@article {pmid40727907,
year = {2025},
author = {Arifah, AQ and Vento, JM and Kurrer, I and Achmedov, T and Beisel, CL},
title = {Cas9-independent tracrRNA cytotoxicity in Lacticaseibacillus paracasei.},
journal = {microLife},
volume = {6},
number = {},
pages = {uqaf013},
pmid = {40727907},
issn = {2633-6693},
abstract = {CRISPR-Cas9 systems are widely used for bacterial genome editing, yet their heterologous expression has been associated with cytotoxicity. The Cas9 nuclease from Streptococcus pyogenes (SpyCas9) has been one common source, with reports of cytotoxicity with the nuclease alone or in combination with a single-guide RNA observed in some bacteria. However, the potential cytotoxic effects of other components of the CRISPR-Cas9 system remain unknown. Here, we report that expression of the short isoform of the trans-activating CRISPR RNA (tracr-S) from the S. pyogenes CRISPR-Cas locus is cytotoxic in Lacticaseibacillus paracasei, even in the absence of SpyCas9. Deleting a putative transcription regulator in L. paracasei alleviates tracr-S cytotoxicity and leads to expression of the long isoform of the trans-activating CRISPR RNA (tracr-L). Furthermore, cytotoxicity was specific to the tracr-S sequence and was linked to direct interactions with host RNAs. This work thus reveals that additional CRISPR components beyond Cas9 can interfere with the use of heterologous CRISPR-Cas systems in bacteria, with potential implications for the evolution of CRISPR immunity.},
}

@article {pmid40726316,
year = {2025},
author = {Alary, B and Mortada, M and Mas, P},
title = {Gene editing of clock components in Solanum lycopersicum: Effects on gene expression, development, and productivity.},
journal = {The Plant journal : for cell and molecular biology},
volume = {123},
number = {2},
pages = {e70383},
doi = {10.1111/tpj.70383},
pmid = {40726316},
issn = {1365-313X},
support = {2021-SGR-01131//Agència de Gestió d'Ajuts Universitaris i de Recerca/ ; PID2022-137770NB-I00//Ministerio de Ciencia, Innovación y Universidades/ ; V6423//Fundación Ramón Areces/ ; CEX2019-000902-S//Ministerio de Economía y Competitividad/ ; },
mesh = {*Solanum lycopersicum/genetics/growth & development/physiology ; *Gene Editing ; *Circadian Clocks/genetics ; Gene Expression Regulation, Plant ; Plant Leaves/genetics/physiology/growth & development ; CRISPR-Cas Systems ; Circadian Rhythm/genetics ; Arabidopsis/genetics ; Plant Proteins/genetics/metabolism ; Fruit/genetics/growth & development/physiology ; },
abstract = {The circadian clock plays a crucial role in regulating key biological processes, including growth and development. While studies in the model plant Arabidopsis thaliana have significantly advanced our understanding of circadian function, recent research has also focused on crop species for improved yield and quality. In this study, we examined the rhythmic behavior and regulatory function of circadian clock components in tomato (Solanum lycopersicum). Time course analyses of gene expression over the circadian cycle revealed robust rhythmic oscillations in tomato leaves under free-running conditions. Comparative analyses showed similar peak phases for several clock genes in Arabidopsis and tomato, suggesting functional conservation. Rhythms in tomato fruits, however, showed reduced amplitude, slight phase changes, or arrhythmia, indicating organ-specific circadian variations. By using CRISPR-Cas9 gene editing strategies (clock[crispr]), we also showed that proper clock gene expression is essential for setting the phase in tomato plants. Leaf movement analyses also showed a phase change in the clock[crispr] lines, correlating with shorter or longer periods. The clock[crispr] lines also displayed distinct growth and developmental phenotypes that differ from those reported in the Arabidopsis clock mutant counterparts. Our transcriptomic analyses identified species-specific regulation of key target genes. The results offer mechanistic insights into the conserved and divergent molecular pathways governing circadian phenotypic variations between Arabidopsis and tomato plants.},
}

*Solanum lycopersicum/genetics/growth & development/physiology
*Gene Editing
*Circadian Clocks/genetics
Gene Expression Regulation, Plant
Plant Leaves/genetics/physiology/growth & development
CRISPR-Cas Systems
Circadian Rhythm/genetics
Arabidopsis/genetics
Plant Proteins/genetics/metabolism
Fruit/genetics/growth & development/physiology

@article {pmid40725481,
year = {2025},
author = {Cosiquien, RJS and Whalen, IJ and Wong, P and Sorensen, RJ and Shetty, AV and Liang, SQ and Steer, CJ},
title = {Detecting Methylation Changes Induced by Prime Editing.},
journal = {Genes},
volume = {16},
number = {7},
pages = {},
doi = {10.3390/genes16070825},
pmid = {40725481},
issn = {2073-4425},
mesh = {Humans ; *DNA Methylation/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; CpG Islands/genetics ; *Epigenesis, Genetic ; },
abstract = {While prime editing offers improved precision compared to traditional CRISPR-Cas9 systems, concerns remain regarding potential off-target effects, including epigenetic changes such as DNA methylation. In this study, we investigated whether prime editing induces aberrant CpG methylation patterns. Whole-genome bisulfite sequencing revealed overall methylation similarity between Cas9-edited, and PE2-edited cells. However, localized epigenetic changes were observed, particularly in CpG islands and exon regions. The PE2-edited group showed a higher proportion of differentially methylated regions (DMRs) in some coding sequences compared to controls and Cas9-edited samples. Notably, CpG island methylation reached 0.18% in the PE2 vs. Cas9 comparison, indicating a higher susceptibility of these regulatory elements to epigenetic alterations by prime editing. Molecular function analyses including Gene Ontology and KEGG pathway analyses further revealed enrichment in molecular functions related to transcriptional regulation and redox activity in PE2-edited cells. These findings suggest that prime editing, while precise, may introduce subtle but functionally relevant methylation changes that could influence gene expression and cellular pathways. In summary, prime editing can induce localized DNA methylation changes in human cells, particularly within regulatory and coding regions. Understanding these epigenetic consequences is critical for the development of safer and more effective therapeutic applications of genome editing technologies.},
}

Humans
*DNA Methylation/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
CpG Islands/genetics
*Epigenesis, Genetic

@article {pmid40725400,
year = {2025},
author = {Kim, JY and Jung, YJ and Kim, DH and Kang, KK},
title = {Transcriptomic Insights into GABA Accumulation in Tomato via CRISPR/Cas9-Based Editing of SlGAD2 and SlGAD3.},
journal = {Genes},
volume = {16},
number = {7},
pages = {},
doi = {10.3390/genes16070744},
pmid = {40725400},
issn = {2073-4425},
support = {2021R1I1A4A01057295//the National Research Foundation of Korea (NRF)/ ; },
mesh = {*Solanum lycopersicum/genetics/metabolism/growth & development ; *gamma-Aminobutyric Acid/metabolism/genetics ; *CRISPR-Cas Systems ; *Glutamate Decarboxylase/genetics/metabolism ; Gene Editing/methods ; *Transcriptome/genetics ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Fruit/genetics/metabolism ; Plant Leaves/genetics/metabolism ; Plants, Genetically Modified/genetics ; },
abstract = {BACKGROUND: γ-Aminobutyric acid (GABA) is a non-proteinogenic amino acid with key roles in plant metabolism, stress responses, and fruit nutritional quality. In tomato (Solanum lycopersicum), GABA levels are dynamically regulated during fruit development but decline in the late ripening stages.

METHODS: To enhance GABA accumulation, we used CRISPR/Cas9 to edit the calmodulin-binding domain (CaMBD) of SlGAD2 and SlGAD3, which encode glutamate decarboxylases (GADs). The resulting truncated enzymes were expected to be constitutively active. We quantified GABA content in leaves and fruits and performed transcriptomic analysis on edited lines at the BR+7 fruit stage.

RESULTS: CaMBD truncation significantly increased GABA levels in both leaves and fruits. In gad2 sg1 lines, GABA levels increased by 3.5-fold in leaves and 3.2-fold in BR+10 fruits; in gad3 sg3 lines, increases of 2.8- and 2.5-fold were observed, respectively. RNA-seq analysis identified 1383 DEGs in gad2 #1-5 and 808 DEGs in gad3 #3-8, with 434 DEGs shared across both lines. These shared DEGs showed upregulation of GAD, GABA-T, and SSADH, and downregulation of stress-responsive transcription factors including WRKY46, ERF, and NAC. Notably, total free amino acid content and fruit morphology remained unchanged despite elevated GABA.

CONCLUSIONS: CRISPR/Cas9-mediated editing of the CaMBD in SlGAD genes selectively enhances GABA biosynthesis in tomato without adverse effects on development or fruit quality. These lines offer a useful platform for GABA-centered metabolic engineering and provide insights into GABA's role in transcriptional regulation during ripening.},
}

*Solanum lycopersicum/genetics/metabolism/growth & development
*gamma-Aminobutyric Acid/metabolism/genetics
*CRISPR-Cas Systems
*Glutamate Decarboxylase/genetics/metabolism
Gene Editing/methods
*Transcriptome/genetics
*Plant Proteins/genetics/metabolism
Gene Expression Regulation, Plant
Fruit/genetics/metabolism
Plant Leaves/genetics/metabolism
Plants, Genetically Modified/genetics

@article {pmid40725376,
year = {2025},
author = {Kumar, H and Gal'chinsky, N and Sweta, V and Negi, N and Filatov, R and Chandel, A and Ali, J and Oberemok, V and Laikova, K},
title = {Perspectives of RNAi, CUADb and CRISPR/Cas as Innovative Antisense Technologies for Insect Pest Control: From Discovery to Practice.},
journal = {Insects},
volume = {16},
number = {7},
pages = {},
doi = {10.3390/insects16070746},
pmid = {40725376},
issn = {2075-4450},
support = {FZEG-2024-0001//This research results obtained within the framework of a state assignment V.I. Vernadsky Cri-mean Federal University for 2024 and the planning period of 2024-2026/ ; },
abstract = {Pest management is undergoing a transformative shift with the development of the cutting-edge antisense technologies: RNA interference (RNAi), contact unmodified antisense DNA biotechnology (CUADb), and the CRISPR-associated proteins (CRISPR/Cas). These approaches function by facilitating sequence-specific pairing of nucleic acids followed by nuclease-mediated cleavage, offering exceptional precision for targeted pest control. While RNA-guided mechanisms such as RNAi and CRISPR/Cas were initially characterized in non-insect systems, primarily as innate defenses against viral infections, the DNA-guided CUADb pathway was first identified in insect pests as a functional pest control strategy. Its broader role in ribosomal RNA (rRNA) biogenesis was recognized later. Together, these discoveries have revealed an entirely new dimension of gene regulation, with profound implications for sustainable pest management. Despite sharing a common principle of sequence-specific targeting RNAi, CUADb, and CRISPR/Cas differ in several key aspects, including their mechanisms of action, target specificity, and applicability. Rather than serving as universal solutions, each technology is likely to be optimally effective against specific pest groups. Moreover, these technologies allow for rapid adaptation of control strategies to overcome target-site resistance, ensuring long-term efficacy. This review summarizes the core functional characteristics, potential applications, and current limitations of each antisense technology, emphasizing their complementary roles in advancing environmentally sustainable pest control. By integrating foundational biological discoveries with applied innovations, this work provides a new perspectives on incorporating antisense-based strategies into next-generation integrated pest management systems.},
}

@article {pmid40725259,
year = {2025},
author = {Wolny, E and Mur, LAJ and Ohmido, N and Yin, Z and Wang, K and Hasterok, R},
title = {Thriving or Withering? Plant Molecular Cytogenetics in the First Quarter of the 21st Century.},
journal = {International journal of molecular sciences},
volume = {26},
number = {14},
pages = {},
doi = {10.3390/ijms26147013},
pmid = {40725259},
issn = {1422-0067},
mesh = {*Cytogenetic Analysis/methods ; Genome, Plant ; Chromosomes, Plant/genetics ; *Plants/genetics ; In Situ Hybridization, Fluorescence/methods ; *Cytogenetics/methods/trends ; Polyploidy ; Genomics/methods ; },
abstract = {Nearly four decades have passed since fluorescence in situ hybridisation was first applied in plants to support molecular cytogenetic analyses across a wide range of species. Subsequent advances in DNA sequencing, bioinformatic analysis, and microscopy, together with the immunolocalisation of various nuclear components, have provided unprecedented insights into the cytomolecular organisation of the nuclear genome in both model and non-model plants, with crop species being perhaps the most significant. The ready availability of sequenced genomes is now facilitating the application of state-of-the-art cytomolecular techniques across diverse plant species. However, these same advances in genomics also pose a challenge to the future of plant molecular cytogenetics, as DNA sequence analysis is increasingly perceived as offering comparable insights into genome organisation. This perception persists despite the continued relevance of FISH-based approaches for the physical anchoring of genome assemblies to chromosomes. Furthermore, cytogenetic approaches cannot currently rival purely genomic methods in terms of throughput, standardisation, and automation. This review highlights the latest key topics in plant cytomolecular research, with particular emphasis on chromosome identification and karyotype evolution, chromatin and interphase nuclear organisation, chromosome structure, hybridisation and polyploidy, and cytogenetics-assisted crop improvement. In doing so, it underscores the distinctive contributions that cytogenetic techniques continue to offer in genomic research. Additionally, we critically assess future directions and emerging opportunities in the field, including those related to CRISPR/Cas-based live-cell imaging and chromosome engineering, as well as AI-assisted image analysis and karyotyping.},
}

*Cytogenetic Analysis/methods
Genome, Plant
Chromosomes, Plant/genetics
*Plants/genetics
In Situ Hybridization, Fluorescence/methods
*Cytogenetics/methods/trends
Polyploidy
Genomics/methods

@article {pmid40725213,
year = {2025},
author = {Covache-Busuioc, RA and Toader, C and Rădoi, MP and Șerban, M},
title = {Precision Recovery After Spinal Cord Injury: Integrating CRISPR Technologies, AI-Driven Therapeutics, Single-Cell Omics, and System Neuroregeneration.},
journal = {International journal of molecular sciences},
volume = {26},
number = {14},
pages = {},
doi = {10.3390/ijms26146966},
pmid = {40725213},
issn = {1422-0067},
mesh = {*Spinal Cord Injuries/therapy/genetics/physiopathology ; Humans ; *Artificial Intelligence ; Animals ; Gene Editing/methods ; *Nerve Regeneration ; Single-Cell Analysis/methods ; *CRISPR-Cas Systems ; Recovery of Function ; Regenerative Medicine/methods ; Precision Medicine/methods ; },
abstract = {Spinal cord injury (SCI) remains one of the toughest obstacles in neuroscience and regenerative medicine due to both severe functional loss and limited healing ability. This article aims to provide a key integrative, mechanism-focused review of the molecular landscape of SCI and the new disruptive therapy technologies that are now evolving in the SCI arena. Our goal is to unify a fundamental pathophysiology of neuroinflammation, ferroptosis, glial scarring, and oxidative stress with the translation of precision treatment approaches driven by artificial intelligence (AI), CRISPR-mediated gene editing, and regenerative bioengineering. Drawing upon advances in single-cell omics, systems biology, and smart biomaterials, we will discuss the potential for reprogramming the spinal cord at multiple levels, from transcriptional programming to biomechanical scaffolds, to change the course from an irreversible degeneration toward a directed regenerative pathway. We will place special emphasis on using AI to improve diagnostic/prognostic and inferred responses, gene and cell therapies enabled by genomic editing, and bioelectronics capable of rehabilitating functional connectivity. Although many of the technologies described below are still in development, they are becoming increasingly disruptive capabilities of what it may mean to recover from an SCI. Instead of prescribing a particular therapeutic fix, we provide a future-looking synthesis of interrelated biological, computational, and bioengineering approaches that conjointly chart a course toward adaptive, personalized neuroregeneration. Our intent is to inspire a paradigm shift to resolve paralysis through precision recovery and to be grounded in a spirit of humility, rigor, and an interdisciplinary approach.},
}

*Spinal Cord Injuries/therapy/genetics/physiopathology
Humans
*Artificial Intelligence
Animals
Gene Editing/methods
*Nerve Regeneration
Single-Cell Analysis/methods
*CRISPR-Cas Systems
Recovery of Function
Regenerative Medicine/methods
Precision Medicine/methods

@article {pmid40725041,
year = {2025},
author = {Fizikova, A and Prokhorova, A and Churikova, D and Konstantinov, Z and Ivanov, R and Karabelsky, A and Rybtsov, S},
title = {Hepatocytes as Model for Investigating Natural Senotherapeutic Compounds and Their Effects on Cell Cycle Dynamics and Genome Stability.},
journal = {International journal of molecular sciences},
volume = {26},
number = {14},
pages = {},
doi = {10.3390/ijms26146794},
pmid = {40725041},
issn = {1422-0067},
support = {Agreement No. 18-03 on 10 September 2024//the state program of the "Sirius" Federal Territory "Scientific and technological development of the "Sirius" Federal Territory"/ ; },
mesh = {Humans ; *Genomic Instability/drug effects ; Animals ; *Senotherapeutics/pharmacology ; *Cell Cycle/drug effects ; *Hepatocytes/drug effects/metabolism/cytology ; Cellular Senescence/drug effects ; DNA Damage ; },
abstract = {DNA is inherently unstable and is susceptible to damage from both endogenous sources (such as reactive oxygen species) and exogenous factors (including UV, ionizing radiation, and chemicals). The accumulation of DNA damage manifests as genetic mutations, chromosomal instability, and the stalling of DNA replication and transcription processes. Accumulated DNA damage influences apoptosis and cell cycle checkpoints, serving as one of the key triggers for the manifestation of the senescent phenotype. Both aging and cancer are associated with the accumulation of mutations in somatic cells. Disruption of cell cycle control and uncontrolled proliferation are fundamental characteristics of any cancer cell, with the majority of anticancer drugs acting as inhibitors of cyclin-dependent kinases, thereby inducing a transition of cells into a senescent state. Consequently, disturbances in the dynamics and regulation of inflammatory responses, oxidative stress, cell proliferation, DNA damage repair, and epigenetic anomalies, along with the influence of retroviruses and transposons, lead to the accumulation of senescent cells within the human body, characterized by blocked replication and cell cycle, as well as a distinct secretory phenotype. The age-related or disease-associated accumulation of these senescent cells significantly alters the physiology of tissues and the organism as a whole. Many secondary metabolites of higher plants exhibit senolytic and senomorphic activities, although most of them are not fully characterized. In this review, we will explore the principal signaling pathways in mammalian cells that govern the cell cycle and cellular senescence, with a particular emphasis on how their dynamics, expression, and regulation have been modified through the application of senotherapeutic compounds. The second section of the review will identify key target genes for the metabolic engineering, primarily aimed at enhancing the accumulation of plant secondary metabolites with potential therapeutic benefits. Lastly, we will discuss the rationale for utilizing liver cells as a model system to investigate the effects of senolytic compounds on human physiology and health, as well as how senotherapeutic substances can be leveraged to improve gene therapy approaches based on CRISPR/Cas9 and prime-editing technologies.},
}

Humans
*Genomic Instability/drug effects
Animals
*Senotherapeutics/pharmacology
*Cell Cycle/drug effects
*Hepatocytes/drug effects/metabolism/cytology
Cellular Senescence/drug effects
DNA Damage

@article {pmid40724990,
year = {2025},
author = {Montagna, C and Maiani, E and Pieroni, L and Consalvi, S},
title = {Duchenne Muscular Dystrophy: Integrating Current Clinical Practice with Future Therapeutic and Diagnostic Horizons.},
journal = {International journal of molecular sciences},
volume = {26},
number = {14},
pages = {},
doi = {10.3390/ijms26146742},
pmid = {40724990},
issn = {1422-0067},
abstract = {Duchenne muscular dystrophy (DMD) is a severe X-linked disorder characterized by progressive muscle degeneration due to mutations in the dystrophin gene. Despite major advancements in understanding its pathophysiology, there is still no curative treatment. This review provides an up-to-date overview of current and emerging therapeutic approaches-including antisense oligonucleotides, gene therapy, gene editing, corticosteroids, and histone deacetylases(HDAC) inhibitors-aimed at restoring dystrophin expression or mitigating disease progression. Special emphasis is placed on the importance of early diagnosis, the utility of genetic screening, and the innovations in pre-and post-natal testing. As the field advances toward personalized medicine, the integration of precision therapies with cutting-edge diagnostic technologies promises to improve both prognosis and quality of life for individuals with DMD.},
}

@article {pmid40724964,
year = {2025},
author = {Ruiz-Moreno, HA and Valderrama-Rincon, JD and Cala, MP and Fernández-Niño, M and Valderruten Cajiao, M and Villegas-Torres, MF and González Barrios, AF},
title = {Enhanced Outer Membrane Vesicle Production in Escherichia coli: From Metabolic Network Model to Designed Strain Lipidomic Profile.},
journal = {International journal of molecular sciences},
volume = {26},
number = {14},
pages = {},
doi = {10.3390/ijms26146714},
pmid = {40724964},
issn = {1422-0067},
support = {822-2017//Colciencias/ ; },
mesh = {*Escherichia coli/metabolism/genetics ; *Lipidomics/methods ; *Metabolic Networks and Pathways ; *Bacterial Outer Membrane/metabolism ; Mutation ; Escherichia coli Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Knockout Techniques ; *Extracellular Vesicles/metabolism ; Bacterial Outer Membrane Proteins/metabolism/genetics ; Gene Editing ; },
abstract = {Bacterial structures formed from the outer membrane and the periplasm components carry biomolecules to expel cellular material and interact with other cells. These outer membrane vesicles (OMVs) can encapsulate bioactive content, which confers OMVs with high potential as alternative drug delivery vehicles or as a platform for novel vaccine development. Single-gene mutants derived from Escherichia coli JC8031 were engineered to further enhance OMV production based on metabolic network modelling and in silico gene knockout design (ΔpoxB, ΔsgbE, ΔgmhA, and ΔallD). Mutants were experimentally obtained by genome editing using CRISPR-Cas9 and tested for OMVs recovery observing an enhanced OMV production in all of them. Lipidomic analysis through LC-ESI-QTOF-MS was performed for OMVs obtained from each engineered strain and compared to the wild-type E. coli JC8031 strain. The lipid profile of OMVs from the wild-type E. coli JC8031 did not change significantly confirmed by multivariate statistical analysis when compared to the mutant strains. The obtained results suggest that the vesicle production can be further improved while the obtained vesicles are not altered in their composition, allowing further study for stability and integrity for use in therapeutic settings.},
}

*Escherichia coli/metabolism/genetics
*Lipidomics/methods
*Metabolic Networks and Pathways
*Bacterial Outer Membrane/metabolism
Mutation
Escherichia coli Proteins/genetics/metabolism
CRISPR-Cas Systems
Gene Knockout Techniques
*Extracellular Vesicles/metabolism
Bacterial Outer Membrane Proteins/metabolism/genetics
Gene Editing

@article {pmid40724866,
year = {2025},
author = {Nidhi, F and Tomatsu, S},
title = {Integrase-Deficient Lentiviral Vector as a Platform for Efficient CRISPR/Cas9-Mediated Gene Editing for Mucopolysaccharidosis IVA.},
journal = {International journal of molecular sciences},
volume = {26},
number = {14},
pages = {},
doi = {10.3390/ijms26146616},
pmid = {40724866},
issn = {1422-0067},
support = {1R01HD102545-01A1/GF/NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Mice ; *Lentivirus/genetics ; *Mucopolysaccharidosis IV/therapy/genetics ; *Genetic Vectors/genetics ; Humans ; NIH 3T3 Cells ; Genetic Therapy/methods ; *Chondroitinsulfatases/genetics/metabolism ; *Integrases/genetics/deficiency ; Fibroblasts/metabolism ; Disease Models, Animal ; },
abstract = {Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disorder causing systemic skeletal dysplasia due to a deficiency of N-acetyl-galactosamine-6-sulfate sulfatase (GALNS) enzyme activity, leading to the impaired degradation and accumulation of glycosaminoglycans (GAGs), keratan sulfate (KS) and chondroitin-6-sulfate. While treatments such as enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT) are available, they have significant limitations regarding efficacy in skeletal tissues and long-term safety, highlighting the need for more effective therapies. We evaluated a novel gene therapy approach using a dual Integrase-deficient lentiviral vector (IDLV) to deliver an expression cassette that includes human GALNS cDNA and Cas9 sgRNA, targeting the upstream region of the mouse Galns initial codon. This approach leverages the endogenous promoter to drive transgene expression. We assessed in vitro transduction, editing, and functional correction in NIH3T3 and MPS IVA mouse fibroblasts. In vivo efficacy was successfully evaluated via the facial vein injection in MPS IVA newborn mice. In vitro, this IDLV platform demonstrated supraphysiological GALNS activity in cell lysate, resulting in the normalization of KS levels. In vivo direct IDLV platform in newborn MPS IVA mice led to sustained plasma GALNS activity, reduced plasma KS, and favorable biodistribution. Partial correction of heart and bone pathology was observed, with no vector toxicity and minimal antibody responses. This dual IDLV-CRISPR/Cas9 approach effectively mediated targeted GALNS knock-in, yielding sustained enzyme activity, reduced KS storage, and partial pathological amelioration in MPS IVA mice. In conclusion, IDLVs represent an efficient, safe platform for delivering the CRISPR/Cas9 gene editing system for MPS IVA.},
}

Animals
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Mice
*Lentivirus/genetics
*Mucopolysaccharidosis IV/therapy/genetics
*Genetic Vectors/genetics
Humans
NIH 3T3 Cells
Genetic Therapy/methods
*Chondroitinsulfatases/genetics/metabolism
*Integrases/genetics/deficiency
Fibroblasts/metabolism
Disease Models, Animal

@article {pmid40722665,
year = {2025},
author = {Zhang, A and Zhang, I and Liu, F},
title = {Applications of CRISPR-Cas-Based Genome Editing Approaches Against Human Cytomegalovirus Infection.},
journal = {Biomedicines},
volume = {13},
number = {7},
pages = {},
doi = {10.3390/biomedicines13071590},
pmid = {40722665},
issn = {2227-9059},
support = {Start-Up Fund 101//University of California, Berkeley/ ; },
abstract = {Human cytomegalovirus (HCMV), a globally ubiquitous herpesvirus with the ability to carry out both lytic productive and lifelong latent infections, is a major cause of congenital infections, often leading to intellectual disabilities and neurological disorders. Moreover, HCMV is an opportunistic pathogen commonly found in immunocompromised individuals such as organ transplant recipients, HIV-positive individuals, and cancer patients, causing severe and life-threatening complications. While effective in inhibiting viral lytic infection, current FDA-approved compounds cannot eliminate the latent viral genome and have little effect on viral latent infection. Developing novel antiviral therapeutic approaches to eliminate HCMV lytic and latent infections is a major public health priority for controlling HCMV infection and preventing viral-associated diseases. The genome-editing technology based on the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) RNA-guided nuclease system represents a novel and promising antiviral approach through modifying or destroying the genetic material of human viruses. This review summarizes the recently published progress in using the CRISPR-Cas approach to study and inhibit HCMV infections and discusses prospects for developing the CRISPR-based genome-editing technology for therapeutic applications against HCMV infection and associated diseases.},
}

@article {pmid40721002,
year = {2025},
author = {Ahi, EP and Khorshid, M},
title = {Potentials of RNA biosensors in developmental biology.},
journal = {Developmental biology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ydbio.2025.07.011},
pmid = {40721002},
issn = {1095-564X},
abstract = {RNA-based/associated biosensors represent a rapidly expanding area of research, providing highly sensitive tools for detecting and monitoring RNA in diverse biological contexts. These sensors offer the ability to track RNA localization, modifications, and interactions in real-time, making them particularly well-suited for developmental biology research. Despite their demonstrated utility in fields such as diagnostics, synthetic biology and environmental science, the application of RNA biosensors in developmental biology has only begun to emerge within the past decade. This gap is notable given the potential of these tools to address key questions about spatiotemporal RNA regulation and cellular signaling during development. This perspective review presents a selection of RNA biosensors, including fluorescent RNA aptamers, CRISPR-Cas-based systems, riboswitches, and catalytic RNA sensors, which have gained attraction in other scientific disciplines. These tools can be used not only to study intrinsic RNA biology, such as RNA expression, splicing, and localization, but also to detect the effects of extrinsic physical and chemical factors, including pH, temperature, redox state, and mechanical stress, on RNA behavior during developmental processes. These examples illustrate how RNA biosensors could be adapted to study developmental mechanisms in model organisms, enabling investigations into RNA dynamics and their role in shaping developmental processes. By revisiting these underutilized tools, this review highlights their relevance for advancing the understanding of molecular mechanisms in developmental biology studies.},
}

@article {pmid40716775,
year = {2025},
author = {Nalefski, EA and Hedley, S and Rajaraman, K and Kooistra, RM and Parikh, I and Sinan, S and Finklestein, IJ and Madan, D},
title = {Unleashing high trans-substrate cleavage kinetics of Cas12a for nucleic acid diagnostics.},
journal = {Nucleic acids research},
volume = {53},
number = {14},
pages = {},
doi = {10.1093/nar/gkaf712},
pmid = {40716775},
issn = {1362-4962},
support = {//College of Natural Sciences Catalyst/ ; //Global Health Labs/ ; F-1808//Welch Foundation/ ; },
mesh = {*CRISPR-Associated Proteins/metabolism/genetics ; *CRISPR-Cas Systems ; Kinetics ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; DNA/metabolism/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Guide, CRISPR-Cas Systems/genetics ; Substrate Specificity ; *Nucleic Acids/genetics/analysis ; Ribonucleoproteins/metabolism ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats)-based nucleic acid diagnostics enable rapid, sensitive pathogen detection. Cas12a is frequently used in these assays because target-activated trans cleavage of a reporter molecule generates an easily detectable signal. However, variable activity across assays suggests that the catalytic potential of Cas12a has been limited via unknown mechanisms. Here, we show that Cas12a trans-nuclease activity is auto-inhibited by long PAM-proximal DNA (>120 bp) following cis-cleavage of targets. Short targets (<100 bp), optimized trans cleavage substrates, and low salt buffers unleash high catalytic efficiency (≈108 M-1 s-1) and turnover (≈1 s-1) across Cas12a orthologs. Pooling multiple Cas12a ribonucleoproteins (RNPs) targeting clustered protospacers overcomes cis-cleavage auto-inhibition, further boosting sensitivity. Optimized CRISPR RNA pools enable sub-femtomolar sensitivity for target detection without any pre-amplification. This mechanistic insight and mitigation strategy broaden the application of CRISPR-Cas enzymes for nucleic acid diagnostics.},
}

*CRISPR-Associated Proteins/metabolism/genetics
*CRISPR-Cas Systems
Kinetics
*Endodeoxyribonucleases/metabolism/genetics
*Bacterial Proteins/metabolism/genetics
DNA/metabolism/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
RNA, Guide, CRISPR-Cas Systems/genetics
Substrate Specificity
*Nucleic Acids/genetics/analysis
Ribonucleoproteins/metabolism

@article {pmid40716264,
year = {2025},
author = {Souza-Neves, M and Pórfido, JL and Crispo, M and Menchaca, A},
title = {Electroporation of sheep zygotes as an alternative to microinjection for the generation of CRISPR/Cas genome edited models.},
journal = {Theriogenology},
volume = {248},
number = {},
pages = {117603},
doi = {10.1016/j.theriogenology.2025.117603},
pmid = {40716264},
issn = {1879-3231},
abstract = {Zygote microinjection is considered the most suitable technique to introduce CRISPR/Cas9 reagents for efficient genome editing in livestock. In this study, zygote electroporation was evaluated as an alternative to microinjection for CRISPR/Cas9-mediated genome editing in sheep. Four experiments were conducted on 3548 cumulus-oocyte complexes. Acid Tyrode's solution (AT) was used to partially degrade the zona pellucida (ZP) to improve reagent entry, resulting in ZP thinning with longer AT exposure (P < 0.05). Although early embryo development was impaired by AT exposure (P < 0.05), blastocyst rates were similar across all groups by day 8. Electroporation conditions were optimized by testing pulse length (1 or 3 ms), with the best results from 6 pulses of 20 V for 3 ms with AT during 60 s. Electroporation with 500 ng/μL Cas9 and 300 ng/μL sgRNA with AT during 60 s achieved a 38.5 % mutation rate. When compared with conventional microinjection, electroporation had higher developmental rates but a lower mutation rate (21.4 % vs. 60.0 %; P < 0.05). These findings suggest that electroporation is a viable, cost-effective technique for genome editing in sheep. Nevertheless, further research will be required to fine-tune electroporation conditions and enhance efficiency in terms of mutation rate.},
}

@article {pmid40715925,
year = {2025},
author = {Westarp, P and Keller, T and Brand, J and Horvat, S and Albrecht, K and Beisel, C and Groll, J},
title = {Efficient encapsulation of CRISPR-Cas9 RNP in bioreducible nanogels and release in a cytosol-mimicking environment.},
journal = {Discover nano},
volume = {20},
number = {1},
pages = {119},
pmid = {40715925},
issn = {2731-9229},
abstract = {CRISPR/Cas9-mediated programmable gene editing has disrupted the biotechnology industry since it was first described in 2012. Safe in vivo delivery is a key bottleneck for its therapeutic use. Viral vector-mediated delivery raises concerns due to immunogenicity, long-term expression, and genomic disruption. Delivery of pre-complexed ribonucleoprotein (RNP) reduces off-target effects, and recombinant Cas9 production is more cost-effective than viral vector synthesis. CRISPR-Cas RNPs do not possess intrinsic cell entry mechanisms, and physical delivery methods are confined to ex vivo editing, necessitating non-viral delivery approaches. Nanogels (NG) are biocompatible polymeric nanoparticles capable of entrapping proteins. Here, we report the first proof of principle that NGs from thiol-functionalized polyglycidol can entrap active RNPs with high efficiency (60 ± 2%). We call these particles CRISPR-Gels. A commercially available E. coli lysate for cell-free transcription and translation (TXTL) was used to mimic the intracellular reductive degradation of NGs while providing a real-time fluorescence readout of RNP activity. Degradation and RNP activity were observed within 30-90 min. The described TXTL assay can be utilized to evaluate the release of RNP in a cytosol-mimicking environment from redox-sensitive nanoparticles in a high-throughput and cost-effective way. Further studies are needed to assess the in vitro and in vivo performance of CRISPR-Gels.},
}

@article {pmid40715906,
year = {2025},
author = {Garg, A and Chauhan, P and Kaur, C and Arora, PK and Garg, SK and Singh, VP and Singh, KP and Srivastava, A},
title = {Comprehensive heavy metal remediation mechanisms with insights into CRISPR-Cas9 and biochar innovations.},
journal = {Biodegradation},
volume = {36},
number = {4},
pages = {69},
pmid = {40715906},
issn = {1572-9729},
support = {DST/INSPIRE/03/2021/002510//DST-INSPIRE INDIA/ ; },
mesh = {*Metals, Heavy/metabolism ; *Charcoal/chemistry ; *CRISPR-Cas Systems ; Biodegradation, Environmental ; *Soil Pollutants/metabolism ; Plants/metabolism/genetics ; Gene Editing ; Bacteria/metabolism/genetics ; },
abstract = {Heavy metal contamination of the environment is a serious issue, and more efficient and effective bioremediation techniques are needed. This review introduces current heavy metal bioremediation techniques, with focus on phytoremediation and microbial remediation, and recent developments in biochar and CRISPR-Cas9 technology. Phytoremediation employs the natural process of plants to accumulate and detoxify metals as an eco-friendly and sustainable technique. Microbial remediation by fungi and bacteria provides an additional approach through reduction, sequestration, and transformation of metals. Biochar as a high-carbon value-added pyrolytic biomass product improves soil quality, increases microbial activity, and adsorbs heavy metals, making bioremediation more effective. The discovery of CRISPR-Cas9 revolutionized gene engineering by allowing gene editing of plants and microbes to improve their metal tolerance and degradation. This review outlines recent developments, synergistic uses of biochar and CRISPR-Cas9, and how they might enhance phytoremediation and microbial remediation. By combining such novel technologies, strong, sustainable, and scalable solutions could be built for curbing heavy metal pollution and safeguarding environmental health.},
}

*Metals, Heavy/metabolism
*Charcoal/chemistry
*CRISPR-Cas Systems
Biodegradation, Environmental
*Soil Pollutants/metabolism
Plants/metabolism/genetics
Gene Editing
Bacteria/metabolism/genetics

@article {pmid40720830,
year = {2025},
author = {Šimečková, H and Bárdy, P and Kuntová, L and Macháčová, E and Botka, T and Bíňovský, J and Houser, J and Farka, Z and Plevka, P and Pantůček, R and Mašlaňová, I},
title = {CRISPR-Cas10-Assisted Structural Modification of Staphylococcal Kayvirus for Imaging and Biosensing Applications.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00387},
pmid = {40720830},
issn = {2161-5063},
abstract = {Recent advances in genome editing techniques based on CRISPR-Cas have opened up new possibilities in bacteriophage engineering and, thus, enabled key developments in medicine, nanotechnology, and synthetic biology. Although staphylococcal phage genomes have already been edited, the modification of their structural proteins has not yet been reported. Here, the structure of Staphylococcus phage 812h1 of the Kayvirus genus was modified by inserting a poly histidine tag into an exposed loop of the tail sheath protein. A two-strain editing strategy was applied, utilizing homologous recombination followed by CRISPR-Cas10-assisted counter-selection of the recombinant phages. The His-tagged phage particles can be recognized by specific antibodies, enabling the modified bacteriophages to be employed in numerous techniques. The attachment of the engineered phage to bacteria was visualized by fluorescence microscopy, and its functionality was confirmed using biolayer interferometry biosensing, enzyme-linked immunosorbent assay, and flow cytometry, demonstrating that the genetic modification did not impair its biological activity.},
}

@article {pmid40720017,
year = {2025},
author = {Maruyama, R and Yokota, T},
title = {Creation of DMD Muscle Cell Model Using CRISPR-Cas9 Genome Editing to Test the Efficacy of Antisense-Mediated Exon Skipping.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2964},
number = {},
pages = {157-162},
pmid = {40720017},
issn = {1940-6029},
mesh = {Humans ; *CRISPR-Cas Systems ; *Muscular Dystrophy, Duchenne/genetics/therapy/pathology ; *Gene Editing/methods ; *Exons/genetics ; *Dystrophin/genetics ; *Oligonucleotides, Antisense/genetics ; Cell Line ; Mutation ; },
abstract = {Duchenne muscular dystrophy (DMD) is a devastating muscle disorder caused by mutations in the DMD gene. Antisense-mediated exon skipping is a promising strategy to treat DMD. The approval of Exondys 51 (eteplirsen) targeting exon 51 was the most noteworthy accomplishment in 2016. To evaluate and optimize the sequence of antisense oligonucleotides (AOs), muscle cell lines with DMD mutations are useful tools. However, there are several immortalized muscle cell lines with DMD mutations available that can be used to test the efficacy of exon skipping in vitro. In addition, an invasive muscle biopsy is required to obtain muscle cells from patients. Furthermore, many DMD mutations are very rare and it is hard to find a patient with a specific mutation for muscle biopsy in many cases. Here, we describe a novel approach to create an immortalized muscle cell line with a DMD deletion mutation using the human rhabdomyosarcoma (RD) cell line and the CRISPR/Cas9 system that can be used to test the efficacy of exon skipping.},
}

Humans
*CRISPR-Cas Systems
*Muscular Dystrophy, Duchenne/genetics/therapy/pathology
*Gene Editing/methods
*Exons/genetics
*Dystrophin/genetics
*Oligonucleotides, Antisense/genetics
Cell Line
Mutation

@article {pmid40719726,
year = {2025},
author = {Deivarajan, HR and Senthilkumar, K and Sekar, HV and Elamurugan, V and Pandian, J and Venugopal, A and Kuppamuthu, D and Prajna, L and Prajna, VN and Narendran, S},
title = {Rapid one-tube RPA-coupled CRISPR/Cas12a-based RID-MyC assay for the diagnosis of fungal keratitis.},
journal = {Indian journal of ophthalmology},
volume = {73},
number = {8},
pages = {1208-1212},
doi = {10.4103/IJO.IJO_1613_24},
pmid = {40719726},
issn = {1998-3689},
mesh = {Humans ; *Eye Infections, Fungal/diagnosis/microbiology ; Prospective Studies ; Cross-Sectional Studies ; Female ; Male ; *CRISPR-Cas Systems ; Middle Aged ; *Nucleic Acid Amplification Techniques/methods ; *Keratitis/diagnosis/microbiology ; Adult ; *Cornea/microbiology/pathology ; *DNA, Fungal/analysis/genetics ; *Fungi/genetics/isolation & purification ; Aged ; *Recombinases/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {PURPOSE: This study introduces and evaluates the single-tube rapid identification of mycoses using CRISPR (ST-RID-MyC) assay. This novel diagnostic tool combines recombinase polymerase amplification (RPA) with CRISPR/Cas12a for the rapid and precise diagnosis of fungal keratitis (FK).

DESIGN: Prospective cross-sectional study.

METHODS: Corneal scrapings from 61 patients with suspected microbial keratitis were collected at the Cornea Department of a Tertiary Eye Care Center. The study assessed the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the ST-RID-MyC assay. Additional measures included concordance rates with traditional diagnostic methods and the time to diagnosis.

RESULTS: The ST-RID-MyC assay exhibited a sensitivity of 90% and a specificity of 90.48%, with a PPV of 94.74% and an NPV of 82.61%. The ST-RID-MyC showed substantial agreement with culture and microscopy and perfect concordance with conventional RID-MyC. The mean time to diagnosis was significantly reduced (P < 0.001) using the ST-RID-MyC assay, compared to the traditional RID-MyC assay (6 vs. 32 minutes). Visual assessments demonstrated a high level of inter-observer agreement (kappa = 0.832).

CONCLUSIONS: The ST-RID-MyC assay, combining RPA and CRISPR/Cas12a in a single-tube system, offers a rapid, accurate, and resource-efficient diagnostic method for FK, potentially transforming clinical management of this condition by enabling faster therapeutic decisions.},
}

Humans
*Eye Infections, Fungal/diagnosis/microbiology
Prospective Studies
Cross-Sectional Studies
Female
Male
*CRISPR-Cas Systems
Middle Aged
*Nucleic Acid Amplification Techniques/methods
*Keratitis/diagnosis/microbiology
Adult
*Cornea/microbiology/pathology
*DNA, Fungal/analysis/genetics
*Fungi/genetics/isolation & purification
Aged
*Recombinases/genetics
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40715281,
year = {2025},
author = {Lee, S and Kyung, M and Park, M and Park, S and Lee, J and Kim, S and Lee, S and Jo, M and Jung, ST and Lee, HW},
title = {Advanced human FcRn knock-in mice for pharmacokinetic profiling of therapeutic antibodies.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {27186},
pmid = {40715281},
issn = {2045-2322},
support = {2020M3F7A1094089//National Research Foundation of Republic of Korea/ ; 2020M3F7A1094089//National Research Foundation of Republic of Korea/ ; 2020M3F7A1094089//National Research Foundation of Republic of Korea/ ; 2020M3F7A1094089//National Research Foundation of Republic of Korea/ ; 2020M3F7A1094089//National Research Foundation of Republic of Korea/ ; 2023-00224201//Ministry of Science and ICT, South Korea/ ; 2023-00224201//Ministry of Science and ICT, South Korea/ ; 2023-00224201//Ministry of Science and ICT, South Korea/ ; 2023-00224201//Ministry of Science and ICT, South Korea/ ; 2023-00224201//Ministry of Science and ICT, South Korea/ ; },
mesh = {Animals ; *Receptors, Fc/genetics/metabolism ; *Histocompatibility Antigens Class I/genetics/metabolism ; Humans ; Mice ; *Gene Knock-In Techniques ; *Immunoglobulin G ; Mice, Transgenic ; CRISPR-Cas Systems ; },
abstract = {IgG-based therapeutic antibodies are increasingly adopted for diverse human diseases, such as cancer and autoimmune disorders displaying remarkable therapeutic performance. A key factor in their success lies in the extended half-life of IgG molecules, which is regulated by the pH-dependent interaction between IgG and neonatal Fc receptor (FcRn). This interaction prevents lysosomal degradation of IgG. Despite the frequent use of humanized rodent models expressing human FcRn (hFcRn) in preclinical studies, these models often fail to accurately replicate human antibody pharmacokinetics (PK) due to the use of non-native promoters that influence FcRn expression. To overcome this limitation, we developed an innovative humanized FcRn knock-in (hiFcRn) mouse model using CRISPR/Cas9 technology. This model integrates hFcRn cDNA into the endogenous locus of the mouse Fcgrt gene, completely replacing native mouse FcRn (mFcRn) expression. The hiFcRn mouse model offers a more human-relevant platform for the preclinical evaluation of therapeutic antibodies and Fc-fusion proteins.},
}

Animals
*Receptors, Fc/genetics/metabolism
*Histocompatibility Antigens Class I/genetics/metabolism
Humans
Mice
*Gene Knock-In Techniques
*Immunoglobulin G
Mice, Transgenic
CRISPR-Cas Systems

@article {pmid40715076,
year = {2025},
author = {Bell, HW and Feng, R and Shah, M and Yao, Y and Douglas, J and Doerfler, PA and Mayuranathan, T and O'Dea, MF and Li, Y and Wang, YD and Zhang, J and Mackay, JP and Cheng, Y and Quinlan, KGR and Weiss, MJ and Crossley, M},
title = {Removal of promoter CpG methylation by epigenome editing reverses HBG silencing.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6919},
pmid = {40715076},
issn = {2041-1723},
support = {2020861//Department of Health | National Health and Medical Research Council (NHMRC)/ ; K01DK132453//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; R01 156647//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; },
mesh = {Humans ; *Promoter Regions, Genetic/genetics ; *DNA Methylation/genetics ; *CpG Islands/genetics ; *Gene Silencing ; *Gene Editing/methods ; CRISPR-Cas Systems ; Ubiquitin-Protein Ligases/genetics/metabolism ; CCAAT-Enhancer-Binding Proteins/genetics/metabolism ; *gamma-Globins/genetics/metabolism ; *Epigenome/genetics ; Cell Line ; DNA (Cytosine-5-)-Methyltransferase 1/metabolism/genetics ; DNA-Binding Proteins/genetics/metabolism ; Epigenome Editing ; },
abstract = {β-hemoglobinopathies caused by mutations in adult-expressed HBB can be treated by re-activating the adjacent paralogous genes HBG1 and HBG2 (HBG), which are normally silenced perinatally. Although HBG expression is induced by global demethylating drugs, their mechanism is poorly understood, and toxicity limits their use. We identify the DNMT1-associated maintenance methylation protein UHRF1 as a mediator of HBG repression through a CRISPR/Cas9 screen. Loss of UHRF1 in the adult-type erythroid cell line HUDEP2 causes global demethylation and HBG activation that is reversed upon localized promoter re-methylation. Conversely, targeted demethylation of the HBG promoters activates their genes in HUDEP2 or primary CD34[+] cell-derived erythroblasts. Mutation of MBD2, a CpG-methylation reading component of the NuRD co-repressor complex, recapitulates the effects of promoter demethylation. Our findings demonstrate that localized CpGmethylation at the HBG promoters facilitates gene silencing and identify a potential therapeutic approach for β-hemoglobinopathies via epigenomic editing.},
}

Humans
*Promoter Regions, Genetic/genetics
*DNA Methylation/genetics
*CpG Islands/genetics
*Gene Silencing
*Gene Editing/methods
CRISPR-Cas Systems
Ubiquitin-Protein Ligases/genetics/metabolism
CCAAT-Enhancer-Binding Proteins/genetics/metabolism
*gamma-Globins/genetics/metabolism
*Epigenome/genetics
Cell Line
DNA (Cytosine-5-)-Methyltransferase 1/metabolism/genetics
DNA-Binding Proteins/genetics/metabolism
Epigenome Editing

@article {pmid40613420,
year = {2025},
author = {Yuan, T and Du, J and Hu, J and Luo, J and Pu, X and Zhu, X and Chen, B and Teng, Y and Li, H and Li, S and Jiang, L and Xiong, E},
title = {CRISPR-Cas13a-based dual-channel AND-logic gated biosensor for the simultaneous assay of APE1 and miRNA-224.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {62},
pages = {11673-11676},
doi = {10.1039/d5cc03222a},
pmid = {40613420},
issn = {1364-548X},
mesh = {*MicroRNAs/analysis/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *DNA-(Apurinic or Apyrimidinic Site) Lyase/analysis/genetics ; Humans ; },
abstract = {Simultaneous detection of multiple targets is of great significance for the precise diagnosis of diseases. Herein, we report a dual-channel AND-logic gated biosensing platform based on the CRISPR-Cas13a system for the simultaneous detection of APE1 and miRNA-224, which showed superior specificity, sensitivity and potential for practical applications. Our study not only expands the CRISPR toolbox beyond nucleic acid assay but also establishes a new paradigm for multi-analyte diagnostic systems.},
}

*MicroRNAs/analysis/genetics
*Biosensing Techniques/methods
*CRISPR-Cas Systems
*DNA-(Apurinic or Apyrimidinic Site) Lyase/analysis/genetics
Humans

@article {pmid40495486,
year = {2025},
author = {Jeong, YY and Han, JH and Yu, J and Bae, S and Seo, PJ},
title = {Identification of optimal adenine and cytosine base editors for genome editing in Arabidopsis and soybean.},
journal = {BMB reports},
volume = {58},
number = {7},
pages = {288-292},
pmid = {40495486},
issn = {1976-670X},
mesh = {*Gene Editing/methods ; *Arabidopsis/genetics ; *Glycine max/genetics ; *Cytosine/metabolism ; *Adenine/metabolism ; Genome, Plant/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Base editors, including adenine base editors (ABEs) and cytosine base editors (CBEs), are widely used in numerous organisms to introduce site-specific sequence modifications in genomic DNA without causing double-strand breaks (DSBs). However, these editors exhibit low editing efficiencies, particularly in dicot plants, thereby limiting their application in dicot plant genome engineering. In this study, we assessed the editing efficiencies of various base editors to identify those optimal for base editing in dicot plants. We discovered that ABE8e, an ABE variant, demonstrated superior A-to-G base editing efficiency within A5-A8 windows, and A3A/Y130F-V04, a CBE variant, exhibited the highest C-to-T base editing efficiency within C4-C15 windows in both Arabidopsis and soybean protoplasts. Overall, we recommend these two base editors as prime choices for efficient genome engineering in a range of crop plants. [BMB Reports 2025; 58(7): 288-292].},
}

*Gene Editing/methods
*Arabidopsis/genetics
*Glycine max/genetics
*Cytosine/metabolism
*Adenine/metabolism
Genome, Plant/genetics
CRISPR-Cas Systems/genetics

@article {pmid40441113,
year = {2025},
author = {Shen, C and Chen, X and Yu, Y and Qin, L and Xu, G and Wei, F and Yang, J and Hu, Q and Cen, Y},
title = {A novel fluorescent sensing platform for miRNA-210 detection based on signal amplification via SDA and self-assembly Cas12a system via RCT.},
journal = {Talanta},
volume = {295},
number = {},
pages = {128382},
doi = {10.1016/j.talanta.2025.128382},
pmid = {40441113},
issn = {1873-3573},
mesh = {*MicroRNAs/analysis/genetics ; Humans ; *Biosensing Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; *Endodeoxyribonucleases/metabolism/genetics ; Limit of Detection ; Spectrometry, Fluorescence ; *Fluorescent Dyes/chemistry ; Fluorescence ; Bacterial Proteins ; },
abstract = {In this work, a biosensing platform for miR-210 which is a potent biomarker for the early diagnosis of breast cancer was innovatively designed based on CRISPR/Cas12a by integrating strand displacement amplification and rolling circle transcription. The target opened the hairpin through toe-hold, allowing polymerization, incision and extension to occur which involved SDA. This process produced two chains: cycle chain and trigger. The cycle chain then complemented to the loop of the hairpin to open it, repeating the above process to generate additional trigger circularly. The trigger was bound to the notched dumbbell through base complementary pairing and then the dumbbell became intact by T4 DNA ligase. The closed dumbbell strand served as the initiator of transcription and the template for crRNA under the influence of T7 enzyme, and was responsible for the transcription of numerous crRNA sequences in a process called RCT. These sequences subsequently bound to Cas12a proteins, forming a binary complex. In the transcriptional state, the dumbbell was in an unwinding configuration, with the stem portion served as activator binding to the binary complex to facilitate trans-cleavage activity of Cas12a, which resulted in cleavage of the F-Q, generating fluorescent signals. The above platform could sensitively detect miR-210 with a detection limit of 6.67 fM. The platform has the advantages of being easy to use and flexible to sequence according to different target, making it feasible to detect different biomarkers in clinic settings.},
}

*MicroRNAs/analysis/genetics
Humans
*Biosensing Techniques/methods
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
*CRISPR-Associated Proteins/metabolism/genetics/chemistry
*Endodeoxyribonucleases/metabolism/genetics
Limit of Detection
Spectrometry, Fluorescence
*Fluorescent Dyes/chemistry
Fluorescence
Bacterial Proteins

@article {pmid40435758,
year = {2025},
author = {Ren, K and Ding, S and Shi, J and Dong, J and Du, F and Tang, Z},
title = {Detection of lead contamination using DNAzyme and split activator-triggered CRISPR/Cas12a.},
journal = {Talanta},
volume = {295},
number = {},
pages = {128385},
doi = {10.1016/j.talanta.2025.128385},
pmid = {40435758},
issn = {1873-3573},
mesh = {*Lead/analysis ; *DNA, Catalytic/metabolism/chemistry ; *CRISPR-Cas Systems ; *Endodeoxyribonucleases/metabolism/chemistry/genetics ; *Biosensing Techniques/methods ; *CRISPR-Associated Proteins/metabolism/chemistry ; *Bacterial Proteins/metabolism/chemistry ; Limit of Detection ; *Water Pollutants, Chemical/analysis ; Humans ; Drinking Water/analysis ; },
abstract = {Widespread Pb[2+] contamination represents a significant global health threat, particularly to children, highlighting the critical need for accurate monitoring and quantification to mitigate its adverse effects. The integration of DNAzymes with the programmable nuclease Cas12a has emerged as a promising approach for achieving specific and ultrasensitive detection of Pb[2+]. However, conventional DNAzyme-Cas12a systems suffer from inevitable background signals caused by dynamic instability between DNAzymes and Cas12a activators, which compromises analytical reliability. Herein, we present a novel DNAzyme-Cas12a assay featuring a split activator-based Cas12a switch. We demonstrate that the split activator containing a flap region effectively prevents Cas12a activation, thereby suppressing background noise induced by "DNA breathing" phenomena. Upon Pb[2+]-dependent activation of the GR-5 DNAzyme, the flap is cleaved, enabling reconstitution of the Cas12a activator and triggering trans-cleavage activity for signal amplification. This strategy achieves a detection limit of 615 pM for Pb[2+] while maintaining high specificity against interfering metal ions. Notably, the assay eliminates requirements for DNA amplification or nanoparticle modification, enabling rapid Pb[2+] detection at ambient temperature. The method demonstrated high accuracy in detecting contaminated tap and drinking water, suggesting its potential as a reliable analytical tool for monitoring Pb2+ contamination in practical samples.},
}

*Lead/analysis
*DNA, Catalytic/metabolism/chemistry
*CRISPR-Cas Systems
*Endodeoxyribonucleases/metabolism/chemistry/genetics
*Biosensing Techniques/methods
*CRISPR-Associated Proteins/metabolism/chemistry
*Bacterial Proteins/metabolism/chemistry
Limit of Detection
*Water Pollutants, Chemical/analysis
Humans
Drinking Water/analysis

@article {pmid40398044,
year = {2025},
author = {Xu, J and Zhang, Y and Yuan, B and Wang, Y and Wang, J and Yuan, Y},
title = {A novel one-tube RPA/CRISPR melting curve detection sensing system based on unique 3'-toehold nucleic acid aptamer for Bacillus anthracis detection.},
journal = {Talanta},
volume = {295},
number = {},
pages = {128306},
doi = {10.1016/j.talanta.2025.128306},
pmid = {40398044},
issn = {1873-3573},
mesh = {*Bacillus anthracis/isolation & purification/genetics ; *Aptamers, Nucleotide/chemistry/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Spores, Bacterial/isolation & purification ; },
abstract = {Developing effective detection methods for Bacillus anthracis is essential for our public health system to accurately detect hidden anthrax outbreaks. Herein, we introduce a unique 3'-toehold nucleic acid aptamer (probes) into Cas12a biosensor, combined with RPA, to establish a rapid (1h), specific, and sensitive (1copy/μL) detection method for B. anthracis. The design behind this approach is that the target sequence is amplified via RPA, and the amplification product triggers the crRNA/Cas12a complex to degrade the 3' toehold probes, which are analyzed using melting curve analysis on a specific instrument, naming a one-tube RPA/CRISPR melting curve detection (ORCMD) sensing system. Furthermore, ORCMD is used to detect the B. anthracis spores-positive or negative soil samples from the location of world War-II site (Harbin, China), B. anthracis was precisely identified as other methods, suggesting its significant practical application potential. This system enriches the CRISPR detection technology toolbox, compared to other CRISPR-based sensing strategies, the concept of the 3' toehold probes offers distinct advantages in the development of CRISPR-based multi-target detection methods.},
}

*Bacillus anthracis/isolation & purification/genetics
*Aptamers, Nucleotide/chemistry/genetics
*Biosensing Techniques/methods
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
Spores, Bacterial/isolation & purification

@article {pmid40185317,
year = {2025},
author = {Lee, EG and Kim, KH},
title = {Transposition of transposable element IS1 in Edwardsiella piscicida mutant generated by CRISPR/Cas9 along with λ-Red recombineering system.},
journal = {Research in microbiology},
volume = {176},
number = {5-6},
pages = {104297},
doi = {10.1016/j.resmic.2025.104297},
pmid = {40185317},
issn = {1769-7123},
mesh = {*CRISPR-Cas Systems ; *DNA Transposable Elements/genetics ; *Gene Editing/methods ; *Edwardsiella/genetics ; Mutation ; Recombination, Genetic ; Genome, Bacterial ; },
abstract = {This study aimed to investigate unintended mutations introduced by the CRISPR/Cas9 genome editing system in Edwardsiella piscicida. Whole-genome sequencing was conducted on the wild-type E. piscicida NH1 and its alanine racemase knockout mutants (E. piscicida Δalr325 NH1 and E. piscicida Δalr50 NH1) generated using CRISPR/Cas9 with a λ-Red recombineering system. Comparative genomic analyses revealed that the insertion sequence 1 (IS1) transpositions occurred in the CRISPR/Cas9-edited mutants, disrupting the type I restriction-modification system subunit M gene, in addition to the targeted gene deletion. Interestingly, no IS1 transpositions were detected in mutants produced via conventional plasmid-based allelic exchange, indicating the potential link between CRISPR/Cas9-mediated editing and transposition events. These results suggest that genome editing via CRISPR/Cas9 could trigger IS1 transposition, potentially due to double-stranded DNA breaks. The lack of sequence similarity between the single guide RNA (sgRNA) and the transposed regions suggests that transpositions are not CRISPR/Cas9 off-target effects. This study provides evidence of interactions between mobile genetic elements and genome editing systems, requiring further investigation into their underlying mechanisms.},
}

*CRISPR-Cas Systems
*DNA Transposable Elements/genetics
*Gene Editing/methods
*Edwardsiella/genetics
Mutation
Recombination, Genetic
Genome, Bacterial

@article {pmid40719891,
year = {2025},
author = {Rather, GA and Ayzenshtat, D and Kumar, M and Aisemberg, E and Bocobza, S},
title = {Direct haploid formation in Arabidopsis using transgenic CENH3-based inducers.},
journal = {Plant cell reports},
volume = {44},
number = {8},
pages = {182},
pmid = {40719891},
issn = {1432-203X},
support = {20-01-0245//Chief Scientist - Ministry of Agriculture and Rural Development/ ; },
abstract = {This study introduces a streamlined transgenic method for generating haploid inducers using a single T-DNA construct, combining CENH3 disruption, functional complementation, and a visual marker for efficient haploid screening. The development of doubled haploid lines is crucial for plant breeding programs, but conventional inbreeding methods are laborious and costly. Centromere-mediated genome elimination using modified CENH3 histones offers an efficient single-generation approach to induce haploidy. However, this approach necessitates the generation of haploid inducer lines, which typically involves cumbersome random mutagenesis screens. In this study, we implemented a transgenic strategy to circumvent this and directly create haploid inducers in Arabidopsis. This was achieved by knocking out endogenous AtCENH3 using CRISPR/Cas while complementing it with mutated AtCENH3 variants on the same T-DNA. Four constructs with truncated or full-length AtCENH3 harboring the G83E mutation alone or with the L130F mutation, and one negative control without mutations, were transformed into Arabidopsis. Stable homozygous transgenic lines were obtained and pollinated with a glabra mutant (Atgl1). Progenies lacking RFP fluorescence and exhibiting a glabrate phenotype were recovered, and flow cytometry analyses showed their haploidy, suggesting genome elimination. Comparatively, the G83E variants showed the highest haploid induction rate. This transgenic approach directly generated haploid inducer lines in Arabidopsis while avoiding random mutagenesis. This novel transgenic strategy provides a powerful tool to rapidly establish haploid inducer lines in additional transformable crops.},
}

@article {pmid40719158,
year = {2025},
author = {Torres-Barceló, C and Boyer, C and Garneau, JR and Poussier, S and Robène, I and Moury, B},
title = {A Phylogenetic Host-Range Index Reveals Ecological Constraints in Phage Specialisation and Virulence.},
journal = {Molecular ecology},
volume = {},
number = {},
pages = {e70052},
doi = {10.1111/mec.70052},
pmid = {40719158},
issn = {1365-294X},
abstract = {Phages are typically known for having a limited host range, targeting particular strains within a bacterial species, but accurately measuring their specificity remains challenging. Factors like the genetic diversity or population dynamics of host bacteria are often disregarded despite their potential influence on phage specialisation and virulence. This study focuses on the Ralstonia solanacearum species complex (RSSC), which comprises genetically diverse bacteria responsible for a major plant disease. It uses a diversified collection of RSSC phages to develop new host-range analysis methods and to test ecological and evolutionary hypotheses on phage host range. We introduce a new 'phylogenetic host-range index' that employs an ecological diversity index to account for the genetic diversity of bacterial hosts, allowing systematic classification of phages along a continuum between specialists and generalists. We propose and provide evidence that generalist phages are more likely to be represented in CRISPR-Cas immune system of bacteria than specialist phages. We explore the hypothesis that generalist phages might exhibit lower virulence than specialist ones due to potential evolutionary trade-offs between host-range breadth and virulence. Importantly, contrasted correlations between phage virulence and host range depend on the epidemiological context. A trade-off was confirmed in a context of low bacterial diversity, but not in a context of higher bacterial diversity, where no apparent costs were detected for phages adapted to a wide range of hosts. This study highlights the need for genetic analyses in phage host range and of investigating ecological trade-offs that could improve both fundamental phage knowledge and applications in biocontrol or therapy.},
}

@article {pmid40717612,
year = {2025},
author = {Bagi, M and Jamalzadegan, S and Steksova, A and Wei, Q},
title = {CRISPR-Cas based platforms for RNA detection: fundamentals and applications.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5cc03257a},
pmid = {40717612},
issn = {1364-548X},
abstract = {The detection of RNA biomarkers is crucial for diagnosing many urgent diseases such as infections and cancer. Conventional RNA detection techniques such as RT-PCR, LAMP, and microarrays are effective, but often face limitations in terms of speed, sensitivity, and equipment demands. In recent years, CRISPR/Cas systems have emerged as versatile platforms for RNA detection, which offer high specificity, programmability, and adaptability across a wide range of diagnostic applications. This review first categorizes different CRISPR-based RNA detection systems according to the CRISPR effectors employed, including Cas13, Cas12, Cas14, Cas9, and newly characterized enzymes such as Cas7-11 and Cas10, detailing their mechanisms of target recognition, cleavage activity, and signal generation. The CRISPR detection platforms are coupled with or without pre-amplification steps to meet the different sensitivity needs. Preamplification-based systems integrate CRISPR with methods like RT-PCR and isothermal amplification to enhance sensitivity. In parallel, preamplification-free strategies, such as split-crRNA or split-activator systems, are gaining attention for their balanced assay performance and simplicity, which are especially attractive for point-of-care (POC) settings. Then, the diagnostic applications of these technologies are explored across two major domains: infectious disease detection and cancer biomarker identification via miRNAs, demonstrating the clinical potential of CRISPR-based RNA detection platforms. In addition, we explore ongoing challenges such as improving sensitivity in amplification-free formats, and developing field-deployable, cost-effective systems. The review concludes by outlining emerging trends and future directions in CRISPR-based RNA diagnostics, emphasizing their transformative potential in clinical settings.},
}

@article {pmid40713745,
year = {2025},
author = {Jiang, M and Chen, X and Song, Y and Wei, M and Cao, J and Lu, W and Lan, F and Bai, Y and Cui, M},
title = {NEXN deficiency leads to dilated cardiomyopathy in human pluripotent stem cell-derived cardiomyocytes.},
journal = {Stem cell research & therapy},
volume = {16},
number = {1},
pages = {402},
pmid = {40713745},
issn = {1757-6512},
support = {7232088//Beijing Natural Science Foundation/ ; BRWEP2024W014090201//Beijing Research Ward Excellence Clinical Study Program/ ; 2024003//Key Clinical Projects of Peking University Third Hospital/ ; 82070272//National Natural Science Foundation of China/ ; },
mesh = {*Myocytes, Cardiac/metabolism/pathology/cytology ; Humans ; *Cardiomyopathy, Dilated/pathology/metabolism/genetics ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Cell Differentiation ; CRISPR-Cas Systems ; Oxidative Stress ; },
abstract = {BACKGROUND: Dilated cardiomyopathy (DCM) constitutes a major cause of heart failure, characterized by high mortality rates and a limited availability of effective therapeutic options. A substantial body of evidence indicates that mutations in the Nexilin (NEXN) gene are significant pathogenic contributors to DCM, but the pathogenic mechanism for dilated cardiomyopathy is unclear.

METHODS: A human NEXN homozygous knockout cardiomyocyte model was established by combining CRISPR/Cas9 gene editing technology and human induced pluripotent stem cells (hiPSCs)-directed differentiation technology. Cell model phenotypic assays were done to characterize the pathological features of the resulting NEXN-deficient cardiomyocytes.

RESULTS: NEXN gene knockout did not affect the pluripotency and differentiation efficiency of hiPSCs. NEXN-deficient cardiomyocytes showed disordered junctional membrane complexes, abnormal excitation-contraction coupling, increased oxidative stress and decreased energy metabolism level. Moreover, levo-carnitine and sarcoplasmic reticulum calcium ATPase (SERCA2a) Activator 1 were identified as promising therapeutic agents for the treatment of DCM.

CONCLUSION: We demonstrated that NEXN was one of the important components in maintaining the structure and function of cardiomyocyte junctional membrane complexes (JMCs), excitation-contraction coupling and energy metabolism of cardiomyocytes, while the loss of its function would lead to DCM. This model represents an important tool to gain insight into the mechanism of DCM, elucidate the gene-phenotype relationship of NEXN deficiency and facilitate drug screening.},
}

*Myocytes, Cardiac/metabolism/pathology/cytology
Humans
*Cardiomyopathy, Dilated/pathology/metabolism/genetics
*Induced Pluripotent Stem Cells/metabolism/cytology
Cell Differentiation
CRISPR-Cas Systems
Oxidative Stress

@article {pmid40713739,
year = {2025},
author = {Amina, S and Wei, M and Zhang, S and Jiaqi, F and Ma, S and Abbas, MK and Lan, F and Jiang, H},
title = {Metabolism-related gene PDK1 regulates myocardial cell remodeling and its mechanism.},
journal = {Stem cell research & therapy},
volume = {16},
number = {1},
pages = {404},
doi = {10.1186/s13287-025-04518-9},
pmid = {40713739},
issn = {1757-6512},
support = {NSFC 82370440//Beijing Natural Science Foundation/ ; },
mesh = {*Myocytes, Cardiac/metabolism/cytology ; Humans ; *Pyruvate Dehydrogenase Acetyl-Transferring Kinase/genetics/metabolism ; Induced Pluripotent Stem Cells/metabolism/cytology ; Apoptosis ; Cell Differentiation ; Oxidative Stress ; Gene Knockout Techniques ; Cell Hypoxia ; *Myocardium/metabolism ; },
abstract = {BACKGROUND: The progression of ischemic heart disease results from various forms of cardiomyopathies, which begin with cardiac remodelling. Pyruvate Dehydrogenase Kinase 1 (PDK1) is one of the basic kinase family components responsible for oxidative phosphorylation. However, due to the lack of a suitable research model, there is no evidence that remodelling pathogenesis in humans causes death by PDK1 knockout (KO). In the current study, we established a PDK1-deficient human cardiomyocyte (CM) model under conditions imitating the human PDK1-KO model. We determined the role of PDK1 in myocardial apoptosis induced by hypoxia and its implicit mechanism.

METHODS: A human PDK1-KO CM's model was established by combining CRISPR/Cas-9 gene-editing and human induced pluripotent stem cells (hiPSC) directed differentiation technology. The pathological features of PDK1-KO cardiomyocytes were assessed using a phenotypic cell model under basal and hypoxic conditions.

RESULTS: We found that pluripotency and differentiation efficiency of hiPSCs after PDK1 knockout remain intact. Cardiomyocytes with a PDK1 gene knockout showed hypoxia-induced myocardial apoptosis by disturbing mitochondrial metabolism, increased oxidative stress levels, and decreased cell energy and viability. In addition, lentivirus transfection significantly improved the metabolism and cell viability in PDK1-deficient cardiomyocytes.

CONCLUSIONS: Our study established a PDK1 knockout model under hypoxia that exhibits mitochondrial metabolism dysregulation, elevated oxidative stress, and decreased cell viability. This model is an important tool for understanding the mechanism of hypoxia-induced myocardial apoptosis, elucidating the gene-phenotype relationship of PDK1 deficiency, and providing evidence to mitigate the damage against hypoxia.},
}

*Myocytes, Cardiac/metabolism/cytology
Humans
*Pyruvate Dehydrogenase Acetyl-Transferring Kinase/genetics/metabolism
Induced Pluripotent Stem Cells/metabolism/cytology
Apoptosis
Cell Differentiation
Oxidative Stress
Gene Knockout Techniques
Cell Hypoxia
*Myocardium/metabolism

@article {pmid40711918,
year = {2025},
author = {Roberts, A and Adler, BA and Cress, BF and Doudna, JA and Barrangou, R},
title = {Phage-based delivery of CRISPR-associated transposases for targeted bacterial editing.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {30},
pages = {e2504853122},
doi = {10.1073/pnas.2504853122},
pmid = {40711918},
issn = {1091-6490},
support = {DE-AC02-05CH11231//U.S. Department of Energy (DOE)/ ; },
mesh = {*Gene Editing/methods ; *Transposases/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics/virology ; *Bacteriophage lambda/genetics ; Genome, Bacterial ; },
abstract = {Phage λ, a well-characterized temperate phage, has been recently leveraged for bacterial genome editing by selectively delivering base editors into targeted bacterial species. We extend this concept by engineering phage λ to deliver CRISPR-guided transposases, accomplishing large insertions and targeted gene disruptions. To achieve this, we engineered phage λ using homologous recombination paired with Cas13a-based counterselection for precise phage modifications. Initially, we established the utility of Cas13a in phage λ by conducting minimal recoding edits, deletions, and insertions. Subsequently, we scaled up the engineering to embed the comprehensive DNA-editing CRISPR-Cas transposase (DART) system within the phage genome, creating λ-DART phages. These modified λ-DART phages were then employed to infect Escherichia coli, generating CRISPR RNA-guided transposition events in the host genome. Applying our engineered λ-DART phages to monocultures and a mixed bacterial community comprising three genera led to efficient, precise, and specific gene knockouts and insertions in the targeted E. coli cells, achieving editing efficiencies surpassing 50% of the population. This research enhances phage-mediated genome editing by enabling efficient in situ gene integrations in bacteria, offering an avenue for further application in microbial community contexts. This scalable method enables flexible microbial genome editing in situ to manipulate the function and composition of diverse ecosystems.},
}

*Gene Editing/methods
*Transposases/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Escherichia coli/genetics/virology
*Bacteriophage lambda/genetics
Genome, Bacterial

@article {pmid40709174,
year = {2025},
author = {Han, B and Liu, W and Wang, W and Li, Z and You, B and Liu, D and Nan, Y and Ding, T and Dai, Z and Zhang, Y and Zhang, W and Liu, Q and Li, X},
title = {CRISPR/Cas9-based discovery of ccRCC therapeutic opportunities through molecular mechanism and immune microenvironment analysis.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1619361},
pmid = {40709174},
issn = {1664-3224},
mesh = {Humans ; *Tumor Microenvironment/immunology/genetics ; *Carcinoma, Renal Cell/genetics/immunology/drug therapy/pathology ; *Kidney Neoplasms/genetics/immunology/drug therapy/pathology ; *CRISPR-Cas Systems ; Biomarkers, Tumor/genetics ; Gene Expression Regulation, Neoplastic ; Prognosis ; Mutation ; },
abstract = {INTRODUCTION: Clear cell renal cell carcinoma is a common and aggressive form of renal cell carcinoma. Its incidence continues to rise, and metastatic recurrence leads to poor clinical outcomes. Current prognostic biomarkers lack reliability. We integrated multi-omics data to discover key ccRCC genes and build a prognostic model to improve risk prediction and guide treatment decisions.

METHODS: Our study integrated genome-wide CRISPR screening data from DepMap and transcriptomic profiles from TCGA to identify key genes associated with ccRCC pathogenesis. Initial screening identified 11 candidate genes through differential expression analysis and CRISPR functional validation. Using LASSO and Cox regression, we selected five key genes (GGT6, HAO2, SLPI, MELK, and EIF4A1) for model construction. The functional role of MELK was tested by knockdown experiments. Additional analyses included tumor mutation burden, immune microenvironment assessment, and drug response prediction.

RESULTS: The model stratified patients into high-risk and low-risk groups with distinct survival outcomes. High-risk cases showed higher mutation loads, immunosuppressive features, and activated cytokine pathways, whereas low-risk cases displayed metabolic pathway activity. MELK knockdown reduced cancer cell proliferation and migration. High-risk patients exhibited better responses to targeted drugs such as pazopanib and sunitinib.

DISCUSSION: Our study demonstrates the pivotal role of MELK in ccRCC progression. This multi-omics-driven model elucidates MELK-mediated mechanisms and their interactions with the tumor microenvironment, providing novel strategies for risk stratification and targeted therapy. Future studies will validate these findings in independent cohorts and investigate the regulatory networks of MELK to identify potential therapeutic targets.},
}

Humans
*Tumor Microenvironment/immunology/genetics
*Carcinoma, Renal Cell/genetics/immunology/drug therapy/pathology
*Kidney Neoplasms/genetics/immunology/drug therapy/pathology
*CRISPR-Cas Systems
Biomarkers, Tumor/genetics
Gene Expression Regulation, Neoplastic
Prognosis
Mutation

@article {pmid40707831,
year = {2025},
author = {Sereika, M and Mussig, AJ and Jiang, C and Knudsen, KS and Jensen, TBN and Petriglieri, F and Yang, Y and Jørgensen, VR and Delogu, F and Sørensen, EA and Nielsen, PH and Singleton, CM and Hugenholtz, P and Albertsen, M},
title = {Genome-resolved long-read sequencing expands known microbial diversity across terrestrial habitats.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {40707831},
issn = {2058-5276},
support = {130690//Villum Fonden (Villum Foundation)/ ; 50093//Villum Fonden (Villum Foundation)/ ; },
abstract = {The emergence of high-throughput, long-read DNA sequencing has enabled recovery of microbial genomes from environmental samples at scale. However, expanding the terrestrial microbial genome catalogue has been challenging due to the enormous complexity of these environments. Here we performed deep, long-read Nanopore sequencing of 154 soil and sediment samples collected during the Microflora Danica project, yielding genomes of 15,314 previously undescribed microbial species, recovered using our custom mmlong2 workflow. The recovered microbial genomes span 1,086 previously uncharacterized genera and expand the phylogenetic diversity of the prokaryotic tree of life by 8%. The long-read assemblies also enabled the recovery of thousands of complete ribosomal RNA operons, biosynthetic gene clusters and CRISPR-Cas systems. Furthermore, the incorporation of the recovered genomes into public genomic databases substantially improved species-level classification rates for soil and sediment metagenomic datasets. These findings demonstrate that long-read sequencing allows cost-effective recovery of high-quality microbial genomes from highly complex ecosystems, which remain an untapped source of biodiversity.},
}

@article {pmid40661409,
year = {2025},
author = {Hoffmann, FT and Wiegand, T and Palmieri, AI and Glass-Klaiber, J and Xiao, R and Tang, S and Le, H and Meers, C and Lampe, GD and Chang, L and Sternberg, SH},
title = {Exapted CRISPR-Cas12f homologs drive RNA-guided transcription.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {40661409},
issn = {2692-8205},
abstract = {Bacterial transcription initiation is a tightly regulated process that canonically relies on sequence-specific promoter recognition by dedicated sigma (σ) factors, leading to functional DNA engagement by RNA polymerase (RNAP) [1] . Although the seven σ factors in E. coli have been extensively characterized [2] , Bacteroidetes species encode dozens of specialized, extracytoplasmic function σ factors (σ [E]) whose precise roles are unknown, pointing to additional layers of regulatory potential [3] . Here we uncover an unprecedented mechanism of RNA-guided gene activation involving the coordinated action of σ [E] factor in complex with nuclease-dead Cas12f (dCas12f). We screened a large set of genetically-linked dCas12f and σ [E] homologs in E. coli using RIP-seq and ChIP-seq experiments, revealing systems that exhibited robust guide RNA enrichment and DNA target binding with a minimal 5'-G target-adjacent motif (TAM). Recruitment of σ [E] was dependent on dCas12f and guide RNA (gRNA), suggesting direct protein-protein interactions, and co-expression experiments demonstrated that the dCas12f-gRNA-σ [E] ternary complex was competent for programmable recruitment of the RNAP holoenzyme. Remarkably, dCas12f-RNA-σ [E] complexes drove potent gene expression in the absence of any requisite promoter motifs, with de novo transcription start sites defined exclusively by the relative distance from the dCas12f-mediated R-loop. Our findings highlight a new paradigm of RNA-guided transcription (RGT) that embodies natural features reminiscent of CRISPRa technology developed by humans [4,5] .},
}

@article {pmid40661357,
year = {2025},
author = {Henriques, WS and Bowman, J and Hall, LN and Gauvin, CC and Wei, H and Kuang, H and Zimanyi, CM and Eng, ET and Santiago-Frangos, A and Wiedenheft, B},
title = {Structures reveal how the Cas1-2/3 integrase captures, delivers, and integrates foreign DNA into CRISPR loci.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {40661357},
issn = {2692-8205},
abstract = {UNLABELLED: Cas1 and Cas2 are the hallmark proteins of prokaryotic adaptive immunity. However, these two proteins are often fused to other proteins and the functional association of these fusions often remain poorly understood. Here we purify Cas1 and the Cas2/3 fusion protein from Pseudomonas aeruginosa . We determine multiple structures of the Cas1-2/3 complex at distinct stages of CRISPR adaptation. Collectively, these structures reveal a prominent, positively charged channel on one face of the integration complex that captures short fragments of foreign DNA. Foreign DNA binding triggers conformational changes in Cas2/3 that expose new DNA binding surfaces necessary for homing the DNA-bound integrase to specific CRISPR loci. The length of the foreign DNA substrate determines if Cas1-2/3 docks completely onto the CRISPR repeat to successfully catalyze two sequential transesterification reactions required for integration. Taken together, these structures clarify how the Cas1-2/3 proteins orchestrate foreign DNA capture, site-specific delivery, and integration of new DNA into the bacterial genome.

HIGHLIGHTS: - A positively charged channel on the Cas1-2/3 complex captures fragments of DNA- A loop in the RecA1 domain controls access to the Cas3 nuclease active site- Foreign DNA binding allosterically regulates access to additional DNA binding sites- Distortion of the CRISPR repeat sequence licenses complete foreign DNA integration.},
}

@article {pmid40652511,
year = {2025},
author = {Wei, T and Li, J and Lei, X and Lin, R and Wu, Q and Zhang, Z and Shuai, S and Tian, R},
title = {Multimodal CRISPR screens uncover DDX39B as a global repressor of A-to-I RNA editing.},
journal = {Cell reports},
volume = {44},
number = {7},
pages = {116009},
doi = {10.1016/j.celrep.2025.116009},
pmid = {40652511},
issn = {2211-1247},
mesh = {*RNA Editing/genetics ; *DEAD-box RNA Helicases/metabolism/genetics ; Humans ; *Adenosine/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; HEK293 Cells ; *Inosine/metabolism/genetics ; RNA-Binding Proteins/metabolism/genetics ; },
abstract = {Adenosine-to-inosine (A-to-I) RNA editing is a critical post-transcriptional modification that diversifies the transcriptome and influences various cellular processes, yet its regulatory mechanisms remain largely unknown. Here, we present two complementary CRISPR-based genetic screening platforms: CREDITS (CRISPR-based RNA editing regulator screening), which enables genome-scale identification of editing regulators using an RNA recorder-based reporter system, and scCREDIT-seq (single-cell CRISPR-based RNA editing sequencing), which provides multiplexed single-cell characterization of transcriptome and editome changes for pooled perturbations. By screening 1,350 RNA-binding proteins, we identified a series of A-to-I editing regulators. Mechanistic investigation revealed DDX39B as a global repressor of A-to-I editing, which functions by preventing double-stranded RNA accumulation through its helicase activity. Targeting DDX39B significantly enhances the efficiency of RNA-editing-based tools, such as CellREADR (cell access through RNA sensing by endogenous ADAR) and LEAPER (leveraging endogenous ADAR for programmable editing of RNA), and disrupts hepatitis D virus (HDV) RNA editing homeostasis. These technological advances not only expand our understanding of RNA editing regulation but also provide powerful tools for exploring tissue-specific and context-dependent RNA modification mechanisms, with broad implications for therapeutic development.},
}

*RNA Editing/genetics
*DEAD-box RNA Helicases/metabolism/genetics
Humans
*Adenosine/metabolism/genetics
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
HEK293 Cells
*Inosine/metabolism/genetics
RNA-Binding Proteins/metabolism/genetics

@article {pmid40600880,
year = {2025},
author = {Kristof, A and Karunakaran, K and Ferry, Y and Briggs, S and Allen, C and Mizote, P and Jian, Z and Arvanitis, C and Blazeck, J},
title = {Dissection of the MeCP2 repressor protein enables CRISPR platform optimization via localization engineering.},
journal = {Protein engineering, design & selection : PEDS},
volume = {38},
number = {},
pages = {},
doi = {10.1093/protein/gzaf008},
pmid = {40600880},
issn = {1741-0134},
mesh = {*Methyl-CpG-Binding Protein 2/genetics/metabolism/chemistry ; Humans ; *CRISPR-Cas Systems ; HEK293 Cells ; *Protein Engineering/methods ; Nuclear Localization Signals/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Knockdown Techniques ; Animals ; Protein Domains ; },
abstract = {Clustered regularly interspaced short palindromic repeat interference (CRISPRi), the fusion of nuclease-inactive Cas9 with transcriptional repressor domains, is a powerful platform enabling site-specific gene knockdown across diverse biological contexts. Previously described CRISPRi systems typically utilize two distinct domain classes: (1) Krüppel-associated box domains and (2) truncations of the multifunctional protein, MeCP2. Despite widespread adoption of MeCP2 truncations for developing CRISPRi platforms, individual contributions of subdomains within MeCP2's transcriptional repression domain (TRD) toward enhancing gene knockdown remain unclear. Here, we dissect MeCP2's TRD and observe that two subdomains, the expected NcoR/SMRT interaction domain (NID) and an embedded nuclear localization signal (NLS), can separately enhance gold-standard CRISPRi platform performance beyond levels attained with the canonical MeCP2 protein truncation. Incorporating side-by-side analyses of nuclear localization and gene knockdown for over 30 constructs featuring MeCP2 subdomains or virus-derived NLS sequences, we demonstrate that appending C-terminal NLS motifs to dCas9-based transcriptional regulators, both repressors and activators, can significantly improve their effector function across several cell lines. We also observe that NLS placement greatly impacts CRISPRi repressor performance, and that modifying the subdomain configuration natively found within MeCP2 can also enhance gene suppression capabilities in certain contexts. Overall, this work demonstrates the interplay of two complimentary chimeric protein design considerations, transcriptional domain 'dissection' and NLS motif placement, for optimizing CRISPR-mediated transcriptional regulation in mammalian systems.},
}

*Methyl-CpG-Binding Protein 2/genetics/metabolism/chemistry
Humans
*CRISPR-Cas Systems
HEK293 Cells
*Protein Engineering/methods
Nuclear Localization Signals/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
Gene Knockdown Techniques
Animals
Protein Domains

@article {pmid40466269,
year = {2025},
author = {Jiang, C and Lei, D and Xu, B and Wang, Z and Fang, R and Tang, Y and Wang, H},
title = {High-efficient molecular detection system termed RAA-based CRISPR-Cas13a for novel duck orthoreovirus.},
journal = {Poultry science},
volume = {104},
number = {8},
pages = {105327},
pmid = {40466269},
issn = {1525-3171},
mesh = {Animals ; *Poultry Diseases/diagnosis/virology ; *Ducks ; *Reoviridae Infections/veterinary/diagnosis/virology ; *Orthoreovirus, Avian/isolation & purification/genetics ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Sensitivity and Specificity ; Recombinases/metabolism ; },
abstract = {The novel duck orthoreovirus (NDRV) is an immunosuppressive pathogen that significantly impacts the health of waterfowl breeding. Accurate, efficient, and convenient detection techniques are crucial for the prevention and control of NDRV, particularly in terms of field detection. By employing recombinase aided amplification (RAA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a, we have developed a highly sensitive enzymatic molecular system that combines Cas13a with T7 in vitro transcription and RAA, enabling efficient and accurate detection of NDRV at a sensitivity level of 10° copies/μL. Furthermore, the integration of portable lateral flow dipstick can effectively reduce the point-of-care testing time to 40 min, while exhibiting no cross-reactivity with duck hepatitis a virus, Tembusu virus and novel duck-origin goose parvovirus. Both this system and the reverse-transcriptase real-time quantitative polymerase chain reaction (RT-qPCR) method demonstrated a consistent 100% accuracy in clinical samples. This study facilitated the development of an optimized assay, which enables specific detection of NDRV through a simplified procedure and significantly reduces the risk of contamination. This highlights the potential applicability of this assay for point-of-care testing.},
}

Animals
*Poultry Diseases/diagnosis/virology
*Ducks
*Reoviridae Infections/veterinary/diagnosis/virology
*Orthoreovirus, Avian/isolation & purification/genetics
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/veterinary/methods
Sensitivity and Specificity
Recombinases/metabolism

@article {pmid40449106,
year = {2025},
author = {Sun, D and Zhu, Y and Wang, M and Wang, J and Cheng, W and Li, Z and Deng, Y and Ou, X and Jia, R and Chen, S and Zhu, D and Liu, M and Zhao, X and Yang, Q and Wu, Y and Zhang, S and Huang, J and He, Y and Wu, Z and Cheng, A},
title = {A RT-ERA-CRISPR/Cas12a assay for rapid point-of-care duck hepatitis A virus detection.},
journal = {Poultry science},
volume = {104},
number = {8},
pages = {105316},
pmid = {40449106},
issn = {1525-3171},
mesh = {Animals ; *Ducks ; *Poultry Diseases/diagnosis/virology ; *Hepatitis Virus, Duck/isolation & purification ; Point-of-Care Systems ; *Hepatitis, Viral, Animal/diagnosis/virology ; *Picornaviridae Infections/veterinary/diagnosis/virology ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Sensitivity and Specificity ; CRISPR-Cas Systems ; },
abstract = {Duck hepatitis A virus (DHAV) is a severe pathogen that threatens the duck industry. DHAV is transmitted primarily through the respiratory and gastrointestinal tracts. Therefore, developing accurate and rapid diagnostic technologies is crucial to prevent the spread of this infectious disease. Currently, the most widely used DHAV detection methods in clinical diagnosis include PCR, real-time PCR (RT-PCR) and ELISA, which require specialized equipment or trained professionals. Isothermal amplification technologies are often combined with lateral flow assays or visual readout methods for detecting pathogen nucleic acids in non-laboratory settings. CRISPR nucleases have accelerated the development of nucleic acid detection, increasing the sensitivity to a higher degree. Here, we applied reverse transcription-enzymatic recombinase amplification (RT-ERA) assisted by a Cas12a-fluorescence assay and a Cas12a-lateral flow assay for the detection of DHAV-1. Based on the sequence of DHAV-1, RT-ERA primers and crRNAs were designed, and different concentrations of ssDNA/Cas12a/crRNA were established to optimize the CRISPR reaction. The LoD for the Cas12a-fluorescence assay was 10 copies/μL, and this assay effectively differentiated DHAV-1 from other avian pathogens, exhibiting high sensitivity and specificity. Additionally, the Cas12a-lateral flow assay is user-friendly and can achieve point-of-care detection. Sixty-four clinical samples were tested and compared with quantitative real-time PCR (RT-PCR). This accurate and rapid point-of-care assay has significant potential for detecting DHAV-1 in clinical applications, especially for duck farms in rural areas.},
}

Animals
*Ducks
*Poultry Diseases/diagnosis/virology
*Hepatitis Virus, Duck/isolation & purification
Point-of-Care Systems
*Hepatitis, Viral, Animal/diagnosis/virology
*Picornaviridae Infections/veterinary/diagnosis/virology
*Nucleic Acid Amplification Techniques/veterinary/methods
Sensitivity and Specificity
CRISPR-Cas Systems

@article {pmid40707593,
year = {2025},
author = {Mahajan, D and Chia, HM and Lu, L},
title = {Knocking out ARL13B completely abolishes primary ciliogenesis in cell lines.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {26980},
pmid = {40707593},
issn = {2045-2322},
support = {Tier 1 RG 25/22//Ministry of Education - Singapore/ ; },
mesh = {*Cilia/metabolism/genetics ; *ADP-Ribosylation Factors/genetics/metabolism ; CRISPR-Cas Systems ; Hedgehog Proteins/metabolism ; Signal Transduction ; Cell Line ; Animals ; Humans ; Gene Knockout Techniques ; Mice ; Smoothened Receptor/metabolism ; },
abstract = {Small G protein ARL13B localizes to the cilium and plays essential roles in cilium biogenesis, organization, trafficking, and signaling. Here, we established multiple ARL13B knockout cell lines using the CRISPR/Cas9 system. Surprisingly, all our cell lines lost their cilia completely, in contrast to the reported short cilium and reduced ciliogenesis phenotype. We found that multiple regions of ARL13B are necessary for a complete rescue. Additionally, we found that ARL13B knockout cells also lost their response to SMO-mediated hedgehog stimulation. Our work demonstrates the critical requirement of ARL13B for ciliogenesis and hedgehog signaling, at least in cultured cells, and suggests that ARL13B plays a more crucial role in ciliary function than previously understood.},
}

*Cilia/metabolism/genetics
*ADP-Ribosylation Factors/genetics/metabolism
CRISPR-Cas Systems
Hedgehog Proteins/metabolism
Signal Transduction
Cell Line
Animals
Humans
Gene Knockout Techniques
Mice
Smoothened Receptor/metabolism

@article {pmid40707336,
year = {2025},
author = {Li, J and Lau, CH and Zhu, H},
title = {CRISPR/Cas-based personal glucose meters for nucleic acid detection.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2025.07.006},
pmid = {40707336},
issn = {1879-3096},
abstract = {Through integration with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems, personal glucose meters (PGMs) have been repurposed to detect non-glucose targets. PGMs enable the intuitive readout of electrochemical signals, while CRISPR/Cas offers rapid detection and signal amplification capability. Their integration can realize point-of-care (POC) diagnostics. Herein, we critically discuss their advances, pitfalls, and future perspectives.},
}

@article {pmid40704512,
year = {2025},
author = {Baig, MMFA and Chien, WT and Chair, SY},
title = {Nanotechnological approaches for the targeted delivery of CRISPR-Cas systems for genomic modifications, biomolecular sensing, and precision medicine.},
journal = {Biomaterials science},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5bm00711a},
pmid = {40704512},
issn = {2047-4849},
abstract = {The integration strategies of CRISPR-Cas gene editing systems with nanotechnological approaches have achieved synergistic effects in targeting genes; correcting genetic disorders; and treating, sensing, and diagnosing a variety of cancers and metabolic, immunological, and complex infectious diseases-all having connectivity with distinct genetic cues and mutations. Numerous recent studies have demonstrated the use of the nano-scale properties of nanomaterials to tremendously improve the genomic-editing efficiencies of CRISPR/Cas systems for achieving 50% enhanced bioavailability, improved cell targetability, and gene-level specificity while minimizing immunogenicity, compared with conventional/ordinary delivery techniques. Thus, nano-delivery methods utilizing the unique properties of nanomaterials, molecular interactions, biocompatibility, targeted cellular uptake, and nuclear delivery capability effectively overcame the challenges of inefficient biomolecular delivery, and off-target effects were effectively overcome. Nano -carriers made up of materials such as DNA lattices, lipids, dendrimers, polymers, peptides, and metals (gold, silver, etc.) that were explored for facilitating the precise delivery of CRISPR/Cas components, sensing biomolecules, and diagnostic purposes are discussed in this review report. The ability of DNA scaffold materials to incorporate nano-CRISPR systems, to sense biomolecules, and for targeted cellular delivery of payloads (e.g., Cas9, Cas12, Cas13, and Cas14 proteins and single-guide RNAs (sgRNAs)) maximized gene targeting and improved therapeutic outcomes while achieving up to 90% efficiency compared with common/trivial delivery methods.},
}

@article {pmid40704023,
year = {2025},
author = {Niu, M and Dong, Z and Yu, L and Dong, X and An, J and Han, Y and Yan, Y and Yi, W and Sun, Y and Li, H},
title = {Anti-RNA virus crRNA targets efficient screening platform based on bioinformatics and CRISPR detection.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {3},
pages = {102619},
pmid = {40704023},
issn = {2162-2531},
abstract = {The mutation and evolution of RNA viruses pose significant challenges in treatment efforts. The CRISPR-Cas system is a promising antiviral tool because of its powerful programmability. However, traditional cell screening methods for CRISPR targets are time-consuming, limiting their application. Here, we developed a rapid and efficient screening platform for crRNA targets by combining the CaSilico-based bioinformatics method with CRISPR in vitro detection technology. Using a bioinformational approach to design and screen crRNAs, the characteristics of crRNAs and the corresponding target sequences can be rapidly determined. CRISPR is used for secondary screening in vitro, enabling swift identification of the target site with optimal cleavage efficiency. This method significantly reduces the screening time for antiviral targets compared with traditional cell screening. We successfully designed and screened effective crRNA targeting SARS-CoV-2 conserved N gene regions and demonstrated its inhibition function in HEK 293T cells. We also designed and screened crRNAs targeting DENV to validate the feasibility of the platform. E-2330 crRNA reduced more than 90% of the DENV RNA load in multiple mammalian cell lines and effectively inhibited the replication of all four DENV serotypes. This study provides a new approach for screening antiviral crRNAs for antivirus research.},
}

@article {pmid40702552,
year = {2025},
author = {Vahabi, M and Xu, G and Sarkisjan, D and Hassouni, BE and Mantini, G and Donati, V and Wang, B and Lencioni, G and Honeywell, RJ and Deng, D and Strano, S and Peters, GJ and Blandino, G and Giovannetti, E},
title = {Unraveling resistance mechanisms to the novel nucleoside analog RX-3117 in lung cancer: insights into DNA repair, cell cycle dysregulation and targeting PKMYT1 for improved therapy.},
journal = {Journal of experimental & clinical cancer research : CR},
volume = {44},
number = {1},
pages = {217},
pmid = {40702552},
issn = {1756-9966},
support = {IG 24444//Fondazione AIRC per la ricerca sul cancro ETS/ ; IG 24444//Fondazione AIRC per la ricerca sul cancro ETS/ ; IG 24444//Fondazione AIRC per la ricerca sul cancro ETS/ ; IG 24444//Fondazione AIRC per la ricerca sul cancro ETS/ ; IG 24444//Fondazione AIRC per la ricerca sul cancro ETS/ ; IG 24444//Fondazione AIRC per la ricerca sul cancro ETS/ ; },
mesh = {Humans ; *Lung Neoplasms/drug therapy/genetics/pathology ; *Drug Resistance, Neoplasm ; *DNA Repair ; Cell Line, Tumor ; Cell Cycle/drug effects ; *Carcinoma, Non-Small-Cell Lung/drug therapy/genetics/pathology ; *Cytidine/analogs & derivatives/pharmacology ; *Nucleoside-Phosphate Kinase/antagonists & inhibitors ; CRISPR-Cas Systems ; *Nucleosides/pharmacology ; },
abstract = {BACKGROUND: Nucleoside analogues are crucial in treating non-small cell lung cancer (NSCLC), but resistance hampers patient outcomes. The cytidine analogue RX-3117 shows promise in gemcitabine-resistant cancers, yet mechanisms underlying acquired resistance to this drug remain unexplored. This study includes a comprehensive investigation into RX-3117 resistance mechanisms by leveraging new preclinical models and cutting-edge genomic tools, including a CRISPR-Cas9 knockout screen and transcriptomics.

METHODS: NSCLC cell lines A549 and SW1573 were exposed to stepwise increasing concentrations of RX-3117 to establish stable resistant subclones, confirmed by SRB and clonogenic assays. Intracellular RX-3117 nucleotide levels were measured via LC/MS-MS, prompting the evaluation and modulation of the expression of key metabolic enzymes by Western blot and siRNA. A CRISPR-Cas9 screen identified genes whose loss increased RX-3117 sensitivity, while RNA-sequencing with differential expression analyses revealed resistance-related pathways, further investigated through cell cycle distribution, knock-out, and ELISA assays.

RESULTS: Resistant clones exhibited decreased accumulation of RX-3117 nucleotides, which however, was not associated to reduced expression of activation enzymes (UCK2, UMPK, CMPK, NME1/NDPK, RR1 and RR2). Instead, increased expression was observed in certain DNA repair and deactivation enzymes (NT5C3) but pharmacological inhibition and silencing of the latter did not circumvent resistance. Remarkably, a comprehensive approach with CRISPR-Cas9 screen highlighted DNA-repair and cell cycle determinants as key sensitizing genes. XL-PCR and RNA-sequencing confirmed aberrations in DNA-repair and pathways involved in cell cycle regulation. Knock-out and pharmacological inhibition validated the role of PKMYT1, a protein kinase involved in G2/M transition and genomic stability. RX-3117-resistant A549 cells showed enhanced sensitivity to the PKMYT1 inhibitor lunresertib and its synergism with RX-3117, suggesting further studies, especially in patients with high PKMYT1 expression who have significantly shorter survival rates, as observed in public databases and validated in an internal cohort of NSCLC patients.

CONCLUSION: By integrating CRISPR-Cas9 with functional assays and transcriptomics, our study established a framework for decoding resistance mechanisms and highlights potential therapeutic strategies to enhance RX-3117 efficacy in NSCLC. We demonstrated for the first time that aberrant DNA repair and cell cycle dysregulation led resistance, identifying PKMYT1 as a promising target.},
}

Humans
*Lung Neoplasms/drug therapy/genetics/pathology
*Drug Resistance, Neoplasm
*DNA Repair
Cell Line, Tumor
Cell Cycle/drug effects
*Carcinoma, Non-Small-Cell Lung/drug therapy/genetics/pathology
*Cytidine/analogs & derivatives/pharmacology
*Nucleoside-Phosphate Kinase/antagonists & inhibitors
CRISPR-Cas Systems
*Nucleosides/pharmacology

@article {pmid40702188,
year = {2025},
author = {Baglaenko, Y and Mu, Z and Curtis, M and Mire, HM and Jayanthi, V and Al Suqri, M and Liu, C and Agnew, R and Nathan, A and Mah-Som, AY and Liu, DR and Newby, GA and Raychaudhuri, S},
title = {Precisely defining disease variant effects in CRISPR-edited single cells.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {40702188},
issn = {1476-4687},
abstract = {Genetic studies have identified thousands of individual disease-associated non-coding alleles, but the identification of the causal alleles and their functions remains a critical bottleneck[1]. CRISPR-Cas editing has enabled targeted modification of DNA to introduce and test disease alleles. However, the combination of inefficient editing, heterogeneous editing outcomes in individual cells and nonspecific transcriptional changes caused by editing and culturing conditions limits the ability to detect the functional consequences of disease alleles[2,3]. To overcome these challenges, we present a multi-omic single-cell sequencing approach that directly identifies genomic DNA edits, assays the transcriptome and measures cell-surface protein expression. We apply this approach to investigate the effects of gene disruption, deletions in regulatory regions, non-coding single-nucleotide polymorphism alleles and multiplexed editing. We identify the effects of individual single-nucleotide polymorphisms, including the state-specific effects of an IL2RA autoimmune variant in primary human T cells. Multimodal functional genomic single-cell assays, including DNA sequencing, enable the identification of causal variation in primary human cells and bridge a crucial gap in our understanding of complex human diseases.},
}

@article {pmid40617066,
year = {2025},
author = {Wang, L and He, X and Tian, T and Cheng, J and Cao, R and Hou, J and Lin, H and Li, Y},
title = {Functional characterization of CEL3C reveals its critical role in regulating cellulase gene expression in Trichoderma reesei Rut C30.},
journal = {Enzyme and microbial technology},
volume = {190},
number = {},
pages = {110706},
doi = {10.1016/j.enzmictec.2025.110706},
pmid = {40617066},
issn = {1879-0909},
mesh = {*Cellulase/genetics/metabolism ; *Gene Expression Regulation, Fungal ; *Fungal Proteins/genetics/metabolism ; *Hypocreales/genetics/enzymology ; *beta-Glucosidase/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {The nuclear-localized β-glucosidase CEL3C in Trichoderma reesei plays a pivotal role in cellulase regulation, though its mechanism remains poorly understood. To address this, we disrupted CEL3C in the hypercellulolytic strain T. reesei Rut C30 via CRISPR-Cas9 and evaluated cellulase production under sophorose-rich MGD induction. Deletion of CEL3C significantly enhanced total cellulase activity by 31.28 % (p < 0.05), with β-glucosidase, endoglucanase, and cellobiohydrolase activities increasing by 94.97 %, 19.40 %, and 28.99 %, respectively. These improvements were driven by transcriptional upregulation of core cellulase genes (CEL7A: 2.01-fold; CEL6A: 1.5-fold; CEL12A: 2.0-fold; CEL5A: 1.32-fold) and β-glucosidases (CEL3A: 6.41-fold; CEL3B: 5.02-fold), confirming transcriptional-level control as the dominant regulatory mechanism. Transcriptomic profiling identified 688 differentially expressed genes (399 upregulated, 299 downregulated), with key changes including activation of transcriptional activators XYR1 (59.6 % increase), ACE3 (75.49 % increase), and RXE1 (161.56 % increase), suppression of repressors RCE1 (65.86 % decrease) and RCE2 (65.23 % decrease), and induction of sugar transporters (TrireC30_133589: 13.41-fold) and ER chaperones (BIP1: 1.26-fold; PDI1: 1.55-fold). These alterations collectively enhanced inducer uptake, enzyme maturation, and secretion while alleviating MAPK-mediated repression (TMK2: 110.54 % decrease). Intracellular sugar profiling revealed that gentiobiose and cellobiose were undetectable in the T. reesei ΔCEL3C, whereas glucose and sophorose levels increased by 31.71 % and 13.45 % (p < 0.05), respectively. These results suggest that CEL3C deletion enhances β-glucosidase-mediated hydrolysis of disaccharides into glucose and possibly promotes sophorose formation via transglycosylation. In parallel, the upregulation of disaccharide transporters may facilitate sophorose uptake. Together, these two mechanisms contributed to the intracellular enrichment of sophorose, thereby amplifying cellulase gene induction and enzyme production. Our findings establish CEL3C as a dual-function nuclear regulator that balances cellulase synthesis through transcriptional and enzymatic pathways, providing actionable targets for engineering T. reesei with optimized industrial cellulase yields.},
}

*Cellulase/genetics/metabolism
*Gene Expression Regulation, Fungal
*Fungal Proteins/genetics/metabolism
*Hypocreales/genetics/enzymology
*beta-Glucosidase/genetics/metabolism
CRISPR-Cas Systems

@article {pmid40541708,
year = {2025},
author = {Yoon, DS and Byeon, E and Yun, SC and Jeong, H and Lee, JS and Sun, Y and Won, EJ and Park, HG and Yang, Z and Hagiwara, A and Lee, MC and Lee, JS},
title = {Effects on life parameters and fatty acid expression profiles in response to elevated temperatures in CRISPR/Cas9-mediated gene-targeted mutants of two desaturase (Δ9-3 and Δ5/6) genes in the water flea Daphnia magna.},
journal = {Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology},
volume = {279},
number = {},
pages = {111119},
doi = {10.1016/j.cbpb.2025.111119},
pmid = {40541708},
issn = {1879-1107},
mesh = {Animals ; *Daphnia/genetics/enzymology/metabolism/physiology ; *Fatty Acid Desaturases/genetics/metabolism ; *CRISPR-Cas Systems ; *Fatty Acids/metabolism/genetics ; *Mutation ; *Hot Temperature ; Daphnia magna ; },
abstract = {Fatty-acid desaturation is central to membrane homeostasis and thermal performance in ectotherms. We disrupted the Δ9-3 (MUFA-specific) and Δ5/6 (long-chain PUFA-specific) desaturase genes in Daphnia magna with CRISPR/Cas9 and compared wild-type and knockout lines at 23 °C and 28 °C. Loss of Δ9-3 substantially depleted monounsaturated fatty acids, especially oleic and palmitoleic acids, with a compensatory rise in total polyunsaturates. These lipid shifts coincided with enhanced growth and fecundity under benign temperature but translated into reduced reproductive output when heat stress was imposed, indicating a context-dependent trade-off. In contrast, deletion of Δ5/6 selectively impaired the n-6 pathway at high temperature, leading to pronounced reductions in total PUFA reserves and a severe decline in offspring production; the usual growth benefit of warming was also abolished. Together, the data reveal that Δ9-3 supports MUFA supply needed for baseline metabolism, whereas Δ5/6 maintains PUFA reserves critical for reproduction under thermal challenge. Divergent desaturase functions thus play distinct roles in lipid remodeling and contribute to climate resilience in freshwater zooplankton.},
}

Animals
*Daphnia/genetics/enzymology/metabolism/physiology
*Fatty Acid Desaturases/genetics/metabolism
*CRISPR-Cas Systems
*Fatty Acids/metabolism/genetics
*Mutation
*Hot Temperature
Daphnia magna

@article {pmid40476445,
year = {2025},
author = {Xu, R and Zhu, S and Zhang, W and Xu, H and Tu, C and Wang, H and Wang, L and He, N and Liu, T and Guo, X and Ren, X and Li, Z},
title = {A Dual Approach with Organoid and CRISPR Screening Reveals ERCC6 as a Determinant of Cisplatin Resistance in Osteosarcoma.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {28},
pages = {e2500632},
doi = {10.1002/advs.202500632},
pmid = {40476445},
issn = {2198-3844},
support = {82172500//National Natural Science Foundation of China/ ; 82272664//National Natural Science Foundation of China/ ; 82103228//National Natural Science Foundation of China/ ; CX20230372//Hunan Provincial Innovation Foundation for Postgraduate/ ; },
mesh = {*Cisplatin/pharmacology/therapeutic use ; *Osteosarcoma/genetics/drug therapy/metabolism/pathology ; Humans ; *Drug Resistance, Neoplasm/genetics ; Mice ; *Poly-ADP-Ribose Binding Proteins/genetics/metabolism ; Animals ; *Organoids/metabolism/drug effects ; *Bone Neoplasms/genetics/drug therapy/metabolism ; Cell Line, Tumor ; Antineoplastic Agents/pharmacology ; CRISPR-Cas Systems ; DNA Helicases ; DNA Repair Enzymes ; },
abstract = {Osteosarcoma (OS), the most prevalent primary bone malignancy in adolescents, is typically treated with cisplatin-based chemotherapy. However, the development of cisplatin resistance often leads to relapse or metastasis, significantly impairing therapeutic efficacy. To tackle this issue, patient-derived osteosarcoma organoids (OSOs) is established that accurately reflect the cellular composition and heterogeneity of the original tumors, as validated by single-cell RNA sequencing, bulk RNA sequencing, and histopathology analysis. Cisplatin resistance is successfully induced in these OSOs, creating a clinically relevant model for investigating chemoresistance. Utilizing RNA sequencing in cisplatin-resistance OSOs and CRISPR screening in OS cell line, ERCC6 is identified as a pivotal regulator of cisplatin resistance. Knockdown of ERCC6 markedly enhanced cisplatin sensitivity in vitro and in vivo. Mechanistically, ERCC6 interacts with HNRNPM, influencing the PI3K/AKT signaling pathway and alternative splicing of pre-mRNA for BAX. Notably, the knockdown of ERCC6 and HNRNPM increased expression of full-length BAX and reduced skipping of exon 2, thus promoting apoptosis. This exon skipping in BAX results in a frameshift and introduces a premature stop codon (TGA) within the BH3 domain. These findings underscore the utility of OSOs in elucidating resistance mechanisms and highlight ERCC6 and HNRNPM as potential therapeutic targets.},
}

*Cisplatin/pharmacology/therapeutic use
*Osteosarcoma/genetics/drug therapy/metabolism/pathology
Humans
*Drug Resistance, Neoplasm/genetics
Mice
*Poly-ADP-Ribose Binding Proteins/genetics/metabolism
Animals
*Organoids/metabolism/drug effects
*Bone Neoplasms/genetics/drug therapy/metabolism
Cell Line, Tumor
Antineoplastic Agents/pharmacology
CRISPR-Cas Systems
DNA Helicases
DNA Repair Enzymes

@article {pmid40701722,
year = {2025},
author = {Long, K and Han, T and Hu, W and Yang, M and Huo, D and Huang, Z and Hou, C},
title = {Double special bodyguard: Selenium-enhanced loop-mediated isothermal amplification combined with CRISPR/Cas12a biosensing strategy.},
journal = {Analytica chimica acta},
volume = {1369},
number = {},
pages = {344353},
doi = {10.1016/j.aca.2025.344353},
pmid = {40701722},
issn = {1873-4324},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Selenium/chemistry ; Humans ; *DNA, Viral/analysis/genetics ; Limit of Detection ; Papillomaviridae/genetics/isolation & purification ; Molecular Diagnostic Techniques ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Cervical cancer is closely associated with human papillomavirus (HPV) infection, underscoring the necessity for effective HPV detection. Various biosensors utilizing isothermal amplification techniques have been developed for HPV DNA detection. These methods offer high sensitivity and specificity, making them suitable for point-of-care testing. However, the in vitro DNA polymerases used in these isothermal amplification methods lack a repair system, may leading to polymerization errors. This limitation highlights the need for improved detection methods that can enhance accuracy and reliability in HPV testing. Here, a sensing strategy called selenium nucleic acid-enhanced loop-mediated isothermal amplification (Se-LAMP) combined with CRISPR/Cas12a (SLC) was developed. The SLC has two specific bodyguards, which ensure the accuracy and specificity of biosensors through two aspects. One bodyguard is dNTPαSe, a supplement added to the initial Se-LAMP step, which guarantees the accuracy of DNA polymerase and reduces background signals. The other bodyguard is CRISPR system, integrating three functions of specific verification, signal amplification and signal output. CRISPR activation can only rely on Se-LAMP products successfully rechecked by crRNA. As a readable signal of SLC, the detection limit was as low as 0.38 copies/μL (0.64 fM) within 65 min, which was one order of magnitude lower than that of non-selenium-modified methods and two orders of magnitude lower than that of qPCR. The SLC is the first to use CRISPR system as the output mode of Se-LAMP, proving the CRISPR/Cas12a system has good recognition ability for selenium-modified nucleic acid. The ultrasensitive dual-specificity guard enables SLC, ensuring accuracy in biodetection. This innovation offers novel clinical testing approaches and holds significance for diagnosing and monitoring disease progression.},
}

*Biosensing Techniques/methods
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
*Selenium/chemistry
Humans
*DNA, Viral/analysis/genetics
Limit of Detection
Papillomaviridae/genetics/isolation & purification
Molecular Diagnostic Techniques
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40699345,
year = {2025},
author = {Schöllkopf, AI and Ehrenreich, A and Liebl, W},
title = {SMC-like Wadjet system prevents plasmid transfer into Clostridium cellulovorans.},
journal = {Applied microbiology and biotechnology},
volume = {109},
number = {1},
pages = {170},
pmid = {40699345},
issn = {1432-0614},
support = {161B0930//German Federal Ministry of Education and Research/ ; 161B0930//German Federal Ministry of Education and Research/ ; 161B0930//German Federal Ministry of Education and Research/ ; },
mesh = {*Plasmids/genetics ; *Conjugation, Genetic ; *Gene Transfer, Horizontal ; *Clostridium cellulovorans/genetics ; *Chromosomes, Bacterial/genetics ; },
abstract = {This study demonstrates the impact of a Structure Maintenance of Chromosome (SMC)-like Wadjet system on the horizontal gene transfer of plasmids by conjugation to a recipient that naturally containing such a system for the first time. A Clostridium cellulovorans mutant with dramatically improved efficiency to receive plasmid DNA by conjugation was isolated and sequenced. Three spontaneous chromosomal deletions included a type II restriction-modification system, a putative CRISPR system, and a cluster of ORFs named jetABCD encoding a putative Wadjet system. Since nearly nothing is known about the role of naturally occurring Wadjet systems in their native host bacteria, markerless chromosomal deletion of jetABCD in the C. cellulovorans wildtype strain 743B was achieved and the effect on conjugative plasmid uptake was studied. The transconjugation frequency of the jetABCD mutant was increased by about five orders of magnitude compared to wildtype C. cellulovorans recipient cells. Bioinformatic analysis of genome sequences of the Bacillota phylum revealed near-complete mutually exclusive possession of either plasmids < 40 kb or jetABCD genes, indicating high efficiency of Wadjet systems in small plasmid prevention in bacteria. Importantly, the implications of this study go beyond the case of C. cellulovorans. Our study demonstrates that the eradication of Wadjet systems can dramatically improve the uptake of recombinant plasmids and thereby enhance genetic engineering of bacterial strains of interest for biotechnological applications. KEY POINTS: • Native Wadjet system inhibits plasmid transfer by conjugation in C. cellulovorans • Deleting jetABCD increased plasmid uptake by about five orders of magnitude • Possession of Wadjet systems efficiently block plasmid maintenance in Bacillota.},
}

*Plasmids/genetics
*Conjugation, Genetic
*Gene Transfer, Horizontal
*Clostridium cellulovorans/genetics
*Chromosomes, Bacterial/genetics

@article {pmid40698645,
year = {2025},
author = {Shah, KM and Crozier, AFF and Assaraf, A and Arya, M and Grevitt, P and Mardakheh, F and Plevin, MJ and Sharp, TV},
title = {C-terminal tagging impairs AGO2 function.},
journal = {RNA biology},
volume = {22},
number = {1},
pages = {1-24},
doi = {10.1080/15476286.2025.2534028},
pmid = {40698645},
issn = {1555-8584},
mesh = {*Argonaute Proteins/metabolism/genetics/chemistry ; Humans ; CRISPR-Cas Systems ; A549 Cells ; Protein Binding ; Autoantigens/metabolism/genetics ; DNA-Binding Proteins/metabolism/genetics ; RNA Interference ; MicroRNAs/genetics/metabolism ; Recombinant Fusion Proteins/genetics/metabolism ; Animals ; RNA-Binding Proteins ; },
abstract = {MicroRNA-mediated gene silencing is a conserved mechanism of post-transcriptional gene regulation across metazoans. It depends on base pairing between small RNAs and mRNAs, and on protein complexes including the RNA-induced silencing complex (RISC), where Argonaute 2 (AGO2) plays a central role. A full understanding of RNA silencing requires reliable molecular tools to study AGO2 and RISC. Affinity tagging and antibody-based methods can introduce artefacts, and both the N- and C-terminal domains of AGO2 are critical for its function. While N-terminal tags are frequently used, and a recent study in mice showed altered activity in N-terminal HaloTag-AGO2 fusions, the consequences of C-terminal tagging remain underexplored. CRISPaint, a CRISPR-Cas9-based technique, enables endogenous C-terminal tag fusions without requiring homology arms. Using this system, we generated the first C-terminal HaloTag fusion of AGO2 (AGO2HALO) in human A549 cells. We found that the AGO2HALO fusion protein exhibits reduced binding with TNRC6A, with no effect on cell viability. However, it significantly impairs RNA cleavage, silencing activity, and nuclear localization. We further compared AGO2-EGFP and EGFP-AGO2 using transient transfection. N-terminally tagged AGO2 retained wild-type-like function and localization, while C-terminally tagged AGO2 was impaired in siRNA and miRNA silencing, nuclear import, and P-body localization. These results demonstrate that a C-terminal HaloTag compromises AGO2 functionality and is unsuitable for studying RISC biology. Our findings highlight the importance of validating tagging strategies to avoid misleading conclusions due to tag-induced functional defects. Pre-print, bioRxiv.},
}

*Argonaute Proteins/metabolism/genetics/chemistry
Humans
CRISPR-Cas Systems
A549 Cells
Protein Binding
Autoantigens/metabolism/genetics
DNA-Binding Proteins/metabolism/genetics
RNA Interference
MicroRNAs/genetics/metabolism
Recombinant Fusion Proteins/genetics/metabolism
Animals
RNA-Binding Proteins

@article {pmid40698183,
year = {2025},
author = {Sailer, AL and Wörtz, J and Smith, V and Stachler, AE and Blau, F and Daratha, M and Maier, LK and Allers, T and Marchfelder, A},
title = {CRISPR-Cas induced self-targeting identifies key players in archaeal microhomology-mediated end joining.},
journal = {microLife},
volume = {6},
number = {},
pages = {uqaf015},
pmid = {40698183},
issn = {2633-6693},
abstract = {DNA repair processes are the foundation for genome integrity and survival, especially in extreme environments where DNA damage occurs more frequently and where archaea are found. Nevertheless, first-hand experimental information on repair pathways in archaea is scarce, and assignment of repair proteins is currently largely based on homology. We showed previously that DNA lesions induced by clustered regularly interspaced short palindromic repeats Cas (CRISPR-Cas) self-targeting are repaired by microhomology-mediated end joining (MMEJ). To identify proteins involved in the archaeal MMEJ pathway, we used deletion strains devoid of proteins assigned to the key steps of MMEJ, to examine changes in the repair outcome. In addition, we used aphidicolin to inhibit the activity of the essential PolB1 protein. For the first time, we were thereby able to experimentally identify proteins involved in this repair pathway in the euryarchaeal model organism Haloferax volcanii. This study confirms that Mre11, Rad50, Fen1, PolB1, LigA, and LigN take part in MMEJ, as previously inferred. In addition, we show that Cas1 and Hel308a are also involved in the MMEJ pathway.},
}

@article {pmid40697666,
year = {2025},
author = {Han, H and Sun, X and Guo, X and Wen, J and Zhao, X and Zhou, W},
title = {CRISPR/Cas9 technology in tumor research and drug development application progress and future prospects.},
journal = {Frontiers in pharmacology},
volume = {16},
number = {},
pages = {1552741},
pmid = {40697666},
issn = {1663-9812},
abstract = {The CRISPR/Cas9 system is an acquired immune defense mechanism that has evolved in bacteria and archaea to protect against viral and plasmid attacks. It consists of regularly spaced clusters of short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas). By adapting the simplest type II CRISPR system to utilize special small guide RNA (sgRNA) and Cas9 nucleic acid endonuclease, precise cuts can be made at specific locations in double-stranded DNA, facilitating gene knockout or knock-in. Due to its efficient gene editing capabilities, CRISPR/Cas9 technology has been widely adopted across various biological and scientific research fields, demonstrating significant potential in tumor research and drug development. This article reviews the progress and future prospects of CRISPR/Cas9 technology in tumor genome editing, drug target screening and validation, and new drug development. It details the fundamental role of this technology in cancer biology research, encompassing various aspects such as gene transcription editors, epigenetic editors, precision genome engineering, and CRISPR-Cas systems targeting RNA. Additionally, the article discusses key applications of CRISPR/Cas9 in anticancer drug discovery, including drug target identification, drug target screening and validation, combinatorial genetic screening, screening of small molecules to overcome resistance to CAR-T therapies, and multimodal functional genomics integration strategies. Finally, although CRISPR/Cas9 has demonstrated great potential for efficient gene editing, precise target discovery, and promotion of personalized therapy and drug screening in oncology research, its application still faces technical bottlenecks such as off-target effects, genomic instability, and low editing efficiency in solid tumors, as well as ethical controversies in gene editing, safety assessment of delivery systems and immune responses in clinical translation, and other ethical and translational challenges.},
}

@article {pmid40695321,
year = {2025},
author = {Ara, PS and Casacuberta, E and Scazzocchio, C and Ruiz-Trillo, I and Najle, SR},
title = {CRISPR-Cas9 genome editing in Corallochytrium limacisporum,a key species for understanding animal origins.},
journal = {Open biology},
volume = {15},
number = {7},
pages = {250066},
doi = {10.1098/rsob.250066},
pmid = {40695321},
issn = {2046-2441},
support = {4973.01//Moore Foundation - MMI Experimental Model Systems/ ; PID2023-153273NB-I00//MICIU/ AEI /10.13039/501100011033/ ; "ERDF A way of making Europe", PID2023-153273NB-I00//MICIU/AEI/10.13039/501100011033/ ; exp. 2021 SGR 00751//Departament de Recerca i Universitats de la Generalitat de Catalunya/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Animals ; Point Mutation ; },
abstract = {Microbial holozoans are the closest unicellular relatives of animals. They share a substantial gene repertoire with animals and exhibit complex life cycles. Studying these organisms is crucial for understanding the evolution of multicellularity, and significant progress has been made in uncovering key aspects of the biology of the four microbial holozoans lineages: choanoflagellates, filastereans, ichthyosporeans and corallochytreans. However, reverse genetic tools are still lacking in corallochytreans, one of the earliest-branching holozoan lineages and the only known group with both coenocytic and binary fission development. Here, we present CRISPR-Cas9-mediated gene inactivation and point mutation methodologies in the corallochytrean Corallochytrium limacisporum. As a proof of concept, we inactivated the fkb12 gene, a component of the mTOR pathway, conferring rapamycin resistance, and introduced a point mutation in sdhB, encoding a subunit of succinate dehydrogenase, conferring carboxin resistance. Our results demonstrate the presence of both non-homologous end-joining and homology-directed repair pathways in C. limacisporum and shows an editing efficiency of approximately 2%. Furthermore, simultaneous gene targeting revealed a co-editing frequency of approximately 20%. Finally, this study establishes unequivocally that C. limacisporum is haploid, making it an ideal model for genetic studies and gene editing applications to unravel the molecular mechanisms involved in animal origins.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
Animals
Point Mutation

@article {pmid40695225,
year = {2025},
author = {Birocchi, F and Maus, MV},
title = {Cas12a knockin mice expand the toolbox for in vivo and ex vivo multiplexed genome editing.},
journal = {Developmental cell},
volume = {60},
number = {14},
pages = {1931-1933},
doi = {10.1016/j.devcel.2025.05.012},
pmid = {40695225},
issn = {1878-1551},
mesh = {Animals ; *Gene Editing/methods ; Mice ; *Gene Knock-In Techniques/methods ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; Mice, Transgenic ; Bacterial Proteins ; },
abstract = {Multiplex genome editing is critical for advancing transformative gene therapies and unraveling complex genetic interactions. In a recent issue of Nature Biomedical Engineering, Tang et al. introduced Cas12a knockin mice, providing a versatile platform for disease modeling and the development of multiplexed therapeutic strategies.},
}

Animals
*Gene Editing/methods
Mice
*Gene Knock-In Techniques/methods
*CRISPR-Cas Systems/genetics
*CRISPR-Associated Proteins/genetics/metabolism
*Endodeoxyribonucleases/genetics/metabolism
Mice, Transgenic
Bacterial Proteins

@article {pmid40694851,
year = {2025},
author = {Song, G and Li, J and Han, J and Gao, X and Tian, C and Zhang, F and Tian, Y},
title = {Identification of regulatory sequences in Aca11 and Aca13 for detection of anti-CRISPR and protein-protein interaction.},
journal = {Nucleic acids research},
volume = {53},
number = {14},
pages = {},
doi = {10.1093/nar/gkaf694},
pmid = {40694851},
issn = {1362-4962},
support = {2022YFF0710700//National Key R&D Program of China/ ; 2024YFF0728700//National Key R&D Program of China/ ; 2021YFF0702800//National Key R&D Program of China/ ; 2020YFA0803501//National Key R&D Program of China/ ; 32270567//National Natural Science Foundation of China/ ; 32070533//National Natural Science Foundation of China/ ; KFJ-BRP-005//Biological Resource Program of Chinese Academy of Sciences/ ; },
mesh = {Humans ; *Bacterial Proteins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic ; CRISPR-Associated Protein 9/metabolism/genetics ; Operon ; *CRISPR-Associated Proteins/genetics/metabolism ; Streptococcus/genetics ; *Regulatory Sequences, Nucleic Acid ; Protein Binding ; },
abstract = {Anti-CRISPR (Acr) proteins are frequently co-encoded with the anti-CRISPR associated (Aca) proteins, which act as repressors for regulating Acr expression within acr-aca operons. We previously identified three aca genes (aca11-13) from Streptococcus mobile genetic elements, but their regulatory mechanisms remained unclear. Here, we showed that Aca11 and Aca13 mediate bidirectional regulation in acr-aca operons through recognition of their inverted repeat (IR) sequences within the acr promoters. Based on the bioinformatics search using Aca13 with its IR sequences, we discovered a novel type II-A Acr (named AcrIIA35). AcrIIA35 exhibits a potent inhibitory activity against St1Cas9 by interfering with DNA recognition of Cas9 in bacterial and human cells. We also developed a novel Aca-driven protein-protein interaction detection (APID) system by integrating Aca-tagged target proteins with fluorescently labeled IR-DNA probes. The APID system enables efficient detection of protein-protein interaction using proteins or crude cell lysates. Utilizing the APID system, we have further elucidated the mechanism of AcrIIA24, which can interact with the HNH nuclease domain of St3Cas9 to inhibit the DNA cleavage activity of Cas9. Collectively, our work expands the understanding of Aca functions to modulate Acrs and expands the potential for Aca-based applications in CRISPR technologies.},
}

Humans
*Bacterial Proteins/genetics/metabolism
*CRISPR-Cas Systems/genetics
Promoter Regions, Genetic
CRISPR-Associated Protein 9/metabolism/genetics
Operon
*CRISPR-Associated Proteins/genetics/metabolism
Streptococcus/genetics
*Regulatory Sequences, Nucleic Acid
Protein Binding

@article {pmid40694850,
year = {2025},
author = {Eleveld, TF and Ylstra, B and Looijenga, LHJ},
title = {Low-amplitude copy number gains shape cancer through known and novel oncogenes with associated therapeutic vulnerabilities.},
journal = {Nucleic acids research},
volume = {53},
number = {14},
pages = {},
doi = {10.1093/nar/gkaf689},
pmid = {40694850},
issn = {1362-4962},
support = {KWF 20161-1-10271//Dutch Cancer Society/ ; //KIKA/ ; },
mesh = {Humans ; *Oncogenes/genetics ; *Neoplasms/genetics/drug therapy ; *DNA Copy Number Variations ; Drug Resistance, Neoplasm/genetics ; *Gene Dosage ; Biomarkers, Tumor/genetics ; CRISPR-Cas Systems ; },
abstract = {Large chromosomal copy number gains are ubiquitous throughout cancer types. However, which genes drive their selective advantage is not well established, and therefore they are hardly utilized in clinical practice. Our analysis of copy number patterns in pan-cancer datasets suggests that the selective advantage of copy number gains is largely driven by known oncogenes. Analysis of CRISPR screening data identifies a list of 101 genes that are likely to mediate the effect of these gains, which is highly enriched in annotated oncogenes but also contains genes that have not been implicated in cancer so far. Moreover, we show that specific gains are associated with drug sensitivity or resistance, with a strong enrichment of gains of oncogenes with increased sensitivity to inhibitors targeting these specific genes. Finally, we provide examples where gains can function as relevant clinical biomarkers for diagnosis and treatment. Thus, large copy number gains exert their selective advantage through known and novel oncogenes, and their systematic analysis could advance precision oncology.},
}

Humans
*Oncogenes/genetics
*Neoplasms/genetics/drug therapy
*DNA Copy Number Variations
Drug Resistance, Neoplasm/genetics
*Gene Dosage
Biomarkers, Tumor/genetics
CRISPR-Cas Systems

@article {pmid40693713,
year = {2025},
author = {Piccolo, D and Sladen, P and Guarascio, R and Ziaka, K and Cheetham, ME},
title = {Investigation of ABCA4 Missense Variants and Potential Small Molecule Rescue in Retinal Organoids.},
journal = {Investigative ophthalmology & visual science},
volume = {66},
number = {9},
pages = {58},
doi = {10.1167/iovs.66.9.58},
pmid = {40693713},
issn = {1552-5783},
mesh = {Humans ; *Mutation, Missense ; *Organoids/metabolism/drug effects ; *ATP-Binding Cassette Transporters/genetics/metabolism ; Induced Pluripotent Stem Cells/metabolism ; *Retinal Diseases/genetics/metabolism/pathology ; *Retina/metabolism ; CRISPR-Cas Systems ; Cells, Cultured ; Cell Differentiation ; },
abstract = {PURPOSE: ABCA4-related retinopathy is the most common monogenic eye disorder in the world and is currently untreatable. Missense variants in ABCA4 constitute ∼60% of causal ABCA4-related retinopathy variants, often resulting in misfolded or dysfunctional protein products. Despite their prevalence, the molecular mechanisms by which these missense mutations impair ABCA4 function are not fully understood, primarily due to limitations in suitable cellular models. In this study, we investigated the cellular and molecular consequences of ABCA4 missense variants using a human photoreceptor-like model system.

METHODS: We used CRISPR/Cas9 technology to introduce two ABCA4 missense misfolding variants, T983A and R2077W, which are associated with ABCA4-associated retinopathy, into control induced pluripotent stem cells (iPSCs). The iPSCs were differentiated into retinal organoids, characterized and treated with small molecules.

RESULTS: The expression level of ABCA4 missense proteins was reduced compared to WT ABCA4 suggesting the variants were degraded in a photoreceptor-like environment. The localization of the missense variants was also altered with negligible ABCA4 detectable in the retinal organoid outer segments compared to the isogenic control. Two small molecule compounds, AICAR and 4-PBA, previously identified as potential ABCA4 folding correctors in vitro, were tested for their ability to enhance ABCA4 traffic to the outer segment. The compounds did not appear to promote ABCA4 folding and traffic in photoreceptors and instead led to a decrease in ABCA4 transcript levels and protein.

CONCLUSIONS: These data highlight that retinal organoids are an exquisite model to investigate pathogenic variants in ABCA4 and test small compounds for translation to the human retina.},
}

Humans
*Mutation, Missense
*Organoids/metabolism/drug effects
*ATP-Binding Cassette Transporters/genetics/metabolism
Induced Pluripotent Stem Cells/metabolism
*Retinal Diseases/genetics/metabolism/pathology
*Retina/metabolism
CRISPR-Cas Systems
Cells, Cultured
Cell Differentiation

@article {pmid40690290,
year = {2025},
author = {Huang, J and Zhao, X and Zang, X and Jin, Z and Zhang, X and Huang, Y and Zhang, L and Xue, X and Zhang, P},
title = {Identification of ATM1 gene involved in antifungal resistance based on CRISPR/Cas9 technology in Cryptococcus gattii.},
journal = {Medical mycology},
volume = {63},
number = {7},
pages = {},
doi = {10.1093/mmy/myaf061},
pmid = {40690290},
issn = {1460-2709},
support = {2021YFC2302100//National Key Research and Development Program of China/ ; 31900130//National Foundation of Science of China/ ; },
mesh = {*Cryptococcus gattii/genetics/drug effects/growth & development/pathogenicity ; *CRISPR-Cas Systems ; *Drug Resistance, Fungal/genetics ; *Antifungal Agents/pharmacology ; Cryptococcosis/microbiology ; Animals ; *ATP-Binding Cassette Transporters/genetics/metabolism ; Mice ; *Fungal Proteins/genetics/metabolism ; Virulence ; Humans ; },
abstract = {Cryptococcus gattii is a fungal pathogen that poses significant threats to human health, affecting both immunocompromised and immunocompetent individuals. Treatment of C. gattii infections typically involves the use of antifungal agents, such as azoles. However, the increasing emergence of antifungal resistance in C. gattii is a growing concern, highlighting the critical need for novel therapeutic strategies. In our previous study, we identified a mitochondrial ATP-binding cassette (ABC) transporter, Atm1, as potentially involved in antifungal resistance in C. gattii through transcriptome sequencing, but its function remains unclear and requires additional confirmation and investigation. In this study, we developed a "suicide" clustered regularlyinterspaced short palindromic repeats-CRISPR-associated protein 9 system in C. gattii, based on the system used in C. neoformans, and successfully validated its functionality by targeting the ADE2 gene. We subsequently generated C. gattii mutants lacking ATM1 and assessed their growth under various stress conditions. Our data suggest that Atm1 is involved in the iron-sulfur cluster biosynthesis process. Besides, disruption of ATM1 resulted in various growth impairments, including reduced stress tolerance, impaired capsule formation, and diminished virulence. Importantly, we observed compromised antifungal drug resistance in the atm1∆ mutant and performed RNA sequencing-based transcriptome analysis and gene ontology analysis with and without antifungal treatment for further investigation. In conclusion, our findings indicate that ATM1 plays a role in iron homeostasis and is critical for antifungal resistance in C. gattii, offering new insights into potential drug development strategies for the clinical treatment of cryptococcosis.},
}

*Cryptococcus gattii/genetics/drug effects/growth & development/pathogenicity
*CRISPR-Cas Systems
*Drug Resistance, Fungal/genetics
*Antifungal Agents/pharmacology
Cryptococcosis/microbiology
Animals
*ATP-Binding Cassette Transporters/genetics/metabolism
Mice
*Fungal Proteins/genetics/metabolism
Virulence
Humans

@article {pmid40674406,
year = {2025},
author = {Dinesh, RK and Wang, C and Qu, Y and Rustagi, A and Cousins, H and Zengel, J and Wang, X and Barnard, TR and Johnson, WA and Xu, G and Zhang, T and Magazine, N and Beck, A and Heilbroner, LM and Peters-Schulze, G and Wilk, AJ and Wang, M and Huang, W and Howitt, BE and Carette, J and Altman, R and Blish, CA and Cong, L},
title = {Membrane-wide screening identifies potential tissue-specific determinants of SARS-CoV-2 tropism.},
journal = {PLoS pathogens},
volume = {21},
number = {7},
pages = {e1013157},
doi = {10.1371/journal.ppat.1013157},
pmid = {40674406},
issn = {1553-7374},
mesh = {Humans ; *SARS-CoV-2/physiology ; *COVID-19/virology/metabolism ; *Virus Internalization ; *Viral Tropism/physiology ; Angiotensin-Converting Enzyme 2/metabolism/genetics ; HEK293 Cells ; Organ Specificity ; CRISPR-Cas Systems ; Antigens, Differentiation ; },
abstract = {While SARS-CoV-2 primarily infects the respiratory tract, clinical evidence indicates that cells from diverse cell types and organs are also susceptible to infection. Using the CRISPR activation (CRISPRa) approach, we systematically targeted human membrane proteins in cells with and without overexpression of ACE2, thus identifying unrecognized host factors that may facilitate viral entry. Validation experiments with replication-competent SARS-CoV-2 confirmed the role of newly identified host factors, particularly the endo-lysosomal protease legumain (LGMN) and the potassium channel KCNA6, upon exogenous overexpression. In orthogonal experiments, we show that disruption of endogenous LGMN or KCNA6 decreases viral infection and that inhibitors of candidate factors can reduce viral entry. Additionally, using clinical data, we find possible associations between expression of either LGMN or KCNA6 and SARS-CoV-2 infection in human tissues. Our results identify potentially druggable host factors involved in SARS-CoV-2 entry, and demonstrate the utility of focused, membrane-wide CRISPRa screens in uncovering tissue-specific entry factors of emerging pathogens.},
}

Humans
*SARS-CoV-2/physiology
*COVID-19/virology/metabolism
*Virus Internalization
*Viral Tropism/physiology
Angiotensin-Converting Enzyme 2/metabolism/genetics
HEK293 Cells
Organ Specificity
CRISPR-Cas Systems
Antigens, Differentiation

@article {pmid40639395,
year = {2025},
author = {Ge, Y and Zheng, W and Hou, Z and Zhang, Y and Du, B and Wei, S and Liu, X and Chen, Z and Zhang, H},
title = {Ultra-sensitive detection of microRNA in intraocular fluid using optical fiber sensing technology for central nervous system lymphoma diagnosis.},
journal = {Reports on progress in physics. Physical Society (Great Britain)},
volume = {88},
number = {7},
pages = {},
doi = {10.1088/1361-6633/adee44},
pmid = {40639395},
issn = {1361-6633},
mesh = {*MicroRNAs/analysis/genetics/metabolism ; Humans ; *Lymphoma/diagnosis/genetics ; *Optical Fibers ; *Central Nervous System Neoplasms/diagnosis/genetics ; Limit of Detection ; *Aqueous Humor/metabolism ; *Fiber Optic Technology ; Surface Plasmon Resonance ; *Biosensing Techniques ; CRISPR-Cas Systems ; },
abstract = {MicroRNA (miRNA) in aqueous humor holds significant promise as a non-invasive biomarker of primary central nervous system lymphoma (PCNSL), enabling early diagnosis and prognosis. However, current methods for miRNA detection often suffer from limitations affecting sensitivity, specificity, and clinical applicability. This study introduces a novel black phosphorus (BP)-enhanced fiber-optic surface plasmon resonance sensor integrated with a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a system for ultrasensitive and single-base-specific detection of PCNSL-associated miRNA in aqueous humor. The BP nano-interface significantly enhances the surface plasmon resonance signal, while the CRISPR-Cas13a technology enables highly specific detection of miRNA, down to single nucleotide mismatches. This system achieves a detection limit as low as 21 aM without the need for amplification and demonstrates robust performance in analyzing clinical samples. With its unparalleled sensitivity, specificity, label-free operation, and potential for portability, this biosensing platform offers transformative capabilities for early PCNSL diagnosis, prognosis, and treatment monitoring.},
}

*MicroRNAs/analysis/genetics/metabolism
Humans
*Lymphoma/diagnosis/genetics
*Optical Fibers
*Central Nervous System Neoplasms/diagnosis/genetics
Limit of Detection
*Aqueous Humor/metabolism
*Fiber Optic Technology
Surface Plasmon Resonance
*Biosensing Techniques
CRISPR-Cas Systems

@article {pmid40615675,
year = {2025},
author = {Chan, PY and Alexander, D and Mehta, I and Matsuyama, LSAS and Harle, V and Olvera-León, R and Park, JS and Arriaga-González, FG and van der Weyden, L and Cheema, S and Iyer, V and Offord, V and Barneda, D and Hawkins, PT and Stephens, L and Kozik, Z and Woods, M and Wong, K and Balmus, G and Vinceti, A and Thompson, NA and Del Castillo Velasco-Herrera, M and Wessels, L and van de Haar, J and Gonçalves, E and Sinha, S and Vázquez-Cruz, ME and Bisceglia, L and Raimondi, F and Choudhary, J and Patiyal, S and Venkatesh, A and Iorio, F and Ryan, CJ and Adams, DJ},
title = {The synthetic lethal interaction between CDS1 and CDS2 is a vulnerability in uveal melanoma and across multiple tumor types.},
journal = {Nature genetics},
volume = {57},
number = {7},
pages = {1672-1683},
pmid = {40615675},
issn = {1546-1718},
support = {220540/Z/20/A//Wellcome Trust (Wellcome)/ ; 1/CX/CSRD VA/United States ; 1/CX/CSRD VA/United States ; 1/CX/CSRD VA/United States ; 1/CX/CSRD VA/United States ; 1/CX/CSRD VA/United States ; 1/CX/CSRD VA/United States ; },
mesh = {Humans ; *Melanoma/genetics/pathology ; *Uveal Neoplasms/genetics/pathology ; Uveal Melanoma ; Cell Line, Tumor ; CRISPR-Cas Systems/genetics ; Apoptosis/genetics ; Gene Expression Regulation, Neoplastic ; *Synthetic Lethal Mutations/genetics ; Animals ; },
abstract = {Metastatic uveal melanoma is an aggressive disease with limited effective therapeutic options. To comprehensively map monogenic and digenic dependencies, we performed CRISPR-Cas9 screening in ten extensively profiled human uveal melanoma cell line models. Analysis involved genome-wide single-gene and combinatorial paired-gene CRISPR libraries. Among our 76 uveal melanoma-specific essential genes and 105 synthetic lethal gene pairs, we identified and validated the CDP-diacylglycerol synthase 2 gene (CDS2) as a genetic dependency in the context of low CDP-diacylglycerol synthase 1 gene (CDS1) expression. We further demonstrate that CDS1/CDS2 forms a synthetic lethal interaction in vivo and reveal that CDS2 knockout results in the disruption of phosphoinositide synthesis and increased cellular apoptosis and that re-expression of CDS1 rescues this cell fitness defect. We extend our analysis using pan-cancer data, confirming increased CDS2 essentiality in diverse tumor types with low CDS1 expression. Thus, the CDS1/CDS2 axis is a therapeutic target across a range of cancers.},
}

Humans
*Melanoma/genetics/pathology
*Uveal Neoplasms/genetics/pathology
Uveal Melanoma
Cell Line, Tumor
CRISPR-Cas Systems/genetics
Apoptosis/genetics
Gene Expression Regulation, Neoplastic
*Synthetic Lethal Mutations/genetics
Animals

@article {pmid40615674,
year = {2025},
author = {Arnoldus, T and van Vliet, A and Bleijerveld, OB and de Groot, AFH and Piao, Q and Blomberg, N and Schatton, D and Dong, J and van Hal-van Veen, SE and Harkes, R and Grootemaat, AE and Proost, N and Cabukusta, B and Frezza, C and van de Ven, M and van der Wel, NN and Giera, M and Altelaar, M and Peeper, DS},
title = {Cytidine diphosphate diacylglycerol synthase 2 is a synthetic lethal target in mesenchymal-like cancers.},
journal = {Nature genetics},
volume = {57},
number = {7},
pages = {1659-1671},
pmid = {40615674},
issn = {1546-1718},
mesh = {Humans ; *Synthetic Lethal Mutations/genetics ; *Neoplasms/genetics/pathology ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; Apoptosis/genetics ; },
abstract = {Synthetic lethal interactions (SLIs) based on genomic alterations in cancer have been therapeutically explored. We investigated the SLI space as a function of differential RNA expression in cancer and normal tissue. Computational analyses of functional genomic and gene expression resources uncovered a cancer-specific SLI between the paralogs cytidine diphosphate diacylglycerol synthase 1 (CDS1) and CDS2. The essentiality of CDS2 for cell survival is observed for mesenchymal-like cancers, which have low or absent CDS1 expression and account for roughly half of all cancers. Mechanistically, the CDS1-2 SLI is accompanied by disruption of lipid homeostasis, including accumulation of cholesterol esters and triglycerides, and apoptosis. Genome-wide CRISPR-Cas9 knockout screens in CDS1-negative cancer cells identify no common escape mechanism of death caused by CDS2 ablation, indicating the robustness of the SLI. Synthetic lethality is driven by CDS2 dosage and depends on catalytic activity. Thus, CDS2 may serve as a pharmacologically tractable target in mesenchymal-like cancers.},
}

Humans
*Synthetic Lethal Mutations/genetics
*Neoplasms/genetics/pathology
Cell Line, Tumor
Gene Expression Regulation, Neoplastic
CRISPR-Cas Systems
Apoptosis/genetics

@article {pmid40570838,
year = {2025},
author = {Wang, L and Zhou, Y and Yu, Z and Wu, P and Lu, Z and Ma, L},
title = {TAS-seq enables subcellular single-stranded adenosine profiling by signal peptide-assisted adenosine deamination.},
journal = {Cell reports methods},
volume = {5},
number = {7},
pages = {101087},
doi = {10.1016/j.crmeth.2025.101087},
pmid = {40570838},
issn = {2667-2375},
mesh = {*Adenosine/metabolism ; Deamination ; Humans ; *Protein Sorting Signals ; Single-Cell Analysis/methods ; *Sequence Analysis, RNA/methods ; RNA Stability ; Adenosine Deaminase/metabolism ; HEK293 Cells ; CRISPR-Cas Systems ; },
abstract = {RNA structure plays a crucial role in its function and undergoes dynamic changes throughout its life cycle. To study these dynamics, we developed TAS sequencing (TAS-seq), which expresses the deaminase TadA-8e in specific subcellular compartments to modify single-stranded adenosines, particularly within hairpin loops. We applied TAS-seq to the nucleus, cytosol, and endoplasmic reticulum membrane, identifying adenosine structural variations and compartment-specific regulation of RNA stability. Single-cell TAS-seq revealed structural heterogeneity of cytosolic RNAs. Additionally, adenosines labeled by TAS-seq contribute to guide RNA optimization in the CRISPR-Cas13d system. Our method provides insights into compartment-specific RNA structural dynamics, cell-specific heterogeneity, and their functional implications.},
}

*Adenosine/metabolism
Deamination
Humans
*Protein Sorting Signals
Single-Cell Analysis/methods
*Sequence Analysis, RNA/methods
RNA Stability
Adenosine Deaminase/metabolism
HEK293 Cells
CRISPR-Cas Systems

@article {pmid40543637,
year = {2025},
author = {Shanaka, KASN and Madushani, KP and Kim, MJ and Jung, S and Lee, J},
title = {Nlrc5 ablation interferes with MHC-I gene expression and immune cell migration.},
journal = {Developmental and comparative immunology},
volume = {169},
number = {},
pages = {105406},
doi = {10.1016/j.dci.2025.105406},
pmid = {40543637},
issn = {1879-0089},
mesh = {Animals ; *Zebrafish/immunology/genetics ; *Zebrafish Proteins/genetics/metabolism ; Cell Movement/genetics ; *Novirhabdovirus/immunology/physiology ; CRISPR-Cas Systems/genetics ; *Neutrophils/immunology ; *Fish Diseases/immunology ; Gene Knockout Techniques ; *Histocompatibility Antigens Class I/genetics ; Gene Expression Regulation ; Animals, Genetically Modified ; *Genes, MHC Class I/genetics ; },
abstract = {Nucleotide-binding domain and leucine-rich repeat-containing receptors (NLRs) have complex and diverse functions. Studies in mammalian models have indicated a clear role of NLRC5 in the regulation of MHC-I gene expression. The nlrc5 KO zebrafish model was generated using CRISPR/Cas9 technology to understand its function with respect to VHSV. The VHSV infection experiment demonstrated higher mortality in nlrc5 KO fish with higher VHSV copy numbers. Virus-infected pathology, such as tissue swelling, hemorrhage, and eye bulging, was significantly higher in the nlrc5 KO fish. Expressional analysis of ifn system genes indicated the significant upregulation of ifnφ1. Gene expression analysis during VHSV infection indicated a significant downregulation of MHC-I genes in the nlrc5 KO model compared to the wild-type (WT). Owing to the impact of MHC-I, the viral copy number may increase with higher mortality under VHSV infections. For neutrophil migration analysis, a nlrc5 KO and a neutrophil-labeled model were developed (nlrc5[-/-] Tg(mpx:mcherry)). Injury was generated in the caudal fin, and the injury site was stimulated with poly I: C. The number of neutrophils were reduced in the nlrc5 KO fish. Levels of critical cytokines responsible for neutrophil migration were significantly reduced in nlrc5 KO fish during VHSV infection. The data from the current study in zebrafish reconfirm the observations in mammalian models, and neutrophil migration analysis suggests that Nlrc5 may be associated with inflammatory activation in the presence of VHSV.},
}

Animals
*Zebrafish/immunology/genetics
*Zebrafish Proteins/genetics/metabolism
Cell Movement/genetics
*Novirhabdovirus/immunology/physiology
CRISPR-Cas Systems/genetics
*Neutrophils/immunology
*Fish Diseases/immunology
Gene Knockout Techniques
*Histocompatibility Antigens Class I/genetics
Gene Expression Regulation
Animals, Genetically Modified
*Genes, MHC Class I/genetics

@article {pmid40532693,
year = {2025},
author = {Modell, SM and Smith, JA and Kardia, SLR},
title = {Progress and Criteria in Public Health Applications of Gene Therapy and Gene Editing: Beyond the White Paper.},
journal = {Public health genomics},
volume = {28},
number = {1},
pages = {241-251},
doi = {10.1159/000546850},
pmid = {40532693},
issn = {1662-8063},
mesh = {*Genetic Therapy/methods/trends ; Humans ; *Gene Editing/methods ; *Public Health ; Anemia, Sickle Cell/therapy/genetics ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: In 2023, the FDA approved two gene therapies for sickle cell disease (SCD), one of which follows a standard gene therapy protocol and the other a gene editing (CRISPR/Cas9) approach. Other gene therapy protocols for conditions relating to public health continue to advance and are being discussed in academic and professional circles. This review examines the pace of public health-related gene therapy and gene editing development since the publication of a key British white paper dealing with the pace of fruition in this field.

SUMMARY: Gene therapy developments related to public health fit into three overarching baskets: (1) gene therapy and editing for rare, single-gene disorders (e.g., homozygous familial hypercholesterolemia and hereditary amyloidosis polyneuropathy); (2) gene therapy and editing for high prevalence conditions (e.g., SCD); and (3) genetic engineering and gene editing of mosquitoes transmitting tropical disease. While the protocols listed in this purposive inspection largely center around phase III (comparing treatments), with several in phase II (establishing efficacy) and phase I (assessing safety), costs of actual administration can span USD 2.1 to 3.1 million. By comparison, conventional SCD treatment runs between USD 22,500 and USD 200,000 per year for its most severe forms. Expert and public buy-in of gene editing of mosquitoes to reduce tropical disease and for human germline gene editing contain many caveats, with public health serving a useful monitoring and filtering role for how a technology might be deemed permissible.

KEY MESSAGES: Gene therapy has advanced beyond the stage where possible consequences serve as an automatic barrier to mainstream use, moving it closer to British white paper objectives. Ethical and feasible adoption by public health, taking into account population needs, will most likely happen through a combination Medicaid and Medicare, as opposed to the system governing newborn screening, under arrangements similar to the Centers for Medicare and Medicaid Services' coverage under evidence development program. Vector gene drives to alleviate tropical disease should remain privately financed, with this type of financing also being used for the vast majority of gene therapies entering the market. Though the criteria for germline applications continue to evolve, in the end such applications do not serve public health purposes. Academic public health has a monitoring role to play as relevant gene therapy and gene editing trials evolve; public health practice a referral and field monitoring role in the T3 (implementation) and T4 (population outcomes) translational research phases for the few applications that could justifiably receive public funding and public health support.},
}

*Genetic Therapy/methods/trends
Humans
*Gene Editing/methods
*Public Health
Anemia, Sickle Cell/therapy/genetics
CRISPR-Cas Systems

@article {pmid40530850,
year = {2025},
author = {Chen, Y and Shi, Y and Zuo, X and Dong, X and Xiao, X and Chen, L and Xiang, Z and Ren, L and Zhou, Z and Wei, W and Lei, X and Wang, J},
title = {UNC0638 inhibits SARS-CoV-2 entry by blocking cathepsin L maturation.},
journal = {Journal of virology},
volume = {99},
number = {7},
pages = {e0074125},
pmid = {40530850},
issn = {1098-5514},
mesh = {*SARS-CoV-2/physiology/drug effects ; Humans ; *Cathepsin L/metabolism/antagonists & inhibitors/genetics ; *Virus Internalization/drug effects ; COVID-19/virology ; *Antiviral Agents/pharmacology ; HEK293 Cells ; CRISPR-Cas Systems ; Vero Cells ; Animals ; Chlorocebus aethiops ; },
abstract = {Since the outbreak of SARS-CoV-2, viral mutations have posed significant challenges in identifying therapeutic targets and developing broad-spectrum antiviral drugs. Post-translational modifications of genes involved in interferon production and signaling pathways play a crucial role in regulating interferon responses. In this study, we employed CRISPR-Cas9 screening based on adenine base editors to investigate functional amino acids in 1,278 innate immune-related genes. This approach, which converts A-T base pairs into G-C base pairs to probe the functional importance of specific amino acids, allowed us to identify 17 vital factors involved in SARS-CoV-2 infection. Among the candidate genes, genetic knockdown of EHMT2 exhibited the strongest antiviral effect. Further analysis revealed that UNC0638, a selective inhibitor of EHMT2, significantly reduced the endosomal entry of SARS-CoV-2 in pseudovirus assays. The observed inhibitory effect was consistently observed across multiple SARS-CoV-2 variants, including Alpha, Beta, Delta, and Omicron. Mechanistically, UNC0638 reduced mature cathepsin L (CTSL) levels, impairing the proteolytic cleavage of SARS-CoV-2 spike protein and subsequent membrane fusion, a critical step for viral entry. Our findings uncover EHMT2 as a host dependency factor and reveal the antiviral mechanism of EHMT2 inhibitors through CTSL maturation blockade. These results advance the understanding of host factors in SARS-CoV-2 infection and provide a strategic framework for developing host-targeted antiviral therapies.IMPORTANCEIn this study, we demonstrated that knockdown or knockout of EHMT2 inhibited SARS-CoV-2 infection, and inhibitors of EHMT2, including UNC0638, UNC0642, and BIX01294 showed similar restrictive effects. Mechanistically, the EHMT2 inhibitor UNC0638 restricts spike-mediated cell entry by inhibiting the maturation of CTSL, a critical protease required for SARS-CoV-2 entry via the endosomal pathway. Importantly, CTSL is not only essential for SARS-CoV-2 but also plays a key role in the entry of other coronaviruses that utilize similar pathways. Therefore, EHMT2 inhibitors could have broader applications as pan-coronavirus therapeutic agents.},
}

*SARS-CoV-2/physiology/drug effects
Humans
*Cathepsin L/metabolism/antagonists & inhibitors/genetics
*Virus Internalization/drug effects
COVID-19/virology
*Antiviral Agents/pharmacology
HEK293 Cells
CRISPR-Cas Systems
Vero Cells
Animals
Chlorocebus aethiops

@article {pmid40522367,
year = {2025},
author = {Akiyama, K and Momobayashi, A and Okano, M},
title = {Direct detection of CRISPR-Cas9 ribonucleoprotein gene doping using RNA immunoprecipitation and quantitative PCR.},
journal = {Analytical and bioanalytical chemistry},
volume = {417},
number = {19},
pages = {4449-4460},
pmid = {40522367},
issn = {1618-2650},
mesh = {*CRISPR-Cas Systems/genetics ; *Doping in Sports ; *Ribonucleoproteins/genetics ; Animals ; Myostatin/genetics ; Humans ; Erythropoietin/genetics ; *Immunoprecipitation/methods ; Mice ; Gene Editing/methods ; *Real-Time Polymerase Chain Reaction/methods ; Actinin/genetics ; *RNA/genetics ; Limit of Detection ; Male ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Gene doping, using technologies such as CRISPR-Cas9, poses a considerable threat to the integrity of sports. In 2018, the World Anti-Doping Agency implemented a ban on genome editing, which highlighted the need for sensitive and specific detection methods. Detection techniques that are currently available have shown effectiveness in specific contexts, but are limited by low sensitivity and short detection windows. To overcome these limitations, this study presents a new detection method for CRISPR-Cas9 ribonucleoprotein (RNP) complexes, termed RNA immunoprecipitation followed by quantitative PCR (RIP-qPCR). The primary focus of this research was the in vitro development of a detection method targeting genes critical for doping, including myostatin (MSTN), α-actinin 3 (ACTN3), erythropoietin receptor (EPOR), and erythropoietin (EPO), with in vivo proof-of-concept demonstrated using MSTN. The RIP-qPCR method demonstrated sensitive performance, with a limit of quantification of 0.1 ng/mL in plasma. This method successfully detected single guide RNA targeting MSTN, ACTN3, EPOR, and EPO, along with two types of Cas9 proteins in RNP complexes in vitro. Additionally, the detection capabilities of RIP-qPCR were maintained for up to 30 days when plasma samples were stored at 4 °C. In vivo experiments were performed where RNPs were administered via intramuscular and intravenous injections to target the murine Mstn gene. CRISPR-Cas9 RNPs remained detectable for up to 24 h following intramuscular injection and 12 h after intravenous injection. This study underscores the potential of RIP-qPCR as a powerful tool for anti-doping analysis, with future efforts on expanding the target gene panel to enhance the detection of gene editing in sports doping.},
}

*CRISPR-Cas Systems/genetics
*Doping in Sports
*Ribonucleoproteins/genetics
Animals
Myostatin/genetics
Humans
Erythropoietin/genetics
*Immunoprecipitation/methods
Mice
Gene Editing/methods
*Real-Time Polymerase Chain Reaction/methods
Actinin/genetics
*RNA/genetics
Limit of Detection
Male
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40521885,
year = {2025},
author = {Choe, D and Lee, E and Song, Y and Kim, SC and Jeong, KJ and Palsson, B and Cho, B-K and Cho, S},
title = {CRISPRi screening reveals E. coli's anaerobic-like respiratory adaptations to gentamicin: membrane depolarization by CpxR.},
journal = {mSystems},
volume = {10},
number = {7},
pages = {e0035325},
pmid = {40521885},
issn = {2379-5077},
support = {2021R1A2C1012589//National Research Foundation of Korea/ ; 2018M3A9H024759//National Research Foundation of Korea/ ; RS-2023-00246928//National Research Foundation of Korea/ ; NNF16CC0021858//Novo Nordisk Fonden/ ; },
mesh = {*Escherichia coli/drug effects/genetics/metabolism/physiology ; *Gentamicins/pharmacology ; *Escherichia coli Proteins/genetics/metabolism ; *Anti-Bacterial Agents/pharmacology ; Anaerobiosis ; Gene Expression Regulation, Bacterial/drug effects ; Adaptation, Physiological/drug effects ; Membrane Potentials/drug effects ; Drug Resistance, Bacterial/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; },
abstract = {Bacterial genes serve diverse cellular functions and many affect fitness in response to environmental challenges. We employed CRISPR interference screening to investigate the fitness effect of each gene in Escherichia coli exposed to gentamicin, aiming to understand the cellular defense mechanisms. Our findings revealed that ribosomal proteins, ribosome-associated proteins, toxin-antitoxin systems, and outer membrane proteins strongly influence the fitness of E. coli in gentamicin. Notably, gentamicin-induced fitness changes resembled those under anaerobic conditions, where resistance to gentamicin was observed. Specifically, genes related to the biosynthesis of cofactors and electron carriers, crucial for the respiratory system, showed reduced essentiality under both gentamicin and anaerobic conditions, suggesting a disruption in membrane potential leading to limited gentamicin uptake. Transcriptomic and genome-wide binding analyses identified the two-component system CpxR as a key regulator of respiratory systems in response to gentamicin. Our study provides insights into cellular defense mechanisms, offering potential strategies for combating antibiotic resistance.IMPORTANCEBacteria can adapt to a variety of stressful environments, including antibiotic exposure. The mechanisms underlying antibiotic resistance remain an active area of investigation. Clustered regularly interspaced short palindromic repeats (CRISPR) interference enables specific silencing of gene expression, allowing researchers to assess the fitness effects of gene knockdowns under given conditions. Using genome-wide CRISPR interference screening on Escherichia coli exposed to gentamicin, we identified anaerobic-like fitness effects of genes involved in respiration and the maintenance of membrane potential-key processes that facilitate gentamicin entrance into the cell. Transcriptomic analysis and immunoprecipitation assays further indicated that the two-component system CpxR modulates respiratory adaptations in response to gentamicin challenge. These findings shed light on the development of antibiotic resistance in bacteria and may offer new insight into strategies for treating gentamicin-resistant pathogens.},
}

*Escherichia coli/drug effects/genetics/metabolism/physiology
*Gentamicins/pharmacology
*Escherichia coli Proteins/genetics/metabolism
*Anti-Bacterial Agents/pharmacology
Anaerobiosis
Gene Expression Regulation, Bacterial/drug effects
Adaptation, Physiological/drug effects
Membrane Potentials/drug effects
Drug Resistance, Bacterial/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Cas Systems

@article {pmid40505881,
year = {2025},
author = {Shihan, MTA and Hyodo, T and Fujino, T and Rahman, ML and Hasan, MN and Biswas, M and Vu, LQ and Jahan, N and Mihara, Y and Karnan, S and Ota, A and Tsuzuki, S and Toyoda, A and Hosokawa, Y and Kasahara, M and Konishi, H},
title = {Characterization of targeted knock-in achieved via tandem paired nicking mediated by CRISPR/Cas9 nickases.},
journal = {Methods (San Diego, Calif.)},
volume = {241},
number = {},
pages = {184-195},
doi = {10.1016/j.ymeth.2025.06.004},
pmid = {40505881},
issn = {1095-9130},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; *Gene Editing/methods ; Humans ; *Deoxyribonuclease I/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Targeted knock-in of specific DNA sequences using CRISPR/Cas9 is an advanced technology that enables programmed genome alterations including insertions, deletions, and base substitutions exactly as designed. Despite its utility in life sciences and promise for medical and industrial applications, it remains critical to establish a methodology for highly precise and efficient targeted knock-in to facilitate the practical use of this technology. Tandem paired nicking (TPN) is a genome editing methodology leveraging nicking variants of CRISPR/Cas9 nucleases (Cas9 nickases) to create site-specific nicks within the homologous region of the genome and donor DNA. Such nicking configuration promotes precise and efficient targeted knock-in while repressing the formation of unintended insertions and deletions and p53-mediated DNA damage response. In this study, we conducted a detailed characterization of TPN-based targeted knock-in by performing genome editing assays with various nicking configurations modified from TPN. Our results demonstrated that genomic nicks remarkably contribute to TPN-based targeted knock-in, whereas donor nicks play a less critical role. The introduction of additional nicks beyond the standard two-nick configuration did not further improve the efficiency of TPN-based targeted knock-in. Comparison with other Cas9 nickase-based methodologies for targeted knock-in demonstrated largely equivalent knock-in efficiencies achieved by these methodologies. High-throughput long-read sequencing confirmed a lower incidence of undesired insertions and deletions of various lengths by TPN, in comparison with a conventional Cas9 nuclease-based approach. These findings underscore TPN as a methodology for precise and efficient targeted knock-in, and highlight the broad potential of Cas9 nickase-based targeted knock-in for clinical and industrial applications.},
}

*CRISPR-Cas Systems/genetics
*Gene Knock-In Techniques/methods
*Gene Editing/methods
Humans
*Deoxyribonuclease I/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40503884,
year = {2025},
author = {Liang, Z and Huang, C and Li, Y and Yang, C and Wang, N and Ma, X and Huo, Y-X},
title = {A recombineering-based platform for high-throughput genomic editing in Escherichia coli.},
journal = {Applied and environmental microbiology},
volume = {91},
number = {7},
pages = {e0019325},
doi = {10.1128/aem.00193-25},
pmid = {40503884},
issn = {1098-5336},
support = {11116022401//The foundation of Muyuan laboratory/ ; C2024105020//Natural Science Foundation of Hebei Province/ ; },
mesh = {*Escherichia coli/genetics ; *Gene Editing/methods ; *Genome, Bacterial ; CRISPR-Cas Systems ; Recombination, Genetic ; },
abstract = {UNLABELLED: Functional analysis of bacterial genes or genomic fragments in vivo primarily relies on the analysis of knockout strains. Although various methods have successfully generated bacterial knockout mutants, the parallel operation of multiple sites, especially in biofoundries, remains challenging. New technological refinements of existing methods are necessary for high-throughput genomic editing in bacteria. In this study, to modify numerous sites in parallel, we optimized the linear donor DNA by adding modification at the different positions and achieved high-efficiency recombination with chemical transformation. Then, by combining with the CRISPR system, we established a guide sequence-independent and donor DNA-mediated genomic editing (GIDGE) method, enabling efficient and scarless engineering of common E. coli strains as well as wild-type strains such as E. coli MG1655, with particularly marked advantages demonstrated in E. coli Nissle 1917. This method allows for high-throughput genomic engineering in a 96-well format and is useful for sequence deletion with a wide range of lengths, sequence insertion, sequence replacement, and point mutation. As a proof-of-concept study, we constructed 96 single-gene knockout mutants and a genomic large-fragment deletion library in E. coli K-12 MG1655 using the GIDGE method. This high-throughput and easy-to-use method has great potential for automation and can be adapted for use in biofoundries.

IMPORTANCE: With the increasing demand in the microbiology field and the expansion of its application scope, the urgency for genome editing techniques that are not only efficient and versatile but also capable of high-throughput processing and even automation has become increasingly critical. In this study, we enhanced the efficiency of recombination engineering by incorporating modifications and integrated it with the CRISPR system to develop an advanced gene editing method. This method allows for various gene editing events such as insertion, replacement, and long fragment knockout without the need for plasmid construction. It not only demonstrated high efficiency in common E. coli strains but also exhibited marked advantages in the probiotic strain E. coli Nissle 1917. This method is a versatile, efficient approach capable of high-throughput parallel gene editing. Using this method, we successfully constructed a large-scale strain library, significantly accelerating the process of microbial engineering.},
}

*Escherichia coli/genetics
*Gene Editing/methods
*Genome, Bacterial
CRISPR-Cas Systems
Recombination, Genetic

@article {pmid40373837,
year = {2025},
author = {Vandendriessche, B and Huyghebaert, J and Rossem, KV and Cremers, TC and Man, K and Sieliwonczyk, E and Boen, H and Akdeniz, D and Rabaut, L and Schippers, J and Ponsaerts, P and Kooy, RF and Loeys, B and Schepers, D and Alaerts, M},
title = {An NGS-based approach for precise and footprint-free CRISPR-based gene editing in human stem cells.},
journal = {Methods (San Diego, Calif.)},
volume = {241},
number = {},
pages = {33-42},
doi = {10.1016/j.ymeth.2025.05.004},
pmid = {40373837},
issn = {1095-9130},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *High-Throughput Nucleotide Sequencing/methods ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Human Embryonic Stem Cells/metabolism/cytology ; Transfection/methods ; },
abstract = {Precise gene editing with conventional CRISPR/Cas9 is often constrained by low knock-in (KI) efficiencies (≈ 2-20 %) in human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs). This limitation typically necessitates labour-intensive manual isolation and genotyping of hundreds of colonies to identify correctly edited cells. Fluorescence- or antibiotic-based enrichment methods facilitate the identification process but can compromise cell viability and genomic integrity. Here, we present a footprint-free editing strategy that combines low-density seeding with next-generation sequencing (NGS) to rapidly identify cell populations containing precisely modified clones. By optimising the transfection workflow and adhering to CRISPR/Cas9 KI design principles, we achieved high average editing efficiencies of 64 % in hiPSCs (introducing a Brugada syndrome-associated variant) and 51 % in hESCs (introducing a neurodevelopmental disorder (NDD)-associated variant). Furthermore, under suboptimal CRISPR design conditions, this approach successfully identified hESC clones carrying a second NDD-associated variant, despite average KI efficiencies below 1 %. Importantly, genomic integrity was preserved throughout subcloning rounds, as confirmed by Sanger sequencing and single nucleotide polymorphism (SNP) array analysis. Hence, this NGS-based enrichment strategy reliably identifies desired KI clones under both optimal and challenging conditions, reducing the need for extensive colony screening and offering an effective alternative to fluorescence- and antibiotic-based selection methods.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*High-Throughput Nucleotide Sequencing/methods
*Induced Pluripotent Stem Cells/metabolism/cytology
*Human Embryonic Stem Cells/metabolism/cytology
Transfection/methods

@article {pmid40324704,
year = {2025},
author = {Quintanilla, I and Azeroglu, B and Sagar, MAK and Stracker, TH and Denchi, EL and Pegoraro, G},
title = {Optical pooled screening for the discovery of regulators of the alternative lengthening of telomeres pathway.},
journal = {Methods (San Diego, Calif.)},
volume = {241},
number = {},
pages = {1-12},
doi = {10.1016/j.ymeth.2025.05.001},
pmid = {40324704},
issn = {1095-9130},
mesh = {Humans ; *Telomere Homeostasis/genetics ; *Telomere/genetics/metabolism ; *In Situ Hybridization, Fluorescence/methods ; CRISPR-Cas Systems ; Telomerase/genetics/metabolism ; },
abstract = {Telomere elongation is essential for the proliferation of cancer cells. Telomere length control is achieved either by the activation of the telomerase enzyme, or by the recombination-based Alternative Lengthening of Telomeres (ALT) pathway. ALT is active in about 10-15% of human cancers, but its molecular underpinnings remain poorly understood, preventing the discovery of potential novel therapeutic targets. Pooled CRISPR-based functional genomic screens enable the unbiased discovery of molecular factors involved in cancer biology. Recently, Optical Pooled Screens (OPS) have significantly extended the capabilities of pooled functional genomics screens to enable sensitive imaging-based readouts at the single cell level and large scale. To gain a better understanding of the ALT pathway, we developed a novel OPS assay that employs telomeric native DNA FISH (nFISH) as an optical quantitative readout to measure ALT activity. The assay uses standard OPS protocols for library preparation and sequencing. As a critical element, an optimized nFISH protocol is performed before in situ sequencing to maximize the assay performance. We show that the modified nFISH protocol faithfully detects changes in ALT activity upon CRISPR knock-out (KO) of the FANCM and BLM genes, which were previously implicated in ALT. Overall, the OPS-nFISH assay is a reliable method that can provide deep insights into the ALT pathway in a high-throughput format.},
}

Humans
*Telomere Homeostasis/genetics
*Telomere/genetics/metabolism
*In Situ Hybridization, Fluorescence/methods
CRISPR-Cas Systems
Telomerase/genetics/metabolism

@article {pmid40143710,
year = {2025},
author = {Ji, C and Han, Y and Li, J and Wei, J and Yang, W and Cai, X and Tian, S and Chen, C and Wang, Y and Zhao, P and Cao, S and Zhang, W and Xu, J and Gu, B and Li, F and Liu, P and Pu, J},
title = {DNA Nanoflower-Powered CRISPR/Cas12a Biosensing Platform for Ultrasensitive Protein Detection in Clinical Samples.},
journal = {Small methods},
volume = {9},
number = {7},
pages = {e2402130},
doi = {10.1002/smtd.202402130},
pmid = {40143710},
issn = {2366-9608},
support = {32371468//National Natural Science Foundation of China/ ; 22204104//National Natural Science Foundation of China/ ; 22474077//National Natural Science Foundation of China/ ; 23ZR1461400 22ZR1459600//Shanghai Municipal Natural Science Foundation/ ; YG2023ZD07 YG2021QN23//Medical-Engineering Joint Funds from the Shanghai Jiao Tong University/ ; 20234Y0201//Foundation of Shanghai Municipal Health Commission/ ; 2022JC002//Foundation of Shanghai Municipal Health Commission/ ; TMSK-2024-203//National Key Scientific Infrastructure for Translational Medicine (Shanghai)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *DNA/chemistry ; *Lipocalin-2/urine/blood ; Biomarkers/blood/urine ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Protein markers secreted by various human cells provide crucial insights for the early diagnosis and prognostic assessment of clinical diseases. However, restricted by efficient protein marker signal amplification in real clinical samples with complex compositions, accurate, sensitive, and rapid detection of protein markers remains largely challenging. Herein, a DNA nanoflower (DNF)-powered CRISPR/Cas12a biosensing platform (DNF-CRISPR) is presented that employs the DNF in upstream to amplify input signals for protein markers, while utilizing the CRISPR system in downstream to amplify output signals by trans cleavage. This upstream and downstream cascade amplification sensing platform exhibits high sensitivity (500 fg mL[-1]), rapid (≤2 h), and a broad dynamic range (2.5 pg mL[-1] to 25 ng mL[-1]). As a proof of concept, DNF-CRISPR biosensing platform enables the quantitative detection of neutrophil gelatinase-associated lipocalin (NGAL) biomarkers in blood and urine samples from kidney injury patients with 91% accuracy. This study provides a powerful and versatile approach for the accurate diagnosis of protein markers in clinical settings, facilitating the application of CRISPR/Cas12a-based sensing platforms for non-nucleic acid markers.},
}

Humans
*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
*DNA/chemistry
*Lipocalin-2/urine/blood
Biomarkers/blood/urine
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40140561,
year = {2025},
author = {Kufrin, V and Seiler, A and Brilloff, S and Rothfuß, H and Küchler, S and Schäfer, S and Rahimian, E and Baumgarten, J and Ding, L and Buchholz, F and Ball, CR and Bornhäuser, M and Glimm, H and Bill, M and Wurm, AA},
title = {The histone modifier KAT2A presents a selective target in a subset of well-differentiated microsatellite-stable colorectal cancers.},
journal = {Cell death and differentiation},
volume = {32},
number = {7},
pages = {1259-1272},
pmid = {40140561},
issn = {1476-5403},
support = {WU977/2-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 70114086//Deutsche Krebshilfe (German Cancer Aid)/ ; },
mesh = {Humans ; *Colorectal Neoplasms/genetics/pathology/metabolism ; Animals ; Mice ; Cell Line, Tumor ; *Histone Acetyltransferases/metabolism/genetics ; Cell Differentiation/genetics ; *Microsatellite Instability ; Cell Proliferation ; Histones/metabolism ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; },
abstract = {Lysine acetyltransferase 2 A (KAT2A) plays a pivotal role in epigenetic gene regulation across various types of cancer. In colorectal cancer (CRC), increased KAT2A expression is associated with a more aggressive phenotype. Our study aims to elucidate the molecular underpinnings of KAT2A dependency in CRC and assess the consequences of KAT2A depletion. We conducted a comprehensive analysis by integrating CRISPR-Cas9 screening data with genomics, transcriptomics, and global acetylation patterns in CRC cell lines to pinpoint molecular markers indicative of KAT2A dependency. Additionally, we characterized the phenotypic effect of a CRISPR-interference-mediated KAT2A knockdown in CRC cell lines and patient-derived 3D spheroid cultures. Moreover, we assessed the effect of KAT2A depletion within a patient-derived xenograft mouse model in vivo. Our findings reveal that KAT2A dependency is closely associated with microsatellite stability, lower mutational burden, and increased molecular differentiation signatures in CRC, independent of the KAT2A expression levels. KAT2A-dependent CRC cells display higher gene expression levels and enriched H3K27ac marks at gene loci linked to enterocytic differentiation. Furthermore, loss of KAT2A leads to decreased cell growth and viability in vitro and in vivo, downregulation of proliferation- and stem cell-associated genes, and induction of differentiation markers. Altogether, our data show that a specific subset of CRCs with a more differentiated phenotype relies on KAT2A. For these CRC cases, KAT2A might represent a promising novel therapeutic target.},
}

Humans
*Colorectal Neoplasms/genetics/pathology/metabolism
Animals
Mice
Cell Line, Tumor
*Histone Acetyltransferases/metabolism/genetics
Cell Differentiation/genetics
*Microsatellite Instability
Cell Proliferation
Histones/metabolism
Gene Expression Regulation, Neoplastic
CRISPR-Cas Systems

@article {pmid40691444,
year = {2025},
author = {Tian, T and Xiao, H and Guo, X and Chen, Y and Qiu, Z and Zhang, T and Chen, M and Qi, W and Cai, P and Cheng, M and Zhou, X},
title = {Identification of a key nucleotide influencing Cas12a crRNA activity for universal photo-controlled CRISPR diagnostics.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6694},
pmid = {40691444},
issn = {2041-1723},
support = {32150019//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2023M741238//China Postdoctoral Science Foundation/ ; },
mesh = {*CRISPR-Associated Proteins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Endodeoxyribonucleases/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Bacterial Proteins/genetics/metabolism ; Mutation ; Humans ; *Nucleotides/genetics/metabolism ; },
abstract = {Developing a one-pot assay is a critical strategy for enhancing the applicability of CRISPR-based molecular diagnostics; however, it is hindered by CRISPR cleavage interfering with nucleic acid amplification templates. Photo-regulation strategies provide an ideal solution to suppress undesired CRISPR cleavage while maintaining detection efficiency. However, existing photo-controlled CRISPR diagnostic methods face limitations in universality, cost, and detection efficiency. In this study, we systematically examine the impact of mutations in the repeat recognition sequence (RRS), a four-nucleotide segment within the Cas12a crRNA direct repeat (DR) region, on cleavage activity. We observe that mutations at positions 3 or 4 nearly abolished crRNA activity. Based on this discovery, we introduce 6-nitropiperonyloxymethyl (NPOM) photo-caging modifications at positions 3 and 4. Photo-caging at position 4 demonstrates the most effective suppression of enzymatic activity and optimal light-mediated activation. We leverage this finding to develop a photo-controlled CRISPR diagnostic method, enabling a universally adaptable one-pot detection strategy. Furthermore, by incorporating a crRNA splinting strategy, this pre-preparable reagent can be adapted for the detection of virtually any target gene.},
}

*CRISPR-Associated Proteins/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Endodeoxyribonucleases/genetics/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Bacterial Proteins/genetics/metabolism
Mutation
Humans
*Nucleotides/genetics/metabolism

@article {pmid40688512,
year = {2025},
author = {Kanazhevskaya, LY and Zhdanova, PV and Chernonosov, AA and Koval, VV},
title = {Off-target interactions in the CRISPR-Cas9 Machinery: mechanisms and outcomes.},
journal = {Biochemistry and biophysics reports},
volume = {43},
number = {},
pages = {102134},
pmid = {40688512},
issn = {2405-5808},
abstract = {The in vivo editing of genetic information necessitates tools of unprecedented accuracy. CRISPR-Cas-based systems have emerged as leading technologies for precisely targeting the genome. The Cas9 endonuclease derived from Streptococcus pyogenes is the most commonly used instrument for targeted DNA cleavage. The development of engineered and chimeric Cas9 variants with enhanced activity and specificity has enabled not only the simple knockout of target genes but also the sophisticated engineering of the epigenome. This advancement has broadened the potential applications of CRISPR-Cas9 technology for the treatment of various disorders characterized by a combination of mutations, deletions, and duplications in the coding and non-coding regions of the genome. The inherent simplicity and predictability of the CRISPR-Cas9 targeting mechanism have led to an explosive growth in the development of prototype gene-editing drugs. However, their therapeutic application is still challenged by potential off-target effects. The erroneous editing of tumor suppressors and oncogenes could lead to adverse outcomes that mitigate the benefits of CRISPR therapy. The evolution of DNA-targeting technologies requires a comprehensive understanding of the mechanisms underlying CRISPR-Cas9 off-target binding and cleavage. The use of massive libraries of DNA targets and guide RNAs, coupled with high-throughput sequencing, contributes significantly to the analysis of mismatch tolerance. Nevertheless, the detection of ultra-low levels of off-target activity is hindered by the sensitivity limitations of current technologies. This review focuses on the mechanisms responsible for off-target interactions during CRISPR-Cas9-mediated gene editing. We discuss the influence of various factors, including nucleotide context, enzyme concentration, guide RNA structure, and the energetics of the RNA-DNA hybrid on mismatch tolerance in vitro and in vivo. Recent advances in the development of technologies for predicting off-target effects are briefly summarized. Particular emphasis is placed on the role of the Cas9 protein structure in the allosteric regulation of the specific and non-specific activity of the Cas9-sgRNA complex.},
}

@article {pmid40687858,
year = {2025},
author = {Bi, Q and Liu, M and Yan, L and Cheng, J and Sun, Q and Dai, Y and Zou, L},
title = {Progress in the application of isothermal amplification technology in the diagnosis of infectious diseases.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1601644},
pmid = {40687858},
issn = {1664-302X},
abstract = {Rapid detection of infectious diseases is critical for global public health prevention and control. However, the use of traditional molecular diagnostic methods, including PCR, has been limited because of their cumbersome procedures, complex equipment requirements, operation at different temperatures, and the level of expertise required for operation. Isothermal amplification technology (IAT) provides a rapid, sensitive, specific, simple and less costly method for diagnosing infectious diseases, which has led to revolutionary breakthroughs in molecular diagnostics. This paper summarizes recent progress in IAT technology, which focuses on the principles and applications of core technologies such as NASBA, LAMP, RPA, and RAA. In addition, the combination of IATs with the CRISPR/Cas system, which further revolutionizes nucleic acid detection technology, is explored in this review.},
}

@article {pmid40617444,
year = {2025},
author = {Zhu, Y and Kuang, S and Yao, Z and Sun, Y and Gao, H and Gao, Q and Ding, W and He, H and Li, Y and Qiu, L},
title = {Doublesex knockout via CRISPR/Cas9 disrupts fertility and sexual dimorphism of wings in the rice stem borer, Chilo suppressalis.},
journal = {Insect biochemistry and molecular biology},
volume = {182},
number = {},
pages = {104356},
doi = {10.1016/j.ibmb.2025.104356},
pmid = {40617444},
issn = {1879-0240},
mesh = {Animals ; *Moths/genetics/growth & development/physiology ; Female ; Male ; *Insect Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Fertility/genetics ; Sex Characteristics ; Wings, Animal/growth & development ; Gene Knockout Techniques ; Phylogeny ; Larva/growth & development/genetics ; Amino Acid Sequence ; },
abstract = {The doublesex (dsx) gene has a conserved role in sex determination in insects, controlling sexual development and mating behavior. Although dsx is known to participate in these critical functions, its role in insect sex determination remains not fully elucidated. Given the economic importance of the rice pest Chilo suppressalis, we employed this species as a model to investigate the function of its dsx homolog. We cloned and characterized the Csdsx gene, which is 1123 bp in length and encodes four sex-specific proteins: three female-specific isoforms of 252, 258, and 254 amino acids, respectively, and one male-specific isoform of 290 amino acids. Phylogenetic analysis revealed that Csdsx is highly conserved within Lepidoptera, containing two domains: DM DNA binding domain and DSX dimer domain. Analysis of transcripts produced from a mini-dsx construct transfected into human HEK293T cells indicates that the female-splicing pattern is the default mode. Quantitative real-time PCR showed that among various developmental stages, Csdsx expression peaked at the first instar larval stage and showed tissue-specific, stage-dependent patterns, with notably high levels in the larval midgut, pupal fat body, and adult thorax in both sexes. In comparison to wild-type (WT) adults, Csdsx-knockout individuals exhibited malformations in their external genitalia, and female wing patterns became masculinized. Dissections revealed that knockout females had a reduced number of mature oocytes, while knockout males displayed a smaller testis area. Furthermore, when Csdsx-knockout females were paired with wild-type males, their mating behavior was significantly impaired. RNA-seq revealed that Csdsx disruption led to sex-biased gene expression shifts, including upregulation of male-associated genes (e.g., OBPs, PBPs, trypsin) and downregulation of female-specific genes (vitellogenin, FAS), indicating partial masculinization at the transcriptional level. These findings underscore the critical role of the dsx gene in reproductive development and sexual dimorphism in C. suppressalis.},
}

Animals
*Moths/genetics/growth & development/physiology
Female
Male
*Insect Proteins/genetics/metabolism
CRISPR-Cas Systems
Fertility/genetics
Sex Characteristics
Wings, Animal/growth & development
Gene Knockout Techniques
Phylogeny
Larva/growth & development/genetics
Amino Acid Sequence

@article {pmid40607956,
year = {2025},
author = {Shi, K and Zhang, J and Yuan, R and Xiang, Y},
title = {Target-induced recycling and self-folding hairpin primer-mediated LAMP activation of CRISPR/Cas12a for highly sensitive aptamer-based therapeutic antibody assay.},
journal = {The Analyst},
volume = {150},
number = {15},
pages = {3475-3480},
doi = {10.1039/d5an00458f},
pmid = {40607956},
issn = {1364-5528},
mesh = {*Aptamers, Nucleotide/chemistry/genetics ; *Trastuzumab/analysis/blood ; Humans ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; *CRISPR-Associated Proteins/genetics/metabolism ; DNA Primers/chemistry/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; Molecular Diagnostic Techniques ; },
abstract = {Owing to their high affinity and specificity for antigen target molecules, therapeutic monoclonal antibodies (mAbs) have been increasingly used for the treatment of different diseases. The sensitive and accurate detection of mAbs is crucial for the evaluation of their efficacy and safety. With a new design of a thiophosphate-modified and self-folding hairpin primer, herein, we described the establishment of an aptamer-based, highly sensitive and simple fluorescent trastuzumab mAb assay method via target-induced recycling and low-temperature LAMP activation of CRISPR/Cas12a signal amplifications. Target trastuzumab molecules bound with and changed the conformation of the hairpin aptamer probes to trigger Bst polymerase-mediated recycling and LAMP reactions with the assistance of hairpin primers to form long dsDNAs containing many protospacer-adjacent motif (PAM) segments. Cas12a/crRNA subsequently associated with these PAMs to exhibit trans-cleavage property for cyclically cutting ssDNA reporter molecules and yielding considerably magnified fluorescence recovery for trastuzumab detection. Owing to target-recycling, LAMP and Cas12a/crRNA-integrated signal amplifications, a low picomolar detection limit (4.17 pM) for trastuzumab was achieved. This assay could also be applied to trace trastuzumab in diluted human serums. With the distinct advantages of low-temperature LAMP activation with minimal primer involvement and the integration of an amplification cascade, this sensing methodology could be employed as a robust signal enhancement methodology for detecting various molecular biomarkers for diverse biomedical and biological applications.},
}

*Aptamers, Nucleotide/chemistry/genetics
*Trastuzumab/analysis/blood
Humans
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
Limit of Detection
*CRISPR-Associated Proteins/genetics/metabolism
DNA Primers/chemistry/genetics
Bacterial Proteins
Endodeoxyribonucleases
Molecular Diagnostic Techniques

@article {pmid40568758,
year = {2025},
author = {Konde, MV and Inchanalkar, S and Sherkhane, TM and Deshpande, N and Virmani, M and Singh, K and Balasubramanian, N},
title = {CRISPR-Cas9 mediated RALA knockout and reconstitution: insights into the detection and role of RALA S194 phosphorylation in Ras-dependent and Ras-independent cancers.},
journal = {Biology open},
volume = {14},
number = {7},
pages = {},
doi = {10.1242/bio.061884},
pmid = {40568758},
issn = {2046-6390},
support = {35/03/2019-NANO/BMS//Indian Council of Medical Research/ ; 35/03/2019-NANO/BMS//Indian Council of Medical Research (ICMR)/ ; //IISER Pune: Indian Institute of Science Education Research Pune./ ; },
mesh = {Phosphorylation ; Humans ; *CRISPR-Cas Systems ; *Neoplasms/metabolism/genetics/pathology ; Cell Line, Tumor ; Gene Knockout Techniques ; *ras Proteins/metabolism/genetics ; Animals ; },
abstract = {Downstream of oncogenic RAS, RALA is critical for cancer tumorigenesis, possibly regulated by phosphorylation of its Serine194 residue. We made CRISPR-Cas9 RALA knockout (RALA KO) in three RAS-dependent and two RAS-independent cancer cells. Detection of RALA S194 phosphorylation using the commercial anti-phospho-RALA antibody lacks specificity in all three RAS-dependent cancers. siRNA knockdown of RALA and AURKA inhibition by MLN8237 (VMLN) also did not affect pS194RALA detection in these cancers. RALA KO MiaPaCa2 (RAS-dependent) and MCF7 (RAS-independent) cells, stably reconstituted with WT-RALA and S194A-RALA mutants, showed no effect on RALA activation. Tumor growth was, however, restored partly by WT-RALA, but not S194A-RALA mutant. Thus, RALA S194 phosphorylation is needed for tumor formation, not affecting its activation, but possibly through its localization.},
}

Phosphorylation
Humans
*CRISPR-Cas Systems
*Neoplasms/metabolism/genetics/pathology
Cell Line, Tumor
Gene Knockout Techniques
*ras Proteins/metabolism/genetics
Animals

@article {pmid40523854,
year = {2025},
author = {Munusamy, S and Zheng, H and Chen, J and Zhou, S and Kong, J and Jahani, R and Zhao, Y and Guan, X},
title = {Gold Nanoparticle-Assisted CRISPR-Cas12a-Based Activity Assay for Highly Sensitive Detection of Trypsin.},
journal = {ACS applied bio materials},
volume = {8},
number = {7},
pages = {6379-6387},
doi = {10.1021/acsabm.5c00854},
pmid = {40523854},
issn = {2576-6422},
mesh = {*Trypsin/analysis/metabolism ; *Gold/chemistry ; *Metal Nanoparticles/chemistry ; *CRISPR-Cas Systems ; Humans ; *Biocompatible Materials/chemistry/chemical synthesis ; Particle Size ; Materials Testing ; Cattle ; Surface Properties ; Biosensing Techniques/methods ; Animals ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Proteases play important roles in diverse physiological processes, and their malfunction has been implicated in various conditions and diseases. Therefore, development of sensitive methods for protease detection in clinical samples is highly desired for disease diagnosis. Herein, we report an ultrasensitive and selective CRISPR-Cas12a based fluorescent assay for trypsin activity measurement. By taking advantage of the signal amplification brought by a unique magnetic bead-gold nanoparticle assembly, which carries the peptide substrate and tens to hundreds of DNA molecules per peptide molecule, trypsin can be detected with a limit of detection reaching as low as 0.13 ng/mL. Furthermore, the sensor selectivity study was performed by examining several biomolecules commonly present in biological samples, including bovine serum albumin (BSA), human serum albumin (HSA), DNase, RNase, chymotrypsin, elastase, and thrombin. Moreover, trypsin inhibition and serum sample analysis were successfully carried out. Given the ultrahigh sensitivity, the CRISPR-based trypsin activity assay developed in this work can be used as a generic platform for developing sensors for other proteases, offering the potential as a noninvasive/minimally invasive tool for clinical diagnosis.},
}

*Trypsin/analysis/metabolism
*Gold/chemistry
*Metal Nanoparticles/chemistry
*CRISPR-Cas Systems
Humans
*Biocompatible Materials/chemistry/chemical synthesis
Particle Size
Materials Testing
Cattle
Surface Properties
Biosensing Techniques/methods
Animals
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40398570,
year = {2025},
author = {Zou, Y and Ye, A and Dong, M and Zhou, Y and Wu, W and Tang, Y and Hu, H and Dai, F and Tong, X},
title = {Silkworm mutagenesis using a ribonucleoprotein-based CRISPR/Cas12a system.},
journal = {Insect biochemistry and molecular biology},
volume = {182},
number = {},
pages = {104329},
doi = {10.1016/j.ibmb.2025.104329},
pmid = {40398570},
issn = {1879-0240},
mesh = {*Bombyx/genetics/metabolism ; Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Ribonucleoproteins/genetics/metabolism ; *Mutagenesis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The development of highly efficient genome editing tools has revolutionized developmental biology and genetic studies in silkworm. Although methods based on CRISPR/Cas9 are currently popular, the Cas12a system has emerged as a promising option. However, it has not yet been applied to target the silkworm genome in vivo, and its activity in silkworm has not yet been characterized. In this study, we established a ribonucleoprotein-based CRISPR/Cas12a system, and compared it to the CRISPR/Cas9 system using 19 crRNA and 17 sgRNAs to target three different genes in vivo. Although Cas12a generates mutants less efficiently than Cas9, we used it successfully to generate transmissible indels, and demonstrated its application by targeting the FibH and mp genes to produce mutants with the expected phenotypes. We also assessed the influence of temperature (37 °C vs. 25 °C) on Cas12a activity, and demonstrated that the effects are target dependent. In summary, we have established a ribonucleoprotein-based CRISPR/Cas12a system in silkworm that offers a practical alternative to CRISPR/Cas9 and extends the genome editing tool box available for silkworm research.},
}

*Bombyx/genetics/metabolism
Animals
*CRISPR-Cas Systems
*Gene Editing/methods
*Ribonucleoproteins/genetics/metabolism
*Mutagenesis
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40320697,
year = {2025},
author = {Batzel, G and Wang, Y and Bock, A and Chen, E and Neal, S and Lopez-Anido, RN and Lee, Y and Tjeerdema, E and Ignatoff, E and Patil, T and Ramirez, G and Lesoway, MP and Hamdoun, A and Lyons, DC},
title = {CRISPR/Cas9 Knockout of Shell Matrix Protein 1 in the Slipper-Snail Crepidula atrasolea.},
journal = {Journal of experimental zoology. Part B, Molecular and developmental evolution},
volume = {344},
number = {5},
pages = {266-283},
doi = {10.1002/jez.b.23293},
pmid = {40320697},
issn = {1552-5015},
support = {R35 GM133673/GM/NIGMS NIH HHS/United States ; //This study was supported by grants to D.C.L. from the National Science Foundation Faculty Early Career Development (CAREER) Award (1943606) and the National Institute of General Medical Sciences Maximizing Investigators' Research Award (MIRA) (R35GM133673). Additional funding from the National Institutes of Health (OD034075) to A.H./ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Snails/genetics/metabolism ; Animal Shells/metabolism ; Gene Editing ; Gene Knockout Techniques ; Gastropoda/genetics ; },
abstract = {Over the course of hundreds of millions of years, biomineralization has evolved independently many times across all kingdoms of life. Among animals, the phylum Mollusca displays a remarkable diversity in biomineral structures, particularly the molluscan shell, which varies greatly in shape, size, pigmentation, and patterning. Shell matrix proteins (SMPs) are key components of these shells, and are thought to drive the precipitation of calcium carbonate minerals and influence shell morphology. However, this structure-function relationship has rarely been studied directly because tools for knocking out genes did not exist in molluscs until recently. In this study, we report the first successful use of CRISPR/Cas9 gene editing to target an SMP in gastropod molluscs. Using the emerging model gastropod Crepidula atrasolea, we generated knockouts of the SMP1 gene. Successful gene editing was confirmed by Sanger and MiSeq sequencing, and loss of SMP1 expression was validated through high-content imaging of crispant embryos. This study establishes C. atrasolea as a valuable model for investigating the genetic basis of shell formation and provides a framework for applying CRISPR/Cas9 technology in other molluscan species. Our approach will enable future studies to thoroughly test the role of SMPs in shaping the diverse array of molluscan shell structures.},
}

Animals
*CRISPR-Cas Systems
*Snails/genetics/metabolism
Animal Shells/metabolism
Gene Editing
Gene Knockout Techniques
Gastropoda/genetics

@article {pmid40072468,
year = {2025},
author = {Chen, Q and Su, C and Li, S and Zhang, Z and Yang, Y and Yang, Y and Tao, D and Xie, S and Gong, P and Feng, Y},
title = {A sensitive and rapid visual method of chicken sexing based on LAMP-CRISPR/Cas12a system.},
journal = {British poultry science},
volume = {66},
number = {4},
pages = {531-538},
doi = {10.1080/00071668.2025.2454963},
pmid = {40072468},
issn = {1466-1799},
mesh = {Animals ; *Chickens/genetics ; *Sex Determination Analysis/veterinary/methods ; *Nucleic Acid Amplification Techniques/veterinary/methods ; *CRISPR-Cas Systems ; Female ; Male ; Chick Embryo ; Sensitivity and Specificity ; *Molecular Diagnostic Techniques/veterinary ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {1. Accurate sex identification of one-day-old chicks is crucial in layer poultry production. Establishing an early sexing method during the chicken embryonic period is essential for animal welfare. However, PCR-based sexing has limitations in terms of specialised equipment and is time-consuming.2. This study presents a rapid, simple and fluorescent visual technique for chicken sex identification based on Loop-mediated isothermal amplification (LAMP)-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a). It targets the chicken Z chromosome gene DMRT1 and W chromosome-specific fragment EE0.6 using designed primers and sgRNA. The LAMP amplicon is cleaved by Cas12a, producing a fluorescent product detectable by a portable light apparatus.3. The method has high sensitivity, capable of detecting as few as two copies per microlitre of the EE0.6 template and 20 copies per microlitre of the DMRT1 template. This has significant potential for distinguishing chicken embryo gender very early in embryonic development.},
}

Animals
*Chickens/genetics
*Sex Determination Analysis/veterinary/methods
*Nucleic Acid Amplification Techniques/veterinary/methods
*CRISPR-Cas Systems
Female
Male
Chick Embryo
Sensitivity and Specificity
*Molecular Diagnostic Techniques/veterinary
CRISPR-Associated Proteins/genetics
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid39702861,
year = {2025},
author = {Su, P and Miao, YL},
title = {A Genome-Wide CRISPR/Cas9 Screen Identifies Regulatory Genes for Stem Cell Aging.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2960},
number = {},
pages = {159-170},
pmid = {39702861},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; *Cellular Senescence/genetics ; Animals ; Cell Differentiation/genetics ; Mice ; Humans ; Gene Editing/methods ; Mesenchymal Stem Cells/cytology/metabolism ; *Embryonic Stem Cells/cytology/metabolism ; },
abstract = {Aging is a ubiquitous biological phenomenon, characterized by a gradual decline in physiological functions and an increased risk of various diseases. Although it is known that aging involves extensive changes in gene expression and disruptions in cellular metabolism, the molecular mechanisms underlying these processes remain incompletely understood. The CRISPR/Cas9 technology provides an efficient method for gene editing. In recent years, this technique has been successfully applied in various cellular and animal models to identify key genes involved in biological processes such as cancer and genetic diseases, which makes it possible to screen genes that affect cell senescence in the whole genome. Here, we describe a method that involves differentiating embryonic stem cells into mesenchymal progenitor cells and employing CRISPR/Cas9 for genome-wide functional screening to identify genes that regulate aging. Further analysis of the functions and regulatory mechanisms of these genes may provide new targets and strategies for anti-aging research and stem cell therapy.},
}

*CRISPR-Cas Systems/genetics
*Cellular Senescence/genetics
Animals
Cell Differentiation/genetics
Mice
Humans
Gene Editing/methods
Mesenchymal Stem Cells/cytology/metabolism
*Embryonic Stem Cells/cytology/metabolism

@article {pmid40685330,
year = {2025},
author = {Far, BF and Akbari, M and Habibi, MA and Katavand, M and Nasseri, S},
title = {CRISPR Technology in Disease Management: An Updated Review of Clinical Translation and Therapeutic Potential.},
journal = {Cell proliferation},
volume = {},
number = {},
pages = {e70099},
doi = {10.1111/cpr.70099},
pmid = {40685330},
issn = {1365-2184},
abstract = {CRISPR-Cas9 technology has rapidly advanced as a transformative genome-editing platform, facilitating precise genetic modifications and expanding therapeutic opportunities across various diseases. This review explores recent developments and clinical translations of CRISPR applications in oncology, genetic and neurological disorders, infectious diseases, immunotherapy, diagnostics, and epigenome editing. CRISPR has notably progressed in oncology, where it enables the identification of novel cancer drivers, elucidation of resistance mechanisms, and improvement of immunotherapies through engineered T cells, including PD-1 knockout CAR-T cells. Clinical trials employing CRISPR-edited cells are demonstrating promising results in hematologic malignancies and solid tumours. In genetic disorders, such as hemoglobinopathies and muscular dystrophies, CRISPR-Cas9 alongside advanced editors like base and prime editors show significant potential for correcting pathogenic mutations. This potential was affirmed with the FDA's first approval of a CRISPR-based therapy, Casgevy, for sickle cell disease in 2023. Neurological disorders, including Alzheimer's, ALS, and Huntington's disease, are increasingly targeted by CRISPR approaches for disease modelling and potential therapeutic intervention. In infectious diseases, CRISPR-based diagnostics such as SHERLOCK and DETECTR provide rapid, sensitive nucleic acid detection, particularly valuable in pathogen outbreaks like SARS-CoV-2. Therapeutically, CRISPR systems target viral and bacterial genomes, offering novel treatment modalities. Additionally, CRISPR-mediated epigenome editing enables precise regulation of gene expression, expanding therapeutic possibilities. Despite these advances, significant challenges remain, including off-target effects, delivery methodologies, immune responses, and long-term genomic safety concerns. Future improvements in editor precision, innovative delivery platforms, and enhanced safety assessments will be essential to fully integrate CRISPR-based interventions into standard clinical practice, significantly advancing personalised medicine.},
}

@article {pmid40684030,
year = {2025},
author = {Lam, HT and Nhi, NHH and Lan, VTH and Van Hau, N and Nghia, NH},
title = {A single-vector CRISPR/Cas9 system for genome editing and heterologous enzyme secretion in Saccharomyces cerevisiae: a case study on pectate lyase for coffee mucilage removal.},
journal = {Biotechnology letters},
volume = {47},
number = {4},
pages = {78},
pmid = {40684030},
issn = {1573-6776},
support = {36-2024-18-02//Viet Nam National University Ho Chi Minh City/ ; },
mesh = {*Polysaccharide-Lyases/genetics/metabolism ; *Saccharomyces cerevisiae/genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Coffee/chemistry ; Pectins/metabolism ; Bacillus subtilis/enzymology/genetics ; Genetic Vectors/genetics ; },
abstract = {The CRISPR/Cas9 system facilitates precise genome editing in various organisms. In this study, a single-vector CRISPR/Cas9 system was developed for Saccharomyces cerevisiae, employing a type II Cas9 enzyme from Streptococcus pyogenes and a single-guide RNA cassette targeting CAN1.Y locus on chromosome V. This system is broadly applicable across yeast strains, as it utilizes G418 selection, eliminating the need for auxotrophic markers. The efficiency of the CRISPR/Cas9 system was demonstrated, with editing efficiencies ranging from 70 to 100%. This system was utilized to integrate a cassette encoding secretory pectate lyase (PL) from Bacillus subtilis 168 into the yeast genome. The engineered S. cerevisiae strain secreted active PL, which exhibited pectin-degrading activity characterized by significant reductions in residual pectin and increased production of reducing sugars. Since pectin constitutes a major component of coffee mucilage, the secreted PL was applied to coffee beans for mucilage removal. The treated beans presented noticeably reduced residual mucilage, a purer green color, and decreased viscosity. These findings suggest the potential of the engineered S. cerevisiae strain for applications in coffee processing, particularly in efficient mucilage removal.},
}

*Polysaccharide-Lyases/genetics/metabolism
*Saccharomyces cerevisiae/genetics/metabolism
*CRISPR-Cas Systems
*Gene Editing/methods
*Coffee/chemistry
Pectins/metabolism
Bacillus subtilis/enzymology/genetics
Genetic Vectors/genetics

@article {pmid40683967,
year = {2025},
author = {Chang, W and Zhang, B and Yang, S and Zhang, X and Zhang, L and Zhang, S and Lu, J and Wang, W and Shang, P and Yue, Z},
title = {Genome-wide CRISPR/Cas9 screening identifies PTGR2 as a potential therapeutic target for sunitinib resistance in clear cell renal cell carcinoma.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {26263},
pmid = {40683967},
issn = {2045-2322},
support = {PR5124025//the Special Fund Project for Doctoral Training Program of Lanzhou University Second Hospital/ ; CY2021-MS-A11//Cuiying Science and Technology Innovation Plan Project of Lanzhou University Second Hospital/ ; 22JR5RA942//Gansu Provincial Science Fund for Distinguished Young Scholars/ ; 22JR5RA1009//Gansu Provincial Natural Science Foundation/ ; lzuyxcx-2022-105//Medical Innovation and Development Project of Lanzhou University/ ; },
mesh = {Humans ; *Sunitinib/pharmacology/therapeutic use ; *Carcinoma, Renal Cell/genetics/drug therapy/pathology/metabolism ; *Drug Resistance, Neoplasm/genetics ; *Kidney Neoplasms/genetics/drug therapy/pathology/metabolism ; *CRISPR-Cas Systems ; Histone Demethylases/metabolism/genetics ; Animals ; Cell Line, Tumor ; Mice ; Xenograft Model Antitumor Assays ; Gene Expression Regulation, Neoplastic/drug effects ; Antineoplastic Agents/pharmacology ; },
abstract = {Acquired and intrinsic resistance to sunitinib is a major obstacle to improving the therapeutic efficacy of treatment for clear cell renal cell carcinoma (ccRCC). This study aimed to identify novel therapeutic targets and the potential molecular mechanisms to overcome sunitinib resistance in ccRCC. Utilizing genome-wide CRISPR/Cas9 screening and resistant transcriptomics, we identified that prostaglandin reductase 2 (PTGR2) is a novel therapeutic target to overcome sunitinib resistance in ccRCC. The silencing of PTGR2 enhanced the cytotoxic effects of sunitinib in ccRCC cells, as measured by cell viability assays, and suppressed tumor growth in xenograft models. Mechanistically, PTGR2 physically interacts with lysine specific demethylase 6A (KDM6A) via endogenous/exogenous co-immunoprecipitation. PTGR2 knockdown reduced KDM6A protein expression, while KDM6A overexpression partially reversed the sensitization effect of PTGR2 silencing, suggesting KDM6A is a major downstream effector. Our findings establish the PTGR2-KDM6A axis as a potential target for overcoming sunitinib resistance in ccRCC. Pharmacological inhibition of PTGR2 or targeted modulation of KDM6A activity represents a promising combination strategy to overcome sunitinib resistance and improve patient outcomes.},
}

Humans
*Sunitinib/pharmacology/therapeutic use
*Carcinoma, Renal Cell/genetics/drug therapy/pathology/metabolism
*Drug Resistance, Neoplasm/genetics
*Kidney Neoplasms/genetics/drug therapy/pathology/metabolism
*CRISPR-Cas Systems
Histone Demethylases/metabolism/genetics
Animals
Cell Line, Tumor
Mice
Xenograft Model Antitumor Assays
Gene Expression Regulation, Neoplastic/drug effects
Antineoplastic Agents/pharmacology

@article {pmid40683956,
year = {2025},
author = {Zhang, C and Li, H and Poluektova, LY and Gendelman, HE and Dash, PK},
title = {Unique molecular signatures in rebound viruses from antiretroviral drug and CRISPR-treated HIV-1-infected humanized mice.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1077},
pmid = {40683956},
issn = {2399-3642},
support = {R01MH115860//U.S. Department of Health & Human Services | NIH | National Institute of Mental Health (NIMH)/ ; R01MH121402//U.S. Department of Health & Human Services | NIH | National Institute of Mental Health (NIMH)/ ; R24OD018546//U.S. Department of Health & Human Services | NIH | NIH Office of the Director (OD)/ ; },
mesh = {Animals ; *HIV-1/genetics/drug effects ; *HIV Infections/virology/drug therapy/genetics ; Mice ; Humans ; *CRISPR-Cas Systems ; *Anti-HIV Agents/pharmacology/therapeutic use ; Mutation ; *Anti-Retroviral Agents/pharmacology/therapeutic use ; Pyridones ; Heterocyclic Compounds, 3-Ring ; Oxazines ; Piperazines ; },
abstract = {HIV-1 elimination from a subset of virus-infected humanized mice (hu-mice) is reported following sequential dual treatment with long-acting (LA) antiretroviral (ART) and CRISPR-Cas9 therapies. However, viral rebound is observed in > 50% of the dual-treated animals. The molecular signatures of the rebound virus, recovered from plasma, have now been determined. The LA-ART treatment contains nanoformulated dolutegravir, lamivudine, abacavir, and rilpivirine combinations, and the HIV-1 excision treatment is CRISPR-Cas9 targeting the HIV-1-LTR-gag. One-step reverse transcriptase polymerase chain reaction, which avoids spontaneous preparatory mutations, is performed on plasma-derived RNA. Sanger and Next-Generation Sequencing are employed targeting the HIV-1-gag, pol, and env genes. HIV-1 env shows the most divergence. LA-ART, with or without CRISPR, is responsible for the new mutations. The primary and accessory mutations are detected by deep sequencing. Viral evolution reflects changes in the virus as reported by ART-treated and HIV-1-infected patients. No major CRISPR-specific mutations are observed. The molecular viral signatures demonstrate an accelerated HIV-1 drug resistance escape from ART rather than from the generation of CRISPR mutants. These define viral rebound in the dual-treated hu-mice. The data underscores the limited role of CRISPR excision in generating these rebound HIV-1 mutants from dual-treated hu-mice.},
}

Animals
*HIV-1/genetics/drug effects
*HIV Infections/virology/drug therapy/genetics
Mice
Humans
*CRISPR-Cas Systems
*Anti-HIV Agents/pharmacology/therapeutic use
Mutation
*Anti-Retroviral Agents/pharmacology/therapeutic use
Pyridones
Heterocyclic Compounds, 3-Ring
Oxazines
Piperazines

@article {pmid40683724,
year = {2025},
author = {Chen, J and Lu, Y and Meng, X and Chen, D and Wu, C and Song, D},
title = {Visual and self-contained diagnosis of Salmonella using RPA-CRISPR on a centrifugal force driven microfluidic system.},
journal = {Food microbiology},
volume = {132},
number = {},
pages = {104844},
doi = {10.1016/j.fm.2025.104844},
pmid = {40683724},
issn = {1095-9998},
mesh = {*Salmonella/genetics/isolation & purification ; *Nucleic Acid Amplification Techniques/methods/instrumentation ; *CRISPR-Cas Systems ; Food Microbiology/methods ; Recombinases/genetics/metabolism ; Food Contamination/analysis ; Lab-On-A-Chip Devices ; Bacterial Proteins/genetics ; DNA, Bacterial/genetics ; },
abstract = {Rapid and sensitive nucleic acid detection is very important for the detection of Salmonella in food. Convenient detection methods show great potential in food safety. CRISPR-Cas12a system has been widely used for nucleic acid detection. In one-pot reaction, Cas12a can perform cis- and trans-cleavage of amplicons and primers, which reduces the sensitivity of the reaction and has certain limitations. In this study, we integrated the Cas12a cutting system with recombinase polymerase amplification (RPA) into one microfluidic chip to avoid cap opening pollution. A portable observation device was integrated to enable visual detection of signals with the naked eye. The detection technology could specifically detect 1 × 10° CFU/mL Salmonella in 50 min at 37 °C. In the field test, there was no need for professional equipment or high-tech means.},
}

*Salmonella/genetics/isolation & purification
*Nucleic Acid Amplification Techniques/methods/instrumentation
*CRISPR-Cas Systems
Food Microbiology/methods
Recombinases/genetics/metabolism
Food Contamination/analysis
Lab-On-A-Chip Devices
Bacterial Proteins/genetics
DNA, Bacterial/genetics

@article {pmid40682826,
year = {2025},
author = {Zheng, H and Mao, C and Chen, S and Hou, S and Sun, D},
title = {A quorum sensing-controlled type I CRISPRi toolkit for dynamically regulating metabolic flux.},
journal = {Nucleic acids research},
volume = {53},
number = {14},
pages = {},
doi = {10.1093/nar/gkaf693},
pmid = {40682826},
issn = {1362-4962},
support = {32170083//National Natural Science Foundation of China/ ; 31670084//National Natural Science Foundation of China/ ; 2020C02031//Research and Development Program of Zhejiang Province/ ; 32170083//National Natural Science Foundation of China/ ; 31670084//National Natural Science Foundation of China/ ; },
mesh = {*Quorum Sensing/genetics ; *Bacillus subtilis/genetics/metabolism ; *CRISPR-Cas Systems ; *Metabolic Engineering/methods ; Gene Expression Regulation, Bacterial ; Riboflavin/biosynthesis ; Synthetic Biology/methods ; Bacterial Proteins/genetics/metabolism ; },
abstract = {Synthetic biology advances have enabled dynamic metabolic regulation via quorum sensing (QS) and CRISPR systems. However, the integration of QS with CRISPR-based systems for dynamic control remains largely underexplored. Here, we developed a QS-controlled type I CRISPR interference (QICi) toolkit that modulates target gene expression in response to cell density. By streamlining CRISPR RNA (crRNA) vector construction and optimizing key QS components PhrQ and RapQ, we achieved a twofold enhancement in QICi efficacy. We subsequently implemented the optimized QICi to reprogram Bacillus subtilis for d-pantothenic acid (DPA) and riboflavin (RF) biosynthesis. Dynamic regulation of the citrate synthase gene citZ by QICi, coupled with pantoate pathway engineering, cofactor supply enhancement, and suppression of sporulation, elevated DPA titers to 14.97 g/l in 5-l fed-batch fermentations without precursor supplementation. QICi-mediated metabolic rewiring of key nodes boosted RF production by 2.49-fold. Together, our work provides a robust tool for reprogramming microbial metabolism and advancing sustainable biomanufacturing.},
}

*Quorum Sensing/genetics
*Bacillus subtilis/genetics/metabolism
*CRISPR-Cas Systems
*Metabolic Engineering/methods
Gene Expression Regulation, Bacterial
Riboflavin/biosynthesis
Synthetic Biology/methods
Bacterial Proteins/genetics/metabolism

@article {pmid40682401,
year = {2025},
author = {Moso, MA and Roche, M and Cevaal, PM and Lewin, SR},
title = {CRISPR/Cas9 for achieving postintervention HIV control.},
journal = {Current opinion in HIV and AIDS},
volume = {},
number = {},
pages = {},
doi = {10.1097/COH.0000000000000963},
pmid = {40682401},
issn = {1746-6318},
abstract = {PURPOSE OF REVIEW: Recent advances in gene therapy have led to the first clinically approved CRISPR/Cas9 therapy for β-thalassaemia and sickle cell disease. Gene therapy could play an important role in targeting HIV persistence and achieving postintervention HIV control. Here, we review recent updates in CRISPR/Cas9-based HIV gene therapy approaches, including CCR5-editing (protect), proviral targeting (excise or modify), and immune cell engineering (attack).

RECENT FINDINGS: Recent studies provide additional safety data for use of CRISPR/Cas9-based gene therapies, however low in vivo editing efficiency highlights the need for improved delivery methods. This is particularly relevant for strategies requiring transfection of all HIV-infected cells containing intact proviruses, such as proviral excision. For ex vivo editing approaches, poor engraftment and durability of edited cells present additional challenges. Newer methods such as lipid nanoparticle delivery could provide a mechanism to overcome current limitations with ex vivo and in vivo delivery. Several studies have demonstrated proof-of-concept of combination gene therapy approaches, including gene editing strategies to generate HIV-resistant cells with immune effector functions, providing novel approaches to control and durably suppress viral replication.

SUMMARY: Several studies have demonstrated feasibility of gene therapy approaches in achieving postintervention HIV control. Improvements in both ex vivo and in vivo delivery methods are required to progress current gene therapy approaches to the clinic.},
}

@article {pmid40682144,
year = {2025},
author = {Motosugi, N and Hasegawa, K and Kurosaki, N and Kawaguchi, E and Izumi, K and Iida, Y and Higashiseto, M and Yokoyama, K and Sasaki, A and Nakabayashi, K and Fukuda, A},
title = {Highly efficient XIST reactivation in female hPSC by transient dual inhibition of TP53 and DNA methylation during Cas9 mediated genome editing.},
journal = {Stem cell research & therapy},
volume = {16},
number = {1},
pages = {389},
pmid = {40682144},
issn = {1757-6512},
support = {22bm0804030h0002//Japan Agency for Medical Research and Development/ ; },
mesh = {Humans ; *RNA, Long Noncoding/genetics/metabolism ; Female ; *Gene Editing/methods ; *DNA Methylation/genetics ; *Tumor Suppressor Protein p53/metabolism/genetics/antagonists & inhibitors ; *CRISPR-Cas Systems/genetics ; X Chromosome Inactivation/genetics ; *Pluripotent Stem Cells/metabolism/cytology ; },
abstract = {The irreversible erosion of X-chromosome inactivation (XCI) due to repression of the long non-coding RNA XIST presents a major challenge for disease modeling and raises safety concerns for the clinical application of female human pluripotent stem cells (hPSCs) due to the aberrant overexpression of X-linked genes. While Cas9-mediated non-homologous end joining (NHEJ) targeting the XIST promoter can induce DNA demethylation and restore XCI by reactivating XIST, its efficiency remains low. Here, we introduce a highly efficient strategy for XIST reactivation by combining TP53 inhibition with suppression of DNA methylation maintenance during Cas9-mediated NHEJ. This dual-inhibition approach increased the proportion of XIST-positive hPSCs from ~ 5 to ~ 43.7%, providing a robust method for stabilizing XCI in female hPSCs for diverse applications.},
}

Humans
*RNA, Long Noncoding/genetics/metabolism
Female
*Gene Editing/methods
*DNA Methylation/genetics
*Tumor Suppressor Protein p53/metabolism/genetics/antagonists & inhibitors
*CRISPR-Cas Systems/genetics
X Chromosome Inactivation/genetics
*Pluripotent Stem Cells/metabolism/cytology

@article {pmid40681749,
year = {2025},
author = {de Lima Balico, L and Gaucher, EA},
title = {Genomic insertion of ancestral uricase into human liver cells to determine metabolic consequences of pseudogenization.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {26093},
pmid = {40681749},
issn = {2045-2322},
mesh = {Humans ; *Urate Oxidase/genetics/metabolism ; Uric Acid/metabolism ; *Hepatocytes/metabolism ; Animals ; Triglycerides/metabolism ; Fructose/metabolism ; *Liver/metabolism/cytology ; CRISPR-Cas Systems ; },
abstract = {The biological role of urate (uric acid) during primate evolution has been unclear ever since it was discovered over 100 years ago that humans have unusually high levels of the small molecule compared to most other mammals. Humans (including all apes) are uncharacteristically susceptible to the build-up of urate because we no longer have a functional uricase enzyme capable of oxidizing this highly insoluble molecule. We have now utilized CRISPR technology to insert functional ancestral uricase into the genome of human liver cells to address recent metabolic hypotheses that our ancestral primates inactivated uricase as a mechanism to increase triglyceride production in response to fructose and/or starvation. Uricase expression is confirmed in both hepatocyte monolayer and spheroid tissue cultures, and its expression reduces intracellular urate levels. The presence of uricase is also shown to prevent an increase in triglyceride production upon cellular uptake of fructose in both culture conditions. Our results make progress that further describes a potential advantageous biological role of urate during primate evolution.},
}

Humans
*Urate Oxidase/genetics/metabolism
Uric Acid/metabolism
*Hepatocytes/metabolism
Animals
Triglycerides/metabolism
Fructose/metabolism
*Liver/metabolism/cytology
CRISPR-Cas Systems

@article {pmid40681400,
year = {2025},
author = {Huang, HC and Wu, LF and Liu, K and Ma, BG},
title = {Opto-CRISPR: new prospects for gene editing and regulation.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2025.06.018},
pmid = {40681400},
issn = {1879-3096},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) technology represents a landmark advance in the field of gene editing. However, conventional CRISPR/Cas systems are limited by inadequate temporal and spatial control. In recent years, the development of optically controlled CRISPR (Opto-CRISPR) technology has offered a novel solution to this issue. As a combination of optogenetics and the CRISPR technology, the Opto-CRISPR technology enables dynamic space-time-specific gene editing and regulation in cells and organisms. In this review, we concisely introduce the basic principles of Opto-CRISPR, summarize its operational mechanisms, and discuss its applications and recent advances across various research fields. In addition, this review analyzes the limitations of Opto-CRISPR, aiming to provide a reference for the development of this emerging field.},
}

@article {pmid40680677,
year = {2025},
author = {Li, X and Liu, J and Wang, R and Fu, H and Kim, M and Li, X and Peng, W and Man, S and Gao, Z and Ma, L},
title = {CRISPR-based one-pot detection: A game-changer in nucleic acid analysis.},
journal = {Biosensors & bioelectronics},
volume = {288},
number = {},
pages = {117786},
doi = {10.1016/j.bios.2025.117786},
pmid = {40680677},
issn = {1873-4235},
abstract = {The CRISPR/Cas system, originally developed as a gene-editing tool, has rapidly emerged as a powerful platform for nucleic acid detection due to its remarkable specificity, programmability, and robust trans-cleavage activity. While conventional CRISPR-based assays typically require separate amplification and detection steps, introducing complexity, manual intervention, and contamination risks. Recent innovations have led to the development of one-pot detection strategies that integrate target amplification with Cas-mediated signal generation within a single reaction vessel. These streamlined systems not only reduce handling steps and turnaround time but also enhance sensitivity and enable multiplexed detection of low-abundance nucleic acid targets. As such, one-pot CRISPR diagnostics are poised to transform point-of-care testing, pathogen surveillance, and clinical diagnostics. In this review, we summarize the latest advances in one-pot CRISPR-based detection technologies, evaluate their current limitations, and discuss strategies for optimization. Future efforts should focus on refining reaction kinetics, improving reagent stability, reducing costs, and expanding the repertoire of detectable targets. Furthermore, integration with portable platforms, AI-assisted signal interpretation, and the discovery of novel Cas effectors will be pivotal in realizing the full potential of CRISPR-based one-pot systems in diverse real-world applications.},
}