@article {pmid42135407,
year = {2026},
author = {Bazick, HO and James, LM and Zylka, MJ},
title = {Nickase NmCas9 unsilences paternal Ube3a in a mouse model of Angelman syndrome without causing AAV vector integration.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42135407},
issn = {2045-2322},
support = {631904//Simons Foundation/ ; 1R01NS109304/NS/NINDS NIH HHS/United States ; 1R01NS109304/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; *Ubiquitin-Protein Ligases/genetics/metabolism ; Mice ; *Dependovirus/genetics ; *Angelman Syndrome/genetics/therapy/metabolism ; Genetic Vectors/genetics ; Disease Models, Animal ; Neurons/metabolism ; *Deoxyribonuclease I/metabolism/genetics ; Gene Editing/methods ; Male ; Virus Integration ; CRISPR-Cas Systems ; },
abstract = {Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by loss of maternal UBE3A. In neurons, the paternal (pat)UBE3A allele is silenced by a long non-coding antisense transcript called Ube3a-ATS. Previous genome-editing approaches used active nucleases to unsilence patUbe3a by disrupting Ube3a-ATS. However, these methods create DNA double-strand breaks (DSBs) and promote integration of adeno-associated virus (AAV) vector genomes, both of which raise potential safety concerns. Here, we found that a nickase Neisseria meningitidis Cas9 variant (nNmCas9-D15A) disrupted Ube3a-ATS transcription when targeted to the non-template strand and unsilenced patUbe3a in cultured mouse neurons without generating DSBs or causing AAV integration. Intracerebroventricular delivery of AAV9-nNmCas9-D15A in AS model mice potently and durably reduced Ube3a-ATS and elevated Ube3a throughout the cerebral cortex and hippocampus for at least 6 months. Further, this vector restored UBE3A expression in ~ 87% of cortical neurons, which compares favorably to previously reported efficiencies with active Cas9, dead Cas9, and zinc finger nuclease vectors. These results demonstrate that nNmCas9 is a highly effective and potentially safer genome editor for the treatment of AS.},
}

Animals
*Ubiquitin-Protein Ligases/genetics/metabolism
Mice
*Dependovirus/genetics
*Angelman Syndrome/genetics/therapy/metabolism
Genetic Vectors/genetics
Disease Models, Animal
Neurons/metabolism
*Deoxyribonuclease I/metabolism/genetics
Gene Editing/methods
Male
Virus Integration
CRISPR-Cas Systems

@article {pmid42303099,
year = {2026},
author = {Zhou, Z and Dong, S and Li, S and Tan, X and Zhou, Z and Fan, L and Liang, J and Li, G},
title = {CRISPR/Cas12a and nanocomposite-based electrochemical/ colorimetric parallel dual-channel aptasensor for highly sensitive LDL detection.},
journal = {Nanomedicine : nanotechnology, biology, and medicine},
volume = {75},
number = {},
pages = {102981},
doi = {10.1016/j.nano.2026.102981},
pmid = {42303099},
issn = {1549-9642},
mesh = {*Nanocomposites/chemistry ; *Aptamers, Nucleotide/chemistry ; *Biosensing Techniques/methods ; *Lipoproteins, LDL/analysis/blood ; Humans ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Colorimetry/methods ; Graphite/chemistry ; DNA, Single-Stranded/chemistry ; },
abstract = {Atherosclerotic cardiovascular disease (ASCVD) remains a leading global health threat, necessitating precise monitoring of low-density lipoprotein (LDL) as a key risk biomarker for assessing ASCVD risk. Herein, an electrochemical/colorimetric dual- channel aptasensor was developed by integrating nitrogen-doped reduced graphene oxide-Hemin-trimanganese tetroxide nanoparticles (NrGO-Hemin-Mn3O4 NPs) with the CRISPR/Cas12a system. The CRISPR/Cas12a system introduces a powerful signal amplification cascade: a single target binding event activates the trans-cleavage of numerous ssDNA probes, translating into a highly amplified electrical and optical response. The NrGO-Hemin-Mn3O4 NPs serves as a conductive redox probe and exhibits superior peroxidase-like activity through the synergistic effect between Hemin and Mn3O4. Mechanistically, surface-bound single-stranded DNA (ssDNA) initially induces steric hindrance, which obstructs electron transfer and suppresses the enzyme-mimicking performance of the NrGO-Hemin-Mn3O4 NPs. Upon the target LDL binding, the released activator DNA triggers the trans-cleavage activity of Cas12a to degrade the ssDNA, thereby restoring both the electroactivity and catalytic performance of the probe. Experimental results demonstrated that the dual-channel aptasensor achieved a wide linear range from 0.01 to 1000 nM with a detection limit of 0.01 nM, demonstrating that CRISPR integration is pivotal for achieving high sensitivity in complex biological matrices. This dual-channel strategy offers a sensitive, intuitive tool for early clinical screening of ASCVD diseases.},
}

*Nanocomposites/chemistry
*Aptamers, Nucleotide/chemistry
*Biosensing Techniques/methods
*Lipoproteins, LDL/analysis/blood
Humans
*Electrochemical Techniques/methods
*CRISPR-Cas Systems/genetics
*Colorimetry/methods
Graphite/chemistry
DNA, Single-Stranded/chemistry

@article {pmid42420735,
year = {2026},
author = {Rijal, S and Zhang, R and Tian, XJ},
title = {Harnessing CRISPRi Competition to Develop Multimodule Controllers for Resource-Aware Circuit Design.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3041},
number = {},
pages = {305-318},
pmid = {42420735},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Gene Regulatory Networks ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Genetic Engineering/methods ; },
abstract = {Cellular resource limitations give rise to resource competition, undermining the modularity and predictability of engineered genetic circuits. In systems containing positive feedback, such competition can drive Winner-Takes-All (WTA) dynamics, resulting in severe imbalances in resource allocation across circuit modules. In this chapter, we present an experimental implementation of a Negatively Competitive Regulatory (NCR) controller based on CRISPR interference (CRISPRi) in dual self-activation (DSA) circuits. We describe a detailed workflow for chromosomal integration of a tunable dCas9 expression cassette, as well as the design of self-activation modules and module-specific guide RNAs that induce self-repression through competition for limiting dCas9. This architecture introduces effective negative feedback to the more active module while reallocating resources to the less active one, thereby promoting balanced module activity. Finally, we provide guidelines for quantitatively assessing the regulation of resource competition between DSA modules using the NCR strategy. Overall, these guidelines demonstrate how CRISPRi can be leveraged to implement NCR strategy in gene circuits, thereby enhancing circuit modularity and predictability.},
}

*CRISPR-Cas Systems
*Gene Regulatory Networks
RNA, Guide, CRISPR-Cas Systems/genetics
*Genetic Engineering/methods

@article {pmid42423795,
year = {2026},
author = {Ata, A and Topuz Ata, D},
title = {A comprehensive review of CRISPR-Cas9-mediated genome editing in Leishmania strains: methodologies, applications, challenges and future directions.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42423795},
issn = {1573-4978},
mesh = {*Leishmania/genetics ; *Gene Editing/methods/trends ; *CRISPR-Cas Systems/genetics ; Animals ; Genome, Protozoan ; Leishmaniasis/parasitology/genetics ; Humans ; },
abstract = {Genome editing employing CRISPR-Cas9 has rapidly transformed experimental research in Leishmania, providing opportunities to investigate the genetic factors responsible for parasite survival, response to drugs and pathogenic traits. This review provides a comprehensive synthesis of CRISPR-based systems implemented across Leishmania species, spanning Cas9-mediated gene deletion, precise genome editing, endogenous locus tagging and pooled screening strategies. Furthermore, we highlight the emergence of Cas variants and next-generation CRISPR systems which expand the range of targetable genomic regions, improve editing precision and reduce the need for generation of double-strand DNA breaks (DSBs). Particular emphasis is placed on conditional and inducible genome-editing platforms, cytosine base-editing technologies, and recently developed CRISPR-based approaches such as prime editing, CRISPR activation/interference and Cas12-associated implementations. This review also discusses the principal biological and technical constraints influencing CRISPR-based studies in Leishmania, including genome plasticity, multicopy gene families, required genes, guide RNA design limitations and off-target considerations. Notably, the review also addresses CRISPR-Cas implementations in sand-fly vector biology, drawing on a foundational study in Phlebotomus papatasi. Through systematic compilation of published studies into comparative tables, we evaluate the strengths, limitations, experimental utility, delivery strategies, experimental workflows and representative applications of major CRISPR platforms. Together, these advances highlight the transition of CRISPR-Cas systems from proof-of-concept tools to versatile platforms for functional genomics, target validation and translational research in Leishmania, while offering a consolidated guide for selecting suitable CRISPR-Cas technologies and underscoring important considerations for their continued development in leishmaniasis research.},
}

*Leishmania/genetics
*Gene Editing/methods/trends
*CRISPR-Cas Systems/genetics
Animals
Genome, Protozoan
Leishmaniasis/parasitology/genetics
Humans

@article {pmid42424815,
year = {2026},
author = {Tang, Q and Zhang, Y and Garza, DR and Ruan, C and Liu, B and Rocha, U and Shen, P and Wei, Y and Deng, Y and Zhang, J and Richnow, HH},
title = {Virus-mediated fate of antimicrobial resistance genes in livestock manure anaerobic digestion.},
journal = {Water research},
volume = {305},
number = {},
pages = {126401},
doi = {10.1016/j.watres.2026.126401},
pmid = {42424815},
issn = {1879-2448},
abstract = {Antimicrobial resistance (AMR) poses a critical global health challenge, with livestock manure acting as a significant environmental reservoir for antimicrobial resistance genes (ARGs). Anaerobic digestion (AD) is a pivotal process for mitigating ARG dissemination at the livestock-environment-human interface. This study aims to elucidate the global dynamics of ARGs in AD systems, focusing on virus-host interactions and arms race, to identify actionable strategies for AMR control. We analyzed 205 metagenomic (4.5 Tb) and 36 meta-transcriptomic (640 Gb) datasets, including 15 newly generated datasets, revealing that pig manure AD harbors the highest ARG abundance (0.668 ARGs/16S rRNA), while AD systems generally exhibit limited transcriptional activation of ARGs. We constructed a viral dataset for livestock manure AD (GVD_LMAD), comprising 59,316 DNA and 727 RNA viral operational taxonomic units (vOTUs). Virus-host interactions established by CRISPR-Cas spacer, tRNA and homology matches revealed 889 lytic infections of antimicrobial-resistant bacteria (ARB) compared to only 18 ARG transduction events. Further analysis showed that the relative abundance of vOTUs assigned to the reduction role (4.11% ± 3.19%) was substantially higher than that of reproduction (0.72% ± 0.64%) and transduction (0.19% ± 0.30%), demonstrating that, among viral processes, lysis outweighs transduction in contributing to ARG abundance reduction in AD. Furthermore, an antiviral defense system (ADS) catalogue (GADSC_LMAD), derived from 2760 high-quality metagenome-assembled genomes (MAGs) containing 39,307 ADS, with ADS prevalence in ARB (7.8 ± 6.0 per MAG), indicating an intensified virus-host arms race in AD that may shield ARB from phage lysis. The resulting CRISPR-Cas immune network with expressed spacers targets foreign ARG-carrying sequences (primarily plasmids and ICEs), suggesting a mechanism that restricts horizontal gene transfer (HGT) via conjugation and transformation, despite shielding ARB from phage lysis. Collectively, these findings highlight that viral communities significantly contribute to ARG reduction through phage lysis relative to transduction, while the ADS-mediated arms race, despite protecting ARB, constructs a biological firewall that potentially limits HGT of ARGs. This study provides novel insights into virus-host dynamics as a key mechanism for controlling ARG dissemination in AD systems.},
}

@article {pmid42424969,
year = {2026},
author = {Samad, MA and Ahmad, I and Jabir, NR and Rehan, M and Zaidi, SK and Al-Abbasi, F and Tabrez, S},
title = {Role of long non-coding RNAs in therapeutic resistance and clinical applications in cancer.},
journal = {European journal of medicinal chemistry},
volume = {317},
number = {},
pages = {119090},
doi = {10.1016/j.ejmech.2026.119090},
pmid = {42424969},
issn = {1768-3254},
abstract = {Cancer is one of the leading causes of mortality worldwide and is recognized as a complex, multifactorial disease with no clearly defined etiology for its onset and progression. Long non-coding RNAs (lncRNAs) are widely distributed across the human body and play varied roles in regulating cellular processes. In recent years, they have gained the attention of the scientific community as key regulators of cancer due to their diverse functional roles and complex regulatory mechanisms. Aberrant expression of lncRNAs contributes to tumor progression, functioning as oncogenes that modulate various pathways through different mechanisms. Early technologies could not study lncRNAs effectively and considered it as "junk" RNA. Studies using gene-expression analyses, functional experiments, and animal-based models have shown that dysregulated lncRNAs are implicated in the maintenance of cancer stem cells (CSCs) and in driving therapeutic resistance. Additionally, lncRNAs have shown promise as valuable biomarkers for cancer diagnosis, prognosis, predicting patient outcomes, and guiding treatment strategies. Moreover, therapeutic strategies targeting lncRNAs, such as antisense oligonucleotides (ASOs), RNA interference (RNAi), exosome-based delivery systems, nanomedicine, virus-mediated therapy, and CRISPR-Cas technologies, have opened new avenues for cancer treatment. This review highlights the diverse roles of lncRNAs in therapeutic resistance and emphasizes their clinical potential as diagnostic and prognostic tools and emerging therapeutic strategies.},
}

@article {pmid42425355,
year = {2026},
author = {V, MS and Chaudhary, N and Hasan, M and Kumar, A and Tripathi, MK},
title = {Filamentous fungi as microbial cell factories for lignocellulosic biomass valorization: A comprehensive review.},
journal = {International journal of biological macromolecules},
volume = {375},
number = {},
pages = {153415},
doi = {10.1016/j.ijbiomac.2026.153415},
pmid = {42425355},
issn = {1879-0003},
abstract = {The transition toward a sustainable bioeconomy requires efficient conversion of lignocellulosic biomass (LCB), the most abundant renewable biological macromolecular resource on Earth, into fuels, chemicals, and other high-value products. However, the complex architecture of cellulose, hemicellulose, and lignin imparts significant recalcitrance, limiting biomass deconstruction and industrial utilization. Although recent reviews have examined fungal biorefineries, lignocellulolytic enzymes, or fungal strain engineering separately, an integrated synthesis linking lignocellulosic biomass characteristics, fungal deconstruction mechanisms, hydrolysate utilization, and cell-factory engineering remains limited. This review presents an integrated framework for lignocellulosic biomass valorization using filamentous fungi as microbial cell factories. We examine biomass composition, recalcitrance, and pretreatment strategies, followed by the fungal macromolecular machinery responsible for biomass deconstruction, including cellulases, hemicellulases, lignin-active oxidoreductases, and auxiliary activity enzymes. Particular emphasis is placed on the regulatory networks and engineering strategies that govern fungal performance, including transcription factor engineering, promoter engineering, metabolic rewiring, heterologous pathway engineering, RNA interference, and CRISPR-Cas-based genome editing. The review further discusses the conversion of lignocellulose-derived hydrolysates into biofuels, organic acids, industrial enzymes, and other high-value compounds, together with emerging advances in co-culture fermentation, downstream processing, and integrated biorefinery design. Collectively, this review highlights how the integration of fungal enzymatic systems, strain engineering, and process-level innovations can overcome biomass recalcitrance and improve lignocellulosic bioconversion efficiency. These insights provide a framework for developing robust fungal platforms for the sustainable production of high-value bioproducts from renewable biomass.},
}

@article {pmid42426280,
year = {2026},
author = {Grigg, S and Shembrey, C and Fareh, M and Blombery, P and Corn, JE and Seymour, JF and Casan, JML},
title = {CRISPR in clinical oncology: translational advances from molecular diagnostics to therapeutics.},
journal = {Nature reviews. Clinical oncology},
volume = {},
number = {},
pages = {},
pmid = {42426280},
issn = {1759-4782},
abstract = {Cancer care is increasingly driven by molecular classification, yet many key oncogenic drivers remain undruggable, and intrinsic or acquired resistance to treatment frequently limits durable clinical benefit. CRISPR-Cas technologies provide a modular, programmable platform to interrogate and directly manipulate cancer biology via sequence-specific targeting of DNA or RNA and have advanced from experimental tools to the early stages of clinical translation. In this Review, we outline how CRISPR-enabled functional genomics approaches can reveal unexpected cancer dependencies and resistance mechanisms. We discuss emerging applications of CRISPR-based diagnostics in oncology that convert precise nucleic acid sequence recognition into rapid mutation detection. We also discuss applications of CRISPR in therapeutic strategies ranging from ex vivo immune cell engineering to nascent in vivo interventions that directly target tumour-related sequences such as fusion junctions or single-nucleotide variants. Finally, we highlight technological and regulatory challenges, including effective delivery of the editing machinery to cells in vivo, safety and platform-level regulatory frameworks, that will determine the clinical utility of CRISPR-based diagnostics and therapies in oncology.},
}

@article {pmid42427176,
year = {2026},
author = {Łakomy, W and Myślińska, M and Tarnawska, E and Rogóż, W and Kulig, K and Owczarzy, A and Maciążek-Jurczyk, M},
title = {Biotechnological strategies to combat antibiotic resistance.},
journal = {Polimery w medycynie},
volume = {56},
number = {1},
pages = {41-51},
doi = {10.17219/pim/218777},
pmid = {42427176},
issn = {0370-0747},
mesh = {Humans ; Antimicrobial Peptides/pharmacology ; Phage Therapy ; *Biotechnology ; CRISPR-Cas Systems ; Gene Editing ; *Drug Resistance, Microbial ; *Drug Resistance, Bacterial ; Anti-Bacterial Agents/pharmacology ; Nanoparticles ; Nanotechnology ; Animals ; },
abstract = {This article aims to present the current state of knowledge on four major biotechnological antimicrobial strategies and to evaluate their potential clinical applications in the context of increasing antibiotic resistance. Approaches such as phage therapy, CRISPR-Cas9 gene editing, nanoparticles, and antimicrobial peptides (AMPs) may significantly contribute to limiting the spread of resistance genes. Particular attention is given to advances in genetic engineering that enable precise targeting and elimination of resistance determinants, as well as to the therapeutic potential of the microbiome. A literature review of studies published between 2010 and 2025 was conducted using the following keywords: antimicrobial resistance, phage therapy, CRISPR-Cas9, AMPs, and nanotechnology. Both review articles and original studies, including preclinical and clinical data, were considered. Phage therapy demonstrates high efficacy against antibiotic-resistant pathogens, particularly in the form of phage cocktails and genetically engineered phages. Antimicrobial peptides exhibit broad-spectrum activity and can be structurally optimized to improve stability and selectivity. CRISPR-Cas9 systems enable targeted elimination of resistance genes or direct disruption of pathogen genomes, while nanotechnology facilitates drug delivery, biofilm penetration, and bactericidal activity, particularly through metal-based nanoparticles. Notably, all approaches show potential for synergistic use with conventional antibiotics. Biotechnological treatment strategies may become a key component in combating antibiotic resistance. However, their clinical implementation requires further research, comprehensive safety evaluation, regulatory development, and integration into medical practice. Advances in these areas could significantly reduce the global burden of infectious diseases.},
}

Humans
Antimicrobial Peptides/pharmacology
Phage Therapy
*Biotechnology
CRISPR-Cas Systems
Gene Editing
*Drug Resistance, Microbial
*Drug Resistance, Bacterial
Anti-Bacterial Agents/pharmacology
Nanoparticles
Nanotechnology
Animals

@article {pmid42428244,
year = {2026},
author = {Zhang, X and Shi, H and Yang, J and Du, L and Zhang, X and Li, X},
title = {The application of CRISPR gene-editing technology in influenza prevention and control.},
journal = {Frontiers in genome editing},
volume = {8},
number = {},
pages = {1844919},
pmid = {42428244},
issn = {2673-3439},
abstract = {Influenza A virus (IAV) and influenza B virus (IBV) remain major global public health threats because of their rapid antigenic evolution and efficient human-to-human transmission. In contrast, influenza C virus (ICV) and influenza D virus (IDV) generally exhibit narrower host ranges and milder pathogenicity, yet their potential for interspecies transmission and zoonotic spillover still warrants attention. Conventional prevention strategies, such as inactivated and live-attenuated vaccines, suffer from prolonged development timelines and diminished efficacy against rapidly evolving viral strains. However, antiviral drugs are increasingly limited by the rapid emergence of drug-resistant variants. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) gene-editing technology has emerged as a promising platform for influenza prevention and control owing to its programmability and precise targeting capability. In this paper, we summarize recent advances in CRISPR-based strategies for influenza prevention and control. The RNA-targeting CRISPR-associated protein 13 (Cas13) system can recognize conserved viral RNA sequences and suppress replication across influenza subtypes, whereas the DNA-targeting CRISPR-associated protein 9 (Cas9) system can edit host susceptibility genes and thereby reduce cellular permissiveness to infection. In addition, lipid nanoparticle (LNP)-based delivery systems have become important tools for improving the in vivo delivery and expression of CRISPR components by enhancing targeting efficiency and reducing immunogenicity. CRISPR-based diagnostics, such as Specific High-sensitivity Enzymatic Reporter unLOCKing (SHERLOCK), further expand the clinical utility of this technology by enabling rapid and sensitive detection of influenza viruses. Despite these advances, substantial challenges remain, including delivery inefficiency, off-target activity, long-term safety concerns, and the risk of viral escape. With continued technological refinement and careful translational development, CRISPR may become a versatile tool for influenza prevention, diagnosis, and therapy.},
}

@article {pmid42429865,
year = {2026},
author = {Preetam, S and Rath, P and Al-Enazi, NM and Sharaf, AAM and Jumah, JB and Govindarajan, RK and Goud, P and Thiruvengadam, M and Mathivanan, K},
title = {Engineering extracellular vesicle biogenesis for therapeutic gene delivery: emerging genetic programming strategies and translational prospects.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42429865},
issn = {1573-4978},
mesh = {Humans ; *Extracellular Vesicles/metabolism/genetics ; CRISPR-Cas Systems/genetics ; *Gene Transfer Techniques ; Gene Editing/methods ; *Genetic Therapy/methods ; *Genetic Engineering/methods ; Animals ; },
abstract = {Extracellular vesicles (EVs) have emerged as promising biological nanocarriers for gene therapy due to their intrinsic ability to transport nucleic acids, proteins, and lipids between cells. Advances in EV biology have revealed complex regulatory mechanisms governing vesicle biogenesis, cargo sorting, secretion, and uptake, offering multiple opportunities for therapeutic engineering. Concurrently, modern genetic technologies, including the CRISPR-Cas9 genome editing system and synthetic biology tools, have enabled precise manipulation of EV composition and functionality. This review integrates current knowledge of EV biogenesis with emerging genetic engineering strategies to transform EVs into programmable gene delivery systems. We discuss recent advances in genetic tools for studying EV dynamics, methods for engineering EV cargo and targeting specificity, and the application of EV platforms for RNA and genome-editing therapies. Furthermore, key challenges related to vesicle heterogeneity, large-scale production, and clinical translation are examined. Finally, we highlight future perspectives on programmable EV therapeutics and their potential role in next-generation precision medicine.},
}

Humans
*Extracellular Vesicles/metabolism/genetics
CRISPR-Cas Systems/genetics
*Gene Transfer Techniques
Gene Editing/methods
*Genetic Therapy/methods
*Genetic Engineering/methods
Animals

@article {pmid42429888,
year = {2026},
author = {Thevendran, R and Maheswaran, S and Lee, SY},
title = {Development of attenuated and inactivated Dengue strains using advanced gene editing tools.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42429888},
issn = {1573-4978},
support = {FRGS/1/2023/SKK06/AIMST/03/2//Ministry of Higher Education, Malaysia/ ; },
mesh = {Humans ; *Dengue Virus/genetics/immunology/pathogenicity ; *Dengue Vaccines/immunology/genetics ; *Dengue/prevention & control/immunology/virology/genetics ; *Gene Editing/methods ; Vaccines, Attenuated/immunology/genetics ; Animals ; CRISPR-Cas Systems ; Vaccine Development/methods ; },
abstract = {Dengue fever remains a persistent viral threat, affecting millions of families every year, turning a simple mosquito bite into a potentially life-threatening emergency. This disease remains a constant burden on our global healthcare system, demanding innovative solutions to protect worldwide communities. While many researchers discuss general treatments, preventions and modern medical interventions, there is often a lack of focus on how current, cutting-edge molecular and genetic tools are employed to engineer dengue strains as vaccine candidates. Therefore, in this paper, we explore the recent genetic strategies, such as targeted virulent gene deletions, CRISPR-Cas inactivation, and viral codon deoptimization approaches used to attenuate or inactivate Dengue viruses specifically. Assays and techniques used in validating Dengue viral attenuation or inactivation are also discussed in detail, highlighting the importance of the balance between safety and immunogenicity for Dengue vaccine uses. The article also briefly elaborates the complex biological challenges and safety concerns that centre on Dengue vaccine developments. By bridging the gap between advanced genetics and public health, this review provides readers with a comprehensive understanding of how modern genetics is paving the way for the next generation of safe and effective Dengue vaccines.},
}

Humans
*Dengue Virus/genetics/immunology/pathogenicity
*Dengue Vaccines/immunology/genetics
*Dengue/prevention & control/immunology/virology/genetics
*Gene Editing/methods
Vaccines, Attenuated/immunology/genetics
Animals
CRISPR-Cas Systems
Vaccine Development/methods

@article {pmid42431872,
year = {2026},
author = {Kaya, NH and Abukhalaf, M and Fuentes, G and Taubenheim, J and Hentschel, U and Tholey, A and Fraune, S},
title = {c-JUN controls microbial colonization via selective phagocytosis in the sea anemone Nematostella.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42431872},
issn = {2041-1723},
support = {CRC 1182, Project B1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CRC 1182, Project B1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CRC 1182, Project Z3//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CRC 1182, Project B1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CRC 1182, Project B1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CRC 1182, Project Z3//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {Animals ; *Sea Anemones/microbiology/immunology/genetics/metabolism ; *Phagocytosis/immunology ; Immunity, Innate ; Vibrio/immunology ; *Proto-Oncogene Proteins c-jun/genetics/metabolism/immunology ; Microbiota/immunology ; CRISPR-Cas Systems ; Lysosomes/metabolism ; },
abstract = {Innate immunity is traditionally viewed as a broad defense system with limited specificity. However, increasing evidence suggests that innate immune cells can discriminate between distinct microbial partners. How such specificity arises in early-diverging animals remains unclear. Here, we identify in the sea anemone Nematostella vectensis a selective host innate immune mechanism mediated by nematosomes, motile multicellular bodies that differentially process bacterial cells. Nematosomes preferentially engulf non-native Vibrio isolates while showing reduced uptake of native host-associated strains. We identify the transcription factor cJUN as a key regulator of this process. CRISPR/Cas9-mediated knockout of cJUN reduces nematosome abundance, impairs lysosomal response, alters microbiome assembly, and increases susceptibility to bacterial infection. These results link immune gene function to microbial selectivity and demonstrate that even early-diverging animals exhibit sophisticated innate immunity mechanisms for microbiome regulation. Our findings support the idea that immune specificity can arise through repurposing deeply conserved pathways and may have deep evolutionary origin.},
}

Animals
*Sea Anemones/microbiology/immunology/genetics/metabolism
*Phagocytosis/immunology
Immunity, Innate
Vibrio/immunology
*Proto-Oncogene Proteins c-jun/genetics/metabolism/immunology
Microbiota/immunology
CRISPR-Cas Systems
Lysosomes/metabolism

@article {pmid42163455,
year = {2026},
author = {Xu, Z and Qiu, S and Tan, Y and Kuang, Y and Yang, C and Yan, F and Zhou, X and Zhou, H},
title = {Optimized tRNA processing and TREX2-SpCas9 fusion enable high-efficiency multiplex genome editing in plants.},
journal = {Plant communications},
volume = {7},
number = {7},
pages = {101921},
doi = {10.1016/j.xplc.2026.101921},
pmid = {42163455},
issn = {2590-3462},
mesh = {*Gene Editing/methods ; *Oryza/genetics ; *RNA, Transfer/genetics/metabolism ; CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism ; *Genome, Plant/genetics ; Plants, Genetically Modified ; Plant Proteins/genetics/metabolism ; },
abstract = {Multiplex genome editing is a powerful approach for dissecting gene networks and engineering complex traits in crops because it enables the simultaneous modification of multiple genomic loci. However, achieving high editing efficiency across multiple targets remains a significant challenge. To address this, we developed an optimized CRISPR system for rice that combines a monomeric TREX2-SpCas9 fusion with a novel array of tRNA-based gRNA processing elements. The TREX2-SpCas9 fusion significantly enhanced editing performance, resulting in higher editing efficiency, larger deletions, and increased mutation frequencies compared with wild-type SpCas9 and other exonuclease fusions. By systematically evaluating 38 endogenous rice tRNA genes, we identified 13 high-performing candidates, including tRNA[Leu-1] and tRNA[Pro-1], that outperformed the widely used tRNA[Gly] and tRNA[Met] elements, enabling highly efficient processing of multiplexed gRNA arrays. Incorporating these top-performing tRNAs into our system enabled simultaneous editing of up to 29 OsCPK genes in a single rice plant. Furthermore, we demonstrated the cross-species applicability of this platform in the dicot Nicotiana benthamiana using transient expression, where rice-derived tRNA elements facilitated high-efficiency editing. This optimized multiplex gene-editing system provides a robust, scalable platform for accelerating plant functional genomics and engineering complex agronomic traits.},
}

*Gene Editing/methods
*Oryza/genetics
*RNA, Transfer/genetics/metabolism
CRISPR-Cas Systems
*CRISPR-Associated Protein 9/genetics/metabolism
*Genome, Plant/genetics
Plants, Genetically Modified
Plant Proteins/genetics/metabolism

@article {pmid42240651,
year = {2026},
author = {Ma, SH and Yu, G and Park, S and Sung, H and Jeong, UJ and Kim, HH and Cho, J and Yoon, SS},
title = {Adapting prime editing with split prime editors in Escherichia coli and its application to Staphylococcus aureus genome editing.},
journal = {Applied microbiology and biotechnology},
volume = {110},
number = {1},
pages = {},
pmid = {42240651},
issn = {1432-0614},
mesh = {*Escherichia coli/genetics ; *Gene Editing/methods ; *Methicillin-Resistant Staphylococcus aureus/genetics ; *Staphylococcus aureus/genetics ; *Genome, Bacterial ; RNA, Guide, CRISPR-Cas Systems/genetics ; Streptococcus pyogenes/genetics/enzymology ; },
abstract = {Prime editing is a precise and rapid genome-editing technique that modifies short DNA sequences using tailored guide RNAs. To implement this technique in bacteria, we used Prime Editor 2 (PE2) with the DeepPrime gRNA design tool and assessed its gene-editing efficiency in Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) cells. Our findings indicate that a split PE2, comprising a reverse transcriptase and two Cas9 nickase domains, exhibited gene-editing efficiency comparable to that of the intact PE2. The efficiency observed in E. coli was significantly affected by the target sites, edit type, and the presence of exonucleases. In MRSA, which serves as a model to evaluate the applicability in non-model bacterial species, Streptococcus pyogenes PE2 (SpPE2) exhibited superior performance relative to Staphylococcus aureus PE2 (SaPE2). Furthermore, the split SpPE2 lacking the reverse transcriptase successfully induced the intended mutation in MRSA. This study demonstrates the feasibility of prime editing within bacterial systems.},
}

*Escherichia coli/genetics
*Gene Editing/methods
*Methicillin-Resistant Staphylococcus aureus/genetics
*Staphylococcus aureus/genetics
*Genome, Bacterial
RNA, Guide, CRISPR-Cas Systems/genetics
Streptococcus pyogenes/genetics/enzymology

@article {pmid42310097,
year = {2026},
author = {Fajardo, AF and Gowda, CP and Johnson, E and Petroni, R and Tomar, VS and Liu, Z and Andres Blanco, M and Janssen, J and Elcheva, IA and Lanza, M and Fuchs, SY and Spiegelman, VS},
title = {In vivo CRISPR knockout screen identifies Polr1a as a key driver and a potential therapeutic target for melanoma metastasis.},
journal = {Oncogene},
volume = {45},
number = {29},
pages = {2978-2987},
pmid = {42310097},
issn = {1476-5594},
support = {CA243167//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; CA288849//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; CA304343//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {*Melanoma/genetics/pathology ; Humans ; Animals ; Cell Movement/genetics ; Mice ; Cell Line, Tumor ; *DNA-Directed RNA Polymerases/genetics/metabolism ; Neoplasm Metastasis ; CRISPR-Cas Systems ; Gene Expression Regulation, Neoplastic ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Identification and characterization of novel mechanisms driving melanoma metastases and ways to target them are paramount for the development of effective treatment modalities. Here, we employed in vivo CRISPR knockout screening targeting the genes associated with poor prognosis to identify Polr1a as a potent driver of melanoma metastasis. High Polr1a levels correlate with increased metastasis and reduced survival in patients. Polr1a inhibition suppressed migration, invasion, and the ability of melanoma cells to colonize lungs. Ribo-seq analysis revealed that Polr1a is involved in regulating the non-canonical NF-κB pathway. Indeed, targeting Polr1a decreased levels of RelB and p52 and suppressed non-canonical NF-κB transcriptional activity; this suppression was responsible for the effects of Polr1a on melanoma cell migration. Accordingly, pharmacological inhibition of Polr1/Polr1a suppressed cell migration, tumor growth, and metastases. We discuss the potential utilization of Polr1 inhibitors for neoadjuvant treatment of melanoma.},
}

*Melanoma/genetics/pathology
Humans
Animals
Cell Movement/genetics
Mice
Cell Line, Tumor
*DNA-Directed RNA Polymerases/genetics/metabolism
Neoplasm Metastasis
CRISPR-Cas Systems
Gene Expression Regulation, Neoplastic
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42366688,
year = {2026},
author = {Wu, J and Yan, J and Li, C and Liu, X and Zhao, K and Wu, T},
title = {CRISPR/Cas12a-Enhanced Cascade Amplification for Ultra-sensitive DNA Ligase Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {27},
pages = {20113-20121},
doi = {10.1021/acs.analchem.6c00404},
pmid = {42366688},
issn = {1520-6882},
mesh = {*DNA Ligases/analysis/metabolism ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/metabolism ; Limit of Detection ; Humans ; *Endodeoxyribonucleases/metabolism ; *Bacterial Proteins/metabolism ; },
abstract = {DNA ligases are essential enzymes for maintaining genomic integrity, serving as critical biomarkers for the early diagnosis of various malignancies. However, current detection paradigms are often hindered by laborious workflows, high costs associated with chemical modifications, and insufficient sensitivity for low-abundance targets. In this study, we developed an integrated, label-free detection system where DNA ligase serves as a molecular gatekeeper to initiate CRISPR/Cas12a activity. This strategy exploits the discovery that nicked activators exhibit significantly attenuated affinity for the Cas12a-crRNA ribonucleoprotein complex, whereas ligase-mediated repair restores backbone continuity to create a high-affinity intact activator. Upon this ligation-gated activation, the system triggers a subsequent circular DNA-mediated autocatalytic cascade, exponentially amplifying the initial enzymatic signal. Through this dual-stage amplification, we achieved an ultimate limit of detection (LOD) of 2.59 × 10[-6] U/mL. Notably, the platform can reach the analytical sensitivity of established methods in as little as 30 min (LOD of 6.12 × 10[-5] U/mL), significantly compressing the diagnostic time frame. The system demonstrates high selectivity against diverse physiological interferents and has been successfully validated for quantifying endogenous DNA ligase in MC38 tumor cell extracts. This innovative ligase-gated CRISPR cascade provides a modular and robust framework for rapid clinical diagnostics and advanced enzymology research.},
}

*DNA Ligases/analysis/metabolism
*CRISPR-Cas Systems/genetics
*CRISPR-Associated Proteins/metabolism
Limit of Detection
Humans
*Endodeoxyribonucleases/metabolism
*Bacterial Proteins/metabolism

@article {pmid42381600,
year = {2026},
author = {Dong, P and Gao, Y and Zhao, W and Fan, L and Wang, Y},
title = {A real-time microfluidic surveillance system for multiplex detection of heavy metal contamination in wastewater.},
journal = {Lab on a chip},
volume = {26},
number = {14},
pages = {4229-4234},
doi = {10.1039/d6lc00331a},
pmid = {42381600},
issn = {1473-0189},
mesh = {*Wastewater/chemistry/analysis ; *Metals, Heavy/analysis ; *Water Pollutants, Chemical/analysis ; *Microfluidic Analytical Techniques/instrumentation ; *Lab-On-A-Chip Devices ; Limit of Detection ; CRISPR-Cas Systems ; },
abstract = {Water pollution, particularly from heavy metals, poses a critical threat to ecosystems and human health. This study integrates the CRISPR-Cas12a system with MOF-based bio-barcode technology to create a platform for the rapid, real-time and on-site detection of multiple heavy metal ions, demonstrating exceptional sensitivity and selectivity. The detection limits for Cu[2+], Pb[2+], and Hg[2+] are 0.26 nM, 0.06 nM, and 0.80 nM, respectively. Inductively coupled plasma-mass spectrometry analysis of real water samples confirmed the high accuracy and reliability of this method. Furthermore, a mobile phone-assisted portable device paired with a microfluidic chip facilitates real-time, rapid multi-channel metal ion detection in resource-limited settings.},
}

*Wastewater/chemistry/analysis
*Metals, Heavy/analysis
*Water Pollutants, Chemical/analysis
*Microfluidic Analytical Techniques/instrumentation
*Lab-On-A-Chip Devices
Limit of Detection
CRISPR-Cas Systems

@article {pmid42393911,
year = {2026},
author = {Zhu, L and Liao, L and Huang, Y and Nie, Z and Lei, C},
title = {Label-Free Electrochemical CRISPR Platform Gated by Allosteric Transcription Factors for Ultrasensitive Small-Molecule Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {27},
pages = {20617-20627},
doi = {10.1021/acs.analchem.6c02971},
pmid = {42393911},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; *Transcription Factors/metabolism/chemistry/genetics ; Allosteric Regulation ; *Tetracycline/analysis ; Animals ; Copper/analysis ; Milk/chemistry ; *CRISPR-Cas Systems ; G-Quadruplexes ; DNA Probes/chemistry ; Parabens/analysis ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Limit of Detection ; },
abstract = {The highly sensitive analysis of small-molecule targets holds profound significance across diverse fields, ranging from clinical diagnosis and environmental monitoring to food safety. Herein, we developed a label-free electrochemical CRISPR platform gated by allosteric transcription factors (aTFs) for the ultrasensitive detection of various small molecules. In this system, the specific binding of target analytes to their cognate aTFs induces the release of programmable DNA adaptors, which subsequently trigger Cas12a to trans-cleave DNA probes anchored to the electrode surface. Consequently, the truncated DNA probes serve as initiators to form electroactive G-quadruplex/hemin complexes in situ via terminal deoxynucleotidyl transferase (TdT)-mediated elongation, generating a robust electrochemical response signal. Using TetR as a model aTF, this integrated electrochemical CRISPR biosensor achieved tetracycline detection with picomolar sensitivity. Furthermore, the versatility of this platform was demonstrated by extending its application to p-hydroxybenzoic acid and copper ions through the simple substitution of the aTF modules. The practical utility of the assay was further demonstrated by the robust detection of tetracycline in complex matrices such as milk. Ultimately, this study not only provides a novel strategy for constructing universal, label-free electrochemical CRISPR platforms but also paves the way for the sensitive detection of low-abundance non-nucleic acid targets.},
}

*Biosensing Techniques/methods
*Electrochemical Techniques/methods
*Transcription Factors/metabolism/chemistry/genetics
Allosteric Regulation
*Tetracycline/analysis
Animals
Copper/analysis
Milk/chemistry
*CRISPR-Cas Systems
G-Quadruplexes
DNA Probes/chemistry
Parabens/analysis
*Clustered Regularly Interspaced Short Palindromic Repeats
Limit of Detection

@article {pmid42397942,
year = {2026},
author = {Tong, Z and Huang, Z and Liu, J and Su, J and Zhu, R and Xiong, R and Yang, Y and Xie, W and Xiao, R},
title = {Heterojunction-Enhanced Interfacial Evanescent-Tunable Fiber Optic Probe for Amplification-free CRISPR/Cas12a-Based Rapid and Ultrasensitive Detection of MPXV.},
journal = {Analytical chemistry},
volume = {98},
number = {27},
pages = {20429-20441},
doi = {10.1021/acs.analchem.6c02129},
pmid = {42397942},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; Gold/chemistry ; *Fiber Optic Technology ; Zinc Oxide/chemistry ; *Optical Fibers ; Metal Nanoparticles/chemistry ; Surface Plasmon Resonance ; Limit of Detection ; },
abstract = {Conventional polymerase chain reaction (PCR)-based detection methods suffer from time-consuming procedures, reliance on specialized equipment, and difficulty in achieving early viral diagnosis. In this study, interferometric fiber-optic sensing is integrated with the CRISPR/Cas12a system for the first time. With sensitivity further enhanced by immobilizing ZnO@Au on the fiber surface, the platform enables rapid, amplification-free detection of monkeypox virus (MPXV) at the single-molecule level. Whispering-gallery modes (WGMs) excited in the fiber probe provide high sensitivity to ambient refractive-index changes, while the ZnO@Au layer induces localized surface plasmon resonance (LSPR) and coupled plasmon-waveguide resonance (CPWR) on the fiber surface. By controlling the AuNPs occupancy on ZnO, the LSPR and CPWR absorption peaks can be tuned to match the demodulation spectral band. Moreover, the ZnO-Au heterojunction further strengthens the LSPR, thereby improving the sensitivity of the fiber probe. The resulting sensing probe achieves amplification-free detection of plasmid targets from both MPXV subtypes down to 10° copies/μL, with the entire assay completed within 9 min. The detection capability was validated using real clinical MPXV samples, showing complete agreement with qPCR results. The strategy proposed in this work offers a feasible approach for early and rapid viral detection.},
}

*CRISPR-Cas Systems/genetics
Gold/chemistry
*Fiber Optic Technology
Zinc Oxide/chemistry
*Optical Fibers
Metal Nanoparticles/chemistry
Surface Plasmon Resonance
Limit of Detection

@article {pmid42420723,
year = {2026},
author = {Kim, G and Kim, HJ and Seo, SW},
title = {Construction of a Tl-CRISPRi Genetic Circuit in Bacteria for Translation-Level Gene Knockdown.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3041},
number = {},
pages = {47-57},
pmid = {42420723},
issn = {1940-6029},
mesh = {*Gene Knockdown Techniques/methods ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Protein Biosynthesis ; *Bacteria/genetics ; Plasmids/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Regulatory Networks ; Genetic Engineering/methods ; },
abstract = {The Tl-CRISPRi system, which harnesses the specific RNA-binding activity of CRISPR-dCas13, has been recently developed for translation-level gene knockdown in bacteria. By introducing spacers complementary to the translation initiation region of the mRNA, dCas13 can be directed to block the ribosome and inhibit the translation of that mRNA. Here, we discuss how to construct the Tl-CRISPRi genetic circuit and implement this system for gene knockdown. This chapter describes how to design spacer sequences and install them into the guide RNA expression plasmid. Also, we describe how to mutate the handle of gRNA to achieve tunable knockdown of a target gene. By following the method described in this chapter, we anticipate that a precise and controllable knockdown of a target gene in bacterial cells can be performed in a programmable manner.},
}

*Gene Knockdown Techniques/methods
*CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
*Protein Biosynthesis
*Bacteria/genetics
Plasmids/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
*Gene Regulatory Networks
Genetic Engineering/methods

@article {pmid42420724,
year = {2026},
author = {Santos-Moreno, J},
title = {Design of CRISPRi-Based Synthetic Gene Circuits in Bacteria.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3041},
number = {},
pages = {59-83},
pmid = {42420724},
issn = {1940-6029},
mesh = {*Gene Regulatory Networks ; *Synthetic Biology/methods ; *CRISPR-Cas Systems ; *Bacteria/genetics ; *Genes, Synthetic ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Engineering/methods ; Gene Expression Regulation, Bacterial ; Escherichia coli/genetics ; },
abstract = {Synthetic gene circuits are key elements of engineered biological systems that allow us to control and program cellular behavior. Yet, circuit design can be challenging to newcomers due to the numerous design choices and the abundance and variety of parameters that can influence circuit performance. While transcription factors have dominated the circuit construction toolbox for two decades, CRISPRi-based tools offer important benefits-especially for large circuits-but also require unique design considerations. Here I provide a detailed guide for designing CRISPRi circuits in bacteria, using the CRISPRlator, the first CRISPRi oscillator, as an example that illustrates the design process.},
}

*Gene Regulatory Networks
*Synthetic Biology/methods
*CRISPR-Cas Systems
*Bacteria/genetics
*Genes, Synthetic
*Clustered Regularly Interspaced Short Palindromic Repeats
Genetic Engineering/methods
Gene Expression Regulation, Bacterial
Escherichia coli/genetics

@article {pmid42420725,
year = {2026},
author = {Park, D and Shin, W and Kang, H and Kim, J},
title = {Design of Conditional Guide RNAs for the Logical Regulation of Gene Expression.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3041},
number = {},
pages = {85-107},
pmid = {42420725},
issn = {1940-6029},
mesh = {Escherichia coli/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; Synthetic Biology/methods ; *CRISPR-Cas Systems ; *Gene Expression Regulation, Bacterial ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The CRISPR interference (CRISPRi) is an RNA-guided regulator that silences gene expression by binding to its cognate DNA target, halting transcription in both prokaryotic and eukaryotic cells. Recent advances in RNA synthetic biology have endowed CRISPR guide RNAs (gRNAs) with conditional functionality: these so-called conditional guide RNAs (cgRNAs) fold into strong hairpins that block their activity until a specific trigger RNA is present. Upon introduction of the cognate trigger RNAs, the hairpin structure unfolds, allowing the activated cgRNA to direct transcriptional repression with large dynamic ranges, minimal crosstalk, expanded tunability, and logic-gated signal processing. Furthermore, cgRNAs can be integrated into endogenous gene circuits to achieve sophisticated and logical regulation of gene expression. This chapter describes the design of cgRNAs and provides detailed protocols for their in vivo characterization in E. coli.},
}

Escherichia coli/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics/chemistry
Synthetic Biology/methods
*CRISPR-Cas Systems
*Gene Expression Regulation, Bacterial
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42420731,
year = {2026},
author = {Pujar, A and Sharma, A and Kushwaha, M},
title = {Intercellular CRISPRi for Distributed Genetic Circuits.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3041},
number = {},
pages = {229-243},
pmid = {42420731},
issn = {1940-6029},
mesh = {*Gene Regulatory Networks ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Bacteria/genetics ; Synthetic Biology/methods ; Escherichia coli/genetics ; },
abstract = {Microbial communities and multicellular organisms employ diverse strategies for allocation of available resources, achieved through task distribution among specialized cells. Drawing inspiration from nature, several synthetic multicellular circuits have been recently constructed where a larger circuit is distributed into several cells in order to reduce the burden on individual cells. Here, we describe the implementation of multicellular logic-gate circuits in bacterial co-cultures that combine DNA messaging with CRISPRi regulation. Leveraging the easily programmable and information-dense DNA molecules, our system is composed of sender bacteria that transmit DNA messages encoding guide RNAs and receiver bacteria that receive them and express the guide RNAs to regulate transcription by CRISPR interference. We demonstrate several functional multicellular circuits representing digital logic gates that operate on timescales comparable to small molecule signaling: NOT, YES, AND, and AND-AND-NOT. The receiver cells process the inputs received to perform computations and generate a logical output.},
}

*Gene Regulatory Networks
*CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
Bacteria/genetics
Synthetic Biology/methods
Escherichia coli/genetics

@article {pmid42340184,
year = {2026},
author = {Kawai-Harada, Y and You, S and Scarborough, T and Siraj, N and Yedla, J and Rennells, T and Walton, SP and Chan, C and Harada, M},
title = {Generation of Cellular Biofactories for the Scalable Production of Surface-Engineered Extracellular Vesicles via CRISPR Genome Editing.},
journal = {ACS biomaterials science & engineering},
volume = {12},
number = {7},
pages = {3821-3831},
doi = {10.1021/acsbiomaterials.6c00782},
pmid = {42340184},
issn = {2373-9878},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Extracellular Vesicles/metabolism/genetics/chemistry ; HEK293 Cells ; },
abstract = {Extracellular vesicles (EVs) are versatile biological nanoparticles with applications in therapeutics, diagnostics, and biotechnology. Current production methods relying on transient transfection or chemical conjugation suffer from high variability, limited scalability, and heterogeneous EV populations. Here, we present a synthetic-biology-based biomaterial manufacturing platform that uses CRISPR-Cas9 genome editing to generate stable HEK293T cell lines for continuous production of surface-functionalized EVs. A fusion construct encoding mCherry-C1C2 was site-specifically integrated into the AAVS1 safe-harbor locus, enabling consistent and heritable expression of EV membrane proteins without repeated transfection. Engineered cells produced EVs with uniform size (120-130 nm), preserved canonical markers (CD63 and ALIX), and enhanced surface-display efficiency compared with transiently transfected controls. These vesicles exhibited robust cellular uptake and maintained structural and functional stability for over 25 passages (∼3 months), confirming durable genome-encoded production. Overall, this platform eliminates batch-to-batch variability inherent to transient systems and provides a genetically defined route to biofunctional nanomaterial fabrication. This approach links genetic design to nanoscale surface functionality, establishing a versatile foundation for reproducible biomanufacturing of engineered EVs for biomaterial, therapeutic, and diagnostic applications.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Extracellular Vesicles/metabolism/genetics/chemistry
HEK293 Cells

@article {pmid42416053,
year = {2026},
author = {Xia, C and Lian, M and Ma, B and Yu, H and Zhang, R and Wen, L and Wang, X and Zhao, Y and Ouyang, Z and Ye, Y and Feng, X and Wu, H and Lai, L},
title = {Development of a BM7G(TKO/hCD46/hCD55/hTHBD/hEPCR) donor pig with endogenous promoter-driven transgenes for xenotransplantation.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1827497},
pmid = {42416053},
issn = {1664-3224},
mesh = {Animals ; *Transplantation, Heterologous/methods ; *Promoter Regions, Genetic ; Animals, Genetically Modified ; Humans ; Swine ; Galactosyltransferases/genetics ; *Transgenes ; *Thrombomodulin/genetics ; *Membrane Cofactor Protein/genetics ; Graft Rejection/immunology/genetics/prevention & control ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Heterografts ; Mixed Function Oxygenases/genetics ; Graft Survival ; N-Acetylgalactosaminyltransferases ; },
abstract = {INTRODUCTION: Xenotransplantation holds promise for addressing the organ shortage crisis. Multi-genetic modification of pigs, such as knockout of three carbohydrate antigen-related genes and expression of immunoprotective proteins, can significantly improve xenograft survival. However, existing multi-gene modification strategies face challenges: transposon-based transgenic technology may lead to unstable expression, while exogenous promoters used in site-specific integration strategies are susceptible to epigenetic silencing, making it difficult to maintain long-term, stable expression levels. Therefore, developing a donor pig model capable of achieving stable and long-lasting multi-gene expression is a critical need in the field.

METHODS: CRISPR-Cas9 technology was used to knockout three major glycan antigen genes (GGTA1, CMAH, β4GalNT2) to eliminate hyperacute rejection. Subsequently, four human protective genes (hCD55, hCD46, hTHBD, hEPCR) were site-specifically integrated into the porcine Rosa26 safe-harbor locus. Their expression was driven by the porcine endogenous Rosa26 promoter and the THBD core promoter, respectively, to ensure long-term stable and tissue-specific expression. Furthermore, the selection marker gene was efficiently removed using the Cre/loxP system.

RESULTS: The three glycan antigens were completely absent at both cellular and tissue levels in BM7G genetically modified pigs. What's more, four protective proteins were stably expressed in vascular endothelial cells and major organs such as the heart, liver, and kidneys. Among them, hCD55 and hCD46 were widely expressed, while hTHBD and hEPCR were specifically expressed in the vascular region. In-vitro functional assays confirmed that BM7G porcine vascular endothelial cells significantly reduced the binding of human antibodies, effectively inhibited complement-dependent cytotoxicity, and decreased the formation of thrombin-antithrombin (TAT) complexes.

CONCLUSION: In summary, by combining the knockout of xenoantigens with the use of endogenous promoters to drive the expression of multiple human protective genes, we successfully constructed a seven-gene modified pig model with low immunogenicity and synergistic protective functions. This provides an important donor resource for preclinical research in xenotransplantation.},
}

Animals
*Transplantation, Heterologous/methods
*Promoter Regions, Genetic
Animals, Genetically Modified
Humans
Swine
Galactosyltransferases/genetics
*Transgenes
*Thrombomodulin/genetics
*Membrane Cofactor Protein/genetics
Graft Rejection/immunology/genetics/prevention & control
CRISPR-Cas Systems
Gene Knockout Techniques
Heterografts
Mixed Function Oxygenases/genetics
Graft Survival
N-Acetylgalactosaminyltransferases

@article {pmid42416071,
year = {2026},
author = {Akula, S and Wernersson, S and Hellman, L},
title = {Immunity: defense against infections essential for all living organisms.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1840774},
pmid = {42416071},
issn = {1664-3224},
mesh = {Animals ; Humans ; *Adaptive Immunity ; Immunity, Innate ; Host-Pathogen Interactions/immunology ; },
abstract = {All organisms need protection against infection. Bacteria are often primarily seen as infectious agents, but they also need protection against bacterial viruses, so-called bacteriophages. To this end, bacteria have developed very complex defense systems, including apoptosis-like mechanisms, restriction enzymes, and even adaptive-type mechanisms involving immunological memory of immune responses through a system called CRISPR-Cas. An earlier dominating view was that adaptive immunity in eukaryotes only exists in jawed vertebrates, as their immune system includes the classical and highly variable immunoglobulins (Igs) and T-cell receptors (TCR). However, other types of variable molecules, which may be involved in immunity, have also been identified in insects, snails, lancelets, plants, sea urchins, and jawless fishes. Interestingly, fishes without jaws, such as the hagfish and lamprey, have a very complex adaptive immunity built on lymphocyte-like cells and variable lymphocyte receptors (VLRs). Notably, the variability of these VLRs has been estimated to be in the same range as Igs and T-cell receptors. This illustrates that very diverse strategies have been used to create an adaptive immune system in different organisms, indicating potent convergent evolution. Vertebrate immunity includes both adaptive and non-adaptive components, which work closely together to form a very powerful immune system for defense against infections. In contrast to adaptive immunity, the majority of the non-adaptive innate defense mechanisms, such as pattern recognition receptors, antimicrobial peptides (AMPs), iron-binding proteins, the complement system, and lysozymes, can be traced back to early eukaryotes. Immunity of invertebrates seems to rely almost entirely on innate defense mechanisms, while the presence of complex adaptive mechanisms in invertebrates, such as the VLRs of jawless fishes and Igs and TCR of jawed vertebrates, is questionable. This review summarizes old and recent findings of importance for our understanding of how immunity became an integrated part of all living organisms, from bacteria to humans, and the very different strategies that different organisms use in the protection against infection.},
}

Animals
Humans
*Adaptive Immunity
Immunity, Innate
Host-Pathogen Interactions/immunology

@article {pmid42416743,
year = {2026},
author = {Raj D, D and Maurya, AK and Singh, J and Kumar G, M and Ramani, A and Khurana, AK and Purwar, S and Biswas, D},
title = {Off-Target activity as a Translational Barrier in Programmable Gene-Editing Strategies for Nontuberculous Mycobacteria: Narrative Review.},
journal = {Maedica},
volume = {21},
number = {2},
pages = {495-503},
pmid = {42416743},
issn = {1841-9038},
abstract = {OBJECTIVES: To review the clinical and translational implications of off-target activity associated with clustered regularly interspaced short palindromic repeats (CRISPR)-based approaches in nontuberculous mycobacteria (NTM) and discuss current strategies aimed at specificity and safety.

MATERIALS AND METHODS: The relevant published literature on the application of CRISPR-Cas systems, including Cas9, Cas12a and CRISPR interference (CRISPRi), in NTM research was reviewed. Particular attention was given to off-target mechanisms, mycobacteria-specific genomic challenges, computational predictions, experimental detection methods, high-fidelity nucleases and delivery optimisation approaches.

RESULTS: Nontuberculous mycobacteria infections often require prolonged treatment and are frequently associated with relapse and rising antimicrobial resistance, particularly in Mycobacterium abscessus infections. CRISPR-based technologies provide advantages in precision diagnostics, functional genomics and therapeutic development; however, high guanine-cytosine (GC) content, repetitive PE/PPE gene families, mismatch tolerance and unique DNA repair mechanisms contribute considerably to off-target effects. Emerging high-fidelity nucleases, guide RNA optimisation, artificial intelligence (AI)-assisted prediction platforms and alternative editing systems demonstrate considerable potential for improving editing specificity and translational safety.

CONCLUSIONS: Advances in nuclease engineering, computational modelling, delivery systems, and genome-wide validation approaches may improve therapeutic precision and diagnostic reliability. Addressing these challenges through interdisciplinary innovation will be essential for the future clinical integration of CRISPR-based antimycobacterial strategies.},
}

@article {pmid42417038,
year = {2026},
author = {Abdullah, N and Lewis, J and Arumugam, P},
title = {Genome-wide CRISPR/Cas9 screening reveals lipid metabolism and inflammatory signalling as modulators of ganoderic acid DM cytotoxicity.},
journal = {Journal of genetics},
volume = {105},
number = {},
pages = {},
pmid = {42417038},
issn = {0973-7731},
mesh = {Humans ; *Triterpenes/pharmacology ; *Lipid Metabolism/drug effects/genetics ; Signal Transduction/drug effects ; *CRISPR-Cas Systems/genetics ; *Inflammation/genetics/metabolism ; Cell Line, Tumor ; Sterol Regulatory Element Binding Proteins/genetics/metabolism ; *Melanoma/genetics/drug therapy/pathology/metabolism ; NF-kappa B/genetics/metabolism ; Cholesterol/biosynthesis ; },
abstract = {Ganoderic acid DM (GA-DM), a triterpenoid derived from Ganoderma lucidum, exhibits anti-cancer and anti-diabetic activities, but the underlying mechanisms of action remain unclear. To identify genetic modulators of the GA-DM response, we conducted a genome-wide CRISPR/Cas9 knockout screen in human melanoma cells. The screen revealed key roles for genes regulating lipid metabolism and inflammatory signalling, particularly those involved in the SREBP (sterol regulatory element-binding protein) and NF-jB (nuclear factor kappa-light-chain-enhancer of activated B cells) pathways, in the cellular response to GA-DM. While loss of genes involved in the regulation of cholesterol biosynthesis conferred resistance to GA-DM, disruption of genes involved in ubiquitin-mediated proteolysis and the Hippo pathway sensitised cells to GA-DM. Inflammatory genes enriched at later time points suggests that a delayed cellular response contributes to cytotoxicity. Our findings propose a mechanistic model wherein GA-DM perturbs lipid and inflammatory pathways to exert cytotoxic effects and highlight potential targets to enhance its therapeutic efficacy. This work demonstrates the utility of functional genomics in elucidating the mechanisms of action of natural products and guiding rational drug development.},
}

Humans
*Triterpenes/pharmacology
*Lipid Metabolism/drug effects/genetics
Signal Transduction/drug effects
*CRISPR-Cas Systems/genetics
*Inflammation/genetics/metabolism
Cell Line, Tumor
Sterol Regulatory Element Binding Proteins/genetics/metabolism
*Melanoma/genetics/drug therapy/pathology/metabolism
NF-kappa B/genetics/metabolism
Cholesterol/biosynthesis

@article {pmid42417866,
year = {2026},
author = {Tang, Y and Zhang, L and Wang, W and Yang, X},
title = {An integrated signal amplification strategy based on catalytic hairpin assembly and hybridization chain reaction for driving a CRISPR/Cas12a biosensor toward ultrasensitive detection of microRNAs.},
journal = {Mikrochimica acta},
volume = {193},
number = {8},
pages = {},
pmid = {42417866},
issn = {1436-5073},
mesh = {*MicroRNAs/blood/genetics/analysis ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Nucleic Acid Hybridization ; Metal Nanoparticles/chemistry ; Gold/chemistry ; Humans ; Limit of Detection ; *Nucleic Acid Amplification Techniques/methods ; DNA Probes/chemistry/genetics ; },
abstract = {A novel biosensing platform is proposed that integrates catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) cascade isothermal amplification with the CRISPR/Cas12a system, enabling ultrasensitive detection of microRNAs (miRNAs) targets. Within this platform, two modules are integrated: a target recognition and signal amplification module constructed by the cascade of CHA and HCR, and a signal transduction module in which the CRISPR/Cas12a system acts in concert with DNA probes loaded onto gold nanoparticles (AuNPs). This design achieves cascaded amplification from target recognition to signal output, thereby conferring high signal gain. Experimental results demonstrate that the proposed biosensor had high sensitivity toward the target miRNA, with a detection limit as low as 37 fM. Moreover, it shows remarkable single-base discrimination capability, effectively distinguishing sequences with single-nucleotide mismatches. Notably, the sensor maintains stable and reliable performance in complex biological matrices, including serum samples and lysates from various tumor cells. This strategy effectively couples signal amplification with the CRISPR system, achieving both high sensitivity and specificity, making it a useful tool for miRNA detection and early cancer screening.},
}

*MicroRNAs/blood/genetics/analysis
*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
Nucleic Acid Hybridization
Metal Nanoparticles/chemistry
Gold/chemistry
Humans
Limit of Detection
*Nucleic Acid Amplification Techniques/methods
DNA Probes/chemistry/genetics

@article {pmid42420722,
year = {2026},
author = {Irvine, TCT and Bailey, AM and Gorochowski, TE},
title = {A Golden Gate-Compatible CRISPR-Associated Transposon Tool for Multiplexed Bacterial Genome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3041},
number = {},
pages = {33-45},
pmid = {42420722},
issn = {1940-6029},
mesh = {*Genome, Bacterial ; Escherichia coli/genetics ; *Gene Editing/methods ; *DNA Transposable Elements/genetics ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; Synthetic Biology/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The insertion of large genetic circuits and metabolic pathways into bacterial genomes is becoming increasingly common within the field of synthetic biology due to the improved robustness and stability that come with genome integration. CRISPR-associated transposases (CASTs) enable RNA-guided DNA insertion without introducing double-stranded breaks and have been shown to function across diverse bacterial species. Here, we present an improved tool called pSPIN-GG and supporting protocols for simplified CAST-based genome engineering. The pSPIN-GG system includes Golden Gate-compatible promoter, guide, and cargo modules for simple assembly, a green fluorescent protein dropout cassette for rapid verification of guide replacement, and a set of tested sites within the Escherichia coli BL21 chromosome to enable gene dosing of genetic cargoes. These refinements support accelerated library construction, reduce assembly and screening burden, and expand the accessibility of CAST systems for multiplexed bacterial genome engineering.},
}

*Genome, Bacterial
Escherichia coli/genetics
*Gene Editing/methods
*DNA Transposable Elements/genetics
*CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
Synthetic Biology/methods
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42415313,
year = {2026},
author = {Solanki, M and Yousuf, F and Srivastava, A and Vaikuntapu, PR and Molla, K and Neeraja, CN and Thakur, V and Barbadikar, KM and Sundaram, RM and Monhannath, G and Mangrauthia, SK},
title = {Powering Genome Editing in Rice by Harnessing Promising Gene Resources: A Comprehensive Roadmap.},
journal = {Physiologia plantarum},
volume = {178},
number = {4},
pages = {e71005},
doi = {10.1111/ppl.71005},
pmid = {42415313},
issn = {1399-3054},
support = {NASF/CRISPR-Cas-7003/2017-18//Indian Council of Agricultural Research/ ; NASF/BGAM-9021/2022-23//Indian Council of Agricultural Research/ ; },
mesh = {*Oryza/genetics ; *Gene Editing/methods ; Plant Breeding/methods ; *Genome, Plant/genetics ; Plants, Genetically Modified/genetics ; },
abstract = {The imprecise breeding methods including recombination breeding, physical/chemical mutagenesis, and marker-assisted breeding have been extensively utilized for trait improvement of rice crop. Despite tremendous progress made through these breeding methods, the critical issues, such as linkage drag, unintended phenotype, and longer duration of time required to breed a cultivar, have been the major limitations. Among the new breeding technologies, genome editing (GE) has become the most promising approach because of its specificity, precision, and speed. Despite its transformative potential, genome editing continues to face several limitations in crop improvement. These include well-recognized policy challenges, such as biosafety regulations and intellectual property constraints, alongside technical barriers like inefficient tissue culture and transformation systems. Additionally, researchers remain constrained by the limited availability of precise gene information necessary for accurate targeted editing and effective trait enhancement. This review presents an analysis of genes that regulate abiotic and biotic stresses, yield, grain quality and nutrition, plant architecture, nutrient absorption and use efficiency, and other agronomically important traits of rice. The trait-wise probable target genes for genome editing have been discussed in detail. This review will serve as a ready reckoner for rice researchers and funding agencies.},
}

*Oryza/genetics
*Gene Editing/methods
Plant Breeding/methods
*Genome, Plant/genetics
Plants, Genetically Modified/genetics

@article {pmid41115768,
year = {2026},
author = {Jordan, AJ and Balmforth, C and Craig, N and Dhaun, N and Baker, A and Dweck, MR and Newby, DE},
title = {Ribonucleic acid and gene therapies in cardiovascular disease: clinical applications, delivery challenges and emerging precision tools.},
journal = {Heart (British Cardiac Society)},
volume = {112},
number = {15},
pages = {828-837},
doi = {10.1136/heartjnl-2024-325280},
pmid = {41115768},
issn = {1468-201X},
mesh = {Humans ; *Cardiovascular Diseases/therapy/genetics ; *Genetic Therapy/methods ; *RNA, Small Interfering/therapeutic use ; *Precision Medicine/methods ; Gene Therapy Agents ; Gene Editing/methods ; *RNAi Therapeutics/methods ; Gene Transfer Techniques ; CRISPR-Cas Systems ; },
abstract = {Cardiovascular diseases remain a leading cause of global mortality despite advancements in pharmacotherapies, with current treatments facing challenges related to efficacy, tolerability and patient adherence. In response, advanced therapies, such as RNA and gene therapies, have emerged as a promising alternative for addressing both acquired and monogenic cardiovascular conditions. This review explores the current landscape of RNA and gene therapies for cardiovascular disease, focusing on RNA-based therapeutics such as small-interfering RNAs (siRNAs), antisense oligonucleotides and clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease (CRISPR-Cas9)-based gene editing systems. Recent European Medicines Agency and Food and Drug Administration-approved RNA therapies, including patisiran, vutrisiran and inclisiran, which employ lipid nanoparticle delivery systems, highlight the clinical potential of siRNAs for targeting hepatic molecular pathways. Emerging CRISPR-Cas9 technologies are poised to address genetic mutations at their source, offering permanent correction of pathogenic variants and the potential to treat a broad range of hereditary cardiovascular conditions. Together, these therapies represent a major leap forward in precision medicine, offering long-lasting therapeutic effects and improved patient care and adherence. However, many challenges remain, particularly in targeting such therapies to cardiac tissues and optimising delivery systems. This review discusses the current state of the art in cardiovascular RNA and gene therapies, including current evidence, delivery challenges and the current landscape of gene and RNA therapies in phase I clinical trials and beyond.},
}

Humans
*Cardiovascular Diseases/therapy/genetics
*Genetic Therapy/methods
*RNA, Small Interfering/therapeutic use
*Precision Medicine/methods
Gene Therapy Agents
Gene Editing/methods
*RNAi Therapeutics/methods
Gene Transfer Techniques
CRISPR-Cas Systems

@article {pmid42224782,
year = {2026},
author = {Dong, Z and Liu, Y and Wu, X and Ban, W and Zhao, L and Liu, X and Ding, J},
title = {Rapid multiplex detection of Echinococcus granulosus and Echinococcus multilocularis using a one-pot RPA-assisted CRISPR-Cas12a/Cas13a assay in a portable multi-tube device.},
journal = {Biosensors & bioelectronics},
volume = {311},
number = {},
pages = {118857},
doi = {10.1016/j.bios.2026.118857},
pmid = {42224782},
issn = {1873-4235},
mesh = {Animals ; *Echinococcus multilocularis/isolation & purification/genetics ; *Echinococcosis/parasitology/diagnosis ; *Biosensing Techniques/instrumentation ; CRISPR-Cas Systems ; *Echinococcus granulosus/isolation & purification/genetics ; Dogs ; Rapid Diagnostic Tests ; DNA, Helminth/genetics/isolation & purification ; Limit of Detection ; Humans ; Equipment Design ; },
abstract = {Echinococcosis, caused by Echinococcus granulosus and Echinococcus multilocularis, remains a significant zoonotic threat, particularly in pastoral regions where rapid environmental surveillance is essential yet technically constrained. Here, we report a rapid and integrated one-pot recombinase polymerase amplification -assisted, orthogonal CRISPR-Cas12a/Cas13a platform for rapid and specific discrimination of these two species in environmental samples. Coupled with a simplified NaOH-based DNA extraction method, the assay enables a streamlined workflow completed within 60 min, achieving a detection limit of as low as 1 copy/μL without observable cross-reactivity. To facilitate point-of-care deployment, we further developed a low-cost, miniaturized handheld device capable of parallel analysis of up to eight samples with dual-target readout. The platform was validated using field samples, including canine feces, pasture grass, and vegetables, demonstrating complete agreement with quantitative PCR results, with 100% sensitivity and specificity. This integrated CRISPR-based biosensing system provides a robust and field-deployable solution for on-site echinococcosis surveillance and offers a scalable framework for multiplex environmental pathogen detection.},
}

Animals
*Echinococcus multilocularis/isolation & purification/genetics
*Echinococcosis/parasitology/diagnosis
*Biosensing Techniques/instrumentation
CRISPR-Cas Systems
*Echinococcus granulosus/isolation & purification/genetics
Dogs
Rapid Diagnostic Tests
DNA, Helminth/genetics/isolation & purification
Limit of Detection
Humans
Equipment Design

@article {pmid42247945,
year = {2026},
author = {Fan, Z and Yin, X and Ma, M and Du, B and Liu, Z and Xu, J and Liu, B and Tong, Z},
title = {Cas12a2-based multiplexed screen-printed electrode electrochemiluminescence biosensor for amplification-free SARS-CoV-2 detection in aerosols.},
journal = {Biosensors & bioelectronics},
volume = {311},
number = {},
pages = {118885},
doi = {10.1016/j.bios.2026.118885},
pmid = {42247945},
issn = {1873-4235},
mesh = {*Biosensing Techniques/instrumentation/methods ; *SARS-CoV-2/isolation & purification/genetics ; Humans ; Aerosols/analysis ; *COVID-19/diagnosis/virology ; Electrochemical Techniques/instrumentation/methods ; RNA, Viral/analysis/genetics ; Luminescent Measurements/instrumentation ; CRISPR-Cas Systems ; *CRISPR-Associated Proteins/chemistry ; Electrodes ; Limit of Detection ; *Endodeoxyribonucleases/chemistry ; Equipment Design ; Rapid Diagnostic Tests ; Bacterial Proteins ; },
abstract = {Airborne transmission of respiratory viruses poses a severe public health threat, urgently requiring portable and sensitive techniques for viral aerosol monitoring. CRISPR-Cas12 technology has brought extensive innovations to the field of nucleic acid detection. Among them, Cas12a2 exhibits unique RNA-triggered trans-cleavage activity, showing prominent advantages in the amplification-free detection of respiratory RNA viruses. Herein, we developed an amplification-free and electrode-modification-free electrochemiluminescence biosensing platform based on screen-printed electrodes by integrating the specific recognition capability of Cas12a2 and the synergistic activation effect of multiple crRNAs. The optimized Cas12a2-based system achieves ultrasensitive detection of SARS-CoV-2 RNA with a low limit of detection of 76 aM. Furthermore, we constructed a stable viral aerosol generation and collection device and successfully validated the practical capability of the proposed platform for SARS-CoV-2 aerosol detection. This rapid and portable detection strategy offers a promising alternative for on-site monitoring of airborne pathogens and further expands the application scope of CRISPR biosensing technology in viral detection.},
}

*Biosensing Techniques/instrumentation/methods
*SARS-CoV-2/isolation & purification/genetics
Humans
Aerosols/analysis
*COVID-19/diagnosis/virology
Electrochemical Techniques/instrumentation/methods
RNA, Viral/analysis/genetics
Luminescent Measurements/instrumentation
CRISPR-Cas Systems
*CRISPR-Associated Proteins/chemistry
Electrodes
Limit of Detection
*Endodeoxyribonucleases/chemistry
Equipment Design
Rapid Diagnostic Tests
Bacterial Proteins

@article {pmid42250350,
year = {2026},
author = {Wang, Q and Choi, S and Heo, W and Kim, MW and Park, S and Park, SJ and Shin, J and Hyun, KA and Kim, J and Lim, CS and Jung, HI},
title = {Electrochemical-sensor-assisted lab-in-a-cartridge (EC-LIC) for on-site detection of SARS-CoV-2 with a self-contained heating system.},
journal = {Biosensors & bioelectronics},
volume = {311},
number = {},
pages = {118899},
doi = {10.1016/j.bios.2026.118899},
pmid = {42250350},
issn = {1873-4235},
mesh = {*SARS-CoV-2/isolation & purification/genetics ; Humans ; *COVID-19/diagnosis/virology ; *Biosensing Techniques/instrumentation ; *Electrochemical Techniques/instrumentation ; Rapid Diagnostic Tests ; Equipment Design ; Limit of Detection ; CRISPR-Cas Systems ; Sensitivity and Specificity ; Heating/instrumentation ; COVID-19 Nucleic Acid Testing/instrumentation ; },
abstract = {Rapid and accurate detection of respiratory viruses is essential for controlling disease transmission and enabling effective public health responses, particularly in resource-limited settings. In this study, we present an electrochemical-sensor-assisted lab-in-a-cartridge (EC-LIC) platform for on-site detection of SARS-CoV-2 featuring a self-contained chemical heating system. The device incorporates rotational and gravity-driven fluid handling along with exothermic heating using calcium oxide and a flameless ration heater to generate controlled temperature gradients. Coupled with a CRISPR-Cas13a-based electrochemical sensor, the system enables direct detection of the SARS-CoV-2 N gene without nucleic acid amplification, achieving high sensitivity and specificity. Integrated with a handheld electrochemical reader, the EC-LIC operates as a fully automated sample-to-answer system, completing the assay within 40 min over a wide dynamic range from 1.0 × 10° to 1.0 × 10[5] fg/mL with a limit of detection as low as 1.21 × 10[-1] fg/mL. Clinical validation using samples from 102 individuals (60 positive and 42 negative) demonstrated a sensitivity of 98% and a specificity of 90%. These results establish the EC-LIC as a robust nucleic acid detection platform for rapid clinical screening and early epidemic response.},
}

*SARS-CoV-2/isolation & purification/genetics
Humans
*COVID-19/diagnosis/virology
*Biosensing Techniques/instrumentation
*Electrochemical Techniques/instrumentation
Rapid Diagnostic Tests
Equipment Design
Limit of Detection
CRISPR-Cas Systems
Sensitivity and Specificity
Heating/instrumentation
COVID-19 Nucleic Acid Testing/instrumentation

@article {pmid42269453,
year = {2026},
author = {Ren, K and Yu, C and Wu, L and Ma, J and Xu, X and Jia, L and Yang, Q},
title = {Construction of an amplification-free dual-mode sensor based on CRISPR/Cas12a-mediated and dual-mode integrated reporter FU for ultrasensitive detection of non-nucleic acid target deoxynivalenol.},
journal = {Biosensors & bioelectronics},
volume = {311},
number = {},
pages = {118879},
doi = {10.1016/j.bios.2026.118879},
pmid = {42269453},
issn = {1873-4235},
mesh = {*Trichothecenes/analysis/isolation & purification/chemistry ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; Colorimetry/methods ; Gold/chemistry ; DNA, Single-Stranded/chemistry ; },
abstract = {Sensitive and accurate detection of deoxynivalenol (DON) is crucial for public health. The CRISPR/Cas12a system exhibits high specificity and efficiency in biosensing, but challenges remain in non-nucleic acid detection, such as reliance on multiple reporters for dual-mode signal output and low detection sensitivity without amplification. In this study, leveraging the magnetic and fluorescence quenching properties of Fe3O4/Au/PDA and the fluorescence/catalytic capabilities of UiO-66-NH2, we developed a multimodal integrated reporter (FU) as Fe3O4/Au/PDA-ssDNA-UiO-66-NH2, enabling dual-mode signal output via a single reporter. In the presence of DON, the DON-Ab-aDNA complex activates CRISPR/Cas12a, which then indiscriminately cleaves the single-stranded DNA in FU, releasing free UiO-66-NH2. Consequently, the fluorescence signal of UiO-66-NH2 is restored while it catalyzes TMB to produce a blue color reaction. The CRISPR/Cas12a-based fluorescence-colorimetric dual-mode biosensor (CrisprFU) achieved a colorimetric limit of detection (LOD) for DON of 2.15 × 10[-3] ng/mL (detection range: 2-100 ng/mL) and a fluorescence LOD of 7.96 × 10[-4] ng/mL (detection range: 0.5-40 ng/mL). Successful application in real samples demonstrated average recovery rates of 97.04%-104.4% for fluorescence detection and 96.4%-101.8% for colorimetric detection, confirming its practical potential. Furthermore, by replacing the recognition antibody, the CrisprFU system can be extended to detect other analytes.},
}

*Trichothecenes/analysis/isolation & purification/chemistry
*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
Limit of Detection
Colorimetry/methods
Gold/chemistry
DNA, Single-Stranded/chemistry

@article {pmid42269938,
year = {2026},
author = {Wei, E and Tang, Y and Lei, Y and Liu, S and Xu, B and Du, B and Wang, Y and Liao, Y and Wang, Y and Zhao, J},
title = {Development and application of a fast and efficient CRISPR/Cas12f -based genetic toolkit in Bacillus cereus GW-01.},
journal = {Journal of microbiological methods},
volume = {247},
number = {},
pages = {107584},
doi = {10.1016/j.mimet.2026.107584},
pmid = {42269938},
issn = {1872-8359},
mesh = {*Bacillus cereus/genetics/metabolism ; *CRISPR-Cas Systems ; Pyrethrins/metabolism ; *Gene Editing/methods ; Gene Knockout Techniques ; Bacterial Proteins/genetics ; Probiotics ; Gene Deletion ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Bacillus cereus GW-01, an efficient degrader of β-cypermethrin (β-CY), has a high safety profile and probiotic potential for regulating intestinal flora and fermented foods, which is difficult to genetically engineer for modification due to its restrictive modification system. This study successfully developed a CRISPR/enCas12f-based genome editing system, first selecting the plcR gene for proof-of-concept validation with 100% knockout efficiency. Subsequently, this system was utilized to delete the virulence gene nheABC in GW-01, yielding a safer probiotic strain. Compared with the wild-type strain GW-01, the probiotic-related indicators of the ΔnheABC mutant, including cell surface hydrophobicity, auto-aggregation ability and biofilm formation ability, were 80%, 90% and 2.9 (OD595), respectively. There were no significant differences in these indicators between the mutant and the wild type. Meanwhile, the ΔnheABC mutant still maintained a high β-cypermethrin degradation efficiency of 80% at the concentration of 30 μg/mL. This work facilitates functional genomic research and genetic modification of Bacillus cereus GW-01. The established CRISPR/enCas12f system enables targeted gene deletion to explore gene functions and phenotypic mechanisms, and paves the way for its development into safe probiotics and excellent microbial chassis.},
}

*Bacillus cereus/genetics/metabolism
*CRISPR-Cas Systems
Pyrethrins/metabolism
*Gene Editing/methods
Gene Knockout Techniques
Bacterial Proteins/genetics
Probiotics
Gene Deletion
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42308857,
year = {2026},
author = {Li, L and Guo, X and Yang, X and Qin, S and Wang, W},
title = {High-sensitivity and portable detection of oral pathogens based on CRISPR/Cas13a combined with exonuclease-assisted cycling amplification and lateral flow assay.},
journal = {Biosensors & bioelectronics},
volume = {311},
number = {},
pages = {118935},
doi = {10.1016/j.bios.2026.118935},
pmid = {42308857},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *RNA, Ribosomal, 16S/genetics/isolation & purification ; Rapid Diagnostic Tests ; *Bacteria/isolation & purification/genetics/pathogenicity ; Limit of Detection ; Exonucleases/chemistry ; *Mouth/microbiology ; },
abstract = {Infectious diseases caused by oral pathogens represent a significant threat to human health. Current diagnostic technologies for oral pathogens lack the characteristics of speed, sensitivity, and convenience, making it difficult to meet the needs of rapid testing in laboratories and on-site. Consequently, the development of novel high-sensitivity and high-specificity pathogen analysis methods and sensing systems is imperative. In this study, we established a high-throughput CRISPR/Cas13a method for identifying pathogenic bacteria 16S rRNA, which we combined with isothermal enzyme cycling amplification technology (CRIE) to improve sample detection resolution, sensitivity, and speed. Furthermore, based on the characteristics of dopamine catalyzed by G4/hemin to form polydopamine and combined with CRIE, we developed lateral flow assay (CRIEC) for simple, portable, and rapid detection of pathogenic bacteria. Preliminary experiments were performed to verify its analytical performance and application potential. The obtained data may lay a basic foundation for the subsequent research and clinical application in the field of oral pathogen detection.},
}

*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
Humans
*Nucleic Acid Amplification Techniques/methods
*RNA, Ribosomal, 16S/genetics/isolation & purification
Rapid Diagnostic Tests
*Bacteria/isolation & purification/genetics/pathogenicity
Limit of Detection
Exonucleases/chemistry
*Mouth/microbiology

@article {pmid42323937,
year = {2026},
author = {Zhang, Y and Li, L and Li, Y and Zeng, Y and Liu, H and He, M},
title = {Functionalized carbon nanotube-assisted dual-mode CRISPR/Cas12a detection of hepatitis C virus via catalytic assembly circuit-driven Y-shaped dsDNA activators.},
journal = {Biosensors & bioelectronics},
volume = {311},
number = {},
pages = {118946},
doi = {10.1016/j.bios.2026.118946},
pmid = {42323937},
issn = {1873-4235},
mesh = {*Nanotubes, Carbon/chemistry ; *Hepacivirus/isolation & purification/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Hepatitis C/virology/diagnosis ; *RNA, Viral/genetics/isolation & purification/analysis ; Colorimetry/methods ; Limit of Detection ; Hydrogen Peroxide/chemistry ; Catalysis ; DNA/chemistry ; Endodeoxyribonucleases/chemistry ; CRISPR-Associated Proteins/chemistry ; },
abstract = {Hepatitis C virus (HCV) is a major etiological agent of liver diseases and remains a serious global health threat. Herein, we report a dual-modal HCV biosensing platform for ultrasensitive HCV RNA detection by integrating near-infrared fluorescence and colorimetric readouts. In this system, the presence of HCV RNA initiates a catalytic assembly circuit (CAC) that forms a Y-shaped DNA structure, exposing two double-stranded DNA activators with complete protospacer adjacent motif (PAM) to trigger CRISPR/Cas12a nuclease activity. The combination of CRISPR/Cas12a-driven signal amplification and hemin-binding aptamer-functionalized single-walled carbon nanotubes (HeApt-SWCNTs) enables highly sensitive target quantification. Upon exposure to hydrogen peroxide (H2O2), ferric ion in hemin catalyzes a Fenton-like reaction, generating hydroxyl radicals (·OH) that quench SWCNT fluorescence and oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to its blue oxidized form (oxTMB). In the presence of target HCV RNA, CRISPR/Cas12a-mediated HeApt cleavage inhibits ·OH generation, resulting in SWCNT fluorescence recovery and suppresses TMB oxidation. Under optimal conditions, detection limits of 0.23 fM and 4.1 fM are achieved for the fluorescence and colorimetric modes, respectively. This integrated CAC-Cas12a-HeApt-SWCNTs (CCHS) biosensing strategy offers high specificity, dual-mode signal reliability, and broad potential for early diagnosis of HCV and other RNA viruses.},
}

*Nanotubes, Carbon/chemistry
*Hepacivirus/isolation & purification/genetics
*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
Humans
*Hepatitis C/virology/diagnosis
*RNA, Viral/genetics/isolation & purification/analysis
Colorimetry/methods
Limit of Detection
Hydrogen Peroxide/chemistry
Catalysis
DNA/chemistry
Endodeoxyribonucleases/chemistry
CRISPR-Associated Proteins/chemistry

@article {pmid42330664,
year = {2026},
author = {Zhao, J and Xu, H and Fei, S and Xu, C and Yin, W and Wei, Q and Lin, J and Liu, G and Feng, S and Gao, F and Wang, Y},
title = {Smartphone-integrated RPA-CRISPR/Cas12a detection system with microneedle sampling for early point-of-care diagnosis of potato late blight.},
journal = {Biosensors & bioelectronics},
volume = {311},
number = {},
pages = {118943},
doi = {10.1016/j.bios.2026.118943},
pmid = {42330664},
issn = {1873-4235},
mesh = {*Solanum tuberosum/microbiology/parasitology ; *Plant Diseases/microbiology/parasitology ; Smartphone ; *Phytophthora infestans/isolation & purification/genetics/pathogenicity ; *Biosensing Techniques/instrumentation ; Point-of-Care Systems ; CRISPR-Cas Systems ; Rapid Diagnostic Tests ; Nucleic Acid Amplification Techniques/instrumentation ; Plant Leaves/microbiology ; Equipment Design ; Needles ; },
abstract = {Potato late blight, caused by the oomycete pathogen Phytophthora infestans (P. infestans), is one of the most devastating diseases threatening global potato production. Conventional plant disease detection methods rely on a labor-intensive and time-consuming workflow and require bulky and expensive benchtop equipment, limiting their in-field applications. Here, we report a portable RPA-CRISPR/Cas12a-based diagnostic platform integrated with a polyvinyl alcohol (PVA) microneedle (MN) patch, which allows rapid in-field sampling, and smartphone-based fluorescence acquisition and analysis to detect P. infestans in potato at the early stage. The PVA MN enables leaf sampling rapidly within 1 min, and yields efficient DNA extraction of 56.3 ± 4.2 ng/mg, which is ∼3-fold higher than the traditional CTAB method (18.1 ± 2.1 ng/mg). The RPA-CRISPR/Cas12a isothermal assay achieved specific detection of P. infestans with no cross-reactivity against closely-related species Phytophthora sojae or Phytophthora capsici. The smartphone-based point-of-care test (POCT) system demonstrates a detection limit of 4 pg/μL for P. infestans genomic DNA, which is comparable to that acquired with commercial laboratory equipment. The method enables early-stage diagnosis of potato late blight as early as Day 2 post-inoculation, with detection rates of 37.5% on Day 2 and 75% on Day 3, prior to the development of visible symptoms on leaves. This portable "sample-to-result" platform provides a promising strategy for rapid, field-deployable early diagnosis and surveillance of plant disease.},
}

*Solanum tuberosum/microbiology/parasitology
*Plant Diseases/microbiology/parasitology
Smartphone
*Phytophthora infestans/isolation & purification/genetics/pathogenicity
*Biosensing Techniques/instrumentation
Point-of-Care Systems
CRISPR-Cas Systems
Rapid Diagnostic Tests
Nucleic Acid Amplification Techniques/instrumentation
Plant Leaves/microbiology
Equipment Design
Needles

@article {pmid42330669,
year = {2026},
author = {Zheng, X and Li, H and Yao, S and Wang, X and Wang, J and Yin, C and Wang, J and Zhao, C},
title = {Multiple DNA cycle amplification-assisted one-pot isothermal Cas12a for ultrasensitive nucleic acid detection.},
journal = {Biosensors & bioelectronics},
volume = {311},
number = {},
pages = {118953},
doi = {10.1016/j.bios.2026.118953},
pmid = {42330669},
issn = {1873-4235},
mesh = {*SARS-CoV-2/isolation & purification/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Biosensing Techniques/methods ; Humans ; Gold/chemistry ; *COVID-19/diagnosis/virology ; Metal Nanoparticles/chemistry ; Limit of Detection ; CRISPR-Cas Systems/genetics ; CRISPR-Associated Proteins ; Colorimetry ; *COVID-19 Nucleic Acid Testing/methods ; Endodeoxyribonucleases/chemistry ; DNA Probes/chemistry ; DNA ; Bacterial Proteins ; Coronavirus Nucleocapsid Proteins ; Phosphoproteins ; Spike Glycoprotein, Coronavirus ; },
abstract = {Rapid and ultrasensitive nucleic acid detection is essential for environmental monitoring and biomedical diagnostics. Here, we report a modular one-pot isothermal platform that integrates catalytic hairpin assembly (CHA), rolling circle amplification (RCA), and CRISPR-Cas12a to construct a self-reinforcing multilayer DNA circuit (CRC). In this system, Cas12a cis-cleavage generates short DNA fragments that recursively activate CHA and RCA, forming a self-sustained cascade amplification loop, while trans-cleavage enables real-time fluorescence signal readout. Using this one-pot platform, ultralow detection limits of 62 aM and 58 aM were achieved for the SARS-CoV-2 S and N genes, respectively, with a total assay time ranging from 20 to 120 min depending on the required sensitivity. Furthermore, functionalizing single-stranded DNA probes on gold nanoparticles (AuNPs) allowed the cleaved DNA to restore fluorescence of fluorophore-quencher reporters, and freeze-thaw-induced AuNP aggregation produced visible colorimetric changes and measurable photothermal signals, enabling trimodal readout without sophisticated instruments. The system demonstrated effective discrimination in controlled experiments, indicating its potential suitability for point-of-care applications. This integrated, one-pot, and scalable platform provides a versatile strategy for fast, sensitive, and multimodal nucleic acid detection applicable to diverse targets.},
}

*SARS-CoV-2/isolation & purification/genetics
*Nucleic Acid Amplification Techniques/methods
*Biosensing Techniques/methods
Humans
Gold/chemistry
*COVID-19/diagnosis/virology
Metal Nanoparticles/chemistry
Limit of Detection
CRISPR-Cas Systems/genetics
CRISPR-Associated Proteins
Colorimetry
*COVID-19 Nucleic Acid Testing/methods
Endodeoxyribonucleases/chemistry
DNA Probes/chemistry
DNA
Bacterial Proteins
Coronavirus Nucleocapsid Proteins
Phosphoproteins
Spike Glycoprotein, Coronavirus

@article {pmid42333535,
year = {2026},
author = {Labun, K and Rio, O and Dahal-Koirala, S and Komisarczuk, AZ and Valen, E and Haapaniemi, E},
title = {SNIPSNP: precision design of CRISPR/Cas9 knock-in reagents for variant correction and disease modeling.},
journal = {Nucleic acids research},
volume = {54},
number = {W1},
pages = {W145-W153},
doi = {10.1093/nar/gkag409},
pmid = {42333535},
issn = {1362-4962},
support = {331912//Norges Forskningsråd/ ; 190290//Kreftforeningen/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing/methods ; *Gene Knock-In Techniques/methods ; *Software ; Recombinational DNA Repair ; Computational Biology/methods ; Alleles ; RNA, Guide, CRISPR-Cas Systems/genetics ; INDEL Mutation ; },
abstract = {We present SNIPSNP (crisprtools.org/snipsnp), a comprehensive bioinformatics pipeline for designing experiments for CRISPR-induced homology-directed repair (HDR). The tool addresses the critical challenge of Cas9 re-cleavage by simplifying the selection of "blocking" silent variants that are effective at inhibiting RNP binding upon donor-templated editing. SNIPSNP handles complex edits, including indels, and uses multi-objective optimization to balance editing efficiency with biological safety. From user-defined wild-type and desired HDR alleles, the pipeline identifies candidate guides, annotating them with integrated efficiency scores and genome-wide off-target assessments. Uniquely, SNIPSNP evaluates guide binding against the post-edit genome to determine whether the therapeutic variant alone disrupts repeated Cas9 recognition. When necessary, it introduces synonymous blocking variants, prioritizing PAM and seed regions to minimize re-cleavage probability and editing of the wild-type (WT) allele when editing heterozygous variants. All candidate modifications undergo safety profiling and prioritization of known benign variants from dbSNP. We experimentally validated SNIPSNP and benchmarked it on pathogenic inborn error of immunity variants in primary patient T-cells. Across loci, SNIPSNP-designed templates outperform standard "correction-only" strategies, demonstrating enhanced precision editing, and reduced re-cleavage, establishing SNIPSNP as a robust platform for genome editing and disease modeling.},
}

*CRISPR-Cas Systems/genetics
Humans
*Gene Editing/methods
*Gene Knock-In Techniques/methods
*Software
Recombinational DNA Repair
Computational Biology/methods
Alleles
RNA, Guide, CRISPR-Cas Systems/genetics
INDEL Mutation

@article {pmid42342132,
year = {2026},
author = {Eskandani, NA and Mirzaee, D and Ramezani Farani, M and Hatami, A and Hatami, M and Ghasemzaei, M and Ghoreishian, SM and Hwang, SK and Huh, YS},
title = {Light-controlled CRISPR-dCas9 epigenome editing: advanced drug-delivery strategies and oncology applications.},
journal = {Advanced drug delivery reviews},
volume = {236},
number = {},
pages = {115921},
doi = {10.1016/j.addr.2026.115921},
pmid = {42342132},
issn = {1872-8294},
mesh = {Humans ; *Epigenome Editing/methods ; *Neoplasms/genetics/therapy/drug therapy ; *Drug Delivery Systems ; Animals ; *CRISPR-Cas Systems/genetics ; Light ; Optogenetics/methods ; Epigenesis, Genetic ; },
abstract = {Cancer is increasingly recognized as a disease of the dysregulated epigenome; however, current epi-drugs are blunt, systemically toxic instruments. Catalytically dead CRISPR nucleases (dCas9) linked to chromatin effectors have now made it possible not only to write and erase epigenetic marks at specified loci without double-strand breaks but also to add an element of optogenetics, or reversible and light-encoded control over the timing and localization of the editors. In this review, the technological underpinnings of light-controlled CRISPR-dCas9 epigenome editing, which include architectures of dCas9 scaffold and guide, blue-to-near-infrared photoswitches, and high-gain epigenetic effector designs, are synthesized, and viral, non-viral, and stimuli-responsive delivery platforms, which have to be co-optimized with clinical light interfaces, are discussed. We then outline four functional routes by which opto-epigenome editors may be used therapeutically in cancer: tumor suppressor reactivation; oncogene and super-enhancer repression with metabolic rewiring; control of cancer stem cell differentiation; and immunomodulation of the tumor microenvironment. Lastly, a translational roadmap is defined in terms of preclinical model tiers, biomarker strategies, regulatory and manufacturing factors, and future directions, including NIR and bioluminescent actuation, implantable μLED devices, and AI-guided closed-loop illumination. Together, these aspects constitute design principles for advancing light-addressable epigenome editors toward first-in-human studies and for integrating them into combination regimens as a new class of precision cancer therapeutics.},
}

Humans
*Epigenome Editing/methods
*Neoplasms/genetics/therapy/drug therapy
*Drug Delivery Systems
Animals
*CRISPR-Cas Systems/genetics
Light
Optogenetics/methods
Epigenesis, Genetic

@article {pmid42379043,
year = {2026},
author = {Zheng, Y and Tian, X and Wang, J and Zhang, Y and Liu, T and Huang, J and Guo, Y and Liang, S and Wang, C},
title = {CRISPR/Cas12a-based dual-modal signal platform using MIL-101(Fe) for colorimetric and electron spin resonance detection of HPV-16 nucleic acid.},
journal = {Biosensors & bioelectronics},
volume = {311},
number = {},
pages = {118976},
doi = {10.1016/j.bios.2026.118976},
pmid = {42379043},
issn = {1873-4235},
mesh = {*Human papillomavirus 16/isolation & purification/genetics ; Humans ; Colorimetry/methods ; *CRISPR-Cas Systems/genetics ; Electron Spin Resonance Spectroscopy/methods ; *Biosensing Techniques/methods ; *DNA, Viral/genetics/isolation & purification/analysis ; *Papillomavirus Infections/virology/diagnosis ; *Metal-Organic Frameworks/chemistry ; Iron/chemistry ; Limit of Detection ; },
abstract = {Human papillomavirus (HPV) infection is a leading cause of cervical cancer and other malignancies, underscoring the urgent need for accurate and rapid early diagnostic strategies. Herein, we report a dual-mode colorimetric and electron spin resonance (ESR) method for the qualitative detection of HPV-16, based on the integration of the CRISPR/Cas12a system with a metal-organic framework (MOF). A peroxidase-mimicking iron-based MOF, designated MIL-101(Fe), was conjugated to magnetic beads via a single-stranded DNA linker to serve as a signal probe. Upon recognition of the target nucleic acid, MIL-101(Fe) catalyzes the decomposition of hydrogen peroxide to generate hydroxyl radicals, which oxidize a chromogenic substrate to produce a visible color change. Meanwhile, the generated radicals are captured by a spin trap and detected by ESR spectroscopy. The assay enables sensitive and rapid detection of HPV-16, with clear discrimination even in mixtures containing both HPV-16 and HPV-18. Importantly, when evaluated with clinical specimens, the method achieved 100% sensitivity and specificity. Overall, this work provides a feasible and promising strategy for ultrasensitive nucleic acid detection and offers a new avenue for advancing CRISPR-based multimodal diagnostic platforms toward practical applications.},
}

*Human papillomavirus 16/isolation & purification/genetics
Humans
Colorimetry/methods
*CRISPR-Cas Systems/genetics
Electron Spin Resonance Spectroscopy/methods
*Biosensing Techniques/methods
*DNA, Viral/genetics/isolation & purification/analysis
*Papillomavirus Infections/virology/diagnosis
*Metal-Organic Frameworks/chemistry
Iron/chemistry
Limit of Detection

@article {pmid42402242,
year = {2026},
author = {Wang, F and He, C and Lin, Y and Zhou, X},
title = {One-Tube RPA-CRISPR-Cas13a assay with rational design for single-molecule detection of waterborne viruses in drinking water treatment.},
journal = {Biosensors & bioelectronics},
volume = {311},
number = {},
pages = {118983},
doi = {10.1016/j.bios.2026.118983},
pmid = {42402242},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; *CRISPR-Cas Systems/genetics ; *Drinking Water/virology ; *Norovirus/isolation & purification/genetics ; *Rotavirus/isolation & purification/genetics ; *Nucleic Acid Amplification Techniques/methods ; Water Purification ; Limit of Detection ; Water Microbiology ; Humans ; Rapid Diagnostic Tests ; },
abstract = {The global rise in waterborne viral infections has created an urgent need for portable, highly efficient environmental virus detection technologies. CRISPR-based nucleic acid detection coupled with isothermal amplification (e.g., Recombinase Polymerase Amplification, RPA) shows great promise for field applications. However, most reported designs fail to achieve the single-molecule sensitivity, which significantly limits their practical applications. To bridge the gap, we proposed a rational design strategy for the RPA primer and the CRISPR-Cas13a crRNA, suggesting that sensitivity can be enhanced by simplifying the secondary structure of the crRNA spacer region. Subsequently, we established a portable, one-tube CRISPR-Cas13a bioassay to detect two major waterborne viruses, achieving ultrasensitive detection limits of 5/8 aM for norovirus and 2/3 aM for rotavirus within 40 min. Thereafter, seasonal sampling across different treatment stages of a drinking water treatment plant was conducted, and water samples were analyzed using the one-tube CRISPR-Cas13a bioassay in comparison with qPCR and dPCR, revealing a positive detection rate of 15.79% (6/38) for the one-tube CRISPR-Cas13a bioassay, 18.42% (7/38) for qPCR, and 15.79% (6/38) for dPCR. The assay's modular design allows for broad applicability to other pathogens by simply modifying the target nucleic acid sequence, offering high sensitivity and specificity. This innovation paves the way for deployable point-of-care testing and large-scale spatiotemporal virus monitoring.},
}

*Biosensing Techniques
*CRISPR-Cas Systems/genetics
*Drinking Water/virology
*Norovirus/isolation & purification/genetics
*Rotavirus/isolation & purification/genetics
*Nucleic Acid Amplification Techniques/methods
Water Purification
Limit of Detection
Water Microbiology
Humans
Rapid Diagnostic Tests

@article {pmid42411270,
year = {2026},
author = {Baldenweck, L and Berg, N and Djisalov, M and Efremov, V and Novakovic, Z and Auger, S and Vidic, J},
title = {Isothermal amplification techniques for rapid bacterial detection: alternatives to culturing and PCR-based methods.},
journal = {Analytical methods : advancing methods and applications},
volume = {},
number = {},
pages = {},
doi = {10.1039/d6ay00756b},
pmid = {42411270},
issn = {1759-9679},
abstract = {Rapid identification of bacteria and their virulence factors is essential for global public health. Isothermal amplification has become a cornerstone of point-of-care diagnostics, enabling genetic testing to be faster, simpler, and more accessible than culturing or polymerase chain reaction (PCR). This review examines recent advances in some of the most commonly used isothermal amplification methods for bacterial detection: SDA, LAMP, HDA, RPA, RCA, and NASBA. The integration of isothermal amplification with the CRISPR/Cas system or microfluidic devices is also highlighted as an advanced gene detection technology. We present various readout methods used to detect gene amplification products or processes, including colorimetric, fluorescent, electrochemical, and quartz microbalance techniques. These integrated approaches can detect very small amounts of bacterial DNA, in under an hour, providing rapid, sensitive, versatile, and portable tools for health control.},
}

@article {pmid42411453,
year = {2026},
author = {Ono, Y and Peterka, M and Love, M and Khan, A and Bowers, F and Bhandari, A and Gordon, E and Ball, JS and Hammond, C and Tyler, CR and Rees, S and Bohlooly-Y, M and Maresca, M and Scholpp, S},
title = {Optimised genome editing for precise DNA insertion and substitution using prime editors in zebrafish.},
journal = {eLife},
volume = {14},
number = {},
pages = {},
pmid = {42411453},
issn = {2050-084X},
support = {BB/X008401/1//UK Research and Innovation/ ; BB/X001458/1//UK Research and Innovation/ ; DA 8438235/WT_/Wellcome Trust/United Kingdom ; NC/X001407/1//National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs)/ ; 29317//Versus Arthritis Senior fellowship/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *CRISPR-Cas Systems ; *Genome ; *Mutagenesis, Insertional/methods ; *DNA/genetics ; Deoxyribonuclease I/metabolism ; *Gene Editing/methods ; },
abstract = {CRISPR/Cas9-mediated genome editing has rapidly become a popular tool for studying gene functions and generating genetically modified organisms. However, using this system, stochastic integration of random insertions and deletions restricts precise genome manipulation. Advanced CRISPR/Cas9 technologies using Prime Editors (PEs), Cas9 proteins fused with reverse transcriptase, enable programmed integration of short DNA modifications into the genome. However, its application in precise genome editing in animal models is challenging. Here, we utilise a nickase- and a nuclease-based PE to perform programmed short DNA substitutions and insertions at various loci in the zebrafish genome. Whereas nickase-based PE2 mediated a higher ratio of precise prime edits to the total edits, nuclease-based PEn was more efficient for short DNA modifications, achieving up to 27.3% precise insertion. To further evaluate our approach, we inserted a nuclear localisation signal into a reporter transgene to incorporate longer fragments by prime editing. These gene modifications were transmitted to the next generation. We show that PE-mediated prime editing can efficiently manipulate genome information in zebrafish without using exogenous donor DNA.},
}

Animals
*Zebrafish/genetics
*CRISPR-Cas Systems
*Genome
*Mutagenesis, Insertional/methods
*DNA/genetics
Deoxyribonuclease I/metabolism
*Gene Editing/methods

@article {pmid42412324,
year = {2026},
author = {Adiga, U and Vasishta, S and Adiga, S and Augustine, AJ},
title = {Targeting the Gut-Heart Axis in Atherosclerosis: Microbial Metabolites, Molecular Mechanisms, and Precision Therapeutics.},
journal = {Probiotics and antimicrobial proteins},
volume = {},
number = {},
pages = {},
pmid = {42412324},
issn = {1867-1314},
abstract = {Despite advances in lipid-lowering and anti-inflammatory medications, atherosclerotic cardiovascular disease (ASCVD) continues to be the leading cause of morbidity and mortality worldwide. Recent studies have identified the gut microbiota as a key modulator of cardiovascular health via the gut-heart axis. This review investigates the molecular processes by which microbial metabolites affect atherogenesis. Proatherogenic substances like trimethylamine-N-oxide (TMAO), which are produced from dietary precursors through gut microbial and hepatic metabolism, aggravate foam cell production, platelet aggregation, and vascular inflammation. Short chain fatty acids (SCFAs), such as butyrate and propionate, have been shown to protect against atherosclerosis by activating G-protein-coupled receptors, regulating gene expression, and improving endothelial function. Additionally, secondary bile acids, tryptophan derivatives, and phenylacetylglutamine have emerged as important microbial metabolites involved in vascular disease. The review also summarizes various therapeutic strategies such as use of probiotics, prebiotics, postbiotics, precision microbiome editing (using bacteriophages and CRISPR-Cas systems), and fecal microbiota transplantation (FMT) for targeting gut-heart axis. Multi-omic systems combined with artificial intelligence can now detect disease-specific microbial signatures, improving risk stratification and paving the way for precision microbiome-based therapeutics. However, challenges such as determining causality, regulatory intricacies, and inter-individual variability in host-microbiome interactions remain. Despite these obstacles, the gut-heart axis provides a disruptive paradigm in preventive cardiology by emphasizing tailored microbiome therapies as a complement to traditional ASCVD care.},
}

@article {pmid42412819,
year = {2026},
author = {Tziony, I and Orenstein, Y},
title = {CROP: a feature-independent context-aware method for CRISPR-Cas9 frameshift prediction.},
journal = {Bioinformatics (Oxford, England)},
volume = {42},
number = {Supplement_1},
pages = {},
pmid = {42412819},
issn = {1367-4811},
support = {358/21//Israel Science Foundation/ ; },
mesh = {*CRISPR-Cas Systems ; *Frameshift Mutation ; *Software ; *Computational Biology/methods ; Prediction Algorithms ; },
abstract = {MOTIVATION: The CRISPR-Cas9 complex has revolutionized genome-editing technologies. By designing a 20 nt-long guide RNA, a Cas9 nuclease can be guided to cleave almost any genomic target site (followed by NGG). The cleavage induces double-stranded DNA breaks, which are then repaired by cellular pathways. Accurate CRISPR-Cas9 repair-outcome prediction is essential for designing guide RNAs with desired genomic effects, such as gene knockout. A central challenge is quantifying the rate of frameshifts, i.e. repair-outcomes that lead to a change in the local length that is not a multiple of three. Previous methods for frameshift-rate prediction were trained on only a few experimental or cellular contexts, mostly relied on manually defined microhomology features, and were limited by sparse features and class labels.

RESULTS: We developed CROP, a feature-independent context-aware repair-outcome prediction method. By aggregating specific repair outcomes as Δlength classes, CROP overcomes class sparsity. We designed CROP to work with variable input sequence lengths and output classes to utilize multiple datasets simultaneously. We benchmarked CROP against state-of-the-art repair-outcome prediction methods over 18 datasets, which we curated and standardized from various studies. Across all datasets, CROP outperformed all competing methods in frameshift-rate prediction. We performed cross-experiment and cross-cellular frameshift-rate predictions to investigate the generalizability of repair mechanisms. Finally, we show that CROP learned microhomology principles from raw sequences without explicit feature engineering, establishing an end-to-end architecture for CRISPR-Cas9 repair-outcome prediction that learns from multiple datasets.

CROP is available at https://github.com/OrensteinLab/CROP.},
}

*CRISPR-Cas Systems
*Frameshift Mutation
*Software
*Computational Biology/methods
Prediction Algorithms

@article {pmid42413622,
year = {2026},
author = {Yu, Q and Waheed, A and Hanioui, M and Ma, P and Wang, X and Liu, K and Li, X and Xue, Z and Zhang, G and Zhao, M},
title = {Engineering a tyrosine-auxotrophic Escherichia coli chassis for residue-specific in vivo DOPA incorporation into mussel foot protein mimics.},
journal = {Journal of biotechnology},
volume = {418},
number = {},
pages = {47-56},
doi = {10.1016/j.jbiotec.2026.07.003},
pmid = {42413622},
issn = {1873-4863},
abstract = {Mussel foot proteins (Mfps) achieve exceptional marine adhesion through post-translational conversion of tyrosine to 3,4-dihydroxyphenylalanine (DOPA). Recombinant production, however, is limited by poor solubility, low yields, and insufficient DOPA incorporation. We generated a genetically stable tyrA-deficient Escherichia coli chassis using CRISPR/Cas, thereby abolishing endogenous tyrosine biosynthesis and enabling residue-specific in vivo incorporation of exogenously supplied DOPA into mussel foot protein (MFP) mimics through selective pressure incorporation (SPI). A two-stage cultivation strategy decoupled biomass accumulation from DOPA-dependent protein synthesis, yielding 18.01 mg L[-1] of purified FP1. Residue-specific DOPA incorporation was verified by the characteristic + 16 Da shift in the [M-H][-] ion, accompanied by the loss of the tyrosine signal following exogenous DOPA supplementation in M9 medium. FP3 was largely soluble (65%) in crude extracts, while FP5 expression remained minimal. Fusion to thioredoxin (TrxA) and magnetoreceptor protein (MagR) further enhanced FP3 solubility to 90% and 85%, respectively. Proteins expressed in minimal M9 medium displayed exceptional shear stability, with viscosity fluctuations limited to ±2.2%, reflecting preserved catechol chemistry and structural integrity. This integrated strategy overcomes recurring trade-offs between DOPA incorporation, solubility, and yield, providing a basis for the potentially scalable production of functional, catechol-rich Mfps. Collectively, these findings support the development of next-generation mussel-inspired adhesives and catechol-based biomaterials.},
}

@article {pmid42413656,
year = {2026},
author = {Zhang, J and Shi, X and Ding, Y and Zheng, J and Li, H and Zhao, H and Zhang, T and Xing, Q and Zhao, C and Yao, S and Wang, J},
title = {Dynamic bidirectional diffusion-controlled multi-enzyme system for one-pot viral detection.},
journal = {Journal of advanced research},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jare.2026.07.019},
pmid = {42413656},
issn = {2090-1224},
abstract = {INTRODUCTION: Integrating isothermal amplification with CRISPR-based detection in a single reaction vessel holds significant promise for rapid and sensitive point-of-care virus diagnostics. However, conventional one-pot methods often suffer from mutual inhibition between amplification and CRISPR-Cas reactions, which compromises detection performance and limits their practical application.

OBJECTIVES: This study aims to overcome the inhibitory interactions between isothermal amplification and CRISPR-based detection by developing an integrated reaction system that enables efficient multi-enzyme coordination within a single tube.

METHODS: We designed a dynamic bidirectional diffusion-controlled RPA/CRISPR-Cas12a multi-enzyme system based on a dual-phase separation strategy. In this system, glycerol was used to modulate viscosity and accelerate the RPA reaction, while sucrose was introduced to create a density gradient that enables spatial separation. This configuration effectively coordinates the activities of multiple enzymes within one reaction vessel. Furthermore, a 3D-printed nucleic acid extraction device was integrated to simplify sample preparation and enhance overall detection efficiency.

RESULTS: The developed system achieved single-copy sensitivity and completed detection within 30 min, exhibiting over 100-fold higher sensitivity than conventional one-pot assays. The method was further validated by detecting Norovirus in both clinical and food samples, confirming its robustness and accuracy.

CONCLUSION: The proposed dual-phase RPA/CRISPR-Cas12a system provides a simple, rapid, and highly sensitive platform for nucleic acid detection. Its operational simplicity, compatibility with low-resource settings, and potential for home-based diagnostics highlight its strong applicability for decentralized virus detection.},
}

@article {pmid41930411,
year = {2026},
author = {Tyagi, DS and Banoo, H and Jha, DK and Meena, V and Joon, R and Agrwal, K and Yadav, P and Kumar, A and Satbhai, SB and Long, T and Pandey, AK},
title = {CRISPR/Cas9 Editing of the Wheat Iron Sensor TaHRZ1 Confirms Its Conserved Role in Iron Homeostasis and Allocation in Grains.},
journal = {Plant, cell & environment},
volume = {49},
number = {8},
pages = {4975-4991},
doi = {10.1111/pce.70516},
pmid = {41930411},
issn = {1365-3040},
support = {//NABI-CORE/ ; },
mesh = {*Triticum/genetics/metabolism ; *Homeostasis ; *Iron/metabolism ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; *CRISPR-Cas Systems/genetics ; Phylogeny ; Plants, Genetically Modified ; *Seeds/metabolism/genetics ; Arabidopsis/genetics ; Amino Acid Sequence ; Edible Grain/metabolism/genetics ; },
abstract = {Plants rely on specialized sensing systems, including transcriptional regulators, to maintain iron (Fe) homeostasis. Among these, Hemerythrin RING Zinc finger (HRZ) proteins have emerged as key regulators of Fe homeostasis. In this study, six Triticum aestivum L. (wheat) HRZ homoeologs referred to as TaHRZ1 and TaHRZ2, were identified by BLAST searches using rice (Oryza sativa) HRZ sequences and mapped to chromosomes 1 and 3. These encode for proteins with conserved N-terminal Hemerythrin (HHE) domains and C-terminal CHY-RING and Zn-ribbon motifs. Phylogenetic analysis grouped these genes into distinct clades, while expression profiling revealed strong root-specific and Fe-responsive expression patterns, indicating roles in nutrient sensing. Functional conservation was demonstrated by complementation of the Arabidopsis thaliana bts-1 mutant, where both wheat genes restored normal Fe regulation. Full-length TaHRZ1 and TaHRZ2 interacted with members of wheat bHLH IVc transcription factors, while truncated versions lacking the RING domain did not, emphasizing their conserved role in protein interactions. CRISPR-Cas9 editing of the conserved HHE3 domain of TaHRZ1, coupled with devlopmental regulators GRF4-GIF1 chimeric protein, achieved 6.4%-8.8% regeneration efficiency in wheat. Elemental analysis indicated enhanced Fe loading in the grains of the edited lines, particularly in the scutellum, suggesting improved Fe partitioning compared to the non-edited plants. Additionally, qRT-PCR revealed upregulation of TaFIT and TaIRO3, and downregulation of IDEF1 in edited lines, supporting an important regulatory role for TaHRZ1 in Fe homeostasis signalling. These findings position TaHRZ1 as a valuable target for biofortification strategies to enhance Fe content in wheat grains.},
}

*Triticum/genetics/metabolism
*Homeostasis
*Iron/metabolism
*Plant Proteins/genetics/metabolism
Gene Expression Regulation, Plant
*CRISPR-Cas Systems/genetics
Phylogeny
Plants, Genetically Modified
*Seeds/metabolism/genetics
Arabidopsis/genetics
Amino Acid Sequence
Edible Grain/metabolism/genetics

@article {pmid42010781,
year = {2026},
author = {Yue, K and Liang, X and Wang, Y and Jiang, N and Hu, Y and Su, J and Lu, C and Gao, H and Gai, X and Guo, W},
title = {A field-deployable RPA-CRISPR/Cas12a dual-mode assay for rapid detection of Fusarium oxysporum in Nicotiana tabacum.},
journal = {Pest management science},
volume = {82},
number = {8},
pages = {7610-7619},
doi = {10.1002/ps.70825},
pmid = {42010781},
issn = {1526-4998},
support = {2024530000241006//Yunnan Academy of Tobacco Agricultural Sciences/ ; 202405AD350100//Yunnan Applied Fundamental Research Projects/ ; 110202401006(JY-06)//China National Tobacco Corporation (CNTC)/ ; },
mesh = {*Fusarium/isolation & purification/genetics ; *Nicotiana/microbiology ; *CRISPR-Cas Systems ; *Plant Diseases/microbiology ; Rapid Diagnostic Tests ; *Nucleic Acid Amplification Techniques/methods ; },
abstract = {BACKGROUND: Fusarium oxysporum is a devastating soil-borne pathogen that causes severe economic losses in tobacco and other crops, necessitating rapid and accurate detection methods for effective disease management. Herein, we developed a dual-mode RPA-Cas12a platform incorporating both lateral flow dipstick (LFD) and fluorescence-based detection (FBD), targeting the CYP51C gene to enable rapid and equipment-facilitated detection of F. oxysporum under isothermal conditions.

RESULTS: The entire assay from sample to result can be completed within 68 min. The platform offers two complementary detection formats. The LFD mode provides visual qualitative results with a detection limit of 360 copies, making it ideally suited for rapid on-site screening. In contrast, the FBD mode achieves a detection limit of 3.6 copies, demonstrating 100-fold higher sensitivity and enabling precise quantitative analysis.

CONCLUSION: The assay successfully identified F. oxysporum in both artificially inoculated and field-collected tobacco samples, showing high concordance with fluorescence intensity. This work provides a sensitive, rapid, and practical diagnostic tool for the on-site detection and monitoring of tobacco root rot caused by F. oxysporum. © 2026 Society of Chemical Industry.},
}

*Fusarium/isolation & purification/genetics
*Nicotiana/microbiology
*CRISPR-Cas Systems
*Plant Diseases/microbiology
Rapid Diagnostic Tests
*Nucleic Acid Amplification Techniques/methods

@article {pmid42092617,
year = {2026},
author = {Richter, E and Klöhn, M and Nocke, MK and Friedrich, ME and Todt, D and Steinmann, E and Brüggemann, Y},
title = {Development of a CRISPR-Cas13-based antiviral strategy against hepatitis E virus.},
journal = {JHEP reports : innovation in hepatology},
volume = {8},
number = {7},
pages = {101885},
pmid = {42092617},
issn = {2589-5559},
mesh = {*Hepatitis E virus/genetics/drug effects ; Humans ; *CRISPR-Cas Systems ; Virus Replication/drug effects/genetics ; *Antiviral Agents/pharmacology ; *Hepatitis E/virology/therapy ; Genome, Viral ; },
abstract = {BACKGROUND & AIMS: Effective antiviral drugs remain unavailable for many clinically relevant pathogens, including the hepatitis E virus (HEV). This study aimed to evaluate the CRISPR/Cas13d system as a potential antiviral strategy against HEV.

METHODS: We developed a reporter assay to screen CRISPR RNAs (crRNAs) targeting conserved regions of the HEV genome and tested their antiviral activity in human hepatoma cells using a robust HEV cell culture model. HEV replication was assessed using a subgenomic replicon, infectious particle production was quantified by immunofluorescence and titration assays. A bioinformatic analysis was performed to identify a minimal set of crRNAs capable of broadly targeting circulating human pathogenic HEV strains.

RESULTS: A crRNA screen identified multiple functional crRNAs targeting HEV-3, with ORF1-targeting crRNAs significantly reducing viral capsid expression (p <0.01) and the number of HEV-infected cells (p <0.01). Cas13d-mediated targeting led to robust reduction of HEV replication and markedly lowered infectious virus production in vitro (p <0.001). Bioinformatic analysis revealed that just three distinct crRNAs could cover ∼94% of known HEV genomes with zero mismatches, while four crRNAs achieved complete coverage.

CONCLUSIONS: Our findings demonstrate that CRISPR/Cas13d can target HEV replication and viral progeny production in vitro. The identification of a minimal crRNA set capable of broadly targeting circulating HEV strains suggests that the CRISPR/Cas13d system may offer an antiviral strategy to address challenges related to viral evolution and treatment escape.

IMPACT AND IMPLICATIONS: This study establishes CRISPR/Cas13d as a proof-of-concept antiviral strategy against hepatitis E virus (HEV), demonstrating suppression of viral replication and particle production in vitro. By identifying a minimal set of broadly effective crRNAs, we provide a framework for targeting diverse HEV variants and buffering against viral evolution. These findings highlight the potential of CRISPR-based systems as innovative antiviral strategies.},
}

*Hepatitis E virus/genetics/drug effects
Humans
*CRISPR-Cas Systems
Virus Replication/drug effects/genetics
*Antiviral Agents/pharmacology
*Hepatitis E/virology/therapy
Genome, Viral

@article {pmid42115764,
year = {2026},
author = {Lv, G and Li, Y and Chen, J and Wu, Z and Wu, W and Wu, X and Lin, H and Wang, T},
title = {CRISPR/Cas9-mediated knockout of ZmHMA3 reveals its essential role in zinc homeostasis and high-zinc stress tolerance in maize.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42115764},
issn = {2045-2322},
support = {2023YFD1201105//National Key Research and Development Program of China/ ; },
mesh = {*Zea mays/genetics/metabolism/physiology ; *Zinc/metabolism/toxicity ; *Homeostasis ; *CRISPR-Cas Systems ; *Plant Proteins/genetics/metabolism ; *Stress, Physiological/genetics ; Gene Expression Regulation, Plant ; Plant Leaves/metabolism/genetics ; Gene Knockout Techniques ; Plant Roots/metabolism/genetics ; *Adenosine Triphosphatases/genetics/metabolism ; Oxidative Stress ; Plants, Genetically Modified ; },
abstract = {Excessive Zn is toxic to maize (Zea mays L.). The heavy metal ATPase gene ZmHMA3 is associated with heavy metal transport, but its function in maize tolerance to high Zn stress has not been fully characterized. In this study, CRISPR/Cas9 technology was used to generate zmhma3 knockout mutants to investigate its function under high Zn stress. High Zn stress significantly induced the expression of ZmHMA3 in maize leaves and roots. Phenotypic analysis showed that, compared to the WT plants, the zmhma3 mutants exhibited significantly reduced tolerance to excessive Zn, manifested as severe growth inhibition, impaired root structure, decreased activity of key antioxidant enzymes (CAT, POD, SOD), and aggravated membrane damage. Furthermore, the mutants accumulated significantly higher levels of Zn in both roots and leaves, accompanied by disordered subcellular Zn distribution, indicating disrupted intracellular Zn homeostasis. Our results demonstrate that ZmHMA3 is a key positive regulator in maize's response to high Zn stress, likely by coordinating Zn compartmentalization and alleviating oxidative damage. This study provides new genetic and physiological insights into the molecular mechanisms of Zn stress tolerance in maize and offers a potential target for breeding new maize varieties tolerant with improved high-Zn-efficiency.},
}

*Zea mays/genetics/metabolism/physiology
*Zinc/metabolism/toxicity
*Homeostasis
*CRISPR-Cas Systems
*Plant Proteins/genetics/metabolism
*Stress, Physiological/genetics
Gene Expression Regulation, Plant
Plant Leaves/metabolism/genetics
Gene Knockout Techniques
Plant Roots/metabolism/genetics
*Adenosine Triphosphatases/genetics/metabolism
Oxidative Stress
Plants, Genetically Modified

@article {pmid42302780,
year = {2026},
author = {Ueki, H and Tomita, Y and Duong, C and Mitake, H and Kiso, M and Furusawa, Y and Zhao, D and da Silva Lopes, TJ and Wu, L and Feng, H and Yamayoshi, S and Fukuyama, S and Yamashita, M and Ozawa, M and Ikawa, M and Yoshida, N and Watanabe, T and Kawaoka, Y},
title = {A CRISPR knockout mouse library for functional genomics in influenza research.},
journal = {Cell},
volume = {189},
number = {14},
pages = {4471-4488.e7},
doi = {10.1016/j.cell.2026.05.032},
pmid = {42302780},
issn = {1097-4172},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Mice ; *Influenza A virus/genetics ; *Orthomyxoviridae Infections/genetics/virology ; *Genomics/methods ; Mice, Knockout ; Cytoskeletal Proteins/genetics/metabolism ; Adaptor Proteins, Signal Transducing/genetics/metabolism ; Guanine Nucleotide Exchange Factors/genetics/metabolism ; Humans ; RNA, Small Interfering/metabolism/genetics ; Host-Pathogen Interactions/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Library ; Mice, Inbred C57BL ; },
abstract = {Functional validation of host factors in whole-animal models is a major bottleneck in virology; it hinders the translation of data from in vitro studies into a deeper understanding of the viral life cycle and pathogenesis. To address this challenge, we developed a systematic in vivo screening platform for influenza A virus. This platform comprises a library of 84 CRISPR-Cas9-generated gene-modified mouse lines targeting host factors prioritized from the literature and in vitro small interfering RNA (siRNA) screening studies. Using this resource, we identified 17 host factors whose genetic ablation conferred resistance to influenza A virus infection. Further studies of two of these factors, Arhgef28 and Lasp1, revealed distinct protective mechanisms against influenza A virus. We offer this mouse library to the research community as a powerful platform for studying virus-host interactions in a physiologically relevant context.},
}

Animals
CRISPR-Cas Systems/genetics
Mice
*Influenza A virus/genetics
*Orthomyxoviridae Infections/genetics/virology
*Genomics/methods
Mice, Knockout
Cytoskeletal Proteins/genetics/metabolism
Adaptor Proteins, Signal Transducing/genetics/metabolism
Guanine Nucleotide Exchange Factors/genetics/metabolism
Humans
RNA, Small Interfering/metabolism/genetics
Host-Pathogen Interactions/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
Gene Library
Mice, Inbred C57BL

@article {pmid42409605,
year = {2026},
author = {Huang, YW and Hu, CC and Cho, YH and Tsai, CH and Lin, NS and Hsu, YH and Dinesh-Kumar, SP},
title = {Efficient CRISPR-Cas9 delivery and transgene-free multiplex genome editing in plants using cymbidium mosaic virus-derived vectors.},
journal = {The Plant journal : for cell and molecular biology},
volume = {127},
number = {1},
pages = {e71031},
pmid = {42409605},
issn = {1365-313X},
support = {NSTC 114-2313-B-005-052-MY2//National Science and Technology Council/ ; //Ministry of Education in Taiwan/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Nicotiana/genetics ; Genetic Vectors/genetics ; Plants, Genetically Modified/genetics ; Oxidoreductases/genetics ; Genome, Plant/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Transgenes/genetics ; Potexvirus ; },
abstract = {Virus-induced genome editing (VIGE) has become a useful method by enabling transient delivery of gene-editing reagents; however, many viral systems face limitations in cargo size, host range, or reliance on transgenic Cas9-expressing plants. In this study, we developed a cymbidium mosaic virus (CymMV)-based VIGE platform that enables simultaneous expression of Streptococcus pyogenes Cas9 (SpCas9) and one or more guide RNAs (gRNAs) from a single viral RNA. In Nicotiana benthamiana, this system induced editing in the Phytoene desaturase (PDS) gene, with indel rates exceeding 50% within 6 days after inoculation, outperforming traditional delivery methods by about fivefold. Notably, over 80% of regenerated plants contained targeted mutations, and 82% of these were both transgene- and virus-free, including tetra-allelic knockouts directly in the M0 generation. Adding a Ruby-based visual counterselection marker enabled rapid, reliable identification of transgene-free, edited plants without antibiotic selection. When adapted to Phalaenopsis aphrodite orchids, the platform efficiently edited the PaPDS gene, achieving a 47% indel frequency at 20 days post-inoculation, with visible bleaching in leaf tissue from inoculated protocorm-like bodies. Additionally, expressing multiple gRNAs from a single CymMV replicon enabled multiplex editing in orchid tissues, demonstrating the system's versatility for complex, polyploid crops. Our findings broaden the use of VIGE in orchids and provide a reliable framework for precision plant breeding.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Nicotiana/genetics
Genetic Vectors/genetics
Plants, Genetically Modified/genetics
Oxidoreductases/genetics
Genome, Plant/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Transgenes/genetics
Potexvirus

@article {pmid42409731,
year = {2026},
author = {Shangguan, YT and Xie, LL and Liu, WB and Gu, RX and Xu, YX and Wang, M and Wang, JX},
title = {[Novel CD6-targeted CAR-T cell therapy for T-cell acute lymphoblastic leukemia: a safe and efficient strategy to prevent fratricide through gene editing].},
journal = {Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi},
volume = {47},
number = {5},
pages = {433-441},
doi = {10.3760/cma.j.cn121090-20260130-00068},
pmid = {42409731},
issn = {0253-2727},
support = {82341213, 82570279, 82570278//National Natural Science Foundation of China/ ; 23JCYBJC01060//Tianjin Natural Science Foundation Project/ ; },
mesh = {Humans ; *Immunotherapy, Adoptive ; *Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; *Gene Editing ; Receptors, Chimeric Antigen ; *Antigens, CD ; *Antigens, Differentiation, T-Lymphocyte ; CRISPR-Cas Systems ; T-Lymphocytes ; Receptors, Antigen, T-Cell ; },
abstract = {Objective: To explore a novel strategy that addresses the dual challenges of fratricide and on-target off-tumor toxicity in current chimeric antigen receptor T-cell (CAR-T) therapy for T-cell acute lymphoblastic leukemia (T-ALL) and to develop a safe and efficacious anti-T-ALL CAR-T product by identifying a new target and compatible gene-editing approach. Methods: Public single-cell RNA sequencing (scRNA-seq) datasets were utilized to analyze bone marrow cells extracted from patients with T-ALL and healthy donors, evaluating the differential expression profiles of CD6 and CD7. In investigating the endogenous role of CD6 in CAR-T cells, the CRISPR/Cas9 RNP system was first employed in a CD19 CAR-T model to evaluate the impact of CD6 knockout on the phenotype and activation status of CAR-T cells. Subsequently, CD6-knockout, CD6-targeted CAR-T cells (6KO-6CAR) were constructed, and their functional activities were evaluated. Results: scRNA-seq analysis revealed that CD6 is broadly expressed in T-ALL. Compared with the traditional target CD7, which is also expressed in a subset of normal hematopoietic stem/progenitor cells and myeloid cells, CD6 exhibits a more restricted expression profile, exhibiting superior safety characteristics. Studies on the CD19 CAR-T model indicated that CD6 knockout enables CAR-T cells to maintain a superior functional state: their baseline activation level (CD25 expression) was reduced (P<0.05) while generating a higher proportion of TNF-α(+)IFN-γ(+) cells (P<0.05) upon antigen stimulation. The further constructed 6KO-6CAR cells exhibited potent specific activation (significantly upregulated CD107a expression level, all P<0.001) and cytotoxicity (all P<0.05) against multiple CD6(+) T-ALL cell lines (MOLT-4, CCRF-CEM, and Jurkat) in vitro. Conclusion: CD6 is a novel therapeutic target for T-ALL with high coverage and a favorable safety profile, and knocking out endogenous CD6 globally optimizes the intrinsic functional state of CAR-T cells. Constructing 6KO-6CAR based on the CRISPR/Cas9 technology addresses fratricide in CAR-T cells while enhancing their antitumor functionality, thereby providing a novel immunotherapy regimen with safety and clinical translational potential for relapsed/refractory T-ALL.},
}

Humans
*Immunotherapy, Adoptive
*Precursor T-Cell Lymphoblastic Leukemia-Lymphoma
*Gene Editing
Receptors, Chimeric Antigen
*Antigens, CD
*Antigens, Differentiation, T-Lymphocyte
CRISPR-Cas Systems
T-Lymphocytes
Receptors, Antigen, T-Cell

@article {pmid42409995,
year = {2026},
author = {Goudarzi, F and Salehipour, P and Modarressi, MH and Hosseini, M},
title = {A "turn-on" CRISPR-mediated method using enhanced fluorescent bimetallic DNA nanoclusters for EGFR mutation detection in non-small cell lung cancer.},
journal = {Scientific reports},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41598-026-61115-3},
pmid = {42409995},
issn = {2045-2322},
support = {981234//University of Tehran/ ; },
abstract = {An affordable, precise detection of mutations is critical for guiding targeted cancer therapies and improving patient outcomes. Epidermal growth factor receptor (EGFR), a protein on the surface of cells that regulates growth and division, is frequently mutated in non-small cell lung cancer (NSCLC). Early identification of these mutations enables clinicians to select the most effective tyrosine kinase inhibitors, thereby enhancing treatment response and survival rates. Recent studies have focused on developing CRISPR-based detection strategies incorporating nanomaterials to achieve more accurate results. In this study, we present a CRISPR-based "turn-on" detection platform that leverages the cleavage of a novel enhanced bimetallic DNA nanocluster to measure EGFR exon 19 deletion in non-small cell lung cancer (NSCLC). The system is innovatively designed using guide RNAs (gRNAs) rationally derived from the normal EGFR gene, enabling the determination of exon 19 deletion through CRISPR-Cas activation in samples containing the normal and mutant. Upon recognition of the normal EGFR gene, the Cas12a enzyme induces cleavage of the Spermiform-designed Ag/Au DNA nanocluster and fluorescence quenching. At the same time, fluorescence signal retention depends on mutation frequency, with higher mutation frequencies resulting in greater or "turn-on" fluorescence signals. This approach achieves a detection limit (LOD) of approximately 0.35 nM, which is capable of detecting about 1.5% mutation, offering a cost-effective, label-free diagnostic tool and a promising strategy for future detection of deletion-related subtypes in PCR products by targeting normal sequences. The integration of bimetallic nanocluster-based reporters with CRISPR precision provides an emerging platform for next-generation molecular diagnostics targeting EGFR and other clinically relevant mutations.},
}

@article {pmid42410413,
year = {2026},
author = {Fan, Q and Stevanie, S and Frielingsdorf, S and Neubauer, P and Lenz, O and Gimpel, M},
title = {Genomically integrated orthogonal translation system in Escherichia coli enables production of functional modified [NiFe]-hydrogenases.},
journal = {Microbial cell factories},
volume = {25},
number = {1},
pages = {},
pmid = {42410413},
issn = {1475-2859},
mesh = {*Hydrogenase/genetics/metabolism/biosynthesis ; *Escherichia coli/genetics/metabolism ; Cupriavidus necator/genetics/enzymology ; CRISPR-Cas Systems ; *Protein Biosynthesis ; Plasmids/genetics ; Gene Editing ; },
abstract = {The functional diversification of O2-tolerant [NiFe]-hydrogenases using orthogonal translation systems (OTSs) offers a promising strategy for developing advanced biocatalysts and biohybrid energy platforms. However, plasmid-based OTSs frequently impose metabolic burdens and suffer from plasmid instability during fermentation, particularly when co-produced with complex metalloenzymes. To overcome these bioprocess limitations, we employed CRISPR/Cas9-mediated genome editing to integrate a psychrophilic pyrrolysyl-tRNA synthetase/tRNA pair into the Escherichia coli BL21 genome. The resulting strain provided a plasmid-free orthogonal translation background that supported amber suppression-mediated expression of the regulatory [NiFe]-hydrogenase (RH) of Cupriavidus necator. Using this genomically integrated OTS, we achieved the production of a full-length, catalytically active RH variant. Our results demonstrate that chromosomal OTS is compatible with the efficient production and maturation of complex metalloenzymes. The present work lays the groundwork for the bio-orthogonal engineering of hydrogenases and related hybrid biocatalysts.},
}

*Hydrogenase/genetics/metabolism/biosynthesis
*Escherichia coli/genetics/metabolism
Cupriavidus necator/genetics/enzymology
CRISPR-Cas Systems
*Protein Biosynthesis
Plasmids/genetics
Gene Editing

@article {pmid41419637,
year = {2026},
author = {Meng, X and Reis, N and Bassik, MC and Pașca, SP},
title = {CRISPR screens in human neural organoids and assembloids.},
journal = {Nature protocols},
volume = {21},
number = {7},
pages = {3127-3147},
pmid = {41419637},
issn = {1750-2799},
mesh = {Humans ; *Organoids/cytology/metabolism ; *CRISPR-Cas Systems ; Neurodevelopment ; *Neurons/cytology/metabolism ; Induced Pluripotent Stem Cells/cytology ; *Gene Editing/methods ; Cell Differentiation ; },
abstract = {Studying the molecular mechanisms underlying the assembly of the human nervous system remains a significant challenge. The ability to generate neural cells from pluripotent stem cells, combined with advanced genome-editing techniques, provides unprecedented opportunities to uncover the biology of human neurodevelopment and disease. Organoids and assembloids enable the in vitro modeling of previously inaccessible developmental processes, such as the specification and migration of human neurons, including the integration of cortical interneurons from the ventral into the dorsal forebrain. Here, we present a detailed protocol that combines pooled CRISPR-Cas9 screening with neural organoid and assembloid models and illustrate how it can be applied to map hundreds of disease genes onto cellular pathways and specific aspects of human neural development. Our protocol outlines key steps, from planning and optimizing genetic perturbations to designing readouts for neuronal generation and migration, conducting the screening and validating candidate genes. The screening experiments take ~3 months to complete and require expertise in stem cell culture and neural differentiation, genetic engineering of human induced pluripotent stem cell lines, fluorescence-activated cell sorting and next-generation sequencing and analyses. The integration of genetic screening and human cellular models constitutes a powerful platform for investigating the mechanisms of human brain development and disease, paving the way for the discovery of novel therapeutics.},
}

Humans
*Organoids/cytology/metabolism
*CRISPR-Cas Systems
Neurodevelopment
*Neurons/cytology/metabolism
Induced Pluripotent Stem Cells/cytology
*Gene Editing/methods
Cell Differentiation

@article {pmid41832622,
year = {2026},
author = {Yuan, C and Chen, F and Gao, X and Yusufu, A and Lu, D and Wu, X and Ni, L},
title = {UBE2M Identified by CRISPR Screening as a Key Regulator of Cisplatin-Induced Acute Kidney Injury via the p53 Pathway.},
journal = {Endocrine, metabolic & immune disorders drug targets},
volume = {26},
number = {},
pages = {e18715303410982},
pmid = {41832622},
issn = {2212-3873},
mesh = {*Cisplatin/toxicity ; *Acute Kidney Injury/chemically induced/genetics/metabolism/pathology ; Animals ; *Ubiquitin-Conjugating Enzymes/genetics/metabolism ; *Tumor Suppressor Protein p53/metabolism/genetics ; Signal Transduction/drug effects/physiology ; Humans ; *CRISPR-Cas Systems ; *Antineoplastic Agents/toxicity ; Mice ; Apoptosis/drug effects ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {INTRODUCTION: Acute kidney injury caused by cisplatin (Cis-AKI) is a major limitation in its clinical use, primarily due to the lack of effective therapeutic targets to mitigate nephrotoxicity. Although several molecular pathways are involved in Cis-AKI, identifying reliable and actionable therapeutic targets has been challenging. Through a CRISPR-based genome-wide screening approach, UBE2M was identified as a novel gene involved in cellular survival during cisplatin-induced stress. However, its expression, biological function, and underlying mechanism in Cis-AKI have not been thoroughly investigated. This study aims to identify key therapeutic targets for Cis- AKI and investigate the role of UBE2M in this condition.

METHODS: A CRISPR-Cas9 genome-wide screening approach was employed to identify key genes involved in cisplatin-induced renal tubular epithelial cell injury. UBE2M, identified as a critical survival factor, was further investigated using both gain- and loss-of-function strategies to explore its biological function and underlying regulatory mechanisms in the Cis-AKI model.

RESULTS: CRISPR screening identified UBE2M as a key regulator of cellular survival in Cis-AKI, and subsequent validation experiments confirmed its suppression in cisplatin-induced renal injury models. UBE2M overexpression alleviated apoptosis and renal injury by reducing p53 activation. In contrast, UBE2M knockdown exacerbated these effects, leading to increased apoptosis and renal injury.

DISCUSSION: This study reveals that UBE2M is a critical regulator of cisplatin-induced renal tubular epithelial cell injury. By regulating the p53-mediated apoptotic pathway, UBE2M protects against Cis-AKI.

CONCLUSION: UBE2M could serve as a novel therapeutic target for the prevention and treatment of cisplatin-induced nephrotoxicity.},
}

*Cisplatin/toxicity
*Acute Kidney Injury/chemically induced/genetics/metabolism/pathology
Animals
*Ubiquitin-Conjugating Enzymes/genetics/metabolism
*Tumor Suppressor Protein p53/metabolism/genetics
Signal Transduction/drug effects/physiology
Humans
*CRISPR-Cas Systems
*Antineoplastic Agents/toxicity
Mice
Apoptosis/drug effects
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42135732,
year = {2026},
author = {Xiao, WT and He, JY and Yang, D and Xun, Y},
title = {Construction of a novel signature based on CRISPR-Cas9 screening for prognostic prediction in breast cancer.},
journal = {BMC cancer},
volume = {26},
number = {1},
pages = {},
pmid = {42135732},
issn = {1471-2407},
support = {82304083//National Natural Science Foundation of China/ ; 2023JJ40584//Natural Science Foundation of Hunan Province/ ; },
mesh = {Humans ; *Breast Neoplasms/genetics/mortality/pathology ; Female ; Prognosis ; *CRISPR-Cas Systems ; *Biomarkers, Tumor/genetics ; Gene Expression Regulation, Neoplastic ; Gene Expression Profiling ; Transcriptome ; },
abstract = {BACKGROUND: Breast cancer (BC) is a highly heterogeneous malignancy and remains the leading cause of cancer-related mortality among women worldwide. Although advances in molecular classification and targeted therapies have improved outcomes for certain subtypes, robust prognostic biomarkers applicable across clinical contexts are still lacking. The CRISPR-Cas9 system offers a powerful platform for identifying cancer cell vulnerabilities and may facilitate the development of clinically relevant prognostic models.

METHODS: We integrated genome-wide CRISPR-Cas9 screening data from the DepMap database with transcriptomic and clinical data from TCGA and GEO datasets to identify BC cell survival-dependent genes (CSDGs). CSDGs prognostic signature was constructed using univariate Cox regression, LASSO, and stepwise multivariate Cox regression analyses. The model was validated in internal and external cohorts. Functional enrichment analyses, including GO, KEGG, WGCNA, and GSEA, were performed to explore the biological mechanisms underlying the signature. Random forest analysis and functional experiments were conducted to investigate the role of key gene in CSDGs signature.

RESULTS: A total of 1,622 CSDGs were identified, and a nine-gene prognostic CSDGs signature (BRD4, CHORDC1, COPZ1, HNRNPC, NUP43, RAD1, RBBP8, TUBA1B, and VPS28) was developed. This signature effectively stratified patients into high- and low-risk groups with significantly different overall survival, and its robustness was confirmed across multiple internal and external cohorts. High-risk patients exhibited a significant association with multiple adverse clinical features. A nomogram that combined the risk score with clinical variables showed robust predictive performance, and its C-index surpassed those of individual predictors, underscoring the enhanced accuracy of the integrated model. Functional analyses revealed enrichment of oncogenic pathways (e.g., MYC targets, G2/M checkpoint, mTORC1 signaling) in high-risk patients, while low-risk patients exhibited immune and hormone response signatures. CHORDC1 was identified as the most critical gene in the model. Knockdown of CHORDC1 significantly inhibited proliferation, migration, and invasion of BC cells. Transcriptomic profiling further linked CHORDC1 to oncogenic pathways, including EMT, mTORC1 signaling, and TNF-α/NF-κB signaling activation.

CONCLUSION: We developed a CRISPR-Cas9 screening-based prognostic signature for BC that effectively stratifies patient risk and demonstrates robust predictive performance across cohorts. CHORDC1 was identified as a key oncogenic driver, promoting tumor progression via pathways such as EMT and mTORC1 signaling, highlighting its potential as a therapeutic target. These findings may contribute to the development of personalized prognostic tools and therapeutic strategies in BC.},
}

Humans
*Breast Neoplasms/genetics/mortality/pathology
Female
Prognosis
*CRISPR-Cas Systems
*Biomarkers, Tumor/genetics
Gene Expression Regulation, Neoplastic
Gene Expression Profiling
Transcriptome

@article {pmid42235446,
year = {2026},
author = {Olfati Sumar, M and Mohammadi, F and Khoshbin, Z and Abbasi Ghaeni, F and Abnous, K and Taghdisi, SM},
title = {A CRISPR-driven aptasensor for colorimetric monitoring of lead (II) ion assisted by rolling circle amplification process: Effective in controlling food and health safety.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {362},
number = {},
pages = {128167},
doi = {10.1016/j.saa.2026.128167},
pmid = {42235446},
issn = {1873-3557},
mesh = {*Colorimetry/methods ; *Lead/analysis ; *Aptamers, Nucleotide/chemistry ; Gold/chemistry ; Metal Nanoparticles/chemistry ; *Biosensing Techniques/methods ; Limit of Detection ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Food Safety/methods ; Food Contamination/analysis ; },
abstract = {Herein, an efficient colorimetric aptasensor has been introduced for highly sensitive measurement of lead (II) ions (Pb[2+]) by integrating the advantages of clustered regularly interspaced short palindromic repeats (CRISPR) system, the rolling circle amplification (RCA) process, and the catalytic activity of gold nanoparticles (AuNPs) for the first time. The presence of Pb[2+] inactivates the CRISPR system, making it unable to cleave the complementary sequence (CS) on the surface of ferrofluids (FFDs) and the formation of RCA product. By trapping AuNPs inside the RCA mass and subsequent magnetic separation of FFDs, the supernatant color remains yellow after adding 4-nitrophenol (4-NP). In the absence of Pb[2+], the supernatant color changes to colorless, due to the activation of CRISPR-Cas12a and the lack of large DNA structures. The colorimetric aptasensor can monitor Pb[2+] ions in the concentration ranges of 0.1 pM-20 nM and 20 nM-800 nM with a detection limit of 0.024 pM. It can also quantify Pb[2+] in the biological, cosmetic, and marine samples.},
}

*Colorimetry/methods
*Lead/analysis
*Aptamers, Nucleotide/chemistry
Gold/chemistry
Metal Nanoparticles/chemistry
*Biosensing Techniques/methods
Limit of Detection
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems/genetics
*Food Safety/methods
Food Contamination/analysis

@article {pmid42284937,
year = {2026},
author = {Yang, Q and Cao, Y and Yuan, J and Lu, W and Guo, Y and Sun, X and Wang, X and Li, X and Li, J and Zhang, N and Cao, L and Gong, P},
title = {A rapid and specific strategy for detecting Orientobilharzia turkestanicum, a water-associated schistosome of veterinary and environmental concern.},
journal = {Water research},
volume = {303},
number = {},
pages = {126262},
doi = {10.1016/j.watres.2026.126262},
pmid = {42284937},
issn = {1879-2448},
mesh = {Animals ; Cattle ; *Schistosoma/isolation & purification ; *Water/parasitology ; CRISPR-Cas Systems ; Environmental Monitoring ; },
abstract = {Orientobilharzia turkestanicum (O. turkestanicum) is a water-associated schistosome parasite widely distributed in pastoral regions of Asia and Europe, where freshwater systems act as key interfaces linking livestock hosts, snail intermediate hosts, and environmental transmission pathways. Despite its strong environmental dependency, effective surveillance of O. turkestanicum in water-related settings remains limited by conventional diagnostic approaches that are time-consuming, equipment-dependent, and poorly suited for field-based monitoring. In this study, a rapid and sensitive detection assay based on clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a (RPA-CRISPR/Cas12a) was developed for on-site identification of O. turkestanicum. The ITS1-5.8S region was selected as the molecular target to enable reliable species discrimination from closely related schistosomes, particularly Schistosoma japonicum (S. japonicum). The assay operates under isothermal conditions at 37 °C and allows result interpretation through visual fluorescence and lateral flow strip (LFS) readouts without the need for sophisticated instrumentation. The established RPA-CRISPR/Cas12a assay exhibited high analytical specificity, showing no cross-reactivity with a range of non-target parasites and bacterial species. Sensitivity evaluation using serially diluted standard plasmids demonstrated analytical limit of detection of 0.16 copies/μL by visual fluorescence and 160 copies/μL by LFS. Field applicability was validated using snail samples, cattle feces, liver, intestinal tissues and simulated infected water samples, yielding an overall concordance rate of 97.55 % compared with qPCR. Collectively, these results indicate that the proposed RPA-CRISPR/Cas12a assay provides a practical and field-deployable tool for livestock and water-associated transmission environment of O. turkestanicum and offers a useful framework for improving field surveillance and risk assessment of livestock schistosomiasis in endemic regions.},
}

Animals
Cattle
*Schistosoma/isolation & purification
*Water/parasitology
CRISPR-Cas Systems
Environmental Monitoring

@article {pmid42300772,
year = {2026},
author = {Chen, J and Le, Y and Yang, L and Qin, H and Yang, W},
title = {Dual mode analysis of lead ions in regulation of intestinal flora via split-DNA re-assembly catalyzed colorimetric reaction and trans-cleavage activity of Cas12a/crRNA.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {26},
pages = {5441-5448},
doi = {10.1039/d6ay00799f},
pmid = {42300772},
issn = {1759-9679},
mesh = {*Colorimetry/methods ; *Lead/analysis/blood ; Humans ; *Gastrointestinal Microbiome/drug effects ; *Bacterial Proteins/metabolism/chemistry ; *Endodeoxyribonucleases/metabolism/chemistry ; DNA, Catalytic/chemistry/metabolism ; *CRISPR-Associated Proteins/metabolism/chemistry ; Limit of Detection ; Biosensing Techniques/methods ; CRISPR-Cas Systems ; },
abstract = {Chronic exposure to lead ions (Pb[2+]) disrupts intestinal flora homeostasis, underscoring the need for sensitive and practical detection methods. Herein, we report a dual-mode analytical strategy combining split-DNA re-assembly-driven colorimetric reaction with CRISPR/Cas12a trans-cleavage activity for Pb[2+] analysis. A bifunctional magnetic probe is constructed, where Pb[2+]-specific DNAzyme recognition triggers cleavage and release of crRNA, which subsequently activates Cas12a to generate fluorescence by cleaving a fluorophore-quencher reporter. Concurrently, the remaining magnetic bead-anchored trigger initiates split G-quadruplex re-assembly, catalyzing a visible colorimetric reaction. Under optimized conditions, the fluorescence mode achieves a detection limit of 1.03 fM with a linear range of 5 fM to 100 pM, while the colorimetric mode offers a limit of 62.1 fM from 100 fM to 500 pM. Both modes exhibit excellent specificity against competing metal ions. The platform's clinical feasibility is validated using human serum samples, showing strong correlation with ICP-MS, satisfactory recoveries (97.1-103.4%), and good repeatability (CV = 4.1%). Magnetic separation effectively minimizes matrix interference, making the assay suitable for complex biological specimens such as intestinal contents and fecal samples. This dual-mode design integrates fluorescence for precise quantification and colorimetry for equipment-free visual detection, holding great promise for point-of-care testing, early risk assessment of lead-induced gut microbiota dysbiosis, and environmental monitoring.},
}

*Colorimetry/methods
*Lead/analysis/blood
Humans
*Gastrointestinal Microbiome/drug effects
*Bacterial Proteins/metabolism/chemistry
*Endodeoxyribonucleases/metabolism/chemistry
DNA, Catalytic/chemistry/metabolism
*CRISPR-Associated Proteins/metabolism/chemistry
Limit of Detection
Biosensing Techniques/methods
CRISPR-Cas Systems

@article {pmid42400707,
year = {2026},
author = {Wang, Y and Chen, M and Wang, Y and Yu, Y},
title = {Simultaneous Detection of Human Norovirus GI, GII and Hepatitis A Virus Using CRISPR-Cas12a-Based RT-RPA and Lateral Flow Strip Method.},
journal = {Food and environmental virology},
volume = {18},
number = {3},
pages = {},
pmid = {42400707},
issn = {1867-0342},
support = {22N31900700//Plan of Action for Scientific and Technological innovation of Science and Technology Commission of Shanghai Municipality/ ; 31601570//National Natural Science Foundation of China/ ; },
mesh = {*Norovirus/isolation & purification/genetics/classification ; Humans ; *Hepatitis A virus/isolation & purification/genetics/classification ; CRISPR-Cas Systems ; Shellfish/virology ; Rapid Diagnostic Tests ; *Nucleic Acid Amplification Techniques/methods ; RNA, Viral/genetics ; Animals ; *Caliciviridae Infections/virology ; Sensitivity and Specificity ; Hepatitis A/virology ; },
abstract = {Human norovirus (HuNoV) and hepatitis A virus (HAV) are highly prevalent and contagious foodborne pathogens that pose a significant threat to global public health. Current molecular detection methods such as RT-qPCR and RT-ddPCR are highly sensitive and specific but time-consuming, require specialized equipment, and are unsuitable for on-site detection. We developed a multiplex reverse transcription recombinase polymerase amplification (RT-RPA) assay coupled with CRISPR-Cas12a and lateral flow detection for rapid, simultaneous identification of HuNoV GI, GII, and HAV. Through rigorous in silico design and experimental validation, we optimized primer pools and crRNAs to ensure broad genotype coverage and high specificity. Using 2 µL of input per target per 50 µL reaction, the assay achieved limits of detection of 10[1] copies/µL (2 × 10[1] copies/reaction) for HAV, 10[3] copies/µL (2 × 10[3] copies/reaction) for GI HuNoV, and 10[2] copies/µL (2 × 10[2] copies/reaction) for GII HuNoV, with a total assay time of 50 min from purified RNA to final readout. No cross-reactivity occurred with other common foodborne viruses. Validation using total RNA extracted from shellfish digestive glands artificially spiked with RNA standards provided preliminary evidence supporting the feasibility of the method under laboratory conditions. This portable system shows strong potential as a rapid multiplex molecular detection platform.},
}

*Norovirus/isolation & purification/genetics/classification
Humans
*Hepatitis A virus/isolation & purification/genetics/classification
CRISPR-Cas Systems
Shellfish/virology
Rapid Diagnostic Tests
*Nucleic Acid Amplification Techniques/methods
RNA, Viral/genetics
Animals
*Caliciviridae Infections/virology
Sensitivity and Specificity
Hepatitis A/virology

@article {pmid42401475,
year = {2026},
author = {Luo, G and Wei, L and Wang, Q and Guo, Y and Liu, Y and Wang, J and Deng, Y and Li, S and Nie, L and He, N and Chen, Z},
title = {A dual-modal RPA-CRISPR/Cas12a biosensor for rapid and ultrasensitive detection of Staphylococcus aureus in bloodstream infections.},
journal = {Analytica chimica acta},
volume = {1416},
number = {},
pages = {345800},
doi = {10.1016/j.aca.2026.345800},
pmid = {42401475},
issn = {1873-4324},
mesh = {*Staphylococcus aureus/isolation & purification/genetics ; *Biosensing Techniques/methods ; Humans ; *Nucleic Acid Amplification Techniques ; *Staphylococcal Infections/blood/diagnosis/microbiology ; Limit of Detection ; Electrochemical Techniques ; *CRISPR-Cas Systems/genetics ; Recombinases/metabolism ; Rapid Diagnostic Tests ; },
abstract = {BACKGROUND: Bloodstream infections (BSIs) caused by Staphylococcus aureus (S. aureus) require rapid and accurate diagnosis to guide effective antimicrobial therapy and improve patient outcomes. However, current diagnostic methods often struggle to balance speed, sensitivity, portability, and cost.

RESULTS: After systematic optimization of reaction parameters (including primer design, reaction temperature and time, buffer composition, and probe concentration), the fluorescence assay demonstrated high specificity and completed detection within 35 min. Under optimized conditions (1.0 μM HS-ssDNA-MB and 10 min CRISPR incubation), the electrochemical sensor achieved a detection limit of 138 copies/mL, with a wide linear dynamic range from 4.37 × 10[0] to 10[5]copies/μL, and showed high specificity against non-target pathogens. Furthermore, the platform demonstrated reliable performance in spiked artificial blood samples, with recovery rates ranging from 100.7% to 107.3%. This dual-modal recombinase polymerase amplification (RPA)-CRISPR/Cas12a biosensor combines the rapid amplification capability of isothermal methods with the high specificity of CRISPR-based detection.

SIGNIFICANCE: This platform provides both a high-throughput fluorescence detection mode and a portable, low-cost electrochemical detection mode, offering an efficient and flexible solution for the rapid point-of-care diagnosis and large-scale screening of BSIs caused by S. aureus, with promising potential for clinical translation.},
}

*Staphylococcus aureus/isolation & purification/genetics
*Biosensing Techniques/methods
Humans
*Nucleic Acid Amplification Techniques
*Staphylococcal Infections/blood/diagnosis/microbiology
Limit of Detection
Electrochemical Techniques
*CRISPR-Cas Systems/genetics
Recombinases/metabolism
Rapid Diagnostic Tests

@article {pmid42402276,
year = {2026},
author = {Pradhan, RR and Pati, S and Samal, SK},
title = {Artificial intelligence and CRISPR-based approaches for targeted delivery of bacteriophages.},
journal = {International journal of pharmaceutics},
volume = {},
number = {},
pages = {127155},
doi = {10.1016/j.ijpharm.2026.127155},
pmid = {42402276},
issn = {1873-3476},
abstract = {The rapid emergence of multidrug-resistant (MDR) bacteria has increased interest in bacteriophage therapy as a promising alternative to conventional antibiotics. Bacteriophages are host-specific bacterial viruses that selectively infect and destroy pathogenic bacterial strains. Recent developments in artificial intelligence (AI) and CRISPR-based technologies offer innovative approaches to address challenges such as narrow host range, rapid immune clearance, phage instability, bacterial resistance, and biofilm penetration barriers. By integrating AI-driven structural modeling with CRISPR-mediated genome editing, these methods enable the targeted delivery of bacteriophages. This review focuses on next-generation approaches that combine AI-assisted phage identification, host prediction, and therapeutic optimization with CRISPR-based genome engineering for targeted phage delivery and improved safety. Overall, this review highlights the potential of AI- and CRISPR-assisted phage therapy for the treatment of MDR bacterial infections. This review provides a systematic overview of bacteriophage biology, life cycle, and mechanisms of action, highlighting the influence of phage morphology on therapeutic performance, recent advances, current clinical and preclinical studies, and future perspectives. Although phage therapy shows considerable potential against MDR bacterial infections, several challenges related to delivery, safety, and clinical translation remain. The integration of AI and CRISPR technologies may improve phage selection, targeting specificity, and therapeutic performance. Continued research, clinical validation, and regulatory development will be essential for translating these advances into practical antimicrobial therapies.},
}

@article {pmid42402524,
year = {2026},
author = {Verupanda, S and Chakraborty, A and Shrivastava, M and Pati, SK and Nandi, D},
title = {Harnessing CRISPR-Cas technology to enhance rice resilience under abiotic and biotic stress.},
journal = {Journal of applied genetics},
volume = {},
number = {},
pages = {},
pmid = {42402524},
issn = {2190-3883},
abstract = {The CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats) genome-editing technology has become an effective and accurate tool for crop development, enabling targeted changes to genes linked to stress tolerance, agronomic performance, and yield. CRISPR-Cas-based technologies are increasingly used in rice (Oryza sativa L.), one of the world's most significant staple crops, to mitigate the negative impacts of disease stress and climate change on productivity. Abiotic factors, including drought, salinity, heat, and cold, as well as biotic challenges such as rice blast, bacterial blight, sheath blight, and insect pests, have a significant impact on rice cultivation and cause substantial yield losses globally. Recent advances in CRISPR/Cas9, base editing, and prime editing have enabled precise manipulation of stress-responsive genes, facilitating the development of climate-resilient and disease and pest-resistant rice varieties. This review summarizes the current progress in CRISPR-Cas-mediated rice improvement, highlighting key genes and molecular pathways involved in tolerance to abiotic and biotic stresses. It also discusses emerging approaches such as transgene-free editing via ribonucleoprotein (RNP) delivery, and high-fidelity Cas variants that enhance editing efficiency and minimize off-target effects. Overall, CRISPR-Cas-based genome editing represents a promising and efficient approach for accelerating the development of high-yielding, climate-resilient, and stress-tolerant rice cultivars, thereby contributing significantly to sustainable rice production and global food security under changing environmental conditions.},
}

@article {pmid42405245,
year = {2026},
author = {XiuJuan, W and Faisal, M and Muhammad, S and Aslam, A and Razzaq, MK and Masroor, A and Yefang, S and Quronfulah, AS and Al-Malki, MA and Osman, HES},
title = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas genome editing transforming crop stress tolerance for global food security.},
journal = {PeerJ},
volume = {14},
number = {},
pages = {e21450},
pmid = {42405245},
issn = {2167-8359},
mesh = {*Gene Editing/methods ; *Crops, Agricultural/genetics ; *Stress, Physiological/genetics ; *CRISPR-Cas Systems ; *Food Security ; Plant Breeding/methods ; Plants, Genetically Modified ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genome, Plant ; Climate Change ; },
abstract = {Climate change increasingly threatens global crop productivity by intensifying drought, salinity, temperature extremes, and biotic stresses. Developing climate-resilient cultivars has therefore become a central objective in modern crop breeding programs. Conventional breeding approaches are often limited by complex trait inheritance and long selection cycles, particularly for polygenic stress-adaptive traits. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein (Cas) genome editing genome editing provides a precise and efficient platform for targeted manipulation of genes controlling stress tolerance, yield stability, and adaptive performance. This review synthesizes recent advances in CRISPR mediated improvement of resilience to major abiotic stresses (drought, salinity, heat, and cold) and biotic stresses (fungi, bacteria, viruses, and insects) across important cereal, legume, and horticultural crops. Emphasis is placed on the editing of transcription factors, signaling regulators, susceptibility genes, and redox-associated pathways that enhance physiological and molecular stress adaptation. Furthermore, the integration of CRISPR with genomics, transcriptomics, proteomics, metabolomics, genome-wide association studies, high-throughput phenotyping, and artificial intelligence-driven prediction tools is accelerating precision breeding strategies. Despite remaining challenges related to off-target effects, delivery systems, and regulatory frameworks, genome editing represents a transformative approach for advancing climate-resilient crop development and sustainable agricultural production.},
}

*Gene Editing/methods
*Crops, Agricultural/genetics
*Stress, Physiological/genetics
*CRISPR-Cas Systems
*Food Security
Plant Breeding/methods
Plants, Genetically Modified
*Clustered Regularly Interspaced Short Palindromic Repeats
Genome, Plant
Climate Change

@article {pmid42405767,
year = {2026},
author = {Zhuang, S and Luo, W and Lan, B and Liu, Y},
title = {Strategic Design and Engineering of CRISPR/Cas-Powered Sensing Platforms for Enhanced Nucleic Acid Detection.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.6c01710},
pmid = {42405767},
issn = {2379-3694},
abstract = {Rapid and accurate nucleic acid detection is fundamental to effective disease management. While PCR remains the gold standard, its requirement for sophisticated instrumentation limits its application in point-of-care settings. CRISPR-Cas systems have emerged as a disruptive diagnostic technology, leveraging the programmable specificity and unique trans-cleavage activity of Cas effectors to revolutionize biosensing. This review systematically evaluates the evolution of CRISPR-Cas-powered sensing platforms, categorized by their signal transduction modalities. We first discuss the expanding biochemical landscape of Cas nucleases, highlighting recent discoveries where conventional boundaries of Cas9, Cas12, and Cas13 have been transcended to enable versatile DNA/RNA targeting. Subsequently, we provide a comprehensive analysis of four primary sensing architectures: (1) Fluorescence-based platforms, exploring diverse strategies from target and signal amplification with dual-labeled ssDNA probes to nanomaterial-based probes; (2) Naked-eye visual platforms, encompassing both solid-phase lateral flow assays and solution-phase colorimetric strategies that facilitate rapid, instrument-free screening; (3) Electrochemical biosensors, which transduce biological recognition events into measurable electrical parameters, offering high sensitivity and seamless integration with miniaturized electronics; and (4) Electronic and Optoelectronic systems, including field-effect transistors and plasmonic sensors, which offer high-sensitivity, label-free detection. Despite significant progress, the translation of CRISPR-Dx from laboratory proof of concepts to clinical reality faces several bottlenecks. We critically analyze current challenges, including the need for integrated "sample-to-answer" workflows, high-throughput multiplexing, and digital quantification. Finally, we envision future trends such as AI-assisted signal processing and wearable sensing interfaces. By bridging the gap between molecular biology and advanced engineering, CRISPR-powered platforms are poised to make precision molecular diagnostics universally accessible.},
}

@article {pmid42405957,
year = {2026},
author = {Soto-Serrano, A and Vincze, T and Roberts, RJ and Krych, L and Mahony, J and Deptula, P},
title = {Comparative genomics and methylome profiling of Pseudolactococcus laudensis reveal signatures of niche adaptation and strain-level variation in mobile genetic elements and phage defence.},
journal = {Microbial genomics},
volume = {12},
number = {7},
pages = {},
pmid = {42405957},
issn = {2057-5858},
mesh = {*Bacteriophages/genetics ; Genome, Bacterial ; Milk/microbiology ; *Interspersed Repetitive Sequences ; Genomics/methods ; Animals ; Gene Transfer, Horizontal ; DNA Methylation ; Adaptation, Physiological/genetics ; *Lactococcus/genetics/virology ; Phylogeny ; },
abstract = {Pseudolactococcus laudensis (formerly named Lactococcus laudensis) is an emerging lactic acid bacterium first isolated from raw milk in 2015 and subsequently detected in vegetables and dairy mesophilic starter cultures. Despite its recurrent isolation from diverse environments, the genetic basis of its niche adaptation, horizontal gene transfer and phage defence remains unexplored. Here, we perform the first comparative genomic and epigenomic analysis of P. laudensis using complete genomes of a plant-derived isolate (MCRI-603), a milk isolate (DSM 28961) and 20 strains from a Danish dairy mesophilic starter culture. Genomes were annotated and analysed using pangenomics, Clustering of Orthologous Genes and methylome profiling. Average nucleotide identity, pangenome and Clustering of Orthologous Genes analyses revealed niche-associated structure: dairy starter strains formed a tight cluster, while the plant isolate MCRI-603 and milk isolate DSM 28961 were more similar to each other than to the starter culture group. The pangenome comprised 4,946 genes, with 1,396 core genes. Dairy starter strains showed markedly elevated numbers of insertion sequences, pseudogenes, plasmids and genomic islands relative to MCRI-603, which was plasmid-free and carried very few insertion sequence elements or genomic islands. DSM 28961 displayed pseudogene count similar to the dairy starter strains but markedly fewer transposases. These patterns are consistent with a plant-associated origin of P. laudensis and progressive dairy specialization via mobile genetic element acquisition. The P. laudensis mobilome was found to carry key niche-related traits. Lactose utilization operons were plasmid-encoded, whereas exopolysaccharide-encoding loci, opp oligopeptide transport systems and several defence loci, including clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas), were consistently encoded within chromosomal integrative elements. All strains harboured prophage-like elements, including putatively intact prophages in 13 of them, and ~67% of 238 predicted antiphage systems resided on mobile genetic elements, underscoring their central role in phage defence. Restriction-modification systems dominated the defensome, and three strains encoded CRISPR-Cas systems (including type III-A and type I-C), indicating a higher prevalence than has been reported for Lactococcus lactis and Lactococcus cremoris, where CRISPR-Cas has rarely been observed. Methylome analysis identified 43 distinct motifs, of which 25 were novel. The P. laudensis methylome was overwhelmingly dominated by N[6]-methyladenine, and most motifs were short, non-palindromic and largely associated with type III restriction-modification systems and some type I and II subtypes. Nearly all strains exhibited distinct methylation profiles, including those isolated from the same dairy starter culture, highlighting extensive epigenetic diversification in dairy environments. Altogether, the data reveals a highly dynamic genomic and epigenomic landscape in P. laudensis, greatly shaped by mobile genetic elements, and provides a foundation for future work in this species and other Pseudolactococci.},
}

*Bacteriophages/genetics
Genome, Bacterial
Milk/microbiology
*Interspersed Repetitive Sequences
Genomics/methods
Animals
Gene Transfer, Horizontal
DNA Methylation
Adaptation, Physiological/genetics
*Lactococcus/genetics/virology
Phylogeny

@article {pmid42406963,
year = {2026},
author = {Carballar-Lejarazú, R and Dong, Y and Pham, TB and Tushar, T and Stillinger, D and Nguyen, DN and Winokur, L and Tavadia, M and Tao, M and Dimopoulos, G and James, AA},
title = {Long-term stability and performance of Cas9/guide RNA-based gene drives in anopheline mosquitoes.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {28},
pages = {e2605739123},
doi = {10.1073/pnas.2605739123},
pmid = {42406963},
issn = {1091-6490},
support = {AI170692//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; INV-043645/GATES/Gates Foundation/United States ; NA//University of California, Irvine, Malaria Initiative/ ; },
mesh = {Animals ; *Anopheles/genetics/parasitology ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Drive Technology/methods ; *Mosquito Vectors/genetics/parasitology ; *CRISPR-Cas Systems ; Malaria/prevention & control ; },
abstract = {Gene-drive population modification strategies are being developed to control the transmission by anopheline mosquitoes of the parasites that cause human malaria. These approaches are designed to reduce disease prevalence and incidence by spreading dominant antiparasite effector genes throughout vector populations. The strains must sustain drive and parasite suppression properties over extended periods of time to have an epidemiological impact. Three gene-drive strains, AcTP13 and AcTP43 in Anopheles coluzzii and AgTP13 in Anopheles gambiae, carrying autonomous Cas9/guide RNA-based drive systems linked to multiple antiparasite effector genes were remarkably stable in all A. coluzzii replicates over a 2-y (35 generation) period in laboratory cage trials. Two of three A. gambiae replicates performed equally well. Stability was assessed as a function of population dynamics (size), molecular integrity of the gene-drive cassettes, maintenance of drive efficiency (gene conversion), generation and accumulation of mutant drive-resistant target alleles, drive system-generated off-target effects, and effector gene parasite suppression activity. All lines met stability requirements with the exception of one AgTP13 cage replicate that was affected by drive-resistant target-site mutations. Notably, all strains retained parasite suppression activity and high drive efficiencies throughout the duration of the trials. These results support the further development and deployment of these strains for malaria control.},
}

Animals
*Anopheles/genetics/parasitology
*RNA, Guide, CRISPR-Cas Systems/genetics
*Gene Drive Technology/methods
*Mosquito Vectors/genetics/parasitology
*CRISPR-Cas Systems
Malaria/prevention & control

@article {pmid42407157,
year = {2026},
author = {Mu, Y and Yang, Y and Niu, Y and Yan, Z and Lv, X and Li, X},
title = {CRISPR-based diagnostics for ESKAPE drug-resistant bacteria: From proof-of-concept to point-of-care.},
journal = {Talanta},
volume = {311},
number = {},
pages = {130240},
doi = {10.1016/j.talanta.2026.130240},
pmid = {42407157},
issn = {1873-3573},
abstract = {The ESKAPE pathogens, including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species, represent a major challenge in the antimicrobial resistance (AMR) crisis. These pathogens are progressively acquiring pan-resistance and spreading beyond traditional healthcare settings, yet conventional diagnostic methods remain ill-equipped for point-of-care (POC) deployment due to slow processing times and limited adaptability. CRISPR/Cas systems feature programmable target specificity and intrinsic signal amplification, enabling rapid and accurate nucleic acid detection. Despite these advantages, the translation of CRISPR-based assays for ESKAPE pathogens from proof-of-concept to practical POC tools faces major challenges. Current research is fragmented, and key trade-offs between sensitivity, multiplexing performance and operational simplicity have not been fully addressed. This review offers a critical assessment of the field, moving beyond a simple summary of existing studies to analyze how various CRISPR systems (Cas9, Cas12, Cas13, and Cas14) and amplification strategies address the demands of POC testing. We identify key barriers to clinical application, particularly sample preparation, multiplex detection, reagent stability, and discuss emerging solutions such as microfluidic integration, lyophilized reagents development, and artificial intelligence-driven data interpretation. By focusing on the central question of how to transition from benchtop research to bedside application, this review provides a strategic framework for advancing next-generation CRISPR diagnostics capable of rapid, precise, and real-time detection of drug-resistant ESKAPE pathogens in the fight against AMR.},
}

@article {pmid42409192,
year = {2026},
author = {Shamim, S and Singh, AP and Sharma, H and Gohri, S and Taumar, D and Chaudhary, V},
title = {Programmable gene modulation networks for Parkinson's disease using nanotechnology enabled CRISPR/Cas brain delivery.},
journal = {International journal of pharmaceutics},
volume = {},
number = {},
pages = {127151},
doi = {10.1016/j.ijpharm.2026.127151},
pmid = {42409192},
issn = {1873-3476},
abstract = {Parkinson's disease is a progressive neurodegenerative disorder driven by interconnected molecular pathways, including α-synuclein aggregation, mitochondrial dysfunction, impaired proteostasis, and neuroinflammation. Current therapies are primarily symptomatic and have not consistently demonstrated prevention of disease progression. This review introduces Programmable gene modulation networks, a systems-level framework that integrates CRISPR/Cas technologies with nanotechnology-enabled brain delivery for precision intervention in Parkinson's disease. Advanced CRISPR modalities, including CRISPR interference, activation, base editing, prime editing, and epigenetic editing, are evaluated for reversible and targeted modulation of disease-relevant gene networks. Non-viral nanocarrier platforms, such as lipid nanoparticles, polymeric systems, and exosome-mimetic vesicles, are discussed for overcoming blood-brain barrier limitations and improving brain-specific delivery. The review further emphasizes translational challenges, including delivery efficiency, off-target effects, long-term safety, manufacturing scalability, and regulatory considerations. By integrating molecular network biology, programmable gene regulation, and translational decision-making, this review provides a roadmap for developing next-generation disease-modifying therapies for Parkinson's disease.},
}

@article {pmid42409502,
year = {2026},
author = {Wang, X and Gu, Z and Xu, Q and Dong, M and Duan, N and Wang, Z and Wu, S},
title = {A dual-mode electrochemical-colorimetric aptasensor based on HCR-CRISPR/Cas12a cascade amplification for MTGase detection.},
journal = {Food research international (Ottawa, Ont.)},
volume = {240},
number = {},
pages = {119676},
doi = {10.1016/j.foodres.2026.119676},
pmid = {42409502},
issn = {1873-7145},
mesh = {*Transglutaminases/analysis ; *Colorimetry/methods ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *Aptamers, Nucleotide/chemistry ; Metal Nanoparticles/chemistry ; Gold/chemistry ; Limit of Detection ; Triticum ; },
abstract = {Microbial transglutaminase (MTGase) is widely used in food processing, but its potential food safety risks cannot be neglected. MTGase-treated wheat products show immunoreactivity, which may induce celiac disease and damage intestinal barrier function. Therefore, trace detection of MTGase is of great significance. In this study, an electrochemical-colorimetric dual-mode aptasensor for MTGase was developed based on HCR-CRISPR/Cas12a cascade amplification. The system employs high-affinity aptamers for specific recognition of MTGase and significantly enhances the detection signal through HCR-Cas12a cascade amplification, achieving highly sensitive responses. The platform incorporates MXene@AuNPs and Co-Fc-MOF composite materials, where MXene@AuNPs provides excellent conductivity and a stable loading interface, and Co-Fc-MOF offers both electrochemical activity and peroxidase-like activity, enabling synchronous electrochemical and colorimetric signal output. Experimental results demonstrate a wide linear range of 0.5-2000 ng/mL, with a detection limit of 0.12 ng/mL for the electrochemical mode and 0.14 ng/mL for the colorimetric mode. The platform exhibits excellent stability and reproducibility, and achieves satisfactory recovery in real sample analysis. This dual-mode sensing strategy efficiently integrates aptamer recognition, bifunctional nanomaterials, and cascade amplification, providing a rapid and reliable method for trace MTGase detection in food matrices, and offering guidance for the development of multi-mode aptasensor.},
}

*Transglutaminases/analysis
*Colorimetry/methods
*Electrochemical Techniques/methods
*CRISPR-Cas Systems
*Biosensing Techniques/methods
*Aptamers, Nucleotide/chemistry
Metal Nanoparticles/chemistry
Gold/chemistry
Limit of Detection
Triticum

@article {pmid41588790,
year = {2026},
author = {Favaratto, L and da Silva, ML and Buss, DS and Quadros, OF and Tapia-Tussell, R and Ventura, JA and Fernandes, AAR and Fernandes, PMB},
title = {The regulatory frameworks surrounding CRISPR-edited papaya and their impact on international commerce.},
journal = {Journal of the science of food and agriculture},
volume = {106},
number = {11},
pages = {6262-6270},
pmid = {41588790},
issn = {1097-0010},
support = {# 404972/2021-7 and 441499/2023-6//Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/ ; # 1132/2024//Fundação de Amparo à Pesquisa do Espírito Santo/ ; #301052/25-5 and #307905/2020-9//Conselho Nacional de Desenvolvimento Científico e Tecnológico/ ; },
mesh = {*Carica/genetics/virology/immunology/growth & development ; *Plant Diseases/virology/economics/genetics ; *Gene Editing/economics/legislation & jurisprudence ; *Commerce/legislation & jurisprudence/economics ; CRISPR-Cas Systems ; *Plants, Genetically Modified/genetics/virology/immunology ; Plant Proteins/genetics/metabolism/immunology ; Fruit/genetics/virology/economics ; },
abstract = {The papaya tree (Carica papaya L.), native to the Americas, is cultivated in tropical regions and holds substantial economic importance, with an estimated export volume of 365 000 t in 2023. However, diseases caused by viruses, fungi, bacteria, and nematodes can lead to severe losses. Among the more than 38 known viral diseases affecting papaya, only a few poses serious threats to cultivation, notably Papaya Ringspot, Papaya Mosaic, and Papaya Sticky Disease (PSD). Emerging technologies, particularly CRISPR/Cas9 gene editing, offer promising avenues to enhance plant resistance. This study examines regulatory paradigms in key papaya-producing and importing countries, highlighting the need for international regulatory harmonization to reduce trade barriers and improve market access for CRISPR-edited cultivars. We demonstrate the feasibility of CRISPR-based genome editing in papaya (Carica papaya L.) by targeting phytoene desaturase as a proof-of-concept marker gene and β-1,3-glucanase, a resistance gene identified through proteomic profiling of host-pathogen interactions during infection by the papaya meleira virus (PMeV and PMeV2) complex. This virus complex causes PSD, a major threat to papaya production, rendering the fruit commercially unviable due to negative effects on texture and flavor as well as inhibiting the formation of benzyl isothiocyanate (BITC), and the fruits become susceptible to fruit flies, which are quarantine pests. Despite extensive traditional breeding efforts, resistant papaya genotypes have yet to be identified, underscoring the need for innovative approaches. However, translating advancements into commercial applications remains challenging due to the diverse and often inconsistent regulatory frameworks governing genome-edited crops across different jurisdictions. © 2026 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.},
}

*Carica/genetics/virology/immunology/growth & development
*Plant Diseases/virology/economics/genetics
*Gene Editing/economics/legislation & jurisprudence
*Commerce/legislation & jurisprudence/economics
CRISPR-Cas Systems
*Plants, Genetically Modified/genetics/virology/immunology
Plant Proteins/genetics/metabolism/immunology
Fruit/genetics/virology/economics

@article {pmid42116141,
year = {2026},
author = {Ford, H and Dalvie, NC and Lorgeree, TR and Barry, RM and Sibel, AN and Ahlmark, RA and Narayanan, H and Sunday, BC and Whittaker, CA and Acharya, R and Elenberger, CM and Love, JC},
title = {Deletion of low-essentiality, secretion-associated genes enhances recombinant protein production in Komagataella phaffii.},
journal = {Microbial cell factories},
volume = {25},
number = {1},
pages = {},
pmid = {42116141},
issn = {1475-2859},
mesh = {*Recombinant Proteins/biosynthesis/genetics ; *Saccharomycetales/genetics/metabolism ; CRISPR-Cas Systems ; *Gene Deletion ; Antibodies, Monoclonal/biosynthesis/genetics ; *Genes, Essential ; },
abstract = {BACKGROUND: Komagataella phaffii (K. phaffii) is used to manufacture biologic medicines, food proteins, reagents, and materials. Despite its increasing prevalence, further improvements to its productivity would enhance its economic and operational benefits. Genomic engineering represents one approach to increase its cell-specific productivity. We hypothesized that combining the metrics for the relative essentiality of genes with biological inference for relevance to protein secretion could identify genes that, when disrupted, would improve specific productivity in the resulting strains.

RESULTS: The essentiality of genes in K. phaffii (NRRL Y-11430) were predicted through a genome-wide knockout screen using CRISPR-Cas9. Based on the results from this screen, we selected and subsequently disrupted the least essential genes from two gene groups heavily associated with secretion, namely those relating to the cell wall and vacuolar transport. Strains of K. phaffii with single gene disruptions from these gene sets showed significantly improved production of a monoclonal antibody (mAb). These strains exhibited no discernible differences in growth or apparent profiles of host cell proteins when compared to the parental strain. The best-performing strains consistently showed 2-3x enhancements in specific productivity and titers across scales (3-150 mL), culture formats (plates, flasks, bioreactors), and processing operations (batch and fed-batch).

CONCLUSIONS: This study demonstrates how combining data on gene essentiality and prior knowledge of biological pathways related to a phenotypic trait of interest (here protein secretion) can inform strain engineering to enhance the trait. This study expands the catalog of genetically engineered strains of K. phaffii with improved productivity. These strains support the long-term goal of achieving low-cost, high-volume production of recombinant proteins using this host. Further engineering of these strains and optimization of fermentation processes could enable volumetric productivities comparable to those of other established hosts used to produce mAbs and other complex recombinant proteins.},
}

*Recombinant Proteins/biosynthesis/genetics
*Saccharomycetales/genetics/metabolism
CRISPR-Cas Systems
*Gene Deletion
Antibodies, Monoclonal/biosynthesis/genetics
*Genes, Essential

@article {pmid42148575,
year = {2026},
author = {Kang, B and Kim, J-Y and Oh, S and Kweon, J and Park, H and Kim, M and Choi, I-G and Chang, IS},
title = {A CRISPR interference system for tunable gene expression integrated with a promoter library for Eubacterium callanderi KIST612, an acetogen of functional diversity and versatility.},
journal = {Microbiology spectrum},
volume = {14},
number = {7},
pages = {e0377925},
pmid = {42148575},
issn = {2165-0497},
support = {RS-2021-NR060081//National Research Foundation of Korea/ ; RS-2023-00249146//National Research Foundation of Korea/ ; },
mesh = {*Promoter Regions, Genetic/genetics ; Gene Expression Regulation, Bacterial ; Gene Library ; *CRISPR-Cas Systems ; Metabolic Engineering/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins/genetics/metabolism ; },
abstract = {UNLABELLED: Acetogens are key biocatalysts for carbon-neutral biorefineries, yet their metabolic engineering is limited by the lack of tunable transcriptional regulation systems. Here, we developed a synthetic promoter library for Eubacterium callanderi KIST612 and integrated it with a CRISPR interference (CRISPRi) system to establish precise and scalable gene regulation. Motif analysis of 3,109 putative native promoters revealed conserved and semi-conserved -35 and -10 elements, which were used to construct a promoter library spanning a >20-fold dynamic range of transcriptional strengths. The system was validated by knockdown of pyrF, where promoter strength directly determined repression efficiency (R[2] = 0.92), with high-strength promoters achieving near-complete gene silencing. Application to lactate dehydrogenase (ldh) revealed that increasing promoter strength progressively reduced lactate production from 93.3% to 0.0% of control. This study establishes a versatile synthetic promoter-CRISPRi platform tailored for acetogens, enabling precise control of gene expression and mechanistic dissection of redox metabolism.

IMPORTANCE: Transitioning to a carbon-neutral economy requires biocatalysts that can efficiently convert waste-derived substrates into valuable products. Acetogens are industrially relevant organisms for gas fermentation, but the lack of genetic toolkits tailored to their physiology has constrained metabolic engineering. We present the first synthetic promoter-CRISPRi platform specifically optimized for Eubacterium callanderi KIST612, a model acetogen with high industrial potential. This system provides tunable and predictable regulation of gene expression, extending from mild repression to a near-complete knockdown that could alternate gene deletion systems. This system could be used for not only advancing fundamental understanding of acetogen physiology but also providing a broadly applicable genetic toolbox for precision engineering of sustainable microbial biorefineries.},
}

*Promoter Regions, Genetic/genetics
Gene Expression Regulation, Bacterial
Gene Library
*CRISPR-Cas Systems
Metabolic Engineering/methods
Clustered Regularly Interspaced Short Palindromic Repeats
Bacterial Proteins/genetics/metabolism

@article {pmid42148800,
year = {2026},
author = {Zheng, J and Wen, Z and Li, Y and Zou, W and Ge, P and Peng, H},
title = {Rapid detection of Enterococcus faecalis using RPA-CRISPR/Cas12a-assisted technology.},
journal = {Microbiology spectrum},
volume = {14},
number = {7},
pages = {e0016826},
pmid = {42148800},
issn = {2165-0497},
support = {2023A1515011733//Natural Science Foundation of Guangdong Province/ ; 82272364, 82330072//National Natural Science Foundation of China/ ; },
mesh = {*Enterococcus faecalis/genetics/isolation & purification ; Humans ; *CRISPR-Cas Systems/genetics ; Bacterial Proteins/genetics ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; *Gram-Positive Bacterial Infections/microbiology/diagnosis ; Rapid Diagnostic Tests ; Recombinases ; CRISPR-Associated Proteins/genetics ; },
abstract = {Enterococcus faecalis (E. faecalis) is an opportunistic pathogen capable of causing various life-threatening infections, including urinary tract infections, bloodstream infections, infective endocarditis, and meningitis. As a major etiological agent of healthcare-associated infections (HAIs), its global prevalence continues to rise, a trend closely linked to the increasing problem of multidrug resistance driven by overuse of antibiotics. Therefore, rapid and accurate detection is essential for timely treatment and improved prognosis. In this study, the pheS gene of E. faecalis was rapidly amplified using recombinase polymerase amplification (RPA), and detection was achieved via a CRISPR/Cas12a system. The Cas12a-crRNA complex specifically recognized the amplification product and triggered nonspecific cleavage of a single-stranded DNA (ssDNA) reporter, generating a fluorescent signal that could be quantified in a real-time PCR system or visualized directly under ultraviolet (UV) light. After optimization of key parameters-including RPA primers, reaction conditions, crRNA sequence, and the crRNA/Cas12a combination-the assay achieved a limit of detection (LOD) of 10[-2] ng/μL within a short turnaround time, and showed no cross-reactivity with other common pathogen detection results from clinical isolates and spiked samples were fully consistent with those obtained through PCR/qPCR, confirming high reliability. In summary, the RPA-CRISPR/Cas12a detection method established in this study is sensitive, specific, and reliable. Its simplicity, minimal equipment requirements, and cost-effectiveness make it a promising tool for rapid clinical detection of E. faecalis.IMPORTANCEEnterococcus faecalis is a major opportunistic pathogen responsible for severe healthcare-associated infections, with rising prevalence linked to antibiotic resistance. Rapid and accurate detection is critical for timely treatment and infection control. Conventional methods are often time-consuming or require complex laboratory infrastructure, limiting their use at the point of care. This study developed a rapid detection assay by integrating recombinase polymerase amplification with the CRISPR/Cas12a system, targeting the pheS gene of E. faecalis. The method is sensitive and specific, providing visual results under UV light within a short turnaround time. It offers a simple, cost-effective, and requires minimal equipment, suitable for clinical and resource-limited settings, potentially improving diagnostic efficiency and supporting antimicrobial stewardship.},
}

*Enterococcus faecalis/genetics/isolation & purification
Humans
*CRISPR-Cas Systems/genetics
Bacterial Proteins/genetics
Sensitivity and Specificity
*Nucleic Acid Amplification Techniques/methods
*Gram-Positive Bacterial Infections/microbiology/diagnosis
Rapid Diagnostic Tests
Recombinases
CRISPR-Associated Proteins/genetics

@article {pmid42155443,
year = {2026},
author = {Liang, WW and Mueller, SJ and Hart, SK and Wessels, HH and Méndez-Mancilla, A and Sookdeo, A and Choi, O and Caragine, CM and Corman, A and Lu, L and Kolumba, O and Williams, B and Sanjana, NE},
title = {Essential lncRNAs in the human transcriptome.},
journal = {Cell genomics},
volume = {6},
number = {7},
pages = {101253},
doi = {10.1016/j.xgen.2026.101253},
pmid = {42155443},
issn = {2666-979X},
mesh = {Humans ; *RNA, Long Noncoding/genetics/metabolism ; *Transcriptome/genetics ; Gene Expression Profiling/methods ; CRISPR-Cas Systems/genetics ; Neoplasms/genetics ; Apoptosis/genetics ; Gene Expression Regulation, Neoplastic ; Cell Line, Tumor ; Cell Cycle/genetics ; },
abstract = {Mammalian genomes host a diverse array of RNAs, including protein-coding and noncoding transcripts. However, the functional roles of most long noncoding RNAs (lncRNAs) remain elusive. Using RNA-targeting CRISPR-Cas13 screens, we probed how the loss of ∼5,500 lncRNAs impacts cell fitness across five human cell lines and identified 788 lncRNAs with context-specific or broad essentiality. We confirm their essentiality through individual perturbations and find that the majority of essential lncRNAs operate independently of their nearest protein-coding genes. Using transcriptome profiling in single cells, we discover that loss of essential lncRNAs impairs cell cycle progression and drives apoptosis. Many essential lncRNAs demonstrate dynamic expression across tissues during development. Using ∼9,000 primary tumors, we pinpoint those lncRNAs whose expression in tumors correlates with survival, yielding new biomarkers and potential therapeutic targets. This transcriptome-wide survey of functional lncRNAs advances our understanding of noncoding transcripts and demonstrates the potential of transcriptome-scale noncoding screens with Cas13.},
}

Humans
*RNA, Long Noncoding/genetics/metabolism
*Transcriptome/genetics
Gene Expression Profiling/methods
CRISPR-Cas Systems/genetics
Neoplasms/genetics
Apoptosis/genetics
Gene Expression Regulation, Neoplastic
Cell Line, Tumor
Cell Cycle/genetics

@article {pmid42214793,
year = {2026},
author = {Kim, GH and Kim, MM},
title = {CRISPR/Cas9 system-mediated p21 knockout impairs the MITF signaling pathway.},
journal = {Journal of biotechnology},
volume = {417},
number = {},
pages = {81-90},
doi = {10.1016/j.jbiotec.2026.05.014},
pmid = {42214793},
issn = {1873-4863},
mesh = {*Microphthalmia-Associated Transcription Factor/metabolism/genetics ; Animals ; *Signal Transduction/genetics ; *Cyclin-Dependent Kinase Inhibitor p21/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Mice ; Melanogenesis ; Gene Knockout Techniques/methods ; Melanins/metabolism ; Cell Line, Tumor ; },
abstract = {The CRISPR/Cas9 method facilitates targeted disruption of gene sequences, providing a reliable means to analyze gene-dependent regulatory pathways. This study aims to investigate melanogenesis in p21-knockout B16F1 cells generated by the CRISPR/Cas9 system. The mutation was confirmed by DNA Sanger sequencing, which identified frameshift-inducing indels in the p21 locus. The protein structure of p21 in KO cells was predicted by the α-Fold2 and ChimeraX models. The expression level of the p21 gene was completely reduced in RT-PCR and qPCR assays. Notably, while p21-knockout cells exhibited significantly reduced SA-β-galactosidase activity, this was not indicative of cellular rejuvenation. Instead, it correlated with a loss of melanocytic functionality, as evidenced by the concurrent decrease in melanin synthesis and collagen production. Western blotting and immunofluorescence analyses were performed to examine cell cycle and melanogenesis-associated proteins in p21-deficient cells. Loss of p21 resulted in reduced expression of p21, phosphorylated p21, p53, acetylated p53, CDK2, Cyclin D, Cyclin E, MITF, TRP-1, TRP-2, TYR, and p-ERK. Collectively, these findings indicate that p21 is essential for maintaining MITF-driven melanogenic signaling.},
}

*Microphthalmia-Associated Transcription Factor/metabolism/genetics
Animals
*Signal Transduction/genetics
*Cyclin-Dependent Kinase Inhibitor p21/genetics/metabolism
*CRISPR-Cas Systems/genetics
Mice
Melanogenesis
Gene Knockout Techniques/methods
Melanins/metabolism
Cell Line, Tumor

@article {pmid42284907,
year = {2026},
author = {Xiao, S and Zhou, L and Tian, Z and Zhou, S and Kong, J and Zhang, X},
title = {CRISPR-Cas12a-regulated photo-ATRP for ultrasensitive detection of lipopolysaccharide.},
journal = {Food chemistry},
volume = {522},
number = {},
pages = {149992},
doi = {10.1016/j.foodchem.2026.149992},
pmid = {42284907},
issn = {1873-7072},
mesh = {*Lipopolysaccharides/analysis ; *Biosensing Techniques/methods/instrumentation ; *CRISPR-Cas Systems ; Limit of Detection ; *Bacterial Proteins/genetics/chemistry ; Food Contamination/analysis ; *Electrochemical Techniques/methods ; *CRISPR-Associated Proteins/chemistry/genetics ; *Endodeoxyribonucleases/chemistry/genetics ; Beverages/analysis ; },
abstract = {Lipopolysaccharide (LPS), a primary virulence factor produced by Gram-negative bacteria, demands rapid and ultrasensitive detection for safeguarding food safety and supporting clinical diagnosis. This study developed a novel electrochemical biosensing platform by integrating the programmable recognition of CRISPR-Cas12a with the high-gain signal amplification of photoinduced ATRP. In this mechanism, the binding of LPS to its aptamer regulated Cas12a activity, which controlled an N3-DNA initiator probe. This probe, in turn, guided in-situ photopolymerization via click chemistry, generating a dramatically enhanced signal. The sensor exhibited a linear range from 10 fg/mL to 1 ng/mL with a detection limit of 2.48 fg/mL, along with high selectivity, reproducibility, and stability. Tests in spiked beverage samples showed high recovery rates and strong anti-interference capability. This work not only achieves ultrasensitive LPS detection but also provides a new approach for extending CRISPR-based biosensing to non-nucleic acid targets.},
}

*Lipopolysaccharides/analysis
*Biosensing Techniques/methods/instrumentation
*CRISPR-Cas Systems
Limit of Detection
*Bacterial Proteins/genetics/chemistry
Food Contamination/analysis
*Electrochemical Techniques/methods
*CRISPR-Associated Proteins/chemistry/genetics
*Endodeoxyribonucleases/chemistry/genetics
Beverages/analysis

@article {pmid42297231,
year = {2026},
author = {Fatima, SW},
title = {Cellular plasticity and epigenetic instability in cancer: Mechanistic insights and functional dissection with CRISPR-based epigenome editing.},
journal = {Cancer letters},
volume = {656},
number = {},
pages = {218679},
doi = {10.1016/j.canlet.2026.218679},
pmid = {42297231},
issn = {1872-7980},
mesh = {Humans ; *Epigenome Editing ; *Cell Plasticity/genetics ; *Neoplasms/genetics/pathology/therapy ; *Epigenesis, Genetic ; Tumor Microenvironment/genetics ; *CRISPR-Cas Systems ; Animals ; Gene Expression Regulation, Neoplastic ; DNA Methylation ; },
abstract = {Cellular plasticity is a fundamental driver of tumor heterogeneity, cancer stemness, immune evasion, therapeutic resistance, and disease progression. In malignancies such as breast cancer and glioblastoma, tumor cells undergo reversible phenotypic transitions between proliferative, stem-like, invasive, and drug-tolerant states in response to intrinsic regulatory programs and extrinsic signals from the tumor microenvironment. These adaptive dynamics are governed by complex interactions among signaling pathways, transcriptional networks, chromatin remodeling, DNA methylation, histone modifications, non-coding RNAs, and immune-mediated microenvironmental cues. Such epigenetic instability enables stochastic and therapy-induced shifts between alternative cellular states, thereby contributing to tumor evolution, metastasis, resistance to targeted therapies, and variable responses to immunotherapy. Understanding the mechanisms that govern epigenetic plasticity remains a central challenge in cancer biology. Recent advances in CRISPR/dCas9-based epigenome editing have provided powerful experimental tools for investigating the functional consequences of locus-specific chromatin modifications without altering the underlying DNA sequence. Catalytically inactive Cas9 (dCas9) fused to epigenetic effector domains, including DNMT3A, TET1, KRAB, and p300, enables targeted modulation of gene expression programs implicated in cell-state transitions, lineage specification, and adaptive resistance. These technologies offer a versatile platform for interrogating causal relationships between chromatin states and cellular phenotypes and for modeling mechanisms of tumor adaptation. This review examines the molecular basis of epigenetic plasticity in cancer, evaluates current CRISPR-based epigenome editing strategies, and discusses their application in studying tumor heterogeneity, microenvironment-driven adaptation, immune escape, and therapy resistance. This study highlights emerging opportunities and persistent challenges associated with epigenome editing, including delivery barriers, durability of epigenetic modifications, context-dependent biological responses, and translational limitations. Collectively, these approaches provide valuable experimental frameworks for dissecting the regulatory logic of cancer cell plasticity while informing future therapeutic development.},
}

Humans
*Epigenome Editing
*Cell Plasticity/genetics
*Neoplasms/genetics/pathology/therapy
*Epigenesis, Genetic
Tumor Microenvironment/genetics
*CRISPR-Cas Systems
Animals
Gene Expression Regulation, Neoplastic
DNA Methylation

@article {pmid42303162,
year = {2026},
author = {Durairaj, S and Durairaj, S and Krishnan, S and Raju, A},
title = {CRISPR Cas9 revolutionizing genetic engineering and therapeutic applications.},
journal = {Journal of biotechnology},
volume = {417},
number = {},
pages = {246-264},
doi = {10.1016/j.jbiotec.2026.06.012},
pmid = {42303162},
issn = {1873-4863},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods ; *Gene Editing/methods ; Animals ; *Genetic Engineering/methods ; },
abstract = {Genetic engineering has been transformed by CRISPR-Cas9 technology, offering high precision and adaptability in biological research and therapeutic innovation. Originating from a bacterial defense system, CRISPR-Cas9 enables targeted DNA editing through guide RNA-directed Cas9 nuclease activity, allowing gene modification, mutation correction, and disease mechanism analysis. This has opened new avenues in personalized medicine and gene therapy, particularly for cancer and inherited disorders, alongside applications in agriculture. In oncology, CRISPR-Cas9 demonstrates strong potential in oncogene targeting, immune cell engineering, and CAR-T-based immunotherapy, supported by substantial preclinical success. Delivery efficiency is enhanced through systems such as exosomes, liposomes, and nanoparticles, improving stability and tumor targeting. However, clinical translation remains constrained by off-target effects, delivery limitations, and ethical concerns in human genome editing, particularly germline modification. CRISPR shows therapeutic promise for muscular dystrophy, sickle cell disease, and cystic fibrosis. Emerging platforms including base editing, prime editing, and dCas9-based epigenome editing enable precise genome and gene regulation without double-strand breaks, reducing toxicity and expanding therapeutic scope in cancer and genetic diseases. Regulatory frameworks remain heterogeneous, affecting translation. The United States leads in approvals and clinical progress, the European Union emphasizes safety and ethics, China shows rapid expansion in clinical trials, and India remains in early stages due to regulatory and infrastructure constraints. Public perception influences adoption, shaped by misinformation and limited awareness. Persistent gaps in long-term safety, clinical efficacy, and population diversity remain challenges. Overall, CRISPR-Cas9 represents a transformative but carefully regulated platform for advancing biotechnology and medicine.},
}

Humans
*CRISPR-Cas Systems/genetics
*Genetic Therapy/methods
*Gene Editing/methods
Animals
*Genetic Engineering/methods

@article {pmid42398757,
year = {2026},
author = {Wang, X and Dong, W and Shen, R and Yu, X and Zhang, Y and Wang, W and Yin, X and Hu, Y and Peng, X and Yang, G and Rao, Q and Deng, X and Wang, R and Tang, F and Huang, Y and Jin, Z and Cai, Q and Xu, H and Tang, Y and Du, D},
title = {Development of optimized fluorogenic DNA aptamers for a portable one-pot CRISPR-Cas12a platform for rapid and sensitive detection of monkeypox virus and chikungunya virus.},
journal = {Journal of advanced research},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jare.2026.07.003},
pmid = {42398757},
issn = {2090-1224},
abstract = {INTRODUCTION: The recent global outbreaks of monkeypox virus (MPXV) and chikungunya virus (CHIKV) underscore the urgent need for rapid, accessible, and cost-effective diagnostic methods. Conventional CRISPR/Cas fluorescence assays rely on trans-cleavage of ssDNA/RNA reporters labeled with expensive fluorophores and quenchers, which limits widespread application.

OBJECTIVES: This study aims to develop and optimize a label-free, fluorogenic DNA aptamer-based reporter for a portable, one-pot Cas12a detection system capable of highly sensitive detection of MPXV and CHIKV directly from clinical specimens.

METHODS: We evaluated commonly used ssDNA aptamers for their fluorescence emission upon Thioflavin T (ThT) binding and their cleavage efficiency by Cas12a. Through systematic mutagenesis targeting G-rich regions, we enhanced fluorescence emission. Additionally, poly-A linkers were introduced between G-rich motifs to promote Cas12a cleavage efficiency. Circular dichroism (CD) spectroscopy confirmed G-quadruplex (G4) formation in the aptamers. The assay's sensitivity and specificity were assessed using simulated clinical samples, followed by validation with actual clinical specimens. The performance of direct detection from simulated clinical samples was compared to qRT-PCR. A battery-powered heating-pad, a mini-centrifuge, and a flashlight were used to validate its POCT applicability.

RESULTS: We designed and optimized a cost-effective, stable fluorogenic ssDNA aptamer that specifically binds to ThT. The aptamer ThT-3-5.1 exhibited the highest fluorescence enhancement and cleavage efficiency by Cas12a. Leveraging this aptamer, we developed a rapid, portable, one-pot detection platform (ROD-ThT) capable of detecting as few as 1 copy/reaction of MPXV and CHIKV nucleic acids within 35 min. Validation with clinical samples confirmed the assay's reliability without the need for nucleic acid purification.

CONCLUSION: Our simple, efficient, portable, and affordable ROD-ThT platform holds great promise for disease diagnostics and management, particularly in resource-limited settings.},
}

@article {pmid42399052,
year = {2026},
author = {Kim, J and Kovacs, H and Wisnovsky, S},
title = {CRISPR screens to identify and characterize ligands for glycan-binding proteins.},
journal = {Methods in enzymology},
volume = {732},
number = {},
pages = {219-263},
doi = {10.1016/bs.mie.2026.03.005},
pmid = {42399052},
issn = {1557-7988},
mesh = {Ligands ; *Polysaccharides/metabolism ; Humans ; *Lectins/metabolism/genetics ; Flow Cytometry/methods ; *CRISPR-Cas Systems ; Protein Binding ; HEK293 Cells ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Cell surface glycans regulate key biological processes including immune signaling, cell communication, and pathogen recognition. Glycan-driven signaling is primarily mediated by glycan-binding proteins (lectins), whose functions depend on the identity and presentation of their glycoprotein ligands. However, identifying ligands for glycan-binding proteins remains challenging due to the structural complexity of carbohydrates and the importance of cellular context in determining binding specificity. Here, we describe a fluorescence-activated cell sorting (FACS)-based pooled CRISPR screening workflow for systematic identification of genetic factors that regulate lectin binding in living cells. The protocol covers lentiviral transduction of pooled sgRNA libraries and phenotypic selection of high- and low-lectin-binding populations by flow cytometry. Genomic DNA extraction, sequencing, and computational sgRNA enrichment analysis enable identification of genes influencing ligand biosynthesis and presentation. Subsequent analysis of these genetic factors can provide a comprehensive view of the structural determinants that govern lectin-glycan binding. The approach is compatible with CRISPR knockout, interference, and activation strategies, allowing broad interrogation of both loss- and gain-of-function effects. Key considerations for maintaining library coverage, optimizing sorting parameters, and performing robust statistical analysis are highlighted to maximize screening performance. Overall, this workflow offers a scalable framework for mapping glycan ligand landscapes in health and disease.},
}

Ligands
*Polysaccharides/metabolism
Humans
*Lectins/metabolism/genetics
Flow Cytometry/methods
*CRISPR-Cas Systems
Protein Binding
HEK293 Cells
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42399613,
year = {2026},
author = {Wei, R and Wang, S and Li, Y and Li, N and Pan, W and Tang, B},
title = {DNAzyme-Enhanced CRISPR/Cas12a Cascade Enables Isothermal, One-Pot RNA Diagnostics.},
journal = {ACS applied materials & interfaces},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsami.6c05456},
pmid = {42399613},
issn = {1944-8252},
abstract = {While integrating DNAzymes with Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas systems offers a promising route to enhance CRISPR/Cas12a-based molecular diagnosis via enzyme-coupled cascade amplification, their implementation in simple, specific, and sensitive nucleic acid detection remains challenging, largely due to reliance on complex, multistep workflows. Here, we report an RNA-triggered DNAzyme circuit integrated with CRISPR/Cas12a that serves as a universal nucleic acid preamplifier, enabling one-pot and homogeneous detection. The catalytic activity of DNAzyme, initially suppressed by a complementary blocker strand, was restored upon the recognition of the target analyte. The activated DNAzyme then cleaved a hairpin-shaped substrate, liberating multiple activators that triggered a secondary CRISPR/Cas amplification reaction. This cascade generated a visible red band signal on a lateral flow assay via the collateral cleavage of a reporter. By employing the DNAzyme as a signal amplifier, the system efficiently converted a single RNA molecule into numerous initiators, breaking the one-to-one activation relationship between the target and Cas12a ribonucleoprotein and thereby greatly enhancing the detection sensitivity. Additionally, the system exhibited high programmability and universality, as a biosensor for a given target could be easily constructed by simply customizing the corresponding region of the blocker strand that is complementary to the target sequence. This integrated cascade system enables efficient signal amplification within a simple one-pot format and holds significant promise for practical applications.},
}

@article {pmid42400295,
year = {2026},
author = {Liang, D and Guo, H and Wei, J and Zhu, F and Zhang, Y and Green, J and Ji, Y and Jin, H and Zhang, X and Gui, H and Dan, H and Liu, Y and Zhang, Y and Wang, H and Jiang, Y and Geng, L and Lv, J and Cai, W and Song, W and Kelliher, T and Chen, X and Egger, R},
title = {A Robust Framework for Maize Elite Line Genome Editing Through Enhanced HI-Edit via LbCas12a Activity Optimization.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.70715},
pmid = {42400295},
issn = {1467-7652},
abstract = {Haploid induction coupled with genome editing (HI-Edit) enables direct modification of commercial crop varieties, bypassing the need for trait introgression or direct transformation of elite lines with CRISPR machinery. However, its widespread application has been constrained by low haploid editing rates (HER), the proportion of haploids carrying edits within the short window between double fertilization and uniparental chromosome elimination. Here, we report substantial improvements in maize HI-Edit efficiency through three complementary strategies: (1) driving an optimized LbCas12a variant (LbCas12aV) using promoters that are highly active in sperm cells and early zygotes; (2) applying a post-pollination heat treatment; and (3) fusing LbCas12aV with the UBA2 domain (ubiquitin-associated domain-2 of Arabidopsis thaliana RAD23) to enhance protein stability during haploid induction. Post-pollination heat treatment alone increased HER to 19.1% (up to 12-fold improvement depending on the target site), providing a simple and effective method to boost the yield of edited doubled haploid (DH) plants. UBA2 fusion improved HER by 6-fold at the Waxy1 (Wx1) locus and 4.5-fold at the Glossy2 (Gl2) locus under normal conditions. Strikingly, combining UBA2 fusion with heat treatment raised the average HER to 25% across multiple events targeting Wx1, with the highest HER reaching 33%. Collectively, these findings demonstrate that increasing CRISPR-Cas protein abundance and modulating environmental conditions can overcome key bottlenecks in HI-Edit. We establish a robust, scalable framework that is readily transferable to other crops for elite-line genome editing.},
}

@article {pmid42400407,
year = {2026},
author = {Wan, L and Zhou, J and Yu, L and Huang, X and Fu, J and Xiao, F and Jia, N and Zhang, Y and Chen, M and Feng, Z and Wang, Y},
title = {A One-Pot CRISPR-Cas12b Assay for Rapid Detection of Human Adenovirus Serotypes 3 and 7.},
journal = {Journal of medical virology},
volume = {98},
number = {7},
pages = {e71032},
doi = {10.1002/jmv.71032},
pmid = {42400407},
issn = {1096-9071},
support = {7262007//Beijing Natural Science Foundation/ ; L234047//Beijing Natural Science Foundation-Changping Innovation Joint Fund Project/ ; XZDX-2025-002//Research Foundation of Capital Institute of Pediatrics/ ; BJRID2025-009//Beijing research center for respiratory infectious diseases project/ ; BJRID2026-011//Beijing research center for respiratory infectious diseases project/ ; 2024-0040//Pathogen spectrum and host marker analysis in respiratory tract infection of children/ ; MEKLCEPP/SXMU-202412//Key Laboratory of Coal Environmental Pathogenicity and Prevention (Shanxi Medical University), Ministry of Educatio/ ; },
mesh = {Humans ; *Adenoviruses, Human/isolation & purification/genetics/classification ; *CRISPR-Cas Systems ; *Adenovirus Infections, Human/diagnosis/virology ; Serogroup ; Sensitivity and Specificity ; Rapid Diagnostic Tests ; *Nucleic Acid Amplification Techniques/methods ; China ; *Molecular Diagnostic Techniques/methods ; Respiratory Tract Infections/virology/diagnosis ; },
abstract = {Human adenovirus (HAdV) is a leading cause of acute respiratory tract infections (ARTIs) in children. The high prevalence of HAdV serotypes 3 and 7 in regions such as China presents a significant public health challenge. Here, we propose a one-pot assay that integrates multiple cross displacement amplification (MCDA) with CRISPR-Cas12b for the detection of HAdV-3 and HAdV-7, termed HAdV-MCDA-One. In this system, MCDA provides exponential target amplification, while the collateral cleavage activity of Cas12b enables secondary signal amplification. The entire reaction is performed isothermally at 60°C in a single tube, providing a fluorescent readout within 50 min, making the assay suitable for point-of-care testing (POCT). Leveraging the single-base recognition capability of CRISPR-Cas12b, the assay demonstrates high specificity, with no cross-reactivity observed against the other 13 identified pathogens. The limit of detection was determined to be 1.59 copies per reaction using target plasmids. Moreover, when evaluated with 96 clinical pharyngeal swabs, the assay showed 100% concordance with quantitative PCR (qPCR), confirming its clinical reliability. These results demonstrate HAdV-MCDA-One as a rapid and robust tool for HAdV-3 and HAdV-7 detection, with significant potential for clinical diagnosis and public health surveillance.},
}

Humans
*Adenoviruses, Human/isolation & purification/genetics/classification
*CRISPR-Cas Systems
*Adenovirus Infections, Human/diagnosis/virology
Serogroup
Sensitivity and Specificity
Rapid Diagnostic Tests
*Nucleic Acid Amplification Techniques/methods
China
*Molecular Diagnostic Techniques/methods
Respiratory Tract Infections/virology/diagnosis

@article {pmid42400607,
year = {2026},
author = {Liu, F and Jiang, T and Tanwir, SE and Ardi, WH and Huo, H},
title = {CRISPR/Cas9-mediated DFR disruption suggests coordinated changes in flavonoid flux and development in Petunia × hybrida.},
journal = {Plant cell reports},
volume = {45},
number = {7},
pages = {},
pmid = {42400607},
issn = {1432-203X},
support = {2019-67013-29236//National Institute of Food and Agriculture/ ; FLA-MFC-006387//USDA HATCH/ ; },
mesh = {*Petunia/genetics/metabolism/growth & development/enzymology ; *Flavonoids/metabolism ; *CRISPR-Cas Systems/genetics ; *Alcohol Oxidoreductases/genetics/metabolism ; Gene Expression Regulation, Plant ; Anthocyanins/metabolism ; *Plant Proteins/genetics/metabolism ; Flowers/metabolism/genetics ; Pigmentation/genetics ; Phenotype ; Plant Leaves/metabolism/genetics ; Plants, Genetically Modified ; Chlorophyll/metabolism ; Carotenoids/metabolism ; Oxidoreductases/metabolism ; },
abstract = {Loss of DFR function in petunia alters pigment metabolism and reduces organ size, suggesting previously underexplored associations among flavonoid biosynthesis, plastidial pigments, and development. Dihydroflavonol 4-reductase (DFR) occupies a critical branch point in flavonoid metabolism, channeling dihydroflavonol substrates toward anthocyanin biosynthesis in competition with flavonol synthase. While DFR's role in floral pigmentation is well established, the broader physiological and transcriptional consequences of its disruption remain poorly characterized, particularly in commercially important ornamental species. Here, we report the generation and comprehensive phenotyping of five independent CRISPR/Cas9-mediated DFR-edited lines in the commercial Petunia × hybrida cultivar 'Carmine Velour'. The edited lines showed a spectrum of floral pigmentation loss that was broadly consistent with the representative editing patterns inferred from Sanger sequencing, supporting the major contribution of DFR-A to corolla anthocyanin accumulation. Beyond pigmentation, dfr mutants exhibited unexpected reductions in floral dimensions (20-40%), leaf biomass (30-50%), and plastidial pigment content, with chlorophyll and carotenoid levels declining 35-60% in petals despite unchanged leaf anthocyanins. Stem anatomy remained unaffected, indicating organ-specific associations between DFR disruption and growth-related traits. Transcriptional profiling uncovered feedback reprogramming within the flavonoid pathway: chalcone synthase A (CHSA) and chalcone isomerase A (CHIA) were downregulated while the competing branch enzyme flavonol synthase (FLS) was upregulated almost twofold, consistent with the possibility of altered flux partitioning toward flavonol biosynthesis. Strikingly, protochlorophyllide oxidoreductase A (PORA), encoding a key chlorophyll biosynthetic enzyme, was severely suppressed by 60-75%, suggesting a possible connection between flavonoid disruption and tetrapyrrole metabolism. Correlation analyses suggested coordinated variation, with floral anthocyanin content positively associated with leaf chlorophyll and carotenoid levels across genotypes. These findings support the view that DFR acts as a functionally important metabolic node whose disruption is associated with effects across pigment classes and organ types, with implications for precision trait engineering in floriculture.},
}

*Petunia/genetics/metabolism/growth & development/enzymology
*Flavonoids/metabolism
*CRISPR-Cas Systems/genetics
*Alcohol Oxidoreductases/genetics/metabolism
Gene Expression Regulation, Plant
Anthocyanins/metabolism
*Plant Proteins/genetics/metabolism
Flowers/metabolism/genetics
Pigmentation/genetics
Phenotype
Plant Leaves/metabolism/genetics
Plants, Genetically Modified
Chlorophyll/metabolism
Carotenoids/metabolism
Oxidoreductases/metabolism

@article {pmid42400669,
year = {2026},
author = {Tamrakar, VK and Sharma, K and Singh, P and Bhargava, A and Thakur, P and Negi, SS},
title = {Development of a CRISPR/dCas9-based membrane-assisted colorimetric assay for detection of high-risk HPV16 and HPV18: a proof-of-concept study.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42400669},
issn = {1573-4978},
support = {Extramural Grant IIPR-2030-3960/F1.//Indian Council of Medical Research/ ; },
mesh = {*Colorimetry/methods ; *Human papillomavirus 18/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; *Human papillomavirus 16/genetics/isolation & purification ; Humans ; *Papillomavirus Infections/diagnosis/virology ; Proof of Concept Study ; Genotype ; Female ; Sensitivity and Specificity ; Rapid Diagnostic Tests ; Genotyping Techniques/methods ; },
abstract = {BACKGROUND AND AIMS: Persistent infection with high-risk human papillomavirus (HR-HPV), particularly HPV16 and HPV18, is the leading cause of cervical cancer. While molecular diagnostics offer high sensitivity, their deployment in decentralized settings remains limited. This study presents a proof-of-concept CRISPR/dCas9-based membrane-assisted detection platform for HR-HPV genotyping.

METHODS: A membrane-based assay integrating recombinase polymerase amplification (RPA) with CRISPR/dCas9-mediated sequence-specific recognition was developed. FAM-labeled amplicons were captured by immobilized dCas9-sgRNA ribonucleoprotein complexes and detected via antibody-mediated colorimetric readout.

RESULTS: The assay enabled specific detection of HPV16 and HPV18 using genotype-specific sgRNAs, producing visually interpretable signals on a nitrocellulose membrane. No signal was observed in negative controls, demonstrating high analytical specificity. Semi-quantitative signal assessment confirmed clear differentiation between positive and negative samples.

CONCLUSION: This study demonstrates the feasibility of a CRISPR/dCas9-based membrane-assisted detection system for HR-HPV genotyping. While not yet configured as a fully integrated lateral flow device, the platform provides a foundation for future development of simplified, point-of-care molecular diagnostics.},
}

*Colorimetry/methods
*Human papillomavirus 18/genetics/isolation & purification
*CRISPR-Cas Systems/genetics
*Human papillomavirus 16/genetics/isolation & purification
Humans
*Papillomavirus Infections/diagnosis/virology
Proof of Concept Study
Genotype
Female
Sensitivity and Specificity
Rapid Diagnostic Tests
Genotyping Techniques/methods

@article {pmid41981786,
year = {2026},
author = {Munir, F and Zaheer, U and Asad, M and Yang, G},
title = {Knockout of Ku70 and Ku80 elevates homology-directed repair efficiency in Plutella xylostella.},
journal = {Insect molecular biology},
volume = {35},
number = {4},
pages = {434-447},
doi = {10.1111/imb.70039},
pmid = {41981786},
issn = {1365-2583},
support = {2024L3004//Fujian Province Science and Technology Project/ ; 2024-P-002//Fuzhou-Xiamen-Quanzhou National Independent Innovation Demonstration Zone RNAi Microbial Pesticide Collaborative Innovation Platform Project/ ; 2024I0007//the External Cooperation Project of Fujian Province/ ; KRA16001A//fund of the "111" program/ ; 2023J02009//Fujian Province Natural Science Foundation/ ; W2533068//Overseas Young Researcher Project of National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Moths/genetics/metabolism ; *Ku Autoantigen/genetics/metabolism ; Female ; Male ; Phylogeny ; Amino Acid Sequence ; *Insect Proteins/genetics/metabolism ; Gene Knockout Techniques ; *Recombinational DNA Repair ; CRISPR-Cas Systems ; Sequence Alignment ; },
abstract = {The suppression of Ku70 and Ku80 has been verified to increase homology-directed repair (HDR) efficiency in fruit fly, silkworm and mosquito, but not in other insects. In this study, PxKu70 and PxKu80 were identified from the Plutella xylostella genome. Domain analysis revealed that PxKu70 contained three conserved domains: Ku N, Ku and Ku C, while PxKu80 comprised the Ku N, Ku and Ku PK bind domains. Phylogenetic analysis and multiple-sequence alignment indicated strong conservation of both proteins among lepidopteran insects. RT-qPCR analysis showed that PxKu70 and PxKu80 were highly expressed in adult stages, particularly in reproductive tissues such as the ovary and testis, suggesting their role in maintaining genomic stability during gametogenesis. Two homozygous knockout lines (ΔPxKu70 and ΔPxKu80) were successfully generated through CRISPR/Cas9-mediated genome editing. These knockout lines remained viable and fertile without observable fitness effects. A donor construct carrying an EGFP cassette designed for insertion at the PxKmo locus was generated to assess HDR-mediated integration. The HDR insertion rate was significantly elevated in both knockout lines compared with the wild-type. These findings demonstrate that suppression of either PxKu70 or PxKu80 can enhance HDR in P. xylostella, offering an effective approach for precise genome editing in lepidopteran species.},
}

Animals
*Moths/genetics/metabolism
*Ku Autoantigen/genetics/metabolism
Female
Male
Phylogeny
Amino Acid Sequence
*Insect Proteins/genetics/metabolism
Gene Knockout Techniques
*Recombinational DNA Repair
CRISPR-Cas Systems
Sequence Alignment

@article {pmid42062284,
year = {2026},
author = {Sun, J and Noss, S and Smolen, C and Bhavana, VH and Banerjee, D and Das, M and Giardine, B and Prabhu, A and Amor, DJ and Pope, K and Lockhart, PJ and Girirajan, S},
title = {Functional impact of genetic background on variable expressivity in neurodevelopmental disorders.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42062284},
issn = {2041-1723},
support = {R01 GM121907/GM/NIGMS NIH HHS/United States ; R21 NS122398/NS/NINDS NIH HHS/United States ; GM121907//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; NS122398//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; },
mesh = {*Neurodevelopmental Disorders/genetics ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Forkhead Transcription Factors/genetics/metabolism ; Phenotype ; Neurons/metabolism ; Chromatin/metabolism ; Signal Transduction/genetics ; Neural Stem Cells/metabolism ; Neurodevelopment ; Cell Proliferation ; CRISPR-Cas Systems ; Animals ; },
abstract = {Disease-associated variants can lead to variable phenotypic outcomes in neurodevelopmental disorders, but the biological mechanisms underlying this variability remain poorly understood. Here, we develop a framework to investigate this phenomenon using the 16p12.1 deletion as a paradigm of variable expressivity. Using induced pluripotent stem cell models from affected families and CRISPR-edited lines with the 16p12.1 deletion, we find that the deletion and rare variants in the genetic background jointly influence chromatin accessibility and expression of neurodevelopmental genes. Cellular analyses identify family-specific phenotypes, including altered inhibitory neuron production and neural progenitor cell proliferation, which correlate with head-size variation. CRISPR activation of individual 16p12.1 genes variably rescue these defects by modulating key developmental signaling pathways. Integrative analyses further identify regulatory hubs, including transcription factors FOXG1 and JUN, as mediators of these effects. Our study provides a functional framework for investigating how individual genetic architectures contribute to phenotypic variability in neurodevelopmental disorders.},
}

*Neurodevelopmental Disorders/genetics
Humans
Induced Pluripotent Stem Cells/metabolism
Forkhead Transcription Factors/genetics/metabolism
Phenotype
Neurons/metabolism
Chromatin/metabolism
Signal Transduction/genetics
Neural Stem Cells/metabolism
Neurodevelopment
Cell Proliferation
CRISPR-Cas Systems
Animals

@article {pmid42091648,
year = {2026},
author = {Chandrasekaran, J and Suthanthiram, B and Selvaraj, EP and Swaminathan, S and Chandran, SA and Ramasamy, S},
title = {Targeting a conserved functional motif in the PDS gene enables efficient CRISPR/Cas9 editing in banana.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42091648},
issn = {2045-2322},
support = {DST/WOS-A/LS-202/2021(G)//Department of Science and Technology, Ministry of Science and Technology, India/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; *Musa/genetics ; *Oxidoreductases/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Base Sequence ; *Gene Editing ; Conserved Sequence ; },
abstract = {Incomplete editing and chimeric phenotypes are major challenges in CRISPR/Cas9-mediated genome editing of polyploid crops. In this study, a single guide RNA (gRNA) was designed to target a conserved dinucleotide-binding motif within exon 3 of the phytoene desaturase (PDS) gene in 'Grand Naine' banana. The gRNA was carefully selected for GC content, guanine residues near the PAM, and predicted secondary structure to enhance Cas9 cleavage efficiency. Agrobacterium-mediated transformation of embryonic cell suspensions produced 102 putative transgenic plants, all exhibiting altered phenotypes, with 91% displaying albino and 9% pale green coloration, indicating efficient PDS gene knockout and absence of chimerism. Sequencing confirmed tri-allelic editing, with all edited plants consistently showing two identical and one distinct mutation. Notably, small in-frame deletions of two to six amino acids within the conserved motif were sufficient to abolish PDS function, confirming its critical role in carotenoid biosynthesis. This strategy is adaptable to clonally propagated polyploid crops, providing a practical framework for achieving high-efficiency, uniform genome edits and supporting the development of precise, non-chimeric CRISPR/Cas9 editing approaches.},
}

*CRISPR-Cas Systems/genetics
Plants, Genetically Modified/genetics
*Musa/genetics
*Oxidoreductases/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
Base Sequence
*Gene Editing
Conserved Sequence

@article {pmid42229614,
year = {2026},
author = {Kalmotia, V},
title = {Enhancing statistical accuracy in gene perturbation studies.},
journal = {Bio Systems},
volume = {266},
number = {},
pages = {105819},
doi = {10.1016/j.biosystems.2026.105819},
pmid = {42229614},
issn = {1872-8324},
mesh = {Humans ; *Gene Expression Profiling/methods ; *Single-Cell Analysis/methods ; High-Throughput Nucleotide Sequencing/methods ; CRISPR-Cas Systems/genetics ; Genomics/methods ; },
abstract = {Accurately analysing gene expression changes in high-throughput perturbation studies remains a challenge due to confounding technical factors. This paper evaluates and extends the SCEPTRE (Single-Cell PerTurbation screens via Conditional REsampling) framework, originally introduced by Barry et al. (2021), demonstrating its applicability to high-multiplicity-of-infection (MOI) CRISPR screens. By leveraging a resampling-based methodology, our approach effectively adjusts for sequencing biases, reducing false discoveries while maintaining statistical power.},
}

Humans
*Gene Expression Profiling/methods
*Single-Cell Analysis/methods
High-Throughput Nucleotide Sequencing/methods
CRISPR-Cas Systems/genetics
Genomics/methods

@article {pmid42335458,
year = {2026},
author = {Yang, L and Zhou, Y and Li, H and Chen, X and Zhang, S and Zhu, L},
title = {An Entropy-Driven Autocatalysis-Regulated Signal-On CRISPR/Cas12a Biosensor Supported by DNA Triangular Prism Scaffold.},
journal = {Analytical chemistry},
volume = {98},
number = {26},
pages = {19636-19651},
doi = {10.1021/acs.analchem.6c01471},
pmid = {42335458},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; *DNA/chemistry ; *Entropy ; *MicroRNAs/blood/analysis ; *CRISPR-Cas Systems/genetics ; *alpha-Fetoproteins/analysis ; Humans ; Electrochemical Techniques ; Limit of Detection ; DNA Nanostructures ; Carcinoma, Hepatocellular/diagnosis/blood ; },
abstract = {Accurate and sensitive detection of low-abundance biomarkers in complex matrices remains challenging due to the inherent trade-off between amplification efficiency and background suppression in conventional biosensing strategies. Herein, a synergistic amplification strategy was constructed by integrating an entropy-driven autocatalysis (EDAC), a signal-on CRISPR/Cas12a assay, and a DNA triangular prism (DTP) interface. In this strategy, EDAC achieved exponential signal amplification through the recycling of target molecules and reaction byproducts, and its output strands simultaneously served as specific inhibitors of CRISPR/Cas12a. Based on this mechanism, the signal-on CRISPR/Cas12a assay strictly coupled signal generation to the presence of the target, thereby fundamentally circumventing the high background interference inherent to conventional signal-off modes. As a rigid three-dimensional interfacial scaffold, DTP provided high-density and well-ordered nucleic acid assembly sites, reduced steric hindrance through a solution-like microenvironment, suppressed nonspecific adsorption, and efficiently initiated downstream hybridization chain reaction for robust electrochemical readout via methylene blue intercalation. With hepatocellular carcinoma-associated biomarkers alpha-fetoprotein and microRNA-122 as model targets, the biosensor achieved detection limits as low as 11.37 fg/mL and 18.13 aM, respectively. In clinical serum sample assays, the biosensor showed strong agreement with the classical ELISA method, with an area under the curve value of 1.00, demonstrating its promising potential for the diagnosis of hepatocellular carcinoma. With its modular architecture and adaptable recognition elements, this strategy establishes a versatile framework for ultrasensitive biosensing and holds promise for clinical translation in early disease diagnosis.},
}

*Biosensing Techniques/methods
*DNA/chemistry
*Entropy
*MicroRNAs/blood/analysis
*CRISPR-Cas Systems/genetics
*alpha-Fetoproteins/analysis
Humans
Electrochemical Techniques
Limit of Detection
DNA Nanostructures
Carcinoma, Hepatocellular/diagnosis/blood

@article {pmid42342147,
year = {2026},
author = {Yuan, J and Ma, Y and Li, J and Chen, X and Wen, Y and Wu, R and Hang, X and Huang, H and Du, S and Wang, Y and Yan, QG and Huang, X and Zhao, F and Yi, Z and Cao, SJ and Zhao, Q},
title = {CXCL8 is associated with aflatoxin B1-triggered injury and caspase-3 activation in porcine kidney epithelial PK15 cells: integrated transcriptomics and CRISPR/Cas9 knockout.},
journal = {Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association},
volume = {215},
number = {},
pages = {116236},
doi = {10.1016/j.fct.2026.116236},
pmid = {42342147},
issn = {1873-6351},
mesh = {Animals ; *Aflatoxin B1/toxicity ; Swine ; *Caspase 3/metabolism/genetics ; *Interleukin-8/genetics/metabolism ; *Epithelial Cells/drug effects/metabolism ; Cell Line ; *Kidney/drug effects/cytology/metabolism ; CRISPR-Cas Systems ; Apoptosis/drug effects ; Gene Knockout Techniques ; *Transcriptome/drug effects ; Reactive Oxygen Species/metabolism ; },
abstract = {Aflatoxin B1 (AFB1) is a prevalent food- and feed-borne mycotoxin, and growing evidence indicates that the renal epithelium is a vulnerable target. However, host determinants that modify epithelial susceptibility remain poorly defined. Here, we investigated AFB1-triggered injury in porcine kidney epithelial PK15 cells and examined the contribution of CXCL8. PK15 cells were exposed to AFB1 (0-32 μM) to define dose-dependent cytotoxicity, and sub-IC50 conditions (4 and 8 μM for 24 h) were used for mechanistic analyses; RNA sequencing at 4 μM for 24 h was used as an exploratory screen to prioritize candidate susceptibility factors. CXCL8 emerged as the most strongly induced transcript and was subsequently evaluated using CRISPR/Cas9-mediated knockout. CXCL8 deficiency attenuated AFB1-induced loss of viability, reduced Annexin V/PI-positive cells, and alleviated mitochondrial ultrastructural injury. In parallel, CXCL8 knockout decreased ROS accumulation, partially restored intracellular GSH and the BCL2/BAX transcriptional ratio, and reduced caspase-3 induction and cleavage. Collectively, these data support CXCL8 as an AFB1-inducible susceptibility factor associated with oxidative stress amplification and caspase-3 activation in PK15 cells, while indicating that the upstream regulatory axis and the precise downstream signaling route require further validation in physiologically relevant renal models.},
}

Animals
*Aflatoxin B1/toxicity
Swine
*Caspase 3/metabolism/genetics
*Interleukin-8/genetics/metabolism
*Epithelial Cells/drug effects/metabolism
Cell Line
*Kidney/drug effects/cytology/metabolism
CRISPR-Cas Systems
Apoptosis/drug effects
Gene Knockout Techniques
*Transcriptome/drug effects
Reactive Oxygen Species/metabolism