@article {pmid42063231,
year = {2026},
author = {Hou, Z and Zhao, Y and Sun, Z and Zhan, Y and Dai, X and Wang, H},
title = {One-Pot RAA-CRISPR/Cas12a Assay for Rapid Detection of Infectious Hypodermal and Haematopoietic Necrosis Virus (IHHNV) in Shrimp Aquaculture.},
journal = {Journal of fish diseases},
volume = {49},
number = {10},
pages = {e70196},
doi = {10.1111/jfd.70196},
pmid = {42063231},
issn = {1365-2761},
support = {202205//Agriculture Research System of Shanghai, China/ ; },
mesh = {Animals ; *Densovirinae/isolation & purification ; *Penaeidae/virology ; Aquaculture ; *Nucleic Acid Amplification Techniques/methods/veterinary ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; Rapid Diagnostic Tests ; *Molecular Diagnostic Techniques/methods/veterinary ; },
abstract = {The infectious hypodermal and haematopoietic necrosis virus (IHHNV) represents a significant viral threat to global shrimp aquaculture, leading to considerable economic losses. In this study, we have developed a one-step, one-pot isothermal assay for the detection of IHHNV, employing recombinase-aided amplification in conjunction with clustered regularly interspaced short palindromic repeats-Cas12a (RAA-CRISPR/Cas12a). The assay is performed at a constant temperature of 37°C, achieving a detection limit of 10 copies per reaction for the fluorescence assay and 1 copy per reaction for the lateral flow dipstick (LFD) assay within a 60-min timeframe. Additionally, we evaluated the assay against four other prevalent shrimp pathogens (WSSV, DIV1, EHP, VpAHPND) and observed no cross-reactivity. This straightforward detection method exhibits high sensitivity and specificity for IHHNV, offering a promising approach for early and rapid field diagnosis.},
}

Animals
*Densovirinae/isolation & purification
*Penaeidae/virology
Aquaculture
*Nucleic Acid Amplification Techniques/methods/veterinary
Sensitivity and Specificity
*CRISPR-Cas Systems
Rapid Diagnostic Tests
*Molecular Diagnostic Techniques/methods/veterinary

@article {pmid42322407,
year = {2026},
author = {Sigrist, R and Chen, T and Montané, MR and Gockel, P and Qiao, Y and Jönsson, M and Yang, Z and Weber, T and Ding, L and Özdemir, E and Yang, L},
title = {Heterologous production of a plant biostimulant in Streptomyces albidoflavus.},
journal = {Applied microbiology and biotechnology},
volume = {110},
number = {1},
pages = {},
pmid = {42322407},
issn = {1432-0614},
support = {NNF20CC0035580//Novo Nordisk Fonden/ ; NNF22OC0079928//Novo Nordisk Fonden/ ; NNF23OC0082882//Novo Nordisk Fonden/ ; },
mesh = {*Streptomyces/genetics/metabolism ; *Metabolic Engineering/methods ; Promoter Regions, Genetic ; Multigene Family ; Polyketide Synthases/genetics/metabolism ; Fermentation ; CRISPR-Cas Systems ; Cloning, Molecular ; Biosynthetic Pathways/genetics ; },
abstract = {Climate change-associated abiotic stresses threaten agricultural productivity, creating a need for sustainable strategies that improve plant resilience. Pteridic acids F and H (PTA-F and PTA-H), originally isolated from Streptomyces iranensis HM 35, are plant growth-promoting polyketides with reported activity under drought and salinity stress. However, reported production was extremely low (~ 0.08 and 0.02 mg/L), limiting further development and application. Here, we established a heterologous production platform for PTA biosynthesis by cloning the 68-kb type I polyketide synthase biosynthetic gene cluster using Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination (CAPTURE), followed by CRISPR-Cas9-mediated genomic integration and promoter engineering in Streptomyces hosts. Initial heterologous expression resulted in detectable elaiophylin production but not PTA, whereas BGC engineering with the strong constitutive kasOp* promoter enabled PTA production (although below the limit of quantification). Genome-scale metabolic model-guided media optimization further improved production and fed-batch fermentation yielded 1.7 mg/L PTA in J1074-PTA-kasOp* and 2.8 mg/L PTA in NBC1270-PTA-kasOp*. These titers represent a more tha n 20-fold increase compared with the native producer under comparable conditions. This work provides the first functional heterologous platform for PTA biosynthesis and demonstrates how synthetic biology and genome-scale metabolic modeling can be combined to improve production of complex plant-beneficial polyketides. KEY POINTS: • Direct BGC cloning and engineering enabled production of PTA in heterologous host. • Genome-scale metabolic models (GEMs) guided media optimization for PTA production. • Fed-batch fermentation achieved > 20-fold PTA titer improvement over native strain.},
}

*Streptomyces/genetics/metabolism
*Metabolic Engineering/methods
Promoter Regions, Genetic
Multigene Family
Polyketide Synthases/genetics/metabolism
Fermentation
CRISPR-Cas Systems
Cloning, Molecular
Biosynthetic Pathways/genetics

@article {pmid42508370,
year = {2026},
author = {Kruisselbrink, AB and Wilpshaar, TAH and Palubeckaitė, I and Dijkland, RC and Belova, T and Cardoso, S and Wijers-Koster, PM and Briaire-de Bruijn, IH and van Zeijl, RJM and Dalebout, H and Kuijjer, ML and Mei, H and Heijs, B and Szuhai, K and Bovée, JVMG and Venneker, S},
title = {Targeting vulnerabilities in IDH mutant tumours: The model matters.},
journal = {Neoplasia (New York, N.Y.)},
volume = {80},
number = {},
pages = {101338},
pmid = {42508370},
issn = {1476-5586},
mesh = {Humans ; *Isocitrate Dehydrogenase/genetics ; *Mutation ; *Chondrosarcoma/genetics/pathology/metabolism ; Cell Line, Tumor ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {INTRODUCTION: Synthetic lethal interactions with IDH1 and IDH2 (IDH) mutations were identified in non-endogenous IDH mutant (IDH[MUT]) AML and glioma models, but are absent in endogenous IDH[MUT] chondrosarcoma cell lines. The translation into successful clinical applications has remained challenging, implying artificially created models do not fully recapitulate endogenous IDH[MUT] tumour biology. The aim of this study was to elucidate if the model system is indeed an important factor to consider when studying therapeutic vulnerabilities in IDH[MUT] tumours.

METHODS: Vector-based and CRISPR-Cas9 approaches were used to introduce or revert the IDH1 mutation in chondrosarcoma cell lines. These isogenic cell line pairs were used to examine the presence of known therapeutic vulnerabilities and their underlying biological mechanisms.

RESULTS: Vector-based IDH[MUT] chondrosarcoma models showed the previously reported synthetic lethal interactions, but these treatment sensitivities were absent in the CRISPR-edited models. Interestingly, not all vector-based IDH[MUT] cell lines displayed the same therapeutic vulnerabilities. Differences in treatment response were associated with multiple factors, including IDH[MUT] protein expression and D-2-HG levels, in line with the fact that therapeutic vulnerabilities could be induced in the CRISPR-edited models by enhancing D-2-HG levels.

CONCLUSION: Our findings demonstrate that synthetic lethal interactions observed in vector-based models are often a consequence of IDH[MUT] protein overexpression and supra-physiological D-2-HG levels. These results highlight that relying on artificially created IDH[MUT] models may lead to the identification of therapeutic vulnerabilities that are not present in IDH[MUT] tumours, potentially explaining the poor translation of preclinical findings to clinical trials.},
}

Humans
*Isocitrate Dehydrogenase/genetics
*Mutation
*Chondrosarcoma/genetics/pathology/metabolism
Cell Line, Tumor
CRISPR-Cas Systems
Gene Editing

@article {pmid42521833,
year = {2026},
author = {Zaalberg, A and Lacoste, A and Minnee, E and Mayayo-Peralta, I and Schuurman, K and Gregoricchio, S and van Schaik, TA and Hoekman, L and Li, D and Corey, E and Janssen, H and Lieftink, C and Prekovic, S and Proost, N and van de Ven, M and Zander, S and Altelaar, M and Nelson, PS and Beijersbergen, RL and Zwart, W and Bergman, AM},
title = {A genome-wide CRISPR screen in human prostate cancer cells reveals drivers of macrophage-mediated cell killing and positions AR as a tumor-intrinsic immunomodulator.},
journal = {Oncogene},
volume = {45},
number = {36},
pages = {3797-3811},
pmid = {42521833},
issn = {1476-5594},
mesh = {Humans ; Male ; *Prostatic Neoplasms/genetics/immunology/pathology ; *Receptors, Androgen/genetics/metabolism/immunology ; *Macrophages/immunology/metabolism ; NF-kappa B/metabolism/genetics ; Cell Line, Tumor ; Signal Transduction ; CRISPR-Cas Systems ; Nitriles ; Tumor Microenvironment/immunology/genetics ; I-kappa B Kinase/genetics ; Benzamides ; Phenylthiohydantoin/analogs & derivatives/pharmacology ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Macrophages are the most abundant immune cells in the prostate tumor microenvironment and capable of killing tumor cells, but tumor intrinsic modulators of resistance to the innate immune system are unknown. To identify genes essential for macrophage-mediated killing, we performed a genome-wide co-culture CRISPR screen and identified Androgen Receptor (AR), PRKCD, and multiple components of the NF-κB pathway (IKBKB/IKBKG/CHUK) as tumor-intrinsic essential factors to allow for macrophage-mediated killing. Mechanistically, both AR and NF-κB directly drive expression of PRKCD within cancer cells, functionally implicating all hits within one molecular pathway. Importantly, androgen deprivation and AR-inhibition both rendered tumor cells resistant to macrophage-mediated killing, which positions tumor-intrinsic AR signaling as a bona fide immunomodulatory pathway. Proteomic analyses showed a selective downregulation of the oxidative phosphorylation pathway in PRKCD- and IKBKG-KO cells, suggesting impaired mitochondrial function, which was confirmed by electron microscopy analyses. Finally, phosphoproteomic analyses revealed that all hits perturbing macrophage-mediated tumor cell eradication, impaired ferroptosis signaling in the tumor cells, which was confirmed transcriptionally using samples from a neoadjuvant phase II clinical trial with the AR-inhibitor enzalutamide. These data reveal immune protection from macrophages as an adverse consequence of hormonal therapy in prostate cancer patients.},
}

Humans
Male
*Prostatic Neoplasms/genetics/immunology/pathology
*Receptors, Androgen/genetics/metabolism/immunology
*Macrophages/immunology/metabolism
NF-kappa B/metabolism/genetics
Cell Line, Tumor
Signal Transduction
CRISPR-Cas Systems
Nitriles
Tumor Microenvironment/immunology/genetics
I-kappa B Kinase/genetics
Benzamides
Phenylthiohydantoin/analogs & derivatives/pharmacology
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42586255,
year = {2026},
author = {Ivanova, E and Ramp, P and Zimmer, N and Mund, M and Antonov, E and Schiklenk, C and Degreif, D},
title = {Inducible flocculation in Komagataella phaffii enables enhanced biomass separation for biopharmaceutical production.},
journal = {Metabolic engineering},
volume = {98},
number = {},
pages = {102523},
doi = {10.1016/j.ymben.2026.102523},
pmid = {42586255},
issn = {1096-7184},
mesh = {Flocculation ; *Saccharomycetales/genetics/metabolism ; *Biomass ; Saccharomyces cerevisiae Proteins/genetics/biosynthesis ; CRISPR-Cas Systems ; Mannose-Binding Lectins/genetics/biosynthesis ; },
abstract = {Biomass separation represents a critical bottleneck in Komagataella phaffii-based biopharmaceutical processes, as typically high cell densities of 40 - 50 % create significant operational, technical and economic challenges for harvest operations. Yeast cell aggregation (flocculation) provides a solution to accelerate cell sedimentation by increasing particle size, thus allowing to improve biomass-supernatant separation efficiency during both natural gravity settling and (continuous) centrifugation operations. This study demonstrates successful engineering of K. phaffii strains with an inducible flocculation phenotype using CRISPR/Cas9-based genome editing to integrate the Saccharomyces cerevisiae FLO1 (ScFLO1) gene under control of various regulatory elements, including methanol-inducible and derepressible promoters. Flocculation strength could be enhanced by implementing transcriptional positive feedback circuits based on the methanol-inducible AOX1 promoter. To address methanol-free production requirements, we developed alternative systems to retrofit PAOX1-based ScFLO1 expression and exploited the derepressible PDF promoter, offering broader compatibility with biopharmaceutical manufacturing facilities. Flocculating cells cultivated in a bioreactor demonstrated significantly improved sedimentation behavior, with considerably lower supernatant turbidity after short low-speed centrifugation or gravity sedimentation compared to non-flocculating controls. Crucially, cell flocculation had no negative impact on product amount and quality when expressing a multivalent NANOBODY® VHH molecule with pharmaceutical relevance. Thus, this work establishes the first genetically engineered flocculation system in K. phaffii compatible with recombinant protein production, providing the basis for an innovative approach to streamline harvest operations in biopharmaceutical processes.},
}

Flocculation
*Saccharomycetales/genetics/metabolism
*Biomass
Saccharomyces cerevisiae Proteins/genetics/biosynthesis
CRISPR-Cas Systems
Mannose-Binding Lectins/genetics/biosynthesis

@article {pmid42595042,
year = {2026},
author = {Yu, Z and Chen, K and Maimaitirexiati, G and Bai, Z and Li, S and Yu, A and Yu, T and Guo, S},
title = {A dual-dimensional CRISPR toolkit enables one-step high-efficiency multiplex genome editing in Komagataella phaffii.},
journal = {Metabolic engineering},
volume = {98},
number = {},
pages = {102525},
doi = {10.1016/j.ymben.2026.102525},
pmid = {42595042},
issn = {1096-7184},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Saccharomycetales/genetics/metabolism ; },
abstract = {Against the backdrop of green biomanufacturing, engineering methanol-utilizing Komagataella phaffii (K. phaffii) represents an effective strategy to expand the one carbon (C1) product profile and speed up the industrialization of C1-based bioeconomy. To address the technical challenges of low efficiency and cumbersome experimental procedures for multiplex gene editing and precise large-fragment integration during the reconstruction of complex metabolic pathways in K. phaffii, this study established a CRISPR toolkit - Efficient Multi-Gene Editing System 3.0 (EMGES 3.0) - which enabled one-step large-fragment integration coupled with multiplex gene knockout. EMGES 3.0 was constructed through the synergistic optimization of a repair-engineered chassis and an episomal CRISPR vector. For chassis engineering, five DNA repair modules: Δlig4 (DNA Ligase IV, non-homologous end joining end ligation), ppMRE11(The endogenous MRE11 gene from Pichia pastoris) overexpression (The Meiotic Recombination 11, DNA double-strand break end resection), Δrad9 (Radiation-Sensitive 9, DNA damage checkpoint regulation), Δmph1 (Mutator Phenotype Helicase 1, improvement of homologous recombinant strand extension), and PapRecT-PaSSB co-expression (stabilization of recombination intermediates) were integrated to generate the highly recombinogenic strain Y09. For vector engineering, cenARS was replaced by panARS and the endogenous promoter PGAP was employed to drive the double hammerhead ribozyme-single guide RNA-hepatitis delta virus ribozyme (double HH-sgRNA-HDV: dHgH)-mediated sgRNA expression, yielding the optimized vector Nov_pGAP_panARS_pLAT1_Cas9. These two features on K. phaffii together enhanced the EMGES 3.0 to a higher standard of transformation rate and editing efficiency. According to our results, EMGES 3.0 achieved dual-functional gene knockout efficiencies between 76.6% and 100%. For insertion of medium-long fragments (>4.5 kb), the efficiency achieved 93.3%. In addition, the one-step integration of ultra-long fragments (>16 kb) achieved 14.8%, which was reported for the first time. Furthermore, the efficiency of simultaneous long-fragment integration at three neutral loci reached 38.4% (>15 kb). We applied the system for one-step production of free fatty acids (FFAs, yield: 5.82 ∼ 7.30 mg/L/OD600) and resveratrol (yield: 1.14 ∼ 1.28 mg/L) using methanol as the sole carbon source. EMGES 3.0 provides a robust technical foundation for complex compounds biosynthesis and high-yield industrial strains, while also advancing K. phaffii as an industrial synthetic biology chassis for efficient C1 utilization.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
*Saccharomycetales/genetics/metabolism

@article {pmid42628890,
year = {2026},
author = {Hassibian, S and Esmaelpourfarkhani, M and Alibolandi, M and Ramezani, M and Abnous, K and Dehnavi, SM and Taghdisi, SM},
title = {CRISPR/Cas12a-based tag-free fluorescent biosensor using G-quadruplex specific thioflavin T for detection of oncogenic microRNAs.},
journal = {Methods (San Diego, Calif.)},
volume = {255},
number = {},
pages = {37-47},
doi = {10.1016/j.ymeth.2026.08.006},
pmid = {42628890},
issn = {1095-9130},
mesh = {*Biosensing Techniques/methods ; *G-Quadruplexes ; *Benzothiazoles/chemistry ; Humans ; *MicroRNAs/blood/genetics ; *CRISPR-Cas Systems ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins/genetics ; Fluorescence ; Fluorescent Dyes/chemistry ; Endodeoxyribonucleases ; },
abstract = {MicroRNAs (miRs) are central regulators of tumor initiation and progression, and their aberrant expression patterns have been identified as clinically valuable biomarkers for the early diagnosis of malignancies and prognostic evaluation. Here, we report tag-free fluorescence biosensing platform for the detection of circulating miRs in serum, targeting microRNA-21 (miR-21) and microRNA-10b (miR-10b) as clinically relevant oncogenic markers. The assay integrates CRISPR/Cas12a-mediated signal regulation with complementary strand (CS)-mediated target recognition. In this strategy, target miRs hybridize with the CS, thereby preventing CS-mediated activation of the Cas12a-crRNA complex. As a result, Cas12a collateral cleavage is suppressed, the G-quadruplex reporter remains intact, and Thioflavin T fluorescence is enhanced. The platform demonstrated excellent sequence discrimination capability, effectively distinguishing closely related and mismatched targets. Sensitive quantification was achieved with limits of detection of 1.4 nM for miR-21 and 852 pM for miR-10b. Importantly, robust analytical performance was maintained in complex biological matrices, confirming its applicability to serum samples. Collectively, this CRISPR/Cas12a-enabled fluorescent biosensor provides a simple approach for circulating miR detection.},
}

*Biosensing Techniques/methods
*G-Quadruplexes
*Benzothiazoles/chemistry
Humans
*MicroRNAs/blood/genetics
*CRISPR-Cas Systems
CRISPR-Associated Proteins/genetics
Bacterial Proteins/genetics
Fluorescence
Fluorescent Dyes/chemistry
Endodeoxyribonucleases

@article {pmid42652212,
year = {2026},
author = {Vanapilli Nursimulu, T and Ali, M and Shin, JA},
title = {Exploring the Evolutionary Landscape with Targeted In Vivo Hypermutations.},
journal = {Biomedicines},
volume = {14},
number = {8},
pages = {},
pmid = {42652212},
issn = {2227-9059},
support = {Discovery Grant 04846//Natural Sciences and Engineering Research Council of Canada/ ; Operating Grant 1050460//Cancer Research Society/ ; },
abstract = {Directed evolution has revolutionized protein engineering by applying the principles of natural selection to the laboratory. However, traditional in vitro methods are quite labor-intensive, while common in vivo methods suffer from low mutation rates and high rates of off-target mutations. To address these issues, researchers have developed targeted mutagenesis tools for rapid in vivo evolution of biomolecules. In this review, we discuss recent in vivo hypermutation tools that enable rapid sampling of the vast evolutionary landscape, all while supporting simultaneous selection of the best proteins within living organisms. We focus on three main mechanisms of hypermutation: (i) orthogonal replication, which uses error-prone replication machinery to replicate the target gene with low fidelity; (ii) CRISPR-Cas-guided mutators, where mutagenic proteins are localized to virtually any user-defined loci; and (iii) transcription-coupled mutagenesis, a simple, yet elegant tool that exploits the innate processivity of orthogonal ribonucleic acid (RNA) polymerases to guide mutagenic proteins along the target gene during transcription. We highlight key advantages of these systems, as well as some clinically- and biotechnology-relevant applications. We discuss important limitations and how they could be addressed in the future to make hypermutation tools with broad mutational spectra and windows that span entire genes with minimal off-target effects.},
}

@article {pmid42652949,
year = {2026},
author = {Hamimed, S and Merazka, R and Kamah, A and Kamah, FZ and Keroui, M},
title = {Emerging Frontiers in CRISPR-Based Strategies for the Detection and Degradation of Microplastics.},
journal = {Life (Basel, Switzerland)},
volume = {16},
number = {8},
pages = {},
pmid = {42652949},
issn = {2075-1729},
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats)-based genome engineering is reshaping how environmental contamination can be interrogated and remediated, offering a level of programmability and specificity that conventional physicochemical workflows seldom match. Microplastics polymer fragments below 5 mm that now pervade virtually every ecosystem are especially difficult to monitor and remove because of their chemical heterogeneity, sub-millimeter size, and capacity to adsorb co-pollutants. This review examines how the molecular logic of CRISPR-Cas systems is being repurposed for two complementary goals: sensitive analytical detection and microbially driven degradation of plastic particles. We first outline the biochemistry of Cas-mediated cis- and trans-cleavage that underpins isothermal, amplification-free biosensing, and then survey direct strategies, in which polymer-binding DNA (deoxyribonucleic acid) aptamers are coupled to Cas12a (CRISPR-associated protein 12a), alongside indirect strategies that read out the molecular stress signatures provoked by microplastic exposure in sentinel organisms and plastisphere communities. On the remediation side, we discuss how targeted editing, CRISPR interference, and rationally assembled microbial consortia enhance enzymatic depolymerization and redirect carbon flux toward valuable bioproducts. By integrating detection and remediation within a single conceptual framework, we identify the principal bottlenecks, aptamer selectivity in complex matrices, reagent stability under field conditions, and host metabolic burden, and outline research priorities for translating these tools from proof of concept toward deployable environmental technologies.},
}

@article {pmid42653084,
year = {2026},
author = {Du, L and Dong, Y and Yang, J and Zhang, Z and Liu, Z},
title = {DNA Methylation as a Programmable Information Layer: From Molecular Marks to Disease State Engineering.},
journal = {International journal of molecular sciences},
volume = {27},
number = {16},
pages = {},
pmid = {42653084},
issn = {1422-0067},
support = {12302397//National Natural Science Foundation of China/ ; },
mesh = {*DNA Methylation ; Humans ; Epigenome Editing ; *Epigenesis, Genetic ; Animals ; CRISPR-Cas Systems ; Epigenomics/methods ; },
abstract = {DNA methylation has long been regarded as a stable, maintenance-based epigenetic marker. However, this classical binary model struggles to fully explain the dynamic and situational dependence of methylation regulation at the multi-biological level. This review defines DNA methylation as a programmable information layer that systematically integrates the latest advances in three interrelated dimensions of molecular coding, disease status indication, and epigenomic engineering. At the molecular level, this paper describes how the chemical diversity of cytosine modification, the writing-erasing enzyme network, and the three-dimensional structure of chromatin jointly construct a methylated polymorphic coding system and evaluates the performance of emerging sequencing technologies in DNA integrity, reading length, modification resolution, and analytical complexity through a multidimensional scoring framework. At the cellular and clinical levels, this paper comprehensively demonstrates methylation as a quantifiable indicator of cell identity, biological aging and disease status, covering circulating free DNA biomarkers and spatial heterogeneity analysis. Critically, this paper evaluates how the clustered regularly interspaced short palindromic repeats (CRISPR)-based epigenome editing platform achieves causal inference and promotes the transformation of methylation from related biomarkers to functional therapeutic targets. At the same time, persistent challenges such as off-target specificity, in vivo delivery, and spatiotemporal regulation encountered in epigenetic gene editing are discussed. This review reveals the paradigm shift of DNA methylation from passive observation markers to actively engineered regulatory parameters, which has direct therapeutic application prospects.},
}

*DNA Methylation
Humans
Epigenome Editing
*Epigenesis, Genetic
Animals
CRISPR-Cas Systems
Epigenomics/methods

@article {pmid42653108,
year = {2026},
author = {Wang, X and Zhou, H and Lim, TS and Węgrzyn, G},
title = {Phage Therapy Enhanced by Using Engineered Bacteriophages: A Powerful Antibacterial Tool to Address the Dilemma Posed by Multidrug-Resistant Bacterial Infections.},
journal = {International journal of molecular sciences},
volume = {27},
number = {16},
pages = {},
pmid = {42653108},
issn = {1422-0067},
mesh = {*Phage Therapy/methods ; Humans ; *Bacteriophages/genetics/physiology ; *Bacterial Infections/therapy/microbiology ; *Drug Resistance, Multiple, Bacterial ; CRISPR-Cas Systems ; Anti-Bacterial Agents/therapeutic use/pharmacology ; Genetic Engineering ; Animals ; Synthetic Biology/methods ; Gene Editing ; Bacteria/virology ; Biofilms ; },
abstract = {The continuous slowdown in the research and development of new antibiotics and antibiotic overuse have turned the problem of antibacterial resistance into a global public health crisis. As a very promising alternative to multi-drug-resistant bacterial infection, phage therapy is receiving renewed attention. However, the inherent biological limitations of natural phages restrict their extensive clinical application. This review examines how synthetic biology can be harnessed to transform phages and to build the next generation of antibacterial therapies. We outline the main advantages of natural phages, including high host specificity, self-amplification, bactericidal activity and the ability to degrade biofilms. We also point out the bottlenecks of clinical applications of bacteriophages, such as narrow host range, rapid removal in the body and potential genetic safety risks. Moreover, we elaborate on the core synthetic biological tools used to overcome the above limitations, including CRISPR-Cas gene editing, receptor-binding protein reprogramming, functional load delivery and immunogenic regulation, and summarize the recent clinical progress and personalized treatment process. The increasing clinical evidence shows that synthetic biology can effectively overcome the inherent defects of natural bacteriophages, confirming the safety and initial efficacy of bacteriophage therapy. Engineered phages provide a practical strategy to meet the antimicrobial resistance challenge. Clinical applications of such phages will mainly depend on progress in production standardization, regulatory framework construction and scientific and reasonable joint treatment program development.},
}

*Phage Therapy/methods
Humans
*Bacteriophages/genetics/physiology
*Bacterial Infections/therapy/microbiology
*Drug Resistance, Multiple, Bacterial
CRISPR-Cas Systems
Anti-Bacterial Agents/therapeutic use/pharmacology
Genetic Engineering
Animals
Synthetic Biology/methods
Gene Editing
Bacteria/virology
Biofilms

@article {pmid42653124,
year = {2026},
author = {Tang, H and Li, H and Tai, Y and Yang, X and Zhang, L and Ma, Y and Cai, G and Zhao, H and Zeng, T and Ai, X and He, S and Wang, J and Gu, Z and Deng, X},
title = {Heterogeneous CRISPR/Cas9 Editing of HMOX1 Is Associated with Altered Heme-Biliverdin Metabolism and Basal Stress-Associated Transcriptional Programs in Chicken LMH Cells.},
journal = {International journal of molecular sciences},
volume = {27},
number = {16},
pages = {},
pmid = {42653124},
issn = {1422-0067},
support = {2022LZGCQY016//Department of Science and Technology of Shandong Province/ ; 32472896//National Natural Science Foundation of China/ ; 2022JXCQZY05//Jiangxi Provincial Joint Research Project/ ; //the National Joint Breeding Research Project. Additional support was provided by the 2115 Talent Development Program of China Agricultural University/ ; },
mesh = {Animals ; Chickens/genetics ; *Heme Oxygenase-1/genetics/metabolism ; *Biliverdine/metabolism ; *Heme/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Stress, Physiological/genetics ; Transcription, Genetic ; Cell Line, Tumor ; },
abstract = {Heme oxygenase-1 (HO-1), encoded by HMOX1, catalyzes the rate-limiting step of heme degradation and generates biliverdin, carbon monoxide, and ferrous iron, thereby linking heme turnover with redox regulation and stress-associated signaling. In birds, biliverdin is retained as a major heme-derived product, but the cellular consequences of HMOX1 perturbation remain insufficiently defined. Here, CRISPR/Cas9-mediated editing was used to generate a heterogeneous HMOX1-edited population in Chicken hepatocellular carcinoma-derived cells. The selected sgRNA reduced HO-1 protein abundance by approximately 47%, and no detectable cleavage was observed at the seven predicted high-risk off-target loci examined. Compared with vector-control cells, HMOX1-edited cells exhibited intracellular heme accumulation, reduced biliverdin levels, increased oxidation-sensitive fluorescence, and reduced CCK-8 absorbance values, indicating disruption of heme-biliverdin metabolic and redox homeostasis. RNA sequencing identified 2650 differentially expressed genes, including 951 upregulated and 1699 downregulated genes. Downregulated genes were mainly enriched in immune, cytokine, MAPK/stress, and extracellular signaling-associated pathways, whereas DNA replication and cell-cycle-related genes were increased. Enrichment-term association and STRING functional-association analyses further identified a coordinated module involving IL1B, JUN, NFKBIA, IRF1, TGFB1, IL10, CCL5, and PTGS2. Independent RT-qPCR analysis confirmed selected expression trends. These findings show that heterogeneous HMOX1 editing and reduced HO-1 abundance are associated with disruption of the avian heme-biliverdin metabolic axis and coordinated remodeling of basal immune, stress, extracellular signaling, and cell-cycle-associated transcriptional programs in Chicken hepatocellular carcinoma-derived cells.},
}

Animals
Chickens/genetics
*Heme Oxygenase-1/genetics/metabolism
*Biliverdine/metabolism
*Heme/metabolism
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Stress, Physiological/genetics
Transcription, Genetic
Cell Line, Tumor

@article {pmid42442654,
year = {2026},
author = {Baruah, A and Wimmer, T and Stieger, K and Ponnam, SPG},
title = {Knockdown of XRCC5 and XRCC6 activity using CRISPR/Cas9 technology enhances homology-directed DNA repair at the CHST6 locus in HEK293 cells.},
journal = {Experimental eye research},
volume = {271},
number = {},
pages = {111165},
doi = {10.1016/j.exer.2026.111165},
pmid = {42442654},
issn = {1096-0007},
mesh = {Humans ; HEK293 Cells ; *Ku Autoantigen/genetics ; *CRISPR-Cas Systems/genetics ; *DNA-Binding Proteins/genetics ; Blotting, Western ; DNA End-Joining Repair ; Gene Knockdown Techniques ; *DNA Helicases/genetics ; DNA Breaks, Double-Stranded ; DNA Repair ; },
abstract = {In mammalian cells, DNA double-strand breaks (DSBs) are repaired by two competing pathways-homologous recombination (HR) and non-homologous end-joining (NHEJ)-that act on the same DNA ends. Downregulation of NHEJ has been shown to enhance HR-mediated repair. Macular corneal dystrophy (MCD) is an autosomal recessive disorder characterized by progressive corneal opacity and vision loss in humans. More than 180 mutations in the CHST6 gene are linked to MCD, with over 70% occurring in exon 3, making it a promising target for genome editing. In this study, we performed in vitro editing of exon 3 of CHST6 using CRISPR/Cas9 in Human Embryonic Kidney (HEK293) cells. To promote HR, the NHEJ genes XRCC6 and XRCC5, encoding KU70 and KU80, were knocked down individually or in combination. A homologous donor template was also introduced, and HR efficiency was assessed by Western blot analysis. Results demonstrated a significant increase in HR activity following downregulation of these NHEJ components, as indicated by elevated RAD51 expression. As proof of concept, partial restoration of CHST6 protein expression was observed in edited cells compared with CHST6 knockdown controls after suppression of XRCC6 and XRCC5 along with donor template delivery. These findings suggest that targeting NHEJ to enhance HR may represent a promising therapeutic strategy for MCD.},
}

Humans
HEK293 Cells
*Ku Autoantigen/genetics
*CRISPR-Cas Systems/genetics
*DNA-Binding Proteins/genetics
Blotting, Western
DNA End-Joining Repair
Gene Knockdown Techniques
*DNA Helicases/genetics
DNA Breaks, Double-Stranded
DNA Repair

@article {pmid42649367,
year = {2026},
author = {Zahn, V and Sievers, AJ and Kersten, B and Fladung, M and Bruegmann, T},
title = {Genetic transformation and CRISPR/Cas12a-mediated gene editing of European beech (Fagus sylvatica L.) employing a transient protoplast system.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {42649367},
issn = {2399-3642},
support = {2219NR359//Fachagentur Nachwachsende Rohstoffe (Agency for Renewable Resources)/ ; 2219NR359//Fachagentur Nachwachsende Rohstoffe (Agency for Renewable Resources)/ ; 2219NR359//Fachagentur Nachwachsende Rohstoffe (Agency for Renewable Resources)/ ; 2219NR359//Fachagentur Nachwachsende Rohstoffe (Agency for Renewable Resources)/ ; },
mesh = {*Fagus/genetics ; *Protoplasts/metabolism ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Transformation, Genetic ; Plants, Genetically Modified/genetics ; },
abstract = {Fagus sylvatica L. (European beech) is a dominant hardwood forest tree species across Central Europe, supporting diverse ecosystem services and forming the basis of a significant market for high-value timber. However, climate change increasingly threatens beech vitality and productivity, making molecular insights into its stress resilience and functional validation of underlying genes urgently needed. Here, we report a protocol for protoplast isolation from seedling leaves and demonstrate transient genetic transformation and CRISPR/Cas-mediated genome editing in F. sylvatica. PEG-mediated transformation was sequentially optimized, achieving efficiencies of 59 ± 6.19% within distinct seasonal windows. Protoplast yield and transformation efficiency showed pronounced temporal variation throughout the year, indicating a strong seasonal influence on reproducibility of the workflow despite controlled growth conditions. A basic molecular toolkit for functional genomics and future biotechnological applications was established by testing a set of promoters and reporters. For proof-of-concept genome editing, we achieved 4.75 to 32.69% editing efficiencies in the PHYTOENE DESATURASE gene (FsPDS) using temperature-tolerant LbCas12a (ttLbCas12a). Although further optimization of transformation reproducibility and regeneration systems remains necessary, the presented protoplast platform provides a valuable foundation for transient functional assays and genome editing studies in this non-model tree species.},
}

*Fagus/genetics
*Protoplasts/metabolism
*CRISPR-Cas Systems
*Gene Editing/methods
*Transformation, Genetic
Plants, Genetically Modified/genetics

@article {pmid42649517,
year = {2026},
author = {Peng, Z and Zhang, X and Deng, X and Hou, D and Liu, R},
title = {Drivers of the paradigm shift in norovirus diagnostics: technological innovation, contextual demands, and collaborative synergy.},
journal = {Virology journal},
volume = {23},
number = {1},
pages = {},
pmid = {42649517},
issn = {1743-422X},
mesh = {*Norovirus/isolation & purification/genetics ; Humans ; *Caliciviridae Infections/diagnosis/virology ; *Molecular Diagnostic Techniques/methods ; *Gastroenteritis/diagnosis/virology ; Rapid Diagnostic Tests ; Point-of-Care Systems ; Sensitivity and Specificity ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {This paper systematically reviews paradigm shifts in diagnostic technologies for norovirus. Given the substantial burden of acute gastroenteritis caused by this virus and the current lack of licensed antiviral therapies, early and accurate diagnosis is of paramount importance. Diagnostic technologies have evolved from electron microscopy and immunological assays to molecular detection methods, with reverse transcription quantitative polymerase chain reaction (RT-qPCR) currently recognized as the gold standard for molecular diagnostics. However, RT-qPCR is constrained by complex operational procedures, the need for specialized equipment, and limited suitability for rapid point-of-care applications. Emerging technologies, including isothermal amplification and clustered regularly interspaced short palindromic repeats (CRISPR)-based technologies, have significantly improved diagnostic sensitivity, specificity, and turnaround time. Nevertheless, challenges related to standardization, quality control, and contamination prevention remain to be addressed before their widespread implementation. Furthermore, this review proposes a stratified, scenario-adaptive diagnostic framework in which rapid immunochromatographic assays are used for preliminary screening in community settings, highly sensitive PCR-based or multiplex molecular methods are employed for clinical diagnosis in healthcare facilities, and digital PCR (dPCR) is applied for precise quantification in food safety and environmental monitoring. Looking ahead,, advances in norovirus diagnostics will increasingly rely on interdisciplinary collaboration to accelerate the development of integrated and intelligent point-of-care testing (POCT) platforms. In particular, biosensors based on molecular recognition technologies and microfluidic platforms, which offer advantages such as, simple operation, rapid processing and high stability, are among the promising approaches for achieving instrument-free, on-site "sample-in, result-out" diagnostics.},
}

*Norovirus/isolation & purification/genetics
Humans
*Caliciviridae Infections/diagnosis/virology
*Molecular Diagnostic Techniques/methods
*Gastroenteritis/diagnosis/virology
Rapid Diagnostic Tests
Point-of-Care Systems
Sensitivity and Specificity
Nucleic Acid Amplification Techniques/methods

@article {pmid42650019,
year = {2026},
author = {Diamantopoulos, MA and Boti, MA and Kanellopoulos, E and Scorilas, A},
title = {MicroRNAs in Breast Cancer: Biological Functions and Technologies for Experimental and Therapeutic Applications.},
journal = {Cancers},
volume = {18},
number = {16},
pages = {},
pmid = {42650019},
issn = {2072-6694},
abstract = {Breast cancer is a highly heterogeneous malignancy that remains one of the leading causes of cancer-related mortality among women worldwide. Despite significant advances in breast cancer research and therapy, disease heterogeneity, treatment resistance, and metastatic progression remain major obstacles to effective disease management. Among the molecular regulators involved in breast cancer, microRNAs (miRNAs) have been recognized as critical post-transcriptional regulators of gene expression, functioning as either oncogenes or tumor suppressors. By modulating the expression of target RNAs, miRNAs control key biological processes involved in tumor initiation and progression, including cell proliferation, apoptosis, angiogenesis, epithelial-mesenchymal transition (EMT), invasion, and metastasis. To investigate miRNA function and explore their therapeutic potential, a wide range of approaches have been developed to modulate miRNA expression. These include gain-of-function strategies, like miRNA mimics, miRNA expression vectors, and CRISPR activation (CRISPRa), as well as loss-of-function approaches, including anti-miRNA oligonucleotides (AMOs), miRNA sponges, CRISPR-Cas9-mediated gene knockout, and CRISPR interference (CRISPRi). This review provides a comprehensive overview of the biological roles of miRNAs in breast cancer and discusses current technologies for miRNA modulation, their molecular mechanisms, experimental and therapeutic applications, and associated limitations. In addition, it summarizes recent advances in miRNA delivery systems, including viral vectors, organic nanoparticles, and inorganic nanocarriers, highlighting their potential to improve delivery efficiency, target specificity, and facilitate clinical translation. Finally, the review discusses future perspectives, emphasizing the transition from single-target interventions toward network-level regulation and the integration of miRNA-based strategies into precision oncology to support the development of more effective breast cancer therapies.},
}

@article {pmid42650652,
year = {2026},
author = {Toma, EA and Enciu, O and Matache, IM and Porosnicu, AL and Calu, V and Miron, A and Delawan, M and Bydon, M and Popa, MI},
title = {Novel Therapeutic Approaches and Alternatives to Antibiotic Therapy for Drug-Resistant Intra-Abdominal Infections.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {15},
number = {8},
pages = {},
pmid = {42650652},
issn = {2079-6382},
abstract = {Antimicrobial resistance (AMR) among pathogens involved in intra-abdominal infections (IAIs) represents a critical and escalating clinical challenge. The interconnected nature of antimicrobial resistance, spanning human medicine, veterinary practice, agricultural use and environmental reservoirs, has required coordinated international responses based on the 'One Health' principle. This study presents an update on efforts underway worldwide to develop new antibiotics, novel combined antimicrobial agents, and alternatives to classic therapies for IAIs. New antibiotics or compounds with antibacterial activity are currently in various stages of clinical trials, including several fluoroquinolones, beta-lactamase inhibitors, and polymyxin analogues. To reduce the risk of bacterial resistance, various additions to antimicrobial treatments are being explored, such as nanoparticles (NPs), antimicrobial peptides (AMPs), bacteriophages, the CRISPR/Cas system, and probiotics. Each modality offers distinct mechanisms that circumvent established resistance pathways, including multi-target membrane disruption, sequence-specific gene editing, and microbiome restoration. Current preclinical and clinical evidence is synthesized, and key translational barriers, including delivery challenges, safety concerns, regulatory complexity, and the need for IAI-specific pharmacokinetic data are critically examined. In conclusion, the convergence of novel antibiotic agents and non-traditional antimicrobial strategies reviewed herein provides the foundation for a new paradigm in the management of drug-resistant IAIs. The transition from a monotherapy-centric approach to an integrated, multi-modal treatment framework, guided by rapid diagnostics and informed by antimicrobial stewardship, will be essential to preserve therapeutic efficacy against AMR threats of the coming decades.},
}

@article {pmid42650792,
year = {2026},
author = {Luan, H and Yang, R and Qiu, W and Feng, K and Xu, W and Wang, F and Feng, W and Song, P},
title = {Construction of Engineered Escherichia coli and Optimization of Conditions for Carcinine Synthesis via Multi-Enzyme Cascade Catalysis.},
journal = {Biomolecules},
volume = {16},
number = {8},
pages = {},
pmid = {42650792},
issn = {2218-273X},
support = {No. 82304838//National Natural Science Foundation of China/ ; ZR2022MC159//Natural Science Foundation of Shandong Province/ ; },
mesh = {*Escherichia coli/genetics/metabolism ; *Peptide Synthases/metabolism/genetics ; Transferases (Other Substituted Phosphate Groups)/metabolism/genetics ; CRISPR-Cas Systems ; *Aminoimidazole Carboxamide/analogs & derivatives ; Biocatalysis ; Bacterial Proteins ; },
abstract = {Carcinine is an imidazole dipeptide with potent antioxidant and antiglycation properties, although its chemical synthesis currently relies on severely environmentally harmful processes. In this work, a multi-enzyme cascade biotransformation system comprising 4'-phosphopantetheinyl transferase and non-ribosomal peptide synthetase was constructed. To overcome the limitations arising from stochastic spatial distribution and suboptimal mass transfer associated with independent enzymes, a fusion protein strategy was adopted. The two enzymes were fused via a flexible genetic linker within plasmid pET28a-SFP-L-Ebony, which enabled robust soluble expression in Escherichia coli. Concurrently, the endogenous peptidase genes (pepA, pepB, pepD, and pepN) were systematically knocked out using CRISPR/Cas9-mediated gene editing. This quadruple protease-deficient strain (designated SFP-L-Ebony-ΔpepABDN) effectively suppressed product degradation. Subsequent optimization revealed that optimal catalytic performance occurred at 25 °C and pH 7.0. The highest biotransformation efficiency was achieved using 15 g/L crude enzymes, in the presence of 2 mM ATP and 10 mM MgCl2. Through a fed-batch substrate feeding strategy in a 50 mL reaction system, the final carcinine titer reached 7.0 g/L after 48 h. This study, therefore, provides an efficient and sustainable technological pathway for the green biomanufacturing of carcinine as well as other high-value dipeptides.},
}

*Escherichia coli/genetics/metabolism
*Peptide Synthases/metabolism/genetics
Transferases (Other Substituted Phosphate Groups)/metabolism/genetics
CRISPR-Cas Systems
*Aminoimidazole Carboxamide/analogs & derivatives
Biocatalysis
Bacterial Proteins

@article {pmid42650936,
year = {2026},
author = {Akter, R and Ryu, SW and Lee, JH},
title = {Advancements in CRISPR/Cas Technologies for Sensitive Cancer Detection: Mechanisms, Platforms, and Clinical Translation Roadmap.},
journal = {Diagnostics (Basel, Switzerland)},
volume = {16},
number = {16},
pages = {},
pmid = {42650936},
issn = {2075-4418},
abstract = {Early cancer detection is critical for improving patient outcomes, yet current diagnostic approaches often fail to identify malignancies when tumor-specific biomarkers are relatively scarce. Emerging CRISPR/Cas-based technologies have revolutionized the ultra-sensitive detection of cancer biomarkers in liquid biopsies, overcoming the inherent limitations of traditional diagnostic approaches such as tissue biopsies and imaging, which frequently fail to detect early-stage malignancies with sufficient sensitivity. This review explores recent advances in CRISPR/Cas diagnostics (CRISPR/Cas-Dx) that employ programmable CRISPR effectors, including Cas9, Cas12, Cas13, and Cas14. In particular, the collateral (trans-) cleavage activities of Cas12, Cas13, and Cas14 enable signal amplification for highly sensitive detection of circulating tumor DNA, microRNAs, exosomes, and other multi-omics biomarkers, often without the need for extensive nucleic acid amplification. Representative CRISPR/Cas-Dx platforms include amplification-coupled assays, amplification-free frameworks, biosensing and multiplexing capabilities, and new digital or droplet-based configurations that include artificial intelligence to improve analytical precision. These technologies demonstrate single-molecule resolution and adaptability for point-of-care testing in malignancies such as non-small cell lung, colorectal, and breast carcinoma. Finally, we outline a clinical translation roadmap encompassing manufacturability, regulatory and standardization requirements, and real-world implementation challenges. This perspective offers a blueprint for CRISPR-powered, ultra-sensitive liquid biopsy diagnostics that can enable population-scale early cancer screening and truly preventive oncology by bridging molecular insights, engineering innovation, and clinical imperatives.},
}

@article {pmid42651807,
year = {2026},
author = {Athanasopoulou, K and Daneva, GN and Michalopoulou, VI and Stamelou, MR and Tsiakanikas, P and Adamopoulos, PG},
title = {Engineering the Future of Precision Medicine: A Comprehensive Guide to RNA Therapeutics.},
journal = {Current issues in molecular biology},
volume = {48},
number = {8},
pages = {},
pmid = {42651807},
issn = {1467-3045},
abstract = {RNA therapeutics have evolved from passive genetic intermediaries into highly programmable platforms, fundamentally transforming the landscape of precision medicine. This comprehensive review examines the molecular architecture and mechanisms of established platforms in the clinical setting, including mRNA, antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs) and aptamers, alongside next-generation platforms, such as CRISPR-guided systems and circular RNAs (circRNAs). Moreover, we discuss strategies to overcome systemic delivery bottlenecks and evaluate advanced non-viral systems, emphasizing lipid nanoparticles (LNPs), polymers and tissue-specific ligand conjugates that facilitate precise intracellular targeting. Furthermore, we explore the clinical expansion of these platforms across infectious diseases, rare genetic disorders, oncology and cardiovascular conditions. Finally, we highlight how the integration of artificial intelligence (AI) and machine learning (ML) redefines the limits of individualized, programmable RNA therapies by accelerating sequence optimization and nanoparticle formulation.},
}

@article {pmid42364748,
year = {2026},
author = {Gao, QY and Han, D and Liao, XR and Lei, JJ and Qi, LJ and Huang, QY and Fu, FF and Cheng, SX},
title = {Targeting delivery systems with aptamer-conjugated hyaluronic acid for personalized assessment of treatment response to targeted cancer therapy.},
journal = {International journal of biological macromolecules},
volume = {375},
number = {},
pages = {153260},
doi = {10.1016/j.ijbiomac.2026.153260},
pmid = {42364748},
issn = {1879-0003},
mesh = {Humans ; *Hyaluronic Acid/chemistry ; *Neoplasms/genetics/drug therapy/pathology/therapy ; *Aptamers, Nucleotide/chemistry ; Cell Line, Tumor ; GTPase-Activating Proteins/genetics ; Cell Proliferation/drug effects ; Apoptosis/drug effects/genetics ; *Precision Medicine ; CRISPR-Cas Systems ; *Drug Delivery Systems ; Molecular Targeted Therapy ; Gene Knockout Techniques ; },
abstract = {A central objective in oncology is to identify therapeutic targets essential for the survival of proliferating malignant cells. However, a major challenge in pre-clinical research is the scarcity of robust models capable of accurately assessing therapeutic efficacy in patient-specific heterogeneous cancer cells. Here, we developed an ex vivo platform for patient-specific assessment using biomacromolecule-based targeting delivery systems for CRISPR-Cas9 mediated gene knockout and in situ mRNA profiling in patient-derived circulating malignant cells (CMCs). Using a cancer targeting vector, we conducted a personalized evaluation of the impact of Rac GTPase activating protein 1 (RACGAP1) knockout on cell growth in a cancer cell line and patient-derived heterogeneous CMCs. RACGAP1 knockout induced irreversible cytokinesis failure, leading to markedly reduced proliferation and invasion capacity, DNA damage, and increased apoptosis. Our approach establishes a promising strategy for the personalized validation of novel therapeutic targets.},
}

Humans
*Hyaluronic Acid/chemistry
*Neoplasms/genetics/drug therapy/pathology/therapy
*Aptamers, Nucleotide/chemistry
Cell Line, Tumor
GTPase-Activating Proteins/genetics
Cell Proliferation/drug effects
Apoptosis/drug effects/genetics
*Precision Medicine
CRISPR-Cas Systems
*Drug Delivery Systems
Molecular Targeted Therapy
Gene Knockout Techniques

@article {pmid42413679,
year = {2026},
author = {Jangra, A and Tiwari, S and Chhokar, V},
title = {Pioneering the formation of 2-carboxylic anthraquinone: CRISPR/Cas9-mediated functional validation of Octaketide synthase and Polyketide reductase genes in Aloe vera.},
journal = {International journal of biological macromolecules},
volume = {375},
number = {},
pages = {153385},
doi = {10.1016/j.ijbiomac.2026.153385},
pmid = {42413679},
issn = {1879-0003},
mesh = {*Aloe/genetics/enzymology/metabolism ; *Anthraquinones/metabolism/chemistry ; *CRISPR-Cas Systems/genetics ; *Polyketide Synthases/genetics/metabolism ; Gene Editing ; },
abstract = {Aloe vera is an authentic medical plant abundant in aromatic polyketides, including the crucial hexaketides aloenin, aloesin, and barbaloin used in pharmaceuticals, yet the enzymatic basis of their biosynthesis remains incompletely understood. While it has been suggested that octaketide synthase (OKS) initiates anthraquinone biosynthesis, heterologous expression of OKS alone consistently produces shunt polyketide products, and the mechanism underlying this derailment was uncertain. To comprehend the proposed anthraquinone biosynthesis pathway, we combined biochemical constitution, structural characterization, and CRISPR/Cas9-mediated editing of key genes in Aloe vera. The results showed that the inclusion of a PKR (polyketide reductase) altered the reaction profile and supported the formation of a product spectroscopically consistent with 2-carboxy anthraquinone (C16H1205). ESI-MS analysis detected the molecular cation in [M][+] and [M + H][+] forms (m/z 284.2936 and 285.2421, respectively), while FTIR and [1]H NMR analyses indicated the presence of characteristic anthraquinone, carboxyl, hydroxyl, and terminal methyl functionalities. The spectroscopic profile additionally distinguished the characterized product from compounds previously misannotated in the literature. Alongside, CRISPR/Cas9-based genome editing of candidate genes resulted in a significant reduction in aloin content in edited lines (OKS mutant: 2.54-fold, PKR mutant 1 and 2: 1.23 and 1.53-fold, respectively) compared to the non-edited control aloe line. Together, these findings support the involvement of tailoring enzyme ketoreductase for the efficient and appropriate formation of anthraquinones and provide functional insights into polyketide biosynthesis in aloe plants that sustain as an indigenous herb for mankind.},
}

*Aloe/genetics/enzymology/metabolism
*Anthraquinones/metabolism/chemistry
*CRISPR-Cas Systems/genetics
*Polyketide Synthases/genetics/metabolism
Gene Editing

@article {pmid42648842,
year = {2026},
author = {Lin, Z and Luo, Y and Li, H and Shao, B and Yang, X and Wang, J and Wan, Y and Song, F},
title = {CRISPR/Cas12a dual-gRNA assay enables precise detection of single-nucleotide mutations via cis-staggered-cleavage.},
journal = {Analytica chimica acta},
volume = {1420},
number = {},
pages = {345968},
doi = {10.1016/j.aca.2026.345968},
pmid = {42648842},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Staphylococcus aureus/genetics ; *Polymorphism, Single Nucleotide ; Methicillin-Resistant Staphylococcus aureus/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; },
abstract = {Robust discrimination of single-nucleotide mutations (SNMs) remains a central challenge in nucleic acid analysis, particularly under minimal sequence constraints. Here, we report a programmable CRISPR/Cas12a sensing strategy, termed STAND (cis-staggered-cleavage-based dual-gRNA assay), that enables precise SNM discrimination through a structurally gated cleavage cascade. In this system, a primary guide RNA directs Cas12a to perform site-specific cis-staggered cleavage of double-stranded DNA, generating a predictable sticky-end intermediate. This transient structure is subsequently recognized by a secondary guide RNA via strand displacement and branch migration, which reactivates Cas12a for PAM-independent trans-cleavage of reporter substrates. This sequential, structure-mediated activation decouples target recognition from PAM constraints and converts single-nucleotide variations into amplified fluorescence signals with high fidelity, and achieves a detection limit as low as 10[1] CFU/mL. We demonstrate that STAND achieves accurate SNM discrimination in clinically relevant targets, including the nuc gene of Staphylococcus aureus and methicillin-resistant S. aureus, outperforming conventional qPCR in specificity. Owing to its modular design, minimal guide requirements, and programmable architecture, this strategy establishes a generalizable framework for high-resolution genetic analysis and molecular diagnostics.},
}

*CRISPR-Cas Systems/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Staphylococcus aureus/genetics
*Polymorphism, Single Nucleotide
Methicillin-Resistant Staphylococcus aureus/genetics
*CRISPR-Associated Proteins/genetics/metabolism

@article {pmid42649178,
year = {2026},
author = {Chen, Y and Li, H and Duan, L and Liu, Y and Yan, J and Chen, H and Yang, J},
title = {A decoupled transcription platform enables tunable and predictable gene expression in yeast.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42649178},
issn = {2041-1723},
support = {32122006//National Science Foundation of China | Young Scientists Fund/ ; },
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; Promoter Regions, Genetic/genetics ; *Gene Expression Regulation, Fungal ; *Transcription, Genetic ; Lycopene/metabolism ; CRISPR-Cas Systems/genetics ; Lactic Acid/analogs & derivatives/biosynthesis ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Regulatory Networks ; },
abstract = {Predictable control of gene expression is essential for building genetic circuits and improving metabolic pathways, but conventional promoter libraries often behave unpredictably when genes are combined. Here we develop CRISPR-Activated Promoter-based Orthogonal expression (CAPO), a quantitative platform for controlling multiple genes in yeast. CAPO uses synthetic CRISPR-activated promoters that remain silent until matching guide RNAs recruit dCas9-VPR. We tune each gene by varying guide RNA abundance with defined T7 promoters, while keeping regulatory channels orthogonal. CAPO reaches expression levels comparable to strong native yeast promoters, maintains low background activity, and preserves promoter-strength order across different genes. We apply CAPO to program broad fluorescence color outputs and to rapidly optimize lycopene and 3-hydroxypropionic acid biosynthesis. These results establish CAPO as a scalable platform for predictable engineering of eukaryotic gene networks.},
}

*Saccharomyces cerevisiae/genetics/metabolism
Promoter Regions, Genetic/genetics
*Gene Expression Regulation, Fungal
*Transcription, Genetic
Lycopene/metabolism
CRISPR-Cas Systems/genetics
Lactic Acid/analogs & derivatives/biosynthesis
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Regulatory Networks

@article {pmid42649193,
year = {2026},
author = {Ittiprasert, W and Smout, MJ and Mann, VH and Moyle, M and Kinahan, SM and Ackerman, DN and Rivera, DN and Santarpia, JL and Carnes, EC and Mentink-Kane, MM and Costa, MR and Hokke, CH and Roestenberg, M and Bottazzi, ME and Bracken, BK and Rosa, BA and Djuranovic, S and Pickering, DA and Giacomin, PR and Watterson, D and Modhiran, N and Moescheid, MF and Grevelding, CG and Mitreva, M and Loukas, A and Brindley, PJ},
title = {Heritable transgenic schistosomes as a living platform for SARS-CoV-2 neutralizing antibody secretion.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42649193},
issn = {2041-1723},
support = {N66001-21-C-4013//United States Department of Defense | Defense Advanced Research Projects Agency (DARPA)/ ; 107475/Z/15/Z//Wellcome Trust (Wellcome)/ ; CA164719//U.S. Department of Health & Human Services | NIH | NCI | Division of Cancer Epidemiology and Genetics, National Cancer Institute (National Cancer Institute Division of Cancer Epidemiology and Genetics)/ ; GR 1549/12-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {Animals ; *Schistosoma mansoni/genetics/immunology ; *SARS-CoV-2/immunology ; Mice ; *Animals, Genetically Modified ; *Antibodies, Neutralizing/immunology ; COVID-19/immunology ; *Antibodies, Viral/immunology ; Female ; Biomphalaria/parasitology ; Angiotensin-Converting Enzyme 2 ; Humans ; Immunoglobulin G/immunology/genetics ; Immunoglobulin Fc Fragments/genetics/immunology ; },
abstract = {We report the generation and propagation of not only the first heritable transgenic schistosome line but also a line that secretes a functional therapeutic protein in vivo. Using multiplexed CRISPR/Cas-mediated homology-directed knock-in targeted to a predicted genomic safe-harbor, we inserted a VHH-IgG1 Fc (termed C5-Fc) transgene into Schistosoma mansoni eggs. Single-miracidium infections of Biomphalaria glabrata yielded parental P0 lines; serial passage through snail and mouse hosts produced an F2 cohort in which all parasites carried the C5-Fc transgene and secreted C5-Fc into the murine venous circulation. Molecular assays confirmed chromosomal insertion, germline transmission and systemic secretion. Sera from mice harboring C5-Fc transgenic worms neutralized SARS-CoV-2 in vitro with potent activity consistent with the expected ACE2-binding blockade by the C5 variable domain of heavy-chain-only antibody (VHH). These results demonstrate (i) stable, heritable transgenesis of a platyhelminth, (ii) delivery of a biologically active antibody fragment by a live helminth in a mammalian host, and (iii) feasibility of using transgenic schistosomes as sustained, single-dose protein delivery platforms. This technology and delivery system enable new experimental approaches for schistosome biology and motivate exploration of living-foundry therapeutics.},
}

Animals
*Schistosoma mansoni/genetics/immunology
*SARS-CoV-2/immunology
Mice
*Animals, Genetically Modified
*Antibodies, Neutralizing/immunology
COVID-19/immunology
*Antibodies, Viral/immunology
Female
Biomphalaria/parasitology
Angiotensin-Converting Enzyme 2
Humans
Immunoglobulin G/immunology/genetics
Immunoglobulin Fc Fragments/genetics/immunology

@article {pmid42649204,
year = {2026},
author = {Madsen, A and Selfjord, N and Martinez-Lage, M and Loyd, AL and Kurgan, G and Ståhlberg, M and Lindgren, J and Liz Touza, J and Wigge, L and Firth, M and Nordström, K and Collin, J and Jachimowicz, D and Schiffthaler, B and Dillmann, I and Antoniou, P and Emmanouilidi, A and Hellsten, J and Forsström, J and Magnell, K and Jacobi, A and Behlke, M and Porritt, M and Madeyski-Bengtson, K and Maresca, M and Akcakaya, P},
title = {Single-cell and in vivo profiling reveal heterogeneous and organ-specific CRISPR-Cas9 off-target and translocation outcomes.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42649204},
issn = {2041-1723},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Mice ; *Single-Cell Analysis/methods ; *Gene Editing/methods ; Organ Specificity/genetics ; DNA Methylation ; Embryonic Stem Cells/metabolism ; Chromatin/metabolism/genetics ; *Translocation, Genetic ; Mouse Embryonic Stem Cells/metabolism ; Embryo, Mammalian/cytology/metabolism ; },
abstract = {CRISPR-Cas9 holds promise for treating genetic disease, but rare off-target mutations and structural variants remain as key safety concerns, especially at scales relevant to therapy. Here, we establish workflows to resolve Cas9 off-target activity in vitro at single-cell resolution and in vivo across different tissues. Using clonally expanded electroporated mouse embryos and embryonic stem cells, we reveal that individual cells exhibit unique off-target and translocation profiles, including events missed in bulk analyses. Integrating single-cell editing with chromatin accessibility, transcription, and DNA methylation measurements suggest that sequence-independent features modulate Cas9 access and cleavage, with preferential editing in regions characterized by open chromatin and lower methylation. In Cas9-inducible mouse models, editing analyses reveal organ-distinct off-target spectra, DNA repair pathway usage, indel patterns, and markedly varying translocation propensity between tissues. These findings demonstrate that off-target activity is heterogeneous across cells and context-dependent across organs, motivating sensitive single-cell analyses and organ-specific evaluation in preclinical development to more accurately assess risk and improve the safety of CRISPR-based genomic medicines.},
}

Animals
*CRISPR-Cas Systems/genetics
Mice
*Single-Cell Analysis/methods
*Gene Editing/methods
Organ Specificity/genetics
DNA Methylation
Embryonic Stem Cells/metabolism
Chromatin/metabolism/genetics
*Translocation, Genetic
Mouse Embryonic Stem Cells/metabolism
Embryo, Mammalian/cytology/metabolism

@article {pmid42649208,
year = {2026},
author = {Hong, M and Luan, C and Yuan, M and Huang, H and Guo, X and Meng, D and Huang, M and Xu, Y and Zhao, S and Chen, K and Chen, J and Li, D and Chen, L},
title = {High-diversity base mutagenesis via simultaneous adenine, cytosine and guanine editing.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42649208},
issn = {2041-1723},
support = {2024YFC3407900//Ministry of Science and Technology of the People's Republic of China (Chinese Ministry of Science and Technology)/ ; },
mesh = {*Guanine/metabolism ; *Adenine/metabolism ; *Cytosine/metabolism ; *Mutagenesis ; Humans ; Mutation ; CRISPR-Cas Systems ; },
abstract = {Base editors hold great promise in endogenous mutagenesis for genetic screening. However, the development of base editors that induce saturated multi-base conversions with diverse mutation spectrum is challenging. Here, we develop triple base editors (smACGs) that simultaneously mutagenize adenine, cytosine, and guanine within the same allele. Through screening and embedding engineered deaminase and alkyladenine DNA glycosylase variants in Cas9 structure, smACGmax is generated to catalyze robust triple-base conversion efficiencies of up to 41% across varied sequence contexts while maintaining low RNA off-target effects compared to previous dual-base editors. We apply smACGmax to enable high coverage (94%) of targeted HBEGF mutagenesis that identified diphtheria toxin-resistant mutations and to dissect SF3B1 variants with alternative splicing specificity via complex single, double, and triple base conversion screening. smACGmax expands base conversion capability from single and double substrates to trinucleotide level, which facilitates the generation of high-diversity and complex genetic variants, providing a useful platform for mutagenesis-based application.},
}

*Guanine/metabolism
*Adenine/metabolism
*Cytosine/metabolism
*Mutagenesis
Humans
Mutation
CRISPR-Cas Systems

@article {pmid41436006,
year = {2026},
author = {Zhang, H and Chen, B and Gu, L and Wang, C and Xu, L and Ji, X and Wang, J and Wang, Z and Xiao, X and Liu, Y},
title = {A genome-wide CRISPRi screen identifies homologous recombination pathway as potential target for broad-spectrum antibiotic adjuvants.},
journal = {Journal of advanced research},
volume = {87},
number = {},
pages = {947-962},
doi = {10.1016/j.jare.2025.12.015},
pmid = {41436006},
issn = {2090-1224},
mesh = {*Anti-Bacterial Agents/pharmacology ; *Homologous Recombination/drug effects/genetics ; Rec A Recombinases/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Escherichia coli/genetics/drug effects ; Microbial Sensitivity Tests ; Escherichia coli Proteins/genetics/metabolism ; Cisplatin/pharmacology ; Genome, Bacterial ; },
abstract = {INTRODUCTION: The widespread misuse and overuse of antibiotics have driven the emergence of multidrug-resistant and pan drug-resistant bacteria, constituting a formidable global health threat. Antibiotic adjuvants that potentiate the efficacy of existing antibiotics represent a particularly promising avenue to address this challenge.

METHODS: We performed a genome-wide CRISPR interference (CRISPRi) screening to identify potential targets for broad-spectrum antibiotic adjuvants, which highlighted the homologous recombination pathway as a promising candidate. To functionally validate this pathway, we employed three strategies to suppress the expression and function of recA, a key component of homologous recombination, including a CRISPRi system delivered via transconjugation, a RecX-derived peptide (RecX-20) fused to a cell-penetrating motif, and a small-molecule inhibitor cisplatin validated by surface plasmon resonance.

RESULTS: Disruption of the homologous recombination pathway not only significantly increased bacterial susceptibility to multiple classes of antibiotics, including quinolones, β-lactams, aminoglycosides, and nitrofurantoin, but also reduced horizontal gene transfer of antibiotic resistance. In addition, recA deficiency resulted in a cascade of physiological disruptions, including membrane damage, efflux pump dysfunction, oxidative stress imbalance and metabolic disruption. All three recA-targeting strategies enhanced the antibacterial activity, with cisplatin exhibiting the most pronounced potentiating effect both in vitro and in vivo.

CONCLUSIONS: This study reveals that the homologous recombination pathway, particularly RecA, is a viable target for the development of broad-spectrum antibiotic adjuvant. Our findings provide mechanistic insights and practical strategies to restore the effectiveness of existing antibiotics and address the growing threat of antimicrobial resistance.},
}

*Anti-Bacterial Agents/pharmacology
*Homologous Recombination/drug effects/genetics
Rec A Recombinases/genetics/metabolism
*CRISPR-Cas Systems/genetics
Escherichia coli/genetics/drug effects
Microbial Sensitivity Tests
Escherichia coli Proteins/genetics/metabolism
Cisplatin/pharmacology
Genome, Bacterial

@article {pmid42625528,
year = {2026},
author = {van Putten, M and Linssen, M and Tanganyika-de Winter, C and Brouwers, CM and Claassens, JWC and Verwey, N and Walsh, M and Loredan Stan, T and Aartsma-Rus, A and Hohenstein, P},
title = {Four new mouse models of Duchenne muscular dystrophy with clinically relevant exon deletions in the human DMD gene.},
journal = {Disease models & mechanisms},
volume = {19},
number = {8},
pages = {},
doi = {10.1242/dmm.052875},
pmid = {42625528},
issn = {1754-8411},
support = {24745//AFM-Telethon/ ; 24745//AFM-Téléthon/ ; //Leids Universitair Medisch Centrum/ ; //REGENXBIO/ ; },
mesh = {Animals ; *Exons/genetics ; *Muscular Dystrophy, Duchenne/genetics/pathology ; Humans ; *Dystrophin/genetics/metabolism ; Disease Models, Animal ; *Sequence Deletion/genetics ; Mice ; Mice, Inbred mdx ; CRISPR-Cas Systems/genetics ; Base Sequence ; Muscle, Skeletal/pathology ; Male ; Mice, Inbred C57BL ; Oligonucleotides, Antisense ; },
abstract = {Variant-specific therapeutic approaches, such as exon skipping or gene editing, hold promise for the treatment of Duchenne muscular dystrophy (DMD). Translatability of preclinical studies investigating these approaches could greatly be improved through the use of humanized mouse models, as these allow preclinical testing of human-specific sequences. We developed four novel humanized mouse models of DMD with a deletion of exon 44, 45, 51 or 53 in the human DMD gene, in a mouse dystrophin-negative background (mdx mouse; exon 23 nonsense mutation). Our optimized prescreening pipeline allowed us to do so very efficiently with the CRISPR-Cas9 technology. We confirmed either complete lack of dystrophin or expression of trace levels, which led to development of muscle pathology consisting of muscle fiber degeneration and regeneration, inflammation and fibrosis in young adult mice. Intramuscular treatment with vivo-morpholinos targeting a flanking exon induced exon skipping in the DMD strains, which restored the disrupted open reading frame and, subsequently, dystrophin expression. This validates these models as valuable tools for preclinical studies investigating human sequence-specific therapeutic approaches for DMD.},
}

Animals
*Exons/genetics
*Muscular Dystrophy, Duchenne/genetics/pathology
Humans
*Dystrophin/genetics/metabolism
Disease Models, Animal
*Sequence Deletion/genetics
Mice
Mice, Inbred mdx
CRISPR-Cas Systems/genetics
Base Sequence
Muscle, Skeletal/pathology
Male
Mice, Inbred C57BL
Oligonucleotides, Antisense

@article {pmid42644396,
year = {2026},
author = {Dong, Y and Xu, C and Yan, B and Mou, S and Li, C and Liu, Y},
title = {Quantitative and Targeted Regulation of Ferroptosis in Bladder Cancer: Preclinical Study.},
journal = {Journal of cellular and molecular medicine},
volume = {30},
number = {16},
pages = {e71333},
pmid = {42644396},
issn = {1582-4934},
support = {2021YFA0911600//National Key R&D Program of China/ ; RCJC20221008092723011//Shenzhen Science and Technology Program/ ; JCYJ20220818102001002//Shenzhen Science and Technology Program/ ; },
mesh = {*Ferroptosis/genetics ; Humans ; *Urinary Bladder Neoplasms/genetics/pathology/metabolism ; Animals ; Gene Expression Regulation, Neoplastic ; Cell Line, Tumor ; *Beclin-1/genetics/metabolism ; Mice ; Amino Acid Transport System y+/genetics/metabolism ; Phospholipid Hydroperoxide Glutathione Peroxidase/genetics/metabolism ; Cell Proliferation ; CRISPR-Cas Systems/genetics ; Female ; },
abstract = {The activation of ferroptosis, a cell death mechanism driven by excessive ferrous ions (Fe[2+]) and lipid peroxides, has emerged as a promising target for cancer treatment. However, in the case of quantitative regulation of target genes, it remains uncertain whether ferroptosis can be induced in bladder cancer (BCa) cells without affecting normal ones. We investigated this using an innovative CRISPR-dCas9 system to upregulate and downregulate the ferroptosis-related gene BECN1 and OTUB1, respectively. We identified two genes that can affect and promote ferroptosis-related pathways, analysing their expression in bladder tissue through The Cancer Genome Atlas. Our unique CRISPR-dCas9 technology, under the control of an hTERT promoter, selectively adjusted BECN1 and OTUB1 expression exclusively in cancer cells. RT-qPCR and western blotting demonstrated significant alterations in the expression of GPX4 and SLC7A11, proteins strongly associated with ferroptosis, in BCa cells, while normal bladder cells remained unaffected. We developed a quantitative model based on synthetic biology principles to describe the regulatory relationships between the ferroptosis-related genes BECN1 and OTUB1 and their downstream targets GPX4 and SLC7A11 in bladder cancer cells. The model establishes a direct proportional relationship between BECN1 upregulation and decreased GPX4 expression, and between OTUB1 downregulation and decreased SLC7A11 expression. In vitro experiments revealed reduced viability, proliferation, migration, and invasion in UMUC-3 and T24 BCa cells. Importantly, Fer-1 and DFO rescued the viability loss, and C11-BODIPY staining confirmed increased lipid ROS accumulation, supporting ferroptosis-associated cell death following BECN1/OTUB1 regulation. In vivo xenograft experiments showed that BECN1 upregulation or OTUB1 downregulation suppressed tumour growth. Tumour-tissue immunofluorescence further showed reduced GPX4 expression in BECN1-upregulated tumours and reduced SLC7A11 expression in OTUB1-downregulated tumours, supporting suppression of the GPX4/SLC7A11 ferroptosis-protective axis in vivo. The quantitative equation derived from our data suggests that the induction of ferroptosis in bladder cancer cells can be effectively modulated by these two genes, and the experimental results also indicate our system can modulate these two genes to affect the function of BCa cells without affecting the normal cells, offering a promising new direction for the development of targeted therapy for bladder cancer.},
}

*Ferroptosis/genetics
Humans
*Urinary Bladder Neoplasms/genetics/pathology/metabolism
Animals
Gene Expression Regulation, Neoplastic
Cell Line, Tumor
*Beclin-1/genetics/metabolism
Mice
Amino Acid Transport System y+/genetics/metabolism
Phospholipid Hydroperoxide Glutathione Peroxidase/genetics/metabolism
Cell Proliferation
CRISPR-Cas Systems/genetics
Female

@article {pmid42644503,
year = {2026},
author = {Gakpo, JO and Gulabrai, B and Sanders, CE and Parrella, JA and Proudman, J and Berger, T and Mitloehner, F},
title = {U.S. consumers' processing of information about CRISPR-edited pork products.},
journal = {GM crops & food},
volume = {17},
number = {1},
pages = {2719351},
doi = {10.1080/21645698.2026.2719351},
pmid = {42644503},
issn = {2164-5701},
mesh = {Animals ; Humans ; Female ; Swine ; *Consumer Behavior ; Male ; *Gene Editing ; United States ; Adult ; Middle Aged ; Information Seeking Behavior ; Young Adult ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems ; Adolescent ; },
abstract = {The commercialization of CRISPR gene-edited pork is advancing rapidly, following the U.S. Food and Drug Administration's approval of gene-edited pigs resistant to Porcine Reproductive and Respiratory Syndrome (PRRS). As these products move closer to market entry, understanding how consumers seek, process, and avoid information about them is critical for developing effective communication strategies. Guided by the Risk Information Seeking and Processing (RISP) model, this study examined factors influencing information seeking, information avoidance, and information processing related to CRISPR-edited pork products among U.S. consumers (n = 2,006). Results show higher information sufficiency thresholds were associated with greater information seeking and lower information avoidance. Information seeking was strongly and positively correlated with systematic processing. Relevant channel beliefs and perceived information gathering capacities were positively associated across communication channels, suggesting the need for integrated communication approaches. Relevant channel beliefs for news media and social media were positively associated with information seeking, while stronger relevant channel beliefs for Extension were associated with lower information seeking. Respondents with some college education reported higher information seeking than those with only a high school diploma or GED, while older adults and individuals with higher education levels reported lower information avoidance. Results also showed that respondents exhibited high intentions to seek information and low tendencies to avoid information, suggesting openness to learning about CRISPR-edited pork. Participants also reported engaging more in systematic processing than heuristic processing, indicating a preference for careful and analytical evaluation of information. Findings highlight the importance of audience segmentation, multi-channel communication strategies, and evidence-based messaging to support informed public engagement with CRISPR-edited food technologies.},
}

Animals
Humans
Female
Swine
*Consumer Behavior
Male
*Gene Editing
United States
Adult
Middle Aged
Information Seeking Behavior
Young Adult
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
CRISPR-Cas Systems
Adolescent

@article {pmid42645040,
year = {2026},
author = {Amanzholova, M and Akimbekova, A and Shaizadinova, A and Sutimbekova, N and Bissenova, N and Tarlykov, P and Abeldenov, S},
title = {Multiplex RPA-CRISPR/Cas12a Assay for Rapid Detection of Class D OXA-Type Carbapenem-Resistant Acinetobacter baumannii.},
journal = {Biosensors},
volume = {16},
number = {8},
pages = {},
pmid = {42645040},
issn = {2079-6374},
support = {BR24992881//Committee of Science of the Ministry of Science and Higher Education of the Republic of Kazakhstan/ ; },
mesh = {*Acinetobacter baumannii/genetics/isolation & purification/drug effects ; Carbapenems/pharmacology ; *beta-Lactamases/genetics ; Humans ; CRISPR-Cas Systems ; Bacterial Proteins/genetics ; Rapid Diagnostic Tests ; },
abstract = {Acinetobacter baumannii is a critical WHO priority pathogen due to its multidrug resistance and high mortality in carbapenem-resistant infections. Resistance is predominantly mediated by class D carbapenemase genes blaOXA-23 and blaOXA-40, which spread rapidly via horizontal gene transfer in healthcare settings. To address the lack of a rapid assay capable of detecting both blaOXA-23 and blaOXA-40 in a single analytical workflow, we developed a multiplex two-step RPA-CRISPR/Cas12a assay. Since infections caused by strains harboring either gene require identical therapeutic management, their co-detection in a single reaction is clinically justified. Although simultaneous use of two crRNAs within a single CRISPR/Cas12a reaction is often considered technically challenging due to potential inter-crRNA competition, here it advantageously enables dual-target coverage without compromising sensitivity. The assay demonstrated high specificity with no cross-reactivity against a panel of clinically relevant bacterial species, including closely related Acinetobacter spp. Evaluation using genomic DNA extracted from 63 cultured clinical A. baumannii isolates revealed blaOXA-23 in 19 isolates (30.2%), blaOXA-40 in 28 (44.4%), and co-carriage of both genes in 9 (14.3%), with at least one resistance gene detected in 60.3% of isolates. The complete workflow was accomplished within 45 min without specialized equipment, offering a rapid, sensitive, and cost-effective solution for point-of-care molecular surveillance of carbapenem-resistant A. baumannii in clinical and resource-limited settings.},
}

*Acinetobacter baumannii/genetics/isolation & purification/drug effects
Carbapenems/pharmacology
*beta-Lactamases/genetics
Humans
CRISPR-Cas Systems
Bacterial Proteins/genetics
Rapid Diagnostic Tests

@article {pmid42647707,
year = {2026},
author = {Tan, K and Sun, W and Fullwood, MJ and Jia, L and Lyu, H and Zhang, L and Dao, F},
title = {AcrPLMEvo: A Two-Stage Framework Integrating Evolutionary Profiles with Protein Language Models for Anti-CRISPR Prediction.},
journal = {IEEE journal of biomedical and health informatics},
volume = {PP},
number = {},
pages = {},
doi = {10.1109/JBHI.2026.3727674},
pmid = {42647707},
issn = {2168-2208},
abstract = {Anti-CRISPR (Acr) proteins are natural inhibitors of CRISPR-Cas systems and are important regulators for controllable genome-editing applications. However, their computational identification remains challenging because Acrs are sequence-diverse, weakly conserved, and supported by limited labeled data. Here, we present AcrPLMEvo, a two-stage framework that integrates protein language model (PLM) representations with PSSM derived evolutionary profiles for low-homology Acr prediction. We systematically compared four representative PLM backbones, parameter-efficient adaptation strategies, and alternative PSSM-coupling routes. Evolutionary profiles were not universally beneficial; instead, their effects depended on both PLM backbone and the stage at which they were incorporated. A key finding was that evolutionary information was more consistently beneficial when retained at the downstream decision stage than when used only during PLM adaptation. Guided by this observation, AcrPLMEvo combines PSSM-aware DoRA adaptation of ESM-2 with frozen feature extraction and final-stage evolutionary feature reintroduction. In the matched benchmark comparison, AcrPLMEvo achieved the best overall performance among competing Acr predictors, with an AUC of 0.965 and an AUPRC of 0.778. Its predictive reliability was further supported on an independently curated external set of 44 proteins, where it correctly classified 41 proteins and produced no false positives. These results indicate that stage-consistent integration of evolutionary profiles can improve PLM-based Acr prediction and support the prioritization of low-homology Acr candidates.},
}

@article {pmid42648526,
year = {2026},
author = {Gamage, A and Herath, HMLPB and de Silva, KMN and de Silva, RM},
title = {RNA biosensors in oncology: Mechanisms, Cancer-specific applications, and a Hallmark-aligned clinical roadmap.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {},
number = {},
pages = {121306},
doi = {10.1016/j.cca.2026.121306},
pmid = {42648526},
issn = {1873-3492},
abstract = {Cancer diagnosis continues to rely on invasive tissue sampling and static molecular assessments that cannot reflect the real time RNA alterations driving tumour progression. RNA biosensors are genetically encoded or synthetic devices that translate specific RNA markers and tumour microenvironment signals into measurable outputs offer a compelling alternative, particularly for liquid biopsy applications where non-invasive, dynamic monitoring is essential. This review systematically examines key RNA biosensor classes developed for oncology, spanning fluorescence based platforms such as aptamers, FRET probes, and molecular beacons, enzymatic and electrochemical architectures including CRISPR Cas systems and field effect transistors, metabolite responsive designs encompassing riboswitches, RNA thermometers, and reactive oxygen species sensors and sequence specific toehold switches alongside exosomal detectors. For each class, we discuss operating principles, cancer relevant applications, reported detection thresholds reaching into the zeptomolar range, and current limitations. These biosensor capabilities are mapped onto Hanahan's hallmarks of cancer, and practical clinical roadmaps are outlined for three priority applications early population screening, longitudinal therapy response and resistance monitoring, and tumour microenvironment prognostication. Convergence with microfluidic integration, AI assisted interpretation, and multiplexed nanotechnology represents the critical next step in translating these platforms from laboratory tools into routine diagnostic practice.},
}

@article {pmid42648813,
year = {2026},
author = {Zhao, M and Zhuang, Q and Wang, X and Gong, J and Chen, L},
title = {CRISPR/Cas12a-based dual intelligent sensors for home pet detection via personal glucose meters.},
journal = {Analytica chimica acta},
volume = {1420},
number = {},
pages = {345935},
doi = {10.1016/j.aca.2026.345935},
pmid = {42648813},
issn = {1873-4324},
mesh = {*Biosensing Techniques/methods/instrumentation ; Animals ; *CRISPR-Cas Systems/genetics ; DNA, Single-Stranded/chemistry ; *Endodeoxyribonucleases/metabolism/chemistry/genetics ; *Glucose/analysis ; Magnetite Nanoparticles/chemistry ; *CRISPR-Associated Proteins/metabolism/chemistry ; *Bacterial Proteins/metabolism/genetics/chemistry ; Rapid Diagnostic Tests ; Dogs ; },
abstract = {Against the backdrop of iterative upgrades in pet pathogen detection technology, rapid on-site testing (POCT) has become the core technology for on-site identification of deadly pet diseases. Based on the research and development of new materials, intelligent sensors with high sensitivity, fast response, and high design flexibility have demonstrated strong application value and have become an important development direction for the next-generation technology system in the field of pet pathogen detection. Herein, we report two advanced intelligent material-integrated biosensing platforms: a DNA hydrogel-encapsulated glucose amylase-based assay (RC-HGPGA) and a magnetic nanoparticles (MNPs)-based system where single-stranded DNA (ssDNA) serves as a molecular bridge to conjugate MNPs with invertase (RC-MBI). Both systems operate via a cascade reaction: recombinase polymerase amplification (RPA) of target nucleic acids first activates Cas12a nuclease, which then exerts trans-cleavage activity toward the biosensing elements. Subsequent enzymatic hydrolysis generates glucose, whose concentration is quantifiable using a commercial personal glucose meter (PGM). All experimental procedures were conducted at a constant temperature of 37 °C, eliminating the need for complex thermal cycling equipment. Our findings demonstrate that the RC-HGPGA and RC-MBI platforms achieve ultra-sensitive detection of feline panleukopenia virus (FPV) and canine distemper virus (CDV)-two clinically significant pet viruses-with limits of detection (LODs) as low as 10° copies/μL and 10[1] copies/μL, respectively, within a rapid time of 35 min. Both systems exhibit high sensitivity, excellent specificity, broad adaptability, and user-friendliness, thereby showing great potential for on-site detection of pet viruses.},
}

*Biosensing Techniques/methods/instrumentation
Animals
*CRISPR-Cas Systems/genetics
DNA, Single-Stranded/chemistry
*Endodeoxyribonucleases/metabolism/chemistry/genetics
*Glucose/analysis
Magnetite Nanoparticles/chemistry
*CRISPR-Associated Proteins/metabolism/chemistry
*Bacterial Proteins/metabolism/genetics/chemistry
Rapid Diagnostic Tests
Dogs

@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