@article {pmid41397899,
year = {2026},
author = {Chowdhury, A and Garcia, BG and Zahoor, MA and ElMawla, NF and Davidson, AR and Wyatt, HDM and Maxwell, KL and Mahassine, A and Gehring, A and Feld, JJ},
title = {A Rapid Assay for Hepatitis C Virus RNA Detection Using Reverse-Transcription Loop-Mediated Isothermal Amplification-Coupled CRISPR-Cas12b-Based Strategy.},
journal = {The Journal of infectious diseases},
volume = {233},
number = {5},
pages = {840-847},
doi = {10.1093/infdis/jiaf609},
pmid = {41397899},
issn = {1537-6613},
mesh = {Humans ; *Hepacivirus/genetics/isolation & purification ; *Hepatitis C/diagnosis/virology ; *RNA, Viral/genetics/blood ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Genotype ; Limit of Detection ; },
abstract = {BACKGROUND: Hepatitis C virus (HCV) diagnosis usually requires detection of antibody followed by HCV RNA. The requirement for 2 tests leads to major drop-offs in the cascade of care. Existing near-care HCV RNA tests have slow turnaround time and are expensive with limited availability. We aim to develop a cost-effective, rapid, and sensitive test for detection of HCV RNA to enhance screening, particularly in marginalized and remote populations.

METHODS: After RNA extraction from plasma, HCV RNA is reverse-transcribed and amplified using loop-mediated isothermal amplification with HCV-specific primers. The amplified HCV DNA is then detected via CRISPR-Cas12b with a fluorescence readout.

RESULTS: HCV RNA from patient samples with genotypes 1a, 1b, 2, 3a, and 4 was detected with high sensitivity and specificity. The lower limit of detection (LLOD) with HCV JFH1 plasmid (genotype 2) is 250 plasmid copies/mL (approximately 100 IU/mL). For clinical samples, we determined the LLOD for genotypes 1 and 3, the most common in North America. Using 500 μL of plasma, genotype 1 RNA ≥100 IU/mL was detected within 40-45 minutes, while genotype 3 had an LLOD of 5000 IU/mL. The clinical sensitivity was 100% in 72 HCV patient samples, including acute HCV and HCV/hepatitis B virus (HBV) coinfection. The specificity was 100%, with no false-positives in 33 HCV-negative samples, including those with HBV or human immunodeficiency virus/HBV coinfection.

CONCLUSIONS: Our assay shows high specificity and sensitivity to detect HCV RNA directly from plasma within 45 minutes and hence could be used for efficient screening and diagnosis of HCV infection globally.},
}

Humans
*Hepacivirus/genetics/isolation & purification
*Hepatitis C/diagnosis/virology
*RNA, Viral/genetics/blood
Sensitivity and Specificity
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
*Molecular Diagnostic Techniques/methods
Genotype
Limit of Detection

@article {pmid41604419,
year = {2026},
author = {Martínez Rivas, FJ and Smith, MA and Zangishei, Z and Alseekh, S and Usadel, B and Plaxton, WC and Fernie, AR},
title = {Malate matters: disrupting bacterial-type phosphoenolpyruvate carboxylase (BTPC) rewires tomato fruit development.},
journal = {Plant physiology},
volume = {201},
number = {1},
pages = {},
pmid = {41604419},
issn = {1532-2548},
support = {452682775//Deutsche Forschungsgemeinschaft/ ; 390686111//Deutsche Forschungsgemeinschaft/ ; //PlantaSyst/ ; 664621//European Union's Horizon 2020 Research and Innovation Programme/ ; 739582//European Union's Horizon 2020 Research and Innovation Programme/ ; 664620//European Union's Horizon 2020 Research and Innovation Programme/ ; RGPIN-2024-06467//Natural Sciences and Engineering Research Council of Canada/ ; //Queen's Research Chair/ ; },
mesh = {*Solanum lycopersicum/growth & development/genetics/enzymology/metabolism ; *Malates/metabolism ; *Fruit/growth & development/genetics/enzymology/metabolism ; *Phosphoenolpyruvate Carboxylase/metabolism/genetics ; *Plant Proteins/metabolism/genetics ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; },
abstract = {Plant phosphoenolpyruvate carboxylases (PEPCs) are ubiquitously expressed as cytosolic Class-1 PEPC homotetramers composed of 107 kDa plant-type PEPC (PTPC) subunits that are highly sensitive to allosteric inhibition by malate. Class-2 PEPC heterooctameric complexes that are desensitized to malate inhibition also exist in certain sink tissues due to the interaction of a Class-1 PEPC with unrelated 118 kDa bacterial-type PEPC (BTPC) polypeptides. Class-2 PEPCs dynamically associate with the mitochondrial outer envelope and have been hypothesized to support sustained anaplerotic flux and respiratory CO2 refixation in malate-rich sink tissues, including immature tomato fruit. The current study generated CRISPR-Cas9-edited tomato lines with targeted disruption of the BTPC gene and investigated the impact on fruit development, metabolism, and transcriptional regulation. Immunoblotting and co-immunoprecipitation confirmed the absence of BTPC polypeptides and Class-2 PEPC complexes in the edited lines. Fruits from the edited plants were 25% smaller and 40% lighter and required up to 10 additional days to complete ripening compared to the WT. Metabolomic analysis across ripening stages revealed substantial reductions in malate and citrate, with elevated sugars and amino acids, indicating reprogrammed carbon flux. RNA-seq data showed downregulation of genes for cell wall remodeling, sugar transport, and ethylene-responsive transcription factors. These results provide direct evidence that BTPC is essential for organic acid balance, sugar metabolism, and ripening regulation in tomato. Its absence perturbs metabolic homeostasis and developmental progression, positioning BTPC as a strategic target for enhancing fruit quality traits through genetic engineering.},
}

*Solanum lycopersicum/growth & development/genetics/enzymology/metabolism
*Malates/metabolism
*Fruit/growth & development/genetics/enzymology/metabolism
*Phosphoenolpyruvate Carboxylase/metabolism/genetics
*Plant Proteins/metabolism/genetics
Gene Expression Regulation, Plant
CRISPR-Cas Systems

@article {pmid41734599,
year = {2026},
author = {Kim, GH and Kim, MM},
title = {Effect of p53 gene mutation with loss of function on the expression of genes and proteins involved in cell proliferation.},
journal = {Mutation research},
volume = {832},
number = {},
pages = {111931},
doi = {10.1016/j.mrfmmm.2026.111931},
pmid = {41734599},
issn = {1873-135X},
mesh = {Humans ; *Cell Proliferation/genetics ; *Tumor Suppressor Protein p53/genetics/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; *Loss of Function Mutation ; Gene Expression Regulation, Neoplastic ; *Mutation ; Cell Cycle/genetics ; },
abstract = {The tumor suppressor gene TP53 plays a vital role in preserving genomic integrity by regulating cell cycle progression, DNA repair mechanisms, and apoptosis. This study aims to examine how CRISPR/Cas9-induced loss-of-function mutations in the p53 gene influence cellular processes on cell cycle regulation and tumorigenic signaling in HT1080 human fibrosarcoma cells. Successful TP53 gene disruption was confirmed by Sanger sequencing, and its structural modelling using AlphaFold2 and ChimeraX confirmed alterations in the predicted TP53 protein structure compared to that of wild type. Gene expression analyses, conducted via RT-PCR and qPCR, demonstrated a marked decrease in TP53 mRNA expression within the modified cells. Despite the mutation, the edited cells elevated activity of the senescence marker β-galactosidase (SA-β-gal). They decreased the production of collagen, suggesting that the structural disruption caused by CRISPR/Cas9 leads to the loss of functional p53 activity. Western blotting and immunofluorescence assays further revealed a remarkable downregulation of key cell cycle and tumorigenesis-related proteins, including TP53, phosphorylated TP53 (p-TP53), acetylated TP53 (ac-TP53), MMP-2, cyclin D, cyclin E, AKT, BAX, MDM2, and phosphorylated Rb (p-Rb) in the edited cells relative to the wild-type counterpart. Our results suggest that the TP53 mutation may disrupt essential pathways related to cell proliferation and stress responses. This provides new insights into TP53 functionality and underscores its potential as a therapeutic target in cancer biology.},
}

Humans
*Cell Proliferation/genetics
*Tumor Suppressor Protein p53/genetics/metabolism
CRISPR-Cas Systems
Cell Line, Tumor
*Loss of Function Mutation
Gene Expression Regulation, Neoplastic
*Mutation
Cell Cycle/genetics

@article {pmid41882360,
year = {2026},
author = {Smith, QM and Whittle, S and Aramayo, RJ and Rollins, DE and Jalal, ASB and Egharevba, DI and Morris, KL and Pyne, ALB and Rueda, DS},
title = {Structural basis of supercoiling-induced CRISPR-Cas9 off-target activity.},
journal = {Nature},
volume = {653},
number = {8114},
pages = {627-635},
pmid = {41882360},
issn = {1476-4687},
mesh = {*CRISPR-Associated Protein 9/metabolism/chemistry/ultrastructure ; *CRISPR-Cas Systems/genetics ; Cryoelectron Microscopy ; DNA, Circular/chemistry/ultrastructure/genetics/metabolism ; *DNA, Superhelical/chemistry/metabolism/ultrastructure/genetics ; *Gene Editing/methods ; Models, Molecular ; Protein Domains ; RNA/chemistry/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {CRISPR-Cas9 is a powerful genome-editing tool[1], but genome-wide off-target activity can hinder therapeutic applications. Negative supercoiling ((-)SC) has been implicated in off-target activity, but a molecular-level understanding is lacking. Here, using (-)SC DNA minicircles, we observe supercoiling-driven structural defects in the DNA that are resolved by Cas9 binding. Cryo-electron microscopy structures of Cas9 bound in both the on-target and off-target configurations highlight that the Cas9 HNH domain is poised in a more catalytically competent conformation. New DNA-RNA mismatch geometries are accommodated across the protospacer and structural plasticity in the protospacer adjacent motif distal region of the protospacer is topology dependent. Together, our study reveals the molecular basis for (-)SC-induced Cas9 targeting and provides a framework for the design of next-generation high-fidelity CRISPR effectors with topological context.},
}

*CRISPR-Associated Protein 9/metabolism/chemistry/ultrastructure
*CRISPR-Cas Systems/genetics
Cryoelectron Microscopy
DNA, Circular/chemistry/ultrastructure/genetics/metabolism
*DNA, Superhelical/chemistry/metabolism/ultrastructure/genetics
*Gene Editing/methods
Models, Molecular
Protein Domains
RNA/chemistry/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems

@article {pmid41913646,
year = {2026},
author = {Streiber, M and Liu, N and Simon, L and Adermann, F and Bachmann, V and Gath, L and Hoeppener, S and Schubert, S and Werz, O and Lapinte, V and Morille, M and Bauer, M and Press, AT and Schubert, US and Traeger, A},
title = {Extrahepatic Gene Editing In Vivo Using Organic Solvent-Free Lipid Nanoparticles.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {22},
number = {27},
pages = {e11489},
pmid = {41913646},
issn = {1613-6829},
support = {13XP5034A//Bundesministerium für Bildung und Forschung/ ; 03RU2U071H//Bundesministerium für Bildung und Forschung/ ; 2021 FGI 0005//Thüringer Aufbaubank/ ; 2023FGR0077//Thüringer Aufbaubank/ ; //Joachim Herz Stiftung/ ; P2024-02-016//Carl-Zeiss-Stiftung/ ; 57604510//German Academic Exchange Service/ ; 2018FGI0025//European Regional Development Fund/ ; ANR- 20-CE09-0011-01//Agence Nationale de la Recherche/ ; 514006196//Deutsche Forschungsgemeinschaft/ ; 316213987//Deutsche Forschungsgemeinschaft/ ; },
mesh = {*Nanoparticles/chemistry/ultrastructure ; Humans ; Animals ; *Lipids/chemistry ; *Gene Editing/methods ; Mice ; Solvents/chemistry ; CRISPR-Cas Systems/genetics ; Polyethylene Glycols/chemistry ; Transfection ; Liposomes ; },
abstract = {Targeted therapy, which modifies genes and their expression, holds great promise for treating a variety of diseases, including cancer, inborn errors of metabolism, and acute and chronic inflammatory and infectious conditions. However, it also presents challenges related to RNA delivery, immune responses, side effects of delivery vectors, and the need for individualized formulations. To overcome these limitations, the choice of lipids and formulation processes might be re-evaluated, with a focus on eliminating critical components, such as poly(ethylene glycol) (PEG) and ethanol. Thus, a purely water-based formulation for lipid nanoparticles was developed, offering a material-efficient, time-saving process with high reproducibility. Initially, a stealth lipid containing poly(2-methyl-2-oxazoline) (PMeOx) was used, and the formulation was later expanded to include approved lipids. These nanoparticles not only efficiently transfect primary human immune cells but also effectively deliver multiple nucleotides in CRISPR-Cas9 applications. Moreover, an in vivo comparison revealed that the nanoparticles exhibited preferential transfection in extrahepatic tissues. This distinguishes them from conventional cholesterol-rich lipid nanoparticles, which primarily target the liver regardless of the application route.},
}

*Nanoparticles/chemistry/ultrastructure
Humans
Animals
*Lipids/chemistry
*Gene Editing/methods
Mice
Solvents/chemistry
CRISPR-Cas Systems/genetics
Polyethylene Glycols/chemistry
Transfection
Liposomes

@article {pmid41931906,
year = {2026},
author = {Pedersen, A and Blay-Cadanet, J and Storgaard, J and Hernaez, B and Thyrsted, J and Bach-Nielsen, CS and Twayana, K and Jørgensen, SE and Rio-Bergé, C and Poulsen, C and Thielke, AL and Thomsen, EA and Kalucka, J and Olagnier, D and Luo, Y and Reggiori, F and Mogensen, TH and Alcamí, A and Hansen, AL and Holm, CK},
title = {NRF2 controls a diverse network of antiviral effectors with p62 acting as a central restriction factor effective across virus families.},
journal = {Redox biology},
volume = {93},
number = {},
pages = {104135},
pmid = {41931906},
issn = {2213-2317},
mesh = {*NF-E2-Related Factor 2/metabolism/genetics ; Humans ; *SARS-CoV-2/physiology ; Virus Replication/drug effects ; *Sequestosome-1 Protein/metabolism/genetics ; Influenza A virus/physiology ; Herpesvirus 1, Human/physiology ; *COVID-19/virology/genetics/metabolism ; Animals ; Autophagy ; Vaccinia virus/physiology ; Antiviral Agents/pharmacology ; CRISPR-Cas Systems ; Chlorocebus aethiops ; },
abstract = {The transcription factor erythroid 2 (NFE2)-related factor 2 (NRF2) is a key regulator of cellular homeostasis. Recent discoveries have identified agonists of NRF2 as inducers of broad cellular resistance to viral infection including SARS-CoV-2. Nevertheless, it is still unclear to what extent NRF2 itself is an inducer of anti-viral immunity and its downstream antiviral effectors have not been mapped. Here, we first demonstrate through specific genetic activation and silencing that NRF2 restricts SARS-CoV-2 replication. We then used a focused CRISPR-activation screen to map antiviral NRF2-inducible effector genes that restrict replication of SARS-CoV-2, Influenza A virus (IAV), Herpes Simplex virus 1 (HSV1) and Vaccinia virus (VACV). This approach allowed us to identify a range of antiviral effectors each of which restrict members of one or more virus families. Importantly, we identified the NRF2-inducible selective autophagy receptor p62/SQSTM1 as a broadly effective restriction factor across all the tested viruses. Importantly, p62 inhibited SARS-CoV-2 replication in cells treated with the lysosomal inhibitor bafilomycin A1, as well as in cells deficient in the autophagy protein ATG5. Similarly, p62 inhibited replication of HSV1 and IAV independently of ATG5 and ATG16L1 respectively. Thus, NRF2 restricts viral replication through a hitherto underappreciated network of antiviral restriction factors effective across multiple virus families. Importantly, we identify p62 as a broadly acting antiviral effector that restricts viral replication independently of canonical autophagy.},
}

*NF-E2-Related Factor 2/metabolism/genetics
Humans
*SARS-CoV-2/physiology
Virus Replication/drug effects
*Sequestosome-1 Protein/metabolism/genetics
Influenza A virus/physiology
Herpesvirus 1, Human/physiology
*COVID-19/virology/genetics/metabolism
Animals
Autophagy
Vaccinia virus/physiology
Antiviral Agents/pharmacology
CRISPR-Cas Systems
Chlorocebus aethiops

@article {pmid41942711,
year = {2026},
author = {Wang, W and Li, Y and Dong, S and Liu, Y and Guo, C and Su, Y and Tian, W and Hu, X and Wang, Z},
title = {The exonic SNP rs11676272-C risk allele mediates diet-induced obesity and reduces enhancer activation.},
journal = {EMBO reports},
volume = {27},
number = {9},
pages = {2462-2490},
pmid = {41942711},
issn = {1469-3178},
support = {32470645//MOST | National Natural Science Foundation of China (NSFC)/ ; 32070567//MOST | National Natural Science Foundation of China (NSFC)/ ; 32202840//MOST | National Natural Science Foundation of China (NSFC)/ ; C2023201032//| Natural Science Foundation of Hebei Province ()/ ; 050001-5000019//Hebei University ()/ ; ZJ2025118//Zhejiang Province Human Resources and Social Security Department ()/ ; },
mesh = {Animals ; Humans ; *Obesity/genetics/etiology/metabolism ; HEK293 Cells ; Mice ; *Polymorphism, Single Nucleotide ; *Alleles ; *Adenylyl Cyclases/genetics/metabolism ; *Enhancer Elements, Genetic ; Diet, High-Fat/adverse effects ; Genetic Predisposition to Disease ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; Gene Expression Regulation ; Male ; },
abstract = {Genome-wide association studies (GWASs) have identified hundreds of obesity-associated SNPs, but establishing their causality remains challenging. Here, we demonstrate that rs11676272, located in the ADCY3 gene, is a functional causal variant for obesity susceptibility. Bioinformatic analyses and dual-luciferase reporter assays indicate that the rs11676272 region may act as a human-gained enhancer regulating ADCY3 expression. In HEK293T cells, CRISPR-Cas9-mediated single-nucleotide editing of rs11676272 (T > C) reduces ADCY3 expression. Moreover, the rs11676272-T allele is preferentially bound by the transcription factor E2F3 to upregulate ADCY3 expression, whereas the rs11676272-C risk allele loses this binding. In vivo, the rs11676272 T > C variant in human ADCY3 (hADCY3) knock-in mice accelerates weight gain under high-fat diet conditions and shortens primary cilia in the ventromedial hypothalamus (VMH). CRISPRa-mediated activation of the hADCY3 promoter region rescues ciliary length in both the VMH and hypothalamic arcuate nucleus of Mut-hADCY3 mice. Our data reveal a causal role for rs11676272 in obesity, offering insight into potential therapeutic strategies.},
}

Animals
Humans
*Obesity/genetics/etiology/metabolism
HEK293 Cells
Mice
*Polymorphism, Single Nucleotide
*Alleles
*Adenylyl Cyclases/genetics/metabolism
*Enhancer Elements, Genetic
Diet, High-Fat/adverse effects
Genetic Predisposition to Disease
Promoter Regions, Genetic
CRISPR-Cas Systems
Gene Expression Regulation
Male

@article {pmid42020604,
year = {2026},
author = {Calvo Fernández, E and Tomassoni, L and Zhang, X and Wang, J and Obradovic, A and Laise, P and Griffin, AT and Vlahos, L and Minns, HE and Morales, DV and Simmons, C and Gallitto, M and Wei, HJ and Martins, TJ and Becker, PS and Crawford, JR and Tzaridis, T and Wechsler-Reya, RJ and Garvin, J and Gartrell, RD and Szalontay, L and Zacharoulis, S and Wu, CC and Zhang, Z and Califano, A and Pavisic, J},
title = {Systematic design of combination therapy by targeting master regulators of coexisting diffuse midline glioma cell states.},
journal = {Nature genetics},
volume = {58},
number = {5},
pages = {1112-1125},
pmid = {42020604},
issn = {1546-1718},
mesh = {Humans ; *Glioma/genetics/drug therapy/pathology ; Animals ; Mice ; Cell Line, Tumor ; Pyrazoles ; *Brain Neoplasms/genetics/drug therapy/pathology ; *Antineoplastic Combined Chemotherapy Protocols/therapeutic use/pharmacology ; Pyrimidines ; Gene Expression Regulation, Neoplastic/drug effects ; Nitriles ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; },
abstract = {Intratumor heterogeneity fundamentally challenges cancer treatment, as coexisting, molecularly distinct cell states with non-overlapping drug sensitivities can drive therapeutic resistance. We establish and validate a generalizable, network-based framework to systematically identify combination therapies targeting complementary tumor cell states. Applied to diffuse midline glioma (DMG)-a universally fatal pediatric malignancy-this approach identified master regulator protein dependencies in seven coexisting cell states, confirmed by pooled CRISPR-Cas9 assays. Perturbational transcriptional profiles for 372 clinically relevant drugs prioritized candidates predicted to invert state-specific master regulator activity. State-selective drug sensitivity was validated for eight out of nine (89%) drugs in vivo, including avapritinib, ruxolitinib and larotrectinib. Compared with monotherapy, co-administering drugs targeting complementary states significantly prolonged survival across virtually all combinations, with avapritinib plus ruxolitinib extending median survival nearly threefold versus vehicle and 1.5-fold versus avapritinib alone. These findings establish clinically actionable DMG combinations and a tumor-agnostic and mutation-agnostic framework for rational combination therapy design.},
}

Humans
*Glioma/genetics/drug therapy/pathology
Animals
Mice
Cell Line, Tumor
Pyrazoles
*Brain Neoplasms/genetics/drug therapy/pathology
*Antineoplastic Combined Chemotherapy Protocols/therapeutic use/pharmacology
Pyrimidines
Gene Expression Regulation, Neoplastic/drug effects
Nitriles
Xenograft Model Antitumor Assays
CRISPR-Cas Systems

@article {pmid42062574,
year = {2026},
author = {Xu, Y and Stubbendieck, RM and Viswanatha, R and Krč, A and Baik, LS and Suh, WS and Hu, Y and Wang, H and Yin, L and Mameli, E and van der Meij, A and Carlson, JR and Doxey, AC and Stenmark, P and Perrimon, N and Currie, CR and Dong, M},
title = {Streptomyces produce a diphtheria toxin-like exotoxin that targets insects.},
journal = {Nature microbiology},
volume = {11},
number = {5},
pages = {1271-1285},
pmid = {42062574},
issn = {2058-5276},
support = {R01AI170835//U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R01AI189789//U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R01NS080833//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; },
mesh = {Animals ; *Streptomyces/metabolism/genetics ; Drosophila melanogaster/drug effects ; Virulence Factors/metabolism/genetics ; *Diphtheria Toxin/genetics/metabolism/chemistry ; *Insecta/drug effects/microbiology ; Bacterial Proteins/genetics/metabolism/chemistry ; *Exotoxins/metabolism/genetics/chemistry/toxicity ; Phylogeny ; *Insecticides/metabolism ; Peptide Elongation Factor 2/metabolism ; CRISPR-Cas Systems ; Insect Proteins/metabolism ; },
abstract = {Streptomyces and insects engage in complex interactions shaped by millions of years of evolution. While many beneficial relationships are well recognized, it remains unknown whether Streptomyces produce virulence factors targeting insects specifically. Here, through bioinformatic analysis, we identified diphtheria toxin (DT) homologues, which we named Streptomyces antiquus insecticidal proteins (SAIP), within a monophyletic lineage of Streptomyces that emerged more than 100 million years ago. SAIP is cytotoxic to insect cells and lethal to Drosophila melanogaster, suppressing neuronal activity and immune responses in vivo. Structural and functional studies validated that SAIP is homologous to DT and acts by ADP ribosylation of eukaryotic elongation factor 2. CRISPR-Cas9 screening identified the insect protein Flower as the SAIP receptor across a range of insects. Toxigenic Streptomyces can consume dead insects and produce bioactive secondary metabolites while growing on insect carcasses. These findings establish an insecticidal toxin in Streptomyces and demonstrate that Streptomyces have evolved highly specific virulence factors against insects.},
}

Animals
*Streptomyces/metabolism/genetics
Drosophila melanogaster/drug effects
Virulence Factors/metabolism/genetics
*Diphtheria Toxin/genetics/metabolism/chemistry
*Insecta/drug effects/microbiology
Bacterial Proteins/genetics/metabolism/chemistry
*Exotoxins/metabolism/genetics/chemistry/toxicity
Phylogeny
*Insecticides/metabolism
Peptide Elongation Factor 2/metabolism
CRISPR-Cas Systems
Insect Proteins/metabolism

@article {pmid42065511,
year = {2026},
author = {Chen, Y and Tang, D and Zhan, J and Wang, Y and Song, Z},
title = {Study on the detection of prostate cancer using MIRA-CRISPR/Cas12a technology.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {18},
pages = {3848-3857},
doi = {10.1039/d6ay00025h},
pmid = {42065511},
issn = {1759-9679},
mesh = {Humans ; Male ; *Prostatic Neoplasms/diagnosis/urine/genetics ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Antigens, Neoplasm/genetics/urine ; *Molecular Diagnostic Techniques/methods ; Biomarkers, Tumor/urine/genetics ; Sensitivity and Specificity ; },
abstract = {Background: Prostate cancer (PCa) is the most diagnosed cancer among men globally and a leading cause of cancer-related mortality. However, current conventional prostate diagnostic methods fail to meet the growing demands of clinical practice in terms of speed, simplicity, sensitivity, and specificity. To address these limitations, we established a molecular detection system based on MIRA-CRISPR/Cas12a technology. Using reverse transcription-multienzyme isothermal rapid amplification (RT-MIRA) to amplify minute PCA3-specific fragments in samples, we employ CRISPR/Cas12a to detect the fluorescence signal released by these fragments, enabling the detection of trace PCA3 molecules in urine. Methods: PCA3 standard strain cultivation and total RNA extraction; establishment and optimization of the MIRA amplification system using primers designed for the PCA3 molecular marker; design of crRNA targeting optimal sites within the detection sequence, combined with CRISPR/Cas12a technology to establish the detection system; preliminary validation of the technology's sensitivity and specificity. Results: the MIRA-CRISPR/Cas12a technology was successfully established for visual detection of PCA3 molecules in prostate cancer. Primer and crRNA sequences within the reaction system were determined. The detection sensitivity for PCA3 molecules in urine reached 1 × 10[0] copies per µL with excellent specificity. Conclusion: the MIRA-CRISPR/Cas12a technology enables specific detection of PCA3 molecules in urine. This technique features high sensitivity, high specificity, visual results, and simple operation. It does not require specialized laboratory UV imaging equipment; results are visible to the naked eye under LED blue light. Following further optimization, it offers a feasible technical solution for rapid molecular screening of prostate cancer.},
}

Humans
Male
*Prostatic Neoplasms/diagnosis/urine/genetics
*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
*Antigens, Neoplasm/genetics/urine
*Molecular Diagnostic Techniques/methods
Biomarkers, Tumor/urine/genetics
Sensitivity and Specificity

@article {pmid42084031,
year = {2026},
author = {Ma, R and Xiao, Y and Yu, W and Huo, C and Meng, S and Zhao, Z and Guo, Z and Ren, X and Zhang, H and Li, B and Wang, Y and Liu, S and Huang, J},
title = {A novel high-sensitivity fluorescence detection technology for zearalenone based on the PER-triggered crRNA conformational change and CHA-coordinated energy supply.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {18},
pages = {3865-3873},
doi = {10.1039/d6ay00474a},
pmid = {42084031},
issn = {1759-9679},
mesh = {*Zearalenone/analysis ; *Biosensing Techniques/methods ; CRISPR-Cas Systems ; Limit of Detection ; Nucleic Acid Conformation ; Spectrometry, Fluorescence/methods ; Fluorescence ; },
abstract = {Zearalenone (ZEN), frequently encountered in corn, is a hazardous mycotoxin capable of impairing liver and kidney function, compromising immune responses, and potentially inducing carcinogenesis. Current detection methodologies are hampered by elevated costs, complex workflows, limited sensitivity, and poor specificity. There is a pressing need to develop simple, rapid, and ultrasensitive assays that combine high specificity with operational convenience, thereby facilitating precise biotoxin surveillance and control. This study developed a novel biosensing strategy for ultrasensitive detection of zearalenone (ZEN) by engineering a blocked Primer Exchange Reaction (PER) dumbbell-hairpin structure integrated with a Catalytic Hairpin Assembly (CHA)-based DNA machine. We constructed a highly specific and sensitive fluorescence biosensor for zearalenone (ZEN) by integrating a Primer Exchange Reaction (PER) with the trans-cleavage activity of CRISPR/Cas12a. This strategy significantly simplifies the operational procedure compared to conventional techniques. Furthermore, its modular design establishes a versatile and efficient platform adaptable for the detection of various trace analytes, offering a promising proof-of-concept for mycotoxin screening in agricultural products, although further extensive validation across diverse realistic matrices is warranted.},
}

*Zearalenone/analysis
*Biosensing Techniques/methods
CRISPR-Cas Systems
Limit of Detection
Nucleic Acid Conformation
Spectrometry, Fluorescence/methods
Fluorescence

@article {pmid42132069,
year = {2026},
author = {Liu, Z and Qi, J and Sun, L and Su, J and Li, W and Wu, L and Liang, Y and Wei, C and He, F and Han, Y and Sun, Y and Yan, H and Li, H and Xiao, R},
title = {Oxygen-Vacancy-Engineered WOX Nanowire-Based Surface-Enhanced Raman Scattering Biosensor with Lyophilized CRISPR/Cas13a Platform for CHIKV Detection.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.6c00934},
pmid = {42132069},
issn = {2379-3694},
abstract = {The outbreak of Chikungunya virus (CHIKV) has caused widespread acute morbidity with severe polyarthralgia or chronic arthritis, placing a great challenge to public health and socioeconomic development. Establishing a rapid and highly sensitive detection technology is crucial for achieving precise control and prevention. Here, we established a CRISPR/Cas13a-mediated SERS lateral flow immunoassay platform for rapid and highly sensitive detection of CHIKV. One-dimensional nanowires loading Au nanoparticles were used to prepare SERS tags, presenting excellent SERS-enhanced performances and superior applicability to directional flow on the test strip. For the best performance, the structure of WOX nanowires was regulated by adjusting the addition concentration of ascorbic acid during the synthesis process, resulting in a stronger LSPR effect derived from more Au NPs in situ grown on highly reducing WOX. Furthermore, the lyophilized CRISPR/Cas system greatly simplified the workflow. In the optimal conditions, the limit of detection reached 0.56 and 1.03 copies/μL for the CHIKV plasmid and inactivated viruses by this method, respectively. Furthermore, 34 clinical serum samples were accurately diagnosed by our proposed method, 100% consistent with qPCR. This platform with the advantages of simple operation and rapid response provides a reliable technical tool for the early precise identification and efficient monitoring of CHIKV.},
}

@article {pmid42133116,
year = {2026},
author = {Rajan, A and Raveendran, M and Shanmugam, V and Arul, L and Kumar, KK and Subramanian, A and Mannu, J and Eswaran, K},
title = {Engineering crop determinacy: CRISPR/Cas based advances in self-pruning gene function and application.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42133116},
issn = {1573-4978},
support = {BT/INF/22/SP45584/2022//Department of Biotechnology, Government of India/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Crops, Agricultural/genetics/growth & development ; Gene Editing/methods ; Flowers/genetics/growth & development ; Plants, Genetically Modified/genetics ; Plant Breeding/methods ; Genetic Engineering/methods ; Plant Proteins/genetics ; },
abstract = {The transition from indeterminate to determinate growth represents a key achievement in crop improvement, as it enhances agricultural productivity by synchronizing flowering, facilitating uniform harvest, and improving overall efficiency. In tomato and other crops, this shift is largely governed by mutations in the SELF-PRUNING (SP) gene, a key member of the CENTRORADIALIS (CEN), TERMINAL FLOWER 1 (TFL1), and SELF-PRUNING (SP) (CETS) gene family that regulates the vegetative to reproductive phase transition and influences overall shoot architecture. With increasing labour constraints, climate variability and rising global food security challenges, the ability to engineer optimized plant architectures has become increasingly important. CRISPR-based genome editing provides a precise and efficient strategy to modify SP/TFL1 homologs, enabling targeted transition from indeterminate to compact, determinate growth forms that exhibit synchronized flowering and enhanced mechanical harvestability. These genome editing approaches have been successfully applied across diverse crop species, including tomato, legumes, cotton, cereals and horticultural crops. This review consolidates current understanding of the molecular mechanisms governing determinacy, with emphasis on the central role of SP/TFL1 genes and their interactions with hormonal pathways such as auxin and cytokinin. By integrating these insights with recent advances in CRISPR-based editing platforms, this review provides a practical framework for researchers and breeders aiming to leverage CRISPR technology for next-generation crop improvement. Such strategies hold significant potential for enhancing productivity, resilience and sustainability within modern agricultural systems.},
}

*CRISPR-Cas Systems/genetics
*Crops, Agricultural/genetics/growth & development
Gene Editing/methods
Flowers/genetics/growth & development
Plants, Genetically Modified/genetics
Plant Breeding/methods
Genetic Engineering/methods
Plant Proteins/genetics

@article {pmid42133523,
year = {2026},
author = {Nammi, B and Madugula, SS and Jayasinghe-Arachchige, VM and Pham, T and Liu, J and Wang, S},
title = {Robust CRISPR-Cas Protein Identification using Max-Margin Regularized Transformer Models.},
journal = {IEEE transactions on computational biology and bioinformatics},
volume = {PP},
number = {},
pages = {},
doi = {10.1109/TCBBIO.2026.3693528},
pmid = {42133523},
issn = {2998-4165},
abstract = {The discovery of CRISPR-Cas system has significantly advanced genome editing, offering vast applications in medical treatments and life sciences research. Despite their immense potential, the existing CRISPR-Cas systems still face challenges concerning size, delivery efficiency, and cleavage specificity. Addressing these challenges requires a deeper understanding of CRISPR-Cas proteins to advance the design and discovery of novel Cas proteins. Here, we study CRISPR-Cas proteins extensively using deep-learning techniques to build classification models that can differentiate between Cas and non-Cas proteins, as well as identify subfamilies Cas9 and Cas12. We developed two types of deep learning models: 1) a transformer encoder-based classification model, trained from scratch; and 2) a large protein language model fine-tuned on ProtBert, pre-trained on more than 200 million proteins. To boost learning efficiency for the model trained from scratch, we introduced a novel margin-based loss function to maximize inter-class separability and intra-class compactness in protein sequence embedding latent space of a transformer encoder. Our results show that the Fine-Tuned ProtBert-based (FTPB) classification model achieved accuracies of 99.06%, 94.42%, 96.80%, 97.57% for Cas9 vs. non-Cas, Cas12 vs.non-Cas, Cas9 vs. Cas12, and multi-class classification of Cas9 vs. Cas12 vs. non-Cas proteins, respectively. The Latent Space Regularized Max-Margin Transformer (LSRMT) model achieved classification accuracies of 99.81%, 99.81%, 99.06%, and 99.27% for the same tasks, respectively. These results demonstrate the effectiveness of the proposed Max-Margin-based latent space regularization in enhancing model robustness and generalization capabilities. Remarkably, the LSRMT model, even when trained on a significantly smaller dataset, outperformed the fine-tuned state-of-the-art large protein model. The high classification accuracies achieved by the LSRMT model demonstrate its proficiency in identifying discriminative features of CAS proteins, marking a significant step towards advancing our understanding of CAS protein structures in future research endeavors.},
}

@article {pmid42134322,
year = {2026},
author = {Lei, L and Kaufmann, MM and Lao, J and Thoulass, G and Ammann, S and Xiao, H and Rhiel, M and Dettmer-Monaco, V and Grünewald, J and Andrieux, G and Alzubi, J and Miller, BR and Weißert, K and Gräßel, L and Schell, C and Illert, AL and Joung, JK and Boerries, M and Cornu, TI and Ehl, S and Erlacher, M and Aichele, P and Cathomen, T},
title = {Genotoxicity profiling reveals distinct platform-and cell type-specific effects in therapeutic gene editing for genetic hyperinflammation.},
journal = {Cell stem cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.stem.2026.04.014},
pmid = {42134322},
issn = {1875-9777},
abstract = {Base editors enable precise correction of point mutations without requiring DNA double-strand breaks, yet platform- and cell type-specific genotoxicities remain incompletely characterized. Here, we applied cytosine base editing (CBE) to disrupt a cryptic splice-site mutation in the Unc13d locus of Jinx mice, a model of familial hemophagocytic lymphohistiocytosis type 3 (FHL3). Efficient editing (62%-89%) in fibroblasts, T cells, and hematopoietic stem cells (HSCs) restored Unc13d splicing, reconstituted cytotoxic T cell function, and protected mice from virus-triggered hyperinflammation after transplantation of edited HSCs. Comparative genotoxicity profiling revealed distinct platform- and cell type-specific patterns: hyperactive CBE induced broader off-target activity and more structural variants than CRISPR-Cas9. Although off-target sequence edits persisted, the stability of CBE-induced chromosomal translocations differed between cell types. These findings establish base editing as a therapeutic strategy for a genetically predisposed hyperinflammatory syndrome and underscore the importance of context-specific safety profiling to guide the clinical translation of genome editors.},
}

@article {pmid41237872,
year = {2026},
author = {Abdul-Rahman, T and Roy, P and Garg, N and Nazir, A and Mehta, KS and Doshi, NA and Hassnain, S and Reddi, R and Kanagala, SG and Ashinze, P and Lavie, CJ and Gupta, R},
title = {Gene editing for inherited cardiac conditions: A new frontier in cardiology.},
journal = {Trends in cardiovascular medicine},
volume = {36},
number = {4},
pages = {232-244},
doi = {10.1016/j.tcm.2025.11.002},
pmid = {41237872},
issn = {1873-2615},
mesh = {Humans ; *Gene Editing/ethics/methods ; *Genetic Therapy/adverse effects/methods ; Genetic Predisposition to Disease ; *Cardiology/methods/trends ; Animals ; *Heart Diseases/genetics/therapy/diagnosis ; Phenotype ; CRISPR-Cas Systems ; Treatment Outcome ; Epigenesis, Genetic ; Mutation ; Heredity ; },
abstract = {Inherited cardiac conditions (ICCs) such as hypertrophic cardiomyopathy and Marfan syndrome pose significant global health challenges, often rooted in complex genetic mutations. Recent advances in gene editing, particularly the CRISPR/Cas9 system, have opened new avenues for precise, personalized interventions. This review examines the current landscape of gene editing in cardiology, with emphasis on its scientific promise, integration with epigenetics, gene therapy, and artificial intelligence, and its potential to transform clinical outcomes. Key gene editing strategies are analyzed for their efficacy and translational potential. The review also explores ongoing clinical trials and emerging research, offering practical insights for future studies. Ethical implications are critically evaluated, with proposed frameworks to address concerns around safety, equity, and long-term consequences. By synthesizing these developments, this review underscores the urgent need for continued interdisciplinary research in the quest to mitigate inherited cardiac diseases.},
}

Humans
*Gene Editing/ethics/methods
*Genetic Therapy/adverse effects/methods
Genetic Predisposition to Disease
*Cardiology/methods/trends
Animals
*Heart Diseases/genetics/therapy/diagnosis
Phenotype
CRISPR-Cas Systems
Treatment Outcome
Epigenesis, Genetic
Mutation
Heredity

@article {pmid41722572,
year = {2026},
author = {Abadie, FMC and Suiter, CC and Smith, NT and Daza, RM and Rominger, MC and Parrish, P and McDiarmid, TA and Lalanne, JB and Martin, B and Calderon, D and Ellison, A and Berger, AH and Shendure, J and Starita, LM},
title = {A multiplex, prime editing framework for identifying drug resistance variants at scale.},
journal = {Cell genomics},
volume = {6},
number = {5},
pages = {101167},
doi = {10.1016/j.xgen.2026.101167},
pmid = {41722572},
issn = {2666-979X},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Point Mutation/genetics ; *Drug Resistance, Neoplasm/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Protein Kinase Inhibitors/pharmacology ; Cell Line, Tumor ; },
abstract = {CRISPR-based genome editing has revolutionized functional genomics, enabling thousands of perturbations to be concurrently assayed in single experiments. However, for methods such as saturation genome editing (SGE), which aims to generate and assay libraries of point mutations, a challenge is that only one region (e.g., one exon) is studied per experiment. Here, we describe prime-SGE, a prime editing-based framework in which libraries of specific point mutations are installed into genes throughout the genome and then functionally assessed by sequencing of prime editing guide RNAs (pegRNAs) rather than the mutations themselves. We apply prime-SGE in two cell lines to assay thousands of point mutations in eight oncogenes for their ability to confer drug resistance to four tyrosine kinase inhibitors. Our prime-SGE strategy, combined with ongoing improvements in prime editing efficiency, opens the door to efficient positive selection screens of large numbers of point mutations at locations throughout the genome.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Point Mutation/genetics
*Drug Resistance, Neoplasm/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Protein Kinase Inhibitors/pharmacology
Cell Line, Tumor

@article {pmid41872214,
year = {2026},
author = {Khadake, RM and Shinde, K and Rode, AB},
title = {Engineering ligands for theophylline riboswitches expands its regulatory dynamic range in prokaryotic and eukaryotic systems.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41872214},
issn = {2041-1723},
support = {BT/ PR45158/DRUG/134/119/2022//Department of Biotechnology, Ministry of Science and Technology (DBT)/ ; CRG/ 2022/004570//DST | Science and Engineering Research Board (SERB)/ ; },
mesh = {*Theophylline/metabolism/pharmacology/chemistry ; *Riboswitch/genetics ; Ligands ; Aptamers, Nucleotide/metabolism/genetics/chemistry ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Gene Expression Regulation ; Genetic Engineering/methods ; },
abstract = {The theophylline riboswitch has been a foundational tool in synthetic biology for three decades, yet its regulatory performance remains constrained by the modest affinity of its native ligand. Enhancing the dynamic range of riboswitches is critical for precise gene regulation in biotechnological applications. Here, we show that synthetic 4-quinazolinone derivatives, designed through a structure-based approach, are significantly better than theophylline in both binding and functional activation across multiple biological systems. We demonstrate that these derivatives bind the theophylline aptamer with up to 30-fold higher affinity, thereby expanding regulatory performance. In the bacterial system, these ligands enhance "ON" gene expression by up to 380-fold, compared to 75-fold with theophylline. This superior control extends to diverse organisms; in mycobacteria, the activation ratio reached 20-fold, and in eukaryotes, expression increased 11-fold. Furthermore, in riboswitch-mediated conditional CRISPR-Cas9 applications, these ligands achieve 70% genome editing efficiency at 10-fold lower concentrations than theophylline. These results demonstrate that ligand optimization is a crucial driver for enhancing riboswitch performance for advanced biomedical engineering.},
}

*Theophylline/metabolism/pharmacology/chemistry
*Riboswitch/genetics
Ligands
Aptamers, Nucleotide/metabolism/genetics/chemistry
Gene Editing/methods
CRISPR-Cas Systems/genetics
Gene Expression Regulation
Genetic Engineering/methods

@article {pmid41887225,
year = {2026},
author = {Drepanos, LM and Srikanth, S and Kaplan, EG and Shah, ST and Velasco, BE and Merzouk, S and Doench, JG},
title = {Balancing off-target and on-target considerations for optimized CRISPR-Cas9 knockout library design.},
journal = {Cell genomics},
volume = {6},
number = {5},
pages = {101190},
doi = {10.1016/j.xgen.2026.101190},
pmid = {41887225},
issn = {2666-979X},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Animals ; *Gene Library ; Mice ; *Gene Knockout Techniques/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genome, Human/genetics ; },
abstract = {The continued development of high-dimensional CRISPR screen readouts, such as single-cell RNA sequencing and high-content imaging, necessitates compact libraries to enable functional interrogation at genome scale. Improved genome annotations cause library deprecation over time, further motivating an updated genome-wide design effort. Additionally, while on-target efficacy and off-target avoidance are often optimized in isolation, we lack a robust framework for simultaneously weighing and balancing these competing priorities. Here, we present a selection strategy that identifies guides with sufficient off-target activity to justify omission from the library, thus avoiding the unnecessary exclusion of active guides, allowing the inclusion of those with maximal on-target activity. We create, validate, and make available to the community the Jacquere library for knockout screens of the human genome, as well as its mouse counterpart, Julianna, to facilitate gene function discovery at scale.},
}

*CRISPR-Cas Systems/genetics
Humans
Animals
*Gene Library
Mice
*Gene Knockout Techniques/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Editing/methods
Genome, Human/genetics

@article {pmid41985719,
year = {2026},
author = {Liang, G and Li, G and Liang, T and Zhen, Z and Teng, C and Xiong, J and Guangqiang, J and Zheng, M and Pan, Y and Zhong, S and Wu, C and Li, J and Huang, W and Wei, Z},
title = {Construction of an RAA-CRISPR detection platform for differentiation of Brucella abortus A19-∆VirB12 vaccine strain from wild-type strains.},
journal = {Journal of microbiological methods},
volume = {245},
number = {},
pages = {107512},
doi = {10.1016/j.mimet.2026.107512},
pmid = {41985719},
issn = {1872-8359},
mesh = {*Brucella abortus/genetics/isolation & purification/classification ; Animals ; Cattle ; Recombinases/metabolism/genetics ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Brucellosis, Bovine/diagnosis/microbiology ; *Brucella Vaccine/genetics ; *Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Brucella abortus is a primary etiological agent of bovine brucellosis, a zoonosis posing significant threats to livestock industries and public health. The recently developed A19-∆VirB12 vaccine strain, which carries a deletion of the VirB12 gene, complicates serological differentiation from wild-type infections. This study aimed to establish a rapid, accurate, and economical detection strategy targeting the VirB12 gene to distinguish the A19-∆VirB12 vaccine strain from wild-type B. abortus. We developed a recombinase-aided amplification (RAA) coupled with CRISPR/Cas12a-based detection method. Following optimization, primer pair C and crRNA1 were selected as optimal components, with 150 nM each of Cas12a protein and crRNA identified as the ideal concentrations in a 50 μL reaction. The assay demonstrated high analytical specificity, showing no cross-reactivity with six non-target bacterial pathogens. Sensitivity analysis established a limit of detection of 10[2] copies per reaction. When evaluated on 52 clinical samples, the RAA-CRISPR assay detected two positive samples, outperforming conventional PCR which detected only one. Crucially, the method yielded no positive signal when challenged with the A19-∆VirB12 gene-deficient vaccine strain, while successfully detecting wild-type strains A19 and S2, confirming its discriminatory capability. The entire workflow, comprising RAA amplification (30 min) and CRISPR-mediated cleavage (20 min), can be completed within one hour, with results visualized via fluorescence or lateral flow strips. This study successfully establishes a rapid, sensitive, and specific diagnostic method for distinguishing the A19-∆VirB12 vaccine strain from wild-type B. abortus, offering a practical tool for field surveillance and eradication programs.},
}

*Brucella abortus/genetics/isolation & purification/classification
Animals
Cattle
Recombinases/metabolism/genetics
Sensitivity and Specificity
*CRISPR-Cas Systems
*Brucellosis, Bovine/diagnosis/microbiology
*Brucella Vaccine/genetics
*Nucleic Acid Amplification Techniques/methods
Bacterial Proteins/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42003616,
year = {2026},
author = {Theriault, ME and Wong, AI and DeJesus, MA and Pisu, D and Nae Rin Lee, B and Kirukubar, G and Li, S and Wallach, JB and Schnappinger, D and Lê-Bury, G and Russell, DG and Rock, JM},
title = {Utilization of a CRISPRi-based ex vivo challenge model to reveal temporally dependent gene essentiality in intracellular Mycobacterium tuberculosis.},
journal = {mBio},
volume = {17},
number = {5},
pages = {e0061026},
doi = {10.1128/mbio.00610-26},
pmid = {42003616},
issn = {2150-7511},
support = {R01 AI155319/AI/NIAID NIH HHS/United States ; AI155319//National Institute of Allergy and Infectious Diseases/ ; AI162598//National Institute of Allergy and Infectious Diseases/ ; INV-055894//Bill and Melinda Gates Foundation/ ; //Mueller Health Foundation/ ; //Rita Allen Foundation/ ; },
mesh = {*Mycobacterium tuberculosis/genetics/growth & development ; Animals ; Mice ; Macrophages/microbiology ; *Genes, Essential ; *CRISPR-Cas Systems ; *Tuberculosis/microbiology ; Clustered Regularly Interspaced Short Palindromic Repeats ; Mice, Inbred C57BL ; Lung/microbiology ; Disease Models, Animal ; },
abstract = {UNLABELLED: Mycobacterium tuberculosis (Mtb) remains a leading cause of infectious disease mortality worldwide, largely due to its ability to survive within host macrophages. Despite advances in understanding the environmental pressures Mtb encounters in vivo, the genetic requirements for adaptation and survival within the intracellular niche remain incompletely defined. Here, we employed a genome-wide CRISPR interference (CRISPRi) screen in an ex vivo model exploiting single-cell suspensions from Mtb-infected mouse lung homogenates to identify genes critical for intracellular survival at different time points in the infection continuum. Using a library comprising ~20,000 sgRNAs covering >96% of Mtb open reading frames, we identified genes required for growth within the changing immune microenvironment. Mutant depletion patterns varied across immune environments sampled at 2, 4, and 6 weeks post-infection, which revealed a weighted dependency on cell wall biosynthesis genes early and the reliance on cholesterol catabolism and iron acquisition across all time points. Functional validation of three genes-embB, fadE29, and mbtI-confirmed their temporal significance in vivo. This screen provides increased resolution of the differential metabolic vulnerabilities in Mtb in the evolving immune environments during infection, stressing the temporal nature of conditional essentiality in vivo.

IMPORTANCE: Mycobacterium tuberculosis (Mtb) remains a leading cause of infectious disease mortality worldwide, largely due to its ability to survive within host macrophages. Despite advances in understanding the environmental pressures Mtb encounters in vivo, the genetic requirements for adaptation and survival within the intracellular niche remain incompletely defined. Here, we employed a genome-wide CRISPR interference (CRISPRi) screen in an ex vivo model exploiting single-cell suspensions from Mtb-infected mouse lung homogenates to identify genes critical for intracellular survival at different time points in the infection continuum. This novel approach enabled us to identify how different bacterial metabolic pathways were of greater importance to the bacterium at different time points post-infection. The results provide insights into how the evolving immune response to infection shapes the metabolic and replicative status of the bacterium. This information has significance in the design of therapeutic strategies toward cure.},
}

*Mycobacterium tuberculosis/genetics/growth & development
Animals
Mice
Macrophages/microbiology
*Genes, Essential
*CRISPR-Cas Systems
*Tuberculosis/microbiology
Clustered Regularly Interspaced Short Palindromic Repeats
Mice, Inbred C57BL
Lung/microbiology
Disease Models, Animal

@article {pmid42031246,
year = {2026},
author = {Han, T and Long, K and Hu, W and Liu, M and Guo, M and Huo, D and Hou, C},
title = {Dual-locked probes inhibit off-target circularization: Enhancing specificity in rolling circle transcription for nucleic acid detection.},
journal = {International journal of biological macromolecules},
volume = {364},
number = {},
pages = {152180},
doi = {10.1016/j.ijbiomac.2026.152180},
pmid = {42031246},
issn = {1879-0003},
mesh = {CRISPR-Cas Systems/genetics ; *Transcription, Genetic ; *Nucleic Acids/genetics/analysis ; Human papillomavirus 16/genetics ; Humans ; Limit of Detection ; Nucleic Acid Amplification Techniques/methods ; CRISPR-Associated Proteins/genetics ; },
abstract = {Rolling Circle Replication (RCR) holds significant potential for detecting low-abundance nucleic acids. However, its practical application is often hindered by nonspecific ligation, which leads to high background signals and false-positive results. Herein, we designed a novel dual-locked circular template, termed no-bilateral-overhang dual-locked probe (ndRC). This probe employed steric hindrance from its dual hairpins lacking overhangs to create a "dual-locked" mechanism that specifically prevents off-target circularization. By integrating this template with exonuclease purification and a CRISPR/Cas12a module, we constructed the dual-locked RCT-CRISPR/Cas12a (DL-RCT-Cas12a) system, in which RCT generates long RNA transcripts containing crRNA repeats. These transcripts then activate Cas12a-mediated trans-cleavage, thereby amplifying the detection signal. The established assay achieved an ultralow detection limit of 40.31 aM, with a detection range from 100 aM to 1 μM. It demonstrated robust performance in complex matrices for the detection of partial HPV16 L1 gene sequences and exhibited a superior ability to discriminate high-concentration targets compared to qPCR. This work presents a versatile strategy to enhance the specificity of highly sensitive detection, improving performance in low-abundance nucleic acid analysis.},
}

CRISPR-Cas Systems/genetics
*Transcription, Genetic
*Nucleic Acids/genetics/analysis
Human papillomavirus 16/genetics
Humans
Limit of Detection
Nucleic Acid Amplification Techniques/methods
CRISPR-Associated Proteins/genetics

@article {pmid42054542,
year = {2026},
author = {Gu, T and Xue, J and Zhang, Z and Cao, J and Song, J and Li, G and Ming, L and Zhu, Z and Wang, H},
title = {Mechanisms of Resistance to ALS Inhibitors and Bentazone in Fimbristylis littoralis and Rapid Identification of the ALS Trp-574-Leu Mutation Using LAMP-CRISPR/Cas12a.},
journal = {Journal of agricultural and food chemistry},
volume = {74},
number = {18},
pages = {14311-14321},
doi = {10.1021/acs.jafc.5c17149},
pmid = {42054542},
issn = {1520-5118},
mesh = {*Acetolactate Synthase/genetics/antagonists & inhibitors/metabolism ; *Herbicides/pharmacology ; *Plant Proteins/genetics/metabolism/antagonists & inhibitors/chemistry ; Herbicide Resistance ; Mutation ; CRISPR-Cas Systems ; *Enzyme Inhibitors/pharmacology ; },
abstract = {Fimbristylis littoralis Gaudich., a harmful sedge weed in Chinese rice paddy, impairs rice productivity and quality. In this study, we identified a resistant population (FL2) displaying multiple resistance to pyrazosulfuron-ethyl and bentazone, alongside cross-resistance to other acetolactate synthase (ALS)-inhibiting herbicides. The other population (FL6) showed exclusive resistance to bensulfuron-methyl. Sequencing demonstrated that FL2 carried a Trp-to-Leu mutation at codon 574 of ALS, whereas no mutations were detected in the psbA gene of bentazone-resistant FL2 or the ALS gene of bensulfuron-methyl-resistant FL6. Studies on nontarget-site resistance (NTSR) mechanisms indicated that FL2's resistance to pyrazosulfuron-ethyl was associated with neither PBO-inhibited P450s nor NBD-Cl-inhibited GSTs. In contrast, FL6's resistance to bensulfuron-methyl and FL2's resistance to bentazone were linked to P450 activity. A loop-mediated isothermal amplification (LAMP) coupled with CRISPR/FnCas12a assay was established for rapid detection of the Trp-574-Leu mutation, facilitating resistance management. These findings provide insights for managing resistant F. littoralis populations.},
}

*Acetolactate Synthase/genetics/antagonists & inhibitors/metabolism
*Herbicides/pharmacology
*Plant Proteins/genetics/metabolism/antagonists & inhibitors/chemistry
Herbicide Resistance
Mutation
CRISPR-Cas Systems
*Enzyme Inhibitors/pharmacology

@article {pmid42061542,
year = {2026},
author = {Yuan, Y and Ni, B and Tian, X and Cui, J and Zhang, Y and Hu, Y and Zheng, D and Zou, Y and Yu, X and Liu, C and Liu, S and Ren, W and Chang, X and Wang, Y and Ge, S and Wei, R and Chen, Y and Wu, X and Li, J and Wang, Z and Huang, B},
title = {Development of a point-of-care diagnostic method for FMDV SAT2 using RT-RAA-CRISPR technology.},
journal = {International journal of biological macromolecules},
volume = {364},
number = {},
pages = {152271},
doi = {10.1016/j.ijbiomac.2026.152271},
pmid = {42061542},
issn = {1879-0003},
mesh = {*Foot-and-Mouth Disease Virus/genetics/isolation & purification ; Animals ; *Foot-and-Mouth Disease/diagnosis/virology ; *CRISPR-Cas Systems ; Cattle ; Swine ; *Nucleic Acid Amplification Techniques/methods ; *Point-of-Care Systems ; Sensitivity and Specificity ; Recombinases/metabolism ; },
abstract = {In recent years, the foot-and-mouth disease virus (FMDV) serotype SAT2 has expanded beyond its traditionally recognized endemic areas on the African continent, leading to continuous dissemination in Middle Eastern countries and presenting a risk of further spread to Asia. In this study, the highly conserved sequence of the FMDV SAT2 genome was initially selected as the target, and five pairs of reverse transcription-recombinase-aided amplification (RT-RAA) primers were designed. By comparing fluorescence signal intensities, the CRISPR RNA (crRNA) with the highest sensitivity and specificity was identified and subsequently integrated with the CRISPR/Cas13a gene-editing system to establish a novel nucleic acid detection method. This method possesses a robust capacity for differential diagnosis and shows no cross-reaction with other serotypes of FMDV. Moreover, this method demonstrated high specificity and no cross-reactivity with the nucleic acid sequences of various common pathogens in porcine and bovine populations. The test results are readily interpretable and can be directly visualized using a fluorescence reader or lateral flow test strips (LFSs). In simulated clinical samples, this method achieved a concordance rate of 100% with the detection results of fluorescence quantitative RT-PCR. This study successfully developed a highly sensitive and specific FMDV SAT2 nucleic acid detection method based on RT-RAA-CRISPR/Cas13a technology. This method is straightforward to perform, does not require costly experimental equipment, and is suitable for rapid onsite detection, offering a convenient and efficient diagnostic tool for the early diagnosis, prevention, and control of FMDV SAT2 epidemics.},
}

*Foot-and-Mouth Disease Virus/genetics/isolation & purification
Animals
*Foot-and-Mouth Disease/diagnosis/virology
*CRISPR-Cas Systems
Cattle
Swine
*Nucleic Acid Amplification Techniques/methods
*Point-of-Care Systems
Sensitivity and Specificity
Recombinases/metabolism

@article {pmid42061934,
year = {2026},
author = {Gunasekaran, H and Najwa, KV and Nidarshan, NC and Porkodi, M and Singh, LS and Rasal, KD and Brahmane, MP and Goswami, M and Sonwane, AA},
title = {Characterization of Trachinotus blochii mstnb gene and construction of cognate gRNA vector.},
journal = {International journal of biological macromolecules},
volume = {364},
number = {},
pages = {152225},
doi = {10.1016/j.ijbiomac.2026.152225},
pmid = {42061934},
issn = {1879-0003},
mesh = {Animals ; *Myostatin/genetics/chemistry ; *RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; *Genetic Vectors/genetics ; *Fishes/genetics ; Amino Acid Sequence ; Base Sequence ; *Fish Proteins/genetics ; },
abstract = {Skeletal muscle development is negatively regulated by the myostatin b (mstnb) gene in finfishes. CRISPR-Cas9-mediated knockout of mstnb has been used to develop fish strains with increased muscle mass. Trachinotus blochii (Silver Pompano), a high-value, moderately sized (~500 g), and cultivable marine finfish, is a promising candidate for mstnb knockout using CRISPR/Cas9. However, limited studies exist on the characterization of T. blochii mstnb and no CRISPR-based knockout studies have been reported in this species. This study aimed to partially characterize the mstnb gene of T. blochii and develop a CRISPR/Cas9-based guide RNA (gRNA) expression vector for its knockout. The T. blochii mstnb sequence available in NCBI was used as a reference to design exon- and intron-specific primers for PCR amplification and sequencing. The sequence revealed three exons and two introns. A predicted 1131 bp open reading frame encodes a 376 amino acid protein containing conserved domains typical of the TGF-β family which includes an N-terminal signal peptide, a propeptide region, a conserved RARR cleavage motif, and a C-terminal GF domain containing nine conserved cysteine residues. Secondary and 3D structure predictions confirmed the protein's functional integrity. Sequence analysis revealed novel putative polymorphisms, including SNPs and a (CA)n microsatellite. Phylogenetic analysis clustered T. blochii with related Trachinotus and other Carangiformes species. A gRNA targeting exon 1 was designed using CRISPOR and successfully cloned into expression vectors. This novel sequence information can aid population-level studies and genetic marker discovery. The constructed gRNA vectors can facilitate CRISPR/Cas9-mediated mstnb knockout in T. blochii to study gene function and develop a fleshy strain.},
}

Animals
*Myostatin/genetics/chemistry
*RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Cas Systems
*Genetic Vectors/genetics
*Fishes/genetics
Amino Acid Sequence
Base Sequence
*Fish Proteins/genetics

@article {pmid42067160,
year = {2026},
author = {Dai, S and Niu, L and Lv, Y and Zhang, H and Xu, L and Liao, Y and Hu, X and Xie, X and Yan, J and Yan, Y},
title = {Semi-artificial photobiocatalysis via genetically modified sulfur metabolism to in situ assembly of a solar-biohybrid for antibiotic degradation.},
journal = {Bioresource technology},
volume = {454},
number = {},
pages = {134750},
doi = {10.1016/j.biortech.2026.134750},
pmid = {42067160},
issn = {1873-2976},
mesh = {*Sulfur/metabolism ; *Anti-Bacterial Agents/metabolism ; Sulfides/metabolism ; Biodegradation, Environmental ; Nanoparticles/chemistry ; Gene Editing ; CRISPR-Cas Systems/genetics ; Solar Energy ; Tetracycline/metabolism ; },
abstract = {The solar-driven semi-artificial biohybrid system incorporates semiconductor materials with microbial metabolism, affording an innovative strategy for antibiotic degradation via photocatalysis. In this study, the sulfur metabolic pathway of biological cells was rationally engineered using CRISPR-Cas9 and Cre-loxP site-specific gene editing systems, successfully achieving intracellular accumulation of sulfide up to 552.84 ppm. Based on this capability, In(Ⅲ) was adsorbed in situ onto the cell surface, leading to the self-assembly of photosensitive In2S3 nanoparticles (NPs). The resulting inorganic-biological hybrid system of In2S3-cell exhibited a broad-spectrum light-harvesting capability with an ideal optical bandgap of 1.96 eV. Photoelectrochemical analysis confirmed the charge transfer process and the semiconductor biointerface based regeneration mechanism of redox cofactors in the cytosol. Photogenerated electrons directly form ROS for tetracycline oxidation, and are transferred to cells for enhance the regeneration of intracellular reducing cofactors. This light-driven photocatalytic biohybrid system enabled efficient tetracycline degradation of over 98% within 4 h and demonstrated excellent stability over consecutive cycles. Transcriptomic analysis identified key genes involved in solar energy capture, electron transport, and metabolic regulation, elucidating their functional roles in biomanufacturing processes and photocatalytic degradation. This study presents a bottom-up paradigm for the biotic-abiotic system from electronic and molecular perspectives to develop efficient and sustainable technologies for antibiotic remediation and solar energy conversion.},
}

*Sulfur/metabolism
*Anti-Bacterial Agents/metabolism
Sulfides/metabolism
Biodegradation, Environmental
Nanoparticles/chemistry
Gene Editing
CRISPR-Cas Systems/genetics
Solar Energy
Tetracycline/metabolism

@article {pmid42121843,
year = {2026},
author = {Siddika, A and Husseiny, FE and Rousseau, J and Tremblay, JP},
title = {Successful In Vitro Modification of the Dmd Gene Using Prime Editing.},
journal = {Cells},
volume = {15},
number = {9},
pages = {},
doi = {10.3390/cells15090740},
pmid = {42121843},
issn = {2073-4409},
support = {53320215//Defeat Duchenne Foundation/ ; CIHR; Application No. 492510/CAPMC/CIHR/Canada ; },
mesh = {*Gene Editing/methods ; *Dystrophin/genetics ; Animals ; Mice ; *Muscular Dystrophy, Duchenne/genetics ; Cell Line ; RNA, Guide, CRISPR-Cas Systems/genetics ; Myoblasts/metabolism ; Base Sequence ; Mutation/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Duchenne muscular dystrophy (DMD) is a fatal X-linked neuromuscular disorder caused by mutations in the dystrophin gene. Prime editing is a versatile genome editing technology capable of introducing precise nucleotide changes without generating double-strand DNA breaks, making it a promising approach for correcting pathogenic point mutations. In this study, we applied prime editing to modify mdx-4cv and mdx-5cv mutation-equivalent sites in mouse C2C12 myoblasts in vitro. Initial editing efficiencies were unexpectedly low and were associated with the presence of a 5'-TTCT-3' motif within engineered prime editing guide RNAs (epegRNAs). epegRNA designs containing this motif exhibited reduced prime editing efficiency, whereas silent substitution eliminating the motif significantly improved editing outcomes, indicating that specific sequence features within epegRNAs can influence editing performance. Rational redesign of epegRNAs to remove this motif substantially enhanced editing efficiency, achieving up to 20% modification at the 4cv target site using an NGG PAM and 21% editing at the 5cv locus using an NGAG PAM. These findings highlight an important sequence-dependent constraint in epegRNA design and provide practical guidance for optimizing prime editing strategies targeting Dmd mutations in vitro.},
}

*Gene Editing/methods
*Dystrophin/genetics
Animals
Mice
*Muscular Dystrophy, Duchenne/genetics
Cell Line
RNA, Guide, CRISPR-Cas Systems/genetics
Myoblasts/metabolism
Base Sequence
Mutation/genetics
CRISPR-Cas Systems/genetics

@article {pmid42121869,
year = {2026},
author = {Siwak, JF and Connelly, JP and Pruett-Miller, SM},
title = {Essential HDRescue: A Co-Targeting Strategy to Enhance Precision Genome Editing by Co-Editing Essential Genes.},
journal = {Cells},
volume = {15},
number = {9},
pages = {},
doi = {10.3390/cells15090768},
pmid = {42121869},
issn = {2073-4409},
support = {N/A//American Lebanese Syrian Associated Charities/ ; NA//St. Jude Graduate School of Biomedical Sciences/ ; },
mesh = {*Gene Editing/methods ; Humans ; *Recombinational DNA Repair/genetics ; *Genes, Essential/genetics ; DNA End-Joining Repair/genetics ; CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; Induced Pluripotent Stem Cells/metabolism ; Animals ; },
abstract = {Genome editing is widely used and conceptually simple, yet in practice, it is hindered by laborious workflows and high costs. These challenges stem from the difficulty of identifying and isolating cells that contain the desired user-defined modifications, a problem compounded by the wide variability in editing efficiencies across cell types. While homology-directed repair (HDR) provides a mechanism for precise genome modification following nuclease-induced double-strand breaks (DSBs), it is frequently outcompeted by the dominant mutagenic non-homologous end-joining (NHEJ) pathway in mammalian cells. Therefore, we developed a novel enrichment method, Essential HDRescue, to increase the frequency of HDR events at a target site by co-targeting an essential genomic locus. Using both intrinsic positive and negative selection at a common essential gene, we enabled enrichment of precise editing events at a second, unlinked target site. We demonstrated that co-targeting essential genes in cancer cell lines and iPSCs increased HDR rates without the need for an exogenous reporter or selective drug. Analysis of resulting clones revealed that Essential HDRescue produced up to a 6-fold increase in single-allele edits and an ~4-fold increase in homozygous edits relative to single-targeted controls. By harnessing the intrinsic cellular dependencies that arise from DSB repair at essential loci, Essential HDRescue offers a widely applicable method to improve precise genome editing outcomes in mammalian cells, leaving only a minimal, protein-silent scar at the essential gene.},
}

*Gene Editing/methods
Humans
*Recombinational DNA Repair/genetics
*Genes, Essential/genetics
DNA End-Joining Repair/genetics
CRISPR-Cas Systems/genetics
DNA Breaks, Double-Stranded
Induced Pluripotent Stem Cells/metabolism
Animals

@article {pmid42122867,
year = {2026},
author = {Wen, Y and Li, Y and Bao, S and Cao, G and Li, M and Wang, J and Ding, B and Xie, X and Qiu, L},
title = {The Auxin Response Factor TaARF18-A Negatively Regulates Salt Tolerance in Common Wheat (Triticum aestivum L.).},
journal = {Plants (Basel, Switzerland)},
volume = {15},
number = {9},
pages = {},
doi = {10.3390/plants15091375},
pmid = {42122867},
issn = {2223-7747},
support = {32301817//National Natural Science Foundation of China/ ; 22JCQNJC01470//Natural Science Foundation of Tianjin/ ; 26CXNE010//Gansu Provincial Science and Technology Program Project/ ; KLIBMC2507//Open Fund from the Tianjin Key Laboratory of Intelligent Breeding of Major Crops/ ; },
abstract = {Soil salinization is one of the major abiotic stresses that influences agricultural production and the environment. Auxin response factors (ARFs) are key components of the auxin signal transduction pathway, while their role in wheat salt stress responses remains unclear. In this study, we identified TaARF18 as a negative regulator of salt tolerance in wheat. The coding sequences of TaARF18-A, TaARF18-B, and TaARF18-D were 2106, 2088, and 2088 bp, respectively. TaARF18 is a hydrophilic protein featuring typical Auxin-resp and B3 DNA-binding domains and exhibits relatively high evolutionary conservation among Poaceae species. The expression of TaARF18 was upregulated under salt stress. TaARF18 predominantly accumulated in the nucleus. Silencing of TaARF18 via the BSMV-VIGS approach enhanced salt tolerance in wheat seedlings. In addition, haplotype analysis based on resequencing data from 355 wheat accessions identified 25, 31, and 16 haplotypes for TaARF18-A, TaARF18-B, and TaARF18-D, respectively. Fourteen wheat accessions carrying different haplotypes were evaluated under salt stress, and HapIII of TaARF18-A exhibited the highest level of salt tolerance, which can act as a strong selection locus in global wheat breeding. Our findings provide insight into the function of ARFs in salt stress responses and offer a potential target for CRISPR/Cas-mediated salt-tolerant wheat breeding programs.},
}

@article {pmid42123509,
year = {2026},
author = {Li, Y and Yao, Y and Xu, Z and Xiong, Y and Zhang, C and Yu, L and Gao, H and Fei, T},
title = {Genome-Wide CRISPR Screening Identifies Genetic Modulators of Amyloid Precursor Protein Processing.},
journal = {International journal of molecular sciences},
volume = {27},
number = {9},
pages = {},
doi = {10.3390/ijms27093926},
pmid = {42123509},
issn = {1422-0067},
mesh = {Humans ; *Amyloid beta-Protein Precursor/metabolism/genetics ; *CRISPR-Cas Systems ; *Alzheimer Disease/genetics/metabolism ; Amyloid beta-Peptides/metabolism/genetics ; HEK293 Cells ; },
abstract = {The proteolytic processing of the amyloid precursor protein (APP) is a core pathological event in Alzheimer's disease (AD) pathogenesis, yet the global genetic regulatory networks modulating this process have not been fully characterized. To systematically identify novel regulators of APP cleavage, we performed a genome-wide CRISPR/Cas9 knockout screen utilizing an optimized UAS-GAL4-based cellular reporter, and identified genetic modulators governing amyloidogenic and non-amyloidogenic processing. The screen uncovered distinct functional gene clusters regulating the APP, prominently involving cellular metabolism, protein modification, and vesicular trafficking. Specifically, LDHB, PIAS2, CCDC53, and TRIM61 emerged as novel functional modulators. Biochemical validation confirmed that ablating these genes significantly alters the metabolic balance between sAPPα and amyloid-β (Aβ) production. Finally, integration with human AD transcriptomic datasets demonstrated that these identified modulators undergo significant dysregulation in clinics. Together, these findings establish a reporter-based functional screening framework for APP processing and identify candidate regulatory nodes linked to metabolism, protein modification, and vesicular trafficking. These candidates provide a resource for future mechanistic investigation and validation in more disease-relevant AD models.},
}

Humans
*Amyloid beta-Protein Precursor/metabolism/genetics
*CRISPR-Cas Systems
*Alzheimer Disease/genetics/metabolism
Amyloid beta-Peptides/metabolism/genetics
HEK293 Cells

@article {pmid42123512,
year = {2026},
author = {Yuan, Y and Yuan, J and Deng, D and Wu, J and Zhou, X and Jiang, A and Wang, J and Wang, X and Li, M and Long, K and Zhao, L},
title = {CRISPR/Cas9-Mediated Knockout of CGNL1 Confers Resistance to Aflatoxin B1 in Porcine Intestinal Epithelial Cells via Suppressing ROS Generation.},
journal = {International journal of molecular sciences},
volume = {27},
number = {9},
pages = {},
doi = {10.3390/ijms27093928},
pmid = {42123512},
issn = {1422-0067},
support = {XZ202501ZY0147//Tibet Autonomous Region Science and Technology Agency/ ; sccxtd-2025-08-13//the Program for Pig Industry Technology System Innovation Team of Sichuan Province/ ; 32472884//National Natural Science Foundation of China/ ; 32573165//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Aflatoxin B1/toxicity ; *CRISPR-Cas Systems ; Swine ; *Epithelial Cells/metabolism/drug effects ; *Reactive Oxygen Species/metabolism ; Oxidative Stress/drug effects ; *Intestinal Mucosa/metabolism/drug effects ; Gene Knockout Techniques ; Cell Line ; Cell Survival/drug effects ; Signal Transduction ; },
abstract = {Aflatoxin B1 (AFB1) is a prevalent and highly toxic mycotoxin in the food and feed chain and can directly injure the intestinal epithelium. Yet, its upstream determinants linking epithelial stress to cytotoxicity remain insufficiently defined. Here, we used porcine intestinal epithelial IPEC-J2 cells to characterize AFB1-induced cytotoxic and transcriptomic responses and to determine the role of the tight-junction scaffold, Cingulin-like 1 (CGNL1), a candidate gene identified through genome-scale CRISPR knockout library screening. The results showed that AFB1 exposure reduced cell viability in a dose-dependent manner and induced oxidative stress. RNA-seq profiling analysis revealed broad transcriptional remodeling, with activation of inflammatory pathways (including NF-κB and JAK-STAT signaling). Based on our constructed CGNL1-knockout IPEC-J2 cell line (CGNL1-KO IPEC-J2) using CRISPR/Cas9, it was found that CGNL1 deficiency markedly alleviated AFB1-induced cytotoxicity and oxidative stress. Comparative transcriptomics analysis showed that CGNL1 knockout attenuated AFB1-triggered aberrant expression of some CGNL1-dependent AFB1-responsive genes related to immune response under AFB1 challenge. Together, these findings identify CGNL1 as a potential modulator of epithelial susceptibility to AFB1 and support its involvement in the regulation of toxin-induced oxidative response.},
}

Animals
*Aflatoxin B1/toxicity
*CRISPR-Cas Systems
Swine
*Epithelial Cells/metabolism/drug effects
*Reactive Oxygen Species/metabolism
Oxidative Stress/drug effects
*Intestinal Mucosa/metabolism/drug effects
Gene Knockout Techniques
Cell Line
Cell Survival/drug effects
Signal Transduction

@article {pmid42123673,
year = {2026},
author = {Jing, L and Roy, D and Kalischuk, M},
title = {Advances in CRISPR Plant Applications.},
journal = {International journal of molecular sciences},
volume = {27},
number = {9},
pages = {},
doi = {10.3390/ijms27094095},
pmid = {42123673},
issn = {1422-0067},
support = {Alliance #44842//Natural Sciences and Engineering Research Council of Canada/ ; 45402//Solanum International Inc./ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Crops, Agricultural/genetics ; Plants, Genetically Modified/genetics ; *Plants/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The ability to precisely edit genetic characteristics with a CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) immunity complex is a revolutionary advance in science. Originally discovered in bacteria as part of a natural defense mechanism against viruses, CRISPR/Cas provides a precise, efficient, and relatively simple method for editing genes in microbes, plants, animals, and humans. The process relies on the Cas protein, an enzyme that cleaves and unwinds DNA at targeted locations. This process is guided by RNA sequences complementary to the DNA or RNA sequence of interest, allowing for changes to the genome through innate non-homologous end joining (NHEJ) and homology-directed repair (HDR). The potential applications of CRISPR/Cas are immense and, in agriculture, is facilitating crop development with resistance to abiotic, biotic, and agronomic characteristics that improve yield, quality, and food security. Gene editing also facilitates the relatively rapid modification of regulatory and complex pathways that enable studies to advance our understanding of gene function. This review provides an update of the fast-evolving CRISPR/Cas modification of important crops to address emerging global population, as well as environmental and climate challenges.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
*Crops, Agricultural/genetics
Plants, Genetically Modified/genetics
*Plants/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42124669,
year = {2026},
author = {Sanderson, EM and Peralta, J and Nouwens, S and Oriolt, L and Hayes, VM and Kaiser, BK and Meeske, AJ},
title = {Phage-encoded CasPRs transcriptionally silence diverse CRISPR-Cas systems.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.64898/2026.02.23.707548},
pmid = {42124669},
issn = {2692-8205},
abstract = {Anti-CRISPRs (Acrs) are diverse proteins or RNAs that protect invading phages and plasmids from host CRISPR-Cas immunity. Most Acrs neutralize their cognate Cas proteins via direct physical interaction. Here we describe CasPRs, a particularly widespread family of DNA-binding Acrs that recognize specific sequence motifs within cas gene coding regions, thereby blocking RNA polymerase and silencing transcription. We demonstrate that eight diverse CasPRs bind to the cas8b gene to repress the type I-B CRISPR-Cas system in its native host, Listeria seeligeri . Meanwhile, a CasPR from Streptococcus dysgalactiae silences type II-A CRISPR-Cas immunity by binding to the cas9 coding sequence. We found that one CasPR is required to inhibit CRISPR immunity during lysogeny by its host prophage. Taken together, our results indicate that members of the CasPR family have diverged to silence completely unrelated CRISPR types, and suggest transcriptional repression is a common mode of phage-mediated immune antagonism.},
}

@article {pmid42126429,
year = {2026},
author = {Huang, RS and Phung, SK and Sumstad, D and Weis, AJ and Kile, QM and Bendzick, L and Khaw, MJ and Vue, YY and McKenna, DH and Kennedy, PR and Miller, JS and Felices, M},
title = {Reprogramming endogenous NK circuits by highly efficient nonviral genome editing.},
journal = {The Journal of experimental medicine},
volume = {223},
number = {7},
pages = {},
doi = {10.1084/jem.20260192},
pmid = {42126429},
issn = {1540-9538},
support = {R35 CA283892/NH/NIH HHS/United States ; P01 CA111412/NH/NIH HHS/United States ; P01 CA065493/NH/NIH HHS/United States ; P30 CA 77598/NH/NIH HHS/United States ; },
mesh = {*Killer Cells, Natural/immunology/metabolism ; *Gene Editing/methods ; Humans ; Animals ; CRISPR-Cas Systems ; Gene Knock-In Techniques ; Mice ; Gene Regulatory Networks ; Interleukin-12/genetics/metabolism ; Receptors, Chimeric Antigen/genetics/immunology ; },
abstract = {Natural killer (NK) cells are promising platforms for off-the-shelf immunotherapy, yet nonviral precision engineering remains limited by poor HDR efficiency, DNA toxicity, and manufacturing challenges. The aim of this study was to establish a high-yield, nonviral knock-in platform. Through extensive in-depth rational screens, we achieved ∼90% HDR insertion of therapeutic payloads while maintaining 100% postediting recovery. By hijacking endogenous transcriptional programs, we installed genetic circuits into defined genomic loci to tune transgene expression. To enable context-dependent therapeutic responses, we integrated a synthetic positive feedback circuit at the CISH locus, which enhanced NK cell persistence and drove strong expression of anti-CD22/19 dual CAR. A hypoxia-responsive IL-12 circuit gated by the PFKFB4 promoter restored cytotoxicity under environmental stress. Finally, we showed this platform is compatible with GMP manufacturing and supports clinical-scale expansion. These findings provide a scalable framework for programmable, nonviral editing of NK cell effector functions for therapeutic and research applications.},
}

*Killer Cells, Natural/immunology/metabolism
*Gene Editing/methods
Humans
Animals
CRISPR-Cas Systems
Gene Knock-In Techniques
Mice
Gene Regulatory Networks
Interleukin-12/genetics/metabolism
Receptors, Chimeric Antigen/genetics/immunology

@article {pmid42127676,
year = {2026},
author = {Cheng, M and Chen, X and Cheng, H and Gao, X and Ao, H and Bao, X and Song, X and Tai, Y and Jin, D and Zhang, L},
title = {An ultrasensitive CRISPR/Cas12a based electrochemical biosensor for detection of toxigenic Clostridioides difficile.},
journal = {Biosensors & bioelectronics},
volume = {308},
number = {},
pages = {118779},
doi = {10.1016/j.bios.2026.118779},
pmid = {42127676},
issn = {1873-4235},
abstract = {Clostridioides difficile (C. difficile) infection (CDI) represents a formidable global healthcare challenge, necessitating the development of rapid, accurate, and cost-effective diagnostic platforms to mitigate nosocomial transmission and improve patient outcomes. Compared with the conventional methods, CRISPR/Cas systems featured by specific target reorganization by a single chain RNA, coupled with electrochemical technology enables highly sensitive detection of various biomarkers. However, their application to CDI has remained unexplored due to the lack of tailored crRNAs. Herein, we present the integration of CRISPR/Cas12a with electrochemical transduction for the direct detection of C. difficile. A novel crRNA was engineered to specifically recognize the toxin B gene (tcdB), activating the trans-cleavage activity of Cas12a upon target binding. This cascade triggers the cleavage of immobilized ssDNA reporters on the electrode surface, generating measurable amperometric signal changes. The developed biosensor demonstrates exceptional performance, achieving a detection limit of pM level for tcdB DNA within 40 min, while exhibiting high specificity against non-target pathogens and robust stability over 7 days. This work establishes a rapid and reliable CRISPR-electrochemical diagnostic platform, offering significant potential for point-of-care CDI management.},
}

@article {pmid42128325,
year = {2026},
author = {Cai, Y and Yang, J and Hou, M and Su, W and Liang, F and Zhu, M and Wu, T},
title = {Multi-omics precision diagnosis of brucellosis: Advances in biomarker discovery and clinical application.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {},
number = {},
pages = {121074},
doi = {10.1016/j.cca.2026.121074},
pmid = {42128325},
issn = {1873-3492},
abstract = {Brucellosis, a neglected zoonosis caused by intracellular Brucella bacteria, remains a formidable global public health challenge, especially in developing regions. The notorious ability of Brucella to evade host immunity and establish chronic focal infections limits the utility of traditional diagnostic methods like bacterial culture and serology for early detection, therapeutic monitoring, and disease staging. This review comprehensively synthesizes the ongoing paradigm shift from pathogen-centric detection toward multi-omics precision diagnosis. We critically evaluate advances in nucleic acid amplification technologies (NAATs), charting the progression from quantitative PCR to absolute quantification via droplet digital PCR (ddPCR) and examining the transformative potential of CRISPR-Cas biosensing for ultrasensitive, instrument-free detection. The discussion also encompasses the renaissance of serology through immunoproteomics, which has identified novel serodominant antigens and multi-epitope fusion proteins to address the persistent specificity problems arising from cross-reacting bacteria. Furthermore, we analyze the emerging landscape of host-response biomarkers, integrating transcriptomic, metabolomic, and single-cell RNA sequencing data to delineate distinct immune signatures of acute and chronic infection. Finally, we consider how artificial intelligence (AI) can integrate these multi-dimensional datasets to build predictive diagnostic models. This consolidated multi-omics framework charts a course for precision medicine in brucellosis, aiming to bridge the gap between biomarker discovery and point-of-care clinical application. SUMMARY: Multi-omics technologies (genomics, proteomics, metabolomics, transcriptomics) are advancing brucellosis diagnosis via sensitive detection and accurate biomarkers, and improving treatment through novel strategies like nano-delivery, vaccines, and AI integration.},
}

@article {pmid42128554,
year = {2026},
author = {Xu, W and Cheng, Y and Sun, K and Wu, Y and Xu, Y and Ye, J and Li, P and Wu, H},
title = {Inception-level signal amplification: Cascaded DNAzyme-Cas9 nickase achieves sub-nanomolar kanamycin tracking.},
journal = {Analytica chimica acta},
volume = {1408},
number = {},
pages = {345562},
doi = {10.1016/j.aca.2026.345562},
pmid = {42128554},
issn = {1873-4324},
mesh = {*Kanamycin/analysis/metabolism ; *DNA, Catalytic/metabolism/chemistry ; *Biosensing Techniques/methods ; Milk/chemistry ; *Nucleic Acid Amplification Techniques ; *Deoxyribonuclease I/metabolism/chemistry ; Limit of Detection ; Animals ; *Anti-Bacterial Agents/analysis ; CRISPR-Cas Systems ; Water Pollutants, Chemical/analysis ; },
abstract = {BACKGROUND: Kanamycin's persistent contamination in agricultural products poses significant human health risks due to its nephrotoxicity and bioaccumulation via the food chain. Existing methods for on-site kanamycin monitoring lack sufficient sensitivity and portability, limiting their utility in field settings. The urgent need for rapid detection technologies remains unmet. This work addresses the critical gap in developing a field-deployable platform for ultrasensitive kanamycin residue screening.

RESULTS: We engineered a fluorescence biosensor integrating DNAzyme-assisted Cas9 nickase-based amplification reaction (Cas9nAR). Kanamycin binding induces aptamer conformational changes, triggering Cas9nAR-driven cascade amplification that continuously generates DNAzymes. These cleave reporter probes to enable quantitative detection. The system achieved a broad linear range (1 nM - 5 μM) with an ultralow detection limit (0.3 nM), surpassing conventional methods. It exhibited high specificity against interfering antibiotics and delivered consistent recoveries (97% to 103%) in spiked water and milk samples. Following pretreatment, analysis can be completed within 120 min, validating its operational simplicity and robustness for complex matrices.

SIGNIFICANCE AND NOVELTY: This work reports the first CRISPR-Cas9 nickase/DNAzyme cascade amplification platform for small-molecule detection, establishing a new paradigm that integrates programmable nucleic acid amplification with catalytic signal turnover. By using Cas9nAR-generated ssDNA as an in situ template for autonomous DNAzyme assembly, the biosensor achieves ultrasensitive, and homogeneous detection of kanamycin-addressing a critical gap in field-deployable antibiotic residue monitoring. The modular design offers a generalizable strategy for translating non-nucleic acid recognition events into amplified fluorescent outputs, with broad implications for point-of-need diagnostics in food safety and environmental analysis.},
}

*Kanamycin/analysis/metabolism
*DNA, Catalytic/metabolism/chemistry
*Biosensing Techniques/methods
Milk/chemistry
*Nucleic Acid Amplification Techniques
*Deoxyribonuclease I/metabolism/chemistry
Limit of Detection
Animals
*Anti-Bacterial Agents/analysis
CRISPR-Cas Systems
Water Pollutants, Chemical/analysis

@article {pmid42128971,
year = {2026},
author = {Xie, R and Zhu, C and Liang, X and Lu, K and Wu, J},
title = {Knockout of bsal/cel.2 results in growth retardation, reduced lipid digestion and altered energy metabolism in medaka larvae (oryzias latipes).},
journal = {Functional & integrative genomics},
volume = {26},
number = {1},
pages = {},
pmid = {42128971},
issn = {1438-7948},
support = {2024YFD2401502//the National Key Research and Development Program of China/ ; },
mesh = {Animals ; *Oryzias/genetics/growth & development/metabolism ; *Energy Metabolism/genetics ; *Lipid Metabolism/genetics ; *Fish Proteins/genetics/metabolism ; Larva/growth & development/genetics/metabolism ; *Lipase/genetics/metabolism ; CRISPR-Cas Systems ; Lipolysis/genetics ; },
abstract = {To evaluate the significance of bile salt-activated lipase (BSAL) in lipid digestion and metabolism in fish, this study used CRISPR/Cas9 gene editing to generate medaka (Oryzias latipes) bsal mutant lines. Given that the bsal gene comprises three copies (bsal, cel.2, and bsal-like) in the medaka genome, bsal-like variants may lead to functional loss in lipid hydrolysis owing to amino acid residue alterations in the bile salt binding site. Consequently, two types of medaka mutants, bsal[-/-] and bsal[-/-]/cel.2[-/-], were generated for experimental exploration in this study. Compared to wild-type (WT) medaka, the bsal[-]/[-]/cel.2[-]/[-] group showed significant reductions in body length, expression of growth-related genes (gh and igf), total lipase and protease activities, and body composition (cholesterol, triglyceride, and protein levels). The bsal[-]/[-]/cel.2[-]/[-] group also exhibited upregulated expression of lipid synthesis genes (fas, acc1, scd) and downregulated expression of lipolysis-related genes (cpt1, acox1). Notably, key glycolysis genes (pk, gk) and gluconeogenesis-related genes (pck2) were significantly upregulated in the bsal[-]/[-]/cel.2[-]/[-] group. However, the bsal[-/-] group exhibited no significant differences from the WT group in all assays, except for notable reductions in protease activity and expression levels of the cpt1 and gk genes, as well as a significant increase in pck2 gene expression compared to the WT group. Remarkably, the expression level of the cel.2 gene was significantly elevated in the bsal[-/-] group compared to the WT group. In summary, this study demonstrates the pivotal role of the bsal gene in lipid digestion and metabolism in medaka. Furthermore, the presence of multiple copies of the bsal gene aids in fulfilling the demands of lipid digestion in medaka. This conclusion can provide insights into the research on lipid digestion and metabolism in other fish species.},
}

Animals
*Oryzias/genetics/growth & development/metabolism
*Energy Metabolism/genetics
*Lipid Metabolism/genetics
*Fish Proteins/genetics/metabolism
Larva/growth & development/genetics/metabolism
*Lipase/genetics/metabolism
CRISPR-Cas Systems
Lipolysis/genetics

@article {pmid42128985,
year = {2026},
author = {Hou, M and Li, Y and Wu, X and Long, D and Sun, D and Chen, P and Huang, H},
title = {Recent Advances in the Development of CRISPR-Based Live-Cell Molecular Imaging and Sensing.},
journal = {Molecular imaging and biology},
volume = {},
number = {},
pages = {},
pmid = {42128985},
issn = {1860-2002},
support = {2026JJ90110//Natural Science Foundation of Hunan Province Joint Fund for Universities/ ; S202512034126//Hunan Province Undergraduate Innovation Training Program/ ; },
abstract = {Visualizing genome organization and transcriptional dynamics with spatial and temporal precision in living cells is essential for elucidating gene regulation and chromatin-associated disease mechanisms, yet conventional methods confront a fundamental tension between endogenous-sequence targeting and live-cell compatibility. Operator-repressor systems require prior insertion of repetitive arrays at engineered loci, whereas fluorescence in situ hybridization mandates cell fixation and thereby precludes temporal analysis. CRISPR-Cas technologies, originally developed for genome editing, have been re-engineered into a versatile molecular-imaging toolkit capable of interrogating native sequences in living cells. Here, we systematically review CRISPR-based live-cell imaging and sensing platforms, critically evaluating their design principles, mechanistic foundations, and performance limitations. We examine dCas9-based DNA labeling, dCas12a systems for non-repetitive loci, Cas13- and Csm-mediated RNA imaging, novel fluorescent reporters, engineered ribonucleoproteins, and delivery innovations including reagent-based Oligo-LiveFISH. To organize this diverse literature, we distinguish three operationally distinct modalities-live-cell imaging, intracellular sensing, and diagnostic biosensing-and assess each platform through three unifying design trade-offs: sensitivity versus cellular perturbation, multiplexing capacity versus system complexity, and detection threshold versus biological fidelity. Building on this framework, we evaluate the integration of CRISPR imaging with super-resolution microscopy, artificial-intelligence-driven computational analysis, and multimodal spatial omics. Collectively, this synthesis clarifies current capabilities, delineates unresolved constraints, and charts a coherent path toward clinically relevant applications of CRISPR-based live-cell molecular imaging.},
}

@article {pmid42129030,
year = {2026},
author = {Watanabe, K and Ishikawa, M and Ishibashi, K},
title = {Development of Tobamovirus-Resistant Tomato Plants by CRISPR-Cas9-Mediated Knockout of Susceptibility Genes.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3039},
number = {},
pages = {1-10},
pmid = {42129030},
issn = {1940-6029},
mesh = {*Solanum lycopersicum/genetics/virology ; *CRISPR-Cas Systems/genetics ; *Disease Resistance/genetics ; *Plant Diseases/virology/genetics ; *Tobamovirus/pathogenicity/physiology ; Gene Editing/methods ; *Gene Knockout Techniques/methods ; Host-Pathogen Interactions/genetics ; Plants, Genetically Modified/genetics/virology ; },
abstract = {The advent of genome editing technologies such as CRISPR-Cas9 has revolutionized the development of disease-resistant crops, offering precision and efficiency in targeting specific genetic loci responsible for susceptibility. In this protocol, we harness the CRISPR-Cas9 system to disrupt key susceptibility genes in tomato, aiming to fortify resistance against tobamoviruses, particularly the aggressive tomato brown rugose fruit virus (ToBRFV). By systematically knocking out four TOM1 homologs, genes essential for tobamoviral replication, tomato lines with robust and heritable resistance can be developed while minimizing adverse developmental effects. The approach not only underscores the significance of basic research on host-pathogen interactions in modern crop protection but also lays the groundwork for sustainable, gene-driven resistance strategies in commercial tomato breeding.},
}

*Solanum lycopersicum/genetics/virology
*CRISPR-Cas Systems/genetics
*Disease Resistance/genetics
*Plant Diseases/virology/genetics
*Tobamovirus/pathogenicity/physiology
Gene Editing/methods
*Gene Knockout Techniques/methods
Host-Pathogen Interactions/genetics
Plants, Genetically Modified/genetics/virology

@article {pmid42129032,
year = {2026},
author = {Yoshida, T and Ishibashi, K},
title = {Heritable Tissue-Culture-Free Gene Editing in Nicotiana benthamiana Using a Meristem-Invading Virus Vector.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3039},
number = {},
pages = {21-27},
pmid = {42129032},
issn = {1940-6029},
mesh = {*Nicotiana/genetics/virology ; *Gene Editing/methods ; *Genetic Vectors/genetics ; *Meristem/genetics/virology ; CRISPR-Cas Systems ; *Potyvirus/genetics ; Plants, Genetically Modified/genetics ; },
abstract = {Gene editing can be achieved using sequence-specific nucleases. This protocol describes a plant gene editing method that eliminates the need for tissue culture by employing a virus-based delivery system. Tobacco ringspot virus (TRSV) can access meristematic tissues in infected plants, enabling the introduction of site-directed mutations into germline cells. This allows for heritable gene modification in the model plant Nicotiana benthamiana without tissue culture.},
}

*Nicotiana/genetics/virology
*Gene Editing/methods
*Genetic Vectors/genetics
*Meristem/genetics/virology
CRISPR-Cas Systems
*Potyvirus/genetics
Plants, Genetically Modified/genetics

@article {pmid42129103,
year = {2026},
author = {Ash, S and Attianese, GMPG and Kosti, P and Semilietof, A and Stefanidis, E and Triboulet, M and Irving, M},
title = {Generation and Characterization of CAR-T Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2998},
number = {},
pages = {439-504},
pmid = {42129103},
issn = {1940-6029},
mesh = {Humans ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; Animals ; Mice ; *Immunotherapy, Adoptive/methods ; *T-Lymphocytes/immunology/metabolism ; Cell Line, Tumor ; *Receptors, Antigen, T-Cell/genetics/immunology/metabolism ; Neoplasms/therapy/immunology ; Lymphocyte Activation ; Lentivirus/genetics ; Retroviridae/genetics ; CRISPR-Cas Systems ; },
abstract = {CARs are synthetic receptors that link antigen binding to T-cell activation. Most CARs used in the clinic for treating cancer are second generation (2G) and comprise (i) a single chain variable fragment (scFv) that binds the target tumor antigen, (ii) a linker/hinge region, (iii) a transmembrane domain, (iv) a costimulatory endodomain, and (v) the endodomain of CD3 zeta. Our lab is focused on the development of function and safety-enhanced, next-generation CAR-T cells for the treatment of solid tumors. For example, we have designed switchable CARs that can be remotely turned on or off upon small molecule administration in order to mitigate toxicity or exhaustion. To address barriers to CAR-T cells in the solid tumor microenvironment, we are further developing rational coengineering strategies to support their function. While we have implemented non-viral tools like CRISPR/Cas9 knockout and knockin, adenine base editing, and transposon-based systems for T cell engineering in the lab, currently we mostly use lentivirus and retrovirus for our pre-clinical studies. Here, we present our most frequently used protocols, improved over many years in the lab, for the production and titration of lentivirus and retrovirus, as well as the purification, activation, transduction and expansion of both mouse and human CAR-T cells. In addition, we share protocols for our most commonly run in vitro assays for characterizing CAR-T cells, including for evaluating transduction efficiency, proliferation, phenotype, cytokine/chemokine production, cytotoxicity, and resistance to stress. Most of these protocols can also be applied to the production and characterization of T cell receptor (TCR)-engineered T cells. Finally, we explain how to set up and perform CAR-T cell transfer studies in subcutaneous tumor-bearing mice, both for syngeneic and xenograft models, and perform ex vivo analysis on tumor tissues post-treatment.},
}

Humans
*Receptors, Chimeric Antigen/genetics/immunology/metabolism
Animals
Mice
*Immunotherapy, Adoptive/methods
*T-Lymphocytes/immunology/metabolism
Cell Line, Tumor
*Receptors, Antigen, T-Cell/genetics/immunology/metabolism
Neoplasms/therapy/immunology
Lymphocyte Activation
Lentivirus/genetics
Retroviridae/genetics
CRISPR-Cas Systems

@article {pmid42129496,
year = {2026},
author = {Duchêne, C and Craig, RJ and Martinho, C and Luthringer, R and Agullo, F and Hipp, K and Escudeiro, P and Alva, V and Haas, FB and Coelho, SM},
title = {Latent endogenous giant viruses drive active infection and inheritance in a multicellular algal host.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {42129496},
issn = {2058-5276},
support = {101109906//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 Marie Skłodowska-Curie Actions (H2020 Excellent Science - Marie Skłodowska-Curie Actions)/ ; },
abstract = {Endogenous viral elements inserted in host genomes are often regarded as inert relics of past infections. Whether they can retain infective potential and contribute to active viral cycles has remained largely unresolved. Here we demonstrate that giant viral elements in the multicellular alga Ectocarpus can reactivate and drive productive viral infections. Using long-read sequencing and transcriptomics, we identify full-length, transcriptionally active phaeoviruses integrated within the host genome, and we use classical genetics and CRISPR-Cas to demonstrate that these elements are stably inherited through the germline, while their reactivation is precisely regulated by developmental and environmental cues including temperature. We resolve the genomic integration sites and propose a mechanism for phaeovirus integration and replication. Our work provides direct evidence and uncovers the mechanisms by which giant viral elements can reactivate, replicate and transmit both horizontally and vertically in a multicellular eukaryote, establishing a new model of latency, inheritance and evolutionary impact of giant dsDNA viruses.},
}

@article {pmid35851300,
year = {2022},
author = {Droogers, WJ and Willems, J and MacGillavry, HD and de Jong, APH},
title = {Duplex Labeling and Manipulation of Neuronal Proteins Using Sequential CRISPR/Cas9 Gene Editing.},
journal = {eNeuro},
volume = {9},
number = {4},
pages = {},
pmid = {35851300},
issn = {2373-2822},
mesh = {Animals ; *CRISPR-Cas Systems ; *Neurons/metabolism ; *Gene Editing/methods ; Rats ; *Gene Knock-In Techniques/methods ; *Nerve Tissue Proteins/metabolism/genetics ; Synapses/metabolism ; Cells, Cultured ; },
abstract = {CRISPR/Cas9-mediated knock-in methods enable the labeling of individual endogenous proteins to faithfully determine their spatiotemporal distribution in cells. However, reliable multiplexing of knock-in events in neurons remains challenging because of cross talk between editing events. To overcome this, we developed conditional activation of knock-in expression (CAKE), allowing efficient, flexible, and accurate multiplex genome editing. To diminish cross talk, CAKE is based on sequential, recombinase-driven guide RNA (gRNA) expression to control the timing of genomic integration of each donor sequence. We show that CAKE is broadly applicable in rat neurons to co-label various endogenous proteins, including cytoskeletal proteins, synaptic scaffolds, ion channels and neurotransmitter receptor subunits. To take full advantage of CAKE, we resolved the nanoscale co-distribution of endogenous synaptic proteins using super-resolution microscopy, demonstrating that their co-organization correlates with synapse size. Finally, we introduced inducible dimerization modules, providing acute control over synaptic receptor dynamics in living neurons. These experiments highlight the potential of CAKE to reveal new biological insight. Altogether, CAKE is a versatile method for multiplex protein labeling that enables the detection, localization, and manipulation of endogenous proteins in neurons.},
}

Animals
*CRISPR-Cas Systems
*Neurons/metabolism
*Gene Editing/methods
Rats
*Gene Knock-In Techniques/methods
*Nerve Tissue Proteins/metabolism/genetics
Synapses/metabolism
Cells, Cultured

@article {pmid41062702,
year = {2026},
author = {Casagrande Raffi, G and Kuiken, HJ and Lieftink, C and Bernards, R and Beijersbergen, RL and Wang, L},
title = {Inducible CRISPR-Cas9 screening platform to interrogate non-proliferative cellular states.},
journal = {Nature protocols},
volume = {21},
number = {5},
pages = {1896-1926},
pmid = {41062702},
issn = {1750-2799},
support = {19-051-ASP//Bergmark Foundation/ ; KWF-12539//KWF Kankerbestrijding (Dutch Cancer Society)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Cell Proliferation ; Flow Cytometry/methods ; Cellular Senescence/genetics ; },
abstract = {CRISPR screens have revolutionized the study of diverse biological processes, particularly in cancer research. Both pooled and arrayed CRISPR screens have facilitated the identification of essential genes for cell survival and proliferation, drivers of drug resistance and synthetic lethal interactions. However, applying loss-of-function CRISPR screening to non-proliferative states remains challenging, largely because of slower editing and the poor sensitivity of identifying guide RNAs that 'drop out' in a population of non-dividing cells. Here, we present a detailed protocol to accomplish this, using an inducible Cas9 system that offers precise temporal control over Cas9 expression. This inducible system allows gene editing to occur only after the non-proliferative state is fully established. We describe the complete procedure for generating an inducible Cas9-expressing model and for measuring editing efficiency by using flow cytometry. In addition, we discuss how to optimize key parameters for performing successful CRISPR screens in various non-proliferative states. We describe a detailed workflow for performing a screen in senescent cells to identify senolytic targets. This protocol is accessible to researchers with experience in molecular biology techniques and can be completed in 8-12 weeks, from the generation of an inducible Cas9 cell line clone to the analysis of a CRISPR screen for hit identification. These techniques can be applied by researchers across different fields, including stem cell differentiation, immune cell development, aging and cancer research.},
}

*CRISPR-Cas Systems/genetics
Humans
*Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Cell Proliferation
Flow Cytometry/methods
Cellular Senescence/genetics

@article {pmid41258015,
year = {2026},
author = {Longo, GMC and Sayols, S and Roukos, V},
title = {Multilevel characterization of genome editor nuclease activity with BreakTag.},
journal = {Nature protocols},
volume = {21},
number = {5},
pages = {2043-2082},
pmid = {41258015},
issn = {1750-2799},
support = {393547839-SFB 1361//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 402733153-SPP 2202//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 455784893//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; INST 247/845-1 FUGG//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *High-Throughput Nucleotide Sequencing/methods ; DNA Breaks, Double-Stranded ; Humans ; CRISPR-Associated Protein 9/metabolism ; },
abstract = {BreakTag is a scalable next-generation sequencing-based method for the unbiased characterization of programmable nucleases and guide RNAs at multiple levels. BreakTag allows off-target nomination, nuclease activity assessment and the characterization of scission profile, that, in Cas9-based gene editing, is mechanistically linked with the indel repair outcome. The method relies on digestion of genomic DNA by Cas9 and guide RNAs in ribonucleoprotein format, followed by enrichment of blunt and staggered DNA double-strand breaks generated by CRISPR nucleases at on- and off-target sequences. Next-generation sequencing and data analysis with BreakInspectoR allows high-throughput characterization of Cas nuclease activity, specificity, protospacer adjacent motif frequency and scission profile. Here we first describe a detailed BreakTag protocol for the nomination of CRISPR off-targets and multilevel characterization of engineered Cas variants and second, we describe a step-by-step protocol for data analysis using BreakInspectoR. Third, we provide a web interface for XGScission, a machine learning model amenable to training with scission-aware BreakTag data to predict the relative frequency of blunt and staggered double-strand breaks at new sequences unseen by the model. XGScission allows a preselection of target sequences predicted to be cut in staggered configuration that are preferably repaired as single-nucleotide templated insertions. Furthermore, XGScisson can be used to assess sequence determinants of blunt and staggered cleavage by SpCas9 and engineered nuclease variants. As a companion strategy, we describe HiPlex for the generation of hundreds to thousands of single guide RNAs in pooled format for the production of robust BreakTag datasets. The BreakTag library preparation takes ~6 h, and the entire protocol can be completed in ~3 d, including sequencing, data analysis with BreakInspectoR and XGScission model training.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*High-Throughput Nucleotide Sequencing/methods
DNA Breaks, Double-Stranded
Humans
CRISPR-Associated Protein 9/metabolism

@article {pmid41833894,
year = {2026},
author = {Ma, Y and Liao, Y},
title = {CRISPR-mediated cancer therapies: Approaches to direct tumor targeting.},
journal = {Critical reviews in oncology/hematology},
volume = {222},
number = {},
pages = {105277},
doi = {10.1016/j.critrevonc.2026.105277},
pmid = {41833894},
issn = {1879-0461},
mesh = {Humans ; *Neoplasms/therapy/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genetic Therapy/methods ; Tumor Microenvironment/genetics ; Animals ; },
abstract = {CRISPR-Cas9 technologies have opened new possibilities for precision cancer treatment, addressing limitations inherent in conventional therapies such as chemotherapy and radiation. This review examines CRISPR-based strategies for direct tumor targeting, including oncogene inactivation, tumor suppressor gene reactivation, and tumor microenvironment (TME) modification. Key advances include KRAS[G12D] inactivation via base editing, in which engineered deaminases introduce precise single-nucleotide changes without generating double-strand breaks; TP53 correction through homologous recombination, which uses a donor DNA template to repair mutant sequences at the targeted locus; and CDKN2A epigenetic remodeling using CRISPR-dCas9-TET1 demethylation, where catalytically inactive Cas9 guides the TET1 demethylase to hypermethylated promoters to restore gene expression. CRISPR screening has identified synthetic lethal interactions, such as PARP1 dependency in BRCA1[-/-] tumors. TME editing strategies, including modification of cancer-associated fibroblasts, demonstrate enhanced antitumor responses. Delivery challenges are being addressed through viral vectors, including adenovirus, AAV, and lentivirus. Non-viral approaches include lipid nanoparticles, gold nanoparticles, exosomes, and stimuli-responsive systems such as MMP-cleavable and hypoxia-responsive nanoparticles. Clinical trials with CRISPR-engineered T-cells (e.g., CTX130) have demonstrated remission rates in hematologic malignancies. However, significant challenges remain, including cytokine release syndrome, immunotoxicity, tumor heterogeneity, and limited delivery efficiency in solid tumors. Overcoming these barriers requires interdisciplinary innovation, ethical oversight, and technological refinement to support the safe and effective integration of CRISPR-based strategies into precision oncology.},
}

Humans
*Neoplasms/therapy/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
*Genetic Therapy/methods
Tumor Microenvironment/genetics
Animals

@article {pmid42046341,
year = {2026},
author = {Wang, Z and Chen, Y and Wang, Y and Li, W and Zhang, X and Zhang, S},
title = {One-Pot Ligation-Recombinase Polymerase Amplification-Clustered Regularly Interspaced Short Palindromic Repeats/Cas12a-Powered Trimode Lateral Flow Assay for Sensitive MicroRNA Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {18},
pages = {13365-13376},
doi = {10.1021/acs.analchem.5c07208},
pmid = {42046341},
issn = {1520-6882},
mesh = {*MicroRNAs/analysis/genetics ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Gold/chemistry ; Humans ; *Recombinases/metabolism ; Limit of Detection ; Platinum/chemistry ; *Endodeoxyribonucleases/metabolism/genetics ; Metal Nanoparticles/chemistry ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Sensitive detection of microRNAs (miRNAs) holds significant importance for the early diagnosis of cancer. Since current sensitive nucleic acid detection methods like recombinase polymerase amplification-clustered regularly interspaced short palindromic repeats (RPA-CRISPR)/Cas12a are not suitable for detecting short-stranded miRNAs, we introduced a T4 ligase-based ligation process to the RPA-CRISPR/Cas12a system and developed a novel miRNA detection method termed ligation-RPA-CRISPR/Cas12a (LRCC). This assay utilizes a glycerol-enhanced one-pot reaction strategy combined with a lateral flow assay (LFA) to streamline the operation, minimize aerosol contamination, and improve point-of-care testing performance. Kinetic studies have shown that the catalytic efficiency of the glycerol-enhanced one-pot reaction is 3.11 and 2.09 times higher than that of the direct one-pot and stepwise methods, respectively. By synthesizing "three-in-one" Au-Pt nanostars (Au@Pt NSs) as probes and stabilizing them via "click" chemistry modification, this work enabled a trimode detection approach (colorimetric, photothermal, and surface-enhanced Raman spectroscopy (SERS)) with improved accuracy. In the experiment, tetrahedron DNAs were immobilized on the test line of the strip to enhance the capture efficiency of probes, thereby improving the detection sensitivity. The entire detection process was completed in 70 min with detection limits of 23.6 fM for colorimetric (C-LFA), 2.19 fM for photothermal (P-LFA), and 72.29 aM for SERS (S-LFA). The results demonstrate the strong practical applicability of the LRCC strategy, which plays a crucial role in miRNA-based early disease diagnosis.},
}

*MicroRNAs/analysis/genetics
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
Gold/chemistry
Humans
*Recombinases/metabolism
Limit of Detection
Platinum/chemistry
*Endodeoxyribonucleases/metabolism/genetics
Metal Nanoparticles/chemistry
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid42065492,
year = {2026},
author = {Han, R and Xiao, N and Wu, Z and Zhao, Y and Wang, X and Tang, X},
title = {Sensitive and Robust One-Pot RPA-CRISPR/Cas12a Assay with Elimination of cis-Cleavage.},
journal = {Analytical chemistry},
volume = {98},
number = {18},
pages = {13307-13318},
doi = {10.1021/acs.analchem.5c06674},
pmid = {42065492},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems ; Limit of Detection ; DNA, Single-Stranded/genetics/chemistry ; *Replication Protein A/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {The rapid detection technology utilizing CRISPR/Cas12a is characterized by high sensitivity, portability, and efficiency, making it a prominent focus in the field of point-of-care testing (POCT). However, it still has limitations in one-pot detection systems. This study developed a one-pot assay based on CRISPR/Cas12a and RPA (11bp+9ss one-pot assay), which nearly eliminates the cis-cleavage activity of Cas12a while retaining its trans-cleavage activity. Specifically, cis-cleavage was abolished by shortening the complementary length between the crRNA and the target to maintain the double-stranded conformation at the cis-cleavage site, whereas trans-cleavage activity was preserved by using an ssDNA complementary to the remaining region. The trans-cleavage activity was applicable to targets within a 200 bp range and under suboptimal PAM conditions. Moreover, rational design of the ssDNA enables effective discrimination of single-base mutations. The 11bp+9ss one-pot assay achieved a limit of detection (LOD) of 1 × 10[0] copies/μL for various targets, demonstrating robust performance even in suboptimal RPA systems. Furthermore, the assay was successfully applied to the detection of Salmonella and Avian Leukosis Virus subgroup J (ALV-J) samples. Overall, the 11bp+9ss one-pot assay exhibits superior sensitivity and robustness, showing great potential for POCT of bacteria and viruses.},
}

*CRISPR-Cas Systems
Limit of Detection
DNA, Single-Stranded/genetics/chemistry
*Replication Protein A/metabolism/genetics
*Endodeoxyribonucleases/metabolism/genetics
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid42115922,
year = {2026},
author = {Bhowmik, PK and Williams, JT and Polley, B and Chen, N and Kavuri, NR and Zang, W and Barakate, A and Yang, H and Narra, MK and Beattie, AD and Starker, C and Voytas, DF and Baysal, C},
title = {BSMV-mediated genome editing exhibits host-specific heritability: germline transmission in barley and somatic edits in Nicotiana benthamiana.},
journal = {BMC plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12870-026-08866-3},
pmid = {42115922},
issn = {1471-2229},
abstract = {BACKGROUND: Plant RNA virus-mediated guide RNA (gRNA) delivery represents a transformative advance in genome editing technologies. Unlike conventional transformation methods that rely on labor-intensive tissue culture and regeneration for each individual gRNA delivery, viral vectors can rapidly and systemically transmit gRNAs into pre-established Cas-expressing plants, providing an accelerated route for functional genomics and trait discovery directly in planta. However, key design parameters, including subgenomic promoter choice, transcript architecture, and their effects on viral fitness and editing outcomes, remain to be elucidated for most viral platforms.

RESULTS: We developed five Barley stripe mosaic virus (BSMV) vectors, each with distinct subgenomic promoter elements to drive single gRNA expression. These were initially evaluated in Cas9-expressing transgenic Nicotiana benthamiana plants targeting the Phytoene desaturase (PDS) gene to compare their editing efficiencies. Single gRNAs expressed under the duplicated γb subgenomic promoter or when fused directly to the γb genome achieved the highest mutation frequencies (up to 90% at 60 days post-inoculation), whereas β1- and β2-driven sgRNAs produced delayed and reduced editing. Thus, promoter selection critically determines gRNA accumulation and the efficacy of BSMV-mediated genome editing. The top-performing design was then applied to Cas9-expressing barley (Hordeum vulgare) targeting HvCMF7 (conferring green-white variegation) and HvGW2.1 (impacts grain width and weight). BSMV spread systemically throughout barley, inducing somatic and heritable mutations at frequencies up to 100%, with virus-free edited progeny. In contrast, despite robust somatic editing in N. benthamiana, no heritable mutations were detected indicating species-dependent limitations in germline transmission.

CONCLUSION: Our systematic comparison of subgenomic promoter architectures establishes clear design principles for optimizing viral vector-mediated delivery. Promoter choice and transcript structure critically shape editing efficiency and viral stability. The host-specific boundary for germline editing, defined by efficient heritable editing in barley but not N. benthamiana, highlights where BSMV offers advantages and where alternative vectors or hybrid strategies are required, guiding rational platform selection for diverse crop species and applications. Collectively, these findings establish BSMV as a promising next-generation vector for rapid, tissue culture-free, and transformation-independent genome editing in cereals and other recalcitrant monocots.},
}

@article {pmid42116436,
year = {2026},
author = {Sun, L and Yan, J and Xie, J and Wang, XY},
title = {From interactions to applications: the role of microbial communities in shaping the physicochemical, safety, and quality attributes of aquatic products.},
journal = {Food research international (Ottawa, Ont.)},
volume = {236},
number = {},
pages = {119126},
doi = {10.1016/j.foodres.2026.119126},
pmid = {42116436},
issn = {1873-7145},
mesh = {*Food Safety ; *Food Microbiology ; *Microbiota ; *Seafood/microbiology ; *Microbial Interactions ; Bacteria/growth & development ; Animals ; Food Handling ; Food Quality ; Quality Control ; },
abstract = {Aquatic products are regarded as important dietary resources due to their abundant high-quality proteins, while their physicochemical properties, safety, and quality attributes during storage and processing are strongly influenced by microbial communities. Microbial interactions in aquatic products can exacerbate spoilage and pathogenic contamination, thereby intensifying food safety issues. However, previous studies primarily focused on identifying and characterizing individual microbial species in aquatic products, whereas current research increasingly emphasizes the critical role of multi-species interactions and applications in quality and safety evaluation of aquatic products. This review focuses on the interactions, characteristics, and growth models of spoilage and pathogenic microorganisms in aquatic products. The regulation mechanisms underlying microbial interactions and their neutral, negative, and positive effects on aquatic products' quality were summarized. Additionally, the paper analyzes targeted strategies for regulating microbial interactions in aquatic product preservation, providing theoretical and practical support for quality control. The microbiota primarily consists of spoilage and pathogenic bacteria, whose growth dynamics and interactions can be quantitatively modeled using models such as the Baranyi and P-model to predict their behavior. These microorganisms directly regulate the spoilage process and health risks via physical structures (e.g., extracellular polymeric substances, intercellular nanotubes) and chemical signaling networks (e.g., metabolic cross-feeding, quorum sensing). Conventional strategies (e.g., modified atmosphere packaging, ultra-high pressure processing) and precision approaches (e.g., digital twin technology and CRISPR/Cas system) have been applied to regulate the microbial interactions, thereby significantly enhancing the quality control in aquatic products.},
}

*Food Safety
*Food Microbiology
*Microbiota
*Seafood/microbiology
*Microbial Interactions
Bacteria/growth & development
Animals
Food Handling
Food Quality
Quality Control

@article {pmid42117889,
year = {2026},
author = {Wu, W and Jin, F and Xu, H and Liao, R and Fang, Z},
title = {Negative Regulators of Rice Agronomic Traits: Functional Insights and Applications in Genome Editing-Based Breeding.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.70684},
pmid = {42117889},
issn = {1467-7652},
support = {32572249//National Natural Science Foundation of China/ ; qiankehepingtairencai-YQK (2023) 002//Guizhou Provincial Excellent Young Talents Project of Science and Technology/ ; qiankehejichu-ZD(2026)068//Science and Technology Program of Guizhou Province/ ; qiankehechengguo (2024) general 116//Science and Technology Program of Guizhou Province/ ; Qiankehepingtairencai-BQW (2024) 001//Science and Technology Program of Guizhou Province/ ; Qiankehepingtai ZSYS (2025) 037//Guizhou Key Laboratory of High Quality, High Efficiency, and Yield Enhancement in Grain and Oil Crops/ ; Qianjiaoji (2023) 007//Key Laboratory of Functional Agriculture of Guizhou Provincial Higher Education Institution/ ; GZSDCYJSTX-202602//Guizhou Provincial Modern Agricultural Industry Technology System Construction Special Program/ ; },
abstract = {Rice is the staple crop for more than half of the global population, and improving grain yield, grain quality, and stress resistance remain central goals of modern rice breeding. Among current precision breeding strategies, genome editing has created new opportunities for crop improvement, but its success depends heavily on the selection of effective target genes. In this context, negative regulators of agronomic traits are particularly valuable because their disruption or attenuation can relieve constraints on desirable phenotypes and generate beneficial variation. In this review, we summarize recent progress in the identification and functional characterization of negative regulatory genes associated with rice grain yield, grain quality and stress resistance. We further integrate the current knowledge of their molecular functions, regulatory mechanisms, and genetic networks and discuss their potential applications in genome editing-assisted breeding. This review provides a target-oriented framework for understanding negative regulation in rice and facilitating the development of improved varieties with increased productivity, quality and stress resistance.},
}

@article {pmid42119611,
year = {2026},
author = {Ghonse, K and Dandekar, S and Koratkar, S},
title = {Multidrug-resistant Acinetobacter baumannii: Molecular insights, clinical challenges, and therapeutic approaches.},
journal = {Journal of microbiological methods},
volume = {},
number = {},
pages = {107544},
doi = {10.1016/j.mimet.2026.107544},
pmid = {42119611},
issn = {1872-8359},
abstract = {Acinetobacter baumannii has emerged as an agent of potentially life-threatening nosocomial infections, particularly among immunocompromised patients. Its ability to rapidly acquire resistance genes has made traditional antibiotic therapies progressively ineffective. The spread of bacterial contamination in hospital facilities increased due to lack of awareness among healthcare workers and improper management/handling of infectious samples. The persistence of pathogen in the hospital environment is increased with its ability to form biofilms, quorum sensing, and virulence factors. The infections caused by these agents are increasing in incidence and severity; necessitating efficient and timely management. This review highlights the epidemiological trends and molecular mechanisms involved in the pathogenesis and resistance of A. baumannii. The key resistance mechanisms that the organism possess include the activity of efflux pumps, beta-lactamase enzymes, and mobile genetic elements. This review discusses emerging treatment strategies - such as phage therapy - antimicrobial peptides, CRISPR-Cas-based technologies, and nanotechnology-enabled drug delivery- highlighting their respective benefits and limitations, with special emphasis on innovations like phage-antibiotic synergy and precision genome editing approaches. Despite promising advances, challenges remain, including the emergence of resistance pathogen, limited clinical scalability, and concern regarding the safety and toxicity of novel treatment options. Addressing these issues require focus on molecular insights of resistance mechanisms, the development of effective alternative therapies, and implementation of preventive strategies such as vaccines. Furthermore, execution of global antimicrobial stewardship program and robust surveillance systems are critical for effectively control and manage the threat posed by A. baumannii.},
}

@article {pmid41668390,
year = {2026},
author = {Gao, J and Hao, Y and Du, T and Li, K and Qiao, S and Xu, M and Guo, J and Hu, G and Ren, F and Fan, X and Dong, Y},
title = {Establishment of an RT-LAMP-CRISPR/Cas12a detection system for grapevine fabavirus and improvement of grapevine leaf crude extract with alkaline resin for on-site naked-eye detection.},
journal = {Pest management science},
volume = {82},
number = {6},
pages = {5245-5254},
doi = {10.1002/ps.70633},
pmid = {41668390},
issn = {1526-4998},
support = {2024BBF01002//Key Research and Development Project of Ningxia Hui Autonomous Region/ ; CARS-29-bc-1//China Agriculture Research System of MOF and MARA/ ; CAAS-ASTIP-RIP//Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences/ ; },
mesh = {*Vitis/virology ; Plant Leaves/virology/chemistry ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *Plant Diseases/virology ; *Molecular Diagnostic Techniques/methods ; *Plant Viruses/isolation & purification/genetics ; Sensitivity and Specificity ; },
abstract = {BACKGROUND: Grapevine fabavirus (GFabV) induces severe chlorosis and malformation in grapevine leaves, and its infection can substantially compromise both fruit yield and quality. Despite its growing impact on viticulture, no rapid and accurate on-site molecular diagnostic tool is currently available for the detection of GFabV. Thus, developing a reliable and field-deployable detection method is urgently needed.

RESULTS: In this study, we designed six sets of primers targeting conserved regions of GFabV for loop-mediated isothermal amplification (LAMP), and identified one optimal primer set capable of detecting all three GFabV variants. To enhance assay specificity and enable instrument-free visual readout, LAMP and CRISPR/Cas12a were integrated to establish a real-time (RT)-LAMP-CRISPR/Cas12a single-tube assay. The established method demonstrated exceptional specificity and remarkable sensitivity, and it is ≤10 000 times more sensitive than conventional RT-PCR. We streamlined the RNA preparation process by replacing traditional RNA extraction with optimized crude extract protocols. For grapevine leaves, we developed a novel 'Alkaline Resin method', utilizing a newly engineered alkaline resin material that effectively neutralizes the acidic lysate while simultaneously adsorbing chlorophyll, polyphenols and polysaccharides. For grapevine branches, we introduced an extremely simple 'branch-washing method', wherein crude nucleic acids are obtained simply by rinsing the phloem tissue with water. By coupling crude extraction strategies with the RT-LAMP-CRISPR/Cas12a single-tube assay, we achieved on-site detection of GFabV within 50 min.

CONCLUSION: We developed an RT-LAMP-CRISPR/Cas12a single-tube method, along with corresponding crude extraction procedures for grapevine branches and leaves, enabling simple, rapid, accurate and on-site detection of GFabV. © 2026 Society of Chemical Industry.},
}

*Vitis/virology
Plant Leaves/virology/chemistry
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
*Plant Diseases/virology
*Molecular Diagnostic Techniques/methods
*Plant Viruses/isolation & purification/genetics
Sensitivity and Specificity

@article {pmid41674090,
year = {2026},
author = {Han, K and Xie, B and Luo, C and Luo, Y and Yang, M and Lei, L and Jia, MA and Xu, T},
title = {Identification of a novel BBWV2 isolate and a sensitive and rapid RT-RPA-CRISPR/Cas12a-LFD detection method development.},
journal = {Pest management science},
volume = {82},
number = {6},
pages = {5255-5265},
doi = {10.1002/ps.70635},
pmid = {41674090},
issn = {1526-4998},
support = {//Guizhou Provincial Innovation Talents Team/ ; //the Guizhou Province Key Core Technology Research Project for Mountain Agriculture/ ; //the Plateau Characteristic Vegetable Industry Technology System Program of Guizhou Province/ ; //Special Program of China National Tobacco Corporation/ ; //National Natural Science Foundation of China/ ; //Guizhou Provincial Science and Technology Foundation/ ; //Provincial College Students' innovation and entrepreneurship training program/ ; },
mesh = {*Plant Diseases/virology ; *CRISPR-Cas Systems ; *Capsicum/virology ; *Nucleic Acid Amplification Techniques/methods ; China ; Sensitivity and Specificity ; },
abstract = {BACKGROUND: Broad bean wilt virus 2 (BBWV2) is a major viral pathogen causing significant economic losses in vegetable production. Existing detection methods often lack the speed, sensitivity, or simplicity required for effective on-site field diagnosis. This study aimed to identify a novel BBWV2 isolate and develop a rapid, equipment-free visual detection method suitable for field applications.

RESULTS: A novel BBWV2 isolate (BBWV2-GZCa) was identified and characterized from pepper in Guizhou Province, China. We developed a one-step visual detection assay by integrating reverse transcription-recombinase polymerase amplification (RT-RPA) with a CRISPR/Cas12a system, enabling readout via fluorescence or lateral flow dipstick (LFD). The assay demonstrated a detection limit of 7.5 copies/μL, which is 10[5] times more sensitive than conventional RT-PCR. It showed no cross-reactivity with other common pepper viruses and achieved 100% accuracy when validated using 20 field-collected samples.

CONCLUSION: The entire detection process can be completed within one hour without specialized equipment, requiring only visual interpretation. This RT-RPA-CRISPR/Cas12a-LFD method provides a rapid, highly sensitive, specific, and user-friendly platform for on-site detection of BBWV2, offering a practical tool for early diagnosis and disease management in agricultural settings. © 2026 Society of Chemical Industry.},
}

*Plant Diseases/virology
*CRISPR-Cas Systems
*Capsicum/virology
*Nucleic Acid Amplification Techniques/methods
China
Sensitivity and Specificity

@article {pmid41854410,
year = {2026},
author = {Dias, RG and Freitas, FPM and de Almeida, ELM and Fietto, LG and Zsögön, A and Silveira, WBD},
title = {CRISPR/Cas9 Genome Engineering in Non-Conventional Oleaginous Yeasts: Applications, Challenges, and Prospects.},
journal = {Yeast (Chichester, England)},
volume = {43},
number = {3},
pages = {77-88},
doi = {10.1002/yea.70015},
pmid = {41854410},
issn = {1097-0061},
mesh = {*CRISPR-Cas Systems ; *Metabolic Engineering/methods ; *Gene Editing/methods ; *Yeasts/genetics/metabolism ; *Genome, Fungal ; Yarrowia/genetics ; Lipid Metabolism/genetics ; Fatty Acids/biosynthesis ; *Genetic Engineering/methods ; },
abstract = {Given the biotechnological potential of yeast-derived oils for oleochemical production, genes encoding lipid metabolism enzymes are key targets for metabolic engineering. Genetic engineering tools such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, Transcription Activator-Like Effector Nucleases (TALENs), Zinc-Finger Nucleases (ZFNs), RNA interference (RNAi), and integrative plasmids can be used to modulate fatty acid biosynthesis and optimize lipid production. Among them, the CRISPR/Cas9 system, recognized for its simplicity and efficiency, has been deployed as a tool to create oleaginous yeast strains with high lipid productivity and features suitable for application in biorefineries. Species such as Cutaneotrichosporon oleaginosus, Rhodotorula toruloides, Candida spp., and Yarrowia lipolytica have already been engineered using CRISPR/Cas9 to enhance the production of fatty acids and their derivatives. However, designing and constructing an efficient CRISPR/Cas9 platform for oleaginous yeasts faces several hurdles, including low transformation efficiency, difficulties in expressing Cas9 and sgRNAs efficiently and consistently, the lack of well-characterized promoters, limited availability of PAM sequences, and poorly understood DNA repair mechanisms. Here, we address the application of the CRISPR/Cas9 system in oleaginous yeasts, laying out the challenges to developing efficient platforms and highlighting key trends in the field. We compare and discuss alternative CRISPR-Cas9 expression strategies to provide an overview of the current landscape and support the development of new approaches.},
}

*CRISPR-Cas Systems
*Metabolic Engineering/methods
*Gene Editing/methods
*Yeasts/genetics/metabolism
*Genome, Fungal
Yarrowia/genetics
Lipid Metabolism/genetics
Fatty Acids/biosynthesis
*Genetic Engineering/methods

@article {pmid41930821,
year = {2026},
author = {Zheng, P and Zheng, S},
title = {Xenotransplantation: Promise, progress, and risks.},
journal = {Veterinary immunology and immunopathology},
volume = {296},
number = {},
pages = {111110},
doi = {10.1016/j.vetimm.2026.111110},
pmid = {41930821},
issn = {1873-2534},
mesh = {*Transplantation, Heterologous/veterinary/adverse effects/trends ; Animals ; Humans ; Gene Editing ; CRISPR-Cas Systems ; Graft Rejection/prevention & control/immunology ; },
abstract = {Xenotransplantation has seen remarkable progress in recent years, largely driven by the advent of CRISPR-Cas9 gene editing technology. Several patients have received genetically modified kidney and heart transplants, achieving graft and patient survival ranging from days to months. This review offers a brief history of xenotransplantation and highlights advancements in gene editing that represent breakthroughs in minimizing rejection. It also presents recent cases of xenotransplantation and their clinical outcomes. Finally, the review addresses the ethical considerations and risks associated with this emerging field.},
}

*Transplantation, Heterologous/veterinary/adverse effects/trends
Animals
Humans
Gene Editing
CRISPR-Cas Systems
Graft Rejection/prevention & control/immunology

@article {pmid42024428,
year = {2026},
author = {Jia, H and Zhao, P and Pei, J and Tian, P},
title = {Rewiring the morphology and metabolism of Escherichia coli with an engineered CRISPRi system.},
journal = {Journal of applied microbiology},
volume = {137},
number = {5},
pages = {},
doi = {10.1093/jambio/lxag104},
pmid = {42024428},
issn = {1365-2672},
support = {22278022//National Natural Science Foundation of China/ ; },
mesh = {*Escherichia coli/genetics/metabolism/cytology ; *Metabolic Engineering/methods ; DNA Replication/genetics ; Escherichia coli Proteins/genetics ; *CRISPR-Cas Systems ; Lactic Acid/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression Regulation, Bacterial ; },
abstract = {AIMS: Microbial morphology is an increasingly leveraged target in metabolic engineering. To causally investigate the coupling between DNA replication, morphology, and metabolism, we constructed a CRISPR interference (CRISPRi) system in Escherichia coli BL21(DE3) to repress essential replication genes (dnaN, dnaG, polA, and ssb).

METHODS AND RESULTS: Transcriptional repression disrupted cell division and yielded distinct, quantifiable shape changes: knockdown of dnaG (encoding primase) and ssb (encoding single-stranded DNA-binding protein), in particular, induced significant cellular elongation and widening. This morphological reprogramming concurrently reallocated metabolic flux, as evidenced by a substantial increase in lactic acid titer from 0.89 g L⁻¹ to 6.01 g L⁻¹. RNA-seq and subsequent analyses (differential expression, GO/KEGG enrichment, GSEA, and PPI) reveal that replication inhibition drives extensive metabolic reprogramming and cell envelope remodeling, with notable perturbations in peptidoglycan biosynthesis.

CONCLUSION: Our results suggest that targeted replication stress can coordinately reshape bacterial morphology and alter metabolic output, providing a controllable strategy for engineering microbial cell factories.},
}

*Escherichia coli/genetics/metabolism/cytology
*Metabolic Engineering/methods
DNA Replication/genetics
Escherichia coli Proteins/genetics
*CRISPR-Cas Systems
Lactic Acid/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats
Gene Expression Regulation, Bacterial

@article {pmid42024433,
year = {2026},
author = {Sato, R and Maruyama, K and Ara, S and Shibata, M and Shida, Y and Ogasawara, W and Yamazaki, H and Takaku, H},
title = {A CRISPR/Cas9-based genome-editing platform enabling efficient and precise gene replacement in Lipomyces starkeyi.},
journal = {FEMS yeast research},
volume = {26},
number = {},
pages = {},
doi = {10.1093/femsyr/foag014},
pmid = {42024433},
issn = {1567-1364},
support = {JPJS00420240017//JSPS/ ; 25K08907//JSPS/ ; 25K18164//JSPS/ ; JPMJPF2211//Japan Science and Technology Agency/ ; //New Energy and Industrial Technology Development Organization/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Lipomyces/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Genome, Fungal ; CRISPR-Associated Protein 9/genetics ; },
abstract = {Lipomyces starkeyi is a promising oleaginous yeast with industrial potential. However, its genome engineering remains constrained by low gene-targeting efficiency and the requirement for long homologous regions. Herein, we established a CRISPR/Cas9 genome-editing platform for L. starkeyi by expressing codon-optimized Streptococcus pyogenes Cas9 fused to an SV40 nuclear localization signal. Furthermore, in vitro-transcribed single-guide RNAs (sgRNAs) were directly delivered into the host, eliminating the need for endogenous RNA polymerase III-dependent sgRNA expression. CRISPR/Cas9 activity was validated using a codon-optimized Aequorea coerulescens GFP reporter. Cas9-induced frameshift mutations caused GFP disruption, leading to fluorescence loss. Gene replacement at the LsURA3 locus was evaluated using donor constructs with homologous regions ranging from 50-3000 bp. In a Cas9-expressing wild-type background, precise gene replacement was dependent on homology arm length, increasing from 36% with 50-bp arms to 80% with 3000-bp arms. Notably, in a Cas9-expressing Δlslig4 strain with suppressed non-homologous end joining (NHEJ), precise gene replacement was achieved with 100% accuracy using 50-bp homology arms under CRISPR/Cas9-dependent conditions. Together, these results demonstrate that a Pol III-independent CRISPR/Cas9 system combined with NHEJ suppression enables precise genome editing in L. starkeyi, providing a foundation for functional genomics and metabolic engineering.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
*Lipomyces/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
*Genome, Fungal
CRISPR-Associated Protein 9/genetics

@article {pmid42108472,
year = {2026},
author = {Maxim, DS and Sostena, J and Johnson, NS and Wu, DW and Charu, V and Carter, JN and Anand, S and Church, GM and Bhalla, V},
title = {Designing genome editing experiments with EditABLE.},
journal = {Genome biology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s13059-026-04095-x},
pmid = {42108472},
issn = {1474-760X},
support = {1R41DK138689/DK/NIDDK NIH HHS/United States ; },
abstract = {While many computational tools exist for designing CRISPR-Cas experiments, there is a need for a centralized resource that combines individual tools to predict the most efficient genome editing strategy for a given application. To fill this gap, we develop EditABLE (EditABLE-app.stanford.edu), an online resource that provides optimal CRISPR editors and guide RNAs based on user provided sequence data with functionalities for base editing, prime editing, and integrase-mediated editing. We demonstrate the utility of EditABLE by applying it to one of the most common monogenic disorders, autosomal dominant polycystic kidney disease (ADPKD), identifying specific editing tools across the ADPKD mutation landscape.},
}

@article {pmid42109826,
year = {2026},
author = {Sun, A and Jin, SL and Liu, JG},
title = {A practical guide for characterization of novel CRISPR-Cas systems with Pro-CRISPR factors.},
journal = {Biophysics reports},
volume = {12},
number = {2},
pages = {85-99},
pmid = {42109826},
issn = {2364-3420},
abstract = {The emergence of advanced genome editing technologies has revolutionized research in life sciences, offering an unprecedented way to uncover unknown biological functions and innovative therapeutic strategies. Among all genome editing tools, CRISPR-Cas-based technologies play a pivotal role in this revolution, particularly Class 2 effectors such as Cas9 and Cas12, owing to their high efficacy and ease of programmability. With the advancements in genome sequencing and metagenomics, an increasing number of novel CRISPR-Cas systems have been discovered, including those found in extreme environments and viruses. Furthermore, recent studies have revealed an unexpected role of non-Cas accessory genes, such as the Tn7-like transposon and Pro-CRISPR factors (Pcr), in conferring additional functionalities to the CRISPR system, providing new insights into the understanding of CRISPR-mediated bacterial immunity and advancing the development of genome editing technologies. Therefore, it is essential to develop comprehensive methods for characterizing the Cas proteins and Pro-CRISPR factors with a growing diversity. In this protocol, we provide a method encompassing protein purification, biochemical characterization, validation of protein-protein interactions, and preliminary in vivo functional assays in bacteria for Cas nuclease and its associated Pro-CRISPR factor. We hope this protocol will not only assist in the characterization of the CRISPR-Cas system, but also provide valuable guidance for the characterization of other nucleases or nucleic acid modification systems.},
}

@article {pmid42112301,
year = {2026},
author = {Saedi, S and Nezhadi, J and Feizi, H and Memar, MY and Arefi, V and Kadkhoda, H},
title = {A comparative analysis of CRISPR systems, virulence factors, and antibiotic resistance genes in carbapenem-sensitive and carbapenem-resistant Klebsiella pneumoniae.},
journal = {Iranian journal of microbiology},
volume = {18},
number = {1},
pages = {1-13},
pmid = {42112301},
issn = {2008-3289},
abstract = {BACKGROUND AND OBJECTIVES: Klebsiella pneumoniae is a major cause of healthcare-associated infections, particularly in immunocompromised patients. This study compares the CRISPR systems, virulence factors, and antibiotic resistance genes in carbapenem-sensitive (CSKP) and carbapenem-resistant (CRKP) clinical isolates.

MATERIALS AND METHODS: Carbapenemase-producing isolates were identified by mCIM/eCIM. PCR and RT-qPCR detected key genes, including cas3, involved in CRISPR-Cas function. In silico analyses included STRING for protein interactions, CRISPRCasdb for CRISPR subtype distribution, and Phyre2/AlphaFold for cas3 structure prediction.

RESULTS: Among the isolates, 35.2% were resistant to carbapenems. Among CRKP strains, high prevalence of bla-NDM-1 (82%) and bla-OXA-48 (64%) was observed. The cas3 expression was significantly upregulated in resistant isolates (P = 0.002). CRISPR subtype I-E was identified in 16% of CRKP and 36% of CSKP isolates. Structural-functional analysis supported the integrity of Cas3 and revealed interactions with regulatory and iron acquisition proteins. Statistically significant differences in virulence and resistance gene profiles were found between CRKP and CSKP groups (P < 0.05).

CONCLUSION: This study highlights key differences between CRKP and CSKP isolates, particularly in CRISPR-Cas systems, resistance, and virulence. The findings suggest that cas3 plays a critical role in genomic adaptation and resistance mechanisms in K. pneumoniae, offering insights for future therapeutic strategies.},
}

@article {pmid42113695,
year = {2026},
author = {Yao, Y and Li, J and Duan, L and Chen, J and Li, G and Hu, Z},
title = {One-step Overlapping PCR for Rapid Synthesis of Single-guide RNA DNA Templates for the CRISPR System.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {230},
pages = {},
doi = {10.3791/70369},
pmid = {42113695},
issn = {1940-087X},
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics/biosynthesis/chemical synthesis ; *CRISPR-Cas Systems/genetics ; *Polymerase Chain Reaction/methods ; *DNA/genetics/chemical synthesis ; Staphylococcus aureus/genetics/enzymology ; *Gene Editing/methods ; },
abstract = {The CRISPR-Cas system has revolutionized genome editing; however, conventional methods for generating single-guide RNA (sgRNA) often involve time-consuming cloning steps or expensive commercial synthesis kits. An optimized one-step overlapping PCR strategy is presented for the rapid, cost-effective synthesis of DNA templates for in vitro sgRNA transcription. Using four partially overlapping primers spanning the T7 promoter, target-specific guide sequence, and sgRNA scaffold, full-length templates are assembled in a single PCR reaction without cloning. Systematic experimental optimization established an optimal primer ratio (AF1:AF2:AF3:Tracr-R = 50:5:1:50), minimizing non-specific byproducts while maximizing full-length product yield, as confirmed by agarose gel electrophoresis. This approach was successfully extended to generate templates for Staphylococcus aureus Cas9 (saCas9) sgRNA, demonstrating cross-system applicability beyond Streptococcus pyogenes Cas9 (SpCas9). Although direct chemical synthesis of sgRNAs offers advantages such as high purity, chemical modifications to enhance stability, and reduced off-target effects, it remains prohibitively expensive for high-throughput applications or large-scale screens that require numerous sgRNAs. In vitro cleavage assays demonstrated that guide RNAs generated using this method achieve editing efficiencies comparable to those obtained via conventional plasmid-based cloning. Furthermore, ribonucleoprotein complexes assembled with these sgRNAs and delivered into HEK293T cells via electroporation resulted in detectable indel formation at the target locus, confirming functionality in vivo. Cost analysis indicates that this method substantially reduces template preparation costs compared to commercial synthesis kits while reducing turnaround time from days to hours, thereby providing an accessible and scalable approach for laboratories engaged in genetic research.},
}

*RNA, Guide, CRISPR-Cas Systems/genetics/biosynthesis/chemical synthesis
*CRISPR-Cas Systems/genetics
*Polymerase Chain Reaction/methods
*DNA/genetics/chemical synthesis
Staphylococcus aureus/genetics/enzymology
*Gene Editing/methods

@article {pmid42113764,
year = {2026},
author = {Iwata, S and Miura, Y and Iwamoto, T},
title = {Non-viral in vivo electroporation-based chromosomal engineering and repair assessment in the murine uterine epithelium.},
journal = {PloS one},
volume = {21},
number = {5},
pages = {e0348797},
doi = {10.1371/journal.pone.0348797},
pmid = {42113764},
issn = {1932-6203},
mesh = {Animals ; Female ; *Electroporation/methods ; *Uterus/metabolism ; Mice ; CRISPR-Cas Systems ; Epithelium/metabolism ; *DNA Repair ; *Genetic Engineering/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Translocation, Genetic ; },
abstract = {Chromosomal rearrangements generated by CRISPR/Cas systems are valuable for studying genomic architecture and repair mechanisms. However, most in vivo approaches rely on viral vectors, which require specialised production, prolonged nuclease expression, and elevated biosafety containment. Here, we applied Cas9 ribonucleoprotein (RNP) electroporation to the murine uterine epithelium as a simple, non-viral strategy for somatic chromosomal engineering. This method successfully induced defined interchromosomal translocations at multiple loci and enabled the molecular assessment of large-scale inversion repair (57.8 Mb) using paired gRNAs with an ssODN donor. While rearranged alleles were detected at low apparent frequencies in bulk uterine DNA-consistent with epithelial-restricted delivery and somatic mosaicism-high-depth whole-genome sequencing (WGS) and PCR provided nucleotide-resolution confirmation of precise junction formation. Our findings demonstrate that uterine electroporation of CRISPR RNPs is a feasible, rapid approach for evaluating engineered chromosomal rearrangements in vivo, providing a controlled platform for analyzing somatic DNA repair outcomes without viral confounds.},
}

Animals
Female
*Electroporation/methods
*Uterus/metabolism
Mice
CRISPR-Cas Systems
Epithelium/metabolism
*DNA Repair
*Genetic Engineering/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Editing/methods
Translocation, Genetic

@article {pmid42115498,
year = {2026},
author = {Valiñas, MA and Cerrudo, I and Marchetti, F and Villarreal, F and Pagnussat, G and Zabaleta, E},
title = {CRISPR/Cas9-mediated disruption of the gamma carbonic anhydrase 2 gene leads to reduced mitochondrial complex I and growth alterations in tomato.},
journal = {Planta},
volume = {263},
number = {6},
pages = {},
pmid = {42115498},
issn = {1432-2048},
support = {PICT 2020 00013//ANPCyT/ ; },
mesh = {*Solanum lycopersicum/genetics/growth & development/enzymology/metabolism ; *CRISPR-Cas Systems/genetics ; *Electron Transport Complex I/metabolism/genetics ; Mitochondria/metabolism ; *Carbonic Anhydrases/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Germination ; },
abstract = {Despite similar complex I reduction, γCA2 disruption in tomato, unlike in arabidopsis, triggers hormonal and developmental changes, challenging assumptions of conserved mitochondrial responses across plant species. NADH-ubiquinone oxidoreductase [complex I (CI)] is the main entry point of electrons to OXPHOS being essential for metabolism and redox balance. In most organisms, except animals and fungi, CI contains an additional domain composed of gamma carbonic anhydrase (γCA) subunits, termed the CA module. In Arabidopsis thaliana, this module includes AtɣCA1/3, AtɣCA2, and AtɣCAL1/2. AtɣCA2 is critical for CI biogenesis, yet its role in other species remains unclear. In tomato, the γCA family comprises SlɣCA1a, SlɣCA1b, SlɣCA2, and SlɣCAL. Here, we report the inactivation of the tomato SlɣCA2 using CRISPR/Cas9 technology. As in arabidopsis, SlɣCA2-KO tomato plants show comparable reduction in CI levels and activity and a similar decrease in oxygen consumption, yet display increased ATP levels in seeds. However, unlike arabidopsis, mutant tomato plants exhibit delayed seed germination and retarded growth and development. Our results further suggest that abscisic acid and gibberellin homeostasis is altered in SlɣCA2-KO plants. Together, these findings support a connection between mitochondrial respiration and hormonal regulation, by which plants adjust developmental processes to mitochondrial electron transport chain functionality, thereby preventing energy depletion during early growth stages.},
}

*Solanum lycopersicum/genetics/growth & development/enzymology/metabolism
*CRISPR-Cas Systems/genetics
*Electron Transport Complex I/metabolism/genetics
Mitochondria/metabolism
*Carbonic Anhydrases/genetics/metabolism
*Plant Proteins/genetics/metabolism
Germination

@article {pmid41932456,
year = {2026},
author = {Silva, CS and Nascimento, GR and Cruz, PEO and Arancibia, RH and Andrade Belitardo, EMM and Castro, TLP and Villar, LM and Pacheco, LGC},
title = {Design principles for LAMP-CRISPR molecular diagnostics.},
journal = {Methods (San Diego, Calif.)},
volume = {251},
number = {},
pages = {1-22},
doi = {10.1016/j.ymeth.2026.03.014},
pmid = {41932456},
issn = {1095-9130},
mesh = {*Nucleic Acid Amplification Techniques/methods/instrumentation ; *Molecular Diagnostic Techniques/methods/instrumentation ; Humans ; *CRISPR-Cas Systems/genetics ; Point-of-Care Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Pathology, Molecular/methods ; },
abstract = {Nucleic acid detection methods leveraging Cas9, Cas12, and Cas13 enzymes have recently been widely integrated with isothermal amplification techniques, particularly Loop-Mediated Isothermal Amplification (LAMP), to develop CRISPR-based diagnostic assays for a broad range of pathogens. Coupling these systems with portable result-readout platforms such as lateral flow devices, microfluidics, and smartphones offers a promising pathway for deploying LAMP-CRISPR diagnostics at the point-of-care (PoC), especially in settings where conventional, resource-intensive methods like real-time PCR are not feasible. However, the development of LAMP-CRISPR assays presents unique challenges not typically encountered in real-time PCR workflows. These include the need for a larger number of oligonucleotides, the complexity of integrating multiple biochemical conditions, and a heightened risk of false-positive results. Despite the growing number of bioinformatics tools designed to aid assay development, establishing a robust and reproducible workflow for LAMP-CRISPR remains a significant hurdle. In this review, we critically examine current strategies for designing LAMP-CRISPR assays and offer a detailed, step-by-step guide to achieving high-performance diagnostic tools using this approach. We cover key aspects of target sequence selection, oligonucleotide and CRISPR system design, and the strategic choice of readout methods. We further discuss available tools for LAMP primer and CRISPR guide RNA design, providing practical recommendations for optimizing sequence selection. Various probe formats for Cas-mediated trans-cleavage detection are summarized, and we present best practices for assay standardization and minimizing false-positive signals. Finally, we highlight the current limitations and outline future directions for LAMP-CRISPR diagnostics in decentralized and PoC testing environments.},
}

*Nucleic Acid Amplification Techniques/methods/instrumentation
*Molecular Diagnostic Techniques/methods/instrumentation
Humans
*CRISPR-Cas Systems/genetics
Point-of-Care Systems
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Pathology, Molecular/methods

@article {pmid42019303,
year = {2026},
author = {Sun, X and Qian, L and Jin, D and Guo, B and Tao, S and Fang, J and Wang, S and Chen, H and Tian, T and Lei, H},
title = {Bifunctional DNA multivalent structure integrating stable capture of urothelial carcinoma cells with CRISPR/Cas12a signal amplification for bladder cancer detection.},
journal = {Biosensors & bioelectronics},
volume = {306},
number = {},
pages = {118710},
doi = {10.1016/j.bios.2026.118710},
pmid = {42019303},
issn = {1873-4235},
mesh = {Humans ; *Urinary Bladder Neoplasms/diagnosis/urine/genetics/pathology ; *Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; Aptamers, Nucleotide/chemistry/genetics ; *DNA/chemistry ; Nanostructures/chemistry ; Cell Line, Tumor ; Nucleic Acid Hybridization ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Noninvasive detection of bladder cancer remains challenging due to the limited analytical performance of current urine-based assays under realistic detection conditions. Here, we report a linear programmable DNA nanostructure that integrates multivalent aptamer recognition with CRISPR/Cas12a signal transduction for detecting tumor-derived urinary exfoliated cells. Assembled via hybridization chain reaction, the multivalent scaffold was shown to enhance ligand-cell binding stability under mechanically perturbed detection processes, thereby supporting mechanically stable biological recognition and yielding an approximately 14-fold increase in cellular binding affinity compared with monovalent aptamers. The optimized architecture (MAP12) enables detection with a LOD of 1.1 cells/mL in model systems and achieves high diagnostic performance in clinical urine samples (92% sensitivity, 88% specificity; AUC = 0.9424), supporting dual signal readouts via fluorescence and lateral flow devices (LFD). This work establishes a DNA nanostructural strategy for reliable, rapid, and noninvasive cancer cell detection under realistic operational conditions.},
}

Humans
*Urinary Bladder Neoplasms/diagnosis/urine/genetics/pathology
*Biosensing Techniques/methods
CRISPR-Cas Systems/genetics
Aptamers, Nucleotide/chemistry/genetics
*DNA/chemistry
Nanostructures/chemistry
Cell Line, Tumor
Nucleic Acid Hybridization
Limit of Detection
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid42025056,
year = {2026},
author = {Ren, Y and Du, W},
title = {Integrated single-tube detection of miRNAs in subpopulation-specific extracellular vesicles via spatially colocalized dual-module DNA scaffold.},
journal = {Biosensors & bioelectronics},
volume = {306},
number = {},
pages = {118711},
doi = {10.1016/j.bios.2026.118711},
pmid = {42025056},
issn = {1873-4235},
mesh = {*MicroRNAs/isolation & purification/genetics/analysis ; *Extracellular Vesicles/chemistry/genetics ; Humans ; *Biosensing Techniques/methods ; Aptamers, Nucleotide/chemistry ; Limit of Detection ; CRISPR-Cas Systems ; *DNA/chemistry ; Biomarkers, Tumor/genetics ; },
abstract = {Extracellular vesicles (EVs)-derived miRNAs are valuable non-invasive biomarkers for early cancer diagnosis. However, most current methods detect either total EVs-miRNAs or those from a single subpopulation, overlooking subpopulation heterogeneity. Moreover, EVs isolation and miRNA analysis are often conducted in separate workflows, increasing sample handling and compromising reproducibility. Herein, we presented an integrated, single-tube platform for subpopulation-specific miRNA profiling, in which multivalent aptamer-based EVs capture and CRISPR/Cas12a-mediated signal amplification were co-localized on a rationally designed DNA scaffold. The multivalent aptamer system achieved a capture efficiency of 84.3%, significantly outperforming monovalent aptamers (56.4%). Upon in situ lysis of captured EVs subpopulation, the released miRNAs preferentially encountered adjacent recognition probes due to the increased local concentration, thereby efficiently initiating catalytic hairpin assembly (CHA). The resulting abundant duplex subsequently activated Cas12a, achieving a limit of detection (LOD) as low as 1.42 × 10[4] particles/μL. Critically, the modularity of the platform allowed for straightforward reconfiguration to target distinct EVs subpopulations by exchanging the capture aptamer. We demonstrated this flexibility by profiling miR-21 and miR-155 across two specific subpopulations (CD63[+] and MUC1[+]). The results revealed both cell-line-specific expression patterns and marked heterogeneity across CD63/MUC1-captured EVs fractions. Meanwhile, MUC1[+] subpopulation outperformed CD63[+] in clinical diagnosis, with higher AUC values for both miR-21 (0.85 vs 0.75) and miR-155 (0.83 vs 0.73). Overall, our study highlighted the critical importance of EVs subpopulation heterogeneity in cancer diagnostics and provided more informative biomarker readouts.},
}

*MicroRNAs/isolation & purification/genetics/analysis
*Extracellular Vesicles/chemistry/genetics
Humans
*Biosensing Techniques/methods
Aptamers, Nucleotide/chemistry
Limit of Detection
CRISPR-Cas Systems
*DNA/chemistry
Biomarkers, Tumor/genetics

@article {pmid42051315,
year = {2026},
author = {Nolan, M and Aryal, S and Ndayambaje, IS and Cao, M and Lee, P and Hovde, M and Yun, S and Wlaschin, J and Held, A and Beaussant, H and Wymann, B and Zong-Lee, C and Lim, SM and Jiang, X and Ramesh, N and Agra Almeida Quadros, AR and Boulos, A and Zinter, N and Salem, S and El-Tayar, L and Beccari, M and Presa, M and Jourdan Ferreras Reyes, C and Ruan, YY and Griesman, G and Aguilar, C and Hawrot, J and Wheeler, H and Melamed, Z and Kleinstiver, BP and Albers, M and Cleveland, DW and Tanzi, RE and Lutz, CM and Hubbard, RD and Kobayashi, D and Ward, M and R R Alves, C and Wainger, B and Pichon, CL and Lagier-Tourenne, C},
title = {Statins and genetic inhibition of the mevalonate pathway activate an ATF3-STMN2 regenerative program.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {42051315},
issn = {2692-8205},
abstract = {Loss of neuronal regenerative capacity is a common feature of neurodegenerative disease and axonal injury, yet the transcriptional programs governing this state remain poorly defined. Stathmin-2 (STMN2), a tubulin-binding protein essential for axon maintenance and repair, is profoundly depleted following loss of nuclear TDP-43 in neurodegenerative disease. Here, we identify statins as potent inducers of STMN2 expression. Pharmacological and genetic suppression of the mevalonate pathway, and subsequent prevention of protein geranylgeranylation, restored STMN2 levels in TDP-43 deficient cells and promoted neurite growth. STMN2 induction was abrogated when using a statin analogue unable to interact with HMG-CoA reductase, and through co-administration of mevalonate or geranylgeranyl diphosphate substrates. RNA-seq revealed that statins induce a coordinated pro-regenerative transcriptional response, including activation of the AP-1 transcription factor complex gene, ATF3 . Loss of ATF3 attenuated STMN2 induction in vitro , and diminished injury-induced Stmn2 upregulation in spinal motor neurons in vivo . These results demonstrate statins as modulators of ATF3 and STMN2 expression and highlight their therapeutic potential in neurodegenerative disease.},
}

@article {pmid42061344,
year = {2026},
author = {Han, X and Chen, H and Chang, Y and Zha, J and Lam, CYK and Yang, M and Wong, SHD and Yin, B},
title = {Surface-confined CRISPR-Cas12a biosensor with metal-enhanced fluorescence for rapid and ultrasensitive detection of SARS-CoV-2 nucleocapsid protein.},
journal = {Biosensors & bioelectronics},
volume = {306},
number = {},
pages = {118649},
doi = {10.1016/j.bios.2026.118649},
pmid = {42061344},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods/instrumentation ; SARS-CoV-2/isolation & purification ; *CRISPR-Cas Systems ; Gold/chemistry ; Humans ; COVID-19/diagnosis/virology ; Limit of Detection ; *Coronavirus Nucleocapsid Proteins/analysis ; Metal Nanoparticles/chemistry ; Silicon Dioxide/chemistry ; Aptamers, Nucleotide/chemistry ; Fluorescence ; *Phosphoproteins/analysis ; *Coronavirus Infections/diagnosis/virology ; *Nucleocapsid Proteins/analysis ; *Pneumonia, Viral/diagnosis/virology ; CRISPR-Associated Proteins ; },
abstract = {CRISPR-Cas12a integrated with nanomaterials has formulated powerful biosensors for viral protein detection, addressing the urgent need for point-of-care diagnostics. However, existing platforms are hindered by either multi-step separation procedures or insufficient signal amplification, limiting their sensitivity and practicality. Here, we report a one-pot "on-off" biosensor that combines metal-enhanced fluorescence (MEF) and nanoscale spatial confinement by co-localizing both reporter substrates and the CRISPR-Cas12a system on gold-silica core-shell nanoparticles (Au@SiO2 NPs), enabling rapid and ultrasensitive protein detection. Using SARS-CoV-2 nucleocapsid (N) protein as a model analyte, Au@SiO2 NPs are co-functionalized with (i) ssDNA activators blocked by N protein-specific aptamers, (ii) light-up hairpin DNA (DAP) complexed with auramine O (AO) as reporters, and (iii) short polyethylene glycol (PEG) spacers to mitigate steric hindrance. The nanoplatform displays an ultrabright "on-state" fluorescence, with an intensity >860-fold higher than that of free AO, enabled by the interaction with DAP and optimized fluorophore-metal spacing (∼20 nm). Upon target binding, aptamer displacement exposes the activator to locally initiate Cas12a trans-cleavage, disrupting proximal DAP structure and its interaction with AO, thereby producing a distinct "off-state" signal. Within the linear detection range, the system demonstrates up to ∼85% signal reduction relative to the initial signal and a signal-to-noise ratio (SNR) of 83.89, corresponding to a ∼2.5-fold higher SNR than the solution-phase system. The platform attains a limit of detection at 67.2 fg/mL within 30 min, with excellent sensitivity, selectivity, stability, and recovery in bronchoalveolar lavage fluid. By combining MEF-driven signal amplification with surface-confined CRISPR-Cas12a trans-cleavage, this platform establishes an efficient strategy for sensitive N protein detection.},
}

*Biosensing Techniques/methods/instrumentation
SARS-CoV-2/isolation & purification
*CRISPR-Cas Systems
Gold/chemistry
Humans
COVID-19/diagnosis/virology
Limit of Detection
*Coronavirus Nucleocapsid Proteins/analysis
Metal Nanoparticles/chemistry
Silicon Dioxide/chemistry
Aptamers, Nucleotide/chemistry
Fluorescence
*Phosphoproteins/analysis
*Coronavirus Infections/diagnosis/virology
*Nucleocapsid Proteins/analysis
*Pneumonia, Viral/diagnosis/virology
CRISPR-Associated Proteins

@article {pmid42106593,
year = {2026},
author = {Enciso-Rodríguez, F and Barrero, LS and Garzón-Martínez, GA and Kim, JH and Kumam, Y and Pagliai, FA and Jiang, T and Huo, H and Munoz, P},
title = {Overcoming breeding barriers with genome editing in autopolyploid crops.},
journal = {BMC plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12870-026-08862-7},
pmid = {42106593},
issn = {1471-2229},
abstract = {Autopolyploid crops play a central role in global agriculture, yet their complex genomes pose significant barriers to genetic improvement. High allelic diversity, extensive redundancy, and polysomic inheritance impede both conventional breeding and the implementation of modern biotechnological tools. Genome editing offers a powerful alternative by enabling precise, multi-allelic modification of traits associated with yield, quality, and stress resilience. However, progress across autopolyploid crops remains uneven due to low transformation and regeneration efficiencies, limited genomic resources, and challenges in achieving complete allele disruption. This review focuses on recent advances in genome editing across four economically important autopolyploid crops-potato (Solanum tuberosum), alfalfa (Medicago sativa), sugarcane (Saccharum spp.), and blueberry (Vaccinium corymbosum). We highlight the diversity of traits targeted through CRISPR/Cas systems, including reporter and selectable marker validation, tuber and forage quality, biomass composition, stress tolerance, flowering modulation, and plant regeneration. We also describe technical constraints affecting genome editing in autopolyploids including genotype-dependent recalcitrance, low transformation and editing efficiency, multiallelic targeting and chimerism, outlining emerging solutions such as multiplexed designs, endogenous promoters, morphogenic regulators and virus-based approaches, among others. Together, these developments provide a path toward efficient and heritable genome editing in complex polyploid genomes, setting the stage for next-generation precision breeding in crops vital to food, forage, and bioenergy security.},
}

@article {pmid42106633,
year = {2026},
author = {Hannan, MN and Khan, S and Siddique, N and Hossain, MN and Islam, R and Nahar, K and Rahman, GKMM and Shozib, HB and Molla, AH and Hoque, MN and Haque, MM},
title = {Draft genome sequence of Priestia megaterium MHES4, a biofertilizer candidate isolated from tomato rhizosphere in Bangladesh.},
journal = {BMC genomic data},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12863-026-01431-9},
pmid = {42106633},
issn = {2730-6844},
support = {Project number: 002, Financial year: 2024 - 2027//This research was supported by Research Management Wing, Gazipur Agricultural University, Bangladesh./ ; },
abstract = {OBJECTIVE: The genus Priestia has recently gained attention for its plant growth-promoting potential. To examine the genomic traits and biosafety profile for potential field application as a native, climate-smart bioinoculant, we sequenced, assembled and annotated the genome of Priestia megaterium strain MHES4, isolated from the rhizosphere of tomato plant grown in drought-prone ecosystem of Rajshahi, Bangladesh.

DATA DESCRIPTION: Genome assembly data from the shotgun whole genome sequencing (WGS) of the P. megaterium MHES4 revealed 60 contigs with a total length of 5,267,048 bp, an N50 of 446,003 bp and 37.9% G + C content. The mean sequencing depth was 127.58×, with 100% breadth of coverage. Genome completeness assessed was 97.43% with 3.5% contamination, confirming high assembly quality. In total, 5,484 protein-coding genes were annotated. Additionally, 5,445 protein-coding sequences, 28 tRNAs, and 5 rRNAs were identified. Functional analysis identified gene clusters involved in the synthesis of secondary metabolites, such as phytoene synthase and alpha-amylase, and a Type I CRISPR-Cas system. Biosafety assessment using in silico tools detected no virulence factors or transmissible antibiotic resistance genes, indicating its potential safe use in agriculture. Overall, this genomic resource provides valuable insights into the genetic potential of P. megaterium MHES4 for nutrient cycling and adaptation to the rhizosphere environment.},
}

@article {pmid42106677,
year = {2026},
author = {Peng, Z and Duan, W and Fan, Y and Yang, Q and Ye, Y and Xing, Y},
title = {PAM-flexible SpCas9 variants expand the targeting scope for porcine genome editing and cellular disease modeling.},
journal = {BMC biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12896-026-01164-8},
pmid = {42106677},
issn = {1472-6750},
support = {2023YFC3404302//National Key Reaearch and Development Program of China/ ; 32260825//National Natural Science Foundation of China/ ; },
abstract = {BACKGROUND: CRISPR-Cas-mediated gene editing has revolutionized life sciences, yet the targeting scope of the widely used SpCas9 is limited by its strict requirement for the NGG protospacer adjacent motif (PAM). To overcome this limitation, PAM-flexible SpCas9 variants have been developed and characterized in multiple species; however, their potential in pigs (an important biomedical model for humans) remains unexplored. Here, we systematically evaluated the editing performance of three PAM-flexible SpCas9 variants (SpRY, SpG, and SpCas9-NG) and their derived base editors in porcine fetal fibroblasts (PFFs).

RESULTS: Profiling across 228 target sites revealed that SpRY exhibits nearly PAM-less activity, with significantly higher editing efficiency at NRN (15.82%, R = A/G) than at NYN PAMs (5.75%, Y = C/T). SpG and SpCas9-NG preferentially targeted NGN PAMs, achieving mean efficiencies of 14.81% and 16.33%, respectively. PAM‑flexible cytosine base editors (CBEs) mediated efficient C:G‑to‑T:A conversion, with mean efficiencies of 12.01% for SpRY‑BE4max (NNN PAMs), 15.43% for SpG‑BE4max (NGN PAMs), and 18.39% for SpCas9‑NG‑BE4max (NGN PAMs). Similarly, PAM‑flexible adenine base editors (ABEs) mediated efficient A:T‑to‑G:C conversion, with mean efficiencies of 15.66% for SpRY‑ABE8e (NNN PAMs), 24.16% for SpG‑ABE8e (NGN PAMs), and 20.50% for SpCas9‑NG‑ABE8e (NGN PAMs). By exploiting this expanded targeting scope, we successfully introduced 16 pathogenic single‑nucleotide variants (SNVs) at NRN PAM sites in the porcine genome, with editing efficiencies reaching up to 40.68% for CBEs and 61.76% for ABEs.

CONCLUSIONS: PAM-flexible SpCas9 variants and their derived base editors greatly expand the targeting scope for porcine genome engineering, thereby substantially broadening the applicability potential of CRISPR-Cas-mediated genome editing tools in porcine genetic improvement and disease model generation.},
}

@article {pmid42107032,
year = {2026},
author = {Singh, V and Fetoh, MEA and Fetoh, IEA},
title = {Degradation dynamics: an insight into microbial interactions with explosive compounds.},
journal = {Biodegradation},
volume = {37},
number = {3},
pages = {},
pmid = {42107032},
issn = {1572-9729},
mesh = {Biodegradation, Environmental ; *Explosive Agents/metabolism ; *Bacteria/metabolism ; *Fungi/metabolism ; *Microbial Interactions ; },
abstract = {Degradation dynamics is an essential aspect in the field of environmental science and is crucial in understanding the interaction between microbes and explosive compounds. Explosive compounds and their residues, such as nitramines, nitro-substituted aromatics, picric acid, TETRYL, and HEXYL), and aliphatic, RDX, etc.are highly persistent in the environment. These compounds are toxic to many life forms at high concentrations, specific microbial species have evolved resistance and degradation capabilities, though their growth can still be inhibited beyond certain thresholds, The results of microbial biodegradation can range from complete mineralization to only the biotransformation into less toxic or more resistant metabolites. Research using pure cultures of bacteria and fungi has provided insight into the degradation pathways of certain nitro-organic compounds, and some key enzymes (laccases and lignin peroxidases) have been identified and studied. This review mainly aims to provide an overview of the current state of research on the degradation dynamics of explosive compounds Recent advancements have pivoted toward 'Bio-omics' and synthetic biology tools, such as CRISPR/Cas systems, to engineer high-activity microbial strains.},
}

Biodegradation, Environmental
*Explosive Agents/metabolism
*Bacteria/metabolism
*Fungi/metabolism
*Microbial Interactions

@article {pmid42108387,
year = {2026},
author = {Pandya, K and Jaisinghani, LS and Tripathi, A and Kumar, D and Saraf, SK and Sahoo, J and Kumar, D},
title = {Rational Design and Optimisation of CRISPR-Cas9 Delivery Systems for Targeted Genomic Transformation.},
journal = {The journal of gene medicine},
volume = {28},
number = {5},
pages = {e70095},
doi = {10.1002/jgm.70095},
pmid = {42108387},
issn = {1521-2254},
support = {EMDR/SG/11/2023-5582//Indian Council of Medical Research/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Genetic Vectors/genetics ; *Gene Editing/methods ; Animals ; Genetic Therapy/methods ; *Gene Transfer Techniques ; Blood-Brain Barrier/metabolism ; Dependovirus/genetics ; Alzheimer Disease/genetics/therapy ; },
abstract = {The CRISPR-Cas9 genome-editing technique offers a promising therapeutic strategy for genetic disorders, including neurodegenerative diseases like Alzheimer's disease (AD), characterised by inherited susceptibility and progressive cognitive decline, as well as other hallmarks such as amyloid beta (Aβ1-42) plaques and neurofibril tangles (NFTs). However, the blood-brain barrier (BBB) poses a significant challenge to the effectiveness of gene editing components in the affected brain region and impedes clinical translation. This comprehensive review compares various CRISPR-Cas9 delivery vectors, viral, nonviral and physical, with a focus on their efficacy in neurological diseases such as AD. Viral vectors viz., adeno-associated viruses (AAVs) and lentiviruses (LVs) demonstrate high transduction efficiency and BBB permeability. AAVs are preferred for their low immunogenicity, minimal toxicity, high neuronal tropism and episomal persistence, enabling sustained expression without insertional mutagenesis. LVs offer larger genetic payloads but raise concerns about genomic integration and potential oncogenesis, though integration-defective variants mitigate these risks. Nonviral vectors, including peptide and polymer-based nanoparticles, lipid nanoparticles (LNPs) and Inorganic carriers such as gold and silver nanoparticles, are less immunogenic and easier to handle but require further optimisation for in vivo BBB crossing and endosomal escape. Physical methods such as electroporation and microinjection are suitable for in vitro/ex vivo use, while novel CNS-targeted strategies, such as RVG-tagged particles, TfR-directed LNPs and engineered AAV variants, enhance brain penetration via receptor-mediated transcytosis. These preclinical studies show that these technologies can successfully edit genes and provide therapeutic benefits, including amyloid reduction and cognitive improvement in AD models. Yet off-target effects, immune responses and regulatory hurdles persist. Overall, continuous innovation in delivery vector design and safety profile-targeting strategies is crucial for advancing CRISPR-Cas9 towards clinical therapies for AD-based therapies and related neurological disorders.},
}

Humans
*CRISPR-Cas Systems/genetics
Genetic Vectors/genetics
*Gene Editing/methods
Animals
Genetic Therapy/methods
*Gene Transfer Techniques
Blood-Brain Barrier/metabolism
Dependovirus/genetics
Alzheimer Disease/genetics/therapy

@article {pmid41146557,
year = {2026},
author = {Saputra, E and Vellarikkal, SK and Li, L and Sun, H and Nguyen, K and Montano, A and Natarajan, S and Piccioni, F and Tamburino, AM and Yu, X and Olow, AK},
title = {In vitro models of cancer-associated fibroblast heterogeneity uncover subtype-specific effects of CRISPR perturbations.},
journal = {Molecular oncology},
volume = {20},
number = {5},
pages = {1253-1269},
doi = {10.1002/1878-0261.70153},
pmid = {41146557},
issn = {1878-0261},
support = {//Merck/ ; },
mesh = {Humans ; *Cancer-Associated Fibroblasts/pathology/metabolism ; Cell Line, Tumor ; *Pancreatic Neoplasms/genetics/pathology ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Genetic Heterogeneity ; *Models, Biological ; *CRISPR-Cas Systems ; Single-Cell Analysis ; Tumor Microenvironment ; Coculture Techniques ; },
abstract = {Cancer-associated fibroblasts (CAFs) are sought after as potential therapeutic targets due to their pro- and antitumorigenic functions, which are attributed to specializations in CAF subtypes. A precise targeting of specific subtypes would be required to design therapies that effectively modulate CAF phenotypes, necessitating translatable model systems to support target discovery efforts. However, not only is our knowledge of CAF heterogeneity in solid tumors lacking, particularly in pancreatic tumors, but the translatability of CAF models has also not been rigorously evaluated. Here, we develop a coculturing model with primary CAFs and immortalized tumor cell lines that can reliably represent CAF phenotypes observed in tumors, with correlations to immuno-resistant and immunomodulatory phenotypes. Using single-cell transcriptomics, we characterize the CAF subtype heterogeneity in the in vitro CAF cell lines isolated from pancreatic cancer patients and investigate the impact of perturbing potential stromal genes on different CAF subtypes. We also infer the continuum of state changes underlying the interconvertibility of CAF subtypes. Finally, we use immortalized CAF cell lines to perform single-cell CRISPR perturbations of stromal targets, revealing the subtype-specific effects of perturbations and the impact of model-type selection on the translatability of insights.},
}

Humans
*Cancer-Associated Fibroblasts/pathology/metabolism
Cell Line, Tumor
*Pancreatic Neoplasms/genetics/pathology
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Genetic Heterogeneity
*Models, Biological
*CRISPR-Cas Systems
Single-Cell Analysis
Tumor Microenvironment
Coculture Techniques

@article {pmid41958313,
year = {2026},
author = {Wong, E and Souza-Fonseca-Guimaraes, F},
title = {Gain-of-function enhancers optimize CAR-NK cell-based anti-cancer immunotherapy.},
journal = {Immunology and cell biology},
volume = {104},
number = {5},
pages = {494-498},
doi = {10.1111/imcb.70113},
pmid = {41958313},
issn = {1440-1711},
mesh = {*Killer Cells, Natural/immunology/transplantation/metabolism ; Animals ; Humans ; *Immunotherapy, Adoptive/methods ; Mice ; *Receptors, Chimeric Antigen/genetics/metabolism/immunology ; *Neoplasms/therapy/immunology/genetics ; CRISPR-Cas Systems/genetics ; HT29 Cells ; *Immunotherapy/methods ; },
abstract = {Schematic overview of the two-stage screening approach used to identify NK cell fitness genes. (A) CRISPRa mechanism, showing dCas9-VP64-mediated upregulation of target genes. (B) Whole-genome CRISPRa screening in HER2-CAR-NK92 cells transduced with a CRISPR sgRNA library and transferred into mice bearing HT29 tumours, followed by tumour collection and next-generation sequencing (NGS). (C) Barcoded ORF mini-screen in primary peripheral blood NK (PBNK) cells transduced with HER2-CAR and an ORF library, transferred into HT29 tumour-bearing mice, with subsequent tumour collection and NGS analysis.},
}

*Killer Cells, Natural/immunology/transplantation/metabolism
Animals
Humans
*Immunotherapy, Adoptive/methods
Mice
*Receptors, Chimeric Antigen/genetics/metabolism/immunology
*Neoplasms/therapy/immunology/genetics
CRISPR-Cas Systems/genetics
HT29 Cells
*Immunotherapy/methods

@article {pmid41967792,
year = {2026},
author = {Yuan, W and Jiang, Z and Li, F and Chen, H and Zhang, X and Fan, X},
title = {THOC6 deficiency leads to cardiomyopathy by reducing myocardial contractile proteins in cardiomyocytes.},
journal = {Experimental cell research},
volume = {459},
number = {2},
pages = {115025},
doi = {10.1016/j.yexcr.2026.115025},
pmid = {41967792},
issn = {1090-2422},
mesh = {*Myocytes, Cardiac/metabolism/pathology ; *Cardiomyopathies/metabolism/genetics/pathology ; Humans ; Animals ; Rats ; Cell Proliferation/genetics ; CRISPR-Cas Systems ; Apoptosis/genetics ; Actins/metabolism/genetics ; *Myocardial Contraction ; Collagen Type I/metabolism/genetics ; Induced Pluripotent Stem Cells/metabolism ; },
abstract = {BACKGROUND: The THOC6 protein is an essential part of the THO complex. Biallelic loss-of-function variants in the THOC6 gene are linked to Beaulieu-Boycott-Innes syndrome (BBIS; OMIM #613680). Although research predominantly focuses on THOC6's involvement in neurodevelopmental disorders, approximately 80% of BBIS patients present with cardiac anomalies, including structural heart disease, cardiomyopathy, and arrhythmia. Despite this, the connection between THOC6 expression and cardiac development remains underexplored. This study firstly investigates THOC6's role in heart development.

METHODS AND RESULTS: This study we firstly utilized CRISPR/Cas9 to knock out THOC6 in H9C2 cardiomyocytes, revealing a reduction in cell proliferation and an increase in apoptosis. With RNA sequencing (RNA-seq) analysis we found abundant gene changes after THOC6 knockout (KO) in H9C2, which associated with hypertrophic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, and dilated cardiomyopathy. Protein-protein interaction analysis and experimental validation indicated that THOC6 regulates the expression of type I collagen (COL1A1, COL1A2) and cytoskeletal protein (Cardiac α actin 1) in cardiomyocytes. Subsequently, we generated a THOC6 knockout cell lines in human induced pluripotent stem cells (hiPSCs) derived from a healthy individual using CRISPR/Cas9 technology. THOC6 knockout (KO) in hiPSCs-derived cardiomyocytes (hiPSC-CMs) led to the early manifestation of hypertrophic cardiomyopathy and dilated cardiomyopathy phenotypic characteristics, including disrupted sarcomeric organization. Notably, THOC6 KO hiPSC-CMs demonstrated a significant decreased in COL1A2 and β-tubulin expression levels.

CONCLUSION: THOC6 may influence cardiac development by regulating myocardial contractile proteins, primarily type I collagen, cardiac α actin 1 and β-tubulin.},
}

*Myocytes, Cardiac/metabolism/pathology
*Cardiomyopathies/metabolism/genetics/pathology
Humans
Animals
Rats
Cell Proliferation/genetics
CRISPR-Cas Systems
Apoptosis/genetics
Actins/metabolism/genetics
*Myocardial Contraction
Collagen Type I/metabolism/genetics
Induced Pluripotent Stem Cells/metabolism

@article {pmid41997283,
year = {2026},
author = {Zhang, Y and Yu, M and Huang, J},
title = {Arginase 2 regulates cholesterol biosynthesis in endothelial cells.},
journal = {Experimental cell research},
volume = {459},
number = {2},
pages = {115029},
doi = {10.1016/j.yexcr.2026.115029},
pmid = {41997283},
issn = {1090-2422},
mesh = {Humans ; *Arginase/metabolism/genetics ; *Cholesterol/biosynthesis ; *Human Umbilical Vein Endothelial Cells/metabolism ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Arginase 2 (Arg 2) is a mitochondrial enzyme that hydrolyzes L-arginine to L-ornithine and urea, influencing endothelial nitric oxide (NO) bioavailability and vascular function. Although Arg 2 is implicated in endothelial dysfunction under hypercholesterolemic and oxidative stress conditions, its potential role in endothelial cholesterol metabolism remains unknown.

METHODS: Arg 2 was deleted in immortalized human umbilical vein endothelial cells (HUVECs) using CRISPR/Cas9, followed by transcriptomic analyses. Differential gene expression was validated by quantitative RT-PCR and immunoblotting. Overexpression of wild-type Arg 2 and the catalytically inactive Arg 2 (H160F) mutant was achieved using recombinant lentiviral transduction. Arginase activity was quantified by measuring urea production using a colorimetric assay. Cholesterol intermediates were quantified by LC-MS.

RESULTS: RNA sequencing revealed that Arg 2 deletion markedly downregulated genes involved in the mevalonate and steroid biosynthesis pathways, including HMGCS1, FDFT1, FDPS, SQLE, and DHCR7. These transcriptional changes were accompanied by reduced protein levels of key cholesterol biosynthetic enzymes and decreased cellular concentrations of sterols, lanosterol, desmosterol, and cholesterol. Conversely, either overexpression of wild-type Arg 2 or the catalytically inactive Arg 2 (H160F) mutant enhanced the expression of these enzymes.

CONCLUSIONS: These findings identify a previously unrecognized role of Arg 2 in promoting endothelial cholesterol biosynthesis. Beyond competing with endothelial NO synthase for L-arginine, Arg 2 may regulate vascular homeostasis through modulation the mevalonate pathway, independent of its enzymatic activity. This dual function may link amino acid and lipid metabolism in the endothelium and suggests new mechanisms by which Arg 2 contributes to endothelial dysfunction and atherosclerotic progression.},
}

Humans
*Arginase/metabolism/genetics
*Cholesterol/biosynthesis
*Human Umbilical Vein Endothelial Cells/metabolism
CRISPR-Cas Systems

@article {pmid42024482,
year = {2026},
author = {Liang, Q and Cao, Y and Zhang, X and Ye, R and Liu, M and Zhang, S and Wang, Y},
title = {A Rapid and Ultrasensitive Detection of Coxsackievirus A16 Using Reverse Transcription Multiple Cross Displacement Amplification Combined with the CRISPR-Cas12a-Based Biosensing System.},
journal = {ACS infectious diseases},
volume = {12},
number = {5},
pages = {1765-1775},
doi = {10.1021/acsinfecdis.6c00151},
pmid = {42024482},
issn = {2373-8227},
mesh = {*CRISPR-Cas Systems ; Humans ; *Biosensing Techniques/methods ; *Enterovirus/genetics/isolation & purification ; *Nucleic Acid Amplification Techniques/methods ; *Hand, Foot and Mouth Disease/diagnosis/virology ; Sensitivity and Specificity ; Reverse Transcription ; Limit of Detection ; RNA, Viral/genetics ; Child, Preschool ; },
abstract = {Coxsackievirus A16 (CVA16) is one of the primary viral etiological agents of hand, foot, and mouth disease (HFMD) in infants and children under five years of age. Prompt and reliable detection of CVA16 is crucial for guiding immediate therapeutic interventions and for implementing effective epidemic prevention and control strategies, particularly in settings with limited resources. Herein, a diagnostic platform for CVA16 (CVA16-RT-MCDA-CRISPR) was developed by combining reverse transcription multiple cross displacement amplification (RT-MCDA) with CRISPR-Cas12a-based detection. In this system, the CVA16 VP1 gene was preamplified using RT-MCDA technology. The resulting amplicons were then specifically recognized and cleaved by the CRISPR-Cas12a-based detection system. MCDA primers, an engineered CP1 primer, and a specific guide RNA (gRNA) were designed to target the VP1 gene of CVA16. The assay achieved a limit of detection of 2.8 × 10[-1] copies per microliter for CVA16 RNA standard templates and showed no cross-reactivity against non-CVA16 pathogens. Furthermore, the CVA16-RT-MCDA-CRISPR assay's feasibility was validated using 96 clinical samples. Taken together, these results demonstrate that the CVA16-RT-MCDA-CRISPR assay is a reliable diagnostic tool for rapidly and sensitively detecting CVA16.},
}

*CRISPR-Cas Systems
Humans
*Biosensing Techniques/methods
*Enterovirus/genetics/isolation & purification
*Nucleic Acid Amplification Techniques/methods
*Hand, Foot and Mouth Disease/diagnosis/virology
Sensitivity and Specificity
Reverse Transcription
Limit of Detection
RNA, Viral/genetics
Child, Preschool

@article {pmid42100374,
year = {2026},
author = {Schrage, PR and Afonina, U and Wörtz, J and Marchfelder, A and Martens, KJA and Sáenz, JP and Endesfelder, U},
title = {A novel expression system for imaging single-molecule fluorescence in Haloferax volcanii WR806 enables visualization of altered Cas1 dynamics during UV-induced DNA damage response.},
journal = {microLife},
volume = {7},
number = {},
pages = {uqag014},
pmid = {42100374},
issn = {2633-6693},
abstract = {Fluorescence microscopy has become an indispensable tool in biological research, offering powerful approaches to study protein dynamics and cellular processes in vivo. Among archaea, Haloferax volcanii has emerged as a particularly well-suited model organism for imaging studies, with a growing toolkit of established fluorescent markers, plasmids, and promoter systems. Recent advances in single-molecule imaging techniques have created new opportunities through WR806, a carotenoid-free H. volcanii strain providing reduced autofluorescence background. However, existing plasmid-based expression systems in WR806 show critical limitations in protein expression control and challenges with protein aggregation. To address these limitations, we developed pUE001, a novel plasmid system specifically designed for WR806. This system achieves precise expression control by decoupling selection and induction through strategic implementation of the trpA selection marker. Through comprehensive characterization, we demonstrate that pUE001 provides superior control over protein expression compared to the previously established pTA962 system. It enables linear, titratable expression of diverse proteins-from the highly regulated CRISPR-Cas component Cas1 to the abundant structural protein FtsZ1-while preventing protein aggregation that could compromise native cellular functions. Additionally, we performed a comprehensive analysis of WR806 to show that carotenoid depletion does not affect native cellular physiology. Finally, to demonstrate the system's utility, we investigated the role of Cas1 in UV-induced DNA repair using single-particle tracking photoactivated localization microscopy (sptPALM). Our findings reveal Cas1 colocalizing with DNA-dense cellular regions and significant, dose-dependent changes in Cas1 mobility following UV-light-induced damage, providing evidence for its possible involvement in DNA damage response processes and offering new insights into the expanding roles of CRISPR-Cas systems beyond adaptive immunity.},
}

@article {pmid42100688,
year = {2026},
author = {Yadav, J and Gehlot, P and Soni, P and Jain, T},
title = {Plant microbiome engineering: from inoculation to genome editing.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1781381},
pmid = {42100688},
issn = {1664-302X},
abstract = {Plant-associated microbiomes are central to crop productivity, nutrient efficiency, and stress resilience, yet conventional microbiome manipulation strategies, largely based on microbial inoculation and agronomic management, often suffer from inconsistent field performance and limited persistence. Although several recent reviews have discussed CRISPR-mediated plant-microbe engineering and synthetic microbial community (SynCom) design separately, few reviews integrate genome editing, ecological stability of microbiomes, and climate-resilient agricultural applications within a unified conceptual framework. Recent advances in molecular biotechnology are transforming this landscape by enabling precision engineering of plant-microbe interactions at genetic, metabolic, and community levels. In particular, synthetic biology tools including CRISPR/Cas genome editing, RNA interference, and synthetic microbial communities (SynComs), now allow targeted modification of plant traits governing microbial recruitment, microbial pathways underpinning nutrient cycling and stress tolerance, and community-level functional complementarity. This review integrates molecular genetics, microbial ecology, and systems-level microbiome design to frame the plant and its microbiome as an engineerable holobiont. We integrate insights from genome editing in plants and microbes, omics-guided SynCom design, climate-resilience mechanisms, and emerging AI-assisted decision frameworks, including machine learning and ecological modeling approaches used to analyze multi-omics datasets, and predict plant-microbiome interactions across experimental and field-based studies. Importantly, we critically assess limitations related to ecological stability, trait trade-offs, biosafety, and regulatory challenges that constrain large-scale deployment. By bridging genome-enabled microbiome manipulation with ecological design principles, this review proposes an integrative framework for climate-smart microbiome engineering and identifies key research priorities required to transition from empirical inoculation toward predictive, sustainable, and socially responsible agricultural biotechnology.},
}

@article {pmid42100873,
year = {2026},
author = {Kavanagh, EW and Joynt, AT and Pion, AR and Eastman, AC and Parr, AI and Starego, KL and Jain, M and Shannon, SR and Yoo, EJ and Newby, GA and Tzeng, SY and Sharma, N and Green, JJ and Cutting, GR},
title = {Base editing and nanoparticle transfection of airway cell types essential for treatment of cystic fibrosis.},
journal = {JCI insight},
volume = {11},
number = {9},
pages = {},
doi = {10.1172/jci.insight.198563},
pmid = {42100873},
issn = {2379-3708},
mesh = {*Cystic Fibrosis/genetics/therapy ; Humans ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism ; *Nanoparticles/administration & dosage/chemistry ; *Gene Editing/methods ; *Transfection/methods ; Epithelial Cells/metabolism ; Genetic Therapy/methods ; Respiratory Mucosa/metabolism ; Animals ; CRISPR-Cas Systems ; Cells, Cultured ; },
abstract = {Cystic fibrosis (CF) is a life-limiting genetic disorder caused by deleterious variants in the CFTR gene that results in altered mucus impairing the airway epithelia. Durable correction of these variants in airway cells remains a therapeutic challenge for about 10% of individuals unresponsive to CFTR modulators. A common disease-causing CFTR splice site variant, 3120+1G>A, was corrected in primary CF airway cells using base editor RNAs. Single-cell RNA sequencing revealed a remarkable increase in detectable CFTR transcript in most CF airway epithelial cell types resulting in notable enrichment of CFTR-expressing ionocytes and secretory goblet cells. Progenitor basal cell subtypes were edited, but they decreased as a fraction of total cells and CFTR-expressing cells compared with unedited cells. CRISPR base editors delivered by polymeric nanoparticles (PNPs) facilitated functional rescue of CFTR to clinically meaningful levels in immortalized and primary airway cells. PNPs delivered GFP-encoding RNA to progenitor airway cells in fully differentiated airway cultures. Vitronectin was a major component of the PNP corona that formed in vivo, but preincubation with vitronectin did not enhance delivery. Together, these findings validate a scalable, nonviral platform with compelling translational promise for treating CF and other respiratory diseases involving respiratory epithelial cell dysfunction.},
}

*Cystic Fibrosis/genetics/therapy
Humans
*Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism
*Nanoparticles/administration & dosage/chemistry
*Gene Editing/methods
*Transfection/methods
Epithelial Cells/metabolism
Genetic Therapy/methods
Respiratory Mucosa/metabolism
Animals
CRISPR-Cas Systems
Cells, Cultured

@article {pmid42101329,
year = {2026},
author = {Moss, O and Li, X and Kanagarajan, S and Wang, ES and Ivarson, E and Zhu, LH},
title = {Evaluation of Bna.SCT and Bna.REF1 as Target Genes to Reduce Sinapine in Rapeseed Using a Protoplast-Based CRISPR RNP Approach.},
journal = {Physiologia plantarum},
volume = {178},
number = {3},
pages = {e70905},
doi = {10.1111/ppl.70905},
pmid = {42101329},
issn = {1399-3054},
support = {//SLU Grogrund- Centre for Breeding of Food Crops/ ; //Trees and Crops for the Future (TC4F)/ ; //The Royal Physiographic Society of Lund/ ; },
mesh = {Gene Editing/methods ; *Brassica napus/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Protoplasts/metabolism ; *Brassica rapa/genetics/metabolism ; Mutation ; Choline/analogs & derivatives ; },
abstract = {Rapeseed is a major oil crop worldwide, producing both oil and a high amount of protein. However, the use of its seed meal as a protein source for animal feed is limited by antinutritional factors, such as sinapine, which reduces nutrient absorption and affects the palatability. Efforts to reduce sinapine levels through conventional breeding have had limited success. Given the challenges of a changing climate and a growing global population, maximising crop utility, particularly the value of seed meal as a byproduct, is increasingly important. Genetic modification has been successfully used to reduce sinapine in rapeseed, but regulatory restrictions limit its commercial adoption in some regions. CRISPR-Cas gene editing, which is gaining broader global acceptance, offers a promising alternative to directly produce transgene-free mutants. In this study, we build on our previous work by generating transgene-free rapeseed mutants using protoplast-based CRISPR RNP gene editing. We successfully targeted the Bna.SCT and Bna.REF1 genes with editing efficiencies of 22%-63%, frequently achieving mutations in all four alleles of the target genes in T2 plants with a single sgRNA. Seed sinapine content was reduced by up to 38% in Bna.SCT mutants and 77% in Bna.REF1 mutants, with no observed effects on plant growth or development. These findings suggest that Bna.REF1 is the most effective target for sinapine reduction in transgene-free mutants among the genes tested in our lab.},
}

Gene Editing/methods
*Brassica napus/genetics/metabolism
CRISPR-Cas Systems/genetics
*Plant Proteins/genetics/metabolism
Plants, Genetically Modified
Protoplasts/metabolism
*Brassica rapa/genetics/metabolism
Mutation
Choline/analogs & derivatives

@article {pmid42101638,
year = {2026},
author = {Uc-Chuc, MA and Jiménez-Ramírez, IA and Guzmán-Marín, EDS and Chan-Pérez, JI and Acosta-Viana, KY},
title = {Beyond the catalysis of Trypanosoma cruzi trans-sialidases: structure, function, post-translational modifications, intrinsically disordered regions and use of CRISPR/Cas9.},
journal = {Archives of microbiology},
volume = {208},
number = {8},
pages = {},
pmid = {42101638},
issn = {1432-072X},
mesh = {*Trypanosoma cruzi/enzymology/genetics ; *Neuraminidase/metabolism/genetics/chemistry ; Protein Processing, Post-Translational ; *Glycoproteins/metabolism/genetics/chemistry ; *CRISPR-Cas Systems ; *Protozoan Proteins/genetics/metabolism/chemistry ; Chagas Disease/parasitology ; Intrinsically Disordered Proteins/metabolism/genetics/chemistry ; Phylogeny ; Humans ; Gene Editing ; },
abstract = {Chagas disease, caused by the protozoan Trypanosoma cruzi, is a global health problem with limited treatment options. The parasite's trans-sialidase (TS) protein family has been widely described as a key component in its life cycle. In this work, we present a comprehensive review of the TS family, including its molecular structure, phylogenetic relationships, and known functions. Based on in silico analyses, we propose that intrinsically disordered regions (IDRs) present in the TS family may play a role in the spatial organization of these proteins. We also hypothesize that these IDRs could contribute to the formation of biomolecular condensates through liquid-liquid phase separation, providing a potential dynamic platform that is not fully explained by conventional structural models. In addition, we discuss recent advances in the application of the CRISPR/Cas9 gene-editing system to T. cruzi TS proteins. The available evidence indicates the multifunctional nature of these proteins, including enzymatic and non-enzymatic isoforms, as well as the presence of conserved motifs associated with host interactions and post-translational modifications. Taken together, this review integrates previous findings and proposes new hypotheses regarding additional functions of the TS family. The need for experimental studies to validate these proposals and clarify their relevance to the parasite's biology is emphasized. This approach could contribute to evaluating the potential of TS as a therapeutic target in Chagas disease.},
}

*Trypanosoma cruzi/enzymology/genetics
*Neuraminidase/metabolism/genetics/chemistry
Protein Processing, Post-Translational
*Glycoproteins/metabolism/genetics/chemistry
*CRISPR-Cas Systems
*Protozoan Proteins/genetics/metabolism/chemistry
Chagas Disease/parasitology
Intrinsically Disordered Proteins/metabolism/genetics/chemistry
Phylogeny
Humans
Gene Editing

@article {pmid42103069,
year = {2026},
author = {Vikal, A and Maurya, R and Kumar, P and Verma, N and Mishra, AK},
title = {Next-generation gene editing strategies in cancer: Integrating CRISPR, PROTACs, and advanced molecular technologies.},
journal = {Life sciences},
volume = {},
number = {},
pages = {124434},
doi = {10.1016/j.lfs.2026.124434},
pmid = {42103069},
issn = {1879-0631},
abstract = {Cancer is a significant therapeutic problem as tumors are heterogeneous, multidrug-resistant, and oncogenic drivers are undruggable. Genome editing and targeted protein degradation are emerging approaches that are transforming precision oncology by allowing genetic and proteomic interventions. Such technologies as CRISPR-based systems and proteolysis-targeting chimeras (PROTACs) are alternative methods of correcting genes and the selective removal of a protein. Their combination provides them with new possibilities regarding long-lasting and specific cancer treatment and discovery of new therapeutic targets. Although this has good news, there are delivery, off-target effects and safety challenges. The continued advancements in nanotechnology, artificial intelligence (AI), and personalized medicine will be likely to improve clinical translation. Generally, the integration of these technologies is an inducing trend in the treatment of cancer in the next generation.},
}

@article {pmid40231503,
year = {2026},
author = {Shams, F and Sharif, E and Abbasi-Kenarsari, H and Hashemi, N and Hosseini, MS and Heidari, N and Noori, E and Amini, AH and Bazrgar, M and Rouhani, M and Teng, Y},
title = {CRISPR/Cas9 Technology for Modifying Immune Checkpoint in CAR-T Cell Therapy for Hematopoietic Malignancies.},
journal = {Current gene therapy},
volume = {26},
number = {2},
pages = {1-29},
pmid = {40231503},
issn = {1875-5631},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Hematologic Neoplasms/therapy/immunology/genetics ; *Immunotherapy, Adoptive/methods ; *Receptors, Chimeric Antigen/genetics/immunology ; Gene Editing ; Immune Checkpoint Inhibitors/therapeutic use ; T-Lymphocytes/immunology ; Programmed Cell Death 1 Receptor/genetics/immunology ; B7-H1 Antigen/genetics/immunology ; CTLA-4 Antigen/genetics/immunology ; },
abstract = {Hematologic malignancies, which arise from dysregulation of hematopoiesis, are a group of cancers originating in cells with diminished capacity to differentiate into mature progeny and accumulating immature cells in blood-forming tissues such as lymph nodes and bone marrow. Immune- targeted therapies, such as Immune Checkpoint Blockade (ICB), chimeric antigen receptor T (CAR-T) cell therapy, and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system, a precise, popular, and versatile genome engineering tool, has opened new avenues for the treatment of malignancies. Targeting immune checkpoints has revolutionized FDA approval in cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), PD-1 (programmed death-1), and PDL1. According to the ICB and CAR techniques, the production of efficient CAR-T cells depends on the successful genetic modification of T cells, making them less susceptible to immune escape and suppression by cancer cells, which results in reduced off-target toxicity. Therefore, CRISPR/Cas9 has revolutionized the immune checkpoint-based approach for CAR-T cell therapy of hematologic malignancy. Continued research and clinical trials will undoubtedly pave the way for further advances in this field, ultimately benefiting patients and improving outcomes.},
}

Humans
*CRISPR-Cas Systems/genetics
*Hematologic Neoplasms/therapy/immunology/genetics
*Immunotherapy, Adoptive/methods
*Receptors, Chimeric Antigen/genetics/immunology
Gene Editing
Immune Checkpoint Inhibitors/therapeutic use
T-Lymphocytes/immunology
Programmed Cell Death 1 Receptor/genetics/immunology
B7-H1 Antigen/genetics/immunology
CTLA-4 Antigen/genetics/immunology

@article {pmid41109795,
year = {2026},
author = {Ghataora, JS and Ellis, T},
title = {Rewiring holobiont systems with synthetic biology.},
journal = {Trends in biotechnology},
volume = {44},
number = {5},
pages = {1312-1329},
doi = {10.1016/j.tibtech.2025.09.017},
pmid = {41109795},
issn = {1879-3096},
mesh = {*Synthetic Biology/methods ; *Microbiota/genetics ; Bacteria/genetics ; Biotechnology ; CRISPR-Cas Systems ; Biosensing Techniques ; },
abstract = {Holobionts are complex communities comprising a host and its diverse microbiota. Their intricate relationships are crucial for biological processes like nutrient uptake, immune function, and environmental adaptation. However, understanding these complex interactions remains challenging. We review how synthetic biology can help address these challenges. We highlight advances in bacterial biosensor design, engineering interkingdom communication, surface display, and clustered regularly interspaced short palindromic repeats (CRISPR) systems to both understand and manipulate holobiont interactions. We also highlight progress in engineering non-model microbiota members and provide rationale for a new field at the intersection of holobiont research and synthetic biology, which we term de novo holobiont design. The integration of synthetic biology with holobiont research promises to deepen our understanding of host-microbiota relationships and open new frontiers in biotechnology.},
}

*Synthetic Biology/methods
*Microbiota/genetics
Bacteria/genetics
Biotechnology
CRISPR-Cas Systems
Biosensing Techniques

@article {pmid41635086,
year = {2026},
author = {Castelli, JMP and Poljakov, K and Jwa, Y and Cunningham, R and Cassidy, ME and Gray, MD and Sanchez Gaytan, JN and Enstrom, MR and Gastelum, G and Wang, Z and Linton, JD and Rongvaux, A and Taylor, JJ and Adair, JE},
title = {In vivo production of an anti-HIV antibody in mice by non-viral gene knockin in primate hematopoietic stem and progenitor cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {5},
pages = {2754-2769},
pmid = {41635086},
issn = {1525-0024},
support = {D43 TW013052/TW/FIC NIH HHS/United States ; P30 AI027757/AI/NIAID NIH HHS/United States ; R01 AI158728/AI/NIAID NIH HHS/United States ; R01 AI167009/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; *Hematopoietic Stem Cells/metabolism/cytology ; Mice ; *HIV Antibodies/immunology/genetics/biosynthesis ; *Gene Knock-In Techniques ; CRISPR-Cas Systems ; Gene Editing/methods ; Humans ; *HIV Infections/immunology/therapy ; Hematopoietic Stem Cell Transplantation ; Primates ; Transgenes ; },
abstract = {Gene editing strategies that do not rely on viral vectors are being explored for their potential to support durable biologics production. While clinical trials have shown that adeno-associated virus encoding broadly neutralizing antibodies can protect against HIV, these interventions often yield limited, short-lived responses. The development of non-viral gene editing approaches in hematopoietic stem and progenitor cells holds promise for long-term antibody production. In this study, we evaluated CRISPR-Cas9 and CRISPR-Cas12a for gene knockin at the immunoglobulin heavy chain locus in non-human primate (NHP) hematopoietic stem and progenitor cells (HSPCs). Delivering the nuclease as a protein alongside a custom DNA template, we optimized editing with Cas12a and demonstrated higher knockin efficiency and fewer non-specific edits than with Cas9. Transplantation of edited NHP HSPCs into MISTRG mice led to engraftment, B cell differentiation, and transgene expression of a reporter transgene or anti-HIV antibody after gp120 antigen immunization with detectable titers in circulation. These findings demonstrate the feasibility of using non-viral knockin in HSPCs as a potential strategy for sustained biologics production in the treatment of chronic diseases. Future work will assess the efficacy of this approach in an NHP model of HIV infection.},
}

Animals
*Hematopoietic Stem Cells/metabolism/cytology
Mice
*HIV Antibodies/immunology/genetics/biosynthesis
*Gene Knock-In Techniques
CRISPR-Cas Systems
Gene Editing/methods
Humans
*HIV Infections/immunology/therapy
Hematopoietic Stem Cell Transplantation
Primates
Transgenes

@article {pmid41856885,
year = {2026},
author = {Ahmar, S and Zhang, R and Pouramini, P and Janeczko, A and Shafique, MS and Rapacz, M and Reis, RS and Zhu, Q and Hensel, G and Pociecha, E},
title = {Precision harvest: path to genetically modified organism-free crops with CRISPR by 2035.},
journal = {Trends in plant science},
volume = {31},
number = {5},
pages = {719-730},
doi = {10.1016/j.tplants.2025.12.014},
pmid = {41856885},
issn = {1878-4372},
mesh = {*Crops, Agricultural/genetics ; *Plants, Genetically Modified/genetics ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Recent advances in clustered regularly interspaced short palindromic repeats (CRISPR) technology enable precise genetic modifications and produce genetically modified organism -free crops that match consumer preferences. By 2035, we will be able to consume CRISPR-edited crops, addressing food security issues and boosting economies for individual countries. This review highlights the progress of genetically modified crops and the regulatory challenges involved in bringing CRISPR-edited crops to market based on product- and process-based approaches across different regions. We also examine public preferences regarding these technologies and the current status of CRISPR-edited crops in terms of market availability. Furthermore, we stress the importance of establishing clear safety standards, effective patent management, and guidance on regulatory pathways for crop approval, as well as exploring future directions for integrating these technologies with artificial intelligence.},
}

*Crops, Agricultural/genetics
*Plants, Genetically Modified/genetics
*Gene Editing
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid41872289,
year = {2026},
author = {You, HJ and Kim, GY and Kang, MJ},
title = {CRISPR/Cas9-mediated targeted knock-in of human erythropoietin at the β-casein locus results in lactogenic hormone-responsive expression in HC11 mammary epithelial cells.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41872289},
issn = {2045-2322},
support = {RS-2024-00343478//National Research Foundation of Korea (NRF) grant awarded by the Korean government (MSIT)/ ; },
mesh = {*Caseins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Humans ; Animals ; *Epithelial Cells/metabolism ; *Gene Knock-In Techniques/methods ; Mice ; *Erythropoietin/genetics/metabolism ; Female ; *Mammary Glands, Animal/cytology/metabolism ; Cell Line ; Genetic Loci ; Gene Expression Regulation ; },
abstract = {Precise genomic integration strategies are essential for achieving stable and regulated transgene expression. In this study, we established a CRISPR/Cas9-mediated targeted knock-in system to integrate the human erythropoietin (hEPO) gene into the endogenous regulatory region of the mouse β-casein locus in HC11 mammary epithelial cells. A donor vector carrying hEPO was designed for homology-directed repair and successfully introduced into the β-casein locus. A heterozygous single-cell knock-in clone was isolated and validated by genomic analysis. Upon stimulation with lactogenic hormones, the integrated hEPO transgene exhibited hormone-responsive expression, resulting in an approximately 20-fold increase in protein levels compared to non-induced conditions. To enable downstream purification and activity assessment, a GST-tagged hEPO construct was employed. The fusion protein was purified using glutathione affinity chromatography, followed by proteolytic cleavage to obtain recombinant hEPO. The purified protein displayed measurable biological activity with a specific activity of 53.4 mIU/μg. These findings demonstrate that targeted integration at the β-casein locus results in lactogenic hormone-responsive expression driven by endogenous regulatory elements in mammary epithelial cells. This approach provides a controlled gene expression platform that may be applicable to hormone-inducible expression systems for therapeutic protein studies.},
}

*Caseins/genetics/metabolism
*CRISPR-Cas Systems/genetics
Humans
Animals
*Epithelial Cells/metabolism
*Gene Knock-In Techniques/methods
Mice
*Erythropoietin/genetics/metabolism
Female
*Mammary Glands, Animal/cytology/metabolism
Cell Line
Genetic Loci
Gene Expression Regulation

@article {pmid41896732,
year = {2026},
author = {Herrera-Cardoso, ED and Tapia-Cervantes, KA and Cepeda-Negrete, J and Gutiérrez-Vargas, S and León-Galván, MF},
title = {Genome sequencing analysis reveals probiotic potential of Lactiplantibacillus plantarum IGMA4EH isolated from the gut of the white maguey worm (Aegiale hesperiaris).},
journal = {BMC genomics},
volume = {27},
number = {1},
pages = {},
pmid = {41896732},
issn = {1471-2164},
support = {CIIC 147/2023//Dirección de Apoyo a la Investigación y al Posgrado of the Universidad de Guanajuato (DAIP UG)/ ; },
abstract = {BACKGROUND: The use of probiotics is increasingly popular for health applications, and Lactiplantibacillus plantarum strains are among the most widely studied for their potential in promoting gut health. In this study, we present the first genomic characterization of L. plantarum IGMA4EH, isolated from the gut of the maguey worm (Aegiale hesperiaris), an edible insect with traditional significance in Mexico.

METHODOLOGY: Whole-genome sequencing was performed on the L. plantarum IGMA4EH strain. Bioinformatics analyses were conducted to identify probiotic-related genes, antimicrobial potential, and safety features. Genes associated with resistance to environmental stressors, adhesion, and immunomodulation were screened, and safety assessment included the detection of antibiotic resistance genes, virulence factors, and plasmids.

RESULTS: The genomic analysis revealed the presence of multiple probiotic-related genes, including those associated with resistance to temperature, acidity, oxidative stress, and bile. Additionally, gene clusters related to bacteriocins, and secondary metabolites were identified, suggesting strong antimicrobial potential. Safety evaluations showed no evidence of acquired antibiotic resistance genes, virulence factors, or plasmids. Furthermore, elements related to genetic stability, such as CRISPR-Cas systems and prophage regions, were also detected.

CONCLUSIONS: The findings highlight the potential of L. plantarum IGMA4EH as a novel probiotic strain with promising applications.

SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-026-12798-5.},
}

@article {pmid42019854,
year = {2026},
author = {Farheen, J and Iqbal, MZ and Mustaq, A and Kong, X},
title = {A synergistic CRISPR-nano-immunotherapeutic system for targeted Bcl-2 silencing in breast tumour.},
journal = {International journal of biological macromolecules},
volume = {363},
number = {},
pages = {152114},
doi = {10.1016/j.ijbiomac.2026.152114},
pmid = {42019854},
issn = {1879-0003},
mesh = {*Breast Neoplasms/genetics/therapy/pathology/immunology ; Humans ; Female ; Animals ; *Proto-Oncogene Proteins c-bcl-2/genetics ; Cell Line, Tumor ; Mice ; *Immunotherapy/methods ; *Gene Silencing ; Apoptosis/genetics ; *CRISPR-Cas Systems ; Nanoparticles/chemistry ; Metal Nanoparticles/chemistry ; Mice, Inbred BALB C ; Gold/chemistry ; },
abstract = {B-cell lymphoma-2 (Bcl-2) protein has an extensive role in anti-cell death regulation and immuno-response modulation. It seizes apoptosis when Bcl-2 interacts and binds to Bax via its BH domain. Breast tumour (BT) was found to have overexpression of Bcl-2 coupled with mitochondrial membrane deprivation and stumpy immune response. Here, we developed a phyto-nanomedicine (HRP-MET)-based immunotherapeutic system integrating gold di‑manganese tri-oxide nanoparticles (GMNPs) with a BT-directed gene knockdown strategy. The nanoformulation is designed to accumulate in tumour tissue through enhanced permeability and retention (EPR)-mediated passive targeting, followed by activation within the tumour microenvironment. Initially, the expression of the Bcl-2 gene was selectively silenced in BT cells using a one-step advanced GenCRISPR™ Ultra NLS-Cas9 ribonucleoprotein (RNP) system with four designed effective sgRNAs (gene-CRISPR). Subsequently, transfected BT cells were treated with GMNP@HRP-MET phyto-nanomedicine in vitro and in vivo. As a result, this combinatorial strategy significantly induced tumour cell death via a mitochondria-mediated apoptotic signalling cascade (mitochondria → Bh3-only → Bax/Bak → Cycs → Apoptosome → Casp-9/Casp-3 → apoptosis) in various BT cells. Notably, Bcl-2 gene expression was prominently blocked in BALB/c female mice, accompanied by enhanced T-cell activation and sustained immune responses at both proteomic and transcriptomic levels. Furthermore, the gene-CRISPR and phyto-nanomedicine combination significantly inhibited tumour growth, migration, and distant organ metastasis in xenograft and syngeneic mice models. Collectively, this study demonstrates a practical and durable therapeutic strategy based on gene-CRISPR-enhanced apoptosis integrated with microenvironment-responsive phyto-nanomedicine for BT treatment.},
}

*Breast Neoplasms/genetics/therapy/pathology/immunology
Humans
Female
Animals
*Proto-Oncogene Proteins c-bcl-2/genetics
Cell Line, Tumor
Mice
*Immunotherapy/methods
*Gene Silencing
Apoptosis/genetics
*CRISPR-Cas Systems
Nanoparticles/chemistry
Metal Nanoparticles/chemistry
Mice, Inbred BALB C
Gold/chemistry

@article {pmid42094414,
year = {2026},
author = {Yoon, PH and Loi, K and Zhang, Z and Docter, TA and Lopez, SC and Langeberg, CJ and Ur-Rehman, MM and Vohra, K and Zhou, Z and Shi, H and Boger, R and Wang, PY and Adler, BA and Brohawn, SG and Doudna, JA},
title = {A Noncontiguous Code for RNA-Guided DNA Recognition Preceded CRISPR.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.64898/2026.04.26.720920},
pmid = {42094414},
issn = {2692-8205},
abstract = {CRISPR-Cas systems use RNA-guided proteins for adaptive immunity through a mechanism whose origin is unknown. Here we report the discovery of Viral Interference Programmable Repeat (VIPR) systems consisting of a Vipr protein more ancient than CRISPR-Cas and vrRNAs comprising alternating GGY/NN motifs. Unlike canonical guide RNAs that base pair with nucleic acid targets using an uninterrupted sequence, vrRNAs recognize double-stranded DNA through a noncontiguous code in which the variable NNs of each repeat collectively specify a target that itself contains a gapped recognition sequence. Analysis of natural vrRNA targets suggests VIPR acts against competing phages. We demonstrate programmable phage defense by redirecting the complex for transcriptional repression. These results suggest that the roots of adaptive immunity lie in ancient warfare between viruses, and reveal a new logic for programmable genetic control.},
}

@article {pmid42094579,
year = {2026},
author = {Malaluan, RPP and Dy, RLV},
title = {Acquisition of novel arrays via horizontal gene transfer rewire CRISPR-mediated defense in Pseudomonas aeruginosa.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.64898/2026.04.27.721218},
pmid = {42094579},
issn = {2692-8205},
abstract = {CRISPR-Cas systems form the adaptive immunity of prokaryotes, conferring sequence-specific protection against genetic parasites. Here, we functionally characterized the CRISPR-Cas system of Pseudomonas aeruginosa ATCC 10145 (PA10145), which led us to discover the existence of an isolated CRISPR array, unique to this system. PA10145 possesses a type I-F CRISPR-Cas composed of a cas operon flanked by two divergently organized CRISPRs. The isolated CRISPR array, CRISPR3, is located ∼1.3 million bp away from the cas loci. The cas and three CRISPR arrays are active. Plasmids with an engineered protospacer matching any of the three arrays were targeted and stimulated hyperactive adaptation in all CRISPR arrays of PA10145 if the plasmids possessed an intact protospacer adjacent motif (PAM), whereas minimal to no adaptation was observed when PAM was mutated. Spacer acquisition via interference-driven adaptation proceeds through strand-biased priming in PA10145. Interestingly, the isolated CRISPR3 and the cas -adjacent CRISPR2 have nearly identical leader sequences with only 3 bp mismatches. From a survey of CRISPR loci in 1,198 P. aeruginosa genomes, isolated arrays only occur as type I-F with similarly matching leaders to CRISPR2. Highly-transmissible mobile genetic elements (MGEs) associate with CRISPR2 and CRISPR3, suggesting that isolated arrays might have originated from recombination events involving CRISPR2 as facilitated by these MGEs. Tracing evolutionary trajectories of the isolated CRISPR3 relative to cas -adjacent arrays revealed that CRISPR3 is laterally transferred across P. aeruginosa genomes. Taken together, these results implicate the role of horizontally-acquired isolated arrays in CRISPR-mediated pan-immunity as gateways to mobilize genetic memories.},
}

@article {pmid42096148,
year = {2026},
author = {Zhu, D and Wang, S and Sun, X and Britton, RA},
title = {CRISPR-AsCas12a and dAsCas12a-Mediated Gene Knockout and Knockdown in Clostridioides difficile.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3046},
number = {},
pages = {47-55},
pmid = {42096148},
issn = {1940-6029},
mesh = {*Clostridioides difficile/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Gene Knockout Techniques/methods ; *Gene Knockdown Techniques/methods ; Bacterial Proteins/genetics ; CRISPR-Associated Proteins/genetics ; },
abstract = {Clostridioides difficile (C. difficile) is a leading cause of antibiotic-associated diarrhea and severe colitis, yet its genetic manipulation has long been constrained by low DNA transfer efficiency and limited recombination systems. Recent advances in CRISPR-based technologies have revolutionized the genetic toolkit for this pathogen, enabling precise genome editing and transcriptional regulation. Among CRISPR nucleases, Cas12a offers distinct advantages over Cas9 for bacterial applications, including a smaller size, T-rich PAM recognition, single-crRNA requirement, and reduced toxicity, which enhances conjugation efficiency in genetically recalcitrant organisms. AsCas12a-based platforms have enabled large fragment deletions, multiplex editing, and rapid generation of marker-free mutants in C. difficile. Complementing these nuclease-active systems, nuclease-deactivated variants (dCas9 or dAsCas12a) support CRISPR interference (CRISPRi)-a reversible, tunable approach for transcriptional repression without altering genomic sequences. Compared to traditional mutagenesis, CRISPRi greatly accelerates functional genomics by enabling high-throughput screening and drug target discovery. Together, our lab has independently developed CRISPR-AsCas12a-mediated genome editing and dAsCas12a-based CRISPRi tools, providing complementary strategies to overcome longstanding genetic barriers in C. difficile. These tools open new avenues for system-level interrogation of virulence, antibiotic resistance, and host-pathogen interactions.},
}

*Clostridioides difficile/genetics
*CRISPR-Cas Systems/genetics
Gene Editing/methods
*Gene Knockout Techniques/methods
*Gene Knockdown Techniques/methods
Bacterial Proteins/genetics
CRISPR-Associated Proteins/genetics

@article {pmid42096830,
year = {2026},
author = {Yin, Y and Wang, M and Sun, Y and Zhao, P and Ye, J},
title = {Sugarcane viral diseases: Epidemiology, detection, and advanced breeding methods for resistance.},
journal = {Virology},
volume = {621},
number = {},
pages = {110940},
doi = {10.1016/j.virol.2026.110940},
pmid = {42096830},
issn = {1096-0341},
abstract = {Sugarcane viruses threaten yield and sucrose content, imposing substantial global economic losses. This review provides current knowledge on the major sugarcane pathogens, with a focus on virus biology, transmission, and virus-host interactions, and uses this foundation to frame practical control strategies. We first describe the epidemiology and economic impact of sugarcane viral diseases to establish the urgency of robust surveillance and management. Building on these insights, we then evaluate detection and surveillance technologies, ranging from traditional immunoassays and nucleic acid hybridization to molecular diagnostics, isothermal amplification, and CRISPR-based approaches, and discuss how high-throughput sequencing accelerates virus discovery. Next, we assess management strategies that include virus-free planting material, vector control, sanitation, and modern breeding technologies, like RNA interference (RNAi), CRISPR/Cas genome editing, marker-assisted selection (MAS), and genome wide association studies (GWAS) within genomic selection (GS) frameworks. We also consider supplementary strategies such as plant-derived antivirals and environmental management, and discuss how they complement core approaches. Crucially, we identify key knowledge gaps in sugarcane virus-host interactions and resistance breeding, and propose data-driven, multi-omics and AI-assisted approaches to precision breeding and integrated disease management. The review concludes with a practical roadmap for advancing sugarcane virus control and promoting sustainable production.},
}

@article {pmid42097051,
year = {2026},
author = {Liao, J and Rima, J and Sharma, A and Tsade, J and Jiang, F},
title = {Machine learning-enabled smartphone CRISPR-Cas12a lateral flow platform for sensitive detection of circulating HPV DNA.},
journal = {Biosensors & bioelectronics},
volume = {307},
number = {},
pages = {118765},
doi = {10.1016/j.bios.2026.118765},
pmid = {42097051},
issn = {1873-4235},
abstract = {Persistent infection with high-risk human papillomavirus (HPV) is a major cause of cervical cancer, and improved point-of-care (POC) detection is critical for early intervention. Although PCR-based assays are highly sensitive, their reliance on centralized laboratory infrastructure limits accessibility in decentralized settings. CRISPR-Cas diagnostics combined with lateral flow assays (LFA) offer a rapid alternative; however, visual interpretation of faint test bands remains subjective and variable. Here, we developed a smartphone-based CRISPR-Cas12a LFA platform integrated with an interpretable machine learning (ML) framework for quantitative detection of circulating HPV DNA in plasma. Standardized image acquisition was implemented using a light-controlled enclosure, and radiomics-inspired features were analyzed using a multivariable logistic regression model. The system was trained on 150 plasma samples and validated in an independent cohort of 60 samples. The optimized model achieved 96.7% sensitivity and 100% specificity, outperforming visual interpretation, particularly for low-signal samples. Performance remained stable across different smartphone models, lighting conditions, and operators, with rapid on-device inference enabling consistent and reliable operation. This integrated CRISPR-LFA platform demonstrates accurate and reproducible detection of circulating HPV DNA and supports feasibility for POC applications, pending further validation in broader clinical settings.},
}

@article {pmid42097949,
year = {2026},
author = {Fuller, MGA and Foley, M and Barrangou, R},
title = {CRISPR-based technologies for large DNA insertions.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2026.04.004},
pmid = {42097949},
issn = {1879-3096},
abstract = {While the advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based technologies has democratized the genesis of precise mutations, there is a need for more sophisticated tools to enable large-scale DNA manipulations, advancing genome editing across medicine, biotechnology, and agriculture. The success of Cas9 and Cas12 has hinged on the generation of precise DNA nicks and double-stranded breaks (DSBs), enabling local sequence mutagenesis, albeit of a limited size range. Emerging effectors combining Cas with other enzymatic functions, such as CRISPR-associated transposons and site-specific recombinases, enable larger integrations. Sophisticated combinations such as programmable addition via site-specific targeting element (PASTE), prime-editing-assisted site-specific integrase gene editing (PASSIGE), and prime-editing-mediated recombination of opportune target (PrimeRoot) expand payload options and DSB-free editing modalities, with translational potential for next-generation crop breeding in sustainable agriculture and the development of gene and cell therapies in personalized medicine.},
}

@article {pmid42098118,
year = {2026},
author = {Gierisch, ME and Barchi, E and Marogna, M and Wallnöfer, MH and Ankarcrona, M and Naia, L and Salomons, FA and Dantuma, NP},
title = {Mitochondria serve as a holdout compartment for aggregation-prone proteins hindering efficient degradation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42098118},
issn = {2041-1723},
support = {2021-02562//Vetenskapsrådet (Swedish Research Council)/ ; 211653Pj//Cancerfonden (Swedish Cancer Society)/ ; FO2022-0271//Hjärnfonden (Swedish Brain Foundation)/ ; FO2023-0376//Hjärnfonden (Swedish Brain Foundation)/ ; KID grant//Karolinska Institutet (Karolinska Institute)/ ; GI-1329/1-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {*Mitochondria/metabolism ; Humans ; Huntingtin Protein/metabolism/genetics ; Proteolysis ; alpha-Synuclein/metabolism/genetics ; Proteasome Endopeptidase Complex/metabolism ; HEK293 Cells ; *Protein Aggregates ; Neurodegenerative Diseases/metabolism/genetics ; CRISPR-Cas Systems ; Protein Aggregation, Pathological/metabolism ; },
abstract = {The accumulation of protein aggregates has been causatively linked to the pathogenesis of neurodegenerative diseases. Here, we conduct a genome-wide CRISPR-Cas9 screen to identify cellular factors that regulate the degradation of an aggregation-prone reporter. Genes encoding proteins involved in mitochondrial homeostasis, including the translation factor eIF5A, are enriched among suppressors of the degradation of the reporter. Genetic or chemical inhibition of eIF5A leads to dissociation of the aggregation-prone substrate from mitochondria, which is accompanied by enhanced ubiquitin-dependent proteasomal degradation. The presence of an aggregation-prone, amphipathic helix that localizes the reporter to mitochondria is crucial for the stimulatory effect of eIF5A inhibition on proteasomal degradation. Additionally, inhibition of eIF5A also enhances degradation of mutant huntingtin and α-synuclein, two disease-associated proteins that contain amphipathic helices and mislocalize to mitochondria. We propose that mitochondria serve as a holdout compartment for aggregation-prone proteins. Therefore, preventing mitochondrial localization of aggregation-prone proteins may offer a viable therapeutic strategy for reducing disease-associated proteins in neurodegenerative disorders.},
}

*Mitochondria/metabolism
Humans
Huntingtin Protein/metabolism/genetics
Proteolysis
alpha-Synuclein/metabolism/genetics
Proteasome Endopeptidase Complex/metabolism
HEK293 Cells
*Protein Aggregates
Neurodegenerative Diseases/metabolism/genetics
CRISPR-Cas Systems
Protein Aggregation, Pathological/metabolism