@article {pmid41712125,
year = {2026},
author = {Saxena, S and Saxena, S and Gupta, D},
title = {Miniaturized CRISPR: Ultra Compact Systems for In Vivo Delivery and Portable Diagnostics.},
journal = {Annals of biomedical engineering},
volume = {54},
number = {9},
pages = {2859-2872},
pmid = {41712125},
issn = {1573-9686},
mesh = {Humans ; Animals ; *CRISPR-Cas Systems ; Miniaturization ; *Gene Editing/methods ; Point-of-Care Systems ; },
abstract = {Reduced-size CRISPR systems have become a possible remedy to the delivery and size constraints of the traditional SpCas9 (~ 1368 Å). Recently described small nucleases, including Cas12f (400-700 Å) or CasX (~ 980 Å), along with designed mini-Cas9 versions, can efficiently be used in vivo to edit cells as well as to perform point-of-care diagnostics because of their lower molecular weight and less complex structures. This review will sum up progress in compact Cas protein engineering, guide RNA optimization, and delivery vector miniaturization, and point to their influence in therapeutic gene editing and portable diagnostic platforms. We additionally cover the contemporary issues of interest, such as off-target activity, delivery barriers and regulatory requirements, and future opportunities provided through AI-assisted protein design and synthetic biology. The miniaturized CRISPR technology is bound to substantially transform the translational arena of gene editing and world diagnostics.},
}

Humans
Animals
*CRISPR-Cas Systems
Miniaturization
*Gene Editing/methods
Point-of-Care Systems

@article {pmid42504735,
year = {2026},
author = {Luong, LH and Stone, S and Bui, V and Bhattarai, B and Vu, MT and Aydin, EP and Craig, T},
title = {Lonvoguran ziclumeran: a CRISPR-CAS9-based gene therapy for the treatment of hereditary angioedema.},
journal = {Expert opinion on investigational drugs},
volume = {35},
number = {8},
pages = {545-553},
doi = {10.1080/13543784.2026.2710107},
pmid = {42504735},
issn = {1744-7658},
mesh = {Humans ; Animals ; *Genetic Therapy/methods/adverse effects ; *Angioedemas, Hereditary/therapy/genetics/physiopathology ; CRISPR-Cas Systems ; Gene Therapy Agents ; Gene Editing/methods ; Precision Medicine ; Plasma Kallikrein/genetics ; Nanoparticles ; },
abstract = {INTRODUCTION: Hereditary angioedema (HAE) is a rare genetic disorder characterized by recurrent swelling caused by dysregulation of the kallikrein-kinin pathway. Although current therapies effectively reduce attack frequency, treatment remains lifelong. Lonvoguran ziclumeran (Lonvo-z; NTLA-2002) is the first systemically administered in vivo CRISPR/Cas9 gene-editing therapy designed to provide durable suppression of plasma kallikrein through permanent disruption of the KLKB1 gene.

AREAS COVERED: This review summarizes the pathophysiology and current management of HAE, the development of Lonvo-z, its lipid nanoparticle delivery platform, and the technical advances enabling in vivo genome editing. Preclinical studies and clinical evidence, including early-phase trials and the Phase 3 HAELO study, are reviewed with emphasis on efficacy, safety and clinical implications.

EXPERT OPINION: Lonvo-z represents a major milestone in precision medicine and the clinical application of systemic genome editing. A single administration has produced sustained reductions in plasma kallikrein levels and HAE attack frequency. Although long-term follow-up is ongoing, current evidence supports its potential as the first one-time disease-modifying treatment for HAE and a landmark advance in CRISPR-based therapeutics.},
}

Humans
Animals
*Genetic Therapy/methods/adverse effects
*Angioedemas, Hereditary/therapy/genetics/physiopathology
CRISPR-Cas Systems
Gene Therapy Agents
Gene Editing/methods
Precision Medicine
Plasma Kallikrein/genetics
Nanoparticles

@article {pmid42621610,
year = {2026},
author = {Li, J and Zhang, H and Yu, H and Liang, P and Xu, S and Zhong, L and Fu, X and Zhang, Y and Wang, Y},
title = {Application of bacteriophages in the prevention and control of bacterial infectious diseases in animals.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1851321},
pmid = {42621610},
issn = {1664-302X},
abstract = {The global spread of antimicrobial resistance (AMR) has intensified the search for alternatives to conventional antibiotics in animal production systems. Bacteriophages can be engineered beyond narrow-spectrum antibacterial agents into multifunctional biological platforms that integrate direct killing, immune modulation, and antigen delivery. We summarize recent advances across livestock, poultry, and aquaculture, delineating mechanistic distinctions between lytic phage therapy, phage display-derived interventions, and engineered platforms including CRISPR-Cas-enabled theranostic systems. However, as detailed below, most evidence remains preclinical, and translational gaps are substantial. Unlike prior descriptive reviews, we analyze translational bottlenecks-host range constraints, pharmacokinetic limitations, regulatory fragmentation-and assess the existing research evidence for claimed advantages such as microbiota preservation and biofilm penetration while upfront acknowledging inconsistent experimental outcomes and inherent application limitations behind these beneficial effects. We conclude that realizing phages' therapeutic potential in veterinary medicine requires coordinated progress in synthetic biology, scalable manufacturing, and regulatory harmonization within a One Health framework.},
}

@article {pmid42624339,
year = {2026},
author = {Gao, R and Jin, H and Zhang, T and Zhang, H and Huang, P and Wang, H},
title = {CRISPR/Cas trans-cleavage activity in pathogen detection: research progress and innovations.},
journal = {Biotechnology advances},
volume = {93},
number = {},
pages = {109017},
doi = {10.1016/j.biotechadv.2026.109017},
pmid = {42624339},
issn = {1873-1899},
abstract = {The trans-cleavage activity of CRISPR/Cas systems has catalyzed significant progress in molecular diagnostics. Compared with traditional methods such as polymerase chain reaction (PCR) and its derivatives, CRISPR/Cas diagnostics are often credited with high specificity, portability, and visual readout. Among various CRISPR systems, CRISPR/Cas9, CRISPR/Cas12, and CRISPR/Cas13 have been extensively applied in pathogen detection owing to their distinct target-recognition and nucleic acid-cleavage mechanisms. In particular, Cas12- and Cas13-based systems exploit target-activated trans-cleavage activity for sensitive signal amplification, whereas Cas9-based diagnostic platforms generally rely on sequence-specific cis-cleavage. This review assesses the integrated CRISPR/Cas detection workflow from sample collection and processing through final result output, and systematically analyzes the intrinsic characteristics of Cas effector proteins with respect to target enrichment, reporter molecules, readout formats, sample background, and validation design. Based on a practical application-oriented framework, we analyzed the adaptability of various CRISPR/Cas systems in distinct scenarios, including point-of-care screening, quantitative laboratory testing, and multiplex pathogen identification. In addition, we highlight engineering innovations derived from mechanistic investigations of Cas9, Cas12, Cas13 and Class I CRISPR systems, discuss the specific diagnostic bottlenecks these effectors can resolve, and outline remaining challenges requiring further optimization prior to clinical translation.},
}

@article {pmid42624823,
year = {2026},
author = {Sun, J and Liu, M and Zheng, X and Ouyang, G and Chen, A and Qian, H},
title = {Partial deletion in the cuticular protein gene BmorCPR2 results in a body shape mutant in silkworm, Bombyx mori L. (Lepidoptera: Bombycidae).},
journal = {Journal of insect science (Online)},
volume = {26},
number = {4},
pages = {},
pmid = {42624823},
issn = {1536-2442},
support = {2023-YBNY-134//Key Industrial Chain Projects of Shaanxi Provincial/ ; 2023-JC-YB-188//Basic Research Programs of the Shaanxi Provincial Science and Technology Department/ ; CARS-18-ZJ0101//the China Agriculture Research System of MOF and MARA/ ; BE2020418//Key R & D plan of Jiangsu Province/ ; },
mesh = {Animals ; *Bombyx/genetics/growth & development/anatomy & histology ; *Insect Proteins/genetics/metabolism ; Larva/genetics/growth & development/anatomy & histology ; Mutation ; CRISPR-Cas Systems ; Sequence Deletion ; },
abstract = {The Bombyx mori L. (Lepidoptera: Bombycidae) is a significant economic insect used for silk production. A novel body shape mutant, stony^sunken (st^sk), that exhibits a sunken intersegmental membrane was isolated from the wild type of st^sk (WT-n08). Investigation indicated that the mutation had no significant effect on its growth and development. To elucidate the molecular mechanism underlying this body shape mutant, genetic analysis, positional cloning, and the CRISPR/Cas9 gene editing system were performed. Genetic analysis demonstrated that the mutant trait in st^sk is controlled by an autosomal recessive gene and follows Mendelian inheritance. Positional cloning showed that a putative cuticular protein gene, BmorCPR2 on chromosome 8, was the candidate gene. Sequencing analysis revealed partial deletion of BmorCPR2 exon 2 and intron 2 sequences occurred and subsequently resulted in the premature termination of gene expression. Knock-out of BmorCPR2 using the CRISPR/Cas9 gene editing system led to the sunken intersegmental membrane phenotype. These findings highlight the essential role of BmorCPR2 in silkworm cuticular formation, providing a foundation for further research on cuticular protein function.},
}

Animals
*Bombyx/genetics/growth & development/anatomy & histology
*Insect Proteins/genetics/metabolism
Larva/genetics/growth & development/anatomy & histology
Mutation
CRISPR-Cas Systems
Sequence Deletion

@article {pmid42624898,
year = {2026},
author = {Bindu, S and Ash, A and Sarkar, K},
title = {Advanced gene editing technologies for oncology mechanisms, applications, and clinical implementation.},
journal = {Cancer gene therapy},
volume = {},
number = {},
pages = {},
pmid = {42624898},
issn = {1476-5500},
support = {EMDR/SG/15/2023-5901//Indian Council of Medical Research (ICMR)/ ; },
abstract = {Advanced gene editing tools have transformed oncology by facilitating precise molecular therapies aimed at the hereditary basis of cancer. This thorough study examines the mechanisms, applications, and clinical implementation of advanced genome editing technologies in cancer treatment. This review commences with the molecular principles of genome editing and DNA repair mechanisms, systematically analyzing established technologies such as Zinc Finger Nucleases, Transcription Activator-Like Effector Nucleases, and various CRISPR/Cas systems (Cas9, Cas12, Cas13), in addition to novel advancements including base editors, prime editors, and the PASTE system. Additionally, hybrid platforms such as ARCUS, MegaTALs, and modified recombinases are examined, highlighting their amalgamation with artificial intelligence, biosensors, and synthetic biology concepts. The study outlines significant applications including functional genomics, disease modeling, synthetic lethality screening, and direct therapeutic interventions, with a specific focus on CAR-T cell engineering and immune checkpoint regulation. Applications unique to various cancer types are thoroughly examined throughout lung, breast, colorectal, hematologic, liver, pancreatic, head & neck, esophageal, prostate, gastric, and brain cancers. Significant obstacles such as delivery optimization via viral and non-viral vectors, tumor-specific targeting, off-target effects, immunogenicity, and ethical issues related to germline vs somatic editing are comprehensively examined. The translational landscape is analyzed via current clinical trials, regulatory structures, and the incorporation of organoid models and patient-derived xenografts for the advancement of personalized therapies. This review highlights the transformative impact of gene editing on cancer medicine, advancing toward more accurate, effective, and personalized therapeutic approaches.},
}

@article {pmid42627520,
year = {2026},
author = {Lv, Z and Wang, G},
title = {Establishment of a CRISPR/Cas9-mediated system for targeted editing of the MFS gene in mint.},
journal = {Plant cell reports},
volume = {45},
number = {9},
pages = {},
pmid = {42627520},
issn = {1432-203X},
support = {23H010204158//Contract Research Project/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Mentha/genetics/metabolism ; Plants, Genetically Modified ; Protoplasts/metabolism ; Oils, Volatile/metabolism ; Base Sequence ; Plant Proteins/genetics/metabolism ; Transformation, Genetic ; *Gene Editing/methods ; },
abstract = {The key message of this study is that we established a CRISPR/Cas9-mediated genome-editing system for Mentha haplocalyx "738" by optimizing protoplast transient assay and screening effective regulatory elements. Targeted knockout of the MFS gene generated edited mint plants with reduced menthofuran content, offering a strategy for quality improvement of mint essential oil. The commercial value of mint (Mentha spp.) essential oil is often diminished by the presence of undesirable metabolites, notably menthofuran, which impairs flavor and raises safety concerns. This study aimed to develop a robust CRISPR/Cas9 gene editing system for mint 738 (Mentha haplocalyx "738") and apply it to disrupt the menthofuran synthase (MFS) gene, thereby redirecting metabolic flux to enhance oil quality. We established an optimized system for high-efficiency protoplast isolation and transient transformation from young mint leaves. Key parameters for enzymatic digestion (1.5% cellulase R10, 0.2% macerozyme R-10, 3 h) and PEG-mediated transformation (40% PEG6000, 0.4 M mannitol, 0.4 M CaCl2) were systematically determined. Using this platform, we screened endogenous regulatory elements, identifying a truncated mint U6 promoter (HmU6.1-3P) and the tomato SlEF1α promoter as the most effective drivers for sgRNA and Cas9 expression, respectively. A CRISPR/Cas9 vector targeting the MFS gene was constructed and used for Agrobacterium-mediated stable transformation. The positive transgenic mint lines were obtained. Sequencing confirmed heritable mutations at the target sites within the MFS gene in multiple independent lines. The results revealed a substantial decrease in menthofuran content in the essential oil of the edited line #10 compared to the wild-type control, thereby demonstrating a viable strategy for improving mint essential oil quality through genome-editing.},
}

*CRISPR-Cas Systems/genetics
*Mentha/genetics/metabolism
Plants, Genetically Modified
Protoplasts/metabolism
Oils, Volatile/metabolism
Base Sequence
Plant Proteins/genetics/metabolism
Transformation, Genetic
*Gene Editing/methods

@article {pmid42627922,
year = {2026},
author = {Wang, SK and Li, Z and Shah, SH and Edwards, QA and Bouffard, R and Hines, ES and Imventarza, JA and Korte, S and Lawrence, MS and Song, E and Helmy, E and Amaya, L and Kang, NW and Myung, D and Tsai, MC and Greenleaf, WJ and Waymouth, RM and Wang, S and Wender, PA and Chang, HY},
title = {RNA delivery to the corneal endothelium using charge-altering releasable transporters.},
journal = {Science advances},
volume = {12},
number = {34},
pages = {eady8161},
pmid = {42627922},
issn = {2375-2548},
support = {R01 CA245533/CA/NCI NIH HHS/United States ; T32 EY027816/EY/NEI NIH HHS/United States ; },
mesh = {Animals ; *Endothelium, Corneal/metabolism ; Humans ; Mice ; *Gene Transfer Techniques ; *RNA/administration & dosage/genetics ; Nanoparticles/chemistry ; CRISPR-Cas Systems ; },
abstract = {RNA therapies hold tremendous promise for treating genetic eye diseases. However, their development is limited by the lack of non-viral delivery platforms that can target specific ocular cell types. Here, we describe a charge-altering releasable transporter (CART) that delivers RNA selectively to the corneal endothelium, a non-regenerative cell layer whose dysfunction underlies several blinding conditions. We characterize the safety of CART-RNA nanoparticles in mice and show that they facilitate delivery of diverse RNA cargoes to the corneal endothelium, including circular RNA and CRISPR/Cas9. We verify that these nanoparticles can be redosed and apply them to achieve corneal gene editing. We further demonstrate CART transfection of corneal endothelial cells from a human donor in vitro and in a non-human primate in vivo, supporting the feasibility of clinical translation. Our findings establish CARTs as a platform for non-viral gene delivery to the eye, with the potential to treat corneal dystrophies and other vision disorders.},
}

Animals
*Endothelium, Corneal/metabolism
Humans
Mice
*Gene Transfer Techniques
*RNA/administration & dosage/genetics
Nanoparticles/chemistry
CRISPR-Cas Systems

@article {pmid42627995,
year = {2026},
author = {Ripken, L and Hoekman, TD and Willemse, M and Troost, ML and Kenyon, AN and van den Broek, WJAA and Wansink, DG},
title = {CRISPR/dCas9-mediated tuning of DMPK transcription reveals a quantitative relationship between toxic repeat RNA expression and MBNL1 activity in myotonic dystrophy.},
journal = {Human molecular genetics},
volume = {35},
number = {17},
pages = {},
doi = {10.1093/hmg/ddag079},
pmid = {42627995},
issn = {1460-2083},
support = {W.OR18-06//Prinses Beatrix Spierfonds/ ; },
mesh = {Humans ; *Myotonic Dystrophy/genetics/pathology/metabolism ; *Myotonin-Protein Kinase/genetics/metabolism ; *RNA-Binding Proteins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Transcription, Genetic ; Myoblasts/metabolism ; RNA Splicing/genetics ; Trinucleotide Repeat Expansion/genetics ; Cell Line ; Alternative Splicing/genetics ; RNA/genetics ; },
abstract = {Myotonic dystrophy type 1 (DM1) is caused by (CUG)n-expanded DMPK transcripts that sequester the splicing factor MBNL1 in the nucleus, resulting in widespread splicing abnormalities. Although significant progress has been made in understanding DM1 pathogenesis, the contribution of DMPK transcript levels to disease severity, and the variability of these levels across cell types, tissues, and patients, remains poorly understood. To investigate this in a quantitative manner, we developed isogenic human immortalized myoblast models with inducible modulation of DMPK RNA levels using CRISPR activation (CRISPRa) and interference (CRISPRi) guided by synthetic sgRNAs. CRISPRa elevated DMPK RNA levels by more than three-fold, intensifying MBNL1-dependent splicing defects. In contrast, CRISPRi reduced DMPK RNA expression by approximately 80%, partially rescuing splicing abnormalities. These changes were validated by visualizing (CUG)n foci using RNA FISH. Lowering DMPK transcript levels increased the availability of free nucleoplasmic MBNL1, whereas upregulation further depleted MBNL1, reinforcing the central role of MBNL1 sequestration in repeat RNA toxicity. Our findings demonstrate that expanded DMPK transcript levels modulate free MBNL1 concentration and alternative splicing in a dose-dependent manner, underscoring the central role of repeat RNA expression in DM1 pathogenesis. These models provide a powerful platform for dissecting variability in DMPK expression and for defining the therapeutic thresholds required for effective DMPK knockdown, thereby offering critical insights for the design and evaluation of DMPK and MBNL1-directed therapeutic strategies.},
}

Humans
*Myotonic Dystrophy/genetics/pathology/metabolism
*Myotonin-Protein Kinase/genetics/metabolism
*RNA-Binding Proteins/genetics/metabolism
*CRISPR-Cas Systems/genetics
Transcription, Genetic
Myoblasts/metabolism
RNA Splicing/genetics
Trinucleotide Repeat Expansion/genetics
Cell Line
Alternative Splicing/genetics
RNA/genetics

@article {pmid42631783,
year = {2026},
author = {Saleem, M and Syed Khaja, AS and Ahmad, I and Alraey, Y and Azhar, MA and Khan, MS},
title = {Staphylococcus aureus biofilms: molecular mechanisms, resistance determinants, and emerging therapeutic strategies.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42631783},
issn = {1573-4978},
support = {R.G.P.2/503/46//The Deanship of Research and Graduate Studies at King Khalid University, Abha, Saudi Arabia./ ; },
mesh = {*Biofilms/drug effects/growth & development ; Humans ; *Staphylococcus aureus/drug effects/genetics/pathogenicity/physiology ; Anti-Bacterial Agents/pharmacology/therapeutic use ; Quorum Sensing/drug effects ; *Staphylococcal Infections/microbiology/drug therapy ; *Drug Resistance, Bacterial/genetics ; Virulence Factors/genetics ; },
abstract = {S. aureus is a significant opportunistic pathogen that causes a variety of community and healthcare-associated infections. Biofilm formation is one of its many virulence factors and contributes to persistent, recurrent, and device-associated infections through enhancing bacterial survival, immune system evasion, and resistance to antimicrobial agents. The development of biofilms is a complex, highly regulated process influenced by genetic regulators, environmental factors, and intercellular communication, leading to the formation of a structured microbial community with a protective extracellular matrix (ECM). These biofilms undergo large-scale physiological, transcriptomic, and proteomic changes, which help them survive harsh host conditions and reduce their susceptibility to immune responses and standard antibiotics. Biofilm-associated antimicrobial resistance is also facilitated by several complementary mechanisms, including limited penetration of antimicrobials, changes in bacterial physiology, persister cell formation, adaptive stress responses, and the presence of other clinically relevant microorganisms in polymicrobial biofilms. Recent evidence has also emphasized the importance of host-biofilm interactions in the establishment of chronic infections, including dysregulated inflammatory responses and immune evasion. Due to the intrinsic inefficacy of traditional antimicrobial drug treatment against mature biofilms, significant efforts have been made to develop novel anti-biofilm interventions, such as matrix-disrupting agents, quorum-sensing inhibitors, antimicrobial peptides, bacteriophages, nanotechnology-assisted delivery systems, CRISPR-Cas-based therapeutics, and rational combination therapy. This review aims to provide a comprehensive and up-to-date overview of the molecular biology of S. aureus biofilms, biofilm-associated antimicrobial resistance, interactions with the host, polymicrobial interactions, and emerging therapeutic strategies, and to highlight the ongoing challenges and future directions in the prevention and treatment of persistent biofilm-associated infections.},
}

*Biofilms/drug effects/growth & development
Humans
*Staphylococcus aureus/drug effects/genetics/pathogenicity/physiology
Anti-Bacterial Agents/pharmacology/therapeutic use
Quorum Sensing/drug effects
*Staphylococcal Infections/microbiology/drug therapy
*Drug Resistance, Bacterial/genetics
Virulence Factors/genetics

@article {pmid42633712,
year = {2026},
author = {Kulishova, LM and Zharkov, DO},
title = {Glycosylase Base Editors: New Tools for Genome Editing.},
journal = {Biochemistry. Biokhimiia},
volume = {91},
number = {7},
pages = {1093-1113},
doi = {10.1134/S0006297926601073},
pmid = {42633712},
issn = {1608-3040},
mesh = {*Gene Editing/methods ; Humans ; *DNA Glycosylases/metabolism/genetics ; CRISPR-Cas Systems ; Animals ; DNA/genetics/metabolism ; },
abstract = {Genome editing using the CRISPR/Cas9 system has become a staple of modern genome manipulation. In its original form, editing involved introducing double-strand breaks into DNA, which can cause genomic instability. The appearance of the first base editors in 2016 expanded the range of editing technologies and enabled single-nucleotide changes to be introduced into the genome through deamination of nucleobases, bypassing the double-strand break stage. Further development of base editors involves the incorporation of additional modules, DNA glycosylases, that can remove modified or even normal nucleobases and create non-instructive apurinic/apyrimidinic sites in DNA, significantly expanding the range of available single-nucleotide substitutions. This review examines the operating principles of the glycosylase base editors, the main limitations of these genome manipulation tools, and promising areas for the development of this technology.},
}

*Gene Editing/methods
Humans
*DNA Glycosylases/metabolism/genetics
CRISPR-Cas Systems
Animals
DNA/genetics/metabolism

@article {pmid42634244,
year = {2026},
author = {Gulfam, T and Li, W and Han, Z and Gulfam, Y and Li, J and Fan, Z and Zhang, H and Wang, F and Yang, J},
title = {Gene Editing in Forest Tree Breeding for Stress Resistance: From Mechanisms to Future Prospects.},
journal = {Plant, cell & environment},
volume = {},
number = {},
pages = {},
doi = {10.1111/pce.70840},
pmid = {42634244},
issn = {1365-3040},
support = {31870649//National Natural Science Foundation of China/ ; },
abstract = {Forest ecosystems face escalating threats from climate change alongside a surging demand for sustainable bioproducts. While conventional tree breeding is inherently constrained by long generation cycles, high heterozygosity, and complex genomes, CRISPR-based genome editing provides a precision framework for targeted genetic improvement. This review synthesises the fundamental principles and limitations of multiple gene-editing technologies, with a particular emphasis on CRISPR systems (Cas9, Cas12, and Cas13), in the specific context of woody perennial biology. Recent applications in key forest genera, including Populus, Pinus, and Eucalyptus, demonstrate the efficacy of these gene-editing tools in manipulating complex traits, such as rewiring phytohormone signalling networks for drought tolerance or remodelling root system architecture to combat abiotic stress. We critically evaluate persistent translational bottlenecks in forest tree genome editing, with a specific focus on recalcitrant, genotype-dependent regeneration and the multifaceted challenges of long-term field validation. Finally, we highlight how synergising CRISPR technologies with multi-omics, genomic selection, and high-throughput phenomics can accelerate the development and application of climate-resilient woody perennials.},
}

@article {pmid42634480,
year = {2026},
author = {Kawamata, M and Niwa, S and Suzuki, A},
title = {Chemical and Structural Engineering of Guide RNAs for Precision Genome Editing: From Design Principles to Clinical Applications.},
journal = {Chemical biology & drug design},
volume = {108},
number = {2},
pages = {e70377},
pmid = {42634480},
issn = {1747-0285},
support = {JP18H04737//Japan Society for the Promotion of Science/ ; JP20H05041//Japan Society for the Promotion of Science/ ; JP23K18097//Japan Society for the Promotion of Science/ ; JP23K27462//Japan Society for the Promotion of Science/ ; JP25K22439//Japan Society for the Promotion of Science/ ; JP18H05102//Japan Society for the Promotion of Science/ ; JP19H01177//Japan Society for the Promotion of Science/ ; JP19H05267//Japan Society for the Promotion of Science/ ; JP20H05040//Japan Society for the Promotion of Science/ ; JP22H05634//Japan Society for the Promotion of Science/ ; JP22H04698//Japan Society for the Promotion of Science/ ; JP22H00592//Japan Society for the Promotion of Science/ ; JP23K18579//Japan Society for the Promotion of Science/ ; JP25K22908//Japan Society for the Promotion of Science/ ; JP25H00445//Japan Society for the Promotion of Science/ ; JPMXP1323015486//MEXT Promotion of Development of a Joint Usage/Research System Project: Coalition of Universities for Research Excellence Program/ ; //the Center for Clinical and Translational Research of Kyushu University Hospital/ ; //the Fukuoka Financial Group Enterprise Development Foundation (KYUTEC)/ ; //the Medical Research Center Initiative for High Depth Omics/ ; //the Takeda Science Foundation (to M.K., and A.S.), the Uehara Memorial Foundation/ ; //Naito Foundation/ ; },
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems/chemistry/genetics/metabolism ; CRISPR-Cas Systems ; Genetic Engineering ; Machine Learning ; *Gene Editing ; Animals ; },
abstract = {CRISPR-Cas9 has revolutionised genome editing by enabling efficient and programmable modification of defined DNA sequences, with guide RNAs (gRNAs) serving as indispensable elements that direct Cas9 to specific genomic loci. Initially regarded as auxiliary components, gRNAs are now recognized as critical determinants of editing efficiency and specificity and have attracted growing attention as independent targets for engineering. Chemical modification, sequence optimisation, and structural alteration of gRNAs have been shown to enhance on-target activity, suppress off-target effects and cytotoxicity, and even achieve allele-selective precision editing in a programmable manner. Moreover, advances in artificial intelligence and machine learning have markedly improved the predictive accuracy of gRNA design through large-scale data analysis. Despite rapid progress, a consolidated review that integrates chemical, structural, and computational advances in gRNA engineering and highlights their translational potential for therapeutic genome editing has been lacking. This review uniquely addresses that gap by presenting an integrated framework that connects molecular design principles with clinical applicability.},
}

Humans
*RNA, Guide, CRISPR-Cas Systems/chemistry/genetics/metabolism
CRISPR-Cas Systems
Genetic Engineering
Machine Learning
*Gene Editing
Animals

@article {pmid42635641,
year = {2026},
author = {Singh, H and Kumar, P and Sharma, V and Singh, J and Swiecicki, WK and Jedryczka, M and Gawlowska, M and Tiwari, S},
title = {Efficient in-vitro regeneration and transformation for CRISPR/Cas9-mediated genome editing of phytoene desaturase (PDS) gene in pea (Pisum sativum L.).},
journal = {Plant cell reports},
volume = {45},
number = {9},
pages = {},
pmid = {42635641},
issn = {1432-203X},
support = {PPN/ BIN/ 2019/1/00142/U/00002//Polish National Agency for Academic Exchange (NAWA), Poland/ ; DST/INT/POL/P-45/2020//Department of Science and Technology, Ministry of Science and Technology, India/ ; },
mesh = {*Pisum sativum/genetics/enzymology/physiology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Regeneration/genetics ; *Transformation, Genetic ; *Oxidoreductases/genetics/metabolism ; Plants, Genetically Modified ; Plant Proteins/genetics/metabolism ; Plant Shoots/genetics/growth & development ; },
abstract = {The present study addresses optimization of in-vitro regeneration via direct organogenesis and Agrobacterium-mediated genetic transformation, enabling efficient multiplex CRISPR/Cas9-based genome editing of the phytoene desaturase (PsPDS) gene in pea. Pea (Pisum sativum L.) is an important legume crop valued for food, plant-based protein, vegetable, and green manure. Although genome editing offers a precise and rapid strategy for crop improvement, its application in pea remains challenging due to inherent recalcitrance to in-vitro regeneration and genotype-dependent transformation. The regeneration and Agrobacterium-mediated transformation systems were optimized, and the dicotyledonary node (DCN) was identified as the preferred explant for multiplex CRISPR/Cas9-based genome editing in pea. Among three explant types (embryonic axis, DCN and nodal segment), DCN showed the highest regeneration efficiency, producing 100% shoot bud induction and 39.70 shoots per explant on MS medium augmented with 6-benzylaminopurine (BAP; 6.00 mg/L) and kinetin (1.00 mg/L). Shoot elongation and rooting efficiencies were improved using GA3 (1.00 mg/L), BAP (1.00 mg/L), IAA (0.10 mg/L), and NAA (0.5 mg/L), respectively. Manipulating explant type, Agrobacterium optical density, vacuum infiltration, acetosyringone concentration, infection time, and co-cultivation duration improved the transient transformation efficiency. We noted efficiency from 23.33% to 90.00% in DCN and from 6.66% to 93.33% in embryonic axis explants across 10 pea cultivars. Stable transformed lines generated from the DCN of cultivar Kashi Samridhi were confirmed by GUS staining and PCR. The optimized regeneration and transformation system facilitated targeted editing of phytoene desaturase (PsPDS) in pea, achieving ICE-estimated mutation frequencies of upto 97% in independent lines. The study provides a robust platform for functional genomics and accelerates the deployment of genome-editing technologies for pea improvement.},
}

*Pisum sativum/genetics/enzymology/physiology
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Regeneration/genetics
*Transformation, Genetic
*Oxidoreductases/genetics/metabolism
Plants, Genetically Modified
Plant Proteins/genetics/metabolism
Plant Shoots/genetics/growth & development

@article {pmid42638006,
year = {2026},
author = {Sreekanth, D and Singh, C and Pawar, DV and Yadav, MK and Sahadeo, IK and Basavaraj, PS and Kumar, R and Mahesh, S},
title = {CRISPR/Cas-Mediated Genome Editing for Developing Herbicide Tolerant Rice: A Step-by-Step Protocol.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3017},
number = {},
pages = {47-57},
pmid = {42638006},
issn = {1940-6029},
mesh = {*Oryza/genetics/drug effects/growth & development ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Herbicides/pharmacology ; *Herbicide Resistance/genetics ; Acetolactate Synthase/genetics ; Plants, Genetically Modified/genetics ; Benzoates ; Pyrimidines ; },
abstract = {Weed management in rice cultivation has predominantly relied on acetolactate synthase (ALS) and acetyl-CoA carboxylase (ACCase) inhibiting herbicides, particularly against Echinochloa spp. (watergrass). However, these herbicides carry a high risk of resistance evolution, as evidenced by the numerous resistant biotypes reported worldwide. The emergence of herbicide resistance necessitates innovative and sustainable weed control strategies. Genome editing, particularly through the CRISPR/Cas system, provides a precise and efficient platform for introducing targeted genetic modifications to develop herbicide-tolerant (HT) rice cultivars. In this protocol, we present a step-by-step approach for generating bispyribac sodium-tolerant rice using the CRISPR/Cas-mediated editing of the ALS gene. The method encompasses guide RNA design, vector construction, transformation, selection of edited plants, and molecular confirmation of targeted mutations. This approach offers a robust framework for producing HT rice lines, potentially reducing reliance on conventional herbicide regimes and mitigating the risk of resistance development in weed populations.},
}

*Oryza/genetics/drug effects/growth & development
*CRISPR-Cas Systems
*Gene Editing/methods
*Herbicides/pharmacology
*Herbicide Resistance/genetics
Acetolactate Synthase/genetics
Plants, Genetically Modified/genetics
Benzoates
Pyrimidines

@article {pmid42638019,
year = {2026},
author = {Mukherjee, A and Basak, S and Singh, R and Bajani, R and Kundu, P},
title = {Targeted Gene Expression Modulation Using CRISPR/dCas9 to Investigate Pathogenic Outcomes in Tomato.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3017},
number = {},
pages = {221-239},
pmid = {42638019},
issn = {1940-6029},
mesh = {*Solanum lycopersicum/genetics/microbiology ; *CRISPR-Cas Systems ; *Gene Expression Regulation, Plant ; *Plant Diseases/microbiology/genetics ; *Gene Editing/methods ; Streptococcus pyogenes/genetics ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats) has become integral to modern biological research, with the Streptococcus pyogenes CRISPR/Cas9 system serving as the most extensively used tool for precise, site-specific genome editing across a wide range of organisms and cell types. Compared with earlier genome-editing platforms, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR/Cas9 offers greater simplicity, precision, versatility, and scalability. Beyond introducing stable DNA modifications, this system can be reengineered to reversibly activate (CRISPRa) or repress (CRISPRi) the transcription of any gene by employing unique nuclease-deactivated variants of Cas9 (dCas9) fused to transcriptional activators or repressors, respectively, providing a compelling alternative to RNA interference (RNAi) and conventional overexpression techniques. In plants, CRISPR/dCas9-based programmable gene control presents an innovative and transformative framework for rewiring gene regulatory networks to study pathogenic stress-signaling pathways. Notably, its strategic use in orchestrating the simultaneous regulation of multiple defense-related genes sets the stage for developing crops with robust and quantitative disease resistance. In this chapter, we outline a comprehensive methodology for the design, assembly, and functional assessment of CRISPR/dCas9 systems optimized for tomato to investigate pathogen-associated responses.},
}

*Solanum lycopersicum/genetics/microbiology
*CRISPR-Cas Systems
*Gene Expression Regulation, Plant
*Plant Diseases/microbiology/genetics
*Gene Editing/methods
Streptococcus pyogenes/genetics

@article {pmid42638068,
year = {2026},
author = {Yao, M and Li, T and Sun, B and Li, M and Peng, D and Wang, Y and Qiu, HJ and Zhang, D and Li, LF},
title = {[Construction and characterization of a stable Cas9-expressing monoclonal WSL cell line].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {8},
pages = {3736-3748},
doi = {10.13345/j.cjb.250933},
pmid = {42638068},
issn = {1872-2075},
support = {32372983//the National Natural Science Foundation of China/ ; ZD2025C008//the Natural Science Foundation of Heilongjiang Province/ ; },
mesh = {Animals ; Cell Line ; *African Swine Fever Virus/genetics/physiology ; *CRISPR-Cas Systems/genetics ; Swine ; Lentivirus/genetics/metabolism ; Lung/cytology ; Genetic Vectors/genetics ; Clone Cells ; },
abstract = {The molecular mechanism underlying the cellular invasion of African swine fever virus (ASFV) remains incompletely understood, particularly with respect to its key cellular receptors, which constitutes a major bottleneck in the development of effective vaccines and targeted antiviral therapies. To establish a robust experimental platform that supports efficient ASFV replication and enables genome-wide CRISPR screening for systematic identification of host factors involved in viral entry, we employed the CRISPR/Cas9 system to generate stable monoclonal cell lines expressing Cas9 protein based on the wild boar lung (WSL) cell line. Recombinant lentiviruses co-expressing Cas9 and blasticidin resistance gene were packaged via a lentiviral vector system and transduced into WSL cells. Following blasticidin selection, a polyclonal population stably expressing Cas9 was obtained and subjected to fluorescence-activated cell sorting (FACS) to derive monoclonal cell lines. Cas9 expression was determined by Western blotting. To assess the functional gene editing activity of the established clones, we introduced the lentiviruses carrying an EGFP reporter gene along with its specific single-guide RNA (sgRNA) into the monoclonal cell lines, and quantitatively evaluated the editing efficiency via flow cytometry. Furthermore, sgRNAs specifically targeting the ASFV B646L gene and the host TMEM239 gene were designed and synthesized to validate the cell line's capacity for editing both viral and host genomic loci. The results demonstrated the successful establishment of seven stable WSL-Cas9 monoclonal cell lines expressing Cas9 protein, among which clone WSL-Cas9-3# exhibited the highest editing efficiency, enabling effective genetic modification of both ASFV and host genes, while maintaining favorable genetic stability and normal growth properties. This study reports the generation of a WSL-Cas9 monoclonal cell line with high CRISPR/Cas9 editing efficiency, stable proliferation, and permissiveness for robust ASFV replication. This engineered cell line provides a reliable platform for future genome-wide functional screening to systematically identify host factors governing ASFV entry and establishes a critical technical foundation for delving into virus-host interactions.},
}

Animals
Cell Line
*African Swine Fever Virus/genetics/physiology
*CRISPR-Cas Systems/genetics
Swine
Lentivirus/genetics/metabolism
Lung/cytology
Genetic Vectors/genetics
Clone Cells

@article {pmid42640835,
year = {2026},
author = {Rahat, H and Qaiser, D and Maqsood, Q and Mehmood, T},
title = {Engineered Microbial Cellulases for Biomass Conversion: Integrating Omics, Expression Platforms, Fermentation Engineering and Enzyme Reusability.},
journal = {Biotechnology and bioengineering},
volume = {},
number = {},
pages = {},
doi = {10.1002/bit.70337},
pmid = {42640835},
issn = {1097-0290},
abstract = {The conversion of lignocellulosic biomass, which is an abundant renewable carbon source, is limited by the cost, stability, loading requirement and scale-up constraints of cellulase systems for sustainable biomanufacturing. Cellulose deconstruction is catalyzed by microbial cellulases such as endoglucanases, cellobiohydrolases, beta-glucosidases and accessory enzymes, which are used in biorefineries, food and feed processing, textiles, detergents, pulp and paper and waste valorization. This review focuses on cellulase production as a platform for biotechnology rather than as a standalone fermentation process. It connects native cellulase-producing microorganisms, omics-guided enzyme discovery, lignocellulosic substrate selection, pretreatment and inhibitor tolerance, solid-state and submerged fermentation, recombinant expression systems, enzyme engineering, downstream recovery, immobilization, reusability and industrial translation. Focus is given to the transition from conventional microbial producers to engineered platforms that combine CRISPR/Cas systems, transcriptional regulation, base and prime editing, strain improvement, promoter and secretion engineering, synthetic biology, enzyme-cocktail optimization and structure-guided or AI-assisted cellulase design. Despite the advances in cellulase yield, catalytic efficiency, thermostability and substrate specificity, the use of cellulases on a large scale is still hindered by the heterogenicity of the feedstock, catabolite repression, enzyme inhibition, downstream recovery cost and scale-up limitations. The next step will be the integration of microbial diversity, multi-omics, advanced host engineering, process intensification, low-cost recovery strategies and application-specific enzyme cocktails to create robust, economically viable cellulase platforms for sustainable biorefineries and circular bioeconomy applications.},
}

@article {pmid42642425,
year = {2026},
author = {Wu, Y and Xia, Y and Yao, Z and Chen, W and Jia, X and Liang, N and Champer, J},
title = {Finding the perfect promoter for Cas9 in homing gene drives using single cell transcriptome data.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42642425},
issn = {2041-1723},
support = {32270672//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; *Promoter Regions, Genetic/genetics ; *Drosophila melanogaster/genetics ; *CRISPR-Cas Systems/genetics ; Female ; Male ; *Transcriptome/genetics ; Single-Cell Gene Expression Analysis ; Single-Cell Analysis ; Alleles ; *CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {Gene drive can modify or suppress vector populations by spreading drive alleles. In CRISPR homing drives, regulating Cas9 expression has been effective for improving drive performance, but selecting suitable promoters is often a major challenge. Here, we evaluate 35 Cas9 constructs with distinct promoters in Drosophila melanogaster and identify associations between drive performance and single-cell RNA expression patterns of the promoter-associated genes. Our results indicate that higher drive conversion is associated with elevated expression of the promoter-associated gene in reproductive cells, but embryo resistance allele formation correlates with excessive female germline expression. For males, early germline expression produces superior performance. Thus, optimal drive performance requires restricting Cas9 expression to a tight quantitative and spatiotemporal window. Additionally, we find that an in situ construct significantly reduces potentially harmful somatic expression. Based on these results, we propose criteria for selecting promoters, providing a rationale and guidance for optimization of homing gene drives.},
}

Animals
*Promoter Regions, Genetic/genetics
*Drosophila melanogaster/genetics
*CRISPR-Cas Systems/genetics
Female
Male
*Transcriptome/genetics
Single-Cell Gene Expression Analysis
Single-Cell Analysis
Alleles
*CRISPR-Associated Protein 9/genetics/metabolism

@article {pmid42642483,
year = {2026},
author = {Li, TT and Chen, X and Wang, F and Tang, YA and Sim, M and Xiao, L and Jin, WB and Shi, H and Ma, JY and Yang, X and Liu, Y and Sorbara, MT and Guo, CJ},
title = {Transferable genetic toolsets for nonmodel gut Clostridia enable in vivo reversible control of metabolite production.},
journal = {Nature biotechnology},
volume = {},
number = {},
pages = {},
pmid = {42642483},
issn = {1546-1696},
abstract = {Gut Clostridia species, including commensal members of the Clostridiaceae and Lachnospiraceae families, maintain microbiota homeostasis and influence human health and disease; however, adequate genetic toolsets to study abundant but nonmodel gut Clostridia are lacking. Here we present a set of transferable and modular genetic toolsets that function broadly across phylogenetically diverse gut Clostridia. We first identify a panel of strong constitutive promoters that drive robust gene expression across diverse clostridial strains. We then develop an inducible promoter system that enables precise, tunable gene regulation and facilitates the implementation of CRISPR-Cas gene-deletion systems. We apply this system for targeted and reversible control of trimethylamine and deoxycholic acid production, two microbiota-derived metabolites implicated in host lipid metabolism and diseases, in mice. This robust genetic toolkit for nonmodel gut Clostridia enables functional studies to causally link microbiota genes to host physiology and disease, paving the way for therapeutic genetic engineering of microbiota.},
}

@article {pmid42642601,
year = {2026},
author = {Zhou, X and Lu, R},
title = {Dissecting Epigenetic Drug Response Mechanisms Using CRISPR Knockout Screens.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3005},
number = {},
pages = {383-401},
pmid = {42642601},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Humans ; *Epigenesis, Genetic/drug effects ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; Epigenome Editing ; *Neoplasms/genetics/drug therapy ; Gene Editing/methods ; *Antineoplastic Agents/pharmacology ; Animals ; },
abstract = {Epigenetic drugs are widely applied in cancer therapy due to their ability to modify gene expression without altering the DNA sequence. Despite their therapeutic potential, drug resistance frequently occurs, posing a significant challenge in cancer treatment. CRISPR screens have emerged as a powerful tool to address this issue by leveraging the precision of CRISPR-Cas9 gene editing to enable the systematic interrogation of genes. This approach involves the simultaneous targeting of thousands of genes to elucidate their roles in various biological processes, disease mechanisms, and drug responses, providing valuable insights into gene function and potential therapeutic targets. In cancer therapy, CRISPR screens provide a deeper understanding of cancer progression by enabling the identification of essential genes and facilitating the discovery of novel therapeutic targets when combined with epigenetic drugs. Here, we present an overview of the CRISPR screen methodology, which involves introducing guide RNAs targeting specific genes into cells, followed by phenotype selection and analysis.},
}

*CRISPR-Cas Systems
Humans
*Epigenesis, Genetic/drug effects
RNA, Guide, CRISPR-Cas Systems/genetics
*Gene Knockout Techniques/methods
Epigenome Editing
*Neoplasms/genetics/drug therapy
Gene Editing/methods
*Antineoplastic Agents/pharmacology
Animals

@article {pmid42642602,
year = {2026},
author = {Tang, Q and Liu, J},
title = {Tools for Cancer Research: CRISPR/Cas-Based Gene Editing and Auxin-Induced Degron Systems.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3005},
number = {},
pages = {403-415},
pmid = {42642602},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Degrons ; *Indoleacetic Acids/pharmacology/metabolism ; *Gene Editing/methods ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Neoplasms/genetics ; Gene Knock-In Techniques ; },
abstract = {CRISPR/Cas9 and auxin-degron systems represent two powerful and complementary genetic tools that have revolutionized cancer research. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated protein)-based gene editing system has been widely used in numerous biological fields, significantly enhancing the capacity of researchers to elucidate the underlying mechanisms of biological phenomena. The two most prevalent applications for CRISPR-mediated gene editing are knockout and knock-in. This protocol covers both single-gRNA (sgRNA) and dual-gRNA directed knockout systems. Furthermore, we describe the procedure for constructing an Auxin-Inducible Degron (AID) knock-in-mediated system to induce the rapid degradation of a target protein in cells, thereby investigating its function.},
}

*CRISPR-Cas Systems
Degrons
*Indoleacetic Acids/pharmacology/metabolism
*Gene Editing/methods
Humans
RNA, Guide, CRISPR-Cas Systems/genetics
*Neoplasms/genetics
Gene Knock-In Techniques

@article {pmid42643124,
year = {2026},
author = {Zafar, MM and Firdous, H and Siddiqua, A and Naveed, A and Razzaq, A and Munawar, S and Ijaz, A and Anwar, Z and Ercisli, S and Jiang, X and Fei, Q},
title = {Programmable Domestication: CRISPR, Pan-Genomics and System Level Engineering for Next-Generation Crops.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
pmid = {42643124},
issn = {1467-7652},
abstract = {Global agriculture is increasingly challenged by climate instability, genetic erosion, emerging pathogens and rising food demands, exposing the limitations of conventional breeding and traditional domestication strategies. Recent advances in CRISPR-based genome editing, pangenomic, synthetic biology, artificial intelligence (AI)-assisted breeding and predictive phenomics are transforming de novo domestication from a slow evolutionary process into a programmable framework for rational crop redesign. This review synthesises recent advances in programmable de novo domestication and highlights how crop wild relatives and underutilised germplasm can be harnessed to develop resilient, climate-adaptive and sustainable crop systems. The integration of multiplex genome editing, pan-genomic variation discovery, AI-driven genomic prediction and predictive breeding enables precise engineering of key domestication traits governing plant architecture, yield potential, stress resilience and nutritional quality. Furthermore, we propose a trajectory-based framework for programmable domestication comprising Adaptive Rescue, Agronomic Refinement and Novel Chassis Engineering, which illustrates distinct evolutionary pathways, engineering complexity and crop redesign objectives. We also examine the major system level challenges that constrain programmable domestication, including cryptic genetic variation, epistasis, gene regulatory network complexity, genotype phenotype predictability, biodiversity conservation and regulatory considerations. Collectively, programmable domestication represents a transformative shift from conventional crop improvement towards system-level engineering of next-generation crops, providing a strategic foundation for enhancing global food security, agricultural sustainability and environmental resilience in the face of accelerating climate change.},
}

@article {pmid42643189,
year = {2026},
author = {Takata, M and Chikumi, H and Yoshifuji, A},
title = {Molecular Diagnostics of Infectious Diseases.},
journal = {Yonago acta medica},
volume = {69},
number = {3},
pages = {228-247},
pmid = {42643189},
issn = {0513-5710},
abstract = {With the advances in molecular biology, molecular testing has been incorporated into pathogen testing for infectious diseases. There are two main objectives for the molecular diagnosis of infectious diseases- first, to detect pathogen genes by using highly sensitive nucleic acid amplification tests, such as polymerase chain reaction and isothermal amplification, and second, to characterize the properties of the pathogen using next-generation sequencing. Polymerase chain reaction plays a key role in molecular testing. The extensive development of polymerase chain reaction methods is underway, focusing on the acceleration of reaction time (microfluidic polymerase chain reaction), quantification (real-time polymerase chain reaction, digital polymerase chain reaction), full automation, and point-of-care testing. Isothermal amplification is a method for amplifying nucleic acids at a constant temperature. Loop-mediated isothermal amplification, recombinase polymerase amplification, and nucleic acid sequence-based amplification have been developed for isothermal amplification. Isothermal amplification does not require a thermal cycler or simplified temperature control; therefore, it is suitable for point of care testing. CRISPR-based diagnostics are a new method for detecting amplified nucleic acids. CRISPR-Cas reaction proceeds at a constant temperature, it is often combined with isothermal amplification. Next-generation sequencing has a high sequence throughput to rapidly obtain large amounts of genomic information and can be used to detect novel pathogens and diagnose complex infectious diseases. It can also be used to track the sources and transmission routes of outbreaks and monitor pathogen evolution. Furthermore, next generation sequencing has enabled the analysis of microbiomes that can serve as biomarkers for diseases or disease susceptibility. In the future, the molecular diagnosis of infectious diseases will advance by overcoming these shortcomings and integrating various technologies as hybrid platforms. This review describes the developments in molecular diagnostics for the treatment of infectious diseases.},
}

@article {pmid42184634,
year = {2026},
author = {Liu, G and Cao, Z and He, Y and Zhu, X and Ali, MA and Sun, H and Zuo, Q and Niu, Y and Song, J and Han, W and Wei, W and Chen, G and Li, B and Jin, K},
title = {Establishment of an inducible knockout model for the chicken Z-chromosome-linked gene DMRT1.},
journal = {Poultry science},
volume = {105},
number = {9},
pages = {107147},
pmid = {42184634},
issn = {1525-3171},
mesh = {Animals ; Male ; *Chickens/genetics ; *Transcription Factors/genetics/metabolism ; *Gene Knockout Techniques/veterinary/methods ; *Sex Determination Processes/genetics ; Chick Embryo ; Female ; *Avian Proteins/genetics/metabolism ; *Sex Chromosomes/genetics ; CRISPR-Cas Systems ; },
abstract = {Sex determination is a crucial process in animal development, regulated by complex genetic networks. In avian species, Doublesex and mab-3 related transcription factor 1 (DMRT1) plays a vital role in gonadal development and sex determination. To unravel the function of DMRT1 in chicken sex determination, establishing an inducible DMRT1 knockout model is essential. In this study, we constructed an inducible DMRT1 knockout system and verified its efficiency and effects on related genes and physiological indicators. To achieve precise genomic ablation, we screened multiple sgRNAs targeting the DMRT1 locus and integrated the optimal sequence into a doxycycline-responsive (Tet-on) CRISPR/Cas9 architecture. For in vitro experiments, vectors were delivered via cell transfection and induced with 20 µg mL[-1] doxycycline (DOX), achieving an 80% knockout efficiency. Following the administration of polyethylenimine (PEI)-encapsulated plasmids into chicken embryos, we successfully implemented the inducible system in vivo. Quantitative analysis confirmed a mosaic knockout of DMRT1 with an observed efficiency reaching 45%. Following targeted disruption, we evaluated sex-related gene and protein expression alterations via qRT-PCR and Western blot (WB). Furthermore, ELISA was performed to measure testosterone levels in male embryonic gonads across multiple developmental stages (E4.5 to E18.5). qRT-PCR analysis showed that after induction, female-related genes (CYP19A1, FOXL2, ESR1) were significantly upregulated, and male-related genes (DMRT1, SOX9, AMH) were significantly downregulated. WB results revealed increased protein expression levels of CYP19A1 and FOXL2, and decreased protein expression of SOX9 post-induction. ELISA confirmed that testosterone levels in the gonads of induced male embryos were significantly reduced compared to normal and non-induced males. The study successfully established an inducible DMRT1 knockout system in chickens. This system effectively regulates the expression of sex-related genes and reduces testosterone levels in male embryos, providing theoretical and technical support for breeding novel sex-controlled breeding materials.},
}

Animals
Male
*Chickens/genetics
*Transcription Factors/genetics/metabolism
*Gene Knockout Techniques/veterinary/methods
*Sex Determination Processes/genetics
Chick Embryo
Female
*Avian Proteins/genetics/metabolism
*Sex Chromosomes/genetics
CRISPR-Cas Systems

@article {pmid42204401,
year = {2026},
author = {Gao, ZY and Shen, TL and Cheng, CY and Sun, YD and Zhang, LM and Zhang, JP and Zhang, XB},
title = {A Dual-Viral Delivery Platform Enables Efficient Site-Specific Integration of Therapeutic-Length Genes in Human Primary Stem Cells.},
journal = {Human gene therapy},
volume = {37},
number = {17-18},
pages = {858-875},
doi = {10.1177/10430342261453040},
pmid = {42204401},
issn = {1557-7422},
mesh = {Humans ; *Lentivirus/genetics ; *Genetic Vectors/genetics/administration & dosage ; Induced Pluripotent Stem Cells/metabolism/cytology ; *Gene Transfer Techniques ; *Gene Editing/methods ; *Genetic Therapy ; Hematopoietic Stem Cells/metabolism ; CRISPR-Cas Systems ; Integrases/genetics ; },
abstract = {Site-specific integration of large genes in human primary stem cells remains a significant challenge in gene therapy, particularly for treating multiallelic diseases. Gene editing efficiency in primary stem cells is heavily influenced by the delivery strategy, which often faces issues with programmability, efficiency, and specificity. Here, we developed a dual-viral delivery system, targeted integration via virus-like particles and integrase-deficient lentivirus (TIVID). This system combines virus-like Cas9 edit particles for delivering Cas9/sgRNA ribonucleoprotein complexes and integrase-deficient lentiviral vectors for delivering HDR donor templates. The TIVID system achieves a knock-in efficiency of 65% ± 5% in human induced pluripotent stem cells (iPSCs). In erythroid progenitor HUDEP2 cells, TIVID mediates precise integration of a 7.1 kb HBB-GFP cassette (from cut site to cut site) at the AAVS1 locus with 20% efficiency and stable expression. Crucially, we demonstrate that TIVID overcomes stringent packaging constraints to deliver an approximately 6 kb full-length HBB therapeutic cassette into primary human CD34[+] hematopoietic stem and progenitor cells. This platform achieved 5-10% targeted integration efficiency and preserved robust lineage-specific differentiation capacity, demonstrating its potential for treating β-thalassemia and other multiallelic disorders. In head-to-head comparisons, TIVID outperformed lentivirus-derived nanoparticles (∼50% vs. <10% at AAVS1 in K562 with M3814) and plasmid-based eePASSIGE in iPSCs (∼20% vs. ∼1.5%). Compared with traditional electroporation delivery, TIVID offers lower early cytotoxicity, promotes predominantly mono-allelic integration, and exhibits enhanced compatibility with primary stem cells. By decoupling nuclease and donor delivery, TIVID circumvents the payload constraints of single-vector systems and the toxicity of physical transfection, providing a robust ex vivo engineering platform for complex gene replacement therapies.},
}

Humans
*Lentivirus/genetics
*Genetic Vectors/genetics/administration & dosage
Induced Pluripotent Stem Cells/metabolism/cytology
*Gene Transfer Techniques
*Gene Editing/methods
*Genetic Therapy
Hematopoietic Stem Cells/metabolism
CRISPR-Cas Systems
Integrases/genetics

@article {pmid42204417,
year = {2026},
author = {Hu, J and Zhang, J and Shao, Y and Li, H and Yang, Y and Zhang, J},
title = {Ablation of Cbl-b in ROBO1 CAR-NK92 Cells Enhances Their Antitumor Efficacy.},
journal = {Human gene therapy},
volume = {37},
number = {17-18},
pages = {849-857},
doi = {10.1177/10430342261453858},
pmid = {42204417},
issn = {1557-7422},
mesh = {Humans ; *Proto-Oncogene Proteins c-cbl/genetics ; Animals ; Roundabout Proteins ; *Receptors, Immunologic/genetics/metabolism ; Mice ; *Killer Cells, Natural/immunology/metabolism ; *Nerve Tissue Proteins/genetics/metabolism ; Cell Line, Tumor ; *Adaptor Proteins, Signal Transducing/genetics ; Xenograft Model Antitumor Assays ; *Immunotherapy, Adoptive/methods ; Female ; CRISPR-Cas Systems ; *Receptors, Chimeric Antigen/genetics ; *Neoplasms/therapy/genetics/immunology/pathology ; Gene Knockout Techniques ; },
abstract = {Emerging evidence suggests CAR-NK cell therapy shows great promise in cancer treatment. ROBO1 is highly expressed in various cancer types, including glioblastoma, hepatocellular carcinoma, lung cancer, breast cancer, and uterine cancer. Our and other laboratories' studies have shown that ROBO1 CAR-NK cells exhibit promising tumor therapeutic effects. However, the results still have some limitations. Cbl-b, an E3 ubiquitin ligase, has been reported to negatively regulate NK cell activation, homeostasis, and antitumor immunity.[1] Therefore, we attempted to further enhance the antitumor activity of ROBO1 CAR-NK92 cells by knocking out Cbl-b using CRISPR/Cas9 gene-editing technology. In this study, we conjugated Cbl-b sgRNA with Cas9 protein to form ribonucleoprotein complexes, which were then delivered into ROBO1 CAR-NK92 and NK-92 cells (control cells) via electroporation. Through fluorescence-activated cell sorting, limiting dilution, and sequencing, we obtained monoclonal Cbl-b-knock-out (KO) cell lines. Both in vitro cytotoxicity assays and in vivo tumor xenograft experiments were conducted to examine whether Cbl-b knockout enhances the target cell killing and tumor suppression capacities of ROBO1 CAR-NK92 cells. In this study, monoclonal cell lines of ROBO1 CAR-NK92-Cbl-b-KO and NK92-Cbl-b-KO were successfully established. In vitro, at an effector-to-target (E:T) ratio of 0.1:1, ROBO1 CAR-NK92-Cbl-b-KO (50.55%) cells exhibited significantly higher cytolytic activity against ROBO1-positive T47D target cells after 3 h of coculture than ROBO1 CAR-NK92 (34.10%), NK92-Cbl-b-KO (22.22%), and parental NK-92 cells (3.28%). In vivo, tumor volume and weight measurements demonstrated that mice treated with ROBO1 CAR-NK92-Cbl-b-KO cells developed significantly smaller tumors than all control groups, achieving a tumor growth inhibition (TGI) rate of 32.45%, indicating enhanced antitumor efficacy conferred by Cbl-b knockout. In vitro and in vivo data confirmed that Cbl-b knockout potentiates the antitumor efficacy of ROBO1 CAR-NK92 cells. The overall cytotoxic capability ranked as follows: ROBO1 CAR-NK92-Cbl-b-KO > ROBO1 CAR-NK92 > NK92-Cbl-b-KO > NK-92.},
}

Humans
*Proto-Oncogene Proteins c-cbl/genetics
Animals
Roundabout Proteins
*Receptors, Immunologic/genetics/metabolism
Mice
*Killer Cells, Natural/immunology/metabolism
*Nerve Tissue Proteins/genetics/metabolism
Cell Line, Tumor
*Adaptor Proteins, Signal Transducing/genetics
Xenograft Model Antitumor Assays
*Immunotherapy, Adoptive/methods
Female
CRISPR-Cas Systems
*Receptors, Chimeric Antigen/genetics
*Neoplasms/therapy/genetics/immunology/pathology
Gene Knockout Techniques

@article {pmid42214263,
year = {2026},
author = {Han, Y and Woo, SJ and Choi, HJ and Han, JY},
title = {Compact Cas12f enables genome editing in avian cells.},
journal = {Poultry science},
volume = {105},
number = {9},
pages = {107116},
pmid = {42214263},
issn = {1525-3171},
mesh = {Animals ; *Gene Editing/veterinary/methods ; *Chickens/genetics ; *CRISPR-Cas Systems ; Male ; },
abstract = {Precise genome editing in avian species has been constrained by the low delivery efficiency of conventional CRISPR nucleases, such as Cas9 and Cas12a, due to their large molecular sizes. Cas12f (also known as Cas14), a compact CRISPR nuclease, has emerged as a potential genome editing system with enhanced delivery efficiency in mammalian systems. However, its effectiveness in avian systems has not been previously validated. Here, Cas12f showed notable transfection efficiency and intracellular expression in chicken Leghorn male hepatoma (LMH) cells and primordial germ cells (PGCs), with no detectable cytotoxicity. Next-generation sequencing (NGS) revealed that Cas12f achieved locus-dependent on-target editing efficiencies, reaching up to 40% at specific loci in LMH cells. Cas12f consistently generated a deletion-dominant indel profile with minimal insertions, distinct from Cas9-mediated patterns. Off-target analysis using Sanger sequencing and Inference of CRISPR Edits (ICE) revealed a few predicted off-target candidates and no detectable off-target mutations above the detection threshold. Consistent with this observation, cross-species in silico analysis showed only a modest increase in predicted Cas12f off-target proportions with increasing genome size. These findings show that Cas12f is a compact genome editing tool in avian cells, serving as a basis for further improvement in genetic engineering and biotechnological research.},
}

Animals
*Gene Editing/veterinary/methods
*Chickens/genetics
*CRISPR-Cas Systems
Male

@article {pmid42241751,
year = {2026},
author = {Liu, H and Cruvinel, JM and Warren, WC and Ma, W and Chen, PR},
title = {Strain-specific responses of avian influenza virus to disruption of solute carrier family 35 member A1 (SLC35A1) in chicken cells.},
journal = {Poultry science},
volume = {105},
number = {9},
pages = {107178},
pmid = {42241751},
issn = {1525-3171},
mesh = {Animals ; *Chickens ; *Influenza in Birds/virology/genetics ; Cell Line ; Virus Replication ; *Influenza A virus/physiology ; *Avian Proteins/genetics/metabolism ; CRISPR-Cas Systems ; N-Acetylneuraminic Acid/metabolism ; *Nucleotide Transport Proteins/genetics/metabolism ; Influenza A Virus, H5N1 Subtype/physiology ; },
abstract = {Avian influenza virus (AIV) poses a persistent threat to poultry health and food security, with conventional control measures offering limited protection. A promising alternative is the use of gene editing to generate host resistance by ablating viral entry receptors or cellular proteins that are required for completion of the viral life cycle. The solute carrier family 35 member A1 (SLC35A1) gene encodes a Golgi-localized CMP-sialic acid transporter that is a key step in the sialylation of glycoproteins. In this study, we used the CRISPR/Cas9 system to disrupt SLC35A1 in chicken DF-1 fibroblasts and evaluated the effect on sialic acid expression and susceptibility to different strains of AIV. Lectin staining and flow cytometry confirmed a significant reduction in α2,3-linked sialic acids in SLC35A1 knockout cells, while α2,6-linked sialic acids were absent in the cells regardless of genotype. Infection experiments with three avian influenza virus strains (H5N1/PR8, H5N2, and H7N1) revealed that SLC35A1 knockout reduced viral replication in a strain-specific manner. Knockout cells infected with H5N1/PR8 showed the greatest dependence on SLC35A1-mediated sialylation with decreased viral load at 24 hours post-infection (hpi) and 48 hpi compared to wildtype cells and no observable viral growth between the timepoints. Infection of knockout cells with H5N2 resulted in a modest decrease in viral load at both timepoints as well as absence of viral growth. On the other hand, infection of knockout cells with H7N1 resulted in decreased viral load only at 48 hpi compared to wildtype cells, but the amount of virus in knockout cultures increased from 24 hpi to 48 hpi. These results demonstrate that SLC35A1 is a key host factor that supports AIV entry via α2,3-linked sialic acids; however, viral dependency on this host factor may be confounded by strain.},
}

Animals
*Chickens
*Influenza in Birds/virology/genetics
Cell Line
Virus Replication
*Influenza A virus/physiology
*Avian Proteins/genetics/metabolism
CRISPR-Cas Systems
N-Acetylneuraminic Acid/metabolism
*Nucleotide Transport Proteins/genetics/metabolism
Influenza A Virus, H5N1 Subtype/physiology

@article {pmid42283432,
year = {2026},
author = {Li, GH and Xu, Y and Liu, T and Ma, XY and Hou, MT and Pang, SC and Zhang, JP and Cheng, T and Zhang, XB},
title = {AviNP-Seq: A Blindspot-Free Single-Molecule Framework for Unmasking AAV Genome Heterogeneity and Determining Packaging Limits.},
journal = {Human gene therapy},
volume = {37},
number = {17-18},
pages = {887-902},
doi = {10.1177/10430342261454324},
pmid = {42283432},
issn = {1557-7422},
mesh = {*Dependovirus/genetics ; Humans ; *Genome, Viral ; *Nanopore Sequencing/methods ; Genetic Vectors/genetics ; CRISPR-Cas Systems ; Terminal Repeat Sequences ; *Viral Genome Packaging ; },
abstract = {Comprehensive recombinant adeno-associated virus characterization is essential for establishing the knowledge base required to ensure clinical safety and efficacy, yet current long-read methods suffer from library preparation biases that obscure genome integrity. We present AviNP-seq, a blindspot-free nanopore sequencing framework utilizing one-end-sufficient ligation and Cas9-ribonucleoprotein (RNP) linearization to minimize terminal selection. Applied to a 1.5-6.5 kb panel, AviNP-seq delineates a sharp packaging cliff at 5.0-5.2 kb and reveals that sequence structure modulates integrity by 2-5× at fixed lengths. It unmasks covalent head-to-tail tandems in sub-3 kb vectors, detecting them with significantly higher sensitivity than PacBio HiFi. The Cas9-RNP step boosts ligation yield ∼7-fold, providing an unbiased assessment of genome integrity (≥95% inverted terminal repeat [ITR]-to-ITR). In addition, the assay quantifies plasmid impurities down to 0.05% with linear response. By integrating integrity mapping, tandem detection, and impurity profiling into a rapid (<36 h), low-input workflow, AviNP-seq provides a robust analytical tool to guide vector design and de-risk early-stage process development.},
}

*Dependovirus/genetics
Humans
*Genome, Viral
*Nanopore Sequencing/methods
Genetic Vectors/genetics
CRISPR-Cas Systems
Terminal Repeat Sequences
*Viral Genome Packaging

@article {pmid42612103,
year = {2026},
author = {Viskadourou, M and Workman, JN and Burke, EV and Eckley, DM and Dohr, J and Newby, GA},
title = {Genome Editing in Primary Mammalian Cells via Electroporation of Editor RNA.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {233},
pages = {},
doi = {10.3791/71449},
pmid = {42612103},
issn = {1940-087X},
support = {R00 HL163805/HL/NHLBI NIH HHS/United States ; DP2 OD038783/OD/NIH HHS/United States ; T32 GM148383/GM/NIGMS NIH HHS/United States ; },
mesh = {*Electroporation/methods ; Humans ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics/administration & dosage ; *RNA, Messenger/genetics/administration & dosage ; Animals ; Induced Pluripotent Stem Cells ; Fibroblasts ; Electroporation Therapies ; },
abstract = {CRISPR editors including nucleases, base editors, and prime editors can efficiently correct disease-causing genetic variants or disrupt target genes. Editing outcomes are commonly evaluated in cultured primary cells, patient-derived cells, or engineered cell lines to study the impact of genetic variation or as a first step before initiating animal studies or clinical translation. Delivery of editors as mRNA together with synthetic guide RNAs into mammalian cells can improve editing efficiency relative to plasmid-based approaches and prevent issues such as DNA integration or off-target editing from sustained expression. This article presents a workflow to prepare genome editor mRNA by in vitro transcription (IVT), including co-transcriptional capping and chemically-modified nucleotides, electroporate editor mRNA and guide RNAs into primary human fibroblasts, induced pluripotent stem cells (iPSCs), or lymphoblastoid cell lines (LCLs), and quantify editing outcomes by targeted amplicon sequencing on an Illumina platform followed by analysis using CRISPResso2. This workflow enables quantitative benchmarking of guide RNAs, electroporation parameters, and editor variants, and supports downstream applications including single-cell cloning, phenotypic assays, preclinical animal studies, and therapeutic development.},
}

*Electroporation/methods
Humans
*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics/administration & dosage
*RNA, Messenger/genetics/administration & dosage
Animals
Induced Pluripotent Stem Cells
Fibroblasts
Electroporation Therapies

@article {pmid42612424,
year = {2026},
author = {Chen, C and Afshar-Saber, W and Iglesias, I and Kim, K and Lewis, B and Srinivasan, G and Chen, C and Hirsh, R and Guardado, R and Polanco, T and Swanson, A and Norabuena, E and Whye, D and Jain, A and Cai, C and Sun, L and Chopra, M and Chen, I and Iannello, G and Rozumny, B and Hanson, E and Sahin, M and Buttermore, ED},
title = {Development of a human iPSC and patient phenotyping resource for preclinical investigations of neurodevelopmental disorders.},
journal = {Stem cell research},
volume = {95},
number = {},
pages = {104073},
doi = {10.1016/j.scr.2026.104073},
pmid = {42612424},
issn = {1876-7753},
abstract = {In this manuscript, we report the development of a comprehensive resource designed to harness the transformative potential of patient-derived induced pluripotent stem cells (iPSCs) to advance the study of neurodevelopmental disorders (NDDs). Using CRISPR-Cas-mediated genome editing, the Human Neuron Core generated a repository comprising 29 isogenic iPSC pairs, two sex-matched parental control iPSC pairs, and one unmatched patient line representing six monogenic NDDs: Tuberous Sclerosis Complex, PTEN Hamartoma Tumor Syndrome, KCNQ2 Developmental and Epileptic Encephalopathy, FOXG1 Syndrome, Phelan-McDermid Syndrome, and SETBP1 Haploinsufficiency Disorder. In parallel, detailed clinical phenotyping data were collected to enable comparison of cellular phenotypes with clinical severity in future studies. This integrated collection of genetically defined iPSC lines and associated clinical data provides a powerful platform for investigating disease mechanisms and advancing iPSC-based drug discovery for NDDs.},
}

@article {pmid42613564,
year = {2026},
author = {Xu, S and G C, B and Tan, K and Wu, C},
title = {Gene Silencing by CRISPR Interference in Fusobacterium nucleatum.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3055},
number = {},
pages = {67-79},
pmid = {42613564},
issn = {1940-6029},
support = {R01 DE030895/DE/NIDCR NIH HHS/United States ; R21 DE034542/DE/NIDCR NIH HHS/United States ; },
mesh = {*Fusobacterium nucleatum/genetics ; *Gene Silencing ; *CRISPR-Cas Systems ; Plasmids/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins/genetics ; },
abstract = {Fusobacterium nucleatum is a strictly anaerobic bacterium associated with periodontal disease and several systemic conditions, including colorectal cancer and adverse pregnancy outcomes. Genetic manipulation in F. nucleatum has been limited by poor transformation efficiency and difficulty studying essential genes. To overcome these challenges, we developed a CRISPR interference (CRISPRi) system that enables efficient and reversible gene silencing without altering the genome. This system uses an inducible dCas9 and a customizable sgRNA expressed from a pCWU6-based shuttle plasmid (pZP4C). In this chapter, we present a step-by-step protocol for designing sgRNAs, constructing CRISPRi plasmids, transforming F. nucleatum ATCC 23726, and evaluating gene silencing phenotypes. We use the nonessential gene ftsW, which encodes a protein required for peptidoglycan synthesis and cell division, as a model target. This protocol is also applicable to other genetically recalcitrant F. nucleatum strains, offering a versatile tool for investigating both essential and nonessential gene functions.},
}

*Fusobacterium nucleatum/genetics
*Gene Silencing
*CRISPR-Cas Systems
Plasmids/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
Bacterial Proteins/genetics

@article {pmid42614264,
year = {2026},
author = {Keerthi, V and Ravindran, P and Kaliyur, S and Tuttagunta, SUS and Mathpal, S and Joshi, T and Ramaiah, S and Anbarasu, A},
title = {A comprehensive review of emerging therapeutic strategies against methicillin-resistant Staphylococcus aureus.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1815573},
pmid = {42614264},
issn = {1664-302X},
abstract = {Methicillin-resistant Staphylococcus aureus (MRSA) remains one of the most significant multidrug-resistant bacterial pathogens responsible for a broad spectrum of infections ranging from mild skin infections to severe invasive diseases, including bacteremia, pneumonia, endocarditis, osteomyelitis, and sepsis. The rapid global dissemination of MRSA is primarily driven by the acquisition of the mecA gene encoding penicillin-binding protein 2a, which confers resistance to β-lactam antibiotics. In addition to β-lactam resistance, MRSA exhibits resistance to multiple antimicrobial classes through diverse mechanisms, including target-site mutations, efflux pumps, biofilm formation, horizontal gene transfer, and adaptive phenotypic variations. The virulence and persistence of MRSA is further enhanced by numerous virulence factors such as adhesins, toxins, immune evasion proteins, and extracellular enzymes that facilitate colonization, persistence, and host tissue damage. Biofilm formation additionally contributes to chronic infection and antibiotic tolerance. Despite the availability of conventional agents such as vancomycin, linezolid, and daptomycin, the emergence of resistant strains including vancomycin-resistant Staphylococcus aureus has significantly limited current therapeutic options. Consequently, there is an urgent need for innovative therapeutic strategies. This review comprehensively summarizes the evolution, pathogenesis, virulence mechanisms, biofilm biology, and antibiotic resistance mechanisms of MRSA, with particular emphasis on emerging therapeutic approaches. Novel strategies including antimicrobial peptides, nanomedicine, bacteriophage therapy, CRISPR-Cas systems, biomimetic nano-NETs, probiotics, monoclonal antibodies and plant-derived compounds are discussed as promising alternatives or adjuncts to conventional antibiotics. Collectively, these advances highlight the evolving landscape of MRSA management and the potential for next-generation therapeutics to overcome antimicrobial resistance challenges.},
}

@article {pmid42614947,
year = {2026},
author = {Wang, S and Kang, L and Li, M and Zhou, X and Li, B and Wang, F and Meng, J and Li, C and Yang, K},
title = {Comparative genomic analyses provide new insights into phylogenetic and functional diversification in genus Fenollaria.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1862120},
pmid = {42614947},
issn = {1664-302X},
abstract = {The genus Fenollaria has gained attention due to its associations with human prostate cancer, colorectal cancer and other diseases. The higher abundance of Fenollaria was believed to be associated with biochemical recurrence of prostate cancer but remission of colorectal cancer. Owing to the fastidious growth requirements of Fenollaria species in laboratory isolation and culture, the genomes of isolated strains is rarely available. Consequently, only limited comparative genomic studies have been conducted, leaving knowledge gap regarding the genomic diversity, distribution of functional genes, and evolutionary relationships, which hindered the understanding of ecological adaptation and mechanism exploration of Fenollaria. Here, a large-scaled genomic investigation of Fenollaria genus was performed using four high quality MAGs generated in this study and publicly available genomic data. The four MAGs were constructed from urine metagenome samples from bladder cancer patients, which were under conditions of oligotrophy and limited oxygen. Four mono-clades were revealed by phylogenomic analysis, representing for three previously described species (i.e., F. massiliensis, F. timonensis, and F. sporofastidiosus) as well as a novel proposed Fenollaria species. The divergences among these clades were also supported by genome-wide G + C content, ANI and AAI values. The functional difference between clades were revealed by the distribution of clade-specific genes in COG categories, as well as the biased distribution of ARGs, VFs, and CRISPR-Cas systems.},
}

@article {pmid42616864,
year = {2026},
author = {Chou, CW and Sinan, S and Kuo, HC and Chang, YC and Arguello, C and Sahaya, D and Russell, R and Finkelstein, IJ},
title = {Structural basis for target discrimination and activation by Cas13d.},
journal = {Science advances},
volume = {12},
number = {34},
pages = {eaec4221},
pmid = {42616864},
issn = {2375-2548},
support = {R35 GM131777/GM/NIGMS NIH HHS/United States ; },
mesh = {Cryoelectron Microscopy ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/chemistry/metabolism/genetics ; Catalytic Domain ; Models, Molecular ; Protein Binding ; Protein Domains ; },
abstract = {CRISPR-Cas13d is increasingly used for RNA knockdowns, but off-target cleavage of near-cognate RNAs hinders its broader adoption. Here, we solve seven cryo-electron microscopy structures of wild-type Cas13d in complex with matched and mismatched targets. These structures reveal active, intermediate, and inactive states that illustrate a detailed activation mechanism. Upon target RNA binding, the CRISPR RNA undergoes marked conformational changes. The Helical-1 domain transitions from a docked state with the amino-terminal domain to an allosterically switched conformation that stabilizes the RNA duplex. Quantitative kinetics show that a single proximal mismatch preserves the binding rate constant but abolishes nuclease activity by trapping Cas13d in an inactive state. We also identify an active site loop in the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains that regulates substrate accessibility and can be mutated to generate both hypo- and hyperactivated variants. These findings establish the structural basis for Cas13d mismatch surveillance and provide a framework for engineering HEPN nuclease specificity and activity.},
}

Cryoelectron Microscopy
*CRISPR-Cas Systems
*CRISPR-Associated Proteins/chemistry/metabolism/genetics
Catalytic Domain
Models, Molecular
Protein Binding
Protein Domains

@article {pmid42617139,
year = {2026},
author = {Wang, X and Wu, S and Ding, Y and Ding, J and Li, P and Lian, Z},
title = {A genome-wide CRISPR knockout screen identified host genes essential for Brucella invasion and intracellular survival.},
journal = {Emerging microbes & infections},
volume = {15},
number = {1},
pages = {2713322},
pmid = {42617139},
issn = {2222-1751},
mesh = {Humans ; *Macrophages/microbiology ; *Brucella/physiology/genetics ; *Brucellosis/microbiology/genetics ; Gene Knockout Techniques ; *Host-Pathogen Interactions/genetics ; THP-1 Cells ; CRISPR-Cas Systems ; Apoptosis ; Clustered Regularly Interspaced Short Palindromic Repeats ; Microbial Viability ; },
abstract = {For Brucella spp., the ability to invade and survive within host macrophages is essential for causing chronic infections in their mammalian hosts. In this study, a genome-wide CRISPR knockout screen was performed for the first time in human THP-1 macrophages to identify host genes mediating resistance to Brucella invasion and intracellular survival. Results showed that the screening identified 35 candidate genes, 11 of which were selected to generate monoclonal knockout cell lines for functional validation. This study demonstrated that knockout of WDR4, ZNF532, or MTHFD1 significantly restricted Brucella invasion and early intracellular survival. In addition, TRAPPC2 knockout restricted Brucella invasion and, crucially, its intracellular survival throughout infection, exerting the most potent antibacterial effect. Mechanistically, TRAPPC2 deficiency suppresses Brucella infection by inhibiting autophagosome formation in macrophages. Furthermore, TRAPPC2 knockout decreases macrophage apoptosis and improves host cell viability following Brucella infection. These results provide therapeutic targets for combating Brucella infection and offer novel insights into the molecular mechanisms associated with Brucella-induced chronic infections.},
}

Humans
*Macrophages/microbiology
*Brucella/physiology/genetics
*Brucellosis/microbiology/genetics
Gene Knockout Techniques
*Host-Pathogen Interactions/genetics
THP-1 Cells
CRISPR-Cas Systems
Apoptosis
Clustered Regularly Interspaced Short Palindromic Repeats
Microbial Viability

@article {pmid42617808,
year = {2026},
author = {Xiao, J and Liang, M and Lei, Y and Huang, Z and Guo, W and Xue, L and Sun, X and Wang, Y and Chen, K and Cao, X and Fan, J and Huang, Z and Chen, M},
title = {Research Advances in Modern Immunoassay Technologies and Novel Alternative Biotechnologies for Rapid Detection of Foodborne Pathogens and Chemical Contaminants.},
journal = {Journal of food protection},
volume = {},
number = {},
pages = {100896},
doi = {10.1016/j.jfp.2026.100896},
pmid = {42617808},
issn = {1944-9097},
abstract = {The development and application of novel rapid detection technologies are critical for advancing food safety regulation. In recent years, immunology-based methods have played a pivotal role in food safety supervision due to their speed, operational simplicity, and cost-effectiveness. However, conventional colloidal gold techniques exhibit limited sensitivity for trace analytes (e.g., early-stage microbial contamination) and are primarily qualitative, thus failing to meet the quantitative detection requirements for pesticides, veterinary drugs, and food additives, which restricts their practical applicability. Additionally, the difficulty and high cost of obtaining high-quality monoclonal antibodies increase the technical barriers and commercialization costs of immunochromatographic assays. Consequently, modern immunological techniques and novel alternative biotechnologies have emerged as focal points in rapid detection research. This review examines the principles and technical characteristics of advanced immunological methods, including immunofluorescence quantitative chromatography (IF-QCT) and flow cytometry-based immunophenotyping (FCI), as well as emerging antibody-alternative technologies such as aptamers, CRISPR/Cas systems, and phage-based technologies. This review further summarizes the latest research advances of the above-mentioned technologies in the rapid detection of food safety risk factors, including pathogenic microorganisms, biotoxins, residues of pesticides and veterinary drugs, heavy metals, and other chemical contaminants. It critically analyzes their technical advantages and practical limitations, and discusses potential future directions. This review aims to provide insights and a theoretical basis for developing and applying technologies to rapidly detect food safety hazards.},
}

@article {pmid42618112,
year = {2026},
author = {Sanjay, BR and Nishanth, MAD and Vergis, J and Pollumahanti, N and Chatlod, L and Gadekar, Y and Muthukumar, M and Reddy, BP and Malik, SVS and Barbuddhe, SB and Rawool, DB},
title = {LAMP-assisted CRISPR-Cas12a platform for detection of Bacillus anthracis spores in environmental samples.},
journal = {Analytica chimica acta},
volume = {1419},
number = {},
pages = {345904},
doi = {10.1016/j.aca.2026.345904},
pmid = {42618112},
issn = {1873-4324},
mesh = {*Bacillus anthracis/isolation & purification/genetics ; *Spores, Bacterial/isolation & purification/genetics ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Soil Microbiology ; Limit of Detection ; *Molecular Diagnostic Techniques/methods ; },
abstract = {Environmental persistence of Bacillus anthracis spores sustains anthrax transmission, necessitating rapid and field-deployable detection tools. This study aimed to develop and quantitatively evaluate a LAMP-assisted CRISPR-Cas12a assay for sensitive and specific detection of B. anthracis spores in soil and meat meal matrices. Two B. anthracis-specific target genes plasmid-encoded lef and chromosomal SNP locus (CR5)- were selected. Target-specific LAMP primers and CRISPR crRNAs were designed using Primer Explorer, CRISPOR, and RNAfold platforms. The LAMP-CRISPR/Cas12a assay was optimised for reaction conditions and evaluated for analytical sensitivity and specificity using UV-inactivated spores and closely related Bacillus spp. Environmental applicability was assessed via spiking experiments in sterile soil and meat meal using the GABRI recovery method. Field validation was performed on 100 samples from anthrax-endemic regions of India, with performance compared against the WOAH-recommended lef gene-based real-time PCR. LAMP-CRISPR-Cas12a assay achieved detection limits of 10 spores/ml (lef) and 10[2] spores/ml (CR5), with no cross-reactivity against related species. In spiked matrices, spore recovery ranged from 50 to 75%, and detection sensitivity remained consistent. Field evaluation demonstrated a sensitivity of 91.70% and specificity of 100%, with near-perfect agreement (κ = 0.95) relative to real-time PCR. The assay delivered results within ∼70 min, including amplification and detection. The LAMP-assisted CRISPR-Cas12a platform provides a rapid, sensitive, and cost-effective approach for environmental detection of B. anthracis spores. The minimal equipment requirements and high diagnostic accuracy support its applicability for field-level surveillance and biosafety monitoring in resource-limited settings.},
}

*Bacillus anthracis/isolation & purification/genetics
*Spores, Bacterial/isolation & purification/genetics
*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
Soil Microbiology
Limit of Detection
*Molecular Diagnostic Techniques/methods

@article {pmid42618126,
year = {2026},
author = {Tao, Q and Xing, Y and Yang, H and Deng, L and He, M and Cheng, Y and Wang, Q and Gao, Z and Wan, H and Li, D and Ling, S},
title = {Field-deployable RspCas13d platform for rapid and extraction-free detection of giant panda- and canine-derived Canine Distemper virus.},
journal = {Analytica chimica acta},
volume = {1419},
number = {},
pages = {345960},
doi = {10.1016/j.aca.2026.345960},
pmid = {42618126},
issn = {1873-4324},
mesh = {*Distemper Virus, Canine/isolation & purification/genetics ; Animals ; Dogs ; *Ursidae/virology ; *RNA, Viral/genetics/analysis ; Nucleic Acid Amplification Techniques/methods ; *Distemper/diagnosis/virology ; Rapid Diagnostic Tests ; CRISPR-Cas Systems/genetics ; },
abstract = {BACKGROUND: Canine distemper virus (CDV) is a highly contagious RNA virus that causes severe disease in domestic dogs and diverse wildlife species, including endangered giant pandas. Current CDV diagnosis mainly relies on laboratory-based RT-qPCR, which requires nucleic acid extraction, trained personnel, and temperature-controlled instruments, limiting its use in field surveillance and resource-limited settings. Although CRISPR-based diagnostics offer promising alternatives, chemically defined extraction-free workflows compatible with one-pot Cas13d detection remain limited. This study addresses the need for a rapid, visual, extraction-free, and low-infrastructure method for CDV detection.

RESULTS: We developed CLEAR-VISION, an integrated CRISPR diagnostic platform combining CLEAR (Chemical Lysis for Extraction-free Access to RNA) with VISION (Visual Isothermal Single-tube Integrated One-pot Nucleic acid detection). VISION integrates RPA amplification, T7 transcription, and RspCas13d-based detection into a single-tube reaction supported by a chemically defined buffer. CLEAR enabled rapid RNA release at room temperature without extraction kits or heating and was compatible with downstream one-pot detection. The PAM- and PFS-independent property of RspCas13d allowed flexible target selection, while lyophilized reagents improved storage and transportation convenience. CLEAR-VISION enabled CDV detection within 30 min and provided dual visual readouts, including fluorescence and lateral flow assays. Clinical evaluation in the current sample set showed consistent results with RT-qPCR for samples from giant pandas, stray dogs, and pet dogs. The assay also maintained stable performance at physiological temperature (37 °C), reducing reliance on temperature-controlled equipment.

SIGNIFICANCE AND NOVELTY: CLEAR-VISION provides a chemically defined, extraction-free, and low-infrastructure CRISPR diagnostic workflow for rapid CDV detection. Its novelty lies in integrating room-temperature chemical lysis with single-tube RPA-T7-RspCas13d detection, lyophilized reagents, and dual visual readouts. This platform supports the potential application of CRISPR-based diagnostics for on-site CDV surveillance in domestic animals and wildlife.},
}

*Distemper Virus, Canine/isolation & purification/genetics
Animals
Dogs
*Ursidae/virology
*RNA, Viral/genetics/analysis
Nucleic Acid Amplification Techniques/methods
*Distemper/diagnosis/virology
Rapid Diagnostic Tests
CRISPR-Cas Systems/genetics

@article {pmid42618136,
year = {2026},
author = {Shao, LN and Zheng-Luo, and Liu, BL and Tong-Xu, and Wang, YM and Duan, JQ and Lei-Zhao, and Li, YY and Dai, YM and Jia, QR and Zhang, LY and Ling-Zhu, and Xu, ZW},
title = {A lyophilized RPA-CRISPR/Cas13d one-pot platform for rapid detection of porcine circovirus type 3.},
journal = {Analytica chimica acta},
volume = {1419},
number = {},
pages = {345832},
doi = {10.1016/j.aca.2026.345832},
pmid = {42618136},
issn = {1873-4324},
mesh = {*Circovirus/isolation & purification/genetics ; Animals ; Swine ; *CRISPR-Cas Systems/genetics ; Freeze Drying ; *Nucleic Acid Amplification Techniques/methods ; DNA, Viral/genetics ; Rapid Diagnostic Tests ; },
abstract = {Rapid molecular diagnostics are essential for effective surveillance of infectious diseases in swine production systems. Here, we report SHARP (Single-step Hybrid RPA-CRISPR/EsCas13d Platform), a one-pot CRISPR-based platform for detection of porcine circovirus type 3 (PCV3). By integrating rapid nucleic acid release with CRISPR/Cas13d detection, a single-step one-pot detection system was established, enabling detection within 30 min under simplified reaction conditions. In the visual readout mode, the detection limit was 50 copies/μL, with no cross-reactivity observed against common swine viruses. After lyophilization and rehydration, the SHARP system showed consistent detection performance across 50 clinical samples, in agreement with qPCR results. These results indicate that SHARP provides a simplified workflow for rapid PCV3 detection, with potential for decentralized molecular surveillance in livestock production systems.},
}

*Circovirus/isolation & purification/genetics
Animals
Swine
*CRISPR-Cas Systems/genetics
Freeze Drying
*Nucleic Acid Amplification Techniques/methods
DNA, Viral/genetics
Rapid Diagnostic Tests

@article {pmid42620120,
year = {2026},
author = {Brodmann, M and Baca, CF and Chandanani, J and Campbell, EA and Marraffini, LA},
title = {MtvS1 and MtvS2 Interact with RNA Polymerase to Regulate the Francisella Type V-A CRISPR-Cas System.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {42620120},
issn = {2692-8205},
support = {R35 GM151879/GM/NIGMS NIH HHS/United States ; },
abstract = {Bacteria and archaea often harbor multiple CRISPR-Cas loci to defend against mobile genetic elements. Little is known, however, about whether and how different CRISPR-Cas systems are differentially regulated, in many instances due to the impossibility of studying CRISPR immunity in native hosts. Here we investigated the regulation of the endogenous type II-B and type V-A CRISPR-Cas systems present in the opportunistic human pathogen Francisella novicida U112. We found that while the type II-B system is constitutively expressed, the type V-A system is differentially expressed at stationary phase and high cell density. We identified MtvS1 and MtvS2 as factors required for this regulation, as well as for the modulation of many additional genes in stationary phase, some of which are required for Francisella virulence. Both Francisella MtvS proteins bind to RNA polymerase. MtvS1 is predicted to interact with the β' subunit of the RNA polymerase, and MtvS2 with multiple RNA polymerase subunits as well as MtvS1. We propose that MtvS1 and MtvS2 constitute noncanonical alternative sigma factors involved in the regulation of the expression of the type V-A CRISPR locus and other genes in Francisella. Last, we show that the MtvS1 homolog YgfB is required for expression of the type I-E CRISPR-Cas system in E. coli, a result that suggests a broader role in gene regulation for these alternative sigma factors.},
}

@article {pmid42620293,
year = {2026},
author = {Matrishin, CB and Haase, EM and Miles, AK and Steimer, S and Soh, D and Smardz, M and Diaz, PI and Kauffman, KM},
title = {Pervasive integrative and conjugative elements shape Porphyromonas gingivalis gene repertoires.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.64898/2026.08.04.741601},
pmid = {42620293},
issn = {2692-8205},
abstract = {BACKGROUND: Porphyromonas gingivalis (Pg) is an oral pathobiont that contributes to periodontal disease and has been associated with systemic health conditions. Although Pg is recognized as exhibiting extensive strain-level genomic diversity and recombination, the extent to which mobile elements contribute to this variation, and their relevance to its fitness and virulence, remain incompletely understood. Our recent study of the Pg pangenome revealed diverse accessory defense-associated genes, raising the question of whether these are carried by unrecognized mobile genetic elements (MGEs). Integrative and conjugative elements (ICEs) are large autonomous mobile elements that often encode genes for proteins beneficial to their bacterial hosts, including defense systems that protect against phage infection. To date, only one ICE, CTnPg1, has been described in Pg .

RESULTS: Here, we developed a bioinformatic approach integrating ICE prediction and curation, hallmark-gene detection, and genomic-context analysis, to investigate ICEs in Pg . We discovered that ICEs are pervasive in Pg genomes, with >90% of genomes harboring at least one ICE. We found that these elements comprise at least five distinct groups, two of which dominate and frequently co-occur in Pg genomes, inserting into distinct characteristic insertion sites. Using marker-gene analysis of enrichment-culture mini-metagenomes from subjects with periodontal disease we detected representatives of these dominant Pg ICE groups, as well as others, in recent clinical samples. We found that anti-defense and defense genes are common in Pg ICEs, and that these elements commonly encode biosynthetic gene clusters, including for menaquinone synthesis and predicted ribosomally synthesized and post-translationally modified peptides (RiPPs). In contrast to the extensive CRISPR-Cas defense targeting we observed for Pg phages, we detected no exact matches between ICE sequences and Pg CRISPR spacers.

CONCLUSION: This work establishes that ICEs are pervasive contributors to Pg 's pangenome and unique strain-level gene repertoires. Their distinct cargo profiles suggest that ICEs likely impact the virulence and ecology of Pg through the introduction and spread of advantageous traits, including expansion of Pg 's biosynthetic capacity and resistance to phage infection. This work provides a curated framework for investigating ICE diversity in Pg and establishes a foundation for expanded experimental studies of their host ranges and roles in shaping Pg 's interactions with phages, other microbes, and the human host.},
}

@article {pmid40216545,
year = {2026},
author = {Febrianti, RA and Narulita, E and Sulistyaningsih, E and Addy, HS},
title = {Innovations in Bacteriophage Genome Engineering for Combating Multidrug-Resistant Bacterial Infections.},
journal = {Foodborne pathogens and disease},
volume = {23},
number = {10},
pages = {639-647},
doi = {10.1089/fpd.2024.0194},
pmid = {40216545},
issn = {1556-7125},
mesh = {*Bacteriophages/genetics ; *Drug Resistance, Multiple, Bacterial ; *Genetic Engineering/methods ; *Phage Therapy/methods ; *Genome, Viral ; *Bacterial Infections/therapy/microbiology ; CRISPR-Cas Systems ; Humans ; Bacteria/virology ; Anti-Bacterial Agents/pharmacology ; },
abstract = {Bacteriophage engineering is a promising strategy to address multidrug-resistant (MDR) bacterial infections that pose significant challenges to public health due to the overuse of antibiotics. Bacteria can develop resistance mechanisms, such as receptor modification and activation of antiviral defense systems, which further complicates the application of phage therapy. Additionally, long-term phage therapy can result in the production of anti-phage antibodies, which may interfere with treatment. These factors require advanced engineering techniques to improve the efficacy of phages and expand their host range. Recent advances in genome engineering methods, including CRISPR/Cas9, homologous recombination, and other synthetic biology techniques, offer promising solutions to these challenges. By modifying receptor-binding proteins and using high-yield screening methods, researchers can create phages that are better equipped to target MDR bacteria effectively. Furthermore, understanding the intricate interactions between phages and their bacterial hosts is critical to guiding these engineering efforts. Future development perspectives lie in integrating these advanced engineering techniques into clinical practice, potentially putting bacteriophages at the forefront of fighting MDR bacterial infections.},
}

*Bacteriophages/genetics
*Drug Resistance, Multiple, Bacterial
*Genetic Engineering/methods
*Phage Therapy/methods
*Genome, Viral
*Bacterial Infections/therapy/microbiology
CRISPR-Cas Systems
Humans
Bacteria/virology
Anti-Bacterial Agents/pharmacology

@article {pmid42418712,
year = {2026},
author = {Poch, D and Mukherjee, C and Mallik, S and Todorow, V and Kuiper, EFE and Dhingra, N and Surovtseva, YV and Schlieker, C},
title = {Integrative chemical genetics platform identifies condensate modulators linked to neurological disorders.},
journal = {Molecular biology of the cell},
volume = {37},
number = {9},
pages = {ar86},
doi = {10.1091/mbc.E26-06-0256},
pmid = {42418712},
issn = {1939-4586},
mesh = {Humans ; Nuclear Envelope/metabolism ; Drug Discovery/methods ; *Nervous System Diseases/genetics/metabolism ; Molecular Chaperones/metabolism/genetics ; Ubiquitin-Protein Ligases/metabolism/genetics ; CRISPR-Cas Systems ; Animals ; },
abstract = {Dysregulation of biomolecular condensates is implicated across multiple neurological disorders. However, approaches to systematically identify their modulators remain limited. Here, we expand the utility of MLF2 as a versatile condensate biomarker and develop CondenScreen, an integrated high-content screening and bioinformatics pipeline enabling identification of condensate modulators across chemical and genetic space. Screening 1760 bioactive compounds in a cellular DYT1 dystonia model, we validate the platform for condensate-targeted drug discovery, identifying drugs that prevent the accumulation of the MLF2 reporter into nuclear envelope condensates. In parallel, a genome-wide CRISPR/Cas9 screen correlates nuclear condensate abundance with genes implicated in microcephaly and over eight additional neurodevelopmental disorders. Machine learning and confocal imaging resolve distinct condensate phenotypes, with RNF26 deletion provoking nuclear envelope condensates that phenocopy hallmarks of torsin deficiency. Our study provides a scalable platform for identifying modulators of condensates and establishes a correlative connection between nuclear condensate accumulation and genes implicated in neurodevelopmental disorders.},
}

Humans
Nuclear Envelope/metabolism
Drug Discovery/methods
*Nervous System Diseases/genetics/metabolism
Molecular Chaperones/metabolism/genetics
Ubiquitin-Protein Ligases/metabolism/genetics
CRISPR-Cas Systems
Animals

@article {pmid42478827,
year = {2026},
author = {Wang, H and Liu, L and Bao, C and Li, F and He, Y and Liu, X and Yin, Y and Xu, S},
title = {A low-cost CHA-integrated CRISPR/Cas12a-based test strip platform for on-site gene detection.},
journal = {Lab on a chip},
volume = {26},
number = {17},
pages = {4679-4688},
doi = {10.1039/d6lc00394j},
pmid = {42478827},
issn = {1473-0189},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Chorionic Gonadotropin/analysis/genetics ; *Pregnancy Tests/instrumentation ; *Reagent Strips ; Female ; DNA/genetics/analysis ; },
abstract = {On-site nucleic acid detection plays a crucial role in disease diagnosis, biosafety monitoring, and food quality control. This study develops a novel nucleic acid detection platform that integrates catalytic hairpin assembly (CHA) with the CRISPR/Cas12a system and utilizes pregnancy test strips (PTS) for result visualization, addressing the limitations of existing nucleic acid detection methods in balancing sensitivity, specificity, and portability with cost and dependence on a cleanroom. The main mechanism involves the following three steps. The presence of target RNA triggers the CHA reaction, generating double-stranded DNA (dsDNA) as an activation unit. Subsequently, this unit activates the CRISPR/Cas12a system to specifically cleave the single-stranded DNA (ssDNA) that has bridged human chorionic gonadotropin (HCG) to a magnetic bead, ultimately releasing HCG that produces a visual result on the PTS. This dual-signal amplification strategy (CHA cycling and Cas12a trans-cleavage) can detect concentrations as low as 10 pM in approximately 50 min, without the need for pre-amplification of the target nucleic acid. This detection system ensures high sensitivity and specificity while effectively avoiding non-specific activation. In practical applications with transgenic maize samples, the detection results are highly consistent with those of real-time quantitative polymerase chain reaction (qPCR), validating its reliability in real-world scenarios. This innovative method offers advantages such as simple operation and low cost, providing an efficient tool for rapid nucleic acid detection while demonstrating broad potential for application in resource-limited settings.},
}

*CRISPR-Cas Systems/genetics
Humans
Chorionic Gonadotropin/analysis/genetics
*Pregnancy Tests/instrumentation
*Reagent Strips
Female
DNA/genetics/analysis

@article {pmid42611132,
year = {2026},
author = {Ramesh, S and Kumar, N and Ghosh, A},
title = {A novel needleless delivery of Cas9 ribonucleoprotein complex in Bemisia tabaci embryos for gene editing.},
journal = {World journal of microbiology & biotechnology},
volume = {42},
number = {9},
pages = {},
pmid = {42611132},
issn = {1573-0972},
support = {(BT/PR40767/AGIII/103/1277/2020).//Department of Biotechnology, Ministry of Science and Technology, India/ ; },
mesh = {Animals ; *Gene Editing/methods ; *Ribonucleoproteins/genetics/metabolism ; *CRISPR-Cas Systems ; *Hemiptera/genetics/embryology ; RNA, Guide, CRISPR-Cas Systems/genetics ; HSP70 Heat-Shock Proteins/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {The CRISPR/Cas9 system, particularly the Cas9-sgRNA ribonucleoprotein (RNP) complex, offers a highly efficient platform for gene editing. However, traditional microinjection methods for RNP delivery in insect embryos are labor-intensive, technically demanding, and often reduce embryo viability, especially in species with fragile, microscopic embryos. In this study, a novel, non-invasive delivery method for the RNP complex in tiny insect embryos has been optimized. Whitefly, Bemisia tabaci, an invasive insect pest of agricultural importance and vector of plant diseases, was considered as a model organism. A needleless dechorionation-permeabilization method was employed to deliver the RNP complex to the early embryonic stage of B. tabaci embryos. B. tabaci heat shock protein 70 (hsp70) gene, which interacts with the begomovirus coat protein, aiding in its internalization and successful transmission by B. tabaci in a persistent circulative manner, was targeted for knockout. Two sgRNAs were synthesized via in vitro transcription, and the Cas9-sgRNA complexes were validated by in vitro cleavage assays. The localization of the GFP-labelled Cas9-sgRNA complex confirmed successful RNP delivery, as observed through confocal microscopy. A survival rate of 18% of the embryos was recorded post-RNP delivery. Sequencing of treated embryos showed 25- and 28-nucleotide deletions in the hsp70 exon. Synthego ICE analysis revealed up to 84% gene knockout efficiency. This method enables batch processing of embryos, drastically reducing delivery time and associated costs while improving throughput. Hsp70 KO B. tabaci mutants generated in the study are expected to be incompetent begomovirus transmitters, which would help restrict the spread of the virus. Our study overcomes a key bottleneck in CRISPR/Cas delivery to small insect embryos, opening new avenues for rapid, high-throughput, and cost-effective RNP delivery methods in insect embryos. The novel non-invasive methods would be helpful in the deployment of gene editing for sustainable pest control.},
}

Animals
*Gene Editing/methods
*Ribonucleoproteins/genetics/metabolism
*CRISPR-Cas Systems
*Hemiptera/genetics/embryology
RNA, Guide, CRISPR-Cas Systems/genetics
HSP70 Heat-Shock Proteins/genetics
*CRISPR-Associated Protein 9/genetics/metabolism

@article {pmid42611239,
year = {2026},
author = {Zhou, Y and Li, H and Chen, M and Ye, J and Yan, Y and Yang, L and Meng, T and Jiao, D and Wang, D and Zhu, L and Yang, X},
title = {Ligand-Engineered Mn-Cysteine as a Potent Laccase Mimic for CRISPR/Cas12a Electrochemical Biosensing of Hepatocellular Carcinoma Biomarkers.},
journal = {ACS nano},
volume = {20},
number = {32},
pages = {22787-22803},
doi = {10.1021/acsnano.6c07904},
pmid = {42611239},
issn = {1936-086X},
support = {2025ZB395//Jiangsu Provincial Department of Human Resources and Social Security/ ; KY2025014//Sichuan Normal University/ ; 2024NSFSC1129//Department of Science and Technology of Sichuan Province/ ; 22404119//National Natural Science Foundation of China (NSFC)/ ; 32501984//National Natural Science Foundation of China (NSFC)/ ; },
mesh = {*Biosensing Techniques/methods ; *Laccase/chemistry/metabolism ; *Biomarkers, Tumor/analysis/blood ; *Cysteine/chemistry ; *Liver Neoplasms/diagnosis/blood ; Humans ; *Carcinoma, Hepatocellular/diagnosis/blood ; *Manganese/chemistry ; Ligands ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; MicroRNAs/analysis/blood ; alpha-Fetoproteins/analysis ; },
abstract = {Laccase is an environmentally friendly catalyst with water as the sole catalytic byproduct, yet its biomedical detection potential remains underexplored. Herein, a ligand engineering strategy was employed to synthesize Mn-cysteine nanoflowers (Mn-Cys NF) with laccase-mimicking activity via a one-pot method, using manganese (Mn) with rich valence variations as the active center and cysteine (Cys) as the ligand. Spectroscopic characterizations confirmed Cys-modulated Mn electronic structure, and theoretical calculations validated enhanced substrate adsorption and reduced reaction barriers. The specific activity of Mn-Cys NF is approximately 3.56 times that of natural laccase and exhibited excellent stability across pH, temperature, ionic strength, and organic solvent conditions. Leveraging this high-performance nanozyme, a CRISPR/Cas12a electrochemical biosensor was constructed with a DNA triangular prism interface, where a target-triggered catalytic hairpin assembly (CHA)-DNAzyme cascade regulated Cas12a cleavage to enable signal-on detection. This biosensor achieved quantification of hepatocellular carcinoma (HCC) biomarkers alpha-fetoprotein (AFP) and microRNA-122 (miRNA-122), with detection limits as low as 4.47 fg/mL and 6.21 aM, respectively. It also effectively discriminated HCC patients from healthy individuals in clinical serum samples. This work offers a ligand engineering strategy for designing high-performance laccase-mimicking nanozymes and expands the application scope of laccase nanozymes from environmental remediation to biomedical biosensing.},
}

*Biosensing Techniques/methods
*Laccase/chemistry/metabolism
*Biomarkers, Tumor/analysis/blood
*Cysteine/chemistry
*Liver Neoplasms/diagnosis/blood
Humans
*Carcinoma, Hepatocellular/diagnosis/blood
*Manganese/chemistry
Ligands
*Electrochemical Techniques/methods
*CRISPR-Cas Systems
MicroRNAs/analysis/blood
alpha-Fetoproteins/analysis

@article {pmid42611583,
year = {2026},
author = {Chun, L and Quan, Z and Ke, M},
title = {CRISPR/Cas9-Mediated Site-Directed Mutagenesis of Genes in Klebsiella pneumoniae.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {234},
pages = {},
doi = {10.3791/72662},
pmid = {42611583},
issn = {1940-087X},
mesh = {*Klebsiella pneumoniae/genetics ; *CRISPR-Cas Systems ; *Mutagenesis, Site-Directed/methods ; Plasmids/genetics ; },
abstract = {Constructing target-gene mutants with a common genetic background is crucial for elucidating gene function in antimicrobial resistance (AMR) research. Taking advantage of the single-guide RNA (sgRNA) and protospacer adjacent motif (PAM) sequence (3'-NGG) specificity of the Cas9 protein in the CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system and codon degeneracy, the authors design a repair template that incorporates the desired point mutation while excluding the PAM sequence disrupted by a synonymous substitution, thereby preventing re-cleavage by CRISPR/Cas9. This method employs two available plasmids, pCasKP-apr and pSGKP-spe, offering straightforward operation and high screening specificity. As a result, the approach enables efficient generation of genetically defined mutant strains of Klebsiella pneumoniae (K. pneumoniae) and is readily adaptable to routine laboratory settings. Furthermore, the protocol minimizes off-target editing, shortens experimental timelines, reduces screening workload, and provides a reliable platform for investigating resistance mechanisms, validating candidate genes, and supporting functional genomics studies in clinically relevant bacterial pathogens.},
}

*Klebsiella pneumoniae/genetics
*CRISPR-Cas Systems
*Mutagenesis, Site-Directed/methods
Plasmids/genetics

@article {pmid42611961,
year = {2026},
author = {Li, C and Li, L and Chen, Y and Ou, X and Ding, Y and Li, X and Hou, W and Cheng, G},
title = {CRISPR/Cas9-Mediated Generation and Characterization of an Ent2*/CyO Drosophila melanogaster Strain.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {234},
pages = {},
doi = {10.3791/69546},
pmid = {42611961},
issn = {1940-087X},
mesh = {Animals ; *Drosophila melanogaster/genetics ; *CRISPR-Cas Systems ; Female ; Male ; *Nucleotide Transport Proteins/genetics ; },
abstract = {In this study, a CRISPR/Cas9-based genome-editing approach was used to introduce mutations in the equilibrative nucleoside transporter 2 (Ent2) gene in Drosophila melanogaster. Guide RNAs targeting the coding region of Ent2 were designed and co-injected with Cas9 mRNA into w[1118] embryos. Mutant alleles were identified by Sanger sequencing and maintained as a stable Ent2*/CyO heterozygous line using a balancer chromosome. Subsequently, we evaluated body weight, climbing ability, survival rate, and the activities of superoxide dismutase (SOD) and catalase (CAT) in fruit flies at 22 °C and 25 °C, respectively. The results indicate that at both 22 °C and 25 °C, the body length and weight of Ent2*/CyO fruit flies were significantly reduced compared to the w[1118], and their development was delayed. At 22 °C, the overall lifespan of Ent2*/CyO flies was slightly longer than that of the w[1118], whereas at 25 °C, no significant difference was observed. Regarding locomotor ability, the climbing performance of heterozygous flies was significantly lower than that of the w[1118] at both temperatures, with males being more severely affected. In addition, the antioxidant enzyme activities of CAT and SOD in Ent2*/CyO fruit flies were significantly reduced, indicating a clear impairment of antioxidant capacity. These results describe the phenotypic profile of a CRISPR-generated Ent2 mutant line and demonstrate the feasibility of combining genome editing with balancer chromosome strategies in Drosophila. This study provides a methodological framework and a genetic resource for future investigations of genes associated with metabolism and environmental responses.},
}

Animals
*Drosophila melanogaster/genetics
*CRISPR-Cas Systems
Female
Male
*Nucleotide Transport Proteins/genetics

@article {pmid42189126,
year = {2026},
author = {Wang, Z and Liu, H and Wang, X and Teng, J and Zheng, Z and Zhang, J and Xiao, W and Liang, Q and Li, J and Jia, X and Feng, X and Cui, H and Luo, M and Yang, T and Wu, L and Zhao, K and Yang, W and Li, MJ and Huang, D and Yang, J},
title = {Genome-Wide CRISPR Screen Identifies a microRNA Orchestrating Pleiotropic Resistance to Targeted Therapy and T Cell Immunity in Melanoma.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {47},
pages = {e15158},
pmid = {42189126},
issn = {2198-3844},
support = {2024YFC2707700//National Key Research and Development Program of China/ ; 32000640//National Natural Science Foundation of China/ ; 82422038//National Natural Science Foundation of China/ ; 82473477//National Natural Science Foundation of China/ ; 32470671//National Natural Science Foundation of China/ ; 25ZXZSSS00910//National Key Laboratory Major Special Project of Tianjin Science and Technology Plan Project/ ; 24JCZDJC00480//Natural Science Foundation of Tianjin/ ; LZYQ25H090001//Natural Science Foundation of Zhejiang/ ; 2005DKA21300//National Human Genetic Resources Sharing Service Platform/ ; OJQD2024001//Oujiang Laboratory Research Launch Project/ ; 016YFC1201703//National Key Research and Development program of China/ ; //Cancer Biobank of Tianjin Medical University Cancer Institute and Hospital/ ; 20250204//Tianjin Medical University Cancer Hospital 'Clinical-Basic' Co-PI Project/ ; //Major Project of State Key Laboratory of Experimental Hematology in 2025/ ; //Major Project of the National Key Laboratory of Drug Developability Evaluation and Systematic Transformation in 2025/ ; },
mesh = {*MicroRNAs/genetics ; *Melanoma/genetics/immunology/therapy/drug therapy ; Humans ; Animals ; *Drug Resistance, Neoplasm/genetics ; Mice ; Cell Line, Tumor ; CRISPR-Cas Systems/genetics ; *CD8-Positive T-Lymphocytes/immunology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Acquired resistance to both targeted therapies and immunotherapies in cancer presents major clinical challenges, yet the molecular mechanisms underlying cross-resistance remain poorly understood. We hypothesized that loss of specific microRNAs (miRNAs) could potentiate melanoma resistance to both targeted drugs and CD8[+] T cell-mediated cytotoxicity. Through genome-wide miRNA CRISPR knockout screening integrated with cellular models, longitudinal clinical samples, and in vivo experiments, we identified miR-18a as a pivotal upstream regulator of pleiotropic resistance in melanoma. We show that miR-18a deficiency drives resistance through two distinct mechanisms: derepressing AJUBA-regulated Hippo signaling during MAPK inhibition, and enhancing THBS1-CD47 interactions that impair the immunological synapse between tumor cells and CD8[+] T cells. Furthermore, hnRNP A1 plays an essential role in modulating miR-18a expression, thereby mediating cross-resistance. These findings suggest that targeting non-coding RNA vulnerabilities may represent a promising therapeutic strategy to overcome complex resistance mechanisms and improve clinical outcomes in melanoma.},
}

*MicroRNAs/genetics
*Melanoma/genetics/immunology/therapy/drug therapy
Humans
Animals
*Drug Resistance, Neoplasm/genetics
Mice
Cell Line, Tumor
CRISPR-Cas Systems/genetics
*CD8-Positive T-Lymphocytes/immunology
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid42378032,
year = {2026},
author = {Huang, J and Liu, X and Floyd, W and Haugh, W and Sun, Z and Kasiewicz, MJ and Wu, Y and Piening, B and Welle, JT and Rosales, WK and Rajamanickam, V and Kim, SY and Xu, ES and Luo, L and Ma, Y and Patel, R and Zhang, Z and Bernard, B and Redmond, WL and Urba, WJ and Bell, RB and Kirsch, DG},
title = {A tailored in vivo CRISPR screen identifies BAP1 as a potent tumor suppressor of sarcoma.},
journal = {JCI insight},
volume = {11},
number = {16},
pages = {},
doi = {10.1172/jci.insight.192686},
pmid = {42378032},
issn = {2379-3708},
support = {K22 CA248849/CA/NCI NIH HHS/United States ; R35 CA197616/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *Tumor Suppressor Proteins/genetics/metabolism ; Mice ; *Ubiquitin Thiolesterase/genetics/metabolism ; Humans ; *Sarcoma/genetics/pathology/drug therapy/metabolism ; Polo-Like Kinase 1 ; Proto-Oncogene Proteins/metabolism/antagonists & inhibitors/genetics ; Protein Serine-Threonine Kinases/metabolism/antagonists & inhibitors/genetics ; Cell Cycle Proteins/metabolism/antagonists & inhibitors/genetics ; CRISPR-Cas Systems ; Cell Line, Tumor ; Tumor Microenvironment/genetics ; Female ; Disease Models, Animal ; },
abstract = {Undifferentiated pleomorphic sarcoma (UPS) is one of the most common adult soft-tissue sarcomas (STSs), yet therapeutic progress remains limited because of the absence of recurrent oncogenic driver mutations. To identify tumor suppressors contributing to UPS pathogenesis, we performed a customized in vivo CRISPR/Cas9 screen in mice. This approach identified BRCA1-associated protein 1 (BAP1) as a potent tumor suppressor in STS. Integrative analyses using RNA sequencing, multiplex immunohistochemistry, and flow cytometry revealed that Bap1-deficient sarcomas exhibited a markedly immunosuppressive tumor microenvironment. Consistent with these findings, BAP1 protein expression was reduced in human UPS, whereas polo-like kinase 1 (PLK1) expression was elevated. Functional studies demonstrated that PLK1 was required for the growth and survival of Bap1-deficient sarcomas. Pharmacologic inhibition of PLK1 with volasertib significantly suppressed tumor growth in both syngeneic and autochthonous mouse models. Moreover, combining PLK1 inhibition with anti-PD-1 therapy enhanced tumor control and improved survival compared with either treatment alone. Together, these results identify PLK1 as a potential therapeutic vulnerability in BAP1-deficient sarcomas and support further evaluation of combined PLK1 inhibition and immune checkpoint blockade as a treatment strategy for a subset of STSs.},
}

Animals
*Tumor Suppressor Proteins/genetics/metabolism
Mice
*Ubiquitin Thiolesterase/genetics/metabolism
Humans
*Sarcoma/genetics/pathology/drug therapy/metabolism
Polo-Like Kinase 1
Proto-Oncogene Proteins/metabolism/antagonists & inhibitors/genetics
Protein Serine-Threonine Kinases/metabolism/antagonists & inhibitors/genetics
Cell Cycle Proteins/metabolism/antagonists & inhibitors/genetics
CRISPR-Cas Systems
Cell Line, Tumor
Tumor Microenvironment/genetics
Female
Disease Models, Animal

@article {pmid42385356,
year = {2026},
author = {Desterke, C and Jarén, A and Francés, R and Casafont, Í and Barrachina, MD and Esplugues, JV and Mata-Garrido, J},
title = {Integrative transcriptomic and CRISPR dependency analysis identifies hepatoblastoma-specific essential genes and actionable vulnerabilities.},
journal = {Cancer genetics},
volume = {306-307},
number = {},
pages = {165-179},
doi = {10.1016/j.cancergen.2026.06.008},
pmid = {42385356},
issn = {2210-7762},
mesh = {Humans ; *Hepatoblastoma/genetics/pathology ; *Liver Neoplasms/genetics/pathology ; Gene Expression Profiling/methods ; *Genes, Essential ; *Transcriptome ; Gene Expression Regulation, Neoplastic ; *CRISPR-Cas Systems ; *Biomarkers, Tumor/genetics ; },
abstract = {BACKGROUND: Hepatoblastoma (HB) is the most common primary liver malignancy in childhood, yet its molecular determinants, functional dependencies, and therapeutic vulnerabilities remain incompletely characterized. Integrative analyses combining transcriptomic profiling with functional genomic datasets provide a strategy to identify essential genes, biomarkers predictive of tumor behavior and treatment response.

METHODS: Differential expression analysis comparing HB tumors with normal liver was processed on training cohort. These genes were integrated with DepMap CRISPR-Cas9 dependency scores to prioritize HB-essential candidates. Elastic Net regression was used to derive a 16-gene predictive signature, which was validated in an external cohort. Single-cell RNA-seq datasets were analyzed to assess expression patterns across hepatic and tumor-associated cell populations. A supervised deep-learning classifier was trained on single-cell profiles to distinguish tumor cells from hepatocytes, and SHAP values were computed to interpret gene contributions. Drug-gene interactions were queried using curated repressive compounds from DGIdb, and approved drugs were screened for relevance in pediatric cancer clinical trials.

RESULTS: A total of 789 genes were found overexpressed in HB tumors from the training transcriptome cohort. Chronos DepMap analysis identified 73 HB-essential genes that were not essential in adult liver cancer cell lines (hepatocellular carcinoma and cholangiocarcinoma). Elastic-net tuning based on the expression of 16 HB-essential genes in the split training cohort enabled robust tumor-normal discrimination, with AUC = 0.88, specificity = 0.90, and sensitivity = 0.90 in internal validation. This performance was confirmed in an independent external cohort, achieving AUC = 0.99, specificity = 1.00, and sensitivity = 0.98. Single-cell validation further demonstrated tumor-specific enrichment of the signature. The deep-learning classifier (tumor cells vs. normal hepatocytes) reached high accuracy (AUC = 0.99; F1-score = 0.97), with SHAP analysis highlighting PEG10, GREB1, PLCB4, RHOBTB1, CRIM1, FSD1L, CORO2A, KIT, ANKRD50, HDAC11, ZNF233, SEMA7A, and FABP4 as major contributors. Six of these genes were confirmed to be absent or lowly expressed in the background liver microenvironment. Drug-gene interaction analysis identified HDAC11 as a potential therapeutic target of approved drugs used in pediatric oncology.

CONCLUSIONS: This integrative framework combining transcriptomics, CRISPR dependency mapping, machine learning, and pharmacogenomic annotation identifies clinically relevant HB-essential genes and predictive molecular signatures for tumor identity. The derived expression-based scores provide tools for patient stratification, while drug-gene mapping highlights actionable vulnerabilities on HDAC11 with pediatric approved drugs that support rational drug repurposing strategies in hepatoblastoma.},
}

Humans
*Hepatoblastoma/genetics/pathology
*Liver Neoplasms/genetics/pathology
Gene Expression Profiling/methods
*Genes, Essential
*Transcriptome
Gene Expression Regulation, Neoplastic
*CRISPR-Cas Systems
*Biomarkers, Tumor/genetics

@article {pmid42477347,
year = {2026},
author = {Huang, Q and Yang, D and Zhou, X and Li, G and Liu, W and Fan, X and Yuan, F and Chang, X},
title = {Incorporating AI-optimized zinc finger proteins enhances the efficiencies and targeting ranges of miniature base editors.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42477347},
issn = {2041-1723},
support = {2022YFA0807300//Ministry of Science and Technology of the People's Republic of China (Chinese Ministry of Science and Technology)/ ; 2018YFA0801400//Ministry of Science and Technology of the People's Republic of China (Chinese Ministry of Science and Technology)/ ; 2022SDXHDX0002//Science and Technology Department of Zhejiang Province/ ; 82450102//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32025016//National Natural Science Foundation of China (National Science Foundation of China)/ ; 31870927//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Zinc Fingers/genetics ; Animals ; Dependovirus/genetics ; Mice ; Humans ; Genetic Vectors/genetics ; Survival of Motor Neuron 2 Protein/genetics ; Muscular Atrophy, Spinal/therapy/genetics ; Exons/genetics ; CRISPR-Cas Systems ; HEK293 Cells ; },
abstract = {The therapeutic application of base editors is limited by their large sizes, which are beyond the packaging capabilities of adeno-associated viral (AAV) vectors. Despite recent progress that has identified many compact CRISPR proteins, the resulting miniature base editors often exhibit reduced activities and limited targeting scope. Here, we introduce a zinc finger protein (ZFP)-enhanced miniature base editor (zmBE), which integrates programmable ZFPs to improve efficiencies and targeting scopes of miniature base editors, including those based on Un1Cas12f1 and OgeuIscB. Utilizing protein language models to optimize ZFPs designed by modular assembly further simplifies the development of zmBEs. Leveraging these methodologies, we engineer a zmBE that effectively induces the SMN2 exon 7 T:A(6) > C:G conversion, restores the exon 7 inclusion, and improves spinal muscular atrophy in a murine model after being delivered via a single AAV vector. Our study provides a versatile platform for developing miniature base editors for in vivo therapeutic applications.},
}

*Zinc Fingers/genetics
Animals
Dependovirus/genetics
Mice
Humans
Genetic Vectors/genetics
Survival of Motor Neuron 2 Protein/genetics
Muscular Atrophy, Spinal/therapy/genetics
Exons/genetics
CRISPR-Cas Systems
HEK293 Cells

@article {pmid42572954,
year = {2026},
author = {Xu, ZH and Weng, X and Zhang, MP and Lin, RM and Xu, W and Wu, H and Gao, H},
title = {An aggregation-induced electrochemiluminescence sensor for ochratoxin A detection integrating CRISPR-Cas12a and tetrahedral DNA nanostructures.},
journal = {The Analyst},
volume = {151},
number = {17},
pages = {4950-4955},
doi = {10.1039/d6an00771f},
pmid = {42572954},
issn = {1364-5528},
mesh = {*DNA Nanostructures/chemistry ; *Biosensing Techniques/methods ; *Ochratoxins/analysis ; *Luminescent Measurements/methods ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; Limit of Detection ; Endodeoxyribonucleases/chemistry ; },
abstract = {An aggregation-induced electrochemiluminescence (AIECL) biosensor combining CRISPR-Cas12a and tetrahedral DNA nanostructures (TDNs) is fabricated for OTA detection, with polymer dots (Pdots) serving as emitters. The sensor achieves favorable analytical performance, with a limit of detection of 0.41 pg mL[-1], showing promising applications in food security monitoring.},
}

*DNA Nanostructures/chemistry
*Biosensing Techniques/methods
*Ochratoxins/analysis
*Luminescent Measurements/methods
*Electrochemical Techniques/methods
*CRISPR-Cas Systems
Limit of Detection
Endodeoxyribonucleases/chemistry

@article {pmid42610445,
year = {2026},
author = {Arti, and Yadav, G and Mathur, J},
title = {A critical review of mechanistic insights and technological advancement for the amelioration of Pb and Cd through phytoremediation.},
journal = {International journal of phytoremediation},
volume = {},
number = {},
pages = {1-23},
doi = {10.1080/15226514.2026.2716124},
pmid = {42610445},
issn = {1549-7879},
abstract = {Heavy metal contamination is a major environmental concern due to its persistence, bioaccumulation, and long-term impacts on ecosystems and human health. Among toxic metals, cadmium (Cd) and lead (Pb) are particularly harmful because of their high toxicity and carcinogenic potential, posing serious risks to plants, animals, and humans even at low concentrations. These metals often enter soil and water through industrial activities, mining, agricultural inputs, and improper waste disposal. Conventional remediation methods, such as chemical treatment, soil excavation, and stabilization, have been used to manage contaminated sites; however, they are often costly, labour- intensive, and may cause secondary environmental pollution, creating a need for more sustainable alternatives. Phytoremediation has emerged as an eco-friendly and cost-effective approach that utilizes the natural ability of plants to absorb, accumulate, detoxify, or stabilize contaminants from soil, water, and air using solar energy. The objectives of this review are to examine the mechanisms of phytoremediation and evaluate recent advance technologies that enhance its efficiency, with a focus on plant growth-promoting microorganisms, biochar, nanomaterials, CRISPR/Cas9-based genetic engineering, isotope monitoring, and AI/ML tools. The novelty of this review lies in its integrated assessment of these emerging technologies as complementary strategies for advancing sustainable heavy metal remediation. Overall, these developments highlight the growing potential of phytoremediation as a sustainable strategy for environmental cleanup. Nevertheless, challenges related to large-scale application, plant tolerance to heavy metals, and long-term ecological sustainability remain, requiring further research to enhance its practical implementation in environmental management.},
}

@article {pmid42610742,
year = {2026},
author = {Kumar, A and Kumari, P and Mishra, S and Thakur, S and Acharya, S and Chakraborty, D and Maiti, S and Jain, N},
title = {Energetic Determinants of Expanded PAM Recognition in Engineered FnCas9.},
journal = {Biochemistry},
volume = {65},
number = {16},
pages = {2495-2509},
doi = {10.1021/acs.biochem.6c00350},
pmid = {42610742},
issn = {1520-4995},
support = {BT/RLF/Re-entry/HRD/35/2019//Department of Biotechnology, Ministry of Science and Technology, India/ ; OLP2303//Council of Scientific and Industrial Research, India/ ; },
mesh = {*Francisella/enzymology/genetics ; *Bacterial Proteins/genetics/metabolism/chemistry ; Thermodynamics ; *CRISPR-Cas Systems ; Protein Engineering ; Substrate Specificity ; Mutation ; },
abstract = {Expanding protospacer-adjacent motif (PAM) compatibility while preserving specificity remains a central challenge in CRISPR-Cas9 engineering. Francisella novicida Cas9 (FnCas9) exhibits high intrinsic specificity but is constrained by stringent PAM requirements. Here, we quantitatively examine the energetic and catalytic consequences of PAM-interacting mutations in three engineered variants, en1 (E1369R), en15 (E1603H), and en31 (G1243T/E1369R/E1449H), using a VEGFA3 DNA substrate framework. Microscale thermophoresis and isothermal titration calorimetry reveal that the engineered variants enhance binding affinity toward the canonical NGG PAM relative to wild-type FnCas9, with modest gains in binding free energy. Selected noncanonical PAM substrates, particularly TGA and TAG, also show improved binding by en15 and en31, with en31 displaying the strongest overall binding among the substrates tested. Thermodynamic profiles indicate that enhanced affinity is associated with more favorable enthalpic contributions, consistent with altered interactions at the PAM interface; however, the specific molecular contributions underlying these changes remain to be directly established. Despite improved binding, active-site titration reveals reduced fractions of catalytically competent enzyme in engineered variants, particularly en31, necessitating higher enzyme concentrations to achieve cleavage efficiencies comparable to wild-type. Cleavage assays demonstrate that en31 most effectively couples improved recognition of the tested noncanonical PAM substrates to productive catalysis, enabling robust cleavage of both TGA and TAG substrates while maintaining minimal off-target activity under the conditions examined. Together, these results suggest that PAM-interacting mutations in FnCas9 can alter the energetic coupling between DNA binding and catalytic activation within the VEGFA3 substrate framework tested, highlighting the importance of balancing substrate affinity with conformational activation in the design of high-precision genome-editing nucleases.},
}

*Francisella/enzymology/genetics
*Bacterial Proteins/genetics/metabolism/chemistry
Thermodynamics
*CRISPR-Cas Systems
Protein Engineering
Substrate Specificity
Mutation

@article {pmid42610908,
year = {2026},
author = {Liu, H and Chen, J and Xiu, L and Liu, Y and Wei, TT and Wang, Y and Shi, L and Wang, X and Li, X and Yin, K},
title = {Integrated CRISPR/Cas12a-Based Duplex Detection Platform for Species Fingerprinting.},
journal = {Analytical chemistry},
volume = {98},
number = {32},
pages = {23672-23683},
doi = {10.1021/acs.analchem.6c02717},
pmid = {42610908},
issn = {1520-6882},
support = {2025YFC3409100//National Key Research and Development Program of China/ ; 24142201300//Science and Technology Innovation Plan Of Shanghai Science and Technology Commission/ ; 24J22800900//Science and Technology Innovation Plan Of Shanghai Science and Technology Commission/ ; 2024ZZ2012//Shanghai Municipal Health Commission's Seed Program/ ; MDPDMT-2023-02//Key Laboratory of Milk and Dairy Products Detection and Monitoring Technology, State Administration for Market Regulation/ ; YG2024ZD02//Interdisciplinary Program of Shanghai Jiao Tong University/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Milk/chemistry ; Cattle ; Colorimetry ; Lab-On-A-Chip Devices ; Buffaloes ; Food Contamination/analysis ; },
abstract = {Species fingerprinting is crucial to ensure food safety and human health, which requires a rapid, simple, multiplex, and field-deployable detection technique. In response, a lab-on-a-disc microfluidic chip with CRISPR/Cas12a that integrates target preamplification and signal readout enhanced by tetrahedral DNA frameworks (TDFs) has been developed in this study. This platform automates the workflow from recombinase polymerase amplification (RPA) through signal readout. In addition, a portable heating module was developed specifically for colorimetric detection, providing a complete field-deployable solution. The performance of the CRISPR/Cas12a platform was evaluated by detecting cow's milk adulteration in buffalo milk. The platform demonstrated high sensitivity, achieving detection limits of 1% (v/v) and 5% (v/v) for fluorescence and colorimetric detection within 1 h, respectively. The results agree well with those from real-time quantitative polymerase chain reaction (qPCR) in real-sample analysis. The integrated CRISPR/Cas12a-based duplex detection platform features high sensitivity and specificity, reaction automation, minimal aerosol contamination risk, and decentralized operation, which demonstrates significant potential for field-deployable species fingerprinting and risk prediction.},
}

Animals
*CRISPR-Cas Systems/genetics
*Milk/chemistry
Cattle
Colorimetry
Lab-On-A-Chip Devices
Buffaloes
Food Contamination/analysis

@article {pmid42095874,
year = {2026},
author = {Quansah, E and Yang, S and Jia, Y and Yu, L and Zhang, C},
title = {The dCas9-SSAP as a promising genome editing tool in malaria parasites.},
journal = {Critical reviews in microbiology},
volume = {52},
number = {5},
pages = {864-873},
doi = {10.1080/1040841X.2026.2667183},
pmid = {42095874},
issn = {1549-7828},
mesh = {*Gene Editing/methods ; *Plasmodium/genetics ; CRISPR-Cas Systems ; Humans ; *Malaria/parasitology ; Animals ; *CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {Measures to combat the Plasmodium parasites which cause malaria have become compromised because of reliance on a small arsenal of drugs, emerging drug resistance and the lack of effective vaccines. A promising avenue for addressing these challenges is the revolutionary gene-editing technology CRISPR-Cas9, due to its high efficiency and ease of design for genetic manipulation. The catalytically inactive Cas9 (dCas9)-microbial single-stranded annealing proteins (SSAP)(dCas9-SSAP) is a recently emerged next-generation gene editing system added to the ever-growing CRISPR-Cas9-based technologies. While the classical Cas9-nuclease technologies are "double-strand break, damage-repair systems", the dCas9-SSAP is distinctively a "cleavage-free" editing tool. Unlimited to the Plasmodium genome, Cas9-nucleases imprint inheritable genetic scars on the subject genomes when applied. Here, we discussed the DSB genotoxicity pitfalls of existing nuclease-based editing tools, especially CRISPR-Cas9, and how the dCas9-SSAP presents a formidable option to the drawbacks within the context of Plasmodium genome editing. Then, we sought to infer a plausible mechanistic framework that could account for dCas9-SSAP-mediated genome editing. Finally, we discussed how dCas9-SSAP aligns with Plasmodium parasites' biology. This review would set the stage for continued research into the potential of this new, exciting technology in malaria parasites.},
}

*Gene Editing/methods
*Plasmodium/genetics
CRISPR-Cas Systems
Humans
*Malaria/parasitology
Animals
*CRISPR-Associated Protein 9/genetics/metabolism

@article {pmid42610144,
year = {2026},
author = {Taki, AG and Shareef, A and Arora, V and Oweis, R and Jyothi, SR and Singh, U and Sahoo, S and Chauhan, AS and Klebleeva, G and Sameer, HN and Yaseen, A and Athab, ZH and Adil, M},
title = {AI-driven CRISPR strategies in breast cancer: Organoid modeling, adaptive editing, and precision delivery.},
journal = {Iranian journal of basic medical sciences},
volume = {29},
number = {6},
pages = {823-843},
pmid = {42610144},
issn = {2008-3866},
abstract = {Triple-negative breast cancer (TNBC) is defined by profound heterogeneity, dormant metastatic reservoirs, and rapid therapy resistance. Building on our AI-Driven CRISPR Strategies in Breast Cancer framework, CRISPR-Cas9 is emerging as more than a gene-editing tool, capable of restoring circadian integrity, eliminating dormant clones, and re-programming immune surveillance. A structured PubMed, Scopus, and ClinicalTrials.gov review through 2025 integrated mechanistic, preclinical, and early clinical evidence. Beyond standard knockout, base, and prime editing, we highlight chrono-genomic repair of BMAL1/PER2, dormancy-focused synthetic-lethality screens, and genomic-collapse tactics for BRCA1-deficient tumors. Adaptive AI pipelines that iteratively refine guide RNAs and exosome-mimetic carriers, incorporating Boolean logic gates, were also evaluated for self-regulated, tumor-specific delivery. Proof-of-concept studies show that HER2 deletion, TP53 rescue, and ABCB1 silencing enhance chemosensitivity across luminal, HER2-positive, and TNBC models. Circadian restoration expands therapeutic windows and delays relapse in xenografts. Dormancy-directed CRISPR screens reveal unique vulnerabilities in disseminated tumor cells, whereas genomic collapse selectively destroys BRCA1-mutant clones. Integration with CAR-T cells and antibody-drug conjugates amplifies cytotoxicity, and transient nanoparticle or exosome systems improve solid-tumor penetration while minimizing off-target events. CRISPR-Cas9 is transitioning from a molecular scalpel to an adaptive, self-learning therapeutic ecosystem. By uniting AI-guided design, circadian reprogramming, dormancy eradication, and logic-gated delivery, the strategies detailed here define a next-generation precision-oncology paradigm capable of anticipating tumor evolution, overcoming resistance, and preventing metastatic relapse.},
}

@article {pmid42425298,
year = {2026},
author = {Sun, X and Zhang, Y and Lu, R and Dai, T and Qiu, J and Jia, X and Wang, G and Pan, Z and Xu, S and Sima, Y},
title = {Integrated mapping and gene editing identify BmCCP as a regulator of cocoon shape and spinning behaviour in Bombyx mori.},
journal = {Journal of insect physiology},
volume = {173},
number = {},
pages = {105033},
doi = {10.1016/j.jinsphys.2026.105033},
pmid = {42425298},
issn = {1879-1611},
mesh = {Animals ; *Bombyx/genetics/growth & development/metabolism/physiology ; *Insect Proteins/genetics/metabolism ; Gene Editing ; Chromosome Mapping ; *Silk/metabolism ; CRISPR-Cas Systems ; Larva/growth & development/genetics ; },
abstract = {The cocoon shape in Bombyx mori (silkworm) is a construction trait shaped by cocoon-spinning behaviour, but the molecular regulation pathways remain poorly understood. Here, quantitative phenotyping, bulked segregant analysis sequencing (BSA-seq), brain transcriptomics and CRISPR/Cas9 mutagenesis were combined to identify a regulator of cocoon morphogenesis and to assess potential roles in spinning behaviour. Using representative strains L6J5 and J8, which produce short oval and long peanut-shaped cocoons, respectively, showed that cocoon shape, quantified by the cocoon aspect ratio, is a quantitative trait. BSA-seq mapped the trait to a 5.04-Mb candidate interval on chromosome 4 containing 213 annotated genes. Integration of the mapping results with brain transcriptomes from individuals with extreme cocoon phenotypes identified Bombyx mori cocoon shape-correlated protein (BmCCP) as the sole overlapping candidate gene. BmCCP was more highly expressed in the brain of strain J8 than L6J5 at the wandering stage, and the locus contained multiple associated polymorphisms. CRISPR/Cas9-mediated knockout of BmCCP in strain J8 significantly increased the cocoon aspect ratio and cocoon size. In the widely used experimental strain DaZao, which has not been artificially selected for cocoon shape, BmCCP deficiency likewise increased cocoon size and significantly reduced the larval spinning rate, while crosses with J8 further supported the role of BmCCP in regulating the cocoon aspect ratio. Together, these results identify BmCCP as a regulator of cocoon morphogenesis and provide a foundation to investigate the relationship between the cocoon morphology and spinning behaviour of the silkworm.},
}

Animals
*Bombyx/genetics/growth & development/metabolism/physiology
*Insect Proteins/genetics/metabolism
Gene Editing
Chromosome Mapping
*Silk/metabolism
CRISPR-Cas Systems
Larva/growth & development/genetics

@article {pmid42435068,
year = {2026},
author = {Zhang, C and Josyula, NK and Cornejo-Corona, I and Watson, R and Cediel-Becerra, JD and Chevrette, MG and Devarenne, TP and Straight, PD},
title = {Tandem-sgRNA Provides an Effective Reverse Genetic Approach for Suppression of Streptomyces Biosynthetic Gene Clusters and Secondary Metabolism.},
journal = {ACS synthetic biology},
volume = {15},
number = {8},
pages = {3382-3395},
doi = {10.1021/acssynbio.6c00274},
pmid = {42435068},
issn = {2161-5063},
support = {GM141700/GM/NIGMS NIH HHS/United States ; },
mesh = {*Streptomyces/genetics/metabolism ; *Multigene Family/genetics ; *Secondary Metabolism/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Reverse Genetics/methods ; CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic/genetics ; },
abstract = {Bacterial biosynthetic gene clusters (BGCs) encode secondary metabolites with diverse biological activities; however, most BGC products remain uncharacterized. One approach to identifying products and their metabolism is to use reverse genetics to identify metabolite-associated phenotypes. CRISPR interference (CRISPRi) offers a promising approach to disrupt BGC functions in high-GC genomes, typical of Streptomyces species. In many of these organisms, single-guide RNA (sgRNA)-mediated CRISPRi often results in incomplete product suppression, resulting in partial phenotypes that are unsuitable for functional studies. Using Streptomyces sp. Mg1, we found that a tandem-sgRNA configuration for CRISPRi improved the efficiency of target metabolite suppression. We engineered strains to express two sgRNAs to target the same promoter region within a BGC, resulting in greater than 80% metabolite suppression across diverse secondary metabolite classes. We used tandem-sgRNA CRISPRi to identify phenotypes associated with the loss of polyketide linearmycins, the siderophore desferrioxamine, the terpene β-carotene, and an uncharacterized nonribosomal peptide synthetase (NRPS). This approach revealed that β-carotene depletion substantially reduced intrinsic cellular autofluorescence. Targeting the unknown NRPS produced developmental phenotypes and enabled the identification of the biosynthetic genes for the antibiotic lavendomycin, revealing a noncollinear organization of genes in the BGC. We suggest that tandem-sgRNA CRISPRi provides an efficient reverse genetics platform for the functional characterization of Streptomyces BGCs, enabling the correlation of metabolites with the gene function, identification of associated phenotypes, and prioritization of cryptic BGCs for natural product discovery.},
}

*Streptomyces/genetics/metabolism
*Multigene Family/genetics
*Secondary Metabolism/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*Reverse Genetics/methods
CRISPR-Cas Systems/genetics
Promoter Regions, Genetic/genetics

@article {pmid42435429,
year = {2026},
author = {Zhou, Q and Xu, B and Wang, Y and Yang, X and Wang, L and Zheng, X and Lou, Y and Zheng, M and Xu, F and Xiao, X},
title = {LAMP-Based Two-DNA-Fragment Fusion and Its Application in Nucleic Acid Detection.},
journal = {ACS synthetic biology},
volume = {15},
number = {8},
pages = {3300-3309},
doi = {10.1021/acssynbio.6c00187},
pmid = {42435429},
issn = {2161-5063},
support = {JS2026005//Wenzhou Medical University/ ; 22578338//National Natural Science Foundation of China/ ; LTGY24H200005//Natural Science Foundation of Zhejiang Province/ ; 2024YFC2309905//National Key Research and Development Program of China/ ; NA//Key Discipline of Zhejiang Province in Medical Technology/ ; WKJ-ZJ-26018//Medical Science and Technology Project of Zhejiang Province/ ; },
mesh = {*Nucleic Acid Amplification Techniques/methods ; *Methicillin-Resistant Staphylococcus aureus/genetics/isolation & purification ; *DNA, Bacterial/genetics/analysis ; CRISPR-Cas Systems/genetics ; *DNA/genetics ; Molecular Diagnostic Techniques ; },
abstract = {Loop-mediated isothermal amplification (LAMP) continuously generates strand-displaced single-stranded DNA intermediates, providing the possibility of assembling DNA fragments. Here, we developed a novel two-DNA-fragment fusion technique, termed fusion LAMP, which is an isothermal DNA-fusion strategy that enables the fusion of two independent DNA fragments within a single amplification reaction. By combining fusion LAMP with CRISPR/Cas13a, we further established an "AND-gate" nucleic acid detection platform, termed Fusion LAMP-Coupled CRISPR/Cas13a (FLCC), which enables concurrent detection of two targets by reading the fusion product-triggered fluorescence signals. This platform generates signals only when two targets are present simultaneously. To prove this concept, we then employed FLCC to identify the methicillin-resistant Staphylococcus aureus (MRSA). This method achieved a limit of detection of 10 copies/μL of MRSA genomic DNA and showed no cross-reactivity with closely related bacterial strains. Furthermore, we validated its feasibility by detecting 19 clinical isolates, demonstrating a simple and accurate approach for MRSA detection. Collectively, the FLCC platform ensures identifying pathogens accurately and provides a promising diagnostic approach for detecting complex genetic targets.},
}

*Nucleic Acid Amplification Techniques/methods
*Methicillin-Resistant Staphylococcus aureus/genetics/isolation & purification
*DNA, Bacterial/genetics/analysis
CRISPR-Cas Systems/genetics
*DNA/genetics
Molecular Diagnostic Techniques

@article {pmid42454678,
year = {2026},
author = {Feldmann, D and van Beljouw, SPB and Haagsma, AC and Kalogeropoulos, K and Muralidharan, A and Brouns, SJJ},
title = {Craspase Protease Activation Is Sensitive to Oncogenic Single-Nucleotide RNA Mismatches.},
journal = {ACS chemical biology},
volume = {21},
number = {8},
pages = {1877-1882},
doi = {10.1021/acschembio.6c00241},
pmid = {42454678},
issn = {1554-8937},
support = {101003229//European Commission/ ; OCENW.XS23.1.006//Nederlandse Organisatie voor Wetenschappelijk Onderzoek/ ; SUMMIT.1.004//Nederlandse Organisatie voor Wetenschappelijk Onderzoek/ ; 15602//KWF Kankerbestrijding/ ; 4257-00010B//Danmarks Frie Forskningsfond/ ; NNF16OC0020670//Novo Nordisk Fonden/ ; },
mesh = {Humans ; Enzyme Activation ; *RNA/genetics ; *Base Pair Mismatch ; Polymorphism, Single Nucleotide ; CRISPR-Cas Systems ; *Peptide Hydrolases/metabolism/genetics ; },
abstract = {The type III-E CRISPR-controlled protease Craspase is distinguished from other type III systems by its single-subunit RNA-guided protein complex and direct coupling of RNA recognition to protease activation without second messenger signaling, making it an attractive development platform for bioengineering and therapeutics. Here, we identify five positions within the CRISPR RNA (crRNA) of Craspase from Candidatus "Scalindua brodae" (Sb-Craspase) that are sensitive to single-nucleotide mismatches. We leverage these positions to design crRNAs that selectively target clinically relevant single-nucleotide variants (SNVs) in oncogenic RNA transcripts. Using this approach, Sb-Craspase is selectively activated by the "undruggable" KRAS G12D SNV, while the wild-type transcript does not induce protease activation. Collectively, our results establish a framework for designing crRNAs to target clinically relevant SNVs, laying the groundwork for Craspase-based diagnostics and therapeutics against otherwise intractable oncogenic mutations.},
}

Humans
Enzyme Activation
*RNA/genetics
*Base Pair Mismatch
Polymorphism, Single Nucleotide
CRISPR-Cas Systems
*Peptide Hydrolases/metabolism/genetics

@article {pmid42477478,
year = {2026},
author = {Tokgün, O and İnci, K and Gültekin, A and Çelikkaya, B and İrep, N and Akça, H and Tokgün, PE},
title = {Targeting RAB27A-mediated small extracellular vesicle secretion via CRISPR-Cas9 negatively affects proliferation and metastasis in both in vitro and in vivo SCLC models.},
journal = {Cancer gene therapy},
volume = {33},
number = {8},
pages = {1000-1013},
pmid = {42477478},
issn = {1476-5500},
mesh = {Humans ; Animals ; Cell Proliferation ; *rab27 GTP-Binding Proteins/genetics/metabolism ; Mice ; *Lung Neoplasms/pathology/genetics/metabolism ; *Small Cell Lung Carcinoma/genetics/pathology/metabolism/therapy ; *CRISPR-Cas Systems ; *Extracellular Vesicles/metabolism/genetics ; Cell Line, Tumor ; Female ; Neoplasm Metastasis ; Xenograft Model Antitumor Assays ; },
abstract = {Small cell lung cancer (SCLC) comprises 15% of lung cancers with a capacity for early and distant metastatic development, high proliferative capacity, and poor survival rates. Ionizing radiation and chemotherapy are effective against early-stage SCLC. This sensitivity wanes over time, however, making treatment difficult. Different types of neoplasms have demonstrated the pivotal role of small extracellular vesicles (sEVs) in disease progression. However, the role of sEVs development in SCLC remains unclear. In this study, the impact of sEVs secretion in SCLC cells was investigated using the CRISPR-Cas9 system to target the RAB27A. The effects of sEVs release inhibition on tumour growth and metastasis were evaluated using micro-PET-CT analysis. A reduction in cellular proliferation as a consequence of sEVs release, along with diminished expression of proteins and RNA (CD9, CD63, and Tsg101) implicated in sEVs secretion in silenced SCLC cells (p < 0.001, p < 0.0001) was detected. The suppression of sEVs release exhibited significant adverse effects on tumor development and metastatic dissemination in the in vivo tumor model. The present study suggests that the targeting of RAB27A could be a viable cancer therapy for SCLC. Targeting the exosomal pathway has the potential to enhance treatment efficacy, and SCLC may depend on sEVs secretion.},
}

Humans
Animals
Cell Proliferation
*rab27 GTP-Binding Proteins/genetics/metabolism
Mice
*Lung Neoplasms/pathology/genetics/metabolism
*Small Cell Lung Carcinoma/genetics/pathology/metabolism/therapy
*CRISPR-Cas Systems
*Extracellular Vesicles/metabolism/genetics
Cell Line, Tumor
Female
Neoplasm Metastasis
Xenograft Model Antitumor Assays

@article {pmid42567105,
year = {2026},
author = {Hassannia, M and Amirifar, P},
title = {CRISPR-Cas9 gene editing approaches in colorectal cancer: Current progress and future prospects.},
journal = {Cancer treatment and research communications},
volume = {48},
number = {},
pages = {101341},
doi = {10.1016/j.ctarc.2026.101341},
pmid = {42567105},
issn = {2468-2942},
mesh = {Humans ; *Colorectal Neoplasms/genetics/therapy ; *CRISPR-Cas Systems ; *Gene Editing/methods/trends ; *Genetic Therapy/methods ; Animals ; },
abstract = {Colorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide, characterized by genetic heterogeneity and the accumulation of mutations in key oncogenes and tumor suppressor genes. CRISPR-Cas9 technology has greatly advanced genetic research by enabling precise genome editing. This review focuses on the innovative applications of CRISPR-Cas9 in CRC research, particularly its role in identifying novel therapeutic targets, elucidating mechanisms of drug resistance, and uncovering metabolic and stem cell pathway alterations in tumorigenesis. We highlight the diverse CRISPR systems, including Cas9, Cas12, Cas13, and advanced variants such as CRISPR activation (CRISPRa), CRISPR interference (CRISPRi), base editing, and prime editing, which have expanded gene knockout studies and enhanced our understanding of CRC. Despite these breakthroughs, challenges such as off-target effects and delivery limitations remain. Ongoing efforts to refine CRISPR technology aim to enhance its precision and clinical applicability, ultimately paving the way for more effective and personalized treatment strategies for CRC. In this review, we explore these advances and focus on the latest developments in CRISPR-based approaches for CRC treatment.},
}

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

@article {pmid42567230,
year = {2026},
author = {Dewar, CE and King, EFB and Rojas, F},
title = {Optimising electroporation protocols for Trypanosoma brucei using the Amaxa 4D-nucleofector system.},
journal = {Molecular and biochemical parasitology},
volume = {267},
number = {},
pages = {111767},
doi = {10.1016/j.molbiopara.2026.111767},
pmid = {42567230},
issn = {1872-9428},
mesh = {*Trypanosoma brucei brucei/genetics ; *Transfection/methods ; *Electroporation/methods/instrumentation ; Cell Survival ; Animals ; CRISPR-Cas Systems ; },
abstract = {Stable transfection of Trypanosoma brucei remains a cornerstone for functional genetic studies in this model parasite. Although the Amaxa Nucleofector II system dramatically improved transfection efficiency in both monomorphic and pleomorphic bloodstream forms, the more recent 4D Nucleofector platform offers enhanced programmability and buffer flexibility that have yet to be systematically evaluated for T. brucei. Here, we benchmark a range of 4D Nucleofector programs to determine optimal parameters for transfection efficiency, cell viability, and reproducibility in bloodstream forms. Using a CRISPR/Cas9 expressing cell line, we compare stable transfection efficiencies across programs. We further demonstrate the advantages of the 16-well Nucleocuvette™ Strip format, enabling simultaneous processing of multiple experimental conditions in 20 µL reactions, reducing DNA, cell, and reagent requirements while increasing experimental throughput. Our results provide a standardized framework for future genetic manipulation of T. brucei using the 4D-Nucleofector X Unit, facilitating robust and reproducible transfection across life-cycle stages and strains.},
}

*Trypanosoma brucei brucei/genetics
*Transfection/methods
*Electroporation/methods/instrumentation
Cell Survival
Animals
CRISPR-Cas Systems

@article {pmid42609829,
year = {2026},
author = {Al-Azzani, H and Aliouat, H and Cheng, H and Dagah, OMA and Al-Subari, MH and Zhou, W and Wu, S},
title = {Antibacterial Immunotherapy: Mechanistic Insights, Emerging Therapeutic Strategies, and Clinical Translation.},
journal = {Infection and drug resistance},
volume = {19},
number = {},
pages = {619916},
pmid = {42609829},
issn = {1178-6973},
abstract = {Antimicrobial resistance (AMR) continues to compromise the effectiveness of conventional antibacterial therapy, driving the development of therapeutic strategies that extend beyond direct antibiotic-mediated bacterial killing. Multidrug-resistant (MDR) pathogens evade treatment through diverse mechanisms, including enzymatic drug inactivation, target modification, efflux pump overexpression, biofilm formation, and persisters development. AMR results in chronic and recurrent infections, prolonged hospitalization, increased healthcare costs, and elevated morbidity and mortality, underscoring the need for innovative therapeutic approaches that target both the pathogen and the host. To bridge the dynamic interplay between bacterial pathogens and the host immune system with emerging therapeutic innovations, this narrative review first examines the biological mechanisms underlying bacterial resistance. It then explores therapeutic strategies beyond conventional antibiotics, providing an overview of current approaches and their limitations, including drug repurposing, bacteriophage therapy, and CRISPR-Cas technology. The review subsequently focuses on antibacterial immunotherapy, discussing a broad range of emerging approaches, including probiotics, monoclonal antibodies, cell-based therapies, host-directed therapies, aptamers, nanotechnology-based platforms, cytokine-based therapies, and antimicrobial peptides. An integrated overview of preclinical evidence, clinical studies, and FDA-approved therapies is presented to assess the translational potential of immunotherapy strategies in combating AMR. Scientific, regulatory, manufacturing, and implementation challenges that influence their successful translation into clinical practice are discussed throughout. By integrating the biological basis of host-pathogen interactions with emerging antibacterial therapeutics and their translational development, this review provides a comprehensive framework for evaluating innovative strategies against antimicrobial resistance. In contrast to modality-focused reviews, it offers a unified perspective that highlights the complementary roles of pathogen-targeted and host-directed interventions and identifies future opportunities to improve the prevention and management of multidrug-resistant bacterial infections.},
}

@article {pmid42379321,
year = {2026},
author = {Wei, Y and Zhu, G},
title = {Targeted gene editing of heterodisulfide reductase mediated electron bifurcation optimises heterodisulfide reductase-ferredoxin-adenosine triphosphate axis for enhanced methanogenesis in anaerobic granular sludge.},
journal = {Bioresource technology},
volume = {459},
number = {},
pages = {135281},
doi = {10.1016/j.biortech.2026.135281},
pmid = {42379321},
issn = {1873-2976},
mesh = {*Methane/biosynthesis/metabolism ; *Sewage/microbiology ; *Adenosine Triphosphate/metabolism ; *Oxidoreductases/genetics/metabolism ; Anaerobiosis ; *Ferredoxins/metabolism ; *Gene Editing/methods ; *Electrons ; CRISPR-Cas Systems/genetics ; },
abstract = {The thermodynamic bottleneck of syntrophic propionate oxidation constrains the efficiency and stability of anaerobic digestion (AD), which depends on flavin-based electron bifurcation (FBEB) mediated by heterodisulfide reductase (Hdr). Five CRISPR-Cas9 engineered Methanobacterium formicicum strains targeting Hdr, nickel homeostasis, and flavin metabolism were evaluated in 10 % and 20 % granular sludge systems to rewire electron flux toward the energy-conserving ferredoxin (Fd) reduction pathway. Within each experimental tier, results are reported against matched controls. In the enzyme-supplementation tier, the heterodisulfide reductase ABC subunit-F420-reducing hydrogenase A subunit (HdrABC-MvhA) supplement produced the highest cumulative methane yield and elevated intracellular adenosine triphosphate (ATP) to 28.31 ± 1.60 nmol/L versus 16.27 ± 0.90 nmol/L in the matched wild-type control (1.74 ± 0.10-fold; P < 0.01, n = 3). In the genome-editing tier, the Δhpt-nikR strain achieved the highest cumulative methane yield under high sludge loading. Because N[5]-methyltetrahydromethanopterin:coenzyme M methyltransferase (Mtr) and A1A0-ATP synthase activities were not directly measured, and the Hdr activity increment falls within the variance of crude-extract assays, these co-occurring changes are interpreted as correlative support for a putative "Hdr-Fd-ATP" working model rather than direct demonstration of a defined energy-conservation pathway. The engineered strains enriched hydrogenotrophic methanogens (Methanobacterium, 1.08-1.13-fold) and increased the predicted genomic abundance of electron-bifurcation, CO2-reduction, and methyl-transfer pathway genes, as inferred from 16S rRNA-based functional prediction. This study provides correlative evidence that CRISPR-based metabolic engineering of methanogens can modulate the Hdr-Fd-ATP axis within heterogeneous granular sludge communities, establishing a mechanistic framework for in situ bioaugmentation strategies targeting intracellular energy-conservation bottlenecks.},
}

*Methane/biosynthesis/metabolism
*Sewage/microbiology
*Adenosine Triphosphate/metabolism
*Oxidoreductases/genetics/metabolism
Anaerobiosis
*Ferredoxins/metabolism
*Gene Editing/methods
*Electrons
CRISPR-Cas Systems/genetics

@article {pmid42463075,
year = {2026},
author = {Cheng, Y and Niu, S and Zhang, Z and Qian, H and Huang, Z and Jing, H and Liao, L and Man, C and Gao, H and Chen, Q and Du, L and Chen, S and Wang, F},
title = {TBC1D14 positively regulates autophagy induced by Brucella melitensis vaccine strain BA0711 in Sheep Leydig Cells.},
journal = {Microbial pathogenesis},
volume = {219},
number = {},
pages = {108664},
doi = {10.1016/j.micpath.2026.108664},
pmid = {42463075},
issn = {1096-1208},
mesh = {Animals ; *Autophagy/genetics ; *Brucella melitensis/immunology ; Male ; *GTPase-Activating Proteins/genetics/metabolism ; Sheep ; *Leydig Cells/microbiology/metabolism/immunology ; Gene Knockout Techniques ; *Brucellosis/veterinary/immunology ; CRISPR-Cas Systems ; Microscopy, Electron, Transmission ; },
abstract = {Brucella is an intracellular Gram-negative bacterium that primarily infects the host reproductive and immune systems, inducing autophagy and facilitating pathogen replication. TBC (Tre2-Bub2-Cdc16) domain-containing proteins are important in membrane trafficking, cell polarity, and signal transduction as regulators of Rab small GTPases. Previously, we demonstrated that B. melitensis M5-90 modulates the expression of miR-146b-5p, which targets TBC1D14, in RAW264.7 cells. In this study, CRISPR-Cas9 was used to generate TBC1D14-knockout (KO) Sheep Leydig cells (SLCs), and B. melitensis BA0711 treatment experiment was conducted at a multiplicity of infection (MOI) of 100. After confirming that SLCs retain autophagic activity, Western blot, autophagy flux assays, transmission electron microscopy (TEM), and RT-qPCR were performed to identify the function of TBC1D14. We found that autolysosome fluorescence was significantly enhanced in NC-SLCs compared with TBC1D14-KO-SLCs following BA0711 treatment at 8 and 12 hpi. Notably, autophagy flux in NC-SLCs remained consistently higher than that in KO-SLCs from 8 hpi onwards. Consistently, Western blot revealed a decreased LC3-II/LC3-I ratio, and TEM confirmed a reduced number of autolysosomes in KO-SLCs at 12 hpi. These results indicate that TBC1D14 positively regulates autophagy in SLCs by BA0711 stimulation. Although transcriptomic and proteomic analyses indicated activation of autophagy- and phagocytosis-related pathways during BA0711 exposure, this response was significantly attenuated in TBC1D14-KO cells. Furthermore, TBC1D14 knockout led to downregulation of phagosome-related proteins and altered chemotaxis pathways, whereas re-expression of TBC1D14 in KO cells upregulated RAB28. In conclusion, this study demonstrates that TBC1D14 positively regulates autophagy and provides insights into the response of SLCs treated by B. melitensis BA0711. Furthermore, it suggests that RAB28 may serve as a downstream effector of TBC1D14.},
}

Animals
*Autophagy/genetics
*Brucella melitensis/immunology
Male
*GTPase-Activating Proteins/genetics/metabolism
Sheep
*Leydig Cells/microbiology/metabolism/immunology
Gene Knockout Techniques
*Brucellosis/veterinary/immunology
CRISPR-Cas Systems
Microscopy, Electron, Transmission

@article {pmid42492860,
year = {2026},
author = {Lee, JH and Lee, ES and Xiang, XR and Kyung, SM and Seo, J and Lee, W and Park, HE and Shin, MK and Yoo, HS},
title = {Roles of mdh and MAP1981c in Mycobacterium avium subsp. paratuberculosis intracellular survival within bovine monocyte-derived macrophages via CRISPR interference.},
journal = {Microbial pathogenesis},
volume = {219},
number = {},
pages = {108724},
doi = {10.1016/j.micpath.2026.108724},
pmid = {42492860},
issn = {1096-1208},
mesh = {Animals ; *Mycobacterium avium subsp. paratuberculosis/genetics/pathogenicity/growth & development ; Cattle ; *Macrophages/microbiology/immunology ; Paratuberculosis/microbiology ; *Bacterial Proteins/genetics/metabolism ; Gene Expression Profiling ; Host-Pathogen Interactions ; CRISPR-Cas Systems ; Microbial Viability ; Gene Expression Regulation, Bacterial ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease, a chronic enteritis in ruminants, and is capable of persisting within macrophages despite the activation of host immune defenses. Although this intracellular persistence is a key determinant of MAP pathogenicity, the bacterial factors and host responses that regulate this process remain poorly understood. In this study, we established the first CRISPR interference (CRISPRi) platform applied to bovine monocyte-derived macrophages (MDM) to evaluate the functions of MAP genes involved in intracellular survival and to perform an integrative analysis of host transcriptomic responses. MAP mutants were targeted to two genes (mdh and MAP1981c). The optimal concentration of anhydrotetracycline (ATc) was determined to be 2 μg/ml by measuring the survival of the cells and the downregulation of gene expression levels in the cells up to 72 h. The gene expression profiles and intracellular MAP levels were investigated using RNA-seq and colony-forming units, respectively. The survival rates of the MAP mutants significantly decreased with the time course of infection in MAP-mdhKD and MAP1981cKD (KD, knockdown). RNA-seq-based gene expression profiling suggested that target gene silencing in MAP mutants led to altered expression of host genes involved in lipid metabolism, T-cell activation reduction, and antimicrobial response in bovine MDM, contributing to reduced intracellular survival of MAP. Our study demonstrates that the downregulation of mdh and MAP1981c in MAP significantly alters the host transcriptomic landscape in bovine MDM, revealing their critical roles in subverting host immune defenses for intracellular persistence.},
}

Animals
*Mycobacterium avium subsp. paratuberculosis/genetics/pathogenicity/growth & development
Cattle
*Macrophages/microbiology/immunology
Paratuberculosis/microbiology
*Bacterial Proteins/genetics/metabolism
Gene Expression Profiling
Host-Pathogen Interactions
CRISPR-Cas Systems
Microbial Viability
Gene Expression Regulation, Bacterial
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42576388,
year = {2026},
author = {Zhang, Y and Yang, Y and Liu, Z and Li, Y and Xue, Y and Zhang, Z and Chen, G and Liu, J and He, M and Lu, T and Zhang, Y and Zhao, D and Yang, K and Miao, L and Gao, F and Guo, Y},
title = {The DreAM-plus integrative RNA switch enhances transient AAV expression and reduces side effects of gene editing.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2026.08.006},
pmid = {42576388},
issn = {1525-0024},
abstract = {Uncontrolled long-term adeno-associated virus (AAV) expression prohibits therapeutic strategies that require more precise and dynamic regulation. For example, long-lasting expression of gene editors by AAV could augment off-target effects and immunogenicity. Drug-inducible RNA switches are desirable tools to achieve transient AAV expression. However, current RNA switches only target a single mechanism such as transcription or RNA splicing, exhibiting limited capacity in transgene regulation. Here, we report DreAM-plus, a multilayer RNA switch that integrates an aptamer-based poly(A) regulator (pA), a drug-elicitable alternative splicing module (DreAM), and an engineered P2A element with conditional upstream open reading frames (uORFs). The pA-DreAM concatenation enhanced gene inducibility by up to 5-fold more than pA or DreAM alone, with 1.4- to 6.3-fold further improvement by uORFs. DreAM-plus achieved transient expression of an array of gene editors (SpCas9, SaCas9, Un1Cas12f1, OsCas12f1, AcCas12n, and IsDra2 TnpB) with a temporal resolution of less than 24 h, which significantly mitigated off-target effects by 1.4- to 2.8-fold. With lipid-nanoparticle-delivered pre-existing immunity in mice, DreAM-plus attenuated AAV-delivered Cas-specific CD8[+] T cell immune toxicity in the liver and heart. Therefore, the inducible RNA switches could be synergistically integrated to build sophisticated genetic cassettes for enhanced safety of AAV-mediated gene editing.},
}

@article {pmid42603302,
year = {2026},
author = {He, R and Zhang, C and Zhang, J and Zhang, K and Yin, W and Qiao, B and Zhang, L and Zhang, S and Qiao, J and Liu, Y},
title = {A One-Pot Reverse Transcriptase-Mediated, Pre-amplification-Free CRISPR/Cas12a Assay for Ultrasensitive Nucleic Acid Detection.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.6c01202},
pmid = {42603302},
issn = {2379-3694},
support = {2025AFB825//Natural Science Foundation of Hubei Province/ ; WHPU-EIRF-2606//Wuhan Polytechnic University/ ; 2023DJC136//Department of Science and Technology of Hubei Province/ ; },
abstract = {CRISPR/Cas12a holds great promise for biosensing and diagnostics, but conventional methods suffer from low catalytic efficiency, high background, and reliance on pre-amplification. Direct detection of structured RNAs also remains challenging. Herein, we report the development of a reverse transcriptase and LNA probe (LNA-p)-mediated CRISPR/Cas12a positive feedback system (RTLC) for highly efficient, one-pot detection of both DNA and RNA. Without pre-amplification or thermal cycling, the assay achieves a 0.5 aM detection sensitivity within 27 min, exhibits single-base resolution, and allows direct detection of RNAs up to 985 nt in length. Together, RTLC is successfully validated in practical samples by detecting lncRNA HULC and miR-21, offering a robust, versatile tool for high-performance nucleic acid diagnostics.},
}

@article {pmid42604377,
year = {2026},
author = {Su, L and Mara, P and Edgcomb, V and Teske, A},
title = {From CRISPR-Cas to Argonautes: Defense systems of bacteria and archaea in the hydrothermal deep subsurface.},
journal = {ISME communications},
volume = {6},
number = {1},
pages = {ycag200},
pmid = {42604377},
issn = {2730-6151},
abstract = {Microbial defense systems are central to virus-host interactions and thus to microbial survival but remain poorly studied in microbial communities of extreme environments. Here we examine the repertoire and distribution of microbial defense genes in hydrothermally influenced deep subsurface sediments and rocks of the Guaymas Basin (Gulf of California). Restriction-modification and abortive infection systems were broadly distributed across the examined sediment depths, and clustered, regularly interspaced short palindromic repeats-Cas systems were primarily detected within temperate surficial sediments. Prokaryotic Argonaute genes were found mostly in archaeal MAGs at elevated temperatures up to 81.8°C. Overall defense gene repertoire declines downcore, as temperature increases and phylogenetic host range narrows, with phylogeny as the decisive control factor. We suggest that these defense systems, together with DNA repair mechanisms, protein maintenance activities, and RNA modification pathways, constitute a survival toolkit for the hydrothermally influenced subsurface, where energy limitation and temperature extremes select for resilient microbial communities.},
}

@article {pmid42605670,
year = {2026},
author = {Le, S and Thach, T},
title = {Structure- and deep learning-guided engineering of a size-minimized CRISPR/Cas.},
journal = {The FEBS journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/febs.70707},
pmid = {42605670},
issn = {1742-4658},
abstract = {Multidomain proteins play central roles in cellular regulation, yet their intrinsic flexibility and structural instability often hinder optimization for biotechnological applications. Here, we present an integrated structure-guided and deep learning-assisted engineering strategy that combines structure modeling with Protein Message Passing Neural Network (ProteinMPNN)-based sequence design to generate an ultracompact CRISPR activator (uCRISPRa) derived from the miniature CRISPR/Cas12f. Structural and computational analyses identified flexible, nonessential regions within both Cas12f and its single-guide RNA (sgRNA), enabling rational truncation and sequence redesign while preserving DNA-targeting capability. When delivered as mRNA encapsulated in lipid nanoparticles, uCRISPRa achieved selective activation of olfr544 among more than a thousand homologous olfactory receptor genes in skeletal muscle cells, leading to enhanced mitochondrial biogenesis. These findings demonstrate that the integration of structure-based protein engineering with deep learning sequence optimization provides a powerful framework for developing compact and efficient CRISPR effectors, offering broad potential for precise gene regulation and functional studies of complex macromolecular systems.},
}

@article {pmid42608492,
year = {2026},
author = {Majewska, Z and Jursza, G and Dolzblasz, A},
title = {Dexamethasone-inducible LhGR/pOp system: simple and flexible spatiotemporal control of gene expression.},
journal = {Planta},
volume = {264},
number = {4},
pages = {},
pmid = {42608492},
issn = {1432-2048},
support = {BPIDUB.4610.174.2022//Centre of Excellence and Applied Sport Science Research, Queensland Academy of Sport/ ; 501/73/10110/MPK 2599150000/2026//funding provided by the Department of Plant Developmental Biology/ ; },
mesh = {*Dexamethasone/pharmacology ; *Arabidopsis/genetics/drug effects ; *Gene Expression Regulation, Plant/drug effects ; Plants, Genetically Modified/genetics ; *Arabidopsis Proteins/genetics/metabolism ; },
abstract = {Our review illustrates how the dexamethasone-inducible LhGR/pOp system has been used across numerous tissues/organs and plant species, and summarizes the resources and inducer application procedures established to date. The establishment of groundbreaking molecular biology tools has enabled the rapid advancement of research based on the model plant Arabidopsis, which currently strongly benefits non-model but economically important species. Spatiotemporal control of transgene expression via the chemically inducible system GR-LhG4/pOp has proven to be a particularly powerful, universal and non-invasive experimental approach. This review characterizes the mechanism of action of GR-LhG4/pOp and synthesizes information on available GR-LhG4 transgenic lines and inductor (dexamethasone) application procedures across various plant tissues. Moreover, feasible experimental approaches are depicted that range from classical ones aiming at selected genes overexpression or silencing to highly innovative ones, like those integrating the GR-LhG4/pOp system with CRISPR-Cas for targeted ablation of specific cell types. Information about the existing GR-LhG4/pOp lines from non-Arabidopsis plants is also provided, in hope that this sophisticated but universal research tool will become more exploited in crop research.},
}

*Dexamethasone/pharmacology
*Arabidopsis/genetics/drug effects
*Gene Expression Regulation, Plant/drug effects
Plants, Genetically Modified/genetics
*Arabidopsis Proteins/genetics/metabolism

@article {pmid42609038,
year = {2026},
author = {Candelotti, AM and Garzillo, G and Bartolini, S and Di Censo, C and Peruzzi, G and Stabile, H and Fionda, C and Sciumè, G and Pietropaolo, G},
title = {Rapid and Highly Efficient CRISPR-Cas9 RNP Genome Editing in Primary ILC2s.},
journal = {European journal of immunology},
volume = {56},
number = {8},
pages = {e70254},
pmid = {42609038},
issn = {1521-4141},
support = {IG-28719//the Italian Association for Cancer Research (AIRC)-Investigator Grant "Mirella Rizzo"/ ; PNRR-MAD-2022-12375947//the Italian Association for Cancer Research (AIRC)-Investigator Grant "Mirella Rizzo"/ ; //Next Generation EU-PNRR M6C2 - Investimento 2.1 Valorizzazione e potenziamento della ricerca biomedica del SSN/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics ; Mice ; Humans ; },
abstract = {CRISPR-mediated gene editing enables efficient genetic manipulation of ILC2s through ex vivo or in vivo activation and Cas9 RNP delivery. This platform provides a robust approach to dissect gene function in ILC2s, with minimal manipulation.},
}

Animals
*CRISPR-Cas Systems/genetics
*Ribonucleoproteins/genetics
Mice
Humans

@article {pmid40945831,
year = {2025},
author = {Duan, X and Zhou, Z and Zeng, X and Chen, J and Mao, A},
title = {Single-step purification of functional Cas9 protein via the ubiquitin expression system.},
journal = {International journal of biological macromolecules},
volume = {328},
number = {Pt 1},
pages = {147590},
doi = {10.1016/j.ijbiomac.2025.147590},
pmid = {40945831},
issn = {1879-0003},
mesh = {*Ubiquitin/genetics/metabolism/chemistry ; Escherichia coli/genetics/metabolism ; *CRISPR-Associated Protein 9/isolation & purification/genetics/metabolism/chemistry ; Streptococcus pyogenes/enzymology/genetics ; CRISPR-Cas Systems ; Chromatography, Affinity ; Recombinant Fusion Proteins/isolation & purification/genetics ; *Gene Expression ; },
abstract = {The CRISPR/Cas9 system serves as a powerful platform for precise genome editing, with CRISPR/Cas9 ribonucleoprotein (RNP) complexes exhibiting superior editing efficiency compared to alternative delivery modalities. However, current methods for Cas9 production typically involve multiple purification steps. Here, we developed a streamlined single-step purification strategy for preparation of functional Streptococcus pyogenes Cas9 (SpCas9) with dispensable tag removal after inducible expression in Escherichia coli. Notably, N-terminal Ubiquitin (Ub) fusion preserved both accurate nuclear localization and remarkable protein stability, enabling robust production of over 8 mg/L of >95 % pure Ub-Cas9 via single metal affinity chromatography. Comprehensive endonuclease activity assays confirmed that the purified Ub-Cas9 maintained robust DNA cleavage capacity both in vitro and in vivo. Our work provides a convenient and efficient platform for production of highly purified Ub-Cas9 protein for widespread applications in gene function study.},
}

*Ubiquitin/genetics/metabolism/chemistry
Escherichia coli/genetics/metabolism
*CRISPR-Associated Protein 9/isolation & purification/genetics/metabolism/chemistry
Streptococcus pyogenes/enzymology/genetics
CRISPR-Cas Systems
Chromatography, Affinity
Recombinant Fusion Proteins/isolation & purification/genetics
*Gene Expression

@article {pmid42323083,
year = {2026},
author = {Byrnes, C and Clarke, BA and Zhu, H and Lee, YT and Majumder, S and Kono, M and Proia, RL},
title = {1-Deoxysphingolipids require very-long-chain ceramide synthesis to induce ER stress and cytotoxicity.},
journal = {The Journal of biological chemistry},
volume = {302},
number = {8},
pages = {113281},
pmid = {42323083},
issn = {1083-351X},
mesh = {Humans ; *Ceramides/biosynthesis/chemistry ; *Endoplasmic Reticulum Stress/drug effects ; *Sphingolipids/metabolism/chemistry ; Cell Line, Tumor ; Cell Survival/drug effects ; CRISPR-Cas Systems ; },
abstract = {Sphingolipids play key roles in cellular systems both as membrane components and as signaling molecules. Their biosynthesis, which occurs in the endoplasmic reticulum (ER), begins with the condensation of an amino acid, typically serine, and a fatty acyl-CoA. Under certain pathological conditions, alanine can be substituted for serine in the condensation reaction, producing 1-deoxysphingolipids, which lack the 1-hydroxyl group on the sphingoid base. Unlike typical sphingolipids, 1-deoxysphingolipids are unable to accept a head group modification, which alters their metabolic processing and prevents their canonical degradation. The accumulation of these "headless" 1-deoxysphingolipids causes neurotoxicity in various neurological and metabolic disorders. Here, we conducted a genome-wide CRISPR-Cas9 screen to identify pathways leading to 1-deoxysphinganine-induced toxicity in SH-SY5Y cells, a model used to study neurotoxic responses. Our top genetic hits highlighted the pathway involved in synthesizing ceramides with very-long-chain fatty acids (C22-C26). Using CRISPR-Cas9-modified SH-SY5Y cells with loss-of-function (LOF) mutations in the TECR or CERS2 genes-both critical for producing very-long-chain ceramides-we validated that this pathway was essential for 1-deoxysphinganine-mediated toxicity. Furthermore, we demonstrated that the ceramide synthesis pathway is required for 1-deoxysphinganine to trigger ER stress, as evidenced by significantly increased expression of the unfolded protein response in WT, but not TECR or CERS2 LOF mutant, SH-SY5Y cells exposed to 1-deoxysphinganine. Collectively, the data support a model in which ceramide synthase-dependent conversion of 1-deoxysphinganine to very-long-chain 1-deoxyceramide species is required for full ER-stress induction and cytotoxicity. The findings highlight potential therapeutic targets for neuropathological diseases caused by 1-deoxysphingolipid accumulation.},
}

Humans
*Ceramides/biosynthesis/chemistry
*Endoplasmic Reticulum Stress/drug effects
*Sphingolipids/metabolism/chemistry
Cell Line, Tumor
Cell Survival/drug effects
CRISPR-Cas Systems

@article {pmid42459132,
year = {2026},
author = {Wen, Z and Wei, C and Shen, M and Yu, S},
title = {Signal logic gate mediated by a controllable CRISPR/Cas12a system for simultaneous detection of DNA mutation and methylation.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {32},
pages = {6823-6830},
doi = {10.1039/d6ay01060a},
pmid = {42459132},
issn = {1759-9679},
mesh = {*DNA Methylation/genetics ; Humans ; Septins/genetics ; *CRISPR-Cas Systems/genetics ; *Mutation ; Proto-Oncogene Proteins p21(ras)/genetics ; Promoter Regions, Genetic ; Colorectal Neoplasms/genetics/diagnosis ; DNA Mutational Analysis/methods ; },
abstract = {Early detection of both genetic mutations and epigenetic modifications is critical for cancer diagnosis, but current methods often require separate assays or suffer from bisulfite-induced DNA damage. Here, we present a controllable CRISPR/Cas12a-based signal logic gate that enables simultaneous detection of KRAS G12C mutation and Septin9 promoter methylation in a single reaction. The strategy converts target information into two distinct ssDNA activators (T1 for methylation and T2 for mutation) via orthogonal enzymatic cascades (GlaI- and FEN1-mediated cleavage followed by strand displacement amplification). By limiting crRNA concentrations, the CRISPR/Cas12a trans-cleavage activity produces three well-resolved fluorescence kinetic states: low (mutation only), medium (methylation only), and high (both targets), respectively, which could be distinguished using three predefined threshold values. Validation with clinical samples from colorectal cancer patients and healthy controls showed complete concordance with Sanger sequencing and qPCR. And the proposed method successfully detected Septin9 methylation in peripheral blood. This isothermal, single-tube, bisulfite-free strategy offers a simple and reliable platform for simultaneous genetic and epigenetic analysis in point-of-care cancer screening.},
}

*DNA Methylation/genetics
Humans
Septins/genetics
*CRISPR-Cas Systems/genetics
*Mutation
Proto-Oncogene Proteins p21(ras)/genetics
Promoter Regions, Genetic
Colorectal Neoplasms/genetics/diagnosis
DNA Mutational Analysis/methods

@article {pmid42600477,
year = {2026},
author = {Yuan, J and Shen, M and Ding, L and Yang, X and Wu, Y and Yu, S},
title = {Orthogonal Cas13a/Cas12a cascade for one-pot amplification-free detection of miRNA-21.},
journal = {Talanta},
volume = {312},
number = {Pt B},
pages = {130431},
doi = {10.1016/j.talanta.2026.130431},
pmid = {42600477},
issn = {1873-3573},
abstract = {MicroRNAs (miRNAs) are promising biomarkers for clinical diagnosis and disease monitoring. However, current CRISPR/Cas-based miRNA sensors generally require reverse transcription or nucleic acid amplification to improve sensitivity, which complicates the workflow and increases the risk of contamination, nonspecific amplification, and false-positive results. Herein, we developed a one pot amplification-free Cas13a/Cas12a cascade platform based on a designed dual-functional molecular bridge probe, Conv HP-3, which served as both a substrate for Cas13a-mediated trans-cleavage and an activator for Cas12a-mediated trans-cleavage, thereby linking target recognition to cascade signal amplification for miRNA-21 detection. Following the introduction of miRNA-21, Cas13a was specifically activated through target-crRNA recognition and cleaved the Conv HP-3 probe to release a Cas12a-activating DNA fragment. This fragment subsequently triggered Cas12a-mediated cleavage of the ssDNA reporter, generating a markedly enhanced fluorescence signal. This one-pot Cas13a/Cas12a cascade fluorescence biosensor enabled quantitative detection of miRNA-21 over a concentration range of 1-1000 pmol/L within 60 min, with a low detection limit of 0.66 pM. Notably, the assay achieved average recoveries ranging from 95.97% to 108.59%, with a variation between 0.6% and 1.78%, demonstrating its good accuracy and precision. This biosensing platform shows great promise for the rapid and sensitive detection of miRNAs in clinical applications.},
}

@article {pmid42600889,
year = {2026},
author = {Shiroshita, K and Stolz, A and Malouf, C and Tran, VL and Li, J and Weiss, MJ and Naldini, L},
title = {Unlocking the Curative Potential of Gene Transfer and Editing for Hematopoietic Disorders.},
journal = {Experimental hematology},
volume = {},
number = {},
pages = {105493},
doi = {10.1016/j.exphem.2026.105493},
pmid = {42600889},
issn = {1873-2399},
abstract = {Hematopoietic stem and progenitor cells (HSPCs) gene therapy may transform the therapeutic landscape for inherited hematological disorders and already offers potentially curative options for some diseases, including inborn errors of immunity and β-hemoglobinopathies. Its continued success relies on further refinement of gene transfer technologies, gene editing tools such as CRISPR-Cas, and optimized ex vivo HSPC manipulation protocols that ensure robust, long-term engraftment and clonal diversity with reduced-toxicity, non-genotoxic conditioning strategies. Here, we review recent developments and refinements in gene transfer and editing technologies for HSPCs, while also discussing the critical limitations and hurdles to clinical translation, as recently presented at the New Investigator Committee Gene Therapy webinar. Future directions must prioritize integrating technological innovation with the development of equitable and simplified models to reduce costs and ensure that these life-saving cellular therapies reach patients worldwide. Teaser abstract Hematopoietic stem and progenitor cells (HSPCs) gene therapy is advancing rapidly, with lentiviral gene transfer, genome editing, and emerging in vivo delivery approaches expanding the therapeutic landscape for inherited hematologic disorders. Recent clinical successes have demonstrated the potential for durable correction, while ongoing refinements continue to improve safety, efficacy, and feasibility. Key challenges remain in genotoxicity, conditioning toxicity, manufacturing scalability, and equitable global access.},
}

@article {pmid42601370,
year = {2026},
author = {Iqbal, G and Pinto, N and Pawaskar, D and Sonwane, AA and Rasal, KD and Singh, LS and Chikkathimmashetty, NN and Goswami, M},
title = {Design and transfection of CRISPR/Cas9 constructs for the myostatin gene in Labeo rohita muscle cells.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42601370},
issn = {2045-2322},
mesh = {Animals ; *Myostatin/genetics ; *CRISPR-Cas Systems/genetics ; *Transfection/methods ; Cell Line ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Muscle Cells/metabolism ; *Cyprinidae/genetics ; },
abstract = {Myostatin (mstn) is a negative regulator of skeletal muscle growth and is considered as an important target for enhancing aquaculture production. The present study aimed to design and validate single-guide RNAs (sgRNAs) and CRISPR/Cas9 constructs for exon 1 of the mstnb gene in Labeo rohita, and to evaluate their transfection efficiency in the L. rohita dorsal muscle (LRDM) cell line at the 10th, 20th, and 30th passages. sgRNAs were designed and cloned into the pSpCas9(BB)-2A-GFP (PX458) vector using BbsI restriction digestion and ligation. Successful insertion and correct orientation of the sgRNAs were confirmed through Sanger sequencing. LRDM cells were revived and maintained in L-15 medium supplemented with 10% fetal bovine serum. Transfection was performed at the 10th, 20th, and 30th passages. Distinct GFP-positive cells were observed at all passages for both sgRNA constructs, indicating the ability of the developed cell line to successfully express the constructs across different passages. The study successfully established CRISPR/Cas9 plasmid constructs for the mstnb gene in L. rohita and demonstrated their transfection in LRDM cell line across multiple passages. These findings provide a basis for future studies on genome editing approaches using CRISPR/Cas9 constructs in fish muscle cell lines and highlight the potential application of CRISPR/Cas9 technology for genetic engineering applications in fish muscle cells.},
}

Animals
*Myostatin/genetics
*CRISPR-Cas Systems/genetics
*Transfection/methods
Cell Line
RNA, Guide, CRISPR-Cas Systems/genetics
*Muscle Cells/metabolism
*Cyprinidae/genetics

@article {pmid42602771,
year = {2026},
author = {Hina, A and Abbasi, A and Chaudhry, A and Sanaullah, T and Arshad, M and Sarwar, HM and Sarfaraz, S and Karikari, B and Kavhiza, NJ},
title = {A review of flavonoids at the crossroads of plant defense: integrating biotic and abiotic stress tolerance through AI- and CRISPR/Cas-guided metabolic reprogramming.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1865665},
pmid = {42602771},
issn = {1664-462X},
abstract = {Flavonoids are multifunctional phenylpropanoid-derived metabolites that occupy a central position in plant adaptation to environmental stress. Beyond their established roles in antioxidant protection, they contribute to defense against pathogens and herbivores, signaling processes, and physiological acclimation to adverse environmental conditions. Although flavonoid responses to individual biotic or abiotic stresses have been extensively investigated, considerably less attention has been given to how flavonoid-associated regulatory networks function when multiple stresses occur simultaneously. This gap is particularly important because crops in agricultural systems are routinely exposed to overlapping biotic and abiotic challenges that generate distinct physiological, transcriptional, and metabolic responses. This review synthesizes current knowledge of flavonoid biosynthesis, structure-activity relationships, and the regulatory mechanisms governing flavonoid accumulation under diverse stress conditions. Particular emphasis is placed on the reorganization of flavonoid-associated networks under combined stress, including signaling crosstalk, pathway competition, metabolic trade-offs, and flux allocation that collectively shape adaptive responses. This review further evaluates how artificial intelligence can support identification of regulatory targets and pathway bottlenecks, how integration with CRISPR/Cas technologies may facilitate more precise manipulation of flavonoid biosynthesis, and how iterative Design-Build-Test-Learn (DBTL) frameworks could improve predictive flavonoid engineering through continuous integration of computational prediction and experimental validation. By integrating advances in stress biology, computational prediction, genome engineering, and iterative DBTL frameworks, this review outlines a roadmap for predictive reprogramming of flavonoid networks under combined stress and the development of crops with improved resilience to increasingly complex environmental conditions.},
}

@article {pmid42286694,
year = {2026},
author = {Xu, J and Xu, J and Cao, C and Shu, Y and Shen, L and He, X and Huangfu, Q and Sun, C and Wang, W and Wei, J and Cai, M and Wang, B and Li, A and Liu, Y and Wen, J},
title = {IDMME and IDMDE: inducible CRISPR-dCasRx platforms for spatiotemporal RNA m[5]C editing.},
journal = {Genome biology},
volume = {27},
number = {1},
pages = {},
pmid = {42286694},
issn = {1474-760X},
support = {2021YFA0911600//National Key Research and Development Program of China/ ; 82371630//National Natural Science Foundation of China/ ; },
mesh = {*5-Methylcytosine/metabolism ; Humans ; *CRISPR-Cas Systems ; RNA Methylation ; *RNA Editing ; Animals ; Epigenesis, Genetic ; *Gene Editing/methods ; Epigenome Editing ; Carcinoma, Renal Cell/genetics ; Cell Line, Tumor ; },
abstract = {RNA 5-methylcytosine (m[5]C) is a dynamic epigenetic mark implicated in tumorigenesis, however, existing editors lack spatiotemporal regulation and reversibility. Here, we develop abscisic acid (ABA)-inducible CRISPR-dCasRx systems for programmable m[5]C methylation (IDMME) and demethylation (IDMDE). These editors enable site-specific, low off-target m[5]C modification through ligand-dependent assembly of split effector domains. We further integrate photocaged ABA to achieve light-controlled activation. Application in renal carcinoma models shows that targeted m[5]C editing modulates transcript function and suppresses tumor growth in vitro and in vivo. This platform provides a versatile, spatiotemporally controllable approach for dissecting RNA epigenetic mechanisms and advancing RNA-based therapeutic strategies.},
}

*5-Methylcytosine/metabolism
Humans
*CRISPR-Cas Systems
RNA Methylation
*RNA Editing
Animals
Epigenesis, Genetic
*Gene Editing/methods
Epigenome Editing
Carcinoma, Renal Cell/genetics
Cell Line, Tumor

@article {pmid42361799,
year = {2026},
author = {He, W and Huang, JW and Wang, Y and Hayward, SB and Leuzzi, G and Fu, R and Wang, S and Vaitsiankova, A and Chen, Y and Bedford, MT and Guerois, R and Ciccia, A and Xu, H},
title = {Deciphering protein mutation-phenotype linkages from CRISPR-based tiling mutagenesis screens.},
journal = {Cell systems},
volume = {17},
number = {8},
pages = {101651},
pmid = {42361799},
issn = {2405-4720},
support = {R01 CA197774/CA/NCI NIH HHS/United States ; R35 GM137927/GM/NIGMS NIH HHS/United States ; R35 GM153387/GM/NIGMS NIH HHS/United States ; R35 GM156417/GM/NIGMS NIH HHS/United States ; },
mesh = {*Mutagenesis/genetics ; Phenotype ; Mutation/genetics ; Humans ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {CRISPR-based high-throughput mutagenesis screens enable systematic mapping of mutations to phenotypes, yet deciphering mutation-phenotype links remains challenging. Here, we present ProTiler-Mut, a versatile computational framework that leverages tiling mutagenesis screens, which introduce variants across entire protein sequences, to analyze mutation effects at the levels of residues, substructures, and protein-protein interactions (PPIs). Applying ProTiler-Mut to multi-condition base-editing (BE) screens targeting DNA damage response proteins and T cell regulators, we define a separation-of-function (SoF) category beyond the conventional loss-of-function (LoF) and gain-of-function (GoF) classes, where SoF mutations show the strongest enrichment for ClinVar-annotated pathogenic variants. ProTiler-Mut also identifies candidate substructures that enable functional inference of unscreened pathogenic mutations and prioritizes candidate phenotype-associated PPIs potentially disrupted by functional variants. Using ProTiler-Mut, in cells with elevated programmed cell death 1 (PD-1) expression, we identify pathogenic GoF mutations that constitute a substructure that may disrupt mitogen-activated protein kinase (MAPK)1-RSK1 interactions and lead to MAPK activation. Finally, we show that ProTiler-Mut is applicable across different mutagenesis screening platforms. A record of this paper's transparent peer review process is included in the supplemental information.},
}

*Mutagenesis/genetics
Phenotype
Mutation/genetics
Humans
CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid42372725,
year = {2026},
author = {Hu, Y and Li, Q and Li, Y and Zeng, Y and Zheng, L and Shen, J and Gao, X and Zhao, GP and Zhao, W and Dai, L},
title = {Targeted genomic editing of human gut Bacteroides species based on CRISPR-associated transposases.},
journal = {Cell systems},
volume = {17},
number = {8},
pages = {101650},
doi = {10.1016/j.cels.2026.101650},
pmid = {42372725},
issn = {2405-4720},
mesh = {Humans ; *Bacteroides/genetics ; *Gene Editing/methods ; *Transposases/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Gastrointestinal Microbiome/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Gut Bacteroides are abundant and critical to human health, yet most are genetically cumbersome, non-model microbes. A widely applicable editing tool for Bacteroides is essential for gut microbiome manipulation. Here, we develop STIB (ShCAST-based transient insertion system for Bacteroides), an efficient genome-editing tool derived from CRISPR-associated transposases that enables rapid and site-specific insertions independent of homologous recombination. By fusing a nicking homing endonuclease to the transposase and an ATPase to Cas12k, we systematically optimize STIB to minimize plasmid cointegration and achieve >97% on-target insertion. STIB exhibits broad applicability across different genomic loci in diverse Bacteroides species, including non-model species. Finally, we apply STIB to achieve species- and site-specific editing of distinct Bacteroides species within a complex synthetic gut microbiota. Overall, STIB expands the toolbox for the functional investigation and engineering of the human microbiome. A record of this paper's transparent peer review process is included in the supplemental information.},
}

Humans
*Bacteroides/genetics
*Gene Editing/methods
*Transposases/genetics/metabolism
CRISPR-Cas Systems/genetics
*Gastrointestinal Microbiome/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid42595755,
year = {2026},
author = {Saxena, S and Kabra, M and Abdeen, AA and Tabima, DM and Sinha, D and Rawding, PA and Zhu, M and Xie, R and Kulkarni, T and Hanstad, GM and Fernandez Zepeda, MA and Gamm, DM and Pattnaik, BR and Gong, S and Saha, K},
title = {Genome-wide CRISPR screening identifies cellular factors controlling nonviral genome editing efficiency.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42595755},
issn = {2041-1723},
support = {R35 GM119644/GM/NIGMS NIH HHS/United States ; U19 NS132296/NS/NINDS NIH HHS/United States ; U19NS132296//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; R35GM119644//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Connexin 26 ; Connexins/genetics/metabolism ; Potassium Channels, Inwardly Rectifying/genetics/metabolism ; Receptors, GABA-A/genetics/metabolism ; Mutation ; HEK293 Cells ; Genome, Human ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {To systematically map cellular factors constraining nonviral genome editing, influencing uptake and intracellular trafficking, we develop a genome-wide CRISPR screening platform linking perturbation of 19,114 genes to editing outcomes in human cells. We identify six negative regulators of delivery whose depletion increases editing efficiency by up to six-fold across diverse payloads, loci, and cell types. We test the top two factors, GJB2 and BET1L, in two distinct human models: correction of a pathogenic adenine base mutation in KCNJ13 and introduction of a cytosine base mutation in the GABAA receptor gene. Depletion of either improves base-editing outcomes by 6-fold, potentially through effects on delivery. In a patient-derived model of retinal channelopathy, knockdown of either gene improves lipid nanoparticle base editing efficiency by over 3.5-fold. This enables functional restoration of Kir7.1 ion channels in a subset of edited cells, highlighting cellular barriers as actionable targets to enhance the potency of genetic therapies.},
}

Humans
*CRISPR-Cas Systems
*Gene Editing/methods
Connexin 26
Connexins/genetics/metabolism
Potassium Channels, Inwardly Rectifying/genetics/metabolism
Receptors, GABA-A/genetics/metabolism
Mutation
HEK293 Cells
Genome, Human
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42597245,
year = {2026},
author = {Ye, R and Liu, M and Zhang, X and Liang, Q and Cao, Y and Zhu, C and Yu, H and Zhang, S and Wang, Y},
title = {Rapid and ultrasensitive detection of Candida tropicalis using multiple cross-displacement amplification combined with CRISPR/Cas12a.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1796886},
pmid = {42597245},
issn = {2235-2988},
mesh = {*Candida tropicalis/genetics/isolation & purification ; Humans ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Candidiasis/diagnosis/microbiology ; *Nucleic Acid Amplification Techniques/methods ; DNA Primers/genetics ; DNA, Fungal/genetics ; Multiplex Polymerase Chain Reaction ; Rapid Diagnostic Tests ; Endodeoxyribonucleases/genetics ; CRISPR-Associated Proteins/genetics ; *Molecular Diagnostic Techniques/methods ; DNA, Ribosomal Spacer/genetics ; Bacterial Proteins ; },
abstract = {BACKGROUND: Candida tropicalis (C. tropicalis), classified as a World Health Organization "critical priority" pathogen, causes life-threatening invasive infections with high mortality due to diagnostic delays. Therefore, the development of rapid fungal detection platforms is an urgent scientific and clinical priority.

METHODS: We designed multiple cross-displacement amplification (MCDA) primers to target the internal transcribed spacer 2 (ITS II) gene of C. tropicalis for specific amplification. Subsequently, the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-crRNA complex bound to the amplified products, activating its trans-cleavage activity to generate a detectable fluorescent signal. Finally, clinical samples were used to validate the detection results, which were further compared with those obtained using fungal culture and multiplex polymerase chain reaction (Multiplex PCR).

RESULTS: Under optimized conditions, the C. tropicalis-MCDA-CRISPR/Cas12a assay was completed within approximately 53 min, with a limit of detection of 30 fg of genomic DNA per reaction. The assay showed no cross-reactivity with non-C. tropicalis pathogens. Clinical validation using 128 specimens demonstrated a sensitivity of 100.0% (95% CI: 93.4-100.0%) and a specificity of 97.3% (95% CI: 90.6-99.7%) against a composite reference standard, with near-perfect agreement (κ= 0.968).

DISCUSSION: The C. tropicalis-MCDA-CRISPR/Cas12a assay is an efficient, accurate, and practical diagnostic tool suitable for use in resource-limited laboratories.},
}

*Candida tropicalis/genetics/isolation & purification
Humans
Sensitivity and Specificity
*CRISPR-Cas Systems
*Candidiasis/diagnosis/microbiology
*Nucleic Acid Amplification Techniques/methods
DNA Primers/genetics
DNA, Fungal/genetics
Multiplex Polymerase Chain Reaction
Rapid Diagnostic Tests
Endodeoxyribonucleases/genetics
CRISPR-Associated Proteins/genetics
*Molecular Diagnostic Techniques/methods
DNA, Ribosomal Spacer/genetics
Bacterial Proteins

@article {pmid42597591,
year = {2026},
author = {Hermain, S and K S, H and Nimbagal, RN},
title = {Beyond chemotherapy: The rise of nucleic acid nanoformulations in personalized lung cancer therapy.},
journal = {Cancer pathogenesis and therapy},
volume = {4},
number = {5},
pages = {349-361},
pmid = {42597591},
issn = {2949-7132},
abstract = {Lung cancer remains the leading cause of cancer-related mortality worldwide, driven by complex crosstalk among genetic, molecular, and environmental factors. Conventional treatments, including immunotherapies and targeted inhibitors, face three main challenges: tumor heterogeneity, drug resistance, and systemic toxicity. Nucleic acid therapeutics (NATs) encompass a diverse array of DNA- and RNA-based tools, including small interfering RNA (siRNA), microRNA (miRNA), messenger RNA (mRNA), antisense oligonucleotides (ASOs), and clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) systems. These tools are central to developing precision oncology approaches that operate through direct gene regulation, mutation correction, and immune system reprogramming. The clinical application of NATs currently faces three main obstacles, which include their vulnerability to enzymatic degradation, their limited ability to penetrate tissues, and their tendency to cause off-target effects. The field has progressed through the implementation of nanoformulation techniques, which utilize lipid-based polymeric and metallic carriers together with exosomes and DNA origami, and hybrid nanostructures as new platforms to enhance the stability of drugs and their cellular absorption and targeted delivery to tumors. The scientists developed functionalized nanocarriers by combining targeting ligands with materials that could respond to specific environmental changes, which allowed them to manage drug distribution and release patterns throughout the tumor microenvironment. This review focuses on establishing a direct connection between nucleic acid design and nanotechnology through an analysis of mechanistic details and progress in preclinical and clinical research, and the difficulties encountered during the progress to practical applications. The research demonstrates how artificial intelligence and bioinspired nanocarriers and multi-omics data integration create new opportunities for developing personalized adaptive nanogenetic treatment methods, which will treat lung cancer. The current advancements indicate that we are approaching a transformative era in which nanomedicine and nucleic acid therapeutics will enable safe genetic alterations of cancer through targeted therapeutic applications.},
}

@article {pmid42598860,
year = {2026},
author = {Chilamkurthy, R and Sudhakar, S and Pater, AA and Bosmeny, MS and Stabile, F and Katolik, A and Harikrishna, S and El Azzouzi, F and Turk, R and Damha, MJ and Leslie, S and Korolev, S and Pradeepkumar, PI and Gagnon, KT},
title = {CRISPR RNA architecture steers Cas9 catalysis and fidelity via a guide repeat clasp motif.},
journal = {Nucleic acids research},
volume = {54},
number = {15},
pages = {},
pmid = {42598860},
issn = {1362-4962},
support = {R01 GM135646/GM/NIGMS NIH HHS/United States ; R01 GM154323/GM/NIGMS NIH HHS/United States ; 1R01GM135646/NH/NIH HHS/United States ; R01GM154323/NH/NIH HHS/United States ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems/chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; Biocatalysis ; Base Sequence ; },
abstract = {A widely adopted modification of CRISPR-Cas9 is fusion of the naturally occurring two-component dual guide RNA (dgRNA) to create an artificial single guide RNA (sgRNA). Here we find that these guide architectures induce differential catalysis, gene editing, and specificity. Spacer sequence and RNA structural features could not predict guide architecture editing preference across 255 endogenous targets. We used cryo-EM and molecular dynamics to identify a new Cas9 structural motif, the guide repeat clasp (GRC), that checks guide RNA repeat structure and coordinates with R-loop sensing checkpoint mechanisms to help license cleavage. Limited mutagenesis of GRC residues significantly altered Cas9 editing and specificity, supporting a key role in catalysis. To further understand the role of the GRC and guide RNA repeat dynamics, we created guide repeat-truncated sgRNAs, or grtRNAs, which conferred some dgRNA properties onto sgRNA, including generally lower off-target editing for targets with PAM-proximal mismatches. dgRNAs and grtRNAs could be combined with a new high-fidelity Cas9 variant called ZiFY, rationally designed to reduce editing of targets with PAM-distal mismatches, to generate broader mismatched target discrimination. These results uncover a previously unknown mechanism that steers Cas9 catalysis and demonstrate the potential to improve Cas9 fidelity by modulating guide repeat interactions.},
}

*RNA, Guide, CRISPR-Cas Systems/chemistry/genetics/metabolism
*CRISPR-Cas Systems
Biocatalysis
Base Sequence

@article {pmid42599326,
year = {2026},
author = {Musini, A and Yata, VK and Bukke, SPN and Akhila, K and Shivani, T and Narapureddy, BR and Thalluri, C and Mohamed, AOA and Adam, MDM},
title = {Phage and CRISPR based precision antimicrobials: a dual strategy against multidrug-resistant bacteria.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42599326},
issn = {1573-4978},
support = {RGP2/639/46//Deanship of Research and Graduate Studies at King Khalid University (Large Research Project)/ ; },
mesh = {*Bacteriophages/genetics ; *CRISPR-Cas Systems/genetics ; *Drug Resistance, Multiple, Bacterial/genetics ; *Bacteria/genetics/drug effects/virology ; Humans ; *Phage Therapy/methods ; Anti-Bacterial Agents/pharmacology ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Anti-Infective Agents/pharmacology ; },
abstract = {The rapid global emergence of multidrug-resistant (MDR) bacterial pathogens has significantly reduced the effectiveness of conventional antibiotics, creating an urgent need for alternative antimicrobial strategies. Among emerging precision therapeutics bacteriophage therapy and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas systems have shown to have strong potential through highly specific bacterial targeting mechanisms. Bacteriophages have the ability to replicate themselves and penetrate biofilms, and the ability of CRISPR-Cas systems to edit the genes responsible for antimicrobial resistance, virulence factors, and the mobile genetic elements that underlie bacterial resistance. The recent advancement enabled the integration of these technologies through CRISPR-armed bacteriophages, which utilize bacteriophages as delivery mechanisms for CRISPR and address the large populations of MDR bacteria. Compared to administering CRISPR and bacteriophage independently, the current data suggest that the use of these two methods synergistically will lead to greater efficacy of delivery, specific targeting of resistance determinants, decreased risk of resistance development, and minimal impact on the body's beneficial microorganisms. While the potential combination of these approaches holds great promise to help combat the issue of MDR bacteria, there are still numerous barriers to overcome in order to implement these methods which include narrow phage host range, bacterial escape mechanisms, off-target CRISPR activity, anti-CRISPR proteins, host immune responses, and unresolved manufacturing and regulatory limitations. This review critically examines bacteriophage-based antimicrobials, CRISPR-Cas therapeutic systems, and their emerging integration as CRISPR-armed phages, highlighting their comparative advantages, current limitations, and future potential as promising targeted antimicrobial approach platforms requiring further clinical validation.},
}

*Bacteriophages/genetics
*CRISPR-Cas Systems/genetics
*Drug Resistance, Multiple, Bacterial/genetics
*Bacteria/genetics/drug effects/virology
Humans
*Phage Therapy/methods
Anti-Bacterial Agents/pharmacology
Clustered Regularly Interspaced Short Palindromic Repeats
*Anti-Infective Agents/pharmacology

@article {pmid42599328,
year = {2026},
author = {Mengistu, DA and Mekasha, YT and Molla, FW},
title = {Non-Drug therapeutic strategies to combat antimicrobial resistance in livestock: a review.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42599328},
issn = {1573-4978},
mesh = {Animals ; *Livestock/microbiology/genetics ; Humans ; Probiotics/therapeutic use ; Phage Therapy ; CRISPR-Cas Systems ; *Drug Resistance, Microbial ; Anti-Infective Agents ; Fecal Microbiota Transplantation ; Anti-Bacterial Agents ; Genetic Engineering/methods ; },
abstract = {BACKGROUND: Antimicrobial resistance (AMR) represents an escalating global public health crisis, projected to cause 10 million deaths annually by 2050. Pathogens such as bacteria, viruses, fungi, and parasites evade treatments in animals and humans due to overuse and misuse of antimicrobials, particularly antibiotics, in medical and veterinary practices. This drives a worldwide surge in resistant infections, disproportionately burdening livestock health and productivity.

OBJECTIVES: This review examines the global rise of AMR and its impacts on livestock and public health, underscoring the need for non-drug therapeutic alternatives.

CONCLUSION: Promising alternatives include genetic engineering and CRISPR-Cas systems, phage therapy, probiotics, antimicrobial peptides, fecal microbiota transplantation, phytotherapy and essential oils, immunomodulators, nanotechnology, biofilm disruptors, acidifiers, vaccination strategies, and precision livestock farming. These approaches target resistance mechanisms without relying on conventional drugs. Despite challenges like knowledge gaps, regulatory barriers, financial limitations, and policy gaps, stakeholders must prioritize accelerated research, regulatory reforms, investments, and international collaboration. Rapid integration of these technologies into human and veterinary medicine is vital to mitigate AMR's health, economic, and social impacts.},
}

Animals
*Livestock/microbiology/genetics
Humans
Probiotics/therapeutic use
Phage Therapy
CRISPR-Cas Systems
*Drug Resistance, Microbial
Anti-Infective Agents
Fecal Microbiota Transplantation
Anti-Bacterial Agents
Genetic Engineering/methods

@article {pmid42599397,
year = {2026},
author = {Sun, J and Ni, Y and Guo, M and Yu, T},
title = {A Multiplex CRISPR-Cas12a-Based Hydrogel Microarray Platform for the Simultaneous Detection of Common Adenovirus Types in Clinical Samples.},
journal = {ACS infectious diseases},
volume = {12},
number = {8},
pages = {2815-2824},
doi = {10.1021/acsinfecdis.6c00407},
pmid = {42599397},
issn = {2373-8227},
mesh = {Humans ; *CRISPR-Cas Systems ; Hydrogels/chemistry ; Sensitivity and Specificity ; *Adenoviridae/genetics/isolation & purification/classification ; *Microarray Analysis/methods ; CRISPR-Associated Proteins/genetics ; *Adenoviridae Infections/diagnosis/virology ; Rapid Diagnostic Tests ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Adenovirus infections are a leading cause of respiratory and gastrointestinal diseases, representing a significant global health challenge. Rapid and accurate detection of adenovirus types is essential for timely diagnosis and effective management. Traditional diagnostic methods, such as PCR, are often time-consuming and require complex laboratory infrastructure, limiting their application in resource-limited settings. In this study, we present a CRISPR/Cas12a-based assay integrated with a hydrogel microarray for the simultaneous detection of six common adenovirus types (1, 2, 3, 4, 7, and 14). After amplification of adenoviral DNA using recombinase polymerase amplification (RPA), the amplified DNA enters the hydrogel, where it activates the Cas12a-crRNA complex trapped within the gel. This activation leads to the cleavage of an ssDNA reporter, generating a fluorescent signal. The use of a hydrogel microarray enables efficient and multiplexed detection of adenovirus types in a single assay. The method demonstrated high sensitivity, with detection limits ranging from 10 to 50 copies/μL across the six adenovirus types. It also showed excellent specificity, with no cross-reactivity observed with other respiratory viruses. Clinical validation with 30 human adenovirus samples revealed 100% specificity and high concordance with qPCR results. This CRISPR/Cas12a-based hydrogel microarray platform offers a rapid, cost-effective, and highly specific diagnostic tool with significant potential for clinical and public health applications.},
}

Humans
*CRISPR-Cas Systems
Hydrogels/chemistry
Sensitivity and Specificity
*Adenoviridae/genetics/isolation & purification/classification
*Microarray Analysis/methods
CRISPR-Associated Proteins/genetics
*Adenoviridae Infections/diagnosis/virology
Rapid Diagnostic Tests
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid42599816,
year = {2026},
author = {Sun, T and Yuan, A and Xie, W and Jiang, G and Peng, H},
title = {CRISPR RNA Engineering Enables Single Nucleotide Polymorphism Discrimination in Nucleic Acid Detection.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {},
number = {},
pages = {e00004},
doi = {10.1002/anie.202600004},
pmid = {42599816},
issn = {1521-3773},
support = {22276199//National Natural Science Foundation of China/ ; 2023YFA0915102//National Key Research and Development Program of China/ ; XDB0750100//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; 22306195//Youth Fund from National Natural Science Foundation of China/ ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based diagnostic systems have emerged as powerful platforms for sensitive nucleic acid detection, yet their clinical reliability is frequently constrained by off-target activation and insufficient discrimination of closely related sequences. This review synthesizes current advances aimed at enhancing the specificity of CRISPR diagnostics, with particular emphasis on the pivotal role of CRISPR RNA (crRNA) engineering. We detail how structural determinants of crRNA, including spacer length optimization, intentional mismatch design, secondary-structure modulation, chemical modification, strand-displacement gating, and synergistic design frameworks, govern CRISPR-mediated target recognition and define the energetic and kinetic thresholds for accurate cleavage. Key engineering strategies encompassing computational prediction and modeling, high-throughput screening, and hybrid guide architectures are systematically examined for their capacity to elevate single-nucleotide discrimination, stabilize reaction performance, and enable robust multiplexed detection for pathogen profiling and mutation identification. Despite rapid progress, outstanding challenges persist, including interference from complex clinical matrices, lack of unified evaluation standards, and scalability barriers that hinder clinical translation. Addressing these limitations through integrated crRNA design, system-level optimization, and standardized benchmarking will be essential for realizing the promise of CRISPR diagnostics. Ultimately, these advances are poised to support ultrasensitive, highly specific, and portable point-of-care testing, thereby accelerating precision medicine and strengthening infectious disease surveillance and management.},
}

@article {pmid42600441,
year = {2026},
author = {Zhang, X and Wang, Z and Guo, Z and Wang, H and Chen, Y and Dong, Z and Zeng, L and Yu, D and Hong, M and Wang, J and Tian, G and Yang, W},
title = {A Novel CRISPR/Cas13a sensor for highly sensitive detection of DENV based on AuPt/g-C3N4@GO and Ag@NU-1000.},
journal = {Biosensors & bioelectronics},
volume = {313},
number = {},
pages = {119122},
doi = {10.1016/j.bios.2026.119122},
pmid = {42600441},
issn = {1873-4235},
abstract = {Dengue virus, an arthropod-borne pathogen, poses a significant global public health concern, necessitating the development of sensitive and rapid diagnostic assays. This paper introduces a novel biosensing platform designed for the ultrasensitive detection of dengue viral RNA. The platform integrates recombinase polymerase amplification, CRISPR/Cas13a-based recognition, and electrochemiluminescence resonance energy transfer (ECL-RET). The sensor employs a highly efficient luminescent emitter, which is a ternary composite of gold-platinum nanoparticles supported on graphitic carbon nitride and graphene oxide. A suitable energy acceptor, consisting of silver-decorated metal-organic frameworks, effectively quenches the emitter's signal via resonance energy transfer. Upon the introduction of target nucleic acids, RPA rapidly generates numerous amplicons. These amplicons are specifically recognized by the CRISPR/Cas13a system, which subsequently activates the collateral cleavage activity of Cas13a. This enzymatic activity cleaves a DNA linker that tethers the acceptor to the electrode surface, leading to the release of the acceptor and a subsequent restoration of the electrochemiluminescence intensity. The recovered signal exhibits a linear proportionality to the target concentration across a broad dynamic range, spanning from 0.1 fg/mL to 100 ng/mL. The detection limits for the four dengue serotypes range from 1.09 to 2.5 fg/mL. The assay demonstrates excellent specificity, showing no cross-reactivity with Zika, Japanese encephalitis, or West Nile viruses, and also exhibits good reproducibility. By combining isothermal amplification, specific CRISPR targeting, and a sensitive luminescence readout, this work presents a straightforward and robust method for the early diagnosis of dengue and for outbreak surveillance. Furthermore, this platform can be readily adapted for the detection of other pathogens of interest.},
}

@article {pmid42109006,
year = {2026},
author = {Rajabi Zangi, A and Amiri, A and Eskandari, F and Pazooki, P and Heidari, HR and Hamishehkar, H and Javadzadeh, Y},
title = {RNA therapeutics and their delivery methods: a paradigm for haemophilia management.},
journal = {Journal of drug targeting},
volume = {34},
number = {8},
pages = {1394-1413},
doi = {10.1080/1061186X.2026.2638330},
pmid = {42109006},
issn = {1029-2330},
mesh = {Humans ; *Hemophilia A/therapy/genetics ; *RNA, Small Interfering/administration & dosage ; Animals ; Nanoparticles ; RNA, Messenger/administration & dosage ; *Gene Transfer Techniques ; CRISPR-Cas Systems ; Genetic Therapy/methods ; Lipids/chemistry ; Drug Delivery Systems ; },
abstract = {Haemophilia management is currently undergoing a paradigm shift from traditional protein replacement to RNA modalities and their delivery. This review provides a critical analysis of RNA-based therapeutics (specifically small interfering RNA (siRNA), mRNA and CRISPR/Cas9) as a versatile paradigm distinct from DNA ones. We synthesise clinical and preclinical data to contrast these modalities: siRNA strategies (e.g. fitusiran) have indicated ∼90% reductions in bleeding rates by rebalancing haemostasis independent of factor deficiency; lipid nanoparticles (LNPs)-mRNA platforms offer tuneable, transient factor production without genomic integration risks; and CRISPR-based editing aims for permanent correction (up to 170% coagulation factor IX, FIX expression in preclinical studies) but necessitates rigorous monitoring for off-target effects. Crucially, this article dissects the non-viral delivery landscape determining the clinical viability of these cargos. We evaluate LNPs as the current clinical gold standard for hepatic delivery, contrasting them against emerging polymeric systems, aptamer conjugates and exosomes designed to overcome rate-limiting barriers such as endosomal entrapment and renal clearance. By juxtaposing the immunogenic limitations of viral vectors, we conclude that next-generation RNA therapeutics, enabled by LNPs and GalNAc-conjugation, offer a strategic pathway to overcome the durability gap observed in haemophilia A and expand treatment access to patients currently excluded by viral seroprevalence.},
}

Humans
*Hemophilia A/therapy/genetics
*RNA, Small Interfering/administration & dosage
Animals
Nanoparticles
RNA, Messenger/administration & dosage
*Gene Transfer Techniques
CRISPR-Cas Systems
Genetic Therapy/methods
Lipids/chemistry
Drug Delivery Systems

@article {pmid42269411,
year = {2026},
author = {Zhang, Y and Ma, H and Li, W and Wang, Z and Song, Z and Qi, Y},
title = {A CRISPR/Cas12a-hyperbranched rolling circle amplification sensor for ultrasensitive visual bacteria detection.},
journal = {Talanta},
volume = {310},
number = {},
pages = {129967},
doi = {10.1016/j.talanta.2026.129967},
pmid = {42269411},
issn = {1873-3573},
mesh = {Milk/microbiology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Animals ; Smartphone ; *Biosensing Techniques/methods ; *Staphylococcus aureus/isolation & purification/genetics ; Copper/chemistry ; Metal Nanoparticles/chemistry ; Limit of Detection ; Food Microbiology ; Humans ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The swift spread of pathogenic bacteria via food, air, and water poses severe risks to human health. Conventional detection methods often suffer from time-consuming operations, bulky instruments, and insufficient sensitivity for on-site screening, highlighting an urgent demand for sensitive and visual platforms. Herein, a detection platform (Cas12a-HRCA) was constructed for sensitive pathogen quantification by integrating hyperbranched rolling circle amplification (HRCA), CRISPR/Cas12a system, copper fluorescence nanoparticles (CuNPs) and smartphone-based signal readout. In this strategy, pathogen target-activated CRISPR/Cas12a precisely regulates HRCA initiation, which exponentially generates AT-TA-rich sequences. These products serve as templates for the self-assembly of the fluorescence CuNPs, achieving integrated rapid signal amplification without requiring sample preprocessing steps. Combined with smartphone-based RGB analysis, the platform enables direct, on-site quantitative readout. Cas12a-HRCA demonstrated exceptional performance for S. aureus, with a detection limit of 1 CFU/mL, high specificity, and 91%-106% reliable recovery in spiked milk samples without pre-enrichment or purification, showcasing great potential for point-of-need food safety monitoring. the detection of various pathogenic bacteria in food, clinical or environmental settings.},
}

Milk/microbiology
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
Animals
Smartphone
*Biosensing Techniques/methods
*Staphylococcus aureus/isolation & purification/genetics
Copper/chemistry
Metal Nanoparticles/chemistry
Limit of Detection
Food Microbiology
Humans
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid42296867,
year = {2026},
author = {Xie, Z and Wu, Y and Zhou, P and Zhang, Y and Li, X and Li, Y and Cui, H},
title = {Label-free CRISPR/Cas12a biosensor based on G-triplex/Thioflavin T and GlaI-assisted strand displacement amplification for ultrasensitive DNA methylation detection.},
journal = {Talanta},
volume = {310},
number = {},
pages = {130144},
doi = {10.1016/j.talanta.2026.130144},
pmid = {42296867},
issn = {1873-3573},
mesh = {*DNA Methylation ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems ; *Benzothiazoles/chemistry ; DNA/genetics ; *Nucleic Acid Amplification Techniques/methods ; },
abstract = {DNA methylation represents a pivotal epigenetic biomarker for cancer, and precise profiling of carcinogenesis-associated methylation is essential for early diagnosis and prognostic evaluation. Herein, we report a novel label-free, sensitive DNA methylation biosensor by integrating GlaI-assisted double cascade strand displacement amplification with G-triplex-facilitated CRISPR/Cas12a (G-DCSDA/Cas12a). Instead of conventional fluorescent-quenched (FQ) probes, this assay employed G-triplex/Thioflavin T as a simple and efficient signal reporter for CRISPR/Cas12a. The methylation-specific endonuclease GlaI selectively digested methylated DNA to release free 3'-OH ends, which initiated the subsequent DCSDA and triggered Cas12a activation. The double-template cascade amplification system delivers significantly improved sensitivity in comparison with the single-template strategy. Upon activation, the trans-cleavage activity of Cas12a rapidly disrupted G-triplex/Thioflavin T complexes, generating a distinct fluorescence response. By combining the high specificity of GlaI, efficient signal amplification of DCSDA, and robust collateral cleavage of Cas12a, the G-DCSDA/Cas12a platform achieved ultrahigh sensitivity and selectivity, enabling detection of methylation levels as low as 0.005% in a background of excessive unmethylated DNA. Furthermore, this strategy was successfully integrated into a lateral flow assay (LFA), enabling visual and point-of-care testing (POCT) of DNA methylation. Importantly, the developed biosensor achieved sensitive detection of genomic DNA methylation in real samples and accurately discriminates cancer cells from normal cells, as well as between colorectal cancer tissue and adjacent normal tissue. These results highlighted the significant potential of the G-DCSDA/Cas12a platform for clinical early cancer diagnosis.},
}

*DNA Methylation
*Biosensing Techniques/methods
Humans
*CRISPR-Cas Systems
*Benzothiazoles/chemistry
DNA/genetics
*Nucleic Acid Amplification Techniques/methods

@article {pmid42330823,
year = {2026},
author = {Jiang, H and Yang, J and Li, A and Mou, D and Deng, Y and Lv, X},
title = {Competitive kinetic mechanisms in one-pot isothermal amplification-CRISPR systems: From model construction to performance evaluation.},
journal = {Talanta},
volume = {310},
number = {},
pages = {130184},
doi = {10.1016/j.talanta.2026.130184},
pmid = {42330823},
issn = {1873-3573},
mesh = {Kinetics ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Nucleic Acid Hybridization ; },
abstract = {The urgent demand for integrating real-time molecular diagnosis with isothermal amplification and CRISPR-based detection has underscored the critical need for streamlined, single-tube one-pot methodologies. However, such integration is often hindered by temporal incompatibilities, including premature activation of early amplification products by CRISPR components, which leads to primer degradation and reduced amplification efficiency. To address this challenge, we established a dedicated quantitative competitive kinetic framework for enzyme-free isothermal amplification-CRISPR one-pot systems, using hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA) as representative models. Two core inhibitory mechanisms were identified: "pre-activation-degradation inhibition", in which early CRISPR activation degrades amplification intermediates, and "substrate competition inhibition" in which hairpin probes compete with reporter probes for CRISPR trans-cleavage. Corresponding kinetic equations were derived to describe these interactions quantitatively. Systematic experimental validation of key parameters such as DNA activator concentration, reporter probe concentration, hairpin probe concentration, and ribonucleoprotein (RNP) complex concentration, confirmed the reliability and predictive accuracy of the proposed models. These results provide mechanistic insights into the factors governing one-pot HCR/CHA-CRISPR coupling and identify conditions that optimize assay performance. Overall, this study offers a theoretical foundation and experimental guidance for probe design, reaction condition optimization, and sensitivity enhancement in enzyme-free isothermal amplification-CRISPR one-pot platforms, and provides a general reference for the rational integration of isothermal amplification and CRISPR-based detection.},
}

Kinetics
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
Nucleic Acid Hybridization