@article {pmid41681112,
year = {2026},
author = {Hao, X and Yuan, R and Guo, Y and Chen, G and Guo, Y and Liu, L and Lu, F and Bai, Y and Tian, Y},
title = {Host Oxidative Response Capacity Determines Longevity Outcomes of Microbial Interventions.},
journal = {Aging cell},
volume = {25},
number = {2},
pages = {e70418},
pmid = {41681112},
issn = {1474-9726},
support = {2022YFA1303000//National Key Research and Development Program of China/ ; 32225025//National Natural Science Foundation of China/ ; 32321004//National Natural Science Foundation of China/ ; 32430025//National Natural Science Foundation of China/ ; 32225038//National Natural Science Foundation of China/ ; YSBR-076//CAS Project for Young Scientists in Basic Research/ ; //New Cornerstone Science Foundation/ ; },
mesh = {*Longevity/drug effects/genetics/physiology ; Oxidation-Reduction/drug effects ; Quantitative Trait Loci ; CRISPR-Cas Systems/genetics ; Mutation ; Antioxidants/pharmacology ; *Host Microbial Interactions/physiology ; *Microbiota/physiology ; DNA-Binding Proteins/genetics ; Transcription Factors/genetics ; Caenorhabditis elegans Proteins/genetics ; Glycogen Synthase/genetics ; Genetic Variation ; *Caenorhabditis elegans/drug effects/genetics/microbiology/physiology ; Gene Editing ; Animals, Genetically Modified ; Animals ; },
abstract = {Microbial communities profoundly influence host aging, yet how natural genetic variation determines microbiota-driven longevity remains unclear. By screening root-derived bacterial isolates across genetically diverse Caenorhabditis elegans strains, we identified striking phenotypic heterogeneity, ranging from lifespan extension to accelerated aging. Combining classical genetic analysis, quantitative trait locus (QTL) mapping and CRISPR-Cas9 allelic recapitulation, we identify skn-1 (Nrf2) and gsy-1 (glycogen synthase) as key host determinants. We demonstrate that strains with mutations or specific natural variants in these loci exhibit a compromised redox buffering capacity, leading to systemic oxidative stress, loss of tissue integrity, and premature death upon microbial challenge. Conversely, robust hosts utilize the same microbial signals to promote longevity. Notably, lifespan defects in susceptible individuals were rescued by antioxidant supplementation. These findings establish redox homeostasis as a central axis in host-microbe-aging interactions and provide a mechanistic framework for precision microbiome interventions tailored to host genetic backgrounds.},
}

*Longevity/drug effects/genetics/physiology
Oxidation-Reduction/drug effects
Quantitative Trait Loci
CRISPR-Cas Systems/genetics
Mutation
Antioxidants/pharmacology
*Host Microbial Interactions/physiology
*Microbiota/physiology
DNA-Binding Proteins/genetics
Transcription Factors/genetics
Caenorhabditis elegans Proteins/genetics
Glycogen Synthase/genetics
Genetic Variation
*Caenorhabditis elegans/drug effects/genetics/microbiology/physiology
Gene Editing
Animals, Genetically Modified
Animals

@article {pmid42520925,
year = {2026},
author = {Quan, AT},
title = {From one-size-fits-all to on-demand: personalized crispr gene editing for rare genetic liver diseases.},
journal = {Clinics and research in hepatology and gastroenterology},
volume = {50},
number = {8},
pages = {102892},
doi = {10.1016/j.clinre.2026.102892},
pmid = {42520925},
issn = {2210-741X},
mesh = {Humans ; *Liver Diseases/therapy/genetics ; *Gene Editing ; *Genetic Therapy/methods ; *Precision Medicine ; *Rare Diseases/therapy/genetics ; *CRISPR-Cas Systems ; },
abstract = {Rare genetic liver diseases collectively affect millions of individuals worldwide and encompass a heterogeneous group of monogenic disorders including Wilson disease, alpha-1 antitrypsin deficiency, glycogen storage diseases, urea cycle disorders, progressive familial intrahepatic cholestasis, and acute hepatic porphyrias. While conventional management relies on dietary modification, pharmacotherapy, and ultimately liver transplantation, the advent of clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing has opened transformative therapeutic avenues. This review provides a comprehensive and critical appraisal of the current landscape of CRISPR-based therapies for genetic liver diseases, from preclinical proof-of-concept studies to landmark clinical trials. We examine the evolution from conventional Cas9 nuclease-mediated editing to precision tools including base editors and prime editors, which enable single-nucleotide corrections without inducing double-strand DNA breaks. The role of lipid nanoparticle delivery systems in achieving efficient hepatocyte-targeted delivery is discussed, alongside emerging challenges in pediatric dosing and immunogenicity. We highlight the paradigm shift toward personalized, patient-specific CRISPR therapies, exemplified by the first-in-human bespoke gene editing treatment delivered in 2025. Competing nucleic acid technologies, including RNA interference and antisense oligonucleotides, are compared in terms of durability, safety, and cost-effectiveness. Finally, we critically evaluate the evolving regulatory landscape and propose a priority framework for selecting genetic liver diseases most amenable to CRISPR-based correction. This review underscores that CRISPR gene editing is transitioning from experimental promise to clinical reality for genetic liver diseases, with personalized approaches poised to redefine the treatment paradigm.},
}

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

@article {pmid42665571,
year = {2026},
author = {Vuelta, E and Liquori, A and Morín, M and Soletto, L and Marugal, MÁ and Santiago-Balsera, M and Gallardo, M and Moreno-Pelayo, MÁ and Cervera, J and Sanjuan-Pla, A},
title = {Functional assessment of inherited myeloid neoplasm-associated SAMD9L germline variants via Monoallelic CRISPR modelling.},
journal = {British journal of haematology},
volume = {209},
number = {4},
pages = {1538-1548},
doi = {10.1111/bjh.70799},
pmid = {42665571},
issn = {1365-2141},
support = {PID2022-136660OB-I00//Ministerio de Educación y Ciencias/ ; PI22/01633//Instituto de Salud Carlos III/ ; PI25/01629//Instituto de Salud Carlos III/ ; },
mesh = {Humans ; *Germ-Line Mutation ; *CRISPR-Cas Systems ; HL-60 Cells ; *Tumor Suppressor Proteins/genetics/metabolism ; Intracellular Signaling Peptides and Proteins/genetics ; Gene Editing ; Genetic Predisposition to Disease ; Alleles ; },
abstract = {While the majority of myeloid neoplasms are sporadic, the increasing application of germline genetic testing has led the World Health Organization to designate 'Myeloid malignancies with germline predisposition' as a distinct clinical entity, carrying major implications for clinical care and research. Germline variants in the Sterile alpha motif domain-containing 9-like (SAMD9L) gene are specifically associated with an increased risk for these malignancies. In this study, we functionally modelled two novel SAMD9L variants-p.N697Y and p.K1294*-alongside two previously reported variants (p.T233N and p.H880Q). We generated heterozygous knock-in cellular models in the Human leukemia (HL-60 myeloid cell line) for each variant using homology-directed repair-based Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) gene editing. Functional assays, focused on proliferation and protein translation, confirmed that the p.T233N, p.N697Y and p.K1294* variants all caused a decreased rate of protein translation. These results provide functional evidence to fine-tune the classification of these SAMD9L variants and significantly advance our understanding of the molecular mechanisms by which SAMD9L variants drive inherited myeloid neoplasms.},
}

Humans
*Germ-Line Mutation
*CRISPR-Cas Systems
HL-60 Cells
*Tumor Suppressor Proteins/genetics/metabolism
Intracellular Signaling Peptides and Proteins/genetics
Gene Editing
Genetic Predisposition to Disease
Alleles

@article {pmid42680563,
year = {2026},
author = {Dong, C and Xu, Y and Wu, K and Mu, N and Kumar, S and Liang, FS},
title = {Cellular signal-responsive m[5]C editing by an inducible split CRISPR platform.},
journal = {RNA (New York, N.Y.)},
volume = {32},
number = {11},
pages = {1823-1836},
doi = {10.1261/rna.081049.126},
pmid = {42680563},
issn = {1469-9001},
mesh = {RNA Methylation ; Abscisic Acid/pharmacology ; *CRISPR-Cas Systems ; *RNA Editing ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Signal Transduction ; *Cytidine/analogs & derivatives/metabolism ; RNA, Messenger/genetics/metabolism ; },
abstract = {RNA 5-methylcytidine (m[5]C) modification plays an essential role in regulating RNA metabolism and functions in cellular processes. Tools to achieve temporal and transcript-specific m[5]C editing for functional studies are still limited. Furthermore, methods enabling m[5]C editing triggered by specific cellular signals to study m[5]C functions are currently lacking. Here, we present a temporally and conditionally controlled m[5]C writing platform engineered through integrating abscisic acid (ABA)-mediated chemically induced proximity with split-dCas13b-NSUN2/NSUN6 technology. This system enables the writing of m[5]C by reconstituting the split dCas13b-based m[5]C editing complex at the guide RNA (gRNA)-targeted RNA transcript sites under the control of the inducer ABA. The deposition of m[5]C is inducible, reversible and selective. The deposited m[5]C is biologically active and influences the stability of endogenous mRNA transcripts. Moreover, by incorporating ABA prodrugs, the m[5]C writing can be triggered by signals associated with distinct physiological or disease conditions (e.g., tumor microenvironment and senescence). This conditional m[5]C editing strategy provides a new programmable tool for studying m[5]C biology in a context-dependent manner and expands the repertoire of RNA modification editing technologies.},
}

RNA Methylation
Abscisic Acid/pharmacology
*CRISPR-Cas Systems
*RNA Editing
RNA, Guide, CRISPR-Cas Systems/genetics
Humans
Signal Transduction
*Cytidine/analogs & derivatives/metabolism
RNA, Messenger/genetics/metabolism

@article {pmid42862161,
year = {2026},
author = {Yaghoubi, D and V Pillai, V},
title = {Synergistic Integration of Recombinase Polymerase Amplification and CRISPR-Cas12a Enables High-Sensitivity Detection of Mycoplasma bovis.},
journal = {MicrobiologyOpen},
volume = {15},
number = {5},
pages = {e70435},
pmid = {42862161},
issn = {2045-8827},
support = {AgSeed-2025//Purdue University AgSeed/ ; },
mesh = {*Mycoplasma bovis/genetics/isolation & purification ; Animals ; Bison ; Cattle ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Mycoplasma Infections/veterinary/diagnosis/microbiology ; Recombinases/genetics/metabolism ; *Nucleic Acid Amplification Techniques/methods ; *Cattle Diseases/diagnosis/microbiology ; Rapid Diagnostic Tests ; Bacterial Proteins/genetics ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Mycoplasma bovis is a major pathogen of cattle, causing substantial economic losses in the global cattle industry and emerging as a serious threat to North American bison (Bison bison). Effective outbreak management is hindered by the lack of rapid diagnostic methodologies deployable in field settings. Current testing approaches are limited by slow turnaround times for microbial culture and the dependence of qPCR on centralized laboratory infrastructure, both of which are poorly suited for field settings. To address this gap, we developed a highly sensitive, isothermal diagnostic platform coupling Recombinase Polymerase Amplification with CRISPR-Cas12a detection targeting the conserved uvrC gene. The optimized assay requires less than 1 h at 37°-39°C and achieves an analytical limit of detection of approximately 8 copies, comparable to that of the gold-standard quantitative PCR. Clinical validation using 110 samples from bison and cattle demonstrated 100% concordance with PCR and complete differentiation from closely related species, including Mycoplasma canis and Mycoplasma felis. Overall, these data establish the RPA-CRISPR/Cas12a assay as a sensitive and specific isothermal approach for detecting M. bovis in clinical specimens, providing a field-adaptable framework for future instrument-free surveillance in resource-limited agricultural settings and supporting improved herd health management in both commercial cattle and bison conservation operations.},
}

*Mycoplasma bovis/genetics/isolation & purification
Animals
Bison
Cattle
*CRISPR-Cas Systems
Sensitivity and Specificity
*Mycoplasma Infections/veterinary/diagnosis/microbiology
Recombinases/genetics/metabolism
*Nucleic Acid Amplification Techniques/methods
*Cattle Diseases/diagnosis/microbiology
Rapid Diagnostic Tests
Bacterial Proteins/genetics
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid42862306,
year = {2026},
author = {Sahu, S and Jain, P and Dharme, KK and Yadav, K},
title = {CRISPR/Cas9-based nanodelivery systems for precision gene editing in neuroinflammation.},
journal = {Inflammation research : official journal of the European Histamine Research Society ... [et al.]},
volume = {75},
number = {1},
pages = {},
pmid = {42862306},
issn = {1420-908X},
support = {Probecell/Res/2026/124//Probecell Scientific Pvt. Ltd./ ; },
mesh = {Humans ; Animals ; *Neuroinflammatory Diseases/genetics/therapy ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Nanoparticles/administration & dosage ; },
abstract = {BACKGROUND: Neuroinflammation is a characteristic feature of many neurological diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Uncontrolled activation of microglia and astrocytes results in the production of proinflammatory mediators that contribute to neuronal dysfunction and degeneration.

PURPOSE: CRISPR/Cas9 genome-editing technology provides highly precise targeting of genes involved in these pathways, enabling the deletion, correction, or modulation of disease-associated genetic alterations. Nonetheless, effective delivery of CRISPR components to the central nervous system (CNS) remains a major challenge because of protective barriers such as the blood-brain barrier (BBB), as well as concerns regarding genomic instability and immunogenicity.

METHODS: Nanotechnology-guided delivery platforms, such as lipid nanoparticles, liposomes, dendrimers and polymeric carriers, have been developed as promising strategies to address these challenges. These nanosystems can improve biodistribution, pharmacokinetic behavior, and cellular uptake, while potentially reducing off-target effects and systemic toxicity.

RESULTS: The preclinical studies demonstrate successful modulation of key neuroinflammatory targets, including the NLRP3 inflammasome, triggering receptor expressed on myeloid cells 2 (TREM2), Janus kinase (JAK) signaling, and the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, with potential therapeutic relevance to neurodegenerative diseases. In addition, novel techniques, such as mitochondrial base editing, prime editing, and epigenetic modulation, expand the scope of CRISPR-mediated therapeutic strategies. Although outcomes from animal models are promising, translation to the clinical setting requires careful consideration of safety, ethical concerns, and long-term efficacy.

CONCLUSIONS: This review highlights the potential of integrating CRISPR/Cas9 with nanodelivery systems to advance precision medicine for neuroinflammatory disorders, while emphasizing their potential for therapeutic applications in complex CNS conditions. Ongoing studies aimed at optimizing delivery and reducing off-target effects will be essential for advancing these approaches toward clinical practice.},
}

Humans
Animals
*Neuroinflammatory Diseases/genetics/therapy
*Gene Editing/methods
*CRISPR-Cas Systems
Nanoparticles/administration & dosage

@article {pmid42862383,
year = {2026},
author = {Ali, Q and Trujillo, C and Altpeter, F},
title = {mTREX2 co-expression promotes large deletions in CRISPR/Cas12a editing of the highly polyploid sugarcane genome.},
journal = {Plant cell reports},
volume = {45},
number = {11},
pages = {},
pmid = {42862383},
issn = {1432-203X},
support = {DE-SC0018420//U.S. Department of Energy/ ; },
mesh = {*Saccharum/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Polyploidy ; *Genome, Plant/genetics ; *Plant Proteins/genetics/metabolism ; Sequence Deletion/genetics ; },
abstract = {The LbCas12a[i]-mTREX2 system enhances large deletions, and the LbCas12a[i]-RRV enables efficient editing of lw1 in sugarcane. Sugarcane (Saccharum spp. hybrids) is a major feedstock for biofuel production and the primary source of global table sugar; however, its highly polyploid and complex genome constrains the effective deployment of functional genomics and precision breeding strategies. While CRISPR/Cas9-mediated genome editing has been reported in sugarcane, Cas12a-based editing remains unexplored despite its advantages in targeting AT-rich genomic regions. In this study, we report efficient CRISPR/Cas12a-mediated targeted mutagenesis of the lw1 gene in sugarcane. The lemon white1 (lw1) is a key regulator of chlorophyll and carotenoid biosynthesis, and targeted lw1 mutagenesis results in a visible phenotype. We compared the editing performance of intron-containing LbCas12a-R (LbCas12a[i]-R) and LbCas12a-RRV (LbCas12a[i]-RRV) variants and assessed the impact of Mus musculus Three prime repair exonuclease 2 (mTREX2) co-expression on editing efficiency and deletion profiles. Editing efficiencies ranged from 31 to 85%, where LbCas12a[i]-RRV consistently outperformed LbCas12a[i]-R in both overall editing efficiency and allelic editing efficiency. Co-expression of the mTREX2 shifted the mutation spectrum toward larger deletions, increasing the frequency of moderate-to-large deletions. The ≥ 51 bp deletion class increased ~7-fold in both variants. Capillary electrophoresis-based genotyping provided a cost-effective, high-throughput alternative to next-generation sequencing (NGS) and accurately predicted allelic editing efficiency and indel size. This first report of CRISPR/Cas12a-mediated genome editing in sugarcane provides a framework for future engineering of AT-rich cis-regulatory elements in the polyploid sugarcane genome.},
}

*Saccharum/genetics
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Polyploidy
*Genome, Plant/genetics
*Plant Proteins/genetics/metabolism
Sequence Deletion/genetics

@article {pmid42862825,
year = {2026},
author = {Wu, J and Liu, D and Xu, H and Zeng, L and Lin, P and Zhao, P and Shen, G},
title = {The AF-2 domain of estrogen-related receptor interacts with PPRC1 to modulate larval epidermal marking in Bombyx mori.},
journal = {PLoS genetics},
volume = {22},
number = {10},
pages = {e1012339},
pmid = {42862825},
issn = {1553-7404},
mesh = {Animals ; *Bombyx/genetics/metabolism/growth & development ; *Receptors, Estrogen/genetics/metabolism/chemistry ; *Epidermis/metabolism ; Larva/genetics/metabolism/growth & development ; *Insect Proteins/genetics/metabolism ; Protein Domains ; Melanins/metabolism ; Melanogenesis ; ERRalpha Estrogen-Related Receptor ; CRISPR-Cas Systems ; },
abstract = {Estrogen-related receptors (ERRs) have no natural ligands, vertebrate ERRs rely on their activation function-2 (AF-2) structural domain to recruit coactivators for transcriptional regulation. While metabolic regulation in insects differs significantly from that in vertebrates, it remains unclear whether the regulatory function of insect ERR requires coactivators. Using silkworms as the research subject, this study aims to analyze the molecular mechanisms of ERR-regulated metabolism in insects and refine the metabolic regulatory network. This study applied CRISPR/Cas9 gene editing technology to knock out the AF-2 domain of BmERR in the lepidopteran silkworms and examined its effects on functional role, associated interacting factors, and related physiological processes. Disruption of the BmERR AF-2 domain inhibited the expression of melanin metabolism-related genes BmTH, BmDDC, BmAA-NAT, Bmebony, and Bmtan, leading to the accumulation of tyrosine and dopa, thereby causing aberrant melanin metabolism and loss of epidermal markings. By contrast, BmERR overexpression enhanced the pathway, resulting in intensification of epidermal markings. The homologous gene of the ERR coactivator, BmPPRC1, showed highly consistent expression with BmERR in the epidermis. The immunofluorescence signal of BmPPRC1 was localized to the nucleus and highly overlapped with that of BmERR. BmPPRC1 interacts with the AF-2 domain of BmERR, and deletion of this region weakens the regulation of melanin metabolism-related genes in silkworm embryonic cells. The ERR function and mode of action are evolutionarily conserved, and its AF-2 structural domain binds equally well to the coactivator BmPPRC1, providing new perspectives for studying the modulability of insect mottle formation.},
}

Animals
*Bombyx/genetics/metabolism/growth & development
*Receptors, Estrogen/genetics/metabolism/chemistry
*Epidermis/metabolism
Larva/genetics/metabolism/growth & development
*Insect Proteins/genetics/metabolism
Protein Domains
Melanins/metabolism
Melanogenesis
ERRalpha Estrogen-Related Receptor
CRISPR-Cas Systems

@article {pmid42863841,
year = {2026},
author = {Yamamoto, K and Shikata, S and Hao, Y and Tomita, T and Kanai, R and Verhoeyen, E and Saeki, Y and Goyama, S},
title = {Development of CRISPR/Cas13-based analytical tools to study RNA-protein interactions.},
journal = {Nucleic acids research},
volume = {54},
number = {18},
pages = {},
pmid = {42863841},
issn = {1362-4962},
support = {//Japan Society for the Promotion of Science/ ; 22H03100//Grant-in-Aid for Scientific Research/ ; 21H00274//Grant-in-Aid for Scientific Research on Innovative Areas/ ; 22KK0127//Fostering Joint International Research/ ; JP22K16319//JSPS KAKEN/ ; JP24K19216//JSPS KAKEN/ ; JP23H05479//JSPS KAKEN/ ; JP23ama221223//Japan Agency for Medical Research and Development/ ; //Japanese Society of Hematology/ ; //Tokyo Biochemical Research Foundation/ ; //Uehara Memorial Foundation/ ; //Mochida Memorial Foundation for Medical and Pharmaceutical Research/ ; },
mesh = {*RNA-Binding Proteins/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; *RNA, Long Noncoding/metabolism/genetics ; Paraspeckles/metabolism/genetics ; Protein Binding ; *RNA/metabolism ; HEK293 Cells ; },
abstract = {RNA-protein interactions (RPIs) are as important as protein-protein interactions (PPIs) for the formation of membraneless organelles (MLOs) and play vital roles in diverse biological processes. Despite remarkable advances in PPI analysis technologies, the development of tools for RPI analysis has lagged behind. To address this challenge, we integrated three established PPI technologies-bimolecular fluorescence complementation (BiFC), NanoBiT, and split-TurboID-with RNA-targeting CRISPR/dCas13. We applied these platforms to paraspeckles, a representative MLO formed through interactions between the long non-coding RNA NEAT1 and the RNA-binding protein NONO. BiFC-dCas13 functions as a fluorescence-based recorder that enables quantitative detection of NEAT1-NONO interaction events. NanoBiT-dCas13 precisely monitors dynamic changes in the NEAT1-NONO interaction through a reversible luminescence readout. Split-TurboID-dCas13 induces proximity-dependent biotinylation of proteins surrounding paraspeckles, identifying the N6-methyladenosine reader YTHDC1 as a paraspeckle-associated protein. Furthermore, BiFC-dCas13 was readily reconfigured to detect additional RPI pairs, demonstrating the modularity of the platform. Together, these complementary systems provide versatile approaches for recording, monitoring, and discovering RPI-associated biology and molecular environments.},
}

*RNA-Binding Proteins/metabolism/genetics
*CRISPR-Cas Systems/genetics
Humans
*RNA, Long Noncoding/metabolism/genetics
Paraspeckles/metabolism/genetics
Protein Binding
*RNA/metabolism
HEK293 Cells

@article {pmid42864354,
year = {2026},
author = {Kinney, KJ and Jia, K and Zhang, H and Schmaljohn, E and Osborne, T and Thommandru, B and Murugan, K and Sánchez-Peña, A and West, S and Chen, S and Codipilly, R and Sturgeon, M and Turk, R and McNeill, MS and Behlke, M and Jacobi, A and Cromer, MK and Rettig, G and Kurgan, GL},
title = {Author Correction: UNCOVERseq enables sensitive and controlled gene editing off-target nomination across CRISPR-Cas modalities and systems.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
doi = {10.1038/s41467-026-78544-3},
pmid = {42864354},
issn = {2041-1723},
}

@article {pmid42864360,
year = {2026},
author = {Zhao, J and Deng, Y and Chen, H and Zhou, X and Yin, S and Qian, Q and Xie, X and Chen, Q and Zhang, J and Fan, L and Ren, X and Chen, B and Jiang, C},
title = {In vivo CRISPR screens identify dual functions of DNMT3A in mediating tumor immune evasion.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42864360},
issn = {2041-1723},
mesh = {Animals ; DNA Methyltransferase 3A ; Female ; *DNA (Cytosine-5-)-Methyltransferases/genetics/metabolism/immunology ; Mice ; CRISPR-Cas Systems ; Humans ; *Tumor Escape/genetics/immunology ; Cell Line, Tumor ; Cholesterol/biosynthesis ; CD8-Positive T-Lymphocytes/immunology/metabolism ; *Neoplasms/immunology/genetics ; Immunotherapy ; Mice, Inbred C57BL ; Gene Expression Regulation, Neoplastic ; },
abstract = {Cancer immunotherapy has achieved remarkable clinical efficacy across multiple cancers but remains ineffective for most patients, and the mechanisms underlying resistance remain incompletely understood. Here, we perform in vivo CRISPR-Cas9 screens in female mice and identify DNA methyltransferase 3 A (DNMT3A) as a key driver of tumor immune evasion through both methyltransferase-dependent and -independent mechanisms. DNMT3A represses TNFRSF1A and TNFRSF1B expression, enabling tumor cells to evade TNFα-induced killing. Independently of its methyltransferase activity, DNMT3A interacts with NFYA to activate cholesterol biosynthesis, leading to zymosterol accumulation in tumor interstitial fluid. Zymosterol impairs SREBP2 cleavage and promotes HMGCR degradation in CD8[+] T cells, thereby suppressing cholesterol biosynthesis and compromising their antitumor effector functions. Targeting DNMT3A with antisense oligonucleotides overcomes resistance to immune checkpoint blockade. Collectively, these findings establish DNMT3A as an integrated epigenetic and metabolic checkpoint in tumor immune evasion and a potential target for immunotherapy.},
}

Animals
DNA Methyltransferase 3A
Female
*DNA (Cytosine-5-)-Methyltransferases/genetics/metabolism/immunology
Mice
CRISPR-Cas Systems
Humans
*Tumor Escape/genetics/immunology
Cell Line, Tumor
Cholesterol/biosynthesis
CD8-Positive T-Lymphocytes/immunology/metabolism
*Neoplasms/immunology/genetics
Immunotherapy
Mice, Inbred C57BL
Gene Expression Regulation, Neoplastic

@article {pmid42864491,
year = {2026},
author = {Yu, Y and Zhang, Y and Xu, Y and Wen, C and Li, L and Yu, T},
title = {The global patent landscape of CRISPR-Cas applications in medicine.},
journal = {Nature biotechnology},
volume = {44},
number = {10},
pages = {1606-1615},
pmid = {42864491},
issn = {1546-1696},
}

@article {pmid42864492,
year = {2026},
author = {Ummarino, D},
title = {Tracing the origins of CRISPR-Cas.},
journal = {Nature biotechnology},
volume = {44},
number = {10},
pages = {1617},
doi = {10.1038/s41587-026-03353-4},
pmid = {42864492},
issn = {1546-1696},
}

@article {pmid42864601,
year = {2026},
author = {Li, M and Jiao, YG and Tao, R and Hu, Y and Fei, JY and Wang, CT and Yao, SH},
title = {Optimized gGBE enables robust G-to-Y conversion in mammalian cells and zebrafish embryos.},
journal = {Zoological research},
volume = {47},
number = {6},
pages = {1839-1850},
doi = {10.24272/j.issn.2095-8137.2025.361},
pmid = {42864601},
issn = {2095-8137},
mesh = {Animals ; *Zebrafish/genetics/embryology ; Humans ; *Gene Editing/methods ; Embryo, Nonmammalian ; CRISPR-Cas Systems ; Amyotrophic Lateral Sclerosis/genetics ; *Glycoside Hydrolases/genetics/metabolism ; },
abstract = {Base editing is a powerful and precise genome-editing tool capable of introducing single-base substitutions with high resolution. Depending on the base-modification domains employed, base editors can mediate both transition and transversion base editing in mammalian cells. While deaminase-derived base editors have enabled efficient transition editing in the zebrafish genome, glycosidase-derived transversion editing remains a significant challenge. In this study, guided by structural insights into the Cas9 complex, we engineered a series of glycosidase guanine base editor (gGBE) constructs by inlaying the N-methylpurine DNA glycosylase (MPG) domain into Cas9 protein. Among these variants, we identified a high-performance gGBE in which MPG was inserted between residues G1246 and S1247 of the PI domain in Streptococcus pyogenes Cas9, which we named gGBE-PIGS. This variant improved editing efficiency by up to 3.5-fold in mammalian cells and 13-fold in zebrafish embryos, without compromising editing precision. Furthermore, we developed a gGBE-editing-responsive GFP reporter to monitor editing efficiency and enrich for gGBE-edited cells and embryos. Using a combination of the optimized gGBE-PIGS and the GFP reporter, we successfully generated zebrafish models carrying mutations homologous to human amyotrophic lateral sclerosis (ALS)-associated variants. Together, these findings highlight the robustness and versatility of the gGBE platform for precise base editing and underscore its promising potential for in vivo disease modeling and therapeutic genome engineering.},
}

Animals
*Zebrafish/genetics/embryology
Humans
*Gene Editing/methods
Embryo, Nonmammalian
CRISPR-Cas Systems
Amyotrophic Lateral Sclerosis/genetics
*Glycoside Hydrolases/genetics/metabolism

@article {pmid42864652,
year = {2026},
author = {Noer, MG and Christensen, KB and Mikkelsen, NS and Skjelbostad, SF and Rothemejer, FH and Wittenborn, T and Fammé, S and Mikkelsen, K and Barington, T and Thomsen, MK and Barnkob, MB and Møller, BK and Bak, RO},
title = {CRISPR integration of an FLT3 CAR into the TRAC locus generates CAR T cells for AML.},
journal = {Genome medicine},
volume = {18},
number = {1},
pages = {},
pmid = {42864652},
issn = {1756-994X},
support = {4-1612-391/1//The Danish health authorities (SST)/ ; 101041231//HORIZON EUROPE European Research Council/ ; 101057438//Horizon 2020/ ; R238-2016-3349//Lundbeck Foundation/ ; NNF19OC0058238//Novo Nordisk Fonden/ ; 8056-00010B//Innovationsfonden/ ; CF20-0424//Carlsbergfondet/ ; },
mesh = {*fms-Like Tyrosine Kinase 3/genetics/immunology ; Humans ; *Leukemia, Myeloid, Acute/therapy/genetics/immunology ; Animals ; *T-Lymphocytes/immunology/metabolism ; *Immunotherapy, Adoptive/methods ; *Receptors, Chimeric Antigen/genetics ; Mice ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Genetic Loci ; },
abstract = {BACKGROUND: Acute myeloid leukemia (AML) remains a therapeutically challenging hematological malignancy with high relapse rates, especially in patients harboring mutations in the FMS-like tyrosine kinase 3 (FLT3) gene. FLT3, a receptor tyrosine kinase involved in hematopoietic cell proliferation and survival, is frequently mutated or overexpressed in AML, contributing to leukemic proliferation and poor prognosis. Although FLT3 inhibitors have demonstrated clinical benefit, resistance often develops, underscoring the need for alternative treatment strategies.

METHODS: In this study, we developed and evaluated allogeneic anti-FLT3 CAR T cells engineered by CRISPR/Cas9-mediated targeted integration into the T cell receptor α constant (TRAC) locus (TRAC-CAR T). This approach enables CAR expression under the endogenous TRAC promoter while simultaneously disrupting native TCR expression, thereby allowing the use of allogeneic T cells with reduced risk of graft-versus-host disease.

RESULTS: TRAC-CAR T cells demonstrated efficient disruption of TCRαβ expression, robust surface CAR expression, and superior integration efficiency compared to safe harbor AAVS1-targeted and lentivirally transduced CAR T cells (LV-CAR T). Functionally, TRAC-CAR T cells exhibited potent and selective cytotoxicity against FLT3-positive AML cell lines in vitro, achieving comparable killing relative to LV-CAR T cells. In a xenograft AML mouse model, TRAC-CAR T cells effectively reduced leukemia burden and prolonged mouse survival, with efficacy matching that of LV-CAR T cells. Notably, TRAC-CAR T cells exhibited reduced exhaustion marker expression.

CONCLUSIONS: Together, these results highlight TRAC-integrated anti-FLT3 CAR T cells as a promising off-the-shelf immunotherapy candidate, combining precise gene editing, efficient manufacturing, and potent anti-leukemic activity.},
}

*fms-Like Tyrosine Kinase 3/genetics/immunology
Humans
*Leukemia, Myeloid, Acute/therapy/genetics/immunology
Animals
*T-Lymphocytes/immunology/metabolism
*Immunotherapy, Adoptive/methods
*Receptors, Chimeric Antigen/genetics
Mice
*CRISPR-Cas Systems
Cell Line, Tumor
*Genetic Loci

@article {pmid42865039,
year = {2026},
author = {Zhen, X and Hu, J and Wang, X and Qing, J and Wu, J},
title = {Genome-wide CRISPR screens identify shared and context-specific regulators of podocyte resistance to oxidative injury.},
journal = {Renal failure},
volume = {48},
number = {1},
pages = {2729639},
pmid = {42865039},
issn = {1525-6049},
mesh = {*Podocytes/drug effects/metabolism/pathology ; Humans ; *Oxidative Stress/genetics/drug effects ; Doxorubicin/toxicity ; Hydrogen Peroxide/toxicity ; *CRISPR-Cas Systems ; Gene Knockout Techniques ; },
abstract = {Podocyte injury is a convergent driver of glomerular disease progression, yet the genetic determinants that govern podocyte vulnerability to oxidative stress remain incompletely defined. Here, we performed genome-wide CRISPR-Cas9 knockout screens in differentiated human podocytes exposed to two complementary injury models: Adriamycin (ADR), a clinically relevant inducer of podocyte damage, and hydrogen peroxide (H2O2), a direct oxidative stress challenge. Human podocytes were transduced with a genome-wide sgRNA library, differentiated under non-permissive conditions and subjected to optimized stress conditions that imposed selective pressure while preserving library complexity. Across five independent replicates per condition, sequencing-based sgRNA profiling demonstrated high reproducibility, balanced library representation and clear treatment-specific selection. The ADR screen identified 819 depleted and 456 enriched genes, whereas the H2O2 screen identified 277 depleted and 215 enriched genes. Integrated analysis revealed both model-specific dependencies and 27 shared depleted genes across the two injury conditions, implicating stress adaptation, chromatin regulation, mitochondrial metabolism, inflammatory signaling and cytoskeletal organization as candidate processes required for podocyte survival. These genome-wide screens provide a functional framework for prioritizing genes that regulate podocyte resilience to oxidative injury and offer a resource for mechanistic studies of glomerular disease.},
}

*Podocytes/drug effects/metabolism/pathology
Humans
*Oxidative Stress/genetics/drug effects
Doxorubicin/toxicity
Hydrogen Peroxide/toxicity
*CRISPR-Cas Systems
Gene Knockout Techniques

@article {pmid42866118,
year = {2026},
author = {Alsuwat, H and Alvarez, D and Al-Babili, S},
title = {Advances in plant metabolic engineering through synthetic biology.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1957203},
pmid = {42866118},
issn = {1664-462X},
abstract = {The challenges associated with the escalating global population and climate change in crop production and food security are substantial. This underscores the necessity for improvements in crop nutrition, yield, and resilience to environmental stresses. Plant metabolic pathways directly influence growth, nutrient accumulation, and stress responses, rendering them prime targets for crop enhancement. Advances in plant synthetic biology and gene editing are facilitating more precise modifications of genes, regulatory elements, and metabolic pathways. This review highlights the primary technologies used in plant metabolic engineering and focuses on representative examples of their implementation for crop improvement, including but not limited to CRISPR-Cas systems, multiplex genome editing, knock-in and knock-out strategies, CRISPR-mediated gene regulation (CRISPRi/CRISPRa), base editing, prime editing, RNA targeting technologies, synthetic promoters, and gene stacking strategies. Furthermore, the emerging role of artificial intelligence in the design and optimization of metabolic pathways and genome editing tools will be briefly discussed.},
}

@article {pmid42866947,
year = {2026},
author = {Kim, Y and An, S and Choi, J and Choi, M and Lee, G and Takata, KI and Anand, R and Myung, K and Lee, JY},
title = {CRISPR-mediated gap-based replacement enables site-specific DNA lesion insertion into long DNA and nucleosomes.},
journal = {Nucleic acids research},
volume = {54},
number = {19},
pages = {},
pmid = {42866947},
issn = {1362-4962},
support = {RS-2024-00449988//National Research Foundation/ ; RS-2025-21063026//National Research Foundation/ ; RS-2025-25419288//National Research Foundation/ ; IBS-R022-D1//Institute for Basic Science/ ; IBS-R039-D3-2026-a00//Institute for Basic Science/ ; },
mesh = {*Nucleosomes/genetics/metabolism ; *DNA/genetics/metabolism/chemistry ; *CRISPR-Cas Systems/genetics ; *DNA Damage ; Excision Repair ; DNA Repair ; DNA Breaks, Double-Stranded ; },
abstract = {Defined DNA lesions are essential substrates for dissecting DNA repair mechanisms, yet existing approaches remain limited in positional flexibility, lesion diversity, efficiency, or compatibility with long and chromatinized DNA. Here, we present CGRID (CRISPR-mediated Gap-based Replacement for Insertion of DNA damage), a programmable strategy for site-specific installation of DNA lesions into long DNA and nucleosomes. CGRID uses paired Cas9 nickases to generate two adjacent nicks, followed by gap formation and replacement with a synthetic lesion-containing oligonucleotide. This design enables easy and efficient insertion of diverse DNA lesions at user-defined positions without pre-engineered nicking sites or substrate redesign. Applied to long λ-DNA, CGRID supports efficient installation of single lesions and internal double-strand breaks at predefined positions, which are challenging to obtain using existing methods. These molecules enable high-throughput single-molecule analysis of diverse repair pathways including base excision repair (BER), nonhomologous end joining, and microhomology-mediated end joining without hydrodynamic force. CGRID further allows efficient site-specific lesion incorporation into nucleosomal DNA, enabling systematic repositioning of damage within a nucleosome. This shows that nucleosome context and lesion orientation strongly influence APE1 incision efficiency. Together, CGRID establishes a broadly applicable platform for programmable DNA damage engineering, bridging biochemical and single-molecule methodologies, and facilitates mechanistic studies of chromatin-dependent genome maintenance.},
}

*Nucleosomes/genetics/metabolism
*DNA/genetics/metabolism/chemistry
*CRISPR-Cas Systems/genetics
*DNA Damage
Excision Repair
DNA Repair
DNA Breaks, Double-Stranded

@article {pmid42867411,
year = {2026},
author = {Holcomb, EA and Jiang, L and Pearson, AN and James, J and Tate, A and Wang, Z and Dasgupta, A and Clauwaert, J and Armas, ND and Elaimy, AL and Palagama, DSW and Goodwin, SK and Kwiatkowski, MJ and Gijón, M and Morgan, MA and Courtney, AH and Carty, SA and Huber, AK and Zou, W and Prensner, JR and Green, MD},
title = {CRISPR-Cas9 screen identifies eIF4G2 as a regulator of CD8+ T cell dysfunction.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {215},
number = {10},
pages = {},
pmid = {42867411},
issn = {1550-6606},
support = {R01CA276217//National Institutes of Health (NIH)/ ; R01CA217648//National Institutes of Health (NIH)/ ; R01CA123088//National Institutes of Health (NIH)/ ; R01CA099985//National Institutes of Health (NIH)/ ; R01CA193136//National Institutes of Health (NIH)/ ; R01CA152470//National Institutes of Health (NIH)/ ; 2T32-AI007413-31//National Institutes of Health (NIH)/ ; 2T32CA009676-31A1//National Institutes of Health (NIH)/ ; I01BX005267//U.S. Department of Veterans Affairs/ ; //NIH/ ; K08-CA263552-01A1/CA/NCI NIH HHS/United States ; V2024-013//V Foundation for Cancer Research/ ; //CureSearch Foundation/ ; //Hyundai Hope on Wheels Foundation/ ; //ChadTough Defeat DIPG Foundation/ ; CI-127-24//the Ben and Catherine Ivy Foundation Clinical Investigator of the Damon Runyon Cancer Research Foundation/ ; },
mesh = {Animals ; *CD8-Positive T-Lymphocytes/immunology/metabolism ; Mice ; *CRISPR-Cas Systems ; *Eukaryotic Initiation Factor-4G/genetics/metabolism/immunology ; Tumor Microenvironment/immunology ; Mice, Inbred C57BL ; T-Cell Exhaustion ; Cell Proliferation ; Melanoma, Experimental/immunology ; Mice, Knockout ; Humans ; },
abstract = {CD8+ T cells directly target and kill cancer cells and are required for effective antitumor immunity. However, CD8+ T cells may become dysfunctional when exposed to chronic antigen stimulation during cancer. Dysfunction is characterized by reduced proliferation, increased expression of inhibitory receptors, and impaired effector function, yet the genetic mechanisms controlling dysfunction remain poorly understood. Therefore, we developed a murine ex vivo chronic stimulation model that recapitulates features of CD8+ T cell dysfunction observed within the tumor microenvironment. By coupling this system with genome-wide CRISPR-Cas9 loss-of-function screening, we discovered that the progression of murine CD8+ T cells to a dysfunctional state required eIF4G2, a noncanonical mRNA translation factor. Deletion of eIF4G2 restored CD8+ T cell survival and proliferation during chronic stimulation conditions in vitro. Transcriptomic, proteomic, and flow cytometric analyses revealed that eIF4G2 deletion shifts CD8+ T cell phenotype from a dysfunctional state to a proliferative state. eIF4G2 depletion reduced oxidative stress, lipid peroxidation, and accumulation of misfolded protein aggresomes during chronic in vitro stimulation. Finally, eIF4G2-depleted cells mediated enhanced tumor control in a murine model of melanoma. These data suggest that targeting eIF4G2 may promote CD8+ T cell resilience and improve therapeutic antitumor immunity.},
}

Animals
*CD8-Positive T-Lymphocytes/immunology/metabolism
Mice
*CRISPR-Cas Systems
*Eukaryotic Initiation Factor-4G/genetics/metabolism/immunology
Tumor Microenvironment/immunology
Mice, Inbred C57BL
T-Cell Exhaustion
Cell Proliferation
Melanoma, Experimental/immunology
Mice, Knockout
Humans

@article {pmid42867739,
year = {2026},
author = {Mangal, H and Mathur, S and Chaurasia, A and Sharma, S and Kumar, S and Ranjan, R},
title = {CRISPR genome editing in food and feed crops: intellectual property challenges, global accessibility, and the Indian perspective.},
journal = {Transgenic research},
volume = {35},
number = {1},
pages = {},
pmid = {42867739},
issn = {1573-9368},
support = {09/0607(20798)/2025-EMR-I//Council of Scientific & Industrial Research (CSIR), New Delhi/ ; BT/PR40161/BTIS/137/32/2021//Department of Biotechnology, Govt. of India/ ; },
mesh = {*Crops, Agricultural/genetics/growth & development ; *Gene Editing/methods ; *Intellectual Property ; *Plants, Genetically Modified/genetics ; *CRISPR-Cas Systems/genetics ; India ; Humans ; Genome, Plant ; },
abstract = {The ever-increasing global demand for food and feed, coupled with population growth and climate change, necessitates the development of high-yielding and stress-resilient crops. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology has emerged as a transformative genome-editing tool due to its efficiency, precision, and ability to generate targeted genetic modifications. This review highlights recent advances in CRISPR genome editing, including base editing, prime editing, and novel Cas variants, together with emerging approaches such as epigenome editing, mitochondrial genome editing, and hypercompact Cas systems, and their applications in improving crop productivity, stress tolerance, and nutritional quality. In food and feed crops, these applications also include crop-specific priorities such as grain and nutritional quality in food crops and digestibility, forage quality, and mineral bioavailability in feed and forage crops. Particular emphasis is placed on the complex intellectual property (IP) landscape surrounding CRISPR technologies, including patents on editing systems, delivery platforms, and proprietary vectors. These intellectual property barriers can restrict research access, technology transfer, and commercialization, particularly in developing countries. The review further discusses patent limitations, licensing frameworks, and strategies to enhance equitable global access through collaborative approaches such as non-exclusive licensing, humanitarian licensing, indigenous technology development, and international partnerships. Overall, the review underscores the need to balance innovation, intellectual property protection, and global accessibility to ensure that CRISPR-based technologies contribute effectively to worldwide food and feed security and sustainable agriculture.},
}

*Crops, Agricultural/genetics/growth & development
*Gene Editing/methods
*Intellectual Property
*Plants, Genetically Modified/genetics
*CRISPR-Cas Systems/genetics
India
Humans
Genome, Plant

@article {pmid42484294,
year = {2026},
author = {Ma, T and Wu, J and Li, S and Chen, J},
title = {CRISPR Screening Identifies SMARCAL1 and MRN as Modulators of WRN Dependency in MSI-H Colorectal Cancer.},
journal = {Cancer research},
volume = {86},
number = {20},
pages = {5271-5286},
doi = {10.1158/0008-5472.CAN-26-1023},
pmid = {42484294},
issn = {1538-7445},
support = {CA274234//National Cancer Institute (NCI)/ ; CA275712//National Cancer Institute (NCI)/ ; CA278758//National Cancer Institute (NCI)/ ; },
mesh = {Humans ; *Werner Syndrome Helicase/genetics/metabolism/antagonists & inhibitors ; *Colorectal Neoplasms/genetics/metabolism/pathology ; *DNA Helicases/genetics/metabolism ; *MRE11 Homologue Protein/metabolism/genetics ; *Cell Cycle Proteins/metabolism/genetics ; DNA-Binding Proteins/metabolism/genetics ; *Nuclear Proteins/metabolism/genetics ; Cell Line, Tumor ; *Microsatellite Instability ; *DNA Repair Enzymes/metabolism/genetics ; CRISPR-Cas Systems ; Acid Anhydride Hydrolases ; },
abstract = {UNLABELLED: Microsatellite instability-high (MSI-H) colorectal cancer cells depend on the Werner syndrome helicase (WRN) to resolve cruciform DNA structures that arise from expanded TA-dinucleotide repeats. Loss of WRN induces replication stress and double-strand breaks, a vulnerability that can be recapitulated with the selective WRN inhibitor (WRNi) HRO761 in MSI-H cancer cells. To uncover the mechanisms governing sensitivity to WRN inhibition, we conducted genome-wide CRISPR/Cas9 screens in colorectal cancer cell lines treated with or without HRO761. These screens identified SMARCAL1 as a key modulator of WRN dependency. Depletion of SMARCAL1 rendered cells resistant to WRN inhibition, and rescue of this effect required the ATPase/translocase activity of SMARCAL1. Mechanistically, SMARCAL1 antagonized WRN and supported cruciform DNA structures, thereby enhancing cellular reliance on WRN. In addition, the MRE11-RAD50-NBS1 (MRN) complex, rather than MUS81 or ERCC1/XPF, was the principal mediator of cruciform DNA processing following WRN inhibition. Acute disruption of the MRN complex conferred profound resistance to WRN inhibition, whereas ataxia-telangiectasia mutated (ATM) deficiency produced a more modest resistant phenotype. Further genetic and pharmacologic epistasis analyses demonstrated that the MRN complex regulates WRNi sensitivity through both ATM-dependent signaling and MRE11 nuclease-dependent functions. Importantly, the key resistance mechanisms identified in this study were independently validated using a structurally distinct clinical-stage WRNi, VVD-214. Collectively, these findings identify the SMARCAL1-MRN-ATM axis as a critical regulator of WRN dependency and provide mechanistic insights into resistance to WRN-targeted therapy.

SIGNIFICANCE: SMARCAL1 and the MRN-ATM pathway determine sensitivity to WRN inhibition in microsatellite instability-high colorectal cancer, revealing key mechanisms of WRN dependency and potential drivers of resistance to WRN inhibitors.},
}

Humans
*Werner Syndrome Helicase/genetics/metabolism/antagonists & inhibitors
*Colorectal Neoplasms/genetics/metabolism/pathology
*DNA Helicases/genetics/metabolism
*MRE11 Homologue Protein/metabolism/genetics
*Cell Cycle Proteins/metabolism/genetics
DNA-Binding Proteins/metabolism/genetics
*Nuclear Proteins/metabolism/genetics
Cell Line, Tumor
*Microsatellite Instability
*DNA Repair Enzymes/metabolism/genetics
CRISPR-Cas Systems
Acid Anhydride Hydrolases

@article {pmid42489285,
year = {2026},
author = {Tang, X and Li, Z and Lu, Y and Ma, M and Mo, Z and Ke, J and Luan, X and Luan, T and Zhai, J},
title = {Systematic Position Mapping of Split CRISPR-Cas12a Activators Enables Highly Sensitive miRNA Detection and Cancer Cell Stratification.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {57},
pages = {e76576},
pmid = {42489285},
issn = {2198-3844},
support = {22127810//National Natural Science Foundation of China/ ; 22536007//National Natural Science Foundation of China/ ; 2025A1515012805//Guangdong Provincial Basic and Applied Basic Research Fund/ ; },
mesh = {*MicroRNAs/genetics/analysis ; Humans ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; *Neoplasms/genetics/diagnosis ; *Bacterial Proteins/genetics ; *Endodeoxyribonucleases/genetics/metabolism ; Cell Line, Tumor ; },
abstract = {Amplification-free Cas12a diagnostics with split crRNA enable rapid and programmable target recognition, yet insufficient understanding of DNA activator architecture prevents predictable control over trans-cleavage activity and sensitivity. Here we systematically map over 200 split DNA activator configurations by introducing nicks at every position across both strands. The mapping reveals that target strand nicks suppress activity with position-dependent severity, while non-target strand nicks enhance activity. Guided by these rules, we engineer an optimized split activator pair that achieves attomolar microRNA detection (LOD: 112 aM), ∼480-fold higher sensitivity than intact activators. The enhanced sensitivity supports multiplexed live-cell profiling of five miRNAs for machine learning-based cancer cell stratification, and is further generalized to non-nucleic acid targets, including APE1 enzyme (0.0073 U/L) and HClO (2.37 pM) through position-informed cleavable sites. This work provides a generalizable methodology for engineering CRISPR-Cas12a performance across diagnostic and biosensing applications.},
}

*MicroRNAs/genetics/analysis
Humans
*CRISPR-Cas Systems/genetics
*CRISPR-Associated Proteins/genetics/metabolism
*Neoplasms/genetics/diagnosis
*Bacterial Proteins/genetics
*Endodeoxyribonucleases/genetics/metabolism
Cell Line, Tumor

@article {pmid42526438,
year = {2026},
author = {Chen, ACY and Ji, KY and Yerinde, C and Knudsen, NH and Bi, K and Hao, S and Zhabotynsky, V and Martinez, D and Carmona-LaSalle, TJ and Taguchi, K and Xu, KH and Seider, EM and Schwartz, MA and Zschummel, M and Nieman, LT and Yates, KB and Gazzaniga, FS and Miller, BC and Mempel, TR and Manguso, RT and Hacohen, N and Sen, DR},
title = {CRISPR screens identify targets to rescue age-related T cell dysfunction in cancer.},
journal = {Cell},
volume = {189},
number = {21},
pages = {6738-6751.e7},
doi = {10.1016/j.cell.2026.07.016},
pmid = {42526438},
issn = {1097-4172},
support = {R01 AI163223/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Humans ; *CD8-Positive T-Lymphocytes/immunology/metabolism ; *Neoplasms/immunology/genetics/pathology ; Mice ; *Aging/immunology ; Dual-Specificity Phosphatases/genetics/metabolism ; Immunotherapy ; Mice, Inbred C57BL ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; T-Lymphocytes/immunology ; CRISPR-Cas Systems ; Cell Proliferation ; },
abstract = {Immune aging impairs T cell-mediated tumor control as well as cancer immunotherapy outcomes. The most important drivers of T cell dysfunction in aged tumors remain unknown. We performed single-cell CRISPR screens to identify Dusp5 and Zfp219 as key regulators of CD8[+] T cell persistence and effector differentiation within aged tumors. Loss of Dusp5 increased extracellular signal-regulated kinase (ERK) phosphorylation and globally enhanced T cell proliferation. Conversely, Zfp219 deletion induced epigenetic reprogramming and increased expression of cytotoxic molecules, enhancing antitumor immunity specifically in aging. Levels of the human ortholog ZNF219 were higher within intratumoral CD8[+] T cells from older cancer patients, which correlates with worse survival following immunotherapy. Zfp219 ablation synergized with immune checkpoint inhibitors to expand effector-like CD8[+] T cells, leading to tumor clearance in aged mice. Our findings highlight Dusp5 and Zfp219 as critical drivers of age-related T cell dysfunction that can be targeted to rejuvenate antitumor immunity in older cancer patients.},
}

Animals
Humans
*CD8-Positive T-Lymphocytes/immunology/metabolism
*Neoplasms/immunology/genetics/pathology
Mice
*Aging/immunology
Dual-Specificity Phosphatases/genetics/metabolism
Immunotherapy
Mice, Inbred C57BL
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
T-Lymphocytes/immunology
CRISPR-Cas Systems
Cell Proliferation

@article {pmid42543449,
year = {2026},
author = {Chen, Z and You, J},
title = {Cas12a/crRNA Recognition Catalyzed Triple Self-Priming Cascade Amplification Strategy for Sensitive and Reliable Drug Resistance Gene Analysis.},
journal = {Applied biochemistry and biotechnology},
volume = {198},
number = {11},
pages = {9429-9445},
pmid = {42543449},
issn = {1559-0291},
mesh = {*Methicillin-Resistant Staphylococcus aureus/genetics ; *Bacterial Proteins/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Penicillin-Binding Proteins/genetics ; *Biosensing Techniques ; *CRISPR-Cas Systems ; G-Quadruplexes ; },
abstract = {Rapid and accurate detection of methicillin-resistant Staphylococcus aureus (MRSA) is crucial for guiding antimicrobial therapy and improving outcomes in infection management, including postoperative care and surgical site infections. Conventional detection methods often suffer from lengthy turnaround times, operational complexity, or limited specificity. To address these challenges, we developed a novel, integrated biosensing platform termed the Cas12a/crRNA-initiated triple self-priming cascade amplification assay for ultrasensitive and specific analysis of the mecA resistance gene. The innovation of this strategy lies in the synergistic combination of CRISPR-Cas12a for high-fidelity target recognition and an elegantly engineered isothermal amplification circuit. Target-specific activation of Cas12a triggers the trans-cleavage of a designed probe, remaining an RNA initiator that sets off a triple self-priming cascade amplification within a single, multifunctional Y-shaped probe (Y-probe). This integrated probe architecture eliminates the need for multiple independent components, significantly simplifying the workflow and enhancing assay stability. Amplification culminates in the generation of G-quadruplex (G4) structures, which are detected through a label-free turn-on fluorescence signal using thioflavin T (ThT), thereby avoiding the instability associated with covalently labeled probes. The assay demonstrated exceptional analytical performance, achieving a detection limit of 0.41 fM for synthetic mecA DNA and 5.2 CFU/mL for viable MRSA cells, with a linear range spanning five to six orders of magnitude. It exhibited high specificity, successfully discriminating MRSA from methicillin-susceptible Staphylococcus aureus and distinguishing single-base mismatches. By integrating precise CRISPR recognition, a streamlined and stable all-in-one probe design, and a robust label-free readout, the platform presents a powerful, user-friendly molecular tool for resistance genotyping, with potential for future application in clinical settings, such as postoperative infection management.},
}

*Methicillin-Resistant Staphylococcus aureus/genetics
*Bacterial Proteins/genetics
*Endodeoxyribonucleases/metabolism/genetics
*CRISPR-Associated Proteins/metabolism/genetics
*Nucleic Acid Amplification Techniques/methods
*Penicillin-Binding Proteins/genetics
*Biosensing Techniques
*CRISPR-Cas Systems
G-Quadruplexes

@article {pmid42647959,
year = {2027},
author = {Hao, W and Han, L and Wu, Y and Fan, C and Zhang, X and Zhou, Z and Zhang, C},
title = {Design of base editing systems with tunable editing hotspots within a defined window through sgRNA engineering.},
journal = {Microbiological research},
volume = {314},
number = {},
pages = {128697},
doi = {10.1016/j.micres.2026.128697},
pmid = {42647959},
issn = {1618-0623},
mesh = {*Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Escherichia coli/genetics ; *CRISPR-Cas Systems/genetics ; Bacillus subtilis/genetics ; Cytidine Deaminase/genetics ; Genetic Engineering/methods ; Rifampin/pharmacology ; },
abstract = {Base editors (BEs) enable efficient A-to-G or C-to-T conversions without double-stranded DNA cleavage, but their editing windows remain difficult to tune, limiting genome engineering flexibility. Here, we engineered CRISPR/Cas12b sgRNA by introducing MS2 hairpins to recruit an MS2-N55K-cytidine deaminase-UGI complex, enabling programmable control of the editing window. Three modified sgRNAs were generated by replacing two loop regions, each producing distinct editing hotspots in E. coli. The AID*Δ-MSBE system (sgRNA1.1) generated a window near the PAM with peak activity at C7-C9, while the CDA-MSBE system (sgRNA1.2) produced a distal window with peak activity at C20-C23. Both systems exhibited identical editing patterns in Bacillus subtilis. A dual-orthogonal system (MS2 and PP7) was constructed to simultaneously recruit two deaminase complexes, restoring the classic dCas12b CBE editing pattern. Rifampicin resistance assays confirmed high targeting specificity with low off-target effects. As proof of concept, the MSBEs were successfully employed for the flexible reprogramming of sfGFP fluorescence and the targeted evolution of the endogenous gene rpsE, respectively. Collectively, we developed the MSBEs with tunable editing hotspots, providing innovative tools to enhance the flexibility and accessibility of BEs for genome engineering.},
}

*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics
Escherichia coli/genetics
*CRISPR-Cas Systems/genetics
Bacillus subtilis/genetics
Cytidine Deaminase/genetics
Genetic Engineering/methods
Rifampin/pharmacology

@article {pmid42758398,
year = {2026},
author = {Wang, H and Wang, X and Geng, Q and Li, M and Li, Z and Liu, J and Yang, Z and Lan, D and Chen, Z},
title = {Rapid Differential Detection of PRRSV NADC30-like and NADC34-like Strains Using a CRISPR-Cas13a/RT-RAA Assay.},
journal = {Applied biochemistry and biotechnology},
volume = {198},
number = {11},
pages = {9312-9328},
pmid = {42758398},
issn = {1559-0291},
mesh = {*Porcine respiratory and reproductive syndrome virus/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; Animals ; Swine ; *Porcine Reproductive and Respiratory Syndrome/diagnosis/virology ; *Nucleic Acid Amplification Techniques/methods ; Recombinases/metabolism ; },
abstract = {The global swine industry suffers substantial economic losses from Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), which causes reproductive and respiratory disorders. With NADC30- and NADC34-like strains now predominant, differentiation is complicated by their similar biology, despite characteristic Nsp2 gene deletions of 131 aa and 100 aa, respectively, highlighting a need for improved diagnostics. Moving fundamentally beyond conventional broad-spectrum detection, this assay innovatively targets the characteristic Nsp2 deletion signatures. A diagnostic platform integrating CRISPR-Cas13a with recombinase-aided amplification (RT-RAA) was constructed. Utilising aligned Nsp2 sequences, conserved crRNAs, and RT-RAA primers, NADC30- and NADC34-like strains were evaluated for their assay time, specificity, sensitivity, and consistency with RT-qPCR. The assay achieved rapid, specific detection with no cross-reactivity to common swine pathogens, and highly sensitive detection limits of 1.7 × 10[1] and 2.3 × 10[1] copies/µL for NADC30- and NADC34-like strains, respectively. Clinical validation using 30 samples demonstrated complete concordance with RT-qPCR, and the integration of T7 transcription enabled the single-tube differentiation of both strains. In summary, this one-tube, two-step fluorescent system presents a robust alternative for PRRSV diagnostics, combining specificity, sensitivity, and ease of use. It is well-suited for rapid field identification, molecular typing, and surveillance, thereby aiding in the implementation of targeted control measures.},
}

*Porcine respiratory and reproductive syndrome virus/genetics/isolation & purification
*CRISPR-Cas Systems/genetics
Animals
Swine
*Porcine Reproductive and Respiratory Syndrome/diagnosis/virology
*Nucleic Acid Amplification Techniques/methods
Recombinases/metabolism

@article {pmid42759161,
year = {2027},
author = {Carvalho, JP and Moreno, DS and Costa, CE and Domingues, L},
title = {CRISPR-Cas9 engineering for high-level β-galactosidase production in Saccharomyces boulardii.},
journal = {Microbiological research},
volume = {314},
number = {},
pages = {128734},
doi = {10.1016/j.micres.2026.128734},
pmid = {42759161},
issn = {1618-0623},
mesh = {*beta-Galactosidase/genetics/biosynthesis/metabolism ; *CRISPR-Cas Systems ; Lactose/metabolism ; *Saccharomyces boulardii/genetics/enzymology/metabolism/growth & development ; Probiotics ; Aspergillus niger/genetics/enzymology ; Galactose/metabolism ; Hydrogen-Ion Concentration ; *Gene Editing/methods ; Glucose/metabolism ; },
abstract = {Saccharomyces boulardii is a clinically validated probiotic yeast widely used to treat gastrointestinal disorders. Its natural probiotic effects, combined with the capacity of surviving the harsh gastrointestinal conditions, makes it an attractive chassis for therapeutic protein delivery. However, its engineering has relied on a limited set of genetic tools and lacks systematic validation, which has constrained its development as a next generation engineered probiotic. In this study, the compatibility of the EasyClone-MarkerFree vector system with CRISPR-Cas9 editing in S. boulardii was systematically assessed. Eight genomic loci were identified in silico as suitable for integration, and four were used to construct strains with the Aspergillus niger lacA gene integrated at one or multiple genomic loci, encoding an extracellular β-galactosidase capable of hydrolysing lactose into glucose and galactose, an activity of potential therapeutic relevance for lactose intolerance. Engineered strains efficiently hydrolysed lactose, with the strain with the lacA cassette integrated in three genomic loci (Sb-Triple) displaying up to an 11.76-fold increase in specific β-galactosidase activity relative to single-integration strains, without compromising growth. Importantly, Sb-Triple retained the native pH tolerance characteristic of S. boulardii and was able to grow on, and hydrolyse lactose from, cheese whey powder, a food-grade substrate. These results demonstrate a validated CRISPR-Cas9 strategy for high extracellular β-galactosidase secretion in the clinically established S. boulardii CNCM I-745, while evaluating the compatibility of EasyClone-MarkerFree vector set with this strain. Overall, this work provides a foundation for future studies where the potential of the developed strains is evaluated in clinically relevant settings.},
}

*beta-Galactosidase/genetics/biosynthesis/metabolism
*CRISPR-Cas Systems
Lactose/metabolism
*Saccharomyces boulardii/genetics/enzymology/metabolism/growth & development
Probiotics
Aspergillus niger/genetics/enzymology
Galactose/metabolism
Hydrogen-Ion Concentration
*Gene Editing/methods
Glucose/metabolism

@article {pmid42832928,
year = {2027},
author = {Li, Y and Zhao, X and Yang, X and Wang, J and Liu, D and Yu, Q and Liu, Q and Tian, C},
title = {Programmable histone acetylation enables reversible regulation of cellulase production in Myceliophthora thermophila.},
journal = {Microbiological research},
volume = {314},
number = {},
pages = {128747},
doi = {10.1016/j.micres.2026.128747},
pmid = {42832928},
issn = {1618-0623},
mesh = {Acetylation ; *Cellulase/biosynthesis/genetics/metabolism ; Gene Expression Regulation, Fungal ; *Histones/metabolism ; *Sordariales/genetics/metabolism/enzymology ; Promoter Regions, Genetic ; Fungal Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Histone Acetyltransferases/metabolism/genetics ; Epigenesis, Genetic ; },
abstract = {Myceliophthora thermophila is a thermophilic filamentous fungus with great potential for biomass biorefining and industrial protein production. However, the locus-specific epigenetic regulation of the cellulolytic network in this fungus remains poorly understood. Here, we established a CRISPR/dCas9-p300 system that recruits the p300 histone acetyltransferase to target loci and targeted the promoters of the key cellulolytic regulators clr-1, clr-2, and xyr-1. Targeted recruitment of dCas9-p300 increased the transcription of these regulators by up to 5.2-fold and promoter-associated histone acetylation by approximately 2- to 3-fold. Simultaneous targeting of all three regulators resulted in greater increases than single-locus targeting, with extracellular protein production and CMCase activity increasing by up to 5.5- and 3.7-fold, respectively. The dependence of these effects on guide RNA-directed targeting and p300 catalytic activity was confirmed using no-guide controls and the p300 inhibitor A-485. A catalytically inactive p300 mutant targeting clr-2 failed to enhance clr-2 transcription, extracellular protein production, or CMCase activity. We further developed an inducible dCas9-p300 system to examine the persistence and reversibility of the activated state. Following inducer withdrawal, the high cellulase-production phenotype was maintained during the initial propagation but gradually declined over successive passages and was restored upon re-induction. These results demonstrate that targeted histone acetylation can coordinately activate an endogenous cellulolytic regulatory network and establish a reversible high-production state without altering the DNA sequences of the target loci. This work provides a chromatin-based strategy for reversible regulation of endogenous gene networks and protein production in filamentous fungi.},
}

Acetylation
*Cellulase/biosynthesis/genetics/metabolism
Gene Expression Regulation, Fungal
*Histones/metabolism
*Sordariales/genetics/metabolism/enzymology
Promoter Regions, Genetic
Fungal Proteins/genetics/metabolism
CRISPR-Cas Systems
Histone Acetyltransferases/metabolism/genetics
Epigenesis, Genetic

@article {pmid42854678,
year = {2026},
author = {Cancian, M and Margaglione, M},
title = {Genome editing for hereditary angioedema: A landmark trial and important unanswered questions.},
journal = {Med (New York, N.Y.)},
volume = {7},
number = {10},
pages = {101324},
doi = {10.1016/j.medj.2026.101324},
pmid = {42854678},
issn = {2666-6340},
mesh = {Humans ; *Angioedemas, Hereditary/therapy/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems ; *Genetic Therapy/methods ; Bradykinin ; Clinical Trials, Phase III as Topic ; },
abstract = {Hereditary angioedema is a bradykinin-mediated genetic disorder increasingly controlled with effective long-term prophylaxis. In a phase 3 trial, a single dose of lonvoguran ziclumeran, an in vivo CRISPR-Cas9 therapy targeting KLKB1, significantly reduced attack rates. This approach represents a highly promising therapeutic strategy for HAE. However, it also raises important questions that require further investigation.},
}

Humans
*Angioedemas, Hereditary/therapy/genetics
*Gene Editing/methods
CRISPR-Cas Systems
*Genetic Therapy/methods
Bradykinin
Clinical Trials, Phase III as Topic

@article {pmid42854934,
year = {2026},
author = {Pourzangiabadi, M and Barkam, M and Najafi, H and Pouladi, I},
title = {An overview of current technologies in the development of drugs and vaccines against dengue virus.},
journal = {Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases},
volume = {},
number = {},
pages = {106041},
doi = {10.1016/j.meegid.2026.106041},
pmid = {42854934},
issn = {1567-7257},
abstract = {Dengue virus (DENV) is a mosquito-borne flavivirus with four distinct serotypes (DENV-1 to DENV-4) and is endemic in tropical and subtropical regions worldwide. It can cause a range of illnesses, from self-resolving dengue fever to severe dengue hemorrhagic fever or dengue shock syndrome. Dengue fever is a major public health hazard in tropical and subtropical areas. Advances in treatment and prevention have mainly focused on developing vaccines and antiviral medications. Currently, no treatment for dengue fever works for everyone. While some antiviral candidates are in clinical trials, most remain in preclinical or early research stages. Regarding vaccinations, the WHO has approved or prequalified two live-attenuated options: TAK-003 (Qdenga®) and Dengvaxia® (CYD-TDV). However, both vaccines have drawbacks, including limited effectiveness across serotypes and safety issues in seronegative individuals. Current research aims to elucidate the complex host immune responses that underlie dengue pathogenesis and vaccine-induced protection, to develop more effective treatments and preventive measures. Critically, most approaches remain at the experimental or preclinical stage and require further clinical evaluation.},
}

@article {pmid42857429,
year = {2026},
author = {Zhang, D and Cui, X and Zhang, R and Cai, W and Wang, L and Yang, J},
title = {Molecular point-of-care: technological evolution, clinical applications, and future perspectives.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {14},
number = {},
pages = {1908277},
pmid = {42857429},
issn = {2296-4185},
abstract = {The landscape of clinical diagnostics is undergoing a fundamental shift from centralized laboratories toward decentralized point-of-care testing (POCT) models. Conventional molecular diagnostics often suffer from prolonged turnaround times, high operational costs, and a strict reliance on sophisticated infrastructure. To overcome these limitations, modern POCT platforms have deeply integrated advanced molecular techniques. These include diverse isothermal nucleic acid amplification methods (such as LAMP, RPA, and RCA) and highly specific CRISPR/Cas recognition systems. Furthermore, coupling these biochemical reactions with microfluidic chips and smartphone-based digital platforms has enabled fully automated, "sample-in, answer-out" diagnostic capabilities. This review systematically summarizes the technological evolution of these molecular POCT platforms. It highlights their transformative clinical applications across diverse fields, including infectious disease containment, early oncology screening, cardiovascular emergency triage, and metabolic disease management. Despite remarkable progress, several critical bottlenecks still prevent the widespread clinical translation of molecular POCT. These challenges include cross-interference in multiplexed detection, enzyme stability outside strict cold chains, high costs of core components, and the lack of tailored regulatory frameworks. Addressing these barriers via synthetic biology, robust system integration, and artificial intelligence is critical to advancing the core vision of delivering rapid, accessible, and precise healthcare solutions in resource-limited settings.},
}

@article {pmid42858833,
year = {2026},
author = {Abbas, B and Abbas, A and Khan, IN and Nazeer, S},
title = {Advances in genetically engineered and humanized mouse models for dissecting sepsis mechanisms and therapeutic response.},
journal = {Clinical immunology (Orlando, Fla.)},
volume = {},
number = {},
pages = {110782},
doi = {10.1016/j.clim.2026.110782},
pmid = {42858833},
issn = {1521-7035},
abstract = {Sepsis remains a major cause of global mortality, yet translation from preclinical studies to effective targeted therapies has been limited. A key barrier is that conventional animal models incompletely capture human immune circuitry and patient heterogeneity. Recent progress in mouse genetics, CRISPR/Cas-based genome editing, conditional alleles (Cre-loxP), and next-generation humanized platforms has expanded the toolkit for functional interrogation of host pathways that shape sepsis outcomes. Germline and cell-type-restricted perturbations have helped map roles for pattern-recognition signaling, inflammasome modules, cell-death effectors, and immune checkpoints, while also showing that the same pathway can produce different phenotypes depending on cellular context. Cross-species differences in innate immune gene networks and caspase biology motivate validation in humanized systems and integration with human genetic evidence. Emerging "wildling" and genetically diverse panels provide complementary approaches to model environmental exposure and host variation. Coupling these platforms with single-cell multi-omics and endotype stratification can link genotype-to-phenotype mechanisms with biomarker-informed therapeutic testing.},
}

@article {pmid42859304,
year = {2026},
author = {Usmani, F and Ali, SU and Ahad, F and Saifullah, },
title = {Recent trends and innovative strategies in the curative approach for β-thalassemia: a current prospect and future perspective.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42859304},
issn = {1573-4978},
mesh = {Humans ; *beta-Thalassemia/therapy/genetics ; *Genetic Therapy/methods/trends ; *Hematopoietic Stem Cell Transplantation/methods/trends ; Gene Editing/methods ; Blood Transfusion ; CRISPR-Cas Systems ; Gene Therapy Agents ; beta-Globins/genetics ; Lentivirus/genetics ; },
abstract = {β-Thalassemia, a major genetic disease affecting the global population, has seen new strategies being pursued in terms of curative attempts directed at the specific gene defect present. Iron chelation therapy and lifelong blood transfusions have historically been used to treat β-thalassemia, a severe hereditary hemoglobin condition marked by decreased or missing β-globin chain synthesis. These methods have significantly increased patient survival, but they do not offer a permanent solution and are linked to serious side effects and a high cost of care. With a focus on hematopoietic stem cell transplantation (HSCT), lentiviral gene-addition treatment, and future genome-editing techniques, this study outlines new and creative methods to long-term disease correction in β-thalassemia. Although allogeneic HSCT is still a proven treatment option, its use is constrained by the availability of donors, graft-related issues, and transplant-related hazards. Autologous hematopoietic stem cell-based gene therapy has become a significant substitute, provided the possibility of long-term transfusion independence while prevented graft-versus-host disease. With the creation of exagamglogene autotemcel (exa-cel; Casgevy), which reactivates fetal hemoglobin expression through modification of the BCL11A erythroid enhancer and has shown significant rates of sustained transfusion independence in eligible patients with transfusion-dependent β-thalassemia, CRISPR/Cas9-based genome editing has recently marked a significant clinical milestone. Notwithstanding these encouraging developments, myeloablative conditioning, long-term safety and durability, high treatment costs, complicated manufacturing requirements, and restricted accessibility continue to be major obstacles, especially in low- and middle-income nations. All things considered, the therapeutic landscape for β-thalassemia is changing from lifelong supportive management to potentially curative and transformative interventions; however, the wider application of these cutting-edge therapies depends on ongoing long-term evaluation and efforts to increase affordability and global accessibility.},
}

Humans
*beta-Thalassemia/therapy/genetics
*Genetic Therapy/methods/trends
*Hematopoietic Stem Cell Transplantation/methods/trends
Gene Editing/methods
Blood Transfusion
CRISPR-Cas Systems
Gene Therapy Agents
beta-Globins/genetics
Lentivirus/genetics

@article {pmid42861067,
year = {2026},
author = {Thangadurai, K and Bedekar, MK and Manojkumar, C and Namdeo, MS and Limbola, M and Jeena, K and Rasal, KD and Rajendran, KV and Kumar, K},
title = {Comparative Evaluation and Optimization of a CRISPR/Cas-Based Assay for Rapid Detection of Tilapinevirus tilapiae (TiLV) in Indian Isolates, Validated Against Conventional Nucleic acid Detection Methods.},
journal = {Indian journal of microbiology},
volume = {66},
number = {5},
pages = {1204-1216},
pmid = {42861067},
issn = {0046-8991},
abstract = {UNLABELLED: Tilapia lake virus (Tilapinevirus tilapiae; TiLV) remains a significant threat to global tilapia aquaculture, underscoring the urgent need for rapid, sensitive, and field-ready diagnostic approaches. In this study, we developed a CRISPR-Cas12a assisted reverse transcriptase-recombinase polymerase amplification assay (Cr-TiLV) targeting a conserved region of segment 6 of the TiLV genome. The integration of RT-RPA with Cas12a significantly enhanced analytical sensitivity, enabling the detection of viral RNA at levels as low as 10 copies. Diagnostic assessment using 40 clinical samples demonstrated that the Cr-TiLV assay achieved sensitivities of 86.67% and 82.29%, and accuracies of 90% and 85%, respectively, relative to semi-nested PCR and RT-qPCR, with specificity approaching 99.9% for both benchmark methods. Although less sensitive than RT-qPCR for samples with low viral loads, the assay consistently detected low to moderate viral concentrations and showed substantial concordance with semi-nested PCR. Operating isothermally at 39 °C and requiring minimal laboratory resources, the Cr-TiLV assay offers a rapid and field-deployable diagnostic solution with strong potential for routine TiLV surveillance in resource-limited aquaculture settings.

SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at https://doi.org/10.1007/s12088-026-01550-y.},
}

@article {pmid42861495,
year = {2026},
author = {Üstüner, E and Öz, M},
title = {From Sequence to Structure: AlphaFold and the AI Revolution in Aquaculture Drug Discovery and Vaccine Design.},
journal = {Biotechnology and bioengineering},
volume = {},
number = {},
pages = {},
doi = {10.1002/bit.70398},
pmid = {42861495},
issn = {1097-0290},
abstract = {Global aquaculture faces an escalating disease and antimicrobial resistance (AMR) crisis, exacerbated by climate-driven physiochemical shifts and intensive farming practices. The integration of artificial intelligence (AI) and structural biology, spearheaded by AlphaFold, represents a paradigm shift in aquatic pharmacology, transitioning the field from empirical trial-and-error to rational in silico design. This review critically evaluates the application of AI-driven tools in aquaculture drug discovery and vaccine development. We examine the evolution of AlphaFold in predicting complex host-pathogen interactions and facilitating de novo drug design. Concurrently, we highlight critical limitations stemming from the reliance on multiple sequence alignment (MSA) depth rather than mere PDB taxonomic composition, the unreliability of direct molecular docking due to apo conformations, and the dynamic nature of psychrophilic adaptations in aquatic environments. Furthermore, we assess next-generation structural vaccinology, including reverse vaccinology and pre-fusion antigen stabilization, alongside the integration of nanomaterial-based delivery systems to overcome aquatic pharmacokinetic barriers. The synergy of AI with CRISPR/Cas technologies and multimodal large language models (MM-LLMs) for disease management is also explored. Despite unprecedented computational capabilities, significant bottlenecks remain, including a severe lack of species-specific omics data, the indispensability of in vivo wet-lab validation, and lagging regulatory frameworks. Ultimately, realizing the potential of precision aquatic medicine requires developing aquaculture-specific databases, addressing the ecological risks of open-water deployment, and democratizing these technologies for small-scale farmers to ensure sustainable, equitable, and resilient global aquaculture.},
}

@article {pmid42861852,
year = {2026},
author = {Chovanec, P and He, S and Yin, Y},
title = {Genome-wide mapping of Cas9-induced sister chromatid exchange across single and over 200 genomic sites in human cells.},
journal = {eLife},
volume = {15},
number = {},
pages = {},
pmid = {42861852},
issn = {2050-084X},
support = {R35 GM142511/GM/NIGMS NIH HHS/United States ; PC250408//DOD Prostate Cancer Research Program/ ; 1002444//W. M. Keck Foundation/ ; R35GM142511/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *Sister Chromatid Exchange ; *CRISPR-Cas Systems ; *Chromosome Mapping ; DNA Breaks, Double-Stranded ; *Genome, Human ; },
abstract = {CRISPR/Cas9-induced DNA double-strand breaks (DSBs) are widely used for genome engineering, yet their capacity to provoke sister chromatid exchange (SCE) remains incompletely understood because exchanges between identical sisters leave no sequence change and are invisible to conventional sequencing. Using sci-L3-Strand-seq, a scalable single-cell template-strand sequencing platform, we mapped SCE following targeted Cas9 cleavage. Cas9 targeting of a unique genomic locus strongly enriched SCE at the break site, reaching 41% in the same cell cycle and 17% in the subsequent division, indicating frequent non-local inter-sister repair. Targeting 237 repetitive Alu sites produced mild enrichment of on-target SCE, whereas cells with elevated SCE burden showed significant enrichment at programmed cut sites, suggesting context-dependent recombination at repetitive loci. Reciprocal daughter-cell pair analysis revealed large-scale structural alterations on chromosomes with induced SCEs and at SCE junctions, indicating that Cas9-induced SCE does not universally reflect error-free homologous recombination. Disruption of LIG3, LIG4, XRCC1, and XRCC4 at Cas9 cut sites did not measurably alter SCE frequency, consistent with delayed functional loss following editing. Together, these findings identify Cas9-induced DSBs as potent local triggers of SCE and reveal structural outcomes invisible to conventional sequencing.},
}

Humans
*Sister Chromatid Exchange
*CRISPR-Cas Systems
*Chromosome Mapping
DNA Breaks, Double-Stranded
*Genome, Human

@article {pmid42862095,
year = {2026},
author = {Wu, W and Xia, S and Li, D and Lin, J and Cao, C and Kong, J and Lu, M and Xiao, Y and Li, Z},
title = {Structural basis for ITP-selective hydrolysis by a CRISPR-associated Nudix hydrolase.},
journal = {Journal of bacteriology},
volume = {},
number = {},
pages = {e0028026},
doi = {10.1128/jb.00280-26},
pmid = {42862095},
issn = {1098-5530},
abstract = {The nucleoside diphosphates linked to moiety-X (Nudix) hydrolases are a superfamily of proteins defined by the characteristic Nudix box sequence, most of which are involved in clearing the cell of potentially deleterious metabolites and in preventing the accumulation of metabolic intermediates. The Nudix hydrolase associated with CRISPR-Cas-associated adenosine deaminases (CAAD) from Limisphaera ngatamarikiensis (LngNH) mitigates CAAD-induced toxicity by hydrolyzing ITP to IMP and pyrophosphate. Here, we determined crystal structures of LngNH in the apo form and in complex with ITP using catalytic mutants and validated key interactions by site-directed mutagenesis. The hypoxanthine of ITP is deeply buried in a hydrophobic pocket and engages a distinct hydrogen-bond network, whereas the triphosphate tail is coordinated by conserved residues and divalent metal ions. Together, these findings provide a structural framework for CAAD-linked nucleotide sanitization and suggest that CRISPR-associated Nudix hydrolases function as local self-protection modules that limit toxicity from noncanonical inosine nucleotides.IMPORTANCENucleotide quality-control enzymes protect cells from harmful metabolic by-products. Here, we characterize a CRISPR-associated nucleoside diphosphates linked to moiety-X (Nudix) hydrolase that removes ITP, a toxic noncanonical nucleotide generated during antiviral defense. Biochemical and structural analyses show how this enzyme specifically recognizes and hydrolyzes ITP while avoiding related cellular nucleotides. Our findings suggest that CRISPR-associated nucleotide deaminase systems require "self-protection" mechanisms to prevent damage from their own antiviral activities. These results expand our understanding of how bacteria maintain nucleotide homeostasis during immune responses and reveal an additional regulatory layer within CRISPR-associated defense systems. The structural framework reported here also provides a foundation for future studies of nucleotide sanitization enzymes in microbial physiology and antiviral immunity.},
}

@article {pmid41412877,
year = {2026},
author = {Mandrioli, M},
title = {Genomic surveillance: protecting sport in the post-CRISPR era.},
journal = {Trends in biotechnology},
volume = {44},
number = {7},
pages = {1811-1813},
doi = {10.1016/j.tibtech.2025.11.021},
pmid = {41412877},
issn = {1879-3096},
mesh = {*Gene Editing/ethics/methods ; *Doping in Sports/ethics/methods/prevention & control ; *Genomics/ethics/methods ; *Athletic Performance ; Athletes ; Genetic Privacy/ethics/standards ; Whole Genome Sequencing/ethics/standards ; Humans ; Sports/ethics/standards ; CRISPR-Cas Systems ; Trust ; Genome, Human ; },
abstract = {Personal genomics and gene editing raise the prospect of undetectable gene doping. Athletes' genome sequencing should be integrated into the Athlete Biological Passport to deter future abuse. This provocative vision calls for urgent debate on fairness in sport and for new solutions to share genomic data while preserving privacy.},
}

*Gene Editing/ethics/methods
*Doping in Sports/ethics/methods/prevention & control
*Genomics/ethics/methods
*Athletic Performance
Athletes
Genetic Privacy/ethics/standards
Whole Genome Sequencing/ethics/standards
Humans
Sports/ethics/standards
CRISPR-Cas Systems
Trust
Genome, Human

@article {pmid42099090,
year = {2026},
author = {Luo, F and Liao, W and Pei, X and Xing, S and Sun, L and Huang, L and Lu, Y},
title = {Evaluation of EndoSens: A reliable skin sensitization assessment model.},
journal = {ALTEX},
volume = {43},
number = {4},
pages = {602-613},
doi = {10.14573/altex.2602051},
pmid = {42099090},
issn = {1868-8551},
mesh = {Humans ; *Animal Testing Alternatives/methods ; CRISPR-Cas Systems ; Keratinocytes/drug effects ; Heme Oxygenase-1/genetics ; Allergens/toxicity ; Animals ; Skin/drug effects ; },
abstract = {The transition toward non-animal safety assessment has driven the development of in vitro models for skin sensitization, particularly those targeting key event 2 (KE2) within the adverse outcome pathway (AOP). While conventional KE2 assays often rely on randomly integrated reporter systems, the EndoSens model utilizes CRISPR/Cas9-mediated knock-in to precisely insert a luciferase reporter into the endogenous HMOX1 locus in HaCaT keratinocytes, providing a genomically anchored and physiologically relevant system. To enable reproducible adoption across laboratories, we established a standardized protocol with defined quality control criteria, incorporating cinnamyl alcohol as a quantitative positive control and a cytotoxicity pre-screen to identify non-cytotoxic test chemical concentrations. Evaluation using ten OECD TG 442D reference chemicals and ten addi­tional compounds demonstrated the assay’s robust performance in discriminating skin sensitizers with high inter-laboratory consistency, supporting its integration as a reliable in vitro component within defined approaches for animal-free skin sensitization assessment.},
}

Humans
*Animal Testing Alternatives/methods
CRISPR-Cas Systems
Keratinocytes/drug effects
Heme Oxygenase-1/genetics
Allergens/toxicity
Animals
Skin/drug effects

@article {pmid42128824,
year = {2026},
author = {Matsumoto, K and Kuno, A and Mizuno, S},
title = {AltEx-BE: a tool for designing splice-site-targeting gRNAs for base editing-mediated exon skipping with exon-level annotations in diverse experimental animals.},
journal = {Experimental animals},
volume = {75},
number = {4},
pages = {429-438},
doi = {10.1538/expanim.26-0023},
pmid = {42128824},
issn = {1881-7122},
mesh = {*Exons/genetics ; Animals ; *RNA Splice Sites/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *RNA Splicing/genetics ; },
abstract = {Pre-mRNA splicing is a conserved post-transcriptional regulatory process in eukaryotes, and mis-splicing is associated with numerous diseases. Therefore, experimentally induced exon skipping provides an important strategy for exon-level analysis of gene function. Exons can be broadly classified by their usage patterns across transcripts: some are shared by all transcripts, whereas others are selectively used across transcripts. Skipping the former affects all transcripts and often results in gene-level effects, while skipping the latter reflects functional diversity across transcripts. Therefore, considering characteristics of target exons is important to enhance interpretability of exon-level functional analysis. Recent advances in base editing enable exon skipping by introducing point mutations at splice sites, providing an approach for exon-level analysis. Accordingly, several guide RNA (gRNA) design tools have been developed for the purpose of inducing exon skipping. However, existing tools do not provide exon-level properties, including exon usage patterns, reading frame, and coding status. As a result, selecting target exons appropriate for specific research objectives remains difficult. Here we developed AltEx-BE, an automated gRNA design tool for base editing-mediated splice site disruption. AltEx-BE uses transcript annotations and reference genome sequences as inputs to classify each exon by usage pattern and to generate gRNA candidates compatible with several base editing tools. The tool also provides annotations, including coding status, to facilitate interpretation of outcomes of exon skipping. The tool promotes base editing-mediated exon skipping experiments across model organisms and enhances the interpretability of experimental outcomes.},
}

*Exons/genetics
Animals
*RNA Splice Sites/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics
*RNA Splicing/genetics

@article {pmid42294842,
year = {2026},
author = {Cohn, DM and Gurugama, P and Longhurst, HJ and Aygören-Pürsün, E and Craig, TJ and Farkas, H and Jacobs, J and Lumry, WR and Magerl, M and Peter, J and Riedl, MA and Maag, D and Golden, A and Shah, MY and Sutherland, A and Miller, CR and Abdelhady, AM and Xu, Y and Butler, JS and Lebwohl, D and Leonard, J and Banerji, A and , },
title = {Lonvoguran Ziclumeran - In Vivo CRISPR Gene Editing in Hereditary Angioedema.},
journal = {The New England journal of medicine},
volume = {395},
number = {15},
pages = {1471-1482},
doi = {10.1056/NEJMoa2600931},
pmid = {42294842},
issn = {1533-4406},
mesh = {Humans ; Female ; Double-Blind Method ; Adult ; Male ; *Angioedemas, Hereditary/therapy/genetics ; Middle Aged ; Young Adult ; Infusions, Intravenous ; Adolescent ; *CRISPR-Cas Systems ; *Genetic Therapy/adverse effects ; Aged ; *Pentanoic Acids/adverse effects/therapeutic use ; Gene Therapy Agents ; },
abstract = {BACKGROUND: Hereditary angioedema is a rare and potentially life-threatening genetic condition characterized by recurrent and debilitating swelling attacks. Lonvoguran ziclumeran (lonvo-z) - an investigational, in vivo gene-editing treatment based on clustered regularly interspaced short palindromic repeats (CRISPR) - is being developed as a single-dose treatment for hereditary angioedema.

METHODS: In a phase 3, double-blind trial, we randomly assigned, in a 2:1 ratio, patients who were 16 years of age or older and had hereditary angioedema with C1 inhibitor deficiency to receive a single intravenous infusion of lonvo-z (at a dose of 50 mg) or placebo. The primary end point was the number of investigator-confirmed hereditary angioedema attacks per month (the monthly attack rate) from week 5 through week 28 after infusion.

RESULTS: A total of 80 patients underwent randomization; 52 were assigned to receive lonvo-z and 28 to receive placebo. As of February 10, 2026, the median duration of follow-up was 7.5 months (range, 4.9 to 12.8). The least-squares mean monthly attack rate from weeks 5 through 28 was 0.26 (95% confidence interval [CI], 0.15 to 0.45) in the lonvo-z group and 2.10 (95% CI, 1.55 to 2.86) in the placebo group (relative difference, -87%; 95% CI, -93 to -78; P<0.001). Adverse events that occurred during or after infusion of lonvo-z or placebo were reported in 92% and 86% of the patients in the lonvo-z and placebo groups, respectively, from day 1 through week 28. Among the adverse events that occurred during or after infusion and more often in the lonvo-z group than in the placebo group, the most common (occurring in >10% of the patients in the lonvo-z group) included infusion-related reaction, headache, fatigue, back pain, and upper respiratory tract infection. No serious or grade 3 or higher adverse events were reported in the lonvo-z group.

CONCLUSIONS: Among patients with hereditary angioedema, a single intravenous infusion of lonvo-z resulted in a significantly lower rate of hereditary angioedema attacks than placebo. (Funded by Intellia Therapeutics; HAELO ClinicalTrials.gov number, NCT06634420.).},
}

Humans
Female
Double-Blind Method
Adult
Male
*Angioedemas, Hereditary/therapy/genetics
Middle Aged
Young Adult
Infusions, Intravenous
Adolescent
*CRISPR-Cas Systems
*Genetic Therapy/adverse effects
Aged
*Pentanoic Acids/adverse effects/therapeutic use
Gene Therapy Agents

@article {pmid42464218,
year = {2026},
author = {Yu, Y and He, W and Fang, Y and Wang, J and Yu, Z and Guo, Y and Zhao, Y and Gao, H and Zhou, P},
title = {Development of an RT-RAA-CRISPR/Cas12a fluorescent detection method for the detection of bovine viral diarrhea virus 1.},
journal = {BMC veterinary research},
volume = {22},
number = {1},
pages = {},
pmid = {42464218},
issn = {1746-6148},
support = {No. NKYG-25-22//Agricultural Science and Technology Innovation Program in Ningxia Hui Autonomous Region/ ; No. 2024BBF2014//Key R&D Program of Ningxia Hui Autonomous Region/ ; NKYGZL-2026-03//R&D and Application of Prevention and Control Products for Major Cattle and Sheep Diseases and Early Warning Technology for Epidemics/ ; },
mesh = {*Diarrhea Virus 1, Bovine Viral/isolation & purification/genetics ; Animals ; Cattle ; Sensitivity and Specificity ; *Bovine Virus Diarrhea-Mucosal Disease/diagnosis/virology ; CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/veterinary/methods ; 5' Untranslated Regions ; },
abstract = {BACKGROUND: Bovine viral diarrhea virus (BVDV) is a viral pathogen that may cause reproductive disorders, health, and performance. BVDV serotypes have undergone dynamic changes due to sustained genetic diversity, particularly in BVDV type 1 (BVDV1), which has compromised the specificity and sensitivity of diagnostic assays. Furthermore, BVDV1 is prevalent worldwide, causing significant losses to the livestock industry. Therefore, in order to further enhance the control and elimination of BVDV, it is necessary to detect and prevent the spread of BVDV. Therefore, establishing a faster and more accurate on-site detection method is particularly important.

METHODS: Traditional PCR and ELISA methods are limited by cost, sensitivity, and equipment dependence. In this study, we designed primers based on the conserved 5'Untranslated Region (5'UTR) of BVDV1 and developed a method that combines one-step reverse transcription recombinase assisted amplification (RT-RAA) and CRISPR/Cas12a based fluorescent substrates in a mixture to quickly screen and accurately distinguish BVDV1 for the diagnosis and differentiation of common bovine viruses such as Bovine Coronavirus (BCoV) and Bovine Herpesvirus type 1 (BoHV) et al. The established RT-RAA-CRISPR/Cas12a scheme can achieve the entire inspection process in a portable metal bath at 40℃. We further optimized the reaction conditions of RT-RAA-CRISPR/Cas12a to detect its sensitivity, specificity, and repeatability. The established RT-RAA-CRISPR/Cas12a and conventional PCR were used to detect 300 clinical samples of cattle, verifying the actual detection ability.

RESULTS: This method did not show any cross-reactivity with the other nine common bovine viruses, demonstrating excellent specificity. The minimum template concentration required to trigger significant trans cleavage activity without RT-RAA amplification is 10 copies/µL of BVDV template. However, combined with RT‑RAA amplification, the detection limit was significantly decreased to one copy of the BVDV template per reaction. In simulated sample testing, this method achieved 100% accuracy, and the entire detection process can be completed within 30 min. In clinical sample testing, RT-RAA-CRISPR/Cas12a showed a higher sensitivity rate for BVDV1, as the positive detection rate was 1.85 times higher than that of conventional PCR.

CONCLUSION: In summary, the BVDV-1 RT-RAA-CRISPR/Cas12a detection method established in this study offers several advantages, including fast operation, high sensitivity, strong targeting, and good repeatability. Moreover, the same test tube reaction system makes it widely applicable in resource-limited environments, making it of great value in large-scale screening during BVDV outbreaks. This method not only expands the diagnostic toolbox of BVDV but also provides an up-and-coming solution for controlling the spread of BVDV in cattle herds.},
}

*Diarrhea Virus 1, Bovine Viral/isolation & purification/genetics
Animals
Cattle
Sensitivity and Specificity
*Bovine Virus Diarrhea-Mucosal Disease/diagnosis/virology
CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/veterinary/methods
5' Untranslated Regions

@article {pmid42478279,
year = {2026},
author = {Röscheise, J and Eberle, LM and Laux, H and Otte, K},
title = {Engineering Stable Lentiviral Vector Producer Cells Improves LVV Production.},
journal = {Biotechnology and bioengineering},
volume = {123},
number = {11},
pages = {3152-3164},
doi = {10.1002/bit.70318},
pmid = {42478279},
issn = {1097-0290},
mesh = {*Lentivirus/genetics/growth & development ; *Genetic Vectors/genetics ; Humans ; CRISPR-Cas Systems ; Cell Line ; *Cell Engineering/methods ; },
abstract = {Stable lentiviral vector producer cell lines represent a promising platform for scalable and cost-efficient vector manufacturing, yet their productivity is often limited by intrinsic host-cell constraints. In this study, we aimed to identify cellular factors restricting LVV production in stable doxycycline-inducible GPRTG producer cell lines and to evaluate whether targeted host-cell engineering can improve vector yield. Comparative transcriptomic analysis of high- and low-producing clones revealed distinct differences in cellular pathways related to transcription, translation, energy metabolism, lipid homeostasis, stress response, and chromatin regulation. Based on differential gene expression, candidate genes were modified by CRISPR/Cas9-mediated knockout or overexpression and functionally analyzed in a low-producing clone. Single-gene KO screening identified H1-2, ADAMTS1, INSIG1, GADD45B, and HSPA1B to increase cell-specific LVV productivity up to 2.6-fold, with H1-2 showing the strongest effect. In addition, combinatorial disruption further improved productivity. In contrast, overexpression of selected candidates did not enhance LVV production. Overall, our results demonstrate that transcriptomics-guided host-cell engineering is an effective strategy to identify and relieve intrinsic bottlenecks in stable LVV producer cell lines.},
}

*Lentivirus/genetics/growth & development
*Genetic Vectors/genetics
Humans
CRISPR-Cas Systems
Cell Line
*Cell Engineering/methods

@article {pmid42556590,
year = {2026},
author = {Ye, WN and Chen, RY and Xing, Q and Zhang, YC and Wu, SF and Gao, CF},
title = {A natural Nlscarlet insertion underlies the orange-eye phenotype and facilitates efficient multiple-sgRNA genome editing in Nilaparvata lugens.},
journal = {Insect biochemistry and molecular biology},
volume = {195},
number = {},
pages = {104649},
doi = {10.1016/j.ibmb.2026.104649},
pmid = {42556590},
issn = {1879-0240},
mesh = {Animals ; *Hemiptera/genetics ; *Gene Editing ; Phenotype ; CRISPR-Cas Systems ; *Insect Proteins/genetics/metabolism ; *Eye Color/genetics ; Female ; },
abstract = {Eye-color mutants provide easily distinguishable visual markers for genetic analysis and genome editing, yet the molecular basis of naturally occurring eye-color variation in Nilaparvata lugens remains unclear. Here, we combined classical genetic analysis, candidate-gene screening, and CRISPR/Cas9 genome editing to elucidate the genetic basis of a stably inherited orange-eye phenotype and evaluate the editing performance of different sgRNA strategies. The orange-eye phenotype was inherited as a single autosomal recessive trait, and the mutant strain showed only limited fitness differences from the WT strain. A 1287-bp insertion spanning exon 13 and the adjacent intronic region of Nlscarlet introduced a premature stop codon, truncating NlScarlet after the fifth transmembrane helix. The insertion completely cosegregated with the orange-eye phenotype, and CRISPR/Cas9-mediated disruption of Nlscarlet recapitulated the phenotype, demonstrating that this insertion underlies the natural mutation. A separate in-frame deletion of lysine 535 produced a dark-orange-eye phenotype, and structural modeling suggested that this residue may contribute to the interaction between the NlWhite/NlScarlet transporter complex and 3-hydroxykynurenine. Using Nlscarlet as a visually scorable marker, two multiple-sgRNA strategies, each using four sgRNAs, increased the proportions of G0 individuals with complete loss-of-function phenotypes from 30.97% to 84.73% and 92.47%, respectively, and of mutant-eyed G1 progeny from 37.33% to 81.33% and 90.67%, respectively. Heritable mutations generated by both strategies were predominantly large deletions. These findings identify the genetic basis of a natural orange-eye mutation in N. lugens and establish multiple-sgRNA editing as an efficient strategy for rapid functional screening in insects.},
}

Animals
*Hemiptera/genetics
*Gene Editing
Phenotype
CRISPR-Cas Systems
*Insect Proteins/genetics/metabolism
*Eye Color/genetics
Female

@article {pmid42664970,
year = {2026},
author = {Srikanth, S and Zheng, F and Drepanos, LM and Shah, ST and Kaplan, EG and Gibson, D and Lynch, GO and Uebele, AT and Reint, G and Merzouk, S and Doench, JG},
title = {Optimized parameters for CRISPR-Cas9 interference library design.},
journal = {Cell genomics},
volume = {6},
number = {10},
pages = {101338},
doi = {10.1016/j.xgen.2026.101338},
pmid = {42664970},
issn = {2666-979X},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Library ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; },
abstract = {CRISPR interference (CRISPRi) is a powerful technology for studying loss-of-function phenotypes, enabling transient and reversible control of gene expression without the introduction of double-stranded DNA breaks. The cost of conducting large-scale CRISPR screens necessitates the selection of effective and specific single-guide RNAs for the design of compact libraries. While several genome-wide CRISPRi-Cas9 libraries have been created, updates to transcript annotations, the generation of higher-resolution chromatin accessibility datasets, and the development of newer on-target prediction models motivate an updated CRISPRi library design approach. Here, we generate large CRISPRi datasets tiling essential and nonessential genes. We compare the performance of multiple KRAB domain systems, develop an updated CRISPRi-specific on-target scoring scheme, and quantitatively characterize off-target effects associated with seed-sequence patterns. We leverage these findings to design an optimized CRISPRi-Cas9 library, Katsano, and validate its performance with genome-wide viability screens.},
}

*CRISPR-Cas Systems/genetics
*Gene Library
RNA, Guide, CRISPR-Cas Systems/genetics
Humans

@article {pmid42665059,
year = {2026},
author = {Naessens, S and De Rouck, S and Vandenhole, M and Hosseinkhani, M and Tsakireli, D and Vontas, J and Van Leeuwen, T},
title = {Gene knockout of P450s CYP392A16 and CYP392D2 reveals unexpected in vivo effects and transcriptional rewiring in the extreme generalist Tetranychus urticae.},
journal = {Insect biochemistry and molecular biology},
volume = {195},
number = {},
pages = {104666},
doi = {10.1016/j.ibmb.2026.104666},
pmid = {42665059},
issn = {1879-0240},
mesh = {Animals ; *Tetranychidae/genetics/enzymology/drug effects/metabolism ; *Cytochrome P-450 Enzyme System/genetics/metabolism ; *Acaricides/pharmacology ; Gene Knockout Techniques ; *Arthropod Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Drug Resistance/genetics ; },
abstract = {The two-spotted spider mite Tetranychus urticae rapidly evolves resistance to acaricides, frequently through the upregulation of detoxification enzymes such as cytochrome P450 monooxygenases. Among these, CYP392A16 and CYP392D2 have repeatedly been implicated as major resistance determinants, yet their in vivo contributions remain incompletely resolved. Here, we used efficient CRISPR/Cas9-mediated genome editing to generate stable knockouts of CYP392A16 and CYP392D2 in a multi-resistant genetic background. Contrary to expectations, loss of either gene resulted in surprisingly limited effects on acaricide susceptibility, with increased sensitivity observed only for a subset of compounds. Experimental evolution assays revealed a moderate, host-dependent fitness cost associated with disruption of CYP392D2, but not CYP392A16. Transcriptomic analyses further uncovered stark contrasts between the knockouts, with CYP392D2 loss triggering extensive regulatory reprogramming suggestive of compensatory detoxification pathways. Together, our results demonstrate that even resistance-associated detoxification genes that are considered functionally validated in vitro can have strong context-dependent phenotypic effects in vivo, underscoring the complexity of resistance gene networks and highlighting the need to interpret CRISPR/Cas9 knockout phenotypes within the broader genetic and regulatory context.},
}

Animals
*Tetranychidae/genetics/enzymology/drug effects/metabolism
*Cytochrome P-450 Enzyme System/genetics/metabolism
*Acaricides/pharmacology
Gene Knockout Techniques
*Arthropod Proteins/genetics/metabolism
CRISPR-Cas Systems
Drug Resistance/genetics

@article {pmid42734334,
year = {2026},
author = {Tran, A-T and Tang, W-F and Hsieh, C-F and Chiu, Y-F and Huang, C-G and Lee, K-M and Kuo, R-L and Hsiung, K-C and Chiu, C-H and Horng, J-T},
title = {Monitoring kinetic changes and restriction of influenza A virus RNA species during infection using a Flu-Stranded CRISPR platform.},
journal = {mBio},
volume = {17},
number = {10},
pages = {e0178926},
doi = {10.1128/mbio.01789-26},
pmid = {42734334},
issn = {2150-7511},
support = {112-2320-B-182-034-MY3, 114-3114-B-182-001, 115-2321-B-182-005//National Science and Technology Council/ ; BMRPK85, BMRP416, CMRPD1M0881-3//Chang Gung Memorial Hospital/ ; UMRPD1N0071, UMRPD1R0191//Chang Gung University/ ; },
mesh = {*RNA, Viral/genetics ; Humans ; *Influenza A virus/genetics/physiology ; Animals ; *CRISPR-Cas Systems ; Mice ; Clustered Regularly Interspaced Short Palindromic Repeats ; Kinetics ; Influenza, Human/virology ; Cell Line ; Virus Replication ; RNA, Complementary/genetics ; },
abstract = {UNLABELLED: Influenza A virus (IAV) generates three closely related RNA species: viral RNA (vRNA), complementary RNA (cRNA), and messenger RNA (mRNA), whose strand-specific quantification remains limited by sensitivity and quantitative dynamic range, particularly at low RNA abundance. Here, we developed Flu-Stranded CRISPR-Cas12a, a strand-specific detection platform integrating tagged reverse transcription, segment-specific PCR, and Cas12a collateral cleavage to support quantitative analysis of all three RNA species across a broad dynamic range. The assay enables reliable detection down to 10[2] copies per reaction, extending the lower quantitative boundary relative to both SYBR Green and TaqMan reverse transcription quantitative PCR (RT-qPCR) under matched conditions. Validated in infected cell lines, murine lung tissues, and clinical nasopharyngeal specimens, the platform enabled subtype-discriminating, strand-resolved detection, including samples near or below the quantitative range of SYBR Green RT-qPCR. Using finely resolved infection time-course analyses in NP and NA segments, we identified a reproducible early vRNA decline within the early post-infection phase. This decline was partially attenuated in RIG-I knockout A549 cells, while subsequent vRNA accumulation was enhanced, consistent with a modulatory rather than essential role for RIG-I in early viral RNA dynamics. Subcellular fractionation localized this decline to cytoplasmic incoming genomes. In contrast, importazole-mediated inhibition of nuclear import abolished vRNA recovery without affecting the early decline, indicating that nuclear entry functionally separates early genome reduction from subsequent productive replication. These findings establish Flu-Stranded CRISPR-Cas12a as a strand-resolved framework for monitoring IAV RNA dynamics and reveal an early window of genome vulnerability during cytoplasmic transit that shapes infection outcome.

IMPORTANCE: The early fate of incoming influenza virus genomes remains unclear, limiting our understanding of how infection is established or aborted in host cells. We developed Flu-Stranded CRISPR-Cas12a, a strand-specific platform for sensitive and quantitative analysis of influenza viral RNA (vRNA), complementary RNA (cRNA), and messenger RNA (mRNA) across experimental and clinical samples. Using high-resolution time-course analysis, we identified a reproducible early decline in vRNA during the post-entry phase. Our data suggest that this early genome loss arises from multiple processes, with RIG-I acting as a modulatory factor rather than a primary driver. Subcellular fractionation localized this effect to cytoplasmic incoming genomes, whereas importin-β-mediated nuclear entry was required for subsequent vRNA recovery. These findings support a model of an early cytoplasmic phase of genome attrition that is distinct from replication and provide a framework for understanding early influenza RNA kinetics and for guiding strand-resolved diagnostics and antiviral evaluation.},
}

*RNA, Viral/genetics
Humans
*Influenza A virus/genetics/physiology
Animals
*CRISPR-Cas Systems
Mice
Clustered Regularly Interspaced Short Palindromic Repeats
Kinetics
Influenza, Human/virology
Cell Line
Virus Replication
RNA, Complementary/genetics

@article {pmid42748591,
year = {2026},
author = {Patil, SB and Das, A and Inamdar, MS},
title = {Endogenous tagging of the TBXT (Brachyury) gene with 2A-EGFP in BJNhem19 human embryonic stem cells using CRISPR-Cas9 genome editing.},
journal = {Stem cell research},
volume = {96},
number = {},
pages = {104103},
doi = {10.1016/j.scr.2026.104103},
pmid = {42748591},
issn = {1876-7753},
mesh = {Humans ; Brachyury Protein ; *Fetal Proteins/genetics/metabolism ; *Human Embryonic Stem Cells/metabolism/cytology ; *T-Box Domain Proteins/genetics/metabolism ; *Green Fluorescent Proteins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Cell Line ; *Gene Editing ; Cell Differentiation ; },
abstract = {Brachyury (TBXT) is a T-box transcription factor essential for directing human embryonic stem cells (hESCs) toward mesodermal lineage specification. Here, we report the generation of a Brachyury/TBXT-2A-EGFP knock-in reporter line in the BJNhem19 hESC line using CRISPR/Cas9-mediated homology-directed repair. Accurate integration of the reporter cassette was confirmed by DNA sequencing and validated by the comparison of EGFP expression to endogenous TBXT gene expression following directed mesodermal differentiation. This reporter line provides a tractable fluorescence-based platform for real-time monitoring of mesodermal commitment, which will help in high-content screening of differentiation protocols.},
}

Humans
Brachyury Protein
*Fetal Proteins/genetics/metabolism
*Human Embryonic Stem Cells/metabolism/cytology
*T-Box Domain Proteins/genetics/metabolism
*Green Fluorescent Proteins/genetics/metabolism
*CRISPR-Cas Systems/genetics
Cell Line
*Gene Editing
Cell Differentiation

@article {pmid42748592,
year = {2026},
author = {Trobec, A and Lorrain, V and Ilic, D and Raymond, K and Liovic, M},
title = {Homology-directed CRISPR-Cas9 correction of the KRT5 p.E475G mutation in human iPSC line from a patient with severe epidermolysis bullosa simplex.},
journal = {Stem cell research},
volume = {96},
number = {},
pages = {104101},
doi = {10.1016/j.scr.2026.104101},
pmid = {42748592},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/pathology ; *CRISPR-Cas Systems/genetics ; *Keratin-5/genetics/metabolism ; *Epidermolysis Bullosa Simplex/genetics/pathology/metabolism ; *Mutation/genetics ; Cell Line ; *Gene Editing ; Cell Differentiation ; },
abstract = {Severe epidermolysis bullosa simplex is a skin fragility disorder characterized by blistering caused by cytolysis within basal keratinocytes, resulting in compromised epidermal integrity. Here we report the generation of the human induced pluripotent stem cell (hiPSC) line MLi002-A-1, an isogenic control derived from patient-specific MLi002-A line carrying the KRT5 c.1424A > G (p.E475G) mutation. Genome editing restored the wild-type sequence without detectable changes at top-predicted off-target sites. The edited line exhibits a normal karyotype, typical pluripotent morphology, robust pluripotency marker expression, and trilineage differentiation potential. This genetically matched control enables mutation-specific studies and in vitro modeling of epidermolysis bullosa simplex.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/pathology
*CRISPR-Cas Systems/genetics
*Keratin-5/genetics/metabolism
*Epidermolysis Bullosa Simplex/genetics/pathology/metabolism
*Mutation/genetics
Cell Line
*Gene Editing
Cell Differentiation

@article {pmid42753306,
year = {2026},
author = {Ghodrat, R and Ramachandran, H and Hildebrandt, B and Binder, S and Rossi, A and Reichert, AS},
title = {CRISPR/Cpf1-mediated editing of DNM1L in induced pluripotent stem cells.},
journal = {Stem cell research},
volume = {96},
number = {},
pages = {104102},
doi = {10.1016/j.scr.2026.104102},
pmid = {42753306},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Dynamins/genetics/metabolism ; Cell Differentiation ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Cell Line ; *Microtubule-Associated Proteins/genetics/metabolism ; Mutation ; *Mitochondrial Proteins/genetics/metabolism ; },
abstract = {The dynamin-1-like protein (DNM1L), also termed DRP1, is essential for mitochondrial fission. Mutations in DNM1L are associated with neurological disorders and cardiac dysfunction. To decipher the role of DNM1L in human induced pluripotent stem cells (hiPSCs) and in their differentiated counterparts, we used CRISPR/Cpf1 and generated a human iPSC line with a mutation by targeting exon 18 of the DNM1L gene. The generated compound heterozygous (biallelic) DNM1L mutant cell line showed normal cell morphology, genomic stability, and expression of classical stem cell markers. Furthermore, the cells can be differentiated efficiently into the three germ layers meso-, endo-, and ectoderm.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*Dynamins/genetics/metabolism
Cell Differentiation
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Cell Line
*Microtubule-Associated Proteins/genetics/metabolism
Mutation
*Mitochondrial Proteins/genetics/metabolism

@article {pmid42765712,
year = {2026},
author = {Banta, AB and Hall, AN and Myers, KS and Ward, RD and Davis, BC and Cuellar, RA and Place, M and Freeh, CC and Bacon, EE and Peters, JM},
title = {Targeted, genome-scale overexpression in Proteobacteria.},
journal = {mBio},
volume = {17},
number = {10},
pages = {e0234025},
doi = {10.1128/mbio.02340-25},
pmid = {42765712},
issn = {2150-7511},
support = {DE-SC0018409//U.S. Department of Energy/ ; 1R35GM150487/NH/NIH HHS/United States ; 1AYSAX000005//Advanced Research Projects Agency for Health/ ; },
mesh = {*Genome, Bacterial ; Escherichia coli/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; DNA Transposable Elements ; *Proteobacteria/genetics ; Zymomonas/genetics ; Gene Expression Regulation, Bacterial ; Promoter Regions, Genetic ; },
abstract = {Targeted, genome-scale gene perturbation screens using clustered regularly interspaced short palindromic repeats interference (CRISPRi) and activation (CRISPRa) have revolutionized eukaryotic genetics, advancing medical, industrial, and basic research. Although CRISPRi knockdowns have been broadly applied in bacteria, options for genome-scale gene overexpression face key limitations. Here, we develop a facile approach for genome-scale overexpression in bacteria we call CRISPR transposition and overexpression (CRISPRtOE). We first create a platform for comprehensive gene targeting using CRISPR-associated transposons (CASTs) and show that transposition occurs at a higher frequency in non-transcribed DNA. We then demonstrate that CRISPRtOE can upregulate gene expression in Proteobacteria with medical and industrial relevance by integrating synthetic promoters of varying strength upstream of target genes. Finally, we employ CRISPRtOE screening at the genome scale in the model bacterium Escherichia coli and the non-model biofuel producer Zymomonas mobilis, recovering known and novel antibiotic and engineering targets. We envision that CRISPRtOE will be a valuable overexpression tool for antibiotic mode of action, industrial strain optimization, and gene function discovery in bacteria.IMPORTANCESystematic alteration of bacterial gene expression enables identification of genes relevant to diverse fields of study and practical applications. Although many targeted, genome-scale genetic tools exist for reducing or eliminating gene expression, there are few facile approaches for systematic gene overexpression in bacteria. Here, we develop a targeted overexpression approach for Proteobacteria of medical and industrial importance that precisely inserts strong promoters upstream of genes using clustered regularly interspaced short palindromic repeats (CRISPR)-associated transposons. We demonstrate that this approach can be used to systematically overexpress genes in both model (Escherichia coli K-12) and non-model (Zymomonas mobilis) Proteobacteria for the purpose of understanding antibiotic resistance mechanisms and improving strain resilience in biofuel production conditions, respectively.},
}

*Genome, Bacterial
Escherichia coli/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Cas Systems
DNA Transposable Elements
*Proteobacteria/genetics
Zymomonas/genetics
Gene Expression Regulation, Bacterial
Promoter Regions, Genetic

@article {pmid42837420,
year = {2026},
author = {Zhang, J and Tao, Y and Zhao, Y and Lei, M and Shen, Y and Li, Q and Liu, C and Bai, J},
title = {A binding-activated CRISPR/dCas13X imaging system enables precise RNA tracking in plants.},
journal = {PLoS biology},
volume = {24},
number = {10},
pages = {e3004043},
doi = {10.1371/journal.pbio.3004043},
pmid = {42837420},
issn = {1545-7885},
mesh = {*Arabidopsis/genetics/metabolism ; Nicotiana/genetics/metabolism ; Arabidopsis Proteins/metabolism/genetics ; RNA-Binding Proteins/metabolism/genetics ; RNA, Messenger/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; *RNA, Plant/metabolism/genetics ; Degrons ; Plants, Genetically Modified ; RNA Transport ; Plasmodesmata/metabolism ; },
abstract = {Visualizing the spatiotemporal dynamics of native RNA molecules with single-transcript-level sensitivity in living plant cells remains a formidable challenge due to the high background noise caused by the unique cellular architecture of plants. Here, using transiently transformed Nicotiana benthamiana cells and stable Arabidopsis thaliana lines, we present a high-signal-to-noise RNA imaging platform that couples CRISPR-dCas13X with a binding-associated fluorescence stabilization strategy. We identified a novel plant-specific degron that selectively targets unbound probes for proteasomal degradation while preserving target-associated signal in a manner consistent with binding-dependent protection, enabling high-fidelity detection of endogenous mRNA puncta with minimal perturbation. Leveraging this platform, we characterized dynamics of FT mRNA trafficking in living plant cells, including plasmodesmata-targeting, cell-to-cell trafficking, and long-distance movement. Loss-of-function analysis further identified the established RNA-binding protein GRP7 as a factor that contributes to efficient FT mRNA transport. This work establishes a useful toolkit for investigating plant RNA biology and provides mechanistic insight into the systemic transport of florigenic signals.},
}

*Arabidopsis/genetics/metabolism
Nicotiana/genetics/metabolism
Arabidopsis Proteins/metabolism/genetics
RNA-Binding Proteins/metabolism/genetics
RNA, Messenger/metabolism/genetics
*CRISPR-Cas Systems/genetics
*RNA, Plant/metabolism/genetics
Degrons
Plants, Genetically Modified
RNA Transport
Plasmodesmata/metabolism

@article {pmid42850253,
year = {2026},
author = {Sun, X and Sang, Y and He, T and Zheng, M and Zou, Y and Tay, A},
title = {CD73 disruption with tumor-specific CRISPR/Cas9 nanoparticles reduces adenosine-mediated immunosuppression and amplifies response to chemotherapy.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42850253},
issn = {2041-1723},
support = {T2EP30224-0021//National Medical Research Council/ ; },
mesh = {Animals ; *5'-Nucleotidase/genetics/metabolism ; Female ; *Adenosine/metabolism ; Mice ; Tumor Microenvironment/immunology/drug effects/genetics ; *Nanoparticles/chemistry ; Humans ; Doxorubicin/pharmacology/therapeutic use/administration & dosage ; *CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Immunotherapy/methods ; Triple Negative Breast Neoplasms/immunology/drug therapy/genetics/therapy ; Mice, Inbred C57BL ; Immunosuppression Therapy ; Antineoplastic Agents/pharmacology ; },
abstract = {The therapeutic efficacy of cancer immunotherapy is impaired by poor tumor immunogenicity and the immunosuppressive tumor microenvironment (TME), partially driven by reprogrammed adenosine metabolism in the tumor. Here, we develop degradable CRISPR/Cas9 nanoparticles to durably and precisely downregulate CD73 expression across multiple cell subsets within the tumor microenvironment, including tumor cells, endothelial cells, cancer-associated fibroblasts, and tumor-infiltrating immune cells, thereby effectively disrupting adenosine metabolism. In combination with immunogenic cell death induced by the co-delivered chemotherapeutic drug doxorubicin, this strategy synergistically potentiates tumor immunogenicity and reprograms the TME into an immunostimulatory state. This immune remodeling contributes to tumor control, prolonging median survival in preclinical models of melanoma and triple negative breast cancer in female mice. This study provides a strategy for precise and durable regulation of cancer immunometabolism to improve therapeutic effects of chemo-immunotherapy for synergistic cancer therapy.},
}

Animals
*5'-Nucleotidase/genetics/metabolism
Female
*Adenosine/metabolism
Mice
Tumor Microenvironment/immunology/drug effects/genetics
*Nanoparticles/chemistry
Humans
Doxorubicin/pharmacology/therapeutic use/administration & dosage
*CRISPR-Cas Systems/genetics
Cell Line, Tumor
Immunotherapy/methods
Triple Negative Breast Neoplasms/immunology/drug therapy/genetics/therapy
Mice, Inbred C57BL
Immunosuppression Therapy
Antineoplastic Agents/pharmacology

@article {pmid42852014,
year = {2026},
author = {Madani, J and Vellani, Z and Fayed, Y},
title = {Reassessing Viral Gene Therapy for Glioblastoma: Translational Opportunities and Challenges.},
journal = {The Yale journal of biology and medicine},
volume = {99},
number = {2},
pages = {295-301},
pmid = {42852014},
issn = {1551-4056},
mesh = {Animals ; Humans ; *Brain Neoplasms/therapy/genetics ; CRISPR-Cas Systems/genetics ; Gene Therapy Agents ; *Genetic Therapy/methods ; *Glioblastoma/therapy/genetics ; Oncolytic Virotherapy/methods ; Oncolytic Viruses/genetics ; *Translational Research, Biomedical ; },
abstract = {Glioblastoma is a highly invasive primary brain tumor with a remarkably poor prognosis. Resistance to the current gold standard treatment, maximal safe surgical resection followed by radiation and chemotherapy, has led to an investigation into novel therapeutic alternatives, notably gene therapy. This perspective evaluates the translational potential of three prominent viral gene therapies for glioblastoma, highlighting the preclinical challenges facing CRISPR-Cas9-mediated gene editing, suicide gene therapy, and oncolytic virus therapy. We assert that clinical translation depends strongly on the optimization of these approaches to overcome biological and delivery barriers. Prioritizing such research is critical, as gene therapies continue to show promise as a much-needed advancement in the field of neuro-oncology.},
}

Animals
Humans
*Brain Neoplasms/therapy/genetics
CRISPR-Cas Systems/genetics
Gene Therapy Agents
*Genetic Therapy/methods
*Glioblastoma/therapy/genetics
Oncolytic Virotherapy/methods
Oncolytic Viruses/genetics
*Translational Research, Biomedical

@article {pmid42852998,
year = {2026},
author = {Bjornson, M and Hardigan, MA and Debernardi, JM and Brukental, H and Pincot, DDA and Vachev, MV and Famula, RA and Cole, GS and Velasco, D and Al Rwahnih, M and Feldmann, MJ and Knapp, SJ},
title = {CRISPR editing of TERMINAL FLOWER 1 recapitulates perpetual flowering in strawberry, mimicking repression by the PERPETUAL FLOWERING locus.},
journal = {Plant physiology},
volume = {202},
number = {2},
pages = {},
pmid = {42852998},
issn = {1532-2548},
mesh = {*Fragaria/genetics/growth & development/physiology ; *Flowers/genetics/growth & development/physiology ; *Gene Editing ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; *CRISPR-Cas Systems ; Genome-Wide Association Study ; },
abstract = {The development of perpetual flowering (PF) cultivars revolutionized octoploid strawberry (Fragaria × ananassa) cultivation by enabling nearly year-round fruit production and doubling fruit yields compared to historic seasonal flowering (SF) cultivars, which flower in spring. Most commercial PF cultivars result from the introgression of a dominant PERPETUAL FLOWERING (PF) allele at the PERPETUAL FLOWERING RUNNERING (PFRU) locus. PFRU has been genetically and physically mapped to chromosome 4B, but the causal gene(s) remain unknown. Here we used the haplotype-phased "UCD Royal Royce" (PFpf) genome, next-generation sequencing-based k-mer genome-wide association study (k-mer GWAS), and RNAseq to investigate PFRU. We narrowed the locus down to a precise physical address and revealed abundant genetic and transcriptomic differences in the locus and associated with flowering. Outside the PFRU locus, flowering regulation diverges at expression of TERMINAL FLOWER 1 (TFL1) in all 4 subgenomes, analogous to diploid Fragaria vesca. This critical step of the flowering pathway is altered by PF in diverse cultivars, and gene editing knockout of TFL1 is sufficient to induce flowering under long days in a seasonal flowering cultivar. Together, these results shed light on the mechanism of flowering regulation by the PF locus and expand biotechnological approaches to achieve extended flowering in any strawberry cultivar.},
}

*Fragaria/genetics/growth & development/physiology
*Flowers/genetics/growth & development/physiology
*Gene Editing
*Plant Proteins/genetics/metabolism
Gene Expression Regulation, Plant
*CRISPR-Cas Systems
Genome-Wide Association Study

@article {pmid42853350,
year = {2026},
author = {Shruthy, NS and Dong, CD and Chen, YT and Hsieh, SL and Singhania, RR and Patel, AK},
title = {CRISPR-enhanced microalgae for carbon sequestration and carotenoid production: opportunities and fundamental constraints.},
journal = {Bioresources and bioprocessing},
volume = {13},
number = {1},
pages = {},
pmid = {42853350},
issn = {2197-4365},
support = {114-2221-E-992-017//NSTC/ ; 114-2221-E-992-018//NSTC/ ; },
abstract = {Microalgae represent a promising biological platform for CO2 sequestration and the sustainable production of high-value metabolites, particularly carotenoids such as lutein and astaxanthin. However, industrial performance remains constrained by low carbon conversion efficiency, reduced photosynthetic performance, and competing metabolic fluxes that limit product yield. The emergence of CRISPR/Cas-based genome engineering has enabled precise and multiplexed modulation of microalgal metabolic networks, offering new opportunities to overcome these limitations. This review critically evaluates CRISPR-enabled engineering strategies for improving carbon assimilation, photosynthetic efficiency, and carotenoid biosynthesis in microalgae. We discuss advances in transformation and delivery systems across diverse species, CRISPR interference, CRISPR activation, and multiplexed pathway rewiring. Targeted modifications of light-harvesting complexes, carbon concentrating mechanisms, and key carotenoid biosynthetic enzymes are discussed with their reported impacts on biomass accumulation, CO2 fixation, and pigment productivity. Emerging evidence indicates that while pathway-specific genetic interventions can significantly enhance metabolic accumulation, these improvements are often not proportionally reflected in whole-cell carbon fixation, revealing underlying constraints imposed by energy allocation, redox balance, and regulatory network coupling. Finally, we highlight the potential of synthetic regulatory circuits and programmable transcriptional control as next-generation tools for dynamic regulation of metabolic fluxes rather than static gene-level modifications. By integrating molecular engineering strategies with physiological and system-level constraints, this review provides a critical framework for developing robust microalgal cell factories for sustainable bioproduction and climate mitigation.},
}

@article {pmid42853493,
year = {2026},
author = {Li, Y and Wu, Z and Xiang, T and Duan, B and Liu, Z and Liu, P and Wei, Y and Ling, P},
title = {Mycoplasma pneumoniae infections in the Post-COVID era: recent advances from pathogenesis to clinical management.},
journal = {Infection},
volume = {},
number = {},
pages = {},
pmid = {42853493},
issn = {1439-0973},
support = {S202310555045 and S202310555226//Hunan Provincial College Students' innovation and Entrepreneurship Training Program/ ; Z2023090//Health Research Project of Hunan Provincial Health Commission/ ; 2023GZ4043//Science and Technology Planning Project of Shaoyang City/ ; },
abstract = {M.pneumoniae is a major cause of human respiratory infection whose pathogenicity relies on adhesion proteins, secreted virulence factors, and immune evasion mechanisms. This review contextualizes these strategies within the broader phylogeny of clinically relevant Mycoplasma species while addressing the diverse extrapulmonary manifestations of M. pneumoniae. Current challenges in managing M. pneumoniae infections are multifaceted. The post-pandemic resurgence reflects a population-level immune debt resulting from prolonged non-pharmaceutical interventions, with test positivity rising sharply after restrictions were lifted and driving pneumonia outbreaks across age groups. Macrolide resistance remains persistently high in East Asia and is emerging at variable rates in other regions; routine diagnostics cannot detect resistance in real time, complicating first-line empirical therapy. Vaccine development continues to face obstacles including antigenic variation mediated by recombination between repetitive genomic sequences, the IL-17 A paradox wherein protective Th17 responses also drive vaccine-enhanced disease, and the inability of systemic vaccines to induce mucosal secretory IgA and tissue-resident memory T cells. In response, we evaluate emerging diagnostics-including metagenomic sequencing, CRISPR-Cas platforms, and host biomarkers-and discuss resistance-guided therapies involving doxycycline, quinolones, and novel agents like lefamulin and omadacycline. We further analyze vaccine development hurdles alongside the potential of mRNA-LNP platforms targeting conserved adhesin epitopes. Our findings indicate that the lag between mechanistic insight and clinical implementation remains the most pressing challenge. Future research must shift from simple mechanistic elucidation to addressing real-world clinical dilemmas, including distinguishing colonization from infection through integrated pathogen-host evaluation, exploring synergistic use of antibiotics and immunomodulators targeting the IL-17 A pathway, advancing vaccine design that circumvents IL-17 A-mediated immunopathology via conserved epitope selection and mucosal delivery, and establishing real-time surveillance networks integrating genomics and clinical phenotypes. Collectively, this work provides a practical roadmap spanning molecular pathology to clinical practice for the precise management of M. pneumoniae infections.},
}

@article {pmid42853768,
year = {2026},
author = {Wang, C and Wang, J and Huang, T and Yu, B and Zou, G},
title = {Optimizing Perfusates for Ex Vivo Lung Perfusion: A Pharmaceutic Perspective on Formulations, Functions, and Future Innovations.},
journal = {Pharmacology},
volume = {},
number = {},
pages = {1},
doi = {10.1159/pha/aejag006},
pmid = {42853768},
issn = {1423-0313},
abstract = {Ex vivo lung perfusion (EVLP) is now an established normothermic machine-perfusion strategy in lung transplantation, primarily enabling structured donor lung assessment, short-term preservation, and selective rehabilitation outside the recipient. Because the supply of transplantable lungs remains limited, clinically implemented EVLP protocols have increased donor-lung utilization by allowing transplant teams to evaluate marginal grafts under near-physiological ventilation and perfusion rather than relying exclusively on static cold storage. The perfusate is central to this process because it stabilizes colloid osmotic pressure, electrolytes, buffering capacity, metabolic substrates, and oxygen transport according to protocol requirements. This review critically summarizes the development of EVLP perfusates and separates clinically validated roles from experimental repair strategies. We first distinguish EVLP perfusates from cold preservation solutions and compare acellular, red blood cell-containing, and whole-blood approaches with attention to their physiological rationale, logistical constraints, and level of evidence. We then discuss protocol-specific additives, including anti-inflammatory agents, antioxidants, antimicrobials, anticoagulants, hormones, nutritional substrates, and cellular or gene-based therapies. Emerging interventions such as polymerized hemoglobin, cytokine adsorption, mesenchymal stromal cell-derived products, CRISPR-Cas-mediated interleukin-10 modulation, and prolonged or intermittent EVLP illustrate the expanding therapeutic potential of the platform. However, most of these strategies remain preclinical or early translational. Future perfusate development should therefore prioritize standardized outcome measures, evidence-based component selection, and careful separation of organ preservation, organ assessment, and organ rehabilitation.},
}

@article {pmid42752528,
year = {2026},
author = {Liang, M and Shi, X and Xia, D and Zong, W and Hu, H and Xie, N and Li, Y and Lv, S and Yin, J},
title = {A hand-powered positive pressure-driven sample injection digital RPA-CRISPR-Cas12a chip.},
journal = {Lab on a chip},
volume = {26},
number = {20},
pages = {5518-5527},
doi = {10.1039/d6lc00542j},
pmid = {42752528},
issn = {1473-0189},
mesh = {Pressure ; *CRISPR-Cas Systems/genetics ; *Lab-On-A-Chip Devices ; Equipment Design ; Listeria monocytogenes/isolation & purification/genetics ; Animals ; *Microfluidic Analytical Techniques/instrumentation ; *Nucleic Acid Amplification Techniques/instrumentation ; },
abstract = {Chamber-based digital nucleic acid assays allow absolute quantification with high analytical sensitivity. However, sample introduction and digital discretization mainly rely on negative pressure drive, and this injection method limits its application, especially in on-site detection. But their use outside laboratory settings is still limited by the way samples are partitioned, which often depends on vacuum systems, centrifugation or specialized actuation. Positive-pressure loading is simple and well suited to portable operation, yet it is difficult to apply directly to chamber-based digital assays because air backflow and sample trailing can disrupt stable compartmentalization. This study proposed a hand-powered positive pressure-driven sample injection digital RPA-CRISPR-Cas12a chip for on-site food-safety testing. In this study, the two problems were addressed by combining photocurable oil fixation with branch-channel optimization, enabling rapid and stable discretization in a 15 120 chamber-based microfluidic digital chip using only a hand-operated syringe. Sample loading and partitioning were completed within 2 min without vacuum or centrifugal loading equipment. After integration with a one-pot digital RPA-CRISPR-Cas12a assay targeting hlyA, the system achieved absolute quantification of Listeria monocytogenes with a detection limit of 10[0] copies μL[-1] and good linearity across dilutions (R[2] = 0.99973). The tests conducted on the milk samples containing the standard substances further proved that this method could be used for on-site testing under resource-limited conditions. Overall, this study provides a feasible strategy for implementing positive-pressure digitization in a portable chamber-based digital microfluidic system, which expands the utility of chamber-based digital assays for portable and instrument-free on-site food-safety testing.},
}

Pressure
*CRISPR-Cas Systems/genetics
*Lab-On-A-Chip Devices
Equipment Design
Listeria monocytogenes/isolation & purification/genetics
Animals
*Microfluidic Analytical Techniques/instrumentation
*Nucleic Acid Amplification Techniques/instrumentation

@article {pmid42785308,
year = {2026},
author = {Dong, XX and Choi, WWY and Ahmed, M and Nishimura, H and Tian, A and Millar, D and Bhattacharya, A and Aggarwal, A and Vu, L and Weng, OY and Nguyen, R and Chakraborty, P and Wang, LY and Wilson, MD and Shu, Q and Li, X and Wu, YC and Muffat, J and Li, Y},
title = {HOPX regulates the development and lineage progression of human stem cell-derived outer radial glia-like cells.},
journal = {Stem cell reports},
volume = {21},
number = {10},
pages = {103087},
doi = {10.1016/j.stemcr.2026.103087},
pmid = {42785308},
issn = {2213-6711},
mesh = {Humans ; *Homeodomain Proteins/metabolism/genetics ; *Cell Lineage/genetics ; *Ependymoglial Cells/metabolism/cytology ; Cell Differentiation/genetics ; *Neural Stem Cells/cytology/metabolism ; Neurogenesis/genetics ; TOR Serine-Threonine Kinases/metabolism ; Cell Proliferation ; CRISPR-Cas Systems ; Organoids/metabolism/cytology ; Phosphatidylinositol 3-Kinases/metabolism ; Neurons/metabolism/cytology ; Signal Transduction ; },
abstract = {Outer radial glia (oRG) are abundant in the human fetal cortex but rare in rodents, yet their molecular regulation remains poorly understood. Although the homeodomain-only protein X (HOPX) is a well-established oRG marker, its functional role in human cortical development remains undefined. Using CRISPR-Cas9 gene editing in human pluripotent stem cells, we show that HOPX loss impairs the expansion and lineage progression of oRG-like cells in two-dimensional neural cultures and three-dimensional cortical organoids. HOPX knockout precursors exhibit reduced proliferation, diminished neurogenesis, and premature differentiation toward astrocytic and oligodendrocytic lineages. HOPX-deficient organoids display reduced outer subventricular zone size, decreased neuronal output, and early gliogenesis. Mechanistically, HOPX loss impairs PI3K-mTOR signaling. Transcriptomic analysis identifies HOPX-regulated programs governing gliogenic competence, mTOR, and Wnt signaling. Together, these findings identify HOPX as an important regulator of oRG-like cell maintenance and the neurogenic-gliogenic balance, providing insight into the molecular programs governing human neural progenitor expansion.},
}

Humans
*Homeodomain Proteins/metabolism/genetics
*Cell Lineage/genetics
*Ependymoglial Cells/metabolism/cytology
Cell Differentiation/genetics
*Neural Stem Cells/cytology/metabolism
Neurogenesis/genetics
TOR Serine-Threonine Kinases/metabolism
Cell Proliferation
CRISPR-Cas Systems
Organoids/metabolism/cytology
Phosphatidylinositol 3-Kinases/metabolism
Neurons/metabolism/cytology
Signal Transduction

@article {pmid42808805,
year = {2026},
author = {Wu, X and Chen, Q and Zheng, J and Gao, Y and Cao, Y and Li, Z and Chen, Z},
title = {Sensitive fluorescent biosensors based on a cascade signal amplification strategy and a stimuli-responsive hydrogel for the detection of the p53 gene.},
journal = {The Analyst},
volume = {151},
number = {20},
pages = {6023-6029},
doi = {10.1039/d6an00852f},
pmid = {42808805},
issn = {1364-5528},
mesh = {*Biosensing Techniques/methods ; Alkaline Phosphatase/chemistry/metabolism ; Humans ; Hyaluronic Acid/chemistry ; *Tumor Suppressor Protein p53/genetics ; DNA/chemistry/genetics ; *Hydrogels/chemistry ; Silicon Dioxide/chemistry ; Limit of Detection ; Spectrometry, Fluorescence/methods ; Fluorescent Chemosensor Compounds ; Carbon Quantum Dots/chemistry ; Nitrophenols/chemistry ; Fluorescent Dyes/chemistry ; CRISPR-Cas Systems ; *Genes, p53 ; Organophosphorus Compounds ; },
abstract = {As a key tumor suppressor gene, the mutation and abnormal expression of p53 are closely correlated with tumor occurrence and progression, making p53 an important biomarker for early tumor diagnosis. Herein, a novel fluorescent biosensor based on a Cas12a/alkaline phosphatase (ALP) cascade signal amplification strategy and a stimuli-responsive DNA hydrogel was constructed for sensitive quantification of the p53 gene. Highly stable carbon dot/silica (CD/SiO2) composites were adopted as excellent fluorescent signal-sensing elements with outstanding optical stability in complex environments. An ALP-encapsulated hyaluronic acid (HA)-DNA hydrogel was prepared as a stimuli-responsive recognition element. Specific binding of the target p53 gene activates the trans-cleavage activity of CRISPR/Cas12a, which cleaves the HA-DNA hydrogel structure and triggers the release of ALP. The released ALP further catalyzes the hydrolysis of the p-nitrophenyl phosphate (PNPP) substrate to generate p-nitrophenol (PNP). The generated PNP quenches the fluorescence of CD/SiO2 composites through the inner filter effect (IFE), and the fluorescence quenching value is positively correlated with the p53 gene concentration. The biosensor exhibited a favorable linear detection range of 1 pM-100 nM with a low detection limit of 0.074 pM. The proposed strategy eliminates cumbersome pre-amplification procedures for nucleic acids, provides a simple, sensitive and reliable new approach for p53 gene detection in complex biological samples, and exhibits great potential for early tumor auxiliary diagnosis and clinical biological analysis.},
}

*Biosensing Techniques/methods
Alkaline Phosphatase/chemistry/metabolism
Humans
Hyaluronic Acid/chemistry
*Tumor Suppressor Protein p53/genetics
DNA/chemistry/genetics
*Hydrogels/chemistry
Silicon Dioxide/chemistry
Limit of Detection
Spectrometry, Fluorescence/methods
Fluorescent Chemosensor Compounds
Carbon Quantum Dots/chemistry
Nitrophenols/chemistry
Fluorescent Dyes/chemistry
CRISPR-Cas Systems
*Genes, p53
Organophosphorus Compounds

@article {pmid42808823,
year = {2026},
author = {Wu, S and Mu, X and Wu, Z},
title = {A PNA-assisted magnetic bead-confined CbAgo-CRISPR/Cas12a cascade for amplification-free detection of Mycobacterium avium subsp. paratuberculosis F57 DNA.},
journal = {The Analyst},
volume = {151},
number = {20},
pages = {6202-6213},
doi = {10.1039/d6an00843g},
pmid = {42808823},
issn = {1364-5528},
mesh = {*Mycobacterium avium subsp. paratuberculosis/genetics/isolation & purification ; *DNA, Bacterial/analysis/genetics ; *Peptide Nucleic Acids/chemistry ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Limit of Detection ; Animals ; Luminescent Measurements/methods ; Electrochemical Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne's disease in ruminants, has attracted increasing attention because of its potential zoonotic relevance. Sensitive molecular detection of MAP typically relies on nucleic acid amplification to overcome low pathogen abundance; however, amplification may generate nonspecific products and false-positive results. Here, targeting MAP-specific F57 double-stranded DNA, we developed a peptide nucleic acid (PNA)-assisted, magnetic bead-confined Clostridium butyricum Argonaute (CbAgo)-CRISPR/Cas12a cascade electrochemiluminescent (ECL) biosensing platform for amplification-free MAP detection. The platform comprises two functional magnetic-bead microreactors. In the PNA-CbAgo cleavage microreactor (PCCM), PNA and CbAgo/gDNA complexes are co-immobilized on magnetic beads. PNA-mediated local duplex opening facilitates CbAgo cleavage and generation of target-derived DNA fragments (tDNA). In the CRISPR/Cas12a-Ru ECL signaling microreactor (CREM), CRISPR/Cas12a-crRNA complexes and Ru-labeled ssDNA reporters are co-confined on magnetic beads. The generated tDNA activates localized CRISPR/Cas12a trans-cleavage, leading to reporter release and a target-dependent decrease in ECL intensity. Mechanistic studies showed that the PCCM enhances tDNA generation through PNA-assisted duplex opening and bead interfacial confinement, whereas the CREM enables efficient ECL transduction through localized CRISPR/Cas12a-crRNA-reporter coupling. Under optimized conditions, the platform achieved a detection limit of 34.1 CFU mL[-1], exhibited good specificity, agreed well with qPCR in spiked bovine fecal samples, and demonstrated robust overall performance in complex matrices. This work provides an amplification-free ECL strategy for sensitive and specific MAP detection and highlights the potential of bead-confined programmable nuclease cascades for pathogen nucleic acid analysis.},
}

*Mycobacterium avium subsp. paratuberculosis/genetics/isolation & purification
*DNA, Bacterial/analysis/genetics
*Peptide Nucleic Acids/chemistry
*CRISPR-Cas Systems
*Biosensing Techniques/methods
Limit of Detection
Animals
Luminescent Measurements/methods
Electrochemical Techniques/methods
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid42838939,
year = {2026},
author = {Chai, X and Poon, L and Liu, H and Singh, MK and Minami, T and Sashida, G and Fukuda, M and Teh, BT and Tan, P and Hwang, WYK and Lin, J and Koh, AS and Kondoh, T and Xu, L and Osato, M and Liu, J and Li, S},
title = {Telomere variant sequences encode the genetic blueprint for allele-specific telomere length.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42838939},
issn = {2041-1723},
support = {MOH-CIRG23jan-0006//MOH | National Medical Research Council (NMRC)/ ; },
mesh = {Humans ; *Telomere/genetics/metabolism ; Alleles ; *Telomere Homeostasis/genetics ; CRISPR-Cas Systems ; Female ; Cell Line ; Male ; },
abstract = {The ends of human chromosomes are capped by specialized nucleoprotein structures, termed telomeres, which are essential for genome stability. Recent advances in long-read sequencing have enabled allele-specific telomere length measurements at nucleotide resolution, uncovering extreme heterogeneity in telomere length between alleles. The progressive telomere shortening over time makes steady-state telomere length in human somatic cells a promising biomarker for age-associated diseases. However, the mechanisms underlying allele-specific telomere maintenance and its stability remain poorly understood. Here, we developed a high-resolution workflow combining PacBio and Nanopore long-read sequencing platforms to map allele-specific telomere length in human blood samples as well as cultured cell lines. By tracing allele-specific telomeric sequence in family members across multiple generations, we show that the allele-specific telomeric variant sequences (TVSs) are heritable and underlie the extreme heterogeneity of telomere length between alleles. Continuous cell proliferation likely drives the slow but stochastic evolution of allele-specific TVSs, resulting in asymmetry in telomere inheritance from father and mother (p-value = 2.354e-7). Targeted deletion of allele-specific TVSs using CRISPR-Cas9 resets telomere length, further confirming their causal role in the control of allele-specific telomere maintenance. These results indicated that TVSs are heritable genetic elements underlying the allele-specific telomere length.},
}

Humans
*Telomere/genetics/metabolism
Alleles
*Telomere Homeostasis/genetics
CRISPR-Cas Systems
Female
Cell Line
Male

@article {pmid42840375,
year = {2026},
author = {Ma, B and Li, Y and Zhang, D and Wu, Y and Gao, J and Li, H and Jia, S},
title = {Engineering TnpB as a compact RNA-guided genome editor from molecular constraints to design principles.},
journal = {Frontiers in genome editing},
volume = {8},
number = {},
pages = {1945303},
pmid = {42840375},
issn = {2673-3439},
abstract = {Recent discoveries of non-CRISPR RNA-guided nucleases have expanded the landscape of programmable genome editing beyond canonical Cas systems. Among these, TnpB proteins derived from IS200/IS605-family transposable elements are ultra-compact RNA-guided DNA endonucleases with potential for delivery-constrained genome engineering. Here, we review the structural and mechanistic basis of TnpB function, including protein architecture, transposon-associated motif (TAM) recognition, ωRNA/reRNA scaffold organization, RNA-DNA heteroduplex formation, and RuvC-mediated DNA cleavage. We highlight that TnpB activity emerges from coordinated interactions between the nuclease and its RNA scaffold, which jointly influence ribonucleoprotein assembly, target recognition, catalytic activation, and specificity. We further discuss engineering strategies involving ortholog selection, protein and TAM-recognition engineering, RNA scaffold optimization, delivery, and specificity control, and relate these approaches to current applications across biological systems. Although TnpB remains less mature than established CRISPR-Cas platforms, its compact architecture and growing engineering toolkit support its development as a complementary genome editing platform. Future progress will benefit from establishing predictive models that integrate target accessibility, RNA-protein compatibility, activity, and fidelity.},
}

@article {pmid42841232,
year = {2026},
author = {Huang, J and Wei, J and Chen, H and Qiao, X and Liu, N and Lu, S},
title = {A dynamically frustrated allosteric checkpoint consistent with conformational proofreading in CRISPR-Cas9.},
journal = {Nucleic acids research},
volume = {54},
number = {18},
pages = {},
pmid = {42841232},
issn = {1362-4962},
support = {2024ZD0531200//Noncommunicable Chronic Diseases-National Science and Technology Major Project of China/ ; //Ministry of Science and Technology of the People's Republic of China/ ; //Program for Innovative Research Team of High-Level Local Universities in Shanghai/ ; //Shanghai Municipal Education Commission/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Molecular Dynamics Simulation ; Allosteric Regulation ; DNA/chemistry/genetics/metabolism ; DNA Cleavage ; RNA, Guide, CRISPR-Cas Systems/genetics/chemistry/metabolism ; *CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; },
abstract = {CRISPR-Cas9 DNA cleavage requires R-loop extension to activate the histidine-asparagine-histidine (HNH) nuclease domain, yet how heteroduplex maturation gates catalytic commitment remains unclear. Using extensive molecular dynamics simulations across seven experimentally trapped heteroduplex states (6-18 nt), we define a three-stage activation pathway: unlocking, preorganization, and precatalytic gating. At the 18-nt checkpoint, HNH and REC2 regain mobility but do not adopt a fully cleavage-competent orientation; this dynamically frustrated metastate is consistent with a conformational-proofreading model. Here, dynamic frustration is used operationally to describe renewed mobility and weakened directional coupling without commitment to the cleavage-competent state, rather than a formal energetic frustration calculation. Two sensors, Y450 at the sgRNA: DNA hybrid interface and K1200 in the PI domain, respond to distinct maturation milestones, encoding heteroduplex length into domain-specific conformational outputs. Integrating these insights with two deep-learning analyses and deep mutational scanning recovers known high-fidelity positions and prioritizes three unannotated residues for testing. In a cellular cleavage reporter assay, R1210D reproduces the attenuated low-completion phenotype of eSpCas9, identifying it as a candidate for direct specificity testing, while destabilizing L1-linker substitutions increase cleavage efficiency. These results connect atomic-level dynamics to experimentally testable, structure-mechanism-informed engineering hypotheses; direct matched-versus mismatched validation is required to establish effects on discrimination fidelity.},
}

*CRISPR-Cas Systems/genetics
Molecular Dynamics Simulation
Allosteric Regulation
DNA/chemistry/genetics/metabolism
DNA Cleavage
RNA, Guide, CRISPR-Cas Systems/genetics/chemistry/metabolism
*CRISPR-Associated Protein 9/chemistry/genetics/metabolism

@article {pmid42841496,
year = {2026},
author = {Shmrany, HA and Belali, TM and Abu-Alghayth, MH and Binshaya, AS and Ali, AA and Alyahyawi, Y and Belali, OM},
title = {In Silico CRISPR Restoration of F7 Promoter Activity in Hereditary Factor VII Deficiency.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {236},
pages = {},
doi = {10.3791/72870},
pmid = {42841496},
issn = {1940-087X},
mesh = {*Promoter Regions, Genetic/genetics ; *Factor VII Deficiency/genetics ; Humans ; *Factor VII/genetics ; Hepatocyte Nuclear Factor 4/genetics/metabolism ; *CRISPR-Cas Systems ; Computer Simulation ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Hereditary Factor VII (FVII) deficiency is associated with reduced F7 gene expression and impaired coagulation, and regulatory mechanisms controlling F7 transcription remain incompletely characterized. This study aimed to computationally explore whether restoration of predicted HNF4α binding at the F7 promoter, through transcription factor modulation, promoter motif correction, or CRISPR activation (CRISPRa)-mediated epigenetic regulation, could theoretically enhance predicted F7 transcription in hereditary FVII deficiency. An entirely in silico pipeline was developed to characterize the F7 promoter regulatory landscape by identifying conserved transcription factor binding motifs using JASPAR, FIMO, and UCSC Genome Browser datasets. Liver expression correlations were evaluated using GTEx transcriptomic data, while chromatin accessibility and regulatory context were assessed using ENCODE ChIP-seq, ATAC-seq, and ChromHMM/Segway annotations. A hepatic regulatory network was constructed in CellDesigner to simulate the predicted effects of HNF4α loss on F7 transcription over a 24-h period. Six high-confidence HNF4α binding motifs were identified within the proximal F7 promoter, including three regions overlapping known disease-associated variants. Liver expression analyses demonstrated positive correlations between F7 and HNF4A (r = 0.68) and HNF1A (r = 0.61). Computational modeling predicted that CRISPRa targeting of HNF4α-associated regulatory regions could increase simulated F7 transcription toward approximately 87% of wild-type levels under the modeled conditions, with predicted target specificity. Overall, this purely computational study presents an integrated multi-omic framework for prioritizing promoter-targeted regulatory strategies in hereditary FVII deficiency. By combining transcription factor motif analysis, chromatin annotation, transcriptomic correlation, regulatory network modeling, and CRISPR guide prioritization within a unified pipeline, the study generates experimentally testable hypotheses while providing a systematic approach that extends beyond previous single-variant or isolated experimental analyses.},
}

*Promoter Regions, Genetic/genetics
*Factor VII Deficiency/genetics
Humans
*Factor VII/genetics
Hepatocyte Nuclear Factor 4/genetics/metabolism
*CRISPR-Cas Systems
Computer Simulation
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42843423,
year = {2026},
author = {Kour, R and Sharma, A and Mishra, S and Mehrotra, S and Srivastava, V},
title = {Unravelling the multifunctional roles of plant LIM proteins in growth, development, and stress responses.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {374},
number = {},
pages = {113501},
doi = {10.1016/j.plantsci.2026.113501},
pmid = {42843423},
issn = {1873-2259},
abstract = {LIM (Lin-11, Isl-1, and Mec-3 domain) proteins are evolutionarily conserved proteins that regulate diverse cellular processes in plants, including actin cytoskeleton organization, organ size determination, protein stability, and transcription-associated regulation. Therefore, these proteins have emerged as important regulators of plant growth, development, and responses to biotic and abiotic stresses. Numerous genome-wide studies, functional analyses, and mechanistic investigations in diverse plant species have considerably expanded our understanding of this gene family. However, these advances remain dispersed across the literature, present in peace-meal and have not been critically synthesized. This review summarizes recent progress in the classification and evolutionary diversification of plant LIM proteins, genome-wide identification and comparative analyses, and their multifunctional roles in plant growth and development, including organ size regulation, pollen and fibre development, and vascular differentiation. The review further critically evaluates current evidence for the involvement of LIM proteins in plant immunity and abiotic stress responses by distinguishing expression-based, functional, and mechanistic studies. Finally, the review discusses major knowledge gaps and highlights emerging research directions, including single-cell and spatial omics, phosphoproteomics, interactomics, and CRISPR/Cas-based genome editing, to advance mechanistic understanding and facilitate the exploitation of LIM proteins for crop improvement.},
}

@article {pmid42844273,
year = {2026},
author = {Ali, A and Zakhartchenko, V and Güngör, T and Nüske, S and Stöckl, JB and Hecken, S and Hauser, A and Krebs, S and Reese, S and Suchowski, M and Friedl, R and Schwanitz, S and Scholz, AM and Blutke, A and Protschka, M and Büttner, M and Fröhlich, T and Reichenbach, HD and Wolf, E},
title = {Pregnancy establishment in cattle without embryonic interferon tau.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42844273},
issn = {2041-1723},
support = {23CVD01//Fondation Leducq/ ; },
mesh = {Animals ; Female ; Cattle ; *Interferon Type I/genetics/metabolism ; *Pregnancy Proteins/genetics/metabolism ; Pregnancy ; Blastocyst/metabolism ; Nuclear Transfer Techniques ; CRISPR-Cas Systems ; Placentation/genetics ; Endometrium/metabolism ; Embryo, Mammalian/metabolism ; Fibroblasts/metabolism ; Luteolysis ; },
abstract = {In ruminants, embryonic interferon tau (IFNT) is widely considered the indispensable signal for maternal recognition of pregnancy, driving endometrial gene expression to prevent luteolysis. However, this canonical paradigm has never been directly tested using genomic loss-of-function models. Here we show that IFNT is completely dispensable for pregnancy recognition, luteal maintenance, and full-term gestation in cattle. Using CRISPR-Cas9, we generate bovine fibroblasts lacking all functional copies of the multigene IFNT locus for somatic cell nuclear transfer. IFNT-null embryos develop into blastocysts in vitro at normal rates and exhibit typical elongation at day 18 post-transfer without inducing endometrial interferon-stimulated genes. Crucially, these IFNT-null conceptuses successfully prevent luteal regression, establish normal placentation, and progress through full-term gestations, culminating in the birth of healthy female calves. These findings provide the first direct genetic evidence that embryonic IFNT is dispensable for pregnancy recognition and establishment in cattle, fundamentally revising the long-standing model of ruminant reproduction and uncovering alternative embryo-maternal signaling mechanisms.},
}

Animals
Female
Cattle
*Interferon Type I/genetics/metabolism
*Pregnancy Proteins/genetics/metabolism
Pregnancy
Blastocyst/metabolism
Nuclear Transfer Techniques
CRISPR-Cas Systems
Placentation/genetics
Endometrium/metabolism
Embryo, Mammalian/metabolism
Fibroblasts/metabolism
Luteolysis

@article {pmid42844286,
year = {2026},
author = {Willier, S and Färber, J and Ispyrlidou, T and Wilhelm, J and Mayer, E and Stenger, D and Seiferling, J and Brien, AE and Nikolaides, S and Ferrada-Ernst, P and Maile, JD and Vick, B and Carr, A and Blaeschke, F and Busch, DH and Spieler, P and Nerreter, T and Lotfi, R and Rudelius, M and Kaeuferle, T and Klein, C and Escherich, G and Jeremias, I and Burk, U and Feuchtinger, T},
title = {Targeting CD28 on T lineage malignancies with chimeric antigen receptor T cells.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42844286},
issn = {2041-1723},
support = {01EO2103//Bundesministerium für Bildung und Forschung (Federal Ministry of Education and Research)/ ; },
mesh = {Humans ; *CD28 Antigens/genetics/immunology/metabolism ; *Receptors, Chimeric Antigen/immunology/metabolism/genetics ; Animals ; *Immunotherapy, Adoptive/methods ; Mice ; *Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/immunology/therapy/genetics ; Adolescent ; Child ; Female ; Mice, Inbred NOD ; Male ; CRISPR-Cas Systems ; },
abstract = {Currently, no immunotherapy is approved for T cell acute lymphoblastic leukemia (T‑ALL). High initial response rates to CD7‑directed chimeric antigen receptor (CAR) T cells are limited by profound T cell aplasia and CD7‑negative immune escape, underscoring the need for alternative targets. Here, we show that CD28 is overexpressed on T‑ALL blasts from children and adolescents compared with lymphoid progenitors from healthy donors and is uniformly upregulated in nodal T-follicular helper cell lymphomas (nTFHL-AI, nTFHL-F). Using CRISPR/Cas9-mediated CD28 knockout to prevent fratricide, we generate highly functional anti‑CD28 CAR T cells with selective cytotoxicity against CD28[+] T‑ALL. Anti‑CD28 CAR T cells match anti‑CD7 CAR T cells in vitro and in vivo, but cause markedly less lymphodepletion, largely sparing CD8 T cells and NK cells while preferentially depleting CD4 T cells, particularly TH2 and TH17 subsets. These findings establish CD28 as an actionable target for CAR T cell therapy of T cell malignancies.},
}

Humans
*CD28 Antigens/genetics/immunology/metabolism
*Receptors, Chimeric Antigen/immunology/metabolism/genetics
Animals
*Immunotherapy, Adoptive/methods
Mice
*Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/immunology/therapy/genetics
Adolescent
Child
Female
Mice, Inbred NOD
Male
CRISPR-Cas Systems

@article {pmid42844689,
year = {2026},
author = {Wang, C and Liu, J and Liu, C and Liu, C and Cui, J and Wang, J and Mei, X and Song, Z and Li, X},
title = {Hypocotyl acupuncture: an efficient and practical protocol for transformation and genome editing in Salvia miltiorrhiza.},
journal = {The Plant journal : for cell and molecular biology},
volume = {128},
number = {1},
pages = {e71103},
pmid = {42844689},
issn = {1365-313X},
support = {82373991//National Natural Science Foundation of China/ ; 2023TZXD085//The Key R&D Plan Projects in Shandong Province/ ; 2024TZXD056//The Key R&D Plan Projects in Shandong Province/ ; },
mesh = {*Salvia miltiorrhiza/genetics ; *Hypocotyl/genetics ; Transformation, Genetic ; Plants, Genetically Modified/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems ; Plant Roots/genetics ; Genome, Plant/genetics ; },
abstract = {Salvia miltiorrhiza Bunge is a valuable medicinal plant, yet its genetic manipulation has been limited by the lack of efficient and practical transformation methods. Here, we report a simplified, tissue culture-independent strategy enabling Agrobacterium-mediated gene delivery under non-sterile conditions, termed hypocotyl acupuncture. The method employs needle-assisted inoculation of intact seedlings at the hypocotyl, completely bypassing callus induction, regeneration, and repeated subculture. Following inoculation, EGFP-positive hairy roots are selectively cultivated for approximately 4 months and then induced to regenerate shoots within one additional month, yielding transgenic plantlets in approximately 5 months. Systematic optimization achieved a transformation efficiency of approximately 55% (defined as the percentage of explants producing EGFP-positive regenerants). ACCC10060 was selected as the optimal strain based on efficiency and root morphology. CRISPR/Cas9-mediated genome editing generated mutation frequencies of 53.8%-88.9% with no off-target effects. Targeted mutagenesis of SmCPS1, a gene encoding a key rate-limiting enzyme in tanshinone biosynthesis, resulted in altered root pigmentation. The method was successfully extended to Astragalus membranaceus, Platycodon grandiflorus, and Isatis indigotica, achieving transformation efficiencies of 25%-55%. This study provides an efficient, practical, and broadly applicable tool for genome editing and functional genomics in medicinal plants.},
}

*Salvia miltiorrhiza/genetics
*Hypocotyl/genetics
Transformation, Genetic
Plants, Genetically Modified/genetics
*Gene Editing/methods
CRISPR-Cas Systems
Plant Roots/genetics
Genome, Plant/genetics

@article {pmid42846869,
year = {2026},
author = {Hua, ZY and Xia, X and Qaisar, K and Zhong, C and Shehzad, KM and Khan, R},
title = {Antimicrobial peptides in the era of resistance: integrating AI-driven design, host-microbe interactions, and nanotechnological delivery.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1946449},
pmid = {42846869},
issn = {1664-302X},
abstract = {The escalating crisis of antimicrobial resistance (AMR) requires a fundamental transition from traditional antibiotic development to innovative therapeutic strategies rooted in microbial physiology and host defense mechanisms. Antimicrobial peptides (AMPs), evolutionarily conserved effectors of innate immunity, offer a potent alternative due to their rapid, multifaceted modes of action that reduce the likelihood of rapid resistance emergence compared to single-target antibiotics, although bacterial adaptation via membrane remodeling and efflux mechanisms remains a documented challenge. This review synthesizes recent advances in AMP research, focusing on their structural diversity, mechanistic interactions with microbial membranes, and immunomodulatory roles in shaping host-microbe dynamics. We critically examine the transition from empirical isolation to rational design, highlighting the integration of artificial intelligence (AI) and machine learning (ML) algorithms in predicting peptide efficacy and optimizing sequence-activity relationships. Furthermore, we explore the application of CRISPR-Cas technologies in engineering producer organisms and modulating pathogen susceptibility. A significant portion of this review addresses the translational challenges of AMPs, including proteolytic instability and toxicity, by evaluating next-generation nanotechnological delivery systems such as liposomal encapsulation and stimuli-responsive hydrogels. By bridging fundamental microbiological insights with cutting-edge computational and material science innovations, this article establishes a framework for overcoming current limitations. We conclude by outlining future perspectives for integrating AMPs into clinical practice, emphasizing their potential as synergistic agents in combination therapies and their role in extending the clinical utility of existing antimicrobial agents within a One Health context.},
}

@article {pmid42848236,
year = {2026},
author = {Wang, Y and Lan, M and Zhong, M and Jiang, H and Wang, Q and Liu, J and Li, B},
title = {Entropy-driven amplification combined with CRISPR-Cas12a to develop a dual-mode biosensor for breast cancer miR-155 detection.},
journal = {Mikrochimica acta},
volume = {193},
number = {11},
pages = {},
pmid = {42848236},
issn = {1436-5073},
support = {2025XH019//Scientific research and development center of colleges and universities of the Ministry of Education/ ; S20250002//The Project of Medical Association of Sichuan Province/ ; 2025YFHZ0329//Science and Technology Department of Sichuan Province/ ; 2025XH037//Health Commission of Sichuan Province/ ; },
mesh = {Humans ; *MicroRNAs/blood/genetics/analysis ; *Biosensing Techniques/methods ; *Breast Neoplasms/genetics/diagnosis ; *CRISPR-Cas Systems/genetics ; Female ; Entropy ; Colorimetry/methods ; Limit of Detection ; *Nucleic Acid Amplification Techniques/methods ; Biomarkers, Tumor/blood/genetics ; },
abstract = {An entropy-driven amplification (EDA) coupled CRISPR-Cas12a dual-mode biosensor was established for sensitive detection of breast cancer biomarker miR-155. Fluorescent quantitative detection proceeds via synchronous single-tube isothermal incubation without liquid transfer, while colorimetric visual screening requires post-reaction addition of chromogenic substrates. The system eliminates reverse transcription for direct miRNA recognition and realizes cascade dual signal amplification, which greatly accelerates the apparent signal generation rate constant compared with standalone EDA. Under optimized conditions, the fluorescent mode achieves a limit of detection of 660 fM with linear quantification from 60 to 800 pM, and the colorimetric branch provides visual readout with a logarithmic linear range of 4-400 nM. The sensor shows favorable anti-interference performance in diluted healthy human serum spike-recovery tests and can realize intracellular in situ miR-155 fluorescence imaging to distinguish breast cancer cells from normal mammary epithelial cells. We systematically optimized all reaction parameters, supplemented comprehensive specificity verification including mismatched sequences, and supplemented detailed kinetic and analytical characterization to support the reliability of this dual-mode nucleic acid sensing platform.},
}

Humans
*MicroRNAs/blood/genetics/analysis
*Biosensing Techniques/methods
*Breast Neoplasms/genetics/diagnosis
*CRISPR-Cas Systems/genetics
Female
Entropy
Colorimetry/methods
Limit of Detection
*Nucleic Acid Amplification Techniques/methods
Biomarkers, Tumor/blood/genetics

@article {pmid42849638,
year = {2026},
author = {Shareef, Z and Hussain, HI and Aqib, AI and Malik, MSU and Rehman, SU},
title = {Combating Antimicrobial Resistance: Emerging Non-Traditional Therapeutics, Clinical Translation Challenges, and Future Perspectives.},
journal = {Microbial pathogenesis},
volume = {},
number = {},
pages = {108871},
doi = {10.1016/j.micpath.2026.108871},
pmid = {42849638},
issn = {1096-1208},
abstract = {Antimicrobial resistance (AMR) has emerged as one of the most severe global health threats. The rapid expansion of multidrug-resistant (MDR) and extensively drug-resistant (XDR) pathogens compromises the effectiveness of conventional antibiotics. Globally, ESKAPE organisms (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) have led to therapeutic failure, prolonged hospitalization, and increased mortality rates. This review identifies the molecular mechanisms, including enzymatic antibiotic inactivation, efflux pump overexpression, biofilm formation, and horizontal gene transfer, that underline the resistance of disseminated pathogens to antibiotics in clinical, agricultural, and environmental settings. Antibiotic overuse and pharmaceutical and agricultural discharges create persistent reservoirs for the dissemination of AMR genes. Furthermore, we evaluated the non-traditional antibacterial efficacy of antimicrobial peptides, bacteriophage therapy, monoclonal antibodies, probiotics, fecal microbiota transplantation, anti-virulence agents, CRISPR-Cas precision antimicrobials, and nanotechnology-based drug delivery platforms. Current preclinical and clinical evidence indicates the eradication of resistant pathogens, disruption of biofilms, and preservation of host microbiota, thereby reducing the selective pressure associated with broad-spectrum antibiotics. Despite these advances, major translational barriers, such as pharmacokinetic instability, manufacturing limitations, and insufficient clinical validation, restrict the implementation of the framework. In the future, the AMR management framework will require precision medicine, artificial intelligence, and One Health-based integration via multidisciplinary collaboration.},
}

@article {pmid42849713,
year = {2026},
author = {León Agudelo, JA and León Agudelo, DF and Martínez Morales, MC and Vásquez Villamil, VV and Pacheco Ricardo, JD and Enríquez Medina, MP},
title = {Advances in precision medicine for the anterior segment: therapeutic applications using CRISPR-Cas9.},
journal = {Archivos de la Sociedad Espanola de Oftalmologia},
volume = {},
number = {},
pages = {502657},
doi = {10.1016/j.oftale.2026.502657},
pmid = {42849713},
issn = {2173-5794},
abstract = {CRISPR-Cas9 gene editing has emerged as one of the most promising strategies in precision medicine applied to ophthalmology. Although its initial development focused on inherited retinal diseases, recent advances have increasingly targeted anterior segment disorders, including herpetic keratitis, hereditary corneal dystrophies, and genetically associated glaucoma. The ability to modify specific DNA or RNA sequences enables direct intervention in molecular pathways involved in inflammatory, degenerative, and infectious disease progression. This review summarizes the biological basis of CRISPR-Cas systems and discusses the main preclinical and translational findings related to corneal and glaucomatous diseases. Current limitations, including off-target effects, immunogenicity, and challenges associated with molecular delivery into ocular tissues, are also addressed. Available evidence suggests that CRISPR-Cas9 may significantly transform the future management of multiple anterior segment diseases.},
}

@article {pmid42849814,
year = {2026},
author = {Maharajan, T and Ceasar, SA and Thakur, NR and Ahmad, A},
title = {Genome editing of susceptibility and resistance genes for engineering disease-resistant crops: recent advances, challenges and future prospects.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {},
number = {},
pages = {113512},
doi = {10.1016/j.plantsci.2026.113512},
pmid = {42849814},
issn = {1873-2259},
abstract = {Plant diseases caused by bacterial, fungal, oomycete, and viral pathogens remain a major constraint to crop productivity worldwide. Recent advances in genome-editing technologies have enabled precise manipulation of genes involved in plant-pathogen interactions, providing new opportunities for developing disease-resistant crops. In particular, CRISPR/Cas-based systems have facilitated functional dissection and engineering of susceptibility genes, resistance genes, and immune regulatory networks across diverse crop species. In this review, we initially discuss emerging genome-engineering platforms and highlighting their potential for precise disease-resistance engineering. We then summarize current progress in genome-editing strategies for improving resistance against bacterial, fungal, oomycete, and viral pathogens through targeted manipulation of host susceptibility factors, immune receptors, transcription factors, and defense-signaling components, while critically evaluating the limitations and challenges associated with the reported approaches. The review further examines pleiotropic effects and fitness costs associated with susceptibility-gene editing, alongside the functional validation and precision engineering of classical resistance genes and quantitative and multilocus resistance mechanisms. Recent field-validation studies are also discussed in relation to agronomic performance, while ethical, regulatory, and policy considerations governing the adoption of gene-edited crops are addressed. Emerging applications of CRISPR-based transcriptional regulation and de novo domestication are highlighted as complementary strategies for developing disease-resistant crops. Finally, we discuss current challenges and future directions, emphasizing AI-driven target discovery and gRNA design and the transition from single-gene modification toward integrated disease-resistance engineering. Collectively, these advances highlight the potential of genome editing to accelerate the development of durable, agronomically competitive, and precisely engineered disease-resistant crops.},
}

@article {pmid41131153,
year = {2026},
author = {Larsen, BB and Kimchi, O and Dunkley, ORS and Grimm, MS and Siegers, JY and Huang, Y and Vandavasi, VG and Nguyen, LT and Lamb, CH and Aranovich, I and Eggink, D and Meijer, A and Jalal, H and Notterman, DA and Karlsson, EA and Te Velthuis, AJW and Myhrvold, C},
title = {RNA structure modulates Cas13 activity and enables mismatch detection.},
journal = {Nature biotechnology},
volume = {44},
number = {10},
pages = {1684-1692},
pmid = {41131153},
issn = {1546-1696},
support = {R01 AI182281/AI/NIAID NIH HHS/United States ; R01HD103669//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; DGE-2039656//National Science Foundation (NSF)/ ; R01HL149689//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; 206579/17/Z//Wellcome Trust (Wellcome)/ ; T32GM007388//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; T32GM148739//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R21 AI168808/AI/NIAID NIH HHS/United States ; DP2 AI175474/AI/NIAID NIH HHS/United States ; T32 GM007388/GM/NIGMS NIH HHS/United States ; /WT_/Wellcome Trust/United Kingdom ; T32 GM148739/GM/NIGMS NIH HHS/United States ; 75D30122C15113//U.S. Department of Health & Human Services | Centers for Disease Control and Prevention (CDC)/ ; R01 AI170520/AI/NIAID NIH HHS/United States ; R01HD076592//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01 AI182281//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R21 AI168808//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; DP2 AI175474//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01 AI170520//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Humans ; Nucleic Acid Conformation ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; *RNA/chemistry/genetics ; Mutation ; CRISPR-Cas Systems/genetics ; SARS-CoV-2/genetics ; *Base Pair Mismatch ; Proto-Oncogene Proteins p21(ras)/genetics ; COVID-19/virology/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Cas13 is activated by the hybridization of a CRISPR RNA to a complementary single-stranded RNA protospacer in a target RNA. While Cas13 is not activated by double-stranded RNA in vitro, it robustly targets RNA in cellular environments where RNAs are highly structured. The mechanism by which Cas13 targets structured RNAs remains unknown. Here, we systematically probe the effects of secondary structure on Cas13. We find that secondary structure in the protospacer and 3' to it inhibits Cas13 activity and quantitatively explains the former effect through a strand displacement framework. We then harness strand displacement to generate an 'occluded' Cas13, which enhances mismatch discrimination up to 50-fold and enables sequence-agnostic mutation identification at low (<1%) allele frequencies. Using occluded Cas13, we identify human-adaptive mutations in SARS-CoV-2 and human and avian influenza A viruses, as well as oncogenic mutations in KRAS. Our work leverages improved mechanistic understanding of Cas13 to expand the scope of RNA diagnostics and enable structure-informed Cas13 approaches.},
}

Humans
Nucleic Acid Conformation
*CRISPR-Associated Proteins/metabolism/genetics/chemistry
*RNA/chemistry/genetics
Mutation
CRISPR-Cas Systems/genetics
SARS-CoV-2/genetics
*Base Pair Mismatch
Proto-Oncogene Proteins p21(ras)/genetics
COVID-19/virology/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid42835755,
year = {2026},
author = {Kachiyeva, Z and Nazik, N and Shahid, M and Ashraf, R and Ul Hassan, J and Khan, AA and Khan, MH and Mushtaq, H and Saeed, F and Arif, U and Karimov, N and Karimov, A and Gritsenko, D and Bakhsh, A},
title = {Next-generation CRISPR systems and advanced delivery paradigms for precision crop improvement: a comparative case study in sugar beet (Beta vulgaris L.).},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1900057},
pmid = {42835755},
issn = {1664-462X},
abstract = {Novel, climate-resilient, high-yielding crop varieties are urgently needed to address food security concerns for the world's growing population. Genome editing techniques have enabled the development of new crop varieties more cheaply, easily, and quickly than conventional breeding methods. A key concern with transgenic crops is producing transgene-free varieties. Transgene-free crops can be developed using new plant breeding technologies and can play a pivotal role in sustainable agriculture. Several techniques can bypass traditional methods for producing edited plants by using hypercompact nucleases and Leaper technology. This review describes various techniques for producing transgene-free crops using combinations of synthetic biology and advanced genome editing tools, such as genome writers, which insert large DNA payloads, and their roles in improving crop genetics. Further, we discuss how these cutting-edge CRISPR techniques can enable the controlled removal of transgenes. The established protocols for tissue culture remain a major hurdle to producing genome-edited plants. The review also outlines several methods that can be used to bypass this laborious tissue culture step, using the recalcitrant crop sugar beet (Beta vulgaris L.) (~30% of global sugar production), as a case study to classify each strategy as either already demonstrated in this species or still only proposed by analogy to model- and staple-crop work. In this review, we emphasize how these new plant breeding technologies - and their successful applications in model species- serve as a comparative blueprint to speed up the development of climate-smart sugar beet varieties.},
}

@article {pmid42836022,
year = {2026},
author = {Kumar, A and Kumar, M and Kumar, N and Saxena, K and Parveen, R and Singh, D and Singh, SP and Satyendra, and Sultana, R and Sinha, S},
title = {Waterlogging and hypoxia in plant abiotic stress.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1927738},
pmid = {42836022},
issn = {1664-462X},
abstract = {Waterlogging is a major abiotic stress affecting 12-27% of global cultivated land and causing average yield losses of 20-33% in cereals and legumes. This poses a serious threat to food security in flood-prone regions. Soil saturation reduces gas diffusion nearly 10,000-fold compared to air. This rapidly induces hypoxia or anoxia in the root zone within hours to days. This oxygen limitation forces a metabolic shift from efficient aerobic respiration (approximately 36 ATP per glucose) to anaerobic fermentation (approximately 2 ATP per glucose). This shift results in severe energy deficits, cytoplasmic acidosis, and accumulation of toxic metabolites such as ethanol, acetaldehyde, and organic acids. Plants exhibit diverse adaptive responses to flooding stress, including morphological modifications such as aerenchyma formation, adventitious root development, hypertrophied lenticels, and shoot elongation in deepwater rice. These modifications facilitate internal oxygen transport and gas exchange. At the biochemical level, fermentative enzymes including alcohol dehydrogenase and pyruvate decarboxylase are upregulated, while antioxidant defense systems mitigate oxidative damage during reoxygenation. Hormonal crosstalk among ethylene, abscisic acid, auxin, and gibberellins regulates the balance between escape and quiescence strategies. At the molecular level, oxygen sensing is primarily mediated by the N-degron pathway through Group VII Ethylene Response Factors (ERF-VIIs). Plant cysteine oxidases regulate oxygen-dependent degradation under normoxia, while hypoxia stabilizes ERF-VIIs to activate anaerobic gene expression. Nitric oxide further modulates ERF-VII proteolysis and stability. The Sub1A locus in rice exemplifies genetic adaptation, conferring 14-18 days of submergence tolerance and significant yield advantages in flood-prone environments. Despite these advances, major crops such as wheat, maize, and soybean remain highly susceptible, underscoring the need for translating mechanistic insights into field-level solutions. Integrating CRISPR/Cas-based gene editing, genomic selection, multi-omics approaches, and improved agronomic practices offers promising avenues for developing flood-resilient crop varieties under changing climatic conditions.},
}

@article {pmid42836310,
year = {2026},
author = {Haldar, A and Yosef, I and Goren, MG and Globus, R and Yakunina, M and Mahata, T and Qimron, U},
title = {Adaptation-coupled CRISPRi identifies antibiotic-sensitizing genes leading to a novel plasmid selection marker.},
journal = {Nucleic acids research},
volume = {54},
number = {18},
pages = {},
pmid = {42836310},
issn = {1362-4962},
support = {101248381//H2020 European Research Council/ ; 818878//H2020 European Research Council/ ; //ERC/ ; },
mesh = {*Anti-Bacterial Agents/pharmacology ; Escherichia coli/genetics/drug effects ; *Plasmids/genetics ; *Genes, Bacterial ; *Drug Resistance, Bacterial/genetics ; *CRISPR-Cas Systems ; DNA Gyrase/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Salmonella/genetics/drug effects ; Shigella/genetics/drug effects ; },
abstract = {Antibiotic resistance through horizontal gene transfer is a major challenge in microbiology and medicine. We describe a CRISPR-based screening system that couples spacer adaptation with gene repression to identify bacterial genes that sensitize bacteria to antibiotics. We used this screening system in Escherichia coli, on seven different antibiotics to select spacers that conferred resistance to them. The screen identified both known and previously unrecognized genes that modulate antibiotic susceptibility. We defined this collection of genes that sensitize bacteria to antibiotics as the 'sensitasome'. Among the identified targets were the essential genes gyrA and gyrB, whose repression increased resistance to multiple antibiotics. Some sensitization genes exhibited a clear trade-off: their repression conferred resistance to certain antibiotics while increasing susceptibility to others. Sensitasome-targeting spacers were also effective in Shigella and Salmonella, indicating that the resistance mechanism is conserved across species. Finally, we repurposed a selected spacer as a CRISPR-based antibiotic selection marker using the compact Cas12m effector. Together, these findings establish adaptation-coupled CRISPRi as a simple platform for discovering antibiotic sensitization genes and provide an environmentally safer alternative to conventional antibiotic resistance markers.},
}

*Anti-Bacterial Agents/pharmacology
Escherichia coli/genetics/drug effects
*Plasmids/genetics
*Genes, Bacterial
*Drug Resistance, Bacterial/genetics
*CRISPR-Cas Systems
DNA Gyrase/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
Salmonella/genetics/drug effects
Shigella/genetics/drug effects

@article {pmid42836822,
year = {2026},
author = {Malaluan, RPP and Dy, RLV},
title = {Acquisition of novel arrays via horizontal gene transfer rewires CRISPR-mediated defense in Pseudomonas aeruginosa.},
journal = {Journal of bacteriology},
volume = {},
number = {},
pages = {e0033926},
doi = {10.1128/jb.00339-26},
pmid = {42836822},
issn = {1098-5530},
abstract = {UNLABELLED: Clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) systems form the adaptive immunity of prokaryotes, conferring sequence-specific protection against genetic parasites. Here, we functionally characterized the type I-F CRISPR-Cas system of Pseudomonas aeruginosa ATCC 10145 (PA10145), which led us to discover the existence of an isolated CRISPR array unique to this system. Its core CRISPR-Cas system is composed of a cas operon flanked by two divergently organized arrays: CRISPR2 and CRISPR1. Meanwhile, the isolated CRISPR array, CRISPR3, was found ~1.3 million bp away from cas. The cas and three CRISPR arrays together function toward adaptive immunity, eliminating plasmids engineered with protospacer targets. If plasmids possessed an intact protospacer adjacent motif (PAM), hyperactive adaptation was stimulated in all CRISPR arrays of PA10145, whereas minimal to no adaptation was observed when the PAM was mutated. Spacer acquisition via interference-driven adaptation proceeds through strand-biased priming in PA10145. Interestingly, the isolated CRISPR3 and cas-adjacent CRISPR2 have nearly identical leader sequences, with only 3 bp mismatches. Of the 1,196 P. aeruginosa genomes analyzed, all 281 isolated arrays only occur as type I-F with similarly matching leaders to CRISPR2. Integrative conjugative elements and prophages only associate with CRISPR2 and CRISPR3, suggesting that isolated arrays might have originated from recombination events involving CRISPR2 as facilitated by these highly transmissible mobile genetic elements. Tracing evolutionary trajectories of the isolated CRISPR3 relative to cas-adjacent arrays revealed that CRISPR3 is horizontally transferred across P. aeruginosa genomes. Taken together, these results implicate the role of horizontally acquired isolated arrays in CRISPR-mediated pan-immunity as gateways to mobilize genetic memories.

IMPORTANCE: Clustered regularly interspaced short palindromic repeat (CRISPR) arrays are typically located adjacent to cas genes, constituting the adaptive immune system. However, a substantial subset of CRISPR arrays, called isolated arrays, is found distantly located from their cognate cas operons. Despite their prevalence, the functional significance and evolutionary implications of isolated arrays have remained poorly understood. Here, we show that isolated arrays are active components of type I-F CRISPR-Cas systems in Pseudomonas aeruginosa. These arrays are continuously updated through hyperactive strand-biased primed adaptation and are transferred horizontally across P. aeruginosa, acting as conduits of immunological memory and proponents of CRISPR-mediated pan-immunity. We posit that bacterial populations mobilize these isolated arrays to maintain transferable memory in the pangenome while remaining compatible with ongoing horizontal gene transfer.},
}

@article {pmid42836916,
year = {2026},
author = {Kisa, O},
title = {Current laboratory diagnostics for tuberculosis: from conventional approaches to next-generation molecular and immunological technologies.},
journal = {European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology},
volume = {},
number = {},
pages = {},
pmid = {42836916},
issn = {1435-4373},
abstract = {Tuberculosis (TB) remains a major global public health concern despite advances in diagnosis and treatment. Early and accurate diagnosis is essential for timely treatment initiation, interruption of transmission, and effective management of drug-resistant TB.Although microscopy and culture remain the cornerstones of TB diagnosis, their limited sensitivity and long turnaround times have driven the integration of molecular and immunological methods.This review summarizes current TB diagnostic approaches, including conventional methods, nucleic acid amplification tests (NAATs), line probe assay (LPA), next-generation sequencing (NGS), point-of-care systems, and interferon-gamma release assays (IGRAs).Emerging technologies, including host-response biomarkers, multi-omics approaches, artificial intelligence-based imaging, nanotechnology-based systems, and CRISPR/Cas-based diagnostics, are also discussed, with emphasis on their diagnostic performance, clinical applications, and current limitations.Current evidence indicates a shift from pathogen detection alone toward integrated approaches incorporating rapid molecular diagnosis, drug-resistance detection, host-response profiling, and immunological assessment.Future TB diagnostics are likely to increasingly rely on multimodal platforms combining molecular tools, host-response biomarkers, multi-omics data, and artificial intelligence to support more precise and clinically actionable TB management.},
}

@article {pmid42838668,
year = {2026},
author = {Wang, X and Chang, Y and Zhang, Y and Yu, X and Zou, Y and Liu, Z and Qi, F and Ming, J},
title = {Proximity-inducible CRISPR/Cas13a trans-cleavage activity and hydrogel-assisted paper-based distance sensor for the detection of aflatoxin M1.},
journal = {Analytica chimica acta},
volume = {1423},
number = {},
pages = {346156},
doi = {10.1016/j.aca.2026.346156},
pmid = {42838668},
issn = {1873-4324},
mesh = {*Aflatoxin M1/analysis ; *Paper ; *CRISPR-Cas Systems/genetics ; *Hydrogels/chemistry ; *Biosensing Techniques/methods ; DNA, Single-Stranded/chemistry ; Animals ; Aptamers, Nucleotide/chemistry ; Limit of Detection ; },
abstract = {Accurate and rapid determination of aflatoxin M1 (AFM1) in food is crucial for ensuring the safety of the human diet. In view of this, we proposed a proximity-inducible Cas13a activity and hydrogel-assisted paper-based distance sensor for the detection of aflatoxin M1. The single-stranded DNA (ssDNA) and AFM1 aptamer were split into two parts and linked to the terminus of two separate DNA activator strands, respectively. By introducing AFM1, it stimulated these two DNA activator strands to assemble with CRISPR RNA (crRNA) through spatial proximity mechanism between the target and these two DNA activators to activate the Cas13a trans-cleavage towards the RNA-containing site within hairpin probe to release a DNA initiator. The released DNA initiator triggered the catalytic hairpin assembly (CHA) between magnetic beads (MBs)-DNA capture probe and trypsin-DNA detection probe. After hydrolysis of gelatin hydrogel by trypsin, water released from the hydrogel flowed along the pH indicator strip and exhibited a distinct mark. Depending on this portable method, the limit of detection (LOD) was achieved at 23.6 ng L[-1]. Furthermore, our developed strategy can be applied to measure AFM1 in ultra-high temperature (UHT) milk with satisfactory effects, and its excellent specificity and reproducibility also demonstrate that our platform has great potential for application in environmental monitoring.},
}

*Aflatoxin M1/analysis
*Paper
*CRISPR-Cas Systems/genetics
*Hydrogels/chemistry
*Biosensing Techniques/methods
DNA, Single-Stranded/chemistry
Animals
Aptamers, Nucleotide/chemistry
Limit of Detection

@article {pmid42838679,
year = {2026},
author = {Chen, F and Tang, M and Liu, Z and Li, W and Hu, X and Xiang, L and Jiang, L and Ban, Y and Wang, J and Li, J and Cheng, W and Yang, T},
title = {A proximity-allosteric Adapter unlocks Cas12a exponential amplification for versatile homogeneous assay.},
journal = {Analytica chimica acta},
volume = {1423},
number = {},
pages = {346180},
doi = {10.1016/j.aca.2026.346180},
pmid = {42838679},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *Bacterial Proteins/metabolism/genetics/chemistry ; *Endodeoxyribonucleases/metabolism/genetics/chemistry ; Allosteric Regulation ; },
abstract = {BACKGROUND: The integration of proximity-triggered homogeneous assays with CRISPR/Cas12a offers a promising route to "mix-and-read" bioanalysis, yet early efforts have faced substantial limitations. Proximity-based recognition enabled high specificity, but often introduced steric hindrance that severely suppressed Cas12a activation. Meanwhile, conventional Cas12a systems relied on linear trans-cleavage, which struggled to achieve the aM-level sensitivity required for early disease detection. It is clear that a CRISPR/Cas12a-based assay is needed that overcomes steric hindrance, achieves exponential signal amplification, and maintains the simplicity of homogeneous detection.

RESULTS: Herein, we report a dual-functional "Smart Adapter" that seamlessly couples proximity recognition with Cas12a exponential amplification. The Smart Adapter operates as a kinetically trapped molecular cage that effectively sequesters the Cas12a activator to ensure minimal background. Target binding induces a proximity-driven allostery that unlocks this cage, liberating the activator from steric constraints to initiate Cas12a activity. This primary activation serves as a catalytic seed, triggering a cascading destabilization of the remaining Adapters. By acting as both an allosteric activator and a signal propagator, the Adapter ensures high-specificity transduction and ultrasensitive amplification. The resulting Smart Proximity-Activated Cas12a Exponential Amplification (SPACE) platform achieves a detection limit (LOD) as low as 500 aM for the BCR/ABL p210 gene and enables precise discrimination of p210 minor subtypes in clinical samples. Furthermore, the platform attains a detection sensitivity of 100 fg/mL for p-Tau217 protein.

SIGNIFICANCE: This versatile strategy provides a robust framework for precise low-abundance biomarker detection, resolving CRISPR/Cas12a bottlenecks and offering a reliable mix-and-read platform for early diagnosis.},
}

*CRISPR-Cas Systems/genetics
*CRISPR-Associated Proteins/metabolism/genetics/chemistry
*Nucleic Acid Amplification Techniques/methods
Humans
*Bacterial Proteins/metabolism/genetics/chemistry
*Endodeoxyribonucleases/metabolism/genetics/chemistry
Allosteric Regulation

@article {pmid42838687,
year = {2026},
author = {Xie, C and Lv, H and Wang, Y and Abdukayum, A and Li, X and Wu, P},
title = {Highly sensitive detection of exosomal miR-122 with CRISPR-Dx for precise HCC diagnosis.},
journal = {Analytica chimica acta},
volume = {1423},
number = {},
pages = {346190},
doi = {10.1016/j.aca.2026.346190},
pmid = {42838687},
issn = {1873-4324},
mesh = {*MicroRNAs/genetics/blood ; Humans ; *Carcinoma, Hepatocellular/diagnosis/genetics ; *Liver Neoplasms/diagnosis/genetics ; *Exosomes/genetics/metabolism/chemistry ; Biomarkers, Tumor/genetics ; *CRISPR-Cas Systems/genetics ; },
abstract = {Hepatocellular carcinoma (HCC), one of the most common malignancies worldwide, poses a major threat to the global health. Its prognosis depends heavily on early detection. Currently, alpha-fetoprotein (AFP) is the widely used biomarker for HCC diagnosis. Nevertheless, approximately 40% of the HCC cases do not produce AFP. It has been reported that the downregulation of miR-122 is strongly associated with the development and progression of HCC. Here, employing miR-122 as a biomarker, we developed a cascade amplification-based CRISPR-Dx platform for HCC diagnosis. Owing to the strong trans-cleavage activities of Cas13a and Cas12a, the proposed assay enabled sensitive miR-122 detection down to 1 aM. Clinical sample analysis (n = 48) demonstrated a sensitivity of 91.3%, a specificity of 84.0%, and an overall accuracy of 87.5%, which is significantly better than that of the conventional AFP-based diagnosis (sensitivity, 43.5%; specificity, 80%; accuracy, 62.5%). In addition, because CRISPR-Dx eliminates the RNA extraction and reverse transcription steps that may cause target loss, it also showed better performance than miR-122-targeted qPCR. Given its high diagnostic accuracy and simplified workflow, the proposed miR-122-based CRISPR-Dx assay holds great promise for the clinical diagnosis of HCC.},
}

*MicroRNAs/genetics/blood
Humans
*Carcinoma, Hepatocellular/diagnosis/genetics
*Liver Neoplasms/diagnosis/genetics
*Exosomes/genetics/metabolism/chemistry
Biomarkers, Tumor/genetics
*CRISPR-Cas Systems/genetics

@article {pmid42838688,
year = {2026},
author = {Bi, M and Li, A and Jin, Y and Gao, Y and Li, M and Li, C},
title = {Highly efficient RPA product degradation and expanded PAM recognition enable RPA-Cas3 detection of Clonorchis sinensis.},
journal = {Analytica chimica acta},
volume = {1423},
number = {},
pages = {346191},
doi = {10.1016/j.aca.2026.346191},
pmid = {42838688},
issn = {1873-4324},
mesh = {*Clonorchis sinensis/isolation & purification/genetics ; Animals ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Temperature ; },
abstract = {Clonorchis sinensis is a major foodborne pathogen threatening aquatic food safety. Microscopy, the diagnostic standard, is laborious and often lacks sensitivity for low-intensity infections. Molecular strategies coupling isothermal amplification with CRISPR/Cas systems have attracted interest, but their reliability is often undermined by competition between amplification and detection and intrinsic limitations of Cas effectors, notably PAM dependence of Cas12a and nonspecific activation of Cas13a. To address these limitations, we used the type I-A CRISPR-Cas3 system (TsiCas3) from T. siculi. Because TsiCas3 displays trans-cleavage activity at 65 °C, a temperature that completely abolishes RPA polymerase activity, this temperature gap can be exploited to avoid interference. A one-tube, closed-reaction workflow was established where target DNA is first amplified by RPA at 37 °C for 20 min, then the temperature is raised to 65 °C. This shift simultaneously inactivates RPA polymerase and activates TsiCas3-Cascade, enabling specific amplicon recognition, trans-ssDNA cleavage, and fluorescence generation. The sequential temperature control physically separates amplification from detection, eliminates mutual interference, and requires no tube opening or reagent addition. Moreover, Cas3 efficiently and near-completely degrades the RPA amplicons, substantially reducing aerosol carryover risk. This closed-tube, contamination-resistant method provides a rapid, sensitive, field-deployable tool for C. sinensis detection and represents a practical addition to clonorchiasis control.},
}

*Clonorchis sinensis/isolation & purification/genetics
Animals
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
Temperature

@article {pmid42371232,
year = {2026},
author = {Huang, J and Chen, J and Huang, L and Qiu, S and Wang, L and Du, X and Wang, C and Yuan, Z and Li, P and Song, H and Liu, H},
title = {Sensitizing carbapenem-resistant Klebsiella pneumoniae using a compact Cascade-Cas3 system.},
journal = {International microbiology : the official journal of the Spanish Society for Microbiology},
volume = {29},
number = {7},
pages = {1411-1419},
pmid = {42371232},
issn = {1618-1905},
support = {32300080//National Science Foundation of China/ ; 32141003//National Science Foundation of China/ ; 2021YFC2301102//National Science and Technology Major Project of China/ ; },
mesh = {*Klebsiella pneumoniae/drug effects/genetics ; *Anti-Bacterial Agents/pharmacology ; Plasmids/genetics ; *Carbapenems/pharmacology ; Microbial Sensitivity Tests ; *CRISPR-Cas Systems ; Klebsiella Infections/microbiology ; Bacterial Proteins/genetics/metabolism ; *Carbapenem-Resistant Enterobacteriaceae/genetics/drug effects ; beta-Lactamases/genetics ; },
abstract = {BACKGROUND: Klebsiella pneumoniae is a 'superbug' characterized by high virulence and carbapenem resistance. CRISPR-Cas3 is capable of performing large-scale deletions in bacterial genomes. The purpose of this study is to sensitize Klebsiella pneumoniae A324 to carbapenem antibiotics by eliminating the multidrug resistance plasmid pA324-IMP using a compact Cascade-Cas3 system.

METHODS: Recombinant plasmids were constructed using molecular cloning techniques. Three sgRNAs targeting different positions within the blaIMP-38 gene were designed. Recombinant plasmids were separately transformed into host bacteria carrying blaIMP38 genes. The elimination efficiency of each CRISPR-Cas3 system target was determined by PCR and qPCR. Fluorescence confocal microscopy was used to observe the fluorescence intensity. Bacterial drug susceptibility phenotypes were determined using E-test drug sensitivity strips.

RESULTS: In this study, we successfully constructed the high copy number plasmid, the fluorescent plasmid, and the recombinant CRISPR plasmids. PCR and qPCR results demonstrated that the CRISPR-Cas3 system could efficiently eliminate blaIMP-38 genes, with pCas3-T3 showing the highest elimination efficiency. Fluorescence confocal microscopy indicated that the fluorescence intensity of the experimental group significantly decreased compared to the control group. Additionally, drug susceptibility tests revealed that the resistance of Klebsiella pneumoniae to imipenem and meropenem was significantly reduced. Furthermore, CRISPR-Cas3 could enhance the effectiveness of meropenem in killing Klebsiella pneumoniae A324.

CONCLUSION: In conclusion, this work demonstrates that the compact Cascade-Cas3 system can serve as a novel tool to eliminate the resistance gene blaIMP-38 and restore the sensitivity of host bacteria to antibiotics. This strategy shows great potential to combat the spread of the blaIMP-38 gene in Klebsiella pneumoniae and holds significant application value in the field of combating drug-resistant bacteria.},
}

*Klebsiella pneumoniae/drug effects/genetics
*Anti-Bacterial Agents/pharmacology
Plasmids/genetics
*Carbapenems/pharmacology
Microbial Sensitivity Tests
*CRISPR-Cas Systems
Klebsiella Infections/microbiology
Bacterial Proteins/genetics/metabolism
*Carbapenem-Resistant Enterobacteriaceae/genetics/drug effects
beta-Lactamases/genetics

@article {pmid42727495,
year = {2027},
author = {Chai, L and Cui, X and Xu, Q and Li, C and Xie, Y and Tong, K and Le, T},
title = {CRISPR/Cas14a-SNPT: An engineered sgRNA mismatch-enabled platform for ultrasensitive SNP typing in hereditary G6PD deficiency.},
journal = {Biosensors & bioelectronics},
volume = {315},
number = {},
pages = {119217},
doi = {10.1016/j.bios.2026.119217},
pmid = {42727495},
issn = {1873-4235},
mesh = {Humans ; *Polymorphism, Single Nucleotide/genetics ; *CRISPR-Cas Systems/genetics ; *Glucosephosphate Dehydrogenase/genetics ; *Biosensing Techniques/methods ; Genotyping Techniques/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Nucleic Acid Amplification Techniques/methods ; Gene Frequency ; Base Pair Mismatch ; },
abstract = {Single-nucleotide polymorphism (SNP) detection plays a critical role in early screening and genotyping of genetic diseases. In this study, hereditary glucose-6-phosphate dehydrogenase (G6PD) deficiency was used as a model to develop a CRISPR/Cas14a-based SNP genotyping platform (CRISPR/Cas14a-SNPT). The platform integrates recombinase polymerase amplification (RPA) signal amplification with optimized sgRNA design. By introducing a shortened optimized sgRNA (op-sgRNA) to enhance Cas14a activity, together with engineered sgRNA mismatch regulation, the system enables rapid, highly sensitive, and high-resolution SNP detection. The results demonstrated a limit of detection of 1 copy/μL for plasmid templates, and reliable discrimination of variant allele frequencies as low as 0.1%. In clinical validation, the method successfully identified three hotspot mutations (c.95A > G, c.1388G > A, and c.1376G > T) in 13 human blood samples, showing excellent agreement with pyrosequencing results. Furthermore, molecular dynamics simulations revealed the regulatory mechanism of Cas14a mismatch recognition at the molecular level, suggesting that additional mismatches induce duplex relaxation and structural compensation effects, thereby enhancing target discrimination specificity. This study provides an efficient and practical strategy for accurate genotyping and prenatal carrier screening of G6PD deficiency-associated SNPs, demonstrating strong potential for rapid detection of clinically relevant SNPs.},
}

Humans
*Polymorphism, Single Nucleotide/genetics
*CRISPR-Cas Systems/genetics
*Glucosephosphate Dehydrogenase/genetics
*Biosensing Techniques/methods
Genotyping Techniques/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Nucleic Acid Amplification Techniques/methods
Gene Frequency
Base Pair Mismatch

@article {pmid42731312,
year = {2027},
author = {Lu, TC and Tian, HY and Zuo, SY and Lin, CH and Lei, Y and Liu, AL and Li, X},
title = {Thermal-enhanced atypical suboptimal PAM-dependent CRISPR/Cas12a amplification for detecting BCR1-Type PML::RARα fusion and L217F mutation.},
journal = {Biosensors & bioelectronics},
volume = {315},
number = {},
pages = {119213},
doi = {10.1016/j.bios.2026.119213},
pmid = {42731312},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Leukemia, Promyelocytic, Acute/genetics/diagnosis ; Mutation ; *Oncogene Proteins, Fusion/genetics ; *Biosensing Techniques/methods ; *Promyelocytic Leukemia Protein/genetics ; Limit of Detection ; },
abstract = {Current clinical methods for detecting low-abundance PML::RARα fusion and drug-resistant single-base mutations face significant challenges, highlighting the urgent need for a rapid, sensitive, cost-effective genetic detection method for diagnosing acute promyelocytic leukemia. The CRISPR/Cas12a system has emerged as a revolutionary tool in nucleic acid detection. However, its applications are frequently constrained by the requirement for protospacer adjacent motifs (PAMs). In this study, we demonstrated that a moderate temperature increase enhanced CRISPR/Cas12a activity while allowing the use of atypical suboptimal PAMs. Utilizing a PCR-driven, suboptimal PAM-dependent CRISPR/Cas12a cascade, we sensitively detected the BCR1-type PML::RARα fusion with a detection limit of 0.1 aM. This method effectively identified low-abundance mutant cells in the presence of wild-type cells and demonstrated its efficacy in detecting minimal residual disease samples from patients with relapsed acute promyelocytic leukemia. Additionally, we developed a sensitive approach for detecting L217F single-base mutations using a cascade strategy of polymerase chain reaction, ligase chain reaction and CRISPR/Cas12a, achieving a detection limit of 10 aM in various wild-type DNA samples. Consequently, these methods hold considerable promise for the rapid diagnosis of acute promyelocytic leukemia, the sensitive detection of and the low-abundance drug-resistance nucleic acid mutations.},
}

*CRISPR-Cas Systems/genetics
Humans
*Leukemia, Promyelocytic, Acute/genetics/diagnosis
Mutation
*Oncogene Proteins, Fusion/genetics
*Biosensing Techniques/methods
*Promyelocytic Leukemia Protein/genetics
Limit of Detection

@article {pmid42753299,
year = {2027},
author = {Xiao, S and Deng, Q and Wan, Q and Huang, N and Jiang, J and Luo, S and Xia, Y and Liu, Y and Zhang, D and Jiang, G},
title = {Engineering a leakage-resistant CHA-CRISPR/Cas12a biosensor via cis-cleavage-triggered split activator assembly for ultrasensitive miRNA detection.},
journal = {Biosensors & bioelectronics},
volume = {315},
number = {},
pages = {119234},
doi = {10.1016/j.bios.2026.119234},
pmid = {42753299},
issn = {1873-4235},
mesh = {*MicroRNAs/genetics/isolation & purification/blood/analysis ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; CRISPR-Associated Proteins/chemistry/genetics ; Limit of Detection ; Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {MicroRNAs (miRNAs) have emerged as important biomarkers for cancer diagnosis and prognosis, yet their accurate detection remains challenging due to low abundance, short sequences, and sequence homology. Herein, we report a novel biosensing platform based on a cascade cis-cleavage-triggered split activator assembly strategy by integrating catalytic hairpin assembly (CHA) with dual CRISPR/Cas12a amplification for ultrasensitive and selective miRNA detection. In contrast to conventional CHA-CRISPR assays that directly couple CHA products with Cas12a activation and suffer from crRNA-induced leakage and elevated background signals, the proposed system decouples target recognition from signal output through a split activator architecture. Target miRNA initiates CHA to generate a duplex intermediate, which is first processed by Cas12a/crRNA1 via cis-cleavage to release a fragmentary DNA trigger. This fragment subsequently assembles with a predesigned auxiliary strand to form a complete activator, enabling secondary activation of Cas12a/crRNA2 and leading to amplified trans-cleavage signals. This cascade design significantly suppresses nonspecific background while enabling sequential signal amplification, resulting in substantially improved signal-to-noise ratios and enhanced analytical performance. The developed biosensor achieves sensitive miRNA detection with a low detection limit and excellent specificity. Furthermore, the practical applicability of the platform was demonstrated in cell lysates and human plasma samples with satisfactory accuracy and recovery. Owing to its modular and programmable design, this split activator-mediated CHA-CRISPR strategy provides a general and versatile framework for constructing low-background CRISPR biosensors and may facilitate the development of sensitive miRNA sensing platforms.},
}

*MicroRNAs/genetics/isolation & purification/blood/analysis
*Biosensing Techniques/methods
Humans
*CRISPR-Cas Systems/genetics
CRISPR-Associated Proteins/chemistry/genetics
Limit of Detection
Nucleic Acid Amplification Techniques/methods
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid42790075,
year = {2027},
author = {Chang, K and Sun, J and Liu, T and Bao, Q and Huang, H and Wang, JS and Zhang, Y and Lu, K and Sun, X},
title = {Photoactivatable NPOM-caged split-activator CRISPR/Cas12a biosensor for amplification-free imaging of deoxynivalenol-induced miR-155 dynamics and lateral-flow sensing.},
journal = {Biosensors & bioelectronics},
volume = {315},
number = {},
pages = {119258},
doi = {10.1016/j.bios.2026.119258},
pmid = {42790075},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *MicroRNAs/blood/genetics/analysis ; Animals ; Mice ; *Trichothecenes/toxicity ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; Macrophages/drug effects ; Humans ; },
abstract = {MicroRNA-155 (miR-155) is an immune-associated small RNA biomarker involved in macrophage responses to environmental stressors such as deoxynivalenol (DON). However, achieving amplification-free miRNA sensing with low background and precise temporal regulation remains challenging. Here, we develop a photoactivatable split-activator CRISPR/Cas12a biosensor for miR-155 detection without target nucleic acid amplification and for dynamic analysis of toxin-induced cellular responses. The sensing system integrates an NPOM-caged crRNA with a split-complementary activator, enabling optical control of Cas12a activation. Upon brief 365 nm irradiation, miR-155 and ssDNA jointly reconstitute an activation-competent Cas12a complex, enabling target-dependent reporter cleavage after optical triggering. After systematic optimization, the fluorescence biosensor achieved quantitative miR-155 detection over a range of 1 pM-5 nM with an LOD of 0.69 pM. In mouse serum, the LOD was 1.27 pM, with relative matrix responses of 83.4%-92.3% and recoveries of 103.5%-106.2%. For DON-stimulated ANA-1 macrophages, the biosensor successfully resolved dose- and time-dependent miR-155 dynamics through live-cell fluorescence imaging and cell-lysate analysis. The time-course CRISPR signals showed excellent agreement with RT-qPCR measurements (Pearson r = 0.996, R[2] = 0.992). Orthogonal validation using miR-155 inhibition, NPOM/UV controls, component-deletion experiments, and flow cytometry further supported intracellular target responsiveness and sensing reliability. Furthermore, the same architecture was adapted to a lateral-flow assay with an LOD of 11.99 pM and applied as a portable visual/semi-quantitative readout for cell-derived and mouse serum samples. Therefore, this work establishes a programmable light-gated Cas12a biosensing platform for amplification-free miRNA quantification, live-cell molecular imaging, and portable analysis of toxin-induced immune responses.},
}

*Biosensing Techniques/methods
*MicroRNAs/blood/genetics/analysis
Animals
Mice
*Trichothecenes/toxicity
*CRISPR-Cas Systems/genetics
Limit of Detection
Macrophages/drug effects
Humans

@article {pmid42804994,
year = {2027},
author = {Hu, Y and Liao, Y and Wang, X and Yuan, T and Qiao, L and Luo, Y and Pan, S and Li, X and Zhu, F},
title = {A CRISPR/Cas14a-based platform for sensitive and specific single-nucleotide variant detection via short single-strand DNA amplification and split target design.},
journal = {Biosensors & bioelectronics},
volume = {315},
number = {},
pages = {119263},
doi = {10.1016/j.bios.2026.119263},
pmid = {42804994},
issn = {1873-4235},
mesh = {Humans ; *Polymorphism, Single Nucleotide/genetics ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *DNA, Single-Stranded/genetics/chemistry ; *Biosensing Techniques/methods ; Limit of Detection ; *Neoplasms/genetics/diagnosis ; },
abstract = {Single-nucleotide variant (SNV) detection, including tumor-associated mutation detection and pharmacogenetic genotyping, holds significant clinical value. Among current methods, CRISPR/Cas14a-based approaches offer advantages in specificity, sensitivity, and efficiency. However, the inherent single-stranded DNA (ssDNA) targeting property of Cas14a limits its direct application to genomic DNA detection, and its specificity requires further optimization to meet clinical standards. Here, we developed a CRISPR/Cas14a system based on short ssDNA amplification and split target design (SAST), which substantially enhances specificity through split target design while achieving high sensitivity via short ssDNA amplification. Experimental results demonstrate that SAST system achieves a Concentration LOD (limit of detection) of 100 aM for target DNA and a MAF (mutant allele frequency) LOD of 0.00001% in simulated samples. The platform was successfully validated for SNV detection in tumor samples (KRAS G12D, rs121913529, c.35G > A) and pharmacogenetic genotyping (CYP2C19*2, rs4244285, c.681G > A; CYP2C19*3, rs4986893, c.636G > A; and CYP2C19*17, rs12248560, c.806C > T). SAST system offers a novel method for clinical single-base mutation detection and is expected to find broad applications in early cancer diagnosis and precision medicine.},
}

Humans
*Polymorphism, Single Nucleotide/genetics
*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
*DNA, Single-Stranded/genetics/chemistry
*Biosensing Techniques/methods
Limit of Detection
*Neoplasms/genetics/diagnosis

@article {pmid42834039,
year = {2026},
author = {Aleksandrovic, E and Fross, SR and Golomb, SM and Ma, W and Liu, X and Zhao, Z and Das, NM and Reese, TC and Borges, HM and Tenekeci, A and Yu, L and Lopez, J and Zhao, M and Zhong, Z and Dean, KM and Lea, J and Xu, L and Stack, MS and Zhang, S},
title = {Tumor-intrinsic IFNγ signaling and niche adaptation drive early colonization in ovarian cancer metastasis.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42834039},
issn = {2041-1723},
support = {RP230261//Cancer Prevention and Research Institute of Texas (Cancer Prevention Research Institute of Texas)/ ; U54 CA268072/CA/NCI NIH HHS/United States ; U54CA268072//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R35 GM142654/GM/NIGMS NIH HHS/United States ; R21CA263798-01//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; T32GM075762//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; RM1GM145399//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01CA255064-01A1//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01 CA255064/CA/NCI NIH HHS/United States ; T32 GM075762/GM/NIGMS NIH HHS/United States ; RP250571//Cancer Prevention and Research Institute of Texas (Cancer Prevention Research Institute of Texas)/ ; RR220024//Cancer Prevention and Research Institute of Texas (Cancer Prevention Research Institute of Texas)/ ; R35GM142654//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; RM1 GM145399/GM/NIGMS NIH HHS/United States ; R21 CA263798/CA/NCI NIH HHS/United States ; },
mesh = {Female ; *Ovarian Neoplasms/pathology/genetics/metabolism ; *Interferon-gamma/metabolism/genetics ; Animals ; Signal Transduction ; Humans ; Cell Line, Tumor ; Mice ; Receptors, Interferon/genetics/metabolism ; Interferon gamma Receptor ; Neoplasm Metastasis ; Macrophages, Peritoneal/metabolism ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; Single-Cell Analysis ; Gene Expression Profiling ; Anoikis ; *Peritoneal Neoplasms/secondary/genetics ; Single-Cell Gene Expression Analysis ; },
abstract = {The mechanisms enabling disseminating tumor cells to survive and colonize peritoneal niches during ovarian cancer metastasis remain incompletely understood. Here, we present MetTag, a single-cell barcoding and transcriptome profiling approach with time-stamped batch identifiers to resolve temporal dynamics of dissemination. Sequencing of MetTag barcodes reveals enrichment of early-disseminated clones across metastatic sites, and targeted depletion of pioneers diminishes the outgrowth of later arriving cells. MetTag-coupled single-cell RNA sequencing uncovers a distinct interferon-gamma-centric transcriptional trajectory enriched among pioneer clones. CRISPR/Cas9 screening and subsequent interferon-gamma receptor 1 knockout in pioneer cells significantly reduces metastatic burden, highlighting a critical window where active interferon-gamma signaling shapes the post-seeding metastatic niche and outgrowth. Mechanistically, the tumor-intrinsic interferon-gamma response and peritoneal macrophages cooperatively shield disseminating tumor cells from anoikis. Our study defines the temporal clonal architecture of peritoneal metastasis, revealing a first come, first served dissemination principle, where pioneer fitness determines the success of subsequent colonizers.},
}

Female
*Ovarian Neoplasms/pathology/genetics/metabolism
*Interferon-gamma/metabolism/genetics
Animals
Signal Transduction
Humans
Cell Line, Tumor
Mice
Receptors, Interferon/genetics/metabolism
Interferon gamma Receptor
Neoplasm Metastasis
Macrophages, Peritoneal/metabolism
Gene Expression Regulation, Neoplastic
CRISPR-Cas Systems
Single-Cell Analysis
Gene Expression Profiling
Anoikis
*Peritoneal Neoplasms/secondary/genetics
Single-Cell Gene Expression Analysis

@article {pmid42834671,
year = {2026},
author = {Zhao, W and Li, X and Hu, Z and Wang, S},
title = {CRISPR-Cas Tools for Dissecting and Engineering Environmental Responses in Microalgae.},
journal = {Plant, cell & environment},
volume = {},
number = {},
pages = {},
doi = {10.1111/pce.70903},
pmid = {42834671},
issn = {1365-3040},
support = {GuikeAA24263042//Science and Technology Major Project of Guangxi/ ; 42406122//National Natural Science Foundation of China/ ; 32273118//National Natural Science Foundation of China/ ; 2023A1515110870//Guangdong Basic and Applied Basic Research Foundation/ ; 2242025RCB0034//Zhishan Young Scholar Program/ ; GPKLPE202416//Guangdong Provincial Key Laboratory for Plant Epigenetics/ ; 4031002405//Southeast University/ ; 2020B121202014//Science and Technology Program of Guangdong Province of China/ ; 2022B1111070005//Guangdong Key R&D Project/ ; CJGJZD2025090291534011//Shenzhen Science and Technology Program/ ; 2022B010//Shenzhen University 2035 Initiative/ ; },
abstract = {Microalgae are photosynthetic eukaryotes that need to maintain productivity under variable light, temperature, nutrient and CO2 conditions, yet their complex regulatory networks have made relevant engineering difficult. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins (Cas) have changed the situation, shifting microalgal research from random transgene integration toward precise editing of the genes that mediate photosynthesis, stress responses and environmental acclimation. This work reviews how CRISPR-Cas has been applied across microalgal lineages to (i) dissect genes involved in stress response and photosynthesis, (ii) streamline genomes through large-scale deletions, (iii) build stress-tolerant chassis that retain photosynthetic performance under unsatisfactory conditions and (iv) reprogramme lipid, pigment and high-value compound pathways. We then discuss the determinants of editing efficiency, including single-guide RNA design, Cas nuclease choice and engineering, species-specific delivery, host physiology (cell-wall architecture, cell cycle phase and stress-induced repair-pathway shifts) as well as screening workflows using endogenous markers and high-throughput phenotyping. We propose that integrating species-specific multi-omics analyses, artificial intelligence-assisted design and precise and controllable editing strategies will further enhance the ability of CRISPR-Cas systems to elucidate photosynthetic regulatory networks and environmental response mechanisms in microalgae, while facilitating the optimisation of microalgal metabolic traits and the engineering of environmental adaptability.},
}

@article {pmid42835670,
year = {2026},
author = {Rachamala, HK and Atla, S and Angom, RS},
title = {Targeted delivery platforms for in vivo immune cell engineering: emerging strategies for cancer immunotherapy.},
journal = {Frontiers in pharmacology},
volume = {17},
number = {},
pages = {1936688},
pmid = {42835670},
issn = {1663-9812},
abstract = {Cancer immunotherapy is a transformative technology allowing for the elimination of malignant cells through immune system stimulation; however, the large-scale clinical application of adoptive cellular therapy technologies is limited due to difficulties in ex vivo immune cell engineering, high costs, and complicated manufacturing process. Recently, in vivo immune cell engineering was discovered as an emerging approach that allows for the genetic reprogramming of immune cells in patients, thus circumventing many problems inherent to traditional cellular therapy technologies. Breakthroughs in targeted drug delivery systems such as cell-penetrating peptides, antibody- and receptor-mediated delivery approaches, lipid nanoparticles (LNPs), polymeric drug carriers, and biomimetic delivery systems allow for the more effective selective delivery of messenger RNA (mRNA), clustered regularly interspaced short palindromic repeats (CRISPR)/Cas gene editing systems, proteins, and other types of cargo to the target immune cells. In vivo immune cell engineering allows for the efficient generation of chimeric antigen receptor T-cells (CAR-T), CAR- natural killer (NK) cells, engineered macrophages, dendritic cells, and other immune cell types; moreover, transient and permanent immune reprogramming is achievable via vivo technologies. The present review discusses the biological barriers limiting immune cell-targeted delivery, including biodistribution, cellular uptake, intracellular transport, and endosomal escape of delivered drugs, and reviews the latest advancements in targeted delivery technologies addressing these obstacles. We also address the latest applications of in vivo immune cell engineering in hematological malignancies and solid tumors, including mRNA therapeutics, CRISPR-based genome editing, and advanced programmable immunotherapy technologies. Furthermore, we discuss unresolved issues related to the specificity of targeting, efficiency of drug delivery, safety of treatment, manufacturing processes, and regulation of in vivo immune engineering technologies and outline future opportunities arising from the integration of nanomedicine, synthetic biology, genome engineering, and artificial intelligence-assisted materials science.},
}

@article {pmid42458265,
year = {2026},
author = {Zhou, Y and Fang, P and Zeng, J and Li, X and He, Q and Yuan, G and Zhang, W and Deng, H and Shu, F and Sun, P},
title = {Loss of function of OsWRKY53-OsARF18-OsRR22 significantly enhances rice salt tolerance.},
journal = {BMC plant biology},
volume = {26},
number = {1},
pages = {},
pmid = {42458265},
issn = {1471-2229},
support = {2025QK1006//the Top Ten Technical Breakthrough Projects of Hunan Province/ ; },
mesh = {*Oryza/genetics/physiology ; *Salt Tolerance/genetics ; *Plant Proteins/genetics/metabolism ; *Transcription Factors/genetics/metabolism ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; Plants, Genetically Modified ; Loss of Function Mutation ; },
abstract = {BACKGROUND: Salt stress is a key abiotic stress factor limiting rice growth and development. Previous studies have shown that OsWRKY53, OsARF18, and OsRR22 not only serve as important negative regulators of salt tolerance in rice but are also crucial for growth and development. However, the relative strengths of salt tolerance among these three genes and their combined effects within the same rice variety have not yet been reported.

RESULTS: In this study, we employed CRISPR/Cas9-mediated genome editing to simultaneously disrupt OsWRKY53, OsARF18, and OsRR22 in the rice cultivar Shuanghui 459. Salt tolerance increases sequentially in single-gene, double-gene, and triple-gene mutants. Under 1.0% NaCl stress, the triple mutant exhibited approximately 80% survival versus complete lethality in the wild type (WT). Physiologically, ROS accumulation progressively declined, while key antioxidant enzyme activities (CAT, SOD, POD) significantly increased. This suggests that the enhanced salt tolerance of the mutants may be associated with increased antioxidant enzyme activity. More importantly, molecular analysis revealed that the WRKY domain of OsWRKY53 binds to the W-box elements in the promoters of OsARF18 and OsRR22, thereby downregulating their expression. Reciprocally, OsARF18 and OsRR22 knockout downregulated OsWRKY53. Furthermore, OsWRKY53 and OsRR22 directly interact at the protein level.

CONCLUSIONS: Taken together, our results reveal that OsWRKY53, OsARF18, and OsRR22 constitute a reciprocal negative-feedback loop, wherein these three transcriptional regulators mutually antagonize each other. The significant enhancement of salt tolerance in the triple mutant may be related to the relief of this antagonistic interaction. Importantly, the polygenic aggregation of OsWRKY53-OsARF18-OsRR22 not only significantly enhances rice salt tolerance but also does not affect normal plant growth and development. These findings provide new strategies for polygenic aggregation-based genetic improvement of salt-tolerant rice varieties.},
}

*Oryza/genetics/physiology
*Salt Tolerance/genetics
*Plant Proteins/genetics/metabolism
*Transcription Factors/genetics/metabolism
Gene Expression Regulation, Plant
CRISPR-Cas Systems
Plants, Genetically Modified
Loss of Function Mutation

@article {pmid42647693,
year = {2026},
author = {Luboshitz, A and Rahimi, M and Hendel, A and Orenstein, Y},
title = {EpiCRISPR: Improving CRISPR/Cas9 On- and Off-Target Prediction by Flanking Sequences, Epigenetic Marks, and High-Throughput Datasets.},
journal = {IEEE transactions on computational biology and bioinformatics},
volume = {23},
number = {5},
pages = {2281-2294},
doi = {10.1109/TCBBIO.2026.3727752},
pmid = {42647693},
issn = {2998-4165},
mesh = {*CRISPR-Cas Systems/genetics ; *Epigenesis, Genetic/genetics ; *Gene Editing/methods ; Humans ; *Computational Biology/methods ; Prediction Algorithms ; },
abstract = {CRISPR/Cas9 technology has revolutionized gene-editing technologies. However, the endogenous gene-editing efficiency at the target site, unintended off-target sites (OTSs), and the DNA repair outcomes are affected by various factors, among them are the sequence of the site, its flanking sequences, and the epigenetic marks harboring it. Researchers have developed computational methods to predict endogenous efficiency, OTSs, and repair outcomes. However, previous studies have failed to successfully incorporate the factors that affect on- and off-target activity, and repair outcomes, and the methods were trained and evaluated on small datasets. A recent dataset of endogenous on-target efficiencies and repair outcomes of nearly 1,600 measurements in T cells, and a recently preprocessed dataset of 78 OTS experiments in T cells provide a unique opportunity to develop a method to predict endogenous on-target efficiency, OTSs, and repair outcomes. Here, we developed EpiCRISPROn and EpiCRISPROff to improve on-target efficiency and OTSs prediction, respectively, by combining various inputs: the site and its flanking sequences, multiple epigenetic marks, and a high-throughput-based prediction. In EpiCRISPROn and EpiCRISPROff, the additional non-sequence features improved prediction performance from an average Spearman correlation of 0.31 to 0.51 in 5-fold cross-validation and from an average AUPRC of 0.436 to 0.441 on a held-out test set, respectively. Moreover, EpiCRISPROn and EpiCRISPROff were trained on one cell type and successfully generalized to other cell types. In contrast, the inclusion of flanking sequences and epigenetic features did not improve repair-outcome prediction. Furthermore, by interrogating EpiCRISPROn and EpiCRISPROff trained models we shed light on on-target and OTS editing preferences. We expect EpiCRISPROn and EpiCRISPROff to advance the field of gene editing by providing improved cell-type-aware prediction of endogenous CRISPR/Cas9 on- and off-target activity.},
}

*CRISPR-Cas Systems/genetics
*Epigenesis, Genetic/genetics
*Gene Editing/methods
Humans
*Computational Biology/methods
Prediction Algorithms

@article {pmid42671082,
year = {2026},
author = {Zhang, Z and Liao, J and Yu, T and Chen, S and Zeng, L and Chen, G and Shen, Q and Tang, Y and Jiang, M and Chen, J and Wang, L},
title = {Lipopolymer Nanoparticle-Mediated In Vivo CRISPR-Cas9 Editing of Mrg15 Restores Mitochondrial Mitophagy to Alleviate Metabolic-Associated Steatohepatitis.},
journal = {Advanced healthcare materials},
volume = {15},
number = {37},
pages = {e71652},
doi = {10.1002/adhm.71652},
pmid = {42671082},
issn = {2192-2659},
support = {52473328//National Natural Science Foundation of China/ ; 2024B1515040006//Guangdong Basic and Applied Basic Research Foundation/ ; 2023B1212060013//Science and Technology Planning Project of Guangdong Province/ ; 2023HYSPT0703//Guangdong High-Level Youth Talent Project/ ; 1320900066//Sun Yat-sen Clinical Scientist Program/ ; },
mesh = {Animals ; *Nanoparticles/chemistry ; Mice ; *Mitophagy/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Mitochondria/metabolism ; Mice, Inbred C57BL ; Male ; Liver/metabolism/pathology ; *Fatty Liver/metabolism/genetics/pathology ; Humans ; },
abstract = {Metabolic-associated steatohepatitis (MASH) remains difficult to treat due to the lack of interventions capable of targeting upstream disease drivers and achieving durable disease modification. The epigenetic regulator Mrg15 has been implicated in mitochondrial dysfunction and metabolic stress in the liver, suggesting its potential relevance to MASH pathogenesis. Here, we develop a liver-targeted lipopolymer nanoparticle (LPNP)-mediated gene editing platform to enable in vivo disruption of Mrg15 by co-delivery of Cas9 mRNA and Mrg15 sgRNA. The screened P64H/Mrg15 system achieved efficient hepatic delivery and genome editing, resulting in reduced Mrg15 expression in hepatocytes. In the MASH mouse model, P64H/Mrg15 treatment was associated with decreased hepatic lipid accumulation, improved liver injury markers, and attenuation of inflammation and fibrosis. Sequence-level analyses confirmed on-target editing in liver tissue, and systemic histopathological evaluation revealed no overt toxicity in major organs under the tested dosing regimen. Transcriptomic profiling revealed coordinated pathway-level associations involving metabolic, inflammatory, and autophagy-related regulation, while protein-level analyses demonstrated alterations in selected autophagy- and mitophagy-related regulators, including TUFM and LC3B-II. Together, these findings identify Mrg15 as a disease-relevant epigenetic regulator in MASH and highlight liver-directed P64H LPNP/CRISPR delivery as a promising non-viral strategy for modulating upstream regulatory pathways in metabolic liver disease.},
}

Animals
*Nanoparticles/chemistry
Mice
*Mitophagy/genetics
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Mitochondria/metabolism
Mice, Inbred C57BL
Male
Liver/metabolism/pathology
*Fatty Liver/metabolism/genetics/pathology
Humans

@article {pmid42715831,
year = {2027},
author = {Ri, L and Cortez, JV and Bogema, DR and Nevard, R and Day, M and Grupen, CG and McFarlane, GR},
title = {Rapid CRISPR-based bovine embryo sexing to streamline genotype-informed cattle breeding.},
journal = {Theriogenology},
volume = {267},
number = {},
pages = {118163},
doi = {10.1016/j.theriogenology.2026.118163},
pmid = {42715831},
issn = {1879-3231},
mesh = {Animals ; Cattle/genetics/embryology ; *Sex Determination Analysis/veterinary/methods ; Male ; Female ; Genotype ; *Breeding/methods ; Polymorphism, Single Nucleotide ; *CRISPR-Cas Systems ; Genotyping Techniques/veterinary ; *Embryo, Mammalian ; },
abstract = {Cattle in vitro fertilisation and embryo transfer programmes increasingly rely on embryo-level selection to accelerate genetic gain, but current sexing and genotyping workflows can be costly, slow and logistically demanding. This study developed an efficient, low-resource workflow for bovine embryo sexing that combines whole genome amplification (WGA) with recombinase polymerase amplification-CRISPR-Cas12a (RPA-Cas12a). It also assessed whether the same WGA biopsy products could be used for downstream single nucleotide polymorphism (SNP) microarray genotyping. A one-tube RPA-Cas12a assay targeting the bovine Y-chromosome S4 repeat was developed for fluorescence and lateral flow assay (LFA) readouts. Analytical sensitivity was assessed using serially diluted bovine genomic DNA (gDNA), and breed robustness was tested using male and female gDNA from five major beef breeds and Holstein cattle. The workflow was then applied to WGA products from 22 bovine blastocyst biopsies, with sex calls validated against an established real-time PCR melt curve assay and 100K SNP microarray genotyping. The assay detected male bovine gDNA down to 100 pg using both fluorescence and LFA readouts, with no signal from female gDNA. Male-specific detection was consistent across all breeds tested. All WGA-RPA-Cas12a sex calls from blastocyst biopsies were concordant with real-time PCR and SNP microarray sex calls, and WGA biopsy products produced genome-wide SNP call rates above 85%. This workflow provides a practical approach for rapid bovine embryo sex triage and could reduce unnecessary cryopreservation and genotyping while improving the efficiency of genotype-informed cattle breeding programmes.},
}

Animals
Cattle/genetics/embryology
*Sex Determination Analysis/veterinary/methods
Male
Female
Genotype
*Breeding/methods
Polymorphism, Single Nucleotide
*CRISPR-Cas Systems
Genotyping Techniques/veterinary
*Embryo, Mammalian

@article {pmid42759490,
year = {2026},
author = {Li, Z and Xiong, X and Cao, R and Li, JF},
title = {Precision genetic manipulation: CRISPR toolkits reshape plant biology.},
journal = {Plant physiology},
volume = {202},
number = {2},
pages = {},
doi = {10.1093/plphys/kiag696},
pmid = {42759490},
issn = {1532-2548},
support = {32125004//National Natural Science Foundation of China/ ; 2023B0303000022//Guangdong Major Project of Basic Research/ ; 2019YFA0906202//National Key Research and Development Program of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Plants/genetics ; *Genome, Plant/genetics ; *Genetic Engineering/methods ; Plants, Genetically Modified/genetics ; },
abstract = {The emergence of clustered regularly interspaced short palindromic repeats (CRISPR) technology has revolutionized plant genome engineering over the past decade. This review begins by highlighting 4 seminal early CRISPR studies published in Plant Physiology that laid critical foundations for crop genome editing and tool development. We then summarize major advances in optimizing CRISPR performance, expanding targeting scope, and developing strategies for transgene removal and transient editing. We also discuss a wide array of innovative applications beyond gene knockout, such as gene targeting, chromosome engineering, synthetic apomixis, and gene drive. Furthermore, we introduce key CRISPR-derived tools, including base editors, prime editors, and programmable transcriptional and epigenetic regulators. Finally, we outline remaining challenges to stimulate future exploration of the full potential of CRISPR toolkits.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Plants/genetics
*Genome, Plant/genetics
*Genetic Engineering/methods
Plants, Genetically Modified/genetics

@article {pmid42810238,
year = {2027},
author = {Rahimi, M and Miskel, D and Schreiber, M and Habermann, H and Haake, L and Brenig, B and Besenfelder, U and Havlicek, V and Tesfaye, D and Tholen, E and Rings, F and Held-Hoelker, E and Ghanem, N and Benedetti, C and Hoelker, M},
title = {Zygote origin shapes sensitivity to electrical voltage field and editing susceptibility after CRISPR/Cas9-RNP electroporation in bovine embryos.},
journal = {Theriogenology},
volume = {267},
number = {},
pages = {118215},
doi = {10.1016/j.theriogenology.2026.118215},
pmid = {42810238},
issn = {1879-3231},
mesh = {Animals ; Cattle/embryology ; *Electroporation/veterinary/methods ; *CRISPR-Cas Systems ; *Zygote/physiology ; *Gene Editing/veterinary/methods ; Female ; Ribonucleoproteins/genetics ; },
abstract = {Despite major progress in generation of edited embryos and offspring via CRISPR/Cas9-RNP electroporation, editing outcomes remain highly variable. This variability may be due to intrinsic differences between in vivo-derived (IVV) and in vitro-derived (IVT) zygotes, which may play a more decisive role than previously anticipated. Therefore, this study aimed to determine how zygote origin shapes the sensitivity to electrical voltage field and editing susceptibility after CRISPR/Cas9-RNP electroporation in the bovine model. The present study systematically compared subsequent developmental competence and editing events in IVV- and IVT-derived bovine zygotes across defined electroporation voltages and pulse durations. Notably, CRISPR/Cas9-RNP electroporation of IVV-derived bovine zygotes of comparable age and collected by tubal embryo flushing (10-18 h post ovulation, hpo) resulted in markedly lower survival rates at 25 and 30 V compared to 20 V. In contrast, higher (p < 0.05) editing frequencies were observed at 25 V and 30 V. Clear differences, however, were observed between the two zygote groups. Following CRISPR/Cas9 RNP electroporation, IVV-derived zygotes yielded a higher proportion of unedited blastocysts (p < 0.05), absence of full edited embryos, a significantly increased frequency of monoallelic edits and reduced rates of mosaicism compared to IVT-derived zygotes. Collectively, the results of the present study clearly underscore a significant lower editing efficiency to pulse sensitivity ratio in IVV-derived zygotes compared to their IVT counterparts. To our knowledge, this is the first study to use bovine IVV-derived zygotes for genome editing using RNP-electroporation to elucidate how zygote origin shapes sensitivity to electrical voltage field and editing efficiency.},
}

Animals
Cattle/embryology
*Electroporation/veterinary/methods
*CRISPR-Cas Systems
*Zygote/physiology
*Gene Editing/veterinary/methods
Female
Ribonucleoproteins/genetics