@article {pmid42288725,
year = {2026},
author = {Coleman, S and Tye, J and Furniss, D and Sainsbury, R and Rastogi, A and Xi, X and Murnyak, B and Bell, J and Skeate, J and Wang, M and Webber, B and Moriarity, B and Clement, K},
title = {CRISPRessoSea: streamlined analysis and comparison of pooled amplicon CRISPR screens.},
journal = {BMC bioinformatics},
volume = {27},
number = {1},
pages = {},
pmid = {42288725},
issn = {1471-2105},
support = {T15LM007124/NH/NIH HHS/United States ; T32HL007062/NH/NIH HHS/United States ; R21CA237789, R21AI163731, P01CA254849, P50CA136393, U54CA268069, R01AI146009/NH/NIH HHS/United States ; R01AI146009, R01AI161017, P01CA254849, P50CA136393, U24OD026641, U54CA232561, P30CA077598, U54CA268069/NH/NIH HHS/United States ; R00HG011658/NH/NIH HHS/United States ; T15LM007124/NH/NIH HHS/United States ; T15LM007124/NH/NIH HHS/United States ; T32HL007062/NH/NIH HHS/United States ; R21CA237789, R21AI163731, P01CA254849, P50CA136393, U54CA268069, R01AI146009/NH/NIH HHS/United States ; R01AI146009, R01AI161017, P01CA254849, P50CA136393, U24OD026641, U54CA232561, P30CA077598, U54CA268069/NH/NIH HHS/United States ; R00HG011658/NH/NIH HHS/United States ; },
mesh = {*Software ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; High-Throughput Nucleotide Sequencing/methods ; },
abstract = {BACKGROUND: CRISPR genome editing enables precise modification of genomic targets but may also induce unintended edits at off-target sites with similar sequences. Pooled amplicon sequencing can assess on- and off-target editing across many samples, yet analyzing, aggregating, and visualizing results from multiple pooled experiments remains challenging. Tools to simplify and standardize these analyses are needed to provide reproducible and comparable interpretation of editing data.

RESULTS: We developed CRISPRessoSea, a software package that processes, compares, and visualizes genome editing rates from pooled amplicon sequencing experiments. The tool provides standardized workflows for analyzing editing across multiple targets and samples, supports both nuclease- and base-editing modalities, and generates clear, data-rich summaries suitable for downstream interpretation.

CONCLUSIONS: CRISPRessoSea facilitates reproducible, scalable analysis of CRISPR editing outcomes across diverse experimental designs, enabling more efficient and transparent assessment of genome editing specificity. The software is freely available at https://github.com/clementlab/CRISPRessoSea .},
}

*Software
*CRISPR-Cas Systems
*Gene Editing/methods
*Clustered Regularly Interspaced Short Palindromic Repeats
High-Throughput Nucleotide Sequencing/methods

@article {pmid42310064,
year = {2026},
author = {Davoodi, P and Atapour, M and Shahsavari, A and Kiani, R},
title = {Simulation of CRISPR/Cas9-mediated gene editing for the Vitellogenin gene in Apis mellifera.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42310064},
issn = {2045-2322},
support = {ص/3/9/22600, (~1000$ for one year).//University of Kurdistan/ ; },
mesh = {Animals ; *Vitellogenins/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Bees/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Computer Simulation ; Exons ; },
abstract = {CRISPR/Cas9 genome editing provides a powerful framework for interrogating gene function in Apis mellifera. Yet, empirical application remains challenging due to biological constraints, including haplodiploid genetics, narrow embryonic injection window, and the social rearing requirements that complicate functional validation. These constraints necessitate in silico pre-screening to maximize editing success before resource-intensive wet-lab implementation. Within the omnigenic framework, which distinguishes core regulatory genes from peripheral loci buffered by network effects, vitellogenin (Vg) represents an optimal target which is ancestrally dedicated to yolk provisioning; it has been co-opted to orchestrate diverse non-reproductive functions including longevity, stress resistance, immunity, and social behavior. We developed a computational pipeline to design a list of 57 and 56 candidate guide RNAs (gRNA) for targeted Vg knockout, evaluating candidate sites in both functional exons 2 and 3 based on structural accessibility and frameshift efficiency. Comparative analysis revealed complementary strengths in two top-best candidates from initial target pool of predicted gRNAs. The gRNA targeting exon 2 exhibits weaker secondary structure (ΔG = -0.25 kcal/mol versus -2.10 kcal/mol for exon 3), aligning with empirical evidence that sites with ΔG > -1.0 kcal/mol achieve 2-5 × higher Cas9 binding efficiency. This site yielded moderate frameshift frequency (77.8%; 61.9 percentile). Conversely, the predicted editing outcome for the gRNA targeting exon 3, despite stronger structural constraints, demonstrated superior functional disruption metrics demonstrating very high frameshift frequency (88.3%; 95.2 percentile), high in silico editing precision, minimal microhomology-mediated repair bias, and reproducible outcomes wherein nearly all predicted indels disrupt the coding sequence. Protein structure and domain analyses further predict that frameshift edits will generate a truncated protein missing all downstream functional domains. We recommend parallel empirical validation of both exon 2 and exon 3 targets to resolve the trade-off between structural accessibility (favoring higher editing rates) and frameshift efficacy (favoring complete loss-of-function). This dual-target strategy accommodates uncertainty in in vivo performance while maximizing the probability of generating informative phenotypes. Our in silico framework enables rational CRISPR design in non-model organisms by computationally balancing biophysical accessibility with functional impact, accelerating functional genomics in species where empirical optimization faces substantial biological constraints.},
}

Animals
*Vitellogenins/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
Bees/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Computer Simulation
Exons

@article {pmid42485833,
year = {2026},
author = {J, MA and K, AP and A, AE},
title = {Cold-chain-free CRISPR diagnostics enabled by magnesium-tannic acid nanoencapsulation of the complete detection system.},
journal = {Colloids and surfaces. B, Biointerfaces},
volume = {268},
number = {Pt 1},
pages = {115989},
doi = {10.1016/j.colsurfb.2026.115989},
pmid = {42485833},
issn = {1873-4367},
mesh = {*Magnesium/chemistry ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; DNA, Single-Stranded/chemistry/genetics ; *Metal Nanoparticles/chemistry ; Polyphenols ; },
abstract = {Cold-chain dependency remains the major barrier to the widespread deployment of CRISPR-based diagnostics, particularly in resource-limited settings, where the thermal instability of protein and nucleic acid reagents necessitates refrigerated storage and transport from manufacture to point of use. Here we report a one-step aqueous nanoencapsulation strategy that overcomes this limitation using magnesium-tannic acid metal-phenolic nanoparticles (Mg[2][+]-TA MPNs). The platform simultaneously co-encapsulates all functional components of a CRISPR-LbCas12a detection system, including the nuclease, guide RNA, and ssDNA reporter, within a single protective matrix. Magnesium ions serve as a structural coordinator of the metal-phenolic nanoparticles while maintaining biochemical compatibility with the released CRISPR system. Physicochemical characterization confirmed successful particle assembly and efficient cargo incorporation. Functional studies demonstrated that encapsulated CRISPR reagents retained greater than 70% of diagnostic activity after 8 days at 50 °C, whereas non-encapsulated controls were completely inactivated within 24 h under identical conditions. Released formulations also remained fully compatible with both fluorescence- and lateral flow-based detection formats. This approach requires no lyophilization, no specialized equipment, and no cold chain at any stage of production or storage, thereby offering a scalable and readily deployable route to thermally stable molecular diagnostics.},
}

*Magnesium/chemistry
*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics/chemistry
DNA, Single-Stranded/chemistry/genetics
*Metal Nanoparticles/chemistry
Polyphenols

@article {pmid42607405,
year = {2026},
author = {Liu, F and Yang, L and Zhang, J and Wang, Y and Xu, F and Ai, Y and Jiang, X},
title = {High-entropy nanozymes integrated with CRISPR/Cas12a cascade amplification for highly sensitive point-of-care detection of cardiac troponin I.},
journal = {Biosensors & bioelectronics},
volume = {313},
number = {},
pages = {119129},
doi = {10.1016/j.bios.2026.119129},
pmid = {42607405},
issn = {1873-4235},
mesh = {*Troponin I/blood/isolation & purification ; *Biosensing Techniques/methods ; Electrochemical Techniques/methods ; *Myocardial Infarction/diagnosis/blood ; Point-of-Care Systems ; CRISPR-Cas Systems ; Humans ; Limit of Detection ; Nucleic Acid Amplification Techniques/methods ; Reproducibility of Results ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Myocardial infarction necessitates rapid and ultrasensitive point-of-care detection of cardiac troponin I (cTnI). In this work, we develop a HEAzyme-enabled interfacial electrocatalytic transduction strategy integrated with CHA-CRISPR/Cas12a amplification framework for ultrasensitive electrochemical detection of cTnI. Capitalizing on the cocktail effect and carbon-shell confinement, HEAzyme amplifies the electrochemical response by catalyzing the redox reaction of surface-confined methylene blue. Target recognition initiates CHA, generating abundant DNA activators for Cas12a trans-cleavage. The synergistic integration of molecular cascade amplification and interfacial electrocatalysis enables a limit of detection of 0.21 fg/mL across a broad linear range from 1 fg/mL to 100 pg/mL. It exhibits good selectivity, maintains good stability within 7 days (RSD = 3.9%), and shows good batch-to-batch reproducibility in different batches (RSD = 3.44 and 5.8% for intra and inter-batch, respectively). This work establishes an effective strategy for integrating CRISPR-based molecular amplification with HEAzyme-enabled interfacial electrocatalytic transduction, providing a promising electrochemical platform for point-of-care diagnosis of myocardial infarction.},
}

*Troponin I/blood/isolation & purification
*Biosensing Techniques/methods
Electrochemical Techniques/methods
*Myocardial Infarction/diagnosis/blood
Point-of-Care Systems
CRISPR-Cas Systems
Humans
Limit of Detection
Nucleic Acid Amplification Techniques/methods
Reproducibility of Results
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid42612455,
year = {2026},
author = {Ke, J and Zhang, H and Chen, S and Ma, M and Tang, X and Wei, J and Deng, J and Zhai, J and Luan, T},
title = {Engineering bubble structures as Cas12a activators for highly sensitive monitoring of WRN helicase function.},
journal = {Biosensors & bioelectronics},
volume = {313},
number = {},
pages = {119123},
doi = {10.1016/j.bios.2026.119123},
pmid = {42612455},
issn = {1873-4235},
mesh = {*Werner Syndrome Helicase/metabolism/genetics/chemistry ; Humans ; *Biosensing Techniques/methods ; *CRISPR-Associated Proteins/chemistry/metabolism/genetics ; CRISPR-Cas Systems ; DNA/chemistry/genetics ; *Bacterial Proteins/chemistry/metabolism/genetics ; *Endodeoxyribonucleases/chemistry/metabolism/genetics ; R-Loop Structures ; RNA/chemistry/genetics ; DNA Replication ; },
abstract = {The Werner syndrome helicase (WRN) is a critical synthetic lethal target in microsatellite instability cancers, essential for resolving complex genomic structures like replication bubbles and R-loops. However, strategies to simultaneously discriminate WRN activity on DNA versus DNA-RNA substrates in living cells are lacking. Here, we developed a structure-specific CRISPR/Cas12a biosensing strategy to visualize WRN functional activity by engineering bubble-structure probes. These probes were rationally designed to structurally mimic DNA replication bubbles and R-loop associated DNA-RNA hybrids. Upon specific unwinding by WRN, the probes release a sequestered activator strand that triggers Cas12a trans-cleavage, effectively converting the unwinding event into an amplified fluorescent signal. This assay achieves low picomolar sensitivity (LODs: 5.6-6.0 pM) and exceptional selectivity against homologous RecQ helicases. Uniquely, this strategy enables the parallel quantification of WRN activity on both substrate types, providing insights into distinct WRN-mediated pathways for resolving genomic stress. We further demonstrated the strategy's utility by visualizing endogenous WRN dynamics in living cells and profiling the efficacy of small-molecule inhibitors. This work offers a powerful molecular toolkit for dissecting WRN biology and facilitating high-throughput drug screening in targeted cancer therapy.},
}

*Werner Syndrome Helicase/metabolism/genetics/chemistry
Humans
*Biosensing Techniques/methods
*CRISPR-Associated Proteins/chemistry/metabolism/genetics
CRISPR-Cas Systems
DNA/chemistry/genetics
*Bacterial Proteins/chemistry/metabolism/genetics
*Endodeoxyribonucleases/chemistry/metabolism/genetics
R-Loop Structures
RNA/chemistry/genetics
DNA Replication

@article {pmid42641278,
year = {2026},
author = {Fu, R and Zhu, C and Hou, J and Wang, Z and Xianyu, Y},
title = {Click-chemistry-mediated modulation of CRISPR-Cas12a activity through activator modification.},
journal = {Biosensors & bioelectronics},
volume = {313},
number = {},
pages = {119144},
doi = {10.1016/j.bios.2026.119144},
pmid = {42641278},
issn = {1873-4235},
mesh = {*Click Chemistry/methods ; *CRISPR-Cas Systems/genetics ; Azides/chemistry ; DNA/chemistry/genetics ; *CRISPR-Associated Proteins/chemistry/genetics/metabolism ; *Biosensing Techniques ; Cycloaddition Reaction ; Alkynes/chemistry ; Molecular Docking Simulation ; Cyclooctanes/chemistry ; *Bacterial Proteins/chemistry/genetics ; *Endodeoxyribonucleases/chemistry/genetics ; },
abstract = {Chemical modification strategies offer a promising route for spatiotemporal regulation of CRISPR-Cas12a activity in molecular diagnostics. However, existing methods involve CRISPR RNA with photolabile groups that suffer from complexity and RNA instability. To address these limitations, we report a simple and robust strategy using dibenzocyclooctyne (DBCO)-mediated click chemistry to modulate CRISPR-Cas12a activity. The copper-free strain-promoted azide-alkyne cycloaddition reaction enables CRISPR-Cas12a modulation with low toxicity, biocompatibility, and high selectivity. Utilizing azide-modified non-target DNA strand sequences at different locations to react with DBCO, we show that DBCO-modified activators can regulate CRISPR-Cas12a cleavage in three distinct states: maintain, enhance, and suppress. Mechanistic studies through cleavage kinetics and molecular docking reveal that the regulatory outcome depends on the modification position, protospacer adjacent motif composition, and DBCO concentration. We further employ asymmetric polymerase chain reaction to generate azide-modified DNA for click-chemistry-mediated modulation of CRISPR-Cas12a. This strategy could be a promising tool for regulating CRISPR-Cas12a activity in molecular diagnostics.},
}

*Click Chemistry/methods
*CRISPR-Cas Systems/genetics
Azides/chemistry
DNA/chemistry/genetics
*CRISPR-Associated Proteins/chemistry/genetics/metabolism
*Biosensing Techniques
Cycloaddition Reaction
Alkynes/chemistry
Molecular Docking Simulation
Cyclooctanes/chemistry
*Bacterial Proteins/chemistry/genetics
*Endodeoxyribonucleases/chemistry/genetics

@article {pmid42641282,
year = {2026},
author = {Wang, Y and Xu, B and Zeng, Z and Wang, H and Shi, L and Liu, W and Chen, Y and Deng, X and Chen, J and Chen, JX},
title = {Cascade-coupled colorimetric-fluorescent dual-signal detection of EGFR-positive extracellular vesicles via bifunctional MOF@Pt nanozyme integrated with a CHA-CRISPR system.},
journal = {Biosensors & bioelectronics},
volume = {313},
number = {},
pages = {119149},
doi = {10.1016/j.bios.2026.119149},
pmid = {42641282},
issn = {1873-4235},
mesh = {Humans ; *Biosensing Techniques/methods ; ErbB Receptors/genetics ; Colorimetry/methods ; *Extracellular Vesicles/chemistry ; Platinum/chemistry ; *Glioma/diagnosis/genetics/blood ; CRISPR-Cas Systems/genetics ; MicroRNAs/genetics ; Metal-Organic Frameworks/chemistry ; Aptamers, Nucleotide/chemistry ; Limit of Detection ; Metal Nanoparticles/chemistry ; Phthalic Acids ; },
abstract = {Extracellular vesicle (EV)-based liquid biopsy holds great promise for glioma diagnosis, but its clinical translation remains hindered by inefficient isolation of disease-relevant EV subpopulations and insufficiently integrated signal validation. Herein, we develop a platform that integrates a bifunctional nanozyme system (phosphatase-like MOF and peroxidase-like Pt) with a CHA-CRISPR/Cas12a cascade, achieving selective isolation and ultrasensitive detection of EGFR-positive glioma-derived EVs. Defective UiO-66-NH2 loaded with Pt nanoparticles and functionalized with EGFR aptamers (UiO@Pt@Apt) is immobilized on Hook strand-modified glass 96-well plates via Apt-Hook hybridization. Upon introduction of EGFR-positive EVs, they specifically bind to aptamers on UiO@Pt@Apt and induce the release of UiO@Pt@Apt-EV complexes through perturbation and destabilization of the Apt-Hook interface. After EV lysis, EV-derived miRNA-21 activates the CHA-CRISPR/Cas12a cascade to generate a fluorescent signal, while the nucleotide fragments produced by Cas12a trans-cleavage are proposed to be hydrolyzed by phosphatase-like defective UiO-66-NH2 to generate PO4[3-]. The PO4[3-] may contribute to Pt-mediated TMB oxidation, thereby supporting colorimetric signal amplification. The platform achieved a colorimetric EV detection limit of 427 particles/μL after coupling with the CHA-CRISPR system, representing a 6.9-fold improvement over the UiO@Pt@Apt system alone, and enabled a miRNA-21 detection limit of 87.0 fM. In plasma samples from 25 glioma patients and 20 healthy donors, the combined readout achieved an AUC of 0.968, showing numerically better discriminatory performance than either single readout, although the improvement was not statistically significant by DeLong analysis. This work provides a promising strategy for EV-based glioma liquid biopsy and offers a potentially adaptable framework for cascade-coupled dual-signal biosensing.},
}

Humans
*Biosensing Techniques/methods
ErbB Receptors/genetics
Colorimetry/methods
*Extracellular Vesicles/chemistry
Platinum/chemistry
*Glioma/diagnosis/genetics/blood
CRISPR-Cas Systems/genetics
MicroRNAs/genetics
Metal-Organic Frameworks/chemistry
Aptamers, Nucleotide/chemistry
Limit of Detection
Metal Nanoparticles/chemistry
Phthalic Acids

@article {pmid42648078,
year = {2026},
author = {Ye, T and Xue, M and Zhou, B and Kang, S and Chen, Z and Tang, Z and Yang, S and Zhao, Q and Yuan, M and Yu, J and Cao, H and Hao, L and Wu, X and Yin, F and Xu, F},
title = {Biphasic spatiotemporal regulation of Cas12a substrate cleavage enables one-pot autocatalytic CRISPR biosensing of non-nucleic-acid targets.},
journal = {Biosensors & bioelectronics},
volume = {313},
number = {},
pages = {119152},
doi = {10.1016/j.bios.2026.119152},
pmid = {42648078},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Associated Proteins/chemistry/genetics ; *CRISPR-Cas Systems/genetics ; *Endodeoxyribonucleases/chemistry/genetics ; DNA, Single-Stranded/chemistry/genetics ; *Bacterial Proteins/chemistry/genetics ; Limit of Detection ; Aptamers, Nucleotide/chemistry ; Catalysis ; },
abstract = {One-pot autocatalytic CRISPR biosensing offers a promising route for signal amplification without nucleic acid pre-amplification, but its efficiency is limited by an intrinsic readout-amplification conflict: the ssDNA reporter required for signal output competes with the autocatalytic mediator required for feedback amplification. Here, we report a biphasic spatiotemporal regulation strategy (BS-Cas12a system) to resolve this substrate competition for one-pot autocatalytic CRISPR biosensing of non-nucleic-acid targets. Mechanistic studies revealed that Cas12a preferentially cleaved the ssDNA reporter over the autocatalytic circular mediator, leading to insufficient mediator linearization and impaired autocatalytic amplification. A glycerol/water biphasic system was therefore constructed to spatially delay reporter access to activated Cas12a, allowing preferential cleavage of the autocatalytic mediator and subsequent generation of additional Cas12a activators. This biphasic system achieved a 3-fold increase in the autocatalytic amplification efficiency. By integrating an aptamer-mediated target-to-activator conversion module, non-nucleic-acid recognition was programmably converted into Cas12a activation. Using sulfadimethoxine as a model target, the platform achieved a detection limit of 65 pM and showed good recoveries in fish samples. The system was further extended to Cd[2+] and thrombin detection with limits of detection of 0.65 nM and 11.63 pM, respectively. Moreover, by replacing the fluorescent reporter with a FAM/biotin-labeled reporter, a lateral-flow readout was achieved. This work provides a kinetic-regulation strategy for modular one-pot Cir DNA autocatalytic biosensing with an independent and exchangeable reporter and expands CRISPR-based detection toward diverse non-nucleic-acid targets.},
}

*Biosensing Techniques/methods
*CRISPR-Associated Proteins/chemistry/genetics
*CRISPR-Cas Systems/genetics
*Endodeoxyribonucleases/chemistry/genetics
DNA, Single-Stranded/chemistry/genetics
*Bacterial Proteins/chemistry/genetics
Limit of Detection
Aptamers, Nucleotide/chemistry
Catalysis

@article {pmid42669262,
year = {2026},
author = {Han, J and Song, Y and Ko, U and Son, SU and Lim, EK and Kim, E},
title = {One-pot dual-toehold RCA-Cas12a biosensor driven by a preassembled three-arm toehold-gated DNA template for sequence-selective miRNA liquid biopsy.},
journal = {Biosensors & bioelectronics},
volume = {313},
number = {},
pages = {119170},
doi = {10.1016/j.bios.2026.119170},
pmid = {42669262},
issn = {1873-4235},
mesh = {*MicroRNAs/blood/isolation & purification/genetics ; *Biosensing Techniques/methods ; Humans ; Nucleic Acid Amplification Techniques/methods ; Liquid Biopsy/methods ; CRISPR-Cas Systems/genetics ; DNA/chemistry/genetics ; Breast Neoplasms/blood/genetics/diagnosis ; Limit of Detection ; Female ; *Endodeoxyribonucleases/chemistry/genetics ; *Bacterial Proteins/chemistry/genetics ; CRISPR-Associated Proteins ; },
abstract = {MicroRNAs (miRNAs) in blood are promising liquid biopsy biomarkers, yet their short length, low abundance, and high intra-family homology hinder sensitive and specific detection. Combining rolling circle amplification (RCA) with CRISPR-Cas12a enables isothermal detection, but existing methods typically depend on auxiliary enzymes or in-assay ligation and rarely encode sequence discrimination within the template itself. Here, we report a one-pot dual-toehold RCA (dtRCA)-Cas12a biosensor driven by a preassembled three-arm toehold-gated (3TG) DNA template for ultrasensitive and selective miRNA detection. The 3TG template adopts a three-arm dumbbell conformation, eliminating the need for a ligase during the assay, and presents two target-complementary toehold domains with a Cas12a-recognition sequence. Target binding triggers strand displacement, initiating dtRCA via a single polymerase. The resulting amplicons activate Cas12a trans-cleavage for fluorescence or lateral flow assay (LFA) readouts. Crucially, a single-base mismatch within the toehold suppressed amplification, whereas a topology-matched circular template lacking the toehold gate failed to distinguish the target, demonstrating that selectivity arises from the template structure. The one-pot dtRCA-Cas12a system achieved attomolar sensitivity, detecting miR-21, miR-375, and let-7a at 2.5, 114.9, and 8.0 aM, respectively. The paper-based LFA maintained femtomolar sensitivity and enabled an instrument-light readout. In plasma, this platform discriminated breast cancer patients (n = 17) from healthy donors (n = 10) with AUC values of 0.97-0.98. Three-marker classification demonstrated robust performance in leave-one-out cross-validation and correctly classified 30 samples in an independent validation cohort, showing performance comparable to RT-qPCR. By embedding selectivity into a preassembled template, this 3TG-driven dtRCA-Cas12a platform provides a highly sensitive and specific strategy for multi-marker miRNA analysis with simplified readout.},
}

*MicroRNAs/blood/isolation & purification/genetics
*Biosensing Techniques/methods
Humans
Nucleic Acid Amplification Techniques/methods
Liquid Biopsy/methods
CRISPR-Cas Systems/genetics
DNA/chemistry/genetics
Breast Neoplasms/blood/genetics/diagnosis
Limit of Detection
Female
*Endodeoxyribonucleases/chemistry/genetics
*Bacterial Proteins/chemistry/genetics
CRISPR-Associated Proteins

@article {pmid42680688,
year = {2026},
author = {Xhaferri, N and Biswas, S and Davies, B and Lindner, M},
title = {Kcnv2 E151X Mouse Captures Hallmarks of KCNV2-Associated Retinal Dystrophy.},
journal = {Clinical & experimental ophthalmology},
volume = {},
number = {},
pages = {},
doi = {10.1111/ceo.70163},
pmid = {42680688},
issn = {1442-9071},
support = {LI 2846/5-1//Deutsche Forschungsgemeinschaft/ ; LI 2846/6-1//Deutsche Forschungsgemeinschaft/ ; },
abstract = {BACKGROUND: KCNV2-associated retinopathy is a rare inherited retinal dystrophy caused by variants in the KCNV2 gene, leading to disrupted photoreceptor behaviour and progressive deterioration of vision. Patients have characteristic electroretinography abnormalities, including reduced cone response, delayed and reduced rod response to low light flashes and paradoxically large rod-driven response to bright flashes of light. To model this condition, we have generated a Kcnv2 E151X mouse line and assessed its structural and functional retinal features.

METHODS: We have employed CRISPR/Cas 9 gene editing technology to generate a mouse line with an early stop mutation in position E151-orthologous to the commonly encountered E143X mutation in humans-and performed a combination of immunohistochemistry and Western blot to confirm the absence of the full-length KCNV2-encoded protein, Kv8.2. Next, to assess how closely it models the human disease, we have characterised the KCNV2 mutant mouse line at histological and functional levels, via immunohistochemistry and electroretinography experiments, respectively.

RESULTS: Kcnv2 mutant mice showed markedly reduced photopic responses and reproduced the supernormal rod phenotype described in affected individuals. In the morphological context, mutant retinas demonstrated strong glial fibrillary acidic protein upregulation together with reduced cone arrestin positive cell counts and photoreceptor layers, indicating photoreceptor loss.

CONCLUSIONS: The Kcnv2 mutant mouse line replicates key functional and structural hallmarks of KCNV2-associated retinopathy. This model provides a relevant platform for mechanistic studies and preclinical evaluation of gene-based or pharmacological therapies targeting cone and rod photoreceptor dysfunction.},
}

@article {pmid42680732,
year = {2026},
author = {Galal, S and Chaltel Lima, L and Wang, N and Moury, C and Yan, G and Dai, M and Ali, S and Lebrun, JJ},
title = {In vivo CRISPR screening identifies metastasis suppressors in triple-negative breast cancer.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42680732},
issn = {2041-1723},
mesh = {Humans ; Animals ; Female ; *Triple Negative Breast Neoplasms/genetics/pathology ; Cell Line, Tumor ; Mice ; Neoplasm Metastasis/genetics ; CRISPR-Cas Systems ; Gene Expression Regulation, Neoplastic ; Epithelial-Mesenchymal Transition/genetics ; Cell Movement/genetics ; Neovascularization, Pathologic/genetics ; *Genes, Tumor Suppressor ; Neoplasm Invasiveness/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Metastatic cancer remains the leading cause of cancer-related mortality, yet tumor cell-intrinsic mechanisms restraining metastatic dissemination remain incompletely defined. Here, we perform an unbiased in vivo genome-wide CRISPR/Cas9 loss-of-function screen in a breast cancer xenograft model to identify regulators of metastatic progression. This approach uncovers clinically relevant metastasis suppressor genes (MSGs), including VPS45, CMTR2, RBSN, and NF2, whose loss enhances lung colonization. Functional validation demonstrates that depletion of these genes promotes epithelial-to-mesenchymal transition, migration, invasion, intravasation, and angiogenesis, whereas CRISPR-mediated activation suppresses metastatic spread. Integration with patient datasets reveals reduced expression in tumors and associations with advanced disease, with higher expression trending toward improved outcomes. Notably, CMTR2 loss induces vascular remodeling and intratumoral heterogeneity, supporting a role in tumor-vascular interactions. Collectively, this study identifies a network of MSGs that constrain tumor dissemination and highlights the power of in vivo CRISPR functional genomics to uncover regulators of metastatic disease.},
}

Humans
Animals
Female
*Triple Negative Breast Neoplasms/genetics/pathology
Cell Line, Tumor
Mice
Neoplasm Metastasis/genetics
CRISPR-Cas Systems
Gene Expression Regulation, Neoplastic
Epithelial-Mesenchymal Transition/genetics
Cell Movement/genetics
Neovascularization, Pathologic/genetics
*Genes, Tumor Suppressor
Neoplasm Invasiveness/genetics
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42681040,
year = {2026},
author = {Tiefenbacher, J and Sandrock, B},
title = {Generation of Gene Knock-Out Mutants in Ustilago maydis Using Cas9hf Nuclease.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3050},
number = {},
pages = {147-161},
pmid = {42681040},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Ustilago/genetics ; *Gene Knockout Techniques/methods ; Mutation ; Basidiomycota ; },
abstract = {Gene disruption of nonessential genes became more convenient with the adaptation of the CRISPR-Cas9 system for Ustilago maydis by the group of Regine Kahmann (MPI Marburg, Germany) in 2016. In our group, we have developed the system further to create defined marker-free gene deletions. Therefore, we used the CRISPR-Cas9 system together with oligonucleotides composed of 40 nucleotides of upstream and downstream flanking regions. Here, we describe the entire way from the decision, which gene of interest should be deleted, to a marker-free U. maydis mutant strain.},
}

*CRISPR-Cas Systems
*Ustilago/genetics
*Gene Knockout Techniques/methods
Mutation
Basidiomycota

@article {pmid42681148,
year = {2026},
author = {Bao, X and Bornert, O and Nyström, A},
title = {Targeting, Mutagenesis, and Functional Testing Strategies for Large Collagens.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3022},
number = {},
pages = {3-20},
pmid = {42681148},
issn = {1940-6029},
mesh = {Humans ; *Mutagenesis ; *Collagen Type VII/genetics/metabolism/chemistry ; CRISPR-Cas Systems ; Animals ; *Protein Engineering/methods ; *Collagen/genetics/metabolism ; *Gene Targeting/methods ; },
abstract = {Collagens comprise a homogeneous family of cell-surface or extracellular proteins. Many of them are fundamental for health, and consequently, their deficiency or dysregulation occurs in a wide range of diseases, from tissue fragility to neoplastic and fibrosing diseases, to name a few. With the advent of new omics approaches, an increasing number of variants of specific collagens have been discovered in various conditions. Some of these variants lead to a loss of expression, highlighting the need to assess the functions and loss of specific collagens in such conditions, while other variants may involve changes in amino acids of unknown consequences. Thus, with the advent of newer omics technologies, there is an increasing need to engineer specific collagens and their encoding genes. However, for larger collagens, this can be challenging because of their size and repetitive structure. Using collagen VII as an example of a large collagen, we will describe strategies for protein engineering, functional analysis, and collagen gene targeting in cells that naturally express collagens.},
}

Humans
*Mutagenesis
*Collagen Type VII/genetics/metabolism/chemistry
CRISPR-Cas Systems
Animals
*Protein Engineering/methods
*Collagen/genetics/metabolism
*Gene Targeting/methods

@article {pmid42681174,
year = {2026},
author = {Su-Tobon, Q and Niu, J},
title = {CRISPR-Hybrid: Intracellular Selection of CRISPR-Associated Aptamers in Bacteria.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3032},
number = {},
pages = {25-45},
pmid = {42681174},
issn = {1940-6029},
mesh = {*Aptamers, Nucleotide/genetics ; *CRISPR-Cas Systems ; Flow Cytometry ; *SELEX Aptamer Technique/methods ; Gene Library ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Bacteria/genetics ; },
abstract = {CRISPR technologies have evolved from nuclease-based genome editing to programmable systems for transcriptional and epigenetic regulations. Emerging CRISPR systems expand editing versatility by incorporating CRISPR-associated aptamers (CAPs) into single-guide RNAs (sgRNAs), enabling recruitment of RNA-binding proteins (RBPs) fused to diverse effectors. However, the limited availability of orthogonal aptamer-RBP pairs has hindered broad application, as conventional SELEX-based aptamer discovery is time-intensive and often fails to yield aptamers functional in cells. We developed the CRISPR-Hybrid platform, an intracellular selection method that directly evolves CAPs within bacterial cells. This system links aptamer-RBP interactions to a fluorescent reporter readout, allowing fluorescence-activated cell sorting (FACS) to enrich functional variants from libraries exceeding 10[[7]] sequences. Each selection round can be completed in 2 days, enabling rapid enrichment of aptamers that retain activity in both bacterial and mammalian contexts. This protocol details construction of randomized DNA libraries, preparation of host cells, execution of intracellular selection and FACS enrichment, and recovery of aptamer sequences for downstream analysis. By providing a fast, in-cell, and physiologically relevant approach to aptamer discovery, CRISPR-Hybrid expands the repertoire of CAPs available for modular and multiplexed CRISPR editing.},
}

*Aptamers, Nucleotide/genetics
*CRISPR-Cas Systems
Flow Cytometry
*SELEX Aptamer Technique/methods
Gene Library
RNA, Guide, CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Clustered Regularly Interspaced Short Palindromic Repeats
*Bacteria/genetics

@article {pmid42681177,
year = {2026},
author = {Li, M and Chen, C and Tao, P},
title = {CRISPR-Engineered Bacteriophage T4 for Foot-and-Mouth Disease Nanoparticle Vaccine Development.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3032},
number = {},
pages = {87-100},
pmid = {42681177},
issn = {1940-6029},
mesh = {*Bacteriophage T4/genetics/immunology ; *Foot-and-Mouth Disease Virus/immunology/genetics ; Animals ; *Foot-and-Mouth Disease/prevention & control/immunology/virology ; *Vaccine Development/methods ; Epitopes, T-Lymphocyte/immunology/genetics ; *CRISPR-Cas Systems ; Epitopes, B-Lymphocyte/immunology/genetics ; *Viral Vaccines/immunology/genetics ; Nanovaccines ; Nanoparticles/chemistry ; Capsid Proteins/genetics/immunology ; Protein Subunit Vaccines ; },
abstract = {Peptide-based vaccines offer a safer alternative to inactivated vaccines. However, the immunogenicity of the peptides is usually poor, and therefore, adjuvants or delivery systems are required. Bacteriophage T4, with its intrinsic immunostimulatory properties, provides a promising platform for antigen display. Here, we used CRISPR-Cas genome editing to insert a gene encoding a foot-and-mouth disease virus (FMDV) B-cell epitope (VP1130-158) into the C-terminus of the soc gene in the T4 genome. The T4 phage self-assembly system enables the display of epitopes on the capsid surface in vivo, generating VP1130-158-T4 virus-like particles. To further enhance immune activation, CD4[[+]] T-cell epitopes FMDV 3A21-35 or tetanus toxoid P2830-844 were fused downstream of the VP1 epitope. These recombinant T4 phages provide proof-of-concept for the development of safe, epitope-based FMDV vaccines.},
}

*Bacteriophage T4/genetics/immunology
*Foot-and-Mouth Disease Virus/immunology/genetics
Animals
*Foot-and-Mouth Disease/prevention & control/immunology/virology
*Vaccine Development/methods
Epitopes, T-Lymphocyte/immunology/genetics
*CRISPR-Cas Systems
Epitopes, B-Lymphocyte/immunology/genetics
*Viral Vaccines/immunology/genetics
Nanovaccines
Nanoparticles/chemistry
Capsid Proteins/genetics/immunology
Protein Subunit Vaccines

@article {pmid42681180,
year = {2026},
author = {Wang, Y and Li, X and Huang, Y and Zhong, M and Yang, H},
title = {CRISPR-Cpf1-Mediated T4 Phage Genome Editing for One-Step In Vivo Display of Heterologous Protein.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3032},
number = {},
pages = {135-145},
pmid = {42681180},
issn = {1940-6029},
mesh = {*Bacteriophage T4/genetics ; *Capsid Proteins/genetics/metabolism ; *Gene Editing/methods ; Escherichia coli/genetics ; *Genome, Viral ; *CRISPR-Cas Systems ; Recombinant Proteins/genetics ; },
abstract = {The T4 phage is a robust vector for high-density heterologous protein display. It leverages two non-essential outer capsid proteins, i.e., Soc (~870 copies) and Hoc (~155 copies). These two proteins enable the efficient display of target proteins on the capsid of T4. Here, we detail the workflow for one-step in vivo display of a heterologous protein. Specifically, this method utilizes CRISPR-Cpf1-mediated gene editing technology to insert the sequence of interest (using mCherry as an example) downstream of the Soc encoding gene within the T4 phage genome, resulting in a Soc-fused recombinant protein. This engineering method allows for endogenous expression of the Soc-mCherry recombinant protein within Escherichia coli cells during phage replication, after which the recombinant protein spontaneously assembles onto the capsid of the engineered phage. By providing a universal framework suitable for in vivo display, this approach empowers researchers to readily construct tailored T4 nanoparticles for diverse biotechnological applications.},
}

*Bacteriophage T4/genetics
*Capsid Proteins/genetics/metabolism
*Gene Editing/methods
Escherichia coli/genetics
*Genome, Viral
*CRISPR-Cas Systems
Recombinant Proteins/genetics

@article {pmid42681181,
year = {2026},
author = {Mahdavi-Amiri, Y and Kim, SB},
title = {Methods for Applying Prime Editing and Inverse Prime Editing in Mammalian Cell Culture.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3032},
number = {},
pages = {147-161},
pmid = {42681181},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; Animals ; Humans ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; Point Mutation ; Flow Cytometry ; Cell Culture Techniques/methods ; Moloney murine leukemia virus/genetics/enzymology ; High-Throughput Nucleotide Sequencing ; },
abstract = {Prime editing (PE) is a powerful method for introducing point mutations into the genomes of living organisms. PE utilizes a Cas9 nickase fused to an engineered Moloney Murine Leukemia Virus reverse transcriptase (MLV-RT), paired with an extended guide RNA known as pegRNA, which contains a primer binding site (PBS) and a reverse transcriptase template (RTT) complementary to the non-target strand DNA. Recently described inverse prime editing (iPE) also employs reverse transcriptase and pegRNAs; however, it utilizes an RNA template complementary to the target strand, resulting in the polymerization of target-strand DNA in the opposite direction. In this work, we provide a practical protocol for using either PE or iPE to introduce point mutation(s) or small-to-medium sized insertions and deletions in cell culture, and demonstrate how to assess editing efficiency via flow cytometry and next-generation sequencing. We include recommendations for prime editor selection and straightforward guidelines for pegRNA design, intended for researchers unfamiliar with genome editing technologies.},
}

*Gene Editing/methods
Animals
Humans
*CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
Point Mutation
Flow Cytometry
Cell Culture Techniques/methods
Moloney murine leukemia virus/genetics/enzymology
High-Throughput Nucleotide Sequencing

@article {pmid42681182,
year = {2026},
author = {Golla, DA and Daniel, TC and Haugh, L and Gao, X},
title = {Constructing Drive-and-Process (DAP) CRISPR Guide RNA Arrays for Multiplexed Base- and Prime-Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3032},
number = {},
pages = {163-180},
pmid = {42681182},
issn = {1940-6029},
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; HEK293 Cells ; *CRISPR-Cas Systems ; RNA, Transfer/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Advancements in base- and prime editing technologies in recent years have offered researchers a plethora of options for introducing precise insertions, deletions, or substitutions into targeted genomic loci. These precision editors' applications have rapidly expanded to the modeling and treatment of polygenic diseases, as well as into the growing field of functional genomics. This has created a need for compact, modular expression systems capable of producing multiple guide RNAs (gRNAs) from a single transcript while preserving high editing efficiency. To address this, we have developed the drive-and-process (DAP) array, a modular architecture composed of alternating gRNA and tRNA units. The DAP array design exploits the cell's endogenous tRNA processing machinery to cleave each tRNA from the array and release individual gRNAs, thereby enabling simultaneous editing at multiple loci following hybridization with Cas9. Here, we outline key design considerations and experimental steps required for the construction and deployment of DAP arrays as multiplex base- or prime editing tools in human cells (e.g., HEK293T). We also highlight how the DAP array can be leveraged to enable efficient processing of gRNAs along with other RNAs of similar size, such as shRNA, potentially broadening its usage in addressing complex biological questions and therapeutic applications that require coordinated genetic perturbation.},
}

Humans
*RNA, Guide, CRISPR-Cas Systems/genetics
*Gene Editing/methods
HEK293 Cells
*CRISPR-Cas Systems
RNA, Transfer/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42240879,
year = {2026},
author = {Jeong, H and Kim, H and Cho, E and Lee, HK and Kim, DY and Keum, B and Jung, C},
title = {Enhancing Next-Generation Sequencing Sensitivity with High-Recovery Adapter Ligation and Cas9-Mediated Dimer Elimination.},
journal = {Clinical chemistry},
volume = {72},
number = {9},
pages = {973-983},
doi = {10.1093/clinchem/hvag052},
pmid = {42240879},
issn = {1530-8561},
support = {//Technology Innovation Program/ ; 20009356//Ministry of Trade, Industry & Energy/ ; //National Research Foundation of Korea/ ; 2021-NR061248//Korean government/ ; RS-2024-00440975//Korean government/ ; //Bio & Medical Technology Development Program/ ; //National Research Foundation/ ; RS-2022-NR067272//Ministry of Science & ICT/ ; RS-2023-00259824//Ministry of Science & ICT/ ; },
mesh = {Humans ; *High-Throughput Nucleotide Sequencing/methods ; *CRISPR-Cas Systems ; *Circulating Tumor DNA/genetics/blood ; Sensitivity and Specificity ; Gene Library ; Sequence Analysis, DNA/methods ; },
abstract = {BACKGROUND: Accurate detection of ultra-low-frequency variants is a major challenge in clinical liquid biopsy. In early cancer detection and minimal residual disease monitoring, Circulating tumor (ctDNA) may fall below 0.1% variant allele frequency, making sensitivity highly dependent on molecular recovery during library preparation. Losses at early steps, especially adapter ligation, permanently reduce analyzable molecules and cannot be rescued by deeper sequencing or bioinformatic refinement.

METHODS: We developed Powerful Recovery and Improved Dimer Elimination (PRIDE) next-generation sequencing NGS, a library preparation strategy that increases adapter ligation efficiency and removes adapter dimers via sequence-specific Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) cleanup. PRIDE NGS is compatible with standard clinical work flows and requires no added sequencing depth or changes to downstream bioinformatic pipelines. Performance was assessed by targeted sequencing of cell-free (cfDNA) reference standards and clinical plasma samples.

RESULTS: PRIDE NGS improved recovery and detection of low-frequency variants vs conventional preparation. In reference standards, it detected more variants at low allele frequencies, particularly below 0.1%. In clinical plasma samples, it similarly increased detection, including variants predicted to have moderate or high functional impact. These gains occurred at comparable or lower sequencing depth, indicating sensitivity improvements driven by enhanced molecular recovery.

CONCLUSIONS: By overcoming a key bottleneck in library preparation, PRIDE NGS lowers the practical detection threshold for ultra-low-frequency variants in liquid biopsy. This clinically applicable approach improves analytical sensitivity by lowering the detection limit without increasing the sequencing burden, supporting routine testing and longitudinal monitoring.},
}

Humans
*High-Throughput Nucleotide Sequencing/methods
*CRISPR-Cas Systems
*Circulating Tumor DNA/genetics/blood
Sensitivity and Specificity
Gene Library
Sequence Analysis, DNA/methods

@article {pmid42261770,
year = {2026},
author = {Zhang, L and Yang, C and Yao, Q and Du, X and Ding, S and Shi, Y and Sheng, C and Wang, M and Han, Y and Luo, H},
title = {Machine Learning-Enhanced Ultrasensitive Immuno-CRISPR Array Facilitates Early Diagnosis of Alzheimer's Disease by Detecting Multiple Plasma Biomarkers.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {49},
pages = {e75983},
pmid = {42261770},
issn = {2198-3844},
support = {82401859//National Natural Science Foundation of China/ ; XKTP2025B04//First-class Discipline Breakthrough Initiative of Hainan University/ ; 826MS0122//Hainan Provincial Natural Science Foundation of China/ ; 826QN0570//Hainan Provincial Natural Science Foundation of China/ ; 825QN284//Hainan Provincial Natural Science Foundation of China/ ; },
mesh = {*Alzheimer Disease/diagnosis/blood ; Humans ; *Biomarkers/blood ; *Machine Learning ; Early Diagnosis ; *Amyloid beta-Peptides/blood ; Sensitivity and Specificity ; tau Proteins/blood ; CRISPR-Cas Systems/genetics ; },
abstract = {Early and accurate diagnosis of Alzheimer's disease (AD) remains a significant challenge due to the multifactorial and dynamic nature of its pathology. Although plasma-based biomarkers such as amyloid-β (Aβ) and phosphorylated tau (p-tau) have shown promise as diagnostic indicators, current single-biomarker detection techniques lack the requisite sensitivity and specificity for early-stage diagnosis. Here, we present the development of an ultrasensitive CRISPR-based multi-protein detection array (UCMDA) capable of concurrently detecting six core AD biomarkers, including Aβ40, Aβ42, p-tau[181], p-tau[217], p-tau[231], and p-tau[396,404]. By integrating antibody pair-based multiplex recombinase polymerase amplification (RPA) with spatially encoded CRISPR-Cas12a detection, the UCMDA achieves a detection limit of 1 fg/mL, which is 10 000-fold more sensitive than conventional ELISA. Clinical validation in a cohort of 155 plasma samples demonstrated that logistic regression (LR)-based integration of the six biomarkers significantly enhanced diagnostic performance, with the multi-biomarker model substantially outperforming single-biomarker approaches in diagnosing AD-MCI and AD. This platform offers a scalable, cost-effective, and minimally invasive strategy for early detection and disease monitoring. This work highlights the potential of CRISPR-based multiplex protein detection technologies combined with machine learning-assisted analysis to enhance the precision of diagnosing neurodegenerative disorders.},
}

*Alzheimer Disease/diagnosis/blood
Humans
*Biomarkers/blood
*Machine Learning
Early Diagnosis
*Amyloid beta-Peptides/blood
Sensitivity and Specificity
tau Proteins/blood
CRISPR-Cas Systems/genetics

@article {pmid42295796,
year = {2026},
author = {Miller, MA and Vo, N and Utkarsh, and Sokirniy, I and Pritchard, J and Freedman, BS and Medina, SH},
title = {Ultrasound-Actuated Gene Editing in Human Kidney Organoids.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {49},
pages = {e20402},
pmid = {42295796},
issn = {2198-3844},
support = {R21DK128638/GF/NIH HHS/United States ; R35GM142902/GF/NIH HHS/United States ; U01DK127553/GF/NIH HHS/United States ; U01AI176460/GF/NIH HHS/United States ; R21DK128638/DK/NIDDK NIH HHS/United States ; R35GM142902/GM/NIGMS NIH HHS/United States ; U01DK127553/DK/NIDDK NIH HHS/United States ; U01AI176460/AI/NIAID NIH HHS/United States ; },
mesh = {Humans ; *Organoids/metabolism ; *Kidney/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics ; },
abstract = {Efficient delivery of gene editing ribonucleoproteins (RNPs) into the interior of solid tissues remains a key hurdle to the clinical translation of non-viral CRISPR-Cas9 technologies. Here, we report acoustically-actuated peptide nanoemulsions (NPeps) that can be spatiotemporally guided and activated by ultrasound to ballistically deliver RNPs into cells within the bulk of dense 3D cellular structures. Using human kidney organoids as a model, we demonstrate NPep vectors improve the spatial profile of gene editing in the organoid mass relative to commercial lipofection reagents, without disruption of tissue structure or qualitative viability features. This technologic paradigm is poised to advance imaging-guided, deep tissue RNP delivery modalities to expand the clinical diagnostic and therapeutic potential of CRISPR-Cas9 editing strategies.},
}

Humans
*Organoids/metabolism
*Kidney/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Ribonucleoproteins/genetics

@article {pmid42360136,
year = {2026},
author = {Ma, L and Yao, P and Wu, S and Shi, Y and Qin, L and Li, B and Zhu, J and Huang, M and Zhu, Y and Song, Y and Pang, J and Guo, Z and Wu, G and Wang, C and Xu, K and Huang, R and Kuang, Q and Qu, L and Pan, C and Xie, X and Zhu, Q and Huang, J and Lin, Q},
title = {Discovery and Engineering of a Rat Endogenous Retrovirus Reverse Transcriptase for Efficient Prime Editing.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {49},
pages = {e75888},
pmid = {42360136},
issn = {2198-3844},
support = {2024YFC3408200//National Key R&D Program of China/ ; 2024YFF1000800//National Key R&D Program of China/ ; 2023ZD04074//STI 2030-Major Projects/ ; 20253BAC260005//Frontier Technology Program of Jiangxi Provincial Natural Science Foundation/ ; 2023-NJS-00-012//Invigorate the Seed Industry of Guangdong Province/ ; 32422050//National Natural Science Foundation of China/ ; 32401250//National Natural Science Foundation of China/ ; 2023ZT10N019//Young Elite Scientists Sponsorship Program of the China Association for Science and Technology, the Guangdong Provincial "Pearl River Talent Program" Innovation and Entrepreneurship Team Project/ ; AB24153006//Key R&D Program of Guangxi Province/ ; 2025A04J7124//Science and Technology Projects in Guangzhou/ ; 2025A04J3669//Science and Technology Projects in Guangzhou/ ; 2023B10564004//specific university discipline construction project/ ; },
mesh = {Animals ; *Gene Editing/methods ; Rats ; *RNA-Directed DNA Polymerase/genetics/metabolism ; *Endogenous Retroviruses/genetics/enzymology ; *CRISPR-Cas Systems/genetics ; Humans ; },
abstract = {CRISPR-based prime editors (PEs) install precise edits into genomic DNA without generating double-strand breaks. Their editing efficiency is highly dependent on reverse transcriptases (RTs), but efficient RT candidates remain limited. Here, we identified 19 novel active RTs by screening 558 candidates. Among them, RERV-RT, derived from Rattus norvegicus, exhibited the highest activity. Through structure-guided engineering and deep mutational scanning, we developed an optimized variant, enRERV-RT, which outperforms conventional M-MLV-RT-based PE systems by 1.20-fold in mammalian and plant cells, and by 1.88-fold at hard-to-edit loci, while enabling precise multiplex editing of functionally relevant genes. Additionally, we developed a high-throughput platform, TRAP-seq-PE, to systematically evaluate prime editor performance. Across diverse mutation types, we found that PE systems based on enRERV-RT exhibited higher editing efficiencies than those based on M-MLV-RT. Collectively, our work establishes a versatile, high-efficiency PE system, thereby facilitating advances in clinical gene therapy and precise crop breeding.},
}

Animals
*Gene Editing/methods
Rats
*RNA-Directed DNA Polymerase/genetics/metabolism
*Endogenous Retroviruses/genetics/enzymology
*CRISPR-Cas Systems/genetics
Humans

@article {pmid42470685,
year = {2026},
author = {Hou, C and Yang, R and Guan, X and Zhang, J and Guo, C and Zhang, S and Pei, M and Schalper, KT and Schreiber, D and Liu, X and Liu, C},
title = {Wax-in-a-Tube: A Simple, Rapid, One-Pot Platform for Molecular Detection.},
journal = {ACS sensors},
volume = {11},
number = {8},
pages = {6673-6681},
doi = {10.1021/acssensors.5c04476},
pmid = {42470685},
issn = {2379-3694},
support = {U01CA269147/CA/NCI NIH HHS/United States ; R01AI194917//National Institute of Allergy and Infectious Diseases/ ; R01EB023607/EB/NIBIB NIH HHS/United States ; },
mesh = {*DNA, Viral/analysis/genetics ; *Nucleic Acid Amplification Techniques/methods/instrumentation ; *Herpesvirus 2, Human/genetics/isolation & purification ; Humans ; Rapid Diagnostic Tests ; Recombinases/metabolism ; CRISPR-Cas Systems ; },
abstract = {CRISPR technology has emerged as a powerful platform for highly sensitive and specific nucleic acid detection, particularly when coupled with isothermal amplification. However, conventional two-step CRISPR assays still require manual operations, such as shaking, centrifugation, or vortexing, that complicate the workflow and increase the risk of aerosol contamination. Here, we present a wax-in-a-tube (WIAT) platform that enables simple, rapid, one-pot recombinase polymerase amplification (RPA) and CRISPR-based detection by leveraging the phase-change properties of a molded wax separator, thereby eliminating additional manual steps and minimizing contamination risks. Using a molding approach, we directly integrated the wax separator into the reaction tube to physically partition different reaction components. The WIAT platform achieved a detection sensitivity of 10 aM for HSV-2 DNA, comparable to that of standard two-step assays. We further validated its clinical performance using HSV-2 swab samples, demonstrating results comparable to those obtained with PCR. Together, these findings establish the WIAT platform as a simple, rapid, and highly sensitive one-pot RPA-CRISPR assay with strong potential for point-of-care infectious disease detection and early surveillance.},
}

*DNA, Viral/analysis/genetics
*Nucleic Acid Amplification Techniques/methods/instrumentation
*Herpesvirus 2, Human/genetics/isolation & purification
Humans
Rapid Diagnostic Tests
Recombinases/metabolism
CRISPR-Cas Systems

@article {pmid42486986,
year = {2026},
author = {Gao, Y and Ma, Y and Yu, K and Liu, Y and Gu, B and Tang, H and Yan, W and Yang, S and Su, J and Wang, X and Ma, X and Wang, X and Wang, F and Li, Q and Liu, M and Wang, H},
title = {Efficient and precise programmable DNA knock-in without double-strand breaks.},
journal = {Nature},
volume = {657},
number = {8130},
pages = {284-294},
pmid = {42486986},
issn = {1476-4687},
mesh = {*Gene Knock-In Techniques/methods ; *DNA Breaks, Double-Stranded ; *CRISPR-Cas Systems/genetics ; Humans ; Animals ; *Gene Editing/methods ; *DNA/genetics ; Deoxyribonuclease I/metabolism ; Receptors, Chimeric Antigen/genetics ; Mice ; Receptors, Antigen, T-Cell/genetics ; INDEL Mutation/genetics ; },
abstract = {Programmable gene knock-in holds substantial promise for treating genetic diseases and advancing cell therapies. However, achieving precise and efficient kilobase-scale DNA fragment integration remains challenging[1,2]. Here we report CRISPR kilobase-scale nickase-targeting (KNIT) editing for efficient, precise and programmable kilobase-scale DNA insertion without double-strand DNA cleavage, which is enabled through the coupling of a Cas9 nickase with a DNA donor recruiting system. KNIT editing facilitates programmable integration of DNA fragments from 0.7 kb to more than 10 kb and is effective across genomic loci and cell types. It achieves up to 89% efficiency and markedly reduces unintended insertion-deletion mutation (indels) rates, translocations and off-target editing. The system supports repeated insertion editing and multiloci gene knock-in with minimal translocations. Its enhanced version, KNIT editor 2, further improves efficiency via a single transfection. Moreover, in mutant cells with a pathological mutation, KNIT editing restores normal gene expression by inserting a therapeutic gene into a safe harbour locus or its native locus. Notably, KNIT editing enables non-viral and programmable chimeric antigen receptor T cell (CAR-T cell) engineering without double-strand breaks and with clinically relevant efficiencies. Moreover, the engineered CAR-T cells exhibit effective antitumour activity in vitro and in mouse models. Therefore, by achieving programmable and site-specific kilobase-scale DNA insertions without double-strand breaks while reducing unintended outcomes, KNIT editing provides a versatile platform for advancing personalized medicine.},
}

*Gene Knock-In Techniques/methods
*DNA Breaks, Double-Stranded
*CRISPR-Cas Systems/genetics
Humans
Animals
*Gene Editing/methods
*DNA/genetics
Deoxyribonuclease I/metabolism
Receptors, Chimeric Antigen/genetics
Mice
Receptors, Antigen, T-Cell/genetics
INDEL Mutation/genetics

@article {pmid42571623,
year = {2026},
author = {Zhou, M and Du, K and Jiang, M and Xu, X and Su, X and Xie, Y and Zhang, D and Sun, X and Peng, G and Xia, K and Hu, Z},
title = {FOCUS: A Dual-Mismatch crRNA Strategy Unlocks High-Fidelity One-Step SNV Detection with Cas12a.},
journal = {ACS sensors},
volume = {11},
number = {8},
pages = {7335-7348},
doi = {10.1021/acssensors.6c01689},
pmid = {42571623},
issn = {2379-3694},
mesh = {*CRISPR-Cas Systems/genetics ; *Polymorphism, Single Nucleotide/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; Humans ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Nucleic Acid Amplification Techniques/methods ; Base Pair Mismatch ; DNA/genetics ; Rapid Diagnostic Tests ; },
abstract = {CRISPR/Cas12a has emerged as a powerful tool for nucleic acid detection; however, its clinical utility is severely hampered by intrinsic limitations in single-nucleotide variant (SNV) discrimination, reliance on pre-processed single-stranded DNA (ssDNA) templates, and cumbersome multi-step workflows. Here, we report a novel molecular design principle by engineering crRNAs with site-specific dual mismatches (positions 12 and 14 relative to the PAM), which we systematically demonstrate to drastically enhance the SNV discrimination capability of Cas12a. Leveraging this breakthrough, we developed FOCUS (Fast One-step CRISPR-based Universalizable SNV detection system), an all-in-one CRISPR sensing platform that enables isothermal detection of SNVs from double-stranded DNA (dsDNA) by integrating amplification and detection in a single reaction system. FOCUS achieved attomolar-level sensitivity (13.15 aM) and ultrafast readout (< 20 min) for distinguishing the highly homologous survival motor neuron 1 (SMN1) and SMN2 genes-the gold standard challenge for SNV genotyping in spinal muscular atrophy (SMA) diagnostics. To validate its clinical translatability, FOCUS was successfully adapted to a low-cost, equipment-free assay using lateral flow strips and UV visualization, facilitating point-of-care testing (POCT). In a comprehensive validation across 175 clinical samples, FOCUS exhibited 100% diagnostic concordance with gold-standard methods for SMA (21 samples), high-risk HPV 16/18 (27 samples), Staphylococcus aureus (20 samples), and SARS-CoV-2 (107 samples). Collectively, our study establishes a generalizable engineering strategy for Cas12a crRNAs and presents FOCUS as a robust, versatile, and field-deployable solution for precision SNV genotyping, underscoring the translational medicine value of FOCUS in molecular diagnostics.},
}

*CRISPR-Cas Systems/genetics
*Polymorphism, Single Nucleotide/genetics
*CRISPR-Associated Proteins/metabolism/genetics
Humans
*Endodeoxyribonucleases/genetics/metabolism
*Bacterial Proteins/genetics/metabolism
Nucleic Acid Amplification Techniques/methods
Base Pair Mismatch
DNA/genetics
Rapid Diagnostic Tests

@article {pmid42608059,
year = {2026},
author = {Chen, Z and Zhou, J and Galli, M and Iohannes, SD and Clark, T and Debernardi, JM and Dubcovsky, J and Jackson, D and Gallavotti, A},
title = {The combination of morphogenic regulators BABY BOOM and GRF-GIF improves maize transformation efficiency and promotes leaf regeneration.},
journal = {The New phytologist},
volume = {252},
number = {1},
pages = {260-275},
pmid = {42608059},
issn = {1469-8137},
support = {2424271//Division of Molecular and Cellular Biosciences/ ; 1916804//Division of Integrative Organismal Systems/ ; },
mesh = {*Zea mays/genetics/physiology/embryology ; Plants, Genetically Modified ; *Regeneration/genetics ; *Transformation, Genetic ; *Plant Leaves/physiology ; *Plant Proteins/metabolism/genetics ; Gene Expression Regulation, Plant ; *Transcription Factors/metabolism/genetics ; Triticum/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Transformation is an indispensable tool for plant genetics and functional genomics. Although stable transformation in maize is no longer a major obstacle, there remains a need for accessible and efficient methods for academic laboratories. Here, we present the GGB system, a rapid and efficient approach optimized for immature embryo transformation in B104 and other maize lines. This system combines two distinct morphogenetic regulators, the wheat GRF4-GIF1 chimera and the maize BABY BOOM (BBM) transcription factor (hence the name "GGB") with a modified QuickCorn protocol, enabling regeneration of transformed maize plantlets in c. 2 months with an efficiency 7-fold higher than when compared to either morphogenic factor used in isolation. Expression of both regulators did not significantly affect development, eliminating the need to excise them after regeneration. However, transmission of the transgenic GGB construct through pollen was significantly reduced, potentially aiding transgenic line containment. We show that the GGB system is adaptable for CRISPR-Cas9 editing and reporter line generation. Furthermore, stable GGB transformants exhibited high leaf regeneration capacity via somatic embryogenesis. RNA-seq time-course profiling of GGB leaf cultures identified additional factors that could promote regeneration and led to the discovery of asparagine and trehalose as additional media components that significantly enhanced leaf regeneration.},
}

*Zea mays/genetics/physiology/embryology
Plants, Genetically Modified
*Regeneration/genetics
*Transformation, Genetic
*Plant Leaves/physiology
*Plant Proteins/metabolism/genetics
Gene Expression Regulation, Plant
*Transcription Factors/metabolism/genetics
Triticum/genetics
CRISPR-Cas Systems/genetics

@article {pmid42669708,
year = {2026},
author = {Boggess, SC and Gandhi, V and Tsai, MC and Marzette, E and Teyssier, N and Chou, JY and Hu, X and Cramer, A and Yadanar, L and Shroff, K and Jeong, CG and Eidenschenk, C and Hanson, JE and Tian, R and Kampmann, M},
title = {A massively parallel CRISPR-based screening platform for modifiers of neuronal depolarization.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42669708},
issn = {2041-1723},
support = {U54 NS123746/NS/NINDS NIH HHS/United States ; 23AARF-1027616/ALZ/Alzheimer's Association/United States ; EDUC2-12730//California Institute for Regenerative Medicine (CIRM)/ ; U54 NS123746//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; },
mesh = {Humans ; *Neurons/metabolism/physiology ; Induced Pluripotent Stem Cells/cytology/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Calcium/metabolism ; },
abstract = {Understanding the complex interplay between gene expression and neuronal activity is crucial for unraveling the molecular mechanisms underlying cognitive function and neurological disorders. Here, we developed pooled screens using CRISPR interference (CRISPRi) and the fluorescent calcium integrator CaMPARI2 to evaluate genetic modifiers of neuronal depolarization. Using this screening method, we evaluated 1343 genes for their effect on depolarization in a human iPSC-derived neuron model, revealing potential links to neurodegenerative and neurodevelopmental disorders. These genes include known regulators of neuronal excitability, such as TARPs and ion channels, as well as genes associated with autism spectrum disorder and Alzheimer's disease not previously described to affect neuronal depolarization. This CRISPRi-based screening platform offers a versatile tool to uncover molecular mechanisms controlling neuronal function in health and disease.},
}

Humans
*Neurons/metabolism/physiology
Induced Pluripotent Stem Cells/cytology/metabolism
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Calcium/metabolism

@article {pmid42669834,
year = {2026},
author = {},
title = {Correction to "Accurate Molecular Sensing based on a Modular and Customizable CRISPR/Cas-Assisted Nanopore Operational Nexus (CANON)".},
journal = {Angewandte Chemie (International ed. in English)},
volume = {},
number = {},
pages = {e6219762},
doi = {10.1002/anie.6219762},
pmid = {42669834},
issn = {1521-3773},
}

@article {pmid42670258,
year = {2026},
author = {Ng, CF and Krishnamurthy, D and Dextre, A and Chorlay, A and Ott, M and Fletcher, DA},
title = {LUCas: Light-Uncaged Cas13a using photocleavable interfering guide RNAs.},
journal = {Nucleic acids research},
volume = {54},
number = {16},
pages = {},
pmid = {42670258},
issn = {1362-4962},
support = {//Schmidt Science Fellowship/ ; //Rhodes Trust/ ; //Burroughs Wellcome Career Award/ ; //European Molecular Biology Organization/ ; DBI-1548297//National Science Foundation/ ; //Wagner Foundation/ ; 4R33AI140465-04//National Institute of Allergy and Infectious Diseases/ ; //James B. Pendleton Charitable Trust/ ; //Gordon and Betty Moore Foundation/ ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Kinetics ; *CRISPR-Associated Proteins/genetics/metabolism ; Humans ; Ultraviolet Rays ; Photolysis ; Light ; },
abstract = {CRISPR diagnostics enable sensitive detection of infectious diseases, with the RNA endonuclease Cas13a providing specific, amplification-free RNA detection through collateral trans-cleavage of fluorescent reporters. However, background cleavage from unbound enzyme, contaminating nucleases, and unsynchronized initiation of reactions limits assay sensitivity and interpretability. A strategy to precisely control the onset of Cas13a catalytic activity, essentially a molecular "starting gun," would address these challenges. Here, we introduce Light-Uncaged Cas13a (LUCas), a light-controllable system that directly blocks Cas13a trans-cleavage activity using a photocleavable interfering guide RNA, even in the presence of target RNA. Brief UV illumination releases this suppression, restoring full activity. Quantitative kinetic analysis reveals an ~100-fold suppression of trans-cleavage activity prior to photo-uncaging, including suppression of target-independent background activity. Using measured kinetic parameters, we predict and experimentally validate the limit of detection of the LUCas system for direct detection. We further demonstrate a multiplexed detection strategy termed "temporal barcoding," enabling quantitative detection of viral co-infections in a single bulk reaction. Finally, LUCas is shown to be compatible with one-pot isothermal amplification for enhanced sensitivity and direct detection of target RNA spiked into blood plasma. Together, these results establish LUCas as a general framework for mechanistically informed, light-based control of Cas13a activity.},
}

*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*CRISPR-Cas Systems/genetics
Kinetics
*CRISPR-Associated Proteins/genetics/metabolism
Humans
Ultraviolet Rays
Photolysis
Light

@article {pmid42670666,
year = {2026},
author = {Mirzaee, Z},
title = {Advancing antimicrobial peptides: Mechanisms, design, and applications in the post-antibiotic era.},
journal = {Protein and peptide letters},
volume = {34},
number = {1},
pages = {19-30},
doi = {10.1016/j.ppl.2026.07.002},
pmid = {42670666},
issn = {1875-5305},
mesh = {*Antimicrobial Peptides/chemistry/pharmacology/therapeutic use ; Humans ; *Drug Design ; Protein Engineering ; Animals ; Structure-Activity Relationship ; *Anti-Bacterial Agents/chemistry/pharmacology ; Bacteria/drug effects ; *Antimicrobial Cationic Peptides/chemistry/pharmacology ; },
abstract = {The rapid emergence of multidrug-resistant and extensively drug-resistant bacteria has intensified the need for alternative antimicrobial strategies in the post-antibiotic era. Antimicrobial peptides (AMPs), as evolutionarily conserved components of innate immunity, have attracted considerable attention due to their broad-spectrum antimicrobial activity, rapid mechanisms of action, and lower propensity for resistance development. This review summarizes the structural diversity, mechanisms of action, and structure-activity relationships (SAR) of AMPs that underpin their biological activity and guide the rational design of next-generation peptide therapeutics. It further discusses recent advances in peptide engineering, peptidomimetic design, machine learning-assisted discovery, innovative production platforms, and the application of CRISPR-Cas genome editing for production host optimization. In addition, the review highlights synergistic therapeutic strategies, current clinical progress, and the expanding applications of AMPs in medicine, food preservation, agriculture, and aquaculture. Despite these advances, challenges including limited stability, potential toxicity, manufacturing costs, and regulatory barriers continue to hinder widespread clinical translation of AMP-based therapeutics. By integrating recent experimental and computational advances with current translational challenges and future perspectives, this review provides a comprehensive overview of the field and highlights key directions for the rational development and clinical translation of next-generation antimicrobial peptides to combat antimicrobial resistance.},
}

*Antimicrobial Peptides/chemistry/pharmacology/therapeutic use
Humans
*Drug Design
Protein Engineering
Animals
Structure-Activity Relationship
*Anti-Bacterial Agents/chemistry/pharmacology
Bacteria/drug effects
*Antimicrobial Cationic Peptides/chemistry/pharmacology

@article {pmid42671221,
year = {2026},
author = {Xu, Y and He, X and Xu, T},
title = {LAMP-CRISPR Integrated Platforms for Rapid Detection of Microbial Pathogens: Principles, Applied Strategies, and the Road to Field Translation.},
journal = {Journal of applied microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1093/jambio/lxag220},
pmid = {42671221},
issn = {1365-2672},
abstract = {Loop-mediated isothermal amplification (LAMP) integrated with CRISPR-Cas systems has emerged as a promising molecular diagnostic platform for the rapid detection of microbial pathogens. By combining the efficient nucleic acid amplification of LAMP with the sequence-specific recognition capability of CRISPR-Cas effectors, these platforms offer potential advantages in analytical sensitivity, specificity, operational simplicity, and field applicability. In this review, we summarize the principles, assay formats, and recent advances of LAMP-CRISPR technologies for detecting a broad spectrum of microbial pathogens, including bacterial, viral, fungal, and parasitic agents across clinical, veterinary, food safety, environmental, and agricultural applications. Representative studies are compared with attention to pathogen type, sample matrix, assay design, CRISPR-Cas system, readout format, analytical performance, and practical application. We further discuss major technical challenges that continue to hinder practical implementation, particularly complex sample pretreatment, workflow integration, carry-over contamination, reagent stability, multiplexing capability, and platform standardization. Attention is given to sample pretreatment and system-level integration, including current extraction and rapid lysis strategies, closed-tube reactions, portable readouts, and microfluidic or cartridge-based formats, which may support simplified "sample-in, answer-out" diagnostic workflows. Finally, we outline future directions for improving matrix-adapted sample processing, assay robustness, standardized validation, large-scale evaluation, and field deployment. This review provides a structured overview of current LAMP-CRISPR platforms and highlights key technological considerations for translating rapid microbial pathogen detection from laboratory research to real-world applications.},
}

@article {pmid42673787,
year = {2026},
author = {Lv, L and Zhang, Y and Fan, Y and Guo, B and Chen, Y},
title = {Bivalent aptamer-assisted CRISPR-Cas12a sensor for precise vancomycin therapeutic drug monitoring.},
journal = {Talanta},
volume = {312},
number = {Pt B},
pages = {130504},
doi = {10.1016/j.talanta.2026.130504},
pmid = {42673787},
issn = {1873-3573},
abstract = {Therapeutic drug monitoring (TDM) of vancomycin (VAN) is critical for maximizing efficacy and minimizing toxicity, but conventional methods are constrained by high costs, slow turnaround times, and operational complexity. To address these limitations, we developed a novel Bivalent Aptamer-assisted CRISPR-Cas12a Sensor (termed BACS) for rapid and precise VAN detection. Central to this platform is a high-affinity bivalent aptamer (2AP33), engineered via molecular docking-guided truncation and rational linker design, which exhibits significantly enhanced binding avidity compared to its monovalent counterpart. This aptamer was integrated into a CRISPR-Cas12a system based on a competitive binding mechanism, where target binding modulates Cas12a trans-cleavage activity. The optimized BACS achieved a wide linear detection range (1-50 μM) with a low limit of detection (0.64 μM) in clinical serum, fully covering the clinical therapeutic window. Notably, the assay is rapid (within 10 min), cost-effective, and simple. Critically, the clinical practicality and reliability of BACS were rigorously validated with 175 clinical serum samples, showing exceptional concordance with both the gold standard method and a classical method. This work not only provides a reliable tool for VAN TDM but also offers an adaptable strategy for developing high-performance CRISPR-powered biosensors for diverse clinical analytes through a streamlined molecular engineering pipeline.},
}

@article {pmid42674013,
year = {2026},
author = {Liu, H and Liu, Y and Xu, Y and Wang, Z and Yu, Y and Su, G and Qu, G},
title = {Inhibitor-Regulated Cas12a Activation Enables Highly Sensitive and One-Pot Detection of Drug-Resistant Genes in River Water.},
journal = {Analytical chemistry},
volume = {98},
number = {33},
pages = {24024-24034},
doi = {10.1021/acs.analchem.6c00793},
pmid = {42674013},
issn = {1520-6882},
support = {22325606//National Natural Science Foundation of China/ ; 22576049//National Natural Science Foundation of China/ ; 2024HIAS-V001//Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences/ ; 2025ZY01044//Central Guiding Local Science and Technology Development Fund Projects/ ; },
mesh = {*Rivers/microbiology/chemistry ; CRISPR-Cas Systems ; *Bacterial Proteins/genetics/metabolism ; *Drug Resistance, Bacterial/genetics ; Nucleic Acid Amplification Techniques/methods ; *CRISPR-Associated Proteins/metabolism/genetics ; },
abstract = {Monitoring environmental drug-resistance genes (DRGs) plays a pivotal role in preventing the transmission of antimicrobial resistance, thereby reducing public health risks. In this study, a one-pot recombinase polymerase amplification (RPA)/clustered regularly interspaced short palindromic repeat (CRISPR) assay was developed for monitoring DRGs in river water. To overcome compatibility challenges between RPA and CRISPR systems, four glycosaminoglycans (heparin sodium, nadroparin calcium, dalteparin sodium, and chondroitin sulfate A sodium salt) with different molecular weights or negative charge density were evaluated as Cas-enzyme activity modulators. Among them, heparin sodium with the high molecular weight and high strong negative charge density exhibited the best performance in the one-pot DRG detection assay. In the system, CRISPR-Cas12a activity was temporarily inhibited during the RPA amplification phase. When sufficient amplicons were accumulated, Cas12a was activated for signal readout, thereby achieving orderly coupling and precise control of both reactions. To further simplify and improve the reliability of environmental DRG monitoring, a pretreatment method that can eliminate nucleic acid extraction was developed and integrated with the inhibitor-controlled one-pot platform. This assay achieved high sensitivity and specificity when it was applied to river samples, matching the performance of qPCR. The developed assay is simple to operate, has high sensitivity, and is widely adaptable, providing a robust tool for rapid antimicrobial resistance surveillance and exhibiting promise for public health management applications.},
}

*Rivers/microbiology/chemistry
CRISPR-Cas Systems
*Bacterial Proteins/genetics/metabolism
*Drug Resistance, Bacterial/genetics
Nucleic Acid Amplification Techniques/methods
*CRISPR-Associated Proteins/metabolism/genetics

@article {pmid42674033,
year = {2026},
author = {Lin, S and Chen, X and Lang, Z and Lu, B and Jia, Y and Ju, H and Cao, H},
title = {Programmable Versatile Socket: A Tight-Locking and High-Gain CRISPR/Cas12a Molecular Circuit for Ultrasensitive Sensing of Diverse Targets.},
journal = {Analytical chemistry},
volume = {98},
number = {33},
pages = {24577-24592},
doi = {10.1021/acs.analchem.6c04985},
pmid = {42674033},
issn = {1520-6882},
support = {ZDYF2026SHFZ032//Key Research and Development Project of Hainan Province/ ; 2025DNJP0208//International Cooperative Research Project/ ; 22104027//National Natural Science Foundation of China (NSFC)/ ; 22364014//National Natural Science Foundation of China (NSFC)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; DNA/genetics ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Associated Proteins/metabolism/genetics ; *Aflatoxin B1/analysis ; *Biosensing Techniques/methods ; RNA/genetics/chemistry ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/genetics ; },
abstract = {CRISPR/Cas12a has emerged as an important platform for nucleic acid analysis, yet limited catalytic turnover and intrinsic nucleic acid specificity constrain its sensitivity and analyte scope. Herein, a tight-locking and high-gain Cas12a-driven strand displacement amplification (CSDA) molecular circuit is developed as a versatile socket without preamplification for programmable sensing of nucleic acid and non-nucleic acid analytes. CSDA relies on an RNA-DNA three-strand hairpin (RD-TSH) switch containing a 2-nt mismatch. RD-TSH suppresses nonspecific amplification and unintended Cas12a self-activation to ensure tight locking. Screening the number of mismatched bases in RD-TSH and molecular-level mechanistic analyses reveal a DNA breathing-driven two-step unlocking mechanism. Only complete unlocking triggers autocatalytic CSDA, allowing high-gain amplification. The sequence-independent unlocking of RD-TSH confers high orthogonality to CSDA, enabling target-specific modules to be coupled to the CSDA socket as interchangeable plugs via programmable crRNA guides, thus achieving universal detection of both nucleic and non-nucleic analytes. Using Vibrio parahaemolyticus DNA, thermostable direct hemolysin, and aflatoxin B1 as representative targets, CSDA achieved ultrasensitive detection in complex matrices with sensitivity improvements of over 5 orders of magnitude, 42-fold, and 602-fold, respectively. This plug-and-play architecture establishes CSDA as a broadly adaptable and ultrasensitive CRISPR/Cas12a sensing socket, providing a general route toward programmable sensing of diverse analyte classes and a promising strategy for more accurate integrated multitarget analytical platforms.},
}

*CRISPR-Cas Systems/genetics
DNA/genetics
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Associated Proteins/metabolism/genetics
*Aflatoxin B1/analysis
*Biosensing Techniques/methods
RNA/genetics/chemistry
*Endodeoxyribonucleases/metabolism/genetics
*Bacterial Proteins/genetics

@article {pmid42675078,
year = {2026},
author = {Tsui, W and Yang, Y and Wang, C and Li, D and Zhang, Y and Zhao, X and Wu, J and Guo, J and Wang, Y and Cheng, X and Li, X and Kotovskaya, O and Isaev, A and Ma, J and Wang, M},
title = {Dynamic balance of CRISPR-Cas immunity and resistance plasmid anti-immunity mediated by a bifunctional protein AcrIE10.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42675078},
issn = {2041-1723},
support = {W2512095, 81991531, and 32471347//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82402671//National Natural Science Foundation of China (National Science Foundation of China)/ ; 22410710300//Science and Technology Commission of Shanghai Municipality (Shanghai Municipal Science and Technology Commission)/ ; 25-44-02137//Russian Science Foundation (RSF)/ ; },
mesh = {*Klebsiella pneumoniae/genetics/immunology/drug effects/metabolism ; *CRISPR-Cas Systems/immunology/genetics ; *Plasmids/genetics/metabolism ; *Bacterial Proteins/metabolism/genetics/chemistry/immunology ; Promoter Regions, Genetic ; Drug Resistance, Bacterial/genetics ; Gene Expression Regulation, Bacterial ; },
abstract = {Despite targeting by CRISPR-Cas system, antimicrobial resistance plasmids are prevalent in clinical isolates of carbapenem-resistant Klebsiella pneumoniae which represent a major public health threat. A stable co-existence of plasmids and CRISPR-Cas systems is mediated by anti-CRISPR (Acr) proteins. Here, we report that previously identified AcrIE10 encoded by a resistance plasmid combines two functions: it inhibits CRISPR immunity by directly binding Cas7* subunit through its Acr domain, and acts as an Acr-associated (Aca) protein that self-represses the transcription of Acr locus. AcrIE10 is an example of an Aca protein that utilizes N-terminal ribbon-helix-helix (RHH) domain to specifically recognize the inverted repeat (IR) region in its own promoter. Crucially, a dimerization of AcrIE10 dimers is required for the effective binding to the IR and self-repression, while stoichiometry-dependent interaction with Cas7* facilitates transition to de-repressed state. These findings elucidate molecular mechanisms by which AcrIE10 operates as a dual functionAcr-Aca protein to achieve a delicate balance between host CRISPR-Cas immunity and plasmid anti-defense.},
}

*Klebsiella pneumoniae/genetics/immunology/drug effects/metabolism
*CRISPR-Cas Systems/immunology/genetics
*Plasmids/genetics/metabolism
*Bacterial Proteins/metabolism/genetics/chemistry/immunology
Promoter Regions, Genetic
Drug Resistance, Bacterial/genetics
Gene Expression Regulation, Bacterial

@article {pmid42675231,
year = {2026},
author = {Rathna, ARS and Ceasar, SA},
title = {Accelerating iron biofortification in millets: progress, challenges, and future prospects.},
journal = {Planta},
volume = {264},
number = {4},
pages = {},
pmid = {42675231},
issn = {1432-2048},
mesh = {*Biofortification/methods ; *Iron/metabolism ; *Millets/genetics/metabolism ; Gene Editing ; Plant Breeding ; CRISPR-Cas Systems ; },
abstract = {Integrating conventional breeding, omics, and CRISPR-based genome editing can overcome genetic and antinutrient constraints, enabling efficient iron biofortification of millets for sustainable and nutrition-secure food systems. Iron (Fe) deficiency remains one of the most widespread forms of micronutrient malnutrition. Biofortification of staple crops has emerged as a particularly sustainable and scalable strategy to combat this issue. Millets are nutrient-dense staple cereals with exceptional nutritional quality and climate resilience. However, genetic variations and the presence of antinutrients limit Fe content in millets, which highlights the necessity of advancing biofortification strategies. This review examines the present state of multi-dimensional strategies and discusses the future prospects for efficient iron biofortification in millets. We analyzed the efforts made for Fe biofortification in millets, ranging from conventional breeding practices to next-generation molecular approaches. Recent advances in omics have enhanced understanding of Fe uptake, transport, and storage in millets. Furthermore, CRISPR/Cas-based genome editing is discussed for the regulated expression of key Fe-transporter genes and targeted knockout of genes responsible for antinutrients. A multidisciplinary approach is essential to develop high-yielding and Fe-rich millet varieties that can contribute to sustainable nutrition security.},
}

*Biofortification/methods
*Iron/metabolism
*Millets/genetics/metabolism
Gene Editing
Plant Breeding
CRISPR-Cas Systems

@article {pmid42677405,
year = {2026},
author = {Zhang, WY and Pei, WK},
title = {[Cellular barcoding and next-generation lineage tracing: concepts and applications].},
journal = {Sheng li xue bao : [Acta physiologica Sinica]},
volume = {78},
number = {4},
pages = {731-740},
doi = {10.13294/j.aps.2026.0063},
pmid = {42677405},
issn = {0371-0874},
mesh = {*Cell Lineage/genetics ; Humans ; *DNA Barcoding, Taxonomic ; High-Throughput Nucleotide Sequencing ; CRISPR-Cas Systems ; Animals ; Single-Cell Analysis ; },
abstract = {Lineage tracing is a fundamental technique for dissecting cell fate decisions and development process. With recent advances in high-throughput sequencing and single-cell sequencing technologies, cellular barcoding-based lineage tracing strategies have transitioned from low-throughput labeling methods to high-resolution, multidimensional lineage reconstruction. In this review, we systematically summarize four major barcoding paradigms: viral integration-based random integration barcodes, transposon-based random integration barcodes, recombinase-mediated DNA rearrangement (e.g., Cre-loxP), and CRISPR-Cas9-based mutation recording systems. We describe their principles, representative studies, technical advantages, and limitations. Furthermore, we discuss the core bottlenecks in terms of editing precision, integration of spatiotemporal information, and non-invasive lineage tracing, with a focus on cutting-edge advancements such as prime editing, sequential recording systems, strategies for integrating spatial transcriptomics, and epigenetic tracing. Overall, single-cell lineage tracing is evolving from clonal labeling toward the multi-dimensional integration of lineage, state, and space. In the future, the deep integration of precise gene-editing tools with high-resolution spatial omics technologies is expected to enable dynamic and systematic analysis of cellular fate trajectories, thereby providing critical technical support for research in developmental biology and regenerative medicine.},
}

*Cell Lineage/genetics
Humans
*DNA Barcoding, Taxonomic
High-Throughput Nucleotide Sequencing
CRISPR-Cas Systems
Animals
Single-Cell Analysis

@article {pmid42678547,
year = {2026},
author = {Akhtar, MS and Amin, A},
title = {CRISPR-cas systems in pharmacology: functional pharmacogenomics, drug screening, resistance, and therapeutic translation.},
journal = {Functional & integrative genomics},
volume = {26},
number = {1},
pages = {},
pmid = {42678547},
issn = {1438-7948},
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Pharmacogenetics/methods ; Animals ; Precision Medicine ; *Drug Discovery/methods ; },
abstract = {CRISPR-Cas9 gene-editing technology has advanced pharmacological research by enabling targeted genetic modification for disease modeling, therapeutic development, and precision medicine. This review discusses the applications of CRISPR-Cas9 in drug discovery, personalized therapy, cancer drug resistance research, genetic disorders, and antimicrobial resistance. By editing disease-associated genes, CRISPR-Cas9 supports the development of patient-specific therapeutic strategies and more accurate preclinical models. In cancer, CRISPR-Cas9 is used to investigate the target genes involved in treatment resistance, while in genetic disorders, it offers potential mutation-correcting approaches, with the most robust clinical evidence currently seen in selected hemoglobinopathies. CRISPR-based strategies also hold promise for restoring antibiotic susceptibility by targeting genes that confer antibiotic resistance. Despite these advances, clinical translation remains limited by off-target effects, delivery challenges, immune responses, long-term safety concerns, and ethical and regulatory issues. Continued improvements in editing precision, delivery systems, and governance frameworks are essential for responsible clinical integration. Overall, CRISPR-Cas9 represents a vital platform for future pharmacological innovation, but its broad clinical use may require further validation of safety, efficacy, durability, and accessibility.},
}

Humans
*CRISPR-Cas Systems
*Gene Editing/methods
*Pharmacogenetics/methods
Animals
Precision Medicine
*Drug Discovery/methods

@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.},
}