@article {pmid41966922,
year = {2026},
author = {Cui, J and Zhang, L and Zhou, J and Shi, T and Wu, S and Dai, T and Hao, L and Pan, J and Lai, X and Lu, W and Huang, X and Li, Z and Lai, L and Wang, X},
title = {A universal light-controlled highly sensitive one-pot CRISPR/Cas12a diagnostic based on structure-engineered crRNA.},
journal = {Trends in biotechnology},
volume = {44},
number = {9},
pages = {2699-2721},
doi = {10.1016/j.tibtech.2026.03.018},
pmid = {41966922},
issn = {1879-3096},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Nucleic Acid Amplification Techniques/methods ; Human papillomavirus 16/genetics/isolation & purification ; Human papillomavirus 18/genetics/isolation & purification ; Polymorphism, Single Nucleotide ; *Molecular Diagnostic Techniques/methods ; CRISPR-Associated Proteins ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid detection has transformed molecular diagnostics through its speed and accuracy; however, one-pot formats are often limited by sensitivity and field suitability. Herein, we developed a universal light-controlled high-sensitivity one-pot CRISPR/Cas12a testing (ULTRAt) platform based on structure-engineered CRISPR RNA (crRNA) scaffolds. By incorporating photocaged 6-nitropiperonyloxymethyl groups into the crRNA stem-loop, Cas12a activity is transiently suppressed during isothermal amplification via structural modulation, enabling efficient target enrichment. Subsequent UV irradiation removes the protecting groups, restoring the native conformation and activating robust trans-cleavage. ULTRAt achieves a limit of detection of two copies of monkeypox virus per reaction with a 15-min time-to-result, representing a 100-fold sensitivity improvement over conventional assays. The platform further supports single-nucleotide polymorphism discrimination and human papillomavirus 16/18 genotyping. Analysis of 91 clinical samples demonstrates strong concordance between ULTRAt and reference qPCR and sequencing assays. Collectively, ULTRAt enables rapid, ultra-sensitive, and versatile one-pot detection, supporting near-patient diagnostics and genotyping.},
}

*CRISPR-Cas Systems/genetics
Humans
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Nucleic Acid Amplification Techniques/methods
Human papillomavirus 16/genetics/isolation & purification
Human papillomavirus 18/genetics/isolation & purification
Polymorphism, Single Nucleotide
*Molecular Diagnostic Techniques/methods
CRISPR-Associated Proteins
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid42237538,
year = {2026},
author = {Sarko, LE and Givand, D and Rattin, B and Shepley, C and Tommasi, A and Attar, A and Taylor, R and Kutler, B and Traynor, RM and Upadhyaya, A and Mnuk, M and Gehrke, C and Murren, N and Ulland, TK and Capitini, CM and Kotanchek, T and Saha, K},
title = {Directing fratricide within T cell products using an anti-uPAR chimeric antigen receptor to drive the production of potent therapeutic cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {9},
pages = {5190-5206},
doi = {10.1016/j.ymthe.2026.05.029},
pmid = {42237538},
issn = {1525-0024},
support = {P30 CA014520/CA/NCI NIH HHS/United States ; T32 GM135119/GM/NIGMS NIH HHS/United States ; S10 RR025483/RR/NCRR NIH HHS/United States ; R35 GM119644/GM/NIGMS NIH HHS/United States ; R01 CA278051/CA/NCI NIH HHS/United States ; R01 AG083883/AG/NIA NIH HHS/United States ; },
mesh = {Humans ; *Receptors, Chimeric Antigen/genetics/metabolism ; *T-Lymphocytes/metabolism/immunology ; Gene Editing ; *Receptors, Urokinase Plasminogen Activator/antagonists & inhibitors/genetics/immunology/metabolism ; CRISPR-Cas Systems ; Lymphocyte Activation/immunology ; Immunotherapy, Adoptive/methods ; *Receptors, Antigen, T-Cell/genetics/metabolism ; Cell- and Tissue-Based Therapy/methods ; },
abstract = {Cell therapy manufacturing of primary T cells often results in heterogeneous cell populations in the final product, with many cells lacking desired receptor expression or exhausted and other dysfunctional phenotypes. Here, we design a novel cell-intrinsic strategy to genetically reprogram primary human T cells to autonomously detect and eliminate dysfunctional cells. This integrated detection and elimination process, known as directed fratricide, is programmed via non-viral CRISPR genome editing to eliminate the T cell receptor (TCR) alpha chain (TRAC gene knockout) and integrate a chimeric antigen receptor (CAR) against the urokinase-type plasminogen activator receptor (uPAR), also known as CD87. In these cell products, strong T cell stimulation or activation during manufacturing causes a small subset of cells to express uPAR, which triggers CAR-mediated killing within the product. This fratricide induces proliferation in the desired cells and destroys undesired cells, a process that could be modeled computationally and controlled robustly via supplements to the culture media. The strategy enabled enrichment of anti-uPAR and anti-disialoganglioside (GD2) CAR-T cell products up to ≥99% CAR+/TCR-, favoring a memory-like phenotype. Understanding growth dynamics among T cell subsets and reprogramming them via CRISPR could accelerate the biomanufacturing of potent cell products without extensive selection methods.},
}

Humans
*Receptors, Chimeric Antigen/genetics/metabolism
*T-Lymphocytes/metabolism/immunology
Gene Editing
*Receptors, Urokinase Plasminogen Activator/antagonists & inhibitors/genetics/immunology/metabolism
CRISPR-Cas Systems
Lymphocyte Activation/immunology
Immunotherapy, Adoptive/methods
*Receptors, Antigen, T-Cell/genetics/metabolism
Cell- and Tissue-Based Therapy/methods

@article {pmid42427028,
year = {2026},
author = {Ginn, SL and Doroudian, F and Christina, S and Chan, OPY and Lucas, CW and Zhu, E and Yang, SF and Devanapalli, B and Klein, AH and Scott, S and Vitale, J and Cunningham, SC and Liao, SHY and Cabanes-Creus, M and Lisowski, L and Alexander, IE},
title = {Functional editing of the OTC locus by targeted integration with phenotype correction and restoration of endogenous expression patterns.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {9},
pages = {5426-5443},
doi = {10.1016/j.ymthe.2026.06.044},
pmid = {42427028},
issn = {1525-0024},
mesh = {Animals ; Humans ; Mice ; Hepatocytes/metabolism ; *Ornithine Carbamoyltransferase/genetics/metabolism ; Dependovirus/genetics ; *Gene Editing/methods ; Phenotype ; CRISPR-Cas Systems ; *Ornithine Carbamoyltransferase Deficiency Disease/genetics/therapy/metabolism ; Genetic Vectors/genetics/administration & dosage ; Mutation ; Disease Models, Animal ; *Genetic Loci ; Liver/metabolism ; Gene Expression Regulation ; },
abstract = {Here, we report highly efficient functional repair of the ornithine transcarbamylase (OTC) locus in mutant mouse and human hepatocytes in vivo using a dual adeno-associated virus system delivering CRISPR-Cas9 editing reagents and a promoterless donor for targeted integration. The approach was mutation agnostic and targeted intronic sequences to prevent inadvertent inactivation of hypomorphic alleles. Notably, in a murine model, we corrected the metabolic defect and simultaneously achieved liver-wide restoration of physiological metabolic zonation of Otc expression by capturing native cis-acting regulatory elements. The effectiveness of this approach was confirmed using a universally configured therapeutic cassette in patient-derived primary human hepatocytes in vivo. These data provide a powerful template to guide further optimization of this approach and, given the high editing efficacy required for phenotypic effect in OTC deficiency, have broader relevance to other liver disease phenotypes.},
}

Animals
Humans
Mice
Hepatocytes/metabolism
*Ornithine Carbamoyltransferase/genetics/metabolism
Dependovirus/genetics
*Gene Editing/methods
Phenotype
CRISPR-Cas Systems
*Ornithine Carbamoyltransferase Deficiency Disease/genetics/therapy/metabolism
Genetic Vectors/genetics/administration & dosage
Mutation
Disease Models, Animal
*Genetic Loci
Liver/metabolism
Gene Expression Regulation

@article {pmid42556172,
year = {2026},
author = {Khare, G and Duong, A and Hanotaux, J and Shorr, R and Nampoothiri, RV and Maganti, H and Allan, DS},
title = {A scoping review of gene editing in clinical trials: identifying aspects of trial design to accelerate clinical adoption.},
journal = {Cytotherapy},
volume = {28},
number = {10},
pages = {102898},
doi = {10.1016/j.jcyt.2026.102898},
pmid = {42556172},
issn = {1477-2566},
mesh = {Humans ; *Gene Editing/methods ; *Genetic Therapy/methods ; Clinical Trials as Topic ; CRISPR-Cas Systems/genetics ; Adolescent ; Child, Preschool ; Child ; Adult ; Infant ; Leukemia/genetics/therapy ; Aged, 80 and over ; Aged ; Middle Aged ; Research Design ; Lymphoma/therapy/genetics ; },
abstract = {BACKGROUND: Initial clinical trials of gene editing have recently emerged and generated significant interest in this promising therapy. A scoping review is needed to understand aspects of study design that may accelerate further clinical translation.

METHODS: A systematic search of published clinical trials was conducted to May 22, 2025.

RESULTS: Twenty-nine published trials were identified for analysis (420 patients; median ages 6 months to 90 years). The most common gene editing technology used was CRISPR-Cas9 (23 studies, 79%). Hematopoietic cells were targeted most frequently, and leukemias and lymphomas were the most common clinical indications (8 studies; 27.5%), followed by hemoglobin disorders (6; 20.6%), solid tumors (6; 20.6%), rare genetic diseases (5; 17.2%), and others (4; 13.8%). All trials were early-phase (Phase I/II) with only 1 controlled study (29 patients). Six studies used in vivo approaches while all others performed gene editing ex vivo. Gene transfection was by electroporation (9 studies; 31.0%), viral-mediated transfection (6; 20.7%), or by lipid nanoparticles (3; 10.3%). Commonly reported outcomes included early safety and adverse events, mortality, persistence of edited cells, clinical and functional responses. Studies of leukemia/lymphoma reported rates of complete remission in 15-92% while we identified important and sustained increases in mean hemoglobin levels in studies of hemoglobin disorders. Edited cells were detected in most studies at 3 and 6 months but later timepoints were reported less frequently. While early serious adverse events were infrequently observed, reporting of longer-term outcomes was lacking.

CONCLUSION: Gene editing appears feasible and generally safe in humans although important safety outcomes such as long-term oncogenic surveillance outcomes remain to be addressed. A broad range of conditions have been treated, and most often leverages the CRISPR-Cas9 technology with transfection by electroporation. The persistence of edited cells remains ill-defined and longer-term safety outcomes are needed. Studies optimizing CART therapy for leukemia/lymphoma and treatment of hemoglobin disorders appear poised for significant clinical adoption.},
}

Humans
*Gene Editing/methods
*Genetic Therapy/methods
Clinical Trials as Topic
CRISPR-Cas Systems/genetics
Adolescent
Child, Preschool
Child
Adult
Infant
Leukemia/genetics/therapy
Aged, 80 and over
Aged
Middle Aged
Research Design
Lymphoma/therapy/genetics

@article {pmid42560066,
year = {2026},
author = {Hao, Z and Zhao, Q and Zhong, Y and Li, M and Wang, C and Wang, C},
title = {Development and clinical validation of a CRISPR/Cas9-engineered reporter phage cocktail for rapid detection of Escherichia coli in urine.},
journal = {Microbiology spectrum},
volume = {14},
number = {9},
pages = {e0099626},
pmid = {42560066},
issn = {2165-0497},
mesh = {*Escherichia coli/isolation & purification/genetics/virology ; *CRISPR-Cas Systems ; Humans ; *Urinary Tract Infections/diagnosis/microbiology ; *Escherichia coli Infections/diagnosis/microbiology/urine ; Sensitivity and Specificity ; *Urine/microbiology ; Genes, Reporter ; *Bacteriophages/genetics ; *Coliphages/genetics ; },
abstract = {Urinary tract infections are one of the most common infectious diseases, with Escherichia coli as the predominant pathogen. Traditional diagnostic methods fail to meet clinical demands for rapid and specific detection. Here, we developed an efficient urine E. coli detection strategy via a reporter phage cocktail. Four reporter phages (T2::Nluc, T4::Nluc, T5::Nluc, T6::Nluc) were constructed by the CRISPR/Cas9 system combined with homologous recombination. One-step growth curves, optimal multiplicity of infection, and lytic efficiency showed that the Nluc gene block insertion exerted heterogeneous effects on phages. Luminescence assays demonstrated that all five reporter phages (including previously preserved T7::Nluc) and the cocktail offered favorable limits of detection (≥10[3] CFU/mL), high specificity, and no urine matrix interference. However, single phages exhibited limited coverage among 177 clinical E. coli isolates. But the reporter phage cocktail remedies this limitation. In large-scale clinical validation, the cocktail achieved sensitivity 73.15% (63.76%-81.22%), specificity 100.00% (99.53%-100.00%), positive predictive value (PPV) 100.00% (95.44%-100.00%), and negative predictive value (NPV) 96.42% (95.18%-97.36%) (all 95% confidence interval [CI]), and excellent concordance with the gold-standard method (Kappa = 0.83, 95% CI: 0.77-0.89), greatly outperforming single reporter phages (~40.00% sensitivity). This method requires no sample pretreatment, is simple to operate, and completes detection within 4 h, significantly improving diagnostic efficiency. Accordingly, it provides a novel platform for pathogen detection and supports the clinical translation of reporter phage diagnostics.IMPORTANCEUrinary tract infections impose substantial economic and public health burdens. In this study, we successfully constructed Escherichia coli-specific reporter phages T2::Nluc, T4::Nluc, T5::Nluc, and T6::Nluc. Combined with the previously preserved T7::Nluc, these phages formed a reporter phage cocktail. Co-cultivation of this cocktail with clinical samples enabled rapid and specific detection of E. coli in clinical urine, with a significantly shortened detection time (4 h) and good concordance with the gold-standard detection method (Kappa = 0.83), effectively improving detection efficiency and accuracy. This novel pathogen detection platform, integrating specific recognition and signal amplification, not only provides a new technical approach for the rapid and accurate diagnosis of clinical urinary tract infections but also effectively promotes the coordinated improvement of infectious disease diagnosis and treatment in terms of timeliness-precision-cost.},
}

*Escherichia coli/isolation & purification/genetics/virology
*CRISPR-Cas Systems
Humans
*Urinary Tract Infections/diagnosis/microbiology
*Escherichia coli Infections/diagnosis/microbiology/urine
Sensitivity and Specificity
*Urine/microbiology
Genes, Reporter
*Bacteriophages/genetics
*Coliphages/genetics

@article {pmid42599083,
year = {2026},
author = {Li, Y-y and Zhang, H and Shao, L-n and Liu, B-l and Wang, Y-m and Duan, J-q and Lai, S-Y and Xu, Z-w and Zhu, L},
title = {A rapid, sensitive, and field-deployable RAA-EsCas13d platform for detection of porcine adenovirus type 3.},
journal = {Microbiology spectrum},
volume = {14},
number = {9},
pages = {e0129126},
pmid = {42599083},
issn = {2165-0497},
support = {2024YFD1800500//Research and Application of Integrated Prevention, Control and Purification Technologies for Major Swine Epidemic Diseases/ ; 2024YFD1800102//National Key Research and Development Program of China during the 14th Five-Year Plan: Research and Development of Key Technologies for the Prevention and Control of Important Diseases in Wild Animals/ ; sccxtd-2024-08//Sichuan Pig Innovation Team of the National Modern Agricultural Industry Technology System/ ; CARS⁃SVDIP//National agricultural industrial technology system Sichuan veterinary medicine innovation team special/ ; },
mesh = {Animals ; Swine ; *Adenoviruses, Porcine/genetics/isolation & purification/classification ; Rapid Diagnostic Tests ; Sensitivity and Specificity ; *Adenoviridae Infections/veterinary/diagnosis/virology ; *Nucleic Acid Amplification Techniques/methods ; *Swine Diseases/virology/diagnosis ; CRISPR-Cas Systems ; Recombinases/genetics ; },
abstract = {Rapid, sensitive, and RADIANT-deployable nucleic acid diagnostics are essential for the prevention and control of porcine adenovirus type 3 (PAdV-3). In this study, we developed RADIANT (RAA-Cas13d DIagnostic plATform for extractioN-free Testing), an extraction-free platform capable of delivering accurate and portable detection in resource-limited settings. By systematically optimizing the reaction buffer composition, recombinase-aided amplification (RAA), T7 transcription, and CRISPR/EsCas13d-mediated cleavage were integrated into a one-pot reaction, reducing the total time-to-result to within 30 minutes. A simplified nucleic acid release step eliminated the need for laboratory-based extraction or complex heating, enhancing the accessibility of the assay. Incorporation of lyophilized reagents minimized cold-chain requirements and simplified assay preparation, enabling cost-effective storage and transport. The platform demonstrated high sensitivity in detecting PAdV-3 and offered two straightforward readout options, fluorescence under 470 nm blue/UV light and lateral flow assay (LFA), facilitating flexible interpretation under diverse RADIANT conditions. Validation with 56 clinical samples showed 100% concordance with quantitative PCR, confirming RADIANT as a rapid, user-friendly, and reliable on-site diagnostic tool for PAdV-3 detection.IMPORTANCEPorcine adenovirus type 3 (PAdV-3) is an important swine pathogen for which rapid, practical, and field-compatible diagnostic tools are still lacking. Here, we developed RADIANT, a CRISPR/EsCas13d-based platform that expands PAdV-3 detection beyond conventional laboratory workflows and supports accessible molecular testing in resource-limited settings. Its simple operation and adaptable visual readouts enhance its suitability for on-site use without compromising reliability. This study provides a valuable diagnostic approach for PAdV-3 surveillance and disease control and supports the broader application of portable CRISPR-based technologies in veterinary diagnostics.},
}

Animals
Swine
*Adenoviruses, Porcine/genetics/isolation & purification/classification
Rapid Diagnostic Tests
Sensitivity and Specificity
*Adenoviridae Infections/veterinary/diagnosis/virology
*Nucleic Acid Amplification Techniques/methods
*Swine Diseases/virology/diagnosis
CRISPR-Cas Systems
Recombinases/genetics

@article {pmid42653322,
year = {2026},
author = {Li, H and Zhang, X and Wang, X and Zhang, R and Liu, L and Sun, L and Yao, Z and Zhu, H},
title = {CRISPR/Cas9-Mediated Disruption of Duplicated Sizzled Genes Induces Twin-Tail-like Caudal Bifurcation in Goldfish (Carassius auratus).},
journal = {International journal of molecular sciences},
volume = {27},
number = {16},
pages = {},
pmid = {42653322},
issn = {1422-0067},
support = {KJCX20261411//Beijing Academy of Agricultural and Forestry Sciences/ ; 32403015//National Natural Science Foundation of China/ ; KJCX20230216//Beijing Academy of Agricultural and Forestry Sciences/ ; 6262009//National Natural Science Foundation of China/ ; GHPT2026-16//Beijing Academy of Agricultural and Forestry Sciences/ ; PT2025-20//Beijing Academy of Agricultural and Forestry Sciences/ ; NY2401170024//Rural Development Institute/ ; GHPT2026-06//China International Science and Technology Cooperation/ ; },
mesh = {Animals ; *Goldfish/genetics/embryology ; *CRISPR-Cas Systems ; *Fish Proteins/genetics/metabolism ; Body Patterning/genetics ; Gene Expression Regulation, Developmental ; Glycoproteins ; Intercellular Signaling Peptides and Proteins ; },
abstract = {The twin-tail phenotype of goldfish represents a striking domestication-associated remodeling of the vertebrate caudal axial system and is classically linked to disruption of Chordin/BMP-mediated dorsal-ventral patterning. Although previous knockdown studies implicated sizzled (szl) in this process, genetic evidence from targeted disruption of endogenous szl loci remains limited. Here, we used CRISPR/Cas9 to mutate conserved coding regions shared by the duplicated goldfish paralogues szlA and szlB in single-tail embryos. Sanger sequencing and ICE analysis showed that szl-sgRNA2 and szl-sgRNA3 efficiently induced indels at both loci, whereas szl-sgRNA1 was ineffective. Across three independent biological replicates, twin-tail-like caudal bifurcation was observed in 44.63-48.19% of szl-sgRNA2-injected larvae, 69.47-79.61% of szl-sgRNA3-injected larvae, and 64.29-76.19% of larvae injected with the sgRNA mixture; szl-sgRNA1-injected larvae remained single-tailed. Calcein staining further revealed separation of distal caudal fin rays and partial splitting of the caudal skeletal complex in szl-edited larvae. qRT-PCR showed selective remodeling of dorsal-ventral patterning genes, including reduced chdA and eve1 expression and increased bmp2 and nog1 expression. These findings provide direct functional evidence that szl regulates median caudal patterning in goldfish and suggest that szl-dependent modulation of the Chordin/BMP network can generate twin-tail-like caudal morphology.},
}

Animals
*Goldfish/genetics/embryology
*CRISPR-Cas Systems
*Fish Proteins/genetics/metabolism
Body Patterning/genetics
Gene Expression Regulation, Developmental
Glycoproteins
Intercellular Signaling Peptides and Proteins

@article {pmid42653378,
year = {2026},
author = {Santos, ARD},
title = {AI-Assisted Cross-Study Synthesis in Genome Editing: Comparing Long-Context Strategies and Uncovering Latent Contradictions in the CRISPR-Cas9 Guide RNA Prediction Literature.},
journal = {International journal of molecular sciences},
volume = {27},
number = {16},
pages = {},
pmid = {42653378},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Large Language Models ; *Artificial Intelligence ; Humans ; },
abstract = {Predicting CRISPR-Cas9 guide RNA efficiency and off-target activity is a precondition for precise genome editing. Computational models have progressively incorporated chromatin accessibility and epigenetic descriptors into their feature sets, yet synthesising findings from independently published studies-especially when those studies contradict one another-remains an unresolved methodological gap. Large Language Models (LLMs) have been proposed as a route to automate cross-study synthesis, but their utility depends on a constraint that receives less attention than model architecture: how much of the source text actually reaches the model at inference time. Cloud-based models process 48,000-token corpora without hardware limitations, but at the cost of data leaving the local environment and with limited reproducibility across API versions. Local RAG systems avoid the cloud dependency while fragmenting the input, discarding the global context needed to link biological arguments that are distributed across separate papers. We benchmark these strategies using a corpus of four CRISPR-Cas9 efficiency prediction studies and apply the Reduced Interaction Sampling (RIS) engine-a local sparse attention method-to retain the full sequence within the memory envelope of a laboratory server. Preserving that context uncovers three latent inconsistencies. The static epigenetic markers used in DeepCRISPR (CTCF, DNase I) show near-zero Spearman correlations with off-target cleavage (ρ≤0.07), while nucleosome positioning scores from the Block Decomposition Method reach ρ=0.388-0.423. The sequence-only Apindel model was published in June 2022 without incorporating nucleosome descriptors reported in the concurrent literature. The benchmark review by Konstantakos et al. attributed 10-20% of rank correlation to epigenetics-a figure that reflects the weak feature subset evaluated, not a ceiling on chromatin influence. These discrepancies are invisible when papers are read individually or retrieved as chunks; they become traceable only when the full corpus is processed as a single context window. An independent empirical analysis of 2000 CRISPR-Cas9 off-target cleavage events provides evidence consistent with this pattern: static epigenetic markers yield |ρ|≤0.11, whereas computed NuPoP Affinity descriptors reach r=-0.622 (p<10-210). On a 30-question cross-study synthesis benchmark (5 independent seeds), baseline accuracy is 53.33%, RAG 60.00%, and RIS (30 seeds, 3% density) 70.00% (p<0.0001, t-test vs. RAG, σ=0.00% for all configurations).},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics
Large Language Models
*Artificial Intelligence
Humans

@article {pmid42654749,
year = {2026},
author = {Martínez-Álvarez, S and Herrera-Espejo, S and Zarazaga, M and Höfle, U and Pachón-Ibáñez, ME and Torres, C},
title = {Virulence and Invasion Profiles of Escherichia coli Across One Health Reservoirs: Genomic Insights into High-Risk Clones and Their Defense Systems.},
journal = {Pathogens (Basel, Switzerland)},
volume = {15},
number = {8},
pages = {},
pmid = {42654749},
issn = {2076-0817},
support = {PID2022-139591OB-I00//MICIU/AEI/10.13039/501100011033 and ERDF/ EU/ ; },
mesh = {Humans ; Virulence ; Animals ; *Escherichia coli Infections/microbiology/veterinary ; *Escherichia coli/genetics/pathogenicity/classification/isolation & purification ; Bacterial Adhesion ; *One Health ; HEK293 Cells ; Genomics ; *Disease Reservoirs/microbiology ; Genome, Bacterial ; CRISPR-Cas Systems ; Virulence Factors/genetics ; Biofilms/growth & development ; Phenotype ; },
abstract = {Escherichia coli is a genetically diverse species encompassing both commensal and pathogenic lineages capable of transitioning among various hosts. Within a One Health framework, we conducted a targeted screening of 38 E. coli strains isolated from wildlife, livestock, and food reservoirs to characterize their pathogenic potential by integrating genomic and phenotypic approaches. In vitro functional assays, including biofilm formation, surface motility, and adherence and invasion of HEK-293 epithelial cells, were statistically evaluated using the non-parametric Mann-Whitney U test. Phenotypic analyses revealed that extraintestinal pathogenic (ExPEC) and uropathogenic E. coli (UPEC) strains, particularly those belonging to the high-risk ST117 clone, exhibited significantly enhanced adherence and internalization capacities. These virulent phenotypes strongly correlated with specific genetic signatures involved in iron acquisition and epithelial invasion (chuA, fyuA, vat, and tia), underscoring that the convergence of ExPEC/UPEC determinants drives increased colonization potential. Genomic characterization further revealed that despite high virulence and widespread antimicrobial resistance, the CRISPR/Cas subtype I-E system was highly prevalent (93.8%), displaying structural variations frequently driven by insertion sequences. Spacer analyses identified limited homology to plasmids and phages, suggesting past mobilome interactions rather than active restriction of current horizontal gene transfer. Overall, these findings illustrate how phenotypic traits of high-risk clones match their genomic virulence platforms. The convergence of multidrug resistance and pathogenic fitness across human, animal, and environmental interfaces underscores the need for integrated molecular surveillance in a One Health context.},
}

Humans
Virulence
Animals
*Escherichia coli Infections/microbiology/veterinary
*Escherichia coli/genetics/pathogenicity/classification/isolation & purification
Bacterial Adhesion
*One Health
HEK293 Cells
Genomics
*Disease Reservoirs/microbiology
Genome, Bacterial
CRISPR-Cas Systems
Virulence Factors/genetics
Biofilms/growth & development
Phenotype

@article {pmid42655688,
year = {2026},
author = {Dai, Y and Xia, L and Yang, Y and Qiao, G and Jin, X and Mao, X},
title = {Recent Advances in CRISPR/Cas Systems for Respiratory Pathogen Diagnostics.},
journal = {Viruses},
volume = {18},
number = {8},
pages = {},
pmid = {42655688},
issn = {1999-4915},
support = {HB2023111//Xuhua Mao/ ; },
mesh = {*CRISPR-Cas Systems ; Humans ; *Respiratory Tract Infections/diagnosis/microbiology/virology ; Bacteria/genetics/isolation & purification ; *Molecular Diagnostic Techniques/methods ; Viruses/genetics/isolation & purification ; Nucleic Acid Amplification Techniques/methods ; Rapid Diagnostic Tests ; Fungi/genetics/isolation & purification ; },
abstract = {Early, rapid, and accurate detection is essential for clinical management and epidemiological control of acute respiratory infections caused by pathogens. Traditional testing methods such as microbial culture, serological testing, and PCR are restrictive in terms of operation and logistics and are therefore not easily used in point-of-care settings. The CRISPR/Cas system is an adaptive prokaryotic immune system composed of clustered regularly interspaced short palindromic repeats and their associated proteins, which has been used as a nucleic acid diagnostic platform with programmable sequence-specific target recognition and signal-amplifying collateral cleavage activity. Existing reviews have mostly focused on the classification of Cas enzymes or amplification strategies; in this review, a pathogen-centric approach was taken, covering viral pathogens (SARS-CoV-2, influenza virus, RSV, HAdV and VZV), bacterial pathogens (Mycobacterium tuberculosis, Streptococcus pneumoniae, Mycoplasma pneumoniae and Staphylococcus aureus) and fungal pathogens (Aspergillus fumigatus and Pneumocystis jirovecii). Key technological advances, such as isothermal amplification coupling, single-vessel integrated reaction designs, amplification-free digital detection, and electrochemical biosensor integration, are evaluated for Cas9-, Cas12-, and Cas13-based systems, with their mechanistic bases outlined. The current challenges that hinder clinical translation, such as sample matrix interference, multiple signal cross-talk, crRNA off-target effects, and the lack of large-scale validation studies, are critically assessed. At the same time, future pathways for portable, integrated, and inexpensive diagnostic platforms are suggested.},
}

*CRISPR-Cas Systems
Humans
*Respiratory Tract Infections/diagnosis/microbiology/virology
Bacteria/genetics/isolation & purification
*Molecular Diagnostic Techniques/methods
Viruses/genetics/isolation & purification
Nucleic Acid Amplification Techniques/methods
Rapid Diagnostic Tests
Fungi/genetics/isolation & purification

@article {pmid42655725,
year = {2026},
author = {Azad, MAK and Ibnat, N and Chowdhury, SS and Huq, S and Islam, S},
title = {Advances in Molecular Techniques for Detecting Sweet Potato (Ipomoea batatas (L.) Lam) Viruses: A Comprehensive Review.},
journal = {Viruses},
volume = {18},
number = {8},
pages = {},
pmid = {42655725},
issn = {1999-4915},
mesh = {*Ipomoea batatas/virology ; *Plant Diseases/virology ; *Plant Viruses/genetics/isolation & purification/classification ; *Molecular Diagnostic Techniques/methods ; High-Throughput Nucleotide Sequencing ; Nucleic Acid Amplification Techniques ; Biosensing Techniques ; },
abstract = {Sweet potato (Ipomoea batatas (L.) Lam) is an important global food crop, but its production is threatened by numerous viral pathogens. More than 30 RNA and DNA viruses have been reported worldwide, making rapid and accurate detection essential for disease management, epidemiological surveillance, germplasm exchange, and resistance breeding. Although previous reviews have addressed sweet potato viruses and individual diagnostic methods, a comprehensive synthesis of emerging molecular technologies remains limited. This review addresses that gap by critically integrating recent advances from PCR-based and isothermal assays to high-throughput sequencing, CRISPR-based diagnostics, biosensors, nanotechnology, and artificial intelligence-driven detection platforms. Conventional approaches, including symptom observation, biological indexing, electron microscopy, and ELISA, have contributed to early virus identification but often lack the sensitivity, specificity, and speed needed for modern diagnostics. Molecular and isothermal techniques have substantially improved detection accuracy and enabled rapid identification and field-deployable diagnostics of diverse and mixed infections, while sequencing, CRISPR, biosensors, and AI-based platforms offer greater capacity for detecting novel and emerging viruses. This review discusses the comparative evaluation of molecular technologies for sweet potato virus detection in terms of diagnostic performance, cost-effectiveness, speed, and suitability for both laboratory and field applications, while highlighting future priorities for next-generation virus diagnostics. Integrating portable and high-throughput diagnostic platforms will strengthen virus surveillance, support virus-free planting material production, and promote sustainable sweet potato production worldwide.},
}

*Ipomoea batatas/virology
*Plant Diseases/virology
*Plant Viruses/genetics/isolation & purification/classification
*Molecular Diagnostic Techniques/methods
High-Throughput Nucleotide Sequencing
Nucleic Acid Amplification Techniques
Biosensing Techniques

@article {pmid42658322,
year = {2026},
author = {Kara, G and Holcomb, M and Hijazi, AA and Ali, Y and López-Espinosa, J and Cruz-Pineda, L and Park, P and Flinn, H and Taylor, N and Galbraith, T and McMahon, L and Rostomily, R and Leonard, F and Villapol, S},
title = {Intranasal CRISPR lipid nanoparticles targeting MAPK9 attenuate neuroinflammation after traumatic brain injury.},
journal = {Biomedical microdevices},
volume = {28},
number = {3},
pages = {},
pmid = {42658322},
issn = {1572-8781},
support = {R56AG080920/AG/NIA NIH HHS/United States ; R56 AG080920/AG/NIA NIH HHS/United States ; R21NS106640/NS/NINDS NIH HHS/United States ; ission Connect 2026 grant (S.V.). (No. 026-102),//TIRR Foundation/ ; R21 NS106640/NS/NINDS NIH HHS/United States ; },
mesh = {*Brain Injuries, Traumatic/complications/pathology ; Animals ; *Nanoparticles/chemistry ; Mice ; Administration, Intranasal ; *Neuroinflammatory Diseases ; *Lipids/chemistry ; *CRISPR-Cas Systems/genetics ; Male ; Microglia/metabolism ; Mice, Inbred C57BL ; },
abstract = {Traumatic brain injury (TBI) induces a sustained neuroinflammatory response involving activated microglia and infiltrating myeloid cells, contributing to secondary brain damage and long-term neurological dysfunction. Modulating these inflammatory responses toward a more reparative phenotype represents a promising therapeutic strategy, but achieving targeted delivery within the injured brain remains a major challenge. Here, we developed a targeted, non-viral gene-editing platform using lipid nanoparticles (LNPs) encapsulating CRISPR-Cas12a components directed against MAPK9, a key mediator of inflammatory signaling. LNPs were functionalized with an Iba-1 antibody to enhance targeting of Iba-1 + myeloid cells following intranasal administration. In primary bone marrow-derived macrophages and primary microglia, CRISPR-mediated MAPK9 targeting reduced MAPK9 expression and suppressed pro-inflammatory activation, decreasing iNOS, NLRP3, CD80, and CCL2 while increasing the anti-inflammatory/reparative markers CD206 and Arg1. In a mouse model of TBI, intranasally delivered Iba-1-targeted CRISPR-LNPs showed preferential association with Iba-1 + cells compared with NeuN+ neurons in the injured cortex and reduced MAPK9 expression within Iba-1 + cells. CRISPR-LNP treatment attenuated microglial/macrophage activation, reduced pro-inflammatory cytokine expression, and decreased iNOS+/Iba-1 + cells while increasing CD206+/Iba-1 + cells in the peri-contusional cortex, supporting a shift toward a less inflammatory phenotype. Treatment also exhibited a favorable safety profile, with no detectable toxicity in the major organs examined. Together, these findings demonstrate that intranasal delivery of Iba-1-targeted CRISPR-LNPs enables effective MAPK9 modulation in Iba-1 + myeloid cells within the injured brain and attenuates acute neuroinflammation following TBI. This non-invasive therapeutic platform provides a promising approach for targeted modulation of neuroinflammatory responses after brain injury.},
}

*Brain Injuries, Traumatic/complications/pathology
Animals
*Nanoparticles/chemistry
Mice
Administration, Intranasal
*Neuroinflammatory Diseases
*Lipids/chemistry
*CRISPR-Cas Systems/genetics
Male
Microglia/metabolism
Mice, Inbred C57BL

@article {pmid42660250,
year = {2026},
author = {Fiaz, S},
title = {Recent trends in nucleic acid research in plants for future food security.},
journal = {International journal of biological macromolecules},
volume = {},
number = {},
pages = {154241},
doi = {10.1016/j.ijbiomac.2026.154241},
pmid = {42660250},
issn = {1879-0003},
abstract = {The agricultural production system is facing unprecedented pressure of climate change, human population, pressure on renewable and non-renewable resources, and elevation in both biotic and abiotic stress factors. The recent development in nucleic acid based technologies have transformed research in plant sciences through deployment of powerful technologies for understanding complex biological mechanisms controlling climate resilience, nutrition and yield attributes in agriculturally important crops. This editorial documented the key results of 33 articles published in this special issue, covering application of various techniques to improve desirable attributes in agriculturally important crops. Furthermore, the published literature displayed the key findings emerging through the application of CRISPR-Cas based genome editing system, integration of multi-omics, application of machine learning, RNA-based regulations and chloroplast bioengineering with higher precision, efficiency, and reliability. The innovation in plant-microbe interaction, rhizosphere engineering has revealed novel avenues of research for improving the potential of resource use efficiency and sustainability. Altogether, the research published in this special issue may play a transformative role in advancing precision breeding, stress resilience and crop performance under ever-changing climatic conditions. However, there is a requirement for continuous interdisciplinary research, embracing innovation and international collaboration for utilization of full potential of cutting-edge technologies contributing significantly to achieving food security.},
}

@article {pmid42661411,
year = {2026},
author = {Vermeulen, W and van Staden, ADP and Dicks, LMT},
title = {Nucleases and Their Inhibitors: Exploring Biological Roles, Industrial Applications, and Challenges in Heterologous Expression.},
journal = {Biotechnology journal},
volume = {21},
number = {9},
pages = {e70303},
pmid = {42661411},
issn = {1860-7314},
mesh = {Humans ; *Endonucleases/genetics/metabolism/antagonists & inhibitors ; Animals ; CRISPR-Cas Systems ; },
abstract = {Nucleases hydrolyze phosphodiester bonds and participate in numerous cellular and metabolic processes. Intracellular nucleases repair nonfunctional or damaged DNA using DNA base excision repair (BER), mismatch repair (MMR), and homologous recombination (HR). Apoptotic nucleases systematically degrade cellular DNA during programmed cell death (PCD). Non-apoptotic nucleases support DNA repair and replication. Small noncoding RNAs (sncRNAs) degrade the RNA of viral particles. Extracellular and membrane-associated nucleases replenish nucleotides, especially in biofilms where cells rely on additional carbon, phosphorus, and energy. Restriction endonucleases (REs) are indispensable in recombinant DNA technology. Some genetic disorders and cancers have been treated by changing the genetic code of host cells using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated proteins) system. Nucleases are also used in vaccine development. Heterologous expression of nucleases remains challenging, largely due to cytotoxicity and product instability. Some successes have been reported using the T7 promoter-based system. However, due to the formation of inclusion bodies (IBs), the nucleases were insoluble and of low activity. Refolding misfolded nucleases from IBs, tight control (sequestration) of periplasmic secretion, and coexpression with natural inhibitor proteins increased yield, purity, and biological activity. This review addresses the significance of nucleases, heterologous expression, gene regulation, activity inhibition, and product yield.},
}

Humans
*Endonucleases/genetics/metabolism/antagonists & inhibitors
Animals
CRISPR-Cas Systems

@article {pmid42662772,
year = {2026},
author = {Kim, GE and Jin, HB and Kang, YJ and Park, HH},
title = {Structural insights into Cas9 inhibition by AcrIIA17 via bridge helix interaction.},
journal = {iScience},
volume = {29},
number = {9},
pages = {117218},
pmid = {42662772},
issn = {2589-0042},
abstract = {Anti-CRISPR (Acr) proteins have evolved in bacteriophages and mobile genetic elements to counteract CRISPR-Cas immune systems through diverse inhibitory mechanisms. Here, we present the crystal structure of AcrIIA17 and elucidate its mechanism of Staphylococcus aureus Cas9 (SauCas9) inhibition. AcrIIA17 adopts a previously uncharacterized protein fold and exists as a monomer in solution. Biochemical analyses reveal that AcrIIA17 inhibits SauCas9 activity in a strictly order-dependent manner, effectively suppressing DNA cleavage only when it engages Cas9 prior to single guide RNA (sgRNA) loading, whereas pre-assembled Cas9-sgRNA ribonucleoprotein (RNP) complexes are resistant to inhibition. Domain-mapping experiments demonstrate that AcrIIA17 directly binds to the bridge helix (BH) domain of SauCas9, and structure-guided mutagenesis confirms that this interaction is essential for its inhibitory function. Together, our findings identify AcrIIA17 as an Acr protein that targets the Cas9 BH domain and reveal the BH domain as a regulatory checkpoint in Cas9 activation.},
}

@article {pmid42662940,
year = {2026},
author = {Şahin, C and Formica, TM and Silva, A and Della Pelle, G and Davis, MC and Andersen, DG and Çavdar, M and Santos, L and Qiu, L and Olsen, AL and Askou, AL and Köber, M and Jimenez-Mallebrera, C and Gutiérrez, MC and Soerensen, JF and Martín, F and Bak, RO and Sendemir, A and Río, P and Cavazza, A and Benabdellah, K and Nair, RR and Luo, Y},
title = {The landscape and trajectory of global CRISPR therapeutics.},
journal = {Molecular therapy. Nucleic acids},
volume = {37},
number = {3},
pages = {103011},
pmid = {42662940},
issn = {2162-2531},
abstract = {With the first regulatory approval in 2023 of a CRISPR-based therapy for sickle cell disease followed by the recent demonstration of an accelerated, personalized CRISPR treatment for congenital severe carbamoyl-phosphate synthetase 1 deficiency, CRISPR-Cas9 genome editing has progressed from a laboratory technology into a clinical reality in just over a decade. While enthusiasm for deploying CRISPR-Cas9 to treat a range of human genetic diseases continues to grow, broad clinical application remains constrained due to technical, regulatory, manufacturing and economic challenges. Here, scientists from the European Cooperation in Science and Technology (COST) action Genome Editing to Treat Human Diseases (GenE-HumDi) provide a comprehensive global overview of the CRISPR therapeutic landscape. We systematically analyze CRISPR-based therapeutic trials registered on ClinicalTrials.gov and the EU Clinical Trials Register, providing a thorough assessment of the current clinical activity and the trajectory of CRISPR technology as it advances toward routine clinical interventions.},
}

@article {pmid42664690,
year = {2026},
author = {Bastías, DA and Zhang, W and Gundel, PE and Mace, WJ and Prakash, S and Morozova, Y and Jáuregui, R and Maclean, PH and Sprosen, J and Johnson, RD},
title = {Fungal endophytes relieve the growth-defence trade-off of their plant hosts through the production of bioactive alkaloids.},
journal = {Plant physiology and biochemistry : PPB},
volume = {238},
number = {},
pages = {111685},
doi = {10.1016/j.plaphy.2026.111685},
pmid = {42664690},
issn = {1873-2690},
abstract = {A central paradigm in plant biology is that there is a trade-off between growth and defence. We propose that Epichloë fungal endophytes relieve this trade-off in plants by the fungal-derived production of antiherbivore alkaloids. Lolium perenne plants without (nil) and with Epichloë LpTG-3 sp. strain AR37 able (wild type (wt), ΔidtA) and unable (ΔidtM) to produce bioactive indole diterpene alkaloids were subjected to an exogenous application of gibberellin (GA) hormone followed by a challenge with Rhopalosiphum padi aphids. The GA-derived plant growth promotion increased the susceptibility to aphids in both nil plants and ΔidtM-associated plants but did not affect the aphid resistance in either wt- or ΔidtA-associated plants. GA treatment changed the composition of AR37-derived alkaloids, reduced the concentration of AR37-derived epoxyjanthitrem alkaloids in wt-associated plants and reduced the amount of mycelial biomass of AR37 variants. GA treatment generally increased expression of plant genes related to abscisic acid (ABA), auxin, cell division, cell wall and GA (e.g., YUCCA2, GA2ox3), reduced expression of photosynthesis-related genes (e.g., RBCS1), and had mixed effects on the expression of plant immunity-related genes (e.g., PR-1). GA treatment increased concentrations of ABA, salicylic acid, and jasmonic acid, and did not affect auxin concentrations. Additionally, soil derived from GA-treated plants showed increased abundance of putative bacterial taxa that included plant growth-promoting members (e.g., Bryobacter). Our findings demonstrate that the AR37-derived production of indole diterpene alkaloids was the key mechanism that relieved the growth-defence trade-off of plants.},
}

@article {pmid42665203,
year = {2026},
author = {Uti, DE},
title = {Engineered exosomes for CRISPR/Cas delivery to overcome oncogene-driven drug resistance.},
journal = {Experimental cell research},
volume = {462},
number = {2},
pages = {115159},
doi = {10.1016/j.yexcr.2026.115159},
pmid = {42665203},
issn = {1090-2422},
abstract = {Engineered exosomes are emerging as biocompatible nanocarriers for delivering CRISPR/Cas components to resistant tumor cells, enabling targeted disruption of oncogenic drivers and resistance-associated pathways. Engineered exosomes offer several delivery-platform advantages, including biocompatibility, membrane-mediated cargo protection, programmable tumor targeting, and potential tissue penetration. Selection of the CRISPR modality, Cas9 ribonucleoprotein, mRNA, base editor, or prime editor, depends on payload size, stability, editing duration, endosomal escape, and nuclear delivery requirements. Therapeutically, these systems may disrupt oncogenic drivers, inhibit resistance pathways, restore tumor-suppressor activity, and re-sensitize tumors to targeted therapy, chemotherapy, or immunotherapy. Clinical translation will require scalable manufacturing, reproducible cargo loading, standardized characterization, validated potency assays, off-target control, and clearly defined regulatory pathways. The goal of this review is to outline a realistic pathway featuring proof-of-concept research, through discoveries to the creation of manufacturable, safe, and effective exosome/CRISPR therapeutics that can trigger durable therapeutic responses in resistant malignancies.},
}

@article {pmid42666455,
year = {2026},
author = {Handelmann, CR and Skeens, E and Lisi, GP and Buck, MJ},
title = {Evaluating high-fidelity CRISPR-Cas nucleases in nucleosomal contexts using a quantitative framework.},
journal = {Frontiers in genome editing},
volume = {8},
number = {},
pages = {1759382},
pmid = {42666455},
issn = {2673-3439},
support = {R35 GM158384/GM/NIGMS NIH HHS/United States ; },
abstract = {Chromatin presents a significant obstacle to CRISPR-Cas gene editing, as chromatin restricts nuclease access to DNA. Recent advances have produced a wide range of high-fidelity Cas9 and Cas12a variants with enhanced properties. However, their precision in targeting DNA within different contexts remains poorly understood. This gap limits our ability to predict and optimize Cas performance in the dynamic chromatin landscape. To elucidate how chromatin variability impacts Cas editing accuracy, we utilized GEMiNI-seq to systematically profile wild-type and engineered Cas9 and Cas12a nucleases across a range of nucleosome sequences. All nucleases showed reduced cleavage in nucleosomal DNA relative to naked DNA, with the strongest inhibition at dyad-proximal sites. Cleavage within nucleosomes was highly variable, with wtSpCas9 exhibiting up to 65-fold different activity depending on the nucleosome type. Editors with high catalytic activity (wtSpCas9, HIFIv2, LbCas12a ULTRA) consistently outperformed high-fidelity variants such as evoSpCas9, which displayed excellent specificity on naked DNA but poor performance in nucleosomal contexts. ROC and PRC analyses revealed that nucleosome sequence and orientation shape both sensitivity and specificity, with HIFIv1 emerging as the top-performing nuclease for nucleosomal targets, while evoSpCas9 excelled in exposed contexts. Our findings demonstrate that local nucleosome sequence and structure profoundly influence Cas nuclease accessibility and specificity. Variability in cleavage across nucleosome types underscores the need to consider chromatin context during target selection and nuclease design. These results provide a framework for selecting or engineering Cas editors optimized for therapeutic genome editing within chromatin.},
}

@article {pmid42667235,
year = {2026},
author = {Postell, L and Elturk, N and Leonard, H and Mycek, J and Snethen, B and de Noronha, CMC and Sharifi, HJ},
title = {Targeting Cas9 to Perform Rescue Experiments in Stably Transduced Knockout HEK293 Cells.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {234},
pages = {},
doi = {10.3791/72172},
pmid = {42667235},
issn = {1940-087X},
mesh = {Humans ; HEK293 Cells ; *Gene Knockout Techniques/methods ; *CRISPR-Cas Systems ; Cullin Proteins/genetics/biosynthesis ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {CRISPR-Cas9 gene editing technology has revolutionized molecular biology. Often, this technology is employed to delete a gene encoding a protein of interest. The resulting phenotype provides valuable insight into the protein's function. The functional importance of the target protein can be confirmed by reintroducing the protein to restore the lost function (rescue). This is typically accomplished by introducing the protein-coding cDNA in trans using an expression vector. However, in knockout cell lines that stably express the CRISPR-Cas9 system, the newly introduced expression plasmid may also be cleaved by Cas9. The protocol presented here provides a strategy to circumvent this potential barrier to rescue experiments. This approach is demonstrated using HEK293 cells in which the gene encoding the E3 ubiquitin ligase scaffold protein CUL4B was disrupted by CRISPR-Cas9. Transduction of these cells with a guide RNA (gRNA) targeting the integrated Cas9 transgene resulted in the loss of detectable Cas9 protein. Cas9 ablation enabled restoration of CUL4B expression and function following introduction of a CUL4B expression plasmid. These results provide proof of concept for a broadly applicable approach to studying protein function through rescue experiments.},
}

Humans
HEK293 Cells
*Gene Knockout Techniques/methods
*CRISPR-Cas Systems
Cullin Proteins/genetics/biosynthesis
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid42667462,
year = {2026},
author = {da Silveira Fonseca, ML and de Souza Júnior, LC and Dos SantosNascimento, F and de Souza Ramos, AP and Santana, WS and Mascarenhas, MS and Amorim, EP and Ferreira, CF},
title = {A banana susceptibility gene underlying fusarium wilt: validation as a target for disease resistance.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42667462},
issn = {1573-4978},
mesh = {*Musa/genetics/microbiology ; *Fusarium/pathogenicity ; *Plant Diseases/microbiology/genetics ; *Disease Resistance/genetics ; Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Plant Roots/microbiology/genetics ; Genes, Plant ; },
abstract = {BACKGROUND: Fusarium wilt, caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense, remains the principal constraint on banana production, particularly for the widely cultivated cultivar 'Prata-Ana' (AAB) in Brazil. Given the limited efficacy of conventional control strategies, susceptibility (S) genes have emerged as promising targets for developing resistant cultivars. This study investigated the expression of the banana DMR6 gene during the interaction with Foc. DMR6 was selected because it is a conserved plant susceptibility gene that negatively regulates salicylic acid-mediated immunity, making it a promising target for genome editing.

METHODS AND RESULTS: Banana plantlets were inoculated with Foc Subtropical Race 4 under controlled conditions. Temporal expression of the banana DMR6 gene was analyzed by RT-qPCR, and host defense responses were assessed by histochemical and microscopic analyses. DMR6 expression initially decreased and subsequently increased, reaching a 6.5-fold induction at 72 h post-inoculation relative to non-inoculated controls. This expression peak coincided with spore formation and advanced vascular colonization. Although infected roots exhibited callose deposition and phenolic compound accumulation, these defense responses were insufficient to restrict pathogen progression, resulting in severe disease symptoms and a disease severity index of 80% at 90 days after inoculation.

CONCLUSIONS: The findings indicate that banana DMR6 functions as a negative regulator of plant immunity and is closely associated with susceptibility to Fusarium wilt. These results provide a molecular basis for future functional validation and support DMR6 as a potential target for precise genome editing to develop resistant banana cultivars.},
}

*Musa/genetics/microbiology
*Fusarium/pathogenicity
*Plant Diseases/microbiology/genetics
*Disease Resistance/genetics
Plant Proteins/genetics/metabolism
Gene Expression Regulation, Plant
Plant Roots/microbiology/genetics
Genes, Plant

@article {pmid42667516,
year = {2026},
author = {Brancazio, S and Khalili, K and Jacobson, S and Kaminski, R},
title = {CRISPR-mediated excision of HTLV-1 reduces proviral loads in PBMCs from HAM/TSP patients.},
journal = {Journal of neurovirology},
volume = {32},
number = {5},
pages = {},
pmid = {42667516},
issn = {1538-2443},
support = {T32 MH079785/MH/NIMH NIH HHS/United States ; 161664-04400-02//School of Medicine/Center for Neurovirology and Gene Editing internal PI fund/ ; },
mesh = {Humans ; *Human T-lymphotropic virus 1/genetics/growth & development ; *Proviruses/genetics/growth & development ; Viral Load ; *Paraparesis, Tropical Spastic/virology/therapy/genetics ; *Leukocytes, Mononuclear/virology ; *CRISPR-Cas Systems ; Gene Products, tax/genetics/metabolism ; Basic-Leucine Zipper Transcription Factors/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; DNA, Viral/genetics/metabolism ; Genome, Viral ; Terminal Repeat Sequences/genetics ; Retroviridae Proteins ; },
abstract = {CRISPR technology is emerging as a promising therapeutic approach for eliminating chronic viral infections, such as herpesviruses and HIV. Here, for the first time, we demonstrate in vitro that CRISPR can be used to excise the HTLV-1 genome and reduce proviral loads in PBMCs from HAM/TSP (HTLV-1-associated myelopathy/tropical spastic paraparesis) patients. Single treatment with CRISPR-RNP (ribonucleoprotein) complexes composed of two gRNAs targeting the HTLV-1 env gene and 3'LTR sequences resulted in excision of a 2613 bp segment of the proviral genome, spanning tax and HBZ genes, without detectable off-target activity. Furthermore, CRISPR treatment led to over 50% reduction in proviral loads 5 days post-electroporation. Our data indicate that CRISPR-Cas9 gene editing can be used as a therapeutic strategy to eliminate HTLV-1 DNA from infected cells and may serve as a platform for curing HAM/TSP.},
}

Humans
*Human T-lymphotropic virus 1/genetics/growth & development
*Proviruses/genetics/growth & development
Viral Load
*Paraparesis, Tropical Spastic/virology/therapy/genetics
*Leukocytes, Mononuclear/virology
*CRISPR-Cas Systems
Gene Products, tax/genetics/metabolism
Basic-Leucine Zipper Transcription Factors/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
DNA, Viral/genetics/metabolism
Genome, Viral
Terminal Repeat Sequences/genetics
Retroviridae Proteins

@article {pmid42668207,
year = {2027},
author = {Casado, A and Wellner, SM and Quirós, A and Herrero-Fresno, A and Olsen, JE and Alvarez, MA and Ladero, V},
title = {CRISPR-Cas9 mediated adiA knockout in Hafnia paralvei: Implications for agmatine production and acid stress survival in a fermented dairy matrix.},
journal = {Food microbiology},
volume = {141},
number = {},
pages = {105270},
doi = {10.1016/j.fm.2026.105270},
pmid = {42668207},
issn = {1095-9998},
mesh = {*Agmatine/metabolism ; *CRISPR-Cas Systems ; *Carboxy-Lyases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Gene Knockout Techniques ; Fermentation ; *Enterobacteriaceae/genetics/metabolism/enzymology ; *Acids/metabolism ; Hydrogen-Ion Concentration ; Cheese/microbiology ; Fermented Foods/microbiology ; },
abstract = {Agmatine, the product of the decarboxylation of arginine, catalysed by arginine decarboxylase (ADC), is a bioactive compound that functions as a neuromodulator and co-transmitter and has gained increasing attention in recent years due to its therapeutic potential, particularly for its neuroprotective properties. Members of the genus Hafnia are the main agmatine producers in dairy products. In this regard, Hafnia is considered a beneficial microorganism due to its ability to enhance cheese organoleptic properties and its emerging probiotic potential, making it relevant for functional food development, specially agmatine-enriched dairy products. This study aimed to identify the genetic basis for agmatine production in Hafnia paralvei and to assess its role in bacterial fitness. Genomic analysis of the strain H. paralvei IPLA15029 revealed the presence of two genes encoding putative ADC enzymes, adiA and speA, however, organized slightly different than those in other enterobacteria. In some bacteria, ADC exists in two forms: one involved in polyamine biosynthesis, encoded by the constitutive speA gene, and another involved in acid stress resistance, encoded by the adiA gene, which is inducible under acidic conditions. In vivo experiments under controlled pH conditions showed that agmatine accumulation occurs exclusively under acidic conditions, which also stabilize the compound by preventing its catabolism to putrescine. Gene expression analysis revealed that adiA was transcribed as a monocistronic unit, and that in these conditions, adiA is the gene responsible for agmatine production. This was confirmed by generating an adiA knockout mutant after the implementation of the CRISPR-Cas9 system, marking the first successful application of this technology in the genus Hafnia. Moreover, the adiA knockout demonstrated that the encoded arginine decarboxylase is essential for survival under severe acid stress.},
}

*Agmatine/metabolism
*CRISPR-Cas Systems
*Carboxy-Lyases/genetics/metabolism
*Bacterial Proteins/genetics/metabolism
Gene Knockout Techniques
Fermentation
*Enterobacteriaceae/genetics/metabolism/enzymology
*Acids/metabolism
Hydrogen-Ion Concentration
Cheese/microbiology
Fermented Foods/microbiology

@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.},
}