@article {pmid41639251,
year = {2026},
author = {Khan, A and Herring, G and Zhu, JY and Petterson, M and Lister, R},
title = {Designing and testing CRISPRi-based synthetic gene circuits in plants.},
journal = {Nature protocols},
volume = {21},
number = {8},
pages = {3528-3551},
pmid = {41639251},
issn = {1750-2799},
support = {DP240103385//Department of Education and Training | Australian Research Council (ARC)/ ; CE230100015//Department of Education and Training | Australian Research Council (ARC)/ ; GNT2035042//Department of Health | National Health and Medical Research Council (NHMRC)/ ; },
mesh = {Protoplasts/metabolism ; Arabidopsis/genetics ; *Gene Regulatory Networks ; *Genes, Synthetic ; *CRISPR-Cas Systems ; Triticum/genetics ; *Plants/genetics ; Brassica napus/genetics ; Bryopsida/genetics ; },
abstract = {Synthetic gene circuits are powerful tools for precisely programming gene expression and introducing novel cellular functions. However, their development and application in plants has lagged behind other systems, due mainly to the limited availability of modular genetic parts. We recently developed a CRISPR interference (CRISPRi)-based synthetic gene circuit system for programming gene expression in plants. Using a robust and high-throughput protoplast-based dual luciferase assay, we demonstrated the development, testing and functionality of these circuits in various plant species. Here we detail the key design principles and considerations for building and testing programmable and reversible CRISPRi-based gene circuits in plants. We also provide detailed procedures for isolating protoplasts from multiple plant species, including Arabidopsis thaliana, Brassica napus, Triticum aestivum and Physcomitrium patens. Furthermore, we provide step-by-step instructions for the 96-well plate-based protoplast transfection assay for testing genetic parts and synthetic circuits, using a dual luciferase assay. The detailed descriptions of these developed systems will enhance the efficiency and reproducibility of the construction, testing, and implementation of synthetic gene circuits in a variety of plant species. This protocol enables the design and testing of CRISPRi-based gene circuits in plants within ~4 weeks.},
}

Protoplasts/metabolism
Arabidopsis/genetics
*Gene Regulatory Networks
*Genes, Synthetic
*CRISPR-Cas Systems
Triticum/genetics
*Plants/genetics
Brassica napus/genetics
Bryopsida/genetics

@article {pmid41933972,
year = {2026},
author = {Li, Z and Wang, X and Liu, J and Janssen, JM and Hoeben, RC and Gonçalves, MAFV},
title = {Selector adeno-associated viral vectors facilitate on-target precise genome editing and purge off-target chromosomal insertions.},
journal = {Trends in biotechnology},
volume = {44},
number = {8},
pages = {2422-2445},
doi = {10.1016/j.tibtech.2026.01.006},
pmid = {41933972},
issn = {1879-3096},
mesh = {*Dependovirus/genetics ; *Gene Editing/methods ; *Genetic Vectors/genetics ; Humans ; CRISPR-Cas Systems ; },
abstract = {Adeno-associated viral (AAV) vectors are commonly used for genome editing owing to the proclivity with which their single-stranded genomes serve as homologous recombination (donor) substrates during programmable nuclease-assisted gene targeting. However, the highly recombinogenic nature of AAV genomes also facilitates their nonhomologous end joining at off-target chromosomal breaks ('capture') created by said nucleases, mutagens, or DNA metabolic processes. Moreover, AAV donor constructs can equally yield imprecise on-target edits resulting from end-joining recombination pathways. Here, we demonstrate that endowing AAV vectors with exogenous marker-free selectable sequences permits enrichment for cells precisely coedited at endogenous target and ATP1A1 alleles. These selector AAV vectors install ATP1A1 polymorphisms conferring resistance to the small molecule ouabain, yielding high frequencies of on-target and precisely edited cell populations. Crucially, we further report that selector AAV vectors achieve a thorough removal of heterogeneous off-target DNA species resulting from conventional AAV-based genome editing procedures.},
}

*Dependovirus/genetics
*Gene Editing/methods
*Genetic Vectors/genetics
Humans
CRISPR-Cas Systems

@article {pmid42035261,
year = {2026},
author = {Tan, K and Del Bosque Siller, D and Xiong, AY and Wang, AXA and McCallister, TX and Mummadi, S and St John, LA and Lee, TK and Carrillo, AN and Renshaw, DG and Zhou, RH and Lim, CKW and He, J and Fields, CJ and Hayden, MR and Gaj, T},
title = {Treatment of Huntington's disease with a pan-HTT-targeting CRISPR nuclease.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {8},
pages = {4635-4655},
pmid = {42035261},
issn = {1525-0024},
support = {R01 GM141296/GM/NIGMS NIH HHS/United States ; R01 NS123556/NS/NINDS NIH HHS/United States ; U01 NS122102/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; *Huntington Disease/therapy/genetics/metabolism/pathology ; *Huntingtin Protein/genetics/metabolism ; Mice ; Disease Models, Animal ; *CRISPR-Cas Systems ; Humans ; Dependovirus/genetics ; *Genetic Therapy/methods ; Genetic Vectors/genetics/administration & dosage ; Gene Editing ; Trinucleotide Repeat Expansion ; Gene Targeting ; Neurons/metabolism ; },
abstract = {Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an expansion of a CAG trinucleotide repeat in the huntingtin (HTT) gene, which leads to a mutant protein that destroys neurons in the brain. Despite intense effort, there remains no approved disease-modifying therapy for HD. Here, we develop a pan-HTT-targeting CRISPR-Cas9 system that, when delivered to the striatum of R6/2 and YAC128 mice by adeno-associated virus serotype 5 (AAV5), lowered mutant HTT mRNA and protein by 55%-80% via its induction of frameshift-inducing insertion or deletion (indel) mutations in HTT exon 1. Cas9 targeting improved motor coordination and locomotor activity, decreased anxiety-like deficits, reduced clasping and weight loss, limited striatal atrophy, and decreased the formation of intranuclear inclusions immunoreactive for the mutant HTT protein. In Hu21/21 mice, which carry the wild-type human HTT gene in lieu of the mouse ortholog, Cas9 lowered the HTT protein by 44% but induced no measurable behavioral deficits and had no adverse effect on neuronal viability, though its targeting was associated with neuroinflammation. Altogether, our results demonstrate the ability of a newly developed pan-HTT-targeting Cas9 system to affect HD-related phenotypes across models and provide insights into its tolerability.},
}

Animals
*Huntington Disease/therapy/genetics/metabolism/pathology
*Huntingtin Protein/genetics/metabolism
Mice
Disease Models, Animal
*CRISPR-Cas Systems
Humans
Dependovirus/genetics
*Genetic Therapy/methods
Genetic Vectors/genetics/administration & dosage
Gene Editing
Trinucleotide Repeat Expansion
Gene Targeting
Neurons/metabolism

@article {pmid42231480,
year = {2026},
author = {Xu, L and Liang, H and Bai, S and Xu, R and Liu, B and Xu, X and Xu, X and Liu, M},
title = {Protein arginine methyltransferase 5 is essential for virulence in Toxoplasma gondii.},
journal = {Parasites & vectors},
volume = {19},
number = {1},
pages = {},
pmid = {42231480},
issn = {1756-3305},
support = {2021A1515011707//Guangdong Provincial Natural Science Foundation/ ; },
mesh = {*Toxoplasma/pathogenicity/genetics/enzymology ; *Protein-Arginine N-Methyltransferases/genetics/metabolism ; Animals ; Virulence ; Mice ; Mice, Inbred BALB C ; Arginine/analogs & derivatives/metabolism ; *Protozoan Proteins/genetics/metabolism ; Female ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Protein arginine methyltransferase 5 (PRMT5) is a key enzyme responsible for catalyzing symmetric dimethylarginine (SDMA) modifications and plays crucial roles in epigenetic regulation, transcription, and cell cycle progression in eukaryotes. Although our previous study determined the expression and cellular localization of PRMT5 in tachyzoites and bradyzoites, and confirmed its type II PRMT activity, its functional significance in Toxoplasma gondii remains entirely uncharacterized.

METHODS: This study aimed to explore the biological functions of PRMT5 in T. gondii. The prmt5 gene was disrupted in the type I RH strain using the clustered regularly interspaced short palindromic repeats (CRISPR) Cas9 system. The biological roles of PRMT5 were evaluated via multiple functional assays, including plaque formation, intracellular proliferation, host cell invasion, virulence, and tachyzoite to bradyzoite conversion assays. RNA sequencing was further performed to profile transcriptomic alterations induced by prmt5 disruption.

RESULTS: Phenotypic characterization revealed that the ∆prmt5 strain exhibited reduced symmetric dimethylarginine (SDMA) levels as well as severe defects in plaque formation, invasion, intracellular replication, and bradyzoite differentiation. Accordingly, the virulence of the ∆prmt5 strain was dramatically attenuated, as all infected BALB/c mice survived over a 10-day period, in stark contrast to the 100% mortality observed in the wild-type control group within 10 days. RNA-sequencing analysis uncovered the molecular basis for these phenotypes, demonstrating that prmt5 disruption leads to global transcriptional dysregulation. Specifically, we identified a significant downregulation of genes associated with motor protein function and fatty acid metabolism pathways.

CONCLUSIONS: Our research has demonstrated that PRMT5 plays a critical role in the proliferation, survival, pathogenicity, and regulation of gene expression in Toxoplasma gondii.},
}

*Toxoplasma/pathogenicity/genetics/enzymology
*Protein-Arginine N-Methyltransferases/genetics/metabolism
Animals
Virulence
Mice
Mice, Inbred BALB C
Arginine/analogs & derivatives/metabolism
*Protozoan Proteins/genetics/metabolism
Female
CRISPR-Cas Systems

@article {pmid42259916,
year = {2026},
author = {Zeng, J and Cheng, Z and Chen, H and Wang, Z and Thompson, J and Crosby, KT and Han, H and Singhal, A and Ngo, W and Xia, C and Rosas-Rivera, D and Zhang, Z and Kang, MH and Mao, Y and Diolaiti, ME and Lee, GC and Diffley, JFX and Song, Y and Qiu, L and Krah, NM and Murthy, N and Jackson, RN and Liu, Y and Ashworth, A and Doudna, JA},
title = {Targeting cancer-specific mutations with RNA-triggered chromatin shredding.},
journal = {Nature},
volume = {656},
number = {8126},
pages = {199-206},
pmid = {42259916},
issn = {1476-4687},
mesh = {Humans ; *Chromatin/metabolism/genetics ; *Mutation/genetics ; *Neoplasms/genetics/pathology/therapy ; CRISPR-Cas Systems/genetics ; DNA Damage ; Animals ; Cell Line, Tumor ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; },
abstract = {Genetic mutations that drive cancer often occur in tumour-suppressor proteins such as the p53 transcription factor, which is altered in 40-50% of cases[1,2]. However, current therapies often fail to target these mutations because the mutant proteins typically lack defined drug-binding pockets and restoring their endogenous function has proven challenging. Here we program Cas12a2, an RNA-guided CRISPR nuclease with trans-nucleolytic cleavage activity[3,4], to kill cancer cells selectively by targeting cancer-specific transcripts. This approach limited cell growth by inducing trans shredding of chromatin and triggering DNA-damage responses and cell death. In contrast to existing methods, RNA-guided Cas12a2 senses cellular RNA signatures, enabling precise targeting of undruggable mutations. Transcript-activated chromatin shredding provides an innovative approach to precision disease treatments for undruggable targets.},
}

Humans
*Chromatin/metabolism/genetics
*Mutation/genetics
*Neoplasms/genetics/pathology/therapy
CRISPR-Cas Systems/genetics
DNA Damage
Animals
Cell Line, Tumor
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism

@article {pmid42331809,
year = {2026},
author = {Zhou, C and Dong, C and Zhao, W and Liang, FS},
title = {Hierarchical interplay between H3K27ac and H3K4me3 in transcriptional regulation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42331809},
issn = {2041-1723},
support = {R01GM143256//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
mesh = {*Histones/metabolism/genetics ; *Transcription, Genetic ; RNA Polymerase II/metabolism ; Epigenome Editing ; Humans ; Promoter Regions, Genetic ; *Gene Expression Regulation ; CRISPR-Cas Systems ; Chromatin/metabolism ; RNA Stability ; Animals ; },
abstract = {H3K27ac and H3K4me3 are enriched at transcriptional start sites and have been implicated in transcription. However, how these marks concertedly regulate transcription is not fully understood. Here, we develop a dual chemically inducible CRISPR/dCas9-based epigenome editing system that enables independent, temporal and transcription stage-specific modulation of H3K27ac and H3K4me3 at a specific gene locus. Stage-specific removal of H3K4me3 impairs RNA polymerase II recruitment, increases promoter-proximal pausing, reduces productive elongation, and accelerates mRNA decay via increased m[6]A deposition. Losing both H3K27ac and H3K4me3 rapidly abolishes transcriptional activity, while preserving H3K4me3 without H3K27ac can partially sustain transcription. These findings reveal a functional hierarchy and interdependence between H3K27ac and H3K4me3 in different transcription stages at the tested gene loci. This versatile tool will contribute to the functional dissection of the temporal dynamics of chromatin modifications in gene regulation.},
}

*Histones/metabolism/genetics
*Transcription, Genetic
RNA Polymerase II/metabolism
Epigenome Editing
Humans
Promoter Regions, Genetic
*Gene Expression Regulation
CRISPR-Cas Systems
Chromatin/metabolism
RNA Stability
Animals

@article {pmid42358199,
year = {2026},
author = {Gijsbertsen, M and Duarte, FM and Fuentes Manjón, A and Maduro, T and Kassem, M and van der Oost, J and Mathijssen, IMJ and van Leeuwen, JPTM and van de Peppel, J},
title = {Evaluation of Prime Editing Efficiency in Human Immortalized MSC-TERT Cells with Osteogenic Potential for Modeling FGFR2-Linked Craniosynostosis.},
journal = {The CRISPR journal},
volume = {9},
number = {4},
pages = {191-206},
doi = {10.1177/25731599261457618},
pmid = {42358199},
issn = {2573-1602},
mesh = {Humans ; *Mesenchymal Stem Cells/metabolism/cytology ; *Receptor, Fibroblast Growth Factor, Type 2/genetics ; *Osteogenesis/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems ; HEK293 Cells ; *Craniosynostoses/genetics ; Telomerase/genetics/metabolism ; Mutation ; Cell Line ; },
abstract = {Craniosynostosis is a rare congenital bone condition where skull sutures fuse prematurely and is linked to mutations in over 60 genes. Generating mutation-specific in vitro models allows investigation of craniosynostosis-associated mutations without the need for patient-derived material or transgenic gene expression. Here, we developed a human in vitro disease model with the CRISPR-Cas9 prime editing variant, using an immortalized TERT-immortalized mesenchymal bone marrow-derived stem (MSC-TERT) cell line with osteogenic potential. MSC-TERT cells showed a higher resistance to prime editing, compared with HEK293FT cells. Addition of dnMLH1 and epegRNAs resulted in higher editing efficiencies in HEK293FT cells, but not in MSC-TERT cells. Prime editing efficiency varied between targeted loci and was found to be more efficient in nonadherent cells compared with adherent cells. Prime editing continued over 4 days in an isolated nonadherent HEK293FT culture. Our results present a foundation on the use of prime editing to establish FGFR2 mutation-specific in vitro models and their application in MSC-TERT cells.},
}

Humans
*Mesenchymal Stem Cells/metabolism/cytology
*Receptor, Fibroblast Growth Factor, Type 2/genetics
*Osteogenesis/genetics
*Gene Editing/methods
CRISPR-Cas Systems
HEK293 Cells
*Craniosynostoses/genetics
Telomerase/genetics/metabolism
Mutation
Cell Line

@article {pmid42413621,
year = {2026},
author = {Nan, Y and Yan, S and Zhang, L and Yang, F and Qiao, M and Xin, Y},
title = {Markerless large DNA integration in Lactococcus lactis through the coupling of homologous single-crossover and Cre/loxP system.},
journal = {Journal of biotechnology},
volume = {418},
number = {},
pages = {60-68},
doi = {10.1016/j.jbiotec.2026.07.004},
pmid = {42413621},
issn = {1873-4863},
mesh = {*Lactococcus lactis/genetics ; Plasmids/genetics ; *Integrases/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Lactococcus lactis is widely used in food fermentation and has great potential as a microbial cell factory for producing high-value compounds. Currently, heterologous gene expression in this host mainly relies on plasmid-based systems, which suffer from segregational instability without antibiotic selection. To address this problem, several genome integration tools have been developed, yet most are limited to single gene insertions or leave selection markers. In this study, we developed an efficient and markerless platform for integration single genes and large DNA fragments combining a temperature-sensitive plasmid for homologous single-crossover and the Cre/loxP system for plasmid backbone excision. First, using CRISPR/Cas9 assisted ssDNA recombineering, we introduced a loxP site into the ribB gene of L. lactis NZ9000/RecT, creating the chassis strain L. lactis loxP. We then constructed a donor plasmid, pG15AribB-Up-Cat-loxP, which carries a homology arm of ribB, the chloramphenicol resistance gene cat, and a second loxP site. This donor plasmid was integrated into the chromosome next to the existing loxP site through a single-crossover event. Subsequent expression of Cre recombinase then removed the plasmid backbone, leaving only the target gene cat at the insertion site. After 99 generations without chloramphenicol selection, the integrant L. lactis IMT maintained nearly 100% genetic stability. Using this method, we next successfully markerless integrated the ∼4.8 kb crtEBI cluster, and the seven gene tagatose-6-phosphate pathway (∼7.0 kb). This work provides an efficient platform for markerless and stable large DNA integration in L. lactis for constructing microbial cell factory to produce high-value compounds.},
}

*Lactococcus lactis/genetics
Plasmids/genetics
*Integrases/genetics/metabolism
CRISPR-Cas Systems

@article {pmid42456866,
year = {2026},
author = {Qu, ZL and Liu, T and Qin, TK and Yue, HM and Huang, L and Ruan, R and Wu, CS and Ye, H and Li, CJ},
title = {Establishment of a novel brain cell line from grass carp (Ctenopharyngodon idella) with high transfection efficiency and CRISPR/Cas9-mediated genome editing capacity.},
journal = {Fish & shellfish immunology},
volume = {177},
number = {},
pages = {111597},
doi = {10.1016/j.fsi.2026.111597},
pmid = {42456866},
issn = {1095-9947},
mesh = {Animals ; *Carps/genetics/immunology ; Cell Line ; Transfection/veterinary ; *CRISPR-Cas Systems ; *Brain/cytology ; *Gene Editing/veterinary ; Reoviridae/physiology ; Fish Diseases/immunology/virology ; Reoviridae Infections/veterinary/immunology ; },
abstract = {Fish cell lines serve as valuable tools in aquaculture research, particularly in immunology, pathology, and toxicology. In this study, we successfully established a novel cell line derived from brain tissue of grass carp, designated CIB. This cell line has been subcultured over 100 times and exhibits a fibroblast-like morphology. Chromosomal analysis revealed that the diploid chromosome number of CIB cells is 2n = 48, while sequencing of the 18S rRNA gene confirmed the cell line's origin. Notably, CIB cells demonstrated a transfection efficiency of 62.4% with pEGFP-N3, highlighting their potential for studies involving exogenous gene expression. Furthermore, CIB cells were susceptible to grass carp reovirus genotype I (GCRV-I), as evidenced by cytopathic effects (CPE), increased synthesis of viral proteins, and accumulation of viral particles within the cells. Both viral infection and poly(I:C) stimulation significantly increased the expression of intracellular interferon-related signaling molecules. Additionally, electroporation of a gRNA-Cas9 ribonucleoprotein (RNP) complex into CIB cells achieved the first successful large-fragment gene knockout in cultured grass carp cells and generated a homozygous clonal line, thereby enhancing the antiviral response. In summary, this novel cell line represents a significant advancement for studying gene functions, host-virus interactions, and genetic engineering in teleost fish.},
}

Animals
*Carps/genetics/immunology
Cell Line
Transfection/veterinary
*CRISPR-Cas Systems
*Brain/cytology
*Gene Editing/veterinary
Reoviridae/physiology
Fish Diseases/immunology/virology
Reoviridae Infections/veterinary/immunology

@article {pmid42460742,
year = {2026},
author = {Lv, B and Chen, Y and Zhou, R and Liu, R and Li, D},
title = {DNAzyme-mediated synergistic activation of CRISPR/Cas12a for Cd[2+] and Pb[2+] biosensing.},
journal = {Chemical communications (Cambridge, England)},
volume = {62},
number = {61},
pages = {15252-15256},
doi = {10.1039/d6cc04053e},
pmid = {42460742},
issn = {1364-548X},
mesh = {*Lead/analysis ; *DNA, Catalytic/metabolism/chemistry ; *Cadmium/analysis ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *CRISPR-Associated Proteins/metabolism ; },
abstract = {This work reports an isothermal, one-pot assay for Cd[2+] and Pb[2+] based on DNAzyme-triggered synergistic activation of CRISPR/Cas12a.},
}

*Lead/analysis
*DNA, Catalytic/metabolism/chemistry
*Cadmium/analysis
*CRISPR-Cas Systems
*Biosensing Techniques/methods
*CRISPR-Associated Proteins/metabolism

@article {pmid42479891,
year = {2026},
author = {Feng, H and Wang, Y and Zhao, J and Fan, H and Wu, T and Qu, J and Pan, S and Wang, J and Han, Z and Liang, A and Xu, T and Chen, C and Hu, C and Hu, T},
title = {Topology-Gated λ Exonuclease Enables Amplification-Free Signal Boosting.},
journal = {Journal of the American Chemical Society},
volume = {148},
number = {30},
pages = {32782-32792},
doi = {10.1021/jacs.6c11250},
pmid = {42479891},
issn = {1520-5126},
support = {23ZR1450300//Natural Science Foundation of Shanghai Municipality/ ; 23-0178-0002//National University of Singapore/ ; A-8002032-00//Ministry of Education - Singapore/ ; 22304061//National Natural Science Foundation of China/ ; 22304157//National Natural Science Foundation of China/ ; LQ23H200005//Natural Science Foundation of Zhejiang Province/ ; ZR2023MB019//Natural Science Foundation of Shandong Province/ ; 24-0994-P0001//National Centre for Infectious Disease/ ; },
mesh = {*Exodeoxyribonucleases/metabolism/chemistry ; CRISPR-Cas Systems ; *RNA, Viral/analysis ; *Exonucleases/metabolism/chemistry ; Humans ; *Bacteriophage lambda/enzymology ; MicroRNAs/analysis ; Viral Proteins ; },
abstract = {Amplification-free detection remains a fundamental challenge in CRISPR-based RNA diagnostics. Here, we identify a previously unrecognized topological property of λ exonuclease, whereby duplex substrates bearing 5' phosphates at both termini undergo a self-sustained cyclic cleavage-reforming process. This topology-gated behavior enables signal renewal without external amplification. Through systematic biochemical and structural analyses, we elucidate the underlying mechanism and establish λ exonuclease as a topology-driven signal amplifier. Then, we design a topology-gated dumbbell probe that sequesters 5' phosphates within dual RNA hairpin loops. Upon target recognition, CRISPR/Cas13 specifically cleaves the loops, exposing the hidden phosphates and thereby activating the λ exonuclease-mediated cyclic reaction. The resulting cascade, termed Topo-CRISPR (Topology-gated λ exonuclease enables CRISPR amplification-free), achieves attomolar sensitivity within 25 min without preamplification. Applied to clinical samples, Topo-CRISPR enables robust and specific detection of enterovirus RNA, miR-21, and ciR1445, demonstrating performance comparable to RT-qPCR. We further extend the Topo-CRISPR to non-nucleic-acid targets via aptamer-mediated conformational gating. This work uncovers a previously overlooked enzymatic topology, positioning λ exonuclease as a cyclic signal transducer and offering a general framework for ultrasensitive molecular sensing.},
}

*Exodeoxyribonucleases/metabolism/chemistry
CRISPR-Cas Systems
*RNA, Viral/analysis
*Exonucleases/metabolism/chemistry
Humans
*Bacteriophage lambda/enzymology
MicroRNAs/analysis
Viral Proteins

@article {pmid42487445,
year = {2026},
author = {Collantes, JC and Xu, K and Ruiz-Urigüen, M and Samaniego-Villacis, AC and Stombaugh, J and Jin, S},
title = {Development and Characterization of RNA Aptamer-Mediated Modular Base Editors Containing Staphylococcus aureus Cas9 Derivatives and Novel Deaminase Orthologs.},
journal = {The CRISPR journal},
volume = {9},
number = {4},
pages = {207-222},
doi = {10.1177/25731599261467606},
pmid = {42487445},
issn = {2573-1602},
mesh = {*Staphylococcus aureus/genetics/enzymology ; *Gene Editing/methods ; Humans ; *CRISPR-Associated Protein 9/genetics/metabolism ; Animals ; *Aptamers, Nucleotide/genetics ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; Cytidine Deaminase/genetics ; APOBEC-1 Deaminase/genetics ; HEK293 Cells ; Rats ; },
abstract = {Base editing enables precise genome modifications without introducing DNA double-strand breaks. Using Streptococcus pyogenes Cas9 as a prototype, we previously developed a modular base editing platform in which the deaminase is recruited by an RNA aptamer engineered into the gRNA, thereby separating sequence recognition from base modification. Here, we expanded this modular base editor toolbox by engineering Staphylococcus aureus Cas9 (SaCas9) in combination with various vertebrate effectors derived from activation induced cytidine deaminase (AID) and apolipoprotein B mRNA editing enzyme, catalytic subunit 1 (APOBEC1) orthologs, from bat, lizard, human, and rat. Moreover, we adopted the SaCas9 variants with different protospacer adjacent motif requirements. These base editors generally showed high editing efficiency with low on-target indel formation and low-to-undetectable off-target activities. Quantitative and qualitative differences in editing occur among the base editors when applied to diverse loci, allowing sequence-specific optimization. Together, our study demonstrates the effectiveness of the SaCas9 modular base editors, the robustness of the platform's modularity, and its feasibility for convenient screening of target-specific base editors.},
}

*Staphylococcus aureus/genetics/enzymology
*Gene Editing/methods
Humans
*CRISPR-Associated Protein 9/genetics/metabolism
Animals
*Aptamers, Nucleotide/genetics
CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
Cytidine Deaminase/genetics
APOBEC-1 Deaminase/genetics
HEK293 Cells
Rats

@article {pmid42488965,
year = {2026},
author = {Shepard, A and Minones-Moyano, E and Mork, C and Pyhtila, B and Barrangou, R},
title = {The Diversifying Distribution Trends of Maturing CRISPR Technologies by Addgene.},
journal = {The CRISPR journal},
volume = {9},
number = {4},
pages = {184-190},
doi = {10.1177/25731599261470096},
pmid = {42488965},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods/trends ; *Plasmids/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Animals ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; },
abstract = {Since the advent of Cas9-based CRISPR technologies in 2012, there has been a remarkable growth in genome editing research, literature, applications, and translational impact. Much of this research has been fueled by the global dissemination of CRISPR plasmids through nonprofit distribution by Addgene, as both a repository and distributor of enabling biological material. Recently, key milestones have been reached, with over 20,000 plasmids deposited by over 1,000 labs, being distributed over 300,000 times globally. The driving trends reflect multidimensional diversification in terms of effectors (Cas9, Cas12 and beyond), editing modalities (base editing, prime editing, epigenetic modification, CRISPRi/a), and deployment across phylogenetic groups (mammalian, bacterial, plant, yeast, insects, and more). Noteworthy, guide RNA and HDR templates account for the bulk of deposits, while cloning backbones are the most requested, and lentiviral plasmids comprise the majority of expression material. The data reflect a continued diversification of the CRISPR-based toolbox, robust interest in genome editing applications across the tree of life, maturation in terms of adoption, and rising relative distribution beyond the USA and China, with Addgene continuing to play a critical role in access to equitable and disruptive technologies.},
}

*CRISPR-Cas Systems
*Gene Editing/methods/trends
*Plasmids/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
Animals
RNA, Guide, CRISPR-Cas Systems/genetics
Humans

@article {pmid42525182,
year = {2026},
author = {Fang, G and Zheng, S and Miao, J and Zeng, Y and Peng, Y and Zhai, Y and Guo, H and Wang, J and Dong, Y and Tian, X and Liu, M and Liu, Y and Li, X and Wang, M and Zhao, X and Du, J and Wang, Y and Dong, J},
title = {The ApoE-Null Golden Hamster: A Novel Model of Atherosclerosis.},
journal = {Cardiovascular toxicology},
volume = {26},
number = {8},
pages = {},
pmid = {42525182},
issn = {1559-0259},
mesh = {Animals ; *Atherosclerosis/pathology/genetics/metabolism/blood ; Disease Models, Animal ; *Apolipoproteins E/genetics/deficiency ; Mesocricetus ; Diet, High-Fat ; Plaque, Atherosclerotic ; *Aortic Diseases/pathology/genetics/metabolism ; Cholesterol, Dietary ; CRISPR-Cas Systems ; *Aorta/pathology/metabolism ; Male ; Phenotype ; Genetic Predisposition to Disease ; Lipids/blood ; Liver/pathology/metabolism ; Hyperlipidemias/genetics/pathology ; Animals, Genetically Modified ; Fibrosis ; },
abstract = {Atherosclerosis is a chronic, progressive arterial disease characterized by the deposition of lipids on the inner arterial walls, leading to plaque formation and serious cardiovascular events. Traditional mouse models of atherosclerosis require prolonged dietary induction to exhibit arterial lesions due to significant differences in lipid metabolism compared to humans. In contrast, Golden hamsters share a lipid metabolic profile more closely aligned with humans. In this study, we utilized CRISPR/Cas9 to generate ApoE knockout (ApoE[-/-]) hamsters using, which spontaneously developed atherosclerotic lesions in the arterial wall after 8 weeks on a standard chow diet. When fed on a high-cholesterol/high-fat diet, they exhibited even more severe aortic atherosclerosis, fatty liver, and liver fibrosis. Our findings demonstrated that the ApoE[-/-] hamster model is highly valuable tool for translational research, offering significant potential for studying hyperlipidemia and atherosclerosis in a context more relevant to human physiology.},
}

Animals
*Atherosclerosis/pathology/genetics/metabolism/blood
Disease Models, Animal
*Apolipoproteins E/genetics/deficiency
Mesocricetus
Diet, High-Fat
Plaque, Atherosclerotic
*Aortic Diseases/pathology/genetics/metabolism
Cholesterol, Dietary
CRISPR-Cas Systems
*Aorta/pathology/metabolism
Male
Phenotype
Genetic Predisposition to Disease
Lipids/blood
Liver/pathology/metabolism
Hyperlipidemias/genetics/pathology
Animals, Genetically Modified
Fibrosis

@article {pmid42525385,
year = {2026},
author = {Kim, GD and Gu, D and Park, M and Wook Chi, S},
title = {abCRISPR: deep learning-based design of abasic gRNA sequences for specific CRISPR-Cas genome editing.},
journal = {Bioinformatics (Oxford, England)},
volume = {},
number = {},
pages = {},
doi = {10.1093/bioinformatics/btag568},
pmid = {42525385},
issn = {1367-4811},
abstract = {SUMMARY: CRISPR-Cas9 has become a widely used tool for genome editing. However, its off-target cleavage caused by partial sequence matches with guide RNAs (gRNAs) remains a critical limitation. Recently, abasic gRNAs (ØXØ) have been developed to enhance target specificity, but their effects vary depending on the positional sequence context. Here, we present abCRISPR, a deep neural network (DNN) framework for the rational design of ØXØ sequences with minimized off-target activity. abCRISPR leverages informative few-shot training with paired datasets of abasic and unmodified gRNAs, using high-quality random mismatch target libraries, exhaustively sequenced for mismatched off-target substrates (n = 97,583) in in vitro CRISPR-Cas9 cleavage experiments. Predicted off-target activities for both abasic and unmodified gRNAs showed strong correlation with experimental data (r ≥ 0.95, 10-fold cross-validation). Notably, these comprehensive training sets provide robust ground-truth negatives, enabling accurate and sensitive prediction of off-targets. For unmodified gRNAs, abCRISPR (AUC = 0.98) was validated to outperform existing deep learning-based methods (AUC = 0.45-0.68). When applied to the human genome, abCRISPR generated ØXØ sequences, covering 58,875,004 potent CRISPR-targetable sites with improved target specificity. Together, this work provides a comprehensive bioinformatics resource for safe and precise CRISPR-Cas9 genome editing.

The source code for abCRISPR and training data are available at https://doi.org/10.5281/zenodo.20398246. abCRISPR results for the human genome are available at http://clip.korea.ac.kr/abCRISPR/.

SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.},
}

@article {pmid42528148,
year = {2026},
author = {du Plessis, J and Omar, A},
title = {Patient-Derived Organoid-Based CRISPR Screens in Cancer Research: Applications, Advances, and Challenges.},
journal = {Cancer medicine},
volume = {15},
number = {8},
pages = {e72112},
pmid = {42528148},
issn = {2045-7634},
mesh = {Humans ; *Organoids/metabolism ; *Neoplasms/genetics/pathology ; *CRISPR-Cas Systems ; Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genomics/methods ; Animals ; Precision Medicine/methods ; },
abstract = {Patient-derived organoids (PDOs) have emerged as physiologically relevant cancer models that preserve key genetic, histological, and functional features of the tumors from which they are derived. In parallel, CRISPR-based perturbation technologies have transformed functional genomics by enabling scalable interrogation of gene function. Their integration provides a powerful framework for identifying cancer dependencies, modeling oncogenic evolution, and investigating mechanisms of drug response and resistance in patient-relevant settings. This review examines how CRISPR knockout, CRISPR interference/activation, and precision editing approaches have been applied in PDO systems to uncover context-specific vulnerabilities, reconstruct mutational trajectories, and study tumor heterogeneity. We further compare pooled and arrayed screening formats and discuss what is uniquely enabled by performing CRISPR screens in organoids rather than conventional 2D models. Particular emphasis is placed on the technical and analytical constraints of organoid-based screening, including variable editing efficiency, clonal bottlenecks, biological heterogeneity, and limited scalability. We argue that the major value of organoid-based CRISPR screening lies in its ability to identify functionally actionable cancer vulnerabilities in a patient-contextualized model, while also introducing methodological challenges that must be addressed for robust clinical translation.},
}

Humans
*Organoids/metabolism
*Neoplasms/genetics/pathology
*CRISPR-Cas Systems
Gene Editing/methods
*Clustered Regularly Interspaced Short Palindromic Repeats
Genomics/methods
Animals
Precision Medicine/methods

@article {pmid42529880,
year = {2026},
author = {Zhang, W and Hang, Y and Zhan, S and Song, W and Li, B and Chen, N and Lv, M},
title = {Emerging point-of-care technologies for bacterial pathogen detection.},
journal = {Journal of Zhejiang University. Science. B},
volume = {27},
number = {7},
pages = {677-697},
pmid = {42529880},
issn = {1862-1783},
support = {32371439 and 22404026//the National Natural Science Foundation of China/ ; 24ZR1455600 and 24ZR1455900//the Natural Science Foundation of Shanghai/ ; },
mesh = {Humans ; *Point-of-Care Systems ; *Bacteria/isolation & purification/genetics ; *Point-of-Care Testing ; *Bacterial Infections/diagnosis/microbiology ; Rapid Diagnostic Tests ; Immunoassay ; Nucleic Acid Amplification Techniques ; CRISPR-Cas Systems ; },
abstract = {Bacterial infections remain a significant threat to public health worldwide, driving an urgent need for rapid, accurate, and field-deployable diagnostic techniques. Point-of-care testing (POCT) has emerged as a transformative strategy, providing timely detection, operational simplicity, and portability. Recent studies have aimed at enhancing sensitivity, specificity, multiplexing capability, and automation through the integration of molecular diagnostics with microfluidics and lab-on-chip technologies, alongside the development of low-cost, portable devices equipped with smartphone-based readout and cloud connectivity for real-time surveillance in resource-limited settings. Nonetheless, evidence-based frameworks for selecting optimal detection targets-such as genomic sequences, conserved protein epitopes, or viable whole cells-and matching them to appropriate POCT modalities remain notably underrepresented in the literature. This review systematically summarizes recent advances in POCT strategies for bacterial detection, categorized according to three major types of detection targets, including cellular phenotypic characteristics, surface antigens, and nucleic acids. We discuss the principles, advantages, limitations, and representative applications of key POCT platforms, which include microscopy-based visualization, immunoassays, isothermal amplification, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) systems, and microfluidic biosensors. Critical challenges, such as sample pretreatment, detection sensitivity, and operational simplicity, have been partially addressed through recent innovations. Finally, we outline the main future research directions focused on the development of integrated, automated, and intelligent POCT systems for clinical deployment.},
}

Humans
*Point-of-Care Systems
*Bacteria/isolation & purification/genetics
*Point-of-Care Testing
*Bacterial Infections/diagnosis/microbiology
Rapid Diagnostic Tests
Immunoassay
Nucleic Acid Amplification Techniques
CRISPR-Cas Systems

@article {pmid42530034,
year = {2026},
author = {Sharma, N and Tanwar, D and Grewal, U and Keerthi, MM and Ahlawat, YK and Pandey, N and Makvana, CG},
title = {Pectinase-Based Bioprocesses for Circular Bioeconomy and Sustainable Industrial Development.},
journal = {Biotechnology and bioengineering},
volume = {},
number = {},
pages = {},
doi = {10.1002/bit.70312},
pmid = {42530034},
issn = {1097-0290},
abstract = {Pectinases play a vital role in the degradation of pectic part of the plant cell wall and are considered in the group of hydrolytic enzymes. In the present scenario, demand of economically feasible and environment friendly techniques and approaches has significantly led the research on the microbial production, standardization of process parameters and improvements of pectinases. The development of pectinase-based bioprocesses has been found to promote sustainable industrial practices by reducing the use of chemicals, energy, and waste, thereby supporting eco-friendly production systems. The unique aspect of this review is that it explains traditional methods of pectinase production alongside novel methods, which include solid-state and submerged fermentation, recombinant DNA techniques, heterologous gene expression, protein engineering, CRISPR/Cas genome editing technologies, enzyme immobilization, and nanobiotechnological methods, which improve the production, stability, and performance of pectinases. It further demonstrates the increasing importance of the use of pectinases in sustainable manufacturing through decreasing the use of chemicals and energy, as well as reducing waste production from industry. It illustrates that innovations in microbial strain engineering, process optimization, and utilization of low-cost substrates have significantly increased the economic viability and efficiency of producing pectinases. Yet, some issues associated with commercialization, standardization of production processes, recovery, and stability of enzymes still exist. Overall, this review paper gives an overview of the recent advancements and limitations of next-generation pectinases.},
}

@article {pmid42531075,
year = {2026},
author = {Zhou, R and Zhan, Y and Sun, Y and Wang, R and Wang, T and Liu, Z and Shan, Z and Li, X and Zhang, S and Sun, N and Zhang, H and Yuan, Z and Yang, J},
title = {Molecular basis of single-mismatch-induced nuclease-to-nickase conversion in TIGR-TasH.},
journal = {Nucleic acids research},
volume = {54},
number = {14},
pages = {},
pmid = {42531075},
issn = {1362-4962},
support = {2024ZD037//Scientific Research Program of Tianjin Municipal Education Commission/ ; TJYXZDXK-009A//Tianjin Key Medical Discipline/ ; TJYXZDXK-3-004B//Tianjin Key Medical Discipline/ ; 2024ZD037//Scientific Research Program of Tianjin Municipal Education Commission/ ; },
mesh = {Cryoelectron Microscopy ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry ; *Base Pair Mismatch ; *Deoxyribonuclease I/metabolism/genetics/chemistry ; Protein Domains ; Models, Molecular ; },
abstract = {Tandem interspaced guide RNA (TIGR)-Tas systems are a distinct class of RNA-guided double-stranded DNA nucleases that employ dual-spacer guide RNAs (tigRNAs) for PAM-independent target recognition. A single mismatch between the tigRNA and target DNA can convert Salicola phage CGphi29 (Sp)TasH from a double-strand nuclease into a nickase in a position-dependent manner, but the molecular basis underlying this functional switch remains unknown. Here, we combined biochemical analyses and cryo-electron microscopy to investigate tigRNA maturation and mismatched target recognition by the Nop domain of SpTasH. We show that the Nop domain is required for pre-tigRNA processing and stabilizes the mature tigRNA through extensive interactions, thereby establishing a cleavage-competent ribonucleoprotein complex. Structural analyses of SpTasH complexes bound to substrates containing single mismatches reveal that a mismatch at the 5'-most position of spacer A is readily accommodated through Nop domain-mediated stabilization of the spacer-target heteroduplex. In contrast, a mismatch proximal to the cleavage site destabilizes the heteroduplex, preventing recruitment of the corresponding HNH domain, thereby converting the complex into a nickase. Together, these findings establish the structural basis for position-dependent mismatch recognition and reveal how Nop domain-mediated tigRNA-target stabilization enables differential responses to mismatches, providing a foundation for engineering TIGR-Tas systems for genome-editing applications.},
}

Cryoelectron Microscopy
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry
*Base Pair Mismatch
*Deoxyribonuclease I/metabolism/genetics/chemistry
Protein Domains
Models, Molecular

@article {pmid42532990,
year = {2026},
author = {Liu, Y and Zhao, H and Cao, X and Jiang, S and Fu, J and Xue, J and Li, C and Xu, Z and Li, M and Du, W},
title = {Rapid and quantitative measurement of bacteriophage infectivity via fully automated droplet digital PCR.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42532990},
issn = {2041-1723},
support = {XDB0810000//CAS | State Key Laboratory of Microbial Resources (State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences)/ ; 22374016//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32370090//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Bacteriophages/genetics/physiology/pathogenicity ; *Polymerase Chain Reaction/methods ; RNA, Ribosomal, 16S/genetics ; DNA, Viral/genetics ; Automation ; Bacteria/virology/genetics ; Phage Therapy/methods ; },
abstract = {The clinical translation of phage therapy for multidrug-resistant infections is constrained by the lack of rapid, standardized therapeutic phage selection. Here, we introduce digital phage susceptibility testing (dPhaST), an automated droplet digital PCR workflow that quantifies phage-induced DNA release as a molecular signature of lysis. By targeting conserved 16S rRNA regions, dPhaST measures lytic activity across diverse bacterial pathogens within 3 h. Across 122 phage-host combinations involving 19 bacterial strains from six species, dPhaST shows 95.9% concordance with spot tests while resolving weak and heterogeneous lytic activities that are not readily distinguished phenotypically. It remains robust during the early infection window despite phage-encoded nuclease activity and tolerates phage cross-contamination better than spot tests. The method captures defense-mediated interactions involving CRISPR-Cas and Sir2-HerA systems. In this work, we show that automated digital quantification enables rapid and mechanistically informative profiling of early phage lytic efficacy across Gram-positive and Gram-negative pathogens.},
}

*Bacteriophages/genetics/physiology/pathogenicity
*Polymerase Chain Reaction/methods
RNA, Ribosomal, 16S/genetics
DNA, Viral/genetics
Automation
Bacteria/virology/genetics
Phage Therapy/methods

@article {pmid42533766,
year = {2026},
author = {Kong, W and Fu, L and Li, Y and Pan, W and Li, N and Tang, B},
title = {Amplification-free CRISPR/Cas biosensors for point-of-care nucleic acid detection: recent advances and future perspectives.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d6cc02994a},
pmid = {42533766},
issn = {1364-548X},
abstract = {Nucleic acid biomarkers are critical targets for early diagnosis of disease, public health surveillance and environmental safety. However, their low abundance and the complexity of the sample matrix pose strict requirements for the high sensitivity and portability of detection technologies. Although traditional clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems exhibit advantages such as high specificity, operational simplicity, and compatibility with mild reaction conditions, their reliance on pre-amplification steps elevates the risk of false-positive results and hinders their broader application. To overcome these limitations, amplification-free CRISPR/Cas technologies have emerged and undergone extensive development. These approaches enable highly sensitive nucleic acid detection without the need for pre-amplification and are more amenable to integration with portable devices, thereby offering promising avenues for point-of-care testing (POCT). This review systematically examines the fundamental principles and design strategies underlying amplification-free CRISPR/Cas biosensors and summarizes recent advances in detection platforms based on autocatalytic signal enhancement, nanomaterial-coupled amplification, and integrated high-sensitivity readout systems, while also outlining their practical applications in POCT settings. Furthermore, the key technical challenges and future development directions of amplification-free CRISPR technologies are discussed based on current advances in the field. These insights and perspectives aim to provide a systematic reference for further research and to facilitate the expanded application of amplification-free CRISPR/Cas systems in POCT.},
}

@article {pmid42534996,
year = {2026},
author = {Zhu, L and Huang, J and Xie, C},
title = {AI-enhanced framework for optimizing CRISPR-Cas gene editing in crop biotechnology addressing regulatory challenges and opportunities in global agricultural practices.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1770472},
pmid = {42534996},
issn = {1664-462X},
abstract = {INTRODUCTION: The integration of CRISPR Cas genome editing with artificial intelligence (AI) offers significant potential for crop biotechnology by supporting more precise and adaptive strategies for trait improvement under complex agricultural and regulatory conditions. However, the global governance of gene edited crops remains highly heterogeneous, creating major challenges for the development of frameworks that can jointly support optimization, uncertainty management, and regulatory alignment. Conventional approaches often lack the ability to account for evolving regulatory requirements and multi source uncertainties in a unified manner.

METHODS: In this paper, we introduce the Adaptive Regulatory Optimizer (ARO), an AI enhanced framework designed to support CRISPR Cas genome editing in crop biotechnology under biologically, regulatorily, and contextually constrained conditions. The ARO consists of three interconnected modules: the Manifold Constrained Gene Editor, the Agent Driven Regulatory Planner, and the Uncertainty Propagation Filter. Together, these modules embed editing decisions within biologically feasible manifolds, incorporate jurisdiction aware regulatory planning, and model interacting uncertainties associated with gene editing and deployment contexts. The The framework combines constrained optimization refinement, probabilistic uncertainty modeling, and adaptive regulatory planning to provide a structured basis for compliance aware and context sensitive decision support.

RESULTS AND DISCUSSION: Experimental results on the evaluated datasets indicate that the ARO achieves improved performance on the selected metrics relative to the compared methods, while its architecture is explicitly designed to integrate regulatory constraints into the optimization process. These findings suggest that the proposed framework provides a promising foundation for supporting more transparent, adaptive, and analytically grounded decision making in CRISPR Cas applications for crop biotechnology.},
}

@article {pmid42537394,
year = {2026},
author = {Zemmouchi, M and El-Fermawi, A and Benagdi, A and Baaziz, S and Hiram, R},
title = {Translational consistency of gene therapy strategies targeting inflammation in atrial fibrillation's management.},
journal = {Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie},
volume = {202},
number = {},
pages = {119824},
doi = {10.1016/j.biopha.2026.119824},
pmid = {42537394},
issn = {1950-6007},
abstract = {Atrial fibrillation (AF) is the most common cardiac rhythm disorder. AF risk factors include pathological ageing, hypertension, obesity, diabetes, and cardiac conditions such as myocardial infarction. Chronic inflammation is a major pathophysiological profile commonly observed in AF and its risk factors. Clinical and preclinical studies have suggested that increased expression of proinflammatory biomarkers such as the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome, interleukin (IL)-1β, or IL6 is associated with the development and maintenance of cardiac arrhythmias including AF. Current anti-arrhythmic and anti-inflammatory treatments are non-optimal in AF management. In parallel, mounting evidence suggests that new biotechnologies including gene therapy approaches, might help to target specific genes to prevent or promote their expression and their associated protein activity. Applied to cardiac arrhythmias, gene therapy might help to restore normal functions of ion channels, optimal calcium (Ca[2 +])-handling machinery, functional gap junctions, and efficient inflammatory signaling, known to be altered in AF. With an emphasis on the importance of gene therapy strategies targeting inflammation, this narrative review aims to: i) highlight the rationale and clinical relevance of gene therapy as an innovative strategy for the management of AF; ii) evaluate current knowledge regarding gene therapy vectors and delivery platforms applicable to cardiology and AF; iii) review emerging molecular targets explored in the context of AF gene therapy; and iv) identify promising gene-based therapeutic candidates, with a particular focus on inflammation-related pathways in AF.},
}

@article {pmid42538871,
year = {2026},
author = {Wang, X and Trypsteen, W and Anckaert, J and de Bony, E and Mestdagh, P},
title = {An Optimized Workflow for In Vitro Transcription of Single Guide RNAs Minimizes Innate Immune Activation.},
journal = {The CRISPR journal},
volume = {9},
number = {4},
pages = {223-232},
doi = {10.1177/25731599261467974},
pmid = {42538871},
issn = {2573-1602},
mesh = {*Immunity, Innate/genetics ; *Transcription, Genetic ; *RNA, Guide, CRISPR-Cas Systems/genetics/immunology ; Humans ; CRISPR-Cas Systems/genetics ; RNA, Double-Stranded/genetics ; Workflow ; Gene Knockdown Techniques ; Sodium Chloride ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; HEK293 Cells ; },
abstract = {CRISPR interference (CRISPRi) often uses single guide RNAs (sgRNAs) generated by in vitro transcription (IVT); however, IVT-derived RNAs can trigger innate immune responses that confound functional analyses. Here, we evaluate innate immune activation induced by IVT sgRNAs in a CRISPRi setting and show that enzymatic removal of the 5'-triphosphate group alone is insufficient to consistently eliminate this response. We therefore assessed modifications of IVT reaction conditions and found that supplementation with sodium chloride or urea further attenuated immune activation. Based on immune suppression, sgRNA yield, and knockdown efficiency, 0.15 M NaCl was selected for the optimized IVT condition. This condition showed a lower double-stranded RNA (dsRNA) concentration, providing direct support for reduced dsRNA by-products as a contributor to diminished immune activation. By integrating NaCl-supplemented IVT with phosphatase treatment, we establish an optimized and scalable workflow that minimizes innate immune responses while preserving sgRNA-mediated target knockdown efficiency in stable CRISPRi cells.},
}

*Immunity, Innate/genetics
*Transcription, Genetic
*RNA, Guide, CRISPR-Cas Systems/genetics/immunology
Humans
CRISPR-Cas Systems/genetics
RNA, Double-Stranded/genetics
Workflow
Gene Knockdown Techniques
Sodium Chloride
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
HEK293 Cells

@article {pmid40476386,
year = {2026},
author = {Su, S and Xu, Z and Suo, J and Zhou, Y and Zhang, X and Peng, X and Chen, M and Li, F},
title = {Function analysis of flightin gene in the global tortricid fruit borer Grapholita molesta using CRISPR/Cas9.},
journal = {Insect science},
volume = {33},
number = {4},
pages = {1357-1368},
doi = {10.1111/1744-7917.70079},
pmid = {40476386},
issn = {1744-7917},
support = {//the Coordinated Research Project (CRP) of the International Atomic Energy Agency/ ; //the National Key R&D Program of China/ ; //the Science and Technology Partnership Program of Ministry of Science and Technology of China/ ; },
mesh = {Animals ; CRISPR-Cas Systems ; *Moths/genetics/growth & development/physiology/metabolism ; *Insect Proteins/genetics/metabolism ; Flight, Animal ; Larva/growth & development/genetics/physiology ; Female ; Pupa/growth & development/genetics ; Gene Knockout Techniques ; Male ; },
abstract = {Many tortricid moths are significant fruit borers characterized by limited flight capacity. However, some individuals within a population of tortricid species exhibit extended flight capabilities, facilitating gene flow between orchards and enabling host switching. To date, research on the proteins involved in flight among fruit borers is limited. Flightin is recognized as a flight muscle protein, yet its function remains unexplored in lepidopteran insects. In this study, quantitative polymerase chain reaction analysis revealed that the flightin gene is expressed at various developmental stages and tissues of Grapholita molesta, with the highest expression in adults and the thorax. Using clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated nuclease 9 (Cas9) gene editing technology, we successfully generated a homozygous flightin gene knockout strain of G. molesta. The knockout of the flightin gene resulted in contraction of indirect flight muscle fibers, irregularities in the Z-disc of the flight muscles, and a significant elongation of sarcomere length. Additionally, cumulative flight distance and flight time were significantly reduced. The larval period and preoviposition period were significantly prolonged, while larval weight, pupal weight, longevity, and fecundity were all significantly decreased. The results indicate that the flightin gene not only plays an important role in the flight capacity of G. molesta, but also has an effect on the growth and development, and reproduction of the insect, suggesting that flightin may be a potential target for pest management of G. molesta. This is the first investigation into the function of the flightin gene using CRISPR/Cas9.},
}

Animals
CRISPR-Cas Systems
*Moths/genetics/growth & development/physiology/metabolism
*Insect Proteins/genetics/metabolism
Flight, Animal
Larva/growth & development/genetics/physiology
Female
Pupa/growth & development/genetics
Gene Knockout Techniques
Male

@article {pmid41159566,
year = {2026},
author = {Yen, PS and Verkuijl, SANR and Capriotti, P and Del Corsano, G and Yee, CKG and Hoermann, A and Inghilterra, MG and Aramburu-Gonzalez, I and Khan, MA and Vlachou, D and Christophides, GK and Windbichler, N},
title = {The nanosd integral gene drive enables population modification of the malaria vector Anopheles gambiae.},
journal = {G3 (Bethesda, Md.)},
volume = {16},
number = {8},
pages = {},
doi = {10.1093/g3journal/jkaf246},
pmid = {41159566},
issn = {2160-1836},
support = {OPP1158151//Bill and Melinda Gates Foundation/ ; INV-058071//Bill and Melinda Gates Foundation/ ; },
mesh = {Animals ; *Anopheles/genetics/parasitology ; Female ; *Mosquito Vectors/genetics/parasitology ; *Malaria/transmission/parasitology ; Male ; *Gene Drive Technology ; CRISPR-Cas Systems ; Fertility/genetics ; *Insect Proteins/genetics ; },
abstract = {The modification of mosquito populations at scale through CRISPR-Cas9-mediated homing gene drives is a promising route for malaria vector control. Integral gene drives (IGDs) are designed to utilize the regulatory sequences of endogenous genes to reduce the size of the modification required for nuclease and effector expression. In this study, we describe the creation and characterization of the nanosd integral gene drive, which targets and is inserted into the nanos gene of the malaria vector Anopheles gambiae, and show that it achieves high rates of gene drive (98.4% in females, 99.5% in males). We find that homozygous nanosd females but not males show impaired fecundity and exhibit variable degrees of ovary underdevelopment. Transcriptomic analysis of ovaries points to decreased transcript levels of the nanos gene when harboring Cas9 and changes to other fertility-related genes. As a minimal genetic modification, nanosd does not induce widespread transcriptomic perturbations that would affect vector competence, and we show that its susceptibility to Plasmodium spp. and O'nyong nyong virus infection remains similar to wild-type mosquitoes. Importantly, we find that nanosd propagates efficiently in caged mosquito populations and is maintained as a source of Cas9 after the emergence of drive-resistant alleles, whilst also mobilizing a nonautonomous antiparasitic effector modification. The nanosd gene drive shows promise as a genetic tool for malaria vector control via population modification, and we outline steps towards its further optimization.},
}

Animals
*Anopheles/genetics/parasitology
Female
*Mosquito Vectors/genetics/parasitology
*Malaria/transmission/parasitology
Male
*Gene Drive Technology
CRISPR-Cas Systems
Fertility/genetics
*Insect Proteins/genetics

@article {pmid41706533,
year = {2026},
author = {Overton, MS and Guy, SE and Chen, X and Martsul, A and Carolino, K and Akbari, OS and Meyer, JR and Kryazhimskiy, S},
title = {Upper bound on the mutational burden imposed by a CRISPR-Cas9 gene-drive element.},
journal = {G3 (Bethesda, Md.)},
volume = {16},
number = {8},
pages = {},
doi = {10.1093/g3journal/jkaf315},
pmid = {41706533},
issn = {2160-1836},
support = {HR0011-17-2-0047//DARPA/ ; R35GM153242/GF/NIH HHS/United States ; 5T32GM133351-02//Pathways in Biological Sciences NIH T32 program/ ; //Tata Institute for Genetics and Society BS/MS Fellowship/ ; R35GM153242/GM/NIGMS NIH HHS/United States ; },
mesh = {Saccharomyces cerevisiae/genetics ; *CRISPR-Cas Systems ; *Mutation ; Loss of Heterozygosity ; Mutation Rate ; },
abstract = {Homing-based CRISPR-Cas9 gene drives (CCGDs) are powerful tools for genetic control of wild populations, with applications from disease eradication to species conservation. However, Cas9 alone and in a complex with guide RNA can cause double-stranded DNA breaks at off-target sites, which could increase the mutational load and lead to unintended loss-of-heterozygosity (LOH) events. These undesired effects raise potential concerns about the long-term evolutionary safety of CCGDs, but the magnitude of these effects is unknown. To measure how the presence of a CCGD or a Cas9 alone in the genome affects the rates of LOH events and de novo mutations, we carried out a mutation accumulation experiment in yeast Saccharomyces cerevisiae. We found no detectable effects on the genome-wide rates of mutations or LOH events. Our power calculations suggest that CCGD or Cas9 affect these rates by less than 30%, which is much less than natural variation for these traits in yeast. A more detailed examination shows that CCGD or Cas9 may alter the lengths and genomic distributions of LOH events, but the statistical support for these effects is weak. Thus, our results demonstrate that CCGDs impose at most a weak additional mutational burden in the yeast model. Although mutagenic effects of gene drives need to be further evaluated in other systems, our results add credence to the proposition that the evolutionary risks posed by well-designed gene drives may be acceptable.},
}

Saccharomyces cerevisiae/genetics
*CRISPR-Cas Systems
*Mutation
Loss of Heterozygosity
Mutation Rate

@article {pmid41774761,
year = {2026},
author = {Harvey-Samuel, T and Kaur, R and Leftwich, PT and Feng, X and Gantz, V and Alphey, L},
title = {Sequence mismatch between gene-drive and target-site flanking regions significantly impairs homing efficiency in Culex quinquefasciatus.},
journal = {Genetics},
volume = {233},
number = {4},
pages = {},
doi = {10.1093/genetics/iyag054},
pmid = {41774761},
issn = {1943-2631},
support = {BBS/E/I/00007033//UK Biotechnology and Biological Sciences Research Council/ ; BBS/E/I/00007038//UK Biotechnology and Biological Sciences Research Council/ ; BBS/E/I/00007039//UK Biotechnology and Biological Sciences Research Council/ ; },
mesh = {Animals ; *Culex/genetics/virology ; *Gene Drive Technology/methods ; CRISPR-Cas Systems ; Transgenes ; Mosquito Vectors/genetics ; },
abstract = {CRISPR/Cas9-based homing gene-drives (homing-drives) hold enormous potential as control tools for mosquito disease-vectors. These genomically encoded technologies spread themselves through target populations by creating double-stranded DNA breaks on homologous chromosomes, into which the homing-drives are copied ("homed"). Homing is dependent on sequence homology between the genomic regions flanking the transgene insertion and the break site. Homing efficiency (ie copying rate) substantially impacts the power of these systems: less efficient homing-drives spread slower, have fewer applications, and are more resistance-prone. Understanding what influences homing-drive efficiency is therefore vital to the successful use of these technologies. Here we report a novel mechanism by which a homing-drive's efficiency can be significantly impaired by natural sequence variation within a population into which it is spreading. Using a kmo-targeting "split" homing-drive in the West Nile virus mosquito Culex quinquefasciatus, we found that target-site heterology (sequence mismatch between the genomic regions flanking the target cut-site and the homing-drive transgene) of less than 10% reduced homing efficiency by up to 54%. While substantial research effort has been dedicated to increasing homing-drive efficiency through optimization of within-construct components, our results highlight that the real-world efficacy of these systems may in part depend on variation beyond these controllable factors.},
}

Animals
*Culex/genetics/virology
*Gene Drive Technology/methods
CRISPR-Cas Systems
Transgenes
Mosquito Vectors/genetics

@article {pmid42083788,
year = {2026},
author = {Surender, S and Haeusser, LA and Kuhlburger, L and Tsiami, F and Dogan, NO and Maise, L and Nahnsen, S and Beck, S and Merk, DJ and Tabatabai, G},
title = {Molecular modulators of cyclin-dependent kinase 4/6 inhibitor response in experimental glioma identified through genome-wide CRISPR-Cas9 screening.},
journal = {Neuro-oncology},
volume = {28},
number = {8},
pages = {1904-1920},
doi = {10.1093/neuonc/noag093},
pmid = {42083788},
issn = {1523-5866},
support = {2019_Kolleg_14//Else Kröner Fresenius Stiftung/ ; },
mesh = {Humans ; *Cyclin-Dependent Kinase 4/antagonists & inhibitors/genetics ; *Cyclin-Dependent Kinase 6/antagonists & inhibitors/genetics ; Animals ; *Glioma/drug therapy/genetics/pathology ; Mice ; *Protein Kinase Inhibitors/pharmacology ; *CRISPR-Cas Systems ; *Brain Neoplasms/drug therapy/genetics/pathology ; Cell Proliferation ; Checkpoint Kinase 1/genetics/antagonists & inhibitors ; Apoptosis ; Oncogene Proteins/genetics ; Cell Line, Tumor ; Cyclin E ; },
abstract = {BACKGROUND: Glioblastoma harbors frequent alterations in the retinoblastoma pathway, providing a genetic rationale for therapeutic targeting with cyclin-dependent kinase 4/6 (CDK4/6) inhibitors. The NOA-20 trial did not reveal a progression-free survival benefit of CDK4/6 inhibition plus radiation therapy in newly diagnosed, O6-methylguanine DNA methyltransferase (MGMT)-unmethylated glioblastoma. In fact, CDK4/6 inhibitor monotherapy has not demonstrated efficacy in solid tumors. We aimed at discovering response modulators to CDK4/6 inhibition, paving the way for rational combination therapies.

METHODS: We conducted genome-wide CRISPR-Cas9 screens in human glioma cell lines and stem-like cells (LN229, LN18, LNZ308, T98G, and GS-9) under CDK4/6 inhibition, employing knockout (Brunello library) and activation strategies (Calabrese library), followed by genetic and pharmacological validation of selected candidate genes in vitro and ex vivo (primary cultures) as well as the investigation of 1 functionally instructed combination therapy in vivo.

RESULTS: Loss of AMBRA1 and gain of function of CCNE1 reduced sensitivity to CDK4/6 inhibition in glioma cells, whereas disruption of checkpoint kinase 1 (CHEK1) or FAM122A resulted in synthetic lethality in combination with CDK4/6 inhibition. AMBRA1-deficient glioma cells exhibited increased sensitivity to CHK1 inhibition, revealing a context-specific vulnerability. Combined inhibition of CHK1 and CDK4/6 led to synergistic antiglioma activity in vitro, ex vivo, and in vivo.

CONCLUSIONS: Our data identify AMBRA1, CCNE1, CHEK1, and FAM122A as potential molecular modifiers of CDK4/6 inhibition response in experimental glioma and provide a biological rationale for combinatorial targeting with CDK4/6 inhibition in glioblastoma.},
}

Humans
*Cyclin-Dependent Kinase 4/antagonists & inhibitors/genetics
*Cyclin-Dependent Kinase 6/antagonists & inhibitors/genetics
Animals
*Glioma/drug therapy/genetics/pathology
Mice
*Protein Kinase Inhibitors/pharmacology
*CRISPR-Cas Systems
*Brain Neoplasms/drug therapy/genetics/pathology
Cell Proliferation
Checkpoint Kinase 1/genetics/antagonists & inhibitors
Apoptosis
Oncogene Proteins/genetics
Cell Line, Tumor
Cyclin E

@article {pmid42192133,
year = {2026},
author = {Elena, M and Giuliana, N and Giuseppina, R and Valeria, L and Geppino, F and Viola, C and Maria, V and Tiziana, A and Dario, A and Alessandra, P},
title = {Unveiling a novel role for p19Arf (alternative reading frame) in mESC differentiation toward the pancreatic lineage.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42192133},
issn = {2045-2322},
support = {2022R74KBC//MUR- Ministero Università Ricerca-PRIN 2022/ ; },
mesh = {Animals ; *Cell Differentiation/genetics ; Mice ; *Cyclin-Dependent Kinase Inhibitor p16/genetics/metabolism ; Endoderm/cytology/metabolism ; *Mouse Embryonic Stem Cells/cytology/metabolism ; *Pancreas/cytology/metabolism ; *Cell Lineage/genetics ; Gene Expression Profiling ; CRISPR-Cas Systems ; Mutation ; },
abstract = {The tumor suppressor ARF (p14 in human, p19 in mouse), has traditionally been characterized by its pivotal role in tumor surveillance. However, its involvement in an expanding range of cellular processes reveals that its functions are broader and more complex than initially appreciated. Here, we uncover a previously unrecognized role of p19ARF in endodermal differentiation, specifically in pancreatic lineage specification using an in vitro differentiation model of mouse embryonic stem cells (mESCs). Using CRISPR/Cas9-mediated mutagenesis, we show that mESCs with mutations in p19Arf are unable to efficiently differentiate towards pancreatic endoderm. Transcriptomic profiling reveals substantial alterations in gene networks associated not only with lineage commitment but also with cytoskeletal organization and cell morphology. These changes correlate with a disruption in stem cell architecture and suggest the persistence of pluripotency feature when only one copy of functional p19Arf is present. Taken together, our findings highlight a role for p19Arf in modulating endodermal differentiation while maintaining the essential cellular properties of stem cells, thus expanding its relevance beyond tumor suppression.},
}

Animals
*Cell Differentiation/genetics
Mice
*Cyclin-Dependent Kinase Inhibitor p16/genetics/metabolism
Endoderm/cytology/metabolism
*Mouse Embryonic Stem Cells/cytology/metabolism
*Pancreas/cytology/metabolism
*Cell Lineage/genetics
Gene Expression Profiling
CRISPR-Cas Systems
Mutation

@article {pmid42210018,
year = {2026},
author = {Malaiwong, N and Malaiwong, P and Kim, C and O'Donnell, M},
title = {FLInt 2.0: robust and customizable single-shot integration in C. elegans.},
journal = {G3 (Bethesda, Md.)},
volume = {16},
number = {8},
pages = {},
doi = {10.1093/g3journal/jkag138},
pmid = {42210018},
issn = {2160-1836},
support = {DP2 GM154014/GM/NIGMS NIH HHS/United States ; DP2 GM154014/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *Caenorhabditis elegans/genetics ; *Transgenes ; CRISPR-Cas Systems ; Animals, Genetically Modified ; Luminescent Proteins/genetics ; Gene Transfer Techniques ; },
abstract = {Transgenesis in Caenorhabditis elegans has revolutionized biological research by enabling the precise control of expression of both endogenous and exogenous genes. FLInt (Fluorescent Landmark Interference) was developed to integrate transgenes via CRISPR-Cas9 using visible changes in existing fluorescent protein expression strains. While the original FLInt method (FLInt 1.0) enabled a simple visual readout of potential transgene integration, the process was prone to false positives, leading to burdensome screening efforts. Here, we present an alternative FLInt strategy, FLInt 2.0, that reduces false positives by targeted CRISPR-Cas9 cutting of fluorescent protein landing sites in a manner which largely retains fluorescence in nonintegrative repair events but eliminates expression upon transgene integration. We demonstrate that this targeted approach maintains effective integration while significantly decreasing the proportion of false positives. Molecular and transmission analyses confirm that nonfluorescent F2 animals more reliably represent stably integrated multicopy transgenic lines. We show that integration efficiency and array transmission are influenced by DNA structure and composition, with linear DNA substrates promoting more robust array formation and insertion. We further show that multicopy transgene lines can be tailored to desired expression levels using a simple subsequent Cas9 targeting approach, reducing labor-intensive screening and increasing experimental throughput. Our strategy provides a robust, visually guided refinement of FLInt, offering a generalizable framework for improving site-specific transgene integration in C. elegans.},
}

Animals
*Caenorhabditis elegans/genetics
*Transgenes
CRISPR-Cas Systems
Animals, Genetically Modified
Luminescent Proteins/genetics
Gene Transfer Techniques

@article {pmid42226151,
year = {2026},
author = {Huang, M and Li, X and Pan, T and Wu, D and Li, G and Wu, W},
title = {CRISPR-Cas9-mediated construction of a Streptococcus agalactiae vaccine for tilapia and evaluation of its protective efficacy.},
journal = {BMC veterinary research},
volume = {22},
number = {1},
pages = {},
pmid = {42226151},
issn = {1746-6148},
support = {AB22013007//the Fangchenggang Science and Technology Program/ ; AA17204081-1//Guangxi Science and Technology Program/ ; },
mesh = {Animals ; *Streptococcus agalactiae/immunology ; *Fish Diseases/prevention & control/microbiology/immunology ; *Streptococcal Infections/veterinary/prevention & control ; *Tilapia/immunology/microbiology ; *CRISPR-Cas Systems ; *Streptococcal Vaccines/immunology ; },
abstract = {BACKGROUND: Streptococcus agalactiae (GBS) causes severe tilapia streptococcosis with heavy aquaculture losses; existing vaccines have administration or efficacy limitations. This study used CRISPR-Cas9 to construct recombinant Escherichia coli DH5α-ORF4-GFP (targeting GBS scpB gene ORF4 fragment), optimized tilapia immersion immunization doses/frequencies, and evaluated the vaccine's protective efficacy, biosafety and regulatory effects via multi-dimensional assays.

RESULTS: The optimal regimen was single immersion at 1.5 × 10[4] CFU/mL, with a maximum RPS of 73.13% and stable 65.79% in validation. Immunized tilapia showed elevated immune indices (161.40% higher platelets) and numerical increases in globulin, normal liver/kidney function, and improved oxidative stress resistance with no tissue damage. The vaccine did not alter intestinal microbial richness but modulated community structure, enriching beneficial taxa such as Alphaproteobacteria, suggesting a potential interaction between vaccination and gut microbiota that may contribute to enhanced host defense.

CONCLUSIONS: In conclusion, this study successfully developed an effective and safe genetically engineered vaccine against GBS in tilapia. The precise CRISPR-Cas9-mediated construction strategy and confirmed immune protective effect provide a novel technical approach for controlling this disease in aquaculture and offer important references for the development of related genetically engineered vaccines.},
}

Animals
*Streptococcus agalactiae/immunology
*Fish Diseases/prevention & control/microbiology/immunology
*Streptococcal Infections/veterinary/prevention & control
*Tilapia/immunology/microbiology
*CRISPR-Cas Systems
*Streptococcal Vaccines/immunology

@article {pmid42525171,
year = {2026},
author = {Duveneck, S and Ille, K and Melzer, S},
title = {FLC genes control flowering time to varying degrees in a Brassica napus spring cultivar.},
journal = {Plant molecular biology},
volume = {116},
number = {4},
pages = {},
pmid = {42525171},
issn = {1573-5028},
mesh = {*Brassica napus/genetics/physiology/growth & development ; *Flowers/genetics/physiology/growth & development ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; *MADS Domain Proteins/genetics/metabolism ; Vernalization/genetics ; Seasons ; Plant Leaves/genetics ; *Genes, Plant ; Gene Expression Profiling ; CRISPR-Cas Systems ; },
abstract = {In the crop Brassica napus (oilseed rape), distinct growth types have been established that differ mainly in their vernalization requirement for flowering. The need for vernalization in Arabidopsis is controlled by the expression of the floral repressor FLOWERING LOCUS C (FLC), which also regulates cold-responsive flowering in B. napus. Notably, FLC homologs are also retained in spring oilseed rape despite its lack of vernalization requirement. To elucidate the functions of the nine BnFLC homologs in a spring type, we generated CRISPR/Cas9 knockout mutants of all homologs in the cultivar Westar. We show that the loss of BnFLC genes significantly accelerates flowering, demonstrating that BnFLC genes regulate flowering in spring types independently of vernalization. Transcriptomic analyses in leaves revealed distinct expression patterns among the BnFLC genes, with some remaining active during floral transition. Finally, no epigenetic regulation of the BnFLC homologs associated with flowering time was detected, while BnFLC.A03b carried persistent repressive marks and was constitutively silenced. Unexpectedly, additional flowering time regulators, including genes typically active in the shoot apical meristem, were expressed in leaves, with some showing altered expression and chromatin states in the mutant. These findings reveal that BnFLC genes are developmentally regulated and directly control flowering in spring oilseed rape, while also modulating the expression of other floral regulators in B. napus.},
}

*Brassica napus/genetics/physiology/growth & development
*Flowers/genetics/physiology/growth & development
Gene Expression Regulation, Plant
*Plant Proteins/genetics/metabolism
*MADS Domain Proteins/genetics/metabolism
Vernalization/genetics
Seasons
Plant Leaves/genetics
*Genes, Plant
Gene Expression Profiling
CRISPR-Cas Systems

@article {pmid40397277,
year = {2026},
author = {Lodewijk, GA and Kozuki, S and Guiltinan, C and Topacio, BR and Shariati, SA},
title = {Application of CRISPR-Based Epigenome Editing Tools for Engineering Programmable Embryo Models.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3048},
number = {},
pages = {211-238},
pmid = {40397277},
issn = {1940-6029},
support = {R35 GM147395/GM/NIGMS NIH HHS/United States ; },
mesh = {*Epigenome Editing/methods ; Animals ; Mice ; *CRISPR-Cas Systems ; *Embryonic Development/genetics ; Humans ; *Embryo, Mammalian/cytology ; Embryonic Stem Cells/cytology/metabolism ; },
abstract = {Stem cell-based embryo models (SEMs) have the potential to transform our understanding of early human embryogenesis. A critical step in engineering SEMs is the generation of the major cell types that compose preimplantation embryos including two primary extraembryonic lineages: (i) trophoblast cells, which are crucial for implantation and the establishment of maternal-fetal exchange, and (ii) hypoblast cells, which contribute to yolk sac formation. In addition, both cell types provide key signaling cues necessary for embryonic development. CRISPR-based epigenome editors are programmable devices that allow for efficient and precise activation (CRISPRa) or repression (CRISPRi) of cell fate-determining factors by modulating endogenous regulatory elements. Here, we present a step-by-step method to implement CRISPRa for controlling cell fate in embryonic stem cells based on our work in generation of CRISPR-programmed mouse embryo models.},
}

*Epigenome Editing/methods
Animals
Mice
*CRISPR-Cas Systems
*Embryonic Development/genetics
Humans
*Embryo, Mammalian/cytology
Embryonic Stem Cells/cytology/metabolism

@article {pmid42218379,
year = {2026},
author = {Shi, Y and Yin, J and Ning, S and Yuan, J and Yang, D and Chuai, G},
title = {DeepCas12a: a hybrid deep learning framework for accurate AsCas12a efficiency prediction from sequence and epigenetic information.},
journal = {BMC genomics},
volume = {27},
number = {1},
pages = {},
pmid = {42218379},
issn = {1471-2164},
support = {2025080107//Tongji University "Medicine + X" Cross Research Program/ ; 62002265//National Natural Science Foundation of China/ ; },
mesh = {*Deep Learning ; *Epigenesis, Genetic ; *CRISPR-Cas Systems ; Convolutional Neural Networks ; *Gene Editing/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {CRISPR-Cas12a (Cpf1) offers distinct advantages for genome editing due to its flexible, T-rich PAM recognition. However, variable cleavage efficiency-modulated by sequence context and epigenetic features-remains a challenge, with existing tools facing challenges in modeling the high-order interactions between multimodal features. Here, we present DeepCas12a, a hybrid deep learning framework integrating Convolutional Neural Networks (CNNs) and a Vision Transformer (ViT) encoder to capture both local sequence motifs and long-range dependencies. The model fuses DNA sequence data with epigenetic profiles (DNA methylation and chromatin accessibility) in an end-to-end architecture. Benchmarked on an independent test set, DeepCas12a outperformed state-of-the-art predictors, achieving an Average Precision of 0.783, an AUC of 0.868, and a Spearman correlation of 0.630. Furthermore, interpretability analysis via saliency maps confirms the model captures biologically relevant features, including PAM specificity and seed region sensitivity, facilitating rational guide RNA design.},
}

*Deep Learning
*Epigenesis, Genetic
*CRISPR-Cas Systems
Convolutional Neural Networks
*Gene Editing/methods
*CRISPR-Associated Proteins/genetics/metabolism
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid42350674,
year = {2026},
author = {Navalayeu, T and Beer, N and Bebjaková, M and Kalis, RW and Hohmann, U and Stejskal, K and Krššáková, G and Fasching, N and Herzog, VA and Popitsch, N and Roitinger, E and Plaschka, C and Zuber, J and Ameres, SL},
title = {Cellular assembly and functional resilience of the mammalian RNA exosome.},
journal = {The EMBO journal},
volume = {45},
number = {15},
pages = {5423-5456},
pmid = {42350674},
issn = {1460-2075},
support = {LS23-053//Vienna Science and Technology Fund (WWTF)/ ; CoG-866166//EC | European Research Council (ERC)/ ; 10.55776/F80//Austrian Science Fund (FWF)/ ; 10.55776/DOC177//Austrian Science Fund (FWF)/ ; PhD Fellowship//Boehringer Ingelheim Fonds (BIF)/ ; },
mesh = {Animals ; Mice ; *Exosome Multienzyme Ribonuclease Complex/metabolism/genetics ; *Exosomes/metabolism ; RNA-Binding Proteins/metabolism/genetics ; Proteasome Endopeptidase Complex/metabolism ; *Mouse Embryonic Stem Cells/metabolism ; CRISPR-Cas Systems ; RNA/metabolism ; },
abstract = {Most eukaryotic proteins assemble into multisubunit complexes that coordinate essential cellular functions, yet the principles governing their assembly and proteostatic control remain largely undefined. Here, we systematically dissect the cellular assembly and functional organization of the RNA exosome, an essential ribonucleolytic complex, using an inducible dual-guide CRISPR/Cas9 system in mouse embryonic stem cells. We reveal a sequential assembly pathway where Exosc2, Exosc4, and Exosc7 initiate complex formation, facilitating the incorporation of barrel and cap subunits in a defined hierarchy. Unlike other structural subunits, the terminally incorporated cap subunit Exosc1 is dispensable for cell viability, revealing a modular, functionally resilient architecture. We demonstrate that orphan subunits are selectively degraded via the ubiquitin-proteasome system, enforcing stringent quality control over RNA exosome biogenesis. These findings define an assembly logic of of the mammalian exosome and uncover previously unrecognized plasticity in the composition and function of this essential ribonucleolytic complex.},
}

Animals
Mice
*Exosome Multienzyme Ribonuclease Complex/metabolism/genetics
*Exosomes/metabolism
RNA-Binding Proteins/metabolism/genetics
Proteasome Endopeptidase Complex/metabolism
*Mouse Embryonic Stem Cells/metabolism
CRISPR-Cas Systems
RNA/metabolism

@article {pmid42462663,
year = {2026},
author = {Chen, H and Jin, Z and Duan, C and Song, Z and Cheng, Y and Chen, M and Zhang, C and Lan, Y and Shen, W and Fu, Y and Liu, R and Zheng, X},
title = {Effects of the ecpA gene on the biological characteristics and pathogenicity of avian pathogenic Escherichia coli strain FJLY68.},
journal = {Veterinary microbiology},
volume = {320},
number = {},
pages = {111145},
doi = {10.1016/j.vetmic.2026.111145},
pmid = {42462663},
issn = {1873-2542},
mesh = {Animals ; *Escherichia coli/pathogenicity/genetics ; *Escherichia coli Infections/microbiology/veterinary ; *Poultry Diseases/microbiology ; Chickens/microbiology ; Biofilms/growth & development ; Virulence/genetics ; *Escherichia coli Proteins/genetics/metabolism ; Bacterial Adhesion/genetics ; Fimbriae, Bacterial/genetics ; Chick Embryo ; *Fimbriae Proteins/genetics ; Cell Line ; CRISPR-Cas Systems ; Genetic Complementation Test ; Gene Deletion ; },
abstract = {Avian pathogenic Escherichia coli (APEC) is a major cause of colibacillosis in poultry, yet the role of the ecpA gene, which encodes the major structural subunit of the Escherichia coli common pilus (ECP), remains incompletely defined in APEC pathogenesis. To investigate the role of ecpA in the biological characteristics and pathogenicity of Avian Pathogenic Escherichia coli (APEC) strain FJLY68, an ecpA deletion mutant (ΔecpA) and its corresponding complemented strain (CΔecpA) were constructed using the CRISPR/Cas9 system and verified by PCR and Sanger sequencing. Phenotypic analyses revealed that the ΔecpA mutation significantly impaired bacterial motility, biofilm formation, adherence to chicken embryonic fibroblast (DF-1) cells, and fimbriae assembly. Transcriptomic analysis identified 1720 differentially expressed genes in the ΔecpA mutant, significantly enriched in pathways associated with flagellar assembly, chemotaxis, and metabolism, consistent with the observed phenotypic changes. Although in vitro growth was unaffected, the ΔecpA mutant exhibited markedly attenuated virulence in a chick infection model, as indicated by an increased LD50, attenuated clinical signs and pathological lesions, and reduced bacterial colonisation in tissues. Full genetic complementation restored all observed defects to wild-type levels. This study identifies ecpA as a critical determinant of APEC pathogenesis, directly linking its function to bacterial motility, biofilm formation, adhesion, and in vivo virulence, and provides a theoretical basis for developing novel control strategies targeting this virulence factor.},
}

Animals
*Escherichia coli/pathogenicity/genetics
*Escherichia coli Infections/microbiology/veterinary
*Poultry Diseases/microbiology
Chickens/microbiology
Biofilms/growth & development
Virulence/genetics
*Escherichia coli Proteins/genetics/metabolism
Bacterial Adhesion/genetics
Fimbriae, Bacterial/genetics
Chick Embryo
*Fimbriae Proteins/genetics
Cell Line
CRISPR-Cas Systems
Genetic Complementation Test
Gene Deletion

@article {pmid42470400,
year = {2026},
author = {Ang, YS and Yung, LL},
title = {Sequence Engineering of Guide DNA for Precise RNA Targeting by Cas12a.},
journal = {ACS nano},
volume = {20},
number = {30},
pages = {21115-21127},
doi = {10.1021/acsnano.6c02663},
pmid = {42470400},
issn = {1936-086X},
support = {NA//Singapore Ministry of Education Academic Research Fund Tier 1/ ; NA//Singapore Ministry of Health?s National Medical Research Council, Programme for Research in Epidemic Preparedness and Response (PREPARE)/ ; },
mesh = {*CRISPR-Associated Proteins/metabolism/chemistry/genetics ; *DNA/chemistry/genetics/metabolism ; *Endodeoxyribonucleases/metabolism/chemistry/genetics ; *Bacterial Proteins/metabolism/genetics/chemistry ; *RNA/metabolism/chemistry/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Base Sequence ; },
abstract = {Cas12a is highly accommodative toward noncanonical activation pathways to the extent of flipping its identity to be a DNA-guided RNA-targeting effector. A sequence engineering approach was used to systematically identify desirable guide DNA (gDNA) sequence motifs to achieve comparable RNA targeting efficiency as the canonical RNA-guided Cas12a with good selectivity down to single-nucleotide mismatch. Importantly, we introduced a split gDNA design concept with greater energetic differences arising from subtle nucleotide changes to probe the key spacer features for effective Cas12a-gDNA activation. Similar to the canonical RNA-guided activation pathway, Cas12a was found to engage actively in the "seed-like" scaffold-proximal region while the scaffold-distal region was largely hybridization-driven. We further evolved the split gDNA design to enhance the sequence selectivity by up to 21-fold compared to a single gDNA design and achieve single-nucleotide discrimination among representative let-7 family members. This study has established a gDNA sequence design framework to reprogram Cas12a as a precise RNA targeting platform.},
}

*CRISPR-Associated Proteins/metabolism/chemistry/genetics
*DNA/chemistry/genetics/metabolism
*Endodeoxyribonucleases/metabolism/chemistry/genetics
*Bacterial Proteins/metabolism/genetics/chemistry
*RNA/metabolism/chemistry/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics
Base Sequence

@article {pmid42480873,
year = {2026},
author = {McClain, IM and Yigit, NS and Royzen, M},
title = {Non-chromatographic purification of guide RNA for gene-editing experiments.},
journal = {Bioorganic & medicinal chemistry letters},
volume = {140},
number = {},
pages = {130740},
doi = {10.1016/j.bmcl.2026.130740},
pmid = {42480873},
issn = {1464-3405},
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems/isolation & purification/genetics/chemistry ; *Gene Editing/methods ; CRISPR-Cas Systems ; Chromatography, High Pressure Liquid ; Green Fluorescent Proteins/genetics ; },
abstract = {CRISPR-Cas12a gene editing technology is gaining momentum as a powerful tool for many biochemical and medicinal applications. The technology requires guide RNA, which is typically made using solid phase synthesis and purified by HPLC. The latter is often the most complex and time-consuming element of the synthetic process. This communication describes a non-chromatographic method for purification of synthetic RNAs. The method consists of five steps and yields target RNA in over 80% purity, which adheres to the FDA's standard for gene editing applications. The non-chromatographic RNA purification approach was applied to synthesize guide RNA targeting the GFP gene. Its purity was analyzed by analytical HPLC. Its functional fidelity was tested in CRISPR-Cas12a experiments in solution and live mammalian cells.},
}

Humans
*RNA, Guide, CRISPR-Cas Systems/isolation & purification/genetics/chemistry
*Gene Editing/methods
CRISPR-Cas Systems
Chromatography, High Pressure Liquid
Green Fluorescent Proteins/genetics

@article {pmid42518166,
year = {2026},
author = {Kang, J and Kim, HH and Yoon, HM and Choi, Y and Heo, J and Woo, Y and Yu, S and Lee, KH and Lee, Y},
title = {Phenotypic and Whole-Genome Characterization of Enterococcus Isolates from Korean Doenjang and Meju: E. durans Edu-1 as a Food-Grade Probiotic Candidate with Epithelial Wound-Healing Activity.},
journal = {Probiotics and antimicrobial proteins},
volume = {},
number = {},
pages = {},
pmid = {42518166},
issn = {1867-1314},
support = {2026-RISE-09-A32//the Regional Innovation System & Education (RISE) program through the Gyeonggi Province RISE initiative, funded by the Ministry of Education (MOE) and the Gyeonggi Province, the Republic of Korea/ ; },
abstract = {Enterococcus species are widely distributed in traditional fermented foods and have shown probiotic potential. However, their food-grade application has been limited because the European Food Safety Authority excluded the genus from the qualified presumption of safety (QPS) list. Therefore, strain-specific safety assessment is essential. In this study, we characterized Enterococcus isolates from Korean Doenjang and Meju using an integrated phenotypic and whole-genome sequencing approach. Sixteen strains were isolated and identified/determined by 16S rRNA gene sequencing. We evaluated phenotypic safety and probiotic traits, and five candidates were further tested in HT-29 wound-healing scratch assays and a 115-gene host expression array. The two best wound-healing strains were then analyzed by whole-genome sequencing using a multi-database approach (VirulenceFinder, VFDB, ResFinder, CARD/RGI, PlasmidFinder, CRISPRCasFinder, and antiSMASH). The 16 isolates belonged to the five species, among which E. durans Edu-1 showed the highest wound closure ability (18.75 ± 1.24% at 72 h), followed by E. raffinosus Era-1 (16.71 ± 0.49%). Whole-genome sequence analysis showed that Edu-1 had no acquired transmissible antibiotic resistance and virulence factors genes and in addition, contains a plasmid-borne Bacteriocin_II family biosynthetic gene cluster, which can explain/support its broad-spectrum antimicrobial activity. In contrast, Era-1 carried tet(M) gene on the chromosome and ermB gene on an insertion-sequence-rich mobile element, although it also has a functional Type I-B CRISPR-Cas system and three glutamate decarboxylase (gadB) genes for γ-aminobutyric acid biosynthesis. These findings identify/suggest E. durans Edu-1 as a food-grade probiotic candidate with epithelial wound-healing activity, while E. raffinosus Era-1 represents a strain of biological interest for mechanistic wound-healing research rather than food application.},
}

@article {pmid42519596,
year = {2026},
author = {Hess, WR and Marchfelder, A and Randau, L},
title = {Editorial: creative CRISPR-Cas: RNA-guided functions in defence and beyond.},
journal = {microLife},
volume = {7},
number = {},
pages = {uqag026},
pmid = {42519596},
issn = {2633-6693},
}

@article {pmid42521019,
year = {2026},
author = {Dalabehera, M and Chaudhari, S and Kumar, J and Poonia, N and Subudhi, RN and Choonara, YE and Figueiras, A and Shukla, N and Sharma, N and Mascarenhas-Melo, F},
title = {Mechanistic advances in nanomedicine, nucleic acid therapies, and AI-driven research on cervical cancer.},
journal = {Journal of pharmaceutical sciences},
volume = {},
number = {},
pages = {104442},
doi = {10.1016/j.xphs.2026.104442},
pmid = {42521019},
issn = {1520-6017},
abstract = {Cervical cancer pharmacotherapy is significantly limited by physiological and cellular barriers that restrict drug access to therapeutic targets, resulting in suboptimal biodistribution, systemic toxicity, and the emergence of drug resistance. This review provides a mechanistic and biopharmaceutics-centered analysis of how advanced drug delivery systems are being engineered to overcome these limitations. We critically examine the role of nanocarriers, including lipid-based vesicles, polymeric nanoparticles, and inorganic hybrid systems, in modulating absorption, distribution, and tumor-targeting efficiency, with emphasis on their physicochemical properties and interaction with biological barriers such as the tumor microenvironment and cellular uptake pathways. In parallel, we analyze nucleic acid-based therapeutics (CRISPR/Cas systems, miRNA, and antisense oligonucleotides) from a pharmaceutical sciences perspective, focusing on delivery constraints, stability, intracellular trafficking, and their ability to modulate pharmacological response and drug resistance mechanisms. The review also discusses the integration of immunomodulatory strategies within nanodelivery platforms as a means to alter disease-related biological barriers and improve therapeutic index. Finally, we explore the emerging role of AI-assisted models in optimizing formulation design, predicting pharmacokinetic behavior, and supporting precision dosing strategies in drug development workflows. By integrating drug delivery engineering, molecular biopharmaceutics, and computational optimization, this work outlines a translational framework for overcoming key barriers in pharmaceutical intervention design for oncology applications.},
}

@article {pmid42521788,
year = {2026},
author = {Liu, H and Yuan, Z and Han, J and Zhao, Y},
title = {A dual-readout RAA-CRISPR/Cas13a diagnostic platform for rapid and sensitive detection of Eggerthella lenta.},
journal = {Mikrochimica acta},
volume = {193},
number = {8},
pages = {},
pmid = {42521788},
issn = {1436-5073},
support = {2019LJ001//Academic Promotion Program of Shandong First Medical University/ ; 2021ZDSYS27//the Key Research and Development Project of Shandong Province/ ; },
mesh = {*CRISPR-Cas Systems ; *Actinobacteria/isolation & purification/genetics ; Limit of Detection ; Humans ; *Nucleic Acid Amplification Techniques/methods ; Rapid Diagnostic Tests ; Recombinases/metabolism ; },
abstract = {Eggerthella lenta (E. lenta) is an opportunistic anaerobic pathogen associated with severe systemic infections, yet rapid and accurate diagnostic tools remain limited. To address this challenge, we developed a highly sensitive and specific dual-readout diagnostic platform integrating recombinase-aided amplification (RAA) with the CRISPR/Cas13a system, targeting the highly conserved rsmG gene of E. lenta. The assay offers two detection modalities: a real-time fluorescence readout and a visually interpretable lateral flow strip. Analytical evaluation demonstrated that the fluorescence-based assay achieved a limit of detection (LOD) of 4.4 copies per reaction (95% CI: 3.7-5.6 copies/reaction), while the instrument-free lateral flow assay yielded an LOD of 10[4] copies per reaction. The platform exhibited exceptional specificity, showing no cross-reactivity with 10 common non-target bacterial species. Clinical validation was performed using 24 synovial fluid samples, all confirmed positive for E. lenta by Sanger sequencing. The fluorescence assay successfully detected all 24 samples, achieving a detection rate of 100% (24/24). In parallel, the lateral flow assay detected 21 of the 24 positive samples, yielding a detection rate of 87.5% (21/24). The three samples undetected by the lateral flow strip were verified as true positives by sequencing, indicating that the discrepancy was due to the lower analytical sensitivity of the strip format rather than a lack of specificity. In conclusion, this dual-mode RAA-CRISPR/Cas13a platform serves as a robust and practical tool for rapid clinical diagnosis and point-of-care (POC) triaging of E. lenta infections. The fluorescence format is optimal for high-sensitivity laboratory testing, whereas the lateral flow variant provides a deployable alternative for rapid, point-of-care screening in resource-limited environments.},
}

*CRISPR-Cas Systems
*Actinobacteria/isolation & purification/genetics
Limit of Detection
Humans
*Nucleic Acid Amplification Techniques/methods
Rapid Diagnostic Tests
Recombinases/metabolism

@article {pmid42521823,
year = {2026},
author = {Tuo, W and Wang, X and Wu, T and Zhang, S and Guo, P and Lin, X and Lin, Q and Zhai, J and Wu, S},
title = {Establishment of an efficient Agrobacterium-mediated transformation system and CRISPR/Cas9-mediated genome editing of the OvPDS1 gene in Oxalis vulcanicola 'Sunset Velvet'.},
journal = {Planta},
volume = {264},
number = {3},
pages = {},
pmid = {42521823},
issn = {1432-2048},
support = {32471958//National Natural Science Foundation of China/ ; KFB24022A//Study on the Molecular Mechanisms of Leaf Coloration in Three Sulfur Oxalis Varieties, Innovation Project of Fujian Agriculture and Forestry University/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; *Transformation, Genetic ; *Gene Editing/methods ; *Agrobacterium/genetics ; *Plant Proteins/genetics/metabolism ; Acetophenones ; },
abstract = {This study overcomes a key technical barrier by establishing transformation and enabling first CRISPR/Cas9 editing in Oxalis, providing a platform for functional genomics and breeding. The lack of an efficient genetic transformation system has considerably hindered functional genomics studies in Oxalis vulcanicola 'Sunset Velvet'. Here, we established a stable and efficient Agrobacterium-mediated transformation system using stem segments as explants. Key parameters, including pre-culture duration, infection time, Agrobacterium cell density, acetosyringone (AS) concentration, and co-cultivation period, were systematically optimized. Under optimal conditions, the highest transient β-glucuronidase (GUS) expression rate reached approximately 9.0%, and eight stable transgenic lines were successfully obtained. CRISPR/Cas9-mediated genome editing was achieved in Oxalis for the first time. Targeted mutagenesis of OvPDS1, a gene involved in carotenoid biosynthesis, resulted in an albino phenotype, and Sanger sequencing confirmed a base substitution at the target site. Although the editing efficiency was relatively low (0.5%), this result demonstrates the feasibility of genome editing in Oxalis. This study overcomes a major technical bottleneck and provides a robust platform for functional gene analysis, trait improvement, and molecular breeding in O. vulcanicola 'Sunset Velvet' and other non-model ornamental plants.},
}

*CRISPR-Cas Systems/genetics
Plants, Genetically Modified/genetics
*Transformation, Genetic
*Gene Editing/methods
*Agrobacterium/genetics
*Plant Proteins/genetics/metabolism
Acetophenones

@article {pmid42522380,
year = {2026},
author = {Xue, F and Xin, Z and Wang, G and Xing, J and Han, H and Zhao, X and Song, N},
title = {Recent advances of CRISPR-based gene editing technologies and delivery strategies.},
journal = {Artificial cells, nanomedicine, and biotechnology},
volume = {54},
number = {1},
pages = {415-431},
doi = {10.1080/21691401.2026.2702869},
pmid = {42522380},
issn = {2169-141X},
mesh = {Humans ; *Gene Editing/methods ; Animals ; *CRISPR-Cas Systems/genetics ; *Gene Transfer Techniques ; },
abstract = {CRISPR technology is a powerful tool for gene editing, in which the efficient delivery of living target cells allows it to show great clinical potential. At present, the commonly used in vivo delivery strategies mainly include biological methods (AAV, VLP, SEND) and chemical methods (LNP), which subtly deliver gene editors to living target cells safely and efficiently from different ways. However, existing delivery systems have different extents of limitations in terms of editing efficiency, immunogenicity, half-life, etc., so developing optimized delivery systems is the key to fully realizing the potential of CRISPR-Cas system for intracellular gene editing. In order to fully understand the advantages of different delivery strategies to maximize the ability to help CRISPR systems choose delivery methods, we conducted a systematic review. In this paper, we introduce the types, principles and characteristics of gene editing systems in order to understand their requirements for delivery tools. We focus on describing the type, principle, load, immunogenicity, specificity, toxicity, etc. of the delivery system, so as to fully analyse its advantages and disadvantages for the selection of different editing environments. This review aims to provide new insights to facilitate appropriate delivery systems or improve the efficacy of existing systems.},
}

Humans
*Gene Editing/methods
Animals
*CRISPR-Cas Systems/genetics
*Gene Transfer Techniques

@article {pmid42522597,
year = {2026},
author = {Lu, W and Yang, Q and Zhao, P and Cao, Y and Jing, Z and Zhang, N and Li, J and Li, X and Wang, X and Zhang, X and Cao, L and Gong, P},
title = {A One-Pot RPA-CRISPR/Cas12a Assay for Rapid Genus-Level Detection of Babesia spp. in Ticks and Livestock Blood Samples.},
journal = {Transboundary and emerging diseases},
volume = {2026},
number = {1},
pages = {e9289663},
pmid = {42522597},
issn = {1865-1682},
support = {2024YFD1800100//National Key Research and Development Program of China/ ; CARS-39//China Wool-sheep & Cashmere-goat Research System/ ; },
mesh = {Animals ; *Babesia/isolation & purification/genetics ; *Babesiosis/diagnosis/parasitology/blood ; Cattle ; *Cattle Diseases/diagnosis/parasitology/blood ; *Sheep Diseases/diagnosis/parasitology/blood ; CRISPR-Cas Systems ; *Ticks/parasitology ; Sheep ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Rapid Diagnostic Tests ; Sensitivity and Specificity ; Livestock ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Babesiosis, a globally significant tick-borne disease, poses substantial threats to livestock production and public health. Reported cases of human babesiosis in the United States increased from 1742 in 2014 to 3586 in 2023. In livestock, cattle babesiosis causes mortality, reduced meat and milk production, reproductive losses, and substantial control costs, with annual economic losses estimated at hundreds of millions of US dollars in several endemic countries. Rapid and sensitive detection methods are essential for early warning, surveillance, and control of this disease. In this study, we developed a closed-tube, one-pot assay for genus-level detection of Babesia spp. associated with cattle and sheep, based on recombinase polymerase amplification (RPA) coupled with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a. This format effectively minimizes cross-contamination risks associated with repeated tube opening in conventional assays. A three-channel signal readout system, including blue-light fluorescence visualization, ultraviolet (UV) fluorescence visualization, and lateral flow strip (LFS) readout, was integrated to enable flexible endpoint detection under different laboratory and field conditions. The assay targets a conserved region of the Babesia 18S rRNA gene and enables genus-level detection of Babesia spp. within 40 min at 37°C. The established RPA-CRISPR/Cas12a platform exhibited high analytical sensitivity, with a limit of detection of 5 copies/μL for recombinant plasmid templates, high analytical specificity against the tested nontarget pathogens, and low equipment dependency. The detection limit of the LFS format reached 50 copies/μL. Field validation using 71 pooled tick samples and 53 clinical blood samples collected from cattle and sheep yielded positive rates of 15.49% and 9.43%, respectively, with 100% concordance between this assay and conventional polymerase chain reaction (PCR) for both specimen types. In conclusion, this one-pot RPA-CRISPR/Cas12a detection platform provides a rapid, sensitive, and field-applicable molecular screening tool for genus-level detection of Babesia spp. This assay may support early warning and preliminary field monitoring of babesiosis, particularly in resource-limited settings. However, species-level confirmation should be performed by sequencing or other species-specific methods when epidemiological tracing or precise species identification is required.},
}

Animals
*Babesia/isolation & purification/genetics
*Babesiosis/diagnosis/parasitology/blood
Cattle
*Cattle Diseases/diagnosis/parasitology/blood
*Sheep Diseases/diagnosis/parasitology/blood
CRISPR-Cas Systems
*Ticks/parasitology
Sheep
*Nucleic Acid Amplification Techniques/veterinary/methods
Rapid Diagnostic Tests
Sensitivity and Specificity
Livestock
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42524122,
year = {2026},
author = {Rananaware, SR and Narisetty, KV and Shah, RA and Jain, PK},
title = {CRISPR-based ex vivo gene editing of donor organs.},
journal = {Nature reviews bioengineering},
volume = {},
number = {},
pages = {},
pmid = {42524122},
issn = {2731-6092},
support = {R21 AI156321/AI/NIAID NIH HHS/United States ; R21 AI168795/AI/NIAID NIH HHS/United States ; R35 GM147788/GM/NIGMS NIH HHS/United States ; R61 AI181016/AI/NIAID NIH HHS/United States ; },
abstract = {Donor organs are frequently discarded because of concerns about quality or pathogen risk, challenges that could be mitigated through ex vivo gene editing or silencing during machine perfusion. Here, we discuss the development of CRISPR-based approaches for ex vivo gene silencing in human donor organs, from proof of concept in kidney biopsies to the challenges of organ-scale translation.},
}

@article {pmid42524609,
year = {2026},
author = {Martin, L and Bohinc, J and Recchia, A and Gritti, S and Santilli, G and Zeyland, J and Vidaković, M and Grdović, N and Benabdellah, K and Ortiz-Bueno, M and Butuner, BD and Pisaniello, L and Stilhano, R and Benati, D and Hapil, FZ and Khawaja, S and Nair, RR and Giacomelli, C and Atilla, E and Zinghirino, F and Ferrari, T and Corradi, F and Laufer, TJ and Khnykin, D and Aseguinolaza, GG and Skrbinek, M and Mlakar, T and Lapanja, T and Lainšček, D},
title = {In vivo delivery strategies for therapeutic CRISPR genome editing.},
journal = {International journal of biological sciences},
volume = {22},
number = {12},
pages = {6539-6581},
pmid = {42524609},
issn = {1449-2288},
mesh = {Humans ; *Gene Editing/methods ; Animals ; *CRISPR-Cas Systems/genetics ; Epigenome Editing ; Genetic Therapy/methods ; *Gene Transfer Techniques ; },
abstract = {CRISPR-based genome and epigenome editing technologies have rapidly evolved from programmable nucleases into a diverse therapeutic toolbox encompassing conventional CRISPR systems, base editing, prime editing, RNA targeting, and epigenetic modulation. While early clinical successes relied on ex vivo manipulation of patient-derived cells, recent advances in delivery chemistry and vector engineering are enabling direct in vivo editing across multiple organs. Here, we provide a comprehensive review of delivery modalities of CRISPR systems solely in vivo that underpin their therapeutic translation. We examine how anatomical, cellular, and immunological constraints shape organ-specific editing strategies in different organ systems and we highlight key preclinical and clinical milestones that define the current translational landscape. Across indications, delivery remains a critical determinant of efficacy, safety, and scalability, governing editor exposure, tissue selectivity, and risk of unintended genomic or epigenomic perturbation. This review, authored by members of the COST Action Genome Editing to treat Human Diseases (GenE-HumDi) Network, delineates the principles guiding in vivo genome and epigenome editing and outlines the remaining barriers to durable, tissue-selective, and broadly deployable CRISPR therapeutics.},
}

Humans
*Gene Editing/methods
Animals
*CRISPR-Cas Systems/genetics
Epigenome Editing
Genetic Therapy/methods
*Gene Transfer Techniques

@article {pmid41795918,
year = {2026},
author = {Liu, Y and Zhang, Y and Ding, H and Li, J and Gao, T and Wen, Z and Wang, Y and Wu, B},
title = {N-myristoyltransferase 1, a key gene for protein N-myristoylation, is dispensable for fertility in male mice.},
journal = {The Journal of reproduction and development},
volume = {72},
number = {4},
pages = {632-640},
doi = {10.1262/jrd.2025-064},
pmid = {41795918},
issn = {1348-4400},
mesh = {Animals ; Male ; *Acyltransferases/genetics/metabolism ; *Fertility/genetics/physiology ; *Spermatogenesis/genetics ; Mice ; Mice, Knockout ; Testis/metabolism ; Spermatozoa/metabolism ; Meiosis/genetics ; Infertility, Male/genetics ; CRISPR-Cas Systems ; },
abstract = {N-Myristoyltransferase 1 (NMT1), the predominant enzyme catalyzing myristoylation of proteins, is involved in various biological processes, including early embryonic development, immune responses, apoptosis, cellular homeostasis, tumorigenesis, and infection, with therapeutic potential in viral and parasitic infections as well as cancer. Despite the critical functions of NMT1, there have been no reports to date regarding its role in reproduction, especially in spermatogenesis. To investigate the function of NMT1 in this context, we utilized CRISPR/Cas9 technology to create a germ cell-specific Nmt1 knockout mice model for the first time. Surprisingly, male mice lacking NMT1 maintained fertility, exhibiting normal testicular structure and sperm morphology, with no significant differences in spermatogenic tubule structure or germ cell distribution compared to wild-type mice. Additionally, the Nmt1[f/f]; Stra8-Cre male mice showed no notable defects in meiosis. These findings suggest that NMT1 is not critical for spermatogenesis or male fertility in mice. However, further studies have shown that the compensatory role of NMT2 may play an unexpected role in maintaining myristoylation levels, which provides a new perspective for understanding the role of myristoylation in spermatogenesis.},
}

Animals
Male
*Acyltransferases/genetics/metabolism
*Fertility/genetics/physiology
*Spermatogenesis/genetics
Mice
Mice, Knockout
Testis/metabolism
Spermatozoa/metabolism
Meiosis/genetics
Infertility, Male/genetics
CRISPR-Cas Systems

@article {pmid41873043,
year = {2026},
author = {Lee, SJ and Lee, GS and Kim, J and Park, KH and Go, SR and Moon, JH and Woo, EJ},
title = {Mass spectrometry based identification of AMP-O-Tris generated by Thermococcus onnurineus Cas10.},
journal = {FEBS open bio},
volume = {16},
number = {8},
pages = {1593-1601},
pmid = {41873043},
issn = {2211-5463},
support = {Korean Government MSIP RS-2022-NR071772 RS-202//National Research Foundation of Korea/ ; KGM5382632 KGM1062612 KGM1322612//Korea Research Institute of Bioscience and Biotechnology/ ; CRC22024-500//National Research Council of Science and Technology/ ; },
mesh = {*Thermococcus/metabolism/genetics ; Tandem Mass Spectrometry/methods ; *Adenine Nucleotides/metabolism/chemistry ; CRISPR-Cas Systems/genetics ; Adenosine Triphosphate/metabolism ; Chromatography, High Pressure Liquid ; Bacterial Proteins/metabolism ; Adenosine Monophosphate/metabolism ; },
abstract = {Cas10, the catalytic core of type III CRISPR-Csm systems, synthesizes cyclic oligoadenylate (cOA) second messengers to activate downstream immune responses. Although Cas10 activity is regulated by complex assembly, the nucleophile selectivity and off-pathway reactivity of isolated Cas10 remain poorly understood. Here, using HPLC separation and subsequent tandem mass spectrometry (MS/MS) analysis, we identify and structurally characterize AMP-O-Tris as a noncanonical adenylylated product generated by isolated Thermococcus onnurineus Cas10. Our results reveal that purified Cas10 exhibits relaxed nucleophile selectivity, diverting ATP turnover into nonproductive adenylylation involving buffer-derived nucleophiles. This suggests that effector complex assembly constrains Cas10 reactivity to promote efficient cOA synthesis and suppress off-pathway chemistry. Furthermore, interception of reactive intermediates by buffer-derived nucleophiles may represent a potential chemical fail-safe that limits unintended signaling when Cas10 is uncoupled from the complex. Together, our study provides mechanistic insight into Cas10 regulation and informs the development of robust type III-based diagnostic platforms. Impact statement Our study reveals that Cas10 exhibits latent catalytic flexibility when isolated, identifying a noncanonical adenylation reaction. These findings demonstrate how complex assembly constrains enzymatic specificity to prevent aberrant signaling. This mechanistic insight is crucial for improving the fidelity and design of next-generation CRISPR-based diagnostic platforms.},
}

*Thermococcus/metabolism/genetics
Tandem Mass Spectrometry/methods
*Adenine Nucleotides/metabolism/chemistry
CRISPR-Cas Systems/genetics
Adenosine Triphosphate/metabolism
Chromatography, High Pressure Liquid
Bacterial Proteins/metabolism
Adenosine Monophosphate/metabolism

@article {pmid42089437,
year = {2026},
author = {Wei, S and Zhang, K and Deng, S and Chen, J and Huang, X and Guo, J and Wu, Y and Guo, Y and Liang, Z},
title = {Transposase-Assisted Donor Tethering Boosts Large-Fragment HDR in Plants.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {43},
pages = {e75565},
pmid = {42089437},
issn = {2198-3844},
support = {32170410//National Natural Science Foundation of China/ ; 62572289//National Natural Science Foundation of China/ ; 202403021221020//Fundamental Research Program of Shanxi Province/ ; 2023-006//Shanxi Scholarship Council of China/ ; 202203021224002//Natural Science Foundation of Shanxi Province for the Excellent Youth/ ; },
mesh = {*Transposases/genetics/metabolism ; *Recombinational DNA Repair/genetics ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; DNA Breaks, Double-Stranded ; },
abstract = {Precise insertion of large DNA fragments by homology-directed repair (HDR) remains inefficient and poorly reproducible in plants, largely due to limited donor availability at double-strand break sites. Here, we develop a transposase-assisted donor tethering strategy that improves the reliability of HDR-mediated large-fragment insertion. By fusing Cas9 to an integration-defective piggyBac variant that retains sequence-specific DNA-binding activity, donor templates are physically co-localized with Cas9-induced breaks. When combined with a transcription-coupled donor and a repair-pathway-biased Cas9 variant, this system enhances the frequency of accurate large-fragment insertions. Using this approach, we achieved efficient and precise kilobase-scale targeted gene insertions across multiple loci in both dicot and monocot species. These findings establish donor tethering as an effective strategy to improve plant HDR efficiency and provide a general framework for precise large-fragment genome insertion.},
}

*Transposases/genetics/metabolism
*Recombinational DNA Repair/genetics
CRISPR-Cas Systems/genetics
Plants, Genetically Modified/genetics
DNA Breaks, Double-Stranded

@article {pmid42309993,
year = {2026},
author = {Yu, W and Chen, J and Guo, J and Yu, F and Wang, G and Lin, J and Dai, X and Tan, X and Ma, P and Wu, L and Zhang, Y and Huang, S and Lan, P and Bian, Q and Huang, X and Wei, J and Cheng, T and Zheng, X and Qiao, Y},
title = {Guide RNA reprogramming facilitates minimized tracrRNA-dependent off-target and versatile CRISPR/Cas9 engineering.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42309993},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; Animals ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Mice ; *Gene Editing/methods ; HEK293 Cells ; Machine Learning ; Transcriptome ; },
abstract = {While innovative, current CRISPR-Cas9 systems face safety concerns and practical hurdles, notably sequence-independent, noncanonical off-targeting. We demonstrate that the crRNA:tracrRNA duplex in guide RNAs (gRNA) is both splittable and reprogrammable. This property, however, enables endogenous RNAs with crRNA-like sequences to hijack any gRNAs, causing low-frequency yet pervasive tracrRNA-dependent off-target (TDO) effects. Using machine learning trained on high-throughput gRNA variant screens, we derive optimal gRNA-designing rules and engineer crRNA variants mismatched to the human/mouse transcriptomes, thereby minimizing TDO. By leveraging splittability and reprogrammability, we develop reprogrammable tracrRNAs for CRISPRa-based mRNA detection and redesign scaffolds to curb PAM-less Cas9-mediated "self-editing". We further create a separately expressed gRNA (segRNA) platform featuring split tracrRNAs and non-repetitive tandem crRNAs, enabling multiplexed editing of up to six genes and functional enhancer annotation in stem cells. Our findings uncover a previously overlooked off-target mechanism and offer versatile strategies to enhance the safety and utility of CRISPR systems.},
}

*CRISPR-Cas Systems/genetics
Animals
Humans
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Mice
*Gene Editing/methods
HEK293 Cells
Machine Learning
Transcriptome

@article {pmid42310027,
year = {2026},
author = {Busquets, O and Li, H and Syed, KM and Jerez, PA and Dunnack, J and Lo Bu, R and Verma, Y and Pangilinan, GR and Martin, A and Straub, J and Du, Y and Simon, VM and Poser, S and Bush, Z and Diaz, J and Sahagun, A and Gao, J and Hong, S and Hernandez, DG and Levine, KS and Pochet, N and Booth, EO and Blanchette, M and Bateup, HS and Rio, DC and Blauwendraat, C and Hockemeyer, D and Soldner, F},
title = {iSCORE-PD: an isogenic stem cell collection to research Parkinson's disease.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42310027},
issn = {2041-1723},
support = {1R56NS128015//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; 1R01NS138402//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; 1R01NS133140//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; 5F31NS129265//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; P30 CA013330/CA/NCI NIH HHS/United States ; P30 CA013330/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Parkinson Disease/genetics/pathology ; Mutation ; Female ; Gene Editing ; alpha-Synuclein/genetics ; *Induced Pluripotent Stem Cells/metabolism ; Whole Genome Sequencing ; Cell Line ; CRISPR-Cas Systems ; *Pluripotent Stem Cells/metabolism ; Genetic Variation ; },
abstract = {Genome-edited human pluripotent stem cells (hPSCs) provide a powerful platform to study complex diseases such as Parkinson's disease (PD). Here, we describe iSCORE-PD, an isogenic collection of 65 genome-edited hPSC lines carrying disease-causing or high-risk variants in 11 PD-linked genes (SNCA, PRKN, PINK1, DJ1/PARK7, LRRK2, ATP13A2, FBXO7, DNAJC6, SYNJ1, VPS13C, and GBA1). All lines are derived from a well-characterized female hESC line and subjected to extensive quality control. Whole-genome sequencing reveals that genetic variation between lines, largely confined to non-coding regions, is minimal relative to inter-individual differences in patient-derived hiPSCs, with most variation arising from random mutations acquired during cell culture rather than genome-editing-induced off-target effects. Including multiple independently derived clones per mutation can control for this random genetic drift. Our systematic approach ensures high quality of this publicly available iSCORE-PD resource, highlights the advantages of prime editing over conventional CRISPR/Cas9 methods, and establishes best practices for generating disease-modeling hPSC collections.},
}

Humans
*Parkinson Disease/genetics/pathology
Mutation
Female
Gene Editing
alpha-Synuclein/genetics
*Induced Pluripotent Stem Cells/metabolism
Whole Genome Sequencing
Cell Line
CRISPR-Cas Systems
*Pluripotent Stem Cells/metabolism
Genetic Variation

@article {pmid42315520,
year = {2026},
author = {Chen, S and Hsiao, S and Xie, T and Chen, D and Chen, N and Jiang, J and Li, J and Wu, Y and Liao, J},
title = {Deep learning-guided engineering of SpuFz1 and rational miniaturization of ωRNA enables efficient genome editing.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42315520},
issn = {2041-1723},
mesh = {Humans ; Animals ; *Gene Editing/methods ; Mice ; *Deep Learning ; *Protein Engineering/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Endonucleases/genetics/metabolism ; HEK293 Cells ; Hematopoietic Stem Cells/metabolism ; CRISPR-Cas Systems ; *RNA/genetics ; Mutation ; },
abstract = {Advancing the performance of programmable genome editing nucleases remains a key challenge in expanding their research and therapeutic applications. Here, we introduce a scalable deep learning-guided protein engineering framework for improving nuclease activity without requiring experimental training data. As a demonstration, we apply this strategy to SpuFz1, a compact Fanzor nuclease of eukaryotic origin, identifying and validating beneficial mutations that produces a multi-mutant variant with an 11.6-fold increase in editing efficiency. In parallel, we use comparative sequence analysis to design and experimentally validate a 75-nt ultrashort ωRNA scaffold, reducing guide RNA length by 79% while maintaining activity. Integration of these optimized components yields enFanzor, a compact genome editing system that achieves editing efficiencies up to 81.9% in mammalian cells, with strong editing performance in both human hematopoietic stem and progenitor cells (HSPCs) and mouse embryos. The outperforming variant developed through this strategy also supports robust CBE and ABE activity. Notably, the shortened ωRNA not only improves nuclease editing specificity but also leads to a substantial increase in base editing efficiency. Together, this work demonstrates the power of combining AI-guided protein optimization with rational RNA design, and establishes a generalizable strategy for engineering next-generation genome editing tools.},
}

Humans
Animals
*Gene Editing/methods
Mice
*Deep Learning
*Protein Engineering/methods
RNA, Guide, CRISPR-Cas Systems/genetics
*Endonucleases/genetics/metabolism
HEK293 Cells
Hematopoietic Stem Cells/metabolism
CRISPR-Cas Systems
*RNA/genetics
Mutation

@article {pmid42333443,
year = {2026},
author = {},
title = {RNA-Triggered Chromatin Shredding Hits Cancer's Hardest Targets.},
journal = {Cancer discovery},
volume = {16},
number = {8},
pages = {OF1},
doi = {10.1158/2159-8290.CD-NW2026-0072},
pmid = {42333443},
issn = {2159-8290},
mesh = {Animals ; Humans ; *Chromatin/genetics/metabolism ; *Neoplasms/genetics/therapy ; Mice ; CRISPR-Cas Systems ; Mutation ; *RNA/genetics ; Tumor Suppressor Protein p53/genetics ; },
abstract = {RNA-triggered chromatin shredding may offer a new way to attack cancers driven by mutations that have resisted conventional drugs, two new studies show. In mouse models, upon activation by a target transcript, the CRISPR enzyme Cas12a2 can selectively eliminate tumor cells carrying mutations in TP53, MYC, and other hard-to-drug cancer genes.},
}

Animals
Humans
*Chromatin/genetics/metabolism
*Neoplasms/genetics/therapy
Mice
CRISPR-Cas Systems
Mutation
*RNA/genetics
Tumor Suppressor Protein p53/genetics

@article {pmid42511810,
year = {2026},
author = {Zaman, W and Ayaz, A},
title = {Beyond Permanent Genome Editing: Molecular Delivery Strategies for RNA Editing and Epigenome-Editing Therapeutics.},
journal = {International journal of molecular sciences},
volume = {27},
number = {14},
pages = {},
pmid = {42511810},
issn = {1422-0067},
mesh = {Humans ; *Epigenome Editing/methods ; *RNA Editing ; Animals ; *Gene Editing/methods ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Transfer Techniques ; },
abstract = {Reversible genetic medicines are emerging as controllable alternatives to permanent genome editing by enabling programmable modulation of RNA sequence, transcript abundance, chromatin state, and gene expression without irreversible genomic alteration. However, reversibility is not a single binary property: transient editor exposure, decay of the molecular effect, recovery of cellular function, and clinical capacity to stop, redose, or counteract treatment may diverge. This review therefore distinguishes mechanistic, functional, and clinical reversibility while examining targeted delivery systems for RNA-editing and epigenome-editing therapeutics. Key payloads include ADAR-recruiting oligonucleotides, CRISPR-Cas13 RNA editors, guide RNAs, chemically modified RNAs, editor-encoding mRNAs, dCas9 transcriptional regulators, DNA methylation editors, histone-modifying systems, and CRISPRoff-like platforms. We evaluate extracellular and intracellular delivery barriers, including nuclease degradation, immune recognition, renal clearance, liver uptake, cellular entry, endosomal escape, cytoplasmic release, nuclear localization, chromatin access, editing-window duration, off-target activity, immunogenicity, repeat-dosing feasibility, manufacturing, quality control, potency assays, and regulatory translation. Overall, delivery systems for reversible genetic medicines should be judged by tissue selectivity, functional editing, duration of action, reversibility after treatment withdrawal, safety, manufacturability, and clinical controllability.},
}

Humans
*Epigenome Editing/methods
*RNA Editing
Animals
*Gene Editing/methods
CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
*Gene Transfer Techniques

@article {pmid42511812,
year = {2026},
author = {Liu, R and Cong, S and Gao, Y and Xu, J and Shi, X},
title = {Application of CRISPR-Cas9-Based Gene Editing Technology in Inherited Liver Diseases.},
journal = {International journal of molecular sciences},
volume = {27},
number = {14},
pages = {},
pmid = {42511812},
issn = {1422-0067},
support = {82300674//National Natural Science Foundation of China/ ; 2024BSL005//Liaoning Normal University Doctoral Research Start-up Project/ ; },
mesh = {Humans ; *Liver Diseases/genetics/therapy ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; Genetic Therapy/methods ; },
abstract = {Inherited liver diseases are predominantly caused by monogenic mutations, and the vast majority of these conditions currently lack curative treatment options. Although liver transplantation may be used for patients with end-stage disease, it faces numerous challenges, including donor organ shortage, immune rejection, and the need for lifelong immunosuppression. In recent years, CRISPR-Cas9-based gene editing technology has advanced rapidly, offering transformative hope for the treatment of these diseases. This review systematically elucidates the working principles and technical advantages of the CRISPR-Cas9 system and its derived tools (base editing and prime editing), summarizes recent applications of these technologies in the treatment of hereditary liver diseases, and discusses the prospects and challenges of their clinical translation, aiming to provide a theoretical reference for future research in this field.},
}

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

@article {pmid42514096,
year = {2026},
author = {Wu, Y and Huang, X and Gu, Q and Li, Y and Zhou, Y and Sun, L and Wang, X},
title = {Characterization of CRISPR Loci and Antimicrobial Resistance in Foodborne Listeria monocytogenes Isolates.},
journal = {Microorganisms},
volume = {14},
number = {7},
pages = {},
pmid = {42514096},
issn = {2076-2607},
support = {2025KJ29//Nanjing Customs/ ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) are widespread in bacterial and archaeal genomes as an adaptive immune system against invading mobile genetic elements. This study investigated the distribution of CRISPR loci and their potential association with antimicrobial resistance (AMR) in 40 foodborne Listeria monocytogenes isolates. CRISPR analysis showed that 18 isolates harbored CRISPR Locus 1, five carried Locus 2, and five possessed both loci. Antimicrobial susceptibility testing against seven antimicrobial agents indicated that most isolates were highly susceptible to the tested agents. Specifically, all isolates were susceptible to gentamicin, ampicillin, penicillin, and tetracycline, whereas resistance was observed in a small subset of isolates: four were resistant to chloramphenicol, five to levofloxacin, and four to ciprofloxacin. Statistical analysis showed no statistically significant association between the presence of CRISPR loci and antimicrobial susceptibility phenotypes. These findings provide baseline information on CRISPR locus distribution and antimicrobial susceptibility profiles in foodborne L. monocytogenes isolates. Further studies based on larger isolate collections, whole-genome sequencing, and characterization of associated cas genes are needed to clarify the potential role of CRISPR-Cas systems in AMR evolution in this species.},
}

@article {pmid42514545,
year = {2026},
author = {Huiban, FA and Galović, V and Tripon, MR and Orlović, S and Tulcan, C and Camen, D},
title = {Nanotechnology-Enabled CRISPR Delivery: Emerging Opportunities in Agriculture and Forest Biotechnology.},
journal = {Plants (Basel, Switzerland)},
volume = {15},
number = {14},
pages = {},
pmid = {42514545},
issn = {2223-7747},
abstract = {Genome editing is one of the key technologies in contemporary plant biotechnology, which has been revolutionized using CRISPR/Cas systems that offer rapid, flexible, and precise options to improve agricultural characteristics, enhance stress tolerance, and accelerate breeding of crops and trees. Despite the significant benefits of CRISPR/Cas systems, their use is restricted by difficulties in genome-editing materials into plant cells. The conventional approaches include Agrobacterium-mediated transformation, particle bombardment and PEG-mediated transfection; these have contributed significantly to advancements in the field; however, dependent on specific plants and requiring tissue cultures, these methods lead to random transgene insertion and poor transformation efficiency. In addition, nanotechnology represents a novel method of delivering CRISPR cargos into plant cells using minimal invasiveness and potentially without DNA. This review provides a synopsis of the most employed CRISPR/Cas systems within plants, comparing the traditional delivery mechanisms and the various nanotechnological delivery vehicles, such as lipid nanoparticles, carbon nanotubes, DNA nanostructures, mesoporous silica nanoparticles, magnetically responsive nanoparticles and green nanomaterials. This review discusses the present challenges of delivery efficacy, biocompatibility, cargo integrity, and regulatory issues, and provides suggestions for future research directions regarding nanotechnology-assisted genome editing for precision breeding, sustainable agriculture, production of crops tolerant to climate conditions, and forest biotechnology.},
}

@article {pmid42514579,
year = {2026},
author = {Janeeshma, E and Das, S and Bouzroud, S and Sarraf, M and Akhtar, N and Mousavi, H and Ibrahimova, U and Fujita, M and Hasanuzzaman, M},
title = {Reactive Oxygen Species in Crop Plants: Production, Detoxification, Signaling, and Molecular Cross-Talk.},
journal = {Plants (Basel, Switzerland)},
volume = {15},
number = {14},
pages = {},
pmid = {42514579},
issn = {2223-7747},
abstract = {Although excess generation of reactive oxygen species (ROS) is harmful for plants, at mild concentrations, they are able to positively regulate the metabolic pathway and signaling cascades of the cell. The mechanisms functional under the ROS-induced stress tolerance need to be decoded for the development of new abiotic stress-tolerant crop varieties with a clear vision. This study details ROS generation, roles of enzymatic antioxidants as well as non-enzymatic antioxidants, and ROS-induced cellular events. Emphasis is given to crucial topics such as cyclin-dependent kinases and mitogen-activated protein kinases signaling mechanisms, calcium-mediated cellular cross-talk, and activation or inactivation of various transcription factors. Introduction of different genetic and molecular approaches to manipulate the ROS pathway helps to find out novel recombinant plant varieties. Selection of specific biotechnological tools, appropriate omics analysis and implementation of CRISPR/Cas 9 in crop plants assist the designing of mutants. This review highlights the beneficial roles of oxidative stress, spotlighting the molecular mechanisms that support the physiological, morphological and biochemical modifications of plant cells.},
}

@article {pmid42516529,
year = {2026},
author = {Nikravan, M and Heidari, H and Khoshnood, S and Ghafourian, S and Kazemian, H},
title = {Molecular Profiling of CRISPR-Cas System, Virulence Traits, and Antimicrobial Resistance in Enterococcus faecalis Clinical Isolates.},
journal = {International journal of microbiology},
volume = {2026},
number = {},
pages = {5903066},
pmid = {42516529},
issn = {1687-918X},
abstract = {INTRODUCTION: Enterococcus faecalis is responsible for life-threatening enterococcal infections. This study is aimed at investigating virulence factors, antimicrobial resistance patterns, and molecular characteristics of clinical E. faecalis isolates.

MATERIALS AND METHODS: A total of 42 E. faecalis isolates were collected. Antimicrobial resistance and the minimum inhibitory concentrations (MICs) of vancomycin and gentamicin were determined using standard microbroth dilution method. The presence of virulence, antibiotic resistance, and CRISPR-Cas genes was investigated by polymerase chain reaction (PCR). Biofilm formation was also assessed, and the genetic diversity of the isolates was analyzed using enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR).

RESULTS: The highest resistance rates were observed for gentamicin and ampicillin, and all isolates were high-level gentamicin resistant (HLGR) (MICs ≥ 500 μg/mL). Vancomycin resistance was detected in 15 isolates (35.7%) (MICs ≥ 32 μg/mL). Thirty-nine isolates (92.8%) were biofilm producers. The efaA gene was the most frequently detected (95.2%), followed by esp (69%), gelE (66.6%), ace (66.6%), and asa1 (57.1%). The aac(6  ['])-Ie-aph(2  [″])-Ia gene was detected in all isolates (100%), followed by ermB (78.6%), ermA (38.1%), ermC (23.8%), and vanA (19%). CRISPR3 was the most frequently detected locus (80.95%), followed by CRISPR1 (50%), CRISPR1-cas csn1 (23.8%), and CRISPR2 (9.5%). The CRISPR3-cas csn1 gene was not detected in any isolate. High heterogeneity was observed among the isolates, with 35 different ERIC types identified.

CONCLUSION: This study demonstrated notable resistance traits and high genetic diversity among clinical E. faecalis isolates, but no statistically significant association was found between CRISPR-Cas genes and phenotypic or genotypic resistance.},
}

@article {pmid42517263,
year = {2026},
author = {Hu, X and Xu, P and Shen, M and Chai, X and Tan, J and Sun, X and Yang, L and Wang, Q and Tan, F},
title = {A CRISPR-Cas12a and Quantum-Dot Lateral-Flow Assay for Rapid Species-Level Detection of Trichophyton rubrum.},
journal = {Mycoses},
volume = {69},
number = {7},
pages = {e70208},
pmid = {42517263},
issn = {1439-0507},
support = {22Y11905700//Program of the Science and Technology Commission of Shanghai Municipality "Science and Technology Innovation Action Plan" Medical Innovation Research Special Project/ ; SHDC12026111//Shanghai Shen Kang Clinical Technology Promotion Program/ ; YXX2024-KF01-04//The Center for Basic Research and Innovation of Medicine and Pharmacy (MOE)/ ; 2026ms03//Xi'an Municipal Health Commission General Research Project/ ; },
mesh = {Humans ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Quantum Dots ; Rapid Diagnostic Tests ; *Arthrodermataceae/isolation & purification/genetics ; *Tinea/diagnosis/microbiology ; *Trichophyton/isolation & purification/genetics ; *Molecular Diagnostic Techniques/methods ; Real-Time Polymerase Chain Reaction ; DNA, Fungal/genetics ; },
abstract = {BACKGROUND: Accurate species-level diagnosis of dermatophytes, particularly Trichophyton rubrum, is important for guiding targeted antifungal therapy and improving the management of recurrent or atypical superficial mycoses. However, routine microscopy has only moderate sensitivity and cannot resolve fungal species; fungal culture is time-consuming, and real-time quantitative PCR (qPCR) requires costly instrumentation.

OBJECTIVES: To develop and clinically evaluate CRI-RUB, a CRISPR-Cas12a-based assay integrating recombinase-aided amplification (RAA) and a quantum-dot (QD) fluorescent lateral-flow strip for rapid species-level detection of T. rubrum.

PATIENTS/METHODS: Analytical sensitivity and specificity were assessed using plasmid standards and a panel of common cutaneous fungi. A total of 140 clinical specimens were tested by both CRI-RUB and TaqMan qPCR. Discrepant or grey-zone qPCR results (Ct 33-34), together with a subset of concordant cases, were further arbitrated by internal transcribed spacer (ITS) sequencing to establish a composite reference standard.

RESULTS: CRI-RUB achieved a visual limit of detection of 2.3 × 10[1] copies/μL and showed no cross-reactivity with other common cutaneous fungi. Among the 140 clinical specimens, CRI-RUB showed a sensitivity of 96.1% (73/76), specificity of 100.0% (64/64), positive predictive value of 100.0% (73/73), negative predictive value of 95.5% (64/67) and Cohen's κ of 0.96 compared with the composite reference standard, indicating excellent concordance. The complete workflow required approximately 85 min.

CONCLUSIONS: CRI-RUB provides a rapid, sensitive, and specific approach for species-level detection of T. rubrum. By combining CRISPR-Cas12a detection with QD-based lateral-flow readout, this assay shows potential for near-patient or point-of-care dermatophyte diagnosis.},
}

Humans
Sensitivity and Specificity
*CRISPR-Cas Systems
*Quantum Dots
Rapid Diagnostic Tests
*Arthrodermataceae/isolation & purification/genetics
*Tinea/diagnosis/microbiology
*Trichophyton/isolation & purification/genetics
*Molecular Diagnostic Techniques/methods
Real-Time Polymerase Chain Reaction
DNA, Fungal/genetics

@article {pmid42510959,
year = {2026},
author = {Gubaidullin, N and Ospankulova, G and Gajimuradova, A and Syzdykova, A and Zhumalin, A and Dairova, K and Konysbayeva, D and Gorbulya, V and Makangali, K},
title = {ROS-Centered Transcriptomic Regulatory Networks Linking Salinity Stress, Antioxidant Defense and Processability Traits in Salicornia spp.},
journal = {Current issues in molecular biology},
volume = {48},
number = {7},
pages = {},
pmid = {42510959},
issn = {1467-3045},
support = {BR22883587//the ministry of agriculture of Kazakhstan/ ; },
abstract = {Salinity stress affects not only the survival and productivity of halophytic plants, but also the composition, structure and processability of their biomass. In Salicornia spp., salt-induced regulation of ion transport, osmotic adjustment, reactive oxygen species signaling, antioxidant defense, and cell wall remodeling can directly influence residual salinity, water retention, texture, extractability, drying behavior, and oxidative stability of plant biomass. However, most existing transcriptomic studies of Salicornia and related halophytes have focused mainly on salt tolerance mechanisms, whereas the connection between stress-regulated molecular networks and processing-related biomass traits remains insufficiently systematized. This review addresses this gap by proposing a mechanistic framework that links salinity perception, ROS-mediated signaling, ABA and MAPK pathways, antioxidant gene families, transcription factor networks and processing-oriented quality traits. Special attention is given to enzymatic antioxidant systems, including SOD, CAT, APX, POD and components of the ascorbate-glutathione cycle, as well as to non-enzymatic defense mechanisms involving ascorbate, glutathione, phenolic compounds, carotenoids, proline and glycine betaine. The review also discusses the regulatory roles of WRKY, DREB/CBF, NAC, bZIP and MYB transcription factor families as molecular control points connecting salinity stress responses with downstream metabolic and structural traits. Network-based approaches, including WGCNA, pathway signatures and transcript panels, are considered more informative than single-gene markers for predicting complex quality traits in Salicornia biomass. In addition, recent genomic and computational strategies, including CRISPR/Cas-mediated functional validation, GWAS, genomic selection, multi-omics integration and AI-assisted modeling, are discussed as emerging tools for candidate-gene prioritization and predictive assessment of stress-dependent biomass quality. Overall, this review shifts the interpretation of Salicornia transcriptomics from a descriptive salt-tolerance model toward a mechanistic and application-oriented framework for improving halophytic raw materials for food, feed and bioprocessing applications.},
}

@article {pmid42511636,
year = {2026},
author = {Zhan, Y and Luo, T and Sun, Y},
title = {Efficient Gene Editing in Fish Primary Germline Stem Cells.},
journal = {International journal of molecular sciences},
volume = {27},
number = {14},
pages = {},
pmid = {42511636},
issn = {1422-0067},
support = {2022YFD2400101//The National Key R&D Program of China/ ; },
mesh = {Animals ; *Gene Editing/methods ; CRISPR-Cas Systems ; Transfection/methods ; Oryzias/genetics ; *Stem Cells/metabolism/cytology ; *Germ Cells/metabolism/cytology ; *Eels/genetics ; Carps/genetics ; },
abstract = {Genome editing by the CRISPR/Cas9 system is widely used for production of gene-modified animals, including fish. However, efficient gene editing in fish cultured cells, in particular germline stem cells (GSCs), is challenging, likely due to the difficulty in transfecting these cells. The ricefield eel (Monopterus albus), a sequential hermaphroditic species, is a freshwater fish of significant economic value in China. In this work, we report a simple method that can achieve high gene editing efficiency in primary GSCs of fish species, including ricefield eel. High transfection efficiency (50-95%) is achieved in fish GSCs by using a microchannel-based cell transfection system. Gene editing efficiency of up to 60% in primary ricefield eel GSCs is achieved using an integrated CRISPR/Cas9 vector strategy. High gene editing efficiency is also achieved in gibel carp (Carassius gibelio) GSCs and the medaka spermatogonial stem cell line SG3, demonstrating the general applicability of our method. Our data suggest that efficient transfection is key to high gene editing efficiency in fish cultured GSCs. This study establishes an efficient and reliable gene editing system for fish GSCs, which may facilitate the creation of new germplasm of genetically difficult-to-breed fish by combining GSC transplantation with gene editing techniques.},
}

Animals
*Gene Editing/methods
CRISPR-Cas Systems
Transfection/methods
Oryzias/genetics
*Stem Cells/metabolism/cytology
*Germ Cells/metabolism/cytology
*Eels/genetics
Carps/genetics

@article {pmid42480477,
year = {2026},
author = {Toishigawa, K and Magoori, K and Sato, H and Edahiro, T and Ureshino, H and Shindo, T and Suzuki, R and Sakuma, T and Yamamoto, T and Okada, M and Ichinohe, T},
title = {Platinum TALEN-mediated nonviral gene editing facilitates clinical-scale production of cancer antigen-reactive T cells.},
journal = {Cytotherapy},
volume = {28},
number = {9},
pages = {102911},
doi = {10.1016/j.jcyt.2026.102911},
pmid = {42480477},
issn = {1477-2566},
mesh = {Humans ; *Transcription Activator-Like Effector Nucleases/genetics/metabolism ; *Gene Editing/methods ; *Antigens, Neoplasm/immunology/genetics ; *T-Lymphocytes/immunology ; *Receptors, Antigen, T-Cell/genetics ; CRISPR-Cas Systems/genetics ; *Neoplasms/immunology/therapy ; },
abstract = {BACKGROUND AIMS: Recently, target-genome editing has emerged as a next-generation tool for the clinical development of designed cellular products. Although Clustered Regularly Interspaced Short Palindromic Repeats-Cas9 (CRISPR-Cas9) is the most frequently adopted nuclease for therapeutic genome editing, its widespread use is still hampered by potential off-target effects and high patent royalties. Platinum transcription activator-like effector nuclease (TALEN) is a modified TALEN that harbors non-repeat-variable di-residue (non-RVD) variations and confers higher efficiency than conventional TALENs lacking non-RVD variations.

METHODS: In this study, using Platinum TALEN targeting T-cell receptor (TCR) gene loci and a single-stranded DNA homology-directed repair (HDR) template, we aimed to produce TCR-replaced human T cells reprogrammed to recognize a cancer antigen and kill cancer cells.

RESULTS: This system can reproducibly produce TCR-engineered T cells from ∼50 mL of peripheral blood on a clinical scale. The resulting genome-edited T cells retain naïve/naïve-like and memory phenotype cells in both CD4+ and CD8+ fractions and exhibit efficient cytolytic activity against cancer cell lines in vitro.

CONCLUSION: In conclusion, Platinum TALEN-mediated nonviral genome editing facilitates the replacement of the endogenous TCR with a desired TCR and can be applied to the clinical manufacturing of therapeutic T-cell products.},
}

Humans
*Transcription Activator-Like Effector Nucleases/genetics/metabolism
*Gene Editing/methods
*Antigens, Neoplasm/immunology/genetics
*T-Lymphocytes/immunology
*Receptors, Antigen, T-Cell/genetics
CRISPR-Cas Systems/genetics
*Neoplasms/immunology/therapy

@article {pmid42510814,
year = {2026},
author = {Zhang, X and Lu, C and Hu, Z and Geng, X and Li, G and Cai, J},
title = {Complete Genome Analysis of Pectobacterium brasiliense BS1113, a Causal Agent of Cigar Tobacco Soft Rot, with Phenotypic Characterization of Virulence and Copper Tolerance.},
journal = {Genes},
volume = {17},
number = {7},
pages = {},
pmid = {42510814},
issn = {2073-4425},
support = {This research was funded by the Major Research Project of Fuyang Normal University (2025FSKJ31), the Doctoral Talent Introduction Project of Fuyang Normal University (2020KYQD0031), the Major Project of Anhui Huatuo Academy of Traditional Chinese Medicine//Fuyang Normal University/ ; },
mesh = {*Copper/metabolism/toxicity ; *Plant Diseases/microbiology/genetics ; *Nicotiana/microbiology ; Virulence/genetics ; *Genome, Bacterial ; Phenotype ; Whole Genome Sequencing ; },
abstract = {Background:Pectobacterium brasiliense-mediated soft rot severely threatens the production of diverse cash crops worldwide and brings severe yield reduction risks. A virulent strain BS1113 was separated from diseased cigar tobacco plants collected in Yunnan, yet its virulence regulatory genes and copper resistance-related genetic background have not been fully analyzed so far. This study aims to decipher the genomic features of BS1113 and clarify its pathogenic and copper-tolerant characteristics via whole-genome sequencing, comparative genomics and indoor phenotype verification. Methods: Hybrid sequencing strategies combining Illumina short reads and PacBio long reads were adopted to obtain the complete circular genome sequence of strain BS1113. Subsequent comparative genomic analysis and multiple phenotypic identification experiments were conducted to characterize its genetic architecture and physiological traits. Results: Genome assembly results showed that the circular chromosome of BS1113 spans 4,916,962 bp with a GC content of 51.96%, which encodes a total of 4369 functional protein-coding genes. Genomic comparison revealed that BS1113 completely lacks the T3SS gene cluster, while it conserves intact T2SS, T6SS and I-F CRISPR-Cas systems; the chromosomal copper resistance operon copRSAB was also detected in this isolate. Pathogenicity tests validated that BS1113 satisfies all criteria of Koch's postulates on cigar tobacco hosts. In addition, BS1113 displayed prominent tolerance against eight mainstream copper bactericides widely used for tobacco disease management. Conclusions: This research generates the first complete high-quality genome of P. brasiliense isolated from cigar tobacco hosts. The genomic data explain the infection mechanism of this pathogen independent of intact T3SS, and also reveal the genetic basis supporting its persistent survival under long-term copper fungicide pressure in field cultivation environments.},
}

*Copper/metabolism/toxicity
*Plant Diseases/microbiology/genetics
*Nicotiana/microbiology
Virulence/genetics
*Genome, Bacterial
Phenotype
Whole Genome Sequencing

@article {pmid42510829,
year = {2026},
author = {Yoshino, H and Fukuda, I and Enokida, H and Seki, N and Goto, Y},
title = {MEK1 as a Synthetic Lethal Target with Cabozantinib in Renal Cell Carcinoma: Insights from CRISPR/Cas9 Screening.},
journal = {Genes},
volume = {17},
number = {7},
pages = {},
pmid = {42510829},
issn = {2073-4425},
support = {24K12458//Japan Society for the Promotion of Science/ ; 24K11347//Japan Society for the Promotion of Science/ ; 24K12641//Japan Society for the Promotion of Science/ ; },
mesh = {Humans ; *Pyridines/pharmacology ; *Carcinoma, Renal Cell/genetics/drug therapy/pathology ; *Anilides/pharmacology ; Animals ; *Kidney Neoplasms/genetics/drug therapy/pathology ; *MAP Kinase Kinase 1/genetics/antagonists & inhibitors ; CRISPR-Cas Systems/genetics ; Mice ; Cell Line, Tumor ; *Synthetic Lethal Mutations ; Xenograft Model Antitumor Assays ; Protein Kinase Inhibitors/pharmacology ; Cell Proliferation/drug effects ; Cell Survival/drug effects ; },
abstract = {Background/Objectives: Cabozantinib is a tyrosine kinase inhibitor that primarily targets MET. It has become an important drug in the treatment of renal cell carcinoma (RCC); however, many patients do not respond to cabozantinib treatment and there is no effective next-line therapy. In this study, we identified molecular-targeted drugs that exhibit synergistic effects with cabozantinib using CRISPR/Cas9 screening. Methods: A kinome-wide synthetic lethal CRISPR/Cas9 screen was used to identify target molecules using 786-o RCC cells. A library was generated, and treatment with vehicle or cabozantinib was carried out, followed by next-generation sequencing to identify candidate genes. A combination index based on the Chou-Talalay method was used to evaluate the synergistic effect of cabozantinib through cell viability assays. Xenograft assays were conducted to determine the effect in vivo. Results: CRISPR/Cas9-based screening revealed four genes (MEK1, DCLK1, DYRK3, and FGFR1) that were candidates for synthetic lethality by cabozantinib in RCC cells. We focused on MEK1 because the MEK1 inhibitor cobimetinib has been approved for melanoma treatment. In a cell proliferation assay using 786-o and A498 RCC cells, the combination of cobimetinib and cabozantinib exhibited a synergistic effect. A xenograft assay also revealed a significant synergistic effect of cobimetinib and cabozantinib. Conclusions: CRISPR/Cas9 screening identified MEK1 as a candidate for a synthetic lethal target with cabozantinib in RCC. The combined inhibition of MET/VEGFR and MEK1 suppressed compensatory MAPK reactivation and downregulated the PI3K-Akt pathway, including the survival-associated genes PPP2R3B and ATF6B, and produced significant tumor growth suppression in vivo. These findings highlight the potential of cabozantinib plus cobimetinib, an already-FDA-approved MEK inhibitor, as a readily translatable combination strategy to overcome cabozantinib resistance in RCC.},
}

Humans
*Pyridines/pharmacology
*Carcinoma, Renal Cell/genetics/drug therapy/pathology
*Anilides/pharmacology
Animals
*Kidney Neoplasms/genetics/drug therapy/pathology
*MAP Kinase Kinase 1/genetics/antagonists & inhibitors
CRISPR-Cas Systems/genetics
Mice
Cell Line, Tumor
*Synthetic Lethal Mutations
Xenograft Model Antitumor Assays
Protein Kinase Inhibitors/pharmacology
Cell Proliferation/drug effects
Cell Survival/drug effects

@article {pmid42510848,
year = {2026},
author = {Zafar, MA and Harling, LC and Li, Y and Celik, NB and Mukherjee, SK and Rizzo, J and Prendergast, A and Elefteriades, JA},
title = {A Preliminary Zebrafish Model of ACTA2 Deficiency Reveals Increased Larval Phenotype Burden and Suggests Reduced Adult Mutant Survival.},
journal = {Genes},
volume = {17},
number = {7},
pages = {},
pmid = {42510848},
issn = {2073-4425},
support = {N/A//Masone Family/ ; Walter-Benjamin Scholarship//German Research Foundation/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Actins/genetics/deficiency ; Phenotype ; Larva/genetics ; Mutation ; *Zebrafish Proteins/genetics/deficiency ; Disease Models, Animal ; Genotype ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: ACTA2 encodes smooth muscle alpha-actin and is one of the most common genetic causes of inherited non-syndromic thoracic aortic aneurysm and dissection. Although murine models have provided important mechanistic insight, complementary vertebrate systems may enable more rapid in vivo phenotyping and future therapeutic screening.

METHODS: Utilizing a CRISPR/Cas9-based approach, we developed zebrafish acta2 mutant models and assessed phenotypes at larval and adult stages. At 3 days post-fertilization, larvae were screened by brightfield microscopy for edema, axis defects, hemorrhage, and a composite endpoint (any phenotype) under basal conditions and after exposure to 0.2 mM epinephrine. Phenotype occurrence was summarized as group-specific proportions across a 2 × 2 design defined by genotype (wild-type vs. acta2-deficient mutant) and treatment (DMSO control vs. epinephrine), with prespecified pairwise comparisons using Pearson's chi-squared tests. Pooled fish-level logistic regression models were also fit for each endpoint. Adult follow-up was performed by genotyping all available acta2 fish at the facility and tracking staggered tank-level cohorts longitudinally.

RESULTS: Initial gross morphologic assessment did not reveal overt external phenotypic differences between heterozygous or homozygous acta2 mutant larvae and wild-type controls. In larval analyses, the mutant genotype was associated with a greater burden of adverse phenotypes than treatment exposure. For the composite endpoint, mutant larvae demonstrated higher proportions than normal larvae under both control (28.3% vs. 17.4%, p = 0.007) and epinephrine conditions (26.9% vs. 17.4%, p = 0.023), whereas epinephrine did not significantly alter composite phenotype frequency within either genotype. Similar genotype-associated trends were observed for axis defects and hemorrhage. In pooled logistic regression, acta2 deficiency was associated with increased odds of axis defects (OR 2.64, 95% CI 1.48-4.89, p = 0.001), hemorrhage (OR 2.40, 95% CI 1.09-5.68, p = 0.036), and the composite endpoint (OR 1.88, 95% CI 1.19-3.00, p = 0.008), whereas epinephrine exposure did not demonstrate a consistent independent effect across endpoints. Adult staggered follow-up showed greater attrition in homozygous mutant cohorts than in wild-type or heterozygous cohorts, with aggregated losses of 24%, 12%, and 3%, respectively.

CONCLUSIONS: This preliminary zebrafish acta2 model demonstrated that acta2 deficiency is associated with increased adverse larval phenotype burden and suggested reduced long-term persistence of homozygous mutant fish in adulthood. The strongest signal in the current study was genotype-associated phenotype burden rather than a robust epinephrine-dependent effect. These findings support the feasibility of zebrafish-based ACTA2 phenotyping while highlighting the need for more specific vascular endpoints, refined longitudinal follow-up, and future variant-specific modeling.},
}

Animals
*Zebrafish/genetics
*Actins/genetics/deficiency
Phenotype
Larva/genetics
Mutation
*Zebrafish Proteins/genetics/deficiency
Disease Models, Animal
Genotype
CRISPR-Cas Systems

@article {pmid42259495,
year = {2026},
author = {Chandrapalan, T and Kwong, RWM},
title = {DMT1 regulates systemic trace metal handling and developmental outcomes: Insights from targeted mutagenesis in zebrafish.},
journal = {Comparative biochemistry and physiology. Toxicology & pharmacology : CBP},
volume = {308},
number = {},
pages = {110597},
doi = {10.1016/j.cbpc.2026.110597},
pmid = {42259495},
issn = {1532-0456},
mesh = {Animals ; *Zebrafish/genetics/metabolism/growth & development ; *Cation Transport Proteins/genetics/metabolism ; *Zebrafish Proteins/genetics/metabolism ; Solute Carrier Family 11, Member 2 ; *Trace Elements/metabolism ; Mutagenesis ; Gene Expression Regulation, Developmental ; CRISPR-Cas Systems ; Gene Knockout Techniques ; },
abstract = {Divalent metal transporter 1 (DMT1) is thought to be the primary route for non-heme iron absorption in vertebrates, but its systemic role remains poorly understood. Using CRISPR-Cas9 gene editing, we generated a DMT1 knockout (dmt1[-/-]; KO) zebrafish mutant line to examine the developmental and physiological consequences of DMT1 loss. Phenotypic and hematological assessments were performed alongside measurements of whole-body and tissue-specific metal concentrations. Further, to identify potential compensatory pathways during DMT1 loss, the expression profile of candidate metal transporters or ion channels (hcp1, zip4, zip8, zip14, and ecac) was quantified using droplet digital PCR (ddPCR). DMT1 KO larvae exhibited delayed development, anemia, and broad disruption in multiple trace metals (iron, zinc, manganese, cobalt, and selenium). Gene expression analysis during early development revealed higher hcp1 mRNA abundance in the mutant, suggesting a possible compensatory response to maintain metal homeostasis during DMT1 loss. Although viable to adulthood, the mutants had persisting iron dysregulation and red blood cell abnormalities. This study provides the first in vivo evidence of the physiological role of DMT1 in multi-metal balance in fish and offers new insight into compensatory mechanisms underlying DMT1 deficiency.},
}

Animals
*Zebrafish/genetics/metabolism/growth & development
*Cation Transport Proteins/genetics/metabolism
*Zebrafish Proteins/genetics/metabolism
Solute Carrier Family 11, Member 2
*Trace Elements/metabolism
Mutagenesis
Gene Expression Regulation, Developmental
CRISPR-Cas Systems
Gene Knockout Techniques

@article {pmid42288133,
year = {2026},
author = {Abrahms, ZN and Majdi, M and Madsen, SM and Morton, CJ and Sen, AK and Fried, NB and Sawyer, LE and Cegielski, ER and Spezzano, SJ and Jones, JA},
title = {Genome-based optimization of psilocybin and N,N-dimethyltryptamine biosynthetic pathways in E. coli using CRISPR-associated transposases.},
journal = {Metabolic engineering},
volume = {97},
number = {},
pages = {102490},
doi = {10.1016/j.ymben.2026.102490},
pmid = {42288133},
issn = {1096-7184},
mesh = {*Escherichia coli/genetics/metabolism ; *Psilocybin/biosynthesis/genetics ; *Metabolic Engineering/methods ; *Genome, Bacterial ; *CRISPR-Cas Systems ; *Biosynthetic Pathways/genetics ; },
abstract = {Stable, high-level biosynthesis of complex natural products requires precise control of heterologous pathway expression, yet transcriptional architectures optimized on plasmids often fail when transferred to the chromosome. Here, we present ePathIntegrate, a genome-centric pathway engineering strategy that leverages CRISPR-associated transposases (CASTs) to integrate and rebalance multigene metabolic pathways in Escherichia coli. Direct genomic transfer of plasmid-optimized psilocybin and N,N-dimethyltryptamine (DMT) pathways resulted in a loss of productivity, driven by context-dependent promoter behavior. To address this, we developed and characterized a library of mutant T7 promoters that restore mid-range transcriptional control on the genome. Applying ePathIntegrate enabled re-optimization of both pathways, yielding genome-encoded strains that achieve 1.88 g/L psilocybin and 1.62 g/L DMT in fed-batch bioreactors. Whole-genome sequencing of CAST-mediated strains further revealed (i) precise on-target integration, (ii) some off-target pathway integrations, and (iii) small mutations in a subset of strains, highlighting both the power and limitations of CAST-mediated strain engineering.},
}

*Escherichia coli/genetics/metabolism
*Psilocybin/biosynthesis/genetics
*Metabolic Engineering/methods
*Genome, Bacterial
*CRISPR-Cas Systems
*Biosynthetic Pathways/genetics

@article {pmid42297823,
year = {2026},
author = {Adler, KM and Xu, H and Gladstein, AC and Irizarry-Negron, VM and Robertson, MR and Doerig, KR and Petrov, DA and Winslow, MM and Feldser, DM},
title = {Tumor suppressor genotype influences the extent and mode of immunosurveillance in lung cancer.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42297823},
issn = {2041-1723},
support = {R01-CA262619-04//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; R01-CA-279698-01)//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Animals ; *Lung Neoplasms/genetics/immunology/pathology ; Genotype ; *Genes, Tumor Suppressor ; Humans ; *Immunologic Surveillance/genetics ; Immunoediting, Cancer ; Antigens, Neoplasm/immunology/genetics ; Proto-Oncogene Proteins p21(ras)/genetics ; Mice ; CRISPR-Cas Systems ; Cell Line, Tumor ; },
abstract = {The impact of cancer driving mutations on immunosurveillance throughout tumor development remains poorly understood. To better understand the contribution of tumor genotype to immunosurveillance, we generated and validated lentiviral-based vectors that create increasingly immunogenic neoantigens. This vector system is compatible with autochthonous Cre-regulated cancer models, CRISPR/Cas9-mediated somatic genome editing, and tumor barcoding. Here, we show that in the context of oncogenic KRAS-driven lung cancer and strong neoantigen expression, tumor suppressor genotype dictates the degree of immune cell recruitment, positive selection of tumors with neoantigen silencing, and tumor outgrowth. By quantifying the impact of 11 commonly inactivated tumor suppressor genes on tumor growth across neoantigenic contexts, we show that the growth-promoting effects of tumor suppressor gene inactivation correlate with increasing sensitivity to immunosurveillance. Importantly, some genotypes also dramatically changed sensitivity to immunosurveillance independently of their growth-promoting effects. We propose a model of immunoediting in which tumor suppressor gene inactivation works in tandem with neoantigen expression to shape tumor immunosurveillance and immunoediting such that the same neoantigens uniquely modulate tumor immunoediting depending on the genetic context.},
}

Animals
*Lung Neoplasms/genetics/immunology/pathology
Genotype
*Genes, Tumor Suppressor
Humans
*Immunologic Surveillance/genetics
Immunoediting, Cancer
Antigens, Neoplasm/immunology/genetics
Proto-Oncogene Proteins p21(ras)/genetics
Mice
CRISPR-Cas Systems
Cell Line, Tumor

@article {pmid42479086,
year = {2026},
author = {Ma, L and Zhao, J and Chen, Y and Wang, Z and Yan, B and Liu, Z and Ma, N},
title = {RI-augmented TdT-CRISPR fluorescent biosensor for sensitive quantification of DNA breakage in CT-irradiated human sperm.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {29},
pages = {6090-6094},
doi = {10.1039/d6ay01260d},
pmid = {42479086},
issn = {1759-9679},
mesh = {Humans ; *Spermatozoa/radiation effects/metabolism ; *Biosensing Techniques/methods ; Male ; *DNA Damage ; *DNA ; *CRISPR-Cas Systems ; Limit of Detection ; },
abstract = {Featuring a linear range of 0.001-0.4 nM and a limit of detection of 0.12 pM, the RI-enhanced TdT-CRISPR sensor precisely detects radiation-induced trace DNA damage in sperm, outperforming the conventional DFI method.},
}

Humans
*Spermatozoa/radiation effects/metabolism
*Biosensing Techniques/methods
Male
*DNA Damage
*DNA
*CRISPR-Cas Systems
Limit of Detection

@article {pmid42482463,
year = {2026},
author = {Yu, J and Wang, J and Yang, Y and Sun, L and Hu, X and Nie, T and Yang, X and Wang, X and Li, C and You, X},
title = {Multifaceted effects of galU deletion on phenotype and virulence of Pseudomonas aeruginosa in vitro and in vivo.},
journal = {Virulence},
volume = {17},
number = {1},
pages = {2707803},
doi = {10.1080/21505594.2026.2707803},
pmid = {42482463},
issn = {2150-5608},
mesh = {*Pseudomonas aeruginosa/pathogenicity/genetics/drug effects ; Virulence ; Animals ; Pseudomonas Infections/microbiology ; Anti-Bacterial Agents/pharmacology ; *Gene Deletion ; *Bacterial Proteins/genetics/metabolism ; Phenotype ; O Antigens/genetics/biosynthesis ; Polymyxins/pharmacology ; Quorum Sensing ; Microbial Sensitivity Tests ; Mice ; CRISPR-Cas Systems ; },
abstract = {Pseudomonas aeruginosa is a widespread Gram-negative opportunistic pathogen in environmental and hospital settings, frequently causing respiratory diseases such as cystic fibrosis (CF), chronic obstructive pulmonary disorder (COPD), and ventilator-associated pneumonia. In our previous study, a galU-deleted clinical P. aeruginosa was found to exhibit increased susceptibility to polymyxins. The galU gene plays an important role in the biosynthesis of lipopolysaccharide (LPS) O-antigen. Here, we systematically evaluated the effects of galU deletion on the phenotype and virulence of P. aeruginosa PAO1. A galU deletion mutant was successfully constructed in P. aeruginosa PAO1 by CRISPR/Cas9, and the complementation was accomplished by pUCP18 plasmid carrying wild-type galU. The changes in phenotype, virulence, and pathogenicity were systemically studied. The results revealed that knockout of galU led to the loss of O-antigen, which affected growth, virulence, and pathogenicity through various ways in P. aeruginosa, and significantly affected the susceptibility of P. aeruginosa to polymyxins. Mechanism study suggested the involvements of quorum sensing, Entner-Doudoroff pathway, and tyrosine metabolism on bacterial virulence and antibiotic susceptibility changes after galU deletion. galU and the related pathways may serve as effective targets for the treatment of P. aeruginosa infection, providing a theoretical basis for the development of novel antibacterial drugs.},
}

*Pseudomonas aeruginosa/pathogenicity/genetics/drug effects
Virulence
Animals
Pseudomonas Infections/microbiology
Anti-Bacterial Agents/pharmacology
*Gene Deletion
*Bacterial Proteins/genetics/metabolism
Phenotype
O Antigens/genetics/biosynthesis
Polymyxins/pharmacology
Quorum Sensing
Microbial Sensitivity Tests
Mice
CRISPR-Cas Systems

@article {pmid42508244,
year = {2026},
author = {Chen, W and Cao, L and Zhu, X and Kang, W and Qiu, Y and Nie, Z and Huang, Y and Lei, C},
title = {Proteolysis-triggered CRISPR/Cas activation via cascaded enzyme switches for viral protease detection.},
journal = {Biosensors & bioelectronics},
volume = {312},
number = {},
pages = {119073},
doi = {10.1016/j.bios.2026.119073},
pmid = {42508244},
issn = {1873-4235},
abstract = {Sensitive analysis of viral proteases is crucial for early infection diagnosis and antiviral drug development; however, developing activity-based assays with high sensitivity and broad adaptability remains a significant challenge. To address this, we developed a cascade signal amplification strategy that enhances protease detection sensitivity through rational coupling of two modular, protease-responsive enzyme switches. Upon recognition of the target protease, this upstream switch triggers the activation of a downstream, protease-responsive CRISPR/Cas12a effector, which converts the specific proteolytic event into an amplified fluorescence signal. This cascaded enzymatic amplification generates a robust signal output, achieving an order-of-magnitude improvement in detection sensitivity compared to single-stage CRISPR/Cas12a assays. The modular nature of the enzyme switches renders the system highly expandable, allowing for the specific detection of diverse viral proteases. We demonstrated the platform's utility in complex biological samples by sensitively monitoring 3C protease activity within enterovirus 71-infected cells. Furthermore, dose-dependent inhibition analysis using the HRV 3C protease inhibitor rupintrivir validated the system's potential for evaluating antiviral drug efficacy. Collectively, this work establishes a versatile and modular analytical platform for the sensitive detection and functional study of viral proteases.},
}

@article {pmid42508886,
year = {2026},
author = {Tian, S and Tang, X and Zhang, S and Yao, Y and Bi, R and Li, H and Sun, X and Chen, Z},
title = {Construction of the CRISPR/Cas12a-digital integrated immunoassay technology for CD44 detection.},
journal = {Analytica chimica acta},
volume = {1417},
number = {},
pages = {346010},
doi = {10.1016/j.aca.2026.346010},
pmid = {42508886},
issn = {1873-4324},
mesh = {Humans ; Immunoassay/methods ; *Hyaluronan Receptors/blood/analysis ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; },
abstract = {Digital immunoassay allows for the detection of proteins at fg/mL levels by leveraging the principles of Poisson distribution. Nevertheless, the inherent Poisson noise restricts digital sensitivity to targets exceeding 100 molecules, rendering standard digital assays incapable of detecting targets below this threshold. To overcome this constraint, we introduce an integrated CRISPR/Cas12a-digital immunoassay technology. In this system, the CRISPR/Cas12a machinery first acts as a molecular amplifier, converting each target protein into multiple enzymatic reporters. These enzyme molecules are then individually quantified via a digitized readout system, enabling ultrasensitive protein detection. By harnessing the catalytic amplification of CRISPR/Cas12a, the method achieves detection of proteins at copy numbers below 100, effectively surpassing the conventional sensitivity barrier of digital immunoassays. We validated this approach through highly selective and accurate detection of CD44 protein. A linear response was observed across a concentration range of 0.05 to 5 fg/mL, conforming to the calibration model: P(X > 0) = 0.1191c + 0.0036. The limit of detection was determined to be 0.018 fg/mL, equivalent to approximately 36 molecules of CD44. The method was further applied to quantify CD44 in plasma samples from colorectal cancer patients, demonstrating its strong potential for clinical use in early cancer diagnosis and treatment monitoring.},
}

Humans
Immunoassay/methods
*Hyaluronan Receptors/blood/analysis
*CRISPR-Cas Systems/genetics
Limit of Detection

@article {pmid42510584,
year = {2026},
author = {Yang, JW and Kim, HS and Kim, YH},
title = {Redox Regulation of Plant-Root-Knot Nematode Interactions: From ROS-Mediated Immunity to Sustainable Resistance.},
journal = {Antioxidants (Basel, Switzerland)},
volume = {15},
number = {7},
pages = {},
pmid = {42510584},
issn = {2076-3921},
abstract = {Root-knot nematodes (RKNs; Meloidogyne spp.) are among the most destructive plant parasites, causing severe yield losses in diverse crops. Reactive oxygen species (ROS), particularly superoxide radicals (O2•[-]) and hydrogen peroxide (H2O2), are central regulators of plant-RKN interactions. This review synthesizes current molecular, biochemical, genetic, transcriptomic, and translational evidence showing that the outcome of infection is determined by the spatiotemporal regulation of H2O2 rather than by ROS abundance alone. In resistant interactions, nematode perception activates PTI-associated signaling through selected cell-surface receptor complexes, including some BAK1/SERK3-associated pathways, together with BIK1, Ca[2+] signaling, and RBOHD/F, generating a sustained oxidative activity associated with salicylic acid-dependent immune signaling and reduced H2O2-scavenging capacity and coupled to hypersensitive response, lignin and callose deposition, and feeding site restriction. In susceptible interactions, RKNs deploy ROS-targeting effectors such as Mi-CRT, MjTTL5, CATLe, Mj-NEROSs, and CMII to suppress ROS production, enhance antioxidant scavenging, or weaken SA-dependent defense. Evidence from a cyst-nematode system suggests that RBOH-derived ROS can restrict excessive cell death around syncytia; whether an analogous lower-redox requirement exists in RKN-induced giant cells remains unresolved. Finally, redox-based strategies, including CRISPR/Cas editing, host-induced gene silencing, chemical priming, and biocontrol, are discussed as promising approaches for durable and sustainable nematode resistance.},
}

@article {pmid42510769,
year = {2026},
author = {Wyman, SK and Romero, Z and Heo, SJ and Navarrete, M and Krishnappa, N and Kohn, DB and Martin, DIK and Walters, MC and Boffelli, D},
title = {Oligonucleotide Synthesis Errors Are a Source of Untoward Variation in HDR-Mediated Gene Editing.},
journal = {Genes},
volume = {17},
number = {7},
pages = {},
pmid = {42510769},
issn = {2073-4425},
support = {TRAN1-09292//California Institute for Regenerative Medicine/ ; CLIN1-11497//California Institute for Regenerative Medicine/ ; CLIN2-11722//California Institute for Regenerative Medicine/ ; HL151319//National Heart Lung and Blood Institute/ ; OT3HL147741//National Heart Lung and Blood Institute/ ; },
mesh = {Humans ; *Oligonucleotides/genetics ; *Recombinational DNA Repair ; High-Throughput Nucleotide Sequencing ; Anemia, Sickle Cell/genetics/therapy ; *Gene Editing ; beta-Globins/genetics ; CRISPR-Cas Systems ; Hematopoietic Stem Cells/metabolism ; },
abstract = {Background/Objectives: Single-stranded oligonucleotides (ssODNs) are used as donor templates for therapeutic gene editing by CRISPR-Cas9 cleavage and homology-directed repair (HDR). Although ssODN sequence fidelity is critical to the safety and efficacy of editing, standard quality control methods cannot resolve individual nucleotide errors. Methods: We performed deep sequencing of ssODNs from three manufacturers and amplicons from edited hematopoietic stem/progenitor cells. Results: We find that synthesis errors are present in all ssODNs tested at rates that vary more than two-fold among manufacturers, at positions that are dependent on sequence context. These synthesis errors are propagated into the genome by HDR at frequencies proportional to their abundance in the ssODN. In our sickle cell mutation correction protocol, the most prevalent SNEs are predicted to produce benign β-globin variants, while the less frequent frameshift deletions are predicted to generate β-thalassemia-like alleles. Conclusions: Current quality control standards are insufficient to detect these errors, and deep sequencing of ssODNs should be incorporated into regulatory submissions for clinical gene editing programs.},
}

Humans
*Oligonucleotides/genetics
*Recombinational DNA Repair
High-Throughput Nucleotide Sequencing
Anemia, Sickle Cell/genetics/therapy
*Gene Editing
beta-Globins/genetics
CRISPR-Cas Systems
Hematopoietic Stem Cells/metabolism

@article {pmid42510803,
year = {2026},
author = {Gonzales, PR},
title = {Advances in Functional Genomics for Human Health.},
journal = {Genes},
volume = {17},
number = {7},
pages = {},
pmid = {42510803},
issn = {2073-4425},
mesh = {Humans ; *Genomics/methods ; Genome, Human ; },
abstract = {Cytogenomics, including karyotyping, FISH, chromosomal microarrays, and optical genome mapping, has yielded significant results for clinical phenotypes in constitutional and cancer genetics, including intellectual disability, autism spectrum disorders, dysmorphic features, and hematological and solid-tissue neoplasia. However, some of these assays have yielded results of unclear significance because the abnormalities detected were often located in intergenic regions of the genome. Because these abnormalities are within the "dark matter" of the genome, their clinical significance has been a matter of speculation. However, functional genomics can explore the clinical implications of such abnormalities more robustly, whether the abnormalities disrupt topologically associating domains (TADs), delete regulatory regions, etc. Some human genetic diseases associated with these intergenic abnormalities and characterized by functional genomics include preaxial polydactyly (SHH gene), Pierre Robin syndrome (SOX9), and 5q14.3 microdeletion syndrome (MEF2C). While functional genomics is a broad research topic, this review focuses on prior and current efforts to leverage functional genomics within the intergenic regions for human health.},
}

Humans
*Genomics/methods
Genome, Human

@article {pmid41805199,
year = {2026},
author = {Marín-Sanz, M and Berlanga-Torres, JA and Guzmán-López, MH and Sánchez-León, S and Vallés, MP and Castillo, AM and Barro, F},
title = {Engineering ultra-low-gliadin wheat for celiac disease using an integrated RNAi, CRISPR, and doubled haploid strategy.},
journal = {Journal of experimental botany},
volume = {77},
number = {14},
pages = {4417-4434},
doi = {10.1093/jxb/erag131},
pmid = {41805199},
issn = {1460-2431},
support = {QUAL21_023 IAS//Junta de Andalucı́a/ ; //Conexión TRIGO, grant number 202490E049)./ ; },
mesh = {*Triticum/genetics/metabolism ; *Gliadin/genetics/metabolism ; *RNA Interference ; Haploidy ; *Celiac Disease/genetics ; *Gene Editing ; CRISPR-Cas Systems ; Glutens ; },
abstract = {The growing prevalence of gluten-related disorders in humans has driven the development of wheat varieties with reduced immunogenic gluten. This study aimed to integrate RNA interference (RNAi) and CRISPR genome editing within a doubled haploid (DH) platform to overcome challenges of gene redundancy and polyploidy in wheat gliadins. We generated DH lines from crosses between RNAi and CRISPR lines and elite wheat cultivars, enabling stable fixation of multiple genetic modifications in a single generation. Deep sequencing analysis of α-gliadin amplicons was conducted using a custom bioinformatics pipeline optimized for complex, repetitive gene families. Gluten protein profiles were evaluated using RP-HPLC and R5 monoclonal antibody. Several DH lines presented >70% reduction in immunogenic epitopes in α-gliadins, with lines outperforming both parents. Editing frequency was influenced by sgRNA efficiency and parental background. Silencing and editing combined led to nearly depleted gliadins in some lines, often with compensatory increases in other storage proteins linked to bread-making quality, such as high-molecular-weight glutenin subunits. Kernel and specific weight traits were largely maintained. This work demonstrates that combining RNAi and CRISPR in a DH platform enables efficient, heritable reduction of immunogenic gluten, providing a viable strategy for breeding wheat lines safer for individuals with gluten-related disorders.},
}

*Triticum/genetics/metabolism
*Gliadin/genetics/metabolism
*RNA Interference
Haploidy
*Celiac Disease/genetics
*Gene Editing
CRISPR-Cas Systems
Glutens

@article {pmid41928454,
year = {2026},
author = {Tietz, SM and Brenner, K and Moyo, T and Young, PR and Vivier, MA},
title = {Optimized protocol for efficient generation, confirmation, transformation, and CRISPR editing of grapevine hairy roots.},
journal = {Journal of experimental botany},
volume = {77},
number = {14},
pages = {4352-4374},
doi = {10.1093/jxb/erag165},
pmid = {41928454},
issn = {1460-2431},
support = {//South Africa Wine/ ; UID120460//National Research Foundation (NRF)/ ; },
mesh = {*Vitis/genetics/growth & development ; *Plant Roots/genetics/growth & development ; *Transformation, Genetic ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; },
abstract = {Hairy root cultures (HRCs) are powerful tools in plant biotechnology but show variable establishment efficiencies, limiting broader applications. Here, we present a standardized and optimized reference methodology for the routine generation, multiplication, and maintenance of HRCs across diverse grapevine genotypes. Our workflow evaluated three Rhizobium strains, seven grapevine cultivars (three Vitis vinifera cultivars; four Vitis rootstock hybrids), multiple explant types, infection protocols, co-cultivation times, growth media types, and anti-browning agents. The resulting protocol was effective for all grapevine genotypes and, with minor adjustments, also yielded HRCs from two other important South African plant species, namely Sutherlandia frutescens and Aspalathus linearis. Useful molecular tools were developed for transformation and selection of HRCs, including universal multiplex primers for confirmation of transformation, tested antibiotic resistance markers (kanamycin and hygromycin), and fluorescent reporters (DsRed and eyGFPuv), with DsRed found to be particularly versatile. To test the system, we overexpressed the VviMYBA1 transcription factor gene, leading to increased anthocyanin accumulation and red pigmentation in HRCs. Additionally, we achieved CRISPR/Cas9 editing of the VviPUB19 gene, the first report of CRISPR-edited grapevine HRCs. Gene editing combined with HRCs can facilitate rapid gene function studies, offering an efficient alternative or pre-screening system to whole-plant transformations, that could support advanced functional genomics and biotechnological applications in grapevine.},
}

*Vitis/genetics/growth & development
*Plant Roots/genetics/growth & development
*Transformation, Genetic
*Gene Editing/methods
*CRISPR-Cas Systems
Plants, Genetically Modified/genetics

@article {pmid42215861,
year = {2026},
author = {Liu, F and Zhang, X and Yang, Y},
title = {A comparison between CARLIN and DNA Typewriter in CRISPR-mediated lineage tracing.},
journal = {BMC bioinformatics},
volume = {27},
number = {1},
pages = {},
pmid = {42215861},
issn = {1471-2105},
support = {NCI U01-CA253553/CA/NCI NIH HHS/United States ; R01-CA251950/NH/NIH HHS/United States ; NCI U01-CA253553/CA/NCI NIH HHS/United States ; R01-CA251950/NH/NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *DNA/genetics ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Cell Lineage/genetics ; },
abstract = {BACKGROUND: CARLIN and DNA Typewriter are two major breakthroughs in CRISPR-based lineage tracing technology. It is essential to understand the potential and performance of these methods in lineage tracing, which provides important guidance on experimental design.

RESULTS: In this study, we systematically compare these two strategies using a unified stochastic simulation framework with known ground-truth lineages. By explicitly modeling CRISPR editing dynamics, barcode evolution, and cell division processes, the framework enables quantitative benchmarking of lineage reconstruction accuracy across diverse experimental parameter regimes. Both methods are evaluated using multiple accuracy metrics, including Robinson-Foulds accuracy and triplet accuracy, allowing a comprehensive assessment of lineage reconstruction performance under various editing probabilities, sampling depths, and lineage lengths.

CONCLUSIONS: DNA Typewriter consistently outperforms CARLIN in lineage reconstruction accuracy when sufficient numbers of recording targets are used, particularly in more cell divisions. Sequential and ordered recording in DNA Typewriter substantially reduces ambiguity in lineage inference compared to unordered CRISPR barcode editing. CARLIN's lineage-recording potential exhausts rapidly under continuous induction, limiting its effectiveness in long-term lineage tracing. Triplet accuracy provides a more permissive and informative metric than Robinson-Foulds accuracy, especially under partial sampling scenarios.},
}

*CRISPR-Cas Systems
*DNA/genetics
*Gene Editing/methods
*Clustered Regularly Interspaced Short Palindromic Repeats
*Cell Lineage/genetics

@article {pmid42233593,
year = {2026},
author = {Zhao, W and Zheng, Z and Li, R and Xie, H and Yu, H and Zhang, Y and Wu, Y and Yang, Y and Zhang, Z and Gao, H and Li, Y and Zhang, K},
title = {In Situ Amplified Mutational mRNA Imaging Using a Spatially Confined CRISPR Nanoplatform.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {65},
number = {31},
pages = {e7088080},
pmid = {42233593},
issn = {1521-3773},
support = {22377110//National Natural Science Foundation of China/ ; 82402749//National Natural Science Foundation of China/ ; U23A20531//National Natural Science Foundation of China/ ; 22122409//National Natural Science Foundation of China/ ; 252300421073//Natural Science Foundation of Henan Province/ ; 252102311025//Science and Technology Department of Henan Province/ ; 2025SGAQZ-MS-03//State Key Laboratory of Metabolic Dysregulation & Prevention and Treatment of Esophageal Cancer/ ; 261111313300//Henan Provincial Key Research and Development Program/ ; },
mesh = {*RNA, Messenger/genetics/analysis ; Humans ; Mutation ; *CRISPR-Cas Systems/genetics ; },
abstract = {Highly sensitive spatial analysis of RNA mutations is essential for understanding cellular heterogeneity and disease mechanisms. Herein, we developed an integrated CRISPR/Cas13a-based nanoprobe system for rapid detection of RNA in tissue sections (Integrated CRISPR/Cas13a-based RNA Rapid Detection, InCasRD). Unlike conventional "always-on" probes that rely on accumulated probe hybridization, InCasRD leverages the trans-cleavage activity of Cas13a to achieve spatially confined signal amplification and a high signal-to-background ratio (SBR). Using InCasRD, we achieved imaging of multiple target RNAs in tumor cells within 0.5 h of incubation, including mRNA (survivin), microRNA (miR-21), and circular RNA (circ1785). Furthermore, the engineered InCasRD system enabled mapping of RNA mutations, such as the EGFR L858R and ovarian tumor domain (OTUD) single-nucleotide variant (SNV, 23439980 G>T), in tumor tissue sections, thereby facilitating clear tumor boundary delineation. Collectively, InCasRD is a powerful, one-step tool for in situ RNA analysis with potential for diagnosis and precision medicine.},
}

*RNA, Messenger/genetics/analysis
Humans
Mutation
*CRISPR-Cas Systems/genetics

@article {pmid42335403,
year = {2026},
author = {Luo, H and Gao, J and Huang, X and Fang, Y and Huang, T and Xia, Y and Yu, Z and Cao, C and Xiong, Z},
title = {Thermo-Responsive Living Microspheroids Enable a Regenerative Living Disk-Drive System for DNA Data Storage.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {38},
number = {42},
pages = {e73806},
pmid = {42335403},
issn = {1521-4095},
support = {53330200321//Tsinghua University/ ; },
mesh = {*DNA/chemistry/genetics ; *Temperature ; Plasmids/genetics ; *Information Storage and Retrieval/methods ; CRISPR-Cas Systems ; },
abstract = {DNA offers exceptional information density and long-term stability, yet its practical deployment is limited by destructive readout and the absence of a reusable, physically addressable architecture that connects nanoscale molecular information with macroscale device-level data organization. Here, we present a regenerative Living Disk-Drive system based on thermo-responsive engineered living memory microspheroids (ELMMs), in which data-encoded bacteria are encapsulated as discrete, file-level living storage units. Each ELMM contains a clonal bacterial population carrying both an information plasmid, which encodes 26 × 26 pixel icon payloads and one- to three-color intracellular fluorescent retrieval indices, and a help plasmid that enables CRISPR-Cas12a/λ-Red rewriting of the data sequence and retrieval tag. A lyophilized ELMM database forms the Living Disk, which is coupled to an Optical Retriever and desktop-scale Living Drive for closed-loop retrieval, regeneration, and database replenishment. Released bacteria regrow for downstream readout or rewriting, while a fraction is re-encapsulated into new ELMMs. The tested system retains retrieval, regrowth, and sequence recovery after four months of ambient dry storage and 13 lyophilization-rehydration cycles. Model-based performance estimates are reported only as theoretical architecture-level bounds. These results establish an experimentally bounded yet extensible architecture for physically manageable and regenerative DNA memory.},
}

*DNA/chemistry/genetics
*Temperature
Plasmids/genetics
*Information Storage and Retrieval/methods
CRISPR-Cas Systems

@article {pmid42372505,
year = {2026},
author = {Wang, R and Pan, Q and Huang, Y and Dai, W and Ping, Y and Jin, Q},
title = {Versatile hollow Ca[2+]-phenolic nanoparticles for intracellular delivery of diverse bioactive molecules and CRISPR-Cas9 genome editing.},
journal = {Biomaterials},
volume = {335},
number = {},
pages = {124407},
doi = {10.1016/j.biomaterials.2026.124407},
pmid = {42372505},
issn = {1878-5905},
mesh = {Humans ; *Nanoparticles/chemistry/ultrastructure ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; *Calcium/chemistry ; *Phenols/chemistry ; },
abstract = {The development of versatile nanoplatforms capable of universally encapsulating diverse bioactive molecules holds significant promise in biomedicine. In this study, size-tunable hollow Ca[2+]-tannic acid (TA) nanoparticles (HCT NPs) are synthesized as universally applicable drug nanocarriers by simply adding TA into amorphous calcium carbonate nanoparticles. The formation of HCT NPs is identified as a surface-protected self-etching process. A wide range of hydrophobic and hydrophilic small-molecule drugs, metal ions, and biomacromolecules including proteins and nucleic acids can be encapsulated in HCT NPs for efficient intracellular delivery. HCT NPs show rapid and efficient endosomal escape, which is crucial for maintaining the bioactivity of biomacromolecules. Remarkably, a wide array of cargo proteins, spanning different molecular weights and isoelectric points can be delivered into the cytosol by HCT NPs without compromising their bioactivities. The therapeutic potential of HCT NPs for intracellular cargo delivery is exemplified by cytosolic delivery of Cas9 plasmids and Cas9 ribonucleoprotein (RNP) for CRISPR-Cas9 genome editing both in vitro and in vivo. The facile and ultrafast synthesis, versatile cargo encapsulation capabilities, efficient cell uptake and endosomal escape, and excellent biocompatibility make HCT NPs a prominent candidate for intracellular delivery of diverse bioactive molecules, particularly in therapeutic applications such as genome editing.},
}

Humans
*Nanoparticles/chemistry/ultrastructure
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Animals
*Calcium/chemistry
*Phenols/chemistry

@article {pmid42390232,
year = {2026},
author = {Duvenage, L and Chetty, A and Thomson, DD and Ballou, ER and Govender, NP and Rappleye, CA and Hoving, JC},
title = {Tools for genetic manipulation of the endemic fungal pathogen Emergomyces africanus and application of a fluorescent reporter strain in infection models.},
journal = {mSphere},
volume = {11},
number = {7},
pages = {e0018026},
pmid = {42390232},
issn = {2379-5042},
support = {209293/Z/17/Z/WT_/Wellcome Trust/United Kingdom ; 310933/Z/24/Z/WT_/Wellcome Trust/United Kingdom ; AI148561/NH/NIH HHS/United States ; },
mesh = {Animals ; Green Fluorescent Proteins/genetics ; *Genes, Reporter ; Mice ; Plasmids/genetics ; Macrophages/microbiology ; Disease Models, Animal ; *Mycoses/microbiology ; *Ascomycota/genetics/pathogenicity ; CRISPR-Cas Systems ; },
abstract = {UNLABELLED: Emergomyces africanus is a thermally dimorphic fungal pathogen endemic to Southern Africa, which can cause fatal systemic infections in persons with advanced HIV disease. Its mechanisms of pathogenesis are not well understood. Characterization of virulence traits in this pathogen requires appropriate molecular tools for genetic manipulation. Molecular technologies developed for the transformation of Histoplasma capsulatum were adapted for use in E. africanus. Agrobacterium-mediated transformation was used to generate a reporter strain expressing green fluorescent protein (GFP). The E. africanus GFP reporter strain facilitated the study of yeast interaction with macrophages in vitro and allowed the identification of infected phagocyte cell types in the mouse lung by flow cytometry. E. africanus could also maintain episomal plasmids with telomere-like sequences to introduce expression constructs without genome modification. Using this plasmid system, RNA interference constructs were used to knock down the expression of cell wall α(1,3)-glucan by targeting the transcripts of the α-glucan synthase (AGS1). An episomal CRISPR/Cas9 system was evaluated for E. africanus, which effectively disrupted GFP in a reporter strain and enabled the generation of a URA5 uracil auxotroph. These tools and strains will facilitate future studies to elucidate the mechanisms of pathogenesis of E. africanus.

IMPORTANCE: Emergomyces africanus is an opportunistic fungal pathogen affecting persons with advanced HIV disease in South Africa. The biology and pathogenesis of E. africanus are not well understood, as the importance of the disease caused by this fungus (emergomycosis) has only been recognized in recent years, and molecular studies have been impaired by the lack of genetic technologies. In this work, we describe tools and methods for the genetic modification of this pathogen, which will accelerate future studies investigating how the fungus causes disease in the human host. These essential tools include (i) the ability to create fluorescent reporter strains, such as the green fluorescent protein E. africanus strain described here, which facilitates tracking the spread of the fungus during infection and enhances microscopy studies, (ii) methods for knocking down gene expression in E. africanus, and (iii) the permanent disruption of genes through CRISPR/Cas9 gene editing.},
}

Animals
Green Fluorescent Proteins/genetics
*Genes, Reporter
Mice
Plasmids/genetics
Macrophages/microbiology
Disease Models, Animal
*Mycoses/microbiology
*Ascomycota/genetics/pathogenicity
CRISPR-Cas Systems

@article {pmid42492264,
year = {2026},
author = {Shen, H and Nourmohammadi, S and Zhou, Y and Harata-Lee, Y and Qu, Z and Wang, W and Yool, AJ and Adelson, DL},
title = {Target pathway validation for Compound Kushen Injection active against cancer cells.},
journal = {Phytomedicine : international journal of phytotherapy and phytopharmacology},
volume = {159},
number = {},
pages = {158598},
doi = {10.1016/j.phymed.2026.158598},
pmid = {42492264},
issn = {1618-095X},
abstract = {BACKGROUND: Traditional Chinese medicines are based on complex mixtures of natural products and their multi-target mechanism of action. Therefore the discovery and validation of targets and mechanisms have always been challenging. In previous studies, using transcriptomic methods and Compound Kushen Injection (CKI) as a model drug, we identified multiple pathways and candidate target genes for validation, through which CKI exerts its pharmacological effects.

PURPOSE: This study aimed to demonstrate the involvement of multiple genetic targets in different pharmacological activities for natural products in Compound Kushen Injection.

METHODS: In this study, we selected eight key genes from four candidate pathways and used CRISPR/CAS technology to knock out these genes in four cell lines, validating their role in CKI activity.

RESULTS: Although the sensitivity of different cell lines to gene knockout varied, overall, it led to reductions in various cellular activities. After the addition of CKI, we observed that, except for the minor impact of CDKN1A gene knockout on the effect of CKI, knocking out the other genes significantly affected the pharmacological efficacy of CKI in different assays. Among them, knockout of MYD88 and NFkB genes enhanced the efficacy of CKI. At the same time, we found that the genes IL24 and CYP1B1 play a crucial role in CKI inhibition of tumour cell migration, and the CYP1A1 gene is critical for the cell cycle arrest induced by CKI.

CONCLUSIONS: These findings validate the results of our previous transcriptomic analysis and further demonstrate the complexity of pharmacological mechanisms of multi-target synergistic action of natural product mixtures.},
}

@article {pmid42492655,
year = {2026},
author = {Patel, S and Panchal, J and Patel, A and Chauhan, H and Sharma, K and Sabara, P and Vahora, S and Shrimali, M and Shekh, S and Thakor, A and Mohapatra, S and Hati, S},
title = {Unravelling the Resistome of Carbapenem-Resistant E. coli from Bovine Mastitis via Whole-Genome Sequencing.},
journal = {Veterinary journal (London, England : 1997)},
volume = {},
number = {},
pages = {106792},
doi = {10.1016/j.tvjl.2026.106792},
pmid = {42492655},
issn = {1532-2971},
abstract = {Carbapenem-resistant Escherichia coli (CREC) poses a growing threat to public health, particularly when emerging from animal reservoirs such as dairy cattle. This study aimed to characterize CREC isolates recovered from bovine mastitis cases in Gujarat, India, using a combination of phenotypic antibiotic susceptibility testing and whole-genome sequencing (WGS). Out of 130 confirmed E. coli isolates from 790 mastitic milk samples, 33 (25.38%) were resistant to imipenem. Of these, nine exhibited multidrug-resistant (MDR), extensively drug-resistant (XDR), or pan-drug-resistant (PDR) phenotypes. WGS was performed on four representative isolates (SKN144, SKN685, SKN687, SKN926), revealing genome sizes ranging from 4.7 to 5.4Mb and GC content between 50.4% and 50.8%. Annotation identified numerous resistance determinants, including carbapenemase genes (blaNDM, blaOXA-48, blaTEM, blaCMY, blaCTX-M), aminoglycoside-modifying enzymes (APH, AAC), macrolide resistance genes (mphA, ermB), and multiple efflux pump systems (AcrAB-TolC, EmrAB, MdtEF-TolC). Functional genes associated with replication, repair, stress response, and mobile genetic elements (integrases, transposases, CRISPR-Cas) were also detected, indicating high genomic adaptability. Phenotypic testing revealed alarming resistance to key antimicrobials, including ampicillin (56.15%), amikacin (55.38%), ceftazidime (53.08%), and colistin (79.23%, including intermediate strains). Subsystem analysis highlighted metabolic versatility, defence mechanisms, and virulence-associated pathways. Phylogenetic analysis indicated that all isolates clustered within the same clade, suggesting possible clonal dissemination within the bovine population. The presence of CRISPR-Cas elements, integrases, and transposases suggests ongoing horizontal gene transfer and genome plasticity. These findings underscore the alarming prevalence of CREC in dairy environments and the urgent need for enhanced AMR surveillance, prudent antibiotic stewardship, and implementation of a One Health approach to prevent zoonotic transmission. This study contributes valuable genomic insights into livestock-associated CREC and highlights their close genomic parallels with high-risk human clinical clones.},
}

@article {pmid42493436,
year = {2026},
author = {Wang, Y and Song, H and DU, Y and Xu, T},
title = {[CRISPR-Cas9 activation screening identifies candidate chemokine regulators of ter-tiary lymphoid structure formation in bladder cancer].},
journal = {Beijing da xue xue bao. Yi xue ban = Journal of Peking University. Health sciences},
volume = {58},
number = {4},
pages = {707-715},
pmid = {42493436},
issn = {1671-167X},
mesh = {Animals ; Mice ; *Urinary Bladder Neoplasms/genetics/pathology/immunology ; Humans ; *Chemokines/genetics/metabolism ; Cell Line, Tumor ; Mice, Inbred C57BL ; *CRISPR-Cas Systems/genetics ; HEK293 Cells ; Chemokine CCL20/genetics ; },
abstract = {OBJECTIVE: To identify the cytokine genes influencing the formation of tertiary lymphoid structures (TLS) through CRISPR-Cas9 library screening, and to discover potential key regulatory molecules, providing new targets for enhancing the efficacy of bladder cancer immunotherapy.

METHODS: Based on a mouse whole-genome library, 44 chemokine-related genes were identified, and an single-guide RNA (sgRNA) library targeting these genes was designed and constructed, with three sgRNAs assigned to each gene. Using a lentiviral packaging system, the library plasmids were used to transfect HEK293T cells to generate a lentiviral library, which was then used to infect the mouse bladder cancer cell line MB49. Purinomycin selection was performed to obtain the MB49-mCherry cell line stably over-expressing chemokines. The cells were inoculated into the peritoneal cavity of C57BL/6 mice to establish a bladder cancer xenograft model, and tumor growth was monitored. Three weeks later, tumor tissue was excised, genomic DNA was extracted for high-throughput sequencing, and sgRNA enrichment was analyzed to screen for differentially expressed cytokine genes. Concurrently, immunohistochemical staining was performed to detect TLS markers CD20 and CD3, and the number, distribution, and maturity of TLS were assessed. The selected candidate genes were validated individually in vivo to further confirm their impact on TLS formation.

RESULTS: We successfully constructed a cytokine gene library containing 132 sgRNAs, covering 44 chemokine genes. Following lentiviral infection, we obtained the MB49-mCherry cell line, which stably expressed the library, and isolated dead Cas9-positive monoclonal cell lines via flow cytometry to ensure the homogeneity and reproducibility of subsequent experiments. Intratumoral tumor experiments in mice revealed that the number of TLS cells in the experimental group was significantly higher than in the control group, primarily distributed at the tumor margins. High-throughput sequencing results showed that, compared with the control group, in the experimental group, Cxcl16 sgDNA was significantly enriched, while Ccl20 and Cx3lc1 sgDNA levels decreased compared with baseline (P < 0.05). Further validation of the individual roles of each factor via intraperitoneal injection revealed that the number of TLSs in tumors decreased in the group treated with the CX3CL1 chemokine, suggesting that CX3CL1 might negatively regulate TLS formation. Immunohistochemical results showed that in the CX3CL1-treated group, the aggregation of CD20-positive B cells and CD3-positive T cells in the tumor tissue was reduced, and the TLS structure was incomplete.

CONCLUSION: Through CRISPR-Cas9 library screening combined with in vivo validation, this study successfully identified CX3CL1 as a potential negative regulator of TLS formation in bladder cancer. High CX3CL1 expression was associated with a reduction in TLS numbers, suggesting that it might exert an inhibitory role in the immune microenvironment of bladder cancer. This finding provides new clues and research directions for understanding the molecular mechanisms of TLS formation in bladder cancer. However, whether CX3CL1 can serve as an immunotherapeutic target remains to be further validated through clinical specimen analysis, multidimensional mechanistic investigation, and immunotherapy response correlation studies.},
}

Animals
Mice
*Urinary Bladder Neoplasms/genetics/pathology/immunology
Humans
*Chemokines/genetics/metabolism
Cell Line, Tumor
Mice, Inbred C57BL
*CRISPR-Cas Systems/genetics
HEK293 Cells
Chemokine CCL20/genetics

@article {pmid42494101,
year = {2026},
author = {Liu, K and Ren, C and Liu, X and Qi, X and Liu, Y and Ma, S and Zhang, S and Wong, XY and Wang, X and Hu, T and Hu, C},
title = {Shared CRISPR arrays underpin type I-A/I-B coexistence.},
journal = {Protein & cell},
volume = {},
number = {},
pages = {},
doi = {10.1093/procel/pwag050},
pmid = {42494101},
issn = {1674-8018},
abstract = {CRISPR-Cas systems provide adaptive immunity in prokaryotes, yet how multiple CRISPR-Cas subtypes coexist and coordinate within a single genome remains unclear. Comparative genomic analysis revealed that nearly one-third of type I-A CRISPR-Cas3 systems are adjacent to a type I-B system, often sharing a single CRISPR array. Using Thermococcus siculi RG-20 (Tsi) as a model, we show that purified TsiCas6a and TsiCas6b independently recognize and cleave the shared pre-crRNA, producing mature crRNAs with comparable efficiency. Plasmid interference assays further demonstrated that crRNAs produced by either Cas6a or Cas6b enzyme could guide both type I-A and type I-B interference complexes. This interchangeability shows that crRNAs generated by either Cas6a or Cas6b can be loaded into, and function with, both type I-A and type I-B interference complexes. Structural modelling revealed distinct but complementary recognition strategies for Cas6a and Cas6b, and mutational analysis of their RNA-binding residues impaired pre-crRNA cleavage and abolished interference activity. Together, these results uncover a shared-array logic in which Cas6a/Cas6b-compatible processing routes a single pre-crRNA to multiple type I effectors-providing a potential mechanism for subtype co-existence, a plausible explanation for array-less (or "orphan") interference modules, and an evolutionary bet-hedging strategy that prevents Acrs from shutting down immunity wholesale.},
}

@article {pmid42494113,
year = {2026},
author = {Ohtomo, M and Takarabe, S and Namiki, T and Kawata, Y and Kaneko, R and Ozawa, M and Yamada, Y and Mori, H and Kageyama, A and Kamoshita, M and Terakawa, J and Ito, J},
title = {Generation and Characterization of a Rdh1-iCre Line to Study Uterine Glandular Biology.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {64},
number = {4},
pages = {e70068},
pmid = {42494113},
issn = {1526-968X},
support = {JP21K09512//Japan Society for the Promotion of Science/ ; JP24K01950//Japan Society for the Promotion of Science/ ; JP25KJ2187//Japan Society for the Promotion of Science/ ; JP22H04922//Japan Society for the Promotion of Science/ ; JP25K22429//Japan Society for the Promotion of Science/ ; //Azabu University/ ; },
mesh = {Animals ; Female ; *Uterus/metabolism ; Mice ; *Alcohol Oxidoreductases/genetics/metabolism ; *Integrases/genetics/metabolism ; Pregnancy ; Mice, Transgenic ; CRISPR-Cas Systems ; },
abstract = {The uterus is an essential organ for fetal development in most mammals. Uterine glands, highly conserved structures in the mammalian uterus, play critical roles in the establishment and maintenance of pregnancy and have been implicated in the pathogenesis of uterine diseases, including endometrial cancer and endometriosis. Previous studies have shown that Retinol dehydrogenase 1 (Rdh1) is specifically expressed in the glandular epithelium (GE) from the onset of gland formation through adulthood. In this study, to develop a GE-specific Cre driver line, we generated Rdh1-iCre mice by introducing an improved Cre recombinase (iCre) into the Rdh1 locus using the CRISPR/Cas9 system. To evaluate the utility of this model, Rdh1-iCre mice were crossed with ROSA26-H2B-mCherry reporter mice, and Cre-dependent reporter expression was analyzed. Robust mCherry fluorescence was observed throughout the uterine glands at 2 weeks after birth, coinciding with the active elongation and branching of the GE. These results demonstrate that the Rdh1-iCre mouse line is a valuable and highly efficient tool for investigating the physiological roles of uterine glands during development and pregnancy, as well as their contribution to the progression of GE-derived uterine diseases.},
}

Animals
Female
*Uterus/metabolism
Mice
*Alcohol Oxidoreductases/genetics/metabolism
*Integrases/genetics/metabolism
Pregnancy
Mice, Transgenic
CRISPR-Cas Systems

@article {pmid42494486,
year = {2026},
author = {Wu, W and Wang, R and Li, J and Zhang, Z and Yao, W and Zhang, N and Xu, W},
title = {Engineering climate-resilient horticultural crops: advances in transcriptional regulation, genome editing, and synthetic networks.},
journal = {Horticulture research},
volume = {13},
number = {8},
pages = {uhag119},
pmid = {42494486},
issn = {2662-6810},
abstract = {Abiotic stresses-particularly cold, drought, and salinity-pose significant threats to the productivity and sustainability of horticultural crops. Recent studies have revealed conserved and species-specific regulatory mechanisms that allow plants to adapt dynamically to these environmental constraints. This review synthesizes advances in understanding key transcription factor families-such as CBF/DREB, NAC, MYB, WRKY, and bHLH-that orchestrate stress-responsive gene networks and modulate physiological processes, including osmotic regulation, antioxidant defense, and ionic homeostasis. We also discuss the emerging roles of chromatin remodeling, DNA methylation, histone modifications, and noncoding RNAs in conferring transcriptional plasticity and stress memory. Beyond endogenous pathways, we evaluate transgenic strategies, CRISPR/Cas-based genome editing, and synthetic gene circuits for engineering abiotic stress tolerance. Particular attention is given to trade-offs between growth and defense, challenges in horticultural crop transformation, and gaps in field translation. We further examine the regulatory role of secondary metabolites-such as flavonoids and salicylic acid-as biochemical interfaces between signal transduction and adaptive responses. Finally, we propose a forward-looking roadmap integrating multi-omics, ideotype design, and precision breeding toward climate-resilient horticultural systems.},
}

@article {pmid42494498,
year = {2026},
author = {Banik, I and Coppé, JP},
title = {CRISPRing through time: How cutting-edge technology is revolutionizing life sciences and medicine.},
journal = {Molecular therapy. Nucleic acids},
volume = {37},
number = {3},
pages = {103003},
pmid = {42494498},
issn = {2162-2531},
abstract = {Given the plethora of emerging technologies, none have truly captured the minds as CRISPR. From the groundbreaking research, the ultimate battle of the prizes and patents to a number of books, the science of CRISPR continues to be significant in the biomedical field. For many decades now, the emergence of synthetic biology as an intervention to correct diseases has become the foundation of biomedical research. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based genetic editing has become a common place for routine investigation of scientific hypotheses in pre-clinical settings. More recently, CRISPR-based diagnostic testing kits for SARS-CoV-2 have showcased a translational output. Furthermore, a technological landmark was achieved when the Food and Drug Administration (FDA) approved the first CRISPR-based gene therapy (exa-cel) to edit erythroid specific enhancer region of BCL11A in hematopoietic stem cells, introduced in patients suffering from sickle cell anemia to achieve durable remission. In this review, we provide a snapshot into the most important milestones along the journey of CRISPR from its discovery in bacteria to its usage in precision medicine. The intervention of machine learning tools has now intertwined complex biology with high-throughput scalable outputs. Given the vast amount of information on CRISPR, we try to pin down key take-home messages for scientists as well as non-scientist readers. This review article attempts to understand why and how CRISPR remains significant and seamlessly integrates in the emerging era of new technologies.},
}

@article {pmid42496566,
year = {2026},
author = {Krishna, TPA and Harikrishnan, D and Veena, M and Maharajan, T and James, M and Udhayakumar, M and Arockiam Jeyasundar, PGS and David, SJ and Dineshkumar, R and Rajan, R and Rathinapriya, P},
title = {Next-Generation Metabolic Engineering of Capsaicinoids Biosynthesis in Chilli Pepper: Bridging Genomic Insights to Biotechnological Applications.},
journal = {Biotech (Basel (Switzerland))},
volume = {15},
number = {3},
pages = {},
pmid = {42496566},
issn = {2673-6284},
abstract = {Chilli peppers (Capsicum species) have been widely used around the world because of their economic value and distinctive sensory characteristics. They contain abundant functional metabolites, especially a group of vanillylamide compounds belonging to the family of capsaicinoids, which have been exploited for medicinal, nutritional, agricultural, and cosmetic uses. The demand for capsaicinoid molecules is increasing day by day due to their high economic value and wide range of applications. Therefore, increasing bioactive metabolites, especially capsaicinoids in chilli peppers, is a major priority in the current scenario. Multi-omics approaches such as genomics, transcriptomics, proteomics, and metabolomics have substantially contributed to understanding the complex regulatory networks governing capsaicinoid biosynthesis. Key structural genes, transcription factors, and signaling pathways involved in the phenylpropanoid and branched-chain fatty acid pathways have been identified, providing valuable targets for metabolic engineering in chilli pepper. Despite these advances, the integration of genetic modification approaches for the targeted enhancement of capsaicinoid production remains limited in chilli pepper. Recent developments in biotechnology, particularly CRISPR/Cas-mediated genome-editing, enable the precise genetic modification of metabolic pathways and regulatory networks in plants. Therefore, it can contribute to the precise modification of key genes involved in the capsaicinoid biosynthesis pathway, offering potential strategies to enhance the capsaicinoid content in chilli pepper. However, CRISPR/Cas-mediated genome editing in chilli pepper is still in its early stages. There are currently no reports available on the successful enhancement of capsaicinoid content in chilli peppers through CRISPR/Cas-mediated genome editing. To date, no comprehensive review has evaluated the CRISPR-Cas-mediated genome-editing approaches for capsaicinoid metabolic engineering in chilli pepper. This review critically evaluates the recent advances in CRISPR/Cas-mediated metabolic engineering in chilli peppers, with particular emphasis on regulatory genes involved in capsaicinoid biosynthesis. Furthermore, multi-omics approaches are expected to complement these strategies by enabling the identification of key regulatory genes, the optimization of genome-editing targets, and the prediction of metabolic outcomes for enhanced capsaicinoid production. Overall, this review provides insights into improving capsaicinoid accumulation in chilli peppers through advanced genome-editing technologies.},
}

@article {pmid42497250,
year = {2026},
author = {Zhou, Z and Yang, Y and Zhou, F and Liang, K and Gong, T and Zhou, X and Li, J and Luo, J and Li, J and Yang, J},
title = {Energy-assisted CRISPR cleavage and probiotic vesicle signaling platform: Microbiome reprogramming for homeostasis.},
journal = {Science advances},
volume = {12},
number = {30},
pages = {eaef1760},
pmid = {42497250},
issn = {2375-2548},
mesh = {*Probiotics ; *Homeostasis ; *Microbiota/genetics ; *Streptococcus mutans/genetics/metabolism ; Adenosine Triphosphate/metabolism ; *CRISPR-Cas Systems ; Quorum Sensing ; *Extracellular Vesicles/metabolism ; Humans ; Signal Transduction ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Animals ; },
abstract = {Pathogenic infections drive microbial dysbiosis and persistent inflammation, posing therapeutic challenges due to difficulties in precise pathogen eradication and microbiome restoration. Although CRISPR-based therapeutics enable pathogen-specific antibacterial targeting, their effectiveness in treating pathogenic infections is constrained by difficulties in navigating complex microbial ecosystems, penetrating pathogenic barriers, sustaining energy-intensive intracellular cleavage, and, critically, restoring microbial balance after pathogen clearance. Here, we engineer a probiotic vesicle-synergized CRISPR platform by encapsulating gtfB-targeting CRISPR plasmids within hybrid extracellular vesicles from probiotics and pathogenic Streptococcus mutans. The pathogen-derived vesicle component enables targeted uptake by S. mutans, facilitating intracellular cleavage of the virulence gene gtfB. Vesicle-carried endogenous adenosine triphosphate (ATP) boosts CRISPR activity, amplifying targeted DNA cleavage for potent and selective pathogen elimination. Probiotic-derived vesicle components further remodel quorum-sensing networks and immunity, restoring microbial homeostasis. This probiotic vesicle-based strategy integrates ATP-enhanced CRISPR cleavage with microbiome and immune modulation, offering a next-generation therapeutic paradigm for microbiome-associated diseases.},
}

*Probiotics
*Homeostasis
*Microbiota/genetics
*Streptococcus mutans/genetics/metabolism
Adenosine Triphosphate/metabolism
*CRISPR-Cas Systems
Quorum Sensing
*Extracellular Vesicles/metabolism
Humans
Signal Transduction
*Clustered Regularly Interspaced Short Palindromic Repeats
Animals

@article {pmid42497869,
year = {2026},
author = {Terkelsen, T and Yumiceba, V and Kim, J and Melo, US and Axelgaard, E and Febbraro, F and Gunnarsson, AV and Balachandran, S and Dahl-Jessen, M and Christensen, R and Peters, BA and Asan, and Thelle, T and Nyegaard, M and Bak, RO and Denham, M and Spielmann, M and Jensen, UB},
title = {Position effect at the SOX3 locus by an interchromosomal insertion causes hereditary spastic paraplegia.},
journal = {American journal of human genetics},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ajhg.2026.07.001},
pmid = {42497869},
issn = {1537-6605},
abstract = {Pathogenic rewiring of the three-dimensional (3D) genome architecture is increasingly being identified as the cause of genetic diseases, but recognizing the cis-regulatory effects of structural variation remains a challenge. The Xq27.1 region contains a quasi-palindrome identified as a pleiotropic hotspot for disease-causing interchromosomal insertions. In a large Danish family affected by X-linked recessive complex spastic paraplegia, we identified the segregation of a 149-kb interchromosomal insertion at Xq27.1 originating from 4q24. To understand the disease mechanism, we generated induced pluripotent stem cells (iPSCs) from affected individuals. Using CRISPR perturbation and neural differentiation experiments combined with high-throughput chromatin conformation capture (Hi-C) and transcriptomic analyses, we identify a 3D regulatory rewiring of SOX3 and transcriptional dysregulation of SOX3 targets in iPSC-derived neurons. Consistent with regulatory partitioning of the SOX3 topologically associating domain (TAD) in affected individuals, our experiments show that upstream cis-regulatory elements have a reduced ability to activate SOX3 expression and that the observed dysregulation depends on CTCF-binding sites within the insertion. This work provides mechanistic evidence that a position effect at the SOX3 locus can cause hereditary spastic paraplegia.},
}

@article {pmid42500818,
year = {2026},
author = {Li, A and Zhang, X and Li, S and Wang, Y and Xu, C and Lv, C and Zhao, M and Liu, Y and Ding, M and Cao, C},
title = {ETTAS: a modular aptamer-recruited platform for programmable translational activation.},
journal = {Nucleic acids research},
volume = {54},
number = {14},
pages = {},
pmid = {42500818},
issn = {1362-4962},
support = {2021YFA0911600//National Key Research and Development Program of China/ ; A2503021//Shenzhen Medical Research Fund/ ; A2303071//Shenzhen Medical Research Fund/ ; 82403183//National Natural Science Foundation of China/ ; 82303113//National Natural Science Foundation of China/ ; 82360603//National Natural Science Foundation of China/ ; 82560157//National Natural Science Foundation of China/ ; 82300871//National Natural Science Foundation of China/ ; 82474383//National Natural Science Foundation of China/ ; JCYJ20250604180714019//Shenzhen Science and Technology Program/ ; RCJC20221008092723011//Shenzhen Science and Technology Program/ ; JCYJ20240813140522029//Shenzhen Science and Technology Program/ ; RCBS20231211090747077//Shenzhen Science and Technology Outstanding Innovative Talent Training/ ; },
mesh = {Humans ; *Aptamers, Nucleotide/genetics/metabolism ; *Protein Biosynthesis/genetics ; Tumor Suppressor Protein p53/genetics/metabolism ; *CRISPR-Cas Systems ; PTEN Phosphohydrolase/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Animals ; Cell Line, Tumor ; },
abstract = {Precise enhancement of endogenous protein synthesis offers a reversible therapeutic strategy without permanent genomic modification. However, existing Cas13-mediated translational activation systems are limited by modest potency and restricted modular expandability. Here, we developed the Enhanced Targeted Translational Activation System (ETTAS), a modular RNA-guided platform that combines dCas13a, the SINEB2 translational activation element, and an independently recruitable aptamer-mediated auxiliary module. Systematic ortholog screening identified dCas13a as the most effective scaffold for SINEB2-mediated translational activation, whereas direct tandem duplication of SINEB2 elements impaired rather than enhanced activity. To overcome this architectural limitation, we used aptamer-mediated recruitment to spatially separate target recognition from auxiliary activation. A binding-validated, non-interfering dCas13a-binding aptamer enabled construction of a dual-module system in which an aptamer-recruited SINEB2 element enhanced translation without altering target mRNA abundance or stability. Compared with the previously reported dCasRx-SINEB2 system, ETTAS produced stronger reporter activation, stronger endogenous induction of P53 and PTEN, and greater antiproliferative and pro-apoptotic effects in bladder cancer cells. Proteomic analyses showed selective target protein upregulation with limited global perturbation. In vivo, dual-AAV delivery of ETTAS activated endogenous P53 and suppressed tumor growth. ETTAS establishes a programmable framework for modular post-transcriptional upregulation of endogenous proteins.},
}

Humans
*Aptamers, Nucleotide/genetics/metabolism
*Protein Biosynthesis/genetics
Tumor Suppressor Protein p53/genetics/metabolism
*CRISPR-Cas Systems
PTEN Phosphohydrolase/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
Animals
Cell Line, Tumor

@article {pmid42500819,
year = {2026},
author = {Wang, W and Chen, Y and Li, Z and Zhang, L and Gui, K and Wu, Y and Yin, N and Han, X and Zhang, Y and Lu, R and Zhang, Z and Wang, L and Xie, G},
title = {A structural accessibility principle for LbuCas13a activation by noncontiguous DNA.},
journal = {Nucleic acids research},
volume = {54},
number = {14},
pages = {},
pmid = {42500819},
issn = {1362-4962},
support = {82372351//National Natural Science Foundation of China/ ; 82572673//National Natural Science Foundation of China/ ; CSTB2024NSCQ-MSX0521//Natural Science Foundation of Chongqing/ ; CSTB2024NSCQ-MSX1223//Natural Science Foundation of Chongqing/ ; 2024M763898//China Postdoctoral Science Foundation/ ; 2024CQBSHTB3005//Chongqing Postdoctoral Special Funding Project/ ; },
mesh = {*DNA/chemistry/metabolism ; *CRISPR-Associated Proteins/metabolism/chemistry ; Models, Molecular ; Humans ; CRISPR-Cas Systems ; Molecular Dynamics Simulation ; },
abstract = {CRISPR-Cas13a is mainly known as an RNA-guided RNA endonuclease. Recent studies show that Leptotrichia buccalis Cas13a (LbuCas13a) can interact with DNA substrates too, without PAM or PFS constraints, but current understanding of DNA-mediated activation is largely based on continuous target strands. Here, we define a structural accessibility principle for LbuCas13a activation by noncontiguous DNA. We show that activation occurs only when overhang positioning creates an accessible protein-DNA interface. Outer overhangs near the crRNA repeat-adjacent side restore strong trans-cleavage activity by stabilizing key LbuCas13a-DNA contacts, whereas distal outer overhangs support only weak activation. In contrast, inner overhangs cause steric mismatch, destabilize the complex, and block formation of an active conformation. Molecular modeling and molecular dynamics simulations support this structure-dependent rule. Noncontiguous DNA also broadens the single-nucleotide discrimination window of LbuCas13a and enables accurate IDH1 R132H detection in glioma tissues. We further develop a one-step APE1-activated CRISPR-LbuCas13a reaction (ACROSS) for sensitive APE1 detection. Because activated LbuCas13a cleaves RNA reporters but not DNA-triggering products, ACROSS preserves the activating structure and supports stable signaling in vitro, in live cells, and in breast cancer serum samples.},
}

*DNA/chemistry/metabolism
*CRISPR-Associated Proteins/metabolism/chemistry
Models, Molecular
Humans
CRISPR-Cas Systems
Molecular Dynamics Simulation

@article {pmid42501080,
year = {2026},
author = {Gençoğlu, HS and Aydemir, E and Ayaz, F},
title = {Bacterial immune systems.},
journal = {Antonie van Leeuwenhoek},
volume = {119},
number = {8},
pages = {},
pmid = {42501080},
issn = {1572-9699},
mesh = {*Bacteria/immunology/virology/genetics ; Bacteriophages/immunology/physiology ; CRISPR-Cas Systems ; Immunity, Innate ; Adaptive Immunity ; },
abstract = {Bacterial immune systems encompass the multi-layered defense mechanisms that bacteria develop against bacteriophages and mobile genetic elements, such as plasmids. This review covers bacterial innate defense systems (surface defenses, superinfection exclusion, restriction-modification, abortive infection, and toxin-antitoxin systems), CRISPR-Cas-mediated adaptive immunity, and the escape strategies used by phages to overcome these defenses (genome modifications, anti-restriction proteins, and anti-CRISPR factors). Emerging evidence also highlights the role of outer membrane vesicles (OMVs) in anti-phage defense and their translational potential as vaccine and delivery platforms. In this context, a better understanding of bacterial defense systems contributes to the development of biotechnology and medical applications such as CRISPR technologies, diagnostic approaches, and phage therapy.},
}

*Bacteria/immunology/virology/genetics
Bacteriophages/immunology/physiology
CRISPR-Cas Systems
Immunity, Innate
Adaptive Immunity

@article {pmid42503339,
year = {2026},
author = {Yuan, YL and Dai, JL and Xiao, L and Jiang, JG},
title = {Advances in engineering microalgae for heterologous terpenoid synthesis: A review.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108993},
doi = {10.1016/j.biotechadv.2026.108993},
pmid = {42503339},
issn = {1873-1899},
abstract = {Terpenoids are a class of natural products widely distributed in living organisms, with isoprene as their fundamental structural unit. However, traditional plant extraction and chemical synthesis methods are often limited by low product purity, difficult separation, and complex synthetic steps, making it challenging to meet the demands of large-scale production. Conventional hosts such as Escherichia coli and Saccharomyces cerevisiae are utilized for terpenoid synthesis due to their advantages of short growth cycles and controllable cultivation conditions. Nevertheless, the complexity of terpenoid biosynthetic pathways poses significant challenges for these hosts in producing structurally complex terpenoids. In contrast, microalgae as photosynthetic microorganisms, possess well-developed endogenous terpenoid metabolic pathways, abundant precursor pools, and subcellular structures and regulatory mechanisms similar to those of plants, demonstrating significant advantages in the heterologous production of complex terpenoids. This review systematically summarizes recent advances in the production of heterologously synthesized terpenoids in eukaryotic microalgae, ranging from monoterpenes to triterpenes, and provides an in-depth analysis of key engineering strategies, including MEP/MVA pathway regulation, gene expression optimization, subcellular compartmentalization, and cultivation process intensification. In addition, the application potential of advanced tools such as CRISPR/Cas, microalgae-microorganism co-culture, and artificial intelligence is introduced. Finally, the major bottlenecks faced by microalgae as a sustainable green cell factory for terpenoid production are briefly analyzed, and future research directions are proposed.},
}

@article {pmid42503939,
year = {2026},
author = {Yang, XP and Gao, YQ and Huang, L and Zhao, LJ and Xiao, Y and Li, L and Zhang, TS},
title = {[Advances in research on the molecular mechanisms and gene therapy of hereditary hearing impairment].},
journal = {Zhonghua yu fang yi xue za zhi [Chinese journal of preventive medicine]},
volume = {60},
number = {7},
pages = {1138-1149},
doi = {10.3760/cma.j.cn112150-20251230-01243},
pmid = {42503939},
issn = {0253-9624},
support = {82560219//National Natural Science Foundation of China/ ; 2025S129//Kunming Medical University 2025 Master's Degree Education Innovation Fund Project/ ; },
mesh = {Humans ; *Genetic Therapy ; Connexin 26 ; *Hearing Loss/genetics/therapy ; Animals ; Connexins ; },
abstract = {Hereditary hearing impairment represents a significant etiology of language and social dysfunction in both children and adults, primarily caused by genetic factors. To date, over 150 genes have been identified in association with this disorder. The pathogenic mechanisms involve multiple molecular levels, including abnormalities in hair cell cytoskeleton and stereociliary structure, dysfunction of intercellular gap junctions (e.g., GJB2, GJB6), dysregulation of ion channels and transporters (e.g., SLC26A4, KCNQ4), alterations in extracellular matrix composition, and disruption of intracellular signaling pathways. In recent years, research has expanded to investigate the role of the inner ear immune microenvironment in this condition, with emerging evidence suggesting that immune dysregulation may contribute to disease initiation and progression. Therapeutically, novel strategies such as adeno-associated virus (AAV)-based gene replacement therapy, CRISPR/Cas-mediated gene editing systems, and lipid nanoparticle (LNP)-delivered mRNA therapeutics have demonstrated partial restoration of auditory function in animal models of hereditary hearing impairment involving genes such as TMC1, OTOF, and GJB2, with some approaches having advanced to clinical trial stages. This article systematically summarizes recent advances in the molecular mechanisms, immune microenvironment involvement, and gene therapy strategies for hereditary hearing impairment, delineates the research trajectory from gene discovery and mechanistic elucidation to therapeutic development, and discusses future translational research directions and clinical challenges.},
}

Humans
*Genetic Therapy
Connexin 26
*Hearing Loss/genetics/therapy
Animals
Connexins