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

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

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

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

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

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

@article {pmid40460174,
year = {2025},
author = {Tan, YS and Mo, BT and Li, GC and Guo, YR and Zhang, JZ and Wang, CZ},
title = {HarmGR13 mediates myo-inositol taste perception in Helicoverpa armigera larvae.},
journal = {PLoS genetics},
volume = {21},
number = {6},
pages = {e1011744},
doi = {10.1371/journal.pgen.1011744},
pmid = {40460174},
issn = {1553-7404},
mesh = {Animals ; *Inositol/metabolism ; Larva/genetics/physiology ; *Taste Perception/genetics ; *Insect Proteins/genetics/metabolism/chemistry ; Sensilla/metabolism/physiology ; *Moths/genetics/physiology ; CRISPR-Cas Systems ; Phylogeny ; Ribose/metabolism ; *Receptors, Cell Surface/genetics/metabolism ; Taste/genetics ; Helicoverpa armigera ; },
abstract = {Myo-inositol, a sugar alcohol produced by most plants, serves as a nutrient and feeding stimulant for many phytophagous insects. Inositol-sensitive taste sensilla have been characterized in many Lepidoptera larvae, but their molecular bases remain unclear. In this study, we determined the gustatory receptors (GRs) for myo-inositol in larva of Helicoverpa armigera, a worldwide crop pest. First, electrophysiological analyses revealed that medial sensilla styloconica strongly responded to myo-inositol and ribose, with weaker responses to xylose, and one GRN inside sensillum may mediate the response to these three chemicals. Based on phylogenetic analysis of sugar GRs of Lepidoptera insects and previous results on Bombyx mori, we then selected two candidate GRs, HarmGR13 and HarmGR11. Using CRISPR-Cas9, we generated knockout mutants for the two GR genes. Knocking out HarmGR13 abolished the responses of the sensilla to myo-inositol, ribose, and xylose, while knocking out HarmGR11 showed no changes. Behavioral assays confirmed that larvae of HarmGR13 homozygous mutant lost the feeding preference to myo-inositol which the wild-type larvae had. Further functional analysis with Xenopus oocytes expressing system and two-electrode voltage-clamping demonstrated that myo-inositol and ribose specifically induced concentration-dependent currents in HarmGR13-expressing oocytes. Structural predictions and molecular docking of HarmGR13 revealed three amino acid residues might be involved in ligand binding. Mutation of these residues resulted in loss of oocyte responses to myo-inositol and ribose. We reveal that HarmGR13 is a receptor that mediates the activity of the cells sensitive to inositol and ribose in larvae, providing new molecular targets for the strategy of regulating the feeding behavior of pests by modifying taste.},
}

Animals
*Inositol/metabolism
Larva/genetics/physiology
*Taste Perception/genetics
*Insect Proteins/genetics/metabolism/chemistry
Sensilla/metabolism/physiology
*Moths/genetics/physiology
CRISPR-Cas Systems
Phylogeny
Ribose/metabolism
*Receptors, Cell Surface/genetics/metabolism
Taste/genetics
Helicoverpa armigera

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

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

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

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

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

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

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

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

@article {pmid40478881,
year = {2025},
author = {Ratajczyk, EJ and Bath, J and Šulc, P and Doye, JPK and Louis, AA and Turberfield, AJ},
title = {Controlling DNA-RNA strand displacement kinetics with base distribution.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {23},
pages = {e2416988122},
doi = {10.1073/pnas.2416988122},
pmid = {40478881},
issn = {1091-6490},
support = {EP/W524311/1//UKRI | Engineering and Physical Sciences Research Council (EPSRC)/ ; CCF 2211794//National Science Foundation (NSF)/ ; },
mesh = {Kinetics ; *DNA/chemistry/genetics/metabolism ; *RNA/chemistry/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Thermodynamics ; Gene Editing ; },
abstract = {DNA-RNA hybrid strand displacement underpins the function of many natural and engineered systems. Understanding and controlling factors affecting DNA-RNA strand displacement reactions is necessary to enable control of processes such as CRISPR-Cas9 gene editing. By combining multiscale modeling with strand displacement experiments, we show that the distribution of bases within the displacement domain has a very strong effect on reaction kinetics, a feature unique to DNA-RNA hybrid strand displacement. Merely by redistributing bases within a displacement domain of fixed base composition, we are able to design sequences whose reaction rates span more than four orders of magnitude. We extensively characterize this effect in reactions involving the invasion of dsDNA by an RNA strand, as well as the invasion of a hybrid duplex by a DNA strand. In all-DNA strand displacement reactions, we find a predictable but relatively weak sequence dependence, confirming that DNA-RNA strand displacement permits far more thermodynamic and kinetic control than its all-DNA counterpart. We show that oxNA, a recently introduced coarse-grained model of DNA-RNA hybrids, can reproduce trends in experimentally observed reaction rates. We also develop a simple kinetic model for predicting strand displacement rates. On the basis of these results, we argue that base distribution effects may play an important role in natural R-loop formation and in the function of the guide RNAs that direct CRISPR-Cas systems.},
}

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

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

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

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

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

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

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

@article {pmid40435597,
year = {2025},
author = {Kim, HJ and Park, DG and Choi, SJ and Cho, SD},
title = {PCSK9 is a passenger gene in head and neck cancer with minimal pathological influence.},
journal = {Archives of oral biology},
volume = {176},
number = {},
pages = {106302},
doi = {10.1016/j.archoralbio.2025.106302},
pmid = {40435597},
issn = {1879-1506},
mesh = {Humans ; *Proprotein Convertase 9/genetics/metabolism ; *Head and Neck Neoplasms/genetics/pathology ; Cell Proliferation/genetics ; Cell Line, Tumor ; Apoptosis/genetics ; Cell Movement ; CRISPR-Cas Systems ; Blotting, Western ; Gene Expression Regulation, Neoplastic ; },
abstract = {OBJECTIVES: In this study, we aimed to explore the pathological effects of PCSK9 in head and neck cancer (HNC) by ablating its expression using the CRISPR-Cas9 knockout system.

DESIGN: To investigate the clinical importance of PCSK9 in HNC, in silico analysis was performed using datasets from the GEO database and the UALCAN database. To evaluate the role of PCSK9 in HNC pathogenesis, both CRISPR-Cas9 knockout system and pharmacological inhibition were employed to ablate PCSK9 expression in HNC cell lines. The impact of PCSK9 on cellular growth and proliferation was assessed using Cell Counting Kit-8, soft agar, and clonogenic assays in a 2D culture model, as well as a hanging drop spheroid formation assay with live/dead staining in a 3D culture model. The involvement of PCSK9 in apoptosis induction was evaluated by detecting c-PARP expression through Western blotting, measuring the sub-G1 population via cell cycle assay, and verifying the Annexin V-positive population. Finally, changes in metastatic profiles associated with fluctuations in PCSK9 expression were examined using wound healing and transwell migration/invasion assays.

RESULTS: In in silico analysis results, PCSK9 appeared to be related to the progression of HNC. However, experimental results demonstrated that PCSK9 plays a minimal role in cancer cell proliferation, anchorage-independent growth, colony formation capacity, in 2D cultures, as well as spheroidal growth in 3D cultures, and apoptosis induction. Furthermore, PCSK9 marginally influenced wound closure, metastatic potential, and invasive ability.

CONCLUSION: Collectively, these data suggest that PCSK9 serves a neutral role in HNC, functioning as a passenger gene.},
}

Humans
*Proprotein Convertase 9/genetics/metabolism
*Head and Neck Neoplasms/genetics/pathology
Cell Proliferation/genetics
Cell Line, Tumor
Apoptosis/genetics
Cell Movement
CRISPR-Cas Systems
Blotting, Western
Gene Expression Regulation, Neoplastic

@article {pmid40356202,
year = {2025},
author = {Butt, H and Mandava, M and Jacobsohn, D},
title = {Advances in Gene Therapy for Sickle Cell Disease: From Preclinical Innovations to Clinical Implementation and Access Challenges.},
journal = {The CRISPR journal},
volume = {8},
number = {3},
pages = {174-188},
doi = {10.1089/crispr.2024.0101},
pmid = {40356202},
issn = {2573-1602},
mesh = {*Anemia, Sickle Cell/therapy/genetics ; *Genetic Therapy/methods/trends ; Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; Animals ; beta-Globins/genetics ; Mutation ; },
abstract = {Sickle cell disease (SCD) is a hereditary blood disorder caused by a specific mutation in the β-globin gene, leading to the production of hemoglobin S, which deforms red blood cells, causing occlusion in small blood vessels. This results in pain, anemia, organ damage, infections, and increased stroke risk. Treatment options, including disease-modifying therapies and curative hematopoietic stem cell transplants, have limited accessibility. Recently, autologous gene therapy has emerged as a promising curative option, particularly for SCD. Gene editing techniques such as CRISPR, base editing, and prime editing offer potential to correct this mutation. In this review, we discuss recent preclinical studies and clinical trials of gene and cell therapies, focusing on the progress of FDA-approved treatments like Lyfgenia and Casgevy. We also examine the many challenges, including accessibility, safety, and long-term efficacy, which continue to shape the future of SCD gene therapy.},
}

*Anemia, Sickle Cell/therapy/genetics
*Genetic Therapy/methods/trends
Humans
*Gene Editing/methods
CRISPR-Cas Systems
Animals
beta-Globins/genetics
Mutation

@article {pmid40329823,
year = {2025},
author = {Henkel, E and Li, Z and Uvehag, D and Schmierer, B and Henkel, M and Wermeling, F},
title = {Green Listed v2.0: A Web Application for Streamlined Design of Custom CRISPR Screens.},
journal = {The CRISPR journal},
volume = {8},
number = {3},
pages = {216-223},
doi = {10.1089/crispr.2025.0023},
pmid = {40329823},
issn = {2573-1602},
mesh = {*Software ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Internet ; *Gene Editing/methods ; Gene Library ; User-Computer Interface ; Humans ; },
abstract = {Custom CRISPR screens are powerful tools for rapid, hypothesis-driven discovery, but their design is often complex and time-consuming. Green Listed v2.0 simplifies this process with an intuitive workflow for designing custom CRISPR spacer libraries and supports downstream analysis for all users, irrespective of their computational experience. The web application features a user-friendly graphical interface freely accessible at https://greenlisted.cmm.se. Version 2.0 includes significant upgrades to the original 2016 version that were implemented based on user feedback. This includes a new gene synonym tool, expanded library options, optimized output lists, performance improvements, and linked scripts for the rational design of custom CRISPR screen gene sets.},
}

*Software
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Internet
*Gene Editing/methods
Gene Library
User-Computer Interface
Humans

@article {pmid40184611,
year = {2025},
author = {Wu, S and Liu, Y and Zeng, T and Zhou, T and Sun, Y and Deng, Y and Zhang, J and Li, G and Yin, Y},
title = {Enhanced the Trans-Cleavage Activity of CRISPR-Cas12a Using Metal-Organic Frameworks as Stimulants for Efficient Electrochemical Sensing of Circulating Tumor DNA.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {22},
pages = {e2417206},
doi = {10.1002/advs.202417206},
pmid = {40184611},
issn = {2198-3844},
support = {82202294//National Natural Science Foundation of China/ ; 82272667//National Natural Science Foundation of China/ ; 82372091//National Natural Science Foundation of China/ ; JX21817902/008//Collaborative Innovation Center for Tumor Individualization Program/ ; JSSCBS20211476//Doctoral Program for Entrepreneurship and Innovation of Jiangsu Province/ ; PY2021035//Young Scholars Fostering Fund of the First Affiliated Hospital of Nanjing Medical University/ ; },
mesh = {*Metal-Organic Frameworks/chemistry ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Electrochemical Techniques/methods ; *Circulating Tumor DNA/analysis/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; Limit of Detection ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Continued development of clustered regularly interspaced short palindromic repeats (CRISPR)-powered biosensing system on the electrochemical interface is vital for accurate and timely diagnosis in clinical practice. Herein, an electrochemical biosensor based on manganese metal-organic frameworks (MOFs)-enhanced CRISPR (MME-CRISPR) is proposed that enables the efficient detection of circulating tumor DNA (ctDNA). In this design, customized enzyme stimulants (Mn[2+]) are co-assembled with Cas12a/crRNA to form enzyme-MOF composites, which can be released quickly under mild conditions. The MOFs-induced proximity effect can continuously provide adequate Mn[2+] to sufficiently interact with Cas12a/crRNA during the release process, enhancing the trans-cleavage activity of complex available for biosensor construction. The MOFs-based enzyme biocomposites also afford efficient protection against various external stimulus. It is demonstrated that the developed biosensor can achieve ultrasensitive detection of epidermal growth factor receptor L858R mutation in ctDNA with a low detection limit of 0.28 fm without pre-amplification. Furthermore, the engineered mismatch crRNA enables the biosensor based on MME-CRISPR to detect single nucleotide variant with a high signal-to-noise ratio. More importantly, it has been successfully used to detect the targets in clinical practice, requiring low-dose samples and a short time. This strategy is believed to shed new light on the applications of cancer diagnosis, treatment, and surveillance.},
}

*Metal-Organic Frameworks/chemistry
*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
Humans
*Electrochemical Techniques/methods
*Circulating Tumor DNA/analysis/genetics
*Endodeoxyribonucleases/metabolism/genetics
Limit of Detection
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid40019805,
year = {2025},
author = {Li, Q and Yu, H and Du, S and Li, Q},
title = {Optimizing Genome Editing in Mollusks (Crassostrea gigas) in Vitro Validation of sgRNA and Identifying Key Factors Influencing Efficiency.},
journal = {The CRISPR journal},
volume = {8},
number = {3},
pages = {205-215},
doi = {10.1089/crispr.2024.0086},
pmid = {40019805},
issn = {2573-1602},
mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Crassostrea/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Aquaculture ; Gene Knockout Techniques ; CRISPR-Associated Protein 9/genetics ; },
abstract = {CRISPR-Cas9 genome editing holds tremendous potential for accelerating genetic improvements in aquaculture. The success of the CRISPR-Cas9 system relies on the specificity and efficiency of engineered single-guide RNAs (sgRNAs). In this study, we optimized an in vitro validation protocol for sgRNAs to streamline the gene editing process, capitalizing on the limited breeding season of the Pacific oyster (Crassostrea gigas). We evaluated the efficiency of 11 sgRNAs targeting four genes both in vitro and in vivo in C. gigas. In addition, we found that Cas9 protein differs from Cas9 mRNA in gene editing efficiency at various stages of early development. Cas9 protein proved particular efficacy in achieving early and efficient gene knockout, functioning effectively during the first cell division and facilitating biallelic gene knockouts. Statistical analysis showed that in the protein group, the biallelic editing frequency ranged from 12.5% to 57.8%, and the overall editing frequency reached as high as 75-90.6%. The mRNA group exhibited a biallelic editing frequency of 3.1-14.0% and the overall editing frequency spanning 65.6-78.1%. Contrary to expectations, low-temperature incubation (20°C) of oyster embryos prolonged the time window for the first cell division but did not improve gene editing efficiency, likely due to the high temperature sensitivity of Cas9 enzyme activity. Together, this study provides a comprehensive analysis of factors affecting the efficiency of CRISPR-Cas9 gene editing in C. gigas, providing a robust framework for future gene editing endeavors in mollusks and other marine invertebrates.},
}

Animals
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Crassostrea/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics
Aquaculture
Gene Knockout Techniques
CRISPR-Associated Protein 9/genetics

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

@article {pmid40469012,
year = {2025},
author = {Zhu, F and Zhao, Q},
title = {Spherical Nucleic Acids as Modulators of CRISPR/Cas12a by a Steric Barrier Effect for Designing Versatile Biosensors.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c00937},
pmid = {40469012},
issn = {1520-6882},
abstract = {Discovery of CRISPR/Cas12a has revolutionized broad fields, including gene editing, molecular diagnosis, and biosensing. Flexible regulation of Cas12a activity is important for diverse CRISPR/Cas12a applications, especially for biosensing, but it still faces limitations and challenges. We find gold nanoparticles (AuNPs) modified with a single-stranded DNA activator create a huge steric barrier that strongly locks activators in a one-to-many manner and inhibits Cas12a activity. This finding offers a new way to modulate the activity of Cas12a for applications, such as designing versatile biosensors. We report a spherical nucleic acid (SNA)-modulating CRISPR/Cas12a (SNA-Cas) platform using SNAs as signal translators for target sensing. A stimuli-responsive SNA was constructed by modifying AuNPs with a DNA activator containing a specific trigger element, and the target triggers specific reactions (e.g., thiol-exchange chemical reaction and RNA-cleaving by DNAzymes) to release activators into solution. A free activator initiates trans-cleavage activity of CRISPR/Cas12a, scissoring fluorescent DNA reporters to produce amplified signals. To show proof of concept, we demonstrate SNA-Cas to sensitively detect diverse non-nucleic acid targets, including biological thiol cysteine, heavy metal Pb[2+], and biomarkers of O[6]-methylguanine-DNA-methyltransferases (MGMT) and fat mass and obesity-related protein (FTO) demethylases. This work opens one door for SNA modulating CRISPR/Cas activity and shows great potential in designing versatile biosensors for detecting diverse targets.},
}

@article {pmid40468657,
year = {2025},
author = {Jun, JS and Kang, SJ and Hong, KW},
title = {Time-resolved analysis of Bacillus subtilis DB104 Spo0A-mutant transcriptome profile and enhancement of recombinant protein release.},
journal = {Journal of microbiology (Seoul, Korea)},
volume = {63},
number = {5},
pages = {e2411032},
doi = {10.71150/jm.2411032},
pmid = {40468657},
issn = {1976-3794},
support = {2021R1I1A2046284//National Research Foundation of Korea/ ; //Ministry of Education/ ; },
mesh = {*Bacillus subtilis/genetics/metabolism/growth & development ; *Bacterial Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; *Transcriptome ; Spores, Bacterial/genetics/growth & development ; *Recombinant Proteins/genetics/metabolism ; *Transcription Factors/genetics/metabolism ; Gene Expression Profiling ; Biofilms/growth & development ; Mutation ; CRISPR-Cas Systems ; Quorum Sensing/genetics ; Gene Editing ; },
abstract = {Spo0A, the master regulator of sporulation initiation in Bacillus subtilis, controls over 500 genes directly or indirectly in early sporulation stages. Although the effects of Spo0A disruption on sporulation have been extensively studied, a comprehensive understanding of the genomic response throughout growth phases remain elusive. Here, we examined the transcriptomic changes in Spo0A mutant strain, R211E, and wild-type across a time-course RNA-seq to identify impacted biological processes and pathways. The R211E strain, which exhibits sporulation deficiency, was constructed using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein (Cas)9 system, highlighting the critical role of proper Cas9 dosing in gene editing. Functional analysis of 3,010 differentially expressed genes (DEGs) showed significant alterations in sporulation, quorum sensing, metabolism, and biofilm formation. The R211E disrupted the Spo0A-AbrB regulatory pathway, reducing biofilm formation and enhancing flagellar gene expression. Up-regulated metabolic pathways, including glycolysis, histidine, and purine biosynthesis, increased cell numbers during vegetative growth. Further, the mutant displayed elevated vegetative autolysin expression, resulting in reduced cell viability in the stationary phase. We also introduce the novel potential of R211E in a recombinant protein expression system that facilitated protein release into the supernatant, providing valuable insight for future research in metabolic engineering and efficient production systems in B. subtilis.},
}

*Bacillus subtilis/genetics/metabolism/growth & development
*Bacterial Proteins/genetics/metabolism
Gene Expression Regulation, Bacterial
*Transcriptome
Spores, Bacterial/genetics/growth & development
*Recombinant Proteins/genetics/metabolism
*Transcription Factors/genetics/metabolism
Gene Expression Profiling
Biofilms/growth & development
Mutation
CRISPR-Cas Systems
Quorum Sensing/genetics
Gene Editing

@article {pmid40468633,
year = {2025},
author = {Cao, M and Brennan, A and Lee, CM and Park, SH and Bao, G},
title = {Deep Learning Based Models for CRISPR/Cas Off-Target Prediction.},
journal = {Small methods},
volume = {},
number = {},
pages = {e2500122},
doi = {10.1002/smtd.202500122},
pmid = {40468633},
issn = {2366-9608},
support = {RP210116//Cancer Prevention and Research Institute of Texas/ ; R01HG011459/HG/NHGRI NIH HHS/United States ; R01HL169761/HL/NHLBI NIH HHS/United States ; },
abstract = {CRISPR/Cas genome editing technologies enable effective and controlled genetic modifications; however, off-target effects remain a significant concern, particularly in clinical applications. Experimental and in silico methods are developed to predict potential off-target sites (OTS), including deep learning based methods, which can automatically and comprehensively learn sequence features, offer a promising tool for OTS prediction. Here, this work reviews the existing OTS prediction tools with an emphasis on deep learning methods, characterizes datasets used for deep learning training and testing, and evaluates six deep learning models -CRISPR-Net, CRISPR-IP, R-CRISPR, CRISPR-M, CrisprDNT, and Crispr-SGRU -using six public datasets and validates OTS data from the CRISPRoffT database. Performance of these models is assessed using standardized metrics, such as Precision, Recall, F1 score, MCC, AUROC and PRAUC. This work finds that incorporating validated OTS datasets into model training enhanced overall model performance, and improved robustness of prediction, particularly with highly imbalanced datasets. While no model consistently outperforms other models across all scenarios, CRISPR-Net, R-CRISPR, and Crispr-SGRU show strong overall performance. This analysis demonstrates the importance of integrating high-quality validated OTS data with advanced deep learning architectures to improve CRISPR/Cas off-target site predictions, ensuring safer genome editing applications.},
}

@article {pmid40466745,
year = {2025},
author = {Yang, P and Zeng, J and Li, L and Ma, R and Peng, J and Zhou, W and Fu, W and Wu, Y and Zhang, Y},
title = {From Cas Proteins to Cutting-Edge Biosensors: A New Era in Clinical Pathogen Diagnostics.},
journal = {The Journal of infection},
volume = {},
number = {},
pages = {106526},
doi = {10.1016/j.jinf.2025.106526},
pmid = {40466745},
issn = {1532-2742},
abstract = {Infectious pathogens exert a profound impact on global health and socio-economic stability, positioning them as a critical focus of scientific inquiry. To safeguard public health, propel advancements in medical diagnostics, and ensure food safety, the development of efficient technologies for rapid, onsite detection of pathogens is imperative. In light of recent research breakthroughs, CRISPR/Cas-based technologies for pathogen biosafety and molecular diagnostics have emerged as particularly promising in the realm of infectious disease detection. This review succinctly introduces the working principles of CRISPR/Cas systems and thoroughly discusses the design and development of various CRISPR/Cas-based biosensors. Importantly, this paper explores the robust applications of CRISPR/Cas-assisted biosensing for emerging infectious diseases, highlighting its potential in pathogen diagnostics with features like cost-effectiveness, multiplex detection and POCT applications. Furthermore, challenges and future developments of CRISPR/Cas-based biosensors for rapid and accurate pathogen detection in specialized settings are also summarized, integrating CRISPR detection with portable POCT biosensors, nanomaterials and novel colorimetric materials. As it builds on a lot of foundational work and offers new insights and detailed reference to advance the development and application of CRISPR technologies in clinical pathogens diagnostics, opening new avenues in medical diagnostics and the prevention and control of infectious diseases.},
}

@article {pmid40466420,
year = {2025},
author = {Jiang, J and Kan, X},
title = {A smartphone-enabled colorimetric tumor-derived exosomes sensing based on multi-enzyme catalysis and dual-recognition triggered CRISPR/Cas12a trans-cleavage.},
journal = {Biosensors & bioelectronics},
volume = {286},
number = {},
pages = {117644},
doi = {10.1016/j.bios.2025.117644},
pmid = {40466420},
issn = {1873-4235},
mesh = {Humans ; *Exosomes/chemistry ; *Biosensing Techniques/instrumentation/methods ; *Colorimetry/instrumentation/methods ; *Smartphone ; CRISPR-Cas Systems ; A549 Cells ; Aptamers, Nucleotide/chemistry ; Limit of Detection ; *Lung Neoplasms/diagnosis ; Tetraspanin 30/chemistry ; Biomarkers, Tumor ; Epithelial Cell Adhesion Molecule/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Detection of protein profiling on exosomes exhibits great promise for early non-invasive and accurate diagnosis of tumor in clinical diagnostics. However, it still faces multiple challenges, such as expensive instruments requirement and weak specificity by single biomarker. Herein, based on a dual-recognition strategy, a ternary hybrid of a trigger DNA (TDNA), EpCAM aptamer, and CD63 aptamer was used to capture A549 cells-derived exosomes to release TDNA, which initiated the trans-cleavage activity of CRISPR/Cas12a to nonspecifically cleave single-stranded DNA (ssNDA) and then resulted in the isolating of ssDNA linked nanozyme of Zr/Fe-CeO2@Ir@CaO2@HA (ZFCIrCH). ZFCIrCH not only achieved H2O2/O2 self-supply, but also possessed high peroxidase-like, oxidase-like, and superoxide dismutase-like activities, thereby generating a sensitive colorimetric signal for A549 cells-derived exosomes detection with a low limit of detection (LOD) of 31 particles/mL. Using a smartphone to analyze colorimetric images, exosome concentration also can be precisely quantified with a LOD of 29 particles/mL, which could also successfully distinguish healthy people from lung cancer patients. With the advantages of high sensitivity, good specificity, low cost, and convenient on-site detection of tumor-derived exosomes, the present colorimetric sensor has great promise in the accurate diagnosis of diseases.},
}

Humans
*Exosomes/chemistry
*Biosensing Techniques/instrumentation/methods
*Colorimetry/instrumentation/methods
*Smartphone
CRISPR-Cas Systems
A549 Cells
Aptamers, Nucleotide/chemistry
Limit of Detection
*Lung Neoplasms/diagnosis
Tetraspanin 30/chemistry
Biomarkers, Tumor
Epithelial Cell Adhesion Molecule/chemistry
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40465697,
year = {2025},
author = {Vo, QD},
title = {Gene editing therapy as a therapeutic approach for cardiovascular diseases in animal models: A scoping review.},
journal = {PloS one},
volume = {20},
number = {6},
pages = {e0325330},
pmid = {40465697},
issn = {1932-6203},
mesh = {Animals ; *Gene Editing/methods ; *Cardiovascular Diseases/therapy/genetics ; Disease Models, Animal ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; Mice ; Humans ; Dependovirus/genetics ; },
abstract = {BACKGROUND: Cardiovascular diseases (CVDs) are the leading cause of mortality worldwide, with hereditary genetic factors contributing substantially to disease burden. Current treatments, including lifestyle modifications, pharmacotherapy, and surgical interventions, focus primarily on symptom management but fail to address underlying genetic causes, often resulting in disease progression or recurrence. Gene therapy has emerged as a transformative approach, offering a potential treatment. This review explores its efficacy and safety in animal models, identifying opportunities for future advancements.

METHODS: This review investigated studies on gene editing interventions in animal models of CVDs, retrieved from PubMed, ScienceDirect, and Web of Science up to December 2024.

RESULT: A total of 57 studies were included in this review. Mice (86%) were the predominant model, with CRISPR-Cas9 (53%) and AAV vectors (80%) as the most used tools. Key targets included PCSK9 (32%), LDLR (9%), and MYH6/7 (7%), achieving 25-85% editing efficiency in liver/heart tissues. Base editors (ABE/CBE) showed superior safety, with <1% off-targets versus CRISPR-Cas9's 2-5 off-targets per guide. Reported toxicity risks included liver injury (AAVs, 23%) and transient cytokine elevation (LNPs, 14%).

CONCLUSION: Gene editing therapy shows great potential for treating CVDs, with high efficiency, strong therapeutic outcomes, and favorable safety in animal models. Continued innovation and rigorous evaluation could transform cardiovascular treatment, benefiting patients with untreatable conditions.},
}

Animals
*Gene Editing/methods
*Cardiovascular Diseases/therapy/genetics
Disease Models, Animal
*Genetic Therapy/methods
CRISPR-Cas Systems
Mice
Humans
Dependovirus/genetics

@article {pmid40446796,
year = {2025},
author = {Yan, J and Luo, R and Rosen, BP and Liu, D and Wong, W and Leslie, CS and Huangfu, D},
title = {Discovery of NANOG enhancers and their essential roles in self-renewal and differentiation in human embryonic stem cells.},
journal = {Stem cell reports},
volume = {20},
number = {6},
pages = {102511},
doi = {10.1016/j.stemcr.2025.102511},
pmid = {40446796},
issn = {2213-6711},
mesh = {Humans ; *Human Embryonic Stem Cells/cytology/metabolism ; *Cell Differentiation/genetics ; *Enhancer Elements, Genetic ; *Nanog Homeobox Protein/genetics/metabolism ; *Cell Self Renewal/genetics ; Animals ; Mice ; Cell Line ; CRISPR-Cas Systems ; },
abstract = {Human embryonic stem cells (hESCs) are notable for their ability to self-renew and to differentiate into all tissue types in the body. NANOG is a core regulator of hESC identity, and dynamic control of its expression is crucial to maintain the balance between self-renewal and differentiation. Transcriptional regulation depends on enhancers, but NANOG enhancers in hESCs are not well characterized. Here, we report two NANOG enhancers discovered from a CRISPR interference screen in hESCs. Deletion of a single copy of either enhancer significantly reduced NANOG expression, compromising self-renewal and increasing differentiation propensity. Interestingly, these two NANOG enhancers are involved in a tandem duplication event found in certain primates including humans but not in mice. However, the duplicated counterparts do not regulate NANOG expression. This work expands our knowledge of functional enhancers in hESCs and highlights the sensitivity of the hESC state to the dosage of core regulators and their enhancers.},
}

Humans
*Human Embryonic Stem Cells/cytology/metabolism
*Cell Differentiation/genetics
*Enhancer Elements, Genetic
*Nanog Homeobox Protein/genetics/metabolism
*Cell Self Renewal/genetics
Animals
Mice
Cell Line
CRISPR-Cas Systems

@article {pmid40446005,
year = {2025},
author = {Shi, L and Zhang, Y and Duan, Y and Sun, M and Yuan, C and Cao, L and Kong, X and Zhang, W and Zheng, H and Wang, Q},
title = {Genome-scale CRISPR screen identifies TMEM198 driving double membrane vesicle formation in swine alphacoronavirus and murine betacoronavirus infected cells.},
journal = {PLoS pathogens},
volume = {21},
number = {5},
pages = {e1013211},
doi = {10.1371/journal.ppat.1013211},
pmid = {40446005},
issn = {1553-7374},
mesh = {Animals ; Mice ; Swine ; *Membrane Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Virus Replication ; *Alphacoronavirus/physiology/genetics ; Humans ; *Coronavirus Infections/virology/metabolism/genetics ; SARS-CoV-2 ; COVID-19/virology ; },
abstract = {COVID-19 pandemic caused by the SARS-CoV-2 which is well-publicized cross-species transmissibility. SARS-CoV-2 belongs to genus Betacoronavirus, several pathogenic alphacoronaviruses have shown similar patterns of emergence. Much less attention paid to host factors required for alphacoronavirus replication compared to those of betacoronaviruses. Here, we utilized a genome-wide CRISPR-Cas9-based screen to identify TMEM198 as a critical host protein for double-membrane vesicle (DMVs) formation during the replication of swine alphacoronavirus. Gene deletion of TMEM198 led to a reduction in the levels of viral infection in cells, whereas the ectopic expression of TMEM198 correspondingly resulted in an increase in infection levels. At the mechanistic level, TMEM198 directly binds to the C-terminal of nonstructural protein 3 (nsp3c) and nonstructural protein 4 (nsp4) to participate in the formation of DMVs. The first 35 amino acids at the N-terminal of TMEM198 are critical for the formation of DMVs and viral replication. Moreover, mice with a gene deletion of TMEM198 exhibit reduced susceptibility to the Betacoronavirus MHV. These results identify the function of TMEM198 in the formation of DMVs during the replication of swine alphacoronavirus and murine betacoronavirus.},
}

Animals
Mice
Swine
*Membrane Proteins/genetics/metabolism
CRISPR-Cas Systems
Virus Replication
*Alphacoronavirus/physiology/genetics
Humans
*Coronavirus Infections/virology/metabolism/genetics
SARS-CoV-2
COVID-19/virology

@article {pmid40414079,
year = {2025},
author = {Lyu, H and Haag, C and Schwarz, N and Löffler, H and Mau-Holzmann, UA and Lerche, H and Rosa, F},
title = {Generation of an induced pluripotent stem cell (iPSC) line carrying a KCNA2 homozygous (p.Arg294His, R294H) mutation related to hereditary spastic paraplegia.},
journal = {Stem cell research},
volume = {86},
number = {},
pages = {103737},
doi = {10.1016/j.scr.2025.103737},
pmid = {40414079},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *Spastic Paraplegia, Hereditary/genetics/pathology ; Homozygote ; Cell Line ; *Mutation ; CRISPR-Cas Systems ; Kv1.2 Potassium Channel ; },
abstract = {The KCNA2 gene encodes the voltage-gated potassium channel Kv1.2, which is essential for repolarization of action potential. The R294H variant in KCNA2 represents the only potassium channel gene variant linked to hereditary spastic paraplegia (HSP) to date. However, this variant has been observed exclusively in heterozygous individuals. Here, we generated a homozygous KCNA2 R294H (c.881G > A) induced pluripotent stem cell (iPSC) line from a healthy individual iPSC line. The variant was introduced into both alleles using the CRISPR/Cas9 system. The resulting iPSC line has a normal karyotype, expresses key pluripotency markers, and is able to differentiate into all three germ layers.},
}

*Induced Pluripotent Stem Cells/metabolism/cytology
Humans
*Spastic Paraplegia, Hereditary/genetics/pathology
Homozygote
Cell Line
*Mutation
CRISPR-Cas Systems
Kv1.2 Potassium Channel

@article {pmid40375406,
year = {2025},
author = {Rhode, J and Edwards, S and Tzvetkova, A and Jensen, LR and Hossain, MF and Nowack, B and Hagenau, L and Kuss, AW},
title = {Two iPSC lines with a heterozygous frameshift mutation in the floating-harbour syndrome locus of the SRCAP gene.},
journal = {Stem cell research},
volume = {86},
number = {},
pages = {103730},
doi = {10.1016/j.scr.2025.103730},
pmid = {40375406},
issn = {1876-7753},
mesh = {Humans ; *Frameshift Mutation/genetics ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Heterozygote ; Cell Line ; *Intellectual Disability/genetics/pathology ; CRISPR-Cas Systems ; },
abstract = {We present two CRISPR/Cas9-modified human iPSC lines with a heterozygous frameshift mutation (NM_006662.3:c.7300_7301insA) in the FLHS-locus of the SRCAP gene, which is associated with Floating-Harbor syndrome, a congenital neurodevelopmental disorder with symptoms including short stature and intellectual disability. The iPSCs express the pluripotency markers OCT4, SOX2, NANOG and TRA 1-60. They show differentiation into cells from all 3 germ layers, no chromosomal abnormalities and no off-target mutations in the tested regions. The mutation leads to a stop codon previously found in patients. Thus, either cell line can serve as disease-specific model for studying SRCAP in the context of FLHS.},
}

Humans
*Frameshift Mutation/genetics
*Induced Pluripotent Stem Cells/metabolism/cytology
Heterozygote
Cell Line
*Intellectual Disability/genetics/pathology
CRISPR-Cas Systems

@article {pmid40373980,
year = {2025},
author = {Deng, X and Wang, W and Tao, Y and Yao, L and Li, Y and Huang, X and He, J},
title = {Application of a rapid and sensitive RPA-CRISPR/Cas12a assay for BCSP31-based Brucella detection.},
journal = {Journal of microbiological methods},
volume = {235},
number = {},
pages = {107148},
doi = {10.1016/j.mimet.2025.107148},
pmid = {40373980},
issn = {1872-8359},
mesh = {*Brucella/genetics/isolation & purification ; *Brucellosis/diagnosis/microbiology ; *CRISPR-Cas Systems/genetics ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; Humans ; Recombinases/genetics/metabolism ; DNA, Bacterial/genetics ; *Molecular Diagnostic Techniques/methods ; Animals ; Bacterial Proteins/genetics ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Brucellosis, a zoonotic disease caused by Brucella species, poses significant health risks to humans and animals. Due to the limitations of current diagnostic methods, such as serological testing and PCR, in terms of sensitivity, specificity, and speed, this study explores the potential of integrating recombinase polymerase amplification (RPA) with the CRISPR-Cas12a system for Brucella detection. This combination leverages the strengths of both technologies for rapid, sensitive, and specific molecular diagnostics. RPA primers and CRISPR RNA (crRNA) targeting the Brucella-specific conserved sequence BCSP31 were designed, followed by optimization of the RPA-CRISPR/Cas12a system. Its performance was evaluated using genomic DNA from Brucella and non-Brucella species. The system's capabilities were assessed on clinical blood samples, demonstrating high sensitivity (detection limit of 10 copies per reaction and 16.6 attomoles for Brucella DNA) and excellent specificity. Testing on clinical samples showed strong agreement with qPCR results and an improvement over the RBT. The RPA-CRISPR/Cas12a platform represents a rapid, ultra-sensitive, and accurate method for Brucella detection and holds promise as a valuable tool for brucellosis control.},
}

*Brucella/genetics/isolation & purification
*Brucellosis/diagnosis/microbiology
*CRISPR-Cas Systems/genetics
Sensitivity and Specificity
*Nucleic Acid Amplification Techniques/methods
Humans
Recombinases/genetics/metabolism
DNA, Bacterial/genetics
*Molecular Diagnostic Techniques/methods
Animals
Bacterial Proteins/genetics
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40347545,
year = {2025},
author = {Wang, S and Guo, X and Yan, H and Zhou, T and Liu, J},
title = {Generation of an anti-CD5 CAR knock-in human induced pluripotent stem cell line using CRISPR/Cas9 technology.},
journal = {Stem cell research},
volume = {86},
number = {},
pages = {103731},
doi = {10.1016/j.scr.2025.103731},
pmid = {40347545},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques ; *CD5 Antigens/immunology/metabolism ; *Receptors, Chimeric Antigen/genetics/metabolism ; Cell Line ; Cell Differentiation ; },
abstract = {Theanti-CD5 chimeric antigen receptor (CAR)isa genetically engineered immune cell therapydevelopedto targetCD5-associatedhematologic malignancies,such asT-cell lymphoma and leukemia.UsingCRISPR/Cas9-mediated gene targeting, wegeneratedananti-CD5 CAR knock-in human induced pluripotent stem cell (iPSC) linethat stably expresses the CAR constructand is detectable via FLAG tag and GFP markers. Thisengineeredcell linemaintainsstem cell morphology,displaysa normal karyotype, andexhibits robustexpression ofpluripotency markers while retaining differentiation potential.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
*Gene Knock-In Techniques
*CD5 Antigens/immunology/metabolism
*Receptors, Chimeric Antigen/genetics/metabolism
Cell Line
Cell Differentiation

@article {pmid40345204,
year = {2025},
author = {Wu, Y and Zhong, A and Evangelisti, A and Sidharta, M and Danwei, H and Studer, L and Zhou, T},
title = {Leveraging CRISPR activation for rapid assessment of gene editing products in human pluripotent stem cells.},
journal = {Stem cell reports},
volume = {20},
number = {6},
pages = {102499},
doi = {10.1016/j.stemcr.2025.102499},
pmid = {40345204},
issn = {2213-6711},
mesh = {Humans ; *Gene Editing/methods ; *Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Transcriptional Activation ; DNA Methylation ; Cell Line ; },
abstract = {Verification of genome editing in human pluripotent stem cells (hPSCs), particularly at silent loci, is desirable but challenging, as it often requires complex and time-intensive differentiation to induce their expression. Here, we establish a rapid and effective workflow for verifying genome-edited hPSC lines targeting unexpressed genes using CRISPR-mediated transcriptional activation (CRISPRa). We systematically compared the efficiency of various CRISPRa systems and identified the synergistic activation mediator (SAM) system as the most potent for activating silent genes in hPSCs. Furthermore, combining SAM with TET1, a demethylation module, enhanced the activation of methylated genes. By inducing targeted gene activation in undifferentiated hPSCs using CRISPRa, we successfully verified single- and dual-reporter lines, functionally tested degradation tag (dTAG) knockins, and validated silent gene knockouts within 48 h. This approach bypasses the need to induce target gene expression through differentiation, providing a rapid and effective assay for verifying silent gene editing at the hPSC stage.},
}

Humans
*Gene Editing/methods
*Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
Cell Differentiation/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Transcriptional Activation
DNA Methylation
Cell Line

@article {pmid40344932,
year = {2025},
author = {Talanaite, D and Wang, H and Qi, M and Lan, F},
title = {Generation of a human embryonic stem cell line (WAe009-A-3B) carrying homozygous TNNT2 gene knockout by CRISPR/Cas9 editing.},
journal = {Stem cell research},
volume = {86},
number = {},
pages = {103729},
doi = {10.1016/j.scr.2025.103729},
pmid = {40344932},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; *Troponin T/genetics/metabolism ; *Gene Knockout Techniques ; *Gene Editing ; Cell Line ; Homozygote ; },
abstract = {The TNNT2 gene encodes cardiac troponin T (cTnT), a critical protein in cardiac muscle contraction. Mutations in TNNT2 are associated with various cardiomyopathies, including hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM), which contribute to significant morbidity and mortality. In this study, we established a novel TNNT2 knockout human embryonic stem cell (hESC) line, WAe009-A-3B, utilizing the CRISPR/Cas9 genome editing system. This novel hESC line provides an important tool for investigating the molecular mechanisms underlying TNNT2-related cardiomyopathies and may serve as a promising in vitro model for the development of therapeutic strategies targeting TNNT2 mutations in cardiac diseases.},
}

Humans
*CRISPR-Cas Systems/genetics
*Human Embryonic Stem Cells/metabolism/cytology
*Troponin T/genetics/metabolism
*Gene Knockout Techniques
*Gene Editing
Cell Line
Homozygote

@article {pmid40318521,
year = {2025},
author = {Chen, H and Liang, Y and Liang, Y and Chen, Y and Li, X and Zhang, R and Duan, C and Li, W and Cui, Z and Gu, J and Ding, C and Sun, X and Chen, J},
title = {Generation of a gene-corrected isogenic human iPS cell line (CSUASOi006-A-2) from a retinitis pigmentosa patient using CRISPR/Cas9 technology.},
journal = {Stem cell research},
volume = {86},
number = {},
pages = {103727},
doi = {10.1016/j.scr.2025.103727},
pmid = {40318521},
issn = {1876-7753},
mesh = {Humans ; *Retinitis Pigmentosa/genetics/pathology/metabolism ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; Cell Line ; Gene Editing ; },
abstract = {Retinitis pigmentosa (RP) is a heterogeneous group of hereditary eye disorders characterized by a progressive degeneration of the light-sensing photoreceptor cells in the retina. Currently, there are no effective treatments. In a previous study, we generated a human induced pluripotent stem (iPS) cell line (CSUASOi006-A) from an RP patient carrying a PRPF8 (c.C5792T) mutation. In this study, we corrected the c.5792C > T mutation in the PRPF8 gene using CRISPR/Cas9 technology and generated an isogenic control cell line (CSUASOi006-A-2). This provides an important cellular resource for RP research.},
}

Humans
*Retinitis Pigmentosa/genetics/pathology/metabolism
*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
Cell Line
Gene Editing

@article {pmid40311328,
year = {2025},
author = {Wang, S and Feng, Y and Xing, Q and Zhou, T and Liu, J},
title = {Generation of an anti-CD19 CAR knock-in human induced pluripotent stem cell line using CRISPR/Cas9 technology.},
journal = {Stem cell research},
volume = {86},
number = {},
pages = {103721},
doi = {10.1016/j.scr.2025.103721},
pmid = {40311328},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Antigens, CD19/immunology/metabolism ; *Gene Knock-In Techniques ; Cell Line ; Cell Differentiation ; *Receptors, Chimeric Antigen/genetics/metabolism ; },
abstract = {Chimeric antigen receptor T cell (CAR-T) therapy represents a major breakthrough in the field of tumor immunotherapy. CD19-targeted CAR-T cells (CD19 CAR-T) have emerged as an important therapeutic approach for treating B-cell malignancies. We successfully constructed a human induced pluripotent stem cell (iPSC) line with an anti-CD19 CAR knock-in using CRISPR/Cas9-mediated gene targeting technology. This cell line can stably express the CAR gene while maintaining its typical stem cell morphology and normal karyotype. Furthermore, this cell line possesses multilineage differentiation potential and can be directionally differentiated into various chimeric antigen receptor-expressing immune cells for targeting CD19-positive tumors.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
*Antigens, CD19/immunology/metabolism
*Gene Knock-In Techniques
Cell Line
Cell Differentiation
*Receptors, Chimeric Antigen/genetics/metabolism

@article {pmid40311326,
year = {2025},
author = {Bai, R and Fu, W and Hou, X and Wan, J},
title = {Generation of a MYL3 knockout stem cell line (WAe009-A-1H) by episomal vector-based CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {86},
number = {},
pages = {103723},
doi = {10.1016/j.scr.2025.103723},
pmid = {40311326},
issn = {1876-7753},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Myosin Light Chains/genetics/metabolism ; Cell Line ; Gene Knockout Techniques ; *Human Embryonic Stem Cells/metabolism/cytology ; Genetic Vectors/genetics/metabolism ; *Plasmids/genetics/metabolism ; },
abstract = {The Myosin light chain 3 (MYL3) gene encodes the ventricular essential light chain isoform, which is an important modulator of sarcomeric myosin cross-bridge kinetics. Variants in MYL3 are a cause of hypertrophic cardiomyopathy and dilated cardiomyopathy with cardiac failure and sudden cardiac death (SCD). To further elucidate the involvement of MYL3 in the pathogenesis of cardiomyopathies, we have created a MYL3 knockout human embryonic stem cell line using the CRISPR/Cas9 system. Notably, this MYL3-knockout cell line retains normal morphology, pluripotency, and karyotype. This resource provides a valuable tool for investigating MYL3-related health and disease.},
}

*CRISPR-Cas Systems/genetics
Humans
*Myosin Light Chains/genetics/metabolism
Cell Line
Gene Knockout Techniques
*Human Embryonic Stem Cells/metabolism/cytology
Genetic Vectors/genetics/metabolism
*Plasmids/genetics/metabolism

@article {pmid40286604,
year = {2025},
author = {Härting, N and Ludwik, KA and Jahn, R and Kaiser, FJ and Stachelscheid, H},
title = {Generation of hiPSC lines with fusion tagged thyroid hormone receptor α isoforms to study isoform-specific actions of TRα1 and TRα2.},
journal = {Stem cell research},
volume = {86},
number = {},
pages = {103720},
doi = {10.1016/j.scr.2025.103720},
pmid = {40286604},
issn = {1876-7753},
mesh = {Humans ; *Thyroid Hormone Receptors alpha/metabolism/genetics ; Protein Isoforms/metabolism/genetics ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Male ; Cell Line ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {The role of the nuclear thyroid hormone receptor TRα1 as a mediator of thyroid hormone action on target gene expression is well understood. However, the function of the TRα2 splicing isoform, which does not bind thyroid hormones, remains unexplored. As no reliable antibodies are available to investigate TRα1 and TRα2 specifically, we introduced small fusion tags into the THRA locus of the male healthy donor iPSC lines BIHi001-B and BIHi005-A by CRISPR/Cas9-mediated genome editing. Consequently, the modified lines express C-terminally tagged TRα1-2xHA or TRα2-3xFLAG. These genome-edited lines facilitate the investigation of isoform-specific actions of TRα in different cell types.},
}

Humans
*Thyroid Hormone Receptors alpha/metabolism/genetics
Protein Isoforms/metabolism/genetics
*Induced Pluripotent Stem Cells/metabolism/cytology
Male
Cell Line
CRISPR-Cas Systems
Gene Editing

@article {pmid40247765,
year = {2025},
author = {Nie, H and Jin, M and Wang, Z and Zheng, J and Zhang, Y and Li, X},
title = {Heterologous Expression of Candida antarctica Lipase B in Aspergillus niger Using CRISPR/Cas9-mediated Multi-Gene Editing.},
journal = {Biotechnology and bioengineering},
volume = {122},
number = {7},
pages = {1770-1779},
doi = {10.1002/bit.29000},
pmid = {40247765},
issn = {1097-0290},
support = {//This study was supported by Application Development Special Key Project of Chongqing (cstc2021jscx-jbgsX0002) and Science and Technology Project Founded by the Education Department of Jiangxi Province (GJJ202305)./ ; },
mesh = {*Lipase/genetics/biosynthesis/metabolism ; *Aspergillus niger/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Fungal Proteins/genetics/metabolism/biosynthesis ; Recombinant Proteins/genetics/metabolism/biosynthesis ; },
abstract = {Aspergillus niger, a filamentous fungus, is known as a cell factory due to its ability to produce large amounts of organic acids and industrial enzymes. Lipase B from Candida antarctica (CALB) is one of the most widely used lipases in industrial applications, including oil processing, papermaking, food, pharmaceuticals, and personal care products. In this study, the CRISPR/Cas9 technique was employed to knock out the pyrG and kusA genes in A. niger. The CALB gene was integrated into the high-production protein gene loci, such as glaA and amyA, to construct a multi-copy CALB production engineered strain. Additionally, the pepA, aglU, and bglA genes were deleted, which minimized the background level of secreted proteins in A. niger and increased the production of CALB. After two rounds of gene editing, the A. niger with multi-copy CALB was created, and the engineered A. niger CCTCC 206047.09 with high CALB yield was isolated. After 120 h of liquid fermentation, the lipase activity reached 17.84 U/mL and the protein yield reached 10.21 mg/mL. In summary, an engineered A. niger strain with high lipase activity was successfully isolated by employing a CRISPR/Cas9 system to integrate CALB into high-expression loci, while simultaneously knocking out the host's highly expressed protein genes. These results provide an effective strategy for the high expression of both heterologous and homologous enzymes in A. niger.},
}

*Lipase/genetics/biosynthesis/metabolism
*Aspergillus niger/genetics/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Fungal Proteins/genetics/metabolism/biosynthesis
Recombinant Proteins/genetics/metabolism/biosynthesis

@article {pmid40222082,
year = {2025},
author = {Karl-Schöller, F and Breyer, M and Klopocki, E and Üçeyler, N},
title = {Generation of a gene-corrected human isogenic iPSC line from a patient with Fabry disease carrying the GLA variant c.1069C>T using CRISPR/Cas9-mediated homology directed repair.},
journal = {Stem cell research},
volume = {86},
number = {},
pages = {103711},
doi = {10.1016/j.scr.2025.103711},
pmid = {40222082},
issn = {1876-7753},
mesh = {Humans ; *Fabry Disease/genetics/pathology/metabolism ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *alpha-Galactosidase/genetics/metabolism ; Cell Line ; Male ; Cell Differentiation ; Gene Editing ; Mutation ; },
abstract = {Fabry disease (FD) is an X-linked genetic disorder caused by mutations in the GLA gene, leading to α-galactosidase A deficiency and intracellular globotriaosylceramide (Gb3) accumulation. To study FD-associated pathomechanisms, we generated an isogenic control induced pluripotent stem cell (iPSC) line (IsoFD-1) from a patient-derived FD-iPSC line (FD-1) carrying the GLA c.1069C>T mutation. Using CRISPR/Cas9 gene correction, we restored the wild-type sequence, confirmed by Sanger sequencing and absence of Gb3 deposits. IsoFD-1 exhibited typical pluripotency markers, normal karyotype, and trilineage differentiation capacity. This line provides a valuable tool for investigating Gb3-related cellular dysfunction in FD.},
}

Humans
*Fabry Disease/genetics/pathology/metabolism
*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
*alpha-Galactosidase/genetics/metabolism
Cell Line
Male
Cell Differentiation
Gene Editing
Mutation

@article {pmid38517013,
year = {2025},
author = {Mboowa, G and Sserwadda, I and Kanyerezi, S and Tukwasibwe, S and Kidenya, B},
title = {The dawn of a cure for sickle cell disease through CRISPR-based treatment: A critical test of equity in public health genomics.},
journal = {Annals of human genetics},
volume = {89},
number = {4},
pages = {188-194},
pmid = {38517013},
issn = {1469-1809},
support = {U2R TW012116/TW/FIC NIH HHS/United States ; },
mesh = {Humans ; *Anemia, Sickle Cell/therapy/genetics ; COVID-19/epidemiology ; *Public Health ; *Genomics/methods ; *Genetic Therapy/methods ; SARS-CoV-2 ; Gene Editing ; *CRISPR-Cas Systems ; *Health Equity ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Equity in access to genomic technologies, resources, and products remains a great challenge. This was evident especially during the coronavirus disease 2019 (COVID-19) pandemic when the majority of lower middle-income countries were unable to achieve at least 10% population vaccination coverage during initial COVID-19 vaccine rollouts, despite the rapid development of those vaccines. Sickle cell disease (SCD) is an inherited monogenic red blood cell disorder that affects hemoglobin, the protein that carries oxygen through the body. Globally, the African continent carries the highest burden of SCD with at least 240,000 children born each year with the disease. SCD has evolved from a treatable to a curable disease. Recently, the UK medical regulator approved its cure through clustered regularly interspaced short palindromic repeat (CRISPR)-based treatment, whereas the US Food and Drug Administration has equally approved two SCD gene therapies. This presents a remarkable opportunity to demonstrate equity in public health genomics. This CRISPR-based treatment is expensive and therefore, a need for an ambitious action to ensure that they are affordable and accessible where they are needed most and stand to save millions of lives.},
}

Humans
*Anemia, Sickle Cell/therapy/genetics
COVID-19/epidemiology
*Public Health
*Genomics/methods
*Genetic Therapy/methods
SARS-CoV-2
Gene Editing
*CRISPR-Cas Systems
*Health Equity
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid40465692,
year = {2025},
author = {Cai, Y and Zhao, P and Wu, F and Zhao, H and Shao, H and Marra, A and Patel, P and O'Connell, E and Fink, E and Miele, MM and Li, Z and De Stanchina, E and Cocco, E and Razavi, P and Toska, E and Fanning, SW and Xu, G and Sablina, AA and Scaltriti, M and Chandarlapaty, S},
title = {Inhibition of NR2F2 restores hormone therapy response to endocrine refractory breast cancers.},
journal = {Science translational medicine},
volume = {17},
number = {801},
pages = {eadk7786},
doi = {10.1126/scitranslmed.adk7786},
pmid = {40465692},
issn = {1946-6242},
mesh = {Humans ; *Breast Neoplasms/drug therapy/genetics/metabolism/pathology ; Female ; *COUP Transcription Factor II/metabolism/antagonists & inhibitors/genetics ; Animals ; Cell Line, Tumor ; *Drug Resistance, Neoplasm/drug effects/genetics ; Neurofibromin 1/metabolism/genetics ; Gene Expression Regulation, Neoplastic/drug effects ; Mice ; Receptors, Estrogen/metabolism ; CRISPR-Cas Systems/genetics ; },
abstract = {Endocrine resistance is frequently encountered in estrogen receptor-positive (ER+) breast cancer, often because of somatic mutations such as neurofibromin 1 (NF1) loss. The mechanisms by which ER-directed proliferation is lost in such cases are unknown, limiting the potential use of additional endocrine treatments. Here, we performed CRISPR-Cas9 knockout (KO) screens and found that nuclear receptor subfamily 2 group F member 2 (NR2F2), an orphan nuclear receptor, was essential for NF1 loss-induced endocrine resistance. Induction of NR2F2 was observed in ER+ cell line models and patient samples and occurred via activation of the mitogen-activated protein kinase (MAPK) pathway upon NF1 loss or other MAPK pathway genetic alterations. Mechanistically, increased NR2F2 orchestrated a repressed ER transcriptional program by repartitioning the ER cistrome, altering the balance of its associated transcriptional coregulators, and modifying global chromatin accessibility. Accordingly, genetic depletion or pharmacologic inhibition of NR2F2 restored sensitivity to hormone therapies in multiple models, including ER+ cell lines, patient-derived xenografts, and patient-derived organoid-based xenografts harboring diverse endocrine-resistance mechanisms such as NF1, AT-rich interactive domain-containing protein 1A (ARID1A), phoshatase and tensin homolog (PTEN) loss, or Kirsten rat sarcoma virus (KRAS) overexpression. Together, these findings underscore NR2F2 as a critical modulator of the hormone response pathway and suggest its inhibition as a promising strategy to overcome endocrine resistance in breast cancer.},
}

Humans
*Breast Neoplasms/drug therapy/genetics/metabolism/pathology
Female
*COUP Transcription Factor II/metabolism/antagonists & inhibitors/genetics
Animals
Cell Line, Tumor
*Drug Resistance, Neoplasm/drug effects/genetics
Neurofibromin 1/metabolism/genetics
Gene Expression Regulation, Neoplastic/drug effects
Mice
Receptors, Estrogen/metabolism
CRISPR-Cas Systems/genetics

@article {pmid40465008,
year = {2025},
author = {Jin, Y and Liang, X and Wang, X},
title = {Alternative splicing in stem cells and development: research progress and emerging technologies.},
journal = {Cell regeneration (London, England)},
volume = {14},
number = {1},
pages = {20},
pmid = {40465008},
issn = {2045-9769},
support = {YD9110002078//USTC Research Funds of the Double First-Class Initiative/ ; 32170557//National Natural Science Foundation of China/ ; 32400436//National Natural Science Foundation of China/ ; 2023IHM01034//Research Funds of Centre for Leading Medicine and Advanced Technologies of IHM/ ; 2024C879//Anhui Postdoctoral Scientific Research Program Foundation/ ; },
abstract = {Alternative splicing is a key regulatory mechanism that generates transcriptomic diversity by selectively splicing pre-RNA molecules in different ways, leading to the production of multiple RNA isoforms from a single gene. This process is crucial for the fine-tuning of gene expression and is tightly regulated during various biological processes. Recent studies have highlighted how alternative splicing contributes to stem cells self-renewal and differentiation, as well as how dysregulation of splicing factors can impact stem cells behavior and lead to developmental abnormalities or diseases. This review summarizes the current understanding of alternative splicing in stem cells and development, focusing on the molecular mechanisms that govern alternative splicing regulation, the role of splicing factors, and the impact of splicing isoforms on stem cell fate determination and developmental processes. We also discuss emerging technologies, such as CRISPR/Cas-based tools, single-cell long-read RNA sequencing, imaging technologies and 3D culture systems, which are advancing our ability to study alternative splicing in vitro and in vivo. Overall, this field is rapidly evolving, revealing new insights into how alternative splicing shapes the molecular landscape and functions of stem cells and developmental processes.},
}

@article {pmid40464988,
year = {2025},
author = {Waqas, MAB and Awan, MJA and Amin, I and Arif, M and Mukhtar, Z and Mansoor, S},
title = {Engineering high yield basmati rice by editing multiple negative regulators of yield.},
journal = {Molecular biology reports},
volume = {52},
number = {1},
pages = {545},
pmid = {40464988},
issn = {1573-4978},
mesh = {*Oryza/genetics/growth & development ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; Plant Proteins/genetics ; Genes, Plant/genetics ; Quantitative Trait Loci/genetics ; Edible Grain/genetics ; Gene Expression Regulation, Plant ; },
abstract = {BACKGROUND: Yield improvement in Basmati rice is important to meet the growing demand of Basmati and pivotal to world food security. Yield in rice is a complex quantitative trait controlled by many genes and yield-determining factors. Although dominant genes introgression through breeding have been extensively used but it is a laborious and time-consuming process. Yield genes OsD27, OsGW2, OsTGW6 and OsGN1a have been shown to negatively regulate an increase in the number of tillers, grain width, grain weight, and number of grains per panicle respectively in different genetic backgrounds of rice separately. The aim of our study is to establish a multiplex system in Super Basmati to check if it acts similarly and what is their combinatorial effect on yield enhancement.

METHODS: Here we generated Super Basmati quadruple edited lines for all four genes OsD27, OsGW2, OsTGW6 and OsGN1a through CRISPR-Cas9 polycistronic tRNA-gRNA (PTG) endogenous processing efficient multiplex editing system. Quadruple edited lines were characterized for key yield parameters such as number of tillers, number of grains, grain weight, overall yield increase, lodging resistance and cooking quality related traits.

RESULTS: We found that all quadruple edited plants produced more tillers, a greater number of grains with high grain weight in field evaluations along with decreased plant height. Overall, the contribution of all four genes was additive resulting in boosting the yield in Super Basmati remarkably. Our data hence demonstrated a promising multiplex genome editing approach for rapid generation of superior alleles for all four yield contributing factor genes in elite Basmati variety with 30% co-editing efficiency.

CONCLUSIONS: We conclude that multiplex CRISPR-Cas9 genome editing of yield-negative regulators may boost the yield of elite Basmati rice in a short time without causing any drastic effect on other agronomic and quality traits such as aroma, chalkiness, and cooking qualities. Data suggests that the tRNA-gRNA multiplexing CRISPR-Cas9 system is efficient in the Basmati rice background for simultaneously editing multiple genes. Due to its capability of rapid generation of beneficial alleles, this multiplex system is well suited for pyramiding of multiple alleles in Basmati rice and may prove a promising approach to enhance yield in Basmati rice while it may provide useful germplasm resource for high-yielding future rice breeding programs.},
}

*Oryza/genetics/growth & development
*Gene Editing/methods
CRISPR-Cas Systems/genetics
Plant Breeding/methods
Plants, Genetically Modified/genetics
Plant Proteins/genetics
Genes, Plant/genetics
Quantitative Trait Loci/genetics
Edible Grain/genetics
Gene Expression Regulation, Plant

@article {pmid40464976,
year = {2025},
author = {Sharma, SS and Pandey, A and Kashyap, A and Goyal, L and Garg, P and Kushwaha, R and Sharma, J and Tripathi, S and Kumari, S and Thomas, G and Verma, M and Gupta, NC and Gupta, AK and Bhattacharya, R and Sharma, S and Rao, M},
title = {CRISPR/Cas9: efficient and emerging scope for Brassica crop improvement.},
journal = {Planta},
volume = {262},
number = {1},
pages = {14},
pmid = {40464976},
issn = {1432-2048},
support = {NASF/BGAM-9021/2022-23/NIPB//ICAR - National Agricultural Science Fund/ ; 2001-2019//Indian Council of Agricultural Research/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Brassica/genetics ; *Gene Editing/methods ; *Crops, Agricultural/genetics ; Genome, Plant ; Plant Breeding/methods ; Plants, Genetically Modified ; },
abstract = {CRISPR/Cas9 revolutionizes Brassica crop improvement by enhancing yield, quality, and stress resistance, providing a precise and versatile tool for genetic and agronomic advancements. The rapidly advancing CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) technologies are being employed in both diploid and polyploid species of Brassica for gene functions and precise genetic improvements. CRISPR/Cas technology has sparked significant attention among the scientific community due to its affordability, precision, and effectiveness compared to other genome editing techniques. The recent discoveries highlight the diverse applications of the CRISPR/Cas9 genome editing tool in enhancing agriculturally important traits in Brassica species. This technology has been utilized to improve yield, quality, and resistance to both biotic and abiotic stresses globally. Here, we present an overview that encourages researchers to explore and improve the functionality and genetic progress of Brassica U-triangle species utilizing genome editing technologies. In addition, ethical considerations and concerns associated with CRISPR technologies are addressed, providing valuable insight into how CRISPR/Cas9 tools and have revolutionized crop improvement with special emphasis on Brassica for various agronomically and nutritionally important traits.},
}

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

@article {pmid40464068,
year = {2025},
author = {Liu, Y and Zhang, S and Hu, C},
title = {Cas7 meets Cas14: a strategic partnership in the type VII CRISPR-Cas.},
journal = {Protein & cell},
volume = {16},
number = {2},
pages = {79-82},
pmid = {40464068},
issn = {1674-8018},
support = {23-0178-A0001//National University of Singapore Presidential Young Professorship (PYP)/ ; 23-1065-P0001//Singapore Ministry of Education Tier 1/ ; },
}

@article {pmid40461565,
year = {2025},
author = {Yang, C and Fang, Q and Li, M and Zhang, J and Li, R and Zhou, T and Wang, K and Deng, J and Wang, X and Huang, C and Feng, Y and Zhang, X and Shi, L and Bi, C and Zhang, X and Yu, J and Hao, J},
title = {Prime editor with rational design and AI-driven optimization for reverse editing window and enhanced fidelity.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5144},
pmid = {40461565},
issn = {2041-1723},
support = {32471478//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82203238//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; *Artificial Intelligence ; },
abstract = {Prime editing (PE) is a precise tool for introducing genetic mutations in eukaryotes. Extending the efficient editing scope and mitigating undesired byproducts are possible. We introduce reverse PE (rPE), a SpCas9-directed variant that enabled DNA editing at the 3' direction of HNH-mediated nick site. The rPE leveraging nCas9-D10A and rPE gRNA targeting the 5' direction of HNH-mediated nick site inscribes genetic alterations, achieving a reverse editing window and potentially high fidelity. HNH and reverse transcriptase engineered using protein language models in conjunction with La facilitate circular erPEmax and erPE7max, achieving editing efficiency up to 44.41% without nick gRNA or positive selection. Furthermore, our findings underscore the capability of rPE in inserting functionally enhanced variant (PIK3CD[E527G]) for cell therapy. By expanding the editing scope and enhancing genomic manipulability, rPE represents a meaningful advancement in prime editing, improving its utility for research and therapeutic applications.},
}

*Gene Editing/methods
Humans
CRISPR-Cas Systems/genetics
CRISPR-Associated Protein 9/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
HEK293 Cells
*Artificial Intelligence

@article {pmid40461259,
year = {2025},
author = {Esser, SP and Turzynski, V and Plewka, J and Nuy, J and Moore, CJ and Banas, I and Soares, AR and Lee, J and Woyke, T and Probst, AJ},
title = {Differential Expression of Core Metabolic Functions in Candidatus Altiarchaeum Inhabiting Distinct Subsurface Ecosystems.},
journal = {Environmental microbiology reports},
volume = {17},
number = {3},
pages = {e70096},
pmid = {40461259},
issn = {1758-2229},
support = {DFG PR1603/2-1//Ministerium für Kultur und Wissenschaft des Landes Nordrhein- Westfalen ("Nachwuchsgruppe Dr. Alexander Probst") and the German Research Foundation/ ; //Aker B.P.: GeneOil Project/ ; //U.S. Department of Energy Joint Genome Institute/ ; //DOE Office of Science User Facility/ ; //the Office of Science of the U.S. Department of Energy operated/ ; },
mesh = {*Ecosystem ; Germany ; Groundwater/microbiology ; Symbiosis ; Transcriptome ; Gene Expression Profiling ; Genome, Archaeal ; Multigene Family ; },
abstract = {Candidatus Altiarchaea are widespread across aquatic subsurface ecosystems and possess a highly conserved core genome, yet adaptations of this core genome to different biotic and abiotic factors based on gene expression remain unknown. Here, we investigated the metatranscriptome of two Ca. Altiarchaeum populations that thrive in two substantially different subsurface ecosystems. In Crystal Geyser, a high-CO2 groundwater system in the USA, Ca. Altiarchaeum crystalense co-occurs with the symbiont Ca. Huberiarchaeum crystalense, while in the Muehlbacher sulfidic spring in Germany, an artesian spring high in sulfide concentration, Ca. A. hamiconexum is heavily infected with viruses. We here mapped metatranscriptome reads against their genomes to analyse the in situ expression profile of their core genomes. Out of 537 shared gene clusters, 331 were functionally annotated and 130 differed significantly in expression between the two sites. Main differences were related to genes involved in cell defence like CRISPR-Cas, virus defence, replication, transcription and energy and carbon metabolism. Our results demonstrate that altiarchaeal populations in the subsurface are likely adapted to their environment while influenced by other biological entities that tamper with their core metabolism. We consequently posit that viruses and symbiotic interactions can be major energy sinks for organisms in the deep biosphere.},
}

*Ecosystem
Germany
Groundwater/microbiology
Symbiosis
Transcriptome
Gene Expression Profiling
Genome, Archaeal
Multigene Family

@article {pmid40460129,
year = {2025},
author = {Jiang, W and Wang, S and Ahlheit, D and Fumagalli, T and Yang, Z and Ramanathan, S and Jiang, X and Weber, T and Dahlin, J and Borodina, I},
title = {High-throughput metabolic engineering of Yarrowia lipolytica through gene expression tuning.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {23},
pages = {e2426686122},
doi = {10.1073/pnas.2426686122},
pmid = {40460129},
issn = {1091-6490},
support = {NNF20CC0035580//Novo Nordisk Fonden (NNF)/ ; NNF20OC0060809//Novo Nordisk Fonden (NNF)/ ; NNF21OC0072559//Novo Nordisk Fonden (NNF)/ ; No. 101123257//EC | H2020 | PRIORITY 'Excellent science' | H2020 European Research Council (ERC)/ ; },
mesh = {*Yarrowia/genetics/metabolism ; *Metabolic Engineering/methods ; Transcription Factors/genetics/metabolism ; Promoter Regions, Genetic ; *Gene Expression Regulation, Fungal ; CRISPR-Cas Systems ; Plasmids/genetics ; Gene Library ; Fungal Proteins/genetics/metabolism ; },
abstract = {The challenge of accurately predicting which genetic alternations lead to the desired phenotype necessitates high-throughput metabolic engineering approaches where numerous hypotheses can be tested simultaneously. We describe the CRISPR-Cas9-based method TUNE[YALI] that enables high-throughput tuning of gene expression in the common industrial yeast Yarrowia lipolytica. The method is based on replacing the promoters of the target genes with native Y. lipolytica promoters of varying strengths or removing the promoters entirely. To demonstrate the method's capabilities, we created a plasmid library that targets 56 transcription factors (TFs) and changes the expression of each TF to seven different levels. We transformed this library into reference and betanin-producing strains of Y. lipolytica and screened the resulting clones for changes in morphology, thermotolerance, or improved betanin production. The genetic markup of the yeast clones with the desired phenotypic changes was determined by sequencing the inserted plasmids. We identified multiple TFs whose regulatory changes increased thermotolerance, two TFs that eliminated pseudohyphal growth, and several TFs that increased betanin production. Analogous libraries can be designed to target any chosen group of genes and even all the genes. The libraries can be shared and reused, accelerating applied strain development projects and fundamental functional genomics research (TUNE[YALI]-TF kit and TUNE[YALI]-TF library are available via AddGene under catalog numbers #1000000255 and #217744).},
}

*Yarrowia/genetics/metabolism
*Metabolic Engineering/methods
Transcription Factors/genetics/metabolism
Promoter Regions, Genetic
*Gene Expression Regulation, Fungal
CRISPR-Cas Systems
Plasmids/genetics
Gene Library
Fungal Proteins/genetics/metabolism

@article {pmid40411798,
year = {2025},
author = {Shi, K and Zhang, Y and Tao, Y and Wang, Y and Yang, J and Deng, R and Yang, H},
title = {Preamplification-Free Detection of RNA N6-Methyladenosine Modification at Single-Base Resolution Using the CRISPR Tandem Assay.},
journal = {Analytical chemistry},
volume = {97},
number = {22},
pages = {11454-11461},
doi = {10.1021/acs.analchem.4c06782},
pmid = {40411798},
issn = {1520-6882},
mesh = {*Adenosine/analogs & derivatives/analysis/metabolism ; Humans ; *CRISPR-Cas Systems ; RNA, Long Noncoding/genetics ; *RNA ; Lung Neoplasms/diagnosis/genetics ; },
abstract = {N6-Methyladenosine (m[6]A) ranks among the most prevalent modifications in RNA, which serves as a biomarker for diseases, such as lung cancer. Herein, we developed a CRISPR/Cas13a-Csm6 tandem assay (termed CRISPRm[6]A assay) allowing for preamplification-free, sensitive, and rapid detection of RNA m[6]A modifications. The coupling of Cas13a-Csm6 tandem with MazF endoribonuclease enables the assay to identify m[6]A RNA with single-base resolution. Compared to the CRISPRm[6]A assay using Cas13a alone, the tandem CRISPRm[6]A assay yielded an improved sensitivity for RNA detection by ∼22 times, thus enabling preamplification-free detection of RNA m[6]A. Particularly, the proposed assay enabled quantification of m[6]A abundance down to 0.5% at the picomole level in lncRNA MALAT1 and demonstrated a 100% correlation in diagnosing nonsmall cell lung cancer. In summary, the CRISPRm[6]A assay supports two key applications in biological samples: (1) precise determination of m[6]A sites and (2) quantitative measurement of m[6]A fractions. Therefore, the CRISPR tandem method presents a promising tool for RNA epigenetics-based diagnostics.},
}

*Adenosine/analogs & derivatives/analysis/metabolism
Humans
*CRISPR-Cas Systems
RNA, Long Noncoding/genetics
*RNA
Lung Neoplasms/diagnosis/genetics

@article {pmid40411467,
year = {2025},
author = {Wang, X and Zhong, L and Zhang, W and Wu, P and Wang, M and Li, D and Dong, L and Wang, G},
title = {CRISPR Digital Sensing: From Micronano-Collaborative Chip to Biomolecular Detection.},
journal = {ACS nano},
volume = {19},
number = {22},
pages = {20427-20451},
doi = {10.1021/acsnano.5c03474},
pmid = {40411467},
issn = {1936-086X},
mesh = {Humans ; *COVID-19/diagnosis/genetics/virology ; *CRISPR-Cas Systems/genetics ; SARS-CoV-2/genetics/isolation & purification ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Biosensing Techniques/methods ; *Lab-On-A-Chip Devices ; },
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) sensing technology proved to be valuable during the COVID-19 pandemic through its sensitivity, specificity, robustness, and versatility. However, issues such as overreliance on amplification, susceptibility to false positives, lack of quantification strategies, and complex operation procedures have hindered its broader application in bioanalysis and clinical diagnostics. The collision between micronano-collaborative chips and CRISPR technology has effectively addressed these bottlenecks, offering innovative solutions for diagnosis and treatment. Unlike conventional micronano chips, micronano digital chips enhance CRISPR's response to trace amounts of target molecules by leveraging highly controllable local environments and compartmentalized microreactors. This advancement improves detection efficiency and revolutionizes traditional in vitro bioanalytical processes. First, the working principles, fabrication techniques, and performance metrics of CRISPR-based digital droplet microfluidics and microarray chips are examined. Then, the applications of CRISPR digital sensing chips in bioassays are reviewed, emphasizing their importance in advancing in vitro detection systems for gene editing. Finally, the prospects of CRISPR digital sensing technology are explored, particularly its potential for body surface biomonitoring and its broader development opportunities in the biomedical field.},
}

Humans
*COVID-19/diagnosis/genetics/virology
*CRISPR-Cas Systems/genetics
SARS-CoV-2/genetics/isolation & purification
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Biosensing Techniques/methods
*Lab-On-A-Chip Devices

@article {pmid40390640,
year = {2025},
author = {Zhang, C and Chen, Y and Chen, X and Lin, X and Huang, Z and Zhang, L and Liu, R and Lv, Y},
title = {Single nanoparticle analysis-based CRISPR/Cas12 bioassay for amplification-free HIV detection.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {48},
pages = {8759-8762},
doi = {10.1039/d5cc01716e},
pmid = {40390640},
issn = {1364-548X},
mesh = {Humans ; *CRISPR-Cas Systems ; Limit of Detection ; *Nanoparticles/chemistry ; *Biological Assay/methods ; *HIV/isolation & purification ; *Biosensing Techniques/methods ; *HIV-1/isolation & purification ; *HIV Infections/diagnosis ; *Metal Nanoparticles/chemistry ; },
abstract = {To reduce the "window period" in HIV detection, most analytical methods require additional enzymes for signal amplification. Exempting challenges like primer interference and false positives in amplification strategies, we developed an amplification-free bioassay that uses CRISPR's potent cleavage activity and the competent sensitivity of single-nanoparticle analysis. An attomolar detection limit was achieved with adequate selectivity. Serum and cell tests confirm the bioassay's accurate and sensitive HIV detection.},
}

Humans
*CRISPR-Cas Systems
Limit of Detection
*Nanoparticles/chemistry
*Biological Assay/methods
*HIV/isolation & purification
*Biosensing Techniques/methods
*HIV-1/isolation & purification
*HIV Infections/diagnosis
*Metal Nanoparticles/chemistry

@article {pmid40326747,
year = {2025},
author = {Chen, Y and Yu, K and Jiang, Z and Yang, G},
title = {CRISPR-based genetically modified scaffold-free biomaterials for tissue engineering and regenerative medicine.},
journal = {Biomaterials science},
volume = {13},
number = {12},
pages = {3149-3175},
doi = {10.1039/d5bm00194c},
pmid = {40326747},
issn = {2047-4849},
mesh = {*Tissue Engineering/methods ; Humans ; *Regenerative Medicine/methods ; *Biocompatible Materials/chemistry ; Animals ; Gene Editing ; *CRISPR-Cas Systems ; Tissue Scaffolds ; },
abstract = {CRISPR-based genetically modified scaffold-free biomaterials, including extracellular vehicles, cell sheets, cell aggregates, organoids and organs, have attracted significant attention in the fields of regenerative medicine and tissue engineering in recent years. With a wide range of applications in gene therapy, modeling disease, tissue regeneration, organ xenotransplantation, modeling organogenesis as well as gene and drug screening, they are at a critical juncture from clinical trials to therapeutic applications. Xenografts have already been tested on non-human primates and humans. However, we have to admit that a series of obstacles still need to be addressed, such as immune response, viral infection, off-target effects, difficulty in mass production, and ethical issues. Therefore, future research should pay more attention to improving their safety, accuracy of gene editing, flexibility of production, and ethical rationality. This review summarizes various types of CRISPR-based genetically modified scaffold-free biomaterials, including their preparation procedures, applications, and possible improvements.},
}

*Tissue Engineering/methods
Humans
*Regenerative Medicine/methods
*Biocompatible Materials/chemistry
Animals
Gene Editing
*CRISPR-Cas Systems
Tissue Scaffolds

@article {pmid40287111,
year = {2025},
author = {Yang, Q and Zhang, Y and Chen, L and Fang, L and Xiao, S},
title = {Isolation, pathogenicity, and an infectious cDNA clone of the prevalent G2c variant of porcine epidemic diarrhea virus in China.},
journal = {Microbial pathogenesis},
volume = {205},
number = {},
pages = {107637},
doi = {10.1016/j.micpath.2025.107637},
pmid = {40287111},
issn = {1096-1208},
mesh = {Animals ; Swine ; *Porcine epidemic diarrhea virus/genetics/pathogenicity/isolation & purification ; China/epidemiology ; *Swine Diseases/virology/epidemiology ; Chlorocebus aethiops ; Vero Cells ; *Coronavirus Infections/veterinary/virology/epidemiology ; *DNA, Complementary/genetics ; Virulence ; Diarrhea/veterinary/virology/epidemiology ; Spike Glycoprotein, Coronavirus/genetics ; Disease Outbreaks ; Mutation ; Phylogeny ; CRISPR-Cas Systems ; },
abstract = {Porcine epidemic diarrhea virus (PEDV) is a major enteric coronavirus causing diarrhea in piglets, and the recent prevalence of its G2c variant poses a significant threat to the pig industry in China. In this study, we successfully isolated a G2c variant strain (designated EHuB4) from a pig farm experiencing an outbreak of severe diarrhea in China, and constructed its full-length cDNA infectious clone using CRISPR/Cas9 technology. The parental EHuB4 strain and its rescued virus, rEHuB4, exhibited similar biological characteristics in Vero cells, including rapid cytopathic effects and the formation of large syncytia, suggesting strong cell fusion ability and replication efficiency. Pathogenicity experiments in piglets demonstrated that both EHuB4 and rEHuB4 caused severe diarrhea and mortality, further confirming that the prevalence of the G2c variant may be the primary reason for the current high incidence of PED outbreaks. We also analyzed the mutation frequency and variation tendency in the amino acids encoded by the EHuB4 Spike (S) gene and identified multiple high-frequency mutation sites located not only in the S1 subunit but also in the intermediate region between heptad repeat 1 (HR1) and HR2. Furthermore, these mutations have persisted since 2011 and may be closely related to virus prevalence, host adaptation, and pathogenicity. In summary, this study not only characterizes the genetic evolution and pathogenicity of the prevalent G2c strain but also provides a valuable molecular tool for developing novel vaccines through the established full-length cDNA infectious clone.},
}

Animals
Swine
*Porcine epidemic diarrhea virus/genetics/pathogenicity/isolation & purification
China/epidemiology
*Swine Diseases/virology/epidemiology
Chlorocebus aethiops
Vero Cells
*Coronavirus Infections/veterinary/virology/epidemiology
*DNA, Complementary/genetics
Virulence
Diarrhea/veterinary/virology/epidemiology
Spike Glycoprotein, Coronavirus/genetics
Disease Outbreaks
Mutation
Phylogeny
CRISPR-Cas Systems

@article {pmid40459995,
year = {2025},
author = {Wu, JL and Zheng, SS and Wang, L and Xiang, X and Li, F and Lv, ML and Wu, Q and Huang, ZX and Miao, HB},
title = {CRISPR-Cas System-Mediated Genetic Modification in Bacillus spp.: Current Status and Future.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c03140},
pmid = {40459995},
issn = {1520-5118},
abstract = {Bacillus spp. are a group of Gram-positive bacteria that have shown significant potential for development in recent years. It is capable of utilizing low-cost substrates to produce various high-value-added compounds, making it widely applicable in fields such as feed, pharmaceuticals, and food. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system-mediated genetic modification is recognized as one of the most efficient technologies. The application of this technology for the genetic improvement of Bacillus spp. greatly enhanced the production performance of these strains. In this review, we summarize the various CRISPR-Cas systems that have been applied to Bacillus spp., with a particular focus on systematically outlining the strategies for implementing CRISPR-Cas-mediated genetic modification in these bacteria. Notably, homologous recombination is the most widely used strategy, while base editing is emerging as a novel and precise approach. Additionally, we discuss the importance of expression regulation strategies in establishing Bacillus spp. as a cell factory. Finally, we propose potential solutions to current technical challenges, providing insights for the development of high-performance genetically modified Bacillus spp. production strains.},
}

@article {pmid40459840,
year = {2025},
author = {Keerthana, R and Rakshana, P and Salunkhe, SR and Sakthi, AR and Kokiladevi, E and Saraswathi, T and Pushpam, R and Raveendran, M and Sudha, M},
title = {CRISPR-Cas9 mediated enhancement of abiotic stress resilience in tomato: a comprehensive review of target genes.},
journal = {Molecular biology reports},
volume = {52},
number = {1},
pages = {538},
pmid = {40459840},
issn = {1573-4978},
mesh = {*Solanum lycopersicum/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Stress, Physiological/genetics ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; },
abstract = {Tomato (Solanum lycopersicum L.), a major vegetable crop grown worldwide, is consumed as both fresh and processed products. Concerns about the impact of abiotic stresses on tomato production are growing worldwide as climate change alters global weather patterns, adversely affecting crop yield and produce quality due to stresses like salt, heat, cold, and drought. Conventional breeding approaches such as hybridization, marker-assisted selection (MAS), and mutation breeding, have long been utilized to improve tomato resilience against abiotic stresses. These approaches are often hampered by extensive field trials, and require multiple generations limiting their efficiency in rapidly developing stress-tolerant cultivars. The efforts of traditional breeding systems are hindered by the narrow genetic base of tomatoes which poses a major bottleneck. Researchers have utilized CRISPR-Cas genome-editing technology to address this challenge to offer a precise and accelerated alternative for enhancing stress resilience in tomato. This versatile tool has gained attention for its simple, precise, and effective gene-editing capabilities. CRISPR-Cas based genome editing has successfully modified key genes related to stress-response pathways, enhancing abiotic stress resilience. Developing resistant cultivars help mitigate the impact of abiotic stress, thereby contributing to increased food production and food security. This review highlights recent progress in use of CRISPR-Cas9 gene editing to enhance tomato resilience to abiotic stresses.},
}

*Solanum lycopersicum/genetics
*CRISPR-Cas Systems/genetics
Gene Editing/methods
*Stress, Physiological/genetics
Plant Breeding/methods
Plants, Genetically Modified/genetics

@article {pmid40459690,
year = {2025},
author = {Lakhani, H and Kumar, N and Jangra, A and Negi, S and Dholariya, T and Tiwari, S},
title = {Streamlined protoplast transfection system for in-vivo validation and transgene-free genome editing in Banana.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {28},
pmid = {40459690},
issn = {1573-9368},
support = {BT/PR25789/GET/119/97/2017//Department of Biotechnology, Ministry of Science and Technology, India/ ; BIRAC/Tech Transfer/08/I2/QUT-BBF//Biotechnology Industry Research Assistance Council/ ; },
mesh = {*Musa/genetics/growth & development ; *Protoplasts/metabolism ; *Gene Editing/methods ; *Transfection/methods ; *Plants, Genetically Modified/genetics/growth & development ; CRISPR-Cas Systems/genetics ; Transgenes/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome, Plant ; },
abstract = {The advancement in the CRISPR/Cas system has significantly streamlined genome editing in plants, rendering it simple, reliable, and efficient. However, the development of transgene-free crops is a challenging task for vegetatively propagated plants like banana. In the present study, we established banana protoplasts-based versatile and efficient platform for genome editing to overcome this limitation. Herein, a protocol has been optimized for protoplast isolation by considering leaf and embryogenic cell suspension (ECS) of banana cultivar Grand Naine. Freshly prepared ECS was identified as the best source for protoplast isolation. The protoplast viability and competency were checked by transfection with plasmid and RNP complex. Polyethylene glycol (PEG)-mediated protoplast transfection using pCAMBIA1302 and pJL50TRBO vectors showed GFP expression with 30 and 70% efficiency, respectively, eventually proving the protocol's efficacy. Further, gRNAs targeting banana β-carotene hydroxylase gene are validated by in-vitro cleavage test and subsequently used for RNP complex formation with varied ratios (1:1, 1:2, 1:5, and 1:10) of SpCas9 to gRNA1. Among these, a 1:2 molar ratio proved best to generate indel frequency with 7%. Sequencing analysis of the target amplicon revealed mutations upstream of the PAM region, specifically with gRNA1, among the three in-vitro validated gRNAs. This study evaluated the effectiveness of gRNAs in-vitro and in-vivo, yielding inconsistent results that highlight the need for comprehensive in-vivo validation of their functionality. Conclusively, the optimized protocol for banana transfection has the potential to be harnessed for the generation of transgene-free genetically improved banana.},
}

*Musa/genetics/growth & development
*Protoplasts/metabolism
*Gene Editing/methods
*Transfection/methods
*Plants, Genetically Modified/genetics/growth & development
CRISPR-Cas Systems/genetics
Transgenes/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Genome, Plant

@article {pmid40457455,
year = {2025},
author = {Zhang, T and Meng, Z and Yu, H and Zhang, Z and Liu, G and Qiu, A and Zheng, W and Ding, P and Kai, T},
title = {An integrated multi-mode detection platform based on CRISPR/Cas 12a and aptamers for ultra-sensitive identification of sulfamethazine and genes associated with sulfonamide resistance.},
journal = {Journal of nanobiotechnology},
volume = {23},
number = {1},
pages = {408},
pmid = {40457455},
issn = {1477-3155},
support = {2025ZZTS0167//Graduate Research Innovation Program of Central South University/ ; 82373635//the National Natural Science Foundation of China/ ; 2022RC1206//the Science and Technology Innovation Program of Hunan Province/ ; 2023QYJC013//the Central South University Research Program of Advanced Interdisciplinary Studies/ ; },
mesh = {*Sulfamethazine/analysis/pharmacology ; *Aptamers, Nucleotide/chemistry ; *CRISPR-Cas Systems/genetics ; *Sulfonamides/pharmacology ; Biosensing Techniques/methods ; *Anti-Bacterial Agents/analysis/pharmacology ; Electrochemical Techniques ; *Drug Resistance, Bacterial/genetics ; Metal-Organic Frameworks/chemistry ; Benzidines/chemistry ; Limit of Detection ; },
abstract = {The production and buildup of sulfamethazine (SMZ) and resistance genes for sulfonamide antibiotics (sul1) pose a serious risk to environmental and public health safety. Creating advanced sensing systems that are both highly sensitive and selective for the prolonged observation of SMZ concentrations in the environment, along with the quantification of sul1 gene prevalence, aims to identify trends in resistance, posing a considerable challenge. Here, we devised a platform (SMZ-sul1 multi-mode detection platform) that allows for the fluorescence detection of SMZ in environmental samples. This is achieved through the competition for the aptamer between the complementary base and SMZ, along with the colorimetric, photothermal, and electrochemical tracking of sul1, using a magnetic separation unit (FP@cDNA). MOF-818@PtPd (MPP) nanozymes with high peroxide mimetic enzyme activity were linked to FP@cDNA through Zr-O-P bond and employed as a catalyst for the 3,3',5,5'-tetramethylbenzidine (TMB) oxidation, as well as for electrocatalytic hydrogen peroxide (H2O2) reduction. The ability of Cas12a to perform trans cleavage was activated by its precise identification of the sul1, leading to the non-selective cutting of single-stranded DNA (ssDNA). Thereafter, the MPP nanoparticles were released into the supernatant, where they catalyzed the oxidation of TMB. Alternatively, the functioning CRISPR/Cas12a system specifically targeted and cleaved ssDNA present on the electrode, resulting in altered loading of MPP nanozymes and a decrease in the current associated with the catalytic reduction of H2O2. The remarkable magnetic separation capabilities of FP@cDNA, combined with the superior target recognition features of CRISPR/Cas12a and aptamer, facilitated the creation of a highly sensitive detection system, achieving detection limits of 0.67 pM for SMZ and 7.6 fM for sul1, and exhibit great potential for monitoring and prediction in the field of public health.},
}

*Sulfamethazine/analysis/pharmacology
*Aptamers, Nucleotide/chemistry
*CRISPR-Cas Systems/genetics
*Sulfonamides/pharmacology
Biosensing Techniques/methods
*Anti-Bacterial Agents/analysis/pharmacology
Electrochemical Techniques
*Drug Resistance, Bacterial/genetics
Metal-Organic Frameworks/chemistry
Benzidines/chemistry
Limit of Detection

@article {pmid40456823,
year = {2025},
author = {Hong, D and Shu, M and Liu, J and Liu, L and Cheng, H and Zhu, M and Du, Y and Xu, B and Hu, D and Liu, Z and Zhao, Y and Dai, J and Lu, F and Huang, J},
title = {Divergent combinations of enhancers encode spatial gene expression.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5091},
pmid = {40456823},
issn = {2041-1723},
support = {92474104//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32370586//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; *Enhancer Elements, Genetic/genetics ; Mice ; Humans ; Brain/metabolism/embryology ; Chromatin/metabolism/genetics ; Basic Helix-Loop-Helix Transcription Factors/genetics/metabolism ; Breast Neoplasms/genetics ; Spinal Cord/metabolism/embryology ; Gene Expression Regulation, Developmental ; Melanoma/genetics ; CRISPR-Cas Systems ; },
abstract = {Spatial transcriptomics and epigenomics have enabled mapping gene regulation in the tissue context. However, it remains poorly understood how spatial gene expression patterns are orchestrated by enhancers. Here we build eSpatial, a computational framework that deciphers spatially resolved enhancer regulation of gene expression by integrating spatial profiles of gene expression and chromatin accessibility. Applying eSpatial to diverse spatial datasets, including mouse embryo and brain, as well as human melanoma and breast cancer, we reveal a "spatial enhancer code", in which divergent combinations of enhancers regulate the same gene in spatially segregated domains. We validate the spatial enhancer code using public spatial datasets such as VISTA, Allen in situ hybridization (ISH), and H3K27ac MERFISH. Moreover, we conduct transgenic reporter assays and in vivo CRISPR/Cas9-mediated perturbation experiments to confirm the Atoh1 spatial enhancer code in determining Atoh1 spatial expression in mouse embryonic spinal cord and brain. Our study establishes the spatial enhancer code concept, revealing how combinations of enhancers dynamically shape gene expression across diverse biological contexts, providing insights into tissue-specific regulatory mechanisms and tumor heterogeneity.},
}

Animals
*Enhancer Elements, Genetic/genetics
Mice
Humans
Brain/metabolism/embryology
Chromatin/metabolism/genetics
Basic Helix-Loop-Helix Transcription Factors/genetics/metabolism
Breast Neoplasms/genetics
Spinal Cord/metabolism/embryology
Gene Expression Regulation, Developmental
Melanoma/genetics
CRISPR-Cas Systems

@article {pmid40425072,
year = {2025},
author = {Nair, A and Rao, AS and Surabhi, MA and Gnanika, M and More, SS},
title = {Unravelling fungal pathogenesis: Advances in CRISPR-Cas9 for understanding virulence and adaptation.},
journal = {Fungal genetics and biology : FG & B},
volume = {179},
number = {},
pages = {104006},
doi = {10.1016/j.fgb.2025.104006},
pmid = {40425072},
issn = {1096-0937},
mesh = {*CRISPR-Cas Systems ; *Fungi/pathogenicity/genetics ; Virulence/genetics ; Gene Editing ; Genome, Fungal ; *Adaptation, Physiological/genetics ; Humans ; Mycoses/microbiology ; Virulence Factors/genetics ; },
abstract = {Fungi, with their billion-year evolutionary history, have adapted to diverse ecological niches, including pathogenic roles that threaten global health, agriculture, and ecosystems. Fungal pathogenicity is shaped by the dynamic evolution of genetic traits that enable fungi to infect hosts, evade immune defenses, and develop resistance to antifungal treatments. Despite their significant clinical and ecological impact, the evolutionary processes underlying fungal virulence and adaptation remain incompletely understood. This review emphasizes the transformative role of CRISPR-Cas9 genome editing in revealing these mechanisms. By allowing precise manipulation of fungal genomes, CRISPR technologies have provided key insights into virulence factors, stress response mechanisms, immune evasion, and antifungal resistance pathways. These advances demonstrate how fungi adapt to selective pressures, repurpose conserved genetic pathways, and exploit genomic plasticity to thrive in host environments. This review explores the intersection of CRISPR technology and fungal biology, shedding light on its implications for understanding fungal pathogenesis and the potential to develop innovative therapeutic strategies against fungal infections. The integration of CRISPR applications into mycology holds promise for furthering our understanding of fungal evolutionary trajectories and enhancing the development of novel therapeutic approaches.},
}

*CRISPR-Cas Systems
*Fungi/pathogenicity/genetics
Virulence/genetics
Gene Editing
Genome, Fungal
*Adaptation, Physiological/genetics
Humans
Mycoses/microbiology
Virulence Factors/genetics

@article {pmid40413994,
year = {2025},
author = {Liu, Y and Liu, Y and Wu, S and Cao, R and Pan, Y and Zhou, F},
title = {Engineered Cas12a-based one-tube detection of DNMT3A R882 H/C mutation in acute myeloid leukemia.},
journal = {Biosensors & bioelectronics},
volume = {286},
number = {},
pages = {117609},
doi = {10.1016/j.bios.2025.117609},
pmid = {40413994},
issn = {1873-4235},
mesh = {DNA Methyltransferase 3A ; *Leukemia, Myeloid, Acute/genetics/diagnosis ; Humans ; *DNA (Cytosine-5-)-Methyltransferases/genetics ; *CRISPR-Cas Systems/genetics ; Mutation ; *Biosensing Techniques/methods ; *Endodeoxyribonucleases/genetics/chemistry ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Advances in sequencing technologies have identified numerous genetic alterations associated with acute myeloid leukemia (AML), many of which play critical roles in diagnosis, classification, and prognosis. Among these, mutations in the DNA methyltransferase 3 alpha (DNMT3A) gene are particularly prevalent, with the R882H and R882C variants being the most common. Accurate and sensitive detection of DNMT3A mutations is crucial for prognosis, treatment guidance, and early intervention in AML. However, existing detection methods often fail to achieve an optimal balance among sensitivity, turnaround time, and operational simplicity. To address this limitation, we aimed to develop a rapid and highly sensitive method for detecting DNMT3A mutations. The CRISPR/Cas12a system shows promise for genetic detection due to its high sensitivity and single-base specificity. Here, we established a Cas12a-based one-tube assay for the detection of DNMT3A R882 H/C mutations. We utilized the mismatch tolerance of enAsU-R Cas12a to design crRNA for DNMT3A R882 H/C mutation and integrated CRISPR/Cas12a system with ERA. The entire detection process can be completed within 1 h at 37 °C. The optimized detection system demonstrated a sensitivity of 0.1 % when analyzing genomic DNA. To validate its clinical applicability, we tested samples from 49 AML patients and successfully identified all DNMT3A R882H/C-positive cases, including one with a mutation rate as low as 0.24 %. These results highlight the potential of our Cas12a-based one-tube detection system as a rapid, sensitive, and cost-effective method for detecting DNMT3A R882 H/C mutation. This approach could serve as a valuable tool for both diagnostic and therapeutic monitoring.},
}

DNA Methyltransferase 3A
*Leukemia, Myeloid, Acute/genetics/diagnosis
Humans
*DNA (Cytosine-5-)-Methyltransferases/genetics
*CRISPR-Cas Systems/genetics
Mutation
*Biosensing Techniques/methods
*Endodeoxyribonucleases/genetics/chemistry
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid40408898,
year = {2025},
author = {Li, M and Tao, C and Tang, Z and Li, K and Wang, Y and Zhao, C and Tao, C and Geng, J and Sun, K},
title = {Ultrasensitive clinical identification of hepatitis B surface antigen (HBsAg) by CRISPR-assisted nanopore sensing.},
journal = {Biosensors & bioelectronics},
volume = {286},
number = {},
pages = {117579},
doi = {10.1016/j.bios.2025.117579},
pmid = {40408898},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Humans ; *Hepatitis B Surface Antigens/blood/isolation & purification/genetics ; *Nanopores/ultrastructure ; *Hepatitis B virus/isolation & purification/genetics ; *CRISPR-Cas Systems/genetics ; *Hepatitis B/diagnosis/virology/blood ; Limit of Detection ; Aptamers, Nucleotide/chemistry ; },
abstract = {Hepatitis B virus (HBV) infection is a major global health issue. The underdiagnosis of HBV contributes to the increasing mortality from hepatitis B-related complications. Hepatitis B surface antigen is a biomarker guiding the clinical management of chronic hepatitis B, and its disappearance from the blood is a key sign of functional cure. There is a need for highly sensitive detection methods for early intervention and prevention of disease recurrence. We presented a new CRISPR-assisted nanopore sensing method for ultrasensitive detection of hepatitis B surface antigen. It uses the high specificity and turnover efficiency of the CRISPR-Cas12a system. The system is activated by the competitive binding between hepatitis B surface antigen and its aptamer, followed by restriction enzyme digestion. The products are detected by a nanopore for precise quantification at very low concentrations. The result achieves the limit of quantification (LOQ) of 10 fM, outperforming conventional assays. Clinical validation with patient samples confirms its superiority. This integrated technology is a powerful tool for HBV early diagnosis, treatment monitoring, and disease assessment, and paves the way for nanopore technology in clinical diagnostics.},
}

*Biosensing Techniques/methods
Humans
*Hepatitis B Surface Antigens/blood/isolation & purification/genetics
*Nanopores/ultrastructure
*Hepatitis B virus/isolation & purification/genetics
*CRISPR-Cas Systems/genetics
*Hepatitis B/diagnosis/virology/blood
Limit of Detection
Aptamers, Nucleotide/chemistry

@article {pmid40408895,
year = {2025},
author = {Xu, Y and Deng, R and Liu, X and Zhou, Y},
title = {CRISPR/Cas12a-triggered electrochemiluminescence biosensor to ultrasensitive detect herpes simplex virus via self-enhanced near-infrared selenium-based polymer dots.},
journal = {Biosensors & bioelectronics},
volume = {286},
number = {},
pages = {117597},
doi = {10.1016/j.bios.2025.117597},
pmid = {40408895},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; *Polymers/chemistry ; Humans ; Limit of Detection ; *Simplexvirus/isolation & purification/genetics ; *Selenium/chemistry ; Luminescent Measurements/methods ; *Herpes Simplex/virology/diagnosis ; Electrochemical Techniques/methods ; *Quantum Dots/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Herpes simplex virus (HSV) as one of the most popular herpesviruses exhibits nearly identical clinical symptoms among the family of herpesvirus species and it is urgent to develop high specific biosensors to realize accurate detection of HSV. CRISPR/Cas12a systems with programmability and high specificity could serve an essential role in accurate diagnosis of HSV in complex samples. Herein, a novel near-infrared selenium-based polymer dots with self-enhanced effect were unprecedentedly designed and successfully synthesized in this work, and an ultrasensitive biosensor has been constructed together with the CRISPR/Cas12a system for the specific detection of HSV. Notably, the as-prepared selenium-based polymer dots exhibited near-infrared emission with a peak at 760 nm, which could significantly minimize background noise and achieve high sensitivity. The proposed biosensor demonstrated a wide linear range from 1 fM to 1 nM and the limit of detection as low as 0.1 fM (S/N = 3). Undoubtedly, the CRISPR/Cas12a-triggered ECL biosensor proposed in this work could provide a simple, sensitive, and environmentally friendly approach to detect HSV in clinical applications.},
}

*Biosensing Techniques/methods
CRISPR-Cas Systems/genetics
*Polymers/chemistry
Humans
Limit of Detection
*Simplexvirus/isolation & purification/genetics
*Selenium/chemistry
Luminescent Measurements/methods
*Herpes Simplex/virology/diagnosis
Electrochemical Techniques/methods
*Quantum Dots/chemistry
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40354126,
year = {2025},
author = {Lv, B and Liang, P and Chang, C and Li, D},
title = {Sensitive aptasensing of tobramycin through a rational design of catalytic hairpin assembly and hybridization chain reaction amplification monomers for CRISPR/Cas12a activation.},
journal = {The Analyst},
volume = {150},
number = {12},
pages = {2580-2590},
doi = {10.1039/d5an00267b},
pmid = {40354126},
issn = {1364-5528},
mesh = {*CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Limit of Detection ; *Aptamers, Nucleotide/chemistry/genetics ; *Nucleic Acid Amplification Techniques/methods ; Milk/chemistry ; *Tobramycin/analysis ; Nucleic Acid Hybridization ; Animals ; *Anti-Bacterial Agents/analysis ; DNA/chemistry/genetics ; Inverted Repeat Sequences ; *CRISPR-Associated Proteins/metabolism/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {The catalytic hairpin assembly (CHA) and hybridization chain reaction amplification (HCR) are enzyme-free isothermal DNA amplification methods based on the self-assembly of hairpin monomers. Recently, CRISPR/Cas12a-based biosensors in combination with CHA or HCR signal amplification have shown promising performance. Herein, several design strategies for hairpin monomers in CHA and HCR were evaluated in the context of CRISPR/Cas12a-based biosensor construction. The SL-HCR strategy, in which the CRISPR/Cas12a target strand is blocked in the loop of one hairpin monomer DNA and released in the duplex HCR products, demonstrated superior performance in terms of a low background signal, wide linear detection range, and high signal-to-noise ratio. With the assistance of an aptamer-containing probe, a highly sensitive aptasensor was constructed for tobramycin detection, whereby the SL-HCR served the function of signal amplification, whereas the CRISPR/Cas12a system acted to cleave the FQ probes, thereby resulting in the production of a fluorescent signal. After optimization, the aptasensor enables linear detection of tobramycin concentrations ranging from 125 pM to 2500 nM, with a limit of detection (LOD) of 92.87 pM. Moreover, the aptasensor was utilized to detect tobramycin in beef and milk samples, yielding satisfactory results. The assay is concise and cost-effective due to the absence of nanomaterial DNA labeling and magnetic separation procedures. Furthermore, the entire detection workflow operates under isothermal conditions, which makes it suitable for use in food safety control and environmental monitoring. In addition, the results presented here may shed new light on the design of CRISPR/Cas12a-based biosensors in combination with CHA or HCR.},
}

*CRISPR-Cas Systems
*Biosensing Techniques/methods
Limit of Detection
*Aptamers, Nucleotide/chemistry/genetics
*Nucleic Acid Amplification Techniques/methods
Milk/chemistry
*Tobramycin/analysis
Nucleic Acid Hybridization
Animals
*Anti-Bacterial Agents/analysis
DNA/chemistry/genetics
Inverted Repeat Sequences
*CRISPR-Associated Proteins/metabolism/genetics
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid40456709,
year = {2025},
author = {Hanlon, MB and Shohet, JM and Wolfe, SA},
title = {Selective targeting of genome amplifications and repeat elements by CRISPR-Cas9 nickases to promote cancer cell death.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5126},
pmid = {40456709},
issn = {2041-1723},
support = {R01 CA275945/CA/NCI NIH HHS/United States ; R01CA275945//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Amplification ; *Neuroblastoma/genetics/pathology ; Cell Line, Tumor ; N-Myc Proto-Oncogene Protein/genetics ; Cell Death/genetics ; *Deoxyribonuclease I/metabolism/genetics ; DNA Breaks, Double-Stranded ; Neoplasms/genetics ; },
abstract = {Focal gene amplification serves as an oncogenic driver during tumorigenesis and is a hallmark of many forms of cancer. Oncogene amplifications promote genomic instability, which is integral to cancer cell survival and evolution. However, focal gene amplification potentially affords an opportunity for therapeutic exploitation. As a proof-of-concept, we leverage CRISPR-Cas9 nickase to selectively promote cancer cell death in MYCN-amplified neuroblastoma in a gene amplification-dependent manner. Our analysis demonstrates that CRISPR-Cas9 nickase can generate a lethal number of highly toxic, replication-dependent double-strand breaks in cells harboring amplified loci. Furthermore, we demonstrate that Cas9 nickase-mediated toxicity can be modulated in combination with small molecule inhibitors targeting key regulators of the DNA-damage response or cell death pathways. Importantly, our findings in MYCN-amplified neuroblastoma translate to other cancer types with distinct oncogene amplifications.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Amplification
*Neuroblastoma/genetics/pathology
Cell Line, Tumor
N-Myc Proto-Oncogene Protein/genetics
Cell Death/genetics
*Deoxyribonuclease I/metabolism/genetics
DNA Breaks, Double-Stranded
Neoplasms/genetics

@article {pmid40412122,
year = {2025},
author = {Wang, R and Yang, Y and Wang, Z and Ma, C and Wu, M and Du, Y and Zhang, X and Cao, M and Xu, H},
title = {Stimuli-responsive peptide nanocarriers for tumor-specific CRISPR/Cas9 delivery and precision genome editing.},
journal = {Journal of colloid and interface science},
volume = {697},
number = {},
pages = {137932},
doi = {10.1016/j.jcis.2025.137932},
pmid = {40412122},
issn = {1095-7103},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; *Nanoparticles/chemistry ; *Peptides/chemistry ; HeLa Cells ; *Drug Carriers/chemistry ; Particle Size ; *Ribonucleoproteins/chemistry/genetics ; Cell Survival/drug effects ; *Neoplasms/genetics ; Surface Properties ; },
abstract = {CRISPR/Cas9 ribonucleoprotein (RNP) delivery remains a critical challenge due to its large size, instability, and off-target effects. Here, we report a stimuli-responsive cationic amphiphilic peptide, (CR3)3C, designed for cancer-targeted delivery of CRISPR/Cas9 RNP. The peptide integrates three functional domains: (1) a naphthyl-diphenylalanine (Nap-FF) motif enabling self-assembly into stable nanoparticles via aromatic interactions, (2) a matrix metalloproteinase-7 (MMP7)-cleavable linker (GPLGLA) for tumor microenvironment-specific activation, and (3) a redox-responsive cationic domain ((CRRR)3-C) for electrostatic RNP binding and glutathione (GSH)-triggered intracellular release. The (CR3)3C/RNP nanocomplexes (108.8 nm diameter, ζ = +10.89 mV) demonstrate exceptional stability and cellular uptake efficiency. Mechanistic studies reveal caveolae-mediated endocytosis and lipid raft-associated pathways, proton sponge effect-driven endosomal escape, and nuclear localization facilitated by Cas9's nuclear localization signal. In HeLa-EGFP cells, (CR3)3C/RNP shows 33.8 % gene editing efficiency at 100 nM RNP with >90 % cell viability. This work establishes a programmable, non-viral platform that synergizes enzymatic and redox responsiveness for tumor-targeted genome editing, addressing critical barriers in CRISPR therapeutics.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Editing
*Nanoparticles/chemistry
*Peptides/chemistry
HeLa Cells
*Drug Carriers/chemistry
Particle Size
*Ribonucleoproteins/chemistry/genetics
Cell Survival/drug effects
*Neoplasms/genetics
Surface Properties

@article {pmid40403777,
year = {2025},
author = {Wu, HJ and Yang, JP and Ma, WJ and Li, ZH and Feng, HY and Yang, ZN and Xu, HJ},
title = {A CRISPR/Cas9-induced point mutation on the GABA receptor subunit RDL confers high resistance to phenylpyrazole insecticides in the rice planthopper Laodelphax striatellus.},
journal = {Insect biochemistry and molecular biology},
volume = {181},
number = {},
pages = {104327},
doi = {10.1016/j.ibmb.2025.104327},
pmid = {40403777},
issn = {1879-0240},
mesh = {Animals ; *Hemiptera/genetics/drug effects/metabolism ; *Insecticide Resistance/genetics ; *Insecticides/pharmacology ; *Pyrazoles/pharmacology ; CRISPR-Cas Systems ; *Receptors, GABA/genetics/metabolism ; *Point Mutation ; *Insect Proteins/genetics/metabolism ; Female ; },
abstract = {The Delphacidae planthopper Laodelphax striatellus (Order: Hemiptera) is one of the most damaging insect pests of rice crops in Asia. The phenylpyrazole insecticide fipronil was introduced in China in the mid-1990s to control these pests, but its widespread use has led to the development of high levels of resistance. Field sampling coupled with in vitro assays indicated that an A2'N-point mutation in the gamma-aminobutyric acid receptor RDL has been linked to fipronil resistance; however, genetic evidence supporting this association has been lacking. Here, we generated an A2'N-point mutation (LsRDL[A/N]) in L. striatellus using CRISPR/Cas9-mediated homologous recombination. Bioassay revealed that the LsRDL[A/N] mutants conferred a 1211-fold higher resistance to fipronil compared to wild-type planthoppers. Furthermore, these mutants also showed significant resistance (>1000-fold) to ethiprole but not to imidacloprid, demonstrating that the A2'N mutation confers resistance specific to phenylpyrazole insecticides. However, the LsRDL[A/N] mutants displayed reduced fecundity, lower egg hatching rates, and prolonged egg and nymph stages, indicating that fipronil resistance carries a substantial fitness cost. These findings enhance our understanding of the genetic mechanisms underlying phenylpyrazole resistance and provide valuable insights for developing effective resistance management strategies for planthoppers.},
}

Animals
*Hemiptera/genetics/drug effects/metabolism
*Insecticide Resistance/genetics
*Insecticides/pharmacology
*Pyrazoles/pharmacology
CRISPR-Cas Systems
*Receptors, GABA/genetics/metabolism
*Point Mutation
*Insect Proteins/genetics/metabolism
Female

@article {pmid40368251,
year = {2025},
author = {Ramadan, NK and Gaber, N and Ali, NM and Amer, OSO and Soliman, H},
title = {SHERLOCK, a novel CRISPR-Cas13a-based assay for detection of infectious bursal disease virus.},
journal = {Journal of virological methods},
volume = {337},
number = {},
pages = {115185},
doi = {10.1016/j.jviromet.2025.115185},
pmid = {40368251},
issn = {1879-0984},
mesh = {*Infectious bursal disease virus/isolation & purification/genetics ; Animals ; Chickens/virology ; *Birnaviridae Infections/diagnosis/veterinary/virology ; *Poultry Diseases/diagnosis/virology ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; RNA, Viral/genetics ; },
abstract = {Infectious bursal disease (IBD) is an extremely contagious viral infection that primarily affects young chicks, leading to significant economic losses in the poultry industry. The disease is caused by a double-stranded RNA virus of the genus Avibirnavirus, family Birnaviridae, namely, the infectious bursal disease virus (IBDV). Unfortunately, current methods for detecting IBDV lack adequate sensitivity. Accordingly, the advantages of the Specific High Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) assay were employed to develop an ultrasensitive assay (IBD-SHERLOCK assay) for the detection of IBDV in clinical chicken tissues. The assay comprises two steps: isothermal preamplification of the target RNA through reverse transcription recombinase polymerase amplification (RT-RPA) and a subsequent detection step, which is based on the CRISPR-Cas13a system. The integration of lateral flow (LFD) visual detection of the IBD-SHERLOCK products strengthens the feasibility of the assay for use as a point-of-care test in chicken farms. Compared with RT-qPCR, this method exhibited ultra-analytical and clinical sensitivity. The assay has a lower detection limit of 5 aM, which is equivalent to three IBDV-RNA molecules. The assay demonstrated the ability to detect IBDV-RNA in 70 clinical field samples, 15 of which tested negative by RT-qPCR. This evidence highlights its superior sensitivity and potential for early detection of IBDV in chicken tissues. This study effectively established and verified a CRISPR-based diagnostic test for the early detection of IBDV in clinical chicken tissues, demonstrating remarkable specificity and sensitivity. The IBD-SHERLOCK assay can be used as a highly sensitive point-of-care diagnostic tool in chicken farms.},
}

*Infectious bursal disease virus/isolation & purification/genetics
Animals
Chickens/virology
*Birnaviridae Infections/diagnosis/veterinary/virology
*Poultry Diseases/diagnosis/virology
Sensitivity and Specificity
*CRISPR-Cas Systems
*Molecular Diagnostic Techniques/methods
*Nucleic Acid Amplification Techniques/methods
RNA, Viral/genetics

@article {pmid40456230,
year = {2025},
author = {Huda, NU and Hasan, KA and Saleem, F and Naz, S and Khan, S and Mirani, ZA and Syed, MN and Alswat, AS and Sohail, M},
title = {Coriander borne Salmonella superbug: genomic assessments of chromosomal and plasmid-associated resistance, virulence and MGEs of XDR Salmonella enterica Typhi NH1.},
journal = {Pathogens and global health},
volume = {},
number = {},
pages = {1-15},
doi = {10.1080/20477724.2025.2513769},
pmid = {40456230},
issn = {2047-7732},
abstract = {The drug-resistant strains of Salmonella enterica serovar Typhi (S. Typhi) are serious threats to health sectors worldwide. This study focuses on genomic characterization of environmentally derived an extensively drug resistant (XDR) strain of Salmonella Typhi, highlighting its potential to cause significant outbreak. The XDR strain (labeled as NH1) was isolated from fresh coriander, and characterized through whole-genome sequencing to investigate its lineage diversity, niche adaptation, sources and resistant mechanism. The NH1 strain exhibits phylogenomic association with clinical lineages S. Typhi NCTC8385 and ASM3025476. Its genome, assembled in 67 contigs, exhibited expected coverage and GC content, harboring a mega-plasmid, virulence factors, and intrinsic and extrinsic antimicrobial resistance genes. Plasmid annotation revealed IncQ1 and IncY plasmids responsible for the acquired resistance. SPIFinder identified SPI1-10 pathogenicity islands, and the CRISPR-Cas locus, associated with bacterial defense, bile salt resistance and biofilm formation, was present. NH1 strain also possesses numerous mobile elements (proteins and transposases), 12 prophages, and secretion systems (types I-IV, VI), aiding its survival in environment like vegetables. The genomic characterization of S. Typhi NH1 highlights the emergence of coriander-borne XDR strain, underscoring the need for targeted prevention to mitigate its public health impact.},
}

@article {pmid40455764,
year = {2025},
author = {De Pablo-Moreno, JA and González-Brusi, L and Miguel-Batuecas, A and Bermejo-Álvarez, P and Revuelta, L and Liras, A},
title = {Development of a novel and viable knock-in factor V deficiency murine model: Utility for an ultra-rare disease.},
journal = {PloS one},
volume = {20},
number = {6},
pages = {e0321864},
pmid = {40455764},
issn = {1932-6203},
mesh = {Animals ; Mice ; *Disease Models, Animal ; *Factor V Deficiency/genetics/pathology ; *Factor V/genetics/metabolism/chemistry ; *Gene Knock-In Techniques ; Humans ; Female ; Male ; CRISPR-Cas Systems ; Phenotype ; Homozygote ; Mutation, Missense ; *Rare Diseases/genetics ; Partial Thromboplastin Time ; Prothrombin Time ; },
abstract = {Factor V deficiency is a congenital coagulation disorder characterized by the absence or malfunction of factor V (FV). The purpose of this study was to develop a viable FV-deficient mouse model using CRISPR/Cas9 technology. A viable pathological model of the disease was not available to develop new therapies. A previous in silico study was performed to select a mutation causing a mild disease phenotype in humans (Thr1898Met missense). Such mutation was replicated in mice by CRISPR-mediated homology directed repair. Following crossing, homozygous individuals were subjected to coagulometry assays, including FV levels, prothrombin time (PT), and activated partial thromboplastin time (aPTT). The in silico study suggested that the mutation destabilizes FV structure of both mouse and human variants, putatively producing a mild phenotype of the disease in mice. Mendelian inheritance was observed in the offspring. No spontaneous signs of blood clotting disturbances, premature deaths or gestational dysfunctions were observed. FV levels in homozygous animals were 24.5% ± 5.1; 39.7 sec ± 2.8; PT was 61.8% ± 6.3; 23.4 sec ± 1.6 (INR = 1.47 ± 0.12); and aPTT was 46.9 sec ± 3.2. A viable FV-deficient mouse model was generated by introducing a missense mutation in FV. The model exhibits a mild phenotype of the disease, akin to that observed in humans.},
}

Animals
Mice
*Disease Models, Animal
*Factor V Deficiency/genetics/pathology
*Factor V/genetics/metabolism/chemistry
*Gene Knock-In Techniques
Humans
Female
Male
CRISPR-Cas Systems
Phenotype
Homozygote
Mutation, Missense
*Rare Diseases/genetics
Partial Thromboplastin Time
Prothrombin Time

@article {pmid40455288,
year = {2025},
author = {Hu, J and Yu, W and Cui, J and Zhang, L and Yu, W},
title = {Recent advances in diagnostic technologies for postoperative central nervous system infections: a review.},
journal = {Neurological sciences : official journal of the Italian Neurological Society and of the Italian Society of Clinical Neurophysiology},
volume = {},
number = {},
pages = {},
pmid = {40455288},
issn = {1590-3478},
abstract = {Postoperative central nervous system infections (PCNSIs), including meningitis, cerebral abscesses, and implant-associated infections, represent critical complications following neurosurgical procedures. These infections pose significant risks to patient outcomes due to delayed diagnosis, escalating antimicrobial resistance, and limited therapeutic efficacy. Conventional diagnostic approaches, such as cerebrospinal fluid (CSF) analysis, microbial cultures, and neuroimaging, exhibit notable limitations in sensitivity, specificity, and rapidity. This review highlights transformative technologies reshaping PCNSI diagnostics, including molecular assays (e.g., quantitative PCR, digital droplet PCR), metagenomic next-generation sequencing (mNGS), CRISPR-based pathogen detection platforms, metabolomics, and advanced molecular imaging modalities. Furthermore, we address translational challenges in clinical adoption, including cost barriers, standardization gaps, and the need for interdisciplinary collaboration. Emerging artificial intelligence (AI)-driven strategies are proposed to optimize pathogen identification, predict antimicrobial resistance profiles, and tailor personalized therapeutic regimens.},
}

@article {pmid40398759,
year = {2025},
author = {Zhao, S and Zhang, J and Yu, S and He, D and Li, B and Fan, Y and Liu, G and Tang, Y and Diao, Y},
title = {Integration of RPA and CRISPR-Cas13a collateral activity for one-step detection of DHAV-3: A biological macromolecule-enabled diagnostic platform.},
journal = {International journal of biological macromolecules},
volume = {314},
number = {},
pages = {144400},
doi = {10.1016/j.ijbiomac.2025.144400},
pmid = {40398759},
issn = {1879-0003},
mesh = {*CRISPR-Cas Systems/genetics ; Animals ; *Recombinases/metabolism ; RNA, Viral/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Picornaviridae/genetics/isolation & purification ; Ducks/virology ; },
abstract = {Duck hepatitis A virus type 3 (DHAV-3), which is families of the Picornaviridae, poses severe threats to waterfowl industries due to rapid antigenic evolution and limitations in conventional diagnostics. Herein, we engineered a CRISPR-Cas13a-mediated RNA detection system by leveraging the intrinsic HEPN domain-dependent collateral cleavage activity of Cas13a, synergistically integrated with recombinase polymerase amplification (RPA) to target DHAV-3 RNA. This biological macromolecule-driven platform achieved ultrasensitive detection (1 copies/μL) within 35 min through sequence-specific crRNA guidance and Cas13a-triggered fluorescent/lateral flow signal amplification. Rigorous validation against four avian pathogens (ARV, H9N2 AIV, TMUV, AstV) confirmed 100 % specificity, highlighting the precise macromolecular interactions between Cas13a and target RNA. Clinical evaluation of 30 field samples demonstrated complete concordance with RT-qPCR. By harnessing the programmable functionality of Cas13a and the thermostable enzymes in RPA, this study provides a novel paradigm for RNA-guided biological macromolecule applications in point-of-care diagnostics, bridging molecular mechanisms with agricultural biosecurity needs.},
}

*CRISPR-Cas Systems/genetics
Animals
*Recombinases/metabolism
RNA, Viral/genetics
*Nucleic Acid Amplification Techniques/methods
*Picornaviridae/genetics/isolation & purification
Ducks/virology

@article {pmid40347696,
year = {2025},
author = {Martin, OL and Lynch, CRH and Fleming, R},
title = {Advancing forensic body fluid identification: A comparative analysis of RT-LAMP+CRISPR-Cas12a and established mRNA-based methods.},
journal = {Forensic science international. Genetics},
volume = {78},
number = {},
pages = {103297},
doi = {10.1016/j.fsigen.2025.103297},
pmid = {40347696},
issn = {1878-0326},
mesh = {Humans ; *RNA, Messenger/genetics ; Female ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Associated Proteins/genetics ; Sensitivity and Specificity ; Cervix Mucus/chemistry ; Male ; Forensic Genetics/methods ; Semen/chemistry ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {In forensic science, the analysis of body fluid evidence determines the cellular origin of a sample, aiding in the reconstruction of a potential crime. Messenger ribonucleic acid (mRNA) based confirmatory tests address limitations of current conventional methods, providing increased specificity and sensitivity, minimal sample consumption, and the detection of a broader range of body fluids. However, they require expensive instrumentation, longer reaction times, and lack portability. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) coupled with clustered regular interspaced short palindromic repeats (CRISPR) with CRISPR-associated protein 12a (Cas12a) has the potential to overcome these challenges. This approach offers reduced testing time and cost, while potentially providing equivalent sensitivity and specificity, as observed in the field of viral diagnostics. Visual detection capabilities enable the development of rapid, portable screening tests suitable for testing at the crime scene. In the context of a sexual assault investigation, RT-LAMP+CRISPR-Cas12a could potentially increase the efficiency and detection rate. This study compares this novel method to two other mRNA-based methods, endpoint reverse transcription polymerase chain reaction (RT-PCR) multiplex assay CellTyper 2, and a real-time reverse transcription quantitative PCR (RT-qPCR) multiplex assay. The tests' sensitivity and specificity were evaluated on single-source and mixed body fluid samples, including rectal mucosa, a fluid which is minimally explored in forensic literature. The RT-qPCR assay demonstrated the highest sensitivity, specificity, and precision in mixed samples. In addition, RT-qPCR offers a greater linear dynamic range, faster processing time and easier methodology compared to CellTyper 2, only limited by its expensive nature. Notably, rectal mucosa samples exhibited non-specific marker expression of CellTyper 2 markers and expression of CYP2B7P (vaginal fluid) for all methods. This emphasises the need for a dedicated rectal mucosa marker. RT-LAMP+CRISPR-Cas12a exhibited a high specificity, displaying off-target expression of CYP2B7P in two fluid types. However, the method lacked sensitivity and precision for most markers except MMP3 (menstrual blood), demonstrating detection down to 1:10,000 with 100 % specificity. RT-LAMP+CRISPR requires further development, but its quick, inexpensive nature and high specificity suggest it has potential as a confirmatory test that could reduce the limitations of existing methods.},
}

Humans
*RNA, Messenger/genetics
Female
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Associated Proteins/genetics
Sensitivity and Specificity
Cervix Mucus/chemistry
Male
Forensic Genetics/methods
Semen/chemistry
*Clustered Regularly Interspaced Short Palindromic Repeats
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid40294708,
year = {2025},
author = {Gao, X and Zhou, C and Feng, Y and Ye, B and Zhao, Z and Qi, L and Hu, L and Deng, Y and Lin, C and Ding, Q and Liu, G and Wang, C and Song, C and Qian, B and Wu, T and Wang, X and Liu, Z and Lin, Z and Zhang, M},
title = {Research progress of gene editing technology in neurological diseases.},
journal = {Gene},
volume = {962},
number = {},
pages = {149534},
doi = {10.1016/j.gene.2025.149534},
pmid = {40294708},
issn = {1879-0038},
mesh = {*Gene Editing/methods/trends ; Humans ; *Nervous System Diseases/therapy/genetics ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; Animals ; },
abstract = {Gene editing (GE) technology is a genetic manipulation technique based on artificial nucleases that enables the precise modification of DNA or RNA. With the development of technology, GE in disease treatment is becoming increasingly widespread, playing an essential role in haematology, cancer, and neurological disorders (ND). This review describes the principles, advantages, and limitations of four GE technologies, focusing on the fourth generation of GE (next-generation GE). The next-generation GE technology breaks the limitations of traditional GE technology, makes GE more precise and stable, and broadens the scope of gene technology applications. Additionally, this review explores the latest gene therapy strategies for ND, focusing on the application of next-generation GE technologies to examine the prospects for the application of GE technologies. This study discusses and analyses the great advantages and potential of GE technology for treating ND and elucidates the shortcomings of GE in this field.},
}

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

@article {pmid40286513,
year = {2025},
author = {Tang, Q and Liao, Q and Huang, X and Huang, H and Tang, Q and Zhang, K and Liao, X},
title = {A highly sensitive MiRNA detection sensor powered by CRISPR/Cas13a and entropy-driven amplification.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {165},
number = {},
pages = {108992},
doi = {10.1016/j.bioelechem.2025.108992},
pmid = {40286513},
issn = {1878-562X},
mesh = {*MicroRNAs/analysis/genetics ; *CRISPR-Cas Systems ; *Entropy ; Humans ; *Biosensing Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Electrochemical Techniques/methods ; Luminescent Measurements/methods ; },
abstract = {MicroRNAs (miRNAs) are critical regulators of numerous physiological and pathological processes, influencing gene expression and playing essential roles in cellular development, differentiation, and disease progression. Their sensitive and specific detection is vital for advancing biomedical research and clinical diagnostics, particularly for early disease detection and biomarker discovery. However, traditional miRNA detection methods often face significant challenges, such as limited sensitivity, insufficient specificity, and the inability to detect low-abundance miRNAs in complex biological samples. To overcome these limitations, we present a novel miRNA detection electrochemiluminescence (ECL) platform that integrates entropy-driven amplification with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a-mediated RNA cleavage. The entropy-driven amplification strategy exploits the thermodynamic advantages of nucleic acid hybridization, driving spontaneous molecular reorganization to amplify detection signals and achieve ultralow detection limits. CRISPR/Cas13a, an RNA-targeting nuclease, provides exceptional sequence specificity by recognizing and binding to target miRNA sequences, activating a collateral cleavage mechanism. This activity cleaves hairpin (HP) structure, generating a signal that further triggers EDA over DNA tetrahedron (DT) to induce a vigorous ECL response. Based on this strategy, we achieve rapid and precise quantification of miR-17 at femtomolar levels. Experimental results demonstrate high sensitivity, specificity, and the ability to analyze complex biological samples in the laboratory. This innovative approach holds great promise for advancing molecular diagnostics and personalized medicine.},
}

*MicroRNAs/analysis/genetics
*CRISPR-Cas Systems
*Entropy
Humans
*Biosensing Techniques/methods
*Nucleic Acid Amplification Techniques/methods
Limit of Detection
Electrochemical Techniques/methods
Luminescent Measurements/methods

@article {pmid40273641,
year = {2025},
author = {Bhoir, K and Prakash, G and Odaneth, A},
title = {Genetic Engineering of Yarrowia lipolytica for 1,8-cineole production: A sustainable approach.},
journal = {Enzyme and microbial technology},
volume = {189},
number = {},
pages = {110659},
doi = {10.1016/j.enzmictec.2025.110659},
pmid = {40273641},
issn = {1879-0909},
mesh = {*Yarrowia/genetics/metabolism ; *Eucalyptol/metabolism ; *Metabolic Engineering/methods ; Streptomyces/enzymology/genetics ; CRISPR-Cas Systems ; Mevalonic Acid/metabolism ; Genetic Engineering ; Intramolecular Lyases/genetics/metabolism ; Squalene/metabolism ; Diterpenes ; Diphosphates ; },
abstract = {1,8-Cineole, a monoterpene with diverse industrial and pharmaceutical applications, has garnered significant interest due to its unique properties. This study aims to achieve sustainable production of 1,8-cineole from Yarrowia lipolytica through metabolic and media engineering strategies. The heterologous 1,8-cineole synthase from Streptomyces clavuligerus was integrated through CRISPR-Cas9, along with overexpression of key genes in the mevalonate pathway and a double mutation in the Erg20p to enhance flux towards geranyl pyrophosphate. The modified strain was further investigated for varying carbon and nitrogen sources with MgSO4 addition. The above approaches achieved a titer of 4.68 mg/L of 1,8-cineole along with 1108.53 mg/L of intracellular squalene when grown on 5 % WCO, marking the first report of genetic engineering of Y. lipolytica for 1,8-cineole production. Further studies are in progress to redirect internal fluxes to 1,8-cineole for improvising yields and productivities. This work shows a sustainable and innovative approach to biotechnology improvements in terpene biosynthesis and waste valorization.},
}

*Yarrowia/genetics/metabolism
*Eucalyptol/metabolism
*Metabolic Engineering/methods
Streptomyces/enzymology/genetics
CRISPR-Cas Systems
Mevalonic Acid/metabolism
Genetic Engineering
Intramolecular Lyases/genetics/metabolism
Squalene/metabolism
Diterpenes
Diphosphates

@article {pmid40221070,
year = {2025},
author = {Khademi, Z and Mottaghi-Dastjerdi, N and Morad, H and Sahebkar, A},
title = {The role of CRISPR-Cas9 and CRISPR interference technologies in the treatment of autoimmune diseases.},
journal = {Autoimmunity reviews},
volume = {24},
number = {7},
pages = {103816},
doi = {10.1016/j.autrev.2025.103816},
pmid = {40221070},
issn = {1873-0183},
mesh = {Humans ; *Autoimmune Diseases/therapy/genetics/immunology ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genetic Therapy/methods ; Animals ; },
abstract = {Autoimmune disorders can be described as inappropriate immune responses directed against self-antigens, which account for substantial healthcare concerns around the world. Immunosuppression or immune modulation are the main therapeutic modalities for autoimmune disorders. These modalities, however, impair the ability of the immune system to fight against infections, thereby predisposing to opportunistic diseases. This review explores existing therapies for autoimmune disorders, highlighting their limitations and challenges. Additionally, it describes the potential of CRISPR-Cas9 technology as a novel therapeutic approach to address these challenges.},
}

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

@article {pmid40175650,
year = {2025},
author = {Guo, P and Song, S and Niu, Y and Kuang, X and Zhou, D and Zhou, Z and Zhang, Y and Ma, X},
title = {Alternative splicing of bunched confers a dual role in hippo pathway-dependent growth and tumorigenesis.},
journal = {Oncogene},
volume = {44},
number = {24},
pages = {1949-1960},
pmid = {40175650},
issn = {1476-5594},
support = {32322027//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32170824//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32370710//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; *Alternative Splicing/genetics ; *Drosophila Proteins/genetics/metabolism ; *Protein Serine-Threonine Kinases/metabolism/genetics ; *Carcinogenesis/genetics ; Signal Transduction/genetics ; Protein Isoforms/genetics ; *Intracellular Signaling Peptides and Proteins/metabolism/genetics ; Drosophila melanogaster/genetics ; YAP-Signaling Proteins ; Hippo Signaling Pathway ; Nuclear Proteins/genetics/metabolism ; Trans-Activators/genetics/metabolism ; Transcription Factors/genetics/metabolism ; Cell Proliferation/genetics ; CRISPR-Cas Systems ; },
abstract = {Alternative splicing is a fundamental mechanism that generates functionally distinct proteins from individual genes, contributing to gene regulation and proteomic diversity. In Drosophila, the bunched (bun) gene, a member of the TSC-22 domain gene family, undergoes alternative splicing, yielding diverse protein isoforms involved in crucial biological processes. Nevertheless, the specific roles and regulatory mechanisms of each isoform remain elusive. Here, we employed CRISPR/Cas9 technology to introduce targeted deletions within the endogenous locus of the bun gene, resulting in the removal of either long or short isoforms. We discovered that the short isoforms demonstrated a growth-suppressive role, whereas the long isoforms exhibited a growth-promoting effect. Surprisingly, the long isoforms exhibited a remarkable dual functionality, as both deletion and amplification of long isoform expression impede the excess growth induced by Hippo pathway inactivation. Mechanistically, ectopically expressed Bun long isoforms act as the transcriptional suppressor by competitively binding to targets' promoter regions in conjunction with Yorkie/Scalloped (Yki/Sd), thereby inhibiting its transcriptional outputs and ultimately leading to the growth suppression. These findings unveil the intricate interaction between distinct spliced isoforms of Bun and oncogenic outcomes, highlighting Bun long isoforms as the critical transcription suppressor regulating Hippo pathway inactivation-mediated growth and tumorigenesis in Drosophila.},
}

Animals
*Alternative Splicing/genetics
*Drosophila Proteins/genetics/metabolism
*Protein Serine-Threonine Kinases/metabolism/genetics
*Carcinogenesis/genetics
Signal Transduction/genetics
Protein Isoforms/genetics
*Intracellular Signaling Peptides and Proteins/metabolism/genetics
Drosophila melanogaster/genetics
YAP-Signaling Proteins
Hippo Signaling Pathway
Nuclear Proteins/genetics/metabolism
Trans-Activators/genetics/metabolism
Transcription Factors/genetics/metabolism
Cell Proliferation/genetics
CRISPR-Cas Systems

@article {pmid40156991,
year = {2025},
author = {Tang, Q and Zhang, J and Pang, J and Huang, Y and Guan, Y and Gong, Y and Tang, Q and Zhang, K and Liao, X},
title = {Hybridization chain reaction and CRISPR/Cas12a-integrated biosensor for precise Ago2 detection.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {165},
number = {},
pages = {108975},
doi = {10.1016/j.bioelechem.2025.108975},
pmid = {40156991},
issn = {1878-562X},
mesh = {*Argonaute Proteins/analysis/genetics/metabolism ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Humans ; *Nucleic Acid Hybridization ; Luminescent Measurements/methods ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {This study introduces an innovative electrochemiluminescence (ECL) biosensor for the highly sensitive and specific detection of Argonaute 2 (Ago2) activity. Ago2, a key enzyme in the RNA interference (RNAi) pathway, plays a crucial role in gene regulation, and its dysregulation is associated with diseases such as cancer and viral infections. The biosensor integrates hybridization chain reaction (HCR) amplification and the CRISPR/Cas12a system, leveraging a multi-stage signal amplification strategy. The detection mechanism begins with Ago2-mediated cleavage of a designed hairpin RNA (HP-RNA), releasing single-stranded RNA (ssRNA) that triggers HCR. This amplification step generates long DNA polymers, which serve as activators for the CRISPR/Cas12a system. Cas12a's collateral cleavage activity amplifies the signal further by cleaving a DNA reporter labeled with a ruthenium-based luminophore, enhancing the ECL output. This dual amplification strategy achieves exceptional sensitivity, with a detection limit of 0.126 aM. The biosensor demonstrates excellent specificity, distinguishing Ago2 from other Argonaute proteins, and maintains high reproducibility and stability, retaining 94 % of its signal after two weeks of storage. Real-world applicability was confirmed by accurately detecting Ago2 in spiked cell lysates, with recovery rates exceeding 100 %. The combination of HCR, CRISPR/Cas12a, and ECL establishes a robust platform for biomarker detection, offering superior sensitivity and adaptability for clinical diagnostics, disease monitoring, and therapeutic evaluation. This biosensor represents a significant advancement in the development of next-generation diagnostic tools.},
}

*Argonaute Proteins/analysis/genetics/metabolism
*Biosensing Techniques/methods
*CRISPR-Cas Systems
Humans
*Nucleic Acid Hybridization
Luminescent Measurements/methods
Limit of Detection
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40118066,
year = {2025},
author = {Lodewijk, GA and Kozuki, S and Han, CJ and Topacio, BR and Lee, S and Nixon, L and Zargari, A and Knight, G and Ashton, R and Qi, LS and Shariati, SA},
title = {Self-organization of mouse embryonic stem cells into reproducible pre-gastrulation embryo models via CRISPRa programming.},
journal = {Cell stem cell},
volume = {32},
number = {6},
pages = {895-913.e8},
doi = {10.1016/j.stem.2025.02.015},
pmid = {40118066},
issn = {1875-9777},
mesh = {Animals ; Mice ; *Mouse Embryonic Stem Cells/cytology/metabolism ; Cell Differentiation/genetics ; CDX2 Transcription Factor/genetics/metabolism ; *Embryo, Mammalian/cytology/metabolism ; GATA6 Transcription Factor/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Models, Biological ; Cell Lineage ; },
abstract = {Embryonic stem cells (ESCs) can self-organize into structures with spatial and molecular similarities to natural embryos. During development, embryonic and extraembryonic cells differentiate through activation of endogenous regulatory elements while co-developing via cell-cell interactions. However, engineering regulatory elements to self-organize ESCs into embryo models remains underexplored. Here, we demonstrate that CRISPR activation (CRISPRa) of two regulatory elements near Gata6 and Cdx2 generates embryonic patterns resembling pre-gastrulation mouse embryos. Live single-cell imaging revealed that self-patterning occurs through orchestrated collective movement driven by cell-intrinsic fate induction. In 3D, CRISPRa-programmed embryo models (CPEMs) exhibit morphological and transcriptomic similarity to pre-gastrulation mouse embryos. CPEMs allow versatile perturbations, including dual Cdx2-Elf5 activation to enhance trophoblast differentiation and lineage-specific activation of laminin and matrix metalloproteinases, uncovering their roles in basement membrane remodeling and embryo model morphology. Our findings demonstrate that minimal intrinsic epigenome editing can self-organize ESCs into programmable pre-gastrulation embryo models with robust lineage-specific perturbation capabilities.},
}

Animals
Mice
*Mouse Embryonic Stem Cells/cytology/metabolism
Cell Differentiation/genetics
CDX2 Transcription Factor/genetics/metabolism
*Embryo, Mammalian/cytology/metabolism
GATA6 Transcription Factor/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Models, Biological
Cell Lineage

@article {pmid38997889,
year = {2025},
author = {Yin, E and Esbin, MN},
title = {Optimized CRISPR-based knockout in BeWo cells.},
journal = {Placenta},
volume = {166},
number = {},
pages = {71-76},
doi = {10.1016/j.placenta.2024.07.005},
pmid = {38997889},
issn = {1532-3102},
mesh = {Humans ; *Gene Knockout Techniques/methods ; *CRISPR-Cas Systems ; Female ; *Trophoblasts/metabolism ; *Gene Editing/methods ; Pregnancy ; Cell Line, Tumor ; Ribonucleoproteins/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Electroporation ; Pregnancy Proteins/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {CRISPR genome editing is a widely used tool to perturb genes of interest within cells and tissues and can be used as a research tool to study the connection between genotypes and cellular phenotypes. Highly efficient genome editing is limited in certain cell types due to low transfection efficiency or single-cell survivability. This is true for BeWo cells, an in vitro model of placental syncytiotrophoblast cell-cell fusion and hormone secretion. Here we describe an optimized and easy-to-use protocol for knockout in BeWo cells using CRISPR Cas9 ribonucleoprotein (RNP) complexes delivered via electroporation. Further, we describe parameters for successful guide RNA design and how to assess genetic knockouts in BeWo cells so that users can apply this technique to their own genes of interest. We provide a positive control for inducing highly efficient knockout of the cell-cell fusion protein Syncytin-2 (ERVFRD-1) and assessing editing efficiency at this locus. We anticipate that efficient RNP-mediated genetic knockouts in BeWo cells will facilitate the study of new genes involved in cell-cell fusion and hormone secretion in this important cellular model system. Furthermore, this strategy of optimized nucleofection and RNP delivery may be of use in other difficult-to-edit trophoblast cells or could be applied to efficiently deliver transgenes to BeWo cells.},
}

Humans
*Gene Knockout Techniques/methods
*CRISPR-Cas Systems
Female
*Trophoblasts/metabolism
*Gene Editing/methods
Pregnancy
Cell Line, Tumor
Ribonucleoproteins/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Electroporation
Pregnancy Proteins/genetics
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40455261,
year = {2025},
author = {Zhang, M and Zhou, L and Afridi, M and Guo, H and Cheng, H},
title = {An efficient hairy root system for validation of CRISPR/Cas system activities in cotton.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {116},
pmid = {40455261},
issn = {1438-7948},
support = {NK2022010303//the Ministry of Agriculture/ ; B23CJ0208//Hainan Seed Industry Laboratory/ ; YSPTZX202502//Hainan Province Academician Innovation Platform/ ; },
mesh = {*Gossypium/genetics/growth & development/microbiology ; *Plant Roots/genetics/growth & development ; *CRISPR-Cas Systems ; Agrobacterium/genetics ; *Gene Editing/methods ; Plants, Genetically Modified/genetics ; },
abstract = {The hairy root induction system has been widely applied in studying gene expression and function in plant species due to its rapidity and efficiency. The hairy root system is an efficient tool for evaluating the activities of CRISPR/Cas systems. Cotton hairy roots were primarily induced through cotton tissue culture under aseptic conditions and by injecting cotton stem tips under non-aseptic conditions. However, both methods are lab-intensive and time-consuming. In this study, an efficient cotton hairy root induction procedure was established via infecting cotton hypocotyls with Agrobacterium rhizogenes under non-sterile conditions. Cotton seedlings with expanded cotyledons were decapitated with a slanted cut, and the residual hypocotyl (maintained 1 cm apical portion) was inoculated with A. rhizogenes. Over 90% of the infected explants from all three tested varieties could produce hairy roots after 8 days of inoculation. The effictiveness of the method was tested by overexpressing two reporter genes (eGFP and GUS). The transformation efficiency of the GUS and eGFP were ranged from50-68.18% and 40.9-68.18%. In addition, the editing efficiency of target sites in different CRISPR/Cas systems were also tested in hairy root. This method provided technical support for screening suitable target sites for cotton gene editing.},
}

*Gossypium/genetics/growth & development/microbiology
*Plant Roots/genetics/growth & development
*CRISPR-Cas Systems
Agrobacterium/genetics
*Gene Editing/methods
Plants, Genetically Modified/genetics

@article {pmid40454797,
year = {2025},
author = {Wu, J and Yan, Q and Qiu, H and Gao, EB},
title = {Integrating Enzyme-DNA Complex and CRISPR/Cas12a for Robust Norovirus Detection.},
journal = {Journal of medical virology},
volume = {97},
number = {6},
pages = {e70426},
doi = {10.1002/jmv.70426},
pmid = {40454797},
issn = {1096-9071},
support = {//The Ningbo Clinical Research Center for Children's Health and Diseases (no. 2019A21002), the Ningbo public welfare project (no. 2022S127), the Ningbo key discipline Pediatrics (no. 2022-B17), the Innovation Project of Distinguished Medical Team in Ningbo (no. 2022020405). Ningbo Medical and Health Brand Discipline (PPXK2024-06)./ ; },
mesh = {*Norovirus/isolation & purification/genetics ; Humans ; *Caliciviridae Infections/diagnosis/virology ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Gastroenteritis/virology/diagnosis ; *Nucleic Acid Amplification Techniques/methods ; RNA, Viral/genetics ; *Molecular Diagnostic Techniques/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Human norovirus (NoV) is a primary cause of acute gastroenteritis in children, making accurate and rapid detection essential for effective disease prevention and control. In this study, we developed a sensitive and efficient platform for pathogen nucleic acid detection by integrating asymmetric nucleic acid sequence-based amplification (asymmetric NASBA), enzyme-DNA molecular complex, and the clustered regularly interspaced short palindromic repeats (CRISPR) system, namely an A-enDMC platform. The target recognition capability of the enzyme-DNA complex operates independently from the signal amplification function of the CRISPR system. By decoupling the CRISPR reaction from the dependence on specific target sequences, the platform's universality and modularity are enhanced. The assay is fast (< 1.5 h), highly sensitive (< 5 copies/µL), and demonstrates no cross-reactivity with other common viruses. Compared to the widely used RT-qPCR method, the platform demonstrates high consistency in detection results, with the detection coincidence rate of 96.77% and a kappa value of 0.87. This platform provides a versatile technological tool for highly sensitive and specific RNA detection, demonstrating its extensive potential in real sample analysis.},
}

*Norovirus/isolation & purification/genetics
Humans
*Caliciviridae Infections/diagnosis/virology
*CRISPR-Cas Systems
Sensitivity and Specificity
*Gastroenteritis/virology/diagnosis
*Nucleic Acid Amplification Techniques/methods
RNA, Viral/genetics
*Molecular Diagnostic Techniques/methods
Clustered Regularly Interspaced Short Palindromic Repeats
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40453079,
year = {2025},
author = {Lund, S and Gong, C and Yu, X and Staudt, LM and Hodson, DJ and Scheich, S},
title = {Strategies for CRISPR-based knock-ins in primary human B cells and lymphoma cell lines.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1589729},
pmid = {40453079},
issn = {1664-3224},
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Knock-In Techniques/methods ; *Gene Editing/methods ; *B-Lymphocytes/metabolism/immunology ; Cell Line, Tumor ; *Lymphoma, Large B-Cell, Diffuse/genetics ; },
abstract = {Since its advent about ten years ago, the CRISPR-Cas9 system has been frequently used in biomedical applications. It has advanced various fields, and CRISPR-Cas9-based therapeutics have shown promising results in the treatment of specific hematological diseases. Furthermore, CRISPR gene editing technologies have revolutionized cancer research by enabling a broad range of genetic perturbations, including genetic knockouts and precise single nucleotide changes. This perspective focuses on the state-of-the-art methodology of CRISPR knock-ins to engineer immune cells. Since this technique relies on homology-directed repair (HDR) of double-strand breaks (DSBs) induced by the Cas9 enzyme, it can be used to introduce specific mutations into the target genome. Therefore, this methodology offers a valuable opportunity to functionally study specific mutations and to uncover their impacts not only on overall cell functions but also on the mechanisms behind cancer-related alterations in common signaling pathways. This article highlights CRISPR knock-in strategies, protocols, and applications in cancer and immune research, with a focus on diffuse large B cell lymphoma.},
}

Humans
*CRISPR-Cas Systems
*Gene Knock-In Techniques/methods
*Gene Editing/methods
*B-Lymphocytes/metabolism/immunology
Cell Line, Tumor
*Lymphoma, Large B-Cell, Diffuse/genetics

@article {pmid40451886,
year = {2025},
author = {Okamoto, M and Sasamoto, K and Takahashi-Nakaguchi, A and Zhao, F and Yamaguchi, M and Chibana, H},
title = {CRISPR-Cas9 RNP-Mediated Deletion of ERG25 in Non-albicans Candida Species, Including Candida auris.},
journal = {Medical mycology journal},
volume = {66},
number = {2},
pages = {35-43},
doi = {10.3314/mmj.24-00023},
pmid = {40451886},
issn = {2186-165X},
mesh = {*CRISPR-Cas Systems ; Candida glabrata/genetics ; *Candida auris/genetics ; *Fungal Proteins/genetics ; *Gene Deletion ; *Candida/genetics ; Candidiasis/microbiology ; Antifungal Agents/pharmacology ; },
abstract = {The incidence of infections caused by non-albicans Candida (NAC) species, including Candida glabrata and Candida tropicalis, has recently increased. Furthermore, Candida auris, a multidrug-resistant yeast, poses a serious threat to global health. The development of novel antifungal agents with alternative mechanisms of action is necessary to combat these fungi. However, genetic analyses of the virulence factors in these NAC species are insufficient for this purpose. Recent advancements in the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system have facilitated enhanced the genetic analysis of NAC species. The RNP-based system, in which the Cas9-gRNA complex is assembled in vitro and introduced into cells, offers a simplified approach to genetic modification, eliminating the need for species-specific plasmids. Previous our research identified the ERG25 gene, which encodes C-4 sterol methyl oxidase, as a promising antifungal target in C. glabrata. This study demonstrated deletion of the ERG25 homolog in C. glabrata and C. auris using an RNP-based CRISPR-Cas9 system. The deletion of ERG25 in C. auris and C. glabrata indicated that Erg25 is crucial for the survival of these pathogenic yeasts within the host. Furthermore, we have successfully deleted the ERG25 alleles in C. tropicalis and Candida parapsilosis, demonstrating the effectiveness of using both the CRISPR-Cas9 and Cre-loxP systems in these species for the first time.},
}

*CRISPR-Cas Systems
Candida glabrata/genetics
*Candida auris/genetics
*Fungal Proteins/genetics
*Gene Deletion
*Candida/genetics
Candidiasis/microbiology
Antifungal Agents/pharmacology

@article {pmid40347811,
year = {2025},
author = {Wang, R and He, B and Liang, Z and Liu, Y and Yang, J and Jin, H and Wei, M and Ren, W and Suo, Z and Xu, Y},
title = {A dual-mode sensor for rapid detection of procymidone: "Dark box" qualitative analysis and electrochemical quantification mediated by PdHPCN-222/PEI-rGO and CRISPR/Cas12a.},
journal = {Food chemistry},
volume = {486},
number = {},
pages = {144586},
doi = {10.1016/j.foodchem.2025.144586},
pmid = {40347811},
issn = {1873-7072},
mesh = {*Electrochemical Techniques/methods/instrumentation ; *Biosensing Techniques/methods/instrumentation ; CRISPR-Cas Systems ; Graphite/chemistry ; Limit of Detection ; Polyethyleneimine/chemistry ; Fluorescence ; Food Contamination/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {This study presents an integrated dual-mode sensing strategy, in which qualitative fluorescence screening is followed by quantitative electrochemical detection, improving detection efficiency and enabling rapid PCM analysis. It develops a novel fluorescence electrochemical aptasensor that combines in vitro "Dark-box" applications with CRISPR/Cas12a system electrode surface sensing technology. PCM activates the DNA walker, and the DNAzyme induces cyclic cleavage of DNA strands bearing the Carboxyfluorescein (FMA) group. After magnetic separation, the fluorescence reaction combined with the "Dark box" enables the preliminary qualitative analysis of procymidone (PCM). Following the preliminary qualitative detection, the solution is introduced to the electrochemical aptasensor platform integrated with the CRISPR/Cas12 system. The Cas12a system triggers cyclic cleavage, producing a signal change that enables the electrochemical quantitative detection of PCM. An fluorescence (FL) response occurs when the PCM concentration in spiked samples is at or above 1 pg·mL[-1], allowing for qualitative fluorescence detection. The EC platform's detection limit is 8.51 × 10[-6] ng·mL[-1], with a range of 1 × 10[-2] ng·mL[-1] to 1 × 10[4] ng·mL[-1]. The designed dual-mode sensor provides reliable monitoring of PCM in real samples.},
}

*Electrochemical Techniques/methods/instrumentation
*Biosensing Techniques/methods/instrumentation
CRISPR-Cas Systems
Graphite/chemistry
Limit of Detection
Polyethyleneimine/chemistry
Fluorescence
Food Contamination/analysis
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid36847138,
year = {2025},
author = {Baerwald, MR and Funk, EC and Goodbla, AM and Campbell, MA and Thompson, T and Meek, MH and Schreier, AD},
title = {Rapid CRISPR-Cas13a genetic identification enables new opportunities for listed Chinook salmon management.},
journal = {Molecular ecology resources},
volume = {25},
number = {5},
pages = {e13777},
doi = {10.1111/1755-0998.13777},
pmid = {36847138},
issn = {1755-0998},
support = {4600012328//Department of Water Resources/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Salmon/genetics/classification ; California ; *Genotyping Techniques/methods ; },
abstract = {Accurate taxonomic identification is foundational for effective species monitoring and management. When visual identifications are infeasible or inaccurate, genetic approaches provide a reliable alternative. However, these approaches are sometimes less viable (e.g., need for near real-time results, remote locations, funding concerns, molecular inexperience). In these situations, CRISPR-based genetic tools can fill an unoccupied niche between real-time, inexpensive, but error-prone visual identification and more expensive or time-consuming, but accurate genetic identification for taxonomic units that are difficult or impossible to visually identify. Herein, we use genomic data to develop CRISPR-based SHERLOCK assays capable of rapidly (<1 h), accurately (94%-98% concordance between phenotypic and genotypic assignments), and sensitively (detects 1-10 DNA copies/reaction) distinguishing ESA-listed Chinook salmon runs (winter- and spring-run) from each other and from unlisted runs (fall- and late fall-run) in California's Central Valley. The assays can be field deployable with minimally invasive mucus swabbing negating the need for DNA extraction (decreasing costs and labour), minimal and inexpensive equipment needs, and minimal training to conduct following assay development. This study provides a powerful genetic approach for a species of conservation concern that benefits from near real-time management decision-making but also serves as a precedent for transforming how conservation scientists and managers view genetic identification going forward. Once developed, CRISPR-based tools can provide accurate, sensitive, and rapid results, potentially without the prohibitive need for expensive specialty equipment or extensive molecular training. Further adoption of this technology will have widespread value for the monitoring and protection of our natural resources.},
}

Animals
*CRISPR-Cas Systems
*Salmon/genetics/classification
California
*Genotyping Techniques/methods

@article {pmid40451196,
year = {2025},
author = {Smirnov, AV and Yunusova, AM},
title = {Novel CRISPR/Cas9-Based Approaches for Quantitative Study of DSB Repair Mechanics.},
journal = {Biochemistry. Biokhimiia},
volume = {90},
number = {4},
pages = {437-456},
doi = {10.1134/S0006297924601813},
pmid = {40451196},
issn = {1608-3040},
mesh = {*CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; *DNA Repair ; Humans ; Animals ; },
abstract = {This review examines modern approaches to studying double-strand break (DSB) DNA repair in mammalian cells, employing the CRISPR/Cas9 system. Due to its flexibility and efficacy, the Cas9 nuclease is used in numerous genetic reporters. We discuss various fluorescence-based genetic reporters used to monitor the repair process. In addition, among the innovative Cas9-based methods, special attention is given to the techniques that examine both single and multiple DSBs, including approaches such as DSB-TRIP and ddXR. These methods open new possibilities for investigating structural rearrangements or analyzing random genomic sites. Additionally, the review considers how DSBs induced by Cas9 differ from those made by other nucleases and how these peculiarities could impact DNA repair mechanisms. Understanding these differences is crucial for planning experiments aimed at studying DSB repair.},
}

*CRISPR-Cas Systems
*DNA Breaks, Double-Stranded
*DNA Repair
Humans
Animals

@article {pmid40451118,
year = {2025},
author = {Piñeiro-Silva, C and Gadea, J},
title = {Optimizing gene editing in pigs: The role of electroporation and lipofection.},
journal = {Animal reproduction science},
volume = {278},
number = {},
pages = {107874},
doi = {10.1016/j.anireprosci.2025.107874},
pmid = {40451118},
issn = {1873-2232},
abstract = {The production of genetically modified pigs is becoming increasingly important in both the agricultural and biomedical fields. Optimization of these processes is a key objective to improve the precision, scalability and viability of genetically modified animals for research and commercial applications. Among the available techniques, electroporation and lipofection have emerged as promising alternatives to traditional methods such as microinjection and somatic cell nuclear transfer (SCNT) due to their simplicity, cost-effectiveness, and potential for high-throughput applications. These methods allow the direct delivery of CRISPR/Cas components into zygotes and embryos, reducing the technical expertise required and bypassing some of the challenges associated with cloning. This review examines the application, efficacy, and outcomes of electroporation and lipofection as gene editing techniques in porcine gametes and embryos. We provide a comprehensive synthesis of recent advances, compare their efficacy, and discuss their potential in agricultural and biomedical research. The principles and mechanisms of both methods are reviewed, highlighting their advantages, such as cost-effectiveness and ease of implementation, over traditional approaches such as microinjection. In addition, we address their limitations, including variability in efficiency, and discuss recent protocol optimizations aimed at improving reproducibility and applicability. By analyzing these developments, this review provides valuable insights into the evolving role of electroporation and lipofection in porcine genetic modification strategies.},
}

@article {pmid40450609,
year = {2025},
author = {Pal, S and Krishna, R and Dedhia, L and Panwar, HS and Karkute, SG and Rai, N and Kumar, R and Pandey, S and Singh, AK},
title = {CRISPR mediated gene editing for economically important traits in horticultural crops: progress and prospects.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {26},
pmid = {40450609},
issn = {1573-9368},
mesh = {*Gene Editing/methods ; *Crops, Agricultural/genetics/growth & development ; *CRISPR-Cas Systems/genetics ; *Plants, Genetically Modified/genetics/growth & development ; Plant Breeding ; Genome, Plant ; },
abstract = {Horticultural crops, with their cost-effectiveness, rich mineral and vitamin content, and high yield potential, have become indispensable worldwide for ensuring food and nutritional security. With the world's population on the rise and climate change becoming more prominent, it is crucial to focus on creating resilient, high-yielding crop varieties that can withstand the changing climate. Genetic improvement of different horticultural crops using conventional tools is both time-consuming and labourious. However, the breeding period can be cut short by adopting modern breeding techniques, including CRISPR/Cas-mediated genome editing. In the present review, we discuss the progress made so far through genome editing to improve several horticultural crops for various traits like stress resistance, morphology, nutritional attributes, quality, shelf life, male sterility, architecture and economic yield. We have also discussed the emerging CRISPR technologies like base editing, epigenome editing, CRE editing, transposon-based editing, prime editing etc., along with their pros and cons and the future prospects. The ethical considerations for commercialization and current regulatory frameworks for gene-edited products have also been discussed.},
}

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

@article {pmid40450056,
year = {2025},
author = {Ortiz-Rodríguez, LA and Cabanzo, R and Jaimes-Dueñez, J and Mendez-Sanchez, SC and Duque, JE},
title = {TropD-detector a CRISPR/LbCas12a-based system for rapid screening of Trypanosoma cruzi in Chagas vectors and reservoirs.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {19107},
pmid = {40450056},
issn = {2045-2322},
mesh = {*Trypanosoma cruzi/genetics/isolation & purification ; *Chagas Disease/parasitology/diagnosis ; Animals ; *CRISPR-Cas Systems ; *Disease Reservoirs/parasitology ; Rhodnius/parasitology ; Cytochromes b/genetics ; Endodeoxyribonucleases/genetics ; Histones/genetics ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; },
abstract = {Chagas disease, also known as American Trypanosomiasis, is a zoonosis with global distribution caused by the parasite Trypanosoma cruzi, primarily transmitted through the feces of infected triatomines. The emergence of new cases highlights the importance of early pathogen detection in vectors and reservoirs to generate effective control strategies and establish preventive policies. The objective of this study was to design and validate a detection system of T. cruzi based on specific DNA cleavage, activation of Cas12a and trans-cleavage, targeting the genes Cytochrome B (Cytb), 18 S ribosomal subunit (SR18 s), and histone (H2 A). This system was validated for their uses in both vectors and reservoirs of the parasite. The initial step involved performing a bioinformatic analysis of the target genes, followed by the design of RNA guides specific to each cleavage site, along with primers for amplifying the target region through PCR and RPA. Subsequently, we sequenced the amplified DNA target and validated the detection system using T. cruzi DNA extracted from naturally infected Rhodnius pallescens in the metropolitan area of Bucaramanga, Colombia. After standardizing the method, we tested the CRISPR/Cas system with Silvio X10 laboratory strain of T. cruzi and scaled up to blood samples of naturally infected Didelphis marsupialis. As a result, we observed DNA cleavage using the CRISPR/Cas system with the Cytb guide, achieving a detection sensitivity of 118 parasite equivalents/mL in PCR and 116 parasite equivalents/mL with RPA amplification. Sequencing of the Cytb gene showed no mutations in the cleavage site. However, point mutations and indels were found in SR18S and H2 A, avoiding the formation of the CRISPR/LbCas12 complex. Furthermore, we introduce the design of a fluorescent detection prototype with CRISPR/LbCas12a called "Tropical Diseases Detector" (TropD-Detector). This device operates with an excitation wavelength of 480 nm emitted by an LED and a high-pass light filter with a cutoff wavelength of 500 nm. We detected positive samples using any photographic camera system. The TropD-Detector provides a visual, viable, and sensitive method for detecting T. cruzi in both vectors and reservoirs from endemic areas.},
}

*Trypanosoma cruzi/genetics/isolation & purification
*Chagas Disease/parasitology/diagnosis
Animals
*CRISPR-Cas Systems
*Disease Reservoirs/parasitology
Rhodnius/parasitology
Cytochromes b/genetics
Endodeoxyribonucleases/genetics
Histones/genetics
CRISPR-Associated Proteins/genetics
Bacterial Proteins

@article {pmid40449999,
year = {2025},
author = {Schweitzer, AY and Adams, EW and Nguyen, MTA and Lek, M and Isaacs, FJ},
title = {Precision multiplexed base editing in human cells using Cas12a-derived base editors.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5061},
pmid = {40449999},
issn = {2041-1723},
support = {R01 GM117230/GM/NIGMS NIH HHS/United States ; GM117230//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; CF22-1046//Carlsbergfondet (Carlsberg Foundation)/ ; },
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/metabolism/genetics ; Genome, Human ; HEK293 Cells ; Cell Line ; Bacterial Proteins ; },
abstract = {Base editors enable the direct conversion of target nucleotides without introducing DNA double strand breaks, making them a powerful tool for creating point mutations in a human genome. However, current Cas9-derived base editing technologies have limited ability to simultaneously edit multiple loci with base-pair level precision, hindering the generation of polygenic phenotypes. Here, we test the ability of six Cas12a-derived base editing systems to process multiple gRNAs from a single transcript. We identify base editor variants capable of multiplexed base editing and improve the design of the respective gRNA array expression cassette, enabling multiplexed editing of 15 target sites in multiple human cell lines, increasing state-of-the-art in multiplexing by three-fold in the field of mammalian genome engineering. To reduce bystander mutations, we also develop a Cas12a gRNA engineering approach that directs editing outcomes towards a single base-pair conversion. We combine these advances to demonstrate that both strategies can be combined to drive multiplex base editing with greater precision and reduced bystander mutation rates. Overcoming these key obstacles of mammalian genome engineering technologies will be critical for their use in studying single nucleotide variant-associated diseases and engineering synthetic mammalian genomes.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*CRISPR-Associated Proteins/genetics/metabolism
*Endodeoxyribonucleases/metabolism/genetics
Genome, Human
HEK293 Cells
Cell Line
Bacterial Proteins

@article {pmid40449852,
year = {2025},
author = {Kolesova, E and Pulone, S and Kostyushev, D and Tasciotti, E},
title = {CRISPR/Cas bioimaging: From whole body biodistribution to single-cell dynamics.},
journal = {Advanced drug delivery reviews},
volume = {},
number = {},
pages = {115619},
doi = {10.1016/j.addr.2025.115619},
pmid = {40449852},
issn = {1872-8294},
abstract = {This review explores the transformative role of CRISPR/Cas systems in optical bioimaging, emphasizing how advancements in nanoparticle (NP) technologies are revolutionizing the visualization of gene-editing processes both in vitro and in vivo. Optical imaging techniques, such as near-infrared (NIR) and fluorescence imaging, have greatly benefited from the integration of nanoformulated contrast agents, improving resolution, sensitivity, and specificity. CRISPR/Cas systems, originally developed just for gene editing, are now being coupled with these imaging modalities to enable real-time monitoring and quantitative measurements of metabolites, vitamins, proteins, nucleic acids and other entities in specific areas of the body, as well as tracking of CRISPR/Cas delivery, editing efficiency, and potential off-target effects. The development of CRISPR/Cas-loaded NPs allows for enhanced imaging and precise monitoring across multiple scales with multiplexed and multicolor imaging in complex settings, including potential in vivo diagnostics. CRISPR/Cas therapeutics as well as diagnostics are hindered by the lack of efficient and targeted delivery tools. Biomimetic NPs have emerged as promising tools for improving biocompatibility, enhancing targeting capabilities, and overcoming biological barriers, facilitating more efficient delivery and bioimaging of CRISPR/Cas systems in vivo. As the design of these NPs and delivery mechanisms improves, alongside advancements in endolysosomal escape, CRISPR/Cas-based bioimaging will continue to advance, offering unprecedented possibilities in precision medicine and theranostic applications.},
}

@article {pmid40448764,
year = {2025},
author = {Jin, H and Sophocleous, A and Azfer, A and Ralston, SH},
title = {Analysis of Transcriptional Regulation in Bone Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2885},
number = {},
pages = {247-269},
pmid = {40448764},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems ; *Transcription, Genetic ; Promoter Regions, Genetic ; *Gene Expression Regulation ; Chromatin Immunoprecipitation/methods ; Humans ; Electrophoretic Mobility Shift Assay/methods ; Animals ; *Bone and Bones/cytology/metabolism ; *Osteoblasts/metabolism ; Mice ; Genes, Reporter ; },
abstract = {Transcription is a process by which the rate of RNA synthesis is regulated. Here we describe the techniques for carrying out promoter-reporter assays: electrophoretic mobility shift assays, chromosome conformation capture (3C) assays, chromatin immunoprecipitation assays, and CRISPR-Cas9 assay-five commonly used methods for studying and altering gene transcription.},
}

CRISPR-Cas Systems
*Transcription, Genetic
Promoter Regions, Genetic
*Gene Expression Regulation
Chromatin Immunoprecipitation/methods
Humans
Electrophoretic Mobility Shift Assay/methods
Animals
*Bone and Bones/cytology/metabolism
*Osteoblasts/metabolism
Mice
Genes, Reporter

@article {pmid40448746,
year = {2025},
author = {Kamli, H and Khan, NU},
title = {Revolutionising cancer intervention: the repercussions of CAR-T cell therapy on modern oncology practices.},
journal = {Medical oncology (Northwood, London, England)},
volume = {42},
number = {7},
pages = {228},
pmid = {40448746},
issn = {1559-131X},
mesh = {Humans ; *Neoplasms/therapy/immunology ; *Immunotherapy, Adoptive/methods ; *Receptors, Chimeric Antigen/immunology ; Gene Editing/methods ; Tumor Microenvironment/immunology ; CRISPR-Cas Systems ; Medical Oncology/methods/trends ; },
abstract = {Chimeric Antigen Receptor T-cell (CAR-T) therapy represents a groundbreaking advance in oncology, leveraging patient-specific immune cells to target malignant tumours precisely. By equipping T cells with synthetic receptors, CAR-T therapy achieves remarkable antitumor effects and offers hope for durable cancer control. However, several limitations persist, including antigen scarcity, immunosuppressive tumour microenvironments, and T-cell exhaustion. CRISPR-Cas9 gene editing has enhanced CAR-T potency by knocking out immune checkpoints (PD-1, CTLA-4) and improving persistence, while RNA interference (RNAi) silences immune-evasion genes (e.g. SOCS1). Nanozyme-based delivery systems enable precise CRISPR-Cas9 delivery (> 70% editing efficiency) and tumour targeting, overcoming instability and off-target effects. Innovations like SUPRA CARs, armoured CAR-T cells (e.g. IL-12/IL-21-secreting TRUCKs), and dual checkpoint inhibition synergize to reprogram the tumour microenvironment, reducing relapse by 40% in trials. Despite progress, high costs, manufacturing hurdles, and ethical concerns (e.g. germline editing risks) remain critical barriers. Emerging solutions include universal off-the-shelf CAR-Ts, hybrid nano-CRISPR systems, and AI-driven design, paving the way for scalable, personalised immunotherapy. This review highlights breakthroughs in CRISPR, RNAi, and nanotechnology, underscoring CAR-T therapy's transformative potential while addressing translational challenges for broader clinical adoption.},
}

Humans
*Neoplasms/therapy/immunology
*Immunotherapy, Adoptive/methods
*Receptors, Chimeric Antigen/immunology
Gene Editing/methods
Tumor Microenvironment/immunology
CRISPR-Cas Systems
Medical Oncology/methods/trends

@article {pmid40447846,
year = {2025},
author = {Blaskovich, MAT and Cooper, MA},
title = {Antibiotics re-booted-time to kick back against drug resistance.},
journal = {npj antimicrobials and resistance},
volume = {3},
number = {1},
pages = {47},
pmid = {40447846},
issn = {2731-8745},
abstract = {After decades of neglect and a decline in antibiotic research and development, we are now finally witnessing the advent of new funding programs dedicated to new therapies. In addition to traditional new chemical entities that directly kill or arrest the growth of bacteria, alternative approaches are being identified and advanced towards proof-of-concept trials in the clinic. We briefly review the current pipeline of conventional new antibiotics and highlight in more depth promising alternatives, including potentiators of antibiotic action, bacteriophage, lysins and microbiome modulation. More innovative approaches, such as adaptive and innate immune modulators, CRISPR-Cas and diagnostic guided 'theranostics' are discussed and contrasted. Such exploratory therapies may require the development of alternative regulatory and clinical development pathways, but represent a potential circuit breaker from the current 'arms race' between bacteria and traditional antibiotics.},
}

@article {pmid40447637,
year = {2025},
author = {Schwaemmle, H and Soldati, H and Lykoskoufis, NMR and Docquier, M and Hainard, A and Braun, SMG},
title = {CRISPR screen decodes SWI/SNF chromatin remodeling complex assembly.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5011},
pmid = {40447637},
issn = {2041-1723},
support = {PCEFP3_194305//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; },
mesh = {Animals ; Mice ; *Chromatin Assembly and Disassembly/genetics ; *Chromosomal Proteins, Non-Histone/metabolism/genetics ; *Transcription Factors/metabolism/genetics ; Chromatin/metabolism/genetics ; RNA-Binding Proteins/metabolism/genetics ; Mouse Embryonic Stem Cells/metabolism ; *CRISPR-Cas Systems ; Humans ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The SWI/SNF (or BAF) complex is an essential chromatin remodeler, which is frequently mutated in cancer and neurodevelopmental disorders. These are often heterozygous loss-of-function mutations, indicating a dosage-sensitive role for SWI/SNF subunits. However, the molecular mechanisms regulating SWI/SNF subunit dosage to ensure complex assembly remain largely unexplored. We performed a CRISPR KO screen, using epigenome editing in mouse embryonic stem cells, and identified Mlf2 and Rbm15 as regulators of SWI/SNF complex activity. First, we show that MLF2, a poorly characterized chaperone protein, promotes SWI/SNF assembly and binding to chromatin. Rapid degradation of MLF2 reduces chromatin accessibility at sites that depend on high levels of SWI/SNF binding to maintain open chromatin. Next, we find that RBM15, part of the m[6]A writer complex, controls m[6]A modifications on specific SWI/SNF mRNAs to regulate subunit protein levels. Misregulation of m[6]A methylation causes overexpression of core SWI/SNF subunits leading to the assembly of incomplete complexes lacking the catalytic ATPase/ARP subunits. These data indicate that targeting modulators of SWI/SNF complex assembly may offer a potent therapeutic strategy for diseases associated with impaired chromatin remodeling.},
}

Animals
Mice
*Chromatin Assembly and Disassembly/genetics
*Chromosomal Proteins, Non-Histone/metabolism/genetics
*Transcription Factors/metabolism/genetics
Chromatin/metabolism/genetics
RNA-Binding Proteins/metabolism/genetics
Mouse Embryonic Stem Cells/metabolism
*CRISPR-Cas Systems
Humans
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40446748,
year = {2025},
author = {Zuo, Z and Liang, R and Fan, S and Shan, H and Zhang, H and Wang, X and Fei, T},
title = {Genome-wide CRISPR screens identify key regulators of adipogenesis and glucose uptake in beige adipocytes.},
journal = {Biochemical and biophysical research communications},
volume = {774},
number = {},
pages = {152093},
doi = {10.1016/j.bbrc.2025.152093},
pmid = {40446748},
issn = {1090-2104},
mesh = {*Adipogenesis/genetics ; *Glucose/metabolism ; *Adipocytes, Beige/metabolism/cytology ; Animals ; Humans ; Mice ; *CRISPR-Cas Systems ; Insulin Resistance ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {The beiging of white adipocytes enhances energy expenditure by utilizing fatty acids and glucose, offering therapeutic potential against obesity and type 2 diabetes. However, the genetic mechanisms driving this process remain unclear. Here, we performed multiple fluorescence-activated cell sorting (FACS)-based genome-wide CRISPR loss-of-function screens in beige adipocytes with or without insulin resistance (IR) induction, and systematically identified functional regulators of beige adipocyte adipogenesis and glucose metabolism. We further integrated transcriptomics and human genetics data to pinpoint key genes for adipogenesis and glucose metabolism in beige adipocytes. Moreover, we validated SULT2B1 and ATP1B2 as key adipogenesis genes for beige adipocytes, and COMMD7 gene as important regulator for glucose uptake against IR. These findings not only provide a comprehensive and valuable resource for cataloguing candidate functional genes underlying lipid and glucose homeostasis in beige adipocytes, but also offer potential therapeutic targets against metabolic disorders.},
}

*Adipogenesis/genetics
*Glucose/metabolism
*Adipocytes, Beige/metabolism/cytology
Animals
Humans
Mice
*CRISPR-Cas Systems
Insulin Resistance
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics