@article {pmid41629010,
year = {2026},
author = {Saini, A and Sharma, N and Sharma, N and Kumari, N and Sharma, M and Singh, B and Thakur, AK},
title = {Precision pest management: Genome editing tools, specifically CRISPR/Cas9 and future prospects.},
journal = {Pesticide biochemistry and physiology},
volume = {218},
number = {},
pages = {106941},
doi = {10.1016/j.pestbp.2026.106941},
pmid = {41629010},
issn = {1095-9939},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; Insecta/genetics ; *Pest Control, Biological/methods ; *Pest Control/methods ; Insect Control/methods ; },
abstract = {The growing resistance to synthetic insecticides and Bt toxins, alongside persistent crop losses despite heavy pesticide application, highlights the urgent need for safer, sustainable and efficient pest management strategies. This review presents genome editing as a precise and versatile approach to reduce pest impact by altering fertility, feeding patterns or vulnerability, while protecting beneficial organisms. Among the genome editing tools, CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) is one of the most promising genome editing techniques in insects. It facilitates targeted functional studies, integration with RNAi and dual-expression systems and gene drive applications. Deployment is envisioned in two phases, initial laboratory modification followed by regulated field release, with a strong emphasis on biosafety through terminator genes, marked individuals for gene flow monitoring, optimized dosages, stringent screening and long-term ecological surveillance, along with transparency and adherence to international safety protocols. Significant challenges encompass delivery efficiency, identification of edits, off-target mutations, dose-related efficacy and sterility, unstable transmission and resistance development. Innovations such as base and prime editing minimize unintended mutations by circumventing double-stranded breaks (DSBs), while paratransgenic strategies targeting gut symbionts offer supplementary avenues; plant-mediated insect gene editing emerges as a promising frontier. Overall, carefully regulated trials aligned with policy frameworks and stakeholder involvement are vital to assess effectiveness in natural environments and achieve targeted, dependable and ecologically responsible pest control.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
Animals
Insecta/genetics
*Pest Control, Biological/methods
*Pest Control/methods
Insect Control/methods

@article {pmid41628563,
year = {2026},
author = {Liang, L and Xu, B and Xiao, S and Mu, X and Zhao, S and Tian, J},
title = {A new split DNA-based activation of CRISPR/Cas12a for amplification-free and dual-stimulus responsive detection and precise imaging of miRNA-221.},
journal = {Talanta},
volume = {303},
number = {},
pages = {129443},
doi = {10.1016/j.talanta.2026.129443},
pmid = {41628563},
issn = {1873-3573},
abstract = {The CRISPR/Cas12a system is a genome editing technology that has been widely applied in biosensing and molecular diagnostics. However, the detection and regulation of its core components remain challenging. Therefore, we constructed a new split DNA-based activation method for the regulation of CRISPR/Cas12a, and based on that, an APE1-assisted activation CRISPR/Cas12a system for miRNA detection and a precise imaging method was also developed without amplification and complex design. Two split DNA were used as activators and embedded in two hairpins. When APE1 and miRNA-221 were simultaneously input, the DNA logic gate was started, thus releasing the determinant activation chain to activate the trans-shearing activity of the CRISPR/Cas12a system, so that the fluorescent probe signal can be significantly recovered. Different cleavage-activated chain hairpins were designed, and the influence on the trans-shear activity of CRISPR/Cas12a and the activation effect were discussed. And the method was successfully applied to detect the expression levels of miRNA-221 in cell lysates. The detection limit for miRNA-221 is 9.71 pmol/L (S/N = 3). At the same time, the method was applied for precise imaging of miRNA-221 within different cells and can effectively distinguish tumor cells. This study combines the regulation of the CRISPR/Cas system by split activators with the advantages of dual-responsive DNA logic circuits. The dual-response activation design effectively reduces false positive signals, thereby enhancing the detection and imaging accuracy. This method provides a novel design concept for utilizing split-DNA activation of the CRISPR/Cas system for nucleic acid detection and cell imaging.},
}

@article {pmid41627753,
year = {2026},
author = {Arnould, K and El Kadri, M and Hervé, P and Asencio, C and Plazolles, N and Monic, S and Morel, CA and Rivière, L and Bringaud, F and Tetaud, E},
title = {Precision Without Selection: A Marker-Free CRISPR/Cas9-Based Protocol for Multiplexed Genome Editing in Trypanosomatids.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3014},
number = {},
pages = {65-78},
pmid = {41627753},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Genome, Protozoan ; Electroporation ; *Trypanosomatina/genetics ; Ribonucleoproteins/genetics ; },
abstract = {The CRISPR-Cas9 system has become a valuable tool for genome editing in trypanosomatid parasites such as Trypanosoma and Leishmania species. Although these organisms have been genetically engineered for a long time using homologous recombination, CRISPR/Cas9 offers improved efficiency for genome editing. However, conventional strategies employing stable Cas9 expression require the persistent use of a specific genetic background (i.e., strains expressing Cas9), depend on selectable resistance markers, compromise genomic stability, and are not readily applicable to diverse strain backgrounds. Herein, we report an optimized marker-free CRISPR/Cas9 method based on transient ribonucleoprotein (RNP) delivery that overcomes these drawbacks. Our method eliminates the need for plasmid integration or antibiotic selection while maintaining high editing efficiency. The protocol comprises the following steps: (1) design of the guide RNA (gRNA), (2) design of the repair template (cassette), (3) assembly of the ribonucleoprotein (RNP) complex, (4) delivery by electroporation, and (5) clonal screening through PCR and sequencing. The procedure permits rapid (≤3 weeks) production of homozygous mutant lines in wild-type strains, including low-density culture strains. The reproducibility and ease of the technique render it particularly suited for multiplexed editing of polyploid genomes, multi-gene families, and several different genes at once, as well as validation of the essential nature of genes. Although designed for trypanosomatids, the workflow can be adapted to other kinetoplastids, offering a flexible platform for functional genomics.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
*Genome, Protozoan
Electroporation
*Trypanosomatina/genetics
Ribonucleoproteins/genetics

@article {pmid41627331,
year = {2026},
author = {Roy, N and Debnath, P and Srivastava, S and Gaur, HS},
title = {Recent developments in CRISPR/Cas9 genome editing research for edible fungiculture.},
journal = {Functional & integrative genomics},
volume = {26},
number = {1},
pages = {36},
pmid = {41627331},
issn = {1438-7948},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Genome, Fungal ; *Agaricales/genetics/growth & development ; },
abstract = {Fungiculture refers to the deliberate cultivation or agricultural practice involving the growth and management of fungi. The practice encompasses the intentional culture of diverse species of macrofungi, including mushrooms and truffles, within controlled habitats or under specified conditions, in order to fulfill human requirements especially for food purpose. As the global market for edible mushrooms grows quickly, it is becoming increasingly necessary to grow novel and improved strains of edible fungi. Growing and breeding edible fungi using traditional methods is both time-consuming and difficult. So, there is a need for evolving advanced techniques at a molecular level which can help breeding of edible fungi with much better efficiency. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9) system is one of the most effective techniques for accurately cutting and modifying the genomes of edible fungi. In this review, we discuss how genome editing using CRISPR/Cas9 has been utilized in many edible fungal species such as Pleurotus ostreatus, Agaricus bisporus, Cordyceps militaris, Ganoderma lucidum, Flammulina filiformis, Lentinula edodes, and others for their target specific breeding. We also discuss the working mechanism of the above-mentioned system in these mushroom species, and also the advantages and limitations of using this system in mushrooms.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
*Genome, Fungal
*Agaricales/genetics/growth & development

@article {pmid41627751,
year = {2026},
author = {Nagamine, S and Oishi, R and Nakazawa, M},
title = {Engineering Wax Ester Composition in Euglena gracilis Using Genome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3014},
number = {},
pages = {37-49},
pmid = {41627751},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *Euglena gracilis/genetics/metabolism ; CRISPR-Cas Systems ; *Metabolic Engineering/methods ; *Esters/metabolism/chemistry ; *Waxes/metabolism/chemistry ; },
abstract = {Genome editing technologies have significantly expanded the potential for metabolic engineering in non-model organisms. In Euglena gracilis, genome editing methods using Cas9 and Cas12a were reported in 2019 and 2024, respectively, and are increasingly being applied to modify metabolic functions. This chapter provides a detailed protocol for CRISPR/Cas9-based genome editing that enables stable modification of wax ester composition under anaerobic conditions. By targeting key enzymes in the reversed β-oxidation pathway, the method allows the generation of knockout mutants with altered wax ester chain lengths. Beyond this application, the protocol supports reproducible and stable genetic modification of E. gracilis metabolism. It can be extended to the engineering of other biosynthetic pathways and is compatible with future integration of knock-in strategies. The approach offers a practical basis for the broader use of E. gracilis as a green chassis organism in synthetic biology and biomanufacturing.},
}

*Gene Editing/methods
*Euglena gracilis/genetics/metabolism
CRISPR-Cas Systems
*Metabolic Engineering/methods
*Esters/metabolism/chemistry
*Waxes/metabolism/chemistry

@article {pmid41581816,
year = {2026},
author = {Xiao, Y and Yang, J and Yang, W and Yuan, M and Zhang, Y and Liu, J and Zhang, Y and Zhu, H and Luo, G},
title = {A rapid on-site diagnostic method for goose parvovirus disease based on recombinase polymerase amplification and CRISPR/AsCas12a.},
journal = {International journal of biological macromolecules},
volume = {343},
number = {Pt 2},
pages = {150496},
doi = {10.1016/j.ijbiomac.2026.150496},
pmid = {41581816},
issn = {1879-0003},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Geese/virology ; *Parvoviridae Infections/diagnosis/veterinary/virology ; *Parvovirus/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases/metabolism ; *Poultry Diseases/diagnosis/virology ; Ducks/virology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Goose parvovirus (GPV) is a highly pathogenic and lethal virus responsible for Derzsy's disease in goslings and ducklings, significantly influencing the economic viability of waterfowl farming. This necessitates the development of rapid diagnostic techniques for effective disease management. In this study, an optimized clustered regularly interspaced short palindromic repeats (CRISPR)/Acidaminococcus sp. CRISPR associated nuclease 12a (AsCas12a) system was developed for the diagnosis of GPV. The study determined that the optimal conditions for the CRISPR/Cas12a-based fluorescence assay were 20 nM AsCas12a, 5 nM crRNA, and 5 nM single-stranded DNA (ssDNA), whereas the lateral flow assay (LFA) required 20 nM AsCas12a and 4 nM crRNA. Moreover, the fluorescence-based assay and LFA achieved minimum detection limits of 7.8 copies/μL and 78 copies/μL, respectively, representing 1000-fold and 100-fold improvements over conventional PCR methods. Both detection methods exhibited high specificity and demonstrated no cross-reactivity with other prevalent waterfowl pathogens, such as duck plague virus, duck hepatitis viruses, H5 avian influenza virus, waterfowl astrovirus, reovirus, Muscovy duck parvovirus, and novel GPV. The results of the LFA were in complete concordance with laboratory qPCR analyses, thereby affirming their reliability for clinical diagnostics. In conclusion, we have successfully developed a dual-readout GPV detection system utilizing CRISPR/Cas12a technology, which holds significant promise for the early surveillance and containment of GPV outbreaks.},
}

Animals
*CRISPR-Cas Systems/genetics
*Geese/virology
*Parvoviridae Infections/diagnosis/veterinary/virology
*Parvovirus/genetics
*Nucleic Acid Amplification Techniques/methods
*Recombinases/metabolism
*Poultry Diseases/diagnosis/virology
Ducks/virology
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41577322,
year = {2026},
author = {Wang, L and Miao, M and Bao, L and Chu, J and Zhou, J and Song, W and Cai, P and Cheng, C and Xu, H and Wang, T and Zhao, R and Wang, H and Liu, F and Xu, M and Tian, G},
title = {CRISPR/Cas9-based genome-wide screen reveals a synergistic effect of Irinotecan and USP1 inhibitor in colorectal cancer.},
journal = {European journal of pharmacology},
volume = {1015},
number = {},
pages = {178558},
doi = {10.1016/j.ejphar.2026.178558},
pmid = {41577322},
issn = {1879-0712},
mesh = {Humans ; *Irinotecan/pharmacology/therapeutic use ; *Colorectal Neoplasms/drug therapy/genetics/pathology ; Animals ; Mice ; *CRISPR-Cas Systems/genetics ; *Ubiquitin-Specific Proteases/antagonists & inhibitors/metabolism ; Drug Synergism ; Cell Proliferation/drug effects ; Xenograft Model Antitumor Assays ; Cell Line, Tumor ; Drug Resistance, Neoplasm/drug effects ; HCT116 Cells ; Gene Expression Regulation, Neoplastic/drug effects ; Antineoplastic Combined Chemotherapy Protocols/pharmacology ; },
abstract = {Irinotecan resistance remains a significant challenge in metastatic colorectal cancer (mCRC) therapy. To address this, we identified USP1 as a synthetic lethal partner of Irinotecan through genome-wide CRISPR/Cas9 screening in HCT-116 cells. Combining the USP1 inhibitor I-138 with Irinotecan in HCT-116, HT-29, and SW620 cell lines significantly reduced IC50, suppressed proliferation, and diminished colony formation compared to monotherapy, demonstrating a synergistic effect (combination index CI < 1). The synergistic therapeutic efficacy was further validated in the xenograft mouse model. Mechanistic studies revealed that I-138 significantly upregulated pCREB (Ser133), concurrently dynamically regulating the activity of USP1, FANCD2/FANCI, and PCNA upon DNA damage response and repair. RNA sequencing further highlighted the enrichment of cAMP, PI3K-AKT, and Wnt pathways, which are all linked to CREB activity in the combination group. These findings establish USP1 inhibition as a promising strategy to overcome Irinotecan resistance through the combination strategy, providing a novel therapeutic avenue for CRC.},
}

Humans
*Irinotecan/pharmacology/therapeutic use
*Colorectal Neoplasms/drug therapy/genetics/pathology
Animals
Mice
*CRISPR-Cas Systems/genetics
*Ubiquitin-Specific Proteases/antagonists & inhibitors/metabolism
Drug Synergism
Cell Proliferation/drug effects
Xenograft Model Antitumor Assays
Cell Line, Tumor
Drug Resistance, Neoplasm/drug effects
HCT116 Cells
Gene Expression Regulation, Neoplastic/drug effects
Antineoplastic Combined Chemotherapy Protocols/pharmacology

@article {pmid41526335,
year = {2026},
author = {Gao, J and Bader, A and Linder, MI and Cheng, J and Richter, M and da Costa, R and Zehrer, A and Mitt, K and Popper, B and Meissner, F and Wei, X and de Vega Gómez, E and Tatematsu, M and Rohlfs, M and Frenz-Wiessner, S and Kiziltug, M and Somekh, I and Yacobovich, J and Steinberg-Shemer, O and Somech, R and Soehnlein, O and Schmid, B and Klein, C and Walzog, B and Maier-Begandt, D},
title = {Mutations in VPS18 lead to a neutrophil maturation defect associated with disturbed vesicle homeostasis.},
journal = {Cell death & disease},
volume = {17},
number = {1},
pages = {180},
pmid = {41526335},
issn = {2041-4889},
support = {CRC914 (project A02)//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; TRR332 (#449437943; project C03)//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CRC914 (project A13)//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; TRR332 (#449437943; project A02)//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; TRR332 (#449437943; project B01)//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CRC914 (projects A02 and Z03)//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {*Neutrophils/metabolism/pathology ; Animals ; Humans ; *Vesicular Transport Proteins/genetics/metabolism ; Zebrafish ; *Mutation/genetics ; *Homeostasis ; Cell Differentiation ; Induced Pluripotent Stem Cells/metabolism ; Autophagy ; CRISPR-Cas Systems ; Neutropenia/genetics/pathology ; Autophagosomes/metabolism ; Male ; },
abstract = {Neutrophils, the first cells to arrive at the site of inflammation, are rather short-lived cells and thus have to be constantly replenished. During neutrophil development, vesicle dynamics need to be fine-tuned and impaired vesicle trafficking has been linked to failure in neutrophil maturation. Here, we characterized the role of VPS18 as a central core component of CORVET & HOPS tethering complexes for neutrophil development. Using CRISPR/Cas9-engineered Hoxb8 cells with heterozygous mutations in Vps18, we found that VPS18 deficiency interfered with neutrophil development due to tethering complex instability. As a result, vesicle dynamics were impaired with a strong increase in LC3B-II and p62 levels, indicating autophagosome accumulation and reduced autophagic flux. With transmission electron microscopy, we verified the increase in autophagosomes and also found irregularly shaped vesicular structures in Vps18 mutants. Subsequently, Vps18 mutant neutrophil progenitors underwent premature apoptosis. We described a novel patient with a heterozygous stop-gain mutation in VPS18 suffering from neutropenia and recurrent infections. To verify our findings in the human system, we used human induced pluripotent stem cells (iPSCs). Upon differentiation into neutrophils, loss of VPS18 resulted in an almost complete absence of iPSC-derived developing neutrophils. Heterozygous VPS18 mutant and patient mutation-harboring iPSCs were characterized by strongly reduced numbers of developing neutrophils. Zebrafish larvae with heterozygous mutations in vps18 were also characterized by significantly reduced neutrophil numbers. This study shows the pivotal impact of VPS18 for adequate vesicle dynamics during neutrophil development which might be relevant in the context of vesicle trafficking during granulopoiesis and congenital neutropenia.},
}

*Neutrophils/metabolism/pathology
Animals
Humans
*Vesicular Transport Proteins/genetics/metabolism
Zebrafish
*Mutation/genetics
*Homeostasis
Cell Differentiation
Induced Pluripotent Stem Cells/metabolism
Autophagy
CRISPR-Cas Systems
Neutropenia/genetics/pathology
Autophagosomes/metabolism
Male

@article {pmid41520927,
year = {2026},
author = {Nishizawa, H and Daimon, T},
title = {DIL-CRISPR: a practical approach to mitigate G0 mosaic lethality in insect gene editing.},
journal = {Insect biochemistry and molecular biology},
volume = {188},
number = {},
pages = {104492},
doi = {10.1016/j.ibmb.2026.104492},
pmid = {41520927},
issn = {1879-0240},
mesh = {Animals ; *Gene Editing/methods ; *Spodoptera/genetics/growth & development ; *CRISPR-Cas Systems ; *Mosaicism ; Larva/genetics/growth & development ; Genes, Lethal ; },
abstract = {Genome editing in insects is typically conducted by injecting genome editing reagents into early embryos, producing generation zero (G0) individuals that develop as genetic mosaics. Targeting genes whose disruption induces mosaic lethality is therefore challenging, since most or all G0 individuals frequently fail to survive to adulthood, preventing germline transmission of edited alleles. Here, we present a straightforward and practical approach, DIL-CRISPR, to mitigate G0 mosaic lethality by systematically diluting the CRISPR/Cas9 injection mix. Using the tobacco cutworm Spodoptera litura and the juvenile hormone receptor gene Met1 as a benchmark, we demonstrate that dilution of the injection mix lessens the severity of larval-pupal mosaic phenotypes and increases G0 survival in a dose-dependent manner. Amplicon sequencing further showed that somatic mutation frequencies decline with dilution, while germline mutation rates remain sufficient to establish mutant lines. Notably, we detected a substantial discrepancy between somatic and germline editing efficiencies, likely reflecting selective loss of highly edited, lethal mosaics before they reach adulthood. We conclude that DIL-CRISPR therefore offers a reliable means to balance G0 survival with germline editing, converting an empirically used dilution practice into a generalizable strategy. Overall, this approach provides a practical solution for generating mutant lines of mosaic lethal genes and is broadly applicable across diverse insect species, facilitating functional genetic studies in non-model insects.},
}

Animals
*Gene Editing/methods
*Spodoptera/genetics/growth & development
*CRISPR-Cas Systems
*Mosaicism
Larva/genetics/growth & development
Genes, Lethal

@article {pmid41519853,
year = {2026},
author = {Michael Deans, PJ and Retallick-Townsley, KG and Li, A and Seah, C and Johnson, J and Garcia Gonzalez, J and Cao, E and Schrode, N and Yu, A and Cartwright, S and Voloudakis, G and Zhang, W and Wang, M and Fullard, JF and Girdhar, K and Stahl, E and Akbarian, S and Zhang, B and Roussos, P and O'Reilly, P and Huckins, LM and Brennand, KJ},
title = {Functional implications of polygenic risk for schizophrenia in human neurons.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1355},
pmid = {41519853},
issn = {2041-1723},
support = {R01ES033630//U.S. Department of Health & Human Services | NIH | National Institute of Environmental Health Sciences (NIEHS)/ ; R56MH101454//U.S. Department of Health & Human Services | NIH | National Institute of Mental Health (NIMH)/ ; },
mesh = {Humans ; *Schizophrenia/genetics ; *Multifactorial Inheritance/genetics ; *Neurons/metabolism ; Induced Pluripotent Stem Cells/metabolism ; *Genetic Predisposition to Disease ; Genome-Wide Association Study ; CRISPR-Cas Systems ; Transcriptome ; Polymorphism, Single Nucleotide ; },
abstract = {Genome wide association studies of schizophrenia reveal a complex polygenic risk architecture comprised of hundreds of risk variants; most are common in the population, non-coding, and act by genetically regulating the expression of one or more gene targets ("eGenes"). It remains unclear how the myriad genetic variants that are predicted to confer individually small effects combine to yield substantial clinical impacts in aggregate. Here, we demonstrate that convergence (i.e., the shared downstream transcriptomic changes with a common direction of effect), resulting from one-at-a-time perturbation of schizophrenia eGenes, influences the outcome when eGenes are manipulated in combination. In total, we apply pooled and arrayed CRISPR approaches to target 21 schizophrenia eGenes in human induced pluripotent stem cell-derived glutamatergic neurons, finding that functionally similar eGenes yield stronger and more specific convergent effects. Points of convergence constrain additive relationships between polygenic risk loci: consistent with a liability threshold model, combinatorial perturbations of these same schizophrenia eGenes reveal that pathway-level convergence predicts when observed effects will fail to sum to levels predicted by an additive model. Targeting points of convergence as novel therapeutic targets may prove more efficacious than individually reversing the effects of multiple risk loci.},
}

Humans
*Schizophrenia/genetics
*Multifactorial Inheritance/genetics
*Neurons/metabolism
Induced Pluripotent Stem Cells/metabolism
*Genetic Predisposition to Disease
Genome-Wide Association Study
CRISPR-Cas Systems
Transcriptome
Polymorphism, Single Nucleotide

@article {pmid41507381,
year = {2026},
author = {Gonzales, F and Schneider, C and Alexe, G and Lin, S and Khalid, D and Alvarez, M and Basanthakumar, A and Ellegast, JM and Merickel, L and Salhotra, S and Taillon, A and Giaimo, M and Wunderlich, M and Ansari, M and Perry, JA and Degar, B and Pikman, Y and Stegmaier, K},
title = {Identifying targeted therapies for CBFA2T3::GLIS2 acute myeloid leukemia.},
journal = {Leukemia},
volume = {40},
number = {2},
pages = {383-396},
pmid = {41507381},
issn = {1476-5551},
mesh = {Humans ; *Leukemia, Myeloid, Acute/drug therapy/genetics/pathology/metabolism ; Animals ; Mice ; CRISPR-Cas Systems ; Janus Kinase 2/antagonists & inhibitors/genetics ; Nitriles ; *Molecular Targeted Therapy ; Pyrazoles/pharmacology ; Protein Kinase Inhibitors/pharmacology ; Xenograft Model Antitumor Assays ; Cell Proliferation ; Pyrimidines ; Cell Line, Tumor ; Apoptosis ; Drug Resistance, Neoplasm ; },
abstract = {CBFA2T3::GLIS2-positive pediatric acute myeloid leukemia (AML) remains one of the worst prognostic AML subgroups. To uncover innovative targeted therapy approaches in this disease subtype we performed genome-scale CRISPR-Cas9 screening that highlighted a strong, selective dependency on JAK2 compared to other types of cancer. Using a doxycycline-inducible JAK2 knockout (KO) system, we validated JAK2 dependency in CBFA2T3::GLIS2 cell lines, observing impaired proliferation in vitro and in vivo and apoptosis induction in vitro. Both type I (ruxolitinib) and type II (CHZ868) JAK2 inhibitors showed selective in vitro activity in CBFA2T3::GLIS2-positive AML models. To identify resistance and sensitizer mechanisms to JAK2 inhibitors, we used CRISPR-Cas9 ruxolitinib anchor screening in CBFA2T3::GLIS2 AML. sgRNAs targeting negative regulators of the MAPK pathway were enriched in the ruxolitinib-treated cells. Similarly, CBFA2T3::GLIS2 AML sublines grown to resistance under chronic ruxolitinib treatment expressed pathogenic NRAS mutations. Both approaches converged on MAPK pathway activation as a resistance mechanism to ruxolitinib treatment. Combining ruxolitinib with MEK inhibitors showed a synergistic effect in cell lines and patient-derived xenograft (PDX) cells expressing the fusion and in vivo activity in a CBFA2T3::GLIS2 AML PDX, suggesting a potential approach to target this signaling circuitry in this poor outcome AML subtype.},
}

Humans
*Leukemia, Myeloid, Acute/drug therapy/genetics/pathology/metabolism
Animals
Mice
CRISPR-Cas Systems
Janus Kinase 2/antagonists & inhibitors/genetics
Nitriles
*Molecular Targeted Therapy
Pyrazoles/pharmacology
Protein Kinase Inhibitors/pharmacology
Xenograft Model Antitumor Assays
Cell Proliferation
Pyrimidines
Cell Line, Tumor
Apoptosis
Drug Resistance, Neoplasm

@article {pmid41501085,
year = {2026},
author = {Singh, P and D'Rozario, R and Chakraborty, B and Meher, S and Raychaudhuri, D and Tannir, AJ and Li, Y and Majumdar, A and Hawkins, J and Xiong, Y and Lorenzi, P and Sharma, P and Akdemir, K and Pilie, P and Jain, AK and Lee, BHL and Goswami, S},
title = {Loss of KDM6A-mediated genomic instability and metabolic reprogramming regulates response to therapeutic perturbations in bladder cancer.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1382},
pmid = {41501085},
issn = {2041-1723},
mesh = {*Urinary Bladder Neoplasms/genetics/drug therapy/metabolism ; Animals ; *Histone Demethylases/genetics/metabolism ; Humans ; Mice ; *Genomic Instability/genetics ; Cell Line, Tumor ; Cisplatin/therapeutic use/pharmacology ; Drug Resistance, Neoplasm/genetics ; CRISPR-Cas Systems ; DNA Repair/genetics ; Female ; Mice, Knockout ; Loss of Function Mutation ; Histones/metabolism ; Metabolic Reprogramming ; },
abstract = {Mutations in epigenetic regulators are common in bladder cancer, yet their impact on therapeutic responses remains unclear. Here, we identify that loss-of-function mutations in KDM6A, a histone demethylase altered in about 26% of advanced bladder cancers, are associated with poor survival after cisplatin chemotherapy, whereas they correlate with improved outcomes with anti-PD-1 therapy. Using CRISPR-Cas9-engineered murine and human bladder cancer models, we show that KDM6A deficiency increases formation of extrachromosomal circular DNA carrying chemoresistance loci, promoting cisplatin resistance. In parallel, KDM6A loss impairs DNA repair and rewires tumor metabolism, reducing glycolysis and lactate output. This metabolic shift diminishes histone lactylation in regulatory T cells, suppressing immunoregulatory genes and limiting expansion of PD-1[hi] regulatory T cells. Collectively, our findings establish KDM6A mutation as a key regulator of therapeutic responses, providing a foundation for its use in guiding precision therapy in advanced bladder cancer.},
}

*Urinary Bladder Neoplasms/genetics/drug therapy/metabolism
Animals
*Histone Demethylases/genetics/metabolism
Humans
Mice
*Genomic Instability/genetics
Cell Line, Tumor
Cisplatin/therapeutic use/pharmacology
Drug Resistance, Neoplasm/genetics
CRISPR-Cas Systems
DNA Repair/genetics
Female
Mice, Knockout
Loss of Function Mutation
Histones/metabolism
Metabolic Reprogramming

@article {pmid41495049,
year = {2026},
author = {Li, X and Shang, X and Liu, J and Zhang, Y and Jia, X and Li, H and Wang, Y and Gao, J and Ma, X and Zhang, X and Rong, X and Gan, W and Zhang, Y and Chen, J and Wang, L and Bao, Z and He, L and Yan, X and Liu, Y and Shao, J and Xiao, Z and Wang, Z and Zhu, H and Wang, Z and Wu, Y and Huang, Y},
title = {Intrathecal CRISPR-edited allogeneic IL-13Rα2 CAR T Cells for recurrent high-grade Glioma: preclinical characterization and phase I trial.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1362},
pmid = {41495049},
issn = {2041-1723},
mesh = {Humans ; Female ; Male ; *Immunotherapy, Adoptive/methods/adverse effects ; *Interleukin-13 Receptor alpha2 Subunit/genetics/immunology ; Middle Aged ; Adult ; *Glioma/therapy/immunology/pathology ; CRISPR-Cas Systems/genetics ; *Receptors, Chimeric Antigen/genetics/immunology ; *T-Lymphocytes/immunology/transplantation/metabolism ; *Brain Neoplasms/therapy/immunology/pathology ; Gene Editing ; Neoplasm Recurrence, Local/therapy ; Animals ; Graft vs Host Disease/prevention & control/immunology ; Glioblastoma/therapy/immunology ; Mice ; Killer Cells, Natural/immunology ; Aged ; Treatment Outcome ; Cell Line, Tumor ; },
abstract = {Patients with recurrent high-grade glioblastoma have a median survival of 6-8 months, with limited therapeutic options. In recent years, interest has grown in applying chimeric antigen receptor T (CAR-T) cells to solid cancers, including advanced gliomas. Here we generated off-the-shelf CRISPR-Cas9-edited IL-13Rα2-specific allogeneic universal CAR-T cells (MT026) by disrupting the endogenous TCR to prevent graft-versus-host disease and knocking out HLA class I molecules to mitigate the host-versus-graft response, and observed minimal NK-cell-mediated rejection in preclinical studies. In a first-in-human, single-center, open-label investigator-initiated trial (ChiCTR2000028801) in patients with high-grade glioma with prior therapy failure and short life expectancy, intrathecal injection of MT026 via lumbar puncture (1.0-3.0×10^7 cells per dose) demonstrated favorable tolerability and safety (primary outcome), pharmacokinetic characteristics, and preliminary clinical activity (secondary outcomes). Among the five patients enrolled, one achieved a complete response and three achieved partial responses. No grade ≥3 adverse events were observed; the predominant treatment-related toxicities were grade 1-2 pyrexia, hypoxia, and vomiting. Trial enrolment was halted after enrolment of the first five patients, however these preliminary clinical data support the potential benefit of locally administered allogeneic universal CAR-T cell therapy for recurrent glioblastoma.},
}

Humans
Female
Male
*Immunotherapy, Adoptive/methods/adverse effects
*Interleukin-13 Receptor alpha2 Subunit/genetics/immunology
Middle Aged
Adult
*Glioma/therapy/immunology/pathology
CRISPR-Cas Systems/genetics
*Receptors, Chimeric Antigen/genetics/immunology
*T-Lymphocytes/immunology/transplantation/metabolism
*Brain Neoplasms/therapy/immunology/pathology
Gene Editing
Neoplasm Recurrence, Local/therapy
Animals
Graft vs Host Disease/prevention & control/immunology
Glioblastoma/therapy/immunology
Mice
Killer Cells, Natural/immunology
Aged
Treatment Outcome
Cell Line, Tumor

@article {pmid41494573,
year = {2026},
author = {Pu, Z and Wang, X and Chen, Y and Li, J and He, X and Chen, W and Zhao, C},
title = {Application of CRISPR/Cas9 gene editing system in microalgal metabolic engineering and synthetic strategies of functional food ingredients.},
journal = {Biotechnology advances},
volume = {87},
number = {},
pages = {108796},
doi = {10.1016/j.biotechadv.2026.108796},
pmid = {41494573},
issn = {1873-1899},
mesh = {*Microalgae/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Metabolic Engineering/methods ; *Functional Food ; *Food Ingredients ; },
abstract = {Microalgae are natural and sustainable biological resources rich in high-value nutrients such as lipids, proteins, and functional pigments, which show great potential in the fields of functional foods, dietary supplements, and natural colorants. However, the yields of target components in natural microalgae are often insufficient to meet commercialization demands. The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) gene editing system, a revolutionary technology, provides a precise and effective means for targeted improvement of microalgae to enhance their nutritional value and yields. This review first outlines the basic principles of the CRISPR/Cas9 system, including its core components and gene editing mechanism. It then summarizes the application of this technology in microalgae, focusing on successful cases of modifying metabolic pathways to enrich specific nutrients, such as increasing the unsaturated fatty acid content of lipids, increasing the proportion of edible proteins, and enriching natural pigments with antioxidant properties. In addition, this review discusses the main challenges faced when applying this technology to microalgae, including delivery difficulties due to strong cell walls, low efficiency of genetic transformation, and the risk of off-target effects. Finally, the paper describes cutting-edge strategies to address these challenges, such as the development of high-fidelity Cas9 enzymes and the optimization of a single-guide RNA (sgRNA) design. Continued advances in these technologies are propelling microalgae into efficient and sustainable "cell factories", providing the food industry with more natural, healthy, and high-value functional ingredients.},
}

*Microalgae/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Metabolic Engineering/methods
*Functional Food
*Food Ingredients

@article {pmid41430241,
year = {2025},
author = {Nieto-Sanchez, A and Martinez-Lage, M and Puig-Serra, P and Carpintero, S and Alonso-Yanez, A and Ojeda-Walczuk, P and Ibañez-Navarro, M and Pita, G and Moya, FJ and Moreno, C and Martin, MC and Alonso, R and Nuñez-Torres, R and Sanchez-Arevalo Lobo, VJ and Alonso-Guirado, L and Malats, N and Gonzalez-Neira, A and Fernandez, L and Roda-Navarro, P and Torres-Ruiz, R and Rodriguez-Perales, S},
title = {Selective genome editing of amplified oncogenes triggers immunogenic cell death and tumor remodeling.},
journal = {Molecular cancer},
volume = {25},
number = {1},
pages = {21},
pmid = {41430241},
issn = {1476-4598},
mesh = {Humans ; Animals ; *Gene Editing/methods ; CRISPR-Cas Systems ; Mice ; *Oncogenes/genetics ; Cell Line, Tumor ; *Immunogenic Cell Death/genetics ; *Gene Amplification ; *Neoplasms/genetics/pathology/immunology ; Tumor Microenvironment/genetics ; Xenograft Model Antitumor Assays ; },
abstract = {Oncogene amplifications fuel some of the most lethal, therapy‑refractory cancers, yet remain clinically untargeted. We report a single‑guide CRISPR/Cas9 strategy that converts the sheer copy‑number excess of oncogene amplicons into an Achilles' heel. A solitary intronic double‑strand break is innocuous in diploid genomes but collapses oncogene amplification‑positive cells across neuroblastoma, small‑cell lung and colorectal carcinoma models, driving > 90% loss of viability, G2/M blockade and catastrophic DNA‑damage signalling. Amplified‑locus cleavage rewires transcription toward cell death activation, necroptosis and cGAS-STING-mediated immunogenic cell death, enabling dendritic‑cell cross‑priming and T‑cell activation and proliferation. In xenografts, delivery of the intronic sgRNA shrinks tumours by 90%, prolongs survival and remodels the innate tumour microenvironment. Deep sequencing confirms negligible off‑target editing, and combination with doxorubicin achieves supra‑additive killing. These findings establish amplification density, not sequence content, as a tractable, tumour‑exclusive target and unveil a dual‑action platform that is simultaneously cytotoxic and immunostimulatory. Editing of tumor amplifications therefore offers a blueprint for translating copy‑number aberrations into precision genome‑editing therapies for treatment‑resistant cancers.},
}

Humans
Animals
*Gene Editing/methods
CRISPR-Cas Systems
Mice
*Oncogenes/genetics
Cell Line, Tumor
*Immunogenic Cell Death/genetics
*Gene Amplification
*Neoplasms/genetics/pathology/immunology
Tumor Microenvironment/genetics
Xenograft Model Antitumor Assays

@article {pmid41254880,
year = {2026},
author = {Zhang, L and Xiang, C and Zheng, N and Kang, H and Kong, L and Huang, W and Peng, H and Lian, Y and Lu, W and Gao, M and Wang, J and Meksem, K and Peng, D and Yu, F and Liu, S},
title = {GmSHMT08-Mediated Soybean Cyst Nematode Resistance Is Negatively Modulated by Two Heat Shock Protein 70s in Soybean.},
journal = {Plant, cell & environment},
volume = {49},
number = {3},
pages = {1214-1228},
doi = {10.1111/pce.70296},
pmid = {41254880},
issn = {1365-3040},
support = {//This work was supported by the Joint Fund for S&T Research and Development Programme of Henan, China (grant no. 222301420028), the China Postdoctoral Science Foundation (grant no. 2023M741140), the Postdoctoral Fellowship Programme of CPSF (grant no. GZC20230772), the National Natural Science Foundation of China (grant no. 31972248), and the National Key Research and Development Programme of China (grant no. 2023YFD1400400)./ ; //This work was supported by the National Key Research and Development Programme of China(grant no. 2023YFD1400400), the China Postdoctoral Science Foundation (grant no. 2023M741140), the Postdoctoral Fellowship Programme of CPSF (grant no. GZC20230772), the Joint Fund for S&T Research and Development Programme of Henan, China (grant no. 222301420028), and the National Natural Science Foundation of China (grant no. 31972248)./ ; },
mesh = {*Glycine max/genetics/parasitology/metabolism/immunology ; *Disease Resistance/genetics ; Animals ; *Plant Diseases/parasitology/immunology/genetics ; *HSP70 Heat-Shock Proteins/metabolism/genetics ; *Plant Proteins/metabolism/genetics ; *Tylenchoidea/physiology ; Plants, Genetically Modified ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; },
abstract = {The soybean GmSHMT08 (Rhg4) is a major gene conferring resistance to soybean cyst nematode (SCN), which is a devastating pathogen in soybean, yet its underlying resistance mechanism remains elusive. Heat shock protein 70 (HSP70) mainly functions in maintaining protein homoeostasis and regulating plant immunity. In this study, CRISPR/Cas9-mediated knockout of two highly similar GmHSP70s [GmHSP70-13a (Glyma.13g254900) and GmHSP70-15a (Glyma.15g059900)] enhanced, whereas overexpressing either of them suppressed, SCN resistance. Both GmHSP70-13a and GmHSP70-15a interacted with the key one-carbon metabolism enzyme GmSHMT08, and promoted GmSHMT08 degradation via 26S proteasome pathway, but neither of them altered GmSHMT08 transcript levels in soybean. Furthermore, both GmHSP70-13a and GmHSP70-15a were strongly induced in susceptible soybeans, while both of them still remained low in resistant soybeans, after SCN infection. Taken together, GmSHMT08-mediated SCN resistance is shown to be negatively modulated by both GmHSP70-13a and GmHSP70-15a. The regulation is facilitated through interacting with and influencing 26S proteasome system-involved degradation of GmSHMT08. This study elucidates a SCN-resistance mechanism of GmSHMT08, expands functions of HSP70s and provides two GmHSP70s gene-editing targets for soybean SCN resistance breeding.},
}

*Glycine max/genetics/parasitology/metabolism/immunology
*Disease Resistance/genetics
Animals
*Plant Diseases/parasitology/immunology/genetics
*HSP70 Heat-Shock Proteins/metabolism/genetics
*Plant Proteins/metabolism/genetics
*Tylenchoidea/physiology
Plants, Genetically Modified
Gene Expression Regulation, Plant
CRISPR-Cas Systems

@article {pmid41174881,
year = {2026},
author = {Hierl, M and Bischof, J and Liemberger, B and Kocher, T and Hainzl, S and Gruner, S and Trattner, L and Reichl, V and Klausegger, A and Aussel, C and Ammann, S and Raab, R and Andrieux, G and Wolf, M and Hochmann, S and Ebner, P and Wally, V and Zauner, R and Guttmann-Gruber, C and Gratz, IK and Hofbauer, JP and Cathomen, T and Strunk, D and Bauer, JW and Koller, U},
title = {Prime editing as a promising therapeutic strategy for junctional epidermolysis bullosa.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {2},
pages = {817-831},
doi = {10.1016/j.ymthe.2025.10.054},
pmid = {41174881},
issn = {1525-0024},
mesh = {*Epidermolysis Bullosa, Junctional/therapy/genetics ; Humans ; *Gene Editing/methods ; Collagen Type XVII ; *Non-Fibrillar Collagens/genetics/metabolism ; Keratinocytes/metabolism ; Animals ; Mice ; *Genetic Therapy/methods ; *Autoantigens/genetics/metabolism ; Disease Models, Animal ; CRISPR-Cas Systems ; },
abstract = {Gene therapies offer new possibilities for the precise correction of monogenic disorders. Here, we present the first prime editing (PE)-based gene repair strategy for pathogenic COL17A1 variants that cause junctional epidermolysis bullosa (JEB). Type XVII collagen (C17), encoded by COL17A1, plays a critical role in skin aging, regeneration, and the maintenance of epidermal stem cell integrity. Treatment of primary human JEB keratinocytes with PE mRNAs resulted in COL17A1 editing efficiencies of up to 60% in bulk-treated cells, leading to the restoration of full-length, accurately shed C17. Chromosomal aberrations analysis by single targeted linker-mediated PCR sequencing analysis of gene-edited JEB keratinocytes confirmed the absence of unintended chromosomal rearrangements at potential off-target sites and only minimal on-target aberrations. Remarkably, in a xenograft model, in which C17[+] cells represented only 55.9% of the input population, COL17A1-corrected cells populated 92.2% of the basal keratinocyte layer in the resulting skin grafts after 6 weeks. These observations highlight a potential selective advantage imparted by C17 restoration, in line with its canonical role in anchoring hemidesmosomes to the basement membrane and preserving the structural integrity of the interfollicular epidermal stem cell niche. Based on our results, we envision PE as an efficient and safe option to restore gene function in EB and other genodermatoses.},
}

*Epidermolysis Bullosa, Junctional/therapy/genetics
Humans
*Gene Editing/methods
Collagen Type XVII
*Non-Fibrillar Collagens/genetics/metabolism
Keratinocytes/metabolism
Animals
Mice
*Genetic Therapy/methods
*Autoantigens/genetics/metabolism
Disease Models, Animal
CRISPR-Cas Systems

@article {pmid41173724,
year = {2026},
author = {Chen, Y and Duan, Q and Wang, L and He, Q and Fang, L and Xiao, L and Song, H and Cao, Y},
title = {Genome-scale CRISPRi and base-editing libraries for genetic decoding and strain engineering in Shewanella.},
journal = {Trends in biotechnology},
volume = {44},
number = {2},
pages = {496-520},
doi = {10.1016/j.tibtech.2025.10.005},
pmid = {41173724},
issn = {1879-3096},
mesh = {*Shewanella/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Genome, Bacterial/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Library ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based libraries with diverse gene-editing functions, such as gene knockdown and mutation, can significantly accelerate our understanding of complex metabolic networks in microorganisms, particularly for species beyond classical model organisms. Here, three distinct CRISPR-based libraries were designed in the electroactive microorganism Shewanella oneidensis MR-1: a CRISPR interference (CRISPRi) library covering 99.6% of genes in the genome, a protein mutation library focused on genes involved in carbon metabolism, and an inactivation library, with sizes of 30 804, 5963, and 4072 single guide (sg)RNAs, respectively. The principles for the design and construction of libraries were validated, and a conjugation-based library transformation method with high coverage and uniformity was developed. For the first time, we explored the potential essential genes of S. oneidensis MR-1, and expanded the substrate spectrum available for electricity generation, including glucose and chitin. These efforts enable deeper genomic interrogation of Shewanella, and provide a framework for applying genome-scale CRISPR-based tools to other undercharacterized microbial species.},
}

*Shewanella/genetics/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Genome, Bacterial/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Gene Library

@article {pmid41130838,
year = {2026},
author = {Crooke, H and Schwindt, S and Fletcher, SL and Isken, O and Harding, S and Berkley, N and Tait-Burkard, C and Warren, C and Whitelaw, CBA and Tautz, N and Lillico, SG},
title = {DNAJC14 gene-edited pigs are resistant to classical pestiviruses.},
journal = {Trends in biotechnology},
volume = {44},
number = {2},
pages = {570-586},
doi = {10.1016/j.tibtech.2025.09.008},
pmid = {41130838},
issn = {1879-3096},
mesh = {Animals ; Swine/genetics ; *Gene Editing/methods ; *Classical Swine Fever Virus/genetics/physiology/pathogenicity ; CRISPR-Cas Systems ; *Classical Swine Fever/genetics/virology ; Diarrhea Viruses, Bovine Viral ; *Disease Resistance/genetics ; },
abstract = {Infectious diseases remain a major impediment to livestock production, negatively impacting both productivity and welfare. Where key interactions between viruses and host proteins have been identified, it is possible to rationally devise intervention strategies. In vitro studies have identified the host protein DNAJC14 as a core component of the replicative cycle of classical pestiviruses. Outbreaks caused by this group of viruses cause enormous losses in stock farming due to culling and export restrictions. Using CRISPR/Cas9 gene editing, we produced a cohort of pigs with altered DNAJC14. Primary cells from these animals did not support replication of either classical swine fever virus (CSFV) or bovine viral diarrhoea virus (BVDV) in vitro. In vivo challenge with CSFV revealed that the edited pigs displayed complete resistance to infection. This establishes gene editing as an additional strategy that can contribute to the control of classical pestiviruses.},
}

Animals
Swine/genetics
*Gene Editing/methods
*Classical Swine Fever Virus/genetics/physiology/pathogenicity
CRISPR-Cas Systems
*Classical Swine Fever/genetics/virology
Diarrhea Viruses, Bovine Viral
*Disease Resistance/genetics

@article {pmid40915965,
year = {2026},
author = {Leech, D and Previtera, DA and Zhang, Y and Botella, JR and Crisp, PA},
title = {Precision plant epigenome editing: what, how, and why.},
journal = {Trends in plant science},
volume = {31},
number = {2},
pages = {192-204},
doi = {10.1016/j.tplants.2025.08.009},
pmid = {40915965},
issn = {1878-4372},
mesh = {*Gene Editing/methods ; *Epigenome/genetics ; *Plants/genetics ; *Genome, Plant/genetics ; *Epigenesis, Genetic ; CRISPR-Cas Systems ; Epigenome Editing ; },
abstract = {Advances in genome engineering have paved the way for targeted epigenome engineering, providing fundamental insights into the role of epigenetic modifications in trait inheritance. Engineered epialleles have already delivered stable, heritable changes in agronomic traits. Despite this capacity, progress in the field has not yet achieved its potential, leaving many avenues of research unexplored. In this review we examine the factors influencing this progress, including the advances in current epigenome editing techniques, the key research goals and translational applications, and the challenges in the selection of ideal target loci. We propose that improved tools for the selection of target loci, particularly in large and complex genomes, are needed to propel the field forward.},
}

*Gene Editing/methods
*Epigenome/genetics
*Plants/genetics
*Genome, Plant/genetics
*Epigenesis, Genetic
CRISPR-Cas Systems
Epigenome Editing

@article {pmid40713217,
year = {2026},
author = {Getachew, H and Metais, T and Daheron, L and Garita-Hernandez, M},
title = {Advances and optimization strategies in prime editing of human pluripotent stem cells.},
journal = {Trends in biotechnology},
volume = {44},
number = {2},
pages = {351-364},
doi = {10.1016/j.tibtech.2025.06.017},
pmid = {40713217},
issn = {1879-3096},
mesh = {Humans ; *Gene Editing/methods ; *Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; },
abstract = {Prime editing, first introduced in 2019, is a precise and efficient genome-editing technique with applications across various organisms and cell lines, including human pluripotent stem cells (hPSCs). Despite its revolutionary potential, prime editing of hPSCs often shows low efficiency, hindered by current delivery methods and DNA repair mechanisms. In this review, we explore prime editing of hPSCs, emphasizing the optimization necessary for creating ex vivo and in vitro disease models, which are critical for developing personalized therapeutics. We discuss key prime-editing methods for hPSCs, optimization strategies, tools available for prime editing, and the rigorous quality control required before and after genome engineering for downstream applications.},
}

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

@article {pmid41626318,
year = {2025},
author = {Sustek-Sánchez, F and Eelmets, E and Nigul, L and Kärblane, K and Laasmaa, M and Balode-Sausina, M and Berzina, SA and Ducis, D and Kaktina, E and Jaškūne, K and Rognli, OA and Rostoks, N and Sarmiento, C},
title = {Isolation and transformation of perennial ryegrass (Lolium perenne L.) protoplasts for the in vivo assessment of guide RNAs editing efficiency.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1744085},
pmid = {41626318},
issn = {1664-462X},
abstract = {Protoplasts are broadly used to perform different cellular and genetic assays. Transformation of protoplasts requires isolation methods that generate a large number of intact cells suitable for downstream applications. Lolium perenne L. is an important forage grass species for which gene editing techniques are in their early stages. Using protoplasts has previously been reported as a suitable approach to test the genome editing efficiency of guide RNAs in important grass species like wheat and rice. This approach can speed up and increase the chances of generating edited plants, especially when working with species for which stable transformation methods have not been established yet. Testing two different approaches regarding the processing of L. perenne L. tillers showed that using a blender for disintegrating the tissue was easier and faster than cutting the tillers with a razor blade. Conversely, the more classical strategy (cutting with a razor) provided a higher number of viable protoplasts. The use of an enzyme solution containing 2% cellulase during 8 h was shown to be the best experimental condition for protoplast isolation. The addition of a sucrose cushion improved the purification of alive cells, which were then positively transformed with guide RNA encoding vectors using polyethylene glycol. The presence of indels induced by these vectors was then confirmed through decomposition-based analysis of their sequenced genomic DNA. These results demonstrated the suitability of using protoplasts for the in vivo assessment of guide RNAs editing efficiency.},
}

@article {pmid41579807,
year = {2026},
author = {Zhang, Y and Wu, M and Geng, F and Wang, Y and Lü, J},
title = {Deciphering functional redundancy of lysine decarboxylases in probiotic E. coli Nissle 1917 via an integrated CRISPR-FTIR phenomics platform.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {351},
number = {},
pages = {127508},
doi = {10.1016/j.saa.2026.127508},
pmid = {41579807},
issn = {1873-3557},
mesh = {*Escherichia coli/enzymology/genetics ; Spectroscopy, Fourier Transform Infrared/methods ; *CRISPR-Cas Systems ; *Probiotics/metabolism ; *Carboxy-Lyases/metabolism/genetics ; Mutation ; *Escherichia coli Proteins/metabolism/genetics ; Phenotype ; },
abstract = {Fourier transform infrared (FTIR) microspectroscopy is a rapid, label-free tool for microbial metabolic phenotyping. Here, we integrate synchrotron-based FTIR microspectroscopy with CRISPR-Cas9 editing to decipher the functional redundancy of lysine decarboxylases (LdcC1 and LdcC2) in probiotic Escherichia coli Nissle 1917. Under lysine stress, isogenic mutants (ΔldcC1, ΔldcC1ΔldcC2) exhibited distinct FTIR fingerprints. Spectral analysis revealed: (i) CH shifts (2950-2850 cm[-1]) indicating ΔldcC1-specific membrane remodeling; (ii) Amide I band profile alterations (∼1650 cm[-1]) suggesting protein structural perturbations; and (iii) a constitutive elevation in 1220-1260 cm[-1] band area in the double mutant, revealing a basal state of metabolic frailty. Principal component analysis of second-derivative spectra revealed clear separation trends among strain phenotypes. We establish LdcC2 as a crucial functional complement, while LdcC1 uniquely contributes to membrane homeostasis. The compensatory stress response activated in the double mutant underscores metabolic redundancy as a cornerstone of intrinsic cellular robustness. Collectively, this work validates a CRISPR-FTIR phenomics platform that bridges targeted genetics with global biochemistry, offering a rapid alternative for functional genomics and metabolic engineering in microbes.},
}

*Escherichia coli/enzymology/genetics
Spectroscopy, Fourier Transform Infrared/methods
*CRISPR-Cas Systems
*Probiotics/metabolism
*Carboxy-Lyases/metabolism/genetics
Mutation
*Escherichia coli Proteins/metabolism/genetics
Phenotype

@article {pmid41557883,
year = {2026},
author = {Jiang, Y and Gao, H and Ran, Q and Xian, Y and Zhang, C},
title = {Near-Infrared Light-Inducible CRISPR-Cas12a/Tetrahedral DNA Nanosystem for Spatiotemporal Control of mtDNA Mutation Imaging and Gene Therapy.},
journal = {Nano letters},
volume = {26},
number = {4},
pages = {1438-1448},
doi = {10.1021/acs.nanolett.5c05621},
pmid = {41557883},
issn = {1530-6992},
mesh = {*CRISPR-Cas Systems/genetics ; Animals ; Infrared Rays ; *DNA, Mitochondrial/genetics ; Mice ; Humans ; *Genetic Therapy/methods ; *Nanoparticles/chemistry ; Mutation ; Gene Editing/methods ; Cell Line, Tumor ; *Neoplasms/therapy/genetics ; },
abstract = {Mitochondrial DNA (mtDNA) mutations are associated with tumor progression and metabolic dysregulation, yet their spatiotemporal visualization and selective therapeutic modulation remain challenging owing to their low abundance and mitochondrial double-membrane barrier. We report a near-infrared (NIR) light-inducible CRISPR-Cas12a/tetrahedral DNA nanosystem for spatiotemporally resolved imaging and editing of mtDNA mutations. The nanosystem integrates upconversion nanoparticles as NIR to ultraviolet converters, photocleavable DNA linkers, and Cas12a/crRNA modules. NIR irradiation triggers linker cleavage, activates Cas12a trans-cleavage, and generates amplified fluorescence for mtDNA mutation detection with a detection limit of 0.83 pM. In living cells and tumor-bearing mice, the nanosystem enables high-spatiotemporal resolution imaging of mtDNA mutations while inducing DNA self-assembly and CRISPR-based mtDNA editing. These disrupt the mitochondrial membrane potential, increase the level of reactive oxygen species, and promote apoptosis, resulting in effective tumor suppression. This nanosystem enables the controllable imaging of mutant mtDNA and gene modulation, offering new opportunities for spatiotemporal cancer theranostics.},
}

*CRISPR-Cas Systems/genetics
Animals
Infrared Rays
*DNA, Mitochondrial/genetics
Mice
Humans
*Genetic Therapy/methods
*Nanoparticles/chemistry
Mutation
Gene Editing/methods
Cell Line, Tumor
*Neoplasms/therapy/genetics

@article {pmid41484984,
year = {2026},
author = {Forbes, CA and Shaw, NC and Chen, KG and Hedges, M and Er, TS and Hool, L and Ward, M and Poulton, C and Baynam, G and Lassmann, T and Fear, VS},
title = {A precision medicine approach to interpret a GATA4 genetic variant in a paediatric patient with congenital heart disease.},
journal = {Human genomics},
volume = {20},
number = {1},
pages = {29},
pmid = {41484984},
issn = {1479-7364},
support = {APP2004282//National Health and Medical Research Council/ ; UWA-VCCRI Chair in Cardiovascular Research//Wesfarmers/ ; Telethon 7 Ball, Gift of Giving, 2020.//McCusker Charitable Foundation/ ; Lassmann//Feilman Foundation/ ; Perron People//Stan Perron Charitable Foundation/ ; Perron Programs and Partnership, 2025//Stan Perron Charitable Foundation/ ; },
mesh = {Humans ; *GATA4 Transcription Factor/genetics ; Induced Pluripotent Stem Cells/metabolism/pathology ; Myocytes, Cardiac/metabolism/pathology ; *Heart Defects, Congenital/genetics/pathology ; *Precision Medicine/methods ; Gene Editing ; *Genetic Variation ; Cell Differentiation/genetics ; CRISPR-Cas Systems ; Male ; Child ; Female ; },
abstract = {BACKGROUND: Patients with congenital heart disease are identified in 1% of live births. Improved surgical intervention means many patients now survive to adulthood, the corollary of which is increased mortality in the over-65-year-old congenital heart disease (CHD) population. In the clinic, genetic sequencing increasingly identifies novel genetic variants in genes related to CHD. Traditional assays for interpreting novel genetic variants are often limited by gene-specificity, whereas animal models are cumbersome and may not accurately reflect human disease. This study investigates CRISPR gene editing in induced pluripotent stem cells and cardiomyocyte-directed differentiation as a human disease model to investigate novel genetic variants identified in association with CHD.

METHODS AND RESULTS: We identified a GATA4 p.Arg284His genetic variant in a paediatric patient. This genetic variant was introduced into induced pluripotent stem cells (iPSCs) using CRISPR gene editing with homology-directed-repair. GATA4 genetic variant and isogenic control iPSCs were selected and differentiated into cardiomyocytes. Expression of the GATA4 p.Arg284His variant resulted in altered calcium transients, indicative of CHD and consistent with the patient's clinical phenotype. Transcriptomics revealed cellular pathway changes in cardiac development, calcium handling, and energy metabolism that contribute to disease aetiology, mechanism and identification of potential treatments.

CONCLUSION: Directed differentiation of iPSCs harbouring the GATA4 p.Arg284His genetic variant recapitulated the CHD phenotype, indicated disease mechanisms, and pointed to potential sites for targeting with therapy. The study highlights the utility of transcriptomics for the functional interpretation of cardiac genetic variants and is an exemplar for precision medicine approaches for the investigation of CHD.},
}

Humans
*GATA4 Transcription Factor/genetics
Induced Pluripotent Stem Cells/metabolism/pathology
Myocytes, Cardiac/metabolism/pathology
*Heart Defects, Congenital/genetics/pathology
*Precision Medicine/methods
Gene Editing
*Genetic Variation
Cell Differentiation/genetics
CRISPR-Cas Systems
Male
Child
Female

@article {pmid41466059,
year = {2025},
author = {Chen, W and He, P and Ding, L and Lou, W and Wang, Y and Shi, W and Fan, Z and Sheng, Y and Luo, J and Tan, Z and Wang, Y and Di, W and Ke, X and Yu, B},
title = {EPI-SauriCas9-based mouse ovarian cancer models recapitulating pten deletion in patients.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {159},
pmid = {41466059},
issn = {2399-3642},
support = {82403651//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; Female ; *PTEN Phosphohydrolase/genetics ; *Ovarian Neoplasms/genetics/pathology/drug therapy ; Mice ; Disease Models, Animal ; Humans ; Gene Deletion ; Tumor Suppressor Protein p53/genetics ; CRISPR-Cas Systems ; },
abstract = {Ovarian cancer remains a deadly gynecological malignancy, with PTEN loss and TP53 mutations frequently implicated in its progression. However, suitable models for studying ovarian cancers with PTEN and TP53 deletions are rare. Here we develop and validate the mouse ovarian epithelium with Pten and Trp53 deletions (MEPP) model using the EPI-SauriCas9 system. We demonstrate the role of Pten loss in promoting tumorigenicity and metastasis. Single-cell RNA sequencing reveals distinct epithelial subpopulations with varying metastatic potential. MEPP also recapitulates key features of human ovarian cancer, including its immune landscape and therapeutic responses. High-throughput drug screening identifies FK228 and thioguanine as promising therapeutic candidates, both of which show in vivo efficacy and are validated in PTEN-deleted organoids. Together, these results establish MEPP as a platform for studying PTEN-deleted ovarian cancer and provide a strategy for generating clinically relevant tumor models through targeted gene editing.},
}

Animals
Female
*PTEN Phosphohydrolase/genetics
*Ovarian Neoplasms/genetics/pathology/drug therapy
Mice
Disease Models, Animal
Humans
Gene Deletion
Tumor Suppressor Protein p53/genetics
CRISPR-Cas Systems

@article {pmid41451985,
year = {2026},
author = {Wang, Z and Hu, T and Liu, W and Zhou, H and Lv, X and Li, H and Li, X and Huang, X and He, L},
title = {Development and clinical validation of an ERA-CRISPR/Cas12a assay for the rapid detection of 14 high-risk HPV types.},
journal = {Microbiology spectrum},
volume = {14},
number = {2},
pages = {e0303625},
doi = {10.1128/spectrum.03036-25},
pmid = {41451985},
issn = {2165-0497},
support = {82100344//National Natural Science Foundation of China/ ; 2024A22//Tongji Hospital/ ; },
mesh = {Humans ; *Papillomavirus Infections/diagnosis/virology ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Female ; *Molecular Diagnostic Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; *Papillomaviridae/genetics/isolation & purification/classification ; Uterine Cervical Neoplasms/virology/diagnosis ; Reproducibility of Results ; DNA, Viral/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {UNLABELLED: Persistent infection with high-risk human papillomavirus (HR-HPV) is the leading cause of cervical cancer, highlighting the critical need for early detection to improve prevention. Although real-time quantitative polymerase chain reaction (RT-qPCR) remains the gold standard for HR-HPV detection, its dependence on sophisticated equipment, complex procedures, and trained personnel limits accessibility. Here, we developed a simplified assay for 14 HR-HPV types by integrating direct lysis, enzyme-mediated isothermal rapid amplification (ERA), and CRISPR-Cas12a-mediated cleavage into a streamlined workflow that requires only a basic isothermal heating device. The optimized system achieved a sensitivity of 50 copies per reaction with no cross-reactivity, while a refined lysis buffer containing 20% Chelex-100 minimized inhibition from vaginal swab samples, thereby enhancing detection performance. Validation with 152 clinical samples demonstrated 97.62% sensitivity and 100% specificity, confirming the reliability of the method. This user-friendly and cost-effective assay requires minimal equipment, enabling rapid and field-deployable HR-HPV detection, and offers a practical alternative to conventional laboratory-based approaches, particularly in resource-limited settings.

IMPORTANCE: High-risk human papillomavirus (HR-HPV) is the principal etiological agent of cervical cancer, and early detection remains central to effective disease prevention. Current PCR-based assays, however, rely on specialized laboratories and trained personnel, limiting their deployment in many settings. Here, we report a streamlined CRISPR-Cas12a assay that integrates direct sample lysis, ERA, and CRISPR-based detection into a single workflow operable with only a simple heating device to determine the presence of 14 HR-HPV types. The assay achieves high analytical sensitivity, strong specificity, and robust clinical performance while maintaining low cost and ease of use. This platform enables rapid HR-HPV detection and scalable screening, particularly in resource-constrained environments, with the potential to facilitate earlier intervention and reduce cervical cancer incidence.},
}

Humans
*Papillomavirus Infections/diagnosis/virology
*CRISPR-Cas Systems
Sensitivity and Specificity
Female
*Molecular Diagnostic Techniques/methods
*Nucleic Acid Amplification Techniques/methods
*Papillomaviridae/genetics/isolation & purification/classification
Uterine Cervical Neoplasms/virology/diagnosis
Reproducibility of Results
DNA, Viral/genetics
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41442816,
year = {2026},
author = {Zhu, Y},
title = {The potential and innovative applications of CRISPR gene editing technology in enzyme gene development.},
journal = {Enzyme and microbial technology},
volume = {195},
number = {},
pages = {110799},
doi = {10.1016/j.enzmictec.2025.110799},
pmid = {41442816},
issn = {1879-0909},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Humans ; *Enzymes/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The CRISPR gene editing technology is simple in design and highly efficient, making it the most widely used gene editing tool today. At present, CRISPR gene editing technology has shown a certain application value in enzyme development, but its application potential has not been fully developed. CRISPR gene editing technology can not only be used to knockin enzyme genes and knockout genes that are not conducive to enzyme expression, but can also be applied to single-base editing of enzyme genes, tandem sgRNA for multi-enzyme gene editing, sgRNA library for enzyme screening, endogenous enzyme gene modification, transcriptional activation or inhibition of enzyme gene expression, and fluorescence imaging of enzyme genes. Especially, this review innovatively proposes for the first time that CRISPR gene editing technology can be used for site specific fusion of enzyme genes, cell surface display of endogenous enzymes, and knockin of super long DNA for simultaneous expression of multiple enzymes, providing new ideas for maximizing the value of CRISPR gene editing technology in enzyme development in the future.},
}

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

@article {pmid41416796,
year = {2026},
author = {Athipanyasilp, N and Saowpak, S and Chaimayo, C and Angkasekwinai, N and Pattama, A and Athipanyasilp, A and Patchsung, M and Aphicho, K and Uttamapinant, C and Horthongkham, N},
title = {CRISPR-Cas13a SHERLOCK assay for rapid and sensitive detection of chikungunya virus.},
journal = {Microbiology spectrum},
volume = {14},
number = {2},
pages = {e0229825},
doi = {10.1128/spectrum.02298-25},
pmid = {41416796},
issn = {2165-0497},
support = {R016034012//Faculty of Medicine Siriraj Hospital, Mahidol University/ ; },
mesh = {*Chikungunya virus/genetics/isolation & purification ; *Chikungunya Fever/diagnosis/virology ; Humans ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; RNA, Viral/genetics ; *Molecular Diagnostic Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Real-Time Polymerase Chain Reaction/methods ; },
abstract = {Chikungunya virus (CHIKV), a major cause of acute febrile illness and joint pain, remains a significant public health threat in tropical regions. Rapid and accurate detection is essential for timely clinical management and outbreak control, particularly in resource-limited settings where real-time PCR (RT-qPCR) is often impractical. We developed and validated a SHERLOCK assay coupled with recombinase polymerase amplification for CHIKV RNA detection. Analytical performance was assessed by determining the limit of detection (LOD), cross-reactivity, clinical sensitivity and specificity, and predictive values. The assay achieved an LOD of 215 copies/reaction with no cross-reactivity against other alphaviruses or flaviviruses. Clinical testing of 146 plasma samples showed a sensitivity and specificity of 94.52% and 100% with lateral-flow readout and 97.26% and 100% with fluorescence readout, respectively. This study establishes a promising CRISPR-Cas13a-based SHERLOCK platform for CHIKV detection, demonstrating high analytical performance, rapid turnaround time, and potential for future adaptation to resource-limited settings.IMPORTANCEEarly and accurate detection of chikungunya virus (CHIKV) is critical for outbreak control, especially in resource-limited settings, where real-time PCR is not feasible. This study demonstrates that the CRISPR-Cas13a-based SHERLOCK platform, combined with RPA, achieves high diagnostic accuracy and a low detection limit, comparable to RT-qPCR. The assay's rapid turnaround time and simple lateral-flow readout make it a promising tool for point-of-care diagnostics during CHIKV outbreaks, potentially improving disease surveillance and clinical decision-making.},
}

*Chikungunya virus/genetics/isolation & purification
*Chikungunya Fever/diagnosis/virology
Humans
Sensitivity and Specificity
*CRISPR-Cas Systems
RNA, Viral/genetics
*Molecular Diagnostic Techniques/methods
Nucleic Acid Amplification Techniques/methods
Limit of Detection
Real-Time Polymerase Chain Reaction/methods

@article {pmid41367295,
year = {2026},
author = {Wyer, CAS and Amaro, IA and Pitcher, S and Ponlawat, A and Harrington, LC and Wolfner, MF and Hollis, B and Cator, LJ},
title = {Pickpocket315 affects male mating behavior in the yellow fever mosquito Aedes aegypti.},
journal = {G3 (Bethesda, Md.)},
volume = {16},
number = {2},
pages = {},
doi = {10.1093/g3journal/jkaf297},
pmid = {41367295},
issn = {2160-1836},
support = {R01-AI095491/NH/NIH HHS/United States ; //L.C.H and M.F.W and International Atomic Energy Association/ ; //Reproductive Biology of Male Aedes Mosquitoes for SIT Applications/ ; NE/S007415/1//Natural Environment Research Council/ ; },
mesh = {Animals ; *Aedes/genetics/physiology ; Male ; *Sexual Behavior, Animal ; Female ; CRISPR-Cas Systems ; *Insect Proteins/genetics/metabolism ; RNA Interference ; Mosquito Vectors/genetics ; Copulation ; Yellow Fever/transmission ; },
abstract = {The molecular basis of mating behavior in the important disease vector mosquito, Aedes aegypti, remains poorly characterized. We investigated the functional role of a pickpocket gene, ppk315, in male mating behavior using both RNAi-mediated knockdown and CRISPR/Cas9 approaches. Behavioral assays revealed that RNAi-treated males (dsPPK315) made fewer mating attempts, were less responsive to female acoustic cues, and were less likely to achieve copulation, though their latency to initiate contact when attempts were made was comparable to controls. Males with a CRISPR/Cas9-induced disruption to ppk315 exhibited reduced success in inseminating multiple females, consistent with previous reports from RNAi knockdown males, ruling out off-target effects as the source of behavioral changes. In contrast to the results of behavioral assays with RNAi, ppk315 mutant males (ppk315-/-) attempted copulation as frequently as wild-type males (ppk315+/+) but were slower to contact females. Despite these impairments in one-on-one interactions, both dsPPK315 and ppk315-/- males displayed normal mating success under competitive swarm-like conditions, potentially due to the socially facilitated activation of mating behavior. Collectively, our findings support a role for ppk315 in the initiation of mating behaviors via sensory detection, with context-dependent consequences for reproductive success.},
}

Animals
*Aedes/genetics/physiology
Male
*Sexual Behavior, Animal
Female
CRISPR-Cas Systems
*Insect Proteins/genetics/metabolism
RNA Interference
Mosquito Vectors/genetics
Copulation
Yellow Fever/transmission

@article {pmid40748748,
year = {2026},
author = {Teubner, JP and Tümen, D and Kandulski, A and Heumann, P and Mester, P and Aschenbrenner, E and Pollinger, K and Gunckel, M and Volz, B and Hein, T and Beltzig, PL and Tengler, L and Voll, F and Kreutz, M and Kunst, C and Nicolay, JP and Müller, M and Gülow, K},
title = {CRISPR-Cas9 screen reveals that inhibition of enhancer of zeste homolog 2 sensitizes malignant T cells to dimethyl-fumarate-induced cell death.},
journal = {The FEBS journal},
volume = {293},
number = {3},
pages = {749-765},
doi = {10.1111/febs.70208},
pmid = {40748748},
issn = {1742-4658},
support = {2022.096.1//Wilhelm Sander-Stiftung/ ; },
mesh = {*Enhancer of Zeste Homolog 2 Protein/antagonists & inhibitors/genetics/metabolism ; Humans ; *CRISPR-Cas Systems ; *Dimethyl Fumarate/pharmacology ; Pyridones/pharmacology ; Cell Death/drug effects ; *Lymphoma, T-Cell, Cutaneous/drug therapy/genetics/pathology/metabolism ; Cell Line, Tumor ; *Sezary Syndrome/drug therapy/genetics/pathology ; Benzamides/pharmacology ; Morpholines/pharmacology ; T-Lymphocytes/drug effects/metabolism/pathology ; Histones/metabolism ; NF-kappa B/genetics/metabolism ; Biphenyl Compounds ; },
abstract = {Constitutive activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway is a hallmark of many lymphocyte-associated cancers, including cutaneous T-cell lymphoma (CTCL) and its leukemic variant, the Sézary syndrome. Dimethyl fumarate (DMF) has been identified as a promising NF-κB-targeted therapy and has shown positive outcomes in a phase II clinical trial involving patients with Sézary syndrome. However, limited responsiveness remains a significant challenge. Through a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screen, we identified enhancer of zeste homolog 2 (EZH2; also known as histone-lysine N-methyltransferase) as a critical target for enhancing DMF-induced cell death. EZH2, the catalytic subunit of Polycomb Repressive Complex 2 (PRC2), is responsible for the methylation of histone H3 (H3K27). Combining DMF with the US Food and Drug Administration (FDA)-approved EZH2 inhibitor tazemetostat significantly increases cell death in patient-derived CTCL cells, offering a promising strategy to improve therapeutic outcomes and overcome limited responsiveness to DMF.},
}

*Enhancer of Zeste Homolog 2 Protein/antagonists & inhibitors/genetics/metabolism
Humans
*CRISPR-Cas Systems
*Dimethyl Fumarate/pharmacology
Pyridones/pharmacology
Cell Death/drug effects
*Lymphoma, T-Cell, Cutaneous/drug therapy/genetics/pathology/metabolism
Cell Line, Tumor
*Sezary Syndrome/drug therapy/genetics/pathology
Benzamides/pharmacology
Morpholines/pharmacology
T-Lymphocytes/drug effects/metabolism/pathology
Histones/metabolism
NF-kappa B/genetics/metabolism
Biphenyl Compounds

@article {pmid41623654,
year = {2026},
author = {Beh, JQ and Muzahid, NH and Mar, JH and Goh, CBS and Huët, MAL and Lim, SY and Rahman, S},
title = {Characterization of the CRISPR1-Cas array and its subtyping potential in Enterococcus faecalis from Malaysia.},
journal = {Access microbiology},
volume = {8},
number = {1},
pages = {},
pmid = {41623654},
issn = {2516-8290},
abstract = {Enterococcus faecalis is a gram-positive bacterium and a common cause of hospital-associated infections. Three major CRISPR loci have been discovered in this species, namely, CRISPR1-cas, CRISPR2 and CRISPR3-cas. We developed novel primers which target the CRISPR1-cas loci in E. faecalis and tested these primers on 26 E. faecalis isolates isolated from diverse settings from Segamat, Malaysia. Half of the isolates were found to carry the CRISPR1-cas9 locus, and the CRISPR1 array was successfully amplified in 12 out of 13 isolates that contained the cas9 gene. Characterization of the CRISPR array shows that CRISPR1-cas shares similar array length and typical repeat sequences with CRISPR2 but differs significantly in terms of spacer identities and terminal repeat (TR) sequences. Most CRISPR spacers encode for chromosomal DNA sequences. Genotype characterization based on ancestral spacer (AS) and TR sequences indicates that E. faecalis with the same CRISPR1-AS genotype do not always harbour the same CRISPR2-AS genotypes and vice versa. A combined CRISPR1-cas and CRISPR2 typing offers comparable discriminatory power to MLST, suggesting its potential to be used in short-term strain identification and epidemiological surveillance at a lower sequencing cost. Our study provides a genetic reference for future studies in Southeast Asia.},
}

@article {pmid41623178,
year = {2026},
author = {Hill, SF and Goldberg, EM},
title = {A hit for base editing: treatment of developmental epilepsy in a mouse model.},
journal = {The Journal of clinical investigation},
volume = {136},
number = {3},
pages = {},
pmid = {41623178},
issn = {1558-8238},
mesh = {Animals ; Mice ; Disease Models, Animal ; *Gene Editing ; *CRISPR-Cas Systems ; *Epilepsy/genetics/therapy ; *NAV1.6 Voltage-Gated Sodium Channel/genetics/metabolism ; Humans ; Mutation, Missense ; *Genetic Therapy ; },
abstract = {CRISPR/Cas9 base editing holds the potential to treat disease caused by single-nucleotide variants. In contrast with conventional CRISPR/Cas9 approaches, base editing enzymatically induces precise DNA alterations and can directly correct disease-causing variants. In this issue of JCI, Reever et al. used base editing to treat a mouse model of a severe neurodevelopmental disorder caused by a pathogenic missense variant in the voltage-gated sodium channel gene SCN8A. This work represents a starting point for the further refinement of base editing to treat genetic epilepsy.},
}

Animals
Mice
Disease Models, Animal
*Gene Editing
*CRISPR-Cas Systems
*Epilepsy/genetics/therapy
*NAV1.6 Voltage-Gated Sodium Channel/genetics/metabolism
Humans
Mutation, Missense
*Genetic Therapy

@article {pmid41621837,
year = {2026},
author = {Patraskaki, M and Seyedkatouli, N and Schlicker, L and Warmoes, MO and Cordero-Maldonado, ML and Heins-Marroquin, U and Linster, CL},
title = {Unveiling Immune System Perturbations in Early Development Through Zebrafish Models of NADHX Repair Deficiency.},
journal = {Journal of inherited metabolic disease},
volume = {49},
number = {2},
pages = {e70149},
pmid = {41621837},
issn = {1573-2665},
support = {PRIDE17/12244779/PARK-QC//Fonds National de la Recherche Luxembourg/ ; PRIDE19/14063202/ACTIVE//Fonds National de la Recherche Luxembourg/ ; C22/BM/17198760//Fonds National de la Recherche Luxembourg/ ; //Fondation Juniclair/ ; },
mesh = {Animals ; Zebrafish ; Disease Models, Animal ; *Zebrafish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Mutation ; NAD/metabolism ; Phenotype ; *Immune System/metabolism ; *Racemases and Epimerases/genetics/deficiency ; },
abstract = {The vital cofactors NADH and NADPH are prone to hydration, forming hydroxylated redox-inactive derivatives (NADHX and NADPHX) in cells. These damaged metabolites are repaired by two highly conserved enzymes, an NAD(P)HX dehydratase (NAXD) and an NAD(P)HX epimerase (NAXE). Mutations in NAXE or NAXD cause early onset progressive encephalopathy (PEBEL1 or PEBEL2), typically induced by fever or other triggers, and leading to premature death. To advance our comprehension of the disease mechanism and investigate potential therapeutic strategies, we generated zebrafish lines deficient in naxe or naxd using CRISPR/Cas9 technology. While both models accumulated NADHX, only naxd[-/-] larvae developed a severe phenotype, showing reduced locomotion and early death, which was partially rescued by nicotinic acid supplementation. Both mutant lines displayed signs of dysregulated immune function based on gene expression analyses and increased neutral red staining in the head region, indicating an increased number or activation of microglial cells. Our findings suggest that immune system perturbations play a role in PEBEL disease development, aligning with its inflammatory trigger-induced nature in humans. The naxd[-/-] model's responsiveness to nicotinic acid underscores its utility for preclinical drug screening. Overall, these models will be instrumental in furthering our understanding of PEBEL disease mechanisms and enhancing translational research efforts.},
}

Animals
Zebrafish
Disease Models, Animal
*Zebrafish Proteins/genetics/metabolism
CRISPR-Cas Systems
Mutation
NAD/metabolism
Phenotype
*Immune System/metabolism
*Racemases and Epimerases/genetics/deficiency

@article {pmid41620285,
year = {2026},
author = {Sohn, M and Pantsulaia, G and Brody, J},
title = {Mouse and human T cell Cas9-RNP/nucleofection-mediated gene-editing.},
journal = {Methods in cell biology},
volume = {201},
number = {},
pages = {91-107},
doi = {10.1016/bs.mcb.2025.03.020},
pmid = {41620285},
issn = {0091-679X},
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; Animals ; Mice ; *CD8-Positive T-Lymphocytes/metabolism ; *Ribonucleoproteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; *Transfection/methods ; },
abstract = {CRISPR/Cas9 technology has revolutionized genome editing, providing a precise and expeditious means of genetic modification. This breakthrough has enhanced our understanding of gene function, including T cell immunology. Efficient gene editing in primary T cells not only offers a robust investigative tool for dissecting gene function but also holds promise for advancing T cell-based immunotherapies, including next-generation chimeric antigen receptor T cells. In this article, we introduce a highly efficient gene editing protocol for unstimulated human CD8 T cells and unstimulated and stimulated murine CD8 T cells, utilizing transient nucleofection of ribonucleoprotein complexes composed of synthesized modified single guide RNAs (sgRNAs) and purified Cas9 protein. This protocol, initially devised for primary CD8 T cells, can be readily adapted to other primary cell cultures through optimization of nucleofection conditions as well. In essence, our method provides a practical and powerful approach for achieving precise and swift gene knockout in primary CD8 T cells.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems/genetics
Animals
Mice
*CD8-Positive T-Lymphocytes/metabolism
*Ribonucleoproteins/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Associated Protein 9/genetics/metabolism
*Transfection/methods

@article {pmid41618915,
year = {2026},
author = {Liu, Q and Huang, Y and Zhou, R and Ding, H and Nie, Q and Gong, X and Zuo, T and Wang, S and Liang, Y and Li, Y},
title = {A Minimal and Portable CRISPR Platform Based on Bifidobacterial Cas9 Enables Genome Editing in E. coli Nissle 1917.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00911},
pmid = {41618915},
issn = {2161-5063},
abstract = {Genetic manipulation of core gut probiotics remains challenging due to endogenous cellular barriers and a scarcity of efficient molecular tools, limiting progress in live biotherapeutic development. Here, we characterized the native type II-C CRISPR-Cas system in Bifidobacterium longum subsp. longum GNB (B. longum GNB). Through integrated bioinformatic analysis and high-throughput protospacer adjacent motif (PAM) screening, we identified a novel 5'-NNRMAT-3' (where R = A/G, M = A/C) motif recognized by its compact Cas9 nuclease (BLCas9). The stringent PAM dependency of BLCas9 was unequivocally confirmed by in vitro cleavage assays. Leveraging this endogenous mechanism, we developed a dual-plasmid editing platform for robust and multiplex genome engineering in the probiotic strain Escherichia coli Nissle 1917 (EcN). Application of this system notably enhanced extracellular γ-aminobutyric acid (GABA) production in EcN through targeted metabolic engineering. Our work provides the first molecular dissection of a type II-C system in Bifidobacterium longum and establishes a generalizable framework for the discovery and application of compact programmable nucleases, suggesting a viable strategy for modulating host physiology via the gut-brain axis.},
}

@article {pmid41617991,
year = {2026},
author = {Nagahata, N and Kato, K and Yamada, S and Kannan, S and Okazaki, S and Isayama, Y and Hiraizumi, M and Yamashita, K and Koonin, EV and Zhang, F and Nishimasu, H},
title = {Structural visualization of the molecular evolution of CRISPR-Cas9.},
journal = {Nature structural & molecular biology},
volume = {},
number = {},
pages = {},
pmid = {41617991},
issn = {1545-9985},
abstract = {RNA-guided DNA nucleases Cas9 and IscB (insertion sequences Cas9-like OrfB) are components of type II CRISPR-Cas adaptive immune systems and transposon-associated OMEGA (obligate mobile element-guided activity) systems, respectively. Sequence and structural comparisons indicate that IscB (~500 residues) evolved into Cas9 (~700-1,600 residues) through protein expansion coupled with guide RNA miniaturization. However, the specific sequence of events in this evolutionary transition remains unknown. Here, we report cryo-electron microscopy structures of four phylogenetically diverse RNA-guided nucleases-two IscBs and two Cas9s-each in complex with its cognate guide RNA and target DNA. Comparisons of these four complex structures to previously reported IscB and Cas9 structures indicate that evolution from IscB to Cas9 involved the loss of the N-terminal PLMP domain and the acquisition of the zinc-finger-containing REC3 domain, followed by bridge helix extension and REC1 domain acquisition. These structural changes led to expansion of the REC lobe, increasing the target DNA cleavage specificity. Additionally, the structural conservation of the RNA scaffolds indicates that the dual CRISPR RNA (crRNA) and trans-activating crRNA guides of CRISPR-Cas9 evolved from the single ωRNA guides of OMEGA systems. Our findings provide insights into the succession of structural changes involved in the exaptation of transposon-associated RNA-guided nucleases for the role of effector nucleases in adaptive immune systems.},
}

@article {pmid41614935,
year = {2026},
author = {El Semary, NAH and Fadiel, A and Eichenbaum, KD and Alhusayni, SA},
title = {Prokaryotic Molecular Defense Mechanisms and Their Potential Applications in Cancer Biology: A Special Consideration for Cyanobacterial Systems.},
journal = {Current issues in molecular biology},
volume = {48},
number = {1},
pages = {},
pmid = {41614935},
issn = {1467-3045},
support = {KFU253867//Vice Presidency of Higher studies and Scientific research, Deanship of Scientific research, King Faisal University, AlHufuf, Al-Ahsa, Kingdom of Saudi Arabia, postcode: 31982/ ; },
abstract = {Cyanobacteria harbor sophisticated molecular defense systems that have evolved over billions of years to protect against viral invasion and foreign genetic elements. These ancient photosynthetic organisms possess a diverse array of restriction-modification (R-M) systems and CRISPR-Cas arrays that present challenges for genetic engineering, but also offer unique opportunities for cancer-targeted biotechnological applications. These systems exist in prokaryotes mainly as defense mechanisms but they are currently used in molecular applications as gene editing tools. Moreover, latest developments in nucleases such as zinc finger nucleases (ZFNs), TALENs (transcription-activator-like effector nucleases) are discussed. A comprehensive genomic analysis of 126 cyanobacterial species found 89% encode multiple R-M systems, averaging 3.2 systems per genome, creating formidable barriers to transformation but also providing molecular machinery that could be harnessed for precise recognition and targeting of cancer cells. This review critically examines the dual nature of these defense systems, their ecological functions, and the emerging strategies to translate their molecular precision into advanced anticancer therapeutics. Hence, the review main objectives are to explore the recent understanding of these mechanisms and to exploit the knowledge gained in opening new avenues for cancer-focused targeted interventions, while acknowledging the significant challenges to translate these systems from laboratory curiosities to practical applications.},
}

@article {pmid41614578,
year = {2026},
author = {Barragán-Borrero, V and de Santana Lopes, A and Rodrigues Batista, ED and Höfer, M and Elias, R and Chakraborty, A and Ponce-Mañe, A and Descombes, C and Diezma-Navas, L and Petraki, L and Huber, M and Xu, S and Marí-Ordóñez, A},
title = {Strain, procedures, and tools for reproducible genetic transformation and genome editing of the emerging plant model Spirodela polyrhiza.},
journal = {The New phytologist},
volume = {},
number = {},
pages = {},
doi = {10.1111/nph.70919},
pmid = {41614578},
issn = {1469-8137},
support = {512079118//Emmy Noether Programme/ ; //Österreichischen Akademie der Wissenschaften/ ; 14002787004056//Erasmus+/ ; //Johannes Gutenberg-Universität Mainz/ ; 407023052//Deutsche Forschungsgemeinschaft/ ; 427577435//Deutsche Forschungsgemeinschaft/ ; 438887884//Deutsche Forschungsgemeinschaft/ ; },
abstract = {Duckweeds (Lemnaceae) have excellent potential for fundamental and applied research due to ease of cultivation, small size, and continuous fast clonal growth. However, their usage as model organisms and platforms for biotechnological applications is often limited by the lack of universal genetic manipulation methods necessary for transgene expression, gene editing, and other methods to modify gene expression. To identify suitable strains for genetic manipulation of the giant duckweed, Spirodela polyrhiza, we screened several genotypes for callus induction and regeneration and established genetic transformation. We identified SP162 to be amenable to Agrobacterium-mediated transformation via tissue culture. The procedure is robust and reproducible across laboratories, allowing stable expression of different reporter genes and selectable markers, enabling CRISPR/Cas9-mediated genome editing. In addition, due to a weak small RNA-based silencing response, S. polyrhiza sustains prolonged periods of transgene activity in transient expression assays. To promote duckweed research and encourage the adoption of S. polyrhiza, we have made SP162 (ID#: 5676) and its genome publicly available and provide here detailed procedures for its cultivation and transformation. Furthermore, we created a web server to explore its genome, retrieve gene sequences, and implement orthologous gene search and a gRNA design function for diverse CRISPR/Cas-based applications (https://agxu.uni-mainz.de/SP162/).},
}

@article {pmid41613863,
year = {2025},
author = {Safrygina, AA and Orlov, YL},
title = {Genetically engineered approaches to the treatment of cystic fibrosis.},
journal = {Biophysical reviews},
volume = {17},
number = {5},
pages = {1333-1358},
pmid = {41613863},
issn = {1867-2450},
abstract = {Failure of functions of CFTR (cystic fibrosis transmembrane conduction regulator) gene, which encodes a protein of a selective ion channel, is causing cystic fibrosis. Cystic fibrosis is a severe systemic monogenic disease with an autosomal recessive type of inheritance, which significantly reduces the duration and quality of life of patients. It is one of the most common hereditary diseases. Studying of molecular functions of CFTR protein in different types of cells, its structural and functional network interactions are critically important for the development of a new and more effective pathogenetic therapy. We are reviewing papers on the structure of the CFTR protein and its pathogenic genetic variants, as well as methods of pathogenetic therapy of cystic fibrosis by CFTR modulators and gene engineering. Recent gene engineering approaches to keep CFTR functions are discussed, such as gene-replacement therapy and genome editing, as well as viral and non-viral delivery systems and strategies of genomic editors.},
}

@article {pmid41613398,
year = {2025},
author = {Mehmood, MA and Iqbal, MM and Ashfaq, M and Chen, S and Wang, J},
title = {Advanced molecular tools for surveillance and management of tobamoviruses.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1718133},
pmid = {41613398},
issn = {1664-462X},
abstract = {Tobamoviruses are a group of plant viruses that can cause yield losses of up to 70% and reduce fruit quality by 30-50%. Historically, tobamoviruses were dominated by tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV). However, the landscape is rapidly shifting with the emergence of economically significant viruses such as tomato mottle mosaic virus (ToMMV) and tomato brown rugose fruit virus (ToBRFV). Both can circumvent the previously durable Tm-2[2] resistance in tomato and spread across multiple continents. This shift coincides with dramatic leaps in diagnostic tools, which have enhanced surveillance capabilities. Sensitive detection of tobamoviruses in the field with minimal sample preparation can be achieved using latest technologies such as isothermal amplification, CRISPR/Cas-hybrid assays or next-generation sequencing. Virus-host interactions underscore that viral proteins, including replicase components, are potent suppressors of RNA silencing (VSRs). Small RNA profiling and network analyses of viral movement proteins reveal complex mechanisms of immune evasion and resistance breakdown. These findings are largely based on dominant NB-LRR genes such as L, Tm-1, and Tm-2[2] . However, evidence indicates that ToBRFV can bypass this resistance via mutation in the movement protein, so supplementary methods should be considered. This review covers latest approaches, such as genome editing with CRISPR, targeting susceptibility genes, RNA interference (RNAi), and multi-omics approaches (transcriptomics, proteomics, metabolomics, ionomics), that can facilitate real-time surveillance and breeding for enhanced resilience. Moreover, the use of bio-formulations and nano-formulations as eco-friendly alternatives against tobamoviruses is discussed in detail. Climate change further complicates disease dynamics by undermining temperature-sensitive resistance, altering virus prevalence, and exacerbating yield losses. The rapid emergence of new tobamoviruses, which threatens the economy, necessitates a comprehensive approach. The integration of molecular diagnostics using CRISPR, omics technologies, designed protective systems, and climate-augmented disease prediction offers a detailed blueprint for the sustainable control of tobamoviruses and crop protection.},
}

@article {pmid41548240,
year = {2026},
author = {Fan, Y and Shen, S and Su, M and Yan, H and Dong, J and Li, Y and Li, N and Gao, Z and Xia, F},
title = {Synergistic CRISPR/Cas12a-Nanozyme System for Iontronic Sensing of Site-Specific Septin9 Methylation.},
journal = {Analytical chemistry},
volume = {98},
number = {4},
pages = {2870-2881},
doi = {10.1021/acs.analchem.5c05637},
pmid = {41548240},
issn = {1520-6882},
mesh = {*Septins/metabolism/genetics ; Humans ; *DNA Methylation ; *CRISPR-Cas Systems ; Gold/chemistry ; *DNA, Catalytic/chemistry/metabolism ; *Biosensing Techniques/methods ; *Endodeoxyribonucleases/metabolism ; Electrochemical Techniques ; Silver/chemistry ; Metal Nanoparticles/chemistry ; Platinum/chemistry ; Colorectal Neoplasms/diagnosis ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Methylated septin9 (septin9-mC) is a well-validated biomarker for colorectal cancer screening, and accurate detection of such site-specific methylated DNA holds significant clinical value for early disease diagnosis. However, conventional methods suffer from cumbersome pretreatment, DNA degradation risks, and poor performance in low-abundance samples. Herein, we report a synergistic iontronic sensing platform integrating methylation-sensitive restriction enzyme (AciI), CRISPR/Cas12a, Ag-DNAzyme, and Au/Pt heterometallic nanozyme for highly sensitive and specific detection of septin9-mC. AciI selectively cleaves unmethylated septin9 (septin9-C) while sparing septin9-mC, and intact septin9-mC activates Cas12a trans-cleavage activity to trigger catalytic hairpin assembly (CHA), generating Ag-DNAzyme. Activated Ag-DNAzyme induces detachment of Au/Pt nanoparticles from anodic aluminum oxide membranes, reducing the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to positively charged oxTMB and altering ion transport fluxes in nanochannels, which is read out via current-voltage characteristics. The linear range is 100 aM to 10 nM with a detection limit of 34.0 aM. This method effectively distinguishing colorectal cancer cells from human colonic epithelial cells and colorectal cancer patients from healthy individuals, showing excellent performance in real sample analysis. The proposed method provides a dependable tool for site-specific methylation detection with promising applications in biological research and clinical diagnosis.},
}

*Septins/metabolism/genetics
Humans
*DNA Methylation
*CRISPR-Cas Systems
Gold/chemistry
*DNA, Catalytic/chemistry/metabolism
*Biosensing Techniques/methods
*Endodeoxyribonucleases/metabolism
Electrochemical Techniques
Silver/chemistry
Metal Nanoparticles/chemistry
Platinum/chemistry
Colorectal Neoplasms/diagnosis
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid41510755,
year = {2026},
author = {Wang, L and Su, L and Gu, W and Nie, S and Luo, J and Li, Y and Pan, A and Li, Y},
title = {A photonic crystal sensing array based on a tandem CRISPR/Cas13a system for ultra-sensitive and high-throughput detection of the CVA6 virus.},
journal = {The Analyst},
volume = {151},
number = {3},
pages = {903-912},
doi = {10.1039/d5an01178g},
pmid = {41510755},
issn = {1364-5528},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Limit of Detection ; Photons ; *Biosensing Techniques/methods ; *RNA, Viral/genetics/analysis ; Fluorescent Dyes/chemistry ; High-Throughput Screening Assays/methods ; },
abstract = {Coxsackievirus A6 (CVA6) has emerged as a major cause of hand-foot-mouth disease (HFMD), yet no standardized detection method for it is currently available. Developing a simple, sensitive, and specific CVA6 test is crucial for HFMD control and safeguarding the health of at-risk children. Herein, a photonic crystal (PC) sensing array based on a tandem CRISPR/Cas13a system has been proposed for highly specific and ultra-sensitive analysis of CVA6 RNA, without the need for reverse transcription and amplification procedures. In this strategy, two crRNAs targeting CVA 6 RNA were designed and screened, and the fluorescence signal of the tandem CRISPR/Cas13a system was found to be up to 4.2 times higher than that of the non-tandem CRISPR system. The PC array with periodic nanostructures was prepared through self-deposition and further enhanced the fluorescent signal output from the tandem CRISPR system, owing to the match of the emission wavelength of the fluorescent dyes and the photonic band gap (PBG) of the PC. Benefitting from the synergistic effect of the tandem CRISPR system and PC array, as well as the high trans-cleavage activity of Cas13a protein, this engineered sensing array enables ultra-sensitive detection with a limit of detection (LOD) as low as 24.9 fM for CVA6. Meanwhile, this sensing strategy also achieved high-throughput and rapid analysis with a detection frequency of about 96 samples every 3.4 minutes. Therefore, the proposed strategy offers a simple workflow without reverse transcription or amplification, along with high sensitivity and high throughput, demonstrating strong potential for applications in biometrics and clinical diagnostics.},
}

*CRISPR-Cas Systems/genetics
Humans
Limit of Detection
Photons
*Biosensing Techniques/methods
*RNA, Viral/genetics/analysis
Fluorescent Dyes/chemistry
High-Throughput Screening Assays/methods

@article {pmid41469506,
year = {2025},
author = {Wang, T and Tian, Y and Yin, R and Li, M and Luo, J and Yang, Y and Zhang, C and Chen, H and Wang, Y and Lu, D},
title = {In vivo genome editing with a novel Cj4Cas9.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {152},
pmid = {41469506},
issn = {2399-3642},
support = {82070258//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Gene Editing/methods ; Animals ; Mice ; *CRISPR-Cas Systems ; Proprotein Convertase 9/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; Humans ; },
abstract = {Natural CRISPR-Cas9 systems provides diverse properties for genome editing, yet finding compact variants remains a priority. In this study, we screened a panel of 11 CjCas9 orthologous using a GFP activation assay and identified seven active nucleases. Among these, Cj4Cas9 stood out as particularly noteworthy due to its compact genome size (985 amino acids) and unique PAM preference (5'-NNNGRY-3'). Cj4Cas9 demonstrates efficient disruption of the Tyr gene in mouse zygotes, resulting in an albino phenotype. Furthermore, when delivered via AAV8, Cj4Cas9 achieves efficient genome editing of the Pcsk9 gene in mouse liver, leading to reduced serum cholesterol and LDL-C levels. Seeking to further expand its utility, we engineered Cj4Cas9 for higher activity by introducing L58Y/D900K mutations, resulting in a variant termed enCj4Cas9. This variant exhibits a two-fold increase in nuclease activity compared to the wild-type Cj4Cas9 and recognizes a simplified N3GG PAM, considerably expanding its targeting scope. These findings establish Cj4Cas9 and its engineered variants for fundamental research and therapeutic applications.},
}

*Gene Editing/methods
Animals
Mice
*CRISPR-Cas Systems
Proprotein Convertase 9/genetics
*CRISPR-Associated Protein 9/genetics/metabolism
Humans

@article {pmid40972958,
year = {2026},
author = {Nishino, T and Ono, K},
title = {Precision cardiology: Integrating gene therapy, genome editing, and single-cell genomics.},
journal = {Journal of cardiology},
volume = {87},
number = {2},
pages = {121-127},
doi = {10.1016/j.jjcc.2025.09.009},
pmid = {40972958},
issn = {1876-4738},
mesh = {*Gene Editing/methods ; Humans ; *Genetic Therapy/methods ; *Genomics/methods ; *Precision Medicine/methods ; *Cardiology/methods/trends ; *Single-Cell Analysis ; *Cardiovascular Diseases/therapy/genetics ; CRISPR-Cas Systems ; },
abstract = {Gene therapy is poised to revolutionize cardiovascular medicine by targeting the molecular roots of disease. This review examines the evolution of gene therapy, highlighting its past progress and future potential with emerging technologies. We first assess foundational gene addition and silencing strategies, noting clinical progress for monogenic cardiomyopathies alongside significant setbacks in multifactorial heart failure, driven mainly by the central challenge of vector delivery. We then discuss the evolution of delivery platforms, from engineered adeno-associated virus capsids to targeted lipid nanoparticles, which are designed to enhance cardiac specificity and safety. Concurrently, the gene editing revolution-progressing from the foundational Clustered Regularly Interspaced Short Palindromic Repeats-Cas9 system to high-fidelity base and prime editors-is enabling the direct correction of pathogenic mutations with increasing precision. Catalyzing these therapeutic platforms is the recent explosion in single-cell genomics, which provides an unprecedented resolution of cardiac pathology, revealing novel cell-specific targets previously obscured by bulk analysis. We conclude that the synergistic convergence of these pillars-genomics-driven discovery, precision genome editing, and targeted delivery-is creating a new paradigm of precision cardiology, moving the field from chronic management towards durable, curative interventions.},
}

*Gene Editing/methods
Humans
*Genetic Therapy/methods
*Genomics/methods
*Precision Medicine/methods
*Cardiology/methods/trends
*Single-Cell Analysis
*Cardiovascular Diseases/therapy/genetics
CRISPR-Cas Systems

@article {pmid41612768,
year = {2026},
author = {Wang, Y and Zhang, X and Lin, Y and Zhang, X and Yang, Z and Wan, Y and Negahdary, M},
title = {Single-Tube Dual-Gene Detection of Methicillin-Resistant Staphylococcus aureus via Selective Trans-Cleavage Preferences of Cas9 and Cas12a.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c05789},
pmid = {41612768},
issn = {1520-6882},
abstract = {Rapid and accurate detection of methicillin-resistant Staphylococcus aureus (MRSA) is essential for guiding clinical treatment and preventing infections. Current dual-gene detection methods based on CRISPR-Cas systems often require additional transcription steps, which increase the complexity of the assay and extend the turnaround time. Here, we report a single-tube dual-gene detection platform that leverages the orthogonal trans-cleavage preferences of Cas9 and Cas12a. By exploiting the loss of RNA cleavage activity in Cas12a when guided by split crRNA, and the inability of Cas9 to cleave DNA hairpin probes, we established a single-tube assay capable of simultaneously detecting the MRSA resistance gene (mecA) and the S. aureus-specific nuclease gene (nuc). The platform achieved attogram-level sensitivity and single-cell detection with high specificity against non-MRSA strains. Validation in an in vivo tilapia infection model demonstrated complete concordance with qPCR, reaching 100% positive and negative percent agreements. This work presents a streamlined, accurate, and practical approach for dual-gene pathogen detection, expanding the potential of Cas protein orthogonality for multiplex diagnostics.},
}

@article {pmid41612423,
year = {2026},
author = {Nizovtseva, I and Rezaykin, A and Korenskaia, A and Zakhartsev, M and Chigireva, A and Starodumov, I and Chernushkin, D},
title = {Identification and comparative genomic analysis of prophage sequences and CRISPR‒Cas immunity in Methylococcus genomes: insights into industrial methane bioconversion.},
journal = {Biotechnology for biofuels and bioproducts},
volume = {},
number = {},
pages = {},
doi = {10.1186/s13068-026-02738-6},
pmid = {41612423},
issn = {2731-3654},
support = {24-24-00454//Russian Science Support Foundation/ ; },
abstract = {BACKGROUND: Methylococcus species utilize methane as the sole carbon and energy source, converting it into biomass and other metabolic end products. Owing to this metabolic capacity, they hold particular promise in industrial C1 biotechnology, especially for the production of protein-rich feed. However, the industrial cultivation of Methylococcus-based consortia on methane is inherently nonsterile, exposing the process to potential biological risks that may compromise the stability, duration and productivity of cultivation. One of the most critical threats is bacteriophage infection, whose triggers for rapid phage-mediated lysis and resulting economic losses remain incompletely understood. Elucidating these processes is paramount for devising strategies to mitigate or prevent detrimental outcomes.

RESULTS: In this investigation, nine publicly accessible genomes of Methylococcus species were examined, culminating in the identification of eleven prophage sequences distributed variably among the genomes. Sequence annotations revealed that nine prophages are potentially functional and intact, whereas the rest carry incomplete gene sets indicative of nonviability. Phylogenetic analyses corroborated the substantial diversity of prophages, which formed distinct clusters related to γ-proteobacteria phages. Furthermore, comparative genomic analyses demonstrated a high degree of structural conservation despite the presence of rearrangements. The annotation of the CRISPR‒Cas systems provided insights into additional dimensions of phage‒bacteria interactions. Examination of prophage integration sites did not reveal any disruption of metabolic gene structures, thus suggesting minimal risk of deleterious phenotypic outcomes.

CONCLUSIONS: These findings considerably advance the current understanding of the genetic diversity and biological properties of prophages infecting Methylococcus species, underscoring the importance of holistic approaches for the detection and analysis of these elements. Our findings underscore the need for routine prophage monitoring in industrial methanotrophic consortia, with the pipeline established here serving as a foundational framework for future refinement and industrial adaptation.},
}

@article {pmid41611506,
year = {2026},
author = {Ji, Y and Bian, X and Feng, Z and Li, F and Hu, Z and Hu, Y and Liu, P and Ma, B and Chen, X},
title = {[Map-based cloning and functional analysis of the golden panicle and brown rice gene gpr1].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {1},
pages = {356-366},
doi = {10.13345/j.cjb.250438},
pmid = {41611506},
issn = {1872-2075},
support = {32272096//the National Natural Science Foundation of China/ ; LZ24C130003//the Natural Science Foundation of Zhejiang Province/ ; 2023R404039//the Zhejiang Provincial Undergraduate Science and Technology Innovation Activity Plan ("Xinmiao" Talents Program)/ ; 2024-2-017 and 2023-2-014//the Science and Technology Key Projects of Jinhua City/ ; },
mesh = {*Oryza/genetics/metabolism ; Cloning, Molecular ; *Plant Proteins/genetics ; Chromosome Mapping ; *Genes, Plant ; CRISPR-Cas Systems ; Pigmentation/genetics ; Flavonoids/biosynthesis ; },
abstract = {The colors of panicle hull and brown rice are crucial agronomic traits in rice, which are widely used in breeding and genetic research. Here, a rice variety named 'LAL SAR' characterized by golden panicle and brown rice was identified from Nepal. Genetic analysis revealed that these phenotypes were controlled by a single recessive nuclear gene, which was designated as gpr1 (golden panicle and brown rice 1). Using the strategy of map-based cloning, we mapped gpr1 into a 47 kb interval on the rice chromosome 3 and identified its candidate gene as LOC_Os03g60509. This gene encodes a chalcone isomerase, a key enzyme in the flavonoid biosynthetic pathway. Through PCR sequencing and RT-PCR analysis, a long-fragment insertion was found in the promoter of gpr1 in 'LAL SAR', which completely suppressed the expression of this gene. Using CRISPR/Cas9 technology, we successfully knocked out GPR1 (the dominant allele of gpr1) from a Oryza sativa subsp. japonica variety 'Zhonghua 11'. The knockout mutant plants exhibited golden panicles and brown rice, with significantly increased naringenin chalcone content in the hull. Our results indicate that GPR1 participates in the flavonoid biosynthesis, providing a critical theoretical foundation and a gene resource for rice quality improvement and genetic enhancement.},
}

*Oryza/genetics/metabolism
Cloning, Molecular
*Plant Proteins/genetics
Chromosome Mapping
*Genes, Plant
CRISPR-Cas Systems
Pigmentation/genetics
Flavonoids/biosynthesis

@article {pmid41611487,
year = {2026},
author = {Huang, X and Zeng, G and Shen, X},
title = {[Research progress and prospects in CRISPR-Cas9 gene editing in sorghum].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {1},
pages = {53-61},
doi = {10.13345/j.cjb.250419},
pmid = {41611487},
issn = {1872-2075},
support = {ZLGC201902-15//the Innovation-driven Assistance Project of China Association for Science and Technology/ ; },
mesh = {*Sorghum/genetics/growth & development ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Plant Breeding/methods ; },
abstract = {Sorghum is an important grain and cash crop in China, and the promotion and application of this crop have long been constrained by a shortage of genetic resources. Issues such as narrow genetic background and outdated breeding techniques have severely hindered the development and dissemination of new sorghum varieties. Although gene editing has demonstrated significant potential in the genetic improvement of crops since its inception, the application of this technology in sorghum remains lagging. This paper provides a systematic review of the latest breakthroughs in CRISPR-Cas9 in sorghum. Focusing on abiotic stress, growth and development, and quality, we explore the innovative applications of this technology in expanding genetic diversity, improving stress tolerance, optimizing plant architecture and yield potential, and enhancing quality characteristics. Additionally, we analyze the main technical challenges including low genetic transformation efficiency and insufficient adaptability of editing tools facing the gene editing in sorghum. Finally, we make an outlook on the future prospects of next-generation gene editing technologies in the genetic improvement of sorghum. This paper can provide important theoretical references for sorghum molecular breeding, and hold significant practical significance for safeguarding China's food security and enhancing the competitiveness of the sorghum industry.},
}

*Sorghum/genetics/growth & development
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Plants, Genetically Modified/genetics
Plant Breeding/methods

@article {pmid41611486,
year = {2026},
author = {Lu, Q and Teng, W and Liang, Y and Wang, Y},
title = {[Advances in genome editing and lodging resistance in sugarcane].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {1},
pages = {33-52},
doi = {10.13345/j.cjb.250555},
pmid = {41611486},
issn = {1872-2075},
support = {XDA0450000//the Strategic Priority Research Program of the Chinese Academy of Sciences/ ; },
mesh = {*Saccharum/genetics ; *CRISPR-Cas Systems/genetics ; *Genome, Plant/genetics ; *Gene Editing/methods ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; Genetic Engineering/methods ; Zinc Finger Nucleases/genetics ; },
abstract = {Sugarcane (Saccharum spp.) is an important cash crop that provides about 90% of sugar and 40% of bioethanol in China. Due to its large genome and complicated genetic background, conventional breeding is difficult to achieve efficient genetic improvement of sugarcane. Genome editing is a disruptive technology in life sciences, enabling precise and efficient modification of target genes. From zinc-finger nucleases (ZFNs) to transcription activator-like effector nucleases (TALENs), the CRISPR/Cas system and the derived base editing and prime editing, these technologies have greatly advanced genetic research and upgraded biological breeding. With the decoding of the sugarcane genome, genome editing has provided a new technical means for the genetic improvement of polyploid sugarcane. This article provides a comprehensive review of the trajectory of genome editing in plants, the optimization of the CRISPR/Cas system, the genetic transformation status of sugarcane, the development of sugarcane genomics, and the application of genome editing in sugarcane. It focuses on exploring the application prospects of genome editing in breeding lodging-resistant sugarcane varieties. This review aims to provide valuable references for promoting the use of genome editing in sugarcane breeding.},
}

*Saccharum/genetics
*CRISPR-Cas Systems/genetics
*Genome, Plant/genetics
*Gene Editing/methods
Plant Breeding/methods
Plants, Genetically Modified/genetics
Genetic Engineering/methods
Zinc Finger Nucleases/genetics

@article {pmid41611404,
year = {2026},
author = {Park, S and Koo, B and Kim, MG and Lee, EY and Lee, HJ and Roh, Y and Lee, M and Bae, CE and Lee, SW and Kang, YA and Shin, Y},
title = {CADEM: Species-level detection of mycobacterial cfDNA via CRISPR for pulmonary disease diagnosis.},
journal = {Analytica chimica acta},
volume = {1388},
number = {},
pages = {345085},
doi = {10.1016/j.aca.2026.345085},
pmid = {41611404},
issn = {1873-4324},
mesh = {Humans ; *CRISPR-Cas Systems ; *Cell-Free Nucleic Acids/genetics/blood/analysis ; *Lung Diseases/diagnosis/microbiology ; *DNA, Bacterial/genetics/blood ; Lab-On-A-Chip Devices ; Mycobacterium avium Complex/genetics/isolation & purification ; Microfluidic Analytical Techniques ; },
abstract = {BACKGROUND: Pulmonary infections caused by Mycobacterium tuberculosis (MTB) and nontuberculous mycobacteria (NTM) present significant clinical challenges due to overlapping symptoms and different treatments. In particular, accurate identification of NTM species such as Mycobacterium avium complex (MAC) and Mycobacterium abscessus complex (MABC) is essential, as these species show drug susceptibility profiles that differ markedly from MTB. However, conventional culture-based diagnostics are time-consuming, and current molecular assays often lack resolution and rely heavily on sputum specimens. To address these limitations, liquid biopsy using bacterial-derived circulating cell-free DNA (cfDNA) offers a minimally invasive alternative, and CRISPR/Cas12a technology provides the sensitivity required to detect its low levels.

RESULTS: We developed CADEM (CRISPR-Assisted Detection via Enrichment of Mycobacterium-derived cfDNA using Microfluidic technology), a streamlined diagnostic system that integrates microfluidic cfDNA enrichment, targeted amplification, and CRISPR/Cas12a-based detection. The microfluidic platform enables high-yield recovery of cfDNA from large-volume clinical samples without the need for cell lysis. Optimized Cas12a-crRNA complexes enable highly sensitive and specific detection of MAC-, MABC-, and MTB -specific amplicons, achieving 10- to 100-fold greater sensitivity than end-point PCR and probe-based real-time PCR. In a validation set of 20 clinical specimens (7 positives and 13 healthy controls), CADEM identified all MAC, MABC, and MTB cases with full accuracy and no false positives. The CRISPR detection step produced a clear fluorescence readout within 20 min and, together with enrichment and amplification, delivered species-level results within a 2-h workflow.

SIGNIFICANCE: CADEM offers an accurate and streamlined molecular approach for distinguishing MAC, MABC, and MTB at the species-level to support appropriate diagnosis and treatment. By combining microfluidic cfDNA enrichment with CRISPR-based detection, CADEM enables efficient analysis from liquid biopsy samples for pulmonary disease diagnosis. The system is also compatible with isothermal amplification, supporting future adaptation for point-of-care testing in resource-limited settings.},
}

Humans
*CRISPR-Cas Systems
*Cell-Free Nucleic Acids/genetics/blood/analysis
*Lung Diseases/diagnosis/microbiology
*DNA, Bacterial/genetics/blood
Lab-On-A-Chip Devices
Mycobacterium avium Complex/genetics/isolation & purification
Microfluidic Analytical Techniques

@article {pmid41611402,
year = {2026},
author = {Hou, J and Wang, Y and Yuan, W and Gong, Y and Yu, Y and Qin, X and Li, H and Zhang, Y and Shi, H and Chen, Y and Zhang, X},
title = {Thermosensitive hydrogel-enhanced RPA-CRISPR/Cas12a biosensor for ultrasensitive detection of methylated loci in breast cancer ctDNA.},
journal = {Analytica chimica acta},
volume = {1388},
number = {},
pages = {345101},
doi = {10.1016/j.aca.2026.345101},
pmid = {41611402},
issn = {1873-4324},
mesh = {Humans ; *Biosensing Techniques/methods ; *Breast Neoplasms/genetics/blood/diagnosis ; *CRISPR-Cas Systems ; *DNA Methylation ; Female ; *Circulating Tumor DNA/genetics/blood ; *Hydrogels/chemistry ; *Temperature ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BACKGROUND: Methylation differences exist between breast cancer tissues and normal tissues. The release of methylated circulating tumor DNA (ctDNA) by tumor cells provides a foundation for breast cancer liquid biopsy using methylated ctDNA. However, detection of low-abundance methylated loci in ctDNA remains a significant challenge to date. Existing one-tube detection systems cannot avoid target depletion caused by CRISPR/Cas12a cleavage, leading to reduced sensitivity.

RESULTS: This study developed a thermosensitive hydrogel-based one-tube RPA-CRISPR/Cas12a detection system, combined with methylation-sensitive restriction endonucleases (MSRE), for the detection of specific methylated loci in breast cancer ctDNA. This method achieves spatial separation while maintaining connectivity of reaction phases in a single tube for the first time, and the thermosensitive hydrogel does not exert inhibitory effects on either system. The system can specifically recognize methylated target molecules with a limit of detection (LOD) as low as 1 × 10[-8] ng/μL (≈70 copies/μL), outperforming the current glycerol-enhanced one-tube reaction system. It is capable of distinguishing methylated fractions as low as 0.05 %, with a sensitivity twice that of the gold standard methylation-specific quantitative PCR (Methylight). Detection of genomic DNA (gDNA) from tumor tissues and paired plasma ctDNA of 15 clinical patients using this method showed both sensitivity and specificity reaching 100 %.

SIGNIFICANCE: This novel, highly sensitive, efficient, and portable detection method innovatively resolves the target depletion issue caused by CRISPR/Cas12a cleavage in traditional RPA-CRISPR/Cas12a systems via thermosensitive hydrogel-mediated single-tube phase separation technology. It provides a new technical pathway for the accurate detection of low-abundance methylated circulating tumor DNA (ctDNA), will significantly enhance the level of breast cancer liquid biopsy, and offer strong support for the diagnosis and differential diagnosis of breast cancer.},
}

Humans
*Biosensing Techniques/methods
*Breast Neoplasms/genetics/blood/diagnosis
*CRISPR-Cas Systems
*DNA Methylation
Female
*Circulating Tumor DNA/genetics/blood
*Hydrogels/chemistry
*Temperature
Limit of Detection
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41610742,
year = {2026},
author = {Jiao, R and Ni, Q and Zhao, R and Zhu, X and Wang, L and Jiao, J and Jiang, H and Wu, Q and Yao, S and Yao, L and Liu, K and Qin, P},
title = {Dual CRISPR/Cas-driven amplification-free surface-enhanced Raman scattering biosensor combined with a smartphone for simultaneous detection of total and live target bacteria.},
journal = {Biosensors & bioelectronics},
volume = {299},
number = {},
pages = {118446},
doi = {10.1016/j.bios.2026.118446},
pmid = {41610742},
issn = {1873-4235},
abstract = {Simultaneous detection of total and live counts of target bacteria is significant but challenging. To address this challenge, here we proposed a dual CRISPR/Cas-driven amplification-free surface-enhanced Raman scattering (SERS) biosensor, termed cc-SERS. The biosensor was constructed based on the property that DNA remains stable while some RNA degrades rapidly after bacterial death. Briefly, the target DNA and RNA from live bacteria could activate both CRISPR/Cas12a and CRISPR/Cas13a, while dead bacteria could only activate CRISPR/Cas12a through the target DNA. In the absence of the target bacteria, neither CRISPR/Cas12a nor CRISPR/Cas13a could be activated. Therefore, the characteristic Raman signal at 1079 cm[-1] generated by the target DNA-activated CRISPR/Cas12a indicated the presence of the target bacteria (the sum of dead and live), while the characteristic Raman signal at 593 cm[-1] produced by the target RNA-activated CRISPR/Cas13a indicated the presence of the live target bacteria. With this unique signaling pattern, the biosensor is capable of detecting both total and live target bacteria in a single tube with a detection limit of ∼10 CFU/mL. The introduction of a rapid pre-processing procedure and a smartphone-assisted portable Raman spectrometer enabled the entire process to be completed in the field within 45 min. Thanks to the excellent programmability of CRISPR/Cas systems, the biosensor has been successfully applied to the detection of Staphylococcus aureus and Cronobacter sakazakii, respectively. As a proof-of-concept, this work opens a promising avenue for the simultaneous detection of total and live target bacteria.},
}

@article {pmid41610326,
year = {2026},
author = {Guo, W and Cheng, Y and Yin, H and Li, S and Wan, Y and Li, C and Jiang, C and Zhou, J and Yuan, X and Wang, J},
title = {CRISPR-AD: Combinational Detection of Blood Protein and miRNA with Digital CRISPR-Based Assay Enable to Improve the Diagnostic Performance of Alzheimer's Disease.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c06080},
pmid = {41610326},
issn = {1520-6882},
abstract = {Blood-based biomarkers present a noninvasive approach for detecting and assessing Alzheimer's disease (AD) pathophysiology, always by using sophisticated instrumentation for accurate detection. Here, we introduce CRISPR-AD, a CRISPR/Cas-based digital assay designed for the combined detection of protein and microRNA in blood. This method achieves a limit of detection (LOD) as low as 60 fg/mL for phosphorylated tau217 (p-tau217) and 0.5 fM for microRNA-34a-5p (miRNA34a), enabling successful detection in both AD patients and healthy individuals. We find that the combined use of these biomarkers improves the ability to distinguish between AD patients and healthy participants, particularly in individuals with mild cognitive impairment (MCI). Additionally, we have developed a portable device that integrates a smartphone as an imaging system for point-of-care testing (POCT), offering the potential for early stage AD screening. This study represents the first effort to evaluate the combined detection of blood protein and microRNA biomarkers for AD, underscoring the potential of multiple biomarker combinations for more accurate AD diagnosis.},
}

@article {pmid41609044,
year = {2026},
author = {Anschuetz, A and Listyono, R and Vorley, T and Platt, B and Harrington, CR and Riedel, G and Schwab, K},
title = {The Icelandic Mutation in the Murine APP Gene, mAPP[A673T], on Amyloid-β Plaque Burden in the 5×FAD Alzheimer Model.},
journal = {Journal of integrative neuroscience},
volume = {25},
number = {1},
pages = {48581},
doi = {10.31083/JIN48581},
pmid = {41609044},
issn = {0219-6352},
support = {PAR1577//TauRx Therapeutics Ltd., Singapore/ ; PAR2074//TauRx Therapeutics Ltd., Singapore/ ; },
mesh = {Animals ; *Alzheimer Disease/genetics/pathology/metabolism ; *Amyloid beta-Protein Precursor/genetics/metabolism ; Disease Models, Animal ; *Plaque, Amyloid/pathology/genetics/metabolism ; Mice, Transgenic ; Male ; *Amyloid beta-Peptides/metabolism ; Mice, Inbred C57BL ; Female ; Presenilin-1/genetics ; Mice ; Humans ; Mutation ; *Brain/pathology/metabolism ; Iceland ; },
abstract = {BACKGROUND: The protective Icelandic mutation in the amyloid precursor protein (APP) gene, APP[A673T], identified in Icelandic and other Nordic populations is associated with a significantly lower risk of developing Alzheimer's disease (AD). Conflicting results have been reported for the human APP[A673T] mutation in various knock-in models of AD, but the effect of the mouse APP[A673T] form in 5× familial AD (5×FAD) mice has never been investigated.

METHODS: We crossed C57Bl6/J mice expressing a single point mutation edited into the murine APP gene via Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated (CRISPR-Cas) gene editing, termed mAPP[A673T], with 5×FAD mice that overexpress human APP carrying the Swedish (K670N/M671L), Florida (I716V), and London (V717I) mutations as well as human presenilin-1 (PS1) with two mutations (M146L and L286V); the resulting mice were termed 5×FAD × mAPP[A673T] mice. We investigated amyloid beta-protein (Aβ) pathology in 5×FAD × mAPP[A673T], 5×FAD and their respective controls, mAPP[A673T], and C57Bl6/J wild type mice, at 6-months of age using immunohistochemistry, immunoblotting, and enzyme-linked immunosorbent assay (ELISA).

RESULTS: We found a moderate yet significant reduction in Aβ plaque size in male 5×FAD × mAPP[A673T] compared with 5×FAD mice. No differences were observed for soluble/insoluble Aβ40 and Aβ42 levels per se, but lower plaque count/area was found in 5×FAD × mAPP[A673T] mice when Aβ42/Aβ40 ratios were low, suggesting a genotype-dependent sensitivity to Aβ aggregation and accumulation.

CONCLUSIONS: The mAPP[A673T] mutation has the potential to modify Aβ pathology in 5×FAD mice at the age of 6 months.},
}

Animals
*Alzheimer Disease/genetics/pathology/metabolism
*Amyloid beta-Protein Precursor/genetics/metabolism
Disease Models, Animal
*Plaque, Amyloid/pathology/genetics/metabolism
Mice, Transgenic
Male
*Amyloid beta-Peptides/metabolism
Mice, Inbred C57BL
Female
Presenilin-1/genetics
Mice
Humans
Mutation
*Brain/pathology/metabolism
Iceland

@article {pmid41592354,
year = {2026},
author = {Du, T and Ding, F and Ma, X and Luo, Y and Zhu, D and Wang, L and Su, S},
title = {Ultrasensitive detection of monkeypox virus: harnessing synergistic CRISPR-driven signal amplification on a DNA tetrahedron-mediated sensing interface.},
journal = {Biosensors & bioelectronics},
volume = {298},
number = {},
pages = {118441},
doi = {10.1016/j.bios.2026.118441},
pmid = {41592354},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Humans ; Limit of Detection ; CRISPR-Cas Systems/genetics ; Electrochemical Techniques/methods ; *Monkeypox virus/isolation & purification/genetics ; *Mpox, Monkeypox/diagnosis/virology ; Nanostructures/chemistry ; *DNA, Viral/genetics ; Hydrogen Peroxide/chemistry ; DNA/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Rapid and ultrasensitive detection of emerging infectious diseases is critical for public health security. Herein, an electrochemical biosensor was developed for ultrasensitive detection of monkeypox virus (MPXV) by integrating CRISPR/Cas12a-driven signal amplification strategy with tetrahedral DNA nanostructure (TDN)-based sensing interface. The added MPXV DNA can efficiently activate the cleavage activity of Cas12a protein, thereby mediating the CRISPR-driven hybridization chain reaction (HCR) on TDN sensing interface. The horseradish peroxidase (HRP)-labeled HCR product can catalyze 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) reaction to generate an amplified electrochemical signal. Based on the signal change, the CRISPR-driven electrochemical biosensor exhibited better detection performance comparable to those of pre-amplification CRISPR-based biosensors for MPXV detection, including wide linear range, an ultralow detection limit, exceptional selectivity against non-target viruses (CPXV, ETCV, VZV, HSV), high reproducibility and accepted stability. Integrated with a smartphone-based portable device, the designed point-of-care testing (POCT) electrochemical biosensor can accurately detect MPXV in 10 % human saliva. This work provides a promising sensing platform for rapid, accurate and on-site detection of infectious diseases.},
}

*Biosensing Techniques/methods
Humans
Limit of Detection
CRISPR-Cas Systems/genetics
Electrochemical Techniques/methods
*Monkeypox virus/isolation & purification/genetics
*Mpox, Monkeypox/diagnosis/virology
Nanostructures/chemistry
*DNA, Viral/genetics
Hydrogen Peroxide/chemistry
DNA/chemistry
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41581366,
year = {2026},
author = {Yu, Y and Zhang, J and Xie, G and Lin, Y and Huang, Y and Rao, C and Hou, Y and Chen, H},
title = {A DNAzyme-CRISPR cascade strategy for preamplification-free detection of Mycobacterium tuberculosis.},
journal = {Biosensors & bioelectronics},
volume = {298},
number = {},
pages = {118428},
doi = {10.1016/j.bios.2026.118428},
pmid = {41581366},
issn = {1873-4235},
mesh = {*Mycobacterium tuberculosis/isolation & purification/genetics ; *Biosensing Techniques/methods ; *DNA, Catalytic/chemistry/genetics ; *Tuberculosis/diagnosis/microbiology ; Humans ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; *DNA, Bacterial/genetics/isolation & purification ; Nucleic Acid Amplification Techniques ; Reproducibility of Results ; },
abstract = {Tuberculosis (TB) remains one of the most fatal infectious diseases worldwide, successful treatment is often limited by insufficient diagnostic capabilities. It creates a pressing need for diagnostic methods that combine high sensitivity, specificity, and operational robustness. In this work, we developed a DNAzyme-CRISPR cascade strategy (Dz-CRISPR) for the specific identification of the IS6110 sequence of Mycobacterium tuberculosis (MTB). This system integrated a thermodynamically stabilized hairpin probe, an Arch-shaped signal transduction switch, and an allosterically activated CRISPR-Cas12a cascade. The design enabled direct target recognition and subsequent signal amplification without a preamplification step, offering a simplified workflow with enhanced stability. The assay demonstrated a detection limit of 211.3 fM and exhibited high specificity by accurately discriminating the IS6110 from specific DNA sequence of non-tuberculous mycobacteria and other common respiratory pathogens. Validation using clinical bronchoalveolar lavage fluid samples further confirmed the method's reliable performance, reproducibility, and satisfactory recovery rates. Current Dz-CRISPR detection strategy provides a reliable and practical solution for tuberculosis diagnosis with high sensitivity, high specificity, and operational robustness, thus demonstrating potential for practical use in resource-constrained areas.},
}

*Mycobacterium tuberculosis/isolation & purification/genetics
*Biosensing Techniques/methods
*DNA, Catalytic/chemistry/genetics
*Tuberculosis/diagnosis/microbiology
Humans
*CRISPR-Cas Systems/genetics
Limit of Detection
*DNA, Bacterial/genetics/isolation & purification
Nucleic Acid Amplification Techniques
Reproducibility of Results

@article {pmid41576437,
year = {2026},
author = {Wei, L and Cheng, Z and Xu, M and Chen, H and Lan, W and Long, W and She, Y and Fu, H},
title = {Bulge DNA-driven CRISPR/Cas12a dynamic activation circuit enables highly sensitive and versatile biosensing.},
journal = {Biosensors & bioelectronics},
volume = {298},
number = {},
pages = {118412},
doi = {10.1016/j.bios.2026.118412},
pmid = {41576437},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Methicillin-Resistant Staphylococcus aureus/isolation & purification/genetics ; Limit of Detection ; *DNA/chemistry/genetics ; *Mycotoxins/analysis/isolation & purification ; Aptamers, Nucleotide/chemistry/genetics ; Bacterial Proteins/genetics/chemistry ; *Endodeoxyribonucleases/chemistry/genetics ; Humans ; CRISPR-Associated Proteins ; },
abstract = {CRISPR/Cas12a has emerged as an innovative biosensing tool; however, its intrinsically linear accumulation of cleavage signals limits detection sensitivity. Herein, we report a bulge DNA (BD)-driven CRISPR/Cas12a dynamic activation circuit, termed CBD, as a highly sensitive and versatile biosensing platform for both nucleic acid and non-nucleic acid targets. The BD structure was rationally engineered to undergo programmable structural and functional transformation upon bulge degradation, thereby initiating exponential, self-amplifying activation of the Cas12a circuit. For nucleic acid targets, direct Cas12a recognition triggers BD cleavage and a positive feedback loop, enabling highly sensitive detection with a limit of 14 CFU/mL for methicillin-resistant Staphylococcus aureus. For non-nucleic acid targets, a universal single-stranded DNA activator was linked to the aptamer-complementary strand, enabling target-responsive release and subsequent initiation of the CBD system without altering the crRNA or BD sequence. This strategy enabled the detection of pesticides and mycotoxins at the picogram-per-milliliter level. Furthermore, an "OR" logic gate was constructed for the simultaneous detection of dual mycotoxins, highlighting the platform's capability for multiplexed hazard monitoring. Overall, CBD demonstrates significant potential as a new paradigm for next-generation biosensing technologies.},
}

*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
*Methicillin-Resistant Staphylococcus aureus/isolation & purification/genetics
Limit of Detection
*DNA/chemistry/genetics
*Mycotoxins/analysis/isolation & purification
Aptamers, Nucleotide/chemistry/genetics
Bacterial Proteins/genetics/chemistry
*Endodeoxyribonucleases/chemistry/genetics
Humans
CRISPR-Associated Proteins

@article {pmid41554314,
year = {2026},
author = {Paulo, BS and Romanowski, SB and Kadjo, AE and Lourenzon, VB and Eustáquio, AS},
title = {Genome minimization of a Burkholderia bacterial host.},
journal = {Metabolic engineering},
volume = {94},
number = {},
pages = {305-314},
doi = {10.1016/j.ymben.2026.01.008},
pmid = {41554314},
issn = {1096-7184},
mesh = {*Burkholderia/genetics/metabolism ; *Genome, Bacterial/genetics ; Plasmids/genetics ; *Metabolic Engineering/methods ; CRISPR-Cas Systems ; },
abstract = {Genome minimization, including the deletion of endogenous gene clusters that encode natural products, is a common strategy to improve the yield of heterologous products. We have been interested in developing Burkholderia sp. FERM BP-3421 as an alternative bacterial host. Instead of indiscriminately deleting gene clusters, which may have deleterious effects, we guided our efforts using transcriptomics data from production cultures. The genome of FERM BP-3421 is subdivided into two chromosomes and two plasmids. The top transcribed gene clusters were those encoding polyketide-nonribosomal peptide spliceostatins on plasmid p1 and nonribosomal peptide selethramide on chromosome 1. Deletion of the spliceostatin cluster had been shown to improve titers of the ribosomal peptide capistruin, whereas we showed that deletion of the selethramide cluster had no effect on capistruin titers. We next targeted the two endogenous plasmids using a CRISPR-Cas12a strategy, resulting in an 11 % reduction in genome size. The plasmid cured strains showed improved growth and 20-40 % increased production of capistruin depending on whether one or both plasmids were deleted. However, deletion of p2 alone negatively affected the heterologous production of two distinct polyketide-nonribosomal peptides. The p2[-] strain produced only 5-23 % of the glidobactin A and megapolipeptin A titers compared to the wild type, respectively, whereas titers were restored to wild type levels in the p1[-] p2[-] strain. The observation that p2 appears to contain functions that support polyketide-nonribosomal peptide biosynthesis was unexpected and sets the stage for future studies aimed at identifying these functions and further enabling engineering efforts that may be widely applicable to other strains.},
}

*Burkholderia/genetics/metabolism
*Genome, Bacterial/genetics
Plasmids/genetics
*Metabolic Engineering/methods
CRISPR-Cas Systems

@article {pmid41547318,
year = {2026},
author = {Beckley, J and Barrangou, R},
title = {Phage-mediated delivery of CRISPR payloads.},
journal = {Current opinion in microbiology},
volume = {89},
number = {},
pages = {102704},
doi = {10.1016/j.mib.2025.102704},
pmid = {41547318},
issn = {1879-0364},
mesh = {*Bacteriophages/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Bacteria/genetics/virology ; Genetic Engineering/methods ; },
abstract = {Over the past decade, improvements in sequencing technologies and computational tools have advanced our understanding of the composition and function of microbial communities in various environments. Now, in order to manipulate and engineer these communities, we need technologies that enable broadly applicable and specific alterations to establish and modulate the molecular basis for their functional roles. Recent advances in bacteriophage engineering strategies, synthetic biology techniques, and in silico approaches have greatly expanded our ability to perform in situ perturbations. Clustered regularly interspaced short palindromic repeats-Cas systems in particular can provide an efficient means of engineering phages, and can also be delivered as a recombinant payload to perform precision genome editing directly in the host environment. Modified Cas effectors have been developed that allow for increasingly diverse edits with applications in the fields of medicine, food, and agriculture. In this review, we discuss recent advances in using bacteriophages to deliver various clustered regularly interspaced short palindromic repeats-Cas effectors. While challenges remain regarding the phylogenetic breadth of deployment, recombinant phages generally present a unique and effective means to rationally engineering microbial community function and composition.},
}

*Bacteriophages/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
*Clustered Regularly Interspaced Short Palindromic Repeats
*Bacteria/genetics/virology
Genetic Engineering/methods

@article {pmid41443126,
year = {2026},
author = {Rahimi, A and Rahimmanesh, I and Abedpoor, N and Boshtam, M and Bidram, E and Javanmard, SH and Khanahmad, H and Rafiee, L and Bigham, A and Rafienia, M and Karbasi, S and Shariati, L},
title = {The MCM/Lys-Cys nanodevices for the efficient gene delivery: An approach towardsMCP1gene manipulation using CRISPR technology.},
journal = {Colloids and surfaces. B, Biointerfaces},
volume = {260},
number = {},
pages = {115377},
doi = {10.1016/j.colsurfb.2025.115377},
pmid = {41443126},
issn = {1873-4367},
mesh = {Humans ; *Silicon Dioxide/chemistry ; *Gene Transfer Techniques ; *Nanoparticles/chemistry ; *CRISPR-Cas Systems/genetics ; *Chemokine CCL2/genetics ; Cell Proliferation ; Cell Line, Tumor ; Female ; *Cysteine/chemistry ; Breast Neoplasms/genetics/pathology/therapy ; *Lysine/chemistry ; Cell Movement ; Particle Size ; Surface Properties ; Gene Editing ; Plasmids/genetics ; },
abstract = {Breast cancer continues to be the most common malignancy among women worldwide, requiring novel therapeutic approaches. This research investigates an innovative gene delivery strategy employing mesoporous silica nanoparticles (MCM-41) modified with lysine and cysteine (Lys-Cys) for the effective delivery of CRISPR-Cas9 plasmids aimed at the monocyte chemoattractant protein-1 (MCP-1/CCL2) gene. Bioinformatics analysis of the TCGA-BRCA dataset revealed substantial deregulation of CCL2 in breast cancer, underscoring its involvement in tumor growth and inflammation. The MCM/Lys-Cys nanocarrier demonstrated remarkable biocompatibility and effectively encapsulated a plasmid containing GFP, promoting superior cellular uptake in MDA-MB-231 breast cancer cells compared to conventional techniques. Functional experiments demonstrated that CRISPR/Cas9-mediated suppression of CCL2 markedly decreased cell proliferation, migration, and invasion, highlighting the promise of this targeted gene therapy strategy in breast cancer management. The findings indicate that the MCM/Lys-Cys nanosystem presents a viable non-viral approach for precise gene editing, potentially boosting therapeutic efforts against breast cancer by modulating inflammatory pathways.},
}

Humans
*Silicon Dioxide/chemistry
*Gene Transfer Techniques
*Nanoparticles/chemistry
*CRISPR-Cas Systems/genetics
*Chemokine CCL2/genetics
Cell Proliferation
Cell Line, Tumor
Female
*Cysteine/chemistry
Breast Neoplasms/genetics/pathology/therapy
*Lysine/chemistry
Cell Movement
Particle Size
Surface Properties
Gene Editing
Plasmids/genetics

@article {pmid41431758,
year = {2026},
author = {Hwang, S and Ko, H and Lee, HY and Choi, J},
title = {Nanocarriers for the delivery of the CRISPR/Cas9 system.},
journal = {Nanomedicine (London, England)},
volume = {21},
number = {3},
pages = {429-448},
doi = {10.1080/17435889.2025.2598332},
pmid = {41431758},
issn = {1748-6963},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; Animals ; *Nanoparticles/chemistry ; *Gene Transfer Techniques ; *Drug Carriers/chemistry ; },
abstract = {The clustered regularly interspaced short palindromic repeat/associated protein 9 (CRISPR/Cas9) system has been used for the precise manipulation of target DNA, making efficient genome editing in cells a reality. The CRISPR/Cas9 system has shown great potential in biomedical applications, such as disease treatment, transcription regulation, and genome-wide screening, and is opening a new era in biotechnology. However, the efficient and selective delivery of the CRISPR/Cas9 system remains a critical obstacle. Literature search conducted using Web of Science, Scopus, PubMed and Google Scholar for articles published from 2015 to 2024. In this review, we discuss several delivery methods for the CRISPR/Cas9 system, focusing on techniques using nanocarriers. Specifically, we comprehensively discussed the challenges, future directions, and potential of various delivery methods for the CRISPR/Cas9 system.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Humans
Animals
*Nanoparticles/chemistry
*Gene Transfer Techniques
*Drug Carriers/chemistry

@article {pmid41259745,
year = {2026},
author = {Liu, L and Glaser, A and Isiaku, AI and Fairfax, K and Casolari, DA and Ristovski, A and Murphy, V and van Twest, S and Henrikus, SS and Heraud-Farlow, J and Granger, E and Novakovic, S and Monks O'Byrne, SF and Tsui, V and Conyers, R and Gonda, TJ and Crismani, W and D'Andrea, RJ and Heierhorst, J and Deans, AJ},
title = {Fancl-mutant mice reveal central role of monoubiquitination in Fanconi anemia and a model for therapeutic gene editing.},
journal = {Blood advances},
volume = {10},
number = {3},
pages = {821-836},
doi = {10.1182/bloodadvances.2025017217},
pmid = {41259745},
issn = {2473-9537},
mesh = {Animals ; *Fanconi Anemia/genetics/therapy/metabolism/pathology ; Mice ; *Gene Editing/methods ; *Ubiquitination ; Disease Models, Animal ; *Fanconi Anemia Complementation Group L Protein/genetics/metabolism ; *Mutation ; Fanconi Anemia Complementation Group D2 Protein/metabolism ; Humans ; CRISPR-Cas Systems ; },
abstract = {Fanconi anemia (FA) is a rare genetic disorder causing the progressive loss of hematopoietic stem cells (HSCs) and bone marrow failure. Most cases result from deficient monoubiquitination of FANCD2 by the FA core complex. However, given that additional functions for the complex have been proposed, it remains unclear whether loss of FANCD2 monoubiquitination is the sole cause of all FA phenotypes. Here, we generated a murine allele (FanclTATΔ) that mimics an allele from a patient with FA. This 3-bp deletion removes a catalytic cysteine in the E3 RING ligase domain of the FANCL subunit. Biochemical assays show that the mutant FA core complex retains structural integrity but lacks FANCD2 monoubiquitination activity. Homozygous FanclTATΔ/TATΔ mice phenocopy classical human FA features, including infertility, craniofacial anomalies, DNA damage hypersensitivity, and progressive HSC loss with age. Correcting the mutation using CRISPR-Cas9 or prime editing technology restores FANCD2 monoubiquitination and normal DNA damage resistance in myeloid cells. Collectively, our mouse model demonstrates that loss of RING E3 ubiquitin ligase activity of the FA core complex explains developmental defects and hematopoietic failure in FA and provides a new animal model for testing potentially therapeutic gene editing.},
}

Animals
*Fanconi Anemia/genetics/therapy/metabolism/pathology
Mice
*Gene Editing/methods
*Ubiquitination
Disease Models, Animal
*Fanconi Anemia Complementation Group L Protein/genetics/metabolism
*Mutation
Fanconi Anemia Complementation Group D2 Protein/metabolism
Humans
CRISPR-Cas Systems

@article {pmid41205860,
year = {2026},
author = {Yunus, IS and Carruthers, DN and Chen, Y and Gin, JW and Baidoo, EEK and Petzold, CJ and Garcia Martin, H and Adams, PD and Mukhopadhyay, A and Lee, TS},
title = {Predictive CRISPR-mediated gene downregulation for enhanced production of sustainable aviation fuel precursor in Pseudomonas putida.},
journal = {Metabolic engineering},
volume = {94},
number = {},
pages = {67-76},
doi = {10.1016/j.ymben.2025.11.007},
pmid = {41205860},
issn = {1096-7184},
mesh = {*Pseudomonas putida/genetics/metabolism ; *CRISPR-Cas Systems ; *Metabolic Engineering/methods ; *Down-Regulation ; *Bacterial Proteins/genetics/biosynthesis/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {CRISPR interference (CRISPRi) has emerged as a valuable tool for redirecting metabolic flux to enhance bioproduction. However, its application is often constrained by two challenges: (i) rationally identifying effective gene targets for downregulation and (ii) efficiently constructing multiplexed CRISPRi systems. In this study, we address both challenges by integrating a computational prioritization tool with a versatile assembly method for building multiplexed CRISPRi systems. FluxRETAP (Flux-Reaction Target Prioritization) accurately identified gene targets whose knockdown led to substantial increase of isoprenol titers in Pseudomonas putida KT2440, outperforming a conventional non-computational, pathway-guided target selection. The highest isoprenol titer of nearly 1.5 g/L was achieved by knocking down PP_4118 (a gene encoding α-ketoglutarate dehydrogenase). The use of VAMMPIRE (Versatile Assembly Method for MultiPlexing CRISPRi-mediated downREgulation) enabled accurate assembly of CRISPRi constructs containing up to five sgRNA arrays, reducing context dependency and achieving uniform, position-independent gene downregulation. The integration of FluxRETAP and VAMMPIRE has the potential to advance metabolic engineering by rapidly identifying CRISPRi-mediated knockdowns and knockdown combinations that enhance bioproduction titers, with potential applicability to other microbial systems.},
}

*Pseudomonas putida/genetics/metabolism
*CRISPR-Cas Systems
*Metabolic Engineering/methods
*Down-Regulation
*Bacterial Proteins/genetics/biosynthesis/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41201956,
year = {2026},
author = {Sinha, R and Patil, RV and Romano, R and Sharma, D and Lee, E and Perriman, R and Takeda, S and Lesch, BJ and Yao, Z and Liu, YL and Cromer, MK and Porteus, MH and Bertaina, A},
title = {Novel humanized loss-of-function NF1 mouse model of juvenile myelomonocytic leukemia.},
journal = {Blood advances},
volume = {10},
number = {3},
pages = {837-849},
doi = {10.1182/bloodadvances.2024015191},
pmid = {41201956},
issn = {2473-9537},
mesh = {Animals ; *Leukemia, Myelomonocytic, Juvenile/genetics/pathology/metabolism/therapy ; Mice ; Disease Models, Animal ; Humans ; *Neurofibromin 1/genetics/metabolism ; *Loss of Function Mutation ; Granulocyte-Macrophage Colony-Stimulating Factor ; Hematopoietic Stem Cells/metabolism ; CRISPR-Cas Systems ; },
abstract = {Juvenile myelomonocytic leukemia (JMML) is a fatal pediatric cancer characterized by classical features such as splenomegaly, monocytosis, and granulocyte-macrophage colony-stimulating factor (GM-CSF) hypersensitivity, with RAS pathway mutations being the major drivers. Mutations causing loss-of-function of the Neurofibromin 1 gene (NF1LOF) occur in ∼20% of patients with JMML. NF1LOF drives upregulation of RAS/MAPK/PI3K pathways that leads to aggressive proliferation/differentiation of immature myeloid cells. Hematopoietic stem cell transplantation is the only curative option, but relapse occurs in ∼50% of patients, indicating an urgent need for novel and targeted therapeutic strategies. However, low patient sample availability and a lack of reliable disease models have made it difficult to study and treat JMML. Using CRISPR/Cas9, we have generated NF1LOF in human umbilical cord blood-derived hematopoietic stem and progenitor cells (HSPCs). We achieved a high gene knockout rate of ∼89% and concomitant loss of NF1 protein in the modified HSPCs. Importantly, NF1LOF cells displayed marked GM-CSF hypersensitivity in in vitro colony-forming unit assays, mirroring JMML. When transplanted into NSG-SGM3 mice, they caused rapid lethality (median survival of 32 days), myeloid expansion, tissue infiltration (spleen, liver, and lungs), and specific upregulation of RAS/MAPK pathway and STAT5 genes, consistent with patient profiles. This first humanized NF1LOF mouse model recapitulates key JMML features, enabling investigation of disease mechanisms and targeted therapies.},
}

Animals
*Leukemia, Myelomonocytic, Juvenile/genetics/pathology/metabolism/therapy
Mice
Disease Models, Animal
Humans
*Neurofibromin 1/genetics/metabolism
*Loss of Function Mutation
Granulocyte-Macrophage Colony-Stimulating Factor
Hematopoietic Stem Cells/metabolism
CRISPR-Cas Systems

@article {pmid40135929,
year = {2026},
author = {Ming, Z and Liu, F and Moran, HR and Lalonde, RL and Adams, M and Restrepo, NK and Joshi, P and Ekker, SC and Clark, KJ and Friedberg, I and Sumanas, S and Yin, C and Mosimann, C and Essner, JJ and McGrail, M},
title = {Lineage labeling with zebrafish hand2 Cre and CreERT2 recombinase CRISPR knock-ins.},
journal = {Developmental dynamics : an official publication of the American Association of Anatomists},
volume = {255},
number = {1},
pages = {86-105},
pmid = {40135929},
issn = {1097-0177},
support = {R01 HL168087/HL/NHLBI NIH HHS/United States ; K99 HL168148/HL/NHLBI NIH HHS/United States ; //Iowa State University of Science and Technology/ ; R24 OD036201/RI/ORIP NIH HHS/United States ; T32 GM141742/GM/NIGMS NIH HHS/United States ; R01 HL153005/HL/NHLBI NIH HHS/United States ; R24 OD020166/OD/NIH HHS/United States ; F31 HL167580/HL/NHLBI NIH HHS/United States ; R24 OD036201/OD/NIH HHS/United States ; R01 DK117266/DK/NIDDK NIH HHS/United States ; //PFIC Research Center, Cincinnati Children's Hospital/ ; //Children's Hospital Colorado/ ; R24 OD020166/RI/ORIP NIH HHS/United States ; R01 HL168097/HL/NHLBI NIH HHS/United States ; R01 DK117266/DK/NIDDK NIH HHS/United States ; T32 GM141742/GM/NIGMS NIH HHS/United States ; R24 OD020166/RI/ORIP NIH HHS/United States ; R24 OD036201/RI/ORIP NIH HHS/United States ; F31 HL167580/HL/NHLBI NIH HHS/United States ; K99 HL168148/HL/NHLBI NIH HHS/United States ; R01 HL153005/HL/NHLBI NIH HHS/United States ; R01 HL168087/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; *Zebrafish/embryology/genetics ; *Zebrafish Proteins/genetics/metabolism ; *Integrases/genetics/metabolism ; *Cell Lineage/genetics ; *Basic Helix-Loop-Helix Proteins/genetics/metabolism ; Gene Knock-In Techniques/methods ; CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Developmental ; },
abstract = {BACKGROUND: The ability to generate endogenous Cre recombinase drivers using CRISPR-Cas9 knock-in technology allows lineage tracing, cell type-specific gene studies, and in vivo validation of inferred developmental trajectories from phenotypic and gene expression analyses. This report describes endogenous zebrafish hand2 Cre and CreERT2 drivers generated with GeneWeld CRISPR-Cas9 precision targeted integration.

RESULTS: hand2-2A-cre and hand2-2A-creERT2 knock-ins crossed with ubiquitous loxP-based Switch reporters led to broad labeling in expected mesodermal and neural crest-derived lineages in branchial arches, cardiac, fin, liver, intestine, and mesothelial tissues, as well as enteric neurons. Novel patterns of hand2 lineage tracing appeared in venous blood vessels. CreERT2 induction at 24 h reveals hand2-expressing cells in the 24- to 48-h embryo contribute to the venous and intestinal vasculature. Induction in 3 dpf larvae restricts hand2 lineage labeling to mesoderm-derived components of the branchial arches, heart, liver, and enteric neurons.

CONCLUSIONS: hand2 progenitors from the lateral plate mesoderm and ectoderm contribute to numerous lineages in the developing embryo. At later stages, hand2-expressing cells are restricted to a subset of lineages in the larva. The endogenous hand2 Cre and CreERT2 drivers establish critical new tools to investigate hand2 lineages in zebrafish embryogenesis and larval organogenesis.},
}

Animals
*Zebrafish/embryology/genetics
*Zebrafish Proteins/genetics/metabolism
*Integrases/genetics/metabolism
*Cell Lineage/genetics
*Basic Helix-Loop-Helix Proteins/genetics/metabolism
Gene Knock-In Techniques/methods
CRISPR-Cas Systems/genetics
Gene Expression Regulation, Developmental

@article {pmid41608958,
year = {2026},
author = {Zhang, Y and Walker, RSK and Sunna, A and Barber, TJ and Li, M},
title = {Droplet Digital CRISPR for Nucleic Acid Detection.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e17470},
doi = {10.1002/advs.202517470},
pmid = {41608958},
issn = {2198-3844},
support = {DP200102269//Australian Research Council/ ; DP240100795//Australian Research Council/ ; GNT2017679//National Health and Medical Research Council/ ; },
abstract = {Droplet digital (dd) clustered regularly interspaced short palindromic repeats (CRISPR) integrates the high sequence specificity of CRISPR-based nucleic acid detection with the absolute quantification capability of digital droplet microfluidics, offering high sensitivity, precision, and scalability. By partitioning samples into thousands to millions of picoliter microdroplets, ddCRISPR enables single-molecule resolution and minimizes background interference. This review summarizes the principles of droplet generation, manipulation, and detection in ddCRISPR platforms, as well as recent advances in amplification-based and amplification-free detection strategies. Representative applications are highlighted for viral, bacterial, and other DNA/RNA biomarker detection. Current challenges, including workflow automation, droplet stability, multiplexing, and assay portability, are discussed alongside future perspectives such as artificial intelligence (AI)-assisted analysis, point-of-care integration, and high-throughput multiplexed detection. These insights aim to guide the translation of ddCRISPR technologies from laboratory research to robust, scalable, and accessible diagnostic solutions.},
}

@article {pmid41608579,
year = {2026},
author = {Sanchez-Pupo, RE and Kelly, JJ and Shalaby, N and Xia, Y and Martinez-Santiesteban, FM and Lau, J and Verriet, IE and Fox, MS and Hicks, JW and Thiessen, JD and Ronald, JA},
title = {Imaging CRISPR-edited CAR-T cell therapies with optical and positron emission tomography reporters.},
journal = {Theranostics},
volume = {16},
number = {7},
pages = {3227-3245},
pmid = {41608579},
issn = {1838-7640},
mesh = {*Positron-Emission Tomography/methods ; Animals ; Humans ; *Receptors, Chimeric Antigen/genetics/metabolism ; *Immunotherapy, Adoptive/methods ; Mice ; Gene Editing/methods ; *T-Lymphocytes/immunology/metabolism/transplantation ; CRISPR-Cas Systems ; Genes, Reporter ; Cell Line, Tumor ; Symporters/genetics ; Female ; Dependovirus/genetics ; Optical Imaging/methods ; },
abstract = {Rationale: Chimeric antigen receptor (CAR) T cell therapies have shown remarkable success in treating hematological cancers and are increasingly demonstrating potential for solid tumors. CRISPR-based genome editing offers a promising approach to enhance CAR-T cell potency and safety, yet challenges such as inefficient tumor homing and toxicities in normal tissues, limit broader adoption. Advanced imaging technologies, including bioluminescence imaging (BLI) and positron emission tomography (PET), provide real-time visualization of CAR-T cell behavior in vivo. Here, we developed Trackable Reporter Adaptable CRISPR-Edited CAR (tRACE-CAR) T cells, a modular system for site-specific integration of CARs and imaging reporters. Methods: The luciferase reporter AkaLuciferase (AkaLuc) or the human sodium iodide symporter (NIS) was cloned downstream of the CAR in adeno-associated virus (AAV) donors for imaging. CAR-reporter cassettes were inserted into the T-cell receptor α constant locus of primary human T cells via CRISPR editing and AAV transduction. Editing efficiency was assessed by flow cytometry. In vitro cytotoxicity was evaluated across multiple effector-to-target ratios. In vivo, BLI and PET imaging were used for tracking CAR-T cells in tumor-bearing immunodeficient mice. Results: T cell receptor (TCR) knockout efficiency exceeded 85% and CAR expression reached 70-80%. Reporter-engineered CAR-T cells exhibited significant cytotoxicity and outperformed naïve T cells. In vivo, AkaLuc BLI and [18]F-tetrafluoroborate PET enabled non-invasive tracking of viable CAR-T cells. Administration route (intravenous, peritumoral, or intraperitoneal) significantly influenced CAR-T cell distribution and therapeutic effectiveness. Conclusion: tRACE-CAR enables precise optical and PET tracking of CRISPR-edited CAR-T cells in models of leukemia and ovarian cancer, allowing dynamic, non-invasive monitoring of cell distribution in both tumors and off-target tissues. This imaging-enabled platform could lead to more personalized, effective CRISPR-edited CAR cell therapies.},
}

*Positron-Emission Tomography/methods
Animals
Humans
*Receptors, Chimeric Antigen/genetics/metabolism
*Immunotherapy, Adoptive/methods
Mice
Gene Editing/methods
*T-Lymphocytes/immunology/metabolism/transplantation
CRISPR-Cas Systems
Genes, Reporter
Cell Line, Tumor
Symporters/genetics
Female
Dependovirus/genetics
Optical Imaging/methods

@article {pmid41513195,
year = {2026},
author = {Luo, Q and Huang, Y and Zheng, H and Ke, W and Zheng, H and Sun, Q and Wang, M and Weng, Z},
title = {CRISPR-engineered zebrafish expression system for human type III collagen: Therapeutic efficacy in wound healing.},
journal = {International journal of biological macromolecules},
volume = {340},
number = {Pt 1},
pages = {150161},
doi = {10.1016/j.ijbiomac.2026.150161},
pmid = {41513195},
issn = {1879-0003},
mesh = {Animals ; *Zebrafish/genetics ; Humans ; *Wound Healing/drug effects/genetics ; Animals, Genetically Modified ; *CRISPR-Cas Systems/genetics ; *Collagen Type III/genetics/pharmacology ; Mice ; Recombinant Proteins/pharmacology/genetics ; Cell Proliferation ; },
abstract = {Human type III collagen (Col III) is a critical component for skin tissue repair and anti-aging, yet its heterologous expression often faces challenges such as incomplete structure and poor thermostability. Here, we established a transgenic zebrafish expression system via CRISPR/Cas9 technology, integrating the human Col3a1 gene into a non-functional region of zebrafish chromosome 4. The extraction yield of total zebrafish collagen (Col III-TC), a composite material comprising both recombinant human Col III and endogenous zebrafish collagens, was 45.76%. Structural analysis revealed intact fibrous architecture and a thermal shrinkage temperature of 71.3 °C, significantly superior to conventional systems. Functionally, Col III-TC exhibited remarkable free radical-scavenging capacity and suppressed LPS-induced inflammation in 3T3-L1 cells (downregulating Tnfα, Il1b, and Il6, while upregulating Il10), alongside promoting fibroblast proliferation. In a murine acute wound model, Col III-TC-based dressings achieved outstanding healing efficacy (>95% closure within 15 days), with histological analysis showing improved neoskin thickness and collagen deposition. The Col3a1 transgenic zebrafish system developed in this study not only provides a novel strategy for heterologous expression of fully functional human proteins, but also highlights the broad application potential of its high-yield collagen in biomedical fields, particularly in wound healing and anti-aging therapies.},
}

Animals
*Zebrafish/genetics
Humans
*Wound Healing/drug effects/genetics
Animals, Genetically Modified
*CRISPR-Cas Systems/genetics
*Collagen Type III/genetics/pharmacology
Mice
Recombinant Proteins/pharmacology/genetics
Cell Proliferation

@article {pmid41062834,
year = {2026},
author = {Zhang, A and Sun, X and Wu, Y and Gao, P and Zhang, R and Zhang, M and Xie, S and Fan, W and Zhang, Y and Yin, H},
title = {Efficient and precise inversion of genomic DNA from large to chromosomal scale.},
journal = {Nature chemical biology},
volume = {22},
number = {2},
pages = {328-339},
pmid = {41062834},
issn = {1552-4469},
mesh = {Humans ; *Chromosome Inversion/genetics ; *Gene Editing/methods ; *DNA/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; Plasmids/genetics ; HEK293 Cells ; Genome, Human ; },
abstract = {Chromosomal inversion is a key structural variation impacting cellular fitness and genomic integrity. Here we developed prime-editing-based inversion with enhanced performance (PIE) to efficiently induce large-scale inversions in mammalian cells. PIEv1 uses a prime-editing guide RNA (pegRNA) pair but yields one imprecise junction. PIEv2 and PIEv3 add a second pegRNA pair for precise inversion, with PIEv3b further enhancing coupling precise inversion through improved plasmid design. PIEv3b achieves inversion efficiencies up to 61.7% for 1 Mb and 14.2% for 50 Mb segments and shows 4-20-fold higher efficiency compared to twin prime editing with integrase, across ranges of 100 kb to 30 Mb. Additionally, PIEv3b outperforms nuclease-based approaches in both inversion efficiency and precision. Using PIE, we convert human chromosomes from metacentric to telocentric configurations by inverting 30-Mb and 100-Mb chromosomal segments. Our work represents a powerful tool for engineering chromosomal structural variations, with broad implications for medicine and biotechnology.},
}

Humans
*Chromosome Inversion/genetics
*Gene Editing/methods
*DNA/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Cas Systems
Plasmids/genetics
HEK293 Cells
Genome, Human

@article {pmid41608243,
year = {2026},
author = {Mitrofanov, A and Beisel, CL and Baumdicker, F and Alkhnbashi, OS and Backofen, R},
title = {Comprehensive analysis of CRISPR array repeat mutations reveals subtype-specific patterns and links to spacer dynamics.},
journal = {microLife},
volume = {7},
number = {},
pages = {uqaf050},
pmid = {41608243},
issn = {2633-6693},
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated CRISPR-associated protein (Cas) systems are adaptive immune mechanisms in bacteria and archaea that protect against invading genetic elements by integrating short fragments of foreign DNA into CRISPR arrays. These arrays consist of repetitive sequences interspersed with unique spacers, guiding Cas proteins to recognize and degrade matching nucleic acids. The integrity of these repeat sequences is crucial for the proper function of CRISPR-Cas systems, yet their mutational dynamics remain poorly understood. In this study, we analyzed 56 343 CRISPR arrays across 25 628 diverse prokaryotic genomes to assess the mutation patterns in CRISPR array repeat sequences within and across different CRISPR subtypes. Our findings reveal, as expected to some extent, that mutation frequency is substantially higher in terminal repeat sequences compared to internal repeats consistently across system types. However, the mutation patterns exhibit an unexpected amount of variation among different CRISPR subtypes, suggesting that selective pressures and functional constraints shape repeat sequence evolution in distinct ways. Understanding these mutation dynamics provides insights into the stability and adaptability of CRISPR arrays across diverse bacterial and archaeal lineages. Additionally, we elucidate a novel relationship between repeat mutations and spacer dynamics, demonstrating that hotspots for terminal repeat mutations coincide with regions exhibiting higher spacer conservation. This observation corroborates recent findings indicating that spacer deletions occur at a frequency 374 times greater than that of mutations and are significantly influenced by repeat misalignment. Our findings suggest that repeat mutations might play a pivotal role in spacer retention or loss, or vice versa, thereby highlighting an evolutionary trade-off between the stability and adaptability of CRISPR arrays.},
}

@article {pmid41606942,
year = {2025},
author = {Fraiture, MA and D'aes, J and Gobbo, A and Delvoye, M and Meunier, AC and Frouin, J and Guiderdoni, E and Deforce, D and De Vogelaere, C and De Keersmaecker, SCJ and Vanneste, K and Roosens, NHC},
title = {Genetic fingerprints derived from genome database mining allow accurate identification of genome-edited rice in the food chain via targeted high-throughput sequencing.},
journal = {Food research international (Ottawa, Ont.)},
volume = {221},
number = {Pt 1},
pages = {117218},
doi = {10.1016/j.foodres.2025.117218},
pmid = {41606942},
issn = {1873-7145},
mesh = {*Oryza/genetics ; *High-Throughput Nucleotide Sequencing/methods ; *Plants, Genetically Modified/genetics ; *Genome, Plant ; *Gene Editing/methods ; Polymorphism, Single Nucleotide ; *Food, Genetically Modified ; Databases, Genetic ; *DNA Fingerprinting/methods ; CRISPR-Cas Systems ; },
abstract = {Genome-edited (GE) organisms are currently classified as GMOs according to European legislation, requiring traceability and labelling in the food and feed supply chain. However, unambiguous identification of a specific GE organism with one or more induced single nucleotide variations (SNVs) dispersed across the genome remains challenging. This study explored whole-genome sequencing-based characterization, public genome databases, and machine learning tools to select key genetic elements and create a unique fingerprint for distinguishing a specific GE line. As a case study, a GE Nipponbare rice line containing a single CRISPR-Cas-induced SNV was used. To experimentally assess the detection of this fingerprint, a targeted high-throughput sequencing approach, including multiplex PCR-based enrichment of key genetic elements, was developed and successfully tested. This promising proof-of-concept demonstrates the potential of combining a unique genetic fingerprint with targeted high-throughput sequencing to facilitate the accurate detection of GE organisms, thereby supporting food traceability and regulatory compliance for the development of new GE lines, as well as protecting associated intellectual property.},
}

*Oryza/genetics
*High-Throughput Nucleotide Sequencing/methods
*Plants, Genetically Modified/genetics
*Genome, Plant
*Gene Editing/methods
Polymorphism, Single Nucleotide
*Food, Genetically Modified
Databases, Genetic
*DNA Fingerprinting/methods
CRISPR-Cas Systems

@article {pmid41606937,
year = {2025},
author = {Jiang, W and Zhu, T and Zhou, S and Pan, L and Qiao, Z and Wang, M and Yang, D},
title = {Recent advances in electrochemical-based CRISPR/Cas biosensing for nucleic acid and non-nucleic acid pathogenic microorganism detection.},
journal = {Food research international (Ottawa, Ont.)},
volume = {221},
number = {Pt 1},
pages = {117213},
doi = {10.1016/j.foodres.2025.117213},
pmid = {41606937},
issn = {1873-7145},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Electrochemical Techniques/methods ; *Nucleic Acids/analysis ; *Food Microbiology/methods ; *Bacteria/genetics/isolation & purification ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The widespread presence of pathogenic microorganisms in food and environmental sources poses a persistent threat to public health. Conventional detection methods-including culture, microscopy, and biochemical assays-are limited by low sensitivity, cross-reactivity, and prolonged turnaround times, particularly when microbial loads are low or phenotypic overlap occurs. These limitations underscore the urgent need for diagnostic platforms that combine speed, specificity, and sensitivity. The advent of CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) systems has revolutionized microbial diagnostics, driving the emergence of electrochemical CRISPR/Cas (EC-CRISPR/Cas) biosensors. This review surveys four principal electrochemical CRISPR/Cas (EC-CRISPR/Cas) platforms-Cas9, Cas12a, Cas13a, and Cas14a-emphasizing their structural characteristics, biosensing mechanisms, and signal amplification strategies for both nucleic acid and non-nucleic acid pathogen detection. We first outline the molecular architecture and functional mechanisms of each Cas protein in the context of biosensing. EC-CRISPR/Cas detection strategies are classified as nucleic acid-based (either amplification-free or amplification-dependent) or non-nucleic acid-based, the latter primarily relying on aptamer-mediated recognition. We also provide a comparative analysis of signal enhancement techniques and application scenarios across bacterial, viral, fungal, and parasitic pathogens. Importantly, we identify key limitations of current systems-such as poor reusability, signal drift, and challenges in point-of-care deployment-and present emerging solutions including crRNA engineering, nanomaterial integration, and artificial intelligence-guided biosensor design. These innovations hold strong potential to enhance sensitivity, specificity, and real-time performance, offering a foundation for next-generation, scalable EC-CRISPR/Cas diagnostics.},
}

*Biosensing Techniques/methods
*CRISPR-Cas Systems
*Electrochemical Techniques/methods
*Nucleic Acids/analysis
*Food Microbiology/methods
*Bacteria/genetics/isolation & purification
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41605509,
year = {2026},
author = {Li, J and Chen, X and Yang, Y and Xu, Y and Lai, M and Shi, L and Lin, X and Chen, W and Peng, H},
title = {Electron Transfer Mechanism-Mediated Host-Guest Nanoswitch Powered Amplification-Free CRISPR/Cas12a-Electrochemiluminescence Bioassay for Alzheimer's Disease Diagnosis.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c06614},
pmid = {41605509},
issn = {1520-6882},
abstract = {Developing a high-performance amplification-free electrochemiluminescence (ECL) assay platform that operates at a low trigger potential is a promising strategy for broadening the applications of ECL sensing. In this work, we present a host-guest interaction-mediated split-type CRISPR/Cas12a-ECL assay platform by using the highly sensitive host-guest recognition between the β-cyclodextrin-functionalized gold nanoclusters (β-CD-AuNCs) probe and methylene blue (MB) system as a proof of concept. Efficient ECL quenching of β-CD-AuNCs by MB is achieved via an electron transfer mechanism based on host-guest recognition between them. By integrating the high-specific recognition and cleavage activity of the CRISPR/Cas technology, the high quantum yield, and low trigger potential β-CD-AuNCs-based ECL probes, together with the highly sensitive and selective host-guest recognition-based split-type assay design, a novel "trinity" detection platform has been successfully constructed. Using Amyloid-β oligomers (AβOs), a key biomarker for Alzheimer's disease (AD) diagnosis and therapy, as the analyte, this amplification-free CRISPR/Cas-ECL biosensing platform enables ultrasensitive and accurate detection of AβO without requiring additional signal amplification strategies. The proposed sensing platform exhibits a linear detection range from 1.0 × 10[-8] to 1.0 × 10[-1] μg/mL for AβO detection, with a detection limit as low as 0.2 fg/mL (S/N = 3). This sensitivity approaches single-molecule levels and is 3-4 orders of magnitude lower than that of traditional ELISA. Furthermore, owing to its outstanding performance including high specificity, excellent selectivity, superior sensitivity, and strong anti-interference capability, the platform demonstrates remarkable detection performance in monitoring AβO in clinical AD blood samples, showing a good Pearson's correlation between the method and ELISA results. This work provides a powerful tool for clinical diagnosis and paves the way for therapeutic development, while also offering a rational design strategy for next-generation ECL biosensing platforms.},
}

@article {pmid41605395,
year = {2026},
author = {Zhang, J and Han, B and Zhang, X and Xie, X and Zhao, F and Zhang, W and Jiang, Y and Zhang, X},
title = {A CRISPR/Cas12a-mediated marker-free fluorescent biosensor constructed based on an automated 3D DNA walker-enabled signal amplification for sensitive detection of aflatoxin B1.},
journal = {International journal of biological macromolecules},
volume = {344},
number = {Pt 2},
pages = {150549},
doi = {10.1016/j.ijbiomac.2026.150549},
pmid = {41605395},
issn = {1879-0003},
abstract = {The efficient and sensitive detection of mycotoxins is critical to ensure food safety and maintain public health worldwide. In this study, a CRISPR/Cas12a-mediated marker-free fluorescent biosensor based on an automated 3D DNA walker-enabled 'one-to-many' signal amplification was developed for sensitive detection of aflatoxin B1. Due to the efficient amplification effect of DNA walkers and the strong fluorescence properties of silver nanoclusters, a sensitive output of the amplified signal was produced by precisely regulating the activated trans-cleavage activity of the specific target DNA of Cas12a. In the established fluorescence biosensor, a small amount of aflatoxin B1 promoted the production of a large amount of activator by the established 3D DNA walker, which stimulated the trans-cleavage activity of Cas12a to degrade the single-stranded DNAs for synthesis of silver nanoclusters, leading to a decreased fluorescent signal. The established biosensor was able to achieve the sensitive detection of aflatoxin B1 under optimal conditions, obtaining a detection limit of 45.38 pg/mL in a linear range of 0.05 to 10 ng/mL. In addition, the developed biosensor showed good recoveries in spiked food samples (peanut milk and drinking water) at different concentrations. This work provided new insights for the applications of DNA walker and the development of a marker-free fluorescent biosensing platform based on CRISPR/Cas for the detection of mycotoxins.},
}

@article {pmid41605290,
year = {2026},
author = {Shi, L and Zhang, M and Zheng, R and Kwok, LY and Zhang, W},
title = {Comparative genomics reveals two major lineages of Bifidobacterium adolescentis in the human gut, driven by divergent adaptation in China and the United States.},
journal = {Journal of advanced research},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jare.2026.01.071},
pmid = {41605290},
issn = {2090-1224},
abstract = {INTRODUCTION: Bifidobacterium adolescentis is a key beneficial member of the human gut microbiota, but its genomic diversity and evolutionary drivers across human populations remain poorly characterized.

OBJECTIVES: Understanding genomic functional heterogeneity and evolutionary patterns in human gut-derived B. adolescentis.

METHODS: We performed a comparative genomic analysis of 395B. adolescentis, mainly from China (n = 169) and the U.S. (n = 146), with smaller sets from Australia, Italy, and the United Kingdom, to investigate functional heterogeneity and evolutionary mechanisms. Our analysis integrated core and pan-genome architecture, phylogenomics, single nucleotide polymorphism (SNP)-based population structure, carbohydrate-active enzyme profiles, CRISPR-Cas systems, antibiotic resistance genes, and recombination dynamics.

RESULTS: The pan-genome was open and highly plastic. Phylogenetic reconstruction identified two major clades with strong geographic stratification: Chinese isolates predominantly clustered in Clade B, while U.S. isolates grouped in Clade A. Functional annotation showed regional specialization in carbohydrate-active enzymes, with Chinese isolates enriched in glycosyltransferase families and U.S. isolates in carbohydrate-binding module and carboxylesterase families, likely reflecting dietary adaptations. Genomic islands were hotspots for horizontal gene transfer, harboring region-specific carbohydrate-active enzymes and antibiotic resistance genes such as tet(W/32/O) and ermX, which were frequently located in Chinese isolates. Recombination was found to be the primary driver of genetic diversity, with recombination-to-mutation ratios approaching and exceeding 3.0 in Chinese and U.S. isolates. Linkage disequilibrium decay further supported higher recombination rates in these populations.

CONCLUSION: B. adolescentis has diverged into two major genomic lineages, primarily associated with isolates from China and the U.S. This divergence reflects adaptation to distinct host-associated ecological factors, such as diet, antibiotic exposure, and lifestyle, and is predominantly driven by extensive homologous recombination rather than point mutations. These findings highlight how regional selective pressures shape the genomic and functional landscape of this key gut symbiont.},
}

@article {pmid41604096,
year = {2026},
author = {Almufarriji, FM},
title = {Nanocarrier-mediated CRISPR-Cas delivery: a novel approach against antibiotic-resistant superbugs.},
journal = {Saudi pharmaceutical journal : SPJ : the official publication of the Saudi Pharmaceutical Society},
volume = {34},
number = {1},
pages = {5},
pmid = {41604096},
issn = {1319-0164},
abstract = {Antibiotic resistance (ABR) is a leading cause of death and a major public health threat globally. Without appropriate interventions, annual ABR-associated deaths have been projected to reach 10 million by 2050 worldwide. Hence, it is critical to develop novel therapeutic interventions that would be able to tackle ABR by targeting mainly the pathogenic microbes, while lessening harm to beneficial microbes. There is an increasing research interest in CRISPR-Cas (CC) systems owing to their potential in controlling and preventing horizontal gene transfer and spread of antibiotic resistance. In addition, CC systems offer several advantages, including high efficiency, rapid turnaround time, low cost, and easy design, which allow these systems to effectively and precisely target antibiotic-resistant bacteria. CRISPR-based gene therapy offers numerous benefits; however, the major limitation in clinical translation is the safe and effective delivery of CRISPR components to target organs or cells, thus hindering its potential in therapeutic interventions. Nanocarriers (NCs) can help the CC systems to overcome their off-target effects by precisely delivering the systems to the target cells. NCs can also be engineered for target site release, payload protection, and high specificity, which can further ensure delivery of the components of CC in the target cells or regions without harming surrounding tissues. This review summarizes the principles and mechanisms of CC systems, highlights their applications against antibiotic-resistant bacteria, and discusses emerging nanocarrier-based delivery strategies that may enhance the clinical utility of CRISPR-Cas technologies in managing ABR.},
}

@article {pmid41546867,
year = {2026},
author = {Nebenfuehr, B and Sanford, L and Taylor, ER and Ball, K and Woods-Killam, CE and Ghasemi, HI and Proctor, B and Ortega, R and Sempeck, C and Dowell, RD and Arnoult, N},
title = {Uncovering genetic interactions in the DNA repair network in response to endogenous damage and ionizing radiation.},
journal = {Cell reports},
volume = {45},
number = {1},
pages = {116850},
doi = {10.1016/j.celrep.2025.116850},
pmid = {41546867},
issn = {2211-1247},
mesh = {*Radiation, Ionizing ; *DNA Repair/genetics/radiation effects ; Humans ; *DNA Damage/genetics ; Ku Autoantigen/metabolism/genetics ; CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded/radiation effects ; MRE11 Homologue Protein/metabolism/genetics ; Ubiquitin-Protein Ligases/metabolism/genetics ; },
abstract = {Genomic integrity relies on a complex network of DNA damage response (DDR) pathways that repair endogenous and exogenous lesions, yet how individual factors operate within this broader landscape remains unclear. We performed a large-scale combinatorial CRISPR-Cas9 knockout screen targeting 461 DNA repair genes, disrupting over 100,000 gene combinations under basal conditions and after ionizing radiation (IR). This approach uncovered thousands of genetic interactions spanning pathways that respond to endogenous damage and those specific to double-strand break repair. From this dataset, we validated both positive and negative interactions under basal and irradiated conditions, including a synthetic lethal relationship between MRE11A and the E3 ligase UBR5, a role for Ku70/80 in preventing unscheduled nuclease activity at telomeres, an IR-specific vulnerability upon co-disruption of CYREN and PARG, and a link between CYREN-mediated radioresistance and innate immunity. This resource enables mechanistic insight and reveals therapeutic vulnerabilities in DNA-repair-deficient cancers.},
}

*Radiation, Ionizing
*DNA Repair/genetics/radiation effects
Humans
*DNA Damage/genetics
Ku Autoantigen/metabolism/genetics
CRISPR-Cas Systems/genetics
DNA Breaks, Double-Stranded/radiation effects
MRE11 Homologue Protein/metabolism/genetics
Ubiquitin-Protein Ligases/metabolism/genetics

@article {pmid41447528,
year = {2026},
author = {Liu, P and Yuan, Q and Yang, X and Wang, Q and Chang, T and Bi, Y and Wu, P and Zhang, T and Yang, J and Guo, S and Xue, C and Zheng, Z and Xin, B and Ma, H and Wang, Y},
title = {A synthetic biology toolkit for the plasmid-dependent and thermophilic methylotroph Bacillus methanolicus.},
journal = {Cell reports},
volume = {45},
number = {1},
pages = {116788},
doi = {10.1016/j.celrep.2025.116788},
pmid = {41447528},
issn = {2211-1247},
mesh = {*Plasmids/genetics/metabolism ; *Bacillus/genetics/metabolism ; *Synthetic Biology/methods ; CRISPR-Cas Systems/genetics ; Gene Editing ; Methanol/metabolism ; Arginine/metabolism ; Metabolic Engineering ; },
abstract = {Bacillus methanolicus, a unique plasmid-dependent and thermophilic methylotroph, is an ideal chassis for one-carbon (C1) biomanufacturing. Despite its evolutionary uniqueness and industrial promise, the synthetic biology toolkit remains limited in comparison to that of conventional model microorganisms. Here, we present a comprehensive toolkit comprising a high-efficiency electroporation protocol, a CRISPR-Cas9 method enabling robust and multiplex genome editing, diverse neutral loci for gene integration, and a cloud-based genome-scale metabolic model iBM822 for user-friendly biodesign. Leveraging this toolkit, we systematically dissected plasmid-dependent methylotrophy, restriction-modification machinery, and the functional significance of chromosomal methylotrophic genes. To address plasmid loss-induced strain degeneration, we integrated the large endogenous plasmid pBM19 into the chromosome for stable and intact methylotrophic growth. Finally, by integrating metabolic modeling with CRISPR-Cas9 editing, we engineered L-arginine feedback regulation to achieve L-arginine overproduction from methanol. This study establishes a synthetic biology framework for B. methanolicus, promoting mechanistic exploration of methylotrophy and C1 biomanufacturing.},
}

*Plasmids/genetics/metabolism
*Bacillus/genetics/metabolism
*Synthetic Biology/methods
CRISPR-Cas Systems/genetics
Gene Editing
Methanol/metabolism
Arginine/metabolism
Metabolic Engineering

@article {pmid41428486,
year = {2026},
author = {Ribeiro Gomes, AR and Hamel, N and Mastwal, S and Wright, N and Ide, DC and Richie, CT and Usdin, TB and Wang, KH and Leopold, DA},
title = {Targeted gene transfer into developmentally defined cell populations of the primate brain.},
journal = {Cell reports},
volume = {45},
number = {1},
pages = {116756},
doi = {10.1016/j.celrep.2025.116756},
pmid = {41428486},
issn = {2211-1247},
mesh = {Animals ; *Gene Transfer Techniques ; *Brain/metabolism/embryology ; Callithrix ; Female ; Rats ; Dependovirus/genetics ; Transgenes/genetics ; Gene Editing/methods ; Genetic Vectors ; Male ; CRISPR-Cas Systems ; },
abstract = {The primate brain possesses unique physiological and developmental features, yet its systematic investigation has been hampered by a paucity of transgenic germline models and tools. Here, we present a minimally invasive method to introduce transgenes widely across the primate cerebral cortex using ultrasound-guided fetal intracerebroventricular viral injections (FIVIs). FIVI enables efficient and long-lasting transgene expression following intrauterine delivery of recombinant adeno-associated viruses (rAAVs). In the marmoset, we demonstrate that adjusting gestational timing, rAAV serotype, and transcriptional regulatory elements enables selective targeting of defined cell populations, including layer-restricted labeling and Cre-dependent intersectional access. Pilot experiments in rats further demonstrate the potential of FIVIs for prenatal CRISPR-based gene editing and labeling of peripheral somatosensory and retinal pathways. By mimicking key desirable features of germline transgenic models, this efficient and targeted method for gene transfer into the fetal primate brain expands the experimental opportunities for basic and translational neuroscience research across the lifespan.},
}

Animals
*Gene Transfer Techniques
*Brain/metabolism/embryology
Callithrix
Female
Rats
Dependovirus/genetics
Transgenes/genetics
Gene Editing/methods
Genetic Vectors
Male
CRISPR-Cas Systems

@article {pmid41417728,
year = {2026},
author = {Huber, A and Djajawi, TM and Rivera, IS and Vervoort, SJ and Kearney, CJ},
title = {CRISPR screens define unified hallmarks of cancer cell-autonomous immune evasion.},
journal = {Cell reports},
volume = {45},
number = {1},
pages = {116738},
doi = {10.1016/j.celrep.2025.116738},
pmid = {41417728},
issn = {2211-1247},
mesh = {Humans ; *Neoplasms/immunology/genetics/pathology ; *Tumor Escape/genetics ; *CRISPR-Cas Systems/genetics ; Tumor Microenvironment/immunology ; Immunotherapy ; Animals ; *Immune Evasion ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CD8-Positive T-Lymphocytes/immunology ; },
abstract = {Cancer immunotherapy has transformed cancer treatment, yet only a minority of patients achieve durable benefit. Although early efforts to enhance immunotherapy focused on boosting immune effector function, reversing T cell exhaustion, or altering the tumor microenvironment, it is now clear that cancer cell-autonomous mechanisms play a major role in immune escape. Such programs, driven by the cancer cell genome, transcriptome, and epigenome, include desensitization to cytokine signaling, such as interferon (IFN)γ and tumor necrosis factor (TNF); impaired antigen presentation; upregulation of suppressive ligands such as programmed cell death ligand 1 (PD-L1); and epigenetic silencing of immunogenic pathways. The rise of high-throughput functional genomics, especially in vitro and in vivo CRISPR-based screening, has greatly expanded our ability to map these pathways and define how tumors evade CD8[+] T cell-mediated pressure. A deeper understanding of these cancer cell-autonomous immune-evasion mechanisms will be essential for developing new therapeutic strategies that broaden the impact of immunotherapy across diverse cancers.},
}

Humans
*Neoplasms/immunology/genetics/pathology
*Tumor Escape/genetics
*CRISPR-Cas Systems/genetics
Tumor Microenvironment/immunology
Immunotherapy
Animals
*Immune Evasion
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
CD8-Positive T-Lymphocytes/immunology

@article {pmid41417727,
year = {2026},
author = {Higginson, LA and Wang, X and He, K and Torstrick, M and Kim, M and Wijeratne, HRS and Runnebohm, AM and Benayoun, BA and MacLean, A and Chanfreau, GF and Morton, DJ},
title = {The RNA exosome maintains cellular RNA homeostasis by controlling transcript abundance in the brain.},
journal = {Cell reports},
volume = {45},
number = {1},
pages = {116729},
doi = {10.1016/j.celrep.2025.116729},
pmid = {41417727},
issn = {2211-1247},
mesh = {Animals ; *Brain/metabolism ; *Homeostasis ; *Exosome Multienzyme Ribonuclease Complex/metabolism/genetics ; Drosophila melanogaster/metabolism/genetics ; Drosophila Proteins/metabolism/genetics ; *Exosomes/metabolism ; Humans ; CRISPR-Cas Systems/genetics ; *RNA/metabolism/genetics ; Transcriptome ; Mutation ; },
abstract = {Intracellular ribonucleases (RNases) are essential for maintaining accurate RNA levels. Inherited mutations in genes encoding ubiquitous RNases are associated with human diseases, primarily affecting the nervous system. Recessive mutations in genes encoding the evolutionarily conserved RNA exosome, an RNase complex, cause syndromic neurodevelopmental disorders, such as pontocerebellar hypoplasia type 1b (PCH1b), characterized by progressive neurodegeneration. Here, we establish a CRISPR-Cas9-engineered Drosophila model of PCH1b to investigate the cell-type-specific post-transcriptional regulatory functions of the RNA exosome complex in vivo. Pathogenic variants in Rrp40, a subunit of the complex, disrupt RNA exosome activity, leading to widespread transcriptomic dysregulation in brain-enriched cell populations, including defective rRNA processing. These molecular defects coincide with progressive neurodegeneration and behavioral impairments that track with allele severity. Our findings provide a cell-type-resolved view of RNA exosome function in a fully developed animal brain and underscore the critical role of RNA surveillance in safeguarding transcriptome homeostasis and neuronal integrity.},
}

Animals
*Brain/metabolism
*Homeostasis
*Exosome Multienzyme Ribonuclease Complex/metabolism/genetics
Drosophila melanogaster/metabolism/genetics
Drosophila Proteins/metabolism/genetics
*Exosomes/metabolism
Humans
CRISPR-Cas Systems/genetics
*RNA/metabolism/genetics
Transcriptome
Mutation

@article {pmid41411128,
year = {2026},
author = {Mochida, T and Fujimoto, N and Asahina, M and Asano, S and Araki, S and Inukai, N and Hotta, A},
title = {Muscle satellite cell editing by LNP-CRISPR-Cas9 to resist muscle injury.},
journal = {Cell reports},
volume = {45},
number = {1},
pages = {116695},
doi = {10.1016/j.celrep.2025.116695},
pmid = {41411128},
issn = {2211-1247},
mesh = {Animals ; *Satellite Cells, Skeletal Muscle/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Mice ; *Muscular Dystrophy, Duchenne/genetics/therapy/pathology ; *Muscle, Skeletal/injuries/metabolism/pathology ; *Nanoparticles/chemistry ; Mice, Inbred C57BL ; RNA, Guide, CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Mice, Inbred mdx ; Male ; Exons/genetics ; Liposomes ; },
abstract = {Muscle satellite cells are essential for skeletal muscle regeneration and represent an attractive therapeutic target for gene delivery in Duchenne muscular dystrophy (DMD). However, efficient in vivo transduction of these cells has remained challenging. Here, we demonstrate that lipid nanoparticle (LNP)-mediated delivery of Streptococcus pyogenes CRISPR-Cas9 mRNA and guide RNA (LNP-CRISPR) induces exon skipping in Pax7-positive satellite cells more efficiently than adeno-associated virus (AAV) vectors following intramuscular or intravenous administration in a DMD mouse model. Furthermore, unlike AAV-CRISPR, LNP-CRISPR-mediated genome editing showed greater resistance to repeated muscle injuries, indicating successful editing of regenerative satellite cells. These results highlight the potential of LNPs as a non-viral platform for durable genome editing in skeletal muscle and lay the foundation for developing safe and sustainable genome-editing therapies for DMD.},
}

Animals
*Satellite Cells, Skeletal Muscle/metabolism
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Mice
*Muscular Dystrophy, Duchenne/genetics/therapy/pathology
*Muscle, Skeletal/injuries/metabolism/pathology
*Nanoparticles/chemistry
Mice, Inbred C57BL
RNA, Guide, CRISPR-Cas Systems/genetics
Disease Models, Animal
Mice, Inbred mdx
Male
Exons/genetics
Liposomes

@article {pmid41173524,
year = {2026},
author = {Dai, J and Molloy, EK},
title = {StarCDP: Dynamic Programming Algorithms for Fast and Accurate Cell Lineage Tree Reconstruction from CRISPR-Based Lineage Tracing Data.},
journal = {Journal of computational biology : a journal of computational molecular cell biology},
volume = {33},
number = {1},
pages = {48-66},
doi = {10.1177/15578666251386082},
pmid = {41173524},
issn = {1557-8666},
mesh = {*Cell Lineage/genetics ; *Algorithms ; Animals ; Mice ; Humans ; Phylogeny ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Software ; *Computational Biology/methods ; Lung Neoplasms/genetics/pathology ; Single-Cell Analysis/methods ; Dynamic Programming ; },
abstract = {CRISPR-based lineage tracing, coupled with single-cell RNA sequencing, has emerged as a promising approach for studying development and disease progression at the cellular level. Thus, cell lineage tree (CLT) reconstruction has attracted significant attention in recent years, including the introduction of Star Homoplasy Parsimony (SHP) to model the unique properties of CRISPR-induced mutations, along with the Startle family of methods. However, CLT reconstruction continues to be challenged by technological limitations in producing consistent phylogenetic signals across CLTs. To address these issues, we present Star-CDP, a collection of dynamic programming algorithms that enable researchers to seek, count, sample, and build consensus trees from solutions to SHP within a constrained search space, defined by subsets of cells from which a solution must draw its clades. When using our procedure to construct clade constraints, Star-CDP runs in polynomial time, enabling scalability to larger numbers of cells than Startle-ILP (integer linear programming), the leading method for SHP. In simulations, Star-CDP's strict consensus achieved the same or higher accuracy (f1-score) compared to the leading parsimony methods, with the greatest gains in accuracy occurring when the phylogenetic signal was limited due to the high ratio of cells to mutations. On lineage tracing data from a mouse model of lung adenocarcinoma, Star-CDP's strict consensus achieved the lowest SHP score and comparable numbers of metastatic reseedings compared to PAUP*'s strict consensus and Startle-NNI (nearest neighbor interchange), all benchmarked on a standard data processing pipeline (although our study also revealed that the pipeline can impact relative performance for migrations/reseedings). Star-CDP is available on GitHub: https://github.com/molloy-lab/Star-CDP.},
}

*Cell Lineage/genetics
*Algorithms
Animals
Mice
Humans
Phylogeny
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Software
*Computational Biology/methods
Lung Neoplasms/genetics/pathology
Single-Cell Analysis/methods
Dynamic Programming

@article {pmid40914889,
year = {2025},
author = {Beran, K and Park, SH and Van den Bergh, A and Dressman, J and Hermans, E and Holm, R and Lin, Y and Sepassi, K and Yang, B and Evers, R},
title = {Canine Mdr1 Knockout MDCK Cells Reliably Estimate Human Small Intestinal Permeability (Peff) and Fraction Absorbed (fa).},
journal = {Molecular pharmaceutics},
volume = {22},
number = {10},
pages = {6067-6082},
pmid = {40914889},
issn = {1543-8392},
mesh = {Animals ; Dogs ; ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics/metabolism ; Madin Darby Canine Kidney Cells/metabolism ; Permeability ; *Intestinal Absorption/physiology ; Intestine, Small/metabolism ; Gene Knockout Techniques ; Voriconazole/pharmacokinetics ; Acetaminophen/pharmacokinetics ; Intestinal Barrier Function ; Humans ; Purines/pharmacokinetics ; Pyridines/pharmacokinetics ; Pyrrolidines/pharmacokinetics ; Benzenesulfonamides/pharmacokinetics ; Benzaldehydes/pharmacokinetics ; CRISPR-Cas Systems ; Administration, Oral ; *Pharmaceutical Preparations/metabolism ; Pyrazines ; Pyrazoles ; Pyrimidines ; ATP Binding Cassette Transporter, Subfamily B ; },
abstract = {Human intestinal permeability is a key determinant of the oral fraction absorbed (fa) of active pharmaceutical ingredients (APIs). This study evaluated the ability of an in-house canine Mdr1 (cMdr1) knockout (KO) Madin-Darby Canine Kidney (MDCK) cell line to correlate in vitro apparent permeability (Papp) with human small intestinal permeability (Peff). In vitro Papp values of 16 reference compounds with high, medium, or low permeabilities were measured in the in-house cMdr1 KO MDCK protocol under pH gradient (6.5 ⇒ 7.4) and pH equivalent conditions (7.4 ⇒ 7.4) and correlations with human Peff were established (R[2] > 0.8). The correlations were subsequently used to estimate Peff and fa for six test APIs: acetaminophen, voriconazole, fedratinib, voxelotor, lemborexant, and istradefylline. The results for these APIs were compared against literature and permeability data from other methods routinely used in drug discovery and development. The projected Peff and fa values for the test APIs aligned well with literature permeabilities derived using other methods and clinical pharmacokinetic studies, respectively. This work highlights the usefulness of cMdr1 KO MDCK cells in permeability classification, especially for highly permeable APIs, and supports its broader use in both research and regulatory contexts.},
}

Animals
Dogs
ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics/metabolism
Madin Darby Canine Kidney Cells/metabolism
Permeability
*Intestinal Absorption/physiology
Intestine, Small/metabolism
Gene Knockout Techniques
Voriconazole/pharmacokinetics
Acetaminophen/pharmacokinetics
Intestinal Barrier Function
Humans
Purines/pharmacokinetics
Pyridines/pharmacokinetics
Pyrrolidines/pharmacokinetics
Benzenesulfonamides/pharmacokinetics
Benzaldehydes/pharmacokinetics
CRISPR-Cas Systems
Administration, Oral
*Pharmaceutical Preparations/metabolism
Pyrazines
Pyrazoles
Pyrimidines
ATP Binding Cassette Transporter, Subfamily B

@article {pmid41603863,
year = {2026},
author = {Calderón, L and Schäfer, M and Rončević, M and Rauschmeier, R and Jaritz, M and Schwickert, TA and Sun, Q and Pauli, A and Zuber, J and Busslinger, M},
title = {In vivo CRISPR/Cas9 screens identify new regulators of B cell activation and plasma cell differentiation.},
journal = {The Journal of experimental medicine},
volume = {223},
number = {3},
pages = {},
pmid = {41603863},
issn = {1540-9538},
support = {//Boehringer Ingelheim/ ; 740349/ERC_/European Research Council/International ; LT00427/2013//Human Frontier Science Program/ ; },
mesh = {Animals ; *Cell Differentiation/genetics/immunology ; *CRISPR-Cas Systems/genetics ; *Plasma Cells/immunology/cytology ; *B-Lymphocytes/immunology/cytology ; Mice ; *Lymphocyte Activation/genetics/immunology ; Mice, Inbred C57BL ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Immune responses to pathogens lead to the generation of plasma cells through a complex interplay of B cells with their microenvironment in lymphoid organs. To identify new regulators of B cell activation and plasmablast differentiation in the context of the splenic microenvironment, we established an in vivo system for pooled sgRNA CRISPR/Cas9 screens in immunized mice. To improve the infection efficiency of naïve B cells, we generated Cd23-Cre Rosa26LSL-EcoR/+ mice exhibiting increased expression of the ecotropic lentivirus receptor EcoR on naïve B cells. Upon adoptive B cell transfer and immunization of recipient mice, 379 sgRNAs, targeting genes with high expression in plasma cells, were analyzed for their effects on plasmablast generation. Gene hits, encoding 23 positive and 18 negative regulators of B cell activation, plasmablast differentiation, or homeostasis, were uniquely identified in these in vivo screens. Validated genes encoded proteins involved in cell adhesion, signal transduction, protein folding, iron transport, and enzymatic processes. Hence, our in vivo screening system identified novel regulators controlling B cell-mediated immune responses.},
}

Animals
*Cell Differentiation/genetics/immunology
*CRISPR-Cas Systems/genetics
*Plasma Cells/immunology/cytology
*B-Lymphocytes/immunology/cytology
Mice
*Lymphocyte Activation/genetics/immunology
Mice, Inbred C57BL
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41603755,
year = {2026},
author = {Yuan, X and Yang, F and Chen, X and Xiao, P and Shen, L},
title = {Single-Nucleotide Variation Analysis in Oral Squamous Cell Carcinoma-Related ctDNA by dCas9/sgRNA Recognition-Mediated Proximity Ligation-Triggered Terminal Hairpin Formation and Self-Priming Amplification.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c07087},
pmid = {41603755},
issn = {1520-6882},
abstract = {Circulating tumor DNA (ctDNA) represents a promising noninvasive biomarker for cancer diagnosis, including oral cancer. However, its clinical translation is currently limited by the lack of precise and reliable detection techniques. In this study, we developed a novel fluorescent biosensor for the detection of single-nucleotide variations in ctDNA, which integrates dual dCas9/sgRNA complexes for target recognition, proximity ligation-initiated terminal hairpin formation and self-priming amplification (PS-THSP), and Cas12a/crRNA-mediated signal output. A key innovation of this design is its multilayered specificity strategy, combining mutation-specific recognition by dual dCas9/sgRNA, proximity-dependent ligation, and Cas12a/crRNA-assisted verification of PS-THSP amplicons. This integrated approach offers a significant advance over existing CRISPR/Cas-based methods that rely primarily on signal amplification. Furthermore, the biosensor achieves high sensitivity through the synergistic coupling of PS-THSP amplification and Cas12a trans-cleavage activity, enabling a broad dynamic range spanning 6 orders of magnitude and a detection limit as low as 0.12 fM within 120 min. When applied to serum samples, the biosensor reliably detected ctDNA with high accuracy, demonstrating its strong potential for clinical cancer diagnostics.},
}

@article {pmid41603736,
year = {2026},
author = {Fuglsang, A and Rout, SS and Koutna, EB and Sofos, N and Gallego, AR and Montoya, G},
title = {Conformational dynamics of CRISPR-Cas type I-F-HNH inform nickase engineering in a cascade scaffold.},
journal = {Nucleic acids research},
volume = {54},
number = {3},
pages = {},
pmid = {41603736},
issn = {1362-4962},
support = {NNF14CC0001//Novo Nordisk Foundation Center for Protein Research (CPR)/ ; NNF14CC0001//Novo Nordisk Foundation/ ; NNF24SA0098829//Novo Nordisk Foundation/ ; NNF0024386//Novo Nordisk Foundation/ ; NNF17SA0030214//Novo Nordisk Foundation/ ; NNF18OC0055061//Novo Nordisk Foundation/ ; NNF24SA0098829//Novo Nordisk Foundation/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; DNA Cleavage ; *Deoxyribonuclease I/genetics/chemistry/metabolism ; *CRISPR-Associated Proteins/chemistry/genetics/metabolism ; Catalytic Domain ; Protein Engineering ; Humans ; Cryoelectron Microscopy ; Gene Editing ; Models, Molecular ; DNA, Single-Stranded/metabolism/genetics/chemistry ; DNA/metabolism/chemistry ; Protein Conformation ; },
abstract = {The type I-FHNH CRISPR-Cas system is a non-canonical Class 1 effector complex distinguished by the replacement of the Cas3 recruitment domain with a catalytic HNH domain in Cas8, enabling autonomous DNA cleavage without accessory nucleases. Using cryo-EM, we determined high-resolution structures of the effector complex in three catalytic states-precatalytic, NTS-cleaved, and post-catalytic-revealing a dynamic trajectory of the HNH domain through inward, middle, and outward conformations. Biochemical assays demonstrated that the complex cleaves the nontarget strand (NTS) prior to the target strand (TS), consistent with a sequential cleavage mechanism similar to Cas12 effectors but notably lacking trans-cleavage activity on single-stranded DNA. Structural comparisons confirmed a minimal PAM requirement (5'-CN) and a constrained HNH catalytic site poised for precise strand scission. We engineered a ΔLinker variant of Cas8 that repositions the HNH domain, selectively abolishing TS cleavage and converting the system into a programmable NTS-specific nickase. Importantly, we validated the functionality of both wild-type and mutant complexes in human cells. While the wild-type system induced indels and base substitutions, the ΔLinker variant triggered targeted single-strand nicks without double-stranded breaks. Together, our work establishes type I-FHNH as a compact and precise genome editing platform with in vivo efficacy.},
}

*CRISPR-Cas Systems/genetics
DNA Cleavage
*Deoxyribonuclease I/genetics/chemistry/metabolism
*CRISPR-Associated Proteins/chemistry/genetics/metabolism
Catalytic Domain
Protein Engineering
Humans
Cryoelectron Microscopy
Gene Editing
Models, Molecular
DNA, Single-Stranded/metabolism/genetics/chemistry
DNA/metabolism/chemistry
Protein Conformation

@article {pmid41603733,
year = {2026},
author = {Kwon, H and Kim, J and Zhou, L and Dean, A},
title = {LDB1 regulates gene expression and chromatin structure in pluripotency and lineage differentiation.},
journal = {Nucleic acids research},
volume = {54},
number = {3},
pages = {},
pmid = {41603733},
issn = {1362-4962},
support = {//Intramural Program of the National Institute of Diabetes/ ; //Digestive and Kidney Diseases/ ; 075033/GF/NIH HHS/United States ; },
mesh = {Animals ; *Cell Differentiation/genetics ; Mice ; Kruppel-Like Factor 4 ; *Chromatin/chemistry/metabolism/genetics ; Cell Lineage/genetics ; *DNA-Binding Proteins/genetics/physiology/metabolism ; SOXB1 Transcription Factors/metabolism/genetics ; Embryoid Bodies/metabolism/cytology ; Erythroblasts/metabolism/cytology ; Mouse Embryonic Stem Cells/metabolism/cytology ; Kruppel-Like Transcription Factors/genetics/metabolism ; Gene Expression Regulation, Developmental ; Mice, Knockout ; CRISPR-Cas Systems ; *Pluripotent Stem Cells/metabolism/cytology ; Enhancer Elements, Genetic ; Embryonic Stem Cells/metabolism/cytology ; *Gene Expression Regulation ; LIM Domain Proteins ; },
abstract = {Chromatin organization is a pivotal factor in stem cell pluripotency and differentiation. However, the role of enhancer looping protein LIM domain-binding 1 (LDB1) in stem cells remains to be fully explored. We generated Ldb1(-/-) embryonic stem cells (ESCs) using CRISPR/Cas9 editing and observed a reduction in key stem cell factors SOX2 and KLF4 upon LDB1 loss. Differential gene expression, including of the Lin28-mediated self-renewal pathway genes, was observed between wild-type and Ldb1(-/-) ESC. LDB1 occupied super enhancers, including those of pluripotency genes, in ESC together with pluripotency factors, and LDB1 loss resulted in loss of Sox2 interactions with the SCR enhancer. Embryoid bodies (EBs) derived from Ldb1(-/-) ESC displayed reduced expression of lineage-specific markers. Ldb1(-/-) ESC had impaired ability to undergo terminal differentiation to erythroblasts, and gene dysregulation was very pronounced in Ldb1(-/-) erythroblasts. Conditional LDB1-deficient mice displayed reduced hematopoietic stem cell markers on bone marrow cells and dysregulation of the Lin28 pathway. Thus, LDB1 function is critical for ESC and EB development and becomes progressively more important for normal gene expression during differentiation to erythroblasts.},
}

Animals
*Cell Differentiation/genetics
Mice
Kruppel-Like Factor 4
*Chromatin/chemistry/metabolism/genetics
Cell Lineage/genetics
*DNA-Binding Proteins/genetics/physiology/metabolism
SOXB1 Transcription Factors/metabolism/genetics
Embryoid Bodies/metabolism/cytology
Erythroblasts/metabolism/cytology
Mouse Embryonic Stem Cells/metabolism/cytology
Kruppel-Like Transcription Factors/genetics/metabolism
Gene Expression Regulation, Developmental
Mice, Knockout
CRISPR-Cas Systems
*Pluripotent Stem Cells/metabolism/cytology
Enhancer Elements, Genetic
Embryonic Stem Cells/metabolism/cytology
*Gene Expression Regulation
LIM Domain Proteins

@article {pmid41603729,
year = {2026},
author = {Huang, Y and Xu, H and Zhang, T and Liao, Y and Hu, J and Huang, Z and Hu, H and Li, P and Fan, L and Xie, J},
title = {Mycobacterial non-homologous end joining is required for antiphage defense.},
journal = {Nucleic acids research},
volume = {54},
number = {3},
pages = {},
pmid = {41603729},
issn = {1362-4962},
support = {82472325//National Natural Science Foundation/ ; 82072246//National Natural Science Foundation/ ; CSTB2024NSCQ-MSX0703//Natural Science Foundation of Chongqing/ ; //Chongqing Public Health Key Specialty (Discipline) Construction Project/ ; CSTB2024NSCQ-MSX0703//Natural Science Foundation of Chongqing/ ; },
mesh = {*DNA End-Joining Repair ; *Mycobacterium smegmatis/virology/genetics ; DNA Breaks, Double-Stranded ; Oxidative Stress ; Homologous Recombination ; },
abstract = {In the ongoing arms race with phages, bacteria have evolved diverse defense systems, such as CRISPR-Cas and restriction-modification systems. The DNA double-strand break repair system represents a core mechanism for maintaining genomic integrity and is vital for cell survival. However, it remains unknown whether and how these repair systems contribute to phage resistance. This study systematically investigates the role of the non-homologous end joining (NHEJ) during phage infection in Mycobacterium smegmatis. We found that NHEJ deficiency compromises host resistance to phage SWU1, as evidenced by increased plaque counts and reduced bacterial survival. Mechanistically, phages exploit host NHEJ for genomic repair; however, the error-prone nature of NHEJ leads to imperfect repair at phage cos sites, thereby blocking replication. The host modulates the balance between NHEJ and homologous recombination (HR) to control repair fidelity: NHEJ loss shifts the balance toward high-fidelity HR, which in turn promotes phage survival. Furthermore, NHEJ deficiency exacerbates infection-induced oxidative stress, leading to a compromise in bacterial viability. Our findings reveal the multifaceted functions of NHEJ in mycobacterium-phage interactions and provide new insights into how DNA repair systems shape antiphage defense and coevolution.},
}

*DNA End-Joining Repair
*Mycobacterium smegmatis/virology/genetics
DNA Breaks, Double-Stranded
Oxidative Stress
Homologous Recombination

@article {pmid41603018,
year = {2026},
author = {Dolder, RE and Friedman, CE and Loiben, AM and Yang, KC and Glazer, AM},
title = {High-Throughput Methods for Variant Functional Assessment in Cardiac Disease.},
journal = {Circulation. Genomic and precision medicine},
volume = {},
number = {},
pages = {e005239},
doi = {10.1161/CIRCGEN.125.005239},
pmid = {41603018},
issn = {2574-8300},
abstract = {In vitro functional modeling of genetic variants has revolutionized our understanding of which variants can cause cardiac disorders, providing insights into their molecular underpinnings. This review provides an overview of high-throughput methods used for the functional assessment of variants implicated in inherited cardiac diseases. Advances in gene-editing technology now enable the efficient generation of cells expressing individual genetic variants or libraries of variants for robust functional studies. We discuss innovative assays that can evaluate dozens or hundreds of variants sequentially. For example, the electrophysiological properties of numerous cardiac ion channel variants in genes linked to inherited arrhythmias can be characterized using automated patch clamping. The mechanical properties of cardiomyocytes expressing candidate cardiomyopathy variants can be assessed using techniques such as atomic force microscopy, traction force microscopy, and impedance-based methods. Multiplexed assays of variant effect are an emerging family of techniques that use gene-specific or general assays, combined with next-generation sequencing, to characterize hundreds or thousands of pooled genetic variants. We examine the key advantages and limitations of each method and outline future goals for the field. Innovative in vitro studies of cardiac genetic variants will enhance our understanding of variant-disease relationships and improve diagnosis, screening, and treatment options for these disorders.},
}

@article {pmid41601654,
year = {2025},
author = {Yin, L and He, W and Wang, Y and Zhang, H and Huang, M and Yan, Y and Li, S and Feng, X and Saenz, F and Zhang, J and Zhu, D and Yang, C and Ma, T and Fu, J and Chen, J},
title = {FACS-based genome-wide CRISPR screening platform identifies modulators of CD47.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1684539},
pmid = {41601654},
issn = {1664-3224},
mesh = {*CD47 Antigen/genetics/metabolism/immunology ; Animals ; Mice ; Cell Line, Tumor ; *CRISPR-Cas Systems ; Humans ; Flow Cytometry/methods ; Phagocytosis ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Macrophages/immunology/metabolism ; Female ; Immunity, Innate ; },
abstract = {BACKGROUND: CD47 is a key innate immune checkpoint that enables tumor cells to evade macrophage-mediated clearance.

METHODS/RESULTS: To systematically identify genetic regulators of CD47 surface expression, we performed FACS-based genome-wide CRISPR screens in three murine cancer cell lines B16 (melanoma), MC38 (colon adenocarcinoma), and EMT6 (breast carcinoma).

RESULTS: Comparative analysis of cells with high or low CD47 surface expression using DrugZ revealed CD47 itself as the top hit, validating the screens. Notably, DNAJC13 emerged as a consistent and robust regulator of CD47 expression across all three cell lines. Functional validation using DNAJC13-knockout cells confirmed a significant reduction in CD47 surface levels. Furthermore, in co-culture assays with macrophages, DNAJC13-deficient tumor cells exhibited increased susceptibility to phagocytosis, supporting a functional role for DNAJC13 in innate immune evasion. Finally, we verify that DNAJC13-knockout decrease tumor burden when treated with CD47 blockade.

CONCLUSIONS: Overall, this study highlights a previously unrecognized regulator of CD47 and demonstrates the utility of high-throughput FACS-based CRISPR screening to uncover modulators of key immune checkpoint pathways.},
}

*CD47 Antigen/genetics/metabolism/immunology
Animals
Mice
Cell Line, Tumor
*CRISPR-Cas Systems
Humans
Flow Cytometry/methods
Phagocytosis
*Clustered Regularly Interspaced Short Palindromic Repeats
Macrophages/immunology/metabolism
Female
Immunity, Innate

@article {pmid41601145,
year = {2026},
author = {Jiao, Y and Liu, Y and Sun, F and Wang, W and Li, H and Gao, Q and Li, Y and Lu, N and Tian, X and Ding, X and Du, J},
title = {Genetically Modified Plant Beneficial Microorganisms: A Sustainable Solution or a New Challenge for Agriculture.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c14342},
pmid = {41601145},
issn = {1520-5118},
abstract = {Plant diseases significantly impact crop yield and quality, while conventional pesticide treatments often disrupt beneficial plant microbiota essential for pathogen prevention and immune regulation. Although plant beneficial microorganisms (PBMs) show promise as disease control agents, their effectiveness is constrained by strain-dependent variations, survival challenges, and inconsistent immune responses. Recent advances in genetic engineering, particularly CRISPR-Cas systems combined with complementary technologies like RecE/T, enable precise modifications of PBMs to enhance their protective potential. Enhanced PBMs improve functionality via multiple mechanisms: targeted gene-expression-mediated colonization, specific antimicrobial activity, and immune regulation. Studies demonstrate that genetically modified PBMs can prevent and control plant diseases through competitive exclusion, antibiotic production, barrier reinforcement, and immune modulation. We analyzed the considerations for the environmental release of engineered PBMs to reduce risks. Future research should focus on optimizing PBMs for specific applications while addressing biosafety concerns, thereby unlocking their full potential in safeguarding plant health.},
}

@article {pmid41600812,
year = {2025},
author = {Tian, C and Feng, L and Zhou, X and Huang, K and Wang, F and Luo, R and Meng, F and Yang, H and Qiao, C and Wang, X and Shi, J and Chen, Y},
title = {A Portable One-Tube Assay Integrating RT-RPA and CRISPR/Cas12a for Rapid Visual Detection of Eurasian Avian-like H1N1 Swine Influenza Virus in the Field.},
journal = {Viruses},
volume = {18},
number = {1},
pages = {},
pmid = {41600812},
issn = {1999-4915},
support = {2021YFD1800200//Nnational Key Research and Development Program of China/ ; },
mesh = {*Influenza A Virus, H1N1 Subtype/isolation & purification/genetics ; Animals ; *CRISPR-Cas Systems ; Swine ; Sensitivity and Specificity ; *Orthomyxoviridae Infections/diagnosis/virology/veterinary ; *Swine Diseases/virology/diagnosis ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Humans ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The widespread circulation of Eurasian avian-like H1N1 (EA H1N1) swine influenza virus poses significant zoonotic and pandemic risks worldwide. However, current diagnostic methods are difficult to deploy in the field, as they generally require specialized laboratory infrastructure and trained personnel. Here, we present a novel dual-signal detection platform that combines reverse transcription recombinase polymerase amplification (RT-RPA) with CRISPR/Cas12a technology for rapid, on-site EA H1N1 detection. We established an integrated one-tube assay by designing and optimizing RT-RPA primers targeting a conserved region of the hemagglutinin (HA) gene, together with engineered CRISPR/Cas12a guide RNAs exhibiting high specificity. The platform incorporates two complementary readout modes: real-time fluorescence monitoring and visual colorimetric detection using a smartphone. The assay shows excellent analytical specificity, with no cross-reactivity observed against other swine influenza virus subtypes or common swine pathogens, (including CSFV, PRRSV, PEDV, PCV, TGEV, and RV). The detection limit is 2 copies/μL, and the entire procedure can be completed within 30 mins using simple portable equipment. When evaluated on 86 clinical samples, the assay demonstrated 94.18% concordance with RT-qPCR. Compared with conventional diagnostic methods, this RT-RPA-CRISPR/Cas12a assay offers greater convenience and cost-effectiveness. Its strong potential for field-based rapid testing underscores promising application prospects in swine influenza surveillance and control programs.},
}

*Influenza A Virus, H1N1 Subtype/isolation & purification/genetics
Animals
*CRISPR-Cas Systems
Swine
Sensitivity and Specificity
*Orthomyxoviridae Infections/diagnosis/virology/veterinary
*Swine Diseases/virology/diagnosis
*Nucleic Acid Amplification Techniques/methods
*Molecular Diagnostic Techniques/methods
Humans
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41599334,
year = {2026},
author = {Wang, X and Chen, X and Zhou, Y and Zhao, Y and Shi, C and Li, R and Liu, L and Mu, C and Song, W and Wang, C},
title = {Establishment of CRISPR-Cas9-Mediated Gene Editing in the Swimming Crab Portunus trituberculatus.},
journal = {Molecules (Basel, Switzerland)},
volume = {31},
number = {2},
pages = {},
pmid = {41599334},
issn = {1420-3049},
support = {2022YFD2400104//the National Key R&D Program of China/ ; 32341058 and 31972783//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Brachyura/genetics ; Electroporation ; Myostatin/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; INDEL Mutation ; },
abstract = {Portunus trituberculatus is an economically important marine crustacean in East Asia's aquaculture industry. Nevertheless, precise genome modification has not yet been established. In this study, we evaluated the applicability of the CRISPR-Cas9 gene editing system in P. trituberculatus using electroporation for efficient delivery of the Cas9-sgRNA complex into zygotes. We systematically investigated electroporation parameters, including buffer composition, voltage, capacitance, and pulse times. Our results showed that artificial seawater was a superior buffer to phosphate-buffered saline (PBS) and identified an effective electroporation condition of 600 V, 1 μF capacitance, and two pulses, resulting in approximately 72.7% fluorescent zygotes. Under these electroporated conditions, we detected gene indels and putative insertion events at the targeted locus of myostatin (mstn) gene. These results demonstrate the feasibility of Cas9-based genome editing in P. trituberculatus and provide a proof-of-concept for functional genomics studies and future genetic improvement of this species.},
}

Animals
*Gene Editing/methods
*CRISPR-Cas Systems
*Brachyura/genetics
Electroporation
Myostatin/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
INDEL Mutation

@article {pmid41599202,
year = {2026},
author = {Yoon, B and Kim, JA and Kang, YK},
title = {CRISPR-Cas-Mediated Reprogramming Strategies to Overcome Antimicrobial Resistance.},
journal = {Pharmaceutics},
volume = {18},
number = {1},
pages = {},
pmid = {41599202},
issn = {1999-4923},
support = {grant number RS-2024-00417430//National Research Foundation of Korea (NRF)/ ; grant number RS-2024-00399808//Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET)/ ; 2025-RISE-16-001//Regional Innovation System & Education (RISE) program/ ; A26234, CTRQQR-2021\100009//CRUK Convergence Science Centre at The Institute of Cancer Research, London, and Imperial Col-lege London/ ; },
abstract = {Antimicrobial resistance (AMR) is escalating worldwide, posing a serious threat to global public health by driving infections that are no longer treatable with conventional antibiotics. CRISPR-Cas technology offers a programmable and highly specific therapeutic alternative by directly targeting the genetic determinants responsible for resistance. Various CRISPR systems can restore antibiotic susceptibility and induce selective bactericidal effects by eliminating resistance genes, disrupting biofilm formation, and inhibiting virulence pathways. Moreover, CRISPR can suppress horizontal gene transfer (HGT) by removing mobile genetic elements such as plasmids, thereby limiting the ecological spread of AMR across humans, animals, and the environment. Advances in delivery platforms-including conjugative plasmids, phagemids, and nanoparticle-based carriers-are expanding the translational potential of CRISPR-based antimicrobial strategies. Concurrent progress in Cas protein engineering, spatiotemporal activity regulation, and AI-driven optimization is expected to overcome current technical barriers. Collectively, these developments position CRISPR-based antimicrobials as next-generation precision therapeutics capable of treating refractory bacterial infections while simultaneously suppressing the dissemination of antibiotic resistance.},
}

@article {pmid41599009,
year = {2025},
author = {Lv, YR and Liu, YY and Zhang, R and Yang, B and Xue, SY and Ding, YL and Jia, JT and Bayaer, H and Bagen, A and Chen, RB and Tunala, S and Zhao, L and Liu, YH},
title = {Rapid and Simple Detection of Mycobacterium avium subsp. paratuberculosis Using a Lateral Flow Assay Based on CRISPR-Cas12a Combined with Recombinase Polymerase Amplification or Nested PCR.},
journal = {Pathogens (Basel, Switzerland)},
volume = {15},
number = {1},
pages = {},
pmid = {41599009},
issn = {2076-0817},
support = {1.(YF20240007) 2.(BR251303) 3.(YLXKZX-NND-012)//1.Ordos Science & Technology Plan 2.Special Project for the Construction of Scientific Research and Innovation Teams, Class B Team 3.Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; },
mesh = {*CRISPR-Cas Systems ; *Mycobacterium avium subsp. paratuberculosis/genetics/isolation & purification ; *Paratuberculosis/diagnosis/microbiology ; *Polymerase Chain Reaction/methods ; Animals ; Sensitivity and Specificity ; Feces/microbiology ; Recombinases/genetics ; DNA, Bacterial/genetics ; Bacterial Proteins/genetics ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Paratuberculosis (PTB), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic intestinal disease in ruminants. PTB is difficult to diagnose, control, and eradicate, leading to substantial economic losses. Thus, sensitive and specific detection methods are urgently required. crRNA and primers targeting the MAP ATPase FtsK gene were designed for recombinase polymerase amplification (RPA) and nested PCR. Fecal DNA was amplified using RPA or nested PCR, purified with Tris-saturated phenol-chloroform-isoamyl alcohol, and detected via CRISPR-Cas12a. Moreover, signals were read using a qPCR instrument, fluorescence reader, or lateral flow strips. RPA-CRISPR-Cas12a and nested PCR-CRISPR-Cas12a assays were optimized and validated on 50 clinical samples and 7 MAP cultures. The limits of detection were 1 × 10[-10] μg/μL for RPA-CRISPR-Cas12a and 1 × 10[-14] μg/μL for nested PCR-CRISPR-Cas12a. Efficient cleavage of the ssDNA reporter occurred at DNA concentrations of ≥1 × 10[-4] μg/μL, producing a strong fluorescent signal. All three detection methods showed perfect agreement with reference assays across both sample sets. This study presents the first integration of RPA or nested PCR with CRISPR-Cas12a for MAP detection, enabling rapid, specific, and highly sensitive diagnosis. Flexible detection options allow adaptation to available resources and bacterial loads, supporting practical use in PTB control.},
}

*CRISPR-Cas Systems
*Mycobacterium avium subsp. paratuberculosis/genetics/isolation & purification
*Paratuberculosis/diagnosis/microbiology
*Polymerase Chain Reaction/methods
Animals
Sensitivity and Specificity
Feces/microbiology
Recombinases/genetics
DNA, Bacterial/genetics
Bacterial Proteins/genetics
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41598917,
year = {2026},
author = {Pei, X and Xie, J and Liang, C and Utkina, AO},
title = {Next-Generation Precision Breeding in Peanut (Arachis hypogaea L.) for Disease and Pest Resistance: From Multi-Omics to AI-Driven Innovations.},
journal = {Insects},
volume = {17},
number = {1},
pages = {},
pmid = {41598917},
issn = {2075-4450},
abstract = {Peanut (Arachis hypogaea L.) is a globally important oilseed and food legume, yet its productivity is persistently constrained by devastating diseases and insect pests that thrive under changing climates. This review aims to provide a comprehensive synthesis of advances in precision breeding and molecular approaches for enhancing disease and pest resistance in peanut. Traditional control measures ranging from crop rotation and cultural practices to chemical protection have delivered only partial and often unsustainable relief. The narrow genetic base of cultivated peanut and its complex allotetraploid genome further hinder the introgression of durable resistance. Recent advances in precision breeding are redefining the possibilities for resilient peanut improvement. Multi-omics platforms genomics, transcriptomics, proteomics, and metabolomics have accelerated the identification of resistance loci, effector-triggered immune components, and molecular cross-talk between pathogen, pest, and host responses. Genome editing tools such as CRISPR-Cas systems now enable the precise modification of susceptibility genes and defense regulators, overcoming barriers of conventional breeding. Integration of these molecular innovations with phenomics, machine learning, and remote sensing has transformed resistance screening from manual assessment to real-time, data-driven prediction. Such AI-assisted breeding pipelines promise enhanced selection accuracy and faster deployment of multi-stress-tolerant cultivars. This review outlines current progress, technological frontiers, and persisting gaps in leveraging precision breeding for disease and pest resistance in peanut, outlining a roadmap toward climate-resilient, sustainable production systems.},
}

@article {pmid41552883,
year = {2026},
author = {Xu, Q and Ji, M},
title = {A Bst-driven Cas12a cascade amplification strategy for microRNA detection.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {4},
pages = {899-906},
doi = {10.1039/d5ay01957e},
pmid = {41552883},
issn = {1759-9679},
mesh = {*MicroRNAs/analysis/genetics/blood ; Humans ; *Nucleic Acid Amplification Techniques/methods ; CRISPR-Cas Systems ; Limit of Detection ; *Endodeoxyribonucleases/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; },
abstract = {Quantification of trace microRNAs is crucial for early disease diagnosis but remains technically challenging. Herein, we developed an ultrasensitive fluorescence platform for microRNA-21 (miR-21) detection by integrating Bst DNA polymerase - assisted target recycling with CRISPR/Cas12a-mediated signal amplification. In this design, the target miRNA triggers toehold-mediated opening of a hairpin probe, followed by Bst-driven primer extension that enables efficient target recycling and the generation of abundant DNA duplex activators. Subsequently, these activators induce strong trans-cleavage activity of Cas12a, producing markedly enhanced fluorescence responses. Benefiting from the dual amplification of enzymatic recycling and Cas12a activation, the proposed assay exhibits high sensitivity toward miR-21 with a detection limit down to 9.25 × 10[-12] M. Furthermore, the platform exhibited excellent sequence selectivity and was successfully applied to monitor miR-21 in both cell lysates and clinical serum samples. Considering its convenient operation, strong analytical performance, and simple readout mode, this method holds great potential for trace biomarker analysis in clinical diagnostics.},
}

*MicroRNAs/analysis/genetics/blood
Humans
*Nucleic Acid Amplification Techniques/methods
CRISPR-Cas Systems
Limit of Detection
*Endodeoxyribonucleases/metabolism/genetics
*CRISPR-Associated Proteins/metabolism/genetics
*Bacterial Proteins/metabolism/genetics

@article {pmid41534496,
year = {2026},
author = {Gogate, A and Chahrour, MH},
title = {Recent advances in the neurogenomics of autism spectrum disorder.},
journal = {Current opinion in genetics & development},
volume = {96},
number = {},
pages = {102431},
doi = {10.1016/j.gde.2025.102431},
pmid = {41534496},
issn = {1879-0380},
mesh = {Humans ; *Autism Spectrum Disorder/genetics/pathology ; *Gene Regulatory Networks/genetics ; *Genomics ; Induced Pluripotent Stem Cells/metabolism ; *Genetic Predisposition to Disease ; CRISPR-Cas Systems/genetics ; Gene Editing ; },
abstract = {Neurogenomics has provided exceptional insights into the genetic architecture underlying autism spectrum disorder (ASD), which is increasingly understood as a collection of individually rare disorders. This review synthesizes current advancements in the field, examining how both rare and common genetic variants contribute to ASD etiology. To functionally interpret the convergence on biological pathways that has emerged despite this genetic heterogeneity, multiomic approaches have been applied to identify gene regulatory networks disrupted in ASD. High-throughput technologies, such as clustered regularly interspaced short palindromic repeats (CRISPR) editing and massively parallel reporter assays, have been employed in human induced pluripotent stem cells and organoids to bridge the gap between genetic association and biological function. Finally, machine learning methods play a pivotal role in integrating and leveraging these complex datasets to inform personalized interventions.},
}

Humans
*Autism Spectrum Disorder/genetics/pathology
*Gene Regulatory Networks/genetics
*Genomics
Induced Pluripotent Stem Cells/metabolism
*Genetic Predisposition to Disease
CRISPR-Cas Systems/genetics
Gene Editing