@article {pmid41806414,
year = {2026},
author = {Kim, H and Kim, D and Han, H and Lee, C and Roh, YH and Han, TS and Lim, EK and Park, J and Ahn, JK and Kang, T and Jung, J and Lee, CY},
title = {On-site microRNA detection with 'off-the-shelf' glucose meter empowered by chimeric probe connecting CRISPR/Cas13a activation to kinases-driven glucose phosphorylation.},
journal = {Biosensors & bioelectronics},
volume = {304},
number = {},
pages = {118568},
doi = {10.1016/j.bios.2026.118568},
pmid = {41806414},
issn = {1873-4235},
abstract = {MicroRNAs (miRNAs) are promising biomarkers for cancer diagnosis due to their stability in body fluids and disease-specific expression profiles. However, current detection methods suffer from limitations including cumbersome workflows, heavy instrumentation for signal readout, or vulnerability in minimizing instrumentation. To address these challenges, we describe a novel point-of-care miRNA detection platform executable with "off-the-shelf", personal glucose meter (PGM), termed 'KEY-FACT (Kinases Ensemble-driven glucose phosphorYlation upon Fuel-Aided CRISPR acTivation)'. Upon recognition of target miRNA, a fuel-assisted toehold-mediated strand displacement reactions liberate guide RNAs (gRNAs) to activate Cas13a to cleave a chimeric reporter probe, producing 2',3'-cyclic adenosine monophosphates (cAMP). Subsequent dephosphorylation and kinases ensemble-mediated phosphorylation/dephosphorylation cycles lead cAMP to consume a large amount of glucose. A user can immediately measure resulting glucose level change with PGM on the spot. This strategy allows sensitive, prompt detection of miR-135b, a gastric cancer (GC) biomarker, with a limit of detection (LOD) of 1.4 pM within 2 h. KEY-FACT is specific to the target miRNA and is applicable to body fluids such as human serum with dilution (95.2% < recovery rates <104.3%, coefficients of variation ≤13%). Owing to its simple probe design, KEY-FACT was readily expanded to detect another GC biomarker, miR-21, with comparable sensitivity (LOD = 1.5 pM). The proposed platform fulfills minimal instrumentation and thus enables cost-effective, field-deployable analysis, paving the way for practical, on-demand miRNA diagnostics.},
}

@article {pmid41806413,
year = {2026},
author = {Jeong, Y and Lee, J and Choi, S and Shin, D and Jang, S and Son, SU and Kang, T and Jung, J and Hwang, J and Lim, EK},
title = {On-site detection of airborne foodborne pathogens using a field-deployable recombinase polymerase amplification and CRISPR/Cas12a cleavage activity assay.},
journal = {Biosensors & bioelectronics},
volume = {304},
number = {},
pages = {118571},
doi = {10.1016/j.bios.2026.118571},
pmid = {41806413},
issn = {1873-4235},
abstract = {With the global increase in single-person households, the demand for meal kits is increasing, leading to the development of large-scale food production systems and complex supply chains. However, under the influence of global warming, these systems can be susceptible to food contamination, particularly by airborne foodborne bacteria. Conventional methods for detecting airborne bacteria involve complex, time-consuming, and labor-intensive processes, which limit their applicability for field use and rapid food hygiene surveillance. In the present study, we developed a field-deployable diagnostic platform by combining recombinase polymerase amplification with CRISPR/Cas12a cleaVage Activity (RCCVA assay) for the rapid and sensitive identification of airborne foodborne bacteria. Airborne bacteria were collected using a self-developed electrostatic air sampler and analyzed using a portable isothermal amplification device. The RCCVA assay was designed to detect four major foodborne pathogens: Staphylococcus aureus, Salmonella enteritidis, Listeria monocytogenes, and Bacillus cereus. The limit of detection was measured as 274.9, 4.5, 9.5, and 28.5 culture-forming units (CFU)/mL, respectively, within 45 min. This platform enables early on-site detection of airborne pathogens within approximately 1 h (for the analytical phase) and shows potential for real-time monitoring in food processing environments, thereby contributing to improved public health and food safety.},
}

@article {pmid41805869,
year = {2026},
author = {Moghe, AS and Nandi, SS and Bhonde, RR and Kamyab, SS and Sawant, SA and Karandikar, MN},
title = {Engineering of cell line assembled enteric organoid for enterovirus infection.},
journal = {Archives of virology},
volume = {171},
number = {4},
pages = {},
pmid = {41805869},
issn = {1432-8798},
support = {5/3/8/57/2020-ITR Date:11.03.2021//Indian Council of Medical Research/ ; },
mesh = {Humans ; *Organoids/virology ; Virus Replication ; Cell Line ; *Enterovirus Infections/virology ; *Enterovirus A, Human/physiology ; Epithelial Cells/virology ; *Enterovirus/physiology ; CRISPR-Cas Systems ; Coculture Techniques ; },
abstract = {The non-polio-enteroviruses are ubiquitous pathogens infecting over a billion people in the world. An alarming number of enterovirus-associated acute flaccid paralysis, encephalitis, hand, foot, and mouth disease, conjunctivitis and diarrhoea cases are reported worldwide. Despite their clinical significance, vaccine development has been hindered due to lack of suitable in vitro models for preclinical investigations. The present study was undertaken to develop a cell line assembled organotypic model of human intestine for replication of enteroviruses. An enterovirus specific PSGL1 receptor was introduced in intestinal epithelial HCT-8 cell line employing CRISPR/cas9 gene editing. It was co-cultured with human colon (CCD-18) and endothelial (HUVEC) cell lines with peripheral blood mononuclear cells in hanging drops and rotating wall vessel bioreactor to yield three-dimensional organoids. Histological analysis of the organoids showed presence of columnar epithelium cells with prominent intracytoplasmic mucin, hyperchromatic nuclei and presence of CK, CK20, MUC 2 and Villin markers characteristic of epithelial cells. Infection with Enterovirus A71 (EV-A71) demonstrated significantly higher viral titre in organoids compared to individual cell lines. Collectively, these findings determine, for the first time, a cell line-derived enteric organoid model that supports robust enterovirus replication, offering a cost-effective and physiologically relevant system for virology research and preclinical applications.},
}

Humans
*Organoids/virology
Virus Replication
Cell Line
*Enterovirus Infections/virology
*Enterovirus A, Human/physiology
Epithelial Cells/virology
*Enterovirus/physiology
CRISPR-Cas Systems
Coculture Techniques

@article {pmid41805349,
year = {2026},
author = {Han, F and Xu, Y and Wang, W and Li, Z and Zhang, Z and Du, R and Xu, Q},
title = {Methylobacterium as a Dual-Function Platform: Advances in C1-Based Biomanufacturing and Plant-Associated Applications.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c08026},
pmid = {41805349},
issn = {1520-5118},
abstract = {One-carbon (C1) substrates are promising feedstocks for microbial bioproduction. Methylobacterium, known for its exceptional C1 utilization capacity, has emerged as a model microbial chassis for sustainable biomanufacturing. In this review, we first outline the C1 assimilation pathways in Methylobacterium and underscore its potential for producing valuable native metabolites. Furthermore, we then survey the genetic tools available for engineering this genus, including plasmid-based methods, transposon mutagenesis, homologous recombination, and CRISPR/Cas systems. Notably, recent advances in metabolic engineering have significantly expanded its biosynthetic scope, enabling the biosynthesis of diverse non-native compounds. Beyond its biomanufacturing potential, Methylobacterium also serves as a versatile plant growth-promoting bacterium, enhancing plant health and productivity through hormone synthesis, nutrient mobilization, stress mitigation, and induced systemic resistance. Collectively, this work highlights the dual potential of Methylobacterium as a sustainable microbial cell factory for biomanufacturing and a beneficial bioinoculant for agriculture.},
}

@article {pmid41805130,
year = {2026},
author = {Yu, W and Yuan, L and Zhou, W and He, L and Huang, X and Yu, J and Deng, J and Zhang, T and Hu, Y and Zhang, Y and Chen, S},
title = {Orn-mediated c-di-GMP regulates the CRISPR-Cas system to confer stress response in Mycobacterium tuberculosis.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41805130},
issn = {1362-4962},
support = {2021YFA1300901//National Key R&D Program of China/ ; 2022YFA1303500//National Key R&D Program of China/ ; GZNL2024A01024//Guangzhou National Laboratory/ ; //National Key Research and Development Program of China/ ; },
mesh = {*Mycobacterium tuberculosis/genetics/metabolism/drug effects ; *CRISPR-Cas Systems/genetics ; *Cyclic GMP/analogs & derivatives/metabolism ; Bacterial Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; Oxidative Stress/genetics ; *Stress, Physiological/genetics ; Promoter Regions, Genetic ; CRISPR-Associated Proteins/genetics/metabolism ; },
abstract = {Mycobacterium tuberculosis (Mtb) possesses a type III-A CRISPR-Cas system and has anti-plasmid immune activity. However, whether this system exerts other additional functions remains to be characterized. Here, we investigated the in vivo roles of the Mtb CRISPR-Cas system. We show that this system is transcriptionally dependent and exhibits limited ability to counteract exogenous nucleic acids, primarily through the Csm6 protein rather than the Cas10 HD domain. We further demonstrate that this system plays a role in mitigating oxidative stress and antibiotic treatment, a function mainly mediated by the Cas10 HD domain. Importantly, through transposon library screening, we identified oligoribonuclease (Orn) as a regulatory protein of the Mtb CRISPR-Cas system. Deletion of the orn gene resulted in elevated c-di-GMP levels. A subsequent biotin-labeled c-di-GMP pull-down assay identified the transcriptional regulator Rv3058. Knockdown of rv3058 significantly increased cas6 promoter activity, and its transcriptional repressor function was directly modulated by c-di-GMP. This regulatory pathway enhances stress defense by activating multiple protective pathways, including DNA repair, cell envelope maintenance, and iron homeostasis regulation. Together, we conclude that the regulation of the CRISPR-Cas system by Orn-mediated c-di-GMP contributes to oxidative and antibiotic stress responses in Mtb.},
}

*Mycobacterium tuberculosis/genetics/metabolism/drug effects
*CRISPR-Cas Systems/genetics
*Cyclic GMP/analogs & derivatives/metabolism
Bacterial Proteins/genetics/metabolism
Gene Expression Regulation, Bacterial
Oxidative Stress/genetics
*Stress, Physiological/genetics
Promoter Regions, Genetic
CRISPR-Associated Proteins/genetics/metabolism

@article {pmid41804827,
year = {2026},
author = {Parada, F and Cabedo-Díaz, P and Cerda, A and Osorio-Navarro, C and Toledo, JA and Villalobos-González, L and Handford, M and Pimentel, P},
title = {CRISPR/dCas9-Mediated BRL3 Activation Enhances Growth and Metabolic Resilience Under Osmotic Stress in Nicotiana tabacum.},
journal = {Physiologia plantarum},
volume = {178},
number = {2},
pages = {e70816},
doi = {10.1111/ppl.70816},
pmid = {41804827},
issn = {1399-3054},
support = {3240290//ANID-FONDECYT Postdoctoral Project/ ; 3210631//ANID-FONDECYT Postdoctoral Project/ ; 1231417//ANID-FONDECYT Regular Project/ ; RF23F0002//ANID Fortalecimiento de Centros Regionales Project/ ; NCN2024_047//ANID-Millennium Science Initiative Program/ ; },
mesh = {*Nicotiana/genetics/growth & development/metabolism/physiology ; Osmotic Pressure/physiology ; Gene Expression Regulation, Plant ; *Plant Proteins/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified ; Promoter Regions, Genetic/genetics ; Brassinosteroids/metabolism ; Plant Leaves/genetics ; },
abstract = {Brassinosteroids (BRs) are crucial plant hormones that influence growth and stress adaptation. However, the specific function of the BR receptor BRL3 under osmotic stress remains largely unexplored outside Arabidopsis thaliana. In this study, we used a CRISPR/dCas9-based transcriptional activation (CRISPRa) system to upregulate the Nicotiana tabacum BRASSINOSTEROID INSENSITIVE-LIKE 3 receptor (NtBRL3) and assessed its impact on osmotic stress tolerance. Synthetic activation vectors were constructed using Loop Assembly, featuring dCas9-6TAL-VP128 modules driven by either a constitutive (CaMV35S) or ABA-inducible (SlAREB) promoter, paired with dual sgRNAs targeting the NtBRL3 promoter. Transient Agrobacterium-mediated transformation followed by PEG treatment was used to impose osmotic stress. RT-qPCR confirmed a 3- to 4-fold activation of NtBRL3 transcripts in CRISPRa-infiltrated leaves. The stress-inducible SlAREB promoter produced the strongest improvements, yielding nearly four-fold higher leaf biomass and a five-fold increase in root biomass relative to PEG-stressed controls. Both constructs reduced malondialdehyde (MDA) accumulation, indicating diminished oxidative damage, and modulated osmoprotectant balance, including reduced root proline and increased total soluble solids, particularly under SlAREB-driven activation. Histological segmentation revealed promoter-dependent anatomical remodeling, with NtBRL3-activated plants exhibiting a higher frequency of enlarged leaf cells and expanded tissue domains, consistent with brassinosteroid-mediated structural plasticity. Collectively, these findings demonstrate that CRISPR/dCas9-mediated transcriptional activation of NtBRL3 enhances osmotic stress resilience in tobacco through coordinated biomass recovery, oxidative stress mitigation, osmolyte homeostasis, and tissue remodeling. This transient, non-integrative CRISPRa approach provides a robust synthetic biology framework for dissecting BR signaling and engineering stress-tolerant crops.},
}

*Nicotiana/genetics/growth & development/metabolism/physiology
Osmotic Pressure/physiology
Gene Expression Regulation, Plant
*Plant Proteins/metabolism/genetics
*CRISPR-Cas Systems/genetics
Plants, Genetically Modified
Promoter Regions, Genetic/genetics
Brassinosteroids/metabolism
Plant Leaves/genetics

@article {pmid41803497,
year = {2026},
author = {Perez Taboada, V and Wu, Y and Cassidy, R and Medvedev, KE and Loeff, L and Nemudraia, A and Nemudryi, A},
title = {Bacterial Schlafen proteins mediate phage defence.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {41803497},
issn = {2058-5276},
support = {R00AI171893//U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; 1T32GM156737-01//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
abstract = {Human Schlafen proteins restrict viral replication by cleaving tRNA, thereby suppressing protein synthesis. Although the ribonuclease domain of Schlafen proteins is conserved across all domains of life, its function in prokaryotes has remained unclear. Here we demonstrate that prokaryotic Schlafen nucleases are widespread antiviral effectors that protect bacteria from bacteriophages and are fused to a diverse array of phage-sensing domains. We expressed seven Enterobacterales Schlafen systems in Escherichia coli, identifying two that confer defence against coliphages. We focused on a system where Schlafen nuclease is fused to a previously unknown immunoglobulin-like sensor domain and demonstrated that it recognizes tail assembly chaperones of T5-like phages. Upon activation, the Schlafen nuclease cleaves both E. coli and phage-encoded tRNAs and restricts T5 phage by reducing its burst size. Our findings redefine Schlafens as an ancient, mechanistically conserved family of immune effectors, revealing the deep evolutionary origin of tRNA-targeting antiviral immunity in humans.},
}

@article {pmid41803127,
year = {2026},
author = {Zhou, R and Liu, Y and Zhang, Q and Yin, Z and Tong, J and Zhang, C and Zhang, L and Li, X and Zhao, Y and Zhang, S and Liu, Z and Chen, W and Ji, N and Zhang, H and Li, Z and Yin, H and Zuo, S and Wei, Y},
title = {Structural and mechanistic insights into the dual-nuclease defense protein Upx as an anti-phage system.},
journal = {Nature communications},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41467-026-70435-x},
pmid = {41803127},
issn = {2041-1723},
support = {2022YFC3400400//National Science Foundation of China | Key Programme/ ; 23ZYCGSY00750//Tianjin Science and Technology Committee (Tianjin Municipal Science and Technology Commission)/ ; },
abstract = {Nucleic acid degradation is a common strategy for prokaryotic anti-phage systems, as exemplified by the CRISPR-Cas system. The PD-(D/E)-XK nucleases constitute a widely distributed family in these defenses. Notably, most members exhibit a single nuclease domain, while variants containing dual nuclease domains within a single polypeptide remain underexplored, and their molecular mechanisms largely obscure. Here, we biochemically and functionally study a single-protein system containing an uncharacterized PD-(D/E)-XK defense protein (Upx). As revealed by single-particle electron cryo-microscopy (cryo-EM) structure, the C-terminal domain (CTD) harboring the conserved PD-(D/E)XK catalytic core is buttressed by the N-terminal domain (NTD) and the middle domain (MD). Functional assays demonstrate that the nucleic acid binding capability of the CTD is enhanced by the MD. The NTD also displays a noncanonical, basal exonuclease activity that is auto-inhibited by MD. IP-MS experiments identify Upx-interacting phage proteins, and substrate profiling defines its physiological preferences, collectively pointing to its potential physiological targets. Notably, the phage protein gp16 was found to relieve MD-mediated inhibition of the NTD, suggesting a virus-triggered mechanism for activating Upx's dual nuclease activity. Together, these findings establish Upx as a single-protein dual-nuclease anti-phage system, expanding our understanding of bacterial immunity and informing antiviral strategy development.},
}

@article {pmid41802999,
year = {2026},
author = {Hao, M and Zhou, M and Pan, F and Liu, T and Li, Y and Su, N and Ashfaq, A and Song, M and Wang, H and Wang, W and Liu, J and Li, C and Fu, L and He, P and Hu, Q and Mei, D and Cheng, H},
title = {Efficient CRISPR/Cas-SF01 genome editing tools with high editing efficiency in allotetraploid oilseed rape.},
journal = {Journal of integrative plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1111/jipb.70221},
pmid = {41802999},
issn = {1744-7909},
support = {2025BEA003//the Major Program (JD) of Hubei Province/ ; CAAS-CSNCB-202303//Innovation Program of Chinese Academy of Agricultural Sciences/ ; 2025AFB468//Hubei Provincial Natural Science Foundation of China/ ; CARS-12//Earmarked Fund for China Agriculture Research System/ ; },
abstract = {CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 has been widely utilized for plant genome editing, but the protospacer adjacent motif (PAM) requirement limits its editing scope. CRISPR/Cas12i3 belongs to the type-VI Cas system that has gained extensive attention due to its smaller size and less restricted canonical TTN PAM sequence. In this study, we explored the newly developed Cas-SF01 system (Cas12i3 variant) for genome editing in oilseed rape. We established an efficient protoplast transformation system in oilseed rape to compare editing efficiency between Cas-SF01 and Cas9. Cas-SF01 shows cleavage activities at the tested 5'-TTN-3' PAM sites with editing outcomes sharing considerable similarities with the CRISPR-Cas9 system in protoplast. Cas-SF01 also induces high efficiency mutagenesis for multiple target sites in stable transformed oilseed rape lines, generating mutants with multilocular silique and male sterile phenotypes. Furthermore, Cas-SF01-derived cytosine base editors (CBEs) were developed to produce targeted C-to-T base edits. Compared to SpCas9, Cas-SF01 has an expanded PAM range and effectively recognizes TTN PAMs, which has substantially broadened the scope of editable sites within the rapeseed genome. No mutations were identified at the putative off-target sites among the edited plants. This study developed a robust, first-of-its-kind Cas12 system in the allotetraploid Brassica napus, expanding the scope of editing and enriching genome-editing toolkits for biological research and genetic improvement.},
}

@article {pmid41747377,
year = {2026},
author = {Pan, MX and Lv, MM and Nie, YG and Su, M and Zha, CJ and Mei, RY and Ying, ZM},
title = {Ultrasensitive miRNA detection via magnetic bead-confined catalytic hairpin assembly enabling transcription-driven crRNA assembly and CRISPR/Cas12a activation.},
journal = {Biosensors & bioelectronics},
volume = {302},
number = {},
pages = {118559},
doi = {10.1016/j.bios.2026.118559},
pmid = {41747377},
issn = {1873-4235},
mesh = {*MicroRNAs/genetics/isolation & purification/analysis ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *CRISPR-Associated Proteins/genetics/chemistry ; DNA/chemistry/genetics ; *Endodeoxyribonucleases/chemistry/genetics ; Limit of Detection ; *Bacterial Proteins/chemistry/genetics ; Nucleic Acid Hybridization ; Transcription, Genetic ; },
abstract = {The integration of CRISPR/Cas12a with catalytic hairpin assembly (CHA), a strategy that predominantly relies on CHA to generate dsDNA activators for direct Cas12a activation, has emerged as a powerful tool in molecular diagnostics. However, two major challenges remain: the strict protospacer adjacent motif (PAM) dependence of the dsDNA and background leakage from hairpin hybridization. Herein, we report a bead-confined platform that transcription mediates crRNA reassembly and template activation of Cas12a for ultrasensitive miRNA detection. The target-triggered CHA assembly dynamically constructed a T7 transcription template from three initially locked hairpins (H1, H2, and H3), which not only transcribed scaffold RNA but also hybridized with its own product to form a DNA/RNA complex that activates Cas12a. The integration of the split T7 promoter with CHA effectively suppressed background suppression and enhanced detection sensitivity. Additionally, the magnetic beads increase local concentration and reaction kinetics, collectively contributing to a substantially enhanced detection sensitivity. Moreover, a crRNA assembly strategy designed for transcription-powered Cas12a not only circumvents the conventional PAM-dependent dsDNA activation pathway of Cas12a but also enables self-supplied crRNA without requiring additional activators. We demonstrated that the biosensor exhibits exceptional sensitivity for miRNA-21 detection, achieving a limit of 65.3 aM. Furthermore, the practicality of this method was preliminarily confirmed through accurately quantifying target levels in cell lines and human serum. Our method presents a viable solution with transformative potential, designed to address complex challenges in contemporary diagnostic applications.},
}

*MicroRNAs/genetics/isolation & purification/analysis
*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
Humans
*CRISPR-Associated Proteins/genetics/chemistry
DNA/chemistry/genetics
*Endodeoxyribonucleases/chemistry/genetics
Limit of Detection
*Bacterial Proteins/chemistry/genetics
Nucleic Acid Hybridization
Transcription, Genetic

@article {pmid41710969,
year = {2026},
author = {Xu, Z and Wei, M and Jiang, M and Wang, Y and He, K},
title = {Single-step duplex CRISPR coupled with lateral flow assay for point-of-care detection of human immunodeficiency virus and Treponema pallidum.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {10},
pages = {1996-2004},
doi = {10.1039/d5ay02168e},
pmid = {41710969},
issn = {1759-9679},
mesh = {Humans ; *Treponema pallidum/genetics/isolation & purification ; *Point-of-Care Systems ; *HIV Infections/diagnosis/virology ; *Syphilis/diagnosis/microbiology ; *CRISPR-Cas Systems/genetics ; *HIV/genetics/isolation & purification ; Point-of-Care Testing ; Sensitivity and Specificity ; },
abstract = {Continued acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV) and syphilis caused by Treponema pallidum (TP) pose great challenges for global health, highlighting the need for rapid and sensitive diagnostics. Here, we introduced DIAL (Duplex Integrated All-in-one CRISPR Lateral flow assay), a simplified CRISPR-based diagnostic system for simultaneous detection of HIV and TP. We developed a single-step duplex CRISPR assay for dual-target recognition, which incorporated reverse transcription, multiplex recombinase polymerase amplification, transcription, CRISPR/Cas12a detection, and CRISPR/Cas13a detection in one pot. Then, we combined CRISPR detection assay with a "line-elimination" dual-line lateral flow assay for low-cost and equipment-free visual readout. We further developed reagent lyophilization and extraction-free sample lysis techniques to meet the point-of-care testing. The optimized DIAL system detected as low as 100 copies per µL of both HIV RNA and TP DNA within 45 minutes. In a clinical evaluation of 46 samples, it demonstrated 100% sensitivity and 97.5% specificity for HIV and 90% sensitivity and 100% specificity for TP, relative to quantitative PCR. The DIAL provided a scalable, accessible approach for decentralized screening of sexually transmitted infections or other pathogens in resource-limited settings.},
}

Humans
*Treponema pallidum/genetics/isolation & purification
*Point-of-Care Systems
*HIV Infections/diagnosis/virology
*Syphilis/diagnosis/microbiology
*CRISPR-Cas Systems/genetics
*HIV/genetics/isolation & purification
Point-of-Care Testing
Sensitivity and Specificity

@article {pmid41707429,
year = {2026},
author = {Nong, J and Pan, Z and Li, Y and Wei, J and Gong, Y and Li, J and Zhang, K and Liao, X},
title = {COF-confined CsPbBr3 nanocomposite with CRISPR/Cas12a-driven DNA walking for ultrasensitive electrochemiluminescent detection of circulating tumor DNA.},
journal = {Biosensors & bioelectronics},
volume = {302},
number = {},
pages = {118536},
doi = {10.1016/j.bios.2026.118536},
pmid = {41707429},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Humans ; *Nanocomposites/chemistry ; Luminescent Measurements/methods ; CRISPR-Cas Systems/genetics ; Electrochemical Techniques/methods ; Limit of Detection ; *Circulating Tumor DNA/blood/isolation & purification/genetics ; Endodeoxyribonucleases/chemistry ; DNA Probes/chemistry ; Metal-Organic Frameworks/chemistry ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {A highly sensitive electrochemiluminescence biosensor was developed for circulating tumor DNA detection by integrating a covalent organic framework-confined CsPbBr3 nanocomposite with a CRISPR/Cas12a-driven amplification strategy. The covalent organic framework not only stabilizes CsPbBr3 nanocrystals in aqueous environments but also regulates interfacial charge transfer and enables programmable immobilization of DNA probes. Upon recognition of the target sequence, Cas12a is activated and progressively cleaves surface-tethered quencher-modified DNA strands, leading to stepwise restoration of electrochemiluminescence emission. This surface-confined signal amplification eliminates the need for polymerase chain reaction or isothermal preamplification. Under optimized conditions, the biosensor exhibited a linear response over a concentration range from 10 fM to 10 nM, with a detection limit of 5.4 fM. The method demonstrated good selectivity toward single-base mismatches and satisfactory performance in diluted serum and clinical plasma samples. These results highlight a synergistic material-enzyme strategy for sensitive and robust nucleic acid detection and provide a proof-of-concept platform for electrochemiluminescence-based circulating tumor DNA analysis.},
}

*Biosensing Techniques/methods
Humans
*Nanocomposites/chemistry
Luminescent Measurements/methods
CRISPR-Cas Systems/genetics
Electrochemical Techniques/methods
Limit of Detection
*Circulating Tumor DNA/blood/isolation & purification/genetics
Endodeoxyribonucleases/chemistry
DNA Probes/chemistry
Metal-Organic Frameworks/chemistry
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid41707427,
year = {2026},
author = {Tu, Z and Wang, Y and Qian, X and Chen, J and Li, L and Wang, T and Chen, H and Wei, H and Yang, P and Pan, J and Wang, S and Rong, Z},
title = {Plasmonic magnetic nanoparticles-enabled universal enrichment, photothermal lysis, and duplex CRISPR detection of bacteria in urine samples.},
journal = {Biosensors & bioelectronics},
volume = {302},
number = {},
pages = {118518},
doi = {10.1016/j.bios.2026.118518},
pmid = {41707427},
issn = {1873-4235},
mesh = {Humans ; *Escherichia coli/isolation & purification/genetics ; *Magnetite Nanoparticles/chemistry ; *Enterococcus faecalis/isolation & purification/genetics ; *Biosensing Techniques/methods ; *Urinary Tract Infections/microbiology/urine/diagnosis ; CRISPR-Cas Systems ; Surface Plasmon Resonance/methods ; *Escherichia coli Infections/urine/microbiology/diagnosis ; Limit of Detection ; Boronic Acids/chemistry ; },
abstract = {Urinary tract infections (UTIs), exacerbated by antibiotic resistance and evolving pathogen diversity, demand rapid and sensitive diagnostics. This study introduces an integrated platform combining magnetic enrichment, photothermal lysis, and CRISPR-based detection (ME-CRISPR) for simultaneous identification of Escherichia coli (E. coli) and Enterococcus faecalis (E. faecalis) in UTIs. We engineered plasmonic magnetic nanoparticles functionalized with 4-mercaptophenylboronic acid for broad-spectrum bacterial capture within 10 min. Subsequent near-infrared laser irradiation leveraged localized surface plasmon resonance to lyse captured bacteria in situ. The released nucleic acids were directly analyzed in a single-step duplex RPA-CRISPR/Cas12a-Cas13a assay. This 40-min workflow achieved a limit of detection of 10 CFU/mL for both pathogens. Validation with 90 clinical samples (39 E. coli, 26 E. faecalis, and 25 negative controls) demonstrated 100% sensitivity and specificity, matching qPCR performance while significantly reducing turnaround time. The platform overcomes limitations of centrifugation and Gram-class-dependent lysis, offering a rapid and ultrasensitive point-of-care testing tool to curb antibiotic misuse.},
}

Humans
*Escherichia coli/isolation & purification/genetics
*Magnetite Nanoparticles/chemistry
*Enterococcus faecalis/isolation & purification/genetics
*Biosensing Techniques/methods
*Urinary Tract Infections/microbiology/urine/diagnosis
CRISPR-Cas Systems
Surface Plasmon Resonance/methods
*Escherichia coli Infections/urine/microbiology/diagnosis
Limit of Detection
Boronic Acids/chemistry

@article {pmid41707424,
year = {2026},
author = {Zhao, L and Liu, Z and Ding, G and Zhu, Y and Wang, H and Liu, R and Qu, F and Ao, Q and Zhu, X and Zhang, Y and Yang, G and Wang, Z},
title = {Dual-readout aptasensor based on CRISPR/Cas12a and nanozyme for accurate detection of KIM-1 and its application in kidney transplant prognosis.},
journal = {Biosensors & bioelectronics},
volume = {302},
number = {},
pages = {118533},
doi = {10.1016/j.bios.2026.118533},
pmid = {41707424},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Humans ; *Hepatitis A Virus Cellular Receptor 1/isolation & purification ; *Aptamers, Nucleotide/chemistry ; CRISPR-Cas Systems ; *Kidney Transplantation ; Limit of Detection ; Prognosis ; Metal Nanoparticles/chemistry ; Silver/chemistry ; Colorimetry/methods ; Metal-Organic Frameworks/chemistry ; SELEX Aptamer Technique ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Kidney injury molecule-1 (KIM-1) has emerged as a pivotal prognostic biomarker for renal allograft function. However, its detection remains challenging due to sensitivity and accuracy limitations. An innovative biosensing platform synergizing aptamer recognition, CRISPR trans-cleavage and nanozyme amplification for dual-readout KIM-1 detection has been presented in this work. This platform employs a meticulously selected high-affinity aptamer with capillary electrophoresis SELEX for specific target recognition, leverages the CRISPR/Cas12a system for signal transduction and cascade amplification, and utilizes engineered FeNi MOF@AgNPs nanozyme for dual-signal output. With the presence of KIM-1, whose binding with aptamer, effectively inhibits the trans-cleavage activity of the CRISPR/Cas12a system, and uninhibited Cas12a subsequently cleaves nanozyme-conjugated magnetic probes, releasing FeNi MOF@AgNPs nanozymes that catalyze a TMB-based reaction to generate intense colorimetric and fluorescent dual-readout signals. The as developed aptasensor demonstrates satisfied sensitivity achieving detection limits of 58.7 pg/mL (colorimetric) and 34.4 pg/mL (fluorometric), and dependable accuracy achieving average relative deviation of -2.7% (colorimetric) and 3.2% (fluorometric) with commercial ELISA kit in urine samples from patients with acute kidney injury. Moreover, longitudinally track the dynamic changes in urinary KIM-1 concentrations over the first 8 days following renal transplantation was successfully realized. This work not only provides a robust analytical tool for KIM-1 detection but also establishes a generic research approach for extending CRISPR-based systems to the precise detection of proteins.},
}

*Biosensing Techniques/methods
Humans
*Hepatitis A Virus Cellular Receptor 1/isolation & purification
*Aptamers, Nucleotide/chemistry
CRISPR-Cas Systems
*Kidney Transplantation
Limit of Detection
Prognosis
Metal Nanoparticles/chemistry
Silver/chemistry
Colorimetry/methods
Metal-Organic Frameworks/chemistry
SELEX Aptamer Technique
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41702192,
year = {2026},
author = {Lu, L and Zhang, Y and Liu, B and Zhou, N and Yu, DJ and Wang, Y},
title = {Magnetic Fe3O4-Au@UIO-66-NH2@toehold probe mediated fluorescent sensor for detecting ovarian cancer-specific circRNA coupled with hybridization chain reaction and the CRISPR-Cas12a system.},
journal = {Biosensors & bioelectronics},
volume = {302},
number = {},
pages = {118535},
doi = {10.1016/j.bios.2026.118535},
pmid = {41702192},
issn = {1873-4235},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; *RNA, Circular/genetics/isolation & purification ; *Biosensing Techniques/methods ; Female ; *Ovarian Neoplasms/genetics/diagnosis ; Gold/chemistry ; Fluorescent Dyes/chemistry ; Nucleic Acid Hybridization ; Limit of Detection ; Magnetite Nanoparticles/chemistry ; Ferrosoferric Oxide/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Circular RNAs (circRNAs) represent an emerging family of noncoding transcripts defined by closed-loop architecture, which are now established as key participants in the etiology of tumorigenesis and malignant progression. While circRNAs show potential as therapeutic targets and biomarkers, the accurate detection of circRNAs remains challenging due to interference from homologous linear RNAs. In this study, an ultrasensitive method for detecting circ_0051240 based on the sulfhydrated toehold capture probe-initiator assembly-mediated hybridization chain reaction (HCR) and the CRISPR-Cas12a system was developed. The capture probe (with a toehold domain)-initiator strand duplex was ingeniously fabricated to identify the back-splice junction (BSJ) of circRNA. After magnetic enrichment and separation by the Fe3O4-Au@UIO-66-NH2 nanocomposite, the initiator strand (H0) was delivered to trigger HCR. The HCR product dsDNA concatemers contained multiply repeated CRISPR-targetable DNA sites that were readily recognized by the CRISPR RNA (crRNA). This specific recognition and binding activated the CRISPR-Cas12a system's collateral endonuclease activity, leading to cleavage of the fluorophore-quencher (FQ) reporters and fluorescence emission at a characteristic wavelength. This design eliminated linear RNA-related interference and enhanced the detection of fluorescence intensity (FI). Under optimal conditions, the proposed HCR/CRISPR-Cas12a method exhibited a wide quantitative measurement range spanning from 45 pM up to 180 nM, achieving a notable limit of detection (LOD) of 0.03 pM. In this study, a novel circRNA sensing strategy capable of accurate and highly sensitive quantification of ovarian cancer-specific circRNA was reported. The proposed method exhibits acceptable performance when compared to present approaches.},
}

Humans
CRISPR-Cas Systems/genetics
*RNA, Circular/genetics/isolation & purification
*Biosensing Techniques/methods
Female
*Ovarian Neoplasms/genetics/diagnosis
Gold/chemistry
Fluorescent Dyes/chemistry
Nucleic Acid Hybridization
Limit of Detection
Magnetite Nanoparticles/chemistry
Ferrosoferric Oxide/chemistry
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41677799,
year = {2026},
author = {Boubakri, H},
title = {CRISPR-Cas9-mediated genome editing in fungi: applications, challenges, and future directions.},
journal = {Journal of applied microbiology},
volume = {137},
number = {3},
pages = {},
doi = {10.1093/jambio/lxag046},
pmid = {41677799},
issn = {1365-2672},
mesh = {*Gene Editing/methods/trends ; *CRISPR-Cas Systems ; *Fungi/genetics ; *Genome, Fungal ; },
abstract = {The clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been shown to be an effective genome-editing tool in many organisms, including fungi. It enables precise modifications to the DNA of fungal species, facilitating advancements in research, agriculture, and biotechnology. CRISPR-Cas9-edited non-pathogenic antagonists have emerged as a promising alternative for biocontrol. Several filamentous fungi have been engineered to produce secondary metabolites. Furthermore, the CRISPR-Cas9 system has been used to improve the quality of several edible fungi. However, the application of CRISPR-Cas9 technology for fungal genome editing is still facing some challenges that researchers must address. This review highlights the major approaches and applications of genome editing in fungi, as well as the associated challenges.},
}

*Gene Editing/methods/trends
*CRISPR-Cas Systems
*Fungi/genetics
*Genome, Fungal

@article {pmid41670347,
year = {2026},
author = {Sullivan, JR and Ferrara, KM and Barrick, R and Romano, KP and Warrier, T and Hung, DT},
title = {An inducible CRISPRi system for phenotypic analysis of essential genes in Pseudomonas aeruginosa.},
journal = {mBio},
volume = {17},
number = {3},
pages = {e0276725},
pmid = {41670347},
issn = {2150-7511},
support = {U19 AI142780/AI/NIAID NIH HHS/United States ; U19AI142780/NH/NIH HHS/United States ; },
mesh = {*Pseudomonas aeruginosa/genetics/drug effects/growth & development ; *Genes, Essential ; Phenotype ; *CRISPR-Cas Systems ; Plasmids ; Gene Expression Regulation, Bacterial ; Rhamnose/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Promoter Regions, Genetic ; },
abstract = {Precise and tunable genetic tools are essential for high-throughput functional genomics. To address this need in the important gram-negative pathogen Pseudomonas aeruginosa, we developed and characterized a tightly regulated CRISPR-interference (CRISPRi) system that enables precise and tunable repression of essential genes. The system utilizes a rhamnose-inducible promoter to control both the Streptococcus pasteurianus-derived dCas9 and gene-specific sgRNAs, each encoded on separate plasmids for modularity and efficiency. The combination of tight regulation and high conjugation efficiency facilitated the rapid and facile construction of strains with regulated depletion of 16 essential genes spanning diverse pathways. Comparison of phenotypes across the different genetically depleted strains, including growth rate, susceptibility to antibiotics, and changes in transcriptional programs, revealed novel aspects of gene function or small-molecule mechanism of action. Finally, the rhamnose-inducible CRISPRi system supports the generation and stable maintenance of pooled mutant libraries, thereby paving the way for future genome-wide, systematic assessment of individual gene vulnerabilities, which will provide critical insights for target prioritization in antibiotic discovery efforts against this recalcitrant pathogen.IMPORTANCECRISPR-interference (CRISPRi) has become an invaluable tool for studying genetics. In particular, the ability to knockdown (KD) genes enables the study of essential genes and their role in cell survival. However, a tightly regulated gene KD system is required to gain valuable insights into these vulnerable genes by virtue of their essentiality. We report a tightly regulated CRISPRi system to study the biology of essential gene perturbations in Pseudomonas aeruginosa, an important gram-negative pathogen that causes severe infections and is increasingly resistant to current antibiotics. This system enables characterization of both chemical genetic interactions between small molecules and specific gene depletions and the impact of genetic perturbations on transcriptional networks. Genetic perturbations using CRISPRi can thus further our understanding of basic biology with translation toward future antimicrobial development.},
}

*Pseudomonas aeruginosa/genetics/drug effects/growth & development
*Genes, Essential
Phenotype
*CRISPR-Cas Systems
Plasmids
Gene Expression Regulation, Bacterial
Rhamnose/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats
Promoter Regions, Genetic

@article {pmid41642947,
year = {2026},
author = {Pelea, O and Tálas, A and Carrera, JF and Mathis, N and van de Venn, L and Yeh, CD and Kulcsár, PI and Marquart, KF and Weber, Y and Gerecke, SE and Harvey-Seutcheu, IF and Mailänder, D and Pfleiderer, MM and Chanez, C and Corn, JE and Schwank, G and Jinek, M},
title = {Programmable genome editing in human cells using RNA-guided bridge recombinases.},
journal = {Science (New York, N.Y.)},
volume = {391},
number = {6790},
pages = {eadz1884},
doi = {10.1126/science.adz1884},
pmid = {41642947},
issn = {1095-9203},
mesh = {Humans ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; Plasmids ; Recombination, Genetic ; Genome, Human ; *DNA Nucleotidyltransferases/chemistry/metabolism/genetics ; CRISPR-Cas Systems ; DNA/genetics ; },
abstract = {Site-specific insertion of gene-sized DNA fragments remains an unmet need in the field of genome editing. IS110-family serine recombinases have recently been shown to mediate programmable DNA recombination in bacteria by using a bispecific RNA guide (bridge RNA) that simultaneously recognizes target and donor sites. In this work, we have shown that the bridge recombinase ISCro4 is highly active in human cells and provided structural insights into its enhanced activity. Using plasmid- or all-RNA-based delivery, ISCro4 supports programmable multikilobase excisions and inversions and facilitates donor DNA insertion at genomic sites with efficiencies that exceed 6%. Last, we assessed ISCro4 specificity and off-target activity. These results establish a framework for the development of bridge recombinases as next-generation tools for editing modalities that are beyond the capabilities of current technologies.},
}

Humans
*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics
HEK293 Cells
Plasmids
Recombination, Genetic
Genome, Human
*DNA Nucleotidyltransferases/chemistry/metabolism/genetics
CRISPR-Cas Systems
DNA/genetics

@article {pmid41524478,
year = {2026},
author = {Kim, M and Kim, K and Lee, J and Lee, S and Choi, S and Park, SA and Jeong, E and Choi, SY and Park, HH and Park, TE and Kwon, T and Myung, K and Yoo, J and Cho, SW and Joo, J},
title = {FAST-CRISPR: Fusogenic Association and Secured Transfection of CRISPR/Cas9 Ribonucleoproteins Using Lipid-Silica Hybrid Nanoparticles for Therapeutic Genome Editing.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {22},
number = {15},
pages = {e11362},
pmid = {41524478},
issn = {1613-6829},
support = {RS-2024-00512120//Korean ARPA-H Project through the KHIDI/ ; 22A0102L1-11//Korean Fund for Regenerative Medicine (KFRM)/ ; RS-2024-00509412//National Research Foundation (NRF)/ ; RS-2023-00209822//National Research Foundation (NRF)/ ; RS-2023-00207746//National Research Foundation (NRF)/ ; IBS-R022-D1//Institute for Basic Science/ ; 1.250006.01//UNIST research fund/ ; RS-2024-00403508//Korea Basic Science Institute/ ; 1.250006.01//Ulsan National Institute of Science and Technology/ ; },
mesh = {*Nanoparticles/chemistry ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/metabolism/genetics ; *Silicon Dioxide/chemistry ; Humans ; *Lipids/chemistry ; Animals ; *Transfection/methods ; Mice ; Cell Line, Tumor ; },
abstract = {Clinical translation of CRISPR/Cas9 therapeutics is challenged by inefficient cytosolic delivery and toxicity issues associated with viral vectors and nanoparticle-based carriers. To overcome these concerns, herein we report a lipid-silica hybrid nanoparticle platform for fusogenic association and secured transfection of CRISPR/Cas9 (FAST-CRISPR), designed for rapid cytosolic delivery of CRISPR/Cas9 ribonucleoproteins, followed by efficient gene editing. Through direct fusion with the plasma membrane and bypassing conventional endocytic barriers, FAST-CRISPR nanoparticles displayed superior intracellular delivery efficacy. Optimizing lipid compositions, we discovered that a 1:1 weight mixture of cationic DOTAP and ionizable DODMA lipids, combined with tailored large-pore silica nanoparticles, enables enhanced loading capacity, rapid cytosolic dispersion, and significant nuclear transport of Cas9/gRNA complexes. FAST-CRISPR nanoparticles efficiently delivered multiplex genome-targeting ribonucleoproteins to induce targeted double-strand DNA breaks, triggering apoptosis in cancer cells and significantly suppressing tumor growth in a mouse xenograft model without systemic toxicity. Our findings demonstrate the therapeutic efficacy and translational potential of FAST-CRISPR nanoparticles as a safe and versatile non-viral delivery platform for precision genome editing.},
}

*Nanoparticles/chemistry
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Ribonucleoproteins/metabolism/genetics
*Silicon Dioxide/chemistry
Humans
*Lipids/chemistry
Animals
*Transfection/methods
Mice
Cell Line, Tumor

@article {pmid41270742,
year = {2026},
author = {Sun, J and Atiş, IS and Empke, SLL and Khokha, MK and Breslow, DK},
title = {A microscopy-based CRISPR screening platform enables organellar functional genomics and illuminates ciliary biology.},
journal = {Developmental cell},
volume = {61},
number = {3},
pages = {687-705.e9},
pmid = {41270742},
issn = {1878-1551},
support = {S10 OD023651/OD/NIH HHS/United States ; R35 GM137956/GM/NIGMS NIH HHS/United States ; S10 OD018034/OD/NIH HHS/United States ; R01 HD102186/HD/NICHD NIH HHS/United States ; S10 OD030363/OD/NIH HHS/United States ; },
mesh = {*Cilia/metabolism/genetics ; Humans ; Animals ; *Genomics/methods ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Microscopy, Fluorescence/methods ; Xenopus ; },
abstract = {Microscopy offers an indispensable technique for visualizing biological processes and for defining cytological abnormalities characteristic of disease. However, combining microscopy with the power of pooled CRISPR screening presents considerable technical challenges, hindering application of systematic genetic analysis to imaging-defined phenotypes. Here, we establish a fluorescence microscopy-based CRISPR screening platform that combines ease of implementation with flexible analysis of live-cell or antibody-based molecular markers, including post-translational modifications. Applying this methodology, we systematically identify regulators of primary cilium structure and function in human cells through targeted and genome-wide screens. We further show that integration of screens focused on distinct ciliary phenotypes yields multi-dimensional profiles that delineate precise gene functions. Among the identified hits, TZMP1 (SMIM27) encodes a microprotein at the ciliary transition zone that is required for ciliogenesis in human cells and for ciliary function in Xenopus embryos. More broadly, our approach provides a technological and conceptual strategy for microscopy-based functional genomics.},
}

*Cilia/metabolism/genetics
Humans
Animals
*Genomics/methods
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Microscopy, Fluorescence/methods
Xenopus

@article {pmid37751223,
year = {2023},
author = {Ji, S and Wang, X and Wang, Y and Sun, Y and Su, Y and Lv, X and Song, X},
title = {Advances in Cas12a-Based Amplification-Free Nucleic Acid Detection.},
journal = {The CRISPR journal},
volume = {6},
number = {5},
pages = {405-418},
doi = {10.1089/crispr.2023.0023},
pmid = {37751223},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; Humans ; *Endodeoxyribonucleases/genetics/metabolism ; *Nucleic Acids/genetics/analysis ; Biosensing Techniques/methods ; *Bacterial Proteins/genetics ; Electrochemical Techniques/methods ; },
abstract = {In biomedicine, rapid and sensitive nucleic acid detection technology plays an important role in the early detection of infectious diseases. However, most traditional nucleic acid detection methods require the amplification of nucleic acids, resulting in problems such as long detection time, complex operation, and false-positive results. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR) systems have been widely used in nucleic acid detection, especially the CRISPR-Cas12a system, which can trans cleave single-stranded DNA and can realize the detection of DNA targets. But, amplification of nucleic acids is still required to further improve detection sensitivity, which makes Cas12a-based amplification-free nucleic acid detection methods a great challenge. This article reviews the recent progress of Cas12a-based amplification-free detection methods for nucleic acids. These detection methods apply electrochemical detection methods, fluorescence detection methods, noble metal nanomaterial detection methods, and lateral flow assay. Under various optimization strategies, unamplified nucleic acids have the same sensitivity as amplified nucleic acids. At the same time, the article discusses the advantages and disadvantages of each method and further discusses the current challenges such as off-target effects and the ability to achieve high-throughput detection. Amplification-free nucleic acid detection technology based on CRISPR-Cas12a has great potential in the biomedical field.},
}

*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Associated Proteins/genetics/metabolism
Humans
*Endodeoxyribonucleases/genetics/metabolism
*Nucleic Acids/genetics/analysis
Biosensing Techniques/methods
*Bacterial Proteins/genetics
Electrochemical Techniques/methods

@article {pmid37739193,
year = {2024},
author = {Mori, AA and Malaquias, VB and Bonjour, K and Ferreira, GM and Bortolin, RH and Borges, JB and Oliveira, VF and Gonçalves, RM and Faludi, AA and Bastos, GM and Thurow, H and Sampaio, MF and Ciconelli, RM and Cury, AN and Fajardo, CM and Hirata, RDC and Hirata, MH},
title = {Effects of LDLR variants rs5928, rs750518671 and rs879254797 on protein structure and functional activity in HepG2 cells transfected with CRISPR/Cas9 constructs.},
journal = {Gene},
volume = {890},
number = {},
pages = {147821},
doi = {10.1016/j.gene.2023.147821},
pmid = {37739193},
issn = {1879-0038},
mesh = {Humans ; Hep G2 Cells ; *Receptors, LDL/genetics/chemistry/metabolism ; *CRISPR-Cas Systems/genetics ; Mutation, Missense ; *Hyperlipoproteinemia Type II/genetics ; Female ; Male ; Polymorphism, Single Nucleotide ; Transfection ; Middle Aged ; },
abstract = {Familial Hypercholesterolemia (FH) is a genetic disorder associated with premature atherosclerosis and increased risk of cardiovascular diseases. LDLR deleterious mutations are associated with FH, however the role of some missense variants in FH pathogenicity remains to be elucidated. This study explored the predictive impact of LDLR missense variants on protein structure and investigated their functional effects on LDLR expression in HepG2 cells transfected with CRISPR/Cas9 constructs. FH (n = 287) and non-FH patients (n = 45) were selected, and lipid profile was obtained from medical records. LDLR variants were identified using an exon-targeted gene sequencing strategy, considering its cost-effective to increase accuracy in the identification step of the most likely FH-related variants in a less laborious process. LDLR variants were selected based on conflicting pathogenicity results found in Clinvar, in silico prediction tools, affected LDLR domains, and less common variants considering minor allele frequency < 0.05. Molecular modeling studies were used to predict the effects of LDLR missense variants on protein structure. Recombinant LDLR variants were constructed using CRISPR/Cas9 system and were used to transfect HepG2 cells. Functional assays in transfected cells were performed to assess LDLR expression using flow cytometry and western blotting, and LDLR activity using flow cytometry and confocal microscopy. The variants rs121908039 (c.551G>A, p.C184Y), rs879254797 (c.1118G>A, p.G373D), rs28941776 (c.1646G>A, p.G549D), rs750518671 (c.2389G>C, p.V797L), rs5928 (c.2441G>A, p.R814Q) and rs137853964 (c.2479G>A, p.V827I) were selected for molecular docking analysis. The p.C184Y exhibited a favorable energy change for protein stability due to its interaction with EGF-A/EGF-B regions; p.G373D and p.G549D displayed intermediate energy changes; and p.R814Q and p.V827I showed smaller energy changes. The results of functional assays showed that p.G373D, p.V797L and p.R814Q reduced LDLR expression and activity (p < 0.05). Microscopic analysis of the p.V797L and p.G373D variants revealed altered lipid localization and accumulation in transfected HepG2 cells. Carriers of p.G549D, p.V797L and p.R814Q had higher LDL cholesterol levels than non-FH group, and (p < 0.05). p.G373D and p.G549D were associated with clinical manifestations of FH. In conclusion, the p.C184Y, p.G373D, p.G549D and p.R814Q variants alter protein stability and intramolecular interactions, while p.V797L has a minimal impact on protein stability, and p.V827I has no significant intramolecular interactions. p.G373D, p.V767L and p.R814Q are associated with impaired LDLR expression and activity.},
}

Humans
Hep G2 Cells
*Receptors, LDL/genetics/chemistry/metabolism
*CRISPR-Cas Systems/genetics
Mutation, Missense
*Hyperlipoproteinemia Type II/genetics
Female
Male
Polymorphism, Single Nucleotide
Transfection
Middle Aged

@article {pmid41802204,
year = {2026},
author = {de Paula, JA and de Araújo, MRB and Guimarães Sousa, E and Prates, FD and Castro, DLC and Fonseca, PAS and Brenig, B and Felice, AG and Pacheco, LGC and Viana, MVC and Azevedo, VAC and Soares, S},
title = {Clonal clusters of multidrug-resistant Brazilian Corynebacterium striatum strains reveal putative virulence traits.},
journal = {Journal of applied microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1093/jambio/lxag070},
pmid = {41802204},
issn = {1365-2672},
abstract = {CORYNEBACTERIUM STRIATUM: Has been increasingly associated with nosocomial outbreaks and antimicrobial resistance.

OBJECTIVES: This study presents the comparative analysis of 26 multidrug-resistant (MDR) C. striatum strains isolated in Brazil.

METHODS: Additional genomes from international sources were incorporated. The analyses encompassed in vitro antimicrobial susceptibility testing and an in silico workflow for genomic similarity comparison, phylogenetic reconstruction, genomic clustering, pangenome analysis, mobilome content, virulence prediction, and functional annotation of unique proteins and putative virulence clusters.

RESULTS: Strong in silico evidence of clonality among several Brazilian isolates was obtained at the same time that some strains consistently indicated a divergent genomic profile. There are 196 unique coding sequences (CDSs) across the Brazilian IHPs. Of particular interest, strain IHP2030 carried an exclusive fimbria, sharing less than 50% similarity with other fimbriae in the dataset. Yet, structural predictions suggested conservation of key structural domains typically associated with fimbrial proteins. Mobilome content analysis revealed that IHPs strains were overall similar, differing primarily in the number of insertion sequences and in the presence or absence of CRISPR-Cas defense systems. Regarding virulence, an exclusive cluster in IHP2050 and IHP2060 suggests adaptive advantages associated with their respective environments of isolation.

CONCLUSION: This study reveals a complex genomic landscape among Brazilian MDR C. striatum strains, marked by clonal dissemination alongside strain-level genetic variation in accessory genomes, mobilome composition, and virulence-associated gene repertoires, providing genomic evidence of diversification within hospital-associated lineages.},
}

@article {pmid41799755,
year = {2026},
author = {Vadrot, N and Moulin, M and Ferreiro, A and Richard, P and Buendia, B},
title = {LAP2 Isoform Profile in Heart Ageing and in Cardiac Cell Proliferation and Differentiation: Input From CRISPR-Cas9-mediated LAP2a Knockdown in H9C2.},
journal = {International journal of medical sciences},
volume = {23},
number = {3},
pages = {741-757},
pmid = {41799755},
issn = {1449-1907},
mesh = {Animals ; Cell Differentiation/genetics ; *Myocytes, Cardiac/metabolism ; Cell Proliferation/genetics ; CRISPR-Cas Systems/genetics ; Mice ; *Membrane Proteins/genetics/metabolism ; MEF2 Transcription Factors/genetics/metabolism ; Protein Isoforms/genetics/metabolism ; Rats ; *Aging/genetics ; Cell Line ; Gene Knockdown Techniques ; Myocardium/metabolism ; Humans ; Heart/growth & development/physiology ; DNA-Binding Proteins ; },
abstract = {Haploinsufficiency of Lap2 alpha (LAP2a), a nuclear partner of Lamins A/C, has been associated with cardiac disease in rare cases, but LAP2a function remains largely unknown. To investigate the functional role of LAP2a in cardiomyocytes, we generated clones of embryonic myocardium-derived H9C2 cells in which LAP2a expression was specifically reduced through gene editing of the LAP2a gene Tmpo by CRISPR-Cas9. Downregulation (+/-) and absence (-/-) of LAP2a expression led to a decreased proliferation capacity of cardiomyocytes in vitro. Upon differentiation, the expression of myocardial markers (alpha cardiac Actin 1/Actc1, cardiac Troponin T2/Tnnt2, Myosin-2/Myh2 and Myosin-7/Myh7) was higher in LAP2a -/- cells compared to LAP2a +/- or LAP2a +/+ cells, with consistently higher expression of their upstream regulator Mef2c in LAP2a-devoid cells. These results suggest that LAP2a promotes cardiomyocyte proliferation and negatively modulates cardiomyocyte differentiation, through mechanisms including Mef2c regulation. Accordingly, normal protein expression of LAP2a was downregulated upon cardiomyocyte differentiation, contrary to LAP2b and a LAP2b-related shorter isoform. The latter tended to increase upon differentiation in all cells, most significantly in the LAP2a -/- clone. In postnatal mouse hearts, LAP2a levels were higher in the right than in the left ventricle, and lowest in the septum. The LAP2a:LAP2b ratio was much lower in murine hearts than in H9C2 cells, and decreased significantly upon ageing, specifically in the left ventricle. Finally, our data show that expression of the nuclear envelope proteins LEMD2 and Lamin A might be influenced by LAP2a upon cardiac differentiation. Our results show that LAP2 expression is finely regulated upon cardiac differentiation in vitro and is dependent on age and heart compartment in vivo. They contribute to clarifying the potential impact of genetic LAP2a defects and their connection with heart disease, possibly including reduced cardiomyoblast proliferation, increased cardiomyocyte differentiation and altered nuclear envelope remodelling.},
}

Animals
Cell Differentiation/genetics
*Myocytes, Cardiac/metabolism
Cell Proliferation/genetics
CRISPR-Cas Systems/genetics
Mice
*Membrane Proteins/genetics/metabolism
MEF2 Transcription Factors/genetics/metabolism
Protein Isoforms/genetics/metabolism
Rats
*Aging/genetics
Cell Line
Gene Knockdown Techniques
Myocardium/metabolism
Humans
Heart/growth & development/physiology
DNA-Binding Proteins

@article {pmid41799190,
year = {2026},
author = {Zhao, P and Li, H and Cai, Z and Zhang, X and Wen, X and Liu, Z and Jiang, S and Jiang, X and Wang, J and Dang, Z and Liu, M and Xie, F and Ma, X},
title = {Molecular hydrogen triggers TRPC4-TRPC4AP-dependent reversible calcium transients via extracellular influx.},
journal = {Theranostics},
volume = {16},
number = {9},
pages = {4843-4864},
pmid = {41799190},
issn = {1838-7640},
mesh = {Animals ; Mice ; *TRPC Cation Channels/metabolism/genetics ; *Calcium/metabolism ; Mice, Inbred C57BL ; *Calcium Signaling/drug effects ; *Hydrogen/metabolism/pharmacology ; Humans ; Cell Movement/drug effects ; Molecular Dynamics Simulation ; Molecular Docking Simulation ; Brain/metabolism ; CRISPR-Cas Systems ; HEK293 Cells ; },
abstract = {RATIONALE: Hydrogen gas (H2) produces pleiotropic therapeutic actions, but the exact molecular targets and ion-channel-based signaling cascades that underlie these benefits remain elusive. H2 may regulate calcium ion (Ca[2+])-dependent processes, but the direct involvement of H2 in Ca[2+] signaling and its underlying molecular mechanisms are unknown. We propose that H2 functions as a gaseous messenger that selectively opens a plasma-membrane Ca[2+] channel to evoke Ca[2+] transients ([Ca[2+] i]t) while avoiding cytotoxic overload, thereby offering a mechanism for its diverse biological effects.

METHODS: This study employed real-time calcium imaging and CRISPR-Cas9 gene editing, with live-cell imaging to monitor real-time calcium signal intensity in living cells. Two-photon in vivo imaging was applied to detect real-time Ca[2+] signals in the brain and dorsal skin of C57BL/6 mice carrying adeno-associated virus-delivered calcium sensors. Live-cell F-actin staining and a wound healing (scratch) assay were used to assess the effects of H2 on cell motility. Protein-protein docking and molecular dynamics simulations were performed to analyze the interaction interface and binding forces between TRPC4 and TRPC4AP in three-dimensional space. Additionally, RNA sequencing was performed to validate downstream biological effects and transcriptional regulation triggered by H2.

RESULTS: H2 elicited rapid and reversible [Ca[2+] i]t across multiple cell types in a Ca[2+]- and concentration-dependent manner, an effect that was absent in TRPC4⁻/⁻ or TRPC4AP⁻/⁻ cells. In vivo imaging in mice expressing a genetically encoded Ca²⁺ sensor showed that H2 inhalation elevated Ca[2+] signals in the motor cortex (M1 region) and dorsal skin. Functionally, live-cell imaging and wound-healing assays confirmed that H2-induced Ca[2+] transients enhanced cell motility. Mechanistically, protein docking revealed a dual-arginine cluster within the CIRB domain of TRPC4; its interaction with TRPC4AP was essential for H2-evoked Ca[2+] influx. Mutating these arginines to alanine residues completely abolishing the response. H2 triggered proton efflux and increased intracellular pH. Molecular dynamics simulations indicated that altered pH modulates the binding force between TRPC4 Arg730/Arg731 and TRPC4AP. Transcriptomic analysis further demonstrated that H2 activates calcium-related channels and promotes cytoskeletal remodeling and cell migration.

CONCLUSIONS: This study identifies H2 as a novel gaseous signaling molecule that can regulate Ca[2+] channels via the TRPC4-TRPC4AP axis. The 730Arg-731Arg motif in TRPC4 serves as a critical H2-sensitive site, enabling dynamic calcium homeostasis without overload. These findings provide a mechanistic framework for developing gas-controlled H2 regenerative therapeutics.},
}

Animals
Mice
*TRPC Cation Channels/metabolism/genetics
*Calcium/metabolism
Mice, Inbred C57BL
*Calcium Signaling/drug effects
*Hydrogen/metabolism/pharmacology
Humans
Cell Movement/drug effects
Molecular Dynamics Simulation
Molecular Docking Simulation
Brain/metabolism
CRISPR-Cas Systems
HEK293 Cells

@article {pmid41797538,
year = {2026},
author = {Fang, M and Yap, J and Fei, M and Gong, M and Li, N and Lu, Y and Yu, M and Xu, Y and Wu, F and Gao, H and Sun, D},
title = {LysR-type regulator LrhA promotes CRISPR-Cas immunity in Escherichia coli.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41797538},
issn = {1362-4962},
support = {32170083//National Natural Science Foundation of China/ ; 31670084//National Natural Science Foundation of China/ ; 31930003//National Natural Science Foundation of China/ ; 2020C02031//Key Research and Development Program of Zhejiang Province/ ; LHDMY23H160003//Natural Science Foundation of China/ ; YS2022005//Natural Science Foundation of China/ ; 2026C02A1080//Zhejiang Lingyan Research and Development Program/ ; LMRY26H200010//Joint Funds of the Zhejiang Provincial Natural Science Foundation of China/ ; },
mesh = {*Escherichia coli/genetics/immunology/virology ; *CRISPR-Cas Systems/genetics ; *Escherichia coli Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; Promoter Regions, Genetic ; *Transcription Factors/genetics/metabolism ; Operon ; Plasmids/genetics ; Trans-Activators/genetics ; },
abstract = {The CRISPR-Cas defense system safeguards prokaryotes against foreign genetic elements. Its activity is determined by the combined effects of adaptation and interference. However, the dynamic regulation of these two processes remains not fully understood. In this study, we identify the LysR-type transcriptional regulator LrhA, which is differentially expressed in various Escherichia coli strains, as a novel CRISPR-Cas activator that plays a critical role in modulating host defense levels. In a representative strain expressing a high level of LrhA, the regulator enhances CRISPR-Cas-mediated adaptive immunity against bacteriophage infection by promoting cas gene transcription through direct interaction with the promoter of the cas operon. Moderate activation of cas genes by weakly expressed LrhA in another representative strain efficiently accelerates the clearance of horizontally transferred CRISPR-targeted plasmids by enhancing spacer acquisition via interference-driven adaptation. This divergence, likely a result of genome evolution, suggests that adaptive immunity is optimized with intermediate transcription levels of cas genes by triggering positive feedback between adaptation and interference. Collectively, our findings highlight the crucial role of LrhA in fine-tuning host defense responses.},
}

*Escherichia coli/genetics/immunology/virology
*CRISPR-Cas Systems/genetics
*Escherichia coli Proteins/genetics/metabolism
Gene Expression Regulation, Bacterial
Promoter Regions, Genetic
*Transcription Factors/genetics/metabolism
Operon
Plasmids/genetics
Trans-Activators/genetics

@article {pmid41796733,
year = {2026},
author = {Lin, J and Wang, Y and Zeng, B and Chen, Z and Lin, X and Zeng, T},
title = {CRISPR-Cas12a/Cas13a in cancer molecular diagnosis.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {},
number = {},
pages = {120934},
doi = {10.1016/j.cca.2026.120934},
pmid = {41796733},
issn = {1873-3492},
abstract = {Cancer remains a leading cause of global mortality, with early diagnosis being pivotal for improving treatment outcomes. Traditional tissue biopsy is limited by its invasiveness, inability to capture tumor heterogeneity, and failure to support dynamic monitoring. Liquid biopsy has emerged as a non-invasive alternative, enabling the analysis of circulating tumor biomarkers (e.g., ctDNA, miRNAs, exosomes) in bodily fluids. However, current liquid biopsy technologies (e.g., NGS, ddPCR) suffer from high costs, complex workflows, poor standardization, and insufficient sensitivity for low-abundance biomarkers. The CRISPR-Cas systems, particularly Cas12a and Cas13a, have revolutionized molecular diagnostics due to their programmable sequence recognition, robust signal amplification via trans-cleavage/collateral cleavage activity, and compatibility with point-of-care testing (POCT). Cas12a targets DNA molecules, enabling sensitive detection of gene mutations and DNA methylation, while Cas13a specifically recognizes RNA, facilitating direct analysis of miRNAs and viral RNAs. Additionally, these systems have been extended to non-nucleic acid biomarkers (e.g., proteins, exosomes) through signal conversion strategies. This review summarizes the latest advances in CRISPR-Cas12a/Cas13a-based biosensors for cancer molecular diagnosis, including the detection of gene mutations, epigenetic modifications, miRNAs, tumor-associated viruses, and non-nucleic acid biomarkers. We critically analyze current challenges (e.g., PAM dependence, matrix interference, multiplexing limitations, clinical validation gaps) and discuss future perspectives, such as engineering PAM-less Cas variants, integrating nanotechnology, microfluidics, and artificial intelligence/artificial intelligence (AI), and advancing clinical standardization. This review aims to provide a comprehensive reference for the development and clinical translation of CRISPR-based cancer diagnostic technologies.},
}

@article {pmid41795439,
year = {2026},
author = {Zhu, L and Yang, C and Bernards, R and Wang, C},
title = {CLIM-TIME links genetic cancer drivers to immune landscapes.},
journal = {Cell},
volume = {189},
number = {5},
pages = {1263-1265},
doi = {10.1016/j.cell.2026.01.014},
pmid = {41795439},
issn = {1097-4172},
mesh = {Humans ; *Neoplasms/genetics/immunology/therapy ; Immunotherapy ; Tumor Microenvironment/immunology/genetics ; Animals ; CRISPR-Cas Systems ; Genes, Tumor Suppressor ; },
abstract = {Immunotherapy resistance is associated with immune-privileged microenvironments, yet the interacting role of tumor-intrinsic genetics remains unclear. In this issue of Cell, Wang et al. introduce CLIM-TIME, a spatially resolved in vivo CRISPR screening platform linking loss of tumor suppressor genes to distinct metastatic immune architectures and divergent responses to immunotherapy.},
}

Humans
*Neoplasms/genetics/immunology/therapy
Immunotherapy
Tumor Microenvironment/immunology/genetics
Animals
CRISPR-Cas Systems
Genes, Tumor Suppressor

@article {pmid41794473,
year = {2026},
author = {Safenkova, IV and Kamionskaya, MV and Serchenya, TS and Sviridov, OV and Dzantiev, BB and Zherdev, AV},
title = {CRISPR/Cas12a and fork-shaped probe enhance LAMP-LFT integration for equipment-free detection of Listeria monocytogenes.},
journal = {Food research international (Ottawa, Ont.)},
volume = {230},
number = {},
pages = {118592},
doi = {10.1016/j.foodres.2026.118592},
pmid = {41794473},
issn = {1873-7145},
mesh = {*Nucleic Acid Amplification Techniques/methods ; *Listeria monocytogenes/isolation & purification/genetics ; *CRISPR-Cas Systems ; *Food Microbiology/methods ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; Limit of Detection ; DNA, Bacterial/genetics ; Food Contamination/analysis ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Listeria monocytogenes is an important foodborne pathogen associated with high mortality rates, especially among vulnerable populations, and therefore requires diagnostic methods that are not only highly sensitive and rapid but also suitable for use in resource-limited settings. In this study, we developed an isothermal amplification assay integrated with a lateral flow test (LFT) for reliable detection of L.monocytogenes. Two assay formats were designed and compared: (1) loop-mediated isothermal amplification (LAMP) with LFT detection of fluorescein- and biotin-labeled amplicons, and (2) LAMP combined with CRISPR/Cas12a, using LFT to detect a cleaved fork-shaped enhanced probe labeled with three fluoresceins. Both LFT formats utilized a common conjugate of gold nanoparticles and anti-fluorescein antibodies (anti-FAM), but differed in the test zone immobilization strategy: streptavidin for LAMP, and anti-FAM for LAMP-CRISPR/Cas12a. Among 12 tested (primer - label) combinations, the most effective was identified, but the sensitivity of the LAMP-LFT format was limited by high signal variability. In contrast, the LAMP-CRISPR/Cas12a assay, targeting LAMP amplicons with guide RNA, achieved a detection limit of 0.9 copies/reaction-representing > 20,000-fold improvement in detectable DNA concentration compared with LAMP-LFT-and comparable to fluorescence-based detection techniques. The LAMP-CRISPR/Cas12a-LFT assay was first reported to detect L.monocytogenes cells following thermal lysis (10 min at 95 °C), with a single-cell detection limit (0.2 cells/reaction in buffer, 1 cells/reaction in spiked milk) and an analysis time of 80 min. These results demonstrate the potential of the approach for sensitive, equipment-free detection of foodborne pathogens in complex food matrices.},
}

*Nucleic Acid Amplification Techniques/methods
*Listeria monocytogenes/isolation & purification/genetics
*CRISPR-Cas Systems
*Food Microbiology/methods
*Molecular Diagnostic Techniques/methods
Sensitivity and Specificity
Limit of Detection
DNA, Bacterial/genetics
Food Contamination/analysis
Gold/chemistry
Metal Nanoparticles/chemistry
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41793913,
year = {2026},
author = {Li, X and Liu, L and Luo, C and Chen, Z and Shu, B},
title = {Efficient CRISPR/Cas9 system established via co-cultivation of plantlets and Agrobacterium tumefaciens for positive transgenic calluses generation and regeneration in cultivated strawberry (Fragaria × ananassa).},
journal = {Plant physiology and biochemistry : PPB},
volume = {232},
number = {},
pages = {111195},
doi = {10.1016/j.plaphy.2026.111195},
pmid = {41793913},
issn = {1873-2690},
abstract = {Recently, an Agrobacterium-mediated CRISPR/Cas9 editing system was successfully applied in a gene function analysis, highlighting its great value for improving strawberry genetics. However, the resulting low transformation rates and long regeneration cycles have limited its extensive application. Based on the biological characteristics of crown branching, an Agrobacterium tumefaciens-mediated CRISPR/Cas9 gene editing system was developed to increase the transformation rate and decrease the regeneration time of cultivated strawberry. Two single guide (sg)RNAs were designed for the strawberry anthracnose-related transcription factor, WRKY (FxaC_17g55530), and its alleles. These sgRNAs were inserted into pKSE401G using pCBC-DT1T2; sgRNAs for subtilisin-like protease (FxaC_22g21540) were designed and cloned in a similar manner. After 10 days of co-cultivating plantlets (without media supply of carbon) and GV3101, 65 (61.9%) and 72 (68.6%) GFP-positive calluses for the two genes were respectively obtained from the crown of 105 plantlets. The positive calluses were removed from the crown and placed on Murashige and Skoog media containing 3 mg/L thidiazuron and 0.2 mg/L indole-3-butyric acid. After 50-80 days, 3-5 positive shoots were obtained from different positive calluses for each gene. The three T0 lines for FxaC_17g55530 and FxaC_22g21540 were found to be successfully edited at the target sites of both sgRNA1 and sgRNA2 or either sgRNA1 or sgRNA2. Overall, a quick and effective CRISPR-Cas 9 gene editing system was developed for cultivated strawberry, highlighting the applicability of gene editing in breeding and gene function analysis.},
}

@article {pmid41791397,
year = {2026},
author = {Low, SJ and O'Neill, MT and Fernando, JA and Kerry, WJ and Prestedge, J and Wild, N and Chahal, S and Pollock, GL and Papadakis, G and Krysiak, M and Williams, E and Azzato, F and Tran, T and Fairley, C and Bradshaw, C and Chen, MY and Lim, CK and Williamson, DA and Pasricha, S},
title = {CRISPR-Cas-based diagnostics for point-of-care detection of sexually transmitted infections: a laboratory development and evaluation study.},
journal = {The Lancet. Microbe},
volume = {},
number = {},
pages = {101289},
doi = {10.1016/j.lanmic.2025.101289},
pmid = {41791397},
issn = {2666-5247},
abstract = {BACKGROUND: Timely, point-of-care diagnosis of sexually transmitted infections (STIs) is crucial for enabling prompt treatment and reducing transmission. We aimed to develop a portable, multiplexed, CRISPR-based assay panel for the detection of Neisseria gonorrhoeae (including the ciprofloxacin resistance marker gyrA S91F), Chlamydia trachomatis, Treponema pallidum, and herpes simplex virus (HSV).

METHODS: In this laboratory development and evaluation study, we developed and optimised four multiplexed, CRISPR-based, diagnostic STI assays for point-of-care use. The complete assay panel comprised a CRISPR TP-HSV (cTP-HSV) panel for the detection of T pallidum and pan-HSV, with reflex testing to distinguish HSV-1 from HSV-2, and a CRISPR NG-CT (cNG-CT) panel for the detection of N gonorrhoeae and C trachomatis, with reflex testing to detect N gonorrhoeae using two additional genome regions and to identify the gyrA S91F mutation. Each pathogen was targeted at two independent genomic regions by isothermal amplification and CRISPR-Cas reaction using Cas12a and Cas13a, each with distinct fluorescent reporters. Analytical specificity and limits of detection (LODs) were determined, and a retrospective, masked concordance study was conducted on genomic DNA from 900 clinical samples (400 for cTP-HSV and reflex testing and 500 for cNG-CT and reflex testing), using quantitative PCR as the reference standard. The diagnostic accuracy of the test was assessed by analysis of receiver operating characteristic curves.

FINDINGS: The overall sensitivity of the TP-HSV CRISPR assay was 82·5% (95% CI 74·0-88·7) for T pallidum and 94·4% (90·2-97·0) for pan-HSV; LODs were 6·2 copies per μL for T pallidum and 7·8 copies per μL for HSV. Reflex testing gave sensitivities of 97·0% (91·1-99·3) for HSV-1 and 96·0% (89·7-98·7) for HSV-2. The NG-CT CRISPR assay had an overall sensitivity of 80·0% (74·0-84·9) for N gonorrhoeae and 73·0% (65·5-79·3) for C trachomatis, with a LOD of 3·9 copies per μL for both pathogens. Reflex testing for the detection of the gyrA S91F mutation in N gonorrhoeae showed an overall sensitivity of 63·1% (55·1-70·4); however, this was dependent on sample type, with a sensitivity of 85·7% (46·7-99·5) in genital samples and 61·2% (52·8-68·9) in extragenital samples. For all pathogens, assay sensitivity was positively correlated with pathogen load. Area under the curve (AUC) values were 0·90 for T pallidum and 0·99 for pan-HSV in the TP-HSV assay, with values of 0·99 for HSV-1 and 0·97 for HSV-2 obtained in the reflex HSV-1-HSV-2 assay. For the cNG-CT assay, AUC values were 0·90 for N gonorrhoeae and 0·85 for C trachomatis, with a value of 0·72 obtained for gyrA S91F in the reflex cNG-gyrA assay.

INTERPRETATION: Our multiplexed, CRISPR-based, point-of-care platform achieved performance consistent with WHO target product profiles for N gonorrhoeae and T pallidum. Proof-of-concept detection of the gyrA S91F resistance marker highlights its potential for resistance-guided therapy. Although optimisation is required before large-scale deployment, this suite offers a promising approach for rapid, decentralised, and resistance-informed STI diagnosis, particularly in resource-limited settings.

FUNDING: Victorian Government Department of Health, Australian Government Department of Health, Disability and Ageing and Aged Care, and Australian Research Council.},
}

@article {pmid41789142,
year = {2026},
author = {Xu, C and Zeng, C and Wang, M and Wei, X and Song, M and Liu, X and Wang, W and Chen, Q and Ji, X and Luo, P and Ma, L and Sun, Y and Gou, H and Zhu, Z and Li, X and Lv, YX and Liu, P and Zhu, JK},
title = {mRNA-engineered CRISPR-Cas epigenetic editors enable durable and efficient gene silencing in vivo.},
journal = {Innovation (Cambridge (Mass.))},
volume = {7},
number = {3},
pages = {101151},
pmid = {41789142},
issn = {2666-6758},
abstract = {Programmable epigenetic editors (EEs) that achieve long-term gene expression modulation without altering the DNA sequence hold immense therapeutic potential. However, the clinical translation of current CRISPR-based epigenome editors is impeded by substantial challenges, particularly their large molecular size, which limits efficient in vivo delivery. Here, we report the rational design and engineering of compact, mRNA-delivered EEs (CRISPR OFF-EE) using Streptococcus pyogenes Cas9 (SpCas9), intein-split-SpCas9, or the smaller Cas-SF01 (a Cas12i3 variant). Combined with optimized mRNA architecture and lipid nanoparticle (LNP) delivery, a single intravenous LNP administration of the optimized OFF-EE V2 mRNA, along with selected guide RNAs (gRNAs) targeting Pcsk9 in mice, resulted in an ∼83.2% reduction in circulating PCSK9 levels and a corresponding ∼51.4% reduction in low-density lipoprotein cholesterol (LDL-C) levels, persisting for at least 180 days. SF01-based EEs showed higher specificity with fewer off-target methylation events than SpCas9-based counterparts. Our optimized LNP formulation also demonstrated a favorable safety profile with predominantly liver-tropic activity. These findings establish a robust and versatile platform for advancing in vivo therapeutics based on precise and durable epigenetic silencing using transiently delivered, engineered mRNA editors.},
}

@article {pmid41785880,
year = {2026},
author = {Petersen, AØ and Damholt, B and Grove, M and Hink, J and Marotte-Hurbon, T and Söderqvist, J and Troy, A and Zdravkovic, M and Bayer, L and Brunner, K and Bryde, T and Clube, J and Gencay, YE and Gram, A and Haaber, JK and Hallström, B and Jasinskytė, D and Pascal, R and Petersen, M and Semsey, S and Torio, AS and Turcu, IC and Smrekar, F and Taur, Y and Satlin, MJ and Sommer, MOA and van der Helm, E and Grøndahl, C},
title = {Safety, recovery, and pharmacodynamics of CRISPR-Cas therapeutic SNIPR001: a phase 1, randomised, double-blind, first-in-human, dose-escalation study.},
journal = {The Lancet. Microbe},
volume = {},
number = {},
pages = {101257},
doi = {10.1016/j.lanmic.2025.101257},
pmid = {41785880},
issn = {2666-5247},
abstract = {BACKGROUND: Patients with haematological cancer who receive stem-cell transplantation are at risk of bloodstream infections, often caused by multidrug resistant gut pathogens such as Escherichia coli. SNIPR001 is a cocktail of four CRISPR-Cas-armed bacteriophages that reduce colonisation of E coli in the gastrointestinal tract in animal models and is designed to not affect other members of the commensal microbiota. We aimed to investigate the safety and tolerability of SNIPR001 in healthy participants.

METHODS: In this randomised, placebo-controlled, double-blind, first-in-human, dose-escalation trial conducted at a single centre (Medpace Clinical Pharmacology Unit; Cincinnati, OH, USA), we sequentially enrolled healthy participants (aged 18-65 years) with more than 10[7]E coli colony-forming units per gram of stool into cohorts 1, 2, and 3, pending a safety review of the previous enrolment group where applicable. Participants in each cohort were randomly assigned to treatment or placebo using a unique three-digit participant identification number. Participants were orally administered 10[8] plaque-forming units (PFU) per dose (cohort 1), 10[10] PFU per dose (cohort 2), and 10[12] PFU per dose (cohort 3) of SNIPR001 or placebo (phosphate-buffered saline buffer), twice daily for 7 days. All personnel, except for a pharmacy staff member who prepared both SNIPR001 and placebo vials, were masked to the administered dose and assignment; masking was ensured by fully covering the surface of each vial. Participants were followed up to day 187. The primary outcome was the incidence and severity of adverse events and medically attended adverse events from the first administration of the study drug until 4 weeks after the last dose administration on day 35 of the study. Recovery and biodistribution of SNIPR001 in faeces, blood, and urine; pharmacodynamics, including the ability of SNIPR001 to reduce E coli levels in stool (assessed using a linear mixed-effects model); and microbiome composition (using Bray-Curtis dissimilarity) were secondary outcomes. Primary safety analyses were assessed per-protocol (ie, all enrolled participants who received at least one administration of the study drug). This trial was conducted under an Investigational New Drug application from the US Food and Drug Administration, is registered with ClinicalTrials.gov (NCT05277350), and is closed to new participants.

FINDINGS: The trial was carried out between March 24, 2022, and Nov 30, 2022. 36 eligible participants were randomly assigned to receive SNIPR001 or placebo in cohorts 1 (six assigned to 10[8] PFU per dose and two assigned to placebo), 2 (six to 10[10] PFU per dose and two to placebo), and 3 (12 to 10[12] PFU per dose and eight to placebo). The mean age of participants was 42·1 years (SD 13·8), with 14 (39%) female participants and 22 (61%) male participants. During the trial and 4-week follow-up period, only mild and moderate adverse events were observed, with most adverse events occurring in the placebo group (13, six, one, and nine for participants receiving either placebo or SNIPR001 at 10[8], 10[10], and 10[12] PFU twice a day, respectively). The number of participants who had adverse events was not significantly higher in treatment groups than in the placebo group (p=0·94, one-sided Fisher's exact test). The most frequently reported adverse events were headaches and diarrhoea. No grade 3-4 adverse events were reported and no serious adverse events were reported in the SNIPR001 dose groups. During and after the dosing period, the gut microbiota composition did not significantly differ between the treatment and placebo groups (p>0·05, two-sided Mann-Whitney U test of Bray-Curtis distances, false discovery rate [FDR]-corrected). Functional SNIPR001 was recovered from stool samples in concentrations proportional to the administered dose but was not meaningfully detected in plasma (only one sample) or urine (only one sample). SNIPR001 was undetected in all samples 6 months after the last dosing, which is a favourable pharmacokinetic property and meets regulatory expectations. We observed the largest reduction in E coli levels compared with placebo 2 weeks after treatment initiation at day 14 (78%; -0·65 log10 [SE 0·64] for 10[12] PFU SNIPR001 twice-daily group), according to a linear mixed-effects model for the highest dose population; however, this change was not statistically significant (p=0·811, linear mixed-effects model, FDR-corrected).

INTERPRETATION: This first-in-human study of SNIPR001 supported its safety, tolerability, and restriction to the gastrointestinal tract, while not systemically disrupting the gut microbiome. These results justify further clinical development of SNIPR001 in an ongoing phase 1b/2a trial.

FUNDING: Combating Antibiotic-Resistant Bacteria Biopharmaceutical Accelerator (CARB-X) and SNIPR Biome.},
}

@article {pmid41721525,
year = {2026},
author = {Chen, N and Gao, M and Bai, Y and Wang, M and Liu, M and Xiong, W and Mo, B},
title = {5'UTR Editing of the ribosomal protein UL3Z gene unveils its critical roles in pre-rRNA processing and global mRNA translation dynamics.},
journal = {Plant physiology},
volume = {200},
number = {3},
pages = {},
doi = {10.1093/plphys/kiag073},
pmid = {41721525},
issn = {1532-2548},
support = {32470592//National Natural Science Foundation of China/ ; 32270595//National Natural Science Foundation of China/ ; 2023B001//SZU 2035 Excellence Research Program/ ; },
mesh = {*5' Untranslated Regions/genetics ; *Ribosomal Proteins/genetics/metabolism ; *Arabidopsis/genetics/metabolism ; *RNA Precursors/metabolism/genetics ; *Arabidopsis Proteins/genetics/metabolism ; *Protein Biosynthesis/genetics ; RNA, Messenger/metabolism/genetics ; Gene Expression Regulation, Plant ; *RNA Processing, Post-Transcriptional/genetics ; Gene Editing ; CRISPR-Cas Systems ; Plants, Genetically Modified ; },
abstract = {The EMBRYO DEFECTIVE 2207 (EMB2207) gene, encoding ribosomal protein UL3Z, is critical for embryonic development in Arabidopsis, with loss of function resulting in embryo lethality. Despite its importance, the role of UL3Z in the complicated protein translation machinery in plants remains poorly understood due to the lack of viable hypomorphic alleles. In this study, we utilized CRISPR/Cas9 to edit the 5' untranslated region (5'UTR) of UL3Z, generating 5 ul3z mutants with varying degrees of reduced expression levels of UL3Z proteins. The mutant with the lowest expression exhibited the most severe developmental defects. In contrast, null mutants of its paralog UL3Y displayed no observable phenotypes. Interestingly, expression of UL3Y driven by the UL3Z/EMB2207 promoter successfully rescued the phenotypes of ul3z, demonstrating that these 2 paralogous ribosomal proteins actually possess functionally interchangeable roles. GUS staining results showed that UL3Z was constitutively expressed in all examined tissues, while UL3Y was only appreciably expressed in specific tissues. Molecular analysis further revealed the accumulation of ribosomal RNA (rRNA) maturation intermediates and increased polysome levels in ul3z mutants, indicating compromised pre-rRNA processing and disturbed global mRNA translation. Interestingly, 3' ends of many rRNA precursors in ul3z had higher frequency of non-encoded tails compared with Col-0. This study demonstrates that CRISPR/Cas9-mediated 5'UTR editing is an effective tool for generating viable hypomorphic alleles of lethal genes and uncovers the critical roles of UL3Z/EMB2207 in pre-rRNA processing and the maintenance of appropriate mRNA translation on ribosomes, underscoring its importance in plant development.},
}

*5' Untranslated Regions/genetics
*Ribosomal Proteins/genetics/metabolism
*Arabidopsis/genetics/metabolism
*RNA Precursors/metabolism/genetics
*Arabidopsis Proteins/genetics/metabolism
*Protein Biosynthesis/genetics
RNA, Messenger/metabolism/genetics
Gene Expression Regulation, Plant
*RNA Processing, Post-Transcriptional/genetics
Gene Editing
CRISPR-Cas Systems
Plants, Genetically Modified

@article {pmid41650957,
year = {2026},
author = {Shi, B and Li, J and Wang, X and Liu, D and Xiang, J and Wang, H and Xu, C and Zou, X and Wang, Z and Huang, T and Min, Q and Wang, K and Yang, Y and Li, J and Wang, B and Zhao, C and Pei, D},
title = {Generating high-quality porcine iPSCs with the new medium cocktail LACID.},
journal = {Stem cell reports},
volume = {21},
number = {3},
pages = {102790},
doi = {10.1016/j.stemcr.2026.102790},
pmid = {41650957},
issn = {2213-6711},
mesh = {Animals ; *Induced Pluripotent Stem Cells/cytology/metabolism/drug effects ; Swine ; Cellular Reprogramming ; *Culture Media/pharmacology/chemistry ; Blastocyst/cytology/metabolism ; *Cell Culture Techniques/methods ; CRISPR-Cas Systems ; Nuclear Transfer Techniques ; Cells, Cultured ; Cell Differentiation ; },
abstract = {Pigs are important for disease model generation, xenotransplantation, and interspecies organogenesis. Porcine induced pluripotent stem cells (piPSCs) should enable these efforts, but have not been generated to meet the attributes, such as feeder-free culture, robust development potential, and blastocyst generation through nuclear transfer. We report an improved strategy to generate such piPSCs. We show that chemically defined medium 3 promotes the formation of epithelium-like colonies in porcine reprogramming, which allows further reprogramming under the new medium cocktail LACID. The resulting piPSCs have key features, including flat morphology with feeder-free culture, generating robust teratoma and blastoids, forming chimeric blastocysts, and readily edited with CRISPR-Cas9. Lastly, nuclear transfer with piPSCs can develop into blastocysts. Despite maintaining a primed pluripotent state, our results suggest that the newly established LACID piPSCs may be ideal for applications in regenerative medicine. This method may be further improved to generate naive or totipotent stem cells.},
}

Animals
*Induced Pluripotent Stem Cells/cytology/metabolism/drug effects
Swine
Cellular Reprogramming
*Culture Media/pharmacology/chemistry
Blastocyst/cytology/metabolism
*Cell Culture Techniques/methods
CRISPR-Cas Systems
Nuclear Transfer Techniques
Cells, Cultured
Cell Differentiation

@article {pmid41517973,
year = {2026},
author = {Jin, G and Li, H and Yu, H and Gu, Z and Cui, H and Chen, J and Li, X},
title = {Asp-2078-Gly mutation in ACCase confers quizalofop-p-ethyl resistance in Eleusine indica and establishment of a LAMP-CRISPR/Cas12a visual genotyping assay for the target mutation.},
journal = {Pest management science},
volume = {82},
number = {4},
pages = {3374-3383},
doi = {10.1002/ps.70468},
pmid = {41517973},
issn = {1526-4998},
support = {//the Key Project in Soybean Bio breeding and Commercialization/ ; //the China Agriculture Research System/ ; 2022ZD04021//the Sci-Tech Innovation 2030 Agenda/ ; },
mesh = {*Herbicide Resistance/genetics ; *Acetyl-CoA Carboxylase/genetics/metabolism ; *Eleusine/genetics/drug effects/enzymology ; *Nucleic Acid Amplification Techniques/methods ; CRISPR-Cas Systems ; *Herbicides/pharmacology ; Mutation ; *Genotyping Techniques/methods ; *Plant Proteins/genetics/metabolism ; Molecular Diagnostic Techniques ; },
abstract = {BACKGROUND: Eleusine indica is a widespread, competitive weed causing yield losses in major crops. Repeated use of acetyl-CoA carboxylase (ACCase)-inhibiting herbicides in cotton fields has led to the evolution of resistant populations, posing a growing threat to cotton production in China. This study aimed to elucidate the target-site resistance mechanism of E. indica to quizalofop-p-ethyl and establish a rapid visual detection method based on the identified mutation.

RESULTS: The NJC-R population showed resistance to quizalofop-p-ethyl (resistance index = 5.5). Gene sequencing revealed that an Asp-2078-Gly mutation in ACCase was one of the mechanisms underlying resistance. Loop-mediated isothermal amplification (LAMP) combined with the CRISPR/Cas12a system was developed to detect this mutation in E. indica. This method not only enabled genotype discrimination (wild-type, heterozygous, homozygous mutant), but also provided visual results within 70 min, exhibiting superior performance compared with the derived cleaved amplified polymorphic sequences assay. In addition, this method eliminated false positives from nonspecific LAMP amplification, was ~100-fold more sensitive than a polymerase chain reaction, and the assay results were 100% concordant with Sanger sequencing for the 50 samples tested.

CONCLUSION: This study confirmed that the Asp-2078-Gly mutation confers quizalofop-p-ethyl resistance in E. indica from the cotton field in China, and LAMP-CRISPR/Cas12a was first applied for detecting ACCase target-site mutations in E. indica. Given its rapidity and high accuracy, this technique has the potential to be applied for resistance monitoring and to guide rational herbicide application. © 2026 Society of Chemical Industry.},
}

*Herbicide Resistance/genetics
*Acetyl-CoA Carboxylase/genetics/metabolism
*Eleusine/genetics/drug effects/enzymology
*Nucleic Acid Amplification Techniques/methods
CRISPR-Cas Systems
*Herbicides/pharmacology
Mutation
*Genotyping Techniques/methods
*Plant Proteins/genetics/metabolism
Molecular Diagnostic Techniques

@article {pmid41445368,
year = {2026},
author = {Wei, J and Jiang, C and Chen, Y and Yang, X and Li, Q},
title = {Functional characterization of Hsk1 and Chit1 genes in the virulence of Metarhizium guizhouense Xct1 via CRISPR-Cas9-mediated gene editing.},
journal = {Pest management science},
volume = {82},
number = {4},
pages = {3625-3639},
doi = {10.1002/ps.70484},
pmid = {41445368},
issn = {1526-4998},
support = {//SCCXTD-2024-04/ ; },
mesh = {*CRISPR-Cas Systems ; Virulence/genetics ; Gene Editing ; Animals ; *Metarhizium/genetics/pathogenicity ; *Fungal Proteins/genetics/metabolism ; Spodoptera/growth & development/microbiology ; *Protein Serine-Threonine Kinases/genetics/metabolism ; Larva/growth & development/microbiology ; Pest Control, Biological ; },
abstract = {BACKGROUND: The entomopathogenic fungus Metarhizium guizhouense Xct1 exhibits high virulence against early-instar Spodoptera frugiperda larvae (>90% mortality in preliminary studies), yet the molecular mechanisms, particularly the roles of key genes such as the chitin-degrading enzyme (Chit1) and serine/threonine kinase (Hsk1) are poorly understood. Functional studies using CRISPR-Cas9 are lacking, limiting its biocontrol application.

RESULTS: Chit1 and Hsk1 genes were amplified from M. guizhouense Xct1. Chit1 showed high homology to M. anisopliae, whereas Hsk1 exhibited greater genetic diversity. Expression analysis revealed peak Chit1 expression on Day (D)4 and peak Hsk1 expression on D2. A CRISPR-Cas9 system was established, and knockout of Chit1 resulted in thickened cell walls [119 nm versus 87 nm in wild-type (WT)] and reduced virulence [median lethal time (LT50) = 7.4 days versus 4.8 days in WT]. Overexpression of Chit1 improved virulence (LT50 = 3.3 days). Hsk1 knockout was lethal, confirming its essential role, while overexpression did not alter virulence (LT50 = 4.8 days).

CONCLUSIONS: Chit1 is a critical virulence factor, influencing cell-wall integrity and insecticidal activity, while Hsk1 is essential for fungal viability. This study presents the first CRISPR-Cas9-mediated functional analysis of these genes, revealing that Chit1 overexpression enhances biocontrol efficacy against S. frugiperda. © 2025 Society of Chemical Industry.},
}

*CRISPR-Cas Systems
Virulence/genetics
Gene Editing
Animals
*Metarhizium/genetics/pathogenicity
*Fungal Proteins/genetics/metabolism
Spodoptera/growth & development/microbiology
*Protein Serine-Threonine Kinases/genetics/metabolism
Larva/growth & development/microbiology
Pest Control, Biological

@article {pmid41057363,
year = {2025},
author = {Li, J and Pan, Z and Peng, X and Feng, Y and Wu, J and Liang, F and Feng, Q and Yu, X and Deng, H},
title = {Ecdysone signaling-induced dumpless1 expression controls nurse cell dumping in Drosophila oogenesis.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {8917},
pmid = {41057363},
issn = {2041-1723},
support = {32170494//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Oogenesis/genetics/physiology ; *Ecdysone/genetics/metabolism ; *Drosophila Proteins/genetics/metabolism ; Animals ; Signal Transduction ; *Drosophila melanogaster/genetics/metabolism ; rho GTP-Binding Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Ovarian Follicle/cytology/metabolism ; Integrin beta Chains/genetics/metabolism ; Transcription Factors/genetics/metabolism ; Oocytes/cytology/metabolism ; Female ; },
abstract = {Nurse cell (NC) dumping, a process essential for oocyte development, involves the rapid cytoplasmic transfer from germline-derived NCs into the oocyte. However, its regulatory mechanism remains unclear. Here, we report that ecdysone signaling in stretch follicle cells (SFCs) regulates NC dumping through dumpless1, a ZAD-C2H2 zinc finger transcription factor, in Drosophila. Ecdysone induced dumpless1 expression in SFCs, and CRISPR/Cas9-mediated knockout of dumpless1 or its functional domain ZAD suppresses NC dumping. Depletion of dumpless1 upregulates integrin βPS expression in SFC plasma membrane, while reducing cortical enrichment of Rho1 signaling-dependent phosphorylated myosin light chain (p-MLC) and disrupting actin cables organization in NCs. SFC-specific overexpression of integrin βPS reduces p-MLC enrichment in the NC cortex, whereas its knockdown in SFCs of dumpless1[-/-] mutants partially rescues NC dumping defect. Our findings identify dumpless1 as a critical effector of ecdysone signaling, bridging somatic-germline communication through the integrin βPS-Rho1-p-MLC axis, revealing a multicellular regulatory mechanism in Drosophila oogenesis.},
}

*Oogenesis/genetics/physiology
*Ecdysone/genetics/metabolism
*Drosophila Proteins/genetics/metabolism
Animals
Signal Transduction
*Drosophila melanogaster/genetics/metabolism
rho GTP-Binding Proteins/genetics/metabolism
CRISPR-Cas Systems
Ovarian Follicle/cytology/metabolism
Integrin beta Chains/genetics/metabolism
Transcription Factors/genetics/metabolism
Oocytes/cytology/metabolism
Female

@article {pmid37766223,
year = {2023},
author = {Zhang, H and Duan, K and Du, Y and Xiao, S and Fang, L and Zhou, Y},
title = {One-Step Assembly of a PRRSV Infectious cDNA Clone and a Convenient CRISPR/Cas9-Based Gene-Editing Technology for Manipulation of PRRSV Genome.},
journal = {Viruses},
volume = {15},
number = {9},
pages = {},
pmid = {37766223},
issn = {1999-4915},
support = {32130103//National Natural Science Foundation of China/ ; 32002279//National Natural Science Foundation of China/ ; },
mesh = {*Porcine respiratory and reproductive syndrome virus/genetics ; Animals ; *CRISPR-Cas Systems ; *Genome, Viral ; *Gene Editing/methods ; Swine ; *DNA, Complementary/genetics ; Porcine Reproductive and Respiratory Syndrome/virology ; Reverse Genetics ; Cell Line ; },
abstract = {Porcine reproductive and respiratory syndrome (PRRS) has been a persistent challenge for the swine industry for over three decades due to the lack of effective treatments and vaccines. Reverse genetics systems have been extensively employed to build rapid drug screening platforms and develop genetically engineered vaccines. Herein, we rescued recombinant PRRS virus (rPRRSV) WUH3 using an infectious cDNA clone of PRRSV WUH3 acquired through a BstXI-based one-step-assembly approach. The rPRRSV WUH3 and its parental PRRSV WUH3 share similar plaque sizes and multiple-step growth curves. Previously, gene-editing of viral genomes depends on appropriate restrictive endonucleases, which are arduous to select in some specific viral genes. Thus, we developed a restrictive endonucleases-free method based on CRISPR/Cas9 to edit the PRRSV genome. Using this method, we successfully inserted the exogenous gene (EGFP gene as an example) into the interval between ORF1b and ORF2a of the PRRSV genome to generate rPRRSV WUH3-EGFP, or precisely mutated the lysine (K) at position 150 of PRRSV nsp1α to glutamine (Q) to acquire rPRRSV WUH3 nsp1α-K150Q. Taken together, our study provides a rapid and convenient method for the development of genetically engineered vaccines against PRRSV and the study on the functions of PRRSV genes.},
}

*Porcine respiratory and reproductive syndrome virus/genetics
Animals
*CRISPR-Cas Systems
*Genome, Viral
*Gene Editing/methods
Swine
*DNA, Complementary/genetics
Porcine Reproductive and Respiratory Syndrome/virology
Reverse Genetics
Cell Line

@article {pmid37722405,
year = {2023},
author = {Low, SJ and O'Neill, MT and Kerry, WJ and Krysiak, M and Papadakis, G and Whitehead, LW and Savic, I and Prestedge, J and Williams, L and Cooney, JP and Tran, T and Lim, CK and Caly, L and Towns, JM and Bradshaw, CS and Fairley, C and Chow, EPF and Chen, MY and Pellegrini, M and Pasricha, S and Williamson, DA},
title = {Rapid detection of monkeypox virus using a CRISPR-Cas12a mediated assay: a laboratory validation and evaluation study.},
journal = {The Lancet. Microbe},
volume = {4},
number = {10},
pages = {e800-e810},
doi = {10.1016/S2666-5247(23)00148-9},
pmid = {37722405},
issn = {2666-5247},
mesh = {*CRISPR-Cas Systems ; Humans ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; *Monkeypox virus/isolation & purification/genetics ; *Molecular Diagnostic Techniques/methods ; *Mpox, Monkeypox/diagnosis/virology ; Limit of Detection ; Proof of Concept Study ; Point-of-Care Systems ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BACKGROUND: The 2022 outbreak of mpox (formerly known as monkeypox) led to the spread of monkeypox virus (MPXV) in over 110 countries, demanding effective disease management and surveillance. As current diagnostics rely largely on centralised laboratory testing, our objective was to develop a simple rapid point-of-care assay to detect MPXV in clinical samples using isothermal amplification coupled with CRISPR and CRISPR-associated protein (Cas) technology.

METHODS: In this proof-of-concept study, we developed a portable isothermal amplification CRISPR-Cas12a-based assay for the detection of MPXV. We designed a panel of 22 primer-guide RNA sets using pangenome and gene-agnostic approaches, and subsequently shortlisted the three sets producing the strongest signals for evaluation of analytical sensitivity and specificity using a fluorescence-based readout. The set displaying 100% specificity and the lowest limit of detection (LOD) was selected for further assay validation using both a fluorescence-based and lateral-flow readout. Assay specificity was confirmed using a panel of viral and bacterial pathogens. Finally, we did a blind concordance study on genomic DNA extracted from 185 clinical samples, comparing assay results with a gold-standard quantitative PCR (qPCR) assay. We identified the optimal time to detection and analysed the performance of the assay relative to qPCR using receiver operating characteristic (ROC) curves. We also assessed the compatibility with lateral-flow strips, both visually and computationally, where strips were interpreted blinded to the fluorescence results on the basis of the presence or absence of test bands.

FINDINGS: With an optimal run duration of approximately 45 min from isothermal amplification to CRISPR-assay readout, the MPXV recombinase polymerase amplification CRISPR-Cas12a-based assay with the selected primer-guide set had an LOD of 1 copy per μL and 100% specificity against tested viral pathogens. Blinded concordance testing of 185 clinical samples resulted in 100% sensitivity (95% CI 89·3-100) and 99·3% specificity (95% CI 95·7-100) using the fluorescence readout. For optimal time to detection by fluorescence readout, we estimated the areas under the ROC curve to be 0·98 at 2 min and 0·99 at 4 min. Lateral-flow strips had 100% sensitivity (89·3-100) and 98·6% specificity (94·7-100) with both visual and computational assessment. Overall, lateral-flow results were highly concordant with fluorescence-based readouts (179 of 185 tests, 96·8% concordant), with discrepancies associated with low viral load samples.

INTERPRETATION: Our assay for the diagnosis of mpox displayed good performance characteristics compared with qPCR. Although optimisation of the assay will be required before deployment, its usability and versatility present a potential solution to MPXV detection in low-resource and remote settings, as well as a means of community-based, on-site testing.

FUNDING: Victorian Medical Research Accelerator Fund and the Australian Government Department of Health.},
}

*CRISPR-Cas Systems
Humans
Sensitivity and Specificity
*Nucleic Acid Amplification Techniques/methods
*Monkeypox virus/isolation & purification/genetics
*Molecular Diagnostic Techniques/methods
*Mpox, Monkeypox/diagnosis/virology
Limit of Detection
Proof of Concept Study
Point-of-Care Systems
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid37721041,
year = {2023},
author = {Gentili, HG and Pignataro, MF and Olmos, J and Pavan, MF and Ibañez, LI and Santos, J and Velazquez Duarte, F},
title = {CRISPR/Cas9-based edition of frataxin gene in Dictyostelium discoideum.},
journal = {The Biochemical journal},
volume = {480},
number = {19},
pages = {1533-1551},
doi = {10.1042/BCJ20230244},
pmid = {37721041},
issn = {1470-8728},
mesh = {*Dictyostelium/genetics/metabolism/growth & development ; Frataxin ; *Iron-Binding Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Humans ; *Gene Editing/methods ; *Protozoan Proteins/genetics/metabolism ; Mutation ; },
abstract = {In this paper, we describe the development of a Dictyostelium discoideum strain deficient in frataxin protein (FXN). We investigated the conservation of function between humans and D. discoideum and showed that DdFXN can substitute the human version in the interaction and activation of the Fe-S assembly supercomplex. We edited the D. discoideum fxn locus and isolated a defective mutant, clone 8, which presents landmarks of frataxin deficiency, such as a decrease in Fe-S cluster-dependent enzymatic functions, growth rate reduction, and increased sensitivity to oxidative stress. In addition, the multicellular development is affected as well as growing on bacterial lawn. We also assessed the rescuing capacity of DdFXN-G122V, a version that mimics a human variant present in some FA patients. While the expression of DdFXN-G122V rescues growth and enzymatic activity defects, as DdFXN does, multicellular development defects were only partially rescued. The results of the study suggest that this new D. discoideum strain offers a wide range of possibilities to easily explore diverse FA FXN variants. This can facilitate the development of straightforward drug screenings to look for new therapeutic strategies.},
}

*Dictyostelium/genetics/metabolism/growth & development
Frataxin
*Iron-Binding Proteins/genetics/metabolism
*CRISPR-Cas Systems
Humans
*Gene Editing/methods
*Protozoan Proteins/genetics/metabolism
Mutation

@article {pmid37718190,
year = {2023},
author = {Kalinna, BH},
title = {Programming schistosomes - a crisper approach to transgenesis.},
journal = {Trends in parasitology},
volume = {39},
number = {11},
pages = {896-897},
doi = {10.1016/j.pt.2023.09.003},
pmid = {37718190},
issn = {1471-5007},
mesh = {Animals ; *Schistosoma mansoni/genetics ; *Gene Transfer Techniques ; CRISPR-Cas Systems ; *Schistosoma/genetics ; Transgenes ; },
abstract = {Ittiprasert and colleagues identified genomic safe harbour (GSH) sites in Schistosoma mansoni using computational methods and inserted a transgene into one of the sites through clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-assisted homology-directed repair. This study outlines a promising strategy for functional genomics to study this parasite that causes a debilitating and neglected tropical disease.},
}

Animals
*Schistosoma mansoni/genetics
*Gene Transfer Techniques
CRISPR-Cas Systems
*Schistosoma/genetics
Transgenes

@article {pmid37702151,
year = {2023},
author = {van Schie, JJM and de Lint, K and Molenaar, TM and Moronta Gines, M and Balk, JA and Rooimans, MA and Roohollahi, K and Pai, GM and Borghuis, L and Ramadhin, AR and Corazza, F and Dorsman, JC and Wendt, KS and Wolthuis, RMF and de Lange, J},
title = {CRISPR screens in sister chromatid cohesion defective cells reveal PAXIP1-PAGR1 as regulator of chromatin association of cohesin.},
journal = {Nucleic acids research},
volume = {51},
number = {18},
pages = {9594-9609},
pmid = {37702151},
issn = {1362-4962},
support = {10701//Dutch Cancer Society/ ; },
mesh = {Cohesins ; Humans ; *Cell Cycle Proteins/metabolism/genetics ; *Chromosomal Proteins, Non-Histone/metabolism/genetics ; *Chromatin/metabolism/genetics ; *Chromatids/genetics/metabolism ; CRISPR-Cas Systems ; DNA-Binding Proteins/genetics/metabolism ; Mitosis/genetics ; DNA Replication/genetics ; Nuclear Proteins/genetics/metabolism ; Sister Chromatid Exchange/genetics ; Cell Line ; Acetyltransferases ; DNA Helicases ; DEAD-box RNA Helicases ; },
abstract = {The cohesin complex regulates higher order chromosome architecture through maintaining sister chromatid cohesion and folding chromatin by DNA loop extrusion. Impaired cohesin function underlies a heterogeneous group of genetic syndromes and is associated with cancer. Here, we mapped the genetic dependencies of human cell lines defective of cohesion regulators DDX11 and ESCO2. The obtained synthetic lethality networks are strongly enriched for genes involved in DNA replication and mitosis and support the existence of parallel sister chromatid cohesion establishment pathways. Among the hits, we identify the chromatin binding, BRCT-domain containing protein PAXIP1 as a novel cohesin regulator. Depletion of PAXIP1 severely aggravates cohesion defects in ESCO2 mutant cells, leading to mitotic cell death. PAXIP1 promotes global chromatin association of cohesin, independent of DNA replication, a function that cannot be explained by indirect effects of PAXIP1 on transcription or DNA repair. Cohesin regulation by PAXIP1 requires its binding partner PAGR1 and a conserved FDF motif in PAGR1. PAXIP1 co-localizes with cohesin on multiple genomic loci, including active gene promoters and enhancers. Possibly, this newly identified role of PAXIP1-PAGR1 in regulating cohesin occupancy on chromatin is also relevant for previously described functions of PAXIP1 in transcription, immune cell maturation and DNA repair.},
}

Cohesins
Humans
*Cell Cycle Proteins/metabolism/genetics
*Chromosomal Proteins, Non-Histone/metabolism/genetics
*Chromatin/metabolism/genetics
*Chromatids/genetics/metabolism
CRISPR-Cas Systems
DNA-Binding Proteins/genetics/metabolism
Mitosis/genetics
DNA Replication/genetics
Nuclear Proteins/genetics/metabolism
Sister Chromatid Exchange/genetics
Cell Line
Acetyltransferases
DNA Helicases
DEAD-box RNA Helicases

@article {pmid37695673,
year = {2023},
author = {Zhang, H and Li, X and Wang, Y and Liu, X and Guo, J and Wang, Z and Zhang, L and Xiong, S and Dong, C},
title = {Genome-Wide CRISPR/Cas9 Screening Identifies That Mitochondrial Solute Carrier SLC25A23 Attenuates Type I IFN Antiviral Immunity via Interfering with MAVS Aggregation.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {211},
number = {9},
pages = {1406-1417},
doi = {10.4049/jimmunol.2300187},
pmid = {37695673},
issn = {1550-6606},
mesh = {Animals ; Mice ; *Interferon Type I/immunology/metabolism ; CRISPR-Cas Systems/genetics ; *Adaptor Proteins, Signal Transducing/metabolism/immunology/genetics ; Humans ; Ubiquitin-Protein Ligases/metabolism ; *RNA Virus Infections/immunology ; Signal Transduction ; Tripartite Motif Proteins/metabolism ; Ubiquitination ; Immunity, Innate ; HEK293 Cells ; Mice, Inbred C57BL ; *Mitochondrial Membrane Transport Proteins/genetics/metabolism ; Mitochondria/metabolism ; },
abstract = {Activation of the mitochondrial antiviral signaling (MAVS) adaptor, also known as IPS-1, VISA, or Cardif, is crucial for antiviral immunity in retinoic acid-inducible gene I (RIG-I)-like receptor signaling. Upon interacting with RIG-I, MAVS undergoes K63-linked polyubiquitination by the E3 ligase Trim31, and subsequently aggregates to activate downstream signaling effectors. However, the molecular mechanisms that modulate MAVS activation are not yet fully understood. In this study, the mitochondrial solute carrier SLC25A23 was found to attenuate type I IFN antiviral immunity using genome-wide CRISPR/Cas9 screening. SLC25A23 interacts with Trim31, interfering with its binding of Trim31 to MAVS. Indeed, SLC25A23 downregulation was found to increase K63-linked polyubiquitination and subsequent aggregation of MAVS, which promoted type I IFN production upon RNA virus infection. Consistently, mice with SLC25A23 knockdown were more resistant to RNA virus infection in vivo. These findings establish SLC25A23 as a novel regulator of MAVS posttranslational modifications and of type I antiviral immunity.},
}

Animals
Mice
*Interferon Type I/immunology/metabolism
CRISPR-Cas Systems/genetics
*Adaptor Proteins, Signal Transducing/metabolism/immunology/genetics
Humans
Ubiquitin-Protein Ligases/metabolism
*RNA Virus Infections/immunology
Signal Transduction
Tripartite Motif Proteins/metabolism
Ubiquitination
Immunity, Innate
HEK293 Cells
Mice, Inbred C57BL
*Mitochondrial Membrane Transport Proteins/genetics/metabolism
Mitochondria/metabolism

@article {pmid37689217,
year = {2023},
author = {Colaco, JC and Chandrasekaran, AP and Karapurkar, JK and Gowda, DAA and Birappa, G and Rajkumar, S and Suresh, B and Ko, N and Hong, SH and Oh, SJ and Kim, KS and Ramakrishna, S},
title = {E3 ubiquitin ligase APC/C[Cdh1] regulates SLC35F2 protein turnover and inhibits cancer progression in HeLa cells.},
journal = {Biochimica et biophysica acta. General subjects},
volume = {1867},
number = {11},
pages = {130454},
doi = {10.1016/j.bbagen.2023.130454},
pmid = {37689217},
issn = {1872-8006},
mesh = {Humans ; HeLa Cells ; Ubiquitination ; *Ubiquitin-Protein Ligases/metabolism/genetics ; Disease Progression ; *Neoplasms/metabolism/pathology/genetics ; CRISPR-Cas Systems ; Cell Proliferation ; Adenomatous Polyposis Coli Protein ; },
abstract = {BACKGROUND: The solute carrier family 35 F2 (SLC35F2), belongs to membrane-bound carrier proteins that control various physiological functions and are activated in several cancers. However, the molecular mechanism regulating SLC35F2 protein turnover and its implication in cancer progression remains unexplored. Therefore, screening for E3 ligases that promote SLC35F2 protein degradation is essential during cancer progression.

METHODS: The immunoprecipitation and Duolink proximity ligation assays (PLA) were used to determine the interaction between APC/C[Cdh1] and SLC35F2 proteins. A CRISPR/Cas9-mediated knockdown and rescue experiment were used to validate the functional significance of APC/C[Cdh1] on SLC35F2 protein stabilization. The ubiquitination function of APC/C[Cdh1] on SLC35F2 protein was validated using in vitro ubiquitination assay and half-life analysis. The role of APC/C[Cdh1] regulating SLC35F2-mediated tumorigenesis was confirmed by in vitro oncogenic experiments in HeLa cells.

RESULTS: Based on the E3 ligase screen and in vitro biochemical experiments, we identified that APC/C[Cdh1] interacts with and reduces SLC35F2 protein level. APC/C[Cdh1] promotes SLC35F2 ubiquitination and decreases the half-life of SLC35F2 protein. On the other hand, the CRISPR/Cas9-mediated depletion of APC/C[Cdh1] increased SLC35F2 protein levels. The mRNA expression analysis revealed a negative correlation between APC/C[Cdh1] and SLC35F2 across a panel of cancer cell lines tested. Additionally, we demonstrated that depletion in APC/C[Cdh1] promotes SLC35F2-mediated cell proliferation, colony formation, migration, and invasion in HeLa cells.

CONCLUSION: Our study highlights that APC/C[Cdh1] is a critical regulator of SLC35F2 protein turnover and depletion of APC/C[Cdh1] promotes SLC35F2-mediated tumorigenesis. Thus, we envision that APC/C[Cdh1]-SLC35F2 axis might be a therapeutic target in cancer.},
}

Humans
HeLa Cells
Ubiquitination
*Ubiquitin-Protein Ligases/metabolism/genetics
Disease Progression
*Neoplasms/metabolism/pathology/genetics
CRISPR-Cas Systems
Cell Proliferation
Adenomatous Polyposis Coli Protein

@article {pmid37688599,
year = {2023},
author = {Minnaar, L and den Haan, R},
title = {Engineering natural isolates of Saccharomyces cerevisiae for consolidated bioprocessing of cellulosic feedstocks.},
journal = {Applied microbiology and biotechnology},
volume = {107},
number = {22},
pages = {7013-7028},
pmid = {37688599},
issn = {1432-0614},
support = {137967//National Research Foundation/ ; },
mesh = {*Saccharomyces cerevisiae/genetics/metabolism/isolation & purification/growth & development ; *Cellulose/metabolism ; Ethanol/metabolism ; Fermentation ; *Cellulases/genetics/metabolism ; CRISPR-Cas Systems ; *Metabolic Engineering ; },
abstract = {Saccharomyces cerevisiae has gained much attention as a potential host for cellulosic bioethanol production using consolidated bioprocessing (CBP) methodologies, due to its high-ethanol-producing titres, heterologous protein production capabilities, and tolerance to various industry-relevant stresses. Since the secretion levels of heterologous proteins are generally low in domesticated strains of S. cerevisiae, natural isolates may offer a more diverse genetic background for improved heterologous protein secretion, while also displaying greater robustness to process stresses. In this study, the potential of natural and industrial S. cerevisiae strains to secrete a core set of cellulases (CBH1, CBH2, EG2, and BGL1), encoded by genes integrated using CRISPR/Cas9 tools, was evaluated. High levels of heterologous protein production were associated with a reduced maximal growth rate and with slight changes in overall strain robustness, compared to the parental strains. The natural isolate derivatives YI13_BECC and YI59_BECC displayed superior secretion capacity for the heterologous cellulases at high incubation temperature and in the presence of acetic acid, respectively, compared to the reference industrial strain MH1000_BECC. These strains also exhibited multi-tolerance to several fermentation-associated and secretion stresses. Cultivation of the strains on crystalline cellulose in oxygen-limited conditions yielded ethanol concentrations in the range of 4-4.5 g/L, representing 35-40% of the theoretical maximum ethanol yield after 120 h, without the addition of exogenous enzymes. This study therefore highlights the potential of these natural isolates to be used as chassis organisms in CBP bioethanol production. KEY POINTS: • Process-related fermentation stresses influence heterologous protein production. • Transformants produced up to 4.5 g/L ethanol, ~ 40% of the theoretical yield in CBP. • CRISPR/Cas9 was feasible for integrating genes in natural S. cerevisiae isolates.},
}

*Saccharomyces cerevisiae/genetics/metabolism/isolation & purification/growth & development
*Cellulose/metabolism
Ethanol/metabolism
Fermentation
*Cellulases/genetics/metabolism
CRISPR-Cas Systems
*Metabolic Engineering

@article {pmid37683439,
year = {2024},
author = {Liu, B and Li, Y and Du, L and Zhang, F and Liu, Y and Sun, J and Zhang, Q and Li, C and Li, X and Xue, Q},
title = {"One-to-many" signal-output strategy-based CRISPR/Cas12a system for sensitive label-free fluorescence detection of HBV-DNA.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {304},
number = {},
pages = {123338},
doi = {10.1016/j.saa.2023.123338},
pmid = {37683439},
issn = {1873-3557},
mesh = {*CRISPR-Cas Systems/genetics ; *DNA, Viral/analysis/genetics ; *Hepatitis B virus/genetics ; *Biosensing Techniques/methods ; Spectrometry, Fluorescence/methods ; Limit of Detection ; Dendrimers/chemistry ; CRISPR-Associated Proteins/metabolism ; Fluorescence ; Nucleic Acid Amplification Techniques ; Humans ; Benzothiazoles ; },
abstract = {Although CRISPR/Cas12a systems significantly enhance the analytical accuracy and flexibility of fluorescent biosensors, their sensitivity is limited by traditional "one-to-one" mediation types and ineffective signal-output turnover routes. Herein, we demonstrate a "one-to-many" signal-output strategy-based CRISPR/Cas12a systems resembling a "seaweed" to enhance the sensitivity. Based on dendrimer DNA from high-dimensional hybridization chain (HCR) of three hairpin-free DNA building blocks, the 3D magnetic DNA machine was created. The HBV-DNA initiates the rolling circle amplification (RCA) reaction and produces DNA nanowires to activate the CRISPR/Cas12a system. The trans-cleavage of the "seaweed root" by CRISPR/Cas12a system left dendrimer DNA in solution, thus, adding SYBR Green I (SG I) to the high-density DNA duplexes, achieving multiple-turnover label-free fluorescence signal output demonstrated and a low LOD (1.502 pM). However, in the absence of target, the blocked RCA failed to activate the CRISPR/Cas12a system, resulting in complete separation from substrate and negligible fluorescence signals. Moreover, the mandatory RCA-based pre-amplification of the DNA activator could efficiently trigger the multiple-turnover trans-cleavage activity of Cas12a. it can cleave one single-stranded linker of "seaweed-like" DNA machine, thereby releasing massive DNA duplex-enriched dendrimer DNA with a "one-to-many" signal-output turnover. By coupling the periodically extended Cas12a activator generated by RCA with hyperbranched DNA duplex by high-dimensional HCR, compact 3D extension structures were formed, achieving high-density fluorescence distribution in focal volume, avoiding signal dilution and ensuring high enhancement. Additionally, spiked recoveries in physiological media exceeded 95%, demonstrating the potential application of such platforms in clinical diagnosis.},
}

*CRISPR-Cas Systems/genetics
*DNA, Viral/analysis/genetics
*Hepatitis B virus/genetics
*Biosensing Techniques/methods
Spectrometry, Fluorescence/methods
Limit of Detection
Dendrimers/chemistry
CRISPR-Associated Proteins/metabolism
Fluorescence
Nucleic Acid Amplification Techniques
Humans
Benzothiazoles

@article {pmid37673694,
year = {2023},
author = {Awan, MJA and Awan, MRA and Amin, I and Mansoor, S},
title = {Fanzor: a compact programmable RNA-guided endonuclease from eukaryotes.},
journal = {Trends in biotechnology},
volume = {41},
number = {11},
pages = {1332-1334},
doi = {10.1016/j.tibtech.2023.08.003},
pmid = {37673694},
issn = {1879-3096},
mesh = {*Endonucleases/genetics/metabolism ; *Gene Editing/methods ; *Eukaryota/genetics/enzymology ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; DNA Transposable Elements/genetics ; },
abstract = {The IS200/605 transposons in prokaryotes are known to harbor programmable endonucleases. Despite carrying their own transposable elements, no such effector has been characterized in eukaryotes. Saito et al. recently reported compact and programmable RNA-guided eukaryotic endonucleases, called Fanzors, that can induce targeted genetic modifications, thus expanding the genome-editing toolbox.},
}

*Endonucleases/genetics/metabolism
*Gene Editing/methods
*Eukaryota/genetics/enzymology
*RNA, Guide, CRISPR-Cas Systems/genetics
Humans
DNA Transposable Elements/genetics

@article {pmid37667014,
year = {2023},
author = {Hooghvorst, I and Altpeter, F},
title = {dCas9-3xSRDX-mediated transcriptional repression in sugarcane.},
journal = {Plant cell reports},
volume = {42},
number = {11},
pages = {1837-1840},
pmid = {37667014},
issn = {1432-203X},
support = {DE-SC0018420//Biological and Environmental Research/ ; },
mesh = {*Saccharum/genetics/metabolism ; Gene Expression Regulation, Plant ; Plants, Genetically Modified ; Plant Proteins/genetics/metabolism ; 5' Untranslated Regions/genetics ; *Transcription, Genetic ; CRISPR-Cas Systems/genetics ; },
abstract = {Targeting dCas9 fused with the 3xSRDX effector to the 5'UTR leads to strong repression of magnesium chelatase in highly polyploid sugarcane.},
}

*Saccharum/genetics/metabolism
Gene Expression Regulation, Plant
Plants, Genetically Modified
Plant Proteins/genetics/metabolism
5' Untranslated Regions/genetics
*Transcription, Genetic
CRISPR-Cas Systems/genetics

@article {pmid37666660,
year = {2023},
author = {Agostini, V and Tessier, A and Djaziri, N and Khonsari, RH and Galliani, E and Kurihara, Y and Honda, M and Kurihara, H and Hidaka, K and Tuncbilek, G and Picard, A and Konas, E and Amiel, J and Gordon, CT},
title = {Biallelic truncating variants in VGLL2 cause syngnathia in humans.},
journal = {Journal of medical genetics},
volume = {60},
number = {11},
pages = {1084-1091},
doi = {10.1136/jmg-2022-109059},
pmid = {37666660},
issn = {1468-6244},
mesh = {Humans ; Zebrafish/genetics ; Animals ; Male ; Female ; *Transcription Factors/genetics ; Alleles ; Pedigree ; Homozygote ; *Craniofacial Abnormalities/genetics/pathology ; *Jaw Abnormalities/genetics/pathology ; Phenotype ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Syngnathia is an ultrarare craniofacial malformation characterised by an inability to open the mouth due to congenital fusion of the upper and lower jaws. The genetic causes of isolated bony syngnathia are unknown.

METHODS: We used whole exome and Sanger sequencing and microsatellite analysis in six patients (from four families) presenting with syngnathia. We used CRISPR/Cas9 genome editing to generate vgll2a and vgll4l germline mutant zebrafish, and performed craniofacial cartilage analysis in homozygous mutants.

RESULTS: We identified homozygous truncating variants in vestigial-like family member 2 (VGLL2) in all six patients. Two alleles were identified: one in families of Turkish origin and the other in families of Moroccan origin, suggesting a founder effect for each. A shared haplotype was confirmed for the Turkish patients. The VGLL family of genes encode cofactors of TEAD transcriptional regulators. Vgll2 is regionally expressed in the pharyngeal arches of model vertebrate embryos, and morpholino-based knockdown of vgll2a in zebrafish has been reported to cause defects in development of pharyngeal arch cartilages. However, we did not observe craniofacial anomalies in vgll2a or vgll4l homozygous mutant zebrafish nor in fish with double knockout of vgll2a and vgll4l. In Vgll2 [-/-] mice, which are known to present a skeletal muscle phenotype, we did not identify defects of the craniofacial skeleton.

CONCLUSION: Our results suggest that although loss of VGLL2 leads to a striking jaw phenotype in humans, other vertebrates may have the capacity to compensate for its absence during craniofacial development.},
}

Humans
Zebrafish/genetics
Animals
Male
Female
*Transcription Factors/genetics
Alleles
Pedigree
Homozygote
*Craniofacial Abnormalities/genetics/pathology
*Jaw Abnormalities/genetics/pathology
Phenotype
CRISPR-Cas Systems

@article {pmid37650631,
year = {2023},
author = {Mohammad, N and Talton, L and Hetzler, Z and Gongireddy, M and Wei, Q},
title = {Unidirectional trans-cleaving behavior of CRISPR-Cas12a unlocks for an ultrasensitive assay using hybrid DNA reporters containing a 3' toehold.},
journal = {Nucleic acids research},
volume = {51},
number = {18},
pages = {9894-9904},
pmid = {37650631},
issn = {1362-4962},
support = {1944167//National Science Foundation/ ; },
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; *Endodeoxyribonucleases/metabolism/genetics ; *DNA/metabolism/chemistry/genetics ; DNA, Single-Stranded/metabolism/genetics ; DNA Cleavage ; *Bacterial Proteins/metabolism/genetics ; },
abstract = {CRISPR-Cas12a can induce nonspecific trans-cleavage of dsDNA substrate, including long and stable λ DNA. However, the mechanism behind this is still largely undetermined. In this study, we observed that while trans-activated Cas12a didn't cleave blunt-end dsDNA within a short reaction time, it could degrade dsDNA reporters with a short overhang. More interestingly, we discovered that the location of the overhang also affected the susceptibility of dsDNA substrate to trans-activated Cas12a. Cas12a trans-cleaved 3' overhang dsDNA substrates at least 3 times faster than 5' overhang substrates. We attributed this unique preference of overhang location to the directional trans-cleavage behavior of Cas12a, which may be governed by RuvC and Nuc domains. Utilizing this new finding, we designed a new hybrid DNA reporter as nonoptical substrate for the CRISPR-Cas12a detection platform, which sensitively detected ssDNA targets at sub picomolar level. This study not only unfolded new insight into the trans-cleavage behavior of Cas12a but also demonstrated a sensitive CRISPR-Cas12a assay by using a hybrid dsDNA reporter molecule.},
}

*CRISPR-Cas Systems
*CRISPR-Associated Proteins/metabolism/genetics/chemistry
*Endodeoxyribonucleases/metabolism/genetics
*DNA/metabolism/chemistry/genetics
DNA, Single-Stranded/metabolism/genetics
DNA Cleavage
*Bacterial Proteins/metabolism/genetics

@article {pmid37642940,
year = {2023},
author = {Schramm, T and Lubrano, P and Pahl, V and Stadelmann, A and Verhülsdonk, A and Link, H},
title = {Mapping temperature-sensitive mutations at a genome scale to engineer growth switches in Escherichia coli.},
journal = {Molecular systems biology},
volume = {19},
number = {10},
pages = {e11596},
pmid = {37642940},
issn = {1744-4292},
support = {715650//European Research Council (ERC)/ ; },
mesh = {*Escherichia coli/genetics/growth & development/metabolism ; *Mutation ; Temperature ; Escherichia coli Proteins/genetics/metabolism ; Genome, Bacterial ; Metabolomics/methods ; CRISPR-Cas Systems ; Metabolome ; DNA Polymerase III/genetics/metabolism ; Metabolic Engineering/methods ; },
abstract = {Temperature-sensitive (TS) mutants are a unique tool to perturb and engineer cellular systems. Here, we constructed a CRISPR library with 15,120 Escherichia coli mutants, each with a single amino acid change in one of 346 essential proteins. 1,269 of these mutants showed temperature-sensitive growth in a time-resolved competition assay. We reconstructed 94 TS mutants and measured their metabolism under growth arrest at 42°C using metabolomics. Metabolome changes were strong and mutant-specific, showing that metabolism of nongrowing E. coli is perturbation-dependent. For example, 24 TS mutants of metabolic enzymes overproduced the direct substrate metabolite due to a bottleneck in their associated pathway. A strain with TS homoserine kinase (ThrB[F267D]) produced homoserine for 24 h, and production was tunable by temperature. Finally, we used a TS subunit of DNA polymerase III (DnaX[L289Q]) to decouple growth from arginine overproduction in engineered E. coli. These results provide a strategy to identify TS mutants en masse and demonstrate their large potential to produce bacterial metabolites with nongrowing cells.},
}

*Escherichia coli/genetics/growth & development/metabolism
*Mutation
Temperature
Escherichia coli Proteins/genetics/metabolism
Genome, Bacterial
Metabolomics/methods
CRISPR-Cas Systems
Metabolome
DNA Polymerase III/genetics/metabolism
Metabolic Engineering/methods

@article {pmid37642675,
year = {2023},
author = {Guo, WF and Guo, DD and Li, F and Shang, SZ and Li, TW and Tang, YC and Jiang, M and Xu, FC and Gao, W},
title = {Efficient genome editing in cotton using the virus-mediated CRISPR/Cas9 and grafting system.},
journal = {Plant cell reports},
volume = {42},
number = {11},
pages = {1833-1836},
pmid = {37642675},
issn = {1432-203X},
support = {202203021222310//Shanxi Basic Research Program/ ; },
mesh = {*Gossypium/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified ; *Genome, Plant/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Plant Viruses/genetics ; },
abstract = {The extensive application of CRISPR in cotton was limited due to the labor-intensive transformation process. Thus, we here established a convenient method of CRISPR in cotton by CLCrV-mediated sgRNA delivery.},
}

*Gossypium/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Plants, Genetically Modified
*Genome, Plant/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
*Plant Viruses/genetics

@article {pmid37634280,
year = {2023},
author = {Han, X and Abdallah, MOE and Breuer, P and Stahl, F and Bakhit, Y and Potthoff, AL and Pregler, BEF and Schneider, M and Waha, A and Wüllner, U and Evert, BO},
title = {Downregulation of MGMT expression by targeted editing of DNA methylation enhances temozolomide sensitivity in glioblastoma.},
journal = {Neoplasia (New York, N.Y.)},
volume = {44},
number = {},
pages = {100929},
pmid = {37634280},
issn = {1476-5586},
mesh = {Humans ; *Glioblastoma/genetics/drug therapy/pathology ; *Temozolomide/pharmacology ; *DNA Methylation ; *DNA Modification Methylases/genetics/metabolism ; *Tumor Suppressor Proteins/genetics/metabolism ; *DNA Repair Enzymes/genetics/metabolism ; Drug Resistance, Neoplasm/genetics ; Cell Line, Tumor ; Promoter Regions, Genetic ; Gene Expression Regulation, Neoplastic/drug effects ; *Gene Editing/methods ; Antineoplastic Agents, Alkylating/pharmacology ; Down-Regulation ; *Brain Neoplasms/genetics/drug therapy ; CRISPR-Cas Systems ; },
abstract = {Glioblastoma is the most common and aggressive primary tumor of the central nervous system with poor outcome. Current gold standard treatment is surgical resection followed by a combination of radio- and chemotherapy. Efficacy of temozolomide (TMZ), the primary chemotherapeutic agent, depends on the DNA methylation status of the O6-methylguanine DNA methyltransferase (MGMT), which has been identified as a prognostic biomarker in glioblastoma patients. Clinical studies revealed that glioblastoma patients with hypermethylated MGMT promoter have a better response to TMZ treatment and a significantly improved overall survival. In this study, we thus used the CRISPRoff genome editing tool to mediate targeted DNA methylation within the MGMT promoter region. The system carrying a CRISPR-deactivated Cas9 (dCas9) fused with a methyltransferase (Dnmt3A/3L) domain downregulated MGMT expression in TMZ-resistant human glioblastoma cell lines through targeted DNA methylation. The reduction of MGMT expression levels reversed TMZ resistance in TMZ-resistant glioblastoma cell lines resulting in TMZ induced dose-dependent cell death rates. In conclusion, we demonstrate targeted RNA-guided methylation of the MGMT promoter as a promising tool to overcome chemoresistance and improve the cytotoxic effect of TMZ in glioblastoma.},
}

Humans
*Glioblastoma/genetics/drug therapy/pathology
*Temozolomide/pharmacology
*DNA Methylation
*DNA Modification Methylases/genetics/metabolism
*Tumor Suppressor Proteins/genetics/metabolism
*DNA Repair Enzymes/genetics/metabolism
Drug Resistance, Neoplasm/genetics
Cell Line, Tumor
Promoter Regions, Genetic
Gene Expression Regulation, Neoplastic/drug effects
*Gene Editing/methods
Antineoplastic Agents, Alkylating/pharmacology
Down-Regulation
*Brain Neoplasms/genetics/drug therapy
CRISPR-Cas Systems

@article {pmid37625131,
year = {2023},
author = {Ueki, K and Nishida, Y and Aoyama, S and Uzawa, H and Kanai, A and Ito, M and Ikeda, K and Iida, H and Miyatsuka, T and Watada, H},
title = {Establishment of Pancreatic β-Cell-Specific Gene Knockout System Based on CRISPR-Cas9 Technology With AAV8-Mediated gRNA Delivery.},
journal = {Diabetes},
volume = {72},
number = {11},
pages = {1609-1620},
doi = {10.2337/db23-0445},
pmid = {37625131},
issn = {1939-327X},
support = {20H03735//Japan Society for the Promotion of Science/ ; },
mesh = {Animals ; *Insulin-Secreting Cells/metabolism ; *CRISPR-Cas Systems/genetics ; *Dependovirus/genetics ; Mice ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; Homeodomain Proteins/genetics ; Trans-Activators/genetics ; Mice, Transgenic ; Mice, Knockout ; },
abstract = {The Cre-loxP system provides valuable resources to analyze the importance of tissue-specific gene knockout (KO), including pancreatic β-cells associated with the pathogenesis of diabetes. However, it is expensive and time consuming to generate transgenic mice harboring floxed genes of interest and cross them with cell-specific Cre expression mice. We establish a βCas9 system with mice expressing Cas9 in pancreatic β-cells and adeno-associated virus 8 (AAV8)-mediated guide RNA (gRNA) delivery based on CRISPR-Cas9 technology to overcome those shortcomings. Interbreeding CAG-loxP-STOP-loxP (LSL)-Cas9 with Ins1-Cre mice generates normal glucose-tolerant βCas9 mice expressing Cas9 with fluorescent reporter EGFP specifically in β-cells. We also show significant β-cell-specific gene KO efficiency with AAV8-mediated delivery of gRNA for EGFP reporter by intraperitoneal injection in the mice. As a proof of concept, we administered AAV8 to βCas9 mice for expressing gRNA for Pdx1, a culprit gene of maturity-onset diabetes of the young 4. As reported previously, we demonstrate that those mice show glucose intolerance with transdifferentiation of Pdx1 KO β-cells into glucagon-expressing cells. We successfully generated a convenient β-cell-specific gene KO system with βCas9 mice and AAV8-mediated gRNA delivery.},
}

Animals
*Insulin-Secreting Cells/metabolism
*CRISPR-Cas Systems/genetics
*Dependovirus/genetics
Mice
*RNA, Guide, CRISPR-Cas Systems/genetics
*Gene Knockout Techniques/methods
Homeodomain Proteins/genetics
Trans-Activators/genetics
Mice, Transgenic
Mice, Knockout

@article {pmid37517884,
year = {2023},
author = {Ahmar, S and Gruszka, D},
title = {CRISPR/Cas9 boosts wheat yield by reducing brassinosteroid signaling.},
journal = {Trends in biochemical sciences},
volume = {48},
number = {11},
pages = {917-919},
doi = {10.1016/j.tibs.2023.07.005},
pmid = {37517884},
issn = {0968-0004},
mesh = {*Brassinosteroids/metabolism ; *CRISPR-Cas Systems/genetics ; *Triticum/genetics/metabolism/growth & development ; *Signal Transduction ; },
abstract = {A modern green revolution is needed to ensure global food security. Recently, Song et al. reported a new strategy to create high-yielding, semi-dwarf wheat varieties with improved nitrogen-use efficiency by inhibiting brassinosteroid (BR) signaling through clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein9 (Cas9)-mediated knockout of the ZnF-B gene encoding a zinc-finger RING-type E3 ligase.},
}

*Brassinosteroids/metabolism
*CRISPR-Cas Systems/genetics
*Triticum/genetics/metabolism/growth & development
*Signal Transduction

@article {pmid37463293,
year = {2023},
author = {Deng, W and Feng, S and Stejskal, V and Opit, G and Li, Z},
title = {An advanced approach for rapid visual identification of Liposcelis bostrychophila (Psocoptera: Liposcelididae) based on CRISPR/Cas12a combined with RPA.},
journal = {Journal of economic entomology},
volume = {116},
number = {5},
pages = {1911-1921},
doi = {10.1093/jee/toad139},
pmid = {37463293},
issn = {1938-291X},
mesh = {Animals ; *Insecta/genetics/classification ; *CRISPR-Cas Systems ; Insect Proteins/genetics ; NADH Dehydrogenase/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae) is a booklouse pest that is a threat to commodity storage security worldwide. Accurate and sensitive methods of L. bostrychophila on-site identification are essential prerequisites for its effective management. Evidence suggests that L. bostrychophila contains 3 intraspecific biotypes that are morphologically indistinguishable but can be discriminated at the level of mitochondrial genome organization and sequences. The traditional molecular identification methods, such as DNA barcoding and PCR-RFLP, are instrumentally demanding and time-consuming, limiting the application of the identification in the field. Therefore, this study developed a new CRISPR/Cas12a-based visual nucleic acid system based on the mitochondrial gene coding for NADH dehydrogenase subunit 2 (nad2), combined with recombinase polymerase amplification (RPA) to accurately identify L. bostrychophila from 4 other common stored-product booklice, and also differentiate 3 biotypes of this species at the same time. The entire identification process could be completed at 37 °C within 20 min with high sensitivity. The system could stably detect at least 1 ng/μl of DNA template. The green fluorescence signal produced by the trans-cleaving of the single-stranded DNA reporter could be observed by the naked eye under blue light. Additionally, the suggested system combined with the crude DNA extraction method to extract DNA rapidly, enabled identification of all developmental stages of L. bostrychophila. With crude DNA, this novel diagnostic system successfully identified an unknown booklouse by holding the reaction tubes in the hand, thus can be considered as an accurate, rapid, highly sensitive, and instrument-flexible method for on-site visual identification of L. bostrychophila.},
}

Animals
*Insecta/genetics/classification
*CRISPR-Cas Systems
Insect Proteins/genetics
NADH Dehydrogenase/genetics
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid37258389,
year = {2023},
author = {Zaman, QU and Raza, A and Gill, RA and Hussain, MA and Wang, HF and Varshney, RK},
title = {New possibilities for trait improvement via mobile CRISPR-RNA.},
journal = {Trends in biotechnology},
volume = {41},
number = {11},
pages = {1335-1338},
doi = {10.1016/j.tibtech.2023.05.001},
pmid = {37258389},
issn = {1879-3096},
mesh = {*CRISPR-Cas Systems/genetics ; *Plants, Genetically Modified/genetics ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {CRISPR/Cas9 gene technology is transported as RNA from transgenic roots to distal parts of unmodified grafted scion, where it is translated into proteins to induce heritable mutagenesis at desired loci. This technique has the potential to produce transgene-free and genetically stable plants in difficult-to-propagate and near-extinct species.},
}

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

@article {pmid41785636,
year = {2026},
author = {Xue, S and Sun, H and Hou, X and Li, N and Xue, L and Dai, E and Wan, J},
title = {An off-target exonuclease activity in AsCpf1 undermines CRISPR diagnostics.},
journal = {Biosensors & bioelectronics},
volume = {303},
number = {},
pages = {118578},
doi = {10.1016/j.bios.2026.118578},
pmid = {41785636},
issn = {1873-4235},
abstract = {The extensive utilization of CRISPR-Cas systems in molecular diagnostics stems from their crRNA-guided trans-cleavage capabilities. However, AsCpf1-based detection systems frequently exhibit unexplained sensitive variations. This research reveals that AsCpf1 maintains a crRNA-independent function, similar to exonuclease I, when utilized in standard buffers containing Mg[2+]. From a structural perspective, this exonuclease activity is independent of the RuvC domain-mediated canonical trans-cleavage activity. It is predicted by structural modeling to be potentially localized within the WED-PI domain. In the context of diagnostics, the effective target concentration is diminished by AsCpf1-mediated degradation of the free 3' ends of target DNA, which impairs detection sensitivity. To mitigate this interference, we demonstrate that 3' end capping effectively restores detection performance. This approach was validated in a CRISPR-EXPAR-based microRNA biosensor, which exhibited approximately 10-fold improvement in sensitivity following 3' end capping. Overall, this investigation characterizes a previously unidentified exonuclease activity within the AsCpf1 system and establishes practical design criteria to improve the robustness and accuracy of CRISPR-based diagnostic tools.},
}

@article {pmid41785320,
year = {2026},
author = {Zhang, X and Richart, D and McFarlin, S and Cheng, F and Park, SY and Zhang-Chen, A and McFarlane, R and Xiao, C and Yan, Z and Qiu, J},
title = {Identification of CD164 as an essential entry receptor for divergent adeno-associated viruses.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {10},
pages = {e2525865123},
doi = {10.1073/pnas.2525865123},
pmid = {41785320},
issn = {1091-6490},
support = {AI166293//HHS | National Institutes of Health (NIH)/ ; AI180416//HHS | National Institutes of Health (NIH)/ ; HL174593//HHS | National Institutes of Health (NIH)/ ; AI182645//HHS | National Institutes of Health (NIH)/ ; HL174593//HHS | National Institutes of Health (NIH)/ ; AI182645//HHS | National Institutes of Health (NIH)/ ; GM129525//HHS | National Institutes of Health (NIH)/ ; YAN23G0//Cystic Fibrosis Foundation (CFF)/ ; AH-2126-20220331//Welch Foundation (The Welch Foundation)/ ; },
mesh = {*Dependovirus/genetics/physiology/metabolism ; Animals ; Mice ; Humans ; Mice, Knockout ; *Virus Internalization ; *Receptors, Cell Surface/metabolism/genetics ; HEK293 Cells ; *Antigens, CD/metabolism/genetics ; Genetic Vectors/genetics ; Transduction, Genetic ; CRISPR-Cas Systems ; *Receptors, Virus/metabolism/genetics ; Capsid/metabolism ; },
abstract = {Recombinant adeno-associated viruses (rAAVs) are widely used for in vivo gene delivery. While KIAA0319L, known as AAV receptor (AAVR), is essential for the transduction of multiserotype AAVs, it is dispensable for AAV4-related (Clade G) AAVs. We conducted a genome-wide CRISPR/Cas9 screen and identified CD164, a type I transmembrane sialomucin, as an essential entry receptor for Clade G AAVs. Ablation of CD164 expression substantially impaired both entry and transduction of Clade G AAVs. CD164-targeting antibodies and soluble CD164 ectodomain effectively blocked transduction. AAV4 capsids colocalized with CD164 at the plasma membrane and in endosomal compartments. In vitro, CD164 interacted with AAV4 or AAVrh32.33 capsids at high affinity. Importantly, systemic administration of rAAV4 or rAAVrh32.33 in CD164 knockout (KO) mice resulted in nearly complete loss of transgene expression. These findings establish CD164 as an essential entry receptor for Clade G AAV vectors and uncover a distinct AAVR-independent mechanism of AAV tropism.},
}

*Dependovirus/genetics/physiology/metabolism
Animals
Mice
Humans
Mice, Knockout
*Virus Internalization
*Receptors, Cell Surface/metabolism/genetics
HEK293 Cells
*Antigens, CD/metabolism/genetics
Genetic Vectors/genetics
Transduction, Genetic
CRISPR-Cas Systems
*Receptors, Virus/metabolism/genetics
Capsid/metabolism

@article {pmid41785318,
year = {2026},
author = {Weber, LI and Timpen, LE and Egger-Hörschinger, AS and Schöpf, P and Ayhan, ND and Demmel, D and Hotze, M and Zhang, Y and Mehrabi, M and Puglisi, K and Stefan, E and Ghaffari-Tabrizi-Wizsy, N and Ramos-Pittol, JM and Kwiatkowski, M and Hartl, M},
title = {Reactivation of the silenced BASP1 gene suppresses oncogenic WNT signaling in human colorectal cancer cells.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {10},
pages = {e2524159123},
doi = {10.1073/pnas.2524159123},
pmid = {41785318},
issn = {1091-6490},
support = {P33662//Austrian Science Fund (FWF)/ ; },
mesh = {Humans ; *Wnt Signaling Pathway/genetics ; *Colorectal Neoplasms/genetics/metabolism/pathology ; Gene Expression Regulation, Neoplastic ; Cell Line, Tumor ; Proto-Oncogene Proteins c-myc/genetics/metabolism ; *Nerve Tissue Proteins/genetics/metabolism ; *Membrane Proteins/genetics/metabolism ; beta Catenin/metabolism/genetics ; Promoter Regions, Genetic ; Gene Silencing ; Cell Movement ; },
abstract = {Starting from human colon cancer cells showing aberrant WNT/β-catenin/TCF signaling, hyperactivated MYC, and silenced BASP1, we generated stable cell lines overexpressing BASP1, either ectopically, or by reactivating the dormant BASP1 promoter using a lentiviral CRISPR-based system. BASP1 encodes a neuronal signaling protein and transcriptional corepressor, from which tumor-suppressive functions have been described in avian cell systems and in multiple human cancer cell types. Proteome and transcriptome analyses revealed activation of several tumor and metastasis suppressors in BASP1-expressing cells, which also show strong repression of the transformed phenotype in terms of contact inhibition, anchorage-independent growth, and tumor formation. Cells with reactivated BASP1 display a flat and differentiated morphology with enhanced migratory potential, accompanied by expression of multiple genes implicated in actin polymerization, focal adhesion, and neuronal migration. Furthermore, MYC protein expression is substantially repressed due to BASP1-mediated transcriptional MYC downregulation involving BASP1 interaction with β-catenin and binding to the MYC promoter. Upon BASP1 activation, multiple key proteins of the canonical WNT signaling pathway become suppressed. One of these BASP1 targets is the protein kinase TNIK catalyzing phosphorylation of TCF7L2, the latter required for transcriptional MYC activation. Results obtained with a preclinical TNIK inhibitor in human colorectal cancer cells show efficient abrogation of MYC expression and consequently impaired dimerization with its interaction partner MAX. The antagonistic BASP1 effect on MYC and the MYC dependency on TNIK could enhance the development of strategies to interfere with oncogenic functions of the cancer driver MYC.},
}

Humans
*Wnt Signaling Pathway/genetics
*Colorectal Neoplasms/genetics/metabolism/pathology
Gene Expression Regulation, Neoplastic
Cell Line, Tumor
Proto-Oncogene Proteins c-myc/genetics/metabolism
*Nerve Tissue Proteins/genetics/metabolism
*Membrane Proteins/genetics/metabolism
beta Catenin/metabolism/genetics
Promoter Regions, Genetic
Gene Silencing
Cell Movement

@article {pmid41784822,
year = {2026},
author = {Eweda, MA and Li, J and Hassan, U and Jalil, S and Jin, X},
title = {Peroxisomal acyl-CoA oxidase OsACX4 negatively regulates salt and drought stress tolerance by modulating cellular redox homeostasis in rice.},
journal = {Plant cell reports},
volume = {45},
number = {3},
pages = {},
pmid = {41784822},
issn = {1432-203X},
support = {517000-N12502ZJ//National Natural Science Foundation of China/ ; 2025ZDXT01-4//The Major Agricultural Technology Collaborative Extension Project/ ; LZ25C130006//Natural Science Foundation of Zhejiang Province/ ; 2021C02063-6//Science and Technology Department of Zhejiang Province/ ; },
mesh = {*Oryza/genetics/physiology/enzymology/metabolism ; Homeostasis ; Oxidation-Reduction ; *Plant Proteins/metabolism/genetics ; Droughts ; Gene Expression Regulation, Plant ; Plants, Genetically Modified ; Peroxisomes/metabolism/enzymology ; *Acyl-CoA Oxidase/metabolism/genetics ; Reactive Oxygen Species/metabolism ; Stress, Physiological/genetics ; *Salt Tolerance/genetics ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Oxidative Stress ; },
abstract = {OsACX4 knockout reduces peroxisomal oxidative stress, enhancing rice drought and salt tolerance through metabolic-redox rebalancing for climate-resilient breeding. Climate change is intensifying the frequency and severity of abiotic stress, such as salt and drought stresses, which severely limit rice productivity worldwide, necessitating the identification of molecular targets for crop improvement. This study provides the first comprehensive functional characterization of the peroxisomal acyl-CoA oxidase OsACX4 in rice (Oryza sativa L.) drought and salinity tolerance, revealing its unexpected role as a negative regulator of stress tolerance through modulation of cellular redox homeostasis. Through genome editing using CRISPR/Cas9-mediated knockout and overexpression approaches, we generated transgenic lines to investigate the function of OsACX4 under salt and drought stress. Knockout lines exhibited superior stress tolerance compared to the wild-type (WT) and overexpression lines, demonstrating significantly higher survival rates under severe stress conditions. Enhanced tolerance correlated with coordinated upregulation of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) activities. Knockout lines accumulated substantially higher proline (Pro) levels while maintaining markedly reduced reactive oxygen species (ROS) compared to overexpression lines. Transcriptional analysis revealed that OsACX4 disruption triggered upregulation of stress-responsive genes, including OsSOD1, OsDREB2A, OsDREB1B, and OsAPX1 under severe stress. Subcellular localization confirmed peroxisomal targeting of OsACX4, where its β-oxidation activity generates hydrogen peroxide (H2O2) as a metabolic by-product. Our results reveal a metabolic trade-off whereby stress-induced OsACX4 expression mobilizes energy reserves but compromises cellular redox homeostasis. The superior performance of knockout lines under both stresses demonstrates that strategic OsACX4 disruption enhances plant resilience, identifying this gene as a promising breeding target for developing climate-resilient rice varieties through precision genome editing.},
}

*Oryza/genetics/physiology/enzymology/metabolism
Homeostasis
Oxidation-Reduction
*Plant Proteins/metabolism/genetics
Droughts
Gene Expression Regulation, Plant
Plants, Genetically Modified
Peroxisomes/metabolism/enzymology
*Acyl-CoA Oxidase/metabolism/genetics
Reactive Oxygen Species/metabolism
Stress, Physiological/genetics
*Salt Tolerance/genetics
Gene Knockout Techniques
CRISPR-Cas Systems
Oxidative Stress

@article {pmid41784340,
year = {2026},
author = {Zhou, C and Cheng, T and Zhou, J and Zhang, B and Liu, L and Jiang, G and Li, W and Wang, C},
title = {Synthetic Biofilms for Green Membranes: Engineering Low-Energy Filtration Systems.},
journal = {Environmental science & technology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.est.5c15661},
pmid = {41784340},
issn = {1520-5851},
abstract = {Membrane filtration is a key technology to modern water purification, yet its sustainability is compromised by biofouling, which increases energy consumption and ecological impacts. Conventional control strategies often struggle to balance efficacy and environmental footprint. In this study, an inducible, engineered quorum-quenching (QQ) bacterium was constructed via a genomic integration strategy, thereby achieving control over the biofilm structure in membrane filtration biofouling layers. By using the clustered regularly interspaced short palindromic repeat (CRISPR-Cas) targeted gene editing technology, the engineered bacteria that were constructed to express aiiO under l-ribose induction have achieved the regulation of biofilms. Validation using a gravity-driven membrane ultrafiltration system (UF-GDM) model showed that the engineered bacterium effectively reduced extracellular polymeric substances (EPS) components, increased the hydrophilic porosity of the residual biofilm, and decreased its stickiness. This approach reduced transmembrane pressure by 64.5%, increased total organic carbon (TOC) removal by 13.2%, and extended membrane lifespan by 16.1%. A technical-economic analysis indicates that the 100,000 m[3]/day treatment plant achieves an annual net profit increase of 31.52%, reaching 1.55 × 10[7] CNY, while reducing its net carbon footprint by 27.43%, with an annual net reduction of 2.96 × 10[5] kg CO2eq. This study provides a novel solution strategy for achieving biofouling resistance and sustainable, low-energy operation in membrane filtration processes, which contributes to the broader application and adoption of this technology.},
}

@article {pmid41784267,
year = {2026},
author = {Wang, Z and Wu, Y and Wang, Z and Zhang, S and Liu, H and Nie, Y and Chen, K and Huang, Y and Zhou, Y and Cao, Y and Sun, L and Hao, R},
title = {crRNA scaffold remodeling controls CRISPR-Cas12a activity for enhanced performance.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41784267},
issn = {1362-4962},
support = {//Prevention and Control of Emerging/ ; 2025ZD01903403//National Science and Technology Major Project/ ; L234051//Beijing Municipal Natural Science Foundation/ ; L246011//Beijing Municipal Natural Science Foundation/ ; 2024-03-18//Training Plan for High-Level Public Health Technical Talents of Beijing Municipal Health Commission/ ; 2025ZD01903403//Prevention and Control of Emerging and Major Infectious Diseases-National Science/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; *Endodeoxyribonucleases/metabolism/genetics/chemistry ; Mycobacterium tuberculosis/genetics/isolation & purification ; Klebsiella pneumoniae/genetics/isolation & purification ; Nucleic Acid Conformation ; *Bacterial Proteins/genetics/metabolism ; },
abstract = {CRISPR-Cas12a has transformative potential in molecular diagnostics owing to its robust signal amplification, but its sustained activity state severely limits temporal programmability and precise nuclease control in complex detection workflows. Here, we demonstrate that the conserved crRNA scaffold secondary structure itself can be repurposed as a reversible and programmable conformational switch to regulate Cas12a activity. By introducing short complementary DNA blockers of tunable length, we achieved length-dependent disruption and remodeling of scaffold secondary structure, shifting LbCas12a into an inactive conformation. Scaffold structure was subsequently reinstated through either single or cooperative strand displacement activation, enabling time-resolved and on-demand restoration of Cas12a activity. The conserved scaffold ensures intrinsic assay universality, while its programmable rewiring markedly improves SNVs discrimination and enables compatibility with one-pot isothermal amplification assays, delivering analytical sensitivity comparable to conventional two-step assays. This regulatory framework was further demonstrated in the detection of Klebsiella pneumoniae and Mycobacterium tuberculosis. By validating the crRNA scaffold as a practical and programmable switch for Cas12a activity control, this work establishes a universal and reversible framework for scaffold rewiring to modulate CRISPR nucleases and offers mechanistic insight to guide future assay engineering.},
}

*CRISPR-Cas Systems/genetics
*CRISPR-Associated Proteins/metabolism/genetics/chemistry
*Endodeoxyribonucleases/metabolism/genetics/chemistry
Mycobacterium tuberculosis/genetics/isolation & purification
Klebsiella pneumoniae/genetics/isolation & purification
Nucleic Acid Conformation
*Bacterial Proteins/genetics/metabolism

@article {pmid41783940,
year = {2026},
author = {Berti, M and Ceriotti, S and Santi, L and Alberti, G and Beretta, S and Degl'Innocenti, S and Ruatti, C and Savoia, EO and Jofra-Hernandez, R and De Ponti, G and Bolamperti, S and Villa, I and Galeotti, F and Romano, A and Visigalli, I and Norata, R and Rocchi, M and Cristofori, P and Cossutta, M and Consiglieri, G and Tucci, F and Santorelli, L and Grumati, P and Ronfani, L and D'Adamo, P and Giustina, A and Angelozzi, M and Settembre, C and Mortellaro, A and Scala, S and Sanvito, F and Volpi, N and Aiuti, A and Bernardo, ME and Crippa, S},
title = {Development and characterization of a model of mucopolysaccharidosis type IVA for evaluating therapies targeting bone disease.},
journal = {Disease models & mechanisms},
volume = {19},
number = {2},
pages = {},
doi = {10.1242/dmm.052540},
pmid = {41783940},
issn = {1754-8411},
support = {20228H9T82//Ministero dell'Istruzione, dell'Università e della Ricerca/ ; P20223MF7X_001//Ministero dell'Istruzione, dell'Università e della Ricerca/ ; CN_00000041 - CUP G83C22000270001//NextGenerationEU/ ; TTAGTXEKFA//Else Kröner-Fresenius-Zentrum für Ernährungsmedizin/ ; TELE-MB//Fondazione Telethon/ ; TELE-AA//Fondazione Telethon/ ; //Ospedale San Raffaele/ ; },
mesh = {Animals ; Disease Models, Animal ; *Mucopolysaccharidosis IV/therapy/pathology/blood/complications ; *Bone Diseases/therapy/pathology/complications ; Chondroitinsulfatases/metabolism/deficiency ; Glycosaminoglycans/metabolism ; Keratan Sulfate/metabolism/urine ; Chondroitin Sulfates/metabolism/urine ; Mice ; Humans ; Genetic Therapy ; Mice, Knockout ; Bone and Bones/pathology ; CRISPR-Cas Systems/genetics ; },
abstract = {Mucopolysaccharidosis type IVA (MPSIVA) is a lysosomal storage disease (LSD) caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), which causes the accumulation of keratan sulphate (KS) and chondroitin sulphate (CS). Patients with MPSIVA typically present with severe skeletal and joint disorders, which are not addressed by conventional therapies. Currently, no animal model accurately replicates the human disease, hindering the development of novel therapeutic interventions. To overcome this limitation, we established, by CRISPR-Cas9 technology, a Galns-/- mouse model that expresses a non-functional enzyme and accumulates CS and KS in the urine, plasma and distinct tissues, and glycosaminoglycans in the spleen. The mice exhibit shortened long bones, trabecular bone alterations and skeletal abnormalities in the growth plate. Additionally, we observed increased levels of inflammatory and oxidative markers in visceral organs and plasma. Our newly developed model of MPSIVA demonstrates clear and quantifiable signs of skeletal alterations, providing novel means of assessment of the safety and efficacy of innovative therapies, including hematopoietic stem and progenitor cell gene therapy, which has recently been shown to provide a beneficial effect on skeletal alterations in Hurler syndrome.},
}

Animals
Disease Models, Animal
*Mucopolysaccharidosis IV/therapy/pathology/blood/complications
*Bone Diseases/therapy/pathology/complications
Chondroitinsulfatases/metabolism/deficiency
Glycosaminoglycans/metabolism
Keratan Sulfate/metabolism/urine
Chondroitin Sulfates/metabolism/urine
Mice
Humans
Genetic Therapy
Mice, Knockout
Bone and Bones/pathology
CRISPR-Cas Systems/genetics

@article {pmid41781627,
year = {2026},
author = {Hoffmann, FT and Wiegand, T and Palmieri, AI and Glass-Klaiber, J and Xiao, R and Tang, S and Le, HC and Meers, C and Lampe, GD and Chang, L and Sternberg, SH},
title = {Exapted CRISPR-Cas12f homologues drive RNA-guided transcription.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {41781627},
issn = {1476-4687},
abstract = {Bacterial transcription initiation is a tightly regulated process that canonically relies on sequence-specific promoter recognition by dedicated sigma (σ) factors, leading to functional DNA engagement by RNA polymerase (RNAP)[1]. Although the seven σ factors in Escherichia coli have been extensively characterized[2], Bacteroidetes species encode dozens of specialized, extracytoplasmic function σ factors (σ[E]) whose precise roles are unknown, pointing to additional layers of regulatory potential[3]. Here we uncover a mechanism of RNA-guided gene activation involving the coordinated action of σ[E] factor in complex with nuclease-dead Cas12f (dCas12f). We screened a large set of genetically linked dCas12f and σ[E] homologues in E. coli using RNA and chromatin immunoprecipitation experiments, revealing systems that exhibit robust guide RNA enrichment and DNA target binding with a minimal 5'-G target-adjacent motif. Recruitment of σ[E] was dependent on dCas12f and guide RNA, suggesting direct protein-protein interactions, and co-expression experiments demonstrated that the dCas12f-gRNA-σ[E] ternary complex was competent for programmable recruitment of the RNAP holoenzyme. Remarkably, dCas12f-RNA-σ[E] complexes drove potent gene expression in the absence of any requisite promoter motifs, with de novo transcription start sites defined exclusively by the relative distance from the dCas12f-mediated R-loop. Our findings highlight a new paradigm of RNA-guided transcription that embodies natural features reminiscent of CRISPR activation (CRISPRa) technology[4,5].},
}

@article {pmid41781609,
year = {2026},
author = {Xiao, R and Hoffmann, FT and Xie, D and Wiegand, T and Palmieri, AI and Sternberg, SH and Chang, L},
title = {Structural basis of RNA-guided transcription by a dCas12f-σ[E]-RNAP complex.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {41781609},
issn = {1476-4687},
abstract = {In both natural and engineered biological systems, RNA-guided proteins have emerged as critical transcriptional regulators by modulating RNA polymerase (RNAP) and its associated factors[1-3]. In bacteria, diverse clades of repurposed TnpB and CRISPR-associated proteins repress gene expression by blocking transcription initiation or elongation, enabling non-canonical modes of regulatory control and adaptive immunity[1,4,5]. A distinct class of nuclease-dead Cas12f homologues (dCas12f) instead activates gene expression through its association with unique extracytoplasmic function sigma factors (σ[E])[6], although the molecular basis has remained elusive. Here we reveal a new mode of RNA-guided transcription initiation by determining the cryo-electron microscopy structures of the dCas12f-σ[E] system from Flagellimonas taeanensis. We captured multiple conformational and compositional states, including the DNA-bound dCas12f-σ[E]-RNAP holoenzyme complex, revealing how RNA-guided DNA binding leads to σ[E]-RNAP recruitment and nascent mRNA synthesis at a precisely defined distance downstream of the R-loop. Rather than following the classical paradigm of σ[E]-dependent promoter recognition, these studies show that recognition of the -35 element is largely supplanted by CRISPR-Cas targeting, whereas the melted -10 element is stabilized through unusual stacking interactions rather than insertion into the typical recognition pocket. Collectively, this work provides high-resolution insights into an unexpected mechanism of RNA-guided transcription, expanding our understanding of bacterial gene regulation and opening new avenues for programmable transcriptional control.},
}

@article {pmid41747766,
year = {2026},
author = {Cai, Y and Zhuang, L and Wang, Z and He, L and Li, X and Liu, BF and Li, T and Zhang, G and Zhou, H and Huang, X and Li, Y},
title = {Gravity-Driven Formation of Water-in-Wax Spheres for Efficient One-Pot CRISPR Diagnostics.},
journal = {ACS nano},
volume = {20},
number = {9},
pages = {8055-8067},
doi = {10.1021/acsnano.6c01232},
pmid = {41747766},
issn = {1936-086X},
mesh = {*Gravitation ; *Water/chemistry ; Humans ; *Waxes/chemistry ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Microspheres ; CRISPR-Cas Systems ; Human papillomavirus 16/genetics/isolation & purification ; },
abstract = {Rapid, decentralized molecular diagnostics are urgently needed for effective infectious disease control. Here, we present "Wax-Sphere CRISPR" (WS-CRISPR), a paradigm-shifting platform for CRISPR-based diagnostics centered on a gravity-driven, interfacial phase-change self-encapsulation mechanism. This system fundamentally decouples bioreagent engineering from specific reaction vessels, transforming conventional, labor-intensive manual encapsulation into a standardized, physics-driven assembly process that generates discrete wax microspheres. Guided by fluid mechanics and interfacial thermodynamics, aqueous CRISPR droplets spontaneously traverse air/molten wax/ethanol to self-encapsulate and solidify, enabling standardized, high-throughput fabrication without manual wax handling. Upon temperature modulation, the wax phase change triggers sequential recombinase polymerase amplification (RPA) and CRISPR detection within a sealed, one-pot vessel. As a clinically oriented demonstration, WS-CRISPR enables multiplexed detection and risk stratification of all 14 high-risk HPV genotypes (HPV16/18 vs others) with a detection limit of 1 × 10[-18] M in under 30 min. Integrated with widely accessible devices─including a thermocycler, hand-held fluorescence reader, and microfluidic platform─the system demonstrated 97.4% sensitivity and 100% specificity across 70 clinical samples. By solving the engineering bottlenecks of scalability and universality, WS-CRISPR offers a robust tool for decentralized, large-scale pathogen surveillance.},
}

*Gravitation
*Water/chemistry
Humans
*Waxes/chemistry
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Microspheres
CRISPR-Cas Systems
Human papillomavirus 16/genetics/isolation & purification

@article {pmid41742365,
year = {2026},
author = {Yao, W and Li, Y and Du, Y and Gao, R and Tan, J and Fan, J and Liu, Y},
title = {Aptamer-Mediated Protein Corona In Situ Multiplex Microfluidic Detection for Cancer Diagnosis.},
journal = {Analytical chemistry},
volume = {98},
number = {9},
pages = {7123-7135},
doi = {10.1021/acs.analchem.6c00170},
pmid = {41742365},
issn = {1520-6882},
mesh = {Humans ; *Aptamers, Nucleotide/chemistry ; *Neoplasms/diagnosis/blood ; *Protein Corona/chemistry ; *Microfluidic Analytical Techniques ; *Biomarkers, Tumor/blood ; *Lab-On-A-Chip Devices ; CRISPR-Cas Systems ; Silicon Dioxide/chemistry ; },
abstract = {Liquid biopsy faces critical limitations in detecting low-abundance protein biomarkers due to serum complexity, single-target constraints, and insufficient sensitivity. Here, we present an integrated microfluidic protein aptamer corona-CRISPR (PACC) platform that synergizes nanoparticle protein corona (PC) enrichment, multiplexed aptamer recognition, and CRISPR-Cas12a-driven signal generation for facile and rapid point-of-care testing. Fe3O4@SiO2 nanoparticles capable of streamlining PC isolation via magnetic separation were employed to concentrate cancer-associated proteins from serum. A 6-plex aptamer panel, which selectively bound target proteins via forming protein-aptamer coronas (PACs), was used to initiate the in situ activation of CRISPR-Cas12a via complementary crRNAs for fluorescence signal generation. This entire workflow was miniaturized into a 3D-printed microfluidic chip and portable reader, achieving a fast sample-to-answer process. Clinical validation with 112 serum specimens demonstrated excellent diagnostic performance, achieving an area under the curve (AUC) of 0.95 and 88.24% accuracy. By integrating these modalities, the PACC platform overcomes serum matrix interference and biomarker scarcity, providing a scalable and low-cost avenue for detecting low-abundance protein biomarkers and advancing liquid biopsy toward precision oncology.},
}

Humans
*Aptamers, Nucleotide/chemistry
*Neoplasms/diagnosis/blood
*Protein Corona/chemistry
*Microfluidic Analytical Techniques
*Biomarkers, Tumor/blood
*Lab-On-A-Chip Devices
CRISPR-Cas Systems
Silicon Dioxide/chemistry

@article {pmid41565063,
year = {2026},
author = {Wu, Q and Du, F and Zhang, X and Lu, Z and Zheng, X and Li, A and Zhang, X and Zhang, R and Wang, Q},
title = {Development and evaluation of a one-pot CRISPR/Cas13-based assay for syphilis detection across multiple clinical sample types.},
journal = {International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases},
volume = {164},
number = {},
pages = {108414},
doi = {10.1016/j.ijid.2026.108414},
pmid = {41565063},
issn = {1878-3511},
mesh = {Humans ; Sensitivity and Specificity ; *Treponema pallidum/genetics/isolation & purification ; *Syphilis/diagnosis/microbiology ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; *CRISPR-Cas Systems ; },
abstract = {OBJECTIVES: To develop and evaluate a rapid, one-pot molecular assay for the detection of Treponema pallidum subspecies pallidum (TPA), addressing the limitations of current diagnostic methods influenced by sample type and pathogen load.

METHODS: A one-pot assay integrating recombinase polymerase amplification (RPA) and Cas13a-based collateral cleavage activity was established for isothermal detection of TPA. The assay targeted the tpp47/tp0574 gene and was validated using 186 clinical specimens, including whole blood, lesion exudate, and cerebrospinal fluid (CSF) samples.

RESULTS: The one-pot RPA-Cas13a assay demonstrated high analytical sensitivity and specificity for TPA detection. Clinical sensitivities were 58.97% in whole blood, 84.21% in lesion exudate, and 57.14% in CSF, with 100% specificity across all sample types.

CONCLUSION: This one-pot isothermal assay enables rapid and accurate detection of T. pallidum directly from diverse clinical samples. Its high specificity and field-friendly format make it a promising complementary tool to conventional diagnostic approaches, particularly for point-of-care testing and screening in resource-limited or high-risk settings.},
}

Humans
Sensitivity and Specificity
*Treponema pallidum/genetics/isolation & purification
*Syphilis/diagnosis/microbiology
*Nucleic Acid Amplification Techniques/methods
*Molecular Diagnostic Techniques/methods
*CRISPR-Cas Systems

@article {pmid41491239,
year = {2026},
author = {Amelan, A and Collins, SC and Damseh, NS and Hamada, N and Salim, A and Dvir, E and Monderer-Rothkoff, G and Harel, T and Nagata, KI and Yalcin, B and Shifman, S},
title = {CRISPR knockout screens reveal genes and pathways essential for neuronal differentiation and implicate PEDS1 in neurodevelopment.},
journal = {Nature neuroscience},
volume = {29},
number = {3},
pages = {592-603},
pmid = {41491239},
issn = {1546-1726},
support = {466/21//Israel Science Foundation (ISF)/ ; 1863/24//Israel Science Foundation (ISF)/ ; ANR-18-CE12-0009//Agence Nationale de la Recherche (French National Research Agency)/ ; },
mesh = {Animals ; Mice ; *Cell Differentiation/genetics ; *Neurons/physiology ; *Neurogenesis/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Humans ; *Neurodevelopmental Disorders/genetics ; Gene Knockout Techniques/methods ; Mice, Knockout ; CRISPR-Cas Systems ; Male ; },
abstract = {Neurodevelopmental disorders (NDDs) arise from disruptions in brain development, yet the underlying pathways remain incompletely understood. Here we demonstrate that genome-wide CRISPR knockout screens in mouse embryonic stem cells differentiating into neural lineages identify hundreds of essential genes, only a minority of which are currently implicated in NDDs. Dominant NDD genes were enriched for transcriptional regulators, whereas recessive NDD genes were predominantly involved in metabolic processes. Mouse models for eight genes (Eml1, Dusp26, Dynlrb2, Mta3, Peds1, Sgms1, Slitrk4 and Vamp3) revealed marked neuroanatomical abnormalities, including microcephaly in half of the cases. Focusing on PEDS1, a key enzyme in plasmalogen biosynthesis, we identified a bi-allelic variant in individuals with microcephaly, global developmental delay and congenital cataracts. In mice, Peds1 deficiency led to accelerated cell-cycle exit and impaired neuronal differentiation and migration. These pathways required for neural differentiation provide a genetic framework for discovering additional NDD genes.},
}

Animals
Mice
*Cell Differentiation/genetics
*Neurons/physiology
*Neurogenesis/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Humans
*Neurodevelopmental Disorders/genetics
Gene Knockout Techniques/methods
Mice, Knockout
CRISPR-Cas Systems
Male

@article {pmid41413662,
year = {2026},
author = {Green, NFO and Sutton, GJ and Pérez-Burillo, J and Wang, J and Bagot, S and Danon, HG and Walsh, K and Gokool, A and Miles, SA and Yang, G and Herring, CA and Liang, Y and Pfundstein, G and Sytnyk, V and Alinejad-Rokny, H and Lister, R and Rosenbluh, J and Gagnon-Bartsch, JA and Voineagu, I},
title = {CRISPRi screening in cultured human astrocytes uncovers distal enhancers controlling genes dysregulated in Alzheimer's disease.},
journal = {Nature neuroscience},
volume = {29},
number = {3},
pages = {703-716},
pmid = {41413662},
issn = {1546-1726},
support = {2020814//Department of Health | National Health and Medical Research Council (NHMRC)/ ; },
mesh = {Humans ; *Astrocytes/metabolism ; *Alzheimer Disease/genetics/metabolism ; *Enhancer Elements, Genetic/genetics ; Cells, Cultured ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Single-Cell Analysis ; Machine Learning ; },
abstract = {Genetic variants associated with complex traits often lie in distal enhancers. While candidate enhancers have been mapped genome wide, their functional state and gene targets in specific cell types remain unclear. Here we present AstroREG, a resource of enhancer-gene interactions in human primary astrocytes, generated by combining CRISPR inhibition (CRISPRi), single-cell RNA-seq and machine learning. By functionally testing nearly 1,000 PsychENCODE enhancers, we identified more than 150 regulatory interactions, revealing enhancers that control key astrocyte functions and genes implicated in Alzheimer's disease. The CRISPRi screen also provided valuable ground-truth data from a primary cell type for training and benchmarking prediction models of enhancer activity. We thus developed EGrf, a random forest (RF) model trained on these data, and applied it genome wide to predict regulatory interactions with high specificity. Together, our data provide a comprehensive functional map of enhancer-mediated regulation in a key glial cell type, shedding light on brain function and disease.},
}

Humans
*Astrocytes/metabolism
*Alzheimer Disease/genetics/metabolism
*Enhancer Elements, Genetic/genetics
Cells, Cultured
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Single-Cell Analysis
Machine Learning

@article {pmid37762360,
year = {2023},
author = {Mak, MCE and Gurung, R and Foo, RSY},
title = {Applications of Genome Editing Technologies in CAD Research and Therapy with a Focus on Atherosclerosis.},
journal = {International journal of molecular sciences},
volume = {24},
number = {18},
pages = {},
pmid = {37762360},
issn = {1422-0067},
mesh = {*Gene Editing/methods ; Humans ; *Coronary Artery Disease/therapy/genetics ; Animals ; *Atherosclerosis/genetics/therapy ; CRISPR-Cas Systems ; Genetic Therapy/methods ; },
abstract = {Cardiovascular diseases, particularly coronary artery disease (CAD), remain the leading cause of death worldwide in recent years, with myocardial infarction (MI) being the most common form of CAD. Atherosclerosis has been highlighted as one of the drivers of CAD, and much research has been carried out to understand and treat this disease. However, there remains much to be better understood and developed in treating this disease. Genome editing technologies have been widely used to establish models of disease as well as to treat various genetic disorders at their root. In this review, we aim to highlight the various ways genome editing technologies can be applied to establish models of atherosclerosis, as well as their therapeutic roles in both atherosclerosis and the clinical implications of CAD.},
}

*Gene Editing/methods
Humans
*Coronary Artery Disease/therapy/genetics
Animals
*Atherosclerosis/genetics/therapy
CRISPR-Cas Systems
Genetic Therapy/methods

@article {pmid37762332,
year = {2023},
author = {Akhtar, J and Imran, M and Wang, G},
title = {CRISPR/Cas9-Mediated CtBP1 Gene Editing Enhances Chemosensitivity and Inhibits Metastatic Potential in Esophageal Squamous Cell Carcinoma Cells.},
journal = {International journal of molecular sciences},
volume = {24},
number = {18},
pages = {},
pmid = {37762332},
issn = {1422-0067},
support = {32070681//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Alcohol Oxidoreductases/genetics/metabolism ; *Esophageal Squamous Cell Carcinoma/genetics/pathology/drug therapy/metabolism ; Cell Line, Tumor ; *Drug Resistance, Neoplasm/genetics ; *CRISPR-Cas Systems/genetics ; *Esophageal Neoplasms/genetics/pathology/drug therapy/metabolism ; Paclitaxel/pharmacology ; *DNA-Binding Proteins/genetics/metabolism ; Cell Proliferation/drug effects/genetics ; *Gene Editing/methods ; Gene Expression Regulation, Neoplastic/drug effects ; Cell Survival/drug effects ; Neoplasm Metastasis ; },
abstract = {Innovative therapeutic strategies for esophageal squamous cell carcinoma (ESCC) are urgently required due to the limited effectiveness of standard chemotherapies. C-Terminal Binding Protein 1 (CtBP1) has been implicated in various cancers, including ESCC. However, the precise expression patterns and functional roles of CtBP1 in ESCC remain inadequately characterized. In this study, we aimed to investigate CtBP1 expression and its role in the resistance of ESCC to paclitaxel, an effective chemotherapeutic agent. Western blotting and immunofluorescence were applied to assess CtBP1 expression in the TE-1 and KYSE-50 cell lines. We observed the marked expression of CtBP1, which was associated with enhanced proliferation, invasion, and metastasis in these cell lines. Further, we successfully generated paclitaxel resistant ESCC cell lines and conducted cell viability assays. We employed the CRISPR/Cas9 genome editing system to disable the CtBP1 gene in ESCC cell lines. Through the analysis of the drug dose-response curve, we assessed the sensitivity of these cell lines in different treatment groups. Remarkably, CtBP1-disabled cell lines displayed not only improved sensitivity but also a remarkable inhibition of proliferation, invasion, and metastasis. This demonstrates that CtBP1 may promote ESCC cell malignancy and confer paclitaxel resistance. In summary, our study opens a promising avenue for targeted therapies, revealing the potential of CtBP1 inhibition to enhance the effectiveness of paclitaxel treatment for the personalized management of ESCC.},
}

Humans
*Alcohol Oxidoreductases/genetics/metabolism
*Esophageal Squamous Cell Carcinoma/genetics/pathology/drug therapy/metabolism
Cell Line, Tumor
*Drug Resistance, Neoplasm/genetics
*CRISPR-Cas Systems/genetics
*Esophageal Neoplasms/genetics/pathology/drug therapy/metabolism
Paclitaxel/pharmacology
*DNA-Binding Proteins/genetics/metabolism
Cell Proliferation/drug effects/genetics
*Gene Editing/methods
Gene Expression Regulation, Neoplastic/drug effects
Cell Survival/drug effects
Neoplasm Metastasis

@article {pmid37755318,
year = {2023},
author = {Ravichandran, K and Khargonkar, T and Samaddar, S and Banerjee, S},
title = {CRISPR Based Programmable RNA Editing in Primary Hippocampal Neurons.},
journal = {Current protocols},
volume = {3},
number = {9},
pages = {e886},
doi = {10.1002/cpz1.886},
pmid = {37755318},
issn = {2691-1299},
mesh = {*Hippocampus/cytology/metabolism ; *Neurons/metabolism/cytology ; *RNA Editing/genetics ; Animals ; Mice ; *CRISPR-Cas Systems/genetics ; Cells, Cultured ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Investigating the RNA regulation landscape primarily relies on understanding how RNA-protein interactions are governed in various cell types, including neurons. Analysis of RNA-protein interactions in physiological environments warrants the development of new tools that rely on RNA manipulation. Recently, a CRISPR-based RNA-editing tool (dCas13b-ADAR2DD) was developed to mitigate disease-associated point mutations in cell lines. Here, we explored the targeted sequence editing potential of the tool (dCas13b-ADAR2DD system) by adapting it to manipulate RNA function to visualize RNA editing in primary hippocampal neurons. This two-component system includes a programmable guide RNA (gRNA) complementary to the target RNA and a catalytically dead version of the Cas13b enzyme fused to ADAR. The RNA editing protocol outlined in this article relies on gRNA-dependent targeting of the dCas13b-ADAR fusion protein to the mutant form of the Dendra2 transcript. Dendra2 is not required for intrinsic cellular functioning. It was ectopically expressed for fluorescent detection as a proof-of-principle demonstration of targeted RNA editing. We first abrogated the fluorescence of Dendra2 by introducing a nonsense mutation that precludes the formation of the functional protein. To visualize the efficacy of the RNA editing in neurons, we used the dCas13b-ADAR2DD system to edit specific nucleotides within the Dendra2 mRNA to restore the amino acid codes critical for Dendra2 fluorescence. This method lays the foundation for future studies on the dynamics of activity-induced RNA-protein interactions in neurons and can be extended to manipulate the endogenous RNome in diverse neuronal subtypes. Furthermore, this methodology will enable investigators to visualize the spatial and temporal resolution of RNA-protein interactions without altering the genomes via conventional methods. © 2023 Wiley Periodicals LLC. Support Protocol: Preparation of mouse primary hippocampal culture Basic Protocol: Targeted editing of RNA.},
}

*Hippocampus/cytology/metabolism
*Neurons/metabolism/cytology
*RNA Editing/genetics
Animals
Mice
*CRISPR-Cas Systems/genetics
Cells, Cultured
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid37741117,
year = {2023},
author = {Lee, J and Kim, DH and Lee, K},
title = {Research Note: Injection of adenoviral CRISPR/Cas9 system targeting melanophilin gene into different sites of embryos induced regional feather color changes in posthatch quail.},
journal = {Poultry science},
volume = {102},
number = {11},
pages = {103087},
pmid = {37741117},
issn = {1525-3171},
mesh = {Animals ; *CRISPR-Cas Systems ; *Feathers/physiology ; *Gene Editing/veterinary/methods ; *Coturnix/genetics/physiology ; Adenoviridae/genetics ; *Pigmentation/genetics ; *Avian Proteins/genetics/metabolism ; },
abstract = {Poultry species is an important animal model in both avian research and the poultry industry. To advance our understanding of genetic factors and benefit both fields, a gene of interest can be genetically edited, and consequential phenotypic changes can be investigated. Injection of adenovirus containing the CRISPR/Cas9 system into avian blastoderm induced genome editing in blastodermal cells randomly, including primordial germ cells, which results in generation of whole-body knockout in the offspring of the virus-injected quail. However, to observe phenotypic and functional changes in whole-body, homozygous knockout of genes using this genome editing technology requires at least 2 generations of breeding of chimeric, and heterozygotes birds. In the current study, we developed a strategy to investigate the gene function in 1-generation by inducing regional genome editing around the injection sites with CRISPR/Cas9 adenovirus. The adenoviral CRISPR/Cas9 vector targeting the melanophilin (Mlph) gene, regulating feather pigmentation, was injected into 2 different regions of embryos, the cervical flexure of quail embryos at HH stage 13 to 15 and the tip of the upper limb bud of embryos at HH stage 22 to 24, to induce genome editing in those regions. Indel mutations in the target loci of the Mlph gene were detected by extracting genomic DNA from the embryonic tissues, and consequential phenotypes, feather color changes, were analyzed at 1 mo after hatch. Injection of the adenovirus into the cervical flexure and the tip of the upper limb bud of embryos resulted in 8 to 21% efficiency of indel mutation in the embryonic cells of the injected regions. In the posthatch quail, gray feathers were shown on their upper back and primary wing feathers, corresponding to the injection sites at embryos. Successful validation of this strategy for inducing genome editing in parts of tissues within 1-generation will accelerate studies on genetic functions with advantages of less time and cost, facilitating avian research and providing foundations for future application for the poultry industry.},
}

Animals
*CRISPR-Cas Systems
*Feathers/physiology
*Gene Editing/veterinary/methods
*Coturnix/genetics/physiology
Adenoviridae/genetics
*Pigmentation/genetics
*Avian Proteins/genetics/metabolism

@article {pmid37718698,
year = {2023},
author = {Kumar, A and Stirling, PC},
title = {Turning up the heat on essential E. coli genes.},
journal = {Molecular systems biology},
volume = {19},
number = {10},
pages = {e11933},
pmid = {37718698},
issn = {1744-4292},
support = {RGPIN 2020-04360//Gouvernement du Canada | Natural Sciences and Engineering Research Council of Canada (NSERC)/ ; },
mesh = {*Escherichia coli/genetics ; *Genes, Essential ; CRISPR-Cas Systems ; Alleles ; Escherichia coli Proteins/genetics ; Hot Temperature ; Gene Expression Regulation, Bacterial ; Gene Editing/methods ; },
abstract = {Temperature-sensitive (TS) alleles create tunable thermoswitches to deplete essential cellular activities and are used to dissect gene function. In their recent study, Link and colleagues (Schramm et al 2023) use a CRISPR-based approach to systematically create TS alleles across essential genes in E. coli.},
}

*Escherichia coli/genetics
*Genes, Essential
CRISPR-Cas Systems
Alleles
Escherichia coli Proteins/genetics
Hot Temperature
Gene Expression Regulation, Bacterial
Gene Editing/methods

@article {pmid37702604,
year = {2023},
author = {Pescod, P and Bevivino, G and Anthousi, A and Shelton, R and Shepherd, J and Lombardo, F and Nolan, T},
title = {Measuring the Impact of Genetic Heterogeneity and Chromosomal Inversions on the Efficacy of CRISPR-Cas9 Gene Drives in Different Strains of Anopheles gambiae.},
journal = {The CRISPR journal},
volume = {6},
number = {5},
pages = {419-429},
doi = {10.1089/crispr.2023.0029},
pmid = {37702604},
issn = {2573-1602},
mesh = {*Anopheles/genetics ; Animals ; *Chromosome Inversion/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Drive Technology/methods ; Gene Editing/methods ; *Genetic Heterogeneity ; Mosquito Vectors/genetics ; },
abstract = {The human malaria vector Anopheles gambiae is becoming increasingly resistant to insecticides, spurring the development of genetic control strategies. CRISPR-Cas9 gene drives can modify a population by creating double-stranded breaks at highly specific targets, triggering copying of the gene drive into the cut site ("homing"), ensuring its inheritance. The DNA repair mechanism responsible requires homology between the donor and recipient chromosomes, presenting challenges for the invasion of laboratory-developed gene drives into wild populations of target species An. gambiae species complex, which show high levels of genome variation. Two gene drives (vas2-5958 and zpg-7280) were introduced into three An. gambiae strains collected across Africa with 5.3-6.6% variation around the target sites, and the effect of this variation on homing was measured. Gene drive homing across different karyotypes of the 2La chromosomal inversion was also assessed. No decrease in gene drive homing was seen despite target site heterology, demonstrating the applicability of gene drives to wild populations.},
}

*Anopheles/genetics
Animals
*Chromosome Inversion/genetics
*CRISPR-Cas Systems/genetics
*Gene Drive Technology/methods
Gene Editing/methods
*Genetic Heterogeneity
Mosquito Vectors/genetics

@article {pmid37493514,
year = {2023},
author = {Wei, H and Li, J and Liu, Y and Cheng, W and Huang, H and Liang, X and Huang, W and Lin, L and Zheng, Y and Chen, W and Wang, C and Chen, W and Xu, G and Wei, W and Chen, L and Zeng, Y and Lu, Z and Li, S and Lin, Z and Wang, J and Lin, M},
title = {Rapid and Ultrasensitive Detection of Plasmodium spp. Parasites via the RPA-CRISPR/Cas12a Platform.},
journal = {ACS infectious diseases},
volume = {9},
number = {8},
pages = {1534-1545},
doi = {10.1021/acsinfecdis.3c00087},
pmid = {37493514},
issn = {2373-8227},
mesh = {Humans ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Plasmodium/genetics/isolation & purification ; Sensitivity and Specificity ; *Malaria/diagnosis/parasitology ; Recombinases/metabolism/genetics ; Plasmodium falciparum/genetics/isolation & purification ; *Molecular Diagnostic Techniques/methods ; Point-of-Care Testing ; Malaria, Falciparum/diagnosis ; },
abstract = {Microscopic examination of thick and thin blood smears stained with Giemsa dye is considered the primary diagnostic tool for the confirmation and management of suspected clinical malaria. However, detecting gametocytes is relatively insensitive, particularly in asymptomatic individuals with low-density Plasmodium infections. To complement existing diagnostic methods, a rapid and ultrasensitive point-of-care testing (POCT) platform for malaria detection is urgently needed and necessary. A platform based on recombinase polymerase amplification (RPA) followed by CRISPR/Cas12a (referred to as RPA-CRISPR/Cas12a) was developed and optimized for the determination of Plasmodium spp. parasites, particularly Plasmodium falciparum, using a fluorescence-based assay (FBDA), lateral flow test strips (LFTS), or naked eye observation (NEO). Then, the established platform was assessed with clinical malaria isolates. Under optimal conditions, the detection threshold was 1 copy/μL for the plasmid, and the limit of detection was 3.11-7.27 parasites/μL for dried blood spots. There was no cross-reactivity against blood-borne pathogens. For the accuracies of RPA-CRISPR/Cas12a, Plasmodium spp. and P. falciparum testing were 98.68 and 94.74%, respectively. The method was consistent with nested PCR results and superior to the qPCR results. RPA-CRISPR/Cas12a is a rapid, ultrasensitive, and reliable platform for malaria diagnosis. The platform requires no or minimal instrumentation for nucleic acid amplification reactions and can be read with the naked eye. Compared with similar diagnostic methods, this platform improves the reaction speed while reducing detection requirements. Therefore, this platform has the potential to become a true POCT for malaria parasites.},
}

Humans
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
*Plasmodium/genetics/isolation & purification
Sensitivity and Specificity
*Malaria/diagnosis/parasitology
Recombinases/metabolism/genetics
Plasmodium falciparum/genetics/isolation & purification
*Molecular Diagnostic Techniques/methods
Point-of-Care Testing
Malaria, Falciparum/diagnosis

@article {pmid37482112,
year = {2023},
author = {Liu, L and Zhang, S and Zhi, F and Song, Y and Li, B and Gao, P and Zhang, Y and Ma, K and Xu, J and Jiang, B and Chu, Y and Li, Y and Qin, J},
title = {RNA helicase DExD/H-box 5 modulates intestinal microbiota in mice.},
journal = {Microbial pathogenesis},
volume = {182},
number = {},
pages = {106265},
doi = {10.1016/j.micpath.2023.106265},
pmid = {37482112},
issn = {1096-1208},
mesh = {Animals ; *Gastrointestinal Microbiome/genetics ; *DEAD-box RNA Helicases/genetics/metabolism ; Mice, Knockout ; Mice ; RNA, Ribosomal, 16S/genetics ; Ileum/microbiology/pathology ; Feces/microbiology ; Mice, Inbred C57BL ; CRISPR-Cas Systems ; Bacteria/classification/genetics/isolation & purification ; },
abstract = {The RNA helicase DExD/H-box (DDX) family of proteins plays a central role in host cellular RNA metabolism, including mRNA regulation, microRNA biogenesis, and ribosomal processing. DDX5, also known as p68, promotes viral replication and tumorigenesis. However, there have been no studies on the regulation of the intestinal microbiota by DDX family proteins. We constructed DDX5 knockout mice (Ddx5[+/-]) using CRISPR/CAS9 technology. Subsequently, DDX5 knockout mice were analyzed for PCR products, mRNA levels, protein expression, immunohistochemistry, and histopathological lesions. Fecal (n = 12) and ileum (n = 12) samples were collected from the Ddx5[+/-] and wild-type (Ddx5[+/+]) mice. The diversity, richness, and structural separation of the intestinal microbiota of the Ddx5[+/-] and Ddx5[+/+] mice were determined by 16S rRNA sequencing and analysis. Ddx5[+/-] mice were successfully established, and the ileum had normal morphology, a clear layer of tissue structures, and neatly arranged cupped cells. DDX5 knockout mice did not exhibit adverse effects on the ileal tissue. Microbial diversity and abundance were not significantly different, but the microbial structure of the intestinal microbiota was clustered separately between Ddx5[+/+] and Ddx5[+/-] mice. Furthermore, we found that the relative abundance of Akkermansia and Clostridium_sensu_stricto_1 in the Ddx5[+/-] mice was significantly lower than in the Ddx5[+/+] mice. These analyses indicated specific interactions between the intestinal microbiota and DDX5 protein. Our results indicate that DDX5 has a significant effect on the composition of the intestinal microbiota in mice, suggesting its potential as a promising novel target for the treatment of inflammation and tumorigenesis in the intestine.},
}

Animals
*Gastrointestinal Microbiome/genetics
*DEAD-box RNA Helicases/genetics/metabolism
Mice, Knockout
Mice
RNA, Ribosomal, 16S/genetics
Ileum/microbiology/pathology
Feces/microbiology
Mice, Inbred C57BL
CRISPR-Cas Systems
Bacteria/classification/genetics/isolation & purification

@article {pmid37477766,
year = {2023},
author = {Hu, J and Guan, X and Zhao, M and Xie, P and Guo, J and Tan, J},
title = {Genome-wide CRISPR-Cas9 Knockout Screening Reveals a TSPAN3-mediated Endo-lysosome Pathway Regulating the Degradation of α-Synuclein Oligomers.},
journal = {Molecular neurobiology},
volume = {60},
number = {11},
pages = {6731-6747},
pmid = {37477766},
issn = {1559-1182},
mesh = {Humans ; *Lysosomes/metabolism ; *alpha-Synuclein/metabolism ; HEK293 Cells ; *Tetraspanins/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; *Proteolysis ; *Endosomes/metabolism ; *Gene Knockout Techniques ; *Protein Multimerization ; },
abstract = {Misfolding and aggregation of α-Synuclein (α-Syn), which are hallmark pathological features of neurodegenerative diseases such as Parkinson's disease (PD) and dementia with Lewy Bodies, continue to be significant areas of research. Among the diverse forms of α-Syn - monomer, oligomer, and fibril, the oligomer is considered the most toxic. However, the mechanisms governing α-Syn oligomerization are not yet fully understood. In this study, we utilized genome-wide CRISPR/Cas9 loss-of-function screening in human HEK293 cells to identify negative regulators of α-Syn oligomerization. We found that tetraspanin 3 (TSPAN3), a presumptive four-pass transmembrane protein, but not its homolog TSPAN7, significantly modulates α-Syn oligomer levels. TSPAN3 was observed to interact with α-Syn oligomers, regulate the amount of α-Syn oligomers on the cell membrane, and promote their degradation via the clathrin-AP2 mediated endo-lysosome pathway. Our findings highlight TSPAN3 as a potential regulator of α-Syn oligomers, presenting a promising target for future PD prevention and treatment strategies.},
}

Humans
*Lysosomes/metabolism
*alpha-Synuclein/metabolism
HEK293 Cells
*Tetraspanins/metabolism/genetics
*CRISPR-Cas Systems/genetics
*Proteolysis
*Endosomes/metabolism
*Gene Knockout Techniques
*Protein Multimerization

@article {pmid37451121,
year = {2023},
author = {Hu, Y and Qiao, Y and Li, XQ and Xiang, Z and Wan, Y and Wang, P and Yang, Z},
title = {Development of an inducible Cas9 nickase and PAM-free Cas12a platform for bacterial diagnostics.},
journal = {Talanta},
volume = {265},
number = {},
pages = {124931},
doi = {10.1016/j.talanta.2023.124931},
pmid = {37451121},
issn = {1873-3573},
mesh = {*CRISPR-Associated Proteins/metabolism/genetics ; *Deoxyribonuclease I/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; *Methicillin-Resistant Staphylococcus aureus/isolation & purification/genetics ; CRISPR-Cas Systems ; DNA, Single-Stranded/genetics ; *Bacterial Proteins/genetics/metabolism ; Humans ; },
abstract = {Rapid, efficient, specific and sensitive diagnostic techniques are critical for selecting appropriate treatments for drug-resistant bacterial infections. To address this challenge, we have developed a novel diagnostic method, called the Dual-Cas Tandem Diagnostic Platform (DTDP), which combines the use of Cas9 nickase (Cas9n) and Cas12a. DTDP works by utilizing the Cas9n-sgRNA complex to create a nick in the target strand's double-stranded DNA (dsDNA). This prompts DNA polymerase to displace the single-stranded DNA (ssDNA) and leads to cycles of DNA replication through nicking, displacement, and extension. The ssDNA is then detected by the Cas12a-crRNA complex (which is PAM-free), activating trans-cleavage and generating a fluorescent signal from the fluorescent reporter. DTDP exhibits a high sensitivity (1 CFU/mL or 100 ag/μL), high specificity (specifically to MRSA in nine pathogenic species), and excellent accuracy (100%). The dual RNA recognition process in our method improves diagnostic specificity by decreasing the limitations of Cas12a in detecting dsDNA protospacer adjacent motifs (PAMs) and leverages multiple advantages of multi-Cas enzymes in diagnostics. This novel approach to pathogenic microorganism detection has also great potential for clinical diagnosis.},
}

*CRISPR-Associated Proteins/metabolism/genetics
*Deoxyribonuclease I/metabolism/genetics
*Endodeoxyribonucleases/metabolism/genetics
*Methicillin-Resistant Staphylococcus aureus/isolation & purification/genetics
CRISPR-Cas Systems
DNA, Single-Stranded/genetics
*Bacterial Proteins/genetics/metabolism
Humans

@article {pmid37421327,
year = {2023},
author = {Yang, M and Wan, S and Chen, J and Chen, W and Wang, Y and Li, W and Wang, M and Guan, R},
title = {Mutation to a cytochrome P450 -like gene alters the leaf color by affecting the heme and chlorophyll biosynthesis pathways in Brassica napus.},
journal = {The Plant journal : for cell and molecular biology},
volume = {116},
number = {2},
pages = {432-445},
doi = {10.1111/tpj.16382},
pmid = {37421327},
issn = {1365-313X},
support = {32171974//National Natural Science Foundation of China/ ; 2022YFF1003500//the National Key R&D Program of China/ ; },
mesh = {*Chlorophyll/biosynthesis/metabolism ; *Brassica napus/genetics/metabolism ; *Plant Leaves/genetics/metabolism/physiology ; *Cytochrome P-450 Enzyme System/genetics/metabolism ; *Heme/biosynthesis/metabolism ; *Plant Proteins/genetics/metabolism ; Mutation ; CRISPR-Cas Systems ; Photosynthesis ; Biosynthetic Pathways ; Gene Expression Regulation, Plant ; Gene Editing ; },
abstract = {The regulated biosynthesis of chlorophyll is important because of its effects on plant photosynthesis and dry biomass production. In this study, a map-based cloning approach was used to isolate the cytochrome P450 -like gene BnaC08g34840D (BnCDE1) from a chlorophyll-deficient mutant (cde1) of Brassica napus obtained by ethyl methanesulfonate (EMS) mutagenization. Sequence analyses revealed that BnaC08g34840D in the cde1 mutant (BnCDE1[I320T]) encodes a substitution at amino acid 320 (Ile320Thr) in the conserved region. The over-expression of BnCDE1[I320T] in ZS11 (i.e., gene-mapping parent with green leaves) recapitulated a yellow-green leaf phenotype. The CRISPR/Cas9 genome-editing system was used to design two single-guide RNAs (sgRNAs) targeting BnCDE1[I320T] in the cde1 mutant. The knockout of BnCDE1[I320T] in the cde1 mutant via a gene-editing method restored normal leaf coloration (i.e., green leaves). These results indicate that the substitution in BnaC08g34840D alters the leaf color. Physiological analyses showed that the over-expression of BnCDE1[I320T] leads to decreases in the number of chloroplasts per mesophyll cell and in the contents of the intermediates of the chlorophyll biosynthesis pathway in leaves, while it increases heme biosynthesis, thereby lowering the photosynthetic efficiency of the cde1 mutant. The Ile320Thr mutation in the highly conserved region of BnaC08g34840D inhibited chlorophyll biosynthesis and disrupted the balance between heme and chlorophyll biosynthesis. Our findings may further reveal how the proper balance between the chlorophyll and heme biosynthesis pathways is maintained.},
}

*Chlorophyll/biosynthesis/metabolism
*Brassica napus/genetics/metabolism
*Plant Leaves/genetics/metabolism/physiology
*Cytochrome P-450 Enzyme System/genetics/metabolism
*Heme/biosynthesis/metabolism
*Plant Proteins/genetics/metabolism
Mutation
CRISPR-Cas Systems
Photosynthesis
Biosynthetic Pathways
Gene Expression Regulation, Plant
Gene Editing

@article {pmid36960824,
year = {2023},
author = {Makrogkikas, S and Cheng, RK and Lu, H and Roy, S},
title = {A conserved function of Pkhd1l1, a mammalian hair cell stereociliary coat protein, in regulating hearing in zebrafish.},
journal = {Journal of neurogenetics},
volume = {37},
number = {3},
pages = {85-92},
doi = {10.1080/01677063.2023.2187792},
pmid = {36960824},
issn = {1563-5260},
mesh = {Animals ; Zebrafish ; *Zebrafish Proteins/genetics/metabolism/physiology ; *Hair Cells, Auditory/metabolism/physiology ; *Hearing/genetics/physiology ; *Stereocilia/metabolism ; Mice ; Hearing Loss/genetics ; Animals, Genetically Modified ; CRISPR-Cas Systems ; },
abstract = {Pkhd1l1 is predicted to encode a very large type-I transmembrane protein, but its function has largely remained obscure. Recently, it was shown that Pkhdl1l1 is a component of the coat that decorates stereocilia of outer hair cells in the mouse ear. Consistent with this localization, conditional deletion of Pkhd1l1 specifically from hair cells, was associated with progressive hearing loss. In the zebrafish, there are two paralogous pkhd1l1 genes - pkhd1l1α and pkhd1l1β. Using CRISPR-Cas9 mediated gene editing, we generated loss-of-function alleles for both and show that the double mutants exhibit nonsense-mediated-decay (NMD) of the RNAs. With behavioural assays, we demonstrate that zebrafish pkhd1l1 genes also regulate hearing; however, in contrast to Pkhd1l1 mutant mice, which develop progressive hearing loss, the double mutant zebrafish exhibited statistically significant hearing loss even from the larval stage. Our data highlight a conserved function of Pkhd1l1 in hearing and based on these findings from animal models, we postulate that PKHD1L1 could be a candidate gene for sensorineural hearing loss (SNHL) in humans.},
}

Animals
Zebrafish
*Zebrafish Proteins/genetics/metabolism/physiology
*Hair Cells, Auditory/metabolism/physiology
*Hearing/genetics/physiology
*Stereocilia/metabolism
Mice
Hearing Loss/genetics
Animals, Genetically Modified
CRISPR-Cas Systems

@article {pmid36948605,
year = {2023},
author = {Deng, Y and Han, X and Chen, H and Zhao, C and Chen, Y and Zhou, J and de The, H and Zhu, J and Yuan, H},
title = {Ypel5 regulates liver development and function in zebrafish.},
journal = {Journal of molecular cell biology},
volume = {15},
number = {3},
pages = {},
pmid = {36948605},
issn = {1759-4685},
support = {31371479//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Zebrafish/genetics/metabolism/embryology ; *Zebrafish Proteins/metabolism/genetics ; *Liver/metabolism/embryology/growth & development ; Hepatocyte Nuclear Factor 4/metabolism/genetics ; Cell Proliferation/genetics ; Hepatocytes/metabolism ; Gene Expression Regulation, Developmental ; CRISPR-Cas Systems ; Signal Transduction ; },
abstract = {YPEL5 is a member of the Yippee-like (YPEL) gene family that is evolutionarily conserved in eukaryotic species. To date, the physiological function of YPEL5 has not been assessed due to a paucity of genetic animal models. Here, using CRISPR/Cas9-mediated genome editing, we generated a stable ypel5-/- mutant zebrafish line. Disruption of ypel5 expression leads to liver enlargement associated with hepatic cell proliferation. Meanwhile, hepatic metabolism and function are dysregulated in ypel5-/- mutant zebrafish, as revealed by metabolomic and transcriptomic analyses. Mechanistically, Hnf4a is identified as a crucial downstream mediator that is positively regulated by Ypel5. Zebrafish hnf4a overexpression could largely rescue ypel5 deficiency-induced hepatic defects. Furthermore, PPARα signaling mediates the regulation of Hnf4a by Ypel5 through directly binding to the transcriptional enhancer of the Hnf4a gene. Herein, this work demonstrates an essential role of Ypel5 in hepatocyte proliferation and function and provides the first in vivo evidence for a physiological role of the ypel5 gene in vertebrates.},
}

Animals
*Zebrafish/genetics/metabolism/embryology
*Zebrafish Proteins/metabolism/genetics
*Liver/metabolism/embryology/growth & development
Hepatocyte Nuclear Factor 4/metabolism/genetics
Cell Proliferation/genetics
Hepatocytes/metabolism
Gene Expression Regulation, Developmental
CRISPR-Cas Systems
Signal Transduction

@article {pmid41779860,
year = {2026},
author = {Zhou, X and Pan, D and Zhou, J and Chen, W and Han, G and Zhang, R and Wang, C and Mao, Y and Du, Z and Zhang, F and Yue, H and Ma, J and Li, Z and Shen, RJ and Wang, B and Zhu, W and Peng, Y and Jin, K and Wu, DD and Wang, W and Zhou, B and Jin, ZB and Chen, L},
title = {Cis-regulatory evolution reveals sensory trade-offs as a genetic basis for temporal niche evolution in tapirs.},
journal = {Science advances},
volume = {12},
number = {10},
pages = {eadz4758},
pmid = {41779860},
issn = {2375-2548},
mesh = {Animals ; *Evolution, Molecular ; Mice ; *Regulatory Sequences, Nucleic Acid ; Biological Evolution ; CRISPR-Cas Systems ; Humans ; },
abstract = {Evolutionary shifts in diel activity patterns shape sensory remodeling across mammals, yet the genetic basis remains poorly understood. Tapirs represent a unique natural experiment, having reverted from a cathemeral ancestor to a nocturnal niche characterized by reduced vision but enhanced hearing and olfaction. Here, we investigate the genetic basis of this phenomenon by generating high-quality chromosome-level genomes for Tapirus terrestris and Tapirus indicus. Comparative analyses revealed extensive lineage-specific remodeling of genes and cis-regulatory elements linked to sensory pathways. Notably, functional validation via CRISPR-Cas9 editing of a tapir-specific conserved noncoding element (CNE74) upstream of the FLT1 gene in mice revealed coordinated sensory effects, including retinal degeneration and reduced visual acuity, yet enhanced auditory sensitivity. These findings suggest that regulatory element evolution may induce pleiotropic effects on competing sensory modalities, offering genetic insights into sensory evolution during temporal niche adaptation and potential relevance to human retinal vascular diseases.},
}

Animals
*Evolution, Molecular
Mice
*Regulatory Sequences, Nucleic Acid
Biological Evolution
CRISPR-Cas Systems
Humans

@article {pmid41779781,
year = {2026},
author = {Ju, X and Dong, L and Liu, T and Zhang, F and Sun, X and Schwoerer, MP and Ren, W and Gong, M and Ploss, A and Qin, W and Wu, X and Wang, L and Ding, Q},
title = {EIF4H and YBX1 are essential host factors for hepatitis E virus replication and pathogenesis.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {10},
pages = {e2529289123},
doi = {10.1073/pnas.2529289123},
pmid = {41779781},
issn = {1091-6490},
support = {2023YFC2306900//National Key Research and Development Plan of China/ ; 82341084//MOST | National Natural Science Foundation of China (NSFC)/ ; 82272302//MOST | National Natural Science Foundation of China (NSFC)/ ; 82522053//MOST | National Natural Science Foundation of China (NSFC)/ ; 20251080029//Tsinghua University Dushi Program/ ; Not applicable//SXMU-Tsinghua Collaborative Innovation Center for Frontier Medicine/ ; Not applicable//High Meadows Environmental Institute/ ; T32 GM007388/GM/NIGMS NIH HHS/United States ; },
mesh = {*Hepatitis E virus/physiology/pathogenicity/genetics ; *Virus Replication ; Humans ; *Hepatitis E/virology/metabolism/pathology/genetics ; Animals ; Rats ; *Y-Box-Binding Protein 1/metabolism/genetics ; *Eukaryotic Initiation Factors/metabolism/genetics ; Host-Pathogen Interactions ; CRISPR-Cas Systems ; Hepatocytes/virology ; RNA-Binding Proteins ; },
abstract = {Hepatitis E virus (HEV) is a leading cause of acute viral hepatitis worldwide, responsible for approximately 20 million infections annually. Despite the availability of a vaccine in China, no direct-acting antivirals are approved, and host factors required for HEV replication remain poorly defined. Here, using a genome-wide CRISPR/Cas9 knockout screen in a replicon system, we identified Eukaryotic Translation Initiation Factor 4H (EIF4H) and Y-Box Binding Protein 1 (YBX1) as essential host factors for HEV replication and pathogenesis. Knockout of either factor markedly impaired replication of HEV genotypes 1, 3, and 4, as well as HEV infection and production in hepatocellular carcinoma cells and human induced pluripotent stem cell-derived hepatocyte-like cells, while leaving SARS-CoV-2, hepatitis B virus, hepatitis C virus, and Zika virus unaffected, underscoring their HEV-specific roles. Mechanistically, EIF4H interacts with ORF1 via its methyltransferase-Y-papain-like protease region, and EIF4H deficiency alters the composition of the ORF1-associated replication complex. By contrast, YBX1 is dispensable for ORF1 translation and RNA binding but is specifically required for ORF1 proteolytic processing, a prerequisite for assembling a functional replication machinery. EIF4H knockout rats and liver-specific YBX1 knockout rats were largely resistant to rat HEV-C1 infection, showing profound reductions in viral shedding, suppressed hepatic and intestinal viral loads, and protection from liver pathology. Together, our findings establish EIF4H and YBX1 as essential host factors for HEV infection and pathogenesis and reveal potential targets for antiviral intervention.},
}

*Hepatitis E virus/physiology/pathogenicity/genetics
*Virus Replication
Humans
*Hepatitis E/virology/metabolism/pathology/genetics
Animals
Rats
*Y-Box-Binding Protein 1/metabolism/genetics
*Eukaryotic Initiation Factors/metabolism/genetics
Host-Pathogen Interactions
CRISPR-Cas Systems
Hepatocytes/virology
RNA-Binding Proteins

@article {pmid41777069,
year = {2026},
author = {Zamperin, G and Palumbo, E and Castellan, M and Marciano, S and Fusaro, A and Monne, I},
title = {Metagenomic sequencing of zoonotic viruses: evaluation of a CRISPR-Cas-based rRNA depletion system.},
journal = {Veterinaria italiana},
volume = {62},
number = {2},
pages = {},
doi = {10.12834/VetIt.3908.38985.2},
pmid = {41777069},
issn = {1828-1427},
mesh = {*CRISPR-Cas Systems ; Animals ; *Metagenomics/methods ; *RNA, Ribosomal/genetics ; *Zoonoses/virology ; Genome, Viral ; },
abstract = {Pathogen-agnostic diagnostics are crucial for the early detection of emerging viruses. Shotgun metagenomic sequencing enables unbiased detection of viral genomes but is frequently constrained by the abundance of host and microbial ribosomal RNA (rRNA), which reduces sensitivity and increases sequencing costs. CRISPR-Cas9-based rRNA depletion has emerged as an alternative to enzymatic methods; however, its performance for the characterization of zoonotic viruses across diverse animal hosts and tissues remains underexplored. We compared CRISPR-Cas9 (Jumpcode CRISPRclean™ Plus) and RNase H-based enzymatic depletion (Ribo-Zero Plus, Illumina) using 12 samples positive for rabies lyssavirus, influenza A virus, West Nile virus or norovirus, from multiple host species and tissues, including both high-quality and degraded RNA. CRISPR-Cas9 efficiently reduced rRNA content (14.5%) but recovered fewer viral reads than Ribo-Zero, which achieved up to 60.7× enrichment. Both methods produced complete viral consensus genomes when RNA quality and viral load were sufficient. However, based on the data generated here, enzymatic depletion currently remains more efficient and cost-effective for viral metagenomics. Further optimization of CRISPR-Cas9 workflows could enhance its utility for viral surveillance and diagnostics.},
}

*CRISPR-Cas Systems
Animals
*Metagenomics/methods
*RNA, Ribosomal/genetics
*Zoonoses/virology
Genome, Viral

@article {pmid41773018,
year = {2026},
author = {Yang, T and Tang, M and Xu, L and Jiang, L and Jiang, L and Zou, Y and Wang, J and Liu, Z and Chen, F and Ban, Y and Ren, W and Cheng, W},
title = {A tailored phosphorothioate coordinator enables CRISPR/Cas in-situ amplification.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41773018},
issn = {1362-4962},
support = {U24A20751//National Natural Science Foundation of China/ ; 82372334//National Natural Science Foundation of China/ ; 82502827//National Natural Science Foundation of China/ ; CSTB2023NSCQ-LZX0022//Chongqing Education Commission/ ; CSTB2024NSCQ-QCXMX0006//New Chongqing Youth Innovative Talents Project/ ; //National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Phosphorothioate Oligonucleotides/chemistry/genetics/metabolism ; *Nucleic Acid Amplification Techniques/methods ; Human papillomavirus 16/genetics ; CRISPR-Associated Proteins/metabolism/genetics/chemistry ; RNA, Viral/genetics/metabolism ; RNA, Messenger/genetics/metabolism ; Uterine Cervical Neoplasms/virology/diagnosis ; Female ; HeLa Cells ; Endodeoxyribonucleases/metabolism/genetics ; },
abstract = {The CRISPR/Cas system is a powerful tool for molecular diagnostics, but its reliance on linear amplification constrains sensitivity, particularly for in situ imaging. Here, we discovered that phosphorothioate (PS)-modified activators can modulate Cas enzyme conformation via hydrophobic anchoring. By adjusting the PS modification sites, we achieved precise control over Cas activation and trans-cleavage resistance. Guided by this mechanism, we proposed a tailored design strategy featuring a "scattered" PS modification to engineer a linear "Coordinator" probe. This design effectively decouples Cas enzyme activation from substrate trans-cleavage resistance, enabling the construction of a Scattered PS Nucleic Acid-driven Cas Autocatalytic system (SACA). SACA achieves exponential amplification without external enzymes, enhancing Cas12a and Cas13a sensitivity by 50 000-fold and 10 000-fold, respectively. Furthermore, the superior biostability and structural simplicity of these linear probes endow SACA with excellent compatibility, facilitating precise in situ imaging of HPV16 and HPV18 mRNA in cervical cancer cells. This study not only advances the understanding of Cas enzyme regulation by chemically modified nucleic acids but also establishes a new paradigm for precise and efficient molecular diagnostics.},
}

*CRISPR-Cas Systems/genetics
Humans
*Phosphorothioate Oligonucleotides/chemistry/genetics/metabolism
*Nucleic Acid Amplification Techniques/methods
Human papillomavirus 16/genetics
CRISPR-Associated Proteins/metabolism/genetics/chemistry
RNA, Viral/genetics/metabolism
RNA, Messenger/genetics/metabolism
Uterine Cervical Neoplasms/virology/diagnosis
Female
HeLa Cells
Endodeoxyribonucleases/metabolism/genetics

@article {pmid41773016,
year = {2026},
author = {Park, H and Yun, J and Lee, K and Kim, JH and Park, JH and Park, YJ and Park, JH and Lee, H and Kim, MG},
title = {Functional decoupling of crRNA enables customizable CRISPR diagnostics.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41773016},
issn = {1362-4962},
support = {//National Research Foundation of Korea/ ; RS-2025-16063091//National Research Council of Science and Technology/ ; RS-2024-00411137//National Research Council of Science and Technology/ ; CRC22024-500//National Research Council of Science and Technology/ ; //National Research Foundation of Korea/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Nucleic Acid Amplification Techniques/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {One-pot CRISPR-based diagnostics have transformed nucleic acid testing, yet their design customizability remains constrained. Because target programming and cis-cleavage activity are simultaneously determined during CRISPR RNA (crRNA) design, optimizing cleavage activity to match isothermal amplification inevitably requires altering the programmed crRNA sequence. This requirement fundamentally constrains the range of compatible target sequences, imposing limitations on the flexible design of diagnostic assays. Here, we establish a customizable one-pot system by decoupling the dual functions inherent in crRNA design to enable their independent control. In this strategy, target programming remains defined by the crRNA sequence, whereas cis-cleavage activity is regulated by the reaction energy barrier. We selectively modulate this energy barrier through the introduction of a crRNA-complementary RNA oligonucleotide, achieving cleavage regulation without altering the crRNA sequence. Consequently, this approach ensures that cis-cleavage activity matches isothermal amplification conditions independent of the programmed target sequence, thereby realizing a customizable CRISPR diagnostic system. We validated the clinical applicability of this system using 120 patient-derived samples, achieving sensitivity and specificity comparable to quantitative polymerase chain reaction. Collectively, this work resolves a fundamental constraint of CRISPR diagnostics and establishes a customizable and clinically deployable platform for next-generation nucleic acid testing.},
}

*CRISPR-Cas Systems/genetics
Humans
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Nucleic Acid Amplification Techniques/methods
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41772759,
year = {2026},
author = {Donega, S and Gorospe, M and Harries, LW and Ferrucci, L},
title = {Loss of Splicing Homeostasis as a Hallmark of Aging.},
journal = {Molecular and cellular biology},
volume = {},
number = {},
pages = {1-19},
doi = {10.1080/10985549.2026.2627235},
pmid = {41772759},
issn = {1098-5549},
abstract = {Alternative splicing is a fundamental mechanism that ensures accurate gene expression, supports cellular adaptability, and expands protein diversity beyond the limits of a fixed gene pool. With aging, splicing fidelity weakens, contributing to decline in RNA homeostasis and disrupting essential cellular functions, including mitochondrial oxidative phosphorylation, genome stability, and immune regulation, and in turn accelerating tissue and organ dysfunction. Evidence from senescent cells, aged tissues, and model organisms shows that altered levels of splicing factors and increased RNA polymerase II elongation rates impair co-transcriptional splicing and promote mis-spliced isoforms that reinforce senescence and drive pathology. Dysfunction of RNA-binding proteins further contributes to aberrant splicing, linking splicing defects to age-related diseases such as atherosclerosis, osteoarthritis, sarcopenia, and neurodegenerative disorders like Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Therapeutic strategies to correct splicing defects, such as antisense oligonucleotides, RNA interference, CRISPR-Cas systems, ADAR-mediated editing, and RNA aptamers, can restore a homeostatic balance of mRNA isoforms. However, major challenges remain, including distinguishing adaptive physiological from pathological splicing 'noise' and achieving targeted delivery to tissues. Despite these obstacles, RNA splicing dysregulation represents a promising avenue to extend health span by reestablishing homeostatic RNA programs, and reinforces the idea that "transcriptomic instability" is a hallmark of aging.},
}

@article {pmid41771871,
year = {2026},
author = {Becerra, B and Wittibschlager, S and Patel, ZM and Kutschat, AP and Delano, J and Che, E and Tauber, A and Wu, T and Starrs, M and Horstmann, CS and Müller, S and Whittaker, MN and Sylvander, E and Lehner, M and Love, MI and Kleinstiver, BP and Jankowiak, M and Bauer, DE and Seruggia, D and Pinello, L},
title = {Nucleotide-resolution mapping of regulatory elements via allelic readout of tiled base editing.},
journal = {Nature communications},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41467-026-69918-8},
pmid = {41771871},
issn = {2041-1723},
abstract = {CRISPR tiling screens have enabled the characterization of regulatory sequences but are limited by low resolution arising from the indirect readout of editing via guide RNA sequencing and enrichment analysis. This study introduces an end-to-end experimental assay and computational pipeline, which leverages targeted sequencing of CRISPR-introduced alleles at the endogenous target locus following dense base-editing mutagenesis. As a proof of concept, we studied a putative CD19 enhancer, an immunotherapy target in leukemia, and identified alleles and single nucleotides crucial for CD19 regulation. Our visualization tools revealed transcription factor motifs corresponding to the top-ranked nucleotides. Validation experiments confirmed that mutations in MYB, PAX5, and EBF1 binding sites reduce CD19 expression. Critically, editing MYB and PAX5 motifs conferred resistance to CD19 CAR-T cell therapy, revealing how non-coding variants can drive immunotherapy escape. Taken together, this approach achieves nucleotide-resolution genotype-phenotype mapping at regulatory elements beyond conventional gRNA-based screens.},
}

@article {pmid41771837,
year = {2026},
author = {Schoger, E and Kim, R and Bleckwedel, F and Peralta, T and Priesmeier, L and Fischer, J and Stengel, L and Rocha, C and Santos, GL and Lutz, S and Boileau, E and Baumgarten, N and Schulz, MH and Dieterich, C and Müller, OJ and Cyganek, L and Cabrera-Orefice, A and Eberl, H and Maack, C and Streckfuss-Bömeke, K and Pavez-Giani, M and Doroudgar, S and Sossalla, ST and Zelarayán, LC},
title = {Enhancing KLF15 activity in cardiomyocytes: a novel approach to prevent pathological reprogramming and fibrosis via nuclease-deficient dCas9VPR.},
journal = {Signal transduction and targeted therapy},
volume = {11},
number = {1},
pages = {},
pmid = {41771837},
issn = {2059-3635},
mesh = {*Myocytes, Cardiac/metabolism/pathology ; *Kruppel-Like Transcription Factors/genetics/metabolism ; Animals ; CRISPR-Cas Systems/genetics ; *Fibrosis/genetics/pathology ; *Cellular Reprogramming/genetics ; Humans ; Mice ; Fibroblasts/metabolism/pathology ; Gene Expression Regulation/genetics ; *Transcription Factors/genetics ; },
abstract = {Transcriptional activity perturbation holds promise for selectively modulating harmful transcriptional networks, but its therapeutic potential remains largely unexplored. We employed a network-based analysis of single-cell heart transcriptomes to identify transcription factor activities linked to pathological cardiomyocytes in vivo. This analysis revealed that transcriptional activity of Krüppel-like factor 15 (KLF15) exhibited the most significant change in pathological cardiomyocytes, characterized by less effective repression of disease-associated genes in stressed hearts, which correlated with reduced KLF15 expression. To restore KLF15 activity, we utilized CRISPR/nuclease-dead (d)Cas9-based transcriptional enhancement (CRISPRa) in cardiomyocytes, which effectively abolished fetal reprogramming by simultaneously suppressing pathological gene expression and restoring metabolic homeostasis under sustained stress conditions. Furthermore, we identified a novel cell-nonautonomous anti-fibrotic effect mediated by cardiomyocyte-fibroblast crosstalk, and revealed the contribution of KLF15-dependent Alpha-2-glycoprotein 1, zinc-binding (AZGP1) regulation in this process. We also elucidated the upstream mechanisms of KLF15 regulation, highlighting its role as a cell-specific downstream target of the broad TGF-β canonical signaling pathway, along with its downstream-dependent mechanisms in human cardiomyocytes. Finally, to enhance the therapeutic potential of this approach, we engineered and validated an adeno-associated viral (AAV) vector with a small CRISPRa system for endogenous regulation in human cardiomyocytes suitable for clinical applications. Overall, we elucidated a regulatory circuit involving TGF-β, KLF15, and AZGP1, which coordinates critical pathological responses through cellular crosstalk between cardiomyocytes and fibroblasts. Importantly, we demonstrated the efficacy of CRISPRa as an epigenetic intervention restoring a critical transcriptional function disrupted in non-genetic heart failure. This approach provides a promising blueprint for future adaptation targeting additional non-hereditary pathologies.},
}

*Myocytes, Cardiac/metabolism/pathology
*Kruppel-Like Transcription Factors/genetics/metabolism
Animals
CRISPR-Cas Systems/genetics
*Fibrosis/genetics/pathology
*Cellular Reprogramming/genetics
Humans
Mice
Fibroblasts/metabolism/pathology
Gene Expression Regulation/genetics
*Transcription Factors/genetics

@article {pmid41769381,
year = {2026},
author = {Mandal, S and Baloch, AR and Yuan, X and Chen, J and Saribas, AS and Zhu, Y and Zhang, D and Jaijyan, D and Xu, J and Hossain, R and Sisto, I and Wang, H and Yang, X and Li, Q and Hu, W},
title = {Bipolar CD4-targeted dual-DARPin-55/57 lipid nanoparticle enables efficient CRISPR/Cas-mediated HIV-1 DNA excision and reactivation blockade in latent CD4 T cell lines.},
journal = {Materials today. Bio},
volume = {37},
number = {},
pages = {102939},
pmid = {41769381},
issn = {2590-0064},
abstract = {The persistence of HIV-1 latent reservoirs remains the principal barrier to a cure, as viral rebound occurs upon interruption of antiretroviral therapy. CRISPR/Cas genome editing offers a promising strategy to excise proviruses from host genome; however, the absence of a targeted and clinically viable delivery platform has hindered its translational application. Here, we report a chemistry-driven, CD4-targeted lipid nanoparticle (LNP) delivery platform employing a unique bipolar conjugation strategy to decorate dual CD4-targeted Designed Ankyrin Repeat Proteins (DARPins-55 and -57) on LNP (dual-DARPin-LNP). The N- and C-terminally modified DARPin-55/57 was thiolated stepwise, then bipolar maleimide-thiol coupling conjugated the thiolates to the maleimide-functionalized LNP surface. This coupling strategy ensured DARPin proper orientation on the LNP surface for efficient uptake by resting CD4 T cells. This dual-DARPin-LNP system was engineered for selective and efficient co-delivery of spCas9-GFP mRNA (Sp9m) and HIV-1-specific single-guide RNAs (sgRNAs) targeting LTR and Gag (LGsg) into HIV-1 latently infected CD4 T cells. In widely used HIV-1 latency models with defined proviral modifications (J-Lat 10.6 and 2D10 cell lines), dual-DARPin-LNP loaded with Sp9m/LGsg efficiently excised integrated HIV-1 proviral DNA, as confirmed by standard PCR genotyping, absolute digital PCR quantification, confocal microscopy, and flow cytometry. Importantly, proviral excision functionally blocked HIV-1 reactivation following stimulation with latency-reversing agents suberoylanilide hydroxamic acid (SAHA) and TNFα. Together, these findings establish a modular, non-viral, receptor-guided delivery platform for CD4 T cell targeting and provide proof-of-concept for precise HIV-1 DNA excision and reactivation blockade in established latency models. This new strategy represents a step toward next-generation curative interventions against persistent HIV-1 infection.},
}

@article {pmid41766888,
year = {2026},
author = {Li, X and Zhao, Y and Guo, X and Bai, Y and Wang, J},
title = {Characterization and diversity of defense systems in Providencia pathogen.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1755933},
pmid = {41766888},
issn = {1664-3224},
mesh = {*Providencia/genetics/immunology/virology ; Phylogeny ; Genome, Bacterial ; *Enterobacteriaceae Infections/microbiology/immunology ; CRISPR-Cas Systems ; Bacteriophages ; Humans ; },
abstract = {INTRODUCTION: Providencia species are emerging opportunistic pathogens associated with multidrug-resistant infections, yet their molecular defense mechanisms against phage or mobile genetic elements remain poorly characterized.

METHODS: We present a comprehensive pan-genomic analysis of antiviral defense systems across 73 complete genomes (or chromosomes) of Providencia stuartii (n = 31) and Providencia rettgeri (n = 42), using DefenseFinder and CRISPRCasFinder. We further expanded analysis of contig/scaffold assemblies to confirm conservation of core defense profiles across assembly types. BacMGEnet was employed to derive spacer-MGE interaction networks. Phylogenetic reconstruction and gene gain and loss modeling were performed to assess evolutionary patterns. To validate functionality, we experimentally tested the anti-phage activity of Gabija and Septu in heterologous E. coli assays, including point mutation analysis of conserved residues.

RESULTS: We reveal a diverse and complex defense repertoire dominated by restriction-modification systems and CRISPR-Cas Class 1 Type I-F, with significant contributions from toxin-antitoxin, GAPS2, PsyrTA, and Mokosh systems. Notably, defense genes are non-randomly distributed, often clustering into genomic islands suggestive of horizontal acquisition. Expanded analysis confirms conservation of core defense profiles across assembly types, supporting the utility of lower-quality data when complete genomes are scarce. Comparative analysis uncovers species-specific differences, with P. rettgeri harboring a higher abundance of non-CRISPR systems. BacMGEnet-derived spacer-MGE interaction networks further highlight species-specific dynamics, dense, hub-driven networks in P. stuartii versus sparser networks in P. rettgeri. Correlation analysis indicates potential associations between specific defense systems and virulence or antibiotic resistance genes. Phylogenetic reconstruction and gene gain and loss modeling further highlight dynamic evolutionary patterns. Both Gabija and Septu systems conferred robust, phage-specific protection; point mutations in conserved residues (GajA E465K and PtuB H53K) abolished defense.

DISCUSSION: Our findings unveil a multi-layered, modular immune architecture in Providencia, providing crucial insights into its genome plasticity, phage resistance, and adaptation in clinical environments. This work establishes a foundation for understanding the role of defense systems in the evolution and pathogenicity of the Providencia genus.},
}

*Providencia/genetics/immunology/virology
Phylogeny
Genome, Bacterial
*Enterobacteriaceae Infections/microbiology/immunology
CRISPR-Cas Systems
Bacteriophages
Humans

@article {pmid41764730,
year = {2026},
author = {D'Souza, LJ and Young, JN and Coffman, H and Petrow, EP and Bhattacharya, D},
title = {A genome-wide CRISPR screen reveals novel determinants of long-lived plasma cell secretory capacity.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {215},
number = {2},
pages = {},
doi = {10.1093/jimmun/vkaf354},
pmid = {41764730},
issn = {1550-6606},
support = {R01AI129945//National Institutes of Health (NIH)/ ; P30CA023074//Research, Innovation & Impact (RII) of the University of Arizona and National Cancer Institute/ ; S10 OD028466/GF/NIH HHS/United States ; },
mesh = {Animals ; *Plasma Cells/immunology/metabolism ; Mice ; Humans ; Myeloid Differentiation Factor 88/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Multiple Myeloma/immunology/genetics ; Mice, Inbred C57BL ; Mice, Knockout ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Plasma cell subsets vary in their lifespans and ability to sustain humoral immunity. We conducted a genome-wide CRISPR-Cas9 screen in myeloma cells for factors that promote surface expression of CD98, a marker of longevity in mouse plasma cells. A large fraction of genes found to promote CD98 expression in this screen are involved in secretory and other vesicles, including subunits of the V-type ATPase complex. Genetic ablation and chemical inhibition of V-type ATPases in myeloma cells and primary plasma cells, respectively, reduced antibody secretion. Mouse and human long-lived plasma cells had greater numbers of acidified vesicles than their short-lived counterparts, and this correlated with increased antibody secretory capacity. The screen also revealed a requirement for the signaling adapter MYD88 in CD98 expression. Plasma cell-specific deletion of Myd88 led to reduced survival and antibody secretion by antigen-specific cells in vivo and unresponsiveness to BAFF and APRIL ex vivo. These data reveal novel regulators that link plasma cell secretory capacity and lifespan.},
}

Animals
*Plasma Cells/immunology/metabolism
Mice
Humans
Myeloid Differentiation Factor 88/genetics/metabolism
CRISPR-Cas Systems/genetics
*Multiple Myeloma/immunology/genetics
Mice, Inbred C57BL
Mice, Knockout
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41628535,
year = {2026},
author = {Xie, K and Ren, H and Ban, D and Chen, L and Xin, X and Zhang, J and Tang, Q and Huang, L and Wei, J and Zhang, K and Liao, X},
title = {Silica-detoxified perovskite ECL: Cas13a-triggered signal-on sensing with CsPbBr3@SiO2@Au.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {170},
number = {},
pages = {109243},
doi = {10.1016/j.bioelechem.2026.109243},
pmid = {41628535},
issn = {1878-562X},
mesh = {*Silicon Dioxide/chemistry ; *Gold/chemistry ; *Biosensing Techniques/methods ; *Titanium/chemistry ; *MicroRNAs/analysis/blood ; *Oxides/chemistry ; *Calcium Compounds/chemistry ; Electrochemical Techniques/methods ; Limit of Detection ; Humans ; Luminescent Measurements/methods ; CRISPR-Cas Systems ; Nanocomposites/chemistry ; },
abstract = {Perovskite nanocrystals are attractive ECL emitters but suffer from poor water stability and potential toxicity. Here we report a signal-on electrochemiluminescent biosensor that integrates CsPbBr3@SiO2@Au nanocomposites with a CRISPR/Cas13a-Nb.BbvCI amplification cascade for ultrasensitive microRNA detection. The CsPbBr3 core provides bright emission, a conformal SiO2 shell enhances water compatibility and suppresses ion leakage, and surface Au nanoparticles offer abundant sites for thiolated ferrocene-hairpin (Fc-HP) immobilization. In the resting state, proximal Fc efficiently quenches the CsPbBr3 ECL. Target miRNA activates Cas13a to cleave a dumbbell probe and release an intermediate strand that hybridizes with Fc-HP; subsequent Nb.BbvCI nicking removes Fc from the electrode and is recycled, producing robust signal restoration. Morphology (TEM), composition (EDS/XPS), and stepwise electrochemistry (CV/EIS) verify a core-shell-Au architecture and a reliably assembled interface that follows the expected quench→restore behavior. Under optimized conditions (0.5 mg mL[-1] CsPbBr3@SiO2@Au, 2.0 μM Fc-HP, 40 min target incubation, 100 mM TPrA, 120 s pre-reaction), the assay affords a 1 aM-1.0 × 10[9] aM linear range with an estimated limit of detection (LOD) of 1.86 aM. The sensor shows high specificity against homologous sequences and achieves 95.22%-104.61% recoveries with RSD < 5% in spiked serum. Pilot measurements distinguish patient serum samples from healthy controls, underscoring clinical potential. This modular platform couples stable perovskite ECL emission with programmable CRISPR chemistry, offering a sensitive, selective, and water-compatible route for microRNA analysis and readily extensible nucleic-acid diagnostics.},
}

*Silicon Dioxide/chemistry
*Gold/chemistry
*Biosensing Techniques/methods
*Titanium/chemistry
*MicroRNAs/analysis/blood
*Oxides/chemistry
*Calcium Compounds/chemistry
Electrochemical Techniques/methods
Limit of Detection
Humans
Luminescent Measurements/methods
CRISPR-Cas Systems
Nanocomposites/chemistry

@article {pmid41534449,
year = {2026},
author = {Kirino, Y and Takeno, M},
title = {A critical look at animal and cellular models in autoinflammatory diseases.},
journal = {Current opinion in immunology},
volume = {99},
number = {},
pages = {102719},
doi = {10.1016/j.coi.2025.102719},
pmid = {41534449},
issn = {1879-0372},
mesh = {Animals ; Humans ; Disease Models, Animal ; *Autoimmune Diseases/immunology/genetics/etiology ; Gene Editing ; Induced Pluripotent Stem Cells ; *Inflammation/immunology ; CRISPR-Cas Systems ; *Hereditary Autoinflammatory Diseases ; },
abstract = {Autoinflammatory diseases (AIDs) comprise a diverse group of conditions arising from dysregulated immune control due to congenital or acquired genetic abnormalities in innate immune pathways, and patients typically require lifelong treatment. Owing to their rarity, access to patient samples is limited, making animal and cellular models indispensable for elucidating pathogenesis and advancing therapeutic development. Nevertheless, robust animal and cell-based models remain scarce. Recent advances in precision genome editing now enable lineage- and cell type-specific modeling of autoinflammation, steadily improving the fidelity with which disease phenotypes are recapitulated. In this review, we survey the current landscape of CRISPR-enabled knock-in/knock-out animal models, engineered cell lines, and patient-derived induced pluripotent stem cells for AIDs, and discuss how these platforms can be leveraged to dissect disease mechanisms and accelerate drug discovery.},
}

Animals
Humans
Disease Models, Animal
*Autoimmune Diseases/immunology/genetics/etiology
Gene Editing
Induced Pluripotent Stem Cells
*Inflammation/immunology
CRISPR-Cas Systems
*Hereditary Autoinflammatory Diseases

@article {pmid41506441,
year = {2026},
author = {Mokhles, F and Moosavi, MA and Gutierrez-Uzquiza, A and Velasco, G and Li, M and Cordani, M},
title = {Unraveling stress-adaptation pathways in cancer: Functional dissection through CRISPR-based genetic screens.},
journal = {Cancer letters},
volume = {644},
number = {},
pages = {218246},
doi = {10.1016/j.canlet.2026.218246},
pmid = {41506441},
issn = {1872-7980},
mesh = {Humans ; *Neoplasms/genetics/pathology/metabolism ; *CRISPR-Cas Systems ; Animals ; Endoplasmic Reticulum Stress/genetics ; *Genetic Testing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Signal Transduction ; Tumor Microenvironment ; Unfolded Protein Response/genetics ; *Stress, Physiological ; Adaptation, Physiological/genetics ; },
abstract = {Cancer cells face a hostile microenvironment characterized by hypoxia, nutrient deprivation, endoplasmic reticulum (ER) stress, and oxidative imbalance. To cope with these challenges, they activate an interconnected network of adaptive pathways including autophagy, the unfolded protein response, metabolic reprogramming, and the integrated stress response., which promote cell survival, therapy resistance, immune evasion, and metastasis. CRISPR-based functional genomics has emerged as a powerful strategy to systematically dissect these stress-adaptive networks, enabling the identification of key regulators and vulnerabilities across diverse contexts. In this review, we first summarize tumor progression in major stress conditions and then highlight how CRISPR screening strategies ranging from genome-wide loss-of-function studies to single-cell and combinatorial platforms, are unraveling critical stress regulators. We further discuss emerging tools, model systems, and translational perspectives, underscoring how the integration of CRISPR technologies with multi-omics, artificial intelligence, and advanced preclinical models is reshaping our understanding of cancer stress biology and guiding the development of novel therapeutic strategies. Finally, we addressed how these novel dissection technologies influence translational opportunities, specifically in the context of combining stress-pathway modulators with immunotherapy and targeted therapy drugs.},
}

Humans
*Neoplasms/genetics/pathology/metabolism
*CRISPR-Cas Systems
Animals
Endoplasmic Reticulum Stress/genetics
*Genetic Testing/methods
*Clustered Regularly Interspaced Short Palindromic Repeats
Signal Transduction
Tumor Microenvironment
Unfolded Protein Response/genetics
*Stress, Physiological
Adaptation, Physiological/genetics

@article {pmid41763755,
year = {2026},
author = {Ma, C and French, N and Wu, X and Gupta, SK and Gupta, TB},
title = {Molecular detection of Clostridium and Bacillus species in foods: recent advances and applications.},
journal = {Food research international (Ottawa, Ont.)},
volume = {229},
number = {},
pages = {118370},
doi = {10.1016/j.foodres.2026.118370},
pmid = {41763755},
issn = {1873-7145},
mesh = {*Clostridium/isolation & purification/genetics ; *Bacillus/isolation & purification/genetics ; *Food Microbiology/methods ; Nucleic Acid Amplification Techniques/methods ; Polymerase Chain Reaction ; CRISPR-Cas Systems ; In Situ Hybridization, Fluorescence ; Humans ; Food Contamination/analysis ; },
abstract = {Spore-forming bacteria, especially Clostridium spp. and Bacillus spp., are ubiquitous in food systems, and their ingestion can cause serious diseases in humans and animals. Their persistence in diverse food matrices and resistance to conventional treatments make rapid and accurate detection essential for effective monitoring and control. Traditional culture-based and biochemical assays remain the standard for identifying these bacteria but are often time-consuming, labor-intensive and limited in sensitivity. In contrast, nucleic acid-based methods provide rapid, specific and sensitive alternatives by directly targeting genetic markers of pathogenic or spoilage strains. This review summarizes how nucleic acid methods, including PCR, FISH, LAMP, RPA, WGS, and the emerging CRISPR/Cas systems, have been applied specifically to detect Clostridium spp. and Bacillus spp. in food systems. Each method offers unique advantages and limitations. PCR-based methods enable accurate quantification but require thermal cycling. FISH-based methods are simple but require microscopy and have limited validation in food. WGS-based methods provide strain-level characterization but depend on informatics and specialized equipment. Isothermal techniques such as LAMP- and RPA-based methods allow rapid field detection but involve complex primer design or poor discrimination of closely related genes. CRISPR/Cas-based platforms further enhance simplicity, specificity, sensitivity for on-site detection, though the validation for spore-forming bacteria remains limited. Overall, this review provides an overview of gene targets, methodological adaptations, and analytical performance of nucleic acid-based assays for detecting Clostridium spp. and Bacillus spp., highlighting current progress and future opportunities for improving food safety monitoring.},
}

*Clostridium/isolation & purification/genetics
*Bacillus/isolation & purification/genetics
*Food Microbiology/methods
Nucleic Acid Amplification Techniques/methods
Polymerase Chain Reaction
CRISPR-Cas Systems
In Situ Hybridization, Fluorescence
Humans
Food Contamination/analysis