@article {pmid40814875,
year = {2026},
author = {Rana, AJ and Hussain, MS and Hanbashi, A and Kamli, F and Khan, G and Qadri, M and Al-Qahtani, SA and Khan, MG and Burle, SS and Jakhmola, V and Gupta, G},
title = {CRISPR-Edited Cell Lines: A New Era in Functional Oncology Research.},
journal = {Current pharmaceutical design},
volume = {32},
number = {13},
pages = {1027-1034},
pmid = {40814875},
issn = {1873-4286},
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; *Neoplasms/genetics/therapy/pathology/drug therapy ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Animals ; },
abstract = {The use of CRISPR-Cas9 to engineer cancer cell lines has made it possible to precisely examine how cancer cells react to different drugs and therapies. Some of the key improvements are in the use of Mediator Complex Subunit 12 (MED12)-knockout cells to study cell resistance to BRAF inhibitors, CRISPR models of epithelial-mesenchymal transition for breast cancer, and pharmacogenomic analysis in various cancer cell lines. CRISPR is used in immunotherapy to help Chimeric Antigen Receptor T (CAR-T) cells function better by disrupting the immune checkpoints like Programmed Cell Death Protein 1 (PD-1) and Cytotoxic T-lymphocyte- associated protein 4 (CTLA-4) and to adapt T cells to react with various antigens. As a result of these innovations, it is now possible to track how cancers like non-small cell lung cancer (NSCLC) and ovarian cancer evolve, change their epigenetic features, and find strategies to reverse their resistance. Moving forward, integrating AI analytics, single-cell multi-omics, patient-derived organoids, and CRISPR mechanisms will help improve precision oncology and speed up effective treatment planning.},
}

Humans
*Gene Editing
*CRISPR-Cas Systems/genetics
*Neoplasms/genetics/therapy/pathology/drug therapy
Cell Line, Tumor
*Clustered Regularly Interspaced Short Palindromic Repeats
Animals

@article {pmid41101730,
year = {2026},
author = {Zhang, H and Tonini, MR and Martires, LCM and Jenkins, HN and Pratt, CB and Gordon, EN and Zhao, S and Choi, AH and Meier, SR and Khendu, T and Liu, S and Shen, B and Stowe, H and Pashiardis, K and Pan, X and Bandi, M and Zhang, M and Yu, Y and Min, C and Huang, A and Andersen, JN and Nicholson, HE and Teng, T},
title = {Tumor suppressor collateral damage screens reveal mRNA homeostasis protein HBS1L as a novel vulnerability in ch9p21 driven FOCAD deleted cancer.},
journal = {Biochimica et biophysica acta. Molecular cell research},
volume = {1873},
number = {1},
pages = {120070},
doi = {10.1016/j.bbamcr.2025.120070},
pmid = {41101730},
issn = {1879-2596},
mesh = {*GTP-Binding Proteins/genetics/metabolism ; *RNA, Messenger/metabolism ; Chromosome Deletion ; Chromosomes, Human, Pair 9 ; Genes, Tumor Suppressor ; Synthetic Lethal Mutations ; *Endonucleases/genetics/metabolism ; *Nuclear Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; Humans ; *Tumor Suppressor Proteins/genetics/metabolism ; *Neoplasms/genetics/pathology ; Cell Line, Tumor ; Xenograft Model Antitumor Assays ; Animals ; Mice ; Female ; },
abstract = {Chromosomal deletion of tumor suppressor genes often occurs in an imprecise manner, leading to co-deletion of neighboring genes. This collateral damage can create novel dependencies specific to the co-deleted context. One notable example is the dependency on PRMT5 activity in tumors with MTAP deletion, which co-occurs with CDKN2A/B loss, leading to the development of MTA-cooperative PRMT5 inhibitors. To identify additional collateral damage context/target pairs for chromosome 9p and other common loci of chromosomal deletions, we conducted a combinatorial CRISPR screen knocking out frequently co-deleted genes in combination with a focused target library. We identified the gene encoding the ribosome rescue factor PELO as synthetic lethal with the SKI complex interacting protein FOCAD, which is frequently co-deleted alongside MTAP and CDKN2A/B on chromosome 9p. A genome-wide screen in FOCAD isogenic cells further identified the ribosome rescue GTPase and PELO binding partner HBS1L as the top synthetic lethal target for FOCAD loss. Analysis of publicly available data and genetic manipulation of HBS1L using orthogonal modalities validated this interaction. HBS1L dependency in FOCAD-deleted cells was rescued by FOCAD re-expression, and FOCAD intact cells could be rendered HBS1L-dependent by FOCAD knockout, demonstrating the context specificity of this interaction. Mechanistically, HBS1L loss led to translational arrest and activated the unfolded protein response in FOCAD-deleted cells. In vivo, HBS1L deletion eliminated growth of FOCAD-deleted tumors. Here we propose a model where the FOCAD/SKI complex and HBS1L/PELO work together to resolve aberrant mRNA-induced ribosomal stalling, making the HBS1L/PELO complex an intriguing novel target for treating FOCAD-deleted tumors.},
}

*GTP-Binding Proteins/genetics/metabolism
*RNA, Messenger/metabolism
Chromosome Deletion
Chromosomes, Human, Pair 9
Genes, Tumor Suppressor
Synthetic Lethal Mutations
*Endonucleases/genetics/metabolism
*Nuclear Proteins/genetics/metabolism
CRISPR-Cas Systems/genetics
Gene Knockout Techniques
Humans
*Tumor Suppressor Proteins/genetics/metabolism
*Neoplasms/genetics/pathology
Cell Line, Tumor
Xenograft Model Antitumor Assays
Animals
Mice
Female

@article {pmid41159851,
year = {2026},
author = {Coppola, MR and Bellitto, D and Asgari, E and Bazzurro, V and Casucci, G and Piacente, F and Bozzo, M and Ceresa, D and Parisi, C and Winata, C and Candiani, S and Tonetti, M},
title = {Zebrafish as a model for Catel-Manzke syndrome-identification and characterization of the zebrafish TGDS ortholog.},
journal = {The FEBS journal},
volume = {293},
number = {8},
pages = {2248-2269},
doi = {10.1111/febs.70307},
pmid = {41159851},
issn = {1742-4658},
support = {GMR22T1065//Fondazione Telethon/ ; PRIN 2022 - grant 22ETJJES//Ministero dell'Università e della Ricerca/ ; },
mesh = {Animals ; *Zebrafish/genetics/embryology/metabolism ; Disease Models, Animal ; *Zebrafish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Mutation ; Gene Expression Regulation, Developmental ; Humans ; },
abstract = {Catel-Manzke syndrome (CMS) is a rare genetic disorder associated with mutations in the TDP-glucose 4,6-dehydratase (TGDS) gene, the function of which in vertebrates remains unclear. This study investigated the zebrafish ortholog tgds to assess its suitability for modeling the disease. During development, the tgds transcript exhibits a conserved biphasic expression pattern with an initial maternal contribution followed by a second wave of expression after gastrulation. Recombinant zebrafish Tgds expressed in Escherichia coli demonstrated UDP-D-glucose 4,6-dehydratase (EC4.2.1.76) activity, similar to TGDS orthologs in lower eukaryotes, where it acts as the first step in the L-rhamnose biosynthetic pathway. This finding suggests the presence of a yet unidentified pathway in vertebrates. Furthermore, CMS-associated mutations in conserved residues significantly impair enzyme activity and stability. CRISPR/Cas9-mediated F0 knockout of tgds resulted in a range of developmental defects in zebrafish. In particular, craniofacial cartilage alterations, associated with a decrease in sulfate glycosaminoglycan content, mirrored some skeletal features observed in humans with CMS. These findings establish the zebrafish as a relevant model to further explore CMS pathogenesis and the in vivo function of tgds.},
}

Animals
*Zebrafish/genetics/embryology/metabolism
Disease Models, Animal
*Zebrafish Proteins/genetics/metabolism
CRISPR-Cas Systems
Mutation
Gene Expression Regulation, Developmental
Humans

@article {pmid41851456,
year = {2026},
author = {Nyberg, WA and Bernard, PL and Ngo, W and Wang, CH and Ark, J and Rothrock, A and Borgo, GM and Kimmerly, GR and Jung, JH and Allain, V and Hamilton, JR and Baldwin, A and Stickels, R and Wyman, S and Khan, SH and Lang, S and Marsh, D and Almudhfar, N and Novick, C and Mortazavi, Y and Zhang, S and AbdElwakil, MM and Sandoval, LR and Hwang, S and Chu, SN and Jung, H and Liu, C and Sharma, D and McCreary, T and Li, Z and Satpathy, AT and Carnevale, J and Rutishauser, RL and Cromer, MK and Roybal, KT and Dodgson, SE and Doudna, JA and Asokan, A and Eyquem, J},
title = {In vivo site-specific engineering to reprogram T cells.},
journal = {Nature},
volume = {652},
number = {8110},
pages = {712-721},
pmid = {41851456},
issn = {1476-4687},
mesh = {Animals ; Mice ; *T-Lymphocytes/metabolism/cytology/immunology ; Receptors, Chimeric Antigen/genetics/metabolism/immunology ; CRISPR-Cas Systems/genetics ; Humans ; Transgenes/genetics ; *Gene Editing/methods ; Dependovirus/genetics ; Female ; *Cellular Reprogramming/genetics ; Genetic Vectors/genetics ; *Cell Engineering/methods ; Immunotherapy, Adoptive/methods ; Male ; },
abstract = {Engineered T cells, reprogrammed to express chimeric antigen receptors (CAR) or T cell receptors (TCR), have transformed cancer treatment and are being explored as therapeutics for autoimmune and infectious diseases. Enhancing T cell function through genome editing, either by disrupting endogenous genes or precisely inserting DNA payloads, has shown considerable promise[1]. However, the ex vivo manufacturing process is lengthy and costly, limiting accessibility of these therapies. In vivo generation of CAR T cells could overcome these barriers, but current methods rely either on transient expression with limited durability, or on random integration of DNA payloads that lack specificity. Here we demonstrate that stable and cell-specific transgene expression can be achieved through in vivo site-specific integration of large DNA payloads. We developed a two-vector system to deliver CRISPR-Cas9 ribonucleoproteins and a DNA donor template, using enveloped delivery vehicles and adeno-associated viruses, respectively. We optimized both vectors for T cell-specific delivery and gene-targeting efficiency. By integrating a CAR transgene into a T cell-specific locus, we generate therapeutic levels of CAR T cells in vivo in humanized mouse models of B cell aplasia, and haematological and solid malignancies. These findings offer a pathway to more efficient, precise and widely accessible T cell therapies.},
}

Animals
Mice
*T-Lymphocytes/metabolism/cytology/immunology
Receptors, Chimeric Antigen/genetics/metabolism/immunology
CRISPR-Cas Systems/genetics
Humans
Transgenes/genetics
*Gene Editing/methods
Dependovirus/genetics
Female
*Cellular Reprogramming/genetics
Genetic Vectors/genetics
*Cell Engineering/methods
Immunotherapy, Adoptive/methods
Male

@article {pmid41854269,
year = {2026},
author = {Fernandes, LGV and Putz, EJ and Nally, JE},
title = {The Leptospira immunoglobulin-like protein LigB from Leptospira borgpetersenii serovar Arborea is not required for either acute or chronic infection.},
journal = {Infection and immunity},
volume = {94},
number = {4},
pages = {e0066225},
doi = {10.1128/iai.00662-25},
pmid = {41854269},
issn = {1098-5522},
mesh = {Animals ; *Leptospirosis/microbiology/immunology ; *Leptospira/genetics/pathogenicity/immunology ; *Bacterial Proteins/genetics/metabolism ; Virulence Factors/genetics/metabolism ; Rats ; Virulence ; Persistent Infection/microbiology ; CRISPR-Cas Systems ; },
abstract = {Leptospirosis is a globally important zoonotic disease caused by more than 40 pathogenic Leptospira spp. that are responsible for more than 1 million human cases and almost 60,000 deaths annually. The disease also affects many companion, domestic, and wild animal species. Leptospiral immunoglobulin-like proteins (Lig), particularly LigA and LigB, are well-established surface membrane proteins that have been extensively studied due to their interactions with the host immune system. Silencing expression of both ligA and ligB in L. interrogans serovar Copenhageni results in attenuation of virulence, confirming their role as virulence factors. To examine the role of Lig proteins in other pathogenic species of Leptospira, we applied the CRISPR-Prime Editing (CRISPR-PE) strategy to engineer a one-nucleotide frameshift deletion in ligB, generating a knockout mutant in L. borgpetersenii serogroup Ballum serovar Arborea strain LR131, a pathogenic species that lacks ligA. Despite a complete loss of LigB expression, the mutant strain maintained its ability to cause acute lethal infection in hamsters and the ability to establish renal colonization in rats. These findings demonstrate that ligB alone is dispensable for both acute and chronic infection in the L. borgpetersenii strain LR131 background. This work represents the first targeted disruption of ligB in L. borgpetersenii as facilitated by CRISPR-PE and prompts a reevaluation of LigB as a universal virulence determinant in different genetic backgrounds. These insights are critical for advancing our understanding of leptospiral pathogenesis and guiding the design of broadly protective subunit vaccines.},
}

Animals
*Leptospirosis/microbiology/immunology
*Leptospira/genetics/pathogenicity/immunology
*Bacterial Proteins/genetics/metabolism
Virulence Factors/genetics/metabolism
Rats
Virulence
Persistent Infection/microbiology
CRISPR-Cas Systems

@article {pmid41954981,
year = {2026},
author = {Sentmanat, MF and Wang, ZT and Kouranova, E and Peters, ST and Chan, WC and Lin, J and Miao, Y and White, JM and Wallace, M and Cui, X},
title = {Efficient multi-kilobase knock-ins in mice and cell lines using CRISPR/Cas9 and rAAV donors with unbiased whole-genome characterization by LOCK-seq.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41954981},
issn = {1362-4962},
support = {//Alvin J. Siteman Cancer Center/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Mice ; *Gene Knock-In Techniques/methods ; *Dependovirus/genetics ; *Gene Editing/methods ; Cell Line ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Genome ; },
abstract = {Multi-kilobase knock-ins (KIs) are a necessary, yet challenging type of genome editing to create and characterize in cell lines and animals. The combination of rAAV donor transduction and electroporation of single-cell mouse embryos with Cas9/gRNA ribonucleoprotein complex enables highly efficient KI, but the insert size is limited by the viral packaging capacity. Here, we report the creation of up to 6.7 kb precise KI achieved in one step by using three rAAVs designed to insert one after the other. To fully characterize the edited genome with large KIs, we developed LOCK-seq (LOng-read sequencing of Captured Kilo-base targets), where relevant genomic regions are enriched via hybridization, achieving over 100-fold greater coverage compared with other long-read methods with enrichment. LOCK-seq simultaneously detects the presence of precise KI alleles, imprecision in the insert and donor concatenation, genotypes of non-KI alleles, and more importantly, uniquely identifies and localizes random integration of the full or partial donor(s). Additionally, the multi-rAAV donor approach is successfully applied to cell lines, including lines intolerant of plasmid DNA, whereas LOCK-seq reliably and efficiently screens for KI clones. Together, the two approaches significantly improve the creation and precision of knock-in models.},
}

Animals
*CRISPR-Cas Systems
Mice
*Gene Knock-In Techniques/methods
*Dependovirus/genetics
*Gene Editing/methods
Cell Line
RNA, Guide, CRISPR-Cas Systems/genetics
Humans
Genome

@article {pmid41954985,
year = {2026},
author = {Zhang, S and Liu, Y and Wu, W and Liu, Z and He, Q and Wang, T and Yang, J and Yin, H and Yuan, Z and Zhang, H},
title = {Allosteric activation mechanism of the type VII CRISPR-Cas system.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41954985},
issn = {1362-4962},
support = {32571682//National Natural Science Foundation of China/ ; 25JCZDJC00740//Natural Science Foundation of Tianjin Municipal Science and Technology Commission/ ; TJYXZDXK-009A//Tianjin Key Medical Discipline (Specialty) Construction Project/ ; TJYXZDXK-3-004B//Tianjin Key Medical Discipline (Specialty) Construction Project/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Allosteric Regulation ; *CRISPR-Associated Proteins/chemistry/metabolism/genetics ; Cryoelectron Microscopy ; RNA/metabolism/chemistry/genetics ; Catalytic Domain ; Models, Molecular ; Ribonucleases/chemistry/metabolism/genetics ; },
abstract = {Type VII CRISPR-Cas system, evolutionarily associated with type III systems, utilizes a Cascade complex formed by Cas5 and catalytically inactive Cas7 copies for target RNA binding, but instead incorporates a specialized Cas14 ribonuclease for target cleavage. Here, we report a high-quality cryo-EM structure at the target engagement state with a shortened crRNA and elucidate how the recruited Cas14 captures the target RNA and undergoes target-mediated activation. The signature Cas14 is homologous to eukaryotic CPSF73 and prokaryotic RNase J, comprising two conserved subdomains, MβL and β-CASP. Different from canonical type III systems, 5'-end target RNA, rather than 3'-end, is bent into the positively charged binding channel formed by the two subdomains to access the conserved catalytic pocket on Cas14. Two special structural features, α1 helix from Cas7 and α10 helix from Cas14, promote the bent target RNA docking into the catalytic pocket of Cas14 nuclease in concert. A dual-functional loop, displaced by the entering target RNA, induces a closed-to-open transition between the two subdomains for nuclease activation. More importantly, the flipped dual-functional loop also maintains the stabilization of incoming target RNA. Altogether, our work provides a more comprehensive understanding of type VII system mechanism, laying a mechanistic foundation for RNA-targeting tool development.},
}

*CRISPR-Cas Systems/genetics
Allosteric Regulation
*CRISPR-Associated Proteins/chemistry/metabolism/genetics
Cryoelectron Microscopy
RNA/metabolism/chemistry/genetics
Catalytic Domain
Models, Molecular
Ribonucleases/chemistry/metabolism/genetics

@article {pmid41956064,
year = {2026},
author = {Wang, M and Sternberg, SH},
title = {'Insane in the membrane': 2',3'-cGAMP triggers filamentous phage defense.},
journal = {Cell host & microbe},
volume = {34},
number = {4},
pages = {556-558},
doi = {10.1016/j.chom.2026.03.009},
pmid = {41956064},
issn = {1934-6069},
mesh = {*Nucleotides, Cyclic/metabolism ; *Inovirus/physiology ; Signal Transduction ; Humans ; Membrane Proteins/metabolism ; Nucleotidyltransferases/metabolism ; *Cell Membrane/metabolism/virology ; *Bacteria/metabolism/virology ; Animals ; },
abstract = {In this issue, Tak et al. report that bacteria produce 2',3'-cGAMP, a signaling molecule once considered unique to metazoans. This cyclic dinucleotide activates a SAVED-domain effector that polymerizes into membrane-disrupting filaments, inducing abortive infection and preventing phage replication, broadening the evolutionary scope of cGAS-STING-like signaling.},
}

*Nucleotides, Cyclic/metabolism
*Inovirus/physiology
Signal Transduction
Humans
Membrane Proteins/metabolism
Nucleotidyltransferases/metabolism
*Cell Membrane/metabolism/virology
*Bacteria/metabolism/virology
Animals

@article {pmid41956072,
year = {2026},
author = {Decloquement, M and Macauley, MS},
title = {Revealing cancer glycome drivers using CRISPR activation screens.},
journal = {Cell genomics},
volume = {6},
number = {4},
pages = {101215},
pmid = {41956072},
issn = {2666-979X},
mesh = {Humans ; *Neoplasms/genetics/metabolism ; Glycosylation ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Glycomics/methods ; },
abstract = {In this study, Wisnovsky and colleagues[1] investigate the genetic determinants underlying cancer-associated glycome remodeling. Using genome-wide CRISPR-based approaches, they identified regulators of cell surface glycosylation. Specifically, genetic networks are discovered that upregulate cancer-related ligands of the sialic acid-binding immunomodulatory Siglecs.},
}

Humans
*Neoplasms/genetics/metabolism
Glycosylation
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Glycomics/methods

@article {pmid41959582,
year = {2026},
author = {Carrasco, E and Gutierrez-Marcos, J},
title = {Novel genome editing approaches to manipulate apical meristem activity for crop yield.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1743528},
pmid = {41959582},
issn = {1664-462X},
abstract = {Meristem function underlies organogenesis and yield potential in crop species, and its regulation depends on the crosstalk of genetic and hormonal networks that balance stem-cell niche maintenance and differentiation. During the shoot apical meristem (SAM) transition, developmental reprogramming shifts the meristem from a vegetative to a reproductive state, referred to as inflorescence meristem (IM). Major regulatory events in this transition include the cytokinin-gibberellin crosstalk, that regulate the expression of the CLAVATA/WUSCHEL (CLV/WUS) negative feedback loop and key transcription factor families like KNOTTED-LIKE HOMEOBOX (KNOX) and SHOOT MERISTEMLESS (STM). Despite the basic principles of apical meristem differentiation are well-described nowadays, major phenotypic bottlenecks were reached in major staple crops during the artificial selection process, known as domestication, leading to a final reduction in total crop yield. This review aims to describe the key processes and genes that play a role in this transition and how they can be artificially targeted to overcome these limitations. Major bioengineering approaches are covered, ranging from classical random mutagenesis with chemicals like ethyl methanesulfonate (EMS) to targeted genome editing using diverse Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins (CRISPR/Cas) systems. Finally, emerging strategies such as epibreeding are considered as promising tools to achieve precise, reversible modulation of meristem activity and to unlock new routes for crop yield enhancement.},
}

@article {pmid41123029,
year = {2026},
author = {Burnase, N and Kumar, B and Kumar, PS and Dharshini, RS and Das, R and Barapatre, A},
title = {Advances in Antiviral Strategies for Oral Herpes Infections in Immunocompromised Patients.},
journal = {Oral diseases},
volume = {32},
number = {2},
pages = {300-321},
doi = {10.1111/odi.70090},
pmid = {41123029},
issn = {1601-0825},
mesh = {Humans ; *Immunocompromised Host ; *Antiviral Agents/therapeutic use ; *Herpesviridae Infections/drug therapy/immunology ; Genetic Therapy ; *Stomatitis, Herpetic/drug therapy/immunology ; Drug Resistance, Viral ; RNA Interference ; CRISPR-Cas Systems ; *Herpes Simplex/drug therapy ; },
abstract = {OBJECTIVE: This review explores conventional antiviral therapies and innovative treatment strategies for managing oral herpes simplex virus (HSV) or human herpesvirus (HHV) infections, particularly in immunocompromised patients. It aims to evaluate clinical outcomes, address challenges such as drug resistance, and assess the sustainability of current and emerging therapeutic approaches.

SUBJECT: A descriptive review of current treatment strategies for oral HSV infections in immunosuppressed individuals is presented. Relevant studies are analyzed for their contributions, highlighting therapeutic advancements, limitations of existing drugs, and novel approaches in development.

RESULTS: Oral herpesvirus infections can lead to considerable morbidity and mortality in immunocompromised patients. Resistance to standard antivirals often develops due to prolonged or inconsistent use and impaired cell-mediated immunity. To overcome these issues, innovative strategies like gene therapy, RNA interference (RNAi), and CRISPR-Cas9 have emerged. These approaches aim to suppress viral gene expression, strengthen innate immunity, and genetically modify host cells to resist viral entry and replication.

CONCLUSION: Emerging antiviral therapies show promise in addressing the limitations of conventional treatments, particularly drug resistance and non-specific targeting. These novel approaches offer more effective, precise, and safer options for treating oral viral infections in immunosuppressed populations at heightened risk for severe outcomes.},
}

Humans
*Immunocompromised Host
*Antiviral Agents/therapeutic use
*Herpesviridae Infections/drug therapy/immunology
Genetic Therapy
*Stomatitis, Herpetic/drug therapy/immunology
Drug Resistance, Viral
RNA Interference
CRISPR-Cas Systems
*Herpes Simplex/drug therapy

@article {pmid41296247,
year = {2026},
author = {Aksoy, MO and Rozynek, J and Stachowiak, M and Szczerbal, I},
title = {The role of the CEBPB gene in porcine adipogenesis: a study using CRISPR/Cas9-edited mesenchymal stem cells.},
journal = {Journal of applied genetics},
volume = {67},
number = {2},
pages = {477-486},
pmid = {41296247},
issn = {2190-3883},
support = {2018/29/B/NZ2/00956//Narodowe Centrum Nauki/ ; },
mesh = {Animals ; *Adipogenesis/genetics ; *Mesenchymal Stem Cells/cytology/metabolism ; *CRISPR-Cas Systems/genetics ; Swine/genetics ; *CCAAT-Enhancer-Binding Protein-beta/genetics ; *Gene Editing ; Cell Differentiation/genetics ; Promoter Regions, Genetic ; Adipocytes/cytology/metabolism ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 is a powerful tool for gene editing and the regulation of gene expression. It enables the introduction of targeted mutations, thereby facilitating functional studies of specific genes in various cellular processes. In this study, we aimed to generate a deletion in the promoter region of the CEBPB gene, which encodes a transcription factor involved in adipogenesis, and to evaluate the impact of this modification on the adipogenic differentiation potential of porcine mesenchymal stem cells (MSCs). A 575-bp deletion was introduced in the target region, resulting in the generation of both homozygous and heterozygous mutant cells. Adipogenic differentiation was assessed by quantifying transcript levels of adipocyte marker genes (GATA2, CEBPA, PPARG, and FABP4) at days 0, 4, 6, 8, and 10 of the differentiation process. Disruption of CEBPB expression led to the downregulation of these adipogenic markers, indicating impaired adipocyte differentiation. Additionally, to assess the proliferative capacity of the edited cells, the expression levels of proliferation-associated genes (CCND1, MCM2, and PCNA) were measured. A reduction in their transcript levels was observed in both homozygous and heterozygous mutant cells. These findings indicate that both homozygous and heterozygous deletions in the CEBPB promoter completely block adipogenesis and alter MSC proliferation, highlighting the pivotal role of CEBPB not only in adipogenic differentiation but also in the regulation of cell proliferation in porcine mesenchymal stem cells. These results provide new insights into the molecular mechanisms underlying adipose tissue development and have implications for pig breeding strategies aimed at optimizing carcass composition, as well as for biomedical research focused on adipose tissue biology.},
}

Animals
*Adipogenesis/genetics
*Mesenchymal Stem Cells/cytology/metabolism
*CRISPR-Cas Systems/genetics
Swine/genetics
*CCAAT-Enhancer-Binding Protein-beta/genetics
*Gene Editing
Cell Differentiation/genetics
Promoter Regions, Genetic
Adipocytes/cytology/metabolism

@article {pmid41885168,
year = {2026},
author = {Hanzelova, P and Baird, C and Keshinro, B and Kadhom, R and Lalonde, RL and Akimenko, MA},
title = {An essential role for actinotrichia in zebrafish fin patterning and courtship behavior.},
journal = {Development (Cambridge, England)},
volume = {153},
number = {16},
pages = {},
doi = {10.1242/dev.204990},
pmid = {41885168},
issn = {1477-9129},
support = {RGPIN-2024-06801//Natural Sciences and Engineering Research Council of Canada/ ; PJT 166139/CAPMC/CIHR/Canada ; PJT 166139/CAPMC/CIHR/Canada ; //University of Ottawa/ ; },
mesh = {Animals ; *Zebrafish/embryology/genetics/physiology ; *Animal Fins/embryology/metabolism ; Male ; *Zebrafish Proteins/genetics/metabolism ; *Body Patterning/genetics ; Female ; *Courtship ; CRISPR-Cas Systems ; *Microfilament Proteins/genetics/metabolism ; Gene Expression Regulation, Developmental ; Cell Movement ; *Sexual Behavior, Animal/physiology ; },
abstract = {A key difference between tetrapod limb buds and teleost fin buds is the presence of rigid actinotrichia fibers that guide the migrating cells contributing to ray formation. Major structural components of actinotrichia are encoded by fish-specific actinodin (And) genes, which were lost in tetrapods. To investigate the consequences of this loss during the fin-to-limb transition, we generated deletions in zebrafish and1 and and2 using CRISPR/Cas9 mutagenesis. Double mutants (and1-/-and2-/-) lack actinotrichia. Embryos and larvae have reduced fin fold size, with disorganized cell migration. In adults, all fin fold-derived skeletal structures are disrupted, including the rays of all fins, as well as the caudal fin endoskeleton. Surprisingly, double mutant males fail to breed, despite being fertile. Video analysis revealed that defects in the fins of males impair their ability to stimulate egg release. Our findings highlight the role of actinotrichia in both fin patterning and zebrafish courtship. We propose that actinodin gene maintenance is under strong selection in fish with similar courtship. We speculate that the loss of actinodin genes and a shift in courtship strategy may have coincided during tetrapod evolution.},
}

Animals
*Zebrafish/embryology/genetics/physiology
*Animal Fins/embryology/metabolism
Male
*Zebrafish Proteins/genetics/metabolism
*Body Patterning/genetics
Female
*Courtship
CRISPR-Cas Systems
*Microfilament Proteins/genetics/metabolism
Gene Expression Regulation, Developmental
Cell Movement
*Sexual Behavior, Animal/physiology

@article {pmid41911023,
year = {2026},
author = {Ye, J and Tang, X and Chen, Y and Guo, R and Zhang, W and Wang, M and Yang, X and Deng, W and Zhang, Y and Feng, X and Hu, N and Zhang, D},
title = {Conformationally Regulated CRISPR/Cas12a Activation Enabled by a Programmable DNA Dumbbell for Electrochemical SNP Genotyping.},
journal = {Analytical chemistry},
volume = {98},
number = {14},
pages = {10814-10823},
doi = {10.1021/acs.analchem.6c00333},
pmid = {41911023},
issn = {1520-6882},
mesh = {*Polymorphism, Single Nucleotide ; *CRISPR-Cas Systems/genetics ; *Electrochemical Techniques/methods ; *DNA/chemistry/genetics ; *Genotyping Techniques/methods ; *CRISPR-Associated Proteins/metabolism/genetics ; *Biosensing Techniques/methods ; Genotype ; *Endodeoxyribonucleases/metabolism/genetics ; Bacterial Proteins ; },
abstract = {Single-nucleotide polymorphism (SNP) genotyping is crucial for genetic research and precision medicine, yet reliable discrimination of single-base variants in complex genomic backgrounds remains analytically challenging. Although CRISPR/Cas12a-based biosensing offers high sequence specificity, its intrinsic mismatch tolerance often leads to nonspecific activation by wild-type sequences, thereby compromising SNP fidelity. Herein, we report a highly sensitive electrochemical sensing platform based on a programmable DNA dumbbell (Dum) probe that functions as a conformational energy-barrier regulator of CRISPR/Cas12a activation. The closed-loop dumbbell architecture sterically shields the crRNA-activating sequence, establishing a high activation threshold that suppresses nonspecific Cas12a triggering. Only precise SNP hybridization induces a thermodynamically favorable conformational rearrangement, releasing the mediator and transitioning the CRISPR system from an inactive to an active state. To further enhance analytical sensitivity and reliability, nucleic acid-functionalized FeCo nanozymes were incorporated as catalytic signal transducers, enabling a self-validating dual-mode signal electrochemical readout through intrinsic metal redox and H2O2 electrocatalysis. The resulting platform achieved reliably discriminates mutation abundances down to 0.1%. Validation using soybean genomic DNA samples demonstrates the robustness and practical applicability of the proposed strategy. This work establishes a conformational energy-barrier-regulated CRISPR activation paradigm, providing a generalizable analytical framework for high-fidelity SNP genotyping in molecular breeding and clinical diagnostics.},
}

*Polymorphism, Single Nucleotide
*CRISPR-Cas Systems/genetics
*Electrochemical Techniques/methods
*DNA/chemistry/genetics
*Genotyping Techniques/methods
*CRISPR-Associated Proteins/metabolism/genetics
*Biosensing Techniques/methods
Genotype
*Endodeoxyribonucleases/metabolism/genetics
Bacterial Proteins

@article {pmid41952451,
year = {2026},
author = {Mengistu, G},
title = {CRISPR-Cas Systems in Human Disease Therapy: Advances, Clinical Applications, Limitations, and Future Directions.},
journal = {The journal of gene medicine},
volume = {28},
number = {4},
pages = {e70091},
doi = {10.1002/jgm.70091},
pmid = {41952451},
issn = {1521-2254},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genetic Therapy/methods/trends ; Precision Medicine ; Animals ; },
abstract = {CRISPR-Cas systems have emerged as versatile platforms for targeted genome and transcriptome engineering, enabling precise manipulation of disease-associated genetic pathways. Continued advances in CRISPR technologies including base editing, prime editing, and epigenome modulation have expanded therapeutic possibilities beyond nuclease-mediated DNA cleavage, allowing programmable gene correction and regulation. Early clinical studies demonstrate sustained therapeutic benefit in selected monogenic disorders and highlight the feasibility of both ex vivo and in vivo editing strategies. However, clinical translation remains constrained by challenges such as off-target activity, delivery inefficiency, immune responses to Cas proteins, editing heterogeneity, and uncertainties regarding long-term safety. This review critically synthesizes recent advances in CRISPR-Cas systems for human disease therapy, integrating molecular innovations, delivery strategies, clinical progress, and ethical considerations. By evaluating both technological achievements and unresolved limitations, this article outlines key priorities for advancing CRISPR-based therapeutics toward safe, effective, and equitable precision medicine.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Genetic Therapy/methods/trends
Precision Medicine
Animals

@article {pmid41952472,
year = {2026},
author = {Martinkienė, J and Cui, T and Ciotta, G and Kupčinskas, J and Pažėraitis, D},
title = {RP-HPLC-based purification of long single-stranded DNA for CRISPR knock-in applications.},
journal = {BioTechniques},
volume = {78},
number = {1-12},
pages = {149-157},
doi = {10.1080/07366205.2026.2649857},
pmid = {41952472},
issn = {1940-9818},
mesh = {*DNA, Single-Stranded/isolation & purification/genetics/chemistry ; Chromatography, High Pressure Liquid/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; *Chromatography, Reverse-Phase/methods ; },
abstract = {BackgroundLong single-stranded DNA (ssDNA; >200 nucleotides) is valuable for DNA nanotechnology, precision medicine, and as a CRISPR-Cas9 knock-in donor template, but existing preparation methods are laborious, low-yield, or difficult to scale. MethodsWe developed a workflow combining enzymatic digestion with high-temperature reversed-phase high-performance liquid chromatography (RP-HPLC) to purify kilobase-length ssDNA. The method was evaluated across analytical and semi-preparative formats. ResultsThe approach enables clean resolution of linear and circular ssDNA species ranging from 1.5 to 4.5 kb and is scalable across formats. A 1.5 kb ssDNA donor supported efficient CRISPR knock-in at the T-cell receptor alpha constant (TRAC) locus in primary human CD8[+] T cells, without adversely affecting viability or expansion. ConclusionsThis RP-HPLC workflow provides a scalable and reproducible method for generating high-purity long ssDNA suitable for genome engineering applications.},
}

*DNA, Single-Stranded/isolation & purification/genetics/chemistry
Chromatography, High Pressure Liquid/methods
Humans
*CRISPR-Cas Systems/genetics
*Gene Knock-In Techniques/methods
*Chromatography, Reverse-Phase/methods

@article {pmid41954204,
year = {2026},
author = {Zheng, S and Wang, C and Huang, Y and Meng, X and Zhao, D and Guan, E and Ma, F and Chen, A and Zheng, L},
title = {Dual-Mode CRISPR/Cas13a Assay for the Detection of Human Metapneumovirus in Clinical Respiratory Samples.},
journal = {Journal of medical virology},
volume = {98},
number = {4},
pages = {e70921},
doi = {10.1002/jmv.70921},
pmid = {41954204},
issn = {1096-9071},
support = {2025YFC2311704//National Key R&D Program of China/ ; },
mesh = {Humans ; *Metapneumovirus/isolation & purification/genetics ; Sensitivity and Specificity ; *Paramyxoviridae Infections/diagnosis/virology ; *Respiratory Tract Infections/diagnosis/virology ; *Molecular Diagnostic Techniques/methods ; *CRISPR-Cas Systems ; Reproducibility of Results ; *Nucleic Acid Amplification Techniques/methods ; Real-Time Polymerase Chain Reaction ; RNA, Viral/genetics ; },
abstract = {Human metapneumovirus (HMPV) is a significant pathogen, causing widespread acute respiratory infections (ARIs). Rapid and accurate detection is crucial for timely diagnosis and outbreak control. To address the limitations of real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR)-based HMPV detection, we developed a novel method integrating recombinase-aided amplification (RAA) with CRISPR-Cas13a technology. Based on the alignment of 335 HMPV whole-genome sequences, a conserved region of the nucleocapsid (N) gene was selected for RAA primer and CRISPR RNA (crRNA) design. The concentrations of Cas protein, crRNA, and probe were optimized for specificity, sensitivity, and repeatability. A dual-mode platform was established, combining a fluorescence assay for quantitative detection and a lateral flow assay (LFA) for visual, on-site readout. Both methods were validated using 106 clinical samples and compared with RT-qPCR. Both methods demonstrated high specificity (no cross-reactivity with other respiratory viruses), a detection limit of 1 copy/μL, and excellent repeatability. Clinical validation showed complete concordance (κ = 1, p < 0.001) between the fluorescence assay and RT-qPCR, and high agreement for LFA (κ = 0.924, p < 0.001), with a sensitivity of 94.23%, specificity of 98.15%, positive predictive value (PPV) of 98%, and negative predictive value (NPV) of 94.6%. This study presents an RAA-CRISPR/Cas13a dual-mode platform for HMPV detection, providing a robust molecular diagnostic framework that paves the way for the future development of rapid testing assays.},
}

Humans
*Metapneumovirus/isolation & purification/genetics
Sensitivity and Specificity
*Paramyxoviridae Infections/diagnosis/virology
*Respiratory Tract Infections/diagnosis/virology
*Molecular Diagnostic Techniques/methods
*CRISPR-Cas Systems
Reproducibility of Results
*Nucleic Acid Amplification Techniques/methods
Real-Time Polymerase Chain Reaction
RNA, Viral/genetics

@article {pmid41954977,
year = {2026},
author = {Duan, X and Zhang, H and Lv, J and Fan, X and Liu, X and Wang, Q and Zhang, F and Zhang, Q and Liu, L and Liang, L and Liu, R},
title = {PE-STAR: prime editing with SOS-triggered and RecJ-augmented repair enables high-efficiency editing in Escherichia coli.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41954977},
issn = {1362-4962},
support = {2023YFC3402300//National Key R&D Program of China/ ; SRICSPYF-ZY2025107//Scientific Research Innovation Capability Support Project/ ; 22278058//National Natural Science Foundation of China/ ; 22578048//National Natural Science Foundation of China/ ; XLYC2203075//Xingliao Talent Plan/ ; 2025JH2/101330156//Natural Science Foundation of Liaoning Province/ ; 2024-MSBA-09//Natural Science Foundation of Liaoning Province/ ; DUT25LAB105//Fundamental Research Funds for the Central Universities/ ; DUT24YG131//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Gene Editing/methods ; *Escherichia coli/genetics ; *Escherichia coli Proteins/genetics/metabolism ; *SOS Response, Genetics/genetics ; *DNA Repair ; CRISPR-Cas Systems ; Exodeoxyribonucleases/genetics/metabolism ; Plasmids/genetics ; },
abstract = {Prime editing enables precise genome modifications without DNA double-strand breaks, yet bacterial applications are limited by low efficiency and small edit sizes. Here, we develop PE-STAR, Prime Editing with SOS-Triggered and RecJ-Augmented Repair, to enhance prime editing in Escherichia coli. Removing three inhibitory 3'→5' exonucleases (SbcB, ExoX, and XseA) improved edited-strand retention, and extending post-transformation outgrowth increased editing efficiency. RecJ overexpression strengthened 5'-directed processing during flap resolution and gap expansion, biasing repair toward incorporation of the reverse-transcribed edited strand. To enrich edited cells, we integrated an SOS-responsive counter-selection circuit that links PE3-associated dual nicking to LexA-dependent gRNA expression targeting a plasmid encoding the toxin CcdB, thereby eliminating unedited cells. PE-STAR achieved up to 80%-90% editing efficiency for short-fragment modifications, representing up to 16-fold improvement across loci. The platform supported insertions, deletions, and replacements of up to 46 bp with high efficiency. Furthermore, installing an attB site by prime editing, followed by Bxb1 integrase recombination, enabled chromosomal integration of 3.2 and 8.0 kb cassettes with 100% recombination efficiency among screened colonies, including GFP reporter and riboflavin biosynthetic pathway. PE-STAR expands both the efficiency and functional scope of bacterial prime editing for programmable genome engineering.},
}

*Gene Editing/methods
*Escherichia coli/genetics
*Escherichia coli Proteins/genetics/metabolism
*SOS Response, Genetics/genetics
*DNA Repair
CRISPR-Cas Systems
Exodeoxyribonucleases/genetics/metabolism
Plasmids/genetics

@article {pmid41168408,
year = {2026},
author = {Ke, Z and Zhang, Q and Chen, X and Jin, R and Han, G and Cao, Q and Zhu, K and Wei, S and Chen, J and Wang, Q and Zhang, M and Huang, W and Li, K and Xiong, K and Lu, K and Chen, Y and Cao, C},
title = {CRISPR/Cas9 library screening uncovered CCT2 as a critical driver of acquired resistance to EGFR-targeted therapy by stabilizing TMX1 in non-small cell lung cancer.},
journal = {Cell death and differentiation},
volume = {33},
number = {4},
pages = {860-876},
pmid = {41168408},
issn = {1476-5403},
mesh = {*Carcinoma, Non-Small-Cell Lung/drug therapy/genetics/metabolism/pathology ; Humans ; *Lung Neoplasms/drug therapy/genetics/pathology/metabolism ; *Drug Resistance, Neoplasm/drug effects/genetics ; *CRISPR-Cas Systems/genetics ; ErbB Receptors/metabolism/antagonists & inhibitors ; Animals ; Mice ; Protein Kinase Inhibitors/pharmacology/therapeutic use ; Cell Line, Tumor ; Mice, Nude ; Protein Stability ; *Thioredoxins/metabolism ; *Membrane Proteins/metabolism ; },
abstract = {In the treatment of non-small cell lung cancer (NSCLC) with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), the emergence of acquired resistance remains a significant challenge. Elucidating the underlying mechanisms of resistance is crucial for developing novel strategies to overcome or delay therapeutic escape. To this end, this study aimed to identify key drivers of EGFR-TKIs resistance and explore actionable targets for intervention. We investigated resistance mechanisms by integrating CRISPR/Cas9-based genome-wide screening with tandem mass tag (TMT) proteomic analysis, and virtually screened bioactive small molecule libraries to identify compounds capable of restoring EGFR-TKIs sensitivity. The multi-omics approach revealed that CCT2 is a critical mediator of resistance to third-generation EGFR-TKIs in lung cancer, with higher expression of CCT2 observed in resistant cells compared to sensitive cells. Mechanistically, CCT2 recruits tripartite motif-containing protein 28 (TRIM28) to catalyze SUMO2 modification of thioredoxin-related transmembrane protein 1 (TMX1), inhibiting its ubiquitination and enhancing protein stability. This post-translational modification (PTM) promotes TMX1-dependent reactive oxygen species (ROS) clearance, thereby conferring resistance. Importantly, pharmacological inhibition with the compound HY-10127, identified through virtual screening, effectively restored EGFR-TKIs sensitivity in resistant cell lines and delayed the development of resistance in xenograft models. The findings establish the CCT2/TRIM28/TMX1/ROS axis as a novel resistance mechanism in EGFR-mutated lung cancer, and targeting this pathway with HY-10127 represents a promising strategy to overcome resistance to third-generation EGFR-TKIs, providing preclinical rationale for clinical translation. These discoveries advance our understanding of molecular resistance mechanisms and offer potential therapeutic targets for improving lung cancer prognosis.},
}

*Carcinoma, Non-Small-Cell Lung/drug therapy/genetics/metabolism/pathology
Humans
*Lung Neoplasms/drug therapy/genetics/pathology/metabolism
*Drug Resistance, Neoplasm/drug effects/genetics
*CRISPR-Cas Systems/genetics
ErbB Receptors/metabolism/antagonists & inhibitors
Animals
Mice
Protein Kinase Inhibitors/pharmacology/therapeutic use
Cell Line, Tumor
Mice, Nude
Protein Stability
*Thioredoxins/metabolism
*Membrane Proteins/metabolism

@article {pmid41546459,
year = {2026},
author = {Niu, C and Fang, S and Zeng, B and Liu, D and Huang, K and Zhang, G and Qian, N and Li, D and Ye, C},
title = {Development of a PCR-Cas12a-LFD visual detection system for highly sensitive and specific detection of Ralstonia sp., Phytophthora sp., Alternaria sp., and Pseudomonas sp. in tobacco.},
journal = {Pest management science},
volume = {82},
number = {5},
pages = {4169-4182},
doi = {10.1002/ps.70528},
pmid = {41546459},
issn = {1526-4998},
support = {//Youth Support Program of Zhengzhou Tobacco Research Institute, China National Tobacco Corporation (CNTC) (No. 702024CR0070)/ ; },
mesh = {*Nicotiana/microbiology ; *Plant Diseases/microbiology ; *Pseudomonas/isolation & purification/genetics ; *Alternaria/isolation & purification/genetics ; *Phytophthora/isolation & purification/genetics ; *Ralstonia/isolation & purification/genetics ; *Polymerase Chain Reaction/methods ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; },
abstract = {Ralstonia sp., Phytophthora sp., Alternaria sp., and Pseudomonas sp. are the major pathogens responsible for tobacco diseases and severe threats to the sustainable development of the tobacco industry. Traditional detection methods, such as plate isolation and polymerase chain reaction (PCR), have improved in sensitivity and timeliness but remain limited by operational complexity, heavy reliance on high expertise requirements for result interpretation. This study focuses on the trans-cleavage activity of the CRISPR-Cas12a system and develops a series of novel detection methods for Ralstonia sp., Phytophthora sp., Alternaria sp., and Pseudomonas sp., respectively. By screening specific genomic targets, designing highly sensitive primers and CRISPR RNA (crRNA), and integrating PCR with lateral flow dipstick (LFD), the PCR-Cas12a-LFD detection system was established with a sensitivity of 100 pg μL[-1] for Ralstonia sp., 10 pg μL[-1] for Phytophthora sp., 5000 pg μL[-1] for Alternaria sp., and 0.1 pg μL[-1] for Pseudomonas sp., respectively, and excellent specificity without cross-reactivity. Furthermore, the combination of this method with LFD enables visual interpretation of results. This study provides an efficient and visualized diagnostic tool for the early prevention and control of tobacco diseases during field cultivation while offering a theoretical reference for the molecular detection of other plant pathogens. © 2026 Society of Chemical Industry.},
}

*Nicotiana/microbiology
*Plant Diseases/microbiology
*Pseudomonas/isolation & purification/genetics
*Alternaria/isolation & purification/genetics
*Phytophthora/isolation & purification/genetics
*Ralstonia/isolation & purification/genetics
*Polymerase Chain Reaction/methods
*CRISPR-Cas Systems
Sensitivity and Specificity

@article {pmid41778754,
year = {2026},
author = {Hoyle, C and Pino, RD and Lai, SM and Green, JP and Adamson, A and Coutts, G and Lawrence, CB and Travis, M and Brough, D and Lopez-Castejon, G},
title = {Inflammatory responses following CRISPR modification of the nuclear localisation sequence in endogenous interleukin-1 alpha.},
journal = {Disease models & mechanisms},
volume = {19},
number = {4},
pages = {},
doi = {10.1242/dmm.052705},
pmid = {41778754},
issn = {1754-8411},
support = {Fellowship//Dowager Countess Eleanor Peel Trust/ ; BB/Y004876/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; SRB1013//Dowager Countess Eleanor Peel Trust/ ; MR/T016515/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; *Interleukin-1alpha/metabolism/genetics/chemistry ; *Nuclear Localization Signals/genetics/metabolism ; *Inflammation/genetics/pathology ; Lipopolysaccharides/pharmacology ; *Cell Nucleus/metabolism/drug effects ; Macrophages/metabolism/drug effects ; Mice ; Mice, Inbred C57BL ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; Peritonitis/genetics/pathology ; },
abstract = {Interleukin (IL)-1α is a pro-inflammatory member of the IL-1 cytokine superfamily and is important for inflammatory responses to infection and injury. Unlike pro-IL-1β, pro-IL-1α is mainly localised to the nucleus upon expression. This is mediated by a nuclear localisation sequence (NLS) responsible for its importin-dependent transport into the nucleus. This nuclear localisation and the presence of histone acetyl transferase (HAT)-binding domains within the pro-domain suggest a role of this cytokine in gene transcription regulation. In addition, nuclear trafficking of pro-IL-1α is proposed to regulate its secretion. To date, studies on the nuclear role of pro-IL-1α have used overexpression systems. Here, we generated a mouse strain in which the endogenous Il1a gene was edited using CRISPR to disrupt the NLS, yielding a mutated NLS (mNLS). Using an in vitro approach with murine macrophages we found that this NLS mutation did not affect pro-IL-1α RNA expression levels in response to lipopolysaccharide (LPS) but increased its protein expression levels. Moreover, we found that the transcriptional signature induced by LPS was not altered between WT and mNLS macrophages. Release of IL-1α in response to different stimuli such as ionomycin was not negatively impacted by disrupted nuclear localisation, although higher levels of IL-1α release were detected, potentially due to increased levels of pro-IL-1α. Inflammatory responses in an in vivo model of peritonitis and an influenza infection model were comparable between WT and mNLS mice. Thus, we have established a mouse model in which pro-IL-1α nuclear localisation is disrupted, although future research is required to reveal the importance of this nuclear localisation for IL-1α function.},
}

Animals
*Interleukin-1alpha/metabolism/genetics/chemistry
*Nuclear Localization Signals/genetics/metabolism
*Inflammation/genetics/pathology
Lipopolysaccharides/pharmacology
*Cell Nucleus/metabolism/drug effects
Macrophages/metabolism/drug effects
Mice
Mice, Inbred C57BL
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*CRISPR-Cas Systems/genetics
Peritonitis/genetics/pathology

@article {pmid41926379,
year = {2026},
author = {Boy, RL and Zampieri, RA and Aoki, JI and Coelho, AC and Floeter-Winter, LM and Laranjeira-Silva, MF},
title = {Glycosomal Aquaglyceroporin 1 dual role in iron homeostasis and antimony susceptibility in Leishmania amazonensis.},
journal = {PLoS neglected tropical diseases},
volume = {20},
number = {4},
pages = {e0014141},
doi = {10.1371/journal.pntd.0014141},
pmid = {41926379},
issn = {1935-2735},
mesh = {Animals ; *Antimony/pharmacology ; Mice ; Homeostasis ; *Iron/metabolism ; Macrophages/parasitology ; *Leishmania mexicana/drug effects/metabolism/genetics ; *Antiprotozoal Agents/pharmacology ; *Protozoan Proteins/genetics/metabolism ; Female ; *Aquaporin 1/genetics/metabolism ; Leishmaniasis/parasitology ; Mice, Inbred BALB C ; *Leishmania/drug effects/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {Leishmania parasites cause a spectrum of diseases known as leishmaniases and must acquire nutrients like iron while surviving host defenses. Aquaglyceroporin 1 (AQP1) is a membrane channel that, in L. major, localizes to the flagellum and mediates antimony uptake and cell-volume regulation. Here, we show that in L. amazonensis AQP1 is instead targeted to glycosomes and that its expression is modulated by iron availability. A CRISPR-Cas9-mediated knockout of AQP1 in L. amazonensis revealed its multifunctional importance. AQP1-null promastigotes displayed a significant growth defect, particularly under iron-depleted conditions, and were impaired in regulating cell volume under osmotic stress. The mutant parasites contained approximately 50% less intracellular iron than wild-type cells and showed an increase in total superoxide dismutase activity, underscoring a role for AQP1 in iron homeostasis and oxidative stress management. AQP1 deletion also markedly reduced virulence in murine macrophages and in infected mice. Strikingly, loss of AQP1 increased resistance to trivalent antimony (SbIII), a first-line antileishmanial drug. AQP1-knockout promastigotes showed a 70% increase in SbIII IC50 and accumulated more Sb intracellularly than wild-type, suggesting an altered antimony handling. Altogether, L. amazonensis AQP1 is a glycosomal protein that links iron metabolism, osmoregulation, and antimony susceptibility. Its glycosomal targeting and multifaceted roles differ from those of AQP1 orthologs in other Leishmania species. These findings suggest the existence of additional antimony uptake mechanisms beyond AQP1, with implications for understanding drug resistance.},
}

Animals
*Antimony/pharmacology
Mice
Homeostasis
*Iron/metabolism
Macrophages/parasitology
*Leishmania mexicana/drug effects/metabolism/genetics
*Antiprotozoal Agents/pharmacology
*Protozoan Proteins/genetics/metabolism
Female
*Aquaporin 1/genetics/metabolism
Leishmaniasis/parasitology
Mice, Inbred BALB C
*Leishmania/drug effects/metabolism/genetics
CRISPR-Cas Systems

@article {pmid41946722,
year = {2026},
author = {Malone, HA and Myers, JA and Gruss, EG and Morgan, MA and Friske, JD and McCarty, TC and Navarro, JJ and Robinson, S and Halliburton, RL and Kietlinska, SJ and De Luna Vitorino, FN and Hansen, BS and Pruett-Miller, SM and Garcia, BA and Roussel, MF and Partridge, JF and Roberts, CWM},
title = {PHIP suppresses NuRD to enable the growth of SWI/SNF-mutant cancers.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41946722},
issn = {2041-1723},
support = {R01-CA-273455//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; R01-CA-172152//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; R01-CA-113794//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; F31-CA-278355//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; P01-CA-96832//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; R01-HD-106051//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; P01-CA-196539//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Humans ; *Neoplasms/genetics/metabolism/pathology ; *Mi-2 Nucleosome Remodeling and Deacetylase Complex/metabolism/genetics ; Cell Line, Tumor ; *Transcription Factors/genetics/metabolism ; Mutation ; *Chromosomal Proteins, Non-Histone/genetics/metabolism ; Chromatin Assembly and Disassembly/genetics ; CRISPR-Cas Systems ; Gene Expression Regulation, Neoplastic ; Chromatin/metabolism ; Promoter Regions, Genetic ; Transcriptional Activation ; Ubiquitination ; },
abstract = {SWI/SNF chromatin remodeling complexes are perturbed in 20% of all cancers and in several developmental disorders, yet the mechanisms by which these mutations dysregulate transcription and drive disease are poorly understood. To both elucidate these mechanisms and identify vulnerabilities caused by these mutations, we leverage genome-wide CRISPR-Cas9 screening in hundreds of cancer cell lines and identify the chromatin reader protein PHIP as a specific dependency in cancers with broadly disrupted SWI/SNF function. Mechanistically, we reveal that PHIP cooperates with SWI/SNF to facilitate transcriptional activation by ubiquitinating and suppressing subunits of the repressive Nucleosome Remodeling and Deacetylase (NuRD) complex. We demonstrate that loss of SWI/SNF results in NuRD complexes accumulating at promoters where they would otherwise cause widespread transcriptional silencing if not antagonized by PHIP. Collectively, we identify PHIP as a regulator of the interplay between distinct chromatin regulators that function in development and disease and as a targetable vulnerability in cancers with broad SWI/SNF inactivation.},
}

Humans
*Neoplasms/genetics/metabolism/pathology
*Mi-2 Nucleosome Remodeling and Deacetylase Complex/metabolism/genetics
Cell Line, Tumor
*Transcription Factors/genetics/metabolism
Mutation
*Chromosomal Proteins, Non-Histone/genetics/metabolism
Chromatin Assembly and Disassembly/genetics
CRISPR-Cas Systems
Gene Expression Regulation, Neoplastic
Chromatin/metabolism
Promoter Regions, Genetic
Transcriptional Activation
Ubiquitination

@article {pmid41946927,
year = {2026},
author = {Fang, GQ and Deng, Y and Lyu, XY and Yin, CQ and Song, J and Zhang, Y and Zhong, J and Shen, EZ and Song, CQ},
title = {Boosting prime editing with engineered non-canonical pegRNAs.},
journal = {Nature biomedical engineering},
volume = {},
number = {},
pages = {},
pmid = {41946927},
issn = {2157-846X},
support = {32471552//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
abstract = {Prime editing (PE) enables precise genetic modifications using canonical prime editing guide RNA (pegRNA), with the reverse transcription template and primer binding site (RTT-PBS) attached to the 3' ends of CRISPR-Cas guide RNAs. Although PE ribonucleoprotein (RNP) delivery holds great therapeutic potential, its weak genomic editing capability limits therapeutic applications. Here we present structure-guided engineering of the PE complex using non-canonical pegRNAs (npegRNAs), with the RTT-PBS integrated within the single guide RNA loops, to improve PE efficiency. This approach demonstrates enhanced precise editing rates across various genomic sites and cell types, and improves therapeutic gene correction in a tyrosinaemia mouse model. Cas9-associated npegRNAs are more resistant to exonuclease degradation, probably enhancing the PE complex's targeting efficiency in living cells. Using PE RNP delivery, npegRNAs achieve increased average editing yields of 26.8-fold over canonical pegRNAs and 5.9-fold over engineered pegRNAs (epegRNAs). Furthermore, npegRNA-mediated RNPs increased the efficiency of installing disease-relevant mutations up to 123-fold in human cell lines, including Jurkat T cells and induced pluripotent stem cells. Collectively, our findings demonstrate a robust PE strategy and highlight the potential of npegRNAs for therapeutic PE applications.},
}

@article {pmid41949766,
year = {2026},
author = {Mostafa, K and Scarano, A and Abdulla, MF and Hacıkamiloğlu, S and Kurt, O and Santino, A and Kavas, M},
title = {A rapid Agrobacterium rhizogenes-mediated transient expression for assessing sgRNA efficiency in CRISPR-Act3.0 in tomato.},
journal = {Plant cell reports},
volume = {45},
number = {5},
pages = {},
pmid = {41949766},
issn = {1432-203X},
support = {121O463//Türkiye Bilimsel ve Teknolojik Araştırma Kurumu/ ; },
mesh = {*Solanum lycopersicum/genetics/metabolism ; *Agrobacterium/genetics ; Plants, Genetically Modified ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Plant ; Fatty Acids/metabolism ; Transformation, Genetic ; Promoter Regions, Genetic ; },
abstract = {CRISPR-Act3.0 is a robust tool for modulating fatty acid profiles in plants. We demonstrate that Agrobacterium rhizogenes-mediated transformation provides a rapid, cost-effective, and equipment-independent platform for validating sgRNA efficiency and metabolic outcomes within a short time. The CRISPR-Act3.0 system offers a powerful strategy for activating endogenous gene expression in plants. However, the labor-intensive and time-consuming nature of stable transformation often hinders the rapid validation of multiple sgRNAs. In this study, we optimized a rapid Agrobacterium rhizogenes-mediated transient expression system in tomato to evaluate sgRNA efficiency within the CRISPR-Act3.0 framework. As a proof-of-concept, we targeted four genes involved in fatty acid biosynthesis: SlFATA, SlFATB-01, SlFATB-02, and SlFATB-03. To ensure precise control, we utilized the root-specific pSMB promoter to drive the CRISPRa components. Our results demonstrate that this system can successfully induce significant transcriptional activation and alter fatty acid compositions specifically increasing palmitic acid levels by up to 45%-within approximately 30 days. This approach bypasses the requirement for whole-plant stable transformation during the initial screening phase and prevents potential pleiotropic effects by restricting activation to root tissues. Overall, this study provides a highly efficient diagnostic pipeline for functional genomics and metabolic engineering in tomato, offering a significant advantage for rapid trait evaluation before committing to stable transgenic line production.},
}

*Solanum lycopersicum/genetics/metabolism
*Agrobacterium/genetics
Plants, Genetically Modified
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*CRISPR-Cas Systems/genetics
Gene Expression Regulation, Plant
Fatty Acids/metabolism
Transformation, Genetic
Promoter Regions, Genetic

@article {pmid41951520,
year = {2026},
author = {He, Y and Ma, Y and Wu, Y and Tang, X and Liu, S and Yin, D and Zheng, X and Qi, Y and Zhang, Y and Zhang, T},
title = {Harnessing diverse tRNAs and AI-guided mining for compact and efficient plant multiplex genome editing.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2026.02.016},
pmid = {41951520},
issn = {1879-3096},
abstract = {The widespread use of CRISPR-Cas9 (Clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) in plants highlights the need for compact and efficient multiplexed genome editing systems. This study optimizes single-guide RNA (sgRNA) expression in CRISPR by leveraging endogenous tRNA processing mechanisms for efficient multiplexed genome editing. Screening in Arabidopsis thaliana and Oryza sativa identified superior tRNAs that outperformed the widely used AtGly-tRgcc. Leveraging tRNA's dual functions in sgRNA processing and their intragenic RNA polymerase III promoter activity, we established a compact multiplexed system for simultaneous editing of at least ten genomic loci in rice and soybean. Moreover, we developed plant tRNA large language models that learn sequence representations to identify both canonical and noncanonical tRNAs, uncovering thousands of tRNAs missed by traditional algorithms and expanding the repertoire for genome editing. This work provides a robust tRNA-based CRISPR platform, an artificial intelligence-guided tRNA mining framework, and a comprehensive tRNA resource for advanced plant genome engineering and germplasm innovation.},
}

@article {pmid41951915,
year = {2026},
author = {Zhu, Y and Moerner, WE and Qi, LS},
title = {CRISPR-Cas-based live cell imaging of genome dynamics.},
journal = {Nature reviews. Genetics},
volume = {},
number = {},
pages = {},
pmid = {41951915},
issn = {1471-0064},
abstract = {The 3D architecture and dynamics of the genome are crucial for regulation of genome stability, transcription and cellular function. CRISPR-based live imaging technologies have enabled real-time visualization of specific genomic loci and transcripts in living cells. These tools harness customized guide RNAs and nuclease-deactivated Cas effectors to achieve precise genomic targeting, and recent methodological advances provide the 3D spatiotemporal resolution required to decipher real-time chromatin communication. These methods are elucidating the biophysical properties of chromatin, linking dynamic enhancer-promoter interactions directly to transcription, and revealing the role of 3D genome dynamics in basic cellular processes and disease. Here, we summarize the development of CRISPR-based live-cell imaging techniques, highlight the complementary 3D microscopy and analysis methods compatible with these methods, and offer perspectives on their applications to uncover fundamental principles that govern genome dynamics and function.},
}

@article {pmid41952056,
year = {2026},
author = {Salum, YM and Chen, J and Dang, J and Qiao, Q and Chen, T and Lin, T and Wei, H and He, W},
title = {Leveraging RNAi and CRISPR/Cas9-based strategies for target gene characterization and control of western flower thrips (Frankliniella occidentalis): Advances and perspectives.},
journal = {Pest management science},
volume = {},
number = {},
pages = {},
doi = {10.1002/ps.70782},
pmid = {41952056},
issn = {1526-4998},
support = {//Major Project of Science and Technology of Fujian Province (2024NZ029029)/ ; //Fujian Provincial Science and Technology Project (2023L3021 and 2025N0081)/ ; },
abstract = {Frankliniella occidentalis, the western flower thrips, is among the most destructive agricultural pests worldwide and a major vector of orthotospoviruses. Escalating insecticide resistance has intensified efforts to develop molecularly informed control strategies. RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome editing have emerged as principal tools for functional genomics in this species, enabling targeted analysis of genes involved in development, metabolism, and insecticide resistance. This review synthesizes recent advances in RNAi and CRISPR applications in F. occidentalis, with particular emphasis on delivery strategies, editing efficiencies, and resulting phenotypes. RNAi studies demonstrate growing technical feasibility through injection, feeding-based approaches, and plant-mediated systems, and emerging CRISPR/Cas investigations confirm the possibility of targeted gene knockouts affecting resistance traits and visible genetic markers. Despite these advances, genetic manipulation of F. occidentalis remains technically challenging, primarily due to biological and technical constraints, including inefficient delivery of ribonucleoprotein to the germline, the extremely small and fragile nature of eggs, and narrow developmental windows for manipulation. We discuss these barriers and highlight methodological innovations required to improve RNAi stability, embryo manipulation, and genome editing efficiency. Finally, we propose how these molecular and genetic tools may be incorporated into existing integrated pest management, emphasizing their potential as complementary, species-specific strategies rather than standalone control solutions toward sustainable F. occidentalis management. © 2026 Society of Chemical Industry.},
}

@article {pmid41952275,
year = {2026},
author = {Das, U and Prasad, SS and Sahoo, T and Paramanik, S and Halder, A and S, P},
title = {Unveiling the potential of banana (Musa spp.) improvement through genetic manipulation: current trends and future implications.},
journal = {Plant signaling & behavior},
volume = {21},
number = {1},
pages = {2656013},
doi = {10.1080/15592324.2026.2656013},
pmid = {41952275},
issn = {1559-2324},
mesh = {*Musa/genetics/microbiology ; Gene Editing ; Plants, Genetically Modified/genetics ; Genetic Engineering/methods ; Disease Resistance/genetics ; Plant Breeding ; Plant Diseases/microbiology/genetics ; },
abstract = {Banana (Musa spp.) is a globally important fruit crop and a staple food for millions of people. However, its narrow genetic diversity and clonal propagation make it highly vulnerable to pests, diseases, and abiotic stresses. Genetic improvement is limited by sterility, and triploid plants are reproduced clonally in most cultivated varieties, preventing traditional breeding based on genetic advancements. Transgenesis and gene editing are among the genetic engineering techniques used to increase yield, improve quality, and enhance resilience. Advances in Agrobacterium-mediated transformation and CRISPR/Cas tools have enabled the development of bananas with enhanced resistance to Fusarium wilt tropical race 4 (TR4), Black Sigatoka, and bacterial wilt, along with increased provitamin A content, longer shelf life, and reduced postharvest losses. DNA-free genome editing provides a promising approach to overcoming certain regulatory barriers and enhancing public acceptance. While challenges such as genotype-specific transformation efficiency, regulatory hurdles, and public perception persist, genetic manipulation holds the potential to both preserve and improve global banana production. This review synthesizes recent progress, key targets, and future prospects for the genetic improvement of banana.},
}

*Musa/genetics/microbiology
Gene Editing
Plants, Genetically Modified/genetics
Genetic Engineering/methods
Disease Resistance/genetics
Plant Breeding
Plant Diseases/microbiology/genetics

@article {pmid41796459,
year = {2026},
author = {Matsuoka, T and Oda, K and Iwashita, K and Watanabe, J},
title = {Identification of DeuA, an Aspergillus oryzae-derived deuterolysin-like metalloprotease, as the predominant thermostable protease in soy sauce.},
journal = {Journal of bioscience and bioengineering},
volume = {141},
number = {6},
pages = {445-449},
doi = {10.1016/j.jbiosc.2026.02.010},
pmid = {41796459},
issn = {1347-4421},
mesh = {*Aspergillus oryzae/enzymology/genetics ; *Soy Foods/microbiology/analysis ; *Metalloproteases/genetics/metabolism/chemistry ; Fermentation ; Enzyme Stability ; *Fungal Proteins/genetics/metabolism/chemistry ; *Peptide Hydrolases/metabolism/genetics ; CRISPR-Cas Systems ; Gene Knockout Techniques ; },
abstract = {The thermostable proteases present in soy sauce can degrade proteins in processed foods, such as boiled eggs and fish cakes, leading to undesirable textural changes in the product. In this study, we identified DeuA, a deuterolysin-like metalloprotease from Aspergillus oryzae, as the major contributor to thermostable protease activity during soy sauce fermentation. Using CRISPR/Cas9-based genome co-editing, we generated deuA-knockout mutants and mutants of the related gene deuB, which encodes a deuterolysin-like metalloprotease, and evaluated their enzymatic activity levels under solid-state culture conditions mimicking soy sauce koji fermentation. The ΔpyrGΔdeuA strain exhibited a marked reduction in thermostable protease activity, with residual activity barely detectable in both the koji extracts and the final soy sauce, whereas knockout of deuB had no significant effect. These results indicate that DeuA is the predominant contributor to thermostable protease activity in soy sauce. The knockout of deuA did not affect other key brewing parameters such as the nitrogen or sugar contents, indicating the potential of this gene as a target for strain improvement. Our findings establish DeuA as an essential thermostable protease in soy sauce and provide a foundation for the development of brewing strains with improved industrial applicability that will not affect the textural stability of processed foods.},
}

*Aspergillus oryzae/enzymology/genetics
*Soy Foods/microbiology/analysis
*Metalloproteases/genetics/metabolism/chemistry
Fermentation
Enzyme Stability
*Fungal Proteins/genetics/metabolism/chemistry
*Peptide Hydrolases/metabolism/genetics
CRISPR-Cas Systems
Gene Knockout Techniques

@article {pmid41812798,
year = {2026},
author = {Huang, G and Zhuge, B and Du, X and Wang, M and Zong, H},
title = {TSA-ultrasound synergy enhances CRISPR-Cas9 gene editing efficiency in diploid yeast.},
journal = {Journal of microbiological methods},
volume = {244},
number = {},
pages = {107449},
doi = {10.1016/j.mimet.2026.107449},
pmid = {41812798},
issn = {1872-8359},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Diploidy ; *Hydroxamic Acids/pharmacology ; Gene Knockout Techniques ; *Candida/genetics/drug effects ; Sonication/methods ; Chromatin ; },
abstract = {The CRISPR-Cas9 system is invaluable for microbial engineering. However, its efficiency remains limited in numerous microorganisms, especially in polyploid yeasts where the compact chromatin structures pose significant physical barriers, which are major constraints in microbial engineering. To address this limitation, we developed and validated a standardized synergistic protocol using the diploid industrial yeast Candida glycerinogenes as a model. This protocol combines TSA-induced chromatin decondensation with brief, low-intensity ultrasonication to enhance Cas9 accessibility. Key parameters were systematically optimized, and 200 nM Trichostatin A (TSA) (10 h) followed by 200 W sonication (3 min) were established as the optimal condition set. Validation results showed that the protocol more than doubled single-gene knockout efficiencies (GPD1, TRP1) compared with conventional methods. Importantly, it enabled complex edits that were previously unattainable in C. glycerinogenes, including the precise deletion of a 7.8-kb fragment and the editing of an 11.4-kb region for functional genomics. In summary, this study establishes a simple and effective workflow that overcomes chromatin-based barriers in the polyploid industrial yeast C. glycerinogenes, providing a practical tool for genetic engineering and functional genomics in this and potentially other recalcitrant yeasts.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
Diploidy
*Hydroxamic Acids/pharmacology
Gene Knockout Techniques
*Candida/genetics/drug effects
Sonication/methods
Chromatin

@article {pmid41887392,
year = {2026},
author = {Wang, H and Shen, T and Yang, S and Zhou, X and Cao, P and Yu, H and He, K and Fu, M and Yu, H and Liu, X and Zhou, T and Wang, J and Huang, M and Qian, X and Wang, X and Wang, Q and Liu, L and Fan, Z and Zhang, Y and Lin, F},
title = {Genome-wide CRISPR/Cas9 screen identified MCL1 as a senolytic target for clearing palbociclib-induced senescent and PD-L1-positive cells in colorectal cancer.},
journal = {Cancer letters},
volume = {647},
number = {},
pages = {218446},
doi = {10.1016/j.canlet.2026.218446},
pmid = {41887392},
issn = {1872-7980},
mesh = {Humans ; *Colorectal Neoplasms/drug therapy/genetics/pathology/metabolism ; *Pyridines/pharmacology ; *Piperazines/pharmacology ; Animals ; *Cellular Senescence/drug effects/genetics ; *Myeloid Cell Leukemia Sequence 1 Protein/genetics/antagonists & inhibitors/metabolism ; CRISPR-Cas Systems ; Mice ; Xenograft Model Antitumor Assays ; *B7-H1 Antigen/metabolism/genetics ; Apoptosis/drug effects ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; Mice, Nude ; Gene Expression Regulation, Neoplastic/drug effects ; Female ; },
abstract = {Colorectal cancer (CRC) is the most prevalent digestive system malignancy worldwide. The development of targeted therapeutics specifically effective for CRC is currently in dire need. Preclinical studies showed that CDK4/6 inhibitor palbociclib suppressed the growth of CRC, but whether this effect is durable is unclear. In this study, we aimed to evaluate the roles of palbociclib-induced senescence and find a new strategy to maximize its effectiveness in CRC treatment. Animal and cellular experiments revealed that palbociclib-induced senescence and the senescence-associated secretory phenotype (SASP) caused drug resistance, anti-apoptosis, PD-L1 upregulation and inhibition of CD8[+] T cells' function. Using CRISPR/Cas9 screening, we identified MCL1 as a senolytic target to eliminate palbociclib-induced senescent CRC cells in the presence of palbociclib. Mechanically, palbociclib-induced senescent cells upregulated ZHX2 and its transcriptional target MCL1, rendered their resistance to apoptosis and T cell-mediated cytotoxicity, whereases combining palbociclib with MCL1 inhibitor markedly induced apoptosis in senescent cells by activating both extrinsic and intrinsic apoptotic pathways. Lastly, we proposed a seno-therapy consisting of a palbociclib pre-treatment plus a combination treatment of palbociclib and MCL1 inhibitor and found it effectively inhibited tumor growth and improved the survival of CRC xenografted mice. Besides its senolytic effect, seno-therapy also reduced PD-L1-positive cells and enhancing the cytotoxic functions of CD8[+] T cells. In conclusion, co-targeting CDK4/6 and MCL1 efficiently eliminates palbociclib-induced senescent CRC cells and offers a promising CDK4/6 inhibitor-based strategy for CRC treatment, ensuring prolonged tumor suppression and reducing the risk of progression or recurrence.},
}

Humans
*Colorectal Neoplasms/drug therapy/genetics/pathology/metabolism
*Pyridines/pharmacology
*Piperazines/pharmacology
Animals
*Cellular Senescence/drug effects/genetics
*Myeloid Cell Leukemia Sequence 1 Protein/genetics/antagonists & inhibitors/metabolism
CRISPR-Cas Systems
Mice
Xenograft Model Antitumor Assays
*B7-H1 Antigen/metabolism/genetics
Apoptosis/drug effects
Cell Line, Tumor
Drug Resistance, Neoplasm
Mice, Nude
Gene Expression Regulation, Neoplastic/drug effects
Female

@article {pmid41887586,
year = {2026},
author = {Menestreau, M and Frostegård, Å and Kjos, M},
title = {Strain-specific challenges in applying CRISPR/Cas9-based genome editing in the novel genus Stutzerimonas.},
journal = {Journal of microbiological methods},
volume = {244},
number = {},
pages = {107478},
doi = {10.1016/j.mimet.2026.107478},
pmid = {41887586},
issn = {1872-8359},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Plasmids/genetics ; *Genome, Bacterial ; Denitrification/genetics ; },
abstract = {Recently reclassified from the genus Pseudomonas, Stutzerimonas comprises metabolically versatile bacteria widely distributed across diverse environments and with a capacity to perform complete denitrification. Here, we evaluated the applicability of CRISPR/Cas9-based genome editing in Stutzerimonas species. Using a two-plasmid pCasPA/pACRISPR system, we achieved efficient deletion of the denitrification-associated narG and dnrE genes in Stutzerimonas decontaminans 19SMN4. On the other hand, Cas9-associated toxicity significantly limited transformation in Stutzerimonas perfectomarina ZoBell. These results highlight both the potential and the limitations of CRISPR/Cas9 editing in Stutzerimonas, emphasizing that genome editing efficiency and tolerance may vary even among closely related strains.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Plasmids/genetics
*Genome, Bacterial
Denitrification/genetics

@article {pmid41905455,
year = {2026},
author = {Meng, Y and Zhang, Z and Jia, K and Wang, W and Wang, H and Hu, Z and Liu, J},
title = {Membrane protein-focused CRISPR screen identifies ATP2A2 as a druggable transcriptional co-regulator of CCND1 (cyclin D1) in lung adenocarcinoma.},
journal = {Life sciences},
volume = {394},
number = {},
pages = {124358},
doi = {10.1016/j.lfs.2026.124358},
pmid = {41905455},
issn = {1879-0631},
mesh = {Humans ; *Lung Neoplasms/genetics/pathology/metabolism/drug therapy ; *Adenocarcinoma of Lung/genetics/metabolism/pathology/drug therapy ; Animals ; *Cyclin D1/genetics/metabolism ; Mice ; Cell Proliferation ; A549 Cells ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; Apoptosis ; Cell Line, Tumor ; Mice, Nude ; Carcinoma, Non-Small-Cell Lung/genetics/pathology/metabolism ; Cell Movement ; Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; },
abstract = {INTRODUCTION: Lung adenocarcinoma (LUAD) is one of the most commonly seen non-small cell lung cancer (NSCLC). Recent progress has highlighted cyclin-dependent kinase (CDK) inhibitors for treating diverse cancers including NSCLC. However, acquired resistance of CDK inhibitors has prompted the exploration of druggable regulatory or compensatory pathways in cell cycle as alternative or combination therapies of CDK inhibitors.

AIM: This study aims to discover the upstream regulatory proteins in LUAD using a membrane protein-focused CRISPR screen in A549 cells and dissect their functions.

MATERIALS AND METHODS: The top hit ATP2A2, an endoplasmic reticulum (ER)-localized protein, was analyzed for its function in LUAD using cell proliferation assay, migration and invasion assays and apoptosis assay. The mechanistic studies of ATP2A2 were conducted using RT-qPCR, Western blotting, RNA-Seq, cell cycle assay, protein immunoprecipitation, mass spectrometry, immunofluorescence microscopy and others.

KEY FINDINGS: The correlation of ATP2A2 expression with NSCLCs was confirmed with public database and collected clinical samples. ATP2A2 promoted LUAD development by involving in cell cycle regulation. ATP2A2 interacted with another ER protein HACD3 and co-regulated the transcription of CCND1 gene (cyclin D1), which forms complex with CDK4/6. ATP2A2 and HACD3 promoted CCND1 expression by activating NF-κB signaling and the nuclear translocation of p65 protein, which is a known transcription factor of CCND1. Curcumin, an ATP2A2-targeting natural product, could inhibit LUAD both in vitro and in xenografted mouse model.

SIGNIFICANCE: This study revealed ATP2A2-HACD3-NF-κB as a regulatory axis of cyclin D1 expression and shed light on developing ATP2A2-targeted medications for LUAD treatment.},
}

Humans
*Lung Neoplasms/genetics/pathology/metabolism/drug therapy
*Adenocarcinoma of Lung/genetics/metabolism/pathology/drug therapy
Animals
*Cyclin D1/genetics/metabolism
Mice
Cell Proliferation
A549 Cells
Gene Expression Regulation, Neoplastic
CRISPR-Cas Systems
Apoptosis
Cell Line, Tumor
Mice, Nude
Carcinoma, Non-Small-Cell Lung/genetics/pathology/metabolism
Cell Movement
Clustered Regularly Interspaced Short Palindromic Repeats
Female

@article {pmid41905499,
year = {2026},
author = {Fu, T and Wang, F and Ren, J and Chen, S and Peng, N and Zhang, L and Pu, K and Li, Q},
title = {An extraction-free HLPCR-Cas12a assay for ultra-sensitive and rapid detection of Acinetobacter baumannii and Klebsiella pneumoniae following craniotomy.},
journal = {Journal of microbiological methods},
volume = {244},
number = {},
pages = {107480},
doi = {10.1016/j.mimet.2026.107480},
pmid = {41905499},
issn = {1872-8359},
mesh = {*Acinetobacter baumannii/isolation & purification/genetics ; Humans ; *Klebsiella pneumoniae/isolation & purification/genetics ; *Craniotomy/adverse effects ; Sensitivity and Specificity ; *Acinetobacter Infections/diagnosis/microbiology ; *Polymerase Chain Reaction/methods ; *Klebsiella Infections/diagnosis/microbiology ; CRISPR-Cas Systems ; Postoperative Complications/microbiology/diagnosis ; },
abstract = {Postoperative intracranial infection represents a significant complication in neurosurgical procedures, with Acinetobacter baumannii (Ab) and Klebsiella pneumoniae (Kp) frequently identified as common drug-resistant pathogens. In response to this challenge, we have developed an efficient, extraction-free detection method that facilitates the rapid and highly sensitive identification of Ab and Kp. This method employs a single-tube format that integrates heat lysis with PCR, allowing for the concurrent bacterial lysis and target gene amplification. Detection of the amplified products is achieved through the Cas12a/crRNA complex, which, upon activation, exhibits trans-cleavage activity that cleaves reporters to produce a signal detectable by microplate readers or lateral flow test strips (LFTS). The Heat Lysis-PCR-CRISPR/Cas12a (HLPCR-CRISPR/Cas12a) detection platform unifies lysis and amplification in one step, streamlining the process. It exhibits high specificity (no cross-reactivity) and achieves detection limits of 10[2] CFU/μL (fluorescence) and 10[3] CFU/μL (LFTS). When validated on clinical CSF samples, it delivered results in 90 min with perfect concordance with quantitative PCR (qPCR). Combining speed, high sensitivity, specificity, and operational simplicity, this platform provides a powerful diagnostic tool for early detection of Ab and Kp in postoperative intracranial infections.},
}

*Acinetobacter baumannii/isolation & purification/genetics
Humans
*Klebsiella pneumoniae/isolation & purification/genetics
*Craniotomy/adverse effects
Sensitivity and Specificity
*Acinetobacter Infections/diagnosis/microbiology
*Polymerase Chain Reaction/methods
*Klebsiella Infections/diagnosis/microbiology
CRISPR-Cas Systems
Postoperative Complications/microbiology/diagnosis

@article {pmid41921842,
year = {2026},
author = {Liao, YJ and Li, YG and Li, YL},
title = {CRISPR/Cas9-mediated lgp2 knockout and cross-species rescue reveal the immunoregulatory role of LGP2 in zebrafish.},
journal = {Fish & shellfish immunology},
volume = {173},
number = {},
pages = {111308},
doi = {10.1016/j.fsi.2026.111308},
pmid = {41921842},
issn = {1095-9947},
mesh = {Animals ; *Zebrafish/genetics/immunology ; CRISPR-Cas Systems ; *Zebrafish Proteins/genetics/immunology ; Gene Knockout Techniques/veterinary ; *Immunity, Innate/genetics ; *Fish Diseases/immunology ; Gene Expression Profiling/veterinary ; *Gene Expression Regulation/immunology ; Species Specificity ; },
abstract = {LGP2 is a key regulator within the RIG-I-like receptor (RLR) pathway with dual roles in antiviral immunity, but its function under basal conditions and its evolutionary variation among fish species require further investigation. In this study, we constructed a zebrafish lgp2 knockout model using CRISPR/Cas9. Knockout lgp2 led to upregulation of key RLR signaling components (mda5, mavs), type I interferons (ifnphi1, ifnphi3, ifnphi4), and the interferon-stimulated gene mxa. Transcriptomic profiling of 3 dpf embryos identified 3186 differentially expressed genes (DEGs), including significantly elevated expression of five claudin-family genes. Protein-protein interaction (PPI) analysis of DEGs indicated that irf7 and claudin b exhibit an interaction at the protein level. Gene ontology analysis showed that LGP2 was enriched in terms such as helicase activity and ATPase activity, and was also significantly enriched in extracellular structure terms. Comparative sequence analysis revealed conserved DEXDc, HELICc and CTD domains in LGP2 from zebrafish, grass carp and barbel chub, with notable species-specific sequence variations. Functional rescue experiments demonstrated that all three LGP2 orthologs could restore lgp2 expression and modulate downstream interferon responses in lgp2[-/-] embryos; however, induction of the transcription factor irf7 was specifically mediated only by zebrafish-derived LGP2. These results provide insights into the functional diversity of LGP2 in teleost immunity and inform strategies for selective breeding aimed at disease resistance.},
}

Animals
*Zebrafish/genetics/immunology
CRISPR-Cas Systems
*Zebrafish Proteins/genetics/immunology
Gene Knockout Techniques/veterinary
*Immunity, Innate/genetics
*Fish Diseases/immunology
Gene Expression Profiling/veterinary
*Gene Expression Regulation/immunology
Species Specificity

@article {pmid41939725,
year = {2026},
author = {Xu, H and Liu, R and Zhou, H and Kong, B and Shen, K and Zhao, T and Du, X and Zhang, H and Song, H and Guo, D and Gu, X and Wang, Q and Lee, CW and Yin, G and Zhang, Y and Chen, W},
title = {Engineered small extracellular vesicles as bioactive materials: Integrating engineering strategies for cargo loading and targeted delivery systems.},
journal = {Bioactive materials},
volume = {59},
number = {},
pages = {96-134},
pmid = {41939725},
issn = {2452-199X},
abstract = {Small extracellular vesicles (sEVs) are increasingly regarded as a unique class of bioactive materials whose intrinsic membrane composition and nanoscale architecture provide a versatile platform for therapeutic engineering. Rather than passive carriers, sEVs can be actively programmed through diverse strategies to achieve efficient loading, precise targeting, and functional integration with synthetic systems. Endogenous modulation of donor cells-via genetic editing, priming with bioactive glass, cytokine stimulation, or hypoxic cues-enables selective packaging of nucleic acids, proteins, and metabolites into secreted vesicles. Exogenous techniques, including electroporation, sonication, and extrusion, allow controlled incorporation of therapeutic drugs or genome-editing complexes such as CRISPR/Cas. In parallel, surface modifications based on Lamp2b-fusion scaffolds, aptamers, antibodies, and click chemistry confer tissue tropism and extend circulation time. Integration with nanomaterials, scaffolds, and microfluidic platforms further enhances stability, scalability, and reproducibility, positioning sEVs at the intersection of biology and materials science. This review highlights recent advances in engineering sEVs as programmable bioactive materials and discusses their potential to transform regenerative medicine, oncology, and precision therapeutics.},
}

@article {pmid41940952,
year = {2026},
author = {Wang, N and Tang, J and Hou, X and Quan, C and Mai, J and He, C and Chen, R and Tao, B and Gu, Y and Jia, D and Fu, T and Zou, J and Zhao, L and Wen, J and Shen, J},
title = {BnaCIPK9 homoeologs mediate the dosage-dependent regulation of seed oil in allotetraploid Brassica napus L.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {139},
number = {4},
pages = {},
pmid = {41940952},
issn = {1432-2242},
support = {2022ZD04008//Biological Breeding-National Science and Technology Major Project/ ; 2022YFD1200804//National Key Research and Development Program of China/ ; 2021HSZD004//Hubei Hongshan Laboratory research funding/ ; 2662023PY004//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Brassica napus/genetics/metabolism ; *Seeds/genetics/metabolism ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; *Plant Oils/metabolism ; Phylogeny ; CRISPR-Cas Systems ; Tetraploidy ; *Gene Dosage ; Haplotypes ; Arabidopsis/genetics ; Genes, Plant ; },
abstract = {CRISPR/Cas9 mutagenesis and overexpression lines analyses revealed CIPK9 homoeologs function in oil regulation of allotetraploid Brassica napus, cooperative multicopy interactions, and a superior haplotype on chromosome A10. Rapeseed (Brassica napus, B. napus), a globally significant allopolyploid oilseed crop, fulfills substantial annual vegetable oil demand. Evolutionary adaptation in this species is underpinned by gene duplication and homoeolog retention, enhancing plasticity under dynamic environmental stresses. This study focuses on BnaCIPK9, a regulator of seed oil content. Phylogenomic and structural analyses demonstrate that BnaCIPK9 homoeologs underwent duplication followed by consistent evolutionary retention within the Brassica lineage, exhibiting remarkable sequence and structural conservation. Expression profiling revealed tissue-partitioned functional specialization among homoeologs, with BnaA10.CIPK9 and BnaC05.CIPK9 showing seed-preferential expression. CRISPR/Cas9 knockout in B. napus and heterologous overexpression in Arabidopsis demonstrate these homoeologs act as dosage-dependent regulators of oil accumulation, dependent on their distinct expression patterns. They further exhibit expression-driven functional diversification in abiotic stress responses during seedling development. Population genomics reveal differential evolutionary trajectories among duplicates, with intensified selection on chromosome A10 driving adaptive divergence. Crucially, haplotype-trait association identifies hap.qCIPK9.A10.0 as a major haplotype linked to elevated oil content. This work elucidates how homoeolog subfunctionalization fine-tunes critical agronomic traits, oil biosynthesis, and stress resilience, in polyploid crops, establishing haplotype-assisted breeding as imperative for developing crop cultivars. Favorable haplotypes, exemplified by hap.qCIPK9.A10.0, offer precise targets for high-oil crop breeding improvement.},
}

*Brassica napus/genetics/metabolism
*Seeds/genetics/metabolism
Gene Expression Regulation, Plant
*Plant Proteins/genetics/metabolism
*Plant Oils/metabolism
Phylogeny
CRISPR-Cas Systems
Tetraploidy
*Gene Dosage
Haplotypes
Arabidopsis/genetics
Genes, Plant

@article {pmid41942919,
year = {2026},
author = {Behera, L and Samal, KC and C, P and Agrawal, PK and Achary, VMM and Dash, M and Mishra, A and Rani, M and Masika, FB and Goud, GSD and Kesawat, MS and Samantaray, S},
title = {An improved Agrobacterium-mediated transformation method for genome editing using CRISPR/Cas9 in elite indica rice (Oryza sativa L.).},
journal = {BMC genomics},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12864-026-12800-0},
pmid = {41942919},
issn = {1471-2164},
}

@article {pmid41943368,
year = {2026},
author = {Sangeetha, VJ and Pawase, PA and Vasanthkumar, SS and Patrimath, SS and Bashir, O},
title = {Precision-engineered starch: Integrating metabolic engineering and cell-free synthetic biology for sustainable bioplastics and functional foods.},
journal = {Carbohydrate polymers},
volume = {381},
number = {},
pages = {125146},
doi = {10.1016/j.carbpol.2026.125146},
pmid = {41943368},
issn = {1879-1344},
mesh = {*Metabolic Engineering/methods ; *Starch/chemistry/biosynthesis/genetics/metabolism ; *Synthetic Biology/methods ; *Functional Food ; Amylose/chemistry/metabolism ; Gene Editing ; },
abstract = {This review consolidates recent advances that reposition starch development from downstream modification towards deliberate biosynthetic engineering. It critically examines the starch biosynthetic pathway as a foundation for rational design, highlighting the coordinated roles of granule-bound starch synthase, soluble starch synthases, starch branching enzymes, and debranching enzymes in defining granule structure. Clear structure-function-nutrition relationships are delineated, demonstrating how amylose-amylopectin ratio, chain-length distribution, and phosphate esterification govern physicochemical behaviour, digestibility, glycemic response, and resistant starch formation. Insights derived from naturally occurring starch mutants are integrated to elucidate genotype-phenotype-function linkages. It further evaluates the recent progress in planta metabolic engineering, with particular emphasis on CRISPR-Cas-based genome editing as a precise strategy for generating food-relevant starch phenotypes without foreign DNA insertion. Representative applications include high-amylose, low-glycemic staple crops and starches with engineered branching patterns to enhance processing performance. In parallel, emerging cell-free synthetic biology platforms are presented as complementary systems for producing structurally defined glucans with high purity and reproducibility. Collectively, these developments establish engineered starch as a next-generation bio-based material aligned with nutritional quality, technological functionality and sustainability objectives.},
}

*Metabolic Engineering/methods
*Starch/chemistry/biosynthesis/genetics/metabolism
*Synthetic Biology/methods
*Functional Food
Amylose/chemistry/metabolism
Gene Editing

@article {pmid41943836,
year = {2026},
author = {Xu, J and Cheng, L and Ma, S and Gan, C and Chai, J and Zheng, X and Hu, L and Ling, M and Zhang, M and Zhao, B and Cheng, H},
title = {In vivo CRISPR/Cas9 Screening Reveals that UBE2L3 Modulates Autophagic Flux through TSC2 Ubiquitination and Potentiates PD-1 Blockade in Triple-Negative Breast Cancer.},
journal = {International journal of biological sciences},
volume = {22},
number = {6},
pages = {2950-2969},
pmid = {41943836},
issn = {1449-2288},
mesh = {*Triple Negative Breast Neoplasms/metabolism/genetics ; *Ubiquitin-Conjugating Enzymes/metabolism/genetics ; Humans ; *Autophagy/genetics/physiology ; *Tuberous Sclerosis Complex 2 Protein/metabolism/genetics ; Ubiquitination ; *CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Female ; Animals ; Mice ; *Programmed Cell Death 1 Receptor/metabolism/antagonists & inhibitors ; Mice, Nude ; },
abstract = {Triple-negative breast cancer (TNBC), a distinct breast cancer subtype, poses significant challenges to conventional therapeutic approaches, and effective targeted therapies are limited. CRISPR/Cas9 library screening has demonstrated unprecedented efficiency and revolutionary potential in the identification of therapeutic targets. In this study, we performed In vivo CRISPR/Cas9 library screening and identified the E2 ubiquitin-conjugating enzyme UBE2L3 as a critical regulatory factor in the progression of TNBC. Loss of UBE2L3 restricted tumor cell growth by modulating autophagy in TNBC cells. Mechanistically, UBE2L3 downregulation led to increased tuberous sclerosis complex 2 (TSC2) expression, suppressing mTOR activity and altering autophagic processes in tumor cells. This regulation was mediated through the interaction between UBE2L3 and the E3 ubiquitin ligase SMURF2, which together control TSC2 protein ubiquitination and degradation. Autophagy and the tumor microenvironment are closely associated, and we observed that UBE2L3 knockdown in TNBC tumors significantly increased CD8+ T lymphocyte infiltration and enhanced tumor sensitivity to anti-PD-1 therapy. Collectively, our findings provide a theoretical foundation for considering UBE2L3 as a potential therapeutic target in TNBC.},
}

*Triple Negative Breast Neoplasms/metabolism/genetics
*Ubiquitin-Conjugating Enzymes/metabolism/genetics
Humans
*Autophagy/genetics/physiology
*Tuberous Sclerosis Complex 2 Protein/metabolism/genetics
Ubiquitination
*CRISPR-Cas Systems/genetics
Cell Line, Tumor
Female
Animals
Mice
*Programmed Cell Death 1 Receptor/metabolism/antagonists & inhibitors
Mice, Nude

@article {pmid41943869,
year = {2026},
author = {Katayama, R and Hayashi, R and Ueda, M and Sakamoto, T and Nakazawa, M},
title = {Elucidation of Enzymatic Routes Underlying Odd-Chain Fatty Acid Synthesis and Propionate Assimilation in Euglena gracilis.},
journal = {The Journal of eukaryotic microbiology},
volume = {73},
number = {3},
pages = {e70078},
doi = {10.1111/jeu.70078},
pmid = {41943869},
issn = {1550-7408},
support = {JPMJGX23B0//Japan Science and Technology Agency/ ; JP24K01897//Japan Society for the Promotion of Science/ ; //Women Researchers Support Ofice of Osaka Metropolitan University/ ; },
mesh = {*Propionates/metabolism ; *Euglena gracilis/metabolism/enzymology/genetics ; *Fatty Acids/biosynthesis ; Gene Knockout Techniques ; Acyl Coenzyme A/metabolism ; Methylmalonyl-CoA Mutase/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Wax esters (WEs) produced by Euglena gracilis under anaerobic conditions contain significant proportions of odd-chain fatty acids and fatty alcohols (30%-40%). Propionyl-CoA, the primer for odd-chain fatty acid synthesis, is generally thought to be generated via the methylmalonyl-CoA pathway; however, the enzymatic basis of this pathway in E. gracilis remains unclear. Here, we generated knockout (KO) mutants of candidate enzymes in the methylmalonyl-CoA pathway-succinyl-CoA synthetase (SCS), methylmalonyl-CoA mutase (MCM), methylmalonyl-CoA epimerase (MCE), and propionyl-CoA carboxylase (PCC)-using the CRISPR/Cas9 system and examined their roles in odd-chain fatty acid synthesis and propionate assimilation. The methylmalonyl-CoA pathway exhibited direction-dependent functions. KO of MCE and PCC did not affect the proportion of odd-chain WEs, indicating that these enzymes are not required for odd-chain WE synthesis and suggesting an alternative route converting methylmalonyl-CoA to propionyl-CoA. In contrast, both enzymes were required for propionate assimilation. Functional differentiation was observed between SCSβ isozymes: SCSβ1 contributed to odd-chain WE synthesis, whereas SCSβ2 primarily functioned in propionate assimilation. Partial impairment of propionate assimilation in SCSα or SCSβ2 KO strains suggests involvement of additional routes. These findings improve our understanding of direction-dependent roles in the methylmalonyl-CoA pathway in E. gracilis and support further studies of odd-chain lipid biosynthesis.},
}

*Propionates/metabolism
*Euglena gracilis/metabolism/enzymology/genetics
*Fatty Acids/biosynthesis
Gene Knockout Techniques
Acyl Coenzyme A/metabolism
Methylmalonyl-CoA Mutase/genetics/metabolism
CRISPR-Cas Systems

@article {pmid41945389,
year = {2026},
author = {Li, M and Gao, G and Jiao, Y},
title = {A Hierarchical Screening Strategy for Genome-Edited Events in Polyploid Species: A Case Study on Hexaploid Common Wheat.},
journal = {Current protocols},
volume = {6},
number = {4},
pages = {e70354},
doi = {10.1002/cpz1.70354},
pmid = {41945389},
issn = {2691-1299},
support = {2024YFF1000301//National Key R&D Program of China/ ; tsqn202312306//Taishan Scholars Program/ ; 2025HWYQ-071//Shandong Provincial Natural Science Fund for Excellent Young Scientists Fund Program (Overseas)/ ; SYS202206//Shandong Provincial Natural Science Foundation/ ; ZR2022ZD22//Shandong Provincial Natural Science Foundation/ ; ZR2021ZD30//Shandong Provincial Natural Science Foundation/ ; },
mesh = {*Triticum/genetics ; *Gene Editing/methods ; *Polyploidy ; *Genome, Plant ; CRISPR-Cas Systems ; },
abstract = {Genome editing via CRISPR/Cas9 has been widely adopted in cereal crops. In diploid species such as rice and barley, the generation of knockout mutants is relatively straightforward for functional characterization of the genes of interest due to their single-copy nature in the genome. In contrast, common wheat (Triticum aestivum L.) is a hexaploid species comprising three subgenomes (AABBDD); consequently, most genes are present as three homoeoalleles that retained substantial function redundancy during evolution. The generation of a complete set of single, double, and triple mutants is therefore essential for elucidating homoeoallele-specific functions and dissecting their contributions to the developmental and agronomic traits. Moreover, ensuring germplasm purity through the elimination of residual T-DNA is critical for maintaining stable mutation, particularly in single- and double-mutant lines. Here, we describe a hierarchical screening strategy for efficient identification of a comprehensive series of CRISPR/Cas9-induced mutants. This approach integrates high-throughput DNA isolation, selection of T-DNA-free mutants, maintenance of a uniform genetic background via backcrossing, systematic screening of all mutant combinations, and molecular confirmation of genome edits. This screening pipeline has proven effective in hexaploid common wheat and is readily adaptable to other polyploid species that are amenable to crossing. © 2026 Wiley Periodicals LLC. Basic Protocol 1: Wheat cultivation and leaf sample preparation Basic Protocol 2: High-throughput DNA isolation Basic Protocol 3: Genotyping using an optimized, cost-efficient T7E1 assay Alternate Protocol 1: Genotyping using the KASP assay Support Protocol 1: Screening of T-DNA-free triple mutants.},
}

*Triticum/genetics
*Gene Editing/methods
*Polyploidy
*Genome, Plant
CRISPR-Cas Systems

@article {pmid41946009,
year = {2026},
author = {Sutaoney, P and Singh, P and Malakar, S and Arsi, L and Ghosh, P},
title = {Microbial lipases: Catalyzing sustainable solutions for industrial innovations.},
journal = {Enzyme and microbial technology},
volume = {198},
number = {},
pages = {110869},
doi = {10.1016/j.enzmictec.2026.110869},
pmid = {41946009},
issn = {1879-0909},
abstract = {Microbial lipases are multifaceted biological catalyst that have surfaced as a key driver in various industries and are both eco-friendly and cost efficient.In large scale applications, lipases produced from bacteria, fungi and yeasts function better than their equivalents generated from plants and animals due to their wide substrate specificity, catalytic efficacy and stability under physicochemical circumstances. Recent developments in microbial lipase research, including sources, screening techniques, assay procedures, production methods, purification tactics, and biochemical characterisation, are critically examined in this review.The structural and mechanistic elements that control lipase function-such as lid domains, interfacial activation, and catalytic triads-are given special attention since they all have an impact on the stability, specificity, and industrial performance of the enzyme.Large-scale screening is done to check for the production of lipase in Bacillus sp., Achromobacter sp., Alcaligenes sp., Arthrobacter sp., Pseudomonas sp., and Penicillium sp. Additionally, the combination of synthetic biology, metagenomics, CRISPR-Cas technologies, enzyme engineering, and AI-assisted modelling is emphasized as a revolutionary strategy for identifying and customizing lipases with desired characteristics, including extreme environment microbes and application-specific variants.The review also highlights the growing industrial uses of microbial lipases in the bio-fuel, food and beverage, detergent, textile, leather, pharmaceutical, and medical industries, highlighting their contribution to the development of economically feasible and ecologically safe bioprocesses. All things considered, microbial lipases are an important biotechnological tool for developing sustainable industrial innovation and green chemistry.},
}

@article {pmid41938355,
year = {2026},
author = {Ma, G and Xu, H and Zhang, S and Li, X and Liu, J and Xie, J and Yan, F and Zhou, H},
title = {Enhancing the efficiency of nuclease-based prime editing in rice with the Tf1 reverse transcriptase.},
journal = {aBIOTECH},
volume = {7},
number = {2},
pages = {100026},
pmid = {41938355},
issn = {2662-1738},
abstract = {The development of efficient and precise genome-editing tools is crucial for advancing functional genomics and improving crops. Our previously established nuclease-mediated prime editing (NM-PE) system, which combines the SpCas9 nuclease with prime editing based on microhomology-mediated end joining, enables the seamless insertion of small DNA fragments into plant genomes to add tags to genes of interest. However, the efficiency of 3 × FLAG sequence insertion via NM-PE requires further improvement. Here, we report a significant optimization of this system by replacing the M-MLV reverse transcriptase (RT) with evolved variants of the retrotransposon RT Tf1 derived from the mammalian PE6 system. Through codon optimization, we generated the evoTf1M4 variant, which substantially enhanced the efficiency of NM-PE. The optimized construct rPE20aV3 achieved up to 18.75% precise insertion of a 66-bp 3 × FLAG sequence at endogenous loci, representing a three-fold improvement over the original NM-PE system. Our results demonstrate that Tf1-aided optimization of NM-PE serves as an efficient platform for seamless insertion of a 3 × FLAG sequence in rice, offering broad potential for advanced genome engineering in plants.},
}

@article {pmid40813912,
year = {2026},
author = {Mathis, N and Marquart, KF and Allam, A and Krauthammer, M and Schwank, G},
title = {Systematic pegRNA design with PRIDICT2.0 and ePRIDICT for efficient prime editing.},
journal = {Nature protocols},
volume = {21},
number = {4},
pages = {1523-1543},
pmid = {40813912},
issn = {1750-2799},
support = {185293, 214936//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; 201184//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; },
mesh = {*Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Software ; Humans ; CRISPR-Cas Systems ; Machine Learning ; Chromatin/genetics ; *Computational Biology/methods ; Neural Networks, Computer ; },
abstract = {Prime editing is a versatile genome editing technology that enables precise genetic modifications without inducing DNA double-strand breaks. Owing to numerous variables in the prime editing guide RNA (pegRNA) design, experimentally identifying the most efficient pegRNA for a specific locus and edit is laborious. Therefore, we have developed computational tools to streamline this process. Here we present a comprehensive protocol detailing how to use PRIDICT2.0 and ePRIDICT, machine-learning models that assess the influence of the pegRNA design and chromatin context on prime editing. PRIDICT2.0 is an ensemble of attention-based bidirectional recurrent neural networks that predicts pegRNA efficiencies for replacements, insertions or deletions in different cellular contexts. Compared with other pegRNA design tools, PRIDICT2.0 accommodates larger edits-up to 40 base pairs-across diverse edit types, also allowing the introduction of silent bystander edits that can enhance editing efficiency. ePRIDICT, a gradient-boosting algorithm, further accounts for the local chromatin environments and assesses how the genomic location of the target site affects prime editing rates. Both tools are available at www.pridict.it for individual predictions or can be installed locally for batch processing of multiple edits and target sites. The protocol provides step-by-step instructions on using PRIDICT2.0 and ePRIDICT, covering sequence input, prediction generation and interpretation. Web-based predictions take under a minute, while local installation and batch processing may take up to several hours, depending on the dataset size. By streamlining pegRNA selection and chromatin context analysis, these tools promote the adoption of prime editing in basic and translational research.},
}

*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics
*Software
Humans
CRISPR-Cas Systems
Machine Learning
Chromatin/genetics
*Computational Biology/methods
Neural Networks, Computer

@article {pmid40983723,
year = {2026},
author = {Lilja, A and Finkel, Y and Aharon, E and Nachshon, A and Schwartz, M and Stern-Ginossar, N},
title = {Multidimensional analysis of host-virus interactions using the virus-encoded CRISPR-based direct readout system (VECOS).},
journal = {Nature protocols},
volume = {21},
number = {4},
pages = {1429-1465},
pmid = {40983723},
issn = {1750-2799},
support = {CoG-2019-864012//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Cytomegalovirus/genetics/physiology ; Genome, Viral ; *Host-Pathogen Interactions/genetics ; *Host Microbial Interactions/genetics ; HEK293 Cells ; },
abstract = {CRISPR-Cas9 technology has transformed the study of gene function, enabling the systematic investigation of host-virus interactions. However, most CRISPR-based screens in the context of viral infections rely on cell survival as a readout, which limits their sensitivity and biases results toward early infection stages. To address these challenges, we developed the virus-encoded CRISPR-based direct readout system (VECOS), a virus-centric approach in which human cytomegalovirus is engineered to express single-guide RNA (sgRNA) libraries directly from its genome. This system allows sgRNA abundance, embedded in the viral genome, to serve as a direct and quantitative readout of gene-perturbation effects on viral propagation. By tracking sgRNA levels at distinct stages of the viral infection cycle, VECOS enables a detailed, multidimensional analysis of virus-host interactions. Here we present a modular detailed Protocol for (1) constructing and reconstituting complex sgRNA libraries in double-stranded DNA viruses using bacterial artificial chromosomes, (2) performing multipassage screens to investigate perturbation effects on various stages of viral infection and (3) analyzing the multipassage and multistage sgRNA abundance measurements utilizing a comprehensive framework for data analysis. Successful implementation of this full Protocol takes 14-22 weeks and requires proficiency in molecular biology, as well as basic familiarity with Unix-based computing and programming in R for data processing. This Protocol offers researchers a robust tool for uncovering the molecular mechanisms that drive viral propagation and host-virus interactions.},
}

*CRISPR-Cas Systems/genetics
Humans
RNA, Guide, CRISPR-Cas Systems/genetics
*Cytomegalovirus/genetics/physiology
Genome, Viral
*Host-Pathogen Interactions/genetics
*Host Microbial Interactions/genetics
HEK293 Cells

@article {pmid41674076,
year = {2026},
author = {Arrigo, A and Rao, V and Ratan, A and Kulkarni, SS},
title = {Patient-informed CRISPR screen identifies FLNB as a congenital heart disease and ciliopathy gene.},
journal = {HGG advances},
volume = {7},
number = {2},
pages = {100580},
pmid = {41674076},
issn = {2666-2477},
mesh = {Humans ; Animals ; *Ciliopathies/genetics ; *Heart Defects, Congenital/genetics/diagnosis ; *Filamins/genetics ; *CRISPR-Cas Systems ; Male ; Female ; *Heterotaxy Syndrome/genetics/diagnosis ; Xenopus ; Exome Sequencing ; Mutation ; },
abstract = {Heterotaxy (HTX) is a congenital disorder characterized by abnormal left-right organ placement, often leading to severe congenital heart disease (CHD). Despite advances in sequencing, many CHD and HTX-associated genes remain functionally unvalidated, hindering effective clinical diagnosis and management. Here, we leveraged a high-throughput CRISPR-Cas9 screening approach in the Xenopus model to rapidly evaluate candidate genes identified from whole-exome sequencing of human CHD patients. Our screen identified Filamin B (FLNB), an actin-binding protein previously linked to skeletal disorders but not to ciliopathies or CHD. We identified 5 probands with CHD and HTX, 3 with recessive and 2 with damaging heterozygous variants in FLNB. Disrupting flnb in Xenopus reproduced key features of the human HTX phenotype, including defects in cardiac development and impaired motile cilia function. Rescue experiments confirmed the functional conservation of human FLNB, directly implicating actin cytoskeletal disruption in ciliogenesis and left-right patterning defects. Our results provide crucial evidence linking human FLNB dysfunction to ciliopathies and CHD and HTX.},
}

Humans
Animals
*Ciliopathies/genetics
*Heart Defects, Congenital/genetics/diagnosis
*Filamins/genetics
*CRISPR-Cas Systems
Male
Female
*Heterotaxy Syndrome/genetics/diagnosis
Xenopus
Exome Sequencing
Mutation

@article {pmid41776280,
year = {2026},
author = {Shirai, Y and Kao, JA and Kumar, T and Matsuda, N and Nakagawa, R and Nureki, O and Extavour, CG and Daimon, T},
title = {HUH-tagged Cas9 as a platform for efficient ssODN-mediated knock-in via embryo and adult injection in insects.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {41776280},
issn = {2399-3642},
support = {20H02999, 20K21311, 22K19179, 24H00511, 24K21869//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 21J20658//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; Overseas Research Fellow//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; Long-Term Fellow//Human Frontier Science Program (HFSP)/ ; JPJ008000//Cabinet Office, Government of Japan/ ; IOS-2220747//NSF | BIO | Division of Integrative Organismal Systems (IOS)/ ; },
mesh = {Animals ; *Gene Knock-In Techniques/methods ; *Tribolium/genetics/embryology ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism ; DNA End-Joining Repair ; *Oligodeoxyribonucleotides/genetics ; Recombinational DNA Repair ; },
abstract = {Recent advances in adult injection-based insect genome editing have enabled genetic manipulation of a wide range of insect species, including those previously considered difficult or even impervious to genetic modification. However, achieving efficient knock-in remains a significant challenge with this approach. Here, we demonstrate that fusing a HUH endonuclease tag to Cas9 significantly enhances both non-homologous end joining (NHEJ)-mediated knockout and homology-directed repair (HDR)-mediated knock-in via adult injection. This fusion increased knockout efficiency by up to fivefold in the beetle Tribolium castaneum through adult injection, likely due to its previously unrecognized nuclear localization activity. It also improved single-stranded oligodeoxynucleotide (ssODN)-mediated knock-in efficiency, which we attribute to its characteristic ssDNA-tethering activity. To evaluate its versatility, we tested the HUH-tagged Cas9 in conventional embryo injection, which significantly enhanced HDR-mediated knock-in of an epitope tag in cricket and milkweed bug embryos. Our findings establish the HUH-tag as a versatile platform for improving both NHEJ- and HDR-based genome editing, providing a robust framework to advance genetic engineering across a broad spectrum of arthropods.},
}

Animals
*Gene Knock-In Techniques/methods
*Tribolium/genetics/embryology
*Gene Editing/methods
*CRISPR-Cas Systems
*CRISPR-Associated Protein 9/genetics/metabolism
DNA End-Joining Repair
*Oligodeoxyribonucleotides/genetics
Recombinational DNA Repair

@article {pmid41867107,
year = {2026},
author = {Zhuang, S and Li, J and Fang, Z and Zhou, H and Zhang, R and He, J and Zhu, L and Xu, Y and Xu, D and Gu, D and Wang, J},
title = {Phosphorothioate Modification-Regulated One-Pot CRISPR Assay for Arbovirus Detection.},
journal = {ACS infectious diseases},
volume = {12},
number = {4},
pages = {1423-1433},
doi = {10.1021/acsinfecdis.6c00013},
pmid = {41867107},
issn = {2373-8227},
mesh = {*CRISPR-Cas Systems ; *Chikungunya virus/genetics/isolation & purification ; Humans ; Nucleic Acid Amplification Techniques/methods ; *Chikungunya Fever/diagnosis/virology ; *Arboviruses/genetics/isolation & purification ; RNA, Viral/genetics ; *Phosphorothioate Oligonucleotides/chemistry ; CRISPR-Associated Proteins/metabolism/genetics ; Endodeoxyribonucleases/metabolism/genetics ; Sensitivity and Specificity ; *Molecular Diagnostic Techniques/methods ; Bacterial Proteins ; },
abstract = {The ongoing arthropod-borne Chikungunya virus (CHIKV) highlights the requirements of rapid and accurate diagnostic methods to enhance the epidemic control. CRISPR diagnostic (CRISPR-Dx) technology holds promise, but the development of a highly efficient one-pot diagnostic system usually requires fine-tuning of the balance between isothermal amplification and Cas cleavage procedures. Here, we describe a simple method (psHOLMES) to create one-pot, two-step CRISPR-Dx systems, using photocleavable partially phosphorothioate-modified DNA (ppPS-DNA) to regulate Cas12a activity. Cas12a activity is first inactivated via binding of ppPS-DNA during the target sequence amplification procedure, which is then reactivated by ultraviolet (UV)-mediated photolysis of ppPS-DNA after amplification, triggering Cas12a trans-cleavage reactions. psHOLMES demonstrates attomolar sensitivity for CHIKV RNA detection and zero cross-reactivity against other related arboviruses. When applied to clinical samples, psHOLMES achieved 100% (50/50) accuracy and could detect CHIKV within 30 min. As traditional efforts for fine-tuning Cas cis-cleavage activity can be omitted, psHOLMES thus enables rapid development of one-pot CRISPR-Dx systems for clinical applications.},
}

*CRISPR-Cas Systems
*Chikungunya virus/genetics/isolation & purification
Humans
Nucleic Acid Amplification Techniques/methods
*Chikungunya Fever/diagnosis/virology
*Arboviruses/genetics/isolation & purification
RNA, Viral/genetics
*Phosphorothioate Oligonucleotides/chemistry
CRISPR-Associated Proteins/metabolism/genetics
Endodeoxyribonucleases/metabolism/genetics
Sensitivity and Specificity
*Molecular Diagnostic Techniques/methods
Bacterial Proteins

@article {pmid41875952,
year = {2026},
author = {Li, C and Zhang, L and Xu, Q},
title = {Extraction-free, rapid LAMP-CRISPR/Cas12a assay for detection of pseudorabies virus.},
journal = {Journal of virological methods},
volume = {343},
number = {},
pages = {115387},
doi = {10.1016/j.jviromet.2026.115387},
pmid = {41875952},
issn = {1879-0984},
mesh = {*Herpesvirus 1, Suid/isolation & purification/genetics ; Animals ; Swine ; *Pseudorabies/diagnosis/virology ; Sensitivity and Specificity ; *Molecular Diagnostic Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; *Swine Diseases/diagnosis/virology ; *CRISPR-Cas Systems ; DNA Primers/genetics ; DNA, Viral/genetics ; },
abstract = {This study developed a LAMP-CRISPR/Cas12a detection system for rapid and visual identification of porcine pseudorabies virus (PRV). Optimal sgRNA and LAMP-specific primers were designed based on the conserved sequences of the viral pathogenic gene gG. The combined detection system demonstrated superior sensitivity compared to PCR-CRISPR/Cas12a and qPCR methods, achieving a detection limit of 1.0 × 10[-4] copies/μL for the target plasmid DNA. Specificity testing confirmed the selective identification of PRV without cross-reactivity to other porcine pathogens. Parallel comparison of 26 serum samples between LAMP-CRISPR/Cas12a and PCR-CRISPR/Cas12a systems showed 100% concordance for positive results, with both detecting 12 positive samples. The method eliminates the need for viral nucleic acid extraction and requires only a constant temperature device and/or basic fluorescence detection equipment. Results are obtainable within one hour and are readable by the naked eye. This simple, sensitive, and equipment-independent approach is ideal for on-site rapid diagnosis of pseudorabies in pigs, offering significant applications in clinical diagnosis, epidemiological surveillance, and field testing.},
}

*Herpesvirus 1, Suid/isolation & purification/genetics
Animals
Swine
*Pseudorabies/diagnosis/virology
Sensitivity and Specificity
*Molecular Diagnostic Techniques/methods
*Nucleic Acid Amplification Techniques/methods
*Swine Diseases/diagnosis/virology
*CRISPR-Cas Systems
DNA Primers/genetics
DNA, Viral/genetics

@article {pmid41937163,
year = {2026},
author = {Zhou, HR and Doan, DTH and Hartwig, T and Turck, F},
title = {Cis-regulatory architecture downstream of FLOWERING LOCUS T underlies quantitative control of flowering in Arabidopsis thaliana.},
journal = {Genome biology},
volume = {27},
number = {1},
pages = {},
pmid = {41937163},
issn = {1474-760X},
support = {EXC 2048/1 Project ID: 390686111//Cluster of Excellence on Plant Sciences/ ; },
mesh = {*Arabidopsis/genetics/growth & development ; *Flowers/genetics/growth & development ; *Arabidopsis Proteins/genetics/metabolism ; *Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; Enhancer Elements, Genetic ; Gene Editing ; Chromatin/metabolism ; Regulatory Sequences, Nucleic Acid ; },
abstract = {BACKGROUND: The FLOWERING LOCUS T (FT) gene is a central integrator of floral induction in Arabidopsis thaliana, with its precise expression controlled by complex transcriptional networks. While upstream regulatory regions are well-studied, the role of downstream cis-regulatory elements in modulating FT expression remains poorly characterized.

RESULTS: Systematic dissection of the FT downstream region in its native chromosomal context using CRISPR/Cas9-mediated genome editing provides genetic evidence that a 2.3-kb sequence, encompassing the Block E enhancer immediately adjacent to the FT coding sequence, is essential for proper FT expression and timely flowering. Fine-scale deletions within Block E reveal that a 63-bp sequence containing one CCAAT-box and one G-box, both closely spaced, forms a core functional module, whereas other conserved motifs contribute modestly in a context-dependent manner. Strikingly, a cryptic CCAAT-box module downstream of Block E that becomes active when repositioned. This coincides with increased transcription factor occupancy and local chromatin accessibility.

CONCLUSIONS: Our work reveals that quantitative FT expression and flowering time are governed by the spatial organization and chromatin context of downstream cis-regulatory elements. The positional sensitivity and modular logic of these elements provide framework for understanding and engineering quantitative gene regulation through targeted cis-regulatory design, a concept broadly applicable across diverse developmental systems.},
}

*Arabidopsis/genetics/growth & development
*Flowers/genetics/growth & development
*Arabidopsis Proteins/genetics/metabolism
*Gene Expression Regulation, Plant
CRISPR-Cas Systems
Enhancer Elements, Genetic
Gene Editing
Chromatin/metabolism
Regulatory Sequences, Nucleic Acid

@article {pmid41938130,
year = {2026},
author = {Yin, X and Fan, Z and Tong, Z},
title = {CRISPR-based electrochemiluminescence biosensors: Principles, optimization strategies, and translational challenges - A review of recent progress.},
journal = {Food chemistry. Molecular sciences},
volume = {12},
number = {},
pages = {100392},
pmid = {41938130},
issn = {2666-5662},
abstract = {The integration of CRISPR/Cas systems with electrochemiluminescence (ECL) has emerged as a promising strategy for constructing high-performance biosensing platforms. CRISPR systems, particularly Cas12a and Cas13a, offer programmable recognition of nucleic acid targets and activatable trans-cleavage activity. ECL provides sensitive signal readout with low background and wide dynamic range. As a narrative review, this article provides a comprehensive overview of recent advances in CRISPR-ECL biosensors, with an emphasis on optimization strategies and practical applications. We first discuss the working principles of Cas12a and Cas13a relevant to biosensing, highlighting their distinct kinetic properties, crRNA design considerations, and reaction condition requirements. We then examine optimization approaches at three interconnected levels: nucleic acid probe design (signal-on, signal-off, and auxiliary probes), sensing interface engineering (probe structures, luminophores, electrode materials, and magnetic nanomaterials), and cascade signal amplification (PCR, CHA, RCA, SDA, EDA, and RPA). Through cross-study comparison, we evaluate the strengths and limitations of different approaches and identify critical knowledge gaps. Their applications in detecting disease biomarkers, pathogen nucleic acids, environmental contaminants, and enzyme activities are summarized. Despite remarkable sensitivity achieved, challenges remain in assay time, reproducibility in complex matrices, and clinical validation. From industrialization and global health perspectives, regulatory approval, manufacturing scalability, cost control, and deployment in low-resource settings are also discussed. Finally, future directions toward simplified workflows, enhanced matrix robustness, standardized validation, multiplexed detection, and point-of-care compatible platforms are proposed. This review provides a structured reference and critical perspective for researchers working on CRISPR-ECL biosensing and related fields.},
}

@article {pmid41610854,
year = {2026},
author = {Daly, J and Piatnitca, L and Al-Seragi, M and Krishnamoorthy, V and Wisnovsky, S},
title = {CRISPR activation screens map the genomic landscape of cancer glycome remodeling.},
journal = {Cell genomics},
volume = {6},
number = {4},
pages = {101139},
doi = {10.1016/j.xgen.2026.101139},
pmid = {41610854},
issn = {2666-979X},
mesh = {Humans ; Glycosylation ; *Polysaccharides/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Sialic Acid Binding Immunoglobulin-like Lectins/metabolism/genetics ; *Neoplasms/genetics/metabolism ; Sulfotransferases/metabolism/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Glycomics/methods ; Gene Expression Regulation, Neoplastic ; },
abstract = {Many cancers upregulate the expression of sialic acid-containing glycans. These oligosaccharides engage inhibitory sialic acid-binding immunoglobulin-like lectin (Siglec) receptors on immune cells, allowing cancer cells to evade immune surveillance. The genetic mechanisms underlying this process remain poorly defined. In this study, we performed gain-of-function CRISPR activation (CRISPRa) screens to define genetic pathways that regulate expression of Siglec-binding glycans. We show that Siglec ligand expression is controlled through genetic competition between genes that catalyze α2-3 sialylation and GlcNAcylation of galactose residues. Cancer glycome remodeling is also aided by the overexpression of "professional ligands" that facilitate Siglec-glycan binding. Notably, we also find that expression of the CD24 gene is genetically dispensable for cell surface binding of the inhibitory receptor Siglec-10. Finally, we identify the sulfotransferase enzyme GAL3ST4 as a potential driver of immune evasion in glioma cells. Our study provides a unique genomic atlas of cancer-associated glycosylation and identifies immediately actionable targets for cancer immunotherapy.},
}

Humans
Glycosylation
*Polysaccharides/metabolism/genetics
*CRISPR-Cas Systems/genetics
Cell Line, Tumor
Sialic Acid Binding Immunoglobulin-like Lectins/metabolism/genetics
*Neoplasms/genetics/metabolism
Sulfotransferases/metabolism/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Glycomics/methods
Gene Expression Regulation, Neoplastic

@article {pmid41652143,
year = {2026},
author = {Fu, T and Zhao, D and Wang, Q and Peng, N and Zhang, L and Chen, S and Ren, J and Tang, F and Pu, K and Wu, Q and Bi, C and Zhang, X and Li, Q},
title = {A rapid and ultrasensitive CRISPR-Cas12a assay for clinical detection of pathogens and mutations.},
journal = {Analytical and bioanalytical chemistry},
volume = {418},
number = {8},
pages = {2455-2469},
pmid = {41652143},
issn = {1618-2650},
mesh = {*CRISPR-Cas Systems ; Humans ; *Mutation ; Limit of Detection ; Nucleic Acid Amplification Techniques/methods ; Isocitrate Dehydrogenase/genetics ; Glioma/genetics/diagnosis ; DNA/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {CRISPR-Cas12a-driven nucleic acid diagnostics offer considerable potential for highly specific and rapid detection. However, their practical applications are limited by the necessity for pre-amplification of target DNA to enhance sensitivity. To overcome this limitation, we developed Auto-catalyst, a novel one-pot, amplification-free DNA detection platform employing a two-stage autocatalytic Cas12a cascade. This approach integrates a positive feedback amplification mediated by a circular crRNA-DNA nanostructure with an asymmetric CRISPR reaction driven by split crRNA. Without external amplification, this system detects DNA fragments at concentrations as low as 80 aM within 30 min at room temperature and maintains high specificity, accurately distinguishing single-base mutations down to 1 fM. Clinical validation demonstrated successful detection of pathogenic DNA in cerebrospinal fluid samples from patients with intracranial infections, highlighting its potential for rapid bedside diagnostics essential for timely clinical decision-making. Additionally, Auto-catalyst accurately identified the clinically significant isocitrate dehydrogenase 1 (IDH1) gene R132H mutation from glioma tissue samples. This integrated two-stage autocatalytic Cas12a strategy represents a powerful, convenient, and promising diagnostic tool suitable for point-of-care applications.},
}

*CRISPR-Cas Systems
Humans
*Mutation
Limit of Detection
Nucleic Acid Amplification Techniques/methods
Isocitrate Dehydrogenase/genetics
Glioma/genetics/diagnosis
DNA/genetics
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41711853,
year = {2026},
author = {Guo, Y and Zhao, J and Li, X and Zheng, X and Yuan, Y and Deng, J and Ren, Y and Xu, K},
title = {Detection of alkaline phosphatase activity based on ATP hydrolysis and CRISPR/Cas12a.},
journal = {Analytical and bioanalytical chemistry},
volume = {418},
number = {8},
pages = {2337-2344},
pmid = {41711853},
issn = {1618-2650},
support = {2023JJ30430//Natural Science Foundation of Hunan Province/ ; 23A0075//Education Department of Hunan Province/ ; 82173572//National Natural Science Foundation of China/ ; 82574159//National Natural Science Foundation of China/ ; },
mesh = {*Alkaline Phosphatase/metabolism/analysis ; *Adenosine Triphosphate/metabolism/chemistry ; *CRISPR-Cas Systems ; Hydrolysis ; Animals ; Limit of Detection ; Milk/enzymology ; Aptamers, Nucleotide/chemistry ; *CRISPR-Associated Proteins/metabolism ; Biosensing Techniques/methods ; Cattle ; *Endodeoxyribonucleases/metabolism ; Bacterial Proteins ; },
abstract = {Alkaline phosphatase (ALP) activity serves as a crucial biomarker for livestock disease diagnosis, nutritional management, and the evaluation of milk pasteurization efficacy, holding significant implications for food safety and food science. In this study, we developed a novel method for detecting ALP activity in food samples, based on ATP hydrolysis coupled with a CRISPR/Cas12a system. The assay employs a DNA molecular recognition lock probe, comprising an ATP-specific aptamer and an activator strand designed to trigger the CRISPR/Cas12a trans-cleavage activity. In the absence of ALP, ATP acts as a trans-hydrolysis substrate to open the lock structure, releasing the activator strand. This subsequently activates the Cas12a protein, leading to the generation of a fluorescent signal. Conversely, when the target ALP is present, it catalyzes the hydrolysis of ATP via a dephosphorylation reaction. This prevents the opening of the "Aptamer-Activator" molecular lock, thereby inhibiting Cas12a activation and resulting in a corresponding decrease in fluorescence intensity. Under optimized conditions, the assay demonstrated a detection limit of 2.52 mU/mL for ALP activity, with a linear range of 0-18.75 mU/mL. The total detection time was 70 min. The method was successfully applied to detect ALP activity in samples from various livestock (chickens, pigs, sheep, cattle) and in milk, achieving recovery rates between 92 and 99%. In conclusion, we have developed a sensitive, cost-effective, and rapid method for ALP detection. This work provides a promising strategy for the development of point-of-care testing (POCT) devices in food safety monitoring.},
}

*Alkaline Phosphatase/metabolism/analysis
*Adenosine Triphosphate/metabolism/chemistry
*CRISPR-Cas Systems
Hydrolysis
Animals
Limit of Detection
Milk/enzymology
Aptamers, Nucleotide/chemistry
*CRISPR-Associated Proteins/metabolism
Biosensing Techniques/methods
Cattle
*Endodeoxyribonucleases/metabolism
Bacterial Proteins

@article {pmid41787916,
year = {2026},
author = {Saboor, M and Jasem Alblooshi, M and Adel Alkaabi, A and Ramazan Soozaei, F and Hamad Alketbi, M},
title = {CRISPR in Thalassemia: Global Research Trend Analysis.},
journal = {Hemoglobin},
volume = {50},
number = {2},
pages = {141-155},
doi = {10.1080/03630269.2026.2634815},
pmid = {41787916},
issn = {1532-432X},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *beta-Thalassemia/genetics/therapy ; *Thalassemia/genetics/therapy ; Genetic Therapy/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Bibliometrics ; Pattern Analysis, Machine ; },
abstract = {β-Thalassemia is a prevalent inherited disorder of β-globin chains. The clustered regularly interspaced short palindromic repeats (CRISPR) genome editing system has emerged as a potential curative strategy. We conducted a bibliometric analysis to map global research trends in CRISPR-based thalassemia research. Original and review research articles were retrieved from the Scopus database using the search terms [TITLE-ABS-KEY ('βeta thalassemia' OR 'β thalassemia' OR thalassemia*) AND TITLE-ABS-KEY ('gene edit*' OR crispr* OR 'clustered regularly interspaced short palindromic repeats')] AND [LIMIT-TO (DOCTYPE, 're') OR LIMIT-TO (DOCTYPE, 'ar')] for analysis. Bibliometric mapping and network visualization were performed using VOSviewer to analyze publication trends, authorship networks, international collaborations, keyword clusters, and citation metrics. Major CRISPR-based therapeutic strategies for thalassemia were reviewed to place experimental and clinical developments within a translational framework. The analysis demonstrates a clear transition from foundational genomic studies to translational applications, with leading contributions from the United States and China. Two dominant therapeutic strategies have emerged: direct correction of the HBB gene in hematopoietic stem cells and fetal hemoglobin reactivation via BCL11A repression. The latter strategy culminated in regulatory approval of exagamglogene autotemcel (Casgevy). Advances in base editing, prime editing, and strategies to improve engraftment are expected to enhance the precision and long-term efficacy of next-generation approaches. Clustered regularly interspaced short palindromic repeats-based research on thalassemia continues to expand, supported by extensive international collaboration and growing clinical translation. Future large-scale implementation will require advances in bioprocess engineering, cost reduction for ex vivo manufacturing, and adaptable treatment models for diverse healthcare systems.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems
*beta-Thalassemia/genetics/therapy
*Thalassemia/genetics/therapy
Genetic Therapy/methods
*Clustered Regularly Interspaced Short Palindromic Repeats
Bibliometrics
Pattern Analysis, Machine

@article {pmid41791263,
year = {2026},
author = {Cho, ES and Hu, JC and Kim, JW},
title = {Generation and characterization of a murine amelogenesis imperfecta model.},
journal = {Archives of oral biology},
volume = {186},
number = {},
pages = {106563},
doi = {10.1016/j.archoralbio.2026.106563},
pmid = {41791263},
issn = {1879-1506},
mesh = {Animals ; *Amelogenesis Imperfecta/genetics/pathology/diagnostic imaging ; Mice ; *Disease Models, Animal ; *Amelogenin/genetics ; X-Ray Microtomography ; Gene Knock-In Techniques ; Exons/genetics ; Mutation ; Dental Enamel/pathology ; Immunohistochemistry ; Alternative Splicing ; Genotype ; CRISPR-Cas Systems ; Phenotype ; },
abstract = {OBJECTIVE: Amelogenesis imperfecta (AI) refers to a group of rare yet complex genetic disorders that affect the quantity and/or quality of tooth enamel. Recently, in AI patients, we identified mutations that disrupt a conserved alternative splicing pattern of the AMELX gene, which encodes amelogenin, the most abundant enamel matrix protein. These mutations led to the retention of exon 4, which is normally skipped during the pre-mRNA splicing process, resulting in the characteristic pitted, hypoplastic, and hypomineralized enamel defects. To observe the impact of retention of exon 4 within AMELX, a gene edited knock-in mouse model was generated.

DESIGN: A single-nucleotide knock-in mouse model was generated using CRISPR/Cas9 technology to introduce a silent mutation (NM_001415990.1: c.120 T>C, p.(Ala40=)) that abrogated alternative splicing of exon 4. Following genomic sequence validation, the successfully-targeted mice were propagated, and their offspring genotyped for characterization. Micro-computed tomography analysis and immunohistochemistry analysis were performed on the hemi-mandibles of the wild-type and the knock-in mice.

RESULTS: The enamel of the knock-in mice was chalky white and lacked translucency, due to faulty mineralization. This defective enamel broke down soon after tooth eruption. During the maturation stage, the ameloblast layer lost its cellular polarity and homogeneity, and intermingled with adjacent cell types to form disorganized clusters.

CONCLUSIONS: The validated and characterized Amelx c.120 T>C mouse model provides a useful platform for investigating the molecular pathophysiology associated with retention of the exon 4 sequence. Following systemic characterization, this mouse model will serve as an important tool for assessing therapeutic strategies aimed at ameliorating the disease phenotype.},
}

Animals
*Amelogenesis Imperfecta/genetics/pathology/diagnostic imaging
Mice
*Disease Models, Animal
*Amelogenin/genetics
X-Ray Microtomography
Gene Knock-In Techniques
Exons/genetics
Mutation
Dental Enamel/pathology
Immunohistochemistry
Alternative Splicing
Genotype
CRISPR-Cas Systems
Phenotype

@article {pmid41928005,
year = {2026},
author = {Migur, A and Feussner, M and Liao, C and Alkhnbashi, OS and Chauvier, A and Walter, NG and Backofen, R and Weinberg, Z and Beisel, CL},
title = {A leader-repeat hairpin blocks extraneous CRISPR RNA production in diverse CRISPR-Cas13 systems.},
journal = {The EMBO journal},
volume = {},
number = {},
pages = {},
pmid = {41928005},
issn = {1460-2075},
support = {468749960//Deutsche Forschungsgemeinschaft (DFG)/ ; 468749960//Deutsche Forschungsgemeinschaft (DFG)/ ; BA 2168/23-2//Deutsche Forschungsgemeinschaft (DFG)/ ; 865973//EC | Horizon Europe | Excellent Science | HORIZON EUROPE European Research Council (ERC)/ ; MCB 2140320//National Science Foundation (NSF)/ ; },
abstract = {CRISPR RNAs (crRNAs) guide recognition and targeting of intracellular invaders as part of adaptive immunity by CRISPR-Cas systems. crRNAs are transcribed from CRISPR arrays of conserved repeats interlaced with invader-derived spacers. While crRNA production is essential for immunity, its optimization for defense remains poorly understood. Here, we show that, in diverse RNA-targeting type VI CRISPR-Cas systems, the leader RNA encoded upstream of the CRISPR array prevents formation of an invader-independent extraneous crRNA (ecrRNA) by blocking processing of the first repeat. Using the VI-B2 system from Porphyromonas gingivalis as a model, we demonstrate that the leader RNA and first repeat form a conserved inhibitory hairpin that precludes binding and processing by the system's Cas13b nuclease. Disrupting this hairpin enables ecrRNA production, which in turn can deplete invader-derived crRNAs and reduce Cas13b-mediated phage defense. Structure prediction indicates that these leader-repeat hairpins are widespread across diverse type VI subtypes, highlighting a conserved regulatory mechanism. Our findings reveal how a prevalent branch of CRISPR-Cas systems suppresses ecrRNA formation to promote RNA-guided immunity.},
}

@article {pmid41930610,
year = {2026},
author = {Xing, B and Zhang, X and Shen, L and Zhang, X},
title = {CRISPR-mediated MLH1 disruption suppresses endometrial cancer growth via genomic instability induction and Wnt/β-catenin pathway inhibition.},
journal = {Folia histochemica et cytobiologica},
volume = {64},
number = {1},
pages = {75-87},
doi = {10.5603/fhc.111186},
pmid = {41930610},
issn = {1897-5631},
mesh = {Humans ; *Endometrial Neoplasms/genetics/pathology/metabolism ; *MutL Protein Homolog 1/genetics/metabolism ; *Wnt Signaling Pathway/genetics ; Female ; Cell Proliferation/genetics ; Cell Line, Tumor ; Animals ; *Genomic Instability ; Mice ; *CRISPR-Cas Systems ; Apoptosis ; Mice, Nude ; Cell Movement ; Gene Expression Regulation, Neoplastic ; },
abstract = {INTRODUCTION: MutL homolog 1 (MLH1) loss is a defining molecular feature of endometrial cancer (EC) and a principal driver of microsatellite instability (MSI). Ishikawa cells harbor intrinsic MLH1 promoter hypermethylation, resulting in reduced but not abolished MLH1 expression and placing these cells in a vulnerable, partially compromised mismatch repair state. This study explores the effects of MLH1 knockdown (MLH1-KD) on MSI, cellular functions, signaling pathways, and tumor growth in Ishikawa EC cells.

MATERIAL AND METHODS: Using CRISPR/Cas9, we created an MLH1-KD Ishikawa EC cell line, validated through Sanger sequencing, qRT-PCR, western blotting, comet assays, and γ-H2AX analysis. Functional assays assessed proliferation, migration, and cell cycle progression and apoptosis. RNA sequencing identified global transcriptomic changes, and Wnt/β-catenin pathway activity was measured by a dual-luciferase reporter assay. A xenograft model evaluated tumor growth in vivo.

RESULTS: MLH1-KD cells showed MSI-H characteristics, increased DNA damage, and downregulation of key EC-related genes. Functionally, MLH1-KD led to significant reductions in cell proliferation and migration, which was accompanied by cell cycle arrest and a marked increase in apoptosis. RNA sequencing revealed profound alterations in the Wnt signaling pathway. Crucially, this was confirmed by a dual-luciferase reporter assay, which showed a significant inhibition of Wnt/β-catenin signaling activity. In vivo, MLH1-KD significantly decreased tumor weight and size in nude mice.

CONCLUSIONS: In EC cells with pre-existing MLH1 promoter methylation, MLH1-KD leads to MSI-H, enhances genomic instability, disrupts Wnt signaling, impairs cellular functions, and inhibits tumor growth, highlighting Wnt signaling and MSI-H as potential therapeutic targets in EC.},
}

Humans
*Endometrial Neoplasms/genetics/pathology/metabolism
*MutL Protein Homolog 1/genetics/metabolism
*Wnt Signaling Pathway/genetics
Female
Cell Proliferation/genetics
Cell Line, Tumor
Animals
*Genomic Instability
Mice
*CRISPR-Cas Systems
Apoptosis
Mice, Nude
Cell Movement
Gene Expression Regulation, Neoplastic

@article {pmid41931047,
year = {2026},
author = {Hanna, R and Frangoul, H and Pineiro, L and McKinney, C and Mapara, M and Dalal, J and Rangarajan, HG and Atkins, H and Sharma, A and Chang, KH and Jaskolka, MC and Kim, K and Yu, Q and Mei, B and Afonja, O and Walters, MC and , },
title = {CRISPR-Cas12a Gene Editing of HBG1 and HBG2 Promoters to Treat Sickle Cell Disease.},
journal = {The New England journal of medicine},
volume = {394},
number = {13},
pages = {1281-1291},
doi = {10.1056/NEJMoa2415550},
pmid = {41931047},
issn = {1533-4406},
mesh = {Adolescent ; Adult ; Female ; Humans ; Male ; Young Adult ; *Anemia, Sickle Cell/blood/diagnosis/genetics/therapy ; *CRISPR-Cas Systems/genetics ; *Fetal Hemoglobin/analysis/genetics ; *gamma-Globins/genetics ; Gene Editing/methods ; *Genetic Therapy/methods/adverse effects ; *Hematopoietic Stem Cell Transplantation/adverse effects/methods ; Promoter Regions, Genetic/genetics ; Severity of Illness Index ; Follow-Up Studies ; Transplantation, Autologous/adverse effects/methods ; Transplantation Conditioning/adverse effects/methods ; Repressor Proteins/metabolism ; Busulfan/administration & dosage/adverse effects ; Myeloablative Agonists/administration & dosage/adverse effects ; *Vaso-Occlusive Crises/epidemiology/genetics/prevention & control/therapy ; Treatment Outcome ; },
abstract = {BACKGROUND: Renizgamglogene autogedtemcel (reni-cel) is an investigational clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a gene-edited autologous hematopoietic stem-cell therapy. The therapy was designed to disrupt the BCL11A binding sites in the HBG1 and HBG2 promoters to reactivate fetal hemoglobin production for the treatment of sickle cell disease.

METHODS: We conducted a phase 1-2, multicenter, open-label, single-group study involving patients with severe sickle cell disease who were 12 to 50 years of age and had had at least two severe vaso-occlusive events per year in the previous 2 years. The patients received a single infusion of reni-cel after myeloablative conditioning with busulfan. The patients were monitored for engraftment, hemoglobin-related measures, allelic editing levels, vaso-occlusive events, and adverse events over a 24-month period. The study was terminated early on the basis of the sponsor's reassessment of clinical development priorities. Results of an analysis that was not prespecified are reported.

RESULTS: As of October 29, 2024, a total of 28 patients with severe sickle cell disease had been treated with reni-cel. The median duration of follow-up was 9.5 months (range, 0.7 to 25.2). Among 27 patients who had neutrophil and platelet engraftment by the data-cutoff date, neutrophil engraftment occurred after a median of 23 days (range, 14 to 29), and platelet engraftment occurred after a median of 25 days (range, 17 to 51). At month 6, among 18 patients with at least 6 months of available data, the mean (±SD) total hemoglobin level (9.8±1.7 g per deciliter at baseline) had increased to 13.8±1.9 g per deciliter, and the mean percentage of fetal hemoglobin (2.5±2.5% at baseline) had increased to 48.1±3.2%; both measures were maintained at or above these values thereafter. One patient had two severe vaso-occlusive events after infusion. Adverse events were consistent with those that occur after myeloablative busulfan-based conditioning and autologous hematopoietic stem-cell transplantation.

CONCLUSIONS: Treatment with reni-cel led to normalization of the total hemoglobin level and an increase in the percentage of fetal hemoglobin, with no vaso-occlusive events occurring in 27 of 28 patients after infusion. These results support further investigation of this gene-editing approach in the treatment of severe sickle cell disease. (Funded by Editas Medicine; RUBY ClinicalTrials.gov number, NCT04853576.).},
}

Adolescent
Adult
Female
Humans
Male
Young Adult
*Anemia, Sickle Cell/blood/diagnosis/genetics/therapy
*CRISPR-Cas Systems/genetics
*Fetal Hemoglobin/analysis/genetics
*gamma-Globins/genetics
Gene Editing/methods
*Genetic Therapy/methods/adverse effects
*Hematopoietic Stem Cell Transplantation/adverse effects/methods
Promoter Regions, Genetic/genetics
Severity of Illness Index
Follow-Up Studies
Transplantation, Autologous/adverse effects/methods
Transplantation Conditioning/adverse effects/methods
Repressor Proteins/metabolism
Busulfan/administration & dosage/adverse effects
Myeloablative Agonists/administration & dosage/adverse effects
*Vaso-Occlusive Crises/epidemiology/genetics/prevention & control/therapy
Treatment Outcome

@article {pmid41931048,
year = {2026},
author = {Frangoul, H and Hanna, R and Walters, MC and Kao, RL and Carroll, C and McManus, M and Chang, KH and Jaskolka, MC and Kim, K and Yu, Q and Badamosi, N and Mei, B and Afonja, O and Thompson, A and , },
title = {CRISPR-Cas12a Gene Editing of HBG1 and HBG2 Promoters to Treat β-Thalassemia.},
journal = {The New England journal of medicine},
volume = {394},
number = {13},
pages = {1292-1301},
doi = {10.1056/NEJMoa2501277},
pmid = {41931048},
issn = {1533-4406},
mesh = {Adolescent ; Adult ; Female ; Humans ; Male ; Young Adult ; *beta-Thalassemia/therapy/genetics/blood ; *CRISPR-Cas Systems/genetics ; *Fetal Hemoglobin/analysis/genetics ; *gamma-Globins/genetics ; Gene Editing/methods ; *Genetic Therapy/adverse effects/methods ; *Hematopoietic Stem Cell Transplantation/adverse effects/methods ; Neutrophils ; Promoter Regions, Genetic ; Repressor Proteins/metabolism ; Busulfan/administration & dosage/adverse effects ; Myeloablative Agonists/administration & dosage/adverse effects ; Transplantation Conditioning/adverse effects/methods ; *Erythrocyte Transfusion/statistics & numerical data ; Treatment Outcome ; },
abstract = {BACKGROUND: Renizgamglogene autogedtemcel (reni-cel) is an investigational clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a gene-edited autologous hematopoietic stem-cell therapy. The therapy was designed to disrupt the BCL11A binding sites in the HBG1 and HBG2 promoters to reactivate fetal hemoglobin production for the treatment of transfusion-dependent β-thalassemia.

METHODS: We conducted a phase 1-2, multicenter, open-label, single-group study of reni-cel in participants 18 to 35 years of age with transfusion-dependent β-thalassemia. The participants received myeloablative conditioning with busulfan before reni-cel infusion. The primary end points were neutrophil engraftment by 42 days after infusion and frequency and severity of adverse events. Participants were monitored for hemoglobin-related measures and transfusion independence. The study was terminated early on the basis of the sponsor's reassessment of clinical development priorities. Results of an analysis that was not prespecified are reported.

RESULTS: Nine participants with transfusion-dependent β-thalassemia (four β[0]/β[0] or β[0]/β[0]-like and five non-β[0]/β[0] genotypes) received reni-cel and were included in the analysis. The median duration of postinfusion follow-up was 17.5 months (range, 3.8 to 23.4), and six participants could be evaluated for transfusion independence at 12 months or more. All the participants had neutrophil and platelet engraftment by 42 days after infusion. Rapid increases in total and fetal hemoglobin levels resulted in each of the nine participants being transfusion-free at their last follow-up visit. The six participants who could be evaluated at 12 months or later were transfusion-independent. The mean total and fetal hemoglobin levels were greater than 12 g per deciliter and greater than 11 g per deciliter, respectively, between months 6 and 18. A total of 69 grade 3 or 4 adverse events with onset or worsening during or after reni-cel infusion were reported in the nine participants. Six serious adverse events (infections, pyrexia, or pneumonitis) were reported in four participants. Adverse events were generally consistent with myeloablative conditioning. One patient had decreased lymphocyte counts attributed to reni-cel.

CONCLUSIONS: Treatment with reni-cel resulted in rapid neutrophil engraftment, an increase in fetal hemoglobin expression, and transfusion independence. These data support further investigation of Cas12a gene editing of the promoters of HBG1 and HBG2 in the treatment of transfusion-dependent β-thalassemia. (Funded by Editas Medicine; EdiThal ClinicalTrials.gov number, NCT05444894.).},
}

Adolescent
Adult
Female
Humans
Male
Young Adult
*beta-Thalassemia/therapy/genetics/blood
*CRISPR-Cas Systems/genetics
*Fetal Hemoglobin/analysis/genetics
*gamma-Globins/genetics
Gene Editing/methods
*Genetic Therapy/adverse effects/methods
*Hematopoietic Stem Cell Transplantation/adverse effects/methods
Neutrophils
Promoter Regions, Genetic
Repressor Proteins/metabolism
Busulfan/administration & dosage/adverse effects
Myeloablative Agonists/administration & dosage/adverse effects
Transplantation Conditioning/adverse effects/methods
*Erythrocyte Transfusion/statistics & numerical data
Treatment Outcome

@article {pmid41934615,
year = {2026},
author = {Wang, Z and Tamura, Y and Hashimoto, M and Nakazato, I and Yamaguchi-Nishimura, A and Arimura, SI},
title = {Using ngTALEN to improve genome editing efficiency on targets containing 5-methylcytosines.},
journal = {The Plant journal : for cell and molecular biology},
volume = {126},
number = {1},
pages = {e70826},
pmid = {41934615},
issn = {1365-313X},
support = {24H02271//Japan Society for the Promotion of Science/ ; JPJSCCA20230008//Japan Society for the Promotion of Science/ ; },
mesh = {*Gene Editing/methods ; *Arabidopsis/genetics/metabolism ; DNA Methylation/genetics ; *5-Methylcytosine/metabolism ; Arabidopsis Proteins/genetics/metabolism ; *Transcription Activator-Like Effector Nucleases/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic/genetics ; Genome, Plant/genetics ; },
abstract = {We recently discovered distinct methylation patterns between the mitochondrial genome and the nuclear-encoded mitochondrial DNA sequences (NUMTs), with the mitochondrial genome being hypomethylated and NUMTs being hypermethylated. Given that genome editing using mitochondrial targeted transcription activator-like effector nucleases (TALEN) is highly efficient, while editing at NUMT is difficult, we hypothesized that the methylation status might affect editing outcomes. To test this, we attempted to use ngTALEN [employing RVD-NG to recognize 5-methylcytosine (5mC)] to target the Flowering Wageningen (FWA) locus of Arabidopsis thaliana, specifically the promoter and gene body regions with varying levels of cytosine methylation. Comparative analysis using the active epimutant allele fwa-d and wild-type Columbia-0 (Col-0) carrying a naturally silenced allele of FWA revealed that editing was impeded by 5mC at both the promoter and gene body of FWA for both CRISPR/Cas9 and TALEN. Overall, TALEN editing is robust and comparable to that of CRISPR/Cas9 at multiple sites, while ngTALEN showed improved editing at the CG-hypermethylated promoter of FWA compared with TALEN. Additionally, when targeting multiple genomic loci with identical sequences that differ in methylation levels and chromatin states, ngTALEN was less effective to induce edits. Therefore, this study represents the first systematic comparison of editing efficiency between CRISPR/Cas9 and TALEN in dealing with methylated or unmethylated DNA in plants. Furthermore, we have developed ngTALEN as a specific and robust tool for enhancing editing at sites with various levels of CG methylation.},
}

*Gene Editing/methods
*Arabidopsis/genetics/metabolism
DNA Methylation/genetics
*5-Methylcytosine/metabolism
Arabidopsis Proteins/genetics/metabolism
*Transcription Activator-Like Effector Nucleases/genetics/metabolism
CRISPR-Cas Systems/genetics
Promoter Regions, Genetic/genetics
Genome, Plant/genetics

@article {pmid41935002,
year = {2026},
author = {Zhang, H and Zhu, H and Gao, H and Zhou, Y and Que, L and Rong, S and Ma, H and Chang, D and Pan, H},
title = {A colorimetric/fluorescent/electrochemical tri-modal biosensor based on dual CRISPR/Cas12a system for detection of microRNA.},
journal = {Analytica chimica acta},
volume = {1402},
number = {},
pages = {345393},
doi = {10.1016/j.aca.2026.345393},
pmid = {41935002},
issn = {1873-4324},
mesh = {*MicroRNAs/analysis/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Humans ; *Electrochemical Techniques/methods ; *Colorimetry/methods ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BACKGROUND: The precise detection of tumor markers is crucial for early cancer diagnosis and monitoring. Existing unimodal detection methods are susceptible to interference in complex biological samples, making it difficult to simultaneously achieve high sensitivity and reliability. MicroRNAs (miRNAs), as a key class of cancer-related biomarkers, necessitate novel detection methods capable of multi-layered verification. This study aims to develop an biosensing platform with multi-signal outputs to address the critical challenge of balancing sensitivity, specificity, and result credibility in current tumor marker detection.

RESULTS: We successfully constructed a tri-modal biosensor based on a dual CRISPR/Cas12a system for the highly sensitive and specific detection of miRNA let-7a. The sensor generates a trigger strand via an exponential amplification reaction (EXPAR), which concurrently regulates three independent signaling pathways: ① It initiates the first CRISPR/Cas12a to suppress G-quadruplex/hemin DNAzyme (G4/hemin DNAzyme) formation, turning off the ABTS colorimetric signal. ② It also activates duplex-specific nuclease (DSN) to inhibit the hybridization chain reaction (HCR), thereby blocking FAM fluorescence coupling to streptavidin-coated magnetic nanoparticles (SMBs) and turning off the fluorescence signal. ③ The absence of HCR products on the SMBs inhibits the second CRISPR/Cas12a system, thereby preserving the electrode's P1 probe for binding with P2-3D-CdCo-ONSs@AuNPs and maintaining a high "turn-on" electrochemical signal from the nanocomposite. Thus, the concentration of miRNA let-7a, ranging from 50 fM to 1 pM, can be precisely quantified and validated through colorimetric, fluorescent, and electrochemical signals.

SIGNIFICANCE: This study integrates a dual CRISPR/Cas12a system with a tri-modal output strategy encompassing colorimetric, fluorescent, and electrochemical detection, thereby constructing a detection platform featuring a cross-verification mechanism. This design not only significantly enhances detection accuracy and anti-interference capability but also lays a solid foundation for developing next-generation, highly reliable molecular diagnostic tools. It holds considerable application potential in the fields of early cancer screening and precision medicine.},
}

*MicroRNAs/analysis/genetics
*Biosensing Techniques/methods
*CRISPR-Cas Systems
Humans
*Electrochemical Techniques/methods
*Colorimetry/methods
Limit of Detection
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41935011,
year = {2026},
author = {Zhang, Y and Liu, W and Guo, R and Qi, Y and Bai, B and Zhang, J and Hu, N and Gu, Y and Yang, Y and Wang, S},
title = {CRISPR/Cas12a-mediated electrochemiluminescent biosensor integrating Ag modified Co-doped metal-organic frameworks for dual detection of malathion and phorate.},
journal = {Analytica chimica acta},
volume = {1402},
number = {},
pages = {345402},
doi = {10.1016/j.aca.2026.345402},
pmid = {41935011},
issn = {1873-4324},
mesh = {*Malathion/analysis ; *Biosensing Techniques/methods ; *Metal-Organic Frameworks/chemistry ; *CRISPR-Cas Systems ; *Electrochemical Techniques/methods ; Silver/chemistry ; *Luminescent Measurements/methods ; Limit of Detection ; Food Contamination/analysis ; Cobalt/chemistry ; Metal Nanoparticles/chemistry ; CRISPR-Associated Proteins ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {BACKGROUND: With growing concerns about food safety, the detection of pesticide residues in food has become increasingly important. The mixed organophosphorus pesticides (OPs) formulations are commonly employed to maximize crop production; however, the excessive application of OPs has posed severe threats to food safety and human health. Therefore, there is an urgent need to develop rapid and sensitive analytical methods that could simultaneously detect the multiple OPs in food samples. Currently, studies on high-performance electrochemiluminescence (ECL) sensors for two or more target sensing have been rarely reported.

RESULTS: This work reports a CRISPR/Cas12a-mediated ECL biosensor for the dual detection of two organophosphorus pesticides, i.e., malathion and phorate. The composite of metal-organic framework material (Co-PTC) loaded with silver nanoparticles (AgNPs@Co-PTC) serves as the single-signal probe, in which Co-PTC was the ECL emitter and AgNPs function as co-reaction accelerators to amplify ECL signals effectively. The method obtains an off-ECL signal by incubating the biosensor with malathion and DNA labelled with black hole quencher 1 (BHQ1-DNA); subsequently, after converting phorate to activator DNA, the sideloading activity of CRISPR/Cas12a was activated, which resulted in an on-ECL signal. By integrating spherical nucleic acid switching strategy with CRISPR/Cas12a for signal amplification, the method achieved the detection limits of 0.108 pM for malathion and 1.01 pM for phorate (S/N = 3), with satisfactory recovery rates of 95.7%-106.4% in food samples (cabbage and lettuce).

SIGNIFICANCE: The established dual-detection mode eliminates the need for multiple signal reporters, simplifying the detection procedure and effectively avoiding cross-interference. Accordingly, the CRISPR/Cas12a-based ECL biosensors featured high selectivity and stability and offered a novel analytical strategy for food safety monitoring.},
}

*Malathion/analysis
*Biosensing Techniques/methods
*Metal-Organic Frameworks/chemistry
*CRISPR-Cas Systems
*Electrochemical Techniques/methods
Silver/chemistry
*Luminescent Measurements/methods
Limit of Detection
Food Contamination/analysis
Cobalt/chemistry
Metal Nanoparticles/chemistry
CRISPR-Associated Proteins
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid41937789,
year = {2026},
author = {Lv, J and He, X and Wu, Q and Sun, Y and Pu, W and Dai, C},
title = {Research progress and applications of gene activation editing technology in crops.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1787461},
pmid = {41937789},
issn = {1664-462X},
abstract = {In recent years, CRISPR/Cas gene editing technology has become a fundamental method in biological breeding. As a vital tool for overcoming technological obstacles, it is currently widely used in functional gene research and genetic enhancement across a variety of organisms. Currently, CRISPR activation (CRISPRa) technology based on dCas9 fusion transcription activation domains has emerged as a powerful tool for expanding the application of CRISPR/Cas systems in improving traits in plants, animals, and microorganisms. This overview starts by going over the underlying principles and components of gene activation editing technology, as well as the phases of development of its three generations. It summarises the present difficulties and potential directions in this field while concentrating on the use of gene activation editing in important crop traits including growth and development regulation, stress resistance, and quality regulation. The objective is to offer valuable insights for the research and development of crop breeding.},
}

@article {pmid41938062,
year = {2026},
author = {Wang, S and Thach, T and De Vita, A},
title = {Editorial: The role of nano-therapeutics in precision cancer medicine.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {14},
number = {},
pages = {1818253},
pmid = {41938062},
issn = {2296-4185},
}

@article {pmid41549804,
year = {2026},
author = {Yang, J and Yang, J and Liu, K and Wang, S and Hu, Y and Li, J and Wang, H and Jiang, D and Han, X},
title = {Microfluidic-Assisted Metal-Polyphenol Cloaked Bacteria Enable CRISPR/dCas9-Mediated Pyroptosis Cascade Amplification for Effective Tumor Immunotherapy.},
journal = {Advanced healthcare materials},
volume = {15},
number = {13},
pages = {e04837},
doi = {10.1002/adhm.202504837},
pmid = {41549804},
issn = {2192-2659},
support = {2023CSJZN0600//Yangtze River Delta Joint Sci-Tech Innovation and Research Projects/ ; NZYSKL240201//Supporting the Innovation Projects of State Key Laboratory on Technologies for Chinese Medicine Pharmaceutical Process Control and Intelligent Manufacture/ ; XPT 82202347//project of Nanjing University of Chinese Medicine to NSFC/ ; //Jiangsu Key Discipline Construction Fund of the 14th Five-Year Plan (Biology)/ ; 82202347//National Natural Science Foundation of China/ ; 82204361//National Natural Science Foundation of China/ ; 32471475//National Natural Science Foundation of China/ ; },
mesh = {*Pyroptosis/drug effects ; Animals ; *CRISPR-Cas Systems/genetics ; Humans ; *Immunotherapy/methods ; *Salmonella typhimurium/metabolism ; Mice ; Phosphate-Binding Proteins/metabolism/genetics ; Tumor Microenvironment ; Cell Line, Tumor ; *Neoplasms/therapy/immunology ; Tannins/chemistry ; *Microfluidics/methods ; Intracellular Signaling Peptides and Proteins/metabolism/genetics ; },
abstract = {Pyroptosis triggered by pore-forming Gasdermin proteins in cancer cells facilitates anti-tumor immune activation by releasing pro-inflammatory cytokines and immunogenic contents following cellular rupture. However, selectively triggering pyroptosis in tumors still remain limited in clinical applications. Here, it is reported a microfluidic-assisted bacterial delivery system using attenuated Salmonella typhimurium VNP20009 encapsulated with metal-phenolic networks composed of ferric ions (Fe[3] [+]) and tannic acid (TA) to enhance intracellular gasdermin D (GSDMD) expression through targeted CRISPR/dCas9 delivery, thereby inducing robust tumor pyroptosis. Mechanistically, this system achieves cascade amplification of pyroptotic cell death through coordinated multi-modal mechanisms. Following systemic administration, VNP20009 specifically accumulates in hypoxic tumor regions while the coated Fe[3] [+]-TA nanofilm undergoes pH-responsive dissolution in the acidic tumor microenvironment (TME), simultaneously generating ROS through Fenton reaction and releasing CRISPR/dCas9 system to upregulate GSDMD expression. Concurrently, the abundant flagella of VNP activate caspase-1, which in turn cleaves the overexpressed GSDMD proteins into its active form, thereby triggering robust pyroptosis in tumor cells. Taken together, by coupling bacterial adjuvanticity with ROS-mediated stress and CRISPR-driven GSDMD upregulation, this strategy achieves efficient amplification of pyroptosis and promotes antitumor immune activation.},
}

*Pyroptosis/drug effects
Animals
*CRISPR-Cas Systems/genetics
Humans
*Immunotherapy/methods
*Salmonella typhimurium/metabolism
Mice
Phosphate-Binding Proteins/metabolism/genetics
Tumor Microenvironment
Cell Line, Tumor
*Neoplasms/therapy/immunology
Tannins/chemistry
*Microfluidics/methods
Intracellular Signaling Peptides and Proteins/metabolism/genetics

@article {pmid41718886,
year = {2026},
author = {Cao, X and He, Y and Guo, H and Cao, X and Zhang, D and Lai, Y and Yang, W and Ma, Z and Yu, X and Wang, L and Li, D and Zeng, Z},
title = {Optimization of RNP-CRISPR for high-efficiency gene editing in mouse intestinal organoids.},
journal = {Molecular and cellular biochemistry},
volume = {481},
number = {4},
pages = {1817-1827},
pmid = {41718886},
issn = {1573-4919},
support = {2019YFA0110802//National Key Research and Development Program of China/ ; No.32025023//National Natural Science Foundation of China/ ; No.32200732//National Natural Science Foundation of China/ ; 20MC1920400//Shanghai Municipal Commission for Science and Technology/ ; 22YF1437700//Shanghai Municipal Commission for Science and Technology/ ; 2019-01-07-00-05-E00054//Innovation Program of Shanghai Municipal Education Commission/ ; },
mesh = {Animals ; *Organoids/metabolism/cytology ; *Gene Editing/methods ; Mice ; *CRISPR-Cas Systems ; *Ribonucleoproteins/genetics/metabolism ; *Intestines/cytology ; Wnt Signaling Pathway ; Mice, Knockout ; beta Catenin/metabolism ; },
abstract = {Mouse intestinal organoids are ideal models for investigating intestinal development and diseases. The full potential of these models hinges on the ability to precisely engineer their genome, yet traditional methods for CRISPR-based editing in 3D cultures often surfer from low efficiency, high cytotoxicity, and inconsistent post-editing differentiation, which limits their applications. Here, we developed an electroporation approach mediated by ribonucleoprotein (RNP)-CRISPR that achieves over 90% gene editing efficiency in mouse intestinal organoids. Using this optimized method, we generated APC-knockout organoids that exhibit Wnt pathway hyperactivation, demonstrated by R-spondin1-independent growth, increased nuclear β-catenin, and enhanced proliferation. Our method addresses a critical technical gap in murine organoid research, offering a scalable platform for intestinal disease modeling.},
}

Animals
*Organoids/metabolism/cytology
*Gene Editing/methods
Mice
*CRISPR-Cas Systems
*Ribonucleoproteins/genetics/metabolism
*Intestines/cytology
Wnt Signaling Pathway
Mice, Knockout
beta Catenin/metabolism

@article {pmid41741636,
year = {2026},
author = {Mathiowetz, AJ and Meymand, ES and Parlakgül, G and van Hilten, N and Torres, EF and Artico, LL and Deol, KK and Lange, M and Pang, SP and Doubravsky, CE and Roberts, MA and Jorgens, DM and Zalpuri, R and Kang, M and Boone, C and Parks, BW and Zhang, Y and Morgens, DW and Newman, ET and Zhou, Y and Talukdar, S and Grabe, M and Ku, G and Levine, TP and Arruda, AP and Olzmann, JA},
title = {CLCC1 promotes hepatic neutral lipid flux and nuclear pore complex assembly.},
journal = {Nature},
volume = {652},
number = {8109},
pages = {462-470},
pmid = {41741636},
issn = {1476-4687},
support = {R01 HL147097/HL/NHLBI NIH HHS/United States ; P30 DK098722/DK/NIDDK NIH HHS/United States ; R01 DK128099/DK/NIDDK NIH HHS/United States ; R01 GM137109/GM/NIGMS NIH HHS/United States ; R01 GM112948/GM/NIGMS NIH HHS/United States ; R01 DK140367/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Mice ; *Nuclear Pore/metabolism ; *Liver/metabolism/cytology ; Hepatocytes/metabolism ; Lipid Droplets/metabolism ; Humans ; Molecular Dynamics Simulation ; Male ; Fatty Liver/metabolism/pathology/genetics ; Membrane Fusion ; *Lipid Metabolism ; CRISPR-Cas Systems ; Mice, Knockout ; Lipoproteins/metabolism ; Nuclear Envelope/metabolism ; Female ; },
abstract = {Imbalances in lipid storage and secretion lead to hepatic steatosis, the accumulation of lipid droplets in hepatocytes[1,2]. Our understanding of the mechanisms that govern the channelling of neutral lipids in hepatocytes towards cytosolic lipid droplets or secreted lipoproteins remains incomplete[3,4]. Here we performed a series of CRISPR-Cas9 screens under different metabolic states that led to the identification of CLCC1 as a critical regulator of neutral lipid storage and secretion in hepatocytes. Loss of CLCC1 resulted in the buildup of large lipid droplets in hepatoma cells and Clcc1 knockout in mice caused liver steatosis. Lipid droplets were present in the lumen of the endoplasmic reticulum of the Clcc1-knockout hepatocytes and exhibited properties of lipoproteins, indicating a profound shift in neutral lipid flux. The loss of CLCC1 also led to the accumulation of nuclear membrane herniations accompanied by a reduction in nuclear pores. Remote homology searches identified a domain in CLCC1 that is homologous to yeast Brl1 and Brr6, factors that promote nuclear envelope fusion during nuclear pore complex assembly. Molecular dynamics simulations and mutagenesis studies support a model in which CLCC1 mediates membrane bending and fusion. We propose that CLCC1 mediates membrane fusion to promote hepatic neutral lipid flux and nuclear pore complex assembly.},
}

Animals
Mice
*Nuclear Pore/metabolism
*Liver/metabolism/cytology
Hepatocytes/metabolism
Lipid Droplets/metabolism
Humans
Molecular Dynamics Simulation
Male
Fatty Liver/metabolism/pathology/genetics
Membrane Fusion
*Lipid Metabolism
CRISPR-Cas Systems
Mice, Knockout
Lipoproteins/metabolism
Nuclear Envelope/metabolism
Female

@article {pmid41791700,
year = {2026},
author = {Xiang, Z and Guo, K and Xi, J and Xu, S and Duan, J and Wen, S and Liu, Z and Wang, X and Zhao, P and Zhang, X},
title = {CRISPR/Cas9-mediated knockout of SPI51 reveals an essential role of protease inhibitors in silk fiber formation.},
journal = {Journal of insect physiology},
volume = {170},
number = {},
pages = {104962},
doi = {10.1016/j.jinsphys.2026.104962},
pmid = {41791700},
issn = {1879-1611},
mesh = {Animals ; *Silk/metabolism/chemistry ; CRISPR-Cas Systems ; *Bombyx/genetics/metabolism/growth & development ; *Protease Inhibitors/metabolism ; *Insect Proteins/genetics/metabolism ; Fibroins/metabolism ; Gene Knockout Techniques ; },
abstract = {Silkworm silk is a natural protein fiber composed mainly of fibroin and sericin, with protease inhibitors representing an additional abundant constituent. However, the impact of protease inhibitors on the structure and properties of silk fibers has not yet been studied. In this study, we focused on the Kunitz-type protease inhibitor SPI51, the most abundant protease inhibitors of cocoon. CRISPR/Cas9 editing was used to generate a homozygous mutant of SPI51 (SPI51[KO]), resulting in premature translation termination at the 33rd amino acid. After knocking out SPI51, the mechanical properties of silk were significantly reduced compared with those of the wild type. Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) results revealed that this deterioration was associated with significantly reduced β-sheet content and lower crystallinity. Morphological observations showed that the fibroin area of SPI51[KO] silk was significantly smaller than that in the wild type. Further Western blot analysis showed that fibroin heavy chain (Fib-H), fibroin light chain (Fib-L), and fibrohexamerin (P25) were decreased after knocking out SPI51, which resulted in a reduction of silk fibroin layer and affected structure and mechanical properties. Our results provide valuable insights into the balance between proteases and protease inhibitors in the silk gland and reveal for the first time the roles of the protease inhibitor in silk protein synthesis and the structural and mechanical properties of silk fibers.},
}

Animals
*Silk/metabolism/chemistry
CRISPR-Cas Systems
*Bombyx/genetics/metabolism/growth & development
*Protease Inhibitors/metabolism
*Insect Proteins/genetics/metabolism
Fibroins/metabolism
Gene Knockout Techniques

@article {pmid41841731,
year = {2026},
author = {Xiong, L and Yadav, V and Sun, S and Heitman, J},
title = {Dissecting the homeodomain MAT locus and engineering novel tripolar and bipolar mating systems in Cryptococcus amylolentus.},
journal = {mBio},
volume = {17},
number = {4},
pages = {e0005926},
doi = {10.1128/mbio.00059-26},
pmid = {41841731},
issn = {2150-7511},
mesh = {*Genes, Mating Type, Fungal ; *Cryptococcus/genetics/physiology ; CRISPR-Cas Systems ; Fungal Proteins/genetics/metabolism ; Gene Deletion ; Reproduction/genetics ; },
abstract = {Sex in fungi is governed by the mating-type (MAT) locus, which exists as a bipolar, pseudobipolar, or tetrapolar system. The significance and impact of MAT on sexual reproduction, however, remain understudied. Furthermore, the evolution of fungal MAT loci shares features with the evolution of plant and animal sex chromosomes. Pathogenic Cryptococcus species harbor a bipolar system with a large contiguous MAT locus, whereas closely related species, such as the non-pathogen C. amylolentus, possess a tetrapolar system with unlinked P/R and HD loci. The P/R locus encodes pheromones and pheromone receptors that mediate partner recognition and cell-cell fusion, while the HD locus encodes homeobox domain-containing proteins (Sxi1 and Sxi2) that play important and evolutionarily conserved roles in sexual reproduction. Here, we explored the roles of HD genes in sexual reproduction and determined the implications of a tetrapolar to bipolar MAT transition. Using a CRISPR-Cas9 system we developed for C. amylolentus, we generated gene deletion mutants and demonstrated that a single compatible pair of heteroallelic Sxi1 and Sxi2 is both necessary and sufficient for mating. By relocating the HD genes to the P/R locus, we found that the artificially generated bipolar configuration led to defective sexual development, which could be partially alleviated through additional rounds of sexual reproduction. Transcriptomic profiling revealed the Sxi1-Sxi2 heterodimeric complex drives expression of genes required for DNA replication and ergosterol biosynthesis during sexual reproduction. These findings provide the first experimental demonstration of a tetrapolar-to-bipolar transition in a tetrapolar species, illuminating MAT locus evolution and homeodomain protein functions.IMPORTANCESexual reproduction is critical for fungal survival and adaptation, yet the mechanisms driving transitions between mating systems remain unclear. With Cryptococcus amylolentus, we provide the first experimental validation of a mating system transition from its original tetrapolar state, through an intermediate tripolar state, to a derived bipolar state in a tetrapolar species. We show that homeodomain (HD) protein heterodimers phenotypically govern dikaryotic filamentation and also transcriptionally modulate DNA replication. These findings establish a mechanistic basis for how MAT locus reorganization drives bipolar evolution from an ancestral tetrapolar state and reinforce that fertility depends on the coordinated control of MAT locus architecture and regulatory functions.},
}

*Genes, Mating Type, Fungal
*Cryptococcus/genetics/physiology
CRISPR-Cas Systems
Fungal Proteins/genetics/metabolism
Gene Deletion
Reproduction/genetics

@article {pmid41889087,
year = {2026},
author = {Yang, L and Ma, W},
title = {CRISPR/Cas12a: A Comprehensive Review from Structural Foundations to Applications in Nucleic Acid Precision Detection.},
journal = {Journal of agricultural and food chemistry},
volume = {74},
number = {13},
pages = {10689-10708},
doi = {10.1021/acs.jafc.5c16528},
pmid = {41889087},
issn = {1520-5118},
mesh = {*CRISPR-Cas Systems ; *Nucleic Acids/genetics/chemistry/metabolism ; Nucleic Acid Amplification Techniques/methods ; Biosensing Techniques/methods ; *Bacterial Proteins/genetics/chemistry/metabolism ; *Endodeoxyribonucleases/genetics/chemistry/metabolism ; Humans ; *Bacteria/genetics/isolation & purification/enzymology ; CRISPR-Associated Proteins ; },
abstract = {CRISPR/Cas12a technology, characterized by its distinctive trans-cleavage activity, has evolved beyond its gene-editing function to emerge as a powerful tool for molecular detection. This review systematically delineates its structural foundation and molecular mechanism, with a focus on how the technology converts specific nucleic acid recognition into cascade signal amplification. Its applications span pathogen diagnosis, species identification, food safety, and authentication of traditional Chinese medicines. Through integration with isothermal amplification and multimodal detection platforms, Cas12a has driven molecular diagnostics toward portability, visualization, and quantification. The review further discusses challenges related to sensitivity, quantitative accuracy, crRNA design, and standardization, while outlining future directions through convergence with cutting-edge technologies such as microfluidics and artificial intelligence, offering a forward-looking perspective for the development of next-generation precision biosensing platforms.},
}

*CRISPR-Cas Systems
*Nucleic Acids/genetics/chemistry/metabolism
Nucleic Acid Amplification Techniques/methods
Biosensing Techniques/methods
*Bacterial Proteins/genetics/chemistry/metabolism
*Endodeoxyribonucleases/genetics/chemistry/metabolism
Humans
*Bacteria/genetics/isolation & purification/enzymology
CRISPR-Associated Proteins

@article {pmid41925118,
year = {2026},
author = {El-Sokkary, MMA and Ali, IEH},
title = {Association of CRISPR/Cas system with integrons and antibiotic resistance in Kliebsiella pneumoniae.},
journal = {Future microbiology},
volume = {},
number = {},
pages = {1-8},
doi = {10.1080/17460913.2026.2647675},
pmid = {41925118},
issn = {1746-0921},
abstract = {AIMS: The interaction between the CRISPR/Cas system and drug resistance was investigated in this study.

METHODS: In this study, 24 isolates, identified as Klebsiella pneumoniae, were subjected to antimicrobial sensitivity screening and detecting the presence of cas, integrons, and antibiotic resistance genes.

RESULTS: The highest resistance level could be detected in ceftriaxone and amoxicillin by 79% and 70.8%, respectively, while the lowest was 16.7% for chloramphenicol. Additionally, 50% of all isolates exhibited multidrug resistance (MDR); however, extensive drug resistance (XDR) was present in 12.5%. Class 1 integrons with different sizes could be identified. sul1 and sul2 were the most resistance genes identified, followed by aacA4 and aac(3)-II and qnrS, with 58%, 50%, 41.7%, 33%, and 33%, respectively. Concerning CRISPR genes, cas3 could be identified in nine different isolates; however, cas1 could be detected in 11 different isolated strains. In case of CRISPR1, it was found in seven isolates (29.3%), while CRISPR2 could be identified in three different isolates (12.5%).

CONCLUSIONS: Antibiotic resistance genes were mostly correlated with CRISPR genes. Interestingly, BOX-PCR group A, with a lower number of CRISPR positive isolates (p = 0.046) had a limited number of detected genes, compared with other groups indicating a significant correlation between antibiotic resistance genes and cas genes (p = 0.0023).},
}

@article {pmid41925232,
year = {2026},
author = {Farruggio, AP and Jiang, L and Duong, K and Nguyen, C and Kaddoura, R and Tsai, R},
title = {S-SELeCT: a human-evolved serine integrase system for efficient large-cargo genome integration.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41925232},
issn = {1362-4962},
support = {1R44GM136045-01/NH/NIH HHS/United States ; 4R44GM136045-02/NH/NIH HHS/United States ; 5R44GM136045-03/NH/NIH HHS/United States ; //Applied StemCell's Research and Development funds/ ; },
mesh = {Humans ; *Integrases/genetics/metabolism ; *Gene Editing/methods ; HEK293 Cells ; Gene Knock-In Techniques/methods ; CRISPR-Cas Systems ; Plasmids/genetics ; Genome, Human ; },
abstract = {As a consequence of their sizes, many loss-of-function genetic mutations fall within large genes. A major gene-therapy tool that could be used to solve large swaths of the genetic diseases that result from these inherited mutations is large-fragment knock-in. I.e. instead of attempting to create separate treatments for each and every location that these mutations occur in, large groups of patients could be aided via a single safe-harbor integration of the full-length coding sequence. Toward this goal, we have created a set of early stage gene-editing enzymes that can help mediate large cargo integration at a safe harbor locus in human cells. When expressed in stable lines, our S-SELeCT (Site-Specific Large Cargo Targeting) integrase fusions can facilitate integration of a 10 kb plasmid at frequencies up to 32%, and when delivered transiently via plasmid transfection, we were able to achieve up to 13% knock-in. These are the first serine integrase enzymes that have been evolved fully in human cells and the first to recognize an endogenous symmetric non-pseudosite-the first true human serine integrase attachment site.},
}

Humans
*Integrases/genetics/metabolism
*Gene Editing/methods
HEK293 Cells
Gene Knock-In Techniques/methods
CRISPR-Cas Systems
Plasmids/genetics
Genome, Human

@article {pmid41925899,
year = {2026},
author = {Wang, B and Liu, M and Wang, H and Wang, C and Zhang, W and Yan, J},
title = {Research progress on nucleic acid amplification-based detection technologies for phytopathogenic fungi.},
journal = {Applied microbiology and biotechnology},
volume = {110},
number = {1},
pages = {},
pmid = {41925899},
issn = {1432-0614},
support = {CARS-29//China Agriculture Research System of MOF and MARA/ ; JKTD2025002//the Outstanding Scientist Project of Beijing Academy of Agriculture and Forestry Sciences/ ; },
mesh = {*Nucleic Acid Amplification Techniques/methods ; *Plant Diseases/microbiology ; *Fungi/genetics/isolation & purification ; *Molecular Diagnostic Techniques/methods ; CRISPR-Cas Systems ; },
abstract = {Phytopathogenic fungi are highly diverse and globally distributed, posing a major threat to agricultural production worldwide. The annual losses caused by plant diseases can reach up to 30% of global crop yields, with over 80% of infections caused by fungal pathogens. The accurate identification of pathogenic fungal species is crucial for effective disease prevention and control. Thus, establishing accurate and rapid detection technologies for phytopathogenic fungi is crucial for implementing targeted control strategies and reducing agricultural losses. Molecular detection technologies based on nucleic acid amplification have recently become indispensable tools for pathogen detection. This review examines the principles and advancements of nucleic acid-based detection techniques, including thermal cycling-based methods (e.g., conventional PCR, real-time quantitative PCR, and droplet digital PCR) and isothermal amplification platforms (e.g., loop-mediated isothermal amplification and recombinase polymerase amplification), as well as CRISPR/Cas-assisted assays coupled with isothermal amplification (e.g., RPA-CRISPR and LAMP-CRISPR), with the aim of evaluating their strengths, limitations, and practical applicability in the rapid diagnosis and precision management of phytopathogenic fungal diseases. KEY POINTS: Nucleic acid amplification technologies enable rapid and sensitive detection of phytopathogenic fungi. Isothermal amplification and CRISPR/Cas-assisted platforms facilitate field-deployable and low-instrumentation diagnostics. Integrated workflows support early diagnosis and precision management of phytopathogenic fungal diseases.},
}

*Nucleic Acid Amplification Techniques/methods
*Plant Diseases/microbiology
*Fungi/genetics/isolation & purification
*Molecular Diagnostic Techniques/methods
CRISPR-Cas Systems

@article {pmid41927937,
year = {2026},
author = {Zhuravlev, IY and Lyakhovets, AA and Matveenko, AG and Lebedeva, MA and Zhernakov, AI and Simonova, VY and Sulima, AS and Tikhonovich, IA and Zhukov, VA},
title = {CRISPR/Cas9-mediated knockout of PsLykX gene of pea (Pisum sativum L.) leads to loss of symbiotic nodules.},
journal = {Transgenic research},
volume = {35},
number = {1},
pages = {},
pmid = {41927937},
issn = {1573-9368},
mesh = {*Pisum sativum/genetics/microbiology/growth & development ; *Symbiosis/genetics ; *CRISPR-Cas Systems ; *Root Nodules, Plant/genetics/microbiology/growth & development ; *Plant Proteins/genetics ; Plants, Genetically Modified/genetics/growth & development/microbiology ; Gene Knockout Techniques ; Plant Roots/genetics/microbiology/growth & development ; Nitrogen Fixation/genetics ; Rhizobium/genetics ; },
abstract = {Pea (Pisum sativum L.) symbiosis with nodule bacteria supplying plants with additional nitrogen is a very specific plant-microbial interaction. Mutual recognition of the partners occurs through perception of bacterial signal molecules (Nod factors) by plant receptors, enabling bacterial entry via root hairs and formation of nitrogen-fixing nodules. The pea gene Sym2, described but not yet cloned, exists in different allelic forms defining the symbiotic specificity, and is therefore thought to encode a Nod factor receptor. The PsLykX gene is a strong candidate for the Sym2, since its alleles coincide with high or low symbiotic specificity; however, to date, no genetic evidence has been obtained for a role of PsLykX in symbiosis. Here, we knocked-out the PsLykX in European pea cultivar Caméor using Agrobacterium-mediated hairy root transformation and CRISPR-Cas9 editing. The roots with editing events confirmed by sequencing lost the ability to form nodules, providing direct functional evidence that PsLykX is essential, at least, for the symbiosis between pea cultivar Caméor and Rhizobium ruizarguesonis RCAM1026.},
}

*Pisum sativum/genetics/microbiology/growth & development
*Symbiosis/genetics
*CRISPR-Cas Systems
*Root Nodules, Plant/genetics/microbiology/growth & development
*Plant Proteins/genetics
Plants, Genetically Modified/genetics/growth & development/microbiology
Gene Knockout Techniques
Plant Roots/genetics/microbiology/growth & development
Nitrogen Fixation/genetics
Rhizobium/genetics

@article {pmid41152563,
year = {2026},
author = {Erbasan, E and Aliciaslan, M and Erendor, F and Sanlioglu, AD and Sanlioglu, S},
title = {Therapeutic precision gene editing of cholesterol pathways as a gene therapy strategy for cardiovascular disease.},
journal = {Gene therapy},
volume = {33},
number = {2},
pages = {218-237},
pmid = {41152563},
issn = {1476-5462},
mesh = {Humans ; *Gene Editing/methods ; *Genetic Therapy/methods ; *Cardiovascular Diseases/therapy/genetics ; CRISPR-Cas Systems ; Animals ; Proprotein Convertase 9/genetics ; *Hypercholesterolemia/therapy/genetics ; *Cholesterol/metabolism/genetics ; Precision Medicine ; Cholesterol, LDL ; },
abstract = {Hypercholesterolemia, defined by high low-density lipoprotein cholesterol levels, critically increases the risk of atherosclerotic cardiovascular disease, which represents the foremost cause of worldwide morbidity and mortality. While established lipid-lowering therapies, primarily statins, are effective for many patients, a significant proportion either fail to achieve optimal LDL-C targets, experience dose-limiting side effects, or face challenges with the long-term adherence required for sustained cardiovascular benefit. The recent emergence and rapid advancement of precise gene editing technologies most notably CRISPR-Cas9 and its advanced variants like base editing and prime editing offer a revolutionary therapeutic paradigm. These tools have the potential to achieve durable modification of the expression or function of genes fundamentally involved in cholesterol metabolism. This comprehensive overview integrates the current knowledge of critical cholesterol regulatory pathways and the main protein targets that are suitable for gene editing. The fundamental mechanisms, relative advantages, and inherent limitations of gene editing platforms and delivery systems for clinical translation are examined. The expanding preclinical data and groundbreaking clinical evidence highlighting the transformative potential of gene editing to achieve significant and lasting reductions in LDL-C, especially through promising therapies like VERVE base editors targeting PCSK9 and ANGPTL3 are critically evaluated. The challenges including off-target effects, delivery efficiency and specificity, long-term safety and durability, complex ethical considerations, and evolving regulatory landscapes that must be rigorously navigated for these therapies to become mainstream clinical practice are thoroughly addressed. Successfully overcoming these challenges could mark the beginning of a new era of personalized, one-time treatments for hypercholesterolemia.},
}

Humans
*Gene Editing/methods
*Genetic Therapy/methods
*Cardiovascular Diseases/therapy/genetics
CRISPR-Cas Systems
Animals
Proprotein Convertase 9/genetics
*Hypercholesterolemia/therapy/genetics
*Cholesterol/metabolism/genetics
Precision Medicine
Cholesterol, LDL

@article {pmid41535609,
year = {2026},
author = {Gottimukkala, KSV and Lane, DD and Cunningham, R and Malik, HS and Jwa, Y and Cassidy, ME and Castelli, JMP and Enstrom, MR and Poljakov, K and Gastelum, G and Ho, SH and Tassa, C and Adair, JE},
title = {CRISPR-AuNP: physicochemical optimization of a gold nanoparticle platform for cost-effective and modular non-viral gene editing in HSPCs.},
journal = {Gene therapy},
volume = {33},
number = {2},
pages = {188-202},
pmid = {41535609},
issn = {1476-5462},
support = {P30 CA015704/CA/NCI NIH HHS/United States ; R01 AI158728/AI/NIAID NIH HHS/United States ; R01 AI167009/AI/NIAID NIH HHS/United States ; U54 DK106829/DK/NIDDK NIH HHS/United States ; },
mesh = {*Gene Editing/methods/economics ; *Gold/chemistry ; *CRISPR-Cas Systems ; *Metal Nanoparticles/chemistry ; Humans ; *Hematopoietic Stem Cells/metabolism ; Cost-Benefit Analysis ; Polyethylene Glycols/chemistry ; },
abstract = {Efficient delivery of CRISPR ribonucleoproteins into primary hematopoietic stem and progenitor cells (HSPCs) is essential for durable gene editing therapies but remains challenging. Here, we advance a modular, benchtop-assembled gold-polymer hybrid nanoparticle (CRISPR-AuNP) platform that enables non-viral delivery of multiple CRISPR systems into HSPCs. Guided by a mechanistic understanding of Cas9's interaction with gold surfaces, we engineered the formulation by conjugating pre-formed RNP-polymer complexes, assembled using thiolated polyethyleneimine-polyethylene glycol, to gold nanoparticles. This system achieved efficient editing in primary CD34+ HSPCs for Cas9, Cas12a, and Cas12a-M29-1 without compromising cell viability. Notably, the nanoformulation can be assembled in under 2 h in a PCR tube for less than $70/million HSPCs treated. This work establishes a scalable, cost-effective, and accessible gene editing system with the potential to democratize CRISPR applications in HSPC research and therapy.},
}

*Gene Editing/methods/economics
*Gold/chemistry
*CRISPR-Cas Systems
*Metal Nanoparticles/chemistry
Humans
*Hematopoietic Stem Cells/metabolism
Cost-Benefit Analysis
Polyethylene Glycols/chemistry

@article {pmid41637037,
year = {2026},
author = {Hao, J and Gao, X and Light, C and Sun, Y and Lu, S and Tian, Y and Gao, X and Su, Y and Gao, J and Huang, X and Zhang, Q and Wang, J and Hai, R and Hu, W and Wang, G},
title = {Genome-wide CRISPR/Cas9 knockout screen identifies host factors essential for bovine parainfluenza virus type 3 replication.},
journal = {Science China. Life sciences},
volume = {69},
number = {4},
pages = {1301-1316},
pmid = {41637037},
issn = {1869-1889},
mesh = {Animals ; Cattle ; *CRISPR-Cas Systems/genetics ; *Virus Replication/genetics ; *Parainfluenza Virus 3, Bovine/physiology/genetics ; *Respirovirus Infections/virology/veterinary/genetics ; Gene Knockout Techniques ; Cell Line ; *Host-Pathogen Interactions/genetics ; },
abstract = {Bovine parainfluenza virus type 3 (BPIV3) is a leading cause of respiratory illness in cattle and a primary component of the bovine respiratory disease complex (BRDC), resulting in significant economic losses. Understanding the mechanisms of BPIV3 infection, particularly the entry process, is essential for developing effective control measures. Identifying specific host factors that viruses exploit during their life cycle can reveal critical vulnerabilities for potential antiviral targets. We established a genome-wide CRISPR/Cas9 knockout screen in bovine cells to identify host factors involved in viral infections. Our screen identified several key host factors required for BPIV3 infection, including the sialic acid transporter SLC35A1 and the Sm-like protein LSM12. Further mechanistic analysis revealed that these factors played critical roles at distinct stages of the BPIV3 entry process. These findings not only advance our understanding of how BPIV3 infects host cells but also identify potential host targets for inhibiting infection and developing novel antiviral strategies.},
}

Animals
Cattle
*CRISPR-Cas Systems/genetics
*Virus Replication/genetics
*Parainfluenza Virus 3, Bovine/physiology/genetics
*Respirovirus Infections/virology/veterinary/genetics
Gene Knockout Techniques
Cell Line
*Host-Pathogen Interactions/genetics

@article {pmid41653634,
year = {2026},
author = {Jung, KM and Klein, R and Mony, SI and Chen, PR and Lee, K and Lee, HJ},
title = {Highly efficient gene editing via targeted Cas9 insertion into chicken housekeeping gene.},
journal = {Poultry science},
volume = {105},
number = {4},
pages = {106585},
pmid = {41653634},
issn = {1525-3171},
mesh = {Animals ; *Chickens/genetics ; *Gene Editing/veterinary/methods ; *Genes, Essential ; *CRISPR-Cas Systems ; Animals, Genetically Modified/genetics ; Transgenes ; *Avian Proteins/genetics/metabolism ; Cell Line ; Mutagenesis, Insertional ; },
abstract = {Achieving stable and efficient transgene expression is a key challenge in advancing avian genome engineering. Although viral vector-based and piggyBac-mediated transgenesis have been widely used in chickens, both approaches are prone to epigenetic silencing, leading to inconsistent, tissue-specific, and often diminished expression over time. This variability limits used of transgenes requiring robust and long-term expression across multiple tissues. In mammals, site-specific integration into genomic safe harbor loci, such as Rosa26, has enabled stable and predictable transgene expression without disrupting endogenous gene function; however, such strategy has not been established in birds. In this research, we hypothesized that integrating Cas9 into endogenous housekeeping genes (the ACTB and GAPDH) could achieve efficient gene editing in chickens through stable and ubiquitous transgene expression. Using two different approaches, 3'-targeted gene insertion and gene tagging, we inserted Cas9 and GFP cassettes into defined genomic loci in chicken DF-1 cells. Both approaches exhibited stable expression of transgenes in the cells, and functional assays confirmed that Cas9 showed highly efficient nuclease activity following guide RNA delivery. Additionally, we derived single-cell clones stably expressing Cas9, enabling uniform and reproducible genome editing in downstream applications. Targeted insertion of transgenes into active housekeeping genes as candidate safe harbor loci mitigates the limitations of random integration and promoter silencing, offering a robust platform for consistent transgene expression in poultry biotechnology and genome engineering.},
}

Animals
*Chickens/genetics
*Gene Editing/veterinary/methods
*Genes, Essential
*CRISPR-Cas Systems
Animals, Genetically Modified/genetics
Transgenes
*Avian Proteins/genetics/metabolism
Cell Line
Mutagenesis, Insertional

@article {pmid41656178,
year = {2026},
author = {McLamarrah, T and Aral, E and Hoffman, M and Tedstone, J and King, T and Vitko, J and Sebastião, MJ and Escandell, JM and Dias, MM and McCall, I and Machado, D and Cairns, V and DeMaria, C and Scarcelli, JJ},
title = {Evaluation of gene editing in CHO cells using the Cas-CLOVER system.},
journal = {Biotechnology progress},
volume = {42},
number = {2},
pages = {e70108},
doi = {10.1002/btpr.70108},
pmid = {41656178},
issn = {1520-6033},
mesh = {CHO Cells ; Animals ; Cricetulus ; *Gene Editing/methods ; *Glutamate-Ammonia Ligase/genetics ; *CRISPR-Cas Systems/genetics ; Recombinant Proteins/genetics/biosynthesis ; Cricetinae ; Antibodies, Monoclonal/genetics/biosynthesis ; Gene Knockout Techniques ; },
abstract = {Recent advances in gene editing technologies have transformed the genetic engineering of Chinese hamster ovary (CHO) hosts, enabling the development of cell lines with improved stability and productivity. In this study, we employed the programmable nuclease (PN) Cas-CLOVER to precisely target the Glutamine synthetase (GS) locus in CHO cells. A total of 30 unique serum-free, suspension-adapted CHO-K1 candidate host cell lines were subjected to Cas-CLOVER-mediated gene editing, generating over one hundred potential GS knockout (GSKO) clones. A subset of the GSKO clones was subsequently validated using three orthogonal methods to confirm complete knockout of the GS gene in 98 clones. Randomly selected GSKO clones were utilized to produce standard monoclonal antibodies. The resulting pools demonstrated enhanced productivity, with up to a 14.5-fold increase in titer compared to their wild-type parental hosts. These findings highlight the potential of gene editing approaches to significantly improve recombinant protein production in CHO expression systems, offering valuable insights for biopharmaceutical manufacturing applications.},
}

CHO Cells
Animals
Cricetulus
*Gene Editing/methods
*Glutamate-Ammonia Ligase/genetics
*CRISPR-Cas Systems/genetics
Recombinant Proteins/genetics/biosynthesis
Cricetinae
Antibodies, Monoclonal/genetics/biosynthesis
Gene Knockout Techniques

@article {pmid41724722,
year = {2026},
author = {Limia, CG and Steffey, V and Cheng, HC and Machado, D and Hart, T and McHargue, MC and Brizzee, C and Crawford, J},
title = {Sequential, chromosome-specific glutamine synthetase double knockout with Cas-CLOVER establishes enhanced CHO platforms for cell line development.},
journal = {Biotechnology progress},
volume = {42},
number = {2},
pages = {e70113},
doi = {10.1002/btpr.70113},
pmid = {41724722},
issn = {1520-6033},
mesh = {CHO Cells ; Animals ; *Glutamate-Ammonia Ligase/genetics/metabolism ; Cricetulus ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; },
abstract = {Cas-CLOVER is an emerging high-fidelity genome editing system that enables precise and efficient cell engineering. In this study, we applied Cas-CLOVER to establish a robust, gene-edited platform in suspension-adapted CHO-K1 cells supporting cell line development (CLD) for biopharmaceutical production. An attractive strategy for high-yield clone selection is the use of glutamine synthetase (GS) knockout CHO cells. The primary GS gene resides on chromosome 5 (GS5), while a recently identified GS pseudogene is located on chromosome 1 (GS1). To compare editing efficiency, we evaluated Cas-CLOVER and Cas9 at both GS loci using the Neon™ Transfection System. Cas-CLOVER achieved 84% editing at GS5 and 74% at GS1, markedly higher than Cas9. Leveraging Cas-CLOVER's dual-guide RNA design, we generated a GS5 single knockout (GS5-SKO) and subsequently a double knockout (GS-DKO) line at both the GS5 and GS1 loci, both with none detected off-target mutations analyzed in 40 predictably off-target sites. For functional validation, these cell lines were engineered with the proprietary Harbor-IN transposase system to stably express trastuzumab. Using an optimized protocol, the resulting GS-DKO platform, termed CleanCut GS CHO, enabled stringent selection and yielded high-producing clones with cell-specific productivity exceeding 100 pg/cell/day and antibody titers greater than 5 g/L in 24 deep well-plate fed-batch cultures after 14 days. The antibody titer stability analysis showed consistency over 60 generations. Collectively, these findings establish Cas-CLOVER as a versatile genome editing tool for developing high-yield CHO host platforms in CLD.},
}

CHO Cells
Animals
*Glutamate-Ammonia Ligase/genetics/metabolism
Cricetulus
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Gene Knockout Techniques/methods

@article {pmid41735098,
year = {2026},
author = {Naruse, K and Loosli, F and Ansai, S and Birney, E and Wittbrodt, J},
title = {Medaka: a novel model for analyzing genome-environment interactions.},
journal = {Trends in genetics : TIG},
volume = {42},
number = {4},
pages = {350-361},
doi = {10.1016/j.tig.2025.12.005},
pmid = {41735098},
issn = {0168-9525},
mesh = {Animals ; *Oryzias/genetics ; *Gene-Environment Interaction ; Genome-Wide Association Study ; *Genome ; Quantitative Trait Loci ; CRISPR-Cas Systems ; },
abstract = {Medaka is an established vertebrate model system for biological and biomedical research. It possesses unique features that make it particularly suitable for studying genome-environment interactions. Endemic to habitats spanning from 4 to 40°C and varying salinities, it combines broad ecological adaptability with experimental tractability. Its exceptional tolerance to inbreeding enabled the creation of the Medaka Inbred Kiyosu-Karlsruhe panel-80 near-isogenic, fully sequenced lines derived from a single wild population. More than 100 wild-derived, fully sequenced strains, collected throughout East Asia for more than 40 years, show relatively low intra-strain variation (inbreeding coefficient of >0.75) but high inter-strain variability (SNP rates >4%). Advanced quantification methods facilitate genome-wide association studies and quantitative trait locus mapping. The system's amenability to clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 editing and emerging epigenomic profiling enables causal validation and regulatory-mechanism discovery. Collectively, medaka offers an unparalleled vertebrate framework for integrating genetics, environment, and epigenetics-bridging evolutionary, biomedical, and population-level perspectives.},
}

Animals
*Oryzias/genetics
*Gene-Environment Interaction
Genome-Wide Association Study
*Genome
Quantitative Trait Loci
CRISPR-Cas Systems

@article {pmid41869962,
year = {2026},
author = {Yang, L and Tan, H and Wang, Y and Zhang, J and Meng, X and Liu, X and Hou, T and Chen, W and Li, F},
title = {Fluidly Confined CRISPR-Magnetic Microbots Empowered Homogeneous Electrochemical Biosensor for Amplified Detection and Discrimination of Cancer-Derived Extracellular Vesicle Subtypes.},
journal = {Analytical chemistry},
volume = {98},
number = {13},
pages = {10103-10111},
doi = {10.1021/acs.analchem.6c00448},
pmid = {41869962},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; Humans ; *Extracellular Vesicles/chemistry/metabolism ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; Doxorubicin/chemistry ; DNA/chemistry ; *Neoplasms ; Biomarkers, Tumor/analysis ; },
abstract = {Accurate identification and profiling of multiple protein biomarkers on tumor-derived extracellular vesicles (tEVs) are crucial for noninvasive cancer subtyping diagnosis but remain technically challenging due to their high heterogeneity, low abundance in biofluids, and preisolation/purification processes. Herein, we developed a homogeneous electrochemical biosensor empowered by fluidly confined CRISPR-magnetic microbots for the amplified detection and sensitive discrimination of tEV subtypes. The CRISPR-magnetic microbots were constructed by engineering CRISPR/Cas12a and DNA icosahedra/doxorubicin (DNA-ICOS/DOX) on intracellularly gelated magnetic cells (IGMCs). Benefiting from the synergistic effects of spatial confinement and membrane fluidity to elevate the local concentration and collision efficiency, the activity of CRISPR/Cas12a was found to be greatly enhanced on IGMCs. For selective sorting of tEVs, a logic-gated aptamer system was used to orthogonally label tEV subpopulations, which further triggers the trans-cleavage activity of CRISPR/Cas12a, resulting in the release of massive DNA-ICOS/DOX into solution. After magnetic separation, the liberated DOX molecules generate a strong electrochemical signal. Particularly, the CRISPR-magnetic microbots could efficiently reduce the background signal, endowing a significantly improved signal-to-noise ratio. Therefore, by combining the CRISPR-magnetic microbots with the dual-target-guided orthogonal barcoding strategy in a homogeneous electrochemical biosensor, precise identification and sensitive detection of tEVs were successfully achieved. More significantly, this assay achieves accurate cancer subtyping in clinical samples, demonstrating its potential as a robust, noninvasive tool for high-accuracy disease screening, classification, and progression monitoring.},
}

*Biosensing Techniques/methods
Humans
*Extracellular Vesicles/chemistry/metabolism
*Electrochemical Techniques/methods
*CRISPR-Cas Systems
Doxorubicin/chemistry
DNA/chemistry
*Neoplasms
Biomarkers, Tumor/analysis

@article {pmid41870471,
year = {2026},
author = {Zhao, X and Wang, Y and Wang, L and Liao, S and Gong, T and Xiong, M and Yu, B and Song, ZL},
title = {Direct RNA Triggering of Cas12a through the Native crRNA Architecture Enables Clinical Nucleic Acids Diagnostics.},
journal = {Analytical chemistry},
volume = {98},
number = {13},
pages = {9982-9992},
doi = {10.1021/acs.analchem.5c08220},
pmid = {41870471},
issn = {1520-6882},
mesh = {Humans ; Manganese/chemistry/metabolism ; *CRISPR-Associated Proteins/metabolism/chemistry ; *RNA/metabolism/chemistry ; CRISPR-Cas Systems ; *Endodeoxyribonucleases/metabolism/chemistry/genetics ; *MicroRNAs/blood/genetics ; Lung Neoplasms/diagnosis/blood ; *Bacterial Proteins/metabolism/chemistry ; },
abstract = {CRISPR/Cas12a has emerged as a powerful platform for nucleic acid diagnostics, yet its activity is widely considered to be restricted to DNA targets, limiting its applicability for direct RNA detection. Here we report a manganese-ion (Mn[2+])-empowered Cas12a (MEC) platform that overcomes this constraint by allowing the robust RNA-mediated activation of Cas12a. Structural analyses reveal that Mn[2+] strengthens RNA engagement and reorganizes the catalytic center by coordinating RNA phosphates, resulting in an enhancement of trans-cleavage efficiency by 60-fold relative to the Mg[2+] conditions, without compromising sequence specificity. This Mn[2+]-dependent activation mechanism is conserved across multiple Cas12a orthologues (LbCas12a, AsCas12a, FnCas12a), permitting amplification-free detection of RNA with femtomolar sensitivity across diverse targets, particularly the ultrashort abortive transcripts (7 nt). Analysis of clinical serum samples further demonstrates that MEC quantitatively measures circulating miR-21 with performance concordant with reference clinical assays and effectively distinguishes lung cancer patients from healthy individuals. These results reveal an unrecognized role for Mn[2+] in Cas12a biochemistry and establish a simple, versatile, and highly sensitive framework for RNA diagnostics.},
}

Humans
Manganese/chemistry/metabolism
*CRISPR-Associated Proteins/metabolism/chemistry
*RNA/metabolism/chemistry
CRISPR-Cas Systems
*Endodeoxyribonucleases/metabolism/chemistry/genetics
*MicroRNAs/blood/genetics
Lung Neoplasms/diagnosis/blood
*Bacterial Proteins/metabolism/chemistry

@article {pmid41871234,
year = {2026},
author = {Liao, H and Xie, H and Ye, H and Liu, X and Chen, Y and Zhong, R and He, S and Xiao, X and Xie, Z and Shao, Z and Yu, L and Chen, Z},
title = {One-Pot CRISPR/Cas12a Assay Based on Ultrashort HDA for Ultrasensitive and Universal Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {13},
pages = {10004-10014},
doi = {10.1021/acs.analchem.5c08249},
pmid = {41871234},
issn = {1520-6882},
mesh = {*Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Limit of Detection ; Influenza A virus/genetics/isolation & purification ; *Nucleic Acids/analysis ; Staphylococcal Infections/diagnosis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Isothermal amplification techniques, such as helicase-dependent amplification (HDA) combined with CRISPR, are cutting-edge approaches for nucleic acid detection. In this work, we developed a novel ultrashort mesophilic HDA (termed usHDA) for rapid, highly sensitive nucleic acid amplification at 37 °C and constructed a one-pot usHDA-CRISPR/Cas12 assay. The usHDA is specifically designed for rapid amplification of ultrashort sequences (about 40 nt) at 37 °C within 30 min. This usHDA-CRISPR/Cas12a detection can be completed within 1 h, achieving a limit of detection (LOD) of 5 aM. When tested on 58 clinical specimens from patients infected with respiratory pathogens, this assay identified 41 positive and 17 negative samples for influenza A virus. This assay achieved 100% sensitivity, 100% specificity, and a perfect receiver operating characteristic curve (area under the curve value = 1.00; n = 58) compared with PCR analysis. Furthermore, 24 samples of Staphylococcus infection were detected using usHDA-CRISPR/Cas12a, and the same 100% sensitivity and specificity were achieved. These findings highlighted the strong applicability of our proposed assay for universal nucleic acid detection.},
}

*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems/genetics
Humans
Limit of Detection
Influenza A virus/genetics/isolation & purification
*Nucleic Acids/analysis
Staphylococcal Infections/diagnosis
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41873844,
year = {2026},
author = {Wang, Q and Sheng, M and Zheng, Y and Zhang, B and Jin, Z and Zhang, T and Li, Z and Huang, J and Yang, X},
title = {Electrochemiluminescence Biosensing Platform Based on CRISPR/Cas12a and DNA Nanotweezer-Mediated Catalytic Hairpin Assembly Amplification.},
journal = {Analytical chemistry},
volume = {98},
number = {13},
pages = {9832-9841},
doi = {10.1021/acs.analchem.5c07500},
pmid = {41873844},
issn = {1520-6882},
mesh = {*MicroRNAs/analysis/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Electrochemical Techniques/methods ; *Luminescent Measurements/methods ; Humans ; *DNA/chemistry/genetics ; Catalysis ; Nucleic Acid Amplification Techniques ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The detection of microRNAs (miRNAs) biomarkers has great potential in the early diagnosis of acute myocardial infarction (AMI). Herein, we constructed an electrochemiluminescence biosensing platform based on DNA nanotweezer (DNT)-mediated catalytic hairpin assembly (CHA) and CRISPR/Cas12a system for detecting potential AMI biomarker miRNA-133a. DNT, as a programmable molecular scaffold, can precisely organize molecules at the nanoscale and output high signal-to-background ratio detection signals, which is introduced into the construction of sensing platforms. When the target miRNA was presented, the hairpin in DNT was opened, which altered the DNT structure from a closed state to an open state and exposed the catalytic sequence for CHA. Subsequently, a large number of F/A-F duplexes were generated after the addition of fuel strands (F) and antifuel strands (A-F), which served as the target for activating the CRISPR/Cas12a system. The activated Cas12a collaterally cleaved the signal probe (H1) on the electrode surface, causing the labeled Ru(bpy)3[2+] to detach from the electrode surface, resulting in a weakened ECL signal. We found that compared with the general CHA reaction, the DNT-mediated CHA reaction significantly lowers the leakage of the circuit; thus, a high signal-to-background ratio and detection sensitivity can be obtained. Therefore, we developed a highly sensitive biosensing platform for detecting miRNA-133a with a detection limit of 0.12 fM. This sensing strategy provides a new approach for nucleic acid detection and disease diagnosis.},
}

*MicroRNAs/analysis/genetics
*Biosensing Techniques/methods
*CRISPR-Cas Systems
*Electrochemical Techniques/methods
*Luminescent Measurements/methods
Humans
*DNA/chemistry/genetics
Catalysis
Nucleic Acid Amplification Techniques
Limit of Detection
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41881766,
year = {2026},
author = {Li, S and Tang, S and Xu, W and Zhou, J and Li, X and Wang, J},
title = {SIMPLE-CRISPR: A Sample-to-Result Platform for Point-of-Care Detection of Nucleic Acids via a Functionalized Magnetic-Bead-Based CRISPR Assay.},
journal = {Analytical chemistry},
volume = {98},
number = {13},
pages = {9964-9971},
doi = {10.1021/acs.analchem.5c08178},
pmid = {41881766},
issn = {1520-6882},
mesh = {*Point-of-Care Systems ; *CRISPR-Cas Systems/genetics ; Humans ; *Nucleic Acids/analysis/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Smartphone ; *Point-of-Care Testing ; Magnetic Phenomena ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR) systems show great promise for next-generation molecular diagnostics due to their programmability and specificity. Amplification-free CRISPR detection has a strong potential for point-of-care (POC) testing, and the digital format of the assay naturally improves the sensitivity of amplification-free CRISPR detection. However, integrating sample preparation, reaction implementation, and signal readout into a streamlined and user-friendly POC workflow remains a major technical challenge. We address this by developing SIMPLE-CRISPR (Sample-to-Result Integrated platform with Magnetic-bead extraction, Polydisperse droplets, Low-complexity operation, and Engineered smartphone readout), an amplification-free CRISPR-Cas12a system for POC diagnostics. Our innovation features functionalized magnetic-bead extraction for the efficient enrichment of low-abundance nucleic acid targets from large-volume samples with minimal loss, vortex-driven polydisperse emulsification for facile digital droplet generation, and smartphone-integrated fluorescence imaging for on-site portable signal detection. This integrated approach significantly improves sensitivity compared to bulk CRISPR, removes the need for amplification and complex microfluidics, and provides sample-to-result functionality for POC environments. Clinical validation for human papillomavirus type 18 (HPV18) detection confirmed that the assay achieved diagnostic performance consistent with quantitative real-time polymerase chain reaction (qRT-PCR), demonstrating its great potential for accessible POC nucleic acid diagnostics.},
}

*Point-of-Care Systems
*CRISPR-Cas Systems/genetics
Humans
*Nucleic Acids/analysis/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
Smartphone
*Point-of-Care Testing
Magnetic Phenomena

@article {pmid41917051,
year = {2026},
author = {Lu, J and Lai, J and Cheng, L and Zhan, H and Jie, K and Liu, C and Huang, L and Cen, M and Liu, S and Chen, Z and Zhang, Q and Zhang, J and Wu, J and Pan, B and Chen, S and Zhong, J and He, B and Li, H and Chen, X and Lin, T},
title = {Miniature and versatile genome regulation TnpB-ωRNA toolkits facilitate cancer immunotherapy.},
journal = {Nature communications},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41467-026-71327-w},
pmid = {41917051},
issn = {2041-1723},
abstract = {CRISPR‒Cas systems represent powerful tools for genome regulation. However, the large size of Cas proteins limits their efficient delivery via an adeno-associated virus (AAV), thereby restricting their clinical translation. Here, we engineer the IS200/IS605 transposon-encoded nuclease TnpB, along with its ωRNA scaffold, to create an enhanced TnpB system, which serves as a compact toolkit for gene activation, genome editing, and base editing. The gene activator enTnpBa increases expression by 2889-fold with a minimized 93 nt ωRNA and robustly activates endogenous genes in mammalian cells. We develop a single-AAV-based regimen for immune activation (AAV-ImmunAct) that delivers enTnpBa to activate CXCL9, IL-15, and IFN-γ. AAV-ImmunAct effectively enhances T cell migration and activation, increases killing of cancer cell lines and patient-derived organoids, and synergizes with anti-PD-1 therapy in humanized mice. Here, we establish enTnpB as a compact and versatile platform for genome regulation and a promising tool for cancer immunotherapy.},
}

@article {pmid41917262,
year = {2026},
author = {Shi, R and Yang, M and Liu, Y and Gao, H and Lin, Z},
title = {Mechanistic basis for selective Csm6-2 activation by cyclic penta-adenylate in a type III CRISPR-Cas system.},
journal = {The EMBO journal},
volume = {},
number = {},
pages = {},
pmid = {41917262},
issn = {1460-2075},
support = {32471255//MOST | National Natural Science Foundation of China (NSFC)/ ; 32271258//MOST | National Natural Science Foundation of China (NSFC)/ ; 2024J02006//| Natural Science Foundation of Fujian Province (Fujian Natural Science Foundation)/ ; },
abstract = {Type III CRISPR systems generate cyclic oligoadenylate (cOA, 3 to 6 AMPs) messengers upon detecting viral RNA, activating downstream effectors to defend against viral infection. Although cOA-activated effectors have been extensively characterized, the effectors specific to cA5-one of the most abundant cOA species produced during phage infection-have remained unexplored. Here, we report that the CRISPR ribonuclease Csm6 (Csm6-2) from Actinomyces procaprae selectively employs cA5 as its activator. Csm6-2 utilizes its HEPN domain, rather than the CARF domain, to mediate self-limiting cleavage of cOA activators. Cryo-EM structural analyses reveal that Csm6-2 functions as a homotetramer, and disruption of tetramer formation significantly reduces its ribonuclease activity. Although cA6 and cA5 bind Csm6-2 with comparable affinity, only cA5 induces CARF domain closure, stabilizes the tetramer, and remodels the active site in the HEPN domain. In contrast, the sixth AMP of cA6 imposes significant steric hindrance on CARF domain movement, preventing its closure and subsequent allosteric activation. These findings expand our understanding of the cOA signaling diversity and specific cOA recognition mechanisms in type III CRISPR immunity.},
}

@article {pmid41917354,
year = {2026},
author = {Chen, Z and Huang, X and Pi, Y and Jiang, Y},
title = {Method for Generation of adamtsl4 Knock-Out Zebrafish Lines Using CRRISPR/Cas9 System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3027},
number = {},
pages = {73-82},
pmid = {41917354},
issn = {1940-6029},
mesh = {Animals ; *Zebrafish/genetics ; *CRISPR-Cas Systems ; *Gene Knockout Techniques/methods ; *ADAMTS Proteins/genetics ; Disease Models, Animal ; *Zebrafish Proteins/genetics ; Phenotype ; Gene Editing/methods ; Mutation ; Humans ; },
abstract = {Congenital ectopia lentis (CEL) is a rare pediatric ocular disorder characterized by zonular fiber defects leading to lens dislocation and is genetically heterogeneous. Among known causes, biallelic mutations in ADAMTSL-4 represent the second most common genetic contributor, frequently associated with ectopia pupillae (EP)-a distinct and clinically significant feature. However, the mechanisms by which ADAMTSL-4 mutations lead to these ocular abnormalities remain poorly understood, partly due to the lack of effective animal models. In this study, we generated adamtsl-4 knock-out zebrafish lines using the CRISPR/Cas9 system. Through microinjection of sgRNA/Cas9 complexes and multigenerational screening, we established stable homozygous mutant lines. These mutants exhibited consistent phenotypes, including lens dislocation into the vitreous body and marked pupillary displacement, faithfully recapitulating human ADAMTSL-4-related EL and EP. This method provides a practical and scalable strategy for generating loss-of-function zebrafish models, with demonstrated utility in recapitulating phenotypes associated with ADAMTSL-4 mutations. Our approach offers a valuable tool for investigating the molecular mechanisms underlying CEL and EP and may support drug screening and therapeutic discovery in the future.},
}

Animals
*Zebrafish/genetics
*CRISPR-Cas Systems
*Gene Knockout Techniques/methods
*ADAMTS Proteins/genetics
Disease Models, Animal
*Zebrafish Proteins/genetics
Phenotype
Gene Editing/methods
Mutation
Humans

@article {pmid41917356,
year = {2026},
author = {Zheng, L and Wu, Z and Zheng, XL},
title = {Generating adamts13[-/-] Zebrafish via CRISPR/Cas9 Gene Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3027},
number = {},
pages = {93-111},
pmid = {41917356},
issn = {1940-6029},
mesh = {Animals ; *Zebrafish/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *ADAMTS13 Protein/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Knockout Techniques/methods ; Animals, Genetically Modified ; Disease Models, Animal ; *Zebrafish Proteins/genetics ; },
abstract = {The zebrafish (Danio rerio) is a powerful vertebrate model for studying hematologic and thrombotic diseases due to its genetic tractability and conservation of hemostatic pathways with humans. In this chapter, we describe a detailed workflow for generating adamts13 knockout zebrafish using CRISPR/Cas9-mediated genome editing. Methods include the design and preparation of guide RNAs, Cas9 mRNA synthesis, and embryo microinjection at the one-cell stage. Alternative strategies for gRNA generation, including synthetic crRNA:tracrRNA duplexes, are also outlined. We provide protocols for screening founder fish, genotyping, and establishing stable mutant lines through outcrossing, thereby minimizing potential off-target effects. Practical notes on embryo handling, RNA stability, and contamination prevention are highlighted to ensure reproducibility. Together, these procedures establish a robust framework for creating zebrafish loss-of-function models, enabling mechanistic studies of ADAMTS13 function in vivo and advancing the exploration of thrombotic disease pathophysiology. This approach can be readily adapted to knock out other genes or introduce specific mutations in zebrafish simply by altering the gRNA sequence.},
}

Animals
*Zebrafish/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
*ADAMTS13 Protein/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Knockout Techniques/methods
Animals, Genetically Modified
Disease Models, Animal
*Zebrafish Proteins/genetics

@article {pmid41918363,
year = {2026},
author = {Yamada, HY and Rao, CV},
title = {Genome-Wide CRISPR Analysis Uncovers Metformin and NSAID Combination as a Chemopreventive Approach in Smoking-Associated Oral Cancer.},
journal = {Cancer prevention research (Philadelphia, Pa.)},
volume = {19},
number = {4},
pages = {183-185},
doi = {10.1158/1940-6207.CAPR-26-0004},
pmid = {41918363},
issn = {1940-6215},
mesh = {*Metformin/therapeutic use/pharmacology ; Humans ; *Mouth Neoplasms/prevention & control/etiology/genetics ; *Anti-Inflammatory Agents, Non-Steroidal/therapeutic use/pharmacology ; *Squamous Cell Carcinoma of Head and Neck/prevention & control/genetics/etiology ; *Antineoplastic Combined Chemotherapy Protocols/therapeutic use ; CRISPR-Cas Systems ; *Smoking/adverse effects ; Chemoprevention/methods ; Drug Resistance, Neoplasm/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers worldwide and carries substantial morbidity. Metformin, a widely used antidiabetic agent, shows promise for HNSCC prevention, but resistance arises in a subset of tumors. In a recent issue of Cancer Prevention Research, Hoang and colleagues use CRISPR screening to identify key mediators of metformin resistance, including AMPK and protein kinase A (PKA), and demonstrate that the cyclooxygenase 2-prostaglandin E2 axis acts upstream of PKA. Because this pathway is readily inhibited by common nonsteroidal anti-inflammatory drugs (NSAID), the findings support clinical evaluation of combined metformin and NSAID therapy to improve HNSCC chemoprevention. See related article by Hoang et al., p. 79 .},
}

*Metformin/therapeutic use/pharmacology
Humans
*Mouth Neoplasms/prevention & control/etiology/genetics
*Anti-Inflammatory Agents, Non-Steroidal/therapeutic use/pharmacology
*Squamous Cell Carcinoma of Head and Neck/prevention & control/genetics/etiology
*Antineoplastic Combined Chemotherapy Protocols/therapeutic use
CRISPR-Cas Systems
*Smoking/adverse effects
Chemoprevention/methods
Drug Resistance, Neoplasm/genetics
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41919328,
year = {2026},
author = {Singh, P and Sharma, K and Tamrakar, VK and Khare, R and Bhargava, A and Negi, SS},
title = {CRISPR-Cas system: recent advancements in prompt diagnosis of high-risk HPV genotypes in cervical cancer.},
journal = {Expert review of molecular diagnostics},
volume = {},
number = {},
pages = {},
doi = {10.1080/14737159.2026.2654503},
pmid = {41919328},
issn = {1744-8352},
abstract = {INTRODUCTION: The CRISPR/Cas system has emerged as a highly versatile platform for diagnosing infectious diseases, particularly viral pathogens. Human papillomavirus (HPV) comprises of more than 200 types, with persistent infection by 14 high-risk genotypes recognized as the primary cause of cervical cancer worldwide. Early and accurate detection of these High-Risk HPV (HR-HPV) types is essential for effective clinical management and prevention of disease progression.

AREAS COVERED: This narrative review was based on literature searches in PubMed, Scopus, and Google Scholar covering studies published between 2015 and 2024. This review summarizes recent advances in CRISPR/Cas based diagnostics for HR-HPV, including both pre-amplification and amplification-free strategies. Integration of CRISPR systems with diverse readout modalities such as colorimetric, fluorescent, electrochemical, and lateral-flow biosensors has enabled rapid, sensitive, and user-friendly detection suitable for point-of-care testing (POCT), particularly in low-resource settings.

EXPERT OPINION: CRISPR/Cas assays demonstrate high sensitivity, specificity, and speed, offering a promising alternative to conventional molecular techniques for HR-HPV detection and genotyping. The convergence of CRISPR diagnostics with artificial intelligence, microfluidics, and affordable biosensors holds significant potential to transform community-level HPV screening. With continued innovation and regulatory support, CRISPR/Cas systems are poised to become indispensable tools for early HR-HPV detection and cervical cancer prevention.},
}

@article {pmid41923586,
year = {2026},
author = {Oh, Y and Lee, H and Jang, S},
title = {Emerging synthetic biology-assisted technologies for overcoming antibiotic resistance: CRISPR-Cas, bacteriophage, microbiome, and metabolic engineering-based solutions.},
journal = {Journal of microbiology (Seoul, Korea)},
volume = {64},
number = {3},
pages = {e2512002},
doi = {10.71150/jm.2512002},
pmid = {41923586},
issn = {1976-3794},
support = {//National Research Foundation of Korea/ ; RS-2025-02214910//Ministry of Science and ICT/ ; //Incheon National University/ ; },
mesh = {*CRISPR-Cas Systems ; *Synthetic Biology/methods ; *Bacteriophages/genetics ; *Metabolic Engineering/methods ; *Anti-Bacterial Agents/pharmacology ; *Microbiota/genetics ; *Bacteria/drug effects/genetics ; Humans ; *Drug Resistance, Bacterial/genetics ; *Drug Resistance, Microbial/genetics ; },
abstract = {Antibiotic resistance has become a critical global health challenge due to the decreased efficacy of existing antibiotics and the emergence of multidrug-resistant pathogens. In particular, the rapid horizontal transfer of resistance genes and the diverse mechanisms by which bacteria acquire resistance have significantly undermined the effectiveness of conventional therapeutic strategies, revealing fundamental limitations in current infectious disease management. In this context, synthetic biology provides a promising framework to overcome the limitations of conventional antibiotics by integrating engineering principles with bioengineering approaches, thereby enabling precise and programmable control of biological processes. These synthetic biology-based approaches offer substantial potential for developing sustainable and highly specific antimicrobial strategies. This review comprehensively examines recent advances in synthetic biology-assisted antimicrobial strategies, including CRISPR-Cas systems, bacteriophage engineering, microbiome engineering, and metabolic engineering-driven antibiotic discovery. Collectively, these approaches represent a precision antimicrobial paradigm that enables selective targeting of resistant bacteria while preserving microbiome homeostasis. These strategies also provide new directions for limiting resistance dissemination and guiding the development of next-generation therapeutics.},
}

*CRISPR-Cas Systems
*Synthetic Biology/methods
*Bacteriophages/genetics
*Metabolic Engineering/methods
*Anti-Bacterial Agents/pharmacology
*Microbiota/genetics
*Bacteria/drug effects/genetics
Humans
*Drug Resistance, Bacterial/genetics
*Drug Resistance, Microbial/genetics

@article {pmid40676395,
year = {2026},
author = {Fereydani, NM and Galehdari, H and Hoveizi, E and Alghasi, A and Ajami, M and Andashti, B and Malayeri, A},
title = {Investigating the Efficacy of the CRISPR/Cas9 Gene-Editing System for Targeting the HBB FSC 36-37 (-T) Mutation Locus in Hematopoietic Stem Cells.},
journal = {Molecular biotechnology},
volume = {68},
number = {4},
pages = {1851-1866},
pmid = {40676395},
issn = {1559-0305},
support = {EE/1401.2.24.118699/SCU.ac.ir//Shahid Chamran University of Ahvaz/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; HEK293 Cells ; *Hematopoietic Stem Cells/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; beta-Thalassemia/genetics/therapy ; Mutation ; *beta-Globins/genetics ; Iran ; },
abstract = {The emergence of genome editing using the CRISPR/Cas9 system has opened up new possibilities and significantly improved the potential for long-term gene therapy of beta-thalassemia. In Iran, FSC 36/37 (-T) is one of the most common mutations among affected individuals, with the highest frequency in the West region (20.8%) and the South-West region (14%). In the context of a proof-of-concept investigation, we present a comprehensive design and assess the efficacy of a CRISPR construct specifically engineered to employ homology-directed repair for targeting the FSC 36/37 (-T) mutation in the HBB gene. The selected sgRNAs were designed and cloned into an optimized CRISPR plasmid. The guide RNAs were transferred to HEK293 cells. T7EI analysis and a non-denaturing PAGE system were employed to assess the effectiveness of the guide RNAs. Hematopoietic stem cells were isolated using the MACS system, and transfection was performed with the Lonza Nucleofector device. The edited cells were monitored using TaqMan-qPCR and RFLP-PCR techniques. Furthermore, the Cas9 enzyme's off-target cleavage sites were meticulously examined to ensure the specificity of the editing process. This research revealed successful genome editing in 30% of the clones analyzed in HEK293 cells and 23.91% of the examined clones in HSCs. Our findings demonstrate the potential of CRISPR/Cas9-mediated genome editing as a promising strategy for addressing genetic mutations associated with thalassemia and other monogenic diseases.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
HEK293 Cells
*Hematopoietic Stem Cells/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
beta-Thalassemia/genetics/therapy
Mutation
*beta-Globins/genetics
Iran

@article {pmid41316685,
year = {2026},
author = {Megarani, DV and Yang, L and Siler, HJ and Quijano Cardé, EM and Martyniuk, CJ and Hick, PM and Becker, JA and Soto, E and Surachetpong, W and Yanong, RPE and Subramaniam, K},
title = {One-Pot RT-LAMP CRISPR/Cas12b Platform for Rapid Detection of Tilapia Lake Virus.},
journal = {Journal of fish diseases},
volume = {49},
number = {5},
pages = {e70087},
doi = {10.1111/jfd.70087},
pmid = {41316685},
issn = {1365-2761},
support = {2023-67015-39481//National Institute of Food and Agriculture/ ; },
mesh = {Animals ; *Fish Diseases/diagnosis/virology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Sensitivity and Specificity ; *RNA Virus Infections/veterinary/diagnosis/virology ; *Molecular Diagnostic Techniques/veterinary/methods ; Aquaculture ; *RNA Viruses/isolation & purification ; *Tilapia ; },
abstract = {Tilapia Lake Virus (TiLV) is a significant threat to global tilapia aquaculture, highlighting the need for rapid and accurate diagnostic methods to manage outbreaks and minimise economic losses. This study presents the development and partial validation of a one-pot assay integrating RT-LAMP with the CRISPR/Cas12b system for sensitive and specific TiLV detection. This assay amplifies viral RNA using RT-LAMP, while CRISPR/Cas12b enables a real-time detectable signal. Targeting a conserved region in TiLV segment four, the assay achieves results within 75 min at 62°C, with easy visualisation using a portable fluorescence viewer. It demonstrated high sensitivity, with a 95% limit of detection of 79.6 copies (95% CI: 48-132 copies), and high specificity, with no cross-reaction to other fish RNA or DNA viruses. Based on a validation panel of 261 samples from 9 source populations, the assay exhibited 92% diagnostic sensitivity (95% CI: 87%-96%) and 100% diagnostic specificity (95% CI: 97%-100%). When assessed as a non-lethal sample, gills provided a reliable and less invasive alternative despite lower viral loads compared to internal organs. Therefore, this partially validated one-pot assay is potentially practical for enhancing TiLV detection and disease management in aquaculture systems, especially in field settings and resource-limited laboratories.},
}

Animals
*Fish Diseases/diagnosis/virology
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/veterinary/methods
Sensitivity and Specificity
*RNA Virus Infections/veterinary/diagnosis/virology
*Molecular Diagnostic Techniques/veterinary/methods
Aquaculture
*RNA Viruses/isolation & purification
*Tilapia

@article {pmid41328622,
year = {2026},
author = {Ma, C and Zhou, Y and Jiang, N and Ren, X and Xu, C and Su, N and Fan, Y and Liu, W},
title = {Development of an RPA-CRISPR-Cas12a Fluorescence Assay for Rapid and Sensitive Detection of Tilapia Parvovirus (TiPV).},
journal = {Journal of fish diseases},
volume = {49},
number = {5},
pages = {e70095},
doi = {10.1111/jfd.70095},
pmid = {41328622},
issn = {1365-2761},
support = {32202994//the National Natural Science Foundation of China/ ; 2023TD46//the Central Public-interest Scientific Institution Basal Research Fund, CAFS/ ; 2024XT0602//the Central Public-interest Scientific Institution Basal Research Fund, CAFS/ ; },
mesh = {Animals ; *Fish Diseases/diagnosis/virology ; Sensitivity and Specificity ; *Parvoviridae Infections/veterinary/diagnosis/virology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/veterinary/methods ; *Tilapia ; *Parvovirus/isolation & purification/genetics ; },
abstract = {Tilapia parvovirus (TiPV) is an emerging pathogen associated with high mortality rates in farmed tilapia, highlighting the urgent need for rapid and accurate diagnostic tools. In this study, we established an RPA-CRISPR/Cas12a detection system targeting the TiPV NS1 gene. The assay conditions were systematically optimised, including 15-min RPA amplification at 39°C, with reagent concentrations of 200 nM Cas12a, 250 nM crRNA and 200 nM ssDNA reporter. Specificity tests showed no cross-reactivity with other tilapia pathogens (TiLV, S. agalactiae) and other aquatic pathogens (LMBRaV, YcCV, GCRV II, WSSV, CyHV-2, SVCV). Sensitivity evaluation revealed a limit of detection (LoD) of 1.97 × 10[1]copies/μL, which was 100-fold more sensitive than PCR (1.97 × 10[3]copies/μL). Clinical validation with 20 tilapia samples demonstrated a 50% positive detection rate for RPA-CRISPR/Cas12a, 15% higher than PCR (35%). This integrated method combines the advantages of RPA and CRISPR-based signal transduction, offering a field-applicable solution for TiPV monitoring in resource-limited aquaculture environments.},
}

Animals
*Fish Diseases/diagnosis/virology
Sensitivity and Specificity
*Parvoviridae Infections/veterinary/diagnosis/virology
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/veterinary/methods
*Tilapia
*Parvovirus/isolation & purification/genetics

@article {pmid41636766,
year = {2026},
author = {Abbas, W and Hu, J and Zhu, Y and Xu, L and Mo, B and Liu, L},
title = {Decoding MicroRNA Networks in Plant Vegetative and Reproductive Branching: Mechanisms and Applications for Crop Improvement.},
journal = {Plant, cell & environment},
volume = {49},
number = {5},
pages = {2795-2808},
doi = {10.1111/pce.70429},
pmid = {41636766},
issn = {1365-3040},
support = {32270878//National Natural Science Foundation of China/ ; JCYJ20220531101618038//Shenzhen Basic Research General Project/ ; JCYJ20230808105419038//Shenzhen Basic Research General Project/ ; 2024ZY0132//Central Guidance Funds for Local Science and Technology Development of Inner Mongolia/ ; 868-000003011001//Excellence 2035 Research Program of Shenzhen University/ ; 2023DFT005//Research Team Cultivation Program of Shenzhen University/ ; 2025M784036//China Postdoctoral Science Foundation of China/ ; 2025TQ09A772//Guangdong Special Support Program for Young Talents in Innovation Research of Science and Technology/ ; },
mesh = {*MicroRNAs/genetics/metabolism/physiology ; *Crops, Agricultural/genetics/growth & development ; Gene Expression Regulation, Plant ; Gene Editing ; *RNA, Plant/genetics ; Gene Regulatory Networks ; Reproduction/genetics ; Plant Development/genetics ; CRISPR-Cas Systems ; },
abstract = {Plant branching, encompassing both vegetative and reproductive forms, is a complex and crucial process that shapes overall architecture and determines crop yield and biomass. MicroRNAs (miRNAs) have emerged as master regulators in fine-tuning the intricate genetic and hormonal networks that govern plant branching. This review systematically synthesises recent advances in understanding how miRNA-target gene modules regulate essential pathways to orchestrate the branching patterns. We highlight a central insight that specific miRNA families form hierarchical, stage-specific networks that facilitate the independent optimisation of vegetative and reproductive branching. Furthermore, we explore the potential applications of miRNA manipulation in optimising branching architecture to improve crop yield. By critically evaluating strategies such as artificial miRNAs, target mimics and CRISPR/Cas9 genome editing, we discuss how modulating miRNA networks can engineer ideal plant architecture. Finally, we provide a forward-looking perspective on overcoming challenges in miRNA-based crop improvement, emphasising the integration of single-cell omics and epigenetic insights to achieve precise genetic modifications. This review underscores the transformative potential of miRNAs in designing future crops for enhanced productivity.},
}

*MicroRNAs/genetics/metabolism/physiology
*Crops, Agricultural/genetics/growth & development
Gene Expression Regulation, Plant
Gene Editing
*RNA, Plant/genetics
Gene Regulatory Networks
Reproduction/genetics
Plant Development/genetics
CRISPR-Cas Systems