@article {pmid40954078,
year = {2025},
author = {Bexte, T and Wagner, DL},
title = {Next-generation multiplex-edited CAR-NK cells: more edits, more power?.},
journal = {Journal for immunotherapy of cancer},
volume = {13},
number = {9},
pages = {},
doi = {10.1136/jitc-2025-012841},
pmid = {40954078},
issn = {2051-1426},
mesh = {*Killer Cells, Natural/immunology/metabolism/transplantation ; Humans ; *Gene Editing/methods ; Animals ; *Receptors, Chimeric Antigen/genetics/metabolism ; Mice ; *Immunotherapy, Adoptive/methods ; CRISPR-Cas Systems ; },
abstract = {First clinical trials demonstrated the safety of adoptive cell transfer with allogeneic natural killer (NK) cell products from healthy donors, making them an attractive candidate for 'off-the-shelf' chimeric antigen receptor (CAR)-immune cell therapy. However, reduced persistence and inactivation of NK cells by immunosuppressive cues likely limit the performance of CAR-redirected NK cells. Wang and colleagues demonstrate that multiplex CRISPR base editing allows optimization of the intrinsic functionality of CAR-NK cells improving their therapeutic potential. In contrast to conventional CRISPR-Cas nucleases, base editing avoided most double-stranded DNA breaks while enabling highly efficient editing at up to six sites simultaneously. The study further demonstrates the feasibility of a non-viral approach to integrate CAR transgene and multiplex base editing of several immune checkpoints in NK cells using a single electroporation. CAR-NK cells harboring up to three base edits demonstrate improved potency over unedited counterparts in vitro. Xenograft mouse models confirmed increased potency, but also indicated signs of organ toxicity - a phenomenon that will require future studies prior to clinical translation. The study demonstrates that CRISPR base editing is a powerful tool to unleash the full cytotoxic potential of NK cells, but it also warrants the question: How many internal breaks can be removed without hurting CAR-NK cell therapy's impeccable safety record?},
}

*Killer Cells, Natural/immunology/metabolism/transplantation
Humans
*Gene Editing/methods
Animals
*Receptors, Chimeric Antigen/genetics/metabolism
Mice
*Immunotherapy, Adoptive/methods
CRISPR-Cas Systems

@article {pmid40952971,
year = {2025},
author = {Leonova, EI and Chirinskaite, AV and Akhmarov, II and Luganskaya, PS and Kirillov, OA and Kandina, DA and Romanovich, AE and Shkodenko, LA and Fedotov, SA and Rubel, AA and Sopova, YV},
title = {Improved Genome Editing via Oviductal Nucleic Acids Delivery-based In Vivo Electroporation Technique for Knockout Mice Generation.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {222},
pages = {},
doi = {10.3791/68704},
pmid = {40952971},
issn = {1940-087X},
mesh = {Animals ; Female ; *Electroporation/methods ; Mice ; Mice, Knockout ; Pregnancy ; *Gene Editing/methods ; *Fallopian Tubes ; Male ; CRISPR-Cas Systems ; *Nucleic Acids/administration & dosage/genetics ; Oviducts ; RNA, Guide, CRISPR-Cas Systems/administration & dosage/genetics ; *Gene Knockout Techniques/methods ; },
abstract = {Methods for creating knockout mice typically involve three main steps: (1) collecting embryos from donor females, (2) microinjecting genetic constructs into the zygotes ex vivo, and (3) surgically transferring them into the oviduct of pseudo-pregnant females. This process requires a significant number of animals, as it involves not only donor females but also vasectomized males and pseudo-pregnant females. Moreover, microinjections into the cytoplasm or pronucleus of mouse zygotes present challenges such as needle clogging, membrane permeability issues due to high elasticity, and potential embryo death. The development of advanced electroporators, such as the Nepa21, provides a unique opportunity to generate mice with targeted gene knockouts in a single step through a method known as Improved Genome Editing via Oviductal Nucleic Acids Delivery (I-GONAD). This technique involves microinjecting CRISPR-Cas components (Cas9 protein and guide RNA) into the oviducts of pregnant females at 0.7 days post-conception, followed by in vivo electroporation to deliver these components directly into the zygotes. Following the I-GONAD procedure, the pregnant mouse carries and gives birth to pups with the targeted gene knockout. This article provides a detailed, step-by-step protocol for implementing the I-GONAD method in mice, offering a more efficient and accessible alternative to traditional knockout mouse generation techniques.},
}

Animals
Female
*Electroporation/methods
Mice
Mice, Knockout
Pregnancy
*Gene Editing/methods
*Fallopian Tubes
Male
CRISPR-Cas Systems
*Nucleic Acids/administration & dosage/genetics
Oviducts
RNA, Guide, CRISPR-Cas Systems/administration & dosage/genetics
*Gene Knockout Techniques/methods

@article {pmid40952968,
year = {2025},
author = {Datta, A and Nelson, N and Orallo, GK and Krull, S},
title = {Targeted Corneal Sensory Nerve Depletion via Subconjunctival Injection: A Model for Investigating Bacterial Adhesion and Neuroimmune Interactions.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {222},
pages = {},
doi = {10.3791/68614},
pmid = {40952968},
issn = {1940-087X},
mesh = {Animals ; *Cornea/innervation/microbiology/immunology/drug effects ; *Bupivacaine/administration & dosage/pharmacology ; *Bacterial Adhesion/drug effects/physiology ; Pseudomonas aeruginosa/physiology ; Conjunctiva/innervation/microbiology ; Staphylococcus aureus/physiology ; Staphylococcus epidermidis/physiology ; *Sensory Receptor Cells/drug effects/microbiology/immunology ; Mice ; },
abstract = {Corneal sensory nerves play a pivotal role in supporting ocular surface integrity and immune defense mechanisms. Loss of this innervation has been associated with increased vulnerability to microbial invasion, yet the precise contribution of nerve depletion to bacterial adhesion on the cornea remains insufficiently characterized. Here, we present a reproducible and temporally controlled method for selective corneal sensory nerve suppression using bupivacaine, a long-acting sodium channel blocker. By combining subconjunctival and topical delivery routes, this dual-application strategy achieves robust, sustained denervation, allowing for precise investigation of how altered sensory input influences corneal epithelial susceptibility to bacterial colonization. Using this model, we investigate how sensory denervation influences microbial adhesion dynamics for Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa three clinically relevant pathogens with distinct adhesion mechanisms. Standardized bacterial inoculation via the laboratory wipe blotting method ensures uniform deposition on the corneal surface, followed by quantitative assessment of bacterial adhesion. Bupivacaine-induced nerve depletion correlates with reduced corneal nerve density and increased bacterial adhesion, confirming a functional link between sensory depletion and microbial susceptibility. By simulating neuropathic conditions such as diabetic neuropathy and neurotrophic keratitis, this approach provides a novel framework for studying neuroimmune interactions in ocular infections. Beyond infection models, this subconjunctival injection strategy offers a versatile platform for investigating ocular drug pharmacokinetics, neuroprotective interventions, and immune modulation. Furthermore, it can be adapted for gene modification studies, including subconjunctival delivery of CRISPR/Cas constructs or viral vectors, broadening its applications in ophthalmic research and therapeutics.},
}

Animals
*Cornea/innervation/microbiology/immunology/drug effects
*Bupivacaine/administration & dosage/pharmacology
*Bacterial Adhesion/drug effects/physiology
Pseudomonas aeruginosa/physiology
Conjunctiva/innervation/microbiology
Staphylococcus aureus/physiology
Staphylococcus epidermidis/physiology
*Sensory Receptor Cells/drug effects/microbiology/immunology
Mice

@article {pmid40951304,
year = {2025},
author = {Chen, J and Wang, Y and Aikebaier, R and Liu, H and Li, Y and Yang, L and Haiyilati, A and Wang, L and Fu, Q and Shi, H},
title = {RAA-CRISPR/Cas12a-based visual field detection system for rapid and sensitive diagnosis of major viral pathogens in calf diarrhea.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1616161},
pmid = {40951304},
issn = {2235-2988},
mesh = {Animals ; Cattle ; *Diarrhea/veterinary/virology/diagnosis ; Sensitivity and Specificity ; *Cattle Diseases/diagnosis/virology ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; China ; *Nucleic Acid Amplification Techniques/methods ; Feces/virology ; *Virus Diseases/diagnosis/veterinary/virology ; Recombinases/metabolism ; *Viruses/genetics/isolation & purification/classification ; },
abstract = {Calf diarrhea is a complex digestive disorder in cattle that imposes significant economic losses in terms of calf mortality, growth impairment, and treatment costs. Both infectious and non-infectious agents contribute to its aetiology; however, most of the infectious cases are caused by viruses, often accompanied by severe co-infections. To identify viral culprits, we performed viral metagenomic sequencing on three pooled samples from the 150 diarrheal samples from Xinjiang, China, which helped with identification of the following four predominant agents: bovine nepovirus (BNeV), bovine coronavirus (BCoV), bovine viral diarrhea virus (BVDV) and bovine enterovirus (BEV). Currently, the process of diagnosing these pathogens involves time-consuming workflows, limited sensitivity, poor portability, and lack of field applicability. Keeping these diagnostic shortcomings in mind, an integrated platform called RAA-CRISPR/Cas12a system was developed by combining recombinase-aided amplification (RAA) at 37°C with CRISPR/Cas12a-mediated fluorescence detection, which achieved 100-100,000 times higher sensitivity than conventional polymerase chain reaction (PCR) (detection limits: 1-10 copies/μL) and demonstrated 100% specificity against non-target pathogens. Clinical validation of sensitivity and specificity of 252 samples revealed 1.6-4.9 times higher detection rates (239 positives) than PCR (81 positives), which was consistent with PCR-confirmed cases. The assay's 40-min. workflow enables rapid on-site deployment without specialized instrumentation, as it requires only a portable heat block and blue LED transilluminator. Hence, with its laboratory accuracy and field applicability, this method helps in early identification of pathogens, outbreak containment and mitigation of economic loss in the global cattle industry.},
}

Animals
Cattle
*Diarrhea/veterinary/virology/diagnosis
Sensitivity and Specificity
*Cattle Diseases/diagnosis/virology
*CRISPR-Cas Systems
*Molecular Diagnostic Techniques/methods
China
*Nucleic Acid Amplification Techniques/methods
Feces/virology
*Virus Diseases/diagnosis/veterinary/virology
Recombinases/metabolism
*Viruses/genetics/isolation & purification/classification

@article {pmid40950114,
year = {2025},
author = {Saito, A and Tankou, S and Ishii, K and Sakao-Suzuki, M and Oh, EC and Murdoch, H and Namkung, H and Adelakun, S and Furukori, K and Fujimuro, M and Salomoni, P and Maul, GG and Hayward, GS and Tang, Q and Yolken, RH and Houslay, MD and Katsanis, N and Kosugi, I and Yang, K and Kamiya, A and Ishizuka, K and Sawa, A},
title = {DISC1-PML protein interaction for congenital CMV infection-induced cortical neural progenitor deficit: perturbance of host signaling via viral IE1.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {40950114},
issn = {2692-8205},
abstract = {Congenital CMV infection is the most common perinatal infection, affecting up to 0.5% of infants. This elicits long-term disabilities that include neuropsychiatric manifestations, such as intellectual disability, microcephaly. Despite its high prevalence, the underlying mechanism of how congenitally acquired CMV infection causes brain pathology remains unknown. Here we discovered the molecular interplay of key host (DISC1 and PML) and viral (IE1) proteins within the neural progenitor cells, which underlay an attenuated neural progenitor proliferation. Abolishing the viral IE1 protein by delivering IE1-targeting CRISPR/Cas9 to fetal brain rescued this progenitor cell deficit, a key pathology in congenital CMV infection. A selective targeting to a viral-specific protein by the CRISPR/Cas9 system is minimal in off-target effects. Therefore, we believe that a pivotal role of IE1 in an attenuated neural progenitor proliferation in the developing cortex through its interfering with interaction between host DISC1 and PML proteins.},
}

@article {pmid40947195,
year = {2025},
author = {Zhou, G and Ding, J and Li, Y and Xu, Q and Zhang, Y and Tang, H and Qi, P and Deng, M and Ma, J and Chen, G and Wang, J and Lin, N and Wei, Y and Jiang, Q},
title = {Ethylene-responsive factor HvERF72 regulates starch synthesis and B-type starch granules initiation in barley.},
journal = {Carbohydrate polymers},
volume = {368},
number = {Pt 2},
pages = {124167},
doi = {10.1016/j.carbpol.2025.124167},
pmid = {40947195},
issn = {1879-1344},
mesh = {*Hordeum/metabolism/genetics ; *Starch/biosynthesis ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; *Transcription Factors/metabolism/genetics ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; },
abstract = {Starch biosynthesis is a pivotal determinant of barley grain quality and yield, yet its regulatory mechanisms remain incompletely characterized. This study identifies HvERF72, an AP2-domain transcription factor, as a key regulator of starch biosynthesis and granule initiation in barley grains. Comparative analyses of CRISPR/Cas9-generated HvERF72 knockout mutants revealed enhanced B-type granule formation and elevated total starch content, whereas overexpression lines exhibited contrasting phenotypes, including reduced starch accumulation and suppressed B-type granule initiation. Transcriptional profiling at 15 DAF indicated significant upregulation of critical starch biosynthesis genes (HvAGPL1, HvAGPS1, HvSS2a, HvSBEI, HvSBEIIb, and HvGBSSI) in mutants, while overexpression lines showed downregulation of these genes. Mechanistic investigation demonstrated that HvERF72 directly binds to GCC-box motifs in the promoter regions of HvSS2a and HvSBEI, repressing their transcription. These findings establish HvERF72 as dual-function regulator that modulates starch biosynthesis and B-type granule initiation, providing novel molecular targets for optimizing starch yield and industrial quality in barley breeding programs.},
}

*Hordeum/metabolism/genetics
*Starch/biosynthesis
*Plant Proteins/genetics/metabolism
Gene Expression Regulation, Plant
*Transcription Factors/metabolism/genetics
Promoter Regions, Genetic
CRISPR-Cas Systems

@article {pmid40943650,
year = {2025},
author = {Ulloa, D and Núñez, C and Matamala, R and San Martín, A and Páez-De Ávila, D and Mercado-Vides, J and Narváez, J and Aguirre, J and Effer, B and Iturrieta-González, I},
title = {CRISPR-Cas12 Application for the Detection of Pneumocystis jirovecii in Immunodepression Patients Through Fluorescent and Lateral Flow Colorimetric Assay.},
journal = {International journal of molecular sciences},
volume = {26},
number = {17},
pages = {},
pmid = {40943650},
issn = {1422-0067},
support = {21251211//ANID - Chile, Scholarship Programa de Doctorado Nacional 2025/ ; UDM010//FONCIENCIAS - Universidad del Magdalena/ ; },
mesh = {Humans ; *Pneumocystis carinii/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Colorimetry/methods ; *Pneumonia, Pneumocystis/diagnosis/microbiology/immunology ; *Immunocompromised Host ; Sensitivity and Specificity ; Tubulin/genetics ; },
abstract = {Pneumonia caused by Pneumocystis jirovecii poses a serious threat, particularly to immunocompromised patients such as those with HIV/AIDS, transplant recipients, or individuals undergoing chemotherapy. Its diagnosis is challenging because current methods, such as microscopy and certain molecular tests, have limitations in sensitivity and specificity, and require specialized equipment, which delays treatment initiation. In this context, CRISPR-Cas12-based methods offer a promising alternative: they are rapid, highly specific, sensitive, and low-cost, enabling more timely and accessible detection, even in resource-limited settings. We developed a simple and rapid detection platform based on the CRISPR-Cas12 coupled with lateral flow strips. A guide RNA was designed against DHPS, β-tubulin, and mtLSU rRNA genes. The guide corresponding to β-tubulin showed high sensitivity in the detection of P. jirovecii to produce a detectable fluorescence signal within the first 20-30 min. In addition, it demonstrated high specificity for P. jirovecii when DNA from other microorganisms was used. When coupled with lateral flow strips, high sensitivity and specificity were also observed for detecting positive samples, without the need for genetic amplification. CRISPR-Cas12 successfully detected P. jirovecii infection in an initial diagnostic application, demonstrating the potential of this method for integration into public health diagnostic systems, particularly in field, due to its adaptability, speed, and ease of use.},
}

Humans
*Pneumocystis carinii/genetics/isolation & purification
*CRISPR-Cas Systems
*Colorimetry/methods
*Pneumonia, Pneumocystis/diagnosis/microbiology/immunology
*Immunocompromised Host
Sensitivity and Specificity
Tubulin/genetics

@article {pmid40943640,
year = {2025},
author = {Yu, I and Jeong, J},
title = {Advancing Gene Therapy for Phenylketonuria: From Precision Editing to Clinical Translation.},
journal = {International journal of molecular sciences},
volume = {26},
number = {17},
pages = {},
pmid = {40943640},
issn = {1422-0067},
support = {RS-2023-00260529//National Research Foundation of Korea/ ; 2022R1A2C1002884//National Research Foundation of Korea/ ; 2024-0036//Seoul Women's University/ ; },
mesh = {*Phenylketonurias/therapy/genetics ; Humans ; *Genetic Therapy/methods ; *Gene Editing/methods ; Animals ; CRISPR-Cas Systems ; Phenylalanine Hydroxylase/genetics ; Gene Transfer Techniques ; Dependovirus/genetics ; Translational Research, Biomedical ; Nanoparticles/chemistry ; },
abstract = {Phenylketonuria (PKU) is an inherited disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene that result in the amino acid phenylalanine (Phe) building up in the blood. Current therapies suggest low-Phe dietary management and (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) therapy, which are limited in efficacy and require lifelong treatment. Recent advances in gene therapy, including gene editing and viral-mediated gene delivery, produce therapeutic effects. Advancements in gene editing technologies, notably adenine base editors (ABEs) and CRISPR-based systems, in conjunction with enhanced delivery methods such as lipid nanoparticles (LNPs) and recombinant viruses, have demonstrated substantial promise in preclinical studies. This review details the pathophysiology of PKU treatment, and progress in preclinical and clinical gene therapy strategies. Emphasis is on adenine base editing using LNPs, recombinant adeno-associated virus (rAAV)-mediated gene transfer, and the translational challenges associated with these technologies. We also discuss future directions for therapeutic reach and ensuring long-term safety and efficacy.},
}

*Phenylketonurias/therapy/genetics
Humans
*Genetic Therapy/methods
*Gene Editing/methods
Animals
CRISPR-Cas Systems
Phenylalanine Hydroxylase/genetics
Gene Transfer Techniques
Dependovirus/genetics
Translational Research, Biomedical
Nanoparticles/chemistry

@article {pmid40943604,
year = {2025},
author = {Yamaji, M and Nakahara, T and Nakanishi, T and Aoyama-Kikawa, S and Yamaguchi, K and Furukawa, Y and Nakamura, M and Okada, T and Tabata, H and Fuse, R and Shimizu, E and Kasajima, R and Imoto, S and Kukimoto, I and Saito, I and Kiyono, T},
title = {Disruption of Human Papillomavirus 16 E6/E7 Genes Using All-in-One Adenovirus Vectors Expressing Eight Double-Nicking Guide RNAs.},
journal = {International journal of molecular sciences},
volume = {26},
number = {17},
pages = {},
pmid = {40943604},
issn = {1422-0067},
support = {JP21fk0108106//Japan Agency for Medical Research and Development/ ; JP22fk0310523//Japan Agency for Medical Research and Development/ ; JP25K02541//Japan Society for the Promotion of Science/ ; JP19K0647//Japan Society for the Promotion of Science/ ; JP23H02402//Japan Society for the Promotion of Science/ ; (JP22ym0126804//Japan Agency for Medical Research and Development/ ; 20jk0210009h0001//Japan Agency for Medical Research and Development/ ; },
mesh = {Humans ; *Oncogene Proteins, Viral/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Papillomavirus E7 Proteins/genetics ; *Adenoviridae/genetics ; *Genetic Vectors/genetics ; CRISPR-Cas Systems ; *Human papillomavirus 16/genetics ; Gene Editing/methods ; *Repressor Proteins/genetics ; Animals ; Cell Line, Tumor ; Papillomavirus Infections/virology/therapy ; Mice ; Female ; Genetic Therapy ; },
abstract = {Human papillomavirus (HPV) is a prime target for genome-editing therapy as its E6 and E7 oncogenes are crucial for cancer development and maintenance. A key challenge in CRISPR/Cas9 therapy is the off-target effects. This study utilized a double-nicking technique to introduce DNA breaks in the E6 and E7 regions of HPV16. From 146 gRNA candidates, 16 double-nicking pairs were selected. Multiple combinations of double-nicking (DN)-gRNA pairs were delivered to HPV16-positive cells via lentiviruses, followed by Cas9 nickase (Cas9n) expression. Combinations of 3-4 DN-gRNA pairs effectively killed HPV16-positive cells while sparing HPV-negative cells. Off-target effects were reduced by nearly three orders of magnitude. An "all-in-one" adenovirus (AdV) system expressing four gRNA pairs and Cas9n showed promise in inhibiting tumor growth in HPV16-positive cancer models, demonstrating its potential as a safe and effective treatment for HPV-induced tumors.},
}

Humans
*Oncogene Proteins, Viral/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics
*Papillomavirus E7 Proteins/genetics
*Adenoviridae/genetics
*Genetic Vectors/genetics
CRISPR-Cas Systems
*Human papillomavirus 16/genetics
Gene Editing/methods
*Repressor Proteins/genetics
Animals
Cell Line, Tumor
Papillomavirus Infections/virology/therapy
Mice
Female
Genetic Therapy

@article {pmid40943194,
year = {2025},
author = {Qi, Y and Jia, X and Lin, C and Qian, W and Chen, H and Fang, D and Han, C},
title = {CRISPR/Cas9-Mediated Overexpression of HGF Potentiates Tarim Red Deer Antler MSCs into Osteogenic Differentiation.},
journal = {International journal of molecular sciences},
volume = {26},
number = {17},
pages = {},
pmid = {40943194},
issn = {1422-0067},
mesh = {Animals ; *Osteogenesis/genetics ; *Deer/genetics ; *Antlers/cytology/metabolism ; *Mesenchymal Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems ; *Cell Differentiation/genetics ; *Hepatocyte Growth Factor/genetics/metabolism ; Signal Transduction ; Proto-Oncogene Proteins c-akt/metabolism ; Phosphatidylinositol 3-Kinases/metabolism ; Cells, Cultured ; },
abstract = {Previous studies conducted by our research groups have demonstrated that the HGF/c-Met signaling pathway promotes the proliferation and migration of MSCs in the antlers of Tarim red deer. However, the role and mechanism of this gene in the osteogenic differentiation of antler MSCs remain unclear. In this study, we used antler MSCs as experimental materials. CRISPR/Cas9 technology was employed to knock out the HGF gene, and lentivirus-mediated overexpression of the HGF gene was constructed in antler MSCs. Subsequently, antler MSCs were induced to undergo osteogenic differentiation in vitro. Alizarin Red staining was employed to identify calcium nodules, while the expression levels of various osteogenic differentiation marker genes were assessed using immunohistochemistry, RT-qPCR, and Western blotting techniques. The findings indicated that the HGF gene facilitates the osteogenic differentiation of antler MSCs. Analysis of genes associated with the PI3K/Akt and MEK/ERK signaling pathways demonstrated that in antler MSCs with HGF gene knockout, the expression levels of PI3K/Akt and MEK/ERK pathway genes were significantly downregulated on days 7 and 14 of osteogenic differentiation (p < 0.05). In contrast, antler MSCs with HGF gene overexpression exhibited a significant upregulation of the PI3K/Akt and MEK/ERK signaling pathways on days 4 and 6 of osteogenic differentiation (p < 0.01). These findings suggest that the HGF gene in antlers enhances the osteogenic differentiation of MSCs by activating the PI3K/Akt and MEK/ERK pathways.},
}

Animals
*Osteogenesis/genetics
*Deer/genetics
*Antlers/cytology/metabolism
*Mesenchymal Stem Cells/metabolism/cytology
*CRISPR-Cas Systems
*Cell Differentiation/genetics
*Hepatocyte Growth Factor/genetics/metabolism
Signal Transduction
Proto-Oncogene Proteins c-akt/metabolism
Phosphatidylinositol 3-Kinases/metabolism
Cells, Cultured

@article {pmid40943160,
year = {2025},
author = {Givi, S and Lohnes, BJ and Ebrahimi, S and Riedel, S and Khokhali, S and Khan, SA and Keller, M and Wölfel, C and Echchannaoui, H and Bockamp, E and Andre, MC and Abken, H and Theobald, M and Hartwig, UF},
title = {CRISPR/Cas9 TCR-Edited NKp30 CAR T Cells Exhibit Superior Anti-Tumor Immunity to B7H6-Expressing Leukemia and Melanoma.},
journal = {International journal of molecular sciences},
volume = {26},
number = {17},
pages = {},
pmid = {40943160},
issn = {1422-0067},
support = {2022.046.1//Wilhelm-Sander-Stiftung/ ; },
mesh = {Animals ; Humans ; *Melanoma/therapy/immunology/genetics ; Mice ; *CRISPR-Cas Systems ; *Immunotherapy, Adoptive/methods ; *B7 Antigens/genetics/metabolism/immunology ; *Natural Cytotoxicity Triggering Receptor 3/genetics/immunology/metabolism ; Cell Line, Tumor ; *Receptors, Chimeric Antigen/genetics/immunology ; *T-Lymphocytes/immunology/metabolism ; *Leukemia, Myeloid, Acute/therapy/immunology/genetics ; Xenograft Model Antitumor Assays ; *Receptors, Antigen, T-Cell/genetics ; },
abstract = {Chimeric antigen receptor (CAR) T-cell therapy directed to CD19 and B-cell maturation antigen has revolutionized treatment of B-cell leukemia and lymphoma, and multiple myeloma. However, identifying suitable targets for acute myeloid leukemia (AML) remains challenging due to concurrent expression of potential target antigens on normal hematopoietic stem cells or tissues. As the stress-induced B7H6 molecule is rarely found on normal tissues but expressed on many cancers including AML and melanoma, the NKp30-ligand B7H6 emerges as a promising target for NKp30-based CAR T therapy for these tumors. In this study, we report a comprehensive B7H6 expression analysis on primary AML and melanoma as well as on different tumor cell-lines examined by RT-qPCR and flow cytometry, and efficient anti-tumor reactivity of NKp30-CAR T cells to AML and melanoma. To overcome limitations of autologous CAR T-cell fitness-dependent efficacy and patient-tailored production, we generated CRISPR/Cas9-mediated TCR-knockout (TCR[KO]) NKp30-CAR T cells as an off-the-shelf approach for CAR T therapy. Functional studies comparing NKp30-CD28 CAR or NKp30-CD137 CAR TCR[+] and TCR[KO] T lymphocytes revealed superior anti-tumoral immunity of NKp30-CD28 CAR TCR[KO] T cells to AML and melanoma cell lines in vitro, and effective control of tumor burden in an NSG melanoma-xenograft mouse model. In conclusion, these findings highlight the therapeutic potential of NKp30 CAR TCR[KO] T cells for adoptive T-cell therapy to B7H6-expressing cancers, including melanoma and AML.},
}

Animals
Humans
*Melanoma/therapy/immunology/genetics
Mice
*CRISPR-Cas Systems
*Immunotherapy, Adoptive/methods
*B7 Antigens/genetics/metabolism/immunology
*Natural Cytotoxicity Triggering Receptor 3/genetics/immunology/metabolism
Cell Line, Tumor
*Receptors, Chimeric Antigen/genetics/immunology
*T-Lymphocytes/immunology/metabolism
*Leukemia, Myeloid, Acute/therapy/immunology/genetics
Xenograft Model Antitumor Assays
*Receptors, Antigen, T-Cell/genetics

@article {pmid40942109,
year = {2025},
author = {Ferrara, F and Sepe, A and Sguizzato, M and Marconi, P and Cortesi, R},
title = {A Pre-Formulation Study for Delivering Nucleic Acids as a Possible Gene Therapy Approach for Spinocerebellar Ataxia Disorders.},
journal = {Molecules (Basel, Switzerland)},
volume = {30},
number = {17},
pages = {},
pmid = {40942109},
issn = {1420-3049},
mesh = {Humans ; *Genetic Therapy/methods ; Liposomes/chemistry ; *Spinocerebellar Ataxias/therapy/genetics ; Cell Line, Tumor ; Gene Transfer Techniques ; *Nucleic Acids/chemistry/genetics ; Transfection ; CRISPR-Cas Systems ; RNA, Messenger/genetics ; Green Fluorescent Proteins/genetics ; Plasmids/genetics ; DNA/genetics/chemistry ; },
abstract = {Liposomes are lipid bilayer vesicles that are highly biocompatible, able to interact with the cell membrane, and able to release their cargo easily. The improvement of the physicochemical properties of liposomes, such as surface charge, lipid composition, and functionalization, makes these vesicles eligible delivery nanosystems for the gene therapy of many pathological conditions. In the present study, pre-formulation analysis was conducted to develop liposomes that facilitate the delivery of nucleic acids to neuronal cells, with the aim of future delivery of a CRISPR/Cas9 system designed to silence genes responsible for autosomal dominant neurodegenerative disorders. To this aim, different nucleic acid cargo models, including λ phage DNA, plasmid DNA, and mRNA encoding GFP, were considered. Liposomes with varying lipid compositions were produced using the ethanol injection method and analyzed for their dimensional stability and ability to interact with DNA. The selected formulations were tested in vitro using a neuroblastoma cell line (SH-SY5Y) to evaluate their potential toxicity and the ability to transfect cells with a DNA encoding the green fluorescent protein (pCMV-GFP). Among all formulations, the one containing phosphatidylcholine, phosphatidylethanolamine, pegylated 1,2-distearoyl-sn-glycero-3-phosphethanolamine, cholesterol, and dioctadecyl-dimethyl ammonium chloride (in the molar ratio 1:2:4:2:2) demonstrated the highest efficiency in mRNA delivery. Although this study was designed with the goal of ultimately enabling the delivery of a CRISPR/Cas9 system for treating autosomal dominant neurodegenerative disorders such as polyglutamine spinocerebellar ataxias (SCAs), CRISPR/Cas9 components were not delivered in the present work, and their application remains the objective of future investigations.},
}

Humans
*Genetic Therapy/methods
Liposomes/chemistry
*Spinocerebellar Ataxias/therapy/genetics
Cell Line, Tumor
Gene Transfer Techniques
*Nucleic Acids/chemistry/genetics
Transfection
CRISPR-Cas Systems
RNA, Messenger/genetics
Green Fluorescent Proteins/genetics
Plasmids/genetics
DNA/genetics/chemistry

@article {pmid40940741,
year = {2025},
author = {Yao, X and Feng, M and Sun, C and Yang, S and Yuan, Z and Liu, X and Li, Q and Jiang, C and Weng, X and Song, J and Mu, Y},
title = {Establishment of a CRISPR/dCas9 Activation Library for Screening Transcription Factors Co-Regulating OCT4 with GATA4 in Pig Cells.},
journal = {Cells},
volume = {14},
number = {17},
pages = {},
pmid = {40940741},
issn = {2073-4409},
mesh = {Animals ; *GATA4 Transcription Factor/metabolism/genetics ; *Octamer Transcription Factor-3/metabolism/genetics ; Swine ; *CRISPR-Cas Systems/genetics ; *Transcription Factors/metabolism/genetics ; Cell Line ; Promoter Regions, Genetic/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Library ; },
abstract = {OCT4 is a critical transcription factor for early embryonic development and pluripotency. Previous studies have shown that the regulation of OCT4 by the transcription factor GATA4 is species-specific in pigs. This study aimed to further investigate whether there are other transcription factors that co-regulate the transcription of OCT4 with GATA4 in pigs. A CRISPR activation (CRISPRa) sgRNA library was designed and constructed, containing 5056 sgRNAs targeting the promoter region of 1264 transcription factors in pigs. Then, a pig PK15 cell line was engineered with a single-copy OCT4 promoter-driven EGFP reporter at the ROSA26 locus, combined with the dCas9-SAM system for transcriptional activation. The CRISPRa sgRNA lentiviral library was used to screen for transcription factors, with or without GATA4 overexpression. Flow cytometry combined with high-throughput sequencing identified MYC, SOX2, and PRDM14 as activators and OTX2 and CDX2 as repressors of OCT4. In the presence of GATA4, transcription factors such as SALL4 and STAT3 showed synergistic activation. Functional validation confirmed that HOXD13 upregulates OCT4, while OTX2 inhibits it. GATA4 and SALL4 synergistically enhance OCT4 expression. These findings provide new insights into combinatorial mechanisms that control the transcriptional regulation of OCT4 in pigs.},
}

Animals
*GATA4 Transcription Factor/metabolism/genetics
*Octamer Transcription Factor-3/metabolism/genetics
Swine
*CRISPR-Cas Systems/genetics
*Transcription Factors/metabolism/genetics
Cell Line
Promoter Regions, Genetic/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Library

@article {pmid40940375,
year = {2025},
author = {Berger, T and Borisova, E and Gamerschlag, A and Terheyden-Keighley, D and Martins, S and Greber, B},
title = {Sequential factor delivery enables efficient workflow for universal gene editing in clinical grade iPS cells.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {32514},
pmid = {40940375},
issn = {2045-2322},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; Workflow ; },
abstract = {Human induced pluripotent stem cells (iPSCs) are gaining momentum as a powerful starting material in cell therapy. To fully harness their potential, CRISPR technology permits endogenous gene modifications as well as the introduction of advanced features, to increase the immune compatibility of the cells or insert suicide genes for enhancing therapeutic safety, for instance. However, genetic manipulation of iPSCs, in particular the generation of knock-in lines, remains relatively inefficient. Conventional mitigation strategies, such as enriching for positive cells using antibiotic selection or complex instrumentation, may, however, cause conflicts with good manufacturing practice (GMP) requirements. To address this challenge, we have systematically optimized a basic gene editing procedure using both Cas9 and Cas12a-based ribonucleoprotein (RNP) complexes. Based on the sequential delivery of RNPs and donor plasmids as a critical hallmark, this virus-free approach permits knock-ins of full-length transgenes at above 30% efficiency, while readily identifying positive clones through random screening at small scale. We exemplify these advances by creating and characterizing homozygous iPSC lines depleted of HLA class I and carrying an inducible caspase-9 suicide gene. Isolated clones from independent GMP iPSC lines retained genomic integrity, differentiation capability, and functionality of the safety switch in the differentiated state. This improved methodology will form a flexible platform for custom gene editing universally applicable both in basic iPSC research and therapy.},
}

*Induced Pluripotent Stem Cells/metabolism/cytology
Humans
*Gene Editing/methods
CRISPR-Cas Systems
Workflow

@article {pmid40897805,
year = {2025},
author = {Xu, N and Cho, HS and Hackland, JOS and Benito-Kwiecinski, S and Saurat, N and Harschnitz, O and Russo, MV and Garippa, R and Ciceri, G and Studer, L},
title = {Genome-wide CRISPR screen identifies Menin and SUZ12 as regulators of human developmental timing.},
journal = {Nature cell biology},
volume = {27},
number = {9},
pages = {1411-1421},
pmid = {40897805},
issn = {1476-4679},
support = {UM1 HG012654/HG/NHGRI NIH HHS/United States ; R01NS128087//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; R01 NS128087/NS/NINDS NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; UM1HG012654//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; P30 CA08748//U.S. Department of Health & Human Services | NIH | NCI | Division of Cancer Epidemiology and Genetics, National Cancer Institute (National Cancer Institute Division of Cancer Epidemiology and Genetics)/ ; },
mesh = {Humans ; Gene Expression Regulation, Developmental ; Cell Differentiation/genetics ; *Proto-Oncogene Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; *Polycomb Repressive Complex 2/genetics/metabolism ; *Human Embryonic Stem Cells/metabolism/cytology ; Histones/metabolism/genetics ; PAX6 Transcription Factor/genetics/metabolism ; Promoter Regions, Genetic ; Epigenesis, Genetic ; Neurons/metabolism/cytology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Embryonic Development/genetics ; Neoplasm Proteins ; Transcription Factors ; },
abstract = {Embryonic development follows a conserved sequence of events across species, yet the pace of development is highly variable and particularly slow in humans. Species-specific developmental timing is largely recapitulated in stem cell models, suggesting a cell-intrinsic clock. Here we use directed differentiation of human embryonic stem cells into neuroectoderm to perform a whole-genome CRISPR-Cas9 knockout screen and show that the epigenetic factors Menin and SUZ12 modulate the speed of PAX6 expression during neural differentiation. Genetic and pharmacological loss-of-function of Menin or SUZ12 accelerate cell fate acquisition by shifting the balance of H3K4me3 and H3K27me3 at bivalent promoters, thereby priming key developmental genes for faster activation upon differentiation. We further reveal a synergistic interaction of Menin and SUZ12 in modulating differentiation speed. The acceleration effects were observed in definitive endoderm, cardiomyocyte and neuronal differentiation paradigms, pointing to chromatin bivalency as a general driver of timing across germ layers and developmental stages.},
}

Humans
Gene Expression Regulation, Developmental
Cell Differentiation/genetics
*Proto-Oncogene Proteins/genetics/metabolism
*CRISPR-Cas Systems
*Polycomb Repressive Complex 2/genetics/metabolism
*Human Embryonic Stem Cells/metabolism/cytology
Histones/metabolism/genetics
PAX6 Transcription Factor/genetics/metabolism
Promoter Regions, Genetic
Epigenesis, Genetic
Neurons/metabolism/cytology
*Clustered Regularly Interspaced Short Palindromic Repeats
*Embryonic Development/genetics
Neoplasm Proteins
Transcription Factors

@article {pmid40890563,
year = {2025},
author = {Li, X and Liu, C and Guo, G and Xu, Q and Ren, X and Tuerhongjiang, A and Liu, J and Zeng, J and Wen, CY},
title = {CRISPR-Driven Portable Piezoresistive Biosensor with Cascaded Signal Amplification for Ultrasensitive Osteocalcin Detection.},
journal = {Analytical chemistry},
volume = {97},
number = {36},
pages = {19552-19559},
doi = {10.1021/acs.analchem.5c02678},
pmid = {40890563},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; Humans ; *Osteocalcin/analysis/blood ; Limit of Detection ; Platinum/chemistry ; *CRISPR-Cas Systems ; Metal Nanoparticles/chemistry ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Low-turnover osteoporosis diagnosis urgently requires sensitive detection of low-abundance osteocalcin (OC), yet conventional methods remain constrained by insufficient sensitivity, cumbersome instrumentation, and laborious operations. We devise a CRISPR-driven pressure bioassay that synergistically integrates molecular recognition, enzymatic amplification, and signal transduction for dual-amplification-enhanced OC quantification. The system features an engineered "locked-to-activated" molecular switch, where target binding liberates CRISPR-activating DNA strands, initiating Cas14a-catalyzed cleavage of ssDNA tethers on Fe3O4-ssDNA-Pt nanoassemblies. This cascade releases a multitude of platinum nanoparticles (the first amplification stage). Subsequently, the liberated platinum nanoparticles drive the catalytic decomposition of H2O2 within sealed microchambers, generating a massive flux of oxygen gas molecules (O2) (second amplification stage). Coupled with a laboratory-fabricated nanostructured piezoresistive sensor (20 Pa resolution), this two-stage amplification strategy achieves high sensitivity with a 7.31 pg/mL detection limit, 124-fold lower than commercial ELISA, while completing analysis within 60 min. The platform demonstrates remarkable specificity (spike recovery of 113%, 112%, and 110% in human serum), operational robustness across varying environmental temperatures (15-40 °C), and compatibility with miniaturized instrumentation. Clinical validation through serum matrix analysis reveals excellent correlation (R[2] = 0.982) with reference values. By integrating CRISPR programmability, nanozyme-amplified signaling, and portable piezoresistive sensing, this work provides a sensitive point-of-care osteoporosis screening method for resource-limited settings.},
}

*Biosensing Techniques/methods
Humans
*Osteocalcin/analysis/blood
Limit of Detection
Platinum/chemistry
*CRISPR-Cas Systems
Metal Nanoparticles/chemistry
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40887819,
year = {2025},
author = {Pérez-Maroto, J and Sepp-Lorenzino, L and Castaño-Esteban, D and Palacios, D and Sot, B},
title = {Advancements in Nonviral Gene Editing Strategies for Rare Diseases.},
journal = {Human gene therapy},
volume = {36},
number = {17-18},
pages = {1118-1137},
doi = {10.1177/10430342251372056},
pmid = {40887819},
issn = {1557-7422},
mesh = {Humans ; *Gene Editing/methods ; *Rare Diseases/therapy/genetics ; *Genetic Therapy/methods ; Genetic Vectors/genetics ; Gene Transfer Techniques ; CRISPR-Cas Systems ; Animals ; Nanoparticles/chemistry ; Nanomedicine ; },
abstract = {Rare diseases are serious and often chronic conditions that affect a small number of individuals. However, with over 7,000 rare diseases identified, their cumulative global numbers and impact are substantial. A considerable proportion of these conditions is caused by genetic abnormalities. Among these, monogenic disorders are of particular relevance, as they are caused by mutations in specific genes. The development of gene therapy, and more specifically, gene editing, offers innovative approaches to treat these rare diseases. A significant challenge associated with the implementation of such strategies concerns the delivery of gene editing tools. Nonviral vectors based on nanomaterials have demonstrated considerable potential as promising alternatives to viral vectors, thereby overcoming their disadvantages. The biocompatibility and tunability of nanoparticles, along with their potential capacity to target diverse tissues, positions them as a promising therapeutic approach for the treatment of a wide range of organ-specific rare diseases. Here, we review current progress in the development and evaluation of novel nanomedicine strategies for gene editing in rare diseases, highlighting new gene editing approaches, delivery systems, and potential targets.},
}

Humans
*Gene Editing/methods
*Rare Diseases/therapy/genetics
*Genetic Therapy/methods
Genetic Vectors/genetics
Gene Transfer Techniques
CRISPR-Cas Systems
Animals
Nanoparticles/chemistry
Nanomedicine

@article {pmid40848918,
year = {2025},
author = {Coratella, E and Bohnert, R and Fischer, B and Lemmens, M and Alpert, T and Beibel, M and Oertli, M and Naumann, U and Elhajouji, A and Odermatt, A and Libertini, S},
title = {SACF and GILA assays on AML12 cells show limited predictive value for mouse liver genotoxicity.},
journal = {Toxicology and applied pharmacology},
volume = {504},
number = {},
pages = {117529},
doi = {10.1016/j.taap.2025.117529},
pmid = {40848918},
issn = {1096-0333},
mesh = {Animals ; Mice ; Gene Editing ; *Liver Neoplasms/genetics/pathology ; CRISPR-Cas Systems ; *Carcinoma, Hepatocellular/genetics/pathology ; Dependovirus/genetics ; *Liver/pathology/metabolism/drug effects ; Predictive Value of Tests ; Mutagenicity Tests/methods ; Humans ; },
abstract = {Hepatocellular carcinoma (HCC) has been observed in neonatal mice following the integration of recombinant Adeno-Associated Viruses (rAAV) into the Rian locus. rAAV-related oncogenic risk for patients remains unclear, and the lack of relevant in vitro methods hinders its proper assessment. The soft agar colony-forming (SACF) assay and the growth in low attachment assay (GILA) monitor anchorage-independent growth, a hallmark of transformed adherent cells, and have been previously proposed to assess the tumorigenicity of CRISPR/Cas9-edited human MCF10A cells. Here, we introduce murine versions of SACF and GILA as surrogate in vitro systems to evaluate the risk of HCC development following genome editing or rAAV induced insertional mutagenesis. Selected tumor suppressors linked to HCC onset in vivo were edited through CRISPR/Cas9 in the hepatic murine cell line AML12. The knockout of neurofibromin (Nf2) and the dual inactivation of tumor protein p53 (Tp53) and phosphatase and tensin homolog (Pten) induced anchorage-independence, while the editing of Axin1, Ctnnb1 (coding for β-catenin), and tuberous sclerosis complex 1 (Tsc1) did not promote growth in anchorage-free conditions. Additionally, we generated stable AML12 and MCF10A clones with the rAAV genome respectively integrated into Rian and MEG8, the human homolog of Rian; however, these clones did not show anchorage independence when seeded in SACF and GILA. Overall, the murine SACF and GILA exhibit low predictive value for HCC development, failing to detect rAAV- and tumor-suppressors-associated oncogenicity. While further optimization may improve assays performance, these results highlight the need for more appropriate in vitro methodologies to accurately evaluate rAAV genotoxicity.},
}

Animals
Mice
Gene Editing
*Liver Neoplasms/genetics/pathology
CRISPR-Cas Systems
*Carcinoma, Hepatocellular/genetics/pathology
Dependovirus/genetics
*Liver/pathology/metabolism/drug effects
Predictive Value of Tests
Mutagenicity Tests/methods
Humans

@article {pmid40845730,
year = {2025},
author = {Lai, B and Guo, H and Zhang, K and Wang, H and Qiu, X and Lin, L and Shi, R and Sedibe, M and Zou, Y and Norvienyeku, J},
title = {Putative SET domain-containing proteins play significant roles in regulating sporulation and pathogenicity in Phytophthora capsici.},
journal = {Microbiological research},
volume = {301},
number = {},
pages = {128316},
doi = {10.1016/j.micres.2025.128316},
pmid = {40845730},
issn = {1618-0623},
mesh = {*Phytophthora/pathogenicity/genetics/growth & development ; *Plant Diseases/microbiology/parasitology ; Virulence/genetics ; Gene Expression Profiling ; Capsicum/microbiology ; CRISPR-Cas Systems ; *Fungal Proteins/genetics/metabolism ; },
abstract = {Phytophthora capsici is a filamentous oomycete responsible for root rot, fruit rot, leaf blight, and other economically destructive diseases in multiple plant species, including pepper (Capsicum annuum), tomato (Solanum lycopersicum), squash (Cucurbita pepo), eggplant (Solanum melongena), faba bean (Vicia faba), and lima bean (Phaseolus lunatus), among others. The pathogen causes significant yield losses in fruit and vegetable crops globally. Multiple molecular parameters, including effector proteins and epigenetic modulators, play vital roles in modulating the physio pathological development of P. capsici. Here, we identified 56 SET domain-containing proteins in P. capsici, with 35 predicted as potential effectors. Transcriptomic analysis revealed the upregulation of 13 candidate effector genes during infection, suggesting their roles in pathogenicity. We successfully deleted one effector, PcSET-C (DVH05_022087), and two non-effector proteins, PcSET-B (DVH05_004260), and PcSET-A (DVH05_000194), using CRISPR-Cas9 and homologous recombination strategies. Phenotypic analysis showed that targeted deletion of the PcSET-A, and PcSET-B gene significantly impaired vegetative growth, while ΔPcset-C strains displayed growth like wild-type strains. Additionally, we showed that targeted disruption of all three genes resulted in reduced asexual sporulation and almost completely abolished the pathogenicity or virulence of ΔPcset-A, ΔPcset-B, and ΔPcset-C strains on different P. capsici-susceptible plants. However, targeted replacement of the three SET domain-containing genes investigated in this study has no significant adverse effects on zoospore release in P. capsici. These findings provide insights into the crucial role of SET domain-containing proteins in both morphological and pathological development of P. capsici and underscore PcSETs as potential targets for disease control.},
}

*Phytophthora/pathogenicity/genetics/growth & development
*Plant Diseases/microbiology/parasitology
Virulence/genetics
Gene Expression Profiling
Capsicum/microbiology
CRISPR-Cas Systems
*Fungal Proteins/genetics/metabolism

@article {pmid40834922,
year = {2025},
author = {Zhao, JJ and Tian, SN and Peng, ZY and Ren, JX and Zhang, YY and Li, GH and Deng, DH and Zhang, JP and Zhang, XB},
title = {Biomembrane-inspired lipid nanoparticles enhance CRISPR-Cas9 editing for hemophilia A.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {386},
number = {},
pages = {114141},
doi = {10.1016/j.jconrel.2025.114141},
pmid = {40834922},
issn = {1873-4995},
mesh = {Animals ; *CRISPR-Cas Systems ; *Hemophilia A/therapy/genetics ; *Gene Editing/methods ; *Nanoparticles/administration & dosage/chemistry ; Humans ; *Factor VIII/genetics ; Mice ; Mice, Inbred C57BL ; Genetic Therapy/methods ; Dependovirus/genetics ; Male ; *Lipids/chemistry/administration & dosage ; Liposomes ; },
abstract = {Lipid nanoparticles (LNPs) have substantially advanced RNA-based therapies; however, their use for CRISPR-Cas9 remains limited by sub-optimal endosomal escape, innate immune activation, transient nuclease expression, and restricted tissue specificity. Here, we engineered biomembrane-inspired LNPs containing sphingomyelin and C18-galactosyl ceramide (C18-GalCer) to improve liver-targeted CRISPR delivery. The optimized formulation increased in vivo editing efficiency 2.3-fold relative to a benchmark ALC-0315 LNP. Compared with adeno-associated virus (AAV) vectors, transient mRNA delivery reduced the theoretical risk of insertional mutagenesis, lowered innate immune readouts, and allowed dose titration. By pairing AAV-mediated delivery of a therapeutic Factor VIII donor with LNP-mediated CRISPR-Cas9, we achieved efficient and durable gene correction in a hemophilia A mouse model. A single LNP dose restored plasma Factor VIII activity to >50 % of wild-type levels and maintained this correction for >12 weeks, with low systemic cytokine induction, undetectable off-target insertions, and no overt toxicity. The LNPs retained physicochemical properties and editing potency after prolonged storage and multiple freeze-thaw cycles, supporting their translational potential. Collectively, our findings position biomembrane-inspired LNPs as a safe and efficient non-viral CRISPR platform with potential applicability to other gene-editing therapies.},
}

Animals
*CRISPR-Cas Systems
*Hemophilia A/therapy/genetics
*Gene Editing/methods
*Nanoparticles/administration & dosage/chemistry
Humans
*Factor VIII/genetics
Mice
Mice, Inbred C57BL
Genetic Therapy/methods
Dependovirus/genetics
Male
*Lipids/chemistry/administration & dosage
Liposomes

@article {pmid40749819,
year = {2026},
author = {Gaur, A and Bhakhri, H and Mishra, N and Sharma, S and Bansal, T and Kalaivani, M and Brijwal, M and Das, BK and Lodha, R and Sinha, S and Luthra, K},
title = {A rapid CRISPR/Cas12a-based assay for the detection of HIV-1 Clade C in infants.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {578},
number = {},
pages = {120518},
doi = {10.1016/j.cca.2025.120518},
pmid = {40749819},
issn = {1873-3492},
mesh = {Humans ; *HIV-1/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; Infant ; *HIV Infections/diagnosis/virology ; Nucleic Acid Amplification Techniques ; },
abstract = {BACKGROUND: Early detection of HIV-1 infection is essential for initiating antiretroviral therapy (ART) to suppress viremia and prevent disease progression. Timely diagnosis, especially in infants, is critical as rapid antibody-based serology tests are ineffective due to the presence of maternal antibodies.

METHODS: We developed a CRISPR/Cas12a-based HIV-1 detection assay by optimizing components for coupled isothermal preamplification using recombinase polymerase amplification (RPA). The assay targeted the conserved region in the pol gene specific to HIV-1 with the designed CRISPR RNA (crRNA). CRISPR/Cas12a-mediated cleavage of viral cDNA was visualized through the collateral cleavage of a single-stranded DNA-FAM-BQ reporter, enabling rapid and visually detectable outcomes. The performance of the assay was evaluated using plasma from 41 HIV-1 Clade C (HIV-1C) seropositive individuals, including 28 HIV-1C infected infant samples, HIV-1 Indian Clade C and Clade B genome plasmids, viral disease control DNA/RNA samples (Influenza, RSV, Parvovirus, HPIV, CMV, and HBV), and 31 healthy donor plasma samples. Sensitivity and specificity were assessed, and detection was performed using fluorescence, visual readout, and lateral flow dipsticks.

RESULTS: The CRISPR/Cas12a-based HIV-1 Clade C detection assay achieved a sensitivity of 96 % and a specificity of 92.65 %. The assay successfully provided results through both fluorescence and visual readouts and was compatible with lateral flow dipstick formats, facilitating easy and rapid detection.

CONCLUSIONS: The developed CRISPR/Cas12a-based HIV-1C detection assay demonstrates high sensitivity and specificity for Clade C, indicating its potential as a robust point-of-care molecular diagnostic tool for HIV-1C. Additionally, it may serve as a rapid nucleic acid test alternative for detecting mother-to-child transmission of HIV-1C in infants under two years of age, where traditional antibody-based tests are ineffective. This assay holds promise for improving early HIV-1 diagnosis and timely initiation of ART, ultimately contributing to better disease management and outcomes.},
}

Humans
*HIV-1/genetics/isolation & purification
*CRISPR-Cas Systems/genetics
Infant
*HIV Infections/diagnosis/virology
Nucleic Acid Amplification Techniques

@article {pmid40709654,
year = {2025},
author = {Chang, MM},
title = {An Undergraduate Laboratory on Recombineering and CRISPR/Cas9-Assisted Gene Editing in Escherichia coli.},
journal = {Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology},
volume = {53},
number = {5},
pages = {555-562},
doi = {10.1002/bmb.70002},
pmid = {40709654},
issn = {1539-3429},
mesh = {*Escherichia coli/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Laboratories ; Humans ; Students ; Universities ; *Recombination, Genetic ; *Genetic Engineering ; },
abstract = {Laboratory experience is vital to undergraduate science education. It allows students to observe and conduct engaging experiments to enhance their skills and literacy, helps them retain knowledge, and deepens their understanding of related content covered in lectures. This paper reports a 4-week undergraduate laboratory exercise on Escherichia coli gene editing by recombineering, recombination-mediated genetic engineering, with or without clustered regularly interspaced short palindromic repeats and their associated protein 9 (CRISPR/Cas9). Gene editing makes precise modifications to the DNA of living organisms that influence their development and functions. As technology evolves, recombineering and CRISPR/Cas9 have replaced methods that use restriction enzymes and DNA ligase and are applied to a wide variety of research and applications. It is necessary to introduce undergraduates to these two rapidly growing technologies. Student results obtained from the lab indicate that antisense single-stranded oligodeoxynucleotide (ssODN) has a 15-20 times higher recombineering efficiency than the sense strand. Treatment with a plasmid containing the crRNA target of CRISPR/Cas9 increased recombineering efficiency. Instructional assessments, based on student feedback, revealed that the lab had clear objectives, instructions, and explicit protocols, with sufficient time to complete them, and was found to be interesting and worthwhile. Student learning outcomes, assessed by comparing pre-lab questions and post-lab tests, suggested that they learned the underlying principles and detailed molecular mechanisms. Besides learning the technologies and acquiring basic laboratory skills, students practiced key components of scientific research, such as data collection, analysis, and scientific communication.},
}

*Escherichia coli/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Laboratories
Humans
Students
Universities
*Recombination, Genetic
*Genetic Engineering

@article {pmid40694540,
year = {2025},
author = {Knoll, N and Masser, S and Bordas, B and Ebright, RY and Li, G and Kesar, D and Destefanis, E and Kania, N and Rodriguez, DJ and Jen, J and Zagar, SE and Mensah, C and Chen, Z and Moffitt, SJ and Enakireru, EM and He, Y and Feng, B and Chokshi, MK and Jin, CY and Raghavan, S and Sellers, WR and Mulvaney, KM},
title = {CRISPR-Drug Combinatorial Screening Identifies Effective Combination Treatments for MTAP-Deleted Cancer.},
journal = {Cancer research},
volume = {85},
number = {18},
pages = {3518-3539},
doi = {10.1158/0008-5472.CAN-25-1464},
pmid = {40694540},
issn = {1538-7445},
support = {R35GM154987//National Institute of General Medical Sciences (NIGMS)/ ; 1R01CA233626//Center for Cancer Research (CCR)/ ; CA232543//Basic Research Laboratory (BRL)/ ; V Scholar Grant//V Foundation for Cancer Research (VFCR)/ ; Promos Scholarship//German Academic Exchange Service (DAAD)/ ; //Austrian Marshall Plan Scholarship/ ; Travel Scholarship//BioTechMed-Graz (BIOTECHMEDGRAZ)/ ; CA260442//Center for Cancer Research (CCR)/ ; //Hale Family Center for Pancreatic Cancer Research (Hale Family Research Center At Dana-Farber Cancer Institute)/ ; Claudia Adams Barr Program//Dana-Farber Cancer Institute (DFCI)/ ; },
mesh = {Humans ; *Purine-Nucleoside Phosphorylase/genetics ; Animals ; Mice ; *Protein-Arginine N-Methyltransferases/antagonists & inhibitors/genetics ; *CRISPR-Cas Systems ; *Cyclin-Dependent Kinase Inhibitor p16/genetics ; Cell Line, Tumor ; Xenograft Model Antitumor Assays ; *Lung Neoplasms/genetics/drug therapy/pathology ; *Antineoplastic Combined Chemotherapy Protocols/pharmacology/therapeutic use ; },
abstract = {UNLABELLED: Cyclin-dependent kinase inhibitor 2A (CDKN2A)/methylthioadenosine phosphorylase (MTAP) codeletion occurs frequently in non-small cell lung cancer and other solid tumors, including glioblastoma and pancreatic ductal adenocarcinoma. Lung cancer remains the leading cause of cancer-related mortality, and fewer than 15% of patients with glioblastoma or pancreatic cancer survive 5 years, underscoring the need for more effective therapies. Protein arginine methyltransferase 5 (PRMT5) is a synthetic-lethal dependency in MTAP-null tumors and an attractive therapeutic target for CDKN2A/MTAP-deleted cancers. A new revolutionary class of inhibitors, referred to as methylthioadenosine (MTA)-cooperative PRMT5 inhibitors (PRMT5i), has shown promising results in ongoing early-phase clinical trials. Nonetheless, effective cancer treatment typically requires therapeutic combinations to improve response rates and defeat emergent resistant clones. Thus, we sought to determine whether perturbation of other pathways could improve the efficacy of MTA-cooperative PRMT5is (MTAC-PRMT5i). Using a paralog and single gene targeting CRISPR library, we screened MTAP-deleted cancers in the presence or absence of MTAC-PRMT5is. Loss of several genes sensitized cells to PRMT5 inhibition, including members of the MAPK pathway. Chemical inhibition of MAPK pathway members using KRAS, MEK, ERK, and RAF inhibitors synergized with PRMT5 inhibition to kill CDKN2A/MTAP-null, RAS-active tumors. Furthermore, MTAC-PRMT5is combined with either KRAS or RAF inhibitors led to complete responses in vivo, emphasizing the potential benefit for patients. Lastly, cell lines resistant to KRAS inhibition were not resistant to MTAC-PRMT5is and vice versa, suggesting noncross-reactive mechanisms of resistance. Overall, this study identifies therapeutic combinations with MTAC-PRMT5is that may offer significant benefits to patients.

SIGNIFICANCE: Combining PRMT5 and MAPK pathway inhibitors leads to complete, durable responses in lung cancer models, providing an effective therapeutic strategy for the 4-5% of cancer patients harboring CDKN2A/MTAP deletion and MAPK alterations. See related article by Drizyte-Miller et al., p. 3540.},
}

Humans
*Purine-Nucleoside Phosphorylase/genetics
Animals
Mice
*Protein-Arginine N-Methyltransferases/antagonists & inhibitors/genetics
*CRISPR-Cas Systems
*Cyclin-Dependent Kinase Inhibitor p16/genetics
Cell Line, Tumor
Xenograft Model Antitumor Assays
*Lung Neoplasms/genetics/drug therapy/pathology
*Antineoplastic Combined Chemotherapy Protocols/pharmacology/therapeutic use

@article {pmid40684990,
year = {2025},
author = {Gustafsson, O and Krishna, S and Borate, S and Ghaeidamini, M and Liang, X and Saher, O and Cuellar, R and Birdsong, BK and Roudi, S and Estupiñán, HY and Alici, E and Smith, CIE and Esbjörner, EK and Spuler, S and de Jong, OG and Escobar, H and Nordin, JZ and Andaloussi, SEL},
title = {Advanced peptide nanoparticles enable robust and efficient delivery of gene editors across cell types.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {386},
number = {},
pages = {114038},
doi = {10.1016/j.jconrel.2025.114038},
pmid = {40684990},
issn = {1873-4995},
mesh = {Humans ; *Gene Editing/methods ; *Nanoparticles/chemistry/administration & dosage ; HEK293 Cells ; CRISPR-Cas Systems ; *Cell-Penetrating Peptides/chemistry/administration & dosage ; Silicon Dioxide/chemistry ; Induced Pluripotent Stem Cells/metabolism ; *Gene Transfer Techniques ; Integrases/genetics/administration & dosage ; CRISPR-Associated Protein 9/genetics ; },
abstract = {Efficient delivery of the CRISPR/Cas9 system and its larger derivatives, base editors, and prime editors remain a major challenge, particularly in tissue-specific stem cells and induced pluripotent stem cells (iPSCs). This study optimized a novel family of cell-penetrating peptides, hPep, to deliver gene-editing ribonucleoproteins. The hPep-based nanoparticles enable highly efficient and biocompatible delivery of Cre recombinase, Cas9, base-, and prime editors. Using base editors, robust and nearly complete genome editing was achieved in the human cells: HEK293T (96%), iPSCs (74%), and muscle stem cells (80%). This strategy opens promising avenues for ex vivo and, potentially, in vivo applications. Incorporating silica particles enhanced the system's versatility, facilitating cargo-agnostic delivery. Notably, the nanoparticles can be synthesized quickly on a benchtop and stored as lyophilized powder without compromising functionality. This represents an important advancement in the feasibility and scalability of gene-editing delivery technologies.},
}

Humans
*Gene Editing/methods
*Nanoparticles/chemistry/administration & dosage
HEK293 Cells
CRISPR-Cas Systems
*Cell-Penetrating Peptides/chemistry/administration & dosage
Silicon Dioxide/chemistry
Induced Pluripotent Stem Cells/metabolism
*Gene Transfer Techniques
Integrases/genetics/administration & dosage
CRISPR-Associated Protein 9/genetics

@article {pmid40680607,
year = {2025},
author = {Eladl, O},
title = {CRISPR interference-mediated silencing of pro-inflammatory genes in autoimmune models: functional and biophysical evaluation with sustained effects in primary human PBMCs.},
journal = {International immunopharmacology},
volume = {163},
number = {},
pages = {115257},
doi = {10.1016/j.intimp.2025.115257},
pmid = {40680607},
issn = {1878-1705},
mesh = {Humans ; *Leukocytes, Mononuclear/immunology ; *Gene Silencing ; CRISPR-Cas Systems ; *Autoimmune Diseases/genetics/immunology/therapy ; CD40 Antigens/genetics ; Interleukin-6/genetics ; Interferon-gamma/genetics ; *Inflammation/genetics ; Cells, Cultured ; Cytokines/genetics ; },
abstract = {Autoimmune diseases are caused by unregulated immune stimulation and overproduction of inflammatory cytokines, leading to chronic tissue injury and disability. Current treatments are broad-acting and short-lived, highlighting the need for targeted molecular interventions with prolonged immunomodulatory effects. Here, we demonstrate CRISPR interference (CRISPRi), employing a catalytically inactive Cas9 fused to a KRAB repression domain, to potently and selectively silence major inflammatory genes-IL-6, CD40, and IFN-γ-in human immune cell lines. Utilizing biophysical methods such as surface plasmon resonance and fluorescence anisotropy, complemented by flow cytometric analysis, we validated functional interference with inflammatory signaling cascades and significant downregulation of immune activation markers. These findings indicate that CRISPRi not only represses gene expression but also significantly affects downstream cytokine-receptor interactions and immune cell activation. Compared to other gene silencing approaches, like siRNA and antisense oligonucleotides, CRISPRi showed greater specificity and longer duration, with sustained repression lasting up to 72 h post-treatment. Importantly, we confirmed these effects in primary human peripheral blood mononuclear cells (PBMCs), where CRISPRi significantly suppressed pro-inflammatory cytokine release and modulated immune cell phenotypes. These results highlight the potent, programmable, and sustained immunomodulatory potential of CRISPRi as a therapeutic for autoimmune diseases characterized by inflammation and immune dysregulation.},
}

Humans
*Leukocytes, Mononuclear/immunology
*Gene Silencing
CRISPR-Cas Systems
*Autoimmune Diseases/genetics/immunology/therapy
CD40 Antigens/genetics
Interleukin-6/genetics
Interferon-gamma/genetics
*Inflammation/genetics
Cells, Cultured
Cytokines/genetics

@article {pmid40673842,
year = {2025},
author = {Johnson, MJ and Sumstad, D and Folsom, TD and Slipek, NJ and DeFeo, AP and Growe, M and Kadidlo, D and Thyagarajan, B and Starr, TK and Lou, E and Choudhry, M and Moriarity, BS and Webber, BR and McKenna, DH},
title = {Clinical manufacture of CRISPR/Cas9-based cytokine-induced SH2 protein knock-out tumor-infiltrating lymphocytes for gastrointestinal cancers.},
journal = {Cytotherapy},
volume = {27},
number = {10},
pages = {1229-1239},
doi = {10.1016/j.jcyt.2025.06.007},
pmid = {40673842},
issn = {1477-2566},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Lymphocytes, Tumor-Infiltrating/immunology/metabolism ; *Gastrointestinal Neoplasms/immunology/therapy/pathology/genetics ; *Cytokines ; Male ; Female ; Gene Knockout Techniques ; },
abstract = {INTRODUCTION: The prognosis of stage IV gastrointestinal (GI) carcinomas is poor with a 15% five-year survival rate for colorectal carcinomas. To improve efficacy of tumor infiltrating lymphocytes (TIL), we isolated mutation-reactive autologous TIL and employed CRISPR/Cas9 to knockout (KO) the intracellular checkpoint protein CISH, which has been shown to enhance T cell expansion, functional avidity, and cytokine polyfunctionality, with consequent durable regression of established tumors in an animal model.

MATERIALS & METHODS: TIL cultures were initiated from resected tumor fragments and maintained for six weeks before harvest and cryopreservation. Candidate neoantigens were nominated by exome sequencing and peptides were used to identify mutation reactive (MR) TIL. Selected MR TIL were thawed and allowed to recover for 24-36 h in media with 10% AB serum, 6000 IU/mL IL-2, and 5 ng/mL IL-7 and IL-15 followed by stimulation with plate-bound anti-CD3/soluble anti-CD28 for 4 days. CISH KO was performed by electroporation of Cas9 mRNA and chemically modified single guide RNA. Between 5 -7.5 million viable cells were added to each 100 cm[2] G-Rex vessel containing 600 mL expansion media (with allogeneic feeder MNC:TIL = 100:1) and incubated for 6-8 days. Cultures were evaluated and split according to cell concentration criteria (and dose cohort) and incubated for an additional 6-8 days. On day 14, all of the cells were harvested, washed with buffer and cryopreserved (5% DMSO). Lot release testing included: viability, %CD3+, cytology review, Gram stain, sterility, endotoxin, mycoplasma, and interferon gamma (IFN-γ) production. Additional testing included DNA sequencing to determine genomic CISH editing efficiency and a Western blot for determination of CISH protein loss.

RESULTS: Patients with GI cancers (colon [10], rectal [8], pancreatic [1], and esophageal [1]) underwent tumor collection. Nineteen of 22 tumor biopsies sampled from 20 patients total proceeded to KO/expansion. Final TIL product results (mean [SD], median [range]) were: viable count (x 10[10]) -3.25 (3.67), 1.95 (0.018-12.40); viable TIL fold expansion -327.1 (364.8), 153.1 (8-1454); % viability - 76 (13), 78 (43-92); % CD3 -94.4 (5.4), 95.8 (78.6-99.4); % CISH KO efficiency - 75 (29), 87 (0-96); % editing efficiency - 59.9 (24.8), 66.9 (0.4-86). Viability fell below 70% for five TIL products. All other lot release testing has met specification. Thirteen patients have received TIL; six patients were not treated due to disease progression prior to anticipated infusion.

CONCLUSION: The translation of CRISPR/Cas9-based CISH KO MR TIL from the basic research lab to current good manufacturing practices The (cGMP) facility was successful, allowing for optimized, large-scale expansion in support of a first-in-human clinical trial to treat patients with metastatic GI cancers (ClinicalTrials.gov Identifier: NCT04426669).},
}

Humans
*CRISPR-Cas Systems/genetics
*Lymphocytes, Tumor-Infiltrating/immunology/metabolism
*Gastrointestinal Neoplasms/immunology/therapy/pathology/genetics
*Cytokines
Male
Female
Gene Knockout Techniques

@article {pmid40641457,
year = {2025},
author = {Su, P and Wu, L and Li, D and Song, W and Tao, D and Liu, L and Wang, Q and Gao, M and Xu, T and Liu, X and Xie, S and Zhang, X and Zhou, J and Miao, YL},
title = {CRISPR Screening Reveals a Novel Role for FOXH1 in Regulating Pluripotency of Porcine Embryonic Stem Cells.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {34},
pages = {e09495},
doi = {10.1002/advs.202509495},
pmid = {40641457},
issn = {2198-3844},
support = {2024YFA1802400//National Key R&D Program of China/ ; 2022YFD1302200//National Key R&D Program of China/ ; 32425051//National Natural Science Foundation of China/ ; 2662023DKPY001//Fundamental Research Funds for the Central Universities/ ; 2024040801020291//Knowledge Innovation Program of Wuhan-Basic Research/ ; },
mesh = {Animals ; Swine ; Cell Differentiation/genetics ; *CRISPR-Cas Systems/genetics ; *Embryonic Stem Cells/metabolism/cytology ; *Pluripotent Stem Cells/metabolism/cytology ; *Forkhead Transcription Factors/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Porcine extended potential stem cells (pEPSCs), which exhibit both self-renewal and pluripotency, are promising for application in both agricultural biotechnology and regenerative medicine. However, the molecular mechanisms governing these two interconnected properties remain elusive. Here, two types of CRISPR-Cas9 screenings are conducted in pEPSCs. This fitness screening identified several genes essential for cell viability, including PRMT1, MYBL2, and NASP. Concurrently, FACS-based screenings revealed genes crucial for pluripotency, such as SOX2, ZFP42, and FOXH1. Notably, it is demonstrated that FOXH1 is required for maintaining pluripotency in pEPSCs, which complements the understanding of its role in mesendoderm specification. pEPSCs lacking FOXH1 exhibited a flatter and more dispersed clonal morphology, accompanied by downregulation of pluripotency genes and upregulation of lineage-specific genes. Additionally, FOXH1 knockdown significantly impaired blastocyst formation during early pig embryogenesis. Functionally, the dual role of FOXH1 in pluripotency maintenance and cell differentiation is validated: FOXH1 preserves pluripotency by enhancing chromatin accessibility at pluripotency gene loci, while also influencing lineage specification through H3K4me3 modification at developmental related genes. Thus, these findings uncover a novel role of FOXH1 involved in the core regulatory network that orchestrates gene expression programs to maintain the pluripotency state of pEPSCs and provide valuable insights into categorizing gene function.},
}

Animals
Swine
Cell Differentiation/genetics
*CRISPR-Cas Systems/genetics
*Embryonic Stem Cells/metabolism/cytology
*Pluripotent Stem Cells/metabolism/cytology
*Forkhead Transcription Factors/genetics/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid40586425,
year = {2025},
author = {Han, Z and Wang, X and Yu, D and Wang, J and Sun, K and Wang, S and Zhang, Y and Feng, G and Li, W and Hai, T and Ren, J},
title = {Achieving scalable expansion of therapeutic porcine hepatocytes in vivo through serial transplantation.},
journal = {Animal models and experimental medicine},
volume = {8},
number = {7},
pages = {1337-1344},
pmid = {40586425},
issn = {2576-2095},
support = {2021YFA0805905//National Key Research and Development Program of China/ ; 2023YFC3404305//National Key Research and Development Program of China/ ; 2024YFA1107900//National Key Research and Development Program of China/ ; XDB1150000//the Strategic Priority Research Program of the Chinese Academy of Sciences/ ; YSBR-012//the CAS Project for Young Scientists in Basic Research/ ; NYHXGG2023AA01//Bingtuan Science and Technology Project/ ; },
mesh = {*Hepatocytes/transplantation/physiology ; Animals ; Swine ; Liver Regeneration ; CRISPR-Cas Systems ; Liver ; },
abstract = {The clinical application of hepatocyte transplantation has been significantly hindered by the scarcity of primary hepatocytes and the functional immaturity of in vitro-produced hepatocytes. By performing serial allogeneic hepatocyte transplantation in CRISPR/Cas9-mediated Fah-knockout pigs, we successfully achieved large-scale expansion of hepatocytes while maintaining their authentic biological characteristics. Particularly, the established model enables sustained in vivo liver reconstruction, concurrently ameliorating hepatic fibrosis and demonstrating functional microenvironmental remodeling. Moreover, through comprehensive single-cell transcriptomic profiling of 52 418 hepatocytes across transplant generations (F0-F2), we discovered that the cellular composition of these transplanted hepatocytes is similar to that of wild-type hepatocytes. The regenerated liver exhibits all six major hepatic cell types identical to the wild-type counterparts, with the characteristic lobular zonation patterns well preserved. Our research provides valuable insights into the large-scale expansion of physiologically functional hepatocytes in vivo without compromising their biological properties. This finding holds great promise for advancing the clinical application of human hepatocyte transplantation, potentially offering more effective treatment options for patients with liver diseases.},
}

*Hepatocytes/transplantation/physiology
Animals
Swine
Liver Regeneration
CRISPR-Cas Systems
Liver

@article {pmid40432605,
year = {2025},
author = {López-Manzaneda, S and Mencía, Á and Bonafont, J and Bassons-Bascuñana, A and García, M and Nyström, A and Duarte, B and Llames, S and Murillas, R and Modamio-Hoybjor, S and Morín, M and Soletto, L and Escamez, MJ and Moreno-Pelayo, MA and Rio, MD and Larcher, F},
title = {Safe and Efficacious Permanent Removal of Large COL7A1 Exons for Gene Reframing as a Reliable Therapeutic Strategy for Recessive Dystrophic Epidermolysis Bullosa.},
journal = {Human gene therapy},
volume = {36},
number = {17-18},
pages = {1211-1221},
doi = {10.1089/hum.2024.238},
pmid = {40432605},
issn = {1557-7422},
mesh = {*Collagen Type VII/genetics/metabolism ; *Epidermolysis Bullosa Dystrophica/genetics/therapy/pathology ; *Exons ; Humans ; *Gene Editing/methods ; Animals ; CRISPR-Cas Systems ; *Genetic Therapy/methods ; Mice ; DNA End-Joining Repair ; },
abstract = {Mutations leading to premature termination codons in COL7A1 are commonly associated with severe generalized recessive dystrophic epidermolysis bullosa (RDEB). Previous research, including our own, has indicated that removing mutated COL7A1 exons along with the consequent reframing of COL7A1 may not pose noticeable impact on protein function, offering a potential therapeutic strategy. However, investigations into the long-term in vivo effects of genome editing-mediated removal of mutant exons have only focused on the small exon 80 thus far. Hence, this study focuses on exons 73 and 105 of COL7A1 to explore whether targeted exon removal, through a CRISPR/Cas9-assisted, Non-homologous end joining (NHEJ)-mediated approach, could be extended to other larger exons. Introducing ribonucleoprotein complexes carrying Cas9 and optimized sgRNA guide pairs for each exon (73 and 105) through electroporation efficiently led to their removal, consequently restoring type VII collagen (C7) synthesis in RDEB primary patient cells carrying frameshift mutations in these exons. In vitro tests indicated the normal stability of the resulting C7 variants expressed at physiological levels, while in vivo analyses of regenerated skin grafted onto immunodeficient mice using E73 or E105 RDEB edited cells demonstrated the proper deposition of C7 at the basement membrane zone, thereby restoring normal dermo-epidermal adherence. This study enhances the broader potential of the exon deletion approach in the treatment of RDEB.},
}

*Collagen Type VII/genetics/metabolism
*Epidermolysis Bullosa Dystrophica/genetics/therapy/pathology
*Exons
Humans
*Gene Editing/methods
Animals
CRISPR-Cas Systems
*Genetic Therapy/methods
Mice
DNA End-Joining Repair

@article {pmid40252393,
year = {2025},
author = {Almasoudi, HH},
title = {Therapeutic promise of CRISPR-Cas9 gene editing in sickle cell disease and β-thalassemia: A current review.},
journal = {Current research in translational medicine},
volume = {73},
number = {3},
pages = {103513},
doi = {10.1016/j.retram.2025.103513},
pmid = {40252393},
issn = {2452-3186},
mesh = {Humans ; *Anemia, Sickle Cell/therapy/genetics ; *beta-Thalassemia/therapy/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods ; Animals ; Mutation ; beta-Globins/genetics ; },
abstract = {Sickle cell disease (SCD and β-thalassemia (BT) affects millions of people worldwide. In addition, around 500,000 infants are born with SCD and 60,000 people are diagnosed with BT every year. Mutations in the hemoglobin subunit beta (HBB) gene are responsible for causing both BT and SCD. Indeed, the diversity of potential mutations in the HBB gene elucidates the diversity in clinical severity observed in individuals with BT and related morbidities. On the other hand, SCD takes place because of the alteration in a single amino acid at position 6 in the beta-globin chain, where a base substitution occurs from glutamic acid to valine, which eventually results in abnormal sickle hemoglobin. Conventional therapies for BT and SCD including pharmaceutical drugs and blood transfusion might temporarily improve the clinical severity of these diseases, however these therapies cannot cure the diseases. CRISPR-Cas9 (CC9) is revolutionizing genome engineering, offering promising therapeutic avenues for genetic diseases. Therefore, CC9-mediated gene therapy provides great hope in the treatment of both BT and SCD. CC9-mediated gene therapy has already demonstrated its effectiveness in correcting both SCD and BT-causing mutations. Moreover, CC9-mediated gene editing was found to be effective in reactivating the expression of hemoglobin F (HbF) and regulating LRF and BCL11A. A number of clinical trials with CC9 gene-edited therapies are being carried out to elucidate their potential in treating BT and SCD. Genetics and pathophysiological mechanisms of SCD and BT, the mechanism of CC9-mediated gene editing, and common delivery methods of the CC9 system have been discussed in this review. Moreover, an in-depth discussion on applications and the current status of CC9-mediated gene editing in SCD and BT along with current challenges and future perspectives have been provided.},
}

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

@article {pmid39500392,
year = {2025},
author = {Huang, M and Wang, K and Li, A and Zhu, X and Zhou, Z and Yang, C and Bi, C and Zhang, X},
title = {Mini and enhanced CRISPR activators for cancer therapies.},
journal = {Journal of advanced research},
volume = {75},
number = {},
pages = {151-161},
doi = {10.1016/j.jare.2024.10.027},
pmid = {39500392},
issn = {2090-1224},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Neoplasms/therapy/genetics ; Animals ; *Gene Editing/methods ; *Genetic Therapy/methods ; Cell Line, Tumor ; Caenorhabditis elegans/genetics ; Cell Proliferation/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Transcriptional Activation/genetics ; },
abstract = {INTRODUCTION: The RNA-guided nuclease Cas9 can be used as a programmable transcription activator, but there is still room for improvement in its effectiveness in eukaryotes, and its potential in cancer genetic therapy has been poorly investigated.

OBJECTIVES: We aim to construct optimized CRISPRa tools and detect their potential role in cancer therapy by screening 9aa-TAD.

METHODS: We selected a range of transcriptional coactivators for programmable activation and analyzed their effects on the expression of multiple endogenous genes using Flow cytometry and qRT-PCR. In order to improve the activation capacity of the CRISPRa tool, we fused the coactivators with the efficient dCas9-VPR system to construct a new activation system. Utilize RNA-seq to assess the activation specificity of genome-wide. To evaluate the value of the newly constructed activation system in cancer gene therapy, we activated the expression of the tumor suppressor genes PER2 and ZNF382, and performed changes in cancer cell proliferation qRT-PCR and clonal formation analysis.

RESULTS: In this study, we screened the NHR module from C. elegans, which demonstrated a high transcription activation capacity with a compact size compared to VP64. We successfully demonstrated its efficiency in activating endogenous genes in mammalian cells. Furthermore, we developed an enhanced fused variant called NHR-VP64-p65-Rta (NVPR), which showed even higher efficiency compared to the previously established VPR module, making it an effective CRISPRa tool. The dCas9-NVPR complex also exhibited high specificity on a genome-wide scale. Finally, we utilized the dCas9-NVPR tool to restore the expression of tumor suppressor genes PER2 and ZNF382, effectively inhibiting the malignant phenotype of cancer cells.

CONCLUSION: We have successfully developed and demonstrated a breakthrough CRISPRa tool with promising implications for cancer genetic therapy. This innovation expands the range of available gene editing tools and further validates the immense potential of CRISPR-based approaches in precision medicine.},
}

Humans
*CRISPR-Cas Systems/genetics
*Neoplasms/therapy/genetics
Animals
*Gene Editing/methods
*Genetic Therapy/methods
Cell Line, Tumor
Caenorhabditis elegans/genetics
Cell Proliferation/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Transcriptional Activation/genetics

@article {pmid39487265,
year = {2025},
author = {Tycko, J and Van, MV and Aradhana, and DelRosso, N and Ye, H and Yao, D and Valbuena, R and Vaughan-Jackson, A and Xu, X and Ludwig, C and Spees, K and Liu, K and Gu, M and Khare, V and Mukund, AX and Suzuki, PH and Arana, S and Zhang, C and Du, PP and Ornstein, TS and Hess, GT and Kamber, RA and Qi, LS and Khalil, AS and Bintu, L and Bassik, MC},
title = {Development of compact transcriptional effectors using high-throughput measurements in diverse contexts.},
journal = {Nature biotechnology},
volume = {43},
number = {9},
pages = {1525-1538},
pmid = {39487265},
issn = {1546-1696},
support = {K00 DK126120/DK/NIDDK NIH HHS/United States ; R01 EB029483/EB/NIBIB NIH HHS/United States ; R01 HG011866/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; *Transcription Factors/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *High-Throughput Screening Assays/methods ; HEK293 Cells ; Protein Domains ; *Transcription, Genetic ; Gene Expression Regulation ; },
abstract = {Transcriptional effectors are protein domains known to activate or repress gene expression; however, a systematic understanding of which effector domains regulate transcription across genomic, cell type and DNA-binding domain (DBD) contexts is lacking. Here we develop dCas9-mediated high-throughput recruitment (HT-recruit), a pooled screening method for quantifying effector function at endogenous target genes and test effector function for a library containing 5,092 nuclear protein Pfam domains across varied contexts. We also map context dependencies of effectors drawn from unannotated protein regions using a larger library tiling chromatin regulators and transcription factors. We find that many effectors depend on target and DBD contexts, such as HLH domains that can act as either activators or repressors. To enable efficient perturbations, we select context-robust domains, including ZNF705 KRAB, that improve CRISPRi tools to silence promoters and enhancers. We engineer a compact human activator called NFZ, by combining NCOA3, FOXO3 and ZNF473 domains, which enables efficient CRISPRa with better viral delivery and inducible control of chimeric antigen receptor T cells.},
}

Humans
*Transcription Factors/genetics/metabolism
CRISPR-Cas Systems/genetics
*High-Throughput Screening Assays/methods
HEK293 Cells
Protein Domains
*Transcription, Genetic
Gene Expression Regulation

@article {pmid39415058,
year = {2025},
author = {Chen, K and Han, H and Zhao, S and Xu, B and Yin, B and Lawanprasert, A and Trinidad, M and Burgstone, BW and Murthy, N and Doudna, JA},
title = {Lung and liver editing by lipid nanoparticle delivery of a stable CRISPR-Cas9 ribonucleoprotein.},
journal = {Nature biotechnology},
volume = {43},
number = {9},
pages = {1445-1457},
pmid = {39415058},
issn = {1546-1696},
support = {RM1HG009490//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; UH3 NS115599/NS/NINDS NIH HHS/United States ; R01MH125979-01//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01 MH125979/MH/NIMH NIH HHS/United States ; DISC2-14097//California Institute for Regenerative Medicine (CIRM)/ ; DISC2-14045//California Institute for Regenerative Medicine (CIRM)/ ; UG3 NS115599/NS/NINDS NIH HHS/United States ; UM1AI164559//U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; },
mesh = {*Gene Editing/methods ; Animals ; *CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics/metabolism ; Mice ; *Nanoparticles/chemistry ; *Liver/metabolism ; *Lung/metabolism ; *CRISPR-Associated Protein 9/genetics/metabolism ; Humans ; Lipids/chemistry ; Liposomes ; },
abstract = {Lipid nanoparticle (LNP) delivery of clustered regularly interspaced short palindromic repeat (CRISPR) ribonucleoproteins (RNPs) could enable high-efficiency, low-toxicity and scalable in vivo genome editing if efficacious RNP-LNP complexes can be reliably produced. Here we engineer a thermostable Cas9 from Geobacillus stearothermophilus (GeoCas9) to generate iGeoCas9 variants capable of >100× more genome editing of cells and organs compared with the native GeoCas9 enzyme. Furthermore, iGeoCas9 RNP-LNP complexes edit a variety of cell types and induce homology-directed repair in cells receiving codelivered single-stranded DNA templates. Using tissue-selective LNP formulations, we observe genome-editing levels of 16‒37% in the liver and lungs of reporter mice that receive single intravenous injections of iGeoCas9 RNP-LNPs. In addition, iGeoCas9 RNPs complexed to biodegradable LNPs edit the disease-causing SFTPC gene in lung tissue with 19% average efficiency, representing a major improvement over genome-editing levels observed previously using viral or nonviral delivery strategies. These results show that thermostable Cas9 RNP-LNP complexes can expand the therapeutic potential of genome editing.},
}

*Gene Editing/methods
Animals
*CRISPR-Cas Systems/genetics
*Ribonucleoproteins/genetics/metabolism
Mice
*Nanoparticles/chemistry
*Liver/metabolism
*Lung/metabolism
*CRISPR-Associated Protein 9/genetics/metabolism
Humans
Lipids/chemistry
Liposomes

@article {pmid39385008,
year = {2025},
author = {Wang, Y and Liu, KI and Liu, MM and Ooi, KH and Nguyen, TA and Chee, JE and Teo, SXD and He, S and Tay, JWD and Teo, SY and Liew, KS and Ge, XY and Ng, ZJ and Avagyan, H and Liu, H and Yi, Z and Chang, K and Kok, EPL and Chen, R and Yau, CE and Koh, JW and Wan, Y and Tan, MH},
title = {A circularly permuted CasRx platform for efficient, site-specific RNA editing.},
journal = {Nature biotechnology},
volume = {43},
number = {9},
pages = {1548-1561},
pmid = {39385008},
issn = {1546-1696},
support = {NRF2017-NRF-ISF002-2673//National Research Foundation Singapore (National Research Foundation-Prime Minister's office, Republic of Singapore)/ ; SSG 2021-03//National Research Foundation Singapore (National Research Foundation-Prime Minister's office, Republic of Singapore)/ ; T2EP30223-0028//Ministry of Education - Singapore (MOE)/ ; RG50/17//Ministry of Education - Singapore (MOE)/ ; NMRC/OFIRG/0017/2016//MOH | National Medical Research Council (NMRC)/ ; },
mesh = {*RNA Editing/genetics ; Humans ; Adenosine Deaminase/genetics/metabolism ; *Protein Engineering/methods ; *CRISPR-Cas Systems/genetics ; RNA-Binding Proteins/genetics ; *CRISPR-Associated Proteins/genetics ; HEK293 Cells ; },
abstract = {Inactive Cas13 orthologs have been fused to a mutant human ADAR2 deaminase domain at the C terminus to enable programmable adenosine-to-inosine (A-to-I) RNA editing in selected transcripts. Although promising, existing RNA-editing tools generally suffer from a trade-off between efficacy and specificity, and off-target editing remains an unsolved problem. Here we describe the development of an optimized RNA-editing platform by rational protein engineering, CasRx-based Programmable Editing of RNA Technology (xPERT). We demonstrate that the topological rearrangement of a CasRx K940L mutant by circular permutation results in a robust scaffold for the tethering of a deaminase domain. We benchmark our tool against the REPAIR system and show that xPERT exhibits strong on-target activity like REPAIRv1 but low off-target editing like REPAIRv2. Our xPERT platform can be used to alter RNA sequence information without risking genome damage, effect temporary cellular changes and customize protein function.},
}

*RNA Editing/genetics
Humans
Adenosine Deaminase/genetics/metabolism
*Protein Engineering/methods
*CRISPR-Cas Systems/genetics
RNA-Binding Proteins/genetics
*CRISPR-Associated Proteins/genetics
HEK293 Cells

@article {pmid39375445,
year = {2025},
author = {Song, J and Cho, MH and Cho, H and Song, Y and Lee, SW and Nam, HC and Yoon, TH and Shin, JC and Hong, JS and Kim, Y and Ekanayake, E and Jeon, J and You, DG and Im, SG and Choi, GS and Park, JS and Carter, BC and Balaj, L and Seo, AN and Miller, MA and Park, SY and Kang, T and Castro, CM and Lee, H},
title = {Amplifying mutational profiling of extracellular vesicle mRNA with SCOPE.},
journal = {Nature biotechnology},
volume = {43},
number = {9},
pages = {1485-1495},
pmid = {39375445},
issn = {1546-1696},
support = {HR22C1832//Korea Health Industry Development Institute (KHIDI)/ ; U01CA284982//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; DP2CA259675//U.S. Department of Health & Human Services | NIH | NIH Office of the Director (OD)/ ; 2021M3E5E3080379//National Research Foundation of Korea (NRF)/ ; U01 CA230697/CA/NCI NIH HHS/United States ; 2023R1A2C2005185//National Research Foundation of Korea (NRF)/ ; 1U01CA279858//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; 2021R1A2B5B03001416//National Research Foundation of Korea (NRF)/ ; 2021R1A2C1005342//National Research Foundation of Korea (NRF)/ ; R01 CA237500/CA/NCI NIH HHS/United States ; R01 CA239078/CA/NCI NIH HHS/United States ; R01CA264363//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {*Extracellular Vesicles/genetics/metabolism ; Humans ; *RNA, Messenger/genetics ; *Mutation/genetics ; Animals ; Mice ; CRISPR-Cas Systems/genetics ; Colorectal Neoplasms/genetics ; Glioblastoma/genetics ; Lung Neoplasms/genetics ; Cell Line, Tumor ; Liquid Biopsy ; },
abstract = {Sequencing of messenger RNA (mRNA) found in extracellular vesicles (EVs) in liquid biopsies can provide clinical information such as somatic mutations, resistance profiles and tumor recurrence. Despite this, EV mRNA remains underused due to its low abundance in liquid biopsies, and large sample volumes or specialized techniques for analysis are required. Here we introduce Self-amplified and CRISPR-aided Operation to Profile EVs (SCOPE), a platform for EV mRNA detection. SCOPE leverages CRISPR-mediated recognition of target RNA using Cas13 to initiate replication and signal amplification, achieving a sub-attomolar detection limit while maintaining single-nucleotide resolution. As a proof of concept, we designed probes for key mutations in KRAS, BRAF, EGFR and IDH1 genes, optimized protocols for single-pot assays and implemented an automated device for multi-sample detection. We validated SCOPE's ability to detect early-stage lung cancer in animal models, monitored tumor mutational burden in patients with colorectal cancer and stratified patients with glioblastoma. SCOPE can expedite readouts, augmenting the clinical use of EVs in precision oncology.},
}

*Extracellular Vesicles/genetics/metabolism
Humans
*RNA, Messenger/genetics
*Mutation/genetics
Animals
Mice
CRISPR-Cas Systems/genetics
Colorectal Neoplasms/genetics
Glioblastoma/genetics
Lung Neoplasms/genetics
Cell Line, Tumor
Liquid Biopsy

@article {pmid40950596,
year = {2025},
author = {Hadian, S and Smith, DL and Suproniene, S},
title = {Genomic and phenotypic profiling of an Artemisia endophyte: dual biostimulant and biocontrol activities in pea under Fusarium stress.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1643204},
pmid = {40950596},
issn = {1664-302X},
abstract = {OBJECTIVE: To evaluate the plant growth-promoting and disease-suppressing potential of Serratia sp. AR11, an endophytic bacterium isolated from Artemisia absinthium, through phenotypic assessment and whole-genome analysis in pea (Pisum sativum) under normal and Fusarium-stress conditions.

MATERIALS AND METHODS: Greenhouse experiments were conducted to assess the effects of AR11 inoculation on shoot and root growth, biomass, chlorophyll content, and Fusarium-induced stunting. Whole-genome sequencing was performed using the PacBio SMRT platform, followed by functional annotation to identify genes related to nutrient metabolism, secondary metabolite biosynthesis, and stress adaptation. Biosafety assessment included screening for virulence and antibiotic resistance genes.

RESULTS: AR11 inoculation significantly increased shoot and root length and biomass, while reducing Fusarium-induced stunting by over 70%. Under pathogen stress, treated plants showed a 67% increase in SPAD index compared to controls. Genome analysis revealed a complete 5.49 Mb circular genome with 5,175 protein-coding genes, including those for nitrogen metabolism, phosphate solubilization, siderophore production, and antifungal secondary metabolite biosynthesis. Additional features included stress-responsive regulatory systems, CRISPR-Cas loci, and DNA methylation patterns. No high-risk virulence or acquired antibiotic resistance genes were detected.

CONCLUSION: Serratia sp. AR11 is a safe, multifunctional endophytic bacterium with strong genomic and phenotypic evidence for enhancing pea growth and suppressing Fusarium-related diseases, making it a promising candidate for sustainable low-input agriculture.},
}

@article {pmid40946761,
year = {2025},
author = {Uddin, N and Ullah, MW and Li, K and Liu, F and Xin, X},
title = {Plant cell wall biosynthesis: Immune signaling, genome editing, and physiological implications for biomass valorization.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108714},
doi = {10.1016/j.biotechadv.2025.108714},
pmid = {40946761},
issn = {1873-1899},
abstract = {Plants continuously face biotic stress from pathogens, pests, and environmental challenges that threaten their survival and productivity. In response, plants have developed complex immune systems, with the cell wall playing a central role in defense. The plant cell wall not only provides mechanical strength but also acts as a dynamic barrier against pathogens, influencing both plant growth and immune responses. This review discusses the molecular mechanisms of cell wall biosynthesis, facilitated by multi-omics technologies, particularly the synthesis and regulation of lignin and other polysaccharides, which contribute to cell wall integrity and plant immunity. It explores the interplay between cell wall modifications and immune signaling pathways, highlighting the role of pattern recognition receptors in pathogen detection and defense activation. Additionally, the potential of genome editing, especially CRISPR-Cas, in enhancing cell wall characteristics to improve pathogen resistance and biomass utilization is discussed. With growing interest in lignocellulosic biomass as a renewable resource for biofuels and bioproducts, this review also addresses the challenges of biomass recalcitrance, focusing on biotechnological advancements to improve saccharification efficiency. Finally, the review proposes integrated strategies combining genetic modifications, biotechnological innovations, and sustainable practices to optimize lignocellulosic biomass for a bio-based economy, contributing to both agricultural resilience and sustainable energy production. As climate change accelerates, these technologies hold the promise of developing resilient crops and enhancing the capacity of the bioeconomy to mitigate environmental impacts.},
}

@article {pmid40946164,
year = {2025},
author = {Sun, J and Liu, K and Sheng, Y and Zhang, H and Wang, J and Wang, Y and Tian, R and Zhu, X and Tian, SS and Wang, P and Zhao, X and Liu, Y},
title = {High-Fidelity AaCas12b[Max] Enables the Development of an Engineered T Cell Therapy with Enhanced Safety and Functional Fitness.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2025.09.009},
pmid = {40946164},
issn = {1525-0024},
abstract = {CRISPR-Cas systems have transformed genome editing, yet the commonly used Streptococcus pyogenes Cas9 (SpCas9) is limited by off-target effects and chromosomal instability. Here, we characterize AaCas12b[MAX], an engineered Alicyclobacillus acidiphilus Cas12b variant, as a high-precision editing platform optimized for tumor infiltrating lymphocyte (TIL) therapy. Using an FDA-compliant safety assessment framework, we systemically compared AaCas12b[MAX]- and SpCas9-edited TIL products in terms of on-target efficiency, genome-wide off-target activity, and structural variants (SVs) formation. AaCas12b[MAX] achieved >80% on-target editing efficiency with undetectable off-target events and a 3.3-fold reduction in SVs relative to SpCas9. Mechanistic studies revealed different DNA repair kinetics in AaCas12b[MAX]-edited cells, reducing sustained DNA damage responses and chromosomal instability. Structural modeling suggested a more stable enzyme-sgRNA-DNA ternary complex, enabling stringent PAM specificity and minimal mismatch tolerance. Functionally, AaCas12b[MAX]-edited TILs exhibited superior therapeutic potential, including enhanced cellular fitness, a twofold increase in expansion capacity, and enrichment of stem-like tumor-reactive CD39[-]CD69[-]CD8[+] subsets. Together, these results establish AaCas12b[MAX] as a robust, clinically translatable platform that improves the safety and functional limitations of SpCas9, enabling the development of next-generation T cell therapies.},
}

@article {pmid40945731,
year = {2025},
author = {Jain, M and Pattnayak, AK and Aggarwal, S and Rai, P and Kavya, J and Chandrayan, S and Goel, M and Gaur, V},
title = {Branched DNA processing by a thermostable CAS-Cas4 from Thermococcus onnurineus: expanding biochemical landscape of nuclease activity.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {110701},
doi = {10.1016/j.jbc.2025.110701},
pmid = {40945731},
issn = {1083-351X},
abstract = {The adaptive immune function of CRISPR-Cas systems in bacteria and archaea is mediated through CRISPR-Associated Proteins (Cas). The adaptation module, typically involving Cas1, Cas2, and Cas4, helps integrate viral "spacer" sequences into the host genome. Cas4 proteins are classified into two types based on neighboring genes: CAS-Cas4, flanked by other cas genes, and Solo-Cas4, which exist independently. While CAS-Cas4 proteins are implicated in adaptation, they remain biochemically uncharacterized in archaea, unlike archaeal Solo-Cas4 proteins. This study biochemically characterizes TON_0321, a CAS-Cas4 protein from the Type IV-C CRISPR cassette of Thermococcus onnurineus. TON_0321 exhibits 5' to 3' exonuclease activity and unique structure-dependent endonuclease activity, shedding light on CAS-Cas4 functional diversity. A distinct spatial organization of the catalytic site, angled with the positively charged patch on the protein surface, enables TON_0321 to recognize branching points in DNA substrates. Furthermore, this spatial arrangement facilitates cleavage 2 to 3 nucleotides away from the branch point in the 5' direction, demonstrating structure-specific endonuclease activity.},
}

@article {pmid40943511,
year = {2025},
author = {Ma, Z and Ren, J and Liu, Q and Li, J and Zhao, H and Tibesigwa, DG and Matola, SH and Gulfam, T and Yang, J and Wang, F},
title = {Integrating Traditional Breeding and Modern Biotechnology for Advanced Forest Tree Improvement.},
journal = {International journal of molecular sciences},
volume = {26},
number = {17},
pages = {},
pmid = {40943511},
issn = {1422-0067},
abstract = {In the context of global climate change and efforts toward "carbon peak and carbon neutrality," forest resource protection and restoration have become fundamental to ecological civilization. The genetic improvement of trees, as the primary component of forest ecosystems, holds strategic importance for ecological security, resource supply, and carbon neutrality. Traditional tree breeding techniques, including selective and hybrid breeding, have established robust technical systems through extensive practice. However, these methods face limitations such as extended cycles, reduced efficiency, and constrained genetic gains in meeting contemporary requirements. Modern biotechnologies, including genomic selection (GS), gene editing (CRISPR/Cas9), and marker-assisted selection (MAS), substantially enhance the precision and efficiency of genetic improvement. Nevertheless, exclusive reliance on either traditional or modern methods proves insufficient for addressing complex environmental adaptation and rapid breeding requirements. Consequently, the integration of traditional breeding with modern biotechnology to develop intelligent, sustainable, and efficient breeding strategies has emerged as a central focus in tree genetics and breeding. An integrated "step-by-step" approach warrants promotion, supported by a multi-source data sharing platform, an optimized core germplasm repository, and a "climate-soil-genotype" matching model to facilitate the region-specific deployment of improved varieties.},
}

@article {pmid40943296,
year = {2025},
author = {Sheveleva, O and Butorina, N and Protasova, E and Medvedev, S and Grigor'eva, E and Melnikova, V and Kuziaeva, V and Minzhenkova, M and Tatarenko, Y and Lyadova, I},
title = {The Generation of iPSCs Expressing Interferon-Beta Under Doxycycline-Inducible Control.},
journal = {International journal of molecular sciences},
volume = {26},
number = {17},
pages = {},
pmid = {40943296},
issn = {1422-0067},
support = {№ 0088-2024-0013//government basic research program at the Koltzov Institute of Developmental Biology of the Russian Academy of Sciences in 2024/ ; },
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology/drug effects ; Humans ; *Doxycycline/pharmacology ; *Interferon-beta/genetics/metabolism ; Cell Differentiation/drug effects ; CRISPR-Cas Systems ; Cell Line ; },
abstract = {Type 1 interferons (IFN-Is) exhibit significant antiviral, antitumor, and immunoregulatory properties, demonstrating substantial therapeutic potential. However, IFN-Is are pleiotropic cytokines, and the available data on their effect under specific pathological conditions are inconclusive. Furthermore, the systemic administration of IFN-Is can result in side effects. Generating cells that can migrate to the pathological focus and provide regulated local production of IFN-Is could overcome this limitation and provide a model for an in-depth analysis of the biological and therapeutic effects of IFN-Is. Induced pluripotent stem cells (iPSCs) are a valuable source of various differentiated cell types, including human immune cells. In this study, we describe the generation of genetically modified human iPSCs with doxycycline-controlled overexpression of interferon β (IFNB1). Three IFNB1-overexpressing iPSC lines (IFNB-iPSCs) and one control line expressing the transactivator M2rtTA (TA-iPSCs) were generated using the CRISPR/Cas9 technology. The pluripotency of the generated cell lines has been confirmed by the following: (i) cell morphology; (ii) the expression of the pluripotency markers OCT4, SOX2, TRA 1-60, and NANOG; and (iii) the ability to spontaneously differentiate into the derivatives of the three germ layers. Upon the addition of doxycycline, all IFNB-iPSCs upregulated IFNB1 expression at RNA (depending on the iPSC line, 126-816-fold) and protein levels. The IFNB-iPSCs and TA-iPSCs generated here represent a valuable cellular model for studying the effects of IFN-β on the activity and differentiation trajectories of different cell types, as well as for generating different types of cells with controllable IFN-β expression.},
}

*Induced Pluripotent Stem Cells/metabolism/cytology/drug effects
Humans
*Doxycycline/pharmacology
*Interferon-beta/genetics/metabolism
Cell Differentiation/drug effects
CRISPR-Cas Systems
Cell Line

@article {pmid40938447,
year = {2025},
author = {Kang, X and Tian, F and Liu, X and Xiao, G and Cai, Y},
title = {CRISPR/Cas13 system-based entropy-driven DNAzyme switch powered DNA walking system for sensitive and direct rotavirus detection.},
journal = {Mikrochimica acta},
volume = {192},
number = {10},
pages = {663},
pmid = {40938447},
issn = {1436-5073},
support = {No. 2018QN035//Sichuan Provincial Administration of Traditional Chinese Medicine Medical Research Special 2018/ ; },
mesh = {*DNA, Catalytic/chemistry/metabolism/genetics ; *Rotavirus/isolation & purification/genetics ; Entropy ; *CRISPR-Cas Systems ; Limit of Detection ; Metal Nanoparticles/chemistry ; *Biosensing Techniques/methods ; Gold/chemistry ; DNA Probes/chemistry/genetics ; Nucleic Acid Amplification Techniques/methods ; Humans ; },
abstract = {DNA walker-based strategies are confronted with significant challenges in harmonizing design complexity, sequence dependence, and amplification efficiency. This study describes the innovative design of a double-stranded DNA probe, named the "LW probe," which integrates a locked DNAzyme segment, enabling the coupling of the entropy-driven amplification (EDA) process with a DNAzyme-powered DNA walker. In the absence of the target, the "LW probe" remains in an inactive ("OFF") state. Upon encountering target rotavirus sequences, the LW probe receives the trans-cleavage activity of Cas13a/crRNA and undergoes a conformational change, transforming into an activated structure. This structural transition initiates the EDA process continuously, leading to the release of the DNAzyme segment. Subsequently, the released DNAzyme segment acts on the surface of gold nanoparticles (AuNPs), cleaving the "Substrate probe" and consequently liberating fluorescence signals. Distinct from traditional DNA walkers that rely exclusively on the EDA for product amplification, the proposed approach synergistically combines the high-precision target recognition capacity of the EDA process with the potent signal amplification efficiency of DNA walkers. This integration results in remarkable enhancements in both specificity, demonstrated by the ability to discriminate single-base mismatched sequences, and sensitivity, with a detection limit as low as 2.7 fM. By synergizing EDA with the DNAzyme-driven DNA walker, our method achieves high sensitivity, with a detection limit of 2.7 fM, outperforming or matching the performance of previous DNA walker-based systems. This system enables highly sensitive and specific detection of low-abundance rotavirus with robust stability, offering a promising platform for disease diagnosis and biomedical research.},
}

*DNA, Catalytic/chemistry/metabolism/genetics
*Rotavirus/isolation & purification/genetics
Entropy
*CRISPR-Cas Systems
Limit of Detection
Metal Nanoparticles/chemistry
*Biosensing Techniques/methods
Gold/chemistry
DNA Probes/chemistry/genetics
Nucleic Acid Amplification Techniques/methods
Humans

@article {pmid40938388,
year = {2025},
author = {Higa, L and Blank, M and Hampson, E and Matsuyama, J and Wilkes, K and Uehara, A and Bouwman, T and Lee, K and Wang, K and Muszynski, M and Du, ZY},
title = {Rapid assessment of CRISPR gRNAs with optimized protoplast transformation in Maize.},
journal = {Plant cell reports},
volume = {44},
number = {10},
pages = {212},
pmid = {40938388},
issn = {1432-203X},
support = {2121410//National Science Foundation/ ; },
mesh = {*Zea mays/genetics ; *Protoplasts/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Transformation, Genetic ; Plants, Genetically Modified ; Transfection ; },
abstract = {We developed an optimized CRISPR/Cas9 gene editing system using maize mesophyll protoplasts to enable rapid evaluation of guide RNA (gRNA) activity. Using the tropical inbred line Tzi8, we improved protoplast isolation and transfection protocols, achieving high yields of 17.88 × 10[6] viable protoplasts per gram fresh weight while extending post-transfection viability. Etiolated seedlings and vertical leaf cutting significantly enhanced protoplast recovery and viability. A transfection efficiency of ~ 50% was achieved using 10 µg of plasmid DNA; higher DNA inputs did not result in significant gains, resulting in a more resource-efficient approach. Protoplast viability was maintained for up to seven days post-transfection, allowing for downstream applications that require extended incubation. This optimized system was used to assess the editing efficiency of nine gRNAs targeting three key floral repressors (ZmCCT9, ZmCCT10, and ZmRap2.7) across four maize genotypes (Tzi8, CML277, B73, and B104). These floral repressor genes are involved in the photoperiod sensitivity of tropical maize, a major challenge in the effort to introduce tropical maize germplasm into temperate breeding programs. Editing efficiencies ranged from 0.4% to 23.7%, with some variation observed between gRNAs and genotypes. Although protoplast-based assays do not currently enable plant regeneration, this platform offers a rapid method for in vivo gRNA validation, reducing assay time from months to days. This work expands the gene editing toolkit for tropical maize, supporting efforts to overcome breeding barriers through gene editing.},
}

*Zea mays/genetics
*Protoplasts/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics
*Transformation, Genetic
Plants, Genetically Modified
Transfection

@article {pmid40934206,
year = {2025},
author = {Lima, DA and Costa-Silva, HM and Albergaria, KSS and Ribeiro, JM and Resende, DM and Santarossa, BA and Liarte, DB and Calderano, SG and Murta, SMF},
title = {Glycosomal ABC transporter 3 (GAT3) deletion enhances the oxidative stress responses and reduces the infectivity of Trypanosoma cruzi.},
journal = {PLoS neglected tropical diseases},
volume = {19},
number = {9},
pages = {e0013479},
pmid = {40934206},
issn = {1935-2735},
mesh = {*Trypanosoma cruzi/genetics/pathogenicity/drug effects/metabolism ; *Oxidative Stress ; *ATP-Binding Cassette Transporters/genetics/metabolism ; Animals ; *Protozoan Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Mice ; Chagas Disease/parasitology ; Nitroimidazoles/pharmacology ; *Microbodies/metabolism/genetics ; Trypanocidal Agents/pharmacology ; Gene Knockout Techniques ; Gene Deletion ; },
abstract = {Glycosomes, peroxisome-like organelles in Trypanosoma cruzi, contain enzymes involved in various metabolic processes, including glycolysis. Glycosomal ABC transporters (GATs) play a vital role in maintaining metabolic homeostasis by facilitating metabolite exchange between glycosomes and the cytoplasm. GAT3 is a member of the GAT family, which also includes GAT1 and GAT2. GAT3 transcript levels are downregulated in benznidazole-resistant T. cruzi populations; however, its specific functions remain unknown. Therefore, in this study, we generated GAT3 single-knockout and null mutant lines of the T. cruzi Dm28c strain using the CRISPR/Cas9 system to investigate GAT3 roles in parasite biology. RT-qPCR revealed increased GAT2 transcript levels in the GAT3 null mutant line, without any changes in GAT1 levels. Our findings suggest that GAT3 is not essential for T. cruzi survival, as null mutant parasites showed no growth difference compared to the Cas9-expressing controls. Moreover, the GAT3 single-knockout line exhibited increased resistance to benznidazole, whereas the null mutant line exhibited benznidazole susceptibility similar to the control. Furthermore, both GAT3 single-knockout and null mutant lines showed increased tolerance to hydrogen peroxide-induced oxidative stress. In vitro infection assay of L929 murine fibroblasts revealed that the GAT3 null parasites exhibited a significantly lower infection rate and fewer intracellular amastigotes than the controls. Overall, GAT3 is crucial for T. cruzi infectivity and the regulation of oxidative stress responses, playing key roles in the metabolic regulation and pathogenicity of this parasite.},
}

*Trypanosoma cruzi/genetics/pathogenicity/drug effects/metabolism
*Oxidative Stress
*ATP-Binding Cassette Transporters/genetics/metabolism
Animals
*Protozoan Proteins/genetics/metabolism
CRISPR-Cas Systems
Mice
Chagas Disease/parasitology
Nitroimidazoles/pharmacology
*Microbodies/metabolism/genetics
Trypanocidal Agents/pharmacology
Gene Knockout Techniques
Gene Deletion

@article {pmid40934013,
year = {2025},
author = {Zhang, Y and Liu, T and Zhang, P and Ni, B and Wang, X and Bai, L and Sun, W and Guan, Y and Xia, X and Cao, H and Gu, J},
title = {A rapid and accurate method for Helicobacter pylori detection via integrating LAMP assay with CRISPR/Cas12b detection by one-step in one-pot.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1611134},
pmid = {40934013},
issn = {2235-2988},
mesh = {*Helicobacter pylori/genetics/isolation & purification ; Humans ; *Molecular Diagnostic Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; *Helicobacter Infections/diagnosis/microbiology ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Limit of Detection ; },
abstract = {INTRODUCTION: Accurate and timely detection of Helicobacter pylori (HP) is crucial for the diagnosis and management of gastritis and other HP-associated gastrointestinal disorders. Conventional diagnostic methods, such as PCR and culture, require specialized equipment and expertise, limiting their applicability in resource-limited settings. There is a pressing need for a rapid, cost-effective, and user-friendly diagnostic platform for HP detection, particularly in point-of-care settings.

METHODS: We developed an integrated detection platform combining loop-mediated isothermal amplification (LAMP) with the CRISPR/Cas12b system in a single, one-step, one-pot reaction. The assay was optimized to function at a constant temperature of 58 °C and provides results within 45 minutes. The clinical performance of the system was evaluated using 22 clinical samples, and its diagnostic accuracy was compared with conventional PCR.

RESULTS: The LAMP-CRISPR/Cas12b assay demonstrated a limit of detection (LOD) of 14.77 copies per test, with no cross-reactivity observed against potential interfering nucleic acids, ensuring 100% specificity for HP. Clinical validation revealed a concordance rate of 90.91% (20/22) between the LAMP-CRISPR/Cas12b platform and conventional PCR, supporting the diagnostic reliability of the system.

DISCUSSION: The integrated LAMP-CRISPR/Cas12b platform represents a promising alternative for the rapid and sensitive detection of HP. It combines the simplicity and rapidity of LAMP with the specificity of CRISPR/Cas12b, offering a robust, cost-effective, and high\-sensitivity diagnostic tool without the need for complex instrumentation. The method shows great potential for use in point-of-care testing (POCT) and could significantly enhance clinical practice by facilitating timely diagnosis and treatment of HP-related diseases.},
}

*Helicobacter pylori/genetics/isolation & purification
Humans
*Molecular Diagnostic Techniques/methods
*Nucleic Acid Amplification Techniques/methods
*Helicobacter Infections/diagnosis/microbiology
*CRISPR-Cas Systems
Sensitivity and Specificity
Limit of Detection

@article {pmid40825449,
year = {2025},
author = {Chowdhury, R and Roure, A and Darras, S},
title = {Towards functional genetics in the European amphioxus: Efficient CRISPR/Cas9 editing reveals Ascl1/2.1 requirement for peripheral nervous system development.},
journal = {Developmental biology},
volume = {527},
number = {},
pages = {218-225},
doi = {10.1016/j.ydbio.2025.08.012},
pmid = {40825449},
issn = {1095-564X},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Basic Helix-Loop-Helix Transcription Factors/genetics/metabolism ; *Lancelets/genetics/embryology ; *Peripheral Nervous System/embryology/metabolism ; Gene Expression Regulation, Developmental ; },
abstract = {Amphioxus, or cephalochordates, have a key phylogenetic position among chordates and serve as pivotal invertebrate models for investigating the evolutionary origins of vertebrate traits. Although functional genetic tools have recently been developed, their application has been limited to the Floridian and Asian species, Branchiostoma floridae and Branchiostoma belcheri, respectively. In this study, we established a CRISPR/Cas9-based genome editing protocol to generate F0 mosaic mutants (crispants) in the European amphioxus B. lanceolatum. As a proof of concept, we targeted the Bl-Ascl1/2.1 gene, a putative regulator of epidermal sensory neuron (ESN) development in the peripheral nervous system coding for a bHLH transcription factor. Using a novel microinjection method of the sgRNA/Cas9 complex in fertilized eggs and two-cell stage embryos, we demonstrated the disruption of Bl-Ascl1/2.1 that resulted in a partial to complete loss of ESNs. Importantly, this phenotype could be rescued by Bl-Ascl1/2.1 mRNA microinjection. These findings demonstrate the efficiency of CRISPR/Cas9-mediated gene editing in B. lanceolatum and establish a foundation for future functional studies in this emerging EvoDevo model.},
}

Animals
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Basic Helix-Loop-Helix Transcription Factors/genetics/metabolism
*Lancelets/genetics/embryology
*Peripheral Nervous System/embryology/metabolism
Gene Expression Regulation, Developmental

@article {pmid40770575,
year = {2025},
author = {Southard, KM and Ardy, RC and Tang, A and O'Sullivan, DD and Metzner, E and Guruvayurappan, K and Norman, TM},
title = {Comprehensive transcription factor perturbations recapitulate fibroblast transcriptional states.},
journal = {Nature genetics},
volume = {57},
number = {9},
pages = {2323-2334},
pmid = {40770575},
issn = {1546-1718},
support = {GM132083//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; DRG-2462-22//Damon Runyon Cancer Research Foundation (Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation)/ ; DP2 GM140925/GM/NIGMS NIH HHS/United States ; AWD-GC-259296//Damon Runyon Cancer Research Foundation (Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation)/ ; GM140925//U.S. Department of Health & Human Services | NIH | NIH Office of the Director (OD)/ ; HG012103//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {*Fibroblasts/metabolism ; Humans ; *Transcription Factors/genetics/metabolism ; Kruppel-Like Factor 4 ; *Transcription, Genetic ; Animals ; Mice ; Gene Expression Regulation ; Kruppel-Like Transcription Factors/genetics ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; Chromatin/genetics ; },
abstract = {Cell atlas projects have revealed that common cell types often comprise distinct, recurrent transcriptional states, but the function and regulation of these states remain poorly understood. Here, we show that systematic activation of transcription factors can recreate such states in vitro, providing tractable models for mechanistic and functional studies. Using a scalable CRISPR activation (CRISPRa) Perturb-seq platform, we activated 1,836 transcription factors in two cell types. CRISPRa induced gene expression within physiological ranges, with chromatin features predicting responsiveness. Comparisons with atlas datasets showed that transcription factor perturbations recapitulated key fibroblast states and identified their regulators, including KLF2 and KLF4 for a universal state present in many tissues, and PLAGL1 for a disease-associated inflammatory state. Inducing the universal state suppressed the inflammatory state, suggesting therapeutic potential. These findings position CRISPRa as a nuanced tool for perturbing differentiated cells and establish a general strategy for studying clinically relevant transcriptional states ex vivo.},
}

*Fibroblasts/metabolism
Humans
*Transcription Factors/genetics/metabolism
Kruppel-Like Factor 4
*Transcription, Genetic
Animals
Mice
Gene Expression Regulation
Kruppel-Like Transcription Factors/genetics
CRISPR-Cas Systems
Cell Differentiation/genetics
Chromatin/genetics

@article {pmid40748247,
year = {2025},
author = {Zhang, Y and Yang, X and Wang, Y and Chen, F and Liu, Y and Jiang, H and Wang, Y and Hu, Y and Li, S},
title = {Establishment of a CRISPR/Cas12b-Based Multiple Cross Displacement Amplification Assay for the Rapid, Sensitive, and Specific Detection of Brucella ovis.},
journal = {ACS infectious diseases},
volume = {11},
number = {9},
pages = {2446-2456},
doi = {10.1021/acsinfecdis.5c00287},
pmid = {40748247},
issn = {2373-8227},
mesh = {*Brucella ovis/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Brucellosis/diagnosis/veterinary/microbiology ; Animals ; Sheep ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *Sheep Diseases/diagnosis/microbiology ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Brucella ovis (B. ovis), a major pathogenic species within the Brucella genus, causes ovine epididymitis. Although the isolation and identification of B. ovis remain the gold standard for diagnosis, these methods are unsuitable for early detection. The traditional polymerase chain reaction (PCR) offers faster detection but requires specialized equipment such as PCR thermal cyclers and gel electrophoresis imagers, limiting its use in basic laboratories. Thus, developing rapid, sensitive, and specific diagnostic strategies is vital for preventing and controlling the spread of ovine brucellosis. In this study, we developed a diagnostic assay combining clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12b with multiple cross displacement amplification (MCDA)─termed CRISPR/Cas12b-MCDA─for rapid, sensitive, and specific identification of B. ovis. In the CRISPR/Cas12b-MCDA system, MCDA amplicons containing protospacer adjacent motif (PAM) sites are recognized by the Cas12b/gRNA complex, which binds the target region and triggers trans-cleavage of a single-stranded DNA (ssDNA) reporter. The CRISPR/Cas12b-MCDA assay demonstrated a detection limit of 10 fg/μL for synthetic genomic DNA and exhibited 100% specificity for B. ovis, with no cross-reactivity against other Brucella or non-Brucella species. The preamplification for template extraction takes 20 min, then 5 min for uracil DNA glycosylase (UDG) digestion, and 45 min for MCDA amplification. The total detection time was 75 min using real-time fluorescence analysis and 90 min with a lateral flow biosensor (LFB). Additionally, the results were validated using UV visualization to confirm the CRISPR/Cas12b-MCDA results. Notably, both LFB and UV analyses are instrument-free, enhancing their accessibility. In conclusion, the CRISPR/Cas12b-MCDA assay is a simple, rapid, sensitive, specific, and reliable method for detecting B. ovis.},
}

*Brucella ovis/genetics/isolation & purification
*CRISPR-Cas Systems
*Brucellosis/diagnosis/veterinary/microbiology
Animals
Sheep
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
*Sheep Diseases/diagnosis/microbiology
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40437754,
year = {2025},
author = {Xu, Y and Li, J and Wang, Z and Lu, R and Liu, Y and Wang, M and Li, H and Zhao, R and Feng, W},
title = {Ablation of dysmorphic neurons is a safe and effective treatment for focal cortical dysplasia II.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {9},
pages = {4414-4430},
pmid = {40437754},
issn = {1525-0024},
mesh = {*Epilepsy/genetics/pathology/therapy ; *Malformations of Cortical Development, Group I/genetics/pathology/therapy ; Proto-Oncogene Proteins c-akt/genetics ; Disease Models, Animal ; Animals ; Mice ; *Diphtheria Toxin/therapeutic use ; *Ablation Techniques ; *Heparin-binding EGF-like Growth Factor/genetics ; Humans ; HEK293 Cells ; TOR Serine-Threonine Kinases/genetics ; *Neurons/pathology ; CRISPR-Cas Systems ; *Gene Editing/methods ; },
abstract = {Focal cortical dysplasia type II (FCDII) is a leading cause of refractory epilepsy in children, yet treatment options remain limited. The most frequent genetic cause of FCDII is mosaic and somatic variants in genes of the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT)-mammalian target of rapamycin (mTOR) pathway, leading to hyperactivation of mTOR signaling. The presence of dysmorphic neurons (DNs) resulting from hyperactive mTOR signaling is critical for the development of epilepsy in FCDII. One critical therapeutic challenge and opportunity for FCDII is to selectively eliminate DNs. Here, we developed two strategies to specifically ablate DNs in FCDII mouse models, and the results demonstrate that DN ablation is sufficient to both prevent and eliminate epilepsy in mice. Moreover, the associated neurobehavioral abnormalities were also reversed following treatment. Therefore, our study provides proof-of-concept evidence that DN ablation is a highly promising approach for curing FCDII in the future.},
}

*Epilepsy/genetics/pathology/therapy
*Malformations of Cortical Development, Group I/genetics/pathology/therapy
Proto-Oncogene Proteins c-akt/genetics
Disease Models, Animal
Animals
Mice
*Diphtheria Toxin/therapeutic use
*Ablation Techniques
*Heparin-binding EGF-like Growth Factor/genetics
Humans
HEK293 Cells
TOR Serine-Threonine Kinases/genetics
*Neurons/pathology
CRISPR-Cas Systems
*Gene Editing/methods

@article {pmid40943207,
year = {2025},
author = {Gilyazova, I and Korytina, G and Kochetova, O and Savelieva, O and Mikhaylova, E and Vershinina, Z and Chumakova, A and Markelov, V and Abdeeva, G and Karunas, A and Khusnutdinova, E and Gusev, O},
title = {Advances in Genomics and Postgenomics in Poultry Science: Current Achievements and Future Directions.},
journal = {International journal of molecular sciences},
volume = {26},
number = {17},
pages = {},
pmid = {40943207},
issn = {1422-0067},
support = {№ 075-15-2025-484//Government of Russian Federation/ ; },
abstract = {The poultry industry, a globally fast growing agricultural sector, provides affordable animal protein due to high efficiency. Gallus gallus domesticus are the most common domestic birds. Hybrid chicken breeds (crosses) are widely used to achieve high productivity. Maintaining industry competitiveness requires constant genetic selection of parent stock to improve performance traits. Genetic studies, which are essential in modern breeding programs, help identify genome variants linked to economically important traits and preserve population health. Next-generation sequencing (NGS) has identified millions of single nucleotide polymorphisms (SNPs) and insertions/deletions (INDELs), enabling detection of genome-wide regions associated with selection traits. Recent studies have pinpointed such regions using broiler lines, laying hen lines, or pooled genomic data. This review discusses advances in chicken genomic and transcriptomic research focused on traits enhancing meat breed performance and reproductive abilities. Special attention is given to transcriptome studies revealing regulatory mechanisms and key signaling pathways involved in artificial molting, as well as metagenome studies investigating resistance to infectious diseases and climate adaptation. Finally, a dedicated section highlights CRISPR/Cas genomic editing techniques for targeted genome modification in chicken genomics.},
}

@article {pmid40943047,
year = {2025},
author = {Huang, C and Cheng, L},
title = {Unlocking Casein Bioactivity: Lactic Acid Bacteria and Molecular Strategies for Peptide Release.},
journal = {International journal of molecular sciences},
volume = {26},
number = {17},
pages = {},
pmid = {40943047},
issn = {1422-0067},
abstract = {Bioactive peptides encrypted in bovine β-casein display diverse physiological functions, including antihypertensive, antioxidative, antimicrobial, and immunomodulatory activities. These peptides are normally released during gastrointestinal digestion or microbial fermentation, especially by proteolytic systems of lactic acid bacteria (LAB). However, peptide yields vary widely among LAB strains, reflecting strain-specific protease repertoires. To overcome these limitations, the scientific goal of this study is to provide a comprehensive synthesis of how synthetic biology, molecular biotechnology, and systems-level approaches can be leveraged to enhance the targeted discovery and production of β-casein-derived bioactive peptides. Genome engineering tools such as clustered regularly interspaced short palindromic repeats associated system (CRISPR/Cas) systems have been applied to modulate gene expression and metabolic flux in LAB, while inducible expression platforms allow on-demand peptide production. Additionally, cell-free systems based on LAB lysates further provide rapid prototyping for high-throughput screening. Finally, multi-omics approaches, including genomics, transcriptomics, proteomics, and metabolomics, further help pinpoint regulatory bottlenecks and facilitate rational strain optimization. This review provides a comprehensive overview of bioactive peptides derived from bovine β-casein and highlights recent progress in LAB-based strategies-both natural and engineered-for their efficient release. These advances pave the way for developing next-generation functional fermented foods enriched with targeted bioactivities.},
}

@article {pmid40825952,
year = {2025},
author = {Vats, P and Kumar, R and Kumar, R and Kaushik, JK and Mohanty, AK and Kumar, S},
title = {Deciphering the role of MFGE8 in lactation using CRISPR-CAS9 based gene editing in Buffalo mammary epithelial cells.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {30194},
pmid = {40825952},
issn = {2045-2322},
support = {EMR/2017/000152//DST-SERB/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Female ; *Lactation/genetics ; *Epithelial Cells/metabolism ; *Buffaloes/genetics ; *Mammary Glands, Animal/cytology/metabolism ; *Milk Proteins/genetics/metabolism ; Proteomics ; Gene Knockout Techniques ; *Glycoproteins/genetics/metabolism ; Proteome ; },
abstract = {Milk fat globule EGF factor 8 (MFGE8) is a glycoprotein which plays a crucial role in mammary gland remodeling. Our group previously identified MFGE8 as a marker associated with high milk yielding cows. Here, we generated MFGE8 knock-out buffalo mammary epithelial cells (BuMEC) via CRISPR-cas9 technology to decipher its role in lactation. gRNA3 reduced MFGE8 expression with good efficiency which was confirmed at transcriptomic and proteomic level and the stable knock-out cells obtained were named mfge8-/-/gRNA3. The amplicon sequencing of the edited region using next generation sequencing (NGS) showed that 54% of total reads showed indels, 3-4 bp upstream to PAM site in 2nd exon. A total 4282 proteins were identified when proteome level changes were examined and 178 were found to be differentially expressed above and below a threshold of ≥ 1.5 and ≤ 0.6. Major DEPs were found to be associated with regulation of hydrolase activity, endopeptidase activity and cytoskeletal organization and some DEPs including FABP3, FABP4, FABP5, KNG1, MT2A, CD82, SLC7A1 and SERPINH1 belonged to genes associated with milk synthesis. To the best of our knowledge, this is the first study which provides a comprehensive proteome profile of MFGE8 knockout BuMEC and explores downstream effects of disruption of MFGE8 gene.},
}

Animals
*CRISPR-Cas Systems
*Gene Editing/methods
Female
*Lactation/genetics
*Epithelial Cells/metabolism
*Buffaloes/genetics
*Mammary Glands, Animal/cytology/metabolism
*Milk Proteins/genetics/metabolism
Proteomics
Gene Knockout Techniques
*Glycoproteins/genetics/metabolism
Proteome

@article {pmid40825942,
year = {2025},
author = {Countryman, AD and Doherty, CA and Herrera-Perez, RM and Kasza, KE},
title = {Endogenous OptoRhoGEFs reveal biophysical principles of epithelial tissue furrowing.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7665},
pmid = {40825942},
issn = {2041-1723},
support = {F31 HD118793/HD/NICHD NIH HHS/United States ; F31HD118793//U.S. Department of Health & Human Services | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; P40 OD018537/OD/NIH HHS/United States ; R35GM138380//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R35 GM138380/GM/NIGMS NIH HHS/United States ; Sloan Fellowship//Alfred P. Sloan Foundation/ ; Packard Fellowship//David and Lucile Packard Foundation (David & Lucile Packard Foundation)/ ; CAREER Award//National Science Foundation (NSF)/ ; },
mesh = {Animals ; *Drosophila Proteins/metabolism/genetics ; Epithelium/embryology/metabolism ; *Drosophila melanogaster/embryology/genetics/metabolism ; Optogenetics/methods ; Morphogenesis ; Actomyosin/metabolism ; Animals, Genetically Modified ; *Rho Guanine Nucleotide Exchange Factors/metabolism/genetics ; Gastrulation ; Embryo, Nonmammalian/metabolism ; CRISPR-Cas Systems ; Signal Transduction ; Gene Expression Regulation, Developmental ; },
abstract = {During development, epithelia function as malleable sheets that undergo extensive remodeling to shape developing embryos. Optogenetic control of Rho signaling provides an avenue to investigate mechanisms of epithelial morphogenesis, but transgenic optogenetic tools can be limited by variability in expression levels and deleterious effects of transgenic overexpression on development. Here, we use CRISPR/Cas9 to tag Drosophila RhoGEF2 and Cysts/Dp114RhoGEF with components of the iLID/SspB optogenetic heterodimer, permitting light-dependent control over endogenous protein activities. Using quantitative optogenetic perturbations, we uncover a dose-dependence of tissue furrow depth and bending behavior on RhoGEF recruitment, revealing mechanisms by which developing embryos can shape tissues into particular morphologies. We show that at the onset of gastrulation, furrows formed by cell lateral contraction are oriented and size-constrained by basal actomyosin. Our findings demonstrate the use of quantitative, 3D-patterned perturbations of cell contractility to precisely shape tissue structures and interrogate developmental mechanics.},
}

Animals
*Drosophila Proteins/metabolism/genetics
Epithelium/embryology/metabolism
*Drosophila melanogaster/embryology/genetics/metabolism
Optogenetics/methods
Morphogenesis
Actomyosin/metabolism
Animals, Genetically Modified
*Rho Guanine Nucleotide Exchange Factors/metabolism/genetics
Gastrulation
Embryo, Nonmammalian/metabolism
CRISPR-Cas Systems
Signal Transduction
Gene Expression Regulation, Developmental

@article {pmid40825812,
year = {2025},
author = {Netsawang, C and Tongbaen, M and Jearawiriyapaisarn, N and Leecharoenkiat, K},
title = {Precise correction of G6PD Viangchan mutation in iPSCs by prime editing strategy.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {30192},
pmid = {40825812},
issn = {2045-2322},
support = {B05F640126//The NSRF via the Program Management Unit for Human Resources & Institutional Development, Research and Innovation/ ; 196807//Thailand Science research and Innovation Fund Chulalongkorn University/ ; },
mesh = {Humans ; *Glucosephosphate Dehydrogenase/genetics ; *Induced Pluripotent Stem Cells/metabolism ; *Gene Editing/methods ; HEK293 Cells ; *Glucosephosphate Dehydrogenase Deficiency/genetics/therapy ; *Mutation ; CRISPR-Cas Systems ; },
abstract = {Individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency hold a significant risk of severe hemolytic crises under oxidative stress. Currently, the definitive and curative treatment for the disorder has not been developed. Among over 200 G6PD variants, G6PD Viangchan (c.871 G > A) is the most prevalent and has been extensively studied in Southeast Asia. This study assessed the effectiveness of prime editing for correcting the G6PD Viangchan mutation in an established mutant HEK293T cell line and G6PD-deficient induced pluripotent stem cells (iPSCs). Using optimized modalities, prime editing achieved a high correction efficiency of over 25% in the HEK293T cells. In iPSCs, this gene editing tool yielded satisfactory correction outcomes, with approximately 5% corrected alleles. Our findings indicate that prime editing provides high precision, producing minimal by-products below baseline and showing undetectable off-target effects. Overall, prime editing has the potential to correct the G6PD Viangchan mutation, providing a valuable approach for future therapeutic strategies and the generation of isogenic cell lines to promote extensive studies in drug discovery and the pathogenesis of the G6PD variant.},
}

Humans
*Glucosephosphate Dehydrogenase/genetics
*Induced Pluripotent Stem Cells/metabolism
*Gene Editing/methods
HEK293 Cells
*Glucosephosphate Dehydrogenase Deficiency/genetics/therapy
*Mutation
CRISPR-Cas Systems

@article {pmid40825783,
year = {2025},
author = {Calvo-Villamañán, A and Sastre-Dominguez, J and Barrera-Martín, Á and Costas, C and San Millan, Á},
title = {Dissecting pOXA-48 fitness effects in clinical Enterobacterales using plasmid-wide CRISPRi screens.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7700},
pmid = {40825783},
issn = {2041-1723},
support = {ALTF 322-2022//European Molecular Biology Organization (EMBO)/ ; },
mesh = {*Plasmids/genetics ; *beta-Lactamases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; *Enterobacteriaceae/genetics/drug effects ; Humans ; Enterobacteriaceae Infections/microbiology/drug therapy ; *Genetic Fitness ; CRISPR-Cas Systems/genetics ; Drug Resistance, Multiple, Bacterial/genetics ; Anti-Bacterial Agents/pharmacology ; Carbapenems/pharmacology ; },
abstract = {Conjugative plasmids are the main vehicle for the spread of antimicrobial resistance (AMR) genes in clinical bacteria. AMR plasmids allow bacteria to survive antibiotic treatments, but they also produce physiological alterations in their hosts that commonly translate into fitness costs. Despite the key role of plasmid-associated fitness effects in AMR evolution, their origin and molecular bases remain poorly understood. In this study, we introduce plasmid-wide CRISPR interference (CRISPRi) screens as a tool to dissect plasmid-associated fitness effects. We design and perform CRISPRi screens targeting the globally distributed carbapenem resistance plasmid pOXA-48 in 13 different multidrug resistant clinical Enterobacterales. Our results reveal that pOXA-48 gene-level effects are conserved across clinical strains, and expose the key role of the carbapenemase-encoding gene, blaOXA-48, as the main culprit for pOXA-48 fitness costs. Moreover, our results highlight the relevance of postsegregational killing systems in pOXA-48 vertical transmission, and uncover new genes implicated in pOXA-48 stability (pri, korC, DNDJGHEP_13 and 14 and H-NS). This study sheds new light on the biology and evolution of carbapenem resistant Enterobacterales and endorses CRISPRi screens as a powerful method for studying plasmid-mediated AMR.},
}

*Plasmids/genetics
*beta-Lactamases/genetics/metabolism
*Bacterial Proteins/genetics/metabolism
*Enterobacteriaceae/genetics/drug effects
Humans
Enterobacteriaceae Infections/microbiology/drug therapy
*Genetic Fitness
CRISPR-Cas Systems/genetics
Drug Resistance, Multiple, Bacterial/genetics
Anti-Bacterial Agents/pharmacology
Carbapenems/pharmacology

@article {pmid40824957,
year = {2025},
author = {Naes, SM and Ab-Rahim, S and Mazlan, M and Syafruddin, SE and Mohtar, MA and Abuhamad, AY and Abdul Rahman, A},
title = {CRISPR/Cas9 mediated ENT2 gene knockout altered purine catabolic pathway and induced apoptosis in colorectal cell lines.},
journal = {PloS one},
volume = {20},
number = {8},
pages = {e0329501},
pmid = {40824957},
issn = {1932-6203},
mesh = {Humans ; *Apoptosis/genetics ; *CRISPR-Cas Systems ; *Purines/metabolism ; *Colorectal Neoplasms/genetics/metabolism/pathology ; Gene Knockout Techniques ; Cell Line, Tumor ; *Equilibrative-Nucleoside Transporter 2/genetics/metabolism ; Reactive Oxygen Species/metabolism ; Xanthine Oxidase/metabolism ; HT29 Cells ; Hypoxanthine/metabolism ; Gene Expression Regulation, Neoplastic ; },
abstract = {Although purine metabolism is one of the most impacted pathways in colorectal cancer (CRC), little is known about the role of equilibrative nucleoside transporter 2 (ENT2) in CRC development and its association with the altered purine metabolism pathway. This study aimed to determine the role of ENT2 in altered purine metabolism in the early and late stages of CRC using CRISPR/Cas9 gene editing tools and a variety of functional experiments. The expression of ENT2 was significantly higher (P < 0.001) in all CRC cell lines as compared to the normal colon cells. The two CRC cell lines with the highest ENT2 expression, the early stage HT29 cells and the late stage DLD1 cells, were knocked out (KO) using the CRISPR/Cas9 tool. The hypoxanthine (HPX) level and the xanthine oxidase (XO) activity were significantly higher in both HT29/KO and DLD1/KO single cell-derived clones (P < 0.01). The increase in HPX level and XO activity were associated with an elevation in the reactive oxygen species (ROS) level. These data suggest that the ENT2 KO elevated the ROS levels induced apoptosis and impaired the cell proliferation of the early stage of CRC cell line, i.e., HT29/KO clonal cells. In this context, targeting ENT2 gene might be a potential strategy in CRC treatment by increasing the production of ROS and hence, inducing the apoptosis pathway.},
}

Humans
*Apoptosis/genetics
*CRISPR-Cas Systems
*Purines/metabolism
*Colorectal Neoplasms/genetics/metabolism/pathology
Gene Knockout Techniques
Cell Line, Tumor
*Equilibrative-Nucleoside Transporter 2/genetics/metabolism
Reactive Oxygen Species/metabolism
Xanthine Oxidase/metabolism
HT29 Cells
Hypoxanthine/metabolism
Gene Expression Regulation, Neoplastic

@article {pmid40823809,
year = {2025},
author = {Migliori, V and Bruntraeger, MB and Gyulev, IS and Lichou, F and Burgold, T and Gitterman, DP and Iwama, S and Trinh, AL and Washer, SJ and Jones, CP and Trynka, G and Bassett, AR},
title = {ONE-STEP tagging: a versatile method for rapid site-specific integration by simultaneous reagent delivery.},
journal = {Nucleic acids research},
volume = {53},
number = {15},
pages = {},
pmid = {40823809},
issn = {1362-4962},
support = {220540/Z/20/A/WT_/Wellcome Trust/United Kingdom ; //Sanger Translation Committee/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Induced Pluripotent Stem Cells/metabolism ; T-Lymphocytes/metabolism ; Integrases/genetics/metabolism ; },
abstract = {We present a novel, versatile genome editing method termed ONE-STEP tagging, which combines CRISPR-Cas9-mediated targeting with Bxb1 integrase-based site-specific integration for efficient, precise, and scalable protein tagging. Applied in human-induced pluripotent stem cells (hiPSCs), cancer cells and primary T cells, this system enables rapid generation of endogenously tagged proteins. By enhancing the nuclear localization signal of the catalytically superior eeBxb1 integrase and co-delivering a DNA-PK inhibitor, we achieved up to ∼90% integration efficiency at the ACTR10 locus in hiPSCs. ONE-STEP tagging is robust across loci and cell types and supports large DNA cargo integration, with efficiencies reaching 16.6% for a 14.4 kb construct. The method also enables multiplexed tagging of multiple proteins within the same cell and simultaneous CRISPR-based editing at secondary loci, such as gene knockouts or homology-directed repair. Importantly, we demonstrate successful application in primary T cells by targeting the T cell receptor locus while simultaneously knocking out B2M, a key step towards generating immune-evasive, off-the-shelf chimeric antigen receptor T cells. Additionally, we introduce a dual-cassette version of the method compatible with universal donor plasmids, allowing use of entirely off-the-shelf reagents. Together, these advances establish ONE-STEP tagging as a powerful tool for both basic and therapeutic genome engineering.},
}

Humans
*CRISPR-Cas Systems
*Gene Editing/methods
Induced Pluripotent Stem Cells/metabolism
T-Lymphocytes/metabolism
Integrases/genetics/metabolism

@article {pmid40814254,
year = {2025},
author = {Nevot, G and Güell, M and Santos-Moreno, J},
title = {Critical Analysis of Preprints and Inquiry-Based Lessons Improve the Synthetic Biology Learning Experience.},
journal = {ACS synthetic biology},
volume = {14},
number = {8},
pages = {2878-2884},
pmid = {40814254},
issn = {2161-5063},
mesh = {*Synthetic Biology/education ; Humans ; *Problem-Based Learning/methods ; Students ; Universities ; CRISPR-Cas Systems/genetics ; },
abstract = {Synthetic biology is a transformative field crucial to address global challenges. It is highly interdisciplinary, integrating different subjects beyond biology. Therefore, traditional lecture-based teaching methods often fall short in effectively covering the diverse and rapidly evolving advancements in synthetic biology. We developed active learning workflows for complementing classic theoretical lectures in universities to improve the synthetic biology learning experience. We used preprints as an educational resource for the students to critically analyze differences comparing manuscripts and the final published work. In addition, we designed a practical laboratory session where students had to infer the logic behind CRISPRi-based gene circuits that they assembled, thus engaging with every step of the design-build-test-learn cycle. Following these activities, 90% of the students reported having improved critical analysis skills and 80% felt that they had learned a wide range of synthetic biology concepts. These approaches demonstrate the potential of innovative teaching for synthetic biology, which helps students with both technical and soft skills at the same time and has the potential to be adapted to other fields.},
}

*Synthetic Biology/education
Humans
*Problem-Based Learning/methods
Students
Universities
CRISPR-Cas Systems/genetics

@article {pmid40736249,
year = {2025},
author = {Zhao, Y and Guo, G and Sun, Y and Zhang, M and Yang, G and Liu, Z and Song, Y and Ghonaim, AH and Ma, N and Zhang, M and Jongkaewwattana, A and He, Q and Li, W},
title = {Membrane protein CRISPR screen identifies RPSA as an essential host factor for porcine epidemic diarrhea virus replication.},
journal = {Journal of virology},
volume = {99},
number = {8},
pages = {e0064925},
pmid = {40736249},
issn = {1098-5514},
mesh = {Animals ; *Porcine epidemic diarrhea virus/physiology ; *Virus Replication ; Swine ; *Coronavirus Infections/virology/veterinary/metabolism ; CRISPR-Cas Systems ; *Swine Diseases/virology/metabolism/genetics ; Host-Pathogen Interactions ; *Membrane Proteins/genetics/metabolism ; MAP Kinase Signaling System ; Gene Knockout Techniques ; Chlorocebus aethiops ; },
abstract = {UNLABELLED: Porcine epidemic diarrhea, caused by porcine epidemic diarrhea virus (PEDV), is one of the most devastating diseases in the global pig industry due to its high mortality rate in piglets. The host factors required for PEDV replication, including receptors, remain poorly understood. Here, we developed a porcine membrane-protein-scale CRISPR/Cas9 knockout (PigMpCKO) library and performed two rounds of PEDV infection. Ribosomal protein SA (RPSA), the known receptor of dengue virus, was found to be a potent host factor. Moreover, our research revealed that RPSA is involved in the replication stage of PEDV and not in the entry stage. Inhibitor and activator experiments demonstrated that knockout (KO) of RPSA downregulates the ERK1/2 signaling pathway to impair PEDV infection. Additionally, RNA sequencing data indicated that cellular lipid biosynthesis and lipid transport processes were significantly inhibited in the absence of RPSA during PEDV infection. Mechanistic studies revealed that the reduction in total cholesterol and triglyceride levels, resulting from RPSA KO, was partially mediated by the ERK1/2 pathway, leading to impaired lipid accumulation during PEDV replication. Interestingly, RPSA KO also significantly downregulated the expression of aminopeptidase N (APN) and inhibited infection by transmissible gastroenteritis virus (TGEV) and porcine deltacoronavirus (PDCoV), both of which belong to the swine enteric coronavirus group. In summary, our results establish RPSA as a novel host factor that is critical for coronavirus replication. This provides new insights into the mechanisms of virus-host interactions and paves the way for the development of broad-spectrum antiviral therapies.

IMPORTANCE: Swine enteric coronaviruses (SeCoVs) cause severe economic losses to the global swine industry and pose a potential threat to public health. Identification of receptors required for PEDV infection could develop novel targets for drug therapy and disease-resistant breeding. We conducted a CRISPR/Cas9 screen targeting membrane proteins in porcine kidney cells infected with PEDV to identify possible receptors and discovered numerous novel candidate host factors. Considering RPSA's known role as a receptor for multiple viruses, we focused on investigating its potential in coronavirus infection. Our results revealed that RPSA does not contribute to the entry stage but to the replication stage of coronavirus infection. We first reported the role that RPSA plays in the regulation of APN expression and lipid metabolism. RPSA is essential for PEDV and other SeCoVs replication, providing a novel insight into the search for the receptor of PEDV and identifying potential therapeutic targets for coronaviruses.},
}

Animals
*Porcine epidemic diarrhea virus/physiology
*Virus Replication
Swine
*Coronavirus Infections/virology/veterinary/metabolism
CRISPR-Cas Systems
*Swine Diseases/virology/metabolism/genetics
Host-Pathogen Interactions
*Membrane Proteins/genetics/metabolism
MAP Kinase Signaling System
Gene Knockout Techniques
Chlorocebus aethiops

@article {pmid40707359,
year = {2025},
author = {Campbell, RR and Green, M and Choi, EY and Wulff, AB and Siclair, AN and Khatri, S and Virata, G and Barrett, C and Key, S and Patel, S and Rowell, MB and Franco, D and Ganapathy-Kanniappan, S and Mathur, BN and Lobo, MK},
title = {Dopamine Receptor 1 Specific CRISPRa Mice Exhibit Disrupted Behaviors and Striatal Baseline Cellular Activity.},
journal = {eNeuro},
volume = {12},
number = {8},
pages = {},
pmid = {40707359},
issn = {2373-2822},
support = {R01 DA038613/DA/NIDA NIH HHS/United States ; R33 DA052101/DA/NIDA NIH HHS/United States ; },
mesh = {Animals ; *Receptors, Dopamine D1/genetics/metabolism ; Mice, Transgenic ; *Corpus Striatum/metabolism ; Mice ; Male ; Reward ; *Behavior, Animal/physiology ; CRISPR-Cas Systems ; Mice, Inbred C57BL ; Motor Activity/physiology ; },
abstract = {The two main cell types in the striatum, dopamine receptor 1 and adenosine receptor 2a spiny projection neurons (D1-SPNs and A2A-SPNs), have distinct roles in regulating motor- and reward-related behaviors. Cre-selective CRISPR-dCas9 systems allow for cell-type specific, epigenomic-based manipulation of gene expression with gene-specific single guide RNAs (sgRNAs) and have potential to elucidate molecular mechanisms underlying striatal subtype mediated behaviors. Conditional transgenic Rosa26:LSL-dCas9-p300 mice were recently generated to allow for robust expression of dCas9-p300 expression with Cre-driven cell-type specificity. This system utilizes p300, a histone acetyltransferase which regulates gene expression by unwinding chromatin and making that region of the genome more accessible for transcription. Rosa26-LSL-dCas9-p300 mice were paired with Drd1-Cre and Ador2a-Cre mice to generate Drd1-Cre:dCas9-p300 and Ador2a-Cre:dCas9-p300 mouse lines and underwent behavioral phenotyping when sgRNAs were not present. Both Drd1-Cre:dCas9-p300 and Ador2a-Cre:dCas9-p300 have cell-type-specific expression of spCas9 mRNA. Baseline behavioral assessments revealed that, under a sgRNA absent nontargeted state, Drd1-Cre:dCas9-p300 mice display repetitive spinning behavior, hyperlocomotion, and enhanced acquisition of reward learning in comparison with all genotypic littermates. In contrast, Ador2a-Cre:dCas9-p300 do not exhibit any changes in behavior in comparison with their littermates. Electrophysiological recordings of dorsal striatum D1-SPNs revealed that Drd1-Cre:dCas9-p300 mice have increased input resistance and increased spontaneous excitatory postsynaptic current amplitude, together suggesting greater excitatory drive of D1-SPNs. Overall, these data demonstrate the necessity to validate CRISPR-dCas9 lines for research investigations. Additionally, the Drd1-Cre:dCas9-p300 line has the potential to be used to study underlying mechanisms of stereotypy and reward learning.},
}

Animals
*Receptors, Dopamine D1/genetics/metabolism
Mice, Transgenic
*Corpus Striatum/metabolism
Mice
Male
Reward
*Behavior, Animal/physiology
CRISPR-Cas Systems
Mice, Inbred C57BL
Motor Activity/physiology

@article {pmid40941236,
year = {2025},
author = {Liu, Y and Xie, Y and Wang, Z and Gai, Z and Zhang, X and Chen, J and Lei, H and Xu, Z and Shen, X},
title = {A Simple, Rapid, and Contamination-Free Ultra-Sensitive Cronobacter sakazakii Visual Diagnostic Platform Based on RPA Combined with CRISPR/Cas12a.},
journal = {Foods (Basel, Switzerland)},
volume = {14},
number = {17},
pages = {},
doi = {10.3390/foods14173120},
pmid = {40941236},
issn = {2304-8158},
support = {2023YFF1105103//National Key Research and Development Program of China/ ; },
abstract = {CRISPR/Cas systems have made significant progress in the field of molecular diagnostics in recent years. To overcome the aerosol contamination problem brought on by amplicon transfer in the common two-step procedure, the "one-pot method" has become a major research hotspot in this field. However, these methods usually rely on specially designed devices or additional chemical modifications. In this study, a novel "one-pot" strategy was developed to detect the foodborne pathogen Cronobacter sakazakii (C. sakazakii). A specific sequence was screened out from the virulence gene ompA of C. sakazakii as the detection target. Combining with the recombinase polymerase amplification (RPA), a rapid detection platform for C. sakazakii based on the CRISPR/Cas12a system was established for the first time. The sensitivity of this method was determined from three different levels, which are 10[-4] ng/μL for genomic DNA (gDNA), 1.43 copies/μL for target DNA, and 6 CFU/mL for pure bacterial culture. Without any microbial enrichment, the detection limits for artificially contaminated cow and goat milk powder samples were 4.65 CFU/mL and 4.35 CFU/mL, respectively. To address the problem brought on by aerosol contamination in the common RPA-CRISPR/Cas12a two-step method, a novel pipette tip-in-tube (PTIT) method for simple and sensitive one-pot nucleic acid detection was further developed under the inspiration of the capillary principle. The RPA and CRISPR/Cas systems were isolated from each other by the force balance of the solution in a pipette tip before amplification. The detection limits of the PTIT method in pure bacterial culture and the spiked samples were exactly the same as that of the two-step method, but with no false positive cases caused by aerosol contamination at all. Compared with other existing one-pot methods, the PTIT method requires no additional or specially designed devices, or any chemical modifications on crRNA and nucleic acid probes. Therefore, the PTIT method developed in this study provides a novel strategy for realizing one-pot CRISPR/Cas detection easily and holds significant potential for the rapid point-on-care testing (POCT) application.},
}

@article {pmid40940734,
year = {2025},
author = {Cattin, E and Schena, E and Mattioli, E and Marcuzzo, S and Bonanno, S and Cavalcante, P and Corradi, F and Benati, D and Farinazzo, G and Cattaneo, M and De Sanctis, V and Bertorelli, R and Maggi, L and Giannotta, M and Pini, A and Vattemi, G and Cassandrini, D and Cavallo, M and Manferdini, C and Lisignoli, G and Fontana, B and Pace, I and Bruno, C and Roncarati, R and Fiorillo, C and Ferracin, M and Schirmer, EC and Recchia, A and Lattanzi, G},
title = {Profibrotic Molecules Are Reduced in CRISPR-Edited Emery-Dreifuss Muscular Dystrophy Fibroblasts.},
journal = {Cells},
volume = {14},
number = {17},
pages = {},
doi = {10.3390/cells14171321},
pmid = {40940734},
issn = {2073-4409},
support = {20223WFJJ3//Ministero dell'università e della ricerca/ ; CUP B33C22001640007//Associazione Italiana DIstrofia Muscolare di Emery-Dreifuss - AIDMED/ ; CUP B33C22001640007//Associazione Alessandra Proietti OdV/ ; ECOSISTER Project. cod. ECS_00000033-CUP B89I22000650001//EU-funded PNRR/ ; ECOSISTER project ECS_00000033 - CUP E93C22001100001//EU-funded PNRR/ ; TREAT-LMNA 2019-004426-24//AIFA/ ; T3-AN-03 CUP: B53C22002520006 REGINA//Ministero della Salute/ ; RRC//The Italian Ministry of Health/ ; },
abstract = {Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in EMD, LMNA, SYNE1, SYNE2, and other related genes. The disease is characterized by joint contractures, muscle weakening and wasting, and heart conduction defects associated with dilated cardiomyopathy. Previous studies demonstrated the activation of fibrogenic molecules such as TGFbeta 2 and CTGF in preclinical models of EDMD2 and increased secretion of TGFbeta 2 in patient serum. A wide screening of patient cells suggested fibrosis, metabolism, and myogenic signaling as the most affected pathways in various EDMD forms. In this study, we show that alpha-smooth muscle actin-positive myofibroblasts are overrepresented in patient fibroblast cultures carrying EMD, LMNA, or SYNE2 mutations, and profibrotic miRNA-21 is upregulated. Upon CRISPR/Cas correction of the mutated EMD or LMNA sequence in EDMD1 or EDMD2 fibroblasts, respectively, we observe a reduced expression of fibrogenic molecules. However, in patient myoblasts, neither fibrogenic proteins nor miRNA-21 were upregulated; instead, miRNA-21-5p was downregulated along with muscle-specific miRNA-133b and miRNA-206, which have a crucial role in muscle cell homeostasis. These observations suggest that the conversion of laminopathic fibroblasts into a profibrotic phenotype is a determinant of EDMD-associated muscle fibrosis, while miRNA-206-dependent defects of laminopathic myoblasts, including altered regulation of VEGF levels, contribute to muscle cell deterioration. Notably, our study provides a proof-of-principle for the application of gene correction to EDMD1 and EDMD2 and presents EDMD1 isogenic cells that exhibit an almost complete rescue of a disease-specific miRNA signature. These cells can be used as experimental models for studying muscular laminopathies.},
}

@article {pmid40938972,
year = {2025},
author = {Abdirassilova, AA and Yessimseit, DT and Kassenova, AK and Abdeliyev, BZ and Zhumadilova, ZB and Tokmurziyeva, GZ and Kovaleva, GG and Abdel, ZZ and Meka-Mechenko, TV and Umarova, SK and Begimbayeva, EZ and Agzam, SD and Motin, VL and Reva, ON and Rysbekova, AK},
title = {Whole genome sequencing of Yersinia pestis isolates from Central Asian natural plague foci revealed the role of adaptation to different hosts and environmental conditions in shaping specific genotypes.},
journal = {PLoS neglected tropical diseases},
volume = {19},
number = {9},
pages = {e0013533},
doi = {10.1371/journal.pntd.0013533},
pmid = {40938972},
issn = {1935-2735},
abstract = {The genetic diversity and biovar classification of Yersinia isolates from Central Asia were investigated using whole-genome sequencing. In total, 98 isolates from natural plague foci were sequenced using the MiSeq platform. Computational pipelines were developed for accurate assembly of Y. pestis replicons, including small cryptic plasmids, and for identifying genetic polymorphisms. A panel of 99 diagnostic polymorphisms was established, enabling the distinction of dominant Medievalis isolates derived from desert and upland regions. Evidence of convergent evolution was observed in polymorphic allele distributions across genetically distinct Y. pestis biovars, Y. pseudotuberculosis, and other Y. pestis strains, likely driven by adaptation to similar environmental conditions. Genetic polymorphisms in the napA, araC, ssuA, and rhaS genes, along with transposon and CRISPR-Cas insertion patterns, were confirmed as suitable tools for identifying Y. pestis biovars, although their homoplasy suggests limited utility for phylogenetic inference. Notably, a novel cryptic plasmid, pCKF, previously associated with the strain of the population 2.MED0 from the Central-Caucasus high-altitude autonomous plague focus, was detected in a genetically distinct isolate of 2.MED1 population from the Ural-Embi region, indicating potential plasmid transfer across the 2.MED lineage. These findings emphasize the need for ongoing genomic surveillance to monitor the spread of virulence-associated genetic elements and to improve our understanding of Y. pestis evolution and ecology.},
}

@article {pmid40938680,
year = {2025},
author = {Yang, K and Wang, T and Zhu, Q and Shen, C},
title = {CRISPR/Cas-based detection strategies for tumor biomarker detection.},
journal = {Analytical methods : advancing methods and applications},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5ay01150g},
pmid = {40938680},
issn = {1759-9679},
abstract = {Tumor biomarkers, such as nucleic acids, proteins, extracellular vesicles (EVs) and circulating tumor cells (CTCs), can provide valuable information for tumor risk assessment, diagnosis, prognosis and recurrence monitoring. Currently, polymerase chain reaction (PCR)-based approaches and enzyme linked immunosorbent assay (ELISA) are typically used for detecting tumor biomarkers in clinics. However, PCR-based methods have limits in sensitivity and detection channels. Besides, ELISA suffers from cumbersome operation and limited sensitivity. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems are adopted for developing novel detection strategies due to the characteristics of high sensitivity, high specificity, simple operability and flexible programmability. Besides, CRISPR/Cas systems are amenable to combination with isothermal amplification techniques, primarily attributed to their compatibility and stability. Moreover, the combination of different CRISPR/Cas systems enables multiplex target detection. Therefore, CRISPR/Cas-based detection strategies have emerged as highly promising approaches for the sensitive, specific and multiplex detection of tumor biomarkers. In this review, we at first introduced the classification and working mechanisms of CRISPR/Cas systems. And then, we comprehensively summarized recently developed CRISPR/Cas-based detection strategies for tumor biomarkers. Besides, we reviewed detection strategies based on CRISPR/Cas systems for multiplex tumor biomarker detection. Furthermore, the challenges and prospects of existing CRISPR/Cas-based detection strategies were thoroughly discussed.},
}

@article {pmid40935887,
year = {2025},
author = {Alves, CRR and Das, S and Krishnan, V and Ha, LL and Fox, LR and Stutzman, HE and Shamber, CE and Kalailingam, P and McCarthy, S and Lino Cardenas, CL and Fong, CE and Imai, T and Mitra, S and Yun, S and Wood, RK and Benning, FMC and Roh, K and Lawton, J and Kim, N and Silverstein, RA and Ferreira da Silva, J and de la Cruz, D and Richa, R and Xie, J and Gray-Edwards, HL and Malhotra, R and Chung, DY and Chao, LH and Tsai, SQ and Maguire, CA and Lindsay, ME and Kleinstiver, BP and Musolino, PL},
title = {Treatment of a severe vascular disease using a bespoke CRISPR-Cas9 base editor in mice.},
journal = {Nature biomedical engineering},
volume = {},
number = {},
pages = {},
pmid = {40935887},
issn = {2157-846X},
support = {P01HL142494//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01NS125353//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; DP2CA281401//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; K01NS134784//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01NS125353//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01NS125353//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
abstract = {Pathogenic missense mutations in the alpha actin isotype 2 (ACTA2) gene cause multisystemic smooth muscle dysfunction syndrome (MSMDS), a genetic vasculopathy that is associated with stroke, aortic dissection and death in childhood. Here we perform mutation-specific protein engineering to develop a bespoke CRISPR-Cas9 enzyme with enhanced on-target activity against the most common MSMDS-causative mutation ACTA2 R179H. To directly correct the R179H mutation, we screened dozens of configurations of base editors to develop a highly precise corrective A-to-G edit with minimal deleterious bystander editing that is otherwise prevalent when using wild-type SpCas9 base editors. We create a murine model of MSMDS that shows phenotypes consistent with human patients, including vasculopathy and premature death, to explore the in vivo therapeutic potential of this strategy. Delivery of the customized base editor via an engineered smooth muscle-tropic adeno-associated virus (AAV-PR) vector substantially prolongs survival and rescues systemic phenotypes across the lifespan of MSMDS mice, including in the vasculature, aorta and brain. Our results highlight how bespoke mutant-specific CRISPR-Cas9 enzymes can improve mutation correction with base editors.},
}

@article {pmid40934884,
year = {2025},
author = {Gast, K and Barrangou, R},
title = {All systems go: CRISPR crosstalk for enhanced immunity.},
journal = {Cell host & microbe},
volume = {33},
number = {9},
pages = {1470-1472},
doi = {10.1016/j.chom.2025.08.002},
pmid = {40934884},
issn = {1934-6069},
abstract = {In this issue of Cell Host & Microbe, companion manuscripts from Margolis & Meeske[1] and Smith & Fineran[2] demonstrate that CRISPR-Cas systems have an unprecedented level of cooperative crosstalk between different subtypes, which enables primed spacer acquisition. These studies illustrate how CRISPR-Cas systems cooperate to enhance adaptive immunity in bacteria.},
}

@article {pmid40641152,
year = {2025},
author = {Robertson, NR and Lenert-Mondou, C and Leonard, AC and Tafrishi, A and Carrera, S and Lee, S and Aguilar, Y and Sanchez Zamora, L and Nguyen, T and Beltrán, J and Li, M and Cutler, SR and Whitehead, TA and Wheeldon, I},
title = {PYR1 Biosensor-Driven Genome-Wide CRISPR Screens for Improved Monoterpene Production in Kluyveromyces marxianus.},
journal = {ACS synthetic biology},
volume = {14},
number = {8},
pages = {2972-2978},
pmid = {40641152},
issn = {2161-5063},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Kluyveromyces/genetics/metabolism ; *Monoterpenes/metabolism ; Metabolic Engineering/methods ; *Fungal Proteins/genetics/metabolism ; Acyclic Monoterpenes/metabolism ; Genome, Fungal ; },
abstract = {Monoterpenes are valued for their roles as flavors, fragrances, insecticides, and energy-dense fuels. Microorganisms provide sustainable biosynthesis routes for these important molecules, but production levels remain limited. Here, we introduce a biosensor-driven microbial engineering strategy to enhance monoterpene production, specifically targeting geraniol. Using mutagenized libraries of the PYR1 receptor─a versatile biosensor from plant ABA signaling pathways with a malleable binding pocket─we screened 24 monoterpenes and identified PYR1 variants responsive to eight, including geraniol. A low background, highly selective geraniol-sensitive PYR1 variant was expressed in the thermotolerant yeast Kluyveromyces marxianus as a growth-based biosensor circuit, allowing for rapid strain engineering. By coupling the geraniol-sensitive PYR1 sensor with a genome-wide CRISPR-Cas9 mutagenesis approach, we identified six gene knockouts that enhance geraniol production, achieving up to a 2-fold increase in titer. This study demonstrates the power of the PYR1 biosensor platform to enable rapid strain engineering and the identification of mutants that improve the titer of a desired metabolite.},
}

*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
*Kluyveromyces/genetics/metabolism
*Monoterpenes/metabolism
Metabolic Engineering/methods
*Fungal Proteins/genetics/metabolism
Acyclic Monoterpenes/metabolism
Genome, Fungal

@article {pmid40580745,
year = {2025},
author = {Asadi-Sarabi, P and Rismani, E and Shabanpouremam, M and Hendi, Z and Nikoubin, B and Rahimi, S and Taleb, M and Khosravi, A and Zarrabi, A and Hassan, M and Vosough, M},
title = {Hypoimmunogenic pluripotent stem cells: A game-changer in cell-based regenerative medicine.},
journal = {International immunopharmacology},
volume = {162},
number = {},
pages = {115134},
doi = {10.1016/j.intimp.2025.115134},
pmid = {40580745},
issn = {1878-1705},
mesh = {Humans ; *Regenerative Medicine/methods ; *Pluripotent Stem Cells/immunology/transplantation ; Animals ; Gene Editing ; *Cell- and Tissue-Based Therapy/methods ; CRISPR-Cas Systems ; },
abstract = {Hypoimmunogenic pluripotent stem cells (hPSCs) represent a transformative innovation in regenerative medicine, offering solutions to the longstanding challenge of immune rejection in cell-based therapies. Through advanced gene-editing techniques, particularly CRISPR/Cas9, hPSCs are engineered to downregulate or eliminate the expression of major histocompatibility complex (MHC) molecules while upregulating immunomodulatory proteins such as HLA-G, PD-L1, and CD47. These modifications enhance immune evasion and create the foundation for universal donor cells. Compared to conventional cell therapies that rely on lifelong immunosuppression, hPSC-based strategies offer safer, more sustainable, and patient-friendly solutions by minimizing the risks of infection, malignancy, and drug toxicity. Beyond immune compatibility, critical challenges persist, including the risk of tumorigenicity, off-target genetic alterations, and ethical considerations surrounding genome editing. Recent advances, such as the integration of suicide gene systems and sensitive monitoring assays, offer promising strategies to enhance the safety and functional stability of hPSC-derived therapies. This review comprehensively discusses the molecular engineering of hPSCs, their biomedical applications, safety strategies, ethical implications, and the evolving regulatory frameworks needed for clinical translation. By addressing both the scientific and societal dimensions, hPSCs have the potential to revolutionize personalized and off-the-shelf regenerative treatments, provided that rigorous safeguards are implemented.},
}

Humans
*Regenerative Medicine/methods
*Pluripotent Stem Cells/immunology/transplantation
Animals
Gene Editing
*Cell- and Tissue-Based Therapy/methods
CRISPR-Cas Systems

@article {pmid40931807,
year = {2025},
author = {Wang, Y and Phelps, A and Godbehere, A and Evans, B and Takizawa, C and Chinen, G and Singh, H and Fang, Z and Du, ZY},
title = {Revolutionizing Agriculture With CRISPR Technology: Applications, Challenges, and Future Perspectives.},
journal = {Biotechnology journal},
volume = {20},
number = {9},
pages = {e70113},
doi = {10.1002/biot.70113},
pmid = {40931807},
issn = {1860-7314},
support = {//MBBE Molecular Biotechnology Lab/ ; HAW05047-H//USDA National Institute of Food and Agriculture/ ; 6114549//USDA REEU-Technology/ ; 5605280//CTAHR Agricultural Research and Extension Stations (CARES)/ ; //Undergraduate Research Opportunities Program (UROP)/ ; 5605280//U.S. Department of Agriculture/ ; 6114549//U.S. Department of Agriculture/ ; HATCH project HAW05047-H//U.S. Department of Agriculture/ ; },
mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Agriculture/methods/trends ; Crops, Agricultural/genetics ; Livestock/genetics ; Aquaculture ; },
abstract = {CRISPR technologies are rapidly transforming agriculture by enabling precise and programmable modifications across a wide range of organisms. This review provides an overview of CRISPR applications in crops, livestock, aquaculture, and microbial systems, highlighting key advances in sustainable agriculture. In crops, CRISPR has accelerated the improvement of traits such as drought tolerance, nutrient efficiency, and pathogen resistance. In livestock and aquaculture, CRISPR has enabled disease-resistant pigs and poultry, hornless cattle, and fast-growing, stress-tolerant fish. Engineered microbes are also being leveraged to enhance nitrogen fixation and reduce input reliance. We examine the evolution of CRISPR tools, such as base and prime editing, multiplex editing, and epigenome modulation, that expand precision and control beyond traditional gene knockouts. These innovations offer significant advantages over conventional breeding, yet challenges remain, including off-target effects, delivery efficiency, and regulatory variability across countries. The review also explores emerging directions such as novel Cas variants and AI-integrated breeding platforms for high-throughput trait discovery. Together, these developments demonstrate the transformative potential of CRISPR technology to reshape agriculture, not only by enhancing productivity and resilience but also by reducing environmental impacts. With responsible implementation, CRISPR-enabled innovations are well-positioned to support global food security and sustainability targets by 2050.},
}

Animals
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Agriculture/methods/trends
Crops, Agricultural/genetics
Livestock/genetics
Aquaculture

@article {pmid40931602,
year = {2025},
author = {Pan, L and Wang, P},
title = {DNA nanotechnology-enabled bioanalysis of extracellular vesicles.},
journal = {Nanoscale horizons},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5nh00557d},
pmid = {40931602},
issn = {2055-6764},
abstract = {Extracellular vesicles (EVs) have emerged as valuable sources for liquid biopsy in disease diagnostics, given their protein and nucleic acid cargoes (e.g., miRNA, mRNA, glycoRNA) can serve as critical biomarkers. DNA nanotechnology, leveraging its inherent programmability, high specificity, and powerful signal amplification capability, offers a transformative approach for the bioanalysis of EVs. This review summarizes recent advances in DNA nanotechnology-based analytical methodologies for detecting EV-associated proteins and nucleic acids. We detail the underlying principles, applications, and performance of key strategies, including aptamer-based recognition, enzyme-free catalytic amplification circuits (e.g., HCR, CHA), enzyme catalytic amplification techniques (e.g., RCA, CRISPR-Cas systems), and DNA nanostructures-assisted amplification. The integration of these DNA tools into multiplexed detection platforms is also discussed. Finally, current challenges and future perspectives concerning clinical translation of EV detection are presented.},
}

@article {pmid40114032,
year = {2025},
author = {Tang, K and Zhou, L and Tian, X and Fang, SY and Vandenbulcke, E and Du, A and Shen, J and Cao, H and Zhou, J and Chen, K and Kim, HR and Luo, Z and Xin, S and Lin, SH and Park, D and Yang, L and Zhang, Y and Suzuki, K and Majety, M and Ling, X and Lam, SZ and Chow, RD and Ren, P and Tao, B and Li, K and Codina, A and Dai, X and Shang, X and Bai, S and Nottoli, T and Levchenko, A and Booth, CJ and Liu, C and Fan, R and Dong, MB and Zhou, X and Chen, S},
title = {Cas12a-knock-in mice for multiplexed genome editing, disease modelling and immune-cell engineering.},
journal = {Nature biomedical engineering},
volume = {9},
number = {8},
pages = {1290-1308},
pmid = {40114032},
issn = {2157-846X},
support = {RF1DA048811//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; K99CA282989//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; F30CA250249//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; RF1 DA048811/DA/NIDA NIH HHS/United States ; R01 CA231112/CA/NCI NIH HHS/United States ; DP2 CA238295/CA/NCI NIH HHS/United States ; T32 GM007205/GM/NIGMS NIH HHS/United States ; F30 CA250249/CA/NCI NIH HHS/United States ; U54CA209992//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; DP2CA238295//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; K99 CA282989/CA/NCI NIH HHS/United States ; U54 CA209992/CA/NCI NIH HHS/United States ; R33 CA281702/CA/NCI NIH HHS/United States ; T32GM007205//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R33CA281702//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R33 CA225498/CA/NCI NIH HHS/United States ; R01CA231112//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Animals ; *Gene Editing/methods ; Mice ; Mice, Inbred C57BL ; *Gene Knock-In Techniques/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; Disease Models, Animal ; CRISPR-Cas Systems/genetics ; *Cell Engineering/methods ; Dependovirus/genetics ; Humans ; Dendritic Cells ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {The pleiotropic effects of human disease and the complex nature of gene-interaction networks require knock-in mice allowing for multiplexed gene perturbations. Here we describe a series of knock-in mice with a C57BL/6 background and with the conditional or constitutive expression of LbCas12a or of high-fidelity enhanced AsCas12a, which were inserted at the Rosa26 locus. The constitutive expression of Cas12a in the mice did not lead to discernible pathology and enabled efficient multiplexed genome engineering. We used the mice for the retrovirus-based immune-cell engineering of CD4[+] and CD8[+] T cells, B cells and bone-marrow-derived dendritic cells, for autochthonous cancer modelling through the delivery of multiple CRISPR RNAs as a single array using adeno-associated viruses, and for the targeted genome editing of liver tissue using lipid nanoparticles. We also describe a system for simultaneous dual-gene activation and knockout (DAKO). The Cas12a-knock-in mice and the viral and non-viral delivery vehicles provide a versatile toolkit for ex vivo and in vivo applications in genome editing, disease modelling and immune-cell engineering, and for the deconvolution of complex gene interactions.},
}

Animals
*Gene Editing/methods
Mice
Mice, Inbred C57BL
*Gene Knock-In Techniques/methods
*CRISPR-Associated Proteins/genetics/metabolism
Disease Models, Animal
CRISPR-Cas Systems/genetics
*Cell Engineering/methods
Dependovirus/genetics
Humans
Dendritic Cells
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid40930534,
year = {2025},
author = {Lejars, M and Maeda, T and Guillier, M},
title = {EASY-edit: a toolbox for high-throughput single-step custom genetic editing in bacteria.},
journal = {Nucleic acids research},
volume = {53},
number = {17},
pages = {},
doi = {10.1093/nar/gkaf883},
pmid = {40930534},
issn = {1362-4962},
support = {/ERC_/European Research Council/International ; 818750//European Union's Horizon 2020 research and innovation/ ; //CNRS/ ; ANR-11-LABX-0011//Initiative d'Excellence/ ; },
mesh = {*Gene Editing/methods ; *Escherichia coli/genetics ; *CRISPR-Cas Systems ; Operon ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genes, Reporter ; },
abstract = {Targeted gene editing can be achieved using CRISPR-Cas9-assisted recombineering. However, high-efficiency editing requires careful optimization for each locus to be modified, which can be tedious and time-consuming. In this work, we developed a simple, fast and cheap method: Engineered Assembly of SYnthetic operons for targeted editing (EASY-edit) in Escherichia coli. Highly efficient editing of the different constitutive elements of the operons can be achieved by using a set of optimized guide RNAs and single- or double-stranded DNA repair templates carrying relatively short homology arms. This facilitates the construction of multiple genetic tools, including mutant libraries or reporter genes. EASY-edit is also highly modular, as we provide alternative and complementary versions of the operon inserted in three loci which can be edited iteratively and easily combined. As a proof of concept, we report the construction of several fusions with reporter genes confirming known post-transcriptional regulation mechanisms and the construction of saturated and unbiased mutant libraries. In summary, the EASY-edit system provides a flexible genomic expression platform that can be used both for the understanding of biological processes and as a tool for bioengineering applications.},
}

*Gene Editing/methods
*Escherichia coli/genetics
*CRISPR-Cas Systems
Operon
RNA, Guide, CRISPR-Cas Systems/genetics
Genes, Reporter

@article {pmid40930528,
year = {2025},
author = {Jiang, Q and Jin, S and Qin, Z and Zhang, J and He, R and Chen, Z and Qiao, B and Qiao, J and Liu, Y},
title = {CRISPR/Cas12a DTR system: a topology-guided Cas12a assay for specific dual detection of RNA and DNA targets.},
journal = {Nucleic acids research},
volume = {53},
number = {17},
pages = {},
doi = {10.1093/nar/gkaf893},
pmid = {40930528},
issn = {1362-4962},
support = {2022YFC2304304//National Key Research and Development Program of China/ ; 2023DJC136//Science and Technology Innovation Talent Plan of Hubei Province/ ; 2025AFB825//Natural Science Foundation of Hubei Province/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *MicroRNAs/genetics/analysis ; *DNA/genetics/analysis ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; *RNA/genetics/analysis ; *Bacterial Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The CRISPR/Cas12a technology has revolutionized molecular diagnostics. However, existing Cas12a systems depend on continuous target DNA activation, which limits them to single-target detection. In this study, we developed a novel topology-guided Cas12a system, the double-target responsive (DTR) system, capable of being activated by noncontiguous dual RNA/DNA targets. The DTR system employs two split CRISPR RNA (crRNA) fragments and two Cas12a proteins that cooperatively reconstitute upon recognizing two nucleic acid activators. We demonstrated the DTR system's ability to specifically detect dual nucleic acid substrates in a single readout, achieving a detection limit of 78 fM for RNA and exceptional specificity for single-nucleotide variations. Additionally, we successfully applied the DTR system to clinical samples, enabling simultaneous detection of two oral squamous cell carcinoma-related microRNAs (miR-155 and miR-let-7a), thereby distinguishing healthy individuals from patients. This work establishes an efficient Cas12a-based platform for sensitive, simultaneous, and discriminative detection of RNA and DNA targets, enhancing the versatility of Cas12a in analytical detection and clinical diagnosis.},
}

*CRISPR-Cas Systems/genetics
Humans
*MicroRNAs/genetics/analysis
*DNA/genetics/analysis
*CRISPR-Associated Proteins/genetics/metabolism
*Endodeoxyribonucleases/genetics/metabolism
*RNA/genetics/analysis
*Bacterial Proteins/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40929845,
year = {2025},
author = {Chang, Y and Ding, J},
title = {CircRNA knockout/knockdown tools in molecular biology research.},
journal = {Biochemical and biophysical research communications},
volume = {783},
number = {},
pages = {152607},
doi = {10.1016/j.bbrc.2025.152607},
pmid = {40929845},
issn = {1090-2104},
abstract = {Circular RNAs (circRNAs), characterized by their covalently closed circular architecture, represent a unique class of endogenous RNA molecules that serve as pivotal regulators in post-transcriptional gene regulation in organisms. Accumulating evidence has established their potential as promising diagnostic biomarkers across various human pathologies, including but not limited to malignant neoplasms, neurodegenerative disorders, and metabolic dysregulation.By inhibiting circRNA expression, we can better understand their functions and their impact on related biological processes. Over the past decade, remarkable advancements have emerged in circRNAs manipulation technologies, including siRNA, DNAzyme, and CRISPR-Cas systems emerging as powerful tools for precise circRNAs editing in both experimental models and preclinical studies. In this review, we summarize the advantages and identification of these editing methods and discuss future challenges and prospects.},
}

@article {pmid40927181,
year = {2025},
author = {Ziemann, M and Mitrofanov, A and Stöckl, R and Alkhnbashi, OS and Backofen, R and Hess, WR},
title = {Analysis of tracrRNAs reveals subgroup V2 of type V-K CAST systems.},
journal = {microLife},
volume = {6},
number = {},
pages = {uqaf020},
pmid = {40927181},
issn = {2633-6693},
abstract = {Clustered regularly interspaced palindromic repeats (CRISPR)-associated transposons (CAST) consist of an integration between certain class 1 or class 2 CRISPR-Cas systems and Tn7-like transposons. Class 2 type V-K CAST systems are restricted to cyanobacteria. Here, we identified a unique subgroup of type V-K systems through phylogenetic analysis, classified as V-K_V2. Subgroup V-K_V2 CAST systems are characterized by an alternative tracrRNA, the exclusive use of Arc_2-type transcriptional regulators, and distinct differences in the length of protein domains in TnsB and TnsC. Although the occurrence of V-K_V2 CAST systems is restricted to Nostocales cyanobacteria, it shows signs of horizontal gene transfer, indicating its capability for genetic mobility. The predicted V-K_V2 tracrRNA secondary structure has been integrated into an updated version of the CRISPRtracrRNA program available on GitHub under https://github.com/BackofenLab/CRISPRtracrRNA/releases/tag/2.0.},
}

@article {pmid40678944,
year = {2025},
author = {Zhu, C and Huang, Q and Fu, R and Xun, Z and Ou, Q and Xianyu, Y and Liu, C},
title = {A Triple-Modal Biosensing Strategy for Hepatitis B Virus Based on Mg[2+]-Mediated Modulation of CRISPR/Cas12a and Au@Pt Nanoparticles.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {21},
number = {36},
pages = {e05341},
doi = {10.1002/smll.202505341},
pmid = {40678944},
issn = {1613-6829},
support = {82030063 82372317 82372316//National Natural Science Foundation of China/ ; 2023Y4004//University-Industry Research Cooperation Project of Science and Technology of Fujian Province/ ; 2025C02124//"Pioneer" and "Leading Goose" R&D Program of Zhejiang/ ; 2023QH1102//Startup Fund for Scientific Research of Fujian Medical University/ ; 2024QH2043//Startup Fund for Scientific Research of Fujian Medical University/ ; },
mesh = {*Hepatitis B virus/genetics/isolation & purification ; *Biosensing Techniques/methods ; *Metal Nanoparticles/chemistry/ultrastructure ; *Gold/chemistry ; *CRISPR-Cas Systems/genetics ; *Magnesium/chemistry ; DNA, Viral ; *Platinum/chemistry ; Nucleic Acid Amplification Techniques ; Humans ; },
abstract = {Hepatitis B virus (HBV) infection remains a significant global public health issue, and rapid detection of HBV DNA is crucial for disease prevention and control. However, traditional methods for HBV DNA detection are limited by their reliance on precise instruments and single readout, which can hardly meet the requirements of on-site detection. In this study, the Mg[2+]-enhanced trans-cleavage activity of clustered regularly interspaced short palindromic repeats/associated protein 12a (CRISPR/Cas12a) is reported and coupled with loop-mediated isothermal amplification (LAMP) and Au@Pt nanoparticles as a signaling reporter for on-site detection of HBV DNA. This triple-modal biosensing strategy enables multiple signal readouts including UV-vis spectrum, RGB value, and temperature with high sensitivity and accuracy. The linear detection ranges using UV-vis spectroscopy, RGB color recognition, and photothermal modes are from 50 to 10,000 copies µL[-1], with the limitation of detection of 24.07, 39.65, and 23.33 copies µL[-1], respectively. This biosensing strategy is further employed for the qualitative detection of HBV DNA in 48 serum samples, achieving sensitivities of 100%, 100%, and 95.24% for triple modes. This work offers a promising tool of next-generation LAMP-CRISPR/Cas12a for the rapid and portable detection of nucleic acids.},
}

*Hepatitis B virus/genetics/isolation & purification
*Biosensing Techniques/methods
*Metal Nanoparticles/chemistry/ultrastructure
*Gold/chemistry
*CRISPR-Cas Systems/genetics
*Magnesium/chemistry
DNA, Viral
*Platinum/chemistry
Nucleic Acid Amplification Techniques
Humans

@article {pmid40925176,
year = {2025},
author = {He, Z and Zhang, J and Kuang, S and Li, S and Wang, Y and Ding, J and Ma, Z and Zhang, B},
title = {Colloidal gold technology in viral diagnostics: Recent innovations, clinical applications, and future perspectives.},
journal = {Virology},
volume = {612},
number = {},
pages = {110686},
doi = {10.1016/j.virol.2025.110686},
pmid = {40925176},
issn = {1096-0341},
abstract = {Colloidal gold technology has revolutionized viral diagnostics through its rapid, cost-effective, and user-friendly applications, particularly in point-of-care testing (POCT). This review synthesizes recent advancements, focusing on its role in detecting respiratory viruses, hepatitis viruses, and emerging pathogens. The technology leverages the unique optical and physicochemical properties of gold nanoparticles (AuNPs), including localized surface plasmon resonance (LSPR) and high surface-to-volume ratios, to achieve rapid antigen-antibody recognition with visual readouts within 15 min. Innovations such as CRISPR-Cas-integrated lateral flow immunoassays (LFIAs), dual-mode plasmonic biosensors, and nanomaterials like CeO2-colloidal gold composites have enhanced sensitivity and multiplex capability, enabling simultaneous identification of co-circulating pathogens. Case studies highlight its efficacy in dengue serotyping, SARS-CoV-2 neutralizing antibody quantification, and HBV/HCV co-detection, demonstrating high clinical specificity. However, challenges persist, including the need for improved sensitivity; interference of sample matrix with immunity; false positives caused by cross-reactions; and limitations of semi-quantitative analysis. Recent progress in hybrid nanomaterial synthesis, surface functionalization, and device-level multiplexing-coupled with AI-driven data interpretation- promises to address these gaps. Future trends emphasize integration with surface-enhanced Raman scattering (SERS), microfluidics, and portable sensors to achieve sub-zeptomolar sensitivity and scalable deployment. By bridging nanotechnology with precision diagnostics, colloidal gold platforms are poised to redefine global viral surveillance, particularly in resource-limited settings, underscoring their indispensable role in pandemic preparedness.},
}

@article {pmid40922689,
year = {2025},
author = {Zhang, Q and Ren, J and Wu, S and Tan, Y and Wang, W and Feng, C and Zhao, L and Zhu, Z},
title = {Plasmid-Free CRISPR/Cpf1 Genome Editing With In Vivo T7 RNA Polymerase-Transcribed CRISPR RNA From Short Double-Stranded DNA.},
journal = {Biotechnology and bioengineering},
volume = {},
number = {},
pages = {},
doi = {10.1002/bit.70062},
pmid = {40922689},
issn = {1097-0290},
support = {//This study was funded by the National Key Research and Development Program (Grant Number: 2022YFA0911802); the National Natural Science Foundation of China (Grant Number: 22177018); the Liaoning Revitalization Talents Program (Grant Number: XLYC2203057) and the Fundamental Research Funds for the Central Universities (Grant Number: DUT23LAB104)./ ; },
abstract = {Plasmids are commonly employed in the delivery of clustered regularly interspaced shortpalindromic repeats (CRISPR)/CRISPR-associated (Cas) components for genome editing. However, the absence of heritable plasmids in numerous organisms limits the development of CRISPR/Cas genome editing tools. Moreover, cumbersome procedures for plasmid construction and curing render genome editing time-consuming. In this study, we developed a plasmid-free CRISPR/Cpf1 genome editing system for Saccharomyces cerevisiae and Starmerella bombicola. This system leveraged integrative expression of the Cpf1 nuclease and T7 RNA polymerase (T7RNAP), as well as the delivery of linear fragments including (i) a marker cassette for integration and selection, (ii) short double-stranded DNA (crDNA) for in vivo transcription of crRNA by T7RNAP, and (iii) donor DNA for homology-directed repair. We demonstrated that this editing system enabled efficient multiplexed and iterative genome editing without the need for marker recycling and plasmid curing. The use of short crDNA (87 bp) and donor DNA (≤ 120 bp), both readily prepared from ordered oligonucleotides via annealing or overlap extension, dramatically simplified the editing process. Successful implementation in S. bombicola, which lacks heritable plasmids for genetic engineering, highlighted the potential of this approach especially for genome engineering of genetically intractable organisms in a plasmid-free way.},
}

@article {pmid40920944,
year = {2025},
author = {Mangeot, PE and Ohlmann, T},
title = {[Harnessing retroviral engineering for genome reprogramming].},
journal = {Medecine sciences : M/S},
volume = {41},
number = {8-9},
pages = {647-656},
doi = {10.1051/medsci/2025098},
pmid = {40920944},
issn = {1958-5381},
support = {ANRS0516//ANRS/ ; },
mesh = {Humans ; *Retroviridae/genetics/physiology ; *Genetic Engineering/methods ; Genetic Vectors/genetics ; Animals ; CRISPR-Cas Systems ; Gene Editing/methods ; Genome, Human ; *Cellular Reprogramming/genetics ; },
abstract = {The accumulated knowledge on the biology of the HIV-1 virus has led to the emergence of technologies that exploit the architecture of retroviruses and their integration or vectorization properties. This field of study constitutes retroviral vectorology, democratized in laboratories by the use of lentiviral vectors. By hijacking retroviral assembly, other systems are emerging and are increasingly mentioned in recent literature. In particular, defective retroviral particles are capable of transiently delivering effectors that act on the genome: they thus appear to be more suitable tools for delivering genetic scalpels, whose persistence in the target cell or organism is not required. Since the description of the CRISPR Cas9 system in 2012, genome engineering techniques have continued to evolve in terms of capacity and reliability. Several derivatives of the CRISPR system can now modify the human genome with nucleotide-level precision. Introducing these effectors into the cell or organism remains a major technical challenge that vector scientists are striving to overcome. This review describes the major retroviral systems used for genome manipulation. Following an overview of genetic engineering techniques, we will see how researchers have developed a wide range of genomic tools by manipulating different processes in the retroviral architecture.},
}

Humans
*Retroviridae/genetics/physiology
*Genetic Engineering/methods
Genetic Vectors/genetics
Animals
CRISPR-Cas Systems
Gene Editing/methods
Genome, Human
*Cellular Reprogramming/genetics

@article {pmid40920796,
year = {2025},
author = {Nakagata, N and Nakao, S and Mikoda, N and Yamaga, K and Nakagawa, Y and Sakuma, T and Yamamoto, T and Takeo, T},
title = {Improved protocol for the vitrification and warming of rat zygotes by optimizing the warming solution and oocyte donor age.},
journal = {PloS one},
volume = {20},
number = {9},
pages = {e0328718},
pmid = {40920796},
issn = {1932-6203},
mesh = {Animals ; Female ; *Zygote/physiology/drug effects/cytology ; *Vitrification ; Rats ; *Oocytes/cytology ; *Cryopreservation/methods ; Rats, Sprague-Dawley ; Embryo Transfer ; Fertilization in Vitro/methods ; Rats, Inbred F344 ; Male ; CRISPR-Cas Systems ; Sucrose/pharmacology ; },
abstract = {Zygotes are used to create genetically modified animals by electroporation using the CRISPR-Cas9 system. Such zygotes in rats are obtained from superovulated female rats after mating. Recently, we reported that in vivo-fertilized zygotes had higher cryotolerance and developmental ability than in vitro-fertilized zygotes in Sprague Dawley (SD) and Fischer 344 rats. To apply the in vitro-fertilized zygotes in creating genetically modified rats, we need to address their low cryotolerance and developmental ability. Hence, we evaluated the effects of warming solutions containing different sucrose concentrations (0-0.3 M) and the oocyte donor's age (3-7-week-old SD rats) on the viability of vitrified-warmed zygotes after in vitro fertilization and on developmental ability by embryo transfer in SD rats. A warming solution containing 0.1 M sucrose enhanced the survival rate of vitrified-warmed zygotes and their rate of development to two-cell embryos. Additionally, zygotes derived from 6- and 7-week-old female rats had higher cryotolerance and developmental ability than those from 3-week-old ones. Next, vitrified-warmed rat zygotes produced using the optimized protocol underwent genome editing by electroporation with Cas9 ribonucleoprotein and gRNA introduced to disrupt the Tyr gene. We then found that 86.5% of the pups derived from zygotes demonstrated mutation of the targeted gene. Therefore, the improved protocol for vitrifying and warming rat zygotes is useful for preserving and producing genetically modified rats.},
}

Animals
Female
*Zygote/physiology/drug effects/cytology
*Vitrification
Rats
*Oocytes/cytology
*Cryopreservation/methods
Rats, Sprague-Dawley
Embryo Transfer
Fertilization in Vitro/methods
Rats, Inbred F344
Male
CRISPR-Cas Systems
Sucrose/pharmacology

@article {pmid40891143,
year = {2025},
author = {Zheng, X and Zhai, Y and Chathurika, HAW and Ni, X and Lv, R and Wu, C and Sun, Z and Shen, Y and Zhang, CY and Zheng, P and Sun, J},
title = {A Highly Efficient 5S rRNA-CRISPR/Cas9 Genome Editing Toolkit in Acremonium chrysogenum.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {36},
pages = {22607-22616},
doi = {10.1021/acs.jafc.5c06429},
pmid = {40891143},
issn = {1520-5118},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Acremonium/genetics/metabolism ; *RNA, Ribosomal, 5S/genetics/metabolism ; Cephalosporins/biosynthesis ; Fungal Proteins/genetics/metabolism ; },
abstract = {Acremonium chrysogenum is an important industrial producer of cephalosporin C (CPC), and efficient genome editing tools are critical for its exploitation and metabolic engineering. Currently, CRISPR/Cas9 systems for A. chrysogenum employ heterologous promoters, including Aspergillus nidulans PgpdA or Aspergillus fumigatus AfU6p, to drive sgRNA expression. These systems often required additional sgRNA processing elements such as ribozymes or tRNAs, which increased cloning complexity and experimental workload. Here, we developed a simplified and highly efficient CRISPR/Cas9 genome editing system using the endogenous 5S rRNA promoter for sgRNA transcription in A. chrysogenum. This system obviated the need for processing elements and achieved up to 100% gene disruption efficiency, as demonstrated by targeting the sorB gene. Furthermore, this platform enabled 100% single gene deletion and efficient large-scale chromosomal deletion, up to 66.17 kb within the sorbicillinoid biosynthetic gene cluster, without donor DNA. To our knowledge, this represents the largest chromosomal deletion reported in A. chrysogenum to date. Moreover, the system also facilitated precise and iterative gene editing through homologous recombination-mediated marker replacement at the kusA locus. Overall, this 5S rRNA-CRISPR/Cas9 system provides a versatile, powerful, and efficient genome editing toolkit for functional genomics and strain improvement in A. chrysogenum.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*Acremonium/genetics/metabolism
*RNA, Ribosomal, 5S/genetics/metabolism
Cephalosporins/biosynthesis
Fungal Proteins/genetics/metabolism

@article {pmid40877754,
year = {2025},
author = {Singuru, MMR and Bhattacharyya, P and Sriramakrishnan, HP and You, M},
title = {Sensitive Detection of Intercellular Tensile Forces via Cas12a-Assisted Membrane Molecular Probes.},
journal = {Nano letters},
volume = {25},
number = {36},
pages = {13519-13525},
doi = {10.1021/acs.nanolett.5c02983},
pmid = {40877754},
issn = {1530-6992},
mesh = {Cadherins/metabolism ; Humans ; *Cell Membrane/chemistry/metabolism ; CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; Tensile Strength ; Mechanotransduction, Cellular ; Epithelial-Mesenchymal Transition ; *Endodeoxyribonucleases/metabolism/chemistry/genetics ; *Molecular Probes/chemistry ; *Bacterial Proteins/metabolism/chemistry ; },
abstract = {Intercellular forces are critical for shaping cells, driving migration, and guiding tissue development and morphogenesis. However, these transient and low-intensity forces are still challenging to detect. Here, we developed a Force-Responsive Cas12a-assisted Tension Sensor (FRCTS), which leverages the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a technology to enable more reliable detection of cumulative molecular force events generated at cell-cell junctions. FRCTS incorporates a lipid-modified DNA hairpin to spontaneously anchor onto live-cell membranes. The hairpin unfolds upon molecular tension exerted by neighboring cells through an integrin or cadherin receptor and reveals a hidden strand to activate Cas12a. Cas12a activation leads to an irreversible cleavage of a fluorogenic reporter on the cell surface, causing cumulative cell membrane fluorescence signals for recording intercellular force events. After systematic optimization, we applied FRCTS to quantify E-cadherin/N-cadherin mechanical correlations during the epithelial-mesenchymal transition. This modular and sensitive FRCTS platform can potentially be used for assessing various intercellular mechanotransduction processes.},
}

Cadherins/metabolism
Humans
*Cell Membrane/chemistry/metabolism
CRISPR-Cas Systems
*CRISPR-Associated Proteins/metabolism/chemistry/genetics
Tensile Strength
Mechanotransduction, Cellular
Epithelial-Mesenchymal Transition
*Endodeoxyribonucleases/metabolism/chemistry/genetics
*Molecular Probes/chemistry
*Bacterial Proteins/metabolism/chemistry

@article {pmid40816522,
year = {2025},
author = {Meng, XQ and Xu, XL and Gao, Y and Deng, SL},
title = {Establishment of CRISPR/Cas9 lineage tracking technology for pig embryos.},
journal = {Molecular and cellular probes},
volume = {83},
number = {},
pages = {102046},
doi = {10.1016/j.mcp.2025.102046},
pmid = {40816522},
issn = {1096-1194},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Swine/genetics/embryology ; *Cell Lineage/genetics ; *Embryo, Mammalian/cytology/metabolism ; Single-Cell Analysis/methods ; },
abstract = {Understanding tissue development in pigs is critical for biomedical research and genetic engineering, particularly for modeling human disease. However, tracing developmental origins and reconstructing lineage trees for pig cells remains a significant challenge. Here, we present a high-resolution lineage tracing system that combines molecular barcoding with single-cell transcriptomics in pigs. Our system combines two key components: DNA barcodes (three CRISPR/Cas9 target sites and an 8-base pair intBC) integrated into the genome via piggyBac transposition, and a constitutive Cas9-EGFP cassette stably integrated at the Rosa26 locus using CRISPR/Cas12a. By combining lineage barcodes with single-cell RNA sequencing (scRNA-seq), we constructed an evolutionary lineage recorder that captures distinct cell states across developmental or differentiation trajectories. This system provides an essential tool for the subsequent construction of complete porcine cell fate maps. Our work provides a tool for studying porcine developmental biology, but also helps to optimize regenerative medicine strategies and improve the design of genetically engineered animal models.},
}

Animals
*CRISPR-Cas Systems/genetics
Swine/genetics/embryology
*Cell Lineage/genetics
*Embryo, Mammalian/cytology/metabolism
Single-Cell Analysis/methods

@article {pmid40815167,
year = {2025},
author = {Sunshine, S and Puschnik, A and Retallack, H and Laurie, MT and Liu, J and Peng, D and Knopp, K and Zinter, MS and Ye, CJ and DeRisi, JL},
title = {Defining the host dependencies and the transcriptional landscape of RSV infection.},
journal = {mBio},
volume = {16},
number = {9},
pages = {e0101025},
doi = {10.1128/mbio.01010-25},
pmid = {40815167},
issn = {2150-7511},
support = {//Chan Zuckerberg Biohub San Francisco/ ; F31AI150007//National Institute of Allergy and Infectious Diseases/ ; },
mesh = {Humans ; *Respiratory Syncytial Virus Infections/virology/genetics/immunology ; *Respiratory Syncytial Virus, Human/genetics/physiology ; *Host-Pathogen Interactions/genetics ; Single-Cell Analysis ; CRISPR-Cas Systems ; Gene Expression Profiling ; },
abstract = {Respiratory syncytial virus (RSV) is a globally prevalent pathogen, causes severe disease in older adults, and is the leading cause of bronchiolitis and pneumonia in the United States for children during their first year of life. Despite its prevalence worldwide, RSV-specific treatments remain unavailable for most infected patients. Here, we leveraged a combination of genome-wide CRISPR knockout screening and single-cell RNA sequencing to improve our understanding of the host determinants of RSV infection and the host response in both infected cells and uninfected bystanders. These data reveal temporal transcriptional patterns that are markedly different between RSV-infected and bystander-activated cells. Our data show that expression of interferon-stimulated genes is primarily observed in bystander activated cells, while genes implicated in the unfolded protein response and cellular stress are upregulated specifically in RSV-infected cells. Furthermore, genome-wide CRISPR screens identified multiple host factors important for viral infection, findings which we contextualize relative to 29 previously published screens across 17 additional viruses. These unique data complement and extend prior studies that investigate the proinflammatory response to RSV infection, and juxtaposed to other viral infections, provide a rich resource for further hypothesis testing.IMPORTANCERespiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in infants and the elderly. Despite its substantial global health burden, RSV-targeted treatments remain unavailable for the majority of individuals. While vaccine development is underway, a detailed understanding of the host response to RSV infection and identification of required human host factors for RSV may provide insight into combatting this pathogen. Here, we utilized single-cell RNA sequencing and functional genomics to understand the host response in both RSV-infected and bystander cells, identify what host factors mediate infection, and contextualize these findings relative to dozens of previously reported screens across 17 additional viruses.},
}

Humans
*Respiratory Syncytial Virus Infections/virology/genetics/immunology
*Respiratory Syncytial Virus, Human/genetics/physiology
*Host-Pathogen Interactions/genetics
Single-Cell Analysis
CRISPR-Cas Systems
Gene Expression Profiling

@article {pmid40767522,
year = {2025},
author = {Guo, G and Zhang, M and Xu, Z and Xi, P and Zhu, H and Evers, A and Lebbink, RJ and Lang, Y and He, Q and Huang, Y-W and Li, T and Bosch, BJ and Li, W},
title = {Genome-wide CRISPR screen reveals key role of sialic acids in PEDV and porcine coronavirus infections.},
journal = {mBio},
volume = {16},
number = {9},
pages = {e0162825},
doi = {10.1128/mbio.01628-25},
pmid = {40767522},
issn = {2150-7511},
mesh = {*Porcine epidemic diarrhea virus/physiology/pathogenicity/genetics ; Animals ; *Coronavirus Infections/virology/veterinary/metabolism ; Swine ; Humans ; *CRISPR-Cas Systems ; *Sialic Acids/metabolism ; *Swine Diseases/virology/metabolism ; Virus Internalization ; Sialyltransferases/genetics/metabolism ; *N-Acetylneuraminic Acid/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Host-Pathogen Interactions ; Cell Line ; },
abstract = {Porcine epidemic diarrhea virus (PEDV) is a globally distributed alphacoronavirus with economic importance that can cause severe watery diarrhea and even death in piglets. To identify host factors essential for PEDV infection, we performed a genome-wide CRISPR/Cas9 screen in human hepatocellular carcinoma cells (Huh7) using the highly virulent PEDV GIIb strain GDU. Several genes involved in the sialic acid and heparan sulfate biosynthesis pathway and cholesterol metabolism were highly enriched following PEDV selection. We validated that the host factor ST3 beta-galactoside alpha-2,3-sialyltransferase 4 (ST3GAL4), which catalyzes the transfer of sialic acid to sugar chains via α2,3-linked linkages, is important for PEDV infection. To systematically investigate the role of sialic acid in PEDV infection, we knocked out genes related to sialic acid synthesis. This led to a reduced abundance of sialic acid on the cell surface, which in turn inhibited PEDV adsorption and internalization. Furthermore, we found that both α2,3-linked and α2,6-linked sialic acids can serve as cellular attachment factors for PEDV. We conducted a glycan microarray screen to determine which sialoglycans are preferred by the PEDV spike protein. The results revealed that PEDV favors binding to α2,3-sialoglycans. Additionally, we found that not only current circulating PEDV strains but also other porcine coronaviruses rely on sialic acid for efficient infection. Collectively, our findings provide insights into critical host factors involved in PEDV infection and demonstrate that disrupting genes involved in sialic acid biosynthesis negatively affects the infectivity of multiple porcine enteric coronaviruses.IMPORTANCEA wide range of viruses utilize sialic acid as receptors. Sialic acid binding may serve as a key determinant of viral host range. Different viruses exhibit distinct preferences for specific types of sialic acid linkages. However, it remains unclear which specific subtypes of sialic acid are utilized during PEDV infection. In this study, we performed CRISPR-based genome-wide knockout screening and identified ST3GAL4 as a key host factor for PEDV infection. Furthermore, we found that both α2,3-linked and α2,6-linked sialic acids can function as attachment factors for PEDV infection. A glycan microarray screen revealed that PEDV S1 shows the strongest binding preference for α2,3-linked and α2,8-linked sialosides. Sialic acids were also implicated in infections by other porcine enteric coronaviruses. Overall, our findings advance our understanding of viral entry mechanisms of PEDV and other swine coronaviruses and may provide avenues for designing antiviral strategies.},
}

*Porcine epidemic diarrhea virus/physiology/pathogenicity/genetics
Animals
*Coronavirus Infections/virology/veterinary/metabolism
Swine
Humans
*CRISPR-Cas Systems
*Sialic Acids/metabolism
*Swine Diseases/virology/metabolism
Virus Internalization
Sialyltransferases/genetics/metabolism
*N-Acetylneuraminic Acid/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats
Host-Pathogen Interactions
Cell Line

@article {pmid40744238,
year = {2025},
author = {Wen, Z and Yang, D and Yang, Y and Hu, J and Parviainen, A and Chen, X and Li, Q and VanDeusen, E and Ma, J and Tay, F},
title = {The path to biotechnological singularity: Current breakthroughs and outlook.},
journal = {Biotechnology advances},
volume = {84},
number = {},
pages = {108667},
doi = {10.1016/j.biotechadv.2025.108667},
pmid = {40744238},
issn = {1873-1899},
mesh = {Humans ; *Biotechnology/trends ; Gene Editing ; Artificial Intelligence ; Synthetic Biology ; Regenerative Medicine ; CRISPR-Cas Systems ; Brain-Computer Interfaces ; },
abstract = {Fueled by rapid advances in gene editing, synthetic biology, artificial intelligence, regenerative medicine, and brain-computer interfaces, biotechnology is approaching a transformative era often referred to as biotechnological singularity. CRISPR-based gene editing has revolutionized genetic engineering, enabling precise modifications for treating hereditary diseases and cancer. Synthetic biology facilitates sustainable biomaterial production and innovative therapeutic applications. Artificial intelligence accelerates drug discovery, enhances diagnostic accuracy, and personalizes treatment through deep learning models. Driven by stem cell research, regenerative medicine offers promising avenues for reversing aging and treating degenerative diseases. Brain-computer interfaces merge human cognition with technology, enabling direct neural control of prosthetics and expanding human-machine interactions. These breakthroughs, however, raise ethical, regulatory, and societal concerns, including equitable access, biosecurity risks, and the implications of human enhancement. The convergence of biological and computational technologies challenges traditional boundaries, necessitating comprehensive governance frameworks. By embracing responsible innovation, society can harness these advancements for transformative health interventions, environmental sustainability, and extended longevity. The realization of biotechnological singularity depends on interdisciplinary collaboration among scientists, policymakers, and the public to ensure that progress aligns with the well-being of humanity and ethical considerations.},
}

Humans
*Biotechnology/trends
Gene Editing
Artificial Intelligence
Synthetic Biology
Regenerative Medicine
CRISPR-Cas Systems
Brain-Computer Interfaces

@article {pmid40552689,
year = {2025},
author = {Yao, B and Yang, Q and Snijders Blok, CJB and Daniels, MA and Doevendans, PA and Schiffelers, R and Sluijter, JPG and Lei, Z},
title = {Insights into pegRNA design from editing of the cardiomyopathy-associated phospholamban R14del mutation.},
journal = {FEBS letters},
volume = {599},
number = {17},
pages = {2543-2554},
doi = {10.1002/1873-3468.70097},
pmid = {40552689},
issn = {1873-3468},
support = {10250022110004//The Netherlands Organisation for Health Research and Development (ZonMw) Psider-Heart/ ; 202006170055//China Scholarship Council/ ; #725229//European Research Council (ERC), EVICARE project under H2020/ ; 2021/TTW/01038252//Netherlands Organization for Scientific Research (NWO) - Applied and Engineering Sciences (TTW)/ ; },
mesh = {*Gene Editing/methods ; Humans ; *Calcium-Binding Proteins/genetics ; *Cardiomyopathies/genetics/pathology ; CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Mutation ; HEK293 Cells ; },
abstract = {Prime editing (PE) represents a transformative genome-editing technology and enables precise insertions, deletions, and base substitutions without introducing double-strand breaks, thereby reducing undesired indels and off-target effects. Despite advancements in enhanced prime editors and optimized prime editing guide RNAs (pegRNAs), designing effective pegRNAs remains a major challenge. The phospholamban (PLN) R14del mutation is associated with cardiomyopathies, making it a crucial target for precise gene-editing strategies. In this study, we explored pegRNA features that contribute to high editing efficiency using the FluoPEER.PLN R14del reporter cell line. Through systematic screening, we identified three pegRNAs with significantly enhanced editing efficiency. Our findings underscore the importance of pegRNA secondary structure and stability in optimizing prime editing, providing valuable insights into precise gene correction strategies.},
}

*Gene Editing/methods
Humans
*Calcium-Binding Proteins/genetics
*Cardiomyopathies/genetics/pathology
CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/genetics
Mutation
HEK293 Cells

@article {pmid40920775,
year = {2025},
author = {Tan, Y and Kumagai-Takei, N and Shimizu, Y and Yamasaki, A and Hara-Yamamoto, M and Mitani, S and Ito, T},
title = {Targeting the Exon2 splice cis-element in PD-1 and its effects on lymphocyte function.},
journal = {PloS one},
volume = {20},
number = {9},
pages = {e0331468},
pmid = {40920775},
issn = {1932-6203},
mesh = {*Programmed Cell Death 1 Receptor/genetics/metabolism ; Humans ; *Exons/genetics ; CRISPR-Cas Systems ; *CD8-Positive T-Lymphocytes/metabolism/immunology ; *RNA Splicing ; Cytokines/metabolism ; RNA Precursors/genetics ; Lymphocyte Activation ; Cell Proliferation ; },
abstract = {T-cell therapies have proven to be a promising treatment option for cancer patients in recent years, especially in the case of chimeric antigen receptor (CAR)-T cell therapy. However, the therapy is associated with insufficient activation of T cells or poor persistence in the patient's body, which leads to incomplete elimination of cancer cells, recurrence, and genotoxicity. By extracting the splice element of PD-1 pre-mRNA using biology based on CRISPR/dCas13 in this study, our ultimate goal is to overcome the above-mentioned challenges in the future. PD-1 plays an important role in controlling T cell responses and is expressed at the cell surface of T cells following activation. The receptor PD-1 interferes with T cell receptor (TCR) signaling following interaction with PD-L1. The outcome of stimulation via PD-1 leads to decreases in cytokine secretion and cell proliferation. We extracted the RNA region of PD-1 pre-mRNA using CD8+T cell lines and examined the effect of targeting the Exon2 splice cis-element on the production of cytokines in the present study. In particular, the production of IFN-γ, TNF-α, GM-CSF was lower in RNA-targeted cells than in non-targeted cells, but the cytokine secretion capacity and cell proliferation were maintained in RNA-targeted cells. These results suggested that the use of the RNA editing technology, CRISPR/dCas13 strategy offers a novel approach to mitigate genotoxicity in lymphocytes with cytokine production and cell proliferation.},
}

*Programmed Cell Death 1 Receptor/genetics/metabolism
Humans
*Exons/genetics
CRISPR-Cas Systems
*CD8-Positive T-Lymphocytes/metabolism/immunology
*RNA Splicing
Cytokines/metabolism
RNA Precursors/genetics
Lymphocyte Activation
Cell Proliferation

@article {pmid40919784,
year = {2025},
author = {Leite, VLM and Faria, AR and Guerra, CF and Souza, SdSR and Freitas, AdAR and Morais, JM and Merquior, VLC and Planet, PJ and Teixeira, LM},
title = {Hidden diversity in Enterococcus faecalis revealed by CRISPR2 screening: eco-evolutionary insights into a novel subspecies.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0142825},
doi = {10.1128/spectrum.01428-25},
pmid = {40919784},
issn = {2165-0497},
abstract = {Enterococcus faecalis is a commensal bacterium that colonizes the gut of humans and animals and is a major opportunistic pathogen, known for causing multidrug-resistant healthcare-associated infections (HAIs). Its ability to thrive in diverse environments and disseminate antimicrobial resistance genes (ARGs) across ecological niches highlights the importance of understanding its ecological, evolutionary, and epidemiological dynamics. The CRISPR2 locus has been used as a valuable marker for assessing clonality and phylogenetic relationships in E. faecalis. In this study, we identified a group of E. faecalis strains lacking CRISPR2, forming a distinct, well-supported clade. We demonstrate that this clade meets the genomic criteria for classification as a novel subspecies, here referred to as "subspecies B." Through a comprehensive pangenome analysis and comparative genomics, we explored the adaptive ecological traits underlying this diversification process, identifying clade-specific features and their predicted functional roles. Our findings suggest that the frequent isolation of subspecies B from meat products and processing facilities may reflect dissemination routes involving environmental contamination (e.g., water, plants, soil) from avian species. The absence of key virulence traits required for pathogenicity in mammals, particularly humans, and the lack of clinically relevant resistance determinants indicate that subspecies B currently poses minimal threat to public health compared with the broadly disseminated "subspecies A." Nevertheless, the unclear potential for genetic exchange between these subspecies and the frequent association of subspecies B with food sources calls for continued genomic surveillance of E. faecalis from a One Health perspective to detect and mitigate the emergence of high-risk variants in advance.IMPORTANCEExploring intraspecific genetic variability in generalist bacteria with pathogenic potential, such as Enterococcus faecalis, is a key to uncovering stable evolutionary trends. By screening the CRISPR2 locus across a representative set of genomes from diverse sources, this study reveals a previously unrecognized lineage within the population structure of E. faecalis, associated with underexplored nonhuman and nonhospital reservoirs. These findings broaden our knowledge of the species' genetic landscape and shed light on its adaptive strategies and patterns of ecological dissemination. By bridging phylogenetic patterns with variation in genetic defense systems and accessory traits, the study generates testable hypotheses about the genomic determinants and corresponding selective pressures that shape the species' behavior and long-term dissemination. This work offers new perspectives on the eco-evolutionary dynamics of E. faecalis and highlights the value of genomic surveillance beyond clinical settings, in alignment with One Health principles.},
}

@article {pmid40845666,
year = {2026},
author = {Wang, M and Zhou, W and Wang, M and Zhang, K},
title = {A highly sensitive ECL biosensor for NF-κB p50 detection based on entropy-driven amplification and CRISPR/Cas12a signal enhancement.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {167},
number = {},
pages = {109081},
doi = {10.1016/j.bioelechem.2025.109081},
pmid = {40845666},
issn = {1878-562X},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *NF-kappa B p50 Subunit/analysis ; Humans ; *Entropy ; *Luminescent Measurements/methods ; Electrochemical Techniques/methods ; Limit of Detection ; Exodeoxyribonucleases/metabolism ; Nucleic Acid Amplification Techniques/methods ; *CRISPR-Associated Proteins/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Transcription factors, particularly NF-κB p50, play crucial roles in regulating gene expression and are involved in several diseases such as cancer, autoimmune disorders, and chronic inflammation. The sensitive detection of NF-κB p50 is essential for clinical diagnostics and therapeutic monitoring. In this study, we present an electrochemiluminescence (ECL) biosensor designed for the highly sensitive and specific detection of NF-κB p50. The biosensor integrates entropy-driven amplification and CRISPR/Cas12a-based signal enhancement to detect trace amounts of NF-κB p50. Upon detection of NF-κB p50, a ternary complex forms with a double-stranded DNA (dsDNA) probe, which prevents subsequent cleavage by exonuclease III (Exo III) and inhibits the CRISPR/Cas12a system. In the absence of NF-κB p50, Exo III digestion triggers entropy-driven amplification, which activates CRISPR/Cas12a, leading to enhanced electrochemical signals. The ECL biosensor demonstrated a detection limit of 0.56 pM, high selectivity, and excellent reproducibility. Furthermore, the biosensor successfully detected NF-κB p50 in complex biological samples, such as HeLa cell lysates, showcasing its potential for clinical applications in disease diagnostics.},
}

*Biosensing Techniques/methods
*CRISPR-Cas Systems
*NF-kappa B p50 Subunit/analysis
Humans
*Entropy
*Luminescent Measurements/methods
Electrochemical Techniques/methods
Limit of Detection
Exodeoxyribonucleases/metabolism
Nucleic Acid Amplification Techniques/methods
*CRISPR-Associated Proteins/metabolism
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid40845664,
year = {2026},
author = {Zhou, Y and Lv, Z and Geng, Y and Liu, R},
title = {An electrochemical RNA aptasensor based on sheet-like α-Fe2O3/Fe3O4 magnetic nanocomposites and CRISPR/Cas13a system for supersensitive detection of osteopontin.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {167},
number = {},
pages = {109085},
doi = {10.1016/j.bioelechem.2025.109085},
pmid = {40845664},
issn = {1878-562X},
mesh = {*CRISPR-Cas Systems ; *Aptamers, Nucleotide/chemistry/genetics ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; *Nanocomposites/chemistry ; *Osteopontin/analysis/blood ; Limit of Detection ; Humans ; *Ferric Compounds/chemistry ; },
abstract = {Osteopontin (OPN) exhibits markedly elevated expression in malignant tumor tissues, rendering it a crucial tumor marker for cancer prevention and monitoring-underscoring the significance of its detection. This work proposed an electrochemical RNA aptasensor based on a novel sheet-like α-Fe2O3/Fe3O4 magnetic nanocomposites (MNCs) and CRISPR/Cas13a system to effectively detect OPN. The proposed aptasensor used the sheet-like α-Fe2O3/Fe3O4 MNCs as the conduction matrix and applied their magnetic property to accomplish self-assembly of the sensing element onto the electrode. The current change of this aptasensor depended on the activation degree of the CRISPR/Cas13a system, which correlated with the amount of OPN expression. Double-stranded RNA (Apt/Activator) was compelled disassembly due to the OPN's strong affinity of Apt, while single-stranded RNA (Activator) could be guided by crRNA to combine with Cas13a/crRNA and activated the enzymatic activity of Cas13a. Cas13a enzyme affected the current and electrical resistance by shearing the nucleic acid strands (Reporter) on the electrode, ultimately enabling the quantitative detection of OPN. The aptasensor demonstrated excellent selectivity, reproducibility, and stability, with the detection limit (LOD) of 0.33 pg·mL[-1] and the wide linear detection range of 1 pg·mL[-1] - 10 ng·mL[-1]. These results offer a novel idea for advancing tumor marker electrochemical biosensors.},
}

*CRISPR-Cas Systems
*Aptamers, Nucleotide/chemistry/genetics
*Biosensing Techniques/methods
*Electrochemical Techniques/methods
*Nanocomposites/chemistry
*Osteopontin/analysis/blood
Limit of Detection
Humans
*Ferric Compounds/chemistry

@article {pmid40757822,
year = {2025},
author = {Echavarria Galindo, M and Lai, Y},
title = {CRISPR-based genetic tools for the study of host-microbe interactions.},
journal = {Infection and immunity},
volume = {93},
number = {9},
pages = {e0051024},
doi = {10.1128/iai.00510-24},
pmid = {40757822},
issn = {1098-5522},
support = {26100423//Research Grants Council, University Grants Committee/ ; PF22-69790//Hong Kong PHD Fellowship Scheme/ ; R9829//Hong Kong University of Science and Technology (HKUST)/ ; },
mesh = {Humans ; Animals ; *CRISPR-Cas Systems ; *Host Microbial Interactions/genetics ; Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Microbiota/genetics ; *Host-Pathogen Interactions/genetics ; },
abstract = {CRISPR-based genetic tools have revolutionized our ability to interrogate and manipulate genes. These tools can be applied to both host and microbial cells, and their use can enhance our understanding of the dynamic nature of host-microbe interactions by uncovering their genetic underpinnings. As reviewed here, CRISPR-based tools are being used to explore the microbiome in an efficient, accurate, and high-throughput manner. By employing CRISPR screens, targeted genome editing, and recording systems to the study of host cells and microorganisms, we can gain critical insights into host defense mechanisms, potential vulnerabilities, and microbial pathogenesis, as well as essential or condition-specific genes involved in host-microbe interactions. Additionally, CRISPR-based genetic tools are being used in animal models to study host-microbe interactions in vivo. Recent advancements in CRISPR-derived technology can be combined with emerging techniques, such as single-cell RNA sequencing, to examine the complex interactions between hosts and microbes, shedding light on the role of the microbiome in health and disease. This review aims to provide a comprehensive overview of how these cutting-edge genetic tools are being used to investigate host-microbial systems, as well as their current limitations. Current research is likely to yield even more advanced genetic toolkits than those presently available, and these can serve researchers in identifying and exploring new therapeutic targets for diseases related to host-microbe interactions.},
}

Humans
Animals
*CRISPR-Cas Systems
*Host Microbial Interactions/genetics
Gene Editing/methods
*Clustered Regularly Interspaced Short Palindromic Repeats
*Microbiota/genetics
*Host-Pathogen Interactions/genetics

@article {pmid40749504,
year = {2026},
author = {Ji, Z and Cheng, S and Li, W and Xing, Y and Tang, Z and Zhu, X and Wang, D and Hao, C and Wang, B and Shi, M},
title = {Ultrasensitive detection of miR-31 using a signal-on electrochemiluminescence biosensor based on CRISPR/Cas12a and MXene nanocomposites.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {167},
number = {},
pages = {109059},
doi = {10.1016/j.bioelechem.2025.109059},
pmid = {40749504},
issn = {1878-562X},
mesh = {*MicroRNAs/analysis/blood/genetics ; *Biosensing Techniques/methods ; Humans ; *Nanocomposites/chemistry ; *CRISPR-Cas Systems ; *Electrochemical Techniques/methods ; Limit of Detection ; *Luminescent Measurements/methods ; *Endodeoxyribonucleases/metabolism ; *CRISPR-Associated Proteins/metabolism ; Gold/chemistry ; Electrodes ; Metallocenes ; Bacterial Proteins ; Nitrites ; Transition Elements ; },
abstract = {MicroRNAs (miRNAs) have emerged as critical biomarkers for early cancer diagnosis due to their high specificity and stability in biological fluids. In this study, we report a highly sensitive and specific electrochemiluminescence (ECL) biosensor for the detection of miR-31, a miRNA closely associated with non-small cell lung cancer. The sensing platform integrates a PEI-Ru@Ti3C2@AuNPs-modified electrode with a ferrocene-labeled DNA probe (DNA1-Fc) to construct a target-responsive signal-on system. Upon recognition of miR-31, an isothermal strand displacement amplification (ISDA) reaction is initiated, producing abundant double-stranded DNA (dsDNA) that activates the CRISPR/Cas12a complex. The trans-cleavage activity of Cas12a then cleaves the DNA1-Fc probes on the electrode surface, removing the quenching ferrocene moiety and restoring the Ru-based ECL signal. Under optimized conditions, the biosensor exhibited a wide dynamic range from 10 aM to 100 pM and a remarkably low detection limit of 1.67 aM. The system also showed excellent specificity against homologous miRNAs, and its applicability was successfully validated in spiked human serum samples, achieving high recovery and reproducibility. The synergistic combination of nanomaterial-enhanced ECL emission, isothermal nucleic acid amplification, and CRISPR-based enzymatic cleavage provides a powerful strategy for ultrasensitive nucleic acid detection. This work offers a promising approach for early cancer diagnosis and has great potential for clinical translation and point-of-care testing.},
}

*MicroRNAs/analysis/blood/genetics
*Biosensing Techniques/methods
Humans
*Nanocomposites/chemistry
*CRISPR-Cas Systems
*Electrochemical Techniques/methods
Limit of Detection
*Luminescent Measurements/methods
*Endodeoxyribonucleases/metabolism
*CRISPR-Associated Proteins/metabolism
Gold/chemistry
Electrodes
Metallocenes
Bacterial Proteins
Nitrites
Transition Elements

@article {pmid40719649,
year = {2025},
author = {Chen, M and Choi, HK and Goldston, LL and Hou, Y and Jiang, C and Lee, KB},
title = {Advanced Cancer Liquid Biopsy Platform for miRNA Detection in Extracellular Vesicles Using CRISPR/Cas13a and Gold Nanoarrays.},
journal = {ACS nano},
volume = {19},
number = {35},
pages = {31438-31456},
doi = {10.1021/acsnano.5c06940},
pmid = {40719649},
issn = {1936-086X},
mesh = {*MicroRNAs/genetics/analysis/blood ; Humans ; *Extracellular Vesicles/chemistry/metabolism/genetics ; *Gold/chemistry ; *CRISPR-Cas Systems ; Liquid Biopsy/methods ; *Colorectal Neoplasms/diagnosis/genetics ; Biomarkers, Tumor/genetics/blood ; Epithelial Cell Adhesion Molecule/metabolism ; Cell Line, Tumor ; Metal Nanoparticles/chemistry ; },
abstract = {Liquid biopsy is a transformative, noninvasive tool for cancer diagnosis and monitoring, with the potential to revolutionize personalized medicine. In this study, we introduce an advanced liquid biopsy platform for highly sensitive and selective detection of extracellular vesicle (EV) microRNAs (miRNA-21 and miRNA-23a) as biomarkers for colorectal cancer. The platform combines two innovations: (1) gold nanoarrays with epithelial cell adhesion molecule (EpCAM)-specific aptamers to selectively isolate tumor-derived EVs from plasma and (2) CRISPR/Cas13a-encapsulated liposomes that fuse with EVs to form nanoscale reactors. Upon fusion, the CRISPR/Cas13a complex is activated by target miRNA, triggering trans-cleavage of RNA reporters and generating an amplified fluorescence signal for enhanced detection sensitivity. The assay achieves a linear detection range of 10 to 10[6] EVs/μL and a detection limit of 2.5 × 10[1] EVs/μL on the gold nanoarray. Its performance relies on three strategies: (i) EpCAM-mediated tumor EV enrichment, (ii) CRISPR/Cas13a-based collateral activity for ultrasensitive miRNA detection, and (iii) fluorescence signal enhancement via localized nanoreactors. Validation with a 2D SW480 cell model, a 3D vascularized tumor spheroid (VTS) model, and clinical plasma samples confirms diagnostic accuracy, with miRNA quantification comparable to RT-qPCR but without the need for labor-intensive RNA extraction and amplification. By integrating nanotechnology with CRISPR-based diagnostics, this platform bridges research and clinical translation, improving diagnostic precision and streamlining workflows. Future development will focus on multiplexed biomarker detection and single-EV analysis to reveal insights into EV heterogeneity and function in cancer. This technology supports the application in precision oncology, offering a tool for early detection, treatment monitoring, and therapeutic decision-making.},
}

*MicroRNAs/genetics/analysis/blood
Humans
*Extracellular Vesicles/chemistry/metabolism/genetics
*Gold/chemistry
*CRISPR-Cas Systems
Liquid Biopsy/methods
*Colorectal Neoplasms/diagnosis/genetics
Biomarkers, Tumor/genetics/blood
Epithelial Cell Adhesion Molecule/metabolism
Cell Line, Tumor
Metal Nanoparticles/chemistry

@article {pmid40653285,
year = {2025},
author = {Monteiro, JP and Vacante, F and De Pace, AL and Bennett, M and Rodor, J and O'Carroll, D and Wu, JC and Quertermous, T and Baker, AH},
title = {Targeting Cis-regulatory elements for CRISPR-mediated transcriptional activation of the human MIR503HG locus.},
journal = {Vascular pharmacology},
volume = {160},
number = {},
pages = {107521},
doi = {10.1016/j.vph.2025.107521},
pmid = {40653285},
issn = {1879-3649},
mesh = {Humans ; *MicroRNAs/genetics/metabolism ; *Transcriptional Activation ; *CRISPR-Cas Systems ; *RNA, Long Noncoding/genetics/metabolism ; Lentivirus/genetics ; HEK293 Cells ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Vectors ; *Gene Editing/methods ; *Regulatory Elements, Transcriptional ; Human Umbilical Vein Endothelial Cells/metabolism ; },
abstract = {Advances in genome annotation have revealed a striking increase in the number and complexity of non-coding RNA (ncRNA) genes, particularly multi-transcript loci that harbor long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) within the same genomic region. These loci can often function as coordinated regulatory units, with their transcription modulated by shared cis-acting regulatory elements (CREs). Traditional gene manipulation approaches, which typically target individual transcripts, are insufficient to capture the full regulatory and therapeutic potential of these complex loci. Here, we present "cis-ON" a single-vector lentiviral delivery system based on CRISPR activation (CRISPRa), designed to simultaneously upregulate multiple noncoding RNA transcripts by targeting a single CRE. We focused on the evolutionarily conserved MIR503HG locus, which encodes seven lncRNA isoforms and hosts the miR-424/503 cluster, both implicated in various cellular processes, including proliferation, angiogenesis, and endothelial-to-mesenchymal transition. Using integrative analysis of histone marks (H3K27Ac, H3K4Me3), DNase hypersensitivity, and CAGE-seq data, we identified the primary promoter of the MIR503HG locus. A dual fluorescent reporter screen selected optimal single guide RNAs (sgRNAs) for targeting this region. We then engineered cis-ON, a novel lentiviral system combining dCas9-VPR and sgRNA to enable a streamlined single-vector delivery approach. Transduction of primary human endothelial cells with this system robustly activated the entire locus including the MIR503HG isoforms and co-embedded miRNAs miR-424 and miR-503, demonstrating coordinated transcriptional regulation. Additionally, the neighboring LINC00629 lncRNA locus remained unaffected, highlighting regulatory specificity. This approach demonstrates the feasibility of modulating complex ncRNA loci across a ∼ 10 kb genomic region by targeting a single CRE, bypassing limitations of transcriptspecific strategies. By enabling simultaneous upregulation of lncRNAs and miRNAs, the cis-ON platform provides a streamlined strategy for restoring regulatory networks disrupted in disease.},
}

Humans
*MicroRNAs/genetics/metabolism
*Transcriptional Activation
*CRISPR-Cas Systems
*RNA, Long Noncoding/genetics/metabolism
Lentivirus/genetics
HEK293 Cells
*Clustered Regularly Interspaced Short Palindromic Repeats
Genetic Vectors
*Gene Editing/methods
*Regulatory Elements, Transcriptional
Human Umbilical Vein Endothelial Cells/metabolism

@article {pmid39992014,
year = {2025},
author = {Chen, RH and Bai, Y and Shi, LD and Liu, XY and Li, DL and Di, M and Duan, JX and Zhang, ZH and Xu, JL and He, ZQ and Li, K},
title = {Functional exploration of the Sex combs reduced gene in Gryllus bimaculatus.},
journal = {Insect molecular biology},
volume = {34},
number = {5},
pages = {608-618},
doi = {10.1111/imb.12987},
pmid = {39992014},
issn = {1365-2583},
mesh = {Animals ; *Insect Proteins/genetics/metabolism ; Female ; CRISPR-Cas Systems ; Male ; Gene Knockout Techniques ; *Homeodomain Proteins/genetics/metabolism ; },
abstract = {The Hox gene Sex combs reduced (Scr) is recognized as a key factor in the development of the head and thorax in insects. However, its function in the growth, development and morphogenesis of Gryllus bimaculatus remains poorly understood. This study aimed to explore the function of the Scr gene in G. bimaculatus by using CRISPR/Cas9 technology to generate an Scr gene knock-out strain. Intercrossing the G0 generation knock-out individuals with wild-type individuals yielded the G1 generation to screen the mutant strain. It was found that the knock-out of the Scr gene had a severe impact on the growth and development of G. bimaculatus, resulting in high mortality and making it difficult to obtain Scr[-/-] mutants. Therefore, heterozygous individuals (Scr[+/-]) with 1 bp deleted were obtained for investigation. The results showed that the Scr deletion led to ectopic segment formation in the G0 generation. In the G2 generation, it was observed that stable Scr[-/-] strains displayed abnormal embryonic development, characterized by enlarged, blackened and lethal eggs during embryogenesis. During the post-embryonic stage, Scr[-/-] mutants exhibited abnormalities in body segmentation, particularly in the head-thorax region, resulting in a dorsal ridge structure. Furthermore, some Scr[+/-] individuals exhibited a dorsal ridge during the nymphal stage. Notably, this characteristic did not persist into the adult stage. Our findings highlight the distinct but crucial roles of the Scr gene in both embryonic and post-embryonic growth and development of G. bimaculatus.},
}

Animals
*Insect Proteins/genetics/metabolism
Female
CRISPR-Cas Systems
Male
Gene Knockout Techniques
*Homeodomain Proteins/genetics/metabolism

@article {pmid40918426,
year = {2025},
author = {Senbadejo, TY and Ntiamoah Osei, S and Isawumi, A},
title = {Snapshot of Defense Systems in Multidrug Resistant Klebsiella pneumoniae.},
journal = {microPublication biology},
volume = {2025},
number = {},
pages = {},
pmid = {40918426},
issn = {2578-9430},
abstract = {Bacterial defense mechanisms protect pathogens from host immunity, bacteriophages, and harsh environments. This study investigates defense systems in multidrug-resistant Klebsiella pneumoniae from Ghanaian hospital ICUs, focusing on CRISPR-Cas, restriction-modification (R-M), and toxin-antitoxin (TA) systems. Genomes of environmental (NS2) and clinical (PS4) strains were sequenced and analyzed using PADLOC, defensefinder, and TADB3.0. NS2 carries 12 defense systems, including CRISPR-Cas, while PS4 has five. Both possess diverse RM and TA systems. These strains, resistant to six antibiotic classes, encode clinically significant defense systems, suggesting microbial exchange between fomites and humans, potentially increasing infection risks in ICU environments requiring targeted surveillance.},
}

@article {pmid40918304,
year = {2025},
author = {Dash, B and Bhuyan, SS and Sahoo, RK and Swain, N and Jeughale, KP and Sarkar, S and Verma, RL and Parameswaran, C and Devanna, BN and Samantaray, S},
title = {CRISPR/Cas-mediated genome editing: playing a versatile role in mitigating the challenges of sustainable rice improvement.},
journal = {3 Biotech},
volume = {15},
number = {10},
pages = {327},
pmid = {40918304},
issn = {2190-572X},
abstract = {Just as Gregor Mendel's laws of inheritance laid the foundation for modern genetics, the emergence of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas systems has catalyzed a new era in precision genome engineering. CRISPR/Cas has revolutionized rice (Oryza sativa L.) breeding by enabling precise, transgene-free edits to improve yield, nutrition, and stress tolerance. Advanced tools like base and prime editing further refine these capabilities, offering powerful solutions for climate-resilient agriculture and global food security. The review synthesizes the CRISPR-mediated strategies for improving resistance against major biotic (bacterial blight, blast, sheath blight) and abiotic (drought, salinity, submergence, nutrient deficiency) stresses. Additionally, we explore the critical prerequisites for efficient genome editing in rice, ranging from target site design, PAM specificity, delivery systems (like Agrobacterium, RNPs, and nanoparticle-mediated delivery), to screening and validation of mutants. This review also highlights recent breakthroughs in multiplex genome editing for complex traits, including the development of haploid inducer lines and clonal seed technology. Haploid inducers accelerate breeding by producing homozygous lines without tissue culture, while engineered apomixis enables clonal propagation of elite hybrids. Beyond technical dimensions, this review underscores the broader socio-economic and regulatory implications of genome-edited rice, addressing the emerging ethical concerns, intellectual property issues, farmer access, and equitable technology dissemination in resource-limited agricultural regions. As the global policy landscape transitions to accommodate CRISPR-edited crops, transparent regulatory frameworks, stakeholder engagement, and public perception will play pivotal roles in ensuring sustainable, safe, and inclusive adoption of genome editing in agriculture.},
}

@article {pmid40918251,
year = {2025},
author = {Zu, H and Sun, R and Li, J and Guo, X and Wang, M and Guo, W and Wang, X},
title = {Integrated CRISPR-Cas12a and RAA one-pot visual strategy for the rapid identification of Streptococcus equi subspecies equi.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1526516},
pmid = {40918251},
issn = {2235-2988},
mesh = {*Streptococcus equi/genetics/isolation & purification/classification ; Horses ; *CRISPR-Cas Systems ; Animals ; *Streptococcal Infections/veterinary/diagnosis/microbiology ; *Horse Diseases/diagnosis/microbiology ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases/metabolism/genetics ; *Molecular Diagnostic Techniques/methods ; Iceland ; Bacterial Proteins/genetics ; Endodeoxyribonucleases ; Streptococcus ; CRISPR-Associated Proteins ; },
abstract = {Strangles, a highly contagious disease caused by Streptococcus equi subspecies equi (S.equi), significantly impacts horse populations worldwide, with Iceland as the only exception. This disease poses serious threats to equine health and results in considerable economic losses. Consequently, the accurate, sensitive, and rapid detection of S.equi from clinical samples is essential for early warning and effective disease management. This study introduces a novel detection method that integrates recombinase-aided amplification (RAA) with CRISPR/Cas12a technologies. We specifically designed RAA primers and CRISPR RNA to target the eqbE gene of S.equi, and we have carefully optimized the reaction systems for this purpose. The newly established visual diagnostic method has shown to be highly effective, demonstrating 97.14% specificity and 100% sensitivity, with the capability to detect as few as 5.6×10[0] copies of the target. This is the first study to propose the combined application of RAA and CRISPR/Cas12a for the on-site rapid detection of S.equi. This is the first study to propose the combined application of RAA and CRISPR/Cas12a for the on-site rapid detection of S.equi, which enables visual point-of-care diagnosis of Strangles.},
}

*Streptococcus equi/genetics/isolation & purification/classification
Horses
*CRISPR-Cas Systems
Animals
*Streptococcal Infections/veterinary/diagnosis/microbiology
*Horse Diseases/diagnosis/microbiology
Sensitivity and Specificity
*Nucleic Acid Amplification Techniques/methods
*Recombinases/metabolism/genetics
*Molecular Diagnostic Techniques/methods
Iceland
Bacterial Proteins/genetics
Endodeoxyribonucleases
Streptococcus
CRISPR-Associated Proteins

@article {pmid40918142,
year = {2025},
author = {Golinelli, G and Scholler, J and Roussel-Gervais, A and Šakić, A and Ilmjärv, S and Song, D and Gabunia, K and Ji, M and Fan, TJ and Gupta, A and Deshmukh, M and Berjis, A and Cuoghi Costantini, R and Apodaca, K and Sheppard, NC and Kili, S and Dominici, M and Alessandrini, M and June, CH and Levine, BL},
title = {Multiplex engineering using microRNA-mediated gene silencing in CAR T cells.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1647433},
pmid = {40918142},
issn = {1664-3224},
mesh = {*MicroRNAs/genetics ; Animals ; Humans ; *Receptors, Chimeric Antigen/genetics/immunology ; *Immunotherapy, Adoptive/methods ; Mesothelin ; *Gene Silencing ; Mice ; *T-Lymphocytes/immunology/metabolism ; Cell Line, Tumor ; Gene Editing ; CRISPR-Cas Systems ; Receptors, Antigen, T-Cell/genetics ; *Pancreatic Neoplasms/therapy/immunology/genetics ; Xenograft Model Antitumor Assays ; GPI-Linked Proteins/genetics/immunology ; },
abstract = {BACKGROUND: Multiplex gene-edited chimeric antigen receptor (CAR) T-cell therapies face significant challenges, including potential oncogenic risks associated with double-strand DNA breaks. Targeted microRNAs (miRNAs) may provide a safer, functional, and tunable alternative for gene silencing without the need for DNA editing.

METHODS: As a proof of concept for multiplex gene silencing, we employed an optimized miRNA backbone and gene architecture to silence T-cell receptor (TCR) and major histocompatibility complex class I (MHC-I) in mesothelin-directed CAR (M5CAR) T cells. The efficacy of this approach was compared to CD3ζ and β2-microglobulin (β2M) CRISPR/Cas9 knockout (KO) cells. miRNA-expressing cassettes were incorporated into M5CAR lentiviral vectors, enabling combined gene silencing and CAR expression. Antitumor activity was evaluated using in vitro assays and in vivo pancreatic ductal adenocarcinoma models.

RESULTS: Silenced (S) M5CAR T cells retained antitumor functionality comparable to, and in some cases exceeding, that of KO cells. In vivo, S M5CAR T cells achieved tumor control with higher persistence and superior metastasis prevention. In vitro assays demonstrated enhanced resistance to alloreactive natural killer (NK) cells and peripheral blood mononuclear cells (PBMCs).

CONCLUSIONS: Titratable multiplex gene silencing via targeted miRNAs offers an alternative to gene editing for CAR T cells, with potential advantages in potency, persistence, metastasis prevention, and immune evasion for allogeneic products. This strategy may overcome tumor-induced immunosuppression while avoiding the risks associated with DNA double-strand breaks.},
}

*MicroRNAs/genetics
Animals
Humans
*Receptors, Chimeric Antigen/genetics/immunology
*Immunotherapy, Adoptive/methods
Mesothelin
*Gene Silencing
Mice
*T-Lymphocytes/immunology/metabolism
Cell Line, Tumor
Gene Editing
CRISPR-Cas Systems
Receptors, Antigen, T-Cell/genetics
*Pancreatic Neoplasms/therapy/immunology/genetics
Xenograft Model Antitumor Assays
GPI-Linked Proteins/genetics/immunology