@article {pmid41057262,
year = {2026},
author = {Char, SN and Liu, H and Birchler, JA and Wang, K and Yang, B},
title = {CRISPR-Cas9 Toolkit for Maize: Vector Design, Construction, and Analysis of Edited Plants.},
journal = {Cold Spring Harbor protocols},
volume = {2026},
number = {7},
pages = {pdb.prot108659},
doi = {10.1101/pdb.prot108659},
pmid = {41057262},
issn = {1559-6095},
mesh = {*Zea mays/genetics ; *CRISPR-Cas Systems ; *Genetic Vectors/genetics ; Plants, Genetically Modified/genetics ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Genetic toolsets are essential for gene discovery, elucidating biological pathways, and accelerating molecular breeding of superior crops in plant biology and agriculture. Among these, the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) system has emerged as a powerful and indispensable tool for precise genome editing in maize (Zea mays L.). This protocol presents a comprehensive, maize-specific approach to constructing CRISPR vectors and analyzing transgenic plants carrying targeted gene mutations. It is organized into two major sections. The first section provides a step-by-step guide for designing guide RNAs and oligonucleotides (oligos) to construct CRISPR vectors containing one, two, four, or multiplexed (up to eight) single-guide RNAs (sgRNAs). It also describes the modular assembly of these sgRNAs with the Cas9 expression cassette using the Gateway cloning strategy to streamline vector construction. The second section focuses on genotyping CRISPR-edited plants by detecting and characterizing target mutations. Four complementary methods are outlined: (1) the T7 endonuclease I (T7EI) assay, (2) restriction enzyme digestion, (3) Sanger sequencing of PCR amplicons, and (4) high-throughput sequencing. Methods 1 and 2 offer rapid and cost-effective screening for small insertions or deletions (indels), while methods 3 and 4 provide high-resolution and scalable mutation analysis. Together, this workflow offers researchers an efficient, flexible, and reliable system for genome editing and mutation validation in maize, supporting both functional genomics studies and trait improvement applications.},
}

*Zea mays/genetics
*CRISPR-Cas Systems
*Genetic Vectors/genetics
Plants, Genetically Modified/genetics
*Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41318237,
year = {2026},
author = {Shin, SW and Lee, GM and Lee, JS},
title = {CRISPR screen-based mammalian cell engineering for complex biotherapeutics.},
journal = {Trends in biotechnology},
volume = {44},
number = {7},
pages = {1817-1820},
doi = {10.1016/j.tibtech.2025.11.010},
pmid = {41318237},
issn = {1879-3096},
mesh = {Animals ; Humans ; *Cell Engineering/methods ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Mammals ; },
abstract = {The rise of complex biotherapeutics has introduced bottlenecks in production using mammalian cells. Clustered regularly interspaced short palindromic repeats (CRISPR)-based screens enable unbiased discovery of engineering targets that mitigate biomanufacturing-relevant constraints. This forum gives an overview of recent advances and remaining challenges in applying CRISPR screening to build robust, modality-specific cell factories.},
}

Animals
Humans
*Cell Engineering/methods
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Mammals

@article {pmid41421891,
year = {2026},
author = {Durán-Vinet, B and Stanton, JL and Jeunen, GJ and Pochon, X and Zaiko, A and Gemmell, NJ},
title = {CRISPR as a next-generation environmental biosurveillance tool for air, land, and water.},
journal = {Trends in biotechnology},
volume = {44},
number = {7},
pages = {1872-1891},
doi = {10.1016/j.tibtech.2025.11.014},
pmid = {41421891},
issn = {1879-3096},
mesh = {*Environmental Monitoring/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Cas Systems ; Air Microbiology ; Water Microbiology ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based environmental biosurveillance (CRISPR-eBx) offers a portable, specific, sensitive, and cost-effective platform for detecting organisms from environmental nucleic acids. Applications are broad, ranging from pathogen detection to monitoring invasive and endangered species across a range of environmental sources, including water, soil, and air. However, if CRISPR-eBx is to be deployed for novel biological/gene targets and environmental sources, key challenges must be addressed. This review synthesizes recent developments at the intersection of CRISPR technology, computational science, synthetic biology, and biosurveillance. We highlight promising innovations and identify knowledge gaps to present a strategic road map for establishing CRISPR-eBx as a next-generation, frontline biosurveillance solution.},
}

*Environmental Monitoring/methods
*Clustered Regularly Interspaced Short Palindromic Repeats
*CRISPR-Cas Systems
Air Microbiology
Water Microbiology

@article {pmid41513118,
year = {2026},
author = {Amieva, R and Román, LR and Coronado, M and Powell, J and Hassan, MA and Hemphill, A and Boubaker, G and Pfarrer, C and Ortega-Mora, LM and Collantes-Fernández, E and Horcajo, P},
title = {NcROP24 loss attenuates Neospora caninum virulence and alters rhoptry organization.},
journal = {International journal for parasitology},
volume = {56},
number = {7},
pages = {104770},
doi = {10.1016/j.ijpara.2026.104770},
pmid = {41513118},
issn = {1879-0135},
mesh = {*Neospora/pathogenicity/genetics ; Animals ; Female ; Cattle ; *Coccidiosis/parasitology/veterinary ; Virulence ; *Protozoan Proteins/genetics/metabolism ; Pregnancy ; Mice ; Macrophages/parasitology ; Host-Parasite Interactions ; Cattle Diseases/parasitology ; Gene Knockout Techniques ; CRISPR-Cas Systems ; },
abstract = {Neospora caninum is an apicomplexan parasite responsible for bovine neosporosis, a leading cause of abortion and economic loss in cattle worldwide. Despite its veterinary significance, the molecular mechanisms underlying parasite virulence and host-pathogen interaction remain poorly understood. In particular, the contribution of rhoptry proteins, key secretory effectors involved in host cell invasion and immune modulation, has yet to be fully elucidated. Here, we investigate NcROP24, a previously understudied rhoptry protein whose expression correlates with isolate virulence. Using CRISPR/Cas9, we generated NcROP24 knock-out mutants (NcΔROP24) by deleting all three genomic copies and confirmed loss of expression with a single-copy insertion of a selectable marker DHFR-TS. In a pregnant mouse model, NcΔROP24 parasites displayed markedly reduced congenital transmission, higher neonatal survival, and lower maternal brain parasite burdens compared to wild-type controls, demonstrating significant attenuation of systemic and vertical infection. Also, in bovine monocyte-derived macrophages, NcΔROP24 tachyzoites showed impaired intracellular growth. Dual RNA-seq of infected macrophages revealed that NcΔROP24 loss prevents the parasite from reprogramming key host metabolic and degradative pathways, instead promoting a stress-induced, lipogenic state that favours clearance. Concurrently, parasites lacking NcROP24 upregulated stress-associated transcripts and downregulated additional secreted effectors, indicating a shift away from aggressive proliferation. Together, these findings establish NcROP24 as a key factor of N. caninum pathogenicity. By defining its role in host-pathogen interactions, our work highlights NcROP24 as a promising target for next-generation vaccines or therapeutics against bovine neosporosis.},
}

*Neospora/pathogenicity/genetics
Animals
Female
Cattle
*Coccidiosis/parasitology/veterinary
Virulence
*Protozoan Proteins/genetics/metabolism
Pregnancy
Mice
Macrophages/parasitology
Host-Parasite Interactions
Cattle Diseases/parasitology
Gene Knockout Techniques
CRISPR-Cas Systems

@article {pmid41593356,
year = {2026},
author = {Wan, Y and He, Y and Chen, X and Wang, S and Zhou, G and Ying, X and Zhang, H},
title = {Effective delivery of genome editor to cervical cancer targeting Mcl1 for cancer therapy.},
journal = {Cancer gene therapy},
volume = {33},
number = {4},
pages = {378-389},
pmid = {41593356},
issn = {1476-5500},
mesh = {Humans ; Female ; *Uterine Cervical Neoplasms/genetics/therapy/pathology ; *Myeloid Cell Leukemia Sequence 1 Protein/genetics/metabolism/antagonists & inhibitors ; Animals ; *CRISPR-Cas Systems ; Mice ; *Gene Editing/methods ; Apoptosis ; Genetic Therapy/methods ; Cell Line, Tumor ; Extracellular Vesicles/metabolism/genetics ; },
abstract = {CRISPR/Cas9 represents a transformative advancement in precision therapies, offering the promise of more effective and targeted treatment options. However, there are still limitations (including off-target editing as well as unsatisfied delivery tool) which obstruct the wide application of CRISPR/Cas9. Here, an endogenic artificial extracellular vesicles (EVs) system is engineered for effective delivery of Cas9 ribonucleoprotein (RNP). We demonstrated that the endogenic Cas9 RNP were sorted by the Lamp2b and delivered by the artificial EVs, which could markedly inhibit the growth of cervical cancer cells by inducing cell apoptosis. Moreover, artificial endogenic EVs[RNP] (Cas9-Mcl1) could result in remarkable antitumor effects in animal models of cervical cancer through suppressing Mcl1 expression. Our findings indicate that the artificial EVs delivery strategy could deliver Cas9 RNP effectively to inhibit cancer progression, which might be a promising treatment.},
}

Humans
Female
*Uterine Cervical Neoplasms/genetics/therapy/pathology
*Myeloid Cell Leukemia Sequence 1 Protein/genetics/metabolism/antagonists & inhibitors
Animals
*CRISPR-Cas Systems
Mice
*Gene Editing/methods
Apoptosis
Genetic Therapy/methods
Cell Line, Tumor
Extracellular Vesicles/metabolism/genetics

@article {pmid41662723,
year = {2026},
author = {De Silva Weligodage, H and Goenaga, R and Gutierrez, OA and Brown, JK},
title = {Development of a Recombinase Polymerase Amplification-CRISPR/Cas12a Detection System for Cacao Mild Mosaic Virus.},
journal = {Phytopathology},
volume = {116},
number = {7},
pages = {1173-1184},
doi = {10.1094/PHYTO-10-25-0319-R},
pmid = {41662723},
issn = {0031-949X},
mesh = {*Cacao/virology ; *Plant Diseases/virology ; Recombinases/metabolism/genetics ; *Caulimovirus/isolation & purification/genetics ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; DNA Primers/genetics ; Plant Leaves/virology ; Sensitivity and Specificity ; },
abstract = {Plant viruses that cause minimal to no disease symptoms may not support readily detectable virus levels. Such viruses are of concern when they persist in plant germplasm collections or in breeding populations because they can provide an inoculum that can be spread and potentially cause outbreaks in susceptible plant species. The mealybug-transmitted cacao mild mosaic virus (CaMMV) causes symptomatic and asymptomatic infection of cacao trees that varies seasonally. The virus accumulates to low levels in leaves and petioles of at least some cacao genetic groups, which has confounded reliable CaMMV detection. Here, a multiplex recombinase polymerase amplification (RPA) assay was developed to increase the reliability of CaMMV detection. Three RPA primers were designed to amplify two regions of the movement protein (mp) gene of CaMMV, yielding fragments of 362 and 284 bp. To increase the detection sensitivity and specificity of CaMMV, two guide RNAs (20 bp) targeting both the CaMMV RPA amplicons were designed to activate Cas12a-mediated collateral cleavage of a fluorescent reporter. An RPA detection efficiency of 100% was achieved with respect to six known CaMMV mp variants, and the analytical sensitivity ranged from approximately 3 to 40 detectable CaMMV genomes. No signal was observed when cloned cacao-infecting badnavirus sequences or virus-free cacao were used as the template, indicating that this assay is highly specific for CaMMV.},
}

*Cacao/virology
*Plant Diseases/virology
Recombinases/metabolism/genetics
*Caulimovirus/isolation & purification/genetics
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
DNA Primers/genetics
Plant Leaves/virology
Sensitivity and Specificity

@article {pmid41792998,
year = {2026},
author = {Shin, EJ and Choi, Y and Jeon, EJ and Lee, KI and Lee, KJ and Son, YJ and Son, MJ and Kim, S and Cho, SW and Lee, JY},
title = {Targeted KEAP1 disruption enhances antioxidant defense and mesenchymal stromal cell therapy for chronic limb-threatening ischemia.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {7},
pages = {3949-3961},
doi = {10.1016/j.ymthe.2026.03.005},
pmid = {41792998},
issn = {1525-0024},
mesh = {*Kelch-Like ECH-Associated Protein 1/genetics/metabolism ; Animals ; *Mesenchymal Stem Cells/metabolism/cytology ; Humans ; Mice ; *Mesenchymal Stem Cell Transplantation/methods ; NF-E2-Related Factor 2/metabolism/genetics ; Oxidative Stress ; *Antioxidants/metabolism ; Disease Models, Animal ; Gene Editing ; *Chronic Limb-Threatening Ischemia/therapy/metabolism/genetics ; *Ischemia/therapy/metabolism ; CRISPR-Cas Systems ; Signal Transduction ; Reactive Oxygen Species/metabolism ; },
abstract = {Chronic limb-threatening ischemia (CLTI) is a severe vascular disorder characterized by tissue hypoxia and oxidative stress that limit the efficacy of regenerative therapies. Mesenchymal stem/stromal cells (MSCs) hold promise for CLTI treatment through paracrine angiogenic and immunomodulatory signaling, yet their survival and function are compromised in the reactive oxygen species-rich ischemic microenvironment. Here, we utilized CRISPR-Cas9 to generate a targeted knockout of Kelch-like ECH-associated protein 1 (KEAP1), the negative regulator of the antioxidant transcription factor NRF2, in human bone marrow-derived MSCs. KEAP1 editing activated the NRF2 pathway, reduced intracellular oxidative stress, and reprogrammed redox and paracrine gene networks. Edited MSCs exhibited enhanced viability, sustained secretion of proangiogenic cytokines, and improved tissue perfusion and arteriogenesis in a murine model of CLTI. These findings establish KEAP1 gene editing as a permanent, integration-free strategy to augment MSC resistance and therapeutic efficacy in oxidative ischemic environments.},
}

*Kelch-Like ECH-Associated Protein 1/genetics/metabolism
Animals
*Mesenchymal Stem Cells/metabolism/cytology
Humans
Mice
*Mesenchymal Stem Cell Transplantation/methods
NF-E2-Related Factor 2/metabolism/genetics
Oxidative Stress
*Antioxidants/metabolism
Disease Models, Animal
Gene Editing
*Chronic Limb-Threatening Ischemia/therapy/metabolism/genetics
*Ischemia/therapy/metabolism
CRISPR-Cas Systems
Signal Transduction
Reactive Oxygen Species/metabolism

@article {pmid41814651,
year = {2026},
author = {Tao, D and Xu, B and Li, S and Liu, H and Wei, Y and Cao, X and Shi, S and Wang, Y and Jiang, R and Zhang, Y and Zhao, C and Ruan, J and Fu, L and Huang, X and Li, X and Zhao, S and Xie, S},
title = {Structural mining and engineering of metagenome-derived Cas12a orthologs expands the CRISPR genome editing and multiplex diagnostics toolkit.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {7},
pages = {4104-4120},
doi = {10.1016/j.ymthe.2026.03.011},
pmid = {41814651},
issn = {1525-0024},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; *CRISPR-Associated Proteins/genetics/chemistry ; *Metagenome ; Mice ; *Bacterial Proteins/genetics/chemistry/metabolism ; *Endodeoxyribonucleases/genetics/chemistry/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Guide, CRISPR-Cas Systems/genetics ; Swine ; Pathology, Molecular/methods ; },
abstract = {CRISPR-Cas12a is a compact, RNA-guided nuclease widely deployed in genome editing and molecular diagnostics, yet its broader utility is limited by suboptimal cis-cleavage efficiency and incompletely defined trans-cleavage behavior. To overcome these constraints, we developed an artificial intelligence-guided structural discovery pipeline powered by AlphaFold2, which identified 1,261 previously uncharacterized Cas12a orthologs. From this set, 21 structurally conserved but sequence-divergent candidates were selected for biochemical characterization. Using structure-informed engineering, we generated PcuCas12a MAX, a high-fidelity variant that achieves genome-editing efficiencies in human cells comparable to the benchmark AsCas12a Ultra while retaining robust activity in murine and porcine systems. In addition, four orthologs (LcoCas12a, FcaCas12a, EsoCas12a, and Mac2Cas12a), when paired with specifically engineered CRISPR RNAs, exhibited distinct single-stranded DNA trans-cleavage signatures. These properties enabled construction of a multiplex CRISPR sensor capable of simultaneously detecting multiple nucleic acid targets. Together, these findings expand the Cas12a endonuclease repertoire and enhance its utility in genome engineering and next-generation diagnostics.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Animals
*CRISPR-Associated Proteins/genetics/chemistry
*Metagenome
Mice
*Bacterial Proteins/genetics/chemistry/metabolism
*Endodeoxyribonucleases/genetics/chemistry/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats
RNA, Guide, CRISPR-Cas Systems/genetics
Swine
Pathology, Molecular/methods

@article {pmid41814652,
year = {2026},
author = {Iyer, S and Daman, K and Sun, Y and Tutto, A and Holbrook, SE and Joynt, AT and Yan, J and Ambegaokar, P and Guo, D and Liu, P and Stauffer, JE and Maitland, SA and Lee, SM and Xiao, Y and Huang, HC and Zhu, LJ and Gallagher, TL and Cox, GA and Keeler, AM and Siegwart, DJ and Emerson, CP and Wolfe, SA},
title = {SORT LNPs encapsulating Cas9 mRNA achieve efficient editing in skeletal muscle in a dystrophic mouse model.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {7},
pages = {3885-3902},
pmid = {41814652},
issn = {1525-0024},
support = {UH3 TR002668/TR/NCATS NIH HHS/United States ; R01 HL150669/HL/NHLBI NIH HHS/United States ; R01 HL170629/HL/NHLBI NIH HHS/United States ; R01 CA269787/CA/NCI NIH HHS/United States ; R01 EB025192/EB/NIBIB NIH HHS/United States ; P01 HL158506/HL/NHLBI NIH HHS/United States ; R37 AI147868/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Mice ; *Muscle, Skeletal/metabolism ; Disease Models, Animal ; *RNA, Messenger/genetics/administration & dosage ; *Nanoparticles/chemistry ; *Gene Editing/methods ; *CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems/genetics ; *Lipids/chemistry ; Genetic Therapy/methods ; *Muscular Dystrophies, Limb-Girdle/genetics/therapy ; Liposomes ; },
abstract = {Limb girdle muscular dystrophy (LGMD) is the fourth most common type of muscular dystrophy. Gene editing holds promise for treating neuromuscular disorders such as LGMD, but clinical translation remains challenging due to lack of complementary delivery tools for skeletal muscle. Lipid nanoparticles (LNPs) offer a promising platform for transient delivery of gene-editing reagents as mRNA or ribonucleoprotein complexes (RNPs) to skeletal muscle, but editing efficiencies remain modest. While lipid compositions have been optimized to improve delivery to muscle, the impact of cargo type on editing efficiency, biodistribution, and immune response has not been evaluated. Here, we demonstrate that selective organ targeting (SORT) LNPs encapsulating optimized Cas9 cargo facilitate efficient, local delivery to skeletal muscle. Using an LGMDR7 mouse model harboring a mutation in TCAP as a proof-of-concept target, we show that LNP cargo type impacts LNP size, delivery to neighboring muscle groups, and editing efficiency. RNP and mRNA LNPs also provoked distinct innate and adaptive immune responses upon repeated dosing. The optimized SORT LNP platform resulted in 40% restoration of Telethonin expression in treated muscle. Overall, these findings offer valuable insights for the continued development of LNP-based gene-editing reagents to facilitate disease-modifying interventions for neuromuscular diseases.},
}

Animals
Mice
*Muscle, Skeletal/metabolism
Disease Models, Animal
*RNA, Messenger/genetics/administration & dosage
*Nanoparticles/chemistry
*Gene Editing/methods
*CRISPR-Associated Protein 9/genetics
*CRISPR-Cas Systems/genetics
*Lipids/chemistry
Genetic Therapy/methods
*Muscular Dystrophies, Limb-Girdle/genetics/therapy
Liposomes

@article {pmid41877484,
year = {2026},
author = {Ralu, M and Guiraud, S and Dastidar, S and Galbiati, P and Sadaoui, E and Mazed, F and Amor, F and de Cian, A and Richard, I and Mamchaoui, K and Ronzitti, G and Tedesco, FS and Amendola, M},
title = {CRISPR-Cas9-mediated upregulation of utrophin ameliorates Duchenne muscular dystrophy.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {7},
pages = {3903-3923},
doi = {10.1016/j.ymthe.2026.03.025},
pmid = {41877484},
issn = {1525-0024},
mesh = {*Muscular Dystrophy, Duchenne/genetics/therapy/metabolism/pathology ; *Utrophin/genetics/metabolism ; Animals ; Humans ; Mice ; *CRISPR-Cas Systems ; MicroRNAs/genetics ; Genetic Therapy/methods ; Disease Models, Animal ; Up-Regulation ; Dependovirus/genetics ; Gene Editing ; Muscle, Skeletal/metabolism/pathology ; RNA, Guide, CRISPR-Cas Systems/genetics ; Myoblasts/metabolism ; Gene Therapy Agents ; Gene Expression Regulation ; Mice, Inbred mdx ; Binding Sites ; },
abstract = {Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disorder caused by loss of dystrophin. Upregulating utrophin, a dystrophin paralog, is a promising gene therapy approach. Here, we present a CRISPR-Cas9-based strategy to enhance utrophin expression by disrupting repressor binding sites. Using a Cas9/guide RNA (gRNA) ribonucleoprotein complex, we disrupted several such sites in DMD myoblasts and identified microRNA Let-7c binding site as effective in relieving repression of the UTRN gene. Interestingly, Cas9-generated insertions or deletions (indels) were as effective as the complete removal of Let-7c binding site in upregulating UTRN expression, with minimal off-target effects. In a three-dimensional tissue-engineered human skeletal muscle model of DMD, this editing strategy resulted in significant utrophin upregulation and functional improvements of calcium dysregulation and muscle contraction. Finally, in mdx mice, local or systemic delivery of recombinant adeno-associated viruses encoding Cas9 and gRNA targeting the Let-7c binding site resulted in utrophin upregulation and amelioration of muscle histopathology and function. These findings provide the foundations for a mutation-independent, potentially universal gene-editing therapeutic strategy for DMD.},
}

*Muscular Dystrophy, Duchenne/genetics/therapy/metabolism/pathology
*Utrophin/genetics/metabolism
Animals
Humans
Mice
*CRISPR-Cas Systems
MicroRNAs/genetics
Genetic Therapy/methods
Disease Models, Animal
Up-Regulation
Dependovirus/genetics
Gene Editing
Muscle, Skeletal/metabolism/pathology
RNA, Guide, CRISPR-Cas Systems/genetics
Myoblasts/metabolism
Gene Therapy Agents
Gene Expression Regulation
Mice, Inbred mdx
Binding Sites

@article {pmid41935953,
year = {2026},
author = {Fumagalli, M and An, D and Simula, L and Combe, C and Aziez, L and Simoni, Y and Alves-Guerra, MC and Valentini, A and Marchais, M and Vermare, A and Moraly, J and Manni, S and Quadraccia, MC and Quintarelli, C and De Angelis, B and Bercovici, N and Donnadieu, E and Pendino, F},
title = {An in vivo CRISPR screen unveils promising target genes to improve CAR-T cell efficacy in a solid tumor model.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {7},
pages = {3976-4000},
doi = {10.1016/j.ymthe.2026.04.009},
pmid = {41935953},
issn = {1525-0024},
mesh = {Animals ; Humans ; Mice ; *Immunotherapy, Adoptive/methods ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Cell Line, Tumor ; *Receptors, Chimeric Antigen/genetics/metabolism/immunology ; Disease Models, Animal ; *Neoplasms/therapy/genetics/immunology ; Xenograft Model Antitumor Assays ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *T-Lymphocytes/immunology/metabolism ; },
abstract = {CAR-T cell therapies are revolutionizing the treatment of refractory or relapsed hematological malignancies, but many patients do not achieve durable responses, and these therapies remain ineffective against solid tumors. Therapeutic failure is closely associated with a poor persistence of CAR-T cells in patients, highlighting the need to identify strategies promoting in vivo expansion. Although numerous gene-editing strategies have been proposed, comparative studies to identify the most effective ones are still lacking. Here, using a focused CRISPR-knockout library targeting 50 selected gene candidates, we developed a competitive screening that revealed ZC3H12A, SOCS1, PTPN2, and CDKN2A as the most robust targets to improve persistence of EGFR CAR-T cells in human lung tumor-bearing mice. Surprisingly, disruption of other genes previously reported to improve CAR-T cell efficacy in other preclinical models-MED12, PRDM1, and BATF-had a detrimental effect in this context. These results suggest that some gene-editing strategies can yield beneficial, neutral, or even deleterious effects on CAR-T cell persistence, depending on specific conditions. Altogether, these findings highlight the importance of performing context-specific evaluations of genetic modifications to accelerate the clinical translation of the most promising editing strategies for optimizing CAR-T cell therapies.},
}

Animals
Humans
Mice
*Immunotherapy, Adoptive/methods
*CRISPR-Cas Systems
*Gene Editing/methods
Cell Line, Tumor
*Receptors, Chimeric Antigen/genetics/metabolism/immunology
Disease Models, Animal
*Neoplasms/therapy/genetics/immunology
Xenograft Model Antitumor Assays
*Clustered Regularly Interspaced Short Palindromic Repeats
*T-Lymphocytes/immunology/metabolism

@article {pmid41981915,
year = {2026},
author = {Truong, LB and Li, S and Domkofski, C and Lin, J and Co, C and Halwasia, S and Kuefner, M and Hu, Y and Mao, HQ and Wang, TH and Zhu, S},
title = {Messenger RNA and guide RNA distributions in lipid nanoparticles impact gene-editing efficiency in vivo.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {7},
pages = {3962-3975},
pmid = {41981915},
issn = {1525-0024},
support = {R01 AI183336/AI/NIAID NIH HHS/United States ; R01 CA260628/CA/NCI NIH HHS/United States ; R01 CA293906/CA/NCI NIH HHS/United States ; },
mesh = {*Nanoparticles/chemistry ; *RNA, Messenger/genetics/chemistry ; Animals ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; *Lipids/chemistry ; *Gene Editing/methods ; Mice ; Humans ; CRISPR-Cas Systems ; Liposomes ; },
abstract = {Lipid nanoparticles (LNPs) are among the most advanced non-viral vectors for CRISPR-based gene-editing therapeutics. Co-packaging of messenger RNA (mRNA) and guide RNA (gRNA) inherently produces heterogeneous payload distributions. The impact of this heterogeneity on editing performance remains unclear. Here, we utilize cylindrical illumination confocal spectroscopy (CICS) for single-particle interrogation of ALC-0315 and DLin-MC3-DMA LNPs prepared by three different mixing methods. CICS resolves four distinct subpopulations: co-encapsulated (50.7%-60.4%), gRNA only (30.0%-36.5%), mRNA only (2.0%-3.4%), and empty (4.2%-13.8%), and it uncovers broad, particle-to-particle variability in RNA copy number within each class. Structure-function analysis reveals that LNP formulation and mixing processes influence payload distribution, resulting in a negative correlation between the fraction of empty LNPs and RNA loading per particle. We further investigated the correlation between these quality attributes and therapeutic performance. In mice, ALC-0315 LNPs carrying higher cargo loads (9.8 vs. 8.0 mRNA copies and 25.4 vs. 20.3 gRNA copies per co-encapsulated particle) yielded 1.5-fold higher in vivo editing activity (55.4% vs. 36.3% insertions and deletions [indels]) despite nearly identical biophysical characteristics including LNP size and RNA encapsulation. These results establish payload distribution as a potential determinant of gene-editing potency and demonstrate single-particle CICS as a powerful tool for rational design of multi-component nucleic acid-delivery systems.},
}

*Nanoparticles/chemistry
*RNA, Messenger/genetics/chemistry
Animals
*RNA, Guide, CRISPR-Cas Systems/genetics/chemistry
*Lipids/chemistry
*Gene Editing/methods
Mice
Humans
CRISPR-Cas Systems
Liposomes

@article {pmid42015511,
year = {2026},
author = {He, L and Fu, Y and Wang, Z and Zhou, Q and Sun, H and Wang, H},
title = {In vivo systematic detection of the outcomes of CRISPR-Cas9-mediated DNA repair in skeletal muscle stem cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {7},
pages = {3924-3948},
doi = {10.1016/j.ymthe.2026.04.032},
pmid = {42015511},
issn = {1525-0024},
mesh = {*CRISPR-Cas Systems ; Animals ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Mice ; *Muscle, Skeletal/cytology/metabolism ; *Stem Cells/metabolism/cytology ; *DNA Repair ; Dependovirus/genetics ; Genetic Vectors/genetics ; DNA End-Joining Repair ; Humans ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-CRSIPR-associated protein 9 (Cas9) has revolutionized genome editing with broad therapeutic applications, yet its repair patterns in vivo remain poorly understood. Here, we systematically profile CRISPR-Cas9 editing outcomes at 95 loci using our established CRISPR-Cas9/adeno-associated virus (AAV)9-single guide RNA (sgRNA) system in skeletal muscle stem cells (MuSCs). Through comprehensive characterization of the repair outcomes, our findings demonstrate that the general rules governing CRISPR-Cas9-mediated editing in vivo largely align with those observed in vitro. In addition to the anticipated small editing insertions or deletions (indels), such as microhomology-mediated end joining (MMEJ)-mediated deletions and non-homologous end joining (NHEJ)-mediated templated insertions, we uncover a prevalent occurrence of large on-target modifications, including large deletions (LDs) characterized by microhomology (MH) and large insertions (LIs). Notably, the LIs comprise not only exogenous AAV vector integrations but also endogenous genomic DNA fragments (Endo-LIs). Endo-LIs preferentially originate from active genomic regions, with their integration shaped by 3D chromatin architecture. By disrupting key components of the NHEJ and MMEJ repair pathways in vivo, we identify their distinct roles in regulating the large on-target modifications. Together, our work systematically profiles CRISPR-Cas9 repair outcomes in vivo and offers valuable guidance for improving the safety of CRISPR-Cas9-based gene therapies.},
}

*CRISPR-Cas Systems
Animals
RNA, Guide, CRISPR-Cas Systems/genetics
*Gene Editing/methods
Mice
*Muscle, Skeletal/cytology/metabolism
*Stem Cells/metabolism/cytology
*DNA Repair
Dependovirus/genetics
Genetic Vectors/genetics
DNA End-Joining Repair
Humans

@article {pmid42031729,
year = {2026},
author = {Moon, HC and Herschl, MH and Sclip, A and Tran, VQ and Pawluk, A and Konermann, S and Hsu, PD},
title = {A combinatorial domain screening platform reveals epigenetic effector interactions for transcriptional perturbation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42031729},
issn = {2041-1723},
mesh = {Humans ; *Epigenesis, Genetic ; DNA Methylation ; Epigenome Editing ; *Transcription, Genetic ; Protein Domains/genetics ; CRISPR-Cas Systems ; },
abstract = {Epigenetic regulation involves the coordinated interplay of diverse proteins. To systematically explore these combinations, we present COMBINE (combinatorial interaction exploration), a high-throughput platform that tests over 50,000 pairs of epigenetic effector domains up to 2,094 amino acids in length for their ability to modulate endogenous human gene transcription. COMBINE reveals diverse synergistic interactions between epigenetic protein domains, including a potent KRAB-L3MBTL3 fusion that increases the effective targeting window, enhances gene silencing in dose-limited conditions, and enables robust dual-directional CRISPR perturbation. Inducible screening shows DNA methylation modifiers are essential for epigenetic memory, with distinct combinations driving long-term repression and activation. This systematic analysis of pairwise domain interactions advances our understanding of epigenetic crosstalks and the development of next-generation epigenome editing tools. More broadly, COMBINE offers a generalizable platform to functionally characterize combinatorial biological processes at scale.},
}

Humans
*Epigenesis, Genetic
DNA Methylation
Epigenome Editing
*Transcription, Genetic
Protein Domains/genetics
CRISPR-Cas Systems

@article {pmid42049824,
year = {2026},
author = {Galindo-González, L and Dupras, AA},
title = {CRISPR-Cas12a fluorescence assays identify weedy Amaranthus species.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42049824},
issn = {2045-2322},
support = {N-000470//Canadian Food Inspection Agency/ ; },
mesh = {*Amaranthus/genetics/classification ; *CRISPR-Cas Systems ; Fluorescence ; *Plant Weeds/genetics/classification ; *Bacterial Proteins/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; Endodeoxyribonucleases ; },
abstract = {Tall Waterhemp (Amaranthus tuberculatus) and Palmer Amaranth (Amaranthus palmeri) populations have developed multiple-herbicide resistance and impact the production of important crops, including corn, soybean and cotton. Morphological plasticity of these species, their persistence in the soil seed bank and their presence as contaminants during trade require efficient and sensitive methodologies to support their identification. Cas12 enzymes can be directed to a specific genomic region by a crRNA, cutting the DNA double strand and exhibiting collateral enzymatic activity on free-floating single-stranded DNA. This characteristic can be used to generate single-stranded oligonucleotide reporters bearing a fluorophore and a quencher, which will produce fluorescence when cut by Cas12. We designed CRISPR-Cas12 fluorescent assays to differentiate A. tuberculatus, A. palmeri and A. palmeri`s sister species (A. watsonii) from other Amaranthus species. Our assays for identifying the A. palmeri + A. watsonii clade and A. tuberculatus were 100% accurate when presented with blind samples of 14 Amaranthus species. Fluorescence could be detected using a blue light filter on a transilluminator within minutes. A preliminary recombinase amplification step to increase the limit of detection, and the Cas12 reaction, can be performed at room temperature and within an hour once DNA has been isolated.},
}

*Amaranthus/genetics/classification
*CRISPR-Cas Systems
Fluorescence
*Plant Weeds/genetics/classification
*Bacterial Proteins/genetics/metabolism
*CRISPR-Associated Proteins/genetics/metabolism
Endodeoxyribonucleases

@article {pmid42055327,
year = {2026},
author = {Mazur, CM and Kotsalidis, PE and George, M and Whalley, T and Sato, T and Doench, JG and Surface, LE and Wein, MN},
title = {Genome-wide CRISPR interference screen identifies Clip2 as a novel regulator of osteocyte maturation and morphology.},
journal = {The Journal of biological chemistry},
volume = {302},
number = {6},
pages = {113075},
pmid = {42055327},
issn = {1083-351X},
mesh = {*Osteocytes/cytology/metabolism ; Animals ; Mice ; *Microtubule-Associated Proteins/genetics/metabolism ; Cell Differentiation/genetics ; Cell Line ; Adaptor Proteins, Signal Transducing/genetics ; Extracellular Matrix Proteins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Osteocytes play critical roles in bone, making them attractive targets for therapeutics aimed at improving bone mass and strength. The genes driving osteocyte maturation and function are not fully understood. Here, we aimed to identify novel genes responsible for osteocyte differentiation and dendrite development by performing a genome-wide CRISPR-interference (CRISPRi) screen in the Ocy454 osteocyte-like cell line. We identify CD61 (integrin β3) as a marker of osteocyte maturation: surface CD61 expression increases during osteocyte maturation, and CD61[high] cells express higher levels of osteocyte marker genes. We then developed a flow cytometry-based assay to quantify surface CD61 protein levels as a phenotypic endpoint for functional genomic screening. In a genome-wide screen, we identified Clip2, which encodes a microtubule-binding protein, as one of dozens of genes necessary for CD61 expression. Clip2 inhibition decreased surface CD61 expression, reduced expression of osteocyte-specific genes Dmp1 and Sost, and impaired dendrite morphology in vitro. Together, these results highlight the utility of surface CD61 as a marker of osteocyte maturity and identify the role of the microtubule cytoskeleton for osteocyte differentiation, form, and function.},
}

*Osteocytes/cytology/metabolism
Animals
Mice
*Microtubule-Associated Proteins/genetics/metabolism
Cell Differentiation/genetics
Cell Line
Adaptor Proteins, Signal Transducing/genetics
Extracellular Matrix Proteins/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid42134180,
year = {2026},
author = {Lee, H and Jeon, BJ and Jang, HS and Choi, JY and Jang, G},
title = {Non-surgical deep uterine embryo transfer combined with electroporation-based genome editing enables scalable production of CD163-Knockout pigs.},
journal = {Theriogenology},
volume = {263},
number = {},
pages = {117977},
doi = {10.1016/j.theriogenology.2026.117977},
pmid = {42134180},
issn = {1879-3231},
mesh = {Animals ; *Embryo Transfer/veterinary/methods ; *Electroporation/veterinary/methods ; *Gene Editing/veterinary/methods ; Female ; Swine/genetics ; *Antigens, CD/genetics/metabolism ; Animals, Genetically Modified ; *Antigens, Differentiation, Myelomonocytic/genetics/metabolism ; CD163 Antigen ; *Receptors, Cell Surface/genetics ; CRISPR-Cas Systems ; *Gene Knockout Techniques/veterinary ; Pregnancy ; Electroporation Therapies ; },
abstract = {Porcine reproductive and respiratory syndrome (PRRS) remains the most economically devastating viral disease in the global swine industry, causing annual losses exceeding $600 million in the United States alone. While CD163-knockout (KO) pigs have demonstrated complete resistance to the PRRS virus (PRRSV) infection, conventional production methods relying on microinjection-based genome editing and surgical embryo transfer present significant bottlenecks for industrial-scale application. Here, we demonstrate that the integration of electroporation-based CRISPR-Cas9 delivery with non-surgical deep intrauterine embryo transfer enables scalable production of CD163-KO pigs. Our approach achieved successful pregnancies and live births of genome-edited piglets without requiring surgical intervention or specialized microinjection expertise. Genotyping analysis revealed complete biallelic KOs in a subset of offspring, while others exhibited mosaic patterns reflecting the stochastic nature of electroporation-mediated editing. These findings establish a technically accessible and scalable pipeline for producing gene-edited pigs. By eliminating the need for complex surgical facilities and specialized microinjection equipment, this approach provides a practical and field-applicable strategy that can be readily implemented in standard commercial swine farms, representing a critical step toward the widespread deployment of genome-edited livestock in global agriculture.},
}

Animals
*Embryo Transfer/veterinary/methods
*Electroporation/veterinary/methods
*Gene Editing/veterinary/methods
Female
Swine/genetics
*Antigens, CD/genetics/metabolism
Animals, Genetically Modified
*Antigens, Differentiation, Myelomonocytic/genetics/metabolism
CD163 Antigen
*Receptors, Cell Surface/genetics
CRISPR-Cas Systems
*Gene Knockout Techniques/veterinary
Pregnancy
Electroporation Therapies

@article {pmid42319782,
year = {2026},
author = {Liu, Z and Li, M and Wang, J and Chen, B},
title = {Copper-only Superoxide dismutase 6 contributes to reactive oxygen species regulation, genotoxic stress tolerance, and virulence in Candida albicans.},
journal = {Medical mycology},
volume = {64},
number = {7},
pages = {},
doi = {10.1093/mmy/myag063},
pmid = {42319782},
issn = {1460-2709},
support = {2023YXNS187//Development Plan of Jining/ ; 202411000427//Medical and Health Science and Technology Development Project of Shandong, China/ ; SDYWZGKCJHLH2023079//Shandong medical staff science and technology innovation plan project/ ; },
mesh = {*Candida albicans/pathogenicity/genetics/enzymology ; *Reactive Oxygen Species/metabolism ; Virulence ; Animals ; Oxidative Stress ; *Superoxide Dismutase/genetics/metabolism ; Mice ; Candidiasis/microbiology ; *DNA Damage ; Disease Models, Animal ; Gene Expression Profiling ; CRISPR-Cas Systems ; Gene Expression Regulation, Fungal ; },
abstract = {Superoxide dismutase (SOD) is a major antioxidant enzyme that protects cells against reactive oxygen species (ROS)-mediated oxidative stress. The identification of a fungal-specific copper-only SOD family in Candida albicans (C. albicans) has revealed a previously unrecognized component of fungal oxidative stress defence, yet the role of SOD6 remains unclear. Here, we generated a sod6Δ/Δ mutant in C. albicans using a transient CRISPR/Cas9 approach and examined the contribution of SOD6 to virulence, ROS homeostasis, oxidative stress resistance, and genome maintenance. Compared with the wild-type (WT) strain, the sod6Δ/Δ mutant showed attenuated virulence in both the Galleria mellonella (G. mellonell) infection model and a murine systemic candidiasis model. This virulence defect occurred without detectable alterations in hyphal formation or other major pathogenicity-associated traits. Instead, the mutant accumulated higher intracellular and extracellular ROS levels and displayed increased sensitivity to hydrogen peroxide, zeocin, and camptothecin (CPT), consistent with impaired oxidative stress adaptation and genome maintenance. Transcriptomic analysis further revealed downregulation of genes involved in DNA replication and repair, with dpb4, which encodes a subunit of DNA polymerase epsilon required for DNA replication and genome stability, showing the greatest reduction. Together, these findings identify SOD6 as an important determinant of oxidative stress adaptation, genome maintenance, and pathogenic fitness in C. albicans.},
}

*Candida albicans/pathogenicity/genetics/enzymology
*Reactive Oxygen Species/metabolism
Virulence
Animals
Oxidative Stress
*Superoxide Dismutase/genetics/metabolism
Mice
Candidiasis/microbiology
*DNA Damage
Disease Models, Animal
Gene Expression Profiling
CRISPR-Cas Systems
Gene Expression Regulation, Fungal

@article {pmid42349956,
year = {2026},
author = {Dutta, S and Pal, A and Srivatsan, SG},
title = {Chemoenzymatic labeling of RNA using terminal uridylyl transferase and bioorthogonal click chemistry.},
journal = {Methods in enzymology},
volume = {731},
number = {},
pages = {223-252},
doi = {10.1016/bs.mie.2026.05.007},
pmid = {42349956},
issn = {1557-7988},
mesh = {*Click Chemistry/methods ; *RNA Nucleotidyltransferases/metabolism/chemistry ; Azides/chemistry ; Uridine Triphosphate/chemistry/analogs & derivatives ; *RNA, Guide, CRISPR-Cas Systems/chemistry/genetics ; *RNA/chemistry ; Staining and Labeling/methods ; *Nucleotidyltransferases/metabolism ; CRISPR-Cas Systems ; Schizosaccharomyces pombe Proteins ; },
abstract = {The ability of native and engineered nucleic acid-processing enzymes to incorporate clickable nucleotide substrates has greatly advanced bioorthogonal labeling of nucleic acids, overcoming the limitations of conventional solid-phase oligonucleotide (ON) synthesis. In this chemoenzymatic approach, template-dependent polymerases routinely enable the incorporation of nucleotides bearing small reactive handles. The resulting nucleic acids undergo chemoselective reactions, such as azide-alkyne cycloaddition, inverse-electron-demand Diels-Alder, or Staudinger ligation, to install desired functionalities. Alternatively, the promiscuity of template-independent transferases, such as terminal uridylyl transferase (TUTase), provides access to site-specific labeling of RNA ONs at the 3'-end. In this methods chapter, we detail protocols for incorporating azide-modified UTP analogs into short RNA ONs and highly structured CRISPR guide RNAs (sgRNAs) using the terminal uridylyl transferase SpCID1. We describe methods to control the enzyme's incessant incorporation behavior and enable subsequent click functionalization of the RNAs. Finally, we demonstrate remodeling of the CRISPR system via synthesis of azide-modified sgRNAs, which when complexed with dCas9, recruit azide groups to specific gene targets for post-hybridization functionalization.},
}

*Click Chemistry/methods
*RNA Nucleotidyltransferases/metabolism/chemistry
Azides/chemistry
Uridine Triphosphate/chemistry/analogs & derivatives
*RNA, Guide, CRISPR-Cas Systems/chemistry/genetics
*RNA/chemistry
Staining and Labeling/methods
*Nucleotidyltransferases/metabolism
CRISPR-Cas Systems
Schizosaccharomyces pombe Proteins

@article {pmid42351104,
year = {2026},
author = {Zhu, Y and Bi, Z and Zhang, Z and Zhang, M and Du, Q and Hu, M and Zhou, T and Fan, Y and Zhang, S and Wang, G and Liu, G},
title = {Virus-like particles in cancer immunotherapy: bridging human and veterinary medicine through one health.},
journal = {Journal of nanobiotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12951-026-04703-9},
pmid = {42351104},
issn = {1477-3155},
support = {2024M753586//the China Postdoctoral Science Foundation/ ; 2026JB09//the Central Public-interest Scientific Institution Basal Research Fund/ ; 32473001//the National Natural Science Foundation of China/ ; 2023YFD1800700//the National Key Research and Development Program of China/ ; },
abstract = {Virus-like particles (VLPs) are engineered nanoplatforms that mimic viral structures, offering high immunogenicity, biocompatibility, and functional versatility for cancer immunotherapy. While widely explored in human oncology as nanovaccines and targeted delivery systems for chemo-/immuno-therapeutics and genetic payloads (e.g., mRNA, siRNA, and CRISPR/Cas systems), their potential in veterinary oncology remains underexploited. This review synthesizes recent advances in VLP design, including scaffold engineering, antigen display, cargo encapsulation, and surface functionalization, and discusses the mechanistic basis of VLP-induced antitumor immunity, encompassing dendritic cell activation, adaptive immune amplification, and tumor microenvironment remodeling. Importantly, we highlight the emerging role of companion animals with spontaneous tumors-such as lymphoma, melanoma, and mammary carcinoma-as immunocompetent translational models within the One Health framework. Comparative oncology reveals striking parallels in oncogenic pathways, immune landscapes, and therapeutic responses, supporting the use of canine and feline cancers as biologically relevant intermediates between murine studies and human clinical trials. We provide an evidence-based assessment of representative VLP platforms, evaluate their translational readiness, and examine cross-species opportunities for shared target development, biomarker discovery, and regulatory convergence, while also addressing species-specific biological and technical limitations. Finally, we propose a forward-looking roadmap that prioritizes manufacturing standardization, biomarker development, comparative validation, precision engineering, and emerging technologies such as AI-guided design and tumor-on-chip systems. Collectively, we position One Health as an operational strategy to accelerate the bidirectional translation of VLP-based immunotherapies for both human and veterinary cancer patients.},
}

@article {pmid42352260,
year = {2026},
author = {Kuroda, T and Yokota, T},
title = {RNA Therapeutics Targeting Skeletal Muscle: Emerging Antisense and Gene-Modifying Strategies.},
journal = {Biomolecules},
volume = {16},
number = {6},
pages = {},
pmid = {42352260},
issn = {2218-273X},
mesh = {Humans ; *Oligonucleotides, Antisense/therapeutic use/genetics ; *Muscle, Skeletal/metabolism/drug effects ; *Muscular Dystrophy, Duchenne/genetics/therapy ; Animals ; *Genetic Therapy/methods ; RNA, Small Interfering/therapeutic use/genetics ; Morpholinos/therapeutic use ; },
abstract = {RNA-based therapeutics are reshaping the treatment landscape for skeletal muscle disorders by enabling modulation of RNA processing or direct correction of disease-causing alleles. In Duchenne muscular dystrophy (DMD), four antisense oligonucleotides-eteplirsen, golodirsen, viltolarsen, and casimersen-have received FDA approval; these phosphorodiamidate morpholino oligomers (PMOs) induce exon skipping to restore the reading frame and enable expression of internally truncated dystrophin. Beyond splice switching, RNA therapeutics include RNase H-active gapmers and steric-blocking antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs) that mediate post-transcriptional gene silencing, and RNA-guided gene-modifying technologies such as CRISPR systems that can reframe or repair endogenous alleles. Despite major progress in DMD, broader clinical impact remains constrained by inefficient delivery to skeletal and especially cardiac muscle, the need for repeat administration for most modalities, and safety considerations that limit dose escalation and durability. Next-generation approaches aim to overcome these barriers through peptide- or antibody-conjugated oligonucleotides that enhance cellular uptake and tissue distribution, alternative chemistries with improved stability and potency, and viral or non-viral platforms for durable splice modulation. In parallel, CRISPR-based strategies-including base and prime editing-offer the prospect of one-time correction, while raising important questions regarding delivery, immunogenicity, editing specificity, and long-term safety. This review synthesizes recent advances in antisense and gene-modifying strategies for skeletal muscle and highlights practical priorities for translation, including improved muscle/heart delivery, controllable safety mechanisms, scalable manufacturing, and standardized biomarker-to-clinical outcome relationships.},
}

Humans
*Oligonucleotides, Antisense/therapeutic use/genetics
*Muscle, Skeletal/metabolism/drug effects
*Muscular Dystrophy, Duchenne/genetics/therapy
Animals
*Genetic Therapy/methods
RNA, Small Interfering/therapeutic use/genetics
Morpholinos/therapeutic use

@article {pmid42352295,
year = {2026},
author = {McGill, LP and Banas, KH and Tiesi, G and Kmiec, EB},
title = {Genotypic and Phenotypic Diversity as a Function of CRISPR-Directed Gene Knock-Out of NRF2 in Pancreatic Adenocarcinoma Cells, a Feasibility Study.},
journal = {Biomolecules},
volume = {16},
number = {6},
pages = {},
pmid = {42352295},
issn = {2218-273X},
mesh = {Humans ; *NF-E2-Related Factor 2/genetics/metabolism ; *Pancreatic Neoplasms/genetics/pathology ; Cell Line, Tumor ; Phenotype ; *CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques ; Drug Resistance, Neoplasm/genetics ; *Adenocarcinoma/genetics/pathology ; Genotype ; Feasibility Studies ; Gemcitabine ; Deoxycytidine/analogs & derivatives/pharmacology ; *Carcinoma, Pancreatic Ductal/genetics/pathology ; RNA, Guide, CRISPR-Cas Systems/genetics ; Cell Survival/drug effects ; },
abstract = {Pancreatic ductal adenocarcinoma (PDAC) presents unique treatment challenges, often due to the development of anti-cancer drug resistance. Previously, we demonstrated that CRISPR-directed gene ablation disabled the master regulator gene NRF2, a transcription factor known to control drug resistance in squamous cell carcinoma tumor cells, and restored chemosensitivity. In this short study, we evaluated a broad range of CRISPR/Cas9 molecules for their capacity to elicit similar responses in PDAC cells. Synthetic single guide RNAs (sgRNAs) were designed to target multiple functional domains encoded by NRF2. These molecules were delivered to cells via nucleofection, with outcomes analyzed by genotypic, phenotypic, and functional assays. We observed targeting efficiencies ranging from 25% to 100% with a high level of random insertions and deletions (indels). sgRNAs targeting exons 2, 3 and 4 demonstrated a high degree of genotypic, phenotypic and functional outcomes. Targeted disruption of exons 3 and 4 reveals significant loss of cell viability while overcoming drug resistance through the restoration of sensitivity to gemcitabine (>1.75 μM). Our study identifies domain-specific sites within NRF2 that, when disabled, restore sensitivity to gemcitabine, potentiating a more in-depth analysis of this novel augmentative therapeutic approach.},
}

Humans
*NF-E2-Related Factor 2/genetics/metabolism
*Pancreatic Neoplasms/genetics/pathology
Cell Line, Tumor
Phenotype
*CRISPR-Cas Systems/genetics
*Gene Knockout Techniques
Drug Resistance, Neoplasm/genetics
*Adenocarcinoma/genetics/pathology
Genotype
Feasibility Studies
Gemcitabine
Deoxycytidine/analogs & derivatives/pharmacology
*Carcinoma, Pancreatic Ductal/genetics/pathology
RNA, Guide, CRISPR-Cas Systems/genetics
Cell Survival/drug effects

@article {pmid42353201,
year = {2026},
author = {Walflor, HSM and Medeiros, LCS},
title = {Machine Learning for CRISPR-Based Diagnostics.},
journal = {International journal of molecular sciences},
volume = {27},
number = {12},
pages = {},
pmid = {42353201},
issn = {1422-0067},
mesh = {Humans ; *Machine Learning ; *CRISPR-Cas Systems ; Deep Learning ; Predictive Learning Models ; Classification Algorithms ; },
abstract = {CRISPR-based diagnostics now detect viral, bacterial, and cancer-associated nucleic acids with sensitivities approaching quantitative PCR; however, their translation to decentralized care rests on computational design and interpretation that current datasets cannot sustain. Pandemic-era Cas12a assays reached 95% positive predictive agreement against reverse transcription quantitative PCR (RT-qPCR) at 10 copies/μL, and deep neural networks now design Cas13 detection assays spanning 1933 vertebrate-infecting viruses, ranking candidate guides at Spearman correlations of 0.69 to 0.84 across internal and external validation. Generative deep-learning systems improve single-nucleotide discrimination two- to three-fold, computer vision classifies lateral flow outputs at 96.5% accuracy, and multi-biomarker fusion reaches an area under the receiver operating characteristic curve (AUC) of 0.998 in lung cancer detection. These results mask a narrow data foundation. Cas13a guide prediction still draws from a single screening library of 19,209 guide-target pairs, Cas12a has one published diagnostic model, and signal classifiers almost uniformly validate on single-site cohorts. This review synthesizes mechanistic constraints, predictive and generative models, and point-of-care classifiers, and maps the path beyond this data ceiling. Evolutionary pretraining on RNA corpora and lab-in-the-loop agents that convert model failure into targeted data acquisition define the route forward.},
}

Humans
*Machine Learning
*CRISPR-Cas Systems
Deep Learning
Predictive Learning Models
Classification Algorithms

@article {pmid42353287,
year = {2026},
author = {Kim, SJ and Nam, YH and Joo, EY and Park, J and Park, S and Jung, SC and Jin, DK},
title = {Systemic AAV-hGCDH Gene Therapy Alleviates Glutaric Acid Accumulation and Attenuates Chronic Brain Vacuolation in a Novel Mouse Model of Glutaric Aciduria Type I.},
journal = {International journal of molecular sciences},
volume = {27},
number = {12},
pages = {},
pmid = {42353287},
issn = {1422-0067},
support = {75440-1//Inha University/ ; },
mesh = {Animals ; *Dependovirus/genetics ; *Genetic Therapy/methods ; *Glutaryl-CoA Dehydrogenase/deficiency/genetics/metabolism ; Mice ; Disease Models, Animal ; *Amino Acid Metabolism, Inborn Errors/therapy/genetics/metabolism/pathology ; *Brain Diseases, Metabolic/therapy/genetics/metabolism/pathology ; *Brain/pathology/metabolism ; *Glutarates/metabolism ; Genetic Vectors/genetics/administration & dosage ; Gene Therapy Agents ; Male ; CRISPR-Cas Systems ; Liver/metabolism ; },
abstract = {Glutaric aciduria type 1 (GA1) is a rare neurometabolic disorder caused by glutaryl-CoA dehydrogenase (GCDH) deficiency, leading to the accumulation of neurotoxic metabolites that can cause both acute encephalopathic crises and progressive, insidious brain injury. Current management primarily relies on a protein-restricted diet, which remains therapeutically insufficient and burdensome for patients, highlighting the need for disease-modifying therapies. In this study, we established a novel GA1 mouse model using CRISPR/Cas9 technology and evaluated the preclinical efficacy of systemic recombinant adeno-associated virus (rAAV)-mediated gene therapy. Under standard dietary conditions without high-lysine challenge, our GA1 model exhibited sustained cerebral and hepatic glutaric acid (GA) accumulation and distinct chronic vacuolation in the hippocampus and cerebellum, mirroring the insidious-onset GA1 phenotype. Five-week-old mice received a single intravenous injection of rAAV-hGCDH using either rAAV2/8 or rAAV2/9 serotypes. Systemic rAAV-mediated gene therapy significantly reduced GA accumulation and attenuated chronic neuropathological changes in this GA1 mouse model for both serotypes. Our findings support the hypothesis that peripheral metabolic correction may play an important role in preventing the chronic neuropathological changes associated with GCDH deficiency. However, further investigation using tissue-specific expression systems is required to definitively delineate the relative contributions of hepatic versus central GCDH restoration to the observed neuroprotection.},
}

Animals
*Dependovirus/genetics
*Genetic Therapy/methods
*Glutaryl-CoA Dehydrogenase/deficiency/genetics/metabolism
Mice
Disease Models, Animal
*Amino Acid Metabolism, Inborn Errors/therapy/genetics/metabolism/pathology
*Brain Diseases, Metabolic/therapy/genetics/metabolism/pathology
*Brain/pathology/metabolism
*Glutarates/metabolism
Genetic Vectors/genetics/administration & dosage
Gene Therapy Agents
Male
CRISPR-Cas Systems
Liver/metabolism

@article {pmid42353300,
year = {2026},
author = {Afifi, N and Colussi, D and Perez-Leal, O},
title = {CRISPR Gene Tagging for Illuminating Endogenous Protein Dynamics.},
journal = {International journal of molecular sciences},
volume = {27},
number = {12},
pages = {},
pmid = {42353300},
issn = {1422-0067},
support = {1R21HG012241-03/NH/NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; Animals ; *Gene Editing/methods ; *Proteins/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Endogenous gene tagging using CRISPR has changed the understanding of the role played by different proteins due to the ability to track and study proteins in their natural state. With CRISPR-based gene tagging, it is possible to insert fluorescent, luminescent, epitope, affinity, and proximity labels into the target protein at its endogenous genomic location without affecting its physiological expression and dynamics. Here, we discuss the DNA-repair mechanisms employed in endogenous gene tagging, including homology-dependent repair, NHEJ-based integration, and alternative approaches that can be used with challenging cell types. Key aspects of efficient CRISPR tagging experiments are also described. Additionally, we review recent advances in the increasing array of protein tag technologies, including fluorescent proteins, split-reporter technologies, NanoLuc/HiBiT, peptide epitopes, and proximity biotinylation enzymes. Lastly, we review the scalability of endogenous tagging approaches using multiplex editing, atlas-scale proteome tagging, iPSC-based disease modeling, and drug discovery platforms for assessing target engagement, protein degradation, phenotype screening, and mechanism of action of compounds. Although difficult in primary and pluripotent cells, new methods based on avoiding double-strand breaks, such as prime editing, PASTE, and CRISPR associated transposases, will drive the future expansion of endogenous tagging approaches. Such developments firmly set up CRISPR gene tagging as a fundamental technology in quantitative cell biology and translational pharmacology.},
}

Humans
*CRISPR-Cas Systems
Animals
*Gene Editing/methods
*Proteins/genetics/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42353328,
year = {2026},
author = {Jan, R and Iqbal, S and Ali, S and Almalki, MA and Alfredan, M and Ibrahim, RIH and Asaf, S and Kim, KM},
title = {Early Flowering (ELF) Gene Integrates Vegetative Growth, Flowering Regulation, and Reproductive Development in Arabidopsis thaliana.},
journal = {International journal of molecular sciences},
volume = {27},
number = {12},
pages = {},
pmid = {42353328},
issn = {1422-0067},
mesh = {*Arabidopsis/genetics/growth & development ; *Flowers/genetics/growth & development ; *Arabidopsis Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Plants, Genetically Modified ; Reproduction/genetics ; *Transcription Factors/genetics/metabolism ; CRISPR-Cas Systems ; Germination/genetics ; Gene Editing ; },
abstract = {Early flowering-related factors play pivotal roles in coordinating plant growth and reproductive development. In this study, we investigated the biological function of early flowering gene (ELF) in Arabidopsis thaliana using CRISPR/Cas9-mediated genome editing and construction of overexpression approaches. Two independent ELF overexpression (OE-ELF) and genome-edited (ge-elf) lines were generated and systemically analyzed. ELF overexpression significantly enhanced early seedling performance, increasing germination rate and seedling fresh weight by up to 8.7%, while genome-edited lines exhibited a marked reduction. Root growth was strongly promoted in OE-ELF plants, with root length increase of 85% and 75%, whereas ge-elf lines showed a reduction of up to 48%. At later developmental stages, OE-ELF plants displayed enhanced vegetative growth, including increased leaf length (32%), leaf area (91%), and accelerated flowering (21% earlier than wild type). In contrast, ge-elf delayed flowering by up to 25% and resulted in compact plant architecture. Reproductive development was severely compromised in ge-elf plants, which exhibited malformed inflorescences, reduced pollen germination, shortened silique (45%), and a drastic decrease in seed number per silique (70%). Conversely, OE-ELF plants showed increased silique number and seed per silique. Molecular analysis revealed that ELF positively regulates key flowering-related genes, including FLC, SOC1, AP1, and LFY, which correlated strongly with growth and reproductive traits. Our results demonstrate that ELF functions as a central regulator integrating vegetative growth, floral development, male fertility, and seed production in Arabidopsis thaliana.},
}

*Arabidopsis/genetics/growth & development
*Flowers/genetics/growth & development
*Arabidopsis Proteins/genetics/metabolism
Gene Expression Regulation, Plant
Plants, Genetically Modified
Reproduction/genetics
*Transcription Factors/genetics/metabolism
CRISPR-Cas Systems
Germination/genetics
Gene Editing

@article {pmid42087135,
year = {2026},
author = {Ouyang, M and Wang, J and Luo, X and Tian, R},
title = {CaRPOOL: a pooled calcium‑recording CRISPR screening platform identifies CCR7 as a modulator of cellular osmomechanosensing.},
journal = {Cell communication and signaling : CCS},
volume = {24},
number = {1},
pages = {},
pmid = {42087135},
issn = {1478-811X},
support = {2024YFA0919800//National Key Research and Development Program of China/ ; A2303039//Shenzhen Medical Research Fund/ ; 2023B151502007//Guangdong Basic and Applied Basic Research Foundation/ ; },
mesh = {Humans ; *Receptors, CCR7/metabolism/genetics ; *Calcium/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Calcium Signaling ; *CRISPR-Cas Systems ; HEK293 Cells ; },
abstract = {Cells must continuously sense and respond to environmental changes by translating physical and chemical cues into intracellular signals. However, systematic discovery of genes governing these sensory processes has been limited by the transient nature of signaling events and the low throughput of measurement assays. Here, we present CaRPOOL, a pooled, high‑throughput genetic screening platform that integrates the calcium‑activity recorder CaMPARI2 with CRISPR interference (CRISPRi), enabling stable capture of transient calcium signals for genome‑scale functional screening. Using osmomechanical stimulation as a model, we demonstrate that CaMPARI2 photoconversion faithfully reports stimulus‑dependent calcium responses and supports pooled fluorescence‑activated cell sorting (FACS)-based screening. A CRISPRi library targeting membrane‑associated genes identified both known and previously uncharacterized regulators of osmomechanosensing, including the chemokine receptor CCR7. Mechanistic analyses revealed that CCR7 promotes osmomechanical calcium signaling through a PIEZO1‑dependent Gαs-cAMP-PKA pathway, establishing it as a GPCR regulator of osmomechanical response. Notably, osmotic stress upregulated CCR7 expression in immune cell lines and enhanced osmomechanical responsiveness, suggesting a role in osmomechanical adaptation. Together, these findings introduce a broadly applicable platform for high‑throughput discovery of genes controlling dynamic signaling responses and reveal a GPCR-ion channel crosstalk mechanism in osmomechanotransduction with potential implications for immune cell mechanoadaptation.},
}

Humans
*Receptors, CCR7/metabolism/genetics
*Calcium/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Calcium Signaling
*CRISPR-Cas Systems
HEK293 Cells

@article {pmid42258637,
year = {2026},
author = {Wei, G and Huang, Z and Wang, S and Zheng, F and Yao, J and Jin, Q and Zhang, Y and Luo, H and Zhang, X and He, Y and Zhou, M and Gu, M and Mao, C and Wang, Z},
title = {Improving low-phosphate tolerance via tissue-specific CRISPR/Cas9 knockout to balance growth and stress responses in rice.},
journal = {The Plant cell},
volume = {38},
number = {6},
pages = {},
doi = {10.1093/plcell/koag170},
pmid = {42258637},
issn = {1532-298X},
support = {2021YFF1000402//National Key Research and Development Program of China/ ; LR24C150001//Natural Science Foundation of Zhejiang Province, China/ ; 92581113//National Natural Science Foundation of China/ ; 32170262//National Natural Science Foundation of China/ ; 226-2024-00102//Fundamental Research Funds for the Central University/ ; },
mesh = {*Oryza/genetics/physiology/growth & development/metabolism ; *Phosphates/metabolism/deficiency ; *CRISPR-Cas Systems/genetics ; Stress, Physiological/genetics ; Gene Expression Regulation, Plant ; Plants, Genetically Modified ; Gene Knockout Techniques ; Plant Proteins/genetics/metabolism ; Plant Roots/genetics/metabolism ; },
abstract = {Balancing growth and stress responses is critical for improving crop stress tolerance. Inorganic phosphate (Pi) deficiency reduces agricultural yields. Plants have evolved a Pi-starvation response (PSR) network that coordinates growth and responds to fluctuating environmental Pi levels. Null mutations or whole-plant knockdown of PSR repressor genes, such as PHOSPHATE2 (OsPHO2) and OsSPX (Syg1, Pho81, XPR1) family genes, enhance Pi absorption and transfer but disrupt Pi homeostasis, inhibiting growth and reducing yields. To overcome this, we developed a CRISPR/Cas9 tissue-specific knockout (TSKO) system for efficient, vascular-specific somatic knockout of OsPHO2 in rice (Oryza sativa) cv. "Nipponbare" across several generations. The plants showed moderately increased Pi concentrations, maintained Pi homeostasis in hydroponic culture, and increased effective tiller number and grain yield in a Pi-deficient paddy. Vascular-specific OsPHO2 knockout moderately increased OsPHO2-repressed, vascular-expressed, Pi-starvation-induced signaling in roots and alleviated disordered PSR in roots and leaves. Vascular-specific knockout of OsPHO2 or OsSPX1/2 in the Zhonghua 11 background gave similar results. Field trials confirmed the enhanced low-Pi tolerance of TSKO plants in a Pi-deficient paddy and these plants showed normal growth in a Pi-sufficient paddy. This highlights the utility of improving rice low-Pi tolerance via a tissue-specific CRISPR/Cas9 knockout, provides insights into the role of vascular tissues in PSR, and offers a promising spatial-targeting strategy for crop improvement.},
}

*Oryza/genetics/physiology/growth & development/metabolism
*Phosphates/metabolism/deficiency
*CRISPR-Cas Systems/genetics
Stress, Physiological/genetics
Gene Expression Regulation, Plant
Plants, Genetically Modified
Gene Knockout Techniques
Plant Proteins/genetics/metabolism
Plant Roots/genetics/metabolism

@article {pmid42347182,
year = {2026},
author = {Feng, C and Yin, J},
title = {Advances in CRISPR-Cas for Diagnosis and Treatment of Klebsiella pneumoniae.},
journal = {Pathogens (Basel, Switzerland)},
volume = {15},
number = {6},
pages = {},
pmid = {42347182},
issn = {2076-0817},
mesh = {*Klebsiella pneumoniae/genetics/drug effects/isolation & purification ; Humans ; *CRISPR-Cas Systems ; *Klebsiella Infections/diagnosis/therapy/drug therapy/microbiology ; Anti-Bacterial Agents/pharmacology/therapeutic use ; Gene Editing/methods ; Drug Resistance, Bacterial ; },
abstract = {Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a significant pathogen for both hospital-acquired and community-acquired infections, characterized by its strong epidemic potential and high mortality rate, posing a severe threat to global public health. CRKP spreads widely across the globe through the horizontal transfer of plasmid-mediated resistance genes such as *blaKPC*, *blaNDM*, and *blaOXA-48*. The clinical treatment options for this bacterium are limited, and its resistance has been increasing year by year, urgently necessitating the development of new antimicrobial drugs or alternative strategies. In recent years, the CRISPR-Cas system has shown great potential in the diagnosis and treatment of CRKP, including rapid detection and identification, gene editing, antimicrobial strategies, and resistance inhibition. For instance, CRISPR-Cas12a/13a can be used for the rapid detection and identification of CRKP, while CRISPR-Cas9/Cas3 can target resistance genes to reverse the resistance of strains. With the advancement of delivery and biotechnologies, the CRISPR-Cas system is expected to become an important tool against drug-resistant CRKP. This review focuses on the application of the CRISPR-Cas system in the detection and treatment of CRKP, analyzing its technical advantages, limitations, and future development directions.},
}

*Klebsiella pneumoniae/genetics/drug effects/isolation & purification
Humans
*CRISPR-Cas Systems
*Klebsiella Infections/diagnosis/therapy/drug therapy/microbiology
Anti-Bacterial Agents/pharmacology/therapeutic use
Gene Editing/methods
Drug Resistance, Bacterial

@article {pmid42348283,
year = {2026},
author = {Ma, W and Shi, S and Ding, G and Ye, X and Wang, S and Xu, F},
title = {CRISPR/Cas9-mediated mutation of BnaA5.JAR1 alleviates boron deficiency stress by enhancing calcium-pectin cross-linking in rapeseed.},
journal = {The Plant journal : for cell and molecular biology},
volume = {126},
number = {6},
pages = {e71006},
doi = {10.1111/tpj.71006},
pmid = {42348283},
issn = {1365-313X},
support = {32372805//National Natural Science Foundation of China/ ; 31972483//National Natural Science Foundation of China/ ; 2022YFD1900705//National Key Research and Development Program of China/ ; },
mesh = {*Pectins/metabolism ; *Boron/deficiency/metabolism ; *Calcium/metabolism ; *Brassica napus/genetics/metabolism/physiology ; CRISPR-Cas Systems ; Cell Wall/metabolism ; Cyclopentanes/metabolism ; *Plant Proteins/genetics/metabolism ; Mutation ; Oxylipins/metabolism ; Gene Expression Regulation, Plant ; },
abstract = {Boron (B) is an essential micronutrient critical for plant growth and reproductive development, primarily through its role in the cell wall. Calcium (Ca[2+]) similarly stabilizes cell wall architecture by forming cross-links with de-esterified pectin. In Arabidopsis thaliana, B deficiency rapidly induces JASMO0NATE RESISTANT 1 (JAR1) and jasmonic acid (JA) accumulation, which negatively regulates growth. However, whether and how JAR1-mediated JA signaling modulates cell wall B and Ca[2+] partitioning to confer B-deficiency tolerance remains unclear. Here, we characterized the function of BnaA5.JAR1 in rapeseed (Brassica napus L.) under B deficiency. Pharmacological inhibition of JA biosynthesis with ibuprofen partially alleviated B-deficiency-induced shoot growth inhibition. BnaA5.JAR1 transcript levels were rapidly and strongly induced by B deprivation. CRISPR/Cas9-mediated knockout of BnaA5.JAR1 enhanced tolerance to B deficiency, whereas overexpression increased sensitivity, despite unchanged leaf B concentrations. Notably, mutant lines maintained robust tolerance throughout the reproductive stage, effectively rescuing the 'flowering without seed setting' phenotype characteristic of B deficiency. Under B limitation, these knockout lines retained higher B and Ca[2+] concentrations in alkali-soluble pectin, exhibited a lower degree of pectin methylesterification, and maintained thinner, more structurally normal cell walls compared with overexpression lines. Exogenous Ca[2+] mitigated B deficiency symptoms without increasing leaf B concentration. Collectively, these findings establish BnaA5.JAR1 as a negative regulator of B-deficiency tolerance and demonstrate that suppressing its activity enhances Ca[2+]-mediated cell wall stabilization across vegetative and reproductive stages, offering a targeted molecular strategy for breeding B-efficient rapeseed cultivars.},
}

*Pectins/metabolism
*Boron/deficiency/metabolism
*Calcium/metabolism
*Brassica napus/genetics/metabolism/physiology
CRISPR-Cas Systems
Cell Wall/metabolism
Cyclopentanes/metabolism
*Plant Proteins/genetics/metabolism
Mutation
Oxylipins/metabolism
Gene Expression Regulation, Plant

@article {pmid42348614,
year = {2026},
author = {Lee, S and Song, J and Kim, Y},
title = {Broad-spectrum antifungal activity and genome-guided characterization of Paenibacillus polymyxa CACC1094 isolated from the bovine rumen.},
journal = {PloS one},
volume = {21},
number = {6},
pages = {e0350885},
pmid = {42348614},
issn = {1932-6203},
mesh = {Animals ; Cattle ; *Antifungal Agents/pharmacology/metabolism ; *Paenibacillus polymyxa/genetics/isolation & purification/metabolism ; *Rumen/microbiology ; Phylogeny ; *Genome, Bacterial ; Multigene Family ; },
abstract = {While Paenibacillus polymyxa is widely recognized for its biocontrol capabilities, most characterized strains originate from soil or rhizosphere environments, leaving animal-associated populations largely unexplored. In this study, we report the isolation of P. polymyxa strain CACC1094 from the bovine rumen and its genome-guided characterization to investigate its biosynthetic potential and antifungal activity. Whole-genome sequencing yielded a complete circular chromosome of 5.55 Mb with a GC content of 45.36%, comprising 5,099 coding sequences, 39 rRNA genes, and 111 tRNA genes. Comparative phylogenomic analysis placed CACC1094 within the P. polymyxa species complex, clustering most closely with the rumen-associated strain ND24 (ANI: 98.31%) and strain 188 (ANI: 96.92%), while forming a distinct branch within the species. Genome mining identified 13 biosynthetic gene clusters, including those associated with fusaricidin and tridecaptin biosynthesis, a paenicidin-like lanthipeptide cluster, and a hybrid NRPS-PKS cluster. In dual-culture assays, CACC1094 showed broad in vitro antifungal activity against multiple plant-pathogenic fungi and oomycetes, as well as selected fungal and yeast pathogens of clinical and veterinary relevance. LC-QTOF/MS analysis of culture supernatants confirmed the production of fusaricidin A and fusaricidin B, providing direct experimental validation of genome-derived predictions. Additionally, genome annotation revealed a complete CRISPR-Cas adaptive immune system, suggesting an integrated ecological strategy that combines antimicrobial biosynthesis with defense against mobile genetic elements. Together, these findings identify CACC1094 as a rumen-associated P. polymyxa strain with broad-spectrum antifungal activity and experimentally validated fusaricidin production, highlighting its potential as a source of antifungal metabolites for agricultural and veterinary applications.},
}

Animals
Cattle
*Antifungal Agents/pharmacology/metabolism
*Paenibacillus polymyxa/genetics/isolation & purification/metabolism
*Rumen/microbiology
Phylogeny
*Genome, Bacterial
Multigene Family

@article {pmid41944382,
year = {2026},
author = {Guan, X and Guo, C and Zhang, J and Yang, R and Ramachandra, Y and Hou, C and Pei, M and Zhang, S and Schalper, KT and Liu, X and Wu, Q and Bulsara, KR and Liu, C},
title = {SDS-CRISPR for Single-Nucleotide Variant Detection.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {36},
pages = {e75149},
doi = {10.1002/advs.75149},
pmid = {41944382},
issn = {2198-3844},
support = {U01AI148306/GF/NIH HHS/United States ; R01AI194917/GF/NIH HHS/United States ; //UConn Research Excellence Program award/ ; },
mesh = {Humans ; *Polymorphism, Single Nucleotide/genetics ; *CRISPR-Cas Systems/genetics ; *Glioma/genetics/diagnosis ; Isocitrate Dehydrogenase/genetics ; High-Throughput Nucleotide Sequencing/methods ; Mutation/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {The CRISPR-Cas12a system offers a promising platform for simple and sensitive nucleic acid diagnostics, including tumor-associated variant detection and infectious agent identification. However, its intrinsic mismatch tolerance limits its ability to accurately detect single-nucleotide variants (SNVs). Here, we introduce Structure-Disruption-Sensitive CRISPR (SDS-CRISPR), a programmable CRISPR-Cas12a approach that achieves highly precise allele discrimination. Guided by AlphaFold3 modeling and bioinformatic analysis, we uncover how split structural design and ionic modulation reconfigure Cas12a conformations, elucidating the structural basis of SNV discrimination in SDS-CRISPR. We apply SDS-CRISPR to detect IDH1[WT] and IDH1[R132H] alleles with attomole sensitivity and 0.01% variant frequency. To facilitate intraoperative use, we combine SDS-CRISPR with a lateral-flow strip and an artificial intelligence-assisted smartphone reader, enabling on-site detection within 20 min. Clinical validation with 43 glioma tissue samples shows high concordance with immunohistochemistry, while plasma cfDNA testing demonstrates mutation fractions consistent with next-generation sequencing. Beyond glioma, SDS-CRISPR generalizes across molecular targets, discriminating microRNA isoforms and identifying HIV-1 drug-resistance mutations. Together, these results establish SDS-CRISPR as a universal, mechanistically informed, and clinically actionable framework for precision molecular diagnostics.},
}

Humans
*Polymorphism, Single Nucleotide/genetics
*CRISPR-Cas Systems/genetics
*Glioma/genetics/diagnosis
Isocitrate Dehydrogenase/genetics
High-Throughput Nucleotide Sequencing/methods
Mutation/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid42069060,
year = {2026},
author = {Cobos-Figueroa, L and Pintor-Poveda, A and Mir, C and Melamed, D and Admon, A and Lauzurica, P and Lorente, E},
title = {Innovative CRISPR/Cas9-Based Strategy for Allele-Specific HLA Peptidome Analysis Using a Pan-HLA Antibody.},
journal = {Molecular & cellular proteomics : MCP},
volume = {25},
number = {6},
pages = {101578},
pmid = {42069060},
issn = {1535-9484},
mesh = {Humans ; Alleles ; *CRISPR-Cas Systems/genetics ; *Peptides/metabolism ; *Antibodies/metabolism/immunology ; *Proteomics/methods ; Tandem Mass Spectrometry ; Cell Line ; *HLA Antigens/genetics/metabolism ; Proteome/metabolism ; },
abstract = {Human leukocyte antigen (HLA) immunopeptidomics is restricted by the limited availability of allele-specific antibodies and by potential artifacts introduced by HLA overexpression systems. To address these challenges, we developed a CRISPR/Cas9-based strategy that selectively deletes undesired classical class I alleles while preserving a single endogenous allele, thereby enabling allele-resolved peptidome profiling with a pan-HLA class I antibody. As a proof of concept, we edited JY cells to eliminate HLA-B∗07:02 and HLA-C∗07:02 while retaining HLA-A∗02:01 (ΔBC clones). Peptide-HLA complexes were immunoprecipitated from WT and ΔBC clones using either the pan-HLA class I antibody W6/32 or the A∗02:01-specific antibody PA2.1, followed by nanoLC-MS/MS and computational HLA assignment. Deletion of HLA-B and HLA-C alleles caused an expected ∼55% reduction in total class I surface expression. Despite this, W6/32 immunoprecipitation from ΔBC clones recovered a comparable peptide yield to PA2.1 in WT cells. Binding predictions showed that most peptides identified in ΔBC clones using W6/32 were assigned to HLA-A∗02:01, with near-complete loss of HLA-B∗07:02- and HLA-C∗07:02-derived peptides. Sequence logo analysis confirmed the canonical A∗02:01 motif across conditions. The ΔBC W6/32 immunopeptidome exhibited a high degree of overlap (∼88%) with the WT PA2.1 repertoire, supporting the specificity and fidelity of the approach. These findings establish CRISPR-based editing of HLA alleles as a viable strategy for allele-specific immunopeptidome analysis using pan-HLA antibodies, supporting its potential application beyond this proof-of-concept system, reducing reliance on allele-specific reagents and facilitating the study of underrepresented HLA alleles.},
}

Humans
Alleles
*CRISPR-Cas Systems/genetics
*Peptides/metabolism
*Antibodies/metabolism/immunology
*Proteomics/methods
Tandem Mass Spectrometry
Cell Line
*HLA Antigens/genetics/metabolism
Proteome/metabolism

@article {pmid42148916,
year = {2026},
author = {Zhou, S and Gu, T and Deng, L and Wang, X and Zhu, S and He, X and Yang, M and Huo, D and Hou, C},
title = {One-step digestion-ligation-activation universal strategy for ultrasensitive detection of DNA methylation.},
journal = {The Analyst},
volume = {151},
number = {13},
pages = {3744-3754},
doi = {10.1039/d5an01270h},
pmid = {42148916},
issn = {1364-5528},
mesh = {*DNA Methylation ; Humans ; *Nucleic Acid Amplification Techniques/methods ; CRISPR-Cas Systems ; Limit of Detection ; CpG Islands ; DNA Restriction Enzymes/metabolism ; DNA/chemistry/genetics ; },
abstract = {DNA methylation and its associated methyltransferases play pivotal roles in epigenetic regulation and are regarded as important biomarkers for early cancer diagnosis; however, the development of a universal platform capable of sensitively detecting both targets remains challenging. Herein, we report a one-step digestion-ligation-activation universal strategy integrating rolling circle extension-assisted loop-mediated isothermal amplification with CRISPR/Cas12a (DL-RLAMP/Cas12a) for ultrasensitive methylation analysis. In this design, a padlock probe hybridizes at CpG sites and undergoes simultaneous digestion and ligation, enabling methylation-dependent circular DNA formation. The use of methylation-sensitive restriction endonucleases (HhaI/BstUI) ensures selective survival of methylated or methyltransferase-modified targets, which subsequently initiate RLAMP amplification to generate abundant double stem-loop DNA structures for Cas12a-mediated trans-cleavage signal amplification. By integrating multistep reactions into a single workflow, the proposed strategy achieves efficient cascade amplification while maintaining an ultralow background signal. The assay discriminates methylation levels down to 0.1% and enables sensitive detection of CpG methyltransferase (M.SssI) with a detection limit of 6.92 × 10[-4] U mL[-1]. Furthermore, the platform demonstrates applicability in cellular methylation analysis and methyltransferase inhibitor screening. This DL-RLAMP/Cas12a system provides a versatile and highly sensitive analytical framework for epigenetic biomarker detection and holds promise for early disease diagnostics.},
}

*DNA Methylation
Humans
*Nucleic Acid Amplification Techniques/methods
CRISPR-Cas Systems
Limit of Detection
CpG Islands
DNA Restriction Enzymes/metabolism
DNA/chemistry/genetics

@article {pmid42214710,
year = {2026},
author = {Yang, X and Sang, R and Hutvagner, G and Hewitt, AW and Li, D and Li, Y and Deng, W},
title = {An optimised lipid nanoparticle platform enables efficient CRISPR/Cas9 genome editing in hard-to-transfect cells.},
journal = {Acta biomaterialia},
volume = {218},
number = {},
pages = {432-444},
doi = {10.1016/j.actbio.2026.05.044},
pmid = {42214710},
issn = {1878-7568},
mesh = {Humans ; *Lipids/chemistry ; *CRISPR-Cas Systems/genetics ; *Nanoparticles/chemistry ; HEK293 Cells ; *Gene Editing/methods ; *Transfection/methods ; Cell Line, Tumor ; Liposomes ; },
abstract = {CRISPR/Cas9 is a powerful tool for genome editing and functional gene studies, but its therapeutic potential is often hampered by inefficient transfection, particularly in hard-to-modify cell types. In this study, we developed and optimised a lipid nanoparticles (LNPs) platform that enhances CRISPR/Cas9-mediated genome editing across diverse cell types, including those that are difficult to modify using commercially available lipid-based delivery agents. Our engineered LNPs exhibit consistent particle size below 100 nm, low polydispersity and high encapsulation efficiency. Using this platform, GFP knockout in HEK-293 cells reached 78.7%, and maintained consistent efficiency after lyophilisation and reconstitution. Knockout of the LCN2 gene in MDA-MB-231 cells resulted in a 90.8% reduction in mRNA expression, outperforming the 51.1% reduction achieved using CRISPRMAX Lipofectamine, and functional assays confirmed that LCN2 disruption significantly inhibited cell proliferation and migration. Co-delivery of CRISPR/Cas9 and a GFP HDR template enabled precise knock-in, achieving >20% efficiency in HEK-293 cells and >8% in MSCs and DC2.4 cells, significantly outperforming Lipofectamine 3000 transfection reagent. Given the rapid expansion of CRISPR applications in biomedical research, our LNP-based delivery system represents a promising non-viral platform with broad potential for therapeutic applications, particularly in hard-to-modify cell types. STATEMENT OF SIGNIFICANCE: CRISPR/Cas9 is a transformative gene-editing technology for functional genomics and therapeutic development; however, its widespread application is constrained by the lack of safe and efficient delivery systems, particularly for hard-to-transfect cell types. Lipid nanoparticles (LNPs) represent a promising non-viral delivery strategy, yet their transfection efficiency varies substantially across cell lines due to differences in cellular membrane properties. Here, we developed and optimized an LNP platform that enables highly efficient Cas9/sgRNA-mediated gene knockout and knock-in across multiple challenging cell types, consistently outperforming commercial transfection reagents. The findings establish this LNP system as a versatile and effective non-viral platform for CRISPR/Cas9-mediated genome editing, overcoming key limitations of existing commercial lipid-based delivery agents in hard-to-modify cell types and offering a promising strategy for future clinical translation.},
}

Humans
*Lipids/chemistry
*CRISPR-Cas Systems/genetics
*Nanoparticles/chemistry
HEK293 Cells
*Gene Editing/methods
*Transfection/methods
Cell Line, Tumor
Liposomes

@article {pmid42344907,
year = {2026},
author = {Oh, JH and Yang, LF and Tanaka, E and de Brito Monteiro, L and Wi, D and Archambault, AS and Klein Geltink, RI},
title = {Versatile electroporation protocols enable reproducible CRISPR-RNP delivery across multiple primary mouse cells of the hematopoietic lineage.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1820963},
pmid = {42344907},
issn = {1664-3224},
mesh = {Animals ; *Electroporation/methods ; Mice ; *CRISPR-Cas Systems ; *Hematopoietic Stem Cells/metabolism ; *Ribonucleoproteins/genetics ; Macrophages/metabolism ; Cell Lineage ; *Gene Editing/methods ; CD8-Positive T-Lymphocytes/metabolism ; Gene Transfer Techniques ; Mice, Inbred C57BL ; Cell Differentiation ; Electroporation Therapies ; Cells, Cultured ; },
abstract = {Genetic engineering of primary hematopoietic cells is essential for mechanistic immunology studies, however the development of cell-based therapies yet remains constrained by two major factors: the fragility of many primary lineages and challenges of viral delivery platforms that are costly, time-intensive, and biologically confounding. Here, we optimized scalable CRISPR-Cas9 electroporation workflows using the ExPERT platform across three primary mouse hematopoietic cell types: OT-I CD8[+] T cells, bone marrow-derived macrophages (BMDMs), and hematopoietic stem/progenitor cells (HSPCs). In activated OT-I CD8[+] T cells, two electroporation programs supported high-efficiency mRNA and RNP delivery with minimal impact on cell viability or proliferative capacity, with subtle activation-state-dependent sensitivity at higher energy settings. Extending optimization to myeloid and stem cell lineages, we found that BMDMs maintained high viability following electroporation, and a high-performing electroporation program supported robust RNP delivery and efficient target gene knockout while preserving macrophage differentiation. In HSPCs, the same program enabled consistent RNP delivery, sustained viability, and reproducible gene knockout. Together, these findings establish ExPERT electroporation as a robust, reproducible, and modular platform for genome editing across primary mouse hematopoietic lineages, lowering barriers to rapid genetic perturbation for both discovery and translational applications. This manuscript has been posted on BioRxiv (https://www.biorxiv.org/content/10.64898/2026.01.27.702081v1).},
}

Animals
*Electroporation/methods
Mice
*CRISPR-Cas Systems
*Hematopoietic Stem Cells/metabolism
*Ribonucleoproteins/genetics
Macrophages/metabolism
Cell Lineage
*Gene Editing/methods
CD8-Positive T-Lymphocytes/metabolism
Gene Transfer Techniques
Mice, Inbred C57BL
Cell Differentiation
Electroporation Therapies
Cells, Cultured

@article {pmid42345443,
year = {2026},
author = {Fiori, S and Adragna, C and Malvicini, E and Basini, T and Galgano, D and Scarpa, E and Jovic, S and Schmacke, NA and Hornung, V and Sallusto, F and Bruno, L and Lanzavecchia, A and Albanese, M},
title = {CRISPR/Cas9-Mediated Gene Knockout Reveals a Nonredundant Role for p16[INK4A] in Controlling TCR-Dependent and Independent CD8 T Cell Expansion.},
journal = {European journal of immunology},
volume = {56},
number = {6},
pages = {e70224},
pmid = {42345443},
issn = {1521-4141},
support = {//Joachim Herz Foundation/ ; 885539/ERC_/European Research Council/International ; 28809//MFAG AIRC/ ; },
mesh = {Humans ; *Cyclin-Dependent Kinase Inhibitor p16/genetics/immunology ; *CRISPR-Cas Systems ; *CD8-Positive T-Lymphocytes/immunology ; Cell Proliferation/genetics ; *Receptors, Antigen, T-Cell/immunology/metabolism ; Gene Knockout Techniques ; Lymphocyte Activation/immunology ; },
abstract = {The possibility of enhancing T cell function by deleting specific genes represents a long-sought goal in preclinical studies and ultimately for clinical applications. Using CRISPR/Cas9 genome editing, we report that, in human cytotoxic CD8 T cell clones, the cell cycle checkpoint gene CDKN2A, encoding p16[INK4A], plays a nonredundant role in controlling T cell receptor (TCR)-dependent and independent cell expansion. Deletion of CDKN2A dramatically enhanced antigen-driven and homeostatic proliferation, while preserving effector functions. In contrast, the deletion of other cell cycle inhibitors (CDKN1B, CDKN2C, and CDKN2D), alone or in combination, had no impact on T cell proliferation. We also report that mediator complex subunit 12 (MED12) and the E3 ubiquitin ligase CBL-B deletions did not affect proliferative capacity of CD8 T cell clones. Interestingly, deletion of the negative regulator of Ras signaling, RASA2, increased antigen sensitivity and cytotoxic activity, while not improving in vitro expansion. Collectively, these findings reveal a unique and critical nonredundant role for p16[INK4A] in regulating CD8 T cells. Deletion of CDKN2A offers a promising strategy to enhance CD8 T cell expansion ex vivo, thereby improving TCR discovery pipelines and, potentially, therapeutic applications.},
}

Humans
*Cyclin-Dependent Kinase Inhibitor p16/genetics/immunology
*CRISPR-Cas Systems
*CD8-Positive T-Lymphocytes/immunology
Cell Proliferation/genetics
*Receptors, Antigen, T-Cell/immunology/metabolism
Gene Knockout Techniques
Lymphocyte Activation/immunology

@article {pmid42345496,
year = {2026},
author = {Schubert, AJ and Meng, Q and Hoffmann, J and Rau, J and Abele, F and Greensmith, R and Cordero, C and Ibel, A and Mandler, JM and Eckardt, KU and Halbritter, J and Dighe, AS and Tan, X and Hwang, DY and Petzold, T and Enghard, P and Kaminski, MM},
title = {CRISPR-Based Assay for Point-of-Care Pharmacogenetic CYP2C19 Genotyping.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.6c00841},
pmid = {42345496},
issn = {2379-3694},
abstract = {Pharmacogenetic testing enables personalized drug dosing by accounting for genetic variation in drug metabolism. Although there is increasing evidence that genetically guided dosing improves therapeutic efficacy and reduces adverse effects, clinical implementation remains limited as genotyping methods suffer from slow turnaround times and are not optimized for point-of-care use. Here, we present a rapid, multiplexed CRISPR-based assay for genotyping the key CYP2C19 polymorphisms *2, *3, and *17. These variants can alter enzyme activity, impacting the metabolism of drugs such as clopidogrel and mavacamten. Our assay combines isothermal amplification with a dual guide RNA detection strategy, where LwaCas13a selectively detects mutant alleles and LbaCas12a identifies wild-type alleles. In a validation study of 110 participants, the assay showed high concordance with Sanger sequencing, the current gold standard, with variant-specific accuracies of 97.3% (*2), 100% (*3), and 99.1% (*17), demonstrating robust performance across a diverse clinical cohort. The workflow supports both column-based DNA extraction and a simplified, crude extraction protocol. Genotyping results are obtained through either fluorescence detection or multi-analyte lateral-flow readouts, allowing visual interpretation of genotypes with minimal equipment. In conclusion, our findings show that CRISPR-based genotyping can deliver accurate CYP2C19 pharmacogenetic testing, facilitating broader adoption of genotype-guided drug dosing. Furthermore, the programmability of CRISPR-Cas systems enables adaptation to other pharmacogenetic targets relevant to drug metabolism.},
}

@article {pmid42345883,
year = {2026},
author = {Li, X and Han, X and Han, Q and He, X and Huang, Y and Liu, A},
title = {Advancements in Nanomaterial-Based Biosensors for Neuropsychiatric and Neurodegenerative Diagnostics: From Biomarker Discovery to Clinical Translation.},
journal = {Biosensors},
volume = {16},
number = {6},
pages = {},
pmid = {42345883},
issn = {2079-6374},
support = {82401622//National Natural Science Foundation of China/ ; ZR2023QH131//Natural Science Foundation of Shandong Province/ ; },
mesh = {*Biosensing Techniques ; Humans ; Biomarkers/analysis ; *Neurodegenerative Diseases/diagnosis ; *Nanostructures ; *Mental Disorders/diagnosis ; },
abstract = {Nanobiosensors, with their unique physicochemical properties, are transformative tools for diagnosing and monitoring neurodegenerative diseases and mental disorders. This article systematically reviews the latest progress of nanomaterial systems and integrated sensing modalities in neurological disease diagnosis. First, we clarify the multiple functional roles of nanomaterials in biosensors, including signal amplification, interface optimization, and spatial positioning, and compare the applicable scenarios of various sensing principles based on different nanomaterials. Second, we evaluate the design and integration strategies of molecular recognition elements (antibodies, nucleic acid aptamers, molecularly imprinted polymers, and CRISPR-Cas systems) and discuss their synergistic integration mechanisms for improving detection performance. In terms of detection targets, we focus on three applications: high-sensitivity quantification of established protein biomarkers, real-time monitoring of dynamic neurochemicals (dopamine, serotonin, glutamate), and emerging liquid biopsy targets such as exosomal cargo and circulating microRNAs. Finally, to address the core challenges of biofouling, sensitivity-selectivity trade-offs, and multiplex detection in complex matrices, we propose three breakthrough directions for next-generation diagnostics: deep integration of multimodal and multiplexing platforms, closed-loop chemical brain-computer interfaces (cBCIs), and AI-driven predictive diagnostic models, collectively enabling a transition from passive detection to active sensing and intervention for precise, rapid, and non-invasive neurological disease management.},
}

*Biosensing Techniques
Humans
Biomarkers/analysis
*Neurodegenerative Diseases/diagnosis
*Nanostructures
*Mental Disorders/diagnosis

@article {pmid42345886,
year = {2026},
author = {Hong, S and Park, CS and Been, KW and Kang, S and Hong, J and Kim, JW and Hur, JK},
title = {Sequential CRISPR-EspCas9-Mediated Wild-Type Depletion Enhances the Detection Sensitivity of Rare Mutations for Canine Liquid Biopsy Application.},
journal = {Biosensors},
volume = {16},
number = {6},
pages = {},
pmid = {42345886},
issn = {2079-6374},
support = {RS-2023-NR076663//National Research Foundation of Korea/ ; RS-2021-NR056589//National Research Foundation of Korea/ ; RS-2023-00261114//National Research Foundation of Korea/ ; RS-2025-02218918//National Research Foundation of Korea/ ; RS-2024-00468036//National Research Foundation of Korea/ ; 2023R1A6C101A009//Korea Basic Science Institute/ ; },
mesh = {Animals ; Dogs ; Female ; Cell Line, Tumor ; *CRISPR-Cas Systems ; High-Throughput Nucleotide Sequencing ; *Liquid Biopsy/veterinary ; Mutation ; },
abstract = {One of the major obstacles in early cancer detection in dogs is the limited sensitivity in detecting circulating tumor DNAs (ctDNAs) with low abundances. Standard next-generation sequencing (NGS) without error correction typically achieves detection limits around ~1% mutant allele frequency (MAF). We sought to improve the detection sensitivity using a sequential CRISPR-EspCas9 enrichment strategy in which iterative in vitro cleavage (IVC) was combined with PCR amplification to selectively deplete wild-type DNA and enrich rare tumor mutations. Applying the strategy to genomic DNA and cell-free DNA mimics from canine mammary gland tumor cell lines demonstrated that IVC enrichment enabled the detection of cancer-associated PIK3CA H1047R mutations that were undetectable by conventional Sanger sequencing. To evaluate detection sensitivity, we characterized enrichment using synthetic templates for PIK3CA H1047R and other cancer-related mutations, BRAF V596E, and KRAS G12C. We observed that three iterations of sequential IVC achieved ~160, ~15, and ~2.2-fold enrichment for PIK3CA H1047R, BRAF V596E, and KRAS G12C, respectively. Under the present synthetic-template conditions, the analytical LOD reached 0.001% MAF for PIK3CA and 0.01% MAF for BRAF, whereas KRAS showed only modest enrichment and remained practically limited under the current guide design. Together, the results show that the CRISPR-EspCas9 IVC strategy enables selective enrichment of low-frequency single-nucleotide mutant alleles. We anticipate that the finding could be utilized to develop a highly sensitive veterinary liquid biopsy application with further optimization and validation using canine plasma cfDNA.},
}

Animals
Dogs
Female
Cell Line, Tumor
*CRISPR-Cas Systems
High-Throughput Nucleotide Sequencing
*Liquid Biopsy/veterinary
Mutation

@article {pmid42346156,
year = {2026},
author = {Rimskaya, B and Kropocheva, E and Ponomareva, I and Karchemkina, L and Lisitskaya, L and Gelfenbein, D and Ulashchik, E and Shmanai, V and Kulbachinskiy, A and Mazunin, I},
title = {Activity of DNA- and RNA-Guided Prokaryotic Argonautes in Human Mitochondria.},
journal = {Cells},
volume = {15},
number = {12},
pages = {},
pmid = {42346156},
issn = {2073-4409},
support = {25-24-00078//Russian Science Foundation/ ; },
mesh = {Humans ; *Mitochondria/metabolism/genetics ; *DNA, Mitochondrial/metabolism/genetics ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; Neurospora crassa/genetics ; CRISPR-Cas Systems ; },
abstract = {Precise manipulation of mitochondrial DNA (mtDNA) by CRISPR-Cas systems remains challenging, largely due to inefficient import of guide RNAs, motivating the exploration of alternative programmable nucleases. Here, we show that prokaryotic Argonaute nucleases (pAgos) of various classes can be efficiently targeted to human mitochondria. Using the Su9 mitochondrial targeting sequence from Neurospora crassa, we achieved robust mitochondrial import of four pAgos-DecAgo, CbuAgo, KmaAgo and RslAgo. As a functional readout of their activity in cells, we targeted the single-stranded D-loop region, which plays a central role in mtDNA replication and maintenance, reasoning that cleavage at this site was expected to potentially result in a reduction in mtDNA copy number. Of the four enzymes, only RNA-guided DecAgo induced a pronounced reduction in mtDNA levels, decreasing copy number approximately fivefold within 48 h. Unexpectedly, this effect occurred independently of exogenous guides, suggesting that DecAgo may utilize endogenous mitochondrial guide RNAs. These findings identify DecAgo as an active nuclease in human mitochondria and reveal a previously unrecognized mode of targeting, highlighting the need to further investigate the underlying mechanism and the potential role of endogenous guide molecules, as well as improving targeting specificity.},
}

Humans
*Mitochondria/metabolism/genetics
*DNA, Mitochondrial/metabolism/genetics
*RNA, Guide, CRISPR-Cas Systems/metabolism/genetics
Neurospora crassa/genetics
CRISPR-Cas Systems

@article {pmid42347171,
year = {2026},
author = {Syrym, N and Yespembetov, B and Kokanov, S and Nakhanov, A and Bulatov, Y and Abdimukhtar, A and Toleukhan, A and Serikbay, Y and Terebay, A and Anarbekova, A and Tileukhanov, K and Alpysbayeva, S and Sarmykova, M and Yerzhigit, B and Zinina, N and Suleimenov, M and Abdykalyk, A},
title = {Bacteriophage-Based Therapeutics for Bacterial Sexually Transmitted Infections: From Biological Barriers to Translational Strategies.},
journal = {Pathogens (Basel, Switzerland)},
volume = {15},
number = {6},
pages = {},
pmid = {42347171},
issn = {2076-0817},
support = {AP23489672//Ministry of Science and Higher Education of the Republic of Kazakhstan/ ; },
mesh = {Humans ; *Bacteriophages/physiology/genetics ; *Phage Therapy/methods ; *Sexually Transmitted Diseases, Bacterial/therapy/microbiology ; Biofilms ; Anti-Bacterial Agents/therapeutic use ; },
abstract = {Bacterial sexually transmitted and sexually associated infections remain a major global health concern, increasingly complicated by antimicrobial resistance and the limited effectiveness of existing therapies. In this context, bacteriophage-based and phage-derived approaches have re-emerged as potential alternative antibacterial strategies. This narrative review examines their applicability across key bacterial pathogens associated with sexually transmitted infections, including Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Treponema pallidum and biofilm-associated bacterial vaginosis, with a particular focus on pathogen-specific biological barriers. Available evidence indicates that the success of phage-based interventions is strongly dependent on factors such as intracellular localisation, structural characteristics of the bacterial envelope and the presence of polymicrobial biofilms. While phage-derived platforms, including endolysins, depolymerases and engineered phages, demonstrate antibacterial activity in experimental settings, their effectiveness is uneven across different pathogens. Biofilm-associated infections appear more accessible to these approaches, whereas intracellular and structurally atypical bacteria are currently considered more challenging targets based on available mechanistic and experimental evidence. These observations highlight the need for pathogen-specific engineering strategies and delivery systems. Overall, phage-based therapeutics in this field should be considered within a framework that integrates biological constraints with targeted antimicrobial design.},
}

Humans
*Bacteriophages/physiology/genetics
*Phage Therapy/methods
*Sexually Transmitted Diseases, Bacterial/therapy/microbiology
Biofilms
Anti-Bacterial Agents/therapeutic use

@article {pmid41793486,
year = {2026},
author = {Wu, J and Zhang, Y and Guo, H and Zhang, Y and Zhang, Y},
title = {CRISPR/Cas9 knockout of DDX5 facilitates foot-and-mouth disease virus replication in PK-15 cells.},
journal = {Archives of virology},
volume = {171},
number = {4},
pages = {},
pmid = {41793486},
issn = {1432-8798},
support = {23JRRA551, 24JRRA012//the Natural Science Foundation of Gansu Province/ ; 2023M733819, 2024M763620//China Postdoctoral Science Foundation Funded Project/ ; 24CXNA030//Technology innovation guidance program of Gansu Province/ ; },
mesh = {*Foot-and-Mouth Disease Virus/physiology/genetics ; *Virus Replication ; Animals ; Cell Line ; *CRISPR-Cas Systems ; *DEAD-box RNA Helicases/genetics/metabolism ; Swine ; Gene Knockout Techniques ; *Foot-and-Mouth Disease/virology/genetics ; Immunity, Innate ; Cytokines/genetics ; },
abstract = {Foot-and-mouth disease virus (FMDV) is a highly contagious pathogen that is controlled mainly by the use of inactivated vaccines, but vaccine production is limited by inefficient cell culture systems. The RNA helicase DDX5 has been implicated in viral replication, but its role in FMDV infection remains unclear. Here, we generated a DDX5-knockout PK-15 cell line using CRISPR/Cas9 to investigate its impact on FMDV replication. DDX5 knockout cells exhibited enhanced FMDV replication, with increased viral protein expression, RNA levels, and titers compared to wild-type cells. Meanwhile, RNA sequencing (RNA-seq) analysis indicated that DDX5 knockout suppressed key proinflammatory cytokines (CXCL2/8/14, CCL2/4/5) and impaired IFN-α/β and ISG (ISG15/20, IRF3, IFIT3) responses postinfection. RT-qPCR was performed to determine the expression level of differentially expressed genes, and the results were consistent with the RNA-seq data. Altogether, the results of this study suggest that DDX5 restricts FMDV replication by modulating host innate immunity. The DDX5 knockout cell line provides a useful model for studying FMDV pathogenesis and improving vaccine development.},
}

*Foot-and-Mouth Disease Virus/physiology/genetics
*Virus Replication
Animals
Cell Line
*CRISPR-Cas Systems
*DEAD-box RNA Helicases/genetics/metabolism
Swine
Gene Knockout Techniques
*Foot-and-Mouth Disease/virology/genetics
Immunity, Innate
Cytokines/genetics

@article {pmid41876983,
year = {2026},
author = {Chen, X and Qin, Y and Dong, S and Jia, C and Li, Y and Liu, Y and Zhao, Q and Zhou, Q},
title = {Nanoparticle-based biosensor integrated with CRISPR/Cas12b platform for sensitive and visual identification of hepatitis B virus pregenomic RNA in chronic hepatitis B patients.},
journal = {BMC microbiology},
volume = {26},
number = {1},
pages = {},
pmid = {41876983},
issn = {1471-2180},
support = {82460140//the National Natural Science Foundation of China/ ; Qian Ke He Support [2023] General 242//the Guizhou Provincial Key Technology R&D Program/ ; Zhu Ke He (2024) 2-34//the Program of Scientific and Technological of Guiyang City/ ; Qiankehe Platform Talent ZSYS [2025]040//Guizhou Provincial Key Laboratory of Integrated Traditional Chinese and Western Medicine for Mechanism and Clinical Application Research/ ; LMS25H200006//the Natural Science Foundation of Zhejiang Province/ ; 2025KY1182//the Medical Scientific Research Foundation of Zhejiang Province/ ; },
mesh = {Humans ; *Hepatitis B virus/genetics/isolation & purification ; *Biosensing Techniques/methods ; *Hepatitis B, Chronic/virology/diagnosis/blood ; *RNA, Viral/genetics/blood ; Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Metal Nanoparticles/chemistry ; *Molecular Diagnostic Techniques/methods ; Gold/chemistry ; Rapid Diagnostic Tests ; },
abstract = {BACKGROUND: Chronic hepatitis B (CHB) represents a leading driver of hepatocellular carcinoma and end-stage liver disease. Serum hepatitis B virus pregenomic RNA (HBV-pgRNA) has emerged as a new bioindicator strongly related to the efficacy and prognosis of CHB treatment. Seeking ultrasensitive, rapid, highly specific, and straightforward HBV-pgRNA detection, we constructed an innovative CRISPR-HBV-pgRNA platform through the integration of a CRISPR/Cas12b system with loop-mediated isothermal amplification (LAMP). Then, we interpreted the detection results via either real-time fluorescence (RTF) or a gold nanoparticle-based lateral flow biosensor (AuNPs-LFB). METHODS: Herein, the AuNPs-based biosensor used was manufactured following our design. The unique LAMP primers and guide RNA (gRNA) were designed against the HBV-pgRNA gene, ensuring optimized diagnostic conditions: Reaction temperature and time. Both assay sensitivity and specificity were validated, and the feasibility was validated via clinical specimens from patients having chronic HBV infection. RESULTS: The developed AuNPs-based biosensor was successfully fabricated. Primers LAMP and gRNA were specifically designed to target the HBV pgRNA sequence. The integrated assay protocol, comprising RNA extraction (45 min), RT-LAMP amplification (30 min), CRISPR/Cas12b cleavage (5 min), and visual readout (2 min), was completed in 85 min without reliance on costly instrumentation. The method achieved a detection limit of 10 copies/reaction and demonstrated no cross-reactivity with other tested pathogens. CONCLUSIONS: The CRISPR-HBV-pgRNA assay is a powerful diagnostic tool and exhibits considerable potential for POC testing for the evaluation of chronic HBV infection status and antiviral drug efficacy, especially for resource-limited regions.},
}

Humans
*Hepatitis B virus/genetics/isolation & purification
*Biosensing Techniques/methods
*Hepatitis B, Chronic/virology/diagnosis/blood
*RNA, Viral/genetics/blood
Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
Sensitivity and Specificity
Metal Nanoparticles/chemistry
*Molecular Diagnostic Techniques/methods
Gold/chemistry
Rapid Diagnostic Tests

@article {pmid41902966,
year = {2026},
author = {Wu, Y and Li, Y and Zhang, H and Wu, L and Wei, Y and Wu, K and Yang, Y and Chen, QH},
title = {TdT cascaded CRISPR/Cas12a integrated MOF-on-MOF nanozyme for ultrasensitive electrochemical detection of acetamiprid.},
journal = {Mikrochimica acta},
volume = {193},
number = {4},
pages = {},
pmid = {41902966},
issn = {1436-5073},
support = {No. 2023KF005//Open Research Fund of State Key Laboratory of Virology and Biosafety/ ; 82272960//National Natural Science Foundation of China/ ; },
mesh = {*Neonicotinoids/analysis/chemistry ; *Electrochemical Techniques/methods ; *Biosensing Techniques/methods ; Limit of Detection ; *Metal-Organic Frameworks/chemistry ; *CRISPR-Cas Systems ; Food Contamination/analysis ; Electrodes ; DNA, Single-Stranded/chemistry ; },
abstract = {An electrochemical biosensor was developed for the detection of acetamiprid (ACE) by integrating a terminal deoxynucleotidyl transferase-(TdT)-cascaded CRISPR/Cas12a with a NH2-MIL-88@PCN-224 nanozyme. ACE triggered the release of a DNA primer, initiating TdT-catalyzed generation of long poly-T strands. These strands activated the trans-cleavage activity of Cas12a, leading to the hydrolysis of phosphate-modified ssDNA on the electrode. Consequently, the adsorption of the nanozyme onto the electrode inversely correlated with the ACE concentration. The nanozyme efficiently catalyzed the oxidation of 3,3’,5,5’-tetramethylbenzidine (TMB), generating a measurable electrochemical signal. This synergistic combination of nucleic acid amplification and nanozyme catalysis achieved exceptional sensitivity, with a wide linear range from 100 fM to 10 nM and a LOD of 9.7 fM. The biosensor demonstrated high specificity, satisfactory stability and reproducibility, and was successfully applied to detect ACE in real food samples, showing excellent consistency (Pearson’s r = 0.99) with HPLC-MS results. This work provides a powerful and reliable platform for monitoring pesticide residues in food.},
}

*Neonicotinoids/analysis/chemistry
*Electrochemical Techniques/methods
*Biosensing Techniques/methods
Limit of Detection
*Metal-Organic Frameworks/chemistry
*CRISPR-Cas Systems
Food Contamination/analysis
Electrodes
DNA, Single-Stranded/chemistry

@article {pmid41912740,
year = {2026},
author = {Aggarwal, F and Jatwani, S and Jadhav, RR and Yadav, R and Sarma, SJ and Khare, D},
title = {A potassium titanate whisker-assisted method for genetic transformation of Phanerochaete chrysosporium using a Cas9-sGFP expression vector.},
journal = {World journal of microbiology & biotechnology},
volume = {42},
number = {4},
pages = {},
pmid = {41912740},
issn = {1573-0972},
mesh = {*Phanerochaete/genetics/metabolism ; *Transformation, Genetic ; Genetic Vectors/genetics ; *Titanium/chemistry ; Green Fluorescent Proteins/genetics ; CRISPR-Cas Systems ; Hygromycin B/pharmacology/analogs & derivatives ; *CRISPR-Associated Protein 9/genetics ; Genes, Reporter ; },
abstract = {Phanerochaete chrysosporium, a model white-rot fungus, plays a central role in lignin degradation but has remained recalcitrant to genetic manipulation due to inherently low transformation efficiencies. In this study, we present a novel whisker-assisted transformation method employing potassium titanate whiskers combined with freeze–thaw pretreatment, L-ornithine supplementation, and sonication to enable efficient DNA delivery into P. chrysosporium. Using the Cas9-sGFP construct as a reporter system, transformants were selected on hygromycin and validated through PCR and fluorescence microscopy. Our protocol consistently generated hygromycin-resistant transformants, achieving a reproducible transformation efficiency of ~ 0.26%, outperforming Agrobacterium-mediated transformation and comparable to shockwave-based approaches. The successful integration and functional expression of the sGFP reporter confirmed the stability and reliability of this approach. This study provides the first evidence of whisker-mediated delivery and stable integration of exogenous DNA into the genomic DNA of a white-rot fungus. By providing a simple, cost-effective, and reproducible transformation strategy, this work addresses a longstanding bottleneck in fungal biotechnology. The method provides a scalable alternative for CRISPR-based genome engineering in ligninolytic fungi and unlocks opportunities for metabolic engineering of P. chrysosporium, including targeted gene disruption, overexpression of ligninolytic enzymes, and expression of heterologous biosynthetic pathways for the sustainable production of industrially valuable compounds.},
}

*Phanerochaete/genetics/metabolism
*Transformation, Genetic
Genetic Vectors/genetics
*Titanium/chemistry
Green Fluorescent Proteins/genetics
CRISPR-Cas Systems
Hygromycin B/pharmacology/analogs & derivatives
*CRISPR-Associated Protein 9/genetics
Genes, Reporter

@article {pmid41931156,
year = {2026},
author = {Kumar, VK and Thamodaran, V},
title = {A genome-edited isogenic human embryonic stem cell model of pompe disease recapitulates cardiac and skeletal muscle pathology.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {41931156},
issn = {1573-4978},
mesh = {Humans ; *Glycogen Storage Disease Type II/genetics/pathology/metabolism ; *Human Embryonic Stem Cells/metabolism ; Cell Differentiation/genetics ; alpha-Glucosidases/genetics/metabolism ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Muscle, Skeletal/pathology/metabolism ; Cell Line ; Induced Pluripotent Stem Cells/metabolism ; Myocardium/pathology/metabolism ; Glycogen/metabolism ; Myocytes, Cardiac/metabolism/pathology ; Lysosomes/metabolism ; },
abstract = {BACKGROUND: Pompe disease is an autosomal recessive lysosomal storage disorder caused by mutations in the GAA gene, leading to acid alpha-glucosidase deficiency and pathological glycogen accumulation, primarily in cardiac and skeletal muscle. While enzyme replacement therapy (ERT) has improved clinical outcomes, its limited efficacy especially in skeletal muscle underscores the need for improved disease models and novel therapeutic strategies. Induced pluripotent stem cells (iPSCs) from Pompe patients have facilitated mechanistic studies; however, their utility is restricted by limited patient sample availability. METHODS: To address this limitation, we employed CRISPR-Cas9 genome editing to disrupt GAA in a well-characterized human embryonic stem cell (hESC) line, BJNhem20, thereby generating a Pompe disease model independent of patient material. RESULTS: The edited hESC line exhibited markedly reduced GAA enzymatic activity while maintaining pluripotency and trilineage differentiation potential. Upon directed differentiation, cardiac and skeletal muscle cells displayed pronounced lysosome and glycogen accumulation. CONCLUSIONS: These findings demonstrate that genome-edited hESC for Pompe disease can recapitulate key pathological features, providing a robust and scalable platform for disease modelling and therapeutic screening. This approach offers a valuable alternative to patient-derived iPSCs for studying rare genetic disorders and for the development of targeted interventions.},
}

Humans
*Glycogen Storage Disease Type II/genetics/pathology/metabolism
*Human Embryonic Stem Cells/metabolism
Cell Differentiation/genetics
alpha-Glucosidases/genetics/metabolism
*Gene Editing/methods
CRISPR-Cas Systems/genetics
*Muscle, Skeletal/pathology/metabolism
Cell Line
Induced Pluripotent Stem Cells/metabolism
Myocardium/pathology/metabolism
Glycogen/metabolism
Myocytes, Cardiac/metabolism/pathology
Lysosomes/metabolism

@article {pmid41952234,
year = {2026},
author = {Manosalva, I and Charles-Alfred, M and Torres, M and Roca Paixao, JF and Spicuglia, S},
title = {Validation of a CD81-based flow cytometry assay to assess dCas9 silencing activity.},
journal = {BMC research notes},
volume = {19},
number = {1},
pages = {},
pmid = {41952234},
issn = {1756-0500},
mesh = {*Flow Cytometry/methods ; Humans ; *Gene Silencing ; *Tetraspanin 28/genetics/metabolism ; Cell Line, Tumor ; Reproducibility of Results ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {OBJECTIVE: The efficiency of CRISPR interference (CRISPRi) depends on functional dCas9 activity, yet practical and reproducible validation of dCas9-expressing cell lines remains limited. Here, we describe a simple and reproducible assay to assess dCas9 functionality using a single sgRNA targeting the nonessential and ubiquitously expressed surface protein CD81. RESULTS: We evaluated this approach in multiple hematological and solid tumor cell lines expressing the dCas9-KRAB-MeCP2 repressor complex. In all tested models, CD81 targeting resulted in a consistent reduction of surface protein levels, quantified by flow cytometry. This assay provides a rapid and quantitative functional readout of dCas9 activity without the need for reporter constructs or transcriptional assays. The CD81-targeting sgRNA and validated cell lines are made available to support reproducibility and technical standardization in CRISPRi experiments. This strategy can be readily implemented in any laboratory using CRISPRi-based approaches.},
}

*Flow Cytometry/methods
Humans
*Gene Silencing
*Tetraspanin 28/genetics/metabolism
Cell Line, Tumor
Reproducibility of Results
*CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41957080,
year = {2026},
author = {Hwang, RW and Khalil, Y and Baumann, E and Huang, J and Charlebois, C and Rukhlova, M and Renner, TM and Liu, Z and Jezierski, A and Kærn, M and Costain, WJ},
title = {Accelerated reprogramming of hiPSCs into functional brain endothelial-like cells using multiplexed CRISPR activation.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41957080},
issn = {2045-2322},
support = {OISB-NRC Scholarship//Ottawa Institute for Systems Biology/ ; NBR1-129//National Research Council Canada/ ; },
mesh = {Humans ; *Induced Pluripotent Stem Cells/cytology/metabolism ; *Endothelial Cells/cytology/metabolism ; *Cellular Reprogramming/genetics ; Cell Differentiation/genetics ; Blood-Brain Barrier/cytology/metabolism ; *Brain/cytology ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Transcription Factors/genetics/metabolism ; },
abstract = {Human induced pluripotent stem cells (hiPSCs) offer a renewable and scalable source for generating brain endothelial cells (BECs), enabling the development of in vitro blood-brain barrier (BBB) models that closely reflect human physiology. In this study, we demonstrate a streamlined differentiation strategy for producing hiPSC-derived BEC-like cells (iBECs) using multiplex CRISPR/dCas9 activation (CRISPRa) to transcriptionally reprogram iPSCs by selectively and simultaneously upregulating BEC transcription factors (ERG, ETV2, FLI1) and BEC genes (CLDN5, CDH5, PECAM1, KDR). We observe that this approach significantly accelerates differentiation from 13 to five days while maintaining BBB characteristics. CRISPRa iBECs exhibit high transendothelial electrical resistance (TEER ~ 400 Ω·cm2), low transcellular permeability (< 0.027 × 10[-]3 cm/min), and expression of key BBB markers including Claudin-5, ZO-1, CDH5, PECAM1, Occludin, and GLUT-1, as well as receptor-mediated transporters TfR1, IGF1R, and TMEM30A. Our study demonstrates a novel CRISPRa-directed differentiation strategy that not only accelerates BEC differentiation but also demonstrates the utility of CRISPR gene regulation as an option in modifying the phenotype of iPSC-derived cells.},
}

Humans
*Induced Pluripotent Stem Cells/cytology/metabolism
*Endothelial Cells/cytology/metabolism
*Cellular Reprogramming/genetics
Cell Differentiation/genetics
Blood-Brain Barrier/cytology/metabolism
*Brain/cytology
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Transcription Factors/genetics/metabolism

@article {pmid41968299,
year = {2026},
author = {Liu, Y and Yin, X and Jin, P and Zhang, H},
title = {CRISPR/Cas9-based knockout of BnaLYK compromises pattern-triggered immunity and resistance to Sclerotinia sclerotiorum in Brassica napus.},
journal = {BMC plant biology},
volume = {26},
number = {1},
pages = {},
pmid = {41968299},
issn = {1471-2229},
support = {KJ2021A0189//Anhui Provincial Department of Education Scientific Research Project/ ; 32472531//National Natural Science Foundation of China/ ; 2024YFD1400800 Subject 3 (2024YFD1400803)//National Key Research and Development Program of China/ ; },
mesh = {*Brassica napus/immunology/genetics/microbiology ; *Ascomycota/physiology ; *Plant Diseases/microbiology/immunology/genetics ; *Disease Resistance/genetics ; CRISPR-Cas Systems ; *Plant Immunity/genetics ; *Plant Proteins/genetics/metabolism ; Reactive Oxygen Species/metabolism ; Gene Knockout Techniques ; },
abstract = {Rapeseed (Brassica napus) is severely threatened by Sclerotinia sclerotiorum, the causal agent of Sclerotinia stem rot (SSR). The lack of fully resistant cultivars, combined with the genetic complexity of rapeseed as an allopolyploid species, has hindered the identification of major resistance genes for molecular breeding strategies. This study identifies the involvement of BnaLYK, a LysM receptor-like kinase, in disease resistance and immune signaling pathways. The two homologs, BnaA05.LYK and BnaC05.LYK, function as orthologs of AtLYK1 in B. napus. Both genes were induced upon S. sclerotiorum infection and were localized to the plasma membrane. Heterologous expression of either gene in the atlyk1 mutant restored chitin-triggered reactive oxygen species (ROS) burst, callose deposition, and defense gene expression. CRISPR/Cas9-mediated knockout of BnaLYK significantly compromised resistance against S. sclerotiorum, characterized by increased necrotic lesion formation and decreased expression levels of defense-related genes. BnaLYK was shown to be indispensable for chitin- and peptidoglycan-induced pattern-triggered immunity, regulating ROS production, callose accumulation, and defense gene expression. Our findings demonstrate BnaLYK is essential for chitin-induced immunity and basal resistance against S. sclerotiorum in rapeseed, which underscore the conserved functionality of LysM receptors in polyploid crops and offer a promising target for breeding SSR-resistant rapeseed varieties.},
}

*Brassica napus/immunology/genetics/microbiology
*Ascomycota/physiology
*Plant Diseases/microbiology/immunology/genetics
*Disease Resistance/genetics
CRISPR-Cas Systems
*Plant Immunity/genetics
*Plant Proteins/genetics/metabolism
Reactive Oxygen Species/metabolism
Gene Knockout Techniques

@article {pmid41998240,
year = {2026},
author = {Buhl, N and Pfister, ED and Oliveira, DV and Turetti, F and Lurz, E and Baumann, U and Di Donato, N and Illig, T and Skawran, B and Andersson, ER and Mašek, J and Stalke, A},
title = {Improved functional JAG1 and NOTCH2 variant testing in patients with clinical or suspected Alagille syndrome using new low-Notch activity cells.},
journal = {Human genetics},
volume = {145},
number = {1},
pages = {},
pmid = {41998240},
issn = {1432-1203},
mesh = {Humans ; *Alagille Syndrome/genetics/diagnosis/metabolism ; *Jagged-1 Protein/genetics/metabolism ; *Receptor, Notch2/genetics/metabolism ; Mutation, Missense ; HEK293 Cells ; Signal Transduction/genetics ; Female ; CRISPR-Cas Systems ; Male ; },
abstract = {The autosomal dominant multisystemic Alagille Syndrome (ALGS) is an important cause of pediatric cholestasis. ALGS is associated with pathogenic variants in JAGGED1 (JAG1) or NOTCH2, ligand and receptor components of the Notch-signaling pathway, respectively. The detected missense variants are commonly classified as variants of uncertain significance, hindering ALGS diagnosis. To overcome this issue, we have developed a CRISPR/Cas9-engineered Low-Notch activity (LNA) cells allowing for selective testing of JAG1-NOTCH2 signaling activity. We tested this approach on 9 patients with pediatric hepatopathies with phenotypes ranging from the full clinical ALGS spectrum to isolated neonatal cholestasis and atypical ALGS abnormalities who carried 5 JAG1 and 3 NOTCH2 missense variants of interest. Additionally, western blot analyses revealed an effect on protein expression for two JAG1 missense variants, one of which had altered glycosylation, potentially indicating pathogenic effects. For this JAG1 and one NOTCH2 de novo missense variant, luciferase-based Notch reporter activity was significantly reduced in LNA cells, while no change was observed in commonly used HEK293T, Huh7, or Hep2G cells. These findings allow independent confirmation of recently classified c.53T>G p.(Leu18Arg) in JAG1, and a re-classification of c.1235G>T p.(Cys412Phe) in NOTCH2 as likely pathogenic based on ACMG criteria. Collectively, we provide evidence that selective testing of JAG1-NOTCH2 interaction in the newly developed CRISPR-engineered cells, combined with a glycosylation assay, enables robust functional evaluation of ALGS-associated JAG1 and NOTCH2 variants.},
}

Humans
*Alagille Syndrome/genetics/diagnosis/metabolism
*Jagged-1 Protein/genetics/metabolism
*Receptor, Notch2/genetics/metabolism
Mutation, Missense
HEK293 Cells
Signal Transduction/genetics
Female
CRISPR-Cas Systems
Male

@article {pmid42000804,
year = {2026},
author = {Tamrakar, VK and Sharma, K and Singh, P and Khan, R and Bhargava, A and Thakur, P and Negi, SS},
title = {Probable hindrance of visible colour due to excess biotin with CRISPR-dCas9-sgRNA lateral flow assay detection of HPV16 and HPV18: a negative finding.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42000804},
issn = {2045-2322},
support = {IIRP-2023-3960//Indian Council of Medical Research/ ; },
mesh = {*Biotin/chemistry ; *Human papillomavirus 16/genetics/isolation & purification ; Humans ; *CRISPR-Cas Systems ; *Human papillomavirus 18/genetics/isolation & purification ; Streptavidin/chemistry ; Colorimetry/methods ; Rapid Diagnostic Tests ; *Papillomavirus Infections/diagnosis/virology ; Color ; },
abstract = {To develop a CRISPR-dCas9-based lateral flow assay (LFA) for the detection of human papillomavirus 16 (HPV16) and human papillomavirus 18 (HPV18) genotypes for point-of-care molecular diagnosis. A CRISPR-dCas9-based LFA was planned to be developed by immobilizing biotin-tagged dCas9-sgRNA assembly on streptavidin-coated nitrocellulose membranes. The assay protocol involved the sequential addition of biotinylated HPV16 and HPV18 PCR amplicons, streptavidin-alkaline phosphatase conjugate, and BCIP/NBT substrate for colorimetric detection. No colour development was observed in the experimental setup, in contrast to the positive control (biotin, streptavidin-alkaline phosphatase conjugate and BCIP/NBT substrate). The failure was attributed to steric hindrance caused by excess biotin present on both the dCas9-sgRNA complex and the HPV PCR amplicons, leading to competition for streptavidin binding sites, improper binding configurations, disrupted protein folding, and interference with biotin-streptavidin interactions. The study demonstrates that using multiple biotinylated molecules in CRISPR-dCas9-based LFAs can lead to assay failure due to competitive binding and steric hindrance. The results advocate the use of two different molecule for immobilization and signal generation and emphasize the necessity for independent optimization of binding and reporting components to establish a functional CRISPR-dCas9-based LFA platform.},
}

*Biotin/chemistry
*Human papillomavirus 16/genetics/isolation & purification
Humans
*CRISPR-Cas Systems
*Human papillomavirus 18/genetics/isolation & purification
Streptavidin/chemistry
Colorimetry/methods
Rapid Diagnostic Tests
*Papillomavirus Infections/diagnosis/virology
Color

@article {pmid42001168,
year = {2026},
author = {Lei, L and Tan, L and Chen, Y and Peng, X and Wang, Y and Liao, F and Yuan, Y and Chen, Y and Wang, K and Huang, X and Zhan, Y and Wang, N and Duan, D and Yang, Y and Wang, A},
title = {A crRNA/Cas12a complex-driven rapid and visual detection method for four porcine diarrhea viruses.},
journal = {BMC veterinary research},
volume = {22},
number = {1},
pages = {},
pmid = {42001168},
issn = {1746-6148},
support = {2021NK1030//Research and development of protein particle vaccines for African Swine Fever and other major viral diseases/ ; },
mesh = {Animals ; Swine ; *Swine Diseases/virology/diagnosis ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Porcine epidemic diarrhea virus/isolation & purification/genetics ; Transmissible gastroenteritis virus/isolation & purification/genetics ; Sensitivity and Specificity ; Deltacoronavirus/isolation & purification/genetics ; *Molecular Diagnostic Techniques/veterinary/methods ; Coronavirus Infections/veterinary/diagnosis/virology ; Rotavirus/isolation & purification/genetics ; CRISPR-Cas Systems ; Rapid Diagnostic Tests ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BACKGROUND: Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine delta-coronavirus (PDCoV), and porcine rotavirus-A (PoRV) G9 are major swine pathogens primarily responsible for gastrointestinal diseases, particularly affecting lactating piglets and resulting in significant economic losses, especially in China. This study reports a novel CRISPR-based nucleic acid detection method that integrates the high specificity of huLbCas12a with the sensitivity of loop-mediated isothermal amplification (LAMP) technology. Central to this method, the crRNA/Cas12a complex, enhances diagnostic accuracy through targeted gene editing. In this approach, the nucleic acids of the four viruses are amplified in parallel by LAMP and subsequently detected in four singleplex CRISPR–Cas12a reactions performed in separate tubes, with the incorporation of fluorescent reporter probes and a lateral flow dipstick assay establishing a visual detection system capable of separately identifying each of the four viruses. RESULTS: It enables the visual detection of viral genomes from as low as 1 copy/µL without cross-reactivity. In comparative testing of 95 clinical samples, our quadruplex LAMP-CRISPR assay demonstrated 100% concordance with RT-qPCR for the three porcine coronaviruses and 98.9% concordance with RT-qPCR for PoRV G9. CONCLUSIONS: Offering a robust and reliable tool for on-site virus detection, this method significantly aids in the timely prevention of virus spread and mitigates its impact on the pig farming industry, demonstrating its potential role in enhancing biosecurity and disease management in veterinary contexts.},
}

Animals
Swine
*Swine Diseases/virology/diagnosis
*Nucleic Acid Amplification Techniques/veterinary/methods
Porcine epidemic diarrhea virus/isolation & purification/genetics
Transmissible gastroenteritis virus/isolation & purification/genetics
Sensitivity and Specificity
Deltacoronavirus/isolation & purification/genetics
*Molecular Diagnostic Techniques/veterinary/methods
Coronavirus Infections/veterinary/diagnosis/virology
Rotavirus/isolation & purification/genetics
CRISPR-Cas Systems
Rapid Diagnostic Tests
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid42018089,
year = {2026},
author = {Satpathy, MM and Kumar, B and Thomas, P and Abhishek, and Singh, KP and Vinodhkumar, OR},
title = {Development of a high-copy target enhanced multiplexed crRNA-based, amplification-free detection assay (HiTECT) for Brucella spp.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42018089},
issn = {1573-4978},
mesh = {*Brucella/genetics/isolation & purification ; *Brucellosis/diagnosis/microbiology/genetics ; CRISPR-Cas Systems/genetics ; Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Animals ; Humans ; *Molecular Diagnostic Techniques/methods ; Rapid Diagnostic Tests ; },
abstract = {BACKGROUND: Brucellosis, a zoonotic disease caused by Brucella species, continues to pose major health challenges for humans and livestock, along with significant economic losses. Traditional serological tests are widely used but are limited, as they cannot reliably distinguish between active infection and past exposure. Molecular tools such as PCR and real-time PCR offer high sensitivity and specificity; however, their dependence on sophisticated equipment and trained personnel restricts their use outside well-equipped laboratories. More recently, isothermal techniques such as LAMP and RPA have emerged as faster, simpler alternatives, and their integration with CRISPR-based platforms has further enhanced detection capabilities. Nevertheless, these methods still face challenges, including the requirement for pre-amplification and the risk of contamination, which limit their practical application in field conditions. METHODS AND RESULTS: In this study, we developed an amplification-free CRISPR-Cas12a assay—HiTECT (High-copy Target Enhanced CRISPR Test)—targeting the high-copy IS711 insertion element for thermocycler free rapid detection of Brucella spp. Using a multiplexed crRNA strategy, HiTECT enabled robust visual detection without nucleic acid amplification. Under optimal conditions (100 nM LbCas12a with a 1:1 Cas12a–crRNA ratio) and employing three crRNAs, fluorescence was significantly enhanced. The assay demonstrated an analytical sensitivity of 461.71 ag/µL of genomic DNA (0.92 fg/ reaction) and approximately 6*10[4] CFU/mL for Brucella suis 1330. HiTECT was found very specific as it could detect all 16 field isolates and four reference Brucella strains, with no cross-reactivity to any of the non Brucella bacterial species, and showed substantial concordance with real-time PCR (κ = 0.65). CONCLUSION: HiTECT provides a robust, amplification-free, field-deployable platform for rapid and sensitive detection of Brucella spp., offering clear advantages for resource-limited settings.},
}

*Brucella/genetics/isolation & purification
*Brucellosis/diagnosis/microbiology/genetics
CRISPR-Cas Systems/genetics
Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
Animals
Humans
*Molecular Diagnostic Techniques/methods
Rapid Diagnostic Tests

@article {pmid42032034,
year = {2026},
author = {Yasui, R and Suzuki, S and Fujii, T and Yabu, Y and Maeda, Y and Murata, S and Kataoka, K and Tanaka, T},
title = {Highly efficient genome editing using CRISPR/Cas9 ribonucleoprotein in the marine oleaginous diatom Fistulifera solaris.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42032034},
issn = {2045-2322},
support = {JPNP17005//the New Energy and Industrial Technology Development Organization/ ; 24H00392//a JSPS KAKENHI Grant-in-Aid for Scientific Research A/ ; JPJS00420230003//JSPS Program for Forming Japan's Peak Research Universities (J-PEAKS)/ ; },
mesh = {*Diatoms/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics/metabolism ; Adenine Phosphoribosyltransferase/genetics ; Mutation ; Genetic Engineering/methods ; *Gene Editing/methods ; },
abstract = {Microalgae are promising hosts for sustainable aviation fuel production due to their rapid growth and their advantage of not competing with agricultural crops. The oleaginous diatom Fistulifera solaris is notable for its exceptionally high lipid content. However, genetic engineering tools for this species remain limited, partly due to its allodiploid genome structure. Here, we report the successful development of a CRISPR/Cas9 genome editing method using ribonucleoprotein (RNP) complexes in F. solaris. We first targeted the adenine phosphoribosyl transferase (apt) genes as a selectable marker. Delivery of RNPs via particle bombardment to conserved regions of the two homoeologous apt genes resulted in 87% biallelic editing efficiency. Next, we demonstrated multiplex genome editing by co-targeting the apt genes and diadinoxanthin de-epoxidase (dde) genes as representative targets for improving valuable compound production in diatoms. Among 27 co-edited clones, 85% showed mutations in at least one dde homoeolog, and 63% exhibited biallelic mutations in both genes. This study demonstrates the applicability of RNP-mediated genome editing in diatoms. Furthermore, the success of this method suggests its broader applicability to non-model diatom species, providing a valuable tool for genome engineering in diatoms.},
}

*Diatoms/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Ribonucleoproteins/genetics/metabolism
Adenine Phosphoribosyltransferase/genetics
Mutation
Genetic Engineering/methods
*Gene Editing/methods

@article {pmid42032156,
year = {2026},
author = {Yu, M and Xiao, Y and Zeng, B},
title = {Optimization of CRISPR/Cas9-based multi-gene editing system in Aspergillus oryzae and its application in α-amyrin biosynthesis.},
journal = {World journal of microbiology & biotechnology},
volume = {42},
number = {5},
pages = {},
pmid = {42032156},
issn = {1573-0972},
support = {32200606//the National Natural Science Foundation of China/ ; 20213AAG02020//the Project of the Department of Science and Technology of the Jiangxi Province/ ; 2021YFA1301302//the National Key Research and Development Plan of China/ ; },
mesh = {*Aspergillus oryzae/genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Promoter Regions, Genetic ; Metabolic Engineering/methods ; Plasmids/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Aspergillus oryzae, with its high protein secretion capacity, post-translational modification capabilities, and safety, is a promising host for producing natural products and recombinant proteins. However, the lack of efficient genetic tools and precise genome modification methods has significantly slowed progress in the metabolic engineering of A. oryzae. Although the CRISPR/Cas9-mediated genome editing system has been applied in A. oryzae, the low efficiency of gene disruption and heterologous gene integration limits its widespread application as a chassis cell for industrial strain development. In this study, the CRISPR/Cas9-mediated genome editing system in A. oryzae RIB40 was optimized to significantly improve its editing efficiency. By evaluating the effect of promoters on sgRNA expression, it was determined that the Ao (Up338)5SrRNA promoter effectively enhances the gene disruption efficiency. Utilizing the Ao (Up338)5SrRNA promoter, a multiplex gene editing system based on the tRNAGly-sgRNA array was developed, achieving dual-gene disruption efficiency of 78.84% and triple-gene disruption efficiency of 41.15%. Additionally, when the ratio of the genome-editing plasmid to the circular donor DNA was 1:3, the site-specific integration efficiency of the exogenous gene reached 61.36%. Based on the developed CRISPR/Cas9 genome editing system, the heterologous α-amyrin synthetic gene (CADDS) was precisely integrated into the wA locus to achieving the biosynthesis of α-amyrin in Aspergillus oryzae. Furthermore, acetyl-CoA supply was improved by knocking out the competing ethanol metabolic pathway, resulting in an engineered strain that produced 0.69 mg/g of α-amyrin.},
}

*Aspergillus oryzae/genetics/metabolism
*CRISPR-Cas Systems
*Gene Editing/methods
Promoter Regions, Genetic
Metabolic Engineering/methods
Plasmids/genetics
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid42032748,
year = {2026},
author = {Vondracek, K and Lee, MB and Liu, T and Lee, S},
title = {Rapid screening of genome edited strawberry (Fragaria ×ananassa) regenerants using high-resolution melting analysis followed by Amplicon sequencing.},
journal = {BMC research notes},
volume = {19},
number = {1},
pages = {},
pmid = {42032748},
issn = {1756-0500},
support = {#2022-51181-38328-0//National Institute of Food and Agriculture/ ; },
mesh = {*Fragaria/genetics ; CRISPR-Cas Systems/genetics ; *Genome, Plant/genetics ; *Gene Editing/methods ; Mutation ; *High-Throughput Nucleotide Sequencing/methods ; Plants, Genetically Modified/genetics ; Sequence Analysis, DNA/methods ; },
abstract = {OBJECTIVE: Plant transformation frequently results in large quantities of regenerant plant materials which must undergo screening prior to advancement into subsequent experiments. In-depth sequencing of such quantities for detection of editing events is costly, and preliminary selection of mutant lines based on phenotypic impacts can cause significant delays. This study aimed to develop a rapid, high-throughput workflow for early detection and genotyping of CRISPR/Cas9-mediated edits in strawberry. RESULTS: High-resolution analysis reliably identified lines containing distinct mutation profiles, and subsequent Amplicon sequencing genotyping confirmed the presence of editing events at the targeted sites, with editing efficiencies varying among subgenomes of octoploid strawberry. This workflow offers a scalable, low-cost solution for early detection and prioritization of genome-edited cultivated strawberry lines.},
}

*Fragaria/genetics
CRISPR-Cas Systems/genetics
*Genome, Plant/genetics
*Gene Editing/methods
Mutation
*High-Throughput Nucleotide Sequencing/methods
Plants, Genetically Modified/genetics
Sequence Analysis, DNA/methods

@article {pmid42046128,
year = {2026},
author = {Du, W and Zhang, T and Guo, L and Zheng, Y and Zhang, H and Zhu, X and Tang, D and Hu, H and Chen, L and Liu, C},
title = {Deep learning-driven prediction of on-target activity, off-target risk, and repair outcomes in CRISPR/Cas9: current landscape and multi-scale perspectives.},
journal = {Journal of translational medicine},
volume = {24},
number = {1},
pages = {},
pmid = {42046128},
issn = {1479-5876},
support = {SZSM202411026//Guangdong Provincial and National Key Clinical Specialty Construction Project and National Key Clinical Specialty Construction Project, Sanming Project of Medicine in Shenzhen/ ; 82371901//National Natural Science Foundation of China/ ; 82471919//National Natural Science Foundation of China/ ; },
mesh = {*Deep Learning ; *CRISPR-Cas Systems/genetics ; Humans ; Gene Editing ; Prediction Algorithms ; Animals ; },
abstract = {The CRISPR/Cas9 system has emerged as a transformative tool in genome editing, playing a pivotal role in enabling precise genetic engineering. Achieving high on-target efficiency while minimizing off-target activity is critical for translating CRISPR/Cas9 into reliable experimental and therapeutic applications. Conventional off-target detection methods are labor-intensive and cost-prohibitive, limiting their scalability. The integration of artificial intelligence has markedly reduced detection costs and substantially increased throughput. Early shallow learning models in the CRISPR/Cas9 domain, although effective in basic classification tasks, exhibited limited feature representation and poor generalization. With advances in algorithms and computational power, deep learning architectures have significantly improved off-target prediction accuracy. However, a critical blind spot remains, most current models operate predominantly at the sequence level, overlooking the downstream functional consequences of genome edits. This review summarizes the current landscape of AI-driven CRISPR/Cas9 prediction methods and proposes a forward-looking “three-layer framework” that integrates molecular, cellular, and tissue dimensions. By linking nucleotide-level edits to protein alterations, cellular functional changes, and tissue-specific responses, this framework aims to bridge the gap between sequence-based predictions and phenotypic outcomes, thereby advancing the precision and translational potential of CRISPR/Cas9 technologies.},
}

*Deep Learning
*CRISPR-Cas Systems/genetics
Humans
Gene Editing
Prediction Algorithms
Animals

@article {pmid42068385,
year = {2026},
author = {Wang, J and Huang, T and Zhao, W and Hu, M and Liu, M and Song, Z and Xing, L and Wang, N},
title = {Development of a one-tube RPA/Cas12a platform for dual-mode detection of Vibrio alginolyticus.},
journal = {World journal of microbiology & biotechnology},
volume = {42},
number = {5},
pages = {},
pmid = {42068385},
issn = {1573-0972},
support = {SF2350//Lianyungang Social Development Project Key R&D Programme Funding/ ; KD2024KYJJ151//Nanjing Medical University Kanda College Key Projects/ ; },
mesh = {*Vibrio alginolyticus/genetics/isolation & purification ; Bacterial Proteins/genetics ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Rapid Diagnostic Tests ; CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems ; *Vibrio Infections/diagnosis/microbiology/veterinary ; DNA-Binding Proteins/genetics ; Recombinases ; Transcription Factors/genetics ; DNA Primers/genetics ; Animals ; Endodeoxyribonucleases ; },
abstract = {Vibrio alginolyticus is a halophilic marine bacterium that threatens aquaculture and food safety. Rapid and reliable detection is vital for early intervention and outbreak prevention. Recombinase polymerase amplification (RPA) coupled with CRISPR/Cas12a offers a sensitive, specific, and isothermal alternative suitable for portable detection. We developed a one-tube RPA/Cas12a assay for rapid and sensitive V. alginolyticus detection, which integrates fluorescence and lateral flow strip (LFS) readouts for flexible visualization. An optimal primer pair (F1/R1) and crRNA2 targeting the toxR gene enabled efficient amplification and Cas12a-mediated trans-cleavage. All key components—RPA mixture, Cas12a, and crRNA—were essential for signal generation. Reaction optimization achieved a detection limit of 0.747 copies/µL for fluorescence and 100 copies/µL for LFS. Specificity assays confirmed the exclusive detection of V. alginolyticus without cross-reactivity to other Vibrio spp. or marine bacteria. The method validation was carried out using multiple types of samples, including artificially contaminated clinical samples, aquatic products, as well as clinical isolated of V. alginolyticus, demonstrating robustness. This dual-mode system simplifies operation, minimizes contamination, and enables field-deployable, real-time monitoring of V. alginolyticus in aquaculture environments, offering a practical, rapid, and instrument-free tool for pathogen surveillance and coastal biosecurity.},
}

*Vibrio alginolyticus/genetics/isolation & purification
Bacterial Proteins/genetics
Sensitivity and Specificity
*Nucleic Acid Amplification Techniques/methods
Limit of Detection
Rapid Diagnostic Tests
CRISPR-Associated Proteins/genetics
CRISPR-Cas Systems
*Vibrio Infections/diagnosis/microbiology/veterinary
DNA-Binding Proteins/genetics
Recombinases
Transcription Factors/genetics
DNA Primers/genetics
Animals
Endodeoxyribonucleases

@article {pmid42095660,
year = {2026},
author = {Xie, Q and Wang, Q and Noettger, S and Gosálbez, G and Betzler, AC and Volcic, M and Kmiec, D and Krebs, S and Graf, A and Gülensoy, D and Weidinger, G and Sparrer, KMJ and Kirchhoff, F},
title = {Replication-competent SIVcpz CRISPR screen identifies barriers to successful cross-species transmission.},
journal = {Journal of virology},
volume = {100},
number = {6},
pages = {e0031426},
pmid = {42095660},
issn = {1098-5514},
support = {101054456/ERC_/European Research Council/International ; KI 548/21-1, VO 2829/2-1//Deutsche Forschungsgemeinschaft/ ; },
mesh = {*Simian Immunodeficiency Virus/genetics/physiology ; Humans ; Animals ; *Virus Replication/genetics ; *CRISPR-Cas Systems ; *Simian Acquired Immunodeficiency Syndrome/transmission/virology ; HIV-1/genetics ; Pan troglodytes ; },
abstract = {UNLABELLED: Simian immunodeficiency viruses (SIVs) have crossed from apes to humans at least four times, but only one event gave rise to the AIDS pandemic. The host barriers that pandemic HIV-1 group M (major) strains overcame to spread efficiently in humans remain poorly understood. To identify such barriers, we performed CRISPR-Cas9 screens driven by the replication efficiency of SIVcpz, the chimpanzee precursor of HIV-1. Guide RNA libraries targeting more than 500 human genes encoding potential antiviral factors were inserted into the replication-competent SIVcpz MB897 molecular clone, which is phylogenetically closely related to HIV-1 group M strains. Propagation in Cas9-expressing human SupT1 T cells significantly enriched for sgRNAs targeting AXIN1, CEACAM3, CD72, EHMT2, GRN, HMOX1, HMGA1, ICAM2, IFITM2, MEFV, PCED1B, SGOL2, SMARCA4, SUMO1, and TMEM173. These hits only partially overlapped with those identified in analogous HIV-1-based screens, indicating virus-specific restriction profiles. Functional analyses confirmed that IFITM2 (interferon-induced transmembrane protein 2), PCED1B (PC-esterase domain-containing protein 1B), MEFV (Mediterranean fever protein, pyrin/TRIM20), and AXIN1 (Axis inhibition protein 1) restrict replication of the analyzed SIVcpz strains but not HIV-1 group M strains in primary human CD4[+] T cells. These findings reveal previously unrecognized host factors that limit SIVcpz replication in human cells and highlight barriers that at least some HIV-1 group M strains overcame during adaptation for pandemic spread.

IMPORTANCE: Four independent transmission events of simian immunodeficiency viruses from chimpanzees and gorillas to humans gave rise to human immunodeficiency virus type 1, but only one led to the global AIDS pandemic. Understanding which adaptations allowed the pandemic HIV-1 M strains to spread efficiently in humans remains a key question in virus evolution and public health. In this study, we engineered replication-competent SIVcpz constructs carrying more than 1,500 single-guide RNAs to identify antiviral genes in Cas9-expressing cells. This approach revealed several cellular factors that restrict SIVcpz but not the pandemic HIV-1 M strains analyzed in primary human T cells. These findings provide new insights into antiviral defense mechanisms and the adaptations that most likely contributed to the efficient spread of HIV-1.},
}

*Simian Immunodeficiency Virus/genetics/physiology
Humans
Animals
*Virus Replication/genetics
*CRISPR-Cas Systems
*Simian Acquired Immunodeficiency Syndrome/transmission/virology
HIV-1/genetics
Pan troglodytes

@article {pmid40481608,
year = {2025},
author = {Liu, H and Sun, N and Liu, Z and Li, J and Zhang, X},
title = {Knockout of bcas3 gene causes neurodevelopment defects in zebrafish.},
journal = {Biological research},
volume = {58},
number = {1},
pages = {34},
pmid = {40481608},
issn = {0717-6287},
support = {2023YFC2706302//National Key R&D Program of China/ ; 81000079, 81170165, and 81870959//National Natural Science Foundation of China/ ; 2016QYTD02//Program for HUST Academic Frontier Youth Team/ ; },
mesh = {Animals ; *Zebrafish/genetics ; Neurodevelopment/genetics ; *Zebrafish Proteins/genetics ; Gene Knockout Techniques ; Disease Models, Animal ; *Neurodevelopmental Disorders/genetics ; CRISPR-Cas Systems ; Apoptosis/genetics ; },
abstract = {BACKGROUND: Neurodevelopmental disorders manifest in early childhood and are characterized by cognitive deficits, intellectual disabilities, motor disorders, and social dysfunction. Mutations in BCAS3 gene are associated with syndromic neurodevelopmental disorders in humans, while the detailed pathological mechanism is still unknown. METHODS: CRISPR/Cas9 technology was used to generate a bcas3 knockout zebrafish model. To investigate the effects of bcas3 on development, morphological evaluations were conducted. Locomotor behaviors, including performance in the light-dark test, novel tank test, mirror test, shoaling test, and social test, were assessed through video tracing and quantitative analysis of movement parameters. Transcriptome sequencing analysis was used to identify dysregulated pathways associated with development process. Additionally, Acridine Orange staining was employed to evaluate apoptosis. Western blot and real-time RT-PCR were used to analyze the expression levels of genes. RESULTS: Bcas3 knockout zebrafish exhibited early larval phenotypes resembling clinical features of patients with BCAS3 mutations, including global delayed development at early embryonic development, microcephaly and reduced body length. Behavior analysis revealed abnormal motor dysfunction, such as social impairment, increased anxiety and heightened aggression. Notably, human BCAS3 rescued the developmental defects and motor disorders in bcas3 knockout larvae. Transcriptomic analysis identified substantial downregulation of genes related to embryonic development and startle response, brain development and neuron migration in bcas3 knockout zebrafish, such as rpl10, cyfip2, erbb3b, eya4a, nr2f1b, prkg1b and ackr3b. Additionally, increased apoptosis was observed in bcas3 knockout zebrafish, which was further confirmed by Acridine Orange staining and a decreased Bcl2/Bax ratio in western blot analysis. The increased apoptosis observed in the brain of bcas3 knockout larvae could contribute to the developmental and locomotor deficits. CONCLUSION: The bcas3 knockout zebrafish model recapitulates the clinical features observed in patients with BCAS3 mutations. Our results suggest that increased apoptosis may underlie the developmental deficits and motor disorders in these patients. The bcas3 knockout zebrafish model provides a valuable tool to identify dysregulated molecular targets for therapeutic intervention during the early stages of disease progression.},
}

Animals
*Zebrafish/genetics
Neurodevelopment/genetics
*Zebrafish Proteins/genetics
Gene Knockout Techniques
Disease Models, Animal
*Neurodevelopmental Disorders/genetics
CRISPR-Cas Systems
Apoptosis/genetics

@article {pmid41258590,
year = {2025},
author = {Wang, X and Xia, C and Zhang, X},
title = {Genetic screening identifies Ube2v1 as a suppressor of immunoglobulin class switch recombination in CH12F3 cells.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {101},
pmid = {41258590},
issn = {1573-4978},
support = {31900655//National Natural Science Foundation of China/ ; },
mesh = {*Ubiquitin-Conjugating Enzymes/genetics/metabolism ; Animals ; *Immunoglobulin Class Switching/genetics ; Ubiquitination ; B-Lymphocytes/metabolism/immunology ; CRISPR-Cas Systems ; Mice ; Cell Line ; Genetic Testing/methods ; Humans ; },
abstract = {BACKGROUND: Antibodies are essential mediators of adaptive immunity, providing defense against pathogens and serving as critical tools in therapeutics of infection and cancer. Antibody diversification, including class switch recombination (CSR), is tightly regulated by ubiquitination, a post-translational modification, that modulates protein stability and functional activity. METHODS AND RESULTS: Using CRISPR/Cas9 screening in B cells, we investigated the role of ubiquitin-related enzymes in CSR. Validation experiments with LentiCRISPR-sgAID stable cell lines, which showed significant inhibition of CSR, confirmed that it works in the CSR model CH12F3 cell. Screening of 35 E2 ubiquitin-conjugating enzymes and 85 deubiquitinases (DUBs) identified Ube2v1, an E2 enzyme, as a potent suppressor of CSR. Specifically knockdown of Ube2v1 significantly enhanced CSR efficiency, whereas its overexpression inhibited CSR without affecting germline transcripts (GLTs) or activation-induced cytidine deaminase (AID). Intriguingly, although Ube2v1 canonically functions with Ube2n to mediate polyubiquitination, overexpression and knockdown of Ube2n had no detectable effect on CSR. CONCLUSIONS: Our CRISPR screening identified multiple components of ubiquitin pathway that regulate CSR and established Ube2v1 as a novel inhibitor. Ube2v1 functions independently of expression of GLTs and AID as well as its canonical partner Ube2n, revealing a non-canonical role. These findings underscore the complexity of post-translational regulation of humoral immunity and suggest Ube2v1 as a potential therapeutic target for modulating antibody responses.},
}

*Ubiquitin-Conjugating Enzymes/genetics/metabolism
Animals
*Immunoglobulin Class Switching/genetics
Ubiquitination
B-Lymphocytes/metabolism/immunology
CRISPR-Cas Systems
Mice
Cell Line
Genetic Testing/methods
Humans

@article {pmid41286760,
year = {2025},
author = {Wu, G and Ren, Y and Wang, Y and Zhao, Y and Wu, Y and Sun, H and Sun, Z and Wang, R and Du, Z},
title = {Detection of rifampin-resistant Mycobacterium tuberculosis using CRISPR/Cas14a-enabled molecular techniques.},
journal = {BMC infectious diseases},
volume = {25},
number = {1},
pages = {1813},
pmid = {41286760},
issn = {1471-2334},
mesh = {*Mycobacterium tuberculosis/genetics/drug effects/isolation & purification ; *Rifampin/pharmacology ; Humans ; Bacterial Proteins/genetics ; *Drug Resistance, Bacterial/genetics ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; DNA-Directed RNA Polymerases/genetics ; *Tuberculosis, Multidrug-Resistant/diagnosis/microbiology ; Microbial Sensitivity Tests ; RNA, Ribosomal, 16S/genetics ; *Molecular Diagnostic Techniques/methods ; *Antitubercular Agents/pharmacology ; },
abstract = {BACKGROUND: The emergence of drug-resistant Mycobacterium tuberculosis (MTB) strains highlights the urgent need for precise and timely diagnostic methods to prevent prolonged and complex treatment regimens. This study aims to develops a CRISPR/Cas14a-based assay for accurate identification of MTB and rifampin-resistant MTB (RR-MTB) strains. METHOD: The 16 S rDNA sequence and rifampin resistance-determining region (RRDR) of rpoB gene were chosen for the detection of MTB and RR-MTB, respectively. Several sgRNAs were designed for each target and evaluated for their performance. The platform was then systematic optimized by adjusting the concentrations of different components, followed by the evaluation of its sensitivity for the detection of MTB and RR-MTB. The system’s efficacy was further validated through a double-blind test on 16 clinical MTB isolates, and the results were compared with genomic sequencing. RESULTS: Through a meticulous screening process, we identified optimal single-guide RNAs (sgRNAs) capable of distinguishing MTB from nontuberculous mycobacteria (NTM) and the eight predominant mutation types associated with rifampin resistance. Our refined CRISPR/Cas14a platform demonstrated a remarkable sensitivity, with a limit of detection (LOD) of 200 copies/µL for MTB and 2 copies/µL for RR-MTB, respectively. This platform demonstrated a 100% accuracy rate in identification of RR-MTB using clinical MTB isolates. CONCLUSIONS: The CRISPR/Cas14a-based platform we developed exhibited superior performance for the detection of MTB and RR-MTB, with significant implications for the diagnosis and management of tuberculosis, particularly in regions with high prevalence of drug-resistant strains.},
}

*Mycobacterium tuberculosis/genetics/drug effects/isolation & purification
*Rifampin/pharmacology
Humans
Bacterial Proteins/genetics
*Drug Resistance, Bacterial/genetics
*CRISPR-Cas Systems
Sensitivity and Specificity
DNA-Directed RNA Polymerases/genetics
*Tuberculosis, Multidrug-Resistant/diagnosis/microbiology
Microbial Sensitivity Tests
RNA, Ribosomal, 16S/genetics
*Molecular Diagnostic Techniques/methods
*Antitubercular Agents/pharmacology

@article {pmid41354677,
year = {2025},
author = {Rangaraj, A and Kaur, H and Mejia, L and Hansen, J and Biswas, A and Tuteja, G},
title = {Characterization of a cis-regulatory element upstream of matrix metalloproteinase-9.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {1463},
pmid = {41354677},
issn = {2045-2322},
support = {R01 HD096083/HD/NICHD NIH HHS/United States ; R01HD096083//Eunice Kennedy Shriver National Institute of Child Health and Human Development/ ; },
mesh = {Humans ; *Matrix Metalloproteinase 9/genetics/metabolism ; Female ; *Enhancer Elements, Genetic ; Pregnancy ; Placenta/metabolism/cytology ; Gene Expression Regulation ; Cell Line ; CRISPR-Cas Systems ; *Regulatory Sequences, Nucleic Acid ; },
abstract = {Dysregulated enhancer activity disrupts gene expression, contributing to disease. However, the structural and functional complexity of enhancers hinders their characterization. Here, we investigate a cis-regulatory element upstream of matrix metalloproteinase-9 (MMP9), a gene implicated in cancer, cardiovascular disease, inflammation, and pregnancy complications. Using luciferase assays and CRISPR-Cas9 mediated knockout in a human placental cell line, we define a one kilobase segment that enhances MMP9 expression. Further dissection reveals two activating sub-segments and, unexpectedly, one repressive sub-segment. Molecular assays suggest transcription factors that mediate these opposing effects. This work adds to the growing understanding that enhancers can integrate both activation and repression, revealing a more complex regulatory architecture than previously appreciated. Together, these findings underscore the importance of enhancer dissection for understanding gene regulation across tissues and diseases.},
}

Humans
*Matrix Metalloproteinase 9/genetics/metabolism
Female
*Enhancer Elements, Genetic
Pregnancy
Placenta/metabolism/cytology
Gene Expression Regulation
Cell Line
CRISPR-Cas Systems
*Regulatory Sequences, Nucleic Acid

@article {pmid41392248,
year = {2025},
author = {Liang, J and Liang, T and Wei, C and Li, L and Li, Y and He, S and Liao, Z and Cui, L},
title = {CRISPR/Cas9-engineered Bacillus subtilis chassis for tailored chitooligosaccharide production from marine waste chitosan.},
journal = {Microbial cell factories},
volume = {25},
number = {1},
pages = {36},
pmid = {41392248},
issn = {1475-2859},
support = {2023GXNSFAA026505//Natural Science Foundation of Guangxi Province/ ; gxkllst-20241009//Guangxi Key Laboratory of Longevity Science and Technology/ ; },
mesh = {*Bacillus subtilis/genetics/metabolism ; *Chitosan/metabolism ; Glycoside Hydrolases/genetics/metabolism ; *CRISPR-Cas Systems ; *Chitin/analogs & derivatives/biosynthesis ; Fermentation ; Oligosaccharides ; Bacterial Proteins/genetics/metabolism ; },
abstract = {BACKGROUND: This study establishes a sustainable bioprocess for converting chitosan from marine waste into high-value chitooligosaccharides (COSs), offering an eco-friendly alternative to conventional methods that often generate chemical waste. We achieved heterologous production of chitosanase in an engineered Bacillus subtilis chassis by knocking out its endogenous chitosanase, leveraging the dual advantages of this bacterium as a robust synthetic biology platform and an industrial microorganism. RESULTS: The endogenous chitosanase gene (BsCsn) in Bacillus subtilis WB800N was deleted via CRISPR/Cas9-mediated editing, generating the chassis strain B. subtilis WB800N ΔBsCsn. A codon-optimized GH46 chitosanase (CsnA) from Streptomyces coelicolor, fused to the AprE signal peptide, was then expressed in this host. Response surface methodology optimized the fermentation process, enabling a high extracellular CsnA activity of 540.08 ± 6.20 U/mL, in a 5-L bioreactor under DO-stat-controlled fed-batch conditions. This process achieved a productivity of 11.25 U/(mL·h) and a carbon conversion efficiency of 1682.86 U/g glycerol. Furthermore, MALDI-TOF MS analysis confirmed that CsnA produces COSs with defined degrees of polymerization (DP2-DP4). CONCLUSION: This integrated platform enables the upcycling of marine waste into high-value COSs, establishing B. subtilis as an eco-efficient cell factory and providing a valuable framework for the heterologous expression of other chitosanases in this host.},
}

*Bacillus subtilis/genetics/metabolism
*Chitosan/metabolism
Glycoside Hydrolases/genetics/metabolism
*CRISPR-Cas Systems
*Chitin/analogs & derivatives/biosynthesis
Fermentation
Oligosaccharides
Bacterial Proteins/genetics/metabolism

@article {pmid41405764,
year = {2025},
author = {Mansoor, MJ and Al-Taie, SF and Al-Khafaji, ZA and Alkhathami, AG and Renuka, JS and Panigrahi, R and Negi, H and Jassal, P and Mustafa, YF and Hamzah, HF},
title = {Overcoming barriers in CAR-NK immunotherapy: CRISPR-Driven advances in checkpoint editing and allogeneic design.},
journal = {Functional & integrative genomics},
volume = {26},
number = {1},
pages = {4},
pmid = {41405764},
issn = {1438-7948},
mesh = {Humans ; *Killer Cells, Natural/immunology/transplantation ; *Gene Editing/methods ; *Immunotherapy, Adoptive/methods ; *Neoplasms/therapy/immunology/genetics ; *CRISPR-Cas Systems ; Animals ; *Receptors, Chimeric Antigen/genetics/immunology ; Immunotherapy ; Tumor Microenvironment ; },
abstract = {Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells are emerging as an exciting avenue in cancer immunotherapy due to their potent cytotoxicity to malignant cells and lower risk of graft-versus-host disease (GvHD) than conventional T cell therapies. The new technology of CRISPR/Cas9 genome editing has significantly expedited the engineering of CAR-NK cells by enabling easy, multiplex, and precise changes to enhance their efficacy, persistence, and specificity to tumors. This review focuses on the incorporation of CRISPR technology into CAR-NK cell development. It examines uses of knockout of inhibitory checkpoint genes (CISH, PD-1, and TGFBR2), as well as knock-in of CAR into safe genomic locations and multiplex editing of CAR-NK cells to improve cytotoxicity against cancer while resisting suppression from the tumor microenvironment (TME). We further explore immuno-cytokine armoring strategies by knock-in of IL-15 or IL-12, to ensure prolonged proliferation and survival of NK cells, and investigate CRISPR-mediated knockouts of immune inhibitors like NKG2A and TIGIT, to evade immune strategies used by the tumor to evade immune destruction. Furthermore, CRISPR-mediated upregulation of the homing receptor enhances NK cell tumor infiltration, addressing a major obstacle in treating solid tumors. It is significant to mention the progress in generating off-the-shelf products, which is a key step supporting the pursuit of allogeneic therapies. While substantial progress has been made, challenges remain related to optimizing CRISPR delivery, off-target effects, and enhancing in vivo persistence. Future directions of CAR-NK studies will likely capitalize on next-generation genome editing tools and synthetic biology for the development of tunable and logic-gated CAR-NK cells. Overall, this review illustrates the revolutionary capacity of combining CRISPR technology with CAR-NK immunotherapy to develop next-generation programmable and efficacious treatments for hematologic and solid malignancies.},
}

Humans
*Killer Cells, Natural/immunology/transplantation
*Gene Editing/methods
*Immunotherapy, Adoptive/methods
*Neoplasms/therapy/immunology/genetics
*CRISPR-Cas Systems
Animals
*Receptors, Chimeric Antigen/genetics/immunology
Immunotherapy
Tumor Microenvironment

@article {pmid41430619,
year = {2025},
author = {Le Reun, J and Salvioli, Z and Croux, C and Esque, J and André, I and Bordes, F},
title = {A workflow to explore elongase diversity and extend the repertoire of fatty acids produced by Yarrowia lipolytica.},
journal = {Microbial cell factories},
volume = {25},
number = {1},
pages = {18},
pmid = {41430619},
issn = {1475-2859},
mesh = {*Yarrowia/metabolism/genetics/enzymology ; *Fatty Acid Elongases/metabolism/genetics ; *Fatty Acids/biosynthesis/metabolism ; Substrate Specificity ; Workflow ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Fatty acids display highly diverse structures that confer these molecules unique chemical properties and distinct physiological functions. Identifying the substrate specificity of enzymes active on fatty acids is crucial, both for understanding their function in natural organisms and for developing efficient cell factories to produce original fatty acids. However, these enzymes are often membrane-bound and/or act on esterified substrates and studying them in vitro is thus challenging. This is why in vivo characterization of these enzymes’ specificity is an interesting approach. Herein, we harness the industrially relevant oleaginous Yarrowia lipolytica as a chassis for characterizing heterologous enzymes active on fatty acids, which can be used to diversify its fatty acid composition. As a case study, we investigated fatty acid elongases (ELO) responsible for the synthesis of very long-chain fatty acids (> 20 carbons), which are specific of given chain lengths and/or unsaturation patterns. Despite their interest, investigation and utilization of these membrane enzymes remain largely underexplored. RESULTS: We developed a workflow for characterizing heterologous elongases in Y. lipolytica, addressing several limitations to increase throughput. First, we set up a strain engineering strategy to easily integrate the ELO cassettes into targeted loci using CRISPR-Cas9, where screening of homologous recombination events is facilitated by fluorescence. We demonstrated that the native elongase YlELO2, responsible for the elongation of saturated and monounsaturated fatty acids up to 26 carbons, has to be inactivated to avoid functional redundancy and finely characterize heterologous elongase specificity. As it is an essential gene, we designed an optimized strategy for YlELO2 Knock-Out by a Knock-In of the elongase cassette. We then miniaturized cultures and fatty acid extraction in 96-well plates format. Using this workflow, we characterized seven human elongases on endogenous fatty acids and on five exogenous polyunsaturated fatty acids in a single series of experiments. CONCLUSION: We have developed tools and methods to characterize elongase specificity, from strain design to fatty acid production and analysis. Applicable to any fatty acid–modifying enzymes, these methodological developments will be useful to expand the repertoire of enzymes usable in Y. lipolytica and pave the way to produce new original fatty acids in this chassis.},
}

*Yarrowia/metabolism/genetics/enzymology
*Fatty Acid Elongases/metabolism/genetics
*Fatty Acids/biosynthesis/metabolism
Substrate Specificity
Workflow
CRISPR-Cas Systems

@article {pmid41508022,
year = {2026},
author = {Fan, T and Zhou, B and Chen, H and Liu, LP and Ni, Y and Zhang, W and Ye, L and Chen, Y and Zhang, D and Yang, S and Bai, Y and Liu, F and Zhi, C and Xu, G and Zheng, B and Lu, S and Qiu, C and Ding, Z and Chen, Y and Jiang, Y},
title = {Novel serum small extracellular vesicle miRNAs with multi-target RCA-CRISPR sensor for liver cancer detection.},
journal = {Journal of translational medicine},
volume = {24},
number = {1},
pages = {144},
pmid = {41508022},
issn = {1479-5876},
support = {21310051//Startup Fund from Shenzhen Bay Laboratory/ ; 2019156//Development and Reform Commission of Shenzhen Municipality/ ; },
mesh = {*MicroRNAs/blood/genetics ; Humans ; *Liver Neoplasms/blood/diagnosis/genetics ; *Extracellular Vesicles/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Biomarkers, Tumor/blood/genetics ; *Biosensing Techniques ; Base Sequence ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Reproducibility of Results ; },
abstract = {BACKGROUND: Detecting liver cancer (LC) remains a significant challenge in clinical practice. Small extracellular vesicle (sEV) miRNAs show promise as non-invasive biomarkers for LC detection, yet their diagnostic potential remains largely unexplored. This study aimed to identify specific sEV miRNA signatures for LC detection and develop a novel synchronized multi-miRNA detection platform to enhance diagnostic efficiency and sensitivity. METHODS: High-throughput sequencing was conducted across four distinct cohorts: normal controls (NC), hepatitis B virus (HBV) patients, liver cirrhosis patients, and LC patients. This sequencing process identified miRNAs with differential expression, followed by RT-qPCR validation in serum sEV miRNAs from LC patients and NC. An innovative detection method, RCA-CRISPR, was introduced, combining rolling circle amplification (RCA) with CRISPR/Cas12a (RCA-CRISPR) for quick and sensitive miRNAs detection. RESULTS: Sequencing results showed a consistent elevation of hsa-miR-203b-5p, hsa-miR-4661-5p, and hsa-miR-219a-2-3p across all cohorts. RT-qPCR validations confirmed significant upregulation of these miRNAs in serum sEVs from LC patients, and the combined three-miRNA panel exhibited high diagnostic accuracy (p = 0.0003; AUC = 0.81). The RCA-CRISPR method demonstrated a detection limit of 3.12 pM for simultaneous multi-target miRNA detection, highlighting its exceptional sensitivity. CONCLUSIONS: Our study identifies hsa-miR-203b-5p, hsa-miR-4661-5p, and hsa-miR-219a-2-3p as promising sEV miRNA biomarkers for LC detection. The developed RCA-CRISPR sensor provides a robust tool for multi-miRNA analysis, potentially advancing non-invasive LC diagnostics. Future validation in larger, prospectively collected cohorts is essential to establish the clinical utility and performance of this biomarker panel and RCA-CRISPR sensor.},
}

*MicroRNAs/blood/genetics
Humans
*Liver Neoplasms/blood/diagnosis/genetics
*Extracellular Vesicles/metabolism/genetics
*CRISPR-Cas Systems/genetics
Biomarkers, Tumor/blood/genetics
*Biosensing Techniques
Base Sequence
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Reproducibility of Results

@article {pmid41530441,
year = {2026},
author = {Li, W and Sun, Y and Ye, M and Liang, Y and Ouyang, J and Xu, W and Su, Y and Huang, X and Nie, D and Ouyang, S},
title = {Rapid visual detection of Treponema pallidum using the RPA-CRISPR/Cas12a system.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {5120},
pmid = {41530441},
issn = {2045-2322},
mesh = {*Treponema pallidum/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; Humans ; *Syphilis/diagnosis/microbiology ; *Nucleic Acid Amplification Techniques/methods ; Rapid Diagnostic Tests ; Sensitivity and Specificity ; DNA, Bacterial/genetics ; },
abstract = {Syphilis, caused by Treponema pallidum, is a sexually transmitted infection that has re-emerged globally over the past decade, posing significant public health challenges. Conventional diagnostic methods are limited by lengthy processing times, operational complexity, and moderate sensitivity, highlighting the urgent need for rapid, sensitive, and user-friendly detection strategies. In this study, we developed a visual detection platform for T. pallidum DNA by integrating recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology. The assay can be completed within one hour, with results directly interpreted via fluorescence readout. It demonstrated a detection limit as low as 11.34 copies/µL and high specificity, accurately distinguishing T. pallidum without cross-reactivity with common blood-borne pathogens, including HIV, HBV, HCV, and DENV. The clinical sample verification showed a consistency rate of 96.6% with the actual diagnosis. To enhance suitability for point-of-care applications, the RPA-CRISPR/Cas12a system was further adapted to a lateral flow assay (LFA) format, achieving a detection sensitivity of 5.56 × 10[2] copies/µL while minimizing reliance on specialized instrumentation. Overall, this platform provides a rapid, sensitive, and robust approach for point-of-care syphilis diagnosis and offers a reference framework for detecting other pathogenic organisms.},
}

*Treponema pallidum/genetics/isolation & purification
*CRISPR-Cas Systems/genetics
Humans
*Syphilis/diagnosis/microbiology
*Nucleic Acid Amplification Techniques/methods
Rapid Diagnostic Tests
Sensitivity and Specificity
DNA, Bacterial/genetics

@article {pmid41535756,
year = {2026},
author = {Park, HR and Park, S and Jun, JM and Shin, YJ and Hwang, Y and Jeong, KY and Kim, MY and Kim, ST and Park, S and Yoo, YH and Lee, E and Park, G and Kim, SG and Park, SK},
title = {CRISPR/Cas9 editing of β-Conglycinin subunits reduces IgE binding in soybean [Glycine max (L.) Merr.].},
journal = {BMC plant biology},
volume = {26},
number = {1},
pages = {265},
pmid = {41535756},
issn = {1471-2229},
support = {PJ016784022024//Agricultural Research Program/ ; },
mesh = {*Glycine max/genetics/immunology/metabolism ; *Seed Storage Proteins/genetics/metabolism/immunology ; *Globulins/genetics/metabolism/immunology ; *Soybean Proteins/genetics/metabolism/immunology ; *Immunoglobulin E/metabolism/immunology ; *Antigens, Plant/genetics/metabolism/immunology ; *CRISPR-Cas Systems ; *Gene Editing ; Protein Subunits/genetics ; Food Hypersensitivity/immunology ; Allergens/genetics/immunology ; Plants, Genetically Modified ; },
abstract = {BACKGROUND: Soybean [Glycine max (L.) Merr.] is a major source of plant-based protein, yet the seed storage protein β-conglycinin (7 S globulin) is a prominent allergen. The αʹ, α, and β subunits contain IgE-binding epitopes, and their high sequence similarity enables simultaneous genome editing. The development of soybean lines with reduced β-conglycinin-specific IgE-binding capacity could enhance food safety for individuals with soy allergies. RESULTS: We employed CRISPR/Cas9 to disrupt the αʹ (Glyma.10G246300) and α (Glyma.20G148300, Glyma.20G148400), subunit genes and to target the β subunit genes (Glyma.20G146200, Glyma.20G148200) of β-conglycinin, generating four edited lines: SP1 (αʹ-null), SP2 (αʹα-null), SP3 (β-null), and SP4, which shows an αʹα-edited genotype and a β subunit-null protein phenotype. SDS-PAGE and DNA sequencing confirmed complete or near-complete loss of the targeted proteins across the T0 to T6 generations, demonstrating stable inheritance of the edited seed protein profiles. IgE immunoblotting and inhibition ELISA using pooled sera from soy-allergic individuals revealed distinct IgE-binding inhibition profiles among the edited lines. At the highest inhibitor concentration, SP4 showed the lowest IgE-binding inhibition (70.0%) compared with the wild type (87.7%), whereas SP1-SP3 exhibited inhibition values similar to or only slightly lower than those of the wild type. CONCLUSIONS: CRISPR/Cas9-mediated elimination of β-conglycinin subunits reduces IgE binding to soybean seed proteins and yields lines with stably inherited seed protein phenotypes. These results highlight the potential of targeted genome editing to generate soybean lines with reduced β-conglycinin-specific IgE recognition, supporting the application of precise genome modification in crop improvement for safer soy-based foods.},
}

*Glycine max/genetics/immunology/metabolism
*Seed Storage Proteins/genetics/metabolism/immunology
*Globulins/genetics/metabolism/immunology
*Soybean Proteins/genetics/metabolism/immunology
*Immunoglobulin E/metabolism/immunology
*Antigens, Plant/genetics/metabolism/immunology
*CRISPR-Cas Systems
*Gene Editing
Protein Subunits/genetics
Food Hypersensitivity/immunology
Allergens/genetics/immunology
Plants, Genetically Modified

@article {pmid41547741,
year = {2026},
author = {Wei, Y and Jiang, J and Gao, Y and Zhu, Y and He, W and Wu, F and Xie, H and Chen, L and Cai, Q and Zhang, J},
title = {Accelerated breeding for early-maturing and aromatic glutinous restorer lines with CRISPR/Cas9-mediated targeted editing for hybrid rice.},
journal = {BMC plant biology},
volume = {26},
number = {1},
pages = {284},
pmid = {41547741},
issn = {1471-2229},
support = {2024R1054//the Fundamental Research Project of Fujian Provincial Research Institute for Public Welfare, China/ ; CARS-01-08//the National Rice Industry Technology System of Modern Agriculture for China/ ; XTCXGC2021001//he "5511" Collaborative Innovation project for High-quality Development and Surpasses of Agriculture between Government of Fujian and Chinese Academy of Agricultural Sciences/ ; 2024NZ029027//Key program of Science and Technology in Fujian province, China/ ; },
mesh = {*Oryza/genetics/growth & development ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Plant Breeding/methods ; Plants, Genetically Modified ; Edible Grain/genetics ; Mutation ; },
abstract = {Hybrid rice breeding depends on the development of elite parental lines with favorable traits such as grain quality, heading time, and plant architecture. However, improving restorer lines through conventional breeding is time-consuming and labor-intensive. Here, we employed a multiplex CRISPR/Cas9 editing strategy to simultaneously target Wx, Badh2, and Se14 in the elite restorer line FH676, aiming to generate glutinous, aromatic, and early-maturing lines. Through Agrobacterium-mediated transformation, we obtained Wx/Badh2 double mutants (Dm) and Wx/Badh2/Se14 triple mutants (Tm). Grain quality analysis revealed significantly reduced amylose content and enhanced aroma content in the edited lines, consistent with Wx and Badh2 knockouts. The triple mutants also exhibited significantly earlier heading compared to the wild type. The early-maturing Tm lines and the Dm lines achieved grain yields of 39.2 ~ 39.4 g and 42.9 ~ 43.0 g per plant, respectively, both exceeding yields of conventional glutinous cultivars used at present. To evaluate hybrid performance, we crossed Tm and Dm lines with two sterile lines: LX (aromatic) and NX (glutinous). The LX/Tm hybrid headed 4.8 days earlier than its wild-type counterpart, with no reduction in plant height or yield. The NX/Tm hybrid showed an advance of ~ 6.3 days in heading but a 6.2 ~ 7.0% reduction in yield due to decreased grain number per panicle. The Se14 knockout likely relieves repression of RFT1 expression during the floral transition under long-day conditions. Variations in phenotypic response across different maternal backgrounds suggest epistatic interactions affecting the dosage response of Se14. In summary, Se14 is a promising target for engineering early-maturing hybrid rice. The edited FH676 lines provide valuable germplasm resource for developing early-maturing, aromatic, and glutinous hybrids through CRISPR-based genome editing.},
}

*Oryza/genetics/growth & development
*CRISPR-Cas Systems
*Gene Editing/methods
*Plant Breeding/methods
Plants, Genetically Modified
Edible Grain/genetics
Mutation

@article {pmid41549083,
year = {2026},
author = {Khayer, A and Ye, P and Eti, FS and Sakif, TI and Azad, RB and Ali, J and Gupta, DR and Asuke, S and Pan, Q and Moni, MA and Kang, H and Islam, T},
title = {Field pathogenomics and evolutionary conservation unveil CRISPR-targetable susceptibility genes for wheat blast resistance.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {5677},
pmid = {41549083},
issn = {2045-2322},
support = {32261143468//National Natural Science Foundation of China/ ; OFANS project (Project Code No: FT-92-FNS/21)//Krishi Gobeshona Foundation/ ; Grant Code: V0156.01//Bill and Melinda Gates Foundation/ ; },
mesh = {*Triticum/genetics/microbiology ; *Plant Diseases/microbiology/genetics ; *Disease Resistance/genetics ; *Magnaporthe/pathogenicity ; Gene Expression Regulation, Plant ; *CRISPR-Cas Systems ; Genes, Plant ; Evolution, Molecular ; Ascomycota/pathogenicity ; Plant Proteins/genetics ; Transcriptome ; Gene Expression Profiling ; Host-Pathogen Interactions/genetics ; },
abstract = {Wheat blast, caused by Magnaporthe oryzae pathotype Triticum (MoT), threatens global wheat production, yet durable resistance mechanisms remain elusive. Current strategies relying on race-specific resistance genes or fungicides are vulnerable to pathogen evolution and inefficacy. Here, we investigated field-derived transcriptomes from the 2016 Bangladesh wheat blast epidemic, a catastrophic event devastating all local varieties to identify host susceptibility (S) genes co-opted by MoT. By analyzing RNA-seq data from infected and healthy plants across geographically distinct regions, we pinpointed 273 consistently upregulated wheat genes, enriched in defense-related pathways. Ortholog analysis with rice, a model for blast resistance, identified three conserved susceptibility (S)-gene candidates: TaSULTR3-3B (an ortholog of a rice bacterial blight susceptibility gene), TaSTP3-4D (associated with stripe rust), and TaMLO1-5A (a wheat powdery mildew susceptibility gene). While all three candidates exhibited significant expression correlation with M. oryzae Triticum (MoT) effectors in field-derived samples, in planta spike assays revealed distinct expression dynamics. Only TaMLO1-5A was significantly upregulated in the susceptible cultivar BARI Gom 26 following MoT inoculation, with no induction observed in the resistant cultivar S-615 (carrying Rmg8). Conversely, TaSULTR3-3B and TaSTP3-4D did not show significant induction under the specific conditions and time points of the in planta spike assays. This discrepancy potentially arises from tissue-specific regulation (spike vs. leaf), environmental variations, or differences in sampling time points between the field and greenhouse experiments. Disruption of such S genes, validated in other cereals for durable resistance, offers a transformative strategy to engineer non-race-specific wheat blast resilience. Our findings shift the paradigm from transient resistance genes to foundational susceptibility networks, proposing CRISPR-based editing of the candidate gene as an actionable target. This approach, resilient to pathogen evolution, could preempt epidemics in climate-vulnerable regions, safeguarding global wheat security. By bridging field pathogenomics and evolutionary genomics, we provide a roadmap for sustainable disease management in an era of expanding fungal threats.},
}

*Triticum/genetics/microbiology
*Plant Diseases/microbiology/genetics
*Disease Resistance/genetics
*Magnaporthe/pathogenicity
Gene Expression Regulation, Plant
*CRISPR-Cas Systems
Genes, Plant
Evolution, Molecular
Ascomycota/pathogenicity
Plant Proteins/genetics
Transcriptome
Gene Expression Profiling
Host-Pathogen Interactions/genetics

@article {pmid41620642,
year = {2026},
author = {Kumar, S and Murugan, B and Das, M and Sanan-Mishra, N and Sahoo, L},
title = {Geminiviral-CR-gRNA expressed in cowpea efficiently edited MYMV and MYMIV genome to provide resistance against cowpea yellow mosaic disease without hampering plant growth and yield.},
journal = {BMC plant biology},
volume = {26},
number = {1},
pages = {},
pmid = {41620642},
issn = {1471-2229},
mesh = {*Vigna/genetics/virology/growth & development ; *Begomovirus/genetics ; *Plant Diseases/virology/genetics ; Plants, Genetically Modified ; *Disease Resistance/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing ; *Genome, Viral ; *Geminiviridae/genetics ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Cowpea is an economically important grain legume widely cultivated in Africa, Latin America, and Southeast Asia. In Southeast Asia, two of the most devastating viral diseases affecting cowpea are cowpea golden mosaic and severe leaf curl disease, both caused by Mungbean yellow mosaic India virus (MYMIV). Despite the availability of various molecular breeding strategies to manage viral infections, progress in cowpea improvement remains limited due to the lack of resistant germplasm, the absence of a reliable transformation system, and the restricted availability of efficient tools for viral gene inactivation. RESULTS: In this study, we employed CRISPR/Cas9-mediated genome editing technology to efficiently disrupt the common region (CR) of the single-stranded DNA-A component of legume-infecting geminiviruses, using cowpea as a test system. Transgenic cowpea plants expressing Cas9 and a guide RNA (gRNA) targeting the CR of MYMV/MYMIV were evaluated for resistance to yellow mosaic disease (YMD). Agrobacterium tumefaciens strain EHA105 carrying pXSE901B-Cas9 and CR-gRNA cassettes was used to generate the transgenic plants. PCR and Southern blot analyses confirmed the integration of transgenes into the cowpea genome. Transgenic lines in the T1 and T2 generations were tested for YMD resistance via agroinfiltration using MYMV and MYMIV agroinfectious clones. Accumulation of AV2 and AC2 transcripts was drastically reduced in T2 lines, which also displayed either no or minimal mosaic symptoms. Mutation analysis of the viral genome revealed frameshift mutations near the PAM region of the targeted CR sequence, with editing frequencies of 28%, 34%, 22%, and 33% in MYMV/MYMIV-infected cowpea lines #L2, #L4, #L7, and #L11, respectively. The transgenic cowpea plants exhibited a normal phenotype and did not show any yield reduction under greenhouse conditions. CONCLUSION: To the best of our knowledge, this is the first report of transgenic cowpea plants stably expressing a geminiviral common region (CR)–targeting gRNA via the CRISPR/Cas9 system, leading to efficient editing of the MYMV/MYMIV genome and conferring durable resistance to Yellow Mosaic Disease without adversely affecting plant growth or yield. These findings demonstrate the potential of CRISPR/Cas9 as a precise and robust platform for developing virus-resistant cowpea and other legume crops.},
}

*Vigna/genetics/virology/growth & development
*Begomovirus/genetics
*Plant Diseases/virology/genetics
Plants, Genetically Modified
*Disease Resistance/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics
*Gene Editing
*Genome, Viral
*Geminiviridae/genetics
CRISPR-Cas Systems

@article {pmid41634544,
year = {2026},
author = {Velangani, HG and Ghosh, A and Singh, S and Kiran, S},
title = {Strategies and considerations for the generation of ssDNA-Based HDR templates for CRISPR-based genome editing.},
journal = {BMC genomics},
volume = {27},
number = {1},
pages = {},
pmid = {41634544},
issn = {1471-2164},
support = {[EMDR/SG/15/2024-01-03223 (E-File No. 222379)]//Indian Council of Medical Research/ ; FILE NO. CRG/2023/007695//ANRF, India/ ; },
mesh = {*DNA, Single-Stranded/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Recombinational DNA Repair ; },
abstract = {The usefulness of genome editing lies in the ability to induce any desirable change in the target genome. It is most efficiently achieved using ssDNA (single-stranded DNA) as the HDR template and CRISPR-Cas9 targeted DNA breaks. However, the low efficiency of HDR integration is a challenge for achieving efficient edits. Among different HDR templates, ssDNA has the highest efficiency in inducing repair after CRISPR-induced DNA breaks. Several methods are used to generate the ssDNA-HDR template. However, each method has limitations in terms of feasibility for different ssDNA types, efficiency, time required, expenses incurred, etc. Often, these factors are overlooked, confusing users regarding the most appropriate method for generating ssDNA. This study describes and compares methods for generating ssDNA and outlines considerations for designing an efficient ssDNA template. Most frequently used methods are PCR-based, utilizing modified primers (phosphorylation, biotinylation, or phosphorothioate-based) or those utilizing the IVT-RT (In vitro transcription- reverse transcriptase) method. Asymmetric PCR and M13-based methods of ssDNA generation have been used as non-PCR methods. The IVT-RT method is widely adopted as it provides a middle-ground in yield and ease. The advantages and shortcomings of these methods, based on reported studies and our own results, are discussed.},
}

*DNA, Single-Stranded/genetics
*Gene Editing/methods
*CRISPR-Cas Systems
*Recombinational DNA Repair

@article {pmid41701371,
year = {2026},
author = {Yi, F and Li, Z and Jiang, F and Zhang, Z and Chen, Y and Zhao, Z and Deng, X and Chen, H and Xu, S and Tao, Y},
title = {An electrochemiluminescence biosensor governed by a CRISPR-actuated electrostatic gate for ultrasensitive aflatoxin B1 detection.},
journal = {Mikrochimica acta},
volume = {193},
number = {3},
pages = {163},
pmid = {41701371},
issn = {1436-5073},
support = {22264018//National Natural Science Foundation of China/ ; 20232BAB203019, 20232BAB216118//Jiangxi Provincial Natural Science Foundation/ ; 202410412021, 202410412246, 202410412114//the College Students' Innovation and Entrepreneurship Training Program Project of Jiangxi Province/ ; },
mesh = {*Aflatoxin B1/analysis/chemistry ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; *Luminescent Measurements/methods ; Static Electricity ; *CRISPR-Cas Systems ; Limit of Detection ; Aptamers, Nucleotide/chemistry/genetics ; Food Contamination/analysis ; Electrodes ; Nucleic Acid Hybridization ; },
abstract = {Aflatoxin B1 (AFB1), a potent mycotoxin, poses a critical threat to global food safety, demanding analytical methods with exceptional sensitivity. Here, we introduce a homogeneous electrochemiluminescence (ECL) biosensor that operates on a novel CRISPR-actuated electrostatic gating mechanism. The core of our strategy relies on controlling the access of ECL reporters to a positively charged electrode surface (PAH-ITO). In the absence of AFB1, cationic Ru(phen)32+ reporters are electrostatically repelled from the electrode, resulting in a low background signal. The presence of AFB1 triggers a CRISPR/Cas12a enzymatic cascade, which activates its trans-cleavage activity to release a highly anionic hybridization chain reaction (HCR) scaffold from magnetic beads (MB). This scaffold serves as a nanocarrier, capturing the Ru(phen)32+ reporters and, by virtue of its strong negative charge, shuttling them to the electrode through potent electrostatic attraction. This action effectively “opens” the electrostatic gate, switching on a robust ECL signal. By synergistically integrating the high specificity of the aptamer-CRISPR system with the immense signal amplification of the HCR scaffold, all under the control of a charge-dominant switch, our biosensor achieves an outstanding limit of detection of 0.121 fg/mL and a broad linear range from 1 fg/mL to 100 pg/mL. Its successful application in spiked food samples validates its practicality and robustness, presenting a powerful new paradigm for designing minimal-background, high-gain ECL sensors for mycotoxin determination.},
}

*Aflatoxin B1/analysis/chemistry
*Biosensing Techniques/methods
*Electrochemical Techniques/methods
*Luminescent Measurements/methods
Static Electricity
*CRISPR-Cas Systems
Limit of Detection
Aptamers, Nucleotide/chemistry/genetics
Food Contamination/analysis
Electrodes
Nucleic Acid Hybridization

@article {pmid41721389,
year = {2026},
author = {Umashankar, P and Choi, B and Nygård, Y},
title = {Towards the development of a CRISPR-Cas9 based kill switch for Saccharomyces cerevisiae.},
journal = {Microbial cell factories},
volume = {25},
number = {1},
pages = {},
pmid = {41721389},
issn = {1475-2859},
mesh = {*Saccharomyces cerevisiae/genetics/growth & development ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; Saccharomyces cerevisiae Proteins/genetics ; },
abstract = {BACKGROUND: Advancements in synthetic genetic circuits have enabled programmable and condition-dependent control of microbial cell growth. CRISPR-Cas9-based kill switches, genetic systems that program cells to lose viability in response to specific conditions, have recently been demonstrated for bacterial cell factories but not yet in yeast. RESULTS: In this study, we present a foundational demonstration for a CRISPR-based kill switch in Saccharomyces cerevisiae, CRISPR KiSS. The CRISPR KiSS employs inducible CRISPR targeting essential genes to elicit growth inhibition. The activation of the KiSS system is achieved through conditional expression of a guide RNA (gRNA) upon anhydrotetracycline (ATc) induction, thereby activating CRISPR-mediated gene disruption. We demonstrate that targeting the essential genes (ERG13, PGA3, TPI1 or CDC19) leads to severe growth inhibition upon ATc induction. Still, the current set up does not allow complete killing of the cells due to system inactivation, e.g. escape from CRISPR based cutting. We studied reasons for system inactivation and substantially improved the system by simultaneous expression of two different gRNAs. Sequencing escape mutants revealed mutations in both the gRNA sequences and target genes as potential sources of system inactivation. CONCLUSIONS: This work highlights the potential of harnessing a CRISPR-based kill switch in S. cerevisiae. Cells expressing the system were able to escape growth inhibition through mutations and further optimization of the KiSS system is still needed for it to be used in various cell factory applications.},
}

*Saccharomyces cerevisiae/genetics/growth & development
*CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
Saccharomyces cerevisiae Proteins/genetics

@article {pmid41739318,
year = {2026},
author = {Joshi, D and Kshatri, P and Tiwari, A and Bhardwaj, U and Patel, K},
title = {CRISPR/Cas9 in Cancer Therapy: Precision Genome Editing Approaches Targeting Hematological Malignancies and Solid Tumors Through Cellular, Biochemical, and Molecular Mechanisms.},
journal = {Cell biochemistry and biophysics},
volume = {84},
number = {2},
pages = {1759-1788},
pmid = {41739318},
issn = {1559-0283},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Neoplasms/therapy/genetics ; *Hematologic Neoplasms/therapy/genetics ; Genetic Therapy/methods ; },
abstract = {Being extremely precise in terms of genetic material modifications, CRISPR/Cas9 technology has very rapidly become the cornerstone in the field of precision oncology. This review is focused on the great potential offered by CRISPR/Cas9 in terms of cancer treatment, emphasizing its cellular, biochemical, and molecular mechanisms of action. We provide a short historical background and description of its operational principles, followed by an in-depth analysis of how CRISPR/Cas9 reprograms oncogenic signaling networks by selectively modifying cancer-associated genes such as KRAS, MYC, BRAF, and EGFR, and restoring the function of tumor suppressors including TP53, RB1, and PTEN. The review further explores its ability to remodel cellular pathways involved in apoptosis, DNA repair, and cell-cycle regulation, alongside its modulation of key biochemical cascades. We analyse the technology’s application to epigenetic modifications and the regulation of non-coding RNAs as arising therapeutic targets. It also considers the deployment of CRISPR/Cas9 across various cancers, including haematological malignancies such as leukaemia and lymphoma and solid tumors such as breast, lung, and colorectal cancer, where it is being contextualized to disease-specific outcomes and limitations. To overcome delivery issues, recent advances in various viral vectors (AAV, lentivirus) and non-viral methods such as lipid nanoparticles, polymeric nanotechnologies, exosomes and magnetic nanoparticles are explained. In addition, findings from completed and ongoing clinical trials that demonstrate the clinical translation of CRISPR are presented. Lastly, important issues are considered, including immune reactions, off-target effects, and integration with precision oncology. When combined, these viewpoints provide a thorough understanding of the current state of CRISPR/Cas9 and its potential to revolutionize cancer treatment in the future.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems
*Neoplasms/therapy/genetics
*Hematologic Neoplasms/therapy/genetics
Genetic Therapy/methods

@article {pmid41742232,
year = {2026},
author = {Deng, H and Wang, J and Meng, F and Ma, C and Li, J and Wang, J and Wang, X and Zhao, C and Zhang, Y and Wang, R and Chen, N},
title = {Combining computer-aided enzyme design and chromosomal integration for plasmid-free biosynthesis of 1,5-pentanediol in Escherichia coli.},
journal = {Microbial cell factories},
volume = {25},
number = {1},
pages = {},
pmid = {41742232},
issn = {1475-2859},
support = {22JCQNJC01310//the Natural Science Foundation of Tianjin/ ; 2023YFD13000700//the National Key Research and Development Program of China/ ; 2021ZDSYS10//the Key Research and Development Program of Shandong Province/ ; },
mesh = {*Escherichia coli/metabolism/genetics ; *Glycols/metabolism ; Plasmids/genetics ; Nocardia/genetics/enzymology ; *Metabolic Engineering/methods ; Alcohol Dehydrogenase/metabolism/genetics ; CRISPR-Cas Systems ; Computer-Aided Design ; Biosynthetic Pathways ; Pentanes ; },
abstract = {BACKGROUND: 1,5-Pentanediol (1,5-PDO) is a high-value chemical with broad uses in polymer, cosmetic, and pharmaceutical industries. Although diverse biosynthetic pathways have been constructed, current recombinant strains typically rely on plasmid-based overexpression, which necessitates antibiotics and hinders industrial-scale production. RESULTS: We developed a robust, plasmid-free Escherichia coli platform for de novo 1,5-PDO synthesis by integrating pathway genes (davB, davA, gabT, yahK, car, sfp and yqhD) into the chromosome of a lysine-hyperproducing strain via CRISPR/Cas9. Screening of carboxylic acid reductases identified Nocardia iowensis CAR-Ni as the most effective, yielding a base strain (D13) that produced 0.672 g/L 1,5-PDO. Integrated analysis confirmed the alcohol dehydrogenase (ADH)-mediated reduction of 5-hydroxypentanal (5-HP) as an underappreciated bottleneck. We subsequently screened ten endogenous ADHs and selected YjgB for computational optimization. Docking-guided saturation mutagenesis at position E205 yielded the variant YjgB(E205C), which exhibited a 3.34-fold increase in in vitro activity, reduced 5-HP accumulation, and elevated the titer to 0.935 g/L. Enhancing NADPH supply by integrating pntAB further raised the shake-flask titer to 1.5 g/L. In a 5-L fed-batch bioreactor, the final strain (D91) achieved 12.1 g/L 1,5-PDO (yield of 0.225 mol/mol glucose) without antibiotics or inducers. To our knowledge, this is the highest reported 1,5-PDO titer in E. coli. CONCLUSION: This study establishes a scalable, sustainable biosynthetic platform through synergistic metabolic engineering and computational enzyme optimization.},
}

*Escherichia coli/metabolism/genetics
*Glycols/metabolism
Plasmids/genetics
Nocardia/genetics/enzymology
*Metabolic Engineering/methods
Alcohol Dehydrogenase/metabolism/genetics
CRISPR-Cas Systems
Computer-Aided Design
Biosynthetic Pathways
Pentanes

@article {pmid41746436,
year = {2026},
author = {Chen, N and Sun, X and Liang, S and Cao, C and Lai, X and Song, C and Yan, Z and Ge, C},
title = {Single particle mediated CRISPR/Cas13a ultrasensitive direct detection of miRNA-21 at femtomolar levels without nucleic acid amplification.},
journal = {Mikrochimica acta},
volume = {193},
number = {3},
pages = {},
pmid = {41746436},
issn = {1436-5073},
mesh = {*MicroRNAs/blood/genetics ; Humans ; Gold/chemistry ; Metal Nanoparticles/chemistry ; *CRISPR-Cas Systems ; Limit of Detection ; *Biosensing Techniques/methods ; Nucleic Acid Hybridization ; DNA Probes/chemistry/genetics ; },
abstract = {A novel biosensing platform has been developed that couples the collateral cleavage activity of the CRISPR/Cas13a system with single-particle inductively coupled plasma mass spectrometry (sp-ICP-MS) using gold nanoparticle (AuNP)-DNA reporter probes. In the presence of target microRNA-21, Cas13a is activated and specifically cleaves RNA bases within the DNA-RNA hybrid linkers on AuNPs, preventing their hybridization with biotinylated capture probes on magnetic beads. As a result, the cleaved AuNPs remain in the supernatant and are directly quantified by sp-ICP-MS. The number of detected AuNPs correlates linearly with the concentration of miRNA-21 (y = 0.2537logCmiRNA + 0.2477 with a correlation coefficient of R2 = 0.9978), enabling a detection limit as low as 68 fM with excellent single-base mismatch discrimination. The assay demonstrated high recoveries (97–108%) and reproducibility in spiked human serum samples, confirming its reliability in complex matrices. This amplification-free strategy combines the high specificity of CRISPR/Cas13a with the single-particle sensitivity of sp-ICP-MS, providing a robust, quantitative, and versatile platform for miRNA detection with promising potential for early cancer diagnostics and precision medicine.},
}

*MicroRNAs/blood/genetics
Humans
Gold/chemistry
Metal Nanoparticles/chemistry
*CRISPR-Cas Systems
Limit of Detection
*Biosensing Techniques/methods
Nucleic Acid Hybridization
DNA Probes/chemistry/genetics

@article {pmid41762386,
year = {2026},
author = {Duran, T and Karaselek, MA and Dagdelen, B and Kuccukturk, S and Guner, SN and Keles, S and Reisli, I},
title = {CRISPR-Cas9-based gene editing as a proof-of-concept approach in an inborn error of immunity caused by a DCLRE1C variant.},
journal = {Immunologic research},
volume = {74},
number = {1},
pages = {},
pmid = {41762386},
issn = {1559-0755},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Severe Combined Immunodeficiency/genetics/therapy ; *Endonucleases/genetics ; DNA-Binding Proteins/genetics ; *Nuclear Proteins/genetics ; Interleukin-2 Receptor alpha Subunit/metabolism/genetics ; },
abstract = {Hypomorphic DCLRE1C variants impair T and B cell development, leading to combined immunodeficiency (CID) or leaky severe combined immunodeficiency (SCID). Current treatment options, such as allogeneic hematopoietic stem cell transplantation (aHSCT), are associated with significant risks, highlighting the need for alternative therapeutic strategies. In this study, we report the first a proof-of-concept CRISPR-Cas9–mediated correction of a hypomorphic DCLRE1C variant (c.194 C > T; p.T65I) in CD4 + helper T (Th) cells using CRISPR-Cas9 gene-editing technology. CD4 + Th cells were isolated, and the variant region was edited with sgRNA and donor DNA. Gene editing efficiency was confirmed by Sanger sequencing, revealing successful restoration of the target region to its wild-type sequence. Functional analyses showed a significant increase in CD25 activation and Artemis protein expression post-editing, although DCLRE1C mRNA levels remained unchanged. The approximately 6–8% increase in CD25 expression was statistically significant but did not reach healthy control levels. These findings suggest that CRISPR-Cas9 –mediated gene editing may enable precise correction and induce measurable cellular-level functional changes, supporting biological feasibility rather than therapeutic efficacy. This study provides a foundation for future research on HSCs and underscores the potential role of CRISPR-Cas9–based approaches in the treatment of inborn errors of immunity (IEIs) associated with DCLRE1C variants.},
}

Humans
CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Severe Combined Immunodeficiency/genetics/therapy
*Endonucleases/genetics
DNA-Binding Proteins/genetics
*Nuclear Proteins/genetics
Interleukin-2 Receptor alpha Subunit/metabolism/genetics

@article {pmid41764471,
year = {2026},
author = {Chen, H and Luo, W and Ma, N and Li, S},
title = {CRISPR/Cas9-mediated B2m knockout paves the way for allogeneic basal cell transplantation.},
journal = {Respiratory research},
volume = {27},
number = {1},
pages = {},
pmid = {41764471},
issn = {1465-993X},
support = {2023B111105006//Guangdong Key Area R&D Program Key Project/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/physiology ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mice ; *beta 2-Microglobulin/genetics/deficiency ; Transplantation, Homologous/methods ; Mice, Knockout ; *Gene Knockout Techniques/methods ; Cells, Cultured ; *Gene Editing/methods ; },
abstract = {BACKGROUND: Autologous transplantation of basal cells (BCs) has shown promise in treating respiratory diseases, but disease-specific subpopulations among BCs probably diminish the treatment efficacy. An alternative approach involves generating universal and healthy BCs, which offers a potentially more efficient and accessible solution for avoiding using abnormal BCs. However, such hypoimmunogenic BCs have not yet been transplanted into the airways of immunocompetent animals. METHODS: Before producing hypoimmunogenic BCs, the predominant transplantation antigen in BCs was explored through RT-qPCR and flow cytometry to identify the key target of CRISPR/Cas9‐mediated editing. The proliferation and expression of specific markers of BCs were evaluated after gene editing by CCK‐8 and RT‐qPCR, respectively. These gene‐edited BCs and wild‐type (WT) BCs, which were both derived from the same BALB/c mouse, were subsequently allogeneically transplanted into C57BL/6 mice with polidocanol‐induced airway injury to evaluate the differentiation and immune response in the recipient mice via immunehistological staining. RESULTS: In the present study, it was demonstrated that major histocompatibility complex class I (MHC-I) is the predominant transplantation antigen in BCs. The hypoimmunogenic BCs were generated through editing beta‐2 microglobulin (B2m) participating in the encoding of MHC‐I. The knockout of B2m in BCs did not affect their proliferation or the expression of specific markers in vitro. Both WT BCs and B2m‐edited BCs (B2m‾ BCs) successfully differentiated into ciliated and secretory cells in the tracheas following allogeneic transplantation and did not elicit an immune response during the 26‐day observation period in the tracheas. However, WT BCs, compared to B2m‾ BCs, induced severe lung injury by provoking an immune response in the lower airways and alveolar regions, as indicated by increased infiltration of CD45+ immune cells, epithelial cell shedding in the bronchi, and obvious alveolar hyperemia with collapse. CONCLUSIONS: The hypoimmunogenic BCs generated through CRISPR/Cas9-mediated B2m gene editing retained their ability to differentiate and maintained viability in the allogeneic respiratory system, supporting the potential application in the cell regeneration therapy of airway diseases.},
}

Animals
*CRISPR-Cas Systems/physiology
Mice, Inbred BALB C
Mice, Inbred C57BL
Mice
*beta 2-Microglobulin/genetics/deficiency
Transplantation, Homologous/methods
Mice, Knockout
*Gene Knockout Techniques/methods
Cells, Cultured
*Gene Editing/methods

@article {pmid41766009,
year = {2026},
author = {Vargas-Reyes, M and Alcántara, R and Alfonsi, S and Peñaranda, K and Petrelli, D and Spurio, R and Pajuelo, MJ and Milon, P},
title = {Versatile and portable Cas12a-mediated detection of antibiotic resistance markers.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41766009},
issn = {2045-2322},
support = {C-004-2021-2//Universidad Peruana de Ciencias Aplicadas/ ; D43TW001140//Forgarty International Center/ ; PE501079419-2022//PROCIENCIA - CONCYTEC/ ; },
mesh = {*Escherichia coli/genetics/drug effects ; Anti-Bacterial Agents/pharmacology ; *CRISPR-Cas Systems ; *Drug Resistance, Bacterial/genetics ; Genetic Markers ; Microbial Sensitivity Tests ; *Drug Resistance, Microbial/genetics ; },
abstract = {Antibiotic-resistant bacteria are spreading in clinical, industrial, and environmental ecosystems. The spreading dynamics to and from the environment are unknown, largely due to the lack of appropriate (robust, fast, low-cost) analytical assays. In this study, we developed C12a, a versatile molecular toolbox to detect genetic markers of antibiotic resistance using CRISPR/Cas12a. Biochemical characterization show that the C12a toolbox can detect less than 100 attoMolar of pure DNA fragments from the blaCTX-M15 and floR genes, conferring resistance to b-lactams and amphenicols, respectively important for human and veterinary uses. In microbiological assays, C12a detected less than 102 CFU/mL and high concordance was observed if compared to antibiotic susceptibility tests, PCR, or to whole genome sequencing. Additionally, C12a confirmed a high prevalence of the integrase/integron system in E. coli isolates containing multiple antibiotic resistance genes (ARGs). The C12a toolbox shows equivalent detection performance in diverse laboratory settings, results readout (Fluorescence vs. FLA) or input sample. Altogether, this work presents a comprehensive proof-of-concept, development description, and biochemical characterization of a collection of molecular tools to detect antibiotic resistance markers in a one health setup.},
}

*Escherichia coli/genetics/drug effects
Anti-Bacterial Agents/pharmacology
*CRISPR-Cas Systems
*Drug Resistance, Bacterial/genetics
Genetic Markers
Microbial Sensitivity Tests
*Drug Resistance, Microbial/genetics

@article {pmid41770417,
year = {2026},
author = {Melamed, J and Barnoy, S},
title = {Understanding the public's intention to adopt CRISPR-Cas9: the effect of beliefs, knowledge, and innovativeness.},
journal = {Human genetics},
volume = {145},
number = {1},
pages = {},
pmid = {41770417},
issn = {1432-1203},
mesh = {Humans ; Adult ; *CRISPR-Cas Systems ; Female ; Male ; *Gene Editing ; Young Adult ; Middle Aged ; *Intention ; Surveys and Questionnaires ; *Health Knowledge, Attitudes, Practice ; },
abstract = {CRISPR-Cas9 is a gene editing technology with wide-ranging medical potential and significant ethical implications. This study examined how personality traits, familiarity with CRISPR, knowledge about CRISPR, and beliefs concerning its applications are connected to the public’s willingness to adopt this technology, drawing on Rogers’ Diffusion of Innovations framework. A sample of 500 young adults aged 20–45 completed questionnaires assessing innovativeness as well as familiarity, knowledge, beliefs, and willingness to adopt CRISPR-Cas9 for therapeutic and non-therapeutic purposes. Results showed that only 24% of participants were familiar with CRISPR and that knowledge levels were generally low. Based on Rogers’ typology, 18.8% were identified as innovators, and 26.8% as early adopters, and only 3.2% as laggards. Beliefs were strongly associated with willingness to adopt CRISPR-Cas9 (r = .63, p < .001), moderating the weak associations of personality traits with adoption intentions. These findings suggest that beliefs are an important factor in CRISPR-Cas9 adoption and they appear to have more influence on willingness to adopt CRISPR-Cas9 than knowledge and innovativeness in this sample. Efforts to promote informed public discussion on CRISPR should be made, with an emphasis on ethical aspects alongside scientific information. This is critical for the responsible adoption of this technology.},
}

Humans
Adult
*CRISPR-Cas Systems
Female
Male
*Gene Editing
Young Adult
Middle Aged
*Intention
Surveys and Questionnaires
*Health Knowledge, Attitudes, Practice

@article {pmid41772313,
year = {2026},
author = {Li, Y and Ye, Z and Zhao, C and Tan, Y and Chen, J and Wu, Z and Zhang, Y and Guo, H and Cheng, Y and Wang, R and Wang, J and Wang, D},
title = {Integrated on-site detection of Fusarium temperatum based on a droplet digital CRISPR-based platform.},
journal = {Mikrochimica acta},
volume = {193},
number = {3},
pages = {},
pmid = {41772313},
issn = {1436-5073},
support = {2024A1515011281//Guangdong Basic and Applied Basic Research/ ; 2023C043-5, 2022C044-9//Technology Research and Development Project from the Development and Reform Commission in Jilin Province of China/ ; },
mesh = {*Fusarium/genetics/isolation & purification ; *CRISPR-Cas Systems ; Zea mays/microbiology ; DNA, Fungal/genetics/analysis ; Rapid Diagnostic Tests ; Smartphone ; Microscopy, Fluorescence/methods ; },
abstract = {Fusarium temperatum (F. temperatum) is a fungus whose infection can cause various diseases in maize plants, leading to premature death, and F. temperatum mycotoxin poses a serious threat to human and animal health. Rapid and early on-site detection of F. temperatum infection facilitates the prevention of disease progression, which is an unmet need. In this study, a droplet digital CRISPR-Cas12a-based platform (DD-Cas), combined with a rapid extraction procedure, was developed for amplification-free on-site detection of F. temperatum. The DD-Cas assay can rapidly detect F. temperatum genomic DNA in infected maize samples with high sensitivity (102 CFU/mL) and specificity. Furthermore, we developed a smartphone-based fluorescence microscope integrating the heating module, that could accurately detect infected samples within 30 min, enabling low-cost point-of-care testing (POCT). This platform can avoid cross-contamination and amplification bias, thus having great potential for on field detection of pathogenic bacteria in agriculture.},
}

*Fusarium/genetics/isolation & purification
*CRISPR-Cas Systems
Zea mays/microbiology
DNA, Fungal/genetics/analysis
Rapid Diagnostic Tests
Smartphone
Microscopy, Fluorescence/methods

@article {pmid41775739,
year = {2026},
author = {Li, X and Wang, S and Qiu, Z and Sun, R and Wang, T and Ren, X and Lv, B and Ma, X and Cheng, L and Liu, Y and Jiang, J},
title = {Construction and initial validation of key gene network for progesterone resistance in endometrial cancer based on genome-wide CRISPR screening.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41775739},
issn = {2045-2322},
support = {2023M731847//China Postdoctoral Science Foundation/ ; 2022YFC2704304//National Key Technology Research and Development Programme of China/ ; },
mesh = {Female ; *Endometrial Neoplasms/genetics/drug therapy/pathology ; Humans ; Animals ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Gene Regulatory Networks ; *Drug Resistance, Neoplasm/genetics ; Mice ; Gene Expression Regulation, Neoplastic/drug effects ; Progestins/pharmacology ; *Progesterone/pharmacology ; Apoptosis/drug effects ; Endometrium/abnormalities ; Uterine Diseases ; },
abstract = {Endometrial carcinoma, a prevailing malignancy of the female reproductive system, exhibits escalating incidence and a trend towards early onset. Hormone therapy serves as a primary choice for fertility preservation and is also considered for advanced and recurrent cases. However, a considerable number of patients fail to respond favorably to progestin treatments. We employed CRISPR/Cas9 technology to establish a comprehensive human genome library in the Ishikawa cell line. Subsequent exposure to medroxyprogesterone was followed by high-throughput sequencing, and differential gene expression and enrichment analyses were conducted using Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout (MAGeCK) Robust Rank Aggregation (RRA) and MAGeCK Maximum-Likelihood Estimation (MLE) algorithms. An iterative data intersection approach was employed, utilizing sequenced data from progestin-resistant cell lines, to identify pivotal genes associated with progestin resistance. The top 10 identified genes were functionally validated in our previously established progestin-resistant cell model through Cell Counting Kit-8 (CCK-8) assays and apoptosis detection. The progestin-resistant gene NNMT and the progestin-sensitive gene SOX17 were validated in vivo in xenograft mouse models. The constructed library exhibited high quality, meeting sequencing standards. Employing RRA and MLE algorithms, we identified 332 and 829 negative selection genes, as well as 3438 and 5098 positive selection genes. Enrichment analysis implicated pathways linked to DNA and RNA synthesis, metabolism, and related processes. After multiple data intersections, we identified a total of 5 genes promoting progestin resistance and 20 genes inhibiting resistance, with functional experiments confirming their roles. Employing CRISPR/Cas9 technology enables the construction of a relatively reliable network of pivotal genes associated with progestin resistance in endometrial carcinoma. Processes involving DNA and RNA synthesis, metabolism, and related mechanisms appear to significantly impact the progestin sensitivity of endometrial carcinoma.},
}

Female
*Endometrial Neoplasms/genetics/drug therapy/pathology
Humans
Animals
*CRISPR-Cas Systems
Cell Line, Tumor
*Gene Regulatory Networks
*Drug Resistance, Neoplasm/genetics
Mice
Gene Expression Regulation, Neoplastic/drug effects
Progestins/pharmacology
*Progesterone/pharmacology
Apoptosis/drug effects
Endometrium/abnormalities
Uterine Diseases

@article {pmid41792204,
year = {2026},
author = {Kim, Y and Jun, Y and Han, J and Choi, S and Kim, H and Lee, M and Kim, SL and Kang, SH and Suh, EJ and Park, SR and Mo, Y},
title = {CRISPR/Cas9-mediated mutagenesis of SMXL4 alters plant height and yield-related traits in rice (cv. Samkwang).},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41792204},
issn = {2045-2322},
support = {RS-2024-00322166//Rural Development Administration/ ; },
mesh = {*Oryza/genetics/growth & development ; *CRISPR-Cas Systems ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; *Mutagenesis ; Plants, Genetically Modified ; Gene Editing ; Phenotype ; },
abstract = {Recent climate change and frequent extreme weather events during the maturation period of rice exacerbate lodging and threaten stable production. Samkwang, a widely cultivated rice variety in Korea, is particularly vulnerable to lodging due to its tall stature. To improve lodging tolerance while preserving Samkwang’s elite genetic background, we identified an SMXL4-edited line (smxl4) with reduced culm length and stable growth from a CRISPR/Cas9-edited Samkwang population. The biological function of SMXL4, a clade Ⅳ member of the SMXL (SUPPRESSOR OF MAX2 1-LIKE) family, has not been well characterized in rice. Compared to Samkwang, the smxl4 plants showed reduced plant height, internode length, panicle length, grain number per panicle, and grain weight, while panicle number per plant increased. Transcriptome profiling of elongating internodes at booting and heading stages revealed upregulation of genes associated with cell wall remodeling and defense responses in smxl4 relative to Samkwang. These findings highlight the broad involvement of SMXL4 in rice growth and development and provide insights for breeding lodging tolerant rice cultivars.},
}

*Oryza/genetics/growth & development
*CRISPR-Cas Systems
*Plant Proteins/genetics/metabolism
Gene Expression Regulation, Plant
*Mutagenesis
Plants, Genetically Modified
Gene Editing
Phenotype

@article {pmid41803678,
year = {2026},
author = {Jiang, Q and Zeng, X and Zhang, Q and Yang, F and Lv, T and Zhang, Y and Wang, J and Li, F and Xu, D},
title = {Development and application of a rapid detection system for Aspergillus fumigatus based on ERA/CRISPR-Cas12a.},
journal = {BMC microbiology},
volume = {26},
number = {1},
pages = {},
pmid = {41803678},
issn = {1471-2180},
support = {2024AH051688, 2024AH051225//the University Natural Science Research Key Projects in Anhui Province/ ; },
mesh = {*Aspergillus fumigatus/genetics/isolation & purification ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Humans ; Rapid Diagnostic Tests ; *Aspergillosis/diagnosis/microbiology ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases/genetics ; Sputum/microbiology ; Limit of Detection ; },
abstract = {Aspergillus fumigatus (AF) is the predominant pathogen implicated in invasive aspergillosis (IA) in humans; therefore, prompt and accurate detection is critical for the effective prevention and management of IA. This study developed a rapid detection system targeting the AF-specific anxC4 gene by integrating enzymatic recombinase amplification (ERA) with CRISPR/Cas12a. The reaction proceeds at a stable temperature of 37 °C, with amplification and detection systems separately positioned in the tube lid and bottom, respectively, effectively minimizing aerosol contamination typically associated with product transfers. To enhance sensitivity, the One-Pot method was optimized. Consequently, the fluorescence detection limit reached 1 fg/µL, and the sensitivity of the test strip reached 10 fg/µL, with no cross-reactivity observed against other fungi. Detection of AF was completed within 60 min, and results were visually displayed through fluorescence signals and nucleic acid test strips. Clinical practicality was further evaluated using aspergillosis samples, which demonstrated satisfactory performance. Pure culture results confirmed that out of 62 sputum samples, 32 were positive and 30 negative. Evaluation of 62 clinical samples using the One-Pot ERA-CRISPR/Cas12a system demonstrated sensitivity and specificity rates of 93.75% and 93.33%, respectively, via fluorescence detection, and 90.63% sensitivity and 96.67% specificity using lateral flow strips.},
}

*Aspergillus fumigatus/genetics/isolation & purification
*CRISPR-Cas Systems
Sensitivity and Specificity
Humans
Rapid Diagnostic Tests
*Aspergillosis/diagnosis/microbiology
*Nucleic Acid Amplification Techniques/methods
*Recombinases/genetics
Sputum/microbiology
Limit of Detection

@article {pmid41808028,
year = {2026},
author = {Grubben, J and Bijsterbosch, G and Visser, RGF and Schouten, HJ},
title = {Influence of gRNA efficiency and inversion size on the frequency of CRISPR/Cas9-induced chromosomal inversions in tomato protoplasts.},
journal = {BMC plant biology},
volume = {26},
number = {1},
pages = {},
pmid = {41808028},
issn = {1471-2229},
support = {GSGT.2019.016//Nederlandse Organisatie voor Wetenschappelijk Onderzoek/ ; TU18048//Top Sector Agri and Food/ ; },
mesh = {*Solanum lycopersicum/genetics ; *Protoplasts/metabolism ; *Chromosome Inversion/genetics ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {BACKGROUND: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 enables induction of chromosomal inversions from hundreds of base pairs to millions of base pairs, but the factors influencing inversion frequency are not well understood. Prior reports differ in species, detection methods, and delivery strategies, making direct comparisons difficult. We addressed this by introducing a normalisation strategy based on a reference guide RNA (gRNA) as an internal standard and testing inversion sizes spanning kilobases to tens of megabases in tomato protoplasts. RESULTS: Tomato (Solanum lycopersicum) protoplasts were transfected with constructs encoding a fixed “reference” gRNA and a second “variable” gRNA positioned at increasing genomic distances, creating potential inversions from 1 kilobase to 37.5 megabases. Using the reference gRNA to normalise across samples, we found that up to ~ 1 megabase, inversion frequency tracked the cutting efficiency of the less efficient gRNA, consistent with gRNA activity being a major contributor within the chromosome tested. For these intervals, the inversion frequencies reached up to 1.24% when both gRNAs were efficient. Above ~ 1 megabase, inversion frequencies declined sharply despite efficient cutting, suggesting a size-dependent barrier to inversion formation; for example, 37.5 megabase inversions occurred at substantially lower frequency (up to 0.18%) despite efficient gRNAs. Because each interval corresponds to a distinct genomic location. Inversions were only observed when both gRNAs were active, and large deletions were more frequent than inversions when dual breaks were induced. CONCLUSIONS: In our experiments, the gRNA cutting efficiency was a major determinant of inversion frequency in our experiment up to ~ 1 megabase, while locus-specific genomic context may also contribute., Larger inversions may be limited by an additional, size-dependent constraint. These findings inform the design of edits aimed at reverting breeding-relevant inversions (for example, those linked to resistance loci) and suggest that achieving high efficiency for multi-megabase inversions will require strategies that overcome spatial or repair-related constraints. The internal reference gRNA normalises sample-to-sample variability in DNA delivery and Cas9 activity, enabling direct comparison of the performance of different gRNAs across samples on a shared, ratio-based scale. This ratio-to-reference strategy may likewise be used to benchmark gRNA performance and edit yields (inversions, deletions, translocations, and base/prime edits) across transfections in plant protoplasts, and may be extended to additional cell systems beyond plants.},
}

*Solanum lycopersicum/genetics
*Protoplasts/metabolism
*Chromosome Inversion/genetics
*CRISPR-Cas Systems/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41817895,
year = {2026},
author = {Prins, TJ and Lai, TJ and Li, T and Fisher, A and Eldred, BSC and Mostafavi, R and Liau, LM and Chong, RA and Nghiemphu, PL and Cloughesy, TF and Nathanson, DA and Lai, A},
title = {MGMT downregulation by CRISPR/Cas13 RNA-guided RNA targeting enhances glioma cell sensitivity to TMZ chemotherapy.},
journal = {Journal of neuro-oncology},
volume = {177},
number = {1},
pages = {},
pmid = {41817895},
issn = {1573-7373},
support = {P50 CA211015/CA/NCI NIH HHS/United States ; P50 CA211015-01A1/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; Temozolomide ; *Tumor Suppressor Proteins/metabolism/genetics ; *Glioma/drug therapy/metabolism/genetics ; *Antineoplastic Agents, Alkylating/pharmacology ; *DNA Modification Methylases/metabolism/genetics ; *DNA Repair Enzymes/metabolism/genetics ; *Brain Neoplasms/drug therapy/metabolism/genetics ; Down-Regulation ; Cell Line, Tumor ; RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; *Dacarbazine/analogs & derivatives/pharmacology ; Gene Expression Regulation, Neoplastic/drug effects ; Drug Resistance, Neoplasm ; RNA, Messenger/metabolism ; Cell Survival/drug effects ; },
abstract = {BACKGROUND: Current standard of care for glioblastoma involves fractionated radiotherapy administered with Temozolomide (TMZ), a DNA-alkylating agent. Inhibition of the DNA repair enzyme, O[6]-methylguanine-DNA methyltransferase (MGMT), promotes sensitivity to TMZ, particularly in tumors that repress MGMT mRNA transcription through promoter methylation. Novel strategies to inhibit MGMT are a promising avenue to improve therapeutic outcomes to TMZ. We hypothesized that CRISPR-Cas13-mediated RNA regulatory silencing of MGMT mRNA enhances response of immortalized and primary patient-derived gliomaspheres to TMZ in vitro. METHODS: We utilized the Cas13x and Cas13d variants to target MGMT mRNA in the MGMT-expressing LN18 glioma cell line and in two patient-derived gliomasphere lines (GS104, GS081). Cas13-guide RNA ribonucleoproteins were delivered via lipofection, and stable knockdown was achieved using a lentiviral all-in-one system. MGMT mRNA and protein downregulation were assessed by RT-PCR and Western blot, respectively. Cell viability and chemosensitivity to TMZ were evaluated using MTT assays. RESULTS: Both Cas13x and Cas13d systems, directed by specific guide CRISPR RNAs, achieved rapid and potent knockdown of MGMT mRNA and protein in all tested cell lines. This downregulation of MGMT expression led to an increase in the cytotoxic effects of TMZ, sensitizing previously resistant glioma cells and patient-derived gliomaspheres to standard chemotherapy. The lentiviral Cas13d system established stable chemosensitization in gliomasphere models. CONCLUSION: CRISPR-Cas13-mediated targeting of MGMT mRNA is an effective strategy for overcoming TMZ resistance in in vitro glioblastoma models. This RNA regulatory editing approach offers a proof-of-principle for CRISPR mediated therapeutics in patients with MGMT unmethylated gliomas.},
}

Humans
Temozolomide
*Tumor Suppressor Proteins/metabolism/genetics
*Glioma/drug therapy/metabolism/genetics
*Antineoplastic Agents, Alkylating/pharmacology
*DNA Modification Methylases/metabolism/genetics
*DNA Repair Enzymes/metabolism/genetics
*Brain Neoplasms/drug therapy/metabolism/genetics
Down-Regulation
Cell Line, Tumor
RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems
*Dacarbazine/analogs & derivatives/pharmacology
Gene Expression Regulation, Neoplastic/drug effects
Drug Resistance, Neoplasm
RNA, Messenger/metabolism
Cell Survival/drug effects

@article {pmid41839945,
year = {2026},
author = {Yanguas-Casás, N and Pedrosa, L and Horcajo, B and Gómez, S and Garcia-Grande, A and Muñoz-Viana, R and Fernández-Miranda, I and Pérez-Aguilera, M and Torres-Ruiz, R and Rodríguez-Perales, S and Sánchez-Beato, M},
title = {Splice-site mutations in POU2AF1 are associated with B-cell lymphomagenesis and therapeutic response.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41839945},
issn = {2045-2322},
support = {POSTD18029SANC//Fundación Científica de la Asociación Española Contra el Cáncer/ ; iPFIS predoctoral fellowship (IFI18/0004)//ISCIII-MINECO AES-FEDER, Plan Estatal I+D+I 2014-2020/ ; PEJ-2020-TL/BMD-19530//Plan de Empleo Juvenil de la CM/ ; PEJ-2023-AI/SAL-GL-28806//Plan de Empleo Juvenil de la CM/ ; B2017/BMD-3778//Dirección General de Universidades e Investigación de la Consejería de Educación e Investigación de la Comunidad de Madrid (CM)/ ; PI17/00272, PI20/00591, PI23/01587//Spanish Ministry of Economy and Competence (MINECO) and Instituto de Salud Carlos III (ISCIII), ISCIII-MINECO AES-FEDER/ ; },
mesh = {Humans ; Cell Line, Tumor ; *RNA Splice Sites/genetics ; *Lymphoma, B-Cell/genetics/pathology/drug therapy ; Cell Proliferation/genetics ; *Mutation ; Cell Movement/genetics ; *Trans-Activators/genetics ; Point Mutation ; CRISPR-Cas Systems ; },
abstract = {BOB.1, encoded by POU2AF1, is one of many factors regulating physiological B-cell maturation in the germinal center. Recently, several studies have described recurrent mutations in a three-nucleotide region in the POU2AF1 splice site in the two most common B-cell non-Hodgkin lymphomas: diffuse large B-cell lymphoma and, more frequently, follicular lymphoma. In this study, we introduced a C→G mutation at the + 1 position of the POU2AF1 splice site in two B-cell lymphoma cell lines (WSU-NHL and SUDHL4) using CRISPR/Cas9 gene editing. Our results demonstrate how point mutations in the POU2AF1 splice site decreased BOB.1 expression levels. The mutation did not produce significant changes in cell proliferation, migration, or invasiveness, but did affect cell morphology, aggregation, and cell survival in a cell-line-dependent manner. Lastly, we found that the POU2AF1 mutation c.16 + 1G > C increased BCR activation, especially in SUDHL4 cells, downregulated oxidative phosphorylation (OxPhos) metabolism, and modified therapy sensitivities in both cell lines. Mutated B-cells were more sensitive to the BTK inhibitor ibrutinib. In conclusion, mutations in the POU2AF1 splice site impact B-cell lymphomagenesis at multiple levels and represent a potential therapeutic target for patients with tumors harboring this mutation.},
}

Humans
Cell Line, Tumor
*RNA Splice Sites/genetics
*Lymphoma, B-Cell/genetics/pathology/drug therapy
Cell Proliferation/genetics
*Mutation
Cell Movement/genetics
*Trans-Activators/genetics
Point Mutation
CRISPR-Cas Systems

@article {pmid41839984,
year = {2026},
author = {Taguchi, YH and Turki, T},
title = {Gene and cell line efficiency of CRISPR computed by tensor decomposition in genome-wide CRISPR-Cas9 knockout screens.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41839984},
issn = {2045-2322},
support = {Deanship of Scientific Research, IPP: 70-611-2025//King Abdulaziz University/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Knockout Techniques/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Cell Line ; Algorithms ; },
abstract = {Genome-wide CRISPR-Cas9 knockout screens are often used to experimentally evaluate gene function. However, the efficacy of individual sgRNAs targeting unique genes varies and is difficult to integrate. In this study, tensor decomposition (TD) was used to integrate multiple sgRNAs and sgRNA profiles simultaneously. Thus, TD can discriminate between essential and non-essential genes with the performance comparative to that of Joint analysis of CRISPR/Cas9 knockout screens (JACKS), a type of SOTA that previously outperformed various other SOTA. In addition, although TD uses simple linear algebra, it can achieve good performance even without control samples, without which JACKS cannot be performed. Moreover, because raw and logarithmic values can achieve similar performances through TD for the largest dataset among the tested datasets, taking logarithmic values as has been done frequently, which is questioned. In conclusion, TD is the first method that can integrate multiple sgRNAs attributed to single a target and sgRNA profiles at the beginning simultaneously and can achieve a performance comparable to that of JACKS.},
}

*CRISPR-Cas Systems
*Gene Knockout Techniques/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Humans
Cell Line
Algorithms

@article {pmid41888883,
year = {2026},
author = {Yuan, G and Deng, S and Dai, Z and Hofstad, BA and Pomraning, KR},
title = {Expanding the genetic toolkit: adenine and cytosine base editors for gene disruption in Aspergillus niger.},
journal = {Microbial cell factories},
volume = {25},
number = {1},
pages = {},
pmid = {41888883},
issn = {1475-2859},
mesh = {*Aspergillus niger/genetics ; *Cytosine/metabolism ; *Gene Editing/methods ; *Adenine/metabolism ; CRISPR-Cas Systems ; },
abstract = {Despite revolutionizing fungal genetic engineering, conventional CRISPR/Cas9-mediated knockouts rely on DNA double-strand breaks (DSBs), which can cause unwanted insertions and deletions, chromosomal abnormalities, and cytotoxicity. Base editors such as adenine base editors (ABEs), which convert A‧T to G‧C, and cytosine base editors (CBEs), which convert C‧G to T‧A, offer a safer alternative by enabling predictable, target-specific single-nucleotide changes without introducing DSBs. To overcome the limitations of traditional genome editing in filamentous fungi, we developed efficient base-editing systems in Aspergillus niger. For the first time, we constructed an ABE in A. niger, achieving up to 80% editing efficiency and inducing predictable A-to-G mutations at the intended intron sites, disrupting gene function through mRNA mis-splicing. We also developed a highly efficient CBE system, capable of introducing premature stop codons with 50–100% efficiency. To broaden the editing scope, we implemented a Cas9-NG variant recognizing a relaxed PAM sequence requiring only a single guanine (G), enabling editing at start codons and splice sites. Leveraging this expanded scope, we established gene disruption approaches by targeting start codons via ABE-mediated A-to-G conversions (ATG-to-GTG and ATG-to-ACG) and CBE-mediated C-to-T conversion (ATG-to-ATA). Additionally, our base-editing systems enable multiplex gRNA delivery and marker-free editing of multiple genes. Collectively, the scope-expanding strategies increase the number of genes targetable for disruption by base-editing in A. niger by 26.3% and enable near-complete coverage of 96% of the coding genes. Overall, this work demonstrates the potential of ABE and CBE systems as versatile, efficient, and safer alternatives to DSBs-based gene disruption in filamentous fungi.},
}

*Aspergillus niger/genetics
*Cytosine/metabolism
*Gene Editing/methods
*Adenine/metabolism
CRISPR-Cas Systems

@article {pmid41961403,
year = {2026},
author = {Sun, J and Hu, Z and Yang, Y},
title = {An ATMT-CRISPR/Cas9 system for genome editing in Monascus purpureus.},
journal = {World journal of microbiology & biotechnology},
volume = {42},
number = {4},
pages = {},
pmid = {41961403},
issn = {1573-0972},
mesh = {*Monascus/genetics/metabolism ; *CRISPR-Cas Systems ; *Agrobacterium tumefaciens/genetics ; *Gene Editing/methods ; Transformation, Genetic ; Homologous Recombination ; Genome, Fungal ; Gene Knockout Techniques ; Protoplasts ; Biosynthetic Pathways/genetics ; Fungal Proteins/genetics ; },
abstract = {Monascus purpureus is a filamentous fungus of significant economic value in the food and pharmaceutical industries, capable of producing a diverse array of secondary metabolites. Although CRISPR/Cas9 systems have been extensively utilized in filamentous fungi, predominantly employing protoplasts as recipients, the genetic manipulation of M. purpureus remains challenging due to the inherent difficulties associated with protoplast preparation. In addition, it has been reported that the Cas9 protein may demonstrate toxicity to cells. To overcome this limitation, this study developed a CRISPR/Cas9 gene-editing system based on Agrobacterium tumefaciens -mediated transformation (ATMT). This system utilizes M. purpureus spores as recipients and employs a homologous recombination strategy to achieve stable, site-specific integration of the Cas9 expression cassette into non-coding regions of the host genome, thereby avoiding the complexity of protoplast preparation and the uncertainty of random integration events. System evaluations indicate that the stable expression of Cas9 protein has no significant adverse effects on the nutritional growth, reproductive development, or characteristic pigment biosynthesis of M. purpureus. The system demonstrated high efficacy in single-gene editing, achieving a knockout efficiency of 74% for the key pigment biosynthetic pathway gene pksPT. Although efficiency decreased when performing double-gene and triple-gene editing (4.8% and 1.7%, respectively), this study successfully validated the system’s potential for multi-gene genome engineering. The ATMT-CRISPR/Cas9 system established herein demonstrates the feasibility of genetic manipulation of M. purpureus, providing a methodological foundation with the potential to facilitating functional genomic studies and the targeted regulation of secondary metabolism in this industrially important fungus.},
}

*Monascus/genetics/metabolism
*CRISPR-Cas Systems
*Agrobacterium tumefaciens/genetics
*Gene Editing/methods
Transformation, Genetic
Homologous Recombination
Genome, Fungal
Gene Knockout Techniques
Protoplasts
Biosynthetic Pathways/genetics
Fungal Proteins/genetics

@article {pmid42000863,
year = {2026},
author = {Piñeiro-Silva, C and Bermejo-Álvarez, P and García-Purriños, FJ and Gadea, J},
title = {Gene editing of the GJB2 locus in porcine embryos using CRISPR/Cas9 and cytosine base editors: toward a model of congenital deafness.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42000863},
issn = {2045-2322},
support = {22065/PI/22//Fundación Séneca-Agencia de Ciencia y Tecnología de la Región de Murcia/ ; 22545/PDC/24//Fundación Séneca-Agencia de Ciencia y Tecnología de la Región de Murcia/ ; 23031/GERM/25 supported by FSRM/10.13039/100007801//Fundación Séneca-Agencia de Ciencia y Tecnología de la Región de Murcia/ ; R-496/2022//Universidad de Murcia predoctoral fellowship/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Connexin 26/genetics ; *Cytosine/metabolism ; Swine ; *Deafness/genetics/congenital ; *Connexins/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Female ; Mutation ; Disease Models, Animal ; Oocytes/metabolism ; },
abstract = {Mutations in the GJB2 gene, which encodes Connexin 26 (Cx26), are responsible for the majority of cases of non-syndromic congenital hearing loss in humans. While murine GJB2 knockout models have provided mechanistic insight, anatomical and physiological differences limit their translational relevance. Pigs represent a valuable large-animal model because their auditory anatomy and maturation closely resemble those of humans. This study compared two genome-editing approaches to disrupt GJB2 in porcine oocytes before fertilization: (1) electroporation with CRISPR/Cas9 ribonucleoprotein and (2) microinjection with cytosine base editor (BE3) and single-guide RNAs (sgRNAs). Electroporation produced high mutation rates (70–90%) across three concentrations of Cas9/sgRNA but yielded mostly heterozygous or mosaic blastocysts, with limited homozygous knockouts (< 4%). BE3 achieved precise cytosine-to-thymine conversions that introduced premature stop codons, reaching up to 47% total editing and 20% homozygous nonsense alleles. However, blastocyst formation declined at higher component concentrations. Overall, BE3 produced more predictable mutations than conventional CRISPR/Cas9, although embryo developmental competence was dose-dependent. Both methods effectively targeted GJB2 and demonstrated feasibility of pre-fertilization genome editing in porcine oocytes. These findings establish the groundwork for generating GJB2-deficient pigs as translational models of Cx26-related congenital deafness and for future evaluation of gene-therapy strategies in a large-animal system.},
}

Animals
*CRISPR-Cas Systems
*Gene Editing/methods
*Connexin 26/genetics
*Cytosine/metabolism
Swine
*Deafness/genetics/congenital
*Connexins/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Female
Mutation
Disease Models, Animal
Oocytes/metabolism

@article {pmid42012544,
year = {2026},
author = {Shen, W and Xiao, R and Li, J and Zheng, S and Wang, C and Hao, R and Yin, J},
title = {CRISPR-based dual-mode lateral flow assay driven by magnetic SERS tags for highly sensitive detection of respiratory viruses.},
journal = {Mikrochimica acta},
volume = {193},
number = {5},
pages = {},
pmid = {42012544},
issn = {1436-5073},
support = {82372348//National Natural Science Foundation of China/ ; },
mesh = {*Spectrum Analysis, Raman/methods ; *SARS-CoV-2/isolation & purification/genetics ; Humans ; *Influenza A Virus, H1N1 Subtype/isolation & purification/genetics ; Gold/chemistry ; Colorimetry/methods ; Rapid Diagnostic Tests ; Limit of Detection ; CRISPR-Cas Systems ; COVID-19/diagnosis/virology ; },
abstract = {Effective epidemic control hinges on rapid point-of-care detection of respiratory viruses, but the sensitivity of current screening technologies remains inadequate. Here, we developed a CRISPR-activated, colorimetric Surface-Enhanced Raman Scattering (SERS) dual-mode nucleic acid lateral flow assay (LFA) that enables highly sensitive, flexible, and simultaneous detection of two common respiratory viruses, influenza A (H1N1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). By coupling recombinase polymerase amplification with CRISPR for sensitive amplification and specific target recognition, viral targets activate Cas13 trans-cleavage to efficiently cleave reporter probes. In parallel, a dual-functional magnetic SERS tag (Fe3O4@Au/Au-SA) was introduced, which captures two biotinylated reporter molecules through streptavidin modification, and generates strong and stable SERS signals through built-in hotspot effects. The resulting CRISPR-magnetic SERS-LFA enables rapid qualitative screening of H1N1 and SARS-CoV-2 through reduced colorimetric signal intensity on two test lines and accurate quantification via SERS signal changes. The establish method achieves a detection limit of 7–9 copies/µL for the two target viruses and shows good agreement with quantitative reverse transcription polymerase chain reaction (qRT-PCR) in validation with 74 clinical samples. Both the sensitivity and specificity for clinical samples reach 100%, highlighting its potential for field deployment.},
}

*Spectrum Analysis, Raman/methods
*SARS-CoV-2/isolation & purification/genetics
Humans
*Influenza A Virus, H1N1 Subtype/isolation & purification/genetics
Gold/chemistry
Colorimetry/methods
Rapid Diagnostic Tests
Limit of Detection
CRISPR-Cas Systems
COVID-19/diagnosis/virology

@article {pmid42015157,
year = {2026},
author = {Yang, P and Ma, R and Zeng, J and Li, L and Peng, J and Zhou, M and Qu, M and Li, X and Lai, T and Zhou, W and Wu, Y and Lu, Y and Zhang, Y},
title = {Simple and rapid profiling of tumor EVs for differential diagnosis of NSCLC via orthogonal barcode-driven CRISPR/Cas12a.},
journal = {Journal of nanobiotechnology},
volume = {24},
number = {1},
pages = {},
pmid = {42015157},
issn = {1477-3155},
support = {82172374//National Natural Science Foundation of China/ ; 2023GDRC005//Chongqing Science and technology joint major funding program/ ; 2022CDJYGRH-010//Natural Science Foundation Project of Chongqing, Chongqing Science and Technology Commission/ ; 2021M693730//China Postdoctoral Science Foundation/ ; },
mesh = {Humans ; *Carcinoma, Non-Small-Cell Lung/diagnosis ; *Lung Neoplasms/diagnosis ; *CRISPR-Cas Systems/genetics ; *Extracellular Vesicles/metabolism ; Biomarkers, Tumor/blood ; Diagnosis, Differential ; Cell Line, Tumor ; },
abstract = {Tumor-derived extracellular vesicles (tEVs), a class of nanoscale vesicles actively released by malignant cells, have emerged as attractive biomarkers for non-invasive cancer diagnosis. However, their clinical translation remains challenging due to low abundance, molecular heterogeneity, and the requirement for multiplexed surface marker discrimination. Here, we report a dual aptamer-mediated CRISPR/Cas12a-assisted sensing platform (DA-CAS) for rapid and orthogonally programmable dual-marker profiling of tEVs, enabling differential diagnosis of non-small cell lung cancer (NSCLC) using only 10 µL of plasma within 100 min. The DA-CAS system integrates proximity ligation-based dual-marker recognition with hyperbranched rolling circle amplification (HRCA) to generate programmable DNA barcodes, which selectively trigger Cas12a trans-cleavage in an orthogonal manner. Using EpCAM and PD-L1 as representative surface markers, the platform achieves subtype-specific detection of NSCLC-derived tEVs with minimal background activation and a detection limit as low as 75 particles/mL. Moreover, a portable lateral flow readout enables accurate, instrument-free visual detection at concentrations down to 406 particles/mL. Under the condition of free-ultracentrifugation, clinical validation using a cohort of 45 plasma samples demonstrated a sensitivity of 97%, specificity of 88%, and overall diagnostic accuracy of 96%, outperforming conventional ELISA assays and multi-marker serum panels in both analytical sensitivity and subtype resolution. In addition, this platform demonstrated preliminary potential for discriminating between benign and malignant lung diseases and for dynamically monitoring radiotherapeutic responses. The orthogonal barcode design effectively eliminates inter-channel crosstalk and enzymatic interference, enabling orthogonal dual-target recognition with high subpopulation specificity. Overall, DA-CAS provides a robust, rapid, and point-of-care-compatible strategy for tEV-based cancer diagnostics, offering strong translational potential for non-invasive tumor profiling and dynamic immune status monitoring.},
}

Humans
*Carcinoma, Non-Small-Cell Lung/diagnosis
*Lung Neoplasms/diagnosis
*CRISPR-Cas Systems/genetics
*Extracellular Vesicles/metabolism
Biomarkers, Tumor/blood
Diagnosis, Differential
Cell Line, Tumor

@article {pmid42031935,
year = {2026},
author = {Hao, D and Xu, X and Li, P and Liu, Y and Liu, G and Deng, S},
title = {A method for CRISPR/Cas9-induced genetic barcoding and lineage tracing in sheep.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42031935},
issn = {2045-2322},
support = {2023-PT180-01//Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Sheep/genetics/embryology ; *Cell Lineage/genetics ; DNA Transposable Elements/genetics ; *Gene Editing/methods ; *DNA Barcoding, Taxonomic/methods ; Embryonic Development/genetics ; },
abstract = {Sheep development involves continuous dynamic processes in which cells propagate, differentiate and orchestrate. However, the method for tracing cell fate during sheep (Ovis aries) embryogenesis, cell differentiation and tissue regeneration remains largely undeveloped. Here, we developed a CRISPR/Cas9-based lineage barcode recording method that directly acts on target cells. With this method, several contiguous CRISPR/Cas9 targeting arrays were synthesized and introduced into multiple loci in the sheep genome using the PiggyBac transposon vector to form the barcode region. Cas9 integrated at the Rosa26 gene locus was used to generate edits in the barcode region at multiple timepoints during early sheep embryonic development. We detected multiple integrated barcodes (intBCs) that were stably inherited in the developing embryos, confirming that the method can generate heritable clonal markers. The method could enable lineage tracing in sheep when combined with single-cell sequencing technologies. Our method establishes a foundation for ruminant lineage tracing technology by combining PiggyBac transposons and CRISPR/Cas9 gene editing tools, providing a new platform for analyzing sheep embryonic development, organ regeneration, and disease mechanisms.},
}

Animals
*CRISPR-Cas Systems
Sheep/genetics/embryology
*Cell Lineage/genetics
DNA Transposable Elements/genetics
*Gene Editing/methods
*DNA Barcoding, Taxonomic/methods
Embryonic Development/genetics

@article {pmid42050530,
year = {2026},
author = {Liao, X and Zhou, J and Shan, Y and Li, X and Peng, Y},
title = {CRISPR/Cas12a-assisted visual and on-site detection of porcine circovirus type 2.},
journal = {BMC veterinary research},
volume = {22},
number = {1},
pages = {},
pmid = {42050530},
issn = {1746-6148},
support = {2024NSFSC0761//the Science and Technology Foundation of Sichuan Province, China/ ; 2021R52041//High-level Talents Special Support Plan of Zhejiang Province/ ; ZYGD23027//the 1·3·5 Project for Disciplines of Excellence, West China Hospital of Sichuan University/ ; },
mesh = {*Circovirus/isolation & purification/genetics ; Animals ; Swine ; *CRISPR-Cas Systems ; *Circoviridae Infections/veterinary/diagnosis/virology ; *Swine Diseases/diagnosis/virology ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Rapid Diagnostic Tests ; Sensitivity and Specificity ; DNA, Viral ; },
abstract = {BACKGROUND: Porcine circovirus type 2 (PCV2) is a globally prevalent viral pathogen that causes substantial economic losses in the swine industry. Rapid and accurate on-site diagnosis is critical for controlling the spread of PCV2. In recent years, RNA-guided CRISPR/Cas12a nucleases combined with recombinase polymerase amplification (RPA) have emerged as a promising approach for nucleic acid detection. This study aimed to develop a novel RPA-CRISPR-based method for the rapid and sensitive detection of PCV2 in field settings. RESULTS: We designed and optimized CRISPR RNAs (crRNAs) targeting conserved regions of the PCV2 Cap and Rep genes. Upon recognition of the target sequence, the Cas12a nuclease was activated to cleave a single-stranded DNA-fluorophore quencher (ssDNA-FQ) reporter, generating a fluorescent signal detectable either by a fluorescence detector or via visual readout. The entire procedure was performed at 37 °C and completed within one hour. The assay achieved a detection limit as low as 10 copies/µL and showed no cross-reactivity with other major porcine viruses. Furthermore, a rapid-release reagent was used to replace conventional DNA extraction from serum samples, facilitating efficient on-site detection. The assay was validated using clinical samples, and the results showed strong concordance with those obtained by PCR. CONCLUSIONS: The RPA-CRISPR-based assay developed in this study is highly sensitive and specific, enabling detection of PCV2 within one hour. Its simplicity, rapidity, and ease of use in the field offer significant practical advantages, making it a valuable tool for the on-site diagnosis of PCV2. This method represents a promising alternative for the early and rapid detection of PCV2 infections and holds potential for contributing to the prevention and control of the disease in the swine industry.},
}

*Circovirus/isolation & purification/genetics
Animals
Swine
*CRISPR-Cas Systems
*Circoviridae Infections/veterinary/diagnosis/virology
*Swine Diseases/diagnosis/virology
*Nucleic Acid Amplification Techniques/veterinary/methods
Rapid Diagnostic Tests
Sensitivity and Specificity
DNA, Viral

@article {pmid40506596,
year = {2025},
author = {Sajjad, MW and Muzamil, F and Naqvi, RZ and Amin, I},
title = {QBEmax redefines the precise base editing in crop plants.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {127},
pmid = {40506596},
issn = {1438-7948},
mesh = {*Crops, Agricultural/genetics ; *CRISPR-Cas Systems ; Genome, Plant ; Cytidine Deaminase/genetics/chemistry/metabolism ; *Gene Editing ; },
abstract = {Hu et al.‘s new study, published in Nature Biotechnology, introduces QBEmax; a tiny, conformationally sound editing tool with a cytidine deaminase buried within a looping permuted Cas9 (cpCas9). Supported by molecular dynamics models and AlphaFold3 structural predictions, this unique internal fusion creates a structurally protected complex that improves editing accuracy and lowers typical artifacts such as indels and impure base conversions (Hu et al. Nature Biotechnology:1-7, 2025). High precision editing (up to 99.8% purity), lower indels, and lower off target impacts well suit imminent plant transformation events. Its tiny, stable architecture and wider editing window at PAM sites increase its ability for precise and adaptable trait change in complex plant genomes.},
}

*Crops, Agricultural/genetics
*CRISPR-Cas Systems
Genome, Plant
Cytidine Deaminase/genetics/chemistry/metabolism
*Gene Editing

@article {pmid40999513,
year = {2025},
author = {Lee, S and Yu, Y and Kim, DH and Bock, M and Kim, Y and An, SB and Choi, H and Shin, HE and Hwang, DY and Han, I},
title = {Enhanced disc regeneration through CRISPR/Cas9-mediated SOX9 and TGFβ1 coexpression in tonsil-derived mesenchymal stromal cells.},
journal = {Stem cell research & therapy},
volume = {16},
number = {1},
pages = {501},
pmid = {40999513},
issn = {1757-6512},
support = {RS-2024-00347403//National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT: Ministry of Science and ICT)/ ; RS-2023-KH141187//Korean Cell-Based Artificial Blood Project funded by the Korean government (The Minis-try of Science and ICT, The Ministry of Trade, Industry and Energy, the Ministry of Health & Welfare, the Ministry of Food and Drug Safety)/ ; },
mesh = {*SOX9 Transcription Factor/genetics/metabolism ; *Transforming Growth Factor beta1/genetics/metabolism ; *Mesenchymal Stem Cells/metabolism/cytology ; Animals ; Rats ; Humans ; *Intervertebral Disc Degeneration/therapy/genetics/pathology/metabolism ; *CRISPR-Cas Systems/genetics ; *Regeneration ; Rats, Sprague-Dawley ; *Palatine Tonsil/cytology/metabolism ; *Intervertebral Disc/physiology/metabolism ; Male ; Cell Differentiation ; Chondrogenesis ; },
abstract = {BACKGROUND: Intervertebral disc (IVD) degeneration, a primary cause of chronic low back pain, currently lacks treatments that target its underlying pathological mechanisms. Tonsil-derived mesenchymal stromal cells (ToMSCs) have shown promise for IVD regeneration; however, their therapeutic potential is limited by the harsh microenvironment of degenerated discs. This study investigated whether ToMSCs engineered to co‐overexpress SOX9 and TGFβ1 using a tetracycline‐off (Tet‐off) regulatory system could enhance extracellular matrix (ECM) restoration and reduce inflammation in degenerative IVDs. METHODS: We used CRISPR/Cas9 technology to generate ToMSCs that express SOX9, TGFβ1, or both factors under Tet-off regulation. Gene expression was confirmed by Western blot and qRT-PCR analyses. In vitro studies assessed chondrogenic differentiation capacity, while in vivo assessments were performed using a rat tail needle puncture model of IVD degeneration. After administering the CRISPR-engineered ToMSCs, we monitored mechanical allodynia with the von Frey test over six weeks. Therapeutic outcomes were evaluated through T2‐weighted MRI and histological analysis. RESULTS: In vitro experiments showed that ToMSCs co-expressing SOX9 and TGFβ1 exhibited superior chondrogenic differentiation compared to cells expressing a single factor. In vivo studies demonstrated that dual-factor expressing ToMSCs significantly improved disc hydration (as confirmed by MRI), enhanced ECM synthesis—particularly aggrecan and type II collagen—and reduced inflammation compared to single-factor treatments. These improvements were accompanied by reduced mechanical allodynia, indicating functional recovery. CONCLUSION: Our study demonstrates that ToMSCs engineered to co-express SOX9 and TGFβ1 effectively promote IVD regeneration by enhancing ECM production and reducing inflammation. This dual-factor approach represents a promising therapeutic strategy for treating degenerative disc disease and warrants further investigation for clinical application.},
}

*SOX9 Transcription Factor/genetics/metabolism
*Transforming Growth Factor beta1/genetics/metabolism
*Mesenchymal Stem Cells/metabolism/cytology
Animals
Rats
Humans
*Intervertebral Disc Degeneration/therapy/genetics/pathology/metabolism
*CRISPR-Cas Systems/genetics
*Regeneration
Rats, Sprague-Dawley
*Palatine Tonsil/cytology/metabolism
*Intervertebral Disc/physiology/metabolism
Male
Cell Differentiation
Chondrogenesis