli Proteins/genetics/metabolism
*Metabolic Engineering/methods
Genome, Bacterial
RNA, Guide, CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40841511,
year = {2025},
author = {Potts, MA and Mizutani, S and Deng, Y and Vaidyanathan, S and Ting, KE and Giner, G and Sridhar, S and Shenoy, G and Liao, Y and Diepstraten, ST and Kueh, AJ and Pal, M and Healey, G and Tai, L and Wang, Z and König, C and Kaloni, D and Whelan, L and Milevskiy, MJG and Coughlan, HD and Pomilio, G and Wei, AH and Visvader, JE and Papenfuss, AT and Wilcox, S and Jeyasekharan, AD and Shi, W and Lelliott, EJ and Kelly, GL and Brown, KK and Strasser, A and Herold, MJ},
title = {Genome-wide in vivo CRISPR screens identify GATOR1 complex as a tumor suppressor in Myc-driven lymphoma.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7582},
pmid = {40841511},
issn = {2041-1723},
mesh = {Animals ; *Lymphoma/genetics/metabolism/pathology ; Mice ; Mechanistic Target of Rapamycin Complex 1/metabolism/genetics ; *Proto-Oncogene Proteins c-myc/genetics/metabolism ; Humans ; CRISPR-Cas Systems ; TOR Serine-Threonine Kinases/metabolism ; *Genes, Tumor Suppressor ; Mice, Knockout ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cell Line, Tumor ; Signal Transduction ; Tumor Suppressor Proteins/genetics/metabolism ; GTPase-Activating Proteins/genetics/metabolism ; Mice, Transgenic ; },
abstract = {Identifying tumor suppressor genes is predicted to inform on the development of novel strategies for cancer therapy. To identify new lymphoma driving processes that cooperate with oncogenic MYC, which is abnormally highly expressed in ~70% of human cancers, we use a genome-wide CRISPR gene knockout screen in Eµ-Myc;Cas9 transgenic hematopoietic stem and progenitor cells in vivo. We discover that loss of any of the GATOR1 complex components - NPRL3, DEPDC5, NPRL2 - significantly accelerates c-MYC-driven lymphoma development in mice. MYC-driven lymphomas lacking GATOR1 display constitutive mTOR pathway activation and are highly sensitive to mTOR inhibitors, both in vitro and in vivo. These findings identify GATOR1 suppression of mTORC1 as a tumor suppressive mechanism in MYC-driven lymphomagenesis and suggest an avenue for therapeutic intervention in GATOR1-deficient lymphomas through mTOR inhibition.},
}

Animals
*Lymphoma/genetics/metabolism/pathology
Mice
Mechanistic Target of Rapamycin Complex 1/metabolism/genetics
*Proto-Oncogene Proteins c-myc/genetics/metabolism
Humans
CRISPR-Cas Systems
TOR Serine-Threonine Kinases/metabolism
*Genes, Tumor Suppressor
Mice, Knockout
Clustered Regularly Interspaced Short Palindromic Repeats
Cell Line, Tumor
Signal Transduction
Tumor Suppressor Proteins/genetics/metabolism
GTPase-Activating Proteins/genetics/metabolism
Mice, Transgenic

@article {pmid40838896,
year = {2025},
author = {Losier, TT and King, KE and Rousseaux, MWC and Russell, RC},
title = {Identification of organelle-specific autophagy regulators from tandem CRISPR screens.},
journal = {The Journal of cell biology},
volume = {224},
number = {10},
pages = {},
doi = {10.1083/jcb.202405138},
pmid = {40838896},
issn = {1540-8140},
support = {#153034/CAPMC/CIHR/Canada ; PJT-169097/CAPMC/CIHR/Canada ; #2023-05587//Natural Sciences and Engineering Research Council of Canada/ ; RGPIN-2019-04133//Natural Sciences and Engineering Research Council of Canada/ ; DGECR-2019-00369//Natural Sciences and Engineering Research Council of Canada/ ; 201911CGV-434032-74238//Natural Sciences and Engineering Research Council of Canada/ ; //Natural Sciences and Engineering Research Council Postgraduate Scholarship/ ; },
mesh = {*Autophagy/genetics ; Humans ; Signal Transduction ; Endoplasmic Reticulum/metabolism/genetics ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Organelles/metabolism ; *Autophagy-Related Proteins/metabolism/genetics ; HeLa Cells ; },
abstract = {Autophagy is a conserved degradative process that promotes cellular homeostasis under stress conditions. Under nutrient starvation, autophagy is nonselective, promoting indiscriminate breakdown of cytosolic components. Conversely, selective autophagy is responsible for the specific turnover of damaged organelles. We hypothesized that selective autophagy may be regulated by signaling pathways distinct from those controlling starvation-induced autophagy, thereby promoting organelle turnover. To address this question, we conducted kinome-wide CRISPR screens to identify distinct signaling pathways responsible for the regulation of basal autophagy, starvation-induced autophagy, and two types of selective autophagy, ER-phagy and pexophagy. These parallel screens identified both known and novel autophagy regulators, some common to all conditions and others specific to selective autophagy. More specifically, CDK11A and NME3 were further characterized to be selective ER-phagy regulators. Meanwhile, PAN3 and CDC42BPG were identified as an activator and inhibitor of pexophagy, respectively. Collectively, these datasets provide the first comparative description of the kinase signaling that defines the regulation of selective autophagy and bulk autophagy.},
}

*Autophagy/genetics
Humans
Signal Transduction
Endoplasmic Reticulum/metabolism/genetics
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Organelles/metabolism
*Autophagy-Related Proteins/metabolism/genetics
HeLa Cells

@article {pmid40828859,
year = {2025},
author = {Saunier, M and Humbert, A and Kreis, V and Peltier, J and Tisba, A and Auxilien, S and Blum, M and Caldelari, I and Lucier, JF and Ueda, J and Gautheret, D and Toffano-Nioche, C and Andreani, J and Fortier, LC and Soutourina, O},
title = {Deciphering the RNA-based regulation mechanism of the phage-encoded AbiF system in Clostridioides difficile.},
journal = {PLoS genetics},
volume = {21},
number = {8},
pages = {e1011831},
doi = {10.1371/journal.pgen.1011831},
pmid = {40828859},
issn = {1553-7404},
mesh = {*Clostridioides difficile/genetics/virology/pathogenicity ; Prophages/genetics ; CRISPR-Cas Systems/genetics ; *Bacteriophages/genetics ; Toxin-Antitoxin Systems/genetics ; Clostridium Infections/microbiology/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Bacterial Proteins/genetics ; Humans ; RNA, Bacterial/genetics ; Gene Expression Regulation, Bacterial ; },
abstract = {Clostridioides difficile is the major cause of nosocomial infections associated with antibiotic therapy. The severity of C. difficile infections increased worldwide with the emergence of hypervirulent strains, including 027 ribotype epidemic strains. Many aspects of C. difficile adaptation strategies during pathogenesis remain poorly understood. This pathogen thrives in gut communities that are rich in microbes and phages. To regulate horizontal transfer of genetic material during its infection cycle, C. difficile relies on diverse mechanisms. More specifically, CRISPR (clustered regularly interspaced short palindromic repeats)-Cas and Toxin-Antitoxin (TA) systems contribute to prophage maintenance, prevention of phage infection, and stress response. Abortive infection (Abi) systems can provide additional lines of anti-phage defense. RNAs have emerged as key components of these systems including CRISPR RNAs and antitoxin RNAs within type I and type III TA. We report here the identification of a new AbiF-like system within a prophage of the hypervirulent C. difficile strain R20291. It is associated with an Abi_2/AbiD/F protein family largely distributed in Bacillota and Pseudomonadota with structural links to ancestral Cas13 proteins at the origin of the RNA-targeting CRISPR-Cas13 systems. We demonstrated toxic activity of the AbiFCd protein in C. difficile and in Escherichia coli and negative regulation of the abiFCd expression by an associated non-coding RNA RCd22. RCd22 contains two conserved abiF motifs and is active both in cis and in trans to neutralize the toxin by direct RNA-protein interaction, similar to RNA antitoxin in type III TA. A mass spectrometry interactomics analysis of protein fractions from MS2-Affinity Purification coupled with RNA sequencing (MAPS) revealed the AbiFCd protein among the most enriched RCd22 partners in C. difficile. Structural modeling of the RNA-protein complex and mutagenesis analysis revealed key positions on both protein and RNA partners for this interaction and toxic activity. In summary, these findings provide valuable insights into the mechanisms of interaction between bacteria and phages, which are pertinent to the advancement of phage therapy, genome editing, epidemiological surveillance, and the formulation of novel therapeutic approaches.},
}

*Clostridioides difficile/genetics/virology/pathogenicity
Prophages/genetics
CRISPR-Cas Systems/genetics
*Bacteriophages/genetics
Toxin-Antitoxin Systems/genetics
Clostridium Infections/microbiology/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Bacterial Proteins/genetics
Humans
RNA, Bacterial/genetics
Gene Expression Regulation, Bacterial

@article {pmid40728278,
year = {2025},
author = {Joung, Y and Han, DK and Jang, H and Kang, T and Chen, L and Choo, J},
title = {Dual-Pathway Lateral Flow Assay for Rapid and Sensitive SARS-CoV-2 RNA Detection via CRISPR/Cas13a-Mediated SERS.},
journal = {ACS sensors},
volume = {10},
number = {8},
pages = {6253-6262},
doi = {10.1021/acssensors.5c02084},
pmid = {40728278},
issn = {2379-3694},
mesh = {*Spectrum Analysis, Raman/methods ; *SARS-CoV-2/genetics/isolation & purification ; *RNA, Viral/analysis/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; *COVID-19/diagnosis/virology ; *COVID-19 Nucleic Acid Testing/methods ; },
abstract = {Reverse transcription-polymerase chain reaction (RT-PCR) has been the gold standard for SARS-CoV-2 detection during the COVID-19 pandemic. However, its requirement for RNA-to-DNA conversion, reliance on centralized laboratory infrastructure, and lengthy turnaround times have limited its application in point-of-care (POC) settings. CRISPR/Cas13a-mediated lateral flow assays (LFAs) have emerged as promising alternatives for direct RNA analysis, yet their two-step workflows introduce procedural complexity and reduce sensitivity. To overcome these limitations, we developed a dual-pathway LFA strip based on surface-enhanced Raman scattering (SERS), which integrates CRISPR/Cas13a-mediated RNA cleavage and SERS detection into a single, portable platform. The device utilizes five vertically stacked paper layers with distinct geometries, enabling sequential CRISPR reaction and SERS quantification through two independent pathways. When tested with SARS-CoV-2 ORF1ab RNA targets, the system exhibited an 80-fold increase in sensitivity and a 10 min reduction in assay time compared to conventional fluorescence assays. Clinical validation using 18 samples (13 positives and 5 negatives) demonstrated high diagnostic accuracy, fully consistent with RT-PCR results. By unifying CRISPR-based RNA recognition and SERS signal amplification in a user-friendly format, this dual-pathway LFA strip offers a rapid, ultrasensitive, and practical diagnostic tool for infectious diseases in POC settings.},
}

*Spectrum Analysis, Raman/methods
*SARS-CoV-2/genetics/isolation & purification
*RNA, Viral/analysis/genetics
*CRISPR-Cas Systems/genetics
Humans
*COVID-19/diagnosis/virology
*COVID-19 Nucleic Acid Testing/methods

@article {pmid40675426,
year = {2025},
author = {Li, W and Tang, Z and Zhu, X and Wang, B and Zhang, X and Ji, Z and Cheng, S},
title = {Ultrasensitive detection of MMP-2 via T7 RNA polymerase and CRISPR/Cas13a-Enhanced electrochemiluminescence biosensor for COPD diagnosis.},
journal = {Methods (San Diego, Calif.)},
volume = {242},
number = {},
pages = {80-88},
doi = {10.1016/j.ymeth.2025.07.005},
pmid = {40675426},
issn = {1095-9130},
mesh = {*Matrix Metalloproteinase 2/analysis/blood/genetics ; *Biosensing Techniques/methods ; Humans ; CRISPR-Cas Systems/genetics ; Limit of Detection ; Luminescent Measurements/methods ; *Electrochemical Techniques/methods ; *Pulmonary Disease, Chronic Obstructive/diagnosis/blood ; *DNA-Directed RNA Polymerases/genetics/metabolism ; *Viral Proteins/genetics/metabolism ; Metal Nanoparticles/chemistry ; Gold/chemistry ; Biomarkers ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {In this work, an electrochemiluminescence (ECL) biosensor integrating T7 RNA polymerase amplification and CRISPR/Cas13a-mediated signal enhancement was developed for the ultrasensitive detection of matrix metalloproteinase-2 (MMP-2), a key biomarker associated with chronic inflammatory diseases such as COPD. A peptide nucleic acid (PNA) probe was designed to respond specifically to MMP-2 cleavage, enabling the release of DNA templates for subsequent T7 RNA polymerase-driven transcription amplification. The generated RNA triggers the collateral cleavage activity of CRISPR/Cas13a, resulting in a significant amplification of the ECL signal. The biosensor's surface was constructed using a AuNPs/Ti3C2Tx/Ru(II)-PEI nanocomposite, which enhanced signal transduction and stability. Under optimized conditions, the proposed biosensor achieved a detection limit as low as 62.05 fM, demonstrating superior sensitivity compared to conventional methods, as summarized in Table 1. The platform also exhibited excellent specificity and anti-interference capability, ensuring reliable detection of MMP-2 in complex biological samples. This study provides a simple yet highly efficient strategy for enzymatic biomarker detection, offering great potential for clinical applications in early disease diagnosis and monitoring.},
}

*Matrix Metalloproteinase 2/analysis/blood/genetics
*Biosensing Techniques/methods
Humans
CRISPR-Cas Systems/genetics
Limit of Detection
Luminescent Measurements/methods
*Electrochemical Techniques/methods
*Pulmonary Disease, Chronic Obstructive/diagnosis/blood
*DNA-Directed RNA Polymerases/genetics/metabolism
*Viral Proteins/genetics/metabolism
Metal Nanoparticles/chemistry
Gold/chemistry
Biomarkers
Nucleic Acid Amplification Techniques/methods

@article {pmid40626419,
year = {2025},
author = {Jacobebbinghaus, N and Bigge, F and Saudhof, M and Hübner, W and Kruse, O and Baier, T},
title = {Transcriptional gene fusions via targeted integration at safe harbors for high transgene expression in Chlamydomonas reinhardtii.},
journal = {The New phytologist},
volume = {247},
number = {6},
pages = {2665-2677},
doi = {10.1111/nph.70368},
pmid = {40626419},
issn = {1469-8137},
support = {2221NR063X//Federal Ministry of Agriculture, Food and Regional Identity/ ; },
mesh = {*Chlamydomonas reinhardtii/genetics/metabolism ; *Transgenes/genetics ; *Gene Fusion ; *Transcription, Genetic ; CRISPR-Cas Systems/genetics ; Gene Editing ; Terpenes/metabolism ; },
abstract = {Conventional genetic engineering in green microalgae employs error-prone nonhomologous end joining to integrate recombinant DNA at double-strand breaks generated at random positions across the nuclear genome. This typically results in variable transcription strength and requires a labor-intensive screening procedure to identify transformants with sufficient expression. Current advances in genome editing enable scar-less integration of DNA at any desired locus for engineered bioproduction. We optimized construct design for predictable transgene expression at a high level, significantly improved scar-less integration rates into the nuclear genome via homology arm length optimization and quantified endogenous gene expression in vivo. Subsequently, endogenous genes were successfully targeted via Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9 (CRISPR/Cas9) to evaluate their capacity for high transgene expression and terpenoid production. Highest scar-less homology-directed repair efficiency was achieved with 50 bp homology arms. The Light harvesting Chl a/b binding protein of LHCII (LHCBM1) locus was found to be differentially expressed under several light intensities and allows an 8.6-fold increase in transgenic protein accumulation compared with random insertion approaches. Co-expression of a functional sesquiterpene synthase achieved a 60-fold increase in valencene production compared with previous attempts. We showed LHCBM1 locus constitutes a genetic safe harbor for transgene expression and demonstrates the potential of C. reinhardtii as a green cell factory.},
}

*Chlamydomonas reinhardtii/genetics/metabolism
*Transgenes/genetics
*Gene Fusion
*Transcription, Genetic
CRISPR-Cas Systems/genetics
Gene Editing
Terpenes/metabolism

@article {pmid40338215,
year = {2025},
author = {Wu, S and Yuan, J and Xi, X and Wang, L and Li, Y and Wang, Y and Lin, J},
title = {A Colorimetric Biosensor Integrating Rotifer-Mimicking Magnetic Separation with RAA/CRISPR-Cas12a for Rapid and Sensitive Detection of Salmonella.},
journal = {ACS sensors},
volume = {10},
number = {8},
pages = {5473-5483},
doi = {10.1021/acssensors.4c03356},
pmid = {40338215},
issn = {2379-3694},
mesh = {*Salmonella/isolation & purification/genetics ; *Biosensing Techniques/methods ; *Colorimetry/methods ; *CRISPR-Cas Systems ; Limit of Detection ; Milk/microbiology ; *Rotifera/chemistry ; Gold/chemistry ; Animals ; Food Microbiology ; Nucleic Acid Amplification Techniques ; Metal Nanoparticles/chemistry ; Food Contamination/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Efficient detection of foodborne bacteria is crucial for ensuring food safety, yet current methods often fall short in balancing speed, accuracy, sensitivity, and cost. This study presents an integrated biosensing platform for the rapid and sensitive detection of Salmonella in large-volume food samples. The platform incorporates a Rotifer-Mimicking Magnetic Separator (RMMS) that enhances the sample pretreatment by effectively mixing and isolating the bacteria from the sample. Coupled with this, the colorimetric biosensor utilizes a streamlined one-pot system that combines Recombinase Aided Amplification (RAA), betaine, and CRISPR-Cas12a to enable efficient pathogen detection. Initially, phenylboronic acid-modified magnetic beads (PBA-MBs) capture Salmonella, forming bacteria-PBA-MB complexes, which are then isolated using the RMMS. Target DNA amplicons activate ribonucleoprotein complexes, and Au@PtNPs-MBs with linker single DNAs are cleaved to release Au@PtNPs. The Au@PtNPs catalyze the H2O2-3,3',5,5'-tetramethylbenzidine, producing a visible blue color that indicates Salmonella concentration. This biosensor successfully detects Salmonella in 40 mL spiked milk samples within 75 min, achieving a detection limit of 89 CFU/mL. This work offers a simple, sensitive, low-cost detection method with potential applications in on-site testing, significantly enhancing food safety monitoring.},
}

*Salmonella/isolation & purification/genetics
*Biosensing Techniques/methods
*Colorimetry/methods
*CRISPR-Cas Systems
Limit of Detection
Milk/microbiology
*Rotifera/chemistry
Gold/chemistry
Animals
Food Microbiology
Nucleic Acid Amplification Techniques
Metal Nanoparticles/chemistry
Food Contamination/analysis
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40838720,
year = {2025},
author = {Ferronato, GA and Silveira, JC and Ferraz, MAMM},
title = {Potential of small extracellular vesicles as Cas9 delivery tool: A promising approach for gene editing livestock gametes and embryos.},
journal = {Biology of reproduction},
volume = {},
number = {},
pages = {},
doi = {10.1093/biolre/ioaf195},
pmid = {40838720},
issn = {1529-7268},
abstract = {Genome editing is a rapidly advancing technology with transformative potential in livestock, offering opportunities that range from enhanced production traits to the generation of biomedical models for human disease and xenotransplantation. The CRISPR/Cas9 system, originally identified as a bacterial defense mechanism, has become the most widely used tool for precise genome editing. In this review, we first summarize the potential applications of CRISPR/Cas9 in livestock and highlight notable successes to date. We then address the ongoing challenges associated with delivering CRISPR/Cas9 into gametes and embryos, as current methods such as microinjection and electroporation often result in high mosaicism and cellular damage. We subsequently introduce extracellular vesicles (EVs) as a promising alternative delivery system. Secreted by virtually all cell types, EVs can efficiently transport bioactive molecules and are readily internalized by gametes and embryos. Although EV-mediated delivery of CRISPR/Cas9 has shown success in somatic cells, its use in reproductive cells remains largely unexplored. We review emerging strategies for loading EVs with CRISPR/Cas components and discuss the potential advantages of combining this approach with recently developed smaller Cas variants to overcome delivery barriers. Collectively, these innovations support the promise of EVs as a biologically compatible, efficient, and minimally invasive system for targeted genome editing in livestock reproduction.},
}

@article {pmid40838475,
year = {2025},
author = {},
title = {RETRACTION: Spider Eye Development Editing and Silk Fiber Engineering Using CRISPR-Cas.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {},
number = {},
pages = {e202517416},
doi = {10.1002/anie.202517416},
pmid = {40838475},
issn = {1521-3773},
}

@article {pmid40835841,
year = {2025},
author = {Watson, LC and Sala, KA and Bernitz, N and Baumgärtel, L and Pallett, MA and Marzook, NB and Straker, LC and Peng, D and Collinson, L and Sateriale, A},
title = {Targeted CRISPR screens reveal genes essential for Cryptosporidium survival in the host intestine.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7749},
pmid = {40835841},
issn = {2041-1723},
support = {CC2063/WT_/Wellcome Trust/United Kingdom ; CC2063/WT_/Wellcome Trust/United Kingdom ; CC2063/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; *Cryptosporidiosis/parasitology ; *Cryptosporidium/genetics/pathogenicity ; *CRISPR-Cas Systems ; *Intestines/parasitology ; Mice ; *Protozoan Proteins/genetics/metabolism ; *Genes, Essential ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Humans ; Gene Knockout Techniques ; Cryptosporidium parvum/genetics ; Protozoan Vaccines/immunology ; },
abstract = {The Cryptosporidium parasite is one of the leading causes of diarrheal morbidity and mortality in children, and adolescent infections are associated with chronic malnutrition. There are no vaccines available for protection and only one drug approved for treatment that has limited efficacy. A major barrier to developing new therapeutics is a lack of foundational knowledge of Cryptosporidium biology, including which parasite genes are essential for survival and virulence. Here, we iteratively improve the tools for genetically manipulating Cryptosporidium and develop a targeted CRISPR-based screening method to rapidly assess how the loss of individual parasite genes influence survival in vivo. Using this method, we examine the parasite's pyrimidine salvage pathway and a set of leading Cryptosporidium vaccine candidates. From this latter group, using inducible knockout, we determined the parasite gene known as Cp23 to be essential for survival in vivo. Parasites deficient in Cp23 were able to replicate within and emerge from infected epithelial cells, yet unable to initiate gliding motility which is required for the reinfection of neighbouring cells. The targeted screening method presented here is highly versatile and will enable researchers to more rapidly expand the knowledge base for Cryptosporidium infection biology, paving the way for new therapeutics.},
}

Animals
*Cryptosporidiosis/parasitology
*Cryptosporidium/genetics/pathogenicity
*CRISPR-Cas Systems
*Intestines/parasitology
Mice
*Protozoan Proteins/genetics/metabolism
*Genes, Essential
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Humans
Gene Knockout Techniques
Cryptosporidium parvum/genetics
Protozoan Vaccines/immunology

@article {pmid40835811,
year = {2025},
author = {Luzics, S and Baka, E and Otto, M and Kosztik, J and Szalontai, H and Bata-Vidács, I and Nagy, I and Tóth, Á and Táncsics, A and Pápai, M and Nagy, I and Orsini, M and Kukolya, J},
title = {High-quality de novo genome assembly and functional genomic insights into Thermobifida alba DSM43795[T], a mesophilic actinobacterium isolated from garden soil.},
journal = {Biologia futura},
volume = {},
number = {},
pages = {},
pmid = {40835811},
issn = {2676-8607},
support = {K142686//National Research, Development and Innovation Office/ ; EKÖP- 24- VI/MATE-3//Ministry for Culture and Innovation from the source of the National Research, Development and Innovation Fund/ ; EKÖP-MATE/2024/25/D//Ministry for Culture and Innovation from the source of the National Research, Development and Innovation Fund/ ; },
abstract = {Thermobifida alba DSM43795[T], a mesophilic actinobacterium isolated from garden soil, plays a vital role in lignocellulose degradation and holds biotechnological and pharmaceutical potential. We present a high-quality, complete de novo genome assembly of T. alba DSM43795[T] using combined PacBio long-read and Illumina short-read sequencing, resulting in a single circular chromosome of 4.9 Mbp with 72.1% GC content. Comparative genomics with the thermophilic relative T. fusca YX revealed 83.39% average nucleotide identity and extensive genome synteny alongside niche-specific differences. Functional annotation identified 4345 genes, including a rich complement of carbohydrate-active enzymes (CAZymes) such as glycoside hydrolases (GHs), esterases, and polysaccharide lyases, supporting versatile plant biomass degradation. GH gene sets were largely conserved between the species in both gene number and distribution, but T. alba uniquely encodes a novel GH10 endo-xylanase near a characterised palindrome regulatory sequence, indicating species-specific regulation. We hypothesise that thermophilic adaptation in T. fusca requires more proteins for ribosome integrity and amino acid metabolism, with reduced emphasis on carbohydrate metabolism and defence compared to T. alba. Moreover, T. alba harbours a broader array of defence-related genes and mobile genetic elements, including integrases and transposases. Although lacking a complete CRISPR-Cas system, two CRISPR arrays were detected, suggesting alternative immune strategies. Virulence factor homologs shared by both species likely reflect environmental survival rather than pathogenicity. This genomic characterisation elucidates T. alba's metabolic versatility and ecological adaptations, laying the groundwork for its potential applications in biomass conversion, environmental biotechnology, and drug discovery.},
}

@article {pmid40835333,
year = {2025},
author = {Sanders, J and Lin, S},
title = {Molecular techniques for understanding harmful algal blooms: A review.},
journal = {Harmful algae},
volume = {148},
number = {},
pages = {102909},
doi = {10.1016/j.hal.2025.102909},
pmid = {40835333},
issn = {1878-1470},
mesh = {*Harmful Algal Bloom ; Dinoflagellida/classification/genetics ; Cyanobacteria/classification/genetics ; Seawater ; Fresh Water ; *Water Pollution/analysis/prevention & control ; Molecular Probe Techniques ; Enzyme-Linked Immunosorbent Assay ; Molecular Diagnostic Techniques ; CRISPR-Cas Systems ; In Situ Hybridization, Fluorescence ; DNA Barcoding, Taxonomic ; *Molecular Typing/methods ; },
abstract = {Harmful algal blooms (HABs) are intricate ecological events caused by diverse algal species and are influenced by a myriad of biotic and abiotic factors. The urgently needed development of effective prevention and control techniques face two primary challenges. The first challenge is the technical shortfalls in rapidly identifying and monitoring the causative species. The second challenge is the absence of research frameworks and technologies for accurately diagnosing the primary drivers of these blooms. Molecular techniques offer promising solutions to these issues, and research in this field has seen significant growth over the past two decades. Previous reviews have predominantly focused on species identification and monitoring, leaving the status of bloom driver studies less clear. This review provides a comprehensive overview of molecular techniques for HAB identification and driver analysis. HAB-specific use cases of techniques and comparison between them based on technical specifications are provided. Nucleic acid-based techniques presently dominate over antibody-based techniques due to their tunable taxon-specificity and ease to prepare probes. In situ applications and monitoring platforms still have a large room for improvement. The omics approach is the most promising choice for unraveling HAB drivers but requires a framework and a quantitative model for estimating the contribution of potential responsible factors. Future prospects relating to particular needs in HAB research and emerging technologies are also discussed.},
}

*Harmful Algal Bloom
Dinoflagellida/classification/genetics
Cyanobacteria/classification/genetics
Seawater
Fresh Water
*Water Pollution/analysis/prevention & control
Molecular Probe Techniques
Enzyme-Linked Immunosorbent Assay
Molecular Diagnostic Techniques
CRISPR-Cas Systems
In Situ Hybridization, Fluorescence
DNA Barcoding, Taxonomic
*Molecular Typing/methods

@article {pmid40782800,
year = {2025},
author = {Traxler, P and Reichl, S and Folkman, L and Shaw, L and Fife, V and Nemc, A and Pasajlic, D and Kusienicka, A and Barreca, D and Fortelny, N and Rendeiro, AF and Halbritter, F and Weninger, W and Decker, T and Farlik, M and Bock, C},
title = {Integrated time-series analysis and high-content CRISPR screening delineate the dynamics of macrophage immune regulation.},
journal = {Cell systems},
volume = {16},
number = {8},
pages = {101346},
doi = {10.1016/j.cels.2025.101346},
pmid = {40782800},
issn = {2405-4720},
mesh = {*Macrophages/immunology/metabolism ; Animals ; Mice ; Signal Transduction ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Expression Regulation ; },
abstract = {Macrophages are innate immune cells involved in host defense. Dissecting the regulatory landscape that enables their swift and specific response to pathogens, we performed time-series analysis of gene expression and chromatin accessibility in murine macrophages exposed to various immune stimuli, and we functionally evaluated gene knockouts at scale using a combined CROP-seq and CITE-seq assay. We identified new roles of transcription regulators such as Spi1/PU.1 and JAK-STAT pathway members in immune cell homeostasis and response to pathogens. Macrophage activity was modulated by splicing proteins SFPQ and SF3B1, histone acetyltransferase EP300, cohesin subunit SMC1A, and mediator complex proteins MED8 and MED14. We further observed crosstalk among immune signaling pathways and identified molecular drivers of pathogen-induced dynamics. In summary, this study establishes a time-resolved regulatory map of pathogen response in macrophages, and it describes a broadly applicable method for dissecting immune-regulatory programs through integrative time-series analysis and high-content CRISPR screening. A record of this paper's transparent peer review process is included in the supplemental information.},
}

*Macrophages/immunology/metabolism
Animals
Mice
Signal Transduction
CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Gene Expression Regulation

@article {pmid40781772,
year = {2025},
author = {Kweon, J and Park, S and Jeon, MY and Lim, K and Jang, G and Jang, AH and Lee, M and Seok, C and Lee, C and Park, S and Ahn, J and Jang, J and Kim, N and Sung, YH and Kim, D and Kim, Y},
title = {High-efficiency base editing for nuclear and mitochondrial DNA with an optimized DYW-like deaminase.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2025.08.007},
pmid = {40781772},
issn = {1525-0024},
abstract = {CRISPR-based cytosine base editors enable precise genome editing without inducing double-stranded DNA breaks yet traditionally depend on a limited selection of deaminases from the APOBEC/AID or TadA families. Here, we present SsCBE, a CRISPR-based cytosine base editor utilizing SsdAtox, a DYW-like deaminase derived from the toxin of Pseudomonas syringae. Strategic engineering of SsdAtox has led to remarkable improvements in the base editing efficiency (by up to 8.4-fold) and specificity for SsCBE, while concurrently reducing cytotoxicity. Exhibiting exceptional versatility, SsCBE was delivered and efficiently applied using diverse delivery methods, including engineered virus-like particles. Its application has enabled targeted cytosine base editing in mouse zygotes and pioneering edits in mitochondrial DNA. SsCBE expands the genome editing toolbox by introducing a distinct deaminase scaffold with broad utility for both basic research and potential therapeutic applications.},
}

@article {pmid40763746,
year = {2025},
author = {Sun, Q and Ma, X and Ning, Q and Li, S and Wang, P and Tan, X and Jin, Q and Zheng, J and Li, Y and Dong, D},
title = {Systematic screening for functional exon-skipping isoforms using the CRISPR-RfxCas13d system.},
journal = {Cell systems},
volume = {16},
number = {8},
pages = {101351},
doi = {10.1016/j.cels.2025.101351},
pmid = {40763746},
issn = {2405-4720},
mesh = {Humans ; *Exons/genetics ; Protein Isoforms/genetics ; *Alternative Splicing/genetics ; Colorectal Neoplasms/genetics/pathology ; *CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Animals ; Mice ; RNA, Guide, CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cell Proliferation/genetics ; },
abstract = {Exon skipping (ES) is the most prevalent form of alternative splicing and a hallmark of tumorigenesis, yet its functional roles remain underexplored. Here, we present a CRISPR-RfxCas13d-based platform for transcript-specific silencing of ES-derived isoforms using guide RNAs (gRNAs) targeting exon-exon junctions. We designed a transcriptome-wide gRNA library against 3,744 human ES events and conducted loss-of-function screens in colorectal cancer (CRC) cells in vitro and in vivo. This screen uncovered multiple ES events essential for CRC growth, notably HMGN3 Δ6, an isoform arising from exon 6 skipping, which enhanced tumor proliferation. Functional validation confirmed the oncogenic role of HMGN3 Δ6 and its necessity for CRC progression. Our study establishes CRISPR-RfxCas13d as a powerful tool for isoform-specific functional genomics and reveals a widespread, previously uncharacterized layer of tumor biology driven by ES. These findings position ES-derived transcripts as promising targets for therapeutic intervention in cancer.},
}

Humans
*Exons/genetics
Protein Isoforms/genetics
*Alternative Splicing/genetics
Colorectal Neoplasms/genetics/pathology
*CRISPR-Cas Systems/genetics
Cell Line, Tumor
Animals
Mice
RNA, Guide, CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Cell Proliferation/genetics

@article {pmid40623538,
year = {2025},
author = {Peng, Z and Yang, T and Xu, S and Yang, B and Zhang, Z and Ding, M and Gu, W and Zheng, L},
title = {Aerobic exercise ameliorates skeletal muscle atrophy in atic knockout zebrafish through the oxidative phosphorylation pathway.},
journal = {Free radical biology & medicine},
volume = {238},
number = {},
pages = {653-668},
doi = {10.1016/j.freeradbiomed.2025.07.007},
pmid = {40623538},
issn = {1873-4596},
mesh = {Animals ; Zebrafish/genetics ; *Muscular Atrophy/genetics/metabolism/pathology/therapy ; Oxidative Phosphorylation ; *Muscle, Skeletal/metabolism/pathology ; Aminoimidazole Carboxamide/analogs & derivatives/metabolism ; Reactive Oxygen Species/metabolism ; Ribonucleotides/metabolism ; *Physical Conditioning, Animal ; *Zebrafish Proteins/genetics/metabolism ; *Hydroxymethyl and Formyl Transferases/genetics/metabolism ; Gene Knockout Techniques ; *Nucleotide Deaminases/genetics/metabolism ; Mice ; Mitochondria/metabolism ; CRISPR-Cas Systems ; Purines/metabolism ; Multienzyme Complexes ; },
abstract = {The mechanisms linking purine metabolism disorders to skeletal muscle pathology are unclear. This study constructed a CRISPR/Cas9-mediated zebrafish atic knockout model and a siRNA-interfered C2C12 myoblast cell model. We revealed a novel mechanism by which ATIC (5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase) deletion drove the atrophy of skeletal muscle through the downregulation of the oxidative phosphorylation of mitochondria (OXPHOS) pathway. It was found that atic/Atic knockout/knockdown led to the interruption of purine de novo synthesis, abnormal 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) accumulation, and blockage of inosine monophosphate (IMP) synthesis, which in turn triggered mitochondrial structural damage, dysfunction of complex I-V function, and a burst of reactive oxygen species (ROS), and ultimately triggered muscle atrophy through activation of the ubiquitin-proteasome system. The progressive aerobic intervention revealed that 8 weeks of training significantly restored skeletal muscle function in zebrafish atic[-/-] mutants, and the mechanism was related to the enhancement of mitochondrial biogenesis, up-regulation of the core complex expression of the OXPHOS pathway, and the improvement of ROS scavenging ability. These findings reveal that ATIC deficiency disrupts mitochondrial function through purine metabolism dysregulation, linking aberrant AICAR accumulation to OXPHOS impairment, which provides a theoretical basis for the early warning of muscular toxicity of targeted purine metabolizing drugs and lays a molecular foundation for the exercise rehabilitation strategy of metabolic myopathies.},
}

Animals
Zebrafish/genetics
*Muscular Atrophy/genetics/metabolism/pathology/therapy
Oxidative Phosphorylation
*Muscle, Skeletal/metabolism/pathology
Aminoimidazole Carboxamide/analogs & derivatives/metabolism
Reactive Oxygen Species/metabolism
Ribonucleotides/metabolism
*Physical Conditioning, Animal
*Zebrafish Proteins/genetics/metabolism
*Hydroxymethyl and Formyl Transferases/genetics/metabolism
Gene Knockout Techniques
*Nucleotide Deaminases/genetics/metabolism
Mice
Mitochondria/metabolism
CRISPR-Cas Systems
Purines/metabolism
Multienzyme Complexes

@article {pmid40456437,
year = {2025},
author = {Yoshida, S and Onozawa, M and Yokoyama, S and Matsukawa, T and Goto, H and Hirabayashi, S and Kondo, T and Hashimoto, D and Onodera, Y and Teshima, T},
title = {Peposertib suppresses generation of FLT3-internal tandem duplication formed by contralateral double nicks.},
journal = {Experimental hematology},
volume = {149},
number = {},
pages = {104819},
doi = {10.1016/j.exphem.2025.104819},
pmid = {40456437},
issn = {1873-2399},
mesh = {*fms-Like Tyrosine Kinase 3/genetics/antagonists & inhibitors ; Humans ; Mice ; Animals ; Phthalazines/pharmacology ; *Gene Duplication/drug effects ; *Leukemia, Myeloid, Acute/genetics/drug therapy ; *Tandem Repeat Sequences/drug effects ; Piperazines/pharmacology ; *DNA Breaks, Double-Stranded/drug effects ; CRISPR-Cas Systems ; Cell Line, Tumor ; },
abstract = {Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) is the most frequent gene mutation in acute myeloid leukemia. The consequences of FLT3-ITD have been analyzed in detail; however, the molecular mechanisms underlying the generation of FLT3-ITD remain to be elucidated. We analyzed FLT3-ITDs in clinical samples using deep sequencing and identified not only oligoclonal ITDs but also rare deletion clones clustered at the palindrome-like sequence at FLT3 exon 14. We hypothesized that FLT3 exon 14 is genetically unstable due to the palindrome-like sequence at the region and that genomic damage at the site initiates FLT3-ITD formation. We used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to induce DNA damage for creating artificial FLT3-ITDs in human and mouse cell lines. We found that double nicks on the adjacent contralateral strand most efficiently generate ITDs. The artificial ITDs resembled clinical ITDs in the length distribution and characteristics at the joint. We further compared the inhibitory effects of olaparib and peposertib, specific inhibitors of single-strand break (SSB) and double-strand break (DSB) repair, respectively. Peposertib remarkably reduced ITD formation, but olaparib did not affect the mutation pattern. The findings indicated that nonhomologous end joining has a crucial role in the generation of ITDs. Our data shed light to the new role of peposertib, which potentially suppresses the generation of de novo FLT3-ITDs caused by mis-repair events of the DNA damages in a clinical course.},
}

*fms-Like Tyrosine Kinase 3/genetics/antagonists & inhibitors
Humans
Mice
Animals
Phthalazines/pharmacology
*Gene Duplication/drug effects
*Leukemia, Myeloid, Acute/genetics/drug therapy
*Tandem Repeat Sequences/drug effects
Piperazines/pharmacology
*DNA Breaks, Double-Stranded/drug effects
CRISPR-Cas Systems
Cell Line, Tumor